Synthesis and biological evaluation of novel 99mtc-labelled bisphosphonates as superior bone imaging agents

Article abstract of DOI:10.3390/molecules16086165

A series of novel zoledronic acid (ZL) derivatives 1-hydroxy-3-(2-methyl- 1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (MIPrDP), 1-hydroxy-4-(2-methyl-1H-imidazol-1-yl)butane-1,1-diyldiphosphonic acid (MIBDP), and 1-hydroxy-5-(2-methyl-1H-imidazol-1-yl)pentane-1,1-diyldiphosphonic acid (MIPeDP) were prepared and successfully labeled with 99mTc in high labeling yields. The in vitro stability and in vivo biodistribution of 99mTc-MIPrDP, ^99mTc-MIBDP and ^99mTc-MIPeDP were investigated and compared. The biodistribution studies indicate that the radiotracer ^99mTc-MIPrDP has highly selective uptake in the skeletal system and rapid clearance from soft tissues. The present findings indicate that 99mTc-MIPrDP holds great potential for use in bone imaging.

Full text of DOI:10.3390/molecules16086165

Molecules 2011, 16, 6165-6178; doi:10.3390/molecules16086165
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
9
9m
Synthesis and Biological Evaluation of Novel Tc-Labelled
Bisphosphonates as Superior Bone Imaging Agents
Ling Qiu, Wen Cheng, Jianguo Lin *, Shineng Luo, Li Xue and Jing Pan
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular
Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China
*
Author to whom correspondence should be addressed; E-Mail: jglin@yahoo.cn;
Tel.: +86-510-85514482; Fax: +86-510-85513113.
Received: 10 June 2011; in revised form: 15 July 2011 / Accepted: 19 July 2011 /
Published: 25 July 2011
Abstract:
A
series
of
novel
zoledronic
acid
(ZL)
derivatives
1
-hydroxy-3-(2-methyl-1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (MIPrDP),
1
-hydroxy-4-(2-methyl-1H-imidazol-1-yl)butane-1,1-diyldiphosphonic acid (MIBDP), and
1
-hydroxy-5-(2-methyl-1H-imidazol-1-yl)pentane-1,1-diyldiphosphonic acid (MIPeDP)
9
9m
were prepared and successfully labeled with
Tc in high labeling yields. The in vitro
9
9m
99m
99m
stability and in vivo biodistribution of
Tc-MIPrDP,
Tc-MIBDP and
Tc-MIPeDP
were investigated and compared. The biodistribution studies indicate that the radiotracer
9
9m
Tc-MIPrDP has highly selective uptake in the skeletal system and rapid clearance from
9
9m
soft tissues. The present findings indicate that Tc-MIPrDP holds great potential for use
in bone imaging.
99m
Keywords: zoledronic acid derivative;
Tc-labelled diphosphonates; in vitro stability;
biodistribution; bone imaging agent
1
. Introduction
Technetium-99m, with its excellent physical characteristics and easy availability from a generator,
has become the most important nuclide for organ imaging in nuclear medicine. Since Fleisch et al.
99m
described that bisphosphonates had high affinity for bone mineral in 1968, several
Tc-labeled

Molecules 2011, 16
6166
phosphate compounds have been developed for skeletal imaging, including pyrophosphate [1],
polyphosphates [2,3], and diphosphonates [4,5].
99m
Complexes of technetium-99m labeled with methylenediphosphonate ( Tc-MDP) and
9
9m
hydroxymethylenediphosphonate ( Tc-HMDP) have been widely used, both experimentally and
clinically, for detection of skeletal metastases and other focal bone lesions [4,6,7]. However, an
interval of 2–6 h is needed between injecting these agents into the patient and bone scanning [8].
Shorting this interval would lessen the burden on patients in terms of the total length of the
examination. To enable imaging at an earlier time after injection, a radiopharmaceutical with higher
affinity for bone, larger ratio of bone-to-soft tissue uptake and more rapid clearance from blood is
required, accordingly [9]. Consequently, the nature of ligands (diphosphonic acid) is a key factor to
determine the advantages of the radiopharmaceuticals.
Diphosphonates have a fundamental P–C–P backbone structure, and have high affinity for bone
mineral. ZL [1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-bisphosphonic acid], one kind of the typical
third-generation DPs, is currently the most potent bisphosphonate. In preclinical models of bone
resorption, for example, ZL is at least 100 times more potent than clodronate and pamidronate, and it is
at least 1,000 times more potent than etidronate [10].
In the previous work of our group, a series of technetium-99m labeled DPs with the alkyl
substituent in the imidazole ring of ZL have been prepared and their in vivo biological properties
were systematically investigated [11-14]. However, to the best of our knowledge, extension and
optimization of the linker chain between the imidazolyl and geminal bisphosphonate group to develop
novel bone imaging agent has been largely unexplored.
Based on our primary studies, we found that optimization of imidazolyl ring or extension of the
linker chain between the imidazolyl and geminal bisphosphonate group in the ZL to be labeled with
technetium-99m can bring significant influence on the biological properties of the bone resorption and
the clearance from blood and soft tissues [15,16]. For the purpose of developing novel bone imaging
agent with excellent biological properties, we continue to extend the number of the methylene chain
between the methylimidazolyl and geminal bisphosphonate group in the diphosphonate acid (see
9
9m
Figure 1). In this work, a series of novel Tc-labeled bisphosphonates were prepared and reported,
9
9m
99m
99m
i.e.,
Tc-MIPrDP,
Tc-MIBDP and
Tc-MIPeDP. Their in vitro stability and in vivo
biodistribution were also studied.
Figure 1. Structures of MIDP and its derivatives.

