Improved methods for thermal rearrangement of alicyclic α-hydroxyimines to α-aminoketones: Synthesis of ketamine analogues as antisepsis candidates

Article abstract of DOI:10.3390/molecules17066808

Ketamine is an analgesic/anesthetic drug, which, in combination with other drugs, has been used as anesthetic for over 40 years. Ketamine induces its analgesic activities by blocking the N-methyl-D-aspartate (NMDA) receptor in the central nervous system (CNS). We have reported that low doses of ketamine administrated to patients before incision significantly reduced post-operative inflammation as reflected by reduced interleukin-6 (IL-6) sera-levels. Our data demonstrated in a rat model of Gram-negative bacterial-sepsis that if we inject a low dose of ketamine following bacterial inoculation we reduce mortality from approximately 75% to 25%. Similar to what we have observed in operated patients, the levels of TNF-α and IL-6 in ketamine-treated rats were significantly lower than in septic animals not treated with ketamine. On the base of these results, we have designed and synthesized series of new analogues of ketamine applying a thermal rearrangement of alicyclic α-hydroxyimines to α-aminoketones in parallel arrays. One of the analogues (compound 6e) displayed high activity in down-regulating the levels of IL-6 and TNF-α in vivo as compared to ketamine.

Full text of DOI:10.3390/molecules17066808

Molecules 2012, 17, 6808-6820; doi:10.3390/molecules17066808
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Radiolabeling and Biological Evaluation of Propylene
Amine Oxime Complexes Containing Nitrotriazoles as
Hypoxia Markers
Huafan Huang, Lei Mei and Taiwei Chu *
Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key
Laboratory of Fundamental Science, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China
* Author to whom correspondence should be addressed; E-Mail: twchu@pku.edu.cn;
Tel./Fax: +86-10-6275-4319.
Received: 10 May 2012; in revised form: 28 May 2012 / Accepted: 29 May 2012 /
Published: 4 June 2012
Abstract: Two propylene amine oxime (PnAO) complexes, 1, containing a 3-nitro-1,2,4-
triazole and 2, containing two 3-nitro-1,2,4-triazoles, were synthesized and radiolabeled
with 99mTc in high labeling yields. Cellular uptakes of 99mTc-1 and 99mTc-2 were tested
using a S180 cells line. Under anoxic conditions, the cellular uptakes of 99mTc-1 and 99mTc-2
were 33.7 ± 0.2% and 35.0 ± 0.7% at 4 h, whereas the normoxic uptakes of the two
complexes were 6.0 ± 1.6% and 4.6 ± 0.9%, respectively. Both 99mTc-1 and 99mTc-2
displayed significant anoxic/normoxic differentials. The cellular uptakes were highly
dependent on oxygen and temperature. Biodistribution studies revealed that both 99mTc-1
and 99mTc-2 showed a selective localization in tumor and slow clearance from it. At 4 h, the
tumor-to-muscle ratios (T/M) were 3.79 for 99mTc-1 and 4.58 for 99mTc-2. These results
suggested that 99mTc-labeled PnAO complexes 99mTc-1 and 99mTc-2 might serve as novel
hypoxia markers. By introducing a second nitrotriazole redox center, the hypoxic
accumulation of the marker was slightly enhanced.
Keywords: nitrotriazole; technetium-99m; tumor; hypoxia marker; cellular accumulation

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1. Introduction
Owing to the vascular deficiencies, hypoxic cells exist in solid tumors and are resistant to radiation [1].
To improve the efficacy of radiotherapy, numerous radiosensitizers have been developed. One
important strategy to develop hypoxia markers is the use of hypoxic radiosensitizers. A variety of
nitroimidazole derivatives had been synthesized and evaluated as hypoxia markers [2], especially
2-nitroimidazole derivatives. For instance, the 18F labelled 2-nitroimidazole derivative, [18F]fluoro-
misonidazole ([18F]FMISO) had been used for the imaging of tumor hypoxia [3], stroke [4] and
ischemic myocardium [5]. Recently, 4- and 5-nitroimidazolyl compounds had also been developed as
hypoxia markers [6,7].
