Novel multifunctional 18F-labelled PET tracer with prostate-specific membrane antigen-targeting and hypoxia-sensitive moieties

Journal Pre-proof 18 Novel multifunctional F-labelled PET tracer with prostate-specific membrane antigen-targeting and hypoxia-sensitive moieties Young-Do Kwon, Jun Young Lee, Minh Thanh La, Sun Joo Lee, Sun-Hwa Lee, Jeong Hoon Park, Hee-Kwon Kim PII:

S0223-5234(20)30066-0

DOI:

https://doi.org/10.1016/j.ejmech.2020.112099

Reference:

EJMECH 112099

To appear in:

European Journal of Medicinal Chemistry

Received Date: 10 November 2019 Revised Date:

19 January 2020

Accepted Date: 22 January 2020

Please cite this article as: Y.-D. Kwon, J.Y. Lee, M.T. La, S.J. Lee, S.-H. Lee, J.H. Park, H.-K. Kim, 18 Novel multifunctional F-labelled PET tracer with prostate-specific membrane antigen-targeting and hypoxia-sensitive moieties, European Journal of Medicinal Chemistry (2020), doi: https:// doi.org/10.1016/j.ejmech.2020.112099. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Novel multifunctional 18F-labelled PET tracer with prostatespecific membrane antigen-targeting and hypoxia-sensitive moieties

Young-Do Kwon a,b,1, Jun Young Lee c,1, Minh Thanh Lab, Sun Joo Lee d, Sun-Hwa Lee d, Jeong Hoon Park c,*, and Hee-Kwon Kim b,e,*

[a]

Department of Nuclear Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.

[b]

Department of Nuclear Medicine, Molecular Imaging & Therapeutic Medicine Research Center, Biomedical

Research Institute, Jeonbuk National University Medical School and Hospital, Jeonju 54907, Republic of Korea. [c]

Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212,

Republic of Korea. [d]

New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic

of Korea. [e]

Research Institute of Clinical Medicine of Jeonbuk National University-Biomedical Research Institute of

Jeonbuk National University Hospital, Jeonju 54907, Republic of Korea.

1

These authors contributed equally to this work.

* Corresponding author. Tel: +82 63 250 2768; Fax: +82 63 255 1172. E-mail address: [email protected] (H-K Kim). Postal address: Department of Nuclear Medicine, Jeonbuk National University Medical School, Jeonju 54907, Republic of Korea. Tel: +82 63 570 3571 E-mail address: [email protected] (J-H Park). Postal address: Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea.

Abstract Prostate cancer is one of the most frequently found cancers in men worldwide. Prostatespecific membrane antigen (PSMA) is typically highly expressed in prostate cancer, and the Glu-Urea-Lys (GUL) structure has recently received considerable attention as a key unit of PSMA-targeting agents. Additionally, one of the common characteristics of many solid tumors, such as prostate cancer, is hypoxia. In this study, novel multifunctional PSMA inhibitors containing a PSMA-targeting moiety either with or without a hypoxia-sensitve moiety (18F-PEG3-ADIBOT-2NI-GUL and 18F-PEG3-ADIBOT-GUL, respectively; ADIBOT: azadibenzocyclooctatriazole, 2NI: 2-nitroimidazole) were designed and synthesized, and their feasibility as PET tracers for prostate cancer imaging studies was examined. The compounds labeled with

18

F via the copper-free click reaction were stable in human serum and showed

nanomolar binding affinities in in vitro PSMA binding assays. Micro-PET and biodistribution studies indicated that both 18F-labelled inhibitors successfully accumulated in prostate cancer regions, and

18

F-PEG3-ADIBOT-2NI-GUL showed a 2-fold higher tumor-to-total non-target

organ ratio than that of

18

F-PEG3-ADIBOT-GUL, suggesting that the synergistic effects of

the PSMA-targeting GUL moiety and the hypoxia-sensitive 2-nitroimidazole moiety can increase tumor uptake of the novel PET tracers in prostate cancer. These findings suggest that this novel multifunctional PET tracer with an

18

F-labelled PSMA inhibitor and a 2-

nitroimidazole moiety is a potent candidate to provide better diagnosis of prostate cancer via PET imaging studies.

Keyword: Prostate cancer; Prostate-specific membrane antigen; fluorine-18; positronemission tomography; Cupper-free click reaction.

1. Introduction Prostate-specific membrane antigen (PSMA), a class II transmembrane glycoprotein, is a zinc-dependent enzyme that hydrolyzes N-acetylaspartylglutamate to glutamate and Nacetylaspartate [1]. Because its expression is significantly high in most prostate cancers and is augmented in proportion to tumor grade and aggressiveness [2-4], PSMA is a promising biomarker target for prostate cancer which is common in men regardless of race [5]. Several studies aimed to develop effective strategies to target PSMA: glutamate-urea-glutamate [6], cysteine-ureaglutamate [6], phosphoramidate peptidomimetic derivatives [7], poly-gamma glutamate derivatives [8], and glutamate-urea-lysine (GUL) [9-14]. Among them, GUL have been reported as promising inhibitors with high affinity to PSMA [9-14]. Recent studies have revealed that cancers including prostate cancers are significantly associated with hypoxia due to chaotic and poorly organized vasculature in the tumor [15-20]. Hypoxia appears in various solid tumors and can result in several impediments to treatment of cancer, such as chemotherapy- and radiotherapy-resistance [21,22]. Thus, it is important to identify hypoxic regions in cancers. Nitroimidazole, especially 2-nitroimidazole, is a widely used structure for detecting hypoxia [23-29], and numerous hypoxia-imaging probes utilizing 2-nitroimidazole, such as [18F]fluoromisonidazole ([18F]FMISO), [18F]fluoroazomycin arabinoside ([18F]FAZA), and [18F]fluoroetanidazole (([18F]FETA), have been developed and extensively evaluated [30-32]. Tissue hypoxia is quantified through reduction of 2nitroimidazole, which is regulated by nitroreductase in the cell, because it produces reactive intermediate species only under hypoxic conditions, and the species created irreversibly bind to intracellular components [33,34]. Thus, diagnostic agents containing 2-nitroimidazole accumulate in hypoxic areas but not in normoxic areas. Positron emission tomography (PET) is a widely used and powerful technique that allows visualization of biochemical events in human subjects using radioligands labelled with

positron emitters. Among the various positron emitters, fluorine-18 is the most widely used for PET due to its nuclear properties, such as half-life of 109.8 min, and its chemical properties. In this study, we prepared a novel PSMA inhibitor containing a GUL structure and 2-nitroimidazole, as well as a PSMA inhibitor bearing only a GUL structure without 2nitroimidazole (Fig. 1). Fluorine-18 was introduced into these tracers via a bioorthogonal strain-promoted click reaction, which is a copper-free version of 1,3-dipolar cycloaddition [35]. The feasibility of these novel compounds as PSMA PET tracers was evaluated in terms of in vitro binding properties in prostate cancer cells, as well as through in vivo micro-PET imaging and ex vivo biodistribution studies in mice bearing 22Rv1 tumors.

