Novel Meta-iodobenzylguanidine-Based Copper Thiosemicarbazide-1-guanidinomethylbenzyl Anticancer Compounds Targeting Norepinephrine Transporter in Neuroblastoma

Article abstract of DOI:10.1021/acs.jmedchem.9b00386

Meta-iodobenzylguanidine (MIBG) is a ligand with high affinity against norepinephrine transporter (NET) that has been used for diagnostic imaging and radionuclide therapy of NET-expressing tumors, such as neuroblastoma. We hypothesize that MIBG can be used as a ligand for development of new anticancer drugs targeting NET-expressing neuroblastoma (NB). To test our hypothesis, we synthesized two MIBG-based anticancer copper complexes [Cu(m-TSBG)₂ and Cu(p-TSBG)₂] by conjugation of a thiosemicarbazone copper group onto MIBG ligand. Both Cu(m-TSBG)₂ and Cu(p-TSBG)₂ compounds showed potent anticancer activity against NB cells (BE2C and SK-N-DZ cells). The NB-specific anticancer activity of Cu(m-TSBG)₂ and Cu(p-TSBG)₂ was further demonstrated by the reduced anticancer activities when nonconjugated MIBG ligand was used to competitively block binding of Cu(m-TSBG)₂ or Cu(p-TSBG)₂ onto NET-expressing NB cells. Both Cu(m-TSBG)₂ or Cu(p-TSBG)₂ compounds hold potential as promising new drugs for targeted therapy of neuroblastoma and other NET-expressing tumors.

Full text of DOI:10.1021/acs.jmedchem.9b00386

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Article
Novel Meta-iodobenzylguanidine-based Copper Thiosemicarbazide-1-
guanidinomethyl-benzyl Anticancer Compounds
Targeting Norephinephrine Transporter in Neuroblastoma
Haiyuan Zhang, Fang Xie, Muhua Cheng, and Fangyu Peng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00386 • Publication Date (Web): 08 Jul 2019
Downloaded from pubs.acs.org on July 9, 2019
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Novel Meta-iodobenzylguanidine-based Copper
Thiosemicarbazide-1-guanidinomethyl-benzyl Anticancer Compounds
Targeting Norephinephrine Transporter in Neuroblastoma
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Haiyuan Zhang1, Fang Xie2, Muhua Cheng3, and Fangyu Peng1,2,4
*
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Carman & Ann Adams Department of Pediatrics1, School of Medicine, Wayne State
University, Detroit, MI 48201, USA
Department of Radiology2, University of Texas Southwestern Medical Center, Dallas,
Texas 735390, USA;
Department of Nuclear Medicine3, Third Affiliated Hospital of Sun Yat-sen University,
Guangzhou, Guangdong 510630, China
Harold C. Simmons Comprehensive Cancer Center4, University of Texas Southwestern
Medical Center, Dallas, Texas 75390, USA;
ABSTRACT
Meta-iodobenzylguanidine (MIBG) is a ligand with high affinity against norephinephrine
transporter (NET) that has been used for diagnostic imaging and radionuclide therapy of
NET-expressing tumors, such as neuroblastoma. We hypothesize that MIBG can be used
as a ligand for development of new anticancer drugs targeting NET-expressing
neuroblastoma (NB).
anticancer copper complexes [Cu(m-TSBG)2 and Cu(p-TSBG)2] by conjugating of a
thiosemicarbazone-copper group onto MIBG ligand. Both Cu(m-TSBG)2 and
To test our hypothesis, we synthesized two MIBG-based
Cu(p-TSBG)2 compounds showed potent anticancer activity against NB cells (BE2C and
SK-N-DZ cells). The NB-specific anticancer activity of Cu(m-TSBG)2 and Cu(p-TSBG)2 was
further demonstrated by the reduced anticancer activities when non-conjugated MIBG
ligand was used to competitively block binding of Cu(m-TSBG)2 or Cu(p-TSBG)2 onto
NET-expressing NB cells. Both Cu(m-TSBG)2 or Cu(p-TSBG)2 compounds hold potential
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as promising new drugs for targeted therapy of neuroblastoma and other NET-expressing
tumors.
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INTRODUCTION
Neuroblastoma is the most common extracranial cancer in childhood and often occurs in
very young children.1, 2 Chemotherapy plays a major role in the treatment of high-risk
neuroblastoma.
