Synthesis of 14C-labeled and 13C-, 15N-labeled dasatinib and its piperazine N-dealkyl metabolite

Article abstract of DOI:10.1002/jlcr.1469

Dasatinib (SPRYCEL) is a multiple kinase inhibitor approved for the treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia in patients with resistance to prior therapy, including imatinib mesylate (Glee

Full text of DOI:10.1002/jlcr.1469

Research Article
Received 30 August 2007,
Revised 26 September 2007,
Accepted 3 October 2007
Published online 8 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1469
14
13 15
Synthesis of C-labeled and C-, N-labeled
dasatinib and its piperazine N-dealkyl
metabolite
Ã
Alban J. Allentoff,a Michael W. Lago,a Marc Ogan,a Bang-Chi Chen,a
Rulin Zhao,a Ramaswarmy A. Iyer,b Lisa J. Christopher,b J. Kent Rinehart,a
Balu Balasubramanian,a and Samuel J. Bonacorsi, Jra
Dasatinib (SPRYCELs) is a multiple kinase inhibitor approved for the treatment of chronic myelogenous leukemia and
Philadelphia chromosome-positive acute lymphoblastic leukemia in patients with resistance to prior therapy, including
imatinib mesylate (Gleevecs). Radiolabeled dasatinib and its piperazine N-dealkyl metabolite were synthesized to
investigate absorption, distribution, metabolism, and elimination of the compounds in humans and animals. These
compounds were prepared following a three-step sequence, which included thiazole carboxamide formation via cyclization
of labeled thiourea with a brominated oxyacrylamide precursor. In the final step a common intermediate was converted to
either [14C]dasatinib or the radiolabeled piperazine N-dealkyl metabolite with labeling in the aminothiazole ring. Syntheses
of both compounds were achieved with radiochemical purities in excess of 98%. Stable-labeled dasatinib and the
piperazine N-dealkyl metabolite were also needed for use as mass spectral internal standards in support of bioanalytical
assays. By following the same route used for the carbon-14 synthesis, [13C4, 15N2]dasatinib and the [13C4, 15N2]metabolite
were prepared with labeling in both the dichloropyrimidine and thiazole ring systems. This convergent process introduced
stable isotope labeling through (1, 2, 3-13C3) diethyl malonate and [13C,15N2]thiourea.
Keywords: BMS-354825; dasatinib; SPRYCELs; carbon-14 labeling; stable labeling; thiazole; kinase inhibitor; metabolite
bioanalytical mass spectral analyses in support of clinical and
toxicological studies.
Introduction
Dasatinib (1, Figure 1), a novel 2-aminothiazole-containing
compound known commercially as SPRYCELs, is a potent
inhibitor of multiple tyrosine kinases1–3 including SRC family
kinases and Bcr-Abl. It has been approved in the US and EU, for
the treatment of chronic myloid leukemia and Philadelphia
chromosome-positive acute lymphoblastic leukemia in patients
with resistance to prior therapy including imatinib mesylate
(Gleevecs).4 Dasatinib is effective in tumors resistant to imatinib
mesylate. X-ray data suggest that there may be differences in
the mechanism of binding between these compounds.2,5,6
During preliminary investigations of the cross-species bio-
transformation of 1, a metabolite, 2, resulting from N-deal-
kylation of the hydroxyethyl group attached to the piperazine
ring was observed (Scheme 1).7,8 Reaction phenotyping studies
have shown that this metabolic transformation was primarily
mediated by CYP3A4.9 Recent X-ray crystallography5 and SAR
investigations3 have suggested that when dasatinib is bound to
Bcr-Abl or the ATP-binding site of SRC kinase, the hydroxyethyl
group in 1 does not contribute to binding interactions.
Therefore, metabolite 2 was of considerable interest since it
was anticipated to exhibit kinase inhibition comparable to 1. A
radiolabeled analog of metabolite 2 was desired for in vitro
binding studies and a stable-labeled analog of 2 was needed as
an MS internal standard to monitor and quantify levels in
bioanalytical assays.
