Synthesis and biological evaluation of novel 99mTcN-labeledbisnitroimidazole complexes containing monoamine-monoamidedithiol as potential tumor hypoxia markers

Lei Mei • Wenjing Sun • Taiwei Chu

Received: 6 January 2014

� Akademiai Kiado, Budapest, Hungary 2014

Abstract Tumor hypoxia can decrease the efficacy of

clinical therapy due to resistance toward radiation damage

and chemotherapy, thus detection of tumor hypoxia by

radiolabeled hypoxia markers is important for the control

of tumor. Radiopharmaceuticals with two bioreductive

groups, such as propylene amine oxime-bisnitroimidazole

or monoamine-monoamide dithiol (MAMA) -bisnitroimi-

dazole, have potential to improve hypoxia selectivity. In

order to obtain radiopharmaceuticals with better features,

we synthesized two novel [99mTcN]2? complexes with

bisnitroimidazole moieties and MAMA ligand for targeting

tumor hypoxia. Their physicochemical characters and

biodistribution were also investigated. Both the [99m-

TcN]2? complexes show good stability and hydrophilicity.

They show faster clearance from blood and soft tissues,

better tumor retention and favorable tumor-to-tissue ratios

compared with a control complex without nitroimidazole

group. In addition, both of them show more favorable

biodistribution patterns than the corresponding [99mTcO]3?

complexes. These results indicate that the 99mTcN-labeled

MAMA-bisnitroimidazole complexes would have potential

to image tumor hypoxia in vivo.

Keywords Tumor hypoxia � Radiolabeling � MAMA �Bisnitroimidazole � Technetium–nitrido complex

Introduction

Tumor hypoxia, which results from excessive growth of

tumor beyond the capacity of accompanying blood vascu-

lature to supply adequate quantities of oxygen [1], is a

characteristic of many solid tumors. Tumor cells in hypoxic

regions show resistance toward radiation damage and

chemotherapy, and decrease the efficacy of clinical therapy

[2, 3]. Moreover, tumor hypoxia is related with malignant

progression and metastasis of tumor [4, 5]. Therefore, the

detection and evaluation of tumor hypoxia, especially by

non-invasive imaging, will be of great importance for the

control of tumor. Nuclear medicine imaging [6, 7] based on

hypoxia-targeting radiopharmaceuticals, is mostly applied

to detect hypoxia for their sensitivity and penetrability. To

date, various hypoxia markers which always employ radi-

olabeled bioreductive pharmacophores as bioactive moiety

(such as nitroimidazole) have been developed for PET and

SPECT imaging of tumor hypoxia [8–15].

For most of hypoxia markers, only one bioreductive

group is contained in the molecular structure. Bonnitcha

et al. [16] reported three nitroimidazole-bis(thio -semi-

carbazonato)copper(II) conjugates comprising two biore-

ductive pharmacophores including a nitroimidazole moiety

and a Cu-ATSM moiety. All the three conjugates displayed

greater selectivity than a simple propyl derivative of Cu-

ATSM used as control. In our previous study, three 99mTc-

labeled propylene amine oxime derivatives containing two

nitroimidazole groups were synthesized [17]. Better

hypoxia selectivity was observed for these complexes than

the corresponding compound with only one nitroimidazole

group. Recently, a further work [18] by our group reported

two bisnitroimidazole compounds with monoamine-

monoamide dithiol (MAMA) ligand as the chelating agent

for 99mTc-oxo [99mTcO]3? core. Both the 99mTcO-labeled

Lei Mei and Wenjing Sun contributed equally to this work.

L. Mei � W. Sun � T. 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,

People’s Republic of China

e-mail: twchu@pku.edu.cn

123

J Radioanal Nucl Chem

DOI 10.1007/s10967-014-3235-6

bisnitroimidazole complexes showed rapider excretion,

lower background activity in liver and higher tumor-to-

tissue ratios than the mononitroimidazole complexes. Ini-

tial results demonstrate that combination of two bioreduc-

tive groups with the same biological target has the potential

to improve hypoxia selectivity. However, the unfavorable

physicochemical characters and relatively low tumor-to-

tissue ratios of 99mTcO-MAMA markers make them sub-

optimal for imaging hypoxia. In order to obtain radio-

pharmaceuticals with better features, further modifications

of their molecular structures are needed.

