Novel β- And γ-Amino Acid-Derived Inhibitors of Prostate-Specific Membrane Antigen

Article abstract of DOI:10.1021/acs.jmedchem.9b02022

Prostate-specific membrane antigen (PSMA) is an excellent biomarker for the early diagnosis of prostate cancer progression and metastasis. The most promising PSMA-targeted agents in the clinical phase are based on the Lys-urea-Glu motif, in which Lys and Glu are α-(l)-amino acids. In this study, we aimed to determine the effect of β- and γ-amino acids in the S1 pocket on the binding affinity for PSMA. We synthesized and evaluated the β- and γ-amino acid analogues with (S)- or (R)-configuration with keeping α-(l)-Glu as the S1′-binding pharmacophore. The structure-activity relationship studies identified that compound 13c, a β-amino acid analogue with (R)-configuration, exhibited the most potent PSMA inhibitory activity with an IC₅₀ value of 3.97 nM. The X-ray crystal structure of PSMA in complex with 13c provided a mechanistic basis for the stereochemical preference of PSMA, which can guide the development of future PSMA inhibitors.

Full text of DOI:10.1021/acs.jmedchem.9b02022

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Article
Novel #- and #-amino acid derived inhibitors
of prostate-specific membrane antigen
Kyul Kim, Hongmok Kwon, Cyril Ba#inka, Lucia Motlova, SangJin Nam, Doyoung Choi, Hyunsoo Ha,
Hwanhee Nam, Sang-Hyun Son, Il Minn, Martin G Pomper, Xing Yang, Zsófia Kutil, and Youngjoo Byun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02022 • Publication Date (Web): 25 Feb 2020
Downloaded from pubs.acs.org on March 1, 2020
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Novel - and -amino acid derived inhibitors of
prostate-specific membrane antigen
Kyul Kim,†, # Hongmok Kwon,†, # Cyril Barinka, Lucia Motlova, SangJin Nam, Doyoung
‡
‡
†
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Choi, Hyunsoo Ha, Hwanhee Nam, Sang-Hyun Son, Il Minn, Martin G. Pomper, Xing
§
,*
‡,*
†,,*
Yang, Zsofia Kutil, Youngjoo Byun
†College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
‡Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595,
252 50 Vestec, Czech Republic
Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins
University School of Medicine, Baltimore, 21205, MD, USA
§Department of Nuclear Medicine, Peking University First Hospital, Beijing, 100034, China.
Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu,
Seoul 08308, Republic of Korea
#
Equal contribution
*
Corresponding authors
Youngjoo Byun, Ph. D.
College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
Tel.: +82-44-860-1619; Fax: +82-44-860-1607; E-mail: yjbyun1@korea.ac.kr
Zsofia Kutil, Ph. D.
Institute of Biotechnology CAS, v.v.i., Laboratory of Structural Biology, Prumyslova 595, 252
0 Vestec, Czech Republic; phone: +420-325-873-736; e-mail: zsofia.kutil@ibt.cas.cz
5
Xing Yang, Ph. D.
Department of Nuclear Medicine, Peking University First Hospital, Beijing, 100034, China.
Tel: 86 10 83572928; Email: yangxing2017@bjmu.edu.cn
Key words: PSMA, Prostate cancer, -amino acid, X-ray crystallography, Absolute
configuration
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ABSTRACT
Prostate-specific membrane antigen (PSMA) is an excellent biomarker for the early
diagnosis of prostate cancer progression and metastasis. Most promising PSMA-targeted
agents in clinical phase are based on the Lys-Urea-Glu motif, in which Lys and Glu are -
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(L)-amino acids. In this study, we aimed to determine the effect of - and -amino acids in
the S1 pocket on the binding affinity for PSMA. We synthesized and evaluated - and -
amino acid analogs with (S)- or (R)-configuration with keeping -(L)-Glu as the S1'-binding
pharmacophore. Structure-activity relationship studies identified that compound 13c, an -
amino acid analog with (R)-configuration, exhibited the most potent PSMA-inhibitory
activity with an IC value of 3.97 nM. The X-ray crystal structure of PSMA in complex
5
0
with 13c provided a mechanistic basis for the stereochemical preference of PSMA which
can guide the development of future PSMA inhibitors.
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INTRODUCTION
Prostate cancer (PCa) is the most commonly diagnosed cancer and the second
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leading cause of cancer-related deaths, only behind lung cancer in USA. Although the
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overall 5-year survival rate for patients with PCa in USA is 97.3%, this drops drastically to
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9% among patients with advanced stage PCa. PCa usually has no physical symptoms until
2
it progresses to an advanced stage. Hence, early and accurate diagnosis of PCa is critical for
successful treatment. Prostate-specific membrane antigen (PSMA) is an attractive biomarker
3
for the diagnosis of primary and metastatic PCa. PSMA is a type II integral membrane
metalloprotease which shows restricted expression on the cell surface of prostate carcinomas,
particularly in androgen-independent, advanced, and metastatic state.4, 5 Furthermore, its
expression levels positively correlates with tumor aggressiveness and metastasis. 6, 7
There are many studies on the identification of PSMA-targeted small molecule
inhibitors.7-14 These small molecules are labeled with radionuclides for the imaging and
therapy of PCa.7, 8, 15-18 In parallel with the discovery of PSMA inhibitors, X-ray crystal
structures of PSMA in complex with small molecules have provided mechanistic insights
into the binding modes in the active site.19-23 According to the PSMA crystal structures, the
PSMA active site consists of three distinct sub-pockets, including a ‘glutamate-sensor’ S1’
site (pharmacophore), a ‘zinc-binding’ site, and an amphiphilic S1 site (non-pharmacophore).
In particular, the structural freedom provided by the S1 site allows diverse structural
modifications to PSMA inhibitors.20, 24, 25 Since the pioneering work by Kozikowski and
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colleagues , the most common scaffold of PSMA inhibitors is a lysine–urea–glutamate
(Lys–Urea–Glu) motif that has a high binding affinity and specificity for PSMA despite the
diverse structural variation in the P1 site. The ε-amine group of the lysine in Lys-Urea-Glu
has been conjugated with a variety of imaging prosthetic groups and diverse linkers.27-31
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Some excellent and comprehensive recent reviews on PSMA-targeted imaging probes
derived from the Lys-Urea-Glu motifs were reported.32-36
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O
O
NH
NH
F
N
I
O
COOH
O
COOH
COOH
O
COOH
COOH
HO
HO
HO
N
N
COOH
N
N
N
N
H
H
H
H
H
H
O
O
O
1
(DCFPyL)
2 (DCIBzL)
3 (ZJ-43)
Figure 1. Representative -(L)-amino acid PSMA inhibitors.
Although diverse amino acids (e.g, Lys, Leu, and Cys) have been introduced to the
P1 site, all of these are -(L)-amino acids (Figure 1). The insertion of methylene (-CH -) or
2
ethylene (-CH CH -) group between COOH and NH in the P1 site was expected to enhance
2
2
2
the conformational freedom. In addition, determining the relationship of PSMA-binding
affinity with the absolute configuration of - and -amino acids will provide valuable
information for understanding PSMA-targeted inhibitors in the P1 region. In this study, we
aimed to investigate the effect of - and -amino acids with (S)- or (R)-configuration in the
S1 pocket on the binding affinity for PSMA. We synthesized and evaluated - and -amino
acid analogs with (S)- or (R)-configuration, and determined PSMA X-ray structures with the
most potent - and -amino acid inhibitors.
