Studies on the synthesis and biological properties of non-carrier-added [125I and 131I]-labeled arylalkylidenebisphosphonates: Potent bone-seekers for diagnosis and therapy of malignant osseous lesions

Article abstract of DOI:10.1021/jm021107v

Arylalkylidenebisphosphonates labeled with nca [¹²⁵I or ¹³¹I] have been synthesized and their biological function investigated. The label was attached to the aromatic group in high yield and under mild conditions by means of iododesi

Full text of DOI:10.1021/jm021107v

J . Med. Chem. 2003, 46, 3021-3032
3021
Stu d ies on th e Syn th esis a n d Biologica l P r op er ties of Non -Ca r r ier -Ad d ed
[
125I a n d 131I]-La beled Ar yla lk ylid en ebisp h osp h on a tes: P oten t Bon e-Seek er s for
Dia gn osis a n d Th er a p y of Ma lign a n t Osseou s Lesion s
Erik Årstad,† Per Hoff,*,† Lars Skattebøl,† Arne Skretting,‡ and Knut Breistøl‡
Department of Chemistry, University of Oslo, Norway, Department of Nuclear Medicine, Norwegian Radium Hospital,
Montebello, Norway
Received December 9, 2002
Arylalkylidenebisphosphonates labeled with nca [125I or 131I] have been synthesized and their
biological function investigated. The label was attached to the aromatic group in high yield
and under mild conditions by means of iododesilylation. The bone affinities of the radioactive
compounds were investigated in normal Balb/C mice. The compound 1-hydroxy(m-iodo[125,131I]-
phenylethylidene)-1,1-bisphosphonate was found to possess superior bone affinity compared
to others, and its in vivo deiodination was insignificant. The uptake in femur 24h after injection
was 850 ( 265% and 986 ( 118% of injected dose per gram tissue times gram body weight in
mice and rats, respectively. The therapeutic potential of the compound was investigated in
two tumor models in athymic (nude) rats, one model for mixed lytic/sclerotic metastatic bone-
lesions originating from breast cancer and the other model simulating osseous osteosarcoma.
The effects in these models compare favorably to those observed for established treatment
modalities. The experiments demonstrate that radioiodinated bisphosphonates may have a
potential for diagnosis and therapy of malignant osseous lesions.
Ch a r t 1
In tr od u ction
The treatment of skeletal metastases is a serious
problem in practical clinical oncology as the majority
of patients with advanced carcinomas of the breast,
prostate, or lung suffer from the condition.1 The prog-
nosis of patients with bone metastases varies from only
a few months to some years, and the number of patients
achieving complete cure is negligible.2 At present, the
current clinical practice for treatment includes external
radiotherapy, hormone therapy, and chemotherapy.
Severe side effects often limit the use of these methods.
Consequently, there is a strong need for improved
therapeutic methods to slow down tumor progression
and alleviate pain. Bone-seeking radiopharmaceuticals
are an attractive alternative to external beam irradia-
tion,3 because these compounds tend to concentrate with
preference to areas of tumor growth.4 As a result,
targeted radiotherapy may spare healthy tissue from
damage and thereby provide an opportunity to increase
the radiation dose to tumor.
So far, the US Food and Drug Administration has
approved only three bone-seeking radiopharmaceuticals,
[32P] sodium orthophosphate, [89Sr] strontium chloride,
and [153Sm] samarium EDTMP (1).5 For many years
[32P] sodium orthophosphate was the most widely used
compound, but because of severe side effects5 it has been
replaced by [89Sr] strontium chloride (Metastron). Sr2+
is preferentially taken up at sites of new bone formation,
and it provides pain relief in up to 80% of the patients,6,7
whereas [153Sm] samarium EDTMP has shown potential
for treatment of osseous osteosarcoma.8,9 The limiting
factor in the use of either [32P] sodium orthophosphate
or [89Sr] strontium chloride is bone marrow depression,
probably caused by penetration into the bone marrow
space of the highly energetic â-radiation. Looking for
more favorable decay modes, the most successful ap-
proach has been the use of metal complexes of radio-
nuclides decaying by emission of medium- to low-energy
â-particles or conversion electrons. [153Sm] samarium
EDTMP(1), [117mSn] tin DTPA (2), and [186Re] rhenium
HEDP(3) are examples of this category.10-12 More-
over, bisphosphonates incorporating 131I such as com-
pounds 5 and 6 (Chart 1) have also shown considerable
promise.
* To whom correspondence should be addressed: P. H., Department
of Chemistry, University of Oslo, P. O. 1033 Blindern, N-0315 Oslo,
Norway. Tel: +4722855484, Fax: +4722855477. E-mail: Per.Hoff@
kjemi.uio.no.
† Department of Chemistry, University of Oslo.
‡ Department of Medical Physics and Technology and Department
of Nuclear Medicine [A.S.]; Department of Tumor Biology [K.B.], The
Norwegian Radium Hospital, N-0310 Oslo, Norway.
10.1021/jm021107v CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/30/2003

3022 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Årstad et al.
Sch em e 1a
a
Reagents and conditions: i, NBS, AIBN, PhCl, 80-90 °C; ii, LiCH2P(O)(OEt)2, THF, -10 °C; iii, LDA, CIP(O)(OEt)2, THF, -78 °C; iv,
TMSBr, 20 °C; v, EtOH, H2O; vi, nca NaI, AcOH, TFA, NCS; vii, ICl, AcOH, 20 °C; viii, (a) TMSBr, 20 °C, (b) EtOH, H2O.
131I is extensively used for treatment of thyroid
cancer.13 The past decade has also seen an increased
use of 131I in radioimmunotherapy (RIT) because of its
favorable chemical properties, low cost, and emission
of low energy â-particles. In particular, the treatment
of non-Hodgkins lymphoma patients has been a suc-
cess.14 For similar reasons the incorporation of 131I into
a bone-seeking compound, e.g., a bisphosphonate de-
rivative is appealing for the treatment of bone-related
cancer.
Eisenhut was first to report the potential use of 131I-
labeled bisphosphonates for palliative treatment of
bone-related cancer.15,16 In these studies, several ben-
zylidenebisphosphonates (5, Chart 1) were synthesized
and evaluated. The bone affinity and selectivity of the
compounds were found to be comparable to that of other
bone-seeking radiopharmaceuticals; however, because
of their low in vivo stability these compounds have not
been used clinically. Recently, the synthesis of several
radiohalogenated bisphosphonates 6 (Chart 1) with
improved bone affinity as well as stability was re-
ported.17,18
our intention to optimize the molecular structure in
order to improve the bone affinity.
Ch em istr y
It is well established that bisphosphonates accumu-
late in bone structures. A benzene ring was included in
the molecule because we envisioned ways of combining
it with radioactive iodine through a halodesilylation
reaction. This approach required m-trimethylsilylphe-
nylethylidene-1,1-bisphosphonic acid (9), and the route
to this compound and the iodinated derivatives 10 is
outlined in Scheme 1. m-Chlorotoluene was transformed
to the Grignard reagent in THF, and the reaction with
trimethylchlorosilane gave the silane 11 in 92% yield.
This was converted to the bromide 12 in 99% yield using
NBS and 2,2′-azobis(isobutyronitrile) in chlorobenzene
as solvent. Treatment of the bromide with the lithium
salt of diethyl methylphosphonate afforded diethyl
m-trimethylsilylphenylethylphosphonate (13) in 53%
yield. The phosphonate was then converted to the
corresponding lithium salt using 2 equiv of LDA, and
the subsequent reaction with diethyl chlorophosphate
gave the tetraethyl bisphosphonate 14 in 87% yield.
