Radiosynthesis, biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Article abstract of DOI:10.1016/j.bmcl.2018.10.035

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3 ± 5.11% of radiochemical yield and 99.7 ± 0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96 ± 0.06) and good stability in human serum over 97% for 2 h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.

Full text of DOI:10.1016/j.bmcl.2018.10.035

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiosynthesis, biological evaluation and preliminary microPET study of
18F-labeled 5-resorcinolic triazolone derivative based on ganetespib
targeting HSP90
⁎
Julie Kanga,1, Jun Young Leeb,1, İsa Taşa, Kunal N. Morea, Hangun Kima, Jeong-Hoon Parkb,
,
⁎
Dong-Jo Changa,
a
College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon 57922,Republic of Korea
b
Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid
tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 in-
hibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences
validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In
this study, we synthesized and biologically evaluated a novel 18F-labeled 5-resorcinolic triazolone derivative (1,
HSP 90
Tumor hypoxia
PET imaging
Ganetespib
TNBC breast cancer
[
18F]PTP-Ganetespib) based on ganetespib. [18F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-
PEG-Alkyne (10) and [18F]PEG-N3 (11) with 37.3
5.11% of radiochemical yield and 99.7
0.09% of
radiochemical purity. [18F]PTP-Ganetespib showed proper LogP (0.96
0.06) and good stability in human
serum over 97% for 2 h. [18F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple ne-
gative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell
lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib
exhibited significantly decreased accumulation of [18F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, in-
dicating the specific binding of [18F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 ex-
pression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in
other thoracic and abdominal organs, but [18F]PTP-Ganetespib was retained relatively longer in the tumor than
other organs. The uptake of [18F]PTP-Ganetespib in tumors was not sufficient for further development as a
tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [18F]PTP-Ganetespib can
be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.
Since the emergence of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) as
a PET imaging agent for the diagnosis of cancer, a large number of PET
imaging agents have been developed and entered in clinical trials.
However, only a few PET imaging agents such as [11C]choline and [18F]
fluciclovine have been used in clinics for imaging of cancer, and the
number of clinically approved PET imaging agents for cancer is very
limited as compared to the development of anticancer agents for
therapy.1 Currently, most PET tracers for imaging of cancer under
preclinical and clinical trials are predominantly ligand-targeted ima-
ging agents. Numerous targeted anticancer agents under clinical trials
have been developed to target various classes of oncogenic proteins,
including cell membrane receptors, nuclear membrane receptors,
cytosolic proteins, cytoskeletons, and macromolecules in diverse orga-
nelles such as the mitochondria and ribosomes, as well as genetic en-
tities in the nucleus such as histone and DNA.2 On the other hand, most
targeted imaging agents, including PET tracers, under preclinical and
clinical trials have been developed to target oncogenic membrane re-
ceptors such as folate receptor (FR), prostate-specific membrane an-
tigen (PSMA), αvβ3 integrin, somatostatin receptor 2 (SSTR2), and
glucagon-like peptide 1 receptor (GLP1R).3 As examples, [99mTc]etar-
folatide,4
[
68Ga]DOTANOC,5,6 and [111In]exendin-4,7 currently devel-
oped in Phase III clinical trials target FR, SSTR2, and GLP1R, respec-
tively. As a number of anticancer drugs targeting various classes of
oncogenic proteins have been developed, cytoplasmic oncogenic
⁎
Corresponding authors.
E-mail addresses: parkjh@kaeri.re.kr (J.-H. Park), djchang@sunchon.ac.kr (D.-J. Chang).
1
The first two authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2018.10.035
Received 16 August 2018; Received in revised form 21 October 2018; Accepted 22 October 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Kang,J.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2018.10.035

J. Kang et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. A. Molecular docking in the N-terminal ATP binding pocket of human HSP90. A. X-ray co-crystal structure of ganetespib (H-bonds, green color); B. Molecular
docking of [19F]PTP-Ganetespib (H-bonds, green color); C. Superimposition of [19F]PTP-Ganetespib (green color) with ganetespib (red color). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
proteins such as HSP90 can be also potential targets for the develop-
ment of clinically available PET imaging agents.
inhibitor with a triazole scaffold called STA-9090 (Ganetespib).31–34
Gantespib exhibits a high affinity to HSP90, and shows promising an-
ticancer activity against solid tumors and hematological malignancies.
