Cyclic Phosphopantothenic Acid Prodrugs for Treatment of Pantothenate Kinase-Associated Neurodegeneration

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Cyclic Phosphopantothenic Acid Prodrugs for Treatment of

Pantothenate Kinase-Associated Neurodegeneration

Giulio Auciello, Annalise Di Marco, Odalys Gonzalez Paz, Savina Malancona, Steven Harper,

Maria Beconi, Ilaria Rossetti, Alina Ciammaichella, Paola Fezzardi, Andrea Vecchi, Elena Bracacel,

Daniel Cicero, Edith Monteagudo, and Daniel Elbaum *

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ABSTRACT: Mutations in the human PANK2 gene are

implicated in neurodegenerative diseases such as pantothenate

kinase-associated neurodegeneration (PKAN) and result in low

levels of coenzyme-A (CoA) in the CNS due to impaired

production of phosphopantothenic acid (PPA) from vitamin B5.

Restoration of central PPA levels by delivery of exogenous PPA is a

recent strategy to reactivate CoA biosynthesis in PKAN patients.

Fosmetpantotenate is an oral PPA prodrug. We report here the

development of a new PANk2^−/−knockout model that allows CoA

regeneration in brain cells to be evaluated and describe two new

series of cyclic phosphate prodrugs of PPA capable of regenerating excellent levels of CoA in this system. A proof-of-concept study in

mouse demonstrates the potential of this new class of prodrugs to deliver PPA to the brain following oral administration and

conﬁrms incorporation of the prodrug-derived PPA into CoA.

mitochondrial deﬁciency of CoA in PKAN patients. As CoA

plays a vital role in a myriad of cellular processes, including

metabolism of fatty acids and post-translational modiﬁcation of

proteins, its deﬁciency is believed to be a root cause of

undesirable phenotypes seen in individuals suﬀering from

PKAN.⁸

Although the synthesis of PPA is defective in the CNS of

PKAN patients, the subsequent processing steps to CoA are

functional, and a potential therapy for PKAN might therefore

stem from administration of either PPA itself, or one of its

downstream products in the CoA biosynthetic pathway. While

this approach is conceptually appealing, poor blood−brain

barrier (BBB) penetration for these highly polar and acidic

compounds poses a signiﬁcant limitation. To circumvent this

issue, approaches that mask the polar phosphoric acid and

carboxylic acid moieties in PPA with biochemically labile

functionality provide an attractive strategy toward brain

penetrant prodrugs with the potential to function as PPA

precursors.⁹The biopharmaceutical company Travere, Inc.

recently reported work in this direction, targeting compounds

capable of delivering viable CoA precursors to the CNS of

INTRODUCTION

■

Neurodegeneration with brain iron accumulation (NBIA)

disorders comprise a group of neurological conditions of which

the most common is pantothenate kinase-associated neuro-

degeneration (PKAN) with a prevalence of 1−3/1 000 000.¹⁻³

Classic and atypical PKAN are autosomal recessive disorders

with onset in childhood or early adolescence, respectively.

Post-onset disease progression leads to a steady increase in

muscle disorders, such as dystonia, rigidity, and dysphagia, and

ultimately to movement disorders, cardiac and opthalmic

complications, and death. The disease results in the

accumulation of iron in the globus pallidus, which is seen as

an “eye-of-the-tiger” pattern in the T₂-weighted magnetic

resonance imaging (MRI).^1,4Although PKAN is rare, its high

toll in terms of patient suﬀering and the current poor standard

of care create a strong case for the development of new

therapies.

PKAN is caused by mutations in the human PANK2 gene

that encodes pantothenate kinase-2 (PanK2), a key enzyme in

coenzyme-A (CoA) biosynthesis.^5,6PanK2 is predominantly

expressed in brain mitochondria, where it catalyzes the mono-

phosphorylation of the primary hydroxyl group of pantothenic

acid (vitamin B5, Chart 1) to generate 4′-phosphopantothenic

acid (PPA). PPA is further processed to 4′-pantetheine by

phosphopantothenoyl cysteine synthetase and phosphopanto-

thenoyl cysteine decarboxylase and ultimately converted to

CoA by phosphopantetheine adenylyltransferase and dephos-

pho-CoA kinase.⁷Loss of Pank2 function leads to a

Received: September 1, 2020

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Chart 1. Biosynthetic Intermediates in CoA Synthesis and New PPA Prodrugs

PKAN patients following oral administration.¹⁰A ﬁrst

candidate from this “kinase-bypass” PPA prodrug approach is

Fosmetpantotenate (Chart 1), a phosphoramidate prodrug of

PPA that recently completed a clinical trial (NCT 03041116)

(https://clinicaltrials.gov/ct2/show/NCT03041116).¹¹Un-

fortunately, this trial did not meet its primary endpoint.

Other eﬀorts in this area include work on 4′-phophopante-

theine.^12,13In addition, another therapeutic strategy is to

increase the activity of other PanK isoforms.¹⁴

set of compounds in which the pantothenate ester was varied

were also explored as a means to modulate absorption,

distribution, metabolism, and excretion (ADME) properties.

Finally, a series of simpliﬁed analogues in which an

arylphosphate (3) serves to mask the phosphoric acid of a

cyclic PPA carboxylic ester that also proved to be viable PPA

prodrugs are described. These compounds proved to be

eﬀective at restoring CoA levels in a cellular model, and oral

dosing of isotopically labeled 2a led to incorporation of the

labels in the CoA found in the CNS. This provides a proof of

principle for the use of this compound class to restore CoA

levels in the CNS of PKAN patients.

In follow-up to Fosmetpantotenate, a backup program was

initiated to develop new prodrugs of PPA for the treatment of

PKAN. The program had three goals: (1) provide a crystalline

compound. Fosmetpantotenate is a viscous oil, which greatly

complicates handling; (2) provide a more stable compound.

Fosmetpantotenate was prone to intramolecular cyclization in

which the 2′ hydroxyl displaced the phenoxy, resulting in cyclic

phoshoramidate, 1 in Chart 1; and (3) provide a more

eﬃcacious compound. Treatment with Fosmetpantotenate

results in a modest 2−3-fold increase in CoA in our cellular

models. While it was uncertain if this would be suﬃcient, the

urgent unmet need dictated rapid progression to the clinic.

As a starting point, we were intrigued by compounds based

on a 1,3,2-dioxaphosphinane heterocyclic core 1 the cyclization

product of Fosmetpantotenate. Cyclic compounds were also

found as circulating metabolites following administration of

Fosmetpantotenate to rodents (data not shown). PPA release

from cyclic phosphoramidates like 1 is potentially possible

following phosphoramidase-mediated cleavage of the P−N

bond¹⁵(and esterase-mediated hydrolysis of the carboxylic

ester) but replacing the exocyclic phosphoramidate with more

biochemically labile groups ought to favor the release of PPA.

Several intramolecularly cyclized phosphate prodrugs have

been reported in the nucleoside area,¹⁶⁻¹⁹and application of

the pivaloyloxymethyl (POM) as a prodrug moiety has been

documented.²⁰Here, we report work on three sets of

analogues. This work started with an exploration of the

pivaloyloxymethyl (POM)¹⁶group as a means to trigger

release of PPA from 1,3,2-dioxaphosphinane-based prodrugs,

and that resulted in a series of compounds (2) with attractive

properties as second-generation PPA prodrugs. In addition, a

RESULTS AND DISCUSSION

■

Synthesis. Synthesis of 1,3,2-dioxaphosphinane-based PPA

prodrug 1 was readily achievable by cyclization of Fosmetpan-

totenate under basic conditions (triethylamine in CH₂Cl₂) in

50% yield. Alkoyl and aroyl-oxymethyl prodrugs from Tables 1

and 4 were prepared by direct alkylation of the cyclic-PPA

analogue 6 (Scheme 1). Synthesis of compound 6 began with

methyl pantothenate 4 with POCl₃followed by in situ

displacement of the chloride from the intermediate 2-chloro-

1,3,2-dioxaphosphinane 2-oxide with benzyl alcohol to give

intermediate 5 in 25% yield. Removal of the benzyl group

proceeded in 96% yield by hydrogenolysis over Pd/C.

Alkylation of 6 could be accomplished by treatment with a

chlormethyl ester¹⁶with either diisopropylethylamine (DIEA)

or Ag₂O (in the case of 2o) to provide the analogues 2a, 2b,

2d, 2f, 2i, 2j, and 2n in unoptimized yields ranging from less

than 10 to 39% as a mixture of cis and trans diastereoisomers

in an approximately 1:1 ratio. An alternative route to the target

prodrugs involved conversion of 6 to the chloride intermediate

7 using chloromethyl sulfurochloridate,²¹which proceeded in

only 10% yield. Compound 7 was then treated with the

required carboxylic acid in the presence of Cs₂CO₃to provide

compounds 2c, 2e, 2g, 2h, 2k−m, and 2p−s, again in

unoptimized yields ranging from less than 10% to higher than

30%. This approach generally favored the formation of the cis-

diastereoisomer. Stable isotope labeled 2a (+6 amu) was

prepared by construction of methyl pantothenate 4 (+4 amu)

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by ring opening²²of commercial (R)-pantolactone with β-

Table 1. CoA Regeneration in PanK2 Knockdown Cells

Treated with 1,3,2-Dioxaphosphinane Heterocycles Based

on Alkoxymethyl and Aryloxymethyl Side Chains

alanine methyl ester (+4 amu) in 67% yield, followed by

23

cyclization employing P¹⁸OCl₃(Scheme 2). The resulting

intermediate 12 was converted to 2a (+6 amu) using the same

conditions to construct 2a in yields of 99 and 33% for the two

remaining steps.

Analogues 9c−g were prepared by cyclization of (R)-

pantothenic acid tert-butyl ester, 8, generated from tert-butyl 3-

amino-propionate and (3R)-4-dimethyl-3-oxindanyl-oxolane-2-

one in 88% yield, as outlined in Scheme 3. Construction of the

cyclic POM analogue from 8 (as previously described for

compound 2a) in 28% overall yield for the three steps,

followed by triﬂuoroacetic acid (TFA)-mediated ester

deprotection gave the advanced carboxylic acid intermediate

9a, in 87% yield. Introduction of the pantothenic ester

functionality in compounds 9c−g was achieved by con-

densation of 9a with the required alcohols using 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraﬂuorobo-

rate (TBTU) as the coupling reagent, in 29−97% yield.

