[18F]fluoroethyltriazolyl monocyclam derivatives as imaging probes for the chemokine receptor CXCR4

Article abstract of DOI:10.3390/molecules24081612

Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [⁶⁸Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of ⁶⁸Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [¹⁸F]RPS-534 (7.2 ± 0.3 %ID/g) and [¹⁸F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [¹⁸F]RPS-547, and especially [¹⁸F]RPS-534, are promising ¹⁸F-labeled candidates for imaging CXCR4 expression.

Full text of DOI:10.3390/molecules24081612

molecules
Article
[18F]Fluoroethyltriazolyl Monocyclam Derivatives as
Imaging Probes for the Chemokine Receptor CXCR4
Alejandro Amor-Coarasa 1,2,† , James M. Kelly 1,2, Pradeep K. Singh 3,4
,
Shashikanth Ponnala 1,2, Anastasia Nikolopoulou 1,5, Clarence Williams Jr. 1,2
,
Yogindra Vedvyas 2, Moonsoo M. Jin 2,6, J. David Warren 3,4,6 and John W. Babich 1,2,5,6,
*
1
Division of Radiopharmaceutical Sciences, Department of Radiology, Weill Cornell Medicine, New York,
NY 10065, USA; alejandro.amor@einstein.yu.edu (A.A.-C.); jak2046@med.cornell.edu (J.M.K.);
shp2018@med.cornell.edu (S.P.); ann2010@med.cornell.edu (A.N.); clw2012@med.cornell.edu (C.W.J.)
Molecular Imaging Innovations Institute (MI3), Department of Radiology, Weill Cornell Medicine, New York,
NY 10065, USA; yov2002@med.cornell.edu (Y.V.); moj2005@med.cornell.edu (M.M.J.)
Milstein Chemistry Core Facility, Weill Cornell Medicine, New York, NY 10065, USA;
prs2016@med.cornell.edu (P.K.S.); jdw2003@med.cornell.edu (J.D.W.)
2
3
4
5
6
Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA
Citigroup Biomedical Imaging Center, Weill Cornell Medicine, New York, NY 10065, USA
Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA
Correspondence: job2060@med.cornell.edu; Tel.: +1-646-962-6148; Fax: +1-646-962-0577
Present address: Radiochemistry Laboratory, Department of Radiology, Albert Einstein College of Medicine,
Bronx, NY 10461, USA; alejandro.amor@einstein.yu.edu.
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 February 2019; Accepted: 22 April 2019; Published: 24 April 2019
Abstract: Determining chemokine receptor CXCR4 expression is significant in multiple diseases
due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a
promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited
by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural
derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression
by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently
achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by
click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and
PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low,
but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2
[18F]RPS-547 (3.1
0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle,
±
0.3 %ID/g) and
±
and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did
the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding
radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially
[18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression.
Keywords: chemokine receptor; CXCR4; positron emission tomography; molecular modeling;
drug discovery
1. Introduction
The transmembrane G-protein coupled receptor C-X-C chemokine receptor type 4 (CXCR4),
which specifically binds to the chemokine CXCL12, is expressed on many cell types, including various
types of cancer cells. Activation of CXCR4 by its natural ligand stromal cell-derived factor 1 (SDF-1)
is known to be involved in a number of vital physiological processes such as inflammation [
1],
cell migration [ ], and tumorigenesis [ ]. CXCR4 plays a key role in organ-specific metastasis, directing
2
3
Molecules 2019, 24, 1612; doi:10.3390/molecules24081612
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1612
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the migration of malignant cells expressing this receptor toward microenvironments where the cognate
ligand is secreted [4–10].
To date, there is no clinically approved imaging agent to assess CXCR4 expression in patients.
The radiotracer, [68Ga]Pentixafor, a cyclic pentapeptide incorporating the chelate, DOTA, is under
clinical investigation for imaging CXCR4 expression in Europe [11–
18]. Although [68Ga]Pentixafor
accumulates in CXCR4-positive tumors with acceptable tumor avidity and contrast, the short half-life
of 68Ga (t1/2 = 68 min) limits its utility. In addition, as 68Ga is obtained from the 68Ge/68Ga generator the
maximum produced activity of 68Ga is typically low (1.85 GBq) [19] precluding large-scale production
for regional distribution.
The DOTA moiety in Pentixafor was added to conserve the presence of the high affinity cyclam
ring [20] found in the small molecule CXCR4 antagonists AMD-3100 and AMD-3465. However,
the CXCR4 affinity of the 68Ga complex was much greater than the complexes of other PET or
SPECT radiometals such as 111In, precluding the substitution of longer-lived isotopes for 68Ga.
Bicyclam-containing [64Cu]AMD-3100 was taken up in tumors in a CXCR4-dependent manner,
but high signal in liver and bone resulted in modest tumor-to-background ratios [21]. The higher
affinity radioligand, [64Cu]AMD-3465, accumulated in tumors to a greater extent than [64Cu]AMD-3100,
although liver and kidney uptake remained significant and persistent [22]. Nevertheless, this early
study supported the monocyclam structure of AMD-3465 as a scaffold for the design of novel
CXCR4-targeting radioligands.
The positron emitting radionuclide, 18F (t1/2 = 109 min), can be produced in large quantity
(>185 GBq) and at high molar activities, making it a more practical choice for CXCR4 probes if
higher volume clinical use is to be achieved [23]. Recently we introduced, [18F]RPS-544, a derivative
of the monocyclam AMD-3465, which demonstrated CXCR-dependent tumor uptake. However,
accumulation of [18F]RPS-544 in the liver and intestines was substantial [24]. We hypothesized that
further modification of the monocyclam structure may lead to a tracer with improved tumor uptake
and/or normal tissue kinetics compared with [18F]RPS-544.
Our strategy was to create a library of fluorine-containing derivatives of AMD-3465, and select
leading candidates for synthesis and in vitro and in vivo evaluation on the basis of an in silico screen.
To generate structural diversity, we considered modifications and substitutions of the phenyl and pyridyl
rings of the parent compound. We present herein a novel series of [18F]fluoroethyltriazolyl-containing
monocyclam derivatives and highlight in particular [18F]RPS-534 and [18F]RPS-547, which demonstrate
improved CXCR4-dependent tumor uptake and tumor-to-background ratios over [18F]RPS-544.
2. Results
2.1. Structural Design and Molecular Modeling
AMD-3465 contains three rings in its structure: Ring A is a cyclam, ring B is a phenyl ring, and ring
C is a pyridine ring (Figure 1). We elected to retain the cyclam moiety, and therefore considered the
possibility of fluorination of rings B and C. Nucleophilic radiofluorination of pyridine rings is feasible
only at the 2- and 4-positions. Therefore we considered other nitrogen-containing heteroaromatic
rings at ring C as a method of introducing fluorine into the structure. We also examined the effect of
transferring the fluorine label from the pyridine ring to ring B. This could be achieved either by direct
fluorination or by the derivatization of ring B with an appropriate prosthetic group. The length of
the linker between the rings B and C was also varied by insertion or deletion of a methylene group
(X, Figure 1).
We created an initial library of over 200 fluorine-containing monocyclam derivatives and performed
an in silico screen to identify those compounds with the highest predicted binding affinity. To give
context to the docking scores, we included AMD-3100, AMD-3465, RPS-544, and 1c, a fluorobenzamide
analogue of Pentixafor, in the in silico screen (Table 1). Pentixafor was synthesized by solution
phase chemistry (Supplemental Data S002) to serve as a comparator for the monocyclam derivatives.

