Synthesis, characterization, and in vivo skeletal localization of a new 99mTc-based multidentate phosphonate chelate: 5-Amino-1,3-bis(ethylamine-(N,N dimethyl diphosphonic acid) acetamido) benzene

Article abstract of DOI:10.1016/j.bmc.2006.10.005

The tetraphosphonate ligand, 5-amino-1,3-bis(ethylamine-(N,N-dimethyl diphosphonic acid) acetamido) benzene (IPTMP) used in the present study was prepared from 5-nitroisophthalate dimethylester to label with radionuclide for targeted diagnosis and therapy

Full text of DOI:10.1016/j.bmc.2006.10.005

Bioorganic & Medicinal Chemistry 15 (2007) 1138–1145
Synthesis, characterization, and in vivo skeletal localization
of a new 99mTc-based multidentate phosphonate chelate:
5-Amino-1,3-bis(ethylamine-(N,N dimethyl diphosphonic
acid) acetamido) benzene
Puja Panwar, Sweta Singh, Nitin Kumar, Harish Rawat and Anil K. Mishra*
Division of cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences,
Brig SK Mazumdar Road, Delhi 110054, India
Received 24 August 2006; revised 5 October 2006; accepted 6 October 2006
Available online 10 October 2006
Abstract—The tetraphosphonate ligand, 5-amino-1,3-bis(ethylamine-(N,N-dimethyl diphosphonic acid) acetamido) benzene
(IPTMP) used in the present study was prepared from 5-nitroisophthalate dimethylester to label with radionuclide for targeted diag-
nosis and therapy. The synthesized multidentate phosphonate ligand was characterized on the basis of spectroscopic techniques,
which exhibited good metal ion control properties when complexed to 99mTc with high in vitro and in vivo stability. Excellent quality
bone images of rabbit were imaged showing rapid clearance of background activity and visualization of skeleton at 1 h.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
phatase), and active bone mineralization of hydroxyap-
atite crystals. In bone imaging we attempt to image the
uncontrolled change of the mineralization process.
Radiotracers have reported to be localized in these sites
of increased mineralization activity in disease states.
Amorphous calcium phosphate present in many bone le-
sions in high concentrations, which has a greater affinity
for bone imaging agents, is also known to play a part in
radionuclide accumulation.1–4 The –OH on the phos-
phonate groups are suggestive of the bone matrix affin-
ity.5 There are other mechanisms of localization which
involves calcium fixed to mitochondria rendering site
for the deposition of the bone imaging agents are report-
ed in the literature.6–8 Phosphonic acid derivatives and
more elaborate derivatives are widely used as complex-
ing agents for calcium, zinc,9 and/or a number of biva-
lent metals.10 They are involved in a large number of
biochemical processes and disease treatments8 or diag-
nosis (scintigraphy in the case of 99mTc)11–15 and therapy
Bone metastases occur frequently in patients with
advanced breast, prostate, kidney, thyroid, and skin
cancers. The challenge of treating bone metastasis is to
deliver high doses of antitumor agents directly to tumor
areas with minimal exposure to the rest of the body.
This work is focused on development of the drug deliv-
ery to specifically target cancer within the skeleton
where bone is being destroyed through the use of specific
targeting molecules attached to the chelating agent that
already has the affinity for bone. The utility of this work
is that it could be used to target bone metastasis from
any cancer. It is thus an object of the present work to
provide a convenient synthetic route for the preparation
of a new multidentate chelating agent to obtain more
stable phosphonic acid based derivative for radiodiag-
nosis and radiotherapy.
188
Skeletal localization of phosphonate based bone imag-
ing agents depends on several factors including bone–
blood flow, enzyme activity (especially alkaline phos-
(90Y, 153Sm, and Re).18–20
Thus, the binding of polyphosphates and bisphosphonates
to calcified tissues is the basis for the use of these com-
pounds as skeletal markers in nuclear medicine when linked
to 99mTc. 99mTc-labeled compound used clinically in nucle-
ar medicine are methylenediphosphonate (MDP), dica-
rboxypropanediphosphonate (DPD), hydroxyethylidine
Keywords: IPTMP; 5-Aminoisophthalate tetramethylene phosphonic
acid; Chelating agent; 99mTc; Bone seeking agent.
*
Corresponding author. Tel.: +91 11 23914374; fax: +91 11
23919509; e-mail: akmishra@inmas.org
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.005

P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
1139
diphosphonate (HEDP), and ethylenediaminetetramethyl-
ene phosphonate (EDTMP).14–17 Multidentate phospho-
nate derivatives containing more than two –C–PO3–H2
groups are effective chelating agents and are hydrophilic
in nature. Greater number of phosphonate groups per mol-
ecule possess high chemical stability and produce 1:1 che-
lates than the conventional methylenediphosphonate.