Nitrotriazole is cheaper and more readily available than 2-nitroimidazole, and nitrotriazole
derivatives had also been widely used as radiosensitizers [8–10]. Several nitrotriazole derivatives have
been investigated as hypoxia markers [11–14]. The biodistribution studies of 99mTc labeled cyclam
N-2'-methoxyethyl-2-(3'-nitro-1'-triazole) acetamide (cyclam AK 2123) [11] and 177Lu labeled
nitrotriazole derivative [12] revealed high tumor to muscle ratios. But no in vitro study was
conducted. The compound 99mTc-1-(3-1,2,4-nitrotriazole-1-yl)-propanhydroxyiminoamide (99mTc-NTPA)
continuously accumulated in hypoxic cells, but not in aerobic cells, and the biodistribution results
indicated that 99mTc-NTPA could localize in tumors [13]. Recently, 3-nitro-1,2,4-triazole derivative
[18F]3-NTR had been synthesized and evaluated as a hypoxia marker. However, its hypoxic cellular
uptake was lower than that of [18F]FMISO, and the biodistribution studies and the PET imaging
showed that the tumor to muscle ratios of [18F]3-NTR were also lower than those of [18F]FMISO [14].
99mTc is an ideal diagnostic radionuclide with versatile chemistry [15]. In most technetium labeled
nitroazole derivatives, such as oxo[[3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diaza-
undecane-2,10-dione dioximato] (3-)-N,N',N'',N''']-technetium (BMS181321) [16,17] and
oxo-[[3,3,9,9-tetramethyl-5-oxa-6-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioximato]
(3-)-N,N',N'',N''']-technetium (BRU59-21) [18], the redox center nitroazole was combined with
chelating groups to coordinate with 99mTc. The chemical structure of BMS181321 was characterized
using 99TcO(PnAO-1-(2-nitroimidazole). 99TcO(PnAO-1-(2-nitroimidazole) was synthesized and
analyzed with X-ray crystallography [16]. It had square-pyramidal geometry about the five-coordinate
99Tc metal core, and oxygen occupied an apical position.
In our previous work, several 99mTc-labeled PnAO complexes containing nitroimidazoles were
prepared, and the effect of a second nitroimidazole redox center on the hypoxic accumulation was
investigated. It was very interesting that by introducing a second 2-nitroimidazole redox center to
BMS181321, the hypoxic accumulation increased significantly. However, a second 4-nitroimidazole
showed little effect [19].
To study the effect of a second nitrotriazole, another redox center on the hypoxic accumulation, in
this study, using nitrotriazole as redox center and propylene amine oxime (PnAO) as chelating group,
we synthesized PnAO complexes 1, containing a 3-nitro-1,2,4-triazole and 2, containing two 3-nitro-
1,2,4-triazoles (Figure 1). Compounds 1 and 2 were labeled with 99mTc. The in vitro and in vivo
evaluation was also investigated.

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Figure 1. The structure of PnAO complexes 1 and 2.
NO2
N
NO2
N
O2N
NH HN
NH HN
N
N
N
N
N
N
N
N
N
N
N
OH OH
OH OH
2
1
2. Results and Discussion
2.1. Synthesis
The synthesis routes of PnAO complexes 1 and 2 are shown in Scheme 1. Alkylation of 3-nitro-1H-
1,2,4-triazole with bromo olefin provided compound 3. Chloronitroso derivative 4 was obtained by the
addition of conc. HCl to the mixture of isoamyl nitrite and olefin 3, and then the reaction between
chloronitroso derivative 4 and diamine mono oxime 5 afforded precursor 1. Precursor 2 was achieved
via the reaction of chloronitroso derivative 4 with 1,3-diaminopropane
Scheme 1. The synthesis of compound 1 and 2.
O2N
NO2
O2N
Cl
K2CO3
N
N
N
N
(CH3)2CHCH2CH2ONO
HCl
N
NH
+
Br
N
N
N
N
OH
3
4
NH NH2
NO2
NO2
N
Cl
NH HN
N
N
N
N
N
N
N
N
N
OH
OH
OH OH
4
5
1
NO2
O2N
NH HN
N
N
1,3-diaminepropane
N
N
N
N
4
N
N
OH OH
2
2.2. Radiolabeling
The 99mTc-complexes were prepared using SnCl2 as a reducing agent. Precursors 1 and 2 were
labeled with 99mTc by ligand exchange from 99mTc-DTPA. The labeling mixture was analyzed by high
performance liquid chromatography (HPLC) [19]. The retention times of 99mTc-DTPA and 99mTcO4
−
were 1.8 and 2.7 min, whereas the radioactivity signals of 99mTc-1 and 99mTc-2 showed a single peak at
13.7 and 13.8 min, respectively (Figure 2). The initial radiochemical purities of 99mTc-1 and 99mTc-2
were above 95%. The radiochemical purities remained above 90% after being kept at room
temperature for 8 h. The stability of 99mTc-2 containing two nitrotriazoles was better than that of
the PnAO analogue containing two 2-nitroimidazoles, for which the purity remained above 80% after
4 h [19].