Molecules 2011, 16
6167
2
. Results and Discussion
2
.1. Chemistry and Radiolabeling
MIPrDP, MIBDP and MIPeDP were synthesized by three step reactions from the starting material
1
2
-methylimidazole. The target compounds were identified by elemental analysis, MS and H-NMR,
9
9m
99m
and the results agree well with the expected chemical structures.
Tc-MIPrDP,
Tc-MIBDP and
9
9m
99m
−
Tc-MIPeDP were labeled with
TcO
4
9
by reduction with stannous chloride. For TLC analysis,
9m
99m
with the H O system, the R values of Na TcO and 5a-5c were about 0.9-1, while
Tc-colloidal
2
f
4
9
9m
impurities remain at 0-0.1. With the acetone system, the Tc-colloidal impurities and 5a-5c remain at
9
9m
4
the origin and Na TcO moves with the solvent front.
9
9m
HPLC analysis revealed the formation of free technetium (Na TcO ) that was eluted at a retention
4
time of 9.8 min, whereas 5a-5c eluted at a retention time of 2.8 ± 0.1 min (see Figure 2). The identical
9
9m
retention times revealed the structural analogy of these Tc-DPs [9]. For each radiolabeled complex,
the single peak in the HPLC-chromatogram clearly shows the formation of only one complex and
9
9m
excludes the possibility of residual Na TcO or other components [17]. That is, the chelation of
4
technetium with the bisphosphonates moiety is unique and complete.
Figure 2. HPLC chromatograms (5a rt = 2.8 min, 5b rt = 2.9 min, 5c rt = 2.9 min and
9
9m
−
TcO4 rt = 9.8 min).
CPM
CPM
9
9m
99m
3
2
2
1
1
0000.0
5000.0
0000.0
5000.0
0000.0
200000.0
Na TcO
4
Tc-MIPrDP
180000.0
160000.0
140000.0
120000.0
100000.0
80000.0
60000.0
40000.0
20000.0
0.0
5
000.0
0.0
0.00
10.00
20.00 mins
0.0
10.00
20.00 mins
CPM
CPM
250000.0
200000.0
150000.0
100000.0
99m
99m
300000.0
250000.0
200000.0
150000.0
100000.0
Tc-MIPeDP
Tc-MIB DP
50000.0
50000.0
0.0
0.0
0.00
10.00
20.00 mins
0.00
10.00
20.00 mins
9
9m
According the TLC and HPLC analysis, the radiochemical purities of these
Tc-DPs were all
9
9m
greater than 97%. This labeling method also meets the clinical requirement of other
Tc-labeled
Tc-MDP. The radiolabeled compounds were used immediately after the
formulation for both in vitro and in vivo studies.
9
9m
bisphosphonates such as