The metal core in the molecule of a radiopharmaceutical

has a dramatic effect on its physical and biological

behaviors. Besides the [99mTcN]2? [17, 18] which is

mostly used in nuclear medicine, other technetium-based

cores, such as Tc-nitrido [99mTcN]2? or Tc-tricarbonyl

[99mTc(CO)3]?, have been also employed [11–15]. The

[99mTcN]2? core, initially developed by Baldas and Bon-

nyman [19], is isoelectronic with [99mTcO]3? and shows

high affinity toward chelating ligands containing sulfur

atoms. The nitrido ligand as a p-electron donor in the Tc-

nitrido core, which can well-stabilize the Tc(V) oxidation

state, endows it with intrinsic structural robustness [20].

These remarkable properties of the Tc-nitrido core make it

a promising tool for the design of novel radiopharmaceu-

ticals as hypoxia markers.

Monoamine-monoamide dithiol (MAMA) ligand is a

‘N2S2’-type ligand and can be used as a chelator for

technetium ([99mTcO]3? or [99mTcN]2?). We have pre-

pared [99mTcO]3? labeled MAMA derivatives, which

contain two nitroimidazoles as the hypoxia-targeting moi-

eties [18]. In this work, we choose the [99mTcN]2? core as

radiolabeling center for MAMA. Herein, two MAMA-

bisnitroimidazole ligands were synthesized and their99mTcN-complexes were prepared. Compound 3, which is a

simple propyl derivative of MAMA, was also prepared and

its 99mTcN-complex was used as a control. A preliminary

evaluation of physicochemical properties and potentiality

as hypoxia imaging agents of both the 99mTcN-MAMA

complexes was presented.

Experimental

Synthesis and radiolabeling

The preparation of MAMA-bisnitroimidazole compounds

MAMA-B4NIL (1), MAMA-B2NIL (2) and MAMA-

mononitroimidazole compound MAMA-Pr (3) (Fig. 1)

were carried out following previously reported methods

[18, 21, 22].

Preparation of the 99mTcN-complexes was performed by

two-steps. Firstly, a certain amount of freshly eluted99mTcO4

-(* 1 mCi) obtained from commercial99Mo-99mTc generator (China Institute of Atom Energy,

Beijing) was added to a succinic dihydrazide (SDH) kit

N

HN

HN

N

O

O

N

NR1

R2

R1R2

HN

O

HN

O

S

N

S

HN

O

CPh3 CPh3

1 MAMA-B4NIL R1=H, R2=NO22 MAMA-B2NIL R1=NO2, R2=H

N

HN

HN

N

O

O

N

NR1

R2

R1R2

HN

O

HN

O

S

N

S

N

O

Tc

N

99mTcN-1 R1=H, R2=NO299mTcN-2 R1=NO2, R2=H

S

N

S

HN

O

CPh3 CPh3

HN

O

3 MAMA-Pr

S

N

S

N

O

Tc

NHN

O

99mTcN-3

Fig. 1 Chemical structures of

MAMA-bisnitroimidazole

ligands, 1 and 2, a control

compound 3 and their

corresponding 99mTcN

complexes, 99mTcN-1, 99mTcN-2 and 99mTcN-3

J Radioanal Nucl Chem

123

containing stannous chloride dehydrate as reducing agent,

SDH as nitride donor, and propylenediamine tetraacetic

acid. The mixture was vortexed and kept at room temper-

ature for 10 min to form a 99mTcN-labeled intermediate

[23]. The radiochemical purity for the precursor is found to

be over 99 % by paper chromatography (using acetone as

the solvent) and radio-HPLC.

For 99mTcN-labeling of ligands 1–3, the thiol groups of

these MAMA derivatives were deprotected in trifluoro-

acetic acid and then incubated with the 99mTcN-interme-

diate at 100 �C for 15 min. The final complexes were

obtained by substitution of deprotected MAMA ligands on

the 99mTcN-precursor. The detailed procedure is as follows.

