Synthesis of imatinib, a tyrosine kinase inhibitor, labeled with carbon-14

Article abstract of DOI:10.1002/jlcr.3830

Imatinib (Gleevec) is a multiple tyrosine kinase inhibitor that decreases the activity of the fusion oncogene called BCR-ABL (breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog) and is clinically used for the treatment of chronic myelogenous leukemia and acute lymphocytic leukemia. Small molecule drugs, such as imatinib, can bind to several cellular proteins including the target proteins in the cells, inducing undesirable effects along with the effects against the disease. In this study, we report the synthetic optimization for ¹⁴C-labeling and radiosynthesis of [¹⁴C]imatinib to analyze binding with cellular proteins using accelerator mass spectroscopy. ¹⁴C-labeling of imatinib was performed by the synthesis of ¹⁴C-labeld 2-aminopyrimidine intermediate using [¹⁴C]guanidine·HCl, which includes an in situ reduction of an inseparable byproduct for easy purification by HPLC, followed by a cross-coupling reaction with aryl bromide precursor. The radiosynthesis of [¹⁴C]imatinib (specific activity, 631 MBq/mmol; radiochemical purity, 99.6%) was achieved in six steps with a total chemical yield of 29.2%.

Full text of DOI:10.1002/jlcr.3830

Chang Dong-Jo (Orcid ID: 0000-0002-2452-4322)
Synthesis of imatinib, a tyrosine kinase
inhibitor, labeled with carbon-14
Julie Kang,1,‡ Jun Young Lee,2, ‡ Jeong-Hoon Park,2,* and Dong-Jo Chang1,*
1 College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon
National University, 255 Junang-ro, Suncheon 57922, Republic of Korea
2
Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute,
Jeongeup 56212, Republic of Korea
‡The first two authors contributed equally to this work.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3830
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Corresponding authors:
*Dong-Jo Chang,
College of Pharmacy, Sunchon National University
Suncheon 57922, Republic of Korea
E-mail: djchang@scnu.ac.kr
* Jeong-Hoon Park,
Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute
Jeongeup 56212, Republic of Korea
E-mail: parkjh@kaeri.re.kr
Abstract
Imatinib (Gleevec®) is a multiple tyrosine kinase inhibitor that decreases the activity of the
fusion oncogene called BCR-ABL (Breakpoint cluster region protein-Abelson murine
leukemia viral oncogene homolog) and is clinically used for the treatment of chronic
myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). Small molecule
drugs, such as imatinib, can bind to several cellular proteins including the target proteins in
the cells, inducing undesirable effects along with the effects against the disease. In this study,
14
we report the synthetic optimization for C-labeling and radiosynthesis of [14C]imatinib to
analyze binding with cellular proteins using accelerator mass spectroscopy (AMS). 14C-
labeling of imatinib was performed by the synthesis of 14C-labeld 2-aminopyrimidine
intermediate using [14C]guanidineHCl, which includes an in-situ reduction of an inseparable
byproduct for easy purification by HPLC, followed by a cross-coupling reaction with aryl
bromide precursor. The radiosynthesis of [14C]imatinib (specific activity, 631 MBq/mmol;
radiochemical purity, 99.6%) was achieved in six steps with a total chemical yield of 29.2%.
