Synthesis and Bioevaluation of Novel [18F]FDG-Conjugated 2-Nitroimidazole Derivatives for Tumor Hypoxia Imaging

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00075

Hypoxia imaging can guide tumor treatment and monitor changes in hypoxia during treatment. However, there is still no ideal hypoxia imaging agent for clinical applications. In this study, two novel 2-nitromidazole derivatives were synthesized and directly radiolabeled by [¹⁸F]FDG in high radiochemical yield and excellent radiochemical purity. Cell experiments, biodistribution, and positron emission tomography (PET) imaging studies were also conducted in mice-bearing S180 or OS732 tumors. [¹⁸F]FDG-2NNC2ON [(2R,3S,4R,E)-2-¹⁸F-fluoro-3,4,5,6-tetrahydroxyhexanal O-3-(2-(2-nitro-1H-imidazole-1-yl)ethylamino)-2-oxopropyl oxime] and [¹⁸F]FDG-2NNC5ON [(2R,3S,4R,E)-2-¹⁸F-fluoro-3,4,5,6-tetrahydroxyhexanal-O-3-(5-(2-nitro-1H-imidazole-1-yl)pentylamino)-2-oxopropyl oxime] can be cleared from the blood quickly and specifically target hypoxic tumor cells. The uptake of the probes by hypoxic cells gradually increases with time. After 4 h, the uptake value of [¹⁸F]FDG-2NNC2ON in hypoxic cells is 3.2 times higher than that in normoxia cells. In contrast, there is no difference in the uptake of [¹⁸F]FDG between hypoxic cells and normoxia cells. Biodistribution resulting from two tumor models indicate that the uptake values of the two radiotracers in the tumor are higher at 1 h than those at 2 and 4 h. At 1 and 2 h, the tumors are clearly observed on the PET images and the imaging features of [¹⁸F]FDG-2NNC5ON and [¹⁸F]FDG-2NNC2ON are distinct from those of [¹⁸F]FDG. Compared with [¹⁸F]FDG-2NNC5ON, [¹⁸F]FDG-2NNC2ON has a higher proportion of renal excretion, lower digestive tract uptake, and better imaging contrast because of its higher hydrophilicity. At 2 h, [¹⁸F]FDG-2NNC2ON shows a good tumor-to-blood (T/B) ratio, tumor-to-muscle ratio based on biodistribution (Bio-T/M ratio), and tumor-to-muscle ratio based on regions of interest on the PET images [region of interest (ROI)-T/M ratio] in the two tumor models (T/B, Bio-T/M, and ROI-T/M ratios are 3.2, 2.6, and 3.9 in the S180 tumor model and are 3.4, 4.2, and 4.6 in the OS732 tumor model, respectively). The imaging features visualized with autoradiography mostly coincided with the positive areas of HIF1α staining by immunofluorescence. Meanwhile, the biodistribution study and PET imaging revealed that the uptake of the radiotracers in the tumor cannot be competed by 5% glucose, confirming that [¹⁸F]FDG-2NNC2ON targets the hypoxic regions of the tumors instead of targeting tumors through the glucose metabolism pathway. These results suggest that the new 2-nitroimidazole derivative conjugated with [¹⁸F]FDG, [¹⁸F]FDG-2NNC2ON, has potential as an imaging agent for hypoxia.

Full text of DOI:10.1021/acs.molpharmaceut.9b00075

Subscriber access provided by The University of Melbourne Libraries
Article
Synthesis and bioevaluation of novel 18FDG-conjugated
2-nitroimidazole derivatives for tumor-hypoxia imaging
Xianteng Yang, Fan Wang, Hua Zhu, Zhi Yang, and TaiWei Chu
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00075 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 11, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 42
Molecular Pharmaceutics
1
2
3
4
Graphic abstract
5
6
7
8
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Headline: Two new 2-nitroimidazole derivatives conjugated with 18FDG, 18FDG-2NNC2ON and
18FDG-2NNC5ON, was successfully designed, synthesized and bioevaluated. 18FDG-2NNC2ON has
potential as a hypoxic imaging agent for clinical application.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 42
1
2
3
4
5
6
1
2
Synthesis and bioevaluation of novel 18FDG-conjugated 2-nitroimidazole
derivatives for tumor hypoxia imaging
7
8
9
3
Xianteng Yang1,2#, Fan Wang3#, Hua Zhu4, Zhi Yang4*, Taiwei Chu3*
1. Guizhou University School of Medicine, Guiyang, Guizhou, 550025, China ;
2. Department of Orthopaedics, Guizhou Provincial People’s Hospital, Guiyang, Guizhou,
550002, China;
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
3. Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, College of
Chemistry and Molecular Engineering, Peking University, Beijing 100871, China;
4. Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing),
Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142,
China.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
#：Xianteng Yang and Fan Wang have contributed equally to this work.
*
Corresponding author: Yang Zhi, E-mail: pekyz@163.com; Taiwei Chu, E-mail:
twchu@pku.edu.cn
Abstract: Hypoxia imaging can guide tumor treatment and monitor changes in hypoxia during
treatment. However, there is still no ideal hypoxia imaging agent for clinical applications. In this study,
two novel 2-nitromidazole derivatives were synthesized and directly radiolabeled by [18F]FDG in high
radiochemical yield and excellent radiochemical purity. Cell experiments, biodistribution and PET
imaging studies were also conducted in mice bearing S180 or OS732 tumors. [18F]FDG-2NNC2ON
((2R,3S,4R,E)-2-18F-fluoro-3,4,5,6-tetrahydroxyhexanal O-3-(2-(2-nitro-1H-imidazol-1-yl)ethylamino)
-2-oxopropyl
oxime)
and
[18F]FDG-2NNC5ON
((2R,3S,4R,E)-2-18F-fluoro-3,4,5,6
-tetrahydroxyhexanal-O-3-(5-(2-nitro-1H-imidazol-1-yl)pentylamino)-2-oxopropyl oxime) can be
1
ACS Paragon Plus Environment