Compounds 3 were accessible by one of the two methods

(Scheme 4). Treatment of methyl pantothenate with POCl₃

provided the chloro-1,3,2-dioxaphosphinane intermediate

(99% yield), which was used without puriﬁcation. The crude

material was treated with a phenol and TEA. This approach led

to a mixture of diastereoisomers that were separated using

SiO₂chromatography to provide compounds 3a and 3b in 14

and 5% yields, respectively. The remaining compounds in

Table 6 were prepared using an alternative approach, in which

the phenol was ﬁrst activated by reaction with POCl₃, followed

by trapping of the resulting dichlorophosphate with 4. This

approach consistently gave rise to a single diastereoisomer of

compounds 3c−g with cis-stereochemistry across the ring

junction in yields ranging from 3 to 23%.

Compounds 2a−s, 9a−g, 3a−g, as well as 2a were puriﬁed

on SiO₂or by reversed-phase C₁₈column chromatography or

high-performance liquid chromatography (HPLC) prior to

use. Reversed-phase HPLC was also used to access the

individual trans-diastereomers of compounds 1 as well as 2m,

2r, and 2s (Table 5). Ring stereochemistry for individual

1

stereoisomers was assessed by H−¹H rotating-frame nuclear

Scheme 1

a

Reagents and conditions: (a) POCl₃, triethylamine (TEA), tetrahydrofuran (THF), −78 to 20 °C, then BnOH, TEA, THF, −78 to 20 °C; (b) H₂,

Pd/C, EtOAc; (c) RC(O)OCH₂Cl, DIEA, dimethylformamide (DMF), 0−80 °C; (d) RC(O)OCH₂Cl, Ag₂O, AcCN, 80 °C; (e) chloromethyl

sulfurochloridate, t-Bu₄NHSO₄, NaHCO₃, H₂O, dichloromethane (DCM), 0−20 °C; (f) RCO₂H, Cs₂CO₃, DMF.

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Scheme 2

a

Reagents and conditions: (a) SOCl₂, MeOH, reﬂux, 2 h; (b) (R)-pantolactone, TEA, MeOH, reﬂux, on; (c) PO(18)Cl₃, TEA, THF, −78 to 20

°C, then BnOH, TEA, THF, −78 to 20 °C; (d) H₂, Pd/C, EtOAc; (e) RC(O)OCH₂Cl, DIEA, DMF, 0−80 °C;

Scheme 3

a

Reagents and conditions: (a) (3R)-4,4-dimethyl-3-oxidanyl-oxolan-2-one, 85 °C; (b) POCl₃, TEA, THF, −78 °C, then BnOH, NMI, THF, −78

°C; (c) H₂, Pd/C; (d) chloromethyl-2,2-dimethylpropanoate, DIEA, 80 °C; (e) TFA, DCM; (f) R′OH, TBTU, AcCN.

Scheme 4

a

Reagents and conditions: (a) POCl₃, TEA, THF, −78 °C; (b) R-C6H4-OH, TEA, Et₂O, 0 °C; (c) POCl₃, TEA, Et₂O, −78 to 20 °C; (d) 4, TEA,

THF, −78 to 20 °C.

Overhauser eﬀect spectroscopy (ROESY) NMR experiments

on early compounds in each chemical series and for key

compounds, as well as by small-molecule crystallography for

compound trans-2a (see the Supporting Information). Assign-

ment of stereochemistry was simpliﬁed by conﬁrmation that

the relative ³¹P NMR shift for either the cis or trans-isomer was

a direct predictor of ring stereochemistry.²⁴The ³¹P resonance

for the cis-isomer was in all cases found downﬁeld with respect

to the trans, providing a straightforward means to assign

stereochemistry in cases where both isomers were available.

Biological Activity. To support medicinal chemistry

eﬀorts, a suitable PanK2-deﬁcient cell model amenable to

medium throughput in which to assess the potential of

prodrugs to generate CoA was developed.¹⁰This stable cell

line expressing a PANK2 gene-speciﬁc shRNA allowed for the

screening and characterization of potential drug candidates

able to restore CoA levels. The quantitation of CoA

concentrations in neuroblastoma cells was performed by

HPLC analysis using a method that allowed measurement of

either free CoA or total CoA.¹⁰For analysis of free CoA, cell

lysate samples were dissolved in a moderately acidiﬁed

medium (pH 5) to avoid oxidation of the CoA thiol group

to disulﬁdes. For total CoA (see the Supporting Information),

conversion of acetyl CoA (CoASAc) and other endogenous

acyl CoA’s (of which >250 species are known) into free CoA

was accomplished by a fast alkaline hydrolysis of the cell pellet

and subsequent acidiﬁcation to pH 5. This approach avoided

the need for complex separations or the availability of

authentic/labeled standards for the various CoA analytes. As

there was a clear trend between free CoA and total CoA

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Figure 1. Eﬀect of compound 2a on lipid droplet content in PANk2^−/−cells. (A) Two representative images of wild-type (mock) and PANk2^−/−

cells (untreated and treated with compound 2a at 10 μM for 48 h); cells were ﬁxed and stained with 100 ng/mL Nile Red and imaged by an

inverted axiovert microscope (Zeiss) at 60× magniﬁcation. (B) Fluorimetric quantiﬁcation of lipid droplet content using a Tecan Saﬁre microplate

detection system. Results are reported as mean standard deviation (sd) (two biological replicates, each in duplicate) **p < 0.01, two-tailed paired

Student’s t-test.

response in our PANk2^−/−assay (data not shown), free CoA

levels were used as the standard primary eﬃcacy readout. CoA

levels in the knockdown cells were not restored when cells

were treated with PPA (likely reﬂecting its poor cell

permeability²⁵). In contrast, daily treatment of cells with

Fosmetpantotenate restored free CoA levels supporting the

hypothesis that a precursor capable of delivering PPA

intracellularly following metabolism can indeed generate free

CoA in the absence of functional pantothenate kinase.

Compounds were initially screened for their ability to increase

intracellular CoA levels in PANk2^−/−neuroblastoma cells at a

ﬁxed concentration of 50 μM for 24 h. Secondary assays that

were used to support the restoration of intracellular CoA levels

included measurement of tubulin acetylation levels¹⁰and

determination of the compounds’ eﬀects on lipid accumu-

lation.

Our ﬁrst goal was the generation of more eﬃcacious

compounds as assessed by increases in CoA in our PANk2^−/−

model. POM analogues of 1,3,2-dioxaphosphinane hetero-

cycles were evaluated as a ﬁrst approach based both on the

expectation that they would more eﬃciently release PPA (by

hydrolysis of their alkoyloxymethyl side chain) and because

they oﬀer improved chemical stability with respect to acyclic

prodrugs that, as described above, are susceptible to intra-

molecular cyclization of the secondary alcohol of pantothenic

acid onto phosphorus. Compound 2a (Table 1) was initially

isolated as an approximately 1:1 mixture of two diastereomers

resulting from the presence of epimers at phosphorus together

with a stereo-deﬁned carbon center in the dioxaphosphinane

ring derived from (R)-pantothenic acid. Compound 2a proved

chemically stable and was a remarkably eﬀective precursor to

CoA when tested in PANk2^−/−neuroblastoma cells. At the

standard test concentration of 50 μM, 2a generated CoA levels

around 180-fold higher than in control cells. Generation of

such high levels of CoA had not been achieved using either

linear or cyclic phosphoramidate derivatives of PPA (data not

shown), and these data therefore generated considerable

interest in the POM series as a potential starting point for

backup candidates to Fosmetpantotenate. Consistent with

release of CoA from compound 2a, a reversal of some

phenotypical characteristics of treated PANk2^−/−cells was

observed. Levels of tubulin and histone H3 acetylation were

measured as we have previously described and were found to

be increased (see the Supporting Information). Acetyl-CoA is

also known to inﬂuence levels of neutral lipids, and it has been

reported that PanK2-defective neurons derived from KO mice

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show accumulation of lipids in the cytoplasm.²⁶To assess lipid

content in PANk2^−/−cells, they were stained with 100 ng/mL

of a ﬂuorescent lipophilic dye (Nile Red) and imaged using an

inverted axiovert microscope (Zeiss) at 60× magniﬁcation.

Figure 1 shows that compound 2a was capable of producing a

statistically signiﬁcant decrease in the content of lipid droplet

in PANk2^−/−cells following this 48 h treatment, in line with

the eﬀect on CoA levels. Separation of the individual

diastereomers of 2a generated cis-2a and trans-2a that have

cis and trans-ring geometry, respectively. These compounds

both proved eﬀective precursors to CoA in the PANk2^−/−

assay, with the trans-isomer generating levels approximately 2-

fold higher. This led to a strategy in which compounds were

ﬁrst tested as diastereomeric mixtures with follow-up to

separate or synthesize the individual diastereomers for selected

compounds. All phosphoramidate-based prodrugs explored

during the discovery of Fosmetpantotenate were viscous oils, a

physical property that complicates puriﬁcation, handling, and

drug development. Importantly, while the cis-isomer cis-2a was

also an oil, the trans-isomer trans-2a was a well-behaved solid,

thus providing the ﬁrst PPA prodrug with the preferred

physical state for drug development.

by two pieces of data. First, the crystal structure of trans-2a

shows the amide side chain in the equatorial position putting

the phosphate side chain in the axial position, and thus,

possibly stabilized by an anomeric eﬀect of the ring oxygens.

Second, the stability of members of this series in whole blood

was generally better for the trans isomers (see below). This

could indicate that substituents in the axial position are less

accessible to esterases.

