Synthesis and biological evaluation of 99mTc(CO) 3(His-CB) as a tumor imaging agent

Article abstract of DOI:10.1016/j.bmcl.2012.10.062

The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [^99mTc(CO)₃]⁺ core to form the ^99mTc(CO)₃(His-CB) complex. The radiochemical purity of the complex was over 90%. It had goo

Full text of DOI:10.1016/j.bmcl.2012.10.062

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7406–7409
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and biological evaluation of 99mTc(CO)3(His–CB) as a tumor
imaging agent
Jianjun Wang a,b, , Jing Yang b, Xiaojiang Duan b, Yanhua Zhang a, Wenjiang Yang a, Yu Liu a,
⇑
⇑
a Research Center for Applications of Nuclear Technology, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
b Radiochemistry Lab, School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
The chlorambucil L
-histidine conjugate was synthesized and radiolabeled with [99mTc(CO)3]+ core to form
Article history:
the 99mTc(CO)3(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good
hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention,
fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images
highlighted the potential of 99mTc(CO)3(His–CB) as a tumor imaging agent.
Received 10 August 2012
Revised 25 September 2012
Accepted 13 October 2012
Available online 23 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Chlorambucil
[
L
99mTc(CO)3]+
-Histidine
Tumor imaging
Cancer is one of the leading causes of death all over the world.
The early imaging diagnosis is very valuable in treatment of tumor
in clinic. For this purpose, [18F]fluorodeoxyglucose (FDG) positron
emission tomography (PET) imaging provides high specificity and
sensitivity in several kinds of cancer and has many applica-
in the choice of ligands for designing complexes of desirable size,
charge, and lipophilicity suitable for the specific study.8–10
The nitrogen mustards are an important class of DNA cross-
linking agents, which are utilized in the treatment of many types
of cancer.11 The cytotoxicity of nitrogen mustards is due to their
ability to alkylate DNA, RNA and proteins leading to the formation
of DNA interstrand cross links.12,13 The prototype nitrogen mustard
drug is mustine, which is no longer commonly in use. Other nitro-
gen mustards include cyclophosphamide, chlorambucil, uramus-
tine, ifosfamide, melphalan and bendamustine.
tions.1–4 18F]FDG is the most successful molecular probe in oncol-
[
ogy available for routine use in the nuclear medicine clinics.
However, its clinical application is heavily restricted by the short
half-life, limited availability and high cost of production of 18F iso-
tope by cyclotron.5 Moreover, [18F]FDG is not a ‘specific’ radio-
tracer for imaging malignant disease.1,6
D. Satpati et al. have reported a 99mTc(CO)3-labeled chlorambu-
cil analog which shows good tumor uptake of 3.2 0.3% ID/g at
3 h3 h post-injection (p.i.). However, the complex shows in vivo
instability.14 Melphalan has been labeled with 125I directly to get
By comparison, 99mTc is the most widely used radionuclide for
diagnostic imaging with single photon emission computed tomog-
raphy (SPECT) because of its favorable physical properties
(t1/2 = 6 h, E
c
= 140 keV), low cost, and widespread availability.7
[
125I]Melphalan, which has a high affinity to be localized in the tu-
Therefore, the development of 99mTc-labeled tumor imaging agent
with SPECT would be of great value.
mor site and meets most of the requirements necessary to be used
as a successful diagnostic and therapeutic agent.15 Our previous
studies have confirmed that 99mTc(CO)3-labeled pegylated chlor-
ambucil ([99mTc(CO)3(IDA-PEG3-CB)]À) shows good accumulation
in tumor tissue with high tumor/muscle ratios. Its major disadvan-
tage lies in the slow clearance from normal organs.16
One-step synthesis of [99mTc(CO)3(H2O)3]+ by direct reduction
of 99mTcO4À with sodium borohydride (NaBH4) in aqueous solution
was firstly developed by R. Alberto et al.8 Since then, a large num-
ber of biologically avid molecules have been labeled by
[
99mTc(CO)3]+ for the development of site-specific radiopharma-
In search for a more suitable 99mTc-labeled tumor imaging
probe based on the biomolecule chlorambucil, we report herein
ceuticals. The [99mTc(CO)3]+ core possesses many excellent fea-
tures, such as its small volume, kinetic inertness, and flexibility
the synthesis of 99mTc(CO)3-labeled
L-histidine-chlorambucil con-
jugate and its biodistribution studies and scintigraphic images in
tumor-bearing mice.
