Synthesis and evaluation of novel F-18-labeled pyrimidine derivatives: Potential FAK inhibitors and PET imaging agents for cancer detection

Article abstract of DOI:10.1039/c6ra28851k

Based on computer-assisted drug design, a series of novel pyrimidine derivatives was successfully synthesized and characterized by ¹H NMR, ¹³C HNMR, and MS spectra. All the new compounds were evaluated for their activity against focal adhesion kinase and showed low IC₅₀ values in comparison with control drugs. In particular, for compound 8i, its IC₅₀ value was 0.060 μM, suggesting its advantage as a focal adhesion kinase inhibitor. To evaluate the potentiality of these compounds as PET imaging agents in cancer detection, compounds 8a, 8c, 8h, and 8i were successively labeled with ¹⁸F. The four ¹⁸F-labeled pyrimidine derivatives showed appropriate log P values and high stability in physiological saline and mouse plasma. Noticeably, compound [¹⁸F]-8a with a 4-methoxyl group at the benzene ring exhibited good in vivo biodistribution data in mice bearing the S180 tumor, which promoted a further microPET imaging study of compound [¹⁸F]-8a. The microPET image of [¹⁸F-8a] administered into the S180 tumor-bearing mice acquired at 60 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggested that compound [¹⁸F]-8a might be a new probe for PET tumor imaging.

Full text of DOI:10.1039/c6ra28851k

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Synthesis and evaluation of novel F-18-labeled
pyrimidine derivatives: potential FAK inhibitors
and PET imaging agents for cancer detection†
Cite this: RSC Adv., 2017, 7, 22388
*
Yu Fang, Hang Wang, Xingyu Xu, Jianping Liu and Huabei Zhang
Dawei Wang,
Based on computer-assisted drug design, a series of novel pyrimidine derivatives was successfully
synthesized and characterized by 1H NMR, 13C HNMR, and MS spectra. All the new compounds were
evaluated for their activity against focal adhesion kinase and showed low IC50 values in comparison with
control drugs. In particular, for compound 8i, its IC50 value was 0.060 mM, suggesting its advantage as
a focal adhesion kinase inhibitor. To evaluate the potentiality of these compounds as PET imaging agents
in cancer detection, compounds 8a, 8c, 8h, and 8i were successively labeled with 18F. The four 18F-
labeled pyrimidine derivatives showed appropriate log P values and high stability in physiological saline
and mouse plasma. Noticeably, compound [18F]-8a with a 4-methoxyl group at the benzene ring
exhibited good in vivo biodistribution data in mice bearing the S180 tumor, which promoted a further
microPET imaging study of compound [18F]-8a. The microPET image of [18F-8a] administered into the
S180 tumor-bearing mice acquired at 60 min post-injection illustrated that the uptake in S180 tumor
was obvious. These results suggested that compound [18F]-8a might be a new probe for PET tumor imaging.
Received 8th February 2017
Accepted 2nd April 2017
DOI: 10.1039/c6ra28851k
rsc.li/rsc-advances
integrins and receptor tyrosine kinase signal transduction
pathways, transmitting signals from the extracellular matrix
Introduction
(ECM) to the cell cytoskeleton. A variety of diseases have been
identied to be related with FAK, especially cancers, in which
FAK is highly expressed or over-expressed at both the tran-
scriptional and translational level. FAK signaling pathways can
stimulate tumor progression and metastasis through the regu-
lation of cell migration, invasion, ECM, and angiogenesis.5–7
Thus, this protein has emerged as a promising therapeutic
target,8 and efforts to investigate FAK for an anticancer effect are
under intense investigation.9 Accordingly, the inhibition
against FAK is considered as an effective antineoplastic strategy
by inducing apoptosis and sensitizing tumor cells to
chemotherapy.10,11
TAE-226, PF-562271, and PF-573228 are the classical FAK
inhibitors. TAE-226 is a novel low-molecular-weight ATP-
competitive tyrosine kinase inhibitor targeting FAK with
potent and selective in vitro activity (IC50 ¼ 5.5 nM). It could
inhibit the phosphorylation of FAK, the downstream oncogenic
signals, the extracellular signal-related kinase, and the S6
ribosomal protein.12 TAE-226 shows impressive antitumor
activity against various cancers, such as neuroblastoma,13 breast
cancer,14,15 and ovarian cancer.16 However, the development of
TAE-226 has been discontinued in the preclinical stage due to
its severe effects on glucose metabolism in animal studies and
its inhibition of insulin activity (IC50 ¼ 44 nM).17 PF-562271,
developed by Pzer, is a dual FAK/Pyk2 inhibitor, and is iden-
tied as the rst-in-class and rst-in-human FAK inhibitor (FAK
IC50 ¼ 1.5 nM, PYK2 IC50 ¼ 14 nM) being evaluated in the clinic.
Cancer is now not only the leading cause of death, but also
a major public health problem all around the world.1 The
increasing growth and aging of the population are the main
factors for the occurrence of cancer,2 while in developed coun-
tries, it may be induced by a plethora of both external and
internal factors. To date, many anticancer drugs have been
marketed. The most well-known and widely clinical used type of
anticancer agents is nitrogen mustard (nonspecic bifunctional
DNA-alkylating agents), which has a serious drawback in that it
could cause DNA damage by interrupting DNA biosynthesis.
Melphalan, chlorambucil, cyclophosphamide, and bendamus-
tine are revolutionary discoveries in the treatment of cancers.
However, many drawbacks exist in these derivatives such as low
specicity to tumor cells, eventual loss in activity, high chemical
reactivity, and bone-marrow toxicity.3 Therefore, the develop-
ment of novel anticancer drugs with high specicity, activity
retention, and low chemical reactivity as well as no bone-
marrow toxicity is of great emergency.
Focal adhesion kinase (FAK) is a multi-domain non-receptor
tyrosine kinase and scaffold protein localized to focal adhe-
sions,4 which is uniquely positioned at the convergence point of
Key Laboratory of Radiopharmaceuticals of Ministry of Education, College of
Chemistry, Beijing Normal University, No. 19 Xinjiekouwai Street, Haidian District,
Beijing 100875, People's Republic of China. E-mail: hbzhang@bnu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra28851k
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PF-573228 is another novel small molecule FAK inhibitor that
interacts with FAK in the ATP-binding pocket and blocks the
catalytic activity of recombinant FAK protein or endogenous
FAK. The treatment of cells with PF-573228 blocks FAK phos-
phorylation on Tyr397 and concomitantly reduces the tyrosine
phosphorylation of paxillin. PF-573228 provides an appropriate
tool to dissect the role of FAK in the regulation of cell adhesion
signaling and adhesion dynamics.18
Fig. 2 The key framework structure of the designed novel FAK
inhibitors.
From the above three FAK inhibitors, it can be seen that the
same structural feature is the pyrimidine ring, which attracts
our intensive interest in studying pyrimidine derivatives as
novel FAK inhibitors related to cancers. As we know, the
pyrimidine ring is a cyclic amine with two nitrogen atoms. It is
the main component of many heterocyclic compounds and
plays important roles in many biological processes, such as the
synthesis and function of nucleic acids, several vitamins, co-
enzymes, and purines, etc.19,20 Pyrimidines and their deriva-
tives exhibit a broad spectrum of biological activities including
signicant in vitro activity against DNA and RNA, potential
inhibition property against polio herpes viruses, and as anti-
tumor, anti-HIV, antimicrobial, insecticidal, and antiviral
agents.21,22
In view of the above observations, a series of novel pyrimi-
dine derivatives as FAK-targeted tumor imaging agents were
designed based on the following considerations:
(1) The key framework structure of TAE-226, PF-562271, and
PF-573228 should remain as in Fig. 1 to keep the excellent
inhibitory activity against FAK. While the strong electron-
withdrawing chloro and triuoromethyl groups are changed
into a bromo moiety to explore the effects of electron density at
the frame structure on the bioactivity.
(2) The literature has reported that the aliphatic chain is
usually considered to be a good substituent to regulate the
solubility of target compounds. Given this, aliphatic chains at
different lengths are introduced into the phenyl ring at the 2-
position of pyrimidine ring in order to investigate the physico-
chemical characters of the designed compounds on the ability
against FAK.
(3) To get relatively comprehensive good structure–activity
relationships of the target compounds, another phenyl ring is
modied by an electron-withdrawing or electron-donating
group at different positions, such as methoxyl and carboxyl
moieties (Fig. 2).
To make this design more rational, molecular docking
between the title compounds and FAK was carried out rst (see
ESI†). The total score of compound 8a was demonstrated to be
6.04, and it could form two hydrogen bonds with the FAK
residues GLU430 and CYS502 through the O and N atoms,
Fig. 3 Molecular docking of compound 8a with FAK (PDB code 4c7t):
(left) the lowest energy binding modes; (right) hydrogen bond
interactions.
which motivates our interest in further investigating its poten-
tiality as a small molecular antitumor-agent targeting FAK
(Fig. 3).