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Figure 2. Quality control analysis of labeling mixture. Radioactivity signals of
99mTc-DTPA (A), 99mTcO4− (B), 99mTc-1 (C) and 99mTc-2 (D).
2.3. Octanol/Water Partition Coefficient
The partition coefficients were determined by the reported procedure [20,21]. The partition
coefficients (log Po/w) of 99mTc-1 and 99mTc-2 were 1.74 and 1.42, respectively. 99mTc-1 was more
lipophilic than 99mTc-2.
2.4. In Vitro Study
After labeling, in vitro studies of 99mTc-1 and 99mTc-2 were carried out using S180 cells. The
cellular uptakes of 99mTc-1 and 99mTc-2 under normoxic and anoxic conditions were plotted versus
time in Figure 3. Under normoxic conditions, the cellular uptakes of 99mTc-1 and 99mTc-2 fluctuated at
a range of 3–7%. In contrast, the cellular uptakes under anoxic conditions showed a continued increase
with time. Significant differences between normoxic and anoxic uptakes were observed in both
99mTc-1 and 99mTc-2 (independent t-test, p values <0.01). For 99mTc-1 containing one 3-nitro-1,2,4-
triazole, the initial cellular uptake under anoxic condition was 10.3 ± 0.9% at 30 min, and it reached up
to 14.4 ± 0.5%, 23.2 ± 0.5% and 33.7 ± 0.2% at 1, 2 and 4 h. Whereas the uptakes under normoxic
condition were 4.5 ± 0.9% at 30 min, and there was little significant increase over four hours. For
99mTc-2 containing two 3-nitro-1,2,4-triazoles, the anoxic cellular uptakes were 9.4 ± 0.7%, 13.9 ± 0.3%,
22.3 ± 0.1% and 35.0 ± 0.7% at 30 min, 1, 2 and 4 h. In contrast, the normoxic uptakes were 3.1 ± 1.4%
by 30 min, and the value was not statistically different during the four hours.
BMS181321 was found to selectively accumulate in anoxic CHO cells [17]. In our previous work [19],
BMS181321 also accumulated in S180 cells under anoxic conditions, and the anoxic uptake was
24.4 ± 0.7% at 4 h. In the cells, nitroazoles including nitroimidazole and nitrotriazole undergo a single
electron reduction catalyzed by xanthine oxidases. In the presence of sufficient oxygen, the free radical
anion can be reoxidized by oxygen, and diffuse out of the cells. However, under hypoxic condition, it
can be further reduced and bind to cellular macromolecules [22]. By changing 2-nitroimidazole to

Molecules 2012, 17
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3-nitro-1,2,4-triazole, the anoxic uptake increased to 33.7 ± 0.2% at 4 h. The reduction potential of 3-nitro-
1, 2,4-triazole (−0.55 V) is more negative than 2-nitroimidazole (−0.40 V) [23]. Thus 2-nitroimidazole
was believed to be a more efficient electron acceptor, and it can accept electrons more easily to form
the radical anion. However, 99mTc-1 containing a 3-nitro-1,2,4-triazole showed higher anoxic cellular
accumulation compared with BMS181321 containing a 2-nitroimidazole [19].
Both 99mTc-1 and 99mTc-2 containing nitrotriazoles showed significant anoxic/normoxic differentials.
The uptake ratios (anoxic/normoxic) at 30 min, 1, 2 and 4 h were 2.3, 3.0, 4.7 and 5.6 for 99mTc-1, and
3.0, 3.3, 6.2 and 7.5 for 99mTc-2. 99mTc-2 showed higher anoxic/normoxic uptake ratios compared with
99mTc-1, indicating that the anoxic/normoxic differentials increased by introducing a second 3-nitro-1,
2,4-triazole moiety into the molecule. Both 99mTc-1 and 99mTc-2 exhibited higher anoxic/normoxic
uptake ratios compared with BMS181321 [19]. Thus 3-nitro-1,2,4-triazole moiety can work as a redox
center with appropriate reduction potential.
