Design, synthesis and biological evaluation of PSMA/hepsin-targeted heterobivalent ligands

Article abstract of DOI:10.1016/j.ejmech.2016.04.033

Cell surface biomarkers such as prostate-specific membrane antigen (PSMA) and hepsin have received considerable attention as targets for imaging prostate cancer (PCa) due to their high cell surface expression in such tumors and easy access for imaging pro

Full text of DOI:10.1016/j.ejmech.2016.04.033

Accepted Manuscript
Design, synthesis and biological evaluation of PSMA/hepsin-targeted heterobivalent
ligands
Milan Subedi, Il Minn, Jianbo Chen, YunHye Kim, Kiwon Ok, Yong Woo Jung, Martin
G. Pomper, Youngjoo Byun
PII:
S0223-5234(16)30326-9
10.1016/j.ejmech.2016.04.033
EJMECH 8548
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2016
Revised Date: 14 March 2016
Accepted Date: 12 April 2016
Please cite this article as: M. Subedi, I. Minn, J. Chen, Y. Kim, K. Ok, Y.W. Jung, M.G. Pomper, Y. Byun,
Design, synthesis and biological evaluation of PSMA/hepsin-targeted heterobivalent ligands, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.033.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of PSMA/hepsin-targeted
heterobivalent ligands
a,#
b,#
a,#
a
a
a
Milan Subedi , Il Minn, Jianbo Chen , YunHye Kim , Kiwon Ok , Yong Woo Jung , Martin
b
a,b,c,
G. Pomper , Youngjoo Byun
*
a
College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 339-700, South
Korea
b
Department of Radiology, Johns Hopkins Medical Institution, 1550 Orleans street, Baltimore
2
1287, MD, USA
c
Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu,
Seoul 152-703, South Korea
*
Corresponding author:
Youngjoo Byun, Ph.D.
College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 339-700, South
Korea.
Tel.: 82-44-860-1619; Fax: 82-44-860-1607; E-mail: yjbyun1@korea.ac.kr
#
Equal contribution

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ABSTRACT
Cell surface biomarkers such as prostate-specific membrane antigen (PSMA) and hepsin have
received considerable attention as targets for imaging prostate cancer (PCa) due to their high cell
surface expression in such tumors and easy access for imaging probes. Novel amidine-containing
indole analogs (13-21) as hepsin inhibitors were designed and synthesized. These compounds
showed in vitro inhibitory activity against hepsin with IC50 values from 5.9 to 70 µM. Based on
the SAR of amidine-derived analogs, the novel heterobivalent compound 30, targeting both
hepsin and PSMA, was synthesized by linking compound 18 with Lys-urea-Glu, the key scaffold
for the specific binding to PSMA, followed by the conjugation of the optical dye sulfoCy7.
Compound 30 exhibited inhibitory activities against PSMA and hepsin, with IC values of 28
5
0
nM and 2.8 µM, respectively. In vitro cell uptake and preliminary in vivo optical imaging studies
of 30 showed selective binding and retention in both PSMA and hepsin high-expressing
PC3/ML-PSMA-HPN cells as compared with low-expressing PC3/ML cells.
KEYWORDS: PSMA, Hepsin, Prostate Cancer, Heterobivalent Ligands, Molecular Imaging

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1
. Introduction
Prostate cancer (PCa) is the most common cancer in American men and the second
leading cause of cancer death in this group [1]. The American Cancer Society estimates that there
were approximately 220,800 new cases of PCa and 27,540 deaths from PCa in 2015
(
www.cancer.org). The current gold standard for diagnosis of PCa is an elevated level of
prostate-specific antigen (PSA) and abnormality of the prostate by digital rectal examination
DRE), followed by prostate needle biopsy [2]. However, benign conditions such as prostatitis
(
and benign prostatic hyperplasia (BPH) can increase the level of PSA [3]. A comprehensive
clinical study in Europe reported that PSA screening can lower the death risk from PCa, while a
US study showed no statistical difference following PSA screening [4-6]. 76% of patients with a
raised PSA level are not diagnosed with PCa, and 2% of patients with fast-growing PCa have a
normal level of PSA (www.prostatecanceruk.org). Conversely, the effectiveness of DRE depends
on the skill and experience of the examiner. 72-82% of individuals who undergo biopsy based on
DRE findings are not diagnosed with PCa [7]. Therefore, there is an urgent need to explore novel
biomarkers for the precise and efficient detection of PCa. Cell surface biomarkers including
v 3
prostate-specific membrane antigen (PSMA), hepsin, and integrin-α β have received substantial
attention as target proteins for the imaging of PCa due to their elevated cell surface expression on
cancer cells compared with normal cells and easy access for imaging probes [8-14].
PSMA, a type II zinc-dependent protease, is highly expressed on the surface of prostate
cancer cells as well as on the neovasculature of most solid tumors [15]. The active site of PSMA
consists of two distinct sub-pockets, which form a ‘glutamate-sensor’ S1 site (pharmacophore)
and an amphiphilic S1 site (non-pharmacophore). A cylindrical ~20Å deep tunnel region exists
adjacent to the S1 site and projects towards the hydrophilic surface of the enzyme [16, 17]. The

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ability to detect overexpression of PSMA has provided novel avenues for the diagnosis and
treatment of PCa. Hepsin is also a cell surface serine protease composed of 413 amino acids,
with a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a
scavenger receptor cysteine-rich (SRCR) domain [18]. Hepsin is overexpressed in up to 90% of
prostate tumors, with levels often increased >10 fold in metastatic PCa than in normal prostate or
BPH [19-21]. PSMA and hepsin may represent excellent targets for PCa imaging since both have
an enzymatic active site in the extracellular region. Imaging probes for the targets do not require
penetration of the cell membrane, thus, have less chance of being degraded by metabolic
enzymes.
Due to the heterogeneous and multifactorial nature of PCa, the search for novel
biomarkers is a key to precise diagnosis and efficient treatment [22]. Recent studies have shifted
from the identification of individual biomarkers to the utilization of combinations of known
specific markers [23]. A multi-targeting strategy has several advantages over single-targeting.
Targeting multiple surface markers simultaneously can improve the sensitivity and specificity of
detection through synergistic binding affinities to target proteins. The concept of targeting more
than one cell surface protein for imaging purposes has been demonstrated by combining two
biomarkers [22-24]. Dual-targeting was found to be more predictive with respect to
distinguishing between PCa and BPH than when a single biomarker is targeted [22]. Eder and
coworkers developed heterodimer molecules targeting both PSMA and the gastrin-releasing
peptide receptor (GRPR), which exhibited high binding affinities to a cell line expressing both
GRPR and PSMA [24]. More recently, Shallal and coworkers have developed heterobivalent
agents targeting PSMA and integrin α
v 3
β [14].
We designed and synthesized a heterobivalent ligand which can bind to PSMA as well as

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hepsin. We used 2-(4-hydroxybiphenyl-3-yl)-1H-indole-5-carboximidamide as a hepsin-binding
scaffold while Lys-urea-Glu moiety as a PSMA-binding scaffold. The amidine group was
reported to interact with Asp189, a key amino acid in the hepsin active site [25]. Lys-urea-Glu
moiety was reported to be strong binder to PSMA despite structural modification on the ε-amine
of Lys. For in vitro cell uptake and in vivo imaging studies, we conjugated a PSMA-hepsin
ligand with an optical dye SulfoCy7.
2
. Results and Discussion
2
.1. Synthesis of hepsin-targeted analogs
As shown in Scheme 1, hepsin-targeted amidine compounds (13-21) were synthesized in
six steps using commercially available aniline, acetophenone, and arylboronic acids.
Preparations of the intermediate imine 2 and the indole-5-carbonitrile 3 were achieved by
applying the procedure previously reported by us [26]. Briefly, the N-aryl imine 2 was prepared
by reacting 4-aminobenzonitrile with 5'-bromo-2'-hydroxyacetophenone in toluene under
piperidine-catalyzed basic condition. The indole-5-carbonitrile 3 was prepared from 2 using
Pd(OAc) (20 mol%) and Bu NBr (2 eq) in DMSO.
2
4
<
Scheme 1 >
The amidine functional group had been reported to interact with the carboxylic acid present in
the side chain of Asp 189 in hepsin. Although several methods for directly converting nitrile into
an amidine group have been reported [27, 28], they were not suitable for the synthesis of a