RESULTS AND DISCUSSION
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Scheme 1. Synthesis of dipeptides derived from - and -amino acidsa
O
COOt-Bu
COOt-Bu
O
COOH
COOH
i
ii
HOOC
HOOC
N
N
H
N
H
N
H
H
NHBoc
NH2
NH2
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O
COOt-Bu
COOt-Bu
O
COOH
COOH
i
i
ii
ii
HOOC
HOOC
N
N
H
N
H
N
H
H
NHBoc
COOt-Bu
COOt-Bu
6
7
H N
2
NHBoc
NHCbz
O
COOt-Bu
COOt-Bu
NH2
O
COOH
COOH
HOOC
HOOC
N
H
N
H
HOOC
HOOC
N
N
H
H
8
9
O
COOt-Bu
COOt-Bu
NH2
O
COOH
COOH
i
ii, iii
N
H
N
H
N
H
N
H
10
11
a
Reagents and conditions: (i) Triphosgene, Et N, CH Cl , −78 ºC to rt, 30 min, and then (S)-3
3
2
2
-amino-2-(Boc-amino)propanoic acid (for 4), (R)-3-amino-2-(Boc-amino)propanoic acid
for 6), (S)-4-amino-2-(Boc-amino)butanoic acid (for 8), and (R)-4-amino-2-(Cbz-amino)but
anoic acid (for 10), rt, 24 h; (ii) TFA/CH Cl (1/4, v/v), rt, 2 h; (iii) 10% Pd/C, H , MeOH, 2
(
2
2
2
h.
The synthesis of - and -amino acid PSMA inhibitors was accomplished by covalently
attaching four different p-iodobenzoyl derivatives that varied in the length of the aliphatic
spacer to urea-based - and -amino acid dipeptide scaffolds with (S)- or (R)-configuration. In

-amino acid PSMA inhibitors, the p-iodobenzoyl group was located into the accessory pocket
at the S1 site and interacted with the arginine-patch region to contribute to strong PSMA
2
0
binding. The p-iodobenzoyl group was also expected to increase the PSMA binding affinity
in the - and -amino acid PSMA inhibitors. The synthetic routes are shown in Scheme 1 and
Scheme 2. Synthesis of the two diastereomeric pairs of Glu-urea-β-amino acid (5 and 7) and
Glu-urea-γ-amino acid (9 and 11) was achieved in a two- or three-step protocol by coupling
commercially available (S)-di-tert-butyl-glutamic acid with (S)- or (R)-3-amino-2-((tert-butox
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ycarbonyl)amino)propanoic acid and (S)- or (R)-4-amino-2-((tert-butoxycarbonyl)amino)b
utanoic acid, respectively. Briefly, (S)-di-tert-butyl-glutamic acid was treated with
triphosgene under anhydrous conditions in the presence of triethylamine to afford the
isocyanate intermediate, which reacted subsequently with the appropriate amino acids at room
temperature to produce the protected four urea-based dipeptides (4, 6, 8, and 10). The tert-
butoxycarbonyl (Boc) and tert-butyl ester (Ot-Bu) protecting groups of the dipeptide (4, 6, and
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) were removed by the treatment of 25% trifluoroacetic acid (TFA) solution in
dichloromethane (DCM) to yield the urea-based dipeptides (5, 7, and 9), which were used for
the next amide coupling reaction without further purification. However, the removal of the tert-
butyl protecting groups of the dipeptide (10) with 25% TFA solution in DCM, followed by the
hydrogenation of the carboxybenzyloxy (Cbz) group using 10% Pd/C in the presence of H gas
2
in methanol afforded the urea-based dipeptide (11). The synthesis of four different p-
iodobenzoyl derivatives was carried out via a three-step process using a previously established
method (Supporting Information).37, 38
With four urea-based dipeptide scaffolds (5, 7, 9, and 11) obtained, their assembly toward
1
2a–15d was performed using p-iodobenzoyl derivatives with a variable length of the aliphatic
part and activated N-hydroxysuccinimide (NHS) esters. Briefly, Glu-urea-β-amino acid 5 with
the (S)-configuration was coupled with an aliphatic spacer having a different chain length in
anhydrous N,N-dimethylformamide (DMF) using triethylamine as a base. The progress of the
reaction was monitored by analytical HPLC until the starting material disappeared completely.
The crude product was purified by semi-preparative HPLC to provide the final compounds
(12a–12d) in moderate yield (22% to 55%) with high purity (>97%). The other -amino acid
analogs with (R)-configuration (13a–13d) and -amino acid analogs (14a–14d for (S) and 15a–
1
5d for (R)) were synthesized using a similar protocol.
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Scheme 2. Ring extension and variation of carbon chain linker a
O
COOH
COOH
O
HOOC
O
O
COOH
COOH
mN
H
N
H
N
O
HOOC
NH
O
mN
H
N
i
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+
O
H
NH2
5
9
: m = 1
: m = 2
12a: m = 1 (32%)
14a: m = 2 (27%)
I
I
O
COOH
COOH
O
HOOC
O
O
COOH
COOH
mN
H
N
N
O
H
O
i
HOOC
NH
O
+
mN
H
N
H
NH2
7
1
: m = 1
1: m = 2
13a: m = 1 (9%)
15a: m = 2 (15%)
I
I
O
COOH
COOH
HOOC
O
mN
H
N
H
O
NH
N
O
COOH
COOH
O
H
O
HOOC
O
N
mN
H
N
i
+
12b: m = 1, n = 1 (35%)
12c: m = 1, n = 2 (56%)
2d: m = 1, n = 3 (22%)
4b: m = 2, n = 1 (15%)
14c: m = 2, n = 2 (24%)
14d: m = 2, n = 3 (28%)
HN
O
H
n
n
NH2
1
1
O
5: m = 1
: m = 2
n =1, 2, 3
9
I
I
O
COOH
COOH
O
HOOC
O
mN
H
N
H
N
O
O
COOH
COOH
NH
O
HOOC
H
i
mN
H
N
O
N
+
13b: m = 1, n = 1 (30%)
13c: m = 1, n = 2 (32%)
13d: m = 1, n = 3 (18%)
5b: m = 2, n = 1 (44%)
15c: m = 2, n = 2 (31%)
5d: m = 2, n = 3 (19%)
HN
O
H
n
n
NH2
O
7: m = 1
11: m = 2
n =1, 2, 3
1
I
1
I
a
Reagents and conditions: (i) Et N, DMF, rt, 2 h.
3
To compare the change in lipophilicity based on the length of linker and chirality, we used
two different mobile phases (acetonitrile/water and methanol/water) and determined retention
times of the synthesized compounds. As shown in Figure 2, compounds with longer linkers
had a longer retention time than those with shorter linkers in all series. However, the -amino
acid analogs (14b and 15b), which had the ethylene spacer, eluted earlier than the
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corresponding analogs (14a and 15a) which directly conjugated with the 4-iodobenzoyl group
(
Figure 2), irrespective of mobile mixtures (CH CN/H O or CH OH/H O).
3 2 3 2
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Figure 2. HPLC chromatograms of 16 compounds in two different eluent conditions.
(a) Acetonitrile and water (25:75, v/v, 0.1% FA), flow rate: 1 mL/min, (b) methanol and
water (50:50, v/v, 0.1% FA), flow rate: 0.7 mL/min.
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Table 1. In vitro PSMA-inhibitory activity of the synthesized compounds
O
COOH
COOH
HOOC
m N
H
N
*
X
H
1
1
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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
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Comp. Configur
ation
DCFPyL (1)
m
X
IC50
(nM)
1.56
95% CIa
0.38 – 6.32
12a
2b
12c
2d
13a
3b
13c
3d
14a
4b
4c
4d
5a
5b
5c
5d
DCIBzL (2)
(S)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(S)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(S)
(R)
1
4-IC H CONH–
> 1000
> 1000
> 1000
155
-
6
4
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
0
4-IC H CONH(CH ) CONH–
-
6
4
2 2
4-IC H CONH(CH ) CONH–
-
6
4
2 3
1
4-IC H CONH(CH ) CONH–
90.5 – 268
6.63 – 18.1
7.40 – 19.0
2.47 – 6.36
10.8 – 25.7
140 – 1130
66.8 – 1700
163 – 494
110 – 346
-
6
4
2 4
4-IC H CONH–
11.0
6
4
1
4-IC H CONH(CH ) CONH–
11.9
6
4
2 2
4-IC H CONH(CH ) CONH–
3.97
6
4
2 3
1
4-IC H CONH(CH ) CONH–
16.7
6
4
2 4
4-IC H CONH–
399
6
4
1
4-IC H CONH(CH ) CONH–
107
6
4
2 2
1
4-IC H CONH(CH ) CONH–
284
6
4
2 3
1
4-IC H CONH(CH ) CONH–
195
6
4
2 4
1
4-IC H CONH–
> 1000
> 1000
> 1000
> 1000
0.22
6
4
1
4-IC H CONH(CH ) CONH–
-
6
4
2 2
1
4-IC H CONH(CH ) CONH–
-
6
4
2 3
1
4-IC H CONH(CH ) CONH–
-
6
4
2 4
4-IC H CONH(CH ) –
0.17 – 0.30
-
6
4
2 4
1
7
4-IC H CONH(CH ) –
> 1000
6
4
2 4
a
Confidence interval.