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Keywords: BCR-ABL, imatinib, target identification, protein binding, carbon-14
1. INTRODUCTION
Small molecule drugs, such as kinase inhibitors, interact with many cellular proteins as well
as the target protein. Imatinib is a tyrosine kinase inhibitor used in the treatment of chronic
myelogenous leukemia (CML) and lymphocytic leukemia (ALL).1-4 Imatinib interrupts the
activity of the BCR-ABL fusion protein by binding to the ATP binding site. However, most
kinase inhibitors, including imatinib, are multiple kinase inhibitors and bind to diverse
kinases owing to the high structural homology among the ATP binding sites of different
kinases.5-7 The interaction of these small molecule drugs with a target protein mediates the
therapeutic effect; however, they simultaneously bind to several cellular proteins along with
the target protein, inducing undesirable side effects.8-10 The most important process in drug
discovery and development is the optimization of therapeutic efficacy while minimizing
undesirable side effects. Thus, the analysis of the interactions between small molecular
ligands and their molecular targets is important in innovative drug discovery and the
prediction of drug toxicity.11-13 The importance of analyzing small molecule–protein
interactions has driven various target identification methods, including affinity-based
methods and proteomic target identification.14-17 Affinity-based target identification methods,
such as affinity chromatography using biotin or isotope-tagged small molecules as affinity
reagents and SILAC (Stable Isotope Labeling of Amino acids in Cell culture), have a clear
disadvantage; they require the chemical modification of small molecules, which may induce
responses different from those of the hit or lead compound. 14C-labeled small molecules
could therefore be an ideal choice to identify and analyze binding proteins as 14C-labeling
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does not require any chemical modification.18, 19 Notably, 14C-labeled compounds show the
same physicochemical properties and biological activities as the parent compound.
Furthermore, 14C-labeled compounds can be detected and analyzed by accelerator mass
spectroscopy (AMS) with extremely high sensitivity.20, 21 Hence, we selected imatinib, an
anticancer therapeutic agent targeting BCR-ABL, for the development of an analytical
method evaluating drug–protein interactions. Previously, [14C]imatinib has been used for the
pharmacokinetic and metabolism investigation22, 23; however, to our knowledge, no
radiosynthetic method of [14C]imatinib has been published yet. Here, we report the efficient
14C-labeling of imatinib for easy purification, prior to the analyses of the interaction between
imatinib and the target protein, BCR-ABL.
2. RESULTS AND DISCUSSION
Imatinib is one of the most well-known anticancer agents for targeted therapy, and a variety
of synthetic methods have been reported.24-29 For efficient 14C-labeling and to obtain
14
metabolically stable C-labeled imatinib, the synthetic route reported by Loiseleur et al. was
modified.25 As depicted in Scheme 1, we envisioned that the Pd-catalyzed cross-coupling
14
reaction of aryl bromide precursor 4 with 2-aminopyrimidine 7, labeled with C at the C2
position in the pyrimidine ring, would produce a metabolically stable 14C-labeled imatinib.
We initiated the synthesis of precursors (4, 6) from commercially available benzaldehyde
derivative 2. Reductive amination of aldehyde 2 with 1-methylpiperazine using sodium
triacetoxyborohydride produced para-substituted methyl benzoate 3 in a yield of 87%, which
was subjected to a transamidation reaction with 4-amino-2-bromotoluene using potassium
tert-butoxide to obtain precursor 4 in a moderate yield (65%). The other precursor 6, 3-
dimethylamino-l-(3-pyridyl)-2-propen-l-one, was prepared by the enamination of 3-
acetylpyridine 5 with N,N-dimethylformamide dimethyl acetal in acetic acid to afford a 78%
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yield. On a large scale, the reflux of enaminone 6 (1.5 eq.) with guanidineHCl (1.0 eq.) in
the presence of NaOH (1.5 eq.) proceeded in ethanol to provide the desired 2-
aminopyrimidine 7 in a 72% yield. Prior to 14C labeling using [14C]guanidineHCl, we
attempted to optimize the synthesis of 4-(pyridin-3-yl)pyrimidin-2-amine 7, which was
carried out using 0.1 M stock solutions of enaminone 6, guanidineHCl, and NaOH in ethanol
(Table 1 & Figure 1). First, we used an excess of precursor 6 (1.5 eq.) to maximally reduce
radioactive waste while preparing [14C]4-(pyridin-3-yl)pyrimidin-2-amine 7 (entry 1 in Table
1). In the HPLC analysis, the reaction exhibited a high rate of conversion of 6 to the desired
product (7, 75.7 % area), but showed inseparable peaks of the unreacted 6 (Rt = 4.3 min) and
the desired product 7 (Rt = 4.6 min). Hence, equivalent amounts of 6 and guanidineHCl were
reacted to minimize the unreacted precursor 6 for efficient purification, indicating 95.3%
conversion in the HPLC chromatogram; however, the peak of the unreacted 6 was still
remained in the HPLC chromatogram (4.7 % area, entry 2 in Table 1). To overcome this
obstacle in the HPLC purification, we designed an in-situ reduction by NaBH4, following the
reaction of enaminone 6 with guanidineHCl, to remove the unreacted 6 (entry 3 in Table 1).