Figure 3. The accumulation of 99mTc-1 (A) and 99mTc-2 (B) in S180 cells under anoxic
conditions (black dot), normoxic conditions (black triangle) and anoxic switched to
normoxic after 2 h (blank triangle).
In another series of cellular uptake studies, the anoxic exposure was switched to the normoxic
exposure at 2 h. The cellular uptakes of both 99mTc-1 and 99mTc-2 declined slightly in the next
two hours (Figure 3). Oxygen can reoxidize the free radical anion to the original drug. The entry of
oxygen restrained the cellular uptakes of nitrotriazole derivatives. Therefore, there was no further
accumulation after 2 h. All the metabolism products of markers would be bound to the cellular
components irreversibly under ideal conditions. However, incomplete retention of metabolized
markers had been reported in previous studies. For instance, BMS181321 showed 60% of the counts
were retained after washing, and these counts were continually lost in the later incubation [17]. The
radiolabeled metabolites of BRU59-21 appeared in the supernatants in hypoxic cells [18]. In this study,
the supernatants were also analyzed with radio-HPLC during the cellular studies. The initial
radiochemical purity of 99mTc-1 was above 95%. After 4 h, there was little change in the radio
chromatographic signals under normoxic conditions, the radiochemical purity remained 94%. In
contrast, after 4 h of incubation under anoxic condition, the radiochemical purity of 99mTc-1 was only
44%. The main metabolites showed the same retention time as 99mTcO4−. These results suggested that
the metabolites of 99mTc-1 might be released from the anoxic cells into the medium. Therefore, the
cellular uptakes of 99mTc-1 and 99mTc-2 declined slightly after switching to the normoxic exposure.

Molecules 2012, 17
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To study the effect of temperature, the cellular uptake studies were also performed at 4 C and 25 C
(Figure 4). Under anoxic conditions, the cellular uptakes of 99mTc-1 and 99mTc-2 were increased
dramatically by rising the temperature. At 4 h, the anoxic uptakes of 99mTc-1 at 4 C, 25 C and 37 C
were 3.5 ± 0.4%, 23.1 ± 0.6% and 33.7 ± 0.2%, respectively. In contrast, the anoxic uptakes of 99mTc-2
at 4 C, 25 C and 37 C were 2.3 ± 0.3%, 14.1 ± 1.2% and 35.0 ± 0.7% at 4 h. The anoxic uptakes of
99mTc-1 and 99mTc-2 strongly depended on temperature. Under normoxic conditions, the cellular
uptakes were low and the measurement errors were relatively high, so the normoxic uptakes showed
no significant difference at various temperatures. The differences in anoxic accumulation at 4 C, 25 C
and 37 C might be due to the enzyme activity and membrane fluidity. The reduction of nitrotriazole in
the cell is an enzymatic process. At low temperature, enzymes (xanthine oxidases) are inactive.
Enzyme activity increases gradually with rising temperature until the optimum temperature (35–45 C
for xanthine oxidases). On the other hand, the markers enter the cells by diffusion. High temperatures
increase the fluidity of membranes, and the markers enter the cells more readily.
Figure 4. The anoxic uptakes of 99mTc-1 (A) and 99mTc-2 (B) in S180 cells at 4 C (blank
triangle), 25 C (black triangle) and 37 C (black dot).
Both of 99mTc-1 (log Po/w = 1.74) and 99mTc-2 (log Po/w = 1.42) showed lipophilic nature, and the
high partition coefficients allowed them cross the membrane readily by diffusion. 99mTc-1 was more
lipophilic than 99mTc-2, however, 99mTc-2 exhibited higher anoxic/normoxic uptake ratios. These
results indicated that the lipophilicity may be an important factor in cellular accumulation, but other
factors are of greater importance in this study.
2.5. Biodistribution Study
The in vivo studies were performed in Kunming mice bearing S180 tumor to determine the tumor
specificity. The biodistribution results are tabulated in Tables 1 and 2. For both 99mTc-1 and 99mTc-2,
the liver was the tissue with highest radioactivity, and the radioactivity in the kidneys was low.