Fig. 1. The structures of PSMA inhibitors with (left panel) and without (right panel) 2nitroimdidazole regioisomers N

N

O

N

O2N

18

F

3

N

N

O

N

18 3

F

NH N

N H

O

O

N

O

O

H N

N

regioisomers

N

O

NH O

O

N H

NH CO2H

O

H N

NH

O CO2H O

O HO2C

N H

N H

HO2C

CO2H

18

F-PEG3-ADIBOT-2NI-GUL

N H

N H

CO2H

18

F-PEG3-ADIBOT-GUL

2. Results & Discussion 2.1. Synthesis of PSMA-targeting inhibitors Two fluorinated PSMA inhibitors, one with 2-nitroimdidazole and one without 2nitroimdidazole, were synthesized as shown in Scheme 1. Two PSMA-targeting ligands

based on the GUL structure (compound 2 without 2-nitroimidazole and compound 3 with 2nitroimidazole) were prepared from L-di-tert-butyl glutamate hydrochloride (1) according to our previously reported method [36, 37]. Copper-free click chemistry based on strain-promoted alkyne azide cycloaddition (SPAAC) is a useful radiolabeling technique due to fast and bioorthogonal conjugation. In the SPAAC reaction, the azide reacts with dibenzocyclooctyne (ADIBO) in the absence of metal to produce the target azadibenzocyclooctatriazoles (ADIBOTs) [38]. To perform the copper-free click reaction, a mesylated PEG linker bearing an azide group (5, N3-PEG3-OMs) was synthesized from 11-Azido-3,6,9-trioxa-1-undecanol via tosylation. Fluorination of compound 5 in the presence of TBAF produced the fluorinated PEG linker bearing an azide group (6, N3-PEG3-F). Azadibenzocyclooctyne-N-hydroxysuccinimidyl ester (7, ADIBO-NHS ester) was reacted with compound 2 or 3 to yield compounds 8 (ADIBO-GUL) and 10 (ADIBO-2NI-GUL, 2NI: 2-nitroimidazole), respectively. Copper-free click reactions were conducted between the azadibenzocyclooctyne groups of compound 8 or 10 and azide moiety of compound 6 to produce two fluorinated PSMA-targeting inhibitors 9 (F-PEG3-ADIBOT-GUL) and 11 (FPEG3-ADIBOT-2NI-GUL), respectively. The click reaction is known for yielding regioisomers [39].

N O

OH

O2N O

O H2N

[Ref 30]

OH

HN

[Ref 31]

O NH

O

NH

H2N

O

O

OH O NH

O

O

N H

OH

1 3

a

O

OH

HN

NH

OH 2

N3

O

O

HCl· H2N

O

N H

O

N

O

3 OH

O

N3

81%

4

3O

O S

b O

O

N3

75%

F

3

5

6 N O

O2N

OH O

O

O

N

O O

N H

OH

HN

N

+

O O

O H2N

O

O

NH

O

H2N

+

O

NH O

c

N

N

O

H N

N H

O NH

O

O

N H

O

OH

O

O

O

H N

N

NH

N H

O

OH

N

OH

HN

F

N N N regioisomers

N

O N H

O OH

76%

O2N

3

O

N H

79% O

O

OH O NH

10

d

O

OH

HN

NH O

8

d

O

N

OH

HN O

O OH

66%

OH O2N

O

O

3

64% O

O NH

N H

7

2

c

OH

HN

N

O OH

OH

O O

N H

O NH

O N H

7

N

O

N

H N O

O

O N H

O NH O OH

O

N

OH

regioisomers F O

N N N

3

9 (F-PEG3-ADIBOT-GUL)

O

O

O

H N

N

O

NH

N H

NH O

11 (F-PEG3-ADIBOT-2NI-GUL)

N H

OH

HN O

O NH O OH

Scheme 1. Preparation of PSMA-targeting inhibitors. Reagents and conditions: (a) methanesulfonyl chloride, triethylamine, dichloromethane, rt, 3 h. (b) tetrabutylammonium fluoride, tert-amyl alcohol, 80oC, overnight. (c) triethylamine, dimethyl sulfoxide, rt, 12 h. (d) Compound 6, triethylamine, dimethyl sulfoxide, rt, 1 h.

2.2. In vitro PSMA inhibition assay and lipophilicity In vitro PSMA inhibition assays were performed using a previously reported fluorescencebased NAALADase assay method [13]. The new fluorinated PSMA inhibitors had nano-

molar binding affinities (Ki values) of 6.5 nM for F-PEG3-ADIBOT-GUL and 17 nM for FPEG3-ADIBO-2NI-GUL (Table 1). Inhibition curves are shown in the Supplementary Data (Fig. S1). In comparing the multifunctional PSMA inhibitor (F-PEG3-ADIBOT-2NI-GUL) with the control PSMA inhibitor without a 2-nitroimidazole moiety (F-PEG3-ADIBOT-GUL), the in vitro assay results suggest that the presence of the 2-nitroimidazole moiety resulted in only a modest decrease (~2.6-fold) in affinity toward PSMA. Log P was also measured to determine the lipophilicity of F-PEG3-ADIBOT-GUL and FPEG3-ADIBOT-2NI-GUL. Although F-PEG3-ADIBOT-2NI-GUL showed a slightly higher lipophilicity value than F-PEG3-ADIBOT-GUL, the Log P values of -2.78 and -0.48, respectively, suggest that both compounds have hydrophilic characteristics.

Table 1. Physical and chemical characteristic of new ligands.

a b

Compound

M.W.

Ki (nM)a

Log Pb

F-PEG3-ADIBOT-GUL

983.04

6.5 ± 0.8

-2.78

F-PEG3-ADIBOT-2NI-GUL

1208.24

17.2 ± 0.4

-0.48

Ki values are presented as mean ± SEM. Log P was measured by a conventional shake flask method.

2.3. Modeling docking study To understand the binding affinity of the novel PSMA-inhibitors on PSMA, a docking model is performed. The result clearly showed that the affinity of F-PEG3-ADIBOT-GUL was significantly higher than that of F-PEG3-ADIBOT-2NI-GUL. Based on Fig. 2, the carboxylate group in GUL part is a crucial structure for PSMA inhibition by binding with Zinc atom. The F-PEG3-ADIBOT-2NI-GUL containing 2-nitroimidazole decreased the affinity due to its hindrance. As showed in B and D of Fig. 2, this moiety prevented the GUL part to approach the active site of the protein (docking score: -10.7 kcal/mol for F-PEG3ADIBOT-GUL, and -7.5 kcal/mol for F-PEG3-ADIBOT-2NI-GUL).

Fig. 2. Predicted binding modes of compound F-PEG3-ADIBOT-GUL and F-PEG3-ADIBO2NI-GUL with PSMA (PDB:4X3R).

2.4. Radiosynthesis Following the in vitro inhibition assay, radiosynthesis of the novel PSMA-inhibitor precursors using fluorine-18 produced in a cyclotron was performed in two steps (Scheme 2). Initially, compound 5 (N3-PEG3-OMs) was reacted with the fluorine-18 complex (18F/kryptofix 2.2.2./K2CO3) in acetonitrile to yield the

18

F-labelled azide-PEG compound

[18F]6 (N3-PEG3-18F) with an 81 ± 11% radiochemical yield (RCY) (n = 4; decay-corrected and isolated; radiochemical purity (RCP) = 95%). Copper-free click reactions (SPAAC reactions) between the azide moiety of [18F]6 (N3-PEG3-18F) and the ADIBO moiety of compound 8 (ADIBO-GUL) or 10 (ADIBO-2NI-GUL) were conducted for 15 min to yield the final

18

F-labelled products [18F]9 (18F-PEG3-ADIBOT-GUL; 48 ± 5% n = 3; decay-

corrected and isolated; RCP = 98%) and [18F]11 (18F-PEG3-ADIBOT-2NI-GUL; 58 ± 7% RCY; n = 3; decay-corrected and isolated; RCP = 99%), respectively. In the subsequent preparative HPLC purification, the product peaks for

18

18

F-PEG3-ADIBOT-GUL and

F-

PEG3-ADIBOT-2NI-GUL appeared after approximately 24 min and 20 min, respectively, without co-elution of any impurities. The identity of these isolated radiofluorinated products was confirmed using co-injection with the corresponding authentic compound (Fig. S2). In additions, the specific activity of the products was 53.2 GBq/µmol for [18F]9 and 45.7 GBq/µmol for [18F]11, respectively.