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designed PSMA-hepsin conjugate which has labile groups under the reported conditions.
Therefore, we utilized an oxadiazole ring as the amidine-protecting group, which can be
removed under mild conditions (H , Raney-nickel). Reaction of 3 with hydroxylamine in ethanol
2
afforded the oxime analog 4 in 60% yield. Compound 4 was subsequently cyclized to give the
oxadiazole 5 using ethyl acetate and ethanol as solvents by modifying a reported procedure [29].
Reaction of 5 with appropriate arylboronic acids under Suzuki Coupling conditions at 100°C for
4
hours, with Pd(PPh
afforded the corresponding biphenyls 6-12. The oxadiazole-containing biphenyls were converted
into the final amidines 13-21 by applying hydrogenation conditions (H , Raney-nickel, 50 psi)
3 4 2 3 2
) as a catalyst, Cs CO as a base, and DMF/H O (5:1) as a solvent,
2
for 7 hours in a mixture of methanol and acetic acid (7:1) [30]. Deprotection of the oxadiazole
ring proceeded in two steps. The O-N bond of the oxadiazole ring is first cleaved, followed by
the cleavage of C-N bond. We observed partially-deprotected compounds during HPLC
experiments, which have similar retention time to the fully-deprotected amidine
compounds 13-21. However, the partially-deprotected compounds were hydrolyzed
slowly at room temperature to give the corresponding amidines.
2
.2. Synthesis of the heterobivalent ligand
The synthesis of the heterobivalent ligand 30 is outlined in Schemes 2 and 3. The use of
protecting group for three hydrophilic functional groups (e.g., amidine, carboxylic acid and
amine) was crucial for the preparation of 30. During the synthesis of 30, the oxadiazole ring as a
protecting group of the amidine was introduced in the first step and removed at the final step
because it was highly stable under acidic or basic reaction conditions and also be deprotected by
hydrogenation without affecting the other functional groups. Deprotection of the Boc group of
compound 9 was achieved using trifluoroacetic acid (TFA) to afford compound 22. Reaction of

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2
2 with Fmoc-Lys(Boc)-OH under traditional peptide-coupling conditions (HATU in DMF)
afforded compound 23 in 56% yield. The Fmoc group of 23 was removed by treatment with 25%
piperidine in DMF at room temperature to give compound 24. The free amine of 24 was
conjugated with the tBu-protected Lys-urea-Glu 25, a known PSMA-binding scaffold [31], to
provide compound 26 in 30% yield. Both the tBu and Boc groups of 26 were removed
quantitatively using 50% TFA in dichloromethane for 2 hours at room temperature to afford
compound 27.
<
Scheme 2 >
Transformation of the oxadiazole ring of 27 into the amidine group was achieved under
hydrogenation conditions (H , Raney-nickel, 50 psi) for 4 hours in dioxane/water/acetic acid
2
(6:2:1) by modifying a previously published procedure [30].
<
Scheme 3 >
The hydrogenation reaction of the oxadiazole moiety generated two products, the amidine 28 and
the partially-deprotected 29. The hydrogenation reaction time was critical for the deprotection of
the oxadiazole ring. The optimal reaction time was found to be 4 hours, with reaction time longer
than 4 hours generating more by-products through the cleavage of amide bonds. The
commercially available SulfoCy7 NHS ester was conjugated to a mixture of 28 and 29 in Tris-
HCl buffer (0.1 M, pH 8.5) at room temperature to afford the dye-linked PSMA-hepsin
conjugates 30 and 31. Pure compounds 30 and 31 were obtained by reversed-phase HPLC,

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applying gradient conditions using 0.5% formic acid in acetonitrile (A) and water (B) as mobile
phase (30% to 40% A in 20 min; 40% to 30% A in 25 min at 3.5 mL/min flow rate) with a
retention time of 5.79 min and 6.21 min, respectively. Compound 31 was hydrolyzed slowly at
room temperature to give compound 30.
2
.3. Molecular docking study
The reported hepsin x-ray crystal structure (PDB ID: 1O5E) in complex with the amidine-
containing ligand (CA-14) was utilized as a template for the docking studies of the synthesized
compounds 13-21 [25]. The ligand CA-14 presented strong hydrogen-bonding interactions with
His57 and Ser195, which were known to be a part of the catalytic triad of hepsin. The positively-
charged amidine group made strong interactions with the carboxylic acid present in Asp189.
<
Figure 1 >
Docking studies of compounds 13-21 with hepsin revealed that they were located in the active
site similar to the crystal ligand CA-14 [25]. As shown in Figure 1, the docked pose of
compound 19 exhibited an additional interaction with Ser214 as compared with the original
ligand.
2
.4. In vitro hepsin and PSMA inhibitory activities
For in vitro hepsin assay, the known hepsin substrate Boc-Gln-Ala-Arg-AMC was used to
determine the hepsin inhibitory activities of the synthesized compounds (13-21 and 30). The
crystal ligand CA-14 (Figure 1) was synthesized by following a reported procedure [32] and used
as a reference for in vitro hepsin assay. All of the synthesized compounds (13-21 and 30) showed

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inhibitory activity against hepsin similar to CA-14, with IC50 values from 5.9 to 70 ꢀM (Table 1).
We observed that the amidine moiety is essential for hepsin-binding activity, since none of the
compounds (5-12) with the oxadiazole ring showed hepsin activity. Even the bulky
heterobivalent compound 30 exhibited similar hepsin-inhibitory activity (IC = 2.8 ꢀM) to the
5
0
monovalent compounds 13-21, indicating that the active site of hepsin tolerates the introduction
of bulky groups.
<
Table 1 >
The PSMA inhibitory activities of the PSMA-hepsin conjugates 30 and 31 were
determined by measuring the amount of glutamate released from the hydrolysis of N-acetyl-L-
aspartyl-L-glutamate (NAAG). The IC50 values of 30 and 31 were 38 nM and 25 nM,
respectively (Figure 2). Although they were less potent than the urea-based low molecular
weight compound ZJ-43 presumably due to the addition of bulky fluorophore and hepsin
binding moieties, 30 and 31 were still sufficiently potent for optical imaging. Previously, the
bulky compounds, which consisted of the optical dyes and the PSMA-binding scaffold (Lys-
urea-Glu) with IC values in the double-digit nanomolar concentration range, exhibited strong
5
0
binding affinity for PSMA-expressing tumors in an in vivo study [33].
<
Figure 2 >
2
.5. In vitro cell uptake study

