A benzenesulfonamide derivative as a novel PET radioligand for CXCR4

Article abstract of DOI:10.1016/j.bmc.2019.115240

CXCR4 is involved in various diseases such as inflammation, tumor growth, and cancer metastasis through the interaction with its natural endogenous ligand, chemokine CXCL12. In an effort to develop imaging probes for CXCR4, we developed a novel small mole

Full text of DOI:10.1016/j.bmc.2019.115240

Journal Pre-proofs
A benzenesulfonamide derivative as a novel PET radioligand for CXCR4
Yoon Hyeun Oum, Dinesh Shetty, Younghyoun Yoon, Zhongxing Liang,
Ronald J. Voll, Mark M. Goodman, Hyunsuk Shim
PII:
S0968-0896(19)31585-8
https://doi.org/10.1016/j.bmc.2019.115240
BMC 115240
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
13 September 2019
20 November 2019
25 November 2019
Please cite this article as: Y. Hyeun Oum, D. Shetty, Y. Yoon, Z. Liang, R.J. Voll, M.M. Goodman, H. Shim, A
benzenesulfonamide derivative as a novel PET radioligand for CXCR4, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.115240
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A benzenesulfonamide derivative as a novel PET radioligand for CXCR4
Yoon Hyeun Ouma, Dinesh Shettyb,#1, Younghyoun Yoona, Zhongxing Lianga,c, Ronald J. Vollb,
Mark M. Goodmanb,c, Hyunsuk Shima,b,c,*
a Department of Radiation Oncology, b Department of Radiology and Image Sciences, Emory
University School of Medicine. c Winship Cancer Institute, Atlanta, GA 30322.
#1Current address: Department of Chemistry, Khalifa University, P. O. Box 127788, Abu Dhabi,
United Arab Emirates.
*Corresponding author at: Department of Radiation Oncology, Emory University School of
Medicine. 1365 Clifton Road NE, Building C., Atlanta GA, 30322, USA. Email:
hshim@emory.edu; Tel: 1-404-712-4564
1

Abstract
CXCR4 is involved in various diseases such as inflammation, tumor growth, and cancer metastasis
through the interaction with its natural endogenous ligand, chemokine CXCL12. In an effort to
develop imaging probes for CXCR4, we developed a novel small molecule CXCR4-targeted PET
agent (compound 5) by combining our established benzenesulfonamide scaffold with a labeling
component by virtue of click chemistry. 5 shows nanomolar affinity (IC50 = 6.9 nM) against a
known CXCR4 antagonist (TN14003) and inhibits more than 65% chemotaxis at 10 nM in vitro
assays. Radiofluorinated compound 5 ([18F]5) demonstrates a competitive cellular uptake against
CXCL12 in a dose-dependent manner. Further, microPET images of [18F]5 exhibits preferential
accumulation of radioactivity in the lesions of λ-carrageenan-induced paw edema, human head
and neck cancer orthotopic xenograft, and metastatic lung cancer of each mouse model.
Keywords:
C-X-C chemokine receptor type 4 (CXCR4), CXCL12, molecular imaging probe, positron
emission tomography (PET), inflammation, head and neck cancer, metastasis
Highlights
ꢀ A novel small molecular CXCR4-targeted PET tracer without chelating moiety is
designed based on benzenesulfonamide scaffold via chemoinformatic and molecular
modeling approach.
ꢀ In vitro binding assay and chemotaxis inhibition assay affirm the affinity and functional
activity of the novel agent with CXCR4.
ꢀ The novel CXCR4-targeted F-18 PET agent demonstrates the ability to visualize acute
inflammation, head and neck cancer, and cancer lung metastasis in mouse models.
Abbreviations:
GPCRs (G-protein-coupled receptors), CXCR4 (C-X-C chemokine receptor type 4), CXCL12
(C-X-C chemokine ligand 12), SDF-1 (stromal cell-derived factor-1), HIV (human
immunodeficiency virus), SCCHN (squamous cell carcinoma of head and neck), PDB (protein
databank), EC (effective concentration), DTPA (Diethylenetriamine pentaacetate); DOTA
(dodecane tetraacetic acid), NOTA (1,4,7-Triazacyclononane-1,4,7-triacetic acid)
2

1. Introduction
C-X-C chemokine receptor type 4 (CXCR4) is a G-protein-coupled receptor involved in various
physiological processes in the hematopoietic and immune systems via the interaction with its
endogenous partner, chemokine ligand 12 (CXCL12) that is also known as stromal-derived-
factor-1 (SDF-1).1-4 The CXCR4/CXCL12 axis attracts significant notice due to its critical
functions in the development of refractory diseases such as HIV infection, metastatic cancer, and
autoimmune diseases.5-10 The pivotal roles of CXCR4 in pathogenesis of such diseases have
encouraged an effort to investigate CXCR4-targeted therapeutics.11-13 Along with this effort,
CXCR4-targeted molecules emerge as imaging probes for relevant diseases. For instance,
noninvasive imaging of CXCR4 could work as a complementary diagnostic and prognostic
biomarker for various metastatic cancers including breast, prostate, lung, colon, and multiple
myeloma.14 Positron emission tomography (PET) is recognized as one of major translational
(“bench to bedside”) molecular imaging modalities owing to its unparalleled sensitivity and
diversity of tracer portfolio to monitor key biological processes of diseases. PET radiotracer can
provide early functional data on disease extent, therapy response, identification of recurrence,
and stratification of patients for personalized medicine. PET agents require high binding affinity,
selectivity, rapid clearance, good tissue penetration, and appropriate metabolism for clear
contrast images.
Up to date, various CXCR4-targeted PET radiotracers have been reported.15 Those PET tracers
can be categorized into three classes, based on the nature of targeting groups: i) conjugates of
potent peptides such as T140 consisting of 14 amino acids and one disulfide bridge,16 ii)
conjugates of cyclic pentapeptic analogues including FC131,17 iii) derivatives of nonpeptidic
small molecules such as bicyclam AMD3100.18 A majority of those peptidic PET CXCR4
tracers are the conjugate of metal chelators harnessed to label positron emitting nuclei such as
Ga-68 and Cu-64, showing preferential accumulation of radioactivities in the lesions of CXCR4-
positive tumors.19-23 However, the use of large chelators such as DTPA, DOTA, and NOTA often
resulted in the reduced ligand affinity due to the steric hindrance upon binding to CXCR4.
Moreover, introducing high energy positron emitters such as Ga-68 (βmax =1.92 MeV) and Co-55
(βmax =1.50 MeV) can lead to the radiolysis of peptides which is hard to suppress, undermining
the advantage of chelators that could provide less laborious and time-saving labeling processes.
Furthermore, the peptidic agents might have the pharmacokinetic disadvantage such as metabolic
instability, low permeability, and in vivo demetallation. Nevertheless, superior affinity of
[68Ga]Pentixafor to CXCR4 and favorable pharmacokinetics expedited its clinical translation.19,
24, 25 However, short half-life (t1/2 = 68 min) and low radioproductivity (<1.85 GBq from
68Ge/68Ga generator) of Ga-68 may limit its clinical usage practically,26 given the radiochemical
properties of the current leading PET emitter, F-18 (t1/2 = 109 min, radioproductivity > 185 GBq,
β
max =0.634 MeV). Direct incorporation of F-18 into biomolecules has foundered due to the
interfering functionalities in target molecules, harsh reaction conditions, poor regioselectivity,
and the low specific activity. Accordingly, labeling peptide with F-18 is usually achieved by
3

