Synthesis and evaluation of bifunctional chelating agents derived from bis(2-aminophenylthio)alkane for radioimaging with 99mTc

Article abstract of DOI:10.1016/j.bmc.2005.04.073

Novel bifunctional chelating agents bearing an aromatic rigid backbone have been synthesized and characterized on the basis of spectroscopic techniques. These macrocyclic multidentate chelating agents were conjugated with monoclonal antibody which forms stable complexes with ^99mTc with high radiochemical purity.

Full text of DOI:10.1016/j.bmc.2005.04.073

Bioorganic & Medicinal Chemistry 13 (2005) 4713–4720
Synthesis and evaluation of bifunctional chelating
agents derived from bis(2-aminophenylthio)alkane
for radioimaging with 99mTc
Bhupender S. Chhikara,a,b Nitin Kumar,b Vibha Tandona,* and Anil K. Mishrab,*
aDr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
bDepartment of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences, DRDO,
Brig. S.K. Mazumdar Marg, Delhi 110054, India
Received 31 March 2005; revised 20 April 2005; accepted 28 April 2005
Available online 26 May 2005
Abstract—Novel bifunctional chelating agents bearing an aromatic rigid backbone have been synthesized and characterized on the
basis of spectroscopic techniques. These macrocyclic multidentate chelating agents were conjugated with monoclonal antibody
which forms stable complexes with 99mTc with high radiochemical purity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, many groups have started exploring the aro-
matic ring based chelating agents. In aromatic BFCs,
besides the increased rigidity in framework, the intro-
duction of a nitro group (required for functionalization
by converting in amine group) is comparatively easier.14
Bifunctional chelating agents (BFCs), the central mole-
cules meant for attaching the radionuclide to biomole-
cules, have gained widespread interest because of their
utility in radiodiagnostic and radiotherapeutic applica-
tions.1–3 Bifunctional chelating agent is key to successful
application of receptor–ligand (biomolecule) based
targeted radiopharmaceuticals and there is continued
interest in the design and development of new bifunc-
tional chelating agents for effective coordination of
radioactive metals.4–9 Bifunctionalized EDTA was the
first chelating agent evaluated for scintigraphy.10 The
metal complex with EDTA had very low stability under
in vivo conditions. Alternatively, bifunctionalized mac-
rocyclic chelating agents based on cyclam and cyclene
framework have been successful for in vivo applications,
as complexes of macrocyclic chelating agents are more
stable than acyclic chelating agents.11,12 As a general
rule, complexes with preorganized structures have high-
er stability under physiological conditions. Accordingly,
many modifications have been introduced in the struc-
tures of chelating moieties, such as introduction of bulky
methyl and ethyl groups and synthesis of cyclohexane
and cyclopentane ring based chelating agents.13
Considering the above aspects, we herein report the syn-
thesis and evaluation of macrocyclic bifunctional chelat-
ing agents based on an aromatic ring (5a–c) (N2S2 and
N2S2O system) derived from bis(2-aminophenylthio)al-
kane. The initial complexation studies with these BFCs
were performed with 99mTc radionuclide.
2. Results and discussion
Designing and synthesis of bifunctional chelating agents
(BFCs) are crucial for efficient in vivo application of radi-
olabeled bioconjugates. BFCs, having rigid backbones as
exemplified by the classical trans-cyclohexylethylenedi-
aminetetraacetic acid (CDTA) ligand, provide enhanced
kinetic inertness relative to flexible parent ligands.15 Sim-
ilarly, for a series of Y(III) complexes of eight coordinate
DTPA type ligands which included cyclohexyl and benzyl
moieties in the backbone, the observed acid catalyzed
dissociation rate constant varied by up to 3 orders of
magnitude.16 In this study, macrocyclic BFCs with in-
creased rigidity for complex stability besides requisite
flexibility for complexation along with variation in ring
size and donor atoms were studied for radiodiagnostic
*
Corresponding authors. Tel.: +911123914374; fax: +911123919509
(A.K.M.); e-mail addresses: vibhadelhi@hotmail.com; akmishra@
inmas.org
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.073

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B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
and radiotherapeutic applications. Bifunctional chelating
agents 5a–c were synthesized by two different synthetic
routes as depicted in Schemes 1 and 2.
of K2CO3 (anhydrous) and TBAÆHSO4 (tetrabutylam-
monium hydrogen sulfate as phase transfer catalyst)
gave macrocycle 3a (yield 42%) mp 245 ꢁC. 3a–c were
selectively reduced by diborane to synthesize 4a–c mac-
rocycles. Reduction of the nitro group of 4a–c with
Pd/C/H2 yielded 5a–c in very low yield besides separa-
tion of product from reaction mixture being tedious.