Ganetespib has not shown hepatotoxicity that was observed in the early
HSP90 inhibitors such as the geldanamycin derivatives.24,35,36 Gane-
tespib with a 5-resorcinolic triazolone scaffold has a high affinity for
HSP90 (IC50 ∼ 10 nM) and has shown higher uptake into tumors than
other organs in clinical trials as well as in vivo models,24,34,37,38 sug-
gesting ganetespib can be a potential ligand for the development of PET
imaging agent for cancer and also HSP90 can be a potential target for
imaging of cancer as well as cancer therapy.24,39 However, there are
only a limited number of studies on the development of imaging agents
targeting HSP90, including PET radiotracers. To date, only one study
has been reported for the development of PET imaging agents targeting
HSP90 using the early HSP90 inhibitor, PU-H71, that targets Her2-
positive breast cancer and triple negative breast cancer.40
Heat-shock protein 90 (HSP90) is an ATP-dependent molecular
chaperone that induces a conformational change of its client protein by
associating with other co-chaperones.8,9 HSP90 regulates various on-
cogenic processes, including apoptosis, angiogenesis, invasion, and
tumor growth, stabilizes and activates several oncogenic client proteins
such as HIF-1α, Akt, Her2, and EGFR, which are involved in various
signal transduction pathways in cancer.10–13 HSP90 is a constitutively
expressed chaperone in most cells of normal tissues and it accounts for
1–2% of all cellular proteins. However, specific stimuli such as phos-
phorylation, ligand binding, dimerization, oxidative stress and hypoxia
result in an approximately two-fold increase of HSP90 levels, which has
been found to be essential for oncogenic transformation.12,14 Since the
discovery of geldanamycin as an HSP90 inhibitor, several HSP90 in-
hibitors have been developed and entered in clinical trials.15–17 Early
HSP90 inhibitors, including the ansanamycin-based compounds such as
17-allylamino-17-demethoxy geldanamycin (17-AAG)18,19 and 17-di-
methylamino ethylamino-17-demethoxygeldanamycin (17-DMAG),20
and the purine-based compounds such as CNF2024/BIIB02121,22 and
MPC-0767,23 have shown promising anticancer activity. However, they
were not considered for further development due to their poor phar-
macokinetic properties and mechanism-based side effects such as he-
patic toxicity.24 Based on the evidence from the clinical trials on gel-
danamycin derivatives, several synthetic HSP90 inhibitors with novel
scaffolds such as pyrazole (CCT018159 and VER-49009) and isoxazole
(NVP-AUY922) have been discovered from chemical libraries and
structure-based drug designs,25–27 and have subsequently entered in
clinical trials.28 Of various HSP90 inhibitors, NVP-AUY922 and NVP-
HSP990 developed by Novartis have shown good efficacy and accep-
table safety profiles in Phase I/II clinical trials.29,30 Quite recently,
Synta Pharmaceutical Corp. has discovered another class of HSP90
In this study, we synthesized 18F-labeled tracer (1, [18F]PTP-
Ganetespib) based on an HSP90 inhibitor, ganetespib, and performed a
preliminary PET study in order to develop a new PET imaging agent for
cancer and validate cytoplasmic HSP90, not a membrane receptor, as a
potential target for imaging of cancer. We chose ganetespib as a ligand
targeting HSP90 because it is one of the most promising candidates in
current clinical trials for the development of anticancer agents targeting
HSP90.