Page 3 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
cleared from the blood quickly and specifically target hypoxic tumor cells. The uptake of the probes by
hypoxic cells gradually increases with time. After 4 h, the uptake value of [18F]FDG-2NNC2ON in
hypoxic cells is 3.2 times higher than that in normoxia cells. In contrast, there is no difference in the
uptake of [18F]FDG between hypoxic cells and normoxia cells. Biodistribution results from two tumor
models indicate that the uptake values of the two radiotracers in the tumor are higher at 1 h than at 2
and 4 h. At 1 and 2 h, the tumors are clearly observed on the PET images, and the imaging features of
[18F]FDG-2NNC5ON and [18F]FDG-2NNC2ON are distinct from those of [18F]FDG. Compared with
[18F]FDG-2NNC5ON, [18F]FDG-2NNC2ON has a higher proportion of renal excretion, lower digestive
tract uptake and better imaging contrast due to its higher hydrophilicity. At 2 h, [18F]FDG-2NNC2ON
shows a good tumor-to-blood (T/B) ratio, tumor-to-muscle ratio based on biodistribution (Bio-T/M
ratio), and tumor-to-muscle ratio based on regions of interest on the PET images (ROI-T/M ratio) in the
two tumor models (T/B, Bio-T/M and ROI-T/M ratios are 3.2, 2.6 and 3.9 in the S180 tumor model
and are 3.4, 4.2 and 4.6 in the OS732 tumor model, respectively). The imaging features visualized with
autoradiography mostly coincided with the positive areas of HIF1α staining by immunofluorescence.
Meanwhile, the biodistribution study and PET imaging revealed that the uptake of the radiotracers in
the tumor cannot be competed by 5% glucose, confirming that [18F]FDG-2NNC2ON targets the
hypoxic regions of the tumors instead of targeting tumors through the glucose metabolism pathway.
These results suggest that the new 2-nitroimidazole derivative conjugated with [18F]FDG,
[18F]FDG-2NNC2ON, has potential as an imaging agent for hypoxia.
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Key words: 2-nitroimidazole, [18F]FDG, tumor, hypoxia imaging
2
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 42
1
2
3
4
5
6
7
1
2
1. Introduction
Hypoxia is a typical feature of solid tumors and one of the main reasons for the failure of
tumor-targeted radiotherapy and chemotherapy 1-3. Therefore, identification of tumor hypoxia is crucial
for the treatment of tumors. With the development of molecular imaging technology, there is no doubt
that radioactive imaging with a hypoxia-targeted agent has more advantages compared with traditional
invasive detection. Hypoxia imaging of tumors by positron emission tomography (PET) can clearly
show the hypoxic areas in tumors and improve the clinical efficacy of treatment strategies. However,
the hypoxia imaging quality is determined by the performance of the hypoxia imaging agent. So far,
there has not been an ideal hypoxia imaging agent used in the clinic yet. It is important to develop a
hypoxia-targeted tracer with excellent performance to guide clinical treatment.
8
9
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Many hypoxia agents have been developed, most of which are based on the nitroimidazole
4
analogues . In particular, 2-nitroimidazole derivatives have been considered to be more suitable for
detecting hypoxia because 2-nitroimidazole has a lower single electron reduction potential (SERP)
5
value and may be more effectively restricted to hypoxic cells . 18F is one of the most important
radionuclides for PET imaging, and it causes low amounts of radiation side effect to the human body
because of its suitable half-life (t1/2, 109.7 min). Currently, many 18F-labeled 2-nitroimidazole
derivatives have been prepared for studying hypoxia in tumors 6-18. The representative 18F-labeled
2-nitroimidazole derivative is [18F]FMISO 9, which has been used to detect tumor clinically. However,
[18F]FMISO is limited by its drawbacks, such as the low ratio between the radioactivity in the hypoxic
tissue and in the background tissue, high lipophilicity, long blood retention time, high radioactivity
concentration in the digestive tract; thus, further improvements are required for effective imaging 19. In
an attempt to overcome some of the above drawbacks, second- and third-generation 2-nitroimidazole
3
ACS Paragon Plus Environment

Page 5 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
radiotracers, such as [18F]FETNIM 20, [18F]EF5 21, and [18F]HX4 16, have been developed. However, the
qualities of these hypoxic imaging agents are not better than that of [18F]FMISO. Glycosylation is one
of the most commonly used modification strategies that can increase the hydrophilicity of the probe,
increase the proportion of renal elimination, reduce the physiological uptake of the probe in the
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
digestive system, reduce the clearance of the reticuloendothelial system, accelerate blood clearance,
22
6
and shorten the time interval between injection of the tracer to imaging. In 1999, Kumar et al
.
7
synthesized [18F]FAZA successfully, and the results of the bioevaluation indicated that [18F]FAZA had
23
8
potential as a hypoxia imaging agent
Patt et al. synthesized [18F]FDG-2-NIm in 2002;
.
18
9
[18F]FDG-2-NIm was obtained by the direct coupling of a 2-nitroimidazole derivative to [18F]FDG,
but it cannot specifically target the hypoxic region of the tumor and failed to provide tumor images.
Recently, Cao et al. synthesized several novel PEG-modified nitroimidazole tracers by rapid
10
11
12
13
14
15
16
17
18
19
20
21
22
6
6
click-reactions . Unfortunately, these molecules did not exhibit specific targeting ability for tumor
cells in hypoxic conditions, and no images of the hypoxic regions of tumors have been reported.
Therefore, 18F-labeled nitroimidazole hypoxia imaging agents are worthy of further study.
For the 18F-labeled hypoxic imaging agents, an efficient, rapid, and convenient method is needed
to overcome the challenges of short half-lives and harsh conditions. Compared with direct
introduction of 18F, the strategy for labeling small molecules with 18F may be a more convenient and
efficient method. [18F]FDG (2-18F-fluoro-2-deoxy-D-glucose) has been widely used in clinic for PET
imaging, and it is an ideal small-molecule for indirect 18F-labeling. Some researchers have reported a
method for the radiosynthesis of 18F-labeled peptides using [18F]FDG by chemoselective oxime
formation 24-27. At the same time, 18F-fluorine labeling of a tracer with [18F]FDG as an auxiliary group
can achieve effects similar to those of glycosylation modifications. It is hypothesized that this method
4
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 42
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
can improve synthesis efficiency, increase probe hydrophilicity, overcome some challenges of
nitroimidazole reagents, and enhance the contrast and usefulness of PET images. However, this
hypothesis has not been investigated yet.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Herein, two novel aminooxy-functionalized 2-nitroimidazole derivatives were synthesized and
directly radiolabeled by [18F]FDG (Figure 1). Then, the radiotracers were evaluated by corresponding
experiments to evaluate their potential for hypoxia imaging.
O
O
H
H
N
N
N
O
N
N
O
N
O
N
N
O
H
H
O
O
NO2
NO2
2NNC5ON
2NNC2ON
OH
OH
OH
OH
H
H
N
HO
HO
HO
HO
N
N
N
N
18F
18F
N
N
O
O
N
O
O
NO2
O2N
[
18F]FDG-2NNC2ON
[
18F]FDG-2NNC5ON
8
9
Figure 1. The structures of the compounds.
10
11
12
13
14
15
16
17
18
2. Materials and methods
All chemical reagents and solvents were purchased from commercial suppliers and were used
directly without any further purification. [18F]FDG was obtained from the Peking University School of
Oncology and Beijing Institute for Cancer Research. FDG was purchased from Shanghai Yuanye
Biotechnology Co., Ltd (Shanghai, China). DMEM, fetal bovine serum (FBS), and human serum
albumin (HSA) were purchased from Invitrogen (CA, USA). Rabbit anti-HIF-1 alpha (bs-0737R) was
purchased from Bioss (Beijing, China). Dye-conjugated goat anti-rabbit IgG (Alexa Fluor® 488,
ab150077) was purchased from Abcam (Shanghai, China). Human osteosarcoma OS732 and mouse
S180 cells were provided by the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing,
5
ACS Paragon Plus Environment

