Synthesis and characterization of a novel 99mTc analogue of ?i-: M IBG showing affinity for nor-epinephrine transport positive tumors

Article abstract of DOI:10.1039/c6ra11217j

Radio-iodine (¹²³I/¹³¹I) labeled meta-iodobenzylguanidine (mIBG) is a proven radiopharmaceutical used for diagnosis of neuroendocrine tumors (NET) related to neural crest origin. Here, a ^99mTc analogue of ?I-mIBG using

Full text of DOI:10.1039/c6ra11217j

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Synthesis and characterization of a novel 99mTc
analogue of *I-mIBG showing affinity for nor-
epinephrine transport positive tumors
Cite this: RSC Adv., 2016, 6, 64902
Navin Sakhare,a Soumen Das,a Anupam Mathur,a Shubhangi Mirapurkar,a
a
b
a
*
Shalaka Paradkar, H. D. Sarma and S. S. Sachdev
Radio-iodine (123I/131I) labeled meta-iodobenzylguanidine (mIBG) is a proven radiopharmaceutical used for
diagnosis of neuroendocrine tumors (NET) related to neural crest origin. Here, a 99mTc analogue of *I-mIBG
using 99mTc-4+1 labeling approach has been synthesized and evaluated for its potential in NET imaging.
This involved preparation of benzylguanidine precursor (mono-dentate donor) which was isolated in
a five step synthetic procedure. The precursor was designed such that, in presence of a tetra-dentate
NS3 co-ligand, [99mTcO4]ꢀ and Sn2+ reducing agent, it formed the desired 99mTc-4+1 complex. Complex
formation was identified by radio-HPLC and structural details were affirmed by characterizing its
rhenium analogue. Bio-evaluation studies of the complex were carried, both in vitro and in vivo, and the
results obtained were compared with no-carrier added-125I-mIBG (nca-125I-mIBG). In vitro studies, in
SK-N-SH neuroblastoma cell line showed affinity of the tracer towards nor-epinephrine transporters.
Although the absolute uptake was lower compared to nca-125I-mIBG, its specificity was similar (ꢁ90%)
as the uptake reduced on inhibition with specific transport blocker, desmethylimipramine (DMI).
Biodistribution studies in normal Wistar rats, pre-treated with mIBG and DMI, respectively, showed
reduced myocardial uptake than its control. The results thus obtained merits high potential of
synthesized 99mTc-4+1 complex for NET imaging.
Received 30th April 2016
Accepted 18th June 2016
DOI: 10.1039/c6ra11217j
www.rsc.org/advances
123/131I-meta-iodobenzylguanidine (mIBG),
a
structural
Introduction
analogue of nor-epinephrine, is one such SPECT based radio-
pharmaceutical that has been the mainstay for the diagnosis of
these types of tumors. It enters the cell membrane of sym-
pathomedullary tissues by an active, sodium- and energy-
dependent amine uptake mechanism and gets stored into the
intracellular catecholamine storing granules.14,15 Although the
uptake and retention of this radiopharmaceutical is ideal for
favorable imaging, availability of 123I radionuclide through high
energy beam cyclotron limits its widespread use. 131I, a thera-
nostic radionuclide, widely available through neutron activa-
tion and ssion pathways, is employed as a rational substitute
and labeled 131I-mIBG has now been in the clinics for nearly
three decades, since its introduction by Donald M. Wieland in
the 1980s.14 Although 131I-mIBG is satisfying the needs of the
medical fraternity, the sub-optimal nuclear characteristics of
131I for diagnostic applications demands a SPECT substitute,
preferably 99mTc based, for the aforementioned application.
This is due to the optimal nuclear characteristics of 99mTc, its
ease of availability through a commercial 99Mo–99mTc generator
and ready formulation of the radiopharmaceutical through
‘cold kits’, ensuring the availability of radiopharmaceutical
preparation in clinics round the clock. Limited efforts have
been put forward by researchers in this direction to develop
99mTc labeled guanidine derivatives as an analogue of *I-labeled
Neuroendocrine tumors (NETs) are a group of neoplasms,
either of epithelial or neural origin, comprising mostly well-
differentiated and slow growing tumors, with small pop-
ulation being poorly differentiated and malignant with an
aggressive behavior. NETs are also known to express different
transporters and hormone receptors (e.g. somatostatin recep-
tors) on their cell surfaces, which is responsible for concen-
trating different hormones inside the cell. These cell
parameters form the basis for designing specic radiolabeled
ligands for NET imaging.1–6 In this category, several radio-
labeled nor-epinephrine and peptide analogs based on PET/
SPECT are in clinical use. Peptide radiopharmaceuticals based
on 111In, 99mTc and 68Ga have gained wide acceptance for NET
imaging related to carcinoid tumors,6–10 however, for tumors
related to neural origin such as phaeochromocytoma, para-
ganglioma and neuroblastoma radiolabeled nor-epinephrine
analogues have shown superior distribution behavior.11–13
aRadiopharmaceuticals Program, Board of Radiation and Isotope Technology,
Navi-Mumbai-400703, India. E-mail: sachdev@britatom.gov.in; Fax: +91-22-2788
7218; Tel: +91-22-2788 7201
bRadiation Biology and Health Science Division, Bhabha Atomic Research Centre,
Trombay, Mumbai-400085, India
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mIBG and although 99mTc-radiotracers evaluated exhibited radiometric detector system, using a C18 reversed phase HiQ Sil
affinity in preclinical animal models, the relatively low target (5 mm, 4 ꢂ 250 mm) column. FT-IR spectra were recorded on
uptake values limit their application for NET imaging in JASCO FT/IR-460 Spectrophotometer, Japan. The 1H-NMR
clinics.16,17
spectra were recorded on a 500 MHz Varian spectrophotom-
Over the past two decades, several new strategies for incor- eter, USA. Low resolution mass spectra were recorded on Advion
porating the 99mTc metal into the carrier molecule have Mass Spectrometer, USA using electrospray ionization (ESI) in
emerged.18 These employ several 99mTc-metal cores/fragments both positive and negative modes.
