Dynamic Carbon Isotope Exchange of Pharmaceuticals with Labeled CO2

Article abstract of DOI:10.1021/jacs.8b12140

A copper-catalyzed procedure enabling dynamic carbon isotope exchange is described. Utilizing the universal precursor [¹⁴C]CO₂, this protocol allows to insert, in one single step, the desired carbon tag into carboxylic acids with no need of structural modifications. Reducing synthetic costs and limiting the generation of radioactive waste, this procedure will facilitate the access to carboxylic acids containing drugs and accelerate early ¹⁴C-based ADME studies supporting drug development.

Full text of DOI:10.1021/jacs.8b12140

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Dynamic Carbon Isotope Exchange of Pharmaceuticals with Labeled CO2
Gianluca Destro, Olivier Loreau, Elodie Marcon, Frederic Taran, Thibault Cantat, and Davide Audisio
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b12140 • Publication Date (Web): 26 Dec 2018
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Dynamic Carbon Isotope Exchange of Pharmaceuticals with Labeled
CO2
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Gianluca Destro,† Olivier Loreau,† Elodie Marcon,† Frédéric Taran,† Thibault Cantat,‡* Davide
Audisio†*
† Service de Chimie Bio-organique et Marquage (SCBM), CEA/DRF/JOLIOT, Université Paris Saclay, F-91191, Gif-sur-
Yvette, France. ‡ NIMBE, CEA, CNRS, Université Paris-Saclay, 91191 Gif-sur-Yvette, France
Supporting Information Placeholder
precious reagent in the radiochemical reaction is [14C]CO2. While
ABSTRACT: A copper-catalyzed procedure enabling dynamic
the emergence of hydrogen isotope exchange (HIE) methodologies
for the direct insertion of deuterium and tritium into drugs has
found widespread application in the pharmaceutical industry,14,15,16
the large majority of carbon-13 and carbon-14 labeled
pharmaceuticals are still synthesized in multistep procedures.17
Besides being time consuming, these procedures generate large
amounts of radioactive waste, thus incrementing the overall cost of
the radiolabeling. The development of selective carbon isotope
exchange (CIE) reactions would enable the synthesis of labeled
compounds in a single operation without the need for precursors
design and synthesis. Such processes would be particularly
attractive for the pharmaceutical and agrochemical industry (Figure
1A).18 Carboxylic acids are highly abundant functional groups,
commonly present in many major drug classes, including
nonsteroidal anti-inflammatory drugs (NSAIDs), diuretics, anti-
tumor agents and antibiotics.19 The development of carbon isotope
exchange procedure on carboxylic acid derivatives with CO2 would
be highly beneficial for radiolabeling.
carbon isotope exchange is described. Utilizing the universal
precursor [14C]CO2, this protocol allows to insert, in one single
step, the desired carbon tag into carboxylic acids with no need of
structural modifications. Reducing synthetic costs and limiting the
generation of radioactive waste, this procedure will facilitate the
access to carboxylic acids containing drugs and accelerate early
14C-based ADME studies supporting drug development.
