Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis, radiolabeling, and preliminary biological studies of fluoroethyl analogues of crizotinib, alectinib, and ceritinib

Article abstract of DOI:10.1016/j.ejmech.2019.111571

Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, is a therapeutic target in various cancers, including non-small cell lung cancer. Although several ALK inhibitors, including crizotinib, ceritinib, and alectinib, are approved for cancer treatment, their long-term benefit is often limited by the cancer's acquisition of resistance owing to secondary point mutations in ALK. Importantly, some ALK inhibitors cannot cross the blood-brain barrier (BBB) and thus have little or no efficacy against brain metastases. The introduction of a lipophilic moiety, such as a fluoroethyl group may improve the drug's BBB penetration. Herein, we report the synthesis of fluoroethyl analogues of crizotinib 1, alectinib 4, and ceritinib 9, and their radiolabeling with ¹⁸F for pharmacokinetic studies. The fluoroethyl derivatives and their radioactive analogues were obtained in good yields with high purity and good molar activity. A cytotoxicity screen in ALK-expressing H2228 lung cancer cells showed that the analogues had up to nanomolar potency and the addition of the fluorinated moiety had minimal impact overall on the potency of the original drugs. Positron emission tomography in healthy mice showed that the analogues had enhanced BBB penetration, suggesting that they have therapeutic potential against central nervous system metastases.

Full text of DOI:10.1016/j.ejmech.2019.111571

European Journal of Medicinal Chemistry 182 (2019) 111571
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis,
radiolabeling, and preliminary biological studies of fluoroethyl
analogues of crizotinib, alectinib, and ceritinib
Bhasker Radaram, Federica Pisaneschi, Yi Rao, Ping Yang, David Piwnica-Worms**,
Mian M. Alauddin*
Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, is a therapeutic target in
various cancers, including non-small cell lung cancer. Although several ALK inhibitors, including crizo-
tinib, ceritinib, and alectinib, are approved for cancer treatment, their long-term benefit is often limited
by the cancer's acquisition of resistance owing to secondary point mutations in ALK. Importantly, some
ALK inhibitors cannot cross the blood-brain barrier (BBB) and thus have little or no efficacy against brain
metastases. The introduction of a lipophilic moiety, such as a fluoroethyl group may improve the drug's
BBB penetration. Herein, we report the synthesis of fluoroethyl analogues of crizotinib 1, alectinib 4, and
ceritinib 9, and their radiolabeling with 18F for pharmacokinetic studies. The fluoroethyl derivatives and
their radioactive analogues were obtained in good yields with high purity and good molar activity. A
cytotoxicity screen in ALK-expressing H2228 lung cancer cells showed that the analogues had up to
nanomolar potency and the addition of the fluorinated moiety had minimal impact overall on the po-
tency of the original drugs. Positron emission tomography in healthy mice showed that the analogues
had enhanced BBB penetration, suggesting that they have therapeutic potential against central nervous
system metastases.
Received 12 March 2019
Received in revised form
26 July 2019
Accepted 28 July 2019
Available online 9 August 2019
Keywords:
Lung cancer
Anaplastic lymphoma kinase
Brain metastasis
Targeted therapeutics
PET
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
structure-based design and synthesis of crizotinib, a potent and
selective dual inhibitor of ALK and mesenchymal-epithelial tran-
Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase
and member of the insulin receptor superfamily, is a component of
the nucleophosmin (NPM1)-ALK fusion gene, which is expressed by
60% of anaplastic large-cell lymphomas. ALK is also part of the
sition factor that was approved by the FDA for the treatment of ALK-
mutant NSCLC in 2011 [6,7]. Although many patients have initial
responses to crizotinib, most tend to develop resistance to the drug
within 1 or 2 years [8e10], mainly because their disease acquires
additional ALK mutations that prevent crizotinib from binding to
ALK and inhibiting its activity [11,12]. Moreover, crizotinib has poor
activity against central nervous system (CNS) metastases due to its
inability to cross blood brain barrier (BBB) [13]. Compared with
crizotinib, the second-generation ALK inhibitor alectinib, initially
reported by Kinoshita et al. [14], has much higher potency (1.9 nM)
and has selectivity against wild-type ALK. Alectinib also has activity
against L1196M, one of the common ALK mutations that lead to
crizotinib resistance, and has efficacy against CNS metastases
[15,16]. Ceritinib, another second-generation ALK inhibitor that was
first reported by Marsilje et al. [17], elicits high responses in pa-
tients with crizotinib-resistant disease and was approved for the
treatment of relapsed or refractory NSCLC after crizotinib failure
echinoderm microtubule-associated protein-like
4 (EML4)-ALK
fusion gene, which occurs in 3e7% of non-small cell lung cancers
(NSCLCs) [1e3]. Thus, ALK is an attractive therapeutic target for
cancers that have ALK gene fusions or activating mutations of ALK
[4]. Accordingly, much work has been done to develop ALK-
inhibiting drugs. Cui et al. [5] were the first to report the
* Corresponding author. Department of Cancer Systems Imaging The University
of Texas MD Anderson Cancer Center 1515 Holcombe Blvd, Houston, TX, 77030,
USA.
** Corresponding author.
E-mail addresses: dpiwnica-worms@mdanderson.org (D. Piwnica-Worms),
alauddin@mdanderson.org (M.M. Alauddin).
https://doi.org/10.1016/j.ejmech.2019.111571
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
[18]. Another ALK inhibitor is lorlatinib (PF-06463922), a third-
generation ALK inhibitor recently approved by the FDA for the
treatment of NSCLC [19,20]. Other potent ALK inhibitors, including
X-396, ASP3026, AP26113, PF-06463922, CEP-37440, and TSR-011,
some of which have enhanced specificity for ALK, are currently in
phase I and II clinical trials [21e25]. The structures of several of
these ALK inhibitors are shown in Fig. 1.
the CNS, and thus help shape patient selection criteria for clinical
trials. In principle, PET could also be used to assess whether the
drug has engaged its target and/or to identify suitable drug dosing
regimens [30].
Tyrosine kinase inhibitors (TKIs) have been radiolabeled with
11C and 18F for use with PET [31]. Many FDA-approved TKIs, as well
as their derivatives, have been radiolabeled and tested preclinically
[32e34]. For the majority of these molecules, however, preclinical
findings have not triggered clinical translation. One exception is
Although crizotinib has high clinical efficacy against ALK fusion-
positive NSCLC, the brain is a frequent site of initial crizotinib failure
in NSCLC patients owing to the drug's poor penetration of the CNS.
On the other hand, [14C]labeled alectinib has been shown to have
modest BBB penetration in rodent models. A pharmacokinetic
study in rats showed that alectinib had a high brain-to-plasma
ratio, and an in vitro drug permeability study in Caco-2 colorectal
adenocarcinoma cells showed that alectinib was not transported by
the P-glycoprotein efflux transporter, a key factor in BBB function
[26]. Lorlatinib, which has moderate brain availability [27] and
broad-spectrum ALK inhibitory potency for the treatment of tu-
mors that progress despite crizotinib therapy, overcomes various
resistance mutations and has efficacy against brain metastases [28].
Ceritinib, another second generation ALK inhibitor, also suffers
from crossing BBB. In mice, only 0.4% of the drug was found in the
brain 24 h after its oral administration [29]. These findings suggest
that most of the ALK-inhibiting drugs have limited or poor BBB
penetration.
Despite considerable efforts, developing ALK inhibitors that can
effectively penetrate the BBB remains a challenge, and no diag-
nostic method for assessing molecule-specific pharmacodynamics
and target sensitivity to ALK inhibition in vivo has been reported.
The restricted repertoire of effective ALK inhibitors that can pene-
trate the BBB limits the targeted treatment of lung cancer brain
metastases, and the lack of effective markers and methods for non-
invasively monitoring these drugs’ early efficacy inhibits the se-
lection of optimal settings in which to test and monitor the bio-
logical and therapeutic efficacy of these novel compounds.
Therefore, there is need for development of an ALK inhibiting drug
that have sufficient BBB penetration for treatment of NSCLC brain
metastases. The addition of a fluoroethyl moiety to ALK inhibitors
could give the drugs a more lipophilic character and enhance their
brain penetration ability. Moreover, the replacement of fluorine (F)
with 18F would enable the use of these drugs as positron emission
tomography (PET) tracers to assess the relative levels and hetero-
geneity of ALK protein in tumors throughout the body, including
[
11C]erlotinib [35], which is an epidermal growth factor receptor
(EGFR) inhibitor used to treat patients with NSCLC and certain
types of pancreatic cancer [36,37]. A clinical trial of [11C]erlotinib in
NSCLC patients showed that accumulation of the tracer was
correlated with response to erlotinib therapy, especially in patients
harboring the EGFR-L858R activating mutation [38,39].
Few studies have investigated ALK inhibitor analogues for use
with PET [27,40]. We previously reported the synthesis of a ceritinib
analogue (fluoroethyl ceritinib; compound 9 in the present study)
labeled with 18F [41], and others have synthesized and tested [11C]
and [18F]labeled lorlatinib for brain penetration [27,40]. Analyses of
the structure-activity relationship and molecular modeling of cer-
itinib suggest that substitutions on the drug's piperidine nitrogen
are fairly well-tolerated without compromising the drug's efficacy
[42]; similarly, substitutions on the piperidine nitrogen in crizoti-
nib [5] and on the secondary amine nitrogen in alectinib are also
well-tolerated [43]. We hypothesized that crizotinib, alectinib, and
ceritinib, minimally modified with the attachment of a lipophilic
group and radioisotope, retain their ALK-specific efficacy, have
enhanced BBB penetration, and enable facile PET pharmacokinetic
analysis. Herein, we report the syntheses of fluorinated analogues
of crizotinib ([19F/18F]fluoroethyl crizotinib; [19/18F]1), alectinib
([19F/18F]fluoroethyl alectinib; [19/18F]4), and ceritinib ([19/18F]fluo-
roethyl ceritinib; [19/18F]9) and the preliminary in vitro and PET-
based validation of efficacy and brain permeability in vivo.
2. Results and discussion
2.1. Chemistry and radiochemistry
We used two methods to synthesize fluorinated analogues of
ALK inhibitors. Method 1 involved a reaction between the original
drug and fluoroethyl tosylate [44], and Method 2 involved intro-
ducing the fluorine atom in the last step by displacing a suitable
Fig. 1. Structures of several well-known ALK inhibitors.

