Structure-activity relationship studies of prostate-specific membrane antigen (PSMA) inhibitors derived from α-amino acid with (S)- or (R)-configuration at P1′ region

Article abstract of DOI:10.1016/j.bioorg.2020.104304

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, is considered an excellent target for the diagnosis or treatment of prostate cancer. We previously investigated the effect of β- and γ-amino acids with (S)- or (R)-configuration in the S1 pocket on the binding affinity for PSMA. However, comprehensive studies on the effect of α-amino acid with (R)-configuration in the S1′ pocket has not been reported yet. We selected ZJ-43 (1) and DCIBzL (5) as templates and synthesized their analogues with (S)- or (R)-configuration in the P1 and P1′ regions. The PSMA-inhibitory activities of compounds with altered chirality in the P1′ region were dropped dramatically, with their IC₅₀ values changing from nM to μM ranges. The compounds with (S)-configuration at both P1 and P1′ regions were more potent than the others. The findings of this study may provide insights regarding the structural modification of PSMA inhibitor in the S1′ binding pocket.

Full text of DOI:10.1016/j.bioorg.2020.104304

Bioorganic Chemistry 104 (2020) 104304
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structure-activity relationship studies of prostate-specific membrane
antigen (PSMA) inhibitors derived from α-amino acid with (S)- or
(R)-configuration at P1′ region
Hongmok Kwona, Hyunwoong Lima, Hyunsoo Haa, Doyoung Choia, Sang-Hyun Sona,
,
,
,d,*
Hwanhee Namb c, Il Minnb c, Youngjoo Byuna
a College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong 30019, Republic of Korea
b Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical Institutions, Baltimore, MD 21287, United States
c Institute for NanoBioTechnology (INBT), Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, United States
d Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu, Seoul 08308, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, is considered an excellent target
for the diagnosis or treatment of prostate cancer. We previously investigated the effect of β- and γ-amino acids
with (S)- or (R)-configuration in the S1 pocket on the binding affinity for PSMA. However, comprehensive studies
Absolute configuration
PSMA
SAR
on the effect of α
-amino acid with (R)-configuration in the S1′ pocket has not been reported yet. We selected ZJ-
Prostate cancer
43 (1) and DCIBzL (5) as templates and synthesized their analogues with (S)- or (R)-configuration in the P1 and
P1′ regions. The PSMA-inhibitory activities of compounds with altered chirality in the P1′ region were dropped
dramatically, with their IC50 values changing from nM to μM ranges. The compounds with (S)-configuration at
both P1 and P1′ regions were more potent than the others. The findings of this study may provide insights
regarding the structural modification of PSMA inhibitor in the S1′ binding pocket.
1. Introduction
since the seminal work of Kozikowski and co-workers was first reported
in 2001 [14]. ZJ-43 (1, Fig. 1), a representative compound of the urea-
Prostate-specific membrane antigen (PSMA), also known as gluta-
mate carboxypeptidase II (GCPII), is a zinc metallopeptidase that hy-
drolyzes endogenous N-acetylaspartylglutamate (NAAG) to N-
acetylaspartate (NAA) and glutamate [1,2]. PSMA exhibits restricted
expression with an enzymatic active site in the extracellular region and
on the cell surface of prostate cancer (PCa) cells, and its upregulated
expression levels correlated with tumor progression and metastasis
[3,4]. Thus, PSMA is considered one of the most attractive targets for the
diagnosis of PCa [5]. In addition, since excessive production of gluta-
mate causes glutamate-associated neurotoxicity and neuronal death,
PSMA-mediated release of glutamate by cleavage of NAAG in the brain
has been identified as a potential target for the regulation of intra-
synaptic glutamate concentration [6–10]. The small molecules bearing
functional groups including phosphinate, phosphoramidate, urea,
hydroxamate, and thiol have been identified as key scaffolds of PSMA
inhibitors [11–17]. Those scaffolds interact with Zn2+ at the active site
[12,13,18–22]. Among them, urea scaffold has been in the limelight
based PSMA inhibitors with high potency (Ki = 0.8 nM) [6], has been
widely exploited as a template for development of PSMA inhibitors and
is a prototype molecule for investigating the physiological features of
PSMA. ZJ-43 treatment in various animal models substantiated that
PSMA inhibition results in neuroprotection by regulating the glutamate
levels, indicating a possibility for it to treat schizophrenia, traumatic
brain injury, and neuropathic pain [23–25]. In addition, ZJ-43 also
provided a structural prototype for the development of urea-based
PSMA inhibitors [26,27]. Not only the synthetic accessibility and high
potency of urea-based compounds, but also the chemical stability of urea
group allowed various structural modifications. The versatile interme-
diate Lys-urea-Glu has been considered the most attractive template to
develop imaging or therapeutic agents for PCa. For example, treatment
with DCIBzL (5), one of the potent PSMA inhibitors (Ki = 0.01 nM),
demonstrated that Lys-urea-Glu can be the most useful scaffold for
developing PCa imaging agents including single photon emission
computed tomography (SPECT), positron emission tomography (PET),
* Corresponding author at: College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong 30019, Republic of Korea.