During the development of dasatinib, a radiolabeled analog
was required to profile and measure the absorption, distribu-
tion, metabolism, and elimination of the compound in humans7
and animals8 and for cellular transporter studies. Stable-labeled
dasatinib was also required for use as an internal standard in
aDepartment of Chemical Synthesis, Bristol-Myers Squibb Research and
Development, One Squibb Drive, New Brunswick, NJ 08903, USA
bDepartment of Biotransformation, Bristol-Myers Squibb Research and Devel-
opment, Route 206 and Province Line Road, Princeton, NJ 08543, USA
*Correspondence to: Alban J. Allentoff, Department of Chemical Synthesis,
Bristol-Myers Squibb Research and Development, One Squibb Drive, New
Brunswick, NJ 08903, USA.
Figure 1. Dasatinib.
E-mail: alban.allentoff@bms.com
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A. J. Allentoff et al.
Scheme 1
Scheme 2
We now report on the synthesis of both 14C-labeled and
Aminothiazole 4 (Scheme 2) reacted cleanly with 4,6-dichloro-
stable-labeled analogs of 1 and 2. Unlabeled 1 has been 2-methylpyrimidine in the presence of sodium tert-butoxide in
previously synthesized through several different synthetic THF at 01C affording 6 in 82% yield. Labeled 6 was elaborated to
routes.10 Preparations of labeled analogs of 1 and 2 were based either [14C]dasatinib (1a) or metabolite 2a via substitution
on synthetic methodology used to prepare 1. This approach of the pyrimidinyl chloride in
6 with the appropriately
allowed for the construction of a labeled penultimate inter- substituted piperazine. Treatment of 6 with 5 equivalents of 1-
mediate via aminothiazole ring formation.11 This intermediate (2-hydroxyethyl)piperazine in n-butanol with diisopropylethyla-
was then converted in a final step to either the labeled parent 1 mine afforded the crude n-butanol solvate of 1a. Since dasatinib
or metabolite 2. Furthermore, this route allowed C-14 label was developed as a monohydrate, the labeled n-butanol solvate
introduction in the metabolically stable thiazole ring core of the was converted to a hydrated form by crystallization from a
molecule. The following results detail this sequence for the mixture of hot ethanol and water. [14C]Dasatinib (1a) was
efficient preparation of carbon-14-labeled 1a and 2a. An prepared with a radiochemical purity of 99.4% and a specific
analogous approach is also described using stable-labeled activity of 30.4 mCi/mg in 66% radiochemical yield from 6
precursors to synthesize 13C,15N-labeled 1b and 2b.
and an overall radiochemical yield of 53% from [14C]thiourea. In
a similar fashion, treatment of 6 with 1 equivalent of piperazine
and diisopropylethylamine in NMP afforded the labeled
metabolite 2a in 64% radiochemical yield from 6 (51%
Results and discussion
The synthetic route for the preparation of [14C]dasatinib (1a)
and radiolabeled piperazine N-dealkyl metabolite (2a) is shown
in Scheme 2. The synthesis began with the isobutyl protected
oxy-E-acyl benzamide 311, which was treated with NBS in the
presence of water in tetrahydrofuran (THF), and then reacted
with [14C]thiourea at 651C to afford the labeled thiazolecarbox-
yamide 4 in 98% radiochemical yield.
overall radiochemical yield from [14C]thiourea) with
a
radiochemical purity of 98.3% and specific activity of 33.7 mCi/
mg.
The mechanism for this transformation (Scheme 3) is
proposed to be analogous to the previously reported conver-
sion of b-ethoxyacrylates to 2-amino-thioazole-5-carboxylates.12
The use of water in the reaction suggests the intermediacy of an
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A. J. Allentoff et al.
Scheme 3
a-formyl-a-bromocarboxamide hemiacetal 7.12 Displacement of Similarly, heating 16 with excess piperazine afforded the stable-
bromine with thiourea gives 8, which subsequently cyclizes with labeled metabolite 2b in 76% yield.
the loss of water affording intermediate 9. Internalization of the
In conclusion, we have developed an efficient three-step
C–N double bond leads to a transient existence of 10 that route for the preparation of both carbon-14 and stable-labeled
rapidly aromatizes through loss of isopropanol giving the analogs of the multiple kinase inhibitor dasatinib and its N-
aminothiazole carboxamide product 4.