Finally, hydrolysis of this compound was achieved in
excellent overall yield by transesterification to the
corresponding tetra (trimethylsilyl) ester 15, followed
by addition of aqueous ethanol.19 The resulting bispho-
sphonic acid 9 was neutralized with sodium hydroxide
and collected as the corresponding disodium salt, which
The present study was initiated in order to investigate
further the potential use of bisphosphonates for the
treatment of bone-related cancer, and herein we report
the syntheses and in vivo biological testing of some novel
non-carrier-added (nca) [125I]- and [131I]-labeled deriva-
tives of this class of compounds. Furthermore, it was

[
125I and 131I]-Labeled Arylalkylidenebisphosphonates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3023
Sch em e 2a
a
Reagents and conditions: i, (a) NaOH, PhSH, MeOH, (b) Oxone; ii, LDA, BrCH2CH2P(O)(OEt)2, THF, -78 °C; iii, Na(Hg), MeOH, 0
°C; iv, LDA, CIP(O)(OEt)2, THF, -78 °C; v, (a) TMSBr, 20 °C, (b) EtOH, H2O; vi, nca NaI, AcOH, TFA, NCS; vii, ICl, AcOH, 20 °C; viii,
HCl, ∆.
was subjected to radiohalogenation using commercially
available non-carrier-added [125I]- and the [131I]- sodium
iodide. A modification of the oxidative deiodosilylation
reaction by Vaidyanathan and Zalutsky20 was used
(NCS, 9:1 TFA:AcOH, 5 min, room temperature) result-
ing in a radiochemical yield of >95%. Interestingly,
labeling could also be carried out in a mixture of acetic
acid/urea/water in 70-86% radiochemical yields. The
product of the radiolabeling was identified by compari-
son of the HPLC retention time with that of the
corresponding nonradioactive iodide. HPLC was also
used to purify the products (see Experimental Section).
Thus, using this radiolabeling procedure the [131I]-iodide
10a and the [125I]-iodide 10b were obtained. Further-
more, the trimethylsilyl derivative 14 was treated with
ICl to give the iodo derivative 16. Transesterification
of this compound resulted in the tetrakis(trimethylsilyl)
ester, which upon hydrolysis yielded the iodide 10c.
The analogue 3-(m-trimethylsilylphenyl)propylidene-
1,1-bisphosphonic acid (17) and its iodinated derivatives
18 were prepared as outlined in Scheme 2. The succes-
sive treatment of the bromide 12 with thiophenol and
oxone afforded the sulfone 19 in 67% overall yield. Base-
induced alkylation of the sulfone with diethyl bromo-
ethylphosphonate furnished the phosphonate 20 in 66%
yield. Reductive removal of the benzenesulfonyl group
with a large excess of sodium amalgam (10 equiv) in
methanol provided the phosphonate 21 in excellent
yield, ester hydrolysis being avoided by quenching with
saturated aqueous ammonium chloride. The ester was
then transesterified and hydrolyzed as described for
compound 14, affording the bisphosphonic acid 17,
which was neutralized and collected as the correspond-
ing disodium salt. The disodium salt of 17 was subjected
to radiohalogenation, as described for the analogue 9,
yielding the [131I]-iodide 18a and the [125I]-iodide 18b.
Reaction of the 1-hydroxy(trimethylsilylphenyl)-bispho-
sphonate 22 with ICl in acetic acid afforded the iodo
ester 23, which was hydrolyzed to the acid 18c by
refluxing in concentrated hydrochloric acid.
The syntheses of 1-hydroxy(-trimethylsilylphenyl)-
ethylidene-1,1-bisphosphonic acid (24) and the corre-
sponding iodo derivatives 25 are outlined in Scheme 3.
The bromide 12 was converted to m-trimethylsilylbenzyl
cyanide (26) with potassium cyanide under phase
transfer conditions.21 Hydrolysis gave the carboxylic
acid 27, which was converted with thionyl chloride to
the acid chloride in good overall yield. Ultrasound
assisted reaction of the acid chloride with trimethyl
phosphite in THF (0.5 h at 0 °C) proceeded cleanly in
close to quantitative yield to give the phosphonate 28,
as a semisolid product. The compound is present as the
enol, and the assignment of the E-configuration is based
3
1
on the J
of 13 Hz in the H NMR spectrum.22 The
P-H
subsequent transformation of 28 to the hydroxybispho-
sphonate 29 was hampered by the phosphonate-
phosphate rearrangement, which is both acid- and base-
catalyzed.23 Nicholson and Vaughn reported good yields
of tetraalkyl hydroxybisphosphonates from the base-
catalyzed condensation of dialkyl phosphites with di-

3024 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Årstad et al.
Sch em e 3a
a
Reagents and conditions: i, KCN, Bu3N; ii, NaOH, MeOH, ∆; iii, P(OMe)3, THF, ultrasound; iv, HP(O)(OMe)2, Bu2NH; v, (a) TMSBr,
20 °C, (b) EtOH, H2O; vi, nca NaI, AcOH, TFA, NCS; vii, ICl, AcOH, 20 °C; viii, HCl, ∆.
alkyl acylphosphonates when ether was used as sol-
vent;24 the starting materials are soluble in ether while
the hydroxybisphosphonates precipitate during the
reaction, thereby reducing the degree of rearrangement.
Using ether alone as solvent the hydroxybisphosphonate
29 was obtained in a disappointing 5% yield, but
addition of hexane as cosolvent caused precipitation of
the bisphosphonate, and the yield of 29 increased to
65%. The latter was transesterified and hydrolyzed as
described for compound 14, affording the bisphosphonic
acid 24, which was neutralized and isolated as the
corresponding disodium salt. The disodium salt of 24
was subjected to radiohalogenation, as described for 9,
yielding the [131I]-iodide 25a and the [125I]-iodide 25b.
Reaction of the bisphosphonate 29 with ICl in acetic acid
afforded the iodo ester 30, which was hydrolyzed to the
acid 25c by refluxing in concentrated hydrochloric acid.
We also wanted to prepare 1-amino-(m-trimethylsi-
lylphenyl)ethylidene-1,1-bisphosphonic acid (31), as a
precursor of the corresponding radioactive iodides.
Several routes were tried without success and the
difficulties encountered made us change the choice of
target compound to N-(m-trimethylsilylphenylethyl)-
aminomethylenebisphosphonic acid (32), and its syn-
thesis is depicted in Scheme 4. Treatment of the cyanide
26 with sodium borohydride and zirconium tetrachloride
in THF gave the amine 33 in 50% yield,25 which was
converted with triethyl orthoformate and 2 equiv of
diethyl phosphite at 150 °C to the aminobisphosphonate
ester 34 in 72% yield. Transesterification followed by
hydrolysis provided the bisphosphonic acid 32, which
was isolated as the disodium salt. The disodium salt of
32 was subjected to radiohalogenation as described
above, yielding the [131I]-iodide 36a . However, with
respect to yields, it was advantageous in this case to
convert the ester 34 to the [131I]-iodide 35a , which was
then transesterified and hydrolyzed to the acid 36a .
Furthermore, reaction of the ester 34 with ICl in acetic
acid gave the iodide 35b, which was hydrolyzed to the
acid 36b by refluxing in concentrated hydrochloric acid.
Biology
Biod istr ibu tion in Mice. The tissue distribution in
mice of 10a , 18a , 25a , and 36a is shown in Table 1.
Since radioiodine is known to accumulate in the thyroid,
high concentration in this organ is indicative of deha-
logenation in vivo. Accordingly, tissue samples contain-
ing the thyroid were systematically collected and their
radioactivity levels measured. Administration of the
labeled bisphosphonates resulted in a very low ac-
cumulation in the thyroid and in other soft tissue,
indicating a high stability in vivo. The bisphosphonate
10a accumulated fast in bone with high preference. The
blood clearance was rapid, and the accumulation in soft
tissue was generally low, except for the kidneys. How-
ever, the compound was efficiently excreted from this
organ, and 24 h after administration the amount of
compound present in the kidneys was low as well. There
was no significant leakage of the compound from bone
tissues, and consequently the resulting femur to soft
tissue ratio remained high (>14 after 24 h). The
propylidenebisphosphonate 18a exhibited a similar bone
affinity as that of 10a , and the accumulation in soft
tissue was also very low for this compound. A superior
bone affinity was recorded for the hydroxybisphospho-
nate 25a with an accumulation twice as high as that of
10a ; maximum absorption was reached after 2 h and
remained very high after 24 h. The activity in blood was
initially high, but had declined 2h after injection. The
accumulation in kidney was initially high as well, but
clearance from this organ was fairly rapid. A somewhat
lower accumulation was found in the liver, and it was
negligible in other soft tissue. The tissue distribution
of 25a in rats for a 24 h period is shown in Table 2. The
high selectivity and superior bone affinity previously
found for this compound, was overall confirmed, with a
femur to soft tissue ratio of >45 after 24 h after
injection. Surprisingly, the aminomethylenebisphospho-
nate 36a appeared to lack bone-seeking properties.