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
3
leaving group of a preinstalled ethyl moiety in the original drug.
Both methods were suitable for producing [18F]1; however, only
Method 2 was suitable for producing [18F]4.
treatment of compound 2 with TsCl, compound 3 was obtained in
place of the expected tosylate analogue. This may have been due to
a further nucleophilic attack the chloride perpetrated on the in-
termediate tosylate either directly or in a two-step process, in
which the chloride attacks an aziridine ring formed by anchimeric
assistance by a lone pair of electrons in the nitrogen in the piperidyl
ring of crizotinib. The treatment of alcohols with tosyl chloride in
similar reaction conditions has been reported to yield chloroethyl
derivatives [41]. Ding et al. also reported that tosylate does not
always form when benzyl alcohols are treated with tosyl chloride
[45].
2.1.1. Synthesis of fluoroethyl crizotinib 1
Non-radioactive fluoroethyl crizotinib (compound 1) was pre-
pared using the two methods mentioned above. In Method 1, re-
action of fluoroethyl tosylate with crizotinib (Scheme 1, Method 1)
afforded compound 1 in 51% yield after chromatographic purifica-
tion in a single-step reaction. Compound 1 was characterized by
spectroscopic methods. 1H nuclear magnetic resonance (NMR)
spectroscopy of compound 1 showed two sets of additional peaks:
one at 4.55 ppm with a coupling constant of 47.8 Hz (J FeH,
geminal), and another at 2.96 ppm with a coupling constant of
28.4 Hz (J FeH, vicinal). 13C NMR spectroscopy showed two addi-
tional peaks, and 19F NMR spectroscopy showed one additional
peak at ꢀ217.1 ppm. High resolution mass spectrometry (HRMS)
showed the desired m/z [MþNa]þ as 518.1290, which was consis-
tent with the desired molecule.
In Method 2, reaction of the precursor chloroethyl crizotinib
(compound 3) with tetrabutylammonium fluoride (Bu4NF; Scheme
1, Method 2) afforded compound 1 in 70% yield. Hydroxyethyl
crizotinib (compound 2) was obtained in 74% yield by the reaction
of crizotinib with hydroxyethyl tosylate. 1H NMR spectroscopy of
compound 2 showed four additional protons, 13C NMR showed two
additional peaks, and the mass spectrum showed the desired m/z
[MþH]þ as 494.88. Reaction of compound 2 with p-toluenesulfonyl
chloride (TsCl) in dichloromethane (CH2Cl2) produced compound 3
in 39% yield. 1H NMR spectroscopy showed that compound 3 had
the same number of protons as compound 2 in the aliphatic region
without any additional aromatic peaks, 13C NMR spectroscopy
showed that compound 3 had the same number of carbons as
compound 2, and the HRMS m/z [MþH]þ was 512.1204. Upon
2.1.2. Radiosynthesis of [18F]1
Radiosynthesis of [18F]1 was also achieved by two different
methods. In Method 1 (Scheme 2, Method 1), the prosthetic group
[
18F]fluoroethyl tosylate was first produced with a mean decay-
corrected radiochemical yield of 68% (range, 65e73%; n ¼ 15).
Compound [18F]1 was produced by reaction of crizotinib with [18F]
fluoroethyl tosylate with a mean radiochemical yield of 54% (range,
45e65%; n ¼ 8). The average decay-corrected yield of [18F]1 from
aqueous [18F]fluoride was 24% (range, 20e28%; n ¼ 8). [18F]1 was
>99% radiochemically pure and had a mean molar activity of 24
GBq/mmol. The synthesis time was 130e135 min from end of
bombardment. High performance liquid chromatography (HPLC) of
radioactive [18F]1 co-injected with the non-radioactive standard
compound 1 showed co-elution of the radioactive and ultraviolet
(UV) peaks, confirming the identity and purity of the radiolabeled
tracer. (Supporting Information, Fig. S1).
Radiosynthesis of [18F]1 by Method 2 was a single-step process
(Scheme 2, Method 2), which produced [18F]1 from compound 3
with a 70% decay-corrected yield (n ¼ 3). Unfortunately, [18F]1
could not be easily separated from the starting material on the
semi-preparative HPLC column or by flash chromatography, which
Scheme 1. Synthesis of fluoroethyl crizotinib 1 by Methods 1 and 2.

4
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
Scheme 2. Radiosynthesis of [18F]fluoroethyl crizotinib ([18F]1) by Methods 1 and 2.
affected the compound's apparent molar activity and chemical
purity. Therefore, Method 1 was preferred when producing [18F]1
for biological studies.
(compound 8), formed by elimination, was obtained as the major
product.
2.1.4. Radiosynthesis of [18F]4
Direct coupling of [18F]fluoroethyl tosylate with alectinib in the
presence of K2CO3 (Method 1, Scheme 3) was unsuccessful. Radi-
osynthesis of [18F]4 could be performed only by Method 2 (Scheme
4). Starting from compound 7a, the mean decay-corrected (d. c.)
yield of [18F]4 was 21% (range, 16e26%; n ¼ 11). The radiochemical
yield of [18F]4 from compound 7b was 18% (d. c.; n ¼ 2). [18F]4 was
2.1.3. Synthesis of fluoroethyl alectinib 4
Fluoroethyl alectinib (compound 4) was also prepared using two
different methods (Scheme 3).
In Method 1, the reaction of alectinib with fluoroethyl tosylate in
the presence of potassium carbonate (K2CO3) produced compound
4 in 63% yield. The product was fully characterized by spectroscopic
methods, including 1H, 13C, and 19F NMR spectroscopy and HRMS.
1H NMR spectra were run in dimethyl sulfoxide (DMSO)-d6 and
deuterated chloroform (CDCl3); in DMSO‑d6, four additional peaks
and two sets of doublets were observed at 5.1 ppm (J ¼ 24 Hz) and
4.9 ppm (J ¼ 47 Hz), respectively. Interestingly, in CDCl3, one set of
protons was observed as a doublet of triplets, and the other peak
was a broad singlet. 13C NMR spectroscopy showed 30 peaks, and
19F NMR spectroscopy (decoupled) showed one peak
at ꢀ218.57 ppm. HRMS revealed the m/z [MþH]þ to be 529.2989,
indicating the desired compound.
In Method 2, the precursor methanesulfonylethyl alectinib
(compound 7a) was synthesized in multiple steps. Reaction of
alectinib with t-butyldimethylsilylethyl tosylate produced an alec-
tinib derivative (t-butyldimethylsilylethyl alectinib; compound 5)
in 61% yield. The product was characterized by 1H NMR spectros-
copy and mass spectrometry (MS). Hydroxyethyl alectinib (com-
pound 6) was obtained after acidic hydrolysis of compound 5 in 97%
(quantitative) yield. This compound was characterized by 1H NMR,
MS and HPLC; 13C NMR spectroscopy could not be performed
because the compound was sparingly soluble in common NMR
solvents. Reaction of compound 6 with methanesulfonyl chloride
produced compound 7a in 57% yield. Compound 7a was fully
characterized by spectroscopic methods, whose data were consis-
tent with the desired compound. Toluenesulfonylethyl alectinib
(compound 7b) was prepared using a process similar to that used to
prepare compound 7a and fully characterized by spectroscopic
methods. Reaction of compound 7a or 7b with Bu4NF in acetonitrile
(MeCN) at 103 ꢁC produced compound 4 in 10e12% yield. In this
reaction, the undesired vinyl derivative, n-vinyl alectinib
>99% pure and had a mean molar activity of 23 GBq/mmol. The
synthesis time was 90e95 min from end of bombardment. The
product could be purified by HPLC using a semi-preparative reverse
phase amide column. Semi-preparative and analytical HPLC of [18F]
4 co-injected with non-radioactive standard compound 4 is pre-
sented in Figs. S2 and S3 (Supporting Information).
2.1.5. Radiosynthesis of [18F]9
Compound [18F]9 was produced by coupling ceritinib with [18F]
fluoroethyl tosylate, as we reported previously [41]. The radio-
chemical yield, purity, and molar activity of [18F]9 were similar to
those we reported previously.
2.1.6. Radiotracer formulation
Because [18F]1, [18F]4, and [18F]9 were lipophilic and insoluble in
water or saline, they were dissolved in a small volume of ethanol
(0.1e0.2 mL) and then diluted with 10% Tween 80 and phosphate-
buffered saline (PBS; pH 7.4). The final formulated radiotracers
were filtered through a 0.22-mm Millipore filter before injection
into the animals.
2.1.7. LogP values
The LogP values (assessed by octanol/water partition) were 0.73
for [18F]1, 0.87 for [18F]4, and 0.97 for [18F]9. The LogDpH¼7.4 values
(assessed by octanol/PBS partition) were 1.43 for [18F]1,1.47 for [18F]
4, and 1.51 for [18F]9. Of note, the theoretical LogP values (calculated
with Molinspiration software) for [18F]1, [18F]4, and [18F]9 were
4.81, 6.01, and 6.94, respectively, and those of the parent com-
pounds crizotinib, alectinib, and ceritinib were 4.01, 5.28, and 5.91,

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
5
Scheme 3. Synthesis of fluoroethyl alectinib 4 by Methods 1 and 2.
Scheme 4. Radiosynthesis of [18F]fluoroethyl alectinib ([18F]4) by Method 2.
respectively. Differences in calculated versus experimental LogP
Western blotting (Fig. 2).
values have been previously reported [46,47], and experimental
LogP values of the parent drugs are not reported; therefore, the
lipophilicities of the parent compounds and the fluoroethyl de-
rivatives were compared using the calculated LogP values. The
higher LogP values of the fluoroethyl derivatives indicated that
these compounds have more lipophilicity than their parent com-
pounds; therefore, they are more likely to cross the intact BBB.
H2228 cells were treated with the three fluorinated ALK in-
hibitors, and efficacy was compared with that of their corre-
sponding parent drugs (Table 1, Fig. 3). Compound 1 had a half
maximal inhibitory concentration ([IC50]) of 7.5
mM, whereas cri-
zotinib had an IC50 of 2.8 M, which was less than 3-fold lower than
m
that of the parent drug. Compound 4, which had an IC50 of 10 nM
(versus an IC50 of 6.5 nM for alectinib), was the most potent de-
rivative but had lower efficacy. Nonetheless, its potency confirmed
compound 4 as a potential PET tracer. Compound 9 had an IC50 of
2.2. Biology
24.2
m
M, indicating that it had nearly 10-fold less potency than
M). Thus, the fluoroethyl moiety impacted the
ceritinib (IC50, 2.9
m
2.2.1. Cytotoxicity
potency of ceritinib, but did not have a detrimental effect on the
potency of crizotinib or alectinib. Both alectinib and compound 4
had no substantial effect on ALK-negative H441 cells.
We assessed the cytotoxicity of the fluorinated ALK inhibitors in
ALK-positive H2228 human lung cancer cells, which harbor EML4-
ALK variant 3 (86 kDa) [48], and in ALK-negative H441 lung cancer
cells. The cell lines’ differential expression of ALK was confirmed by

6
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
2.2.2. Live cell uptake of [18F]1, [18F]4, and [18F]9
The cell-associated radioactivity of [18F]1, [18F]4, and [18F]9 in
ALK-positive H2228 and ALK-negative H441 cells is shown in Fig. 4.
After 30 min of incubation, both [18F]1 and [18F]9 showed higher
uptake in the ALK-positive H2228 cells, as expected. In contrast,
[
18F]4 showed higher uptake in the ALK-negative H441 cells. For all
three analogues, nonspecific binding, measured as the radioactivity
retained by the ALK-negative cells, was relatively high. TKI-based
PET tracers tend to interact with cell membranes, potentially ac-
counting for the high background binding; they are also often
substrates for ATP-binding cassette (ABC) transporters, which could
account for the low uptake of [18F]4 in H2228 cells [49]. In fact, P-
glycoprotein-mediated ceritinib resistance in ALK-rearranged
NSCLC has been reported [50]. Although others have reported
that alectinib is not transported by the P-glycoprotein efflux
transporter, a key factor in BBB penetration [26], the three [18F]
labeled molecules all have theoretical LogP values greater than 3,
large surface areas, and molecular weights higher than 400, which
may render them susceptible to efflux by other members of the ABC
transporter family [51].
Fig. 2. Western blotting of ALK expression in lung cancer cells. Lane 1, molecular
weight markers; lane 2, H2228 cell lysate; lane 3, H441 cell lysate. GAPDH was probed
as a loading control.
Table 1
IC50 values of fluoroethyl derivatives and their parent compounds in H2228 cells.
Compound
IC50 value
Crizotinib
2.8
Compound 1
7.5
Alectinib
6.5 nM
Compound 4
10 nM
Ceritinib
2.9
Compound 9
24.2
m
M
m
M
m
M
mM
Fig. 3. Cytotoxicity of compounds 1 (A), 4 (B), and 9 (C) and their parent compounds in ALK-expressing H2228 lung cancer cells, and cytotoxicity of compound 4 and its parent
compound in non-ALK-expressing H441 lung cancer cells (D). Data are means standard deviations.

