Concise site-specific synthesis of DTPA-peptide conjugates: Application to imaging probes for the chemokine receptor CXCR4

Article abstract of DOI:10.1016/j.bmc.2011.03.059

Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as ⁶⁸Ga, ^99mTc and ¹¹¹In, which are applicable to nuclear medicine imaging. In this study, we established a facile synthetic prot

Full text of DOI:10.1016/j.bmc.2011.03.059

Bioorganic & Medicinal Chemistry 19 (2011) 3216–3220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Concise site-specific synthesis of DTPA–peptide conjugates: Application
to imaging probes for the chemokine receptor CXCR4
⇑
⇑
Ryo Masuda, Shinya Oishi , Hiroaki Ohno, Hiroyuki Kimura, Hideo Saji, Nobutaka Fujii
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as 68Ga,
99mTc and 111In, which are applicable to nuclear medicine imaging. In this study, we established a facile
synthetic protocol for the production of mono-DTPA-conjugated peptide probes. A novel monoreactive
DTPA precursor reagent was synthesized in two steps using the chemistry of the o-nitrobenzenesulfonyl
Article history:
Received 17 February 2011
Revised 25 March 2011
Accepted 26 March 2011
Available online 2 April 2011
e
(Ns) protecting group, and under mild conditions this DTPA precursor was incorporated onto an N -
bromoacetylated Lys of a protected peptide resin. The site-specific DTPA conjugation was facilitated by
using a highly acid-labile 4-methyltrityl (Mtt) protecting group for the target site of the bioactive peptide
during the solid-phase synthesis. A combination of both techniques yielded peptides with disulfide
bonds, such as octreotide and polyphemusin II-derived CXCR4 antagonists. DTPA–peptide conjugates
were purified in a single step following cleavage from the resin and disulfide bond formation. This
site-specific on-resin construction strategy was used for the design and synthesis of a novel In-DTPA-
labeled CXCR4 antagonist, which exhibited highly potent inhibitory activity against SDF-1–CXCR4
binding.
Keywords:
CXCR4
DTPA
Molecular imaging
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
the chelating ligands, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
raacetic acid (DOTA) 1a has been most widely utilized, since a vari-
Recent progress in molecular imaging methodologies such as
positron emission tomography (PET), single-photon emission com-
puted tomography (SPECT) and optical imaging technologies has
significantly improved the early detection and diagnosis of malig-
nant tumors. To visualize the specific molecular events involved in
the physiological and/or pathological processes, a number of pep-
tide-based imaging probes have been developed for overexpressed
receptors of peptide hormones and extracellular matrix proteins.1
These probes are usually designed by a combination of three com-
ponents: a target-specific vector peptide, an imaging part such as a
radionuclide or fluorophore, and a linker to covalently or noncova-
lently conjugate the peptide with the imaging moiety. The addition
of a functional moiety onto small-sized bioactive peptides may be
highly susceptible to interaction with receptors or counterpart
molecules. Consequently, there have been many reagents of choice
for appropriate protein/peptide modifications. In addition, to
determine the best labeling position from structure-function rela-
tionship studies, versatile synthetic approaches toward various
types of labeled peptide are desired.
ety of metal radioisotopes for both diagnostic and therapeutic
purposes form complexes with high affinity and kinetic stability
(Fig. 1).2 DOTA-modification of bioactive peptides is facilitated by
commercially available reagents such as DOTA-NHS 1b and
DOTA-maleimide 1c to provide the expected peptides in a single
step.3,4 Alternatively, tris(tert-butyl)-DOTA 2a with a free carboxyl
group is employed for the modification of an amino group of pro-
tected peptides bound to solid-supports.5 Lysine or phenylalanine
derivatives 2b,c possessing a tert-butyl-protected DOTA moiety
are also useful components for the peptide sequence assembly.6
tert-Butyl protecting groups in these reagents are easily removed
during the final side-chain deprotection process of peptide
synthesis.
In contrast to these DOTA derivatives, there has been limited
work exploring the application of the diethylenetriaminepentaace-
tic acid (DTPA) chelating group 3a, although DTPA represents a
promising alternative, especially for 68Ga, 99mTc and 111In (Fig. 1).
