A novel synthesis of [2-14C]2,5-dichloropyrimidine

Article abstract of DOI:10.1002/jlcr.1940

[2-¹⁴ C]2,5-dichloropyrimidine is a useful reagent for labeling biologically active compounds for use in hepatocyte transport studies, protein covalent binding, and metabolic profiling. This paper describes a novel five-step synthesis of [2-¹⁴ C]2,5-dichloropyrimidine from readily available [¹⁴C]urea by way of a boronic acid intermediate. A total of 4.34 mCi of [2-¹⁴ C]2,5-dichloropyrimidine was obtained with a specific activity of 226.0 μCi/mg (33.7 mCi/mmol). The radiochemical purity was 95.8%, and the overall radiochemical yield was 22% based on 20 mCi of [ ¹⁴C]urea starting material.

Full text of DOI:10.1002/jlcr.1940

Research Article
Received 16 June 2011,
Revised 1 September 2011,
Accepted 12 September 2011
Published online 18 October 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1940
A novel synthesis of [2-14C]2,5-dichloropyrimidine
a
a
b
a
*
Scott B. Tran, Brad D. Maxwell, Hong Wu, and Samuel J. Bonacorsi, Jr.
[2-14 C]2,5-dichloropyrimidine is a useful reagent for labeling biologically active compounds for use in hepatocyte
transport studies, protein covalent binding, and metabolic profiling. This paper describes a novel five-step synthesis of
[2-14 C]2,5-dichloropyrimidine from readily available [14C]urea by way of a boronic acid intermediate. A total of
4.34 mCi of [2-14 C]2,5-dichloropyrimidine was obtained with a specific activity of 226.0 mCi/mg (33.7 mCi/mmol). The
radiochemical purity was 95.8%, and the overall radiochemical yield was 22% based on 20 mCi of [14C]urea starting
material.
Keywords: pyrimidine; 2,5-dichloropyrimidine; 14C; isotope labeling; boronic acid
15% B. TLC was performed on 60F254 silica gel plates (Merck) with
Introduction
UV and/or iodine detection. The distillation was performed with a
Pyrimidine and its substituted analogs are present throughout
micro distillation apparatus from Sigma-Aldrich (catalog number:
nature in various forms and are the building blocks of numerous
Z129607). Flash chromatography was conducted using Teledyne Isco
biologically active compounds from antibiotics to vitamins and
RediSep Rf (San Diego, CA, USA) packed columns using an AnaLogix
liposaccharides.1,2 Pyrimidines are bases in RNA and DNA, in which
BSR pump. Radiolabeled products were compared with authentic
the most abundant are cytosine, thymine, or uracil. Substituted
standards when possible. All reagents and solvents were ACS grade
or better. The specific activity of [2-14 C]2,5-dichloropyrimidine was
pyrimidines are also common components of many drugs such
as buspirone (antianxiety agent), dasatinib (tyrosine kinases inhibi-
determined gravimetrically.
tor), bosentan (antihypertensitive agent), and sildenafil (vasodilator
agent).3 For the the drug discovery and development activities
5-Bromopyrimidin-2[14 C]-ol hydrochloride, 2
within Bristol-Myers Squibb to be supported, it was necessary to
prepare [2-14C]2,5-dichloropyrimidine, 5. We herein described a
To a 10-ml reaction flask was added pyrimidin-2[14C]-ol hydrochloride
prepared according to the procedure of Bonacorsi et al.5, 1 (93mg,
0.70 mmol) and concentrated HCl (1.5 ml). To this solution was slowly
added a solution of bromine (64.9 ml, 1.26 mmol) in 0.3 ml concen-
trated HCl. A precipitate that formed after the bromine solution was
completely added. The reaction was stirred at room temperature.
