Synthesis and preliminary biological evaluation of a 99mTc-chlorambucil derivative as a potential tumor imaging agent

Article abstract of DOI:10.1002/jlcr.3481

Technetium-99m-based radiopharmaceuticals have been used widely as diagnostic agents in the nuclear medicine. Chlorambucil (CLB) as one typical alkylating drug exhibits excellent inhibition effects against many human malignancies. To develop and explore a

Full text of DOI:10.1002/jlcr.3481

Synthesis and preliminary biological evaluation of a 99mTc-chlorambucil
derivative as a potential tumor imaging agent
Jianguo Lin*, Ling Qiu, Gaochao Lv, Ke Li, Wei Wang, Guiqing Liu, Xueyu Zhao, and
Shanshan Wang
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of
Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China
*Corresponding author. Tel: +86-510-85514482, Fax: +86-510-85513113, E-mail address:
linjianguo@jsinm.org (J. Lin).
Technetium-99m-based radiopharmaceuticals have been used widely as diagnostic agents in
the nuclear medicine. Chlorambucil (CLB) as one typical alkylating drug exhibits excellent
inhibition effects against many human malignancies. In order to develop and explore a novel
potential imaging agent for early diagnosis of tumors, tricarbonyl technetium-99m and
rhenium complexes based on the tridentate ligand dipicolylamine (DPA) bound to the
chlorambucil pharmacophore were designed and synthesized: 99mTc-DPA-CLB (3) and
Re-DPA-CLB (4). HPLC analyses showed that the retention time of 3 and 4 was 13.5 and
13.6 min, respectively. Radiolabeling efficiency (RE) of the 99mTc-DPA-CLB tracer was 97%
and the radiochemical purity (RCP) was larger than 95% after 6 h stored in phosphate
buffered saline or human serum as observed by TLC and HPLC. Biodistribution studies in a
mouse model of breast cancer showed 99mTc-DPA-CLB exhibited a favorable tumor affinity.
The radiotracer cleared quickly in the first hour via hepatobiliary and renal routes of
excretion, resulted in a very low background at 4 h p.i.. It had moderate uptake ratios of
tumor to blood and tumor to muscle. These results suggested 99mTc-DPA-CLB might be a
promising SPECT imaging agent for tumor diagnosis.
Keywords: tricarbonyl technetium/rhenium complex; dipicolylamine; chlorambucil; SPECT;
tumor imaging
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3481
This article is protected by copyright. All rights reserved.

Introduction
Cancer is one of the leading causes of death all over the world. Early detection is one of
the critical challenges for contemporary clinical cancer care. In particular, detection of small
pre-malignant lesions and early stage primary tumors is crucial for effective cancer therapy.
For this reason, development of highly specific and sensitive non-invasive molecular imaging
methodologies has increased tremendously in recent decades, such as single photon emission
computed tomography (SPECT) and positron emission tomography (PET). Many groups
have also studied the in vivo detection of tumors using contrast agents appropriate for various
imaging modalities.1-3 There are single-modality tumor agents for PET, SPECT, MRI, CT,
fluorescence or ultrasound, and there are also hybrid agents (these possess two or more
signals within a single molecule/material) for PET-CT, PET-MRI, and etc. Methods have
been developed for specific targeting in order to maximize the localization of ligands in the
tumor and to minimize the uptake in the surrounding normal tissues to achieve high
signal-to-noise ratios.4,5
Since the 1950s, technetium-99m-based radiopharmaceuticals have been important
diagnostic agents in nuclear medicine, mainly because the radionuclide has favorable nuclear
properties (Eγ = 142 keV, t1/2 = 6.02 h) for imaging and from the convenient, reusable on-site
99Mo/99mTc generator.6 To date, many 99mTc-agents have been developed and achieved
regulatory approval for routine clinical use.7,8 Technetium(V) and (III) are the more common
ionic forms for coordinating with ligands. The fac-[99mTc(CO)3]+ species is particularly
attractive because of its ease of preparation via an aqueous-based kit, versatile coordination
chemistry, and formation of kinetically inert complexes which can be conjugated with
targeting biomolecules.9 The lipophilic tridentate ligand 2,2′-dipicolylamine (DPA) has been
employed as the chelating scaffold to bind with the fac-[MI(CO)3]+ core that yields stable
complexes in high efficiency.10-12 For example, [99mTc(CO)3]+ labeled with DPA was found
to be stable against trans-chelation by other ligands in vivo.12
This article is protected by copyright. All rights reserved.