Scheme 4 shows the synthetic route used to prepare stable- labeled parent or metabolite from
dealkyl metabolite. This process allowed elaboration of the
single penultimate
a
labeled dasatinib (1b) and its metabolite 2b. Owing to the precursor. The sequence successfully applied a novel method
presence of chlorine in 1 and 2, an MS internal standard for aminothiazole ring formation, allowing the facile incorpora-
required at least five isotopic mass units more than the parent to tion of isotopes into the thiazole core ring system in high yield.
avoid interference with naturally abundant 37Cl. The convergent This route incorporates labeling in a structurally common ring
synthetic paths for stable-labeled 1b and 2b incorporated labels system that may have utility for a wider range of structurally
in both the dichloropyrimidine and thiazole ring systems. suitable molecules.
Labeled 4,6-dihydroxypyrimidine 14 was prepared in quantita-
tive yield from commercially available (1, 2, 3-13C3) diethyl
malonate that was reacted with acetamidine hydrochloride in
the presence of methanolic sodium methoxide.13,14 Treatment
Experimental
All unlabeled reagents were of ACS grade or better. (E)-N-(2-
Chloro-6-methylphenyl)-3-[2-methylpropyl]oxyacrylamide 3 was
provided by Bristol–Myers Squibb Process Research and
Development. [14C]Thiourea was purchased from GE Health-
care (formerly Amersham Biosciences UK Limited). (1, 2, 3-13C3)
Diethyl malonate 11 and [13C, 15N2]thiourea were both obtained
from Isotec Inc. Reactions were run under an inert atmosphere
of nitrogen and magnetically stirred at a constant rate. Column
of 13 with an excess of neat phosphorous oxychloride at reflux
afforded the pyrimidine chloride 14 in 25% yield. The modest
isolated yield of 14 most likely resulted from its volatility and
subsequent loss during purification and removal of excess
phosphorus oxychloride in the workup.14,15 Elaboration of the
stable-labeled aminothiazole portion of the molecule (15)
proceeded in 97% yield via reaction of 3 with 13C2,15N2-thiourea.
Coupling of the dichloropyrimidine 14 and aminothiazole 15
using sodium tert-butoxide gave 16 in 49% yield with unreacted
15 present in the crude product. The lower yield obtained for
this coupling relative to the analogous carbon-14-labeling
reaction is most likely due to the presence of solvent in the
crude 14 used in the reaction. Solvent impurities created
difficulties in quantifying 14. As a result, it was difficult to react
14 with 15 in an equimolar ratio required for a high yield of 16.
Finally, treatment of 16 with excess 1-(2-hydroxyethyl)piper-
azine gave [13C4, 15N2]dasatinib (1b) in 80% yield after
recrystallization of the crude product from aqueous ethanol.
chromatography was performed using
a
Biotages Flash
Chromatography system. Proton NMR spectra were recorded
on a Bruker Spectrospin 300 MHz spectrometer. Mass spectra
were obtained using a Micromass Q-Tof high-resolution mass
spectrometer which was tuned to a resolution of 18 000.
Radiochemical purities were determined by high-performance
liquid chromatography (HPLC) (Rainin Model SD-200, Varian
PDA-2 detector and Beta-Ram detector (IN/US Systems Inc.).
Thin-layer chromatographic (TLC) analyses were performed
using Merck 60 F254 silica-coated plates with radiochemical
detection (QC-Scan, Bioscan Model B-QC). Specific activity was
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A. J. Allentoff et al.
Scheme 4
measured by gravimetric analysis using liquid scintillation (20–100% B) 0–13 min. Isocratic (100% B) 13–19 min. Isocratic
counting (Wallac Model 1409). Reactions were monitored by (20% B) 20–25 min; flow rate: 1 mL/min, injection volume: 10 mL,
HPLC and TLC with comparisons made to authentic unlabeled diluent: 1:5 DMF:CH3CN [v:v]; UV detection: l 5 320 nm.
materials when available.
