Increasing C-Terminal Hydrophobicity Improves the Cell Permeability and Antiproliferative Activity of PACE4 Inhibitors against Prostate Cancer Cell Lines

Article abstract of DOI:10.1021/acs.jmedchem.8b01144

The serine protease, PACE4, is a proprotein convertase that plays a substantial role in malignancy of prostate cancer. Our initial selective PACE4 inhibitor (Ac-LLLLRVKR-NH₂) has evolved to the current lead compound C23 (Ac-dLeu-LLLRVK-Amba), w

Full text of DOI:10.1021/acs.jmedchem.8b01144

Subscriber access provided by University of South Dakota
Article
Increasing C-Terminal Hydrophobicity Improves the
Cell Permeability and Antiproliferative Activity of
PACE4 Inhibitors against Prostate Cancer Cell Lines
Vahid Dianati, Anna Kwiatkowska, Frédéric Couture, Roxane Desjardins, Yves L. Dory, and Robert Day
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01144 • Publication Date (Web): 05 Sep 2018
Downloaded from http://pubs.acs.org on September 7, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Increasing C-Terminal Hydrophobicity Improves the
Cell Permeability and Antiproliferative Activity of
PACE4 Inhibitors against Prostate Cancer Cell Lines
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
‡
‡
‡
†
Vahid Dianati, Anna Kwiatkowska, Frédéric Couture, Roxane Desjardins, Yves L. Dory *and
Robert Day‡*
†Institut de Pharmacologie de Sherbrooke, Département de Chimie, Faculté des Sciences,
Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec, J1H 5N4 (Canada)
‡
Institut de Pharmacologie de Sherbrooke, Département de Chirurgie/Urologie, Université de
Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec,
J1H 5N4 (Canada)
KEYWORDS. Prostate cancer, Proprotein convertases, PACE4, Inhibitor, Cell permeability,
Cell penetrating peptide.
ABSTRACT
The serine protease, PACE4, is a proprotein convertase which plays a substantial role in
malignancy of prostate cancer. Our initial selective PACE4 inhibitor (Ac-LLLLRVKR-NH ) has
2
evolved to our current lead compound C23 (Ac-DLeu-LLLRVK-Amba), which is active both in-
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
vitro and in-vivo. By screening natural residues, except Cys, in C-terminal P1' position, it was
established that increasing hydrophobicity was improving cell permeability, which was directly
translated into PCa cells antiproliferative activity. This cell antiproliferation enhancement seems
independent from effect of P1' residue on PACE4 affinity. Replacement of P1-Amba of C23 by
Acpa ((S)-2-amino-3-(4-carbamimidoylphenyl)propanoic acid) followed by addition of
tryptamine in P1' resulted in compound 32 exhibiting superior PCa cells antiproliferative activity
over the reference compound C23 (3-fold). This study sheds light on key factors that improve
cell penetrating property and antiproliferative activity of PACE4 inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
PACE4 is an enzyme in the family of proprotein convertases (PCs) that activates secretory
protein precursors through peptide bond cleavage at paired basic amino acids (i.e., consensus
cleavage site R-X-R/K-R↓). PACE4 transits within the secretory pathway and is secreted.1-2 Our
3
studies were the first to demonstrate the role of PACE4 in prostate cancer (PCa). We showed
an overexpression of PACE4 in PCa tissues obtained from patients who underwent radical
prostatectomy for clinically localized tumor, while no other PC showed any significant changes.
Using mouse PCa xenograft models, we also showed that only PACE4 inhibition blocks PCa
4
progression, and not the inhibition of other PCs. Recently, we discovered that PCa cells utilize
an alternative splicing mechanism to generate a cancer-specific C-terminally modified PACE4
5
isoform (named PACE4-altCT), which is retained in the cell. RNA interference silencing
studies targeting PACE4-altCT demonstrate that this isoform is responsible for PACE4-
associated cancer progression. PACE4 is also overexpressed and involved in other malignancy
including non-small cell lung carcinoma, ovarian, and breast cancer.3-4, 6-8 The sum of our data
2
ACS Paragon Plus Environment

Page 3 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
provides the proof of concept and the justification to proceed with a development plan for
PACE4 inhibitors for a novel cancer therapy.
In this regard, we developed a specific PACE4 inhibitor named the Multi-Leu peptide (ML-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
peptide).9-10 In enzyme kinetic assays, the ML-peptide (sequence: Ac-LLLLRVKR-NH
potent (K = 22 ± 6 nM) and selective for PACE4 (e.g., 20-fold over furin). In cell-based assays,
the compound 1 blocks the proliferation of PCa cell lines (LNCaP, DU145 and 22Rv1) and
induces G /G cell cycle arrest. We also discovered that the target of the compound 1 is
) 1 is
2
i
0
1
intracellular since a cell impermeable analog (i.e., PEG8-ML-peptide) has no effect on cell
9
proliferation. We believe compound 1 exerts its anti-proliferative effects by reaching PACE4-
altCT (i.e., cell retained PACE4 isoform). While potent in cell-based assays, the compound 1
displays poor stability in vivo, so we developed a stable version named C23 (compound 2;
11
Figure 1). Among the modifications in compound 2, the replacement of the P1 Arg by 4-
amidinobenzylamide (Amba) gives this PACE4 inhibitor with higher potency than the
compound 1 and antiproliferative activity against PCa cell lines and improved stability and
pharmacokinetic profile.10, 12-13 The additional change of P8-Leu to its D-isomer, raised the
plasma stability up to 1.7 hours with almost no loss of potency and anti-proliferative
activity.12,14 In LNCaP xenografted mice, compound 2 (2 mg/kg/day iv) diminished tumor
volumes by 60% over 18 days with a 47% reduction in plasma PSA levels, as well as a
significant decrease in cells progressing through the cell cycle, increased apoptosis and
impaired neovascularization.11
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Structure of lead compound 2 (C23)
While compound 2 represents a significant improvement for PACE4 inhibition in vivo, further
refinements may be needed. Indeed, a systematic scan of positions P5 to P8 of compound 2
revealed that these four residues have to be hydrophobic. It was even shown that charged
residues at those positions reduce antiproliferative activity on PCa cell lines. This may be due to
the disruption of the amphipathic nature of the inhibitor, which affects its cell penetrating
15
properties. Parallel studies on the compound 1 indicated that introduction of a β-branched basic
residue, (2S)-amino-(3S)-guanidinobutyryl (S-i-Agb), in the P3 position led to a compound with
1
6
unprecedented high potency and selectivity towards PACE4. However, further antiproliferative
PCa cells analysis of this compound and related analogs showed very poor activity.
The analysis of PC homology models based on furin crystal structure suggests that the S1 to
S4 pockets are strongly conserved among PCs.17 However, these models also highlight
noticeable differences in the P' region of PC catalytic pockets. Despite few reports, no
comprehensive investigation has been done on the significance of the P' region of the catalytic
site for PACE4 inhibitors.18-19 Accordingly, a screening was conducted with natural residues at
the P1' position of the compound 1. The impact of these modifications was evaluated on the
inhibition of PACE4 and PCa cell lines antiproliferative activity of new compounds. Additional
4
ACS Paragon Plus Environment