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Cell uptake studies revealed that 30 and 31 specifically bound to PC3/ML-PSMA
(PSMA-expressing cell line), PC3/ML-HPN (hepsin-expressing cell line), and PC3/ML-PSMA-
HPN (PSMA- and hepsin-expressing cell line) cells (Figure 3). Both compounds exhibited strong
binding affinity for PSMA-expressing cells even at a concentration of 10 nM as compared with
PC3/ML cells. The weak binding of the two compounds to PC3/ML-HPN cells might be due to
their moderate affinity for hepsin. Both compounds also showed a slightly better uptake at higher
concentrations by cells expressing both targets compared with cells expressing PSMA only,
indicating synergistic affinity enhancement.
<
Figure 3 >
2
.6. Preliminary in vivo animal study
We evaluated whether the dual-targeted heterobivalent compound 30 is capable of
specifically targeting PSMA/hepsin-expressing tumors in vivo. We generated a subcutaneous
xenograft model of PC3/ML-PSMA (red circle in Figure 4) and PC3/ML-PSMA-HPN (white
circle in Figure 4), and injected the animal with 1 nmol of 30. Near infrared (NIR) images were
taken at 24 hr post-injection, showing that 30 exhibited a higher uptake and retention in the
PC3/ML-PSMA-HPN tumor than in the PC3/ML-PSMA tumor.
<
Figure 4 >
3
. Conclusion
In summary, we designed, synthesized, and evaluated diverse amidine-functionalized
analogs as hepsin inhibitors. In vitro studies showed that the amidine-containing compounds 13-
1 exhibited moderate affinity for hepsin. The PSMA-hepsin conjugate 30 was prepared by
2

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linking the identified hepsin-binding amidine scaffold with the PSMA-binding Lys-urea-Glu
scaffold, followed by conjugation of the optical dye SulfoCy7. Compound 30 exhibited strong
binding affinity for PSMA and moderate binding affinity for hepsin. In vitro cell uptake and
preliminary in vivo animal optical imaging studies indicated that 30 have a potential to used as a
lead compound in the development of more potent PSMA-hepsin conjugates which may provide
a more powerful mechanism for imaging metastatic prostate cancer.
4
. Experimental Sections
4
4
.1. Chemistry
.1.1. General
Chemicals and solvents used in the reaction were purchased from Aldrich, Acros, TCI,
1
13
and Lumiprobe. H NMR and C NMR spectra were carried out on BRUKER Biospin AVANCE
3
00 MHz and 75 MHz spectrometer, respectively. Chemical shifts were reported as δ values
downfield from internal TMS in appropriate organic solvents. Mass spectra were obtained on
Agilent 6530 Accurate mass Q-TOF LC/MS spectrometer and Agilent 6460 Triple Quad LC/MS
spectrometer. High performance liquid chromatography (HPLC) was carried out on Agilent 1290
Infinity LC using Gemini-NX C18 column (150 mm x 10 mm, 5 µm, 110 Å). Melting points
were determined on microscope hot stage apparatus (Linkam THMS 6000). Hydrogenation was
carried out using Parr-Hydrogenator (Marathon Electric). Silica gel column chromatography
experiments were carried out using Merck Silica Gel F254
.
4
.1.2. 4-{(E)-[1-(5-Bromo-2-hydroxyphenyl)ethylidene]amino}benzonitrile (2)
To a solution of 4-aminobenzonitrile (1.00 g, 8.4 mmol) in toluene (10 mL) were added 5'-

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bromo-2'-hydroxyacetophenone (1.80 g, 8.4 mmol) and piperidine (1 mL). The reaction mixture
was stirred at 130 °C for 6 hr using Dean-Stark apparatus. The reaction mixture was cooled to
room temperature and excess solvent was concentrated to dryness under reduced pressure. The
residue was washed with a mixture of hexane and ethylacetate (25 mL × 2) to afford compound 2
1
as yellow solid. Yield: 56%, R : 0.52 (hexane/EA = 9/1), mp: 145-148 °C, H NMR (300 MHz,
f
3
CDCl ): δ 13.70 (s, 1H, NH), 7.78-7.70 (m, 3H, Ar-H), 7.50 (dd, 1H, Ar-H, J = 8.7 & 2.4 Hz),
7
.02 (d, 2H, Ar-H, J = 8.7 Hz), 6.95 (d, 2H, Ar-H, J = 9.0 Hz), 2.34 (s, 3H, CH ), HRMS (ESI):
3
-
[
M-H] calcd for C H BrN O: 313.0055, found: 312.9987, ESI-MS/MS: 195.0436 [M-Br-CN-
15 11 2
-
OH-H]
4
.1.3. 2-(5-Bromo-2-hydroxyphenyl)-1H-indole-5-carbonitrile (3)
To a solution of compound 2 (0.90 g, 2.9 mmol) in DMSO (10 mL) were added Pd(OAc)
mg, 0.29 mmol) and Bu NBr (1.87 g, 5.8 mmol). The mixture was stirred at 110 °C for 12 hr.
2
(65
4
Upon cooling to room temperature, the reaction mixture was diluted with water (50 mL) and
ethyl acetate (50 mL). The combined organic phase was dried over MgSO filtered, and
4
,
concentrated under reduced pressure. The crude residue was purified by silica gel
chromatography to afford compound 3 as solid. Yield: 33%, R : 0.23 (hexane/EA = 3/1), mp:
f
1
6
1
1
03-105 °C, H NMR (300 MHz, DMSO-d ): δ 11.84 (s, 1H, NH), 10.65 (s, 1H, OH), 8.07 (s,
H, Ar-H), 7.95 (d, 1H, Ar-H, J = 8.7 Hz), 7.59 (d, 1H, Ar-H, J = 8.7 Hz), 7.45-7.32 (m, 2H, Ar-
-
H), 7.22 (s, 1H, Ar-H), 6.98 (d, 1H, Ar-H, J = 8.7 Hz), HRMS (ESI): [M-H] calcd for
-
C H BrN O: 310.9898, found: 310.9807, ESI-MS/MS: 231.0576 [M-Br-H] , 189.0467 [M-Br-
15
9
2
-
CN-OH-H]

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4
.1.4. (Z)-2-(5-Bromo-2-hydroxyphenyl)-N'-hydroxy-1H-indole-5-carboximidamide (4)
To a solution of compound 3 (0.37 g, 1.2 mmol) in ethanol (20 mL) were added hydroxylamine
hydrochloride (0.71 g, 10.2 mmol) and sodium carbonate (0.55 g, 5.1 mmol). The reaction
mixture was stirred at 90 °C for 7 hr. After the completion of reaction, excess solvent was
evaporated to dryness under reduced pressure. The residue was diluted with water (25 mL) and
ethyl acetate (25 mL). The combined organic phase was dried over MgSO filtered, and
4
,
concentrated under reduced pressure. The crude residue was purified by silica gel
chromatography to afford compound 4 as solid. Yield: 60%, R : 0.12 (hexane/EA = 3/1), mp: >
00 °C, H NMR (300 MHz, DMSO-d ): δ 11.37 (s, 1H, OH), 9.39 (s, 1H, NH), 8.22 (s, 1H,
Ar-H), 7.93 (d, 1H, Ar-H, J = 2.4 Hz), 7.83 (d, 1H, Ar-H, J = 1.2 Hz), 7.49-7.38 (m, 2H, Ar-H),
f
1
6
4
7
.28 (dd, 1H, Ar-H, J = 2.4 & 8.4 Hz), 7.10 (s, 1H, Ar-H), 6.94 (d, 1H, Ar-H, J = 8.7 Hz), 5.71
-
(
s, 2H, NH
2
), HRMS (ESI): [M-H] calcd for C15
H12BrN
3
O
2
: 344.0113, found: 344.0068, ESI-
-
2
MS/MS: 310.9815 [M-NH -OH-H]
4
.1.5. 4-Bromo-2-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl]phenol (5)
To a solution of compound 4 (0.23 g, 0.69 mmol) in ethanol (9 mL) were added sodium ethoxide
90 mg, 1.32 mmol) and ethyl acetate (1 mL). The reaction mixture was stirred at 90 °C for 2 hr.
(
After completion of reaction, excess solvent was evaporated to dryness under reduced pressure.
The residue was diluted with water (25 mL) and ethyl acetate (25 mL). The organic phase was
dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was
f
purified by silica gel chromatography to afford compound 5 as solid. Yield: 42%, R : 0.25
1
6
(
hexane/EA = 3/1), mp: 236-238 °C, H NMR (300 MHz, DMSO-d ): δ 11.61 (s, 1H, NH), 10.59
(s, 1H, OH), 8.22 (s, 1H, Ar-H), 7.95 (d, 1H, Ar-H, J = 2.4 Hz), 7.74 (dd, 1H, Ar-H, J = 8.4 &