2-[14C]-2-Amino-N-(2-chloro-6-methylphenyl)thiazole-5-carbox-
HPLC
amide (4)
HPLC methods described below were used for in-process and To a round bottom flask was added THF (18 mL), water (18 mL),
final product analyses. and (E)-N-(2-chloro-6-methylphenyl)-3-[2-methylpropyl]oxyacryl-
Method 1: Column: YMC Pack Pro C18, 3 mm (4.6 Â 150 mm). amide (3, 1.76 g, 6.56 mmol) and the mixture was stirred at room
Mobile phase A: 90% water/10% CH3CN with 0.075% TFA. Mobile temperature to obtain a colorless biphasic emulsion. To this
phase B: 10% water/90% CH3CN with 0.075% TFA. Program: mixture was added N-bromosuccinimide (1.28 g, 7.22 mmol) and
gradient (0–90% B) 0–20 min; flow rate: 1 mL/min; injection the resulting emulsion was stirred for 2 h at ambient tempera-
volume: 10 mL; diluent: MeOH; UV detection: l 5 254 nm.
ture. The yellow emulsion turned colorless after stirring for 1 h.
Method 2: Column: YMC Pack Pro C18, 3 mm (4.6 Â 150 mm). [14C]Thiourea (0.133 g, 1.75 mmol, 100 mCi, specific activi-
MobilephaseA:90%50 mMNH4OAc(aq, pH 5 5.2)/5%CH3CN/5% ty 5 57 mCi/mmol) and unlabeled thiourea (0.366 g, 4.81 mmol)
MeOH. Mobile phase B: 10% 50 mM NH4OAc (aq, pH 5 5.2)/85% were then added and the reaction mixture was heated at 651C.
CH3CN/5% MeOH. Gradient (16–33% B) 0–18 min. Gradient The colorless, biphasic, cloudy emulsion turned to a colorless
(33–60% B) 18–30 min. Gradient (60–100% B) 30–40 min. Gradient mixture upon initial heating, and then returned to a yellowish
(100–16% B) 40–40.5 min. Isocratic (16% B) 40.5–45 min; flow rate: color after 20 min. TLC analysis (10:90 MeOH:CH2Cl2 [v:v]) of the
1 mL/min, injection volume: 10 mL, diluent: 50% acetic acid (0.25% reaction mixture after 1 h of heating showed no residual 3
aq)/50% CH3CN acetonitrile; UV detection: l 5 320 nm.
remaining. The reaction mixture was cooled to room tempera-
Method 3: Column: YMC Pack Pro C18, 3 mm (4.6 Â 150 mm). ture. Ammonium hydroxide (3.6 mL, 28% NH3 in water) was
Mobile phase A: 90% 10 mM NH4OAc (aq)/10% CH3CN. Mobile slowly added to adjust the pH from 2 to 11 to form a thin
phase B: 5% 10 mM NH4OAc (aq)/95% CH3CN. Program: Gradient suspension. The volatiles were then removed with a stream of
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A. J. Allentoff et al.