The noninvasive imaging of FAK by positron emission
tomography (PET) has the ability to not only provide an inno-
vative pharmacological approach for the diagnosis of diseases
but can also contribute to a better understanding of the physi-
ological and pathophysiological functions of FAK.23 18F is the
most favorable positron emission isotope out of all the
commonly used ones, which is mainly due to its longer physical
half-life of 110 min, its comparable size to the H atom, and its
lower positron energy.24 However, until now, only a few research
studies on 18F-labeled FAK imaging agents have been reported.
Therefore, there is still a need to develop an 18F-labeled FAK
imaging agent with high effect and low toxicity, and for
providing useful diagnostic tools to investigate diseases related
to FAK, including cancer.25
In this paper, all the newly synthesized compounds were
evaluated for their in vitro inhibitory activity against FAK, and
some target compounds were radiolabeled by 18F. The radio-
active pyrimidine derivatives were further investigated via
partition coefficients determination, in vitro stability studies,
and in vivo biodistribution studies in S180-bearing mice to
better support their potency as PET imaging agents for cancer
detection. Importantly, microPET imaging was also studied to
develop new F-18 labeled PET imaging agents based on pyrim-
idine derivatives.
Experimental
General methods
All the chemicals and reagents used in the study were of
commercial quality and were used as purchased. Proton nuclear
magnetic resonance (1H NMR) spectra were acquired using
a BRUKER AVANCE® III HD 400 Spectrometer in CDCl3 solu-
tions at room temperature. Chemical shis were given in
chemical shi (d) as parts per million (ppm) relative to tetra-
methylsilane (TMS), which was used as an internal standard,
and were referenced to the centerline of deuterochloroform
Fig. 1 The structures of some classical FAK inhibitors.
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1
(7.27 ppm H NMR). The ESI-MS spectra were determined on raw material of 1 was completely consumed by that time), the
a Waters Quattro Micro® Quadrupole Mass Spectrometer. Thin reaction mixture was ltered, and the ltrate was concentrated
layer chromatography was performed on glass plates coated in vacuo. The residue was puried by column chromatography
with 60 GF254 silica. Plates were visualized using UV light (254 (dichloromethane to dichloromethane/MeOH, 100/1) (Caution!
nm). Flash column chromatography was carried out using an Using petroleum ether/AcOEt as the eluent would cause the
Interchim Puriash® 4100 medium pressure preparative chro- crystallization of the product in the column, leading to blocking
matography apparatus on silica gel (Bonna-Agela® ash silica, of the column!) to give relatively pure intermediate 2 as a light
˚
40–60 mm, 60 A).
yellow solid (2.94 g). (Note: a small part of the superuous raw
Truncated human FAK(PTK2) [376-1052(end) amino acids material TsO–CH2–CH2–OTs was included and was unable to be
of accession number NP_722560.1] was provided by Carna completely separated from the intermediate 2, since the polarity
Biosciences, Inc. The HTRF KinEASE®-TK kit was purchased of the raw material TsO–CH2–CH2–OTs was close to that of the
from Cisbio Biosciences, Inc.
intermediate 2. Therefore, the 1H NMR analysis was not per-
The [18F]uoride used for the radiosynthesis was produced formed in this step of the reaction. However, the remaining raw
by the 18O(p, n)18F nuclear reaction by irradiation on 97% material TsO–CH2–CH2–OTs included in the relatively pure
enriched H218O at the Nuclear Medicine Department of Peking intermediate 2 had little effect on the next step of reaction, and
Cancer Hospital (Beijing, China). QMA light ion-exchange the mole of 2 used in the next step of the reaction was calculated
cartridges and C-18 light Sep-Pak® cartridges were obtained as the hypothetical mass of pure 2 divided by the relative
from Waters (Milford, MA). We activated the Waters Sep-Pak® molecular mass of 2.)
Accell™ Plus QMA Plus Light Cartridges (130 mg sorbent per
cartridge, 37–55 mm particle size) with 1 N NaHCO3 (10 mL), Synthesis of intermediate 3
followed by deionized water (10 mL), and the Waters Sep-Pak®
C18 Plus Light Cartridges (130 mg sorbent per cartridge, 55–105
mm particle size) with methanol (10 mL) and deionized water
(10 mL) before use. Radiopharmaceuticals were puried and
their radiochemical purity were determined on a Shimadzu®
LC-20AT HPLC apparatus equipped with a SPD-20A UV detector
To a solution of 2 (2.94 g, 8.71 mmol) in dichloromethane (15
mL) and glacial acetic acid (15 mL) was added zinc powder
(2.83 g, 43.57 mmol). The mixture was stirred at ambient
temperature for 12 h under an inert atmosphere. The reaction
mixture was ltered, and the ltrate was concentrated in vacuo.
The crude intermediate 3 (2.02 g) was immediately used for the
next step of the reaction without further purication since the
intermediate 3 was unstable in air. However, according to the
(l ¼ 254 nm) and Bioscan® ow count 3200 NaI/PMT g-radia-
tion scintillation detector. We performed the radiopharmaceu-
tical HPLC separations on an Inertsil® ODS-3 C18 reverse phase
immediate ESI-MS analysis of the reaction mixture, we could
semi-preparative column (GL Sciences, Inc. 5 mm, 10 mm ꢀ 250
obviously nd the characteristic peak of 3: 308.2 (C15H18NO4S,
mm), and we carried out the elution with a binary gradient
[M + H]+), 330.1 (C15H17NO4SNa, [M + Na]+). Furthermore, the
system at a ow rate of 2.0 mL minꢁ1. Their radiochemical
mole of 3 used in the next step of the reaction was also calcu-
lated as the hypothetical mass of pure 3 divided by the relative
molecular mass of 3.
purity determinations were achieved on a Kromasil® 100-5C18
reverse phase column (AkzoNobel, 5 mm, 4.6 mm ꢀ 250 mm),
and elution was carried out with a binary gradient system at
a ow rate of 2.0 mL minꢁ1
.
Synthesis of intermediate 6a
S180 ascites sarcoma mice were purchased from Beijing
Vitalriver Animal Technology Co., Ltd, and normal ICR mice (20–
25 g, female) were provided by Beijing Xinglong Animal Tech-
nology Co., Ltd. 18F radioactivity of the tissues and organs of
interest was measured on a PerkinElmer® 2480 WIZARD2 auto-
matic gamma counter. The microPET imaging was performed on
a SuperArgus PET/CT, which was designed and manufactured by
Here, 4-methoxyphenyl amine (150 mmol) was added to a solu-
tion of 5-bromo-2,4-dichloropyrimidine 4 (100 mmol) in i-PrOH
(200 mL) at ambient temperature. The rst batch of the precip-
itant appeared aer a few minutes of stirring at room tempera-
ture. Aer ltration, to the ltrate was added the solution of
K2CO3 (300 mmol) in water (200 mL), and the reaction mixture
was stirred at room temperature until all of the 5-bromo-2,4-
dichloropyrimidine 4 was consumed completely, as shown by
TLC monitoring. The resulting second batch of the precipitant
was ltered and the lter cake was washed with water and ethyl
acetate in sequence. Aer desiccation, the two batches of
precipitant were combined to give the 5-bromo-2-chloro-N-
phenylpyrimidin-4-amines 6a as a gray solid (16.17 g, 51.4%). 1H
NMR (400 MHz, CDCl3, d ppm): 8.25 (s, 1H), 7.47 (d, J ¼ 8.0 Hz,
2H), 7.16 (br, 1H), 6.93 (d, J ¼ 8.0 Hz, 2H), 3.83 (s, 3H). ESI-MS:
calcd for C11H10BrClN3O ([M + H]+) 314.0, found 314.3 ([M + H]+).
˜
SEDECAL (Sociedad Espanola de Electromedicina y Calidad,
S.A.). All the protocols requiring the use of mice were in accor-
dance with the “guidelines for humane treatment of laboratory
animals” promulgated by the National Health and Family Plan-
ning Commission of China, and were approved by the Animal
Care Committee of Beijing Normal University.
Synthesis of intermediate 2
A mixture of ethane-1,2-diyl bis(4-methylbenzenesulfonate)
(13.58 g, 36.66 mmol, 1.5 equiv.), K2CO3 (12.67 g, 91.65 mmol,
3.75 equiv.) and 18-crown-6 (0.32 g, 1.22 mmol, 0.05 equiv.) was
Synthesis of intermediate 6b
added to a solution of 1 (3.40 g, 24.41 mmol) in acetone (30 mL). A similar reaction to that described above to prepare 6a was
Aer stirring at 56 ꢂC for 3–4 h (TLC monitoring showed that the used to obtain 6b as a gray solid (4.9 g, 55%). 1H NMR (400 MHz,
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DMSO-d6) d: 3.66 (s, 3H), 3.76 (s, 6H), 7.01 (s, 1H), 8.45 (s, 1H), 3.66–3.71 (m, 2H), 2.45 (s, 3H) (the H NMR data of 14a were
9.18 (s, br, 1H). ESI-MS: calcd for C13H14BrClN3O3 ([M + H]+) identical with those in the literature27).
