Design of a radiopharmaceutical for the palliation of painful bone metastases: Rhenium-186-labeled bisphosphonate derivative

Article abstract of DOI:10.1002/jlcr.864

To develop a radiopharmaceutical for the palliation of painful bone metastases based on the concept of bifunctional radiopharmaceuticals, we designed a bisphosphonate derivative attached to a stable ¹⁸⁶Re- monoaminemonoamidedithiol (MAMA) chela

Full text of DOI:10.1002/jlcr.864

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 753–761.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.864
Research Article
Design of a radiopharmaceutical for the palliation
of painful bone metastases: rhenium-186-labeled
bisphosphonate derivative
Kazuma Ogawa1,2, Takahiro Mukai3, Yasushi Arano4, Hirofumi Hanaoka1,
Kazuyuki Hashimoto5, Hiroshi Nishimura2 and Hideo Saji1,*
1 Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical
Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
2 Radioisotope Laboratory, Kyoto Prefectural University of Medicine, Kamigyo-ku,
Kyoto 602-0841, Japan
3 Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of
Medicine, Kyoto University, Kyoto 606-8507, Japan
4 Department of Molecular Imaging and Radiotherapy, Graduate School of
Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan
5 Japan Atomic Energy Research Institute, Tokai-mura, Ibaraki 319-1195, Japan
Summary
To develop a radiopharmaceutical for the palliation of painful bone metastases based
on the concept of bifunctional radiopharmaceuticals, we designed a bisphosphonate
derivative attached to a stable 186Re-monoaminemonoamidedithiol (MAMA) chelate
(
186Re-MAMA-BP) to improve the instability of 186Re-HEDP. The precursor (Tr-
MAMA-BP) of 186Re-MAMA-BP was synthesized by coupling the carboxyl group of
the Tr-MAMA derivative with the amino group of the bisphosphonate derivative.
This 186Re-labeled compound was prepared by a ligand exchange reaction using
186Re-glucoheptonate with a radiochemical yield of 32.0 ꢀ 4.1%. In the incubation
study in buffered solution (pH 7.0), 186Re-MAMA-BP was more stable than 186Re-
HEDP. This suggests that 186Re-MAMA-BP is a potential radiopharmaceutical for
the palliation of painful bone metastases. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: bone metastases; palliation; bisphosphonate; rhenium-186; bifunctional
*Correspondence to: H. Saji, Department of Patho-Functional Bioanalysis, Graduate School of
Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: hsaji@pharm.
kyoto-u.ac.jp
Contract/grant sponsor: Grant-in-Aid for Young Scientists; contract/grant number: 13770500
Contract/grant sponsor: Grant-in-Aid for Young Scientists; contract/grant number: 15790665
Contract/grant sponsor: Exploratory Research; contract/grant number: 13877381
Copyright # 2004 John Wiley & Sons, Ltd.
Received 1 March 2004
Revised 15 June 2004
Accepted 16 June 2004

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K. OGAWA ET AL.
Introduction
Malignant tumors, especially breast and prostate carcinomas, frequently
metastasize to the bone.1 A prominent symptom caused by these metastases is
pain, which has a significant impact on the patients’ quality of life. Localized
radiation therapy is an effective modality in the treatment of bone pain,2
however, a common problem in cases of bone metastases is the development of
multiple sites of metastases, so internal radiotherapy using specifically
localized beta emitters is preferable. Recently, rhemium-186-1-hydroxyethy-
lidene-1,1-diphosphonate (186Re-HEDP), a polynuclear complex, has been
proposed for the palliation of pain resulting from the metastatic bone lesions
of tumors.3ꢁ5 However, this complex showed slow blood clearance and high
gastric uptake of radioactivity upon injection, leading to unnecessary radiation
in the patients. The cause of these findings has been reported to be the in vivo
instability of this 186Re complex leading to the generation of 186ReO4ꢁ.6,7 Thus,
development of a more stable 186Re-labeled compound than 186Re-HEDP for
the palliation of painful bone metastases has been desired. For this purpose,
based on the concept of bifunctional radiopharmaceuticals, we designed a
stable 186Re-labeled bisphosphonate derivative (186Re-MAMA-BP) ð1Þ by the
conjugation of the monoaminemonoamidedithiol (MAMA) moiety to a
bisphosphonate analogue (Figure 1) because MAMA derivatives form stable
chelates with rhenium.8,9 This paper describes the synthesis and stability of
186Re-MAMA-BP.