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1
.5 Hz), 7.58 (d, 1H, Ar-H, J = 8.4 Hz), 7.33 (dd, 1H, Ar-H, J = 2.4 & 8.7 Hz), 7.21 (s, 1H, Ar-
13
H), 6.97 (d, 1H, Ar-H, J = 8.7 Hz), 2.66 (s, 3H, CH ), ), C NMR (75 MHz, DMSO- d ): δ
3
6
117.17, 154.31, 138.36, 135.51, 131.38, 129.78, 128.68, 121.10, 120.72, 120.28, 119.00, 117.82,
-
112.54, 111.16, 103.52, 12.49 (CH
3
), HRMS (ESI): [M-H] calcd for C17
H12BrN
3
O
2
: 368.0113,
-
found: 368.0025, ESI-MS/MS: 327.1152 [M-Ac-OH-H]
4
.1.6. 4'-Chloro-3-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-biphenyl]-4-ol (6)
To a solution of compound 5 (100 mg, 0.27 mmol) in DMF (5 mL) were added 4-chlorophenyl
boronic acid (42 mg, 0.27 mmol), cesium carbonate (182 mg, 0.55 mmol in 1 mL water) and
Pd(PPh ) (16 mg, 0.014 mmol). The reaction mixture was stirred at 100°C for 4 hr. Upon
3
4
cooling to room temperature, the reaction mixture was diluted with water (30 mL) and ethyl
acetate (15 mL). The solution pH was adjusted to 7 by using 1N HCl. The organic phase was
dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified
by silica gel chromatography to afford compound 6. Yield: 30%, R : 0.25 (hexane/EA = 3/1),
f
-
LRMS (ESI): [M-H] calcd for C H ClN O : 400.1, found: 400.1, ESI-MS/MS: 290.1 [M-Ph-
2
3
16
3
2
-
Cl-H]
4
.1.7. 2-[5-(5-Methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl]-4-(thiophen-2-yl)phenol (7)
In the same method as 6, 2-thienylboronic acid was used instead of 4-chlorophenylboronic acid.
-
Yield: 33%, R : 0.25 (hexane/EA = 3/1), LRMS (ESI): [M-H] calcd for C H N O S: 372.1,
f
21 15
3
2
-
found: 372.0, ESI-MS/MS: 315.0 [M-Ac-NH2]
4
.1.8. 4'-Fluoro-3-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl] ][1,1'-biphenyl]-4-ol (8)

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In the same method as 6, 4-fluorophenyboronic acid was used instead of 4-chlorophenylboronic
-
acid. Yield: 34%, R : 0.27 (hexane/EA= 3/1), LRMS (ESI): [M-H] calcd for C H FN O :
f
23 16
3
2
-
3
84.1, found: 384.1, ESI-MS/MS: 290.1 [M-Ph-F-H]
4
.1.9. tert-Butyl({4'-hydroxy-3'-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-
biphenyl]-4-yl}methyl)carbamate (9)
To a solution of 4-aminomethylphenylboronic acid (2.65 mmol) in THF (10 mL) were added di-
tert-butyl dicarbonate (3.97 mmol) and triethylamine (1 mL). The mixture was stirred at room
temperature for 1 hr. After completion of the reaction, excess solvent was evaporated to dryness
under reduced pressure. The residue was diluted with water (30 mL) and ethyl acetate (15 mL).
4
The organic phase was separated, dried over MgSO , filtered, and concentrated under reduced
pressure. The NHBoc-protected boronic acid was then used for Suzuki coupling as described in
1
compound 6 to afford compound 9. Yield: 32%, R : 0.31 (hexane/EA= 3/2), mp: 147-148 °C, H
f
6
NMR (300 MHz, DMSO-d ): δ 11.63 (br s, 1H, NH), 10.39 (br s, 1H, OH), 8.22 (s, 1H, Ar-H), 8.
0
7 (d, 1H, Ar-H, J = 2.4 Hz), 7.76-7.64 (m, 3H, Ar-H), 7.59 (d, 1H, Ar-H, J = 8.4 Hz), 7.51-7.42
(m, 2H, Ar-H), 7.32 (d, 2H, Ar-H, J = 8.1 Hz), 7.23 (s, 1H, Ar-H), 7.10 (d, 1H, Ar-H, J = 8.4 H
-
z), 4.17 (d, 2H, J = 6.0 Hz, CH ), 2.66 (s, 3H, OCH ), 1.41 (s, 9H, (CH ) ), HRMS (ESI): [M-H]
2
3
3 3
-
calcd for C H N O : 495.2111, found: 495.1981, ESI-MS/MS: 439.1448 [M-Ac-NH ] ,
2
9
28
4
4
2
-
3
95.1550 [M-Boc-H]
4
.1.10. 3-[5-(5-Methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-biphenyl]-4-ol (10)
In the same method as 6, phenyboronic acid was used instead of 4-chlorophenylboronic acid.
-
Yield: 37%, R
f
: 0.23 (hexane/EA= 3/1), LRMS (ESI): [M-H] calcd for C23
H
17
N
3
O
2
: 366.1,

ACCEPTED MANUSCRIPT
found:366.1
4
.1.11. 4'-Methoxy-3-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-biphenyl]-4-ol (11)
In the same method as 6, 4-methoxyphenyboronic acid was used instead of 4-
-
chlorophenylboronic acid. Yield: 36%, R
f
: 0.27 (hexane/EA= 3/1), LRMS (ESI): [M-H] calcd
19 3 3
for C24H N O
: 396.1, found: 396.2
4
4
.1.12. 3-[5-(5-Methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl]-4'-(trifluoromethyl)[1,1'-biphenyl]-
-ol (12)
In the same method as 6, 4-trifluoromethyphenyboronic acid was used instead of 4-
-
chlorophenylboronic acid. Yield: 39%, R : 0.26 (hexane/EA= 3/1), LRMS (ESI): [M-H] calcd
f
16 3 3 2
for C24H F N O : 434.1, found: 434.1
4
.1.13. 2-(4'-Cloro-4-hydroxy[1,1'-biphenyl]-3-yl)-1H-indole-5-carboximidamide (13)
To a solution of compound 5 in a mixture of MeOH and AcOH (10 mL, 7:1) was added a
catalytic amount of Raney-nickel. The reaction mixture was shaken under 50 psi of H for 7 hr.
2
The mixture was filtered through Celite and the filtrate was concentrated to dryness under
reduced pressure. The residue was purified by reversed-phase HPLC to afford compound 13.
HPLC: 80/20 water/acetonitrile (0.1% formic acid), flow rate 2 mL/min, R = 9.15 min, Yield:
t
1
2
8
4
1
2%, mp: 126-128°C, H NMR (300 MHz, CD
3
OD): δ 8.58 (s, 2H, NH
2
), 8.11 (s, 1H, Ar-H),
.01 (d, 1H, Ar-H, J = 2.4 Hz), 7.67-7.57 (m, 3H, Ar-H), 7.54-7.45 (m, 1H, Ar-H), 7.45-7.25 (m,
1
3
H, Ar-H), 6.99 (d, 1H, Ar-H, J = 8.4 Hz), C NMR (75 MHz, CD OD): 168.92, 168.03, 158.67,
3
40.43, 139.78, 139.22, 131.78, 129.40, 128.60, 128.37, 127.37, 127.30, 125.45, 120.19, 119.33,

