Synthesis, radio-LC-MS analysis and biodistribution in mice of 99mTc-NIM-BAT

Article abstract of DOI:10.1002/jlcr.698

S,S'-bis-trityl-N-BOC-1,2-ethylenedicysteamine (S,S'-bis-trityl-N-BOC-BAT) was conjugated to 2-nitroimidazole (NIM) through a propylene spacer in order to provide a precursor for a potential technetium-99 m labelled hypoxia tracer. For labelling with tech

Full text of DOI:10.1002/jlcr.698

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 575–585.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.698
Research Article
Synthesis, radio-LC–MS analysis and
biodistribution in mice of 99mTc–NIM–BAT
G. Bormans1, B. Cleynhens2, T.J. de Groot2, L. Mortelmans2,
J.-L. Moretti3 and A. Verbruggen1,*
1 Laboratory of Radiopharmaceutical Chemistry, F.F.W. K.U.Leuven,
Herestraat 49, Leuven B-3000, Belgium
2 Department of Nuclear Medicine, U.Z. Gasthuisberg, Herestraat 49,
Leuven B-3000, Belgium
3Department of Nuclear Medicine, CHU Avicenne, Rue de Stalingrad 125,
93009 Bobigny Cedex, France
Summary
S,S0-bis-trityl-N-BOC-1,2-ethylenedicysteamine
(S,S0-bis-trityl-N-BOC–BAT)
was conjugated to 2-nitroimidazole (NIM) through a propylene spacer in order
to provide a precursor for a potential technetium-99 m labelled hypoxia tracer.
For labelling with technetium-99 m, a two-step one-pot procedure was developed
consisting of deprotection of the ligand by heating in mild acidic conditions and
subsequent exchange labelling in the presence of SnCl2, tartrate and 99mTcO4ꢀ.
The labelling reaction mixture was analyzed using electrospray radio-LC–
MS and the observed mass spectrum corresponding to the main radiometric
peak was in accordance with the predicted structure of oxo–Tc(V)–NIM–BAT.
99mTc–NIM–BAT was purified using RP–HPLC and its biodistribution was
evaluated in normal mice at 10 min and 4 h p.i. 99mTc–NIM–BAT was cleared
from plasma mainly by hepatobiliary excretion. Copyright # 2003 John Wiley
& Sons, Ltd.
Key Words: hypoxia; technetium-99m; bis-amino bis-thiol; bifunctional
chelating agent
*Correspondence to: A. Verbruggen, Laboratory of Radiopharmaceutical Chemistry, U.Z.
Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: alfons.verbruggen@uz.kuleuven.ac.be
Contract/grant sponsor: Fund For Scientific Research–Flanders, Belgium
Contract/grant sponsor: Onderzoeksfonds, University of Leuven
Copyright # 2003 John Wiley & Sons, Ltd.
Received 3 February 2003
Accepted 10 February 2003

576
G. BORMANS ET AL.
Introduction
Accurate measurement of tumor hypoxia can be clinically useful to
predict sensitivity to radiation therapy and to evaluate interventions
that aim to increase tumor oxygenation or to identify tumors that are
amenable to hypoxia-directed gene therapy or hypoxia-selective
therapy, e.g.with tirapazamine.1
Several hypoxia markers contain a nitroimidazole moiety as the
reactive chemical species. Nitroimidazoles are reduced intracellularly in
all cells, but in the absence of an adequate supply of oxygen, they
undergo further reduction to more reactive products which bind to cell
components. In this way, they are trapped in hypoxic tissue.1,2
Examples of PET hypoxia imaging agents are the fluorine-18 labelled
2-nitroimidazole derivatives 18FNIM3, 18FETNIM4 and 18F-EF15
besides 62Cu-ATSM6, which lacks the 2-nitroimidazole moiety. For
SPECT imaging of hypoxia, the 2-nitroimidazole containing iodine-123
labelled compound IAZA has been used clinically.7 In addition, several
technetium-99 m complexes have been proposed and evaluated8–14
among which 99mTc-HL91 (oxo-99mTc(V)-4,9-diaza-3,3,10,10-tetra-
methyldodecan-2,11-dione dioxime, 99mTc-BnAO, Prognox) which does
not contain the bioreductive 2-nitroimidazole moiety.15 In a clinical
study, 99mTc-HL91 was found to be useful for tumor visualization16,17
but despite these encouraging results commercialization of this agent
was not pursued.