Degradation of the alkoyl or aroyl-oxymethyl side chain

ultimately releases one equivalent of formaldehyde, a potential

alkylating agent of nucleic acids and proteins. Although we did

not observe toxicity in our cellular assay at 50 μM or any

untoward eﬀects in our in vivo studies, we decided to explore

the possibility of using a prodrug that would release

acetaldehyde rather than formaldehyde. However, analogues

such as 2j that were designed to release acetaldehyde (a less

eﬀective alkylating agent than formaldehyde and a xenobiotic

that is eﬀectively detoxiﬁed in man³⁰) resulted in much poorer

generation of CoA in the PANk2^−/−model. Overall, the results

in Table 1 indicate a broad tolerance to changes at the terminal

ester functionality, with compounds such as 2g and 2h

suggesting scope to employ changes at this ester group for

modulating the overall physicochemical properties of the

prodrugs without completely abolishing their ability to restore

CoA in cells.

The successful use of a prodrug approach to increase CoA in

the CNS requires a complex interplay between biodistribution

and metabolism. Adequate delivery of the intact prodrug to the

systemic circulation and distribution across the blood−brain

barrier must ultimately be balanced by incorporation of

suﬃciently labile functionality that allows for PPA release in

the target tissue. Compound 2a (and indeed all compounds in

Table 1) showed rather short in vitro half-lives in both human

whole blood and hepatocytes, with both diastereomers being

more than 50% degraded at the ﬁrst time point of the whole

blood/hepatocyte bioassay (10 and 15 min, respectively). This

set of compounds also illustrates the need to assess stability in

multiple matrices. Our initial compound progression tree used

a plasma stability assay, and many of the POM analogues are

quite stable in this matrix, with half-lives >1 h (data not

shown). It thus came as somewhat of a shock when the assay in

whole blood demonstrated limited stability. Rapid turnover of

2a (T_1/2< 15 min) was also measured in mouse plasma and

hepatocytes.

In an attempt to modulate the rate of ester hydrolysis,²⁷and

by extension the eﬀects on CoA generation, changes in the

pivaloyloxymethyl side chain on CoA generation in PANk2^−/−

cells were explored extensively. The remaining compounds

shown in Table 1 illustrate the broad trends uncovered in these

investigations. Apolar lipophilic terminal ester functionality in

the side chain favored the generation of CoA, with extremely

high levels (compared to vehicle-treated cells) being seen for

compounds such as 2b. In contrast, compounds 2c and 2d,

both of which are formally truncated in their side-chain ester

group by two carbons with respect to 2b, were 4−5-fold less

eﬃcient at producing CoA. Introduction of more polar

functionality into the side-chain ester consistently attenuated

the levels of CoA in the PANk2^−/−neuroblastoma assay. For

example, compound 2e generated relatively moderate levels of

CoA with respect to the original lead 2a, and only 3−4-fold

higher than Fosmetpantotenate. Similarly, while replacement

of the pivaloyl group on the oxymethyl side chain with a simple

benzoyl ester 2f led to a substantial increase in CoA, the

introduction of more polar hetereoaryl substituents such as

pyridine 2g or pyrazine 2h led to prodrugs that were somewhat

less eﬃcient CoA precursors. A further change that was

explored was the use of a carbonate group at the terminus of

the prodrug side chain. The use of phosphonyloxycarbonate

(POC) groups²⁸as promoieties has been successfully applied

as an approach to mask nucleoside phosphate groups in

marketed antivirals such as Tenofovir isoproxil,²⁹resulting in a

large increase in the systemic exposure of the parent drug. In

the context of a cyclic PPA methyl ester, the use of a POC

group (compound 2i) led to a large increase in CoA level in

our cellular model, comparable to 2a.

Compound 2a showed levels of permeability close to the

value of the positive control (propranolol) used in a porcine

brain endothelial cell (PBEC) blood−brain barrier (BBB)

model in which both active and passive permeability (as well as

P-glycoprotein (P-gp) and multidrug resistance protein

(MRP)-superfamily counter-transport) can be assessed.

Despite the low stability in whole blood, these data indicate

the potential of this compound class for CNS delivery. The

high levels of CoA generated from 2a and its excellent brain

permeability allowed a stable isotope-labeled isomer of the

compound to be used at a high dose in an in vivo proof-of-

concept study. The goal of this investigation was to evaluate

whether CoA that originated from 2a-derived PPA could be

detected in mouse brain following a single oral dose of the

isotopically labeled 2a (500 mg/kg). The use of compound 2a

labeled both on its β-alanine fragment (with three ¹³C atoms

While the speciﬁc sequence of (bio-catalytic) hydrolysis

events that free the eﬀective PPA precursor of CoA from

compounds 2a−i are not known, esterase-mediated hydrolysis

of the alkoyl or aroyl-oxymethyl side chains in these

compounds is believed to serve as the trigger for release of

the free phosphate group. It remains a mystery as to why the

trans isomer of 2a is more eﬃcacious than the cis.

Paradoxically, it is possible that the trans isomer is the more

stable one, and thus survives to enter the cell and provide PPA

to the CoA synthetic machinery. This hypothesis is bolstered

18

and one ¹⁵N atom) and on its phosphate (one P O group)

allowed insight into the fate of the administered prodrug by

mass spectrometry analysis of brain tissue samples. CoA

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incorporating a 6 atomic mass unit (amu) increase in

molecular ion over endogenous unlabeled CoA ([M + H]⁺=

774.1344 vs 768.1230, respectively) must derive from PPA

formed directly from the administered compound. In contrast,

CoA that incorporates a +4 amu increase in molecular ion over

unlabeled CoA ([M + H]⁺= 772.1301 vs 768.1230,

respectively) may derive either from H₂O-mediated ¹⁸O to

¹⁶O exchange in PPA or may reﬂect complete hydrolysis of the

phosphate group and re-phosphorylation by wild-type PanK (a

pathway that is viable in the wild-type animals used) to

regenerate PPA. In both cases, the pantothenic acid moiety

incorporated into CoA originated from the administered

labeled 2a. As the data in Table 2 highlight, no CoA derived

Table 3. CoA Regeneration in PanK2 Knockdown Cells and

Human Blood Stability for 1,3,2-Dioxaphosphinane

Heterocycles Containing a POM Side Chain and

Structurally Diverse Pantothenate Ester Functionality

Table 2. Percentage of Free CoA Detected in Mouse Brain

Following Oral Administration of Isotopically Labeled

Compound 2a That Is (a) Derived from Endogenous

Phosphopantothenic Acid (Unlabeled); (b) Resulting from

Phosphopantothenic Acid Derived from Compound 2a (+4

amu); (c) Derived from Phosphopantothenic Acid from

Compound 2a (+6 amu)

a

free CoA (%)

time (h)

unlabeled

+4 amu

+6 amu

0

1

6

100

92

0

2.7

12.9

0

5.3

2.1

24

85

a

Values are corrected based on the known % of ¹⁸O content of

Compound 9b is a cis/trans mixture (5:95) of diastereoisomers. All

b

other compounds are single diastereoisomers/enantiomers. Human

plasma stability.

compound 2a dosed.

from the labeled phosphopantothenic acid in the prodrug was

found in brain at the 1 h time point. However, 6 h post dose,

both CoA +6 and CoA +4 amu were detected and together

comprised 15% of the free brain CoA at 24 h. Though no eﬀort

was made to quantitate actual CoA concentrations or to follow

the full pharmacokinetic and metabolic behavior of compound

2a, the ﬁnding that ca. 5% of CoA (at 6 h) retains all labeled

atoms from the administered prodrug was encouraging. These

data demonstrate that compound 2a is competent in supplying

the brain PPA that can ultimately be transformed to CoA,

providing a proof of principle that cyclic phosphate prodrugs

can eﬀectively deliver their payload to the CNS.

The demonstrated ability of prodrugs such as 2a to

regenerate high CoA levels in a PANk2^−/−model and to

deliver intact PPA to the brain following oral administration in

mouse provided additional impetus for the optimization of this

compound class. Although these compounds need to be labile

enough to generate PPA/CoA, the very rapid turnover in the

liver and blood seen for compounds in Table 1 clearly needed

to be addressed. The ﬁnding that the carboxylic acid derivative

9a (Table 3) showed good stability in human blood, but

generated low amounts of CoA in the PANk2^−/−assay, led to

an exploration of changes at the pantothenate methyl ester.

This allowed the lipophilic ester functionality in the

alkoyloxymethyl side chain that had proved highly beneﬁcial

for CoA generation to be retained. These ester analogues were

anticipated to be more resistant to esterase-mediated

hydrolysis. A panel of structurally diverse ester modiﬁcations

was investigated, and a subset of these are illustrated in Table

3. As hypothesized, increasing the steric impact of the ester

moiety increased blood stability (e.g., T_1/2between 1 and 4 h

for the tert-butyl analogue 9b). However, it also signiﬁcantly

attenuated the generation of CoA in the PANk2^−/−cell model.

The relationship between blood stability and CoA production

is also apparent in results for the less sterically encumbered

ethyl analogue 9c that combines good CoA production in

PANk2^−/−neuroblastoma cells with marginally improved

stability in human blood (T_1/2∼ 15 min). Eﬀorts to balance

CoA production and blood stability by changing the

stereochemical impact and physicochemical properties of the

ester ultimately proved unsuccessful. Introduction of hetero-

cyclic systems (e.g., 9d, 9e), basic amines (e.g., 9g), or polar

groups (e.g., sulfone 9f) typically generated prodrugs that

retained good CoA in the PANk2^−/−assay, but disappointingly

none of these compounds improved blood stability with

respect to 2a.