Due to the multiplicity of the ligands and the radionuclides
now currently available, it is of interest to see the variation
in bone uptake with the 99mTc complex of this new phos-
phonic acid derivative to develop suitable imaging agents
to label with radionuclide having excellent radiophysical
properties.
and ethylenediamine (Scheme 1). The tetraacetic acid
was formed by standard amine alkylation technique
employing excess bromoacetic acid and aqueous 10 N
NaOH by pH stat. 5-nitro-1,3-bis(ethylamine-(N,N
dimethyl diphosphonic acid) acetamido) benzene (3)
was prepared by the reaction of phosphorous acid in
the presence of phosphotrichloride and characterized
on the basis of spectral studies. The tetraphosphonate
product was obtained in 89% yield and [M+H+] was
found to be 672.1 in positive mode. In 1H NMR
–CH2–P chemical shift appeared at 3.59–2.88 ppm.
Reduction of NO2 group with Pd/C in basic media
unmasked the amino group with a purity of above 97%.
Phosphonates are synthesized from phosphorous acid
by reaction with formaldehyde and either ammonia or
amines with old methodology yielding only 20% of the
required compound and resulted in the formation of
bridge compound in higher yields (78%).21 In this work
new methodology is employed for producing phospho-
nate directly from carboxylate in one-step process mak-
ing the route of synthesis much more convenient and
producing high yield of phosphonate derivative.22,23
Introduction of functionalized group to phosphonic acid
derivative provides the reactive functionality for the
conjugation of biological vector. The unique functional-
ity of this phosphonic acid derivative departed consider-
ably from the traditional acyclic phosphonate chelators
used in nuclear medicine.
2.2. Quality control
Radiochemical purity of 99mTc-IPTMP was estimated
chromatographically using ITLC-SG (instant thin layer
chromatography-silica gel) paper as the stationary phase
and 100% acetone as the mobile phase. The complex re-
mained at the point of spotting. Under identical condi-
tion 99mTcO4 moved toward the solvent front. Thus
À
the yield of free and complex ligand could be estimated.
The labeling yield was found to be more than 97%.
Metal-binding assay confirmed the radiochemical purity
of IPTMP to be >97%. Phosphor imager showed only a
single spot with Rf = 0.3 (10% ammonium acetate/meth-
anol) which suggests the formation of only one species.
2.3. In vitro and in vivo stability studies
The multidentate agent was derived from p-NO2-iso-
phthalate ester and the NO2 group was further reduced
to reactive amine. The substitution of carboxylic acid
pendant arms to phosphonic and the four nitrogens in
the chelating system provides more strong coordination
for metal–ion complexation. As is known, phosphonic
acids are substantially more acidic than the correspond-
ing carboxylic acids. As anticipated from the more polar
nature of the phosphonic acid moiety, the phosphonic
acids have appreciably lower logP values, indicating
that they concentrate more favorably in the aqueous
phase.24
99mTc-IPTMP showed no transchelation toward serum
proteins in vitro under physiological condition as only
2% detachment of the radiometal ion was observed at
24 h (Fig. 1). Radiolabeled MDP and IPTMP were chal-
lenged with DTPA (25–100 mM) (Fig. 2). Major trans-
complexation of 99mTc to 25 mM DTPA was observed
for 99mTc-MDP that was found to be 45%, whereas
IPTMP showed only 6% transcomplexation of 99mTc
to 25 mM DTPA. The blood activity curves in rabbit
show that the blood clearance of 99mTc-IPTMP is com-
parable to that of 99mTc-MDP. The biological t1/2 (F)
was found to be 28 min and t1/2 (S) was 8 h 25 min for
99mTc-IPTMP and t1/2 (F) = 1 h 5 min; t1/2 (S) = 8 h
for 99mTc-MDP (Fig. 3). The initial clearance rate was
comparable to that of MDP and also there was less
activity present in the blood at 3 h post-injection. This
accounts for high bone/blood and bone/muscles ratio
exhibited by 99mTc-IPTMP.
Moreover, aminophosphonates show a propensity to-
ward the strong chelation of metal ion.10,25 In an effort
to synthesize compounds that can be used in nuclear
medicine applications, a new multidentate phosphonate
derivative, 5-amino-1,3-bis(ethylamineacetamido)-ben-
zene (IPTMP), was synthesized. The purpose was to pre-
pare a synthetically useful phosphonic acid derivative
with higher coordinating ability and a reactive moiety
in the chelating system. Synthesis, radiolabeling, and
biodistribution studies of a novel 99mTc-labeled multid-
entate phosphonate ligand IPTMP are described.
