Influence of the dipeptide linker configuration on the activity of PSMA ligands

Article abstract of DOI:10.1016/j.mencom.2020.11.022

Selective ligands of an urea-based prostate specific membrane antigen with a phenylalanine/tyrosine-based dipeptide linker and with a mingled chiral centers configuration and/or substituted aromatic fragments were prepared in seven steps by liquid- and in

Full text of DOI:10.1016/j.mencom.2020.11.022

Mendeleev
Communications
Mendeleev Commun., 2020, 30, 756–759
Influence of the dipeptide linker configuration on the activity of PSMA ligands
Anastasiya A. Uspenskaya,*a Alexey E. Machulkin,a Ekaterina A. Nimenko,a Radik R. Shafikov,b
Stanislav A. Petrov,a Dmitry A. Skvortsov,b Elena K. Beloglazkinaa and Alexander G. Majougaa,c
a Department of Chemistry, M. V. Lomonosov Moscow State University, 119991 Moscow, Russian Federation.
E-mail: Uspenskaya.n@gmail.com
b A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University,
119991 Moscow, Russian Federation
c D. I. Mendeleev University of Chemical Technology of Russia, 125047 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2020.11.022
R2
Fragment containing
an azido group
R1
Selective ligandsofan urea-based prostate specificmembrane
antigen with a phenylalanine/tyrosine-based dipeptide linker
O
O
O
H
N
H
Cl
N
*
and with a mingled chiral centers configuration and/or
substituted aromatic fragments were prepared in seven steps
by liquid- and in six steps by solid-phase synthesis. In vitro
test for inhibiting the cleavage of N-acetylaspartylglutamate
revealed the optimum linker containing l-phenylalanine in
the structure on the N-terminus of a dipeptide chain.
*
N
O
N
N
H
N3
H
O
HO
O
O
O
R3
O
Aliphatic
fragment
of the linker
R4
N
N
Phenylalanine
and/or tyrosine
based dipeptide
chain
H
H
OH
OH
R1, R3 = OH, H
R2, R4 = OH, H, Cl, Br, NO2
Urea-based vector-molecule
Keywords: peptide synthesis, target drug delivery, prostate cancer, prostate specific membrane antigen, solid-phase synthesis, amides.
Prostate specific membrane antigen (PSMA) is a promising
protein marker for the diagnosis and treatment of prostate
cancer.1 In prostate tumor cells as well as solid vascular tumors,
its expression is significantly increased compared to healthy
tissues, which makes it promising as a target for drug delivery.2,3
Among conjugates for the selective delivery of drugs to
prostate cancer cells, the largest number was synthesized with
urea-based PSMA inhibitors: DUPA, 2-[3-(1,3-dicarboxy-
propyl)ureido]pentanedioic acid, and DCL, N-{N-[(S)-1,3-
dicarboxypropyl]carbamoyl}-(S)-l-lysine.4 Nowadays, the
modification of this structure in order to improve its selective
binding to the active center of PSMA is multiply reported.5,6
One common modification is the introduction of a polypeptide
linker into the structure of the ligand, which would interact
with the hydrophobic tunnel of PSMA and thus increase its
effectiveness.7,8 The best amino acids for introduction into the
peptide chain of the linker were found to be phenylalanine and
tyrosine and their substituted analogues.9,10 However, there are
no documented systematized studies and conclusions on the
influence of the configuration of chiral centers and the effect of
the position of the substituent in the aromatic ring on the
biological activity of the linker.11
The aim of this work was to synthetize a series of urea-based
ligands DCL with an azido group, for which the dependence of
the ligand affinity on the position and nature of the substituents
in the dipeptide linker was clearly shown. For this purpose, the
urea-based fragment of the ligand with the chloro-substituted
aromatic fragment at the lysine atom was not varied (Figure 1).
The aliphatic fragment of the linker of 6-aminohexanoic acid
and succinic anhydride is necessary to ensure the optimal length
of the linker. Combination of these two parts is a fixed fragment.
This choice was based on previous studies, which showed that
such a structure contributes to a high affinity for the active center
of PSMA.12 Phenylalanine- and/or tyrosine-based dipeptide
R2
Fragment containing
an azido group
R1
O
O
O
H
H
N
Cl
N
*
*
N
O
N
N
H
N3
H
O
HO
O
O
O
R3
O
Aliphatic
fragment
of the linker
R4
N
Phenylalanine
and/or tyrosine
based dipeptide
chain
N
H
H
OH
OH
R1, R3 = OH, H
R2, R4 = OH, H, Cl, Br, NO2
Urea-based vector-molecule
Figure 1 General structure of the ligand.
