Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate

Article abstract of DOI:10.1021/acs.jmedchem.9b00833

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac₃ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac₃ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

Full text of DOI:10.1021/acs.jmedchem.9b00833

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Article
Targeting GNE myopathy: A dual prodrug approach
for the delivery of N-acetyl mannosamine 6-phosphate.
Chiara Morozzi, Jana Sedláková, Michaela Serpi, Marialuce Avigliano, Rosangela Carbajo, Lucia
Sandoval, Yadira Valles-Ayoub, Patrick J Crutcher, Stephen Thomas, and Fabrizio Pertusati
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00833 • Publication Date (Web): 06 Aug 2019
Downloaded from pubs.acs.org on August 7, 2019
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Targeting GNE myopathy: A dual prodrug approach for the delivery of N -acetylmannosamine
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6-phosphate.
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Chiara Morozzi , Jana Sedláková , Michaela Serpi , Marialuce Avigliano , Rosangela
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Carbajo , Lucia Sandoval , Yadira Valles-Ayoub , Patrick Crutcher , Stephen Thomas ,
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Fabrizio Pertusati1*
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1School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, King Edward
VII Avenue, Cardiff CF10 3NB, U.K.
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2FirmaLab Bio-Diagnostics, 21053 Devonshire St, 106, Chatsworth, CA 91311, USA
3Cerecor Inc., 540 Gaither Rd, Suite 400, Rockville, MD 20850, USA
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Abstract
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ProTides comprise an important class of prodrugs currently marketed and developed as
antiviral and anticancer therapies. The ProTide technology employs a phosphate masking
groups capable of providing more favorable drug-like properties and an intracellular activation
mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein we describe
the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine
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(Ac ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic
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acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital
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disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of
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ManNAc-6-P. Synthetic methods were developed to provide a library of Ac ManNAc-6-
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phosphoramidates that were evaluated in a series of studies for their potential as a treatment
for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY)
enzymatic assay and favorable ADME properties, while also being more effective than
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ManNAc at increasing sialic acid levels in GNE-deficient cell lines than ManNAc. These
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results provide a potential platform to address substrate deficiencies in GNE myopathy and
other CDGs.
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Introduction
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Congenital Disorders of Glycosylation (CDGs), first described as Carbohydrate Deficient
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Glycoprotein Syndrome by Jaeken et al. in 1980 , are a group of rare, inherited, metabolic
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diseases characterized by defects in protein or lipid glycosylation. CDGs primarily affect
children and can vary in clinical presentation from disabling to life-threatening conditions.
Disease manifestations typically impact multiple organ systems, including liver and
neurological involvement. Over 100 unique CDGs have been identified to date and the number
continues to increase due to advances in the availability and efficiency of diagnostic
techniques, namely genome sequencing.2, 3
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Among the CDGs, Hereditary Inclusion Body Myopathy (HIBM), also known as GNE
myopathy (GNEM), is an autosomal recessive neuromuscular disorder characterized by
symptoms that typically appear during the third decade of life, starting with skeletal muscle
atrophy and weakness that slowly progresses to a significant physical disability. GNEM is
caused by biallelic mutations in the GNE gene resulting in loss-of-function of uridine 5’-
diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase and N-acetylmannosamine
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(ManNAc) kinase (GNE), a key enzyme in sialic acid biosynthesis (Figure 1). Sialic acids
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decorate cell surfaces as terminal component of glycoprotein and glycolipids, where they
mediate or modulate a variety of physiological and pathological processes like signal
transduction, cell adhesion, system embryogenesis, cancer metastasis or viral and bacterial
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infections. GNE is a bifunctional enzyme capable of catalyzing epimerization of UDP-
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GlcNAc at the 2-position of the hexose ring to afford ManNAc and subsequent phosphorylation
at the 6-position to afford ManNAc-6P (Figure 1). As the rate-limiting step in sialic acid
biosynthesis, GNE deficiency results in decreased sialic acid production and consequent
hyposialylation of glycoproteins, particularly in muscle tissue. The prevalence of GNEM is
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estimated to be roughly 1 to 9 in 1,000,000 people. More than 140 mutations in GNE have been
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associated with GNE myopathy and can compromise the enzymatic activity of either catalytic
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domain. Each domain is functional when expressed separately, but evolution of some
intramolecular dependency is exemplified by mutations causing reductions in both enzyme
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activities to different extents in a manner independent of domain localization.9
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Figure 1. Sialic acid biosynthetic cascade: the point of mutation of UDP-GlcNAc 2-
epimerase/ManNAc kinase, responsible for the GNE myopathy, is highlighted in yellow.
To date, there is no approved therapy for GNEM.10 Palliative treatments, such as
physiotherapy, analgesic drugs, mobility devices and psychological support are available to
alleviate patient symptoms and improve quality of life. Therapeutic approaches have focused
on substrate replacement paradigms seeking to re-establish available pools of intracellular
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sialic acid by supplementation of aceneuramic acid (Neu5Ac) or its precursor, ManNAc. Both
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compounds have proven capable of restoring sialic acid biosynthesis and preventing or
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arresting disease progression in mouse models of GNEM. ManNAc is currently in late-stage
clinical trials to assess its safety and efficacy in patients with GNE myopathy,13, 14 whereas
aceneuramic acid, although safe and well-tolerated, failed to demonstrate efficacy in a Phase 3
double-blind, placebo-controlled trial and was subsequently discontinued.15 Intravenous
administration of immunoglobulin (IVIG) and gene therapy are other therapeutic options.
Unfortunately, IVIG, although containing eight micromoles of sialic acid per gram, was not
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capable of improving muscle sialylation in GNE myopathy patients. Correction of GNE
function through liposomal delivery of a wild-type GNE gene demonstrated increased
production of sialic acid in vitro and in vivo. However, data supporting the continued
stabilization of decline in muscle strength in a single patient are limited and no follow-up
studies have been reported thus far.16
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Although ManNAc presents an intriguing therapeutic approach, poor intracellular transport and
feedback inhibition necessitates high daily dosage (from 3g to 12g).13,17 No dose-response
correlation was observed after repeated administration in patients with GNEM. Furthermore,
patients with loss-of-function mutations in the ManNAc kinase domain of GNE may not be
capable of phosphorylating ManNAc, thereby limiting restoration of sialic acid production.
A study conducted in a GNE myopathy mouse model demonstrated that the lipophilic ManNAc
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analog O-tetra-acetylated N-acetylmannosamine (Ac ManNAc) was more effective at
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increasing sialylation and preventing the myopathic phenotype when compared to natural sialic
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acid metabolites after oral administration. Ac ManNAc is a prodrug of ManNAc and can
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more readily cross cellular membranes via passive diffusion, providing evidence that synthetic
sugar analogs may be a practical consideration for the design of therapeutic agents for GNE
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myopathy. However, Ac ManNAc still requires phosphorylation upon release of ManNAc. To
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overcome the deficiency in both UDP-GlcNAc-2-epimerase and ManNAc kinase enzyme
activity observed in GNE myopathy, administration of ManAc-6-P could be envisaged as an
ideal substrate replacement strategy. Due to the high polarity of the phosphate moiety, a
prodrug approach would be required to overcome poor cell permeability and mitigate de-
phosphorylation by extracellular phosphatases.
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Prodrug approaches for delivering phosphate carbohydrate analogs have been reported
previously. Meyer et al designed cyclosaligenyl prodrugs of O-tetra-acetylated mannose-1-
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phosphate whereas Eklund with co-workers synthesized two mannose-1-acetoxymethylated
phosphates.20 Both classes of phosphate prodrugs were designed for the treatment of
phosphomannomutase 2 (PMM2) deficiency, characteristic of the PMM2-CDG condition also
known as CDG-Ia. Despite encouraging results demonstrating intracellular delivery of the
mannose-1-monophosphate capable of correcting the CDG-Ia phenotype in vitro, these
approaches were not explored further, most likely due to toxicity and stability concerns. To
date, no additional studies have been reported.