The PSMA-inhibitory activities of the synthesized 16 inhibitors were determined by applying
the fluorescence-based NAALADase assay.39, 40 We used extracts of the LNCaP cell line,
which is an androgen-sensitive human prostate adenocarcinoma cell line that overexpresses
2
7
PSMA. DCFPyL (1) , a known potent inhibitor of PSMA, was used as the positive control.
Table 1 summarizes the IC values of the synthesized PSMA inhibitors and compound 1. The
50
-amino acid analogs (13a–13d) with (R)-configuration exhibited strong PSMA-inhibitory
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activities with IC values from 3.97 to 16.7 nM. However, the corresponding (S)-analogs were
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inactive (> 1 M IC ) except compound 12d (155 nM). Compound 13c, with a propylene
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spacer, was the most potent out of the -amino acid inhibitors, with IC value of 3.97 nM. In
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the case of the -amino acid analogs, the (S)-analogs (14a–14d) were more potent than the (R)-
analogs (15a–15d). Compound 14b (107 nM), with an ethylene linker, displayed the strongest
PSMA-inhibition in the γ-amino acid series but was less potent than 13c.
Scheme 3. Synthesis of compound 17a
O
NHBoc
NH
COOt-Bu
I
i
ii
O
COOt-Bu
COOt-Bu
H N
2
COOt-Bu
O
COOH
COOH
HOOC
N
N
H
H
HOOC
N
N
H
H
16
17
a
ε
Reagents and conditions: (i) Triphosgene, Et N, CH Cl , −78 ºC to rt, 30 min, and then N -B
3
2
2
oc-D-lysine, (ii) TFA/CH Cl (1/4, v/v), rt, 2 h, and then N-succinimidyl 4-iodobenzoate, Et N,
2
2
3
DMF, rt, 2.5 h.
To investigate the effect of -amino acid stereochemistry on the P1 region, compound 17,
41
the diastereomer of DCIBzL (2) , the most potent PSMA inhibitor based on the Lys-Urea-Glu
scaffold, was prepared as described in Scheme 3. By modifying the synthetic method for
compound 2, compound 17 was synthesized in three steps from commercial (S)-di-tert-butyl-
glutamic acid and (R)-tert-butyl-lysine. Analytical HPLC studies showed a slight difference in
retention time (5.59 min for 2 and 5.84 min for 17) (See supporting information). In vitro
PSMA-inhibitory studies showed a dramatic difference of IC values (0.22 nM for 2 and 1.7
50
M for 17), which indicated that the (S)-configuration in -amino acids on the P1 region is
critical for PSMA-binding affinity. Previously, a significant decrease in the PSMA-inhibitory
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activity was observed when (S)-leucine of ZJ-43 (3) on the P1 region was replaced with (R)-
leucine that is consistent with our result.43
Table 2. Data Collection and Refinement Statistics
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
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1
2
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Data collection statistics
Inhibitor
PDB ID
Space group
13c
6RBC
I222
14b
6S1X
I222
Unit-cell parameters a, b, c (Å)
101.5, 130.5, 159.0
101.4, 130.2, 159.3
Wavelength (Å)
Resolution limits (Å)
No of unique refl.
Redundancy
0.9184
50 – 1.77 (1.88 – 1.77)
102705 (16397)
6.7 (6.5)
0.9184
50 – 1.76 (1.86 – 1.76)
104162 (16420)
6.0 (5.7)
Completeness (%)
I/σ(I)
99.9 (99.3)
14.5 (1.94)
0.083 (0.827)
99.6 (98.4)
15.91 (1.92)
Rmerge
0.072 (0.823)
Refinement statistics
45.00 – 1.77 (1.81 – 1.77)
100601 (7450)
Resolution limits (Å)
Total number of reflections
45.00 – 1.76 (1.80 – 1.76)
102046 (7427)
Number of reflections
in working set
98500 (7297)
99947 (7277)
2099 (150)
Number of reflections
in test set
2
101 (153)
R/Rfree (%)
16.5/19.1 (27.0/27.2)
16.8/19.4 (33.3/34.9)
Total number of non-H atoms
Number of non-H atoms
Number of inhibitor molecules
6603
5802
1
6776
5968
1
Number of water molecules
601
572
Average B-factor (Å2)
Protein
Water molecules
Inhibitor
&Ramachandran Plot (%)
35.65
34.41
43.60
32.60
37.29
35.93
44.55
39.8
Most favored
Additionally allowed
Disallowed
97%
3%
96%
3%
1%
0%
R.M.S deviations:
Bond lengths (Å)
Bond angles (°)
Chiral centers (Ǻ3)
0.011
1.73
0.12
0.012
1.74
0.12
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To elucidate the importance of the absolute configuration and spacer length of - and -amino
acid inhibitors on their potency towards PSMA, we determined the crystal structures of human
PSMA in complex with 13c (-amino acid) and 14b (-amino acid) to the final resolution of
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1
.77 and 1.76 Å, respectively (Table 2). The Glu moiety of both inhibitors occupies the
customary position in the S1’ pocket, identical to the position in a published study on
20
PSMA/urea-based inhibitor complexes (Figure 3). The Urea carbonyl oxygen atoms of 13c
and 14b interact the zinc (II) ion at a distance of 2.29 Å and 2.44 Å, respectively. The
divergence among the binding modes of 13c, 14b, and 2 (PDB code 3D7H) becomes, however,
apparent on their non-prime S1 side. The interactions between P1 residues and the α-
carboxylate of Lys-Urea-Glu scaffold have been shown to be critical for the high affinity of
PSMA inhibitors, and the typical interaction pattern involves three direct and one water-
4
4
mediated contacts with Arg534 and Arg536 comprising the P1 arginine patch. In the case of
3c, the P1 β-(R)-carboxylate interacts with the side chain of Arg534, forming one direct (2.7
1
Å) and one water-mediated (2.5 Å) hydrogen bond (Figure 4). For 14b, the P1 -(S)-
carboxylate forms a direct hydrogen bond with Arg536 (2.7 Å) and water-mediated hydrogen
bonds with Arg534 (2.7 Å and 2.9 Å, respectively). Interestingly, the both P1 β- and γ-
carboxylates of 13c and 14b, respectively, also form hydrogen bonds with the carbonyl/amide
groups of the linker and can, in principle, constrain the positioning of the distal p-iodobenzoyl
moiety. It should be noted that changing the absolute configuration at the carbon atom bearing
the non-prime carboxylate of 13c and 14b to (S,S) and (R,S) diastereomers, respectively, would
place the P1 carboxylate groups in unfavorable positions. In the case of 13c, the change in
chirality made it too close to the p-iodobenzoyl group that was presumably the cause of its
displacement (Supporting information). In the case of 14b, the change in chirality forced the
p-iodobenzoyl group away from the arginine patch (Supporting information), resulting in the
4
4
loss of critical interactions. These results provide a mechanistic rationale for the measured
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inhibition constants where the changes in the absolute configuration at the side carbon atoms
decreased inhibition constants by more than 250-fold (Table 1). It should be noted that the p-
iodobenzoyl group in the - and -amino acid PSMA inhibitors may interact with human serum
1
1
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1
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albumin and affect biodistribution of the parent compound, which was observed with the -
amino acid PSMA inhibitors.45
Figure 3. (A) Fo - Fc difference maps (green) for 13c (PDB ID: 6RBC) and 14b (PDB ID:
6S1X), respectively, are contoured at 3.0 σ and modeled inhibitors are shown in stick
representation with atoms colored yellow and cyan (carbons of 13c and 14b, respectively), red
(oxygen), blue (nitrogen) and magenta (iodine). The active-site zinc ions are shown as orange
spheres. (B, C, D) Comparison of binding modes of 13c and 14b with DCIBzL (grey carbons)
from three different view angles. While the position of the urea-Glu moiety in the S1’ pocket
is identical in all cases (I), the positioning of the linkers (III) and interaction pattern of the P1
carboxylate groups with Arg534 and Arg536 of the arginine patch differ substantially (II). The
rotation of the p-iodobenzoyl group of 14b (IV) results in the displacement of Arg463 (V).
