Radiosynthesis and radiopharmacological evaluation of cyclin-dependent kinase 4 (Cdk4) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.11.020

Tumor cells are characterized by their loss of growth control resulting from alterations in regulating pathways of the cell cycle, such as a deregulated cyclin-dependent kinase (Cdk) activity and/or Cdk expression. Appropriately radiolabeled Cdk4 inhibitors are discussed as promising molecular probes for imaging cell proliferation processes and tumor visualization by PET. This work describes the design, synthesis and radiopharmacological evaluation of two ¹²⁴I-labeled Cdk4 inhibitors as potential radiotracers for imaging of Cdk4 in vivo. Treatment of a solution containing labeling precursors with [¹²⁴I]NaI gave radiolabeled Cdk4 inhibitors [¹²⁴I]CKIA and [¹²⁴I]CKIB in radiochemical yields of up to 35%. ¹²⁴I-labeled radiotracers [¹²⁴I]CKIA and [¹²⁴I]CKIB were used in cell uptake studies as well as biodistribution studies in Wistar rats and small-animal PET in tumor-bearing mice. In vitro radiotracer uptake studies in adherent tumor cells using [¹²⁴I]CKIA showed substantial uptake in HT-29 and FaDu cells (750-850 %ID/mg?protein [¹²⁴I]CKIA and 900-1000?%ID/mg protein [¹²⁴I]CKIB) after 1?h at 37?°C. Biodistribution of [¹²⁴I]CKIA and [¹²⁴I]CKIB showed rapid blood clearance of radioactivity and an accumulation as well as metabolization in the liver. Both radiotracers were administered intravenously to mouse FaDu xenograft tumor model and imaging studies were performed on a small-animal PET scanner. Both imaging techniques showed only little uptake of both radiotracers in the FaDu tumor xenografts.

Full text of DOI:10.1016/j.ejmech.2009.11.020

European Journal of Medicinal Chemistry 45 (2010) 727–737
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Radiosynthesis and radiopharmacological evaluation of cyclin-dependent
kinase 4 (Cdk4) inhibitors
,b,
Lena Koehler a, Franziska Graf a, Ralf Bergmann a, Jo¨rg Steinbach a, Jens Pietzsch a, Frank Wuest a
*
a Institute of Radiopharmacy, Research Center Dresden-Rossendorf, Dresden, Germany
b Department of Oncology, University of Alberta, 11560 University Ave, Edmonton, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Tumor cells are characterized by their loss of growth control resulting from alterations in regulating
pathways of the cell cycle, such as a deregulated cyclin-dependent kinase (Cdk) activity and/or Cdk
expression. Appropriately radiolabeled Cdk4 inhibitors are discussed as promising molecular probes for
imaging cell proliferation processes and tumor visualization by PET. This work describes the design,
synthesis and radiopharmacological evaluation of two 124I-labeled Cdk4 inhibitors as potential radio-
tracers for imaging of Cdk4 in vivo. Treatment of a solution containing labeling precursors with [124I]NaI
gave radiolabeled Cdk4 inhibitors [124I]CKIA and [124I]CKIB in radiochemical yields of up to 35%. 124I-
labeled radiotracers [124I]CKIA and [124I]CKIB were used in cell uptake studies as well as biodistribution
studies in Wistar rats and small-animal PET in tumor-bearing mice. In vitro radiotracer uptake studies in
adherent tumor cells using [124I]CKIA showed substantial uptake in HT-29 and FaDu cells (750–850 %ID/
mg protein [124I]CKIA and 900–1000 %ID/mg protein [124I]CKIB) after 1 h at 37 ꢀC. Biodistribution of
Received 5 August 2009
Received in revised form
30 October 2009
Accepted 12 November 2009
Available online 24 November 2009
Keywords:
Iodine-124
Cell cycle
Cyclin-dependent kinase 4 inhibitor
Positron emission tomography (PET)
[
124I]CKIA and [124I]CKIB showed rapid blood clearance of radioactivity and an accumulation as well as
metabolization in the liver. Both radiotracers were administered intravenously to mouse FaDu xenograft
tumor model and imaging studies were performed on a small-animal PET scanner. Both imaging tech-
niques showed only little uptake of both radiotracers in the FaDu tumor xenografts.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
complexes composed of a catalytic kinase subunit and a regulatory
cyclin subunit. Cdk activity is controlled by association with their
Tumorigenesis is a result of cell-cycle dysregulation leading to
an uncontrolled cellular proliferation [1–4]. An aberrant cell
proliferation is a hallmark of cancer. In healthy tissue, cell division
occurs in the context of a highly regulated concert of molecular
events known as the cell cycle. The cell cycle is divided into four
distinct phases: DNA synthesis (S phase), mitosis (M phase), and
the gaps of varying length between these periods called G1 and G2.
Non-dividing cells remain in a resting or quiescence stage named
G0 before they re-enter into phase G1.
The regulation of the cell cycle is governed and controlled by
specific proteins, which are activated and deactivated mainly
through phosphorylation/dephosphorylation processes in a precise
timely matter. The key proteins that coordinate the initiation,
progression, and completion of cell-cycle program are cyclin-
dependent kinases (Cdks). Cyclin-dependent kinases belong to the
serine–threonine protein kinase family. They are heterodimeric
corresponding regulatory subunits (cyclins) and Cdk inhibitor
proteins (Cip & Kip proteins, INK4s), by their phosphorylation state,
and by ubiquitin-mediated proteolytic degradation [5–8]. In early
to mid G1 phase, when the cell is responsive to mitogenic stimuli,
activation of Cdk4-cyclin D and Cdk6-cyclin D induces phosphor-
ylation of the retinoblastoma protein (pRb). Phosphorylation of pRb
releases the transcription factor E2F, which enters the nucleus to
activate transcription of other cyclins which promote further
progression of the cell cycle [9,10]. Cdk4 and Cdk6 are closely
related proteins with basically indistinguishable biochemical
properties [11]. Genetic analysis of numerous human cancers has
shown that in more than 80% of human neoplasia the cyclin
D-Cdk4/INK4/pRb/E2F pathway has been altered, making Cdk4 an
attractive target for the development of anti-cancer drugs [12,13].
Over the last years, considerable effort has been made to screen
and design small molecule Cdk inhibitors [14]. Several drug
discovery programs have identified potent small molecule Cdk
inhibitors from a variety of chemical classes, which include purine
analogues, pyrimidine analogues, indenopyrazoles, pyridopyr-
imidines, pyrozolopyridines, indolocarbazoles, pyrrolocarbazoles,
* Corresponding author. Tel.: þ1 780 989 8150; fax: þ1 780 432 8483.
E-mail address: frankwue@cancerboard.ab.ca (F. Wuest).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.020

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L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
oxindoles, and aminothiazoles [14]. Although much biochemical
evidence supports targeting Cdk4 as the most important Cdk for
regulating cell proliferation, most drug development programs
have produced compounds which inhibit multiple Cdks, with Cdk2
and Cdk1 being particularly common targets [15,16]. Several of the
compounds are currently in preclinical and clinical trials including
flavopiridol [17–19], staurosporine UCN-01 [20,21], BMS-387032
[19], olomoucine [5,22,23], CYC202 [24,25], NU6102 [26], CR8 [27],
AT7519 [28], and SCH-727965 [29–31] (Fig. 1).
However, evidence has been reported in the literature that
mammalian cells can continue to proliferate in the absence of Cdk2
activity [32]. Hence, Cdk2 activity seems to be not essential for cell
proliferation in various cancers. These results suggest that Cdk2
may not be the ideal target for inhibition by small molecules to
treat cancer. The possible compensation of Cdk2 activity by Cdk4
and the upregulation of Cdk4 activity in many human cancers have
shifted attention of cancer drug discovery towards Cdk4 as the
more important cell-cycle Cdk target.