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
7
Fig. 4. Uptake of [18F]1 (A), [18F]4 (B), and [18F]9 (C) by ALK-positive H2228 cells and ALK-negative H441 cells. Data are means standard deviations.
2.2.3. Biodistribution of [18F]1, [18F]4, and [18F]9 in healthy mice
Non-tumor-bearing healthy mice were injected with [18F]1, [18F]
4, or [18F]9, and the biodistribution of each of the three compounds
was assessed by PET and region of interest analysis. The mean time-
activity curves of [18F]1, [18F]4, and [18F]9 demonstrated that the
tracers were quickly cleared from the heart, accumulated in both
the kidney and liver, and then cleared rapidly from the urinary track
(Fig. 5). The tracers steadily accumulated in the brain from 0.5 to
6.0 min, with a peak of 6e8 %ID/cc, followed by a wash out phase
from the brain. Within 30 min, most of the activity was cleared
from the brain. Most of the radioactivity accumulated in the liver
and was slowly excreted into the bowel with significant tracer
accumulation in the gallbladder. Tracer accumulation in muscle
was low, being somewhat lower than retention in blood.
PET images revealed the whole-body distributions of [18F]1, [18F]
Fig. 6. Maximum intensity projection PET images show the biodistribution of [18F]1 in
a representative animal at 5, 10, 30, and 60 min after injection. B ¼ brain, K ¼ kidney,
H ¼ heart, and L ¼ liver.
4, and [18F]9 (Figs. 6e8, respectively), which complemented the
compounds’ time-activity curves; there was early accumulation of
the tracers in the brain and washout within 30 min. One hour after
injection, most of the activity was cleared from the heart (blood),
and activity remained in the liver, gallbladder, and intestine. The
tracers cleared quickly from the kidneys and accumulated in the
bladder.
compounds quickly washed out of normal brain, except [18F]9,
which was retained at a %ID/cc of about 4 at 1 h. Compounds [18F]1
and [18F]4 cleared the brain rapidly (within 30 min), which may
reflect a susceptibility of these compounds to be substrates for the
ABC transporter family. Nonetheless, initial accumulation of activ-
ity in the normal brain suggests that these compounds, especially
compound l, have an advantage for therapeutic application in CNS
metastasis. For example, therapeutic potential of 1 may exceed the
parent compound, crizotinib, which has relatively poor penetration
of the BBB [32]. Our results are in line with the findings of Collier
et al. [27,40], who reported brain penetration of [11C]lorlatinib in a
rhesus macaque. They reported an activity peak in the cerebellum
at 10 min, with a %ID/g value of approximately 0.020 and a stan-
dardized uptake value almost equal to 2, and a rapid washout
Region of interest analysis confirmed a substantial level of tracer
accumulation in the brain at 10 min, with [18F]4 having the greatest
accumulation (Table 2).
The fluoroethyl group was incorporated into the parent drugs to
increase their lipophilic behavior. As the biodistribution assess-
ments with PET and region of interest analysis show, lipophilicity
also increases their brain permeability, as all three newly synthe-
sized compounds crossed the BBB. Based on the vascular volume of
the brain (1/30 by gram weight whole brain) [52], the 8.1% ID/cc of
[
18F]4 5 min after injection exceeded by 40-fold the value expected
for a tracer confined to the vascular compartment (0.2 %ID/g). The
Fig. 5. Mean time-activity curves and distributions of [18F]1 (A), [18F]4 (B), and [18F]9 (C) in major organs, including brain, heart, liver, kidney, and muscle, in normal nude mice. Data
are means standard deviations.

8
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
high non-specific binding to biological tissues, a trait similar to
those of other compounds of this general class. Compared with
their parent compounds, which already have proven anticancer
efficacy, these analogues may have a better therapeutic effect
against brain metastases owing to their enhanced CNS pharmaco-
kinetic properties.
4. Materials and methods
4.1. Reagents and instrumentation
Reagents and solvents were purchased from ThermoFisher Sci-
entific (Waltham, MA) and Sigma-Aldrich (St. Louis, MO) and used
without further purification. Crizotinib, alectinib, and ceritinib with
greater than 95% purity were purchased from Pure Chemistry Sci-
entific, Inc. (Sugar Land, TX). Silica gel solid-phase extraction car-
tridges (Sep-Pak, 900 mg) and C18 reverse-phase extraction
cartridges (Sep-Pak, 900 mg) were purchased from Alltech Associ-
ates (Deerfield, IL). K[18F]F/Kryptofix 2.2.2 as an aqueous solution
was purchased from Cyclotope, Inc. (Houston, TX) or from MD
Anderson's Center for Advanced Biomedical Imaging (Houston, TX).
Radioactivity was measured using a dose calibrator (model # CRC-
15R, Capintec, Inc., Florham Park, NJ), and tracer activity was
measured using a gamma counter (model # E500300; PerkinElmer,
Waltham, MA).
Fig. 7. Maximum intensity projection PET images show the biodistribution of [18F]4 of
in a representative animal at 5, 10, 30, and 60 min after injection. B ¼ brain, K ¼ kidney,
H ¼ heart, and L ¼ liver.
Thin-layer chromatography (TLC) was performed on pre-coated
Kieselgel 60 F254 glass plates (Merck, Darmstadt, Germany) and
aluminum-backed, pre-coated silica gel plates (Sorbent Technolo-
gies, Inc., Norcross, GA). 1H, 13C, and 19F NMR spectroscopy was
performed at MD Anderson Cancer Center on a 300-, 500-, or 600-
MHz spectrometer (Bruker, Billerica, MA) using tetramethylsilane
as an internal reference or hexafluorobenzene as an external
reference. HRMS was performed at the University of Minnesota on a
Bruker BioTOF II mass spectrometer using electrospray ionization.
Low-resolution MS was performed at MD Anderson Cancer Center
on a liquid chromatography mass spectrometer with an Acquity TQ
detector (Waters Corp., Milford, MA) using electrospray ionization.
HPLC was performed on 1100 Series pumps (Agilent Technolo-
gies, Stuttgart, Germany) with built-in ultraviolet detectors and a
radioactivity detector with a single-channel analyzer (Bioscan,
Washington, DC). Purification of [18F]fluoroethyl tosylate was per-
Fig. 8. Maximum intensity projection PET images show the biodistribution of [18F]9 in
a representative animal at 5, 10, 30 and 60 min after injection. B ¼ brain, K ¼ kidney,
H ¼ heart, and L ¼ liver.
during the subsequent hour. In contrast, the %ID/cc values of our
three novel analogues in mice were almost equal to 7. Similar to the
present study, the study by Collier et al. in H3122 tumor-bearing
mice showed the expected hepatobiliary clearance. The modest
degree of blockable tumor uptake in vivo implied a significant
component of non-specific binding [40].
formed on a reverse-phase C18 semi-prep column (Alltima C18; 10 m,
10 ꢂ 250 mm; Alltech/Grace, Chicago, IL) using a solution of 50%
3. Conclusion
MeCN in water. Purification of [18F]4 was performed on a reverse-
phase semi-prep amide column (Ascentis-RP-Amide; 10 mm,
We synthesized and radiolabeled novel analogues of crizotinib,
alectinib, and ceritinib, ([19/18F]fluoroethyl crizotinib, [19/18F]fluo-
roethyl alectinib, and [19/18F]fluoroethyl ceritinib, respectively).
This series of ALK inhibitors was obtained in good yields with high
purity and good molar activity. The radiolabeled analogues have
potential as PET tracers for quantitative studies in the context of
brain metastasis, but this potential is mitigated by these analogues’
25 cm ꢂ 10 mm; Supelco, Sigma-Aldrich, St. Louis, MO) using a
solution of 38% MeCN in water with 0.1% trifluoroacetic acid (TFA).
Quality control analyses of [18F]1 and [18F]9 were performed on an
analytical C18 column (Econosil C18; 4.6 ꢂ 250 mm; Alltech/Grace)
in solvent systems comprising 60% MeCN/40% water/0.1% TFA; [18F]
4 was analyzed with 40% MeCN/60% water/0.1% TFA on analytical
HPLC.
Table 2
Biodistribution of [18F]1, [18F]4, and [18F]9 in normal nude mice (n ¼ 3 per group) at various times. Data are mean %ID/cc values standard deviations.
Organ
Compound
18F]1
[
[
18F]4
[
18F]9
5 min
10 min
30 min
60 min
5 min
10 min
30 min
60 min
5 min
10 min
30 min
60 min
Brain
Heart
Liver
Kidney
Muscle
6.6 2.6
5.2 1.0
6.8 0.8
16.9 5.5
2.0 0.6
6.5 2.8
3.6 0.8
7.1 1.0
14.2 4.7
2.3 0.8
2.6 0.9
1.9 0.6
4.9 1.9
3.2 0.9
1.6 0.7
1.8 0.6
1.6 0.4
3.7 1.2
2.2 0.7
1.2 0.5
8.1 2.6
6.1 1.3
12.7 4.4
20.7 6.8
2.4 1.2
8.0 2.9
4.2 1.1
14.8 4.9
17.8 7.6
2.5 1.2
2.2 0.1
1.9 0.3
9.6 0.7
4.3 0.4
1.7 0.4
1.5 0.4
2.0 0.7
10.2 5.1
4.9 1.7
2.0 0.6
6.6 1.8
9.1 2.9
16.8 2.8
20.2 7.9
3.6 1.1
6.5 1.6
7.0 2.6
17.9 6.4
18.2 6.6
3.9 1.1
4.6 0.1
3.1 0.3
9.5 1.7
7.2 1.1
2.4 0.3
4.0 0.2
2.6 0.1
6.4 0.5
5.7 0.2
2.1 0.3

