Rapid decomplexation of bis(thiosemicarbazonato)zinc(ll) complexes using citric acid

Article abstract of DOI:10.3184/030823408X380731

Facile and quantitative dissociation of bis(thiosemicarbazonato)zinc(ll) complexes to give the corresponding neutral proligands in solution has been achieved by reaction with citric acid. This reaction provides an elegant solution to a problem encountered in the design, synthesis and purification of potential copper-based radiopharmaceuticals.

Full text of DOI:10.3184/030823408X380731

702 RESEARCH PAPER
DECEMBER, 702-703
JOURNAL OF CHEMICAL RESEARCH 2008
Rapid decomplexation of bis(thiosemicarbazonato)zinc(1I) complexes
using citric acid
Jason P. Hollanda,b*, Jennie A. Hickina, Emma Grenville-Mathersa, ThaoNguyen Nguyena and
Josephine M. Peacha*
aDepartment of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK
bMemorial Sloan-Kettering
Cancer Centre, 1275 York Avenue, New York, NY10021, USA
Facile and quantitative dissociation of bis(thiosemicarbazonato)zinc(lI)
complexes to give the corresponding neutral
proligands in solution has been achieved by reaction with citric acid. This reaction provides an elegant solution to a
problem encountered in the design, synthesis and purification of potential copper-based radiopharmaceuticals.
Keywords: radiopharmaceuticals, bis(thiosemicarbazonato) complexes, citric acid, copper, zinc
In the modem clinical environment, in vivo molecular imaging
plays a vital role in drug discovery and in the diagnosis
and treatment of disease.!-4 In recent years, there has been
intense research into the development of copper-based
radiopharmaceuticals for imaging tumour hypoxia (tissue
oxygenation <10% of normal levels).5,6 In particular, the
diacetyl-2,3-bis( 4-N-methyl-3-thiosemicarbazonato )copper(ll)
complex, [Cu(ll)ATSM] has emerged as the leading hypoxia-
selective agent for positron emission tomography (PET)
imaging and radiotherapy.7-9 Recently, Donnelly et al.lO have
reported that selective intracellular release of copper and zinc
ions from bis(thiosemicarbazonato) complexes reduce levels
of amyloid-~ peptide, which is an important factor in the
regulation of Alzheimer's Disease (AD). These metal chelates
have potential to be used in the diagnosis and treatment of
patients with AD.
Synthetic work has lead to the development of new
intermediates for the facile conjugation of bis( thiosemicarbazo-
nato)zinc(ll) complexes to a range of biologically active
molecules.ll,12 Transmetallation of bis(thiosemicarbazonato)
zinc(II) complexes with copper diacetate, Cu(OAcMaq.) has
also been shown to be an efficient and rapid method for the
preparation of copper-64 radio labelled complexes for PET
imaging.! I
found to be less reactive than their corresponding metal-free
proligands. However, synthesis of the ligands was found to
be best achieved using zinc(ll) ions as a templating agent.
Purification of the zinc(II) complexes was also found to be
easier due to their increased solubility in common organic
solvents in comparison to the corresponding proligands.
Therefore, in order to facilitate these conjugation reactions
we investigated several potential solution and solid-phase
methods for the removal of zinc(ll) ions from bis(thiosemi-
carbazonato )zinc(ll) complexes.
Initial work focused on the use of imidodiacetate resins to
remove the zinc(ll) ions from complex 1, [Zn(ll)ATSM] to
give the neutral proligand 2, H2ATSM. Although the reaction
was found to be successful at room temperature in dimethyl
sulfoxide (DMSO), proligand 2 could only be isolated on the
milligram scale and the kinetics were found to be very slow
(>24 h for the purification approx. 2 mg of 2; the reaction
was found to be even slower in ethyl acetate or methanol).
In addition, recovery of the proligand from DMSO was
found to be difficult. Solid-phase attempts were subsequently
abandoned in favour of more rapid and high yielding solution-
phase methods (vide infra).
It was noted that the characteristic bright yellow colour
of complex 1 (Amax(DMF)= 431 nm, with molar absorption
coefficient, E = 11266 M-I cm-I),12 was lost on addition of
strong acids such as HC1,HN03, H2S04 and HBF4to a solution
of complex 1 in ethyl acetate, ethanol, methanol, DMSO, or
dimethyl formamide (DMF), giving a colourless/pale yellow
solution. Protonation of complex 1 with strong acids leads to
Despite the potential advantages of using zinc(ll) complexes
as precursors for copper radio labelling, during recent efforts
towards the synthesis of bis(thiosemicarbazonato)metal(ll)
complexes conjugated to fluorophores such as pyrene,
dansyl cWoride and fluorescein, the zinc(ll) complexes were
I
[Zn(II)ATSM]
As above
4
3
Scheme
1
Facile and quantitative removal of the zinc(ll) ions from bis(thiosemicarbazonato)zinc(ll)
corresponding proligands.
complexes to give the
* Correspondent. E-mail: hollanj3@mskcc.org