E-mail address: yjbyun1@korea.ac.kr (Y. Byun).
https://doi.org/10.1016/j.bioorg.2020.104304
Received 22 May 2020; Received in revised form 15 September 2020; Accepted 20 September 2020
Available online 24 September 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
and optical scanning in animal models and clinical studies [28–30]. The
p-iodobenzoyl group of DCIBzL interacts with the S1 hydrophobic
accessory pocket, which consists of Arg463, Arg534, and Arg536, to
contribute to the enhancement of PSMA-inhibitory activity [31]. Based
on the structural freedom of S1 pocket, diverse functional groups have
been conjugated to the P1 region of Lys-urea-Glu PSMA inhibitors, tar-
geting the clinical application for PCa diagnosis and treatment
[13,30,32–35].
removed by adding 25% trifluoroacetic acid (TFA) in CH2Cl2. The
deprotected Lys-urea-Glu compounds (13–15) without further purifi-
cation were used for the reaction with N-succinimidyl 4-iodobenzoate to
afford the final compounds 6–8.
All final compounds were purified by semi-preparative reversed-
phase high-performance liquid chromatography (RP-HPLC) with high
purity (>95%) using acetonitrile (ACN) and water containing 0.1%
formic acid (FA) as mobile phase. The chemical structure of the final
compounds were analyzed by NMR spectroscopy and ESI-MS. We
monitored all final compounds in two different eluent conditions (ACN/
water and methanol/water) using analytical RP-HPLC to compare the
differences in lipophilicity between isomers. As shown in Fig. 2, com-
pounds 1 (S/S) and 4 (R/R) displayed faster retention time than their
diastereomers 2 (R/S) and 3 (S/R) in ACN/water eluent condition. The
compounds that are enantiomers (1/4 and 2/3) showed the same
retention times in the HPLC experiment. These results were found to be
consistent for compounds 5–8 irrespective of the mobile phase mixtures.
While researching the structural modification of urea-based PSMA
inhibitors, we recently reported structure–activity relationship (SAR)
studies of PSMA inhibitors derived from β- and γ-amino acid analogues
with (S)- or (R)-configuration at the S1 binding site [36]. The β- and
γ-amino acid-derived compounds presented critical alterations in the
PSMA-inhibitory activities depending on their absolute configuration at
the P1 region. This result prompted us to investigate the effect of the
absolute configuration in the P1′ region that interacts with the S1′
pharmacophore pocket and has not been sufficiently studied yet.
Although Tsukamoto and colleagues reported carbamate-based com-
pounds with (S)- or (R)-configuration at the S1 or S1′ binding sites
derived from ZJ-43 [37], urea-based compounds with (R)-configuration
at the P1′ region have not been studied comprehensively.
2.2. In vitro NAALADase assays
Fluorescence-based NAALADase assay [38,39] was applied to
determine the PSMA-inhibitory activities of the prepared 8 compounds
and the results are summarized in Table 1. Significant decreases in the
PSMA-inhibitory activities of ZJ-43 (1, IC50 = 4.03 nM) and DCIBzL (5,
IC50 = 0.13 nM) were observed when the (S)-configuration in the P1
region was changed to the (R)-configuration (2, IC50 = 197 nM; 6, 1200
nM), which is consistent with the results reported previously [36,37].
Because of the bulky p-iodobenzoyl group at 6, the effect of chirality
change in the P1 region at 6 was greater than that of 2. When the ste-
reochemistry of glutamic acid at the P1′ region was changed from (S)- to
In this study, we synthesized and evaluated a small number of ZJ-43
and DCIBzL analogues to examine the effect of absolute configuration in
the P1 and P1′ region of urea-based compounds on the PSMA-binding
affinity. We selected ZJ-43 (1) to investigate the effect of configura-
tional change in the S1′ site without affecting the S1 pocket much. We
also selected DCIBzL (5) to determine the effect of chirality changes on
the binding leverage of S1 and S1 accessory pocket.