With changes to the pantothenic acid ester functionality

having proven unsuccessful as a strategy to simultaneously

improve both CoA production in the PANk2^−/−cell model

with in vitro drug metabolism and pharmacokinetic (DMPK)

properties, it was apparent that changes in the POM side chain

needed to be found to achieve this goal. Comparison of

compounds 2f and 2h illustrated that introduction of

heteroatoms into the ester functionality could be tolerated

with respect to CoA generation, and a more extensive

exploration of heterocycle containing ester functionality was

made. Table 4 shows a number of ﬁve-membered aromatic

heterocycle replacements for the pivaloyl group that were

initially evaluated as diastereomeric mixtures in the PANk2^−/−

assay and in a human whole blood stability assay. The

consistent chromatographic behavior of the individual cis and

trans-isomers in this series allowed data on the stability of the

individual isomers to be obtained following incubation of the

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Table 4. CoA Regeneration in PanK2 Knockdown Cells and Human Blood Stability for 1,3,2-Dioxaphosphinane Heterocycles

Containing Optimized Heteroaryloxymethyl Side Chains

a

Human plasma stability.

mixture. Thiazole 2k and isothiazole 2l, generated 88- and 37-

fold improved CoA levels, respectively, in the PANk2^−/−assay

but were rapidly degraded in human blood. The introduction

of substituents at the carbon α to the carbonyl of 2k was

explored to create a more sterically encumbered environment

around the ester group. Compounds 2m and 2n highlight the

success of this strategy with regard to blood stability, with the

trans-isomers clearly emerging as more stable than the cis. The

increased blood stability of the more sterically crowded methyl

analogue 2m and tert-butyl analogue 2n came at the expense of

progressively attenuating the production of CoA in the

PANk2^−/−cell model, with the latter compound generating a

signiﬁcantly lower CoA response than 2k. The oxazole

analogue of 2o was prepared and found to show a similar

proﬁle with blood stability somewhat lower for the trans-

isomer and marginally improved for the cis. Of the hetero-

cycles, explored pyrazoles provided the most attractive overall

proﬁles. Analogues 2p−r, which contain an N-methyl

substituent aimed at mimicking the eﬀect of methyl

functionality in 2m, gave good to high levels of CoA.

Furthermore, the trans-isomers of these compounds consis-

tently showed half-lives between 1 and 4 h in human whole

blood. Interestingly, pyrazoles retained good CoA levels and

blood stability even in the absence of methyl functionality on

the ring nitrogen atom, as illustrated with 2s.

Table 5. In Vitro Assay Data and Physical Properties for

Optimized PPA Prodrugs

trans-2r

trans-2m

trans-2s

PANk2^−/−assay CoA (fold)

physical state

human blood T_1/2(h)

human hepatocytes T_1/2(h)

BBB P_app(10⁻⁶cm/s)

14

oil

>1

0.3

11.8

40

101

solid

>1

0.5

5.3

oil

>1

0.5

5.1

neuroblastoma cells from the individual isomers was similar to

that of the mixture for trans-2m and trans-2s but was markedly

lower for trans-2r. Compounds trans-2r and trans-2m were

isolated as oils, while importantly compound trans-2s was

solid. For all three compounds, the human blood stability data

were in line with the values measured for each compound in

the diastereomeric mixture. Human hepatocyte half-lives were

determined to be in the 30 min range, somewhat improved

with respect to the original lead 2a. The presence of more

heterocyclic functionality in place of the pivaloyl group in 2a

somewhat impaired BBB permeability in the PBEC assay, but

good (trans-2r, P_app= 11.8 × 10⁻⁶cm/s) to moderate (trans-

2m, P_app= 5.1 × 10⁻⁶cm/s; trans-2s, P_app= 5.3 × 10⁻⁶cm/s)

apparent permeability (P_app-values) was nonetheless retained.

As an alternative to the POM prodrug strategy, phospho-

triesters were explored. Simplifying the POM fragment in this

way is expected to aﬀect the mechanism by which the prodrug

The single trans-isomers of 2m, 2r, and 2s were isolated and

proﬁled as described in Table 5. CoA production in PANk2^−/−

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Table 6. CoA Regeneration in PanK2 Knockdown Cells and Human Blood Stability for 1,3,2-Dioxaphosphinane Heterocycles

Containing an Aryloxy Side Chain

a

Determined on the diasteroisomers mixture.

releases PPA, while POM-like side chains are expected to

degrade after hydrolysis of their terminal ester functionality,

and simple phosphates more likely release PPA by direct

hydrolytic cleavage at phosphorus. Cyclic 1,3,2-dioxaphosphi-

nane PPA prodrugs containing simple alkyl phosphates in place

of the POM side chain consistently failed to generate CoA in

PanK2-deﬁcient cells (data not shown). This ﬁnding likely

reﬂects the relatively poor leaving group potential for alkoxy

groups in phosphates. Phenoxy leaving groups on phosphorus

that are activated by electron-withdrawing functionality in their

para-position were explored (Table 6) and proved more

promising. Although compounds with a simple phenoxy

leaving group did not show increased generation of CoA in

the PANk2^−/−assay (data not shown), the presence of even

moderately electron-withdrawing substituents, such as F

(compound 3a), were found to be suﬃcient to generate

signiﬁcant increases in CoA level. The markedly higher CoA

increase from the cis-isomer 3a with respect to the trans-3b

was a consistent trend and could reﬂect that hydrolytic

cleavage at phosphorus is more facile from attack at the less

hindered face of the 1,3,2-dioxaphosphinane ring or that the

trans isomer is more stable due to the anomeric eﬀect. No

direct correlation between the electron-withdrawing potential

(Hammet sigma coeﬃcient³¹) of the phenoxy 4-substituent in

compounds 3a−g and the CoA levels generated in the

PANk2^−/−assay could be identiﬁed. The strongly electron-

withdrawing nitrile analogue, 3c, generated relatively low levels

of CoA in this assay, while 3d and 3e generated CoA levels

within 2−3-fold of the simple 4-F analogue 3a despite being

activated with more powerful electron-withdrawing function-

ality. Phenoxy leaving groups that would ultimately generate

known/benign byproducts following their displacement from

the prodrug were explored, and though relatively low levels of

CoA were generated from 3f (that ultimately should release

salicylic acid), a surprisingly robust eﬀect was noted for 3g that

does not contain an electron-withdrawing group on the phenyl

ring and should generate tyrosine methyl ester as a byproduct.

Compound 3g was also the only compound in this series that

could be isolated as a solid. When incubated in human whole

blood, all compounds in Table 6 (aside from 3f which was not

tested) showed human whole blood stability ≥0.5 h, with

longer half-lives (1−4 h) being measured for 3a and 3d.

Further proﬁling of analogue 3c demonstrated that its half-life

in human hepatocytes was comparable to that found in blood

and that it had excellent BBB permeability with P_app= 16 ×

10⁻⁶cm/s, close to that of the assay’s positive control.

CONCLUSIONS

■

We have reported here two new series of cyclic phosphate

prodrugs of PPA and have described optimization work aiming

to balance compound proﬁles between release of PPA for

conversion to CoA together with in vitro stability in blood and

liver. Early compounds were able to generate very high CoA

levels in a PANk2^−/−neuroblastoma cell model but were

rapidly turned over in blood and hepatocytes. Improving in

vitro metabolic stability consistently attenuated the levels of

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harvested, collected in a 15 mL falcon tube, and centrifuged at 200g

for 5 min at 4 °C. The supernatant was removed, and the cell pellet

was washed in ice-cold phosphate-buﬀered saline, centrifuged again,

then rapidly frozen in liquid nitrogen, and stored at −80 °C until

liquid chromatography-tandem mass spectrometry (LC-MS/MS)

analysis for CoA formation. Samples were reconstituted with

ammonium acetate buﬀer (pH 5.1) containing dextrorphan (50 ng/

mL) as the internal standard, stirred for 2 min, sonicated in an

ultrasonic bath for 1 min, and stirred again for 1 min before LC-MS/

MS analysis for determination of intracellular CoA levels. Intracellular

CoA concentration was calculated considering an intracellular volume

of 10⁶cells equal to 2 μL. The measurement of intracellular volume of

cultured cells was performed following the distribution of [¹⁴C]urea

and [¹⁴C]mannitol with time.

In Vivo Mouse Study to Determine Brain Levels of Stable

Isotope Labeled 2a Following Oral Administration. C57BL6

mice (three animals per time point) received a single oral dose of 500

mg/kg isotopically labeled 2a, and brain samples were collected at 0,

1, 6, and 24 h post dose. Samples were analyzed by UPLC-

quadrupole-time-of-ﬂight mass spectrometry, with the electrospray

source scanning in positive-ion mode. The relative percentages of +6

amu CoA, +4 amu CoA, and unlabeled CoA were determined from

the peak intensity of the corresponding molecular ions, where the sum

of the three peak intensities represented 100%. The identity of each

peak was conﬁrmed by matching the fragment ions from the MS/MS

fragmentation pattern to the indicator ions of the CoA standard

accurate mass. Brain tissues were homogenized in a Precellys

homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France)

using 3 mL of water for 1 g of tissue (v/w). Protein was precipitated

by adding 500 μL of MeOH to 100 μL of a homogenate (1:6

dilution), mixed, and centrifuged. The supernatants (200 μL) were

dried under nitrogen, reconstituted with 100 μL of 10 mM

ammonium acetate (pH ∼ 5) and injected (10 μL) into the LC-

MS system.

CoA Determination. An Acquity UPLC (Waters) system was used.

Samples were chromatographed on a Luna C₁₈column (50 × 2.0

mm²; 5 μm), with a 0.4 mL/min ﬂow rate and a 10 μL injection

volume. The mobile phases were (A), 10 mM ammonium acetate in

H₂O, pH 6.8, and (B) acetonitrile/2-propanol (9:1; v/v). The

gradient was started with 2% (B) and was increased linearly to reach

20% (B) at 5 min, the gradient was ramped to reach 90% (B) at 5.1

min, and kept isocratic at 90% (B) through 7.1 min. Conditions were

returned to initial 2% (B) at 7.2 min, and the column was equilibrated

at initial conditions until the run reached 11.2 min. The detector was

a Waters QTOF Synapt mass spectrometer, with an electrospray ion

source operating in the positive scanning mode acquiring in full-scan

mode (accuracy <5 ppm). The following exact masses were

determined: 768.1230 for CoA, 772.1301 for CoA +4 amu, and

774.1344 for CoA +6 amu. The CoA levels obtained by MS/MS

analyses were corrected for ¹⁸O isotopic incorporation but were not

adjusted for the 1 and 2% of ¹²C and ¹⁴N, respectively, in labeled 2a.

The amount of CoA formed after dosing 2a was calculated using the

observed isotopic pattern and correcting for the ¹⁸O content of

labeled 2a as follows: The % CoA derived from intact compound 2a-

PPA = (100/¹⁸O% in prodrug) × % +6 amu, while the % CoA derived

from compound 2a-derived PA (or from oxygen exchange) = % +4

amu − ((100 − ¹⁸O% in prodrug)/¹⁸O% in prodrug) × % +6 amu,

where % +6 and % +4 amu are the percents of the +6 and +4 amu [M

+ H]⁺CoA ions experimentally determined by high-resolution mass

spectrometry (HRMS) from the isotopic pattern in brain samples.