The design of selective Cdk4 inhibitors represents a formidable
challenge due to the common folding pattern of numerous kinases
and their kinase domains including the ATP binding pockets. Over
the last years, various Cdk4 inhibitors have been described in the
medicinal chemistry literature. The structure-based design of
potent and selective Cdk4 inhibitors led to the development of
several classes of compounds, including pyrido[2,3-d]pyrimidines,
2-anilinopyrimidines, diaryl ureas, benzoyl-2,4-diaminothiazoles,
indolo[6,7-a]pyrrolo[3,4-c]carbazoles, and oxindoles [14]. Espe-
cially various compounds based on a pyrido[2,3-d]pyrimidine
scaffold containing a side chain at the C2 position were shown to be
highly potent Cdk4 inhibitors (IC50 < 20 nM) with favorable
selectivity profile over Cdk1, Cdk2, and other kinases (Fig. 2)
[33,34]. Compound PD 0332991 has entered clinical trial for cancer
therapy [35].
Fig. 2. Pyrido[2,3-d]pyrimidines containing a side chain at position C2 as potent and
selective Cdk4 inhibitors [35,36,47].
iodine substituent in compound CKIA represents an attractive site
for isotopic substitution with radioiodine isotopes to give the cor-
responding radiotracers for molecular imaging purposes. In
previous work we showed that Cdk4 inhibitor CKIA and pyridine-
containing analog CKIB act as tumor cell growth inhibitors via
specific inhibition of the Cdk4/INK4/pRb/E2F signaling pathway
and induction of G1 arrest in tumor cells [44].
Within the arsenal of molecular imaging methodologies, posi-
tron emission tomography (PET) is a particularly powerful tech-
nique to provide quantitative kinetic information of metabolic
pathways and physiological processes in vivo [37].
The positron emitter iodine-124 (124I) is an attractive radionu-
clide for the design, synthesis and radiopharmacological evaluation
of iodine-containing compounds. Its convenient 4.18 d half-life
allows extended synthesis protocols, and longitudinal imaging
studies using small-animal PET [38,39]. The labeling technique for
124I is well established, and this approach is continuously being
used in the labeling and PET imaging of various biologically active
compounds.
Currently, an exact and accurate assessment of functional Cdk4
expression in tumors or other tissues can only be achieved by
laborious analyses of invasively acquired biopsies. Moreover,
instability of Cdk4 mRNA and protein leads to further difficulties in
terms of the intervals between tissue sampling and time of analysis.
The development of a PET imaging probe for monitoring Cdk4
expression in vivo would provide data on functional Cdk4 protein
expression in tumor development and progression. Moreover, the
development and in vivo study of appropriately radiolabeled
selective Cdk4 inhibitors would provide pharmacological data,
which may help to further understand their exact physiological
actions and metabolic pathways.
The iodine-containing pyrido[2,3-d]pyrimidine-7-one CKIA has
been reported as a very potent (IC50 (Cdk4) ¼ 5 nM) and selective
Cdk4 inhibitor (IC50 (Cdk1 and Cdk2) >360 nM) [36]. Moreover, the
This work describes the design, synthesis and radiopharmacological
evaluation of two 124I-labeled Cdk4 inhibitors (8-cyclopentyl-6-
[
124I]iodo-5-methyl-2-(4-piperazin-1-yl-phenyl-amino)-8H-pyrido[2,3-d]
pyrimidin-7-one ([124I]CKIA) and 8-cyclopentyl-6-[124I]iodo-5-methyl-
2-(5-(piperazin-1-yl)pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-
one ([124I]CKIB)) as potential radiotracers for imaging Cdk4 expression
in vivo. The radiopharmacological characterization of radiotracers
[
124I]CKIA and [124I]CKIB involved cellular uptake studies, metabolism
and biodistribution studies in normal rats, and small-animal PET
studies in NMRI nu/nu mice bearing the human head and neck squa-
mous cell carcinoma tumor FaDu.
2. Results and discussion
2.1. Chemistry and radiosynthesis
The key step within the synthesis of pyrido[2,3-d]pyrimidine-
Fig. 1. Cdk inhibitors Flavopiridol, UCN-01, BMS-387032, olomoucine, CYC202, CR8,
AT7519, SCH-727965, and NU6102.
based compounds as potent and selective Cdk4 inhibitors is the

L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
729
introduction of amino-substituted side chains by displacement of
the sulfoxide group at the C2 position of the pyrido[2,3-d]pyrimi-
dine-7-one core [33,36]. The overall synthesis scheme for the
preparation of reference compounds CKIA and CKIB, and labeling
precursors 6 and 7 is depicted in Scheme 1.
Introduction of (4-aminophenyl)piperazine-containing side
chain 2 into sulfoxide 1 to give compound 4 was accomplished in
75% yield at elevated temperature in DMSO, whereas application of
DMSO as the solvent failed in the case of (2-amino-pyridin)piper-
azine-containing side chain 3 due to the formation of numerous
side products during the reaction. Replacement of DMSO with
toluene as the solvent gave the desired compound 5 in moderate
chemical yield of 38%.
Removal of the Boc protecting group was easily achieved by the
treatment of piperazines 4 and 5 with aqueous HCl at room
temperature. Subsequent silica-gel purification gave the reference
compounds CKIA and CKIB in excellent chemical yields of 95%.
Several radioiodination reactions involving organometallic
intermediates have been shown to be very effective, rapid and site-
specific methods for the incorporation of iodine isotopes into small
molecules [40]. Especially the use of aromatic and vinylic organo-
stannanes as labeling precursors gave very good results for radio-
iodinations via iodo-destannylation reactions. Thus, we prepared
organostannanes 6 and 7 as precursors for radiolabeling with
iodine-124. Organostannanes 6 and 7 were prepared via Pd-cata-
lyzed cross coupling reaction between iodides 4 and 5, and an
excess of hexamethylditin (Sn2Me6) in 32% and 55% yield, respec-
tively, after silica-gel chromatography.
Scheme 2. Radiosynthesis of 124I-labeled Cdk4 inhibitors [124I]CKIA and [124I]CKIB:
(d) [124I]NaI, DMSO, MeOH, HOAc, IodogenÒ, r.t., 10 min; (e) TFA, 50 ꢀC, 20 min.
Varying amounts of labeling precursor 6 were dissolved in a 1:3
mixture of DMSO and 5% acetic acid in methanol in an Eppendorf
vial. The use of DMSO as a solvent was necessary since compound 6
showed only poor solubility in methanol containing 5% of acetic
acid. Besides the beneficial effect of DMSO to ensure complete
solubility, DMSO was also reported to improve the efficiency of
radioiodination reactions [41]. Polymer-bound chloramine-T
(iodobeads) and IodogenÒ (IodogenÒ-coated tubes) were used as
mild oxidizing agents. The radiochemical yield was determined via
radio-TLC, and is presented as the percentage of the desired radi-
olabeled product Boc-[124I]CKIA in the reaction mixture at
different time points.