JOURNAL OF CHEMICAL RESEARCH 2008 703
with a previously reported procedure. 12Compound 4 was isolated as
a cream/white powder (0.43 g, 1.1 mmol, 79%). Elemental analysis
(%) for C17H2~6S2 (376.15 g mol,l) Calcd C 54.0, H 6.4, N 21.9
and S 17.5; found C 54.2, H 6.4, N 22.3 and S 17.0. IH NMR (300
MHz, CDC13):8/ppm 10.34 (lH, s, NHC( = S)NHN =); 10.17 (lH,
s, NHC( = S)NHN = ); 8.66 (lH, br m, (Ph)CH=CHCH2NH); 8.43
(lH, brm, CH3CH2NH); 7.41 (2H, d, 3JHH= 7.4 Hz, o-Ph); 7.32 (2H,
app. t, 3JHH = 7.3 Hz, m-Ph); 7.21 (lH, ill, 3JHH = 7.3 Hz,p-Ph); 6.52
initial demetallation. However, it was not possible to isolate
proligand 2 after reaction with these strong acids. Therefore,
the reactions of complex 1 with weaker acids, especially citric
acid, were investigated (Scheme I).
Reaction of a solution of complex 1 or 3 in ethyl acetate
with 10% citric acid (aq.) at room temperature, followed by
neutralisation with saturated sodium hydrogen carbonate
solution, organic-layer extraction, drying and removal of the
solvent under reduced pressure, was found to give proligands
2 and 4, respectively, in quantitative yield. The reaction to
give proligand 4 has been successfully scaled up to >400 mg
of complex 3.
(lH, d, 3JHH = 16.0 Hz, (Ph)CH=CHCH2NH); 6.35 (lH, dt, 3JHH
=
16.0 and 5.4 Hz, (Ph)CH=CHCH2); 4.41 (2H, m, (Ph)CH=CHCH2);
3.60 (2H, ill, 3JHH = 6.6 Hz, CH3CH2NH); 2.23 (6H, two overlapping
singlets, CH3C=N); 1.14 (3H, t, 3JHH = 6.9 Hz, CH3CH2NH). 13C{IH}
NMR (75.5 MHz, DMSO-~): 8/ppm 177.82 (C=S); 177.31 (C=S);
148.27 (C=N); 147.85 (C=N); 136.45 «Ph)CH=CHCH2); 130.40
(i-Ph); 128.57 (m-Ph); 127.37 (P-Ph); 126.40 «Ph)CH=CHCH2);
126.Dl (o-Ph); 45.54 «Ph)CH=CHCH2); 38.50 (CH3CH2NH); 14.32
(CH3CH2NH); 11.74 (CH3C=N); 11.69 (CH3C=N). MS (ES+): m/z
399 = {M +Na+} 100%. HPLC: Rt 15.72 min.
In conclusion the facile demetallation of zinc(ll) ions
from bis(thiosemicarbazonato)zinc(II)
complexes by the
reaction with citric acid will facilitate the synthesis and
purification of new ligands for use as potential copper-based
radiopharmaceuticals for PET imaging and radiotherapy.
This reaction is being used frequently in the synthesis of
functionalised bis(thiosemicarbazonato) ligands at Oxford.
Reactions with citric acid
Decomplexation of bis( thiosemicarbazonato )zinc(ll) complexes (lor
3), to give the corresponding neutral proligands (2 or 4) in solution
was achieved using the following general procedure. Bis(thiosemi-
carbazonato)zinc(II) complex (Complex 1, 50.0 mg, 0.Dl5 mmol)
was dissolved in ethyl acetate (100 ml) and 10% aqueous citric acid
solution (100 ml) was added. The solution was stirred for 5 min at
room temperature during which time the colour changed from bright
yellow to colourless/pale yellow. Saturated NaHC03(aq.) (100 ml)
was then added and the organic layer was extracted (3 x 50 ml ethyl
acetate), dried over anhydrous magnesium sulfate, filtered and the
solvent was removed under reduced pressure to give either proligands