Moreover, that compound had a rather high accumula-

[
125I and 131I]-Labeled Arylalkylidenebisphosphonates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3025
Sch em e 4a
a
Reagents and conditions: i, NaBH4, ZrCl4, THF; ii, CH(OEt)3, HP(O)(OEt)2, 150 °C; iii, (a) TMSBr 20 °C, (b) EtOH, H2O; vi, nca NaI,
AcOH, TFA, NCS; v, ICl, AcOH, 20 °C; vi, HCl, ∆.
tion in soft tissue. Because of the superior bone affinity
of the bisphosphonate 25a , and its high selectivity, this
compound was chosen for further studies.
that 65% of the administered radiopharmaceutical is
permanently bound to bone surfaces, 10 percent of it in
trabecular bone, where it remains until an insignificant
amount of 131I is left. This assumption is supported by
the data of Table 1, even though the uncertainties are
large. Furthermore, it is a general observation that
bisphosphonates bind to bone so tightly that the binding
can be considered an irreversible process. This observa-
tion is corroborated by clinical experience from the use
of these compounds in a large number of patients.27
Additionally, deiodination of the compound when at-
tached to bone is not a very likely process. First, we
observed no enhanced enrichment of radioidine in the
thyroid region, showing that there is no rapid deiodi-
nation of the compound in rats. Second, long-term
deiodination is even less likely, since bisphosphonates
do not only deposit on bone surfaces, but are also
incorporated into the bone matrix as it develops, making
the compound out of reach from deiodinase enzymes.
An experimental determination of the biological half-
life of the compound in humans and possible enrichment
of radioiodine in nontarget organs belongs to a future
phase I evaluation.
P h a r m a cok in etics in Mice. After intravenous in-
jection of 25a in mice, dynamic studies were performed
with a gamma camera equipped with a 6 mm pinhole
collimator. The mice were anaesthetized, and their legs
and bodies fixed so that the field of view covered the
entire animal as seen from the posterior direction. A
total of 90 images were obtained during 2h, from which
the content in the kidneys, bladder, and the whole body
was derived. The analysis showed that, as a mean value,
27% of the injected activity had accumulated in the
bladder after 2 h. Furthermore, accumulation in the
kidneys reached a maximum 45 min postinjection,
amounting to 7.5% of the injected dose at this point of
time. From then on the effective halflife in the kidneys
was 50 min. The bladder curve was still increasing after
2h. By extrapolating the kidney curves to infinity and
assuming that the excreted urine ends up in the
bladder, it was estimated that 35% of the initial amount
of the radiopharmaceutical left the body. Hence, 65%
remained in the body, mainly in the skeleton.
The pharmacokinetics of polyphosphonates in differ-
ent species, varying from rodents to humans, are known
to relate to weight by the expression:26 corr factor )
(mass man/mass animal)0.33. In our case, this would
mean that the biological processes of uptake and excre-
tion run 15.1 times slower in humans as compared with
mice. For the estimation of the cumulative activity in
the kidneys, it turns out that the time scale is unim-
portant, given that the same amount of radioactivity
passes through this organ and the excretion curve is
exponential.
Because of the relatively long half-life of 131I, the
uptake phase was neglected in the calculation of resi-
dence times. The mouse kidney and bladder curves,
extrapolated to infinity and taking into account the
differences in time scales between man and mice,28 were
used to estimate the residence times in these organs.
Absorbed doses were calculated with the MIRDOSE 3.1
program, assuming an adult patient. To facilitate
comparison with dose estimates for other radiopharma-
ceuticals, values (Table 3) were expressed in both mGy/
MBq and rad/mCi.
Dose Estim a tes. To get an indication of the absorbed
radiation dose in different organs in humans, the
measured pharmacokinetics of 25a in mice were ex-
trapolated to humans as follows: It was thus assumed
Su r viva l Stu d ies. The hydroxybisphosphonate 25a
was evaluated in two antitumor efficacy studies in
models with human breast cancer cells (MT-1) and

3026 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Årstad et al.
Ta ble 1. Tissue Distributions of nca 131I-Labeled Bisphosphonates in Micea
organ
femur
time postinjection (h)
10a
18a
25a
36a
0.5
2
5
24
0.5
2
18.80 ( 0.51
21.16 ( 3.85
10.00 ( 7.81
15.25 ( 4.89
10.28 ( 2.58
7.64 ( 3.53
6.16 ( 4.82
7.13 ( 2.73
2.22 ( 0.31
0.31 ( 0.25
0.11 ( 0.09
0.03 ( 0.01
0.90 ( 0.15
0.26 ( 0.08
0.07 ( 0.05
0.04 ( 0.02
1.00 ( 0.12
0.35 ( 0.05
0.10 ( 0.08
0.06 ( 0.03
2.98 ( 0.65
2.14 ( 0.11
0.94 ( 0.70
0.23 ( 0.02
8.23 ( 6.39
3.68 ( 2.80
2.26 ( 1.64
1.06 ( 0.10
0.35 ( 0.26
0.26 ( 0.07
0.12 ( 0.09
0.10 ( 0.03
1.14 ( 0.88
1.12 ( 0.50
0.23 ( 0.18
0.05 ( 0.03
0.37 ( 0.29
0.28 ( 0.05
0.24 ( 0.17
0.28 ( 0.30
0.21 ( 0.09
0.14 ( 0.05
0.06 ( 0.01
0.02 ( 0.01
45.03 ( 6.75
45.54 ( 4.10
30.30 ( 8.90
37.79 ( 11.78
24.61 ( 3.85
26.49 ( 0.61
19.14 ( 8.61
21.34 ( 5.62
15.92(12.75
1.36 ( 0.25
0.82 ( 0.19
0.09 ( 0.03
3.35 ( 0.41
0.82 ( 0.21
0.40 ( 0.17
0.18 ( 0.07
5.41 ( 1.67
1.20 ( 0.48
0.61 ( 0.05
0.54 ( 0.36
3.88 ( 0.49
3.36 ( 0.67
2.51 ( 0.07
2.27 ( 0.69
18.64 ( 1.78
13.30 ( 1.70
7.17 ( 0.25
5.16 ( 2.00
2.02 ( 0.357
0.77 ( 0.07
0.47 ( 0.06
0.49 ( 0.20
3.22 ( 1.06
1.79 ( 0.64
0.38 ( 0.13
0.13 ( 0.01
1.07 ( 0.06
1.00 ( 0.20
0.45 ( 0.06
0.16 ( 0.05
0.51 ( 0.19
0.19 ( 0.08
0.49 ( 0.11
0.06 ( 0.04
8.84 ( 0.97
10.36 ( 4.26
0.02 ( 0.02
0.06 ( 0.00
0.21 ( 0.02
0.35 ( 0.06
0.85 ( 0.08
0.17 ( 0.04
0.04 ( 0.01
0.05 ( 0.01
1.39 ( 0.43
0.91 ( 0.08
0.18 ( 0.04
0.19 ( 0.08
0.67 ( 0.19
5.57 ( 0.79
0.40 ( 0.10
2.49 ( 0.37
0.07 ( 0.04
0.10 ( 0.03
0.02 ( 0.01
skull
5
24
0.5
2
blood
5
24
0.5
2
heart
5
24
0.5
2
lungs
5
24
0.5
2
liver
5
24
0.5
2
kidneys
spleen
stomach
intestine
thyroidb
5
24
0.5
2
5
24
0.5
2
5
24
0.5
2
5
24
0.5
2
5
24
0.02 ( 0.01
a
b
Expressed as % injected dose per gram tissue (mean ( 1 SD for three mice). Expressed as % of injected dose.
