Development of a new epidermal growth factor receptor positron emission tomography imaging agent based on the 3-cyanoquinoline core: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2010.08.004

The epidermal growth factor receptor (EGFR/c-ErbB1/HER1) is overexpressed in many cancers including breast, ovarian, endometrial, and non-small cell lung cancer. An EGFR specific imaging agent could facilitate clinical evaluation of primary tumors and/or metastases. To achieve this goal we designed and synthesized a small array of fluorine containing compounds based on a 3-cyanoquinoline core. A lead compound, 16, incorporating 2′-fluoroethyl- 1,2,3-triazole was selected for evaluation as a radioligand based on its high affinity for EGFR kinase (IC₅₀ = 1.81 ± 0.18 nM), good cellular potency (IC₅₀ = 21.97 ± 9.06 nM), low lipophilicity and good metabolic stability. 'Click' labeling afforded [¹⁸F]16 in 37.0 ± 3.6% decay corrected radiochemical yield based on azide [ ¹⁸F]14 and 7% end of synthesis (EOS) yield from aqueous fluoride. Compound [¹⁸F]16 was obtained with >99% radiochemical purity in a total synthesis time of 3 h. The compound showed good stability in vivo and a fourfold higher uptake in high EGFR expressing A431 tumor xenografts compared to low EGFR expressing HCT116 tumor xenografts. Furthermore, the radiotracer could be visualized in A431 tumor bearing mice by small animal PET imaging. Compound [¹⁸F]16 therefore constitutes a promising radiotracer for further evaluation for imaging of EGFR status.

Full text of DOI:10.1016/j.bmc.2010.08.004

Bioorganic & Medicinal Chemistry 18 (2010) 6634–6645
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a new epidermal growth factor receptor positron emission
tomography imaging agent based on the 3-cyanoquinoline core: Synthesis
and biological evaluation
Federica Pisaneschi a,c, , Quang-De Nguyen a, Elham Shamsaei a, Matthias Glaser b, Edward Robins b,
⇑
Maciej Kaliszczak a, Graham Smith a, Alan C. Spivey c, Eric O. Aboagye a,
⇑
a Comprehensive Cancer Imaging Centre, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom
b Medical Diagnostic Discovery (part of GE Healthcare) at Hammersmith Imanet Ltd, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom
c Department of Chemistry, Imperial College London, South Kensington Campus SW7 2AZ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2010
Revised 22 July 2010
Accepted 2 August 2010
Available online 6 August 2010
The epidermal growth factor receptor (EGFR/c-ErbB1/HER1) is overexpressed in many cancers including
breast, ovarian, endometrial, and non-small cell lung cancer. An EGFR specific imaging agent could facili-
tate clinical evaluation of primary tumors and/or metastases. To achieve this goal we designed and synthe-
sized a small array of fluorine containing compounds based on a 3-cyanoquinoline core. A lead compound,
16, incorporating 20-fluoroethyl-1,2,3-triazole was selected for evaluation as a radioligand based on its
high affinity for EGFR kinase (IC50 = 1.81 0.18 nM), good cellular potency (IC50 = 21.97 9.06 nM), low
lipophilicity and good metabolic stability. ‘Click’ labeling afforded [18F]16 in 37.0 3.6% decay corrected
radiochemical yield based on azide [18F]14 and 7% end of synthesis (EOS) yield from aqueous fluoride. Com-
pound [18F]16 was obtained with >99% radiochemical purity in a total synthesis time of 3 h. The compound
showed good stability in vivo and a fourfold higher uptake in high EGFR expressing A431 tumor xenografts
compared to low EGFR expressing HCT116 tumor xenografts. Furthermore, the radiotracer could be visu-
alized in A431 tumor bearing mice by small animal PET imaging. Compound [18F]16 therefore constitutes a
promising radiotracer for further evaluation for imaging of EGFR status.
Keywords:
EGFR
[
18F]-PET imaging
3-Cyanoquinoline
Click chemistry
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
breast cancer6,7 and an arsenal of ATP competitive small molecules
designed to inhibit the intracellular tyrosine kinase activity.8 Cer-
Epidermal growth factor receptor (EGFR/c-ErbB1/HER1) is over-
expressed in breast, ovarian and other human cancers.1,2 EGFR
along with the other three members of the HER family (HER2,
HER3 and HER4) is a transmembrane glycoprotein with an extra-
cellular ligand-binding domain, a transmembrane domain, and an
intracellular domain with tyrosine kinase activity. EGFR is acti-
vated by binding to a variety of ligands including EGF, amphiregu-
tain compounds in this class have already been approved for
clinical use (Gefitinib/Iressa,9 Erlotinib/Tarceva10) whilst others
are under preclinical or clinical investigation [e.g., Neratinib,
pelitinib (1, Fig. 1)].11,12
Molecular imaging techniques, such as PET (Positron Emission
Tomography) have the potential to provide insights into EGFR biol-
ogy. Potentially, PET with EGFR probes can non-invasively deter-
mine whether the target protein is overexpressed in a specific
lin and TGF-a 3
. Once activated it is believed to undergo homo- or
heterodimerization followed by activation of the intrinsic protein
tyrosine kinase, autophosphorylation and activation of intracellu-
lar signal transduction pathways such as phosphatidylinositol-3-
kinase (PI3K)/AKT and ras/raf/MEK/MAPK.4,3,5 Blocking EGFR
activity therefore appears to be a promising strategy in the treat-
ment of cancer. Various anti-EGFR therapies have been developed;
these include monoclonal antibody inhibitors like Cetuximab
(IMC-C225) which is currently in use for the treatment of colorec-
tal and head and neck cancers and in clinical trials for metastatic
⇑
Corresponding authors.
E-mail addresses: f.pisaneschi@imperial.ac.uk (F. Pisaneschi), eric.aboagye@
Figure 1. Structures of pelitinib (EKB-569, 1) and Neumaier’s quinazoline imaging
agent 2.
imperial.ac.uk (E. Aboagye).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.004

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6635
primary tumor or metastasis in vivo, the magnitude and duration
2. Results
of receptor occupancy, and target-drug interactions (including pos-
sibly the functional consequence of mutations that lead to reduced
EGFR-drug interactions).
2.1. Chemistry
A number of previously reported therapeutics have been la-
beled with radionuclides, including radiometals and radiohalo-
gens, as potential ligands for imaging EGFR.13 Among the
monoclonal antibodies, 64Cu-DOTA-cetuximab showed high up-
take in A431 tumors although a significant uptake in the liver
was observed, partly due to the dissociation of the 64Cu from the
DOTA chelator.14,15 Initial attempts to develop small molecule
imaging agents focused on the introduction of radiohalogens into
Pelitinib (EKB-569, 1) was synthesized by the procedure of
Wissner et al.29,31 via the key 3-cyanoquinoline intermediate 3.
This latter compound also provided the starting point for the syn-
thesis of three potential labeling precursors 6, 8, and 10, which
incorporate modified C-6 Michael acceptor units. Compound 6
incorporates a methylamine unit in place of the dimethylamine
unit found in pelitinib (1) so as to allow label introduction via
alkylation or reductive amination. This derivative was prepared
by AlMe3 mediated amidation with methyl (E)-4-(tert-butoxycar-
bonylmethylamino)-but-2-enoate 4 (47%)32 followed by Boc re-
moval using 10% concd HCl in dioxane (78%). Compound 8
incorporates a Boc-propargylamine unit in place of the dimethyl-
amine unit found in pelitinib (1) so as to allow label introduction
via copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition (the
‘click’ reaction).33–36 This derivative was prepared by AlMe3 medi-
ated amidation with ethyl (E)-4-(tert-butoxycarbonyl-prop-2-
ynyl-amino)-but-2-enoate 7 (see Section 4, 68%). Compound 10
incorporates a terminal alkyne in place of the dimethylamino-
methyl unit found in pelitinib (1) so as to allow an alternative
mode of label introduction via ‘click’ cycloaddition. This derivative
was prepared by AlMe3 mediated amidation with methyl (E)-pent-
2-en-4-ynoate 9 (20%)37 (Scheme 1).
Derivatization of N-methyl amine 6 in a manner amenable to
the introduction of an 18F radiolabel was explored by alkylation
and via reductive amination. Alkylation of amine 6 with 1-fluoro-
2-mesyloxy ethane (11) in CH2Cl2 gave the N-fluoroethyl product
12 in 33% yield following difficult chromatographic separation
from unidentified by-products. Alternatively, reductive amination
of 4-fluoro benzaldehyde by quinoline 6 was achieved using NaB-
H(OAc)3 and gave the 4-fluorobenzyl product 13 in 21% yield fol-
lowing rapid chromatography (Scheme 2).
reversible EGFR inhibitors based on
a 4-anilinoquinazoline
core.13,16–18 These showed promise in vitro but displayed inade-
quate signal to noise ratios for PET radioimaging in animal models,
attributable to their relatively high lipophilicity (i.e., Log P) and ra-
pid washout from cells by intracellular ATP. More recent reports by
VanBrocklin et al.,19 Mishani et al.20–24 and Neumaier and co-work-
ers25 have described the evaluation of 11C, 124I and 18F radioligands
based on irreversible EGFR inhibitors in which an electrophilic
function is incorporated at C-6 of the quinazoline core. These
derivatives bind covalently to the Cys 773 located in the tyrosine
kinase binding pocket of EGFR thus preventing washout by intra-
cellular ATP and increasing potency.26 However, despite their
resistance to washout and improved in vitro inhibition profiles,
radioligands of this type have thus far displayed rapid metabolic
degradation20 and/or low specific tumor uptake.27,22 The only
probe that has been evaluated in vivo by imaging in tumor bearing
animals, quinazoline 2 (Fig. 1), gave non-ideal image quality, at
least in part due to these features.25
The aim of the work described here was the synthesis and bio-
logical evaluation of 18F radiolabeled irreversible imaging agents
based on the known 3-cyanoquinoline irreversible inhibitor, pelit-
inib (EKB-569, 1, Fig. 1).28,29 To enable selection of the most suit-
able candidate radiotracer we designed a small array of fluorine
containing derivatives with the intention of selecting the best
candidate on the basis of high affinity to the receptor and ease
of labeling. 18F was chosen in preference to 11C because it’s long
half-life time (t1/2 = 109.8 min) allows more elaborate radiosyn-
thesis and permits washout of non-specific binding. It was
planned to introduce the 18F-containing prosthetic group at the
last stage of the synthesis so as to maximize the radiochemical
yield of the process.