ACCEPTED MANUSCRIPT
+
1
18.53, 117.93, 111.33. HRMS (ESI): [M+H] calcd for C21
3
H16ClN O: 362.0982, found:
+
+
3
62.1053, ESI-MS/MS: 345.0763 [M-NH +H] , 320.0793 [M-NH -CN+H]
3
3
4
.1.14. 2-[2-Hydroxy-5-(thiophen-2-yl)phenyl]-1H-indole-5-carboximidamide (14)
In the same method as 13, compound 14 was prepared from 7. HPLC: 70/30 water/acetonitrile
1
(
0.1% formic acid), flow rate 2 mL/min; R = 8.71 min, Yield: 37%, mp: 240-243 °C, H NMR
t
(
300 MHz, CD
3
OD): δ 8.58 (s, 4H), 8.14 (s, 1H, Ar-H), 8.04 (d, 1H, Ar-H, J = 2.1 Hz), 7.66 (d,
1
7
H, Ar-H, J = 8.4 Hz), 7.56-7.50 (m, 1H, Ar-H), 7.47 (dd, 1H, Ar-H, J = 8.4 & 2.4 Hz), 7.37-
.28 (m, 2H, Ar-H), 7.19 (s, 1H, Ar-H), 7.13-7.04 (m, 1H, Ar-H), 7.00 (d, 1H, Ar-H, J = 8.4 Hz),
1
3
C NMR (75 MHz, CD OD): 168.99, 167.98, 154.86, 144.01, 139.61, 138.61, 128.45, 127.59,
3
1
26.45, 124.78, 123.34, 121.80, 120.54, 119.79, 118.51, 118.22, 116.90, 111.54, HRMS (ESI):
+
[
M+H] calcd for C19
H
15
N
3
OS: 334.0936, found: 334.1013, ESI-MS/MS: 317.0727 [M-
+
+
3 3
NH +H] , 292.0788 [M-NH -CN+H]
4
.1.15. 2-(4-Hydroxy[1,1'-biphenyl]-3-yl)-1H-indole-5-carboximidamide (15)
In the same method as 13, compound 15 was prepared from 5. HPLC: acetonitrile(A)/water(B)
(0.1% formic acid), the gradient consisted of 10% A to 90% A over 30 min at 2 mL/min flow
1
rate. R
t
= 9.78 min. Yield: 46%, mp: 181-183 °C, H NMR (300 MHz, CD
3
CN/D
2
O): δ 8.48 (s,
1
7
H, NH), 8.18 (s, 1H, Ar-H), 7.88 (d, 1H, Ar-H, J = 7.8 Hz), 7.73 (d, 1H, Ar-H, J = 8.7 Hz),
.65-7.56 (m, 1H, Ar-H), 7.39-7.29 (m, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 7.15-6.95 (m, 2H, Ar-H),
C NMR (75 MHz, CD CN/D O): 170.52, 167.61, 154.28, 139.82, 139.29, 130.23, 128.61,
1
3
3
2
+
1
21.63, 121.43, 121.04, 120.88, 118.22, 117.18, 112.62, 101.09, HRMS (ESI): [M+H] calcd for
+
C H N O: 252.1059, found: 252.1134, ESI-MS/MS: 235.0863 [M-NH +H] , 210.0902 [M-
15
13
3
3

ACCEPTED MANUSCRIPT
+
3
NH -CN+H]
4
.1.16. 2-(4'-Fluoro-4-hydroxy[1,1'-biphenyl]-3-yl)-1H-indole-5-carboximidamide (16)
In the same method as 13, compound 16 was prepared from 8. HPLC: acetonitrile(A)/water(B)
(0.1% formic acid), the gradient consisted of 40% A to 50% A over 30 min at 2 mL/min flow
1
rate. R = 9.78 min. Yield: 30%, mp: 165-168 °C, H NMR (300 MHz, CD OD): δ 8.14 (s, 1H,
t
3
Ar-H), 8.03 (d, 1H, Ar-H, J = 2.1 Hz), 7.68-7.60 (m, 3H, Ar-H), 7.57-7.45 (m, 2H, Ar-H), 7.37
1
3
(
d, 1H, Ar-H, J = 8.1 Hz), 7.22 (s, 1H, Ar-H), 7.06 (d, 1H, Ar-H, J = 8.4 Hz), C NMR (75
MHz, CD OD): 167.95, 163.79, 154.07, 139.67, 138.65, 137.00, 132.10, 128.56, 128.49, 128.00,
3
1
27.90, 127.33, 125.88, 120.60, 119.83, 118.40, 118.19, 116.55, 115.17, 114.89, 111.53, 101.26,
+
HRMS (ESI) MS: [M+H] calcd for C H FN O: 346.1277, found: 346.1361, ESI-MS/MS:
2
1
16
3
+
+
3
30.2389 [M-NH +H] , 305.2931 [M-NH -CN+H]
3 3
4
.1.17. tert-Butyl{[3'-(5-carbamimidoyl-1H-indol-2-yl)-4'-hydroxy[1,1'-biphenyl]-4-yl] methyl}
carbamate (17)
In the same method as 13, compound 17 was prepared from 9. HPLC: 70/30 water/acetonitrile
1
(
0.1% formic acid), flow rate 2 mL/min, R = 5.94 min, Yield: 40%, mp: 196-199 °C, H NMR
t
(
300 MHz, CD OD): δ 8.14 (s, 1H, Ar-H), 8.00 (d, 1H, Ar-H, J = 2.1 Hz), 7.71-7.63 (m, 3H, Ar-
3
H), 7.57-7.52 (m, 1H, Ar-H), 7.45 (dd, 1H, Ar-H, J = 8.4 & 2.1 Hz), 7.24-7.14 (m, 3H, Ar-H),
1
3
7
.07 (d, 1H, Ar-H, J = 8.4 Hz), 4.23 (s, 2H, CH ), 1.49 (s, 9H, (CH ) ), C NMR (75 MHz,
2 3 3
CD OD): 167.97, 157.22, 154.03, 139.68, 139.33, 138.77, 138.12, 132.80, 128.59, 128.51,
3
1
4
4
27.34, 127.25, 126.19, 125.83, 120.58, 119.79, 118.34, 118.16, 116.51, 111.52, 101.20, 78.82,
+
3.35 (CH ), 27.38 (CH ) HRMS (ESI) MS: [M+H] calcd for C H N O : 457.2162, found:
2
3 3,
27 28
4
3
+
+
57.2225, ESI-MS/MS: 357.3035 [M-Boc+H] 341.2742 [M-Boc-NH +H] , 324.2468 [M-Boc-
2