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Our laboratory was previously successful in applying phosphoramidate chemistry to N-acetyl
glucosamine for the treatment of osteoarthritis21, 22 and to various analogs of deoxyribose-1-
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phosphate as potential anti-HIV agents. The N-acetylglucosamine phosphoramidate prodrugs
generated in these studies showed acceptable biological stability and prodrug activation in
chondrocyte cell lysate. In particular, the (L)-proline amino acid-containing O-3/O-4
phosphoramidates demonstrated good activity at non-cytotoxic concentrations in human
cartilage ex vivo, exceeding that of N-acetylglucosamine and emerging as promising candidate
for further development for the treatment of osteoarthritis and other musculoskeletal diseases.
Among many existing phosphate prodrug strategies,24 the ProTide approach has been
successfully applied to nucleosides25, 26 as evidenced by the development and approval of the
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currently marketed drugs Sofosbuvir and Tenofovir Alafenamide (TAF) (Figure 2). In
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addition, in collaboration with our laboratories, NuCana plc is currently pioneering the ProTide
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technology in oncology, with the clinical candidates NUC-1031 (Acelarin), and NUC-3373,
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respectively in Phase III and Phase I studies for patients with advanced solid tumors (Figure
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nucleoside derivatives.31
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Figure 2. FDA-approved antiviral ProTides, Sofosbuvir and TAF and anticancer clinical
candidates ProTides, NUC-1031 and NUC-3373.
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Given the importance of phosphoramidate prodrug technology in recent drug discovery, we
revived our interest in its application toward the delivery of ManNAc-6-P as a novel approach
to the treatment of GNE myopathy. Rationally designed ManNAc phosphoramidate prodrugs
could enable the bypass of deficient GNE enzyme observed in GNEM patients by directly
providing a source of intracellular ManNAc-6-P. Our approach sought to optimize drug-like
properties of ManNAc phosphoramidates to improve upon the low permeability and plasma
stability of ManNAc-6-P. In this work, we report the design, synthesis and evaluation of a
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family of ManNAc-6-P and Ac ManNAc-6-P ProTide analogues, including ADME assays,
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enzymatic activation studies and in vitro assessment of sialic acid production in cell-based
models of GNE myopathy. These data serve as proof-of-concept that the ProTide platform is a
practical approach to substrate replacement therapies for GNE myopathy and potentially for
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other CDGs characterized by deficiencies in phosphorylated metabolites.
RESULTS AND DISCUSSION
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Chemistry
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Our synthetic efforts began with the synthesis of ManNAc-6-phosphoramidates. We initially
envisaged the application of a synthetic procedure previously reported by our group for the
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preparation of glucosamine phosphoroamidates as feasible approach. Surprisingly, when
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ManNAc (1) was coupled with different phosphorochloridates in the presence of N-methyl
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imidazole (NMI), the formation of a complex mixture of by-products was always observed,
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from which no desired compound could be isolated.
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We then chose to explore an alternative synthetic route, involving protection of the anomeric
hydroxyl prior to attempting the coupling reaction. The benzyl group was selected as the
protecting group given its removal via hydrogenation is highly compatible with the
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phosphoramidate motif, as evidenced by a similar procedure employed by our group in the
synthesis of N-acetylglucosamine phosphoramidate derivatives.22
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ManNAc (1) was fully acetylated (acetic anhydride in pyridine), and selectively benzylated at
the anomeric position with benzyl alcohol and a catalytic amount of BF OEt . Subsequent O-
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deprotection of the acetylated alcohols gave compound 2 as exclusively the -anomer in 87%
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yield over three steps (Scheme 1). Pleasingly, the coupling of phosphorochloridates (3a, 4a-
c) with compound 2 was efficient, affording exclusively O-6 phosphoramidate derivatives 5a
and 6a-c (as a mixture of S and R diastereoisomers) with the same O-6 regioselectivity as
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previously observed for the N-acetyl glucosamine series. Finally, hydrogenation (10% Pd/C
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and H ) of compound 5a in methanol proceeded smoothly and after 12 hours both MS and H-
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NMR analysis of the crude reaction mixture revealed the presence of the desired product with
concomitant disappearance of the starting material. Disappointingly, attempts to isolate pure
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compound 7a via traditional purification techniques (silica gel chromatography and/or reverse
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phase HPLC) were unsuccessful due to partial degradation of the compound. When repeating
the synthesis with phosphorochloridates bearing different promoieties, purification following
hydrogenation consistently led to the observation of partial degradation and hindered isolation
of the desired product. Surprised by these results, we decided to further investigate the
degradation pathway via mass spectrometry. LC-MS analysis of 7a following purification
indicated the presence of the desired compound (m/z 490) and a side product showing a peak
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at m/z 419 [M+Na] consistent with structure IV (Figure 3). Lack of UV absorbance in the LC-
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MS traces for this peak and evidence of phenol release observed in the H-NMR spectra of the
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purified material further supported this hypothesis. LC-MS analysis of other
phosphoramidates, whose purification was attempted after hydrogenation, led to similar results
that indicated the formation of compound IV-like structures (m/z values were in agreement
10
1
1
2
3
1
1
with the amino acid ester present in the parent compound). In addition, P-NMR of these
mixtures showed two peaks at ~9 ppm, indicative of a chiral phosphorodiamidate as in
compound IV (Figure 3).
13
1
1
4
5
R1
HN
O
OR2
Cl P NH OR2
O
O
O
P
R1
O
O
HO
HO
NHAc
O
O
NHAc
O
NHAc
O
R
R
i, ii, iii
87%
3a, 4a-c
HO
HO
HO
HO
HO
HO
iv
OH
5a, 6a-c
1
2
20-45%
OBn
v
OBn
v
R1
HN
R1
R2
R1
OR2
R
OR2
3a,5a,7a
H
CH3
iPr
HN
O
O
O
O
P
P
4
4
4
a,6a,9a OCH3 CH CH(CH )
Et
O
2
3 2
O
O
NHAc
O
O
NHAc
O
MeO
b,6b,9b OCH3
c,6c,9c OCH3
(CH ) Pro
CH3
HO
2 3
HO
HO
HO
CH3
iPr
OH
OH
8a-c
29-42%)
7a
(
not isolated
1
6
7
a
1
Scheme 1. Phosphoramidate synthesis through 1’-O-Bn-N-acetylmannosamine. Reagents and
18
conditions: (i) Ac O, DMAP, Py, rt, 12h; (ii) BnOH, BF OEt cat., ACN, 80 °C, 3h; (iii)
2
3
2
1
9
NaOMe cat. MeOH, rt, 5h; (iv) NMI, THF/pyridine 4/2 v/v, rt, 12h; (v) H , 10% Pd/C, MeOH,
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
rt, 12h.
A plausible mechanism for the formation of by-product IV is proposed in Figure 3.
Deprotection of the benzyl group at the anomeric position leads to the formation of both - (I)
and -anomers (III) in equilibrium with an open-chain form (II). Since the 1-O-benzyl
phosphoramidates were formed exclusively as  anomers and proved to be stable, we then
hypothesized that only the  anomer of II could participate in the degradation pathway. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
6
7
8
9
0
phosphorus atom of the  anomer could be particularly susceptible to intramolecular
nucleophilic attack by the nitrogen atom of the sugar, leading to the corresponding cyclic
adduct IV. Interestingly, this side reaction was not previously observed with any of the N-
2
2
1
acetyl-glucosamine phosphoroamidate derivatives. The difference in stereochemistry at the
2’-position between GlcNAc and ManNAc phosphoramidates may be responsible for the
divergence in reactivity toward the intramolecular cyclization process, further supporting the
proposed mechanism. This could also justify the difficulty in obtaining the ManNAc
phosphoramidate series from O-unprotected ManNAc in our earlier attempts. Although it
would appear reasonable to assume that the phosphorous atom of the more flexible open chain
structure II could undergo the same intramolecular nucleophilic attack by the nitrogen atom,
we have ruled out this possibility given such degradation was not observed with N-acetyl
11
1
1
2
3
14
1
1
5
6
17
22
1
8
glucosamine derivatives. Further studies to confirm this proposed mechanism are in progress.
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1
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2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
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0
1
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4
5
6
7
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9
0
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3
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8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Figure 3. Proposed mechanism for the formation of the side product IV during the purification
step.
