Synthesis of deuterium-labeled crizotinib, a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK)

Article abstract of DOI:10.1002/jlcr.3678

To more accurately and rapidly achieve quantitative detection of clinical crizotinib samples, stable isotope labeled crizotinib was required as an internal standard. We have developed a method to prepare racemic [D₉] crizotinib using a base-catalyzed H/D exchange of both nitroso compound 2 and the acetophenone compound 6 with D₂O and NaBD₄ reduction of 7 as the key steps to introduce the 9 deuterium atoms. Starting with 4-hydroxypiperidine, 14-step synthesis furnished the desired racemic [D₉] crizotinib 18. The deuterium-labeled compound 18 with the chemical purity of 99.62% was applicable for use as internal standards in the drug clinical study.

Full text of DOI:10.1002/jlcr.3678

Synthesis of deuterium-labeled crizotinib , a potent and selective dual inhibitor of
mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma
kinase (ALK)
^
^
Wangwei Ao , Yuan Li , Yinsheng Zhang*
Jiangsu Key Laboratory of Targeted Antiviral Research
Chia Tai Tianqing Pharmaceutical Group Co., LTD
6
99-8 Xuanwu Ave. Building #9, Nanjing, Jiangsu Province, China
*
Correspondence to : Yinsheng Zhang ; Email: zys@cttq.com
Address: Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing
Pharmaceutical Group Co., LTD, 699-8 Xuanwu Ave. Building #9, Nanjing, Jiangsu Province,
China.
^
These authors contributed equally to this work.
Abstract: To more accurately and rapidly achieve quantitative detection of clinical crizotinib
samples, stable isotope labeled crizotinib was required as an internal standard. We have
developed a method to prepare racemic [D ]crizotinib using a base-catalyzed H/D exchange
9
of both nitro compound 2 and the acetophenone compound 6 with D O and NaBD reduction
2
4
of 7 as the key steps to introduce the nine deuterium atoms. Starting with
-hydroxypiperidine, 14 step-synthesis furnished the desired racemic [D ]crizotinib 18. The
4
9
deuterium-labeled compound 18 with the chemical purity of 99.62% was applicable for use
as internal standards in the drug clinical study.
Key words: Deuteration; H-D exchange; Internal Standard; Crizotinib; c-MET; ALK
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.3678
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Introduction
In 2011, crizotinib (brand name Xalkori, Fig.1) became the first ALK inhibitor approved by
the USA Food and Drug Administration (FDA) as a first-line treatment for ALK-positive lung
[
1-4]
cancer patients.
Stable isotope-labeled compounds have been proven to be ideal internal standards for use in a
[
5]
human absorption, distribution, metabolism, and excretion (ADME) studies. Among all of
the internal standard compounds developed, deuterium-labeled compounds are well known to
be the first choice for such applications due to their similarity of physical and chemical
properties with original compounds and easy availability in terms of deuterium source and
[
6]
synthetic approach.
To our knowledge, there were few procedures described in literature for the preparation of
[7-8]
deuterium-labeled crizotinib.
The known approaches either need expensive deuterium
sources, such as CDCl and DCOD, or only can offer one or two deuterium labeled crizotinib.
3
For crizotinib, the abundance of the M+5 (454 amu) isotopologues comes to 1.5%,
35
37
12 13
considering the natural isotope distribution of Cl/ Cl and C/ C. Therefore, a minimum
of six deuterated sites for the internal standard appeared to be sufficient to prevent
unfavorable interferences between signals of non-deuterated and deuterated crizotinib in a
quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS)
assay.
Considering the structure and possible synthetic approach of crizotinib (Fig. 1), a 14-step
labeling process (Scheme 1 , 2 & 3) was developed to prepare the desired deuterium-labeled
R/S-crizotinib 18.
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RESULTS AND DISCUSSION
[
3, 9-10]
Methods for the synthesis of crizotinib have been reported in the literature,
but the key
approach is basically the same. According to the existing synthetic route of the original drug,
we designed the synthetic routes shown in Scheme 1, 2 & 3 to successfully prepare the
deuterated crizotinib 18.