Results and discussion
Tr-MAMA-BP (8), the precursor of 186Re-MAMA-BP, was synthesized by
coupling the carboxyl group of a Tr-MAMA derivative (4) with the amino
group of a bisphosphonate derivative (6). Namely, bisphosphoric tetraethyl
ester (6) as a bisphosphonate analogue containing an amino group was
coupled with a Tr-MAMA derivative (4) in the presence of dicyclohexylcar-
bodiimide (DCC) in chloroform to yield the desired compound (7) in good
yield (65%). After the selective hydrolysis of bisphosphoric tetraethyl ester via
O
OH
O
O
P
OH
OH
H
N
S
N
S
N
O
O
Re
P
OH
Chelate Site
Bisphosphonate Frame
Figure 1. Chemical structure of 186Re-MAMA-BP (1)
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DESIGN OF A RADIOPHARAMACEUTICAL
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trimethylsilylbromide,10 Tr-MAMA-BP was obtained by reversed phase (RP)-
HPLC. Trityl groups of Tr-MAMA-BP were deprotected just before the
ligand exchange reaction by treatment with trifluoroacetic acid (TFA) and
triethylsilane.
MAMA-BP (9) contains a MAMA site as a ligand to form a stable complex
with 186Re. In addition, MAMA-BP also contains a bisphosphonate site that is
capable of chelating with 186Re. Accordingly, to obtain the desired complex,
the reaction condition where Re is chelated not with the bisphosphonate site
but with the MAMA site is required.
Since it has been reported that MAMA chelates with rhenium in oxidation
state (V) to form a stable oxorhenium(V) complex,8,11 186Re(V)-glucohepto-
nate was first prepared by reacting rhenium with glucoheptonate, and then a
ligand exchange reaction was performed between 186Re-glucoheptonate and
MAMA-BP to produce 186Re-MAMA-BP (1).
To be certain that 186Re was selectively bound to the MAMA site in
MAMA-BP by the transchelation reaction with 186Re-glucoheptonate, the
mixture containing MAMA and HEDP at equal concentrations was reacted
with 186Re-glucoheptonate. When this reaction mixture was analyzed by RP-
HPLC, almost all radioactivity was eluted in the fraction with the same
retention time as that of nonradioactive Re-MAMA on UV detection at
254 nm. 186Re-HEDP was, however, eluted in a shorter retention time than
Re-MAMA. These results suggest that 186Re is chelated with the MAMA site
when 186Re was bound to MAMA-BP via 186Re-glucoheptonate.
In the labeling reaction of 186Re-MAMA-BP, the same reaction conditions
were investigated. Since the labeling reaction of 186Re-MAMA-BP was difficult
to proceed at room temperature, we heated the labeling solution in boiling
water. After 5, 20 and 60 min of heating, the sample was drawn, and the
radioactivity was analyzed by RP-HPLC. The reactions proceeded equally on
heating for 5 and 20min, but the radiochemical yield decreased slightly after
60min of heating. Therefore, 10min was used as the reaction time.
Furthermore, to test slightly moderate conditions, the labeling solution was
reacted at 808C, and the radiochemical yield of 186Re-MAMA-BP was improved
from 21.3 ꢀ 4.5 to 32.0 ꢀ 4.1%. After purification by RP-HPLC, 186Re-
MAMA-BP showed a radiochemical purity of over 95% (96.4 ꢀ 1.4%).