A. [18F]-Labelling of N3-PEG3-OTs (5) 18 -

N3

O

3O

O S

F /K 2.2.2./K2CO3 MeCN

O

N3

O o

18 3 F

110 C, 15 min [18F]6

5

B. Cu-free click reaction of [18F]-PEG3-N3 with compound 8 and 10 O

18

3

N N N regioisomers

rt, 15 min N3

O [18F]6

18 3 F

OH

HN

F O

8 EtOH-H2O (1:1)

OH

O N

O N H

O

H N

O

O NH O

N H

O

OH

[18F]9 (18F-PEG3-ADIBOT-GUL)

10 EtOH-H2O (1:1)

N O2N

18

F

regioisomers N N N O 3

N O

O

O

H N O

O

N

rt, 15 min

NH

N H

NH O

N H

OH

OH

HN O

O NH O OH

[18F]11 (18F-PEG3-ADIBOT-2NI-GUL)

Scheme 2. Radiofluorination of 18F-PEG3-ADIBOT-GUL and 18F-PEG3-ADIBOT-2NI-GUL.

2.5. In vitro stability studies

In vitro stability tests of the

18

F-labelled products (18F-PEG3-ADIBOT-GUL and

18

F-PEG3-

ADIBOT-2NI-GUL) were carried out in human serum albumin using radio-TLC analysis to examine the stability of the radiofluorinated PET tracers. After 18F-PEG3-ADIBOT-GUL and 18

F-PEG3-ADIBOT-2NI-GUL were incubated in human serum solution at 37 oC, evaluation

of their stability was conducted at various time points. Both 18

18

F-PEG3-ADIBOT-GUL and

F-PEG3-ADIBOT-2NI-GUL had greater than 97% radiochemical purity over the 2h

incubation (Fig. 3). These results indicate that the new PSMA inhibitors were suitable for the subsequent in vivo animal PET study. In addition, in vitro cell uptake studies in PSMA+ 22Rv1 and PSMA‒ PC3 using 18F-PEG3ADIBOT-2NI-GUL and 18F-PEG3-ADIBOT-GUL were conducted, and found that 18F-PEG3ADIBOT-2NI-GUL and while

18

18

F-PEG3-ADIBOT-GUL showed higher uptake in 22Rv1 cells,

F-PEG3-ADIBOT-2NI-GUL and

18

F-PEG3-ADIBOT-GUL showed lower uptake in

PC3 cells (Fig. S3). The result indicated that in vitro cell uptake was affected by PSMA targeting in prostate cancer.

Fig. 3. In vitro stability of human serum albumin.

18

F-PEG3-ADIBOT-GUL and

18

F-PEG3-ADIBOT-2NI-GUL in

2.6. PET imaging study and Biodistribution The behaviors of the new PSMA inhibitors (18F-PEG3-ADIBOT-GUL and

18

F-PEG3-

ADIBOT-2NI-GUL) were assessed using in vivo and ex vivo experiments in mice bearing 22Rv1 tumors. In vivo PET image scans were carried out after intravenous tail injection of 18

F-PEG3-ADIBOT-GUL or

18

F-PEG3-ADIBOT-2NI-GUL in a 22Rv1 xenograft tumor

model. Micro-PET imaging studies demonstrated that

18

F-PEG3-ADIBOT-GUL and

18

F-

PEG3-ADIBOT-2NI-GUL were highly accumulated in the 22Rv1 tumors in mice (Fig. 4 and 5). Ex vivo biodistribution studies were next performed after harvesting the body organs to investigate the pharmacokinetic natures of ADIBOT-2NI-GUL (Table 2 and 3).

18

18

F-PEG3-ADIBOT-GUL and

18

F-PEG3-

F-PEG3-ADIBOT-2NI-GUL exhibited PSMA-

dependent uptake in tumors and had a 1 h tumor uptake of 0.814 ± 0.134%, which reached 0.694 ± 0.097% at 2 h. Non-target organs, such as the heart, lungs, liver, spleen, and stomach, showed high uptake at 15 min and were rapidly cleared within 1 h. Additionally, the intestines, pancreas, and fat showed high uptake at 30 min, which was subsequently rapidly cleared. The higher uptake of

18

F-PEG3-ADIBOT-GUL compared to

18

F-PEG3-ADIBOT-

2NI-GUL in the kidneys at 30 min was presumably due to the 2-fold greater affinity of 18FPEG3-ADIBOT-GUL for PSMA, which is known to be present in the proximal renal tubules [40,41]. The sufficient clearance in the kidneys was observed at 2 h. Ex vivo biodistribution studies using

18

F-PEG3-ADIBOT-GUL showed no noticeable

differences in uptake pattern. Tumor uptake of the

18

F-labeled ligand without 2-

nitroimidazole was observed as 0.646 ± 0.097 at 1 h. Liver and intestinal uptake of 18F-PEG3ADIBOT-GUL was rapidly cleared and was lowest at 2 h, which was similar to what was observed for

18

F-PEG3-ADIBOT-2NI-GUL. However, some non-target organs, such as the

stomach, intestines, pancreas, and kidneys, showed higher uptake of

18

F-PEG3-ADIBOT-

GUL at 1 h compared to 18F-PEG3-ADIBOT-2NI-GUL. The tumor-to-muscle ratio of

18

F-PEG3-ADIBOT-2NI-GUL was 54% higher compared to

that of 18F-PEG3-ADIBOT-GUL at 1 h (7.324 vs. 4.748), but the tumor-to-liver ratio of 18FPEG3-ADIBOT-2NI-GUL was 30% higher compared to that of 18F-PEG3-ADIBOT-GUL at 1 h (5.048 vs. 3.865). In addition, the ratio of localization in tumors compared to the total in non-target organs (i.e., sum of uptake values of the heart, lungs, liver, spleen, stomach, kidneys, muscles, etc.) was approximately 2-fold higher in

18

F-PEG3-ADIBOT-2NI-GUL

compared to 18F-PEG3-ADIBOT-GUL at 1 h. These results indicate that 18F-PEG3-ADIBOT2NI-GUL had higher uptake in prostate cancers and lower non-specific uptake compared to 18

F-PEG3-ADIBOT-GUL, suggesting that a 2-nitroimidazole hypoxia-sensitive moiety of

new PSMA inhibitors helped higher accumulation in hypoxic regions. In addition, PET studies using [18F]FMISO, a PET radiotracer for imaging hypoxia, was conducted to examine the accumulation of

18

F-PEG3-ADIBOT-GUL and

18

F-PEG3-

ADIBOT-2NI-GUL in hypoxic regions, and provided that the location of the tumors found by PET imaging using

18

F-PEG3-ADIBOT-2NI-GUL and

18

F-PEG3-ADIBOT-GUL was

consistent with the location of hypoxia region identified by[18F]FMISO (Fig. S4).