nitrogen resulting in a heavy yellow suspension, which was over 90 min during which time a precipitate formed which was
stirred for 2 h at room temperature and stored at 41C for 36 h. allowed to sit at ambient temperature for 18 h. This precipitate
After carefully removing the yellow supernatant, the resulting was then separated by centrifugation and the resulting solid
amorphous solid residue was compacted by centrifugation and rinsed twice with absolute ethanol:sterile water (1:1 [v:v]) and
the pellet rinsed twice with water. The pellet was dissolved in dried in vacuo yielding [14C]dasatinib (1a) as an off-white solid
THF (20 mL), concentrated to dryness under a stream of nitrogen (0.603 g, 18.3 mCi, 66% radiochemical yield from 6) with
and then dried in vacuo to constant weight affording 4 as an radiochemical purity of 99.4% (HPLC method 2, RT 5 19.2 min)
amorphous yellow solid (1.717 g, 97.6%): 1H NMR (d6-DMSO) and specific activity of 30.4 mCi/mg: 1H NMR (d6-DMSO) d 5 2.24
d 5 2.19 (s, 3H), 7.25 (m, 2H), 7.39 (d, 1H, J 5 7.5 Hz), 7.61 (br s, (s, 3H), 2.40 (s, 3H), 2.42 (t, 2H, J 5 6), 2.49 (dd, 4H, J 5 6.6 Hz),
2H), 7.87 (br s, 1H), 9.65 (br s, 1H) the 1H NMR spectrum was 3.52 (m, 6H), 4.45 (br t, 1H, J 5 5 Hz), 6.05 (s, 1H), 7.28 (m, 2H),
1
consistent with the published spectrum for unlabeled 4.11
7.42 (br d, 1H, J 5 7 Hz), 8.23 (s, 1H), 9.88 (s, 1H). The H NMR
spectrum was consistent with the published spectrum for 1.11
2-[14C]-2-(6-Chloro-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-
methylphenyl) Thiazole-5-carboxamide (6)
N-(2-Chloro-6-methylphenyl)-2-(piperazin-1-yl)-2-(methylpyrimi-
din-4-ylamino)-2-[14C]-thiazole-5-carboxamide (2a)
To 4 (1.66 g, 6.20 mmol, 94.2 mCi) under nitrogen in a round
bottom flask was added anhydrous THF (20 mL) and 4,6- To a solution of 6 (0.10 g, 0.254 mmol, 3.91 mCi) and piperazine
dichloro-2-methylpyrimidine (5, 1.21 g, 7.44 mmol). The resulting (0.21 g, 2.54 mmol) in N-methylpyrrolidone (1.0 mL, anhydrous)
solution was stirred for 30 min at room temperature. The under argon was added N, N-diisopropylethylamine (0.69 g,
reaction mixture was then cooled to 01C with an ice bath and 0.53 mmol) dropwise. The resulting mixture was heated at 1201C
sodium tert-butoxide (2.15 g, 21.7 mmol) was added in portions for 20 min and then allowed to cool to ambient temperature.
over 10 min. The resulting suspension was warmed to ambient Water (5 mL) was added slowly resulting in the formation of a
temperature and was stirred for 8 h during which time it became precipitate. The mixture was stirred for 48 h at room tempera-
a solution. TLC analysis (10:90 MeOH:CH2Cl2 [v:v]) showed clean ture and then subjected to centrifugation. The resulting pellet
formation of 6 (Rf 5 0.70) and no residual 4. Water (5.8 mL) was was rinsed four times with water and dried in vacuo affording 2a
added and the pH was adjusted to 5–6.5 with glacial acetic acid as a white solid (72.4 mg, 2.4 mCi, 64% radiochemical yield) with
(1.0 mL). Water (35 mL) was added resulting in the formation of a radiochemical purity of 98.3% (HPLC method 3, RT 5 11.8 min)
1
thick pale-yellow suspension. The suspension was stirred over- and specific activity 5 33.7 mCi/mg: H NMR (d6-DMSO) d 5 2.25
night at ambient temperature and the solids collected after (s, 3H), 2.40 (s, 3H), 2.76 (m, 4H), 3.44 (m, 4H), 6.01 (s, 1H), 7.25
centrifugation. After repeated rinsing with THF (7 mL), water (m, 2H), 7.41 (d, 1H, J 5 br d, J 5 7 Hz), 8.21 (s, 1H), 9.87 (s, 1H).
(10 mL), and THF (7 mL) the resulting solid was dried in vacuo to
give 6 as a pale yellow solid (1.99 g, 81.5% yield) with a 4, 6-Dihydroxy-2-methyl-[13C3]-pyrimidine (13)
radiochemical purity of 99% (HPLC method 1, RT 5 15.1 min): 1H
To a solution of (1, 2, 3-13C3) diethyl malonate (11, 1.0 g,
NMR (d6-DMSO) d 5 2.25 (s, 3H), 2.60 (s, 3H), 6.95 (s, 1H), 7.25 (m,
2H), 7.41 (d, 1H, J 5 7.5 Hz), 8.32 (s, 1H), 7.87 (s, 1H), 10.1 (s, 1H).