Molecules 2019, 24, 1612
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The affinity of these ligands for CXCR4 ranges from approximately 5 nM to 650 nM [20,24], although
some variation in affinity based on assay conditions is evident. Among the new structures we screened,
those containing the 2-fluoroethyltriazole moiety consistently improved docking scores relative to
other fluorine-containing prosthetic groups or direct incorporation of fluorine onto aromatic rings
(Supplemental Data, Table S1). Interactions with residues Glu-32, Asp-87, Trp-94, His-113, Asp-187,
and Arg-188 most contributed to high docking scores.
Figure 1. AMD-3465, the parent structure for derivatization. Fluorine was introduced by addition or
substitutions of chemical moieties at rings B and C. The resulting library of structures were screened in
silico against human CXCR4 (PDB ID: 3ODU) using Schrodinger.
Table 1. Comparison of docking scores of six candidate CXCR4 ligands with known ligands with high
affinity. Docking score was determined in an extra precision screen against human CXCR4 (PDB ID:
3ODU) using Schrodinger. The highest docking score is reported for each compound.
Name
Structure
Docking Score (kcal/mol)
RPS-545
−8.51
AMD-3100
−8.50
−8.29
1c
RPS-544
RPS-534
−8.18
−8.17

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Table 1. Cont.
Name
Structure
Docking Score (kcal/mol)
RPS-533
−8.08
RPS-547
RPS-552
−7.95
−7.18
AMD-3465
RPS-546
−7.01
−6.22
The 2-fluoroethyltriazole group was either directly conjugated to ring B at the C2 (RPS-534 and
RPS-545) or C3 (RPS-533) positions, or used as ring C, as in RPS-546, RPS-547, and RPS-552 (Table 1).
The docking scores of RPS-533 (
to RPS-544 ( 8.18 kcal/mol), but substantially higher than AMD-3465 (
the strongest binders, the docking scores of ligands with the triazole as ring C, such as RPS-547
7.95 kcal/mol), were lower. Insertion (RPS-552; x = 2; 7.18 kcal/mol) or deletion (RPS-546; x = 0;
−
8.08 kcal/mol) and RPS-534 (
−
8.17 kcal/mol) were nearly identical
−
−
7.01 kcal/mol). Relative to
(
−
−
−6.22 kcal/mol) of a methylene group induced larger decreases in docking score.
2.2. Radiosynthesis
Radiosynthesis of the candidate ligands was achieved by a Cu(I)-catalyzed click reaction between
2-[18F]fluoroethyl azide and the corresponding alkyne precursors. The precursors and 19F standards
were prepared in multistep syntheses from commercially available building blocks. A full description
of the synthesis and structural characterizations of these compounds is available in the Supplemental
Data (S002).
2-[18F]fluoroethyl azide was synthesized and distilled in 0.5 mL DMSO with a decay-corrected
radiochemical yield of 45.8
increased the yield to 51.7
to the second reaction vial. The radiochemical purity of the distillate was 91.9
±
15.4% (n = 5). A change in solvent to 0.1 mL MeCN in 0.4 mL DMSO
9.5 % (n = 20) with concomitant transfer of a maximum of 100 L MeCN
4.3%, with the
±
µ
±
only radiochemical impurity being a substance that co-eluted with the solvent front. No chemical
impurities were evident by UV-Vis HPLC. The distillation was completed within 10 min and the rate
was independent of the solvent system used. The average decay-corrected radiochemical yield of the
click reaction was 85.3
typically lower than the other ligands. Deprotection of the radiolabeled precursors was quantitative
(>98%). Consequently, overall decay-corrected radiochemical yield was 38.6 6.1% (n = 20), and total
±
5.2% (n = 20), as determined by radioHPLC. The yield of [18F]RPS-533 was
±

