Novel 99mTc labelled complexes with 2-nitroimidazole isocyanide: Design, synthesis and evaluation as potential tumor hypoxia imaging agents

Article abstract of DOI:10.1039/c8md00146d

Radiolabelled 2-nitroimidazoles have been used for imaging hypoxia. With the aim of developing novel ^99mTc radiotracers for imaging hypoxia, four novel 2-nitroimidazole isocyanide derivatives (2a, 2b, 2c, and 2d) were synthesized and radiolabelling was carried out for preparing their corresponding ^99mTc complexes. These ^99mTc complexes were stable in vitro and could exhibit good hypoxic selectivity. The partition coefficient results indicated that they were hydrophilic, and an evaluation of biodistribution in mice bearing S180 tumors indicated that all of the complexes could accumulate in the tumors. Among them, ^99mTc-2c exhibited the highest tumor uptake and tumor/blood and tumor/muscle ratios at 2 h post-injection. Further, single photon emission computed tomography (SPECT) imaging studies indicated clear accumulation in tumors, suggesting that ^99mTc-2c was a promising candidate for hypoxia imaging.

Full text of DOI:10.1039/c8md00146d

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ARTICLE TYPE
Novel 99mTc labelled complexes with 2-nitroimidazole isocyanide:
Design, synthesis and evaluation as potential tumor hypoxia imaging
agents
Qing Ruana, Xuran Zhanga, Xiao Lina,b, Xiaojiang Duana and Junbo Zhanga*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Radiolabelled 2-nitroimidazoles have been used for imaging hypoxia. With the aim to develop novel 99mTc
radiotracers for imaging hypoxia, four novel 2-nitroimidazole isocyanide derivatives (2a, 2b, 2c, 2d) were
synthesized and radiolabelling was carried out for preparing their corresponding 99mTc complexes. These
10 99mTc complexes were stable in vitro and could exhibit good hypoxic selectivity. The partition coefficient
results indicated they were hydrophilic, and an evaluation of biodistribution in mice bearing S180 tumors
indicated that all of the complexes could accumulate in tumor. Among them, 99mTc-2c exhibited the highest
tumor uptake, tumor/blood and tumor/muscle ratios at 2 h post-injection. Further, single photon emission
computed tomography (SPECT) imaging studies indicated clear accumulation in tumor, suggesting 99mTc-
15 2c was a promising candidate for hypoxia imaging.
Introduction
45 many 99mTc labelled 2-nitroimidazole analogues have been
reported.12-15 However, properties of these 99m Tc complexes are
not yet ideal and ongoing efforts should be made to develop novel
99mTc labelled 2-nitroimidazole analogues for tumor hypoxia
imaging.
Tumor hypoxia is one of the major causes of resistance to
conventional radiotherapy and chemotherapy. Therefore, the
evaluation of oxygenation status of solid tumor plays an important
20 role in tumor treatment planning.1-4 With the development of
noninvasive imaging techniques, positron emission tomography
(PET) and single photon emission computed tomography (SPECT)
with hypoxia targeted radiopharmaceuticals are considered to be
reasonable alternatives. In the development of hypoxia imaging
25 agent, nitroimidazole derivatives are enzymatically reduced and
accumulated in hypoxic regions, therefore most labelled probes for
tumor hypoxia have been based on nitroimidazole analogues. 5, 6 In
particular, 2-nitroimidazole derivatives have been found be more
suitable for detection of tumor hypoxia, because 2-nitroimidazole
30 has a more positive single electron reduction potential (SERP)
value, which means it can be efficiently reduced and retained in
hypoxic cells.7-9 [18F]Fluoromisonidazole ([18F]FMISO), a 2-
nitroimidazole radiotracer, is one of the most clinically studied
radiotracers for hypoxia imaging.10 However, to some degree, the
35 limited availability of the [18F] isotope and limited PET scanners
restricted its widely clinical use, especially in many developing
countries. Because there have been larger number of SPECT
scanners in the whole world and progresses being made in
hardware and image reconstruction algorithms have optimized the
40 spatial resolution of images of SPECT,11 to develop effective
SPECT tracers for hypoxia imaging still exists great necessity.
