Preparation of asymmetric urea derivatives that target prostate-specific membrane antigen for SPECT imaging

Article abstract of DOI:10.1021/jm400895s

Prostate-specific membrane antigen (PSMA) has been identified as a diagnostic and therapeutic target for prostate cancer. (S)-2-[3-[(R)-1-Carboxy- 2-mercaptoethyl]ureido-pentanedioic acid (Cys-CO-Glu) were used to design novel PSMA targeting probes by nucleophilic conjugate addition between cysteine and maleimide based reagents. 3 ([¹²³I]IGLCE) was synthesized by this strategy and showed high affinity for PSMA. Results of binding inhibition assays of these derivatives suggested the importance of an aromatic group and succinimide moiety for high affinity. [¹²³I]3 was evaluated in vivo with PSMA positive LNCaP and PSMA negative PC-3 human prostate cancer xenograft bearing mice. [¹²⁵I]3 accumulated in LNCaP tumors but not in PC-3 tumors, and the accumulation was inhibited by 2-(phosphonomethyl)pentanedioic acid (2-PMPA). Use of [¹²³I]3 provided positive images of LNCaP tumors in single photon emission tomography scans. These results warrant further evaluation of [¹²³I]3 and its derivatives as radiolabeled probes for the diagnosis of prostate cancer.

Full text of DOI:10.1021/jm400895s

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Article
Preparation of Asymmetric Urea Derivatives that Target
Prostate-Specific Membrane Antigen for SPECT Imaging
Naoya Harada, Hiroyuki Kimura, Masahiro Ono, and Hideo Saji
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400895s • Publication Date (Web): 24 Sep 2013
Downloaded from http://pubs.acs.org on September 30, 2013
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Preparation of Asymmetric Urea Derivatives that Target
Prostate-Specific Membrane Antigen for SPECT Imaging
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Naoya Harada, Hiroyuki Kimura, Masahiro Ono, Hideo Saji*
Department of PathoꢀFunctional Bioanalysis, Graduate School of Pharmaceutical Sciences,
Kyoto University, Kyoto, Japan
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Abstract
Prostateꢀspecific membrane antigen (PSMA) has been identified as a diagnostic and
therapeutic target for prostate cancer.
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(S)ꢀ2ꢀ[3ꢀ[(R)ꢀ1ꢀcarboxyꢀ2ꢀmercaptoethyl]ureidoꢀpentanedioic acid (CysꢀCOꢀGlu) were used
to design novel PSMA targeting probes by nucleophilic conjugate addition between cysteine
and maleimide based reagents.
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3
([ I]IGLCE) was synthesized by this strategy and showed high affinity for PSMA.
Results of binding inhibition assays of these derivatives suggested the importance of an
1
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aromatic group and succinimide moiety for high affinity. [ I]3 was evaluated in vivo with
PSMA positive LNCaP and PSMA negative PCꢀ3 human prostate cancer xenograft bearing
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mice. [ I]3 accumulated in LNCaP tumors but not in PCꢀ3 tumors, and the accumulation was
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inhibited by 2ꢀ(phosphonomethyl)pentanedioic acid (2ꢀPMPA). Use of [ I]3 provided
positive images of LNCaP tumors in single photon emission tomography scans. These results
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warrant further evaluation of [ I]3 and its derivatives as radiolabeled probes for the diagnosis
of prostate cancer.
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Introduction
Prostate cancer is the most commonly diagnosed form of cancer and the second leading
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cause of cancerꢀrelated deaths among men worldwide. Quantitation of concentrations of
prostate specific antigen (PSA) concentrations, a serum tumor marker of prostate cancer, is
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, 3
effective for diagnosing prostate cancer. However, an invasive biopsy is required to confirm
the diagnosis when the results of PSA tests are positive, because the PSA level is prone to
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, 4
elevation from other causes, such as aging and benign prostatic hyperplasia (BPH).
Therefore, nonꢀinvasive diagnosis of prostate cancer using imaging techniques has significant
clinical benefits.
Nuclear imaging techniques such as positron emission tomography (PET) and single photon
emission computed tomography (SPECT) are valuable for noninvasive diagnosis. Currently,
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ꢀdeoxyꢀ2ꢀ[ F]fluoroꢀDꢀglucose ([ F]FDG) is widely used as a PET probe for diagnoses of
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various cancers. However, [ F]FDG is not suitable for diagnosis of prostate cancer because of
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the lack of correlation between [ F]FDG uptake and tumor incidence. Furthermore,
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F]FDG uptake into the prostate of patients with BPH is similar to that of patients with
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prostate cancer. Therefore, imaging probes that target prostate cancerꢀspecific biomarkers are
desired.
Prostateꢀspecific membrane antigen (PSMA) is a useful target molecule for imaging and
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radiotherapy of prostate cancer because of its high expression in this form of cancer and its
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transmembrane location. PSMA is expressed in normal prostate, primary prostate cancer, and
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lymph node metastases of prostate cancer, although PSMA expression in prostate cancer is
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10–100ꢀfold higher than expression in normal prostate tissue. PSMA expression is
upꢀregulated in patients with hormoneꢀrefractory prostate cancer, while PSA expression is
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decreased in the same patient population. Because PSA is a secretory protein, metastases of
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prostate cancer cannot be diagnosed by the PSA test. On the other hand, metastases can be
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diagnosed by PSMA imaging because PSMA is a membraneꢀanchored protein.
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Inꢀlabeled antiꢀPSMA monoclonal antibody [ In]7E11ꢀC5, marketed as ProstaScint, is a
US Food and Drug Administration approved imaging agent for soft tissue metastases in
patients with prostate cancer. It is not in widespread use, in part because it targets an
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intracellular epitope of PSMA and is believed to bind dead or dying prostate cancer cells.
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More recently, Inꢀlabeled monoclonal antibodies that bind to the extracellular domain of
PSMA have been developed and shown to accumulate in prostate cancer xenograft bearing
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mice. However, there have been limited clinical success using monoclonal antibodies
because of their long circulating plasma halfꢀlife and low permeability in solid tumors,
particularly for metastases to bone. Therefore, we believe that small molecule imaging agents
should have a pharmacokinetic advantage.