374.0, found 373.9 ([M + H]+).
Synthesis of intermediate 14b
Synthesis of intermediate 6c
A similar reaction to that described above to prepare 14a was
used to obtain 14b as a colorless oil (15 g, 58%). H NMR (400
1
A similar reaction to that described above to prepare 6a was
1
used to obtain 6c as a white solid (3.5 g, 49%). H NMR (400
MHz, CDCl3): d 7.80 (d, 2H, J ¼ 7.8 Hz), 7.35 (d, 2H, J ¼ 7.8 Hz),
MHz, DMSO-d6) d: 7.74 (d, J ¼ 8.4 Hz, 2H), 7.83 (d, J ¼ 8.8 Hz,
2H), 8.55 (s, 1H), 9.55 (s, br, 1H). ESI-MS: calcd for C11H7-
BrClF3N3 ([M + H]+) 351.9, found 352.1 ([M + H]+).
4.20 (t, 2H, J ¼ 4.0 Hz), 3.66–3.71 (m, 4H), 3.52–3.55 (m, 2H),
2.45 (s, 3H) (the 1H NMR data of 14b were identical with those in
the literature28).
Synthesis of intermediate 6d
Synthesis of intermediate 15a
A similar reaction to that described above to prepare 6a was
used to obtain 6d as a light yellow solid (3.8 g, 54%). H NMR
(400 MHz, DMSO-d6) d: 9.51 (s, br, 1H), 8.42 (s, 1H), 7.92 (d, J ¼
8.4 Hz, 2H), 7.67 (d, J ¼ 8.8 Hz, 2H), 4.29 (q, J ¼ 6.8 Hz, 2H), 1.32
(t, J ¼ 6.8 Hz, 3H). ESI-MS: calcd for C13H12BrClN3O2 ([M + H]+)
356.0, found 356.1 ([M + H]+).
A mixture of 14a (12 g, 0.056 mol), 4-nitrophenol (9.3 g, 0.067
1
mol), and K2CO3 (13.8 g, 0.1 mol) in DMF (50 mL) was heated at
ꢂ
75 C overnight. The mixture was ltered and the ltrate was
concentrated in vacuo. The residue was recrystallized from
methanol to give 15a (4.5 g, 14%) as a light yellow solid. 1H NMR
(CDCl3): d 8.10 (d, 2H, J ¼ 8.0 Hz), 7.01–7.06 (m, 2H), 4.09–4.14
(m, 2H), 3.89–3.92 (m, 2H) (the 1H NMR data of 15a were
identical with those in the literature29).
Synthesis of intermediate 10a
TBAF (36 mL, 1 M in THF) was added to a solution of ethylene
di(p-toluenesulfonate), 9a (11.1 g, 30 mmol), and 80 mL THF
with stirring. The mixture was reuxed overnight and the
solvent was removed under reduced pressure. The residue was
partitioned between water and ethyl acetate. The organic phase
was dried over anhydrous sodium sulfate and ltered. Aer
concentration, the crude product was puried via silica gel
column chromatography by eluting with ethyl acetate/hexane
(1/5, v/v) to give 10a as a slightly yellow oil (3.27 g, 50% yield).
1H NMR (300 MHz, CDCl3): d 7.85 (d, J ¼ 8.2 Hz, 2H), 7.40 (d, J ¼
8.2 Hz, 2H), 2.49 (s, 3H), 4.69 (t, J ¼ 4.5 Hz, 1H), 4.53 (t, J ¼
4.5 Hz, 1H), 4.35 (t, J ¼ 4.5 Hz, 1H), 4.26 (t, J ¼ 4.2 Hz, 1H) (the
1H NMR data of 10a were identical with those in the literature26).
Synthesis of intermediate 15b
A similar reaction to that described above to prepare 15a was
used to obtain 15b as a light yellow solid (10 g, 76%). H NMR
(CDCl3): d 8.10 (d, 2H, J ¼ 8.0 Hz), 7.01–7.06 (m, 2H), 4.09–4.14
(m, 2H), 3.89–3.92 (m, 4H), 3.75–3.78 (m, 2H) (the 1H NMR data
of 15b were identical with those in the literature30).
1
Synthesis of intermediate 11a
A similar reaction to that described above to prepare 15a was
1
used to obtain 11a as a light yellow solid (9 g, 74%). H NMR
(400 MHz, CDCl3): d 8.23 (d, 2H, 3J ¼ 9.0 Hz), 7.00 (d, 2H, 3J ¼ 9.0
Hz), 4.80 (dt, 2H, 3JH–H ¼ 4.4 Hz, 2JH–F ¼ 47.6 Hz), 4.32 (dt, 2H,
Synthesis of intermediate 10b
3
1
3JH–H ¼ 4.0 Hz, JH–F ¼ 27.6 Hz) (the H NMR data of 11a were
identical with those in the literature31).
10b was prepared from 9b (10.25 g, 24.7 mmol) as described
above for 10a. The crude product was puried by silica gel
column chromatography by eluting with ethyl acetate/hexane
Synthesis of intermediate 12a
1
(3/10, v/v) to give 10b as a colorless oil (2.66 g, 41% yield). H
A similar reaction to that described above to prepare 15a was
used to obtain 12a as a light yellow solid (7.5 g, 68%). 1H NMR
(300 MHz, CDCl3): d 4.141 (dt, J ¼ 1.0 Hz, J ¼ 1.0 Hz, 2H), 4.715
(dt, J ¼ 2.0 Hz, J ¼ 1.0 Hz, 2H), 6.650 (d, J ¼ 2.0 Hz, 2H), 6.771 (d,
NMR (300 MHz, CDCl3): d 7.81 (d, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼
8.1 Hz, 2H), 4.57 (t, J ¼ 4.2 Hz, 1H), 4.41 (t, J ¼ 4.2 Hz, 1H), 4.18
(t, J ¼ 4.8 Hz, 2H), 3.69–3.76 (m, 3H), 3.63 (t, J ¼ 4.2 Hz, 1H), 2.45
(s, 3H) (the 1H NMR data of 10b were identical with those in the
literature26).
1
J ¼ 2.0 Hz, 2H) (the H NMR data of 11a were identical with
those in the literature32).
Synthesis of intermediate 14a
Synthesis of intermediate 16a
Briey, to a mixture of 13a (124 g, 2 mol) and triethylamine
(12 g, 0.12 mol) in dry DCM (100 mL) was ꢂadded TsCl (19 g, 0.1 A mixture of 15a (4.5 g, 25 mmol) and 7.5% Pd/C (0.45 g) in
mol) in dry DCM (100 mL) dropwise at 0 C. The solution was ethanol (50 mL) was stirred under a H2 atmosphere for 5 h. The
ꢂ
stirred at 0 C for 3 h. Then, the reaction was quenched with mixture was ltered and the ltrate was concentrated in vacuo.
water. Aer extraction with DCM, the organic phase was washed The crude 16a (2.6 g) was used in the next step without further
with water two times. Aer removal of the solvent, the residue purication since the intermediate 16a was unstable in air.
was puried by silica gel chromatography to give 14a (12 g, Furthermore, the mole of 16a used in the next step of the
37.1%) as a colorless oil. 1H NMR (400 MHz, CDCl3): d 7.81 (d, reaction was also calculated as the hypothetical mass of pure
2H, J ¼ 8.0 Hz), 7.35 (d, 2H, J ¼ 8.0 Hz), 4.15 (t, 2H, J ¼ 4.0 Hz), 16a divided by the relative molecular mass of 16a.
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Synthesis of intermediate 16b
Synthesis of intermediate 17b
A similar reaction to that described above to prepare 16a was A similar reaction to that described above to prepare 7 was used
used to obtain the crude 16b as a red oil (3.2 g), which was then to obtain the crude 17b (0.62 g), which was then used in the next
used in the next step without further purication since the step without further purication since the intermediate 17b was
intermediate 16b was unstable in air. Furthermore, the mole of unstable in air. Furthermore, the mole of the 17b used in the
16b used in the next step of the reaction was also calculated as next step of the reaction was also calculated as the hypothetical
the hypothetical mass of pure 16b divided by the relative mass of pure 17b divided by the relative molecular mass of 17b.
molecular mass of 16b.
Synthesis of intermediate 17c
A similar reaction to that described above to prepare 7 was used
to obtain the crude 17c (0.65 g), which was then used in the next
step without further purication since the intermediate 17c was
unstable in air. Furthermore, the mole of 17c used in the next
step of reaction was also calculated as the hypothetical mass of
pure 17c divided by the relative molecular mass of 17c.