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Figure 4. (A) PSMA represented with its active site (PDB ID: 6RBC) co-crystallized with 13c
(in yellow), (B) PSMA represented with its active site (PDB ID: 6S1X) co-crystallized with
14b (in light blue).
The distal p-iodobenzoyl groups of both 13c and 14b are placed inside the accessory
hydrophobic pocket formed by the opening of the arginine patch as reported previously.20, 44
Although the p-iodobenzoyl ring of 13c overlaps well with the “canonical” position observed
20
for ()-Lys-Urea-Glu molecules comprising the P1 lysine linker, such as compound 2 , the p-
iodobenzoyl moiety of 14b is shifted by 1.4 Å and rotated by approximately 45 degrees.
Concurrently, the side chain of Arg463, which forms the “upper” wall of the accessory
hydrophobic pocket is pushed away by 2.1 Å to accommodate the rotated p-iodobenzoyl ring.
Structurally, the placement of the p-iodobenzoyl ring of 14b into the “canonical”, and
presumably energetically favorable, position is sterically prohibited by the position the P1 -
carboxylate. These observations imply that, in general, when the p-iodobenzoyl ring is to be
used as the distal moiety anchoring the inhibitor at the accessory hydrophobic pocket, the -
amino acid analogs may be excluded due to their positional incompatibility. This is
demonstrated by the fact that the “preferred” (S)-analogs of -amino acid are overall less potent
than the “preferred” (R)-analogs of -amino acid analogs (Table 1).
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The Fo-Fc electron density map for the distal part (the linker and the p-iodobenzoyl group)
of 14b is much weaker compared to the rest of the compound, suggesting its positional
variability and the existence of several (at least two) alternative conformations (although these
cannot be modeled due to the absence of strong positive Fo-Fc electron density peaks). We
speculate that in addition to the binding modeled for 14b, the distal p-iodobenzoyl moiety in
alternative binding mode(s) is placed outside the accessory hydrophobic pocket in the internal
funnel. As the engagement of the accessory hydrophobic pocket by the p-iodobenzoyl group
contributes to higher inhibitor affinity by the avidity effect, its displacement into the internal
funnel can result in a weaker affinity for PSMA, as demonstrated in our inhibition experiments
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
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0
1
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(Table 1). PSMA affinity of the most potent derivative 13c is approximately 18-fold lower
compared to the parent DCIBzL compound (Table 1). However, inhibitory potency of 13c is
in the similar range as that of the PSMA-specific ligand 1 used in clinical trial (3.97 nM vs.
1.56 nM). Consequently, upon radiolabeling new compounds reported here can be in principle
used as diagnostic agents of prostate cancer in vivo.
CONCLUSIONS
We designed and synthesized novel dipeptide urea-based PSMA inhibitors which possess -
and -amino acids with (S)- or (R)-configuration on the P1 side. According to the structure-
activity relationship studies, the -amino acid analogs with (R)-configuration inhibited
PSMA stronger than the corresponding analogs with (S)-configuration, whereas the -amino
acids with (S)-configuration were more active than the corresponding ones with the (R)-
configuration. The -amino acid compound with (R)-configuration, 13c, exhibited the
strongest PSMA-inhibitory activity in this series with an IC value of 3.97 nM. Hydrogen-
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bonding interactions with the arginine patch and proper placement of the p-iodobenzoyl ring
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in the accessory hydrophobic pocket were observed in the X-ray crystal structure, which
explains the strong affinity of 13c for PSMA. Compound 13c has the potential to be a proto-
type molecule for the development of new -amino acid PSMA inhibitors.
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EXPERIMENTAL SECTION
General. 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 precoated silica gel plates (60 F ). Reaction
2
54
progress was monitored by TLC analysis using a UV lamp and/or KMnO staining for
4
detection purposes. Column chromatography was performed on silica gel (230–400 mesh,
1
13
Merck, Darmstadt, Germany). H and C NMR spectra were recorded at room temperature
(298 K) in CDCl (7.26/77.16 ppm), CD CN (1.94/118.26 ppm), D O (4.79 ppm), CD OD
3
3
2
3
(
3.31/49.00 ppm), or (CD ) SO (2.50/39.52 ppm) on either Bruker BioSpin Avance 300
3 2
MHz NMR or 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;
1
m, multiplet; br, broad for H NMR data. High resolution mass spectra (HRMS) were
recorded on an Agilent 6530 Accurate mass Q-TOF LC/MS spectrometer. Low resolution
mass spectra (LRMS) analyses were obtained from an API 150EX ESI-MS spectrometer.
High-performance liquid chromatography purification was performed on Agilent 1260
Infinity (Agilent). The purification of synthesized compounds was performed on a semi-
preparative reverse-phase high-performance liquid chromatography (RP-HPLC; Agilent
1
260 series HPLC instrument) using a semi-preparative column (Phenomenex Gemini-NX
C18, 110 Å, 150 mm × 10 mm, 5 μm) over 30 min at a flow rate of 2 mL/min. A suitably
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adjusted gradient of 5% B to 95% B was used, where solvent A was 0.1% formic acid (FA)
in H O and B was 0.1% FA in acetonitrile (ACN). UV detection was carried out at 220 nm
2
and 254 nm. The structure identification of each HPLC fractions was carried out by an
electrospray ionization PE Biosystems SciexApi 150 EX mass spectrometer single
quadruple equipped with a turbo ion spray interface. The purity of all final compounds was
measured by analytical RP-HPLC on an Agilent 1260 Infinity (Agilent) with a C18 column
1
1
1
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1
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(Phenomenex, 150 mm × 4.6 mm, 3 μm, 110 Å). RP-HPLC was performed on two different
solvent systems using the following isocratic conditions: for method A, mobile phase was
ACN and water (25:75, v/v, 0.1% FA); for method B, mobile phase was methanol and water
(50:50, v/v, 0.1% FA). All compounds were eluted with a flow rate of 1.0 mL/min (method
A) or 0.7 mL/min (method B) and monitored at UV detector: 254 nm. Purity of the tested
compounds was >97%.
Synthesis.
dioxopentan-2-yl)ureido)propanoic acid (4). L-Glutamic acid di-tert-butyl ester hydrochloride
888 mg, 3.10 mmol) was placed in an oven dried 100 mL two-necked round bottom flask that
(S)-2-((tert-Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-
(
was then fitted with a rubber septum and a three-way connected to a balloon filled with argon.
The flask was flushed with argon and anhydrous dichloromethane (10 mL) was added,
followed by triethylamine (3.5 mL, 24.5 mmol). After the mixture was cooled to –78 ºC,
triphosgene (303 mg, 1.02 mmol) in anhydrous dichloromethane (5 mL) was added dropwise
to the mixture. The reaction mixture was slowly warmed up to room temperature and stirred
for 30 min. A solution of (S)-3-amino-2-(Boc-amino)propanoic acid (500 mg, 2.45 mmol) in
anhydrous dichloromethane (10 mL) and triethylamine (2.9 mL, 20.4 mmol) were added to the
reaction mixture. The reaction mixture was stirred at room temperature for 24 h. The reaction
was quenched with ice-cooled 1 N HCl and extracted with dichloromethane (ca. 100 mL × 3).
The combined organic layer was dried over MgSO , and concentrated under reduced pressure.
4
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(
S)-2-((tert-Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)propanoic acid 4 was obtained and used for the next steps without further
purification.