Molecules 2019, 24, 1612
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synthesis time was 2 h. Chemical and radiochemical purity exceeded 97% and molar activity was
greater than 185 GBq/µmol.
2.3. In Vitro Experiments
Contrary to our expectations, the candidate ligands showed lower potency for binding CXCR4
than the relative docking scores would have suggested. The affinities of these ligands were in the
range 200–1500 nM, representing a decrease of approximately 100-fold relative to RPS-544 (Table 2 and
Supplemental Data Figure S7). To control for the possibility that the interaction between the ligands
and [68Ga]Pentixafor is not a simple competitive one, possibly due to the peptide occupying a different
part of the binding pocket, competition binding assays were also performed for RPS-533, RPS-534,
and RPS-545 against [18F]RPS-544. The IC50 values determined in this assay were not significantly
different from the values obtained against [68Ga]Pentixafor (p = 0.85). The binding curves were fitted
to Hill plots with a slope of 1 (Supplemental Data, Table S2), with the exception of [18F]RPS-545,
whose slope of 1.75
±
0.33 suggests a positively cooperative binding interaction. Kd values were
determined in a separate saturation binding experiment. The dissociation constants obtained were in
the range 150–1700 nM (Supplemental Data, Figure S8), and the Hill slopes were consistent with those
derived in the competition binding assay (Supplemental Data, Table S3).
Table 2. Comparison of key parameters in PC3-CXCR4 cells and PC3-CXCR4 xenograft tumors. IC50
was determined by competition binding against [68Ga]Pentixafor. PC3-CXCR4 cell and tumor uptake
was determined at 1 h p.i. Cell-associated activity is defined as the percentage of initial activity (%IA)
remaining after removal of media and washing of cells. Values are expressed as mean ± SEM.
Compound
IC50 (nM)
Kd (nM)
Cell-Associated Activity (%IA)
PC3-CXCR4 Tumor Uptake (%ID/g)
RPS-544
RPS-533
RPS-534
RPS-545
RPS-546
RPS-547
RPS-552
6.3 ± 0.8 (n = 6)
356 ± 100 (n = 9)
218 ± 38 (n = 18)
398 ± 41 (n = 9)
≈ 1500 (n = 3)
5.1 ± 1.0
366 ± 70
176 ± 30
515 ± 68
≈ 1700
8.2 ± 0.3
2.1 ± 0.4
18.5 ± 3.7
0.8 ± 0.1
1.2 ± 0.1
6.7 ± 2.8
3.5 ± 0.1
3.4 ± 1.2 *
1.93 ± 0.18
7.20 ± 0.30
n.d.
n.d.
601 ± 118 (n = 6)
515 ± 68 (n = 6)
261 ± 22
314 ± 48
3.09 ± 0.52
2.52 ± 0.11
* = Previously published data [24]. n.d. = Not determined.
The cell-associated activity of the compounds was generally low, with the exception of [18F]RPS-547
2.8 percent initial activity [%IA]), which was comparable to [18F]RPS-544, and [18F]RPS-534 (18.5
3.7%IA), which was two-fold greater. In comparison, the cell-associated activity of [68Ga]Pentixafor
was 39.7 8.0 %IA. Negligible internalization of the radioligands was observed after 60 min
(6.7
±
±
±
incubation, and approximately 7% of the total cell-associated activity of [18F]RPS-534, corresponding
to approximately 2%IA, was internalized after 135 min incubation (Supplemental Data, Figure S9).
No correlation was observed between affinity for CXCR4 and uptake in PC3-CXCR4 cells in this group
of ligands representing a narrow band of docking scores.
2.4. In Vivo Imaging and Biodistribution Studies
[68Ga]Pentixafor accumulation was primarily evident in PC3-CXCR4 tumors and bladders at 1 h
p.i. (Figure 2), with weaker signal also observed in PC3-WT tumors and kidneys. These findings
were confirmed by our biodistribution studies. Maximum PC3-CXCR4 tumor activity was 14.6
at 1 h p.i., with activity also most evident in PC3-WT tumors and kidneys. The difference in uptake
between the PC3-CXCR4 and PC3-WT tumors was statistically significant (p < 0.001), resulting in a
±
2.7
PC3-CXCR4-to-PC3-WT tumor uptake ratio of 2.0
±
0.2 at 1 h p.i. and 2.8
±
0.1 at 2 h p.i. By 2 h p.i. the
PC3-CXCR4-to-blood ratio was 31.1 ± 0.1, and the ratio to all normal tissue except kidneys (5.3 ± 0.1)
was greater than 10.

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Figure 2. Tissue distribution of [68Ga]Pentixafor in male BALB/C nu/nu mice bearing subcutaneous
bilateral PC3-WT (yellow arrow) and PC3-CXCR4 (white arrow) tumors. ( ) Imaging of CXCR4
expression by microPET/CT. Mice were injected intravenously with 5.5 MBq [68Ga]Pentixafor and
imaged at 1 h p.i. (
) Biodistribution of [68Ga]Pentixafor. Mice were injected with 1.85 MBq and
A
B
sacrificed at 1 h (n = 5) and 2 h (n = 5) p.i.
The PET images of [18F]RPS-534, [18F]RPS-547, and [18F]RPS-552 at 1 p.i. were most promising,
based on high uptake in PC3-CXCR4 tumors, low uptake in PC3-WT tumors, and good contrast
(Figure 3). Co-injection of AMD-3100 confirmed CXCR4-specificity by reducing the tumor uptake
of these ligands without substantially influencing clearance. Low level uptake in PC3-WT tumors
is consistent with basal expression of CXCR4 in these cells [24]. The hepatic uptake of [18F]RPS-547
was lower than [18F]RPS-544. [18F]RPS-547 and [18F]RPS-552 cleared through the kidneys, leading
to a reduction in liver uptake. Uptake of [18F]RPS-533 in the liver and intestines was lower than its
regioisomer [18F]RPS-534, but tumor uptake also decreased. [18F]RPS-545 and [18F]RPS-546 were
excluded from the animal studies due to low cell-associated activity, which reinforced the low affinities
determined in vitro.
Figure 3. Maximum intensity projection µPET/CT images of male BALB/C nu/nu mice bearing PC3-WT
xenograft tumors (left shoulder) and PC3-CXCR4 xenograft tumors (right shoulder). Images were
prepared using Amide, and are horizontally inverted relative to the imaging position. The mice were
administered intravenously with 7.4 MBq of each radioligand and imaged at 1 h p.i. Blocking was
performed by co-injection of AMD-3100 (5 mg/kg).