coupling with prosthetic groups, but that also requires time-consuming radiosynthesis, ending up
with low radiochemical yield. For example, a couple of nonmetallic, peptidic F-18 PET tracers
were reported, but presented with a long labeling time (> t1/2) and low radiochemical yield (<
5%).27, 28
Recently, nonpeptidic F-18 CXCR4-targeted PET tracers bearing macrocyclic cyclam moiety
were reported.26, 29, 30 In those PET tracers, cyclam moiety was used to label PET emitter, Cu-64,
into the established antagonists such as AMD-3100 and AMD-3465.22, 23, 31, 32 These CXCR4
PET tracers show high T/M ratios (~ 50 for 64Cu-AMD-3100 and ~ 360 for 64Cu-AMD-3465).
However, whole-body imaging can be hampered by high liver (36% ID/g) and kidney uptake (
38% ID/g) owing to their high lipophilicity.19 Moreover, the drawbacks of using metal PET
emitters still remain as described above. More recently, F-18 labeled AMD-3100 or AMD-3465
were reported by means of prosthetic groups which were linked in the various positions of the
antagonists. However, these PET tracers display moderate tumor uptake and significant liver and
kidney uptake which is the caveat of the previous CXCR4- PET tracers. Interestingly, the
cationic nature of cyclam is hypothesized to contribute to the unwanted liver and kidney uptake
along the expression of nonspecific organic cation transporters on the tissues.30
The majority of PET agents belongs to small molecules due to their favorable distribution to
targets, rapid clearance, and better penetration. At present, most of reported small molecular
CXCR4-targeted PET tracers possess chelating moieties such as cyclam, DTPA, NOTA, and
DOTA. More importantly, none of the reported tracers has the same structure as their original
agents, but rather they are introduced additional chemical moieties for radiolabeling, which may
alter the biological properties of the original ligand. For example, an introduction of fluorobenzyl
moiety to c-terminal of highly CXCR4-specific TN14003 peptide caused the additional affinity
to CXCR4-negative red blood cells.28 In this context, we sought to develop a novel small
molecular (< 500 Da) CXCR4-targeted F-18 PET agent not by introducing additional moieties
into the established structure of agents for the purpose of labeling, but rather to develop the
authentic structure bearing an isotope, fluorine, that can be directly labeled into the molecule by
adopting rational drug design approach.
4

Scheme 1. Reagents and conditions: (a) DCM, TEA, ice bath to r. t., 6 h, 75%; (b) MeCN, r.t., 12 h, 75%; (c) DMF,
K2CO3, 70 °C, 51%; (d) DMF, NaN3, CuI, DMEDA, EtOH/H2O (7:3), 80 °C, 6 h, 80%; (e) DCM, TEA, DMAP, 0
°C, 30 min; 70%, X = CH3 or C6H4CH3-p or C6H4NO2-p; (f) THF/H2O (10:1), CuSO4, sodium ascorbate, r.t., 3 h,
60%; (g) t-BuOH, CsF, reflux, 3 h, 51%; (h) MeCN, K2CO3, K222, [18F]F-
2. Materials and methods
1.1. General
All chemical reagents were obtained from commercial sources (Sigma Aldrich Co., St Louis
MO, USA) and used without further purification unless otherwise noted. NMR spectra were
recorded on a Varian 400 MHz NMR spectrometer or Inova 400 MHz NMR spectrometer. Mass
spectra for the small molecules were obtained using an Agilent 1100 LC/MSD VL instrument.
Thin Layer Chromatography (TLC) carried out on silica gel 60 (Merck; 230-400 mesh ASTM).
RP-HPLC was performed using Beckman Coulter System Gold® on Waters XTerra® Prep C18
Column (10 µm, 10 × 250 mm).
1.2. Cell Culture
Dulbecco’s modified Eagle’s medium (DMEM) with glutamine, Penicillin/Streptomycin and
0.5% Trypsin-EDTA were purchased from GIBCO (Grand Island, NY). Fetal bovine serum
(FBS) was purchased from HyClone (Logan, UT). All cell lines were maintained in 5% CO2 at
37 °C. MDA-MB-231 (human breast adenocarcinoma) cells were cultured in DMEM
supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin. The human SCC
cell line 686LN and its derivatives (nonmetastatic clones 686LN-Ps and metastatic clones
686LN-Ms) were cultured in DMEM/F-12 (Invitrogen) supplemented with 10% FBS, 50 IU/mL
penicillin, and 50 μg/mL of streptomycin. Metastatic subclones of E3 cells (CXCR4-positive
murine carcinoma) were cultured in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient
Mixture F-12) supplemented with 10% FBS, 50 IU/mL penicillin, 50 μg/mL streptomycin.
5

1.3. Computational modeling (Molecular Docking)
The default parameters in the Maestro docking module of Schrödinger Suite (v. 9.3) were used
unless otherwise noted. The CXCR4 receptors were prepared using the Protein Preparation
Wizard in the Schrödinger Suite that assigns bond orders, adds hydrogen atoms, and creates
disulfide bonds. The hydrogen-bonding network was optimized at neutral pH 7.0. Receptor grids
were constructed with Glide using a 10 × 10 × 10 Å3 boundary box spanning the entire ligand
binding pocket and centered on the centroid of either IT1t (from 3ODU, PDB ID) or the Arg2–
Nal3 residues of CVX15 (from 3OE0, PDB ID).33 The designed ligands were prepared using the
2D Sketcher, Lig-Prep, and the OPLS3 force field. Minimized ligand poses were docked flexibly
into the CXCR4 receptor grids using Glide Standard Precision (Glide SP) and Extra Precision
(Glide XP) and the Epic state-penalties were added to the Glide score. To estimate the binding
affinity of designed ligands to CXCR4, the energy of ligand-receptor complex (MMGBSA dG
Bind) was calculated using Prime MM-GBSA method and compared to the values of known
active CXCR4-ligands.
1.4. Chemical Synthesis
Synthesis of Compound 1. To a solution of 4-(bromomethyl)benzene-1-sulfonyl chloride (1.00
g, 3.71 mmole ) in DCM was added N-methylpropargylamine (0.28 g, 4.08 mmole, 1.1 eqv.)
under cooling. The reaction mixture was stirred at 0 °C for 6 h and monitored by TLC. After
completion of the reaction, the mixture was purified by flash column chromatography, eluting
with ethyl acetate/hexane (1:3, v/v). 1H NMR (400MHz, CD3Cl): δ 7.80 (2H, m), 7.55 (2H, m),
4.51 (2H, s), 4.05 (2H, d, J13 = 2.4 MHz), 2.86 (3H, s), 2.06 (1H, t, J13 = 2.4 MHz) ppm. The
purified compound was obtained as white crystalline powder (835 mg, yield 75%) and dissolved
in acetonitrile, then was added morpholine (267 mg, 1.1 eqv.). The reaction mixture was stirred
at room temperature overnight. After the completion of the reaction, the mixture was filtered
through celite and organic solvent was removed by rotary evaporation. The residue was
dissolved in DCM and washed with water, brine, and dried over MgSO4 and concentrated. The
crude product was purified by flash column chromatography, eluting with DCM/methanol (10:3,
v/v). The purified compound was obtained as a white powder. (724 mg, yield 85%). 1H NMR
(400MHz, CD3Cl): δ 7.80 (2H, m), 7.60 (2H, m), 4.05 (2H, d, J13 = 2.4 MHz), 3.82 (4H, m),
3.73 (2H, s), 2.85 (3H, s), 2.62 (4H, m), 2.07 (1H, t, J13 = 2.4 MHz) ppm.
Synthesis of Compound 2. Compound 2 was synthesized as described previously.34 Briefly, to a
suspension of 4-bromophenol (5.00 g, 28.90 mmol) and K2CO3 (5.99 g, 43.35 mmol) in DMF
(100 mL) was added 3-bromo-1-propanol (6.02g, 43.35 mmol), and the reaction mixture was
heated to 70 °C. After 12 h, the reaction mixture was cooled to room temperature, quenched with
water and aqueous NH4Cl. The organic compound was extracted with ethyl acetate (3 × 100
mL). The combined organic layer was washed with brine (3 × 100 mL), dried over MgSO4, and
then concentrated under reduced pressure. The residue was purified by flash column
6