In general, the radiochemical yield of 186Re-MAMA is very high (over
95%). However, the radiochemical yield of 186Re-MAMA-BP was not high
(32%). The reason for the low radiochemical yield of 186Re-MAMA-BP
compared to that of 186Re-MAMA is not clear, but it may be that the steric
hindrance occurred with the conjugation of the MAMA moiety to a
bisphosphonate analogue.
Figure 2 shows the stability of 186Re-MAMA-BP and 186Re-HEDP in
buffered solutions. After 24 h of incubation, 81.8 ꢀ 1.7% of 186Re-MAMA-
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K. OGAWA ET AL.
100
80
60
40
20
0
0
6
12
18
24
Incubation Time (h)
Figure 2. Stability of 186Re-MAMA-BP (closed circles) and 186Re-HEDP (open
circles) in buffered-solution
BP remained intact, compared to 24.8 ꢀ 0.2% in the case of 186Re-HEDP.
This indicates that the novel compound 186Re-MAMA-BP, based on the
concept of bifunctional radiopharmaceuticals, is more stable than 186Re-
HEDP.
In conclusion, 186Re-MAMA-BP showed much greater stability than 186Re-
HEDP. Although further investigation of its behavior in vivo is required, these
results indicate that 186Re-MAMA-BP is a potential radiopharmaceutical for
the palliation of painful bone metastases.
Experimental
General
Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a
Bruker AC-200 spectrometer (JEOL Ltd., Tokyo, Japan), and the chemical
shifts were reported in ppm downfield from an internal tetramethylsilane
standard. Fast atom bombardment mass spectra (FAB-MS) were obtained
with a JMS-HX/HX 110 A (JEOL Ltd.). 186Re was supplied by the Japan
Atomic Energy Research Institute (Tokai-mura, Japan) as perrhenate
(
186ReOꢁ4 ).12 Cellulose acetate electrophoresis (CAE, Separax-SP; Joko Co.
Ltd., Tokyo, Japan) was run with an electrostatic field of 0.8 mA/cm for
20 min in veronal buffer (I=0.06, pH 8.6). TLC analyses were performed with
silica plates (Merck Art 5553) with acetone as a developing solvent. Other
reagents were of reagent grade and used as received.
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DESIGN OF A RADIOPHARAMACEUTICAL
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Synthesis of 186Re-MAMA-BP (Scheme 1)
N-[2-(Tritylthio)ethyl]-2-[2-(tritylthio)ethylamino]acetamide (Tr-MAMA)
ð2Þ. Compound 2 was synthesized according to the published procedure.13
1H NMR (CDCl3): d 7.42–7.17 (overlapped m,30H), 3.06 (td,2H), 3.02 (s,2H),
2.45(t,2H), 2.38–2.31 (overlapped m,4H). FAB-MS calcd for C44H42N2OS2
(M+ H)+: m/z 679. Found: 679.