These results suggested lipophilic complexes 99mTc-1 and 99mTc-2 were largely cleared through the
hepatobiliary pathway. The tumor uptakes decreased slowly after injection, from 0.47 ± 0.08 %ID/g at
0.5 h to 0.35 ± 0.03 %ID/g at 4 h for 99mTc-1 and 0.39 ± 0.06 %ID/g at 0.5 h to 0.25 ± 0.06 %ID/g at 4 h
for 99mTc-2. The tumor-to-blood ratios (T/B) were 0.39 for 99mTc-1 and 0.31 for 99mTc-2 at 4 h. Both
99mTc-1 and 99mTc-2 showed a slow blood clearance, similar to that observed in BMS181321 [17]. This

Molecules 2012, 17
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might be due to the high octanol/water partition coefficients of these complexes. However, the uptakes
in muscle were low and cleared relatively quickly. The muscle uptakes decreased from 0.19 ± 0.03 %ID/g
at 0.5 h to 0.09 ± 0.01 %ID/g at 4 h for 99mTc-1, and from 0.22 ± 0.04 %ID/g at 0.5 h to
0.05 ± 0.01 %ID/g at 4 h for 99mTc-2. The tumor-to-muscle ratios (T/M) increased with time after
injection, and the ratios were 3.79 for 99mTc-1 and 4.58 for 99mTc-2 at 4 h. Compared with BMS181321
at 4 h after injection (3.53 in KHT tumor, 4.21 in SCC tumor and 3.54 in RIF-1 tumor) [17], 99mTc-1
and 99mTc-2 showed similar or higher tumor specificity. By introducing a second nitrotriazole, 99mTc-2
was a better tumor hypoxia marker compared with 99mTc-1.
Table 1. Biodistribution of 99mTc-1 in mice bearing S180 tumour (%ID/g).
Tissue
Blood
Heart
Lung
Liver
Spleen
Stomach
Kidney
Muscle
Brain
Tumor
T/B
T/M
0.5 h
1 h
2 h
4 h
2.61 ± 0.65
0.57 ± 0.13
1.91 ± 0.74
12.26 ± 5.14
0.52 ± 0.05
1.47 ± 0.36
1.73 ± 0.42
0.19 ± 0.03
0.08 ± 0.01
0.47 ± 0.08
0.16 ± 0.03
2.46 ± 0.29
1.97 ± 0.72
0.43 ± 0.04
1.30 ± 0.12
8.15 ± 0.59
0.43 ± 0.05
1.42 ± 0.27
1.41 ± 0.42
0.16 ± 0.05
0.05 ± 0.01
0.45 ± 0.01
0.26 ± 0.10
2.72 ± 0.55
1.32 ± 0.40
0.28 ± 0.02
1.21 ± 0.23
7.41 ± 2.03
0.37 ± 0.03
1.51 ± 0.19
1.17 ± 0.13
0.11 ± 0.02
0.05 ± 0.01
0.42 ± 0.05
0.34 ± 0.12
3.88 ± 0.40
0.91 ± 0.37
0.24 ± 0.03
0.83 ± 0.12
4.91 ± 0.55
0.30 ± 0.05
1.16 ± 0.18
0.89 ± 0.16
0.09 ± 0.01
0.05 ± 0.01
0.35 ± 0.03
0.39 ± 0.19
3.79 ± 0.59
Table2. Biodistribution of 99mTc-2 in mice bearing S180 tumour (%ID/g).
Tissue
Blood
Heart
Lung
Liver
Spleen
Stomach
Kidney
Muscle
Brain
Tumor
T/B
T/M
0.5 h
1 h
2 h
4 h
1.30 ± 0.14
0.38 ± 0.13
0.57 ± 0.06
15.90 ± 2.06
0.31 ± 0.02
2.30 ± 1.42
1.85 ± 0.16
0.22 ± 0.04
0.03 ± 0.01
0.39 ± 0.06
0.31 ± 0.03
1.80 ± 0.39
1.17 ± 0.11
0.29 ± 0.04
0.49 ± 0.14
18.77 ± 3.03
0.32 ± 0.09
1.54 ± 0.30
1.58 ± 0.28
0.12 ± 0.01
0.03 ± 0.00
0.34 ± 0.08
0.30 ± 0.04
2.87 ± 0.91
0.75 ± 0.38
0.24 ± 0.08
0.41 ± 0.09
13.80 ± 3.58
0.29 ± 0.09
1.00 ± 0.11
1.42 ± 0.15
0.08 ± 0.02
0.02 ± 0.00
0.27 ± 0.06
0.47 ± 0.29
3.35 ± 0.60
0.84 ± 0.25
0.18 ± 0.03
0.31 ± 0.03
9.09 ± 2.40
0.15 ± 0.02
0.75 ± 0.27
0.92 ± 0.15
0.05 ± 0.01
0.02 ± 0.00
0.25 ± 0.06
0.31 ± 0.08
4.58 ± 0.76
3. Experimental
3.1. General
3-Nitro-1H-1,2,4-triazole was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan).