The results in Table 1 clearly demonstrate the importance of
precursor amount on the radiochemical yield. The use of 20
trimethylstannyl compound 6 gave only low radiochemical yields
of 30% after 30 min (entry 1). Increase of the amount of 6–40
(entry 2) and 50 g (entry 3) resulted in an improved radiochemical
yield of 70% and 80%, respectively, after 30 min. Thus, a minimum of
40 g of labeling precursor seems to be necessary to achieve
reasonable radiochemical yields. Further increase of the amount of
mg of
mg
Incorporation of 124I into C-6 position of pyrido[2,3-d]pyrimi-
dine compounds was accomplished under mild conditions through
electrophilic substitution employing a regioselective iodo-destan-
nylation reaction of C-6 trimethylstannyl-substituted compounds 6
and 7 with [124I]NaI in the presence of a mild oxidizing agent
(immobilized chloramine-T or IodogenÒ). Subsequent removal of
the Boc protecting group afforded the desired radiolabeled
compounds. The general reaction scheme for the radiosynthesis of
124I-labeled Cdk4 inhibitors [124I]CKIA and [124I]CKIB is given in
Scheme 2.
m
m
compound 6–100
mg (entry 4) gave the desired radiolabeled
compound Boc-[124]CKIA in radiochemical yields of 82% after
10 min, 89% after 20 min, and very good 94% after 30 min. The use of
more oxidizing agent (2 iodobeads, entries 4 and 6) did not further
improve the radiochemical yield. The use of 200
mg of labeling
precursor 6 afforded 90% of radiolabeled compound Boc-[124]CKIA
after 10 min. The radiochemical yield could slightly be improved
over time, reaching 93% after 20 min, and 95% after 30 min.
In addition to the use of immobilized chloramine-T as the
oxidizing agent we also tested the reaction according to the Iodo-
genÒ method using IodogenÒ-coated tubes (entry 8). The use of
For the optimization of the reaction conditions the influence of
the amount of stannylated labeling precursor and the oxidizing
agent on the radiochemical yield was tested. The optimization was
performed with trimethylstannyl-precursor 6 and 2–3 MBq of
[
124I]NaI (3–4 l of [124I]NaI in 0.25 M NaOH) at room temperature.
m
40 mg of labeling precursor 6 gave radiochemical yield of 20% after
The results are summarized in Table 1.
10 min and 25% after 20 min. Further extension of the reaction time
to 30 min increased the radiochemical yield to 80%.
All reactions were stopped by the addition of 50 ml of saturated
bisulfite solution to the reaction mixture. The radioiodination step
proceeded without formation of any by-products as monitored by
radio-TLC. Since the oxidation agent is immobilized (iodobeads or
IodogenÒ-coated tubes) it can easily be separated from the reaction
mixture for the subsequent deprotection step of the Boc group.
Table 1
Radiolabeling of compound 6 with [124I]NaI.
Entry
Mass
Oxidizing agent
Radiochemical yield [%]
10 min
20 min
30 min
1
2
3
4
5
6
7
8
20
40
50
m
g
g
g
g
g
g
g
g
1 Iodobead
1 Iodobead
1 Iodobead
2 Iodobeads
1 Iodobead
2 Iodobeads
1 Iodobead
IodogenÒ
18
58
55
40
82
56
90
20
20
58
58
52
89
81
93
25
30
70
80
64
94
91
95
80
m
m
m
m
m
m
m
60
100
100
200
40
Scheme 1. Synthesis route for reference substances CKIA and CKIB, and labeling
precursors 6 and 7: (a) DMSO or toluene, 100 ꢀC, 16 h; (b) dioxane, 6 M HCl, r.t., 2 h; (c)
Sn2Me6, tetrakis(triphenyl-phosphine)palladium(0), dioxane, reflux, 8 h.

730
L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
For a larger scale radiosynthesis (40 MBq–275 MBq of [124I]NaI),
250 g of labeling precursor 6 dissolved in 50 l of solvent
The lipophilicity of both radiotracers was measured according to
m
m
the method reported by Wilson et al. [43]. Compound [124I]CKIA
showed a log D value of 2.77 ꢂ 0.13, whereas a lower log D of
1.99 ꢂ 0.03 was determined for compound [124I]CKIB at a pH-value
of 7.4. Thus, substitution of the phenyl ring in compound [124I]CKIA
with a pyridine ring as in compound [124I]CKIB resulted in
a significant decrease of lipophilicity in the order of almost one
magnitude.
precursor was used. However, the use of iodobeads as the oxidizing
agent caused unexpected problems. Most of the radioactivity was
irreversibly attached to the iodobeads. Attempts to remove the
product from the iodobeads by means of various solvents such as
diethyl ether, chloroform, ethyl acetate and methanol, failed. For
this reason, we decided to perform the reaction with IodogenÒ-
coated tubes. The use of the IodogenÒ method in IodogenÒ-coated
tubes proved to be the approach of choice. No undesired loss of
activity was observed. The reaction mixture was incubated at room
temperature for 10 min. Radio-TLC analysis indicated product
formation of greater than 90%. The reaction mixture was quenched
2.3. Stability analysis
The stability of both radiotracers was tested in ethanol, buffer
solution (PBS, pH 7.4), cell culture medium, and rat plasma at 37 ꢀC
at 30 min and 24 h with radio-HPLC. The results are summarized in
Table 3.
by the addition of 50 ml of saturated sodium bisulfite. Deprotection
was performed with trifluoroacetic acid (TFA) at 50 ꢀC. Removal of
the Boc protecting group was accomplished after 20 min to give the
desired compound [124I]CKIA in radiochemical yields of 45–65%
along with the formation of some not further identified side
products (ꢁ40%). Lowering the reaction temperature to 25 ꢀC
resulted in significantly lower radiochemical yield (13%) of the
desired product but with also significantly lower amounts of side
products (7%). Increasing the reaction temperature of the depro-
tection step to 90 ꢀC resulted in complete deprotection of
compound Boc-[124I]CKIA leading to the formation of 60% of
compound [124I]CKIA along with 40% of side products. The results
are summarized in Table 2.
Radio-HPLC analysis showed no substantial decomposition of
both radiotracers after incubation in all media at 37 ꢀC after 30 min.
Comparable results were obtained after 24 h, with a more
pronounced tendency for decomposition (14%) of compound
[
124I]CKIA in buffer at 24 h. For all other media, high stability
(>92%) of both compounds was found at 30 min and 24 h
2.4. Cell uptake studies
In vitro radiotracer cell uptake studies using [124I]CKIA and
[
124I]CKIB showed substantial uptake in HT-29 and FaDu tumor cells
The crude product was subjected onto a semi-preparative HPLC
column for purification. The product fraction (13.2–14.0 min) was
collected, diluted with water and passed through a Waters Sep-Pak-
tC-18 cartridge. The cartridge was washed with water and the final
product [124I]CKIA was eluted with 1 mL ethanol. The solvent was
evaporated in a gentle stream of nitrogen to afford compound
(approximately 750–850 %ID/mg protein
[
124I]CKIA and 900–
1000 %ID/mg protein [124I]CKIB (means) after 1 h) at 37 ꢀC (Fig. 3 A
and B). In both cell lines HT-29 and FaDu the time-dependent cellular
uptake of [124I]CKIA and [124I]CKIB was similar. The early uptake
kinetics of both compounds was comparable in both tumor cell lines.
In posterior phase (>1 h) [124I]CKIA showed a further increasing
cellular uptake compared to [124I]CKIB. On the other hand, [124I]CKIB
uptake level remained nearly constant after 1 h. This is consistent
with the different therapeutic potency of CKIA and CKIB observed in
cell-cycle studies of tumor cell lines. Uptake of both radiolabeled
compounds [124I]CKIA and [124I]CKIB could be blocked with non-
radioactive CKIA or respectively CKIB in FaDu cells dependent on
concentration (Fig. 3C), indicating a specific uptake mechanism.