Ta ble 2. Tissue Distributions of 25a in Rats 24 h after
Ta ble 3. Estimates of Doses Resulting from Administration of
1.00 GBq of 25a in Mana
Injectiona
organ
25a
target organ
mGy/MBq
rad/mCi
femur
skull
blood
heart
lungs
16.44 ( 1.96
5.84 ( 0.35
0.01 ( 0.01
0.04 ( 0.02
0.05 ( 0.01
0.18 ( 0.03
0.36 ( 0.03
0.09 ( 0.01
0.05 ( 0.01
0.03 ( 0.01
0.02 ( 0.01
bone surfaces
red marrow
brain
kidneys
liver
7.
1.10
27.5
4.07
0.249
0.170
0.106
0.0878
0.467
0.921
0.629
0.392
0.325
1.73
liver
urine bladder wall
total body
kidneys
spleen
stomach
intestine
thyroidb
a
The MIRDOSE (3.1) program was used with the adult
phantom, assuming residence times of 28, 153, 10, and 3 h in
trabecular bone, cortical bone, kidneys, and the remainder of body,
respectively.
a
Expressed as % injected dose per gram tissue (mean ( 1 SD
b
for three rats). Expressed as % of injected dose.
However, while this manuscript was in preparation, a
significant antitumor effect was observed after treat-
ment with the R-particle-emitting 223Ra.30 Treatment
with the hydroxybisphosphonate 25a was initiated 7
days after inoculation of the tumor cells, while the
control group received saline only. As shown in Figure
1, the rats in the control group developed tumors and
had a mean survival time of 20.8 days (range 20-21,
one survivor). Survival time refers to the time from cell
inoculation until the animals suffered discomfort be-
cause of their metastatic condition and hence were
sacrificed. The mean survival times for the animals
human osteosarcoma cells (OHS) in immunodeficient
nude rats.
The MT-1 model simulates formation of mixed lytic/
sclerotic skeletal metastasis in breast cancer patients.29
The cell line has an aggressive and metastatic behavior,
and animals infected develop tumors in the brain, lung,
and adrenals as well. Repeated treatments (days 7 and
14) with the chemotherapeutic agents cisplatin and
doxorubicin have not improved survival nor did they
have any significant effect on the metastatic growth.29

[
125I and 131I]-Labeled Arylalkylidenebisphosphonates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3027
F igu r e 1. Survival curves for rats injected (LV) with MT-1
cells and treated with bisphosphonate 25a . A total of 20
animals, allocated in four groups (n ) 5 in each group), were
used. Seven days after cell injection the animals were treated
with 200, 300, or 400 MBq/kg of 25a , whereas the control group
received saline only.
F igu r e 3. Survival curves for rats inoculated with OHS cells
and treated with bisphosphonate 25a . A total of 20 animals
were used, allocated in four groups (n ) 5 in each group).
Seven days after cell inoculation the animals were treated with
100, 200, or 400 MBq/kg of 25a , whereas the control group
received saline only.
cancer. Disease-free latency was defined as the period
between tumor cell inoculation and the time the diam-
eter of the tumor-injected tibia had increased by 2-3
mm compared with that of the noninjected contralateral
leg. As shown in Figure 3, all the untreated, OHS-
injected control animals (5/5) developed palpable bone
tumors after 16-32 days (mean 25.4 days). The mean
disease-free latency was 27, 30, and 31 days for the
animals treated with 100, 200, and 400 MBq/kg of 25a
(day 7), respectively (n ) 5 in each group). The group
receiving the highest dose had a marked increase in
disease-free latency time (P < 0.05, Kruskal-Wallis
test) as compared to the other groups. In addition, 3 of
5 (60%) animals receiving the highest dose were long
time survivors. Furthermore, 5 of 15 (33%) animals
treated in total with the compound were long time
survivors (>100 days), while all controls developed
palpable tumors (5/5, <33 days).
F igu r e 2. The scintigraphy of a rat (114 g), recorded 24 h
after injection of 25a .
Discu ssion
treated with the bisphosphonate 25a were 27.5, 29.0,
and 30.6 days with increasing dose of 200, 300, and 400
MBq/kg, respectively (n ) 5 in each group). One long
time survivor was observed in each of the control groups
and the group receiving the lowest dose. This may be
interpreted as cases where the cell line failed to
establish metastasis in the actual animals, or behaved
atypically. Excluding the survivors from the data, the
observed survival time for the animals treated with 25a
as compared with the controls are statistically signifi-
cantly longer (p < 0.05, Wilcoxon rank-sum test) at all
three dose levels. The results indicate a dose-response
relationship as well, but not statistically significant. As
part of this experiment, a scintigraphy was obtained
from a rat 24 h postinjection. The image shows the
favorable targeting abilities of 25a with particularly
high concentration in the spine and the pelvis and no
visible presence in soft tissue (Figure 2).
Only a few 131I-labeled bisphosphonates have so far
been prepared, and little is known about the relation-
ship of chemical structure with bone affinity and
selectivity. The results of the present study indicate that
the nature of the linker between the aromatic group and
the bisphosphonate moiety is important; the bone af-
finity of 10a is twice that observed for 18a . Substituting
the alpha hydrogen in 10a with a hydroxyl group as in
25a doubles the bone affinity as well. When comparing
these results with previously reported 131I-labeled bis-
phosphonates a similar pattern is revealed. To compare
bone affinity measured in different species one has to
express the uptake as % ID/g tissue × body weight to
correct for difference in weight of the animals used.
When expressed in this fashion, uptake in femur 24 h
after injection was found to be 850 ( 265% in mice, and
985 ( 118% in rats, in the case of 25a . The correspond-
ing values for [89Sr] strontium chloride and [153Sm]
samarium EDTMP are 360% (24 h, mice)30 and 580%
(24 h, rats),33 respectively. The analogue of 25a lacking
the methylene linker has been reported to have an
uptake in femur (47 h, rats) of 300%.34 Recently, Larsen
The OHS model was established by intratibial injec-
tions of the osteogenic sarcoma (OHS) cell line in
immunodeficient rats.31,32 The model has been used
previously to evaluate the antitumor efficacy of several
drugs currently used for treatment of bone-related

3028 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Årstad et al.
et al.18 reported the biodistribution data for a related
131I-labeled hydroxy bisphosphonates, i.e., compound 6a .
In this study uptake in femur (24 h, mice) was reported
to be 415%, substantiating the importance of the linker
in relation to bone affinity. The same authors also found
a reduction in bone affinity when the phenyl group in
6a was replaced by a pyridine ring as in compound 6b.18
Why the amino bisphosphonate 36a lack bone-seeking
abilities is not understood.
uptake in a reaction zone in the bone marrow surround-
ing the tumor.
To the best of our knowledge, compound 25a has the
highest bone-affinity reported for any radiopharmaceu-
tical. The radionuclide employed is readily available at
reasonable cost, and the method developed for radio-
halogenation provides close to quantitative radiochemi-
cal yields in short time with a mild and safe handling
procedure. Most importantly, dose estimates as well as
preliminary survival studies indicate the improved
antitumor efficacy of 25a compared with currently used
bone-seeking radiopharmaceuticals.
The dosimetry part of this work only serves to indicate
what might be expected in humans. Per unit of injected
activity, the bone marrow dose is proportional to the
fraction of injected activity that is accumulated in the
skeleton, and so is the dose to a tumor. An important
unknown factor is the ratio between uptake of the
compound in the new bone matrix surrounding a tumor
and the normal bone, including trabecular bones adja-
cent to the bone marrow. A rapid and high bone uptake
means that a lower fraction of the activity passes
through the kidneys and the bladder and thus results
in lower doses to these organs. The kidney data of Table
1 indicate that the kidney excretion may be slower than
what was measured in vivo. This would give a higher
dose to the kidneys than indicated by Table 3, perhaps
by as much as a factor 2. Nevertheless, this would still
amount to only 4% of the dose to the bone surfaces.