Ying reported the X-ray co-crystal structure of ganetespib bound to
the N-terminal ATP-binding pocket of HSP90, which revealed that 5-
resorcinolic triazolone group of ganetespib makes two hydrogen
bondings with Asp93 and Lys58 in the active site of HSP90, while the
indole group is aligned towards the outside.41 Based on the analysis of
X-ray co-crystal structure of ganetespib, we designed a [18F]PTP-Ga-
netespib (1) with a 18F-labeled PEG linker at indole group. Considering
the efficient synthesis, we planned to introduce [18F] by the click
2

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Scheme 1. Reagents and conditions for synthesis of cold [19F]PTP-Ganetespib (1): (a) NaH, 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl 4-methylbenzene-
sulfonate (3), DMF, 99%; (b) SnCl2·H2O, EtOH, reflux, 80%; (c) Phenyl chloroformate, Et3N, CH2Cl2, 0 °C, 83%; (d) Hydrazine monohydrate, dioxane, mw, 88%; (e)
Chloroacetic acid, NaHCO3, 2,4-dihydroxy-5-isopropylbenzodithioic acid (8), DMF, 68%; (f) KOH, MeOH, 75 °C, 27%; (g) CuSO4·5H2O, sodium ascorbate, [19F]PEG-
N3 (11), 60%.
reaction of ganetespib bearing a PEG-alkyne group (Ganetespib-PEG-
Alkyne) with a 18F-labeled PEG linker bearing an azide ([18F]PEG-
N3).42 Prior to the synthesis and evaluation of [18F]PTP-Ganetespib, a
molecular docking study using the crystal structure of human HSP90
complexed with ganetespib (PDB:3TUH)41 was carried out to in-
vestigate whether tracer 1 with a triazole-PEG linker can bind to HSP90
compared to its parent ligand, ganetespib. The molecular docking re-
vealed that [18F]PTP-Ganetespib (1) rests very well in the N-terminal
ATP binding pocket of HSP90 and the binding mode of 5-resorcinolic
triazolone moiety in tracer 1 is the almost same as ganetespib. A hy-
droxyl group of resorcinol and a carbonyl group of triazolone make two
H-bondings with Asp93 and Lys58, respectively, and the N2 atom of
triazolone facilitates H-bonding with Thr184, similar to ganetespib
(Fig. 1B). Fluorinated PEG linker of tracer 1 protrudes from the pocket
and interacts well in the outside of the ATP-binding pocket. The su-
perimposition of ganetespib with tracer 1 showed that they have the
almost same conformation and alignment in the ATP-binding pocket
(Fig. 1C).
click reaction with a triazolone group was synthesized by the coupling
reaction of the hydrazide 7 with benzodithioic acid (8)43,44 and the
subsequent intramolecular cyclization using potassium hydroxide in
methanol.45 The synthesis of cold [19F]PTP-Ganetespib was completed
by the click reaction of Ganetespib-PEG-Alkyne (10) with [19F]PEG-N3
(11) in the presence of CuSO4·5H2O and sodium ascorbate. 18F-Radi-
olabeling of the tosylated PEG linker bearing an azide group (12, N3-
PEG-OTs) with [2,2,2]-cryptand in acetonitrile successfully provided a
18F-labeled PEG linker with an azide group (11, [18F]PEG-N3) with
57.5
5.95% (n = 4) of radiochemical yield and 99.3
0.03%
(n = 3) of radiochemical purity. Finally, [18F]PTP-Ganetespib (1) was
produced by the click reaction of the precursor 10 with [18F]PEG-N3
(11) in the same condition as synthesis of
[
19F]PTP-Ganetespib
(Scheme 2). It resulted in 37.3
5.11% (n = 4) of radiochemical
yield, 99.7
0.09% (n = 3) of radiochemical purity, and 1.70 GBq/
μmol of the specific activity after the purification by reverse phase
HPLC (Fig. 2).
As presented in Fig. 3, the physicochemical properties of [18F]PTP-
Ganetespib were evaluated by measuring hydrophobicity and stability
Scheme 1 illustrates the synthesis of cold [19F]PTP-Ganetespib (1).