The levels of endogenous CoA in brain samples were estimated using

the standard addition method, where aliquots of the untreated

samples were spiked with diﬀerent known amounts of the analytical

standard (CoA) and all aliquots including the nonspiked sample (pre-

dose) were analyzed in the same way. Due to the limited dynamic

range of the QTOF analyzer, a ﬁve-point calibration curve (1000,

2000, 4000, 8000, and 16 000 ng/mL) was run.

CoA in the cellular model, where the release of PPA is also

dependent on metabolism of the prodrugs. However,

optimized compounds such as 2m and 2s demonstrate

improved capacity to generate CoA in this brain cell model

with respect to ﬁrst-generation prodrugs. In addition, both 2s

and trans-2s are solids addressing another vexing issue of

Fosmetpantotenate. Importantly, given the known deﬁciency

of the PanK2 enzyme in the CNS of PKAN patients, optimized

compounds retained acceptable levels of blood−brain barrier

penetration in a mammalian in vitro model of brain

permeability. The ability of cyclic prodrugs of PPA to both

reach the brain and to liberate PPA for productive biosynthesis

to CoA following oral administration was demonstrated in

mouse for an early second-generation prodrug 2a despite its

suboptimal metabolic stability. The ﬁnding that at least 5% of

CoA in mouse brain 6 h after an oral dose of 2a contains the

intact PPA moiety from this compound provides a validation

for the PANk2^−/−assay, and the screening funnel reported, as

a means to identify viable CoA prodrugs, highlighting the

potential of these new prodrugs as therapeutic agents.

EXPERIMENTAL PROCEDURES

■

All solvents and chemical reagents were purchased with purity grades

>90% from chemical suppliers and, unless otherwise stated, were used

as received. β-Alanine +4 amu (¹⁵Nx1, ¹³Cx3, CAS number: 285978-

07-6) was purchased from the Sigma-Aldrich chemical company

(product code: 490822, 98 atom %) and was used without further

puriﬁcation. All reactions were followed by thin-layer chromatography

(TLC) analysis (TLC plates GF254, Merck) or ultra performance

liquid chromatography (UPLC-MS). The purity was determined

using UPLC-MS analysis conducted on a Waters Acquity UPLC

system with both diode array detection and electrospray (+ve and −ve

ion) MS detection. The stationary phase was a Waters Acquity UPLC

BEH C₁₈1.7 μm, 2.1 × 50 mm². The mobile phase was a mixture of

H₂O containing 0.1% formic acid (A) and AcCN containing 0.1%

formic acid (B) in the following linear gradient: 90% A (0.1 min),

90−0% A (2.5 min), 0% A (0.3 min), 90% A (0.1 min); the ﬂow rate

was 0.5 mL/min. Purity of all compounds is >95% except for

isotopically labeled 2a, which has a purity of 92%. Reversed-phase

(C₁₈) column chromatography was carried out using as mobile phase

H₂O containing 0.1% of TFA and AcCN containing 0.1% of TFA.

NMR spectra were collected on Bruker instruments operating at the

indicated frequencies. Diasteromeric ratios were determined by ³¹P

NMR. Melting points were determined for solid single diaster-

eoisomers using a Buchi B-540 apparatus and are reported as the

mean of three independent measurements. All studies using animals

were conducted in accordance with protocols approved by the

Institutional Animal Care and Use Committee (IACUC) of IRBM

Science Park, in full compliance with the EU Directive 63/2010 and

its Italian transposition as well as with all applicable Italian legislation

and guidelines. IRBM Science Park is authorized by the Italian

Ministry of Health and by local veterinary authority to house, breed,

and use laboratory rodents for scientiﬁc purposes.

In Vitro Studies in PanK2 Knockdown Cell Line. The in vitro

model to evaluate the eﬃcacy of analogues was developed using the

human neuroblastoma IMR32 cell line (ATCC) described in the

Supporting Information. Cells were cultured in minimum essential

medium (MEM) (Invitrogen) supplemented with 10% fetal bovine

serum, 2 mM glutamine, 1% penicillin−streptomycin, 1 mM sodium

pyruvate, 1 mM nonessential amino acids, 1.5 g/L sodium

bicarbonate, and 1 μg/mL puromycin. The cells were plated on 12-

well culture plates at a density of 0.2 × 10⁶cells per well. After 72 h,

test compounds were added as freshly dissolved dimethyl sulfoxide

(DMSO) stock solutions (the ﬁnal DMSO concentration was 0.1%,

v/v). The cells were incubated for 24 h at 37 °C, then the treatment

was repeated after 24 h with newly dissolved test compound or

control vehicle. The cells were further incubated for 24 h, then

Methyl 3-((4R)-2-(((S)-1-Methoxy-1-oxopropan-2-yl)amino)-

5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane-4-

carboxamido)propanoate (1). A stirred solution of methyl 3-

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((2R)-2-hydroxy-4-(((((S)-1-methoxy-1-oxopropan-2-yl)amino)-

(phenoxy)phosphoryl)oxy)-3,3-dimethylbutanamido)propanoate³²

(Fosmetpantotenate, 500 mg, 1.05 mmol) in DCM (10.5 mL) was

treated slowly with Et₃N (0.29 mL, 2.1 mmol), and stirring was

continued for 16 h. The solvent was removed in vacuo, and the

residue was puriﬁed by SiO₂chromatography (0−5% MeOH/EtOAc)

to aﬀord a mixture of diastereoisomers (46:54*) of the title

compound (200 mg, 50%) as a colorless sticky solid. ¹H NMR

(400 MHz, CDCl₃, 300 K) δ 7.28* and 7.14 (bs, 1 H), 4.76 and 4.51*

(d, J = 1.5 Hz, J* = 5.0 Hz, 1H), 4.36 (dd, J = 11.5, 1.6 Hz, 0.5H)

4.15−4.00 (m, 2H), 3.92* (dd, J = 11.57, 20.10 Hz, 0.5 H), 3.79 (s,

3H), 3.71 (s, 3H), 3.61−3.46 (m, 3H), 2.60 (m, 2H), 1.50 and 1.46*

(d and d*, J = 7.2 Hz, J* = 6.8 Hz, 3H), 1.19* and 1.18 (s, 3H), 1.13*

and 1.09 (s, 3H). ³¹P NMR (162 MHz, CDCl₃, 300 K) δ 4.70 and

1.59*; UPLC t_R0.95* min and 1.03 min; MS (ES⁺) m/z 381 (M +

H)⁺.

Methyl 3-((4R)-2-(Benzyloxy)-5,5-dimethyl-2-oxido-1,3,2-di-

oxaphosphinane-4-carboxamido)propanoate (5). Methyl 3-

[[(2R)-2,4-dihydroxy-3,3-dimethyl-butanoyl]amino]propanoate (4)

(10.0 g, 42.9 mmol) was dissolved in THF (112 mL) and treated

with a solution of Et₃N (12.6 mL, 90 mmol) in THF (15 mL). A

solution of POCl₃(4.41 mL, 47.16 mmol) in THF (10 mL) was

added dropwise at −78 °C, and after 0.5 h, the cooling bath was

removed and the reaction mixture was warmed to ambient

temperature over 1 h. The mixture was cooled to −78 °C, then

treated dropwise sequentially with a solution of TEA (12.6 mL, 90

mmol) in THF (15 mL) and benzyl alcohol (5.1 mL, 47.16 mmol) in

THF (10 mL). The reaction was left to reach ambient temperature

and stirred for 12 h. DCM and H₂O were added, and the organic layer

was separated and washed with 5% aqueous citric acid solution, H₂O,

and brine, and then dried over Na₂SO₄. Filtration and solvent removal

in vacuo aﬀorded a residue that was puriﬁed by column

chromatography on SiO₂(0−90% EtOAc in DCM) to aﬀord a single

diastereoisomer of the title compound (4.17 g, 25%) as a white solid.

¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.45−7.39 (m, 5H), 6.70 (bs,

1H), 5.26−5.22 (m, 2H), 4.74 (d, J = 1.5 Hz, 1H), 4.36−4.32 (dd, J =

3.0, 3.2 Hz, 1H), 3.86−3.78 (m, 1H), 3.68 (s, 3H), 3.49−3.47 (m,

2H), 2.51 (t, J = 6.4 Hz, 2H), 1.16 (s, 3H), 1.06 (s, 3H); ³¹P NMR

(162 MHz, CDCl₃, 300 K) δ −3.13; UPLC t_R1.42 min; MS (ES⁺) m/

z 386 (M + H)⁺.

Methyl 3-((4R)-2-Hydroxy-5,5-dimethyl-2-oxido-1,3,2-diox-

aphosphinane-4-carboxamido)propanoate (6). Compound 5

(2.90 g, 7.53 mmol) was dissolved in EtOAc (50 mL) and treated

with Pd/C (5% w/w, 0.58 g). The mixture was stirred 4 h under an

atmosphere of hydrogen gas. The catalyst was ﬁltered, and the solvent

was evaporated to aﬀord the title compound (2.13 g, 96%) as an oil

that was used directly in subsequent steps. ¹H NMR (400 MHz,

CDCl₃, 300 K) δ 7.99 (m, 1H), 4.40 (s, 1H), 4.0−3.98 (m, 1H),

3.77−3.69 (m, 1H), 3.57 (s, 3H), 3.41−3.29 (m, 2H), 2.48 (m, 2H),

0.95 (s, 3H), 0.91 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−6.48 and −6.63.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Pivalate

(2a). A solution of compound 6 (2.12 g, 7.20 mmol) in DMF (29

mL) was cooled to −78 °C and treated with DIEA (3.51 mL, 20.15

mmol) and chloromethyl-2,2-dimethylpropanoate (1.84 g, 12.23

mmol). The cooling bath was removed, and the mixture was warmed

over 1 h to 20 °C and then heated at 80 °C for 16 h. The cooled

mixture was concentrated in vacuo, and the residue was puriﬁed by

ﬂash chromatography over C₁₈using H₂O/MeCN, to furnish a

mixture of diasteroisomers (44:56*) of the title compound (0.89 g,

1

30%) as an oily solid. H NMR (400 MHz, CDCl₃, 300 K) δ 7.13−

7.05* and 7.00−6.85 (m, 1H), 5.78−5.76* and 5.71−5.68 (m, 1H),

4.77* and 4.55 (s, 1H), 4.35−4.27* and 4.09−4.17 (m, 2H), 3.87−

3.81 (m, 1H), 3.7 (s, 3H), 3.64−3.47 (m, 2H), 2.62−2.49 (m, 2H),

1.19* and 1.16 (s, 9H), 1.18* and 1.12 (s, 3H), 1.11 and 1.09* (s,

3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −5.34 and −9.26*;

UPLC t_R1.42 min and 1.46* min; MS (ES⁺) m/z 410 (M + H)⁺.