Proper dosimetric studies should be based on measure-
ments in humans with 123I and are beyond the scope of
this article.
Exp er im en ta l Section
Ch em istr y. Gen er a l. Unless otherwise indicated analytical
grade reagents were used without further purification. Other
grades were purified according to standard procedures before
use. All experiments involving organic solvents were run under
an argon atmosphere. NMR spectra were obtained with Varian
Gemini-200, Bruker DPX 200, and Bruker DRX 500 instru-
ments. Infrared spectra were obtained with a Perkin-Elmer
1310 infrared spectrophotometer or a Nicolet Magna-IR 550
spectrometer. Mass spectra were obtained on a Fision VG pro
spectrometer; for GC-MS, a Fisons 8065 gas chromatograph
with a CP SIL SCB-MS column was attached to the spectrom-
eter. Radioactivity measurements were carried out either with
a Beckman LS 6500 liquid scintillation counter or with a
Capintec CRC-7R radioisotope calibrator. Sodium [131I]-iodide
was obtained from NEN Life Science Products, and sodium
[
125I]-iodide was purchased from Amersham, UK. HPLC
separations were performed with a system consisting of a LC10
AT pump and an SPD-M10A diode array UV detector (both
Shimadzu) combined with a Beckman Model 170 radioactivity
detector. The columns used were PLRP-S (5 µm, 100 Å, 150
× 1.6 mm and 8 µm, 1000 Å, 150 × 4.6 mm, Polymer
Laboratories, UK). Sonification experiments were carried out
with a Transsonic 310 ultrasound bath (Heigar). Melting
points were measured on a Bu¨chi apparatus and are uncor-
rected.
The results from the survival study involving the OHS
cell line indicate that the antitumor efficacy of 400 MBq/
kg of the hydroxybisphosphonate 25a is similar to that
of 800 MBq/kg of [153Sm] samarium-EDTMP.35 How-
ever, the actual dose to bone marrow is estimated to be
2.2-3.6 times as high for 800 MBq/kg [153Sm] sa-
marium-EDTMP as compared to 400 MBq/kg of the
hydroxybisphosphonate 25a . Consequently, it is reason-
able to assume that considerably higher doses of 25a
may be delivered to tumor volumes without increased
detrimental effects as compared with currently used
drugs.
Gen er a l P r oced u r es. Iod in a tion . The trimethylsilyl de-
rivative (x mmol) was added to a solution of ICl (2x mmol) in
AcOH (5x mL), and the mixture was stirred for 5 h at room
temperature. The reaction mixture was evaporated to dryness
under reduced pressure, and the residue was dissolved in
ether. The solution was washed successively with saturated
NaHCO3 (aq), saturated Na2SO3 (aq), water, and brine. The
organic phase was dried (MgSO4) and the ether removed under
reduced pressure to give the iodo derivative. Tr a n sester ifi-
ca tion . To 1 equiv of the parent tetraalkyl bisphosphonate
was added 10 equiv of TMSBr, and the mixture was stirred
for 3 h at room temperature. The volatiles were then removed
under reduced pressure to give the tetrakis(trimethylsilyl)
ester. Hyd r olysis. The tetraalkyl bisphosphonate was added
to concentrated HCl and the mixture heated under reflux for
3 h. The reaction mixture was then concentrated to dryness
under reduced pressure to give the bisphosphonic acid.
m -Tr im eth ylsilyltolu en e (11). A solution of m-chlorotolu-
ene (12.66 g, 0.10 mol) and 1,2-dibromoethane (0.5 mL) in THF
(20 mL) was added to magnesium turnings (2.67 g, 0.11 mol).
The mixture was heated under reflux for 4 h, after which the
heating bath was removed and trimethylchlorosilane (11.08
g, 0.10 mol) was added at a rate maintaining gentle reflux.
The mixture was stirred for another 0.5 h, and 5% aq NaHCO3
(100 mL) was added. The resulting mixture was extracted with
CH2Cl2, and the extract was washed with brine and dried
(MgSO4). Filtration and evaporation provided 15.05 g (92%)
of the silane 11 as a pale, yellow-colored oil. Spectroscopic data
were identical to those of an authentic sample.36
One must recognize that the optimal choice of radio-
nuclide depends on the geometrical relation between the
structures that accumulate the radiopharmaceutical
and the tumor itself (e.g., uptake in a bone reaction zone
surrounding a metastatic lesion). Uptake in the tumor
will only happen in the case of osteosarcoma. Tumor
penetration and cell killing ability of the radiation
depend on the types of radiation emitted. Thus, a large
metastatic lesion would require even higher energy beta
particles than those emitted by 131I. If the radionuclide
is uniformly distributed within an osteosarcoma, a low
beta energy would lead to deposition of most of the
released energy within the tumor. Alpha emitters are
only useful it they are accumulated in the tumor or in
a zone immediately adjacent to a very small tumor.
Furthermore, it should be noted that the bone marrow
compartments and the bones in general are significantly
smaller in rats than in humans. Hence it is possible that
the results of the survival studies reflect dose contribu-
tions to the tumorous sites from activity deposited in
cortical bone. Because of the larger dimensions, this
would seldom be the case in humans, where the effect
of small tumors probably would mainly relate to the
m -Tr im eth ylsilylben zyl Br om id e (12). A mixture of the
silane 11 (8.22 g, 50 mmol), NBS (8.90 g, 50 mmol), AIBN (0.05
g), and chlorobenzene (50 mL) was heated in an oil bath kept
at 110 °C. When the reaction mixture reached 80-90 °C, an

[
125I and 131I]-Labeled Arylalkylidenebisphosphonates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3029
exothermic reaction started and the orange suspension became
a colorless solution within minutes. The mixture was cooled
with the aid of an ice-bath, ice-water was added, and the
resulting mixture was extracted with hexane. The organic
phase was washed with water and brine and dried (MgSO4).
Filtration through a silica plug (10 g) followed by evaporation
of solvents yielded 12.05 g (99%) of the bromide 12 as an oil.
Spectroscopic data were identical with those of an authentic
sample.37
Dieth yl m-Tr im eth ylsilylph en yleth ylph osph on ate (13).
To a solution of diethyl methylphosphonate (2.28 g, 15 mmol)
in THF (20 mL), kept at -78 °C, was added BuLi (17 mmol,
1.6 M solution in hexane). After 45 min, the white suspension
was added to a cold (-78 °C) solution of the bromide 12 (3.65
g, 15 mmol) in THF (20 mL), and the mixture was stirred for
1.5 h. The temperature was then increased to -10 °C, and
after 1 h the deeply red-colored solution was quenched with
AcOH (1 mL). Volatiles were removed under reduced pressure,
and the residue was purified by flash chromatography (silica,
EtOAc) to give 2.48 g (53%) of the phosphonate 13 as a
colorless oil. IR (film): 3473, 2955, 1248, 1058, 1031 cm-1; 1H
NMR (200 MHz, CDCl3): 0.30 (9H, s), 1.31 (6H, t, J ) 7 Hz),
2.10 (2H, m), 2.92 (2H, m), 4.11 (4H, m), 7.29 (4H, m); 13C NMR
(50 MHz, CDCl3): 1.2, 16.4, 26.3, 28.6, 29.0, 61.5, 127.9, 128.4,
131.2, 132.9, 139.9, 140.2, 140.7; HRMS-EI (M+) m/z 314.1453
(calculated for C15H27O3PSi 314.1467).
Tetr a eth yl m -Tr im eth ylsilylp h en yleth ylid en e-1,1-bis-
p h osp h on a te (14). A solution of the phosphonate 13 (2.51 g,
8 mmol) in THF (10 mL) was added to a solution of LDA (16
mmol) at -78 C. After 30 min, diethyl chlorophosphate (1.41
g, 9 mmol) was added, and the mixture was stirred for 45 min.