N-[2-(Tritylthio)ethylaminocarbonylmethyl]-N-[2-(tritylthio)ethyl]-4-amino-
butyric Acid ð4Þ. Compound 2 (3.30 g, 4.86 mmol) was dissolved in 50 ml of
N,N-dimethylformamide (DMF), and diisopropylethylamine (689 mg,
5.34 mmol) and ethyl 4-bromo-n-butyrate (1.04 g, 5.34 mmol) were added to
the solution. After the reaction mixture had been stirred at 908C for 3 h, the
solvent was removed in vacuo. The residue was dissolved in ethyl acetate, and
the solution was washed with brine. The organic layer was dried over
anhydrous CaSO4, and the solvent was removed in vacuo. The oily residue was
purified by flash chromatography on silica gel using ethyl acetate-hexane (1:1)
O
O
O
OEt
OH
NH
N
NH
N
NH
NH
(a)
(b)
O
O
S
S
S
S
S
S
Tr
Tr
Tr
Tr
Tr
Tr
2
3
4
OEt
OEt
O
O
P OEt
O
O
P OEt
(c)
O N
2
H N
2
P OEt
OEt
P OEt
OEt
6
5
O
O
OEt
OH
P OH
H
H
N
O P OEt
O
O
(d)
(e)
(g)
NH
N
N
NH
N
4
6
+
O
O
O P OEt
OEt
P OH
OH
S
Tr
S
Tr
S
S
Tr
Tr
8
7
O
O
OH
OH
H
N
H
N
O
P OH
O P OH
(f)
NH
N
N
N
S
O
O
O
186
Re
O
P OH
OH
O P OH
OH
SH HS
S
9
1
Scheme 1. Synthesis of 186Re-MAMA-BP
Reagents: (a) ethyl 4-bromo-n-butyrate, (i-Pr)2NEt; (b) NaOH; (c) Pd-C; (d)
DCC; (e) (CH3)3SiBr; (f) TFA, triethylsilane; (g) 186Re-glucoheptonate
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K. OGAWA ET AL.
as the eluent to provide compound 3 (2.03 g, 52.9%) as a yellow oil. 1H NMR
(CDCl3): d 7.41–7.15 (overlapped m,30H), 4.06 (q,2H), 3.01 (td,2H), 2.83
(s,2H), 2.38–2.12 (overlapped m,10H), 1.68–1.54 (overlapped m,2H), 1.20
(t,3H). FAB-MS calcd for C50H52N2O3S2 [M + H]+: m/z 793. Found: 793.
After hydrolysis of the ester bond, compound 4 (1.92 g, 98.0%) was
1
obtained as brown crystals. H NMR (CDCl3): d 7.40–7.14 (overlapped
m,30H), 2.99 (td,2H), 2.85 (s,2H), 2.38–2.23 (overlapped m,10H), 1.65–1.60
(overlapped m,2H). FAB-MS calcd for C48H48N2O3S2 (M+ H)+: m/z 765.
Found: 765.
Tetraethyl-3-nitropropylidene-1,1-bisphosphonate ð5Þ. Compound
5
was
synthesized according to the published procedure.14,15 1H NMR (CDCl3): d
4.71 (t,2H), 4.27–4.14 (overlapped m,8H), 2.68–2.40 (overlapped m,3H), 1.36
(t,12H). FAB-MS calcd for C10H22O6P2 (M+ H)+: m/z 362. Found: 362.
Tetraethyl-3-aminopropylidene-1,1-bisphosphonate ð6Þ. Compound 5 (100 mg,
0.28 mmol) was dissolved in a mixture of 1.13 ml of methanol and 0.03 ml of
water, and 70.0 mg of the catalyst (10% Pd on activated carbon) was added to
the solution. The mixture was hydrogenated at room temperature for 6 h. The
catalyst was removed by filtration, and the filtrate was evaporated in vacuo to
provide crude compound 6 as a pale yellow oil. Compound 6 was used in the
next reaction without further purification.
[1-Phosphono-3-[4-[[2-(tritylthio)ethyl]-[[2-(tritylthio)ethylcarbamoyl]methyl]
amino]-butyrylamino]propyl]phosphonic Acid Tetraethyl Ester ð7Þ. Compound 4
(206 mg, 0.27 mmol) and crude compound 6 (90.0 mg, 0.27 mmol) were dissolved
in chloroform. DCC (56.0 mg, 0.27 mmol) in 2 ml of chloroform was added
dropwise to the reaction mixture while the reaction temperature was maintained
below 08C. After 30 min of stirring below 08C, the reaction solution was stirred at
room temperature for 5 h. After filtration, the filtrate was evaporated in vacuo,
and the residue was purified by flash chromatography on silica gel using
chloroform-methanol (10:1) as the eluent to provide compound 7 (190 mg,
1
65.3%) as yellow crystals. H NMR (CDCl3): d 7.40–7.15 (overlapped m,30H),
4.23–4.11 (overlapped m,8H), 3.38 (td,2H), 3.01 (td,2H), 2.83 (s,2H), 2.38–2.02
(overlapped m,13H), 1.67–1.60 (overlapped m,2H), 1.33 (t,12H). FAB-MS calcd
for C59H73N3O8P2S2 (M+ H)+: m/z 1078. Found: 1078.