It is known that the nitrogen mustard drugs, such as cyclophosphamide and
mechlorethamine, share similar reactivity to nucleophilic atoms at the
a
N,N-bis(2-chloroethyl)-amine group. This electrophilic moiety can form covalent adducts
with double-helical DNA, especially via reaction with N7 of guanines, either within the same
strand (intrastrand) or between the two opposite DNA strands (interstrand).13,14 Chlorambucil
(CLB) is a chemotherapy drug classified within the nitrogen mustard group, its mechanism of
action involves alkylation, and has been employed for the treatment of various
malignancies.15,16 Moreover, it has also been used as a template to design drugs for diagnosis
and therapy of tumors.17-22
In this study, a novel SPECT tumor imaging agent was synthesized based on a design
that combined the fac-[99mTc(CO)3]+ core, CLB and DPA. As a mimic of the 99mTc-complex,
the rhenium analogue was also prepared using the same chemistry, and the purified complex
was characterized by spectroscopic techniques. The in vitro stability, lipophilicity and in vivo
biodistribution in the mouse xenograft model bearing a breast cancer cell line (MDA-MB-231)
were studied for the 99mTc-DPA-CLB radiotracer.
Material and Methods
General
2,2'-Dipicolylamine was purchased locally (J&K Scientific Ltd; Shanghai; China),
chlorambucil from another supplier (Sigma-Aldrich; Saint Louis; USA), and other
reagents/solvents from elsewhere (Sinopharm Chemical Reagent Co. Ltd; Shanghai; China).
All the chemicals were analytically pure and used without further purification. Na99mTcO4
99
was eluted from the Mo/99mTc generator, which was obtained from the China Institute of
Atomic Energy. The organometallic precursor [Et4N]2[Re(CO)3Br3]9 and the radioactive
precursor fac-[99mTc(CO)3(H2O)3]+23 were prepared as reported.
Elemental analysis was carried out using an analyzer (Vario EL III; Elementar;
Germany). Electron spray ion mass spectra (ESI-MS) were determined using a Waters
Platform ZMD4000 LC/MS (Waters, USA). FT-IR spectra (4000-400 cm-1) were recorded on
an IR Fourier transform spectrophotometer using KBr pellets (Bruker Vector 22 FT-IR
spectrophotometer; Bruker; Germany). Nuclear magnetic resonance spectrometers (13C-NMR;
This article is protected by copyright. All rights reserved.

Bruker DRX-400; Bruker; Germany) and (1H-NMR; Bruker DRX-400; Bruker; Germany)
were used to obtain spectra of samples dissolved in CDCl3 or CD3OD, and the chemical shift
value was referenced to tetramethylsilane. Chromatography paper (Xinhua; Shanghai; China)
was used for thin layer chromatography (TLC) analysis. The high performance liquid
chromatography (HPLC) system was equipped with a pump (Waters 1525 HPLC; Waters;
USA), connected to reverse phase column (RP-C18; 4.6 × 250 mm; 10 um; Elite Analytical
Instrument Company; Dalian; China), a UV detector (2487 dual wavelength absorbance;
Waters; USA) and a radioactivity detector (Radiomatic 610TR; Perkin Elmer; MA; USA)
which were operated by software programs (Breeze Software; NY; USA) and (proFSA;
Perkin Elmer; USA). The flow rate for HPLC analysis was 1.0 mL/min, and the mobile phase
was gradient conditions: A: H2O + 0.1% TFA, B: CH3CN + 0.1% TFA; 0-3 min 20% B, 3-35
min 20-80% B, 35-45 min 80-20% B. Radioactivity was counted using a counter
(Packard-multi-prias; Perkin Elmer; USA).
BALB/c nude mice (18-20 g; 4-6 week old; female; SLAC Laboratory Animal Co. Ltd;
Shanghai; China) were used for animal experiments. Mice were housed with free access to
food and water and allowed ample time to acclimatize before the experiments. All procedures
and animal protocols were approved by the Animal Care and Ethnics Committee of Jiangsu
Institute of Nuclear Medicine.
Synthesis of the ligand DPA-CLB
Preparation of 4-(4-[bis(2-chloroethyl)amino]phenyl)butanoyl chloride (1).
Chlorambucil (3.03 g, 0.01 mol) was dissolved in CH2Cl2 (75 mL). Then the solution was
treated dropwise with thionyl chloride (SOCl2) (30 g, 0.25 mol) and stirred for 2 h at room
temperature. The reaction mixture was concentrated in rotary evaporator (RE-2000B;
Shanghai Yarong Biochemical Instrument Plant; Shanghai; China) and finally compound 1
was obtained as black grease and used immediately in the next step without additional
purification to avoid hydrolysis.
This article is protected by copyright. All rights reserved.

