Preparation and evaluation of 99mTc-labeled HYNIC-palbociclib analogs for cyclin-dependent kinase 4/6-positive tumor imaging

Article abstract of DOI:10.1016/j.ejmech.2019.112032

Overexpression and amplification of cyclin-dependent kinase 4/6 (CDK4/6) occur in many cancers and may be the cause of resistance to CDK4/6 inhibitors in preclinical models. However, there are few investigations on the assessment of CDK4/6 expression in tumors or other tissues. Palbociclib, which was approved in 2015 to treat ER+/HER2-breast cancer in combination with letrozole, is a selective CDK4/6 inhibitor. In this study, an intermediate (compound 3), which could be hydrolyzed into the ligand (compound L) consisting of palbociclib as the bioactive molecule and 6-hydrazino nicotinamide (HYNIC) as the bifunctional chelator, was synthesized. Compound L was radiolabeled with ^99mTc using tricine/TPPTS or tricine/TPPMS as co-ligands. ^99mTc-tricine-TPPTS-L and ^99mTc-tricine-TPPMS-L were prepared with high radiochemical purity without postlabeling purification. They had great in vitro stability. Both radiotracers were hydrophilic, but ^99mTc-tricine-TPPTS-L had a lower log P value. In vitro cell uptake studies in MCF-7 cells showed that cellular uptake was blocked by preincubation with palbociclib, suggesting a CDK4/6-mediated uptake mechanism. Biodistribution in mice bearing MCF-7 tumors showed that ^99mTc-tricine-TPPTS-L had higher tumor uptake than ^99mTc-tricine-TPPMS-L, while they had comparable tumor-to-muscle and tumor-to-blood ratios. Radioactivity accumulation in tumors was obvious in micro-SPECT/CT images with ^99mTc-tricine-TPPTS-L. When mice were preinjected with palbociclib, tumor uptake of ^99mTc-tricine-TPPTS-L significantly decreased and the tumor accumulation was clearly lost, confirming CDK4/6 specificity. All results in this work indicated that ^99mTc-tricine-TPPTS-L is a promising tumor imaging agent that targets CDK4/6.

Full text of DOI:10.1016/j.ejmech.2019.112032

Journal Pre-proof
99m
Tc-Labeled HYNIC-Palbociclib analogs for cyclin-
Preparation and evaluation of
dependent kinase 4/6-positive tumor imaging
Qianqian Gan, Xiaoqing Song, Xuran Zhang, Junbo Zhang
PII:
S0223-5234(19)31190-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.112032
EJMECH 112032
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 November 2019
Revised Date: 24 December 2019
Accepted Date: 31 December 2019
99m
Tc-
Please cite this article as: Q. Gan, X. Song, X. Zhang, J. Zhang, Preparation and evaluation of
Labeled HYNIC-Palbociclib analogs for cyclin-dependent kinase 4/6-positive tumor imaging, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.112032.
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Preparation and Evaluation of 99mTc-Labeled HYNIC-Palbociclib Analogs for
Cyclin-Dependent Kinase 4/6-Positive Tumor Imaging
Qianqian Gan1, Xiaoqing Song1, Xuran Zhang, Junbo Zhang*
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal
University, Beijing, 100875, P. R. China.
Abstract
Overexpression and amplification of cyclin-dependent kinase 4/6 (CDK4/6) occur in many cancers and may be the cause of
resistance to CDK4/6 inhibitors in preclinical models. However, there are few investigations on the assessment of CDK4/6
expression in tumors or other tissues. Palbociclib, which was approved in 2015 to treat ER+/HER2- breast cancer in
combination with letrozole, is a selective CDK4/6 inhibitor. In this study, an intermediate (compound 3), which could be
hydrolyzed into the ligand (compound L) consisting of palbociclib as the bioactive molecule and 6-hydrazino nicotinamide
(HYNIC) as the bifunctional chelator, was synthesized. Compound L was radiolabeled with 99mTc using tricine/TPPTS or
tricine/TPPMS as co-ligands. 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L were prepared with high radiochemical
purity without postlabeling purification. They had great in vitro stability. Both radiotracers were hydrophilic, but
99mTc-tricine-TPPTS-L had a lower log P value. In vitro cell uptake studies in MCF-7 cells showed that cellular uptake was
blocked by preincubation with palbociclib, suggesting a CDK4/6-mediated uptake mechanism. Biodistribution in mice
bearing MCF-7 tumors showed that 99mTc-tricine-TPPTS-L had higher tumor uptake than 99mTc-tricine-TPPMS-L, while
they had comparable tumor-to-muscle and tumor-to-blood ratios. Radioactivity accumulation in tumors was obvious in
micro-SPECT/CT images with 99mTc-tricine-TPPTS-L. When mice were preinjected with palbociclib, tumor uptake of
99mTc-tricine-TPPTS-L significantly decreased and the tumor accumulation was clearly lost, confirming CDK4/6 specificity.
All results in this work indicated that 99mTc-tricine-TPPTS-L is a promising tumor imaging agent that targets CDK4/6.
Key words: CDK4/6 99mTc palbociclib HYNIC tumor imaging
1. Introduction
There are four distinct phases (G1, S, G2, M) in the cell cycle. Before entering G1 phase, nondividing cells stay in a resting
or quiescence state called G0 phase.[1, 2] Cell cycle dysregulation leads to uncontrolled cell proliferation, subsequently