Synthesis of intermediate 12a
A similar reaction to that described above to prepare 16a was
used to obtain the crude 12a as a red oil (5.4 g), which was then
used in the next step without further purication since the
intermediate 12a was unstable in air. Furthermore, the mole of
12a used in the next step of the reaction was also calculated as
the hypothetical mass of pure 12a divided by the relative
molecular mass of 12a.
Synthesis of intermediate 17d
A similar reaction to that described above to prepare 7 was used
to obtain the crude 17d (0.64 g), which was then used in the next
step without further purication since the intermediate 17d was
unstable in air. Furthermore, the mole of 17d used in the next
step of the reaction was also calculated as the hypothetical mass
of pure 17d divided by the relative molecular mass of 17d.
Synthesis of intermediate 12b
A similar reaction to that described above to prepare 16a was
used to obtain the crude 12b as a red oil (4.1 g), which was then
used in the next step without further purication since the
intermediate 12b was unstable in air. Furthermore, the mole of
12b used in the next step of the reaction was also calculated as
the hypothetical mass of pure 12b divided by the relative
molecular mass of 12b.
Synthesis of intermediate 17e
A similar reaction to that described above to prepare 7 was used
to obtain the crude 17e (0.72 g), which was then used in the next
step without further purication since the intermediate 17e was
unstable in air. Furthermore, the mole of 17e used in the next
step of the reaction was also calculated as the hypothetical mass
of pure 17e divided by the relative molecular mass of 17e.
Synthesis of precursor 7
A mixture of the crude compound 3 (0.91 g, 3.0 mmol) and
intermediate 6a (0.62 g, 2.0 mmol) in 1,4-dioxane (20 mL) using
p-toluenesulfonic acid (0.14 g, 0.8 mmol) as the catalyst was
stirred under reux under an inert atmosphere. When inter-
mediate 6a was completely consumed (as monitored by TLC,
petroleum ether/ethyl acetate, 3/1, v/v), it was concentrated
under reduced pressure. The resulting residue was puried by
silica gel column chromatography by eluting with petroleum
ether/ethyl acetate (6/1–2/1, v/v) to give the pure compound 7 (60
mg) with the yield of 5.2%. 1H NMR (400 MHz, CDCl3, d ppm):
7.94 (s, 1H), 7.81 (d, J ¼ 8.3 Hz, 4H), 7.30–7.40 (m, 8H), 4.36 (t, J
¼ 4.7 Hz, 2H), 4.13 (t, J ¼ 4.5 Hz, 2H), 3.86 (s, 3H), 2.44 (s, 3H);
13C NMR (100 MHz, DMSO-d6, d ppm): 158.71, 157.92, 157.24,
153.07, 153.00, 145.52, 135.06, 134.70, 132.79, 130.65, 128.17,
124.87, 121.18, 116.73, 114.77, 92.81, 69.70, 66.12, 55.65, 21.58;
HRMS (ESI): calcd for C26H26BrN4O5S ([M + H]+) 585.0729,
found 585.0713 ([M + H]+).
Synthesis of intermediate 17f
A similar reaction to that described above to prepare 7 was used
to obtain the crude 17f (0.69 g), which was then used in the next
step without further purication since the intermediate 17f was
unstable in air. Furthermore, the mole of 17f used in the next
step of the reaction was also calculated as the hypothetical mass
of pure 17f divided by the relative molecular mass of 17f.
Synthesis of precursor 18a
Briey, to a mixture of 17a (570 mg, 1.2 mmol), triethylamine
(202 mg, 2 mmol), and 4-dimethylaminepyridine (14 mg, 0.12
mmol) in dry DCM (10 mL) was added TsCl (380 mg, 2 mmol) in
ꢂ
dry DCM (10 mL) dropwise at 0 C. The solution was stirred at
0 ꢂC for 3 h. Then, the reaction was quenched with water. Aer
extraction with DCM, the organic phase was washed with water
two times. Aer removal of the solvent, the residue was puried
Synthesis of intermediate 17a
A similar reaction to that described above to prepare 7 was used by silica gel chromatography to give 18a (0.30 g, 45%) as a red
1
to obtain the crude 17a (0.57 g), which was then used in the next solid. H NMR (400 MHz, DMSO-d6): d 9.18 (s, br, 1H), 8.48 (s,
step without further purication since the intermediate 17a was br, 1H), 8.16 (s, 1H), 7.79 (d, J ¼ 8.0 Hz, 2H), 7.47 (d, J ¼ 8.8 Hz,
unstable in air. Furthermore, the mole of the 17a used in the 4H), 6.91 (s, 2H), 6.63 (d, J ¼ 9.2 Hz, 2H), 4.30 (s, 2H), 4.06 (s,
next step of the reaction was also calculated as the hypothetical 2H), 3.67–3.68 (m, 9H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSO-
mass of pure 17a divided by the relative molecular mass of 17a. d6, d ppm): 158.71, 157.92, 157.24, 153.07, 153.00, 145.52,
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135.06, 134.70, 132.79, 130.65, 128.17, 121.18, 116.73, 114.77,
92.81, 69.70, 66.12, 60.65, 56.22, 21.58; HRMS (ESI): calcd for
C
Synthesis of precursor 18f
A similar reaction to that described above to prepare 18a was
used to obtain 18f as a light yellow solid (0.58 g, 66%). 1H NMR
(DMSO-d6) d: 9.27 (s, br, 1H), 8.81 (s, br, 1H), 8.26 (s, 1H), 7.88–
7.90 (m, 4H), 7.78 (d, J ¼ 8.0 Hz, 2H), 7.41–7.49 (m, 4H), 6.80 (d, J
¼ 9.2 Hz, 2H), 4.29 (q, J ¼ 7.2 Hz, 2H), 4.15 (t, J ¼ 4.0 Hz, 2H),
3.96 (t, J ¼ 4.0 Hz, 2H), 3.64–3.67 (m, 4H), 2.36 (s, 3H), 1.31 (t, J
¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3, d ppm): 166.22,
158.89, 157.44, 155.80, 155.09, 144.91, 142.43, 133.00, 132.43,
130.65, 129.91, 128.07, 125.43, 122.80, 119.87, 114.96, 94.05,
70.00, 69.38, 68.99, 67.81, 60.95, 21.73, 14.49; HRMS (ESI):
calcd for C30H32BrN4O7S ([M + H]+) 671.1097, found 671.1213
([M + H]+).
28H30BrN4O7S ([M + H]+) 645.0940, found 645.1007 ([M + H]+).
Synthesis of precursor 18b
A similar reaction to that described above to prepare 18a was
used to obtain 18b as a light yellow solid (0.53 g, 66%). 1H NMR
(DMSO-d6) d: 9.26 (s, br, 1H), 8.86 (s, br, 1H), 8.26 (s, 1H), 7.89
(d, J ¼ 7.6 Hz, 2H), 7.79 (d, J ¼ 8.0 Hz, 2H), 7.66 (d, J ¼ 8.4 Hz,
2H), 7.43–7.47 (m, 4H), 6.71 (d, J ¼ 8.8 Hz, 2H), 4.32 (t, J ¼
4.0 Hz, 2H), 4.10 (t, J ¼ 4.0 Hz, 2H), 2.40 (s, 3H); 13C NMR (100
MHz, DMSO-d6, d ppm): 158.73, 156.82, 153.34, 145.47, 143.25,
134.27, 132.79, 130.61, 128.16, 125.87, 124.11, 124.06, 123.85,
123.09, 121.70, 114.83, 69.67, 66.04, 21.55; HRMS (ESI):
calcd for C26H23BrF3N4O4S ([M + H]+) 623.0497, found 623.0568
([M + H]+).