Acquired resistance of NB to cisplatin and other anticancer drugs
currently available results in poor prognosis of children diagnosed with late-stage disease
of NB. Despite significant efforts devoted to development of effective drugs for treatment
of NB, side effects from non-specific cytotoxicity of many of anticancer compounds
hampered their clinical application.3-5 There are continued efforts in developing new
anticancer drugs with improved cancer cell targeting capability or improved in vivo
pharmacokinetics.6 Positron emission tomography (PET) could be used to study
biodistribution and in vivo pharmacokinetics of new compounds by radiolabeling of
anticancer compounds with a positron emitting radionuclide.7
Many of metal complexes were found to have potent anticancer activity and hold
potentials as promising new anticancer drugs.8-12 Copper is a nutritional metal required for
many of physiological processes in human body. Many of copper complexes were found
to have strong anticancer activity against NB and other tumors such as prostate cancer.13
Copper
complexes
of
8-hydroxyquinoline-2-carboxaldehyde-thiosemicarbazide
(CuHQTS) is an anticancer copper complex with potent anticancer activity against NB
and prostate cancer cells.14, 15 Recently, it was found that growth of human prostate
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cancer xenograft tumors was inhibited in athymic nu/nu mice treated with CuHQTS.16
However, significant weight loss of the mice treated with CuHQTS was observed, likely
a side effect of non-specific toxicity on body metabolism due to lack of tumor-specific
cytotoxicity of CuHQTS.14-16
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Neuroblastoma cells are derived from the neural crest and reserve many properties
18
such as synthesis, storage, release and reuptake of norephinephrine (NE).17,
Benzylguanidine is a metabolically stable NE analogue derived from the adrenergic
neuron-blocking agents, bretylium and guanethidine. Benzylguanidine can be specifically,
actively and efficiently taken up by neuroblastoma cells through NE transporter (NET), a
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specific transport system located at neuroblastoma cell membrane.19,
Meta-iodobenzylguanidine (MIBG), a small molecule, has been widely applied as an
imaging agent in the diagnostic imaging and radionuclide therapy of neuroendocrine
tumors, such as neuroblastoma, pheochromocytoma and carcinoid tumor, based on its
high affinity binding to NET.21-24 We hypothesize that MIBG may be used as a targeting
ligand for synthesis of new anticancer drugs consisting of MIBG and an anticancer
copper complex such as CuHQTS for targeted therapy of neuroblastoma.
This study aimed to test our hypothesis by synthesizing new CuTSBG anticancer
compounds combining MIBG and CuHQTS for targeted therapy of NB. Cu(m-TSBG)2
and Cu(p-TSBG)2 (Figure 1), were designed to engineer a new compound consisting of
both benzylguanidine and copper-thiosemicarbazone groups for NB specific delivery and
cytotoxicity.
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N
NH2
N
S
Cl
N
+
N
N
N
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N
H
S
N
N
NH2
Cu
Cu
H
N
S
S
NH2
N
N
+
Cl
N
N
NH2
Cu(m-TSBG)2
Cu(m-TSB)2
N
S
N
NH
+
NH2
2 Cl
N
N
N
N
N
S
H
Cu
Cu
H
N
NH2
+
NH2
S
N
S
N
N
N
N
Cl
N
Cu(p-TSBG)2
Cu(p-TSB)2
Figure 1. Schematic presentation of structure of MIBG-based anticancer copper compounds
and control compounds.
In comparison, two control compounds, Cu(m-TSB)2 and Cu(p-TSB)2 (Figure 1), were
designed with similar structures to Cu(m-TSBG)2 and Cu(p-TSBG)2, but without
benzylguanidine group. Benzylguanidine group may represent a critical functional group for
NET-mediated cellular uptake and internalization of Cu(m-TSBG)2 and Cu(p-TSBG)2
(Figure 2).
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Cu(p-TSBG)2
Cu(m-TSBG)2
NH2
N
S
N
S
NH
+
NH2
Cl
+
2 Cl
N
N
N
H
N
N
N
N
NH2
N
H
Cu
Cu
H
N
H
N
NH2
S
NH2
S
N
+
Cl
N
+
N
Cl
N
NH2
NH2
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Cu(p-TSB)2
Cu(m-TSB)2
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N
S
N
S
N
N
N
N
×
×
Cu
Cu
S
N
S
N
N
N
N
N
Figure 2. Schematic presentation of NB-specific uptake of MIBG-based anticancer
copper complexes mediated by NET. Cellular uptake of Cu(m-TSBG)2 and
Cu(p-TSBG)2 by NET-expressing NB cells, but absence of NB-specific cellular uptake of
control Cu(m-TSB)2 and Cu(p-TSB)2 compounds without MIBG ligand.