Table 2. Biodistribution of 18F-PEG3-ADIBOT-2NI-GUL ([18F]11) in 22Rv1 xenograft mice after intravenous injection (n = 6)

blood heart lung liver spleen stomach intestine pancreas kidney muscle fat bone brain tumor

15 min 1.062 ± 0.142 0.687 ± 0.167 0.837 ± 0.164 0.645 ± 0.104 1.084 ± 0.149 0.924 ± 0.171 1.551 ± 0.298 0.951 ± 0.108 17.035 ± 3.114 0.378 ± 0.102 0.815 ± 0.149 1.181 ± 0.238 0.062 ± 0.021 0.806 ± 0.174

30 min 0.582 ± 0.158 0.545 ± 0.132 0.658 ± 0.081 0.504 ± 0.078 0.658 ± 0.111 0.485 ± 0.098 1.564 ± 0.224 1.657 ± 0.268 23.701 ± 1.422 0.275 ± 0.065 0.998 ± 0.234 0.852 ± 0.095 0.069 ± 0.023 0.915 ± 0.186

60 min 0.224 ± 0.047 0.143 ± 0.042 0.251 ± 0.034 0.158 ± 0.028 0.322 ± 0.057 0.150 ± 0.021 0.272 ± 0.034 0.117 ± 0.021 11.191 ± 1.084 0.111 ± 0.012 0.104 ± 0.016 0.234 ± 0.029 0.041 ± 0.009 0.814 ± 0.134

60 minb 0.358 ± 0.058 0.078 ± 0.012 0.175 ± 0.031 0.142 ± 0.028 0.144 ± 0.031 0.102 ± 0.022 0.245 ± 0.038 0.088 ± 0.014 1.852 ± 0.356 0.101 ± 0.018 0.943 ± 0.173 0.185 ± 0.028 0.032 ± 0.005 0.483 ± 0.068

120 min 0.131 ± 0.022 0.104 ± 0.011 0.141 ± 0.022 0.104 ± 0.023 0.254 ± 0.037 0.102 ± 0.021 0.141 ± 0.056 0.080 ± 0.015 4.797 ± 0.101 0.062 ± 0.015 0.087 ± 0.016 0.151 ± 0.031 0.025 ± 0.006 0.694 ± 0.097

a

Values expressed are in % ID/g ± standard deviation. N = 4 for all tissues. bBiodistribution of blocking study from [18F]11 and DCIBzL 60 min after intravenous injection (n = 4).

Fig. 4. Animal PET imaging of 22Rv1 xenografted mice 1 h after intravenous injection of 18 F-PEG3-ADIBOT-2NI-GUL through the tail vein. (A) Uptake of 18F-PEG3-ADIBOT-2NIGUL (left panel). (B) Blocking study using co-injection of DCIBzL (right panel) at 1 h.

Table 3. Biodistribution of 18F-PEG3-ADIBOT-GUL ([18F]9) in 22Rv1 xenograft mice after intravenous injection (n = 4)

blood heart lung liver spleen stomach intestine pancreas kidney muscle fat bone brain tumor

15 min 0.482 ± 0.085 0.214 ± 0.033 0.365 ± 0.046 0.456 ± 0.061 1.482 ± 0.298 0.520 ± 0.121 0.163 ± 0.007 0.179 ± 0.021 29.781 ± 4.278 0.186 ± 0.028 0.390 ± 0.061 0.458 ± 0.037 0.036 ± 0.003 0.559 ± 0.084

30 min 0.312 ± 0.073 0.143 ± 0.041 0.351 ± 0.046 0.330 ± 0.012 1.121 ± 0.194 1.216 ± 0.216 3.294 ± 0.165 0.251 ± 0.027 34.246 ± 2.948 0.197 ± 0.077 1.368 ± 0.231 0.386 ± 0.055 0.041 ± 0.006 0.639 ± 0.073

60 min 0.182 ± 0.028 0.098 ± 0.019 0.163 ± 0.044 0.167 ± 0.023 0.371 ± 0.088 0.390 ± 0.062 1.331 ± 0.331 0.378 ± 0.059 18.763 ± 3.545 0.136 ± 0.008 0.711 ± 0.101 0.413 ± 0.101 0.041 ± 0.009 0.646 ± 0.097

60 minb 0.287 ± 0.054 0.034 ± 0.007 0.045 ± 0.007 0.148 ± 0.022 0.064 ± 0.009 0.105 ± 0.018 0.847 ± 0.138 0.274 ± 0.035 1.842 ± 0.147 0.075 ± 0.011 0.104 ± 0.021 0.321 ± 0.052 0.022 ± 0.005 0.078 ± 0.011

120 min 0.112 ± 0.007 0.052 ± 0.019 0.107 ± 0.009 0.087 ± 0.005 0.166 ± 0.038 0.255 ± 0.033 0.475 ± 0.051 0.123 ± 0.016 4.721 ± 0.542 0.052 ± 0.018 0.073 ± 0.006 0.358 ± 0.051 0.028 ± 0.009 0.517 ± 0.085

a

Values expressed are in % ID/g ± standard deviation. N = 4 for all tissues. bBiodistribution of blocking study from [18F]9 and DCIBzL 60 min after intravenous injection (n = 4).

Fig. 5. Animal PET imaging of 22Rv1 xenografted mice 1 h after intravenous injection of 18 F-PEG3-ADIBOT-GUL through the tail vein. (A) Uptake of 18F-PEG3-ADIBOT-GUL (left panel). (B) Blocking study using co-injection of DCIBzL (right panel) at 1 h.

2.7. Blocking studies Blocking studies in mice bearing 22Rv1 xenograft tumors were also carried out to investigate the roles of the GUL group and 2-nitroimidazole structures in targeting prostate cancer. Although high accumulation of

18

F-PEG3-ADIBOT-GUL was

observed in PSMA-expressing 22Rv1 tumors after injection of the new PSMA inhibitor alone, greater than 85% of that uptake was blocked by co-injection of

18

F-

PEG3-ADIBOT-GUL with an excess amount of (S)-2-(3-((S)-1-carboxy-5-(4iodobenzamido)pentyl)ureido)pentanedioic acid (DCIBzL), a previously reported PSMA-targeting ligand [13] (Fig. 5, Fig. S5 and Table 3). This indicates that the GUL group of

18

F-PEG3-ADIBOT-GUL has binding specificity to PSMAs on prostate

cancer. After co-injection of excess blocking compound with 18F-PEG3-ADIBOT-2NIGUL, which contains the hypoxia-sensitive 2-nitroimidazole moiety, the uptake of 18FPEG3-ADIBOT-2NI-GUL in 22Rv1 tumors remained high (Fig. 4, and Table 2). This suggests that the 2-nitroimidazole group may help detect PSMA-negative hypoxic regions. The result demonstrated that the PSMA inhibitor bearing 2-nitroimidazole (18F-PEG3-ADIBOT-2NI-GUL) was successfully located in the hypoxic tumor regions; however, the PSMA inhibitor without 2-nitroimidazole (18F-PEG3-ADIBOT-GUL) did not accumulate in the hypoxic tumor locations. Blocking studies using FMISO, a hypoxia marker, were also carried out. But the high accumulation of ADIBOT-GUL and

18

F-PEG3-

18

F-PEG3-ADIBOT-2NI-GUL was still observed in PSMA-

expressing 22Rv1 tumors (Table S1 and Table S2). The result suggested that PSMA targeting is more important factor for prostate cancer study rather than hypoxia effect.