Derivatization of N-Boc-propargylamine 8 and enyne containing
quinoline 10 in a manner amenable to the introduction of an 18F
radiolabel was envisaged via ‘click’ cycloaddition with a fluorine
containing azide partner. Propargylamine 8 was therefore reacted
with 1-fluoro-2-ethyl azide (14)38 under Cu(I) catalysis and micro-
wave irradiation39 to give fluorotriazole-containing quinoline 16 as
For quinazoline-based inhibitors (e.g., Neumaier’s quinazoline
2, Fig. 1), a key interaction with the EGFR binding site is the indi-
rect hydrogen bonding of N-3 of the quinazoline core to Thr 830
mediated by a molecule of water.29 However, molecular modeling
studies indicate that in 3-cyanoquinolines (e.g., pelitinib 1, Fig. 1),
the 3-cyano group hydrogen bonds directly to Thr 830 without the
involvement of a water molecule.29 We speculated that this latter,
probably more entropically favored, interaction might be exploited
in a derived imaging agent to allow for higher uptake into the tu-
mor and result in more specific imaging signal. In line with the de-
sign criteria applied previously to irreversible quinazoline-based
tracer candidates, we wanted to retain an aniline moiety at C-4
to optimally occupy the active site hydrophilic pocket and a Mi-
chael acceptor (incorporating a tethered amine) at C-6 to cova-
lently bind to Cys 773.29 Consequently, it was decided that
compound 1 would be modified by the addition of fluorine con-
taining groups at the terminus of the C-6 Michael acceptor (with
minimal structural perturbation) or at the C-7 position (which
points outside the active site and does not appear to play an impor-
tant role in binding). In the interest of maximizing resistance to
metabolic degradation we were also interested in exploring the
reportedly increased stability of Michael acceptors modified with
secondary rather than tertiary amines.30
Scheme 1. Reagents and conditions: (a) AlMe3, CH2Cl2, rt, (5: 47%, 8: 68%, 10: 20%).
(b) (i) dioxane/HCl, 10:1, rt, 30 min; (ii) K2CO3, H2O, 14 h (78%).

6636
F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
Scheme 2. Reagents and conditions: (a) 11, Et3N, CH2Cl2, 18 h at rt and 24 h at
reflux (33%). (b) p-fluorobenzaldehyde, AcOH, NaBH(OAc)3, 1,2-dichloroethane,
14 h (21%).
Scheme 4. Reagents and conditions: (a) BBr3, CH2Cl2, rt (40%); (b) (i) 11, K2CO3,
DMF, 70 °C, 18 h (83%); (ii) concd HCl, H2O, 120 °C, 1.5 h (53%); (c) (i) 4, AlMe3,
toluene, rt, 14 h (61%); (ii) concd HCl, dioxane, rt (67%).
an HCl salt following Boc removal using HCl in 1,4-dioxane. Simi-
larly, enyne 10 reacted with azide 14 to give fluorotriazole-contain-
ing quinoline 15 in 32% yield. In both cases, the final yields were
compromised by the apparent instability of the Michael acceptor
precursors under the reaction conditions (Scheme 3).
by using 2-[18F]fluoroethylazide [18F]14 following a published pro-
cedure (Scheme 5).
2-[18F]Fluoroethylazide [18F]14 was synthesized from the corre-
sponding tosyloxyethylazide 22 and [18F]KF/Kryptofix 222 at 80 °C
for 15 min. The product was purified and collected by distillation in
MeCN; it was obtained with a 43.0 4.1% (n = 12) decay corrected
radiochemical yield. Precursor 21 was dissolved in MeCN/water,
1:1, mixed with the catalytic system and then reacted with the
azide [18F]14 at 80 °C for 15 min. The crude compound [18F]16
was purified by semipreparative HPLC in a 37.0 3.6% (n = 12) de-
cay corrected radiochemical yield from azide [18F]14 (EOS = 7%
from aqueous [18F]fluoride) and >99% radiochemical purity. Com-
pound [18F]16 was formulated by solid-phase extraction with an
efficiency of ꢀ90%. The identity of [18F]16 was confirmed by co-
elution with the non-radioactive compound. The formulated com-
pound was obtained with a specific activity (SA) of 48–685 GBq/
Quinoline 20 was designed as a direct analogue of quinoline 6
but containing a 2-fluoroethoxy group in place of the C-7 ethoxy
group. The synthesis route started from quinoline 17,29,31 which
is an intermediate in the Wissner synthesis of pelitinib (EKB-569,
1), and was not designed for direct translation into a labeling
method but rather as the most convenient entry for SAR purposes.
The C-7 ethyl ether in quinoline 17 was removed by treatment
with BBr3 and the resulting quinoline 18 was alkylated with 1-flu-
oro-2-mesyloxy ethane (11) under basic conditions followed by
acetamide hydrolysis to give 7-(2-fluoroethoxy)quinoline 19.
AlMe3 mediated amidation with methyl (E)-4-(tert-butoxycar-
bonyl-methyl-amino)-but-2-enoate 4 and Boc removal by treat-
ment with HCl in 1,4-dioxane gave the target quinoline 20 as its
HCl salt (Scheme 4).
l
mol at EOS. The radioimaging agent [18F]16 was stable for at least
4 h after formulation in phosphate buffered saline (PBS). The radio-
synthesis including formulation took 3 h in total.
None of the aforementioned ‘cold’ procedures were optimized
as this was not required in order to prepare sufficient material
for subsequent evaluation (see below).
2.3. Biology
A key design goal is the introduction of radiolabeled motifs
without significant detriment to EGFR inhibitory affinity (asurro-
gate for binding) relative to the starting quinoline 1. Therefore,
assessment of the EGFR tyrosine kinase activity of control quino-
lines 1 together with quinolines 6, 12, 13, 15, 16, 20, and 21 was
2.2. Radiochemistry
Alkyne containing quinoline 21 was obtained from compound 8
by treatment with concd HCl in 1,4-dioxane in quantitative yield.
Compound 21 was labeled by cycloaddition under Cu(I) catalysis
Scheme 3. Reagents and conditions: (a) 14, Cu powder, CuSO4, water, MW 125 °C,
15 min (15: 32%, 16: 19%); (b) dioxane/HCl, 10:1, 1 h (99%).
Scheme 5. Reagents and conditions: (a) [18F]KF/Kryptofix 222, 80 °C, 15 min (43%);
(b) CuSO4, Na ascorbate, pH 6, MeCN, H2O (37%).

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6637
carried out using a cell-free kinase activity inhibition assay as de-
tailed in Section 4. 4-[(3-Bromophenyl)amino]-6,7-diaminoqui-
nazoline (BPDQ),40 a quinazoline-based EGFR inhibitor, was also
included in this assay as a further reference standard. Concentra-
tions of the compounds that inhibited EGFR kinase activity by
50% (IC50) were calculated and are reported in Table 1.
incubation and a further 2 h of washing with drug free medium.
Due to the washing steps, the protocol permits the effect of irre-
versible binding activity to be quantified. Typical immunoblots
demonstrating inhibition of EGFR autophosphorylation are shown
in Figure 2. In these studies the drug did not inhibit the expression
of total EGFR protein. The inhibitory activity of compounds 1, 6, 12,
13, 16, 20, and 21 on cellular EGFR autophosphorylation, (Table 1)
translated well from that assessed in the cell-free system, with IC50
values in the low nanomolar range. Interestingly, no cellular activ-
ity was apparent when dosing with quinoline 15. Furthermore, the
cellular activity of quinoline 21 was in the low nanomolar range
indicating that, although potent, the high affinity of this alkyne
in the kinase assay did not directly translate into cellular activity.
As a consequence of the in vitro screening, quinoline 16 was se-
lected for further development because of its high binding affinity,
structural novelty, and the ease of click radiolabeling. The in vivo
All eight compounds inhibited EGFR kinase activity with IC50
values in the low- or sub-nanomolar range which compares well
with that of BPDQ (Table 1). Compound 1 appeared more potent
than previously reported by Wissner et al.29 presumably because
of differences in the assay used. The same authors have shown that
compound 1 functions as an irreversible inhibitor of EGFR. The IC50
values are probably best interpreted in the context of reversible
inhibition, as well as irreversible covalent binding resulting from
interactions with the Michael acceptor (and other reactive) moie-
ties. The sub-nanomolar kinase activity observed with compounds
1 and 6 was retained in compounds 12 and 13 demonstrating tol-
erance for small and large fluorine containing substituents on the
tertiary amine group. Fluorine substitution at the C-7 position
was also tolerated as reflected in the comparably low IC50 valued
measured for compounds 6 and 20. Of interest to the application
of ‘click’ radiochemistry, incorporation of a fluoroethyl triazole
onto the Michael acceptor—exemplified by quinolines 15 and
16—was tolerated, with quinoline 16 being twofold more active
than 15. The activity of triazole derivatives 15 and 16 was reduced
10–20-fold relative to quinoline 1; the reason for this is unclear
since previous modeling studies place the amine substituents at
the edge of the kinase pocket. In addition, a bulky substituent
was tolerated in the case of quinoline 13. Surprisingly, the activity
of the alkyne-containing quinoline 21 was in the picomolar range
(30 pM) possibly due to a previously undocumented
p–p interac-
tion that may also help to explain the high affinity of fluorobenzyl
quinoline 13.
Given the encouraging inhibitory EGFR kinase activity of the
compound series in the cell-free system, we examined cellular
activity—the ability of the compounds to be transported across cell
membranes and to inhibit EGFR autophosphorylation—in highly
EGFR expressing A431 cells.41 Following a protocol previously re-
ported by Rabindran et al.,11 we measured the potency of the com-
pounds to inhibit autophosphorylation of EGFR after 3 h of drug
Figure 2. Cellular activity of quinolines 1 and 16 assessed by Western blots analysis
of phosphorylated EGFR (p-EGFR) and total EGFR (EGFR).