Since the discovery of 99mTc in the late 1930s and its introduction
into nuclear medicine via the 99Mo/99mTc generator in the 1950s,
99mTc has been the most widely used SPECT isotope. Up to date,
50 Isocyanide (CN-R) is regarded as a monodentate ligand to strongly
coordinate with 99mTc(I) core and [99mTc(I)(CO)3]+ core to produce
stable 99mTc complexes([99mTc(CN-R)6]+ and [99mTc(CO)3(CN-
R)3]+) with high yields.15-21 With the aim to develop novel 99mTc
labelled 2-nitroimidazole analogues for tumor hypoxia imaging
55 and explore the influence of different length of the alkyl spacer
between the 2-nitroimidazole moiety and the 99mTc core, in this
study, by using 2-nitroimidazole as the targeting vector and
isonitrile to coordinate with 99mTc, we synthesized four novel 2-
nitroimidazole isocyanide derivatives and labelled it with 99mTc in
60 an effort to develop novel tumor hypoxia imaging agents.
Results and discussion
Chemistry
Synthesis of four novel 2-nitroimidazole isocyanide derivatives
was achieved by reaction of the corresponding amino derivatives
65 of 2-nitroimidazole (1) with different isocyanide-containing active
esters in the presence of triethylamine, as shown in Scheme 1. The
synthesis methods were very convenient to obtain the desired
products. The ligands (2a, 2b, 2c, 2d) were characterized by the 1H
NMR, 13C NMR, IR and HR-MS, and the results obtained were in
70 agreement with the proposed structures.
Radiolabelling and quality control
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99mTc-2a, 99mTc-2b, 99mTc-2c and 99mTc-2d complexes can be
prepared by direct labelling method with high radiochemical
purities (RCP). The preparation of these complexes does not
require heating and it can be easily formed at room temperature,
while the preparation of 99mTc-MIBI (MIBI: 2-methoxy-2-isobutyl
isonitrile) needs heating at 100 °C for 15 min. Just like 99mTc-MIBI,
the proposed structure of these complexes would be a monovalent
cation with a central technetium core that is surrounded by six
same ligands coordinated through the isonitrile carbon [Scheme
View Article Online
DOI: 10.1039/C8MD00146D
5
10 2].19
The RCP of the complexes were assessed by thin layer
chromatography
(TLC)
and
high-performance
liquid
Scheme 2 Preparation procedures for 99mTc-2a, 99mTc-2b,
chromatography (HPLC). By TLC, in methanol, 99mTc complexes
99mTc-2c and 99mTc-2d.
migrated at the front (Rf = 0.8–1.0), while 99mTcO4 ,99mTcO2·2H2O
−
15 remained at the origin (Rf = 0–0.1). By HPLC, single radioactive
peaks were observed with retention times (tR) of 14.35 min, 14.63
min, 14.88 min and 15.38 min corresponding to 99mTc-2a, 99mTc-
2b, 99mTc-2c and 99mTc-2d, respectively. While the retention times
–
of 99mTcO4 was 3.33 min. The mean radiochemical purities of the
20 final products were all over 95%, suggesting they can be used for
in vitro and in vivo studies without further purification (Fig. 1).
As the structural characterization of 99mTc complexes is difficult to
verify at the no-carrier-added level, corresponding rhenium
analogue can be synthesized at macroscopic level.22 Tc and Re
25 belong to the same group and they have similar chemistry. The
structure of 99mTc complex can be studied by using stable rhenium
as a surrogate for 99mTc. The co-injection HPLC pattern of 99m Tc-
2c and Re-2c was showed in Fig. 2. It could be noted that the
radioactivity profile of 99m Tc-2c (tR=14.86 min) nearly matched
30 with the UV-chromatograms (tR=14.48 min) of Re-2c, suggesting
99m Tc-2c possessed the proposed structure.