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, 15, 16
PSMA has 86% sequence homology with glutamate carboxypeptidaseꢀII (GCPꢀII).
Two structured pockets exist at the active site of GCPꢀII, which cleaves Nꢀacetylaspartyl
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7ꢀ19
glutamate to Nꢀacetyl aspartate.
Several asymmetric ureaꢀbased compounds (XꢀCOꢀGlu
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where X is an amino acid) are reported to be GCPꢀII inhibitors. The glutamate moiety of
ureaꢀbased inhibitors interacts with the S1′ pocket, while the other portions interact with the S1
pocket. Because there is a funnelꢀshaped tunnel with a depth of approximately 20 Å and width
of 8–9 Å in the S1 pocket, ureaꢀbased compounds can be radioꢀlabeled by introducing proper
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linkers to the amino acid residue without loss of affinity.
Previously, PSMA probes with
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this motif have been reported, such as 23 ([ I]DCIT) and 24 ([ I]MIPꢀ1072)
(Figure 1).
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While [ I]23 had low affinity for PSMA and low tumor accumulation in LNCaP bearing mice,
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[
I]24 had high affinity for PSMA.
Therefore [ I]24 appeares to be a promising probe
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for detection, prognosis, and monitoring prostate cancer, and further improvement of [ I]24
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would be of benefit to clinical cancer research.
However, [ I]24 is intolerant to
modification because of its simple structure, which has proven difficult to improve by
derivatization. In this study we aimed to find a new design strategy for asymmetric urea
derivatives with high affinity to PSMA that were easy to design and modify.
Nucleophilic conjugate addition reactions between thiols and maleimides proceed under
mild conditions. Utilizing this reaction, we have previously reported the synthesis of the
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maleimideꢀbased radioꢀiodination reagent, [ I]1 ([ I]HML) (Figure 2). [ I]1 can be easily
prepared by iododestannylation, it rapidly reacts with thiols at room temperature, is stable in
human serum, and is a peptidic reagent and thus derivatives are easily prepared by replacement
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of specific amino acids. 1 (CysꢀCOꢀGlu), an asymmetric urea inhibitor of PSMA , has a
reactive thiol (Figure 2). Therefore, using the reaction between 2 and derivatives of 1, we can
design and synthesize various types of PSMA probes. In this study, we prepared 3
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(
[ I]IGLCE) via the nucleophilic conjugate addition reaction between 2 and [ I]1 (Figure 2).
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The affinity of [ I]3 ([ I]IGLCE) for PSMA was evaluated, and a rationale for its high
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affinity to PSMA was investigated. We hypothesized that the high affinity of [ I]3 for PSMA
was attributable to interactions between the 1ꢀderived moiety and PSMA, and eight derivatives
of 3 were designed (Figure 2). We focused on modifying the iodine, two amino acids (Lys and
Gly), the aromatic ring, and the sccinimide in the 1ꢀderived moiety. The moieties important for
high affinity were identified through a structure activity relationship study.
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Additionally, the stability and biodistribution of [ I]3 was assessed, and SPECT/computed
tomography (CT) imaging was performed to assess its utility.
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RESULTS and DISCUSSION
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Chemistry and Radiochemistry
As shown in Scheme 1, 3 was synthesized by a nucleophilic conjugate addition of the
cysteine thiol of the asymmetric urea (2) with the maleimide moiety of 1. The nonradioactive
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product, 3, was analyzed by HꢀNMR and FABꢀHRMS. Its radiolabeled form, [ I]3, was
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prepared from [ I]1. This labeling reaction proceeded under mild conditions (pH 6–8, room
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temperature) to afford high radiochemical yields (total yields were 58–63%). [ I]3 was
prepared with a radiochemical purity greater than 95% after HPLC purification, and analysis
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R R
of the purified [ I]3 showed a single peak (t : 23.1 min) corresponding to the isolated 3 (t :
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3.0 min).
Eight derivatives of 3 were synthesized via the same conjugate addition approach (Schemes
–4) and were employed to investigate the relationship between the structure of 3 and high
2
affinity for PSMA. The appropriate active ester and maleimideꢀbased ammonium 6a or 6b
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known in the literature were condensed to yield the maleimideꢀbased precursors 10a, 10b,
1
5a–15b, 17, and 21. By only selecting the active ester, we could design these precursors.
Introduction of several functional groups into the maleimideꢀbased precursors was not difficult,
such as the amino group of 21. The methylamino group of active ester 20 was protected with
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tertꢀbutoxycarbonyl group but not with a benzyl group because maleimide was resistant to acid
but not to hydrogenation. The condensation product of 20 and 6a was deprotected with 4 N
HCl/ethyl acetate to yield 21. These maleimideꢀbased precursors were reacted with 2 to yield
the final products. All of these reactions proceeded at room temperature, and the final products
were purified by reverse phase HPLC.
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This synthetic strategy appears promising for the design of novel probes, not only for the
derivatives that were synthesized, but also for PET probes, which generally have a short
radiochemical halfꢀlife, as this reaction proceeds rapidly at room temperature.
In Vitro Cell-binding Assay
d
Using Scatchard plots, the affinity of 3 for PSMA was evaluated by obtaining a K value
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using an in vitro cellꢀbinding saturation assay. [ I]23 and [ I]24 were selected as positive
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control compounds to evaluate binding affinity.
As shown in Table 1, [ I]3 had a higher
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affinity (K
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: 7.8 ± 1.4 nM) than [ I]23 (K
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: 143 ± 2 nM) and [ I]24 (K : 20.4 ± 0.6 nM).
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We hypothesized that the high affinity of [ I]3 for PSMA was attributable to interactions
between the 1ꢀderived moiety and PSMA. Eight derivatives (11a, 11b, 16a–16c, 18, 19, and
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2) of 3 were designed to further investigate the interactions between the 1ꢀderived moiety and
PSMA and their affinities for PSMA were compared (Figure 2). The design of these
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derivatives introduced four types of modifications into the 1ꢀderived moiety: substitution of
the iodine for a proton (11a), substitution of a carboxyl group of Lys for a proton (11b),
alteration of the carbon chain length of Gly (16a–16c and 18), and removal of the iodobenzoyl
group (aromatic group) (19 and 22). The affinities of these compounds for PSMA were
evaluated in the binding inhibition assay.