Preparation of 4-(4-[bis(2-chloroethyl)amino]phenyl)-N,N-bis(pyridin-2-ylmethyl)
butanamide (2). Compound 1 (0.636 g, 2.0 mmol) was dissolved in CH2Cl2 (10 mL). While
stirring at room temperature, DPA (0.398 g, 2.0 mmol) in CH2Cl2 (20 mL) was added and
then triethylamine (0.2 mL). After stirring for 1 h water (20 mL) was added to quench the
reaction, the organic layer was separated and washed with HCl (1 N, 2 × 10 mL), saturated
NaCl solution (2 × 10 mL) and then water ( 3 × 10 mL), respectively. The organic layer was
dried over anhydrous Na2SO4, and then the solvent was removed by rotary evaporator.
Finally, compound 2 was obtained as brown viscous oil after purification by silica gel
chromatography with an eluent of ethyl acetate:ethanol [8:1] (Rf = 0.6). The first fraction
(120 mL) of the product was collected. HPLC analysis showed a single peak (tR = 13.6; purity:
98.6%). The combined yield of two steps was 72%.
Anal. Calcd for C26H30Cl2N4O: C, 64.33; H, 6.23; N, 11.54%. Found: C, 64.24; H, 6.41;
1
N, 11.38%. ESI-MS, m/z (%): 485.2(100) = [M+H]+. H-NMR (400 MHz, CDCl3, ppm) δ:
8.59 (d, 2H, J = 4.8 Hz, α-CH, Py), 7.69 (q, 2H, J ＝ 8.0 Hz, γ-CH, Py), 7.14-7.34 (m, 4H, β,
δ-CH, Py), 7.07 (d, 2H, J = 8.8 Hz, H-Ar), 6.61 (d, 2H, J = 8.4 Hz, H-Ar), 4.80 (s, 2H,
N-CH2-Py), 4.70 (s, 2H, N-CH2-Py), 3.73 (m, 4H, J ＝ 6.8 Hz, Cl-CH2), 3.64 (m, 4H, J ＝
6.8 Hz, Ar-N-CH2), 2.58 (t，2H, J ＝7.6 Hz, COCH2), 2.47 (t, 2H, J ＝ 7.6 Hz, Ar-CH2)，
2.02 (m, 2H, CH2). IR (KBr, cm-1): 3448 (m, vC-H, Ar), 2929 (m, vC-H), 1646 (s, vC=O), 1570
(m, vph), 1434 (s, vC-N), 1097 (w, vC-Cl), 750 (m, vph), 654(w, vC-H).
Preparation of 99mTc/Re complexes
The technetium-99m-labeled complex (3) was prepared according to the following
procedure. To a solution of fac-[99mTc(H2O)3(CO)3]+ (500 μL, pH = 6) in water in a glass vial
(10 mL), was added DPA-CLB (2) in ethanol (5 g/L; 100 μL) under a blanket of carbon
o
monoxide gas. The reaction was heated to 75 C for 30 min and then cooled to room
temperature in water. The radiolabeling efficiency (RE) and radiochemical purity (RCP) of
the complex were measured by TLC and HPLC. Two developing solvents,
acetonitrile/methanol = 2/1 (V/V) and ethyl acetate/methanol = 10/1 (V/V), were employed
for the TLC analysis.
This article is protected by copyright. All rights reserved.

The rhenium-complex 4 was synthesized according to the following procedure.
(Et4N)2[Re(CO)3Br3] (55 mg, 0.08 mmol) and the ligand DPA-CLB (2) (48 mg, 0.1 mmol)
were dissolved in methanol (3 mL) and then stirred at room temperature (25 oC) for 1.5 h.
Then the reaction mixture was concentrated in vacuum and the residue was purified by silica
gel chromatography with an eluent of ethyl acetate:ethanol:triethylamine [8:1:0.1] (Rf = 0.4).
The gray powder product was obtained (yield: 75%). HPLC analysis showed a single peak (tR
= 13.6; purity: 99.4%).
Anal. Calcd for C29H31Cl2N4O4Re: C, 46.03; H, 4.13; N, 7.40%. Found: C, 45.91; H,
1
4.26; N, 7.35%. ESI-MS, m/z (%): 756.21(100) = [M+H]+. H-NMR (400 MHz, CD3OD,
ppm) δ: 8.82 (d, 2H, J = 6.4 Hz, α-CH, Py), 7.84-7.88 (td, 2H, J1,2,3 = 1.2 Hz, γ-CH, Py), 7.58
(d, 2H, J = 8.0 Hz，β-CH, Py), 7. 31 (t, 2H, J = 6.4 Hz, δ-CH, Py), 6.94 (m, 2H, H-Ar), 6.56
(m, 2H, H-Ar), 4.72 (s, 4H, N-CH2-Py), 3.61 (m, 4H, Cl-CH2), 3.25 (m, 4H, Ar-N-CH2), 2.43
(t, 2H, J ＝ 7.2 Hz, Ar-CH2), 2.20 (t, 2H, J ＝ 7.2 Hz, CH2 C=O), 1.22 (m, 2H,
CH2CH2CH2). 13C-NMR (100 MHz, CD3OD, ppm) δ: 194.68 (Re(CO)3), 174.28 (C=O),
160.30, 151.29, 139.65, 124.96, 122.866 (Py), 144.169, 137.223, 129.64, 111.76 (Ph), 61.62
(CH2-Py), 52.69 (CH2-Cl), 51.70 (N-CH2), 40.18 (Ph-CH2), 33.10 (-CH2C=O), 26.13
(CH2CH2CH2). IR (KBr, cm-1): 3443 (m, vC-H, Ar), 2922 (m, vC-H), 2027 (s, vCO), 1928 (s, vCO),
1888 (s, vCO), 1610 (m, vC=O), 1580 (m, vph), 1414 (m, vC-N), 1090 (br, vC-Cl), 798 (m, vph), 615
(w, vC-H).
In vitro stability assays
The in vitro stability of the 99mTc-DBA-CLB (3; 3.7 MBq) diluted in saline (100 μL),
was evaluated in the presence of phosphate buffered saline (PBS; 0.1 M; pH = 7; 1 mL), and
also in human serum (1 mL) separately. Then the solutions were incubated (K37
multifunctional incubator, Hangzhou Allsheng Instruments Company; Hangzhou; China) at
37 oC. Samples (50 uL) were removed from each incubating solution at t = 0, then every hour
for 6 hours, and analyzed for %RCP by HPLC.
This article is protected by copyright. All rights reserved.