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
9
4.2. Chemistry and radiochemistry
for C23H25Cl2F2N5ONa, 518.1302; found, 518.1290.
Method 2: Compound
3
(synthesized from crizotinib as
4.2.1. Ethylene glycol monotosylate and ethylene glycol ditosylate
Ethylene glycol monotosylate and ethylene glycol ditosylate
were prepared using a procedure similar to one described previ-
ously [53]. Briefly, ethylene glycol (3.5 mL, 62.9 mmol, 8.0 eq.),
triethylamine (3.3 mL, 23.6 mmol, 3.0 eq.), and dicholor-
omethathane (CH2Cl2, 12 mL) were added to a flask and stirred at
0 ꢁC for 20 min. A solution of TsCl (1.5 g, 7.9 mmol, 1.0 eq.) in CH2Cl2
(13 mL) was added slowly using a syringe over 1 h. The resulting
mixture was warmed to room temperature and stirred for 24 h. TLC
indicated complete consumption of TsCl. The reaction mixture was
washed with water (2 ꢂ 10 mL), and the aqueous layer was
extracted with CH2Cl2. Organic layers were combined and evapo-
rated. The products were purified by flash column chromatography
on silica gel using 30% ethyl acetate (EtOAc)/hexanes (Hex) to yield
ethylene glycol monotosylate as a colorless oil (970 mg, 57% yield)
with >95% purity and ethylene glycol ditosylate as a white crys-
talline solid (490 mg, 17% yield) with >96% purity. Ethylene glycol
monotosylate: Rf ¼ 0.3 in 30% EtOAc/Hex; 1H NMR (CDCl3,
described in sections 4.2.4 and 4.2.5; 3 mg, 0.0058 mmol, 1.0 eq.)
was dissolved in dry MeCN (400 L) in a v-vial, and Bu4NF (15 L,
m
m
0.1 M in THF, 0.0064 mmol, 1.1 eq.) was added. The resulting
mixture was heated at 105e107 ꢁC for 1 h. The mixture was
concentrated under a stream of air, and then the crude product was
analyzed by analytical HPLC, which indicated 70% conversion to
fluoroethyl crizotinib (compound 1). The product was confirmed by
co-injecting it with the reference authentic sample of compound 1;
both the product and the authentic sample had the same retention
time.
4.2.4. 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-
(hydroxyethylpiperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine
(hydroxyethyl crizotinib; compound 2)
3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-
4-yl)-1H-pyrazol-4-yl)pyridin-2-amine
(crizotinib;
50 mg,
0.11 mmol) was dissolved in dry MeCN (2.0 mL), and then trie-
thylamine (0.6 mL, excess) was added. To this mixture, ethyl-
eneglycol monotosylate (48 mg, 0.2 mmol, 2.0 eq.) was added, and
the mixture was refluxed at 95 ꢁC for 5 h. The reaction mixture was
concentrated under vacuum, and the crude product was purified by
flash chromatography on silica gel, eluted with 100% EtOAc to
remove unreacted ethylene glycol monotosylate, and then eluted
with 10% MeOH/CH2Cl2. After solvent evaporation, hydroxyethyl
crizotinib (compound 2) was obtained as an off-white solid (40 mg,
74% yield) with 95% purity. 2: Rf ¼ 0.4 in 10% MeOH/CH2Cl2; 1H
500 MHz)
d: 7.81 (d, J ¼ 7.1, 2H), 7.36 (d, J ¼ 7.1 Hz, 2H), 4.14 (q,
J ¼ 2.3 Hz, 2H), 3.82 (t, J ¼ 1.9 Hz, 2H), 2.45 (s, 3H). Ethylene glycol
ditosylate: Rf ¼ 0.5 in 30% EtOAc/Hex; 1H NMR (CDCl3, 500 MHz)
d:
7.73 (d, J ¼ 8.1 Hz, 2H), 7.34 (d, J ¼ 7.7 Hz, 2H), 4.18 (s, 2H), 2.46 (s,
3H).
4.2.2. Fluoroethyl tosylate
Fluoroethyl tosylate was prepared as described previously [41].
Briefly, ethylene glycol ditosylate (100 mg, 1 eq.) was dissolved in
dry MeCN (4 mL); then, a solution of Bu4NF in tetrahydrofuran (THF,
NMR (CDCl3, 500 MHz), d: 7.76 (s, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.30
(m, 1H), 7.05 (t, J ¼ 8.0 Hz, 1H), 6.87 (s, 1H), 6.07 (q, J ¼ 6.6 Hz, 1H),
4.79 (s, 2H, NH2), 4.14 (m, J ¼ 4.3 Hz,1H), 3.65 (t, J ¼ 4.8 Hz, 2H), 3.07
(d, J ¼ 10.9 Hz, 2H), 2.60 (t, J ¼ 4.8 Hz, 2H), 2.28 (t, J ¼ 11.7 Hz, 2H),
2.17 (d, J ¼ 11.4 Hz, 2H), 2.06 (q, J ¼ 11.9 Hz, 2H), 1.85 (d, J ¼ 6.5 Hz,
300 mL, 1 M, 1.1 eq.) was added, and the reaction mixture was
heated at 95 ꢁC for 1 h. The crude reaction mixture was concen-
trated, purified by flash column chromatography, and eluted with
20% EtOAc/Hex. After evaporation of the solvent, 35 mg of fluo-
roethyl tosylate was obtained as a colorless liquid in a 60% yield. 1H
3H). 13C NMR decoupled (CDCl3, 125 MHz),
d: 148.89,139.85,136.95,
135.72, 135.51, 128.96, 128.93, 122.58, 122.13, 121.93, 119.93, 119.21,
116.81, 116.63, 114.96, 72.42, 59.32, 59.07, 58.01, 52.32, 32.54, 18.90.
MS: m/z [MþH]þ 494.88.
NMR (CDCl3, 600 MHz)
d
: 7.81 (d, J ¼ 8.3 Hz, 2H), 7.36 (d, J ¼ 8.3 Hz,
2H), 4.57 (dt, J ¼ 47.1 Hz & 4.1 Hz, 2H), 4.26 (dt, J ¼ 27.1 Hz & 4.1 Hz,
2H), 2.46 (s, 3H). 19F NMR decoupled (CDCl3, 300 MHz)
(s). MS: m/z [MþH]þ 219.03.
d: ꢀ224.65
4.2.5. 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-
(chloroethylpiperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine
(chloroethyl crizotinib; compound 3)
4.2.3. 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-
(fluoroethylpiperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine
(fluoroethyl crizotinib; compound 1)
Compound 2 (40 mg, 0.0811 mmol, 1.0 eq.) was dissolved in dry
CH2Cl2 (5.0 mL) and cooled to 0 ꢁC under argon, then triethylamine
Method 1: 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-
(160 mL, 0.648 mmol, 8.0 eq) was added. The solution was stirred for
(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine
22.5 mg, 0.054 mmol) was dissolved in dry MeCN (2.0 mL) in a v-
vial, and triethylamine (300 L) was added. To this mixture, fluo-
(crizotinib;
10 min, and then a solution of TsCl (62 mg, 0.324 mmol, 4.0 eq.) in
dry CH2Cl2 (1.0 mL) was added dropwise over 15 min. This mixture
was allowed to slowly warm to room temperature over 3 h, and
then it was stirred continuously for another 35 h. The crude reac-
tion mixture was concentrated under vacuum, re-dissolved in
CH2Cl2 (5 mL), and washed with an aqueous sodium bicarbonate
(NaHCO3) solution (0.1 M, 3 ꢂ 3 mL). The organic layer was isolated,
dried under magnesium sulfate (MgSO4), concentrated, and puri-
fied by chromatography on silica gel and eluted with 4% MeOH/
CH2Cl2. After solvent evaporation, chloroethyl crizotinib (com-
pound 3) was obtained as a semisolid (16 mg, 39% yield) with 97%
purity. 3: Rf ¼ 0.5 in 10% MeOH/CH2Cl2; 1H NMR (CDCl3, 600 MHz),
m
roethyl tosylate (12 mg, 0.06 mmol, 1.1 eq.) was added, and the
reaction mixture was heated for 5.5 h at 95e97 ꢁC. TLC showed a
higher Rf spot in about 75% intensity. The mixture was concen-
trated under a stream of air, and then the crude product was pu-
rified by flash chromatography on a silica gel column and eluted
with 5% methanol (MeOH)/CH2Cl2 to obtain fluoroethyl crizotinib
(compound 1) (14.6 mg, 51% yield) with 99% purity as assessed by
analytical HPLC. 1: Rf ¼ 0.5 in 5% MeOH/CH2Cl2; 1H NMR (DMSO‑d6,
300 MHz)
d
: 7.96 (s, 1H), 7.76 (d, J ¼ 1.8 Hz, 1H), 7.55e7.60 (m, 1H),
7.53 (s, 1H), 7.45 (t, J ¼ 8.3 Hz, 1H), 6.90 (d, J ¼ 1.6 Hz, 1H), 6.09 (q,
J ¼ 6.6 Hz, 1H), 5.65 (s, 2H, NH2), 4.55 (dt, J ¼ 47.8 Hz & 4.9 Hz, 2H),
4.05e4.16 (m, 1H), 3.16 (s, 1H), 2.96 (s, 1H), 2.66 (dt, J ¼ 28.4 Hz &
4.9 Hz, 2H), 2.20 (dt, J ¼ 11.4 and 2.6 Hz, 2H), 1.90e2.01 (m, 4H), 1.81
d
: 7.68 (s, 1H), 7.54 (s, 1H), 7.48 (s, 1H), 7.31 (m, J ¼ 4.8 Hz, 1H), 7.05
(t, J ¼ 7.92 Hz, 1H), 6.87 (s, 1H), 6.07 (q, J ¼ 6.4 Hz, 1H), 5.26 (s, 2H,
NH2), 4.12 (t, J ¼ 11.6 Hz,1H), 3.61 (t, J ¼ 6.8 Hz, 2H, CleCH2), 3.06 (d,
J ¼ 11.1 Hz, 2H), 2.79 (t, J ¼ 6.8 Hz, 2H, NeCH2), 2.30 (t, J ¼ 11.7 Hz,
2H), 2.16 (d, J ¼ 12.2 Hz, 2H), 2.06 (m, 2H), 1.85 (d, J ¼ 6.5 Hz, 3H).
(d, J ¼ 6.6 Hz, 3H). 13C NMR decoupled (DMSO‑d6, 150 MHz)
d:
149.40,138.75,136.80,135.43,134.46,128.72,123.42,121.08,120.95,
119.09, 117.49, 117.38, 117.33, 114.38, 95.38, 82.49, 81.40, 71.91,
58.32, 57.35, 52.17, 32.02, 18.57. 19F NMR, decoupled (DMSO‑d6,
13C NMR decoupled (CDCl3, 125 MHz)
d: 175.13, 148.84, 140.02,
136.75, 135.67, 134.07, 128.96, 122.47, 119.70, 119.05, 116.89, 116.70,
115.23, 72.57, 59.49, 52.46, 41.00, 32.25, 18.87. HRMS: m/z [MþH]þ
calculated for C23H26Cl3FN5O, 512.1187; found, 512.1204.
300 MHz)
d
: ꢀ113.2 (s), ꢀ217.1 (s). HRMS: m/z [MþNa]þ calculated