chromatography, eluting with ethyl acetate/hexane (1:4, v/v) to afford 3-(4-
bromophenoxy)propan-1-ol as a white solid (3.4 g, yield 51%). 1H NMR (400MHz, CD3Cl): δ
7.35 (2H, d), 6.76 (2H, d), 4.04 (2H, t), 3.81 (2H, t), 2.24 (1H, br), 2.06~1.94 (2H, m) ppm. The
3-(4-bromophenoxy)propan-1-ol (1.00 g, 4.33 mmol), sodium azide (563 mg, 8.66 mmol),
sodium ascorbate (44 mg, 0.22 mmol), copper iodide (82 mg, 0.43 mmol), and N, N’-
dimethylethylenediamine (DMEDA, 70 µL, 0.65 mmol) were placed in a two-necked round
bottom flask equipped with a reflux condenser, and 13 mL of ethanol/water (7:3, v/v) was added
under N2 atmosphere. The reaction mixture was stirred at 80 °C for 6 h, and then cooled down to
room temperature, quenched with water, and organic compounds were extracted with ethyl
acetate (3 × 100 mL). The combined organic layer was washed with brine (3 × 100 mL), dried
over MgSO4 and concentrated under reduced pressure. The residue was purified by flash column
chromatography, eluting with ethyl acetate/hexane (2:3, v/v) to afford compound 2 as yellow oil
(669 mg, 80% yield). 1H NMR (400MHz, CD3Cl): δ 6.95~6.84 (4H, m), 4.06 (2H, t), 3.82 (2H,
t), 2.35 (1H, br), 2.07~1.95 (2H, m) ppm.
Synthesis of Compound 3. (compound 3-1: X = CH3 or 3-2: C6H4CH3-p or 3-3: C6H4NO2-p).
To a solution of 3-(4-azidophenoxy)-propan-1-ol (498 mg, 2.58 mmol) and trimethylamine (432
µL, 3.09 mmol) in DCM (10 mL) was added slowly methanesulfonyl chloride for 3-1 or
toluenesulfonyl chloride for 3-2 or 4-nitrobenzenesulfonyl chloride for 3-3 (1.2 eqv.) in DCM at
0 °C over 10 min. After 30 min, the reaction was quenched with water and aqueous NH4Cl, and
organic compound was extracted with DCM (3 × 100 mL). The combined organic layer was
dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by
flash column chromatography, eluting with ethyl acetate/hexane (1:4, v/v) to afford 3-(4-
azidophenoxy) propyl methanesulfonate (3-1, white solid, yield 64%). 1H NMR (400MHz,
CD3Cl): δ 6.95~6.84 (4H, m), 4.42 (2H, t), 4.06 (2H, t), 2.98 (3H, s), 2.20 (2H, dd) ppm; 3-2: 3-
(4-azidophenoxy) propyl toluenesulfonate (3-2, white solid, yield 70%). 1H NMR (400MHz,
CD3Cl): δ 7.75 (2H, m), 7.25 (2H, m), 6.90 (2H, m), 6.72 (2H, m), 4.23 (2H, t), 3.90 (2H, t), 2.37
(3H, s), 2.09 (2H, dd) ppm; 3-(4-azidophenoxy) propyl nitrobenzenesulfonate (3-3, yellow solid,
yield 79%). 1H NMR (400MHz, CD3Cl): δ 8.23~8.20 (2H, m), 8.03~8.01 (2H, m), 6.88~6.85
(2H, m), 6.67~6.64 (2H, m), 4.43 (2H, t), 3.86 (2H, t), 2.13 (2H, dd).
Synthesis of Compound 4. (compound 4-1: X = CH3 or 4-2: C6H4CH3-p or 4-3: C6H4NO2-p).
To a mixture of compound 1 (50 mg, 0.162 mmol) and 3-1 or 3-2 or 3-3 (1.1 eqv.) in THF 2 mL
was added aqueous solution of 1 M CuSO4 100 µL and 1 M sodium ascorbate 100 µL. The
mixture was stirred at room temperature for 3 h. The reaction mixture was extracted with ethyl
acetate (3 × 50 mL) and dried over MgSO4, and then concentrated under reduced pressure. The
residue was purified by flash column chromatography, eluting with DCM/methanol (10:1, v/v) to
1
afford compound 4-1 (X = CH3) (white solid, yield 61%). H NMR (400MHz, DMSO-d6): δ
8.59 (1H, s), 7.76~7.73 (4H, m), 7.50 (2H, d), 7.15 (2H, d), 4.38~4.36 (4H, m), 4.15 (2H, t), 3.54
(4H, m), 3.53 (2H, s), 3.34 (6H, m), 3.20 (3H, s), 2.75 (3H, s), 2.30 (4H, m), 2.16 (2H, m) ppm.
7