[
124I]CKIA in radiochemical yield of 33.6%. Optimized reaction
conditions were also applied to the synthesis of compound
124I]CKIB, which was prepared in radiochemical yield of 28%. The
specific activity was determined to be 35 GBq/ mol for compound
124I]CKIA and 25 GBq/ mol for compound [124I]CKIB. The radio-
[
m
[
m
chemical purity of both compounds exceeded 95% suitable for
further radiopharmacological characterization.
2.2. Lipophilicity
2.5. Tissue biodistribution studies
The distribution of radiotracers [124I]CKIA and [124I]CKIB in
selected tissues and organs was studied in normal Wistar rats at
5 min p.i. as relevant for the perfusion phase, and 60 min p.i. as
relevant for the metabolic phase. The dose of both radiotracers was
in the range of 0.2–0.3 MBq/animal. The results of the standardized
radioactivity concentrations in different organs and tissues are
presented in Fig. 4.
Lipophilicity (log D at pH ¼ 7.4) is an important and funda-
mental physico-chemical property of a compound in the area of
rational drug design and development. It is a key parameter of
structure-activity relationship studies in medicinal chemistry to
predict cell membrane penetration and blood protein binding. In
the field of radiotracer design and development, lipophilicity is
often used to predict delivery of the radiotracer to target tissues or
organs, and to anticipate the clearance rate of the radiotracer from
non-target tissue [42,43]. In the case of compounds [124I]CKIA and
[
124I]CKIA and 124I]CKIB showed similar biodistribution
[
profiles, although they exhibit different lipophilicity at pH of 7.4
(log D ¼ 2.77 for [124I]CKIA and log D ¼ 1.99 for [124I]CKIB). High
initial radioactivity concentrations after 5 min p.i. were detected in
the lung (8.67 ꢂ 2.04 for [124I]CKIA and 8.43 ꢂ 0.66 for [124I]CKIB).
High initial uptake values were also found in the spleen, adrenals,
[
124I]CKIB as potential Cdk4 inhibitors, the radiotracer should
possess an appropriate lipophilicity to enable sufficient cell uptake
while avoiding undesired high non-specific binding to plasma
proteins.
Table 2
Table 3
Stability of [124I]CKIA and [124I]CKIB in different media.
Radiochemical yields for Boc group removal after 30 min.
Compound
Radiochemical yield
25 ꢀC
Compound EtOH
PBS buffer
(pH 7.4)
Cell culture medium Rat plasma
50 ꢀC
90 ꢀC
124I]CKIA
13%
80%
7%
45–65%
ꢁ10%
60%
0%
40%
30 min 24 h 30 min 24 h 24 h
30 min 24 h
[
Boc-[124I]CKIA
[
[
124I]CKIA 93%
124I]CKIB 98%
94% 92%
97% 97%
86% 96%
95% 97%
93%
94%
94%
94%
Side-products
ꢁ40%

L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
731
A
B
1750
1750
1500
1250
1000
750
500
250
0
HT-29
FaDu
HT-29
FaDu
1500
1250
1000
750
500
250
0
0
10 20 30 40 50 60 70 80 90 100 110 120
Time (minutes)
0
10 20 30 40 50 60 70 80 90 100 110 120
Time (minutes)
C
2000
FaDu 60 min
124
[
[
I]CKIA
I]CKIB
124
1500
1000
500
0
0.001
0.01
0.1
1
Concentration CKIA/CIKB (mmol/L)
Fig. 3. Cellular radiotracer uptake of [124I]CKIA (A) and [124I]CKIB (B) over a period of 2 h at 37 ꢀC and blocking experiments with different concentrations non-radioactive CKIA or
CKIB, respectively, in FaDu after 60 min at 37 ꢀC (C) (means ꢂ SD, n ꢃ 8, ANOVA).
Fig. 4. Biodistribution of [124I]CKIA or [124I]CKIB in selected tissues and organs of normal male Wistar rats (body weight 180 ꢂ 20 g) after single intravenous injection at 5 min and
60 min. Data are presented as standardized uptake values (SUV). SUV ¼ (organ activity/organ weight)/(total given activity/rat body weight)/(Bq/g); means ꢂ SD; n ¼ 8.

732
L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
kidneys and liver (SUV > 3.2) after 5 min p.i. The initial high
radioactivity uptake in these organs was decreased at 60 min p.i. in
the case of less lipophilic compound [124I]CKIB, whereas more
lipophilic radiotracer [124I]CKIA did not clear from the liver after
60 min p.i.
No radioactivity uptake was found in the brain at 5 min and
60 min p.i. suggesting that both radiotracers do not sufficiently
penetrate the blood–brain barrier, and/or that they are substrates for
the P-glycoprotein efflux pump. Radioactivity was cleared from most
organs and tissues within 60 min after radiotracer administration,
except for thyroidea. The observed increase of radioactivity concen-
tration in the thyroidea is indicative of an in vivo radiodeiodination
and accumulation of free [124I]iodide in the thyroidea over time. The
determined biodistribution profile of both radiotracers suggests
a predominantly hepatobiliary elimination pathway.
performed in FaDu xenograft bearing mice as models of human
tumors. Representative images for both tumor models are shown in
Fig. 6.
The maximum intensity projections at different time points
further confirm the comparable biodistribution pattern for
[
124I]CKIA and [124I]CKIB in FaDU tumor-bearing mice, as found in
the biodistribution studies with rats. Both radiotracers were
predominantly hepatobiliarly eliminated, with no specific radio-
activity accumulation in other organs and tissues, including the
tumor.
Time activity curves of radiotracers [124I]CKIA and [124I]CKIB
demonstrated rapid clearance of both compounds from the blood,
and accumulation of radioactivity in the liver and thyroid over time
(Fig. 7). More lipophilic compound [124I]CKIA (log D ¼ 2.77,
pH ¼ 7.4) showed higher liver uptake compared with less lipophilic
compound [124I]CKIB (log D ¼ 1.99, pH ¼ 7.4).
The half-life of radioactivity clearance from the blood was
calculated according to a two-phase distribution profile, derived
from ROI’s over the heart, which is representative of the radioac-
tivity clearance from the blood. The data were not partial volume
corrected. The calculated half-life accounting for the late phase was
7.3 min in the case of compound [124I]CKIA and 6.4 min for
compound [124I]CKIB. The half-times accounting for the early
distribution phase were 21 s and 26 s for [124I]CKIA and [124I]CKIB,
respectively.
Biodistribution profile of both radiotracers in FaDu tumor-
bearing mice was further studied by ex vivo autoradiography.
Representative autoradiograms at 60 min after radiotracer admin-
istration and the corresponding histological sections are given in
Fig. 8. Highest radioactivity concentrations were observed in the
liver and intestine for both compounds. No radioactivity was found
in the brain and only marginal uptake in the FaDu tumor. These
finding are consistent with the results obtained from the bio-
distribution and small-animal PET studies.
2.6. In vivo stability
Metabolic stability in vivo of radiotracers
124I]CKIB was studied in rats. Fig. 5 shows the time curves of the
[
124I]CKIA and
[
percentage of original compound at different time points in rat
blood plasma after single intravenous injection.
Both radiotracers showed similar metabolic stability profiles
indicating that both compounds are rapidly metabolized in vivo.
After 10 min, a fraction of 19% ([124I]CKIA) and 21% ([124I]CKIB),
respectively, of intact parent compound was detected. Two new
more hydrophilic peaks appeared, which have not been charac-
terized further. Both radiotracers were almost completely metab-
olized after 60 min p.i., resulting in a remaining fraction of intact
parent compounds of less than 5%. However, the found SUV values
of 3.2 ([124I]CKIA) and 2.2 ([124I]CKIB) for thyroids from the tissue
biodistribution study at 60 min p.i. (Fig. 4) suggest, that radio-
deiodination seems not to be the main catabolic pathway. Obvi-
ously, the shown instability of both compounds in vivo is an
obstacle for performing in vivo analyses. The in vivo stability was
unexpectedly low despite the high in vitro stability shown in Table
3. Further investigations should deal with the identification of the
two new formed metabolites in order to gain information for
stabilizing this compounds.