3
4
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7
8
9
0
1
2
3
4
5
Efforts to block the attack of the nucleophilic nitrogen atom onto the phosphorus center by
introducing hindered amino acid esters in conjunction with bulkier aryloxy moieties failed and
degradation was still observed. We then sought to stabilize the phosphoroamidate moiety by
introducing a para-methoxy functionality within the aryl group, thereby decreasing the
electrophilicity of the phosphorous atom and making it less susceptible to nucleophilic attack.
This resulted in a decreased rate of by-product formation and enabled isolation of the desired
products (8a-c) (Scheme 1). However, these compounds were not pursued further as possible
candidates due to unsatisfactory ADME properties, namely poor cell permeability due to the
presence of several free hydroxyl groups.
1
1
1
1
1
1
Given these results, we turned our attention to the preparation of 1,3,4-O-triacetylated
ManNAc-6-phosphoramidates, which we envisaged as targets with more appropriate drug-like
properties. The acetyl protecting groups would further increase the lipophilicity of the
compounds while allowing for the possibility of in vivo cleavage by plasma esterases following
absorption from the gut, thus acting as a dual prodrug. Additionally, 1-O-acetylation could
16
1
1
7
8
stabilize the prodrug structure and prevent the cyclization side-reaction. 1,3,4-O-triacetylated
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
ManNAc (11) was prepared according to a literature procedure34 outlined in Scheme 2.
ManNAc (1) was tritylated, peracetylated and selectively deprotected at the O-6 position to
yield compound 11 as a mixture of  and  anomers (1:0.3), which was then coupled with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
32
phosphorochloridates 3a-j in the presence of Grignard reagent to afford compounds 12a-j as
a mixture of S and R diastereoisomers of  and  anomers. To avoid the formation of
p
p
regioisomeric phosphoramidates arising from acetyl migration,35, 36 low reaction temperature
was required during phosphoramidate formation.
8
9
Scheme 2. Synthetic strategy for the preparation of 1,3,4-O-triacetylated ManNAc
phosphoramidates. Reagents and conditions: (i) Pyridine, rt, 12h and 60 °C, 1h; (ii) Pyridine,
0 °C to rt, 12h; (iii) glacial acetic acid, 60 °C, 2h; (iv) 1M t-BuMgCl, THF, 0 °C to rt, 12h.
1
1
0
1
12
A small, rationally designed library of Ac ManNAc phosphoramidates was built based on
3
1
1
3
4
predicted log P values and existing knowledge of prodrug activation kinetics, using different
amino acids and ester moieties to sample a diverse set of analogs (Table 1). Furthermore, since
2
2
15
our previous report demonstrated that proline-based ProTides of N-acetyl glucosamine were
1
1
6
7
particularly effective in cell-based models of osteoarthritis, we prepared the proline derivative
12c.
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9
Yieldb
Compound
ID
Ar
R
R1
R2
log Pa
(%)
60
16
19
49
45
1
1
1
1
1
1
1
1
1
1
2a
2b
2c
2d
2e
2f
Ph
CH3
iPr
Et
H
H
H
H
H
H
H
H
1.32
1.94
0.92
1.30
2.28
2.95
2.64
1.95
2.38
3.04
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ph
CH CH(CH )
2
3 2
Ph
(CH ) Pro
Met
Met
Et
2 3
Naph
Ph
H
CH Ph
2
Naph CH3
Bn
Bn
Et
47
2g
2h
2i
Ph
Ph
Ph
Ph
CH(CH3)2
39
CH CH CHCH
34
3
2
3
CH3
Bn
Bn
CH3
64
2j
CH CH(CH )
H
61
2
3 2
a
1
2
3
4
5
6
7
8
Table 2. Library of Ac ManNAc phosphoramidate prodrugs. Predicted log P calculated using
3
SwissADME webservice.37 b Yield of the isolated compounds.
Carboxypeptidase Activation Studies
The ProTide technology has proven to be a valuable strategy capable of delivering membrane-
permeable nucleotide analogs intracellularly. Once across the cell membrane via passive
diffusion, ProTides have been shown to undergo a series of enzymatic reactions leading to the
release of the corresponding monophosphate analog. ManNAc phosphoramidates are believed
to follow a similar activation sequence as depicted in Figure 4.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Figure 4. Proposed enzymatic activation mechanism for Ac ManNAc phosphoramidates: a)
3
3
4
5
6
7
8
9
0
1
carboxylesterase or carboxypeptidase catalyzed ester hydrolysis to metabolite A.
Intramolecular nucleophilic displacement of the aryloxy group provide the cyclic anhydride
intermediate B. c) water-mediated hydrolysis of B to phosphoromonoamidate C. d) P-N bond
cleavage mediated by HINT enzyme or by spontaneous hydrolysis.
To assess whether
a
carboxypeptidase-mediated activation mechanism to
phosphoromonoamidate C is also operative with ManNAc phosphoramidates, compounds 12a-
i (Scheme 2 and Table 2) were subjected to enzymatic studies by incubating each compound
with carboxypeptidase Y (CPY, from Baker’s yeast) in 0.05 M Trizma buffer (pH = 7.6) in
1
1
3
1
deuterated acetone, which allowed monitoring of reaction progress by P-NMR and LC-MS
analyses. Almost all compounds showed activation of the promoiety with less than 50% of the
parent phosphoramidates remaining 15 minutes following the addition of CPY. The only
12
1
1
3
4
3
1
exception was 12c, which showed no activation (up to 24 hours). Figure 5 displays the P-
NMR spectra for the enzymatic activation assays of compounds 12b and 12c. Although the
exact reaction sequence cannot be established from these data alone (and, consequently, the
prodrug half-life), we can assert that compound 12b demonstrates rapid conversion into a
15
1
1
1
6
7
8
species showing two signals at 6.82 and 7.02 ppm. On the other hand, replacement of leucine
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with proline (12b to 12c) completely abolishes prodrug activation under identical incubation
1
2
conditions, as the minor change from ethyl to methyl ester moiety at R is unlikely to cause
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
such a stark contrast in susceptibility to carboxypeptidase-mediated activation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
3
1
Figure 5. Deconvoluted P-NMR spectra of phosphoroamidates 12b (A) and 12c (B) over
6
7
8
9
0
1
time after enzymatic incubation with Carboxypeptidase Y. Conditions: 202MHz, [D ]acetone,
6
0.05M Trizma buffer, pH=7.6.
Crude enzymatic reaction mixture of 12b was further analyzed by LC-MS confirming the
presence of intermediate C (see Figure 31 in Supporting Information). To independently
3
1
1
1
confirm the real structure of intermediate C (two P-NMR peaks at 6.82 and 7.02 ppm), a
sample of this compound was prepared synthetically. Compound 12j was debenzylated via
catalytic hydrogenation to the corresponding acid, which formed the expected intermediate C
as a triethyl ammonium salt (13) (Scheme 3) after treatment with 0.1 M triethylammonium
12
1
1
3
4
3
1
bicarbonate buffer. Comparison of LC-MS (tR = 2.5, 3.7 min) and P-NMR (7.02, 6.97 ppm)
data for compound 13 and those obtained from incubation of compound 12b confirmed the
effective activation of 12b into the corresponding phosphoromonoamidate C. This result
demonstrates for the first time that carbohydrate phosphoramidates undergo a similar activation
15
1
1
1
6
7
8
pathway observed for nucleoside phosphoramidates. In addition, these studies suggest that
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
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5
5
5
5
5
5
6
1
2
3
4
even non-nucleoside phosphoramidates are metabolized intracellularly and can serve as a
substrate for monophosphate formation via enzymatic (i.e. HINT1) or spontaneous activation,
but the selection of the appropriate amino acid side chain is critical towards enabling the
activation mechanism.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
Scheme 3. Synthesis of compound 13. Reagents and conditions: (i) H , 10% Pd/C,
2
7
8
EtOH/EtOAc, 2h at rt; (ii) 0.1 M triethylammonium bicarbonate, 5 min at rt.
ADME Evaluation
9
Having established that these compounds can readily undergo enzymatic activation, we
10
decided to evaluate several Ac ManNAc phosphoramidates for ADME properties to select lead
3
1
1
1
2
candidates for cell-based models of GNE myopathy.
