7
930
A. Mathur et al. / Bioorg. Med. Chem. 16 (2008) 7927–7931
the major fraction (61%) was isolated, by HPLC, and evaluated in
animal model. Biodistribution studies in swiss mice showed up-
take of the complex in the myocardium, but was found to be lower
v/v) = 0.3; IR (neat, cmꢀ1) 3056 (w); 2919 (s); 2849 (m); 1740
(s); 1651 (s). 1H NMR (CDCl
, d ppm) 7.20–7.39 (m, 15H,
(C C); 5.96–6.00 (d, 1H, CONH); 4.58–4.62 (m, 1H, NH–CH–
–S); 4.10–4.20 (q, 2H, COOCH CH ); 2.59–2.63 (m, 2H, –CH–
S); 2.28–2.36 (t, 2H, –CH CH CONH); 2.11–2.18 (t, 2H, –
CH COOH); 1.58 (s, 4H, –CH –CH –COOH and –CH CH CONH);
10& COOCH CH ). The ester and trityl
deprotection of the fatty acid–cysteine conjugate were carried
3
6
5 3
H )
1
25
than that of
I-IPPA. Retention of activity in myocardium was
CH
CH
CH
2
2
2
2
3
also found to be lower for the complex under investigation. An
interesting observation which emerges from the in vivo studies is
the rapid washout from liver and lungs, an important factor that
determines the critical ratios, namely, heart/lung and heart/liver,
which could lead to significant improvement in the quality of the
image. The results indicate the need for synthetic modification of
the parent fatty acid molecule with a view to increasing its resi-
dence time in the myocardium, with retention of its other favor-
able features.
2
2
2
2
2
2
2
1.20–1.24 (s, 23H, (CH
2
)
2
3
1
8
out as reported in the literature. The target ligand formed was
used as such for radiolabeling without further characterization.
4.2. Radiolabeling
4
.2.1. Preparation of 99mTcN(PNP) fatty acid complex
About 5 mg of succinic dihydrazide, 0.1 mg of stannous chlo-
4
4
4
. Experimental
.1. Synthesis
ride, and 250
l
L of ethanol were taken in a vial to which 750
lL
9
9m
ꢀ
of
TcO4 (1.85 GBq; 50 mCi) was added. The reaction mixture
was kept at room temperature for 20 min for formation of
9
9m
2+
[
TcN] intermediate. To this intermediate, ꢁ3
lL of PNP6 li-
.1.1. General
gand and 4 mg of fatty acid cysteine conjugate, each dissolved in
Hexadecanedioic acid, ethyl p-toluene sulfonate, S-trityl cys-
2
50
lL of ethanol (purged with nitrogen before use), were added
teine, triethyl silane, succinic dihydrazide and stannous chloride
were obtained from Aldrich, USA. EDCI was purchased from Fluka,
Germany. Iodophenyl pentadecanoic acid (IPPA) was purchased
from Emka Chemicals, Germany. All other reagents used were of
analytical grade. Solvents used for syntheses were dried as per
the standard procedure prior to use. Sodium pertechnetate
simultaneously and the reaction mixture was heated at 90 °C for
0 min. The final complex was characterized by HPLC.
3
4
.2.2. Preparation of 125I-IPPA
The labeling of IPPA with 125I was carried out by heating 2–3 mg
9
9m
of IPPA with 68
l
g of CuSO4.5H
2
O, 4 mg sodium metabisulfite,
L (ꢁ185 MBq; 5 mCi) of
Na I to a temperature of 160 °C for 35–40 min. After cooling,
mL of 0.76 M sodium acetate solution was added to the reaction
(
Na
TcO
4
) was eluted with saline just before use from a
9
9
Mo–99mTc gel generator supplied by Board of Radiation and Iso-
5
0
l
L of glacial acetic acid, and 50 l
1
25
1
25
tope Technology, India. Na I was obtained from Radiochemicals
section, Radiopharmaceuticals Division, BARC, India. Bis[(diethoxy-
propylphosphanyl)ethyl]ethoxyethylamine (PNP6) was obtained
as a gift as a part of a Coordinated Research Project. Silica gel plates
2
mixture. Labeling yield was ascertained by paper electrophoresis
and HPLC. The labeled product was separated from unreacted io-
dide by solvent extraction, using dichloromethane. The organic ex-
tract was flushed with nitrogen to remove dichloromethane and
reformulated in 5% ethanol. The pure product was again character-
ized by paper electrophoresis and HPLC.