13C NMR (400MHz, DMSO-d6): δ 158.3, 143.4, 142.1, 135.8, 129.8, 127.8, 122.1, 115.9, 88.6,
66.7, 64.3, 62.0, 58.2, 53.5, 44.7, 36.9, 35.4, 29.2 ppm. HRMS (ESI-TOF) m/z: [M+H]+ Calcd
for C25H34N5O7S2 580.18; Found 580.19. Anal. Calcd for C25H33N5O7S2: C, 51.80; H, 5.74; N,
12.08; S, 11.06; F, 3. Found: C, 51.64; H, 5.67; N, 11.99; S, 10.91 or compound 4-2 (X =
C6H4CH3-p) (white solid, yield 71%) 1H NMR (400MHz, DMSO-d6): δ 8.60 (1H, s), 7.51 (2H,
d), 7.39 (2H, d), 7.00 (2H, d), 4.36 (2H, s), 4.20 (2H, t), 3.98 (2H, t), 3.54 (4H, m), 3.50 (2H, s),
2.75 (3H, s), 2.33~2.30 (6H, m), 2.95 (2H, m) ppm. 13C NMR (400MHz, DMSO-d6): δ 158.6,
145.3, 144.0, 142.8, 135.4, 130.6, 130.4, 129.8, 128.0, 127.8, 125.8, 122.6, 122.0, 115.1, 68.0,
66.6, 64.0, 62.0, 53.5, 45.3, 35.4, 28.5, 21.5 ppm. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C31H38N5O7S2 656.21; Found 656.22. Anal. Calcd for C31H37N5O7S2: C, 56.78; H, 5.69; N,
10.68; S, 9.78. Found: C, 56.48; H, 5.59; N, 10.48; S, 9.80. or compound 4-3 (X = C6H4NO2-p)
(yellow solid, yield 74%) 1H NMR (400MHz, DMSO-d6): δ 8.57 (1H, s), 8.31 (2H, d), 8.17 (2H,
d), 7.75 (2H, d), 7.68 (2H, d), 7.51 (2H, d), 6.95 (2H, d), 4.36~4.34 (4H, m), 3.95 (2H, t),
3.54~3.51(6H, m), 2.75 (3H, s), 2.31 (3H, br), 2.10 (2H, m) ppm. 13C NMR (400MHz, DMSO-
d6): δ 158.0, 150.4, 142.3, 140.0, 135.6, 130.0, 129.3, 127.3, 126.9, 124.9, 123.0, 122.1, 121.3,
115.1, 68.8, 66.1, 63.2, 61.6, 53.1, 44.9, 35.0, 27.9 ppm. HRMS (ESI-TOF) m/z: [M+H]+ Calcd
for C30H35N6O9S2 687.18; Found 687.19. Anal. Calcd for C30H34N6O9S2: C, 52.47; H, 4.99; N,
12.24; S, 9.34. Found: C, 52.17; H, 5.00; N, 12.00; S, 9.41.
Synthesis of Compound 5. To a solution of compound 4-1 (100 mg, 0.173 mmol) in tert-
butanol (2 mL) was added Cesium fluoride (131 mg, 5.0 eqv.) and refluxed the reaction mixture
for 3 h. After the completion of the reaction, the reaction solvent was removed by rotary
evaporation. The residue was dissolved in DCM and washed with water, brine, and dried over
MgSO4 and concentrated. The crude product was purified by flash column chromatography,
eluting with DCM/methanol (10:1, v/v). The purified compound was obtained as a white powder.
(44 mg, yield 51%). 1H NMR (400MHz, DMSO-d6): δ 8.59 (1H, s), 7.76~7.72 (4H, m), 7.49
(2H, d), 7.14 (2H, d), 4.69 (1H, t), 4.57 (1H, t), 4.36 (2H, s), 4.15 (2H, t), 3.53 (4H, br), 3.49
(2H, s), 2.75 (3H, s), 2.30 (4H, br), 2.14 (2H, m) ppm. 13C NMR (400MHz, DMSO-d6): δ 158.4,
143.5, 142.3, 135.4, 130.0, 129.3, 127.3, 122.2, 212.6, 115.4, 80.8 (1JC-F = 643 Hz), 66.1, 64.0
(3JC-F = 2.4 Hz), 61.6, 53.1, 44.9, 35.0, 29.6 (2JC-F = 26 Hz) ppm. 19F NMR (300MHz, CD3Cl): δ
7.53 (1F, m). HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C24H31FN5O4S 504.20; Found 504.21.
Anal. Calcd for C24H30FN5O4S: C, 57.24; H, 6.00; N, 13.91; S, 6.37; F, 3.77. Found: C, 57.07;
H, 5.88; N, 13.99; S, 6.46; F, 3.67.
1.5. Radiosynthesis (Synthesis of Compound [18F]5)
No-carrier-added [18F]F- was obtained through the nuclear reaction 18O(p, n)18F by irradiation of
18O-enriched water. After the delivery of [18F]F- from the cyclotron, the radioactivity was passed
through an anion exchange resin cartridge to trap [18F]F-. [18F]F- was then eluted with a
potassium carbonate solution into a vessel containing Kryptofix 2,2,2 (K222) and the mixture was
dried by azeotropic distillation with acetonitrile. Compound 4 (4-1 or 4-2 or 4-3) in anhydrous
acetonitrile was added to the dried K222/K[18F]F- and the mixture was heated at 120 ˚C for 30
8

min to produce [18F]compound 5. The crude reaction mixture was purified by HPLC (Prep
column, methanol/water = 1/1), then the collected fraction including [18F]5 was trapped on C18
solid phase extraction cartridge and eluted by ethanol into a vial containing isotonic saline. A
total 123 mCi of [18F]5 was obtained from 1389 mCi of [18F]- fluoride in a synthesis time of
approximately 100 min. The precursors (4-1, 4-2, and 4-3) provided 16.5 % (from 4-1), 15.7%
(from 4-2), and 27.0% (from 4-3) decay corrected yield (DCY) of [18F]5 respectively (Fig. S6A
using 4-1). We were able to obtain [18F]5 from 3 different precursors (4-1, 4-2, and 4-3). Because
the mesyl precursor provided the highest radiochemical yield, we only showed the
chromatogram of the mesyl precursor. For the chromatogram of standard cold compound 5, we
included in Fig S6. C at the bottom. Because low Signal of [18F]5 in crude mixture in HPLC, we
used the chromatogram of [18F]5 in the dose to confirm the radiotracer identity by co-injecting
standard cold compound 5. Since the specific activity of [18F]5 was not directly determined due
to the detection limit of UV detector in HPLC, we assume that the maximum amount of
nonradioactive material in the final dose arising from the precursor is about 1μg. On the basis of
an HPLC serial dilution injection of the aqueous solution of cold compound 5 (100μg/mL,
10μg/mL, 1μg/mL, 0.1μg/mL, and 0.01μg/mL; starting from 0.1μg/mL, no UV peak
corresponding to 5 was observed on HPLC chromatogram) using the same analytical HPLC
labeling method and a 123 mCi yield in 7.5 mL solution at end of synthesis, the amount of
unlabeled material in the final product would not exceed 0.06 μg/mCi. Quality control HPLC of
the labeled [18F]5 showed chemical and radiochemical purity > 99%. The saline solution of
[18F]5 was sterilized by filtration through a 0.2-micron filter for further study. The cold (radio-
inactive) standard cold compound 5 was co-injected with dose and HPLC traces displayed the
same elution time which supported that the dose was the desired hot compound ([18F]5) (Fig.
S6C).
1.6. In vitro affinity assay
MDA-MB-231 cells were cultured in an eight-well slide chamber for two days. The cells were
pre-incubated with the antagonist (compound 5 or AMD-3100) for 15 min, and then fixed with
4% formaldehyde. The fixed cells were subsequently incubated for 45 min with biotinylated-
TN14003 (CXCR4-specific antagonist peptide). Then, cells were incubated for 30 min in
streptavidin–rhodamine red-x at a 1:150 dilution (Jackson Immuno Research Laboratories, West
Grove, PA, USA) after washing three times with PBS. The slides were washed with PBS and
mounted in an anti-fade mounting solution (Molecular Probes, Eugene, OR, USA). The pictures
of stained cells for each treatment were taken on a Nikon Eclipse E800 microscope. Pictures
were analyzed quantitatively with ImageJ. The inhibition % of the antagonist (compound 5 or
AMD-3100) was tested at 0.01, 0.1, 1, 10, 100, 1000, and 10000 nM and IC50 value for the
antagonist (compound 5 or AMD-3100) was fitted with GraphPad Prism 4.
1.7. In vitro Matrigel invasion assay
Matrigel invasion chambers from BD Biocoat Cellware (San Jose, CA, USA) were used for
invasion assays. MDA-MB-231 cells were cultured on a layer of Matrigel in the upper chamber
9