The temperature was then increased to -25 °C, and the
reaction mixture was quenched by addition of saturated NH4-
Cl (aq). The mixture was concentrated under reduced pressure,
and the residue was extracted with hexane. The extract was
washed with water and brine and dried (MgSO4). Filtration
and evaporation provided 3.22 g (87%) of the bisphosphonate
14 as a pale yellow-colored oil. 1H NMR (200 MHz, CDCl3):
0.19 (9 H, s), 1.21 (12 H, m), 2.61 (1 H, m), 3.19 (2 H, m), 4.03
(8 H, m), 7.26 (4 H, m); 13C NMR (50 MHz, CDCl3): 0.5, 17.6,
32.5, 37.7, 40.3, 42.9, 63.4, 127.9, 129.6, 131.7, 134.0, 138.8,
138.9, 139.1, 140.4; HRMS-EI (M+) m/z 450.1753 (calculated
for C19H36O6P2Si 450.1756).
m -Tr im et h ylsilylp h en ylet h ylid en e-1,1-b isp h osp h on -
ic Acid (9). Transesterification of the ester 14 (0.45 g, 1.0
mmol) was carried out as described under general procedures.
The resulting tetrakis(trimethylsilyl) ester was dissolved in
EtOH (90%, 2.0 mL) at 0 °C and stirred for 30 min. NaOH
(0.08 g, 2 mmol) in EtOH (90%, 4 mL) was added, and removal
of volatiles provided 9 as a white solid (0.43 g).
night. The resulting mixture was extracted with CH2Cl2, and
the extract washed with brine and dried (MgSO4). Filtration
and evaporation gave a solid residue (2.84 g), which was
purified by chromatography on silica (hexane/EtOAc gradient),
furnishing the sulfone 19 (2.05 g, 67%) as a white solid, mp
1
73-74 °C. IR (neat): 2954, 1446, 1310, 1253, 1155 cm-1; H
NMR (200 MHz, CDCl3): 0.18 (9H, s), 4.35 (2H, s), 7.31 (9,
m); 13C NMR (50 MHz, CDCl3): -1.2, 63.1, 127.6, 128.1, 128.8,
128.9, 131.4, 133.7, 135.7, 137.9, 141.1; HRMS-EI (M+): m/z
304.0943 (calculated for C16H20O2SiS 304.0953).
Dieth yl 3-(m-Tr im eth ylsilylph en yl)-3-ben zen esu lfon yl-
p r op yl-1-p h osp h on a te (20). To a solution of the sulfone 19
(3.04 g, 10 mmol) in THF (50 mL) was added LDA (10.5 mmol)
at -78 °C. The mixture was stirred for 0.5 h, and diethyl
2-bromoethylphosphonate (2.45 g, 10 mmol) was then added
neat. After 2 h, the reaction was quenched with AcOH (1 mL)
at 0 °C. Concentration under reduced pressure followed by
chromatography (silica, EtOAc) of the residue gave the phos-
phonate 20 (3.08 g, 66%) as a colorless oil. IR (film): 2982,
2956, 1308, 1249, 1056 cm-1; 1H NMR (200 MHz, CDCl3): 0.13
(9H, s), 1.27 (6H, m), 1.70 (2, m), 4.09 (5H, m) and 7.20 (9H,
m); 13C NMR (50 MHz, CDCl3): 1.4, 16.3 (d, J ) 5 Hz), 20.8,
21.5, 24.4, 61.7 (d, J ) 7 Hz), 70.9, 71.2, 128.0, 128.5, 128.9,
129.5, 130.5, 133.4, 133.8, 135.1, 137.0, 140.9; HRMS-ESI (M
+ 1+): m/z 469.1628 (calculated for C22H34O5SiP 469.1621);
HPLC: (PLRP 100Å, 95:5 MeOH/H2O), tR )3.5 min, >95%
purity.
Dieth yl 3-(m -Tr im eth ylsilylp h en yl)p r op yl-1-p h osp h o-
n a te (21). To a solution of the phosphonate 20 (1.15 g, 2.45
mmol) in MeOH (25 mL) at 0 °C was added Na2HPO4 (3.48 g,
24.5 mmol) followed by Na(Hg) 6% (9.21 g, 24.5 mmol). The
resulting mixture was stirred for 3 h and quenched with
saturated NH4Cl (aq). The mixture was extracted with ether,
and the extract was washed with brine and dried (MgSO4).
Evaporation under reduced pressure provided the phosphonate
21 (0.77 g, 96%) as an oil. IR (film): 2955, 1405, 1259, 1060,
1032 cm-1; 1H NMR (200 MHz, CDCl3): 0.28 (9H, s), 1.33 (6H,
t, J ) 7 Hz), 1.86 (4H, m), 2.73 (2H, t, J ) 7 Hz), 4.10 (4H, m),
7.28 (4H, m); 13C NMR (50 MHz, CDCl3): 1.0, 16.5, 24.3, 25.2
(d, J ) 141 Hz), 36.7 (d, J ) 17 Hz), 61.5, 127.9, 129.0, 131.2,
133.5, 140.3, 140.7; HRMS-EI (M+): m/z 328.1634 (calculated
for C16H29O3P 328.1624); HPLC: (PLRP 100 Å, 95:5 MeOH/
H2O), tR ) 4.2 min, >95% purity
Tetr a eth yl 3-(m -Tr im eth ylsilylp h en yl)p r op yl-1,1-bis-
p h osp h on a te (22). To LDA (8.9 mmol) was added a solution
of the phosphonate 21 (1.40 g, 4.25 mmol) in THF (15 mL) at
-78 °C. After 0.5 h, diethyl chlorophosphate (0.70 g, 4.5 mmol)
was added, and the mixture stirred for 2 h. The reaction was
quenched with AcOH (0.5 mL), and the resulting mixture was
evaporated to dryness under reduced pressure. Flash chro-
matography (silica, Et2O/EtOH gradient) of the residue gave
the bisphosphonate 22 (1.86 g, 94%) as colorless oil. IR (film):
2981, 1249, 1027, 969 cm-1; 1H NMR (200 MHz, CDCl3): 0.25
(9H, s), 1.32 (12H, m), 2.29 (3H, m), 2.91 (2H, t, J ) 7 Hz),
4.17 (8H, m) and 7.29 (4H, m); 13C NMR (50 MHz, CDCl3):
1.3, 16.2, 27.2, 34.6, 35.6 (t, J ) 134 Hz), 62.4, 127.7, 128.9,
131.0, 133.4, 140.0, 140.4; HRMS-EI (M+): m/z 464.1878
(calculated for C20H38O6P2Si 464.1913).
Tetr a eth yl m -Iod op h en yleth ylid en e-1,1-bisp h osp h o-
n a te (16). Iodination of the ester 14 was carried out as
described under general procedures. IR (film): 3350, 2960,
1240, 1015, 960 cm-1; 1H NMR (200 MHz, CDCl3): 1.26 (12H,
m), 2.57 (1H, m), 3.12 (2H, m), 4.07 (8H, m), 7.39 (4H, m); 13
C
NMR (50 MHz, CDCl3): 16.3, 30.7, 36.2, 38.9, 41.5, 62.5, 93.9,
128.1, 129.8, 135.4, 137.8, 141.9; HRMS-EI (M+): m/z 504.0365
(calculated for C16H27O6P2I 504.0328).
m-Iodop h en yleth yliden e-1,1-bisp h osp h on ic Acid (10c).
The ester 16 was transesterified as described under general
procedures. Hydrolysis was achieved by stirring the resulting
tetrakis(trimethylsilyl) ester in EtOH (75%) overnight.
m -Tr im eth ylsilylben zyl p h en yl su lfon e (19). To a solu-
tion of NaOH (0.80 g, 20 mmol) in MeOH (20 mL) was added
thiophenol (2.20 g, 20 mmol). After 10 min was added a
solution of the bromide 12 (4.38 g, 18 mmol) in MeOH (10 mL),
and the resulting mixture was stirred overnight. The crude
product was separated between water and EtOAc, and the
extract was washed with water and brine and dried (MgSO4).