The recent success of DTPA-based probes is exemplified by a
glucagon-like peptide-1 (GLP-1) receptor ligand, [Lys40
(Ahx-DTPA-111In)NH2]-exendin-4, for insulinoma diagnosis.7 The
DTPA group also works as a more favorable functional group than
DOTA to facilitate the biological or biodistribution properties of
several probes.8 For the preparation of DTPA-conjugated imaging
probes, several conjugation reagents have been developed. The
most familiar cyclic diethylenetriaminepentaacetic dianhydride 4
Polyamino polycarboxylate ligands efficiently coordinate metal
radioisotopes to aid the radiolabeling of bioactive peptides. Among
⇑
Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-u.
ac.jp (N. Fujii).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.059

R. Masuda et al. / Bioorg. Med. Chem. 19 (2011) 3216–3220
3217
intermediate 7 in the solution-phase. In addition, a secondary
amine 8 as a nucleophilic precursor for the bromoacetyl group
on peptide resin 11 can directly produce the overall DTPA frame-
work of 12 on the solid support without the additional three-step
modification process of 8 in solution (Scheme 1B).16
Synthesis of DTPA precursor 8 began with mono-Ns protection
of the commercially available diethylenetriamine 5 (Scheme 2).
The Ns-protected intermediate was successively treated with ex-
cess equivalent of t-butyl bromoacetate in a one-pot process.
Although the solvent EtOH has been reported to be effective in pre-
dominantly giving the mono-Ns product,17 concomitant produc-
tion of bis-Ns product 14b was not suppressed as in DMF. The
treatment of excess diethylenetriamine 5 with NsCl in EtOH pro-
vided mono-Ns product 14a in 65% yield (calculated based on
NsCl), which can be readily purified by chromatography. Com-
pound 14a was then subjected to deprotection with mercaptoace-
tic acid and LiOH to provide the expected precursor 8 in 77% yield.
1a R = OH (DOTA)
O
HO2C
COR
N
N
N
N
1b R =
1c R =
O
N
O
O
H
HO2C
CO2H
COR
N (CH2)2 N
O
2a R = OH
H
t-BuO2C
N (CH2)4
CO2H
NHFmoc
N
N
N
N
2b R =
CO2H
2c R =
t-BuO2C
CO2t-Bu
NHFmoc
N
H
CO2R
Using the resulting reagent 8, DTPA-conjugation of
[D-
N
N
3a R = H (DTPA)
Phe1]octreotide was investigated as a model study (Scheme 3),
which is employed as a radionuclide imaging probe for the somato-
statin receptor.14,18,19 After peptide-chain elongation by Fmoc-
based solid-phase peptide synthesis, the N-terminus of 16 was
modified with bromoacetic acid and 1,3-diisopropylcarbodiimide
(DIC). Subsequently, the bromide 17 was treated with the reagent
8 in the presence of (i-Pr)2NEt to provide the fully protected pep-
tide resin 18a. Cleavage from the resin 18a and disulfide formation
RO2C
RO2C
N
N
N
CO2H
3b
R = t-Bu
CO2R
CO2H
O
O
O
O
N
4
O
under air-oxidation conditions provided [DTPA-D
-Phe1]octreotide
19a with high purity. The bromoacetylated peptide 17 was also
O
Figure 1. Structures of radionuclide chelating agents and the precursors.
modified with commercially available DOTA precursor reagent
20, using the identical procedure to provide [DOTA-D
-Phe1]octreo-
tide 19b.20 These suggest that this on-resin modification procedure
is widely applicable to any chelating reagents with nucleophilic
functional groups such as DTPA and DOTA precursors.
is a bifunctional chelating agent, which can conjugate with peptide
hormones and antibodies.9 Using this reagent, concomitant forma-
tions of a bis-conjugated product10 and intra- and intermolecular
cross-linked products11 were unavoidable. Monoreactive DTPA
derivatives have also been developed for the preparation of
DTPA–peptide conjugates without the unfavorable by-product
formations.12,13 For example, we reported the synthesis and
application of 3,6,9,9-tetrakis[(tert-butoxycarbonyl)methyl]-3,6,
9-triazanonanoic acid 3b (mDTPA),14 in which the four carboxyl-
ates were protected with tert-butyl ester. However, a longer pro-
cess from the commercially available reagents is required for the
synthesis of these DTPA-conjugation reagents (Scheme 1A).