Figure 1 HPLC patterns of 99mTc-2a (A), 99mTc-2b (B), 99mTc-2c (C)
and 99mTc-2d (D).
In vitro stability study
The stability of the 99mTc complexes was assayed by measuring the
radiochemical purity (RCP). The RCP of the complexes was more
35 than 90% and all of the complexes did not show decomposition for
6 h in the labelling milieu at room temperature and in the mouse
serum at 37 °C, demonstrating their good in vitro stability.
Figure 2 Co-injection analysis of 99mTc-2c and Re-2c
complexes are in accordance with the structure of the ligands, since
incorporation of more CH2 group increases the lipophilicity.
In vitro cellular uptake
The cellular uptake analysis of 99mTc-2a, 99mTc-2b, 99mTc-2c and
50 99mTc-2d was carried out using S180 cell under both hypoxic and
aerobic conditions. As presented in Fig. 3, the uptake of the four
99mTc complexes in hypoxic cells were persistently higher than that
in aerobic cells, suggesting they had good hypoxia selectivity.
Significant differences between each point in hypoxic and aerobic
55 conditions were determined by Student’s t test (independent, two-
Scheme 1 Synthesis of 2a, 2b, 2c and 2d.
40
Determination of the partition coefficient
The partition coefficient (log P) values of 99mTc-2a, 99mTc-2b,
99mTc-2c and 99mTc-2d were −2.65 ± 0.14, −1.95 ± 0.13, −1.27 ±
0.04 and −0.45 ± 0.05, respectively, suggesting they were
45 hydrophilic. Moreover, the log P values of the corresponding 99mTc
2
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tailed). The criterion for significance was p < 0.05.
Liver
4.11 ± 0.49
0.99 ± 0.06
7.72 ± 0.60
0.53 ± 0.04
0.10 ± 0.02
0.52 ± 0.10
0.21 ± 0.03
1.14 ± 0.49
0.92 ± 0.18
1.17 ± 0.08
0.01 ± 0.00
0.79
3.20 ± 0.35
0.78 ± 0.02
8.15 ± 0.42
2.83 ± 0.26
0.64 ± 0.09
Lung
View Article Online
Kidney
Spleen
Stomach
Bone
8.22 ± 0.29
DOI: 10.1039/C8MD00146D
0.47 ± 0.07
0.39 ± 0.05
0.09 ± 0.02
0.42 ± 0.12
0.12 ± 0.02
0.36 ± 0.09
0.52 ± 0.08
0.53 ± 0.04
0.01 ± 0.01
1.00
0.11 ± 0.04
0.47 ± 0.10
0.14 ± 0.03
0.51 ± 0.15
0.63 ± 0.07
0.81 ± 0.09
0.01 ± 0.00
0.78
Muscle
Intestine
Tumor
Blood
Thyroid(%ID)
T/B
T/N
4.28
4.57
4.32
Table 3 Biodistribution of 99mTc-2c in mice bearing S180 tumor
(n=5, T/B = tumor-to-blood ratio, T/N = tumor-to-muscle)
%ID/g ± s
0.5 h
1 h
2 h
Figure 3 Cellular uptakes of 99mTc-2a (A), 99mTc-2b (B), 99mTc-2c
Heart
0.64 ± 0.07
6.77 ± 0.81
2.31 ± 0.40
5.70 ± 0.38
1.19 ± 0.16
0.35 ± 0.10
1.00 ± 0.04
0.37 ± 0.04
3.64 ± 0.53
1.55 ± 0.19
1.31 ± 0.20
0.02 ± 0.00
1.18
0.35 ± 0.03
4.93 ± 0.37
1.29 ± 0.08
5.18 ± 0.39
0.63 ± 0.06
0.24 ± 0.08
0.67 ± 0.10
0.21 ± 0.03
1.45 ± 0.40
0.93 ± 0.15
0.87 ± 0.01
0.01 ± 0.00
1.06
0.29 ± 0.04
4.10 ± 0.55
1.01 ± 0.16
4.91 ± 0.66
0.69 ± 0.16
0.53 ± 0.20
0.60 ± 0.08
0.16 ± 0.04
0.85 ± 0.21
0.83 ± 0.14
0.63 ± 0.12
0.01 ± 0.00
1.32
(C) and 99mTc-2d (D).