2.4. Toxicity studies
The acute iv LD50 was determined to be 240 mg/kg.
Doses <240 mg/kg were well tolerated and no adverse
reaction was observed. Higher doses >240 g/kg were
toxic and found lethal. At 108–180 mg/kg dose immedi-
ate adverse reaction was observed following tremors and
increased heart rate, these reactions were observed to be
gone completely after 2–3 min. No adverse signs were
observed in these groups after 30 days. At 240 mg/kg
dose 50% of the death was seen in 30 days. Two hundred
and eighty milligrams per kilogram dose was found to
be the lethal dose.
2. Results
2.1. Synthesis
The compound 5-amino-1,3-bis(ethylamine-(N,N-di-
methyl diphosphonic acid) acetamido) benzene (IPTMP)
was synthesized from 5-nitroisophthalate dimethyl ester

1140
P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
Scheme 1. Synthesis of 5-amino-1,3-bis(ethylamine-(N,N dimethyl diphosphonic acid) acetamido) benzene (4). Reagents: (i) BrCH2COOH; (ii)
H3PO3/PCl3; (iii) 5% Pd–C/H2.
100
100
90
80
70
60
99mTc-IPTMP
99mTc-MDP
10
99mTc-IPTMP
99mTc-MDP
50
40
30
20
10
0
1
0
25
50
100
0
5
10
15
20
25
Concentration of DTPA (in mM)
Time in hours
Figure 2. In vitro stability study of 99mTc-labeled MDP and multid-
entate chelate IPTMP challenged with DTPA (25–100 mM).
Figure 1. Human serum stability studies of 99mTc-IPTMP and
99mTc-MDP under physiological conditions.
MDP in normal rabbit of the same age and weight dem-
onstrated comparable quality at 3 h with 99mTc-MDP
and at 1 h with 99mTc-IPTMP post-injection. The scinti-
grams of 99mTc-IPTMP are of comparable quality to
99mTc-MDP scintigram in a 3.1 kg rabbit and the images
also show that non-osseous tissue uptake of 99mTc-
IPTMP is minimal and when assessed with its rapid
blood clearance accounts for the low soft tissue activity
(Fig. 4). In the early images of the 99mTc-IPTMP, major
accumulation was seen in kidneys. This accounts for the
aminophosphonate group in the compound, which has
higher affinity for the kidney cells, but with the period
of time it was cleared from the kidneys.
2.5. Scintigraphy
Scintigraphic images of 99mTc-labeled 5-amino-1,3-
bis(ethylamine-(N,N-dimethyl diphosphonic acid) acet-
amido) benzene in rabbit in 3.3 kg rabbit showed rapid
accumulation of radioactivity in bone. Imaging of ani-
mals was carried out at different time intervals after
administering labeled compound intravenously. Early
images were taken at 30 min and 1 h. Within 15 min la-
beled phosphonate ligand IPTMP accumulated in the
shoulder region. After 1 h the ligand was localized in
the whole skeleton of rabbit. A comparison study was
conducted for the amino derivative (IPTMP) and

P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
1141
studies the tracer is noted to persist in the skeletal sys-
tem even at 24 h and showed less accumulation in kid-
ney. The excretion of the drug (IPTMP) is renal,
therefore the accumulation in the kidneys showed high
uptake (2.32 0.01% ID/g) initially. But with the period
of time it got cleared from the kidneys (0.142 0.12%
ID/g). This accounts for high bone/blood and bone/mus-
cles ratio exhibited by 99mTc-IPTMP, which is compara-
ble to 99mTc-MDP Table 1b.
50
40
30
20
10
0
99mTc-IPTMP
99mTc-MDP
3. Discussion
0
5
10
15
20
25
Entrapment of metal ion into a framework of multiden-
tate poly (aminocarboxylates) such as bifunctional
DTPA covalently attached to mAbs has been proven
to provide thermodynamically stable compounds suit-
able for clinical trials.26 Nevertheless, results obtained
for human patients and for animal models indicate that
the high level of radioactivity in blood, bone marrow,
kidney, and liver limits the applicability of these com-
pounds. This necessitates reduction of in vivo decom-
plexation of metal ions. The fast forming chelates like
those of derivatives of EDTA undergo rapid transchela-
tion to blood proteins.26,27 Tetracarboxylate was pre-
pared by the reaction of bromoacetic acid at high pH
and converted to phosphonate by following reported
Time in Hours
Figure 3. Blood clearance of 99mTc-labeled 5-amino-1,3-bis(ethyla-
mine-(N,N dimethyl diphosphonic acid) acetamido) benzene in normal
rabbit.