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We observed significantly greater (p < 0.01) uptake of [18F]RPS-534, [18F]RPS-547, [18F]RPS-552
(Figure 4), and [18F]RPS-533 (Supplemental Data, Table S2) in PC3-CXCR4 tumors than PC3-WT tumors
in the biodistribution studies at 1 h p.i. [18F]RPS-533 (1.93
±
0.18 %ID/g) and [18F]RPS-552 (2.52
±
0.11
%ID/g) did not reach the levels in PC3-CXCR4 tumors reported for [18F]RPS-544 [24]. The greatest
uptake was observed for [18F]RPS-534 (7.20 0.30 %ID/g), while uptake of [18F]RPS-547 (3.09
0.52
±
±
%ID/g) was comparable to [18F]RPS-544. Uptake of [18F]RPS-534 and [18F]RPS-547 in PC3-CXCR4
tumors at 2 h p.i. decreased but remained significant (p < 0.001) with respect to PC3-WT tumors
(Figure 4). The difference in uptake between PC3-CXCR4 and PC3-WT tumors was not significant at
1 h p.i. following co-administration of AMD-3100. Full tables of biodistribution values are available in
the Supplemental Data (S006).
Figure 4. Biodistribution of (A B C
) [18F]RPS-534, ( ) [18F]RPS-547, and ( ) [18F]RPS-552 at 1 h p.i. and 2 h
(**) p.i. in male BALB/C mice bearing bilateral PC3-WT/PC3-CXCR4 xenograft tumors. Blocking (*)
was performed at 1 h p.i. by co-injection of 5 mg/kg AMD-3100.

Molecules 2019, 24, 1612
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Although [18F]RPS-534 accumulated in the liver at 1 h p.i. (19.14
±
0.42 %ID/g), leading to
a lower tumor-to-liver ratio than [18F]RPS-533, [18F]RPS-547, and [18F]RPS-552, contrast to other
tissues was greater (Table 3). Liver uptake decreased to 12.01
±
4.67 %ID/g upon co-injection
of AMD-3100, suggesting a possible component was related to CXCR4, but a similar degree of
decrease in liver activity was not observed when [18F]RPS-547 was blocked with AMD-3100. As a
consequence of decreased uptake in both PC3-WT and PC3-CXCR4 tumors following blocking,
tumor-to-blood and tumor-to-muscle ratios decreased. These ratios were not significantly different for
[18F]RPS-534, [18F]RPS-547 and [18F]RPS-552 following blocking (p > 0.38). [18F]RPS-533, [18F]RPS-547,
and [18F]RPS-552 cleared primarily through kidneys (12.17
2.66 %ID/g, respectively). Therefore, tumor-to-kidney ratios for these ligands were one order
±
0.78 %ID/g, 10.73
±
1.10 %ID/g, and 7.23
±
of magnitude lower than for [18F]RPS-534. We also note the tumor-to-blood, tumor-to-muscle,
and tumor-to-lung ratios of [18F]RPS-534 and [18F]RPS-547, which are considerably larger than those of
[18F]RPS-544. Washout of [18F]RPS-534 and [18F]RPS-547 from PC3-CXCR4 tumors at 2 h p.i. ensured
that these ratios did not change substantially, with the exception of tumor-to-blood, which continued
to increase.
Table 3. Tumor-to-tissue ratios for candidate CXCR4 ligands at 1 h p.i. (top) and 2 h p.i. (bottom).
[
[
18F]RPS-544 [24] and [68Ga]Pentixafor are included for comparison. The highest values for the
18F]fluorinated compounds are shown in bold for clarity.
1 h p.i.
PC3-CXCR4 Tumor-to-Tissue Ratio
Compound
PC3-WT
Liver
Kidneys
Blood
Muscle
Bone
Lungs
Pentixafor
RPS-544
RPS-533
RPS-534
RPS-547
RPS-552
2.0 ± 0.2
3.3 ± 1.3
2.0 ± 0.4
2.5 ± 0.4
1.9 ± 0.3
2.5 ± 0.3
10.8 ± 0.2
0.1 ± 0.1
0.5 ± 0.1
0.4 ± 0.2
0.5 ± 0.2
0.6 ± 0.1
4.0 ± 0.2
0.1 ± 0.1
0.2 ± 0.1
1.1 ± 0.4
0.3 ± 0.2
0.3 ± 0.3
11.4 ± 0.2
2.5 ± 0.4
6.9 ± 0.2
27.5 ± 0.4
10.1 ± 0.3
11.9 ± 0.1
56.1 ± 0.2
11.1 ± 0.4
2.2 ± 0.3
28.7 ± 0.2
4.2 ± 1.2
1.4 ± 0.7
5.7 ± 0.4
5.9 ± 0.8
4.0 ± 0.2
11.8 ± 0.2
2.0 ± 1.2
3.3 ± 0.4
8.0 ± 0.4
6.2 ± 0.8
5.0 ± 0.1
42.4 ± 0.1
20.3 ± 0.3
16.7 ± 0.1
2 h p.i.
Pentixafor
RPS-544
RPS-534
RPS-547
2.8 ± 0.1
2.6 ± 0.3
2.5 ± 0.2
2.2 ± 0.2
10.8 ± 0.1
0.1 ± 0.1
0.2 ± 0.1
0.4 ± 0.2
5.3 ± 0.1
0.1 ± 0.1
0.9 ± 0.2
0.3 ± 0.1
32.1 ± 0.1
4.8 ± 0.1
101.2 ± 0.1
6.7 ± 0.2
47.2 ± 0.1
2.9 ± 0.3
3.5 ± 0.2
6.3 ± 0.4
17.1 ± 0.1
1.7 ± 0.3
7.1 ± 0.2
8.2 ± 0.4
51.1 ± 0.2
24.0 ± 0.2
38.8 ± 0.2
11.0 ± 0.3
Affinity for CXCR4 in vitro did not correlate to uptake of the radioligands in PC3-CXCR4 xenograft
tumors. However, we found a linear correlation (R2 > 0.99) between total cell-associated activity and
tumor uptake at 1 h p.i. (Figure 5). [68Ga]Pentixafor also fitted this relationship, suggesting that
it is not specific to AMD-3465 and its structural derivatives. To create an alternative parameter for
CXCR4 affinity that more closely correlated to tumor uptake in vivo, we titrated the tumor uptake
of [68Ga]Pentixafor, [18F]RPS-534, [18F]RPS-544, and [18F]RPS-547 in
of increasing masses of AMD-3100. Blocking doses of 0.08 0.01 mg/kg and 0.17
AMD-3100 reduced the uptake of [18F]RPS-547 and [18F]RPS-544, respectively, by 50% in PC3-CXCR4
tumors (Figure 6). However, 1.41
0.13 mg/kg of AMD-3100 was necessary to reduce [68Ga]Pentixafor
binding by the same amount. [18F]RPS-534 uptake was reduced by 50% with 0.31
0.10 mg/kg of
µPET/CT images by co-injection
±
±
0.06 mg/kg of
±
±
AMD-3100. By this measure, affinity for CXCR4 was in the order [68Ga]Pentixafor > [18F]RPS-534
> [18F]RPS-544 > [18F]RPS-547, and the correlation between PC3-CXCR4 tumor uptake and mass of
AMD-3100 required to reduce uptake by 50% was linear (R2 > 0.95).