(
1
Silica Gel 60 F254) were obtained from Merck, India. Whatman No.
chromatography paper was used for paper electrophoresis. Elec-
trophoresis was carried out at 300 V using 0.05 M sodium acetate
solution for 30 min. HPLC characterization of the prepared com-
plex was carried out on a JASCO PU 1580 HPLC system, with a JAS-
CO 1575 tunable absorption detector and radiometric detector
system, using a C18 reversed phase HiQ Sil (5 mm, 4 ꢂ 250 mm)
column and water (A): acetonitrile (B) mixture as the mobile phase
under gradient elution (0 min 90% A, 28 min 10% A, 50 min 10% A).
Characterization of
tion using acetonitrile (0.1% TFA): water (0.1% TFA) (90:10) mix-
ture as the mobile phase. IR spectra of the synthesized ligands
4
4
.3. Stability studies
.3.1. Ligand exchange studies
The stability of the complex toward ligand exchange was stud-
1
25
ied using cysteine as the challenging ligand. For this, ꢁ100
lL
I-IPPA was carried out under isocratic condi-
9
9m
TcN(PNP) fatty acid complex was added to 900 lL of phos-
phate-buffered saline containing 10–500-fold molar excess of cys-
teine over that of the ligand. The samples were incubated at 37 °C
for 60 min and then analyzed by HPLC.
were recorded on a JASCO FT-IT/420 spectrophotometer, Japan.
1
H
NMR spectra were recorded on
a
200 MHz Bruker
spectrophotometer.
4
.3.2. Serum stability
4
.1.2. Synthesis of fatty acid cysteine conjugate
Stability of the 99mTcN(PNP) fatty acid complex in serum was
About 100 mg (0.35 mmol) of hexadecanedioic acid and 136 mg
tested in vitro. About 50
lL of the radiolabeled preparation was
(
0.35 mmol) of S-trityl cysteine ester, synthesized following a re-
added to 450 L serum, and the mixture was incubated at 37 °C
l
1
8
ported procedure, were dissolved in 15 mL of dry dichlorometh-
ane, in 100 mL round-bottomed flask, under nitrogen
atmosphere. The reaction mixture was cooled to 0 °C after which
5 mg (0.38 mmol) of EDCI, dissolved in minimum amount of di-
for 60 min. To this mixture, equal volume of cold ethanol was
added to precipitate the serum proteins and centrifuged at
10,000g (4 °C, 20 min). The supernatant was analyzed by HPLC to
assess stability of the complex in serum. The precipitate was
washed twice with ethanol and counted in the NaI(Tl) scintillation
detector to determine the activity associated with serum protein.
a
7
methyl formamide, was added and the reaction mixure kept stir-
ring at 0 °C for one hour. The reaction mixture was then brought
to room temperature and the reaction continued overnight. The
progress of the reaction was monitored by TLC. Upon completion
of the reaction, the solvent was removed under vacuum, water
was added and extracted with dichloromethane (3 ꢂ 10 mL). The
dichloromethane extracts were pooled, dried over anhydrous so-
dium sulfate, and filtered. The filtrate was then evaporated to yield
the crude coupled product, which was purified on silica gel column
4.4. Biodistribution studies
Normal Swiss mice (20–25 g body weight) were used for the
9
9m
in vivo distribution studies of
and
TcN (PNP)–fatty acid complex
I-IPPA. Prior to the experiment, the mice were fasted over-
night, although water was given ad libitum. About 100
(ꢁ1.85 MBq; 50 Ci) of the prepared complex was administered
intravenously in each animal. Different sets of animals (n = 3) were
1
25
lL
using 10% ethyl acetate–chloroform mixture. The product was
characterized using H NMR. R
l
1
f
(ethylacetate:chloroform, 10:90;
Mathur, Anupam