2.6. Biodistribution studies
In mice the skeletal uptake of 99mTc-IPTMP is higher at
1 h compared to 99mTc-MDP, which may be due to less-
er structural forms and higher stability of complex. The
bone uptake in mice from biodistribution was found to
be 7.3 0.69% ID/g at 4 h Table 1a. In biodistribution
Figure 4. Whole body c-Scintigraphic anterior and posterior images (A). 99mTc labeled 5-amino-1,3-bis(ethylamine-(N,N dimethyl diphosphonic
acid) acetamido) benzene (1 h) (B). 99mTc-MDP in rabbit (3 h).

1142
P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
Table 1a. Biodistribution of 99mTc-labeled 5-amino-1,3-bis(ethylamine-(N,N dimethyl diphosphonic acid) acetamido) benzene in BALB/c mice
Organs
30 min % ID/g
1 h % ID/g
4 h % ID/g
24 h % ID/g
Blood
Heart
0.64 0.01
0.23 0.01
0.16 0.01
0.16 0.03
2.32 0.01
0.18 0.08
0.12 0.01
0.12 0.05
4.69 0.92
0.22 0.02
0.71 0.08
0.21 0.04
0.14 0.02
0.23 0.06
1.85 0.12
0.16 0.03
0.18 0.06
0.13 0.02
6.6 0.46
0.21 0.06
0.14 0.09
0.22 0.08
0.21 0.06
2.47 0.07
0.13 0.02
0.26 0.05
0.24 0.01
7.3 0.69
0.037 0.05
0.087 0.06
0.038 0.08
0.081 0.03
0.142 0.12
0.128 0.03
0.021 0.02
0.06 0.01
2.96 0.06
0.08 0.01
Liver
Lungs
Kidneys
Spleen
Stomach
Intestine
Bone
Muscles
0.17 0.09
0.13 0.05
Data from five animals expressed as % ID/g SD.
Table 1b. Comparative uptake (%ID/g) of 99mTc-multidentate chelates in mice
Organs
99mTc-MDP
99mTc-IPTMP
1 h
4 h
1 h
4 h
Blood
Kidneys
Bone
0.21 0.01
1.46 0.20
4.92 0.15
0.19 0.01
0.98 0.11
5.92 0.36
0.71 0.08
1.85 0.12
6.6 0.46
0.21 0.07
2.47 0.07
7.3 0.69
BALB/c mice, body weight = 22–28 g. Data from groups of five mice are expressed as mean % ID/g SD. Based on estimates assuming: blood
volume = 7% of the body weight.
method of phosphonate synthesis. Phosphonate deriva-
tive produced in a Mannich type reaction by reacting
ethylamines with phosphorous acid and formaldehyde
leads to the formation of by-products which requires
purification.21 The new methodology describes one-step
process for the synthesis of tetraphosphonate chelator
using phosphorous acid and phosphorous trichloride;
this eliminates the formation of unwanted intermediate
product thereby obviating the need for its further purifi-
cation.22 The phosphonate derivative was synthesized
using new methodology that gave product in high yield.
Because of the four-methylene phosphonic acid groups/
ligand molecule should form highly negatively charged
chelates. The polydentate nature of the ligand having a
reactive functionality for the conjugation of biological
vector favors the formation of complex with only one li-
gand per metal atom as determined by metal binding as-
say which is required for in vivo stability and biological
activity.23 The multidentate agent developed included a
p-NO2 benzyl group for conjugation to biological target-
ing moieties, four nitrogens and four phosphonic groups
for metal–ion chelation to wrap transition/lanthanide
metal ions because most of these ions possess minimum
six coordination number which cannot be fulfilled by
MDP. To overcome the problem associated with MDP,
this novel chelating system can be exploited as bone seek-
ing agent for diagnosis and for therapeutic application
since it has multidenticity to bind with b emitters.
study, particularly chelate stability in serum, which de-
pends on various parameters such as medium pH, the
metabolism of the metal, and the presence of binding
proteins. The stability study for 99mTc-IPTMP showed
no transchelation phenomena for the metal toward the
physiological components of the blood because there
was no release of radioactivity to complex in a more
or less stable manner with albumin and transferrin and
at blood pH form colloid substances with high affinity
for carbonates, thus leading to sequestration in Reticu-
lo-Endothelial tissue.29,30 This study showed that
99mTc-IPTMP is highly stable in serum medium.