Filtration and evaporation yielded the sulfide (4.79 g, 98%)
as a pale, yellow-colored oil. The product (2.73 g, 10 mmol)
was dissolved in MeOH (40 mL), and a suspension of oxone
(18.44 g, 30 mmol) in water (40 mL) was added at 0 °C. The
reaction mixture was stirred at ambient temperature over-
Tet r a et h yl 3-(m -Iod op h en yl)p r op yl-1,1-b isp h osp h o-
n a te (23). The bisphosphonate 22 (0.46 g, 1 mmol) was
iodinated as described under general procedures to provide the
bisphosphonate 23 (0.46 g, 86%) as a yellow oil. 1H NMR (200
MHz, CDCl3): 1.26 (12H, m), 2.14 (3H, m), 2.28 (2H, t, J ) 7
Hz), 4.08 (8H, m), 7.20 (4H, m); 13C NMR (50 MHz, CDCl3):
6.3, 26.9, 33.9, 35.5 (t, J ) 133 Hz), 62.4, 94.3, 127.8, 130.0,
135.1, 137.5, 143.2; HRMS-EI (M+): m/z 518.0460 (calculated
for C17H29O6P2I 518.0484).
3-(m -Tr im et h ylsilylp h en yl)p r op ylid en e-1,1-b isp h os-
p h on ic Acid (17). The ester 22 (0.23 g, 0.5 mmol) was
transesterified as described under general procedures. The
tetrakis(trimethylsilyl) ester was then dissolved in EtOH (75%,
1.5 mL) at 0 °C. After 0.5 h, a solution of NaOH (40 mg, 1
mmol) in EtOH (75%, 1 mL) was added. Evaporation under

3030 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Årstad et al.
reduced pressure furnished the disodium salt of 17 (0.20 g) as
a white solid.
acetic acid (2 mL), and the solution was stirred for 5 h. The
solvent was evaporated, and the residue was extracted with
1:1 hexane/ether (2 × 10 mL). Concentration of the extract
under reduced pressure furnished the bisphosphonate 30 (0.38
g, 81%) as an orange-colored oil. IR (film): 3381 (br), 2958,
1643, 1216, 1057 cm-1; 1H NMR (200 MHz, CDCl3): 3.18 (2H,
t, J ) 14 Hz) 3.74 (12H, m), 7.32 (4H, m); 13C NMR (50 MHz,
CDCl3): 39.7, 55.8, 56.3, 73.3, 76.3, 79.3, 94.2, 129.9, 130.8,
136.3, 136.8, 140.2; P (500 MHz; CDCl3; H3PO4): 19.6; HRMS-
EI(M+): m/z 463.9630 (calculated for C12H19O7P2I 463.9651).
3-(m -Iod op h en yl)p r op ylid en e-1,1-bisp h osp h on ic Acid
(18c). The ester 23 was hydrolyzed as described under general
procedures to give the acid 18c as a tanned solid.
m -Tr im eth ylsilylben zyl Cya n id e (26). A mixture of KCN
(5.5 g, 84 mmol), the bromide 12 (5.31 g, 21.8 mmol), tribu-
tylamine (0.11 g, 0.59 mmol), and water (12.5 mL) was stirred
overnight. The product was extracted with CH2Cl2, the extract
was filtered through a silica plug (5 g), and solvents were
removed under reduced pressure. The residue was distilled to
give the cyanide 26 (2.27 g, 55%), bp 134-136 °C (2 mm). IR
1-Hyd r oxy(m -iod op h en yl)eth ylid en e-1,1-bisp h osp h o-
n ic Acid (25c). The bisphosphonate 30 was hydrolyzed as
described under general procedures to give the title compound
as an orange colored solid.
1
(film): 2956, 2255, 1412, 1249, 885, 867 cm-1; H NMR (200
MHz, CDCl3): 0.31 (9H, s), 3.76 (2H, s), 7.42 (4H, m); 13C NMR
(50 MHz, CDCl3): 0.3, 24.3, 117.5, 127.7, 127.9, 128.6, 132.0,
132.3, 141.2; HRMS-EI (M+): m/z 189.0972 (calculated for
m -Tr im eth ylsilylp h en yleth yla m in e (33). To a suspen-
sion of ZrCl4 (4.66 g, 20 mmol) in THF (70 mL) was added
NaBH4 (3.03 g, 80 mmol). This resulted in gas evolution and
the formation of a cream-colored suspension. The cyanide 26
(3.03 g, 16 mmol) was added, and the mixture was stirred for
24 h. After cooling in ice, the reaction was quenched by
addition of water, then 25% NH3 (aq) until basic and extracted
with EtOAc. The organic phase was washed with brine, and
the solvents were removed under reduced pressure to give
crude amine (3.65 g), which was purified by precipitation of
the picric salt from benzene. The salt was dissolved in water,
LiOH was added until pH > 11, and the aqueous phase was
extracted with ether. The picric acid was removed by repeat-
edly washings with NaOH (aq). The organic phase was washed
with brine and dried (MgSO4), and the solvents were evapo-
rated to give the amine 33 (1.53 g, 50%) as pale, yellow-colored
C
11H15NSi 189.0974).
m -Tr im eth ylsilylp h en yla cetic Acid (27). To a solution
of NaOH (4.0 g, 0.1 mol) in MeOH (20 mL) was added the
cyanide 26 (2.84 g, 15.0 mmol), and the mixture was heated
under reflux for 4 h. The solvent was evaporated, and the
residue was dissolved in water (50 mL). The resulting solution
was washed with CH2Cl2, acidified with 85% H3PO4 to pH 2,
and extracted with CH2Cl2. The extract was washed with brine
and dried (MgSO4). Solvents were removed under reduced
pressure to give the acid 27 (2.93 g, 94%) as a yellow-colored
oil. IR (film): 3600-2600, 1717, 1418, 1254, 847 cm-1; 1H NMR
(200 MHz, CDCl3): 0.32 (9H, s), 3.70 (2H, s), 7.42 (4H, m);
13C NMR (50 MHz, CDCl3): 0.5, 41.6, 127.8, 129.6, 132.1,
132.3, 134.1, 140.8, 177.7; HRMS-EI (M+): m/z 208.0918
(calculated for C11H16O2Si 208.0920).
oil. IR (film): 2954, 1248, 858 cm-1 1H NMR (200 MHz,
;
CDCl3): 0.27 (9H, s), 1.49 (2H, br, NH2), 2.76 (2H, d, J ) 7
Hz), 2.98 (2H, d, J ) 7 Hz), 7.28 (4H, m); 13C NMR (50 MHz,
CDCl3): 0.37, 40.4, 43.8, 127.0, 128.4, 130.3, 133.8, 138.0,
139.6; HRMS-EI(M+): m/z 193.1293 (calculated for C11H19NSi
193.1287).
N -(m -Tr im e t h ylsilylp h e n yle t h yl)a m in om e t h yle n e -
bisp h osp h on ic Acid (32). The bisphosphonate 34 (0.48 g, 1
mmol) was transesterified by the general procedure. The
resultant tetrakis(trimethylsilyl) ester was dissolved in a
mixture of CH3CN (2 mL) and MeOH (0.5 mL), and the
solution was left in the cold for 5 h. A solution of NaOH (0.08
g, 2 mmol) in EtOH (2 mL) was then added. The resulting
precipitate was collected by filtration and dried under reduced
pressure to give the disodium salt of 32 (0.38 g) as a white
solid.
Dim eth yl m -Tr im eth ylsilylp h en yla cetylp h osp h on a te
(28). A mixture of the acid 27 (1.04 g, 5.0 mmol) and SOCl2
(0.82 g, 6.9 mmol) was stirred at room-temperature overnight.