Liver
Lung
Biodistribution studies
Kidney
Spleen
Stomach
Bone
Biodistribution studies were performed in kunming mice bearing
S180 tumor, which is a representative hypoxia model of solid
tumors.23-25 We conducted biodistribution studies at 0.5, 1 and 2 h
after 99mTc complexes were injected into mice. The results of
99mTc-2a, 99mTc-2b, 99mTc-2c and 99mTc-2d were shown in Tables
1-4, respectively. They all had a relatively high tumor uptake.
Muscle uptakes were low, hence the tumor/muscle ratios were high.
5
Muscle
Intestine
Tumor
Blood
Thyroid(%ID)
T/B
10 The initial accumulations in blood were high, but the clearance of
activity from blood is faster than that of tumor. For the four 99mTc
complexes, excretion occurred through both the urinary and
hepatobiliary tract. Low uptake in the thyroid and stomach
T/N
4.15
4.39
5.05
25
Table 4 Biodistribution of 99mTc-2d in mice bearing S180 tumor
(n=5, T/B = tumor-to-blood ratio, T/N = tumor-to-muscle)
-
suggested no 99mTcO4 occured.
15
%ID/g ± s
0.5 h
1 h
2 h
able 1 Biodistribution of 99mTc-2a in mice bearing S180
tumor(n=5, T/B = tumor-to-blood ratio, T/N = tumor-to-muscle
Heart
0.70 ± 0.17
15.67 ± 0.37
3.09 ± 0.92
7.09 ± 1.42
1.42 ± 0.12
0.43 ± 0.02
1.06 ± 0.39
0.42 ± 0.13
3.70 ± 0.77
1.30 ± 0.14
1.93 ± 0.21
0.02 ± 0.01
0.67
0.55 ± 0.13
9.49 ± 0.69
3.76 ± 1.60
3.36 ± 0.28
0.94 ± 0.15
0.31 ± 0.07
0.97 ± 0.24
0.29 ± 0.08
1.30 ± 0.16
1.10 ± 0.23
1.47 ± 0.13
0.01 ± 0.01
0.75
0.40 ± 0.08
6.96 ± 0.61
3.73 ± 0.45
2.74 ± 0.41
0.80 ± 0.12
0.40 ± 0.14
0.60 ± 0.15
0.22 ± 0.07
1.47 ± 0.33
0.78 ± 0.06
1.03 ± 0.13
0.01 ± 0.00
0.75
T
Liver
Lung
%ID/g ± s
Heart
0.5 h
1 h
2 h
Kidney
Spleen
Stomach
Bone
1.04 ± 0.18
4.77 ± 0.55
2.26 ± 0.27
8.80 ± 1.15
0.77 ± 0.13
0.33 ± 0.18
1.01 ± 0.15
0.64 ± 0.22
1.96 ± 0.53
1.27 ± 0.37
2.87 ± 0.66
0.03 ± 0.01
0.44
0.32 ± 0.09
1.86 ± 0.30
0.81 ± 0.04
6.80 ± 2.01
0.33 ± 0.07
0.29 ± 0.17
0.49 ± 0.08
0.19 ± 0.02
0.75 ± 0.20
0.83 ± 0.19
0.96 ± 0.08
0.02 ± 0.00
0.86
0.28 ± 0.06
1.52 ± 0.34
0.61 ± 0.13
5.28 ± 1.21
0.24 ± 0.04
0.26 ± 0.05
0.28 ± 0.07
0.16 ± 0.02
0.58 ± 0.26
0.63 ± 0.09
0.70 ± 0.08
0.01 ± 0.01
0.91
Liver
Lung
Kidney
Spleen
Stomach
Bone
Muscle
Intestine
Tumor
Blood
Muscle
Intestine
Tumor
Blood
Thyroid(%ID)
T/B
T/N
3.11
3.77
3.45
Thyroid(%ID)
T/B
The comparison results of biodistribution of the four 99mTc
30 complexes in mice bearing S180 tumor at 2 h post-injection are
shown in Table 5. It was found that with the increase of log P of
the complex, the uptake in the liver was increased and kidney
uptake was decreased. By comparison, 99mTc-2c has a lower
muscle and blood uptake and higher tumor uptake, so the
35 tumor/blood and tumor/muscle ratios of 99mTc-2c is much higher
than that of other three complexes.