As previously mentioned, substrate replacement for GNE myopathy with highly polar small
molecules were hampered by poor passive diffusion across cellular membranes. While the
phosphoramidate promoiety increases cell permeability due to increased lipophilicity, this
effect must be balanced with adequate aqueous solubility to ensure proper compound
dissolution and absorption. Therefore, compounds 12b-e were screened for Caco-2 cell
permeability, kinetic solubility, human plasma stability and metabolic stability as key
parameters that would increase the likelihood of success in cell-based assays. The results of
these studies are displayed in Table 3.
13
1
1
1
4
5
6
17
1
1
8
9
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Compoun Papp (B-A/A- Kinetic
Human
Plasma HLM
(T ,
d
B)
Solubility
(μM, pH 7.4)
125
Stability
1
/2
-6
ID
(10 cm/s)
0.8 / 1.5*
0.1 / 0.6*
0.3 / 0.6*
0.3 / 0.4*
(% remaining at 2 h)
min)
1.3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2b
2c
20
152
15
1.4
12d
2e
129
8.3
9.8
1.2
1
124
0.8
1
2
3
4
5
6
7
8
9
0
1
Table 3. ManNAc phosphoroamidates ADME properties. *with pgp/bcrp inhibitor GF120918
(10 µM). Low permeability control was nadolol (Papp = 0.09) and high permeability control
was metoprolol (Papp = 13.6). Efflux control was digoxin, and efflux ratio reduced from 374 to
1.69 in the presence of GF120918.
Compound 12b showed improved permeability based on the Caco-2 assay relative to all other
compounds. Compound 12c had comparable permeability to 12d and 12e but showed increased
plasma stability. Pleasingly, each compound showed acceptable solubility in aqueous
environment at physiological pH. Hydrolysis of ester side chains via plasma esterases
unsurprisingly causes rapid degradation of the parent compound, and the same phenomenon
holds true for metabolic stability when incubated with human liver microsomes (HLMs) as
evidenced by short half-lives of each compound. Aside from displaying acceptable ADME
properties, 12c did not demonstrate activation in the aforementioned carboxypeptidase assay,
potentially providing a negative control for assessing prodrug efficacy in cell-based assays
models of GNE myopathy. Based on the results of the ADME screening, as well as the
enzymatic activation data, compounds 12b and 12c were selected for further testing.
Sialic Acid Quantification in Cell-Based Models of GNE Myopathy
1
1
12
1
1
3
4
15
1
1
6
7
The evaluation of prodrugs (12b and 12c) to rescue sialic acid deficiencies in vitro was
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1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
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4
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5
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1
2
conducted using two different cell lines: Lec3 CHO cells and GNEM patient-derived
myoblasts. Both cell types present with reduced sialic acid levels due to mutations in GNE that
mimic the hyposialylation defects observed in patients with GNE myopathy. In particular, Lec3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38
4
mutant CHO cells lack any detectable GNE activity , whereas the patient-derived myoblasts
9
5
contained a D378Y mutation that cause an 80 % reduction in GNE activity. Compounds 12b
6
and 12c were initially assessed for cytotoxicity in GNE mutant cell lines relative to ManNAc
using Trypan Blue staining as an assay for cell viability after 48 hours incubation. At 0.1 mM,
12b and 12c induced no observable difference in cell viability relative to controls and at 1 mM,
cell viability was comparable to 1 mM ManNAc (see Supporting Info, Figure 32).
Sialic acid quantification was then carried out in duplicate at two concentrations to assess dose-
response in GNE-deficient cells after 48-hour incubation with 0.1 mM and 1 mM 12b or 12c,
1 and 100 mM for ManNAc, and a negative control with media only. We focused specifically
on assessing total and free sialic acid production in membrane protein fractions, as sialic acid
availability in cell membranes plays a key role in promoting muscle growth, structure and
integrity, a critical issue for GNE myopathy patients. Although clinical studies with therapies
like aceneuramic acid and ManNAc supplementation have shown increases in plasma sialic
acid levels, it is not clear that these data have correlated with increases in sialic acid levels
within tissues.
7
8
9
10
1
1
2
1
13
1
1
1
1
1
1
4
5
6
7
8
9
As shown in Figure 6, 12b induced dose-dependent increases in total and free sialic acid levels
in membrane protein fractions in both Lec3 CHO cells and GNEM patient-derived myoblasts.
Given the lack of activation observed with 12c in the carboxypeptidase assay, the compound
unsurprisingly produced no effects on sialic acid levels in Lec3 CHO cells. The effects of 12b
were more pronounced than ManNAc, producing >30-fold and >50-fold more sialic acid in
Lec3 CHO cell and GNEM patient-derived myoblasts, respectively, at 1 mM.
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Figure 6. Sialic acid levels in membrane protein fraction. Sialic acid levels were determined
using Sialic Acid Assay kit on isolated membrane protein fractions from Lec3 CHO cells (A)
and GNEM patient-derived fibroblasts (B) incubated with 12b, 12c, ManNAc for 48 hours and
normalized to control (media only) (n = 2, +/- SEM).
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CONCLUSION
A substrate replacement paradigm was applied to the design of novel ProTide prodrugs of
ManNAc-6-P as a therapeutic approach for GNE myopathy. A focused library of ManNAc-6-
P phosphoramidates was synthesized and evaluated for ADME properties to select lead
compounds for profiling in cell-based models of GNE myopathy. Two leads, 12b and 12c,
displayed the most promising ADME properties, although 12c did not show activation in a
carboxypeptidase assay. The demonstration and independent confirmation of
carboxypeptidase-mediated activation of 12b serves as the first example of a non-nucleoside
phosphoramidate activation mechanism. The contrast in prodrug activation between 12b and
12c further emphasizes the importance of selecting the appropriate amino acid side chain for
the ProTide moiety. Compounds 12b and 12c were evaluated in two separate GNE-deficient
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cell lines for their ability to increase sialic acid production in membrane protein fractions
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relative to ManNAc. Compound 12b produced >30-fold and >50-fold more sialic acid than
ManNAc in Lec3 CHO cell and GNEM patient-derived myoblasts, respectively, at 1 mM. The
extension of the ProTide platform to monosaccharide prodrugs presents an opportunity to
explore therapeutic options for diseases due to the deficiency of sugar metabolites, such as the
CDGs.
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Experimental Section
General Experimental Information
All chemicals were purchased from Sigma-Aldrich and Carbosynth and used without any
1
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13
further purification. All H-NMR (500MHz), P-NMR (202MHz) and C-NMR (125MHz)
spectra were recorded on a Bruker AVANCE III HD 500 MHz spectrometer at 25 C. The
deuterated solvent used were CDCl and CD OD and the spectra were calibrated using their
1
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12
residual peaks: CDCl , H: =7.26 ppm; C: =77.36 ppm and CD OD, H: =3.34 ppm; C:
3
3
1
1
3
4
=49.86 ppm. Chemical shifts () are reported in parts per million (ppm) and coupling
constants (J) in Hertz. The following abbreviations are used in the assignment of NMR signals:
s (singlet), d (doublet), t (triplet), q (quartet), spt (septet), m (multiplet), br (broad), dd (doublet
15
1
13
1
1
6
7
of doublets) and ddd (doublet of doublet of doublets). The signals in the H and C NMR
spectra were assigned based on analysis of coupling constants and additional two-dimensional
experiments (COSY and HSQC). Some peaks in all NMR analyses may be overlapped.
Reactions followed by thin-layer chromatography (precoated aluminium-backed plates, 60
F254, 0.2 mm thickness, Merck, were visualized under short-wave ultraviolet light (254 nm)
and/or via Pancaldi’s solution (molybdate ammonium cerium sulfate solution).
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Chromatography purifications were performed through ISOLERATM 3.0 BIOTAGE OS 852M
both with the sample being loaded as a concentrated solution in the same eluent and pre-
adsorbed on top of silica. Fractions containing the product were identified by TLC, combined,
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and the solvent was removed in vacuo. HPLC analysis was carried out to confirm the purity of
final compounds by using a Varian Pursuit XRs 5 C18 150 mm  4.6 mm analytical column.
All compounds tested had a purity of >95% as determined by HPLC at two different
wavelengths (220 and 254 nm).