1,3-Diaminopropane (98%) and N,N-diisopropylethylamine (98%) were supplied from Acros Organics

Molecules 2012, 17
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(Geel,Belgium). All other reagents were of analytical grade. NMR spectra were obtained on Bruker
(400 MHz and 500 MHz) spectrometers (Bruker, Faellanden, Switzerland). Chemical shifts are given
in ppm relative to tetramethylsilane used as an internal standard, the coupling constants are expressed
in hertz, and the splitting patterns are designated as s (singlet), d (doublet), t (triplet) and m (multiplet).
Mass spectra were measured on a Bruker APEX IV FTMS, positive mode, ESI. Elemental analyses
were carried out on an Elementar Vario MICRO CUBE (Hanau, Germany). RP-HPLC analyses were
performed on a Waters 1525 binary HPLC pump and a Waters 2487 UV absorbance dual  detector
(Milford, MA USA). The elution was also monitored with a Packard 500 TR flow scintillation
radioactivity detector (Meriden, CT, USA). Murine sarcoma S180 cell line was provided by the
College of Life Sciences, Peking University. Dulbecco’s Modified Eagle’s Medium (DMEM) was
from Gibco BRL Life Technologies (Grand Island, NY, USA) and fetal bovine serum from Hyclone
(Logan, UT, USA). The JPSJ-605 dissolved oxygen meter was purchased from REX Instrument
Factory of Shanghai Precision & Scientific Instrument Co., LTD. (Shanghai, China).
3.2. Synthesis
The synthesis routes for compound 1 and 2 are depicted in Scheme 1. Compound 5 was synthesized
according to the method reported in the literature [24].
1-(3-Methylbut-2-enyl)-3-nitro-1H-1,2,4-triazole (3). 1-Bromo-3-methylbut-2-ene (1.5 mL, 13 mmol)
was added to a mixture of K2CO3 (0.72 g, 5.2 mmol) and 3-nitro-1H-1,2,4-triazole (0.51 g, 4.5 mmol) in
acetone (15 mL). The mixture was refluxed over night. Acetone was evaporated under reduced
pressure and the residue was redissolved in EtOAc. The organic layer was washed with water (3 × 20 mL)
and dried by Na2SO4. Evaporation of solvent gave brown oil. The crude product was purified by
column chromatography (silica gel, petroleum ether–EtOAc = 2:1) to provide 3 as a light yellow
1
oil (0.70 g, yield 85%). H-NMR (CDCl3, 400 MHz) δ 8.16 (s, 1H, triazole-H), 5.47 (t, 1H,
=CHCH2-), 4.88 (d, 2H, =CHCH2-), 1.86 (s, 3H, =C(CH3)2), 1.82 (s, 3H, =C(CH3)2); 13C-NMR
(DMSO-d6, 125 MHz) δ 162.0, 146.0, 139.4, 116.8, 48.3, 25.1, 17.8; HRMS (ESI): calculated for
C7H10N4NaO2 [M+Na]+ 205.06960, found 205.06923.
3-Chloro-3-methyl-1-(3-nitro-1H-1,2,4-triazol-1-yl)butan-2-one oxime (4). Compound 3 (0.98 g,
5.4 mmol) and isoamyl nitrite (1.6 mL, 12 mmol) was cooled to −30 °C in an acetone-dry ice bath and
vigorously stirred. Concentrated hydrochloric acid (1.4 mL) was added dropwise over 30 min. The
mixture was stirred at −30 °C to −20 °C for another 2 h. The precipitated was isolated by filtration and
washed with cold methanol-ether = 1:5 (10 mL) to give 4 as a white solid (1.09 g, yield 82 %).
1H-NMR (DMSO-d6, 400 MHz) δ 11.97(s, 1H, C=N-OH), 8.88 (s, 1H, triazole-H), 5.33 (s, 2H, NCH2-),
1.87 (s, 6H, -C(CH3)2-); HRMS (ESI): calculated for C7H11ClN5O3 [M+H]+ 248.05449, found
248.05444.