Table 1
EGFR kinase activity inhibition profile for quinolines 1, 6, 12, 13, 15, 16, 20 and 21
R1
R2
IC50 (nM)
EGFR kinase activity
A431 EGFR autophosphorylationa
c log Pb
BPDQ
1
6
12
13
0.81 0.01
0.24 0.02
0.25 0.06
0.80 0.04
0.57 0.12
>1000
2.57
4.18
3.80
4.38
6.05
CH2NMe2
CH2NHMe
CH2NH(Me)CH2CH2F
CH2N(Me)(4-fluorobenzyl)
OEt
OEt
OEt
OEt
8.02 0.75
5.35 1.52
23.02 12.00
16.52 8.38
15
OEt
4.05 0.57
>1000
4.45
16
OEt
1.81 0.18
21.97 9.06
3.85
20
21
CH2NHMe
CH„CCH2NHCH2
OCH2CH2F
OEt
0.29 0.03
0.03 0.01
8.12 2.03
60.20 17.10
3.64
3.94
a
Data were extracted from the concentration versus p-EGFR/total EGFR Western blot absorbance ratio. Data are mean sem, n = 3 replicates.
c log P are calculated by ChemAxon’s MarvinSketch, version 5.2.6.
b

6638
F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
evaluation of the imaging agent [18F]16 focused on the assessment
of its metabolic stability and its ability to be taken up into a EGFR
expressing xenograft, A431, in animals. Non-specific binding pla-
gued development of earlier generation radiotracers (quinazolines)
for imaging EGFR.22 We initially assessed two of the likely reasons
for non-specific binding—poor metabolic stability and poor phar-
macokinetics—in the context of [18F]16 to inform the development
of this and the next generation of EGFR imaging agents. We inves-
tigated the in vivo metabolic stability of [18F]16 in non-tumor
bearing mice by analyzing liver and plasma extracts at 2, 30, and
60 min post injection. Sample analysis was accomplished by
radio-HPLC. Typical radiochromatograms are shown in Figure 3
and the results are summarized in Table 2. At 2 min, only parent
compound was observed in liver and plasma. A low level more po-
lar peak was seen in liver at 30 and 60 min and a similar radioac-
tive metabolite was seen in plasma. This metabolic stability data
demonstrates that the parent radiotracer [18F]16 remains a major
component of both liver and plasma even at 60 min post injection;
indicative of relatively good stability in vivo.
Table 2
In vivo metabolism of compound [18F]16 (SA = 685 GBq/
lmol) at
selected time points, showing the proportion of compound [18F]16
present in plasma and liver extractsa
Time (min)
Parent (liver)
Parent (plasma)
2
30
60
95.00 1.00
85.06 1.75
75.45 2.73
98.92 1.06
62.81 1.70
49.75 6.27
a
The extracts were analyzed by radio-HPLC [50% MeCN
(0.085% H3PO4)]. The values are the average of three indepen-
dent studies per time point. Proportion of compound [18F]16 in
plasma and liver were calculated by comparison of compound
[
18F]16 peak to total radioactivity present on chromatogram. The
efficiency of the extraction from plasma was 83.5%.
The 60 min tissue biodistribution A431 tumor bearing mice, ex-
pressed as tissue to blood ratios, has been measured and the re-
sults are shown in Figure 4. Tumor (A431 xenograft) uptake was
approximately fourfold higher than that of muscle (%ID/g in tu-
mor = 0.082 0.022 and %ID/g in muscle = 0.018 0.004, see Sup-
plementary data, Table S1). Low radioactivity was also observed
in other organs including bone, brain, and heart. The low uptake
in bone suggests that the radiotracer does not undergo defluorina-
tion. The radiotracer appeared to be eliminated via both the hepa-
tobiliary and renal routes, as the highest tissue radioactivities were
found in the gallbladder and the urine. Elimination of the radio-
tracer into the gut may also account for the high radioactivity in
the early part of the intestine (Fig. 4).
Figure 4. Tissue distribution of compound [18F]16 in untreated tumor bearing mice
at 60 min (sacrifice time point). Data are SEM; n = 3 mice.
We further assessed the potency of compound [18F]16 to detect
high EGFR expressing A431 xenografts relative to low EGFR
expressing HCT116 xenografts by small animal PET imaging. PET
images from representative A431 and HCT116 tumor bearing mice
with [18F]16 (Fig. 5) demonstrated localization and visualization of
the tumor, particularly in A431. The time activity curves (TACs) of
A431 and HCT116 tumors extracted from the PET data (Fig. 5)
showed more rapid washout of the radiotracer from HCT116 tu-
mors. Significant radiotracer localization was also seen in the
abdominal region consistent with the biodistribution data. The
PET data were corroborated by ex vivo tumor uptake and western
blot analysis of EGFR protein content of the two tumor types
Figure 3. In vivo metabolism of compound [18F]16 in mice liver as assessed by radio-HPLC. Top panel: reference compound and 2 min, respectively. Bottom panel: 30 min
and 60 min, respectively.

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6639
Figure 5. Computed tomography and compound [18F]16 PET images (summed dynamic) of representative A431 and HCT116 xenograft-bearing mice (SA = 48 GBq/
lmol),
together with time versus radioactivity curves and tumor uptake measured by
c-counting. White arrowheads on the PET images indicate the tumor seen on the computed
tomography images. Western blots of the two cell lines are also shown for EGFR and phosphorylated EGFR–p-EGFR; b-actin was used as loading control.
(Fig. 5). There was a fourfold higher uptake in A431 xenografts
compared to HCT116 xenografts.
congeners from readily available radiolabeled prosthetic groups
(i.e., [18F]-1-fluoro-2-ethyltosylate,44 18F]-p-fluorobenzaldehyde45
[
or [18F]-1-fluoro-2-ethylazide46). The route for preparation of the
C-7 modified 2-fluoroethyl ether (quinoline 20) did not share this
anticipated ease of translation into a convenient late-stage labeling
method but gave expedient access to the 19F product in order to
evaluate its properties. The eight compounds were screened for
their EGFR tyrosine kinase activity inhibition using BPDQ as posi-
tive control. The non-fluorinated quinolines 1, 6, and 21 were also
screened; compound 1 was used as a standard and compounds 6
and 21 were the potential radiochemical precursors for those fluo-
rinated quinolines most likely to be translated into imaging agents.
We assessed these latter compounds in order to better understand
the potential impact of residual impurities in an eventual radio-
pharmaceutical formulation.
3. Discussion and conclusion
Previous PET imaging studies with irreversible inhibitors of
EGFR have suggested that selectivity and metabolic stability are
likely to be key attributes of radiotracers if they are to display good
uptake by tumors and give good image quality.13 With this in
mind, we selected the 3-cyanoquinoline therapeutic agent pelitinib
(1), first reported by Tsou et al.,29,31,43 as a template on which to
introduce an 18F label. The 3-cyanoquinoline core, as indicated in
the introduction, was chosen in preference to the quinazoline core
because it was hoped that it might engender higher uptake into the
tumor and result in more specific imaging signal. The C-4 aniline
and C-6 aminocrotonylamide moieties were retained to ensure
selectivity for EGFR and irreversible binding to the active site Cys
773 via Michael addition, respectively.29 A small array of 19F con-
taining 3-cyanoquinolines was therefore synthesized (compounds
12, 13, 15, 16, and 20) and these compounds were tested for their
ability to inhibit the intracellular tyrosine kinase autophosphoryla-
tion activity of EGFR. The fluorine containing groups were intro-
duced onto both the Michael acceptor moiety and the C-7 ether
position of the quinoline core. Previous studies29,31 have indicated
some tolerance for substitution at these sites, which made the C-6
and C-7 positions attractive for modification in the present inves-
tigation. The synthetic routes developed to prepare the C-6 deriv-
atives were designed such that they would be amenable to
As expected, in the cell-free kinase activity inhibition assay, the
sub-nanomolar binding affinity showed by quinolines 1 and 6 was
retained in quinolines 12 and 13 despite the increased bulk of the
tertiary amine substituents. Surprisingly, the activity of the alkyne-
containing quinoline 21 was in the picomolar range, possibly
because of secondary interactions of the alkyne group with the
binding site of the enzyme, a feature unfortunately not retained
in compound 16. Nevertheless, the low nanomolar affinity of com-
pound 16 was considered sufficient to allow further in vivo charac-
terization of EGFR related activities. The slightly higher IC50 of
compound 15 may be attributable to the replacement of the ter-
tiary amine, which acts as internal base, with the significantly less
basic triazole ring (vide infra). Finally, quinoline 20, designed to be
a direct analogue of quinoline 6, but containing a 2-fluoroethoxy
preparation of the corresponding
[
18F]-fluorine containing

6640
F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
group in place of the C-7 ethoxy group, showed the same activity
as compound 6 confirming that the C-7 group is not involved in
the binding with the EGFR tyrosine kinase enzyme. With these pre-
liminary encouraging results in hand, the candidate compounds
were screened for their ability to inhibit autophosphorylation in
the human epithelial carcinoma A431 cell line. All the compounds
were active at nanomolar concentrations except for compound 15
and the positive control BPDQ. Compounds 12, 13, 16, and 21 were,
however, 10-fold less active than reference compounds 1 and 6.
The lack of activity of compound 15 might be accounted for by
its inability to cross the cell membrane, although its physicochem-
ical properties are comparable with the other members of the ser-
ies. Alternatively, the lack of activity of compound 15 in cells might
be a consequence of it having a more reversible interaction with
the kinase, as the methodology we employed for measuring cellu-
lar activity (with washing steps) permits mainly irreversible inhi-
bition to be quantified. This would be consistent with the lack of
activity of BPDQ which is known to be a reversible inhibitor of
EGFR.40 A reversible interaction of compound 15 with EGFR kinase
is possible since replacement of the amine group at the terminus of
the Michael acceptor with a nitrogen atom contained within a tri-
azole ring system may prevent the Michael reaction of the Cys 773
thiol from occurring; this would result in loss of activity in the cel-
lular assay. The low basicity of the triazole nitrogen and/or the lack
of conformational flexibility of this group could well be preventing
the Michael acceptor from reacting efficiently. In particular, the
aromatic nature of the triazole ring combined with the sp2 hybrid-
isation state of the N-3 atom ensures that its basicity is likely
ꢀ109-fold lower than that of the secondary/tertiary amines. More-
injection showed that the radiotracer is probably eliminated via
both the hepatobiliary and renal routes. Elimination of the radio-
tracer into the gut may also account for the high radioactivity in
the early part of the intestine. Compared to other radiotracers re-
ported in the literature,22 tracer [18F]16 shows reduced kidney, li-
ver and lung uptake which may be related to its relatively high
metabolic stability and also suggests that tracer [18F]16 may be a
more suitable candidate for upper torso imaging. The tumor uptake
in A431 xenografts compares favorably with data reported in the
literature for tracers with a quinazoline core.22 The low uptake in
bone suggests that the radiotracer does not undergo defluorina-
tion. We also assessed the potency of compound [18F]16 to detect
A431 and HCT116 xenografts by small animal PET imaging.