resulting in tumorigenesis.[3] Specific proteins are in control of the cell cycle. Cyclin-dependent kinases (CDKs), belonging
to the serine-threonine protein kinase family, are the primary proteins that regulate the cell cycle. They are triggered by their
corresponding cyclins.[4] Among the twenty-one CDKs that have been found already, CDK4 and CDK6 are a pair of CDKs
sharing 71% amino acid homology as well as similar functions.[5] They control progression from G1 to S phase through the
cyclin D-CDK4/6-INK4-pRb pathway.[6-8] The CDK4/6-cyclin D complex phosphorylates pRb, the product of the
antioncogene, causing the dissociation of the pRb-E2F complex. Then, free E2F begins to play the role of a transcription
factor to motivate the cell cycle transition from G1 to S phase.[9, 10] Overexpression of CDK4/6 has been observed in many
cancers, such as breast cancer[11], oral cancer[12] and glioma[13]. Therefore, selective inhibition of CDK4/6 has been an
attractive target for its potential to enhance the chemotherapeutic effects of tumor treatment.[14]
There have also been reports that the amplification of CDK4/6 might be related to the resistance to CDK4/6 inhibitors in
preclinical models.[15] Therefore, an assessment of CDK4/6 expression in tumors or other tissues may help to select patients
that could be responsive to CDK4/6 inhibitors as well as to reveal the mechanisms of drug resistance. Nuclear medicine
technology with the proper tracers can noninvasively achieve this goal at the molecular and cellular levels in living systems.
Disappointedly, there have been few investigations on the development of radiotracers targeting CDK4/6 for the diagnosis
of cancer. The first reported radiotracers of CDK4 imaging were [124I]CKIA and [124I]CKIB. Unfortunately, these
compounds showed poor performance in vivo. They were rapidly metabolized to other substances besides [124I]I2. Their
radioactivity accumulation in FaDu xenografts was unnoteworthy in micro-PET images and only marginal in ex vivo
autoradiography.[16]
With the development of hardware and image reconstruction methods, the quality of SPECT images has greatly improved.
Remarkable physical properties (t1/2 = 6 h, Eγ = 140 keV) and convenient availability make 99mTc still the most widely used
radionuclide for SPECT imaging in the clinic. Furthermore, 99mTc can easily coordinate with biomolecules through a
bifunctional chelator, such as isonitrile (-N≡C) and 6-hydrazino nicotinamide (HYNIC). Thus, it has drawn our attention to
develop 99mTc-labeled radiotracers for CDK4/6 imaging.
In our previous work[17], four isocyano-containing palbociclib derivatives were synthesized and radiolabeled with a

[
99mTc(CO)3]+ core. These 99mTc complexes had good in vitro and in vivo stability. The corresponding Re chelates showed
high affinity for CDK4 and good selectivity for CDK4 versus CDK2 in CDK kinase inhibition assays. A significant reduction
of MCF-7 cellular uptake was observed in the presence of palbociclib, indicating a CDK4/6 specific uptake mechanism.
Biodistribution studies showed considerable tumor uptake and tumor-to-muscle ratios of all four radiotracers, and the tumor
was obviously visible in micro-SPECT/CT images. The results that the tumor uptake of 99mTc-L4 could be blocked by excess
palbociclib, both in its biodistribution and by micro-SPECT/CT imaging, confirmed its specificity for CDK4/6. However, the
highest radioactivity accumulation was found in the abdomen, especially in the liver, which might be due to their good
lipophilicity. In this study, we aimed to lower the lipophilicity of 99mTc-labeled complexes to reduce the uptake in the liver.
HYNIC has been a popular and efficient bifunctional chelator for 99mTc. We maintained the target vector palbociclib but
changed the bifunctional chelator to HYNIC. Two 99mTc-labeled HYNIC-palbociclib complexes using tricine/TPPTS or
tricine/TPPMS as co-ligands were prepared and evaluated for their potential to be tumor imaging agents targeting CDK4/6.
2. Results
2.1. Chemistry and radiochemistry
The overall synthesis of compound 3 and the labeling procedures are shown in Scheme 1. A HYNIC-containing active ester
(compound 2) using hydrazone to protect the hydrazine group was synthesized in three steps. Then, palbociclib substituted
the NHS ester of 2 under basic conditions to obtain compound 3, which could be hydrolyzed to produce ligand L under
acidic conditions (pH 3.5-5.0).[18] All intermediates were characterized by 1H NMR, 13C NMR and MS (spectra are shown
in Supplementary data). 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L were prepared in one pot. The speculative
structures of the complexes are shown in Scheme 1. As shown in Figure 2, the retention time of the 99mTc chelates were
-
completely different from that of 99mTcO4 (14.00 min for 99mTc-tricine-TPPTS-L, 16.29 min for 99mTc-tricine-TPPMS-L
-
and 2.67 min for 99mTcO4 ). The radiochemical purity of both complexes was over 90%, so purification was not performed
in subsequent in vitro and in vivo studies considering the low concentration of compound 3 used during radiolabeling.