bonded through different bi-functional chelates, which affect
the pharmacokinetics in vivo, leading to diverse bio-distribution
Synthesis
characteristics with common targeting vectors or carrier mole-
cules. For designing a 99mTc analogue of *I-mIBG, the selection of
Synthesis of copper(I) complex of 3-(isocyanomethyl)benzyl-
99mTc incorporation approach should be such that the nal 99mTc guanidine (mono-dentate donor)
building block is kept to a minimum. In the present work, 99mTc-
N,N0-Di-Boc-3-(aminomethyl)benzyl guanidine (1). A mixture of
4+1 chemistry approach has been utilized, which is known to m-xylelene diamine (300 mg, 2.2 mmol) and N,N0-di-Boc-S-
contribute little to the overall molecular framework of the methyl isothiourea (580 mg, 2.0 mmol) in THF (15 mL) and
complex. Also, the biomolecule attached to the mono-dentate water (0.2 mL) was stirred overnight at room temperature. Upon
donor leads to minimum distortion and no special care in completion of the reaction (cf. TLC), the reaction mixture was
respect of stereochemistry has to be taken into account.18 The concentrated in vacuo and the crude product obtained was
99mTc-4+1 approach stabilizes the metal in +3 oxidation state by further puried by silica gel column chromatography to yield
coordinating through two different sets of donor ligands, a tetra- the desired product (650 mg, 86%).
dentate (NS3) and a mono-dentate (isonitrile/phosphine) ligand.
The carrier molecule can either be linked to a functionalized
Rf(CHCl3/MeOH 9 : 1 v/v) ¼ 0.3.
dH (500 MHz; CDCl3; Me4Si): 7.18–7.32 (4H, m, Ph); 4.59–4.67
tetra-dentate ligand (e.g., NS3–COOH) or can be a part of the (2H, m, CH2–NH–C); 3.80–3.85 (2H, m, –CH2NH2); 1.45–1.67
mono-dentate ligand for getting attached to the 99mTc(III) (18H, br s, (CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–).
centre.19,20 In the present approach, the active benzylguanidine
dC (125 MHz; CDCl3; Me4Si): 163.6 (]N–COO); 156.1 (NH–
functionality has been introduced via a mono-dentate donor with COO); 153.2 (BocN]C–NH); 138.2 (phenyl HC]C–CH2–NH–
the standard NS3 ligand as tetra-dentate co-ligand. The newly C]); 137.2 (phenyl HC]C–CH2–NH2); 129.1, 128.8, 127.5,
synthesized 99mTc-complex has been evaluated in vitro for its 126.5 (phenyl HC]CH–CH); 83.1 ((CH3)3C–C]O(N])); 80.3
efficacy in neuroblastoma cell line and the results obtained have ((CH3)3C–C]O(NH)); 50.3 (ArCH2NH2); 44.5 (ArCH2NHCO–);
been compared with that of nca-125I-mIBG. Further, the complex 29.6, 29.3, 28.9, 28.3, 28.2, 28.0 ((CH3)3C).
was evaluated in vivo in normal rats to evaluate its efficacy
towards sympathetically innervated myocardium.
ESI-MS (+ve mode): mass (calculated) C19H30N4O4 378.2; m/z
(observed) 379.
N,N0-Di-Boc-3-(formamide N-methyl)benzyl guanidine (2). Ethyl
formate (10 mL) was added slowly to a stirred ice cooled mixture
of compound 1 (300 mg, 0.8 mmol) containing catalytic amount
of p-toluene sulphonic acid. Aer addition is complete, the
Experimental
General
The compounds m-xylylenediamine, N,N0-di-Boc-S-methyl iso- reaction mixture was brought to room temperature and then
thiourea, ethyl formate, p-toluene sulphonic acid, mannitol and reuxed overnight. Upon completion of the reaction, excess
stannous chloride were obtained from Aldrich, USA. Ammo- ethyl formate was removed in vacuo to give the crude product
nium perrhenate and dimethylphenyl phosphine were acquired which on purication by silica gel column yielded the pure
from Alfa Aesar, Germany. Ethylenediamine tetraacetic acid compound 2 (225 mg, 70%).
