Direct Carbon Isotope Exchange through Decarboxylative Carboxylation

Article abstract of DOI:10.1021/jacs.8b12035

A two-step degradation-reconstruction approach to the carbon-14 radiolabeling of alkyl carboxylic acids is presented. Simple activation via redox-active ester formation was followed by nickel-mediated decarboxylative carboxylation to afford a range of complex compounds with ample isotopic incorporations for drug metabolism and pharmacokinetic studies. The practicality and operational simplicity of the protocol were demonstrated by its use in an industrial carbon-14 radiolabeling setting.

Full text of DOI:10.1021/jacs.8b12035

Communication
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Direct Carbon Isotope Exchange through Decarboxylative
Carboxylation
Cian Kingston,† Michael A. Wallace,‡ Alban J. Allentoff,‡ Justine N. deGruyter,† Jason S. Chen,§
Sharon X. Gong,‡ Samuel Bonacorsi, Jr.,‡ and Phil S. Baran*,†
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United
States
‡Radiochemistry, Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey 08543, United States
§Automated Synthesis Facility, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United
States
S
* Supporting Information
ABSTRACT: A two-step degradation−reconstruction
approach to the carbon-14 radiolabeling of alkyl
carboxylic acids is presented. Simple activation via
redox-active ester formation was followed by nickel-
mediated decarboxylative carboxylation to afford a range
of complex compounds with ample isotopic incorpora-
tions for drug metabolism and pharmacokinetic studies.
The practicality and operational simplicity of the protocol
were demonstrated by its use in an industrial carbon-14
radiolabeling setting.
uccessful execution of the metabolic and pharmacokinetic
Sstudies required in a modern drug development campaign
is often predicated on the ability to access useful quantities of
radiolabeled lead analogues.1 The demand for ever more
advanced radiolabeling techniques is illustrated by the recent
reports describing the tritiation of arene-, azaarene-, amine-,
amide-, and thioether-containing compounds through efficient
hydrogen isotope exchange (HIE) processes (Figure 1A, I).2
As the more metabolically stable radiolabel, carbon-14 is
heavily relied upon during all stages of drug development,
including (pre)clinical absorption, distribution, metabolism,
and excretion (ADME) studies.3 In contrast to tritium, the
synthetic methods for introduction of this isotope have
remained relatively inefficient due to the difficulties associated
with C−C bond formation.4 Still, the ubiquity of carboxylic
acids in nature and in drug-like leads has made them a prime
target for carbon-14 radiolabeling, particularly in the syntheti-
cally favorable degradation−reconstruction strategies that
begin from the unlabeled target compound (Figure 1A,
II).1a,5 However, current approaches rely on harsh conditions
Figure 1. (A) A new approach to carbon-14 radiolabeling inspired by
hydrogen isotope exchange. NHPI = N-hydroxyphthalimide. (B)
Potential pitfalls of Ni-mediated carboxylation of redox-active ester
(RAE) substrates.
to facilitate the common activation/substitution/hydrolysis
sequence, thereby severely limiting their application.6
Inspired by the synthetic utility of HIE, we set out to invent
a method to radiolabel alkyl carboxylic acids through direct
carbon isotope exchange, wherein the isotope is incorporated
in the final step. We envisaged an expedient sequence
consisting of mild formation of a redox-active ester7 (RAE),
followed by a chemoselective nickel-mediated reductive
decarboxylative carboxylation (Figure 1A, III).8 As a further
advantage, the use of carbon-14-labeled CO2 would maximize
the radiochemical yields and minimize the handling of
Received: November 8, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b12035
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Journal of the American Chemical Society
Communication
Scheme 1. (A) Proof-of-Concept Using 12CO2, and (B) Development, Optimization, and Analysis with 13CO2
a
b
C
0.1 mmol. 1H NMR yield with 1,3,5-trimethoxybenzene as an internal standard. Calculated from high-resolution mass spectrometry data in
d
MassWorks (Cerno Bioscience) and confirmed by 13C NMR. Isolated yield. DSPR = deck screening pressure reactor.