6.13 mmol) in anhydrous methanol (11.0 mL) under nitrogen was
added sodium methoxide (36.8 mL, 0.5 M in methanol,
1H NMR spectrum was consistent with the published spectrum
18.4 mmol) and acetamidine hydrochloride (12, 0.61 g,
for unlabeled 6.11
6.13 mmol). The resulting mixture was stirred at ambient
temperature for 48 h. After removal of the volatiles in vacuo,
the resulting residue was dissolved in water (8.5 mL). The pH of
this aqueous solution was adjusted to 2 by the addition of conc.
HCl (1.30 mL) affording a thick white suspension which was
N-(2-Chloro-6-methylphenyl)-2-(6-(4-(3-hydroxyethyl)piperazin-1-
yl)-2-methylpyrimidin-4-ylamino)-2-[14C]-thiazole-5-carboxamide
(1a)
To a solution of 6 (1.0278 g, 2.61 mmol, 39.6 mCi) in anhydrous
stirred for 8 h at room temperature and then stored at 41C for
1-butanol (16 mL) under nitrogen was added 1-(2-hydroxyethyl)
piperazine (1.70 g, 13.03 mmol) dissolved in 16 mL of anhydrous
1-butanol. An additional 10 ml of 1-butanol and N, N-diisopro-
pylethylamine (0.673 g, 5.21 mmol) were added and the result-
ing suspension was heated to 1151C for 18 h. During this period
all solids dissolved affording a solution. HPLC (method 2) and
TLC analyses (10:90:0.2 MeOH:CH2Cl2:NH4OH [v:v:v]) showed
clean formation of 1a (RT 5 19.3 min, Rf 5 0.17) with complete
consumption of 6. The reaction mixture was cooled to ambient
temperature and was stirred for 2 h during which time a
precipitate formed. The solids were precipitated by centrifuga-
tion, rinsed repeatedly with 1-butanol, and dried in vacuo to
afford 1.15 g of 1a as its butanol solvate. The beige butanol
solvate (0.80 g, 1.423 mmol) was suspended in absolute ethanol
(13.0 mL) and sterile water (3.40 mL). The suspension was heated
to 801C, causing the solids to slowly dissolve into a uniform
solution. Additional absolute ethanol (2.0 mL) and sterile water
(5.1 mL) were added and the solution was held at 751C for 1 h.
Seed crystals of the hydrated form of unlabeled 1a (10 mg) were
then added. The solution was cooled to ambient temperature
18 h. The solid was collected by vacuum filtration and dried in
vacuo giving 4, 6-dihydroxy-2-methyl-[13C3]-pyrimidine (13,
0.80 g, 100%) as a white solid with a chemical purity of 98.3%
(HPLC method 1, RT 5 2.1 min) by relative UV peak area ratio: 1H
NMR (d6-DMSO) d 5 2.23 (S, 3H), 4.95 (d, 1H, J 5 166 Hz, 13C–H).
4,6-Dichloro-2-methyl[13C3]-pyrimidine (14)
4,6-Dihydroxy-2-methyl-[13C3]-pyrimidine (13, 0.797 g, 6.17 mmol)
and phosphorous oxychloride (13.4 g, 87.4 mmol) were com-
bined under nitrogen and the mixture heated at reflux for 2 h.
The progress of the chlorination was monitored by TLC (90:10
methanol:CH2Cl2 [v:v]). Excess phosphorous oxychloride was
then distilled off under reduced pressure. To the resulting
residue was added ice water and the mixture extracted several
times with ethyl acetate. The combined organic extracts were
washed with brine (4 mL) and concentrated in vacuo yielding
crude 14 (0.77 g) as a slightly viscous, yellow oil. Purification of
the product was achieved by flash chromatography on silica gel
(CH2Cl2) affording pure 14 (0.26 g, 25.4%) with chemical purity
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A. J. Allentoff et al.
of 98% (HPLC method 1, RT 5 10.9 min) by relative UV peak area 18 h during which time the suspension dissolved affording a
ratio: 1H NMR (CDCl3) d 5 2.72 (S, 3H), 7.23 (d, 1H, J 5 181 Hz, uniform solution. HPLC analysis (method 2, RT 5 19.3 min) and
13C–H).