T/N
1.98
4.47
3.86
20 Table 2 Biodistribution of 99mTc-2b in mice bearing S180 tumor
(n=5, T/B = tumor-to-blood ratio, T/N = tumor-to-muscle)
%ID/g ± s
0.5 h
1 h
2 h
Heart
0.47 ± 0.05
0.34 ± 0.02
0.26 ± 0.03
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By the analysis of previously reported data, we can find that the
from the China Institute of Atomic Energy (CIAE). The murine
sarcoma S180 cell line was obtained from Peking University
tumor uptake of 99mTc-2c even the tumor/blood and tumor/muscle
ratios were higher than those of some reported 99mTc-labelled 2-
nitroimidazole derivatives. In 2017, Vats et al. reported a 99mTc-
Health Science Center (Beijing, China). IR sDpOecI:tr1u0m.10w39Va/iseCwo8AbMrttDaici0lne0eO1d4nl6inDe
with an AVATAR 360 FT-IR spectrometer using KBr pellets.
5
‘4+1’ mixed ligand complex of 2-nitroimidazole with isocyanide 40 NMR spectrum was recorded on a 400 MHz Bruker Avance
for hypoxia imaging. The tumor uptake of 99mTc-2c (0.93 ± 0.15)
was more than two times better than that of 99mTc(NS3) (2NimNC)
(0.40 ± 0.07) at 1 h post-injection. As for the uptake of non-target
spectrophotometer. ESI-MS spectrum was recorded on a LC-MS
Shimadzu 2010 series. High performance liquid chromatography
(HPLC) was performed on a Waters 600 binary HPLC pump and
a Waters 2487 UV absorbance dual λ detector (Milford, MA USA)
99m
organs and tissues, 99mTc-2c was much better than that of
10 Tc(NS3) (2NimNC). The liver uptake of 99mTc-2c (4.93±0.37) 45 with a reversed-phase column (Kromasil C18, 250×4.6 mm).
was less than half of that of 99m Tc(NS3) (2NimNC) (12.42±0.42),
Chemistry
and the intestine uptake of 99mTc-2c (1.45 ± 0.40) was merely less
than one thirteenth of that of 99m Tc(NS3) (2NimNC) (20.86 ±0.62)
at 1 h post-injection. As for the tumor/blood and tumor/muscle
15 ratios, 99mTc-2c was superior to 99mTc(NS3 )(2NimNC) at 1 h post-
injection.15
Compound 1 (2-(2-nitro-1H-imidazol-1-yl)ethan-1-amine) was
prepared according to literature previously described.26 The
synthetic procedures and the spectral data for the final products (2a,
50 2b, 2c and 2d) were as follows.
Synthesis of 2a. Compound 1 156 mg (1 mmol), Compound a (2,
3, 5, 6-tetrafluorophenyl 3-isocyanopropanoate) 247 mg (1 mmol)
and triethylamine 0.21 mL (1.5 mmol) were dissolved in 15 mL
methanol. The mixture was stirred at room temperature for 6 h. The
55 solvent was removed under a reduced pressure and the residue was
purified by chromatography (CH2Cl2/CH3OH) to give 2a (3-
isocyano-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl) propanamide) as
white solid (yield, 64%). IR (KBr)/cm −1: 3342.78 (νNH), 1487.18,
Table 5 Comparison of biodistribution of the 99mTc complexes at
2 h post-injection.