2. Results and discussion
(R)-configuration, compounds 3 (IC50 > 10 μM) and 7 (IC50 = 2410 nM)
2.1. Synthesis of urea-based PSMA inhibitors
almost lost the PSMA-inhibitory activities. These results indicate that
the S1′ site is much more sensitive for stereochemistry than the S1 site,
which is in accordance with the argument that S1 site provides more
structural freedom than the S1′ site [13]. Interestingly, compound 7
showed a little stronger PSMA-inhibitory activity than 3 because the
interaction of the p-iodobenzoyl group with accessory hydrophobic
pocket, which is formed by the arginine patch at the S1 accessary site,
Compounds 1–4 were synthesized from commercial L- or D-glutamic
acid di-tert butyl ester by attaching L- or D-leucine tert-butyl esters ac-
cording to the synthetic routes described in Scheme 1. Compound 5 was
also prepared by applying the reported procedure [28] whereas the
other compounds (6–8) were synthesized by modifying the synthetic
method used for synthesizing 5 [36], as shown in Scheme 2. Briefly,
reaction of L- or D-glutamic acid di-tert butyl ester with triphosgene in the
presence of triethylamine, followed by the addition of Leu or Lys, pro-
vided the Leu-urea-Glu (9–12) or Lys-urea-Glu compounds (13–15),
respectively. The tert-butyl group of the urea compounds (9–15) were
′
́
might offset the effect of the chirality change at the P1 region. Both
compounds 4 and 8 with (R/R) configurations at the P1 and P1′ sites
displayed no PSMA-inhibitory activities, demonstrating that the
α
-amino acids with (R)-configuration in the urea-based PSMA inhibitors
negatively affected the interaction with the key residues in both the S1
P1 region
modification
Urea scaffold
introduction
Fig. 1. Design concept of urea-based PSMA inhibitors derived from α-amino acids with (S)- or (R)-configuration.
2

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
Scheme 1. Synthetic route for compounds 1–4a. aReagents and reaction conditions: (i) Triphosgene, L-Leu di-tert butyl ester HCl (for 9 and 11) or D-Leu di-tert butyl
ester HCl (for 10 and 12), Et3N, CH2Cl2, –78℃ to rt, 24 h; (ii) TFA/CH2Cl2 (1/4, v/v), rt, 3 h.
Scheme 2. Synthetic route for compounds 6–8a. aReagents and reaction conditions: (i) Triphosgene, Nε-Boc-L-Lys (for 14) or Nε-Boc-D-Lys (for 13 and 15), Et3N,
CH2Cl2, –78 ℃ to rt, 24 h; (ii) TFA/CH2Cl2 (1/4, v/v), rt, 3 h; (iii) N-succinimidyl 4-iodobenzoate, Et3N, DMF, rt, 2.5 h.
and S1′ sites of PSMA.
docked in the active site of the X-ray crystal structure of PSMA com-
plexed with ZJ-43 (PDB ID: 6FE5) [37], whereas 5–8 were docked in the
X-ray crystal structure of PSMA complexed with DCIBzL (PDB ID: 3D7H)
[13]. Schematic depiction of the key interactions between the synthe-
sized compounds and PSMA active site is provided in Supplementary
data. Docking studies revealed that the chirality change in 1 forced the
urea group away from Zn2+, resulting in the loss of PSMA-inhibitory
2.3. Molecular modeling studies
To elucidate the binding modes of the synthesized compounds, in
silico docking studies were conducted using Surflex-Dock Geom module
using SYBYL-X 2.1.1 software (Tripos Inc, NJ). Compounds 1–4 were
3

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
(A)
(B)
mAU
mAU
1 (S / S)
2 (R / S)
3 (S / R)
4 (R / R)
1 (S / S)
2 (R / S)
3 (S / R)
4 (R / R)
Mixture of 1–4
Mixture of 1–4
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
tR (min)
tR (min)
(C)
(D)
mAU
mAU
5 (S / S)
6 (R / S)
7 (S / R)
8 (R / R)
5 (S / S)
6 (R / S)
7 (S / R)
8 (R / R)
Mixture of 5–8
Mixture of 5–8
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
t
R (min)
tR (min)
Fig. 2. (A) HPLC spectra of compounds 1–4 eluted with ACN and water (20:80, v/v, and 0.1% FA), flow rate: 1 mL/min. (B) HPLC spectra of compounds 1–4 eluted
with methanol and water (50:50, v/v, and 0.1% FA), flow rate: 1 mL/min. (C) HPLC spectra of compounds 5–8 eluted with ACN and water (30:70, v/v, and 0.1% FA),
flow rate: 1 mL/min. (D) HPLC spectra of compounds 5–8 eluted with methanol and water (60:40, v/v, and 0.1% FA), flow rate: 1 mL/min.
Table 1
Table 2
In vitro PSMA-inhibitory activity of the synthesized compounds.
In silico docking data of synthesized compounds.