The Gowda and Gowda18 method for reduction of nitro
group was the only successful method by which the
product was easily obtained in high yield. In Scheme
1, the cyclization was performed under high dilution
conditions to increase the yield of macrocycle. The cycli-
zation reaction was followed by selective reduction
of the carbonyl and nitro groups present on the macro-
cyclic moiety and as both these reactions involved the
low yielded macrocycles, this effectively lowered the final
yield of macrocyclic BFCs. Alternatively another
The bis(2-aminophenylthio)alkanes were prepared from
2-aminothiophenol by the reaction of respective dibro-
moalkane and ditosylates. In one of the earlier attempts
to cyclize the diamines (1a–c) with bromoacetyl bro-
mide, 3-oxo-3,4-dihydro-2H-1,4-benzothiazine was ob-
tained quantitatively.17 Another approach to obtaining
the macrocycles prepared using derivatives of 5-nitro-
isophthalic acid are shown in the schemes. As described
in Scheme 1 the 5-nitroisophthalic acid was converted
into diacidchloride (2) with PCl5 and used for cycliza-
tion of 1a–c to obtain 3a–c. The slow addition of
5-nitroisophthaloyl dichloride (2) to a solution of
diamine 1a in dichloromethane containing a suspension
X
SH
S
S
NH2
NH2 H2N
X = 0 = 1a
X = CH2 = 1b
X = -CH2-O-CH2 = 1c
K2CO3/
CH2Cl2
Cl
Cl
X
O
O
OH
PCl5
OH
S
S
O
O
NO2
NH
HN
2
O
O
NO2
X = 0 = 3a
X = CH2 = 3b
X = CH2-O-CH2 = 3c
NO2
B2H6/THF
X
X
S
S
S
S
Zn dust/
NH2NH2
NH
HN
NH
HN
X = 0 = 4a
X = CH2 = 4b
X = 0 = 5a
X = CH2 = 5b
X = CH2-O-CH2 = 5c
X = CH2-O-CH2 = 4c
NO2
NH2
Scheme 1.

B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
Cl
4715
Cl
OH
OH
R
R
O
O
PX3
NaBH4
NO2
NO2
NO2
7a R = Cl
7b R = Br
2
6
X
S
S
K2CO3/DMF
NH2 H2N
X = 0 = 1a
X = CH2 = 1b
X = CH2-O-CH2 = 1c
X
X
S
S
S
S
Zn dust/ NH2NH2
NH
HN
NH
HN
NO2
NH2
X = 0 = 4a
X = 0 = 5a
X = CH2 = 4b
X = CH2 = 5b
X = CH2-O-CH2 = 4c
X = CH2-O-CH2 = 5c
Scheme 2.
synthetic route was followed in which the reduction of
carbonyl group was done prior to cyclization reaction as
depicted in Scheme 2. The 5-nitroisophthaloyl dichloride
(2) was reduced to dialcohol by sodium borohydride,
which was further converted into dihalide with phospho-
rus trihalides. The synthesis of dihalides from diol, the
dibromo product 7b, was obtained in very high yield
compared to dichloro 7a. The cyclization of 1b with bis-
3,5-bromomethylnitrobenzene in dry DMF at 120 ꢁC in
the presence of K2CO3 and TBAÆHSO4 gives 4b (62%).
vent conditions. The complexes were stable in dilute sal-
ine solution and in serum under physiological conditions
as expected for backbone substituted rigid macrocyclic
chelating agents. In serum, neither transchelation of
radioactive metal ions nor metal ion transfer to serum
proteins was observed. The final chelating agents (5a–
c) are bifunctional bearing a free amine group that
was used for conjugation with monoclonal antibodies
by converting it to –NCS, which can be extended to cou-
ple with other biologically active peptides.
All the chelating agents (5a–c) are stable at room tem-
perature and do not require any specialized condition
for long-term storage. The radiocomplexation studies
of these BFCs (5a–c) with 99mTc were performed in a bi-
phasic state. Complexation of chelating agent with
99mTc was done by reduction of pertechnetate with stan-
nous chloride (SnCl2Æ2H2O) in the presence of chelating
agents. Radiocomplexation was completed in 40–50 min
with a radiochemical purity of 92% (5a), 94% (5b), and
98% (5c) as monitored by ITLC-SG using different sol-
The synthesis of bifunctional chelating agent (5a–c) is
shown in Schemes 1 and 2. Synthesis of bifunctional
chelating agents was performed as described in Section
4.1. The –NCS derivatives were found to be stable when
stored at À20 ꢁC in 0.3 M HCl.
Conjugation of mAbs with chelates was performed in
the ratio of 1:20. UV absorbance and the 57Co binding
assay indicated that 2.1 0.1 chelate molecules were
conjugated per antibody molecule.

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B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
The conjugated antibodies were labeled with a specific
activity 20–30 mCi/mg of protein. Labeling efficiencies
were measured by ascending paper chromatography on
ITLC-SG strips. Results of radiolabeling of the immu-
noconjugates were found to be 98.5 0.30%. ITLC-
SG results in acetone showed that 1.5–2% or less free
pertechnetate ran with the solvent front (Rf = 0.7–1.0).
This indicated that pertechnetate was reduced almost
entirely. Using 10% NH4Ac and methanol 1:1 as a sol-
vent for migration showed all the activity on the base
of ITLC-SG strips indicating the radiolabeled immuno-
conjugate, not the other species.
was carried out on a Bruker 400 MHz NMR instrument
operating near 400 (1H) and 100 (13C) MHz, and a Bru-
ker 300 MHz NMR instrument operating near 300 MHz
(1H) and 75 MHz (13C). The FAB-MS spectra were
recorded at Central Drug Research Institute, India, on
a JEOL SX 102/DA-6000 mass spectrometer using
m-nitrobenzyl alcohol as matrix. EI-MS spectra were
recorded on a JEOL SX102/DA (KV 10 mA) instru-
ment. Elemental analysis was done on the Elementar
Analysensysteme GmbH VarioEL system. Radiocom-
plexation and radiochemical purity were checked by
instant strip chromatography (silica gel impregnated
paper chromatography) with ITLC-SG (Gelman Sci-
ences, Ann Arbor, MI, USA). The gamma scintillation
counting was done on an ECA (Electronic Corporation
of India Ltd) Gamma Ray Spectrometer K2700B.
