An efficient chelator for complexation of thorium-227

Article abstract of DOI:10.1016/j.bmcl.2016.07.034

We present the synthesis and characterization of a highly efficient thorium chelator, derived from the octadentate hydroxypyridinone class of compounds. The chelator forms extremely stable complexes with fast formation rates in the presence of Th-227 (ambient temperature, 20?min). In addition, mouse biodistribution data are provided which indicate rapid hepatobiliary excretion route of the chelator which, together with low bone uptake, supports the stability of the complex in vivo. The carboxylic acid group may be readily activated for conjugation through the ?-amino groups of lysine residues in biomolecules such as antibodies. This chelator is a critical component of a new class of Targeted Thorium Conjugates (TTCs) currently under development in the field of oncology.

Full text of DOI:10.1016/j.bmcl.2016.07.034

Accepted Manuscript
Efficient Chelator for Complexation of Thorium-227
Thomas Ramdahl, Hanne T. Bonge-Hansen, Olav B. Ryan, Åsmund Larsen,
Gunnar Herstad, Marcel Sandberg, Roger M. Bjerke, Derek Grant, Ellen M.
Brevik, Alan S. Cuthbertson
PII:
S0960-894X(16)30747-8
DOI:
Reference:
http://dx.doi.org/10.1016/j.bmcl.2016.07.034
BMCL 24078
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 June 2016
14 July 2016
15 July 2016
Please cite this article as: Ramdahl, T., Bonge-Hansen, H.T., Ryan, O.B., Larsen, a., Herstad, G., Sandberg, M.,
Bjerke, R.M., Grant, D., Brevik, E.M., Cuthbertson, A.S., Efficient Chelator for Complexation of Thorium-227,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.034
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Efficient Chelator for Complexation of Thorium-227
Thomas Ramdahla, Hanne T. Bonge-Hansena, Olav B. Ryana, Åsmund Larsena, Gunnar Herstadb,
Marcel Sandbergb, Roger M. Bjerkea, Derek Granta, Ellen M. Brevika, Alan S. Cuthbertsona*
aThe Department of Thorium Research, Bayer AS, Drammensveien 288, 0283 Oslo, Norway
bSynthetica AS, Gaustadalléen 21, 0349 Oslo, Norway.
We present the synthesis and characterization of a highly efficient thorium chelator, derived from the octadentate
hydroxypyridinone class of compounds. The chelator forms extremely stable complexes with fast formation rates in the presence
of Th-227 (ambient temperature, 20 minutes). In addition, mouse biodistribution data are provided which indicate rapid
hepatobiliary excretion route of the chelator which, together with low bone uptake, supports the stability of the complex in vivo.
The carboxylic acid group may be readily activated for conjugation through the -amino groups of lysine residues in biomole-
cules such as antibodies. This chelator is a critical component of a new class of Targeted Thorium Conjugates (TTCs) currently
under development in the field of oncology.
The clinical development of targeted radioimmunotherapies (RIT) for the treatment of cancer utilizing the high linear energy
transfer (LET) of alpha-particles has, to a large extent, been limited by the availability of suitable radionuclides on a commercial
scale. More recently, with the approval in May 2013 of Xofigo® for the treatment of castration resistant prostate cancer (CRPC),1 a
commercial production line for the alpha-emitting radionuclide radium-223 (Ra-223) was established. However, although the
inherent bone-seeking characteristics of Ra-223 make it well suited for the targeting of bone metastases in CRPC, the paucity of
efficient chelator systems limits its use in radioimmunotherapy. By utilizing the same actinium-227 generators as used in the
manufacture of Ra-223,2 highly purified thorium-227 (Th-227) may be produced. Unlike Ra-223, Th-227 exists in the 4+ oxidation
state and forms stable complexes with chelators such as 1,4,7,10-tetra-azacycloododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA).3
However, in order to achieve sufficient labeling of DOTA-coupled antibodies, the complexation step must either be performed
as a two-step process 4 or directly at elevated temperatures.5 The harsh conditions used for direct labeling are not always com-
patible with the stability of complex biological macromolecules such as antibodies. In addition the slower complex formation
rates affect radiolabeling yield, efficiency and specific activity.6
Figure 1. Drawing of the Octadentate hydroxypyridinone thorium-227 complex 1
Corresponding Author
* alan.cuthbertson@bayer.com