2 or 4 as a white powder. (Proligand 2, 40.1 mg, 100%). Analytical
data were fully consistent with previous reports.12,13Removal of
zinc(II) ions from complex 3 to give proligand 4 was also found to
be quantitative.
Experimental
General experimental apparatus used has been described
elsewhere.ll,12 High performance liquid chromatography was
conducted by using an acetonitrile/water gradient elution method and
unless otherwise stated a flow rate of 1.0 ml/min.ll
Diacetyl-bis( 4-N-methyl-3-thiosemicarbazonato)
zinc (II) [Zn(II)
ATSM] (1): Synthesised in accordance with a previously reported
procedure.13 Proligand 2 (1.50 g, 5.78 mmol) was reacted with
1.1 equiv. Zn(OAch-2H20 (1.40 g, 6.3 mmol) in ethanol (30 ml)
under reflux for 4 h. Complex 1 was isolated as a yellow powder
(1.57 g, 4.85 mm04 84%). Elemental analysis (%) for C8HI~6S2Zn
(323.77 g mol,l) Calcd C 29.7, H 4.4, N 26.0, S 19.8 and Zn 20.2;
foundC29.8,H4.4,N25.9,
S 19.5 andZn20.4.IHNMR(300MHz,
DMSO-~): 8/ppm 7.21 (2H, br, CH3NH); 2.82 (6H, d, CH3NH); 2.20
(6H, s, CH3C=N). MS (ES+): m/z (Calcd) 323.0104 (323.0091) = {M
+ W} 100%. AmaiDMF)/nm 431 (eIM'l cm,l 11266),312 (11978).
HPLC: ~ 10.40 min (Flow rate: 0.9 ml/min).
J.P.H. thanks the Engineering and Physical Sciences Research
Council (EPSRC) and Merton College (Oxford) for financial
support. We thank Prof. Jonathan R. Dilworth and Prof.
Jennifer C. Green for helpful discussions. We are indebted to
Dr Nick Rees, Mr Colin Sparrow and Mrs Maria Marshall for
technical support.
Diacetyl-bis(4-N-methyl-3-thiosemicarbazone)
H2ATSM
(2):
Proligand was synthesised in accordance with a previously reported
procedure.13 Proligand 2 was isolated as a white powder (1.88 g,
7.2 mmol, 76%). Elemental analysis (%) for C8HI~6S2 (260.38 g
mol,l) Calcd C 36.9, H 6.2, N 32.3 and S 24.6; found C 36.9, H 6.3,
N 32.1 and S 24.5. IH NMR (300 MHz, DMSO-~): 8/ppm 10.31 (2H,
br s, 2 x C=SNH); 8.46 (2H, br ill, 2 x CH3NH); 3.09 (6H, d, 3JHH
= 4.4 Hz, 2 x CH3NH); 2.29 (6H, s, 2 x CH3C=N). 13C{IH} NMR
(75.5 MHz, DMSO-~): 8/ppm 178.96 (2 x C=S), 148.52 (2 x C=N),
31.77 (2 x CH3NH), 12.25 (2 x CH3C=N). MS (ES+): m/z (Calcd)
261.0949 (261.0956) = {M + W} 100%. Amax(DMF)/nm344 (e/mol-I
dm3 em-I 36814),334 (37813) and 273 (12174). HPLC: ~ 10.31 min.
Received 22 July 2008; accepted 15 October 2008
Paper 08/0068 doi:l0.3184/030823408X380731
Published online: 10 December 2008
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9
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=
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130.10 (i-Ph); 128.56 (m-Ph); 127.47 (P-Ph);
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36.88 (CH3CH2NH); 14.60 (CH3CH2NH); 13.92 (CH3C=N); 13.78
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100%. Amax(DMSO)/nm 437 (elM' I cm,l 11328) and 319 (13900).
HPLC: ~ 15.91 min.
Diacetyl- 2-(4-N-ethyl-3 -thiosemicarbazone )-3 -(4-N-(I -phenyl-
prop- I (E)-enyl)-3-thiosemicarbazone) (4): Synthesised in accordance