Radiolabeled immunoconjugates were challenged with
(25–100 mM) DTPA and cysteine to test the stability
of the radiolabeled antibodies. Macrocyclic chelating
agents—ior egf/r3—showed only 2–3% transcomplex-
ation. Approximately more than 97% of the radioactiv-
ity remains associated with the antibody after 24 h
challenge at 37 ꢁC with DTPA. Immunoreactivity of
immunoconjugate was determined on a phase grafted
with antigens. The immunoreactivity of 125I-labeled ior
egf/r3 and CB-CEA-1 using the iodogen method19 was
examined as a reference.
4.2. Synthesis
4.2.1. 1,2-Bis(2-aminophenylthio)ethane (1a). The 1,2-
dibromoethane (3.7 g, 20 mmol) was added dropwise
to a refluxing solution of 2-aminothiophenol (5 g,
40 mmol) and sodium ethoxide (3.7 g, 50 mmol) in dry
ethanol (20 mL). The refluxing reaction mixture was
stirred for 6–7 h and it was monitored by TLC. On com-
pletion of reaction the solvent was removed in vacuo
and the reaction mixture was cooled to 0 ꢁC. Water
(50 mL) was added and the reaction mixture was
extracted with dichloromethane (20 mL · 4). The organ-
ic layer was dried over anhydrous Na2SO4 and evapo-
rated to dryness in vacuo to get a solid product, which
on recrystallization from ethanol gave white colored
solid (4.3 g, 78%), mp 72–73 ꢁC. IR (KBr pellets,
cmÀ1) 3385, 3356, 3290, 3018, 2988, 2925, 1617, 1582,
Immunoreactivity was greater than 80% for 99mTc-
labeled anti-EGFr conjugated with chelating agents.
These values are equivalent to those of 125I-labeled
counterparts, indicating that biological activities were
not compromised after modification with a bifunctional
chelating agent.
3. Conclusions
1
1479, 1446, 749. H NMR (60 MHz, CDCl3) d ppm:
In conclusion, we here describe the synthesis of new aro-
matic bifunctional chelating agents (5a–c) with different
central cavity sizes and donor atoms. The complexes
formed by these chelating agents are highly stable under
physiological conditions. New chelating agents can be
effectively used for targeted scintigraphy with 99mTc by
conjugation with biomolecules because biological activ-
ities were not compromised after modification with
bifunctional chelating agent.
6.70–7.50 (m, 8H, ArH), 4.30 (br s, 4H, 2· NH2,
exchanged with D2O), 2.80 (s, 4H, 2· S–CH2). Anal.
Calcd for C14H16N2S2: C, 60.83; H, 5.83; N, 10.13; S,
23.20. Found: C, 60.63; H, 5.76; N, 10.60; S, 23.01.
FAB-MS: Found: m/z 276 [M]+Å. Calcd for C14H16N2S2:
276. EI-MS: 276, 138, 124 (base peak), 94.
4.2.2. 1,3-Bis(2-aminophenylthio)propane (1b). The 1,3-
dibromopropane (4.02 g, 20 mmol) was added dropwise
to a refluxing solution of 2-aminothiophenol (5 g,
40 mmol) and sodium methoxide (3.2 g, 50 mmol) in dry
methanol (20 mL). The refluxing reaction mixture was
stirred for 6–7 h and it was monitored by TLC. On com-
pletion of reaction the solvent was removed in vacuo
and the reaction mixture was cooled to 0 ꢁC. Water
(50 mL) was added and the reaction mixture was ex-
tracted with dichloromethane (20 mL · 4). The organic
layer was dried over Na2SO4 and evaporated to dryness
in vacuo to get a crude product, which was further purified
by column chromatography [column of SiO2 (100 g); pre-
adsorption of the residue at SiO2 (ca. 8 g) with ethyl
acetate; elution with petroleum ether/ethyl acetate 60:40
(v/v)] to obtain transparent oily liquid (4.5 g, 82%). IR
(NaCl plates, cmÀ1) 3434, 3355, 3018, 2988, 2925, 1606,
4. Experimental
4.1. Materials and methods
All chemicals and reagents used in present study were of
analytical grade. TLC was run on the silica gel coated
aluminum sheets (silica gel 60 F254, E. Merck, Germany)
and visualized in UV light (254 nm). Melting point
was determined with a Buchi B540 instrument. The
corresponding 3,5-bis-bromoethylnitrobenzene was
synthesized by following the reported procedure.20
2-Aminothiophenol was procured from Acros Organics
(USA) and distilled prior to use. 5-Nitroisophthalic acid
was synthesized by nitration of isophthalic acid essen-
tially following the reported procedure.21,22 IR spectra
were recorded on the FT-IR Perkin Elmer Spectrum
BX spectrophotometer. NMR spectral characterization
1
1478, 1448, 1301, 748. H NMR (400 MHz, CDCl3) d
ppm: 7.20 (dd, Jab = 7.5 Hz, Jac = 1.5 Hz, 2H, ArH),
7.00 (dt, Jab = 7.6 Hz, Jac = 1.4 Hz, 2H, ArH), 6.68 (dd,
Jab =8.0 Hz, Jac = 1.2 Hz, 2H, ArH), 6.64 (dt, Jab
=

B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
4717
7.6 Hz, Jac = 1.2 Hz, 2H, ArH), 4.10 (br s, 4H, 2· NH2,
exchanged with D2O), 2.80 (t, J = 7.1 Hz, 4H, 2·
S–CH2–), 1.75 (quintet, J = 7.1 Hz, 2H, S–C–CH2–).
Anal. Calcd for C15H18N2S2: C, 62.03; H, 6.25; N, 9.64;
S, 22.08. Found: C, 62.14; H, 6.26; N, 9.82; S, 21.88.