The chlorambucil
by reacting
L-histidine conjugate (His-CB) was prepared
L
-histidine with an equivalent amount of active ester
⇑
Corresponding authors. Tel.: +86 10 88236900 (J.W.); tel.: +86 10 88236945
(Y.L.).
of chlorambucil.17 The reaction is schematically shown in
Scheme 1.
E-mail addresses: wangjianjun@ihep.ac.cn (J. Wang), yuliu@ihep.ac.cn (Y. Liu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.062

J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7406–7409
7407
Cl
Cl
Cl
NHS
DCC
L-histidine
pH = 8~9
O
N
N
N
NH
Cl
O
CO2H
Cl
O
Cl
O
N
N
H
N
O
OH
O
1
2
His-CB
Scheme 1. Synthesis of the chlorambucil
L
-histidine conjugate (His–CB).
Cl
Cl
NH
[
99mTc(CO)3(H2O)3]+
N
Cl
O
N
N
NH
Cl
O
CO2H
NH
100 °C
CO
CO
99mTc
CO
N
H
N
O
O
Scheme 2. Preparation procedure and proposed structure of 99mTc(CO)3(His–CB).
Figure 1. HPLC pattern of 99mTc(CO)3(His–CB).
The 99mTc(CO)3-labeled
L
-histidine-chlorambucil conjugate
The partition coefficient (LogP) was determined by mixing the
complex with an equal volume of n-octanol and phosphate buffer
(25 mM, pH 7.4) in a centrifuge tube. The mixture was vigorously
stirred for 10 min at room temperature, and was then transferred
to an Eppendorf microcentrifuge tube. The tube was centrifuged
at 8000 rpm for 10 min. Samples in triplets from n-octanol and
(
99mTc(CO)3(His–CB)) was prepared by ligand-exchange reaction
of His–CB with [99mTc(CO)3(H2O)3]+ at pH ꢀ7.4 in boiling water-
bath (Scheme 2).18 99mTc(CO)3(H2O)3]+ was synthesized by direct
[
reduction of 99mTcO4À with NaBH4 in aqueous solution in the pres-
ence of CO at 1 atm.8,9 As a tridentate ligand, His–CB was reacted
with the [99mTc(CO)3(H2O)3]+ intermediate very effectively to give
the final complex 99mTc(CO)3(His–CB).
99mTc(CO)3(H2O)3]+ precursor could be prepared over 95%
Table 1
[
Biodistribution study results of 99mTc(CO)3(His–CB) in tumor-bearing mice (ID%/g,
yield, and this precursor was used without further purification.