N2-[4-[(3,3-Diphosphonopropyl)amino]-4-oxobutyl]-N1,N2-bis[2-(tritylthio)ethyl]
glycin-amide (Tr-MAMA-BP) ð8Þ. Hydrolysis of the phosphonic esters of
compound 7 was carried out via trimethylsilylbromide. Compound 7 (100 mg,
92.8 mmol) was dissolved in 2 ml of chloroform, and trimethylsilylbromide
(116 mg, 0.76 mmol) was added to the solution. After 3 h of stirring at 508C,
the reaction solution was stirred at room temperature for 36 h. After the
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DESIGN OF A RADIOPHARAMACEUTICAL
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solvent was removed in vacuo, the residue was recovered in 2 ml of methanol
and stirred at room temperature for 60 min. After the solvent was removed in
vacuo, the residue was purified by RP-HPLC performed with a Cosmosil 5C18-
AR-300 column (10 ꢂ 150 mm; Nacalai Tesque, Kyoto, Japan) at a flow rate
of 4.7 ml/min with a gradient mobile phase from 95% A (0.1% aqueous TFA)
and 5% B (acetonitrile with 0.1% TFA) to100% B in 30 min. A fraction
containing compound 8 was collected, and the solvent was removed by
1
lyophilization to provide compound 8 (35.0 mg, 39.1%) as white crystals. H
NMR (CDCl3): d 7.14–7.37 (overlapped m,30H), 3.36 (m,2H), 3.08 (m,2H),
2.92 (s,2H), 2.84–2.60 (overlapped m,5H), 2.33–2.22 (overlapped m,6H), 2.01
(m,2H), 1.65 (m,2H). FAB-MS calcd for C51H57N3O8P2S2 (M+ H)+ : m/z
966. Found: 966.
[N-[2-[[3-(3,3-Diphosphonopropylcarbamoyl)propyl](2thioethyl)amino]acetyl]-
2-aminoethanethiolate] Oxorhenium(V) (186Re-MAMA-BP) ð1Þ. 186Re-MAMA-
BP ð1Þ was formed by transchelation form 186Re-glucoheptonate. Namely,
186ReOꢁ4 solution was added to stock freeze-dried powder of SnCl2 ꢃ 2H2O and
glucoheptonate (made from 1 g of glucoheptonate and 30 mg of SnCl2 ꢃ 2H2O in
20 ml of aqueous solution pH 8.6) to produce a final freeze-dried powder
concentration of about 125 mg/ml. After heating at 808C for 10 min and standing
at room temperature for 50 min, the formation of 186Re-glucoheptonate with a
radiochemical yield of over 90% (93.3 ꢀ 2.1%) was ascertained by TLC
(Rf=0). Trityl groups of Tr-MAMA-BP (8) (0.3 mg) were deprotected by
treatment with TFA and triethylsilane just before radiolabeling. After removal of
the solvent under a stream of N2, 0.2 ml of 0.2 M acetate buffer (pH 3.0) was
added to the residue. Then, 0.2 ml of 186Re-glucoheptonate solution was added to
this solution, and the reaction mixture was vigorously stirred and allowed to react
at 808C for 10 min. After the reaction mixture was allowed to cool to room
temperature, 186Re-MAMA-BP was purified by RP-HPLC performed with a
Cosmosil 5C18-AR-300 column (4.6 ꢂ 150 mm) at a flow rate of 1 ml/min with a
mixture of 0.2 M phosphate buffer (pH 6.0) and ethanol (80:20) containing
10 mM tetrabutylammoniumhydroxide.