99mTc-2a
99mTc-2b
99mTc-2c
99mTc-2d
Log P
Liver
-2.65
-1.99
-1.27
-0.44
1.52 ± 0.34
2.83 ± 0.26
8.22 ± 0.29
0.52 ± 0.08
1.00
4.10 ± 0.55
4.91 ± 0.66
0.83 ± 0.14
1.32
6.96 ± 0.61
2.74 ± 0.41
0.78 ± 0.06
0.75
Kidney 8.42 ± 1.18
Tumor
T/B
0.63 ± 0.09
0.91
1
1359.87 (νNO2), 1670.43 (νCO), 2152.65 (νNC); H NMR (400
60 MHz, D2O) δ(ppm): 7.29 (s, 1H), 7.03 (s, 1H), 4.49-4.46 (t, 2H),
3.60-3.58 (t, 2H), 3.53-1.3.51 (t, 2H),2.54-2.51 (t, 2H); 13C NMR
(100 MHz, D2O) δ(ppm): 172.62, 151.97, 144.32, 128.72, 127.27,
48.34, 38.67, 38.11, 34.55; HR-MS (ESI): found 238.0938, calcd
238.0934, C9H12N5O3[M+H+].
65 Synthesis of 2b. Following the same procedure used in the
synthesis of 2a, 2b was isolated as white solid (yield, 35%). IR
(KBr)/cm −1: 3254.05 (νNH), 1489.11, 1327.08 (νNO2), 1633.78
(νCO), 2150.72 (νNC); 1H NMR (400 MHz, D2O) δ(ppm): 7.28 (s,
1H), 7.05 (s, 1H), 4.49-4.46 (t, 2H), 3.57-3.55 (t, 2H), 3.32-3.30 (t,
70 2H), 2.19-2.16 (m, 2H), 1.71-1.59 (t,2H); 13C NMR (100 MHz,
D2O) δ(ppm): 175.55, 151.33, 143.70, 128.73, 127.72, 49.83,
40.56, 37.95, 32.43, 22.99; HR-MS (ESI): found 252.1098, calcd
252.1091, C10H14N5O3 [M+H+].
T/N
3.86
4.32
5.05
3.45
20
SPECT/CT imaging studies
As 99mTc-2c had a higher tumor uptake, tumor/blood and
tumor/muscle ratios than the other three complexes, it was selected
as a promising candidate for further SPECT/CT imaging studies.
25 The SPECT/CT imaging results of 99mTc-2c showed the tumor
uptake was observable (Fig. 4). However, the appreciable uptake
of 99mTc-2c in the liver and kidneys is the defect of the complex.
The value of the region-of-interest (ROI) ratio of the uptake for the
tumor site to the corresponding non-tumor region was 5.39±0.67.
30 The imaging data in mice were consistent with the biodistribution
results in mice.
Synthesis of 2c. Following the same procedure used in the
75 synthesis of 2a, 2c was isolated as white solid (yield, 53%). IR
(KBr)/cm −1: 3366.99 (νNH), 1491.04, 1363.73 (νNO2), 1655.00
(νCO), 2146.86 (νNC); 1H NMR (400 MHz, D2O) δ(ppm): 7.26 (s,
1H), 7.03 (s, 1H), 4.46-4.45 (t, 2H), 3.56-3.54 (t, 2H), 3.33-3.31 (t,
2H), 2.05-2.03 (m, 2H),1.44-1.42 (m, 4H); 13C NMR (100 MHz,
80 D2O) δ(ppm): 176.87, 150.82, 144.331, 128.56, 127.58, 49.01,
40.86, 37.65, 34.21, 26.89, 22.83; HR-MS (ESI): found 266.1244
calcd 266.1247, C11H16N5O3 [M+H+].