Compound
Stereochemistry
IC50 (nM)
95% CIa
Cmpd
Stereo-
T-score
H-bonding amino acidsa
Zn2+–O of
chemistry
urea distance
ZJ-43 (1)
(S,S)
(R,S)
(S,R)
(R,R)
(S,S)
(R,S)
(S,R)
(R,R)
4.03
2.96–5.49
123–316
2
197
ZJ-43
(1)
(S,S)
12.3131
R210, N257, G518, N519,
R534, R536, Y552, K699,
Y700b
2.88 Åb
3
>10000
>10000
0.13
4
DCIBzL (5)
0.04–0.40
529–2740
1230–4730
2
3
(R,S)
(S,R)
11.4895
11.8887
R210, N257, G518, N519,
R534, Y552
3.19 Å
3.37 Å
6
7
8
1200
2410
R210, N257, G518, N519,
R534, R536, Y552, K699
G518, N519, R534, Y552
R210, N257, E425, G518,
N519, R534, R536, Y552,
K699, Y700c
>10000
4
(R,R)
(S,S)
11.0603
15.1531
4.38 Å
a
Confidence interval.
DCIBzL
(5)
2.55 Åc
activity (Table 2). Despite insignificant differences in binding poses
between 2 and 3, compound 3 showed longer distance of Zn2+ from
oxygen of urea group than 2. In addition, the γ-carboxylate group of
glutamic acid of 3 was closer to Leu428 than that of 2. Leu428 con-
tributes nonpolar interactions to the inhibitor binding [13], which might
have led to the repulsion of 3 to S1′ site. Compound 4 was docked in the
active site of PSMA in a reverse pose contrary to 2 or 3, which failed to
6
7
8
(R,S)
(S,R)
(R,R)
14.0610
13.3062
13.1755
R210, N257, R463, G518,
Y552, K699
1.99 Å
5.58 Å
4.18 Å
R210, E425, R463, G518,
Y552, K699
R210, N257, G518, N519,
R534, Y552
a
Amino acids within a range of 2.50 Å.
b
c
́
Based on PSMA X-ray crystal structure in complex with 1 (PDB ID: 6FE5).
Based on PSMA X-ray crystal structure in complex with 5 (PDB ID: 3D7H).
present hydrogen bonding interaction with S1 pocket, particularly
Lys699 (Fig. 3).
Because of distorted binding pose, 4 exhibited the longest distance
(4.38 Å) of Zn2+ from oxygen of urea scaffold among 1–4. Most of the
potent urea-based or carbamate-based PSMA inhibitors exhibited
interaction with Zn2+ within 2–3 Å range [13,32,37,40–42], providing
an explanation for the diminished PSMA-inhibitory activity of ZJ-43
analogues. In docking simulation experiments of 5–8, compounds 5
and 8 showed the highest (15.1531) and lowest (13.1755) T-scores,
respectively, correlating with the in vitro assay results. The p-iodo-
benzoyl groups of 5–8 were projected into the S1 hydrophobic accessory
pocket (Fig. 3). While compound 6 exhibited a shorter distance (1.99 Å)
between oxygen of urea and Zn2+ than 5 (2.55 Å), it showed less
hydrogen-bonding interactions with amino acids within a range of 2.50
Å than 5. As the chirality of lysine was changed from (S) to (R), the
carboxylate group in lysine of 6 was located at>2.50 Å range from
Arg534, Arg536, and Asn519. The urea group of 7 was farther away
from Zn2+ (5.58 Å) than 8 (4.18 Å) but the carboxylate of lysine pre-
sented a pose to interact with Zn2+, whereas 8 displayed no interaction
with Zn2+. In addition, the urea group of 8 formed hydrogen bonds with
Gly518 less tightly than 7. Overall, the chirality changes in urea-based
PSMA inhibitors in the α-amino acid region led not only to alterations
in hydrogen bonding interaction with PSMA active site but also relo-
cation of urea group from active site Zn2+, resulting in detrimental ef-
fects on the PSMA-inhibitory activities.