3. Conclusion

In conclusion, we designed, synthesized, and evaluated novel

18

F-labelled PSMA

inhibitors with and without the 2-nitroimidazole moiety. Stability studies indicated that the novel

18

F-labelled PSMA inhibitors were stable in human serum. In vitro

competitive binding assays showed that these inhibitors exhibited high affinity to prostate cancer. Micro-PET and ex vivo biodistribution studies demonstrated that both PSMA inhibitors showed higher uptake in prostate cancer than in the rest of the body. In particular, the

18

F-labelled PSMA inhibitor with 2-nitroimidazole (18F-PEG3-

ADIBOT-2NI-GUL) had a better tumor-to-total non-target organ ratio than the control 18

F-labelled PSMA inhibitor without 2-nitroimidazole (18F-PEG3-ADIBOT-GUL).

These observations suggest that the novel multifunctional strategy utilizing the synergistic effects of tumor-targeting and a hypoxia-sensitive moiety could be a promising approach to develop effective diagnosis and therapy agents for prostate cancers.

4. Experimental All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Azadibenzocyclooctyne-N-hydroxysuccinimidyl ester (ADIBO-NHS ester) and 11-azido-3,6,9-trioxa-1-undecanol were purchased from FutureChem (Seoul, Republic of Korea). Thin-layer chromatography (TLC) was performed using Merck aluminum plates with silica gel 60 F254, and TLC spots were visualized using UV light at 254 nm. For purification of non-radioactive fluorinated products, high-performance liquid chromatography (HPLC) was performed using a Spectra System (Thermo Scientific, Waltham, MA) with a semi-preparative column (YMC-Pack ODS-A, C18 silica gel, 5 µm, 10 × 250 mm). 1H nuclear magnetic resonance (NMR) spectra were obtained on a JEOL JNM-ECA 600 spectrometer (600

MHz) installed in the Center for University-Wide Research Facilities (CURF) at Chonbuk National University, Jeonju, Korea. Chemical shifts (δ units, ppm) were reported relative to residual protonated solvent resonance, and the coupling constants (J) were measured in Hz. Electrospray ionization (ESI) low-resolution mass spectrometry (LRMS) was performed by the Mass Spectrometry Service of the CURF at

Chonbuk

National

University,

Jeonju,

Korea.

ESI

high-resolution

mass

spectrometry (HRMS) was carried out by the Mass Spectrometry Service at Korea Basic Science Institute, Ochang, Republic of Korea. In vitro prostate-specific membrane antigen (PSMA) inhibition studies were performed using NAALADase assays with Amplex Red Glutamic Acid kits at the New Drug Development Center of Daegu-Gyeongbuk Medical Innovation Foundation. [18F]Fluoride was produced using a GE PETtrace cyclotron (16.4 MeV) via irradiation of an [18O]H2O target. To purify the radiofluorinated products, radio-HPLC was performed with an SP930D pump (Young-Lin Inc., Republic of Korea) using a semi-preparative column (YMC-Pack ODS-A, C18 silica gel, 5 µm, 10 × 250 mm). The eluant was observed using a UV730D UV detector (Young-Lin Inc., Republic of Korea) and an FC-3200 high energy gamma detector (Bioscan, USA). A CRC-15R radioisotope calibrator (Capintec, Inc., USA) was utilized for radioactivity measurements. Radio-TLC was performed using an AR-2000 radio-TLC Imaging Scanner (Bioscan, USA). Positronemission tomography (PET) imaging was acquired using a micro-PET scanner (Inveon, Siemens Healthineers, Germany). Biodistribution studies were carried out using a 1480 WIZARD gamma counter (Perkin Elmer, USA). All animal experiments were performed with approval of the Institutional Animal Care and Use Committee (IACUC) of Chonbuk National University (IACUC Number: CBNU 2016-18). In addition, the experiments were conducted in compliance with the guidelines of the

Care and Use of Animals for Scientific Purposes of the Ethics Committee of the Chonbuk National University Laboratory Animal Center and in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011). 4.1. Chemistry 4.1.1. Compound 5 (N3-PEG3-OMs) To a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol (0.43 g, 2.0 mmol) and triethylamine (0.83 mL, 5.9 mmol) in dichloromethane (10 mL) were added methanesulfonyl chloride (0.26 mL, 3.3 mmol) at 0 oC. After being stirred for 3 h at room temperature, the reaction was quenched with H2O (10 mL). The organic phase was separated, and the aqueous phase was extracted with dichloromethane (10 mL). The combined organic phase was dried over with Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexane = 3/1) on silica gel to yield compound 5 (0.47 g, 81%) as a yellowish oil. 1H NMR (400 MHz, CDCl3) δ 4.39–4.12 (m, 2H), 3.81– 3.78 (m, 2H), 3.71–3.67 (m, 10H), 3.42–3.39 (t, J = 5.2 Hz, 2H), 3.09 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 70.71, 70.68, 70.63 (2C), 70.06, 69.25, 69.03, 50.69, 37.70; HRMS (ESI) m/z (M+H)+ calcd for C9H20N3O6S = 298.1073, found 298.1076.

4.1.2. Compound 6 (N3-PEG3-F) Compound 5 (0.30 g, 1.0 mmol) and tetrabultylammonium fluoride hydrate (0.53 g, 2.0 mmol) were dissolved in 2-methyl-2-butanol (10 mL). After being stirred for 10 h at 80 oC, the mixture was diluted with ethyl acetate (20 mL) and washed with H2O (20 mL). The organic phase was dried over with Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexane = 1/2) on silica gel to yield compound 6 (0.17 g, 75%) as a yellowish oil. 1H NMR (400 MHz, CDCl3)

δ 4.65–4.63 (m, 1H), 4.54–4.52 (m, 1H), 3.81–3.78 (m, 1H), 3.74–3.68 (m, 11H), 3.42–3.39 (t, J = 4.8 Hz, 2H);

13

C MR (100 MHz, CDCl3) δ 83.98 & 82.31 (d, JC-F = 167.6 Hz, 1C),

70.82, 70.70 (2C), 70.49, 70.29, 70.04, 50.71; HRMS (ESI) m/z (M+H)+ calcd for C8H17FN3O3 = 222.1254, found 222.1258.

4.1.3. Compound 8 (ADIBO-GUL) To a solution of 2 (17.0 mg, 32.8 µmol) in DMSO (300 µL), triethylamine (20.0 µL, 144 µmol) was added, followed by ADIBO-NHS ester 7 (13.7 mg, 28.9 µmol). After being stirred for 12 h at room temperature, the mixture was purified using HPLC (mobile phase, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 min = 20% B, 3 min = 20% B, 10 min = 40% B, 30 min = 40% B; 2 mL/min) to yield compound 8 (16.0 mg, 64.0%) at tR = 19.5 min. 1

H-NMR (400 MHz, D2O) δ 7.54 (d, J = 7.3 Hz, 1H), 7.36–7.33 (m, 4H), 7.32-7.29

(m, 1H), 7.28–7.24 (m, 1H), 7.19 (dd, J = 7.5, 1.3 Hz, 1H), 4.95 (d, J = 14.4 Hz, 1H), 3.84 (dd, J = 7.9, 5.0 Hz, 2H), 3.66 (d, J = 14.4 Hz, 1H), 3.05–2.91 (m, 6H), 2.24–2.10 (m, 6H), 2.07 (t, J = 7.9 Hz, 4H), 1.86–1.82 (m, 1H), 1.74–1.55 (m, 4H), 1.52–1.40 (m, 1H), 1.37–1.30 (m, 2H), 1.25–1.15 (m, 2H); HRMS (ESI) m/z (M + H)+ calcd for C38H47N6O11 = 763.3303, found 763.3297.