Accordingly, to establish a facile and efficient synthetic method
for DTPA–peptide conjugates, we have investigated the site-
specific and on-resin construction of a DTPA moiety. Herein, we
describe the short-step synthesis of a DTPA precursor using the
o-nitrobenzenesulfonyl (Ns) protecting group and the solid-phase
synthesis of DTPA–peptide conjugates. The design and synthesis
of DTPA–peptide conjugates that potentially target the somato-
statin receptor and chemokine receptor CXCR4 are also
presented.15
2.2. Site-specific DTPA-conjugation of bioactive peptides:
synthesis of CXCR4 receptor probes
It has been reported that a high level of CXCR4 expression in tu-
mors is associated with malignant and metastatic properties.21
Intrinsic SDF-1 release from the potential distal metastatic sites
mediates organ-specific metastasis of CXCR4-expressing cells from
the primary lesions. Since CXCR4-expressing cancer stem cells are
related to the metastatic spread in orthotopic primary tumors,22 it
is of considerable importance to develop potent CXCR4-imaging
probes to detect potential cancer stem cells within malignant tu-
mors, as exemplified by the diagnosis of bladder cancer by a fluo-
rescent CXCR4 probe.23,24
Previously, we reported a DTPA-conjugated CXCR4 antagonist,
DTPA-Ac-TZ14011 26a,25 which was designed from a horseshoe
crab-derived anti-HIV peptide T140. This peptide has b-sheet-like
structures maintained by a disulfide bond, around which the phar-
macophore residues for bioactivity are located.26 For the site-spe-
cific conjugation at D
-Lys8 in the type II’ b-turn region of T140
2. Results and discussion
with a single DTPA group in the solution-phase, a secondary lysine
(Lys7) was substituted with arginine, which cannot be acylated by
standard reagents.25 Although a DTPA group was successfully li-
gated with maintenance of highly potent CXCR4 antagonistic activ-
ity in this case,25 the accompanying substitutions needed for
specific modification of other peptides may possibly lead to a
decrease in the bioactivity. Therefore, we planned the facile site-
specific DTPA conjugation on a solid-support for production of
CXCR4 imaging probes without substitution of the secondary
2.1. Synthesis of a DTPA-conjugation reagent and the
application to octreotide derivatives
The synthetic scheme for the production of mDTPA reagent 10,
as described in our previous study, is presented in Scheme 1A. We
hypothesized that two remedies could significantly improve the
overall synthetic process of DTPA–peptide conjugates. First, the
use of an Ns group in place of the trifluoroacetyl group was ex-
pected to serve as a temporary protecting group and an auxiliary
group for global modification with four tert-butoxycarbonylmethyl
groups. This potentially improves the stepwise synthesis of the
Lys7 residue. To distinguish
the highly acid-labile 4-methyltrityl (Mtt) group was exploited
for temporary protection of the -amino group during solid-phase
peptide synthesis.27 For the other Lys residues such as Lys7 of
D
-Lys8 to be labeled in peptides 26,
e

3218
R. Masuda et al. / Bioorg. Med. Chem. 19 (2011) 3216–3220
(2-chloro)trityl
CO2t-Bu
A
Cl
chloride resin 15
H
N
N
COCF3
a, b
t-BuO2C
t-BuO2C
N
N
R
a
H2N
NH2
5
Trt
Boc Boc t-Bu Trt t-Bu
R-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr
6 R = H
One-pot reaction using
Ns-protecting group
c
7 R = -CH2CO2t-Bu
16 R = H
b
c
CO2t-Bu
8 R = H
17 R = -CO-CH2Br
e
9 R = -CH2CO2Bn
18a
R = DTPA(t-Bu)4
f
N
R
d
3a R = -CH2CO2H
10 R = -CH2CO2Su
t-BuO2C
t-BuO2C
N
N
g
18b R = DOTA(t-Bu)3
CO2t-Bu
d, e
B
Peptide
11
12
Br
HN
R-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol
19a
19b
R = DTPA;
R = DOTA
O
On-resin construction
of DTPA moiety by
using the precursor
8, base
t-BuO2C
t-BuO2C
N
N
HN
N
H
N
Peptide
t-BuO2C
N
O
CO2t-Bu
t-BuO2C
t-BuO2C
N
N
20
CO2t-Bu
Scheme 3. Synthesis of DTPA- and DOTA-conjugated D-Phe-octreotides. Reagents:
(a) Fmoc-based peptide synthesis; (b) BrCH2CO2H, DIC; (c) 8 for 18a, or 20 for 18b,
(i-Pr)2NEt (d) TFA/H2O/1,2-ethanedithiol (EDT) (95:2.5:2.5) for 19a, 1 M TMSBr,
thioanisole/TFA, 1,2-ethanedithiol, m-cresol for 19b; (e) NH4OH (air oxidation).