Synthesis of F-19 standard 8a
To a stirred solution of compound 7 (30 mg, 0.05 mmol) in dry
THF (4 mL), tetrabutylammonium uoride (0.5 mL, 1 M in THF)
was added. The mixture was heated using a digestion high-
pressure tank at 100 ꢂC for 6–7 h. Aer the intermediate 7
was completely consumed (as monitored by TLC, petroleum
ether/ethyl acetate, 6/1, v/v), the reaction was quenched with
water, and then the mixture was extracted with ethyl acetate (3
ꢀ 10 mL). The combined organic phase was dried over anhy-
drous sodium sulfate and concentrated under reduced pres-
sure, which was further puried by silica gel column
chromatography eluted with petroleum ether/ethyl acetate (10/
1–4/1, v/v) to give the pure compound 8a (1.4 mg) as a white
Synthesis of precursor 18c
A similar reaction to that described above to prepare 18a was
used to obtain 18c as a light yellow solid (0.52 g, 60%). 1H NMR
(DMSO-d6) d: 9.26 (s, br, 1H), 8.80 (s, br, 1H), 8.25 (s, 1H), 7.87–
7.90 (m, 4H), 7.79 (d, J ¼ 8.4 Hz, 2H), 7.46 (d, J ¼ 8.4 Hz, 4H),
6.71 (d, J ¼ 8.8 Hz, 2H), 4.29–4.32 (m, 4H), 4.10–4.11 (m, 2H),
2.39 (s, 3H), 1.32 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3,
d ppm): 166.21, 158.61, 157.15, 155.86, 154.43, 145.05, 142.29,
132.94, 132.69, 130.68, 129.97, 128.14, 125.62, 122.62, 119.97,
115.03, 68.26, 65.92, 61.00, 21.55, 14.50; HRMS (ESI): calcd for
1
C
28H28BrN4O6S ([M + H]+) 627.0835, found 627.0901 ([M + H]+).
solid. Yield: 6.3%; H NMR (CDCl3, 400 MHz) d: 7.82 (s, 1H),
7.30 (d, J ¼ 8.8 Hz, 4H), 6.83 (d, J ¼ 8.6 Hz, 2H), 6.75 (d, J ¼
8.5 Hz, 2H), 4.75 (t, J ¼ 4.1 Hz, 1H), 4.63 (t, J ¼ 3.9 Hz, 1H), 4.16
(t, J ¼ 3.9 Hz, 1H), 4.10 (t, J ¼ 4.0 Hz, 1H), 3.78 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d: 158.74, 157.93, 157.24, 153.51, 152.99,
135.07, 134.67, 134.47, 122.67, 121.33, 116.71, 114.74, 92.78,
Synthesis of precursor 18d
A similar reaction to that described above to prepare 18a was
used to obtain 18d as a light yellow solid (0.52 g, 60%). 1H NMR
(DMSO-d6) d: 9.17 (s, br, 1H), 8.46 (s, br, 1H), 8.16 (s, 1H), 7.78
(d, J ¼ 8.4 Hz, 2H), 7.49 (d, J ¼ 9.2 Hz, 2H), 7.44 (d, J ¼ 8.0 Hz,
2H), 6.91 (s, 2H), 6.70 (d, J ¼ 9.2 Hz, 2H), 4.14 (t, J ¼ 4.0 Hz, 2H),
3.92 (t, J ¼ 4.0 Hz, 2H), 3.64–3.68 (m, 13H), 2.38 (s, 3H); 13C NMR
(100 MHz, DMSO-d6, d ppm): 158.73, 157.89, 157.24, 153.75,
152.99, 145.38, 135.07, 134.67, 134.21, 132.93, 130.60, 128.12,
121.30, 114.61, 102.10, 92.72, 70.47, 69.46, 68.53, 67.67, 60.61,
56.21, 55.61, 21.57; HRMS (ESI): calcd for C30H33BrN4O6S ([M +
H]+) 689.1202, found 689.1317 ([M + H]+).
83.58, 81.91, 67.87, 67.68, 55.61; HRMS (ESI): calcd for C19
H19BrFN4O2 ([M + H]+) 433.0597, found 433.0621 ([M + H]+).
-
Synthesis of F-19 standard 8b
A similar reaction to that described above to prepare 7 was used
to obtain 8b (0.46 g, 68%) as a white solid; 1H NMR (400 MHz,
DMSO-d6) d: 9.18 (s, 1H), 8.47 (s, 1H), 8.16 (s, 1H), 7.50 (d, 2H),
6.91 (s, 2H), 6.75 (d, 2H), 4.79–4.75 (m, 1H), 4.67–4.63 (m, 1H),
4.17 (s, 1H), 4.12–4.07 (m, 1H), 3.69–3.65 (m, 10H); 13C NMR
(100 MHz, DMSO-d6) d: 158.74, 157.93, 157.24, 153.51, 152.99,
135.07, 134.67, 134.47, 121.33, 114.74, 102.11, 92.78, 83.58,
81.91, 67.87, 67.68, 60.61, 56.21, 55.61; HRMS (ESI): calcd for
Synthesis of precursor 18e
A similar reaction to that described above to prepare 18a was
used to obtain 18e as a light yellow solid (0.57 g, 61%). 1H NMR
(DMSO-d6) d: 9.25 (s, br, 1H), 8.85 (s, br, 1H), 8.25 (s, 1H), 7.90
(d, J ¼ 8.4 Hz, 2H), 7.78 (d, J ¼ 8.0 Hz, 2H), 7.66 (d, J ¼ 8.8 Hz,
2H), 7.42–7.47 (m, 4H), 6.78 (d, J ¼ 8.8 Hz, 2H), 4.14 (t, J ¼
C
21H23BrFN4O4 ([M + H]+) 493.0808, found 493.0887 ([M + H]+).
Synthesis of F-19 standard 8c
4.0 Hz, 2H), 3.95 (t, J ¼ 4.0 Hz, 2H), 3.64 (q, J ¼ 4.4 Hz, 4H), 2.37 A similar reaction to that described above to prepare 7 was used
1
(s, 3H); 13C NMR (100 MHz, CDCl3, d ppm): 158.92, 157.60, to obtain 8c (0.69 g, 90%) as a white solid; H NMR (400 MHz,
155.92, 155.15, 144.93, 141.38, 132.99, 132.40, 129.91, 128.08, DMSO-d6) d: 9.26 (s, 1H), 8.85 (s, 1H), 8.25 (s, 1H), 7.89 (d, 2H),
126.10, 125.63, 125.41, 125.16, 123.36, 122.87, 120.66, 114.93, 7.66 (d, 2H), 7.47 (d, 2H), 6.83 (d, 2H), 4.80–4.75 (m, 1H), 4.70–
102.10, 93.91, 70.00, 69.35, 68.98, 67.81, 21.72; HRMS (ESI): 4.63 (m, 1H), 4.23–4.17 (m, 1H), 4.15–4.11 (m, 1H); 13C NMR
calcd for C28H27BrF3N4O5S ([M + H]+) 667.0759, found 667.0865 (100 MHz, DMSO-d6) d: 158.81, 156.82, 153.84, 143.26, 134.14,
([M + H]+).
125.82, 124.11, 123.84, 123.10, 121.96, 114.80, 83.27, 82.17,
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67.78, 67.66; HRMS (ESI): calcd for C19H16BrFN4O4 ([M + H]+) stirred at 90 ꢂC. When the compound 8d was completely
471.0365, found 471.0440 ([M + H]+).
consumed (as monitored by TLC, petroleum ether/ethyl acetate,
6/1, v/v), the mixed solution was concentrated under reduced
pressure. The pH of the resulting residue was adjusted to 1–2 by
1 M HCl (aq.), and then ltrated and washed with water to get
Synthesis of F-19 standard 8d
A similar reaction to that described above to prepare 7 was used
to obtain 8d (0.94 g, 78%) as a white solid; 1H NMR (400 MHz,
DMSO-d6) d: 9.26 (s, 1H), 8.80 (s, 1H), 8.26 (s, 1H), 7.94–7.80 (m,
4H), 7.49 (d, 2H), 6.85 (d, 2H), 4.82–4.76 (m, 1H), 4.70–4.63 (m,
1H), 4.30 (q, 2H), 4.24–4.19 (m, 1H), 4.17–4.12 (m, 1H), 1.32 (t,
3H); 13C NMR (100 MHz, CDCl3) d: 166.23, 158.81, 157.33,
155.83, 154.92, 142.35, 132.59, 130.67, 125.55, 122.90, 119.92,
115.08, 94.15, 82.66, 81.52, 67.69, 67.55, 60.97, 14.47; HRMS
(ESI): calcd for C21H21BrFN4O3 ([M + H]+) 475.0703, found
475.0784 ([M + H]+).
1
the pure compound 8h (0.30) as a white solid. Yield: 63%; H
NMR (400 MHz, DMSO-d6) d: 9.26 (s, 1H), 8.75 (s, 1H), 8.25 (s,
1H), 7.95–7.78 (m, 4H), 7.49 (d, 2H), 6.84 (d, 2H), 4.82–4.76 (m,
1H), 4.70–4.64 (m, 1H), 4.24–4.19 (m, 1H), 4.17–4.11 (m,
1H) ppm; 13C NMR (100 MHz, DMSO-d6) d: 167.83, 165.99,
158.81, 158.56, 156.67, 153.80, 144.08, 143.59, 134.14, 130.25,
126.08, 124.63, 121.97, 114.81, 93.35, 83.57, 81.92, 67.84, 67.65,
60.96; HRMS (ESI): calcd for C19H16BrFN4O3 ([M + H]+)
447.0390, found 447.0461 ([M + H]+).