As part of drug discovery and development process, a full
understanding of the fate of drug candidates is strictly required by
most drug regulatory agencies (FDA, EMA) for marketing
applications.1,2 The direct and traceless incorporation of a mass or
radioactive tag, with no significant structural modifications, is of
critical importance to follow the fate of the drug candidate, when
administered to humans and animals. To date, the most effective
and precise method to detect and quantify drugs and their
metabolites both in vivo and in vitro relies on the introduction of
isotopic labels.3 Because of their ubiquitous presence in organic
compounds, carbon and hydrogen isotopes are unique tools to
elucidate the absorption, distribution, metabolism, excretion
(ADME) of the drug candidate.4,5 For human ADME studies and
reactive metabolite screens, the use of carbon-14 (14C) over tritium
(3H) isotopes is generally preferred to circumvent the loss of label
due to the more rapid hydroxylation and exchange reactions in
tritiated compounds, while featuring minimal isotope effects.6 The
insertion of a carbon tag into organic molecules relies on the use
14C-carbon dioxide (CO2),7 the universal precursor for all 14C
labeled compounds; yet, a poorly reactive and remarkably stable
gas.8 Incorporation of 14C usually leans on strong nucleophiles,
such as the carboxylation of organolithium and Grignard reagents
with CO2. This class of reactions, originally developed more than
100 years ago,9 is often not suitable to address modern challenges
in radiochemistry and requires early installation of the carbon tag,
because of functional group incompatibility.10 The hurdles
involved in achieving late-stage carbon labeling of functionalized
organic molecules and pharmaceuticals still represents a major
challenge.11 Recent developments have mainly been focused on
late-stage labeling with [14C]CO2 and the secondary building block
K[14C]CN.12,13 Nevertheless, both strategies require the multistep
synthesis of elaborated precursors, are limited to specific structures
and lack of generality. An additional challenge to be considered is
the stoichiometry of the reactions which must be reversed
compared to the synthesis of the unlabeled materials, as the most
In nature, the γ-resorcylate decarboxylase (γ-RSD) catalyzes the
reversible decarboxylation of γ-resorcylate to resorcinol (1,3-
dihydroxybenzene) and CO2, at a zinc active site (Figure 1B).20,21
This biochemical transformation inspired us to adopt an analogous
approach for the carbon isotopic exchange of carboxylic acids
(Figure 1D). Decarboxylative cross coupling reactions have
recently emerged as a method of choice for carbon-carbon bond
22
formation.
Aromatic carboxylates were shown to undergo
thermal carbon dioxide extrusion to generate a highly reactive aryl-
metal species, in the presence of copper or silver salts. In the
presence of a co-catalyst (i.e. Pd) a rapid transmetallation could
take place and yield high value carbon-carbon bonds.23 We
reasoned that, instead of trapping the aryl-metal species II with an
appropriate co-catalyst, this intermediate could react with a labeled
source of CO2 to yield labeled products, through a new CIE reaction
(Figure 2A). We anticipated that the choice of the metal catalyst
and the ancillary ligand would heavily influence the
thermodynamics of the process as well as the competing proto-
decarboxylation of aryl-metal intermediate. In particular, the higher
reactivity of aryl-metal species II compared to the carboxylate I, as
shown by the relative pKa of o-nitrobenzene and the corresponding
carboxylic acid derivative, should require careful consideration and
delicate balance. Because copper is an active decarboxylation
catalyst and has also been reported as an efficient CO2 fixation
catalyst,24 we postulated that copper complexes could be active
catalyst in a CIE reaction.25
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B
A
Me
O
Me
O
Br
Zn
OH
OH
OH
O
Me3Si
previous
N
N
N
Me
S
Me
S
O
SiMe3
O
O
O
O
approach
this work
CO2
OH
Precursor
(4 steps)
Probenecid
(1 steps)
Labeled Probenecid
Carbon
Isotope
Exchange
Linear
Multi
Step
-RSD

O
O
O
N
O
reversible
F
F
F
decarboxylation
OH
OH
9
NH
Me
OH
H
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N
CO2
Me
Me
Flumequine
(1 steps)
Precursor
(10 steps)
Labeled Flumequine
OH
Challenge: CO2
as carbon-14 source
C
D
O
O
R
R
O
O
R
Cu
M
O
14C
O
*
*
*CO2
C
C
M
or
or
or
OH
OH
Het
OH
OH
Het
Het
CO2
stable gas
harsh conditions required
limiting reagent, high cost
carboxylic acid
organometallic intermediate
labeled carboxylic acid
Figure 1: Catalytic reaction design. (A) Carbon labeling technologies for the insertion of the isotope into pharmaceutically relevant
molecules. Light and dark blue circles represent the positions of isotopically labeled C(sp2) with CIE and linear multi step procedures,
respectively. (B) Enzymatic reversible decarboxylation of γ-resorcylate to resorcinol. (C) Hurdles to overcome in the routine synthesis of
carbon-14 labeled compounds. (D) Proposed hypothesis for the mechanism of the dynamic CIE.