This in-situ one-pot reaction showed a high conversion of 6 to 7 (95.7 % area), and the
unreacted precursor 6 (Rt = 4.3 min) was converted to a more polar byproduct (8, Rt = 3.0
min, 4.3% area), which enabled the easy purification of the desired product by HPLC. Next,
we attempted to optimize the cross-coupling reaction of 2-aminopyrimidine 7 with precursor
4 to complete the synthesis of imatinib for 14C-labeling by the method described by Loiseleur
et al. On a large scale, a mixture of precursors 4 (1 eq.) and 7 (2 eq.) was refluxed in the
presence of Pd2(dba)3CHCl3 (0.05 eq.), rac-BINAP (0.1 eq.), and t-BuONa (1.5 eq.) in
anhydrous xylene and afforded imatinib with a high yield of 92%. For the synthesis of
radioactive [14C]imatinib, the optimization of the cross-coupling reaction of precursor 4 with
7 was performed in the presence of the Pd catalyst and t-BuONa in anhydrous xylene, and
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analyzed by HPLC (Table 2). This optimization of cross-coupling reaction was performed on
a small scale in anhydrous xylene and showed results different from those of the large scale
reaction. The reactions with an excess of 2-aminopyrimidine 7 failed to proceed adequately
and yielded a very small amount of the product, regardless of variations in the amount of Pd
catalyst and temperature (entry 1 to 4 in Table 2). Thus, an excess of precursor 4 (1.2 or 1.5
eq.) with 0.05 or 0.1 eq. of Pd catalyst was subjected to cross-coupling at 140°C; however,
the reaction failed to produce a sufficient yield of desired product 1 (entry 5 to 8 in Table 2).
Finally, we increased the amount of Pd catalyst (0.2 eq.) under the same condition (1.2 eq. of
4, 1 eq. of 7, 140°C); this resulted in a high conversion of precursor 7 (11.5 % area) to
imatinib (1, 74.2 % area, entry 9 in Table 2 & Figure 2b). However, more excess of Pd
catalyst (0.3 eq.) exhibited a decreased conversion of 7 (24.5 % area) to 1 (50.2 % area, entry
10 in Table 2), which correlated with the reactions at 140°C described at entry 3 and 4 in
Table 2.
Based on the optimization using the nonradioactive precursor 4, 7, and guanidineHCl,
we synthesized [14C]imatinib using commercially available [14C]guanidineHCl (specific
activity = 53.3 mCi/mmol, Moravek Biochemicals®, SCHEME 2). Here, 250 µCi of
radioactive [14C]guanidineHCl was used to synthesize [14C]4-(pyridin-3-yl)pyrimidin-2-
amine ([14C]-7) by a reaction with enaminone 6, followed by in-situ NaBH4 reduction, under
the same conditions as those used in the synthesis of nonradioactive 7. The crude [14C]-7 was
purified by filtration on a Sep-Pak silica cartridge and subsequently separated using
preparative HPLC, affording a radiochemical yield of 95% and radiochemical purity of 95.2%
(Figure 3a). Finally, the cross-coupling reaction of 14C-labeled precursor 7 ([14C]-7, 1 eq.)
with an aryl bromide precursor 4 (1.2 eq.) was performed in the presence of Pd2(dba)3CHCl3
(0.2 eq.), rac-BINAP (0.3 eq.) and t-BuONa (2 eq.) in xylene at 140°C, which was the
condition optimized using cold compounds, to yield the desired 14C-labeled 4-[(4-
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methylpiperazin-1-yl)methyl]-N-(4-methyl-3-{[(4-pyridin-3-yl)-pyrimidin-2-
ylamino]phenyl}benzamide ([14C]imatinib). The desired product, [14C]imatinib, was obtained
by HPLC purification with specific activity of 631 MBq/mmol (radiochemical yield, 33%;
radiochemical purity, 99.6%; Figure 3b).