0
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(S)-2-(3-((S)-2-Amino-2-carboxyethyl)ureido)pentanedioic acid (5). (S)-2-((tert-
Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)propanoic
acid 4 (616 mg, 1.3 mmol) was dissolved in anhydrous dichloromethane (8 mL), slowly added
TFA (2 mL) at 0 ºC. The reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was diluted with dichloromethane and poured into iced water. The organic layer was
washed with water and brine. The combined organic phase was dried over MgSO and
4
concentrated
under
reduced
pressure.
(S)-2-(3-((S)-2-Amino-2-
carboxyethyl)ureido)pentanedioic acid 5 was obtained and used for the next steps without
further purification.
(R)-2-((tert-Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)propanoic acid (6). L-Glutamic acid di-tert-butyl ester hydrochloride (870 mg, 3.0
mmol) was placed in an oven dried 100 mL two-necked round bottom flask that was then fitted
with a rubber septum and a three-way connected to a balloon filled with argon. The flask was
flushed with argon and anhydrous dichloromethane (10 mL) was added, followed by
triethylamine (3.3 mL, 23.7 mmol). After the mixture was cooled to –78 ºC, triphosgene (293
mg, 1.00 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to the mixture. The
reaction mixture was slowly warmed up to room temperature and stirred for 30 min. A solution
of (R)-3-amino-2-(Boc-amino)propanoic acid (500 mg, 2.45 mmol) in anhydrous
dichloromethane (10 mL) and triethylamine (2.9 mL, 20.4 mmol) were added to the reaction
mixture. The reaction mixture was stirred at room temperature for 24 h. The reaction was
quenched with ice-cooled 1 N HCl and extracted with dichloromethane (ca. 100 mL × 3). The
combined organic layer was dried over MgSO , and concentrated under reduced pressure. (R)-
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-((tert-Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)propanoic acid 6 was obtained and used for the next steps without further purification.
(S)-2-(3-((R)-2-Amino-2-carboxyethyl)ureido)pentanedioic acid (7). (R)-2-((tert-
Butoxycarbonyl)amino)-3-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)propanoic
acid 6 (1.19 g, 2.4 mmol) was dissolved in anhydrous dichloromethane (8 mL), slowly added
TFA (2 mL) at 0 ºC. The reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was diluted with dichloromethane and poured into iced water. The organic layer was
1
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0
washed with water and brine. The combined organic phase was dried over MgSO and
4
concentrated
under
reduced
pressure.
(S)-2-(3-((R)-2-Amino-2-
carboxyethyl)ureido)pentanedioic acid 7 was obtained and used for the next steps without
further purification
(S)-2-((tert-Butoxycarbonyl)amino)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)butanoic acid (8). L-Glutamic acid di-tert-butyl ester hydrochloride (820 mg, 2.8
mmol) was placed in an oven dried 100 mL two-necked round bottom flask that was then fitted
with a rubber septum and a three-way connected to a balloon filled with argon. The flask was
flushed with argon and anhydrous dichloromethane (10 mL) was added, followed by
triethylamine (3.1 mL, 22.2 mmol). After the mixture was cooled to –78 ºC, triphosgene (280
mg, 0.95 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to the mixture. The
reaction mixture was slowly warmed up to room temperature and stirred for 30 min. A solution
of (S)-4-amino-2-((tert-butoxycarbonyl)amino)butanoic acid (500 mg, 2.3 mmol) in anhydrous
dichloromethane (5 mL) and triethylamine (2.5 mL, 17.9 mmol) were added to the reaction
mixture. The reaction mixture was stirred at room temperature for 24 h. The reaction was
quenched with ice-cooled 1 N HCl and extracted with dichloromethane (ca. 100 mL × 3). The
combined organic layer was dried over MgSO , and concentrated under reduced pressure. (S)-
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2
-((tert-Butoxycarbonyl)amino)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)butanoic acid 8 was obtained and used for the next steps without further purification.
(S)-2-(3-((S)-3-Amino-3-carboxypropyl)ureido)pentanedioic acid (9). (S)-2-((tert-
Butoxycarbonyl)amino)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)butanoic
acid 8 (1.12 g, 2.4 mmol) was dissolved in anhydrous dichloromethane (8 mL) and then slowly
added TFA (2 mL) at 0 ºC. The reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was diluted with dichloromethane and poured into iced water. The organic
layer was washed with water, followed by brine. The combined organic phase was dried over
MgSO4 and concentrated under reduced pressure. (S)-2-(3-((S)-3-Amino-3-
carboxypropyl)ureido)pentanedioic acid 9 was obtained and used for the next steps without
further purification.
0
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(R)-2-(((Benzyloxy)carbonyl)amino)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)butanoic acid (10). L-Glutamic acid di-tert-butyl ester hydrochloride (280 mg, 0.95
mmol) was placed in an oven dried 100 mL two-necked round bottom flask that was then fitted
with a rubber septum and a three-way connected to a balloon filled with argon. The flask was
flushed with argon and anhydrous dichloromethane (5 mL) was added, followed by
triethylamine (1.0 mL, 7.60 mmol). After the mixture was cooled to –78 ºC, triphosgene (303
mg, 1.02 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to the mixture.
The reaction mixture was slowly warmed up to room temperature and stirred for 30 min.
A solution of (R)-4-amino-2-(((benzyloxy)carbonyl)amino)butanoic acid (200 mg, 0.79 mmol)
in anhydrous dichloromethane (5 mL) and triethylamine (880 μL, 6.30 mmol) were added to
the reaction mixture. The reaction mixture was stirred at room temperature for 24 h. The
reaction was quenched with ice-cooled 1 N HCl and extracted with dichloromethane (ca. 100
mL × 3). The combined organic layer was dried over MgSO , and concentrated under reduced
4
pressure. (R)-2-(((Benzyloxy)carbonyl)amino)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-
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-yl)ureido)butanoic acid 10 was obtained and used for the next steps without further
purification.
(S)-2-(3-((R)-3-Amino-3-carboxypropyl)ureido)pentanedioic acid (11). The crude product
1
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0
dissolved in MeOH (10 mL) was added catalytic amount of AcOH and hydrogenated in a Paar
hydrogenation apparatus by using 10% Pd/C under hydrogen gas (40 psi). The reaction mixture
was filtered through a pad of celite and the celite pad was washed with MeOH. The filtrate was
concentrated in vacuo.
To a solution of the intermediate in anhydrous dichloromethane (8 mL) was added dropwise
TFA (2 mL) at 0 ºC. The reaction mixture was stirred vigorously at room temperature for 2 h.
The reaction mixture was diluted with dichloromethane and poured into iced water. The
organic layer was washed with water and brine. The combined organic phase was dried over
MgSO4 and concentrated under reduced pressure. (S)-2-(3-((R)-3-Amino-3-
carboxypropyl)ureido)pentanedioic acid 11 was obtained and used for the next steps without
further purification.
Typical
procedure
for
the
synthesis
of
(S)-2-(3-((S)-2-Carboxy-2-(4-
iodobenzamido)ethyl)ureido)pentanedioic acid (12a). N-Succinimidyl 4-iodobenzoate (270
mg, 0.78 mmol) and (S)-2-(3-((S)-2-amino-2-carboxyethyl)ureido)pentanedioic acid 5 (175 mg,
0
.63 mmol) were placed in an oven dried 100 mL two-necked round bottom flask that was then
fitted with a rubber septum and a three-way connected to a balloon filled with argon. The flask
was flushed with argon and anhydrous dichloromethane (2 mL) was added, followed by
triethylamine (700 μL, 5.05 mmol). The reaction mixture was stirred at room temperature for
2
h. The reaction mixture was filtered through a 0.45 μm PTFE filter and the filter was washed
with a mixture of ACN and water (1:1, v/v, 0.1% FA, 1 mL). The filtrate was purified by RP-
1
HPLC to give compound 12a (101 mg, 32%) as white solid. H NMR (300 MHz, D O-CD CN
2
3
=
1:1 (v/v)) δ 8.28 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz, 2H), 4.99 (q, J = 2.9 Hz, 1H), 4.67
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2
2
2
2
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6
(
q, J = 3.9 Hz, 1H), 4.11 (dd, J = 4.3 Hz and 14.5 Hz, 1H), 3.96 (dd, J = 7.2 Hz and 14.5 Hz,
1
3
1
H), 2.81 (t, J = 7.4 Hz, 2H), 2.57–2.44 (m, 1H), 2.35–2.21 (m, 1H); C NMR (75 MHz, D O-
2
CD CN = 1:1 (v/v)) δ 176.3, 175.5, 173.0, 168.4, 164.8, 159.9, 138.3, 133.1, 129.4, 99.2, 55.0,
3
0
1
2
3
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1
2
3
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0
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9
0
1
2
3
4
5
6
7
8
9
0
–
–
5
2.8, 40.9, 30.4, 27.1. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 506.0066; found,
16 17 3 8
5
06.0064. >97% purity (as determined by RP-HPLC, method A, t = 6.62 min, method B, t =
R R
7.35 min).