Page 5 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
molecules were further designed based on the obtained results to evaluate any potential gain in
terms of PACE4 inhibition and cellular activity.
RESULTS AND DISCUSSION
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Screening of DNA-encoded residues in P1'. So far, the most potent PACE4 inhibitors
including our lead compound 2 have been armed with Amba at their P1 C-terminal. For synthetic
feasibility reasons in the investigation of the P1' position, Amba was replaced by Arg-NH
2
. The
20 natural amino acids, except Cys, have been incorporated in the P1' position of compound 1.
The inhibitory constants (K ) of each peptide have been determined for PACE4 and furin using a
i
fluorometric assay to compare the binding affinities. The efficacy of compounds was further
assessed ex vivo using DU145 and LNCaP PCa cell lines using an MTT cell proliferation assay.
The PACE4 and furin affinities (Table 1) of the new compounds 3−21, compared to the
control compounds 1 and 2, reveal that P1'-Lys (10) and P1'-Arg (16) are the least-favored
residues for the S1' pocket of both enzyme which is in accordance with what has been observed
17
for affinity of PC’s substrates. However, only compounds 10 and 16 remain as selective as
compound 1 (17 and 16 times more selective toward PACE4, respectively). As illustrated in
Figure 2, other P1'-modified peptides, except Pro (14) and Leu (11), inhibit PACE4 in the range
of compound 1 (20-64 nM). Compounds 6, 20 and 21 with aromatic residues (Phe, Trp and Tyr)
in P1' are among the best PACE4 inhibitors in this series, with compound 6 displaying a K value
i
as low as 20 ± 2 nM for PACE4. In general, most of the inhibitors were showing enhanced
affinity for furin and diminished inhibition of PACE4, in comparison to compound 1. As a result,
no gain in selectivity was ever observed with the introduction of residues in position P1'. The
PACE4 inhibition profile of compounds 3−21 suggests that no natural residue could introduce a
favorable interaction with PACE4 in the S1' pocket.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Inhibition profiles and cellular antiproliferative activities of compounds 3−21 with
general structure of Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Xaa-NH
2
compared to control
compounds 1 and 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(nM) ± SEMa
i
IC50 (μM) ± SEMa
K
Selectivity
for PACE4
Entry
Structure
PACE4
22 ± 6
4.9 ± 0.9
37 ± 5
30 ± 2
58 ± 4
20 ± 2
44 ± 5
33 ± 7
64 ± 4
150 ± 20
170 ± 30
32 ± 2
28 ± 3
250 ± 10
43 ± 3
160 ± 5
64 ± 2
22 ± 2
38 ± 4
29 ± 1
29 ± 1
Furin
DU145 LNCaP
100 ± 10 180 ± 60
25 ± 10 45 ± 10
160 ± 10 N.D.c
230 ± 20 N.D.
b
1
2
3
4
5
6
7
8
9
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-NH
2
430 ± 10
9.8 ± 2
20
2
Ac-DLeu-Leu-Leu-Leu-Arg-Val-Lys-Ambab
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Ala-NH
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Asp-NH
2
210±10
6
2
74 ± 8
2
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Glu-NH
2
2
260 ± 30
74 ± 9
4
N.C.d
70 ± 8
N.C.
N.D.
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Phe-NH
4
170 ± 40
N.D.
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Gly-NH
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-His-NH
Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Ile-NH
0 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Lys-NH
1 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Leu-NH
2
500 ± 30
140 ± 10
290 ± 10
2600 ± 100
990 ± 30
170 ± 10
130 ± 20
990 ± 30
190 ± 10
2500 ± 400
330 ± 20
100 ± 10
200 ± 10
110 ± 10
64 ± 5
11
4
2
N.C.
N.D.
2
5
72 ± 8
N.C.
1
1
1
2
17
6
130 ± 10 N.D.
90 ± 10 N.D.
130 ± 20 N.D.
2
2 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Met-NH
2
5
13 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Asn-NH
2
5
N.C.
N.D.
1
1
1
1
4 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Pro-NH
2
4
210 ± 30 N.D.
210 ± 20 N.D.
5 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Gln-NH
2
5
6 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Arg-NH
7 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Ser-NH
2
16
5
71 ± 7
N.C.
2
240 ± 50 N.D.
170 ± 10 N.D.
170 ± 10 N.D.
18 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Thr-NH
9 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Val-NH
20 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Trp-NH
1 Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-Tyr-NH
2
5
1
2
5
2
4
25 ± 2
31 ± 3
2
2
2
100 ± 20 N.D.
aK
and IC50 values are based on at least two independent experiments. Errors reported as SEM
and IC50. For more details of inhibitor concentrations used in the MTT antiproliferative
i
for K
i
b
c
assay see experimental section. Data were adapted from references 10 and 12. N.D.: Not
d
determined. N.C.: Not calculable as the curves did not converge to 50% with doses up to 300
µM.
6
ACS Paragon Plus Environment

Page 7 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
MTT cell proliferation was then investigated to assess the cell activity of inhibitors on PCa
cell lines (Table 1). The compounds 3−21 were first monitored on DU145 cells. Thereafter, the
most active compounds were also examined on LNCaP cells. Clearly, the introduction of
hydrophobic residues in P1' position enhances the antiproliferative activity of peptides. For
instance, compounds with P1' residues Phe (6), Ile (9), Leu (11) and Trp (20) led to the IC50
values of 70 ± 8, 72 ± 8, 90 ± 10 and 24 ± 2 μM, for DU145 PCa cells, respectively. As
exceptions for polar residues, we were at first very surprised by the P1'-Lys (10) and P1'-Arg
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(16) peptides with their respective antiproliferative IC50 values of 130 ± 20 and 71 ± 7 μM while
they are very weak PACE4 inhibitors. Based on these results, it was then uncovered that positive
charge and hydrophobicity in P1' residue are the main factors for enhancing the antiproliferative
activity.
SAR studies based on the screening. Following the encouraging data collected for peptides 6,
16 and 20 (Table 1), the next logical move was to combine the basic nature of Arg and the
aromatic nature of Phe at position P1'. This is how the new residues 4-aminophenylalanine (Apa)
22, 4-guanidinophenylalanine (Gpa) 23 and 4-(2-aminoethyl)benzimidamide (Aeba) 24 were
designed (Figure 2-a). Hence, the corresponding compounds 28−30 were synthesized on solid
support, then tested (Figure 2-b). Only compound 29 inhibited PACE4 with an improved K
11 ± 2 nM but once again, this increased affinity didn’t translate to antiproliferative activity. The
reduced polarity in Aeba 24 with no C-terminal −CONH group, contributed to the
i
of
2
antiproliferative properties of compound 30 (41 ± 9 and 67 ± 7 μM for DU145 and LNCaP cells,
respectively) which is no better than lead compound 2.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. a) The PACE4 affinity and antiproliferative activity of compounds 28−33. K and IC50
i
values are means of at least two independent experiments. Errors reported as SEM for K and
i
IC50. For more details on the concentration of inhibitors in the MTT antiproliferative assay see
experimental section. b) The IC50 values of selected compounds for DU145 and LNCaP PCa cell
lines.
Since the decarboxylated compound 30 (residue 24 at P1') showed better IC50 values than
compounds 28 and 29, P1'-Trp in compound 20 was replaced with tryptamine (Tryp) 25
8
ACS Paragon Plus Environment

