Late-Stage 18O Labeling of Primary Sulfonamides via a Degradation–Reconstruction Pathway

Article abstract of DOI:10.1002/chem.202000484

A late-stage ¹⁸O labeling approach of sulfonamides that employs the corresponding unlabeled molecule as the starting material was developed. Upon deamination of the sulfonamide, a sulfinate intermediate was isotopically enriched using eco-friendly reagents H₂¹⁸O and ¹⁵NH₃(aq) to afford a M+5 isotopologue of the parent compound. This degradation–reconstruction approach afforded isolated yields of up to 96 % for the stable isotope labeled (SIL) sulfonamides, and was compatible with multiple marketed therapeutics, including celecoxib, on a gram scale. The SIL products also exhibited no ¹⁸O/¹⁶O back exchange under extreme conditions, further validating the utility of this green strategy for drug labeling for both in vitro and in vivo use. This procedure was also adapted to include pharmaceutically relevant methyl sulfones by using ¹³CH₃, affording M+5 isotopic enrichment, thereby illustrating the broad utility of this methodology.

Full text of DOI:10.1002/chem.202000484

A Journal of
Accepted Article
Title: Late-Stage 18O Labeling of Primary Sulfonamides via a
Degradation-Reconstruction Pathway
Authors: Sean Reilly, Frank Bennett, Patrick S. Fier, Sumei Ren, and
Neil A. Strotman
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.202000484
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1
8
Late-Stage O Labeling of Primary Sulfonamides via a
Degradation-Reconstruction Pathway
Sean W. Reilly,* Frank Bennett, Patrick S. Fier, Sumei Ren, and Neil A. Strotman[a]
1
8
Abstract: A late-stage O labeling approach of sulfonamides that
employs the corresponding unlabeled molecule as the starting
material was developed. Upon deamination of the sulfonamide, a
sulfinate intermediate was isotopically enriched using eco-friendly
ng stable isotope labeled (SIL) sulfonamides. As such, a late-
stage isotopic labeling strategy, which tolerates sulfonamide
compounds with complex ligand architectures, would constitute a
significant impact in drug development and delivery.
18
15
reagents H
2
O and NH
3
(aq) to afford a M+5 isotopologue of the
Generally, SIL compounds are at least three to four mass
units heavier than the parent congener, with no greater than 0.2%
of the unlabeled compound present, in order to avoid any overlap
between the SIL compound and analyte in bioanalytical mass
spectrometry assays.[ Some compounds containing elements
with a naturally wider occurring isotopic distribution, (i.e., S, Cl, or
Br), can require a SIL standard with a mass enrichment greater
than four in order to avoid signal interference. Developing
complex SIL sulfonamide compounds that meet such SIL mass
requirements typically involve time consuming multi-step
syntheses and extensive chemical derivatization (Figure 2A).
parent compound. This degradation-reconstruction approach afforded
isolated yields of up to 96% for the stable isotope labeled (SIL)
sulfonamides, and was compatible with multiple marketed
therapeutics, including celecoxib, on a gram scale. The SIL products
5]
18
16
also exhibited no O/ O back exchange under extreme conditions,
further validating the utility of this green strategy for drug labeling for
both in vitro and in vivo use. This procedure was also adapted to
13
3
include pharmaceutically relevant methyl sulfones by using CH ,
affording M+5 isotopic enrichment, thereby illustrating the broad utility
of this methodology.
2
Synthetic reagents containing the traditionally used isotopes H,
1
3
15 [4b, 6]
C, and
N
can also be costly and only provide one mass
Sulfonamide moieties are ubiquitous in pharmaceuticals due to
the high metabolic stability, water solubility, and biological
response elicited by these pharmacophores (Figure 1).[1] Of the
numerous sulfur-containing drugs, 30% are known to contain a
sulfonamide functional group.[2] As a result, these motifs have
become increasingly prevalent throughout small molecule
libraries and pharmaceutical discovery programs. Evaluating
the bioavailability, pharmacokinetics, pharmacodynamics, and
metabolites of the sulfonamide drug candidates throughout
development requires the development of internal standards
containing isotopic labels in metabolically stable positions.[4] With
the ever tighter timeline demands in drug discovery, there is a
critical need for more practical and efficient methods for developi-
unit increase for each atom. Although advances have been made
[7]
in utilizing hydrogen-isotope exchange (HIE) catalysts as a late-
[
8] 2
stage labeling option for primary sulfonamides , H isotope is not
typically desirable for bioavailability studies as deuterium is often
[9]
labile to metabolic pathways, and can also exhibit problematic
chromatographic behavior[10] by failing to co-elute with the non-
labeled analyte of interest.[11]
[
3]
Figure 1. Chemical scaffolds of select primary sulfonamide drugs.