disodium salt and triuoroacetic acid (TFA) were procured from
Merck, India. p-Toluene sulphonyl chloride and pyridine were
Rf(EtOAc) ¼ 0.6.
dH (500 MHz; CDCl3; Me4Si): 8.23 (1H, s, NH–CHO); 7.20–
obtained from S.D. Fine chemicals limited, India. 2,20,200-Nitri- 7.31 (4H, m, Ph); 4.59 (2H, d, J ¼ 3 Hz, CH2–NHC]N); 4.46 (2H,
lotris(ethanethiol) (NS3) was synthesized following a reported d, J ¼ 6 Hz, CH2–NHCHO); 3.70 (1H, s, –NHCHO); 1.45–1.50
procedure.21 Cuprous chloride was prepared fresh following (14H, br s, (CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–); 1.25–
a standard procedure from cupric chloride. All other reagents 1.32 (4H, br s, (CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–).
used were of analytical grade and were used without further
dC (125 MHz; CDCl3; Me4Si): 164.2 (–NHCHO); 163.6
purication. Sodium pertechnetate (Na99mTcO4) was eluted (tautomer –N]CHOH); 160.9 (]N–COO); 156.2 (NH–COO);
with normal saline prior to use from a 99Mo–99mTc column 153.1 (BocN]C–NH); 138.1 (phenyl HC]C–CH2–NH–C]);
generator, supplied by Board of Radiation and Isotope Tech- 137.3 (phenyl HC]C–CH2–NH2); 129.3, 127.1, 126.5, 126.2
nology (BRIT), India. nca-125I-mIBG was prepared from m-tri- (phenyl HC]C–CH2); 83.4 ((CH3)3C–C]O(N])); 79.5 ((CH3)3C–
methylsilylbenzylguanidine following a reported procedure.22 C]O(NH)); 61.3 (tautomer CH2–N]CHOH); 44.6 (ArCH2-
Silica gel plates (Silica Gel 60 F254) were obtained from Merck, NHCHO); 41.9 (ArCH2NHC]NBoc); 29.6 29.3, 28.3, 28.2, 28.1,
India. The HPLC of the prepared complex was carried out on 28.0 ((CH3)3C).
a JASCO PU 2080 Plus dual pump HPLC system, Japan, with
ESI-MS (+ve mode): mass (calculated) C20H30N4O5 406.2; m/z
a JASCO 2075 Plus tunable absorption detector and Gina Star (observed) 406.7.
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N,N0-Di-Boc-3-(isocyanomethyl)benzyl guanidine (3). To
a cooled solution of compound 2 (200 mg, 0.5 mmol) in pyridine
(5 mL), p-toluene sulphonyl chloride (190 mg, 1.0 mmol) was
mL) was added to a vial containing EDTA (5.0 mg), mannitol (5.0
mg) and SnCl2$2H2O (100 mg) in 0.1 N HCl (100 mL) and incu-
bated at room temperature for 30 min. The formation of the
intermediate complex was analyzed by TLC (>99%) (Rf ¼ 0,
intermediate EDTA complex; Rf ¼ 1, [99mTcO4]ꢀ; solvent:
acetone).
ꢃ
added and the mixture stirred at 0 C for 15 min. The reaction
mixture was then brought to room temperature and stirred for
another 2 h. Upon completion of the reaction (cf. TLC), the
reaction mixture was poured into water and extracted with
chloroform (3 ꢂ 10 mL). The combined chloroform extracts
were dried over anhydrous sodium sulphate and removed under
vacuo to yield the crude product which was puried using silica
gel column chromatography to give 3 (87 mg, 45%).
99mTc-(4+1) complex (7). Intermediate complex (6) (0.5 mL)
was added to a vial containing a mixture of compound 5 (1–2
mg) and NS3 ligand (1 mg) in methanol (0.5 mL). The vial was
sealed and the reaction mixture heated in a boiling water
bath for 30 min. Aer cooling the vial to room temperature,
the complex 7 was analyzed by TLC (EtOH: Rf ꢁ 0.9 and
CHCl3: Rf ꢁ 0.2) and HPLC. To resolve the identity of the nal
complex 7 from the secondary intermediate 99mTc–NS3 (only
Rf(EtOAc) ¼ 0.5.
IR (neat, nmax/cmꢀ1): 3053 (Ar-H str., m); 2923 (C–H str., s);
2853 (C–H str., m); 2152 (N^C str., s); 1665 (C]O str., bs); 1529
(m); 1440 (m); 1384 (m); 1241 (m); 1064 (m); 788 (m).
dH (500 MHz; CDCl3; Me4Si): 7.0–7.4 (4H, m, Ph); 4.59 (2H, s,
CH2–NHC]N); 4.49 (2H, s, CH2–NC); 1.35–1.69 (14H, br s,
(CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–); 1.22–1.33 (4H,
br s, (CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–).