Determination of octanol/water partition coefficient
The octanol/water partition coefficient of 99mTc-DPA-CLB (3) was determined at two
different pH values of 7.0 and 7.4 by measuring its distribution in n-octanol and PBS,
respectively. A sample of 3 (100 μL) was added to an immiscible liquid containing PBS (900
μL; pH 7.0 or 7.4) and n-octanol (1 mL), then after 2 min vigorous vortex (XH-C vortex,
Nanjing Eastmai Science and Technology Instrument Co., Ltd.; Nanjing; China), the mixture
was incubated for 30 min at room temperature. Centrifugation (PJ-TDL-40B, Wuxi Ruijiang
Analysis Instrument Co. Ltd., Wuxi; China) at 4000 g for 5 min ensured complete separation
of the organic and the aqueous layers. An aliquot (100 uL) from each layer was measured
with a γ counter. The partition coefficient was calculated using the following formula: log P
= log (octanol counts/water counts). The reported value is the average obtained from three
independent measurements.
Biodistribution studies in tumor-bearing mice
In vivo biodistribution of 99mTc-DBA-CLB (3) was evaluated in the mouse xenograft
model bearing breast cancer cell MDA-MB-231 (Cell Bank of the Chinese Academy of
Sciences; Shanghai; China). The breast cancer cells MDA-MB-231 (1×106) in phosphate
buffered saline (0.2 mL) were injected subcutaneously into the left front leg of the mice. Ten
or fifteen days after inoculation, thirty five mice with the tumor size in the range of 0.5-1.0
cm in diameter divided into seven groups were used to study the biodistribution of
99mTc-DBA-CLB (3) in various organs. Each mouse per group was intravenously
administered with the radiotracer (100 μL, 37 kBq) via the tail vein using an insulin syringe.
The mice were anesthesized (2% isoflurane in oxygen at a flow rate of 2 L/min) and
sacrificed at 10, 30, 60, 120 and 240 min post injection. Tissue samples of interest were
collected and weighed, such as bone (femur), muscle from leg, heart, liver, spleen, lung,
kidney, uterus, ovary, intestines, stomach, and brain as well as 200 μL blood taken from the
carotid artery. The radioactivity in each sample was measured by a γ counter. The radiotracer
uptake in different organs was calculated and expressed as the percent uptake of the injected
dose per gram of organ using the formula %ID/g = 100 × [organ counts/(organ weight x
whole body counts)]. And the uptake ratios of tumor to muscle and to blood were calculated
This article is protected by copyright. All rights reserved.

from the %ID/g values. The results were expressed as mean values of five independent
experiments.
Results and discussion
Synthesis and radiolabeling
From the commercially available starting material chlorambucil, suitable
functionalization with a bifunctional chelating group 2,2'-dipicolylamine was possible in a
two-step procedure involving the activation of the carboxyl group and the formation of an
amide (Scheme 1). Briefly, the corresponding acid chloride of chlorambucil (1) was prepared
with excess thionyl chloride, and further reaction with 2,2'-dipicolylamine in the presence of
triethyl amine in CH2Cl2 afforded the compound DPA-CLB (2). The chemical syntheses were
convenient and the overall yield of the product was good. The DPA moiety in DPA-CLB (2)
contained three N-donor atoms for coordination with the [MVII(CO)3(H2O)3]+ core to form
stable octahedral complexes, and the rhenium complex was characterized by the elemental
1
analysis, MS and H/13C-NMR. The results obtained were in agreement with the expected
chemical structure.
Taking advantage of the stability of 99mTc-tricarbonyl reagent and the adducts derived
from it, [99mTc(CO)3(H2O)3]+ was hence selected as a precursor of choice for the
radiolebeling step. The radiolabeled complex 3 was prepared by the ligand-exchange reaction
of [99mTc(CO)3(H2O)3]+ and DPA-CLB in aqueous ethanol solvent in 97.3% RE within 20
minutes. Furthermore, it is acknowledged that the development of technetium-labeled
complexes as radiopharmaceuticals has been facilitated by the use of rhenium, the group
VIIB congener of technetium. Rhenium chemistry is similar to technetium-99m chemistry
and therefore non-radioactive metal complexes are often used as mimics of 99mTc-analogues,
with the practical advantage that larger scale syntheses can yield quantities of product for
structural characterization.23-25 In this study, the rhenium-complex 4 was synthesized in
sufficient quantity from DPA-CLB (2) in 75% yield via Scheme 1, it was characterized by
elemental analysis and a mix of different spectroscopic methods. NMR analysis of the Re
complex in CD3OD allowed the specific binding mode of the precursor to be deduced. The
This article is protected by copyright. All rights reserved.