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Low-resolution mass and LC-MS analyses were performed on a Bruker Daltonics MicroTof-
LC instrument (atmospheric pressure ionization, electrospray mass spectroscopy) in either the
positive- or negative-ion mode. MS parameters were optimized to the following: set capillary
4500 V, set end plate offset -500 V, set nebulizer 1.0 Bar, set dry heater 200 ºC, set dry gas 8.8
l/min, set divert valve source. Full scan mass spectra were recorded over a range of m/z 50-
3000.
1
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1
Standard Procedures. For practical purposes, standard procedures are given. Any variations
from these procedures are discussed individually and described in full.
Synthesis of ()-(2R,3S,4R,5S)-5-Acetamido-2-(acetoxymethyl)-6-(benzyloxy)tetrahydro-
2H-pyran-3,4-diyl diacetate (2): A solution of N-acetyl-D-mannosamine hydrochloride
(45.21 mmol, 10.0 g) and DMAP (4.52 mmol, 0.55 g) in pyridine (200 mL) was reacted with
acetic anhydride (43 mL) and stirred at rt for 12 h. The reaction mixture was diluted with DCM,
washed with 1M HCl, NaHCO and brine. The residue was concentrated in vacuo and purified
3
by column chromatography eluting with DCM/CH OH (2:10) to afford tetra-acetylated-D-
3
19
mannosamine as a yellowish oil (74 %) which was then dissolved in ACN (40 mL) followed
.
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the addition of benzyl alcohol (57.8 mmol, 6 mL) and BF Et O (1.83 mmol, 230 L). The
3
2
21
resulting mixture was heated at 80 C and stirred for 12 h. Afterwards, the mixture was cooled
2
2
2
2
3
4
down to rt, diluted with DCM (400 mL) and washed with NaHCO . The expected product was
3
obtained after column chromatography purification eluted with Hexane/EtOAc (1:4) in 75 %
yield. Final deacetylation was accomplished via treating the latter compound (2.29 mmol, 1.00
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g) dissolved in anhydrous MeOH (50 mL) with a solution of NaOMe (0.23 mmol, 0.012 g) in
300 L of dry CH OH under argon atmosphere. The resulting solution was stirred at rt for 5 h
3
and then Amberlite IR-120 hydrogen form was added leaving the mixture under stirring for
another 1 h. The mixture was then filtered through celite and the solvent was removed under
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reduced pressure. The resulting solid was triturated with Et O and further purified by column
2
chromatography on silica using EtOAc/MeOH (4:1) to obtain the final product (2) in 87 % as
1
two anomers  and  anomers (0.62 g). White solid. H-NMR (500 MHz, CDCl ): δ 7.35-
3
H
7.28 (m, 5H, Ph), 4.81 (s, 1H, H-1), 4.64 (d, 1H, J = 11.7 Hz, CH Ph), 4.47-4.44 (m, 2H,
2a
CH Ph and H-2), 4.11-4.09 (m, 1H, H-3), 3.95 (d, 1H, J = 10.8 Hz, H-4), 3.79-3.730 (m, 2H,
2
b
1
1
H-5, H-6), 3.61 (d, 1H, J = 9.4 Hz, H-6), 2.00 (s, 3H, OCOCH ) ppm. MS (ES+): m/z 334.15
3
[M+Na]+
12
Standard Procedure 1. General Synthesis of Phosphorochloridate (3a-i/4a-c): A solution
of aryl dichlorophosphate (1 equiv) and an appropriate amino acid ester (1 equiv) in anhydrous
1
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4
DCM under argon atmosphere was cooled down to -78 C. Afterwards, Et N (2 equiv) was
3
15
added dropwise and the resulting mixture was stirred for 15 minutes at -78 C. Then, the
reaction mixture was allowed to reach room temperature and left under stirring for 1 hour. The
solvent was removed under reduced pressure, the solid residue was redissolved in diethyl ether
and the resulting mixture was stirred under argon atmosphere for 20 min. The solid was filtered
off and the solvent was evaporated giving the pure compound as a colorless oil which was used
in the next step without further purification.
1
1
6
7
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Isopropyl (chloro(phenoxy)phosphoryl)-L-alaninate (3a): Colorless oil; 99 %, 8.36 g. P-
2
2
2
2
3
4
NMR (202 MHz, CDCl , mixture of R and S diastereoisomers, a:b): δ 8.09 (1P), 7.71 (0.9)
3
P
P
P
1
ppm. H-NMR (500 MHz, CDCl ): δ 7.39-7.36 (m, 2H, CH-Ph), 7.28-7.22 (m, 3H, CH-Ph),
3
H
5.10-5.05 (m, 1H, CH(CH ) ), 4.16-4.08 (m, 1H, NHCHCO), 1.50 (dd, 3H, J = 2.4, 7.1 Hz,
3 2
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NHCHCH ), 1.30-1.26 (m, 6H, CH(CH ) ) ppm.
3 3 2
2
Ethyl (chloro(phenoxy)phosphoryl)-L-leucinate (3b): Prepared according to Standard
procedure 1 in 98 %, yield as a mixture of two diastereoisomers (SS and SR ). Colorless oil;
8
9
1
1
1
1
1
1
1
1
1
1
2
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4
P
P
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4.20 g. P-NMR (202 MHz, CDCl ): δ 8.51 (1P), 8.27 (1P) ppm. H-NMR (500 MHz,
3
P
5
CDCl ): δ 7.38-7.34 (m, 2H, CH-Ph), 7.27-7.21 (m, 3H, CH-Ph), 4.34-4.19 (m, 3H, NH,
3 H
6
OCH CH ), 4.14-4.02 (m, 1H, NHCHCO), 1.86 (spt, 0.5H, J = 6.6 Hz, CH (CH ) ), 1.79 (spt,
2 3 a 3 2
7
0.5H, J = 6.6 Hz, CH (CH ) ), 1.64-1.59 (m, 2H, CHCH CH), 1.30-1.26 (m, 3 H, OCH CH ),
b 3 2 2 2 3
8
0.96-0.93 (m, 6H, CH CHCH ) ppm.
3 3
9
Methyl (chloro(phenoxy)phosphoryl)-D-prolinate (3c): Prepared according to Standard
procedure 1 in 80 %, yield as a mixture of two diastereoisomers (SS and SR ). Light yellow
10
P
P
31
1
1
1
2
oil; 1.20 g. P-NMR (202 MHz, CDCl ): δ 7.95 (1P), 7.76 (0.9P) ppm. H-NMR (500 MHz,
3 P
1
CDCl ): δ 7.38-7.34 (m, 2H, CH-Ph), 7.30-7.28 (m, 1H, CH-Ph), 7.24-7.20 (m, 2H, CH-Ph),
3
H
13
4.50-4.46 (m, 0.5H, NHCH CO), 4.32-4.39 (m, 0.5H, NHCH CO), 3.73 (s, 1.5H, COOCH ),
a b 3
1
1
1
1
1
1
4
5
6
7
8
9
3.72 (s, 1.5H, COOCH ), 3.58-3.49 (m, 2H, H-Pro), 2.28-2.19 (m, 1H, H-Pro), 2.15-2.08 (m,
3
1H, H-Pro), 2.07-1.95 (m, 2H, H-Pro) ppm.
Methyl (chloro(naphthalen-1-yloxy)phosphoryl)glycinate (3d): Prepared according to
Standard procedure 1 in 78 %, yield as a mixture of two stereoisomers (SS and SR ). Light
P
P
3
1
1
yellow oil; 2.30 g. P-NMR (202 MHz, CDCl ): δ 9.08 ppm. H-NMR (500 MHz, CDCl ):
3
P
3
δH 8.06 (d, 1H, J = 8.3 Hz, CH-Naph), 7.87 (d, 1H, J = 7.7 Hz, CH-Naph), 7.72 (d, 1H, J = 8.3
Hz, CH-Naph), 7.60-7.52 (m, 3H, CH-Naph), 7.43 (t, 1H, J = 8.3 Hz, CH-Naph), 4.43-4.40
(br, 1H, NH), 4.00 (dd, 2H, J = 4.2, 9.7 Hz, NHCH CO), 3.80 (s, 3H, COOCH ) ppm.