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The binding inhibition assay data are summarized in Table 2. Introduction of an iodine (11a)
or carboxyl group (11b) into the 1ꢀderived moiety did not alter the affinity for PSMA.
Alteration of the carbon chain length was acceptable since elongated (16a–16c) or shortened
(
18) carbon chain lengths of the 1ꢀderived moiety also did not affect the affinity. On the other
hand, all of these derivatives, even 19, which had a Nꢀmethylsuccinimidyl group appended to 2,
showed higher affinity (2.14–94.8 nM) than 2 (376 nM). This result suggested that the
succinimidyl group of the 1ꢀderived moiety was important for high affinity. This finding
supports the efficacy of the design strategy using 2 conjugated maleimideꢀbased reagents.
Compounds 19 and 22 showed modest affinity (62.1–94.8 nM) compared with the other
derivatives (2.14–8.06 nM). It appears that the aromatic group of the 1ꢀderived moiety was
also important for high affinity because 19 and 22, which did not have an aromatic group, had
poorer affinity than the other derivatives (3, 11a, 11b, 16a–16c, 18), which had an aromatic
group. These results suggested that both a succinimidyl group and aromatic group were
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important for high affinity to PSMA although the distance between these moieties was not
critical for the high affinity. We assessed this seemingly contradictory result using the Xꢀray
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structure of the binding site of PSMA, which had been reported by C. Barinka et al.
In
a previous report, C. Barinka et al. suggested that the glutamate moiety of ureaꢀbased
inhibitors interacts with a pharmacophore (S1′ pocket), and the remainder of the inhibitor
interacts with a lipophilic pocket (S1 pocket), which had been reported as a funnelꢀshaped
tunnel with a depth of approximately 20 Å composed of hydrophobic βꢀsheets. Previously, M.
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Eder et al. had designed Gaꢀlabeled PSMA probes using the lipophilic chelator
N,N ꢀbis[2ꢀhydroxyꢀ5ꢀ(carboxyethyl)ꢀbenzyl]ethylenediamineꢀN,N ꢀdiacetic acid
́ ́
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(
HBEDꢀCC) that targeted this hydrophobic pocket.
Applying the same logic to 3, the
1
ꢀderived moiety should interact with the S1 pocket, and the aromatic ring of the 1ꢀderived
moiety should interact with the hydrophobic βꢀsheets of the S1 pocket. We also believe that the
carbon chain length of 1 was not critical for high affinity because of the elongated structure of
the hydrophobic S1 pocket.
To investigate these hypotheses, we performed a docking simulation study. The docking
models between 3 and recombinant human GCPꢀII (rhGCPꢀII: PDB code 2c6c) are shown in
Supporting Information. In these models, key interactions between the asymmetric urea moiety
and the S1′ pocket were examined, including the cysteine moiety and Arg534, the urea group
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and Gly518, and the glutamate moiety and Asn257, Lys699, and Try700 (Figure S1). These
interactions corresponded with those identified in previous reports. In addition, interactions
between the 1ꢀderived derived moiety and the S1 pocket were examined. As previously
discussed, our results supported the importance of the succinimidyl and aromatic groups for
achieving high binding affinity. In the docking simulation, the aromatic ring of the 1ꢀderived
moiety was oriented to the hydrophobic S1 pocket, and the two oxygen atoms of the
succinimidyl group interacted with Arg536 and Tyr549 (Figure S2). These interactions
confirmed the importance of the aromatic and succinimidyl groups for obtaining high affinity.
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In Vitro Stability
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We investigated the stability of [ I]3 using mouse serum. [ I]3 was expected to be stable
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since [ I]1 had been reported to be a stable radiolabeled reagent in human serum. A portion
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of [ I]3 was degraded in mouse serum after an 18 h incubation period (Figure. 3B). This
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degradation product was probably mꢀ[ I]iodohuppric acid, which would be produced by
cleavage of the GlyꢀLys peptide bond. However, this degradation was not a rapid reaction, as
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no degradation products of [ I]3 were observed after a 6 h incubation period (Figures. 3A and
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B). In accordance with this result, [ I]3 was regarded as being stable. Indeed, long term
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stability would not be required since the majority of [ I]3 was excreted from blood 30 min
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after injection in the subsequent biodistribution study.
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Biodistribution and Blocking Studies
1
25
An in vivo biodistribution study of [ I]3 was performed using LNCaPꢀ and PCꢀ3ꢀbearing
1
25
mouse models to confirm the viability of our design strategy. The biodistribution data of [ I]3
1
25
and the tumor to blood or muscle ratios are shown in Table 3. [ I]3 showed good tumor
uptake by LNCaP tumors (9.5% ID/g at 30 min), and this accumulation was maintained for at
least 6 h, while low tumor uptake was observed in PCꢀ3 tumors (0.94% ID/g at 30 min).
Moreover, this high uptake in LNCaP tumors was significantly inhibited (0.55% ID/g at 30
min) by coinjection of 2ꢀ(phosphonomethyl)pentanedioic acid (2ꢀPMPA) (50 mg/kg body
1
25
weight) (Figure 4B). These results suggested that the tumor uptake of [ I]3 was due to
PSMAꢀspecific binding. Although high accumulation in the kidneys was also observed (89%
ID/g at 30 min), the accumulation was inhibited (3.6%ID /g at 30 min) by coinjection of
2
ꢀPMPA (Figure 4A). GCPꢀII is expressed in mouse kidneys, which is likely responsible for
125
the [ I]3 accumulation in this organ. Similar results have been observed with other probes
2
2, 23, 26, 27, 28, 31 125
targeting PSMA.