Alkylation of 5-nitroindole with alkyne-PEG-sulfonate (3) followed by
reduction using SnCl2·H2O produced the intermediate 5. The formyla-
tion of 5 with phenyl chloroformate and the subsequent substitution
reaction with hydrazine afforded the hydrazide 7.43 The precursor 10 of
in human serum. [18F]PTP-Ganetespib exhibited 0.96
0.06 of LogP
(ESI†, Table S1), which suggests tracer 1 probably has a suitable cell
permeability and in vivo distribution as an imaging agent targeting an
intracellular protein. In the stability test,
[
18F]PTP-Ganetespib
Scheme 2. Reagents and conditions for radiosynthesis of [18F]PTP-Ganetespib (1): A. 18F-Labeling of N3-PEG-OTs (12); B. Click reaction of Ganetespib-PEG-Alkyne
(10) with [18F]PEG-N3 (11).
3

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Fig. 2. Analysis of 18F-labeled precursor of click reaction and [18F]PTP-Ganetespib (1). A. Radio-TLC and HPLC chromatogram of [18F]PEG-N3 (11) after HPLC
purification, B. Radio-TLC and HPLC chromatogram of [18F]PTP-Ganetespib (1).
exhibited a high stability in human serum up to 2 h post-labeling and
purification by HPLC. [18F]PTP-Ganetespib (1) showed the retained
radiochemical purity over 97% in triplicate tests (Fig. 3).
120 min (Fig. 4A and Table S3 in ESI†). Furthermore, we confirmed
specific uptake of [18F]PTP-Ganetespib into MDA-MB-231 and MCF-7
cells by blocking of HSP90 using the pretreatment of ganetespib. To
confirm the uptake capacity of [18F]PTP-Ganetespib to HSP90 in these
two cells, we incubated MDA-MB-231 and MCF-7 cells with 50 μg of
ganetespib and then treated [18F]PTP-Ganetespib (Fig. 4B and 4C,
Table S4 in ESI†). In competitive assay, the uptake of [18F]PTP-Gane-
tespib was dramatically inhibited by the pretreated ganetespib in both
cell lines. Cellular uptake of [18F]PTP-Ganetespib was decreased to
below 1 ID% at the final time interval of 120 min by blocking HSP90
with ganetespib. This result clearly implies that our tracer can specifi-
cally bind to MDA-MB-231 and MCF-7 cells with high HSP90 expression
although we did not determine the binding affinity of radiotracer 1.
Based on the result of cellular uptake study, the biodistribution of
Clinical trials of ganetespib in combination therapies with cytotoxic
agents as well as a single anticancer agent have been conducted against
several solid tumors, including non-small cell lung cancer (NSCLC) and
breast cancer.14 Also, ganetespib has shown potent anticancer effect in
preclinical studies using in vitro and in vivo breast cancer models.46,47
Preclinical studies showed that ganetespib has a strong anticancer effect
on a broad spectrum of breast cancer subtypes, including triple negative
breast cancer (TNBC) and Her2-negative breast cancer such as MDA-
MB-231 and MCF-7 cells, respectively.46 Based on reported preclinical
studies of ganetespib, we carried out cell uptake test of [18F]PTP-Ga-
netespib in MDA-MB-231 cells and MCF-7 cells. The accumulation of
[
18F]PTP-Ganetespib increased linearly with time in both MDA-MB-231
[
18F]PTP-Ganetespib was evaluated on orthotopic breast cancer xeno-
and MCF-7 cells and showed a specific accumulation of 3.30
0.05 ID
grafts with MDA-MD-231 tumors. [18F]PTP-Ganetespib was adminis-
tered to xenografts by intravenous injection (3.7 MBq), followed by
detection of uptake signals at the time intervals of 15, 60, and 120 min
post-injection (p.i). The biodistribution data of tracer 1 for tumors,
blood, muscle, and other peripheral organs such as heart, lung, liver,
spleen, stomach, intestine, pancreas, kidney, tail, skin, and brain is
listed in Fig. 5A. Initially, the abdominal and thoracic regions, in-
cluding intestine, stomach, kidney, liver, and lung, showed much
higher uptake than tumors at 15 min p.i. [18F]PTP-Ganetespib was ra-
pidly eliminated from kidney over time, and showed relatively slow