To activate the ortho-hydrogen of the nitrogen atom, nitroso group was first introduced into
the nitrogen atom of 4-hydroxypiperidine 1 to form nitroso compound 2 in 78% yield, and
then four deuterium atoms can be easily introduced to give compound 3 after the
[11]
hydrogen-deuterium (H-D) exchange with D O under basic (NaOD) conditions. Removal
2
of nitroso group by using aluminum-nickel (Al-Ni) alloy under alkaline condition afforded
4
-[D ]hydroxypiperidine 4. Without isolation of 4, direct N-acylation of 4 with di-tert-butyl
4
dicarbonate furnished 5 with a protection group in overall 25% yield (3 steps). From 2, 5 was
prepared in one pot without the isolation of intermediates 3 & 4 since H-D exchange, (Al-Ni)
alloy reduction and Boc protection reactions used the same basic solution as a solvent.
1
2
Although Hesk et al. has described the three-step approach toward the synthesis of 5, it
needed expensive dueterated formaldehyde and lacked analytical data. Our new approch to 5
uses much cheaper and more readily availabe D O as only deuterium source to offer >95%
2
deuterium enrichment.
Furthermore, four deuterium atoms were introduced into 7 after the H-D exchange in a mixed
[
13-14]
solution of D O and 1,4-dioxane under a basic (NaOD) condition in a 92% yield.
Unlike
2
7
the literature, our NaOD promoted H-D exchange reaction allowed not only deutrium atoms
to be incorpoarted into α methyl group of the ketone 7, but also into 3 position of phenyl ring.
To avoid the back-exchange of 7 with the non-deuterated agents and solvents, deuterated
sodium boron hydride (NaBD ) in a mixed solution of MeOD and THF was applied to reduce
4
the labeled ketone 7 to racemic 8 in a 82% yield. Based on HNMR of 8, it was observed that
the base-catalyzed D-H exchange of 6 with D O gave >95% D at 2 position and >93% D at 3’
2
position, respectively, and NaBD reduction of the ketone 7 offered 98% D enrichment
4
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(Scheme 2). Mitsunobu reaction of 8 with 9 provided 10 in 42.8% yield, and then 10 was
further reduced with zinc powder in acetic acid under reflux to 11. Bromination of 11 with
NBS in acetonitrile produced 12 in 78% yield.
With the key deuterium-labeled intermediates 5 & 12 in hand, deuterated crizotinib was
successfully prepared via a 5-step synthetic sequence starting from 5 as depicted in Scheme 3.
The N-mesylation of 5 with methane sulfonyl chloride under a basic (Et N) condition
3
provided 13 in 86% yield. N-Alkylation of 13 with 3-iodopyrazole 14 generated iodo
compound 15 in 70.7% yield. Palladium-catalyzed coupling reaction of 15 with
bis(pinacilato)diboron gave the boronic ester derivative 16 in 62.9% yield. Suzuki
cross-coupling reaction of two labeled intermediates 12 & 16 offered boc-protected crizotinib
1
7 that was de-protected with 5% HCl methanol solution to generate R/S-[D ]crizotinib 18 in
9
a fair yield. It was not necessary to separated R/S isomers since the mixture of two
enantiomers as an internal standard (Figure 2) does not affect the accuracy in a quantitative
bioanalytical LC/MS/MS assay.
Conclusion
The key points of our deuterium-labeling synthetic approach to R/S-[D ]crizotinib 18 are
9
summarized as follows: 1) activating the adjacent hydrogen atom of the piperidine moiety by
introducing the nitroso group into the secondary amine of 4-hydroxypiperidine, which make
the nitroso compound easily exchanged with D O to incorporate four deuterium atoms into
2
the key intermediate 5; and 2) carrying out H-D exchange of acetophenone 6 with D O
2
followed by reduction of carbonyl with NaBD , which allows five deuterium atoms
4
introduced into another intermediate 8. Although the entire synthetic route is long (14 steps),
the reactions involved are relatively conventional and several steps do not require
post-treatment.
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So far, we have developed a practical method for synthesizing deuterium-labeled crizotinib.
The total yield of the 14-step synthesis was 6.2%, which provided a much-needed stable
isotope internal standard for clinical studies of crizotinib.