Page 7 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
1
2
China). The H NMR and 13C NMR spectra were obtained on a Bruker spectrometer (Bruker,
Germany). Mass spectrometry (including high resolution mass spectra, HRMS) was acquired on a
Fourier-transform ion cyclotron resonance mass spectrometer (Bruker, Germany). IR spectra were
obtained on Fourier-transform infrared spectrometer (ThermoFisher, USA). The radiochemical yield
(RCY) and radiochemical purity (RCP) were determined by a radio-HPLC system (Agilent
Technologies 1200 series of high-performance liquid chromatography systems, California, USA)
equipped with a UV absorption detector and a B-Fc-1000 HPLC radioactivity detector (Bioscan, USA).
The radioactivity of the samples was measured by FH463A- Automatic Scaler (Nuclear Instrument
Factory of Beijing, China) and FJ374 well gamma detector (Nuclear Instrument Factory of Beijing,
China). A JPS-605-dissolved oxygen meter was purchased from REX Instrument Factory of Shanghai
Precision & Scientific Instrument Co., LTD. (Shanghai, China). For autoradiography, the compounds
were exposed overnight to a high-sensitivity imaging film (BAS-SR2025, Fuji Photo Film, Japan). PET
imaging was conducted by a Super Argus PET/CT system (Sedecal, Spain).
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2.1 Chemical synthesis
The compounds 1-10 were prepared according to the method reported with a little modification 24,
28-30, and synthetic procedures are shown in the Scheme 1. The detailed procedures and corresponding
characterization data of the other compounds are provided in the Supporting Information, except for
compound 7-10.
6
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 42
1
2
3
4
5
6
7
8
9
O
O
a
b
N ( )
N
K
Br
n
O
O
1:n=1
2:n=4
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
c
N
N
.
N ( )
N
(
)
NH2 HCl
N
n
n
NO2
NO2
5:n=1
6:n=4
O
3:n=1
4:n=4
O
O
N
O
d
N
(
)
N
N
O
n
H
H
NO2
7:n=1,2NNC2ON
8:n=4,2NNC5ON
OH
OH
e
H
HO
HO
N
N
)
(
N
F
n
N
O
O
NO2
9:n=1, FDG-2NNC2ON
10:n=4, FDG-2NNC5ON
1
2
Scheme 1. The synthesis of compounds 1-10. Reagents and conditions: (a) BrCH2CH2Br or
3
4
5
6
BrCH2CH2CH2CH2CH2Br, K2CO3, MeCN, Reflux, 24 h; (b) 2-nitroimidazole, K2CO3, MeCN, Reflux;
(c) NH2NH2 H2O, ethanol, Reflux, HCl.Et2O; (d) Boc-Aoa, DCM, Et3N, EDCl, HOBt; and (e) FDG,
.
0.1M HCl, MeCN.
7
ACS Paragon Plus Environment

Page 9 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
Synthesis
of
compound
7
(2NNC2ON,
tert-butyl
2-(2-(2-nitro-1H-imidazol-1-yl)ethylamino)-2-oxoethoxycarbamate). Boc-Aoa (Boc-aminooxy
acetic acid, 0.49 g, 2.33 mmol), EDCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride,
0.54 g, 2.8 mmol), HOBt (1-hydroxybenzotriazole hydrate, 0.43 g, 2.8 mmol), and Et3N (0.71 g, 6.99
mmol) were dissolved in 30 mL DCM. The mixture was stirred for 1 h at 0 °C. Afterwards, compound
5 (0.47 g, 2.44 mmol) was added, and the mixture was stirred overnight at room temperature. Then, the
organic phase was washed by 20 mL 1 M HCl, 20 mL saturated sodium bicarbonate solution, and 20
mL saturated saline, consecutively. The organic phase was dried by Na2SO4 and evaporated under
reduced pressure. The crude product was purified by chromatography (ethyl acetate:petroleum ether =
3:2) to obtain compound 7 as a yellow solid (0.47 g, yield: 61%).
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1H NMR (400 MHz, DMSO): δ 10.17 (s, 1H), 8.08 (s, 1H), 7.51 (s, 1H), 7.14 (s, 1H), 4.48 (s, 2H),
4.09 (s, 2H), 3.57 (s, 2H), 1.41 (s, 9H).
13C NMR (101 MHz, CDCl3): δ 169.98, 158.15, 144.75, 128.26, 126.68, 83.62, 76.35, 49.01, 39.04,
28.08.
HRMS-ESI: [M+H+] calculated for C12H20N5O6, 330.14136; found, 330.14066.
+
HRMS-ESI: [M+NH4 ] calculated for C12H23N6O6, 330.14136; found, 347.16710.
HRMS-ESI: [M+Na+] calculated for C12H19N5NaO6, 352.12266; found, 352.12330
IR (KBr)/cm-1: 3334(ν NH), 3173(ν NH), 2981, 2928, 1717(ν C=O), 1650(ν C=O), 1556, 1536.1502,
1482, 1459, 1437, 1417, 1396, 1356 ,1279, 1256, 1161, 1117, 1089, 1062, 867, 847, 832, 808, 777, 667
650, 636 594.
Synthesis of compound
8
(2NNC5ON, tert-butyl 2-(5-(2-nitro-1H-imidazol-1-yl)
pentylamino)-2-oxoethoxycarbamate). Following a similar procedure to that used in the synthesis of
8
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 42
1
2
3
4
5
6
7
8
9
1
2
compound 7, compound 8 was prepared from compound 6 (1.00 g, 4.26 mmol), Boc-Aoa (0.78 g, 4.06
mmol), EDCl (0.93 g, 4.87 mmol), HOBt (0.75 g, 4.87 mmol), and Et3N (1.23 g, 12.18 mmol).
Compound 8 was a yellow oil (1.24 g, yield: 78.5%).
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
1H NMR (400 MHz, DMSO): δ 10.31 (s, 1H), 8.02 (s, 1H), 7.68 (s, 1H), 7.18 (s, 1H), 4.37 (t, J = 7.3
Hz, 2H), 4.13 (s, 2H), 3.13 (q, J = 6.7 Hz, 2H), 1.86 – 1.69 (m, 2H), 1.48 (dd, J = 14.5, 7.1 Hz, 2H),
1.41 (s, 9H), 1.32 – 1.25 (m, 2H).
5
6
7
13C NMR (101 MHz, CDCl3): δ 169.06, 158.03, 128.40, 125.99, 83.21, 76.20, 50.14, 38.42, 30.06,
28.67, 28.09, 23.56.
8
9
HRMS-ESI: [M+H+] calculated for C15H26N5O6, 372.18831; found, 372.18670.
+
10
11
12
13
14
15
16
17
18
19
20
21
HRMS-ESI: [M+NH4 ] calculated for C15H29N6O6, 389.21486; found, 389.21400.
HRMS-ESI: [M+Na+] calculated for C15H25N5NaO6, 394.17025; found, 394.16974
IR (KBr)/cm-1: 3297(ν NH), 3115(ν NH), 2975, 2933, 2867, 1721(ν C=O), 1655(ν C=O), 1537, 1484,
1459, 1394, 1358, 1276, 1251, 1161, 1109, 1049, 975, 920, 835, 776, 743, 650, 633, 585, 516.
Synthesis
of
compound
9
(FDG-2NNC2ON,
2-((((2R,3S,4R,E)-2-fluoro-3,4,5,6-tetrahydroxyhexylidene)amino)oxy)-N-(2-(2-nitro-1H-imidazol
-1-yl)ethyl)acetamide).Compound 7 (2.0 mg, 0.006 mmol) and FDG (3.0 mg, 0.016mmol ) were
dissolved in 2 mL of ACN, then 200 μL of 0.1 M HCl was added. The mixture was heated for 1h at
100 °C. After cooling to room temperature, the solvent was removed under vacuum, and the crude
product was analyzed by the HPLC. The analytical conditions were as follows: (A): water (containing
0.1% TFA); and (B) acetonitrile. The elution gradient: 0-10 min 95% A-40% A; and 10-15 min 40%
9
ACS Paragon Plus Environment