10
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
4.2.6. [18F]fluoroethyl tosylate
higher Rf spot (0.6 in 5% MeOH/CH2Cl2) at about 65% intensity as
[
18F]Fluoroethyl tosylate was prepared as described previously
compared with the starting material (0.5 in 5% MeOH/CH2Cl2). The
mixture was filtered and concentrated under a stream of air, and
then the crude product was purified by flash chromatography on
silica gel and eluted with 3% MeOH/CH2Cl2 to obtain fluoroethyl
alectinib (compound 4; 10 mg, 63% yield) with 99% purity as
assessed by analytical HPLC. 4: Rf ¼ 0.6 in 5% MeOH/CH2Cl2;1H NMR
[41]. Briefly, aqueous K[18F]F/kryptofix was dried under a stream of
argon at 103e105 ꢁC, a solution of ethylene glycol ditosylate
(5e6 mg in 0.5 mL dry MeCN) was added to the dry [18F]fluoride,
and the reaction mixture was heated at 103 ꢁC for 15 min. The crude
reaction mixture was passed through a silica gel Sep-Pak cartridge,
eluted with CH2Cl2 (2 mL), and evaporated under a stream of argon.
The crude product was purified by HPLC on a semi-preparative C18
(DMSO‑d6, 600 MHz)
d
: 8.49 (d, J ¼ 8.0, 1H), 8.29, (s, 1H), 8.01 (s,
1H), 7.64 (d, J ¼ 8.0, 1H), 7.34 (s, 1H), 5.05, (d, JHF ¼ 24.1 Hz, 2H), 4.92
(d, JHF ¼ 47.1 Hz, 2H), 3.61 (s, 5H), 3.23 (d, J ¼ 11.6 Hz, 2H), 2.79 (t,
J ¼ 11.5 Hz, 2H), 2.73 (q, J ¼ 7.1 Hz, 2H), 2.33 (quintet, 1H), 2.49, 2H,
1.93 (d, J ¼ 10.9, 2H), 1.85 (s, 6H), 1.75 (s, 1H), 1.61 (q, J ¼ 10.4 Hz,
column (Alltima C18; 10
m
, 10 ꢂ 250 mm; Alltech/Grace). The
radioactive product [18F]fluoroethyl tosylate was eluted with a
mixture of 50% MeCN in water at a flow of 4 mL/min. The radio-
active fraction was collected, and an aliquot of the product [18F]
2H), 1.29 (t, J ¼ 7.2H, 3H). 1H NMR (CDCl3, 300 MHz)
d: 8.75 (d,
fluoroethyl tosylate was co-injected with
a
non-radioactive
J ¼ 8.2 Hz, 1H), 8.22 (s, 1H), 7.74 (s, 1H), 7.60 (d, J ¼ 6.9 Hz, 1H), 7.16
(s, 1H), 4.93 (dt, J ¼ 24.6 Hz, 4.6 Hz, 2H), 4.82 (bs, 2H), 3.77 (t,
J ¼ 4.4 Hz, 4H), 3.31 (d, J ¼ 10.7 Hz, 2H), 2.75 (m, 4H), 2.63 (t,
J ¼ 4.6 Hz, 4H), 2.36 (m, 1H), 2.01 (d, J ¼ 11.3 Hz, 2H), 1.87 (s, 6H),
1.73 (m, 2H), 1.33 (t, J ¼ 7.5 Hz, 3H). 13C NMR decoupled (CDCl3,
authentic sample of fluoroethyl tosylate into an analytical HPLC
column to verify the product's identity and purity. The solvent from
the rest of the product was partially evaporated under reduced
pressure, and then water (10 mL) was added. The solution was
passed through a C18 reverse-phase cartridge; the cartridge was
dried by vacuum for 5 min and then flashed with argon for 2 min.
The product was eluted with 1.3 mL of dry MeCN and collected into
a v-vial containing crizotinib (5.5 mg).
150 MHz) d: 180.61, 156.85, 156.15, 147.71, 137.71, 136.89, 128.37,
126.76, 125.89, 125.73, 123.43, 120.04, 115.92, 114.45, 114.44, 111.97,
106.44, 81.92, 80.76, 67.33, 61.98, 52.18, 49.99, 45.94, 45.79, 37.59,
29.53, 28.96, 23.20, 14.39. 19F NMR, decoupled (CDCl3, 300 MHz)
d
: ꢀ218.57 (s). HRMS: m/z [MþH]þ calculated for C32H38FN4O2,
4.2.7. [18F]fluoroethyl crizotinib ([18F]1)
529.2979; found, 529.2989.
Method 1: The reaction mixture of crizotinib and [18F]fluo-
roethyl tosylate in the v-vial described in section 4.2.6 was heated
at 103 ꢁC for 1 h, and then an aliquot of the reaction mixture was
injected into the analytical HPLC system, which showed major
consumption of the radioactive [18F]fluoroethyl tosylate. The sol-
vent was evaporated completely under a stream of argon, and the
crude product was purified on a Sep-Pak silica gel cartridge and
eluted with 4% MeOH/CH2Cl2. Fractions (0.5 mL) were collected, the
radioactive fractions were combined, and radioactivity was
measured using a dose calibrator (Capintec) to obtain [18F]1. An
aliquot of the product, [18F]1, was dissolved in MeCN and then co-
injected with the standard compound 1 into an analytical HPLC
column to verify the product's identity and purity. The rest of the
product was evaporated and formulated for further biological
study.
Method 2: Compound 7a or 7b (prepared from alectinib as
described in sections 4.2.10 to 4.2.13; 2.4 mg, 0.004 mmol, 1 eq.) was
dissolved in dry MeCN (0.4 mL); then a solution of Bu4NF in THF
(12 mL, 1M, 0.006 mmol, 1.5 eq.) was added, and the reaction
mixture was heated at 100 ꢁC for 30 min. Analytical HPLC of an
aliquot of the reaction mixture indicated formation of the desired
compound as a minor peak and a new product as a major peak. The
crude reaction mixture was concentrated, purified by column
chromatography, and eluted with 2% MeOH/CH2Cl2 to afford fluo-
roethyl alectinib (compound 4) in 11% yield (Rf ¼ 0.6 in 3% MeOH/
CH2Cl2) with 99% purity as assessed by analytical HPLC. Another off-
white solid was obtained and identified as the elimination product
9-ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-11-
oxo-6,11-dihydro-5-ethenyl-benzo [b]carbazole-3-carbonitrile (n-
vinyl alectinib; compound 8; Rf 0.7 in 3% MeOH/CH2Cl2) in 80%
yield with 97% purity as assessed by analytical HPLC. 8: 1H NMR
Method 2: Compound 3 (3 mg) was placed in a v-vial, and a
solution of dry K[18F]F/kryptofix 2.2.2 in anhydrous MeCN (0.5 mL)
was added. The reaction mixture was heated at 103 ꢁC for 20 min;
analysis of an aliquot of the mixture by HPLC showed high intensity
of the product with insignificant [18F]fluoride remaining. The crude
reaction mixture was passed through a silica gel Sep-Pak cartridge
and eluted with 5% MeOH/CH2Cl2 (2 mL). The solvent from the
eluent was evaporated, and the product was dissolved in CH2Cl2
and loaded onto a silica Sep-Pak cartridge. The product was eluted
with 3% MeOH/CH2Cl2, and 0.5-mL fractions were collected. The
radioactive fractions were combined, and the solvent was evapo-
rated at 40 ꢁC under a stream of argon. An aliquot of the product,
(CDCl3, 600 MHz)
d
: 8.65 (d, J ¼ 8.1 Hz, 1H), 8.23 (s, 1H), 7.87 (s, 1H),
7.59 (d, J ¼ 8.1 Hz, 1H), 7.27 (dd, J ¼ 8.0 Hz, 1H), 7.15 (s, 1H), 5.85 (d,
J ¼ 8.0 Hz, 1H), 5.72 (d, J ¼ 15.48 Hz, 1H), 3.77 (t, J ¼ 4.4 Hz, 4H), 3.31
(d, J ¼ 12.0 Hz, 2H), 2.79e2.72 (m, 4H), 2.63 (t, J ¼ 4.4 Hz, 4H), 2.35
(m, 1H), 2.01 (d, J ¼ 11.7 Hz, 2H), 1.85 (s, 6H), 1.74 (qd, J ¼ 11.8 Hz,
2H), 1.34 (t, J ¼ 7.5 Hz, 3H). 13C NMR decoupled (CDCl3, 75 MHz)
d:
180.90, 156.57, 156.12, 147.54, 137.58, 136.72, 130.91, 128.31, 126.80,
125.96, 125.88, 123.15, 120.11, 117.41, 115.86, 115.59, 111.90, 106.53,
67.33, 61.97, 52.15, 49.98, 37.75, 28.95, 28.50, 23.21, 14.14. MS: m/z
[MþH]þ calculated for C32H37N4O5, 509.2811; found, 509.2819.
Similar results were obtained when compound 7b was reacted with
Bu4NF.
[
18F]1, was dissolved in MeCN and co-injected with the nonradio-
active authentic sample of compound 1 into an analytical HPLC
column to verify the product's identity and purity.
4.2.9. t-Butyldimethylsilylethyl tosylate
t-Butyldimethylsilylethyl tosylate was prepared as described
previously [54] with modification. Briefly, hydroxyethyl tosylate
(200 mg, 0.924 mmol, 1.0 eq.) and t-butyldimethylsilyl chloride
(418 mg, 2.774 mmol, 3.0 eq.) were dissolved in CH2Cl2 (8 mL).
Then, triethylamine (0.64 mL, 4.624 mmol, 5.0 eq.) was added at
0 ꢁC, and the resulting mixture was stirred for 10 min. The mixture
was then warmed to room temperature and stirred for 23 h. The
reaction mixture was washed with water (2 ꢂ 5 mL), and the
aqueous layer was extracted with CH2Cl2. Combined organic layers
were dried over MgSO4, filtered, and evaporated. The crude product
was purified by flash column chromatography on silica gel using
4.2.8. 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-
11-oxo-6,11-dihydro-5(2-fluoroethyl)-benzo[b]carbazole-3-
carbonitrile (fluoroethyl alectinib; compound 4)
Method 1: 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piper-
idin-1-yl]-11-oxo-6,11-dihydro-5H-benzo
[b]carbazole-3-
carbonitrile (alectinib; 14.5 mg, 0.03 mmol, 1.0 eq.) was dissolved
in dry MeCN (2.0 mL) in a v-vial, and anhydrous K2CO3 (29 mg,
0.21 mmol, 7 eq.) was added. To this mixture, fluoroethyl tosylate
(21.8 mg, 0.1 mmol, 3.3 eq.) was added, and the reaction mixture
was heated at 105 ꢁC for 5.5 h. TLC showed that the mixture had a

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
11
12% EtOAc/Hex to obtain t-butyldimethylsilylethyl tosylate as a pale
yellow oil (243 mg, 79% yield) with 95% purity. The compound was
fully characterized by 1H NMR spectroscopy and MS. Rf ¼ 0.9 in 50%
a pale-yellow suspension. The suspended material was cooled to
0 ꢁC, and methane sulfonyl chloride (38
L, 0.5 mmol, 6.0 eq.) was
m
added. The reaction mixture was warmed to room temperature and
stirred for 3 h. Reaction progress was monitored by TLC, which
showed a higher Rf (0.6 in 5% MeOH/CH2Cl2) as compared with the
starting material (0.3 in 5% MeOH/CH2Cl2). The reaction mixture
was diluted with 5 mL of CH2Cl2 and washed with saturated
NaHCO3, and the organic layer was dried over anhydrous MgSO4,
filtered, and evaporated. The crude product was purified by flash
column chromatography on silica gel and eluted with 2% MeOH/
CH2Cl2 to yield methanesulfonyl ethyl alectinib (compound 7a) as a
pale yellow solid (27 mg, 57% yield) with 98% purity as assessed by
analytical HPLC. 7a: Rf ¼ 0.6 in 5% MeOH/CH2Cl2; 1H NMR (CDCl3,
EtOAc/Hex; 1H NMR (CDCl3, 600 MHz)
d: 7.79 (d, J ¼ 8.2 Hz, 2H),
7.32 (d, J ¼ 8.1 Hz, 2H), 4.06 (t, J ¼ 4.9 Hz, 2H), 3.79 (t, J ¼ 5.2 Hz, 2H),
2.44 (s, 3H), 0.83 (s, 9H), 0.016 (s, 6H). MS: m/z [MþH]þ 331.13. The
compound's NMR spectrum was consistent with that reported
previously [55].
4.2.10. 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-
11-oxo-6,11-dihydro-5(2-t-butyldimethylsilylethyl)-benzo[b]
carbazole-3-carbonitrile (t-butyldimethylsilylethyl alectinib;
compound 5)
Alectinib (50 mg, 0.103 mmol, 1.0 eq.) and anhydrous K2CO3
(100 mg, 0.725 mmol, 7.0 eq.) were suspended in dry MeCN
(3.5 mL) in a 5-mL v-vial. To this mixture, t-butyldimethylsilylethyl
tosylate (68 mg, 0.206 mmol, 2.0 eq.) was added, and the reaction
mixture was heated at 100 ꢁC for 23 h. TLC showed a higher Rf spot
in about 75% intensity compared to the starting material. The re-
action mixture was cooled to room temperature and filtered; the
filtrate was washed with 5% MeOH/CH2Cl2 (10 mL). The filtrate was
concentrated under a stream of air, and the crude product was
purified by flash chromatography on silica gel and eluted with 3%
MeOH/CH2Cl2 to obtain t-butyldimethylsilylethyl alectinib (com-
pound 5) (41 mg, 61% yield) with 96% purity. 5: Rf ¼ 0.7 in 5%
600 MHz)
d
: 8.67 (d, J ¼ 8.1 Hz, 1H), 8.22 (s, 1H), 7.78 (s, 1H), 7.61 (d,
J ¼ 8.1 Hz, 1H), 7.17 (s, 1H), 4.91 (t, J ¼ 6.9 Hz, 2H), 4.60 (t, J ¼ 6.8 Hz,
2H), 3.77 (t, J ¼ 4.1 Hz, 4H), 3.31 (d, J ¼ 11.7 Hz, 2H), 3.01 (s, 3H), 2.75
(m, J ¼ 6.8 Hz, 4H), 2.63 (t, J ¼ 4.1 Hz, 4H), 2.36 (m, J ¼ 17.2 Hz, 1H),
2.01 (d, J ¼ 11.7 Hz, 2H), 1.89 (s, 6H), 1.74 (dq, J ¼ 3.0 Hz, 2H), 1.34 (t,
J ¼ 7.5 Hz, 3H). 13C NMR decoupled (CDCl3, 150 MHz)
d: 180.57,
156.72, 156.26, 147.57, 137.77, 136.64, 128.37, 126.77, 126.08, 125.62,
123.64, 119.86, 115.92, 113.99, 112.15, 106.65, 67.30, 65.16, 61.95,
52.14, 49.98, 44.53, 37.82, 37.60, 29.44, 28.93, 23.20, 14.39. HRMS:
m/z [MþH]þ calculated for C33H41N4O5S, 605.2798; found,
605.2802.
MeOH/CH2Cl2; 1H NMR (CDCl3, 600 MHz)
d
: 8.64 (d, J ¼ 8.2 Hz, 1H),
4.2.13. 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-
11-oxo-6,11-dihydro-5(2-p-toluenesulfonylethyl)-benzo[b]
carbazole-3-carbonitrile (p-toluenesulfonylethyl alectinib;
8.23 (s, 1H), 7.80 (s, 1H), 7.56 (dd, J ¼ 7.2 Hz, 1H), 7.17 (s, 1H), 4.65 (t,
J ¼ 6.3 Hz, 2H), 4.07 (t, J ¼ 6.2 Hz, 2H), 3.77 (t, J ¼ 4.4 Hz, 4H), 3.31 (d,
J ¼ 12.1 Hz, 2H), 2.74 (m, J ¼ 7.5 Hz, 4H), 2.63 (t, J ¼ 4.1 Hz, 4H), 2.36
(m, J ¼ 6.7 Hz, 1H), 2.01 (d, J ¼ 11.9 Hz, 2H), 1.88 (s, 6H), 1.74 (q d,
J ¼ 3.8 Hz, 2H), 1.34 (t, J ¼ 7.5 Hz, 3H), 0.87 (s, 9H), ꢀ0.05 (s, 6H). 13C
compound 7b)
Alectinib (30 mg, 0.621 mmol, 1.0 eq.) was added to dry MeCN
(3.0 mL) in a v-vial, and then anhydrous K2CO3 (62 mg, 0.4 mmol,
7.0 eq.) was added. To this mixture, ethylene glycol ditosylate
(32 mg, 0.09 mmol, 1.4 eq.) was added; the suspension became
clear after heating at 105 ꢁC for 10 min. The reaction mixture was
heated at 105 ꢁC for 20 h. TLC (5% MeOH/CH2Cl2) showed a higher Rf
(0.6) spot at about 65% intensity as compared with the starting
material (0.3 in 5% MeOH/CH2Cl2). The reaction mixture was
filtered and washed with 10% MeOH/CH2Cl2 (10 mL), and the filtrate
was evaporated under vacuum. The crude product was purified by
flash chromatography on silica gel and eluted with 2% MeOH/
CH2Cl2 to obtain p-toluenesulfonylethyl alectinib (compound 7b) as
a pale-yellow solid (24 mg, 57% yield). 7b: Rf ¼ 0.6 in 5% MeOH/
NMR decoupled (CDCl3, 150 MHz) d: 180.66, 157.00, 156.00, 147.93,
137.57, 137.19, 128.22, 126.69, 125.83, 125.52, 123.17, 120.20, 115.95,
115.17, 111.59, 105.94, 67.32, 62.00, 61.52, 52.19, 49.98, 47.91, 37.63,
29.48, 28.95, 25.77, 23.10, 18.25, 14.41, ꢀ5.53. MS: m/z [MþH]þ
calculated for C38H53N4O3Si, 641.3887; found, 641.34.
4.2.11. 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-
11-oxo-6,11-dihydro-5(2-hydroxyethyl)-benzo[b]carbazole-3-
carbonitrile (hydroxyethyl alectinib; compound 6)
Compound 5 (50 mg, 0.0778 mmol) was suspended in MeOH
(9 mL); 15 drops of 1N hydrochloric acid (HCl) was added, and the
mixture was heated under reflux for 1.5 h. TLC showed the full
consumption of the starting material and formation of a new lower
Rf spot (Rf ¼ 0.3 in 5% MeOH/CH2Cl2). Most of the MeOH and
aqueous HCl were evaporated under vacuum azeotropically with
MeCN (4 ꢂ 5 mL) and dried under high vacuum to yield hydrox-
yethyl alectinib (compound 6) as a pale yellow solid (40 mg, 97%
yield) with >95% purity. 6: Rf ¼ 0.3 in 5% MeOH/CH2Cl2; 1H NMR
CH2Cl2; 1H NMR (CDCl3, 600 MHz)
d
: 8.61 (d, J ¼ 8.1 Hz, 1H), 8.21 (s,
1H), 7.52 (t, J ¼ 8.1 Hz, 3H), 7.45 (s, 1H), 7.16 (d, J ¼ 7.9 Hz, 3H), 4.80
(t, J ¼ 6.4 Hz, 2H), 4.42 (t, J ¼ 6.3 Hz, 2H), 3.77 (t, J ¼ 3.9 Hz, 4H), 3.31
(d, J ¼ 11.6 Hz, 2H), 2.75 (m, J ¼ 7.6 Hz, 4H), 2.63 (t, J ¼ 4.2 Hz, 4H),
2.38 (s, 3H), 2.01 (d, J ¼ 11.8 Hz, 2H),1.85 (s, 6H),1.74 (dq, J ¼ 11.8 Hz,
2H), 1.64 (s, 1H), 1.34 (t, J ¼ 7.5 Hz, 3H). 13C NMR decoupled (CDCl3,
150 MHz) d: 180.55, 156.80, 156.26, 147.66, 145.78, 137.74, 136.25,
(DMSO‑d6, 500 MHz)
d: 10.97 (br, 1H), 8.45 (d, J ¼ 7.3 Hz, 1H), 8.29
131.29, 130.03, 128.34, 127.57, 126.71, 125.77, 123.50, 119.72, 115.94,
113.74, 112.01, 106.39, 67.34, 65.55, 61.95, 52.16, 49.99, 44.19, 37.60,
29.57, 28.96, 23.20, 21.69, 14.40. MS: m/z [MþH]þ calculated for
(s, 1H), 8.03 (s, 1H), 7.64 (d, J ¼ 7.2 Hz, 1H), 7.39 (s, 1H), 4.70 (s, 2H),
4.01 (d, J ¼ 10.85 Hz, 3H), 3.87 (s, 6H), 3.75 (bs, 2H), 3.33 (d,
J ¼ 8.8 Hz, 2H), 3.14 (br, 1H), 2.88 (m, J ¼ 10 Hz, 1H), 2.70 (d,
J ¼ 6.6 Hz, 1H), 2.24 (d, J ¼ 10 Hz, 2H), 2.06 (s, 1H), 1.88 (s, 7H), 1.28
(s, 3H). MS: m/z [MþH]þ calculated for C32H39N4O3, 527.30; found,
527.64. This compound was used for the next step for the synthesis
of compound 7a without further characterization or purification.
C39H45N4O5S, 681.31; found, 681.31.
4.2.14. [18F]fluoroethyl alectinib ([18F]4)
Method 2: An aqueous solution of K[18F]F/Kryptofix 2.2.2
(110 mCi in 0.5 mL) was transferred into a v-vial. Water was
removed by azeotropic evaporation with MeCN (1.0 mL) at 103 ꢁC
under a stream of argon, and the dry K[18F]F/Kryptofix 2.2.2 was
dissolved in 0.5 mL of anhydrous MeCN and then transferred to a v-
vial containing compound 7a (2 mg) or compound 7b (2.9 mg). The
reaction mixture in the v-vial was heated at 103 ꢁC for 30 min, and
an aliquot was injected into the analytical HPLC system, which
showed the presence of radioactive [18F]4 as the major product,
4.2.12. 9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-
11-oxo-6,11-dihydro-5(2-methanesulfonylethyl)-benzo[b]
carbazole-3-carbonitrile (methanesulfonylethyl alectinib;
compound 7a)
Compound 6 (41 mg, 0.077 mmol, 1.0 eq.) was suspended in
anhydrous CH2Cl2 (6.5 mL), and triethylamine (250
mL, excess) was
added; the mixture was stirred for 15 min, and the solution became
with undetectable
[
18F]fluoride. The solvent was completely