The ligand His–CB could be labeled by [99mTc(CO)3]+ very effec-
tively. The suggested structure of 99mTc(CO)3(His-CB) was drawn
in Scheme 2.9,10 The radiochemical purity (RCP) of the complex
was routinely checked by thin layer chromatography (TLC) and
high performance liquid chromatography (HPLC). When TLC was
performed on a polyamide strip developed by dichloromethane/
methanol = 1/1 (V/V), 99mTc(CO)3(His–CB) moved to the solvent
front (Rf = 0.8ꢀ1.0), 99mTcO2ÁnH2O remained at the origin (Rf = 0)
x
s, n = 6)
Tissure
5 min
30 min
60 min
120 min
240 min
Tumor
Blood
Heart
Liver
Spleen
Lung
3.14 0.52
4.95 1.12
1.86 0.36
6.12 1.43
1.59 0.29
3.81 0.58
2.89 1.00 1.25 0.33 0.72 0.18 0.25 0.08
3.02 1.25 0.75 0.18 0.45 0.19 0.10 0.06
1.37 0.40 0.37 0.17 0.22 0.10 0.05 0.02
4.69 1.26 2.83 1.23 1.81 0.56 0.99 0.35
1.50 0.41 0.75 0.71 0.52 0.14 0.21 0.05
3.07 1.07 1.43 0.50 0.64 0.17 0.24 0.09
Kidneys
Intestine 2.58 0.81
11.47 3.19 5.54 1.27 1.63 0.22 1.39 0.73 0.36 0.06
À
2.84 1.30 1.02 0.26 0.74 0.35 0.03 0.01
0.31 0.10 0.15 0.08 0.14 0.05 0.06 0.02
1.15 0.45 0.26 0.11 0.17 0.09 0.04 0.02
0.28 0.06 0.21 0.07 0.07 0.02 0.06 0.02
0.89 0.08 1.64 0.39 1.67 0.26 1.84 0.73
2.87 0.99 5.02 2.48 5.14 2.68 9.23 3.86
and Rf value for 99mTcO4 and [99mTc(CO)3(H2O)3]+ precursor was
Brain
Muscle
Bone
T/B ratio 0.59 0.03
T/M 3.42 0.18
ratio
0.28 0.07
1.01 0.17
1.28 0.51
about 0.0–0.1. The HPLC pattern of 99mTc(CO)3(His-CB) is shown
in Figure 1. It was observed that the retention time (RT) of
[
99mTc(CO)3(H2O)3]+ was 5.52 min, while that of 99mTc(CO)3(His–
CB) was found to be 15.73 min. The mean radiochemical purity
(RCP) of the complex was over 90% after the preparation.

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J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7406–7409
Figure 2. Scintigraphic images with Ehrlich tumor-bearing mice after injection of 99mTc(CO)3(His–CB).
aqueous layer were obtained, and were counted in a well
c
-coun-
tail vein. Multiple static scans were obtained at 30, 60, 120, 180
and 240 min p.i. The tumor was clearly visualized during the whole
experiment with very high tumor-to-muscle contrast (Fig. 2),
which is consistent with the results from in-vivo biodistribution
studies.
ter. The partition coefficients were calculated using the following
equation: P = (activity concentration in n-octanol)/(activity con-
centration in aqueous layer). The partition coefficient (LogP) of
99mTc(CO)3(His–CB) was obtained to be À0.85 0.05, suggesting
that it was hydrophilic.
The stability of the complex was determined by measuring the
RCP at room temperature (25 °C) at different time points (0, 1, 2, 4,
6 h) after preparation. The RCP of the product was nearly constant
(>90%) over the observed period of 6 h, suggesting that the com-
plex possessed a great stability in the reaction mixture at room
temperature.
In summary, novel His-CB ligand was successfully synthesized
by conjugation of L
-Histidine to chlorambucil, and 99mTc(CO)3
(His–CB) was prepared in high yield by ligand-exchange reaction
with [99mTc(CO)3(H2O)3]+ intermediate. The RCP of the complex
was over 90%. It had good hydrophilicity and was stable at room
temperature. 99mTc(CO)3(His–CB) showed high initial tumor up-
take with certain retention, fast clearance from background, good
T/NT ratios and satisfactory scintigraphic images, suggesting it
would be a promising candidate for tumor imaging.
Biodistribution characteristics of 99mTc(CO)3(His–CB) were eval-
uated using Kunming mice bearing H22 liver cancer xenografts. In
vivo growth was initiated by hypodermic injection of approxi-
mately 106 H22 cells into the left front leg of female Kunming mice.
Seven-eight days after inoculation, the tumor size was in the range
of 0.5–0.8 g, and animals were used for biodistribution studies. A
Acknowledgments
The work was financially supported by National Natural Science
Foundation of China (21001060).
solution of the 99mTc(CO)3(His–CB) (100
l
L, 3.7 Â 105 Bq) was in-
jected into the tumor-bearing mice via the tail vein. The mice were
sacrificed at 5, 30, 60, 120 and 240 min post-injection (pi). The or-
gans of interest and blood were collected, weighed and measured
for radioactivity. The accumulated radioactivity in the tissue of or-
gans was calculated in terms of percentage of injected dose per
gram organ (%ID/g). The biodistribution data and tumor/non-
tumor (T/NT) ratios are reported as an average plus the standard
variation. All biodistribution studies were carried out in compli-
ance with the national laws related to the conduct of animal
experimentation.