4

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
(A)
(B)
Y552
Y552
R536
R536
Y700
Y700
R210
R210
R534
R534
K699
K699
G518
G518
N257
N257
N519
N519
(C)
(D)
Y552
R210
Y552
R210
Y700
Y700
K699
N257
N257
K699
E425
R536
R536
G518
G518
R534
R534
E425
N519
N519
Fig. 3. (A) Bound-pose of ZJ-43 (1) in the active site of PSMA (PDB ID: 6FE5). (B) Docked-pose of 4 in the active site of PSMA. (C) Bound-pose of DCIBzL (5) in the
active site of PSMA (PDB ID: 3D7H). (D) Docked-pose of 8 in the active site of PSMA. The active site zinc and chloride ions are shown as orange and lime green
spheres, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Conclusion
precoated silica gel plates (60 F254). Reaction progress was monitored by
TLC analysis using a UV lamp and/or KMnO4 staining for detection
purposes. Column chromatography was performed on silica gel
(230–400 mesh, Merck, Darmstadt, Germany). 1H and 13C NMR spectra
were recorded at room temperature (298 K) in CDCl3 (7.26/77.16 ppm),
CD3CN (1.94/118.26 ppm), D2O (4.79 ppm) or CD3OD (3.31/49.00
ppm) on Bruker Ultrashield 600 MHz Plus spectrometer and referenced
to an internal solvent. Chemical shifts are reported in parts per million
(ppm). Coupling constants (J) are given in Hertz. Splitting patterns are
indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd,
double of doublet; br, broad for 1H NMR data. High resolution mass
spectra (HRMS) were recorded on an Agilent 6530 Accurate mass Q-TOF
LC/MS spectrometer. High-performance liquid chromatography purifi-
cation was performed on Agilent 1260 Infinity (Agilent). The purifica-
tion of synthesized compounds was performed on a semi-preparative
reverse-phase high-performance liquid chromatography (RP-HPLC;
Agilent 1260 series HPLC instrument) using a semi-preparative column
In this study, we synthesized 8 compounds to investigate the absolute
configuration of the PSMA inhibitors derived from α-amino acids and
their effect on the PSMA-inhibitory activities. Two potent urea-based
PSMA inhibitors, ZJ-43 (1) and DCIBzL (5), were used as molecular
templates for introducing structural variations. The PSMA-inhibitory
activities changed dramatically when changed from (S) to (R)-configu-
ration, and thus substantiating α-amino acids with (S)-configuration are
favorable for both the S1 and S1′ sites of PSMA. Particularly, the
′
́
chirality change of glutamic acid moiety at the P1 region affected the
PSMA-inhibitory activity more than that of the P1 region, demonstrating
that the S1′ site is a pharmacophore pocket. In molecular docking
studies, the synthesized compounds with changed chirality showed
distorted binding poses leading to the shifted position of urea group
from Zn2+ at the PSMA active site. These results support previous studies
showing that the S1′ pocket is structurally rigid and much less permis-
sive than the S1 pocket [12,27,43]. However, despite relatively few
reported cases compared to those of the P1 region, some studies have
reported structural modifications of the P1′ region of PSMA inhibitors
[26,27,44]. Especially, Yang and co-workers proved that there is still
potential for structural optimization of the S1′ site binding [44]. The
results of SARs in this study may provide insights for further modifica-
tion of urea-based PSMA inhibitors.
(Phenomenex Gemini-NX C18, 110 Å, 150 mm × 10 mm, 5 μm) using
water (containing 0.1% FA) and acetonitrile (ACN; containing 0.1% FA)
as mobile phase. UV detection was carried out at 220 nm. The purity of
all final compounds was measured by analytical RP-HPLC on an Agilent
1260 Infinity (Agilent) with a C18 column (Phenomenex, 150 mm × 4.6
mm, 3 μm, 110 Å). RP-HPLC was performed on two different solvent
systems of ACN/water or water/methanol. Purity of the tested com-
pounds was > 95%.
4. Experimental section
4.2. Synthesis
4.1. General methods
4.2.1. (S)-di-tert-butyl 2-(3-((S)-1-(tert-butoxy)-4-methyl-1-oxopentan-2-
yl)ureido)pentanedioate (9).
All the chemicals and solvents used in the reaction were purchased
from Sigma-Aldrich, TCI, or Alfa Aesar, and were used without further
purification. Reactions were monitored by TLC on 0.25 mm Merck
L-Glutamic acid di-tert-butyl ester hydrochloride (150 mg, 0.51
mmol) was dissolved in anhydrous dichloromethane (DCM, 3.0 mL)
5

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
then added triethylamine (0.57 mL, 4.08 mmol) under argon atmo-
sphere. The solution was cooled to –78 ℃. To the reaction mixture was
added dropwise triphosgene (47 mg, 0.16 mmol) dissolved in anhydrous
DCM (1.5 mL). The reaction mixture was slowly warmed up to rt and
stirred for 35 min. L-leucine tert-butyl ester hydrochloride (114 mg, 0.51
mmol) and triethylamine (0.57 mL, 4.08 mmol) dissolved in DCM (3.0
mL) were added in the reaction mixture and stirred at room temperature
for 24 h. The organic layer was washed with water then drop the acidity
level to pH 6 by 3 N HCl. The collected organic layer was dried over
MgSO4 then concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel with DCM–methanol (100:1 to
80:1, v/v) to afford compound 9 in 75% yield (181 mg, 0.38 mmol). 1H
NMR (600 MHz, CD3OD): δ ppm 4.22–4.15 (m, 2H), 2.34–2.28 (m, 2H),
2.08–2.00 (m, 1H), 1.86–1.69 (m, 2H), 1.59–1.51 (m, 1H), 1.48–1.43
(m, 28H), 0.96 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.5 Hz, 3H). ESI-HRMS
4.2.6. (S)-2-(3-((R)-1-carboxy-3-methylbutyl)ureido)pentanedioic acid
(2).