1
aromatic portion of H-NMR spectrum showed that two pyridyl units were equivalent and
shifted downfield with respect to the free precursor. The chemical shift of α-CH was 8.59
ppm in the ligand precursor molecule (2) and 8.82 ppm for the product rhenium-complex (4).
This downfield shift of hydrogens between two molecules was evidence that neighboring
nitrogen atoms coordinated to the transition metal. The chemical shift of the methylene
neighboring pyridyl is 4.72 ppm, while they are 4.70 and 4.80 ppm in the precursor. The
results indicated that the two pyridyl nitrogen atoms are symmetrical at the octahedron
coordination geometry of the Re metal. Interestingly, the 13C data of the complex revealed
that there were only two Re-CO peaks (~195 ppm), due to the simple overlap or those two
CO groups were magnetically equivalent because of the mirror symmetry.
Quality control
The RCP of fac-[99mTc(CO)3(H2O)3]+ and 99mTc-DPA-CLB was checked by TLC and
HPLC, respectively. In the developing solvent of acetonitrile/methanol (V/V = 2/1),
fac-[99mTc(CO)3(H2O)3]+ moved to the solvent front (Rf = 0.9-1.0), while 99mTcO2·nH2O and
Na99mTcO4 remained at the origin (Rf = 0-0.1) in comparison with the corresponding
reference materials. When TLC developed by ethyl acetate/methanol (V/V = 10/1), the Rf
value for 99mTc-DPA-CLB (3) was about 0.6-0.7, while 99mTcO2·nH2O, Na99mTcO4 and
fac-[99mTc(CO)3(H2O)3]+ remained at the origin (Rf = 0-0.1). In two kinds of developed
solvent systems, most of the radioactivity moved to the front and the RCP of
99mTc-DPA-CLB (3) was higher than 95%. About 2% fac-[99mTc(CO)3(H2O)3]+ and 3%
99mTc-pertechnetate along with 99mTcO2 were determined to be the impurities.
The radiotracer 3 was analyzed by the radio-HPLC, as shown in Figure 1. It was
observed that the retention time of [99mTc(CO)3(H2O)3]+ was 4.5 min, while that of
99mTc-DPA-CLB was found to be ~13.5 min (Figure 1a). The reference complex (4) was
checked by HPLC with tR = 13.6 min and high purity (Figure 1b). The retention time of
99mTc-labelled complex was almost identical with that of the corresponding Re complex (4).
According the HPLC analysis, the radiolabeling yield of the 99mTc-labelled complex was 96%.
About 4% impurities were observed at ~3.8 min and ~4.5 min for 99mTc-pertechnetate and
This article is protected by copyright. All rights reserved.

fac-[99mTc(CO)3(H2O)3]+, respectively. The HPLC results were consistent with TLC analysis,
indicating that the quality control methods were feasible and efficient for the radiotracer.
Since the freshly prepared radiotracer was used without further purification for both in vitro
and in vivo studies, the RCP was identical to the RE.
In vitro stability and lipophilicity
In vitro stabilities of the radiotracer 3 in PBS (pH = 7.0) and human serum were both
studied and analyzed by radio-HPLC. As shown in Figure 2, after 6 h of incubation, more
than 95% of the complex still remained intact according to the HPLC analysis. No obvious
decomposition products or reoxidation to pertechnetate were detected during the period of
study, demonstrating that the chemical stability of the radiotracer was high enough for further
biological investigation as a molecular imaging agent.
The partition coefficient logP of 99mTc-DPA-CLB (3) was determined to be 2.34 ± 0.48,
suggesting that it was hydrophobic rather than hydrophilic. In comparison with the
radiolabeling precursor 2 (clog P = 3.47), the positive charge of the tricarbonyl moiety
increased the polarity and aqueous solubility of the overall complex (3). The pyridine
nitrogen atom lone pairs contributed to stabilizing the electropositive metal, and resulted in
an integral molecule for six hours at room temperature in media in vitro.12,26
Biodistribution in tumor-bearing mice
In vivo biodistribution results of the radiotracer 3 were summarized in Figure 3 and
Table 1, in which the initial tumor uptake of 1.2% ID/g accumulated to 1.5% ID/g after 60
minutes and this level was retained for an hour until its gradual decline to 1.2% ID/g at 4
hours post injection. The complex was excreted quickly via hepatobiliary and renal route.
Hence, the liver and the kidney had higher radioactivities than other organs, especially in the
first hour. Moreover, it was cleared from cancer-free organs very fast, thus leading to a very
low background at 4 h p.i.. For example, its blood clearance decreased from 2.1% ID/g at 10
min p.i. to 0.6% ID/g at 30 min p.i., and then to 0.2% ID/g at 4 h p.i.; and the liver clearance
This article is protected by copyright. All rights reserved.