4.1.4. Compound 9 (F-PEG3-ADIBOT-GUL) Compound 8 (8.0 mg, 10.5 µmol) and compound 6 (4.6 mg, 21.0 µmol) were dissolved in DMSO (300 µL). After being stirred for 1 h at room temperature, the mixture was purified using HPLC as described for compound 9 to yield compound 7 (4.7 mg, 79.1%) at tR = 17.5 min. 1H-NMR (400 MHz, D2O) δ 7.69–7.51 (m, 1H), 7.47–7.35 (m, 3H), 7.26–7.17 (m, 3H), 7.14–7.04 (m, 1H), 5.70 (d, J = 17.0 Hz, 1H), 4.58–4.33 (m, 4H), 4.28–3.92 (m, 2H), 3.86– 3.83 (m, 2H), 3.76–3.65 (m, 1H), 3.63–3.58 (m, 1H), 3.54–3.23 (m, 10H), 3.08–2.92 (m, 6H),

2.35–2.27 (m, 4H), 2.10–2.02 (m, 4H), 1.94–1.80 (m, 1H), 1.74–1.52 (m, 5H), 1.51–1.41 (m, 1H), 1.37–1.30 (m, 2H), 1.22–1.15 (m, 2H);

19

F-NMR (400 MHz, D2O) δ -122.4; HRMS

(ESI) m/z (M + Na)+ calcd for C46H62FN9O14Na = 1006.4298, found 1006.4294.

4.1.5. Compound 10 (ADIBO-2NI-GUL) To a solution of 3 (18.0 mg, 24.2 µmol) in DMSO (300 µL), triethylamine (17.0 µL, 122 µmol) was added, followed by ADIBO-NHS ester 7 (11.5 mg, 24.3 µmol). After being stirred for 12 h at room temperature, the mixture was purified using HPLC as described for compound 8 to yield compound 10 (15.8 mg, 66.0%, regioisomer) at tR = 21.0 min. 1H-NMR (400 MHz, D2O) δ 7.54 (d, J = 7.3 Hz, 1H), 7.38–7.15 (m, 4H), 7.31–7.27 (m, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.21 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.98 (s, 1H), 4.96 (d, J = 14.4 Hz, 1H), 4.47–4.37 (m, 2H), 3.87–3.78 (m, 3H), 3.66 (dd, J = 14.4, 2.6 Hz, 1H), 3.59–3.49 (m, 2H), 3.03–2.93 (m, 4H), 2.90 (t, J = 6.4 Hz, 2H), 2.24–2.13 (m, 6H), 2.08 (t, J = 8.4 Hz, 2H), 1.87–1.83 (m, 1H), 1.74–1.66 (m, 1H), 1.60–1.53 (m, 1H), 1.50–1.40 (m, 2H), 1.37–1.30 (m, 5H), 1.21–1.07 (m, 4H); HRMS (ESI) m/z (M + H)+ calcd for C46H58N11O14 = 988.4165, found 988.4162.

4.1.6. Compound 11 (F-PEG3-ADIBOT-2NI-GUL) Compound 10 (8.0 mg, 8.12 µmol) and compound 6 (3.6 mg, 16.3 µmol) were dissolved in DMSO (300 µL). After being stirred for 1 h at room temperature, the mixture was purified using HPLC as described for compound 9 to yield compound 11 (7.4 mg, 75.7%, regioisomer) at tR = 18.5 min. 1H-NMR (400 MHz, D2O) δ 7.72–7.52 (m, 1H), 7.48–7.37 (m, 3H), 7.30– 7.20 (m, 4H), 7.13 (m, 1H), 6.99 (s, 1H), 5.71 (d, J = 17.0 Hz, 1H), 4.57–4.33 (m, 6H), 4.26– 3.93 (m, 2H), 3.86–3.70 (m, 4H), 3.64–3.23 (m, 12H), 3.06–2.92 (m, 6H), 2.33–2.30 (m, 4H), 2.08 (t, J = 8.2 Hz, 2H), 2.04–1.83 (m, 2H), 1.74–1.66 (m, 1H), 1.59–1.40 (m, 4H), 1.33 (m,

5H), 1.19–1.08 (m, 4H);

19

F-NMR (400 MHz, D2O) δ -122.4; HRMS (ESI) m/z (M + Na)+

calcd for C54H73FN14O17Na = 1231.5160, found 1231.5161.

4.2. In vitro PSMA inhibition assay Ki values were determined by the modified fluorescence-based assay previously reported [13]. LNCaP cell lysates were prepared using lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and 1% Triton X-100). The cell lysates were incubated with the inhibitors in the presence of 20 µM N-acetylaspartylglutamate (NAAG) at 37 °C for 2 h. The amount of glutamate produced from NAAG hydrolysis was measured by incubating with a working solution from the AmplexTM Red Glutamic Acid/Glutamate Oxidase assay kit (Invitrogen, USA) at 37 °C for 1 h. Fluorescence was acquired using a Synergy™ NEO HTS multi-mode microplate reader (BioTek Instruments, Inc., USA) with excitation at 530 nm and emission at 590 nm. Inhibition curves were produced from the fluorescence measurements, and IC50 values of the ligands were determined as the concentration at which enzymatic activity of the lysates was inhibited by half. Enzyme inhibitory constants (Ki values) were derived from the IC50 values. Data analysis was performed using GraphPad Prism software, version 7.00 (GraphPad Software, USA).

4.3. Radiofluorination of

18

F-PEG3-ADIBOT-GUL and

18

F-PEG3-ADIBOT-2NI-

GUL. To a reaction vial containing 7.5 mg of Kryptofix 2.2.2 and 1.5 mg of K2CO3 in an acetonitrile–water mixture (1 mL, 9:1 v/v), aqueous [18F]fluoride was added. The solvent was removed by purging with nitrogen gas at 100 oC, and the residue was dried using azeotropic distillation with acetonitrile (3 mL x 3). To the resultant residue, a solution of 2-(2-(2-(2-

azidoethoxy)ethoxy)ethoxy)ethyl methanesulfonate (5; 3.0 mg) in 0.5 mL of acetonitrile was added. The reaction mixture was heated at 110 oC with stirring for 15 min and cooled to room temperature. The crude mixture was injected into a semi-preparative HPLC column system for purification of [18F]6 (mobile phase, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 min = 5% B, 2 min = 5% B, 45 min = 35% B, 55 min = 75% B; 3 mL/min). For identification of the

18

F-labeled product, the collected HPLC fraction for [18F]6 (tR = 38.0

min) was co-injected with cold compound [19F]6. After drying the solution of [18F]6, the residue was solubilized with an ethanol–water mixture (0.15 mL, 1:1 v/v). To the resulting solution, a solution of compound 8 or 10 (0.5 mg) in an ethanol–water mixture (0.05 mL, 1:1 v/v) was added. The reaction mixture was stirred at room temperature for 15 min and purified using semi-preparative HPLC ([18F]9: mobile phase, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 min = 20% B, 3 min = 20% B, 10 min = 35% B, 30 min = 35% B; 2 mL/min; tR = 23.8 min. [18F]11: mobile phase, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 min = 20% B, 3 min = 20% B, 10 min = 40% B, 30 min = 40% B; 2 mL/min; tR = 20.3 min.). The purified radiofluorinated product ([18F]9 or [18F]11) was dried, made isotonic with sodium chloride, and passed through a 0.20-µm membrane filter into a sterile vial for subsequent in vitro and in vivo experiments.