with the antagonist (compound 5 or AMD-3100) at 1, 10, 100, and 1000 nM, while 200 ng/ml of
CXCL12 was added in the lower chamber as a chemoattractant. The Matrigel invasion chamber
was incubated for 22 h in a humidified tissue culture incubator. After noninvading cells were
removed from the top of the Matrigel with a cotton-tipped swab. Invading cells at the bottom of
the Matrigel were fixed in methanol and stained with H&E. The invasion rate was determined by
counting the H&E-stained cells.
1.8. In vitro competition binding assay with hot compound 5
CXCR4-positive metastatic squamous cell carcinoma of head and neck (SCCHN) cells were
preincubated with radiolabeled ligand suspension in binding buffer (BB, 1 mg/mL BSA in PBS,
180 µL of cell suspension of 1 × 107 cells/mL + 5 µL solution of [18F]compound 5 (1 µCi/µL) in
each tube for 15 min, and 20 µL of natural ligand (CXCL12) solutions were added to make up
final concentrations of 11, 33, and 100 nM, and then incubated for 60 min with gentle vortexing
every 10 min. The tubes were centrifuged, and the supernatant was removed, then the cells were
washed with 0.5 mL cold BB twice. 200 µL of cold BB was added to each tube and 50 µL of cell
suspension was taken from each tube (triplicate). Finally, radioactivity of each tube was
measured by a gamma counter.
1.9. In vivo μPET/CT study for inflammation model with hot compound 5
Acute inflammation was induced by subcutaneous injection of 50 μL of λ-carrageenan (1% w/v
in saline) into one of the hind paws of male nude mice (6 weeks, 20 g, Jackson Laboratory,
Strain JAZ 007850 J:NU). An apparent edema response was observed 5 h after the λ-carrageenan
injection. 150 µCi of radiotracer ([18F]compound 5) in 150 µL PBS was injected to mice through
the tail vein (i.v.). Then, mice were anaesthetized with isoflurane (1.0 ~ 2.0%) and 90 min after
injection of radiotracer, PET images were acquired for 20 min using an Inveon micro PET/CT
Preclinical Scanner (Siemens). Subsequent CT images were acquired for 10 min.
2.10. In vivo μPET/CT study for head and neck cancer with hot compound 5
SCCHN (squamous cell carcinoma of head and neck) tumor cells were inoculated (2.5 × 106
cells in 50 µL) to female nude mouse (6 weeks, ~20 g, Harlan Sprague Dawley, Strain: HSD :
Athymic Nude-Foxn1<nu>) in the area of neck (orthotopic), 3 weeks before in vivo test.
PET/CT images were acquired using the same protocol as the paw edema case.
2.11. In vivo μPET/CT study with cold compound 5 (Blocking study)
Before in vivo blocking assay, toxicity of cold compound 5 was tested. 10 mg/kg dose (vehicle:
10% DMSO + 90% PBS including 45% cyclodextrin) of compound 5 was injected to the mouse
(female, 6 weeks, 20g, Jackson Laboratory, JAX000819 B6 Cg-Foxnl <nu>/J.) through the tail
vein (i.v.), which showed no toxicity to mice. For the blocking, 200 µL of cold compound 5
solution (30 mg/kg, 10% DMSO, 90% PBS including 45% cyclodextrin) was administered to the
mouse (i.p.) (male, 6 weeks, 20 g, Jackson Laboratory, Strain JAZ 007850 J:NU) 30 min prior to
10

[18F]5 injection (i.v.). PET/CT images were acquired using the same protocol as the paw edema
case.
2.12. In vivo μPET/CT study for mouse lung metastasis model with hot compound 5
Six-week-old female nude mice (Harlan Sprague Dawley, Strain: HSD : Athymic Nude-
Foxn1<nu>) were given injections of 2.0 × 106 metastatic subclones of E3 (CXCR4-positive
murine carcinoma cells donated by Dr. Zhuo (Georgia) Chen35) through the tail vein. After 8
weeks, PET/CT images were acquired using the same protocol as the paw edema case.
3. Results
3.1. Design and Synthesis of Small Molecule CXCR4 F-18 PET Ligand
Using the benzenesulfonamide scaffold of CXCR4 antagonist which was reported previously,36
we designed novel molecular structures containing a fluorine atom to synthesize CXCR4-
targeted F-18 PET agents. First, we analyzed a series of the reported benzenesulfonamide
compounds by chemoinformatics model (fingerprint-based KPLS in Canvas, Schrödinger Suite)
to access each atom’s contribution to biological activities such as binding affinity and
chemotaxis inhibition. (Fig. S1). The analysis indicated each atom’s positive or negative
contribution to the binding affinities of compounds. Thus, we replaced the negatively
contributing moiety with fluorine-containing group to label the compound with F-18
Figure 1. Docking pose of Compound 5 in the CXCR4 crystal structure (A) Docking pose representing the
interactions of compound 5 with the critical residues of CXCR4 (W94, D97, Y116, D187, R188). Dotted lines show
putative hydrogen bonds. (B) Molecular surface representation of the binding pocket of CXCR4 receptor in the
crystal structure [PDB ID: 3OE0] colored by atom type (carbon and hydrogen: light grey; nitrogen: blue; oxygen:
red). The ligand (Compound 5) is represented by stick models.
radionuclide without losing the affinity. Next, we estimated the binding affinity of the designed
compounds through molecular docking model using Glide (Schrödinger Suite). In order to
validate our docking model first, we conducted “redocking” experiment (Fig. S2) and examined
the correlation between MM-GBSA dG binding energies and in vitro affinity data (IC50) (r2 ≈
0.7). Then, we compared the calculated binding energy of designed compounds with the values
11

of known benzenesulfonamide antagonists. We chose the best structure (compound 5) which
showed competitively high binding energy (-63.7 kcal/mole) and reasonable pose representing
the interactions with the critical residues (W94, D97, Y116, D187, R188)37 of CXCR4 which
were reported previously (Fig. 1, Fig. S3). To synthesize the best compound, we prepared
compound 1 and 3 respectively as described in the previous literatures34, 36, 38 (Scheme 1). Then,
we introduced 3 different leaving groups (mesyl, tosyl, and nosyl) into the precursor of
compound 5 to increase labeling yield and purify the labeled compound from the unlabeled
compounds more effectively. Then, we adopted copper-catalyzed azide-alkyne cycloaddition
(CUAAC, click chemistry) which allows an easy and straightforward synthetic route for the
precursors (compound 4) of the F-18 PET ligand ([18F]compound 5). To optimize radiochemical
synthesis, we prepared [18F]5 from the corresponding 3 different precursors (mesyl, tosyl, and
nosyl). Nosyl precursor provided the best radiochemical yield, but mesyl precursor showed the
best separation of leveled compound from unlabeled impurities as the HPLC profile indicates
(Fig. S6). We also prepared F-19 cold compound 5 as a standard for purification and to test its
biological activities before in vivo PET evaluation of hot compound [18F]5.
3.2. In vitro affinity and chemotaxis inhibition assays
To
Figure 2. In vitro assays of compound 5 (A) Binding assay using the biotinylated CXCR4 antagonist TN14003.
MDA-MB-231 cells on an 8-well slide chamber were treated with various concentrations of the antagonist
(compound 5 or AMD-3100) and biotin-labeled TN14003. After washing, cells were incubated with streptavidin-
rhodamine. Mean fluorescence intensity was quantitated with ImageJ. IC50 (compd 5) = 6.9 nM; IC50 (AMD-3100)
= 66 nM. (B) Matrigel invasion assay induced by CXCR4/CXCL12-mediated chemotaxis using MDA-MB-231
cells in the presence of the antagonist (compound 5 or AMD-3100). The invasion % was determined by counting
the H&E-stained cells. (C) In vitro celluar uptake assay with hot compound [18F]5 and CXCL12 as a competitive
ligand. CXCR4-positive metastatic squamous cell carcinoma of head and neck (SCCHN) cells were preincubated
with radiolabeled ligand and natural ligand (CXCL12) were added to make final concentrations of 11, 33, and 100
nM. After 60 min incubation and washing, radioactivity of each tube was measured by a gamma counter (triplicate).
Error bars indicate standard deviations.
test the affinity of the designed PET ligand, we performed in vitro binding assay as described
previously,39 which showed 6.9 nanomolar IC50 of compound 5 to block CXCR4-specific
antagonist peptide, TN14003. As a positive control, we also tested an established CXCR4
antagonist, ADM-3100, which showed IC50 (66 nM) close to reported values. (Fig. 2A, Fig.
12