decreased from 15.5% ID/g at 10 min p.i. to 12.9% ID/g at 60 min p.i., and then to 5.6% ID/g
at 4 h p.i.. On the whole, the radiotracer 3 had modest uptake ratios of tumor-to-blood (T/B)
and tumor-to-muscle (T/M) after 1 h (3.27 ± 0.35 and 2.44 ± 0.46, respectively), which
increased up to 5.22 ± 0.63 and 7.18 ± 0.80, respectively, at 4 h p.i.. 99mTc-pertechnetate is
characteristically taken up by the stomach in mammals. In this study the stomach uptake in
mice was found to be low to the extent of 0.3-0.7% ID/g over 1-4 hours, indicating no
significant decomposition or metabolism of the complex to 99mTc-pertechnetate occurred in
o
vivo. This result concurs with the stability of the radiotracer in serum at 37 C in vitro. As
reported in a previous study,20 another chlorambucil derivative was synthesized by
conjugating a NNO chelating group [N-(3-(dimethylamino)propyl)amido]acetic acid to
chlorambucil, which was further labeled with 99mTc(CO)3 successfully. The in vivo biological
studies of the reported 99mTc(CO)3-labeled chlorambucil derivative showed increasing uptake
in the stomach with the time (increasing from 3.07% ID/g at 1 h to 17.7% ID/g at 3 h),
indicating that this radiotracer took place an in vivo decomposition even though it was very
o
stable (98% RCP) in vitro experimental study under 37 C incubation in PBS at pH 7.0 as
well as in human serum.20 The difference might originate from the different tridentate
chelating moieties. In our study, the tridentate ligand 2,2′-dipicolylamine (DPA) was chosen
as the chelating scaffold, which exhibited favorable complexation property and kinetic
stability with the fac-[MI(CO)3]+ core.27,28 As a whole, the 99mTc-DPA-CLB radiotracer (3)
has a superior stability and favorable tumor uptake with good T/NT uptake ratios, which
strongly suggests that it has potential for tumor imaging.
Conclusion
A new analogue of the anticancer drug chlorambucil was synthesized after covalent bonding
with the dipicolylamine chelating group. The resulting ligand DPA-CLB was successfully
radiolabeled with 99mTc(CO)3(H2O)3 in high yield (>95% RE), and the resulting
99mTc-DPA-CLB complex was found to be stable in vitro and in vivo. Biodistribution studies
in a mouse model of breast cancer showed rapid clearance from the bloodstream, with the
major route of excretion by the hepatobiliary system. There was peak tumor uptake of 1.5%
ID/g at 60 min pi and this gradually decreased to 1.2% ID/g after 4 hours. In a mouse model
This article is protected by copyright. All rights reserved.

of breast cancer, this agent gave a tumor/blood ratio increase from 3.3 to 5.2, and a tumor to
muscle ratio from 2.4 to 7.2 over that time frame. The 99mTc-complex has the property of
favorable tumor uptake and retention, fast clearance from background and good T/NT ratios,
which is an advantage over another reported CLB analogue. The data presented so far
suggests 99mTc-DPA-CLB (3) has potential as a tumor imaging agent. This laboratory intends
to evaluate its application further.
Acknowledgements
This work was financially supported by National Natural Science Foundation of China
(21371082 and 21001055), Natural Science Foundation of Jiangsu Province (BK20141102
and BK20151118) and Key Medical Talent Project of Jiangsu Province (RC2011097).
References
[1] Gao ZY, Ma TC, Zhao EY, Docter D, Yang WS, Stauber RH, Gao MY. Small is Smarter: Nano MRI
Contrast Agents – Advantages and Recent Achievements. Small. 2016;12:556-576.
[2] Guo WS, Sun XL, Jacobson O, Yan XF, Min K, Srivatsan A, Niu G, Kiesewetter DO, Chang J, Chen
XY. Intrinsically Radioactive [64Cu]CuInS/ZnS Quantum Dots for PET and Optical Imaging:
ImprovedRadiochemical Stability and Controllable Cerenkov Luminescence. ACS Nano. 2015;
9:488-495.
[3] Alsibai W, Hahnenkamp A, Eisenblatter M, Riemann B, Schäfers M, Bremer C, Haufe G, Höltke C.
Fluorescent non-peptidic RGD-mimetics with high selectivity for αVβ3 integrin vs. α11bβ3 receptors:
Novel probes for in vivo optical imaging. J.Med. Chem. 2014;57: 9971-9982.
[4] Alam IS, Arshad MA, Nguyen QD, Aboagye EO. Radiopharmaceuticals as probes to characterize
tumour tissue. Eur. J. Nucl. Med. Mol. Imaging. 2015;42: 537-561.
[5] Cai HC, Conti PS. RGD-based PET tracers for imaging receptor integrin avβ3 expression. J. Labelled
Compd. Radiopharm. 2013; 6: 264-279.
[6] Selivanova SV, Lavallée É, Senta H, Caouette L, Sader JA, van Lier EJ, Zyuzin A, van Lier JE,
Guérin B, Turcotte É, Lecomte R. Radioisotopic Purity of Sodium Pertechnetate 99mTc Produced with
a Medium-Energy Cyclotron: Implications for Internal Radiation Dose, Image Quality, and Release
Specifications. J. Nucl. Med. 2015; 56: 1600-1608.
[7] Arano Y, Recent advances in Tc-99m radio pharmaceuticals. Ann. Nucl. Med., 2002; 16: 79-93.
[8] Gaertner FC, Kessler H, Wester HJ, Schwaiger M, Beer AJ. Radiolabelled RGD peptides for imaging
and therapy. Eur. J. Nucl. Med. Mol. Imaging, 2012;39:S126–S138.
[9] Alberto R, Schibli R, Egli A, Schubiger AP. A Novel Organometallic Aqua Complex of Technetium
for the Labeling of Biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous
Solution and Its Reaction with a Bifunctional Ligand. J. Am. Chem. Soc. 1998; 120: 7987-7988.
[10] Kasten BB, Ma XW, Liu HG, Hayes TR, Barnes CL, Qi SB, Cheng K, Bottorff SC, Slocumb W S,
This article is protected by copyright. All rights reserved.