4
intermediate complex, without mono-dentate donor),
a fresh reaction was carried following the same procedure
without addition of compound 5 and the reaction mixture
was analyzed by HPLC.
Cu(I) complex of N,N0-di-Boc-3-(isocyanomethyl)benzyl guani-
dine (4). Compound 3 (78 mg, 0.4 mmol) was heatedꢃwith CuCl
(10 mg, 0.1 mmol) in anhydrous EtOH (2 mL) at 90 C for 1 h.
The reaction mixture was then allowed to attain ambient
temperature and was ltered through a 0.22 mm membrane
lter (Millipore). The solvent ethanol was then removed under
vacuo to obtain the desired compound 4 (130 mg, 80%).
IR (neat, nmax/cmꢀ1): 3055 (Ar-H str., m); 2923 (C–H str., s);
2853 (C–H str., m); 2185 (N^C str., s); 1667 (C]O str., bs); 1530
(m); 1440 (m); 1384 (m); 1244 (m); 1055 (m); 792 (m).
dH (500 MHz; CDCl3; Me4Si): 7.81 (1H, s, Ph); 7.51–7.62 (1H,
m, Ph); 7.26–7.42 (1H, m, Ph); 7.07–7.13 (1H, m, Ph); 4.68 (2H, s,
CH2–NC); 4.59 (2H, s, CH2–NHC]N); 1.08–1.38 (14H, m,
(CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–); 0.84–0.92 (4H,
m, (CH3)3C–O–CO–N] and (CH3)3C–O–CO–NH–).
dC (125 MHz; CDCl3; Me4Si): 161.3 (–N^C); 161.0 (]N–
COO); 156.2 (NH–COO); 153.1 (BocN]C–NH); 133.7 (phenyl
HC]C–CH2–NH–C]); 131.3 (phenyl HC]C–CH2–NH2); 129.4,
128.3, 126.3, 125.6 (phenyl HC]CH–CH); 83.4 ((CH3)3C–C]
O(N])); 79.5 ((CH3)3C–C]O(NH)); 48.7 (ArCH2NC); 41.5
(ArCH2NHCO–); 29.7, 29.5, 29.3, 28.9 ((CH3)3C).
Cu(I) complex of 3-(isocyanomethyl)benzyl guanidine (5).
Compound 4 (100 mg, 0.06 mmol) was directly treated with
triuoroacetic acid (TFA) (2 mL) and the solution stirred for 2 h
at room temperature. Thereaer, TFA was removed under
vacuum to obtain compound 5 (50 mg, quantitative) and was
analyzed as such without further purication.
Synthesis of Re-4+1 complex (8)
[Re(Me2PPh)NS3] intermediate complex. This intermediate
was prepared following a reported procedure.21
dH (500 MHz; CDCl3; Me4Si): 7.1–7.9 (5H, m, Ph); 2.5–3.1
(12H, br s, –CH2–S– and –CH2N–); 2.18 (3H, s, –PCH3); 2.05 (3H,
s, –PCH3).
ESI-MS (+ve mode): mass (calculated) C14H23NPReS3 516.7,
518.7; m/z (observed) 517.1, 519.1.
[ReNS3 (3)]. A mixture of [Re(Me2PPh)NS3] (50 mg, 0.09
mmol) and compound 3 (37 mg, 0.09 mmol) in chloroform was
reuxed overnight. Thereaer, the reaction mixture was cooled
and solvent was removed under vacuo to give the crude product.
The pure product (18 mg, 25%) was obtained using silica gel
column chromatography with chloroform as the eluting
solvent.
Rf(chloroform) ¼ 0.5.
IR (neat, nmax/cmꢀ1): 3331 (N–H str., m); 2924 (C–H str., s);
2854 (C–H str., m); 1972 (N^C str., bs); 1725, 1641 (C]O str., s);
1441 (m); 1154 (m).
dH (500 MHz; CDCl3; Me4Si): 7.51 (1H, d, J ¼ 6 Hz, Ph); 7.45
(1H, d, J ¼ 8 Hz, Ph); 7.34–7.37 (1H, m, Ph); 7.23–7.26 (1H, m,
Ph); 6.04 (2H, s, CH2NC); 4.66–4.68 (2H, m, CH2–NH–C); 2.8–3.2
(12H, br s, CH2–S– and –CH2N–); 1.45–1.56 (14H, m, (CH3)3C–O–
CO–N] and (CH3)3C–O–CO–NH–); 1.21–1.33 (4H, m, (CH3)3C–
O–CO–N] and (CH3)3C–O–CO–NH–).
ESI-MS (+ve mode): mass (calculated) C26H40N5O4ReS3 767.2,
769.2; m/z (observed) 767.4, 769.4.
IR (neat, nmax/cmꢀ1): 3055 (Ar-H str., m); 2923 (C–H str., s);
2853 (C–H str., m); 2185 (N^C str., s); 1530 (m); 1440 (m); 1384
(m); 1244 (m); 1055 (m); 792 (m).
dH (500 MHz; CDCl3; Me4Si): 7.2–7.7 (1H, m, Ph); 4.68 (2H, s,
CH2–NC); 4.55 (2H, s, CH2–NHC]NH).