Molecules 2019, 24, 1612
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Figure 5. Correlation plot of the PC3-CXCR4 tumor uptake in vivo versus the PC3-CXCR4 cell-associated
activity of the radioligands in vitro. Maximum cell-associated activity was calculated by subtracting
the cell uptake at the highest non-radioactive ligand concentration of 10
µ
M from the cell uptake at
the lowest non-radioactive ligand concentration of 10−5
µM. The equation of the linear fit and the
correlation coefficient are reported.
Figure 6. The effect of co-injection of 0.1, 0.5, 1, or 5 mg/kg of AMD-3100 on uptake of [18F]RPS-534,
18F]RPS-544, [18F]RPS-547, and [68Ga]Pentixafor in PC3-CXCR4 xenograft tumors. (
) Absolute
tumor uptake as a function of the mass of AMD-3100. Uptake was quantified from PET/CT images;
) block50 values determined by binding curves fitted to the in vivo data. For fitting, negligible
blocking was assumed for all compounds at 1 g/kg AMD-3100. Based on the molar activity of the
[
A
µ
(
B
µ
radioligands, the average amount of non-radioactive compound in the radiopharmaceutical injection
was estimated to be 10 ng/kg.
3. Discussion
The novel fluoroethyltriazole-containing derivatives of AMD-3465, [18F]RPS-534 and [18F]RPS-547,
show high and specific uptake in CXCR4-positive tumors, which might be predicted based on their
binding to PC3-CXCR4 cells. While we acknowledge the compounds prepared as part of this work
represent a narrow chemical space, we observed a surprising lack of correlation between the docking
score, CXCR4 affinity, and CXCR4 binding. On the basis of the docking score alone, [18F]RPS-544
(
−
8.18 kcal/mol) and [18F]RPS-534 (
8.17 kcal/mol) would be expected to exhibit similar CXCR4
−
affinities. However, the apparent affinity of [18F]RPS-534 is 30-fold lower. The Hill plots of both
of these ligands support a one site, independent binding model, though we cannot rule out the
possibility that multiple molecules of [18F]RPS-534 can bind CXCR4 independently. This might
explain why, notwithstanding its weaker affinity, uptake of [18F]RPS-534 in PC3-CXCR4 xenograft
tumors is 2-fold higher than [18F]RPS-544. Similarly, although both ligands can be partially displaced
from PC3-CXCR4 tumors by co-injection of AMD-3100, the mass of AMD-3100 required to displace
[18F]RPS-534 is greater than the mass required to displace [18F]RPS-544. An alternative explanation

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is that [18F]RPS-534 may also interact with related chemokine receptors such as CXCR7 to a greater
extent than [18F]RPS-544. The affinity or binding interaction of [18F]RPS-534 for CXCR7 might differ
to AMD-3100, which is an allosteric agonist [25], leading to incomplete blocking of tumor uptake.
We are currently developing methods for determining the affinity of [18F]RPS-534, [18F]RPS-547,
and [18F]RPS-552 for other chemokine receptors.
When we fitted a curve of the docking score versus IC50, we derived linear plot with R2 = 0.76
.
Our choice of AMD-3465 as a parent structure restricted our ligands to a relatively narrow band
of docking scores. Within this narrow band, the docking score proved to have little predictive
value. However, inclusion of RPS-510 (docking score =
−
2.13 kcal/mol; IC50 >10,000 nM in our
assay) [24] as a negative control in the plot improved the fitting (R2 = 0.89; Figure S10). In this context,
perhaps the greatest value of the in silico screen is to exclude structures with extremely poor affinity.
Our experience suggests that structures whose docking score against CXCR4 exceeds 7.0 kcal/mol
−
should be synthesized and comprehensively evaluated. We acknowledge a potential bias in favor of
triazole-containing structures in our choice of candidate CXCR4 ligands due to our familiarity with
synthetic and radiosynthetic strategies to producing these compounds. It is possible that some of the
other monocyclam-containing analogues that we screened in silico might prove to be superior ligands
in vivo. It is apparent that there is a lack of correlation between affinity and tumor uptake which
cannot be attributed solely to compound pharmacokinetics. Given the PC3-CXCR4 tumor uptake of
the radioligands in our bilateral tumor model, we found that the most relevant metric of evaluating
these CXCR4 ligands in vitro is to measure the total cell associated activity (%IA) over 1 h.
The first reported 18F-labeled analogue of [68Ga]Pentixafor, [18F]AlF-NOTA-pentixather,
was limited by relatively high plasma protein binding and in vivo defluorination [26]. The effect of
these properties was to reduce the tumor-to-background contrast. To our knowledge, no 18F-labeled
analogue of pentapeptide 1c [20] has been developed, perhaps due to the challenges of synthesizing
the 4-[18F]fluorobenzoic acid synthon and selectively coupling it to the ornithine side chain. Therefore,
small molecule CXCR4 ligands based around para-xylyl groups, such as AMD-3100, AMD-3465,
and WZ811 [27], have served as the primary platforms for the derivatives of new fluorine-containing
antagonists. [18F]-3 [28], alternatively reported as [18F]RPS-510 [24], a pyrimidine-pyridine amine
compound, had low, but CXCR4-specific tumor uptake. [18F]RPS-544 showed improved tumor uptake
compared to [18F]-3, but failed to address its pharmacokinetic deficiencies.
The tumor-to-background ratios of [18F]RPS-534 and [18F]RPS-547 are noticeably higher than
those of [18F]RPS-544, but generally lower than those of [68Ga]Pentixafor. One exception is the
tumor-to-blood ratio of [18F]RPS-534, 27.5
±
0.4 at 1 h p.i., which is more than double the ratio of
[68Ga]Pentixafor. Rapid clearance of [18F]RPS-534 from the blood is matched by its rapid clearance
from the heart, which is captured by a tumor-to-heart ratio of 28.8 0.3. These properties may be
±
suitable for PET/CT imaging of myocardial infarction, an emerging application of CXCR4 molecular
imaging [29]. Uptake in spleen and bone was evident for [18F]RPS-534 but not observed to the same
extent with [68Ga]Pentixafor, [18F]RPS-547, or [18F]RPS-552. A blocking dose of 5 mg/kg AMD-3100
nearly eliminated radioactivity in these tissues, but only reduced activity in the tumor by approximately
70%. This may indicate that [18F]RPS-534 has a higher affinity than the other radioligands for native
murine CXCR4, which is highly expressed in the bone and spleen [19]. These observations lead us
to suggest that, at a minimum, the tumor-to-spleen and tumor-to-bone ratios of [18F]RPS-534 could
improve upon translation to humans.
4. Materials and Methods
4.1. Schrodinger Molecular Modeling
All molecular modeling was performed using Schrodinger v. 2014-3 (Schrodinger, New York, NY,
USA). Structures were docked against the available protein structure of the human CXCR4 chemokine
receptor in complex with small molecule antagonist (3ODU). The small molecule antagonist was used