Figure 1. (A) Numbering of the pyrido-[2,3-d]pyrimidin-7-one scaffold and (B) the structure of palbociclib.
Scheme 1. Synthetic route and speculative structures of 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L. Reagents and conditions: a.
hydrazine hydrate, 100 °C, 4 h; b. sodium 2-formylbenzenesulfonate, r.t., 3 h; c. N-hydroxysuccinimide, DCC, r.t., 18 h→reflux, 1 h; d.
palbociclib, TEA, DCM, r.t., 6 h; e. tricine, TPPTS, SnCl2·2H2O, pH 4-5, Na99mTcO4, 100 °C, 30 min; f. tricine, TPPMS, SnCl2·2H2O, pH 4-5,
Na99mTcO4, 100 °C, 30 min.

-
Figure 2. Radiochromatograms of (A) 99mTcO4 , (B) 99mTc-tricine-TPPTS-L, and (C) 99mTc-tricine-TPPMS-L.
2.2. Stability assessment and partition coefficient
The results of the in vitro stability assessment (Figure 3) showed that the radiochemical purities of the 99mTc-labeled
radiotracers were still beyond 90% after incubation for 4 h either in the reaction mixture at room temperature or in mouse
serum at 37 °C, indicating good in vitro stability of both complexes. The log P values of 99mTc-tricine-TPPTS-L and
99mTc-tricine-TPPMS-L were -2.951 ± 0.078 and -0.350 ± 0.024, respectively, which demonstrated that they were
hydrophilic.
Figure 3. Radiochromatograms of 99mTc-tricine-TPPTS-L (red) and 99mTc-tricine-TPPMS-L (blue) after incubation in the reaction mixture at (A)
room temperature or (B) in mouse serum at 37 °C for 4 h.
2.3. Cellular uptake studies
The cellular uptake in the presence of 60 µM of palbociclib decreased by approximately 84% and 27% from unblocked
conditions (Figure 4A, p < 0.05), respectively. These results indicated a CDK4/6-mediated uptake mechanism. The binding
affinity of 99mTc-tricine-TPPTS-L measured by saturation binding assay exhibited a Kd value of 4.68 ± 1.84 nM.

Figure 4. (A) In vitro uptake by MCF-7 cells incubated with 99mTc-labeled tracers in the absence (black column) or presence (red column) of a
final concentration of 20 µM palbociclib at 37 °C after 120 min. Statistical analysis was carried out using a bilateral t test of equal variance (* p
< 0.05). (B) Saturation binding study of 99mTc-tricine-TPPTS-L by measuring the cellular uptake at different concentrations as indicated.
2.4. Biodistribution experiments
The biodistribution results of radiotracers 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L in MCF-7 xenograft-bearing
BALB/c nude mice (n = 3) at 1 and 2 h post-injetion. are shown in Figure 5. MCF-7 tumors showed much higher uptake of
99mTc-tricine-TPPTS-L (3.83 ± 1.33% ID/g at 1 h post-injection and 2.70 ± 0.58% ID/g at 2 h post-injection) than that of
99mTc-tricine-TPPMS-L (0.52 ± 0.04% ID/g at 1 h post-injection and 0.61 ± 0.01% ID/g at 2 h post-injection); the muscle
and blood showed similar results, which resulted in comparable ratios of tumor-to-muscle and tumor-to-blood of
99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L. 99mTc-tricine-TPPTS-L had good retention in the tumor, with
approximately 70% radioactivity remaining after 1 h of metabolism. For 99mTc-tricine-TPPMS-L, the tumor uptake even
increased slightly at 2 h post-injection compared with that at 1 h post-injection, of which the tracer was also observed in the
liver, kidney and spleen. Among the nontargeted tissues, the kidney showed the highest uptake of 99mTc-tricine-TPPTS-L,
and the liver had the highest uptake of 99mTc-tricine-TPPMS-L. Low stomach and thyroid (not presented) accumulation
-
implied the absence of 99mTcO4 in vivo. 99mTc-tricine-TPPTS-L had rather high blood uptake at 1 h post-injection but was
cleared quickly, with only 57% of the initial uptake being retained at 2 h post-injection. There were few differences in
99mTc-tricine-TPPMS-L uptake in blood between 1 h post-injection and 2 h post-injection.
To assess the specificity for CDK4/6, a group of mice (n = 3) bearing MCF-7 xenografts received palbociclib 30 min prior