[
a]
Dr. S. W. Reilly, Dr. F. Bennett, Dr. P. S. Fier, Dr. S. Ren, Dr. N.
Strotman
Figure 2. Reported synthetic methods for labeling primary sulfonamides with
stable isotopes.
Department of Process Research & Development
MRL, Merck & Co., Inc.
Rahway, NJ 07065 (USA)
E-mail: sean.reilly@merck.com
Supporting information for this article is given via a link at the end of
the document.
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2
13
C, or 15N isotopes, developing
Compared to H,
unlabeled parent. Therefore, we developed Method 2 (Figure 3C)
which involves a crude isolation of the sulfinate following the
degradation step by washing the insoluble intermediate with
organic solvents over a filter frit (See SI), thereby removing any
remaining non-degraded sulfonamide. Compared to Method 1,
Method 2 still produced the label sulfonamide 4a in 63% isolated
yield, along with comparable M+5 isotopic enrichment of 89%. Of
particular importance, this crude isolation eliminated any potential
for parent compound contamination in the final step of the
degradation-reconstruction method since the sulfinate was
separated from any residual starting sulfonamide.
1
8
sulfonamides tracers with a doubly-labeled oxygen-18 ( O)
sulfonyl would be a far more efficient strategy for achieving the
required mass differential of the SIL compound, due to the overall
four-mass unit increase. Furthermore, the inherent chemical and
metabolic stability of sulfonamides allows the doubly-labeled
sulfonyl oxygens to be “locked” into tracer, thereby eliminating any
potential for degradation or 18O/ O back-exchange of the labeled
handle (vide infra). Inspired by the sulfonamide deamination work
of Fier and Maloney[12], we envisioned utilizing the more reactive
sulfinate to access an 18O-labeled sulfinic acid intermediate,
16
1
5
followed by subsequent S– N bond reformation, to generate the
desired doubly-labeled sulfonamide (Figure 2B). By incorporating
1
5N, this approach would deliver an overall five mass unit increase
and allow the use of the non-labeled parent compound as the
starting material. Herein, we disclose this late-stage 18O labelling
strategy and illustrate the tolerance of this approach towards
complex primary sulfonamides and methyl sulfones.
Unlike carboxylic acid functional groups[13], sulfonamides do
16
18
not undergo direct O/ O exchange under acidic or basic
conditions (Figure 3A). As such, we focused our initial studies on
enriching sulfinates with 18O, as this species would be more
susceptible to 16O/ O exchange. We found that reacting sulfinate
18
1
8
1
2
in a 0.75 M solution of H O with 3 equiv. of HCl at 80 ºC for 90
min consistently provided the doubly-labeled 18O sulfinic acid (2a)
at ~90% enrichment (Figure 3B). Increasing the reaction time or
temperature to further enhance 18O enrichment led to the
formation of disulfoxide side-product 2b, (detected by LC/MS)
along with other impurities, presumably due to disproportionation
of the reactive sulfinic acid.[14]
We next applied the deamination strategy to sulfonamide
a[12] to generate sulfinate intermediate 1 for subsequent
18
O
3
exchange (Figure 3C). Under these conditions, no trace of the
parent compound (3a) was observed by LC/MS, eliminating the
potential for contamination of the sulfonamide product with the
unlabeled isotopologue, therefore, allowing us to directly carry
sulfinate 1 through a one-pot reaction (Method 1, Figure 3C).
Using modified conditions to those previously reported,[15] direct
amination of sulfinic acid 2a was achieved using 15NH
(aq) and I
to generate the desired S– N bond in sulfonamide 4a. We found
that 10 equiv. of 15NH
was required to neutralize the remaining
3
2
1
5
3
1
8
HCl from the O labeling step, as well as any HI produced from
Figure 3. Development of a degradation and reconstruction pathway using 18O-
labeled sulfinic acid intermediates to access isotopically enriched primary
sulfonamides. Reaction was monitored by LCMS to determine total enrichment
for both oxygen atoms.