Re-4+1 complex (8). The nal complex was obtained on
treatment of above Re-complex (10 mg) with TFA (1 mL) for 3 h.
No further purication was carried and pure product (13 mg,
quantitative) was obtained aer TFA removal.
IR (neat, nmax/cmꢀ1): 3333, 3198 (N–H str., m); 2926 (C–H str.,
s); 2854 (C–H str., m); 1966 (N^C str., bs); 1781, 1739, 1681 (C]
N str., bs); 1445 (m); 1203 (bm); 1154 (m); 777 (m).
dH (500 MHz; CDCl3; Me4Si): 7.23–7.52 (4H, m, Ph); 6.04 (2H,
Radiolabeling
99mTc(III)–EDTA complex (6). The synthesis of the reactive
intermediate complex was carried as per the reported proce- s, CH2NC); 4.66–4.68 (2H, m, CH2–NH–C); 2.8–3.2 (12H, br s,
dure.20 Briey, freshly eluted [99mTcO4]ꢀ (30 mCi/1.1 GBq, 1.0 CH2–S– and –CH2N–).
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Fig. 1 Scheme for the synthesis of Cu(I) complex of (3-(isocyanomethyl)benzyl)guanidine.
ESI-MS (+ve mode): mass (calculated) C16H25N5ReS3 567.1, phase analytical column was used for the purication of the
569.1; m/z (observed) 567.7, 569.7.
complex 7. Fraction containing the complex 7 was collected,
subjected to vacuo and reconstituted in 5% ethanol. Around
18.5 MBq (500 mCi) of pure radiolabeled complex was obtained
by this method which was subsequently used for in vitro and in
vivo studies.
The formation of Re-4+1 complex 8 too was conrmed using
the same gradient elution system wherein UV prole of the
complex was monitored to establish its identity in conformity
with 99mTc-4+1 analogue.
Quality control
HPLC. The radiochemical purity (RCP) of the 99mTc-4+1
complex (7) was determined by reversed phase HPLC. Gradient
elution program was followed using water (solvent A) and
acetonitrile (solvent B) as the mobile phase (0 min 90% A, 20
min 0% A, 30 min 0% A). Flow rate of the solvent was main-
tained at 1 mL minꢀ1. Test solution (20 mL) was injected into the
column using a micro-syringe and elution was monitored by
observing the radioactivity prole. The same C18 reversed
Partition coefficient (log Po/w). The HPLC puried complex
7 (0.1 mL, 185 KBq/5 mCi) was mixed with double distilled
water (0.9 mL) and n-octanol (1 mL) and vortexed for 3 min.
Fig. 2 Scheme for the preparation of 99mTc-4+1 complex.
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The mixture was then centrifuged at 3500 g for 3 min to effect
clear separation of the two layers. Equal aliquots from the two
layers were withdrawn in triplicate and measured for associ-
ated radioactivity. The readings thus obtained were used to
calculate the log Po/w value (octanol–water partition) of the
complex.
Bio-distribution studies
All procedures performed herein were in accordance with the
national laws pertaining to the conduct of animal experiments.
Normal Wistar rats (150–200 g body weight) were used for the in
vivo distribution studies. The HPLC puried radiolabeled
preparation 7 (100 mL, 740 KBq/20 mCi) was administered
intravenously through tail vein of each rat. The animal distri-
bution experiment was conducted in three different groups. The
rst study utilized animals (3 ꢂ 3) for normal distribution of
99mTc complex at three different time points (2 min, 30 min and
1 h). Another category of animals (2 ꢂ 3) were pre-treated with
desmethylimipramine (10 mg kgꢀ1 intra-peritoneal injection),
30 min prior to the activity injection and distribution of 99mTc
complex seen at two different time-points (2 min and 1 h). Third
set of animal (2 ꢂ 3) distribution involved co-injection of 99mTc
complex 7 along with cold mIBG at two different time points (2
min and 1 h). At the end of each time point, respective set of
animals were sacriced and the relevant organs and tissue were
excised for the measurement of associated activity. The organs
were weighed and the activity associated with each organ was
measured in a at-bed type NaI(Tl) counter with suitable energy
window for 99mTc (140 keV ꢄ 10%). The activity associated with
each organ/tissue was expressed as a percent injected dose per
gram (%ID per g).
Serum stability and protein association. The puried 99mTc-
4+1 complex 7 (50 mL, 370 KBq/10 mCi) was incubated in human
serum (450 mL) at 37 ꢃC for 1 h and 2 h, respectively. Thereaer,
the serum proteins were precipitated by addition of EtOH (500
mL). The solution was centrifuged and the supernatant was
analyzed by TLC to ascertain the stability of the complex in
serum. The precipitate was washed twice with ethanol and the
activity associated with precipitate was expressed as a percent of
the initial activity.