a
Scheme 2. (A) Scope of the Ni-mediated decarboxylative carboxylation with 13CO2 and (B) Translation to a 14CO2
b
Radiochemistry Setup
a
14C specific activity values were calculated by multiplying the observed percentage of 13C incorporation by the maximum theoretical specific
activity of a molecule containing a single carbon-14 label (62.4 mCi/mmol). Green values represent 13CO2 incorporations that, in the case 14CO2
was used, would afford sufficiently high specific activities for all preclinical/clinical ADME studies. Reaction conditions: RAE (1.0 equiv), NiBr2·
b
glyme (1.0 equiv), neocuproine (2.2 equiv), Mn (2.2 equiv), 13CO2 (50 psi), DMF (0.1 M), rt, 20 h. Reaction conditions: RAE (1.0 equiv),
NiBr2·glyme (1.0 equiv), neocuproine (2.2 equiv), Mn (2.2 equiv), 14CO2 (1 atm, 5.5−32 equiv), DMF (0.03−0.05 M), −25 °C (1 h) to rt, 20 h.
c
1H NMR yield with 1,3,5-trimethoxybenzene as an internal standard. % C = percentage carbon-13 incorporation, SA = specific activity, LSC =
liquid scintillation counting, AMS = accelerator mass spectrometry, HPLC = high-performance liquid chromatography.
B
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Journal of the American Chemical Society
Communication
a
Scheme 3. Comparative Studies on the Synthesis of Biologically Relevant Compounds
a
Reactions conditions as in Scheme 2.
radioactive intermediates.9 A concern with the use of RAE
substrates is the undesired partial re-formation of the unlabeled
target compound via cleavage or incomplete 12CO2/14CO2
exchange (Figure 1B). However, while this concern mecha-
nistically limits the specific activity, the levels of incorporation
should be more than sufficient for practical use in ADME
studies.10
potential application of the methodology for carbon-14
preclinical and clinical ADME studies, the observed 13CO2
incorporations were converted to the theoretical specific
activities which would result from using 14CO2. With the
recent advent of ultrasensitive analytical techniques, the
bottleneck for specific activity in radiolabeling clinical human
ADME studies has shifted to analysis of radiochemical purity.
Industrial standard HPLC with in-line radioactivity detection
requires 10 μCi/mg or higher to allow for sufficient
disintegrations per minute for precise radiochemical analysis
while maintaining column performance.13 Lower isotopic
incorporations can be analyzed by liquid scintillation counting,
microplate scintillation, or accelerator mass spectrometry, but
these are more resource-intensive techniques. In general,
higher incorporations (≥20 μCi/mg) are required for early
preclinical animal ADME studies.14 Gratifyingly, primary and
secondary RAEs generated from acids 14−22 bearing a variety
of functional groups, including ketone, ester, protected amines
and amides, and a phenol, provided excellent isotopic
incorporations suitable for all preclinical and clinical ADME
studies, although no product was observed for a substrate
containing a pyridine ring (see the Supporting Information for
further details). In the case of such radiolabeling studies,
isolated chemical yields are less important than the speed of
preparation and cost of the isotope source. The chemo-
selectivity of the process is also critical so as to avoid extra
functional group/protecting group manipulations on radio-
active material. Thus, the mild degradation−reconstruction
approach described here, in which the unlabeled target
compound is readily available from preliminary scale-up, is
ideal. Compound 23, bearing additional functionality in an
epoxide and free alcohols, was formed with isotopic
incorporation that would be sufficiently high for in-line
HPLC purity analysis for use in carbon-14 clinical ADME
studies. While secondary acids 19, 21,15 and 22 were formed
with excellent isotopic incorporations, no incorporation was
observed for tertiary acid 24.
The desire to evaluate the proposed methodology using
cheap and readily available 12CO2 presented an interesting
problem specific to the development of degradation−
reconstruction radiolabeling methodologies: How can isotope
incorporation be quantified when using an unlabeled reagent?