[ I]3 also showed high accumulation in the spleen of
C.B.ꢀ17/Icr scid/scid Jcl mice which was blocked with 2ꢀPMPA indicating GCPꢀII expression
1
25
in the spleen. [ I]3 showed fast blood clearance (8.9% ID/g at 2 min and 1.5% ID/g at 30 min,
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respectively) and rapid pharmacokinetics as both the tumor/blood and tumor/muscle ratios
1
25
reached high values after only 30 min (5.3 and 11.1, respectively). [ I]3 showed little
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deiodination as accumulation of radioactivity in the thyroid was not observed. These results
1
25
suggested the possibility of the use of [ I]3 as a PSMA targeted probe that possesses good
accumulation in PSMAꢀexpressing prostate cancer tissue with rapid pharmacokinetics, and
high stability.
1
25
125
In addition to [ I]3, we prepared [ I]11b and evaluated it in vivo (its radiosynthesis is
1
25
shown in Supporting Information). [ I]11b showed more rapid clearance from blood and
1
25
125
slightly lower accumulation in LNCaP tumor than [ I]3 (Figure S4). However, [ I]11b
1
25
showed significantly higher accumulation in liver and intestine than [ I]3 (Figure S3). We
1
25
considered that such high hepatic and intestinal accumulations of [ I]11b were not preferable,
125
and we attributed this accumulation to the lower polarity of [ I]11b compared with that of
125
125
[
I]3 (Table S1). This was supported by the fact that [ I]11b had a longer HPLC retention
125
time indicating it was lower in polarity than [ I]3. Moreover, other iodinated derivatives
125
(
16a–16c and 18) also had longer HPLC retention times than [ I]3 (Table S1). Therefore, we
1
23
regarded [ I]3 as the best of the tested derivatives.
1
25
Then using mice, we compared the properties [ I]3 with the previously reported PSMA
1
25
probe [ I]24, for which a phase I clinical trial was completed. In accordance with the
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previous study, high accumulation of [ I]24 in LNCaP tumor was observed (Table S2).
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[
I]24 showed higher accumulation in LNCaP tumor and kidney than [ I]3, although the
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affinity of [ I]24 for PSMA was lower than [ I]3. We consider that rapid clearance of [ I]3
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25
from blood is the cause of this lower accumulation. [ I]3 showed lower radioactivity than
1
25
[
I]24 in blood 30 min after injection. The differences in blood clearance may be attributed to
1
25
125
differences in the excretion routes. [ I]3 was excreted from both kidney and liver. [ I]3
showed high accumulation in kidney, and accumulation in intestine was elevated over time. On
1
25
the other hand, [ I]24 was excreted from kidney but not from liver. The lower hydrophilicity
1
25
125
of [ I]11b compared to [ I]3 is likely the cause of its higher hepatic accumulation of
1
25
[
I]11b. We also believed that lower hydrophilicity is likely the cause of the higher hepatic
1
25
125
125
accumulation of [ I]3 compared to [ I]24. The distribution coefficient (log D) of [ I]3 and
125
125
125
[
I]24 is shown in Table S3. [ I]3 had a larger log D value (log D = ꢀ1.97) than [ I]24 (log
D = ꢀ2.91). Therefore, an improvement of the hydrophilicity would be necessary for lowering
hepatic accumulation and elevating tumor accumulation.
Small Animal SPECT/CT
A SPECT/CT study was performed on the basis of the biodistribution study results. SPECT
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23
scanning commenced 30 min after injection of [ I]3 since the tumor/blood ratio was
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sufficiently high at this time point to obtain SPECT images. Although the tumor/blood ratio
was higher 3 h after injection, the 30 min delay was selected as the time point for SPECT
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scanning in order to determine whether [ I]3 could distinguish LNCaP tumors from PCꢀ3
tumors immediately after injection.
1
23
[
I]3 clearly distinguished LNCaP tumors from PCꢀ3 tumors on SPECT/CT images 30 min
post injection (Figures. 5A and 5C), and tracer accumulation was clearly absent from SPECT
images when coinjected with 2ꢀPMPA (Figure 5B). These results were consistent with those
1
23
from the biodistribution study, and suggested that [ I]3 would be efficient as a PSMA
targeting probe.
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CONCLUSION
The syntheses of PSMA targeting derivatives by a nucleophilic conjugate addition between
the cysteine of the asymmetric urea compound (2) and a maleimide based reagent were
successful, and the aromatic and succinimidyl moieties of these derivatives were found to be
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important for high affinity. [ I]3, which was synthesized by this reaction, exhibited high
affinity for PSMA and gave clear SPECT images with LNCaP bearing mice.
This study clearly established the basis of a new design strategy for PSMA probes. Through
1
25
our investigation of [ I]3, we clarified the importance of the aromatic ring and the
succinimidyl group for high affinity. Moreover, the significance of hydrophilicity for
pharmacokinetics was confirmed.
1
23
These findings warrant further evaluation of [ I]3 as an imaging agent, and support the
benefit of this strategy for design of other PSMA probes.
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EXPERIMENTAL SECTION
General
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WꢀPrep 2XY (Yamazen Corporation, Osaka, Japan) was used for silica gel column
TM
chromatography on a Hi Flash silica gel column (40 mm, 60 Å, Yamazen). Silica gel 60
F254, 0.5 mm (Merck KGaA, Darmstadt, Germany) was used for preparative thin layer
chromatography (PTLC). A LCꢀ20AD (SHIMADZU CORPORATION, Kyoto, Japan)
equipped with a SPDꢀ20A UV detector (ρ; 220 nm and 254 nm) (SHIMADZU) and a
NDWꢀ351 radioisotope detector (Hitachi Aloka Medical, Ltd., Tokyo, Japan) was used for high
performance liquid chromatography (HPLC). The eluent consisted of a binary mixture of 0.1%
2
trifluoroacetic acid (TFA) in H O (solvent A) and 0.1% TFA in methanol (solvent B).
Nanopure water was prepared by MQ Integra15 (Nihon Millipore, Tokyo, Japan). Mass spectra
were recorded on a LCMSꢀ2010 EV (SHIMADZU), GCMSꢀQP2010 Plus (SHIMADZU), or
1
JMSꢀSX 102A QQ (JEOL Ltd., Tokyo, Japan). HꢀNMR spectra were recorded on a
3 3 2
LNMꢀAL500 (JEOL) using CDCl , dimethylsulfoxideꢀd6 (DMSOꢀd6), CD OD, or D O as the
solvent and tetramethylsilane as an internal standard (Eurisoꢀtop, SaintꢀAubin, France). All
chemicals used in the syntheses were reagent grade.