Synthesis of F-19 standard 8i
Synthesis of F-19 standard 8e
Compound 8i (0.37 g) was obtained as a white solid according to
the general procedure described for 8h starting from the
pyrimidine derivative 8g (0.50 g, 0.96 mmol) and potassium
A similar reaction to that described above to prepare 7 was used
to obtain 8e (1.20 g, 82%) as a white solid; H NMR (400 MHz,
1
DMSO-d6) d: 9.16 (s, 1H), 8.46 (s, 1H), 8.16 (s, 1H), 7.60–7.40 (m,
3H), 6.91 (s, 3H), 6.77–6.69 (m, 3H), 4.68–4.55 (m, 1H), 4.52–4.44
(m, 1H), 4.04–3.95 (m, 3H), 3.79–3.72 (m, 4H), 3.69–3.64 (m,
13H); 13C NMR (100 MHz, DMSO-d6) d: 158.76, 157.93, 157.23,
153.80, 152.99, 135.07, 134.67, 134.21, 121.32, 114.63, 102.10,
92.70, 84.39, 82.74, 70.41, 70.22, 69.58, 67.78, 60.61, 56.21,
55.60; HRMS (ESI): calcd for C23H27BrFN4O5 ([M + H]+)
537.1071, found 537.1141 ([M + H]+).
1
hydroxide (2.00 g, 35.7 mmol). Yield: 78%; H NMR (400 MHz,
DMSO-d6) d: 12.72 (s, 1H), 9.25 (s, 1H), 8.75 (s, 1H), 8.25 (s, 1H),
7.91–7.80 (m, 4H), 7.54–7.42 (m, 2H), 6.88–6.78 (m, 2H), 4.64–
4.58 (m, 1H), 4.52–4.46 (m, 1H), 4.06 (t, 2H), 3.80–3.73 (m, 3H),
3.71–3.66 (m, 1H) ppm; 13C NMR (100 MHz, DMSO-d6) d: 167.61,
165.96, 158.83, 158.59, 156.65, 154.13, 143.76, 133.85, 130.28,
130.06, 125.62, 124.63, 122.03, 114.73, 93.28, 84.40, 82.75,
70.40, 70.22, 69.57, 67.75, 60.95; HRMS (ESI): calcd for C19
H16BrFN4O3 ([M + H]+) 491.0652, found 491.0718 ([M + H]+).
-
Synthesis of F-19 standard 8f
A similar reaction to that described above to prepare 7 was used
to obtain 8f (1.08 g, 75%) as a white solid; H NMR (400 MHz,
FAK inhibitory assay
1
The inhibition tests of the title compounds 8a–8i against FAK
were performed using the HTRF® (Homogeneous Time-
Resolved Fluorescence Methodologies) kinEASE™ TK kit. The
concentration of the enzyme used in the assay, as well as the
enzymatic reaction time and the ATP concentration were opti-
mized before the compound testing. Here, 0.11 ng mLꢁ1 enzyme
was chosen to be used with 13.8 mM ATP to react for 50 min at
room temperature.
During the enzymatic reaction step, 4 mL of the compound
and 2 mL TK substrate–biotin were incubated with 2 mL kinase. 2
mL ATP was added to start the enzymatic reaction. Then, the
enzymatic buffer from the HTRF® kinEASE™ TK kit was added,
following by the addition of 5 mM MgCl2, 1 mM DTT, and 25 nM
SEB.
DMSO-d6) d: 9.25 (s, 1H), 8.85 (s, 1H), 8.25 (s, 1H), 7.97–7.78 (m,
2H), 7.70–7.62 (m, 2H), 7.50–7.41 (m, 2H), 6.86–6.76 (m, 2H),
4.65–4.56 (m, 1H), 4.52–4.45 (m, 1H), 4.10–3.99 (m, 2H), 3.82–
3.72 (m, 3H), 3.72–3.64 (m, 1H); 13C NMR (100 MHz, CDCl3) d:
158.84, 157.43, 155.94, 155.32, 141.33, 132.21, 126.16, 122.93,
120.66, 115.02, 83.85, 82.73, 70.76, 70.63, 70.06, 67.93; HRMS
(ESI): calcd for C21H20BrF4N4O2 ([M + H]+) 515.0628, found
515.0704 ([M + H]+).
Synthesis of F-19 standard 8g
A similar reaction to that described above to prepare 7 was used
1
to obtain 8g (1.28 g, 88%) as a white solid; H NMR (400 MHz,
DMSO-d6) d: 9.25 (s, 1H), 8.80 (s, 1H), 8.25 (s, 1H), 7.88 (s, 5H),
7.65–7.36 (m, 2H), 6.95–6.73 (m, 2H), 4.68–4.55 (m, 1H), 4.55–
4.43 (m, 1H), 4.37–4.23 (m, 3H), 4.10–4.01 (m, 3H), 3.82–3.72
(m, 4H), 3.70–3.63 (m, 1H), 1.35–1.30 (m, 4H); 13C NMR (100
MHz, CDCl3) d: 166.24, 158.98, 157.54, 155.77, 155.18, 142.45,
132.42, 130.65, 125.40, 122.84, 119.82, 115.00, 93.98, 83.86,
82.74, 70.76, 70.63, 70.07, 67.91, 60.95, 14.49; HRMS (ESI):
calcd for C23H25BrFN4O4 ([M + H]+) 519.0965, found 519.1032
([M + H]+).
The detection reagents (5 mL Eu3+-cryptate labeled TK-
antibody and 5 mL Steptavidin-XL665) dissolved in the detec-
tion buffer (in the presence of EDTA) were mixed and added to
the reaction system. The TR-FRET signal was proportional to the
phosphorylation level and was detected by a plate reader (BMG
FS) aer incubation for 1 h. For each compound, the IC50 value
was determined from a sigmoid dose–response curve using
GraphPad Prism (GraphPad Soware, San Diego, CA, USA).
Synthesis of F-19 standard 8h
Partition coefficient determination
A mixture of compound 8d (0.50 g, 1.1 mmol) and potassium The determination of the partition coefficients of the radio-
hydroxide (2.00 g, 35.7 mmol) in EtOH/H2O (20 mL, 1/1, v/v) was tracers [18F]-8a, [18F]-8c, [18F]-8h and [18F]-8i was carried out
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according to the following procedure: rst, the same volume of
n-octanol and PBS (phosphate buffer saline, 0.05 M, pH 7.4) was
mixed, and each phase was presaturated with the opposite
phase overnight before use. Then, to a centrifuge tube was
added a solution of 18F-labeled tracer (370 kBq, 10 mCi) in 0.1
mL normal saline, 0.9 mL PBS (presaturated with the opposite
phase overnight), and 1.0 mL n-octanol (presaturated with the
opposite phase overnight) in sequence. The centrifuge tube was
vortexed for 0.5–1.0 min at room temperature, followed by
centrifugation for 5 min at 5000 rpm. Aer standing for a while,
two samples were transferred from the n-octanol (100 mL) and
PBS (100 mL) layers to the counting tubes and were measured in
a gamma counter. The partition coefficient was expressed as the
logarithm of the ratio of the radioactivity from the n-octanol
phase versus the PBS phase. The measurement was done in
triplicate and repeated three times.
MicroPET imaging
The S180-tumor-bearing mice were injected intravenously with
approximately 3.7 MBq of [18F]-8a in 300 mL normal saline,
anesthetized with 1.5% isourane in air (about 1.5 mL minꢁ1),
and xed near the center of the microPET scanner (SuperArgus
microPET/CT). Static scans (for 30 min and 60 min, 10 min
scans for each time point) were obtained on a SuperArgus
microPET/CT. Then, the images were reconstructed with the
SEDECAL reconstruction soware and the images were expor-
ted by MMWKS SUPERARGUS soware.
Results and discussion
Chemistry
The synthetic route used to prepare the pyrimidine derivative 8a
is shown in Scheme 1. The starting material 4-nitrophenol 1 was
treated with ethane-1,2-diyl bis(4-methylbenzenesulfonate) to
give compound 2, which was reduced by Zn to obtain
compound 3. Intermediate 6a was obtained by the reaction of 5-
bromo-2,4-dichloropyrimidine 4 and 4-methoxyaniline 5a in i-
PrOH using K2CO3 as a base at room temperature with a yield
of 78%. Compound 7 could be prepared by compounds 3 and 6a
in 1,4-dioxane at 100 ꢂC using p-toluene sulfonic acid as the
catalyst, which was further reuxed in THF in the presence of
TBAF to provide the target compound 8a.
The synthetic route used to prepare the pyrimidine deriva-
tives 8b–g is shown in Scheme 2. The uorinated products 10a–
b were obtained starting from the commercially available
materials 9a–b, followed by etherication and hydrogenation to
give the key intermediates 12a–b in excellent yields. Another
In vitro stability studies
[18F]-8a, [18F]-8c, [18F]-8h, and [18F]-8i (370 kBq, 10 mCi) aer
HPLC purication were dissolved in 100 mL of normal saline,
and then mixed with 500 mL of normal saline at 37 ꢂC for 1 h and
2 h, respectively. About 100 mL of the solution was analyzed by
radio-HPLC.