This compound was afforded in 44% yield, following the same pro-
cedure described for synthesis of compound 1 with compound 10. 1H
NMR (600 MHz, D2O): δ ppm 4.26 (dd, J = 5.0 and 9.2 Hz, 1H), 4.21 (t,
J = 7.6 Hz, 1H), 2.49–2.43 (m, 2H), 2.20–2.11 (m, 1H), 1.99–1.90 (m,
1H), 1.71–1.62 (m, 1H), 1.59 (t, J = 7.6 Hz, 2H), 0.90 (d, J = 6.5 Hz,
3H), 0.87 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, D2O): δ ppm 179.23,
178.58, 177.59, 160.57, 53.91 53.26 41.27 31.41 27.68, 25.76 23.57,
21.97. ESI-HRMS (m/z): calculated for
C
12H21N2O+7 [M
+
H]+,
305.1343; found, 305.1360.
4.2.7. (R)-2-(3-((S)-1-carboxy-3-methylbutyl)ureido)pentanedioic acid
(3).
This compound was afforded in 52% yield, following the same pro-
cedure described for synthesis of compound 1 with compound 11. 1H
NMR (600 MHz, D2O): δ ppm 4.27 (dd, J = 5.1 and 9.2 Hz, 1H), 4.22 (t,
J = 7.6 Hz, 1H), 2.52–2.44 (m, 2H), 2.21–2.12 (m, 1H), 2.00–1.91 (m,
1H), 1.72–1.63 (m, 1H), 1.60 (t, J = 7.1 Hz, 2H), 0.91 (d, J = 6.5 Hz,
3H), 0.88 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, D2O): δ ppm 179.26,
178.61, 177.62, 160.60, 53.92, 53.27, 41.26, 31.42, 27.68, 25.77,
23.57, 21.96. ESI-HRMS (m/z): calculated for C12H21N2O+7 [M + H]+,
305.1343; found, 305.1354.
(m/z): calculated for
C
24H45N2O+7 [M
+
H]+, 473.3221; found,
473.3231.
4.2.2. (S)-di-tert-butyl 2-(3-((R)-1-(tert-butoxy)-4-methyl-1-oxopentan-2-
yl)ureido)pentanedioate (10).
This compound was afforded in 78% yield, following the same pro-
cedure described for synthesis of compound 9 with D-leucine tert-butyl
ester HCl. 1H NMR (600 MHz, CDCl3): δ ppm 5.36–5.24 (m, 2H),
4.39–4.26 (m, 2H), 2.41–2.24 (m, 2H), 2.12–2.02 (m, 1H), 1.92–1.82
(m, 1H), 1.76–1.67 (m, 1H), 1.61–1.54 (m, 1H), 1.47–1.41 (m, 28H),
0.94 (d, J = 6.6 Hz, 6H). ESI-HRMS (m/z): calculated for C24H45N2O7+
[M + H]+, 473.3221; found, 473.3215.
4.2.8. (R)-2-(3-((R)-1-carboxy-3-methylbutyl)ureido)pentanedioic acid
(4).
This compound was afforded in 51% yield, following the same pro-
cedure described for synthesis of compound 1 with compound 12. 1H
NMR (600 MHz, D2O): δ ppm 4.25 (dd, J = 5.2 and 9.2 Hz, 1H), 4.21 (t,
J = 7.2 Hz, 1H), 2.50 (t, J = 7.3 Hz, 2H), 2.20–2.12 (m, 1H), 2.00–1.91
(m, 1H), 1.73–1.65 (m, 1H), 1.61 (t, J = 7.1 Hz, 2H), 0.92 (d, J = 6.6 Hz,
3H), 0.88 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, D2O): δ ppm 179.31,
178.65, 177.67, 160.72, 53.97, 53.31, 41.21, 31.42, 27.61, 25.74,
23.56, 21.97. ESI-HRMS (m/z): calculated for C12H21N2O+7 [M + H]+,
305.1343; found, 305.1353.
4.2.3. (R)-di-tert-butyl 2-(3-((S)-1-(tert-butoxy)-4-methyl-1-oxopentan-2-
yl)ureido)pentanedioate (11).
This compound was afforded in 65% yield, following the same pro-
cedure described for synthesis of compound 9 with D-glutamic acid di-
tert butyl ester hydrochloride. 1H NMR (600 MHz, CDCl3): δ ppm
5.35–5.22 (m, 2H), 4.40–4.25 (m, 2H), 2.40–2.23 (m, 3H), 2.11–2.03
(m, 1H), 1.93–1.67 (m, 2H), 1.62–1.52 (m, 1H), 1.48–1.41 (m, 27H),
0.94 (d, J = 6.6 Hz, 6H). ESI-HRMS (m/z): calculated for C24H45N2O7+
[M + H]+, 473.3221; found, 473.3202.