Chemistry and Radiochemistry
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3
1
30
27
28, 32
2
was synthesized as previously reported. 1, 23, 24,
and radioiodinated forms of
1
23
these compounds were synthesized by previously reported methods. [ I]3 was prepared by
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incubation of 2 with [ I]1 at room temperature for 15 min. Each radiolabeled compound was
purified by reverseꢀphase HPLC using a COSMOSIL 5C18ꢀARꢀII 4.6 × 150 mm column eluted
with 0.1% TFA in water (solvent A) and 0.1% TFA in methanol (solvent B). An HPLC gradient
with a flow rate of 1 mL/min was initiated from an isocratic system (30% solvent B) over 10
min and adjusted to 30–60% solvent B over 10 min followed by 60% solvent B.
Compounds 11a, 11b, 16a–16c, 18, 19, and 22 were synthesized to investigate the
3
0
relationships between the high affinity of 3 and its structure (Schemes 2–4). Compounds 4–6,
3
0
30
33
34
9
a, 10a, 12, and 20 were synthesized by previously reported methods. All compounds
tested in the inhibition assay were prepared in > 95% purity as judged by HPLC.
Conditions for HPLC purification are defined as follows: condition 1, YMCꢀPack
ODSꢀAQ 20 × 250 mm column (YMC Co. Ltd., Kyoto, Japan), 5 mL/min, 30–80%
gradient of solvent B over 60 min; condition 2, YMCꢀPack ODS 20 × 250 mm column,
mL/min, 40–90% gradient of solvent B over 60 min.
5
(
-
2S)-2-(3-((1R)-1-Carboxy-2-((1-((R)-5-carboxy-5-(2-(3-iodobenzamido)acetamido)pentyl)
2,5-dioxopyrrolidin-3-yl)thio)ethyl)ureido)pentanedioic acid (3)
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To a solution of 2 (6.8 mg, 0.023 mmol) in H O (0.2 mL) was added 1 (11.9 mg, 0.023
2
mmol) in acetonitrile (0.15 mL). 2 N NaOH was added to the mixture to adjust the pH to 7.
The mixture was stirred at room temperature for 2 h. HPLC purification afforded 3 (3.8 mg,
21% yield) under the following conditions: COSMOSIL 5C18ꢀARꢀII column (10 × 250 mm)
eluted with 0.1% TFA in water (solvent A) and 0.1% TFA in methanol (solvent B), at a flow
rate of 2 mL/min, and an HPLC gradient initiated from an isocratic system (30% solvent B)
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over 15 min, adjusted to 30–60% of solvent B over 10 min followed by 60% of solvent B.
1
HꢀNMR (500 MHz, D O) δ; 8.10 (s, 1H), 7.90 (d, J = 8.02 Hz, 1H), 7.71 (d, J = 7.45 Hz, 1H),
2
7
.21 (dd, J = 7.45, 8.02 Hz, 1H), 4.39 (t, J = 5.73 Hz, 1H), 4.28 (broad s, 1H), 4.15 (m, 1H),
.99–4.07 (m, 2H), 3.93 (m, 1H), 3.39 (t, J = 6.87 Hz, 2H), 2.95–3.35 (m, 3H), 2.56 (m, 1H),
.38 (dt, J = 2.86, 7.45 Hz, 2H), 2.05 (m, 1H), 1.83 (m, 2H), 1.65 (m, 1H), 1.48 (m, 2H) and
3
2
1
8
+
+
35 5
.21 (t, J = 7.45 Hz, 2H). ESIꢀMS; 808 [M + H] . HRFAB ꢀMS: calcd for C28H N O13SI,
+
08.0994 [M+H] ; found 808.0997.
4
-Methoxybenzyl 2-(3-iodobenzamido)acetate (7a)
To a solution of 3ꢀiodobenzoic acid (2.8 g, 11 mmol) in N,Nꢀdimethylformamide (DMF) (25
mL) was added 4ꢀmethoxybenzyl 2ꢀaminoacetate hydrochloride (HꢀGlyꢀOPMB·HCl) (2.6 g,
1
1 mmol), water soluble carbodiimide hydrochloride (WSCI·HCl) (2.1 g, 11 mmol), and
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triethylamine (1.1 mL). The mixture was stirred at room temperature for 6 h. After extraction
with ethyl acetate, the organic layer was washed with brine and dried over sodium sulfate. The
solvent was removed under reduced pressure, and the product 7a was purified by silica gel
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column chromatography (hexane/ethyl acetate = 2/1, 3.0 g, 64% yield). HꢀNMR (DMSOꢀd6):
9.06 (m, 1H), 8.20 (m, 1H), 7.92 (m, 1H), 7.87 (m, 1H) 7.31 (m, 2H), 6.93 (m, 2H), 5.08 (s,
+
2
H), 4.03 (d, J = 5.73 Hz, 2H), and 3.75 (s, 3H). EIꢀMS m/z: 425 [M] , 231 (4.7), 121 (100).
4
-Methoxybenzyl 2-benzamidoacetate (7b)
To a solution of benzoic acid (1.6 g, 13 mmol) in DMF (15 mL) was added HꢀGlyꢀOPMB·
HCl (3.0 g, 13 mmol), WSCI·HCl (2.5 g, 13 mmol), and triethylamine (1.8 mL). The mixture
was stirred at room temperature overnight. After extraction with ethyl acetate, the organic layer
was washed with brine and dried over sodium sulfate. The solvent was removed under reduced
pressure, and the product 7b was purified by silica gel column chromatography (hexane/ethyl
1
acetate = 2/1, 2.2 g, 57% yield). HꢀNMR (CDCl
3
): 7.81 (d, J = 7.45 Hz, 2H), 7.51 (m, 2H),
7
4
.45 (t, J = 7.45 Hz, 2H), 7.32 (m, 2H), 6.90 (m, 2H), 6.63 (s, 1H), 5.17 (s, 2H), 4.26 (d, J =
+
.58 Hz, 2H), and 3.82 (s, 3H). EIꢀMS m/z: 299 [M] , 162 (2.0), 137 (4.7), 134 (15.9), 121
(
100), 77 (34.7).