© 2020 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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Mendeleev Commun., 2020, 30, 756–759
R1
*
R1
*
For binding with PSMA, the ligands require the presence of
O
H
N
free carboxyl groups.11 To remove tert-butyl protecting groups,
conversion of tert-butyl esters to carboxylic acids was carried out
in a system: 46.25% TFA, 46.25% DCM, 5% water and 2.5%
TIPS (see Scheme 2, stage ii). Triisopropylsilane acts as a cationic
scavenger and protects the side chains of peptide from undesired
parallel reactions.17,18 Amino acid sequences are sensitive to
strong acid conditions, so the concentration of trifluoroacetic
acid was reduced to 45–50%.19 Hence, a number of PSMA
inhibitor ligands 3a–i was prepared. The obtained compounds
were characterized by NMR spectroscopy, high-resolution mass
spectrometry and high-performance liquid chromatography.
When attempting an introduction of the amino acid tyrosine
from the N-terminus of the dipeptide sequence by liquid phase
synthesis methods, a number of difficulties were found associated
with poor solubility of tyrosine, as well as side reactions from
the hydroxyl group in the para position. We tried a different
approach to produce the desired dipeptide chains, namely, viz., a
solid-phase synthesis (cf. ref. 20).
i, ii
Boc
Boc
OH
*
OH
N
N
53–100%
H
H
O
O
R2
R1 = PhCH2 (N-Boc-L-Phe)
R1 = PhCH2 (N-Boc-D-Phe)
R1 = H (N-Boc-Gly)
1a–i
R1
*
O
H
iii (46–100%)
+
N
*
N
N3
H3N
CF3COO–
iv (53–98%)
H
R2
O
2a–i
a R1 = PhCH2, R2 = H (L-Phe-Gly)
b R1 = H, R2 = PhCH2 (Gly-L-Phe)
c R1 = R2 = PhCH2 (L-Phe-D-Phe)
d R1 = PhCH2, R2 = 4-HOC6H4CH2 (L-Phe-D-Tyr)
e R1 = R2 = PhCH2 (D-Phe-L-Phe)
f R1 = PhCH2, R2 = 4-HOC6H4CH2 (D-Phe-L-Tyr)
g R1 = PhCH2, R2 = 4-O2NC6H4CH2 [L-Phe-L-Phe(4-NO2)]
h R1 = PhCH2, R2 = 3-Br-4-HOC6H3CH2 [L-Phe-L-Tyr(3-Br)]
R1 = PhCH2, R2 = 3,4-(HO)2C6H3CH2 [L-Phe-L-Tyr(3-OH)]
i
In this work, 2-chlorotritile resin in chloride form (2-CTC) was
selected as the solid support. This resin prevents the racemization of
the first amino acid residues and provides the possibility of using a
wide variety of side chains. It is also important to note the conditions
for removing the resulting chains from the resin.21,22 Weakly acidic
conditions allow for maintaining tert-butyl protecting groups
present in the structure of both the molecule vector and the linker.
Fmoc-protected amino acid residues of phenylalanine and
tyrosine were used for synthesis. Removal of Fmoc protection
takes place under basic conditions, which corresponds to the
type of resin chosen. At the first stage, we activated the resin and
introduced the first amino acid residue (tyrosine). Chain building
was performed by creating a peptide bond between the amino
group of the residue on the resin and the carboxyl group of the
following Fmoc-protected amino acid residue.
A modified urea-based vector-molecule was attached to the
obtained dipeptide chain after removal of Fmoc protection group
by creating a peptide bond using activated HOBt and HBTU
esters. Removal from the resin was carried out in a 0.5% solution
of trifluoroacetic acid in dichloromethane. The tert-butyl
protecting groups of the vector-molecule and the tyrosine
fragment in the linker side chain were not affected.
Since the azide–alkyne cycloaddition reaction requires the
presence of an azide group in the ligand structure, the next step
involved the reaction of compound 4 with 3-aminopropyl azide
(Scheme 3, stage i). To avoid racemization and minimize the
amount of by-products, it is important to consider the addition
order of the reagents. The reaction was executed without
preactivation and at a reduced temperature (0°C). The removal
of tert-butyl protecting groups was carried out in the standard
way (stage ii). The obtained compound 5 was characterized by
1H NMR, HPLC-MS and HRMS spectra.