to receiving 99mTc-tricine-TPPTS-L. As shown in Figure 5A, the tumor uptake in the blocked mice showed a significant
decrease compared with that in the unblocked mice (1.49 ± 0.30% ID/g vs 2.70 ± 0.58% ID/g, p = 0.03), confirming the
CDK4/6-mediated tumor uptake mechanism observed in vitro.
Figure 5. Biodistribution in MCF-7 xenograft-bearing BALB/c nude mice (n = 3) at 1 and 2 h post-injection. (A) 99mTc-tricine-TPPTS-L (* p <
0.05) and (B) 99mTc-tricine-TPPMS-L.
Table 1. Comparison of 99mTc-labeled tracers in BALB/c nude mice bearing MCF-7 xenografts at 2 h post-injection (mean ± SDa, % ID/g
, n =
3)
99mTc-tricine-TPPTS-L
-2.951 ± 0.078
4.16 ± 0.33
99mTc-L4
1.616 ± 0.066
62.10 ± 6.71
10.97 ± 1.48
9.88 ± 1.39
0.38 ± 0.10
1.92 ± 0.76
1.43 ± 0.10
5.52 ± 0.92
99mTc-tricine-TPPMS-L
-0.350 ± 0.024
20.84 ± 0.98
3.94 ± 0.40
log P
liver
kidney
14.71 ± 1.09
1.59 ± 0.04
spleen
3.87 ± 0.70
intestine
2.61 ± 0.51
1.66 ± 0.32
muscle
1.34 ± 0.21
0.27 ± 0.01
tumor
2.70 ± 0.58
0.61 ± 0.01
blood
6.92 ± 0.61
1.43 ± 0.14
a SD is the standard deviation.
2.5. Micro-SPECT/CT imaging
According to the biodistribution results, the tumor uptake of 99mTc-tricine-TPPMS-L was indeed much lower than that of
99mTc-tricine-TPPTS-L. Therefore, a micro-SPECT/CT imaging study was performed with 99mTc-tricine-TPPTS-L in
tumor-bearing mice. Because the tumor-to-muscle and tumor-to-blood ratios were enhanced and the uptake decreased over
time in the nontarget tissues and organs, such as the kidney and intestine, scans were carried out at 2 h after
99mTc-tricine-TPPTS-L injection. The SPECT/CT images were consistent with the biodistribution results. As seen in Figure

6A, the radioactivity accumulated clearly in the tumor (ROI of 3.94 ± 0.28). The specificity for CDK4/6 was again
confirmed by the receptor inhibition study, where the pretreatment of palbociclib suppressed the tumor uptake to a ROI of
0.95 ± 0.18 (Figure 6B).
Figure 6. Micro-SPECT/CT images of 99mTc-tricine-TPPTS-L in MCF-7 xenograft-bearing BALB/c nude mice 2 h after radiotracer
administration. (A) An unblocked mouse and (B) a blocked mouse. In the blocking study, one hundred micrograms of palbociclib was injected
intravenously via the tail vein 30 min before the administration of 99mTc-tricine-TPPTS-L. The images are shown as coronal plane (a), sagittal
plane (b) and transverse plane (c).
2.6. Toxicity study
We conducted the MTT assay to determine the cytotoxicity of compound 3, the prodrug of ligand L, in MCF-7 cells. As
shown in Figure 7, no remarkable cell death was detected after incubation with compound 3. In a further abnormal toxicity
study, none of five mice showed abnormality or died after 48 h. If one takes a person weighing 60 kg, the dose received by a
mouse was 250-278 times as much as a human received per kilogram, suggesting the high safety of the solution of
99mTc-tricine-TPPTS-L.

Figure 7. Cell viability after incubation with compound 3 at different concentrations. Each sample was tested in sextuplicate with results given as
the mean ± SD.
3. Discussion
Palbociclib, the first selective CDK4/6 inhibitor approved by FDA in 2015, has the potential to be a good and available
molecular skeleton to obtain novel ligands for CDK4/6 imaging if the modifications had little interference with the affinity
and selectivity. Palbociclib was selected from a subset of pyrido-[2,3-d]pyrimidin-7-one scaffolds (Figure 1A) that had
favorable selectivity for CDK4.[19] The introduction of the C5 methyl group led to improved selectivity for CDK4.[20] The
C6 position appeared to tolerate a range of substituents, but small substituents were preferred. In particular, an acetyl or
ethyl ester substituent at the C6 position cooperated better with the C5 methyl group to give rise to an enhancement in
potency for CDK4. A cycloalkyl group was required at the N8 position, and a cyclopentyl group held the best balance of
potency and selectivity for CDK4.[21] Both the substituents at the C6 and N8 positions occupied the hydrophobic back
pocket of the ATP binding pocket.[22, 23] Although substitution of the aniline with a 2-aminopyridine diminished the potency,
excellent selectivity for CDK4 was achieved.[21] Studies have revealed that the NH at the C2 position and the nitrogen at the
N3 position play a key role in the interaction with the kinase hinge region.[24] The 2-aminopyridine nitrogen is fundamental
for CDK4 selectivity. The piperazine moiety attached to the 2-aminopyridine is preferred for its better solubility, but
substitution of the piperazine for other heterocyclic groups or modification at the terminal NH position have proven
sufferable in light of their small influences on the potency and selectivity.[21, 25] Based on these findings, four 99mTc-labeling