decrease of the coordination of the carboxylate to the metal center
(Figure 3).28
To test our hypothesis and identify systems capable of mediating
CIE in carboxylic acids, 2-nitrobenzoic acid was selected as a
model substrate, the electron withdrawing substituent in ortho
O
C
O
C
A
X+
O-
position favoring a milder decarboxylation step.26 The cesium salt
of the corresponding acid was utilized to limit a possible proto-
decarboxylation. Catalyst evaluation was initially conducted with
[13C]CO2 gas, as a convenient and readily handled [14C]CO2
surrogate. All of the reactions were conducted under standard
conditions employing 20 mol% of copper salt, 20 mol% of ligand
in NMP and DMSO with 3 equivalents of [13C]CO2 at 150 °C. The
extent of isotopic exchange was determined after 2 hours using
mass spectroscopy. Selected results from our screening
experiments are summarized in the supporting information (Table
S1 to S6). After evaluating a variety of phosphines, NHCs and
multi-dentate nitrogen ligands, the proof-of-concept was obtained
in presence of tetramethylethylenediamine (TMEDA, see SI) and
[13C]2 could be isolated in 21% isotopic enrichment (IE), albeit
only trace amounts of material could be isolated due to parasitic
dynamic 12C/13C/14C exchange
no need for re-synthesis
OH
NO2
NO2
HCl
1
2
[Cu]
O
C
O
C
[CuI]
[CuI]
[CuI]
12CO2
*CO2
O
O
NO2
NO2
NO2
I
II
labeled-I
B
CN
O
O
13
O
C
C] O2
[
75%
OH
O
(3 equiv)
OCs
NO2
20 mol% CuBr
20 mol% Ligand
DMSO, 150 °C, 2h
then HCl
proto-decarboxylation. Surprisingly,
a
variety of ligands
N
N
NO2
13C]2
commonly utilized in copper-catalyzed decarboxylative cross
coupling, such as bipyridines were poorly effective in CIE (see
Table S2). After extensive optimization, we found that ligand L1,
featuring a bisoxazoline scaffold, enabled the formation of [13C]2
in 75% IE, the highest possible enrichment at equilibrium.27
Ph
Ph
1
L1
[
, 50%
Figure 2: Reaction development. (A) Proposed catalytic cycle for
the CIE protocol. (B) The colored circles (blue or green) and
numbers denote the positions of the carbon atoms labeled and the
percent incorporation of the carbon isotope, respectively. DMSO,
dimethyl sulfoxide.
With the optimized conditions in hand, we sought to examine the
scope of the CIE protocol with a library of aryl carboxylic acids.
For the labeling of these substrates (Figure 3), the best conditions
were found to be substrate-dependent, given a choice of two ligands
(L1 and L2) and two solvents (DMSO and NMP/DMSO as a 4:1
mixture). As expected, the use of carboxylate salts was crucial to
limit proto-decarboxylation.
Stronger electron-donating methoxy groups were well tolerated and
13C enriched products [13C]7-9 were obtained in 39% to 64% IE.
While the presence of iodo and bromo substituents is not suitable
under the applied reaction conditions, products [13C]10, [13C]11
and [13C]12 bearing a m-chlorine, p-chlorine and m-fluorine were
isolated with excellent IE (66%, 59%, 70%). The presence of a nitro
substituent in meta position was detrimental to the reactivity and
only unlabeled material was recovered, under standard conditions.