clearance in liver. However, most organs exhibited a linear decrease in
signal intensity with time, whereas the radioactivity in tumors was
relatively less decreased or maintained up to 120 min p.i. At the final
interval of 120 min p.i, more than 1 %ID/g of radioactivity was ob-
served in the liver, stomach, intestine, bone, skin, and tumors, whereas
the blood, heart, spleen, pancreas, kidney, muscle, and brain showed
faster elimination than tumors. [18F]PTP-Ganetespib showed 1.13 %ID/
% and 2.99
0.28 ID%, respectively, at the final time interval of
g of uptake in tumors at 60 min p.i, which was retained by 1.08
at 120 min p.i. The ratios of tumor to blood (T/B) and tumor to muscle
(T/M) increased with time, and reached to 1.31 0.34 and
1.78 0.53, respectively, at 120 min p.i., which was correlated with in
vitro cellular uptake. As shown in time-activity curves (TACs) based on
0.16
Fig. 3. A. Stability of [18F]PTP-Ganetespib (1) in human serum.
4

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Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 4. A. Cell uptake of [18F]PTP-Ganetespib (1) in MDA-MB-231 and MCF-7 cells, B. Cell uptake of [18F]PTP-Ganetespib (1) in MDA-MB-231 cells with blocking of
HSP90 by the pretreatment of ganetespib, C. Cell uptake of [18F]PTP-Ganetespib (1) in MCF-7 cells with blocking of HSP90 by the pretreatment of ganetespib.
ex vivo biodistribution study (Fig. 5B), [18F]PTP-Ganetespib showed a
showed very low uptake into bones (< 1% ID/g, ESI†, Table S5).
MicroPET study was performed to evaluate the distribution of [18F]
PTP-Ganetespib in the animal’s whole body, and to investigate tumor-
specific uptake in orthotopic TNBC xenografts bearing MDA-MB-231
tumors (Fig. 6). The imaging was carried out at the intervals of 15, 60
and 120 min p.i. At the first interval of 15 min p.i, a high uptake was
mainly detected in the thoracic and abdominal regions such as the lung,
liver, intestine, and kidney. The signal from tumors was also detected
but it was relatively weak as compared to the background tissues.
high distribution of 1.45
0.40 %ID/g up to 60 min in static flow of
blood and then was slowly decreased, whereas the distribution into the
tumor was slightly increased or maintained up to the final time interval
of 120 min. However, [18F]PTP-Ganetespib does not show enough
tumor-specific distribution for PET imaging although giving promising
results in vitro. The accumulation of tracer 1 tends to increase only in
the bones and tumors. The absorption of free 18F into bones was widely
reported in many previous studies48,49, but in our case, the tracer 1
Fig. 5. Biodistribution of [18F]PTP-Ganetespib (1) on orthotopic TNBC xenograft mice bearing MDA-MB-231 tumors at 15, 60 and 120 min post injection.
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In conclusion, we designed a 18F-labeled 5-resorcinolic triazolone
derivative based on ganetespib targeting HSP90 by molecular mod-
eling, and successfully synthesized [18F]PTP-Ganetespib (1) by the click
chemistry of alkyne (Ganetespib-PEG-Alkyne) with 18F-labeled azide
([18F]PEG-N3). [18F]PTP-Ganetespib showed good stability in human
serum, adequate hydrophobicity for permeation, and high in vitro cell
uptake in TNBC MDA-MB-231 and Her2-negative MCF-7 cells. Specific
binding of [18F]PTP-Ganetespib to two cell lines with HSP90 was
confirmed by blocking test using ganetespib. The biodistribution and
microPET imaging of [18F]PTP-Ganetespib were evaluated in ortho-
topic TNBC xenograft models with MDA-MB-231 tumors. This is the
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This work was supported by the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT & Future
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Appendix A. Supplementary data
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