The MAMA compound (1 mg) was dissolved in 0.5 mL

TFA and the resulting bright yellow solution was stirred for

5 min and cooled to 5 �C. The reaction mixture was then

titrated with triethylsilane until the disappearance of the

yellow color. The solution was evaporated to dryness by

rotary evaporation at room temperature, and then neutral-

ized with 0.1 M NaOH aqueous solution. The deprotected

ligand was resolved in 0.5 mL of water and added to the99mTcN-intermediate solution prepared as above. The

mixture was adjusted to neutral, diluted to a total volume of

1 mL with phosphate buffer solution (0.1 M, pH 7.4), and

incubated at 100 �C for another 15 min (final specific

activity of *1 mCi/mL). After cooled to room tempera-

ture, the radiolabeled solution was analyzed by radio-

HPLC.

Radio-HPLC conditions: reversed-phase column (Agi-

lent HC-C18, 4.6 9 150 mm, size 5 micron), a Waters

2487 dual wavelength absorbance detector (Waters 600E

series) and a radiometric detector (Packard 500TR series)

system. Solvent systems for HPLC analysis: Phase A,

0.1 M ammonium acetate; Phase B, CH3CN. Gradient:

0–2 min 90 %A; 2–15 min 90–50 %A; 15–20 min

50–20 %A; 20–22 min 20 %A; 22–24 min 20–90 %A;

flow rate 1.0 mL/min.

Physicochemical studies

Partition coefficients

Partition coefficients were determined in a similar method

as reported [24]. One milliliter of 1-octanol was added to a

mixture of 0.9 mL of phosphate buffer solution (0.1 M, pH

7.4) and 0.1 mL of the radiolabeled complex. The mixture

was vortexed at room temperature for 5 min and centri-

fuged at 2,000 rpm for 5 min. Equal aliquots (100 lL) of

sample were taken from each phase of water and octanol

and counted for radioactivity. The partition coefficient was

calculated by dividing the radioactivity of the octanol layer

with that of the water layer. This measurement was repe-

ated three times.

Stability and protein binding experiments

In vitro stability was determined in a similar method as

reported [13]. The labeled complexes were dissolved in

phosphate buffer solution (0.1 M, pH 7.4) and incubated at

room temperature after preparation. Certain aliquots of

sample were taken at different time points over a period of

4 h and the radiochemical purity was analyzed by HPLC.

Moreover, in vitro serum stability studies in rat serum were

also performed using a method reported earlier [18]. About

0.1 mL of the radiolabeled complex was added to 0.4 mL

of rat serum and this mixture was incubated at 37 �C for

4 h. At intervals of 1, 2 and 4 h, 0.1 mL of aliquots were

withdrawn and 0.2 mL of ethanol was added to precipitate

the serum protein. The mixture was filtered with a 0.45 lm

Millipore filter and the filtrate was analyzed by HPLC to

assess the radiochemical purity of the complex. Plasmatic

protein binding experiments were also performed accord-

ing to the procedure reported [15].

Biodistribution study

Murine sarcoma (S180) cell line and male Kunming mice

(20–25 g, male) were supplied by Department of Labora-

tory Animal Science, Peking University Health Science

Center. Hypodermic injection of approximately 1.0 9 106

cells into the left front leg of male Kunming mice was

performed to establish the murine sarcoma (S180) model.

Tumor grew to diameters of 10–15 mm during about a

week’s time and could be used in study.

The newly prepared radiolabeled complex (100 lL,

1 MBq, 0.05 mg of a total amount of ligand) was injected

into the mice bearing S180 (weighing 20–25 g) via the tail

vein. The mice were sacrificed in groups of three at various

time intervals after injection and the organs or tissues of

interest were removed and washed, which were then

weighed in plastic tubes and counted in an auto gamma

counting system. The results were calculated as percent-

ages of injected dose per gram tissue (i.e., % ID/g), using a

standard equivalent to 1 % of the injected dose. The final

results were expressed as mean ± S.D.(standard

deviation).