The blood activity curves in rabbits (Fig. 3) show that
the blood clearance of 99mTc-IPTMP initially is faster
than 99mTc-MDP due to which there was less activity
present in the blood at 3 h post-injection. This accounts
for high bone/blood and bone/muscles ratio exhibited by
99mTc-IPTMP, which is comparable to that of 99mTc-
MDP. These findings in rabbits are an indication of
the excellent in vivo stability of the 99mTc-IPTMP. The
images also show that non-osseous tissue uptake of
99mTc-IPTMP is minimal and when assessed with its ra-
pid blood clearance accounts for the low soft tissue
activity. Rapid incorporation of phosphonates into cal-
cified tissue leads to their short presence in the
circulation.1
Biodistribution studies revealed, in mice the skeletal up-
take of 99mTc-IPTMP is higher than 99mTc-MDP at 1 h,
which may be due to lesser structural forms and higher
stability of complex. 1:1 ligand ratio accounts for lesser
structural forms and better-defined preparations of
IPTMP, whereas MDP forms dimers, trimers, and olig-
omers with the metal ion. Four-phosphonate moiety in
the same molecule as compared to MDP is responsible
Kunzmann et al. revealed a novel antitumor effect of
aminobisphosphonates through their stimulation of
cdT cells and the induction of an anti-multiple myeloma
activity in patient samples. Therefore our compound
being an aminophosphonate having several hydroxyl
groups can afford high affinity toward bone matrix.28
Different factors were considered before biodistribution

P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
1143
for the formation of 1:1 complexation as shown by
MBA. The relative uptake in rabbit is similar to the re-
sults obtained in mice from biodistribution.
atmosphere and the reaction mixture was stirred for
24 h. EDA was removed under reduced pressure. The
obtained oily product was dissolved in chloroform and
allowed to evaporate slowly at room temperature.
Considering that the images and selective skeletal
uptake of 99mTc-IPTMP is comparable to a commercial
99mTc-MDP agent (Fig. 4) suggests conjugation of
IPTMP with targeting molecule and further optimiza-
tion of the formulation (e.g., dose, quantity of com-
pound, pH, Sn2+, etc.) of 99mTc could produce even
better results. For therapeutic application like octreo-
tide, the kidneys can be blocked with non-specific agents
to avoid any untoward reaction as it is being used in the
case of petptide-based therapy.31
Yield: 96.8%; IR (KBr) m cmÀ1: 3367, 3086, 1654, 1534,
1
1487, 1348. H NMR (300 MHz): d 8.76 (2H, Ar, s);
8.62 (1H, Ar, s); 3.42 (4H, t); 2.81 (4H, t). Mass: MS+
m/e Calcd 295.2, found [M+H+] 296.26. Elemental Anal.
Calcd for C12H17N5O4: C, 48.81; H,5.80; N,23.72; O,
21.67. Found: C, 48.80; H, 5.81; N, 23.71; O, 21.68.
5.2.2. Synthesis of 5-amino-1,3-bis(ethylamine (N,N-
diacetic acid) acetamido) benzene (2). Bromoacetic acid
(2.12 g; 15.25 mmol) was added to a stirring solution
of 1 (1 g; 3.37 mmol) in 15 mL of distilled water at room
temperature, pH 5. After complete addition, the temper-
ature was raised to 70 °C and pH was maintained at 10
by the solution of 0.1 M NaOH.
4. Conclusion
This investigation shows that tetramethylene phospho-
nate ligand obtained from p-nitroisophthalate is capable
of forming stable 99mTc-chelate that exhibits high and
selective in vivo bone uptake in both mice and rabbit.
99mTc-IPTMP has high bone uptake properties for skel-
etal imaging in terms of physiological and chemical sta-
bility. Its rate of blood clearance is rapid and its skeletal
uptake is significantly higher at 1 h compared to that of
99mTc-MDP in mice and rabbits. These results certainly
warrant further work with this ligand.
Yield: 89% IR (KBr) m cmÀ1 3435, 3013,2850, 1634,
1533, 1451, 1355, 1199, 1145, 722. 1H NMR
(300 MHz) d 8.74 (2H, Ar, s); 8.51 (1H, Ar, s); 3.32
(12 H, m); 2.66 (4H, t). Mass: MS+ m/e Calcd 528.15,
found [M+H+] 528.12, (M+2NaÀH) 570. Elemental
Anal. Calcd for C20H25N5O12: C, 45.54; H, 4.78; N,
13.28; O, 36.40. Found: C, 45.53; H, 4.78; N, 13.27; O,
36.42.