S4). As a secondary functional assay, we employed Matrigel invasion assay39 to test the
antagonistic activity of compound 5 against CXCR4/CXCL12 axis. Chemokine CXCL12 (or
SDF-1) is the natural ligand of CXCR4 and strongly chemotactic for the CXCR4 positive cells.
Compound 5 inhibited 85% of chemotactic invasions at 100 nM in the Matrigel assay, which is
better than AMD-3100 (55%) (Fig. 2B, Fig. S5).
3.3. In vitro cellular uptake assays
To determine whether compound 5 binds to the same target (CXCR4) as endogenous chemokine
ligand (CXCL12), we performed in vitro competition binding assays with radioactive [18F]5 and
CXCL12. At first, CXCR4-positive cells (686LN) were labeled with [18F]5 showed strong
radioactivity due to the retention of [18F]5 in the absence of CXCL12. Then, as we increase the
concentration of CXCL12, the observed radio activity is reduced by 32.4 8.5% (100 nM),
27.9 2.2% (33 nM), 14.7 5.1% (11 nM) in a dose-dependent manner. This result suggests that
[18F]5 competes for the same binding sites of endogenous ligand, CXCL12 (Fig. 2C).
3.4. In vivo imaging study
Figure 3. μPET/CT images of [18F]5 (A) Acute inflammation was induced by subcutaneous injection of λ-
carrageenan into one of the hind paws of male nude mice. An apparent edema response was observed 5 h after the
λ-carrageenan injection. Radiotracer ([18F]5) was injected to mice through the tail vein. Then, mice were
anaesthetized with isoflurane. 90 min after injection of radiotracer, PET images were acquired for 20 min using an
Inveon micro PET/CT Preclinical Scanner (Siemens). Subsequent CT images were acquired for 10 min. (B)
SCCHN (squamous cell carcinoma of head and neck) CXCR4-positive tumor cells were inoculated (2.5 × 106 cells
in 50 µL) to female nude mouse in the area of neck (orthotopic), 3 weeks before in vivo test. PET/CT images were
acquired using the same protocol as the paw edema case. Tumor is indicated by yellow arrows. (C) Six-week-old
female nude mice were given injections of 2.0 × 106 metastatic subclones of E3 (CXCR4-positive murine carcinoma
cell line) through the tail vein. After 8 weeks, PET/CT images were acquired using the same protocol as the SCCHN
cancer study. Representative [18F]5 transverse PET images of 90 min p.i., Yellow arrows and text denote the location
of the tumor (T), lung (L), spinal column (SC). The circles in the bottom figure indicate induced metastatic tumor
nodules. (D) μPET transverse plane images with cold + hot (Blocking, left) compd. vs only hot compd. (right); For
the blocking, 200 µL of cold [19F]5 solution (30 mg/kg, 10% DMSO, 90% PBS including 45% cyclodextrin) was
administered to the mouse (i.p.) (male, 6 weeks, 20 g, Jackson Laboratory, Strain JAZ 007850 J:NU) 30 min prior
to [18F]5 injection (i.v.). PET images were acquired using the same protocol as the paw edema case. This experiment
suggests the specificity of compound 5 to bind CXCR4-positive stem cells in bone marrow.
13

After testing the affinity and antagonistic activity of compound 5, we are encouraged to conduct
in vivo imaging studies using animal models. CXCR4/CXCL12 interaction has been reported to
play an important role in the recruitment of immune cells during early phase of inflammation.39-
43 Therefore, we tested the ability of compound [18F]5 to visualize λ-carrageenan-induced
inflammation in a mouse model as described above, which showed significantly higher
radioactivity in the lesion of paw edema (Fig. 3A). Mean tracer uptake, as determined by injected
dose per gram (%ID/g), was found to be statistically different, using a paired Student’s t test (p =
0.0015, n =5), between paw edema (1.53 0.08 %ID/g) and negative control. Then, to test the
ability to visualize tumor non-invasively, we evaluated the accumulation of [18F]5 in xenograft
SCCHN expressing CXCR4 in orthotopic mouse model. After administration of [18F]5, mice
exhibited preferential accumulation in sites of tumor growth (4.00 0.28 %ID/g), relative to
muscle (p = 0.005, n=5), in our orthotopic xenograft model (Fig. 3B). Further, we evaluated the
ability of [18F]5 to image metastatic tumors arising in the lung using athymic nude mouse model
as described above. After administration, [18F]5 displayed preferential accumulation in lung
metastatic tumors (1.66 0.14 %ID/g). Based on Standardized Uptake Value (%ID/g), a tumor-
to-lung ratio of 3:1 (p = 0.002, n = 5) was achieved by conclusion of the 90-min scan (Fig. 3C).
Of note, we found illuminated bone
Table 1. Biodistribution of [18F]5 at 90 min
marrow presumably due to the enrichment
of non-neoplastic CXCR4-positive stem
cells (not from defluorination and uptake in
the bone). To test our hypothesis, we
performed a blocking experiment with cold
compound 5 (F-19 compound 5). After
administering of cold compound 5, we
observed significant decreased (95% >)
radioactivity in the bone regions of the
blocking models, which suggests specific
binding of compound 5 to CXCR4-positive
stem cells in bone marrow (Fig. 3D) and further supports our hypothesis. However, a trace of
radioactivity was detected in the spinal region of cold compound injected mice. A possible
explanation for this vague uptake is that the small portion of CXCR4 receptors are rapidly
internalized and recycled to the cell surface,44 which lead to the availability of new unoccupied
receptors in the presence of competing compound.
4. Discussion
In this study, we sought to develop a novel small molecular CXCR4-targeted PET radioligand
which is designed through a rational drug design approach not to change its authentic structure.
Because our new tracer doesn’t necessitate the additional labeling moieties such as chelators
which can alter biological properties of the radioligand, we expect our PET tracer could be a
more reliable noninvasive imaging probe to evaluate CXCR4-relavant diseases.
14