ACCEPTED MANUSCRIPT
+
+
2 3 2 3
NH -NH +H] , 299.2524 [M-Boc-NH -NH -CN +H]
4
.1.18. 2-[4'-(Aminomethyl)-4-hydroxy[1,1'-biphenyl]-3-yl]-1H-indole-5-carboximidamide (18)
Compound 17 (10 mg, 0.02 mmol) was dissolved in a mixture of TFA and dichloromethane (2
mL, 1:1). The reaction mixture was stirred at room temperature for 1 hr. The mixture was
concentrated to dryness under reduced pressure. The residue was dissolved in a mixture of water
and acetonitrile and purified by reversed-phase HPLC to give compound 18. HPLC: 70/30
t
water/acetonitrile (0.1% formic acid), flow rate 2 mL/min, R = 2.56 min. Yield: 72%, mp: 126-
1
1
29 °C, H NMR (300 MHz, CD CN/D O): δ 8.49 (d, 2H, Ar-H, J = 7.2 Hz), 8.14 (d, 1H, Ar-H,
3
2
J = 8.1 Hz) , 8.06 (d, 1H, Ar-H, J = 8.7 Hz) , 8.01 -7.87 (m, 4H, Ar-H), 7.61 (s, 1H, Ar-H), 7.53
1
3
(
d, 1H, Ar-H, J = 8.4 Hz), 4.54 (s, 2H, CH ), C NMR (75 MHz, CD CN/D O): 167.57, 161.90,
2 3 2
1
54.34, 141.20, 139.86, 132.61, 131.91, 130.12, 128.62, 127.50, 126.89, 121.53, 121.05, 117.79,
+
112.66, 101.63, 43.15 (CH
2
), HRMS (ESI) MS: [M+H] calcd for C22
H
20
N
4
O: 357.1637, found:
-
+
3
57.1711, ESI-MS/MS: 340.2968 [M-NH +H] , 323.2406 [M-NH -NH +H] , 315.2702 [M-
3
3
3
+
NH -CN+H]
3
4
.1.19. 2-(4-Hydroxy[1,1'-biphenyl]-3-yl)-1H-indole-5-carboximidamide (19)
In the same method as 13, compound 19 was prepared from 10. HPLC: 72/28 water/acetonitrile
1
(0.1% formic acid), flow rate 2 mL/min, R
t
= 8.62 min, Yield: 42%, mp: 220-223 °C, H NMR
(
300 MHz, CD CN/D O): δ 8.76 (s, 1H, NH), 8.48 (s, 1H, Ar-H), 8.43 (s, 1H, Ar-H), 8.10-7.97
3 2
(m, 3H, Ar-H), 7.96-7.80 (m, 5H, Ar-H), 7.78-7.70 (m, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.49 (d,
1
3
1
1
H, Ar-H, J = 8.4 Hz), C NMR (75 MHz, CD CN/D O): 153.94, 140.49, 139.78, 138.84,
3 2
34.31, 133.52, 131.24, 129.45, 128.29, 127.57, 127.36, 126.91, 126.77, 121.41, 120.94, 117.67,

ACCEPTED MANUSCRIPT
+
1
12.98, HRMS (ESI) MS: [M+H] calcd for C21
H
17
N
3
O: 328.1372, found: 328.1435, ESI-
+
+
MS/MS: 311.2381 [M-NH +H] , 286.2379 [M-NH -CN+H]
3
3
4
.1.20. 2-(4-Hydroxy-4'-methoxy[1,1'-biphenyl]-3-yl)-1H-indole-5-carboximidamide (20)
In the same method as 13, compound 20 was prepared from 11. HPLC: 75/25
water/acetonitrile(0.1% formic acid), flow rate 2 mL/min, R = 12.98 min, Yield: 33%, mp: 132-
t
1
1
35 °C, H NMR (300 MHz, CD
3
CN/D
2
O): δ 8.48 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 8.08 (d, 1H,
Ar-H, J = 2.4 Hz), 7.80-7.68 (m, 3H, Ar-H), 7.65-7.52 (m, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.22-
13
7
.10 (m, 3H, Ar-H), 6.92-6.82 (m, 1H, Ar-H), 3.85 (s, 3H, OCH3), C NMR (75 MHz,
CD CN/D O): 172.68, 169.92, 161.54, 156.06, 142.12, 135.61, 135.43, 130.91, 130.83, 130.41,
3
2
1
28.58, 123.75, 123.23, 120.0, 118.67, 117.63, 117.11, 114.88, 103.69, 57.94 (OCH ), HRMS
3
+
(
ESI): [M+H] calcd for C22
H
19
N
3
O
2
: 358.1477, found: 358.1564, ESI-MS/MS: 341.2541 [M-
+
+
+
NH
3
+H] , 326.2285 [M-NH
3
-OH+H] , 316.2550 [M-NH
3
-CN+H] , 298.2271 [M-NH
3
-CN-
+
OH+H]
4
.1.21.
2-[4-Hydroxy-4'-(trifluoromethyl)[1,1'-biphenyl]-3-yl]-1H-indole-5-carboximidamide
(21)
In the same method as 13, compound 21 was prepared from 12. HPLC: 68/32
water/acetonitrile(0.1% formic acid), flow rate 2 mL/min, Rt = 9.92 min. Yield: 28%, mp: 191-
1
1
93 °C, H NMR (300 MHz, CD CN/D O): δ 8.53 (s, 2H, Ar-H), 8.29 (d, 2H, Ar-H, J = 7.5
3
2
Hz), 8.19 (d, 2H, Ar-H, J = 7.8 Hz), 8.12-7.90 (m, 4H, Ar-H), 7.63 (s, 1H, Ar-H), 7.56 (d, 1H,
13
Ar-H, J = 8.4 Hz), C NMR (75 MHz, CD CN/D O): 167.59, 154.84, 144.48, 139.88, 131.86,
3
2
+
1
28.79, 127.48, 127.22, 126.28, 121.56, 121.10, 117.88, 112.68, 101.78, LRMS (ESI): [M+H]

ACCEPTED MANUSCRIPT
+
calcd for C22
H
16
F
3
N
3
3
O: 396.1245, found: 396.1272, ESI-MS/MS: 379.2376 [M-NH +H] ,
+
3
54.2366 [M-NH -CN+H]
3
4
.1.22. 4'-(Aminomethyl)-3-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-biphenyl]-4-
ol (22)
Compound 9 (300 mg, 0.60 mmol) was dissolved in a mixture of TFA and dichloromethane (5
mL, 1:1). The reaction mixture was stirred at room temperature for 1 hr. The mixture was
concentrated under reduced pressure and used for next step without further purification. LRMS
-
(
ESI): [M-H] calcd for C24
H
20
N
4
2
O : 395.1, found: 395.0
4
.1.23. (9H-Fluoren-9-yl)methyl N-{1-[({4'-hydroxy-3'-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-
indol-2-yl]-[1,1'-biphenyl]-4-yl}methyl)carbamoyl]-5-{[(propan-2-yloxy)carbonyl]amino}
pentyl}carbamate (23)
To a solution of compound 22 (100 mg, 0.26 mmol) in DMF (15 mL) were added HATU (192
mg, 0.50 mmol), Fmoc-Lys(Boc)-OH (140 mg, 0.3 mmol), and triethylamine (70 ꢀl , 0.50
mmol). The reaction mixture was stirred at room temperature for 8 hr. The reaction mixture was
diluted with water (30 mL) and ethyl acetate (15 mL). The solution pH was adjusted to 7 by
using 1N HCl. The organic phase was dried over MgSO filtered and concentrated under reduced
4
,
pressure. The crude residue was purified by silica gel chromatography to afford compound 23.
1
6
Yield: 56%, R : 0.41 (hexane/EA= 3/2), H NMR (300 MHz, DMSO-d ): δ 11.84 (s, 1H, NH),
f
8
7
.52-8.45 (m, 1H, Ar-H), 8.26 (s, 1H, Ar-H), 8.09 (d, 1H, Ar-H, J = 12 Hz), 7.96 (s, 4H, Ar-H),
.87 (d, 3H, Ar-H, J =7.5 Hz), 7.81-7.62 (m, 6H, Ar-H), 7.54 (d, 2H, Ar-H, J =8.4 Hz), 7.43-7.20
(m, 9H, Ar-H), 7.06 (s, 1H, Ar-H), 6.82-6.74 (m, 1H, Ar-H), 4.46-4.32 (m, 4H), 4.30-4.20 (m, 4
H), 4.08-3.95 (m, 2H), 2.69 (s, 1H), 2.59 (s, 3H, CH ), 1.35 (s, 9H, (CH ) )
3
3 3