Page 9 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
increasing the C-terminal hydrophobicity. As expected, compound 31 showed promising IC50
values (14 ± 2 μM for DU145 and 18 ± 5 μM for LNCaP cells). To further increase the cell
efficacy, compound 31 was modified in P1 position by substituting Arg with (S)-2-amino-3-(4-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
carbamimidoylphenyl)propanoic
carbamimidoylphenyl)acetic acid (Aca) 27 which have the 4-amidinophenyl side chain similar to
Amba in compound 2. Those modifications led to the compounds 32 (PACE4 K of 41 ± 7 nM)
and 33 (PACE4 K of 248 ± 6 nM). The PACE4 affinity comparison of these compounds with
compound 31 indicates a 2-fold preference for Acpa (32) over Arg (31) in P1 position while Aca
33) is 6 times disfavored. The diminished PACE4 affinity of compound 33 is most likely due to
acid
(Acpa)
26
and
(S)-2-amino-3-(4-
i
i
(
the steric hindrance around the opening of P1 pocket. Overall, there seems to be no correlation
between affinity and antiproliferative activity linked to the nature of the P1' residues (Figure 3-
a). On the other hand, observations indicate that such a correlation may exist at the P1 position,
since PACE4 affinity is well translated into PCa cell antiproliferative activity in both compounds
32 and 33. Compound 32, displaying unprecedented IC50 values (7.5 ± 0.8 and 13 ± 1 μM for
DU145 and LNCaP cells, respectively) for PCa cell lines, is a good candidate for further in vivo
studies (Figure 2-b).
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. a) Linear relation of IC50 values for MTT antiproliferative assay on DU145 PCa cell
line with Wimley-White bilayer scale for hydrophobicity of P1' residues and b) weak correlation
to PACE4 affinity of compounds 3−21.
Cell permeability studies. The relationship between K and IC50 is clearly not linear. It is
i
obvious that PACE4 affinity of inhibitors translates poorly into antiproliferative activity with
introduction of the P1' residue (Figure 3-a). On the other hand, there is a linear relation between
hydrophobicity of P1' residues and antiproliferative activity of compounds (Figure 3-b). The
IC50 values are in respectable agreement with Wimley-White bilayer scale for hydrophobicity of
2
0
amino acids. In this scale, the ΔG(water to bilayer), is calculated for the partitioning of peptides Ac-
1
0
ACS Paragon Plus Environment

Page 11 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
21
WL-X-LL-OH (X: natural amino acid) between water and a POPC lipid bilayer. The ΔG(water to
bilayer) value of each amino acid can be used to evaluate its contribution to membrane penetration.
We, thus, suggest that the preeminent cytostatic activity of compounds with a hydrophobic P1'-
residue originated from enhancement in penetration of such peptides through cell membrane.
Thus, the compounds 35−38 which were FITC-labeled in their N-terminal. After an hour of
incubation of the labeled compounds with DU145 cells, the emitted fluorescence from cells was
analyzed using fluorescence-activated cell sorting (FACS) and compared to compound 34 as
control. The signal of membrane absorbed peptides was eliminated by treatment of cells with
trypan blue prior to analysis. The depicted results in Figure 4 revealed that the higher
permeability achieved with increased P1' hydrophobicity. In comparison to control compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4, uptake of compounds 37 and 38 enhanced by 2 and 3.5-fold, respectively, which totally
correlate with IC50 values for compounds 20 and 31. The better penetration of compounds with
Arg (35) and Lys (36) in P1' position, compared to control, also suggests that lower PACE4
affinity of compounds 16 and 10 were compensated by their amended cell penetration.
Figure 4. Quantitative cell penetration assessment of FITC-labeled compounds 35−38
compared to control compound 34.
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The direct relation between cell penetration and antiproliferative activity has been constantly
observed for compound 1 and its derivatives.10, 12 The cell permeability is required to reach the
intracellular PACE-altCT isoform which is responsible for PACE4-associated cell growth of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
prostate malignant cells. This is consistent with our previous observations with N-terminal
PEGylated derivatives of compound 1 displayed no cell permeability and thus no
9
antiproliferative activity. The amphipathic nature of peptide inhibitors with the charged warhead
and four hydrophobic residues in N-terminal has been considered crucial for cell derivatives of
compound 2.15 Arg residues plays the key role in cell permeability of poly-arginine cell
penetrating peptides (CPPs), including compound 1 derivatives, through bidentate electrostatic
and hydrogen bonds with negatively charged groups of membrane.22 Hydrophobic residues,
when present in a CPP, increase the interactions with lipid bilayer and thus increasing
2
3
translocation of peptides through membrane. Among hydrophobic residues, inclusion of Trp
within basic peptides is a molecular determinant for enhancement of cell uptake efficiency.24-27
For instance, more abundancy of Trp residues in amphiphilic helical CPPs increased their uptake
2
8
in A549 cell lines. The exclusive role of tryptophan in cell penetration of polyarginine peptides
is ascribed to the interaction with sulfated glycosaminoglycans possibly through hydrophobic
and π–anion interactions in addition to its important hydrophobic interactions with lipid
bilayer.29-30
DU145 cell toxicity studies. The toxicity of compounds 10, 16, 20, 31 and 32 on DU145 cells
was evaluated to ascertain the fact that observed fluorescence in the permeability assay is related
to pure cell penetration of peptides and not to cell lysis. Hence, DU145 cells treated with
peptides for 1.5 h and the number of live cells was assessed by addition of PI (propidium iodide)
followed by FACS measurement of fluorescence. The compounds 10 and 16, displayed no
1
2
ACS Paragon Plus Environment