FAB-MS: Found: m/z 290 [M]+Å. Calcd for C15H18N2S2:
290. EI-MS: 290, 166, 138, 124 (base peak), 94.
chromatographed over silica gel (EtOAc/Pet ether
40:60) to obtain a light yellowish solid product (1 g,
70%). mp 91–92 ꢁC. IR (KBr pellets, cmÀ1) 3290,
3207, 1533, 1342, 1035, 773, 746, 678. 1H NMR
(300 MHz, DMSO-d6) d: 8.0 (s, 2H), 7.6 (s, 1H), 5.5
(t, J = 5.5 Hz, 2H, exchanged with D2O), 4.6 (d,
J = 5.5 Hz, 4H). 13C NMR (75 MHz, DMSO-d6) d:
148.7, 145, 131.3, 119.8, 62.7. Anal. Calcd for
C8H9NO4: C, 52.46; H, 4.95; N, 7.65; O, 34.94. Found:
C, 52.28; H, 4.89; N, 7.71. EI-MS: Calcd for C8H9NO4:
183. Found: m/z 183 [M]+Å.
4.2.3. 1,5-Bis(2-aminophenylthio)-3-oxapentane (1c). The
diethyleneglycolditosylate (8.2 g, 20 mmol) was added
dropwise to a refluxing solution of 2-aminothiophenol
(5 g, 40 mmol) and sodium methoxide (3.2 g, 50 mmol)
in dry methanol (20 mL). The refluxing reaction mixture
was stirred for 6–7 h and it was monitored by TLC. On
completion of reaction the solvent was removed in va-
cuo and the reaction mixture was cooled to 0 ꢁC. Water
(50 mL) was added and the reaction mixture was ex-
tracted with dichloromethane (20 mL · 4). The organic
layer was dried over Na2SO4 and evaporated to dryness
in vacuo to get a crude product, which was further puri-
fied by column chromatography [column of SiO2
(100 g); pre-adsorption of the residue at SiO2 (ca. 8 g)
with ethyl acetate; elution with petroleum ether/ethyl
acetate 60:40 (v/v)] to obtain viscous liquid (4.56 g,
72%). IR (KBr pellets, cmÀ1) 3432, 3349, 3058, 2921,
2855, 1607, 1479, 1105, 748. 1H NMR (300 MHz,
CDCl3) d ppm: 7.30 (d, J = 7.5 Hz, 2H, ArH), 7.10 (t,
J = 7.5 Hz, 2H, ArH), 7.00 (d, J = 7.5 Hz, 2H, ArH),
6.60 (t, J = 7.8 Hz, 2H, ArH), 4.00 (br s, 4H, 2· NH2,
exchanges with D2O), 3.30 (t, J = 7.1 Hz, 4H, 2·
O–CH2–), 2.73 (t, J = 7.0 Hz, 4H, 2· S–CH2). 13C
NMR (CDCl3) d ppm: 32.15, 67.32, 112.71, 114.06,
114.97, 127.81, 133.97, 148.19. Anal. Calcd for
C16H20N2OS2: C, 59.96; H, 6.29; N, 8.74; O, 4.99; S,
20.01. Found: C, 59.66; H, 6.08; N, 8.44; S, 20.00.
EI-MS: Calcd for C16H20N2OS2: 320. Found: m/z 320,
319, 227, 195, 136 (base peak), 166, 94, 57.
4.2.6. 3,5-Bis-chloromethylnitrobenzene (7a). 3,5-Bis-
hydroxymethylnitrobenzene (1 g, 5.4 mmol) was dis-
solved in ether (30 mL) with stirring and cooled to
0 ꢁC. PCl3 (1 mL) dissolved in ether (20 mL) was added
to the above reaction mixture and stirred at 30–35 ꢁC for
36 h. The reaction mixture was poured onto crushed ice
and extracted with ether. The organic layer was washed
with Na2CO3 sol and water, and dried over Na2SO4.
Whitish solid product is obtained on removal of solvent
(200 mg, 20%). mp 67–68 ꢁC. IR (KBr pellets, cmÀ1):
2923m, 1529s, 1361s, 1213m, 777w, 746w, 686s. 1H
NMR (60 MHz, CDCl3) d: 8.2 (d, J = 3.6 Hz, 2H), 7.8
(s, 1H), 4.7 (s, 4H).
4.2.7. 3,5-Bis-bromomethylnitrobenzene (7b). 3,5-Bis-
hydroxymethylnitrobenzene (1 g, 5.4 mmol) was dis-
solved in ether (30 mL) with stirring and cooled to
0 ꢁC. PBr3 (1 mL) dissolved in ether (20 mL) was added
to the above reaction mixture and stirred at the same
temperature for 6 h and then at ambient temperature
for 12 h. The reaction mixture was poured onto crushed
ice and extracted with ether (20 mL · 5). The organic
layer was washed with Na2CO3 solution and water,
and dried over Na2SO4. White solid product is obtained
on removal of solvent (1.6 g, 97%). mp 102–103 ꢁC (lit.23
mp 103–104 ꢁC). IR (KBr pellets, cmÀ1): 3067vw,
1
4.2.4. 5-Nitroisophthaloyl dichloride (2). 5-Nitroiso-
phthalic acid (5 g, 23.7 mmol) and phosphorus penta-
chloride (PCl5) (10 g, 48 mmol) were mixed in a dry
flask and heated to 120 ꢁC. The molten solid reaction
mixture was stirred at the same temperature for 2 h.