4.2.9. (R)-6-((tert-butoxycarbonyl)amino)-2-(3-((S)-1,5-di-tert-butoxy-
1,5-dioxopentan-2-yl)ureido)hexanoic acid (14).
4.2.4. (R)-di-tert-butyl 2-(3-((R)-1-(tert-butoxy)-4-methyl-1-oxopentan-2-
yl)ureido)pentanedioate (12).
D-Glutamic acid di-tert-butyl ester hydrochloride (300 mg, 1.01
mmol) was dissolved in anhydrous DCM (5.0 mL) then added triethyl-
amine (1.13 mL, 8.08 mmol) under argon atmosphere. The solution was
cooled to –78 ℃. To the reaction mixture was added dropwise tri-
phosgene (95 mg, 0.32 mmol) dissolved in anhydrous DCM (3.0 mL).
The reaction mixture was slowly warmed up to rt and stirred for 35 min.
Nε-Boc-L-Lys (200 mg, 0.81 mmol) and trimethylamine (0.90 mL, 6.48
mmol) dissolved in DCM (8.0 mL) were added in the reaction mixture
and stirred at room temperature for 24 h. The organic layer was washed
with water then drop the acidity level to pH 6 by 3 N HCl. The collected
organic layer was dried over MgSO4 then concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel with
DCM–methanol (100:1 to 10:1, v/v) to afford compound 14 in 49%
yield. 1H NMR (600 MHz, (CD3)2SO): δ ppm 4.05–3.97 (m, 1H),
3.90–3.80 (m, 1H), 2.85 (q, J = 6.6 Hz, 2H), 2.30–2.12 (m, 2H),
1.88–1.77 (m, 1H), 1.71–1.56 (m, 2H), 1.52–1.44 (m, 1H), 1.39 (s, 18H),
1.36 (s, 9H), 1.34–1.29 (m, 2H), 1.26–1.16 (m, 2H). ESI-HRMS (m/z):
calculated for C25H45N3O9Na+ [M + Na]+, 554.3048; found, 554.3075.
This compound was afforded in 78% yield, following the same pro-
cedure described for synthesis of compound 9 with D-glutamic acid di-
tert butyl ester hydrochloride and D-leucine tert butyl ester hydrochlo-
ride. 1H NMR (600 MHz, CDCl3): δ ppm 5.02–4.72 (m, 2H), 4.37–4.30
(m, 2H), 2.38–2.23 (m, 3H), 2.11–2.03 (m, 1H), 1.90–1.82 (m, 1H),
1.75–1.66 (m, 1H), 1.60–1.52 (m, 1H), 1.55–1.41 (m, 27H), 0.94 (dd, J
= 2.0 and 6.5 Hz, 6H). ESI-HRMS (m/z): calculated for C24H45N2O+7 [M
+ H]+, 473.3221; found, 473.3232.
4.2.5. (S)-2-(3-((S)-1-carboxy-3-methylbutyl)ureido)pentanedioic acid
(1).
To a solution of compound 9 (181 mg, 0.38 mmol) in anhydrous
DCM (6.0 mL) was added TFA (1.5 mL) at 0 ℃. The reaction mixture was
stirred at room temperature for 3 h then concentrated in vacuo. The
residue was purified with semi-preparative RP-HPLC using 0.1% FA in
water and 0.1% FA in ACN as mobile phase. The product was afforded as
colorless solid in 39% yield (45 mg, 0.15 mmol). 1H NMR (600 MHz,
D2O): δ ppm 4.23 (dd, J = 5.2 and 9.2 Hz, 1H), 4.19 (t, J = 7.2 Hz, 1H),
2.47 (t, J = 7.3 Hz, 2H), 2.18–2.02 (m, 1H), 1.98–1.89 (m, 1H),
1.71–1.62 (m, 1H), 1.59 (t, J = 7.1 Hz, 2H), 0.90 (d, J = 6.6 Hz, 3H),
0.86 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, D2O): δ ppm 179.26,
178.60, 177.62, 160.68, 53.94, 53.29, 41.21, 31.40, 27.62, 25.73,
23.55, 21.98. ESI-HRMS (m/z): calculated for C12H21N2O+7 [M + H]+,
305.1343; found, 305.1336.
4.2.10. (R)-6-((tert-butoxycarbonyl)amino)-2-(3-((R)-1,5-di-tert-butoxy-
1,5-dioxopentan-2-yl)ureido)hexanoic acid (15).