Although high non-specific localization was seen in the elimination
organs, the uptake into the tumor was clearly visible, particularly
for A431. Specificity of radiotracer uptake was demonstrated by
the higher uptake of the radiotracer in high EGFR expressing
A431 tumor xenografts compared to low EGFR expressing
HCT116 tumor xenografts. The high level of radioactivity in the
intestine is of particular concern as this would preclude use of this
tracer for imaging, for example, colorectal cancer but should not
limit its application for other EGFR overexpressing cancers, for
example, breast cancer.
In conclusion, compound [18F]16 represents a first generation,
3-cyanoquoline-based selective EGFR radioimaging agent. The 3-
cyanoquinoline core and C-6 Michael acceptor bearing a triazole
functionalized secondary amine terminus appear to confer greater
metabolic stability than has been previously reported for related
EGFR probes. The biodistribution profile in normal tissues, the
selectivity of radiotracer uptake in A431 versus HCT116 and the
ability to detect A431 tumors in animal models by PET imaging
certainly encourage further investigations into the use of this re-
agent for EGFR imaging. The initial results reported herein suggest
that modifications of the structure of compound [18F]16 designed
to modulate the pharmacokinetic properties will be necessary to
further improve uptake into the tumor and reduce non-specific
binding. Further studies are currently ongoing in our laboratories.
over, the triazole ring has an extended planar
p-electron system
conjugated to the amide carbonyl group and consequently will
have a significant barrier to rotation to give the correct orientation
in which this nitrogen lone pair can assist with general base catal-
ysis of the attack of the Cys 773 thiol at the
mide unit.
c position of the ena-
We interpret the lower activity of compounds 12, 13, 16, and 21
relative to compounds 1 and 6 as suggesting that bulkier groups on
the amine of the Michael acceptor have a negative effect on the cel-
lular activity. This effect is particularly apparent for compound 21
whose activity decreases 2000-fold in the cell relative to its activity
in the cell-free assay. This may be due to side reactions of the al-
kyne moiety with the cell surface which could partially prevent
its penetration into the cell, although no specific evidence for this
was obtained. Compound 20, where the Michael acceptor has not
been modified, showed comparable activity to compounds 1 and 6.
Quinoline 16 was then selected for further development as a
first generation 3-cyanoquinoline imaging agent. This compound
balanced high binding affinity in vitro, structural novelty, and a
radiosynthesis method via efficient ‘click’ chemistry. Cognizant of
the importance of a favorable metabolic profile, we decided to test
compound 16 for its stability in mouse and human liver micro-
somes in comparison with compound 6. Interestingly, after
60 min of incubation, only the parent compound was detected by
HPLC (data not shown). The absence of metabolites is consistent
with the notion that secondary amine substituted Michael accep-
tors display relatively high metabolic stability (cf. tertiary amine
analogues).30 The C-7 ethoxy group also appears to be relatively
stable in this regard. The metabolic stability of the radiolabeled
compound [18F]16 was then explored further in vivo; metabolism
studies were carried out by radio-HPLC analysis of liver and plasma
samples of non-tumor bearing mice treated with [18F]16 at three
time points (2, 30, and 60 min). Tracer [18F]16 appeared to be sta-
ble in the liver as the parent compound was by far the major com-
ponent present after 60 min. In plasma, this was the case after
2 min, but a metabolite appeared after 30 min and increased after
60 min. The tissue distribution of tracer [18F]16 at 60 min post
4. Experimental section
4.1. Chemistry and radiochemistry
Solvents were distilled as follows: THF and Et2O over Na-benzo-
phenone ketyl, toluene over Na, and CH2Cl2 over CaH2. Reagents
were used as commercially supplied unless otherwise stated and
handled in accordance with COSHH regulations. Flash chromatog-
raphy (FC) was carried out on Silica gel (BDH Silica Gel for FC).
NMR spectra were recorded at 400 MHz on a Bruker AV-400 or
Bruker DX-400 instrument or at 500 MHz on a Brucker AV-500
instrument. Chemical shifts (d) are given in parts per million
(ppm) as referenced to the appropriate residual solvent peak. The
reference for 19F NMR is CFCl3 (= 0 ppm). Broad signals are assigned
as br. 13C chemical shifts (d) are assigned as s, d, t, and q, for C, CH,
CH2, and CH3, respectively. Coupling constants (J) are given in Hz.
Infrared spectra were recorded as thin films, on Perkin–Elmer
Paragon 1000 Fourier transform spectrometer or as solids, on
Pelkin–Elmer Spectrum 100 Fourier transform spectrometer. Only
selected absorbances (mmax) are reported. Low resolution and
high-resolution mass spectra were recorded on a VG Prospec spec-
trometer, with molecular ions and major peaks being reported.
Intensities are given as percentages of the base peak. HR-MS values
are valid to 5 ppm. Melting points of solid compounds were high-
er than 250 °C and decomposition was observed. [18F]Fluoride was
produced by a cyclotron (PET trace) using the 18O(p,n)18F nuclear
reaction with 16.4 MeV proton irradiation of an enriched
[
18O]H2O target.

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6641
4.2. HPLC methods
(d), 121.1 [s (d, JCF = 18.9)], 116.8 (s), 116.4 [d (d, JCF = 22.3)], 113.1
(s), 109.9 and 109.5 (d), 108.4 (d), 88.3 (s), 79.9 (s), 65.0 (t), 49.9
and 49.3 (t), 34.3 (q), 28.2 (q, 3C), 14.4 (q); IR: mmax 3347, 2928,
2211, 1681, 1535, 1498, 1391, 1145 (MS (ESI): m/z (%) 554 [MH+]
(100); HR-MS (ESI) calcd for C28H30ClFN5O4: 554.1970, found
(a) Preparative radio-HPLC was carried out on a Beckman Sys-
tem Gold equipped with a Bioscan Flowcount FC-3400 PIN diode
detector (Lablogic) and a linear UV-200 detector (wavelength
254 nm). A Phenomenex Luna C18 150 mm ꢁ 10 mm HPLC column
and a isocratic mobile phase of water and 20% acetonitrile (0.085%
H3PO4) and flow rate 3 mL/min. (b) Analytical radio-HPLC was car-
ried out as above but using a Bioscan Flowcount FC3200 sodium io-
dide/PMT gamma detector (Lablogic), a Thermo Spectra SERIES
554.1981 (D 2.0 ppm).
4.3.2.2. {(E)-3-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-
ethoxyquinolin-6-ylcarbamoyl]-allyl}-prop-2-ynyl carbamic acid
tert-butyl ester (8). Colorless oil; 68% yield, Rf = 0.14 (eluent:
AcOEt/Et2O, 10:1); 1H NMR (400 MHz, CDCl3) d 9.18 (s, 1H), 8.39
(s, 1H), 8.17 (br s, 1H), 7.96 (s, 1H), 7.08 (s, 1H), 7.05–6.94 (m,
2H), 6.89 (t, J = 8.6, 1H), 6.68 (m, 1H), 6.08 (d, J = 14.9, 1H), 4.22
(q, J = 6.9, 2H), 4.13 (d, J = 4.2, 2H), 4.05 and 3.90 (br s, 2H), 2.25
(t, J = 2.2, 1H), 1.58 (t, J = 7.0, 3H), 1.48 (s, 9H); 13C NMR
(101 MHz, CDCl3) d 164.3 (s), 155.8 [s (d, JCF = 249.8 Hz], 154.7
(s), 152.1 (d), 151.0 (s), 149.6 (s), 147.3 (s), 142.7 and 142.2 (d),
135.7 (s), 127.5 (s), 126.1 (d), 125.5 (d), 123.43 and 123.36 (d),
121.2 [s (d, JCF = 18.9)], 116.8 (s), 116.5 [d (d, JCF = 22.3)], 113.2
(s), 109.7 (d), 108.5 (d), 88.5 (d), 81.0 (s), 79.1 (s), 72.2 and 71.9
(d), 65.1 (t), 47.0 (t), 36.5 and 35.9 (t), 28.3 (q; 3C), 14.1 (q); IR: mmax
3412, 3307, 2980, 2930, 2252, 2214, 1690, 1682, 1537, 1250,
734 cmꢂ1; MS (ESI): m/z (%) 578 [MH+] (100); HR-MS (ESI) calcd
UV150 (wavelength 254 nm), and
a
Phenomenex Luna
50 mm ꢁ 4.6 mm (3
l
m) column with an isocratic mobile phase
of water and 22% acetonitrile (0.1% TFA), flow rate 1 mL/min.
4.3. Synthesis
Compounds 1,29 3,29 4,32 and 937 were synthesized according to
established literature procedures and 1H NMR spectral data were
consistent with those previously reported.
4.3.1. (E)-4-(tert-Butoxycarbonyl-prop-2-ynylamino)-but-2-
enoic acid methyl ester (7)
4-Bromo methylcrotonate (1 g, 5.6 mmol) was dissolved in dry
THF (10 mL) and propargyl amine (961
lL, 14 mmol) was added
for C30H30N5O4FCl: 578.1970, found 578.1948 (
D
ꢂ3.8 ppm).
dropwise at ꢂ20 °C. The resulting mixture was stirred at ꢂ20 °C
for 4 h then cooled to ꢂ65 °C. Boc2O (4.9 g, 22.3 mmol) and Et3N
(4 mL, 27.9 mmol) were then added in turn and the mixture stirred
at ꢂ65 °C ? rt for 14 h. The white solid was filtered off and the
mother liqueur was concentrated under reduced pressure, dis-
solved in CH2Cl2 (30 mL) and washed with water (20 mL), HCl
1 M (20 mL), water (20 mL), and brine (20 mL) and finally dried
over MgSO4. The crude residue was purified by FC (Et2O/petroleum
ether, 1:4; Rf = 0.12) to give the title compound (681 mg, 49%) as
colorless oil.
