Proton exchange reactions in isotope chemistry (II) synthesis of stable isotope-labeled LCQ908

Article abstract of DOI:10.1002/jlcr.3226

The proton exchange reaction was applied to the preparation of stable isotope-labeled LCQ908. For this synthesis, a suitable intermediate with protons alpha to a carbonyl group was first subjected to the H-D exchange reaction; subsequent coupling of a car

Full text of DOI:10.1002/jlcr.3226

Research Article
Received 28 March 2014,
Revised 2 July 2014,
Accepted 15 July 2014
Published online 14 October 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3226
Proton exchange reactions in isotope chemistry
†
(II) synthesis of stable isotope-labeled LCQ908
Zhigang Jian,* Tapan Ray, Amy Wu, Lawrence Jones, and Ry Forseth
The proton exchange reaction was applied to the preparation of stable isotope-labeled LCQ908. For this synthesis, a suitable
intermediate with protons alpha to a carbonyl group was first subjected to the H–D exchange reaction; subsequent coupling
1
3
of a carbonyl group with [ C ]triethyl phosphonoacetate, followed by hydrogenation and hydrolysis, led to the stable
2
labeled compound. Incorporation of two carbon-13 atoms in the molecule eliminated the presence of undesired M+0.
Keywords: deuterium; exchange; stable isotope
Introduction
LCQ908 starting from either carbon-13-labeled or deuterium-
labeled commercial starting materials.
Proton exchange reactions on target compounds have been
widely used in the preparation of tritium-labeled products but
The non-labeled LCQ908 is prepared by a linear multistep
1
3
synthesis (Figure 1). Three key intermediates were identified
only with limited success in the synthesis of stable labeled
materials suitable for bio-analytical studies. Among the reasons
are the lack of multiple exchangeable protons in the target
molecules, the wide molecule ion mass distribution, and the
presence of significant amounts of M+0 due to the incomplete
exchange of protons and the presence of labile protons. On
the other hand, there are many synthetic intermediates in the
synthesis of a particular compound, and some of those
intermediates may present opportunities for the introduction
of multiple deuterium atoms, for example, a carbonyl group with
alpha protons that can be easily replaced by deuterium. But the
challenge lies on how to stabilize the newly introduced deuterium
because these exchange sites may be labile. One of our strategies
is to remove/inactivate those functional groups used in exchange
reactions so that those sites are no longer labile:
as potential starting materials for the synthesis of stable
isotope-labeled LCQ908: 4-(4-hydroxyphenyl)-cyclohexanone A,
2
[
,5-dibromopyridine B, and 3-amino-6-trifluoromethylpyridine C.
The preparation of carbon-13-labeled A can be achieved from
13
4
6
C ]phenol through a four-step sequence. Carbon-13 labeled
5
13
13
B can be prepared from [ C]acrylic aldehyde and [ C]acetaldehyde
through pyridine and 2-aminopyridine. Carbon-13-labeled
6
7
8
13
compound C can also be prepared from [ C]acrylic aldehyde and
13
6
7
[
C]acetaldehyde through pyridine, 2-aminopyridine, 2,5-dibrom-
5
9
opyridine, and 6-trifluoromethylpyridine-3-carboxylic acid. Routes
to B and C are very long and contain low yields. While deuterium-
2
labeled B or C can be achieved from relatively inexpensive [ H
4
]
2-aminopyridine through similar multistep syntheses, the deuterium
on the pyridine ring could be lost during the synthesis because of the
harsh reaction conditions.
Compound A contains a carbonyl group that allows four protons
to be easily exchanged with deuterium. Furthermore, this carbonyl
functionality will be replaced by an alkyl group in subsequent
reactions, thereby converting the labile deuterium into stable ones
(Figure 2). While this looked attractive, it would be an eight-step
synthesis, and this was deemed too long both for the overall
synthesis and for the reaction steps involving isotopes.
We have previously reported the direct deuterium exchange
approach to labeling target compounds and would like to
report the successful application of this new labeling strategy
to the synthesis of stable isotope-labeled LCQ908.
2
We then decided to reduce the number of reaction steps
involving isotopes by starting the reaction from new
intermediate 1. Based upon our past experiences, deuterium
exchange reactions do not go to completion, which leaves the
final compound with undesired M+0 species in the mass
Results and discussion
LCQ908 is a novel, small-molecule inhibitor of lipid synthesis that
is highly effective in reducing diet-induced weight gain in
preclinical models and is under development for the treatment
Novartis Pharmaceuticals Corp, NIBR-DMPK-DMBA-IL, East Hanover, NJ 07936, USA
of type-2 diabetes. Stable isotope-labeled LCQ908 was required *Correspondence to: Zhigang Jian, NIBR-DMPK-DMBA-IL, Novartis Pharmaceuticals,
B435/R3149, One Health Plaza, East Hanover, NJ 07936, USA.
E-mail: zhigang.jian@novartis.com
for bio-analytical studies. Initial experiments with direct H–D
exchange reactions failed to provide stable isotope-labeled
LCQ908 with mass units greater than or equal to M+3, and we
†
Additional supporting information may be found in the online version of this article
turned our attention to the synthesis of a stable isotope-labeled at the publisher’s web-site.
J. Label Compd. Radiopharm 2014, 57 670–673
Copyright © 2014 John Wiley & Sons, Ltd.