12b–12d were synthesized using similar method as described 12a.
(7S,12S)-1-(4-Iodophenyl)-1,5,10-trioxo-2,6,9,11-tetraazatetradecane-7,12,14-
tricarboxylic acid (12b). This compound was obtained in 35% yield as a white solid, following
the same procedure described for synthesis of 12a with 2,5-dioxopyrrolidin-1-yl 3-(4-
1
iodobenzamido)propanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.85 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 4.51 (q, J = 3.5 Hz, 1H), 4.10 (q, J =
2
3
2
.5 Hz, 1H), 3.73–3.59 (m, 3H), 3.47 (dd, J = 6.8 Hz and 14.5 Hz, 1H), 2.62 (t, J = 6.4 Hz,
1
3
H), 2.39 (t, J = 7.3 Hz, 2H), 2.12–1.99 (m, 1H), 1.89–1.75 (m, 1H); C NMR (75 MHz, D O)
2
δ 177.1, 176.1, 174.1, 173.3, 170.0, 165.0, 159.7, 137.9, 133.0, 128.6, 98.7, 53.5, 52.6, 40.5,
–
–
3
6.4, 35.2, 30.1, 26.2. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 577.0437; found,
19 21 4 9
5
77.0437. >97% purity (as determined by RP-HPLC, method A, t = 6.16 min, method B, t =
R R
8.20 min).
(8S,13S)-1-(4-Iodophenyl)-1,6,11-trioxo-2,7,10,12-tetraazapentadecane-8,13,15-
tricarboxylic acid (12c). This compound was obtained in 56% yield as a white solid, following
the same procedure described for synthesis of 12a with 2,5-dioxopyrrolidin-1-yl 4-(4-
1
iodobenzamido)butanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz, D O)
2
δ 8.35 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 4.96 (dd, J = 4.6 Hz and 7.2 Hz, 1H), 4.76–
4
.72 (m, 1H), 4.12 (dd, J = 4.6 Hz and 14.3 Hz, 1H), 4.02–3.90 (m, 1H), 3.89 (t, J = 7.0 Hz,
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H), 2.92 (t, J = 7.5 Hz, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.70–2.55 (m, 3H), 2.50–2.25 (m, 1H);
1
3
C NMR (75 MHz, D O-CD CN = 1:1 (v/v)) δ 176.5, 175.8, 175.5, 173.4, 168.8, 159.6, 138.3,
2
3
134.1, 129.4, 98.7, 53.8, 52.9, 40.9, 39.6, 33.4, 30.5, 27.2, 25.3. ESI-HRMS (m/z) calcd for
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
–
–
C H IN O [M – H] , 591.0593; found, 591.0593. >97% purity (as determined by RP-HPLC,
2
0
25
4
9
method A, t = 7.32 min, method B, t = 9.37 min).
R
R
(9S,14S)-1-(4-Iodophenyl)-1,7,12-trioxo-2,8,11,13-tetraazahexadecane-9,14,16-
tricarboxylic acid (12d). This compound was obtained in 22% yield as a white solid, following
the same procedure described for synthesis of 12a with 2,5-dioxopyrrolidin-1-yl 5-(4-
1
iodobenzamido)pentanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.87 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 4.50 (dd, J = 4.7 Hz and 7.1 Hz, 1H),
2
4.17 (d, J = 5.1 Hz and 9.1 Hz, 1H), 3.63 (dd, J = 4.6 Hz and 14.4 Hz, 1H), 3.46 (dd, J = 7.4
Hz and 14.4 Hz, 1H), 3.40 (t, J = 6.3 Hz, 2H), 2.47–2.28 (m, 4H), 2.15–1.96 (m, 1H), 1.92–
1
3
1
.75 (m, 1H), 1.65 (brs, 4H); C NMR (75 MHz, D O) δ 177.2, 176.7, 176.3, 173.8, 170.0,
2
137.9, 133.3, 128.6, 98.4, 53.5, 52.7, 44.4, 40.5, 39.5, 35.0, 30.1, 27.8, 26.4, 22.6. ESI-HRMS
–
–
(m/z) calcd for C H IN O [M – H] , 605.0750; found, 605.0752. >97% purity (as
21 25 4 9
determined by RP-HPLC, method A, t = 8.79 min, method B, t = 9.86 min).
R
R
Typical
procedure
for
the
synthesis
of
(S)-2-(3-((R)-2-carboxy-2-(4-
iodobenzamido)ethyl)ureido)pentanedioic acid (13a). N-Succinimidyl 4-iodobenzoate (270
mg, 0.78 mmol) and (S)-2-(3-((R)-2-amino-2-carboxyethyl)ureido)pentanedioic acid 7 (175
mg, 0.51 mmol) were placed in an oven dried 100 mL two-necked round bottom flask that was
then fitted with a rubber septum and a three-way connected to a balloon filled with argon. The
flask was flushed with argon and anhydrous dichloromethane (2 mL), followed by
triethylamine (700 μL, 5.05 mmol). The reaction mixture was stirred at room temperature for
2
h. The reaction mixture was filtered through a 0.45 μm PTFE filter and the filter was washed
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2
2
2
2
2
2
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3
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5
6
with a mixture of ACN and water (1:1, v/v, 0.1% FA, 1 mL). The filtrate was purified by RP-
1
HPLC to give compound 13a (27.6 mg, 9%) as white solid. H NMR (300 MHz, D O-CD CN
2
3
= 1:1 (v/v)) δ 8.30 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 4.98 (dd, J = 4.5 Hz and 7.0
Hz, 1H), 4.63 (dd, J = 5.1 Hz and 8.9 Hz, 1H), 4.08 (dd, J = 4.6 Hz and 14.5 Hz, 2H), 4.00 (dd,
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
3
J = 7.1 Hz and 14.5, 1H), 2.75 (t, J = 7.5 Hz, 2H), 2.57–2.37 (m, 1H), 2.35–2.18 (m, 1H); C
NMR (75 MHz, D O-CD CN = 1:1 (v/v)) δ 176.5, 175.8, 173.3, 168.7, 159.9, 138.3, 133.0,
2
3
–
–
1
5
29.4, 99.3, 55.1, 52.9, 40.9, 30.4, 27.1. ESI-HRMS (m/z) calcd for C H IN O [M – H] ,
16 17 3 8
06.0066; found, 506.0063. >99% purity (as determined by RP-HPLC, method A, t = 6.35
R
min, method B, t = 8.06 min).
R
1
3b–13d were synthesized using similar method as described 13a.