Page 13 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
toxicity to doses up to 100 µM. The results for compounds 20, 31, and 32 revealed that toxicity
for these compounds occurred at concentrations above 10 µM which is more than 10-fold higher
than the tested concentration for FITC-labeled peptides (1µM) in permeability assays (Figure
S1). For compound 32, a few more concentrations were tested to obtain a dose-response curve
for cellular toxicity (DU145 IC50 (PI) = 28 ± 2 µM; Figure 6). In comparison, this compound
inhibited metabolic activity of DU145 cells in 4-fold lower doses (DU145 IC50 (MTT) = 7.5 ±
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.8 µM). The fact that 4-fold more concentration of compound 32 is necessary for DU145
cytotoxicity, suggests that this compound is likely mediating its effects through mechanisms
other than killing the cells in one-digit µM concentrations.
Plasma stability studies. Despite rapid tumor uptake of PACE4-inhibitor, which permits in
vivo efficacy, the half-lives of all-natural residue peptide inhibitors of PACE4 in human plasma
11
is quite short. Accordingly, increasing the peptide stability in biological matrices is essential.
Half-life (t1/2) of compound 31 with DLeu in P8 and Tryp in P1' (0.8 h) was less than half of
compound 2 (1.7 h). Statistically, addition of a residue in P1' increased the number of breakable
amide bonds thus making the modified peptides more prone to the plasma proteases than their 8-
mer counterparts. It is well known that replacement of natural residues with the unnatural one, is
31
a way that could potentially increase the stability of peptides. As a result, modification of P1-
Arg in 31 with Acpa in 32 increased the plasma half-life up to 2.0 h (Figure 5).
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Stability of compounds 28, 31 and 32 in human plasma comparing to lead compounds
1 and 2.
Acute toxicity studies. To assess the safety profile of compound 32, the acute toxicity of this
compound was studied on healthy mice. The experiment included a single intravenous or
intraperitoneal administration dose of compound 32 into healthy mice (CD1 mice as standard for
toxicological and safety evaluations) at various doses to find the toxicity threshold (maximum
tolerated dose, MTD). The results indicated that this compound has an MTD of at least 5 mg/kg
for intravenous administration (Figure 6-b).
In a previous study, we showed that a 2 mg/kg/day dose of compound 2 is sufficient for
11
therapeutic activity in a LNCaP xenografted model of prostate cancer while it displayed safe
1
2
profile in acute toxicity studies (MTDs of 10 mg/kg in healthy mice). The in vivo therapeutic
activity for compound 32 is yet to be evaluated in LNCaP xenografted models.
1
4
ACS Paragon Plus Environment

Page 15 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. The toxicity profile of compound 32. a) The dose-response curve for DU145 cellular
toxicity was performed using PI as a staining reagent. The errors are reported as SEM. For more
details on concentration of inhibitors in this assay see experimental section. b) Table showing
IC50(PI) and MTD values of compound 32 in healthy CD1 mice. Data are representative of at
least two independent experiments.
Synthesis. Compounds 3−21, 28 and 29 with amino acid residues in P1' were prepared with
conventional 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis (SPPS)
on TentaGel® S RAM resin (Scheme S1). The decarboxylated analogues 30, 31, 32 and 33 were
synthesized using a combination of solid and solution phase peptide synthesis (Schemes 1 and
2
). At first crude protected peptide were obtained from Fmoc-SPPS on 2-chlorotrityl chloride
resin, and then the free C-terminal was coupled with the corresponding amines in solution. The
global deprotection with a TFA cocktail (composed of TFA/TIPS/H
desired peptides.
2
O 95:5:5) yielded the
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. Synthesis of P1 residue of compound 30, and P1-P1' adduct of compounds 32 and
3
3.a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aReagents and conditions: (a) CH
(b) Bu
NH
NO
, NaOH, MeOH/H
O, <10-15 C, 15 min then 5 M HCl;
O, K CO , THF/H O, 16 h; (e)
O, DIPEA, THF then 5% Pd/C, AcOH/MeOH
o
3
2
2
o
3
SnH, DCM, rt, 16 h; (c) Zn, HCl(aq), 65 C, 1 h; (d) (Boc)
2
2
3
2
o
2
OH.HCl, DIPEA, MeOH, 60 C, 16 h; (f) Ac
2
o
35 atm H
2
, 16 h; (g) Conc. HCl(aq), MeOH, 0 C to rt, 1 h; (h) tryptamine hydrochloride, EDCI,
o
6
-Cl-HOBt, DIPEA, DCM, 0 C to rt, 16 h; (i) i-BuOCOCl, DIPEA, tryptamine hydrochloride,
o
o
THF,
̶
20 C, 2 h; (j) Pd(PPh
3
)
4
, Zn(CN) , DMF, 100 C, 16 h.
2
For obtaining Aeba 24, aldehyde 39 condensed with nitromethane to give (E)-4-(2-
nitrovinyl)benzonitrile 40 (Scheme 1). The double bond and nitro moiety were reduced using
tributyltin hydride and Zn/HCl, respectively. The resulting amine was protected with t-butyl
1
6
ACS Paragon Plus Environment

Page 17 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
carbamate (Boc) group for more convenient purification in the following steps. Transformation
1
0
of nitrile 41 to amidine in compound 24 was performed as previously reported for Amba. Boc-
Phe(4-CN)-OH 42 was coupled with tryptamine 25 using EDCI coupling reagent. Compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45‘s amino group was protected with Boc and then coupled with tryptamine 25 at low
temperature using acyl chloride strategy. An additional cyanation step was necessary to obtain
compound 46. The conversion of nitrile groups in compounds 43 and 46 to the corresponding
amidines in 44 and 47 was performed with a similar procedure as was used for AEBA 24.
Scheme 2. Synthesis of compounds 30−33.a
aReagents and conditions: (a) Fmoc-Arg(Pbf)-OH, DIPEA, DMF/DCM (1:1); (b) Pip/DMF
(1:4); (c) Fmoc-aa-OH, HATU, DIPEA, DMF; (d) Ac-DLeu-OH, HATU, DIPEA, DMF; (e)
o
HFIP/DCM (1:4); (f) PyBOP, 6-Cl-HOBt, DIPEA, DMF, 24, 0 C to rt, 16 h; (g) PyBOP, 6-Cl-
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
HOBt, DIPEA, DMF, tryptamine hydrochloride, 0 C to rt, 16 h; (h) TFA/TIPS/H
2
O (38:1:1); (i)
o
Fmoc-Lys(Boc)-OH, DIPEA, DMF/DCM (1:1); (j) PyBOP, 6-Cl-HOBt, DIPEA, DMF, 44, 0 C
o
to rt, 16 h; (k) PyBOP, 6-Cl-HOBt, DIPEA, DMF, 47, 0 C to rt, 16 h.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
In conclusion, we developed PACE4 inhibitors by SAR studies in P1 and P1' using ML-
peptide 1 as template. By performing a systematic screening of natural amino acids in P1'
position of peptide 1, compound 20 (Trp in P1') was identified as having improved
antiproliferative effect on PCa cell lines. Further modification of this Trp residue to Tryp (31)
increased the hydrophobicity in the C-terminal region of the resulting peptide. Despite its
diminished affinity for recombinant PACE4, Trp to Tryp replacement improved plasma stability
and cell efficacy. In an attempt to further increase the in vitro cell activity of compound 31, P1-
Arg was replaced with an Acpa residue. This change contributed to the plasma stability and
antiproliferative effect of compound 32 which possesses the best cell efficacy of peptide 1
derivatives reported to date. The enhancement of antiproliferative activity with the incorporation
of Trp or Tryp in the C-terminal region was found to be related to the improved cell penetration
properties of such inhibitors. The cell permeability is the main obstacle in the development of
multi-basic inhibitors of PACE4 as well as similar targets such as furin and some other flaviviral
proteases.32-34 The present study constitutes a significant step forward to address such cell
penetration issues.
EXPERIMENTAL
Chemistry. General Experimental Methods. All chemical reagents and solvents were obtained
from commercial resources and used without further purification. Fmoc-protected amino acids
and coupling reagents, were purchased from ChemPep (Miami, FL, USA) or Chem-Impex
1
8
ACS Paragon Plus Environment