Z. Jian et al.
Figure 1. Synthesis of unlabeled LCQ908.
13
2
species in subsequent reactions. The coupling of 2 with [ C ]
triethyl phosphonoacetate yielded olefin 3 in 64% yield.
Hydrogenation of compound 3 generated a mixture of cis and
trans isomers that were separated by preparative HPLC. Hydrolysis
2
13
of the desired trans isomer 4 afforded [ H
4 2
, C ]LCQ908 in an
overall 22% yield. Although we observed slight deuterium loss
2
13
4 2
during the reactions, mass analysis of [ H , C ]LCQ908 showed
no detectable M+0 and M+1 species (Figure 4).
Figure 2. Proposed synthesis of stable labeled intermediate.
Experimental
1
13
H-NMR spectra were recorded on a 400-MHz Bruker (Bruker Biospin,
Bellrica, MA) NMR spectrometer. Chemical shifts are reported in parts
per million relative to TMS (tetra methyl silane). Hydrogenation reaction
was carried out on a HEL (Lawrenceville, NJ) Automate Reactor System
with pressure control. The LC-MS (Liquid Chromatography-Mass
Spectroscopy) analysis was performed on an LCQ-Fleet (ThermoScientific,
Madison, WI) ion trap mass spectrometer coupled with a Waters (Waters
spectrum. To avoid this, we decided to incorporate a [ C
2
] unit into
the molecule as well (Figure 3).
Thus, a mixture of ketone 1 in deuterium methoxide containing
a catalytic amount of deuterium chloride was refluxed for 6 h.
Solvent removal and neutralization with sodium deuteroxide
2
afforded the [ H
4
]ketone 2 (92%). Mass spectrum analysis showed
the presence of M+0 species (0.6%). We decided to carry the Corp. Milford, MA) Acquity UPLC system with the following conditions:
material on to the next step because we will eliminate the M+0 Waters BEH C18, 1.7 μm, 2.1 × 100 mm; mobile phase A, 0.1% TFA
Figure 3. Synthesis of stable labeled LCQ908.
J. Label Compd. Radiopharm 2014, 57 670–673
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