All experiments were carried out following the princi-

ples of laboratory animal care and the China’s law on the

protection of animals.

Results and discussions

Synthesis and radiolabeling

The products 1–3 are also identified: the accurate molec-

ular formulas are obtained by high-resolution mass spectra;

J Radioanal Nucl Chem

123

the NMR and IR spectra give the further evidences of their

structures and are consistent with the data reported previ-

ously [18].

The radiochemical purities for all the 99mTcN-labeled

complexes were analyzed by radio-HPLC (Fig. 2). The

retention times of 99mTcO4- and 99mTcN-intermediate are

3.20 and 2.00 min, while those of 99mTcN-1, 99mTcN-2

and 99mTcN-3 are found to be 8.00, 8.30 and 6.10 min,

respectively. The HPLC retention times for these 99mTcN-

labeled complexes are different from those for the corre-

sponding 99mTcO-labeled complexes in the same HPLC

condition (99mTcO-1, 99mTcO-2 and 99mTcO-3: 13.30,

13.50 and 14.70 min, respectively [18]) and this result

demonstrates the successful preparation of 99mTcN com-

plexes, other than 99mTcO complexes. The radiochemical

purities of all the radiolabeled complexes are over 95 %

after preparation.

In vitro stability of 99mTcN-1 and 99mTcN-2 in PBS

(0.1 M, pH 7.4) at room temperature was studied at

Fig. 2 HPLC chromatograms

of 99mTcN-labeled complexes:

a 99mTcO4-, tR = 3.20 min;

b 99mTcNint, tR = 2.00 min; c99mTcN-1, tR = 8.00 min;

d 99mTcN-2, tR = 8.30 min;

e 99mTcN-3, tR = 6.10 min (tR:

Retention time in RP-HPLC)

Table 1 Stability and partition coefficients for 99mTcN-1 and99mTcN-2

Complex Stability in plasmaa PBPPb log Pc

99mTcN-1 [98 15.6 ± 0.5 -2.56 ± 0.0199mTcN-2 [98 20.6 ± 0.6 -2.49 ± 0.04

a Radiochemical purities after 4.0 h of incubation at 37 �Cb PBPP percentage of binding to plasmatic proteinsc log P = Radioactivity(octanol)/Radioactivity(PBS buffer)

J Radioanal Nucl Chem

123

different time during 4 h. The radio-HPLC analysis results

demonstrate that both of 99mTcN-1 and 99mTcN-2 show no

detectable decomposition after incubation for 4 h, indi-

cating their stability in PBS (0.1 M, pH 7.4) in physio-

logical environment. The in vitro stability in rat plasma

was also evaluated [18], and the radiochemical purities are

all over 98 % after 4 h (Table 1). Lower protein binding

values (*15.6 %) of 99mTcN-1 was found in the protein

binding experiment (Table 1).

The partition coefficient, which is a typical physico-

chemical parameter and a relevant indication of pharma-

cokinetic behavior for the radiopharmaceutical, was

determined by distributing the radiolabeled complex in a

mixture of 1-octanol and PBS (0.1 M, pH 7.4). The log P(o/

w) values for 99mTcN-1 and 99mTcN-2 are respectively -

2.56 ± 0.01 and -2.49 ± 0.04. Both the 99mTc-nitrido

complexes are more hydrophilic than the corresponding99mTc-oxo complexes, 99mTcO-1 (log P(o/w): 0.12 ± 0.02)

and 99mTcO-2 (log P(o/w): 0.28 ± 0.03). The 99mTcN-3

shows similar hydrophilicity (log P(o/w): -2.12 ± 0.02) to

the bisnitroimidazole complexes. Relatively lower

lipophilicity may lead to faster blood clearance and less

background activity [25, 26]. As the partition coefficients

shown above, the biodistribution patterns of the 99mTc-

nitrido complexes, 99mTcN-1 and 99mTcN-2, can probably

be more favorable than the 99mTc-oxo complexes.