3. CONCLUSION
In summary, we developed an efficient synthetic method to generate 14C-labeled imatinib
using a total of six steps, with a 29.2% chemical yield, accomplished by the modification of
Loiseleur’s method. The key intermediate 7 was synthesized by cyclization of enaminone 6
with guanidineHCl, and crude 7 was easily purified by subsequent in-situ reduction using
NaBH4 followed by HPLC. The optimization of the cross-coupling reaction of 2-
aminopyrimidine 7 with aryl bromide 4 was performed using various equivalences of
Pd2(dba)3CHCl3, rac-BINAP, and t-BuONa. Thus, we applied the optimized 14C-labeling
method to the synthesis of imatinib labeled with 14C, and successfully prepared 14C-labeled 2-
aminopyrimidine intermediate ([14C]-7) with a radiochemical yield of 95% and radiochemical
purity of 95.2% and the desired [14C]imatinib with a specific activity of 631 MBq/mmol,
radiochemical yield of 33%, and radiochemical purity of 99.6%. Currently, we are
performing in vitro experiments for the AMS analysis of imatinib binding to the c-Abl
protein using a complex of purified c-Abl protein and [14C]imatinib.
4. EXPERIMENTAL
Starting materials were obtained from commercial sources and used without purification
(Sigma-Aldrich, Combi-Block, Acros, TCI, Alfa Aesar, Daejung). Anhydrous solvents were
purchased from Sigma-Aldrich and used under dry nitrogen atmosphere. Completion of
reaction was carried out on thin layer chromatography by TLC 60 F254 from Merck. All
synthesized compounds were purified by flash column chromatography using ZEOprep 60
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(40-63) μM silica gel from ZEOCHEM. HPLC analysis of cold compounds was performed
on a Knauer instrument (Azura® analytical HPLC) with AZURA P 6.1L LPG pump and a
DAD 6.1L PDA detector. Radiochemical reactions were monitored by radio-thin layer
chromatography (Radio-TLC, BioScan, INC. Washington D.C. B-AR-2000-1), analytical
reverse phase-HPLC (Agilent Technologies, Tokyo, 1200 series) with Phenomenex Luna 5
μm C18(2) 100 Å of size 150 × 4.6 mm and Beta-RAM flow detector (LabLogic model-5).
1H NMR and 13C spectra were recorded on JEOL JNM-ECZ400s/L1 (400 MHz) and used
CDCl3 or DMSO-d6 as the solvent. Chemical shifts were quoted in parts per million (ppm)
and coupling constants (J) were reported in Hertz (Hz). Chemical shifts (in ppm) were
referenced to tetramethylsilane (δ = 0 ppm) in CDCl3 as an internal standard. 13C NMR
spectra were obtained by using the same NMR spectrometers and chemical shifts were
reported in ppm referenced to the center line of a triplet at 77.0 ppm of CDCl3 or 39.5 ppm of
DMSO-d6.
4.1. Chemistry
Methyl 4-((4-methylpiperazin-1-yl)methyl)benzoate (3)
To the mixture of 1-methylpiperazine (1.0 g, 9.78 mmol) and 4-formylbenzoate (2, 1.8 g,
10.76 mmol) in anhydrous CH2Cl2 (97.8 mL) was added NaBH(OAc)3 (4.37 g, 19.57 mmol).