4.4. Cell lines and tumor models The 22Rv1 cells and PC3 were acquired from professor Lee of Seoul National University Hospital, Seoul, Republic of Korea, and used without authentication. The 22Rv1 cells and PC3 were grown in RPMI 1640 medium containing 10% heatinactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cell cultures were maintained in a humidified incubator with 5% CO2 at 37 °C. Six-week-old female BALB/c nude mice (NARA Biotech, Republic of Korea) were implanted

subcutaneously with 1 × 106 22Rv1 cells in the right side of the back. The mice were used in in vivo imaging and ex vivo biodistribution studies when the xenografted tumors reached 7–10 mm in diameter. The animals used in these experiments were handled using the Guidelines for Animal Experimentation. This study was approved by the Ethics Committee of Chonbuk National University Animal Care and Use (IACUC number: CBNU 2016-18).

4.5. In vivo PET imaging and ex vivo biodistribution studies Before scans, mice were anesthetized with 3% isoflurane and thereafter sustained with 2.5% isoflurane in an atmosphere of 80% O2. Mice bearing 22Rv1 tumors were subjected to tailvein injection of an 18F-labeled ligand, and PET imaging was performed. For blocking studies to confirm the binding specificity of GUL (18F-PEG3-ADIBOT-GUL) and the synergistic effects on ligand accumulation in tumors (18F-PEG3-ADIBOT-GUL), the PSMA-targeting ligand DCIBzL was pre-injected to mice at 100 mg per kg body weight. For ex-vivo biodistribution studies, blood samples were collected from the hearts of anesthetized mice from 15 to 120 min postinjection. In addition, organs and tumors were collected and analyzed using a γ-counter. The percentage of injected dose per gram of tissue (ID/g%) was calculated by comparing the tissue counts to the initial dose.

Acknowledgments This research was supported by Basic Science Research Program through the National Research

Foundation

of

Korea

(NRF)

funded

by

the

Ministry

of

Education

(2018R1D1A1B07047572). This work was supported by Fund of Biomedical Research Institute, Jeonbuk National University Hospital.

Conflict of interest The authors declare no conflict of interest.

Appendix A. Supplementary data Supplementary data related to this article can be found at.

References [1] J.R. Mesters, C. Barinka, W. Li, T. Tsukamoto, P. Majer, B.S. Slusher, J. Konvalinka, R. Hilgenfeld, Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer, EMBO J. 25 (2006) 1375−1384. [2] G.P. Murphy, A.-A. Elgamal, S.L. Su, D.G. Bostwick, E.H. Holmes, Current Evaluation of the Tissue Localization and Diagnostic Utility of Prostate Specific Membrane Antigen, Cancer 83 (1998) 2259−2269. [3] J.S. Ross, C.E. Sheehan, H.A.G. Fisher, R.P. Kaufman, Jr., P. Kaur, K. Gray, I. Webb, G.S. Gray, R. Mosher, B.V.S. Kallakury, Correlation of Primary Tumor Prostate-Specific Membrane Antigen Expression with Disease Recurrence in Prostate Cancer, Clin. Cancer Res. 9 (2003) 6357−6362. [4] S. Minner, C. Wittmer, M. Graefen, G. Salomon, T. Steuber, A. Haese, H. Huland, C. Bokemeyer, E. Yekebas, J. Dierlamm, S. Balabanov, E. Kilic, W. Wilczak, R. Simon, G. Sauter, T. Schlomm, High Level PSMA Expression Is Associated With Early PSA Recurrence in Surgically Treated Prostate Cancer, Prostate 71 (2011) 281−288. [5] R.L. Siegel, K.D. Miller, A. Jemal, Cancer Statistics, 2019, Ca-Cancer J. Clin. 69 (2019) 7−34. [6] X. Wang, S.S. Huang, W.D.W. Heston, H. Guo, B.-C. Wang, J. P. Basilion, Development of Targeted Near-Infrared Imaging Agents for Prostate Cancer. Mol. Cancer Ther. 13 (2014)

2595−2606. [7] S.E. Lapi, H. Wahnishe, D. Pham, L.Y. Wu, J.R. Nedrow-Byers, T. Liu, K. Vejdani, H.F. VanBrocklin, C.E. Berkman, E.F. Jones, Assessment of an

18

F-Labeled Phosphoramidate

Peptidomimetic as a New Prostate-Specific Membrane Antigen−Targeted Imaging Agent for Prostate Cancer. J. Nucl. Med. 50 (2009) 2042−2048. [8] P. Warren, L. Li, W. Song, E. Holle, Y. Wei, T. Wagner, X. Yu, In Vitro Targeted Killing of Prostate Tumor Cells by a Synthetic Amoebapore Helix 3 Peptide Modified with Two γlinked Glutamate Residues at the COOH Terminus Cancer Res. 61 (2001) 6783−6787. [9] Y. Chen, M. Pullambhatla, S.R. Banerjee, Y. Byun, M. Stathis, C. Rojas, B.S. Slusher, R.C. Mease, M.G. Pomper, Synthesis and Biological Evaluation of Low Molecular Weight Fluorescent Imaging Agents for the Prostate-Specific Membrane Antigen, Bioconjugate Chem. 23 (2012) 2377−2385. [10] K. Bao, J.H. Lee, H. Kang, G.K. Park, G. El Fakhri, H.S. Choi, PSMA-targeted contrast agents for intraoperative imaging of prostate cancer, Chem. Commun. 53 (2017) 1611−1614. [11] M. Schäfer, U. Bauder-Wüst, K. Leotta, F. Zoller, W. Mier, U. Haberkorn, M. Eisenhut, M. Eder, A dimerized urea-based inhibitor of the prostate-specific membrane antigen for 68

Ga-PET imaging of prostate cancer, EJNMMI Res. 2 (2012) 23.

[12] M. Benešová, U. Bauder-Wüst, M. Schäfer, K.D. Klika, W. Mier, U. Haberkorn, K. Kopka, M. Eder, Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors, J. Med. Chem. 59 (2016) 1761−1775. [13] Y. Chen, C.A. Foss, Y. Byun, S. Nimmagadda, M. Pullambhatla, J.J. Fox, M. Castanares, S.E. Lupold, J.W. Babich, R.C. Mease, M.G. Pomper, Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)-Based Ureas as Imaging Agents for Prostate Cancer, J. Med. Chem. 51 (2008) 7933−7943.