The in vitro stability of the 18F-labeled tracers in mouse
plasma was determined using the following procedure: (1) rst,
incubate 370 kBq of the HPLC-puried radiotracers in 100 mL of
normal saline with 500 mL of murine plasma at 37 ꢂC for 1 h and
2 h, respectively; (2) aer centrifuging the plasma proteins at
5000 rpm for 5 min at 4 ꢂC, they were precipitated by adding 200
mL of acetonitrile. The supernatant was collected. Approxi-
mately 100 mL of the supernatant solution was loaded for the
HPLC analysis.
In vivo biodistribution studies in S180-bearing mice
An S180 ascites sarcoma mouse was sacriced and the ascites
was immediately drawn off from the carcass and made into an
S180 sarcoma cell suspension (diluted four times by normal
saline). Then, the normal ICR mice were inoculated subcuta-
neously into the right front ank with the diluted ascites
sarcoma cell suspension (100 mL per mouse, containing about
5 ꢀ 106 tumor cells by cell count).
Scheme 1 Reagents and conditions: (i) ethane-1,2-diyl bis(4-meth-
ylbenzenesulfonate), K2CO3, 18-crown-6, 56 ꢂC; (ii) Zn, acetic acid,
room temperature, 12 h; (iii) i-PrOH, K2CO3, rt; (iv) TsOH$H2O, 1,4-
dioxane, 100 ꢂC; (v) TBAF, THF, reflux, 6–7 h.
The in vivo biodistribution studies could be conducted until
the tumors reached a size of 0.5–0.8 cm in diameter (i.e., for
about one week). A normal saline solution containing the 18F-
labeled tracers [18F]-8a, [18F]-8c, [18F]-8h, and [18F]-8i (370 kBq
per 100 mL) was injected via the tail vein for each of the S180-
tumor-bearing mice. The mice (n ¼ 5 for each time point)
were sacriced at 5, 15, 30, 60, and 120 min post-injection. The
tissues and organs of interest were anatomized and weighed,
and the radioactivity was determined with an automatic
g-counter. The percent dose per gram of wet tissue was calcu-
lated by a comparison of the tissue counts to suitably diluted
aliquots of the injected material. The values were expressed as
the mean ꢃ SD (n ¼ 5).
Scheme 2 Reagents and conditions: (i) TBAF, THF, N2, 80 ꢂC, 20 h; (ii)
p-nitrophenol, K2CO3, DMF, N2, 80 ꢂC, 20 h; (iii) CH3OH–THF, 7.5%
Pd–C, H2, rt, 4 h; (iv) i-PrOH, K2CO3 rt; (v) TsOH$H2O, 1,4-dioxane,
100 ꢂC, 3 h.
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important kind of intermediate 6b–d could be easily prepared ester moiety, the inhibitory activity is reduced a lot. Comparing
by compounds 4 and 5b–d, whereby the former further reacted compounds with n ¼ 1 (8a–d, 8h) to n ¼ 2 (8e–g, 8i), the
ꢂ
with compounds 12a–b in 1,4-dioxane at 100 C to provide the pyrimidine derivatives 8e–g and 8i generally showed stronger
target pyrimidine derivatives 8b–g using p-toluene sulfonic acid activity, which indicated that the length of the aliphatic chain
as the catalyst with 68–90% yields.
has an important effect on the FAK inhibitory activity.
The synthetic route used to prepare the pyrimidine deriva-
tives 8h and 8i is shown in Scheme 3. The target compounds 8h
and 8i with moderate yields were easily synthesized by pyrimi- Radiolabeling
dine derivatꢂives 8d and 8g in the mixed solution of ethanol and
Encouraged by the good inhibitory activity and docking results
water at 90 C in the presence of KOH.
of most of the newly prepared pyrimidine derivatives against
FAK, a radiouorination method for the preparation of [18F]-8
was developed with different reactions.
All the newly synthesized target pyrimidine derivatives were
characterized by 1H NMR, 13C NMR, and MS spectra. The
spectral data were in accordance with the assigned structures,
and all the spectral data are listed in the Experimental section.
Scheme 4 describes the synthesis of compound [18F]-8a,
which could be easily obtained with the yield of 20% by the
reaction of the tosylate precursor 7 with [18F]uoride/potassium
carbonate and Kryptox 2.2.2. in anhydrous acetonitrile using
FAK inhibitory assay
ꢂ
a digestion high-pressure tank at 100 C for 15 min.
The prepared compounds 8a–i were evaluated for their in vitro
inhibitory activities against FAK using 16-OTS-1 as the positive
control. The obtained results are shown in Table 1.
The synthetic route for the preparation of the radioactive
derivatives [18F]-8c, [18F]-8d, [18F]-8g, [18F]-8h, and [18F]-8i is
outlined in Scheme 5. The starting materials 13a–b were fol-
lowed by sulfonylation, etherication, and hydrogenation to
give the intermediates 16a–b, which further reacted with 6c and
6d to provide the pyrimidine derivatives 17a–c. Then the sul-
fonylated products 18a–c were treated with [18F]uoride/
potassium carbonate and Kryptox 2.2.2. in anhydrous aceto-
nitrile using a digestion high-pressure tank at 160 ꢂC for 15 min
to give the radioactive derivatives [18F]-8c (16%), [18F]-8d, and
The results in Table 1 demonstrated that all the target
compounds showed superior inhibition against FAK compared
to the reference 18c. The most active compound 8i displayed an
IC50 value of 0.060 mM against FAK, which was more than 500-
fold lower than 18c (IC50 ¼ 33.290 mM). When n ¼ 1, compound
8h with a carboxyl group exhibited strong inhibitory activity
with an IC50 value of 0.285 mM, while when n ¼ 2, the carboxyl-
group-modied derivative 8i was still the most active one. These
outcomes suggested that the carboxyl group was favorable to
exert FAK inhibition for this type of pyrimidine derivatives.
However, with the change of the carboxyl group into a carboxylic
Scheme 4 Reagents and conditions: (i) anhydrous KF, Kryptofix 2.2.2.,
anhydrous acetonitrile, 100 ꢂC, 15 min.
Scheme 3 Reagents and conditions: (i) (a) KOH, EtOH–H2O (1 : 1),
90 ꢂC; (b) 1 M HCl.
Table 1 SAR-table of target pyrimidine derivatives
FAK biochemical assay
Compds
n
R
IC50/mM
8a
8b
8c
8d
8e
8f
8g
8h
8i
1
1
1
1
2
2
2
1
2
4-OCH3
3,4,5-OCH3
4-CF3
4-COOEt
3,4,5-OCH3
4-CF3
4-COOEt
4-COOH
4-COOH
1.085
1.730
3.258
9.423
0.768
0.722
4.419
0.285
0.060
33.290
Scheme 5 Reagents and conditions: (i) TsCl, TEA, DCM, 0–5 ꢂC, 3 h;
(ii) p-nitrophenol, K2CO3, DMF, 75 ꢂC; (iii) EtOH, 7.5% Pd–C, H2, rt, 4 h;
(iv) 6c, 6d, TsOHS$H2O, 1,4-dioxane, 100 ꢂC, 5 h; (v) TsCl, TEA, DMAP,
DCM, room temperature, 3 h; (vi) anhydrous KF, Kryptofix 2.2.2.,
anhydrous acetonitrile, 160 ꢂC, 15 min; (vii) 1 M NaOH, EtOH, 85 ꢂC,
10 min.
18c
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[18F]-8g. Compounds [18F]-8h (7%) and [18F]-8i (9%) were ob-
tained by further treating [18F]-8d and [18F]-8g with 1 M NaOH in
In vitro stability studies
The in vitro stability of [18F]-8a, [18F]-8c, [18F]-8h, and [18F]-8i
were determined in physiological saline and mouse plasma.
Radio-HPLC analysis of the physiological saline and mouse
plasma samples revealed that compounds [18F]-8a, [18F]-8c,
[18F]-8h, and [18F]-8i remained sufficiently stable (>95%) during
incubation at 37 ꢂC for 2 h, demonstrating a high in vitro
stability of these radio-compounds (Fig. 5).
ꢂ
EtOH at 85 C for 10 min.
The radiochemical purity of the radiotracers [18F]-8a, [18F]-
8c, [18F]-8h, and [18F]-8i was greater than 98% aer purication
by high performance liquid chromatography (HPLC). The
identity of the 18F-labeled tracers was veried by a comparison
of their retention times with those of the corresponding
nonradioactive F-19 standards 8a, 8c, 8h, and 8i via co-injection.
The retention times of [18F]-8a, [18F]-8c, [18F]-8h, and [18F]-8i
were 14.576, 19.197, 6.696, and 6.668 min, respectively, which
matched well with the corresponding F-19 standards 8a (14.301
min), 8c (18.899 min), 8h (6.357 min), and 8i (6.313 min) within
admissible error, respectively (Fig. 4).