complexes were prepared successfully and proven to target CDK4/6, which indicated that this combination with a
bifunctional chelator for radiometal labeling at NH-piperazine was also practicable. However, their good lipophilicity might
be the cause of high liver uptake, which sequentially led to poor quality images.[17] Therefore, it is worth an attempt to
lower the lipophilicity by changing the chelator from isocyano-group to others that can chelate with 99mTc and other
hydrophilic co-ligands in further studies. In addition, the bifunctional chelator is expected to form a stable 99mTc complex
with high efficiency at very low amounts of the ligand. The HYNIC moiety is one such chelator that satisfies the above
demands. However, it has been reported that the isolated hydrazine group of the HYNIC-biomolecule conjugate was
unstable, especially under basic conditions.[18, 26, 27] Aldehydes or ketones extracted from various plastic and rubber
materials during preparation could react readily with the hydrazine group to form a hydrazone, which brought about
difficulties in maintaining the purity of the ligand. On the other hand, this characteristic could be used to protect the
hydrazine group if there was already a hydrazone moiety that was inert to reaction with aldehyde and ketone impurities,
formaldehyde in particular, but was not stable enough to hydrolyze under a certain condition to obtain a free hydrazine
during radiolabeling. A hydrazone with a 2-sulfonatobenzaldehyde group has been reported to be a good choice.[18, 27] It is
inert toward formaldehyde and unstable at pH 3.5-5.0 to form enough of the free hydrazine group during 99mTc labeling.[18]
Furthermore, it is easy to prepare and soluble in water. Compound 2 was synthesized according to the literature,18 of which
the NHS ester could be replaced readily by palbociclib under basic conditions. However, the low solubility of palbociclib in
DCM limited the yield of compound 3.
The preparation of 99mTc-HYNIC complexes was easy to operate. Not only was labeling accomplished in one pot, but it was
also purification free postlabeling since the radiochemical purity was greater than 90% and only 10 µg of compound 3, the
potential bioactive molecule for CDK4/6, had been added. Based on this, for the convenience of 99mTc labeling, a kit
formulation is under consideration.
Choosing HYNIC as the bifunctional chelator has another advantage. Although the certain configuration of 99mTc-HYNIC
complexes has not been confirmed, it is clear that HYNIC occupies only part of the radiolabeling spheres of 99mTc.[28-32]
Therefore, co-ligands are required to complete the unoccupied sites of the 99mTc coordination spheres, which provides an

opportunity to adjust the efficiency of the labeling yield, the stability of the 99mTc chelates, and the in vivo performance of
99mTc complexes upon the selection of co-ligands.[31, 33] Tricine is a weakly chelating primary co-ligand, but useful for
ligand exchange to obtain more stable and more homogeneous 99mTc-HYNIC conjugates.[34] TPPTS and TPPMS were used
as the secondary co-ligands in this work. As shown in Figure 1, only one new species that definitely had a different
-
retention time from 99mTcO4 was detected in each labeling mixture by the reversed-phase HPLC method. The speculative
structures of 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L are depicted in Scheme 1. Two more sulfonic groups
make TPPTS more water-soluble than TPPMS. Therefore, it is reasonable that substitution of the co-ligand TPPTS in the
complex 99mTc-tricine-TPPTS-L and the co-ligand TPPMS in the complex 99mTc-tricine-TPPMS-L led to a significant
decrease in hydrophilicity. Additionally, the trend in the log P values was also consistent with the radio-HPLC retention
time of each complex, where the retention time of 99mTc-tricine-TPPTS-L was shorter than that of 99mTc-tricine-TPPMS-L
(Figure 2). As expected, changing the bifunctional chelator from an alkyl isocyanide to the hydrazinonicotinamide
significantly decreased the lipophilicity of the 99mTc-labeling palbociclib derivatives due to the introduction of water soluble
co-ligands, which might reduce the nonspecific uptake in normal organs or tissues such as the liver and small intestine.
The good in vitro stability of 99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L established the foundation for further
studies. An internalization study suggested that about 80% of total uptake was found inside the cells at 120 min, consistent
with the previous observation that CDK 4/6 were mostly located intracellularly.[35] A significant reduction of uptake in
MCF-7 cells was observed with excess treatment with palbociclib, implying a CDK4/6-targeting uptake mechanism. The
results of further in vivo studies, both biodistribution and micro-SPECT/CT imaging, confirmed this conjecture by blocking
experiments. When mice received palbociclib prior to the injection of 99mTc-tricine-TPPTS-L, the tumor uptake and the
ROI value decreased by approximately 45% (from 2.70 ± 0.58% ID/g to 1.49 ± 0.30% ID/g) and 76% (from 3.94 ± 0.28 to
0.95 ± 0.18), respectively.
Consistent with previous results, the higher the lipophilicity of the complex, the higher the uptake of the liver.[17, 36] The
liver uptake of 99mTc-tricine-TPPTS-L was obviously lower than that of 99mTc-tricine-TPPMS-L, while both were
significantly lower than that of the 99mTc-labeled isocyano-containing palbociclib derivatives (Table 1). The uptake of