12
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
evaporated by heating under a stream of argon, and the crude
product was dissolved in CH2Cl2 (0.2 mL) and passed through a
silica Sep-Pak cartridge to remove unreacted fluoride. The cartridge
was eluted with 10% MeOH/CH2Cl2 (2 mL). The solvent was evap-
orated, and the crude product was purified by HPLC using a reverse-
phase amide column and eluted with 38% MeCN/water at 4 mL/
min; the radioactive fraction was collected. An aliquot of the pu-
rified product was co-injected with the standard cold reference
compound 4 into an analytical HPLC column to verify the product's
identity and purity. The solvent was evaporated under reduced
pressure, and the product was dissolved in a small volume of
ethanol and then diluted with 10% Tween 80 in water.
dithiothreitol, 5 mM EDTA, 0.5% NP-40, 1 mM microcystin, 1 mM
sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride,
0.15
mg/ml aprotinin, 20 mM leupeptin, and 20 mM pepstatin). Each
sample (15
mg total protein lysate) was loaded and fractionated on a
4e20% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA). Then, the
gel was transferred onto a polyvinylidene fluoride membrane
(Trans-Blot Turbo Transfer Pack, Bio-Rad) and blocked with 5% milk
in a mixture of Tris-buffered saline solution and polysorbate 20
(Tween 20). The blot was incubated overnight with conjugated
primary antibody
(Alk
D5F3
CP
rabbit
monoclonal
antibodyehorseradish peroxidase conjugate; Cell Signaling Tech-
nology, Beverly, MA) at a 1:3000 dilution.
4.2.15. 5-Chloro-N2e(2-isoprpoxy-5-methyl-4-(1-
4.5.2. Cytotoxicity assay
fluoroethylpiperidin-4-yl)phenyl)-N4-[2-(propane-2-sulfonyl)
phenyl]pyrimidine-2,4-diamine ([18F]fluoroethyl ceritinib; ([18F]9)
The title compound was prepared as we described previously
[38]. Briefly, [18F]fluoroethyl tosylate was produced and reacted
with ceritinib and then purified by chromatography. The product
was assessed by analytical HPLC to determine its identity and pu-
rity. The solvent was evaporated, and the product was dissolved in a
small volume of ethanol and then diluted with 10% Tween 80 so-
lution for formulation and animal injection.
H2228 and H441 cells were seeded in 96-well clear-bottom
black-wall plates at a density of 8000 cells per well in RPMI-1640
medium (100 mL) supplemented with 10% FBS. The next day, the
cells were treated with various concentrations of crizotinib, alec-
tinib, and ceritinib or their corresponding fluoroethyl derivatives
for 72 h. Subsequently, cell viability was measured by adding 100 mL
of Alamar Blue reagent (1:10 dilution) in phenol red-free RPMI
medium with 10% FBS to each well. Cells were incubated for 4 h,
and fluorescence was measured with a Synergy H4 microplate
reader (BioTek, Winooski, VT) at the excitation wavelength of
530 nm and the emission wavelength of 580 nm. IC50 values were
calculated with GraphPad Prism 7.01. Experiments were performed
in triplicate in three independent sets, and results are reported as
means with standard deviations.
4.3. Formulation
Because compounds 1, 4, and 9 were not soluble in saline or PBS
solutions, they were dissolved in DMSO prior to their use in the
cytotoxic assay. The radiolabeled analogues, after evaporation of
the chromatographic solvent (MeOH/CH2Cl2 or MeCN), were dis-
solved in a minimum volume of ethanol (0.1e0.2 mL, depending on
the amount of activity). The alcoholic solutions were diluted with a
10% solution of aqueous Tween 80 to make a stock solution with a
final concentration of 10% ethanol. The stock solution was then
diluted with PBS prior to injection into animals.
4.5.3. Tracer uptake assays
H2228 and H441 cells were seeded in 6-well clear-bottom plates
at a density of 106 cells per well in RPMI-1640 medium (2 mL)
supplemented with 10% FBS. Aliquots of tracer (1 mCi in 30 ml PBS)
were added to each well, and the cells were incubated for 30, 60, or
120 min. At the designated times, the medium was removed, the
wells were washed twice with ice-cold PBS (1 mL), the cells were
trypsinized for 5 min at 37 ꢁC, and the cell suspension was trans-
ferred to gamma-counting tubes and assessed for [18F]radioactivity.
Experiments were performed in triplicate. Radioactivity uptake
was calculated as counts per minute per million cells.
4.4. LogP measurements
LogP values were determined using a standard method [46].
Briefly, [18F]1, [18F]4, or [18F]9 (10
mL, 1 mCi) in ethanol was added to
a mixture of 1-octanol (1.0 mL) and water (1.0 mL) in test tubes in
triplicate. The mixtures in the test tubes were subjected to vor-
texing for 5 min, which was repeated four times; the mixtures were
then subjected to centrifugation (2 ꢂ 5 min, 4000 rpm at 4 ꢁC).
4.5.4. In vivo biodistribution and PET imaging
All animal studies were performed according to a protocol
approved by the Institutional Animal Care and Use Committee of
MD Anderson Cancer Center. PET imaging and image analysis were
performed on an Albira trimodality rodent scanner (Bruker). In
brief, 8- to 10-week-old female nude mice were injected intrave-
From each test tube, three aliquots of the 1-octanol layer (50 mL
each) were transferred into three gamma-counting tubes (nine
tubes total). Similarly, 50- L aliquots of the aqueous layer were
m
transferred into nine separate test tubes. The amount of radioac-
tivity in each tube was counted using a gamma counter. The
partition coefficient (P) was calculated as the ratio of activity in
octanol to activity in water. The LogD value was also measured in
octanol/PBS (pH 7.4) using the same methodology. In addition,
Cheminformatics software (Molinspiration, Slovak Republic) was
used to calculate theoretical LogP values for [18F]1 (4.81), [18F]4
(6.01), and [18F]9 (6.94).
nously with approximately 100 mCi (3.7 MBq) of one of the PET
tracers under anesthesia (2% isoflurane in oxygen) and then imaged
on an Albira trimodality rodent scanner (Bruker). Dynamic PET
scans were acquired 0e10 min after injection under anesthesia.
Static PET scans (10 min) were acquired under anesthesia at 30 and
60 min after injection. For image analysis, PMOD software (PMOD
Technologies Ltd., Zurich, Switzerland) was used to draw regions of
interest over the major organs on decay-corrected whole-body
coronal images. The radioactivity concentration was obtained from
the mean values within multiple regions of interest and then
converted to percent injected dose per cubic centimeter (%ID/cc) of
tissue. Results are reported as means with standard deviations.
4.5. Biology
4.5.1. Cell lines and ALK expression
The human lung cancer cell lines H2228 (ALK-positive, EML4-
ALK v3) and H441 (ALK-negative) were purchased from ATCC
(Manassas, VA). Cells were grown in RPMI-1640 medium (Sigma-
Aldrich) supplemented with 10% fetal bovine serum (FBS). For
Western blot analysis, the cells were lysed with mammalian cell
lysis buffer (50 mM Tris HCl, pH 8.0, 100 mM NaCl, 2 mM
Conflicts of interest
The authors declare no conflict of interest.