In vivo biodistribution studies in S180-bearing mice
In vivo biodistribution data in mice bearing the S180 tumor for
[18F]-8a at 5, 15, 30, 60, and 120 min are shown in Table 1, while
for compounds [18F]-8c, [18F]-8h, and [18F]-8i the date are shown
in the ESI.† The results in Table 1 display that [18F]-8a was
Partition coefficient determination
Selecting potential candidates as FAK targeting tumor imaging
agents for further development requires measuring a range of
physiochemical properties.33 The partition coefficient (log P)
value governs various biological processes, such as the trans-
portation, distribution, metabolism, and secretion of biomole-
cules, which is essential to predict their transportation and
activity. Therefore, the selected compounds [18F]-8a, [18F]-8c,
[18F]-8h, and [18F]-8i were measured for their log P values.
Table 2 shows that the log P values of the four measured
compounds were all over 0. The results indicated that the four
compounds were all liposoluble. Notably, the log P value of
compound [18F]-8a (4.51) was higher than the others. The
difference might result from the substituents, such as tri-
uoromethyl and the carboxyl groups on compounds [18F]-8c,
[18F]-8h, and [18F]-8i, which increased their hydrophilicity.
Fig. 4 HPLC chromatogram of the 18F-labeled radiotracers and the
corresponding 19F standards.
Table 2 log P values of compounds [18F]-8a, [18F]-8c, [18F]-8h, and
[
18F]-8i
Fig. 5 (A) HPLC chromatograms of [18F]-8a in normal saline and
murine plasma at 37 ꢂC after 1 h and 2 h, respectively. (B) HPLC
chromatograms of [18F]-8i in normal saline and murine plasma at 37 ꢂC
after 1 h and 2 h, respectively.
Compds
log P
[
18F]-8a
4.51 ꢃ 0.04
[
18F]-8c
1.63 ꢃ 0.04
[
18F]-8h
1.22 ꢃ 0.06
[
18F]-8i
1.35 ꢃ 0.05
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Table 3 Biodistribution of [18F]-8 in mice bearing the S180 tumora
ID% (gꢁ1
)
5 min
15 min
30 min
60 min
120 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Muscle
Intestb
Stomb
Tumor
Tu/Muc
Tu/Boc
Tu/Blc
1.95 ꢃ 0.32
1.20 ꢃ 0.12
1.23 ꢃ 0.15
3.29 ꢃ 0.02
1.98 ꢃ 0.03
6.98 ꢃ 0.09
0.70 ꢃ 0.09
1.38 ꢃ 0.21
1.18 ꢃ 0.02
1.14 ꢃ 0.21
3.69 ꢃ 0.51
2.67
1.99 ꢃ 0.09
0.78 ꢃ 0.03
2.44 ꢃ 0.22
1.99 ꢃ 0.23
1.65 ꢃ 0.11
8.01 ꢃ 0.17
1.51 ꢃ 0.10
1.47 ꢃ 0.19
1.59 ꢃ 0.03
1.33 ꢃ 0.06
3.39 ꢃ 0.25
2.31
2.19 ꢃ 0.01
1.19 ꢃ 0.18
2.45 ꢃ 0.20
1.87 ꢃ 0.19
2.28 ꢃ 0.12
6.30 ꢃ 0.18
1.80 ꢃ 0.03
1.73 ꢃ 0.20
3.46 ꢃ 0.26
1.49 ꢃ 0.08
3.11 ꢃ 0.22
1.80
1.9 ꢃ 0.10
1.51 ꢃ 0.12
3.12 ꢃ 0.18
1.41 ꢃ 0.08
1.74 ꢃ 0.19
2.26 ꢃ 0.12
1.49 ꢃ 0.13
1.97 ꢃ 0.25
3.51 ꢃ 0.14
1.61 ꢃ 0.04
3.71 ꢃ 0.43
1.88
0.81 ꢃ 0.01
0.91 ꢃ 0.03
1.72 ꢃ 0.08
0.50 ꢃ 0.06
0.75 ꢃ 0.23
1.25 ꢃ 0.06
1.33 ꢃ 0.13
1.59 ꢃ 0.27
3.03 ꢃ 0.19
1.35 ꢃ 0.05
3.23 ꢃ 0.14
2.03
1.96
1.89
1.64
1.70
0.90
1.42
0.95
1.92
0.65
3.99
a
Expressed as % injected dose per gram (% ID gꢁ1) unless otherwise indicated. Data are the average for ve mice ꢃ standard deviation. b Expressed
as % injected dose per organ (% ID), intest ¼ intestine, stom ¼ stomach. c Tu/Mu ¼ tumor/muscle, Tu/Bo ¼ tumor/bone, Tu/Bl ¼ tumor/blood.
accumulated in the tumor with retention values between 3.11 ꢃ bladders of the mice could be clearly observed, which might be
0.22 and 3.71 ꢃ 0.43. At 60 min post-injection, [18F]-8a had the responsible for the metabolism of [18F]-8a in mice kidney. These
highest uptake in the tumor (3.71 ꢃ 0.43 ID% gꢁ1). The uptake by results were in accordance with the data for the in vivo bio-
the tumor of [18F]-8a was found to be at least 2 times more than distribution (Fig. 6).
the uptake in the liver (1.41 ꢃ 0.08 ID% gꢁ1) and in the kidney
(1.49 ꢃ 0.13 ID% gꢁ1). Also, a good to moderate uptake was
Conclusions
observed in other major organs or tissues, such as the intestine
(3.51 ꢃ 0.14 ID% gꢁ1), heart (3.12 ꢃ 0.18 ID% gꢁ1), lung (2.26 ꢃ
In conclusion, a series of novel pyrimidine derivatives were
0.12 ID% gꢁ1), and muscle (1.97 ꢃ 0.25 ID% gꢁ1) at 60 min post-
successfully synthesized and characterized by 1H NMR, 13C
injection. Since most PET-based radiotracer studies are generally
performed within 60 min of the radiotracer administration,34
thus [18F]-8a seems suitable for early tumor imaging (Table 3).
HNMR, and MS spectra. All the new compounds were evaluated
for their activity against FAK, and showed low IC50 values in
comparison with control drugs. Especially for compound 8i, its
IC50 value was 0.060 mM, suggesting its advantage as an FAK
inhibitor. To evaluate the potentiality of these compounds as PET
imaging agents in cancer detection, compounds 8a, 8c, 8h, and 8i
were successively labeled with 18F. The four 18F-labeled pyrimi-
dine derivatives [18F]-8a, [18F]-8c, [18F]-8h, and [18F]-8i showed
appropriate log P values and high stability in physiological saline
and mouse plasma. Noticeably, compound [18F]-8a with a 4-
methoxyl group at the benzene ring exhibited good in vivo bio-
distribution data in mice bearing the S180 tumor, which
promoted its further microPET imaging study. MicroPET image
of [18F]-8a administered into S180-tumor-bearing mice acquired
at 60 min post-injection illustrated that the uptake in the S180
tumor was obvious. These results suggested that compound [18F]-
8a might be a promising PET tracer candidate for tumor detec-
tion. On the other hand, on-going efforts to optimize the struc-
ture of [18F]-8a aimed at enhancing the tumor-to-nontarget ratios
in vivo are under way. Furthermore, in order to enhance the
uptake of the F-18-labeled tracer in tumor and its target/
nontarget ratios, the interaction between the corresponding F-
19 standards and the FAK should be further increased and the
log P should be further properly lowered in future designs.
MicroPET imaging
Compound [18F]-8a was selected as the microPET imaging agent
in the four F-18-labeled pyrimidine derivatives for its good in
vivo biodistribution in mice bearing the S180 tumor. In the
microPET images, it could be seen that the diffusion of the [18F]-
8a in vivo needed a period of time, and with the time increasing,
the accumulation of [18F]-8a increased close to the tissue of the
tumor. It is worth noting that aer 60 min of caudal intravenous
injection for [18F]-8a, the intensity of radioactivity in the urinary
Compliance with ethical standards
Fig. 6 MicroPET images of [18F]-8a at 60 min (left) and 120 min (right)
of S180-bearing nude mice.
All protocols requiring the use of mice were approved by the
Animal Care Committee of Beijing Normal University.
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Acknowledgements
This work was supported by the National Major Scientic and
Technological Special Project for “Signicant New Drugs Devel-
opment” (Grant No. 2014ZX09507007-001 and 2014ZX09507007-
003); the National Science and Technology Support Program
(Grant No. 2014BAA03B03) and the National Natural Science
Foundation of China (Grant No. 21371026). We also thank
the Nuclear Medicine Department of Peking Cancer Hospital
(Beijing, China) for providing the uoride-18 nuclide and the use
of the Micro PET of Peking Union Medical College Hospital
(Beijing, China).
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