(7R,12S)-1-(4-Iodophenyl)-1,5,10-trioxo-2,6,9,11-tetraazatetradecane-7,12,14-
tricarboxylic acid (13b). This compound was obtained in 30% yield as a white solid, following
the same procedure described for synthesis of 13a with 2,5-dioxopyrrolidin-1-yl 3-(4-
1
iodobenzamido)propanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.85 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 4.49 (dd, J = 5.1 Hz and 6.0 Hz, 1H),
2
4
1
.11 (dd, J = 5.3 Hz and 8.8 Hz, 1H), 3.71–3.55 (m, 3H), 3.49 (dd, J = 6.7 Hz and 14.5 Hz,
H), 2.60 (t, J = 6.1 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H), 2.17–1.99 (m, 1H), 1.96–1.75 (m, 1H);
1
3
C NMR (75 MHz, D O) δ 177.2, 176.2, 174.1, 173.5, 170.1, 159.6, 137.9, 133.0, 128.6, 98.6,
2
–
–
5
3.5, 52.6, 40.4, 36.4, 35.2, 30.1, 26.3. ESI-HRMS (m/z) calcd for C H IN O [M – H] ,
19 21 4 9
5
77.0437; found, 577.0431. >97% purity (as determined by RP-HPLC, method A, t = 6.32
R
min, method B, t = 8.50 min).
R
(8R,13S)-1-(4-Iodophenyl)-1,5,10-trioxo-2,6,9,11-tetraazatetradecane-7,12,14-
tricarboxylic acid (13c). This compound was obtained in 32% yield as a white solid, following
the same procedure described for synthesis of 13a with 2,5-dioxopyrrolidin-1-yl 4-(4-
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iodobenzamido)butanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz, D O)
2
δ 7.86 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 4.45 (dd, J = 4.9 Hz and 6.9 Hz, 1H), 4.17
(dd, J = 5.2 Hz and 9.0 Hz, 1H), 3.57 (dd, J = 4.9 Hz and 14.4 Hz, 1H), 3.47 (dd, J = 7.0 Hz
and 14.5 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.47–2.30 (m, 4H), 2.18–2.01 (m, 1H), 1.98–1.80
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
3
(
m, 3H); C NMR (75 MHz, D O) δ 177.3, 176.6, 175.9, 174.1, 170.0, 159.6, 137.9, 133.2,
2
128.6, 98.5, 53.9, 52.9, 40.6, 39.2, 33.0, 30.2, 26.4, 24.6. ESI-HRMS (m/z) calcd for
–
–
C H IN O [M – H] , 591.0593; found, 591.0593. >97% purity (as determined by RP-HPLC,
2
0
25
4
9
method A, t = 7.19 min, method B, t = 9.24 min).
R
R
(
9R,14S)-1-(4-Iodophenyl)-1,7,12-trioxo-2,8,11,13-tetraazahexadecane-9,14,16-
tricarboxylic acid (13d). This compound was obtained in 18% yield as a white solid, following
the same procedure described for synthesis of 13a with 2,5-dioxopyrrolidin-1-yl 5-(4-
1
iodobenzamido)pentanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.89 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 4.45 (brs, 1H), 4.13 (brs, 1H), 3.60
2
(dd, J = 4.4 Hz and 13.7 Hz, 1H), 3.48 (dd, J = 7.0 Hz and 13.8 Hz, 1H), 3.39 (brs, 2H), 2.56–
13
2
.26 (m, 4H), 2.16–1.97 (m, 1H), 1.96–1.78 (m, 1H), 1.65 (brs, 4H); C NMR (75 MHz, D O)
2
δ 177.4, 176.7, 176.5, 174.5, 170.1, 159.6, 137.9, 133.3, 128.6, 98.4, 54.0, 53.0, 40.7, 39.5,
–
–
35.1, 30.2, 27.7, 26.4, 22.5. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 605.0750;
2
1
25
4
9
found, 605.0753. >97% purity (as determined by RP-HPLC, method A, t = 9.10 min, method
R
B, t = 10.29 min).
R
Typical
procedure
for
the
synthesis
of
(S)-2-(3-((S)-3-Carboxy-3-(4-
iodobenzamido)propyl)ureido)pentanedioic acid (14a). N-Succinimidyl 4-iodobenzoate (128
mg, 0.29 mmol) and (S)-2-(3-((S)-3-amino-3-carboxypropyl)ureido)pentanedioic acid 9 (90
mg, 0.25 mmol) were placed in an oven dried 100 mL two-necked round bottom flask that was
then fitted with a rubber septum and a three-way connected to a balloon filled with argon. The
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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
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5
5
5
5
5
5
5
5
5
6
flask was flushed with argon and anhydrous dichloromethane (2 mL) was added, followed by
triethylamine (300 μL, 2.15 mmol). The reaction mixture was stirred at room temperature for
2 h. The reaction mixture was filtered through a 0.45 μm PTFE filter and the filter was washed
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
with a mixture of ACN and water (1:1, v/v, 0.1% FA, 1 mL). The filtrate was purified by RP-
1
HPLC to give compound 14a (42.8 mg, 27%) as white solid. H NMR (300 MHz, D O) δ 7.90
2
(d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 4.61 (dd, J = 4.7 Hz and 8.9 Hz, 1H), 4.18 (dd, J
=
5.1 Hz and 8.6 Hz, 1H), 3.45–3.31 (m, 1H), 3.31–3.13 (m, 1H), 2.41 (t, J = 7.3 Hz, 2H),
1
3
2
1
.28–1.96 (m, 4H), 1.96–1.76 (m, 1H); C NMR (75 MHz, D O) δ 177.3, 176.5, 175.7, 170.1,
2
59.6, 137.9, 132.5, 128.8, 99.0, 52.7, 51.5, 36.6, 30.7, 30.2, 26.4. ESI-HRMS (m/z) calcd for
–
–
C H IN O [M – H] , 520.0222; found, 520.0220. >97% purity (as determined by RP-HPLC,
1
7
19
3
8
method A, t = 6.98 min, method B, t = 8.09 min).
R
R
1
4b–14d were synthesized using similar method as described 14a.
(7S,13S)-1-(4-Iodophenyl)-1,5,11-trioxo-2,6,10,12-tetraazapentadecane-7,13,15-
tricarboxylic acid (14b). This compound was obtained in 15% yield as a white solid, following
the same procedure described for synthesis of 14a with 2,5-dioxopyrrolidin-1-yl 3-(4-
1
iodobenzamido)propanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.88 (d, J = 6.9 Hz, 2H), 7.48 (d, J = 6.9 Hz, 2H), 4.48–4.28 (m, 1H), 4.28–4.11 (m,
2
1H), 3.83–3.56 (m, 2H), 3.19–2.99 (m, 2H), 2.74–2.54 (m, 2H), 2.53–2.34 (m, 2H), 2.28–2.07
13
(
m, 1H), 2.07–1.89 (m, 2H), 1.89–1.69 (m, 1H); C NMR (75 MHz, D O) δ 177.4, 176.5,
2
1
2
75.8, 174.1, 170.0, 159.4, 137.9, 133.0, 128.6, 98.6, 52.8, 50.8, 36.5, 36.2, 35.3, 30.9, 30.2,
–
–
6.5. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 591.0593; found, 591.0593. >97%
20 25
4
9
purity (as determined by RP-HPLC, method A, t = 6.32 min, method B, t = 8.06 min).
R
R
(8S,14S)-1-(4-Iodophenyl)-1,6,12-trioxo-2,7,11,13-tetraazahexadecane-8,14,16-
tricarboxylic acid (14c). This compound was obtained in 24% yield as a white solid, following
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the same procedure described for synthesis of 14a with 2,5-dioxopyrrolidin-1-yl 4-(4-
1
iodobenzamido)butanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz, D O)
2
δ 7.91 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 4.33 (dd, J = 4.7 Hz and 9.1 Hz, 1H), 4.23
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
(dd, J = 5.1 Hz and 9.0 Hz, 1H), 3.43 (t, J = 6.8 Hz, 2H), 3.29–3.03 (m, 3H), 2.48 (t, J = 7.3
1
3
Hz, 2H), 2.41 (t, J = 7.1 Hz, 2H), 2.22–2.08 (m, 1H), 2.06–1.75 (m, 5H); C NMR (75 MHz,
D O) δ 177.3, 176.5, 176.1, 175.7, 170.1, 159.6, 137.9, 133.2, 128.6, 98.5, 52.7, 50.7, 46.7,
2
–
–
3
9.3, 36.3, 32.9, 30.9, 30.2, 24.5. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 605.0750;
21 25 4 9
found, 605.0751. >97% purity (as determined by RP-HPLC, method A, t = 7.54 min, method
R
B, t = 9.19 min).