3. Conclusions
The radiosynthesis of two 124I-labeled Cdk4 inhibitors has been
developed. Both non-carrier added radiotracers were obtained in
reproducible radiochemical yields and purity enabling detailed
radiopharmacological characterization. In vitro experiments
demonstrated substantial cellular uptake in tumor cell lines HT-29
and FaDu and a high in vitro stability in rat plasma and cell culture
medium. In contrast, small-animal PET and autoradiography
studies in vivo showed only very low uptake in FaDu tumors due to
a rather low in vivo stability. So both radiolabeled compounds show
limitations regarding their ability to act as a radiotracer for imaging
of tumor cells in vivo. On the other hand, the corresponding non-
radioactive compounds clearly show inhibitory effects on cell-cycle
regulation in the tumor cells when applied in pharmacological
2.7. Small-animal PET imaging and ex vivo autoradiography
To further assess the metabolic pathway of radiotracers
[
124I]CKIA and
[
124I]CKIB, small-animal PET imaging was
concentrations (50 nM–1 mM) as shown previously by our group
[44]. The analysis of the metabolic fate of the compounds in vivo
should come to the fore to develop more stable compounds
enabling the imaging of tumor cells in vivo. Further studies should
also focus on the development of more hydrophilic and, regarding
the positron-emitting nuclide, fluorine-18 radiolabeled derivatives
of CKIA and CKIB as suitable radiotracers for imaging Cdk4 by
means of PET.
4. Materials and methods
4.1. General methods
Fig. 5. Representative curves of original [124I]CKIA and [124I]CKIB in rat blood plasma
as percentage of the total plasma activity from two rats after single intravenous
injection.
All reagents and solvents were purchased from commercial
suppliers and used without further purification unless otherwise

L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
733
Fig. 6. Maximum intensity projections of small-animal PET studies with [124I]CKIA and [124I]CKIB in FaDu tumor-bearing mice at 1 min, 5 min and 45 min midframe time after single
intravenous injections (b: bladder; g: gall bladder; h: heart; i: intestines; k: kidneys; l: liver).
specified. IodogenÒ precoated tubes were purchased from Thermo
Scientific Pierce Protein Research Products, Iodo-Beads were
Reactions were monitored by thin-layer chromatography (TLC) on
Merck silica-gel 60 F-254 aluminum plates, with visualization
under UV (254 nm). Radio-TLC analyses was performed on Merck
silica-gel RP-18 aluminum plates and Merck silica-gel 60 F-254
aluminum plates, with visualization using a Fuji 2000 BAS reader
and AIDA (Advanced Image Data Analyzer, version 4.10.020) soft-
ware. Melting points were determined with a Galen IIIÔ melting
point apparatus from Cambridge Instruments. The following
compounds were prepared according to literature procedures: 8-
cyclopentyl-6-iodo-5-methyl-2-(methylsulfinyl)-pyrido[2,3-d]pyr-
imidin-7(8H)-one 1 [36], tert.-butyl 4-(4-aminophenyl)piperazine-
purchased from Sigma–Aldrich. [124I]NaI (3 MBq/
ml, 0.1 M sodium
hydroxide solution) in 3 mL KIMAX V vials was purchased from QSA
Global GmbH (Braunschweig, Germany). NMR spectra were recor-
ded on a Varian Inova 400 MHz spectrometer. 1H chemical shifts are
given in ppm and were referenced with the residual solvent reso-
nances relative to tetramethylsilane (TMS). Mass spectra (MS) were
obtained on a Quattro/LC mass spectrometer (MICROMASS) using
electrospray ionization. Silica-gel column chromatography was
performed on MERCK silica-gel 60 (mesh size 230–400 ASTM).

734
L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
Fig. 7. Time activity curves of radioactivity concentration from ROI’s over the heart (A), and liver and thyroid (B) of [124I]CKIA and [124I]CKIB in FaDu tumor-bearing mice after single
intravenous injections. Data are expressed as SUVPET (means ꢂ SD, n ¼ 4).
1-carboxylate 2 [36] and tert.-butyl-4-(4-aminopyridin-3-yl)piper-
azine-1-carboxylate 3 [34]. High performance liquid chromato-
graphy (HPLC) analyses (VWR-Hitachi Elite LaChrom system) were
carried out with a Supelco RP-18 column (Supelco Discovery C18
trifluoroacetic acid (TFA)/CH3CN (containing 0.1% TFA) from
a gradient pump (VWR Hitachi Elite-LaChrom Pump L-2130)
([124I]CKIA: 0 min 60/40, 7 min 60/40, 12 min 20/80, 16 min 20/80;
[
124I]CKIB: 0 min 70/30, 5 min 70/30, 10 min 20/80, 12 min 20/80)
4.6 ꢄ 150 mm,
5
mm) using
a
gradient elution with 0.1%
using a flow of 1 mL/min. The products were monitored by an UV
Fig. 8. Representative ex vivo autoradiograms at 60 min after injection of [124I]CKIA (A) and [124I]CKIB (B), and the corresponding histological sections.

L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
735
detector (VWR Hitachi Elite-LaChrom UV/VIS detector L-2400) at
270 nm and by -detection with a scintillation detector Gabi
(raytest). Semi-preparative HPLC purification was performed on
3.55–3.51 (m, 4H), 3.45–3.40 (m, 4H), 2.80 (s, 3H), 2.24–2.16 (m,
2H), 2.12–2.04 (m, 2H), 1.92–1.80 (m, 2H), 1.72–1.64 (m, 2H). Low-
resolution mass spectrometry electron spray ionization (ESIþ):
532.3 [M þ H].
g
a Supelco RP-18 column (Supelco Discovery C18) 10 ꢄ 250 mm,
5 mm) using gradient elution (Jasco LG 2080-02 Ternary Gradient
Unit) with CH3CN (containing 0.1% TFA)/0.1% TFA (0 min 40–60,
20 min 40/60, 30 min 80/20, 50 min 80/20) at a flow rate of 3.0 mL/
min for [124I]CKIA and 1.5 mL/min for [124I]CKIB (Jasco PU-
2080plus Intelligent Pump System). The products were monitored
by an UV detector (Jasco UV-2075 plus intelligent UV/VIS detector)
4.2.3. General procedure for the synthesis of trimethyl stannyl-
containing compounds
Under nitrogen, hexamethyl distannane (0.617 mmol) and tet-
rakis(triphenyl-phosphine)palladium(0) (0.024 mmol) were dis-
solved in dioxane (10 mL). Compound 4 or 5 (0.475 mmol) was
added, and the reaction mixture was refluxed for 8 h. The reaction
mixture was cooled to room temperature, and the solvent was
evaporated in vacuum. The residue was the purified by silica-gel
chromatography (ethyl acetate/petroleum ether 1/1) to yield 6 and
7 as greyish-yellow solids.
at 270 nm and by
20 046.
g-detection with a scintillation detector Robotron
4.2. Chemical synthesis
4.2.1. General procedure for the introduction of the side chains 2
and 3 to sulfoxide 1 at C2 position
tert-Butyl-4-(6-(8-cyclopentyl-5-methyl-7-oxo-6-trimethyl-
stannyl-7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)phenyl)pi-
perazine-1-carboxylate (6). Yield: 32%. mp ¼ 225–227 ꢀC. 1H NMR
Sulfoxide 1 (420.0 mg, 1.00 mmol) and tert.-butyl 4-(4-amino-
phenyl)piperazine-1-carboxylate
2
or tert.-butyl-4-(4-amino-
(CDCl3, 400 MHz)
d
8.59 (s, 1H), 7.42 (d, 2H, J ¼ 8.9 Hz, Ar-H), 6.92
pyridin-3-yl)piperazine-1-carboxylate 3 (1.1 mmol) were dissolved
in DMSO (5 mL) for synthesis of compound 4 and toluene (5 mL) for
the synthesis of compound 5 and heated at 100 ꢀC for 16 h. The
reaction mixture was cooled to room temperature, diluted with
brine and extracted with ethyl acetate. The organic layer was dried
over sodium sulfate and filtered. The filtrate was concentrated in
vacuum to give compounds 4 and 5 as yellow–orange solids. The
products were used for the next reaction steps without further
purification.