-
I
2
mediated amination of 2a. For easier handling, the solid
1
5
Cl was also found to be a compatible 15N nitrogen-
reagent NH
labeled source when introduced with NaOH. Although commercial
4
15NH 16O, no appreciable
(aq) is provided as a 6N solution in H
3 2
loss of 18O incorporation from sulfinic acid 2a, via 16O back-
exchange, was detected during the amination step. As expected,
After validating the isotopic enrichment strategy of Method 2
with compound 3a, we next examined a broad scope of aryl
sulfonamides with varying functional groups (Table 1). Our
optimized conditions were found to tolerate both electron-rich and
-deficient aryl sulfonamides, consistently providing M+5 isotopic
enrichment values of 85-90% for products 4b-g. Although some
decomposition of the sulfinic acid intermediate for benzylic
substrate 3c was observed under acidic conditions, we still
obtained an isolated yield of 24% with 89% M+5 isotopic
enrichment for 4c. Upon examining substrate 3g, we failed to
detect any discernible hydrolysis of the –CN moiety, illustrating
how these conditions can be applied to sulfonamide systems
bearing sensitive functional groups. Ketone and amide containing
1
8
16
no detectable
O/ O back-exchange was observed with
sulfonamide 4a under acidic or basic aqueous conditions, further
validating how these 18O and 15N enriched sulfonamides are
suitable for studies conducted under physiological, as well as
extreme pH conditions.
While Method 1 allowed for the successful one-pot 18O-
labeling synthesis on compound 3a without observation of any
unlabeled parent compound, we found that quantitative
deamination was not obtained with many of the more complex
substrates examined throughout this study. As a result, using
Method 1 could risk contamination of the final products with the
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systems also reacted smoothly under the outlined conditions to
afford excellent yields and isotopic enrichment for isotopologues
however, the anti-inflammatory pharmaceutical was readily
enriched with 18O and 15N, yielding 6k at 64% with a M+5 mass
increase of 88%. This example was also conducted on a one-
gram scale to demonstrate how these conditions can be
employed to deliver the larger amounts of SIL tracers typically
required for human and animal studies throughout drug
development.
4h-i. Overall, this scope demonstrated Method 2 to be an efficient
1
8
and user-friendly protocol, affording excellent
enrichment values for simple aryl sulfonamide derivatives using
the corresponding unlabeled compound as the precursor.
O isotopic
Table 1. Late-Stage 18O Labeling of Aryl Sulfonamides
Table 2. Late-Stage 18O Labeling of Complex Sulfonamides
Reaction conditions: (i) 3 (1 equiv), NHC precatalyst (3 mol %), K
2 3
CO (2.0
equiv), EtOH (1.0 mL per mmol of 5), and benzaldehyde (1.2 equiv) at 80 ºC for
18
18
1
2 h. (ii) Sulfinate (1 equiv), H
2
O (97 atom% O) (0.75 M), 4M HCl/dioxane (3
15
equiv) at 80 ºC for 90 min. (iii) Sulfinic acid (1 equiv), 6N NH
4
OH (10 equiv), I
2
(
1 equiv), at 23 ºC for 12 h. [a] Isolated yield based on 3. [b] % of the isolated
18
18
compound enriched with M+5. [c] H
2
O (99 atom% O) (0.75 M) was used.
Although a slight increase in overall M+5 isotopic enrichment was achieved, the
more economical 97 atom% 18O-water reagent was employed for the remainder
of this study. In all cases, MS peak corresponding to the unlabeled parent
molecule (M) was not observed in the final compound.
Next, we explored a variety of complex primary sulfonamides,
including several known to be pharmaceutically active (Table 2).
We found this degradation-reconstruction strategy to be tolerant
of molecular frameworks containing basic heterocycles, alkyl and
aryl ethers, amides, halides, tertiary sulfonamides, and free
amines. Marketed drugs such as sulthiame (5b), valdecoxib (5c),
and mefruside (5e) were all conveniently isotopically enriched to
deliver the SIL drugs in good to excellent isolated yields using this
straightforward labeling technique. Both pazopanib (5i) and
tamsulosin (5j), known renal cancer and benign prostatic
hyperplasia (BPH) therapeutics, respectively, were efficiently
enriched with 18O and 15N to afford 6i and 6j in moderate yields.