Scheme 1 Reagents and conditions: i, C6F5OH, ECD-Cl, CH2Cl2;
ii, H2NCH(R2)CO2H, DIPEA, THF/H2O; iii, H2N(CH2)3N3, HBTU, BtOH,
DIPEA, THF; iv, TFA, CH2Cl2.
chain will vary. The fragment containing an azido group is
required for the azide–alkyne addition reaction.
Synthesis of the urea-based vector-molecule fragment was
carried out using previously developed methods.12 The synthesis
of the dipeptide chain began with the setting of the Boc-
protecting group on the nitrogen atom of the N-terminal amino
acid, the removal of this group occurring at the last stage in an
acidic solution.13 We used classical reaction of an amino acid
with di-tert-butyl dicarbonate in an alkaline solution. At the
second step, we synthesized dipeptides 1a–i (Scheme 1, stages i
and ii).14 An activated N-terminal amino acid pentafluorophenyl
ester was initially prepared and subsequently reacted with the
free amino acid.15 The resulting compounds were sufficiently
pure to use for further synthesis steps. In the third step, the
activation the carboxyl group of dipeptides with HOBt and
HBTU activating agents was conducted with the following
reaction with 3-azidopropylamine (see Scheme 1, stage iii). The
substances were purified by extraction followed by column
chromatography. The tert-butoxycarbonyl protecting group was
removed to obtain compounds 2a–i using a 10% solution of
trifluoroacetic acid in dichloromethane (stage iv). In this manner,
nine dipeptide linkers were synthesized, which were further used
to produce highly specific PSMA vectors. These methods
provide high yields and easy purification of the products.
The synthesis of ligands with dipeptide linkers included well-
recognized peptide coupling reaction. Catalytic reagents HOBt
and HBTU led to formation of activated esters, which reacted
readily with amino acids.16 (Scheme 2, stage i).
R1
*
O
O
O
O
O
H
H
H
N
Cl
Cl
N
N
OH
*
R2
N
N
O
N
N
N3
5
5
ButO
O
O
H
H
O
HO
O
O
O
O
i (54–90%)
ii (20–43%)
Urea-based
vector-molecule
O
O
O
N
N
N
N
H
H
H
H
OBut
OH
OH
OBut
3a–i
f R1 = PhCH2, R2 = 4-HOC6H4CH2 (D-Phe-L-Tyr)
a R1 = PhCH2, R2 = H (L-Phe-Gly)
b R1 = H, R2 = PhCH2 (Gly-L-Phe)
c R1 = R2 = PhCH2 (L-Phe-D-Phe)
g R1 = PhCH2, R2 = 4-O2NC6H4CH2 [L-Phe-L-Phe(4-NO2)]
h R1 = PhCH2, R2 = 3-Br-4-HOC6H3CH2 [L-Phe-L-Tyr(3-Br)]
R1 = PhCH2, R2 = 3,4-(HO)2C6H3CH2 [L-Phe-L-Tyr(3-OH)]
d R1 = PhCH2, R2 = 4-HOC6H4CH2 (L-Phe-D-Tyr)
e R1 = R2 = PhCH2 (D-Phe-L-Phe)
i
Scheme 2 Reagents and conditions: i, dipeptide 2a–i, HOBt/HBTU, DIPEA, DMF; ii, TFA–CH2Cl2 (1:1), H2O (5%), TIPS (2.5%).
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Mendeleev Commun., 2020, 30, 756–759
OBut
O
O
O
H
H
Cl
N
N
N
OH
N
H
ButO
O
O
O
O
Ph
O
O
N
N
H
H
OBut
OBut
4 (D-Tyr-L-Phe)
OH
O
O
O
H
N
H
Cl
N
N3
N
O
N
N
H
H
i (59%)
ii (26%)
O
HO
O
O
O
Ph
O
N
N
H
H
OH
OH
5
Scheme 3 Reagents and conditions: i, H2N(CH2)3N3, HOBt, HBTU, DMF, 0 °C; ii, TFA–CH2Cl2 (1:1), H2O (5%), TIPS (2.5%).
The affinity of the synthesized vectors with the linker
correlates with the efficiency of inhibiting their cleavage of
PSMA N-acetylaspartylglutamate. After analyzing several cell
lines available in our laboratory and according to available data,
it was found that the highest level of PSMA was observed in the
LNCaP cell line, which was taken for further work.23,24 Control
test was conducted with the DCL drug analogue synthesized in
our laboratory. Table 1 shows the results of a series of 10 new
ligands by inhibiting the cleavage reaction of N-acetylaspartyl-
glutamate on the cell line LNCaP as well as data on ligands
obtained earlier.12 In the structure of the latter, the molecule
vector is also represented by urea DCL with a m-chloro-
substituted aromatic fragment at a lysine atom.
affects the affinity of the ligand l-Phe-Gly (821.0 162.0 nm).