3-(3-(3-(Hydroxyimino)-2-methylbutan-2-ylamino)propylamino)-3-methyl-1-(3-nitro-1H-1,2,4-triazol-
1-yl)butan-2-one oxime (1). Compound 4 (0.054 g, 0.22 mmol), compound 5 (0.043 g, 0.25 mmol) and
(i-Pr)2NEt (100 μL) were added in dry acetonitrile (2 mL), and the mixture was stirred at room
temperature for 24 h. Acetonitrile was removed on a rotary evaporator. The crude product was purified

Molecules 2012, 17
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by column chromatography (silica gel, CH2Cl2-CH3OH = 10:1) and recrystallized from CH2Cl2 to
1
provide a yellow solid (0.072 g, yield 86%), m. p. 63.5–64.3 °C; H-NMR (DMSO-d6, 400 MHz)
δ 11.32 (s, 1H, C=N-OH), 10.35 (s, 1H, C=N-OH), 8.79 (s, 1H, triazole-H), 5.16 (s, 2H, NCH2), 2.23
(m, 4H, -NHCH2-), 1.69 (s, 3H, N=CCH3), 1.40 (m, 2H, -CH2CH2CH2-), 1.23 (s, 6H, C(CH3)2), 1.11
(s, 6H, C(CH3)2); 13C-NMR (DMSO-d6, 125 MHz) δ 161.4, 160.1, 155.7, 147.9, 56.7, 56.6, 43.1, 41.1,
31.3, 25.6, 25.4, 8.9; HRMS (ESI): calculated for C15H29N8O4 [M+H]+ 385.23063, found 385.23008.
3-(3-(3-(Hydroxyimino)-2-methyl-4-(3-nitro-1H-1,2,4-triazol-1-yl)butan-2-ylamino)propylamino)-3-
methyl-1-(3-nitro-1H-1,2,4-triazol-1-yl)butan-2-one oxime (2). Compound 4 (0.046 g, 0.19 mmol),
1,3-diaminopropane (8 μL, 0.096 mmol) and (i-Pr)2NEt (50 μL) were added in dry acetonitrile (2 mL),
and the mixture was stirred at 40 C for 0.5 h. Acetonitrile was removed. The crude product was
purified by column chromatography (silica gel, CH2Cl2–CH3OH = 10:1) and recrystallized from
1
CH2Cl2 to provide a white solid (0.039 g, yield 84%), m.p. 158.2–159.1 °C; H-NMR (DMSO-d6,
400 MHz) δ 11.32 (s, 2H, C=N-OH), 8.79 (s, 2H, triazole-H), 5.16 (s, 4H, NCH2), 2.25 (t, 4H,
-NHCH2-), 1.40 (m, 2H, -CH2CH2CH2-), 1.23 (s, 12H, C(CH3)2), 0.98 (t, 2H, -NHCH2CH2CH2-).
13C-NMR (DMSO-d6, 125 MHz) δ 161.4, 155.7, 147.8, 56.7, 43.1, 40.9, 31.3, 25.4; HRMS (ESI):
calculated for C17H29N12O6 [M+H]+ 497.23275, found 497.23254.
3.3. Radiolabeling
Na99mTcO4 was obtained from a 99Mo/99mTc generator. The labeling of compound 1 and 2 was
conducted according to the literature methods [17] with minor modification. 99mTcO4− (100 µL, 15.0 MBq),
the ligand solution (100 µL, 2 mg/mL), phosphate buffer solution (400 µL, pH 7.2, 0.2 mol/L), saline
(400 µL), diethylenetriaminepentaacetic acid (DTPA) (2 µL, 2.5 mg/mL) were mixed in a 2 mL
centrifugal tube. The mixture was purged with argon and fresh SnCl2 solution (1.5 µL, 1 mg/mL) was
added. The labeling mixture was stirred in ice water bath for 15 min and filtrated by 0.22 µm filter.
The radio high-performance liquid chromatography (radio-HPLC) method had been described in
previous work [19]. The stabilities of 99mTc-1 and 99mTc-2 were investigated in saline for 8 h at 37 °C.
3.4. Octanol/Water Partition Coefficient
The partition coefficients were determined by the reported procedure [20,21] with minor differences.
Octanol (1.0 mL), 99mTc-labeling solution (0.5 mL) and saline (0.5 mL) were mixed in a 2 mL centrifugal
tube. The tube was vigorously vortexed for 5 min and centrifuged at 3000 rpm for another 5 min. Five
samples (10 µL) of each phase were removed into gamma counter tubes and counted for radioactivity.