The presence of methyl substituents on the ortho-nitro carboxylates
(3-5) did not affect much the reactivity, though a slight decrease of
the IE was observed when the substituent is in ortho ([13C]6, 23%
IE), as the presence of additional steric hindrance results in the
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O
C
O
CN
20 mol% CuBr
20 mol% Ligand
1
2
3
4
O
O
O
O
13
OCs
+
OH
C
C] O2
[
Ph
Ph
R
R
or
DMSO, 150 °C, 2h
then HCl
N
N
N
N
Ph
Ph
Ph
Ph
L1
L2
(hetero)aryl
carboxylate
isotope source
(3 equiv)
carbon labeled
product
5
6
aryl carboxylates
7
8
9
O
O
O
O
Me
O
O
44%
OH
75%
66%
Me
65%
OH
23%
OH
68%
OH
MeO
OH
OH
NO2
10
11
12
13
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NO2
Me
NO2
NO2
[
13C]5
, 45%
NO2
NO2
Me
13C]3
[
13C]6
[
13C]7
, 48%
, 59%
[
13C]2
[
[
13C]4
, 50%
, 16%
, 42%
O
30%
O
O
O
O
O
OH
60%
OH
69%
OH
66%
OH
59%
OH
70%
OH
MeO
MeO
Cl
F
MeO
NO2
, 53%
NO2
, 41%
NO2
, 10%
Cl
NO2
, 15%
NO2
NO2
13C]8
[
13C]11
[
13C]9
[
13C]10
[
13C]12
, 17%
[
[
13C]13a
, 88%
O
48%
OH
O
O
O
F
OMe O
14%
OH
16%
OH
31%
OH
39%
OH
Me
NO2
O2N
O2N
OMe
, 81%
[
13C]14
[
13C]15
[
13C]16
[
13C]17
[
13C]18
, 50%
, 50%
, 65%
, 10%
heteroaryl carboxylates
Me
S
Me
O
O
O
O
O
9%
OH
65%
OH
N
32%
N
20%
OH
52%
OH
N
S
OH
S
Me
Me
[
13C]19
[
13C]20
[
13C]21
[
13C]22b
[
13C]23
, 35%
, 22%
, 36%
, 30%
, 42%
O
O
Me
O
27%
OH
O
O
OH
29%
47%
OH
43%
OH
O
O
O
O
O
[
13C]24
[
13C]25
[
13C]26
[
13C]27
, 53%
, 69%
, 72%
, 56%
pharmaceutical carboxylates
N
O
O
Me
O
54%
49%
OH
O
O
F
34%
OH
Me
N
S
OH
Me
Me
O
N
N
O
Me
S
O
O
O
66%
Me
Flumequine, 42%
HO
[
13C]31
Febuxostat, 10%
[
13C]28a
[
13C]29a
[
13C]30
UBP608, 26%
Probenecid, 50%
Figure 3: Cu-catalyzed carbon-13 labeling of representative arenes, heteroarenes and pharmaceutical compounds. The green colored
circles and numbers denote the positions of the carbon atoms labeled and the percent incorporation of the carbon-13 isotope, respectively.
Reaction conditions: substrate (0.1 mmol, 1eq), ligand (20 mol%) CuBr (20 mol%.), 13CO2 (3eq.), DMSO (0.5 mL). See supporting
a
information for detailed experimental procedures. DMSO, dimethyl sulfoxide. Unless otherwise stated, L1 was utilized. reaction
temperature 190 °C. b L2 was utilized.
The reactivity could be partially restored by increasing the
temperature to 190 °C, and product [13C]13 was isolated with 30%
IE. The importance of the ortho substituent effect is highlighted in
substrate 14, where the presence of the o-methyl allows the
isolation of [13C]14 in 48% IE, without need of higher temperature.
Substrates [13C]15 and [13C]16 bearing a nitro substituent in para
position were isolated with 14% and 15% IE, respectively. Other
substituents than the nitro group are effective under CIE protocol,
however the weaker coordinating fluorine group in [13C]17,
translated in a reduction of enrichment (31% IE). On the other
hand, the use of 1-methoxy naphthalene core [13C]18 gave an
interesting IE of 39%. Current limitations of the procedure are the
presence of sole electrodonating methoxy substituents and
substrates bearing labile protons which are unproductive or
undergo proto-decarboxylation (see SI for details).
