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Aryloxy Triester Phosphoramidates as Phosphoserine
Prodrugs: A Proof of Concept Study
Ageo Miccoli,[a] Binar A. Dhiani,[a] Peter J. Thornton,[b] Olivia A. Lambourne,[a] Edward James,[a]
Hachemi Kadri,[c] and Youcef Mehellou*[a]
The specific targeting of protein-protein interactions by
phosphoserine-containing small molecules has been scarce due
to the dephosphorylation of phosphoserine and its charged
nature at physiological pH, which hinder its uptake into cells. To
address these issues, we herein report the synthesis of
phosphoserine aryloxy triester phosphoramidates as phospho-
serine prodrugs that are enzymatically metabolized to release
phosphoserine. This phosphoserine-masking approach was
applied to a phosphoserine-containing inhibitor of 14-3-3
dimerization, and the generated prodrugs exhibited improved
pharmacological activity. Collectively, this provided a proof of
concept that the masking of phosphoserine with biocleavable
aryloxy triester phosphoramidate masking groups is a viable
intracellular delivery system for phosphoserine-containing mol-
ecules. Ultimately, this will facilitate the discovery of phospho-
serine-containing small-molecule therapeutics.
Protein phosphorylation at serine residues is a fundamental
phenomenon that is used by cells to affect the function,
localization and degradation of proteins.[1] In some cases,
phosphorylated serine residues mediate protein-protein inter-
actions via the docking of the phosphoserine residue into a
positively charged pocket within the partner protein. An
example of this is the serine phosphorylation of the adaptor
protein 14-3-3, which facilitates its homodimerization.[2] At-
tempts at inhibiting these phosphoserine-mediated protein-
protein interactions with small molecules have mostly led, by
design, to small molecules that lack phosphoserine. The move
to discard phosphoserine groups from these molecules was
driven by the fact that phosphoserine carries two negative
charges at physiological pH, which limit its (passive) cellular
uptake.[3] Additionally, phosphoserine is also subject to dephos-
phorylation by alkaline phosphatases, a process that yields
serine-containing derivatives that do not often retain potent
pharmacological activity compared to their parent phosphoser-
ine-containing molecules. Although the masking of the
phosphate group of phosphoserine was previously investigated,
the study was limited to a phosphoserine mimetic
(difluoromethylenephosphoserine) and not the natural phos-
phoserine moiety.[4] With this in mind and in order to improve
the drug-like properties of phosphoserine-containing small
molecules, we explored the application of the aryloxy triester
phosphoramidate technology to phosphoserine. This technol-
ogy has been widely used to mask the 5’-O-monophosphate
groups of nucleotides, and has so far led to two FDA-approved
drugs; sofosbuvir and tenefovir alafenamide.[5]
As a proof of concept, we initially synthesized phosphoser-
ine with the aryloxy triester phosphoramidate masking groups
(Figure 1 a). For this, the N- and C-terminals of phosphoserine
had to be protected[6] first to allow for the selective addition of
the aryloxy triester phosphoramidate moiety to the side chain
hydroxyl group. The synthesis was initiated by the chlorination
of tert-butanol (1) by copper(I) chloride with the peptide
coupling reagent N,N’-dicyclohexylcarbodiimide (DCC).[6] The
generated compound, 2, was then reacted with the commer-
cially available N-Boc L-serine, 3, in DCM to yield the N- and C-
protected L-serine 4.[6] This latter compound was subsequently
reacted with phenyl L-alanine methyl ester phosphorochlor-
idate (7), which had been synthesized according to reported
procedures[7] by reacting L-alanine methyl ester hydrochloride
(6) with the commercially available phenyl dichlorophosphate
(5) in DCM and in the presence of triethylamine (NEt3). This
reaction yielded the desired phosphoserine aryloxy triester
phosphoramidate, compound 8, as a white solid in a good yield
(62 %).
With the phosphoserine aryloxy triester phosphoramidate in
hand, we first studied whether the aryl and amino acid ester
groups that mask the phosphate group of phosphoserine could
be metabolized in vitro to release the unmasked phosphoserine
species. It is now well established that the metabolism of the
aryloxy triester phosphoramidate moieties is initiated by the
cleavage of the ester motif by carboxypeptidase Y to yield
metabolite A(Figure 1 b).[5a,8]
The formed carboxylate group then performs a nucleophilic
attack onto the phosphate group leading to the release of the
aryl group and the formation of a highly unstable five-
membered anhydride ring (metabolite B, Figure 1 b). This is
subsequently opened up by a water molecule to generate the
phosphoramidate metabolite C(Figure 1 b). Finally, the phos-
phoramidase-type enzyme Hint-1[9] cleaves the PN bond of
[a] Dr. A. Miccoli, Dr. B. A. Dhiani, O. A. Lambourne, Dr. E. James,
Dr. Y. Mehellou
School of Pharmacy and Pharmaceutical Sciences
Cardiff University
King Edward VII Avenue, Cardiff, CF10 3NB (UK)
E-mail: MehellouY1@cardiff.ac.uk
[b] Dr. P. J. Thornton
Technology Solutions GBU, Solvay Solutions
Oldbury, B69 4LN (UK)
[c] Dr. H. Kadri
Department of Chemistry, Durham University
South Road, Durham, DH1 3LE (UK)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000034
ChemMedChem
Communications
doi.org/10.1002/cmdc.202000034
671ChemMedChem 2020,15, 671– 674 © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley VCH Dienstag, 14.04.2020
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