Therefore, there remains a need for alternative chelators which complex thorium-227 in near quantitative yield at ambient
temperature in aqueous solutions. To this end we have developed an octadentate chelator utilizing the 3-hydroxy-N-methyl-2-
pyridinone moiety, abbreviated henceforth as Me-3,2-HOPO, reported previously to form extremely stable thorium complexes.7,8
The Me-3,2-HOPO groups are monoprotic acids that complex thorium-227 through the two oxygen atoms on each subunit.9
Figure 1 shows the structural drawing of the target octadentate Me-3,2-HOPO thorium-227 complex 1. We report herein on the
synthesis, labeling and in vivo biodistribution in mice and provide evidence of superiority of this new chelator compared to
DOTA for thorium-227 complexation.
The chelator comprises three key components. The Me-3,2-HOPO moiety, which complexes the thorium; a symmetrical poly-
amine scaffold to which the Me-3,2-HOPO moieties are coupled; and a carboxylic acid group facilitating conjugation to biomol-
ecules such as antibodies.
Scheme 1 shows the synthesis of the polyamine scaffold component. Compound 3 was isolated in 66% yield by the addition of
4-nitrobenzyl bromide to a mixture of dimethyl malonate/ NaH in tetrahydrofuran (THF). Diisobutylaluminium hydride
(DIBAL-H) reduction in THF was performed to generate diol 4 in 46 % yield. Care was taken to stop the reaction before comple-
tion to avoid reduction of the nitro group. Activation by mesylation of 4 with methane sulfonyl chloride and triethylamine in
dichloromethane (DCM) gave compound 7, the crude material after workup being used directly in the next step. The bis-Boc
protected triamine 6 was formed in 56% yield from diethylenetriamine 5, directly using N-Boc-imidazole. The fully protected
precursor was prepared in 44% yield by combining 6 and 7 in acetonitrile using N,N-diisopropylethylamine (DIPEA) as base.
Finally, the deprotected product 9 was isolated in 66% yield by addition of sulfuric acid to compound 8 in methanol.
Scheme 1. The synthesis route to the polyamine scaffold
Scheme 2 shows the synthesis of the activated Me-3,2-HOPO precursor. Construction of building block 11 poses the challenge
of correctly introducing the functional group in the 4-position of the dihydropyridine ring. One approach, previously reported
by Xu et al. relied on the carboxylation of 1-methyl-3-hydroxy-2-pyridinone at 5860 kPa/ CO2.10 We have investigated an alterna-
tive method employing condensation of 2-pyrrolidinone with diethyl oxalate which also yields the correctly substituted
tetrahydropyridine-4-carboxylate through a sequential ring-opening and closure under Claisen conditions.11 In the first step, the
reaction between 2-pyrrolidinone and diethyl oxalate gives compound 11 in 73% yield. Subsequent aromatization using Pd/C has
been described previously and results in a 75% yield of 12.12 N-Methylation was then performed with an excess of methyl iodide
and potassium carbonate. This reaction resulted in the formation of the methyl ether as well as partial trans-esterification. The
O-methyl group was removed using BBr3 in dichloromethane (DCM) and following quenching and isolation by filtration 14 was
treated with benzyl bromide furnishing the desired benzyl ether 15. Further transesterification occurs in this reaction; the com-
bined yield of all three esters being 67%. Ester hydrolysis was then carried out in methanol/water with NaOH, giving carboxylic
acid 16 in 89% yield. Finally conversion of the carboxylic acid with 2-thiazoline-2-thiol with N,N’-dicyclohexylcarbodiimide and

