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Aryloxy Triester Phosphoramidates as Phosphoserine Prodrugs: A Proof of Concept Study

ChemMedChem

March 2020
15(8)

DOI:10.1002/cmdc.202000034

Authors:

Binar Asrining Dhiani

Muhammadiyah University Purwokerto

Show all 7 authorsHide

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 phosphoserine 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 molecules. Ultimately, this will facilitate the discovery of phosphoserine‐containing small‐molecule therapeutics.

Synthesis and in vitro esterase‐mediated metabolism of phosphoserine aryloxy triester phosphoramidates. A. Synthesis of a phosphoserine aryloxy triester phosphoramidate (8). B. Mechanism of aryloxy triester phosphoramidates metabolism. C. ³¹P NMR in vitro enzymatic assay of the breakdown of the phosphoserine phosphoramidate by carboxypeptidase Y.

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A. Synthesis of the phosphoserine‐containing 14‐3‐3 dimerization inhibitor (13), diethyl phosphate (14) and its aryloxy triester phosphoramidates (15 a–c). The serine motif is shown in red; its phosphate and masked phosphate groups are shown in blue. B. Synthesis of the intermediate 2‐((4‐azidobenzoyl)oxy)acetic acid (18). C. Synthesis of phenyl L‐alanine ester phosphorochloridates (7 and 20 a–b).

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Cell viability of the unmasked phosphoserine compound (12), phosphoserine‐containing 14‐3‐3 dimerization inhibitor (13), its diethyl phosphate derivatives (14) and the aryloxy triester phosphoramidates (15 a–c). Cell viability was determined by standard MTT assay. The compounds were incubated with the A549 lung cancer cell line for 48 h (white bars) and 72 h (grey bars) at the indicated concentrations. The percentage of cell viability was calculated and is presented as a normalized value to the control DMSO. Error bars show standard error from triplicate experiments.

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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 PN 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

Wiley VCH Dienstag, 14.04.2020

2008 / 162173 [S. 671/674] 1

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Aryloxy Triester Phosphoramidates as Phosphoserine Prodrugs: A Proof of Concept Study

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