Page 11 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
A-95% A. The product was detected at 254 nm using UV absorption detector. The product was
collected (retention time 5.0-6.0 min) and identified by HRMS directly.
HRMS-ESI: [M+H+] calculated for C13H21FN5O8, 394.13742; found, 394.13708.
HRMS-ESI: [M+Na+] calculated for C13H20FN5NaO8, 416.11936; found, 416.12971.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis
of
compound
10
(FDG-2NNC5ON,
2-((((2R,3S,4R,E)-2-fluoro-3,4,5,6-tetrahydroxyhexylidene)amino)oxy)-N-(5-(2-nitro-1H-imidazol
-1-yl)pentyl)acetamide) Following similar synthetic procedure and same separating condition to that
used in the synthesis of compound 9, compound 10 was prepared from compound 8 (2.0 mg, 0.005
mmol), FDG (3.3 mg, 0.018 mmol). The product was detected at 254 nm using UV absorption detector.
The product was collected (retention time 7.0-8.0 min) and identified by HRMS directly.
HRMS-ESI: [M+H+] calculated for C16H27FN5O8, 436.18437; found, 436.18387.
10
11
12
13
14
15
16
17
18
19
20
21
22
2.2 Radiolabelling and quality control
The radiolabelling of 2NNC2ON and 2NNC5ON was performed according to the method in the
literature 24-27, and the reaction routes are shown in Scheme 2.
Radiolabeling of 2NNC2ON. The precursor 2NNC2ON (2 mg) was dissolved in 20 μL DMSO,
then 140 μL 0.1 M HCl and 200 μL [18F]FDG (180-370 MBq) were added to the solution. The mixture
was heated to 130 °C for 30 min. After cooling to room temperature, the mixture volume was diluted to
1 mL with normal saline (0.9% NS) and loaded onto a Sep-pak C18 cartridge. After being eluted with 2
mL 0.9% NS to remove extra [18F]FDG, the product was collected with 0.5 mL ethanol. The collected
product was evaporated and dissolved in 0.9% NS for other experiments. The product used in the
10
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 42
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
blocking experiment was diluted with 5% glucose. The RCY and RCP of the product after purification
were measured by radio-HPLC, where the flow rate was maintained at 1 mL/min. The analytical
conditions of the radio-HPLC solvents were as follows: (A): water (containing 0.1% TFA); and (B)
acetonitrile. The elution gradient: 0-10 min 95% A-40% A; and 10-15 min 40% A-95% A.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Radiolabelling of 2NNC5ON. The radiolabelling, separating, and analytical methods of
2NNC5ON were similar to those of 2NNC2ON.
OH
OH
O
18F
DMSO/H2O, pH=1~2
HO
HO
OH
OH
O
O
H
HO
HO
N
N
)
(
18F
N
O
n
N
O
N
N
)
(
N
N
O
n
H
H
O
NO2
NO2
n=1, [18F]FDG-2NNC2ON
n=4, [18F]FDG-2NNC5ON
n=1, 2NNC2ON
n=4, 2NNC5ON
8
9
Scheme 2. The radiolabelling of 2NNC2ON and 2NNC5ON.
10
11
12
13
14
15
16
17
18
19
2.3 In vitro experiments
2.3.1 Stability in vitro
In vitro stability of the radiolabeled compounds was evaluated in both phosphate buffer solution
(PBS, 0.01 M, pH 7.4) and 5% human serum albumin (HSA). Approximately 20 μL of the radiolabeled
compound was added to 0.2 mL of PBS or HSA. Then, the mixture was incubated at 37 °C for 4 h. The
stability of the radiolabeled compounds in PBS could be directly measured by radio-HPLC. For HSA,
aliquots (100 μL) were removed and 200 μL of ethanol was added to precipitate the protein. After
centrifugation, the supernatant was filtrated through a 0.22 μm filter, and the filtrate was measured by
radio-HPLC.
2.3.2 Partition coefficient
11
ACS Paragon Plus Environment

Page 13 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
The partition coefficient was measured in a similar method as what was previously reported 31. In
a 5 mL centrifuge tube, 0.1 mL of the radiolabeled compound, 0.9 mL of water and 1 mL of 1-octanol
were mixed together. After shaking on a vortex mixer for 5 min at room temperature, the mixture was
centrifuged at 5000 rpm for 5 min and allowed to settle for another 10 min. Then, 100 μL of samples
were taken from each phase and measured by the gamma counter. The partition coefficient was
calculated as follows: P= (the radioactivity of the 1-octanol phase/ the radioactivity of the water phase),
and log P was the final expression of the partition coefficient. The experiment was repeated five times.
The final results were expressed as the mean ± S.D (standard deviation).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2.3.3 Cell uptake
10
11
12
13
14
15
16
17
18
19
20
21
22
The S180 cell line was used for the cellular uptake experiments32. The cells were diluted to a final
concentration of approximately 107 cells/mL with PBS (0.01 M, pH 7.4); then the cells (1 mL) were
suspended in 20 mL DMEM containing 10% fetal bovine serum at 37 °C with gentle magnetic stirring
under hypoxic (95% N2 + 5% CO2) and aerobic (95% air +5% CO2) conditions. After equilibration, 1
mL of the radiolabeled compound (1 MBq) was added. At different time points, five samples were
removed from the vial. The 200 μL samples were placed in a 0.5 mL centrifuge tube and centrifuged at
1500 rpm for 5 min. Then, 50 μL of the supernatant was removed from the centrifuge tube and
measured as sample A. The residual amount was measured as sample B. The cellular uptake was
calculated as follows: %Uptake = (B-3A) / (B+A) ×100%. In this calculation method, the cell volume
was ignored. The cell viability was assessed by trypan blue after the experiments, and the cell viability
was much higher than 90%. The final results were expressed as the mean ± S.D.
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.4 In vivo experiments
12
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 42
1
2
3
4
5
6
7
8
9
1
2
2.4.1 Animal model and blood retention in mice
The protocols for all performed animal experiments were approved by the Peking University
Cancer Hospital Animal Care and Use Committee. Healthy male Kunming mice were used to assess
blood retention, to evaluate in vivo stability of the radiolabeled compounds and to establish the S180
tumor model; six-week-old BALB/c nude mice were used to establish the OS732 tumor model, and the
specific details are described in the Supporting Information.
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
2.4.2 Stability in vivo
8
In vivo stability of the radiolabeled compounds was also evaluated throught testing urine in
animals. The radiolabeled compounds (29.6 MBq, 200 μL, approximately 3.2 mg/mL) were injected
into the healthy Kunming mice via the tail vein (n=3). The mice were sacrificed at 3 h postinjection and
the urine were collected, filtrated through a 0.22 μm filter and measured by radio-HPLC.
2.4.3 Biodistribution
9
10
11
12
13
14
15
16
17
18
19
20
21
The radiolabeled compounds (1 MBq, 200 μL, approximately 108 μg/mL) were injected into the
S180 or OS732 tumor-bearing mice via the tail vein (n=4). For the competition study, 1 MBq (200 μL)
of the radiolabeled compounds diluted with 5% glucose (GS) were injected into mice bearing S180
tumors (n=4). The mice were sacrificed at 1 h, 1 h-competition, 2 h, and 4 h postinjection. After
washing, the organs and tissues of interest were collected, weighed and measured for activity on a
gamma counter. The results were expressed as percent injected dose per gram of tissue (%ID/g). The
final results were expressed as the mean ± S.D.
2.4.4 Micro-PET imaging
13
ACS Paragon Plus Environment