Cell uptake experiments
SK-N-SH neuroblastoma cell line23,24 was used for the uptake
studies. The cell line was obtained from National Centre for Cell
Science, Pune, India and grown in Eagle’s minimal essential
medium supplemented with 10% fetal calf serum, glutamine (2
mmol), penicillin–streptomycin (100 IU mLꢀ1) and amphotericin
B (2.5 mg mLꢀ1). All other media and supplements used were
purchased from Himedia, India. Cells were seeded in six well
plates at an initial density of 5 ꢂ 105 per well per 3 mL of medium
and were cultured as monolayers for 2–3 days at 37 ꢃC in 5% CO2
until semi-conuent. Triplicate wells in each plate containing
Results
monolayers were pre-incubated with desmethylimipramine (0.45 123/131I-labeled mIBG is a widely used diagnostic agent for
mg, 1.5 mmol) for 4 h. Subsequently, HPLC puried 99mTc neuroendocrine tumors of neural origin. The present work
complex 7 was introduced in wells and incubated for a specic explores the possibility of synthesizing a 99mTc analogue of this
time period. Aer incubation, media was removed and mono- agent for NET imaging. The schematic involved synthesizing
layers were washed twice with phosphate buffered saline. The cell a suitable guanidine precursor and attaching it to 99mTc radi-
bound radioactivity was then extracted with 10% (w/v) tri- ometal via 99mTc-4+1 strategy. The active pharmacophore ben-
chloroacetic acid (2 ꢂ 0.5 mL). The activity of the combined zylguanidine was suitably modied at meta-position, wherein
extracts was measured in a NaI(Tl) well counter with suitable an isonitrile functionality was introduced to link it to 99mTc
energy window for 99mTc (140 keV ꢄ 10%). The uptake via specic metal via mono-dentate linkage in presence of the tetra-dentate
active transport pathway was deduced by calculating the differ- co-ligand (NS3).21 The scheme for the synthesis of Cu(I) complex
ence between the uptakes observed for 99mTc complex in SK-N-SH of benzylguanidine derivative (5) is shown in Fig. 1. The
cells to the uptake seen in same cells with desmethylimipramine synthetic procedure involved ve steps starting from m-xylelene
blocking. The results obtained were compared with nca-125I- diamine, wherein monoguanylation at single amine function-
mIBG evaluated following the identical procedure.
ality with N,N0-di-Boc-S-methyl isothiourea yielded 1 at room
Fig. 3 HPLC profile of (a) 99mTc–NS3 (b) complex 7.
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Fig. 4 Scheme for the synthesis of Re-4+1 complex.
temperature. The monoguanylated product 1 was then for- identity and spectroscopic data substantiate the structure as
mylated in the presence of ethyl formate to give 2, which on proposed in Fig. 4.
subsequent dehydration in the presence of p-toluene sulphonyl
The log Po/w of the complex 7 was found to be 1.9 which
chloride and pyridine resulted in di-Boc protected isonitrile shows high lipophilic nature of the complex. The stability of
ligand 3. Since the isonitriles are prone to degradation, a stable HPLC puried 99mTc-4+1 complex in human serum, aer
species in the form of Cu(I) complex 4 was prepared by treat- protein precipitation, showed insignicant degradation of the
ment of the free isonitrile with freshly prepared CuCl. Finally, complex (>90%, Fig. 6) up to a period of 2 h. Around 30% of the
the target Cu(I) complex 5 was obtained on Boc de-protection activity was found to be associated with the serum proteins.
using TFA. All the intermediates and target ligand were char-
acterized by various spectroscopic methods. The radiolabeling
scheme of 5 with 99mTc is shown in Fig. 2. The procedure
involves generation of 99mTc(III)–EDTA intermediate 6 in the
presence of a reducing agent and EDTA ligand. This interme-
diate was produced in near quantitative yield (characterized by
TLC) which on trans-chelation with tetradentate NS3 ligand (4
donor) and monodentate Cu(I) complex of (3-(isocyanomethyl)
benzyl)guanidine donor yields the desired 99mTc-4+1 complex 7
in more than 80% radiochemical purity as characterized by
HPLC (Rt ꢁ 19.4 min, Fig. 3). The product was puried by HPLC
and used for bio-evaluation studies. The structural character-
ization of complex 7 was carried by preparing its Re-analogue 8
in millimolar amounts and characterizing the same using
HPLC, IR, NMR and MS techniques. Fig. 4 shows the synthetic
scheme followed for the preparation of Re-analogue 8. The
analogue 8 was prepared following a reported protocol,21
wherein the intermediate [Re(Me2PPh)NS3] is rst synthesized.
HPLC elution prole of this labile rhenium intermediate
showed a single sharp peak at 25.6 min which on further
reaction with 3 followed by TFA treatment yielded the desired
complex 8 (Fig. 5). The reproducibility in retention times (19.4
min) of 8 to that of 99mTc-4+1 derivative 7 (Fig. 3) conrm the
In vitro cell uptake studies
*I-mIBG enters tumor cells by an active transport mechanism
via nor-epinephrine transporters (NET) and gets stored in the
Fig. 6 Serum stability of 99mTc-4+1 complex 7.
Fig. 5 HPLC UV profile of (a) [Re(NS3)PMe2Ph] (b) complex 8.