99mTc-tricine-TPPTS-L in the kidney was significantly higher than that of 99mTc-tricine-TPPMS-L. This might suggest their
different metabolism in vivo; renal clearance for the former and a hepatobiliary pathway for the latter. Comparing
99mTc-tricine-TPPTS-L with the most promising CDK4/6 imaging agent 99mTc-L4 among 99mTc-Ln (n = 2, 3, 4, 5),
although the soluble co-ligands tricine and TPPTS occupied several vector molecules, the tumor uptake showed hardly any
interference, while better water solubility (lower log P value), lower hepatic and splenic uptake were observed, as shown in
Table 1. However, 99mTc-tricine-TPPTS-L failed to reduce blood uptake.
4. Conclusion
In this study, a hydrazone-protected HYNIC-containing NHS ester reacted with palbociclib at the NH-piperazine to
successfully obtain compound 3. The hydrolysate of compound 3, L, was labeled with 99mTc in the presence of
tricine/TPPTS and tricine/TPPMS in high radiochemical yield and radiochemical purity. Both complexes,
99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L, were hydrophilic and stable for up to 4 h in vitro. Palbociclib could
significantly suppress the uptake of these complexes in MCF-7 cells as well as the tumor uptake of 99mTc-tricine-TPPTS-L
in an MCF-7 tumor-bearing mouse model, which indicated a CDK4/6 specific uptake mechanism. In mice bearing MCF-7
tumors, 99mTc-tricine-TPPTS-L had much higher tumor uptake than 99mTc-tricine-TPPMS-L. In the micro-SPECT/CT
imaging study, 99mTc-tricine-TPPTS-L successfully visualized the tumor. Based on the current results, we recommend
99mTc-tricine-TPPTS-L as the best choice of tumor imaging agent with CDK4/6 specificity among 99mTc-labeled derivatives
due to its purification-free preparation, good water solubility and favorable biological performance.
5. Experiments
5.1. Materials
All chemicals were purchased from commercial sources and used without further purification. Palbociclib was obtained
from Beijing Lunarsun Pharmaceutical Co. Ltd. N-[Tris(hydroxymethyl)methyl]glycine (tricine) was purchased from Ark
Pharm.
Triphenylphosphine-3,3',3''-trisulfonic
acid
trisodium
salt
(TPPTS)
and
sodium
diphenylphosphinobenzene-3-sulfonate (TPPMS) were purchased from TCI (Shanghai) Development Co., Ltd. NMR
spectra were obtained on a 400 MHz NMR spectrometer (JOEL). Mass spectra (MS) were recorded on an AB SCIEX