Experimental
General
All reagents were purchased from commercial sources and were used as received. Routine
monitoring of reactions was performed by thin layer chromatography (TLC) using pre-coated
Haiyang GF254 silica gel TLC plates. NMR spectra were recorded on a Bruker AVANCE 500
spectrometer at 500 MHz with tetramethylsilane used as an internal reference. High
resolution mass spectra (HRMS) were performed on Agilent Acrrurate-Mass Q-ToF LC/MS
6
520 mass spectrometer with electron spray ionization (ESI) mode. Intermediates 1, 7 & 9
were purchased from Meryer (Shanghai) Co., Ltd. Intermediates 14 were purchased from
Energy chemical Ltd. Deuterium agents, D O (99.9% D) and NaBD (98.5% D) were
2
4
obtained from Sigma-Aldrich.
1
-Nitrosopiperidin-4-ol (2)
A solution of sodium nitrite (20.0 g, 0.29 mol) in water (100 mL) was slowly added at
5 °C to a solution of piperidin-4-ol (1, 25.0 g, 0.25 mol) in water (100 mL) and 4.5 N
-
hydrochloric acid (80 mL, 0.36 mol). The resulting mixture was stirred at room temperature
overnight and then concentrated and purified by flash column chromatography (PE:EA = 1:5)
1
to afford 2 (25.3 g, 77.7% yield) as a light yellow oil. H-NMR (500 MHz, CDCl ) δ
3
4
1
.32-4.36 (m, 1H), 4.03-4.08 (m, 2H), 3.88-3.92 (m, 1H), 3.71-3.78 (m, 1H), 3.69-3.70 (m,
1
3
H), 1.94-1.97 (m, 1H), 1.69-1.79 (m, 2H), 1.50-1.54 (m, 1H); C-NMR (125 MHz, CDCl )
3
δ 65.36, 46.76, 35.92, 33.84, 32.23.
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tert-Butyl 4-[2,2,6,6-D ]hydroxypiperidine-1-carboxylate (5)
4
Sodium methoxide (40.0 g, 0.74 mol) was added to a solution of the compound 2 (19.0 g,
0
.15 mol) in D O (200 mL). The resulting solution was stirred at 100 °C for 24 hours. The
2
solvent was evaporated under reduced pressure to give the residue which was mixed with
D O (200 mL). The resulting solution was stirred at 100 °C for another 14.5 hours. The
2
reaction solution can be used for next reaction without purification.
Aluminum and nickel alloy (14 g) were added slowly to the above reaction solution
(0.15 mol) over a time period of 50 minutes at 80 °C. The resulting reaction mixture was
stirred at 80 °C for 30 minutes, and then cooled to room temperature. The mixture was
filtered and the filtrate (about 200 mL) can be used directly for next reaction.
Di-tert-butyl dicarbonate (36.8 g, 0.17 mol) was added to the above filtrate (200 mL,
0
.15 mol) at room temperature. The resulting reaction mixture was stirred at room
temperature for 4 hours and then extracted with DCM (200 mL x 2). The combined organic
layers were washed with brine, dried with MgSO and evaporated to dryness. The crude
4
residue was purified by flash column chromatography to afford 5 (7.64 g, 25.5% yield for 3
1
steps) as a yellow oil. H-NMR (500 MHz, CDCl ) δ 3.79-3.84 (m, 1H), 2.17 (s, 1H), 1.82
3
1
3
(
dd, J = 13.0 Hz, J = 4.0 Hz, 2H), 1.46 (s, 1H), 1.45 (s, 9H ); C-NMR (125 MHz, CDCl )
1
2
3
+
δ 154.85, 79.51, 67.51, 40.10-41.28 (m, C-D), 33.91, 28.42. HRMS ( ESI+, [M+H] ) m/z:
2
06.1681.
1
-(2,6-Dichloro-3-fluoro[5-D]phenyl) [1,2,2,2-D ]ethan-1-ol (8)
4
Sodium methoxide (0.54 g, 0.01 mol) was added to a solution of the
1
1
-(2,6-dichloro-3-fluorophenyl)ethan-1-one (6, 8.32 g, 0.04 mol) in D O (100 mL) and
2
,4-dioxane (100 mL). The resulting solution was stirred at 100 °C for 6 hours. The solvent
was evaporated under reduced pressure to give the residue which was mixed with D O (100
2
mL) and 1,4-dioxane (100 mL). The resulting solution was stirred at 100 °C for another 6
hours. The solvent was evaporated under reduced pressure to dryness to give crude 7 (7.8 g)
as a colorless oil.