Demonstration of anti-cancer activity of Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds
on NET-expressing NB cells, but not on NET-negative U87 glioblastoma cells or PC-3
prostate cancer cells, may provide evidence of feasibility to support further investigation of
MIBG-mediated anticancer drug delivery.
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RESULTS
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Cu(m-TSBG)2 and Cu(p-TSBG)2 were synthesized through chelation of copper ions with
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3-carbaldehyde-thiosemicarbazide-1-guanidinomethyl-benzyl
4-carbaldehyde-thiosemicarbazide-1-guanidinomethyl-benzyl
(m-TSBG)
(p-TSBG)
and
ligands,
respectively. The m-TSBG and p-TSBG were synthesized through multi-step reactions as
shown in Supporting Information (Scheme 1). Commercially available isophthalaldehyde
(1) and terephthalaldehyde (2) were used as starting materials to prepare
monoaldehyde-monoalcohol
(3)
and
(4)
through
NaBH4
reduction.25
Dimethylthiosemicarbazide in methanolic solution reacted with (3) or (4) to form (5) or (6)
by direct condensation of amine and aldehyde.14 Under Mitsunobu conditions (TPP, DIAD
and THF),21 the hydroxyls in (5) or (6) was further replaced with bis-Boc-guanidine to form
Boc-protected guanidines products (7) or (8). Boc groups in (7) or (8) were readily
removed by treatment with HCl/dioxane to provide m-TSBG (9) and p-TSBG (10) ligands.25
Under weak acidic conditions, dimethylthiosemicarbazide group in m-TSBG (9) or p-TSBG
(10) could chelate copper (II) ion in methanol solution, and the resulting mixtures were
concentrated and subjected to Sephadex LH-20 column to obtain pure copper compounds,
Cu(m-TSBG)2 (11) and Cu(p-TSBG)2 (12).26 Keeping the copper ion chelation reaction
under weak acidic condition prevailed the copper coordination through
dimethylthiosemicarbazide group rather than guanidine group, which was verified by IR
spectra that showed C=S vibration in dimethylthiosemicarbazide group was significantly
influenced while N-H vibration in benzylguanidine group remain unchanged.14
Cu(m-TSBG)2 and Cu(p-TSBG)2 have very similar structures, and the only difference
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between them was the substitution of copper-thiosemicarbazide group at benzylguanidine
group where Cu(m-TSBG)2 have meta- substitution while Cu(p-TSBG)2 has para-
substitution. Purities of Cu(m-TSBG)2 and Cu(p-TSBG)2 synthesized for this study
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were >95% as determined by H NMR spectra of ligands and elemental analyses of
compounds.
Previous study has demonstrated that the substitution at meta- or para-position of
benzylguanidine will not influence the capability of benzylguanidine targeting to
neuroblastoma cells.19, 20, 22 Cu(m-TSBG)2 and Cu(p-TSBG)2 were expected to show
specific antiproliferative activity to NB cells because benzylguanidine group could act as a
targeting ligand to direct the whole molecule specifically internalized into neuroblastoma
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cells
through
NE
transporter,
while
enhancing
anticancer
activity
of
copper-thiosemicarbazone group delivered into NB cells. To further demonstrate the
critical role of benzylguanidine group, two non-targeting control compounds, Cu(m-TSB)2
and Cu(p-TSB)2 (Figure 1), were synthesized as shown in Scheme 2 in Supporting
Information, for comparing the anti-proliferative activity of non-targeting Cu(m-TSB)2 and
Cu(p-TSB)2 without a benzylguanidine group with the anti-proliferative activity of
NET-targeting Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds.