In a search for a new technetium-99 m labelled hypoxia imaging agent
we have conjugated the thiol protected bifunctional chelating agent 1,2-
ethylenedicysteamine (BAT) to 2-nitroimidazole using a propylene
spacer. The corresponding 99mTc-complex was characterized with LC–
MS and its biodistribution was studied in mice.
Results and discussion
The S,S0-bis-trityl-N-BOC protected conjugate of 1,2-ethylenedicystea-
mine and 2-nitroimidazole was synthesized by reaction of protected
BAT with 1-bromo-3-chloropropane and subsequent alkylation of 2-
nitroimidazole using the N-derivatized BAT intermediate (Figure 1).
Deprotection and labelling of the conjugate with 99mTc can be
performed in the conventional way. In this case, thiols and the BOC-
protected amine are first deprotected, e.g. using a mixture of
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 575–585

SYNTHESIS OF 99mTc–NIM–BAT
577
NH
S
NH
S
NH
S-
NH
-S
HCl
Na/NH3
N
(C6H5)3COH
CF3COOH
S
(BOC)2O
Cl
C
OCO
C
OCO
N
S
NH
S
N
S
N
S
BrCH2CH2CH2Cl
N
HN
N
NO2
N
N
N
C
OCO
NO2
N
N
S
O
NO2
N
S
N
1. HCl 0.5M
2. SnCl2, TcO4
Tc
-
S
S
Figure 1. Synthesis of S,S0-bis-trityl-N-BOC–NIM–BAT and Tc–NIM–BAT
(proposed structure)
trifluoroacetic acid, anisole and methansesulfonic acid, and the
deprotected conjugate is then isolated, purified and stored in this form
for labelling at a later moment. However, we have now developed a
simpler one-pot two-step procedure to deprotect and label the S,S0-bis-
trityl-N-BOC protected conjugate. In this procedure, the ligand was
deprotected just before use in a labelling vial by heating in acidic
conditions. The deprotected conjugate was subsequently labelled with
technetium-99 m in a second heating step after addition of solutions of
phosphate buffer pH 7, EDTA and tartrate, SnCl2 and 99mTcO4ꢀ. Using
this two-step one-pot labelling procedure, the 99mTc–NIM–BAT
complex was obtained with a radiochemical purity of 80%, which is
comparable to the radiochemical purity obtained using the two-pot
labelling procedure. The trityl group is thus an interesting protection
group for thiols of technetium chelating agents as it remains stable
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 575–585

578
G. BORMANS ET AL.
MISOBAT20
100
RA NaI
An1
3.02e5
10.05
A
B
%
0
MISOBAT20
1: TOF MS ES+
445.914_446.213
166
10.05
100
%
0
Time
0.00
2.50
5.00
7.50
10.00
12.50
15.00
17.50
20.00
MISOBAT20 596 (10.138) Cm (569:632-(484:552+630:733)x2.000)
1: TOF MS ES+
446.0760
374
100
C
%
436.3057 447.0669
313.2833
585.2134
0
100
m/z
1000
200
300
400
500
600
700
800
900
Figure 2. Radio-LC–MS analysis of Tc–NIM–BAT: (A) Radiometric trace. (B)
Single ion chromatogram (444.914–446.213 Da). (C) Background subtracted
summed spectrum over Rt=9.1 to 10.95 min
during conjugation reaction and can nevertheless be removed in a
convenient way by heating in mild acidic conditions. This allows flexible
coupling of the BAT bifunctional chelating agent to a variety of
biologically interesting molecules which in this way can be labelled
efficiently with technetium-99 m.