4.3.2.3. (E)-Pent-2-en-4-ynoic acid [4-(3-chloro-4-fluorophe-
nylamino)-3-cyano-7-ethoxyquinolin-6-yl]-amide (10). Yellow
solid, 20% yield, Rf = 0.13 (Et2O); 1H NMR (500 MHz, CDCl3) d 9.10
(s, 1H), 8.56 (s, 1H), 8.09 (s, 1H), 7.39 (s, 1H), 7.35 (br s, 1H), 7.24
(dd, J = 6.3, 2.7, 1H), 7.16 (t, J = 8.6, 1H), 7.08 (ddd, J = 2.8, 3.9, 8.7,
1H), 6.82 (dd, J = 1.6, 15.4, 1H), 6.55 (d, J = 15.4, 1H), 4.33 (q,
J = 7.0, 2H), 3.40 (dd, J = 0.4, 2.4, 1H), 1.58 (t, J = 7.0, 3H); 13C
NMR (126 MHz, CDCl3) d 162.6 (s), 156.6 [s (d, JCF = 249.0)], 152.2
(d), 151.3 (s), 150.0 (s), 147.6 (s), 135.6 (s), 134.2 (d), 128.1 (s),
126.9 (d), 124.6 [d (d, JCF = 7.1)], 122.9 (d), 121.9 [s (d,
JCF = 19.1)], 117.2 [d (d, JCF = 22.3)], 116.5 (s), 113.2 (s), 109.9 (d),
108.8 (d), 89.2 (s), 86.3 (d), 80.3 (s), 65.3 (t), 14.5 (q); 19F NMR
(376 MHz, CDCl3) d ꢂ117.0; IR: mmax 3300, 2925, 2361, 2342,
2214, 1670, 1624, 1539, 1498, 1458, cmꢂ1; MS (ESI): m/z (%) 435
[MH+] (100); HR-MS (ESI) calcd for C23H17N4O2FCl: 435.1024,
1H NMR (400 MHz, CDCl3) d 6.90 (dt, J = 15.7, 5.3, 1H), 5.93 (d,
J = 15.7, 1H), 4.19–3.91 (m, 4H), 3.77 (s, 3H), 2.24 (t, J = 2.4, 1H),
1.49 (s, 9H); 13C NMR (101 MHz, CDCl3) d 166.5 (s), 154.6 (s),
143.6 (d), 122.1 and 121.8 (d), 81.0 (s), 78.9 (d), 72.3 (s), 71.9 (s),
51.7 (q), 47.0 and 46.8 (t), 36.4 and 35.9 (t), 28.3 (q, 3C); IR: mmax
3263, 2976, 2361, 1699, 1450, 1273, 1167 cmꢂ1; MS (ESI): m/z
(%) 276 [MNa+] (35); HR-MS (ESI) calcd for C13H19NO4Na:
found 435.1018 (
D
ꢂ1.4 ppm).
276.1211, found 276.1212 (
D
ꢂ0.4 ppm).
4.3.2.4. {(E)-3-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-
(2-fluoroethoxy)-quinolin-6-ylcarbamoyl]-allyl}-methylcarbamic
acid tert-butyl ester (Boc-20). Yellow oil, 61% yield; Rf = 0.38
(AcOEt); 1H NMR (500 MHz, CDCl3) d 9.15 (s, 1H), 8.57 (s, 1H),
8.10 (s, 1H), 7.40 (br s, 2H), 7.31–7.25 (m, 1H), 7.21–7.14 (m,
1H), 7.14–7.07 (m, 1H), 6.93 (dt, J = 5.0, 15.2, 1H), 6.13–5.98 (m,
1H), 4.90 (dm, J = 47.9, 2H), 4.50 (dm, J = 27.3, 2H), 4.06 (br s,
2H), 2.91 (br s, 3H), 1.48 (s, 9H); 13C NMR (126 MHz, CDCl3) d
163.6 (s), 156.6 [s (d, JCF = 284.1)], 155.8 (s), 152.3 (d), 150.6 (s),
150.0 (s), 147.1 (s), 142.6 and 142.2 (d), 135.5 (s), 128.4 (s),
126.9 (d), 124.6 (d), 123.5 (d), 121.8 [s (d, JCF = 19.6)], 117.2 [d (d,
JCF = 22.3)], 116.5 (s), 113.8 (s), 110.0 (d), 109.2 (d), 100.0 (s),
85.4 [t (d, JCF = 174.1)], 80.9 (s), 68.4 [t (d, JCF = 19.5)], 50.0 and
49.4 (t), 34.6 and 34.5 (q), 29.7 (s, 3C); MS (ESI): m/z (%); 572
[MH+] (100); HR-MS (ESI) calcd for C28H29N5O4ClF2: 572.1876,
4.3.2. General procedure for the synthesis of compounds 5, 8,
10, and Boc-20
The amino quinolines 3 or 19 (1 equiv) and the Michael accep-
tor 4, 7 or 9 (1.5 equiv) were suspended and sonicated in dry
CH2Cl2 (0.06 M) and AlMe3 (2.0 M solution in hexane, 2 equiv)
was added dropwise at rt. The mixture was stirred at rt and mon-
itored by TLC until the starting quinoline had disappeared. The
mixture was quenched with a saturated solution of NaHCO3 and
the phases were separated. The aqueous layer was extracted twice
with equal volumes of CH2Cl2 and the combined organic layers
washed with equal volume of brine and dried over MgSO4. The
crude mixture was purified by FC or plate silica gel TLC.
4.3.2.1. {(E)-3-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-
ethoxyquinolin-6-ylcarbamoyl]-allyl}-methylcarbamic acid tert-
butyl ester (5). Yellow semisolid, 47% yield; Rf = 0.12 (petroleum
ether/AcOEt, 1:2); 1H NMR (400 MHz, CDCl3): d 9.20 (s, 1H), 8.53
(s, 1H), 8.07 (s, 1H), 7.91 (br s, 1H), 7.31–7.21 (m, 1H), 7.21–7.13
(m, 1H), 7.12–6.88 (m, 3H), 6.15–6.02 (m, 1H), 4.38–4.27 (m, 2H),
4.08 (br s, 2H), 2.98–2.85 (m, 3H), 1.61 (t, J = 6.9 Hz, 3H), 1.53 (s,
found 572.1868 (
D
ꢂ1.4 ppm).
4.3.3. General procedure for the synthesis of compounds 6, 16,
20 and 21
Quinoline 5, 8, Boc-16 or Boc-20 (1 equiv) were dissolved in
1,4-dioxane (0.03 M) and concd HCl (0.003 M) was added dropwise
at rt. The mixture was stirred 30 min—1 h and the precipitate was
collected and washed with dioxane and Et2O and dried in vacuo to
give the title compounds as HCl salts.
9H); 13C NMR (101 MHz, CDCl3):
d 164.4 (s), 156.6 [s (d,
JCF = 247.8)], 155.6 (s), 152.0 (d), 150.8 (s), 149.4 (s), 147.2 (s),
143.3 and 142.3 (d), 135.7 (s), 127.3 (s), 125.8 (d), 124.3 (d), 123.1

6642
F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
4.3.3.1. (E)-4-(Methylamino)-but-2-enoic acid [4-(3-chloro-4-
fluorophenylamino)-3-cyano-7-ethoxyquinoline-6-yl] amide
hydrochloride (6ꢃHCl). Yellow solid; 78% yield; 1H NMR
(400 MHz, MeOD): d 9.24 (s, 1H), 8.87 (s, 1H), 7.72 (dd, J = 6.5,
2.5 Hz, 1H), 7.52 (ddd, J = 6.5, 8.7, 2.5, 1H), 7.48–7.36 (m, 2H),
7.00 (dt, J = 15.2, 1H), 6.82 (dt, J = 15.2, 1.4, 1H), 4.47 (q, J = 7.0,
2H), 3.92 (d, J = 6.4, 2H), 2.78 (s, 3H), 1.62 (t, J = 7.0, 3H); 13C
NMR (101 MHz, MeOD): d 163.5 (s), 158.1 [s (d, JCF = 250.1)],
155.7 (s), 154.5 (s), 147.5 (d), 146.1 (s), 136.6 (s), 134.6 (d), 133.8
(s), 129.6 (d), 129.4 (d), 127.7 [d (d, JCF = 7.8 Hz)], 121.3 [s (d,
JCF = 19.2)], 117.0 [d (d, JCF = 22.7 Hz)], 114.0 (d), 113.1 (s), 111.9
(s), 100.3 (d), 86.7 (s), 66.8 (t), 48.9 (t), 32.2 (q), 13.2 (q); IR (solid):
mmax 3377, 2938, 2687, 2225, 1634, 1530, 1491, 1258, 1060,
880 cmꢂ1; MS (ESI): m/z (%) 454 [MH+] (48), 248 (100); HR-MS
(d), 74.8 (s), 65.4 (t), 46.2 (t), 35.3 (t), 14.1 (q); IR (solid): mmax
3404, 3291, 2932, 2601, 2424, 2223, 1687, 1578, 1489, 1452,
1258, 1032, 883 cmꢂ1; MS (ESI): m/z (%) 478 [MH+] (100); HR-MS
(ESI) calcd for C25H22N5O2FCl: 478.1446, found 478.1448 (
0.4 ppm).
D
4.3.3.5. (E)-4-(Methylamino)-but-2-enoic acid [4-(3-chloro-4-
fluorophenylamino)-3-cyano-7-ethoxy-quinoline-6-yl] amide
(6). The quinoline hydrochloride 6ꢃHCl (39.0 mg, 0.08 mmol) was
dissolved in water (1 mL) and K2CO3 (55 mg, 0.4 mmol) was added.
The mixture was stirred 14 h at rt and the pale yellow precipitate
was collected, washed with water and dried in vacuo to give the
title compound 6 (27.1 mg, 78%) as a yellow solid.