The anti-proliferative activity of Cu(m-TSBG)2, Cu(p-TSBG)2, Cu(m-TSB)2 and Cu(p-TSB)2
was examined in NB cells (BE2C and SK-N-DZ cells) and NET-negative glioblastoma cells
(U87 cells) and prostate cancer cells (PC-3 cells) by MTT assay. Non-NET-expressing
U87 glioblastoma cells were selected as control cells based on their similarity to NB cells,
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while PC-3 prostate cancer cells were selected as control cells based on prior use for
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testing anticancer activity of CuHQTS.15,16
After 24 h treatment, Cu(m-TSBG)2 and
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Cu(p-TSBG)2 showed strong anti-proliferation activity on NB cells (BE2C and SK-N-DZ
cells) with IC50 of 0.87±0.11 and 0.55±0.08 μM for BE2C cells as well as 1.09±0.24 and
0.83±0.13 μM for SK-N-DZ cells. In contrast, weak inhibition activity on non-neuroblastoma
cells was observed with IC50 of 4.6±0.32 and 4.8±0.24 μM for U87 glioblastoma cells as
well as 5.0±0.35 and 4.7±0.21 μM for PC-3 prostate cancer cells (Figure 3A, and Table S1
in supporting information).
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A
B
Cu(m-TSB)2
Cu(p-TSB)2
Cu(m-TSBG)2
Cu(p-TSBG)2
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BE2C
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U87
U87
PC-3
PC-3
BE2C
BE2C
SK-N-DZ
SK-N-DZ
U87
U87
PC-3
PC-3
Figure 3. NB specific anti-proliferative activity of CuTSBG compounds by
comparison of IC50 values of compounds on growth of NB cells (BE2C, SK-N-DZ) and
non-NB cells (U87 and PC-3 cells). A) IC50 values of Cu(m-TSBG)2 and Cu(p-TSBG)2; B)
IC50 values of Cu(m-TSB)2 and Cu(p-TSB)2.
Similar results were also obtained by evaluation for cytopathological effects of these cells
treated with Cu(m-TSBG)2 and Cu(p-TSBG)2 (Figure S1 in supporting information). The
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data demonstrated that Cu(m-TSBG)2 and Cu(p-TSBG)2 had strong NB-specific
anti-proliferative activity. In contrast, non-targeting Cu(m-TSB)2 and Cu(p-TSB)2 showed
weak inhibition of the proliferation of both NB and non-NB cells with IC50 of 5.7±0.33 and
4.8±0.45 μM for BE2C cells, 6.0±0.31 and 5.2±0.13 μM for SK-N-DZ cells, 6.4±0.42 and
5.8±0.32 μM for U87 cells, 6.0±0.44 and 6.1±0.26 μM for PC3 cells, in which the difference
of anti-proliferative activity between NB and non-NB cells were not significant (Figure 3B,
and Table S1 in supporting information).
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Since Cu(m-TSBG)2 and Cu(p-TSBG)2 have similar structures to Cu(m-TSB)2 and
Cu(p-TSB)2, respectively, except the presence of a benzylguanidine group, their differential
activity profiles can only be attributed to the role of benzylguanidine group. Where the
existence of benzylguanidine groups at Cu(m-TSBG)2 and Cu(p-TSBG)2 will avail
Cu(m-TSBG)2 and Cu(p-TSBG)2 to show specific anti-proliferative activity against NB cells
while the elimination of this group at Cu(m-TSB)2 and Cu(p-TSB)2 will dramatically impair
the specificity of anti-proliferative activity on NB cells. We also noticed that the precursors
(m-TSBG, P-TSBG, m-TSB, P-TSB) without copper ions have minimal anti-proliferative
activity on both NB and non-NB cells, with IC50 between 23.3±1.2 to 142.1±5.7μM (Table
S1 in supporting information). The results of these experiments have demonstrated
essential role of copper ions accounting for anti-proliferative activity of these MIBG-based
anticancer compounds.
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Benzylguanidine and their derivatives have been known capable of being specifically
internalized into cells by NET expressed on tumor cell membrane.17, 19-22 Since most of
NB cells have high NET expression, and Cu(m-TSBG)2 and Cu(p-TSBG)2 have
benzylguanidine groups, the NB-specific anti-proliferative activity of Cu(m-TSBG)2 and
Cu(p-TSBG)2 was probably achieved by targeted cellular uptake by NB cells. To verify this
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hypothesis, Cu-radiolabeled Cu(m-TSBG)2, Cu(p-TSBG)2, Cu(m-TSB)2 and Cu(p-TSB)2
were prepared to assess their cellular uptake by both NB cells (BE2C and SK-N-DZ cells)
and non-NB cells (U87 and PC-3 cells). Radiolabeling of these compounds with radioactive
64Cu radionuclide was performed in a protocol modified from the method reported by Lewis
et al.27 Cells were incubated with 64Cu-radiolabeled copper complexes for 3 hours and the
cellular 64Cu radioactivity was determined by gamma counting using a γ-counter. The
results indicated that the 64Cu radioactivity of the NB cells incubated with 64Cu(m-TSBG)2 or
64Cu(p-TSBG)2 were higher than the 64Cu radioactivity of the non-NB cells incubated with
these two radioactive compounds (Figure 4A). There was no significant difference of
64Cu radioactivity between the NB cells or non-NB cells after incubation with 64Cu(m-TSB)2
or 64Cu(p-TSB)2 compounds (Figure 4B).