The absence of an aromatic fragment in the N-terminal position
also negatively affects affinity, but not so pronounced (Gly-l-Phe,
293.0 37.2 nm). It can be concluded that the number and position
of aromatic fragments in the linker structure has a significant role
in biological activity, so the presence of dipeptide aromatic
fragments is required to maintain a high affinity for PSMA.
Ligands with an unmixed structure show the best results, in
particular, the dipeptide chain l-Phe-l-Tyr reveals the highest
affinity (22.5 6.0 nm). Dipeptide chains with the mixed
structure are inferior in affinity, but it is possible to conclude that
l-phenylalanine has a favorable effect on affinity. The highest
values belong to ligands containing l-phenylalanine from the
N-terminus of the chain: l-Phe-l-Tyr (22.5 6.0 nm), l-Phe-l-Phe
(86.2 23.9 nm), l-Phe-d-Phe (279.0 37.3 nm). For ligand
d-Tyr-l-Phe, the IC50 value of 741.0 123.0 nm indicates that
the introduction of tyrosine into the position 1 of the dipeptide
chain reduces affinity.
According to the obtained data, the presence of an aromatic
fragment in the C-terminus of the linker is critical and strongly
Table 1 Results of IC50 affinity parameter determination by inhibiting the
cleavage reaction of N-acetylaspartylglutamate.
The introduction of the second hydroxy group at the position
3 of the tyrosine moiety turned out to be negative for affinity and
practically makes the molecule inactive. However, the results for
d-Phe-d-Tyr(3-Br) (267.3 151.8 nm) and l-Phe-l-Tyr(3-Br)
(1390.0 341.0 nm) show that the configuration has a strong
effect on affinity.
In conclusion, a series of ten novel PSMA ligands bearing
dipeptide linkers with mixed chiral site configurations and/or
substituted aromatic moieties were obtained. A new solid-phase
synthesis of such ligands proved to be very efficient. Based on
the results of in vitro biological tests, it can be concluded that
ligands with a mixed-configured dipeptide linker are inferior to
ligands with non-mixed ones, the best result for the new series
relating to l-Phe-d-Phe (279.0 37.3 nm) dipeptide chain.
Substituents in aromatic fragments in the linker structure can
also have a significant effect on affinity, but it is inextricably
linked to the configuration of the amino acid residue, which is
promising for further study.
b
b
Linker structure
IC50 SDa/nm
Ki SDa/nm
Control
DCL
2149.0 235.0
224.6 24.6
Ligand Series Gly/Phe
293.0 37.2
Gly-l-Phe (3b)
l-Phe-Gly (3a)
30.2 3.83
84.6 16.7
821.0 162.0
Ligand Series Phe-Tyr(Phe)
22.5 6.0
l-Phe-l-Tyr
2.4 0.6
d-Phe-d-Phe
58.0 16.3
6.06 1.7
9.01 2.5
28.8 3.84
53.7 11.5
85.7 46.7
112.0 18.5
137.0 31.6
l-Phe-l-Phe
86.2 23.9
l-Phe-d-Phe (3c)
d-Phe-l-Phe (3e)
d-Phe-d-Tyr
279.0 37.3
520.0 111.0
820.0 447.0
l-Phe-d-Tyr (3d)
d-Phe-l-Tyr (3f)
1080.0 179.0
1330.0 307.0
Ligand Series Tyr-Tyr(Phe)
741.0 123.0
d-Tyr-l-Phe (5)
76.5 12.7
Ligand Series with substituted Phe/Tyr
267.3 151.8
The reported study was funded by the Russian Foundation for
Basic Research (project no. 19-33-90118).
d-Phe-d-Tyr(Br)
27.9 15.9
100.3 11.2
143.0 35.2
l-Phe-l-Phe(NO2) (3g)
l-Phe-l-Tyr(Br) (3h)
l-Phe-l-Tyr(3-OH) (3i)
959.0 107.0
1390.0 341.0
6030.0 1140.0
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2020.11.022.
622.0 117.0
a SD – standard deviation. b nm – nanomolar.
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Mendeleev Commun., 2020, 30, 756–759
11 S. A. Kularatne, Z. Zhou, J.Yang, C. B. Post and P. S. Low, Mol. Pharm.,
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Received: 26th August 2020; Com. 20/6297
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