To achieve our goal, we determined the modifiable part of our CXCR4 antagonists36 through
highly predictive QSAR model, kernel-based partial least squares (KPLS) method with chemical
fingerprint model (Canvas in Schrödinger Suite) (Fig. S1). At first, a series of our prior
benzenesulfonamide compounds and their binding affinities to CXCR4 were analyzed by the
KPLS/fingerprint model to access each atom’s contribution to binding affinity (IC50). This
chemoinformatic model revealed each atom’s impact on the predicted binding affinity and
elucidated that the benzylamine group exerts a negative effect on affinity whereas the other part
of compound makes a positive contribution to the affinity, suggesting that structural modification
could be employed on this benzylamine moiety without affinity loss. Second, we designed a
series of conceptualized radioligand structures by combining the positively contributing part of
the scaffold with new components bearing fluorine atom in the modifiable part. Third, we
estimated the affinity of each conceptualized structure to CXCR4 through molecular docking
simulation (Glide in Schrödinger Suite), then screened out the compounds with high estimated
affinity worthwhile to synthesize. In the molecular docking simulation, we tested two crystal
structure of CXCR4 (3ODU and 3OE0), and used 3OE0 because it showed the better correlation
with in vitro assay data of the known CXCR4-antagonists possessing benzenesulfonamide
scaffold. 3ODU has higher resolution (2.5 Å) than 3OE0 (2.9 Å), but 3ODU is often unable to
generate a consistent pose with X-ray structure and interactions with critical residues of CXCR4
as described in the previous literature.33
After synthesizing selected compounds (Scheme 1), we assessed actual biological activities of
the compounds by two orthogonal in vitro assays such as receptor binding assay (Fig. S4) and
chemotaxis inhibition assay (Fig. S5). Through these in vitro assays, we identified the compound
5 as the most potent one. In our binding assay, IC50 value of the compound 5 was determined to
be 6.9 nM which is comparable to the affinity of reported prior antagonists (low nanomolar Kd).
Encouraged by the promising in vitro results, we conducted in vivo test using the compound 5
labeled with F-18 to evaluate the ability to visualize CXCR4-associated diseases such as
inflammation, head and neck cancer, and metastatic lung cancer using the corresponding mouse
models. Our mouse models exhibited preferential accumulation of tracers in the lesion of
inflammation and orthotopic xenograft SCCHN, which are consistent with our anatomical
studies (Fig 3). To explore the diagnostic potential of [18F]5 for cancer metastasis as well, we
evaluated [18F]5 in our lung metastasis model and observed higher radioactivity accumulated in
the sites of tumor growth, compared with the normal lung tissue area, and found modest tumor
tissue wash-out over the course of a 90-min uptake period. Of note, the uptake of metabolic
organs (liver: 1.20 0.12 %ID/g and kidney: 3.22 0.60 %ID/g) were relatively low, compared
with prior CXCR4-tracers21, 25, 45 likely due to the absence of the aforementioned cationic
chelating moieties and faster clearance of the tracer.
While the present study provides an initial evaluation of [18F]5 as a PET imaging probe to
visualize metastatic cancer guided along CXCR4 expression, the potential of this imaging probe
to be served as an alternative to established PET agents such as FDG requires further
15

exploration. Wester et al. reported that CXCR4-targeted-cyclic peptide-conjugated chelate
([68Ga]Pentixafor) displays more CXCR4-specific cancer images of multiple myeloma patients
with higher contrast than FDG,24 whereas the same tracer showed a lower radioactivity
accumulation in solid tumors.46 Hence, future studies should include comparing [18F]5 with
established agents in a setting where we can evaluate our imaging probe’s performance more
rigorously using additional cancer models beyond our presented animal models.
5. Conclusion
In conclusion, we have described the synthesis, characterization, and biological evaluation of our
novel PET-radiotracer, [18F]5, as a CXCR4-specific imaging probe. The radiotracer was
synthesized in high radiochemical yield and high radiochemical purity with stability in a
biological buffer. Micro-PET imaging and biodistribution studies showed high accumulation in
inflammatory lesion, human xenograft tumor, and metastatic lung tumor tissue of mice. Taken
together, [18F]5 hold potential to serve as a CXCR4-specific imaging probe to diagnose and
monitor the prognosis of inflammatory diseases, CXCR4-positive tumors, and metastatic cancers
in the clinic.
Acknowledgements
The authors wish to thank Dr. Bryan Cox, Dr. Qi Shi and the late Dr. James P. Snyder for the
help with computational modeling. We also thank Dr. Dennis C. Liotta for allowing us to use
computation facilities.
16

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20

Scheme 1. Reagents and conditions: (a) DCM, TEA, ice bath to r. t., 6 h, 75%;
(b) MeCN, r.t., 12 h, 75%; (c) DMF, K2CO3, 70 °C, 51%; (d) DMF, NaN3, CuI,
DMEDA, EtOH/H2O (7:3), 80 °C, 6 h, 80%; (e) DCM, TEA, DMAP, 0 °C, 30
min; 70%, X = CH3 or C6H4CH3-p or C6H4NO2-p; (f) THF/H2O (10:1), CuSO4,
sodium ascorbate, r.t., 3 h, 60%; (g) t-BuOH, CsF, reflux, 3 h, 51%; (h) MeCN,
K2CO3, K222, [18F]F-

A
B
Figure 1. Docking pose of Compound 5 in the CXCR4
crystal structure (A) Docking pose representing the
interactions of compound 5 with the critical residues of CXCR4
(W94, D97, Y116, D187, R188). Dotted lines show putative
hydrogen bonds. (B) Molecular surface representation of the
binding pocket of CXCR4 receptor in the crystal structure [PDB
ID: 3OE0] colored by atom type (carbon and hydrogen: light
grey; nitrogen: blue; oxygen: red). The ligand (Compound 5) is
represented by stick models.

A
B
C
110
AMD-3100
Compd 5
100
90
80
70
60
50
40
30
20
10
0
-3 -2 -1 0 1 2 3 4 5
(Log nM)
Figure 2. In vitro assays of compound 5 (A) Competition-binding assay using the biotinylated
CXCR4 antagonist TN14003. MDA-MB-231 cells on an 8-well slide chamber were treated with
various concentrations of compound 5 and biotin-labeled TN14003. After washing, cells were
incubated with streptavidin-rhodamine. Mean fluorescence intensity was quantitated with ImageJ
(triplicate). (B) Matrigel invasion assay induced by CXCR4/CXCL12-mediated chemotaxis using
MDA-MB-231 cells in the presence of compound 5. The invasion % was determined by counting
the H&E-stained cells (triplicate). (C) In vitro cellular uptake assay with hot compound [18F]5
and CXCL12 as a competitive ligand. CXCR4-positive metastatic squamous cell carcinoma of
head and neck (SCCHN) cells were preincubated with radiolabeled ligand and natural ligand
(CXCL12) were added to make final concentrations of 11, 33, and 100 nM. After 60 min
incubation and washing, radioactivity of each tube was measured by a gamma counter
(triplicate). Error bars indicate standard deviations.