4-dimethylaminopyridine was performed in dichloromethane yielding the thiazolidino-activated and benzyl protected Me-3,2-
HOPO building block 17 in 87% yield.
Scheme 2. The synthesis route to the activated Me-3,2-HOPO precursor
Scheme 3 details the synthesis of the octadentate Me-3,2-HOPO chelator compound 21. By simply stirring compounds 9 and 17
in DCM for 16-20 hours the protected chelate 18 was formed in 68% yield. Reduction of the nitro group to the amine 19 with
iron/NH4Cl, followed by removal of the benzyl protecting groups with BBr3 gave the desired product 20 in 90% yield. In the final
step, compound 21 was formed by reaction with succinic anhydride as shown in scheme 3. On removal of the benzyl protecting
groups care was taken to avoid contamination of the chelate system with unwanted metal ions. Both compounds 20 and 21,
complex a variety of metals with oxidation states of +2, +3 and +4 and in particular show high affinity for ferric ions. Complex
formation and characterization was confirmed by LC-high resolution mass spectrometry using non-radioactive thorium-232 as
surrogate. The full details of the synthesis and characterization steps are given in the supporting information.
Conjugation of compound 21 to monoclonal antibodies is described in detail elsewhere. 13 However, the chemistry is robust and
reproducible. Briefly, the in situ pre-activation of compound 21 is performed in 4-morpholinoethanesulfonic acid (MES) buffer
pH 5.5 in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. 14 The activated chelator
is then added to a solution of the antibody in phosphate-buffered saline (PBS) at pH 7.4 with gentle mixing for 30 minutes be-
fore purification of the conjugate.
In this study our aim was to investigate the labeling of compound 21 with thorium,-227 (synthesis of complex 1) as well as the
pharmacokinetics of complex 1 in mice. In a typical labeling experiment 19 compound 21 is added directly to a solution of thori-
um and the complexation allowed to proceed for 30 minutes. Complex 1 as the test item for injection had a final chelator con-
centration of 0.0125 mg/ mL (target; 0.0125 mg/ mL), specific activity of 14082 kBq/ mg (target 14000 kBq/ mg, and thorium con-
centration of 188 kBq/ mL (target 175 kBq/ mL). An aliquot of the chelate complex was analysed by iTLC, the strips were divided
into two halves and counted separately in a high performance germanium detector. Free Th-227 is known to migrate with the
solvent front while chelated Th-227 remains at the application point. In this example > 96% of the measured activity remained at
the application point.

Scheme 3. The synthesis route to the octatendate Me-3,2-HOPO thorium chelator, compound 21
The blood clearance and tissue distribution of complex 1 in female C57Bl6 mice was studied (Table 1). Animals were adminis-
tered complex 1 as a single slow bolus injection via the tail vein. The dose volume was 5 mL/kg (0.1 mL/20 g mouse), the target
dose of complex 1 was 0.05 mg/kg, and for the radioactivity, 700 kBq/ kg thorium-227. Animals were sacrificed at the following
post-treatment time-points; 5 minutes, 1 and 4 hours and on days 1, 3 and 7. Samples of blood and tissues were collected and the
radioactivity measured on a high performance germanium detector. Activity was decay corrected to give the activity at time of
sampling/euthanasia.
Complex 1 rapidly cleared from the blood, with t½ alpha approximately 0.4 to 1 hour, and t½ beta in the range of 4.9 to 6.4
hours, with almost complete elimination from the blood at 24 hours. At the early time points the presence of complex 1 was
observed mainly in the liver and small intestine with measured doses of 7.2 % and 14. 4 % ID/g respectively after only 5 minutes.
After 4 hours the large intestine had a measured dose of 48 % ID/ g, indicating that the route of excretion was predominant
biliary, consistent with the hydrophobic nature of complex 1. Although our ultimate aim is to develop antibody-based targeted
thorium conjugates with this chelator system, it is important to know the fate of any free complex 1 formed as a result of meta-
bolic cleavage of the chelate from the antibody in vivo. We anticipate the rapid excretion of free labeled chelate through the liver
will minimize the potential off-target toxicity of this potential metabolite.
It has been previously observed by ourselves and others that free thorium-227 has bone-seeking properties, with the highest
uptake rates in the skeleton. Washiyama et al. demonstrated that following injection of thorium-227 citrate in mice 28.4 and 19.4
%ID/g was measured in the femur after 1 and 7 days respectively.15 In this study we hypothesized that any instability of the com-
plex in vivo would be reflected in high bone uptake values. The finding on days 1, 3 and 7 that femur uptake was measured at 1.8,
1.9 and 1.4 % ID/g, respectively, consistent with in vivo stability of the complex 1.
In conclusion, the discovery of new chelator systems that efficiently complex Th-227 with sufficient in vivo stability is a prereq-
uisite for the development of antibody targeted alpha-pharmaceuticals. The optimal chelator characteristics are governed by the
long half-life of Th-227 (18.7 days) coupled to the long biological half-life of the antibody in vivo. The high Relative Biological
Effectiveness of the alpha particle potentially allows for therapeutic efficacy at lower radiation doses compared to beta-emitters
16,17 as the decay of Th-227 releases in total 5 alpha-particles with a total alpha energy of 32.5 MeV. 18 We provide evidence in this
report that compound 21 is an efficient chelator of Th-227 and is a superior chelator to DOTA as it requires no heating to effect
efficient labeling. In addition the octadentate Me-3,2-HOPO, compound 21, demonstrates rapid complexation, which is a key
requirement for the future commercial development of thorium-227 based alpha-pharmaceuticals.