5.2.3. Synthesis of 5-nitro-1,3-bis(ethylamine-(N,N
dimethyl diphosphonic acid) acetamido) benzene (3). To
a solution of 2 (0.5 g, 0.948 mmol) dissolved in 10 mL
of toluene added phosphorous acid (0.38 g, 4.77 mmol)
under nitrogen atmosphere. The reaction mixture was
refluxed while phosphorous trichloride in a volume
(0.65 g, 4.74 mmol) was added dropwise to the refluxing
mixture. At the end of 3 h toluene was removed follow-
ing addition of water. The reaction mixture was filtered
and concentrated under vacuum. The concentrated
product was precipitated on addition of ethanol.
5. Experimental
5.1. Materials and methods
5.1.1. Chemicals. 5-Nitroisophthalate-1,3-dimethyl ester
was purchased from Fluka, all the chemicals ethylenedi-
amine, bromoacetic acid, phosphorous acid and phos-
phorous trichloride, stannous chloride dihydrate, 5%
Pd/C, ethanol, and methanol (HPLC grade) were pur-
chased from Sigma–Aldrich Co. 99mTc was procured
from Regional Center for Radiopharmaceuticals
(Northern Region), Board of Radiation and Isotope
Technology (BRIT), Department of Atomic Energy,
India. MDP kit is procured from CIS Bio Internation-
al-Schering, France.
Yield: 82% IR (KBr) m cmÀ1 3382, 2999, 2445, 1644,
1542, 1464, 1180, 1014, 939, 530. Mass: MS+ m/e Calcd
671.2, found [M+H+] 672.1, (M+NaÀH) 694.1. 1H
NMR (300 MHz) d 8.78 (2H, Ar, s); 8.56 (1H, Ar, s);
3.34 (4H, t); 2.62 (4H, t), 2.4 (8H, m) Elemental Anal.
Calcd for C16H29N5O16P4: C, 28.63; H, 4.35; N, 10.43;
O, 38.13; P, 18.46. Found: C, 28.62; H, 4.36; N, 10.43;
O, 38.11; P, 18.48.
5.1.2. Instrumentation. The 1H NMR spectra were
recorded by Bruker 300 MHz system. Mass spectrum
was obtained inhouse from ion trap SL 1100 system
(Agilent, Waldbronn, Germany) using ESI positive
and negative mode.
5.2.4. Synthesis of 5-amino-1,3-bis(ethylamine-(N,N
dimethyl diphosphonic acid) acetamido) benzene (4). To
a solution of (3) (100 mg) in water flushed with nitrogen
was taken into schlenk flask under vacuum. Molar
equivalent of 5% Pd/charcoal in nitrogen flushed, water
was added and fitted onto an atmospheric hydrogena-
tor. The apparatus was flushed with H2 (g) twice to fully
saturate the catalyst. The hydrogenation was allowed to
proceed until the uptake of H2 (g) had reached satura-
tion. The reaction mixture was filtered through a bed
of Celite. The filtrate was reduced to dryness by rotary
evaporation.
5.1.3. Animal models. Animal protocols have been ap-
proved by Institutional Animal ethics Committee. New
Zealand Rabbits 2–3.5 kg and BALB/c mice 22–28 g
were used for blood clearance, imaging, and biodistribu-
tion studies.
5.2. Synthesis
5.2.1. Synthesis of 5-nitro-1,3-bis(ethylenediamine) (1).
Dimethyl-5-nitroisophthalate (1 g; 4.18 mmol) was dis-
solved in neat ethylenediamine at 0 °C under nitrogen

1144
P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
Yield: 86% IR (KBr) m cmÀ1 3540, 3350, 3180, 1658,
1551, 1430, 1182, 1010, 928, 510. H NMR (300 MHz)
tered intravenously through the dorsal ear vein of the
animal. At different time interval starting from 5 min
to 24 h persistence of activity in terms of percentage-ad-
ministered dose in samples at different time intervals was
calculated using gamma counter.
1
d 8.2 (2H, Ar, s); 7.4 (1H, Ar, s); 3.30 (4H, t). 2.4 (8H,
m) ESI-MS: Calcd m/e 641.3 for C16H31N5O14P4;
found [M+H+] 642.1, Elemental Anal. Calcd for
C16H31N5O14P4: C, 29.96; H, 4.87; N, 10.92; O, 34.93;
P, 19.32. Found: C, 29.95; H, 4.88; N, 10.93; O, 34.92;
P, 19.31.