Page 19 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
International (Wood Dale, IL, USA). Tryptamine and most of other reagents were bought from
Sigma Aldrich (St. Louis, MO, USA). TentaGel® S RAM resin and 2-chlorotrityl-chloride resin
were received from Rapp Polymer (Tübingen, Germany). Peptide synthesis (Fmoc/tBu strategy)
was accomplished either manually or automatically on a Pioneer peptide synthesizer (Applied
Biosystems). The purification and purity check of peptide inhibitors were performed using
reverse phase high-performance liquid chromatography (RP-HPLC) on an Agilent Technologies
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1100 system (analytical and semi-prep) equipped with a diode array detector (λ = 210, 214, 230,
and 254 nm). Preparative HPLC were done using either a Varian ProStar preparative system
equipped with a UV-Vis detector (λ = 214 nm) or a Waters preparative HPLC system
(Autosampler 2707, Quaternary gradient module 2535, UV detector 2489 (λ = 214 and 230 nm),
fraction collector WFCIII) equipped with an ACE5 C18 column (250 × 21.2 mm, 5 μm spherical
particle size). A gradient of 0.1% TFA in water and acetonitrile was used as eluent. Analytical
HPLC was carried out using C18 columns, either Agilent Eclipse XDB (5 mm, 4.6 μm, 250 mm)
or a Phenomenex Jupiter (5 mm, 4.6 μm, 250 mm). ESI-HRMS (TripleTOF 5600, ABSciex;
Foster City, CA, USA) was used to confirm the identity of the pure products. Water H Class
Acquity UPLC coupled with an SQ Detector 2 and a PDA eλ detector paired with an Acquity
UPLC CSH C18 column (1.7 μm×2.1 mm×50 mm) was also used for synthesis check and
plasma stability assessment (linear gradient from 5 to 95% of ACN containing 0.1% formic acid
in 0.1% aqueous formic acid was used for 1.3 min, flow rate 0.8 mL/min). NMR experiment
1
13
were performed on either AV300 Bruker (300 MHz for H and 75 MHz for C) or an Avance III
1
13
13
hd 400 Bruker (400 MHz for H and 100 MHz for C). C NMR experiments were done with
complete decoupling of protons. Infrared (IR) spectra were recorded using Alpha-Platinum ATR
Bruker, diamond crystal.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Peptide Synthesis. A sequence of washes including DMF 3×, a cycle of MeOH/DCM 3× and
DCM 3× was applied, after every reaction on resin except stated otherwise. TentaGel® S RAM
resin was used for the synthesis of compounds 3−21, 28 and 29. The chain length growth was
performed by standard Fmoc-SPPS. Fmoc group was removed with 20% piperidine in DMF.
Fmoc protected amino acids were coupled using 5 equiv of protected amino acids, 5 equiv of
HATU and 15 equiv of DIPEA in DMF. The cleavage from resin and side chain deprotection
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was carried out in one step with a cocktail of TFA/TIPS/H
2
O 95:2.5:2.5 for 2 h. The crude
peptides were triturated with Et O after evaporation of 50% of cleaving cocktail, centrifuged and
2
the supernatant was discarded. For compounds 30−33 the peptide cores were synthesized on 2-
chlorotrtiyl chloride resin as following. 1.2 equiv of first amino acid was loaded on resin in the
presence of 4 equiv DIPEA in DMF for 3 h. The unreacted groups were capped with a mixture of
DCM/MeOH/DIPEA 85:10:5. Resin, then, was washed by a sequence of DCM 3×, a cycle of
MeOH/DCM 3× and DCM 5×. The protected peptides were cleaved from resin with 20%
hexafluoro-2-propanol in DCM. The solvent was evaporated in vacuo and the residue was
dissolved in a mixture of t-BuOH/H O (1:1) and lyophilized. A mixture of protected peptide, 2.5
2
equiv of corresponding P1' or P1-P1' amine, 2.2 equiv of PyBOP, 7.5 equiv of 6-Cl-HOBt was
dissolved in DMF and cooled in an ice bath, then 7.5 equiv of DIPEA was added to the solution
and the reaction was stirred overnight. Solvent was removed with an air stream and the crude
protected peptide was deprotected with the same TFA cocktail as compounds 3−21. FITC-
1
0
labeled peptides were prepared as reported elsewhere. The crude of compounds 3−21, 28−33
and 35−38 were purified using preparative HPLC. Analytical HPLC was used for confirmation
of purity (>95%) of fractions. These compounds were characterized by high-resolution mass
2
0
ACS Paragon Plus Environment