POCl3 formed was distilled off under vacuum and resid-
ual mixture on cooling gave colorless solid acid chloride
product (Note: in some repeat reactions, acid chloride
remained as a colorless syrupy liquid) (6.5 g, 92%)). IR
(KBr pellets, cmÀ1): 3088m, 1756vs, 1622m, 1535s,
1349s, 1145s, 1005s, 734m, 703s, 680s.
3033vw, 2919w, 1530vs, 1361m, 1214,656m, 607m, H
NMR (300 MHz CDCl3) d: (d, J = 1.5 Hz, 2H), 7.75
(t, J = 1.5 Hz), 4.52 (S, 4H). 13C NMR (75 MHz,
CDCl3) d: 148.6, 140.4, 135.2, 123.6, 30.6. Anal. Calcd
for C8H7Br2NO2: C, 31.10; H, 2.28; N, 4.53. Found:
C, 31.28, 2.44; N, 4.36. EI-MS: Calcd for C8H7Br2NO2:
308. Found: m/z 308 [M]+Å.
4.2.8. General procedure for the synthesis of macrocycles
3a, 3b, and 3c. 1,3-Bis(2-aminophenylthio)propane
(2.9 g, 10 mmol) in 150 mL dry dichloromethane and
5-nitroisophthaloyl dichloride (2.5 g, 10 mmol) in
150 mL dichloromethane were added simultaneously
dropwise to a vigorously stirring reaction mixture of
K2CO3 (2.8 g, 20 mmol) and TBAÆHSO4 (20 mg) in
200 mL dry dichloromethane at ambient temperature.
After complete addition, the reaction mixture was fur-
ther stirred for an additional 1 h. Solvent was removed
under reduced pressure and the obtained solid product
was washed with water and dried under vacuum, which
resulted in a puff-colored solid product.
4.2.5. 3,5-Bis-hydroxymethylnitrobenzene (6). 5-Nitroi-
sophthaloyl dichloride (2 g, 8.0 mmol) in dry diglyme
(10 mL) was added dropwise with stirring (mechanical)
to sodiumborohydride (0.92 g, 24 mmol) solution in dig-
lyme (20 mL) at 0 ꢁC under nitrogen atmosphere. After
complete addition of acid chloride, the reaction mixture
was stirred at ambient temperature for 7 h. The reaction
mixture was cooled to 0 ꢁC and treated with 1 N HCl
(aq) to destroy excess NaBH4. The resulting mixture
was concentrated to remove diglyme, added distilled
water (100 mL), and extracted with ethyl acetate
(40 mL · 5). The organic phase was dried over Na2SO4
and concentrated to afford a thick liquid which was
4.2.8.1. Macrocycle 3a. (1.9 g, 42%.) IR (KBr pellets,
cmÀ1) 3276m (N–H str), 1680s (C@O str), 1653s, 1579s,
1513s, 1436s, 1348s, 757m, 722w, 681w. 1H NMR

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B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
(300 MHz, DMSO) d ppm: 10.1 (s, 1H, ArH), 8.9 (d,
J = 1.24 Hz, 2H, ArH), 8.6 (d, J = 8.32 Hz, 2H, ArH),
7.6 (dd, J = 7.7 Hz, J = 1.48, 2H, ArH), 7.4 (dt,
J = 8.04, J = 1.44 Hz, 2H, ArH), 7.1 (dt, J = 7.4 Hz,
J = 1.44 Hz, 2H, ArH), 2.8 (s, 4H, 2· S–CH2–). Anal.
Calcd for C22H17N3O4S2: C, 58.52; H, 3.97; N, 9.31;
O, 14.17; S, 14.20. Found: C, 58.31; H, 3.89; N, 9.45;
S, 14.19. FAB-MS: Calcd for C22H17N3O4S2: 451.
Found: m/z 452 [M+H]+.
solid left triturated with water thoroughly, and the water
phase decanted away. Residual solid was again washed
with methanol (20 mL) and dried under vacuum to
afford a yellow solid product (4b).
4.2.10.1. Macrocycle 4a. (2.2 g, 52%.) mp 222–224 ꢁC
IR(KBr, cmÀ1): 3384m (N–H str), 1586m, 1524s (NO2
asym str), 1498s, 1350s (NO2 sym str), 1311s, 743s. H
1
NMR (300 MHz, DMSO-d6) d ppm: 8.1 (s, 2H, ArH),
7.5 (s, 1H, ArH), 4.5 (d, J = 6 Hz, 4H, Ar–CH2–N),
7.2 (d, J = 6.9 Hz, 2H, ArH), 6.9 (t, J = 7.44 Hz, 2H,
ArH), 6.4 (t(merged), J = 7.4 Hz, 2H, ArH), 6.3 (d,
J = 8.1 Hz, 2H, ArH), 2.8 (s, 4H, S–CH2–). 13C NMR
(75 MHz, DMSO-d6) d ppm: 147.9, 142.7, 137.0,
130.3, 129.2, 120.3, 115.9, 114.8, 109.8, 44.1, 33.7. Anal.