Additional bromine, 7 ml and 9ml, was added after 1h and 18h,
respectively, to drive the reaction to completion. The solution was
concentrated to near dryness by rotovap. The wet crude product
was partially azeotroped with acetonitrile (2 Â 2 ml) and rinsed with
ether (3 Â 1 ml) then partially dried under vacuum to produce a light
yellow solid as the HCl salt (181.7 mg, >quantitative crude yield due to
the product still containing solvent). HPLC, product retention time =
3.80 min, radiochemical purity = 92.3%. 1H-NMR (300 MHz, DMSO-d6)
d 8.51 (s 2H). MS ESI+ [M + H]+ = 175 (57%), 177 (100%), 179 (44%).
novel five-step synthesis of [2-14 C]2,5-dichloropyrimidine, 5.4 This
sequence improves the existing routes to 5 by being more robust
and higher yielding.
Experimental
General: Radioactivity was measured with a PerkinElmer Liquid
Scintillation Analyzer, Tri-Carb Model 2900TR (PerkinElmer Life
Sciences, Inc., Boston, MA, USA). Mass spectra were obtained with
a Finnigan LXQ mass spectrometer (Thermo Electron Corp.,
Marietta, OH, USA). Proton NMR spectra were recorded on a Bruker
Advance Ultrashield 300 MHz. UV and radiochemical purities were
determined by HPLC (Agilent Technologies 1100 Series HPLC
System and IN/US System b-Ram radiometric flow detector with
a 0.5-ml flow cell). Analytical HPLC method: Phenomenex C18 Luna
(Phenomenex Inc., Torrance, CA, USA), 5 mm, 4.6Â 150 mm,
detected at 220 nm. Mobile phase A: 0.05% trifluoroacetic acid
(TFA) in water, mobile phase B: 0.05% TFA in acetonitrile. Gradient:
0 min 5% B, 6 min 5% B, 11 min 95% B, 13 min 95% B, 15 min 5% B.
Flowrate= 1.0ml/min. Semi-preparative HPLC was performed with
Varian Model 218 PrepStar Pumps with 25-ml heads equipped with
a Varian ProStar Model 320 UV-Vis Detector (Varian Medical
Systems, Inc., Palo Alto, CA, USA) and Rheodyne Model 7725i injec-
tor. Semi-preparative HPLC Conditions: Phenomenex Luna C18,
21.2 Â 250mm. UV detection: 220nm, Flowrate: 15 ml/min. Mobil
phase A: 0.1% TFA in water, Mobile phase B: 0.1% TFA in acetoni-
aDepartment of Chemical Synthesis, Radiochemistry Group, Bristol-Myers Squibb,
Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543, USA
bDiscovery Chemistry, Bristol-Myers Squibb, Pharmaceutical Research and
Development, P.O. Box 4000, Princeton, NJ 08543, USA
*Correspondence to: Scott B. Tran, Department of Chemical Synthesis, Radiochemistry
Group, Bristol-Myers Squibb, Pharmaceutical Research and Development, P.O. Box 4000,
Princeton, NJ 08543, USA.
trile. Gradient: 0 min 15% B, 11 min 95% B, 13 min 95% B, 15 min E-mail: scott.tran@bms.com
J. Label Compd. Radiopharm 2011, 54 813–815
Copyright © 2011 John Wiley & Sons, Ltd.