Biodistribution

The biodistribution studies were performed in male Kun-

ming mice bearing murine sarcoma (S180) to evaluate the

potentiality of 99mTcN-MAMA bisnitroimidazole com-

plexes for targeting tumor hypoxia. Tables 2 and 3 show

tumor uptakes of the newly developed 99mTcN-bisnitro-

imidazole complexes and the control complex, 99mTcN-3,

at different time points post-injection. Though the initial

blood activity of 99mTcN-2 is higher than that of 99mTcN-1

due to slightly different hydrophilicity, both the complexes

show fast blood clearance. The activity is excreted mainly

through the hepatobiliary tract, as demonstrated by the high

activity in intestine and liver, and part of renal tract. It is to

be noted that this phenomenon of high activities in liver

Table 2 Tissue or organ

uptakes of 99mTcN-1 and99mTcN-2 in Kunming mice

bearing murine sarcoma tumor

(% ID/g)

Time post injection

0.5 h 1 h 2 h 4 h

99mTcN-1

Blood 1.31 ± 0.20 0.57 ± 0.07 0.19 ± 0.02 0.13 ± 0.01

Brain 0.12 ± 0.02 0.06 ± 0.00 0.03 ± 0.00 0.03 ± 0.02

Muscle 0.42 ± 0.04 0.22 ± 0.03 0.11 ± 0.01 0.08 ± 0.01

Bone 1.09 ± 0.32 0.58 ± 0.10 0.31 ± 0.02 0.24 ± 0.05

Heart 0.67 ± 0.06 0.37 ± 0.06 0.18 ± 0.04 0.13 ± 0.02

Spleen 0.46 ± 0.02 0.28 ± 0.03 0.16 ± 0.02 0.19 ± 0.05

Lung 2.41 ± 0.55 1.58 ± 0.03 0.71 ± 0.12 0.98 ± 0.03

Stomach 1.33 ± 0.20 1.57 ± 0.05 0.48 ± 0.21 1.11 ± 0.13

Kidney 10.73 ± 2.36 5.02 ± 1.05 3.53 ± 0.45 3.13 ± 0.40

Intestine 51.31 ± 19.39 36.93 ± 1.54 9.18 ± 0.13 1.37 ± 0.27

Liver 7.75 ± 3.96 6.30 ± 3.71 1.71 ± 0.48 1.17 ± 0.12

Tumor 1.38 ± 0.26 0.93 ± 0.08 0.58 ± 0.04 0.25 ± 0.0399mTcN-2

Blood 5.56 ± 0.27 2.44 ± 0.44 1.54 ± 0.24 0.97 ± 0.20

Brain 0.27 ± 0.02 0.19 ± 0.05 0.14 ± 0.03 0.12 ± 0.06

Muscle 1.20 ± 0.28 0.60 ± 0.07 0.42 ± 0.02 0.28 ± 0.06

Bone 2.63 ± 0.32 1.50 ± 0.34 1.24 ± 0.27 0.73 ± 0.03

Heart 2.65 ± 0.48 1.28 ± 0.20 0.98 ± 0.01 0.59 ± 0.12

Spleen 2.16 ± 0.26 0.98 ± 0.15 1.17 ± 0.49 0.65 ± 0.17

Lung 6.69 ± 1.11 2.94 ± 0.04 3.63 ± 0.09 2.30 ± 0.16

Stomach 4.30 ± 1.98 1.68 ± 0.26 2.34 ± 0.33 4.08 ± 1.88

Kidney 14.26 ± 2.30 9.68 ± 0.79 8.48 ± 1.14 6.27 ± 1.31

Intestine 50.64 ± 7.13 19.26 ± 11.25 25.21 ± 6.45 3.54 ± 0.17

Liver 49.90 ± 23.26 41.34 ± 12.02 18.64 ± 5.70 6.07 ± 0.56

Tumor 3.85 ± 0.38 2.15 ± 0.40 1.78 ± 0.26 1.33 ± 0.31

J Radioanal Nucl Chem

123

and intestine is in contrast to high hydrophilicity for both

the labeled compounds 99mTcN-1 and 99mTcN-2 (log P(o/w)

values: *-2.5). The cause for the uncommon retention

has not been determined yet. We speculate that the

uncommon liver and intestine retention is related to the

relatively large molecular size of tracers (showed as

Fig. 1). Detailed study on this issue will be reported in the

later work.