Molecules 2011, 16
.2. In Vitro Stability and Octanol-Water Partition Coefficient
6168
2
9
9m
99m
99m
The in vitro stabilities of
Tc-MIPrDP,
Tc-MIBDP and
Tc-MIPeDP were studied in PBS
(
pH = 7.4) for different time intervals (1, 2, 3, 4, 5, 6 h) at 37 °C. The stability was presented as RCP
9
9m
on the basis of the HPLC analysis. After 6 h of incubation, more than 97% of
9
Tc-MIPrDP,
9m
99m
Tc-MIBDP and
Tc-MIPeDP remained intact in the PBS. The results indicated that the labeling
efficiency of these complexes was high and their stability duration was long enough to allow further
biodistribution and imaging studies.
99m
99m
99m
The octanol-water partition coefficients (logP) for Tc-MIPrDP, Tc-MIBDP and Tc-MIPeDP
were −1.89, −1.93, −2.10 and −1.71, −1.77, −1.89 in PBS at two different pH values of 7.0 and 7.4
9
9m
respectively (see Table 1), which demonstrated that logP decreased from
Tc-MIPrDP to
9
9m
Tc-MIPeDP and the liposolubility at pH = 7.4 was higher than that at pH = 7.0. From
9
9m
99m
Tc-MIPrDP to
Tc-MIPeDP, the longer the carbon chain, the smaller the log P. As well known,
the log P value is a very useful parameter that can be used to understand the behavior of a drug and
predict its distribution in the organism in combination with other parameters [18]. The different
lipotropy among three technetium-99m labeled zoledronic acid derivatives may be attributed to the
change in the linker chain between the methylimidazolyl and the geminal bisphosphonate group.
Table 1. Octanol-water partition coefficient.
Constituent
pH = 7.0
pH = 7.4
9
9m
Tc-MIPrDP
−1.89 ± 0.05
−1.93 ± 0.09
−2.10 ± 0.07
−1.71 ± 0.12
−1.77 ± 0.05
−1.89 ± 0.07
9
9m
Tc-MIBDP
Tc-MIPeDP
9
9m
2
.3. Plasma Protein Binding
The percentage of protein binding was 11.2%, 12.4%, and 14.7% for Tc-MIPrDP, Tc-MIBDP
9
9m
99m
9
9m
and Tc-MIPeDP, respectively. As known, different protein binding rate has significant influence on
the bone uptake of the tracer agent [19]. Here, with the extension of carbon chain, the plasma protein
binding increases and the maximum bone uptake decreases gradually. The structural difference further
affects the blood clearance of the radiolabeled complexes [20], which will be discussed in detail in the
next blood kinetics studies.
2
.4. Blood Kinetics Studies
Pharmacokinetic parameters were listed in Table 2. Figure 3 shows the blood clearance of 5a-5c in
the mice 6 h post injection. Pharmacokinetics of 5a-5c comply with the two-compartment model with
−
0.16t
−0.01t
−0.056t
−0.0034t
the pharmacokinetic equations of C = 2.66e
+ 0.90e
, C = 2.53e
+ 0.282e
and
−0.061t
−0.0067t
C = 2.03e
+ 0.736e
, respectively. The values of CL were 2.58, 2.90 and 3.84 and the AUC
9
9m
99m
99m
were 142.9, 127.4, and 96.2 for Tc-MIPrDP, Tc-MIBDP and Tc-MIPeDP, respectively.

Molecules 2011, 16
Table 2. Pharmacokinetic parameters of the 5a-5c in mice (mean ± SD, n = 5, %ID/g).
6169
9
9m
99m
99m
Parameters
Tc-MIPrDP
0.0266
Tc-MIBDP
0.0286
0.0087
0.0221
2.9035
12.369
202.12
127.43
Tc-MIPeDP
0.0873
0.0493
0.0369
3.8432
4.269
−
1
K (min )
1
2
−
1
K (min )
0.0209
2
1
−
1
K (min )
0.0193
e
CL (%ID/g/min)
T1/2α (min)
2.5887
11.519
T1/2β (min)
102.78
61.64
AUC (%ID/g * min)
142.92
96.21
9
9m
99m
Figure 3. Pharmacokinetic curves in the mice for
Tc-MIPrDP,
Tc-MIBDP and
9
9m
Tc-MIPeDP (n = 5, mean ± SD).
9
9m
99m
In the early phase, the blood clearance of
Tc-MIPrDP was slower than
Tc-MIBDP and
9
9m
Tc-MIPeDP. After 2 h, the radioactivity concentration of three tracer agents in blood reaches an
equilibrium which coincides with the pharmacokinetic parameters CL, AUC and the pharmacokinetic
curves. Ranging from 5 to 10 min, the bone uptake increases with the increased carbon chain. That is,
9
9m
99m
99m
the uptake of
consists with the variation of K12. It was concluded that
eliminated from the blood rapidly. In this study, the blood clearance (see Figure 3) of Tc-MIPrDP,
Tc-MIPeDP in bone increases faster than
Tc-MIPrDP and
Tc-MIBDP, which
9
9m
Tc-MIPeDP can be absorbed quickly and
9
9m
9
9m
99m
Tc-MIBDP and Tc-MIPeDP were very fast, due to low protein binding.
.5. Biodistribution Studies
Biodistributions of 5a-5c were determined in ICR mice, and the data is shown in Table 3 as the
2
percentage administered activity (injected dose) per gram of tissue (%ID/g). Although ZL has been
known to possess low toxicity and can be used therapeutically at a high dose [21,22], the toxicity
9
9m
profile of its derivatives are uncertain. Therefore, after the complexes
Tc-DPs (1.85 MBq) in a
volume of 0.2 mL were given to the normal mice via intravenous injection respectively, the mice were
watched carefully for any sign of adverse reaction. As expected, the mice showed no signs of toxicity
through the overall study period.