20
2
2
1
2
2
3
Ethyl (chloro(phenoxy)phosphoryl)-L-phenylalaninate (3e): Prepared according to
Standard procedure 1 in > 99 %, yield as a mixture of two diastereoisomers (SS and SR ).
23
P
P
31
1
2
4
5
Colorless oil; 6.30 g. P-NMR (202 MHz, CDCl ): δ 8.03 (1P), 7.96 (1P) ppm. H-NMR (500
3 P
2
MHz, CDCl ): δ 7.39-7.15 (m, 10H, CH-Ar), 4.48-4.35 (m, 1H, NHCHCO), 4.23-4.16 (m,
3
H
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2
2H, OCH CH ), 3.21-3.10 (m, 2H, CH Ph), 1.26-1.21 (m, 3H, OCH CH ) ppm.
2
3
2
2
3
Benzyl (chloro(naphthalen-1-yloxy)phosphoryl)-L-alaninate (3f): Prepared according to
Standard procedure 1 in 96 %, yield as a mixture of two diastereoisomers (SS and SR ).
3
P
P
0
1
2
3
4
5
6
7
8
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0
1
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Colorless oil; 7.2 g. P-NMR (202 MHz, CDCl ): δ 8.17 (0.8P), 7.90 (1P) ppm. H-NMR
3 P
5
(500 MHz, CDCl ): δ 8.06-8.04 (m, 1H, CH-Ar), 7.88-7.86 (m, 1H, CH-Ar), 7.74-7.72 (d,
3 H
6
1H, J = 8.3 Hz, CH-Ar), 7.60-7.52 (m, 3H, CH-Ar), 7.44-7.40 (m, 1H, CH-Ar), 7.39-7.35 (m,
7
2H, CH-Ar), 7.34-7.32 (m, 3H, CH-Ar), 5.23-5.15 (m, 2H, OCH Ph), 4.52-4.39 (m, 1H, NH),
2
8
4.37-4.31 (br, 1H, NHCHCO), 1.55 (dd, 3H, J = 7.3, 10.2 Hz, CHCH ) ppm.
3
9
Benzyl (chloro(phenoxy)phosphoryl)-L-valinate (3g): Prepared according to Standard
procedure 1 in 89 %, yield as a mixture of two diastereoisomers (SS and SR ). Light yellow
10
P
P
31
1
1
1
1
2
oil; 4.40 g. P-NMR (202 MHz, CDCl ): δ 9.53 (1P), 9.02 (1P) ppm. H-NMR (500 MHz,
3 P
CDCl ): δ 7.38-7.22 (m, 10H, CH-Ar), 5.32-5.07 (AB system, 2H, J = 7.6 Hz, OCH Ph),
3
H
2
13
4.32-4.16 (m, 1H, NH), 4.04-3.93 (m, 1H, NHCHCO), 2.21-2.13 (m, 1H, 3H, CH(CH ) ), 1.00
3 2
1
1
1
1
1
1
4
5
6
7
8
9
(m, 3H, CH(CH ) ), 0.90 (m, 3H, CH(CH ) ) ppm.
3 2 3 2
Ethyl (chloro(phenoxy)phosphoryl)-L-isoleucinate (3h): Prepared according to Standard
procedure 1 in 98 %, yield as a mixture of two diastereoisomers (SS and SR ). Light yellow
P
P
3
1
1
oil; 8.40 g. P-NMR (202 MHz, CDCl ): δ 9.47 (1P), 8.91 (0.9P) ppm. H-NMR (500 MHz,
3
P
CDCl ): δ 7.39-7.35 (m, 2H, CH-Ph), 7.29-7.22 (m, 3H, CH-Ph), 4.56-4.48 (m, 1H, NH),
3
H
4.28-4.20 (m, 2H, OCH CH ), 4.03-3.93 (m, 1H, NHCHCO), 1.91-1.88 (m, 1H, CH CHCH ),
2
3
3
2
20
1.56-1.48 (m, 1H, CHCH CH ), 1.30 (m, 3H, OCH CH ), 1.27-1.23 (m, 1H, CHCH CH ),
2a 3 2 3 2b 3
2
2
1
2
1.00-0.92 (m, 6H, CH(CH ) ) ppm.
3 2
Benzyl 2-((chloro(phenoxy)phosphoryl)amino)-2-methylpropanoate (3i): Prepared
23
according to Standard procedure 1 in 96 %, yield as a mixture of two stereoisomers (SS and
P
31
1
2
2
4
5
SR ). Light yellow oil; 96 %; 3.84 g. P-NMR (202 MHz, CDCl ): δ 5.62 ppm. H-NMR (500
P 3 P
MHz, CDCl ): δ 7.28-7.11 (m, 10H, CH-Ph), 5.12 (s, 2H, OCH Ph), 1.62 (s, 3H, C(CH ) ),
3
H
2
3 2
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1.60 (s, 3H, C(CH ) ) ppm.
3 2
2
Benzyl (chloro(phenoxy)phosphoryl)-L-leucinate (3j): Prepared according to Standard
procedure 1 in > 99 %, yield as a mixture of two diastereoisomers (SS and SR ). Colorless oil;
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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3
4
P
P
0
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2
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4
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2.43 g. P-NMR (202 MHz, CDCl ): δ 8.31 (0.9P), 8.00 (1P) ppm. H-NMR (500 MHz,
3
P
5
CDCl ): δ 7.30-7.26 (m, 7H, CH-Ar), 7.18-7.13 (m, 3H, CH-Ar), 5.16-5.08 (m, 2H, OCH Ph),
3 H 2
6
4.14-4.01 (m, 2H, NH and NHCHCO), 1.81-1.67 (m, 1H, CH(CH ) ), 1.61-1.49 (m, 2H,
3 2
7
CHCH CH), 0.88-0.85 (m, 6H, CH(CH ) ) ppm.
2
3 2
8
Ethyl (chloro(4-methoxyphenoxy)phosphoryl)-L-leucinate (4a): Prepared according to
Standard procedure 1 in > 99 %, yield as a mixture of two diastereoisomers (SS and SR ).
9
P
P
3
1
1
10
Colorless oil; 4.58 g. P-NMR (202 MHz, CDCl ): δ 9.03 (1P), 8.84 (1P) ppm. H-NMR (500
3 P
1
1
2
MHz, CDCl ): δ 7.18-7.16 (m, 2H, CH-Ph), 6.87-6.85 (m, 2H, CH-Ph), 4.22 (m, 2H,
3 H
1
OCH CH ), 4.15-4.00 (m, 2H, NH and NHCHCO), 3.78 (s, OCH ), 1.86 (spt, 0.5H, J = 6.7
2
3
3
13
Hz, CH (CH ) ), 1.79 (p, 0.5H, J = 6.7 Hz, CH (CH ) ), 1.66-1.51 (m, 2H, CHCH CH), 1.29
a 3 2 b 3 2 2
14
15
16
17
18
19
(m, 3 H, OCH CH ), 0.97-0.94 (m, 6H, CH(CH ) ) ppm.
2 3 3 2
Methyl (chloro(4-methoxyphenoxy)phosphoryl)-D-prolinate (4b): Prepared according to
Standard procedure 1 in > 99 %, yield as a mixture of two diastereoisomers (SS and SR ).
P
P
3
1
1
Light yellow oil; 10.00 g. P-NMR (202 MHz, CDCl ): δ 8.60 (1P), 8.42 (0.9P) ppm. H-
3
P
NMR (500 MHz, CDCl ): δ 7.20 (dd, 1H, J = 1.8, 9.3 Hz, CH-Ph), 7.15 (dd, 1H, J = 1.8, 9.1
3
H
Hz, CH-Ph), 6.86 (dd, 2H, J = 1.7 Hz, 8.9 Hz, CH-Ph), 4.49-4.45 (m, 0.5H, NHCH CO), 4.41-
a
20
4.37 (m, 0.5H, NHCH CO), 3.78 (s, 1.5H, OCH ), 3.77 (s, 1.5H, OCH ), 3.73 (s, 1.5H, OCH ),
b
3
3
3
2
2
1
2
3.72 (s, 1.5H, OCH ), 3.57-3.49 (m, 2H, H-Pro), 2.2-2.18 (m, 1H, H-Pro), 2.15-2.07 (m, 1H,
3
H-Pro), 2.06-1.94 (m, 2H, H-Pro) ppm.