Wang J, Cheng Z, Benny PD. Clickable, Hydrophilic Ligand for fac-[M1(CO)3]+ (M = Re/99mTc)
Applied in an S‑ Functionalized α‑ MSH Peptide. Bioconjugate Chem. 2014; 25: 579-592.
[11] Mallia MB, Mittal S, Sarma HD, Banerjee S. Modulation of in vivo distribution through chelator:
Synthesis and evaluation of a 2-nitroimidazole–dipicolylamine–99mTc(CO)3 complex for detecting
tumor hypoxia Bioorg. Med. Chem. Lett. 2016; 26: 46-50.
[12] Kasten BB, Ma XW, Cheng K, Bu LH, Slocumb WS, Hayes TR, Trabue S, Cheng Z, Benny PD.
Isothiocyanate-Functionalized Bifunctional Chelates and fac-[M1(CO)3]+ (M = Re, 99mTc) Complexes
for Targeting uPAR in Prostate Cancer. Bioconjugate Chem. 2016; 27: 130-142.
[13] Antonio MD, McLuckie IEK, Balasubramanian S. Reprogramming the Mechanism of Action of
Chlorambucil by Coupling to a G‑ Quadruplex Ligand J. Am. Chem. Soc. 2014; 136: 5860-5863.
[14] Hartley JA, Bingham JP, Souhami RL. DNA sequence selectivity of guanine-N7 alkylation by
nitrogen mustards is preserved in intact cells. Nucleic Acids Res. 1992; 20: 3175-3178.
[15] Xu YZ, Gu XY, Peng SJ, Fang JG, Zhang YM, Huang DJ, Chen JJ, Gao K. Design, synthesis and
biological evaluation of novel sesquiterpene mustards as potential anticancer agents. Eur. J. Med.
Chem. 2015; 94: 284-297.
[16] Faguet GB. Chronic Lymphocytic Leukemia: An Updated Review. J. Clin. Oncol. 1994; 12:
1974-1990.
[17] Nazarov AA, Meier SM, Zava O, Nosova YN, Milaeva ER, Hartinger CG, Dyson PJ. Protein
ruthenation and DNA alkylation: chlorambucil-functionalized RAPTA complexes and their anticancer
activity. Dalton Trans. 2015; 44: 3614-3623.
[18] Millard M, Gallagher JD, Olenyuk BZ, Neamati N. A Selective Mitochondrial-Targeted
Chlorambucil with Remarkable Cytotoxicity in Breast and Pancreatic Cancers. J. Med. Chem. 2013;
56: 9170-9179.
[19] Bell RA, Hughes DW, Lock CJL, Valliant JF. The synthesis and high-resolution NMR spectroscopy
of
ethyl
N-(2-triphenylmethylthio)ethanoyl-O-(4'-[4"-(1"-bis(2"'-chloroethyl)amino)phenyl]butanoyl}-L-seryl
-S-benzyl-L-cysteine: a chelate-chlorambucil complex for use as a ligand for 99mTc radio-imaging.
Can. J. Chem. 1996; 74: 1503-1511.
[20] Satpati D, Korde A, Venkatesh M, Banerjee S. Preparation and bioevaluation of a 99mTc-labeled
chlorambucil analog as a tumor targeting agent. Appl. Radiat. Isotopes. 2009; 67: 1644-1649.
[21] Wang JJ, Yang J, Duan XJ, Zhang YH, Yang WJ, Liu Y. Synthesis and biological evaluation of
99mTc(CO)3(His–CB) as a tumor imaging agent. Bioorg. Med. Chem. Lett. 2012; 22: 7406-7409.
[22] Wang JJ, Yang J, Yan ZY, Duan XJ, Tan CM, Shen YL, Wu WS. Synthesis and preliminary
biological evaluation of [99mTc(CO)3(IDA–PEG3–CB)]- for tumor imaging. J. Radioanal. Nucl. Chem.
2011; 287: 465-469.
[23] Alberto R, Schibli R, Egli A, Schubiger AP, Herrmann WA, Artus G, Abram U, Kaden TA. Metal
carbonyl syntheses XXII. Low pressure carbonylation of [MOC14]-and [MO4]-:the technetium(I)and
rhenium(I)complexes [NEt4]2[MC13(CO)3] J. Organomet. Chem. 1995; 493: 119-127.
[24] Makris G, Papagiannopoulou D. Synthesis, characterization, and biological evaluation of new
biotinylated 99mTc/Re-tricarbonyl complexes. J. Labelled Comp. Radiopharm. 2016; 59: 95-102.
[25] Klenc J, Lipowska M, Abhayawardhana PL, Taylor AT, Marzilli LG. Structure and Properties of
fac-[ReI(CO)3(NTA)]2− (NTA3− = Trianion of Nitrilotriacetic Acid) and fac-[ReI(CO)3(L)]n−
Analogues Useful for Assessing the Excellent Renal Clearance of the fac‑ [99mTcI(CO)3(NTA)]2−
Diagnostic Renal Agent. Inorg. Chem. 2015; 54: 6281-6290.
This article is protected by copyright. All rights reserved.