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A
B
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Cu(m-TSB)2
Cu(m-TSBG)2
Cu(p-TSBG)2
Cu(p-TSB)2
2.5
2.0
1.5
1.0
0.5
0
2.5
2.0
1.5
1.0
0.5
0
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BE2C
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SK-N-DZ
U87
U87
PC-3
PC-3
BE2C
SK-N-DZ
SK-N-DZ
U87
U87
PC-3
PC-3
BE2C
BE2C
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Figure 4. Cellular uptake of Cu-labeled compounds by NB cells (BE2C, SK-N-DZ)
and non-NB cells (U87 and PC-3 cells). A) Cellular uptake of 64Cu(m-TSBG)2 or
64Cu(p-TSBG)2 following incubation of cells with 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 (10
µCi/mL) for 3 h, respectively.
B) Cellular uptake of 64Cu(m-TSB)2 or 64Cu(p-TSB)2
following incubation of cells with 64Cu(m-TSB)2 or 64Cu(p-TSB)2 (10 µCi/mL) for 3 h,
respectively.
Moreover, 64Cu radioactivity of NB cells incubated with 64Cu(m-TSBG)2 and 64Cu(p-TSBG)2
was significantly higher than the 64Cu radioactivity of NB cells incubated with 64Cu(m-TSB)2
and 64Cu(p-TSB)2.
The findings from these experiments suggested that NB-specific
anti-proliferative activity of Cu(m-TSBG)2 and Cu(p-TSBG)2 was most likely due to
presence of benzylguanidine group and specific cellular uptake of these two compounds
mediated by NET expressed on NB cells. The benzylguanidine groups at Cu(m-TSBG)2
and Cu(p-TSBG)2 compounds could be recognized by NET on the NB cell membrane, and
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probably could work like the “Trojan horse” to guide the whole Cu(m-TSBG)2 and
Cu(p-TSBG)2 compounds to be internalized into cells, leading to the high cellular uptake
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and enhanced NB-specific cytotoxicity.
In comparison, Cu(m-TSB)2 and Cu(p-TSB)2
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compounds without a benzylguanidine group showed low NB cellular uptake due to lack of
NET-mediated uptake or internalization.
To further confirm the role of NET-mediated cellular uptake in NB-specific anticancer
activity of Cu(m-TSBG)2 and Cu(p-TSBG)2, MIBG blocking assay was performed to
determine whether the cellular uptake of 64Cu(m-TSBG)2 and 64Cu(p-TSBG)2 could be
blocked by MIBG to reduce anti-proliferative activities of Cu(m-TSBG)2 and Cu(p-TSBG)2
on NB cells.
NB cells were incubated with 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 and MIBG ligand for 3
hours, and 64Cu radioactivity of the cells was determined by gamma counting using a
γ-counter.
As expected, 64Cu radioactivity of the NB cells ( BE2C and SK-N-DZ )
incubated with 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 in the presence of MIBG blocking ligand
(Figure 5A) were significantly lower than the 64Cu radioactivity of the NB cells ( BE2C and
SK-N-DZ ) incubated with 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 without presence of MIBG
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blocking ligand (as shown in Figure 4A). In comparison, there was no difference of Cu
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radioactivity of the cells incubated with Cu(m-TSB)2 or Cu(p-TSB)2 in the presence of
MIBG blocking ligand (Figure 5B) or in the absence of MIBG blocking ligand (Figure 4B).
Furthermore, no effect of MIBG blocking ligand on cellular uptake of these radiolabeled
compounds by non-NB cells (Figure 5, and Figure S2 in supporting information).