23
Isopropyl (chloro(4-methoxyphenoxy)phosphoryl)-L-alaninate (4c): Prepared according to
Standard procedure 1 in > 99 %, yield as a mixture of two diastereoisomers (SS and SR ).
2
4
5
P
P
3
1
1
2
Colorless oil: 14.00 g. P-NMR (202 MHz, CDCl ): δ 8.76 (1P), 8.41 (1P) ppm. H-NMR
3
P
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
(500 MHz, CDCl ): δ 7.19-7.16 (m, 2H, CH-Ph), 6.86 (dd, 2H, J = 2.4, 9.0 Hz, CH-Ph), 5.11-
3
H
5.04 (m, 1H, CH CHCH ), 4.17-4.09 (m, 1H, NHCHCO), 3.78 (s, 3H, OCH ), 1.48 (dd, 3H, J
3
3
3
3
= 3.7, 7.1 Hz, CH(CH ) ), 1.28-1.25 (m, 6H, CH(CH ) ) ppm.
3 2 3 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Standard Procedure 2. General Synthesis of 1-OBn-phosphoroamidates (5a/6a-c): To a
solution of compound 2 (1 equiv) in THF/Py (8/2, v/v), N-methylimidazole (2.5 equiv) was
added dropwise under argon atmosphere at room temperature and the resulting solution was
stirred for 30 minutes. A solution of an appropriate phosphorochloridate (2.5 equiv) in THF
was added dropwise to the mixture and stirred overnight at room temperature. After solvent
evaporation, the product was purified by column chromatography on silica using DCM/
5
6
7
8
9
10
CH OH (93:7) yielding the expected products 5-6.
3
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
()-Isopropyl
((((2R,3S,4R,5S)-5-acetamido-6-(benzyloxy)-3,4-dihydroxytetrahydro-2H-
3
1
pyran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (5a): Colorless oil; 45 %, 1.23 g. P-
NMR (202 MHz, CDCl , mixture of R and S diastereoisomers as  anomer): δ 3.86 (1P),
3
P
P
P
1
3.45 (0.6P) ppm. H-NMR (500 MHz, CDCl ): δ 7.37-7.15 (m, 10H, CH-Ar), 4.97-4.90 (m,
3
H
1H, CH(CH ) , overlap with the solvent), 4.78-4.77 (m, 1H, H-1, overlap with the solvent),
3 2
4.67-4.63 (m, 1H, CH Ph), 4.47-4.44 (m, 1H, CH Ph), 4.35-4.30 (m, 1H, H-2), 4.01-3.90 (m,
2
a
2b
3H, NHCHCO, H-5 and H-4), 3.83-3.77 (m, 1H, H-3), 3.63-3.56 (m, 2H, H-6), 1.99 (s, 3H,
1
3
NHCOCH ), 1.37-1.27 (m, 3H, NHCHCH ), 1.23-1.18 (m, 6H, CH(CH ) ) ppm. C-NMR
3
3
3 2
3
3
19
(125 MHz, MeOD): δ 174.63 (d, J = 5.2 Hz, C=O ester), 174.07 (d, J = 7.2 Hz, C=O
C
C-P
C-P
2
2
2
2
0
1
ester), 170.03 (NHCOCH ), 154.66 (d, J = 6.8 Hz, “ipso” OPh), 152.28 (d, J = 6.5 Hz,
3 C-P C-P
“ipso” OPh), 138.57 (“ipso” OCH Ph), 130.72, 130.69, 130.07, 129.41, 129.19, 129.16,
2
3
22
128.85, 128.65, 126.03, 125.99, 123.82 (CHAr), 121.54 (d, J = 4.9 Hz, CH-OPh), 121.45
C-P
3
4
2
2
3
4
(d, J = 4.9 Hz, CH-OPh), 99.57 (C-1), 73.12 (d, J = 6.8 Hz, C-4), 70.55, 70.39 (C-3),
C-P C-P
70.17 (OCH Ph), 70.11, 70.03 (CH(CH ) ), 69.19, 69.07 (NHCHCO), 68.33 (C-5), 68.07 (d,
2
3 2
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J = 5.6 Hz, C-6), 67.94 (d, J = 5.5 Hz, C-6), 54.35 (C-2), 21.98, 21.95 (NHCHCH ), 9.19,
C-P C-P 3
+
2
7.59 (CH(CH ) ) ppm. MS (ES+): m/z 603.22 [M+Na]
3 2
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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5
5
5
5
5
5
5
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6
3
4
5
6
7
8
9
0
1
()-Ethyl ((((2R,3S,4R,5S)-5-acetamido-6-(benzyloxy)-3,4-dihydroxytetrahydro-2H-pyran-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-yl)methoxy)(4-methoxyphenoxy)phosphoryl)-L-leucinate (6a): Prepared according to
Standard procedure 2 in 20 %, yield as a mixture of R and S diastereoisomers as  anomer.
P
P
3
1
1
Light yellow oil; 0.16 g. P-NMR (202 MHz, CDCl ): δ 4.37 (1P), 4.30 (1P) ppm. H-NMR
3
P
(500 MHz, CDCl ): δ 7.35-7.31 (m, 5H, CH-Ar), 7.18-7.13 (m 2H, CH-Ar), 6.87-6.83 (m,
3
H
2H, CH-Ar), 4.79 (d, 1H, J = 10.9 Hz, H-1), 4.68-4.64 (m, 1H, CH Ph), 4.49-4.41 (m, 1H,
2a
CH Ph), 4.35-4.34 (m, 1H, H-2), 4.17-4.12 (m, 2H, OCH CH ), 4.11-4.07 (m, 1H, H-4), 4.00-
2
b
2
3
1
1
4.07 (m, NHCHCO), 3.92-3.87 (m, 1H, H-3), 3.84-3.73 (m, 4H, OCH and H-5), 3.63-3.58 (m,
3
2H, H-6), 2.01 (s, 3H, NHCOCH ), 1.71-1.62 (m, 1H, CH(CH ) ), 1.58-1.50 (m, 2H,
3
3 2
1
3
12
CHCH CH), 1.27-1.19 (m, 3H, OCH CH ), 0.94-0.84 (m, 6H, CH(CH ) ) ppm. C-NMR (125
2 2 3 3 2
3
3
1
1
1
1
1
1
3
4
5
6
7
8
MHz, MeOD): δ 175.45 (d, J = 2.5 Hz, C=O ester), 175.33 (d, J = 2.5 Hz, C=O ester),
C C-P C-P
2
174.11, 174.05 (NHCOCH ), 158.25, 157.59 (“ipso” PhOCH ), 145.83 (d, J = 3.8 Hz,
3
3
C-P
“ipso” POPh), 138.56 (“ipso” OCH Ph), 129.42, 129.39, 129.27, 129.22, 128.86, 122.46,
2
3
3
122.42 (CHAr), 122.32 (d, J = 4.4 Hz, CH-POPh), 122.18 (d, J = 4.5 Hz, CH-POPh),
C-P
C-P
4
115.58, 115.55, 115.31 (CHAr), 99.56, 99.51 (C-1), 73.11 (d, J = 7.5 Hz, C-4), 73.08 (d,
C-P
4
J = 6.3 Hz, C-4), 70.58, 70.37 (NHCHCO), 70.14 (OCH Ph), 68.40, 68.37 (C-5), 68.04 (d,
C-P
2
2
2
19
J = 5.3 Hz, C-6), 67.88 (d, J = 5.4 Hz, C-6), 62.27, 62.22 (OCH CH ), 56.02, 55.99 (C-
C-P C-P 2 3
2
2
0
1
3), 54.56, 54.37 (C-2), 44.15, 43.96 (CHCH CH), 25.55, 25.46 (CH(CH ) ), 23.19, 23.16,
2 3 2
22.56, 22.41, 22.13, 21.90 (NHCOCH and CH(CH ) ) 14.46, 14.41 (OCH CH ) ppm. MS
3
3 2
2
3
22
(ES+): m/z 661.26 [M+Na]+
2
2
3
4
()-Methyl ((((2R,3S,4R,5S)-5-acetamido-6-(benzyloxy)-3,4-dihydroxytetrahydro-2H-pyran-
2-yl)methoxy)(4-methoxyphenoxy)phosphoryl)-D-prolinate (6b): Prepared according to
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5
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6
7
8
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0
1
Standard procedure 2 in 32 %, yield as a mixture of R and S diastereoisomers as  anomer.