References and notes
1. Vallabhajosula, S. Semin. Nucl. Med. 2007, 37, 400.
2. Fletcher, J. W.; Djulbegovic, B.; Soares, H. P.; Siegel, B. A.; Lowe, V. J.; Lyman, G.
H.; Coleman, R. E.; Wahl, R.; Paschold, J. C.; Avril, N.; Einhorn, L. H.; Suh, W. W.;
Samson, D.; Delbeke, D.; Gorman, M.; Shields, A. F. J. Nucl. Med. 2008, 49, 480.
3. Necib, H.; Garcia, C.; Wagner, A.; Vanderlinden, B.; Emonts, P.; Hendlisz, A.;
Flamen, P.; Buvat, I. J. Nucl. Med. 2011, 52, 354.
4. Escalona, S.; Blasco, J. A.; Reza, M. M.; Andradas, E.; Gómez, N. Med. Oncol. 2010,
27, 114.
5. Brock, C. S.; Meikle, S. R.; Price, R. Eur. J. Nucl. Med. 1997, 24, 691.
6. Oriuchi, N.; Higuchi, T.; Ishikita, T.; Miyakubo, M.; Hanaoka, H.; Iida, Y.; Endo, K.
Cancer Sci. 2006, 97, 1291.
The data of biodistribution are summarized in Table 1.
99mTc(CO)3(His–CB) did exhibit tumor affinity with certain accu-
mulation and retention (5 min: 3.14 0.52, 30 min: 2.89 1.00,
60 min: 1.25 0.33, 120 min: 0.72 0.18, 240 min: 0.25 0.08
ID%/g). The complex was excreted via hepatobiliary and renal
route. The clearance from normal organs was very fast, thus lead-
ing to very low background at 4 h p.i. (blood: 0.10 0.06, muscle:
0.04 0.02, liver: 0.99 0.35, kidneys: 0.36 0.06 ID%/g). It had
moderate tumor/blood (T/B) ratio that increased up to 4 h p.i.
(1.84 0.73). And the tumor/muscle (T/M) ratios were very high
during the delayed time period (60 min: 5.02 2.48, 120 min:
5.14 2.68, 240 min: 9.23 3.86). The favorable tumor uptake
and retention with the good T/NT ratios strongly suggest the po-
tential of 99mTc(CO)3(His-CB) for tumor imaging.
7. Jurisson, S. S.; Lydon, J. D. Chem. Rev. 1999, 99, 2205.
8. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P. J. Am. Chem. Soc. 1998, 120, 7987.
9. Alberto, R.; Schibli, R.; Waibel, R.; Abram, U.; Schubiger, A. P. Coord. Chem. Rev.
1999, 190–192, 901–919.
10. Schibli, R.; Bella, R. L.; Alberto, R.; Garcia-Garayoa, E.; Ortner, K.; Abram, U.;
Schubiger, A. P. Bioconjug. Chem. 2000, 11, 345.
11. Panasci, L.; Xu, Z. Y.; Bello, V.; Aloyz, R. Anticancer Drugs 2002, 13, 211.
12. Osborne, M. R.; Wilman, D. E. V.; Lawley, P. D. Chem. Res. Toxicol. 1995, 2, 316.
13. Hurley, L. H. Nat. Rev. Cancer 2002, 2, 188.
14. Satpati, D.; Korde, A.; Venkatesh, M.; Banerjee, S. Appl. Radiat. Isot. 2009, 67,
1644.
15. Amin, A. M.; Soliman, S. E.; El-Aziz, H. A. J. Labelled. Compd. Radiopharm. 2010,
53, 1.