H
Peptide
13
DTPA N
26b,c, a Boc group was employed. This group can be cleaved by the
standard TFA-based treatment in Fmoc chemistry (Scheme 4). After
the construction of the protected peptide resin, the orthogonal Mtt
group at the labeling position was cleaved off using 1,1,1,3,3,3-
Scheme 1. (A) Synthetic scheme for the DTPA-conjugation reagent 10 prepared in
our previous study; (B) synthetic plan for the DTPA-conjugated peptides in this
study. Reagents: (a) CF3CO2Et; (b) BrCH2CO2t-Bu, (i-Pr)2NEt; (c) BrCH2CO2t-Bu,
NaH; (d) NH2NH2, t-BuOH; (e) BrCH2CO2Bn, (i-Pr)2NEt; (f) H2, Pd/C; (g) DCC, HOSu.
hexafluoropropan-2-ol (HFIP). The resulting
e-amino group was
successively modified with bromoacetic acid followed by the re-
agent 8 to provide the fully protected DTPA–peptide resin 25. Final
deprotection, air-oxidation and HPLC purification afforded the ex-
pected DTPA-conjugated CXCR4 antagonists 26a,b. This concise
protocol facilitates the selection of chelating structure and posi-
tion(s) on the peptide chain, and aids structure–activity relation-
ship studies aimed at exploring the more potent peptide probes.
For example, a 4-fluorobenzoyl modification at the N-terminus,
which should increase CXCR4 antagonism,28 was easily appended
to the peptide using this protocol to give the modified peptide
26c. The subsequent treatment with nonradioactive InCl3 in
acidic conditions provided the In-DTPA-labeled CXCR4 antagonists
27a–c.
CO2t-Bu
R
N
Ns
a, b
c
N
N
5
8
t-BuO2C
CO2t-Bu
14a R = -CH2CO2t-Bu
14b R = Ns
Scheme 2. Synthesis of DTPA precursor 8 via a global N-alkylation process using a
Ns-protecting group. Reagents: (a) NsCl; (b) BrCH2CO2t-Bu, K2CO3; (c) HSCH2CO2H,
LiOH.
Scheme 4. Site-specific In-DTPA labeling of CXCR4 antagonists and biological activity. Reagents: (a) Fmoc-based peptide synthesis; (b) CH2Cl2/1,1,1,3,3,3-hexafluoro-2-
propanol (HFIP)/2,2,2-trifluoroethanol (TFE)/triethylsilane (TES) (65:20:10:5); (c) BrCH2CO2H, DIC; (d) 8, (i-Pr)2NEt; (e) TFA/H2O/EDT (95:2.5:2.5); (f) NH4OH (air oxidation);
(g) InCl3. Abbreviations: Mtt: 4-methyltrityl; Cit: L-citrulline, Nal: L-3-(2-naphthyl)alanine, 4FBz: 4-fluorobenzoyl.

R. Masuda et al. / Bioorg. Med. Chem. 19 (2011) 3216–3220
3219
2.3. Bioactivity of In-DTPA-labeled CXCR4 antagonists
4.1.2. Bis(tert-butyl) 3,6-bis[(tert-butoxycarbonyl)methyl]-
3,6,9-triazaundecanedioate (8)
The biological activity of the In-DTPA-labeled peptides 27a–c
was evaluated as the inhibitory potency of [125I]-SDF-1-binding
to CXCR4 membrane extracts (Scheme 4). Peptides 27a,b, with
an N-terminal acetyl group, exhibited similar potency towards
To a solution of compound 14a (0.216 g, 0.29 mmol) in DMF
(0.726 mL), LiOH (0.128 g, 2.90 mmol) and mercaptoacetic acid
(0.101 mL, 1.45 mmol) were added below 0 °C. After stirring for
2 h at room temperature, the mixture was concentrated under re-
duced pressure, and the residue was dissolved in CHCl3. The whole
reaction mixture was washed with saturated NaHCO3, and was
dried over Na2SO4. Concentration under reduced pressure followed
by flash chromatography over silica gel with CHCl3–MeOH gave
compound 8 as a yellow oil (0.124 g, 77%); 1H NMR (CDCl3,
500 MHz) d 3.39 (4H, s), 3.28 (4H, s), 2.72–2.82 (6H, m), 2.63
(2H, t, J = 5.4 Hz), 1.39 (9H, s), 1.38 (27H, s); 13C NMR (CDCl3,
500 MHz) d 170.9, 170.7 (3C), 80.8 (4C), 55.9 (2C), 55.8 (2C),
52.4, 52.3, 51.3, 47.0, 28.2 (3C), 28.1 (9C); HRMS (FAB) m/z calcd
for C28H54N3O8 ([M+H]+): 560.3911, found 560.3910.