Radio-LC–MS analysis of the reaction mixture after labelling of the
BAT–NIM conjugate with 99mTc showed one major peak on the
radiometric channel (Figure 2A). The background subtracted summed
mass spectrum over this peak showed the presence of a molecular ion
peak corresponding to the expected oxo-technetium(V)–NIM–BAT
complex (Figure 2C). The accurate mass (theoretical 446.0137 Da,
found 446.0760 Da) also corresponds to the expected structure (Figure
1) and was determined with a relative error of only 2.9 ppm.
The single ion mass chromatogram (445.914–446.213 Da) further
shows only one peak which has an identical retention time as the peak
of the radiometric trace (Figure 2B). These mass spectrometric data
provide a strong identity confirmation of the supposed structure of Tc–
NIM–BAT.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 575–585

SYNTHESIS OF 99mTc–NIM–BAT
579
Table 1. Biodistribution of 99mTc–NIM–BAT in mice
% of ID (n = 4)
% of ID/g (n = 4)
10 min p.i.
ꢂ
4 h p.i.
SD mean
10 min p.i.
ꢂ
4 h p.i.
mean
mean
ꢂ
SD
7.2
mean
SD
ꢂ
SD
Urine
0.3
1.7
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
0.2
0.2
8.8
0.9
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
}
} }
}
} }
Kidneys
Liver
Spleen
Lungs
Heart
Intestines
Stomach
Cerebrum
0.3
2.9
4.2 17.8
0.01 0.9
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
0.3
1.8
9.0
0.1
0.4
0.2
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
0.6
39.2
0.1
3.2 18.7
0.03 0.02
2.0
0.1
0.3
0.3
2.6
0.0
0.3
0.1
2.7
0.2
0.02
0.04
0.1
0.1
0.5
0.2
0.1
0.0
0.1
0.04
0.1
0.01 1.6
16.0 7.00
0.1 3.1
1.9
25.9
1.5
6.2 59.0
0.8 0.7
0.01 0.01
0.00 0.01
0.59 0.7
1.6 17.7
2.4 1.3
0.02 0.03
0.00 0.05
0.02
0.00 0.07
0.00 0.10
Cerebellum 0.01
Blood
Carcass
3.0
22.2
0.3
1.7
1.2
0.8
0.2
0.1
0.3
0.1
2.9
3.5
The results of the biodistribution study in mice are summarized in
Table 1. As can be expected for a lipophilic compound, 99mTc–NIM–
BAT undergoes extensive hepatobiliary excretion.
At 4 h p.i. about 60% of the injected activity is excreted to the
intestines and about 10% is excreted with the urine. A fraction of 22%
of the injected dose of 99mTc–NIM–BAT is retained in the carcass at
10 min p.i. and is cleared as a function of time, resulting in a residual
activity of only 3.5% of the injected dose at 4 h p.i. From the %ID/g
values it can be concluded that this compound distributes evenly over
the different organs and tissues with exception of the excretory organs
liver (and intestines) and kidneys, the latter however to a minor degree.
Experimental
All reagents were obtained commercially from Acros (Geel, Belgium),
Fluka (Bornem, Belgium) or Merck (Darmstadt, Germany).
1H-NMR spectra were obtained using a Varian 200 MHz spectro-
meter (Varian, Palo Alto, CA, USA). Chemical shifts are reported in
ppm relative to the internal standard tetramethylsilane (TMS, d=0).
Mass spectra were acquired on a Micromass LCT mass spectrometer
(Micromass, Manchester, UK) equipped with an electrospray interface.
HPLC analysis and purification was done using an XTerra RP18 5 m
column (4.6 mm ꢁ 250 mm, Waters, Milford, USA) eluted with gradient
mixtures of ammonium acetate 0.1 M and acetonitrile (linear gradient;
Copyright # 2003 John Wiley & Sons, Ltd.