(d, 2H, J ¼ 9.0 Hz, Ar-H), 5.73–5.69 (m, 1H), 3.68 (s, 3H), 3.60–3.55
(m, 4H), 3.10–3.06 (m, 4H), 2.235–2.25 (m, 2H), 1.90–1.70 (m, 4H),
1.65–1.55 (m, 2H), 1.43 (s, 9H), 0.36 (s, 9H). Low-resolution mass
spectrometry electron spray ionization (ESIþ): 669.3 [M þ H].
tert.-Butyl-4-(6-(8-cyclopentyl-5-methyl-7-oxo-6-trimethyl-
stannyl-7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)pyridin-3-
yl)piperazine-1-carboxylate (7). Yield: 55%. mp ¼ 209–211 ꢀC. 1H
NMR (CDCl3, 400 MHz)
d 8.70 (s, 1H, CH); 8.27–8.20 (m, 1H), 8.02–
7.99 (m, 1H), 7.81–7.75 (m, 1H), 5.82–5.71 (m, 1H), 3.72 (s, 3H),
3.67–3.58 (m, 4H), 3.17–3.09 (m, 4H), 2.09–1.98 (m, 4H), 1.90–1.78
(m, 2H), 1.74–1.64 (m, 2H), 1.49 (s, 9H), 0.36 (s, 9H). Low-resolution
mass spectrometry electron spray ionization (ESIþ): 670.3 [M þ H].
tert-Butyl-4-(6-(8-cyclopentyl-6-iodo-5-methyl-7-oxo-7,8-dihy-
dro-pyrido[2,3-d]pyrimidin-2-ylamino)phenyl)piperazine-1-carb-
oxylate (4). Yield: 75%. 1H NMR (CDCl3, 400 MHz)
d 8.72 (s, 1H),
7.47 and 6.95 (2d of AA’BB’ system, J ¼ 8.8 Hz, 4H, Ar-H),
5.97 quint., J ¼ 8.4 Hz, 1H), 3.63–3.58 (m, 4H), 3.15–3.08 (m, 4H),
2.66 (s, 1H, CH3), 2.30–2.22 (m, 2H), 2.06–1.98 (m, 2H), 1.87–1.78
(m, 2H), 1.68–1.56 (m, 2H), 1.49 (s, 9H). Low-resolution mass
spectrometry electron spray ionization (ESIþ): 631.4 [M þ H].
tert-Butyl-4-(6-(8-cyclopentyl-6-iod-5-methyl-7-oxo-7,8-dihy-
dropyrido[2,3-d]pyrimi-din-2-yl-amino)pyridin-3-yl)piperazine-1-
4.3. Radiosynthesis
Preparation of [124I]CKIA. 50
MeOH 1/3) was placed in a iodotube followed by the desired
quantity of [124I]NaI (36.08 MBq, ca. 3 MBq/
l in 0.1 M sodium
hydroxide). The reaction mixture was diluted with 100 l of 5%
glacial acetic acid in methanol. The Iodotube was shaken for 10 min
at room temperature. After 10 min the conversion was monitored
ml of 6 (5 mg/ml in DMSO/5% HOAc in
m
m
carboxylate (5). Yield: 38%. 1H NMR (CDCl3, 400 MHz)
d
9.41 (s, 1H,
CH); 8.11 (m, 1H), 7.92 (m, 1H), 7.83 (m, 1H), 6.46–6.36 (m, 1H), 3.74
(m, 4H), 3.66 (m, 4H), 2.98 (s, 3H, CH3), 2.50–2.40 (m, 2H) 2.33–2.23
(m, 2H), 2.14–2.04 (m, 2H), 1.93–1.83 (m, 2H), 1.49 (s, 9 H, (CH3)3).
Low-resolution mass spectrometry electron spray ionization (ESIþ):
632.4 [M þ H].
via radio-TLC. The reaction was quenched with 50
sodium bisulfite and transferred into a low-bind eppendorf vial.
200 l trifluoroacetic acid was added. After shaking for 20 min at
ml saturated
m
50 ꢀC the crude product was subjected onto a semi-preparative
HPLC column. The product fraction (13.2–14.0 min) was collected,
diluted with water (20 mL) and passed through a Waters Sep-Pak-
tC-18 cartridge. The cartridge was washed with water (10 mL) and
4.2.2. General procedure for removal of the Boc protecting group
Compound 4 or 5 (0.44 mmol) was stirred at ambient temper-
ature in dioxane (4 mL) and 6 M HCl (4 mL) for 2 h. The solvent was
evaporated in vacuum, and the residue was diluted with methanol.
The product was precipitated by adding diethyl ether. The resulting
solids were filtrated off and re-dissolved in dichloromethane. The
product was purified by silica-gel chromatography (MeOH/CHCl3 1/
1) to give compounds CKIA and CKIB as greyish-yellow solids.
8-Cyclopentyl-6-iod-5-methyl-2-(4-(piperazin-1-yl)phenylamino)-
pyrido[2,3-d]pyri-midin-7(8H)-one (CKIA). Yield: 95%. mp ¼ 200–
[
124I]CKIA was eluted with ethanol (1 mL). The solvent was evap-
orated in a gentle stream of nitrogen to afford 11.8 MBq (33.6%,
decay-corrected) of [124I]CKIA within 104 min, including HPLC
purification. The specific activity was determined to be 35 GBq/
m
mol. Radio-HPLC-analysis: tR ¼ 5.9 min.
Preparation of [124I]CKIB. Starting from 50
ml of 7 (5 mg/ml in
DMSO/5% HOAc in MeOH 1/3) and [124I]NaI (186.1 MBq, ca. 3 MBq/
m
l in 0.1 M sodium hydroxide) 32.66 MBq (17.8%, decay-corrected)
201 ꢀC. 1H NMR (acetone, 400 MHz)
d
9.25 (br, s, 1H); 8.90 (s, 1H), 7.59
of [124I]CKIB was obtained according to the method described for
(d, 2H, J ¼ 9.0 Hz, Ar-H), 7.03 (d, 2H, J ¼ 9.0 Hz, Ar-H), 5.95–5.85 (m,
1H), 3.35–3.31 (m, 4H), 3.25–3.20 (m, 4H), 2.63 (s, 3H), 2.20–2.10 (m,
2H), 1.95–1.85 (m, 2H), 1.80–1.70 (m, 2H), 1.54–1.52 (m, 2H). Low-
resolution mass spectrometry electron spray ionization (ESIþ): 531.2
[M þ H].
radiosynthesis of [124I]CKIA within 107 min, including HPLC puri-
fication. The specific activity was determined to be 25 GBq/mmol.