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concentrations (activity in cpm) each incubated for xed period
of 2 h. The percentage of specic binding with the cells
increased from 4.2 to 5.2% ID per 106 cells with increase in
tracer concentrations. The non-specic bound component
exhibited a constant fraction of the total uptake activity and was
observed to be less than 10%, similar to the behavior observed
for nca-125I-mIBG.
The bio-distribution results carried in normal Wistar rats are
shown in Table 1. Fig. 9 shows the myocardial uptake of
complex 7 and nca-125I-mIBG at two different time points.
Complex 7 showed initial uptake of 1.69 ꢄ 0.42% ID per g in the
myocardium at 2 min p.i., however it was signicantly lower
than nca-125I-mIBG (4.36 ꢄ 0.68% ID per g). Unlike nca-125I-
mIBG, the initial uptake of 7 in the myocardium was not
retained and cleared with time over 60 min p.i. (0.58 ꢄ 0.03%).
The latter behavior indicates that the attachment of 99mTc-
chelate modies the basic benzyl guanidine moiety signi-
cantly, which is non-recognizable by the innervated sympathetic
organ. Hence, the uptake observed may be non-specic in
nature, where other uptake mechanisms such as passive diffu-
sion contribute to the myocardial uptake. To ascertain the
uptake pathway, in vivo NET transport blocking using mIBG and
Fig. 7 Uptake studies of 99mTc-4+1 complex 7 in SK-N-SH cell line at
different time points. The data shown is from a set of representative
experiments.
cells. SK-N-SH is a neuroblastoma cell line with NET positive DMI were carried, respectively. Fig. 9 includes the tracer
expression known to accumulate nor-epinephrine analogues. distribution in the myocardium, co-injected along with cold
Uptake of synthesized 99mTc radiotracer 7 in SK-N-SH cells was mIBG and 30 min DMI pre-treated animals. Signicant differ-
carried out to evaluate the equivalence of the synthesized ence in myocardial uptake was seen at 60 min injection (p >
complex with respect to nca-125I-mIBG. Fig. 7 represents the 0.05) where mIBG/DMI presence affects the tracer distribution,
uptake of 99mTc complex in SK-N-SH cells at different incuba- thereby favoring the uptake-1 pathway. The uptake of 7 in other
tion times. The initial uptake value of 99mTc complex at 1 h was non-target organs such as blood, lungs and liver was low and
signicant (5.08 ꢄ 0.02% ID per 106 cells) but was lower than that too clears over a period. These factors favor high heart/non-
nca-125I-mIBG (12.2 ꢄ 0.08% ID per 106 cells). The uptake of target ratios, but the effect gets minimized as the complex 7 is
99mTc tracer in neuroblastoma cells was found to be specic via not signicantly retained in the myocardium.
NET transporter as the uptake reduced signicantly (ꢁ90%) on
inhibition with DMI, which is a known inhibitor of uptake-1
pathway responsible for nor-epinephrine transport. The initial
Discussion
uptake in the cells of 99mTc complex was found to be retained up
to a study period of 4 h. A small percentage of non-specic
uptake for complex 7 (ꢁ0.48 ꢄ 0.01% ID per 106 cells) was
seen in the cells which may be associated to other mechanistic
pathways such as uptake-2 mechanism. Fig. 8 shows the uptake
of 99mTc tracer in SK-N-SH cells at three different complex 7
Development of 99mTc-agent for NET tumors of neural crest
origin is of high signicance as the use of 99mTc radioisotope
allows ready availability of the tracer in the clinics. Further, even
if the tracer uptake in the target is low, the injected 99mTc
activity content can be increased to obtain high resolution
images without delivering extensive radiation load to the
Fig. 8 Cell uptake studies of 99mTc-4+1 complex (7) and nca-125I-mIBG in SK-N-SH human neuroblastoma cells as a function of tracer
concentration. The data shown is from a set of representative experiments.