Molecules 2011, 16
6170
9
9m
Table 3. Biodistribution of Tc-MIDP and 5a-5c in mice (mean ± SD, n = 5, %ID/g).
Time after injection
Tissue
5
min
15 min
30 min
60 min
120 min
240 min
9
9m
Tc-MIDP [11]
Heart
Liver
3.03 ± 0.18 1.23 ± 0.05 0.84 ± 0.01 0.54 ± 0.02 0.31 ± 0.02 0.20 ± 0.01
1.52 ± 0.03 0.68 ± 0.02 0.56 ± 0.03 0.33 ± 0.03 0.25 ± 0.01 0.16 ± 0.01
Spleen 1.73 ± 0.01 0.60 ± 0.01 0.40 ± 0.02 0.32 ± 0.04 0.32 ± 0.01 0.21 ± 0.02
Kidney 19.7 ± 1.22 12.1 ± 2.11 8.42 ± 0.22 7.82 ± 0.88 4.54 ± 0.52 4.14 ± 0.67
Muscle 1.65 ± 0.34 0.66 ± 0.02 0.34 ± 0.01 0.28 ± 0.01 0.23 ± 0.02 0.26 ± 0.03
Blood 8.54 ± 0.92 3.90 ± 0.11 1.35 ± 0.22 0.63 ± 0.02 0.30 ± 0.01 0.15 ± 0.01
All bone 11.1 ± 0.08 12.8 ± 0.23 13.2 ± 1.11 11.6 ± 1.22 14.2 ± 0.45 14.2 ± 0.23
9
9m
Tc-MIPrDP
Heart
Liver
1.41 ± 0.03 0.71 ± 0.07 0.53 ± 0.02 0.30 ± 0.01 0.12 ± 0.01 0.07 ± 0.00
1.17 ± 0.03 0.87 ± 0.06 1.35 ± 0.09 1.15 ± 0.02 0.32 ± 0.05 0.16 ± 0.00
Spleen 0.86 ± 0.03 0.59 ± 0.03 0.64 ± 0.01 0.38 ± 0.02 0.14 ± 0.00 0.07 ± 0.00
Kidney 8.35 ± 0.11 4.58 ± 0.19 3.44 ± 0.23 2.54 ± 0.05 3.21 ± 0.50 2.78 ± 0.09
Muscle 0.86 ± 0.02 0.58 ± 0.11 0.34 ± 0.03 0.19 ± 0.00 0.10 ± 0.01 0.05 ± 0.00
Blood 5.00 ± 0.02 2.69 ± 0.19 1.54 ± 0.12 0.74 ± 0.05 0.13 ± 0.01 0.08 ± 0.00
All bone 6.43 ± 0.40 10.5 ± 0.12 10.2 ± 0.56 12.2 ± 0.05 19.6 ± 0.87 11.5 ± 0.27
9
9m
Tc-MIBDP
Heart
Liver
1.43 ± 0.04 1.02 ± 0.08 0.63 ± 0.03 0.36 ± 0.02 0.16 ± 0.00 0.11 ± 0.01
1.85 ± 0.08 2.96 ± 0.07 3.59 ± 0.25 1.79 ± 0.12 1.14 ± 0.04 0.81 ± 0.01
Spleen 1.12 ± 0.03 1.05 ± 0.04 0.95 ± 0.03 0.52 ± 0.03 0.41 ± 0.03 0.29 ± 0.01
Kidney 8.36 ± 0.28 6.53 ± 0.33 5.62 ± 0.46 5.21 ± 0.40 4.45 ± 0.22 5.78 ± 0.20
Muscle 0.87 ± 0.08 0.55 ± 0.11 0.41 ± 0.03 0.15 ± 0.00 0.08 ± 0.00 0.05 ± 0.00
Blood 3.79 ± 0.07 1.54 ± 0.06 0.81 ± 0.00 0.35 ± 0.02 0.19 ± 0.00 0.09 ± 0.00
All bone 6.56 ± 0.20 11.1 ± 1.45 12.9 ± 0.42 17.1 ± 1.08 11.2 ± 0.13 16.8 ± 0.88
9
9m
Tc-MIPeDP
Heart
Liver
1.83 ± 0.05 0.99 ± 0.03 0.52 ± 0.02 0.39 ± 0.02 0.32 ± 0.03 0.16 ± 0.00
1.89 ± 0.04 5.31 ± 0.33 4.58 ± 0.16 4.20 ± 0.13 2.25 ± 0.24 0.90 ± 0.01
Spleen 1.07 ± 0.04 1.80 ± 0.14 1.45 ± 0.04 1.27 ± 0.03 0.67 ± 0.02 0.28 ± 0.02
Kidney 8.15 ± 0.26 7.05 ± 0.24 6.16 ± 0.16 5.31 ± 0.08 4.80 ± 0.14 4.06 ± 0.03
Muscle 1.08 ± 0.06 0.53 ± 0.05 0.37 ± 0.04 0.17 ± 0.01 0.11 ± 0.01 0.08 ± 0.01
Blood 3.40 ± 0.14 1.18 ± 0.08 0.91 ± 0.01 0.53 ± 0.01 0.18 ± 0.00 0.12 ± 0.01
All bone 7.22 ± 0.33 11.3 ± 1.76 11.4 ± 0.05 12.6 ± 0.13 17.6 ± 0.42 11.4 ± 0.31
Inspecting Table 3, one can observe that 5a-5c are mainly accumulated in the bone, kidneys, and
liver. The uptake of 5a-5c in bone increased steadily from 5 to 60 min post injection. At 2 h post
9
9m
injection, the bone uptake was 19.6 ± 0.87, 11.2 ± 0.13 and 17.6 ± 0.42 %ID/g for
Tc-MIPrDP,
Tc-MIBDP and Tc-MIPeDP, respectively. From previous work of our group [16], it was found that
the bone uptake increases with the increasing carbon chain between the imidazolyl and geminal
99m
99m
99m
99m
bisphosphonate group in ZL (i.e., from Tc-ZL to Tc-IPrDP). However, in the present work the bone
99m
99m
99m
uptake of Tc-MIPeDP was smaller than those of Tc-MIPrDP and Tc-MIBDP (see Table 3). This
indicates that limitless extension of the carbon chain is not always beneficial to improve the bone
uptake. Furthermore, the bone uptake efficiency of these radiotracers was compared. For example,