These results illustrate how this enrichment protocol can provide
a more practical alternative to the previously disclosed costly and
more involved eight-step synthetic strategies called upon for
preparing deuterium-labeled analogues of 5i-j.[16] Similarly,
development of celecoxib isotopologues were shown to require
multi-step synthetic routes which involve cyanation and reduction
2 3
Reaction conditions: (i) 5 (1 equiv), NHC precatalyst (3 mol %), K CO (2.0
equiv), EtOH (1.0 mL per mmol of 5), and benzaldehyde (1.2 equiv) at 80 ºC for
18
18
1
2 h. (ii) Sulfinate (1 equiv), H
2
O (97 atom% O) (0.75 M), 4M HCl/dioxane (3
15
4 2
equiv) at 80 ºC for 90 min. (iii) Sulfinic acid (1 equiv), 6N NH OH (10 equiv), I
(1 equiv), at 23 ºC for 12 h. [a] Isolated yield from 5. [b] % of the isolated
compound enriched with M+5. In all cases, MS peak corresponding to the
unlabeled parent molecule (M) was not observed in the final compound.
To further expand the scope of this late-stage 18O labeling
strategy, we adapted the degradation-reconstruction approach for
pharmacologically relevant methyl sulfone compounds (Table 3).
18
13CH
Our aim was to incorporate a –S O
2
3
labeled handle in order
of intermediates in order to achieve the desired M+4 enriched
to provide a higher mass and more metabolically stable tracer
than the –SO CD containing congeners. In order to access the
[13
C, H
2
]-labeled drug 5k.[17] Using our late-stage approach,
3
2
3
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Keywords: 18O • late-stage isotopic exchange • synthetic
desired sulfinate, we followed a sulfone demethylation strategy
disclosed by Gauthier and Yoshikawa in 2016[18]. After obtaining
the crude sulfinate intermediate from unlabeled 7a, we employed
methods • stable isotope labeling • sulfonamides • late-stage
functionalization
1
8
13
3
our O enrichment conditions, followed by treatment with CH I
in DMF, to obtain isotopically enriched etoricoxib 8a, a selective
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1
18
13
8
2
8%. We were also able to obtain [ O, C]MK-6482 (7b), a HIF-
α inhibitor, in 22% yield, with an overall 87% M+5 enrichment.
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making this degradation-reconstruction approach
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2
Reaction conditions for 8a: (i) 7a (1 equiv), BnBr (3.6 equiv), 1M t-BuOK in THF
(
H
4 equiv), THF (5 mL per 3 mmol of 7) 40 °C, for 1 h. (ii) Sulfinate (1 equiv),
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18
18
2
O (97 atom% O) (0.75 M), 4M HCl/dioxane (2 equiv) at 80 ºC for 90 min.
I (1.5 equiv), K CO (2 equiv), DMF (3 mL), at 23 ºC for 1 h. Reaction
(
iii) 13CH
3
2
3
conditions for 8b: (i) 7b (1 equiv), 2,5-difluorobenzyl bromide (2.0 equiv), 1M t-
BuOK in THF (6 equiv), THF (7 mL per 1 mmol of 7b) -75 °C to 23 °C. (ii)
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Strittmatter, D. G. Camp II, R. D. Smith, M. G. Pallavicini, Proteomics
18
18
Sulfinate (1 equiv), H
at 80 ºC for 60 min. (iii) CH
2
O (97 atom% O) (0.75 M), 4M HCl/dioxane (1.5 equiv)
3
3
I (3.0 equiv), DIEA (3 equiv), DMF (1 mL), at 23 ºC
for 3 h. [a] % of the isolated compound enriched with M+5. [b] Isolated yield
based on 7. In all cases, MS peak corresponding to the unlabeled parent
molecule (M) was not observed in the final compound.
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In conclusion, we have developed an 18O labeling strategy
involving degradation-reconstruction of complex molecules
containing primary sulfonamides or methyl sulfones. These
conditions were found to tolerate a broad range of functional
groups, and when supplemented with 15N or 13C, consistently
afforded M+5 enrichment for the desired sulfonyl containing
molecule, producing internal standards with no contamination
from the unlabeled compound. The greatest strength of this
strategy is the avoidance of lengthy de novo SIL synthesis in
favour of late-stage functionalization of pharmaceutical
compounds. Thus, we envision this methodology to be highly
useful for the labeled compound synthesis, drug metabolism, and
medicinal chemistry communities throughout the drug
development process.
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Experimental Section
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