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MeOD (5.0 mL, 123.59 mmol) was slowly added to a mixture of 7 (7.50 g, 35.54 mmol)
and boron sodium deuteride (1.76 g, 42.08 mmol) in dry THF (30 mL) at room temperature.
The resulting mixture was stirred at 60 °C for 1 hour, and then quenched with H O (100 mL)
2
at 0 °C. The mixture was extracted with EtOAc (100 mL x 2). The combined organic layers
were dried with MgSO and evaporated to dryness. The crude residue was purified by flash
4
column chromatography (PE:EA = 5:1) to afford 8 (6.27 g, 82.4% yield) as a colorless oil.
1
13
H-NMR (500 MHz, CDCl ) δ 7.26 (d, J = 4.5 Hz, 1H), 2.83 (s, 1H); C-NMR (125 MHz,
3
CDCl ) δ 157.25 (d, J = 247.5 Hz), 140.50, 129.50 (d, J = 7.5 Hz), 128.30, 121.41 (d, J =
3
1
8.75 Hz), 115.11-115.73 (m), 67.66-68.02 (m), 20.00-20.98 (m).
3
-(1-(2,6-Dichloro-3-fluoro[5-D]phenyl) [1,2,2,2-D ]ethoxy)-2-nitropyridine (10)
4
To a stirred solution of triphenylphosphine (11.45 g, 43.66 mmol) and DEAD (7.61 g,
3.70 mmol) in THF (30 mL) at 0 °C was added a solution of
4
1
3
-(2,6-dichloro-3-fluoro[5-D]phenyl) [1,2,2,2-D ]ethan-1-ol (8, 6.18 g, 28.87 mmol) and
4
-hydroxynitropyridine (9, 4.08 g, 29.12 mmol) in THF (30 mL). The resulting bright orange
solution was stirred under a nitrogen atmosphere at ambient temperature for 0.5 hour at
which point all starting materials had been consumed. The solvent was removed, and the
crude residue was purified by flash column chromatography (PE:EA = 2:1) to afford 10 (4.15
1
g, 42.8% yield) as an off-white solid. H-NMR (500 MHz, CDCl ) δ 8.03 (d, J = 5.0 Hz, 1H),
3
7
.39 (dd, J = 8.5, 4.5 Hz, 1H), 7.31 (dd, J = 9.5, 5.0 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H);
1
3
C-NMR (125 MHz, CDCl ) δ 157.51 (d, J = 250.0 Hz), 149.56, 145.12, 139.53, 135.49,
3
1
30.09, 128.88 (d, J = 2.5 Hz), 128.18, 123.88, 121.91 (d, J = 18.8 Hz), 117.15 (d, J = 23.8
Hz), 74.50, 17.94-18.34 (m).
3
-(1-(2,6-Dichloro-3-fluorophenyl-5-[D]) [1,2,2,2-D ]ethoxy-pyridin-2-amine (11)
4
To a stirred mixture of AcOH (25 mL) and 10 (4.10 g, 12.20 mmol) was added zinc
powder (15.7 g, 240.06 mmol). The mixture was heated to 60 °C and allowed to stir for 30
minutes. The mixture was cooled to room temperature and carefully neutralized by the
addition of a saturated Na CO aqueous solution. The mixture was extracted with EtOAc
2
3
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(
100 mL x 2). The combined organic layers were washed with brine, dried with MgSO and
4
evaporated to dryness. The crude residue was purified by flash column chromatography
1
(
PE:EA = 1:1) to afford 11 (3.15 g, 84.5% yield) as an off-white solid. H-NMR (500 MHz,
CDCl ) δ 7.60 (d, J = 5.0 Hz, 1H), 7.28 (d, J = 4.5 Hz, 1H), 6.71 (dd, J = 8.0, 1.0 Hz, 1H),
3
1
3
6
2
1
2
.47 (dd, J = 7.5, 5.0 Hz, 1H), 4.89 (br s, 2H); C-NMR (125 MHz, CDCl ) δ 157.42 (d, J =