Calcd for C22H21N3O2S2: C, 62.39; H, 5.00; N, 9.92; O,
7.55; S, 15.14. Found: C, 62.28; H, 4.98; N, 10.01; S,
15.26. EI-MS: Calcd for C22H21N3O2S2: 423. Found:
m/z 429 [M+Li]+. FAB-MS: Calcd for C22H21N3O2S2:
423. Found: m/z 423 [M]+Å.
4.2.8.2. Macrocycle 3b. (2.4 g, 52%.) IR (KBr pellets,
cmÀ1) 3311m, 1681s, 1580s, 1517s, 1434, 1347m, 756m,
1
733m. H NMR (400 MHz, CDCl3) d ppm: 9.7 (s, 1H,
ArH), 9.1 (d, J = 1.24 Hz, 2H, ArH), 8.6 (d, J =
8.32 Hz, 2H, ArH), 7.6 (dd, J = 7.7 Hz, J = 1.48, 2H,
ArH), 7.4 (dt, J = 8.04, J = 1.44 Hz, 2H, ArH), 7.1 (dt,
J = 7.4 Hz, J = 1.44 Hz, 2H, ArH), 2.7 (t, J = 7.9 Hz,
4H, 2· S–CH2–), 1.8 (quintet, J = (merged), 2H, 2·
S–C–CH2–). Anal. Calcd for C23H19N3O4S2: C, 59.34;
H, 4.11; N, 9.03; O, 13.75; S, 13.78. Found: C, 59.17; H,
4.03; N, 9.23; S, 13.82. FAB-MS: Calcd for
C23H19N3O4S2: 465. Found: m/z 466 [M+H]+.
4.2.10.2. Macrocycle 4b. (2.8 g, 64%.) mp 206–207 ꢁC
IR(KBr, cmÀ1): 3388m (N–H str), 1585s, 1520s (NO2
asym str), 1499s, 1352s (NO2 sym str), 1320s, 745s. H
1
4.2.8.3. Macrocycle 3c. (2.9 g, 59%.) IR (KBr pellet,
cmÀ1) 3430w, 3247w, 2922m, 2858m, 1646s, 1527vs,
1440, 1351m, 731m, 664m. 1H NMR (400 MHz, CDCl3)
d ppm: 9.7 (s, 1H, ArH), 9.1 (d, J = 1.24 Hz, 2H, ArH),
8.6 (d, J = 8.32 Hz, 2H, ArH), 7.6 (dd, J = 7.7 Hz,
J = 1.48, 2H, ArH), 7.4 (dt, J = 8.04, J = 1.44 Hz, 2H,
ArH), 7.1 (dt, J = 7.4 Hz, J = 1.44 Hz, 2H, ArH), 3.6
(t, J = 4.88 Hz, 4H, 2· O–CH2–), 3.0 (t, J = 4.88, 4H,
2· S–CH2–). Anal. Calcd for C24H21N3O5S2: C, 58.17;
H, 4.27; N, 8.48; O, 16.14; S, 12.94. Found: C, 58.02;
H, 4.20; N, 8.62; S, 12.98. FAB-MS: Calcd for
C24H21N3O5S2: 495. Found: m/z 496 [M+H]+.
NMR (300 MHz, CDCl3) d ppm: 8.06 (s, 2H, ArH), 7.6
(s, 1H, ArH), 4.5 (d, J = 5.5 Hz, 4H, 2· Ar–CH2–N),
5.6 (s (broad), 2H, NH), 7.3 (d, J = 7.0 Hz, 2H, ArH),
7.0 (t, J = 7.3 Hz, 2H, ArH), 6.5 (t, J = 7.2 Hz, 2H,
ArH), 6.1 (d, J = 7.9 Hz, 2H, ArH), 2.7 (t, J = 7.9 Hz,
4H, 2· S–CH2–), 1.7 (quintet (merged), 2H, 2· S–C–
CH2–). 13C NMR (75 MHz, CDCl3) d ppm: 142.2,
135.6, 129.9, 128.6, 120.2, 117.5, 109.9, 46.3, 34.7, 30.4.
Anal. Calcd for C23H23N3O2S2: C, 63.13; H, 5.30; N,
9.60; O, 7.31; S, 14.66. Found: C, 62.98; H, 5.21; N,
9.89; S, 14.78. EI-MS: Calcd for C23H23N3O2S2: 437.
Found: m/z 443 [M+Li]+. FAB-MS: Calcd for
C23H23N3O2S2: 437. Found: m/z 437 [M]+Å.
4.2.9. General procedure for the synthesis of macrocycles
4a, 4b, and 4c from 3a, 3b, and 3c. To a solution of 3a
(1.15 g, 2.5 mmol in 20 mL of dry THF) 88 mL of 1 M
diborane in THF at 0 ꢁC was added with syringe under
argon atmosphere and the reaction mixture was stirred
for 1 h. The temperature of the reaction mixture was
raised to 60 ꢁC and the solution allowed to reflux for
24 h. After cooling, 20 mL of water was carefully added
into the solution to destroy the excess diborane. The sol-
vent was evaporated under reduced pressure, the residue
was dissolved in 8 mL of ethanol, and the resultant
solution was saturated with HCl(g) and refluxed for
2 h. On cooling, the precipitated product was collected
and washed with water and methanol (5 mL) to give
compound 4a (522 mg, 49%).
4.2.10.3. Macrocycle 4c. (3.2 g, 68%.) mp 159.5–
160 ꢁC IR(KBr, cmÀ1): 3365m (N–H str), 2921m,
2850w, 1587s, 1529s (NO2 asym str), 1497s, 1447m,
1350s (NO2 sym str), 1317s, 1259m, 1085s, 751s. 1H
NMR (300 MHz, CDCl3) d ppm: 8.1 (s, 2H, ArH), 7.8
(s, 1H, ArH), 7.4 (d, J = 7.35 Hz, 2H, ArH), 7.1 (t,
J = 7.5 Hz, 2H, ArH), 6.6 (t, J = 7.5 Hz, 2H, ArH), 6.6
(t, J = 7.29 Hz, 2H, ArH), 6.3 (d, J = 8.0 Hz, 2H,
ArH), 5.6 (s (broad), 2H, NH), 4.4 (d, J = 5.3 Hz, 4H,
2· Ar–CH2–N), 3.4 (t, J = 6.9 Hz, 4H, 2· O–CH2–),
2.8 (t, J = 6.9, 4H, 2· S–CH2–). 13C NMR (75 MHz,
CDCl3) d ppm: 149.2, 148.3, 142.5, 136.7, 130.4, 120.9,
117.8, 117.1, 110.5, 69.2, 47.9, 34.6. Anal. Calcd for
C24H25N3O3S2: C, 61.65; H, 5.39; N, 8.99; O, 10.26; S,
13.17. Found: C, 61.52; H, 5.28; N, 9.02; S, 13.89. EI-
MS: Calcd for C24H25N3O3S2: 467. Found: m/z 473
[M+Li]+. FAB-MS: Calcd for C24H25N3O3S2: 467.