As a solution, methods for the formation of chemically distinct
“labeled” and “unlabeled” products via different mechanistic
pathways were evaluated (Scheme 1A). Whereas diastereo-
meric inversion (Scheme 1A, I)7l and nickel chain-walking11
(Scheme 1A, II) afforded irreproducible results and no product
formation, respectively, the use of cyclopropylmethyl substrate
7 enabled reproducible differentiation between the carboxy-
lated and hydrolyzed products 9 and 10 via radical-induced
ring-opening (Scheme 1A, III). With a proof-of-concept in
hand, we chose RAE 11 as a model substrate and subjected it
to carboxylation by non-radioactive 13CO2 as a surrogate for
14CO2 (an abbreviated summary of reaction optimization is
depicted in Scheme 1B; see the Supporting Information for
further details). Some initial difficulties encountered using a
traditional pressure vessel were circumvented by switching to
Unchained Labs’ deck screening pressure reactor, which
provided reliable and reproducible results. Screening of the
reaction parameters led to the formation of acid [13C]-12 in
42% yield with 19% 13CO2 incorporation (entry 1).12 If this
level of incorporation could be realized using 14CO2, a specific
activity of 66 μCi/mg would be achieved. Lowering both the
pressure and the nickel/ligand loading had a deleterious effect
upon the isotopic incorporation (entries 2 and 3), and control
reactions showed that the nickel source, ligand, and reductant
were all essential for product formation and/or 13CO2
incorporation (entries 4−6). The major byproducts observed
were consistent with β-hydride elimination, decarboxylative
protonation, and dimerization pathways. Photoinduced elec-
tron-transfer processes were also investigated but failed to
provide the product in meaningful yields and isotopic
incorporations (see the Supporting Information).
With its scope and practicality established, the reaction was
implemented in an industrial radiosynthetic setting using
14CO2. Although only a small amount of pressure was required
under the optimized conditions with 13CO2, efforts were made
to further increase operational simplicity, cost effectiveness,
and safety. It was thought that the requirement for pressure
could be avoided by cooling the reaction to increase the
solubility of 14CO2 in the solvent.16 Gratifyingly, implementa-
tion of an initial temporary cryogenic period at −25 °C under
With an optimized set of conditions in hand, we next
explored the scope of the reaction with a series of primary,
secondary, and tertiary RAEs (Scheme 2A). To gauge the
C
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Communication
atmospheric pressure provided a 14CO2 solubility (0.71 M)
comparable to that at 50 psi/room temperature (0.74 M). This
method facilitated the development of a practical laboratory
setup using standard vacuum manifold techniques to allow safe
application of 14CO2 to the process (see the Supporting
Information for further details). Using only 5.5−32 equiv of
14CO2 (depending on the use of an ampule or in situ formation
from [14C]-BaCO3), a selection of RAEs underwent
decarboxylative carboxylation to afford the carbon-14-radio-
labeled products with sufficient levels of isotopic incorporation
for general use in preclinical and clinical ADME studies
(Scheme 2B). Interestingly, the inverted α-diastereomer of 21
was isolated with a significantly higher specific activity than the
corresponding β-product, presumably due to the intermediacy
of a radical species (as proposed in Scheme 1A, I). To further
explore the advantages of this methodology, it was compared
to two previous radiosyntheses of biologically important
compounds (Scheme 3). Parnes and co-workers previously
reported a seven-step degradation−reconstruction approach to
[14C]-mycophenolic acid 25 which relied upon a decarbox-
ylative halogenation/[14C]-cyanation sequence to introduce
the radiolabel in the second-to-last step.17 In stark contrast, the
developed procedures for 13CO2 and 14CO2 both afforded the
product in just two steps, one (radio)labeled, with sufficient
isotopic incorporation for all ADME studies. In the synthesis of
[14C]-chlorambucil 26, Madelmont and co-workers introduced
the radiolabel in the first step of an eight-step sequence via
[14C]-cyanation of alkyl bromide 27 using low specific activity
K14CN.18 In contrast, decarboxylative carboxylation afforded
the product in an expedient two-step sequence. The differences
between the procedures for 13CO2 and 14CO2 were highlighted
by variation in the isotopic incorporations, although in both
cases excellent levels were observed. The results bode well for
the widespread adoption of this method for carbon-14
radiosynthesis of ubiquitous alkyl carboxylates.19
ACKNOWLEDGMENTS
■
Financial support for this work was provided by Bristol-Myers
Squibb and NSF GRFP (J.N.D.). We are grateful to Dr. Dee-
Hua Huang and Dr. Laura Pasternack (TSRI) for assistance
with nuclear magnetic resonance spectroscopy. We also thank
Dr. Byron K. Peters and Dr. Yu Kawamata for helpful advice
and discussion.
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AUTHOR INFORMATION
■
Corresponding Author
*pbaran@scripps.edu
ORCID
Cian Kingston: 0000-0002-2907-4260
Justine N. deGruyter: 0000-0003-0465-8988
Phil S. Baran: 0000-0001-9193-9053
Notes
The authors declare no competing financial interest.
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