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580
G. BORMANS ET AL.
0 min 0% acetonitrile; 30 min 90% acetonitrile). The eluate was
monitored for radioactivity using a 2-in NaI(Tl) scintillation detector.
Radio-LC–MS
Radio-LC–MS analysis was performed on a system consisting of a
Waters separation module (Waters, Milford USA), an XTerra MS C18
column (50 mm ꢁ 2.1 mm, Waters) and a 2-in NaI(Tl) radiation detector
and a Micromass LCT (time-of-flight) mass spectrometer equipped with
electrospray interface in series. Masslynx software (Micromass) was
used for acquisition and data processing.
The column was eluted at a flow rate of 300 ml/min with gradient
mixtures of acetonitrile and HCOONH4 1% (linear gradient from 0%
acetonitrile at 0 min to 80% at 20 min).
A solution of Kryptofix 2.2.2 (0.01% m/v) in CH3CN/H2O (50:50, v/
v) was added to the column eluent at a flow rate of 1 ml/min and served
as lock mass for accurate mass determination. For each analysis a
volume of 50 ml of the radiolabelling reaction mixture was injected on
the column.
1; 2-Ethylenedicysteamine (2)
Thiazolidine (1, 8.91 g, 100 mmol) was dissolved in liquid ammonia
(60 ml) in a two-necked flask fitted to a Dewar cooler, and the mixture
was stirred vigorously. Sodium (4.84 g, 210 mmol) was added until a
blue color persisted for at least 15 min. Ammonium chloride was then
added in portions until decoloration persisted and the resulting reaction
mixture was kept at room temperature to allow slow evaporation of
ammonia. Cold water (200 ml) was added followed by the addition of
HCl 12 M to pH 1. After washing with three portions of 100 ml ethyl
acetate, the water layer was evaporated at reduced pressure. The white
residue was dried overnight at 608C under vacuum and used without
purification in the next reaction step.
MS ESI+: m/z (M+H)+ found 181 (calculated, C6H17N2S2, 181).
S,S0-Bis-triphenylmethyl-1,2-ethylenedicysteamine (3)
The residue obtained in the previous reaction step was dissolved in
120 ml trifluoroacetic acid at room temperature. Triphenylmethanol
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF 99mTc–NIM–BAT
581
(26.03 g, 100 mmol) was added in portions to the reaction mixture which
was subsequently stirred at room temperature for 15 min. Evaporation
of the reaction mixture under reduced pressure yielded a red oil. The
evaporation was repeated three times after addition of hexane
(3 ꢁ 100 ml) to remove traces of trifluoroacetic acid. The residual oil
was dispersed in a mixture of water (200 ml) and ethyl acetate (200 ml)
and the mixture was neutralized with 10% NaHCO3 solution. The
organic phase was separated, dried over anhydrous magnesium sulfate,
concentrated under reduced pressure and chromatographed on a
Kieselgel 60 column eluted with 5% MeOH in EtOAc. The appropriate
fractions were pooled and evaporated to give 16.6 g (24.9 mmol, 49.9%)
of a cream-colored foam. TLC (5% MeOH in EtOAc): Rf 0.55.
1H-NMR (CDCl3) d 1.79 (br s, 2 H, NH), 2.3–2.6 (m, 4 H,
SCH2CH2N), 2.45 (s, 4 H NCH2CH2N), 2.84 (m, 4 H, SCH2CH2N).
MS ESI+: m/z (M+H)+ found 664.38 (calculated, C44H45N2S2,
664.29).