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2
-(3-Iodobenzamido)acetic acid (8a)
To a solution of 7a (3.0 g, 7.1 mmol) in dichloromethane (24 mL) was added TFA (5.3 mL).
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The mixture was stirred at room temperature for 15 min. The solvent was removed under
reduced pressure, and the product 8a was recrystallized from chloroform (2.1 g, 97% yield).
1
HꢀNMR (CD
3
OD) δ: 8.95 (m, 1H), 8.22 (t, J = 1.72 Hz, 1H), 7.92 (m, 1H), 7.88 (m, 1H), 7.30
ꢀ
(
t, J = 8.02 Hz, 1H), and 3.92 (d, J = 5.73 Hz, 2H). ESIꢀMS m/z: 304 [M] .
2
-Benzamidoacetic acid (8b)
To a solution of 7b (2.2 g, 7.3 mmol) in dichloromethane (25 mL) was added TFA (5.5 mL).
The mixture was stirred at room temperature for 15 min. The solvent was removed under
reduced pressure, and the product 8b was recrystallized from chloroform (1.2 g, 94% yield).
1
HꢀNMR (DMSOꢀd6) δ: 8.81 (t, J = 5.73 Hz, 1H), 7.87 (m, 2H), 7.55 (m, 1H), 7.48 (t, J = 7.45
+
Hz, 2H), and 3.93 (d, J = 5.73 Hz, 2H). EIꢀMS m/z: 179 [M] , 134 (20), 105 (100), 77 (69).
2
,5-Dioxopyrrolidin-1-yl 2-benzamidoacetate (9b)
To a solution of 8b (1.2 g, 6.7 mmol) in DMF (10 mL) was added Nꢀhydroxysuccinimide
(
NHS) (0.78 g, 6.8 mmol) and N,N’ꢀdiisopropylcarbodiimide (DIC) (0.86 mg, 6.8 mmol). The
mixture was stirred at room temperature for 14 h. After extraction with ethyl acetate, the
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organic layer was washed with brine and dried over sodium sulfate. The solvent was removed
under reduced pressure, and the resulting white precipitate was washed with chloroform and
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collected (1.8 g, 95% yield). HꢀNMR (DMSOꢀd6) δ: 9.16 (t, J = 5.73 Hz, 1H), 7.88 (d, J =
7.45 Hz, 2H), 7.58 (t, J = 6.87 Hz, 1H), 7.50 (t, J = 7.73 Hz, 2H), 4.43 (d, J = 5.73 Hz, 2H),
2.82 (s, 4H).
(
(
S)-2-(2-Benzamidoacetamido)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid
10a)
To a solution of 9b (0.1 g, 0.36 mmol) in acetonitrile (3 mL) was added 6a (95 mg, 0.36
mmol) and N,Nꢀdiisopropylethylamine (DIPEA) (0.13 mL). The mixture was stirred at room
temperature for 3 h. After extraction with ethyl acetate and washing with saturated aqueous
ammonium chloride, the organic layer was dried over sodium sulfate. The solvent was
removed under reduced pressure, and the product 10a was purified by PTLC
(
R
chloroform/methanol = 9/1) followed by HPLC using condition 1 (t : 44 min, 6.3 mg, 4.5%
1
yield). HꢀNMR (CD
3
OD) δ: 7.87 (m, 2H), 7.55 (t, J = 7.45 Hz, 1H), 7.47 (t, J = 7.73 Hz, 2H),
6
1
.77 (s, 2H), 4.42 (m, 1H), 4.10 (m, 2H), 3.49 (t, J = 6.87 Hz, 2H), 1.90 (m, 1H), 1.74 (m, 1H),
+
.60 (m, 2H), and 1.38 (m, 2H). ESIꢀMS m/z: 388 [M + H] .
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N-[2-[[5-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl]amino]-2-oxoethyl]-3-iodobenzami
de (10b)
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11
12
13
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23
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25
26
27
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59
60
To a solution of 9a (96 mg, 0.24 mmol) in acetonitrile (3 mL) was added 6b (52 mg, 0.24
mmol) and DIPEA (41 ꢀL). The mixture was stirred at room temperature for 1 h. After
extraction with ethyl acetate, the organic layer was dried over sodium sulfate. The solvent was
removed under reduced pressure, and the product 10b was purified by silica gel column
1
chromatography (chloroform/methanol = 10/1, 51 mg, 46% yield). HꢀNMR (CDCl
3
) δ: 8.18 (t,
J = 1.72 Hz, 1H), 7.85 (m, 1H), 7.78 (dd, J = 1.15, 7.45 Hz, 1H), 7.19 (t, J = 7.73 Hz, 1H),
7
3
.05 (s, 1H), 6.69 (s, 2H), 6.13 (s, 1H), 4.10 (d, J = 5.16 Hz, 2H), 3.52 (t, J = 6.87 Hz, 2H),
+
.43 (m, 2H), 1.62 (m, 4H), and 1.32 (m,2H). ESIꢀMS m/z: 470 [M + H] .
(
2S)-2-[3-[(1R)-2-[[1-[(S)-5-(2-Benzamidoacetamido)-5-carboxypentyl]-2,5-dioxopyrrolidi
n-3-yl]thio]-1-carboxyethyl]ureido]pentanedioic acid (11a)
2
To a solution of 2 (4.8 mg, 0.016 mmol) in H O (0.4 mL) was added 10a (6.3 mg 0.013
mmol) in acetonitrile (0.6 mL). The mixture was neutralized with 2 N NaOH and stirred at
R
room temperature for 1h. 11a was purified by HPLC using condition 1 (t : 37 min, 5.9 mg,
1
5
4% yield). HꢀNMR (CD
3
OD) δ: 7.88 (m, 2H), 7.55 (t, J = 7.73 Hz, 1H), 7.47 (t, J = 7.45 Hz,
2H), 4.61 (m, 1H), 4.42 (m, 1H), 4.32 (m, 1H), 4.11 (m, 2H), 4.01 (m, 1H), 3.50 (m, 2H), 3.19
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1
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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5
5
5
6
(m, 2H), 2.48 (m, 1H), 2.40 (m, 2H), 2.14 (m, 1H), 1.89 (m, 2H), 1.73 (m, 1H), 1.61 (m, 2H),
+
+
36 5
and 1.39 (m, 2H). FABꢀMS m/z: 682 [M + H] . HRFAB ꢀMS: calcd for C28H N O13S,
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
+
682.2037 [M + H] ; found 682.2030.