[14] K.P. Maresca, S.M. Hillier, F.J. Femia, D. Keith, C. Barone, J.L. Joyal, C.N. Zimmerman, A.P. Kozikowski, J.A. Barrett, W.C. Eckelman, J.W. Babich, A Series of Halogenated Heterodimeric Inhibitors of Prostate Specific Membrane Antigen (PSMA) as Radiolabeled Probes for Targeting Prostate Cancer, J. Med. Chem. 52 (2009) 347−357. [15] A.G. Anastasiadis, B.C. Stisser, M.A. Ghafar, M. Burchardt, R. Buttyan, Tumor Hypoxia and the Progression of Prostate Cancer, Current Urology Reports 3 (2002) 222−228. [16] M. Höckel, P. Vaupel, Tumor Hypoxia: Definitions and Current Clinical, Biologic, and Molecular Aspects, J. Natl. Cancer Inst. 93 (2001) 266−276. [17] B. Movsas, J.D. Chapman, E.M. Horwitz, W.H. Pinover, R.E. Greenberg, A.L. Hanlon, R. Iyer, G.E. Hanks, Hypoxic regions exist in human prostate carcinoma, Urology 53 (1999) 11−18. [18] B. Movsas, J.D. Chapman, A.L. Hanlon, E.M. Horwitz, W.H. Pinover, R.E. Greenberg, C. Stobbe, G.E. Hanks, Hypoxia in human prostate carcinoma: an Eppendorf PO2 study. Am J Clin Oncol 24 (2001) 458−461. [19] M. Milosevic, P. Chung, C. Parker, R. Bristow, A. Toi, T. Panzarella, P. Warde, C. Catton, C. Menard, A. Bayley, M. Gospodarowicz, R. Hill, Androgen withdrawal in patients reduces prostate cancer hypoxia: implications for disease progression and radiation response. Cancer Res 67 (2007) 6022−6025. [20] S.R. McKeown, Defining normoxia, physoxia and hypoxia in tumors-implications for treatment response. Br J Radiol 87 (2014) 20130676. [21] J.-P. Cosse, C. Michiels, Tumour Hypoxia Affects the Responsiveness of Cancer Cells to Chemotherapy and Promotes Cancer Progression, Anti-Cancer Agents Med. Chem. 8 (2008) 790−797. [22] J.M. Brown, W.R. Wilson, Exploiting Tumour Hypoxia in Cancer Treatment, Nat. Rev. Cancer 4 (2004) 437−447.

[23] A. Nunn, K. Linder, H.W. Strauss, Nitroimidazoles and imaging hypoxia, Eur. J. Nucl. Med. 22 (1995) 265−280. [24] Y. Joyard, V. Le Joncour, H. Castel, C.B. Diouf, L. Bischoff, C. Papamicaël, V. Levacher, P. Vera, P. Bohn, Synthesis and biological evaluation of a novel

99m

Tc labeled 2-

nitroimidazole derivative as a potential agent for imaging tumor hypoxia, Bioorg. Med. Chem. Lett. 23 (2013) 3704−3708. [25] S. Kizaka-Kondoh, H. Konse-Nagasawa, Significance of nitroimidazole compounds and hypoxia-inducible factor-1 for imaging tumor hypoxia, Cancer Sci. 100 (2009) 1366−1373. [26] M. Takasawa, R.R. Moustafa, J.-C. Baron, Applications of Nitroimidazole In Vivo Hypoxia Imaging in Ischemic Stroke, Stroke 39 (2008) 1629−1637. [27] B.G.M. Youssif, K. Okuda, T. Kadonosono, O.I.A.R. Salem, A.A.M. Hayallah, M.A. Hussein, S. Kizaka-Kondoh, H. Nagasawa, Development of a Hypoxia-Selective NearInfrared Fluorescent Probe for Non-invasive Tumor Imaging, Chem. Pharm. Bull. 60 (2012) 402−407. [28] S.K. Chitneni, G.M. Palmer, M.R. Zalutsky, M.W. Dewhirst, Molecular Imaging of Hypoxia, J. Nucl. Med. 52 (2011) 165−168. [29] M.A. Varia, D.P. Calkins-Adams, L.H. Rinker, A.S. Kennedy, D.B. Novotny, W.C. Fowler, J.A. Raleigh, Pimonidazole: A Novel Hypoxia Marker for Complementary Study of Tumor Hypoxia and Cell Proliferation in Cervical Carcinoma, Gynecol. Oncol. 71 (1998) 270−277. [30] J.S. Rasey, W.-J. Koh, J.R. Grierson, Z. Grunbaum, K.A. Krohn, Radiolabeled fluoromisonidazole as an imaging agent for tumor hypoxia, Int. J. Radiat. Oncol., Biol., Phys. 17 (1989) 985−991. [31] G. Reischl, W. Ehrlichmann, C. Bieg, C. Solbach, P. Kumar, L.I. Wiebe, H.-J. Machulla, Preparation of the hypoxia imaging PET tracer [18F]FAZA: reaction parameters and

automation, Appl. Radiat. Isot. 62 (2005) 897−901. [32] J.S. Rasey, P.D. Hofstrand, L.K. Chin, T.J. Tewson, Characterization of [18F]Fluoroetanidazole, a New Radiopharmaceutical for Detecting Tumor Hypoxia, J. Nucl. Med. 40 (1999) 1072−1079. [33] D.I. Edwards, Nitroimidazole drugs - action and resistance mechanisms, J. Antimicrob. Chemother. 31 (1993) 9−20. [34] J.L. Bolton, R.A. McClelland, Kinetics and Mechanism of the Decomposition in Aqueous Solutions of 2-(Hydroxyamino)imidazoles, J. Am. Chem. Soc. 111 (1989) 8172−8 181. [35] M. Pretze, D. Pietzsch, C. Mamat, Recent Trends in Bioorthogonal Click-Radiolabeling Reactions Using Fluorine-18, Molecules 18 (2013) 8618−8665. [36] Y.-D. Kwon, H.-J. Chung, S.-J. Lee, S.-H. Lee, B.-H. Jeong, H.-K. Kim, Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation, Bioorg. Med. Chem. Lett. 28 (2018) 572−576. [37] Y.-D. Kwon, J.-M. Oh, M.T. La, H.-J. Chung, S.-J. Lee, S. Chun, S.-H. Lee, B.-H. Jeong, H.-K. Kim, Synthesis and Evaluation of Multifunctional Fluorescent Inhibitors with Synergistic Interaction of Prostate-Specific Membrane Antigen and Hypoxia for Prostate Cancer, Bioconjugate Chem. 30 (2019) 90−100. [38] H.L. Kim, K. Sachin, H.J. Jeong, W. Choi, H.S. Lee, D.W. Kim, F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging, ACS Med. Chem. Lett. 6 (2015) 402−407. [39] M.J. Jacobs, G. Schneider, K.G. Blank, Mechanical Reversibility of Strain-Promoted Azide–Alkyne Cycloaddition Reactions. Angew. Chem. Int. Ed. 55 (2016) 2899−2902. [40] D.J. Bacich, J.T. Pinto, W.P. Tong, W.D. Heston, Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane

antigen/NAALADase/folate hydrolase. Mamm. Genome 12 (2001) 117−123. [41] P.D. Gregor, J.D. Wolchok, V. Turaga, J.-B. Latouche, M. Sadelain, D. Bacich, W.D.W. Heston, A.N. Houghton, H.I. Scher, Induction of autoantibodies to syngeneic prostatespecific membrane antigen by xenogeneic vaccination. Int. J. Cancer 116 (2005) 415−421.

Highlights New 18F-labelled PSMA inhibitor with 2-nitroimidazole was synthesized. Ki value of 11 was 17.2 nM. In vivo PET study of [18F]11 showed its high uptake in prostate cancer. Blocking study confirmed selective binding of [18F]11.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Young-Do Kwon , Jun Young Lee, Minh Thanh La, Sun Joo Lee , Sun-Hwa Lee , Jeong Hoon Park , and Hee-Kwon Kim declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. There are no potential competing interests.