ACCEPTED MANUSCRIPT
4
.1.24. tert-Butyl N-{5-amino-5-[({4'-hydroxy-3'-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-
yl][1,1'-biphenyl]-4-yl}tmethyl)carbamoyl]pentyl}carbamate (24)
Compound 23 (50 mg, 0.06 mmol) was dissolved in 25% piperidine in DMF (5 mL) and stirred
at room temperature for 1 hr. The mixture was concentrated to dryness under reduced pressure
+
and used for next step without purification. LRMS (ESI): [M+Na] calcd for C H N O : 647.3,
3
5
40
6
5
found: 647.4
4
.1.25. 1,5-Di-tert-butyl 2-({[1-(tert-butoxy)-6-{7-[(5-{[(tert-butoxy)carbonyl]amino}-1-[({4'-
hydroxy-3'-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-yl][1,1'-biphenyl]-4-yl}methyl)
carbamoyl]pentyl)carbamoyl]heptanamido}-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate
(
26)
To a solution of compound 24 (50 mg, 0.08 mmol) in DMF (10 mL) were added trimethylamine
0.05 mL) and compound 25 (59 mg, 0.08 mmol) which was prepared by applying a reported
(
procedure [31]. The mixture was stirred at room temperature for 1 hr. The reaction mixture was
diluted with water (30 mL) and ethyl acetate (15 mL). The solution pH was adjusted to 7 by
using 1N HCl. The combined organic phase was dried over MgSO4, filtered and concentrated
under reduced pressure. The crude residue was purified by silica gel chromatography to afford
1
compound 26. Yield: 30%, R : 0.38 (ethylacetate/methanol= 10/1), H NMR (300 MHz,
f
CD OD): δ 8.28 (s, 1H), 8.05-7.92 (m, 1H, Ar-H), 7.88-7.54 (m, 1H, Ar-H), 7.52-7.60 (m, 3H,
3
Ar-H), 7.59-7.50 (m, 2H, Ar-H), 7.48-7.32 (m, 3H, Ar-H), 7.17-7.10 (m, 2H, Ar-H), 6.90-6.70
(
m, 1H), 6.52-6.64 (m, 1H), 6.42-6.30 (m, 2H), 4.51-4.41 (m, 3H), 4.39-4.32 (m, 2H), 4.29 (s, 1
H), 4.23-4.09 (m, 7H), 3.22-3.10 (m, 6H), 3.08-2.98 (m, 5H), 2.71-2.62 (m, 4H), 2.45-2.23 (m, 1
H), 2.20-2.10 (m, 8H), 2.08-1.97 (m, 4H), 1.58-1.70 (m, 18H), 1.56-1.10 (m, 36H)
1

ACCEPTED MANUSCRIPT
4
.1.26. 2-[({5-[7-({5-Amino-1-[({4'-hydroxy-3'-[5-(5-methyl-1,2,4-oxadiazol-3-yl)-1H-indol-2-
yl][1,1'-biphenyl]-4-yl}methyl)carbamoyl]pentyl}carbamoyl)heptanamido]-1-carboxypentyl}
carbamoyl)amino]pentanedioic acid (27)
Compound 26 (20 mg, 0.02 mmol) was dissolved in a mixture of TFA and dichloromethane (5
mL, 1:1) and stirred at room temperature for 2 hr. The mixture was concentrated under reduced
pressure. The residue was purified by reversed-phase HPLC to afford compound 27. HPLC:
7
2/28 water/acetonitrile (0.1% formic acid), flow rate 2 mL/min; Rt = 11.68 min. Yield: 25%,
1
mp: 205-207 °C, H NMR (300 MHz, CD CN/D O): δ 8.16 (s, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 7.
3
2
6
0 (d, 1H, Ar-H, J = 8.7 Hz), 7.53 (t, 3H, Ar-H, J = 7.5 Hz), 7.40 (d, 1H, Ar-H, J = 8.4 Hz), 7.25
d, 2H, Ar-H, J = 9.0 Hz), 7.05 (m, 2H, Ar-H), 4.29 (s, 2H), 3.98 (m, 2H), 2.96 (t, 2H, J =6.8 H
z ), 2.81 (t, 2H, J = 7.7 Hz ), 2.53 (s, 3H), 2.29 (t, 2H, J = 7.5 Hz), 2.15 (t, 2H, J =7.4 Hz), 1.98
(
1
3
(
t, 2H, J = 7.2 Hz), 1.58-1.82 (m, 24H), C NMR (75 MHz, CD
3
2
CN/D O): 180.44, 178.72, 178.
3
3
1
3, 176.17, 171.72, 156.20, 141.78, 140.69, 140.03, 139.85, 135.31, 131.01, 130.59, 130.35, 129.
4, 128.79, 123.13, 120.17, 119.94, 114.83, 56.44, 56.21, 56.05, 45.07, 41.88, 41.57, 38.45, 38.2
, 34.19, 33.42, 33.18, 30.98, 30.86, 30.80, 30.19, 28.98, 28.04, 27.98, 25.25, 25.07, 14.37,
+
HRMS (ESI): [M+H] calcd for C H N O : 982.4596, found: 982.4715, ESI-MS/MS:
5
0
63
9
12
+
+
8
35.4162 [M-Boc-CH (C=O)N +H] , 809.4351 [M-Boc-CH (C=O)N-CN+H]
3 3
4
.1.27. 2-({[5-(7-{[5-Amino-1-({[3'-(5-carbamimidoyl-1H-indol-2-yl)-4'-hydroxy-[1,1'-
biphenyl]-4-yl]methyl}carbamoyl)pentyl]carbamoyl}heptanamido)-1-carboxypentyl]carbamoyl}
amino)pentanedioic acid (28) and (Z)-4-(4-aminobutyl)-1-(4'-hydroxy-3'-(5-(N-(1-
hydroxyethylidene)carbamimidoyl)-1H-indol-2-yl)-[1,1'-biphenyl]-4-yl)-3,6,13,21-tetraoxo-
2
,5,14,20,22-pentaazapentacosane-19,23,25-tricarboxylic acid (29)
To a solution of compound 27 in a mixture of dioxane, water, and acetic acid (5 mL, 6:2:1) was
2
added catalytic amount of Raney-nickel. The mixture was shaken under 50 psi of H for 4 hr. The

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mixture was filtered through Celite and the filtrate was concentrated to dryness under reduced
pressure. The crude residue was purified by reversed-phase HPLC to afford a mixture of
compounds 28 and 29. HPLC: acetonitrile (A)/water (B) (0.1% formic acid). The gradient
consisted of 10% A to 35% A over 8 min; 35% A to 10% A over 10 min at 2 mL/min flow rate,
-
Rt =8.36 min, Yield: 30%, HRMS (ESI) compound 28: [M-H] calcd for C H N O : 940.4647,
48
63
9
11
-
found: 940.4680, HRMS (ESI) compound 29: [M-H] calcd for C H N O : 982.4753, found:
5
0
65
9
12
9
82.4783
4
.1.28. 1-(5-{[5-({[3'-(5-Carbamimidoyl-1H-indol-2-yl)-4'-hydroxy-[1,1'-biphenyl]-4-yl]
methylcarbamoyl)-5-{7-[(5-carboxy-5-{[(1,3-dicarboxypropyl)carbamoyl]amino}entyl)
carbamoyl]heptanamido}pentyl]carbamoyl}pentyl)-3,3-dimethyl-2-[(E)-2-[(3E)-3-{2-[(2E)-
1
,3,3-trimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]ethylidene}cyclohex-1-en-1-
yl]ethenyl]-3H-indol-1-ium-5-sulfonate (30) and 1-{5-[(5-{7-[(5-carboxy-5-{[(1,3-
dicarboxypropyl)carbamoyl]amino}pentyl)carbamoyl]heptanamido}-5-({[4'-hydroxy-3'-(5-{[(Z)-
(1-hydroxyethylidene)amino]methanimidoyl}-1H-indol-2-yl)-[1,1'-biphenyl]-4-yl]methyl}
carbamoyl)pentyl)carbamoyl]pentyl}-3,3-dimethyl-2-[(E)-2-[(3E)-3-{2-[(2E)-1,3,3-trimethyl-5-
sulfonato-2,3-dihydro-1H-indol-2-ylidene]ethylidene}cyclohex-1-en-1-yl]ethenyl]-3H-indol-1-
ium-5-sulfonate (31)
To a mixture of compounds 28 and 29 (2 mg in 0.3 mL of DMSO) were added tris-HCl buffer
(0.3 mL, 0.1 M, pH=8.5) and Sulfo-Cyanine 7 NHS ester (2 mg in 0.1 mL of
DMSO). The reaction mixture was stirred at room temperature for 4 hr. The residue was purified
by reversed-phase HPLC to afford compounds 30 and 31, respectively. HPLC: mobile phase
acetonitrile (A)/water (B) (0.1% formic acid). The gradient consisted of 30% to 40% A over 20
min; 40% to 30% A over 25 min at 3.5 mL/min flow rate, Rt = 5.18 min for 30, 6.21 min for 31.
+
Yield: 37% (30), 45% (31), compound 30: LRMS (ESI) [M+H] calcd for C H N O S :
8
5
104 11 18
2
3
+
2+
+
3+
1
631.7 found: [M+3H] 544.8, [M+2H] 816.8, and [M+H] 1631.7; HRMS (ESI): [M+3H]