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A
B
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Cu(m-TSB)2
Cu(p-TSB)2
Cu(m-TSBG)2
Cu(p-TSBG)2
1.0
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0.6
0.4
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BE2C
SK-N-DZ
U87
PC-3
BE2C
SK-N-DZ
U87
PC-3
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Figure 5. MIBG-blocking of NB cellular uptake of Cu(m-TSBG)2 or Cu(p-TSBG)2
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compounds. A) Cellular uptake of Cu(m-TSBG)2 or Cu(p-TSBG)2 (10 µCi/mL) by NB
cells (BE2C, SK-N-DZ) and non-NB cells (U87 and PC-3 cells) in the presence of 10 ng
MIBG ligand (20 ng/mL) ; B) Cellular uptake of 64Cu(m-TSB)2 or 64Cu(p-TSB)2 (10 µCi/mL)
by NB cells (BE2C, SK-N-DZ) and non-NB cells (U87 and PC-3 cells) in the presence of
MIBG ligand (20 ng/mL).
Additionally, MIBG blocking assay were performed to assess the influence of MIBG on
anti-proliferative activity of Cu(m-TSBG)2 and Cu(p-TSBG)2.
Following addition of MIBG
at various concentrations to NB cell culture medium, NB cells (BE2C, SK-N-DZ) were
treated with Cu(m-TSBG)2 or Cu(p-TSBG)2 at their IC50 concentrations for 24 h, and
anti-proliferative activity of the compounds was assessed by MTT assay. Significant
reduction of anti-proliferative activity of Cu(m-TSBG)2 or Cu(p-TSBG)2 on NB cells was
observed, in the presence of gradually increased concentration of MIBG competitive
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blocking ligand. In the presence of 2 µM of MIBG, more than 80% of NB cells were found
to be viable after treatment with IC50 dose of Cu(m-TSBG)2 or Cu(p-TSBG)2 for 24 hours.
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DISCUSSION AND CONCLUSIONS
Children suffered advanced stage neuroblastoma have a poor prognosis due to
acquired resistance to many of anticancer drugs currently available. There is an urgent
need of new effective drugs for targeted treatment of children with drug-resistant NB
metastasis. Continuing from our efforts in developing new anticancer copper complexes
for treatment of cisplatin-resistant NB tumors, we synthesized new anticancer compounds
targeting NET-expressing NB cells by combining MIBG targeting ligand and CuHQTS
anticancer copper complexes that were demonstrated to have potent anticancer activity on
NB and prostate cancer cells. Two novel Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds,
consisting of CuHQTS (anticancer copper complexes) and MIBG ligand, were found to
have MIBG-mediated NB-specific anticancer activity.
The findings from this study
demonstrated that potent anticancer activity of Cu(m-TSBG)2 and Cu(p-TSBG)2 on NB cells
was most likely due to NET-mediated NB cellular uptake and internalization.
.
It was observed that Cu(p-TSBG)2 exhibited higher inhibition activity over
Cu(m-TSBG)2 against NB cells, that could be due to different strength of functional group
of CuHQTS following its connection to MIBG ligand, not due to difference in cellular binding
and internalization of these two compounds.
Indeed, we did not observed positive
correlation between inhibition activity and cellular uptake suggesting no difference of these
two compounds in MIBG-ligand mediated cellular uptake (Figure 4). Both Cu(m-TSBG)2
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and Cu(p-TSBG)2 displayed higher cellular uptake in NB cells (BE2C and SK-N-DZ cells)
over non-NB cells (U87 and PC-3 cells). It was most likely due to difference in NET
expression (high level expression in NB and low level expression in U87 and PC-3 cells),
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not due to difference in cellular uptake of free Cu radionuclide disassociated from the
copper compounds, based on previously demonstrated increase of carrier-free 64CuCl2
uptake by PC-3 prostate cancer cells, 28, 29 U87 glioblastoma cells, 30, 31 and NB cells 32.
Radiolabeled MIBG ligand is clinically used for diagnostic imaging of neuroblastoma 21,
24. NB-specific cellular uptake of Cu(m-TSBG)2 or Cu(p-TSBG)2 demonstrated in this
study (Figure 4) support further study of 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 compounds as
novel theranostic agents for targeted imaging and therapy of NB in children. Moreover,
the findings of this study invite investigation of 64Cu(m-TSBG)2 or 64Cu(p-TSBG)2 for
diagnostic imaging and therapy of other NET-expressing neuroendocrine tumors such as
carcinoid.
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In conclusion, two novel Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds, consisting of
MIBG (targeting ligand) and CuHQTS (anticancer copper complexes), were synthesized
and tested for MIBG-mediated NB-specific anticancer activity.