S. B. Tran et al.
[2-14 C]5-Bromo-2-chloropyrimidine, 3
[2-14 C]2,5-Dichloropyrimidine, 5
To a 10-ml dry reaction flask was added crude 5-bromopyrimidin-2 To a 5-ml flask equipped with a condenser under nitrogen was
[
14 C]-ol hydrochloride, 2 (181.0mg) from the previous step, and added [2-14 C]2-chloropyrimidin-5-ylboronic acid TFA salt, 4 (39 mg,
neat phosphoryl trichloride (801 ml, 8.59 mmol). To the reaction 0.14 mmol) and acetonitrile (750 ml) to produce a suspension. To
was added N,N-dimethylaniline (109ml, 0.86 mmol) slowly at room the suspension was added N-chlorosuccinimide (32.9 mg,
temperature resulting in a clear brown solution. The reaction was 0.25 mmol) and copper (I) chloride (24.4 mg, 0.25 mmol). The mixture
heated to 120ꢀC for 1 h. TLC analysis (25%:75% EtOAc : hexane, was heated to 76ꢀC. After 16 h, HPLC analysis indicated complete
product 2 Rf = 0.40, product 3 Rf =0.85) showed the reaction to formation of the product (retention time = 8.32 min) and loss of
be complete. HPLC analysis showed the disappearance of product boronic acid starting material. The flask was cooled to 0ꢀC in an ice
2 and the formation of product 3 (retention time = 8.45 min). water bath, diethyl ether (3 ml) was added, and pH of the solution
Phosphoryl trichloride was quickly, but carefully distilled at was adjusted to 9 by slowly adding saturated NaHCO3 (6 ml). After
120ꢀC with a microdistillation apparatus. Some of the product separation of the layers, the aqueous layer was extracted with diethyl
sublimed on the upper walls of the flask. The flask was cooled ether (3 Â 3 ml). The combined organic extracts were washed with
to 0ꢀC in an ice water bath, and diethyl ether (3ml) was added brine, dried over Na2SO4, and filtered. The solvent was removed by
followed by saturated NaHCO3 (6ml) and NaOH (20mg). The distillation to afford an off white solid (19 mg, 0.13 mmol, 92% yield).
aqueous layer was extracted with diethyl ether (4 Â 3 ml). The The product radiochemical purity was 95.8%, total activity
combined organic extracts were washed with brine, dried over obtained = 4.34 mCi, and specific activity = 226.0 mCi/mg or
Na2SO4, and filtered. The majority of the ether was removed 33.7 mCi/mmol. 1H-NMR (300 MHz, DMSO-d6) d 8.95 (s, 2H). MS
by distillation to give a brown oil (148 mg, >100% crude yield, (+H2O, ÀHCl) ESI+ [M + H]+ = 131 (98%), 133 (100%), 135 (25%).
95.2% radiochemically pure). The oil was used in the next reac-
1
tion without further purification. H-NMR (300 MHz, DMSO-d6) d
ppm 8.90 (s, 2H).
Results and discussion
2,5-dichloropyrimidine is chemically labile, volatile, and has a low
melting point (57.0–57.5ꢀC). It sublimes slowly upon standing at
[2-14 C]2-Chloropyrimidin-5-ylboronic acid, 4
room temperature. At room temperature under vacuum at 50-m
m mercury, approximately 45% of 2,5-dichloropyrimidine is lost
To an oven-dried 5-ml vial with a stir bar under nitrogen was added after 1 h, and 85% is lost after 2 h. In unlabeled pilot reactions,
THF (240 ml), toluene (960 ml), 5-bromo-2[14C]-chloropyrimidine, 3 we initially chose to follow the work reported by Tsantrizos6
(148 mg) from the previous step, and triisopropyl borate (197 ml, because of its convenience and quick access to the desired
0.842 mmol). The clear solution was cooled to À78ꢀC to form a thick product, 2,5-dichloropyrimidine, after only two steps from
white suspension. n-Butyllithium (478 ml, 0.77 mmol) was added pyrimidin-2-ol. However, on smaller scale, chlorination of
dropwise over 20 min. The reaction was vigorously stirred at pyrimidin-2-ol (95 mg, 0.70 mmol) gave low yields, produced
À78ꢀC for 1.5 h. The reaction was quenched at À78ꢀC with 2N multiple products, and required preparative HPLC to purify
HCl (1.2 ml) and then warmed slowly to room temperature resulting 5-chloropyrimidin-2-ol. Because of the low recovery and difficult
in two layers. HPLC analysis indicated that both layers contained purification and isolation, we developed a new synthetic path-
product with the majority of the product being present in the way that produced the desired product in reasonable yield
aqueous layer. The aqueous layer was collected and evaporated to requiring less challenging purification conditions (Scheme 1).