Molecules 2019, 24, 1612
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to define the binding pocket and generate the docking grid for the modeling experiments. An Extra
Precision (XP) docking study was performed on >200 fluorine-containing compounds structurally
derived from AMD-3465. To calibrate the resulting docking scores, known CXCR4 ligands including
AMD-3100, AMD-3465, cyclic pentapeptide 1c [20], and RPS-544 [24] were included in the modeling.
The compounds were ranked by the docking score, reported in kcal/mol.
4.2. Synthesis of Compounds and Precursors
All precursors and non-radioactive standards were synthesized from commercially available
building blocks purchased from Sigma Aldrich (St. Louis, MO, USA), unless otherwise indicated.
Compounds were characterized by mass spectrometry and nuclear magnetic resonance (NMR) and
were greater than 98% pure. A full description of synthetic procedures and compound characterizations
is available in the Supplemental Data (S002).
4.3. Radiochemistry
4.3.1. General Methods
Reactions were monitored by HPLC and radioHPLC using a PRP polymeric column (50 mm,
Hamilton Company, Reno, NV, USA) and a Symmetry C18, 4.6
×
50 mm column (Waters Corporation,
Milford, MA, USA). Chemical and radiochemical purity of the final compounds were determined
by analytical (radio)HPLC. Compound identity was confirmed by co-injection with non-radioactive
standards. Analytical and semi-preparative HPLC were performed on a dual pump Varian Dynamax
HPLC (Agilent Technologies, Santa Clara, CA, USA) fitted with an Agilent ProStar 325 Dual Wavelength
UV-Vis Detector, and radiochemical purity was determined using a NaI(Tl) flow count detector (Bioscan,
Poway, CA, USA). UV absorption was monitored at 220 nm and 280 nm. A binary solvent system was
used, with solvent A comprising H2O + 0.1% TFA and solvent B consisting of 90% v/v MeCN/H2O +
0.1% TFA. For analytical HPLC the following gradient was used at a flow of 2 mL/min: 0–2 min: 0% B;
2–8 min: 0–100% B; 8–9 min: 100% B; 9–10 min: 100–0% B. Semi-prep HPLC was performed using a
µ
Bondapak® C18, 10
µ
m, 125 Å, 7.8
×
300 mm column (Waters, Milford, MA, USA). The following
gradient was used at a flow of 10 mL/min: 0–1 min: 0% B; 1–16 min: 0–30% B; 16–18 min: 30–100% B;
18–19 min: 100% B; 19–20 min: 100–0% B.
4.3.2. Radiosynthesis of [68Ga]Pentixafor
An ITG 68Ge/68Ga Generator was eluted using 4 mL 0.05M HCl and collected in a sterile 15 mL
centrifuge tube. To this eluate, containing 555–1110 MBq (15–30 mCi) of 68GaCl3, was added 25
µL of a
1 mg/ml solution of pentixafor in DMSO. The pH was adjusted to pH 4–5 by addition of 80
µL of a
3N NaOAc solution. The reaction was then placed in an Eppendorf ThermoMixer® (ThermoFisher
Scientific, Waltham, MA, USA) at 95 ◦C and 600 rpm shaking for 15 min. A sample of the reaction was
taken and analyzed by radio HPLC to confirm the labeling. Immediately after labeling was confirmed,
the reaction was diluted with 9 mL H2O and the product was trapped on a Sep-Pak C18 Plus Lite
(Waters, Milford, MA, USA) cartridge. The cartridge was washed with 5 mL normal saline solution
(VWR International, Radner, PA, USA) and the product was eluted with 200
µL of a 50 % EtOH/saline
solution, followed by 900 L saline to produce a final injectable solution in 9% EtOH. A final QC HPLC
µ
was performed to assess purity.
4.3.3. Radiosynthesis of 18F-Labeled Monocyclam Derivatives
No-carrier-added 18F− was obtained from a (18O(p,n)18F) reaction by irradiating a liquid H218O
target in a 19 MeV EBCO Cyclotron (Vancouver, BC, Canada). H218O containing [18F]F− was passed
™
through a Sep-Pak® Light Waters Accell Plus QMA cartridge (Waters, Milford, MA, USA). The [18F]F−
was eluted using a solution of 10 mg kryptofix (K222) and 5 mg K2CO3 in 80% v/v MeCN/H2O. The eluate
was dried at 100 ◦C under vacuum (25 mmHg) and a constant flow of N2 (99.999%, 30 mL/min flow).