(((2R,4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-di-

methyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Piva-

late (cis-2a). The title compound was obtained by RP-HPLC

1

separation of compound 2a H NMR (400 MHz, CDCl₃, 300 K) δ

7.13 (bs, 1H), 5.79 (dd, J = 4.8, 11.4 Hz, 1H), 5.70 (dd, J = 4.8, 14.0

Hz, 1H), 4.78 (d, J = 1.7 Hz, 1H), 4.34 (dd, J = 3.9, 11.8 Hz, 1H),

3.86 (dd, J = 11.4, 22.8 Hz, 1H), 3.71 (s, 3H), 3.65−3.52 (m, 2H),

2.61−2.58 (m, 2H), 1.27 (s, 9H), 1.20 (s, 3H), 1.10 (s, 3H); ³¹P

NMR (162 MHz, CDCl₃, 300 K) δ −6.69; UPLC t_R1.44 min; MS

(ES⁺) m/z 410 [M + H]⁺.

(((2S,4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-di-

methyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Piva-

late (trans-2a). The title compound was obtained as described above

for compound cis-2a ¹H NMR (400 MHz, CDCl₃, 300 K) δ 6.93 (bs,

1H), 5.74−5.71 (m, 1H), 5.70−5.68 (m, 1H), 4.56 (s, 1H), 4.17 (d, J

= 10.9 Hz, 1H), 3.84 (dd, J =11.4, 24.9 Hz, 1H), 3.72 (s, 3H), 3.65−

3.59 (m, 1H), 3.57−3.50 (m, 1H), 2.59−2.55 (m, 2H), 1.24 (s, 9H),

1.14 (s, 3H), 1.12 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−10.6; UPLC t_R1.48 min; MS (ES⁺) m/z 410 [M + H]⁺; M.P. 65 °C.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 2-Ethylbu-

tanoate (2b). Following the same procedure as 2a, treatment of 6

(0.33 g, 1.11 mmol) with chloromethyl-2-ethylbutanoate (0.31 g, 1.90

mmol) aﬀorded a mixture of diastereomers (11*:89) of the title

1

compound (193 mg, 39%) as a colorless oil. H NMR (400 MHz,

CDCl₃, 300 K) δ 6.93 (bs, 1H), 5.81−5.75* and 5.73−5.70 (m, 2H),

4.77* and 4.57 (s, 1H), 4.34* and 4.17 (dd* and d, J* = 3.6, 10.9 Hz,

J = 10.9 Hz, 1H), 3.83 (dd, J =12.2, 25.5 Hz, 1H), 3.71 and 3.70* (s,

3H), 3.65−3.58 (m, 1H), 3.55−3.47 (m, 1H), 2.59−2.55 (m, 2H),

2.34−2.26 (m, 1H), 1.72−1.53 (m, 4H), 1.19* and 1.13 (s, 3H), 1.12

and 1.09* (s, 3H), 0.91 (t, J = 7.3 Hz, 6H); ³¹P NMR (162 MHz,

CDCl₃, 300 K) δ −5.42* and −9.45; UPLC t_R1.58 and 1.61* min;

MS (ES⁺) m/z 446 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Isobuty-

rate (2c). 2-Methylpropanoic acid (76.9 mg, 0.87 mmol) and

Cs₂CO₃(283.7 mg, 0.87 mmol) were dissolved in DMF (3 mL) and

then treated with compound 7 (250.0 mg, 0.73 mmol). The reaction

mixture was stirred for 5 h, then ﬁltered through a pad of SiO₂, and

concentrated. The residue was puriﬁed by ﬂash chromatography on

C₁₈using H₂O/CH₃CN as an eluent to aﬀord a 78:22* mixture of

diastereoisomers of the title compound (33.6 mg, 12%) as a colorless

oil. ¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.11 and 6.93* (t, J = 5.2

Hz, 1H), 5.79 and 5.76 and 5.73−5.66* (dd and m*, J = 4.8, 11.4 Hz,

2H), 4.76 and 4.56* (d and s*, J = 2.2 Hz, 1H), 4.33 and 4.16* (dd

and d*, J = 3.5, 11.4 Hz, J* = 11.4 Hz, 1H), 3.85 and 3.83* (dd, J

=11.8, 23.2 Hz, J* = 11.4, 24.9 Hz, 1H), 3.71* and 3.70 (s, 3H),

3.63−3.49 (m, 2H), 2.71−2.64 (m, 1H), 2.62−2.55 (m, 2H), 1.23

and 1.21* (s, 3H), 1.22 and 1.20* (s, 3H), 1.19 and 1.13* (s, 3H),

1.11* and 1.09 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−6.64, −10.70*; UPLC t_R1.19 and 1.22* min; MS (ES⁺) m/z 418

[M + Na]⁺.

Methyl 3-((4R)-2-(Chloromethoxy)-5,5-dimethyl-2-oxido-

1,3,2-dioxaphosphinane-4-carboxamido)propanoate (7).

Compound 6 (3.29 g, 11.4 mmol) was dissolved in H₂O (34 mL),

then tetrabutylammonium hydrogen sulfate (0.37 g, 1.11 mmol) and

NaHCO₃(3.74 g, 44.6 mmol) were added sequentially at 0 °C. The

mixture was stirred for 10 min, then a solution of chloromethyl

sulfurochloridate (2.21 g, 13.37 mmol) in DCM (54 mL) was added.

The reaction was warmed to 20 °C and stirred for 16 h. The organic

phase was separated, washed with brine, and dried. Filtration and

solvent removal in vacuo aﬀorded a residue that was puriﬁed by ﬂash

chromatography on SiO₂using petroleum ether (PE)/EtOAc as

eluent, aﬀording a mixture of diastereoisomers (58:42*) of the title

1

compound (510 mg, 10%) as a colorless oil. H NMR (400 MHz,

CDCl₃, 300 K) δ 7.01 and 6.96* (bs, 1H), 5.86−5.72 (m, 2H), 4.80

and 4.68* (d and s*, J = 2.4 Hz, 1H), 4.38 and 4.27 (dd and d*, J =

3.6, 12.2 Hz, J* = 10.9 Hz, 1H), 4.0−3.87 (m, 1H), 3.73 (s, 3H),

3.67−3.55 (m, 2H), 2.62−2.58 (m, 2H), 1.23 and 1.19* (s, 3H), 1.16

and 1.14* (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −5.00,

−8.93*; UPLC t_R1.13* and 1.15 min; MS (ES⁺) m/z 366 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Butyrate

K

https://dx.doi.org/10.1021/acs.jmedchem.0c01531

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

(2d). Following the same procedure as 2a, treatment of 6 (300.0 mg,

1.01 mmol) with chloromethyl butanoate (236.0 mg, 1.72 mmol)

aﬀorded a 23*:77 mixture of diastereomers of the title compound

(128.0 mg, 32%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃, 300

K) δ 7.12* and 6.94 (bs, 1H), 5.80−5.75* and 5.73−5.68 (dd* and

m, J* = 4.9, 8.2 Hz, 2H), 4.77* and 4.57 (d* and s, J* = 2.4 Hz, 1H),

4.34* and 4.17 (dd* and d, J* = 3.6, 12.2 Hz, J = 10.9 Hz, 1H), 3.90−

3.80 (m, 1H), 3.71 and 3.70* (s, 3H), 3.65−3.49 (m, 2H), 2.60−2.55

(m, 2H), 2.43 (t, J = 7.3 Hz, 1H), 2.37 (t, J = 7.3 Hz, 1H), 1.73−1.67

(m, 2H), 1.19* and 1.13 (s, 3H), 1.12 and 1.10* (s, 3H), 1.0−0.95

(m, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −6.68* and −10.84;

UPLC t_R1.34 and 1.36* min; MS (ES⁺) m/z 418 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 1-Carba-

moylcyclopropane-1-carboxylate (2e). Following the same

procedure as 2c, treatment of 7 (200.0 mg, 0.58 mmol) with 1-

carbamoylcyclopropanecarboxylic acid (90.1 mg, 0.70 mmol) aﬀorded

a mixture of diastereomers (86:14*) of the title compound (65 mg,

title compound (82 mg, 6%) as a colorless oil. H NMR (400 MHz,

CDCl₃, 300 K) δ 7.13* and 6.96 (bs, 1H), 5.79−5.69 (m, 2H), 5.03−

4.93 (m, 1H), 4.81* and 4.61 (d* and s, J* = 2.4 Hz, 1H), 4.39* and

4.22 (dd* and d, J* = 3.6, 10.9 Hz, J = 10.9 Hz, 1H), 3.90 (dd, J =

12.2 Hz, 23.1 Hz, 1H), 3.73 (s, 3H), 3.69−3.49 (m, 2H), 2.63−2.58

(m, 2H), 1.37 (d, J = 6.1 Hz, 3H), 1.35 (d, J = 6.1 Hz, 3H), 1.22* and

1.16 (s, 3H), 1.15 and 1.12* (s, 3H); ³¹P NMR (162 MHz, CDCl₃,

300 K) δ −5.89 and −9.78*; UPLC t_R1.31 and 1.35* min; MS (ES⁺)

m/z 434 [M + Na]⁺.