1H NMR (400 MHz, MeOD): d 8.96 (s, 1H), 8.48 (s, 1H), 7.48–
7.41 (m, 1H), 7.41–7.35 (m, 1H), 7.35–7.24 (m, 2H), 7.04
(dt, J = 15.4, 5.8 Hz, 1H), 6.50 (dt, J = 15.5, 1.5 Hz, 1H), 4.37 (q,
J = 6.8 Hz, 2H), 3.44 (dd, J = 5.7, 1.0 Hz, 2H), 2.45 (s, 3H), 1.59 (t,
J = 6.9 Hz, 3H).
(ESI) calcd for C23H22ClFN5O2: 454.1446, found 454.1459 (
2.9 ppm).
D
4.3.3.2. (E)-4-{[1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-ylmethyl]-
amino}-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-
3-cyano-7-ethoxy-quinolin-6-yl]-amide hydrochloride (16). Yellow
solid; 99% yield; 1H NMR (500 MHz, DMSO-d6) d 10.99 (br s, 1H),
9.93 (s, 1H), 9.83 (s, 1H), 9.12 (br s, 1H), 8.97 (s, 1H), 8.33 (s, 1H),
7.72 (d, J = 6.1, 1H), 7.61 (s, 1H), 7.53 (t, J = 9.0, 1H), 7.47–7.41
(m, 1H), 6.87 (dt, J = 15.5, 6.35, 1H), 6.78 (d, J = 15.6, 1H), 4.84
(dm, J = 32.3, 2H), 4.79–4.74 (m, 2H), 4.34 (q, J = 7.0, 2H), 4.29 (t,
J = 4.9, 2H), 3.86 (br dd, J = 11.6, 5.9, 2H), 1.49 (t, J = 7.0, 3H).; 19F
NMR (376 MHz, DMSO-d6) d ꢂ117.9, ꢂ222.0; 13C NMR (101 MHz,
DMSO-d6) d 162.7 (s), 155.3 [s (d, JCF = 422.7)] 155.1 (s), 154.9 (s),
149.3 (d), 138.5 (s), 135.6 (s), 134.6 (d), 129.4 (d), 128.6 (s),
128.4 (s), 127.8 [d (d, JCF = 7.5)], 126.6 (d), 126.0 (d), 119.8 [s (d,
JCF = 18.9)], 117.3 [d (d, JCF = 22.3)], 116.4 (d), 114.8 (s), 112.5 (s),
102.9 (d), 86.9 (s), 81.9 [t, (d, JCF = 168.3)], 65.3 (t), 50.2 [t, (d,
JCF = 19.4)], 46.6 (t), 40.8 (t), 14.1 (q); IR (solid): mmax 3371, 2937,
2732, 1635, 1533, 1493, 1260, 1020, 823 cmꢂ1; MS (ESI): m/z (%)
567 [MH+] (80), 440 (100); HR-MS (ESI) calcd for C27H26N8O2F2Cl:
4.3.3.6. (E)-4-[(2-Fluoroethyl) methyl amino]-but-2-enoic acid
[4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxyquinoline-
6-yl] amide (12). Quinoline 6 (32 mg, 0.07 mmol) was dissolved
in dry CH2Cl2 (0.7 mL) and 1-fluoro-2-mesyloxy ethane (11,
13 mg, 0.09 mmol) and Et3N (20 lL, 0.14 mmol) were added in
turn. The mixture was stirred overnight, concentrated and directly
purified by preparative silica TLC on silica gel (CH2Cl2/MeOH, 20:1;
Rf = 0.28) to give quinoline 12 (11.5 mg, 33%) as a yellow solid.
1H NMR (400 MHz, CDCl3): d 9.17 (s, 1H), 8.54 (s, 1H), 8.11 (s,
1H), 7.53 (s, 1H), 7.33 (s, 1H), 7.21 (dd, J = 6.3, 2.6, 1H), 7.12 (t,
J = 8.6, 1H), 7.08–6.99 (m, 2H), 6.27 (dt, J = 15.2, 1.6, 1H), 4.59
(dt, J = 47.6, 4.8, 2H), 4.32 (q, J = 7.0, 2H), 3.33 (dd, J = 5.6, 3.1,
2H), 2.77 (dt, J = 28.0, 4.8, 2H), 2.39 (s, 3H), 1.60 (t, J = 7.0, 3H);
19F NMR (376 MHz, CDCl3):
d
ꢂ117.5, ꢂ219.4; 13C NMR
(126 MHz, CDCl3): d 164.0 (s), 156.4 [s (d, JCF = 248.7 Hz)], 152.2
(d), 151.2 (s), 149.9 (s), 147.4 (s), 143.9 (d), 135.7 (s), 128.3 (s),
126.7 (d), 125.3 (d), 124.3 [d (d, JCF = 7.3 Hz)], 121.7 [s
JCF = 19.0)], 117.1 (s), 116.8 [d (d, JCF = 24.8 Hz)], 113.3 (s), 109.4
(d), 108.8 (d), 88.9 (s), 82.1 [t, (d, JCF = 168.0 Hz)], 65.2 (t), 58.6
(t), 57.0 [t, (d, JCF = 19.7 Hz)], 42.9 (q), 14.5 (q); IR: mmax 3250,
2924, 2212, 1685, 1622, 1537, 1498, 1458, 1393, 1215 cmꢂ1; MS
(ESI): m/z (%) 500 [MH+] (76), 271 (100); HR-MS (ESI) calcd for
567.1835, found 567.1841 (
D 1.1 ppm).
4.3.3.3. (E)-4-Methylamino-but-2-enoic acid [4-(3-chloro-4-flu-
orophenylamino)-3-cyano-7-(2-fluoroethoxy)-quinolin-6-yl]-
amide hydrochloride (20). Yellow solid; 67% yield; 1H NMR
(400 MHz, MeOD) d 9.27 (s, 1H), 8.93 (br s, 1H), 7.73 (dd, J = 6.5,
2.5, 1H), 7.55–7.48 (m, 2H), 7.48–7.40 (m, 1H), 7.01 (dt, J = 15.1,
6.5, 1H), 6.83 (d, J = 15.4, 1H), 5.06–4.87 (dm, 2H), 4.67 (dm,
J = 28.7, 2H), 3.93 (d, J = 6.5, 2H), 2.79 (s, 3H); 19F NMR (376 MHz,
MeOD) d ꢂ118.1, ꢂ223.9; 13C NMR (101 MHz, MeOD) d 164.8 (s),
159.7 [s (d, JCF = 255.2)], 156.9 (s), 156.2 (s), 149.55 (d), 138.6 (s),
135.5 (d), 135.3 (s), 131.2 (d), 131.0 (s), 130.8 (d), 128.9 [d (d,
JCF = 7.9)], 122.9 [s (d, JCF = 19.3)], 118.6 [d (d, JCF = 22.8)], 116.2
(d), 114.4 (s), 113.8 (s), 102.5 (d), 101.9 (s), 82.7 [t (d,
JCF = 169.7)], 71.0 [t, (d, JCF = 19.8)], 50.2 (t), 33.3 (q); IR (solid): mmax
3395, 2926, 2224, 1532, 1494, 1259, 1059, 835 cmꢂ1; MS (ESI): m/z
(%); 472 [MH+] (52), 257 (100); HR-MS (ESI) calcd for
C25H25N5O2F2Cl: 500.1665, found 500.1662 (
D
ꢂ0.6 ppm).
4.3.3.7. (E)-4-[(4-Fluorobenzyl) methylamino]-but-2-enoic acid
[4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxyquinoline-
6-yl] amide (13). Quinoline 6 (50 mg, 0.10 mmol) was suspended
in dichloroethane (0.3 mL) and p-fluorobenzaldehyde (12
lL,
0.11 mmol) and acetic acid (10 L) were added dropwise at rt.
l
After 30 min, NaBH(OAc)3 (32 mg, 0.15 mmol) was added and the
mixture was stirred 14 h at rt. The mixture was quenched with a
saturated solution of NaHCO3 (1 mL) and extracted with CH2Cl2
(3 ꢁ 2 mL). The combined organic layers were dried over MgSO4.
After purification by plate silica TLC (CH2Cl2/MeOH, 20:1;
Rf = 0.37), the title compound 13 was obtained (12 mg, 21%) as a
yellow solid.
C23H21N5O2ClF2: 472.1354, found 472.1342 (
D
ꢂ2.1 ppm).