Page 21 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
spectroscopy (HRMS). The HPLC chromatograms and HRMS details of these compounds are
provided in supplementary material.
(E)-4-(2-Nitrovinyl)benzonitrile (40). A solution of NaOH (0.84 g, 21.00 mmol) in ice-water
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(40 mL) was added dropwise to a solution of 4-Formylbenzonitrile (2.60 g, 20.00 mmol) and
MeNO (1.08 mL, 20.00 mmol) in MeOH (40 mL). The reaction temperature was kept below 10-
2
o
1
5 C during the addition of the NaOH solution. After stirring for 15 min, the whole reaction
mixture was transferred to a separating funnel and slowly added to 5 M HCl (100 mL). A yellow
solid was obtained almost instantly. The solid was filtered, washed with cold water, dried and
recrystallized from hot EtOH to furnish the title compound as yellow crystal needles (1.66 g,
1
48%). H NMR (400 MHz, CDCl
3
) δ (ppm): 8.00 (d, J = 13.7 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H),
13
7
1
.67 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 13.7 Hz, 1H). C NMR (100 MHz, CDCl
3
) δ (ppm) 139.5,
36.5, 134.4, 133.0, 129.4, 117.8, 115.3.
tert-Butyl (4-cyanophenethyl)carbamate (41). The first step was adopted from a literature
35
method with some modifications . To a solution of compound 40 (1.29 g, 7.40 mmol) in dry
DCM (19 mL) under inert atmosphere was added tributyltin hydride (2.39 mL, 8.88 mmol) and
the reaction mixture was stirred for 16 h. After completion of reaction, the solvent was
evaporated under reduced pressure. The residue was partitioned between MeCN (100 mL) and
hexanes (30 mL). The MeCN phase was then washed two more times with hexanes to remove
the remained tin biproducts and concentrated under reduced pressure. The residue was dissolved
in MeOH (110 mL) and 2 N HCl (110 mL). Zinc powder (6.00 g, 90.00 mmol) was added slowly
o
to the solution prior to stirring for 1 h at 65 C, it was cooled and basified to pH 8 with sodium
carbonate. The solids were filtered off and the filtrate was concentrated to half of its volume. To
this solution was added (Boc) O (1.62 g, 7.44 mmol) in THF (50 mL) and stirred overnight.
2
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
After completion of reaction, THF was evaporated under reduced pressure and the product was
extracted with EtOAc three times. The combined organic phases were washed with brine, dried
over magnesium sulfate and evaporated to dryness. Flash chromatography with hexanes/EtOAc
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(5:1 to 4:1) as eluent furnish title compound as a colorless crystalline solid (1.13 g, 57% for three
1
step). H NMR (300 MHz, CDCl
3
) δ (ppm): 7.59 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H),
1
3
4
.60 (br. s., 1H), 3.38 (ABq, J = 6.5 Hz, 2H), 2.86 (t, J = 6.5 Hz, 2H), 1.42 (s, 9 H). C NMR
(75 MHz, CDCl
3
) δ (ppm) 155.7, 144.7, 132.3, 129.6, 118.8, 110.3, 79.5, 41.3, 36.4, 28.3.
+
HRMS (ESI) calculated for C14
H
18
N
2
O
2
m/z [M+H] 247.1441 found 247.1415.
4-(2-Aminoethyl)benzimidamide (24). To a solution of 41 (0.27 g, 1.07 mmol) in MeOH (20
mL) was added hydroxylammonium chloride (0.11 g, 1.60 mmol) and DIPEA (0.28 mL, 1.60
mmol) and the reaction was stirred at room temperature overnight. The solvent was evaporated
o
under reduced pressure after 16 h stirring at 60 C and the residue was taken into EtOAc, washed
with water and brine and dried over magnesium sulfate. The solvent was evaporated in vacuum
and the residue dissolved in THF (10 mL). DIPEA (0.28 mL, 1.60 mmol) and acetic anhydride
(0.15 mL, 1.60 mmol) were added to the solution. The amidoxime were acetylated within 30 min
according to TLC. Then, the volatiles were evaporated in vacuo and the residue was dissolved in
EtOAc, washed with 0.50 M HCl and brine, dried over magnesium sulfate and the solvent
removed in vacuo. Then the residue was dissolved in 1:1 MeOH/ AcOH and 5% Pd/C (0.050 g)
were added. The hydrogenation was conducted under 35 atm pressure of H in a Parr
2
hydrogenator jar for 16 h. After the completion of reaction (HPLC-MS) reaction mixture was
passed through a pad of diatomaceous earth and purified with preparative HPLC (gradient of 0 to
1
3
0% MeCN in water) to yield a white solid. (0.19 g, 69%). H NMR (300 MHz, D
2
O) δ (ppm):
7.76 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 3.41 (t, J = 7.0 Hz, 1H), 3.09 (t, J = 7.0 Hz,
2
2
ACS Paragon Plus Environment

Page 23 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 165.3, 144.0, 129.3, 128.4, 126.1, 39.2, 32.7
+
HRMS (ESI) calculated for C
9
H
13
N
3
m/z [M+H] 264.1706 found 264.1683. The obtained solid
o
was, then, dissolved in MeOH (3 mL) and conc. HCl (1 mL) was added slowly in 0 C. After
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
stirring for 1 h the solvent was evaporated by an argon stream. The residue was triturated using
1
MeOH/Et
2
O to achieve a white solid in quantitative yield. H NMR (400 MHz, DMSO-d
6
) δ
(ppm): 9.34 (s, 2H), 8.40 (br. s., 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 3.00-
1
3
3
3
.09 (m, 4H). C NMR (100 MHz, DMSO-d6) δ (ppm) 165.3, 144.0, 129.3, 128.4, 126.1, 39.2,
+
2.7. HRMS (ESI) calculated for C
tert-Butyl
9
H
13
N
3
m/z [M+H] 164.1182 found 164.1181.
(S)-(1-((2-(1H-indol-2-yl)ethyl)amino)-3-(4-cyanophenyl)-1-oxopropan-2-yl)-
carbamate (43). To a suspension of Boc-Phe(4-CN)-OH 42 (1.45 g, 5.00 mmol), tryptamine
hydrochloride (0.98 g, 5.00 mmol) and 6-Cl-HOBt (0.93 g, 5.50 mmol) in DCM (15 mL) was
o
added DIPEA (2.61 mL, 15.00 mmol) while the temperature kept 0-5 C. Once a clear solution
obtained, EDCI (1.05 g, 5.50 mmol) was added in the same temperature. The resulting solution
was stirred for 16 h at room temperature. Then, solvent was evaporated, and the residue was
taken into EtOAc and washed with 10% citric acid, saturated NaHCO
over MgSO . The residue was triturated with DCM and hexanes to obtain the pure title
compound as a white solid (1.96 g, 90% yield) H NMR (400 MHz, DMSO-d
br. s., 1H), 8.05 (br. s., 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.54 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.7
3
and brine, then, dried
4
1
6
) δ (ppm) 10.81
(
Hz, 2H), 7.34 (d, J = 7.8 Hz, 1H), 7.15 (s, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.91 - 7.02 (overlapped,
2H), 4.18 (m, 1H), 3.35 (m, 2H), 3.00 (dd, J = 13.0, 3.1 Hz, 1H), 2.80 (overlapped, 3H), 1.28 (s,
13
9
1
H). C NMR (100 MHz, DMSO-d
6
) δ (ppm) 171.0, 155.2, 144.5, 136.3, 131.9, 130.4, 127.2,
22.7, 121.0, 119.1, 118.3, 111.7, 111.4, 109.1, 78.1, 55.3, 41.2, 37.9, 28.1, 25.1. IR (neat) ν
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
(
cm ) 1651, 1682, 2228, 2918, 2956, 3327, 3348, 3407, 3412. HRMS (ESI) calculated for
+
o
C
25
H
28
N
4
O m/z [M+H] 433.2234 found 433.2306. mp; 174-176 C.
3
(S)-N-(2-(1H-indol-2-yl)ethyl)-2-amino-3-(4-cyanophenyl)propanamide
(44).
This
O) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
compound was prepared in similar conditions that was used for 24. H NMR (400 MHz, D
2
(
ppm) 7.68 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.33 (d, J =
7
3
9
1
.9, 1H), 7.08 - 7.12 (m, 1H), 7.06 (s, 1H), 6.97-7.03 (m, 1H), 4.30 (dd, J = 9.0, 5.6 Hz, 1H),
.42 - 3.57 (m, 2H), 3.13 (dd, J = 13.5, 5.6 Hz, 1H), 2.92 (t, J = 7.0 Hz, 2H), 2.86 (dd, J = 13.5,
13
.0 Hz, 1H). C NMR (100 MHz, D
2
O) δ (ppm) 168.3, 157.6, 146.3, 138.3, 131.6, 129.0, 127.9,
-1
23.6, 122.5, 119.8, 119.4, 116.9, 113.2, 112.4, 57.2, 41.4, 39.4, 26.3. IR (neat) ν (cm ) 1666,
1678, 2905, 2943, 2989, 3296, 3393, 3413 HRMS (ESI) calculated for C20
H
23
N
O m/z [M+H]+
5
o
3
50.1975 found 350.1995. mp; decomposed at >300 C.
tert-Butyl (S)-(2-((2-(1H-indol-3-yl)ethyl)amino)-1-(4-cyanophenyl)-2-oxoethyl)carbamate
(46). Compounds 45 (0.92 g, 4.00 mmol) and K CO (0.55 g, 4.00 mmol) were dissolved in H
50 mL). (Boc) O (1.05 g, 4.80 mmol) in THF (25 mL) was added to this solution and stirred
2
3
2
O
(
2
overnight. After completion of reaction, THF was evaporated under reduced pressure. The
solution neutralized with solid citric acid and the product was extracted to EtOAc three times.
The combined organic phases were washed with brine, dried over magnesium sulfate and
evaporated to dryness to give Boc-Phg(4-Br)-OH in quantitative yield. This white solid (1.38 g,
4
.00 mmol) and i-butyl chloroformate (0.52 mL, 4.00 mmol) were dissolved in THF (20 mL) and
o
o
cooled to -20 C prior to addition of DIPEA (0.70 mL, 4.00 mmol, precooled to -20 C). The
suspension was agitated for 10 min before the addition of a mixture of tryptamine hydrochloride
(
0.94 g, 4.80 mmol) and DIPEA (0.84 mL, 4.8 mmol) in THF (5 mL). After stirring for 24 h in -
o
20 C, the solvent was evaporated to dryness and the residue was partitioned between EtOAc and
2
4
ACS Paragon Plus Environment