TripleTOFTM 5600 spectrometer (AB Sciex, Concord, Canada). Fresh Na99mTcO4 eluent was obtained from a
99Mo/99mTc-generator (Chinese Institute of Atomic Energy). Radiochemical purity was analyzed by analytical
high-performance liquid chromatography (HPLC) on a Waters 600 binary HPLC pump equipped with a Raytest Gabi
radioactivity detector and a Waters 2487 UV absorbance dual λ detector (Milford, MA, USA). A C18 column (5 µm, 250 × 4.6
mm, Kromasil) was used with a flow rate of 1 mL/min in the following system: solvent A, H2O with 0.1% TFA; and solvent B,
CH3CN with 0.1% TFA (gradient: 0-2 min 10% B, 2-20 min 10-90% B, 20-28 min 90% B, 28-40 min 90-10% B).
Radioactivity counts were recorded by a PerkinElmer system (WIZARD2, 2480 Automatic Gamma Counter). Imaging studies
were performed on a Triumph SPECT/CT scanner (TriFoil Imaging, California, USA).
5.2. Chemical synthesis
Compounds 1-2 were synthesized according to the reported literature,[27] and the details are shown in the Supplementary
data. The synthesis of compound 3 was as follows: Palbociclib (447 mg, 1 mmol) and compound 2 (440 mg, 1 mmol) were
added to 30 mL of dichloromethane in a 100 mL round-bottom flask, followed by the addition of 200 µL of trimethylamine.
The mixture was allowed to stir at room temperature for 6 h. The solvent was then removed under reduced pressure, and the
residue was purified via silica gel column chromatography (dichloromethane/methanol = 10/1) to obtain compound 3 as
1
yellow powder (178 mg, yield 23%). H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 10.12 (s, 1H), 8.98 (s, 1H), 8.91 (s,
1H), 8.22 (dd, J = 2.3, 0.8 Hz, 1H), 8.05 (d, J = 2.9 Hz, 1H), 7.99 (dd, J = 7.8, 1.4 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.75
(dd, J = 7.7, 1.5 Hz, 1H), 7.70 (dd, J = 8.6, 2.3 Hz, 1H), 7.47 (dd, J = 9.1, 3.0 Hz, 1H), 7.35-7.30 (m, 1H), 7.28-7.21 (m,
2H), 5.78 (p, J = 8.9 Hz, 1H), 3.66 (m, 4H), 3.19 (m, 4H), 2.38 (s, 3H), 2.26 (s, 3H), 2.24-2.15 (m, 2H), 1.90-1.79 (m, 2H),
1.74 (m, 2H), 1.61-1.49 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 203.04, 168.25, 161.28, 159.00, 158.78, 158.39, 155.28,
148.19, 146.36, 145.34, 143.69, 142.62, 141.36, 138.12, 136.53, 132.56, 129.83, 129.27, 128.13, 127.31, 125.96, 125.66,
122.17, 115.51, 107.16, 106.17, 53.49, 49.25, 49.13, 31.83, 28.10, 25.67, 14.16. HRMS (m/z): 749.2618 (calc. 749.2618 for
C37H37N10O6S [M-Na]-).
5.3. Radiosyntheses
The radiosynthesis of 99mTc-tricine-TPPTS-L was as follows: In a clean penicillin bottle, tricine (20 mg) and TPPTS (5 mg)

were dissolved in 0.2 mL of saline. After the addition of 25 µL of SnCl2·2H2O (1 mg/mL in 0.1 N HCl), the pH was
adjusted to 4-5. Then, 10 µL of 3 (1 mg/mL in DMF) and 0.5 mL of freshly eluted Na99mTcO4 (37-370 MBq) solution were
added. The reaction mixture was incubated at 100 °C for 30 min under vacuum. After cooling to room temperature, the
radiochemical yield and purity were analyzed by radio-HPLC. The only difference between the radiosynthesis of
99mTc-tricine-TPPTS-L and 99mTc-tricine-TPPMS-L was the replacement of TPPTS by TPPMS.
5.4. Stability studies
The in vitro stability study of 99mTc-labeled L was in accordance with the literature.[37] In brief, 200 µL of the reaction
mixture was kept at room temperature or incubated in 200 µL of mouse serum at 37 °C. After 4 h, an aliquot of the reaction
mixture was loaded onto the HPLC for assessment of the radiochemical purity.
5.5. Determination of the partition coefficient
To evaluate the hydrophilicity of 99mTc-labeled complexes, the partition coefficient between 1-octanol and phosphate buffer
(0.025 mol/L, pH 7.4) was determined according to the method described previously.[37] The final partition coefficient value
was expressed as log P ± SD.
5.6. Cell culture and tumor models
The human breast adenocarcinoma MCF-7 cell line was purchased from the Chinese Infrastructure of Cell Line Resource
and cultured in DMEM containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin in a humidified incubator
at 37 °C with 5% CO2. Female BALB/c nude mice (18-20 g) were obtained from Beijing Vital River Laboratory Animal
Technology Co., Ltd. The left armpit of the mice were inoculated subcutaneously with nearly 1 × 107 cells in 0.1 mL of
DMEM. After 7-10 days, biodistribution and micro-SPECT/CT imaging studies were carried out when the tumors reached
5-10 mm in diameter.
5.7. Cell studies
To demonstrate whether the uptake of the complexes by the tumor cells was CDK4/6-mediated, MCF-7 cells (2 × 105
cells/well in 24-well plates) were pretreated with palbociclib (60 µM/well) for 30 min. Then, 99mTc-tricine-TPPTS-L or
99mTc-tricine-TPPMS-L (7.4 kBq) was added followed by incubation at 37 °C for 2 h. The cells were then rinsed with 0.5