Page 15 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
The radiolabeled compounds (29.6 MBq, 200 μL, 3.2 mg/mL) were injected into S180 or OS732
tumor-bearing mice. For glucose-competition imaging, a glucose-diluted tracer was injected into S180
tumor-bearing mice. After the injection, the mice were anesthetized with 2.5% isoflurane and
maintained under 1.5% isoflurane throughout the imaging experiment. Then, the mice were placed in
the prone position and fixed on a micro-PET scanner (Sedecal, Spain). Micro-PET imaging was
performed at the indicated time points (1 h, 1 h-competition and 2 h). The parameters for obtaining
PET images were as follows: 80 mm diameter transaxial FOV, 900-1200 s PET acquisition time, and
OSEM 3D reconstruction iterative algorithms with random and attenuation corrections. At the same
time, a semi-quantitative region of interest (ROI) was sketched on the PET image.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
10
11
12
13
14
15
16
17
18
19
20
21
22
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.5 Pathological examination
To verify the expression and distribution of HIF1α in tumor tissues, immunohistochemistry and
immunofluorescence were performed at the end of imaging, and the details are provided in the
Supporting Information.
2.6 Autoradiography of [18F]FDG-2NNC2ON for tissue hypoxia
Tumor specimens of the S180 cell line were selected for autoradiographic analysis. A 30 μm thick
slice of the rapidly frozen tumor sample was dried at 40 °C by air and then exposed overnight on a
high-sensitivity imaging film (BAS-SR2025, Fuji Photo Film, Japan). The autoradiographic images
were analyzed with a computerized imaging analysis system (FLA5100, Fuji Medical System, Stanford,
CT, USA) and compared with the immunofluorescently stained image.
14
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 42
1
2
3
4
5
6
7
1
2
2.7 Statistical Analysis
Quantitative data are described as the mean ± S.D. Significant differences between the two sets of
data were determined by one-way analysis of variance. SPSS 21.0 software (IBM, Armonk, NY, USA)
was used for the statistical analysis. When the P value was <0.05, it was considered statistically
significant.
8
9
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
3. Results
8
3.1 Chemical synthesis and radiolabelling
9
The compounds 7-8 were synthesized via 4 steps from 2-nitroimidazole, and the reaction
equations are shown in Scheme 1. Products 7-8 were identified by 1H NMR spectra, 13C NMR spectra,
HRMS-ESI, and IR ; the results indicate that the target precursors were successfully synthesized, and
the corresponding details are provided in the Supporting Information. The cold reference compounds
9-10 were also prepared and identified by the HRMS-ESI to certify the identity of the corresponding
radiolabeled compounds according to the reaction described in Scheme 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
The RCY and RCP of the products were measured by radio-HPLC. As shown in Figure 2, the
retention time of [18F]FDG is 3.1 min, while the retention times of [18F]FDG-2NNC2ON and
[18F]FDG-2NNC5ON are 6.1 min and 8.1 min, respectively. The RCY of [18F]FDG-2NNC2ON and
[18F]FDG-2NNC5ON are 57.6 ± 8.1% and 59.5 ± 9.5% (n=5), respectively. After purification, the RCP
of the products are higher than 99%, and the specific activity of [18F]FDG-2NNC2ON and
[18F]FDG-2NNC5ON are about 51.8-106.6 and 53.6-110.1 MBq/mg (n=5). Compared with the
corresponding cold reference compounds, the retention times of FDG-2NNC2ON and FDG-2NNC5ON
are 5.6 min and 7.4 min respectively, which are close with that of the [18F]FDG-2NNC2ON and
15
ACS Paragon Plus Environment

Page 17 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
1
2
[18F]FDG-2NNC5ON. The results indicate that the structures of radiolabeled compounds are proposed
structures.
7
8
9
[18F]FDG
[18F]FDG
3.1 min
a
b
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
120000
100000
80000
60000
40000
20000
0
3.1 min
[18F]FDG-2NNON
6.1 min
200000
150000
100000
50000
0
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14
Retention time (min)
Retention time (min)
3
[18F]FDG-2NNC2ON
6.1 min
[18F]FDG
3.1 min
c
d
120000
100000
80000
60000
40000
20000
0
80000
70000
60000
50000
40000
30000
20000
10000
0
[18F]FDG-2NNC5ON
8.1 min
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14
Retention time (min)
Retention time (min)
4
[18F]FDG-2NNC5ON
8.1 min
FDG-2NNC2ON
5.6 min
e
f
600
70000
60000
50000
40000
30000
20000
10000
0
500
400
300
200
100
0
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14
Retention time (min)
Retention time (min)
5
16
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 42
1
2
3
4
5
6
7
8
9
g
FDG-2NNC5ON
7.4 min
600
500
400
300
200
100
0
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
2
4
6
8
10 12 14
Retention time (min)
1
2
Figure 2 Radio-HPLC chromatograms of [18F]FDG and the [18F]FDG-labeled compounds and UV
chromatograms of FDG-2NNC2ON and FDG-2NNC5ON. (a) [18F]FDG; (b) [18F]FDG-2NNC2ON
before purification; (c) [18F]FDG- 2NNC2ON after purification; (d) [18F]FDG-2NNC5ON before
purification;(e) [18F]FDG-2NNC5ON after purification; (f) the UV chromatogram of FDG-2NNC2ON
at 254 nm; and (g) the UV chromatogram of FDG-2NNC5ON at 254 nm.
3
4
5
6
7
3.2 Stability
8
As shown in Figure 3 a-d, the RCP of the labeled compounds is still above 99%, which
indicates that the compounds are stable for over 3h in urine and over 4 h at 37 ºC in PBS or HSA; this
result suggests that the compounds have high stability in vitro and in vivo.
9
10
11
12
13
14
15
16
17
18
3.3 In vitro experiments
Partition coefficients. The log P values of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON are
-1.93±0.02 and -1.25±0.03, respectively. The results show that the two compounds are hydrophilic,
which may be related to the OH group of FDG.
Cellular uptake experiments. The cellular uptake of [18F]FDG, [18F]FDG-2NNC2ON and
[18F]FDG-2NNC5ON in the S180 cell line are shown in Figure 3 c-e. At 1 h, the cellular uptake of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in the hypoxic cells is clearly more than that in the
aerobic cells, and the difference is statistically significant. In the case of hypoxia, the uptake values of
17
ACS Paragon Plus Environment