Page 25 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
water. The phases were separated, and the organic phase was washed with saturated NaHCO ,
3
water, 10% citric acid and brine solutions. The EtOAc was evaporated in vacuum and the residue
was purified with flash chromatography to yield Boc-Phg(4-Br)-Tryp as a white solid (1.13 g, 60
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
% 1
.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.06-7.15 (overlapped, 3H), 6.68
d, J = 2.2 Hz, 1H), 5.93 (overlapped, 2H), 5.02 (br. s, 1H), 3.58 (dt, J = 13.0, 6.5 Hz, 1H), 3.49
) H NMR (400 MHz, CDCl ) δ (ppm) 8.19 (br. s, 1H), 7.49 (d, J = 8.00 Hz, 1H), 7.38 (d, J =
3
8
(
(
(
1
3
dt, J = 13.0, 6.5 Hz, 1H), 2.80 - 2.96 (m, 2H), 1.40 (s, 9H). C NMR (100 MHz, CDCl ) δ
3
ppm) 169.4, 155.1, 137.7, 136.3, 131.9, 128.7, 127.0, 122.2, 122.1, 119.4, 118.4, 112.1, 111.3,
+
8
0.2, 57.9, 39.9, 28.2, 24.9. HRMS (ESI) calculated for C23
found 472.1217. The obtained solid plus Zn(CN) (0.16 g, 1.40 mmol) and Pd(PPh
.11 mmol) were suspended in deoxygenated dry DMF (3 mL) under inert condition. The yellow
H
26BrN
3
O
3
m/z [M+H] 472.1230
2
3
) (0.13 g,
4
0
o
slurry was heated to 100 C for 16 h, then taken into EtOAc and washed three times with 20%
NH
4
OH, dried over MgSO
4
, and purified with flash chromatography to yield the title compound
1
as a white solid (0.39 g, 40%) H NMR (400 MHz, CDCl
3
) δ (ppm) 8.07 (br. s, 1H), 7.46 - 7.57
(overlapped, 3H), 7.38 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.23 (t, J = 8.0 Hz, 1H),
7.12 (t, J = 8.0 Hz, 1 H), 6.79 (d, J = 2.0 Hz, 1H), 5.94 (br. s, 1H), 5.76 (br. s, 1H), 5.05 (br. s,
1
3
1
H), 3.57-3.67 (m, 2H), 2.84 - 2.98 (m, 2H), 1.40 (br. s., 9H). C NMR (100 MHz, CDCl ) δ
3
(ppm) 168.5, 164.8, 143.8, 136.3, 132.6, 127.7, 127.0, 122.4, 122.0, 119.7, 118.4, 118.4, 112.1,
-1
1
12.0, 111.4, 80.5, 60.4, 40.1, 28.2, 24.8. IR (neat) ν (cm ) 1659, 1699, 2235, 2939, 2983, 3234,
+
3290, 3421. HRMS (ESI) calculated for C24
H
26
N O
4 3
m/z [M+H] 419.2077 found 419.2137. mp;
o
9
8-101 C.
(S)-N-(2-(1H-Indol-3-yl)ethyl)-2-amino-2-(4-carbamimidoylphenyl)acetamide 47. The title
1
compound was prepared with the same procedure as 24. H NMR (400 MHz, D
2
O) δ (ppm) 7.54
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
d, J = 8.54 Hz, 2H), 7.44 (d, J = 7.86 Hz, 1H), 7.39 (d, J = 8.20 Hz, 1H), 7.26 - 7.33 (m, 2H),
7
.16 (t, J = 7.2 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.89 (s, 1H), 5.00 (s, 1H), 3.89 (ddd, J = 13.6,
8.7, 5.1 Hz, 1H), 3.39 (dt, J = 13.6, 5.1 Hz, 1H), 2.94 (dt, J = 13.6, 5.1 Hz, 1H), 2.83 (ddd, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
1
1
1
3
3.6, 8.7, 5.1 Hz, 1H). C NMR (100 MHz, D
2
O) δ (ppm) 165.6, 162.8,137.5, 135.9, 128.5,
-1
28.1, 126.9, 123.1, 121.6,118.9, 118.2, 114.9, 111.5, 111.0, 55.8, 39.5, 23.5. IR (neat) ν (cm )
622, 1682, 2914, 2970, 3010, 3265, 3292. HRMS (ESI) calculated for C19
H
21
N
O m/z [M+H]+
5
o
36.1819 found 336.1872. mp; decomposed at >300 C.
Enzyme kinetics. The fluorometric K measurements of PC enzymes was performed using a
i
Gemini EM 96-well spectrofluorometer (Molecular Devices; Sunnyvale, CA, USA) (λEX., 370
nm; λEM., 460 nm; Cut Off, 435 nm). Both PACE4 and furin are human recombinant produced
19
from S2 insect cells and purified by a method described earlier . All the experiments were done
in duplicate or triplicate. Furin inhibition measurements were performed in 100 mM of HEPES
buffer (pH = 7.5), 1 mM CaCl
2
, 1 mM β-mercaptoethanol, and 1.8 g/L BSA, while PACE4
, and 1.8 g/L
inhibition assays were done in a 20 mM Bis-Tris buffer (pH = 6.5), 1 mM CaCl
2
BSA. The competitive substrate was pyroGlu-Arg-Thr-Lys-Arg-AMC peptide (Bachem,
Switzerland) for both furin and PACE4 with the concentration of 100 μM in each well. Inhibitors
were added with various concentrations in the range of 0-10 μM. 2 units of enzyme (final
concentration 100 μL) were added in each well, and the real-time fluorescence was measured
during 1 h of experiments. The K values were determined using Cheng and Prusoff’s equation
i
by SoftMaxPro5 program.36
Cell proliferation. MTT antiproliferative assay has been accomplished for DU145 and LNCaP
9
PCa cell lines as reported earlier. The 96 well plates seeded with approximate density of 1500
cells for DU145 or 2500 cells for LNCaP. The media was changed after 24 h followed by
2
6
ACS Paragon Plus Environment