S,S0-Bis-triphenylmethyl-N-(tert-butoxycarbonyl)-1,2-ethylenedicystea-
mine (4)
To a solution of 3 (7.51 g, 11.26 mmol) and diisopropylethylamine
(DIEA, 2.2 ml, 12 mmol) in 250 ml CH2Cl2 was added dropwise a
solution of (BOC)2O (2.53 g, 11.26 mmol) in CH2Cl2 (50 ml) at 08C. The
mixture was stirred at 08C for 1 h, concentrated and purified by column
chromatography (Kieselgel 60, hexane/EtOAc/NEt3 80:15:5, v/v/v) to
give 4.21 g of a white foam (48.8%).
1H-NMR (CDCl3) d 1.38 (s, 9 H, C(CH3)3), 2.27–2.58 (m, 8 H,
CH2NCH2 +2x SCH2CH2N), 2.97 (m, 4 H, CH2N-BocCH2), 7.1–7.4
(m, 30 H, ArH).
MS ESI+: m/z (M+H)+ found 764.96 (calculated, C49H53N2O2S2,
765.34).
S,S0-Bis-triphenylmethyl-N-(tert-butoxycarbonyl)-N0-(3-chloropropyl)-
1,2-ethylenedicysteamine (5)
The amine 4 (3.45 g, 4.5 mmol) and DIEA (4 ml, 22.5 mmol) were
dissolved in dry acetonitrile (30 ml) and to this solution was added 1-
bromo-3-chloropropane (3.6 g, 22.5 mmol). The resulting solution was
refluxed overnight, concentrated in vacuo and chromatographed
(Kieselgel 60, hexane/ethyl acetate, 4:1, v/v). This yielded 1.5 g (40%)
of the desired product 5 which was isolated as a colorless oil.
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582
G. BORMANS ET AL.
1H NMR (CDCl3) d 1.38 (s, 9 H, C(CH3)3), 1.62 (m, 2 H,
CH2CH2CH2Cl), 2.2–2.3 (m, 8 H, -CH2NCH2- + 2x SCH2CH2N), 2.93
(m, 4 H, CH2N-BocCH2), 3.46 (t, 2 H, CH2Cl), 3.55 (t, 1 H,
NCH2CH2CH2Cl), 3.69 (t, 1 H, NCH2CH2CH2Cl), 7.2-7.4 (m, 30 H, ArH).
MS
ESI+:
m/z
(M+H)+
found
840.92
(calculated,
C52H58ClN2O2S2, 841.35).
S,S0-Bis-triphenylmethyl-N-(tert-butoxycarbonyl)-N0-(3-(20-nitro-10H-
imidazoyl)-propyl)-1,2-ethylenedicysteamine (6)
A solution of 5 (149 mg, 0.18 mmol), 2-nitroimidazole (30.3 mg,
0.18 mmol), KI (30 mg, 0.18 mmol) and K2CO3 (252 mg, 1.8 mmol) in
5 ml DMF was heated at 1108C for 2 h. After removal of DMF, the
residue was purified by chromatography (Kieselgel 60, hexane/EtOAc/
NEt3, 4:1:0.25, v/v/v) to yield 100 mg (55%) of a yellow powder.
1H NMR (CDCl3) d 1.37 (s, 9 H, C(CH3) 3), 1.76 (m, 2 H,
CH2CH2CH2), 2.1–2.4 (m, 10 H, CH2NCH2 + 2x SCH2CH2N +
NCH2CH2CH2Im), 3.0 (m, 4 H, CH2N-BocCH2), 4.34 (m, 2 H,
CH2Im), 7.0–7.4 (m, 32 H, 2 ꢁ ImH, 30 ꢁ ArH).
MS ESI+: m/z (M+H)+ found 918.37 (calculated, C55H60N5O4S2,
918.40).