Molecules 2011, 16
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9
9m
after injection of
Tc-MIPrDP, the bone uptake was up to 6.43 %ID/g at 5 min and it increased
continuously to a maximum of 19.6 %ID/g at 120 min, which was larger than the corresponding values
9
9m
of Tc-IPrDP (5.37 and 11.14 %ID/g respectively) [16]. This implied that the bone uptake efficiency
of the former with a methyl in the imidazole ring is larger than the latter with no substituent in the
9
9m
99m
99m
imidazole ring. Considering that
Tc-IPrDP is better than
99m
Tc-MDP and
Tc-ZL in the bone
imaging, it is therefore concluded that Tc-MIPrDP is the best one among these bone scanning agents.
Compounds 5a-5c also showed predominant kidney and liver uptake, indicating that they were not
only excreted through the kidneys, but also absorbed and eliminated by the liver. It was noted that the
9
9m
99m
uptake of
Tc-MIBDP and
Tc-MIPrDP. For instance, the kidneys and liver uptake of 5a-5c at 2 h post injection were
.21 ± 0.50, 4.45 ± 0.22 and 4.80 ± 0.14 %ID/g for kidneys and 0.32 ± 0.05, 1.14 ± 0.04 and
.25 ± 0.24 %ID/g for liver, respectively. The uptake ratios of bone to liver and kidneys for
Tc-MIPeDP in liver, spleen and kidneys were larger than those of
9
9m
3
2
9
9m
99m
Tc-MIPrDP were 61.25 ± 1.89 and 6.10 ± 0.78 respectively, while those of
Tc-MIBDP and
9
9m
Tc-MIPeDP were 9.81 ± 1.23, 2.51 ± 0.69 and 7.81 ± 0.74, 3.6 ± 1.21 (see Figure 4). Thus, we can
9
9m
99m
see that from
Tc-MIPrDP to
Tc-MIPeDP with the extension of the linker chain between the
methylimidazolyl and geminal bisphosphonates group, the former exhibits a significant advantage in
the clearance from soft tissues and it can better satisfy the clinical requirements of a good bone
imaging agent.
9
9m
Figure 4. Bone to tissue uptake ratio in mice at different time post injection of Tc-MIDP
and 5a-5c.
Radioactivity levels of 5a-5c in the blood were 5.00 ± 0.02, 3.79 ± 0.07 and 3.40 ± 0.14 %ID/g at
5
min post injection, respectively, which followed by a rapid clearance. At 4 h post injection, the