5.4.3. Bone scintigraphy. Imaging of rabbit was done at
different time intervals after administering 10 MBq of
the 99mTc-IPTMP intravenously in normal rabbit. A
comparison study was carried out by administerting
99mTc-MDP in rabbits and scintigrams of both the
radiotracers were compared at different time points.
5.3. Radiochemical synthesis of 99mTc-IPTMP
Phosphonic Acid derivative (10.0 lmol) was taken and
stannous chloride 1.0 lmol was added. The pH of the
reaction mixture was adjusted to 6.0–6.5 with 0.5 M
Na2CO3. The mixture was passed through a 0.22 lm
Millipore filter into a sterile vial. 99mTc Eluate contain-
ing 400–800 MBq activity was added and the complex
was incubated for 30 min at room temperature to get
optimum labeling yield. The labeling efficiency was esti-
mated chromatographically using ITLC-SG (instant
thin-layer chromatography-silica gel) paper as the sta-
tionary phase and 100% acetone as the mobile phase.
Percentage of colloids was determined using pyridine/
acetic acid/water (3:5:1.5) as the mobile phase and
ITLC-SG strip as stationary phase. In vitro stability of
the complex was determined chromatographically by
estimating labeling efficiency at different time intervals
up to 24 h.
5.4.4. Biodistribution studies. This parameter was studied
in male BALB/c mice (22–28 g). An aliquot of 3.7 MBq
in each mouse was injected intravenously through the
tail vein. The animals were sacrificed at 1, 4, and 24 h
post-injection. The animals were dissected and the vari-
ous organs were removed, made free from adhering tis-
sue, weighed, and radioactivity was measured in each
organ. The data were expressed as percent administered
dose per gram of the organ.
Acknowledgments
We thank Dr. R. P. Tripathi, Director INMAS, for pro-
viding necessary facilities. The work was supported by
Defence Research and Development Organization,
Ministry of Defense, under R&D project INM–306.
The complexation was determined by titration of the
chelating groups with 111In. A stock solution of carri-
er-free 111InCl3 (20 lL, in sodium acetate buffer, pH
5.5, adjusted with 1 M sodium acetate) was added to a
stock solution (20 lL of 50 mM, 0.1 lmol). Then
16 lL of 0.1 M sodium acetate buffer, pH 7, was added.
The pH was checked to be 7. After 3 h, the mixture was
analyzed by using a TLC system (1:1 MeOH/10%
NH4OAc).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.10.005
5.3.1. DTPA challenge. 99mTc-MDP and 99mTc-IPTMP
(300 MBq) were incubated at different concentration
25, 50, and 100 mM DTPA, and maintained at 37 °C
upto 24 h. Periodically, samples were removed, spotted
on a 10-cm ITLC-SG strip, and developed in 0.9%
NaCl. Once the solvent front had reached the end of
the strip, it was removed from the solvent and cut at
an Rf of 0.5. The two portions were assayed for radio-
activity, and the amount of intact chelate was
determined.
References and notes
1. Bisaz, S.; Jung, A.; Fleisch, H. Clin. Sci. Mol. Med. 1978,
54, 265.
2. Jung, A.; Bisaz, S.; Fleisch, H. Calcif Tissue Res. 1973, 11,
269.
3. Richards, A. G. J. Nucl. Med. 1974, 15, 1057.
4. Fitton, A.; McTavish, D. Drugs 1991, 41, 289.
5. Francis, M. D.; Ferguson, D. L.; Tofe, A. J.; Bevan, J. A.;
Michaels, S. E. J. Nucl. Med. 1980, 21, 1185.
6. Muhlbauer, R. C. In Bisphosphonate on Bones; Bijvoet, O.
¨
5.4. Animal studies
L. M., Fleisch, H. A., Canfield, R. E., Russell, R. G. G.,
Eds.; Elsevier: Amsterdam, 1995; pp 171–187.
7. Jones, A. G.; Francis, M. D.; Davis, M. A. Semin. Nucl.
Med. 1976, 6, 3.
8. Rodan, G. A.; Fleisch, H. J. Clin. Invest. 1996, 97, 2692.
9. Dufau, C.; Benramdane, M.; Leroux, Y.; El Manouni, D.;
Neuman, A.; Prange, T.; Sylvestre, J. P.; Gillier, H.
Phosphorous, Sulfur Silicon Relat. Elem. 1995, 107, 145.