dryness by azeotroping with acetonitrile (3 Â 10 ml) then further The synthesis of [2-14 C]2,5-dichloropyrimidine, 5 was completed in
dried under vacuum. The crude product was dissolved in 1:1 water : five steps from commercially available [14 C]urea. The specific activity
acetonitrile (1.0 ml) and was purified by preparative HPLC. The pure of [14 C]urea (58 mCi/mmol) was reduced with unlabeled urea to
fractions (retention time = 9.17 min) were pooled, and the solvents approximately 30 mCi/mmol to accommodate the scale of the
were removed by rotovap. The product was dried under vacuum synthesis. It was reacted with 1,1,3,3-tetramethoxypropane in a
to give a white solid as the TFA salt (44 mg, 23% yield after 3 steps). heterocyclic condensation reaction to give pyrimidin[2-14 C]2-ol
HPLC analysis showed that the product (retention time = 5.33 min) hydrochloride, 1 in 97% yield following the procedure of Bonacorsi.5
was 95.0% radiochemically pure. 1H-NMR (300 MHz, DMSO-d6) Conversion of 1 to 5-bromopyrimidin[2-14 C]2-ol hydrochloride, 2
d 8.98 (s, 2H), 8.75 (br s, 2H).
required 2.25 equivalents of bromine in concentrated HCl to ensure
Scheme 1. Preparation of [2-14 C]2,5-dichloropyrimidine, 5.
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J. Label Compd. Radiopharm 2011, 54 813–815

S. B. Tran et al.
complete conversion. After solvent removal by rotovap, the residue 5-dichloropyrimidine be used promptly upon its preparation. In
was partially azeotroped with acetonitrile and purified by trituration our case, it was pure enough to be used directly in the next reaction
with diethyl ether to afford the wet product greater than quantita- without additional purification after aqueous work-up.
tive yield. This and other intermediates were only partially dried to
avoid the loss of product under high vacuum. Crude compound 2
was subsequently used in the chlorination reaction with neat Acknowledgements
phosphoryl trichloride in the presence of N,N-dimethylaniline. The
reaction was completed after 1 h at 120 ꢀC. In the absence of N,N- We gratefully acknowledge Huiping Zhang and Xiaoping Hou of
Bristol-Myers Squibb Discovery Chemical Synthesis and colleagues
at the Biocon Bristol-Myers Squibb Research and Development
Center for providing us with unlabeled 2,5-dichloro-pyrimidine.
We also thank John Hynes of Bristol-Myers Squibb Discovery
Chemistry for providing us with the chlorination methodology
used in the last step of the synthesis.
dimethylaniline, extended reaction times (>36 h) and product
decomposition were observed. Unreacted phosphoryl trichloride
was carefully removed by distillation at 120 ꢀC at ambient pressure.
This step was performed quickly to avoid chemical decomposition
at elevated temperatures. Although the product could have been
purified by sublimation, distillation was preferred to remove residual
phosphoryl trichloride and to avoid loss of product. This was
followed by a basic work-up to afford crude chlorinated product 3
that was determined to be 95.2% radiochemically pure. This was
sufficiently pure for use directly in the subsequent reaction with
n-BuLi and triisopropyl borate to form [2-14 C]2-chloropyrimidin-
5-ylboronic acid TFA salt, 4 in 23% yield after three steps. On larger
scale unlabeled pilot boronation reactions, yields as high as 97%
were obtained for this step. The lower yield obtained during the
radiochemical synthesis was due to residual water being present in
the previous crude product, which inhibited the Li/Br exchange
and boronation. The final step involved the mild conversion of 4 to
[2-14 C]2,5-dichloropyrimidine, 5 with stoichiometric amounts of N-
chlorosuccinimide and copper chloride in 92% yield. This represents
the first-reported heteroaromatic extension of the copper (I)
mediated aromatic boronic acid to chloride conversion reported
by Wu and Hynes.7 HPLC analysis of the reaction progress indicated
the desired product to be 95.8% radiochemically pure. Because of its
chemical instability and volatility, it is recommended that [2-14 C]2,
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Copyright © 2011 John Wiley & Sons, Ltd.
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