Molecules 2011, 16
6172
radioactivity in blood was only 0.08, 0.09 and 0.12 %ID/g for 5a-5c respectively. This indicates that all
these radiotracers can eliminate quickly from the blood, which agrees well with the
pharmacokinetics studies.
For better understanding the biodistributions and metabolism of 5a-5c in vivo, we further compared
9
9m
them with the radiotracer
Tc-MIDP (1-hydroxy-2-(2-methyl-1H-imidazolyl)ethane-1,1-diyl
9
9m
diphosphonic acid) [11]. The biodistribution data also show that Tc-MIDP has high kidney uptake,
indicating that it is excreted quickly through the kidneys. Comparison of the uptake ratios of bone to
soft tissue (including heart, liver, spleen, muscle and kidneys) among these homologues shows that
9
9m
99m
these uptake ratios of Tc-MIDP are all very small while those of Tc-MIPrDP are all larger than
9
9m
others after 120 min post injection (see Figure 4). This showed Tc-MIPrDP was not only superior to
9
9m
99m
its parent complex
studies. In summary, the radiotracer
imaging agent with higher bone uptake and lower background among this kind of homologues.
Tc-MIDP but also superior to
Tc-IPrDP judged from the biodistribution
99m
Tc-MIPrDP better meets the clinical requirement of bone
3
. Experimental
3.1. General
All analytical chemical reagents employed were purchased from commercial sources and used
9
9m
without further purification. Na TcO was supplied by the Affiliated Jiangyuan Hospital of the
4
Jiangsu Institute of Nuclear Medicine. All melting points were measured on a Yanaco MP-500 melting
point apparatus (Shimadzu, Japan). Elemental analysis was carried out using an Elementar Vario EL
III analyzer. Electron spray ion (ESI) mass spectra were determined using a Waters Platform
ZMD4000 LC/MS (Waters, U.S.A.). Nuclear magnetic resonance (NMR) spectra were obtained on a
Bruker DRX-500 spectrometer (Bruker, Germany), and the chemical shift value was given relative to
the internal tetramethylsilane (TMS). Xinhua chromatography paper (Shanghai, China) was used for
thin layer chromatography (TLC). A Packard-multi-prias γ Counter (Perkins Elmer, U.S.A.) was used.
High performance liquid chromatography (HPLC) analysis was performed on a Waters 600-type
high-performance liquid chromatography (Waters, U.S.A.) equipped with a dural k absorbance
detector (Waters 2487), binary HPLC pump (Waters 1525) and Cd (Te) detector equipped with a flow
scintillation analyzer (Perkin Elmer). Normal Institute of Cancer Research (ICR) mice (weighting
1
8~20 g) were supplied by the Shanghai SLAC Laboratory Animal CO., Ltd. (Shanghai, China). The
animal experiment was approved by the Animal Care and Ethics Committee of the Jiangsu Institute of
Nuclear Medicine.
3
.2. Syntheses of Diphosphonic Acids
9
9m
Three
Tc-diphosphonates were synthesized according to the procedure outlined in Scheme 1 as
described previously [16,23].

Molecules 2011, 16
Scheme 1. Syntheses of Tc-MIPrDP, Tc-MIBDP and Tc-MIPeDP.
6173
99m
99m
99m
3
0
.2.1. General Procedure for the Preparation of Compounds 3a-3c
2
-Methylimidazole (1, 8.2 g, 0.1 mol) was dissolved in CH
2 2
Cl (75 mL). Then KOH (8.4 g,
.15 mol), K CO (13.8 g, 0.0835 mol) and tetrabutylammonium bromide (0.7 g, 0.002 mol) were
2
3
added and stirred at room temperature for 30 min. The solution was treated dropwise with the
corresponding ethyl bromoacetate homolog (0.1 mol) at room temperature. The mixture was heated to
reflux for 7 h. The inorganic salt was removed by filtration and the filtrate was washed with saturated
NaCl solution (30 mL × 4). Finally, the organic layer was evaporated under vacuum to give crude
compound 2 as a brown gum, which was used without any further purification for the next step. Water
(
90 mL) and HCl (12 mol/L, 10 mL) were successively added to compound 2, and the mixture was
refluxed 8 h, the solution was concentrated and recrystallized from isopropanol to give the white
crystalline product 3.
3
-(2-Methyl-1H-imidazol-1-yl)propanoic acid (3a): Yield: 34%. mp 125-128 °C; ESI-MS, m/z (%):
+
1
53 (100) = M-H .
4
-(2-Methyl-1H-imidazol-1-yl)butanoic acid (3b): Yield: 42%. mp 120-122 °C; ESI-MS, m/z (%): 167
+
(100) = M-H .
5
-(2-Methyl-1H-imidazol-1-yl)pentanoic acid (3c): Yield: 30%. mp 123-125 °C; ESI-MS, m/z (%):
+
1
81 (100) = M-H .
3
.2.2. General Procedure for the Preparation of Compound 4
Compound 3 (20 mmol) was dissolved in chlorobenzene (25 mL) and heated to 120 °C for 30 min,
then phosphoric acid (85%, 4.2 mL) was added. The solution was treated dropwise with phosphorus
trichloride (7.6 mL), and kept at 120 °C for 4 h. The chlorobenzene was decanted. Then, the yellow
residue was redissolved in HCl (9 mol/L, 20 mL) and heated to reflux for 5 h. The mixture was treated