The mixture was stirred at room temperature for 12 h. The solvent was removed under
reduced pressure, and basified with aqueous NaHCO3 to pH 8~9. The residue was extracted
with EtOAc (1 × 50 mL) and washed with water (1 × 45 mL). The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The product was isolated by flash
column chromatography on silica gel (CH2Cl2: MeOH = 20: 1) to yield 3 as a yellow oil
1
(2.11 g, 87%). H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2
13
Hz, 2H), 3.83 (s, 3H), 3.51 (s, 2H), 2.35 (s, 8H), 2.14 (s, 3H); C NMR (100 MHz, DMSO-
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d6) δ 166.15, 144.21, 129.10, 128.91, 128.26, 61.51, 54.63, 52.50, 52.02, 45.64. ESI-MS: m/z
= 271 (M + Na)+.
N-(3-Bromo-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)benzamide (4)
A solution of compound 3 (1 g, 4.03 mmol) and 3-bromo-4-methylaniline (1.55 mg, 8.05
mmol) in anhydrous THF (60 mL) was added potassium tert-butoxide (2.02 mg, 17.97
mmol). The mixture was stirred under nitrogen atmosphere at room temperature for 17 h. The
mixture was extracted with EtOAc (3 × 60 mL) and washed with water (1 × 50 mL). The
combined organic layers were dried over Na2SO4 and concentrated under reduced pressure.
The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:
1
MeOH = 10: 1) to produce 4 as a red solid (1.05 g, 65%). H NMR (400 MHz, DMSO-d6) δ
10.24 (s, 1H), 8.10 (d, J = 2.3 Hz, 1H), 7.88 (d, J = 8.2 Hz, 2H), 7.65 (dd, J = 8.2, 2.3 Hz,
1H), 7.43 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.7 Hz, 1H), 3.51 (s, 2H), 2.36 (s, 8H), 2.30 (s,
3H), 2.14 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 165.43, 142.42, 138.38, 133.24, 132.02,
130.83, 128.67, 127.59, 123.59, 123.28, 119.40, 61.57, 54.67, 52.53, 45.69, 21.71. ESI-MS:
m/z = 403 (M + H)+.
(E)-3-(Dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (6)
To a solution of 3-acetylpyridine (5, 1.0 g, 8.09 mmol) and 1,1-dimethoxy-N,N-
dimethylmethanamine (1.99 g, 16.18 mmol) in toluene (80 mL) was added acetic acid (0.08
mL), and the mixture was refluxed at 95℃ for 18 h. After completion of the reaction, the
mixture was extracted with CH2Cl2 (2 × 30 mL), washed with water (3 × 25 mL), and dried
with Na2SO4. The crude residue was purified by flash column chromatography on silica gel
(CH2Cl2: MeOH = 10: 1) to give 6 as a red solid (1.11 g, 78%). 1H NMR (400 MHz, DMSO-
d6) δ 9.05 (d, J = 1.8 Hz, 1H), 8.64 (dd, J = 4.9, 1.7 Hz, 1H), 8.20 (dt, J = 7.9, 1.9 Hz, 1H),
7.75 (d, J = 11.9 Hz, 1H), 7.43-7.46 (m, 1H), 5.85 (d, J = 12.3 Hz, 1H), 3.12 (d, J = 21.5 Hz,
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3H), 2.93 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 183.96, 154.54, 151.24, 148.47, 135.12,
134.57, 123.37, 90.91, 44.54, 37.19. ESI-MS: m/z = 177 (M + H)+.
4-(Pyridin-3-yl)pyrimidin-2-amine (7)
A mixture of compound 6 (600 mg, 3.41 mmol) and guanidineHCl (217 mg, 2.27 mmol) in
n-BuOH (4 mL) was added NaOH (139 mg, 3.41 mmol), and the reaction mixture was heated
at 85℃ for 12 h. Then, the reaction was cooled down to room temperature and a white
precipitates appeared. The precipitates were filtered, washed with iced water and dried under
1
reduced pressure to give 7 (283 mg, 1.64 mmol, 72%). H NMR (400 MHz, CDCl3) δ 9.21
(q, J = 1.0 Hz, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.38 (dt, J = 7.9, 1.9 Hz, 1H), 8.34 (d, J =
5.5 Hz, 1H), 7.50-7.53 (m, 1H), 7.19 (d, J = 5.0 Hz, 1H), 6.75 (s, 2H); 13C NMR (100 MHz,
CDCl3) δ 163.81, 161.58, 159.39, 151.14, 147.97, 134.16, 132.49, 123.77, 106.04. ESI-MS:
m/z = 173 (M + H)+.