3
48.8 Hz), 150.56, 139.81, 138.83, 137.06, 129.83, 128.70 (d, J = 41.3 Hz), 121.97 (d, J =
8.8 Hz), 116.97, 116.36 (t, J = 28.8 Hz), 113.38, 71.86-72.04 (t, J = 22.5 Hz), 18.17 (q, J =
0.0 Hz).
5
-Bromo-3-(1-(2,6-dichloro-3-fluoro[5-D]phenyl) [1,2,2,2-D ] ethoxy) pyridin-2-amine
4
(12)
N-Bromosuccinimide (NBS, 1.78 g, 0.01 mol) was added portionwise to a stirred
solution of 11 ( 3.06 g, 0.01 mol) in acetonitrile (50 mL) at 0 °C. After the resulting mixture
was stirred at 0 °C for 10 minutes, it was concentrated to dryness under vacuum. The residue
was diluted with EtOAc (100 mL). The organic layer was washed with brine, dried with
MgSO and evaporated to dryness. The crude residue was purified by flash column
4
chromatography (PE:EA = 1:1) to afford 12 (3.00 g, 77.9% yield) as an off-white solid.
1
H-NMR (500 MHz, CDCl ) δ 7.67 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 4.5 Hz, 1H), 6.85 (d, J =
3
1
3
1
1
1
.5 Hz, 1H), 4.91 (br s, 2H); C-NMR (125 MHz, CDCl ) δ 157.50 (d, J = 248.8 Hz), 149.31,
3
40.03, 139.33, 136.35, 129.93, 128.89 (d, J = 3.8 Hz), 121.98 (d, J = 20.0 Hz), 119.98,
16.76 (t, J = 22.5 Hz) , 106.61, 72.49 (t, J = 22.5 Hz ), 18.12 (dd, J = 32.5, 18.8 Hz ).
+
HRMS ( ESI+, [M+H] ) m/z: 383.9739 .
tert-Butyl 4-((methylsulfonyl)oxy) [2,2,6,6-D ]piperidine-1-carboxylate (13)
4
To a stirred solution of tert-butyl 4-[2,2,6,6-D ]hydroxypiperidine-1-carboxylate (5, 6.10
4
g, 29.71 mmol) in DCM (60 mL) was slowly added Et N (4.21 mL, 30.29 mmol) first and
3
then methane sulfonyl chloride (4.44 g, 38.76 mmol) in DCM (5 mL) at 0 °C. The mixture
was stirred at room temperature for 2 hours and then quenched with water (25 mL). The
aqueous layer was extracted with DCM (100 mL x 2). The combined organic layers were
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washed with brine, dried with MgSO and evaporated to dryness. The crude residue was
4
purified by flash column chromatography (PE:EA = 1:2) to afford 13 (7.24 g, 86.0% yield) as
+
an light yellow solid. HRMS ( ESI+, [M+H] ) m/z: 284.1456.
tert-Butyl 4-(4-iodo-1H-pyrazol-1-yl) [2,2,6,6-D ]piperidine-1-carboxylate (15)
4
To
a
stirred
solution
of
tert-butyl
4-((methylsulfonyl)oxy)
[
2,2,6,6-D ]piperidine-1-carboxylate (13, 2.00 g, 7.06 mmol) and 4-iodopyrazole (14, 1.34 g,
4
6
.91 mmol) in NMP (15 mL) was added cesium carbonate (3.0 g, 9.21 mmol). The mixture
was heated to 80 °C and allowed to stir for 4 hours. The reaction solution was diluted with
EtOAc (200 mL) and washed with water (100 mL). The organic layer was separated and
washed with brine, dried with MgSO and evaporated to dryness. The crude residue was
4
purified by flash column chromatography (PE:EA = 2:1) to afford 15 (1.86 g, 70.7% yield) as
1
a colorless oil. H-NMR (500 MHz, CDCl ) δ 7.53 (s, 1H), 7.47 (s, 1H), 4.26-4.32 (m, 1H),
3
1
3
2
1
.09-2.12 (m, 2H), 1.88 (t, J = 12.0 Hz, 2H), 1.49 (s, 9H); C-NMR (125 MHz, CDCl ) δ
3
1
3
54.54, 144.00, 131.26, 79.96, 59.76, 55.84, 32.09, 28.41 (note: C-NMR peaks for carbons
+
connected with deuterium atoms were not showing up). HRMS ( ESI+, [M+H] ) m/z:
3
82.0867.