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Table 1 Bio-distribution studies of 99mTc-4+1 complex (7) in normal Wistar rats in comparison with nca-125I-mIBGa
30 min
2 min
60 min
Organ
Complex 7
7 + mIBG
7 + DMI
Complex 7
Complex 7
7 + mIBG
7 + DMI
Liver
1.21 (0.05)
0.31 (0.03)
0.34 (0.17)
2.12 (0.01)
1.69 (0.42)
1.95 (0.24)
0.25 (0.12)
0.37 (0.05)
2.55 (0.27)
0.22 (0.21)
0.65 (0.14)
1.42 (0.55)
0.85 (0.15)
1.39 (0.05)
0.37 (0.06)
0.30 (0.13)
2.32 (0.27)
2.28 (0.29)
2.87 (0.37)
0.91 (0.13)
0.33 (0.21)
3.13 (0.37)
0.25 (0.18)
0.73 (0.02)
1.64 (0.36)
0.79 (0.04)
1.35 (0.11)
0.34 (0.03)
0.39 (0.13)
2.27 (0.30)
1.9 (0.31)
2.21 (0.35)
0.77 (0.26)
0.4 (0.05)
3.16 (0.41)
0.29 (0.21)
0.6 (0.06)
1.41 (0.32)
0.86 (0.06)
1.13 (0.09)
1.03 (0.15)
3.76 (0.65)
1.53 (0.06)
0.75 (0.13)
0.82 (0.13)
0.89 (0.13)
0.25 (0.01)
1.18 (0.11)
0.09 (0.05)
0.63 (0.13)
0.66 (0.14)
0.91 (0.03)
1.28 (0.31)
1.35 (0.33)
2.19 (0.06)
1.69 (0.01)
0.58 (0.03)
0.62 (0.07)
0.75 (0.16)
0.18 (0.07)
1.21 (0.08)
0.03 (0.02)
0.48 (0.05)
0.47 (0.09)
0.94 (0.06)
1.25 (0.03)
1.23 (0.33)
3.86 (0.59)
1.66 (0.10)
0.31 (0.03)
0.54 (0.04)
0.84 (0.12)
0.13 (0.05)
0.62 (0.14)
0.14 (0.08)
0.54 (0.21)
0.24 (0.03)
0.57 (0.1)
1.30 (0.05)
1.54 (0.21)
1.11 (0.11)
1.85 (0.14)
0.39 (0.09)
0.60 (0.18)
0.68 (0.32)
0.07 (0.05)
0.84 (0.18)
0.12 (0.08)
0.46 (0.08)
0.29 (0.08)
0.66 (0.15)
Int + GB
Stomach
Kidney
Heart
Lungs
Spleen
Muscle
Blood
Bone
H/Blood
H/Liver
H/Lung
a
All results are expressed as %ID per g ꢄ SD, 3 animals were used for each time point, DMI ¼ desmethylimipramine; mIBG ¼ meta-
iodobenzylguanidine.
frequency at 2185 cmꢀ1 in the Cu(I) complex. The labeling of the
latter ligand was achieved in high yield and the labeled product
was puried using analytical HPLC, to remove excess cold
ligand before carrying out biological studies.
SK-N-SH neuroblastoma cell line was used for assessing the
affinity of the synthesized 99mTc tracer towards nor-epinephrine
transport expression. The specic uptake of the present 99mTc-
4+1 radiotracer (complex 7) in SK-N-SH neuroblastoma cells,
when compared with that of the previously reported 99mTc-2
(N2S2 complex),16 showed signicantly improved results (90%
vs. 60%) with non-specic uptake values being similar to
nca-125I-mIBG (ꢁ10%). The specic uptake values were also
high for the complex 7 (50% of nca-125I-mIBG vs. 10% of
nca-125I-mIBG) in comparison to the previously reported
complex.
For evaluating the potential of the new tracers towards nor-
epinephrine transporter in vivo, myocardium is taken as the
organ of interest. This is because the heart, being a sympathet-
Fig. 9 Myocardial uptake (%ID per g) of complex 7 in comparison with
nca-125I-mIBG in normal Wistar rats.
ically innervated organ, requires transport of nor-epinephrine
hormone for its functioning. 123I-mIBG is a radiopharmaceu-
patients. Also, the superior nuclear imaging characteristics of
tical which is used in sympathetic myocardial imaging and its
99mTc over 131I will further improve the resolution of the images.
uptake is found to correlate with myocardial defect. Though, the
Considering these aspects, designing a 99mTc analogue of *I-
mIBG is of high relevance but this has met with limited success
until now. Diverse complexation chemistry of the 99mTc metal
opens up a number of synthetic strategies for attaching the
vector molecule to the same metal through different labeling
methods. In the present work, 99mTc-4+1 labeling approach has
been followed for introducing the benzylguanidine derivative.
The latter moiety has been modied to introduce an isonitrile
motif to be suitable for complexation through this approach.
Since isonitriles are reactive intermediates and are prone to
degradation, the isonitrile ligand synthesized was transformed
into a stable Cu(I) isonitrile complex 5. The formation of Cu(I)
complex could be easily deduced from the IR spectrum which
shows a shi in the frequency position of isonitrile group
(N^C) from 2152 cmꢀ1 for the free isonitrile ligand to a higher
99mTc tracer under study showed specic avidity (as established
by in vivo inhibition using DMI/mIBG) towards myocardium, the
uptake in the myocardium was observed to be lower. Never-
theless, the efficacy in vivo needs further introspection by
carrying out bio-distribution in tumor bearing athymic mice.
Conclusions
A
99mTc analogue of *I-mIBG was synthesized via 99mTc-4+1
strategy and was biologically evaluated. This involved synthesis
of a suitable Cu(I) complex of benzylguanidine precursor with
isonitrile functionality at the meta-position in moderate yields.
This complexed efficiently with 99mTc, in the presence of tetra-
dentate NS3 ligand, in high yield and purity. The chemical
structure of 99mTc-4+1 complex was ascertained by preparing its
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¨
¨
¨
rhenium analogue. In vitro cell uptake studies of the complex in 11 B. Eriksson, M. Bergstrom, H. Orlefors, A. Sundin, K. Oberg
˚ ¨
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