B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
13
N. Oikawa, Design and synthesis of a highly selective, orally active and potent
anaplastic lymphoma kinase inhibitor (CH5424802), Bioorg. Med. Chem. 20
(2012) 1271e1280. https://doi.org/10.1016/j.bmc.2011.12.021.
Acknowledgments
The authors thank Dr. Seth T. Gammon for editing the figures
and for providing helpful discussion. The authors also thank
Kathryn Hale and Joe Munch in Scientific Publication Services in the
Research Medical Library at MD Anderson for editing the manu-
script. This work was supported by the U.S. National Institutes of
Health through the Washington UniversityꢀMD Anderson Cancer
Center Inter-Institutional Molecular Imaging Center Grant (NCI P50
CA94056; DP-W) and through MD Anderson’s Cancer Center Sup-
port Grant (P30CA016672), which supports the NMR Facility and
Small Animal Imaging Facility used in the study; and by an MD
Anderson Cancer Center Institutional Research Grant (MMA).
[15] J. Wu, J. Savooji, D. Liu, Second- and third-generation ALK inhibitors for non-
small cell lung cancer, J. Hematol. Oncol. 9 (2016) 19. https://doi.org/10.1186/
s13045-016-0251-8.
[16] E. Larkins, G.M. Blumenthal, H. Chen, K. He, R. Agarwal, G. Gieser, O. Stephens,
E. Zahalka, K. Ringgold, W. Helms, S. Shord, J. Yu, H. Zhao, G. Davis, A.E. McKee,
P. Keegan, R. Pazdur, FDA Approval: alectinib for the treatment of metastatic,
ALK-positive nonesmall cell lung cancer following Crizotinib, Clin. Cancer Res.
22 (2016) 5171e5176. https://doi.org/10.1158/1078-0432.CCR-16-1293.
[17] T.H. Marsilje, W. Pei, B. Chen, W. Lu, T. Uno, Y. Jin, T. Jiang, S. Kim, N. Li,
M. Warmuth, Y. Sarkisova, F. Sun, A. Steffy, A.C. Pferdekamper, A.G. Li,
S.B. Joseph, Y. Kim, B. Liu, T. Tuntland, X. Cui, N.S. Gray, R. Steensma, Y. Wan,
J. Jiang, G. Chopiuk, J. Li, W.P. Gordon, W. Richmond, K. Johnson, J. Chang,
T. Groessl, Y. He, A. Phimister, A. Aycinena, C.C. Lee, B. Bursulaya,
D.S. Karanewsky, H.M. Seidel, J.L. Harris, P. Michellys, Synthesis,
structureeactivity relationships, and in vivo efficacy of the novel potent and
selective Anaplastic Lymphoma Kinase (ALK) inhibitor 5-Chloro-N2-(2-
isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)
phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2
clinical trials, J. Med. Chem. 56 (2013) 5675e5690. https://doi.org/10.1021/
jm400402q.
[18] S. Khozin, G.M. Blumenthal, L. Zhang, S. Tang, M. Brower, E. Fox, W. Helms,
R. Leong, P. Song, Y. Pan, Q. Liu, P. Zhao, H. Zhao, D. Lu, Z. Tang, A. Al Hakim,
K. Boyd, P. Keegan, R. Justice, R. Pazdur, FDA approval: ceritinib for the
treatment of metastatic Anaplastic Lymphoma Kinaseepositive nonesmall
cell lung cancer, Clin. Cancer Res. 21 (2015) 2436e2439. https://doi.org/10.
1158/1078-0432.CCR-14-3157.
[19] H.Y. Zou, Q. Li, L.D. Engstrom, M. West, V. Appleman, K.A. Wong, M. McTigue,
Y. Deng, W. Liu, A. Brooun, S. Timofeevski, S.R.P. McDonnell, P. Jiang, M.D. Falk,
P.B. Lappin, T. Affolter, T. Nichols, W. Hu, J. Lam, T.W. Johnson, T. Smeal,
A. Charest, V.R. Fantin, PF-06463922 is a potent and selective next-generation
ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations,
Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 3493e3498. https://doi.org/10.1073/
pnas.1420785112.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111571.
References
[1] S. Morris, M. Kirstein, M. Valentine, K. Dittmer, D. Shapiro, D. Saltman, A. Look,
Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-
Hodgkin's lymphoma, Science 263 (1994) 1281e1284. https://doi.org/10.
1126/science.8122112.
[2] M. Soda, Y.L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa,
S. Fujiwara, H. Watanabe, K. Kurashina, H. Hatanaka, M. Bando, S. Ohno,
Y. Ishikawa, H. Aburatani, T. Niki, Y. Sohara, Y. Sugiyama, H. Mano, Identifi-
cation of the transforming EML4eALK fusion gene in non-small-cell lung
cancer, Nature 448 (2007) 561e566. https://doi.org/10.1038/nature05945.
[3] R.H. Palmer, E. Vernersson, C. Grabbe, B. Hallberg, Anaplastic lymphoma ki-
nase: signalling in development and disease, Biochem. J. 420 (2009) 345e361.
https://doi.org/10.1042/BJ20090387.
[20] N.R. Infarinato, J.H. Park, K. Krytska, H.T. Ryles, R. Sano, K.M. Szigety, Y. Li,
ꢀ
H.Y. Zou, N.V. Lee, T. Smeal, M.A. Lemmon, Y.P. Mosse, The ALK/ROS1 inhibitor
PF-06463922 overcomes primary resistance to Crizotinib in ALK-driven
neuroblastoma, Cancer Discov.
2159-8290.CD-15-1056.
6 (2016) 96e107. https://doi.org/10.1158/
ꢀ
[4] E. Grande, M. Bolos, E. Arriola, Targeting oncogenic ALK: a promising strategy
[21] W.D. Jang, J. Kim, H.Y. Son, S.Y. Park, Y.S. Cho, T. Koo, H. Lee, N.S. Kang, Dis-
covery of Tyk2 inhibitors via the virtual site-directed fragment-based drug
design, Bioorg. Med. Chem. Lett 25 (2015) 3947e3952. https://doi.org/10.
1016/j.bmcl.2015.07.037.
[22] C.M. Lovly, J.M. Heuckmann, E. de Stanchina, H. Chen, R.K. Thomas, C. Liang,
W. Pao, Insights into ALK-driven cancers revealed through development of
novel ALK tyrosine kinase inhibitors, Cancer Res. 71 (2011) 4920e4931.
https://doi.org/10.1158/0008-5472.CAN-10-3879.
[23] M. Mori, Y. Ueno, S. Konagai, H. Fushiki, I. Shimada, Y. Kondoh, R. Saito,
K. Mori, N. Shindou, T. Soga, H. Sakagami, T. Furutani, H. Doihara, M. Kudoh,
S. Kuromitsu, The selective anaplastic lymphoma receptor tyrosine kinase
inhibitor ASP3026 induces tumor regression and prolongs survival in
nonesmall cell lung cancer model mice, Mol. Cancer Ther. 13 (2014)
329e340. https://doi.org/10.1158/1535-7163.MCT-13-0395.
for cancer treatment, Mol. Cancer Ther. 10 (2011) 569e579. https://doi.org/10.
1158/1535-7163.MCT-10-0615.
ꢀ
[5] J.J. Cui, M. Tran-Dube, H. Shen, M. Nambu, P.P. Kung, M. Pairish, L. Jia, J. Meng,
L. Funk, I. Botrous, M. McTigue, N. Grodsky, K. Ryan, E. Padrique, G. Alton,
S. Timofeevski, S. Yamazaki, Q. Li, H. Zou, J. Christensen, B. Mroczkowski,
S. Bender, R.S. Kania, M.P. Edwards, Structure based drug design of crizotinib
(PF-02341066),
a
potent
and
selective
dual
inhibitor
of
mesenchymaleepithelial transition factor (c-MET) kinase and anaplastic
lymphoma kinase (ALK), J. Med. Chem. 54 (2011) 6342e6363. https://doi.org/
10.1021/jm2007613.
[6] S.I. Ou, Crizotinib: a novel and first-in-class multitargeted tyrosine kinase
inhibitor for the treatment of anaplastic lymphoma kinase rearranged non-
small cell lung cancer and beyond, Drug Des. Dev. Ther. 5 (2011) 471e485.
https://doi.org/10.2147/DDDT.S19045.
[24] M. Ceccon, L. Mologni, G. Giudici, R. Piazza, A. Pirola, D. Fontana,
C. Gambacorti-Passerini, Treatment efficacy and resistance mechanisms using
the second-generation ALK inhibitor AP26113 in human NPM-ALKepositive
anaplastic large cell lymphoma, Mol. Cancer Res. 13 (2015) 775e783. https://
doi.org/10.1158/1541-7786.MCR-14-0157.
[7] D. Kazandjian, G.M. Blumenthal, H. Chen, K. He, M. Patel, R. Justice, P. Keegan,
R. Pazdur, FDA approval summary: crizotinib for the treatment of metastatic
non-small cell lung cancer with anaplastic lymphoma kinase rearrangements,
The Oncologist 19 (2014) e5ee11. https://doi:10.1634/theoncologist.2014-
0241.
[25] C. Iragavarapu, M. Mustafa, A. Akinleye, M. Furqan, V. Mittal, S. Cang, D. Liu,
Novel ALK inhibitors in clinical use and development, J. Hematol. Oncol. 8
(2015) 17. https://doi.org/10.1186/s13045-015-0122-8.
[8] R. Katayama, A.T. Shaw, T.M. Khan, M. Mino-Kenudson, B.J. Solomon,
B. Halmos, N.A. Jessop, J.C. Wain, A.T. Yeo, C. Benes, L. Drew, J.C. Saeh,
K. Crosby, L.V. Sequist, A.J. Iafrate, J.A. Engelman, Mechanisms of acquired
[26] T. Kodama, M. Hasegawa, K. Takanashi, Y. Sakurai, O. Kondoh, H. Sakamoto,
Antitumor activity of the selective ALK inhibitor alectinib in models of
intracranial metastases, Cancer Chemother. Pharmacol. 74 (2014) 1023e1028.
https://doi.org/10.1007/s00280-014-2578-6.
[27] T.L. Collier, K.P. Maresca, M.D. Normandin, P. Richardson, T.J. McCarthy,
S.H. Liang, R.N. Waterhouse, N. Vasdev, Brain penetration of the ROS1/ALK
inhibitor Lorlatinib confirmed by PET, Mol. Imaging 16 (2017) 1e3. https://
doi.org/10.1177/1536012117736669.
[28] A.T. Shaw, L. Friboulet, I. Leshchiner, J.F. Gainor, S. Bergqvist, A. Brooun,
B.J. Burke, Y. Deng, W. Liu, L. Dardaei, R.L. Frias, K.R. Schultz, J. Logan,
L.P. James, T. Smeal, S. Timofeevski, R. Katayama, A.J. Iafrate, L. Le, M. McTigue,
G. Getz, T.W. Johnson, J.A. Engelman, Resensitization to crizotinib by the
lorlatinib ALK resistance mutation L1198F, N. Engl. J. Med. 374 (2016) 54e61.
https://doi.org/10.1056/NEJMoa1508887.
[29] A. Kort, R.W. Sparidans, E. Wagenaar, J.H. Beijnen, A.H. Schinkel, Brain accu-
mulation of the EML4-ALK inhibitor ceritinib is restricted by P-glycoprotein
(P-GP/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), Pharmacol.
Res. 102 (2015) 200e207. https://doi.org/10.1016/j.phrs.2015.09.003.
[30] R. Sharma, E. Aboagye, Development of radiotracers for oncology–the inter-
face with pharmacology, Br. J. Pharmacol. 163 (2011) 1565e1585. https://doi.
org/10.1111/j.1476-5381.2010.01160.x.
Crizotinib resistance in ALK-rearranged lung cancers, Sci. Transl. Med.
(2012), 120ra117-120ra117, https://doi.org/10.1126/scitranslmed.3003316.
[9] I. Dagogo-Jack, A.T. Shaw, Crizotinib resistance: implications for therapeutic
strategies, Ann. Oncol. 27 (2016) iii42eiii50. https://doi.org/10.1093/annonc/
mdw305.
[10] Crizotinib resistance arises through multiple mechanisms, Cancer Discov. 2
(2012), 204-204, https://doi.org/10.1158/2159-8290.CD-RW2012-018.
[11] S. Zhang, F. Wang, J. Keats, X. Zhu, Y. Ning, S.D. Wardwell, L. Moran,
Q.K. Mohemmad, R. Anjum, Y. Wang, N.I. Narasimhan, D. Dalgarno,
W.C. Shakespeare, J.J. Miret, T. Clackson, V.M. Rivera, Crizotinib-resistant
mutants of EML4-ALK identified through an accelerated mutagenesis screen,
Chem. Biol. Drug Des. 78 (2011) 999e1005. https://doi.org/10.1111/j.1747-
0285.2011.01239.x.
[12] M. Ceccon, L. Mologni, W. Bisson, L. Scapozza, C. Gambacorti-Passerini, Cri-
zotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated
ALK inhibitors, Mol. Cancer Res. 11 (2013) 122e132. https://doi.org/10.1158/
1541-7786.MCR-12-0569.
4
[13] J.E. Frampton, Crizotinib: a review of its use in the treatment of anaplastic
lymphoma kinase-positive, advanced non-small cell lung cancer, Drugs 73
(2013) 2031e2051. https://doi.org/10.1007/s40265-013-0142-z.
[14] K. Kinoshita, K. Asoh, N. Furuichi, T. Ito, H. Kawada, S. Hara, J. Ohwada,
T. Miyagi, T. Kobayashi, K. Takanashi, T. Tsukaguchi, H. Sakamoto, T. Tsukuda,
[31] P. Slobbe, A.J. Poot, A.D. Windhorst, G.A.M.S. van Dongen, PET imaging with