[26] Hayes TR, Lyon PA, Silva-Lopez E, Twamley B, Benny PD. Photo-initiated Thiol-ene Click
Reactions as a Potential Strategy for Incorporation of [MI(CO)3]+ (M = Re, 99mTc) Complexes. Inorg.
Chem. 2013; 52: 3259-3267.
[27] Liu GZ, Dou SP, He J, Vanderheyden JL, Rusckowski M, Hnatowich DJ. Preparation and Properties
+
of 99mTc(CO)3 -Labeled N,N-Bis(2-pyridylmethyl)-4-aminobutyric Acid. Bioconjugate Chem. 2004;
15: 1441-1446.
[28] Banerjee SR, Schaffer P, Babich JW, Valliant JF, Zubieta J. Design and synthesis of site directed
maleimide bifunctional chelators for technetium and rhenium. Dalton Trans. 2005; 3886-3897.
This article is protected by copyright. All rights reserved.

O
N
N
CH2CH2CH2COOH
CH2CH2CH2COCl
N
SOCl2
CH2Cl2
2,2'-Dipicolylamine
N(CH2CH3)3, CH2Cl2
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
CLB
1
2
O
N
CO
[
99mTc(CO)3(H2O)3]
N
CO
CO
M
N
or [Et4N]2[Re(CO)3Br3]
N
M = 99mTc, 3
M = Re, 4
Cl
Cl
Scheme 1. Synthesis of the ligand DPA-CLB (2) and the complexes 99mTc-DPA-CLB (3), Re-DPA-CLB
(4).
This article is protected by copyright. All rights reserved.

(a)
(b)
13.5 min
0.6
0.5
0.4
0.3
0.2
0.1
0.0
13.6 min
0
5
10
15
Time (min.)
20
25
Figure 1. HPLC chromatogram of the radiotracer 3 (a, tR=13.5 min) and the Re-complex 4 (b, tR=13.6
min).
This article is protected by copyright. All rights reserved.

100
98
96
94
92
90
PBS
Serum
1
2
3
4
5
6
Time (h)
Figure 2. In vitro stability of the radiotracer 3 in PBS and human serum, respectively.
This article is protected by copyright. All rights reserved.

3.0
2.4
1.8
1.2
0.6
0.0
10 min
30 min
60 min
120 min
240 min
(a)
r
s
y
d
n
i
r
va
u
er
o
mo
u
ines
st
Bra
Blo
O
T
Ut
Muscle
e
Int
25
20
15
10
5
1 0 m i n
3 0 m i n
6 0 m i n
1 2 0 m i
2 4 0 m i
(b)
0
y
t
n
r
e
n
g
e
n
n
l e e
i v e
u
m a c h
p
L
L
B o
H e a r
o
S
t
K i d
S
Figure 3. Biodistribution of the radiotracer 3 in the mouse model of breast cancer (n=5).
This article is protected by copyright. All rights reserved.

8
6
4
2
0
T/M
T/B
10
30
60
120
240
Time (min)
Figure 4. Uptake ratios of tumor/blood and tumor/muscle for the radiotracer 3.
This article is protected by copyright. All rights reserved.

Table 1. Biodistribution of 99mTc-DPA-CLB (3) in the mouse model of breast cancer (mean ± SD,
n=5, %ID/g)
Time after administration
Tissues
10 min
30 min
60 min
120 min
1.46±0.06
0.71±0.13
0.33±0.05
0.41±0.05
0.13±0.04
0.23±0.07
0.22±0.02
1.42±0.13
9.24±0.28
1.39±0.17
1.07±0.38
8.86±0.76
0.37±0.08
240 min
1.15±0.10
0.16±0.06
0.22±0.08
0.33±0.06
0.23±0.09
0.32±0.11
0.17±0.03
0.61±0.11
5.59±0.31
1.28±0.13
0.58±0.09
8.82±0.79
0.38±0.07
Tumor
Muscle
Blood
1.19±0.06
0.87±0.04
2.08±0.65
0.56±0.04
0.26±0.05
0.48±0.18
0.33±0.05
4.29±0.05
15.45±4.42
1.80±0.22
5.95±1.48
11.38±2.54
0.30±0.06
1.24±0.07
0.70±0.08
0.68±0.27
0.23±0.08
0.18±0.08
0.33±0.10
0.33±0.02
3.49±0.25
15.15±3.16
1.71±0.25
5.44±0.32
9.60±1.04
0.32±0.19
1.54±0.07
0.63±0.08
0.47±0.10
0.48±0.15
0.16±0.05
0.24±0.04
0.27±0.07
2.71±0.41
12.88±1.32
1.45±0.20
3.10±0.78
8.67±1.28
0.68±0.15
Intestines
Uterus
Ovary
Brain
Heart
Liver
Spleen
Lung
Kidney
Stomach
Bone
0.85±0.05
0.79±0.08
0.89±0.10
0.57±0.09
0.51±0.14
(Femur)
This article is protected by copyright. All rights reserved.