Molecules 2011, 16
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with charcoal before filtration and concentration. Finally, the crude product was recrystallized from
ethanol to give the white crystalline product 4.
1
-Hydroxy-3-(2-methyl-1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (4a): Yield: 65%. mp
1
183-186 °C; H-NMR (400 MHz, D O): δ7.332 (d, 1H, CH-ring), 7.208 (d,1H, CH-ring), 4.378 (t, 2H,
2
1
3
N-CH
2
3 2 2
), 2. 557 (s, 1H, ring-CH ), 2.419 (q, 2H, OH-C-CH ); C-NMR (125 MHz, D O) δ: 144.216,
1
21.349, 117.808 (3C, C-ring), 71.896 (C-OH), 43.167 (N-CH ), 33.599 (CH ), 9.943 (-CH ); ESI-MS,
2
2
3
+
m/z (%): 299 (100) = M-H . Anal. calcd for C
7
H
14
N
2
O
7
P
2
(%): C, 28.01; H, 4.70; N, 9.33; Found (%):
C, 28.22; H, 4.82; N, 9.51.
1
-Hydroxy-4-(2-methyl-1H-imidazol-1-yl)butane-1,1-diyldiphosphonic acid (4b): Yield: 58%. mp
1
142-145 °C; H-NMR (400 MHz, D O): δ7.635 (d, 1H, CH-ring), 7.547 (d, 1H, CH-ring), 4.115 (t, 2H,
2
1
3
N-CH ), 2.592 (s, 1H, ring-CH ), 2.320 (q, 2H, OH-C-CH ), 2.004 (m, 2H, CH -CH -CH ); C-NMR
2
3
2
2
2
2
(
125 MHz, D
2
O) δ: 146.160, 124.981, 120.525 (3C, C-ring), 74.692 (C-OH), 46.669 (N-CH
2
), 31.901,
+
2
5.431 (2C, CH ), 11.813 (-CH ); ESI-MS, m/z (%): 231 (100) = M -83. Anal. calcd for C H N O P
2
3
8
16
2
7
2
(
%): C, 30.61; H, 5.09; N, 8.92; Found (%): C, 30.72; H, 5.24; N, 9.01.
1
-Hydroxy-5-(2-methyl-1H-imidazol-1-yl)pentane-1,1-diyldiphosphonic acid (4c): Yield: 70%. mp
1
2
12-215 °C; H-NMR (400 MHz, D
2
O): δ7.295 (d, 1H, CH-ring), 7.208 (d, 1H, CH-ring), 4.072
), 1.943 (q, 2H, OH-C-CH ), 1.824 (m, 2H, CH -CH -COH),
); C-NMR (125 MHz, D O) δ: 144.388, 121.271, 119.475 (3C, C-ring),
), 33.678, 30.169, 21.143(3C, CH ), 10.396 (-CH ); ESI-MS, m/z (%):
(%): C, 32.93; H, 5.49; N, 8.54; Found (%): C,33.04;
(
t, 2H, N-CH
2
), 2.533 (s, 1H, ring-CH
3
2
2
2
1
3
1
7
2
.606 (m, 2H, ring-CH
2
CH
2
2
4.209 (C-OH), 46.965 (N-CH
2
2
3
+
45 (100) = M -83. Anal. calcd for C
9
H
18
N
2
O
7
P
2
H, 5.52; N, 8.39.
3
.3. Radiochemical Syntheses of 5a-5c
To a 10 mL vial, aqueous solution of 4a-4c (100 μL, 0.25 g DPs dissolved in 5.0 mL 0.2 mol/L
sodium hydroxide solution), freshly prepared solution of stannous chloride dehydrate (100 μL, 10 mg
9
9m
2 2 4
SnCl ·2H O dissolved in 10.0 mL 0.5 mol/L HCl), and 74.0 MBq freshly eluated Na TcO were
added. The reaction solution was adjusted to pH = 6.0 by adding 0.2 mol/L phosphate buffered
solution (PBS) and diluted to 2 mL. It was vortexed adequately and reacted at 70 °C for 30 min.
3
.4. Quality Control of 5a-5c
The radiochemical purity (RCP) and radiolabeling yield (RLY) of 5a-5c was determined by TLC
and HPLC.
3
.4.1. TLC
About 3 μL 5a-5c solutions were spotted with a microcap at 1.5 cm from the bottom of paper strips.
The paper strips were placed in two developing agents, acetone and distilled water. With the distilled
9
9m
99m
water, Tc-colloidal impurities remain at the bottom on the paper strip, while Na TcO and 5a-5c
4