14
B. Radaram et al. / European Journal of Medicinal Chemistry 182 (2019) 111571
small-molecule tyrosine kinase inhibitors: TKI-PET, Drug Discov. Today 17
(2012) 1175e1187. https://doi.org/10.1016/j.drudis.2012.06.016.
ALK-driven lung cancers, Pharmacol. Res. 117 (2017) 343e356. https://doi.
org/10.1016/j.phrs.2017.01.007.
[32] Z. Peng, D.S. Maxwell, D. Sun, B.A. Bhanu Prasad, A. Pal, S. Wang, J. Balatoni,
P. Ghosh, S.T. Lim, A. Volgin, A. Shavrin, M.M. Alauddin, J.G. Gelovani,
W.G. Bornmann, Imatinib analogs as potential agents for PET imaging of Bcr-
Abl and c-KIT expression at a kinase level, Bioorg. Med. Chem. 22 (2014)
623e632. https://doi.org/10.1016/j.bmc.2013.10.040.
[33] D.R. Veach, M. Namavari, N. Pillarsetty, E.B. Santos, T. Beresten-Kochetkov,
C. Lambek, B.J. Punzalan, C. Antczak, P.M. Smith-Jones, H. Djaballah,
B. Clarkson, S.M. Larson, Synthesis and biological evaluation of a fluorine-18
derivative of Dasatinib, J. Med. Chem. 50 (2007) 5853e5857. https://doi.org/
10.1021/jm070342g.
[34] F. Pisaneschi, Q. Nguyen, E. Shamsaei, M. Glaser, E. Robins, M. Kaliszczak,
G. Smith, A.C. Spivey, E.O. Aboagye, Development of a new epidermal growth
factor receptor positron emission tomography imaging agent based on the 3-
cyanoquinoline core: synthesis and biological evaluation, Bioorg. Med. Chem.
18 (2010) 6634e6645. https://doi.org/10.1016/j.bmc.2010.08.004.
[35] A.A. Memon, S. Jakobsen, F. Dagnaes-Hansen, B.S. Sorensen, S. Keiding,
E. Nexo, Positron emission tomography (PET) imaging with [11C]-labeled
Erlotinib: a micro-PET study on mice with lung tumor xenografts, Cancer Res.
69 (2009) 873e878. https://doi.org/10.1158/0008-5472.CAN-08-3118.
[36] C.M. Rocha-Lima, L.E. Raez, Erlotinib (tarceva) for the treatment of non-small-
cell lung cancer and pancreatic cancer, P & T : a peer-reviewed, journal for
formulary management 34 (2009) 554e564. https://www.ptcommunity.com/
journal/article/archives/2009/10/554/erlotinib-tarceva-treatment-non-small-
cell-lung-cancer-and.
[44] T. Kniess, M. Laube, P. Brust, J. Steinbach, 2-[18F]Fluoroethyl tosylate e a
versatile tool for building 18F-based radiotracers for positron emission to-
mography, Med. Chem. Comm. 6 (2015) 1714e1754. https://doi.org/10.1039/
C5MD00303B.
[45] R. Ding, Y. He, X. Wang, J. Xu, Y. Chen, M. Feng, C. Qi, Treatment of alcohols
with tosyl chloride does not always lead to the formation of tosylates, Mol-
ecules 16 (2011) 5665e5673. https://doi.org/10.3390/molecules16075665.
[46] A.A. Wilson, L. Jin, A. Garcia, J.N. DaSilva, S. Houle, An admonition when
measuring the lipophilicity of radiotracers using counting techniques, Appl.
Radiat. Isot. 54 (2001) 203e208. https://doi.org/10.1016/S0969-8043(00)
00269-4.
[47] C. Vraka, L. Nics, K. Wagner, M. Hacker, W. Wadsak, M. Mitterhauser, LogP, a
yesterday's value? Nucl. Med. Biol. 50 (2017) 1e10. https://doi.org/10.1016/j.
nucmedbio.2017.03.003.
[48] M.P. Martelli, G. Sozzi, L. Hernandez, V. Pettirossi, A. Navarro, D. Conte,
P. Gasparini, F. Perrone, P. Modena, U. Pastorino, A. Carbone, A. Fabbri,
ꢀ
A. Sidoni, S. Nakamura, M. Gambacorta, P.L. Fernandez, J. Ramirez, J.K.C. Chan,
W.F. Grigioni, E. Campo, S.A. Pileri, B. Falini, EML4-ALK rearrangement in non-
small cell lung cancer and non-tumor lung tissues, Am. J. Pathol. 174 (2009)
661e670. https://doi.org/10.2353/ajpath.2009.080755.
[49] H.J. Kim, K.Y. Lee, Y.W. Kim, Y.J. Choi, J. Lee, C.M. Choi, I. Baek, J.K. Rho, J.C. Lee,
P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with
an ALK rearrangement, BMC Canc. 15 (2015), 553-553, https://doi.org/10.
1186/s12885-015-1543-z.
[37] C. Gridelli, M.A. Bareschino, C. Schettino, A. Rossi, P. Maione, F. Ciardiello,
Erlotinib in non-small cell lung cancer treatment: current status and future
development, The Oncologist 12 (2007) 840e849. https://doi.org/10.1634/
theoncologist.12-7-840.
[38] I. Bahce, E.F. Smit, M. Lubberink, A.A.M. van der Veldt, M. Yaqub,
A.D. Windhorst, R.C. Schuit, E. Thunnissen, D.A.M. Heideman, P.E. Postmus,
A.A. Lammertsma, N.H. Hendrikse, Development of [11C]-Erlotinib positron
emission tomography for in vivo evaluation of EGF receptor mutational status,
Clin. Cancer Res. 19 (2013) 183e193. https://doi.org/10.1158/1078-0432.CCR-
12-0289.
[39] A.A. Memon, B. Weber, M. Winterdahl, S. Jakobsen, P. Meldgaard,
H.H.T. Madsen, S. Keiding, E. Nexo, B.S. Sorensen, PET imaging of patients with
non-small cell lung cancer employing an EGF receptor targeting drug as
tracer, Br. J. Canc. 105 (2011) 1850e1855. https://doi.org/10.1038/bjc.2011.
493.
[50] R. Katayama, T. Sakashita, N. Yanagitani, H. Ninomiya, A. Horiike, L. Friboulet,
J.F. Gainor, N. Motoi, A. Dobashi, S. Sakata, Y. Tambo, S. Kitazono, S. Sato,
S. Koike, A. John Iafrate, M. Mino-Kenudson, Y. Ishikawa, A.T. Shaw,
J.A. Engelman, K. Takeuchi, M. Nishio, N. Fujita, P-glycoprotein mediates
ceritinib resistance in anaplastic lymphoma kinase-rearranged non-small cell
lung cancer, EBioMedicine 3 (2016) 54e66. https://doi.org/10.1016/j.ebiom.
2015.12.009.
[51] R.L. Slade, F. Pisaneschi, Q. Nguyen, G. Smith, L. Carroll, A. Beckley,
M.A. Kaliszczak, E.O. Aboagye, Identification of ABC transporter interaction of
a novel cyanoquinoline radiotracer and implications for tumour imaging by
positron emission tomography, PLoS One 11 (2016), e0161427. https://doi.
org/10.1371/journal.pone.0161427.
[52] A. Leonidova, C. Foerster, K. Zarschler, M. Schubert, H.J. Pietzsch, J. Steinbach,
R. Bergmann, N. Metzler-Nolte, H. Stephan, G. Gasser, In vivo demonstration
of an active tumor pretargeting approach with peptide nucleic acid bio-
[40] T.L. Collier, M.D. Normandin, N.A. Stephenson, E. Livni, S.H. Liang,
D.W. Wooten, S.A. Esfahani, M.G. Stabin, U. Mahmood, J. Chen, W. Wang,
K. Maresca, R.N. Waterhouse, G. El Fakhri, P. Richardson, N. Vasdev, Synthesis
and preliminary PET imaging of 11C and 18F isotopologues of the ROS1/ALK
inhibitor lorlatinib, Nat. Commun. 8 (2017) 15761. https://doi.org/10.1038/
ncomms15761.
conjugates as complementary system, Chem. Sci.
https://doi.org/10.1039/c5sc00951k.
6 (2015) 5601e5616.
[53] S. Saha, D. Copic, S. Bhaskar, N. Clay, A. Donini, A.J. Hart, J. Lahann, Chemically
controlled bending of compositionally anisotropic microcylinders, Angew.
Chem. Int. Ed. 51 (2012) 660e665. https://doi.org/10.1002/anie.201105387.
[54] M.M. Alauddin, P.S. Conti, Selective alkylation of pyrimidyl dianions II: syn-
thesis, characterization, and comparative reactivity of 30, 50-o-bis- tetrahy-
dropyranyl, trimethylsilyl and tert-butyldimethylsilyl derivatives of 5-bromo-
20-deoxyuridine, Tetrahedron 50 (1994) 1699e1706. https://doi.org/10.1016/
S0040-4020(01)80845-0.
[41] S. Perera, D. Piwnica-Worms, M.M. Alauddin, Synthesis of a [18F]-labeled
Ceritinib analogue for positron emission tomography of anaplastic lymphoma
kinase, a receptor tyrosine kinase, in lung cancer, J. Label. Comp. Radiopharm.
59 (2016) 103e108. https://doi.org/10.1002/jlcr.3373.
[42] P. Wang, J. Cai, J. Chen, M. Ji, Synthesis and anticancer activities of ceritinib
analogs modified in the terminal piperidine ring, Eur. J. Med. Chem. 93 (2015)
1e8. https://doi.org/10.1016/j.ejmech.2015.01.056.
[55] T. Darmanin, F. Guittard, One-pot method for build-up nanoporous super oil-
repellent films, J. Colloid Interface Sci. 335 (2009) 146e149. https://doi.org/10.
1016/j.jcis.2009.03.073.
[43] R. Roskoski, Anaplastic lymphoma kinase (ALK) inhibitors in the treatment of