R
(9S,15S)-1-(4-Iodophenyl)-1,7,13-trioxo-2,8,12,14-tetraazaheptadecane-9,15,17-
tricarboxylic acid (14d). This compound was obtained in 28% yield as a white solid, following
the same procedure described for synthesis of 14a with 2,5-dioxopyrrolidin-1-yl 5-(4-
1
iodobenzamido)pentanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.89 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 4.33 (dd, J = 4.4 Hz and 9.5 Hz, 1H),
2
4
7
.14 (dd, J = 5.0 Hz and 8.8 Hz, 1H), 3.38 (t, J = 5.8 Hz, 2H), 3.28–3.02 (m, 2H), 2.43 (t, J =
.3 Hz, 2H), 2.35 (t, J = 5.9 Hz, 2H), 2.19–1.97 (m, 2H), 1.97–1.77 (m, 2H), 1.66 (brs, 4H);
1
3
C NMR (150 MHz, D O) δ 175.9, 175.7, 175.7, 175.6, 169.6, 159.0, 137.4, 133.1, 128.1,
2
9
7.7, 57.4, 56.0, 39.1, 36.4, 34.9, 31.4, 28.9, 27.7, 27.4, 22.3. ESI-HRMS (m/z) calcd for
–
–
C H IN O [M – H] , 619.0906; found, 619.0905. >97% purity (as determined by RP-HPLC,
2
2
28
4
9
method A, t = 8.74 min, method B, t = 10.00 min).
R
R
Typical
procedure
for
the
synthesis
of
(S)-2-(3-((R)-3-carboxy-3-(4-
iodobenzamido)propyl)ureido) pentanedioic acid (15a). N-Succinimidyl 4-iodobenzoate (65
mg, 0.19 mmol) and (S)-2-(3-((R)-3-amino-3-carboxypropyl)ureido)pentanedioic acid 11 (50
mg, 0.17 mmol) were placed in an oven dried 100 mL two-necked round bottom flask that was
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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
then fitted with a rubber septum and a three-way connected to a balloon filled with argon. The
flask was flushed with argon and anhydrous dichloromethane (2 mL) was added, followed by
triethylamine (700 μL, 5.05 mmol). The reaction mixture was stirred at room temperature for
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
h. The reaction mixture was filtered through a 0.45 μm PTFE filter and the filter was washed
with a mixture of ACN and water (1:1, v/v, 0.1% FA, 1 mL). The filtrate was purified by RP-
1
HPLC to give compound 15a (13.3 mg, 15%) as white solid. H NMR (300 MHz, D O) δ 7.90
2
(d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 4.57 (dd, J = 4.5 Hz and 9.2 Hz, 1H), 4.16 (dd, J
=
5.2 Hz and 8.9 Hz, 1H), 3.29 (t, J = 6.2 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.30–1.98 (m, 3H),
1
3
1
1
.98–1.78 (m, 1H); C NMR (150 MHz, D O) δ 177.4, 176.7, 176.1, 170.1, 159.6, 137.9,
2
–
32.6, 128.8, 98.9, 52.9, 51.8, 36.6, 30.8, 30.2, 26.5. ESI-HRMS (m/z) calcd for C H IN O
8
1
7
19
3
–
[
M – H] , 520.0222; found, 520.0221. >97% purity (as determined by RP-HPLC, method A,
t = 7.29 min, method B, t = 8.17 min).
R
R
1
5b–15d were synthesized using similar method as described 15a.
(7R,13S)-1-(4-Iodophenyl)-1,5,11-trioxo-2,6,10,12-tetraazapentadecane-7,13,15-
tricarboxylic acid (15b). This compound was obtained in 44% yield as a white solid, following
the same procedure described for synthesis of 15a with 2,5-dioxopyrrolidin-1-yl 3-(4-
1
iodobenzamido)propanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (600 MHz,
D O) δ 7.85 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 4.35 (dd, J = 4.5 Hz and 9.0 Hz, 1H),
2
4
3
1
.16 (dd, J = 5.4 Hz and 8.8 Hz, 1H), 3.81–3.67 (m, 1H), 3.68–3.55 (m, 1H), 3.20–2.91 (m,
H), 2.74–2.51 (m, 3H), 2.44 (t, J = 7.9 Hz, 3H), 2.20–2.05 (m, 1H), 2.05–1.86 (m, 3H), 1.88–
1
3
.71 (m, 1H); C NMR (150 MHz, D O) δ 177.4, 176.7, 176.1, 174.0, 169.9, 159.4, 137.9,
2
132.9, 128.6, 98.6, 52.9, 50.9, 36.5, 36.2, 35.2, 31.0, 30.2, 26.5. ESI-HRMS (m/z) calcd for
–
–
C H IN O [M – H] , 591.0593; found, 591.0596. >98% purity (as determined by RP-HPLC,
2
0
25
4
9
method A, t = 5.99 min, method B, t = 7.75 min).
R
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(
8R,14S)-1-(4-Iodophenyl)-1,6,12-trioxo-2,7,11,13-tetraazahexadecane-8,14,16-
tricarboxylic acid (15c). This compound was obtained in 31% yield as a white solid, following
the same procedure described for synthesis of 15a with 2,5-dioxopyrrolidin-1-yl 4-(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
1
iodobenzamido)butanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz, D O)
2
δ 8.24 (d, J = 8.1 Hz, 2H), 8.00 (d, J = 7.9 Hz, 2H), 4.73–4.61 (m, 1H), 4.61–4.42 (m, 1H),
3.72 (t, J = 6.6 Hz, 2H), 3.64–3.30 (m, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.70 (t, J = 7.4 Hz, 2H),
13
2
1
3
6
9
.55–2.29 (m, 2H), 2.29–2.07 (m, 4H); C NMR (150 MHz, D O-CD CN = 1:1 (v/v)) δ 176.8,
2
3
76.1, 175.7, 175.4, 169.1, 159.5, 138.2, 133.8, 129.1, 98.7, 52.8, 50.8, 39.5, 36.6, 33.1, 31.4,
–
–
0.4, 26.9, 25.1. ESI-HRMS (m/z) calcd for C H IN O [M – H] , 605.0750; found,
21 25
4
9
05.0758. >97% purity (as determined by RP-HPLC, method A, t = 7.55 min, method B, t =
R
R
.23 min).
9R,15S)-1-(4-Iodophenyl)-1,7,13-trioxo-2,8,12,14-tetraazaheptadecane-9,15,17-
(
tricarboxylic acid (15d). This compound was obtained in 19% yield as a white solid, following
the same procedure described for synthesis of 15a with 2,5-dioxopyrrolidin-1-yl 5-(4-
1
iodobenzamido)pentanoate instead of N-succinimidyl 4-iodobenzoate. H NMR (300 MHz,
D O) δ 7.86 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 4.35 (dd, J = 4.5 Hz and 9.5 Hz, 1H),
2
4.15 (q, J = 5.2 Hz and 8.8 Hz, 1H), 3.38 (t, J = 5.7 Hz, 2H), 3.29–3.04 (m, 2H), 2.43 (t, J =
7
.3 Hz, 2H), 2.35 (t, J = 6.8 Hz, 2H), 2.19–1.98 (m, 2H), 1.97–1.77 (m, 2H), 1.66 (brs, 4H);
1
3
C NMR (75 MHz, D O) δ 177.4, 176.8, 176.7, 176.2, 170.1, 159.5, 137.9, 133.3, 128.6, 98.4,
2
5
2.9, 50.8, 44.5, 39.6, 36.3, 35.1, 31.0, 30.3, 27.8, 26.5. ESI-HRMS (m/z) calcd for
–
–
C H IN O [M – H] , 619.0906; found, 619.0909. >97% purity (as determined by RP-HPLC,
2
2
28
4
9
method A, t = 8.76 min, method B, t = 10.08 min).
R
R
(
R)-6-((tert-butoxycarbonyl)amino)-2-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-
yl)ureido)hexanoic acid (16)
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