Found: m/z 467 [M]+Å.
4.2.10. General procedure for the synthesis of macrocycles
4a, 4b, and 4c. 1,3-Bis(2-aminophenylthio)propane 1b
(2.9 g, 10 mmol) in 100 mL DMF and bis-3,5-bromom-
ethylnitrobenzene 7b (3.09 g, 10 mmol) in 100 mL DMF
were added simultaneously dropwise from separate
dropping funnels under nitrogen atmosphere at 120 ꢁC
with vigorous stirring (using mechanical stirrer) dry
K2CO3 (2.8 g, 20 mmol) and TBAÆHSO4 (20 mg) in
100 mL DMF. The reaction mixture was stirred at the
same temperature for 7 h and on completion of the reac-
tion (TLC) the DMF was removed under vacuum, the
4.2.11. General procedure for the synthesis of macrocycles
5a, 5b, and 5c. Compound 4b (437 mg, 1 mmol) was sus-
pended in ethyl acetate/tetrahydrofuran 50:50 (25 mL)
in a nitrogen flushed flask. Zinc dust (130 mg) and
hydrazine hydrate 99% (5 mL) were added and the reac-
tion mixture was stirred at room temperature under N2

B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
4719
atmosphere for 8 h. On completion of reaction (TLC),
the reaction mixture was decanted carefully, leaving be-
hind zinc dust adhered to walls of the flask. Organic sol-
vent was removed under reduced pressure and the
reaction mixture kept still for 1 h. The solid precipitated
was filtered through filter paper by decantation so that
most of the solid remained in the flask. Solid so obtained
was triturated in water, filtered, and dried under vacuum
to obtain a pale whitish solid product.
tion vortexed to mix it properly. The vial was allowed to
stand for 50 min at room temperature. The yield of com-
plexation and radiochemical purity of the 99mTc–BFC
complex were determined by ascending thin layer chro-
matography on ITLC-SG strips using 0.9% NaCl aque-
ous solution (saline) as developing solvent and
simultaneously in pyridine/acetic acid/water (PAW)
3:5:1.5 and acetone. Each TLC was cut in 0.5 cm seg-
ments and counts of each segment were taken. By using
this method the percentage of complex formed between
99mTc and BFC could be calculated.
4.2.11.1. Macrocycle 5b. (350 mg, 85%.) mp 159–
160 ꢁC. IR(KBr, cmÀ1) 3373w, 3278m, 2846w, 1585s,
1500s, 1450m, 1319m, 1286m, 746s. 1H NMR
(300 MHz DMSO-d6) d ppm: 7.2 (d, J = 7.4 Hz, 2H,
ArH), 6.9 (t, J = 7.6 Hz, 2H, ArH), 6.3 (d, J = 8.1 Hz,
2H, ArH), 6.4 (merged peaks, 3H, ArH), 5.8 (t,
J = 5.58 Hz, 2H, ArH), 5.05 (s), 4.1 (d, J = 5.59 Hz, 4H,
2· N–CH2–), 2.7 (t, J = 7.7 Hz, 4H, 2· S–CH2–), 1.6
(quintet (merged), 2H, S–C–CH2–). 13C NMR
(75 MHz, DMSO-d6) d ppm: 148.6, 148.5, 140.6, 135.0,
129.4, 115.7, 115.4, 111.0, 110.2, 109.8, 46.1, 33.3, 29.9.
Anal. Calcd for C23H25N3S2: C, 67.77; H, 6.18; N,
10.31; S, 15.73. Found: C, 67.12; H, 6.08; N, 11.04; S,
15.76. FAB-MS: Calcd for C23H25N3S2: 407. Found:
m/z 407 [M]+Å.
4.4. In vitro serum stability assay
The fresh human serum was prepared by allowing blood
collected from healthy volunteers to clot for 1 h at 37 ꢁC
in a humidified incubator maintained at 5% carbon
dioxide, 95% air. Then the samples were centrifuged at
400g and the serum was filtered through 0.22 lm syringe
filter into sterile plastic culture tubes. The above freshly
prepared technetium radiocomplexes 99mTc–5a–c
(10 MBq) were incubated in fresh human serum under
physiological conditions, that is, at 37 ꢁC at a concentra-
tion of 100 nmol/ml and then analyzed by ITLC-SG at
different time intervals to check for any dissociation of
complex. Percentage of free pertechnetate at a particular
time point estimated using saline and pyridine/acetic
acid/water (PAW) 3:5:1.5 as mobile phase represented
percentage dissociation of the complex at that particular
time point in serum.