CXCR4 [IC50(27a) = 1.95 0.64 lM, IC50(27b) = 1.60 0.91 lM],
indicating that the Lys and Arg for the i-position of b-turn were
both tolerant to the bioactivity. In contrast, peptide 27c exerted
much more potent inhibitory activity for the SDF-1 binding to
CXCR4 [IC50(27c) = 0.014 0.010 lM]. These results of In-DTPA-la-
beled peptides 27a–c coincided with our previous report on the
unlabeled peptides.28 The novel potent In-DTPA-labeled CXCR4
antagonist 27c could be a promising imaging probe for CXCR4-
expressing malignant cancer cells.11
3. Conclusions
4.1.3. Standard procedure for solid-phase peptide synthesis
Protected peptide-resins were manually constructed by Fmoc-
based solid-phase peptide synthesis. t-Bu ester for Asp and Glu;
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf) for
Arg; t-Bu for Thr and Tyr; Boc for Lys and Trp; Trt for Cys were em-
ployed for side-chain protection. Fmoc-amino acids were coupled
using three equivalents of reagents [Fmoc-amino acid, 1,3-diiso-
propylcarbodiimide (DIC), and HOBtÁH2O] to the free amino group
in DMF for 1.5 h. Fmoc deprotection was performed by 20% (v/v)
piperidine in DMF (2 Â 1 min, 1 Â 30 min). The protected peptide
resin was treated with a cocktail of deprotection reagents. After re-
moval of the resin by filtration, the filtrate was poured into ice-cold
dry Et2O. The resulting powder was collected by centrifugation and
washed with ice-cold dry Et2O. The crude peptide was dissolved in
H2O, and the pH was adjusted to 8.0 with NH4OH for disulfide bond
formation. After air-oxidation for 1 d, the crude product was puri-
fied by preparative HPLC on a Cosmosil 5C18-ARII preparative col-
umn (Nacalai Tesque, Kyoto, Japan; 20 Â 250 mm, flow rate 10 mL/
min) to afford the expected peptides. All peptides were character-
ized by MALDI-TOF-MS (AXIMA-CFR plus, Shimadzu, Kyoto, Japan)
and the purity was calculated as >95% by HPLC on a Cosmosil
5C18-ARII analytical column (Nacalai Tesque, 4.6 Â 250 mm, flow
rate 1 mL/min) at 220 nm absorbance.
In this study, we have established a novel synthetic method for
the production of DTPA–peptide conjugates. The process includes
facile solid-phase synthesis of a DTPA framework using a novel
precursor substrate and site-specific conjugation using a highly
acid-labile protecting group. Using a temporary Ns protecting
group, the DTPA precursor 8 was obtained through two purifica-
tion steps from commercially available diethylenetriamine. In
addition, the on-resin incorporation of a bromoacetyl group into
the specific free amino group followed by the addition of the nucle-
ophilic DTPA precursors provided the expected DTPA–peptide con-
jugates with high purity. Taking advantage of secondary amine
precursors of choice, these processes represent versatile methods
to prepare a series of peptide conjugates, including DTPA and
DOTA, for optimization of imaging probes. This conjugation meth-
od was applied to the preparation of DTPA-conjugates of octreotide
and CXCR4 antagonist, which have been reported to effectively de-
tect cancer cells. The peptide 27c with highly potent inhibitory
activity of SDF-1 binding to CXCR4 was obtained without any ami-
no acid substitution to avoid multiple modifications on the amino
groups. This peptide represents a promising lead compound as an
imaging probe towards CXCR4-positive metastatic tumors.