5.4.1. Toxicity studies. Toxicity study was carried out in
groups of 5 male BALB/c rats. In the experiment, a sin-
gle dose of IPTMP was administered iv into groups of
five male BALB/c mice of 22–28 g. The dosage levels
were 30–280 mg/kg body weight. The animals are
weighed weekly and observed for clinical signs of toxic-
ity and number of deaths that occurred in 4 weeks was
recorded after treatment.
´
10. Kubicek, V.; Vojtısek, P.; Rudovsky, J.; Hermann, P.;
Lukes, I. Dalton Trans. 2003, 20, 3927.
11. Davis, M.; Jones, A. G. Semin. Nucl. Med. 1976, 6, 19.
12. Arteaga de Murphy, C.; Melendez-Alafort, L.; Montoya-
Molina, C. E.; Sepulveda-Mendez, J. Nucl. Med. Biol.
1997, 24, 27–33.
5.4.2. Blood clearance. In normal rabbit weighing about
2–2.5 kg, 10 MBq of the 99 mTc-IPTMP was adminis-

P. Panwar et al. / Bioorg. Med. Chem. 15 (2007) 1138–1145
1145
13. Volkert, W. A.; Edwards, B.; Simon, J.; Wilson, D. A.;
McKenzie, E. H.; Oberle, P.; Holmes, R. A. Int. J. Radiat.
Appl. Instrum. B 1986, 13, 31.
22. Bailya, T.; Burgada, R.; Lecouvey, M.; Neuman, A.;
´
Prange, T. Phosphorous, Sulfur Silicon Relat. Elem. 1995,
101, 131–140.
14. Pauwels, E. K.; Blom, J.; Camps, J. A.; Hermans, J.;
Rijke, A. M. Eur. J. Nucl. Med. 1983, 8, 118.
15. Vorne, M.; Vahatalo, S.; Lantto, T. Eur. J. Nucl. Med.
1983, 8, 395.
16. de Klerk, J. M. H.; van Dijk, A.; van Rijk, P. P.;
Zonnenberg, B. A.; van het Schip, A. D. J. Nucl. Med.
1991, 32, 1082.
17. Maxon, H. R., III; Deutsch, E. A.; Thomas, S. R.; Libson,
K.; Lukes, S. J.; Williams, C. C.; Ali, S. Radiology 1988,
166, 501–507.
18. Lin, W. Y.; Lin, C. P.; Yeh, S. J.; Hsieh, B. T.; Tsai, Z. T.;
Ting, G.; Yen, T. C.; Wang, S. J.; Knapp, F. F., (Russ)Jr.;
Stabin, M. G. Eur. J. Nucl. Med. 1997, 24, 590.
19. Goeckeler, W. F.; Edwards, B.; Volkert, W. A.; Holmes,
R. A.; Simon, J.; Wilson, D. J. Nucl. Med. 1987, 28, 495.
20. Hamdy, N. A.; Papapoulos, S. E. Semin. Nucl. Med. 2001,
31, 62.
23. Mishra, A. K.; Chopra, M.; Jain, V. Chem. Lett. 2005, 34,
1098–1099.
24. Franz, R. G. AAPS Pharmsci. Tech. 2001, 3, article 10.
25. Even, P.; Guenin, E.; Benramdame, M.; Quidu, P.;
Manouni, D. E.; Lecouvey, M. Lett. Org. Chem. 2004,
1, 75.
26. Ali, M. S.; Quadri, S. M. Bioconjugate Chem. 1996, 7, 576.
27. Sundberg, M. W.; Meares, C. F.; Goodwin, D. A.;
Diamanti, C. I. J. Med. Chem. 1974, 17, 1304.
28. Kunzmann, V.; Bauer, E.; Feurle, J.; Weißinger, F.; Tony,
H.-P.; Wilhelm, M. Blood 2000, 96, 384–392.
29. Schomacker, K.; Mocker, D.; Munze, R.; Beyer, G. J. Int.
J. Rad. Appl. Instrum. 1988, 39, 261–264.
30. Turner, J. H.; Martinadale, A. A.; DeWitt, C.; Webb, J.;
Sorby, P.; Boyd, R. E. Eur. J. Nucl. Med. 1987, 13, 432.
31. Vegt, E.; Wetzels, J. F. M.; Russel, F. G. M.; Masereeuw,
R.; Boerman, O. C.; van Eerd, J. E.; Corstens, F. H. M.;
Oyen, W. J. G. J. Nucl. Med. 2006, 47, 432–436.
21. Moedritzer, K.; Irani, R. J. Org. Chem. 1966, 31, 1603.