Page 19 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
the two probes in the cells gradually increased with time. At 4 h, the uptake of [18F]FDG-2NNC2ON in
the hypoxic cells is 3.2 times higher than that in the aerobic cells, and the uptake of
[18F]FDG-2NNC5ON in the hypoxic cells is 2.4 times higher than that in the aerobic cells. In addition,
the cellular uptake of [18F]FDG is similar in both hypoxic conditions and aerobic conditions. The
results of the in vitro cell experiments indicate that [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON
have obvious hypoxic selectivity.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[18F]FDG-2NNC2ON
[18F]FDG-2NNC5ON
a
b
60000
50000
40000
30000
20000
10000
0
6.1min
140000
120000
100000
80000
60000
40000
20000
0
8.1min
HSA
PBS
PBS
HSA
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14 16
Retention time (min)
Retention time (min)
7
c
[18F]FDG-2NNC2ON in urine
5.99min
d
[18F]FDG-2NNC5ON in urine
120000
100000
80000
60000
40000
20000
0
120000
7.74min
100000
80000
60000
40000
20000
0
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14
Retention time (min)
Retention time (min)
8
18
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 42
1
2
3
4
5
6
7
8
9
[18F]FDG-2NNC2ON
f
[18F]FDG-2NNC5ON
e
20
16
12
8
20
16
12
8
Hypoxic
Aerobic
Hypoxic
Aerobic
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
4
0
0
0
50
100 150 200 250
0
50
100 150 200 250
Time (min)
Time（min)
1
g
[18F]FDG
Hypoxic
Aerobic
30
25
20
15
10
5
0
0
50
100 150 200 250
Time (min)
2
3
Figure 3 a-b. The RCP of [18F]FDG-2NNC2ON and [18F]DG-2NNC5ON after 4 h at 37 ºC in PBS and
5% HSA. (a) [18F]FDG-2NNC2ON; (b) [18F]FDG-2NNC5ON. Figure 3 c-d. The RCP of
[18F]FDG-2NNC2ON and [18F]DG-2NNC5ON in urine after 3 h injection. (c) [18F]FDG-2NNC2ON; (d)
4
5
6
[18F]FDG-2NNC5ON. Figure
3
e-g. The cellular uptake of [18F]FDG-2NNC2ON, and
7
[18F]FDG-2NNC5ON and [18F]FDG in S180 cell lines.
8
9
3. 4 In vivo experiments
10
11
12
13
3.4.1 Blood retention in mice
As shown in the Supporting Information Figure S1, the radioactivity of the two compounds in
the blood rises rapidly and gets eliminates quickly. The results show that the blood retention of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON follows the typical blood retention pattern of small
19
ACS Paragon Plus Environment

Page 21 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
molecule compounds. The curves are fitted with a two-compartment model. The fitted equations of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON are y=20.92 × e-0.23t+0.78 × e-0.01t (R2=0.92) and
y=4.04 × e-0.17t+0.94 × e-0.009t (R2=0.97), respectively. The half-life of the distribution phase (T1/2α) of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON are 3.01 and 4.07 min, respectively, while the half-life
of the elimination phase (T1/2β) of each compound are 69.3 and 77.02 min, respectively.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.4.2 Biodistribution
The biodistribution results of the two radiotracers in mice bearing S180 tumors and bearing
OS732 tumors are shown in Figure 4, and the details are described in the Supporting Information
Table S1-4, respectively. The intestine and liver have the highest uptake values, but the uptake values
of the radiotracers in all organs are very low, which suggests that most of inject dose has been
eliminated from the urinary system and partly excreted from the hepato-intestinal system. At 1 h, the
uptakes values of [18F]FDG-2NNC2ON are 0.4±0.1 %ID/g in S180 tumors and 0.3±0.0 %ID/g in
OS732 tumor model, and the uptakes values of [18F]FDG-2NNC5ON are 0.3±0.0 %ID/g in S180
tumor model and 0.3±0.1 %ID/g in OS732 tumors. Although the uptake values in the tumor are not
high, the blood and muscle uptake values are extremely low, and the radioactive clearance from the
blood and muscle is very fast, so the two compounds exhibit high tumor/blood (T/B) and tumor/muscle
(T/M) ratios (Figure 5 g-h). At 2 h, the T/B and T/M ratios of [18F]FDG-2NNC2ON in the S180
tumor-bearing mouse are 3.2±0.8 and 2.6±0.9 and are 3.4±0.2 and 4.2±0.7 in the OS732
tumor-bearing mice; the T/B and T/M ratios of [18F]FDG-2NNC5ON in the S180 tumor-bearing mouse
are 4.7±0.7 and 5.2±1.0, and are 7.3±2.2 and 5.9±1.5 in the OS732 tumor-bearing mice. After
co-injection with 5% GS, although the uptake of the radiotracers in the tumor is a little higher than that
10
11
12
13
14
15
16
17
18
19
20
21
22
20
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 42
1
2
3
4
5
6
7
8
9
1
2
without co-injection of 5% GS, the difference is not statistically significant (Figure 4 a, c). This result
demonstrates that these two radiotracers do not target the tumors through the glucose metabolism
pathway. The difference between the uptake values of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON
in the tumor is not statistically significant (Figure 4 e), suggesting that the increased lipophilicity of
[18F]FDG-2NNC5ON does not obviously improve tumor uptake. In the kidneys, the uptake value of
[18F]FDG-2NNC2ON is higher than that of [18F]FDG-2NNC5ON. However, in the small intestine, the
uptake value of [18F]FDG-2NNC2ON is significantly lower than that of [18F]FDG-2NNC5ON (Figure
4 f). These results suggest that the proportion of [18F]FDG-2NNC2ON excreted by the kidney is greater
than that of [18F]FDG-2NNC5ON because the lipophilicity of [18F]FDG-2NNC5ON is greater than that
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
of [18F]FDG-2NNC2ON.
[
18F]FDG-2NNC2ON in S180 tumor-bearing mice
1h
a
[
18F]FDG-2NNC2ON in OS732 tumor-bearing mice
1h
b
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0.0
1h-co-injection with 5% GS
2h
4h
6
5
2h
4h
3
2
1
0
t
r
r
r
y
e
n
e
e
e
n
o
M
e
l
c
T
d
H
n
g
n
u
h
n
i
a
t
r
r
r
e
v
o
m
u
y
e
e
e
e
d
H
n
g
h
n
i
e
e
l
p
c
a
m
n
n
o
a
l
e
i
i
t
s
e
t
o
e
e
c
n
n
n
o
n
i
L
S
t
a
o
l
e
r
B
c
s
v
i
i
i
t
s
e
t
a
r
B
e
l
n
a
i
t
o
d
i
s
o
l
e
u
s
m
L
B
u
B
d
s
e
n
i
L
L
B
u
u
p
i
m
o
t
K
B
e
t
t
T
o
n
S
K
M
i
t
S
n
n
l
l
i
S
e
l
a
g
l
e
r
a
g
r
m
a
L
S
m
a
S
L
11
d
[
18F]FDG-2NNC5ON in S180 tumor-bearing mice
c
6
[
18F]FDG-2NNC5ON in OS732 tumor-bearing mice
10.0
9.8
9.6
9.4
9.2
9.0
8.8
1h
1h
2h
4h
1h-co-injection with 5% GS
2h
4h
5
3
2
1
0
4
3
2
1
0
t
r
a
r
r
o
t
r
y
e
e
e
e
n
o
e
l
c
r
r
d
H
n
g
n
u
h
n
i
a
y
e
e
e
e
d
H
n
g
h
n
i
e
l
e
e
e
l
p
c
a
m
n
n
o
m
B
o
o
l
B
e
e
c
n
n
i
n
i
t
s
e
t
o
n
v
i
L
S
i
a
c
v
i
i
a
r
n
t
e
r
B
m
u
T
e
l
n
a
i
s
t
t
o
o
l
e
u
s
d
s
e
t
n
n
L
B
u
M
L
d
s
s
i
L
B
u
u
p
i
m
B
e
t
n
i
e
t
o
t
S
K
T
o
S
K
M
t
i
i
e
n
S
l
l
l
l
e
a
m
L
g
r
a
g
r
a
m
a
S
S
L
12
21
ACS Paragon Plus Environment