Molecules 2019, 24, 1612
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Drying was completed in approximately 20 min following two azeotropic cycles with MeCN (0.5 mL).
To the dried residue was added 0.5 mL of a 10 mg/mL solution of 2-azidoethyltosylate in 20% v/v
MeCN/DMSO, and the reaction was heated for 10 min at 80 ◦C. The reaction mixture was purified by
distillation at 120 ◦C for 10 min. The distillate was collected in a clean V-shaped reaction vial containing
300
in DMSO, followed by a pre-mixed solution of reduced Cu+ consisting of 50
CuSO4 solution, 50 L of a 1.5 M aqueous sodium ascorbate solution, and 100
µ
L of DMF cooled to 0 ◦C. To this vial was added 100
µ
L of a 10 mg/mL solution of alkyne precursor
L of a 0.5 M aqueous
L DMF. The reaction
µ
µ
µ
vial was heated for 10 min at 100 ◦C before it was diluted with H2O (10 mL) and passed through a
pre-activated Sep-Pak® Plus C18 Lite cartridge (Waters, Milford, MA, USA). The cartridge was washed
with H2O (10 mL) and eluted with TFA (1 mL) into a clean V-shaped reaction vial. Deprotection was
completed in 10 min at 100 ◦C, and the remaining TFA was evaporated under N2 flow. The dried
residue was re-dissolved in 200 µL of 1M NH4OAc solution for final purification via semi-prep HPLC.
The peak corresponding to the product was collected and diluted with H2O (20 mL). The product was
trapped on a pre-activated Sep-Pak® C18 Plus cartridge (Waters, Milford, MA, USA) and eluted with
0.5 mL EtOH (190 proof) followed by 5 mL of isotonic saline. The final product solution contained 9%
v/v EtOH and a radioactivity concentration of 185–370 MBq/mL.
4.4. Cell Lines
The human prostate cancer cell line PC3-WT and the stably transduced human PC3-CXCR4 cell
line were cultured as previously described [24].
4.5. Cell-Based Assays
4.5.1. Determination of IC50 by Competition Binding Assay
PC3-CXCR◦4 cells were plated in 12-well plates at a density of 500,000 cells per well and incubated
for 48 h at 37 C/5% CO2. Prior to the binding assay the cells were incubated in fresh media
and 370–555 kBq of [68Ga]Pentixafor or [18F]RPS-544 was added to each well. The corresponding
concentration of [68Ga]Pentixafor in each well was 100 nM, and the concentration of [18F]RPS-544 was
approximately 5 pM. Then non-radioactive ligands were added to give a final ligand concentration in
the range 0.001–10,000 nM. A total of eight concentrations were studied, and each concentration was
tested in triplicate. The cells were incubated for 60 min at 37 ◦C/5% CO2. The media was removed under
suction, the cells were washed with cold PBS 1X and detached and transferred to tubes for counting in
1 M NaOH. The cells were counted in a Wizard2 Gamma Counter (PerkinElmer, Waltham, MA, USA).
Non-specific binding was subtracted, and the IC50 values were determined by assuming a simple
competitive binding interaction and fitting the data to a sigmoidal Hill plot in Origin 9.0 (OriginLab,
Northampton, MA, USA). Potencies are expressed as IC50
Figure S7).
±
standard deviation (Supplemental Data,
4.5.2. Determination of Kd by Saturation Binding Assay
To determine the dissociation constants, saturation binding assays were performed in the
PC3-CXCR4 cells. The cells were plated as described above, and approximately 5 nM of the radioligand
was added to each well. Then the corresponding non-radioactive ligand was added to give a final
concentration in the range 0.001–10,000 nM. A total of eight concentrations were tested, and each
concentration was tested in triplicate. The samples were washed and counted as described above.
Non-specific binding was subtracted, and Kd values were determined by performing a nonlinear one
site binding curve fit with equation y = (Bmax
deviation (Supplemental Data, Figure S8).
·
x)/(Kd + x). Values are expressed as Kd
±
standard

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4.5.3. Determination of Cell Binding and Internalization
Internalization studies were performed for [18F]RPS-534 and [18F]RPS-547 in a time dependent
manner. PC3-CXCR4 cells were plated as described above in 12-well plates and incubated for 48 h prior
to the experiment. The media was exchanged and [18F]RPS-534 or [18F]RPS-547 (10−5 µM; approximately
400 kBq) was added to each well. All experiments were performed in triplicate. The plates were
incubated at 4 ◦C or 37 ◦C for 15, 30, 60, and 120 min. Then the media was removed under suction
at the corresponding temperature, and the cells washed with 1 mL PBS. Cell surface-bound activity
was collected by two successive 5 min incubations of 0.5 mL of a 50 mM glycine buffer (pH = 2.8)
performed at 4 ◦C or 37 ◦C. The cells were then washed with 1 mL ice-cold PBS and detached with
1 mL ice cold 1 M NaOH. Detached cells were collected and counted along with the washes. An aliquot
of the administered radioactivity (10% IA) was also counted and used for quantification. The activity
found in the NaOH fractions of the 4 ◦C plates was subtracted from the activities of the fractions at
37 ◦C to correct for non-specific binding. Internalized activity was expressed as the fraction of activity
in the NaOH fraction relative to the total activity added.
4.5.4. Determination of Cell-Associated Activity
Maximum cell-associated activity was calculated by subtracting the cell uptake of the radioactive
ligand at the highest non-radioactive ligand concentration of 10 µM (which we considered to be
non-specific uptake/binding) from the cell uptake at the lowest non-radioactive ligand concentration
of 10−5 µM.
4.6. Animal Studies
All animal studies were approved by the Institutional Animal Care and Use Committee of Weill
Cornell Medicine (Protocol Number: 2015-0066) and were undertaken in accordance with the guidelines
set forth by the USPHS Policy on Humane Care and Use of Laboratory Animals. Animals were housed
under standard conditions in approved facilities with 12 h light/dark cycles. Food and water was
provided ad libitum throughout the course of the studies. Hairless 6-to-8-week old male BALB/c
inbred athymic nude mice (Jackson Laboratory, Bar Harbor, ME, USA) were inoculated bilaterally
with 0.2 mL of a 1:1 mixture of 107 PC3-CXCR4 cells in PBS and Matrigel (Corning, Tewksbury, MA,
USA) subcutaneously in the left shoulder and 0.2 mL of a 1:1 mixture of 107 PC3-WT cells in PBS and
Matrigel subcutaneously in the right shoulder. Imaging and biodistribution studies were performed
three weeks post-implantation, when tumors were approximately 100–300 mm3.
For the imaging studies, the tumor-bearing mice (n = 2/compound) were injected intravenously
with 100
µL of a 9% v/v EtOH/saline solution containing approximately 3.7 MBq of the
radioligand. Blocking of CXCR4-dependent tissue uptake of [18F]RPS-534, [18F]RPS-547, [18F]RPS-544,
and [68Ga]Pentixafor was evaluated by co-injection of 0.1, 0.5, 1, or 5 mg/kg of AMD-3100 (n = 2 per
compound per mass of AMD-3100). The animals were imaged under isoflurane at from 60–90 min
p.i. in an Inveon µPET/CT (Siemens Medical Solutions, Malvern, PA, USA). Total image acquisition
time was 30 min, and a CT scan was performed directly before the PET imaging for anatomical
co-registration and attenuation correction. Images were reconstructed using the software provided by
the vendor and post-reconstruction analysis, including decay correction, was performed using AMIDE
(Open Source Software) prior to comparison. The images are laterally inverted relative to the imaging
position, such that the PC3-CXCR4 tumor appears on the right shoulder and the PC3-WT tumor on the
left shoulder. Tumor uptake was quantified by the following procedure: CT-aided regions of interest
were drawn, and tissue concentration was determined by comparison with a 3 %ID/cm3 standard
included in the field of view. Uptake was expressed as percent injected dose per centimeter cubed
(%ID/cm3) ± standard deviation.
For the biodistribution studies, tumor-bearing animals (n = 5/compound/time point) were injected
intravenously with 50–100 µL of a 9% v/v EtOH/saline solution containing approximately 1.1 MBq of