4. Experimental
4.1. Synthesis
4.1.4. Preparation of DTPA- and DOTA-conjugated octreotides
(19a,b)
According to the procedure reported previously,18 (2-chloro)tri-
tyl chloride resin 15 (214 mg, 1.4 mmol/g), Fmoc-Thr(t-Bu)-ol
(345 mg, 0.9 mmol), and pyridine (0.145 mL, 1.8 mmol) were agi-
tated for 21 h in dry CH2Cl2–DMF (1:1, 3.94 mL). The loading was
determined by measuring the 290 nm UV absorption of the piper-
idine-treated sample (0.455 mmol/g). After the construction of the
peptide chain (0.017 mmol scale) using a standard procedure,
bromoacetic acid (23.6 mg, 0.17 mmol) with DIC (0.026 mL,
0.17 mmol) in CH2Cl2 was reacted with resin 16 for 2 h at room
temperature. The subsequent treatment of 17 with amines 8
(29.0 mg, 0.51 mmol) and 20 (26.3 mg, 0.51 mmol) with (i-Pr)2NEt
(0.009 mL, 0.51 mmol) in DMF for 12 h at room temperature pro-
vided 18a and 18b, respectively. Cleavage and deprotection of
18a (72.5 mg) and 18b (73.8 mg) was achieved using a TFA/1,2-
ethandithiol (EDT)/H2O (5 mL; 95:2.5:2.5) cocktail for 2 h at room
temperature and by treatment with 1 M TMSBr-thioanisole/TFA in
the presence of EDT/m-cresol (3.3 mL) for 2 h at 0 °C, respectively.
After disulfide formation under air-oxidation conditions, the crude
peptides were purified using the standard procedure, to afford the
desired peptides 19a (8.2 mg, 23%) and 19b (9.5 mg, 26%) as white
powders. Compound 19a: MS (MALDI-TOF) m/z calcd for
4.1.1. Bis(tert-butyl) 3,6-bis[(tert-butoxycarbonyl)methyl]-9-(o-
nitrobenzenesulfonyl)-3,6,9-triazaundecanedioate (14a)
To diethylenetriamine 5 (0.540 mL, 5.00 mmol) in dehydrated
EtOH (5 mL), o-NsCl (0.367 g, 1.67 mmol) was slowly added below
0 °C. After stirring for 2 h, EtOH was removed in vacuo. To dehy-
drated DMF (8 mL), K2CO3 (4.49 g, 32.5 mmol) and BrCH2CO2t-Bu
(4.06 mL, 27.5 mmol) were added at 0 °C. The mixture was stirred
overnight at room temperature, and filtered. The filtrate was con-
centrated under reduced pressure to give an oily residue, and the
residue was dissolved in EtOAc (100 mL). The whole mixture was
washed with saturated NaHCO3, and was dried over MgSO4. Con-
centration under reduced pressure followed by flash chromatogra-
phy over silica gel with n-hexane–EtOAc gave compound 14a as a
yellow oil (0.81 g, 65%); 1H NMR (CDCl3, 500 MHz) d 8.08–8.11 (1H,
m), 7.64–7.69 (2H, m), 7.56–7.60 (1H, m), 4.24 (2H, s), 3.49 (2H, t,
J = 6.9 Hz), 3.42 (4H, s), 3.30 (2H, s), 2.88 (2H, t, J = 6.6 Hz), 2.78
(2H, t, J = 6.9 Hz), 2.77 (2H, t, J = 6.9 Hz), 1.45 (27H, s), 1.36 (9H,
s); 13C NMR (CDCl3, 500 MHz) d 170.6 (3C), 168.0, 133.7, 133.2,
131.6 (2C), 130.9, 123.9, 82.0, 81.0, 80.9 (2C), 56.1 (3C), 53.3,
52.8, 52.4, 49.4, 46.7, 28.1 (9C), 27.9 (3C); HRMS (FAB) m/z calcd
for C34H58N4O12S ([M+H]+): 746.3772, found 746.3779.
C
63H89N13O19S2 ([M+H]+): 1395.6, found 1395.3. Compound 19b:

3220
R. Masuda et al. / Bioorg. Med. Chem. 19 (2011) 3216–3220
MS (MALDI-TOF) m/z calcd for C65H93N14O17S2 ([M+H]+): 1405.6,
Acknowledgments
found 1405.8.