N-(4-Methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-4-((4-methylpiperazin-1-
yl)methyl)benzamide (1, Imatinib)
To a solution of 4 (50 mg, 0.125 mmol) and 7 (43 mg, 0.25 mmol) in anhydrous xylene (2
mL) in two neck round-bottomed flask was added t-BuONa (18 mg, 0.19 mmol). Then,
Pd2(dba)3CHCl3 (6.5 mg, 0.006 mmol) and rac-BINAP (8 mg, 0.0125 mmol) were added,
and the reaction mixture was sonicated for 10 min. The reaction mixture was refluxed at
140℃ for 5 h. The reaction was quenched with H2O (15 mL), extracted with CH2Cl2 (4 × 20
mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced
pressure. The crude residue was purified by flash column chromatography on silica gel
(EtOAc: Hexane = 5: 1) for the removal of unreacted 4, and on silica gel (CH2Cl2: MeOH =
1
10: 1) to produce imatinib (1) as a white solid (55 mg, 0.11 mmol, 92%). H NMR (400
MHz, DMSO-d6) δ 10.14 (s, 1H), 9.26 (d, J = 1.4 Hz, 1H), 8.95 (s, 1H), 8.67 (dd, J = 4.6, 1.4
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Hz, 1H), 8.50 (d, J = 5.5 Hz, 1H), 8.46 (dt, J = 8.1, 1.9 Hz, 1H), 8.07 (d, J = 2.3 Hz, 1H),
7.90 (d, J = 8.2 Hz, 2H), 7.55-7.45 (m, 2H), 7.45-7.38 (m, 3H), 7.19 (d, J = 8.7 Hz, 1H), 3.51
(d, J = 6.9 Hz, 2H), 2.31 (s, 8H), 2.20 (s, 3H), 2.15 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 165.20, 161.56, 161.14, 159.42, 151.33, 148.15, 142.03, 137.75, 137.16, 134.36, 133.73,
132.17, 129.96, 128.57, 127.53, 123.73, 117.18, 116.69, 107.46, 61.54, 54.61, 52.46, 45.60,
17.59. ESI-MS: m/z = 494 (M + H)+.
Optimization of 14C-labeling using cold compounds
Step I
Optimization of the synthesis of 4-(pyridin-3-yl)pyrimidin-2-amine (7)
To a mixture of the stock solutions of guanidineHCl (0.1 M in EtOH, 0.1 mL) and 6 (0.1 M
in EtOH, 0.15 mL) was added NaOH (0.1 M in EtOH, 0.15 mL). The reaction mixture was
capped with screwed cap and heated at 85℃ for 12 h. Then, the mixture was cooled down to
room temperature, added NaBH4 solution (0.5 M in EtOH, 0.1 mL) at room temperature, and
heated to 85℃ for 30 min. The solvent was removed under reduced pressure and filtered
through a Sep-pak silica cartridge using elution solvent (CH2Cl2: MeOH = 10: 1). The solvent
was removed under reduced pressure and dissolved in MeOH (5 mL). The solution was
analyzed by reverse phase HPLC (Rt = 4.6 min, column: Phenomenex Luna 5 μm C18(2) 100
Å of size 150 × 4.6 mm, elution with 20 to 50% MeOH in H2O for 10 min, and then with 50
to 100% MeOH in H2O for 10 min, injection volume: 20 μL, flow rate: 1 mL/min, UV
wavelength: 254 nm).