1-(((2R,4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-di-

methyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)ethyl Piva-

late (2j). Following the same procedure as 2a, treatment of 6

(280.0 mg, 0.95 mmol) with 1-chloroethyl 2,2-dimethylpropanoate

(249.8 mg, 1.52 mmol) aﬀorded a mixture of diastereomers (43*:57)

both as a single trans isomer at phosphorus of the title compound (23

1

mg, 5%) as a colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ

7.02−6.93 (bs, 1H), 6.55−6.48 (m, 1H), 4.57 and 4.55 (s, 1H), 4.20

and 4.15 (d and d, J = 11.2, 11.2 Hz, 1H), 3.83 (dd, J = 11.4, 25.2 Hz,

1H), 3.72 (s, 3H), 3.68−3.58 (m, 1H), 3.56−3.46 (m, 1H), 2.61−

2.55 (m, 2H), 1.63 and 1.62 (s and s, 3H), 1.24 and 1.21 (s and s,

9H), 1.14 and 1.12 (s and s, 6H); ³¹P NMR (162 MHz, CDCl₃, 300

K) δ −12.99 and −12.99; UPLC t_R1.57 min; MS (ES⁺) m/z 446 [M

+ Na]⁺.

1

26%) as a colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 8.41

and 8.33* (bs, 1H), 7.07 and 6.97* (bs, 1H), 5.99−5.93 (bs, 1H),

5.82−5.70 (m, 2H), 4.79 and 4.60* (d and s*, J = 2.4 Hz, 1H), 4.36

and 4.17* (dd and d*, J = 4.87, 12.2 Hz, J* = 12.2 Hz, 1H), 3.91 and

3.90* (dd, J = 12.2, 23.1 Hz, J* = 10.9, 25.5 Hz, 1H), 3.73 (s, 3H),

3.61−3.57 (m, 2H), 2.62−2.59 (m, 2H), 1.88−1.75 (m, 4H), 1.22

and 1.17* (s, 3H), 1.15* and 1.11 (s, 3H); ³¹P NMR (162 MHz,

CDCl₃, 300 K) δ −4.99, −9.69*; UPLC t_R0.96 min; MS (ES⁺) m/z

459 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Benzoate

(2f). Following the same procedure as 2a, treatment of 6 (300.0

mg, 1.02 mmol) with chloromethyl benzoate (294.7 mg, 1.72 mmol)

aﬀorded a mixture of diastereomers (17*:83) of the title compound

(128 mg, 30%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃, 300 K)

δ 8.14−8.07 (m, 2H), 7.67−7.61 (m, 1H), 7.53−7.47 (m, 2H), 7.05*

and 6.86 (bs, 1H), 6.04* and 5.99−5.92 (dd* and m, J* = 4.9, 12.2

Hz, 2H), 4.80* and 4.59 (d* and s, J* = 2.4 Hz, 1H), 4.35* and 4.17

(dd* and d, J * = 3.5, 10.9 Hz, J = 10.9 Hz, 1H), 3.89* and 3.82 (dd, J

=10.9, 24.3 Hz, 1H), 3.71 (s, 3H), 3.58−3.50 (m, 1H), 3.43−3.35 (m,

1H), 2.55−2.45 (m, 2H), 1.20* and 1.10 (s, 6H); ³¹P NMR (162

MHz, CDCl₃, 300 K) δ −5.31* and −9.36; UPLC t_R1.43 and 1.47*

min; MS (ES⁺) m/z 452 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methylthiazole-5-

carboxylate (2k). Following the same procedure as 2c, treatment of

7 (600.0 mg, 1.74 mmol) with thiazole-5-carboxylic acid (270.5 mg,

2.09 mmol) aﬀorded a mixture of diastereomers (88:12*) of the title

1

compound (166 mg, 22%) as a colorless oil. H NMR (400 MHz,

CDCl₃, 300 K) δ 9.08 and 9.07* (s, 1H), 8.69 and 8.64* (s, 1H), 7.08

and 6.96* (bs, 1H), 6.03−5.92 (m, 2H), 4.81 and 4.61* (d and s*, J =

2.4 Hz, 1H), 4.38 and 4.21* (dd and d*, J = 3.6, 10.9 Hz, J* = 10.9

Hz, 1H), 3.91 (dd, J = 10.9, 23.1 Hz, 1H), 3.73* and 3.71 (s, 3H),

3.60−3.55 (m, 2H), 2.59 (t, J = 6.1 Hz, 2H), 1.22 and 1.15* (s, 3H),

1.14* and 1.12 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−5.33, −9.61*; UPLC t_R1.05* and 1.08 min; MS (ES⁺) m/z 459 [M

+ Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Isothia-

zole-5-carboxylate (2l). Following the same procedure as 2c,

treatment of 7 (242.0 mg, 0.70 mmol) with 1,2-thiazole-5-carboxylic

acid (109.1 mg, 0.84 mmol) aﬀorded a mixture of diastereomers

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Isonicoti-

nate (2g). Following the same procedure as 2c, treatment of 7

(300.0 mg, 0.87 mmol) with isonicotinic acid (128.9, 1.05 mmol)

aﬀorded a mixture of diastereomers (81:19*) of the title compound

(42 mg, 25%) as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆, 300

K) δ 8.87−8.85 (m, 2H), 8.20 and 8.13* (m, 1H), 7.88 −7.86 (m,

2H), 5.97−5.90 (m, 2H), 4.71 and 4.61 (d and s*, J = 7.3 Hz, 1H),

4.27−4.13 and 4.05−3.93* (m, 2H), 3.59 (s, 3H), 3.29−3.21 (m,

2H), 2.47 (t, J = 6.1 Hz, 2H), 1.08 and 1.01* (s, 3H), 0.94 and 0.93*

(s, 3H); ³¹P NMR (162 MHz, DMSO-d₆, 300 K) δ −7.77 and

−9.22*; UPLC t_R1.36 min; MS (ES⁺) m/z 453 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Pyrazine-

2-carboxylate (2h). Following the same procedure as 2c, treatment

of 7 (200.0 mg, 0.58 mmol) with pyrazine-2-carboxylic acid (86.6 mg,

0.70 mmol) aﬀorded a mixture of diastereomers (78:22*) of the title

1

(92:8*) of the title compound (17 mg, 5%) as a colorless oil. H

NMR (400 MHz, CDCl₃, 300 K) δ 8.62 (d, J = 1.7 Hz, 1H), 7.94 and

7.90* (d, J = 1.7 Hz, 1H), 7.04 (bs, 1H), 6.03−5.93 and 5.86−5.76*

(m, 2H), 4.80 and 4.60* (d and s*, J = 2.2 Hz, 1H), 4.36 and 4.20*

(dd and d*, J = 3.9, 11.8 Hz, J* = 10.9 Hz, 1H), 3.91 (dd, J = 11.4,

22.8 Hz, 1H), 3.73* and 3.70 (s, 3H), 3.61−3.52 (m, 2H), 2.61−2.53

(m, 2H), 1.22 and 1.14* (s, 3H), 1.15* and 1.12 (s, 3H); ³¹P NMR

(162 MHz, CDCl₃, 300 K) δ −6.58, −11.00*; UPLC t_R1.22*, 1.26;

MS (ES⁺) m/z 459 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 4-Methyl-

thiazole-5-carboxylate (2m). Following the same procedure as 2c,

treatment of 7 (242.0 mg, 0.70 mmol) with 4-methyl-1,3-thiazole-5-

carboxylic acid (121.0 mg, 0.84 mmol) aﬀorded a mixture of

diastereomers (69:31*) of the title compound (116 mg, 37%) as an

1

oily solid. H NMR (400 MHz, CDCl₃, 300 K) δ 8.89 and 8.88* (s,

1

1H), 7.08 and 6.91* (bs, 1H), 6.01−5.89 (m, 2H), 4.80 and 4.60* (d

and s*, J = 1.7 Hz, 1H), 4.36 and 4.20* (dd and d*, J = 11.4 Hz, J* =

10.9 Hz, 1H), 3.90 and 3.87* (dd, J = 11.4, 22.8 Hz, J* = 11.4, 25.8

Hz, 1H), 3.73* and 3.70 (s, 3H), 3.60−3.54 and 3.64−3.42* (m,

2H), 2.85 and 2.83 (s, 3H), 2.60−2.54 (m, 2H), 1.22 and 1.14* (s,

3H), 1.13* and 1.12 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−6.50, −10.87*; UPLC t_R1.18*, 1.21; MS (ES⁺) m/z 473 [M +

Na]⁺. This material was puriﬁed by column chromatography on silica

gel eluting with ethyl acetate/petroleum ether (from 0 to 100% of

EtOac) to aﬀord (((2S,4R)-4-((3-methoxy-3-oxopropyl)carbamoyl)-

5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 4-meth-

compound (28 mg, 11%) as a colorless oil. H NMR (400 MHz,

DMSO-d₆, 300 K) δ 9.26−9.24 (m, 1H), 8.97−8.95 (m, 1H), 7.85−

8.90 (m, 1H), 8.18 and 8.12 (bs, 1H), 6.00−5.94 (m, 2H), 4.71 and

4.64* (d and s*, J = 6.7 Hz, 1H), 4.25−4.14 and 4.02−3.96 (m, 2H),

3.58 (s, 3H), 3.31−3.27 (m, 2H), 2.47 (t, J = 6.7 Hz, 2H), 1.08 and

1.01* (s, 3H), 0.95 and 0.93 (s, 3H); ³¹P NMR (162 MHz, DMSO-

d₆, 300 K) δ −7.69 and −9.29*; UPLC t_R1.00 min; MS (ES⁺) m/z

432 [M + H]⁺.