Page 23 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
Kidney- [18F]FDG-2NNC2ON
e
[
18F]FDG-2NNC2ON
18F]FDG-2NNC5ON
f
Kidney- [18F]FDG-2NNC5ON
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
[
Small intestine- [18F]FDG-2NNC2ON
Small intestine- [18F]FDG-2NNC5ON
10
6
4
2
0
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
1h
2h
4h
1h
2h
4h
1h 1h-block 2h
S180 tumor
4h
1h
2h
4h
OS732 tumor
S180 tumor
OS732 tumor
1
2
Figure 4: Biodistribution of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in both S180 and
OS732 tumor-bearing mice. (a) [18F]FDG-2NNC2ON in S180 tumor-bearing mice; (b)
[18F]FDG-2NNC2ON in OS732 tumor-bearing mice; (c) [18F]FDG-2NNC5ON in S180 tumor-bearing
mice; (d) [18F]FDG-2NNC5ON in OS732 tumor-bearing mice; (e) Comparison of uptake values of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in tumors. (f) Comparison of uptake values of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in the kidney and small intestine.
3
4
5
6
7
8
9
3.4.3 Micro-PET imaging
10
11
12
13
14
15
16
17
18
Whole-body PET images were obtained at 1 and 2 h after the injection of the radiotracers, as
shown in Figure 5. Both the S180 tumor and OS732 tumor could be observed obviously (Figure 5
a-d). The intestine has the highest uptake, followed by the liver. The results of the PET imaging are in
accordance with the results of the biodistribution. Comparing between [18F]FDG-2NNC2ON and
[18F]FDG-2NNC5ON, the PET image of [18F]FDG-2NNC2ON has higher contrast, and the effect of
imaging of [18F]FDG-2NNC2ON is better than that of [18F]FDG-2NNC5ON (Figure 5 a vs c and
Figure 5 b vs d). After competition with 5% GS, the tumor is still clearly visible in the image (Figure
5 e). Moreover, to confirm the imaging results of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON,
imaging was also performed with [18F]FDG in mice bearing OS732 tumors too (Figure 5 f). The PET
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 42
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
image with [18F]FDG is significantly different from that of the [18F]FDG-2NNC2ON and
[18F]FDG-2NNC because the brain and heart have very significant radioactive tracer uptake. In the
S180 tumor model, the tumor to muscle ratio based on ROI (ROI-T/M ratios) of [18F]FDG-2NNC2ON
are 2.4 ± 0.3 at 1 h and 3.9 ± 0.1 at 2 h; the ROI-T/M ratios of [18F]FDG-2NNC5ON are 3.4 ± 0.2 at 1
h and 1.5 ± 0.1 at 2 h (Figure 5 g). In the human OS732 tumor model, the ROI-T/M ratios of
[18F]FDG-2NNC2ON are 3.0 ± 0.4 at 1 h and 4.6 ± 0.6 at 2 h; the ROI-T/M ratios of
[18F]FDG-2NNC5ON are 2.8± 0.6 at 1 h and 1.1 ± 0.3 at 2 h (Figure 5 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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
9
10
23
ACS Paragon Plus Environment

Page 25 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
T/M-[18F]FDG-2NNC2ON
T/M-[18F]FDG-2NNC5ON
In the OS732 tumor model
h
8
g
In the S180 tumor model
T/M-[18F]FDG-2NNC2ON
T/M-[18F]FDG-2NNC5ON
7
6
5
4
3
2
1
0
7
6
5
4
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
2
1
0
1h
2h
4h
1h
2h
1h 1h-block 2h
4h
1h 1h-block 2h 2h-block
ROI-based
Biodistribution-based
ROI-based
Biodistribution-based
1
2
Figure 5. Micro-PET images of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in OS732
3
4
5
6
7
8
9
and S180 tumor-bearing mice at 1 h and 2 h postinjection. (a) Micro-PET images of
[18F]FDG-2NNC2ON in
a
mouse bearing
a
S180 tumor; (b) Micro-PET images of
[18F]FDG-2NNC2ON in a mouse bearing a human OS732 tumor; The images in figures a-b show the
tumor quite clearly. (c) Micro-PET images of [18F]FDG-2NNC5ON in a mouse bearing a S180 tumor;
(d) Micro-PET images of [18F]FDG-2NNC5ON in a mouse bearing an OS732 tumor. Figures c-d show
that the tumor can be seen, but the contrast of the image is not very high. (e) Micro-PET images of
[18F]FDG-2NNC2ON in a mouse bearing a S180 tumor after com with 5% GS, and the tumor is still
clearly visible on the image. (f) Micro-PET images of [18F]FDG in a mouse bearing a human OS732
tumor. Figure 5 f shows that the characteristics of the PET image with [18F]FDG are significantly
different from those of figures 5 a-e. (g) T/M ratios (both based on biodistribution and ROI) of
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in the S180 tumor model. (h) T/M ratios (both based
on biodistribution and ROI) of [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON in the OS732 tumor
model.
10
11
12
13
14
15
16
17
3.5 Histopathological analysis and autoradiography
24
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 42
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
The results of the immunohistochemistry and immunofluorescence staining of HIF1α confirm the
presence of hypoxia in the S180 and OS732 tumors, as shown in Figure 6. From the enlarged
pathology image, the expression of HIF1α is observed to be mainly located in the nucleus and
cytoplasm. Compared with hematoxylin-eosin staining (HE), the positive area of HIF1α staining is
mainly located around the necrotic tumor tissue (Figure 6 a-c). In the two adjacent sections of the
continuous frozen section, the region positive for HIF1α immunofluorescence staining is generally
similar with the imaging area highlighted in autoradiography, suggesting that the radiotracer
specifically targets the hypoxic region (Fig 6 c4-5).
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
10
11
Figure 6. Autoradiography and histopathological analysis images (0.5 × magnification in full
12
landscape image and 30 × magnification in local magnified image). (a, b) Immunohistochemical
25
ACS Paragon Plus Environment