The PO/W was the ratio of the radioactivity of the octanol layer to that of the water layer. Then the
octanol layer (0.8 mL) was moved to another centrifugal tube, in which octanol (0.2 mL) and saline
(1.0 mL) were added. The tube was vortexed and centrifuged. Samples of each phase were removed
and counted. This process was repeated for several times until consistent PO/W was obtained.
3.5. In Vitro Study
The cellular accumulation experiments were performed according to the literature methods [25,26].
Murine sarcoma S180 cells were suspended in a fresh DMEM medium plus 10% (v/v) of fetal bovine

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serum (FBS). The final cell concentration was 2 × 106 cells/mL. Aliquots of 20 mL were added to
glass vials and gently stirred with magnetic spinbars. The glass vials were placed in water baths to
control the temperature. The cells were incubated under normoxic conditions (95% air plus 5% carbon
dioxide) or anoxic conditions (95% nitrogen plus 5% carbon dioxide, below 10 ppm O2). After an
equilibration for 30–45 min, The JPSJ-605 dissolved oxygen meter read 0.00 mg/L, and the
99mTc-labeling marker (5 MBq in 0.5 mL) was added to each glass vial and the final concentration of
unlabeled drug was 5 µg/mL. More than 1 mL sample was removed each 30 minutes. Five aliquots
(200 µL) were pipetted from each sample removed and centrifuged at 1500 rpm for 5 minutes. 180 µL
of supernatant was removed for counting (A), and the cells and medium left were also counted (B).
The cellular uptakes were calculated as % Uptake = [(B − A/9) / (A + B)] × 100%. The trypan blue
exclusion assay revealed that S180 cells maintained more than 90% viability for 4 h.
To imitate the physiological circumstances, the temperature in the accumulation experiments was
37 C. At 1, 2 and 4 h, aliquots of supernatant were filtrated by 0.22 µm filter. Filtered supernatant was
analyzed with radio-HPLC [18]. To investigate the effect of entry of oxygen to the cellular uptakes of
hypoxia markers, the anoxic exposure was switch to normoxic exposure in another series of cellular
accumulation experiments. To study the effort of temperature, the cellular accumulation experiments were
also conducted at 25 C (room temperature) and 4 °C [27].
3.6. Biodistribution Study
All the animal experiments were performed in accordance with the national laws related to the
conduct of animal experimentation. The biodistribution of 99mTc-1 and 99mTc-2 was evaluated in
Kunming male mice (20–25 g) bearing the S180 tumor in the left front leg (diameter of 10–15 mm).
The 99mTc-complex was (1 MBq in 0.1 mL) administered by tail vein injection. The mice were
sacrificed by cervical dislocation at different times after injection. Various organs and tissues were
excised, weighed and counted. The percent injected dose per gram (%ID/g) of each organ/tissue or
tumor was calculated by the above data. The final results were expressed as means ± SD. of five
parallel experiments.
4. Conclusions
Two propylene amine oxime derivatives 1 and 2 containing 3-nitro-1,2,4-triazole moieties were
synthesized and labeled with 99mTc at high labeling efficiencies (95%). Both 99mTc-1 and 99mTc-2 were
stable at 8 h after labeling. In vitro cellular uptake studies, 99mTc-1 and 99mTc-2 displayed significant
anoxic/normoxic differentials. The anoxic cellular uptakes of 99mTc-1 and 99mTc-2 reached to
33.7 ± 0.8% and 35.0 ± 0.7% at 4 h, whereas the normoxic cellular uptakes were 6.0 ± 1.6% and
4.6 ± 0.9%, respectively. The entry of oxygen restrained the cellular uptakes of 99mTc-1 and 99mTc-2,
and the anoxic cellular uptakes were highly dependent on the temperature. In biodistribution
studies, the tumor-to-muscle ratios (T/M) of 99mTc-1 and 99mTc-2 increased with time. At 4 h, the
tumor-to-muscle ratios (T/M) were 3.79 for 99mTc-1 and 4.58 for 99mTc-2. These results suggested that
nitrotriazole derivatives 99mTc-1 and 99mTc-2 might serve as novel hypoxia markers. Moreover, by
introducing a second nitrotriazole redox center, 99mTc-2 showed a higher selective localization in
tumor hypoxia compared with 99mTc-1.

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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 21071010).
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Sample Availability: Samples of the compounds mentioned are available from the authors.
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