To test the relevance of this novel CIE procedure, the labeling of
important substructures in pharmaceuticals was tested. They
include nitrogen-, oxygen- and sulphur-containing heteroarenes
(19-27). In particular, methylthiazole [13C]21, benzofuranes-
[13C]24-25 and indole-2-carboxylates [13C]23 were obtained in 43
to 65% IE. Chromone and coumarine carboxylate derivatives
[13C]26-27 were labeled in 29 and 27% IE, respectively.
Pharmaceutically relevant compounds 28-31 were labeled in a
single step using the CIE procedure with good IE (Figure 3).
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A series of representative, commercially available pharmaceuticals
high degree of chemoselectivity, and is compatible with the
presence of electrophilic moieties such as nitriles, ketones and
sulfonamides.
1
2
3
4
5
6
7
8
and drug precursors were then labeled using [14C]CO2, to evaluate
the utility of the copper-catalyzed CIE over existing multistep
methods (Figure 4A). The results were in good agreement with
those obtained using stable carbon-13 isotope and did not require
any further protocol optimization (Figure 3). 2-Nitrobenzoic acid
2, a synthetic precursor of antipruritic drug crotamitone previously
labeled with 14C in four steps from [14C]CO2,29 was obtained by
means of CIE with a high molar activity of 1506 MBq mmol-1
(Figure 4),30 meeting previously described requirements for use in
preclinical candidate selection studies. Probenecid 29 was labeled
in one single step from [14C]CO2 with a molar activity of 923 MBq
mmol-1, more three times higher than what previous reported by
Ellsworth and co-workers for metabolic disposition studies.31
Fluoroquinolone antibiotic flumequine 30 was successfully labeled
on the carboxylic acid, which is essential for the pharmacological
activity and metabolically stable,32 with a molar activity of 1076
MBq mmol-1. The preparation of [14C]30 had previously involved
a sequence of 10 linear steps from [14C]CO2.33 The current CIE
procedure has the advantage of reducing dramatically the
generation of radioactive waste, which are extremely polluting and
expensive to dispose of. The 14C labeling of benzofuran-2-
carboxylic acid, synthetic precursor of neural nicotinic
acetylcholine receptor agonist bradanicline, and febuxostat
proceeded with good incorporation of the carbon tag.
At this early stage of development, this dynamic carbon isotope
exchange protocol has already opened up new opportunities for the
synthesis of carbon labeled drug intermediates and
pharmaceuticals. Further extensions of the CIE can be expected
from ligand design and the use of a variety of transition metal
catalysts.
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ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge via the
Internet at http://pubs.acs.org.
Experimental procedures and computational details
NMR spectra for obtained compounds
AUTHOR INFORMATIONS
Corresponding Author
* davide.audisio@cea.fr,
thibault.cantat@cea.fr
This work: [Cu]-catalysed carbon-14 exchange with [14C]CO2
A
O
N
O
Me
O
40%
47%
OH
O
65%
OH
F
Notes
OH
The authors declare no competing financial interests.
N
Me
S
NO2
14C]2
O
O
ACKNOWLEDGMENT
[
[
14C]29
[
14C]30
Flumequine
1076 MBq mmol-1, 38% yield
Probenecid
1506 MBq mmol-1, 28% yield
923 MBq mmol-1, 61% yield
This work was supported by CEA and by the European Union’s
Horizon 2020 research and innovation programme under the Marie
Sklodowska-Curie grant agreement N°675071. The authors thank
David-Alexandre Buisson and Céline Chollet for the excellent
analytical support.
(Crotamitone precursor)
N
O
47%
OH
Me
N
O
Me
Me
O
O
S
54%
[
14C]24
HO
REFERENCES
1080 MBq mmol-1, 50% yield
[
14C]31
(Bradanicline precursor)
Febuxostat
1254 MBq mmol-1, 19% yield
(1) Maxwell, B. D.; Elmore, C. S. Eds. Radiosynthesis for ADME
Studies; 461–471 (John Wiley & Sons, Inc: Hoboken, 2012).