4.2.11.2. Macrocycle 5a. mp 205–206 ꢁC. IR (KBr,
cmÀ1) 3367w, 2923w, 1654w, 1586s, 1499s, 1450m,
1
1316m, 746m. H NMR (300 MHz, DMSO-d6) d ppm:
7.3 (d, J = 7.4 Hz, 2H, ArH), 6.9 (t, J = 7.6 Hz, 2H,
ArH), 6.2 (d, J = 8.1 Hz, 2H, ArH), 6.3 (merged peaks,
3H, ArH), 5.8 (t, J = 5.58 Hz, 2H, ArH), 5.05 (s, NH),
4.1 (d, J = 5.59 Hz, 4H, 2· N–CH2–), 2.8 (s,
J = 7.7 Hz, 4H, 2· S–CH2–). 13C NMR (75 MHz,
DMSO-d6) d ppm: 148.6, 148.5, 140.6, 135.0, 129.4,
115.7, 115.4, 111.0, 110.2, 109.8, 46.1, 33.3. Anal. Calcd
for C22H23N3S2: C, 67.14; H, 5.89; N, 10.68; S, 16.29.
Found: C, 66.98; H, 5.68; N, 11.01; S, 16.31. FAB-
MS: Calcd for C22H23N3S2: 393. Found: m/z 393 [M]+Å.
4.5. Conjugation
The amino group of the bifunctional chelating agents
(5a–c) was converted to isothiocyanato group by react-
ing with thiophosgene at pH 2. Compound (5a–c,
1.5 mmol) in 5 mL of 3 M HCl was added to 3 mL
CSCl2 (85% in CCl4) at room temperature. The reaction
was stirred vigorously for 4 h. The aqueous phase was
washed with CHCl3 (4 · 5 mL) under a fume hood to re-
move excess CSCl2 and then purified by reverse phase
HPLC. Conjugation of EGFr monoclonal antibody
was performed by adding the 25 lL of 20 mM chelate
solution to 300 lL of a solution containing 3 mg of anti-
body in 0.1 M sodium phosphate, pH 7. Saturated triso-
dium phosphate solution (40 lL) was added to make the
pH 8.5. The reaction mixture was incubated at 37 ꢁC for
60 min and then subjected to centrifuged column gel
chromatography, which removed the unreacted chelate
and changed the buffer to 0.1 M sodium acetate, pH
5.5. UV absorbance at 280 nm for the centrifuged col-
umn effluent was used to determine the antibody con-
centration, and 57Co assay to obtain the bound chelate
concentration. Briefly, 1 nmol of conjugated antibody,
2 nmol of 59Co, and a tracer of 57Co were mixed and
incubated at pH 5.5 at room temperature for 30 min.
An aliquot was applied to thin layer chromatography
with ITLC-SG (Gelman Sciences, Ann Arbor, MI) with
ammonium acetate/CH3OH 1:1 (v/v) solution as eluent.
Free cobalt migrates to Rf = 1.0 while labeled cobalt
with immunoconjugate stays at Rf = 0. The 57Co bind-
ing assay indicates that 2.1 0.1 chelate molecules were
conjugated per antibody molecule.
4.2.11.3. Macrocycle 5c. IR (KBr, cmÀ1) 3373w,
3278m, 2846w, 1585s, 1500s, 1450m, 1319m, 1286m,
746s. 1H NMR (300 MHz, CDCl3) d ppm: 7.4 (d,
J = 7.4 Hz, 2H, ArH), 7.1 (t, J = 7.6 Hz, 2H, ArH), 6.2
(quintet (merged peaks), 6H, ArH), 6.8 (s, 1H, ArH),
5.4 (br s, 2H, exchanges with D2O), 4.2 (s, 4H, 2· N–
CH2–), 3.4 (t, J = 7.7 Hz, 4H, 2· O–CH2–), 2.8 (t,
J = 7.8 Hz, 4H, 2· S–CH2–). 13C NMR (75 MHz, CDCl3)
d ppm: 148.6, 148.5, 140.6, 135.0, 129.4, 115.7, 115.4,
111.0, 110.2, 109.8, 69.2, 47.9, 34.6. Anal. Calcd for
C24H27N3OS2: C, 65.87; H, 6.22; N, 9.60; O, 3.66; S,
14.65. Found: C, 65.23; H, 6.03; N, 9.89; S, 14.89. FAB-
MS: Calcd for C24H27N3OS2: 437. Found: m/z 437 [M]+Å.
4.3. Radiocomplexation of BFCs (5a–c) with technetium
Macrocycle 5c (100 lg) was dissolved in CHCl3 (100 lL)
in a shielded vial and stannous chloride (100 lg; 1 mg
dissolved in N2 purged 1 mL of 10% acetic acid) was
added followed by addition of (<1 h) freshly eluted sal-
ine solution of sodium pertechnetate (NaTcO4)
(74 MBq, 100 lL). The pH of the reaction mixture was
marked to 7 with 0.1 M NaHCO3 solution and the solu-

4720
B. S. Chhikara et al. / Bioorg. Med. Chem. 13 (2005) 4713–4720
10. Sundberg, M. W.; Meares, C. F.; Goodwin, D. A.;
Acknowledgment
Diamanti, C. I. J. Med. Chem. 1974, 17, 1304.
11. Mishra, A. K.; Chatal, J. F. New J. Chem. 2001, 25,
336.
12. Milenic, D. E.; Garmestani, K.; Chappell, L. L.; Dadach-
ova, E.; Yordanov, A.; Ma, D.; Scholm, J.; Brechbiel, M.
W. Nucl. Med. Biol. 2002, 29, 431.
The senior research fellowship (SRF) provided by the
Council of Scientific and Industrial Research (CSIR),
New Delhi, to one of the authors (B.S.C.) is highly
acknowledged.
13. Li, L.; Tsai, S. W.; Anderson, A. L.; Keire, D. A.;
Raubitschek, A. A.; Shively, J. E. Bioconjugate Chem.
2002, 13, 110.
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