Radio-HPLC-analysis: tR ¼ 4.2 min.
4.4. Lipophilicity
8-Cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)pyridin-
2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (CKIB). Yield: 95%.
Lipophilicity (log D) of [124I]CKIA and [124I]CKIB was deter-
mined at pH 7.4 according to the method reported by Wilson et al.
[43].
m
p > 240 ꢀC. 1H NMR (acetone, 400 MHz)
d
9.15 (s, 1H); 8.25–8.20
(m, 1H), 7.99–7.96 (m, 1H), 7.55–7.51 (m, 1H), 6.11–6.05 (m, 1H),

736
L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
4.5. Stability studies
and hind legs (organs of interest: liver and tumor) in the center of
the field of view. For attenuation correction, a 15 min transmission
scan was performed using a rotating 57Co point source before tracer
injection and collection of the emission scans. Then, approximately
10 MBq of [124I]CKIA and [124I]CKIB were administered intrave-
nously within 15 s in a 0.3 mL volume into the tail vein. Simulta-
neously with tracer application, PET measurement was started for
60 min. Sinogram generation and image reconstruction followed
the protocol reported by our group elsewhere [46]. Three-dimen-
sional tumor regions of interest were determined for subsequent
data analysis. As an index of [124I]CKIA or respectively [124I]CKIB
uptake in vivo, maximum standardized uptake values (SUVs) at
60 min after injection were calculated in regions of interest for each
PET measurement. The SUV value, expressing the ratio of [124I]CKIA
or [124I]CKIB uptake in relation to the injected [124I]CKIA or
In vitro and ex vivo stability studies of the radiotracers
[
124I]CKIA and [124I]CKIB were performed by HPLC analysis. The
radiolabeled compounds (50 kBq) were added to 1 mL of phosphate
buffered saline (PBS, 0.1 M, pH 7.4), ethanol, cell culture medium
(RPMI þ 10% FKS þ 1% penicillin/streptomycin) or fresh rat plasma
and were incubated for 30 min or 24 h at 37 ꢀC. HPLC analysis was
performed on a Hewlett Packard Series 1100 (detector: Raytest
Ramona) with a Zorbax 300SB-C18 column (250 ꢄ 9.4 mm, 5
mm)
using a gradient elution with Soerensen buffer (0.01 M, pH 7.4) and
methanol (in 5 min from 40 to 90% methanol, then 25 min 90%
methanol) by injection of the samples.
4.6. Biological studies
[
124I]CKIB activity normalized to body weight, was calculated using
4.6.1. Cellular studies
the Rover software package (ABX Radeberg).
Human tumor cell lines HT-29 (a colorectal adenocarcinoma cell
line), and FaDu (a head and neck squamous cell carcinoma cell line)
were cultured in McCoy’s 5A medium (HT-29) or RPMI 1640
medium (FaDu) supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin-streptomycin at 37 ꢀC, 5% CO2 and 95% humidity
in a CO2 incubator (Heracell, Heraeus, Hanau, Germany).
For radiotracer uptake experiments, 5 ꢄ 104 cells were seeded in
a cavity of a 24 well plate (Greiner, Frickenhausen, Germany). After
overnight incubation, 0.25 mL of cell culture medium with about
25 kBq of [124I]CKIA or [124I]CKIB was added, and incubation was
continued for several time points at 37 ꢀC. Subsequently, cells were
washed three times with ice-cold PBS and lysed in 0.5 mL of 0.1 M
sodium hydroxide with 1% SDS. Cell lysates were counted with
a Cobra II gamma counter (Canberra-Packard, Meriden, CT, USA).
Protein levels were quantified using the BCA protein assay kit
(Pierce, Rockford, USA) according to the manufacturer’s recom-
mendations and bovine serum albumin as protein standard. Uptake
data for all experiments are expressed as percent of injected dose
per mg protein (%ID/mg protein).
After PET measurements, mice were euthanized and ex vivo
autoradiography of whole mice was performed on a cryostat (Jung
Cryopolycut, Leica)1 h after injection of radiotracers [124I]CKIA and
[
124I]CKIB. The body sections were dried in a freeze-dryer and then
exposed to a phosphor imaging plate for 4 h and scanned (Bio
Imaging Analyzer Fujifilm BAS-5000).
All animal experiments were performed in accordance with the
guidelines of the German laws for animal Welfare. The protocol was
approved by the local Ethical Committee for Animal Experiments.
Acknowledgements
The authors are grateful to Heidemarie Kasper, Regina Herrlich,
Andrea Suhr and Mareike Barth for their excellent technical
assistance.
References
[1] H.X. An, M.W. Beckmann, G. Reifenberger, H.G. Bender, D. Niederacher, Am. J.
Clin. Pathol. 154 (1999) 113–118.
[2] S. Ortega, M. Malumbres, M. Barbacid, Biochim. Biophys. Acta. Rev. Cancer
1602 (2002) 73–87.
[3] A. Perry, K. Anderl, T.J. Borell, D.W. Kimmel, C.H. Wang, J.R. O’Fallon,
B.G. Feuerstein, B.W. Scheithauer, R.B. Jenkins, Am. J. Clin. Pathol. 112 (1999)
801–809.
4.6.2. Biodistribution studies
Biodistribution of radiotracers [124I]CKIA and [124I]CKIB were
studied in male Wistar rats (Unilever, Harlan Germany, 200–230 g
body weight) after 5 min and 60 min post injection. Aliquots (0.2–
0.3 MBq) of each compound were added to 500 ml of E153 infusion
[4] G. Wei, F. Lonardo, T. Ueda, T. Kim, A.G. Huvos, J.H. Healey, M. Ladanyi, Int. J.
Cancer 80 (1999) 199–204.
[5] D.G. Johnson, C.L. Walker, Annu. Rev. Pharmacol. Toxicol 39 (1999) 295–312.
[6] D.O. Morgan, Annu. Rev. Cell Dev. Biol. 13 (1997) 261–291.
[7] C.J. Sherr, Science 274 (1996) 1672–1677.
[8] T. Shimamura, J. Shibata, H. Kurihara, T. Mita, S. Otsuki, T. Sagara, H. Hirai,
Y. Iwasawa, Bioorg. Med. Chem. Lett. 16 (2006) 3751–3754.
[9] J.A. Diehl, Cancer Biol. Ther. 1 (2002) 226–231.
[10] C.J. Sherr, Cell 73 (1993) 1059–1065.
[11] M. Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630–641.
[12] G.I. Shapiro, J. Clin. Oncol 24 (2006) 1770–1783.
[13] H.C. Thoms, M.G. Dunlop, L.A. Stark, Cell Cycle 6 (2007) 1293–1297.
[14] P.S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer Drug Targets 8 (2008) 53–75.
[15] P.M. Fischer, Cell Cycle 3 (2004) 742–746.
solution containing 1% HSA, and the solution was administered
intravenously into the tail vein. After radiotracer application, rats
were anaesthetized with ether at 5 or 60 min p.i., respectively.
Afterwards, blood samples were withdrawn by heart puncture, and
the animals were euthanized. Organs and tissues were removed
and weighted, and the radioactivity was measured in a calibrated
well counter. The data were normalized to the amount of injected
radioactivity, and expressed as standardized uptake values (SUV,
SUV ¼ (organ activity/organ weight)/(total given activity/rat body
weight)).
[16] Y.M. Lee, P. Sicinski, Cell Cycle 5 (2006) 2110–2114.
[17] B.A. Carlson, M.M. Dubay, E.A. Sausville, L. Brizuela, P.J. Worland, Cancer Res.