(2S)-2-[3-[(1R)-1-Carboxy-2-[[1-[5-[2-(3-iodobenzamido)acetamido]pentyl]-2,5-dioxopyrr
olidin-3-yl]thio]ethyl]ureido]pentanedioic acid (11b)
2
To a solution of 2 (14 mg, 0.048 mmol) in H O (0.3 mL) was added 10b (23 mg, 0.059
mmol) in acetonitrile (0.3 mL). The mixture was neutralized with 2 N NaOH and stirred at
room temperature for 1 h. 11b was purified by HPLC using condition 2 (t : 42 min, 16 mg,
R
1
4
1
3% yield). HꢀNMR (CD
3
OD) δ: 8.26 (t, J = 1.72 Hz, 1H), 7.89 (m, 2H), 7.25 (t, J = 8.02 Hz,
H), 4.61 (m, 1H), 4.31 (m, 1H), 3.98 (m, 3H), 3.48 (m, 3H), 3.20 (m, 4H), 2.50 (m, 1H), 2.42
(
m, 2H), 2.14 (m, 1H), 1.89 (m, 1H), 1.56 (m, 4H), and 1.32 (m, 2H). FABꢀMS m/z: 764 [M +
+
+
+
H] . HRFAB ꢀMS: calcd for C H N O SI, 764.1093 [M + H] ; found 764.1099.
2
7
35
5
11
3
-(3-Iodobenzamido)propanoic acid (13a)
To a solution of 12 (600 mg, 1.74 mmol) in acetonitrile (6 mL) was added 3ꢀaminopropionic
acid (170 mg, 1.91 mmol) and DIPEA (0.33 mL). The mixture was stirred at room temperature
for 11 h. After extraction with ethyl acetate and washing with saturated aqueous ammonium
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chloride, the organic layer was dried over sodium sulfate. The solvent was removed under
reduced pressure, and the resulting precipitate was washed with chloroform to afford 13a (214
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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36
37
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43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
mg, 39% yield). HꢀNMR (CD
3
OD) δ: 8.16 (t, J = 1.72 Hz, 1H), 7.88 (d, J = 6.87 Hz, 1H),
7.79 (m, 1H), 7.23 (t, J = 8.02 Hz, 1H), 3.61 (t, J = 6.87 Hz, 2H), and 2.62 (t, J = 6.87 Hz, 2H).
+
EIꢀMS m/z: 319 [M] , 231 (36), 203 (12), 76 (36).
4
-(3-Iodobenzamido)butanoic acid (13b)
To a solution of 12 (500 mg, 1.45 mmol) in acetonitrile (4 mL) was added 4ꢀaminobutanoic
acid (164 mg, 1.59 mmol) and DIPEA (0.27 mL). The mixture was stirred at room temperature
overnight. After extraction with ethyl acetate and washing with saturated aqueous ammonium
chloride, the organic layer was dried over sodium sulfate. The solvent was removed under
reduced pressure, and the resulting precipitate was washed with chloroform to afford 10b (316
1
mg, 65% yield). HꢀNMR (CD
3
OD) δ: 8.17 (t, J = 1.72 Hz, 1H), 7.88 (dt, J = 1.72, 8.02 Hz,
1
H), 7.80 (dt, J = 1.72, 8.02 Hz, 1H), 7.23 (t, J = 8.02 Hz, 1H), 3.41 (t, J = 7.16 Hz, 2H), 2.38
+
(
(
t, J = 7.16 Hz, 2H), and 1.90 (tt, J = 7.16, 7.16 Hz, 2H). EIꢀMS m/z: 333 [M] , 274 (19), 231
36), 102 (50), 76 (51).
5
-(3-Iodobenzamido)pentanoic acid (13c)
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2
2
2
2
2
2
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5
5
6
To a solution of 12 (500 mg, 1.45 mmol) in acetonitrile (6 mL) was added 5ꢀaminopentanoic
acid (188 mg, 1.60 mmol) and DIPEA (0.27 mL). The mixture was stirred at room temperature
for 7 h. After extraction with ethyl acetate and washing with saturated aqueous ammonium
chloride, the organic layer was dried over sodium sulfate. The solvent was removed under
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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9
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reduced pressure, and the resulting precipitate was washed with chloroform to afford 13c (252
1
mg, 50% yield). HꢀNMR (CD
3
OD) δ: 8.17 (t, J = 1.72 Hz, 1H), 7.87 (dt, J = 1.72, 8.02 Hz,
1
2
1
H), 7.80 (m, 1H), 7.23 (t, J = 8.02 Hz, 1H), 3.38 (t, J = 6.87 Hz, 2H), 2.35 (t, J = 6.87 Hz,
+
H), and 1.66 (m, 4H). EIꢀMS m/z: 347 [M] , 288 (27), 260 (12), 231 (100), 203 (23), 116 (18),
04 (14), 76 (36).