mL of cold PBS with 0.2% BSA twice and lysed with 0.5 mL of 1 M NaOH. Radioactivity counts were measured with a
γ-counter. The experiment was repeated in triplicate.
Saturation binding assays were performed as described in the literature.[38] In brief, approximately 2 × 104 MCF-7 cells
were seeded in every well of 96-well plates and allowed to attach overnight. After removal of the medium,
99mTc-tricine-TPPTS-L at different concentrations (15-1000 nM) was added followed by incubation with the cells at 37 °C
for 1 h. Post-incubation treatments were the same as described above. The radioactivity was expressed as the percentage of
applied activity. The resulting data was used for determining the potency of 99mTc-tricine-TPPTS-L against CDK4/6 by
calculating the dissociation constant (Kd), which was calculated by GraphPad Prism 5.0. Each assay was performed four
times in parallel.
5.8. Biodistribution experiments
For biodistribution studies, every MCF-7 tumor-bearing mouse received 0.1 mL of 99mTc-tricine-TPPTS-L or
99mTc-tricine-TPPMS-L (74 kBq) by intravenous injection via the tail vein. Mice were sacrificed by decapitation at 1 or 2 h
post-injection. The tumor, blood and other tissues of interest (liver, lung, kidney, spleen, stomach, small intestine, bone,
muscle and thyroid) were harvested, weighed and measured for their radioactivity via a γ-counter. The radioactivity of every
organ or tissue was expressed as the percent uptake of the injected dose per gram of tissue (% ID/g). To confirm the
CDK4/6-specific uptake by tumors, we performed a blocking study. In the blocking group, mice were injected with 100 µg
of palbociclib 30 min before the injection of 99mTc-tricine-TPPTS-L (74 kBq). Mice were sacrificed at 2 h post-injection.
5.9. Micro-SPECT/CT imaging
MCF-7 tumor-bearing BALB/c nude mice (n = 3) were injected intravenously with 0.1-0.2 mL of 99mTc-tricine-TPPTS-L
(18.5 MBq). Acquisition of SPECT/CT images was performed while the mice were anesthetized with 1.5% isoflurane in air
at 500 mL/min at 120 min post-injection. A blocking study was also carried out in the micro-SPECT/CT imaging study. On
the same day, another mouse was pretreated with 100 µg of palbociclib by i.v. injection via the tail vein, followed by 0.1-0.2
mL of 99mTc-tricine-TPPTS-L (18.5 MBq). Then, a SPECT/CT scan was acquired 2 h later by means of the same method. A
protocol consisting of an SPECT acquisition (peak 140 keV, 20% width, no rotation, MMP930 collimator) for 20 min and a

CT scan (512 views, 2 × 2 binding, 75 kV, exposure time 230 ms) for 4 min was performed. The SPECT/CT images were
acquired using the HiSPECT software and the VivoQuant 2.5 software.
5.10.Toxicity study
To evaluate the cytotoxicity of compound 3, MTT assay were carried out in MCF-7 cells according to the literature.[39]
Briefly, MCF-7 cells were seeded in a 96-well plate (5000 cells/well) and incubated for attachment. After a night, different
concentrations (0.1, 1, 100 µM) of compound 3 were added and incubated with cells for 24 h. Then, after the addition of
MTT solution (5 mg/mL), cells were incubated at 37 °C in darkness for another 4 h. At the end of incubation, the culture
media were removed after centrifugation. 50 µL of DMSO was added to each well to dissolve the purple crystal. The
absorbance of each well at 570 nm was measured using 1420 Multiabel counter. For most radiopharmaceuticals, the
abnormal toxicity was within consideration. Therefore, we studied the toxicity of the solution of 99mTc-tricine-TPPTS-L
according to the regulation of pharmacopoeia of People’s Republic of China (2015 Edition). The total volume of the
reaction mixture was adjusted to 6 mL with saline after radiolabeling. Then, 0.5 mL of this solution were injected to
Kunming mice (n = 5, female, 18-20 g) via the tail vein. The toxicity of the solution was determined by observing the death
and survival of mice in the next 48 h.
Compliance with ethical standards
All animal studies were performed according to the Regulations on Laboratory Animals of Beijing Municipality.
Author Information
Corresponding Author
*zhjunbo@bnu.edu.cn
ORCID: Junbo Zhang: 0000-0003-3549-6483
Present Addresses
No. 19 Xinjiekou Wai Boulevard, Haidian District, Beijing. College of Chemistry, Beijing Normal University, 100875, P. R.
(China).
Author Contributions

1 Qianqian Gan and Xiaoqing Song contributed equally to this work.
Notes
The authors confirm that this article content has no conflicts of interest.
Acknowledgments
The work was financially supported by the National Natural Science Foundation of China (21771023) and the project of the
Beijing Municipal Science and Technology Commission (Z181100002218033).
Appendix A. Supplementary data
Synthesis of compound 1-2, 1H-NMR, 13C-NMR, and MS spectra are listed in the supporting information.
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Highlights
1. Two 99mTc-labeled HYNIC-palbociclib analogs were prepared as
cyclin-dependent kinase 4/6-positive tumor imaging agents.
2. A CDK4/6-mediated uptake mechanism in MCF-7 cells was observed.
3. 99mTc-tricine-TPPTS-L showed specific accumulation in CDK4/6-expressing
tumors at 2 h post-injection.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
The authors declare no conflicts of interest. All authors have given approval to the
final version of the manuscript.