Z. Jian et al.
Figure 4. Mass spectrum of stable labeled LCQ908.
(
Trifluoroacetic Acid) in water; mobile phase B, acetonitrile; linear
J= 7.8Hz), 7.96 (d, 2H, J = 7.8Hz), 8.47 (s, 1H), 8.54 (s, 1H), 9.19 (s, 1H, NH). MS
(ESI): m/z 484 (M+2, 0.5%), 485 (M+3, 8.3%), 486 (M+4, 27.2%), 487 (M+5,
gradient: hold 1 min at 90% A–10% B and then ramping to 10% A–90%
B in 5min; 0.5mL/min; 254 nm. Mass: positive ionization with zoom scan. 55%), 488 (M+6, 100%), 489 (M+7, 39%), 490 (M+8, 8.5%).
Prep-HPLC conditions: Cosmosil Cholester, 5μm, 20 × 250mm; mobile
phase: 80% methanol–20% water; 20mL/min isocratic; 328 nm. Flash
chromatography was conducted on an Analogix BSR system with an
Analogix (Madison, WI) SF25-40 g silica gel cartridge (Sepra SI50), eluted with
2
13
[
H , C ]trans-Ethyl 4-(4-(5-((6-(trifluoromethyl)pyridin-3-yl)
4 2
amino)pyridin-2-yl) phenyl) cyclohexyl) acetate 4
hexane/EtOAc (3/1). The starting material 4-(4-(5-((6-(trifluoromethyl)pyridin- Olefin 3 (420 mg, 0.86 mmol) and 10% Pd/C (234 mg) in ethyl acetate (20 mL)
-yl)amino)pyridin-2-yl)phenyl)cyclohexanone 1 was purchased from Wuxi
were stirred under 27 psi hydrogen atmosphere for 3 days. The mixture was
Pharm. (Wuxi, China), and all other chemicals and solvents were reagent grade filtered through a thin layer of celite and concentrated. The cis and
3
obtained from Sigma Aldrich (St. Louis, MO) without further purification.
trans isomers were separated by preparative HPLC to afford 201 mg
Supporting documents are provided and can be obtained.
of the trans isomer (48%) and 105 mg of the cis isomer (25%).
H-NMR (DMSO-d ) δ 1.2 (t, 3H, J = 7.2Hz), 1.48 (m, 2H), 1.8 (m, 3H), 2.23
6
(dm, 2H, J = 128Hz), 2.5 (m, 1H), 4.07 (m, 2H), 7.32 (d, 2H, J = 7.8 Hz), 7.6
1
2
[
H ]4-(4-(5-((6-Trifluoromethyl)pyridin-3-yl)amino)pyridin-2-yl)
4
(
(
m, 1H), 7.7 (m, 2H), 7.89 (d, 1H, J = 8.6Hz), 7.95 (d, 2H, J = 7.8 Hz), 8.46
s, 1H), 8.54 (s, 1H), 9.19 (s, 1H, NH). MS (ESI): m/z 486 (M+2, 1.4%), 487
phenyl)cyclohexanone 2
4
-(4-(5-((6-(Trifluoromethyl)pyridin-3-yl)amino)pyridin-2-yl)phenyl)cyclohexanone (M+3, 12.8%), 488 (M+4, 21%), 489 (M+5, 62%), 490 (M+6, 100%), 491 (M
(600 mg, 1.46 mmol) was taken up in CH OD (30 mL) containing DCl (0.2 mL),
+7, 39.5%), 492 (M+7, 7%).
and the solution was heated to reflux and stirred for 6 h. Excess solvent was
removed under reduced pressure. CH OD (10 mL) was added and evaporated
1
3
2
13
3
[
4 2
H , C ]LCQ908
to dryness. The residue was dried under vacuum for 2 h and then taken up in
hexanes. The suspension was filtered, and the filter cake dried under vacuum
The trans-ester 4 (201 mg, 0.41 mmol) was taken up in THF (4mL), sodium
for 2h. The resulting solid (the DCl salt of 2) was taken up in a mixture of EtOAc hydroxide (93 mg, 2.3 mmol) in water (1mL) was added, and the mixture
(
5
20 mL) and 7% sodium deuteroxide (sodium deuteroxide in deuterium oxide,
mL) and stirred for 10 min. Layers were separated, and the organic layer was
washed with deuterium oxide and then dried over sodium sulfate. Solvent
was stirred at 50 °C for 12h and then at room temperature for 3 days.
Solvent was removed, and the residue was taken up in water (5mL),
acidified with 1N HCl, and extracted with EtOAc (3× 20 mL). The combined
1
removal afforded 560 mg of 2 (92%). H-NMR (CDCl
m, 2H), 3.10 (m, 1H), 6.7 (br. s, 1H), 7.38 (d, 2H, J= 7.8 Hz), 7.51 (m, 1H), 7.58
m, 1H), 7.67 (m, 1H), 7.75 (m, 1H), 7.95 (d, 2H, J= 7.8 Hz), 8.48 (s, 1H), 8.66
s, 1H). MS (ESI): m/z 412 (M+0, 0.6%), 413 (M+1, 5.5%), 414 (M+2, 20.9%), 415 J= 127 Hz), 2.51 (m, 1H), 7.33 (d, 2H, J = 7.8Hz), 7.71 (m, 3H), 7.9 (d, 1H,
3
) δ 1.98 (m, 2H), 2.26
EtOAc extracts were washed with water and dried over Na
removal and drying under vacuum afforded 147mg of product (78%). H-
NMR (DMSO-d ) δ 1.49 (m, 2H), 1.74 (m, 1H), 1.83 (m, 2H), 2.16 (dm, 2H,
2 4
SO . Solvent
1
(
(
(
6
(
M+3, 57%), 416 (M+4, 100%), 417 (M+5, 20.3%), 418 (M+6, 0.6%).
J= 8.6Hz), 7.96 (d, 2H, J= 7.8Hz), 8.47 (s, 1H), 8.55 (s, 1H), 9.19 (br. s, 1H,
NH), 12.01 (s, 1H). MS (ESI): m/z 458 (M+2, 2.5%), 459 (M+3, 10.3%), 460
(M+4, 30.4%), 461 (M+5, 71%), 462 (M+6, 100%), 463 (M+7, 39%), 464 (9%).
2
13
[
4 2
H , C ]Ethyl 4-(4-(5-((6-(trifluoromethyl)pyridin-3-yl)amino)
pyridin-2-yl)phenyl)-cyclohexylidene)acetate 3
1
3
Acknowledgements
[
C
2
]Triethylphosphonoacetate (1.0 g, 4.42 mmol) in THF (3 mL) was
added dropwise over 10 min to a suspension of 60% sodium hydride
222 mg, 5.55 mmol) in THF (20 mL) at 0 °C. The reaction mixture was
The authors would like to thank Novartis DMPK (Drug Metabolism
and Pharmacokinetics) management for their support and other
Novartis Isotope Lab colleagues for their technical assistance.
(
stirred at room temperature for 60 min and then cooled to 0 °C before
a solution of compound 2 (560 mg, 1.35 mmol) in THF (10 mL) was
added dropwise over 30 min. The mixture was stirred for 3.5 h at room
temperature, cooled to 0 °C, and quenched with deuterium oxide
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J. Label Compd. Radiopharm 2014, 57 670–673
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org