Potent anticancer activity
of Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds on NB cells were demonstrated,
suggesting that these two novel Cu(m-TSBG)2 and Cu(p-TSBG)2 compounds are promising
new drugs for treatment of children suffering advanced stage NB refractory to cisplatin or
other chemotherapeutic drugs currently available.
EXPERIMENTAL SECTION
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1. Chemical Synthesis
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All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Dried THF was
obtained by refluxing with sodium and distilling. Dried CH2Cl2 was obtained by refluxing
with CaH2 and distilling. Other reagents were all commercial and used without further
purification. The synthesis of Cu(m-TSBG)2, Cu(p-TSBG)2, Cu(m-TSB)2 and Cu(p-TSB)2
compounds were described in Supporting Information. 1HNMR spectra and elemental
analysis were used to determine purity of ligands and elemental analyses was used to
determine purity of copper compounds, along with use of IR spectra for characterization.
Purity of copper compounds (> 95%) were confirmed by elemental analysis. IR spectra
were recorded from 4000 to 400 cm-1 as KBr pellets on a Tensor 27 FT-IR
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1
spectrophotometer. HNMR spectra were measured using Varian 400 mHz instruments.
ESI mass spectra were measured in a triple quadrupole Micromass QuattroLC
spectrometer with an electroscopy/APCI source. Elemental analyses were performed by
Midwest Microlab (Indianapolis, IN).
2. MTT Cell Proliferation Assay
MTT assay was performed to evaluate antiproliferative activity of the compounds using a
MTT assay kit from Chemicon (Temecula, CA), as described previously.14, 33 Briefly,
BE2C, SK-N-DZ, U87 and PC-3 cells from ATCC (Manassas, VA) were cultured in
EMEM:F12K (BE2C), DMEM (SK-N-DZ), RP1640 (U87 and PC-3) medium (Biosource
International, Camarillo, CA), respectively, supplemented with fetal bovine serum (10%),
penicillin (100 units /ml), streptomycin (100mg/ml), and glutamine (100 mg/ml), at 37°C in
an atmosphere of 5% CO2. The cells were seeded in a 96-well plate (1×104 /0.1 ml/well)
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and were treated with compounds at noted concentration for 24 h.
Upon end of drug
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treatment, MTT solution was added and optical density (OD) was measured at the
wavelength of 570 nm with a reference wavelength of 630nm. The results of the MTT assay
were recorded as inhibition of cell proliferation (%) calculated from a formula = (OD of the
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cells in treatment group/ OD of the cells in negative control group) ×100%.
Each
experiment was repeated at least three times and each point was determined in triplicate.
MIBG inhibitory assays were performed to evaluate the inhibitory effects of MIBG on the
anti-proliferative activity of compounds. Briefly, the cells were seeded in a 96-well plate
(1×104 /0.1 ml/well) for 12 h, and then were treated with varied MIBG concentrations for 24
h in the presence of compounds at their IC50 concentrations, respectively. The
anti-proliferative activity was evaluated by MTT assay as described above.
3. 64Cu Radiolabeling of Compounds
64Cu(m-TSBG)2, 64Cu(p-TSBG)2, 64Cu(m-TSB)2 and 64Cu(p-TSB)2 were prepared according
to literature methods.28-30 In brief, a 10 μl aliquot of m-TSBG, p-TSBG, m-TSB or p-TSB (1
mg/ml DMSO) was mixed with 150 μl of 64Cu (1M NaOAc, pH 4.0) and vortexed for 30 min.
The mixture was added to an ethanol-water preconditioned C-18 SepPak Light column.
64Cu(m-TSBG)2, 64Cu(p-TSBG)2, 64Cu(m-TSB)2 or 64Cu(p-TSB)2 was eluted off with 500 μl
of ethanol after the initial 150 μl fraction. The radiochemical purity of compounds were
TM
determined by Instant Thin Layer Chromatography using ITLC
SG Impregnated Glass
Fiber Sheets (Pall Life Sciences, Ann Arbor, MI) as the stationary phase and MeOH/10%
NaOAc as the eluent, found to exceed 99%. Through saline dilution, final concentration of
64Cu(m-TSBG)2, 64Cu(p-TSBG)2, 64Cu(m-TSB)2 or 64Cu(p-TSB)2 was provided with 0.5
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μCi/μl.