Synthesis of 2d. Following the same procedure used in the
synthesis of 2a, 2d was isolated as white solid (yield, 69%). IR
85 (KBr)/cm −1: 3342.78 (νNH), 1487.18, 1359.87 (νNO2), 1670.43
(νCO), 2152.65 (νNC); 1H NMR (400 MHz, D2O) δ(ppm): 7.27(s,
1H), 7.05 (s, 1H), 4.48-4.46 (t, 2H), 3.56-3.54 (t, 2H), 3.35-3.32 (t,
2H), 2.04-2.02 (t, 2H),1.52-1.50 (m, 2H), 1.38-1.31 (m, 2H), 1.20-
1.15 (m, 2H); 13C NMR (100 MHz, D2O) δ(ppm): 177.04, 150.33,
90 144.67, 128.53, 127.39, 49.52, 41.04, 37.92, 35.18, 27.80, 25.07,
24.32; HR-MS (ESI): found 280.1406 calcd 280.1404,
C12H18N5O3 [M+H+].
Figure 4 SPECT/CT images of 99mTc-2c at 2 h post-injection,
visual imaging(A), sagittal section(B), coronal section(C) and
transverse section(D).
Experimental section
General
All chemicals were purchased from commercial sources and used
35 without further purification. 99Mo/99mTc generator was obtained
4
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reported methods.29 Briefly, S180 cells were suspended in fresh
Radiolabelling and quality control
DMEM growth medium having 10 % (v/v) of fetal bovine serum
The preparation procedures for 99mTc-2a, 99mTc-2b, 99mTc-2c and
99mTc-2d are as follows: 2 mg 2a (2b, 2c, 2d) was dissolved in
0.3mL saline, then 0.2 mL sodium citrate buffer (0.1mol/L,
pH=4.0) was add. After 0.03 mL SnCl2·2H2O (1mg/mL) was
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at a cell concentration of 2×106 cells / mL.DAOlIi:q1u0o.1t0s3o9f/C280MmDL00w14e6reD
55 put into glass vials and incubated at 37 °C under an atmosphere of
95% nitrogen plus 5% carbon dioxide (hypoxic conditions, oxygen
concentration <10 ppm) or 95% air plus 5% carbon dioxide
(aerobic conditions). After a 30 min equilibration period, 0.2 mL
(3.7 MBq) of the 99mTc labelled complex was mixed to the
60 suspension. 1 mL of cell suspension were pipetted and centrifuged
at 3000 g for 5 min at 0.5, 1, 2 and 4.0 h post-incubation. 0.9 mL
of supernatant was taken for counting (Cout) and the residual
sample containing cells with 0.1 mL of supernatant was also
counted (Cin). The studies were measured five times at each time
65 point. The cell accumulation, A, was calculated as follows: A =
(Cin - Cout/9) / (Cin + Cout). The final results were expressed as the
average of the measurements ± the standard deviation.
5
-
added, 0.5mL of saline containing 99mTcO4 (37–74 MBq) was
added right away. The mixture was kept at room temperature for
30 min. The radiochemical purity (RCP) of 99mTc-2a, 99mTc-2b,
99mTc-2c and 99mTc-2d was checked by TLC and HPLC. The TLC
10 was performed on a polyamide strip and eluted with methanol. For
HPLC, water (A) and acetonitrile (B) mixture were adopted as the
mobile phase at a flow rate of 1.0 mL/min, and the analytical
conditions was described in Table 6.
15 Table 6 The analytical conditions for HPLC
Time/min
0
5
15
30
70
20
30
70
22
10
90
32
10
90
35
90
10
A/%
90
10
90
10
Biodistribution studies
B/%
All biodistribution studies were performed according to the
70 Regulations on Laboratory Animals of Beijing Municipality and
all animal procedures were carried out in compliance with the
Guidelines for Care and Use of Laboratory Animals of Beijing
Normal University and approved by the Animal Ethics Committee
of Beijing Normal University.
75 Experiments were performed using Kunming female mice (18-22
g) bearing S180 tumors.30, 31 0.1 mL of 99mTc complexes (7.4 × 105
Bq mL−1) was injected via a tail vein. The mice were sacrificed in
groups of five at 0.5, 1 and 2 h post-injection. The tumor, muscle,
blood and other organs of interest were collected, weighed and
80 counted using Gamma Counter. The final results were expressed
as the percent uptake of injected dose per gram of tissue ± standard
deviation (% ID/g ± SD).