tert-Butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)
[
2,2,6,6-D ]piperidine-1-carboxylate (16)
4
Bis(pinacilato)diboron (1.78 g, 7.01 mmol) and potassium acetate (1.93 g, 19.67 mmol)
were added sequentially to a solution of 15 (1.86 g, 4.88 mmol) in DMSO (20 mL). After the
mixture was purged with nitrogen three times, dichlorobis(triphenylphosphino)palladium(II)
(0.2 g, 0.28 mmol) was then added to the mixture. The resulting mixture was heated at 80 °C
for 2 hours and then cooled to ambient temperature. The mixture was filtered through a bed
of Celite and the solid residue in a funnel was washed with EtOAc (5 mL x2). The filtrate
was washed with brine, dried with MgSO and evaporated to dryness under vacuum. The
4
crude residue was purified by flash column chromatography (PE:EA = 3:1) to afford 16 (1.17
1
g, 62.9% yield) as a yellow oil. H-NMR (500 MHz, CDCl ) δ 7.82 (s, 1H), 7.75 (s, 1H),
3
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4
.32-4.36 (m, 1H), 2.12-2.15 (m, 2H), 1.89 (t, J = 11.5 Hz, 2H), 1.48 (s, 9H), 1.33 (s, 12H);
1
3
C-NMR (125 MHz, CDCl ) δ 154.58, 144.84, 133.58, 83.40, 79.88, 59.07, 32.15, 28.42,
3
1
3
2
4.86 (note: C-NMR multi-peaks for carbons connected with deuterium and boron atoms
+
are buried in the spectrum background). HRMS ( ESI+, [M+H] ) m/z: 382.2731.
tert-Butyl
4
-(4-(6-amino-5-(1-(2,6-dichloro-3-fluoro[5-D]phenyl)ethoxy-[1,2,2,2-D ])pyridin-3-yl)-1
4
H-pyrazol-1-yl) [2,2,6,6-D ]piperidine-1-carboxylate (17)
4
A
solution
-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)
2,2,6,6-D ]piperidine-1-carboxylate (16, 1.60 g,
of
tert-butyl
4
[
4.20
mmol)
and
4
5
1
-bromo-3-(1-(2,6-dichloro-3-fluorophenyl-5-D) [1,2,2,2-D ] ethoxy) pyridin-2-amine (12,
4
.53 g, 3.97 mmol) in toluene (12 mL) and water (12 mL) was purged with nitrogen gas a
few times. Then, Pd(dppf)Cl (180 mg, 0.25 mmol) , Cs CO (3.90 g, 11.99 mmol) and
2
2
3
tetraethylammonium bromide (70 mg, 0.33 mmol) were added to the above solution under
nitrogen atmosphere. The resulting mixture was purged with nitrogen gas and stirred at 80 °C
for 9 hours. The reaction solution was diluted with EtOAc (200 mL) and washed with water
(
100 mL). The organic layer was separated and washed with brine, dried with MgSO and
4
evaporated to dryness. The crude residue was purified by flash column chromatography
1
(
PE:EA = 1:2) to afford 17 (1.64 g, 73.9% yield) as an off-white solid. H-NMR (500 MHz,
DMSO-d ) δ 7.96 (s, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.54-7.57 (m, 2H), 6.91 (d, J = 2.0 Hz,
6
1
H), 5.62 (s, 2H), 4.30-4.36 (s, 1H), 1.97-2.00 (m, 2H), 1.74 (t, J = 12.0 Hz, 2H), 1.42 (s, 9H);
1
3
C-NMR (125 MHz, DMSO-d ) δ 154.32, 149.97, 139.28, 136.02, 135.13, 124.08, 119.69,
6
1
3
1
17.84, 115.01, 79.28. 58.51, 28.56 (note: C-NMR peaks for carbons connected with
+
deuterium atoms are buried in the background). HRMS ( ESI+, [M+H] ) m/z: 559.2354 .