56 (1996) 2973–2978.
[18] G. Kaur, M. Stetler-Stevenson, S. Sebers, P. Worland, H. Sedlacek, C. Myers,
J. Czeck, R. Naik, E. Sausville, Nat. Cancer Inst. 84 (1992) 1736–1740.
[19] R.N. Misra, J. Med. Chem 47 (2004) 1719–1728.
[20] S. Akinaga, K. Gomi, M. Morimoto, T. Tamaoki, M. Okabe, Cancer Res. 51 (1991)
4888–4892.
[21] K. Kawakami, H. Futami, J. Takahara, K. Yamaguchi, Biochem. Biophys. Res.
Commun. 219 (1996) 778–793.
[22] F.I. Raynaud, S.R. Whittaker, P.M. Fischer, S. McClue, M.I. Walton, S.E. Barrie,
M.D. Garrett, P. Rogers, S.J. Clarke, L.R. Kelland, M. Valenti, L. Brunton, S. Eccles,
D.P. Lane, P. Workman, Clin. Cancer Res. 11 (2005) 4875–4888.
[23] J. Vesely, L. Havlicek, M. Strnad, J.J. Blow, A. Donelladeana, L. Pinna,
D.S. Letham, J. Kato, L. Detivaud, S. Leclerc, L. Meijer, Eur. J. Biochem. 224
(1994) 771–786.
4.6.3. Small-animal PET imaging and ex vivo autoradiography
Small-animal positron emission tomography experiments were
performed on an established mouse tumor xenograft model (NMRI
nu/nu male mice with implanted FaDu tumor, transplantation
procedure described in ref. [45]).
When tumors reached a weight of about 200 mg, imaging
studies were performed on a dedicated small-animal PET scanner
(microPET P4; CTI Concorde Microsystems). The animals were
anesthetized through inhalation of 9% desflurane (v/v) (Suprane;
Baxter) in 40% oxygen/air (gas flow, 1 L/min). Then, the animals
were positioned and immobilized prone with their medial axis
parallel to the axial axis of the scanner and their thorax, abdomen,

L. Koehler et al. / European Journal of Medicinal Chemistry 45 (2010) 727–737
737
[24] S.J. McClue, D. Blake, R. Clarke, A. Cowan, L. Cummings, P.M. Fischer,
M. MacKenzie, J. Melville, J.K. Stewart, S. Wang, N. Zhelev, D. Zheleva,
D.P.I. Lane, Int. J. Cancer 102 (2002) 463–468.
[35] D.W. Fry, P.J. Harvey, P.R. Keller, W.L. Elliott, M. Meade, E. Trachet, M. Albassam,
X.X. Zheng, W.R. Leopold, N.K. Pryer, P.L. Toogood, Mol. Cancer Ther. 3 (2004)
1427–1437.
[25] B.P. Nutley, F.I. Raynaud, S.C. Wilson, P.M. Fischer, A. Hayes, P.M. Goddard,
S.J. McClue, M.Jarman,D.P. Lane, P. Workman, Mol. CancerTher. 4(2005)125–139.
[26] I.R. Hardcastle, C.E. Arris, J. Bentley, F.T. Boyle, Y.H. Chen, N.J. Curtin,
J.A. Endicott, A.E. Gibson, B.T. Golding, R.J. Griffin, P. Jewsbury, J. Menyerol,
V. Mesguiche, D.R. Newell, M.E.M. Noble, D.J. Pratt, L.Z. Wang, H.J. Whitfield,
J. Med. Chem 47 (2004) 3710–3722.
[36] S.N. VanderWel, P.J. Harvey, D.J. McNamara, J.T. Repine, P.R. Keller, J. Quin,
R.J. Booth, W.L. Elliott, E.M. Dobrusin, D.W. Fry, P.L. Toogood, J. Med. Chem. 48
(2005) 2371–2387.
[37] L. Varagnolo, M.P. Stokkel, U. Mazzi, E.K. Pauwels, Nucl. Med. Biol. 27 (2000)
103–112.
[38] K.S. Pentlow, M.C. Graham, R.M. Lambrecht, F. Daghighian, S.L. Bacharach,
B. Bendriem, R.D. Finn, K. Jordan, H. Kalaigian, J.S. Karp, W.R. Robeson,
S.M. Larson, J. Nucl. Med. 37 (1996) 1557–1562.
[27] K. Bettayeb, N. Oumata, A. Echalier, Y. Ferandin, J.A. Endicott, H. Galons,
L. Meijer, Oncogene 27 (2008) 5797–5807.
[28] P.G. Wyatt, A.J. Woodhead, V. Berdini, J.A. Boulstridge, M.G. Carr, D.M. Cross,
D.J. Davis, L.A. Devine, T.R. Early, J. Med. Chem 57 (2008) 4986–4999.
[29] D. Parry, T. Guzi, W. Seghezzi, K. Paruch, R. Doll, A. Nomeir, Y. Wang, M. Oft, P.
Kirschmeier, T. Chen, E. Lees, Presented at the AACR Conference, Los Angeles,
CA, (2007) Poster 4371.
[30] M.A. Dickson, G.K. Schwartz, Curr. Oncol. 16 (2009) 120–127.
[31] G.I. Shapiro, R. Bannerji, K. Small, S. Black, P. Statkevich, M. Abutarif, J. Moseley,
S. Yao, C.H. Takimoto, M.M. Mita, J. Clin. Oncol 26 (2008) 15S.
[32] S. Ortega, I. Prieto, J. Odajima, A. Martı´n, P. Dubus, R. Sotillo, J.L. Barbero,
M. Malumbres, M. Barbacid, Nat. Genet. 35 (2003) 25–31.
[39] S. Vandenberghe, Nucl. Med. Commun. 27 (2006) 237–245.
[40] G.W. Kabalka, R.S. Varma, Tetrahedron 45 (1989) 6601–6621.
[41] K. Wang, S.J. Adelstein, A.I. Kassis, DMSO increases radioiodination yield of
radiopharmaceuticals. Appl. Radiat. Isot. 66 (2008) 50–59.
[42] W.C. Eckelman, Nucl. Med. Biol. Int. J. Radiat. Appl. Instrum. Part B 16 (1989)
233–245.
[43] A.A. Wilson, L. Jin, A. Garcia, J.N. DaSilva, S. Houle, Appl. Radiat. Isot. 54 (2001)
203–208.
[44] F. Graf, L. Koehler, T. Kniess, F. Wuest, B. Mosch, J. Pietzsch [article ID 106378].
J. Oncol. (2009) 1–12.
[33] M. Barvian, D.H. Boschelli, J. Cossrow, E. Dobrusin, A. Fattaey, A. Fritsch, D. Fry,
P. Harvey, P. Keller, M. Garrett, F. La, W. Leopold, D. McNamara, M. Quin,
S. Trumpp-Kallmeyer, P. Toogood, Z.P. Wu, E.L. Zhang, J. Med. Chem. 43 (2000)
4606–4616.
[45] C. Haase, R. Bergmann, F. Fuechtner, A. Hoepping, J. Pietzsch, J. Nucl. Med. 48
(2007) 2063–2071.
[46] M. Berndt, J. Pietzsch, F. Wuest, Nucl. Med. Biol. 34 (2007) 5–15.
[47] D.W. Fry, D.C. Bedford, P.H. Harvey, A. Fritsch, P.R. Keller, Z.P. Wu, E. Dobrusin,
W.R. Leopold, A. Fattaey, M.D. Garrett, J. Biol. Chem. 276 (2001) 16617–16623.
[34] P.L. Toogood, Med. Res. Rev. 21 (2001) 487–498.