16. Wang, J.; Yang, J.; Yan, Z.; Duan, X.; Tan, C.; Shen, Y.; Wu, W. J. Radioanal. Nucl.
Chem. 2011, 287, 465.
17. The synthetic procedures and the spectral data for His–CB are as follows. To
10 mL of acetonitrile was added chlorambucil (compound 1) (500 mg,
1.64 mmol), N-hydroxysuccinimide (NHS) (227.7 mg, 1.98 mmol) and
dicyclohexylcarbodiimide (DCC) (407.9 mg, 1.98 mmol). The reaction mixture
was stirred overnight at room temperature. The white precipitate was filtered.
The solvent was removed under reduced pressure and the residue was purified
by silica gel column to give compound 2 (571 mg, 1.42 mmol, 86.6%) as a light-
The imaging study of 99mTc(CO)3(His-CB) was performed using
Kunming mice bearing H22 liver cancer xenografts with Discovery-
VH SPECT system. A solution of the 99mTc(CO)3(His–CB) (100
lL,
ꢀ1.48 Â 107 Bq) was injected into the tumor-bearing mice via the

J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7406–7409
7409
yellow oil. 1H NMR (400 MHz, CDCl3): d = 2.04 (m, 2 H), 2.62 (m, 4 H), 2.85 (d, 4
according to the procedure published by Alberto and his co-workers.8,9 The pH
of the intermediate was adjusted to ꢀ7.0 with 1 N HCl. A 0.5 mL of the freshly
prepared [99mTc(CO)3(H2O)3]+ precursor (185 MBq) was added into a 5 mL vial
containing His–CB (1 mg) in 0.5 mL 100 mM PBS (pH 7.4). The reaction mixture
was heated at 100 °C for 15 min. After cooling to room temperature, the
radiochemical purity (RCP) of the mixture was evaluated by thin layer
chromatography (TLC) and high performance liquid chromatography (HPLC).
H), 3.63 (t, 4 H), 3.72 (t, 4 H), 6.66 (d, 2 H), 7.10 (d, 2 H), ppm. ESI-MS: 401.6
([M+H]+). To 1 mL of water was added
L-Histidine (38.8 mg, 0.25 mmol), and
the pH value was adjusted to ꢀ9 by 1 N NaOH solution. Then the active ester of
chlorambucil (compound 2) (93.7 mg, 0.234 mmol) dissolved in 1 mL N,N-
dimethylformamide (DMF) was added. The solution was stirred at room
temperature, and the pH value was kept 8–9 by adding NaOH solution for 8 h.
The solvent was removed under reduced pressure and the residue was purified
TLC was performed on
a polyamide strip eluted with dichloromethane/
by silica gel column to give His–CB (0.181 mmol, 77.4%) as a white powder. 1
H
methanol = 1/1 (V/V). HPLC analysis was carried out by using a Waters 600
Controller System with Raytest Gabi Star radio-detector. The column (Kromasil
100–5 C13, 250 Â 4.6 mm) was eluted at a flow rate of 1.0 mL/min. Water (A)
and acetonitrile (B) mixtures were used as the mobile phase and the following
gradient elution technique was adopted for the preparation (0 min 10% B,
5 min 10% B, 20 min 60% B, 25 min 10% B, 30 min 10% B).
NMR (400 MHz, DMSO): d = 1.69 (m, 2 H), 2.07 (t, J = 7.4 Hz, 2 H), 2.38 (t,
J = 7.4 Hz, 2 H), 2.91 (m, 2 H), 3.70 (s, 8 H), 4.12 (t, J = 5.4 Hz, 1 H), 6.64 (t,
J = 7.2 Hz, 2 H), 6.71 (s, 1 H), 6.98 (t, J = 7.2 Hz, 2 H), 7.49 (s, 1 H), 7.65 (br s, 1
H), 11.18 (s, 1 H), ppm. ESI-MS: 439.4 ([MÀH]À).
18. The preparation procedure for 99mTc(CO)3(His–CB) and the TLC, HPLC analysis
conditions are as follows. The tricarbonyl technetium precursor was prepared