This compound was afforded in 53% yield, following the same pro-
cedure described for synthesis of compound 14 with Nε-Boc-D-Lys. 1H
NMR (600 MHz, (CD3)2SO): δ ppm 4.02–3.95 (m, 1H), 3.82 (brs, 1H),
2.84 (q, J = 6.2 Hz, 2H), 2.30–2.14 (m, 2H), 1.86–1.78 (m, 1H),
1.71–1.56 (m, 2H), 1.52–1.44 (m, 1H), 1.39 (s, 18H), 1.36 (s, 9H),
1.34–1.29 (m, 2H), 1.23–1.16 (m, 2H). ESI-HRMS (m/z): calculated for
C
25H45N3O9Na+ [M + Na]+, 554.3048; found, 554.3038.
6

H. Kwon et al.
Bioorganic Chemistry 104 (2020) 104304
4.2.11. (R)-2-(3-((S)-1-carboxy-5-(4-iodobenzamido)pentyl)ureido)
pentanedioic acid (7).
Protein preparation: The protein structures of in PDB format were
downloaded from RCSB protein data bank (PDB ID: 6FE5 for compounds
1–4, 3D7H for compounds 5–8). Structure preparation tool in SYBYL-X
2.1.1 was employed for protein preparation. Water molecules of crystal
structures were removed then conflicted side chains of amino acid res-
idues were fixed. Hydrogen atoms were added under the application of
TRIPOS Force Field as a default setting. Minimization process was per-
formed by POWELL method, initial optimization option and termination
gradient were set to None and 0.5 kcal/(mol*Å), respectively.
The docking studies of all prepared ligands were performed by Sur-
flex-Dock Geom module in 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. Two factors
related with a generation of Protomol are Bloat(Å) and Threshold were
set to 0.5 and 0, respectively. Other parameters were applied with its
default settings in all runs.
To a solution of compound 14 (207 mg, 0.39 mmol) in anhydrous
DCM (8.0 mL) was added TFA (2 mL) at 0 ℃. The reaction mixture was
stirred at room temperature for 3 h and was concentrated in vacuo.
Methanol added to the residue then was evaporated, and this procedure
was conducted several times to remove residual TFA. Without further
purification, the residue was dissolved in anhydrous N,N-dime-
thylformamide (DMF, 2.0 mL). N-Succinimidyl 4-iodobenzoate (160 mg,
0.47 mmol) and trimethylamine (0.43 mL, 3.12 mmol) was added to the
reaction mixture under argon atmosphere. The reaction mixture was
stirred at room temperature for 2.5 h and purified with semi-preparative
RP-HPLC using 0.1% FA in water and 0.1% FA in ACN as mobile phase.
The product was afforded as white solid in 22% yield (47 mg, 0.08
mmol). 1H NMR (600 MHz, CD3CN/D2O = 1/1, v/v): δ ppm 8.41–8.34
(m, 2H), 8.05 (d, J = 8.3 Hz, 2H), 4.65 (brs, 2H), 3.84 (t, J = 6.6 Hz, 2H),
2.87 (brs, 2H), 2.62–2.51 (m, 1H), 2.41–2.59 (m, 2H), 2.23–2.01 (m,
3H), 1.97–1.85 (m, 2H). 13C NMR (150 MHz, CD3CN/D2O = 1/1, v/v): δ
ppm 177.52, 177.07, 169.38, 159.65, 138.90, 134.88, 130.01, 99.19,
55.18, 54.78, 40.64, 32.50, 31.60, 29.38, 28.39, 23.70. ESI-HRMS (m/
z): calculated for C19H24IN3O8Na+ [M + Na]+, 572.0500; found,
572.0486.
Declaration of Competing Interest
The authors declare no conflict of interests.
Acknowledgements
4.2.12. (R)-2-(3-((R)-1-carboxy-5-(4-iodobenzamido)pentyl)ureido)
pentanedioic acid (8).
This work was supported by the National Research Foundation of
Korea (2019R1A6A1A03031807 and 2020R1A2C2005919 to Y.B.).
This compound was afforded in 36% yield, following the same pro-
cedure described for synthesis of compound 7. 1H NMR (600 MHz,
CD3CN/D2O = 1/1, v/v): δ ppm 8.37 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.2
Hz, 2H), 4.73–4.62 (m, 2H), 3.83 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 7.7 Hz,
2H), 2.64–2.54 (m, 1H), 2.44–2.35 (m, 1H), 2.35–2.26 (m, 1H),
2.23–2.15 (m, 1H), 2.14–2.04 (m, 2H), 1.97–1.87 (m, 2H). 13C NMR
(150 MHz, CD3CN/D2O = 1/1, v/v): δ ppm 177.38, 176.65, 169.37,
159.74, 138.90, 134.88, 130.02, 99.20, 54.50, 53.99, 40.63, 32.29,
31.31, 29.38, 28.04, 23.68. ESI-HRMS (m/z): calculated for
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104304.
References
19H24IN3O8Na+ [M + Na]+, 572.0500; found, 572.0489.
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