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Step II
Optimziation of the cross coupling reaction for synthesis of imatinib (1)
Precursor 4 (1.08 mg, 0.0027 mmol), 7 (0.39 mg, 0.0023 mmol), Pd2(dba)3CHCl3 (0.47 mg,
0.00046 mmol), rac-BINAP (0.42 mg, 0.00068 mmol) and t-BuONa (0.45 mg, 0.0046
mmol) were dissolved in anhydrous xylene (0.1 mL). The reaction mixture was capped with
screwed cap and then heated at 140℃ for 4 h. And then, the mixture was cooled down to
room temperature and solvent was removed under reduced pressure. The crude was filtered
through a Sep-pak silica cartridge cartridge using elution solvent (CH2Cl2: MeOH = 10: 1).
The solvent was removed under reduced pressure and dissolved in MeOH (5 mL). The
solution was analyzed by reverse phase HPLC (Rt = 12.7 min, column: Phenomenex Luna 5
μm C18(2) 100 Å of size 150 × 4.6 mm, elution with 20 to 50% MeOH in 0.1% TFA for H2O
for 20 min, and then with 50 to 80% MeOH in 0.1% TFA for H2O for 10 min, injection
volume: 20 μL, flow rate: 1 mL/min, UV wavelength: 235 nm).
4.2. Radiochemistry
14C-4-(Pyridin-3-yl)pyrimidin-2-amine ([14C]-7)
[14C]Guanidinium hydrochloride (250 µCi, Specific activity = 53.3 mCi/mmol) was
purchased from Moravek Biochemicals. To a stirred mixture of [14C]guanidineHCl solution
(125 µCi, 2.35 µmol, 1 eq, 0.1 M in EtOH) and NaOH solution (2.35 µmol, 1 eq, 0.1 M in
EtOH) in 2 mL reaction V-vial was added the precursor 6 solution (2.82 µmol, 1.2 eq, 0.1 M
in EtOH), and the stirring was continued at 85°C for 12 h. After completion of the reaction
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monitored on the radio TLC, the reaction was cooling down to room temperature. Then,
NaBH4 solution (11.7 µmol, 5 eq, 0.5 M in EtOH) was added to the reaction mixture and
stirred at 85°C for 30 min. The volatiles were removed on a rotary evaporator, and the
resulting residue was diluted in 2 mL of MC/MeOH (v/v; 10/1). The 14C-labeled crude
residue was filtered with Sep-Pak silica cartridge (7 mL MC/MeOH, v/v; 10/1). Separation,
purification and radiochemical purity of [14C]-7 were determined by reverse phase HPLC
with a radio detection Beta-RAM system to give 118 μCi of [14C]-7 with a radiochemical
yield of 95% and a radiochemical purity of 95.2% (Rt = 11.1 min, column: Phenomenex Luna
5 μm C18(2) 100 Å of size 150 × 4.6 mm, gradient: 30% of MeOH in 0.1% TFA for H2O for
10 min, 30 to 50% MeOH in 0.1% TFA for H2O for next 10 min, and then 50 to 80% MeOH
in 0.1% TFA for H2O for 10 min, 500 µL flow cell and auxiliary pump to mix scintillation
cocktail).
14C-N-(4-Methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-4-((4-
methylpiperazin-1-yl)methyl)benzamide ([14C]imatinib)
[14C]-7 (1 eq, 0.275 mg, 118 μCi, 0.0016 mmol), 4 (1.2 eq, 0.764 mg 0.0019 mmol),
Pd2(dba)3CHCl3 (0.2 eq, 0.327 mg, 0.00032 mmol), rac-BINAP (0.3 eq, 0.30 mg, 0.00045
mmol) and t-BuONa (2 eq, 0.30 mg, 0.0032 mmol) were added into 2 mL reaction V-vial
with a wing-type spin bar. The mixture was dissolved and stirred in 200 µL of anhydrous
xylene at 140℃ for 4 h. After cooling of the reaction, the volatiles were removed under
reduced pressure with heating to 55℃. The crude residue was diluted with 2 mL of
MC/MeOH (v/v; 10/1), and was filtered with Sep-Pak silica cartridge (7 mL MC/MeOH, v/v;
10/1). Separation, purification and radiochemical purity of 14C-labeled imatinib were
determined by reverse phase HPLC with a radio detection Beta-RAM system to afford 41 μCi
of [14C]imatinib with a specific activity of 631 MBq/mmol, a radiochemical yield of 33% and
a radiochemical purity of 99.6%. (Rt = 15.4 min, column: Phenomenex Luna 5 μm C18(2)
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100 Å of size 150 × 4.6 mm, gradient: 20 to 50% of MeOH in 0.1% TFA for H2O for 20 min,
50 to 80% MeOH in 0.1% TFA for H2O for next 10 min, and then 80 to 20% MeOH in 0.1%
TFA for H2O for next 30 min, 500 µL flow cell and auxiliary pump to mix scintillation
cocktail).