4.3.3.4. (E)-4-Prop-2-ynylaminobut-2-enoic acid [4-(3-chloro-4-
fluorophenylamino)-3-cyano-7-ethoxyquinolin-6-yl]-amide
hydrochloride (21). Yellow solid; 99% yield; 1H NMR (400 MHz,
DMSO-d6) d 11.02 (br s, 1H), 9.98 (s, 1H), 9.83 (s, 2H), 9.14 (s,
1H), 8.98 (s, 1H), 7.75 (d, J = 6.6, 1H), 7.61–7.57 (m, 1H), 7.55 (t,
J = 8.5, 1H), 7.50–7.44 (m, 1H), 6.90–6.78 (m, 1H), 6.81 (t, J = 11.9,
1H), 4.36 (q, J = 7.0, 2H), 3.95 (s, 2H), 3.87 (d, J = 5.4, 2H), 3.76 (t,
J = 2.3, 1H), 1.50 (t, J = 6.9, 3H); 13C NMR (101 MHz, DMSO-d6) d
162.8 (s), 156.3 [s (d, JCF = 247.0)], 155.2 (s), 153.2 (s), 149.3 (d),
139.6 (s), 135.4 (s), 134.3 (d), 129.6 (d), 128.6 (d), 127.9 (s),
126.7 [d (d, JCF = 7.0)], 119.9 [s (d, JCF = 18.9)], 117.4 [d (d,
JCF = 22.3)], 116.3 (d), 114.7 (s), 112.5 (s), 102.9 (d), 86.9 (s), 79.8
1H NMR (400 MHz, CDCl3): d 9.18 (s, 1H), 8.52 (s, 1H), 8.07 (s,
1H), 7.66 (s, 1H), 7.34–7.27 (m, 4H), 7.12 (dd, J = 6.3, 2.5, 1H),
7.12–7.00 (m, 4H), 6.96 (dt, J = 8.3, 3.4, 1H), 6.24 (dt, J = 15.3, 1.6,
1H), 4.32 (q, J = 7.0, 2H), 3.53 (s, 2H), 3.23 (dd, J = 5.7, 1.0, 2H),
2.25 (s, 3H), 1.61 (t, J = 6.7, 3H); 19F NMR (376 MHz, CDCl3): d
ꢂ117.6, ꢂ115.5; 13C NMR (126 MHz, CDCl3): d 163.9 (s), 162.1 [s
JCF = 245.1 Hz], 156.5 [s (d, JCF = 249.2 Hz)], 152.1 (d), 151.2 (s),
149.8 (s), 147.5 (s), 144.5 (d), 135.7 (s), 134.2 (s), 130.3
[d (d, JCF = 7.8 Hz, 2C)], 128.5 (s), 126.8 (d), 125.2 (d), 124.3 [d (d,
JCF = 7.4 Hz)], 121.6 [s (d, JCF = 21.0)], 117.2 (s), 116.9
[d (d, JCF = 43.3 Hz)], 115.2 [d (d, JCF = 21.4 Hz, 2C)], 113.3 (s),

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6643
109.3 (d), 108.8 (d), 89.1 (s), 65.2 (t), 61.3 (t), 57.8 (t), 42.5 (q), 14.6
(q); IR: mmax 3380, 2922, 2220, 1680, 1620, 1537, 1458 cmꢂ1; MS
(ESI): m/z (%) 562 [MH+] (50), 454 (100); HR-MS (ESI) calcd for
1H NMR (400 MHz, MeOD) d 9.12 (s, 1H), 8.79 (s, 1H), 7.68 (dd,
J = 1.5, 6.1, 1H), 7.51–7.45 (m, 1H), 7.41 (t, J = 8.7, 1H), 7.35 (s, 1H),
2.28 (s, 3H); 13C NMR (101 MHz, MeOD) d 170.9 (s), 158.2 [s (d,
JCF = 250.0)], 155.6 (s), 154.7 (s), 147.3 (d), 136.4 (s), 134.0 (s),
130.0 (s), 129.4 (d), 127.6 [d (d, JCF = 7.9)], 121.4 [s (d, JCF = 19.0)],
117.6 [d (d, JCF = 22.8)], 113.7 (d), 113.1 (s), 111.4 (s), 102.3 (d),
86.1 (s), 22.7 (q); IR: mmax 3357, 3018, 2925, 2228, 1613, 1539,
1495, 1469, 1238 cmꢂ1; MS (ESI): m/z (%) 371 [MH+] (100), 144
(25); HR-MS (ESI) calcd for C18H13N4O2ClF: 371.0711, found
C30H27N5O2F2Cl: 562.1821, found 562.1828 (1.2 ppm).
4.3.4. General procedure for the synthesis of compounds 15 and
Boc-16
Quinoline 8 or 10 (1 equiv) was dispersed in water (0.15 M) and
fluoro ethylazide 14 (0.5 M solution in DMF, 2 equiv), CuSO4
(0.3 equiv) and Cu powder (0.3 equiv) were added. The mixture
was heated by microwave irradiation at 125 °C for 15 min and di-
luted with water and AcOEt. The phases were separated and the
aqueous phase was extracted with AcOEt. The combined organic
layers were dried over MgSO4. The crude residue was purified by
FC to give the compounds Boc-16 or 15, respectively.
371.0707 (
D
ꢂ1.1 ppm).
4.3.4.4.
6-Amino-4-(3-chloro-4-fluorophenylamino)-7-(2-flu-
oroethoxy)-quinoline-3-carbonitrile (19). The quinoline 18 (25 mg,
0.06 mmol) was heated with K2CO3 (41.4 mg, 0.30 mmol) and 2-
mesyloxy-1-fluoroethane (17 mg, 0.12 mmol) in DMF (1 mL) at
70 °C overnight. The mixture was cooled at rt, water (1 mL) was
added and yellow solid was collected, washed with water (3 mL)
and Et2O (3 mL) and dried in vacuo. The yellow solid was then re-
fluxed in water (0.5 mL) and concd HCl (0.5 mL) for 2.5 h then
cooled at rt. The crude mixture was concentrated under reduced
pressure, dissolved in water (2 mL) and neutralized with K2CO3.
Quinoline 19 (8 mg, 53%) was collected as a yellow solid.
4.3.4.1. (E)-N-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-eth-
oxyquinolin-6-yl]-3-[1-(2-fluoroethyl)-1H-[1,2,3]triazol-4-yl]-
acrylamide (15). Yellow solid; 32% yield; Rf = 0.28 (AcOEt/MeOH,
1:1); 1H NMR (500 MHz, CDCl3) d 9.20 (s, 1H), 8.55 (br s, 1H), 8.26
(s, 1H), 7.83 (s, 1H), 7.68 (d, J = 15.3, 1H), 7.52 (br s, 1H), 7.48 (s,
1H), 7.29 (dd, J = 6.2, 2.6, 1H), 7.18 (t, J = 8.6, 1H), 7.15–7.09 (m,
1H), 7.05 (d, J = 15.3, 1H), 4.84 (dm, J = 46.7, 2H), 4.73 (dm,
J = 27.1, 2H), 4.35 (q, J = 7.0, 2H), 1.61 (t, J = 7.0, 3H); 19F NMR
(376 MHz, CDCl3) d ꢂ116.1, ꢂ220.7; 13C NMR (126 MHz, CDCl3) d
163.89 (s), 156.73 [s (d JCF = 250.1)], 151.62 (d), 151.38 (s),
150.29 (s), 148.88 (s), 143.57 (s), 135.41 (s), 130.30 (d), 128.75
(s), 127.08 (d), 124.92 (d), 124.72 [d (d, JCF = 6.4)], 122.24 (d),
121.91 [s (d, JCF = 18.9)], 117.23 [d (d, JCF = 22.4)], 116.29 (s),
113.17 (s), 109.54 (d), 108.05 (d), 99.96 (s), 81.36 [t (d,
JCF = 173.0)], 65.49 (t), 50.73 [t (d, JCF = 20.5)], 14.61 (q); IR: mmax
3266, 2954, 2212, 1623, 1538, 1498, 1460, 1224, 1036 cmꢂ1; MS
(ESI): m/z (%) 524 [MH+] (100); HR-MS (ESI) calcd for
1H NMR (400 MHz, MeOD) d 8.30 (s, 1H), 8.25 (s, 1H), 7.33 (dd,
J = 2.6, 6.4, 1H), 7.26 (s, 1H), 7.24–7.21 (m, 1H), 7.18–7.15 (m, 1H),
4.87 (dm, J = 47.7, 2H), 4.46 (dm, J = 28.6, 2H); 19F NMR (376 MHz,
MeOD) d ꢂ122.4, ꢂ225.1; 13C NMR (101 MHz, MeOD) d 157.1 [s (d,
J = 249.0)], 155.8 (s), 153.3 (s), 149.5 (d), 145.0 (s), 140.8 (s), 138.3
(s), 126.9 [d (d, J = 7.0)], 125.0 [d (d, JCF = 7.3)], 122.2 [s (d,
JCF = 19.0)], 118.0 [d (d JCF = 22.3)], 117.1 (s), 113.2 (d), 108.1 (s),
103.5 (s), 102.7 (d), 82.8 [t, (d JCF = 169.2)], 69.4 [t, (d JCF = 20.0)];
IR (solid): mmax 3348, 2926, 2210, 1619, 1568, 1454, 1214, 1047,
869 cmꢂ1; MS (ESI): m/z (%) 375 [MH+] (85), 355 (100); HR-MS
(ESI) calcd for
1.6 ppm).
C18H14N4OClF2: 375.0824, found 375.0830 (D
C25H21N7O2F2Cl: 524.1413, found 524.1404 (
D
ꢂ1.7 ppm).
4.3.4.2. {(E)-3-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-
ethoxyquinolin-6-ylcarbamoyl]-allyl}-[1-(2-fluoroethyl)-1H-[1,2,3]
triazol-4-ylmethyl]-carbamic acid tert-butyl ester (Boc-16). Yellow
semisolid; 19% yield; Rf = 0.36 (eluent: AcOEt/MeOH, 10:1); 1H
NMR (400 MHz, CDCl3) d 9.15 (s, 1H), 8.58 (s, 1H), 8.14 (s, 1H),
7.73 (dd, J = 5.7, 3.3, 1H), 7.53–7.42 (m, 2H), 7.31 (dd, J = 6.2, 2.6,
1H), 7.21 (t, J = 8.5, 1H), 7.17–7.11 (m, 1H), 6.97–6.87 (m, 1H),
6.26–6.06 (m, 1H), 4.81 (dt, J = 46.8, 4.3, 2H), 4.69 (dm, J = 26.7,
2H), 4.58–4.50 (m, 2H), 4.42–4.34 (m, 2H), 4.22–4.13 (m, 2H),
1.63 (t, J = 6.8, 3H), 1.28 (s, 9H); 19F NMR (376 MHz, CDCl3) d
ꢂ59.9, ꢂ115.8; 13C NMR (126 MHz, CDCl3) d 171.1 (s), 163.7 (s),
156.3 [s (d, JCF = 249.0)], 152.1 (d), 151.3 (s), 149.9 (s), 147.3
(s), 145.2 and 144.9 (s), 142.2 and 141.8 (d), 135.6 (s), 128.2 (s),
126.7 (d), 124.4 (d), 124.3 [d (d, JCF = 6.8)], 123.8 and 123.0 (d),
121.6 [s (d, JCF = 19.1)], 117.0 [d (d, JCF = 22.4)], 116.6 (s), 113.2
(s), 109.7 (d), 108.6 (d), 88.9 (s), 81.4 [t (d, JCF = 162.1)], 80.8 (s),
65.2 (t), 50.5 [t (d, JCF = 20.4)], 47.9 and 47.5 (t), 42.0 and 41.5 (t),
28.3 (q, 3C), 14.5 (q); IR: mmax 2925, 2854, 2220, 1688, 1537,
1459, 1163 cmꢂ1; MS (ESI): m/z (%) 689 [MNa+] (100), 667 [M+];
HR-MS (ESI) calcd for C32H34N8O4F2Cl: 667.2360, found 667.2354
4.4. Radiochemistry
Under an atmosphere of nitrogen, a buffered solution (sodium
phosphate buffer, pH 6.0, 250 mM) of sodium ascorbate (50 L,
8.7 mg, 43.2 mol) was added to a Wheaton vial (3 mL) containing
an aqueous solution of copper(II) sulfate (50 L, 1.7 mg pentahy-
drate, 7.0 mol). After 1 min, a solution of alkyne 21 (2.1 mg,
4.4 mol) in MeCN/water, 1:1 (50 L) was added followed by dis-
tilled 2-[18F]fluoroethylazide (94–740 MBq) in acetonitrile
(100 L). The mixture was heated at 80 °C for 15 min, the HPLC
mobile phase [21% MeCN (0.085% H3PO4), 500 L] was added and
l
l
l
l
l
l
l
l
the resulting mixture was purified by preparative radio-HPLC.