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3
+
5
45.3593, [M+3Na]
567.3402, ESI-MS/MS: 370.3259 [M-(Lys-urea-Glu)-2SO3-NH3-
3
+
+
CN+3H] , compound 31: LRMS (ESI) [M+H] calcd for C H N O S : 1674.9, found: [M+3
8
7
106 11 19 2
3
+
2+
+
H] 558.8, [M+2H] 838.0 and [M+H] 1674.7
4
4
.2. Biological evaluation
.2.1 In vitro hepsin-inhibitory assay
The synthesized compounds (10 nM-10 ꢀM) were diluted in 2% DMSO 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 for hepsin was 0.3 nM in TNC buffer
(
25 mM Tris, 150 mM NaCl, 5 mM CaCl , 0.01% Triton X-100, pH 8). After incubation for 30
2
min at room temperature Boc-QAR-AMC substrate (#ES014, R&D Systems, Minneapolis,
Minnesota) was added to the Hepsin assays. The final substrate concentration was 150 ꢀM in
final reaction volume of 100 ꢀL. Changes in fluorescence (excitation at 380 nm and emission at
4
60 nm) were measured at room temperature over time in a Biotek Synergy 2 plate reader
(Molecular devices). From a plot of the mean reaction velocity versus comppound concentration,
a non-linear four parameter curve fit was performed using GraphPad Prism version 4.03 for
Windows (GraphPad Software, SanDiego, CA, www.graphpad.com) to determine inhibitor IC .
5
0
4
.2.2. In vitro PSMA-inhibitory assay
The IC values of compounds 30 and 31 were measured using NAALADase assay where
5
0
the glutamic acid/glutamate released from the natural substrate of PSMA was analyzed by the
fluorescence-based Amplex Red Glutamic Acid/Glutamate Oxidase assay kit (Invitrogen). In
vitro PSMA binding affinities of the synthesized compounds were evaluated by following the

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manufacturer’s procedure (www.lifetechnologies.com). Briefly, in a 96-well half area black flat
bottom polystyrene NBS microplate (Corning, Tewksbury, MA), PSMA (3 nM, 10 µL) and
different concentrations of inhibitor (20 µL) or buffer (TBS, 20 µL) were incubated at 37°C for
3
0 min. Then NAAG (4 µM, 10 µL) was added to the mixture (37°C, 30 min). The fluorescence
signals generated from the Amplex working solution (50 µL) with the NAAG-liberated
glutamate (37°C, 1 h) were then quantified by the Synergy H4 hybrid multi-mode microplate
reader (excitation at 530 nm and emission at 590 nm). Data was analyzed by non-linear
regression with GraphPad Prism 4.03 for Windows to determine inhibitor IC50
.
4
.2.3. In vitro cell uptake study
PC3/ML, PC3/ML-HPN, PC3/ML-PSMA and PC3/ML-PSMA-HPN cells were prepared
at Johns Hopkins Medical Institutions, Baltimore, Maryland. The cells were seeded at a density
5
of 5 × 10 cells/well in 6-well plates for 48ꢁh until total adhesion was achieved. Then the medium
was removed, and 30 and 31 (final concentration: 10ꢁꢀg/mL) in RPMI 1640 medium
supplemented with 10% FBS were added. After another 4ꢁh of incubation, the cells were washed
with cold PBS twice and fixed with 4% paraformaldehyde for 10ꢁmin and the flow cytometry
(
LSRII, BD Biosciences, San Jose, CA) analysis was employed to estimate the cellular uptake at
different cell lines. Data were processed by FlowJo software (Ashland, OR)
4
.2.4. In vivo near infrared (NIR) optical imaging study
One million cells of PC3/ML-PSMA and PC3/ML-PSMA-HPN were subcutaneously
null
injected into upper flanks of six-week old male NSG (NOD/Shi-scid/IL-2Rγ ) mice. Two
weeks after the cell injection, one nanomole of 30 in 100 uL of PBS was injected via tail vein.

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Images were taken 24 hr post injection of 30 using the Pearl Impulse Small Animal Imaging
System (LI-COR, Lincoln, NA).
4
4
.3. In silico molecular docking studies
.3.1. Ligand preparation and optimization
All ligands 13-21 were generated as 2D and 3D structure by ChemBioDraw (ver. 11.0.1)
and Chem3D Pro (ver. 11.0.1), respectively. The prepared compounds and CA-14, the original
crystal ligand of hepsin X-ray crystal structure (PDB ID: 1O5E), were saved as .sdf file. The
process of ligand preparation and optimization was performed by ‘Sanitize’ preparation protocol
in SYBYL-X 2.1.1 (Tripos Inc., St Louis) to clean up the structures involving filling valences,
standardizing, removing duplicates and producing only one molecule per input structure.
4
.3.2 Protein preparation
The hepsin structure of in PDB format was downloaded from RCSB protein data bank
(PDB ID: 1O5E). Structure preparation tool in SYBYL-X 2.1.1 was employed for the protein
preparation. The original crystal ligand and water molecules were removed from the protein-
ligand complexes for docking. Conflicted side chains of amino acid residues were fixed.
Hydrogen atoms were added under the application of TRIPOS Force Field as a default setting.
Minimization process was performed by POWELL method, initial optimization option was
changed to None from a default setting SIMPLEX, and termination gradient and max iteration
were set 0.5 kcal/(mol*Å) and 1000 times, respectively.
4
.3.3. Molecular docking
Docking studies of all prepared ligands were performed by Surflex-Dock Geom module in

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SYBYL-X 2.1.1. Docking was guided by the Surflex-Dock protomol, an idealized representation
of a ligand that makes every potential interaction with the binding site. The binding site of hepsin
X-ray crystal structure (PDB ID: 1O5E) for Surflex-Dock was defined according to the location
information of CA-14, the original ligand. Two factors related with a generation of Protomol are
Bloat (Å) and Threshold were set to 0.5 and 0, respectively. The maximum number of generated
poses per each ligand and the minimum RMSD between final poses were set to 20 and 0.05,
respectively. Other parameters were applied with its default settings in all runs. The Surflex-Dock
scoring function, which contains hydrophobic, polar, repulsive, entropic and solvation terms, was
d d
trained to estimate the dissociation constant (K ) expressed in –log (K ) units. The docked poses
were obtained after running Surflex-Dock and the scores of the docked poses were ranked
according to total score and Cscore (Table 2: Supplementary data).
Acknowledgments
This research supported by the NIH (CA134675 and CA184228 to M.G.P), USA Department of
Defense (W81XWH-10-1-0189 to Y.B), the National Research Foundation of Korea (NRF)
(2014R1A1A2056522 and 2014R1A4A1007304 to Y. B).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://