TLC analysis (10:90 MeOH:CH2Cl2 [v:v]) of the solution showed
clean product formation with no residual 16. The reaction
mixture was cooled to room temperature and stirred for
2 h during which time precipitate was formed. The solids were
separated by centrifugation, the pellet was rinsed twice
with 1-butanol, and dried in vacuo giving the [13C3, 15N2]dasa-
tinib butanol solvate as a pale beige solid (0.251 g). To this
butanol solvate (0.23 g, 0.40 mmol) was added absolute
ethanol (3.7 mL) and water (0.96 mL). The resulting suspension
was heated at 801C and the solids gradually dissolved.
Additional absolute ethanol (0.56 mL) and water (1.46 mL) were
added and the solution was heated at 751C for 1 h. Seed crystals
of unlabeled 1 (approximately 1 mg) were added and the
mixture was allowed to cool to room temperature over
90 min during which time a precipitate formed. The resulting
suspension was maintained at room temperature overnight
without stirring. The precipitate was then filtered, rinsed
with a solution of 1:1 absolute ethanol:water [v:v], and dried in
vacuo affording [13C3, 15N2]dasatinib monohydrate (1b) as an
off-white solid (0.169 g, 80%) with chemical purity of 99.5%
by UV relative peak–area ratio (HPLC method 1, RT 5 9.7 min): 1H
NMR (d6-DMSO) d 5 2.24 (s, 3H), 2.40 (s, 3H), 2.42 (t, 2H,
J 5 6), 2.49 (dd, 4H, J 5 6.6 Hz), 3.52 (m, 6H), 4.42 (br s, 1 H,O–H),
6.05 (d, 1H, J 5 162 Hz, pyrimidine 13C–H), 7.28 (m, 2 H), 7.40 (br
d, 1H, J 5 7 Hz), 8.21 (dd, 1H, J 5 20, 9 Hz, thiazole ring C–H), 9.85
(br s, 1H); MS (positive ion) [M1H]1 5 494 (96.4%, C18 13C4H26N5
15N2O2S1Cl1), 493 (3.6%, C19 13C3H26N5 15N2O2S1Cl1).
2-[13C]-2-[15N]-Amino-N-(2-chloro-6-methylphenyl)-[15N]-thiazole-
5-carboxamide (15)
To a stirred mixture of water (17 mL) and THF (17 mL) was added
(E)-N-(2-chloro-6-methylphenyl)-3-[2-methylpropyl]oxyacryla-
mide (1.69 g, 6.32 mmol) and N-bromosuccinimide (1.24 g,
6.95 mmol), and the resulting emulsion was stirred at room
temperature for 2 h. [13C, 15N2]Thiourea (0.50 g, 6.32 mmol) was
then added and the reaction mixture was heated at 651C for 1 h.
Ammonium hydroxide (7.0 mL) was added slowly to the mixture
to adjust the pH from 2 to 11. During this addition, a cloudy
suspension was formed. The mixture was then concentrated
under a stream of nitrogen, resulting in the formation of more
solids. After stirring the mixture at ambient temperature for 2 h,
it was stored at 41C for 18 h. The precipitate was then collected
by vacuum filtration, rinsed with water (10 mL), and dried in
vacuo affording crude 15 as a pale yellow solid (1.49 g, 87%)
with chemical purity of 87% (HPLC method 1, RT 5 8.1 min) by
relative UV peak area ratio. This material was used directly for
1
the next step: H NMR (d6-DMSO) d 5 2.19 (s, 3H), 7.25 (m, 2H),
7.39 (d, 1H, J 5 7.5 Hz), 7.60 (br d, 2H, J 5 90 Hz, H–15N), 7.88 (dd,
1H, J 5 22, 13 Hz, thiazole ring 13C–H), 9.63 (br s, 1H).