Table 1. Organ distribution of complex 1 as % injected dose per gram (%ID/g) post injection
Th-227 (%ID/g)
Time
5 min.
Average
(n = 3)
1 hr.
4 hr.
Day 1
Average
(n = 3)
Day 3
Average
(n = 3)
Day 7
Average
(n = 3)
Average
(n = 3)
1.1
Average
(n = 3)
0.2
Organ
SD
SD
SD
SD
SD
SD
3.2
0.9
2.4
0.5
0.06
0.4
0.1
0.1
0.02
0.00
0.02
0.20
0.06
0.38
0.06
0.02
0.01
0.21
0.02
0.60
0.00
0.01
0.01
0.06
0.09
0.08
0.00
0.00
0.01
0.61
0.00
0.53
0.00
0.00
0.05
0.17
0.16
0.29
0.01
0.01
0.02
0.53
0.03
0.72
Blood
Lung
Liver
3.9
0.7
0.2
0.10
0.08
0.12
7.2
4.2
1.9
1.3
0.4
1.08
0.29
2.26
0.22
0.10
0.08
1.81
0.14
0.92
0.91
0.33
1.66
0.04
0.04
0.07
1.94
0.13
0.31
0.83
0.39
1.37
0.05
0.04
0.09
1.43
0.12
1.39
1.28
3.89
0.97
14.4
1.61
2.17
1.08
49.9
0.57
2.11
0.60
12.2
0.72
1.47
0.26
45.4
0.37
1.99
12.0
37.8
0.32
1.40
0.84
25.5
0.06
0.3
0.13
1.73
48.1
6.83
0.14
0.33
0.36
16.5
0.10
1.04
19.4
2.11
0.12
0.15
0.24
1.98
Spleen
Kidney
Large Intestine + contents
Small Intestine + contents
Heart
Femur
Residual carcass
Tail
19.9
14.4
0.03
0.35
0.26
24.4
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at
References and Notes
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(19) Chelator labeling protocol: two stock solutions were prepared: Solution 1, comprising 0.5 mg/mL of compound 21 in 30
mM citrate buffer containing 70 mM NaCl, 2 mM EDTA, 0.5 mg/ mL PABA and 5% DMSO, pH 5.5; solution 2 was identi-
cal to solution 1, without the chelate compound 21 and DMSO. A 113 L aliquot of chelator stock solution 1 was transferred
to a vial containing 792 kBq of thorium-227 (2.1 L in 0.05 M HCl) and the volume adjusted to 250 L by addition of stock
solution 2. The labeling reaction, forming complex 1 was allowed to proceed for 30 minutes at ambient temperature be-
fore final dilution to a volume of 3.8 mL with stock solution 2. A 10 L aliquot of the chelate complex was analysed by
iTLC using iTLC-SG chromatography paper eluted with the mobile phase 0.1 M citric acid/ citrate buffer in 0.9% NaCl, pH
5.5.

Graphical Abstract
Efficient Chelator for Complexation of Thorium-227
Molecular model
Keywords: Chelator, Thorium-227, Th-227, Targeted Thorium Conjugates,TTCs, 3,2-
hydroxypyridinone, Me-3,2-HOPO
6