Labelling with 99mTc and purification
Method A. An aliquot of 1 mg of ligand 6 was dissolved in a mixture of
0.3 ml ethanol and 0.5 ml HCl 0.5 M and heated in a boiling water-bath
during 10 min. After cooling, 1 ml of a solution containing 0.5 ml
phosphate buffer 0.5 M pH 7, 0.25 ml Na2EDTA 0.1 M and 0.25 ml
KNa-tartrate solution (40 mg/ml) was added, immediately followed by
the addition of 100 mg SnCl2 ꢃ 2H2O in 25 ml HCl 0.05 N and 1 ml 99mTc-
pertechnetate solution (200 MBq, monday morning eluate). The
labelling vial was heated again in a boiling water-bath for 10 min,
cooled and the solution filtered through a 0.45-mm membrane filter. The
reaction mixture was then analyzed with the HPLC system described
higher (Rt 99mTc–NIM–BAT=23 min).
Method B. An amount of 918 mg (1 mmol) of 6 was dissolved in a
mixture of TFA (1 ml) and anisole (0.1 ml) at room temperature. The
solution was cooled to 08C and methanesulfonic acid (0.4 ml) was added
dropwise. The resulting solution was stirred at room temperature for
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF 99mTc–NIM–BAT
583
1.5 h under N2 and concentrated in vacuo. Water (5 ml) and diethyl
ether (5 ml) were added to the residue, the water layer was separated,
neutralized with 10% NaHCO3 solution and extracted with CH2Cl2
(3 ꢁ 5 ml). The CH2Cl2 layers were combined, dried over anhydrous
MgSO4 and concentrated in vacuo. The yellow solid formed was dried
overnight under vacuum at 608C. Yield: 125 mg (30%).
MS ESI+: m/z (M+H)+ found 334 (calculated, C12H24N5O2S2,
334). For labelling with 99mTc, 100 mg of the deprotected ligand was
added to a solution containing 100 ml ethanol, 100 mg SnCl2 ꢃ 2H2O,
0.5 ml HCl 0.5 N and 0.5 ml generator eluate (containing 370 MBq
99mTcOꢀ4 ). The labelling reaction mixture was heated for 10 min in a
boiling water-bath. The reaction mixture was then analyzed with the
HPLC system described higher (Rt Tc–NIM–BAT=23 min).
Biodistribution in mice
Solutions of 99mTc–NIM–BAT obtained after HPLC purification were
diluted with normal saline to a concentration of 148 kBq/ml. Biodistribu-
tion was studied in normal male NMRI mice (body mass 30–40 g). The
animal studies were performed in accordance with the Belgian code of
practice for the care and use of animals. The mice were sedated by i.m.
injection of 0.1 ml of a solution containing 34 mg/ml fentanyl and 16 mg/ml
ketamine. A volume of 0.1 ml of the diluted tracer solution was injected in
the mice via a tail vein. The mice were sacrificed by decapitation at 10 min
or 4 h post injection (p.i.) (n= 4 at each time point). Blood was collected in
a tared tube and weighed. All organs and other body parts were dissected,
weighed and their radioactivity was counted in a 3 in NaI(Tl) well crystal,
coupled to a multichannel analyzer (Wallac, Turku, Finland). Corrections
were made for background radiation and physical decay during counting.
Results were expressed as percentage of injected dose (% of ID), equal to
the sum of the net counts in all organs and where possible as percentage of
injected dose per gram of tissue (% of ID/g). For calculation of total blood
activity, blood mass was assumed to be 7% of the body mass.
Conclusion
We have conjugated S,S0-bis-trityl-N-BOC protected 1,2-ethylenedicys-
teamine with 2-nitroimidazole. An interesting one-pot two-step method
Copyright # 2003 John Wiley & Sons, Ltd.
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584
G. BORMANS ET AL.
for the labelling with technetium-99 m was developed starting from the
protected ligand, yielding a high yield of the oxo-technetium(V)–NIM–
BAT complex of which the identity was confirmed with radio-LC–MS.
In mice, this lipophilic compound showed extensive hepatobiliary
excretion.
The evaluation of the potential of this new 99mTc-labelled conjugate
as a hypoxia tracer will be published in a separate paper.
Acknowledgements
This study was supported by the Fund for Scientific Research-
Flanders, Belgium and by a research grant from the Onderzoeksfonds,
University of Leuven.
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