Molecules 2019, 24, 1612
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the radioligand. Co-administration of AMD-3100 (5 mg/kg) was used to confirm CXCR4-dependent
uptake of [18F]RPS-534, [18F]RPS-547, and [18F]RPS-552. The mice were euthanized at 1 or 2 h
p.i. and tissues including blood, heart, lungs, liver, small intestine, large intestine, stomach (with
contents), spleen, pancreas, kidneys, muscle, bone, PC3-WT tumors, PC3-CXCR4 tumors, and tail
were collected. All organs were weighed and placed in test tubes for counting in a Wizard2 Gamma
Counter (PerkinElmer, Waltham, MA, USA). For comparison, 1% injected activity standards were
prepared and counted along with the tissue samples. The tails were counted to correct for any dose
extravasation. Activity in each tissue was corrected for decay and for injected activity and expressed
as percent injected dose per gram of tissue (%ID/g) ± standard error.
5. Conclusions
As predicted by their binding to PC3-CXCR4 cells, the fluoroethyltriazole-containing derivatives of
AMD-3465, [18F]RPS-534, and [18F]RPS-547 showed high and specific uptake in CXCR4-positive tumors.
Uptake of [18F]RPS-547 was comparable to [18F]RPS-544, a first-generation AMD-3465 derivative,
but the improved clearance profile of [18F]RPS-547 led to higher tumor-to-normal tissue ratios.
The tumor-to-normal tissue ratios of [18F]RPS-534, particularly tumor-to-blood, tumor-to-muscle,
and tumor-to-lung, were even greater and comparable to [68Ga]Pentixafor. The chemical and
pharmacokinetic properties of [18F]RPS-547 and especially [18F]RPS-534, including high yielding
radiosynthesis, high tumor uptake, and good contrast to background, render these radiotracers
promising candidates for imaging CXCR4 expression by PET.
Supplementary Materials: The following are available online: a selected summary of ligands included in the
in silico screen (S001), and a full description of the synthesis of precursors and non-radioactive fluorinated
standards (S002). Additional data are presented in Table S1: Fluorine-containing derivatives of AMD-3465,
Table S2: Table of tissue activity values from biodistribution studies conducted in male BALB/c mice bearing
bilateral PC3-WT/PC3-CXCR4 tumors; Figure S1: Synthesis of p-RPS-533 and RPS-533, Figure S2: Synthesis of
p-RPS-545 and RPS-545, Figure S3: Synthesis of p-RPS-534 and RPS-534, Figure S4: Synthesis of p-RPS-547 and
RPS-547, Figure S5: Synthesis of p-RPS-552 and RPS-552, Figure S6: Synthesis of p-RPS-546 and RPS-546, Figure S7:
Determination of IC50 in a competition assay vs [68Ga]Pentixafor in PC3-CXCR4 cells, Figure S8: Determination of
dissociation constants (Kd) in PC3-CXCR4 cells, Figure S9: Internalization of [18F]RPS-534 and [18F]RPS-547 in
PC3-CXCR4 cells, Figure S10: Linear plot of docking score and IC50
.
Author Contributions: A.A.-C. and J.W.B. conceived the project and designed the studies. A.A.-C. contributed to
the acquisition and interpretation of data and to editing the final version of the manuscript. J.M.K. contributed to
the synthetic design, acquisition and interpretation of data, and wrote the manuscript. P.K.S. contributed to the
synthetic design and compound synthesis. S.P. contributed to the compound synthesis. A.N. contributed to the
acquisition of data. C.W.J. contributed to the acquisition of data. Y.V. and M.M.J. prepared the cell lines. J.D.W.
contributed to the synthetic design and compound synthesis. J.W.B. also contributed to editing the final version of
the manuscript and approved the final version for submission. All authors have given their approval to the final
version of the manuscript.
Funding: This research was funded in part by an NIH/NCI Kirschstein-NRSA Post-Doctoral Fellowship
F32CA203473 (recipient: Alejandro Amor-Coarasa).
Acknowledgments: The authors wish to acknowledge the help of Thomas M. Jeitner in the manuscript preparation.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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