(2) Penner, N.; Klunk, L. J.; Prakash, C. Human Radiolabeled Mass
Balance Studies: Objectives, Utilities and Limitations. Biopharm. Drug
Dispos. 2009, 30, 185-203.
(3) Isin, E. M.; Elmore, C. S.; Nilsson, G. N.; Thompson, R. A.;
Weidolf, L. Use of Radiolabeled Compounds in Drug Metabolism and
Pharmacokinetic Studies. Chem. Res. Toxicol. 2009, 25, 532–542.
(4) Elmore, C. S.; Bragg, R. A. Isotope Chemistry; a Useful Tool in the
Drug Discovery Arsenal. Bioorg. Med. Chem. Lett. 2015, 25, 167–171.
(5) Marathe, P. H.; Shyu, W. C.; Humphreys, W. G. The Use of
Radiolabeled Compounds for ADME Studies in Discovery and Exploratory
Development. Curr. Pharm. Des. 2004, 10, 2991-3008.
(6) Elmore, C. S., The Use of Isotopically Labeled Compounds in
DrugDiscovery. Ed. Annu. Rep. Med. Chem. 2009, 44, 515–534.
(7) Zwiebel, N.; Turkevich, J.; Miller, W. W. Preparation of
Radioactive C02 from BaCO3. J. Am. Chem. Soc., 1949, 71, 376-377.
(8) Aresta, M. Carbon Dioxide as Chemical Feedstock (John Wiley &
Sons, 2010).
(9) Grignard, V. Compt. Rend. Acad. Sci. 130, 1322-1325 (1900).
(10) Bragg, R. A.; Sardana, M.; Artelsmair, M.; Elmore, C. S. New
Trends and Applications in Carboxylation for Isotope Chemistry J. Label.
Compd. Radiopharm. doi.org/10.1002/jlcr.3633.
B
Previously reported multi-step synthesis
O
N
O
Me
O
O
F
OH
OH
OH
NO2
N
Me
S
O
O
[
14C]2
[
14C]29
248 MBq mmol-1
[
14C]30
Probenecid
Flumequine
162 MBq mmol-1
10 steps from 14CO2
192 MBq mmol-1
4 steps from 14CO2
Ref. 27
4 steps from 14CO2
Ref. 28
Ref. 30
Figure 4: Cu-catalyzed carbon-14 labeling of pharmaceutical
relevant molecules. (A) Reaction conditions: substrate (0.1 mmol,
1eq), ligand (20 mol%) CuBr (20 mol%.), 14CO2 (3eq.), DMSO
(0.5 mL), see supporting information for detailed experimental
procedures. (B) Comparison with multi step methods previously
reported in the literature. The colored circles (dark or light blue)
and numbers denote the positions of the carbon atoms labeled and
the percent incorporation of the carbon isotope, respectively. Molar
activities for each compound are expressed in MBq mmol-1.
Radiolabeled compounds [14C]24 and [14C]31 were obtained with
molar activities of 1080 and 1254 MBq mmol-1, in line with values
acceptable for ADME studies. It is worth noting that compared to
traditional multi-step procedures the current methodology shows a
(11) Kamen, M. D. Early History of Carbon-14: Discovery of this
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TOC
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7
8
Me
O
Me
O
Br
OH
this work
OH
Me3Si
N
N
N
Me
S
Me
S
previous
approach
SiMe3
O
O
O
O
Carbon Isotope Exchange
Precursor
(4 steps)
Probenecid
(1 steps)
Labeled Probenecid
Linear
Multi
Step
CuBr, Ligand
[
13C]CO2 and [14C]CO2
O
O
O
O
F
F
F
OH
OH
9
NH
Me
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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53
54
55
56
57
58
59
60
Me
Me
Labeled Flumequine
Flumequine
(1 steps)
Precursor
(10 steps)
=
13C and 14
C
6
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