ACKKNOLEDGMENT
This research was supported by the National Research Foundation of Korea (NRF), funded
by the Ministry of Science, ICT & Future Planning (NRF-2015M2C2A1047700,
2017M2A2A6A05016600 & 2019R1F1A1063304). We would like to thank Editage
(www.editage.co.kr) for English editing.
CONFLICTS OF INTEREST
The authors declare no competing financial interests.
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TABLE 1. Optimization for synthesis of 7 using guanidineHCl
reaction condition (eq.)
result on HPLC (% area)
entry
guanidineHCl
6
NaOH
NaBH4
6 : 7 : 8
1
2
3
1.0
1.0
1.0
1.5
1.0
1.5
1.5
1.5
1.5
-
-
24.3 : 75.7 : 0
4.7 : 95.3 : 0
0 : 95.7 : 4.3
7
*All reagents were used as 0.1 M of stock solution in ethanol. **The ratios of compounds
were calculated using the % area in the HPLC chromatograms of the reactions.
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TABLE 2. Optimization of the final cross-coupling reaction of 4 with 7 for 14C-labeling
reaction condition (eq.)
result on HPLC (% area)
4 : 7 : 1
entry
4
7
Pd catalyst
0.2
T (℃)
120
120
140
140
140
140
140
140
140
140
1
2
1.0
1.0
1.0
1.0
1.2
1.2
1.5
1.5
1.2
1.2
1.5
1.5
1.5
1.5
1.0
1.0
1.0
1.0
1.0
1.0
35.1 : 53.0 : 10.8
25.0 : 64.5 : 9.4
24.9 : 52.9 : 19.9
35.7 : 50.5 : 2.8
13.6 : 68.7 : 1.7
15.6 : 59.4 : 4.3
15.7 : 64.4 : 4.7
28.9 : 48.6 : 9.5
2.0 : 11.5 : 74.2
18.7 : 24.5 : 50.2
0.3
3
0.2
4
0.3
5
0.05
0.1
6
7
0.05
0.1
8
9
0.2
10
0.3
**
*rac-BINAP (0.3 eq.) and t-BuONa (2 eq.) were used for the cross-coupling reaction. The
ratios of compounds were calculated using the % area in the HPLC chromatograms, deleting
the peaks of the Pd-complexe behind 25 min. (The original chromatograms are shown in ESI.)
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SCHEME 1. Synthesis of cold imatinib.
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SCHEME 2. Radiosynthesis of [14C]imatinib. (*Asterisk means the 14C-labeld position.)
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FIGURE 1. a. Crude HPLC chromatogram of entry 1 in Table 1. b. Crude HPLC
chromatogram of entry 2 in Table 1. c. Crude HPLC chromatogram of entry 3 in Table 1.
*The same concentration (1 mg/mL) of crude samples (entry 1 ~ 3) in MeOH were injected
for HPLC analysis.
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FIGURE 2. a. HPLC chromatograms of purified precursors (4, 7) and imatinib (1). b. Crude
HPLC chromatogram of the cross-coupling reaction of 4 with 7 (entry 9 in Table 2).
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FIGURE 3. HPLC chromatograms of purified precursor [14C]-7 (a) and [14C]imatinib (b) on
a BETA RAM detector.
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