2-[13C]-2-(6-Chloro-2-methylpyrimidin-4-yl-[15N]-amino)-N-(2-
chloro-6-methylphenyl)-[15N]-thiazole-5-carboxamide (16)
Carboxamide 15, (0.43 g, 1.54 mmol) was charged to a round
bottom flask under nitrogen. THF (5 mL, anhydrous) was added
followed by 4,6-dichloro-2-methyl[13C3]-pyrimidine (14, 0.26 g,
1.57 mmol) and the resulting mixture was stirred for 30 min at
room temperature. This mixture was then cooled to 01C and
sodium tert-butoxide (0.54g, 5.50 mmol) was added portionwise
over 10min to form a suspension that was allowed to warm at
room temperature. The suspension was dissolved within 8 h,
giving a uniform solution. TLC analysis (10:90 MeOH:CH2Cl2 [v:v])
showed product formation and some residual 15 remaining. The
reaction was allowed to stir at room temperature for an additional
20 h. Water (1.5 mL) was added and the pH was adjusted to 5–6.5
with glacial acetic acid (300 mL). Additional water (8.5 mL) was
added, resulting in the formation of a thick pale yellow
suspension. The suspension was stirred for 36 h at room
temperature and then concentrated to dryness in vacuo. The
solids were washed with THF (3 mL), water (4 mL), THF (4 mL), and
dried in vacuo affording crude 16 (0.31 g) as a pale beige solid
with some remaining 15 (HPLC method 1). This crude 16 was used
directly for the next step without further purification: 1H NMR (d6-
DMSO) d 5 2.25 (s, 3H), 2.60 (s, 3H), 6.95 (d, 1H, J5 179, pyrimidine
13C–H), 7.25 (m, 2H), 7.41 (d, 1H, J 57.5 Hz), 8.31 (dd, 1H, J5 22,
13 Hz, thiazole ring 13C–H), 10.0 (s, 1H).
N-(2-Chloro-6-methylphenyl)-2-((piperazin-1-yl)-2-methyl-[13C3]-
pyrimidin-4-yl-[15N]-amino)-2-[13C]-[15N]-thiazole-5-carboxamide
(2b)
To a solution of 16 (56 mg, 0.14 mmol) and piperazine (121 mg,
1.40 mmol, 10 eq.) in N-methylpyrrolidone (0.56 mL) under argon
was added N, N-diisopropylethylamine (38 mg, 0.294 mmol). The
reaction mixture was heated to 1201C for 15 min, and then
cooled to room temperature. Water (5.4 mL) was slowly added,
resulting in the formation of a precipitate which was stirred for
16 h at room temperature and separated from the mother liquor
by centrifugation. The resulting pellet was rinsed three times
with water (3 mL) and dried in vacuo yielding 2b as a white solid
(47.6 mg, 76% yield) with a chemical purity of 98.1% by UV
relative peak–area ratio (HPLC method 3, RT 5 11.8 min): 1H NMR
(d6-DMSO). d 5 2.25 (s, 3H), 2.43 (s, 3H), 3.15 (m, 4H), 3.71 (m,
4H), 6.10 (d, 1H, J 5 163, pyrimidine 13C–H), 7.29 (m, 2H), 7.42 (d,
1H, J 5 br d, J 5 7 Hz), 8.25 (dd, 1H, J 5 20, 9 Hz, thiazole ring
13C–H), 9.91 (s, 1H); MS (positive ion) [M1H]1 5 450.1 (95.01%,
C16 13C4H22N5 15N2O1S1Cl1), 449.1 (4.99%, C17 13C3H22N5
15N2O1S1Cl1).
Acknowledgement
N-(2-Chloro-6-methylphenyl)-2-(6-(4-(3-hydroxyethyl)piperazin-1-
yl)-2-methyl-[13C3]-pyrimidin-4-yl-[15N]-amino)-2-[13C]-[15N]-thia-
zole-5-carboxamide (1b)
The authors wish to thank Richard Gedamke for providing
high-resolution MS analyses of stable-labeled dasatinib and
metabolite.
To 16 (0.25 g, 0.625 mmol) under nitrogen was added a solution
of 1-(2-hydroxyethyl) piperazine (0.41 g, 3.12 mmol) dissolved in
1-butanol (4 mL). Additional 1-butanol (2.5 mL) and N, N-
diisopropylethylamine (0.161 g, 1.25 mmol) were then added.
The resulting mixture was heated at 1151C with stirring for
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