The initial uptake of 99mTcN-1 in tumor is

1.38 ± 0.26 % of injected dose per gram (%ID/g), which

is similar to that of 99mTcN-3. However, 99mTcN-1,

exhibits better retention in tumor. The tumor activity is still

0.58 %ID/g at 2 h, which is almost four times as much as

that of 99mTcN-3. Favorable tumor-to-tissue ratios are also

observed for 99mTcN-1 due to good retention in tumor

(Fig. 3). For the 2-nitroimidazole analogue 99mTcN-2, it

has a higher tumor uptake than 99mTcN-1. It is supposed

that the tumor-to-tissue ratios of 4-nitroimidazole tracer,99mTcN-1, are also not as good as those of its 2-nitroimi-

dazole analog because of lower reduction potentials of

4-nitroimidazole derivatives (i.e. relatively inefficient

enzymatic reduction for 4-nitroimidazole) compared with

the corresponding 2-nitroimidazoles. In fact, this is not the

case in the present study. The tumor-to-tissue ratios of99mTcN-2 demonstrate a trend of increase, but are not

better than 99mTcN-1. It is probably due to relatively lower

lipophilicity of 99mTcN-1 (log P(o/w): -2.56 ± 0.01) than

that of 99mTcN-2 (log P(o/w): -2.49 ± 0.04), resulting in

faster plasma clearance. On the other hand, 99mTcN-2

shows a similar biological behavior to 99mTcN-1 when

compared with the control complex: uptake in tumor is

significantly higher and tumor-to-tissue ratios especially

tumor-to-muscle ratios are better; tumor-to-blood ratios are

also up to 1.16 and 1.37 at 2 h and 4 h after injection. In

Table 3 Tissue or organ

uptakes of 99mTcN-3 in

Kunming mice bearing murine

sarcoma tumor (% ID/g)

Time post injection

0.5 h 1 h 2 h 4 h

99mTcN-3

Blood 1.19 ± 0.28 0.29 ± 0.05 0.18 ± 0.03 0.10 ± 0.01

Brain 0.20 ± 0.04 0.10 ± 0.01 0.02 ± 0.00 0.03 ± 0.00

Muscle 0.51 ± 0.12 0.18 ± 0.00 0.14 ± 0.06 0.05 ± 0.00

Bone 0.70 ± 0.16 0.45 ± 0.02 0.24 ± 0.07 0.15 ± 0.02

Heart 0.82 ± 0.13 0.33 ± 0.04 0.19 ± 0.09 0.09 ± 0.02

Spleen 1.08 ± 0.27 0.87 ± 0.10 0.44 ± 0.01 0.33 ± 0.01

Lung 2.56 ± 0.28 0.97 ± 0.16 0.41 ± 0.08 0.38 ± 0.03

Stomach 1.44 ± 0.15 0.62 ± 0.05 0.34 ± 0.09 0.35 ± 0.11

Kidney 15.45 ± 3.39 8.92 ± 3.02 4.99 ± 1.11 3.28 ± 0.25

Intestine 32.54 ± 7.93 1.37 ± 0.36 0.89 ± 0.40 0.33 ± 0.02

Liver 19.14 ± 4.28 4.51 ± 0.27 3.04 ± 0.29 1.73 ± 0.24

Tumor 1.32 ± 0.16 0.48 ± 0.07 0.15 ± 0.01 0.10 ± 0.00

0

1

2

3

4

Tu

mo

r/B

loo

d R

atio

s

Time (h)

99mTcN-199mTcN-299mTcO-199mTcO-299mTcN-3

1 2 40.5

A

0

1

2

3

4

5

6B

Tu

mo

r/M

usc

le R

atio

s

99mTcN-199mTcN-299mTcO-199mTcO-299mTcN-3

Time (h)1 2 40.5

Fig. 3 Tumor-to-blood (a) and tumor-to-muscle (b) ratios of99mTcN-1, 99mTcN-2, 99mTcO-1a nd 99mTcO-2a. aThe data of99mTcO-1 and 99mTcO-2 are cited from the literature [18]

J Radioanal Nucl Chem

123

comparison to the control, the beneficial tumor retention

and tumor-to-tissue ratios of the bisnitroimidazole com-

plexes could be partially due to differential lipophilicity,

but the main factor seems to be bioreductive pharmaco-

phore, nitroimidazole group, as bioactive moiety targeting

hypoxia. As the results shown above, the favorable bio-

distribution patterns of 99mTcN-1 and 99mTcN-2 suggest

their potentiality of tumor hypoxia targeting.