P
P
3
1
1
Light yellow oil; 0.23 g. P-NMR (202 MHz, CDCl ): δ 2.27 (0.1P), 1.87 (1P) ppm. H-NMR
3
P
(500 MHz, CDCl ): δ 7.33-7.28 (m, 5H, CH-Ar), 7.14-7.12 (m 2H, CH-Ar), 6.87-6.84 (m,
3
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2H, CH-Ar), 4.77-4.76 (m, 1H, H-1), 4.61 (d, 1H, J = 11.7 Hz, CH Ph), 4.55-51 (m, 1H,
2
a
NHCHCO), 4.4 (m, 1H, J = 11.7 Hz CH Ph), 4.33-4.31 (m), 4.26-4.23 (m), 3.98-3.95 (m),
2
b
3.81-3.78 (m), 3.71 (s, 3H, COOCH ), 3.69 (s, 3H, PhOCH ), 3.41-3.33 (m), 2.19-2.13 (m, 2H,
3
3
CH Pro), 1.99 (s, 3H, NHCOCH ), 1.96-1.81 (m, 2H, CH Pro) ppm. MS (ES+): m/z 631.21
2
3
2
[M+Na]+
()-Isopropyl
((((2R,3S,4R,5S)-5-acetamido-6-(benzyloxy)-3,4-dihydroxytetrahydro-2H-
1
1
pyran-2-yl)methoxy)(4-methoxyphenoxy)phosphoryl)-L-alaninate (6c): Prepared according to
Standard procedure 2 in 21 %, yield as a mixture of R and S diastereoisomers as  anomer.
P
P
3
1
1
12
Colorless oil; 0.20 g. P-NMR (202 MHz, CDCl ): δ 4.30 (1P), 4.09 (0.8P) ppm. H-NMR
3 P
1
1
3
4
(500 MHz, CDCl ): δ 7.30 (m, 5H, CH-Ar), 7.14 (d, 2H, J = 86 Hz, CH-Ar), 6.84-6.80 (m,
3 H
2H, CH-Ar), 4.98-4.93 (m, 1H, CH(CH ) ), 4.77 (s, 1H, H-1), 4.65-4.64 (m, 1H, CH Ph),
3
2
2a
15
4.44-4.38 (m, 1H, CH Ph), 4.32-4.31 (m, 1H, H-2), 4.29-4.21 (m), 3.98-3.95 (m), 3.93-3.88
2b
1
1
6
7
(m, 1H, NHCHCO), 3.80-3.77 (m), 3.70 (s, 1.5H, PhOCH ), 3.69 (s, 1.5H, PhOCH ), 3.62-
3 3
3.55 (m, 2H, H-6), 1.99 (s, 3H, NHCOCH ), 1.37-1.32 (m, 3H, NHCHCH ), 1.23-1.20 (m, 6H,
3
3
1
3
18
CH(CH ) ) ppm. C-NMR (125 MHz, MeOD): δ 173.34, 173.33 (C=O ester), 172.73, 172.67
3 2 C
1
2
2
9
0
1
(NHCOCH ), 156.87 (“ipso” OPh), 144.38 (“ipso” POPh), 137.21 (“ipso” OCH Ph), 128.05,
3 2
127.87, 127.84, 127.49, 121.00, 120.93, 114.23, 114.20 (CHAr), 98.18, 98.14 (C-1), 73.11 (d,
4
4
J = 7.5 Hz, C-4), 73.08 (d, J = 6.3 Hz, C-4), 70.58, 70.37 (NHCHCO), 70.14 (OCH Ph),
C-P
C-P
2
2
2
22
68.40, 68.37 (C-5), 68.04 (d, J = 5.3 Hz, C-6), 67.88 (d, J = 5.4 Hz, C-6), 62.27, 62.22
C-P C-P
2
2
3
4
(OCH CH ), 56.02, 55.99 (C-3), 54.56, 54.37 (C-2), 44.15, 43.96 (CHCH CH), 25.55, 25.46
2 3 2
(CH(CH ) ), 23.19, 23.16, 22.56, 22.41, 22.13, 21.90 (NHCOCH and CH(CH ) ) 14.46, 14.41
3
2
3
3 2
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7
+
1
(OCH CH ) ppm. MS (ES+): m/z 661.26 [M+Na]
2 3
2
Standard Procedure 3. General Synthesis of Deprotected Phosphoroamidates (8a-c): To a
solution of 5 or 6 (1 equiv) in EtOH/EtOAc (2:1) was added Pd/C (10% Pd on activated carbon)
under inert atmosphere. The resulting mixture was stirred at room temperature for 5 hours
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
5
6
7
8
9
0
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
under H atmosphere (1 atm, ballon). The crude was filtered through celite, concentrated in
2
vacuo and triturated with Et O giving the expected products 8a-c.
2
Ethyl ((((2R,3S,4R,5S)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)(4-
3
1
methoxyphenoxy)phosphoryl)-L-leucinate (8a): Light yellow oil; 29%; 0.17 g. P-NMR (202
MHz, CDCl , mixture of R and S diastereoisomers as  and  anomers): δ 4.41 (1P), 4.31
3
P
P
P
1
1
1
1
(0.7P), 4.19 (0.7P) ppm. H-NMR (500 MHz, CDCl ): δ 7.14 (d, 2H, J = 9.0 Hz, CH-Ar), 6.87
3 H
(d, 2H, J = 9.0 Hz, CH-Ar), 5.03 (s, 1H, H-1), 4.56 (s, 1H, H-2), 4.34-4.26 (m, 1H, CH Ph),
2
4.17-4.12 (m, 2H, OCH CH ), 4.11-4.07 (m, 1H, H-4), 4.00-4.07 (m, NHCHCO), 3.92-3.87
2
3
13
(m, 1H, H-3), 3.84-3.73 (m, 4H, OCH and H-5), 3.63-3.58 (m, 2H, H-6), 2.01 (s, 3H,
3
1
1
4
5
NHCOCH ), 1.71-1.62 (m, 1H, CH(CH ) ), 1.58-1.50 (m, 2H, CHCH CH), 1.27-1.19 (m, 3H,
3
3 2
2
1
3
OCH CH ), 0.94-0.84 (m, 6H, CH(CH ) ) ppm. C-NMR (125 MHz, MeOD): δ 174.20,
2
3
3 2
C
16
174.09 (C=O ester), 172.77, 172.69 (NHCOCH ), 156.92, 156.84 (“ipso” PhOCH ), 144.41 (d,
3 3
1
1
7
8
2JC-P= 6.8 Hz, “ipso” POPh), 121.06, 121.02, 120.81, 120.77, 114.21, 114.16 (CHAr), 93.67,
4
4
93.37 (C-1), 73.11 (d, J = 7.5 Hz, C-4), 73.08 (d, J = 6.3 Hz, C-4), 70.58, 70.37
C-P
C-P
2
2
19
(NHCHCO), 70.14 (OCH Ph), 68.40, 68.37 (C-5), 68.04 (d, J = 5.3 Hz, C-6), 67.88 (d, J
2
C-P
C-
2
2
2
0
1
2
= 5.4 Hz, C-6), 62.27, 62.22 (OCH CH ), 56.02, 55.99 (C-3), 54.56, 54.37 (C-2), 44.15, 43.96
P 2 3
(CHCH CH), 25.55, 25.46 (CH(CH ) ), 23.19, 23.16, 22.56, 22.41, 22.13, 21.90 (NHCOCH
3
2
3 2
+
and CH(CH ) ) 14.46, 14.41 (OCH CH ) ppm. MS (ES+): m/z 571.21 [M+Na]
3 2
2
3
23
Methyl
((((2R,3S,4R,5S)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-
2
4
yl)methoxy)(4-methoxyphenoxy)phosphoryl)-D-prolinate (8b): Prepared according to
29
ACS Paragon Plus Environment