Molecules 2011, 16
6175
9
9m
both migrate with the solvent front. With the acetone, the Tc-colloidal impurities and 5a-5c remain
99m
at the origin and Na TcO
4
moves with the solvent front. The strips were cut into pieces of 1 cm and
the activity of these pieces was counted to determine the RCP value by a well-type γ counter.
3.4.2. HPLC
The RCP of 5a-5c were determined a Waters 600-type high-performance liquid chromatography.
The sample was passed through a millipore filter carefully and injected into the HPLC column
SinoChrom ODS-BP, PN: E2117215-080108, 4.6 mm × 250 mm × 10 μm, DaLian, China). The
absorbance was measured on the UV detector at 210 nm. Radioanalysis of the labeled compound was
(
−1
conducted using a Cd (Te) detector. The flow rate was adjusted to 0.9 mL·min and the isocratic
mobile phase was 70% H O and 30% CH CN.
2
3
3
.5. In Vitro Stability of 5a-5c
9
9m
99m
99m
The in vitro stabilities of
Tc-MIPrDP,
Tc-MIBDP and
Tc-MIPeDP were studied in PBS
(
pH = 7.4) after different interval (1, 2, 3, 4, 5 and 6 h) at physiological temperature of 37 °C. The
RCP values were evaluated by HPLC at different time points to determine if they were stable in vitro.
3
.6. Octanol-Water Partition Coefficients of 5a-5c
Octanol–water partition coefficients were determined for 5a-5c at two different pH values of 7.0
and 7.4 by measuring the distribution of radiolabeled compounds in n-octanol and PBS, respectively.
A sample of radiolabeled compound 5a-5c (100 μL) was diluted with PBS (900 μL). Then, it was
mixed with n-octanol (1 mL) and vortexed at room temperature for 2 min. The mixture was further
centrifuged at 4,000 rpm for 5 min to ensure complete separation of layers. Both layers were collected
and the radioactivity counts from 100 μL aliquots of both the organic and aqueous phases were
measured with a γ counter. Log P values were calculated using the formula of log P = log (CPM of
octanol/CPM of water).
3.7. Plasma protein Binding Assay
The radiolabled compounds (100 μL, 37 KBq) were mixed with the human plasma (100 μL) in the
centrifuge tube. After the mixture was incubated at 37 °C for 2 h, the plasma protein was precipitated
by adding trichloroacetic acid (1 mL, 250 g/L) to the mixture. The supernatant and precipitate were
separated by centrifugation at 2,000 rpm for 5 min. The radioactivities of both phases were measured
separately. The above experimental procedure was repeated three times. The percentage of protein
binding was determined by the following equation: plasma binding % = [plasma (CPM) − supernatant
(
CPM)/plasma (CPM)] × 100 = [precipitate (CPM)/plasma (CPM)] × 100%
3
.8. In Vivo Distribution of 5a-5c
Thirty mice were randomly divided into six groups and injected via the tail vein with the test agent
9
9m
99m
99m
(
Tc-MIPrDP,
Tc-MIBDP and
Tc-MIPeDP) in the volume of 0.2 mL and activity of
approximately 7.4 MBq. Groups of mice were sacrificed by decapitation at 5, 15, 30, 60, 120 and

Molecules 2011, 16
6176
2
40 min after injection. Tissue samples of interest were removed and weighed, as well as 200 μL
blood were taken from carotid artery. The activity for each sample was determined by a γ counter.
Distribution of the radioactivity in different tissues and organs was calculated and expressed as
the %ID/g, where:
organ CPM
organ weight×injected CPM
%
ID/g =
× 100%
3
.9. Blood Kinetics Studies
For pharmacokinetics study, 5a-5c (7.4 MBq, 0.2 mL) was administered to the mice via intravenous
injection in the tail vein respectively. A series of blood samples (20 μL) were collected in the microcap
tube by nicking the tail with a needle at 2, 5, 10, 15, 30, 60, 120, 180, and 240 min after injecting
5
a-5c. The radioactivity of each blood sample was counted and expressed as %ID/g. Pharmacokinetic
parameters were analyzed by the program 3P97, and the radioactivity can be expressed as a function of
−αt
−βt
time with the following equation C = Ae + Be .
4
. Conclusions
A series of novel ZL derivatives MIPrDP, MIBDP and MIPeDP have been prepared and
9
9m
successfully labeled with
Tc in a high labeling yield and good in vitro stability. Compared with
9
9m
99m
99m
Tc-MIDP and Tc-IPrDP, the radiolabeled complex Tc-MIPrDP shows higher selective uptake
9
9m
in the skeletal system and rapider clearance from soft tissues. The results indicate that Tc-MIPrDP
is a better bone-imaging agent among our designed homologous radiotracers, and it is worthy of
further preclinical investigation (such as SPECT imaging studies) to determine if it is better for bone
scintigraphy. This work will be continued later.
Acknowledgment
The authors are very grateful to the National Natural Science Foundation of China (20801024 and
2
1001015), Natural Science Foundation of Jiangsu Province (BK2009077) and Science Foundation of
Health Department of Jiangsu Province (H200963) for their financial support.
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Sample Availability: Samples of the compounds are available from the authors.
©
2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(