Synthesis of [N-[2-[(2-Mercaptoethyl)amino]acetyl]-2-aminoethanethiolate]
rhenium(V) Oxide (Re-MAMA)
A modified procedure reported previously was employed with slight
modification.16 Compound 2 (100 mg, 0.15 mmol) was suspended in 2 ml of
ethanol. SnCl2 ꢃ 2H2O (31 mg, 0.14 mmol) in 0.5 ml of 0.1 N HCl and KReO4
(40 mg, 0.14 mmol) in 0.5 ml of 0.1 N HCl were added to this stirred
suspension, then this reaction mixture was refluxed at 1208C for 12 h. After
the solvent was removed in vacuo, the residue was purified by RP-HPLC
performed with a Cosmosil 5C18-AR-300 column (10 ꢂ 150 mm) at a flow rate
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K. OGAWA ET AL.
of 4.7 ml/min with a gradient mobile phase from 95% A (0.1% aqueous TFA)
and 5% B (acetonitrile with 0.1% TFA) to 100% B in 30 min. A fraction
containing Re-MAMA was collected, and the solvent was removed by
lyophilization to provide Re-MAMA (5.0 mg, 9.1%) as reddish violet crystals.
1H NMR (CDCl3): d 7.98 (m,1H), 4.03 (m,2H) 3.54 (m,2H), 3.20 (m,2H),
3.05 (m,2H), 2.86 (m,2H). FABMS calcd for C6H11N2O2 187ReS2 (Mꢁ H)ꢁ:
m/z 393. Found: 393, C6H11N2O2 185ReS2 (Mꢁ H)ꢁ: m/z 391. Found: 391.
Preparation of 186Re-HEDP
186Re-HEDP was prepared according to the published procedure,7 and used
after confirmation of its radiochemical purity by TLC (Rf=0) and CAE
(3.5 cm anode from the origin).
Analysis of reaction of 186Re-glucoheptonate with the mixture of MAMA and
HEDP
A mixture containing MAMA and HEDP at equal concentrations (15 mM)
was reacted with same volume of 186Re-glucoheptonate solution at room
temperature for 60 min. This reaction mixture was analyzed by RP-HPLC,
performed with a gradient mobile phase from 95% A (0.1% aqueous TFA)
and 5% B (acetonitrile with 0.1% TFA) to 100% B in 30 min at a flow rate of
1 ml/min.
In vitro stability
To evaluate the stability of 186Re complexes in buffered-solution, 186Re-labeled
compounds were diluted 5-fold with 0.1 M phosphate buffered (pH 7.0)
saturated with 95% O2/5% CO2, and the solutions were incubated at 378C for
24 h. After 1, 3, 6, and 24 h of incubation, the samples were drawn, and the
radioactivity was analyzed by RP-HPLC or TLC.
Conclusion
Based on the concept of bifunctional radiopharmaceuticals, we have
developed a novel rhenium-186-labeled bisphosphonate (186Re-MAMA-BP)
for the palliation of painful bone metastases. Precursor (8) was prepared by
the conjugation of MAMA derivative protected thiol groups with a bispho-
sphonate derivative. The rhenium-186-labeled compound was synthesized
from the precursor by simple reactions, followed by purification by RP-HPLC
in high radiochemical purity. 186Re-MAMA-BP showed the greater stability
than 186Re-HEDP when incubated in the phosphate buffer (pH 7.0). These
results suggest that the newly synthesized 186Re-MAMA-BP could become a
potential agent for the palliation of painful bone metastases. The biodistribu-
tion of 186Re-MAMA-BP will be reported elsewhere in the near future.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 753–761

DESIGN OF A RADIOPHARAMACEUTICAL
761
Acknowledgements
This work was supported in part by Grants-in-Aid for Young Scientists
(13770500 and 15790665) and Exploratory Research (13877381) from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.
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