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tert-Butyl
-(4-(6-amino-5-(1-(2,6-dichloro-3-fluoro[5-D]phenyl)ethoxy[1,2,2,2-D ])pyridin-3-yl)-1
4
4
H-pyrazol-1-yl) [2,2,6,6-D ]piperidine-1-carboxylate (18)
4
A solution of tert-Butyl
4
-(4-(6-amino-5-(1-(2,6-dichloro-3-fluoro[5-D]phenyl)ethoxy-[1,2,2,2-D ])pyridin-3-yl)-1H-
4
pyrazol-1-yl) [2,2,6,6-D ]piperidine-1-carboxylate (17, 700 mg, 1.25 mmol) in 5% HCl in
4
MeOH (8 mL) was stirred at room temperature for 18 hours. The mixture was diluted with
water (70 mL) and the aqueous layer was extracted with EtOAc (70 mL). The pH of aqueous
layer was adjusted to about 11 by adding a NaOH solution, and then the aqueous layer was
extracted with DCM (150 mL x 2). The combined organic layers were washed with brine,
dried with MgSO and evaporated to dryness. The crude residue was purified by flash column
4
chromatography (DCM:MeOH = 20:1) to afford 18 (299 mg, 52.0% yield) ad an off-white
1
solid. H-NMR (500 MHz, DMSO-d ) δ 7.91 (s, 1H), 7.76 (d, J = 1.5 Hz, 1H), 7.57 (d, J =
6
5
.0 Hz, 1H), 7.53 (s, 1H), 6.91 (d, J = 2.0 Hz, 1H), 5.62 (s, 2H), 4.14-4.18 (m, 1H), 2.99-3.07
1
3
(
m, 2H), 1.92-1.95 (m, 2H), 1.74 (t, J = 12.0 Hz, 2H); C-NMR (125 MHz, DMSO-d ) δ
6
1
56.30, 149.91, 139.29, 137.29, 135.97, 134.94, 130.98, 129.20, 123.75, 121.53 (d, J = 18.8
1
3
Hz), 119.50, 117.95, 115.00, 59.40, 33.64 (note: C-NMR peaks for carbons connected with
+
deuterium atoms are buried in the background). HRMS ( ESI+, [M+H] ) m/z: 459.1815.
ACKNOWLEDGEMENT
Thanks to Xushi Liu, Yuhan Li and Lu Zhang for their support necessary to successfully
complete this project.
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4
[
1
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Scheme 1. Synthesis of tert-butyl 4-[2,2,6,6-D ]hydroxypiperidine-1-carboxylate 5.
4
reagents and conditions: (i) NaNO , HCl, -5 °C to ambient temperature, overnight; (ii)
2
NaOMe, D O, 100 °C, 24 h; (iii) Al-Ni, 80 °C, 80 min; (iv) (Boc) O, RT, 4 h.
2
2
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Scheme 2. Synthesis of the deuterated intermediate 12. reagents and conditions: (i)
NaOMe, D O, 1,4-dioxane, 100 °C, 6 hours; (ii) NaBD , THF, CH OD, 60 °C, 1 hour; (iii)
2
4
3
Ph P, DEAD, THF, 0 °C, 0.5 h; (iv) Zn, AcOH, 60 °C, 30 minutes; (v) NBS, ACN, 0 °C, 10
3
minutes.
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Scheme 3. Synthesis of R/S-[D ]crizotinib 18. reagents and conditions: (i) MsCl, Et N,
9
3
DCM, RT, 2 hours; (ii) Cs CO , NMP, 80 °C, 4 hours; (iii) Bis(pinacilato)diboron,
2
3
Pd(Ph P) Cl , KOAc, DMSO, 80 °C,
2
hours; (iv) Pd(dppf)Cl2, Cs CO ,
2 3
3
2
2
Tetraethylammonium bromide, Toluene/H O, 80 °C, 9 h; (v) 5% HCl in MeOH, RT, 18 hours.
2
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Figure 1 Structure of crizotinib
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Figure 2 HRMS of crizotinib and [D ]crizotinib
9
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