The isolated HPLC fraction was diluted with water (5 mL) and
loaded onto a SepPak C18-light cartridge (Waters) that had been
conditioned with ethanol (5 mL) and water (10 mL). The cartridge
was subsequently flushed with water (5 mL) and [18F]16 eluted
with ethanol (0.1 mL fractions). The product fraction was diluted
with PBS to provide an ethanol content of 10–20% (v/v).
4.5. EGFR tyrosine kinase enzyme inhibition assay
(D
ꢂ0.9 ppm).
The inhibitory activity of quinolines 1, 6, 12, 13, 15, 16, 20, and
21 against EGFR kinase activity was measured by a time resolved
fluorescence assay (DELFIA, Perkin–Elmer Life Sciences, Boston,
MA, USA). The compounds were dissolved in DMSO and diluted
4.3.4.3. N-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-hydroxy-
quinolin-6-yl]-acetamide (18). The quinoline 17 (500 mg, 1.15
mmol) was suspended in dry CH2Cl2 (50 mL) and BBr3 (1.0 M in
CH2Cl2, 5.7 mL, 5.7 mmol) was added dropwise at rt. The mixture
was stirred 14 h and quenched with water (20 mL). The yellow pre-
cipitate was collected, washed with water (50 mL), and dried in va-
cuo. The title compound 18 was obtained as a yellow solid
(166 mg, 40%) and used in the next step without further
purification.
in DMSO to give final concentrations of 0.0001–100,000 lg/mL.
EGFR protein (E-3641, Sigma) was incubated with the compounds
in a kinase buffer for 15 min at rt in accordance with manufac-
turer’s instructions (DELFIA Tyrosine kinase kit; Perkin–Elmer).
After 15 min at rt the kinase reaction was initiated by addition of
25 lM ATP, 25 mM MgCl2, and 0.25 lM/L of biotinylated poly(Glu,

6644
F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
Ala, Tyr) in 10 mM HEPES buffer, pH 7.4. The reaction proceeded at
rt for 1 h and was stopped by addition of 100 mM EDTA. The en-
zyme reaction solution was diluted and aliquots added to 96-well
ELISA streptavidin plates with shaking for 1 h. The plates were
washed and phosphorylated tyrosine was detected with Eu-la-
belled antiphosphotyrosine antibody (50 ng/well; PT66; Perkin–El-
mer). After washing and enhancement steps, the plates were
assessed in a Victor3 multi-label counter (Perkin–Elmer) using
the EGFR Europium protocol. The concentration of compound that
inhibited 50% of receptor phosphorylation activity (IC50) was esti-
mated by non-linear regression analysis using GraphPad Prism
(Version 4.0 for Windows, GraphPad Software, San Diego California
USA).
within the whole body at each of the time points to obtain the nor-
malized uptake value expressed as %ID/mL. Direct [18F]16 tissue
biodistribution was assessed subsequent to the PET scan. For this,
mice were sacrificed by exsanguination via cardiac puncture under
general anesthesia (isofluorane inhalation) and tissues were ex-
cised, weighted and immediately counted for fluorine-18 radioac-
tivity on a Cobra-II Auto-Gamma counter (Packard Instruments,
Meriden, CTA). Data were expressed as tissue to blood ratios and
% injected dose per gram (%ID/g).
4.8. Metabolism studies
Non-tumor-bearing mice were injected intravenously with
3.7 MBq of radiotracer [18F]16. Plasma and liver were collected at
the indicated time and were snap-frozen in liquid nitrogen for sub-
sequent HPLC analysis. For extraction, ice cold MeOH (1.5 mL) was
added to plasma. The mixture was centrifuged (15,493g, 4 °C,
3 min) and the resulting supernatant was evaporated to dryness
under vacuum at 40 °C using a rotary evaporator. Liver samples
were homogenized with ice cold MeOH (1.5 mL) using an IKA Ul-
tra-Turrax T-25 homogenizer prior to centrifugation. The superna-
tant was then decanted and evaporated to dryness. The samples
were re-suspended in HPLC mobile phase (1.2 mL) and filtered
4.6. Protein immunoblotting
The ability of the compounds to diffuse into cells and to inhibit
EGFR was assessed by measuring inhibition of receptor phosphor-
ylation by quinolines 1, 6, 12, 13, 15, 16, 20, and 21 in A431 human
epidermoid cancer cells (American Type Culture Collection,
Manassas, VA, USA). The cells were maintained in DMEM
(Sigma–Aldrich Company Ltd, Dorset, UK) supplemented with
10% fetal bovine serum (Lonza, UK), and 2 mM
100 U/mL penicillin, 100 g/mL streptomycin and 1
L
-glutamine,
through a Whatman PTFE syringe filter (0.2 lm). The samples
(1 mL) were analyzed by radio-HPLC on an Agilent 1100 series
l
lg/mL fungi-
zone (GIBCO) in six well plates incubated at 37 °C in a humidified
incubator with 5% CO2. The experiments were designed to assess
irreversibility of EGFR inhibition by the compounds. Cells in expo-
nential growth were incubated with quinolines 1, 6, 12, 13, 15, 16,
20, and 21 at various concentrations for 3 h. EGF (100 ng/ml) was
added to the cells during the last 15 min to induce p-EGFR. The
medium was removed and replaced with fresh compound-free
medium for 1 h. The last step was then repeated twice. The cells
were then washed with cold PBS and lysed in RIPA buffer (Invitro-
gen Ltd, Paisley, UK) supplemented with protease and phosphatase
inhibitor cocktails (Sigma–Aldrich Company Ltd, Dorset, UK). Ly-
sates were clarified by centrifugation. The following antibodies
were used: rabbit polyclonal antibody anti-p-EGFR (Cell signalling
Technology, Denver, MA; 1:1000) and rabbit polyclonal antibody
anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000)
and mouse monoclonal antibody anti-b-actin (Abcam, UK;
1:10000) as primary antibodies. The secondary antibodies were
Goat anti-Rabbit IgG HRP (Santa Cruz Biotechnology Santa Cruz,
CA; 1:2000) and Goat anti-Mouse IgG HRP (Autogen Bioclear, UK;
1:2000). The same procedure was used to assess EGFR and phos-
pho-EGFR expression in HCT116 human colon carcinoma cells.
HPLC system (Agilent Technologies, Stockport, UK) equipped with
a
c
-RAM model 3 gamma-detector (IN/US Systems Inc., Florida)
and the Laura 3 software. The stationary phase comprised of a
Waters
l
Bondapak C18 reverse-phase column (300 mm ꢁ 7.8 mm)
and the mobile phase comprised of water (0.085% H3PO4)/acetoni-
trile (0.085% H3PO4) (50:50) running in isocratic mode at a flowrate
of 3 mL/min.
4.9. Extraction efficiency for plasma
To pre-weighed counting tubes (n = 4) Dulbecco’s phosphate
buffered saline (Sigma, Gillingham, UK) (200 lL) was added and
to a further set of tubes (n = 4) mouse plasma extract (Mouse plas-
ma lithium heparin—CD-1- Mixed Gender, pooled, Sera Laborato-
ries International, West Sussex, UK) (200 lL) was added and the
samples stored on ice until radiopharmaceutical addition. Formu-
lated radiotracer [18F]16 was added to a set (n = 4) of blank count-
ing tubes and to the tubes containing PBS or plasma. The samples
were then incubated at 37 °C for 30 min and then snap-frozen
using dry ice. Immediately prior to extraction samples were
thawed on ice and ice cold methanol (1.5 mL) added. The samples
were then centrifuged (15,493g, 4 °C, 3 min). The supernatant was
then decanted and evaporated to dryness. The sample was then re-
suspended in HPLC mobile phase (1.1 mL) and filtered (Whatman
4.7. In vivo PET imaging and biodistribution
A431 and HCT116 xenografts were established by sc injection of
5 ꢁ 106 cells on the back of 6- to 8-week-old female nu/nu Balb/c
mice (Harlan). All animal work was performed by licensed investi-
gators in accordance with the United Kingdom’s ‘Guidance on the
Operation of Animals (Scientific Procedures) Act 1986’ (HMSO,
London, United Kingdom, 1990).42 When tumors reached
ꢀ100 mm3, animals (n = 3) were scanned on a dedicated small ani-
mal CT/PET scanner (Siemens Multimodality Inveon, Siemens
Molecular Imaging Inc., Knoxville, USA) following a bolus iv injec-
tion of 3.7 MBq of [18F]16. Dynamic emission scans were acquired
in list-mode format over 60 min. Cumulative images of the dy-
namic data (30–60 min) were iteratively reconstructed (OSEM3D)
and used for visualization of radiotracer uptake to define the re-
gions of interest (ROIs) with the Siemens Inveon Research Work-
place software (three-dimensional ROIs were defined for each
tumor). The count densities (counts/mL) were averaged for all ROIs
at each of the 19 time points to obtain a time versus radioactivity
curve (TAC). Tumor TACs were normalized to that total counts
PTFE 0.2 lm, 13 mm filters). Total radioactivity for each sample
(control: 100%, PBS extract: 95.4%, and plasma extract: 83.5%)
was then measured on a Cobra-II Gamma Counter.
Acknowledgments
This work was funded by Cancer Research UK programme Grant
C2536/A7602 and UK Medical Research Council core funding Grant
U.1200.02.005.00001.01. We thank Rozanna Slade and Elizabeth
Stevens for their contribution to the biology studies.
Supplementary data
Supplementary data (table of % injected dose per gram tissue of
compound [18F]16 and chromatograms and chromatography pur-
ity of tested compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2010.08.004.

F. Pisaneschi et al. / Bioorg. Med. Chem. 18 (2010) 6634–6645
6645
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