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4. Cellular Radioactivity Assay
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To test cellular uptake of 64Cu(m-TSBG)2, 64Cu(p-TSBG)2, 64Cu(m-TSB)2 and 64Cu(p-TSB)2,
NB cells (BE2C, SK-N-DZ) and non-NB cells (U87 and PC-3 cells) were seeded in 6-well
plates (5×105 /2 ml/well), respectively. After incubated for 12 h, cells were treated with
64Cu(m-TSBG)2, 64Cu(p-TSBG)2, 64Cu(m-TSB)2 or 64Cu(p-TSB)2 at 10 µCi/mL for 3 h. After
treatment, cells were washed with PBS for 3 times, then harvested and counted in a
γ-counter (Packard). MIBG blocking assays were performed to evaluate the inhibitory
effects of MIBG on cellular uptake of these radioactive compounds. Cells were treated with
64Cu-labeled compounds (10 µCi/mL) for 3 h in the presence of MIBG (20 ng/mL). After
treatment, cells were washed with PBS for 3 times, then harvested and counted in a
γ-counter (Packard).
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5. Statistical analysis
A series of two-factor analysis of variance (ANOVA) models were employed to examine
differences in mean values (IC50 or cellular uptake) between study groups by
concentration and cell types. Statistically significant differences were considered achieved
at a P-value ≤0.05, two-tailed. All analyses were conducted using SPSS Version 15.
ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding author: Fangyu Peng, MD, PhD
E-mail: Fangyu.Peng@utsouthwestern.edu.
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Addresses:
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Department of Radiology,
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd, Dallas, Texas 75390, United States
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Author Contributions
H.Z. synthesized and characterized chemical compounds, and performed radiolabeling and
biological assays. F.X. performed biological assays. F.P. analyzed data of radiolabeling
and biological assays. The manuscript was written through the contributions of all authors
(H.Z., F.X., M.C., and F. Peng). All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
CuHQTS, Copper complex of 8-hydroxyquinoline-2-carboxaldehyde-thiosemicarbazide;
Cu(m-TSB)2, 3-(hydrazinecarbothioamide)-1-benzyl-copper (II) chlorate; Cu(p-TSB)2,
4-(hydrazinecarbothioamide)-1-benzyl-copper
(II)
chlorate;
Cu(m-TSBG)2,
(II) chlorate;
3-carbaldehyde-thiosemicarbazide-1-guanidinomethyl-benzyl-copper
Cu(p-TSBG)2, 4-carbaldehyde-thiosemicarbazide-1-guanidinomethyl-benzyl-copper (II)
chlorate; DIAD, diisopropylazodicarboxylate; MIBG, Meta-iodobenzylguanidine; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NaBH4, sodium borohydride;
NB, Neuroblastoma; NE, norephinephrine; NET, norephinephrine transporter; TPP,
triphenylphosphine.
ACKNOWLEDGEMENT
This study was partly funded by a faculty research development fund awarded to F. P. by
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Carman & Ann Adams Department of Pediatrics, School of Medicine, Wayne State
University, Detroit, MI 48201, United States and a faculty research development fund
awarded to F. P. by Harold C. Simmons Comprehensive Cancer Center, University of
Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
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Supporting Information Availability
1. Schemes for synthesis of Cu(m-TSBG)2, Cu(p-TSBG)2, Cu(m-TSB)2 and
Cu(p-TSB)2 and characterization. Scheme 1, Synthesis of Cu(m-TSBG)2; Scheme
2, Synthesis of Cu(p-TSBG)2; and Scheme 3, Synthesis of Cu(m-TSB)2
2. Table S1. IC50 values of copper complexes, ligands, and MIBG in BE2C, SK-N-DZ,
U87 and PC-3 cell lines.
3. Microscopic examination for cytotoxicity on NB cells (Figure S1)
4. Cellular uptake of radiolabeled compounds (Figure S2)
5. Molecular Formula Strings (CSV) is available in supporting information
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NB-specificity of novel Cu(m-
T
TSBG)2 and Cu(p-TSBG)2 anticancer cop
p
p
er complexes
demonstrated by differential celll
ular uptake of radioactive64Cu(m-TSBG)2,64Cu(p-TSBG)2,
64Cu(m-TSB)2 and 64Cu(p-TSB)2 compounds by NB cells (BE2C, SK-N-
DZZ) and non-NB
cells (U87 and PC-3) with or withhout competitive blockage of MIBG binding tto NET.
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