Page 27 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
staining of S180 (a) and human OS732 (b) tumors. The hypoxic regions are shown in brown. (c)
Images from the S180 tumor model of HE staining (c1-3), immunofluorescence staining (c4) to detect
HIF1α and autoradiography (c5). (c2) The necrotic area of tumor tissue. (c3) The region around the
necrotic tumor tissue. Figures c4 and c5 show that the positive region of HIF1α immunofluorescence
staining was mainly consistent with the area highlighted by autoradiography (green area in C4 vs
yellow area in C5). HIF1α is expressed in the nucleus and cytoplasm (a1, a2, d).
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4. Discussion
In this study, two new nitroimidazole derivatives, 2NNC2ON and 2NNC5ON, were successfully
synthesized through 4-step reactions. [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON could be
obtained by one-step reactions within 30 min with a relatively good RCY and are more conveniently
synthesized than some 18F-labeled compounds6,18,23, and show a fast clearance rate in pharmacokinetic
studies. Both radiotracers are stable in vitro, which meets the quality control standards for clinical
applications. In the current study, [18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON demonstrate the
ability to specifically target hypoxic cells or hypoxic regions of the tumor, similar to [18F]FAZA 23 and
18FMISO 23. This ability is crucial because the specific targeting hypoxic regions is a prerequisite for
hypoxia imaging agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
In the in vivo experiments, both radiotracers can be taken up by the tumor, and the tumor can be
clearly observed on the PET image. Compared to [18F]FDG-2NNC5ON, the PET images with
[18F]FDG-2NNC2ON shows the tumor more clearly and has better image contrast. At 2 h, both probes
demonstrate excellent tumor-to-background ratios in both tumor models that are comparable to
26
previously reported results in [18F]FMISO
and seem even better than what is published in other
26
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 42
1
2
3
4
5
6
7
8
9
6,18.
1
2
reports
Interestingly, the T/M and T/B ratios of [18F]FDG-2NNC5ON are higher than those of
[18F]FDG-2NNC2ON, but PET imaging of the tumor with [18F]FDG-2NNC5ON is not as good as with
[18F]FDG-2NNC2ON. The biodistribution analysis reveals that the uptake of [18F]FDG-2NNC5ON in
the intestine is significantly higher than that of [18F]FDG-2NNC2ON, which may be the cause of
poorer imaging with [18F]FDG-2NNC5ON.
3
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
These studies illustrate that the two compounds (especially [18F]FDG-2NNC2ON) have potential
applications in detecting the hypoxic areas of tumors, but the uptake of [18F]FDG-2NNC2ON in the
tumor is slightly low because of the high clearance rate in vivo. Therefore, to overcome the lower
tumor uptake of [18F]FDG-2NNC2ON, a good direction for future research might be prolonging the
residence time of [18F]FDG-2NNC2ON.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Moreover, [18F]FDG-2NNC5ON is more lipophilic than [18F]FDG-2NNC2ON due to the
extension of its carbon chain. Therefore, in biological distribution and PET imaging studies,
[18F]FDG-2NNC2ON demonstrates a higher renal excretion ratio and less digestive tract uptake than
[18F]FDG-2NNC5ON, and [18F]FDG-2NNC2ON achieves better tumor imaging. Because of its higher
hydrophilicity, [18F]FDG-2NNC2ON diffuses into the tissue from the blood slowly, and its
concentration in the blood correspondingly increases in the early stages of drug metabolism, which is
more conducive for the uptake of radiotracers by target tissues. These results indicate that the linking
group between [18F]FDG and the 2-nitroimidazole derivative has an important effect on the in vivo
biodistribution of the radiotracers. Therefore, these findings suggest that altering the structure of the
linker to develop a hypoxic imaging agent may be another strategy.
5. Conclusion
27
ACS Paragon Plus Environment

Page 29 of 42
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
In summary, we have successfully synthesized two new 2-nitroimidazole hypoxic imaging agents,
[18F]FDG-2NNC2ON and [18F]FDG-2NNC5ON, by direct radiolabeling with [18F]FDG. According to
the results of the biodistribution, the effects of [18F]FDG-2NNC2ON are better than those of
[18F]FDG-2NNC5ON, most likely due to the lower lipophilicity of [18F]FDG-2NNC2ON. The PET
imaging qualities of [18F]FDG-2NNC2ON are desirable. These findings suggest that direct
radiolabeling with [18F]FDG for the modification of 2-nitroimidazoles is a feasible and convenient
method, and a new radiotracer, [18F]FDG-2NNC2ON, has potential as a hypoxic imaging agent for
clinical application.
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10
11
12
13
14
Supporting Information
Blood retention experiments in mice, animal models, pathological examinations, experimental
procedures, characterization of the compounds, and the detailed values of the biodistribution are
described in the supporting information.
Acknowledgements
15
16
17
18
19
20
21
22
This work was supported by the National Natural Science Foundation of China (Grant No.
11575010, 21371017, 81571705, 81671733, 81871386, 81560356, and 81871387), Beijing Nova
Program (Z171100001117020), Beijing Excellent Talents Funding (2017000021223ZK33), Beijing
Municipal Administration of Hospitals-Yangfan Project (ZYLX201816). Beijing Natural Science
Foundation, Jing-Jin-Ji special projects for basic research cooperation (H2018206600), Major State
Research Development Program of China (2016YFC0100402). The science and Technology
Foundation of Guizhou (Grant nos. [2015] 2089 and [2019] 1201). Science and Technology
Foundation of Health and Family Planning Commission of Guizhou Province (gzwjkj2018-1-040).
28
ACS Paragon Plus Environment