The biodistribution results of 99mTc-nitrido complexes

are compared with those of the corresponding 99mTc-oxo

complexes in the same tumor model (Fig. 3). Both the99mTc-nitrido complexes have higher tumor-to-blood ratios

than the 99mTc-oxo complexes during the studying period.

Especially for 99mTcN-1, more favorable tumor-to-blood

ratios can be achieved, and a remarkable value increases up

to 3.10 ± 0.30 at 2 h. Meanwhile, tumor-to-muscle ratios

of the 99mTc-nitrido complexes are also better. At 2 h, the

differences of tumor-to-muscle ratios between 99mTc-nitr-

ido complexes and the corresponding 99mTc-oxo com-

plexes are even more significant than other time intervals.

Many factors may affect the biological behavior of a

radiopharmaceutical due to complexity of physiological

environment. Besides tumor and animal model, several

structure-related factors, such as pharmacophores,

molecular structures and lipophilicity, are always taken

into account. Bioreductive pharmacophore, e.g. nitroimi-

dazole, plays a vital role in hypoxia selectivity and tar-

geting. As we have seen, 99mTcN-3, the control complex

without nitroimidazole group, shows poor retention in

tumor while the bisnitroimidazole complexes (99mTcN-1

and 99mTcN-2) affords the favorable biodistribution pat-

terns. For the radiopharmaceuticals with similar bioactive

moieties, biological behavior has more relation with both

of molecular structures and lipophilicity. Although a

lower reduction potential of 4-nitroimidazole leads to

more difficult and inefficient enzymatic reduction than

2-nitroimidazole [21, 24, 27], relatively lower lipophilic-

ity endows the 4-nitroimidazole derivative, 99mTcN-1,

better tumor-to-tissue ratios than 99mTcN-2. Similarly,

change of radio-metal core from 99mTc-oxo to 99mTc-

nitrido alters dramatically the physicochemical characters

of the tracer and its pharmacokinetics subsequently. In

comparison to the 99mTc-oxo MAMA complexes, the99mTc-nitrido MAMA complexes with more hydrophilic-

ity show faster background clearance and more favorable

biodistribution subsequently.

Conclusion

The MAMA-derived ligands 1 and 2 were synthesized

using a modified procedure in high yield. Two novel99mTc-nitrido labeled bisnitroimidazole complexes

99mTcN-1 and 99mTcN-2 were successfully prepared

though a substitution reaction with 99mTcN intermediate as

well as a control complex 99mTcN-3. All the 99mTcN

complexes were obtained with high radiochemical purity

([95 %) and show good stability and hydrophilicity. Bio-

distribution results of 99mTcN-1 and 99mTcN-2 demon-

strate faster clearance from blood and soft tissues, better

tumor retention and favorable tumor-to-tissue ratios than99mTcN-3, suggesting their potentiality of tumor hypoxia

targeting. Compared with the 99mTc-oxo complexes in the

same tumor model, the 99mTcN-bisnitroimidazole com-

plexes show significantly higher tumor-to-blood and

tumor-to-muscle ratios, especially for 99mTcN-1. This

favorable biological behavior makes the 99mTcN-com-

plexes more beneficial to image tumor hypoxia than the99mTcO-complexes. The further investigations on the

chemical structures of 99mTcN-MAMA complexes and

other labeling methods in the search of hypoxia targeting

radiopharmaceutical with improved properties are per-

formed currently.

Acknowledgments The authors would like to thank Pr. Junbo

Zhang for help of providing SDH kit. This work was supported by the

National Science Foundation of China (Grant No. 21071010,

21371017).

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