Toluene (2 mL) was added, and volatiles were removed under
reduced pressure. The residue was dissolved in THF (5 mL),
and trimethyl phosphite (0.69 g, 5.5 mmol) was added at -20
°C. The resulting solution was kept in an ultrasound bath at
0 °C for 30 min. Evaporation of solvent gave the crude
bisphosphonate 28 (1.63 g, 99%), which consisted essentially
of the E-enolate. The unstable product was used further
without purification. An analytical sample was prepared by
washing the crude product with hexane. IR (film): 3600-2600,
1698, 1249, 1036 cm-1; 1H NMR (200 MHz, CDCl3): 0.19 (9H,
s), 3.86 (6H, d, J ) 14 Hz), 6.11 (1H, d, J ) 13 Hz), 7.46 (4H,
m), 7.92 (1H, d, J ) 7 Hz); 13C NMR (50 MHz, CDCl3): 0.7,
54.6, 117.5, 118.1, 128.1, 130.4, 132.9, 133.6, 134.0, 134.9,
138.3, 140.6, 142.3; P (202 MHz; CDCl3; H3PO4): 16.3; HRMS-
EI (M+): m/z 300.0929 (calculated for C13H21O4PSi 300.0947).
Tet r a et h yl N-(m -Iod op h en ylet h yl)a m in om et h ylen e-
bisp h osp h on a te (35). The trimethylsilyl derivative 34 was
iodinated with ICl by the general procedure to give the iodo
ester 35. IR (film): 2982, 1658, 1247, 1042 cm-1; 1H NMR (200
MHz, CDCl3): 1.29 (12H, m), 2.67 (2H, t, J ) 7 Hz), 3.06 (2H,
Tetr a m eth yl 1-Hyd r oxy(m -tr im eth ylsilylp h en yl)eth -
ylid en e-1,1-bisp h osp h on a te (29). To an ice-cold solution of
the phosphonate 28 (1.63 g, 5.0 mmol) in ether (10 mL) was
added a solution of dimethyl phosphite (0.83 g, 7.5 mmol) and
dibutylamine (0.32 g, 2.5 mmol) in hexane (10 mL). A white
solid formed after a few minutes. After 20 min, the mixture
was filtered to give the bisphosphonate 29 (1.34 g, 65%) as a
white solid, mp 114-118 °C. IR (film): 3700-2815, 2958, 1645,
1251 cm-1; 1H NMR (200 MHz, CDCl3): 0.25 (9H, s), 3.38 (2H,
t, J ) 14 Hz), 3.76 (12H, m), 7.39 (4H, m); 13C NMR (50 MHz,
CDCl3): 0.1, 40.1, 54.9, 73.3, 76.3, 79.3, 127.7, 132.1, 132.4,
133.8, 136.6, 140.2; P (202 MHz; CDCl3; H3PO4): 20.7; HRMS-
EI (M+): m/z 410.1080 (calculated for C15H28O7P2Si 410.1080).
m), 3.23 (1H, t, J ) 22 Hz), 4.14 (8H, m), 7.24 (4H, m, Ar); 13
C
NMR (50 MHz, CDCl3): 16.1, 35.8, 50.8, 51.0, 53.9, 56.8, 63.0,
94.0, 127.7, 129.7, 134.8, 137.2, 141.7; HRMS-ESI (M + 1+):
m/z 534.0666 (calculated for C17H31NO6P2I 534.0668).
N -(m -I o d o p h e n y le t h y l)a m in o m e t h y le n e b is p h o s -
p h on ic Acid (36b). Hydrolysis of the iodo ester 35 with
concentrated HCl, as described under general procedures,
furnished the acid 36b.
P r ep a r a tion of [125I a n d 131I] 10a , 10b, 18a , 18b, 25a ,
a n d 25b. To a solution of the radionuclide in water (2 µL) was
added a solution of NCS (400 µg, 3 µmol) in TFA (10 µL),
followed by a solution of the appropriate trimethylsilyl bis-
phosphonate (0.2 µmol) in AcOH (2 µL). The mixture was
sealed, swirled, and left for 5 min at room temperature. In
the case of the hydroxy compound 25a a trace of TFA was
required to dissolve the compound in AcOH.
1-Hyd r oxy(m -tr im eth ylsilylp h en yl)eth ylid en e-1,1-bis-
p h osp h on ic Acid (24). The ester 29 (0.82 g, 2.0 mmol) was
transesterified as described under general procedures. The
resultant tetrakis(trimethylsilyl) ester was dissolved in EtOH
(75%, 8 mL) at 0 °C and stirred for 0.5 h. The mixture was
neutralized by addition of Na2CO3 (aq) (1.0 M, 3 mL) and
concentrated under reduced pressure to give the disodium salt
of 24 (0.75 g), as a white solid.
P u r ifica tion of [125I a n d 131I] 10a a n d 10b. The reaction
mixture was purified by means of HPLC. For this purpose a
PLRP-S column (100Å) was used with
a mobile phase
consisting of 50 mM phosphate buffer with pH 7.2, to which
2% EtOH was added. With a flow rate of 1 mL/min the iodides
10a and 10b eluted after 6.0 min. The radiochemical yield, as
determined by integration, was >95%.
Tetr a m eth yl 1-Hyd r oxy(m -iod op h en yl)eth ylid en e-1,1-
bisp h osp h on a te (30). The bisphosphonate 29 (0.41 g, 1.0
mmol) was added to a solution of ICl (0.32 g, 2.0 mmol) in

[
125I and 131I]-Labeled Arylalkylidenebisphosphonates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3031
P u r ifica tion of [131I a n d 125I] 18a a n d 18b. The reaction
inoculation. The control groups received saline only. Doses
were corrected for the actual weight of each animal and
administered by single bolus tail vein injections.
mixture was purified by means of HPLC. A PLRP-S column
(1000Å) was employed with a mobile phase consisting of 50
mM phosphate buffer with pH 7.3, to which 2% EtOH was
added. With a flow rate of 1 mL/min the iodides 18a and 18b
eluted after 5.9 min. The radiochemical yield as determined
by integration, was >95%.
Ra d ioa ctivity Mea su r em en ts. Radioactivity measure-
ments of the labeled compounds were carried out with a liquid
scintillation counter (Beckman LS 6500, Beckman Instru-
ments) or with a Capintec CRC-7R radioisotope calibrator
(Capintec). The radioactivity level of the excised organs was
measured with an automated gamma counter (LKB Wallac
1282, Turku, Finland).
P u r ifica tion of [131I a n d 125I] 25a a n d 25b. Phosphoric
acid (25%, 30µL) was added to the solution of the crude labeled
compound, and the mixture was placed in an ultrasound bath
for 5 min. HPLC was carried out with a PLRP-S column (5
µm, 100Å) and a mobile phase consisting of 50 mM phosphate
buffer with pH of 7.1, to which 2% EtOH was added. With a
flow rate of 1 mL/min, the iodides 25a and 25b eluted after
4.9 min. The radiochemical yield as determined by integration,
was >95%.
Ack n ow led gm en t. We thank Ms. S. G. Vik for
skilled technical assistance. The financial support of the
Norwegian Research Council is highly acknowledged.
P r ep a r a tion a n d P u r ifica tion of [131I] 36a . To a solution
of Na131I in water (2 µL) was added a solution of NCS (400 µg)
in TFA (10 µL), followed by addition of the ester 34 (100 µg,
0.2 µmol) in AcOH (2 µL). The resulting mixture was sealed,
swirled, and left for 0.5 h. Analysis and purification was
carried out by means of HPLC. For this purpose a PLRP-S
(1000Å) column was used, and the mobile phase consisted of
70:30:1 MeOH/H2O/TFA. With a flow rate of 1 mL/min, the
iodide 35a eluted after 6.8 min. The yield, as measured by
integration, was 50%. The iodide was collected and the eluate
concentrated to dryness. The residue was dissolved in TMSBr
(2.5 mL) and left overnight. Evaporation to dryness, followed
by addition of 75% EtOH (0.25 mL), provided the iodo acid
36a . With a flow rate of 1 mL/min the iodide 36a eluted after
2.7 min.
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