Preparation of rhenium analogue
In order to validate the proposed structure of 99mTc-2c, we
synthesized the stable complex Re-2c. The preparation procedures
for Re-2c were as follows: 100 mg of 2c was dissolved in 0.3 mL
20 saline, then 1 mL of sodium citrate buffer (0.1 mol/L, pH=4.0) was
added. After the addition of 1.5 mg SnCl2·2H2O, 2 mg KReO4 was
added right away. The mixture was kept at room temperature for
30 min. The desire product was purified by HPLC under conditions
described in Table 6. Then the solvent was removed and brown
25 residue was obtained. The product was analyzed by 1H NMR and
HR-MS. 1H NMR (400 MHz, D2O) δ(ppm): 7.30 (s, 1H), 7.08 (s,
1H), 4.52-4.48 (t, 2H), 3.60-3.57 (t, 2H), 3.39-3.36 (t, 2H), 2.10-
2.06 (m, 2H), 1.54-1.47 (m, 4H); HR-MS(ESI):
[(C11H15N5O3)6ReNa]2+: m/z calcd. for C66H90N30O18ReNa
30 900.3249. found 900.3245.
SPECT/CT imaging studies
SPECT/CT images were acquired on a small animal Triumph
85 SPECT/CT system (Trifoil imaging, USA). The SPECT data was
reconstructed with HiSPECT software and analyzed with the 40
vivoquant software. The Kunming female mice (18–20 g) bearing
S180 tumor were injected via a tail vein with 99mTc-2c (0.1 mL,
1.11 × 106 Bq). SPECT/CT images were acquired at 2 h after
90 injection. The ROIs for the tumor site and the corresponding non-
tumor region were selected and determined. The final results were
expressed as mean ± standard deviation(n=3).
In vitro stability study
The stability studies of 99mTc-2a, 99mTc-2b, 99mTc-2c and 99mTc-2d
were performed by checking the RCP in the labelling milieu at
room temperature for 6 h. In vitro serum stability studies in mouse
35 serum were also assessed by a method reported earlier.27 The RCP
was checked by TLC and HPLC.
Determination of the partition coefficient (log P)
The lipophilicity of the four 99mTc complexes were evaluated by
the partition coefficients (log P) between 1-octanol and phosphate
40 buffer (0.025 mol/L, pH 7.4) according to the previously reported
method.28 Briefly, the complex was mixed with an equal volume
of the two phase. The mixture was vortexed for 3 min and then
centrifuged at 10000 g for 5 min. From each phase, 0.1 mL of
aliquot was pipetted and counted in a γ-counter. The partition
45 coefficient, P, was calculated as the mean value of each counts per
minute per milliliter of 1-octanol layer divided by that of the buffer.
The final partition coefficient value was expressed as log P ± SD.
Conclusion
In the present study, four novel 2-nitroimidazole isocyanide
95 derivatives (2a, 2b, 2c, 2d) were synthesized and their
corresponding 99mTc complexes can be prepared by direct labelling
method without the need for heating. These 99mTc complexes
exhibited hydrophilicity and in vitro stability as well as hypoxic
selectivity. The preliminary biological evaluation in mice bearing
100 S180 tumor showed that they could accumulate in the tumor.
Especially for 99mTc-2c, the biodistribution and SPECT imaging
studies in mice bearing S180 tumor showed it had an obvious
tumor uptake and good target/no-target ratios, suggesting it would
a potential tracer for hypoxia imaging in tumor.
In vitro cellular uptake
In vitro uptake of the 99mTc complexes were performed using the
50 murine sarcoma S180 cell lines. The cells were incubated in
hypoxic and aerobic conditions and evaluated by the previously
105 Conflict of interest
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

MedChemComm
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The author(s) confirm that this article content has no conflicts of
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65
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DOI: 10.1039/C8MD00146D
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DOI: 10.1039/C8MD00146D
99mTc-2c can be prepared by direct labelling method without the need for heating and would be a
promising probe for hypoxia imaging.