Page 27 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
addition of different concentrations of peptide inhibitors (For peptides 3−19, 21 and 28, a range
of 300, 200, 100, 50, 25, 10 and 1 μM concentrations; and for peptides 20 and 29−33 a range of
150, 100, 75, 50, 25, 10 and 1 μM were applied). The incubation continued for 72 h prior to
addition of MTT reagent (with final concentration per well of 1 g/L). The incubation continued
for further 4 h. At the time, the media has been removed and cells were treated with 0.10 mL of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-propanol/HCl (24:1 N) to solubilize the formazan produced in the mitochondria. The metabolic
activity of cells assessed relatively by vehicle treated cells (Sterile bi-distilled water 0.1%
DMSO). The maintenance of cells and all the assay steps were performed in RPMI 1640 5% FBS
for DU145 and 10% FBS for LNCaP cells. The IC50 values were calculated using Prism 6.0
(GraphPad software).
Cell permeability. The DU145 cells were plated at a density of 200000 cells per 100 mm petri
o
dish and incubated for 48 h at 37 C. Cells were treated with 1 µM of FITC analogs for 1 h at the
same temperature prior to collection using 0.05% trypsin which later was inactivated with FBS-
containing media. Cell pellets were washed one more time with PBS and were resuspended in
200 µL of fresh PBS after centrifugation. PI was added at a final concentration of 10 µg/mL just
before doing the acquisition. In another set of tubes, fluorophore was quenched using trypan blue
at a final concentration of 0.04%. Cells were analyzed (at least 10000 events) by CytoFLEX 15
flow cytometer (Beckman Coulter, Brea, CA, USA) with following diode lasers: 488 nm and 638
nm, 50 mW each. The emitted fluorescence was split into four channels and detected through
band pass filters (Forward scatter area, side scattered area and side scattered width signals) to
establish the live gates and exclude debris and cell clumps. Dead cells (PI-positive) were
excluded by the gating in the red channel.
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cell toxicity. 200 000 cellules were plated in a 6-well plate and then, were treated with 1, 10,
0 and 100 µM concentrations of compounds 10, 16, 20 and 31 in serum free media for 1.5 h
5
(For compound 32 the concentrations were 1, 10, 15, 20, 30, 50, 100 µM). Cells were collected
using trypsin prior to washing and then resuspension in PBS. PI at a final concentration of 10
µg/mL was added just prior to acquisition of fluorescence by CytoFLEX 15 flow cytometer as
described above. The IC50(PI) value for compound 32 was calculated using Prism 6.0 (GraphPad
software).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Plasma stability. Peptide samples for determination of t1/2 half-lives were prepared as follows;
The total concentration of 50 μM peptide in human serum (from male AB plasma; Sigma
o
Aldrich) were incubated at 37 C. At each indicated timepoint and just following peptide
addition, 25 μL of plasma were precipitated with 100 μL of MeOH containing 20 μM Fmoc-Leu-
OH as internal standard. Samples, then, were subjected to centrifuge at 12000 ×g for 10 min at
room temperature to remove precipitated proteins and clear supernatants were filtered on 0.2 μM
PVDF centrifugal filters (Canadian Life Science) prior to analysis. Quantitative analysis
performed based on the peptide/standard ratio of integrated area under the curve in the total ion
chromatogram and reported from 0 h incubation. The one-phase decay curve-fit of GraphPad
Prism was used to determine t1/2 values.
In vivo acute toxicity. All experimental procedures were performed as reported earlier in
accordance with regulations of the Canadian Council on Animal Care.12
ASSOCIATED CONTENT
Supporting Information
Information for enzyme kinetics and cell toxicity assays, analytical data (HPLC and HRMS
analysis) and molecular formula strings for all biologically tested compounds (3−21, 28−33 and
2
8
ACS Paragon Plus Environment

Page 29 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
5−38) and NMR spectra for small molecule intermediates are provided as supporting
information. This material is available free of charge via the http://pubs.acs.org.
AUTHOR INFORMATIONS
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Corresponding Authors
*
*
(R.D.) Telephone: +1 (819) 821-8000, ext. 75428. E-mail: Robert.Day@USherbrooke.ca and
(Y.L.D.) Telephone: +1 (819) 821 8000 ext. 75299. E-mail: Yves.Dory@USherbrooke.ca
ORCID
Vahid Dianati: 0000-0001-6883-6574
Anna Kwiatkowska: 0000-0002-5659-3680
Yves Dory: 0000-0003-4758-1589
Robert Day: 0000-0001-8123-1991
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge the Canadian Cancer Society Research Institute (701590 to R.D. and Y.L.D.)
and Prostate Cancer Canada (TAG2014-02 to R.D.) for their support. F.C. holds a Banting and
Charles Best Canada Graduate Scholarships (grant#315690) from CIHR and Graduate
Studentship from Prostate Cancer Canada (Grant #GS-2015-07). We also thank Hugo Gagnon
and Jean-Philippe Couture (PhenoSwitch Biosciences Inc.) for HRMS analysis and Sandra
Gagnon for acute toxicity analysis.
ABBREVIATIONS USED
PC, proprotein convertase; PCa, prostate cancer; PACE4, paired basic amino acid cleaving
enzyme 4; GDF-15, Growth/differentiation factor 15; Amba, 4-amidinobenzylamide; S-i-Agb,
2
9
ACS Paragon Plus Environment