15
w/o Blocking %ID/g
3
%ID/g
D
7
%ID/g
A
B
C
10
%ID/g
Inflammation
Control
Blocking
SC
Control
Tumor
Control
Tumor
SC
SC
T
SC
T
L
L
T
T
0
%ID/g
0
%ID/g
19
18
[ F]5 + [ F]5
[18F]5
0
%ID/g
0
%ID/g
Figure 3. μPET/CT images of [18F]5 (A) Acute inflammation was induced by subcutaneous injection of λ-carrageenan into
one of the hind paws of male nude mice. An apparent edema response was observed 5 h after the λ-carrageenan injection.
Radiotracer ([18F]5) was injected to mice through the tail vein. Then, mice were anaesthetized with isoflurane. 70 min after
injection of radiotracer, PET images were acquired for 20 min using an Inveon micro PET/CT Preclinical Scanner (Siemens).
Subsequent CT images were acquired for 10 min. (B) SCCHN (squamous cell carcinoma of head and neck) CXCR4-
positive tumor cells were inoculated (2.5 × 106 cells in 50 µL) to female nude mouse in the area of neck (orthotopic), 3 weeks
before in vivo test. PET/CT images were acquired using the same protocol as the paw edema case. Tumor (T) is indicated
by yellow arrows. (C) Six-week-old female nude mice were given injections of 2.0 × 106 metastatic subclones of E3 (CXCR4-
positive murine carcinoma cell line) through the tail vein. After 8 weeks, PET/CT images were acquired using the same
protocol as the SCCHN cancer study. Representative [18F]5 transverse PET images of 90 min p.i., Yellow arrows and text
denote the location of the tumor (T), lung (L), spinal column (SC). The circles in the bottom figure indicate induced metastatic
tumor nodules. (D) μPET transverse plane images with cold + hot (Blocking, left) compd. vs only hot compd. (right); For the
blocking, 200 µL of cold [19F]5 solution (30 mg/kg, 10% DMSO, 90% PBS including 45% cyclodextrin) was administered to
the mouse (i.p.) (male, 6 weeks, 20 g, Jackson Laboratory, Strain JAZ 007850 J:NU) 30 min prior to [18F]5 injection (i.v.).
PET images were acquired using the same protocol as the paw edema case. This experiment suggests the specificity of
compound 5 to bind CXCR4-positive stem cells in bone marrow.

Table 1. Biodistribution of [18F]5 at 90 min
Organ
SUV (%ID/g)
P
(n = 5)
0.002
0.005
0.002
n/a
Paw edema
SCCHN tumor
Metastatic tumor in lung
Lung
1.53 0.08
4.00 0.28
1.66 0.14
0.50 0.04
1.20 0.12
3.22 0.60
0.16 0.03
0.54 0.08
0.58 0.07
0.68 0.04
0.17 0.04
Liver
n/a
Kidney
n/a
Muscle
n/a
Heart
n/a
Spleen
n/a
Small intestine
Large intestine
n/a
n/a

21

Scheme 1. Reagents and conditions: (a) DCM, TEA, ice bath to r. t., 6 h, 75%; (b)
MeCN, r.t., 12 h, 75%; (c) DMF, K2CO3, 70 °C, 51%; (d) DMF, NaN3, CuI, DMEDA,
EtOH/H2O (7:3), 80 °C, 6 h, 80%; (e) DCM, TEA, DMAP, 0 °C, 30 min; 70%, X = CH3 or
C6H4CH3-p or C6H4NO2-p; (f) THF/H2O (10:1), CuSO4, sodium ascorbate, r.t., 3 h, 60%;
(g) t-BuOH, CsF, reflux, 3 h, 51%; (h) MeCN, K2CO3, K222, [18F]F-
Figure 1. Docking pose of Compound 5 in the CXCR4 crystal structure (A) Docking
pose representing the interactions of compound 5 with the critical residues of CXCR4
(W94, D97, Y116, D187, R188). Dotted lines show putative hydrogen bonds. (B)
Molecular surface representation of the binding pocket of CXCR4 receptor in the crystal
structure [PDB ID: 3OE0] colored by atom type (carbon and hydrogen: light grey; nitrogen:
blue; oxygen: red). The ligand (Compound 5) is represented by stick models.
Figure 2. In vitro assays of compound 5 (A) Competition-binding assay using the
biotinylated CXCR4 antagonist TN14003. MDA-MB-231 cells on an 8-well slide chamber
were treated with various concentrations of compound 5 and biotin-labeled TN14003.
After washing, cells were incubated with streptavidin-rhodamine. Mean fluorescence
intensity was quantitated with ImageJ (triplicate). (B) Matrigel invasion assay induced by
CXCR4/CXCL12-mediated chemotaxis using MDA-MB-231 cells in the presence of
compound 5. The invasion % was determined by counting the H&E-stained cells
(triplicate). (C) In vitro cellular uptake assay with hot compound [18F]5 and CXCL12 as a
competitive ligand. CXCR4-positive metastatic squamous cell carcinoma of head and
neck (SCCHN) cells were preincubated with radiolabeled ligand and natural ligand
(CXCL12) were added to make final concentrations of 11, 33, and 100 nM. After 60 min
incubation and washing, radioactivity of each tube was measured by a gamma counter
(triplicate). Error bars indicate standard deviations.
Figure 3. μPET/CT images of [18F]5 (A) Acute inflammation was induced by
subcutaneous injection of λ-carrageenan into one of the hind paws of male nude mice.
An apparent edema response was observed 5 h after the λ-carrageenan injection.
Radiotracer ([18F]5) was injected to mice through the tail vein. Then, mice were
anaesthetized with isoflurane. 70 min after injection of radiotracer, PET images were
acquired for 20 min using an Inveon micro PET/CT Preclinical Scanner (Siemens).
Subsequent CT images were acquired for 10 min. (B) SCCHN (squamous cell carcinoma
of head and neck) CXCR4-positive tumor cells were inoculated (2.5 × 106 cells in 50 µL)
to female nude mouse in the area of neck (orthotopic), 3 weeks before in vivo test.
PET/CT images were acquired using the same protocol as the paw edema case. Tumor
(T) is indicated by yellow arrows. (C) Six-week-old female nude mice were given
injections of 2.0 × 106 metastatic subclones of E3 (CXCR4-positive murine carcinoma cell
line) through the tail vein. After 8 weeks, PET/CT images were acquired using the same
protocol as the SCCHN cancer study. Representative [18F]5 transverse PET images of 90
min p.i., Yellow arrows and text denote the location of the tumor (T), lung (L), spinal
column (SC). The circles in the bottom figure indicate induced metastatic tumor nodules.
(D) μPET transverse plane images with cold + hot (Blocking, left) compd. vs only hot
compd. (right); For the blocking, 200 µL of cold [19F]5 solution (30 mg/kg, 10% DMSO,
22

90% PBS including 45% cyclodextrin) was administered to the mouse (i.p.) (male, 6
weeks, 20 g, Jackson Laboratory, Strain JAZ 007850 J:NU) 30 min prior to [18F]5 injection
(i.v.). PET images were acquired using the same protocol as the paw edema case. This
experiment suggests the specificity of compound 5 to bind CXCR4-positive stem cells in
bone marrow.
Table 1. Biodistribution of [18F]5 at 90 min
Organ
SUV (%ID/g)
P (n = 5
)
Paw edema
SCCHN tumor
Metastatic tumor in lung
Lung
1.53 0.08
4.00 0.28
1.66 0.14
0.50 0.04
1.20 0.12
3.22 0.60
0.16 0.03
0.54 0.08
0.58 0.07
0.68 0.04
0.17 0.04
0.002
0.005
0.002
n/a
Liver
n/a
Kidney
n/a
Muscle
n/a
Heart
n/a
Spleen
n/a
Small intestine
Large intestine
n/a
n/a
23

Declaration of interests
܈ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
܆The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
24