2
,5-Dioxopyrrolidin-1-yl 3-(3-iodobenzamido)propanoate (14a)
To a solution of 13a (200 mg, 0.63 mmol) in DMF (4 mL) was added NHS (72 mg, 0.63
mmol) and WSCI·HCl (120 mg, 0.63 mmol). The mixture was stirred at room temperature for
7
h. After extraction with ethyl acetate and washing with saturated aqueous sodium
bicarbonate, the organic layer was dried over sodium sulfate. The solvent was removed under
reduced pressure, and the product 14a was purified by silica gel column chromatography
1
(
chloroform/methanol = 10/1, 93 mg, 36% yield). HꢀNMR (CDCl
3
, 500 MHz) δ: 8.18 (t, J =
1.72 Hz, 1H), 7.83 (d, J = 8.02 Hz, 1H), 7.76 (d, J = 8.02 Hz, 1H), 7.17 (t, J = 8.02 Hz, 1H),
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6.95 (broad s, 1H), 3.88 (m, 2H), 2.94 (t, J = 6.30 Hz, 2H), and 2.88 (s, 4H). EIꢀMS m/z: 416
+
[M] , 301 (41), 260 (13), 231 (100), 203 (24), 104 (12), 76 (40).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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49
50
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59
60
2,5-Dioxopyrrolidin-1-yl 4-(3-iodobenzamido)butanoate (14b)
To a solution of 13b (250 mg, 0.75 mmol) in DMF (5 mL) was added NHS (86 mg, 0.75
mmol) and WSCI·HCl (144 mg, 0.75 mmol). The mixture was stirred at room temperature
overnight. After extraction with ethyl acetate and washing with saturated aqueous sodium
bicarbonate, the organic layer was dried over sodium sulfate. The solvent was removed under
reduced pressure, and the product 14b was purified by silica gel column chromatography
1
(
chloroform/methanol = 10/1, 195 mg, 60% yield). HꢀNMR (CDCl
3
, 500 MHz) δ: 8.10 (t, J =
1
6
.72 Hz, 1H), 7.82 (d, J = 7.45 Hz, 1H), 7.73 (d, J = 7.45 Hz, 1H), 7.17 (t, J = 7.45 Hz, 1H),
.51 (broad s, 1H), 3.56 (m, 2H), 2.86 (s, 4H), 2.73 (t, J = 6.87 Hz, 2H), and 2.12 (tt, J = 6.87
+
Hz, 6.87 Hz, 2H). EIꢀMS m/z: 430 [M] , 315 (36), 274 (8), 231 (100), 203 (25), 104 (13), 84
(
26), 76 (42).
2
,5-Dioxopyrrolidin-1-yl 5-(3-iodobenzamido)pentanoate (14c)
To a solution of 13c (133 mg, 0.38 mmol) in DMF (4 mL) was added NHS (44 mg, 0.38
mmol) and WSCI·HCl (73 mg, 0.38 mmol). The mixture was stirred at room temperature for 3
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2
2
2
2
2
2
2
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3
3
3
3
3
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3
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4
4
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6
h. After extraction with ethyl acetate and washing with saturated aqueous sodium bicarbonate,
the organic layer was dried over sodium sulfate. The solvent was removed under reduced
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
pressure, and the product 14c was purified by silica gel column chromatography
1
(chloroform/methanol = 20/1, 83 mg, 49% yield). HꢀNMR (CDCl , 500 MHz) δ: 8.10 (t, J =
3
1.72 Hz, 1H), 7.81 (d, J = 8.02 Hz, 1H), 7.75 (d, J = 8.02 Hz, 1H), 7.17 (t, J = 8.02 Hz, 1H),
6
1
7
.36 (broad s, 1H), 3.50 (m ,2H), 2.86 (s, 4H), 2.69 (t, J = 6.87 Hz, 2H), 1.88 (m, 2H), and
+
.76 (m, 2H). EIꢀMS m/z: 444 [M] , 330 (7), 288 (18), 231 (100), 203 (20), 104 (14), 98 (16),
6 (32).
(
S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(3-(3-iodobenzamido)propanamido)hexan
oic acid (15a)
To a solution of 14a (110 mg, 0.26 mmol) in acetonitrile (3 mL) was added 6a (69 mg, 0.26
mmol) and DIPEA (91 ꢀL). The mixture was stirred at room temperature for 4 h. After
extraction with ethyl acetate and washing with saturated aqueous ammonium chloride, the
organic layer was dried over sodium sulfate. The solvent was removed under reduced pressure,
and the product 15a was purified by PTLC (chloroform/methanol = 10/1) followed by HPLC
1
using condition 1 (t
R
: 60 min, 19 mg, 14% yield). HꢀNMR（CD
3
OD, 500 MHz）δ: 8.17 (t, J =
1.72 Hz, 1H), 7.87 (d, J = 8.02 Hz, 1H), 7.81 (d, J = 8.02 Hz, 1H), 7.22 (t, J = 8.02 Hz, 1H),
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6.78 (s, 2H), 4.36 (m, 1H), 3.64 (m, 2H), 3.44 (t, J = 7.16 Hz, 2H), 2.56 (m, 2H), 1.66–1.89 (m,
+
2H), 1.57 (m, 2H), and 1.35 (m, 2H). ESIꢀMS m/z: 528 [M + H] .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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(S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(4-(3-iodobenzamido)butanamido)hexano
ic acid (15b)
To a solution of 14b (58 mg, 0.13 mmol) in acetonitrile (2 mL) was added 6a (35 mg, 0.13
mmol) and DIPEA (46 ꢀL). The mixture was stirred at room temperature for 4 h. After
extraction with ethyl acetate and washing with saturated aqueous ammonium chloride, the
organic layer was dried over sodium sulfate. The solvent was removed under reduced pressure,
and the product 15b was purified by PTLC (chloroform/methanol = 10/1) followed by HPLC
1
using condition 1 (t
R
: 62 min, 12 mg, 17% yield). HꢀNMR（CD
3
OD, 500 MHz）δ: 8.18 (t, J =
1
6
.72 Hz, 1H), 7.88 (d, J = 8.02 Hz, 1H), 7.82 (d, J = 8.02 Hz, 1H), 7.24 (t, J = 8.02 Hz, 1H),
.78 (s, 2H), 4.34 (m, 1H), 3.49 (t, J = 6.87 Hz, 2H), 3.41 (t, J = 6.87 Hz, 2H), 2.33 (t, J = 7.45
Hz, 2H), 1.92 (t, J = 6.87 Hz, 2H), 1.67–1.91 (m, 2H), 1.59 (m, 2H), and 1.36 (m, 2H).
+
ESIꢀMS m/z: 542 [M + H] .
(
S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(5-(3-iodobenzamido)pentanamido)hexan
oic acid (15c)
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