This work is supported by Grants-in-Aid for Scientific Research
and Molecular Imaging Research Program from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. R.M.
is grateful for Research Fellowships from the JSPS for Young
Scientists.
4.1.5. Preparation of DTPA-conjugated CXCR4 antagonists (26a–
c)
Protected peptide resins were manually constructed according
to the standard procedure using NovaSyn TGR-resin 21 (96.2 mg,
0.025 mmol). 4-Methyltrityl (Mtt) group was employed for the
protection of the
D-Lys e-amino group. The N-terminal amino
Supplementary data
group was acylated by treatment with Ac2O (0.012 mL,
0.125 mmol)/pyridine (0.020 mL, 0.250 mmol) for 1 h at room tem-
perature for peptides 26a,b, and with 4-fluorobenzoic acid
(17.5 mg, 0.125 mmol)/DIC (0.019 mL, 0.125 mmol)/HOBtÁH2O
(19.2 mg, 0.125 mmol) for 1.5 h at room temperature for peptide
26c. Subsequently, the resin 22 was treated with CH2Cl2/
1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP)/trifluoroethanol (TFE)/
triethylsilane (TES) [65:20:10:5; 5 mL] for 2 h at room tempera-
ture. The DTPA group was incorporated using the identical proce-
dure employed for the synthesis of the octreotide derivative 19a.
Treatment of the resins (25a: 178 mg, 25b: 165 mg, 25c: 162 mg)
with a TFA/1,2-ethandithiol(EDT)/H2O (95:2.5:2.5; 5 mL) cocktail
for 2 h at room temperature followed by air oxidation and purifica-
tion provided the peptides Compound 26a (14.6 mg, 15.4%), 26b
(6.67 mg, 8.7%) and 26c (7.4 mg, 9.5%) as white powders.
Compound 26a: MS (MALDI-TOF) m/z calcd for C106H165N38O28S2
([M+H]+): 2482.2, found 2482.5. Compound 26b: MS (MALDI-
TOF) m/z calcd for C106H165N36O28S2 ([M+H]+): 2454.2, found
2453.9. Compound 26c: MS (MALDI-TOF) m/z calcd for
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.059.
References and notes
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111H166FN36O28S2 ([M+H]+): 2534.2, found 2533.8.
4.1.6. Indium chelating for CXCR4 antagonist probes (27a–c)
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To a solution of peptides 26a–c (8 mM in 0.1 N AcOH, 26a:
45.9
0.39
l
L, 0.37
l
mol; 26b: 48.4
l
L, 0.39
l
mol; 26c: 48.8
lL,
l
mol), InCl3 (1 M in 0.02 N HCl, 50
l
L) was added and the
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solution stirred for a further 30 min at room temperature. HPLC
purification using a standard procedure provided the desired pep-
tides 27a (0.43 mg, 36.7%), 27b (0.42 mg, 34.3%) and 27c (0.38 mg,
30.3%) as white powders. Compound 27a: MS (MALDI-TOF) m/z
calcd for C106H165InN38O28S2 ([M+H]+): 2597.1, found 2596.9. Com-
pound 27b: MS (MALDI-TOF) m/z calcd for C106H165InN36O28S2
([M+H]+): 2569.1, found 2569.1. Compound 27c: MS (MALDI-
TOF) m/z calcd for C111H166FInN36O28S2 ([M+H]+): 2649.1, found
2649.0.
4.2. Evaluation of [125I]-SDF-1 binding and displacement
For ligand binding, the CXCR4 membrane was incubated with
0.5 nM of [125I]-SDF-1 and increasing concentrations of compounds
27a–c in binding buffer [50 mM HEPES (pH 7.4), 5 mM MgCl2,
1 mM CaCl2 and 0.1% BSA in H2O] for 1 h at room temperature.
The reaction mixtures were filtered through GF/B filters (Perkin-El-
mer, Wellesley, MA) pretreated with 0.1% polyethyleneimine. The
filter plate was washed with wash buffer [50 mM HEPES (pH
7.4), 500 mM NaCl and 0.1% BSA in H2O] and the bound radioactiv-
ity was measured by TopCount (Packard, Meriden, CT). Inhibitory
activity of test compounds was determined based on the inhibition
of [125I]-SDF-1 binding to the CXCR4 receptor (IC50).