Methyl 3-((4R)-2-(((Isopropoxycarbonyl)oxy)methoxy)-5,5-

dimethyl-2-oxido-1,3,2-dioxaphosphinane-4-carboxamido)-

propanoate (2i). Following the same procedure as 2a, treatment of

6 (1.00 g, 3.39 mmol) with chloromethyl propan-2-yl carbonate (1.03

g, 6.77 mmol) aﬀorded a mixture of diastereomers (43*:57) of the

1

ylthiazole-5-carboxylate (trans-2m) as an oil. H NMR (400 MHz,

CDCl₃, 300 K) δ 8.91 (s, 1H), 6.91 (bs, 1H), 5.94−5.88 (m, 2H),

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01531

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

4.61 (s, 1H), 4.21 (d, J = 11.4 Hz, 1H), 3.87 (dd, J = 11.4, 24.9 Hz,

1H), 3.73 (s, 3H), 3.64−3.56 (m, 1H), 3.51−3.42 (m, 1H), 2.83 (s,

3H), 2.58−2.51 (m, 2H), 1.14 (s, 3H), 1.13 (s, 3H); ³¹P NMR (162

MHz, CDCl₃, 300 K) δ −10.88; UPLC t_R1.18 min; MS (ES⁺) m/z

451 [M + H]⁺.

aﬀorded a mixture of diastereomers (75:25*) of the title compound

(106 mg, 40%) as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆, 300

K) δ 8.18 and 8.12* (bs, 1H), 6.76 and 6.74* (s, 1H), 5.88−5.82 (m,

2H), 4.69 and 4.58* (d and s*, J = 6.7 Hz, 1 H), 4.24-412 and 4.39−

3.92 (m, 2H), 4.03 and 4.02* (s, 3H), 3.58 (s, 3H), 3.21−3.38 (m,

2H), 2.57 (q, J = 7.31 Hz, 2H), 2.48−2.44 (m, 2H), 1.17 (t, J = 7.61

Hz, 3H), 1.07 and 1.01* (s, 3H), 0.94 and 0.93* (s, 3H); ³¹P NMR

(162 MHz, DMSO-d₆, 300 K) δ −7.57 and −9.23*; UPLC t_R1.37*

and 1.40 min; MS (ES⁺) m/z 462 [M + H]⁺. This material was

puriﬁed by column chromatography on silica gel eluting with

dichloromethane/ethyl acetate (from 0 to 100% of EtOac) to aﬀord

(((2S,4R)-4-((3-methoxy-3-oxopropyl)carbamoyl)-5,5-dimethyl-2-

oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 3-ethyl-1-methyl-1H-

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 4-(tert-

Butyl)thiazole-5-carboxylate (2n). Following the same procedure

as 2a, treatment of 6 (270.0 mg, 0.91 mmol) with chloromethyl 4-tert-

butyl-1,3-thiazole-5-carboxylate (363.4 mg, 1.55 mmol) aﬀorded a

mixture of diastereomers (7*:93) of the title compound (15 mg, 3%)

1

as a colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 8.91* and

8.89 (s, 1H), 7.37* and 7.11 (bs, 1H), 5.97−5.93* and 5.91−5.85 (m,

2H), 4.84* and 4.63 (s, 1H), 4.40−4.35 and 4.22−4.19 (m, 1H),

3.91−3.82 (m, 1H), 3.71 and 3.69* (s, 3H), 3.65−3.57 (m, 1H),

3.52−3.43 (m, 1H), 2.64−2.50 (m, 2H), 1.53* and 1.52 (s, 9H), 1.12

and 1.11* (s, 6H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −7.01*,

−10.96; UPLC t_R1.65 min; MS (ES⁺) m/z 493 [M + H]⁺.

1

pyrazole-5-carboxylate (trans-2r) as a colorless oil. H NMR (400

MHz, DMSO-d₆, 300 K) δ 8.13 (bs, 1H), 6.73 (s, 1H), 5.87−5.81 (m,

2H), 4.58 (s, 1H), 4.13 (d, J = 11.0 Hz, 1H), 4.02 (s, 3H), 3.96−3.92

(m, 1H), 3.58 (s, 3H), 3.27−3.22 (m, 2H), 2.56 (q, J = 7.5 Hz, 2H),

2.48−2.44 (m, 2H), 1.17 (t, J =7.5, 3H), 1.00 (s, 3H), 0.93 (s, 3H);

³¹P NMR (162 MHz, DMSO-d₆, 300 K) δ −10.58*; UPLC t_R1.37

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 4-Methyl-

oxazole-5-carboxylate (2o). A mixture of chloromethyl 4-methyl-

1,3-oxazole-5-carboxylate (261.7 mg, 1.49 mmol), compound 6

(400.0 mg, 1.35 mmol), and AgO (471.0 mg, 2.03 mmol) in

MeCN (13.5 mL) was stirred under reﬂux for 3 h. The mixture was

cooled, the solvent was evaporated, and then the residue was puriﬁed

by ﬂash chromatography on SiO₂(0−100% EtOAc/DCM) to obtain

a mixture of diastereomers (10*:90) of the title compound as a

min; MS (ES⁺) m/z 462 [M + H]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 3-(tert-

Butyl)-1H-pyrazole-5-carboxylate (2s). Following the same

procedure as 2c, treatment of 7 (300.0 mg, 0.87 mmol) with 3-

(tert-butyl)-1H-pyrazole-5-carboxylic acid (176.2 mg, 1.05 mmol)

aﬀorded a mixture of diastereomers (79:21*) of the title compound

(34 mg, 8%) as a white solid. ¹H NMR (400 MHz, CDCl₃, 300 K) δ

7.54 and 7.19* (bs, 1H), 6.77 and 6.75* (s, 1H), 6.68 (bs, 1H), 6.12

and 5.94* and 5.80 (dd and d*, J = 5.3, 17.9 Hz, J* = 13.6 Hz, 2H),

4.84 and 4.66* (s, 1H), 4.43 and 4.21* (dd and d*, J = 2.2, 11.8 Hz,

J* = 11.8 Hz, 1H), 3.91 and 3.84* (dd, J = 11.4, 23.6 Hz, J* = 10.9,

24.9 Hz, 1H), 3.76 and 3.72* (s, 3H), 3.65−3.61 and 3.56−3.50* (m,

2H), 2.73−2.69 and 2.57* (m and t*, J* = 6.5 Hz, 2H), 1.39 and

1.38* (s, 9H), 1.21 and 1.14* (s, 3H), 1.15 and 1.11 (s, 3H); ³¹P

NMR (162 MHz, CDCl₃, 300 K) δ −6.77, −10.73*; UPLC t_R1.43*

and 1.46 min; MS (ES⁺) m/z 476 [M + H]⁺. This material was

puriﬁed by RP-HPLC (Waters XBridge C₁₈5 μm, 19 × 150 mm²;

ﬂow rate, 20 mL/min) eluting with water and acetonitrile containing

0.1% TFA (from 10 to 50% of acetonitrile) to aﬀord (((2S,4R)-4-((3-

methoxy-3-oxopropyl)carbamoyl)-5,5-dimethyl-2-oxido-1,3,2-dioxa-

phosphinan-2-yl)oxy)methyl 3-(tert-butyl)-1H-pyrazole-5-carboxylate

1

colorless oil (28.0 mg, 5%). H NMR (400 MHz, CDCl₃, 300 K) δ

7.99* and 7.97 (s, 1H), 7.07* and 6.94 (bs, 1H), 5.98−5.91 (m, 2H),

4.81* and 4.63 (d* and s, J* = 1.7 Hz, 1H), 4.37* and 4.21 (dd* and

d, J* = 3.9, 11.4 Hz, J = 11.4 Hz, 1H), 3.80 (dd, J = 11.4, 24.9 Hz,

1H), 3.73 and 3.71* (s, 3H), 3.67−3.56 (m, 1H), 3.54−3.45 (m, 1H),

2.59−2.56 (m, 5H), 1.22* and 1.15 (s, 3H), 1.14 and 1.12* (s, 3H);

³¹P NMR (162 MHz, CDCl₃, 300 K) δ −6.57*, −10.97; UPLC t_R

1.10; MS (ES⁺) m/z 457 [M + Na]⁺.

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 1-Methyl-

1H-pyrazole-5-carboxylate (2p). Following the same procedure as

2c, treatment of 7 (220.0 mg, 0.64 mmol) with 2-methylpyrazole-3-

carboxylic acid (96.9 mg, 0.77 mmol) aﬀorded a mixture of

diastereomers (85:15*) of the title compound (21 mg, 8%) as a

1

colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 7.54 and 7.53*

1

(trans-2s) as a white solid. H NMR (400 MHz, CDCl₃, 300 K) δ

(d, J = 2.2 Hz, 1H), 7.01 and 6.97* (d, J = 2.2 Hz, 1H), 7.03 and

6.91* (bs, 1H), 6.02−5.90 (m, 2H), 4.81 and 4.61* (d and s*, J = 1.7

Hz, 1H), 4.37 and 4.20* (dd and d*, J = 3.5, 11.4 Hz, J* = 11.4

Hz,1H), 4.25 and 4.23* (s, 3H), 3.90 and 3.86* (dd, J = 11.8, 23.2

Hz, J* = 11.4, 25.4 Hz, 1H), 3.73* and 3.69 (s, 3H), 3.57−3.51 and

3.63−3.42* (m, 2H), 2.58−2.55 (m, 2H), 1.22 and 1.14* (s, 3H),

1.13* and 1.11 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−6.33, −10.87*; UPLC t_R1.17* and 1.20; MS (ES⁺) m/z 434 [M +

H]⁺.

7.20 (bs, 1H), 6.75 (s, 1H), 5.94 (d, J = 13.37 Hz, 2H), 4.66 (s, 1H),

4.22−4.17 (m, 1H) overlay with water, 3.86 (dd, J =11.4, 25.2 Hz,

1H), 3.72 (s, 3H), 3.55−3.52 (m, 2H), 2.57 (t, J = 6.25 Hz, 2H), 1.38

(s, 9H), 1.15 (s, 3H), 1.11 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300

K) δ −10.69; UPLC t_R1.44 min; MS (ES⁺) m/z 476 [M + H]⁺; M.P.

102 °C.

tert-Butyl-(R)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)-

propanoate (8). tert-Butyl 3-aminopropanoate (48.7 g, 335.4 mmol)

was treated with (3R)-4,4-dimethyl-3-oxidanyl-oxolan-2-one (56.8 g,

436.0 mmol) and heated with stirring at 85 °C. This mixture melted

to give a pale yellow oil that after 48 h was cooled and taken up in the

minimum amount of DCM and puriﬁed by ﬂash chromatography on

SiO₂(0−70% EtOAc/PE) to furnish the title compound (80.7 g,

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 1,3-Di-

methyl-1H-pyrazole-5-carboxylate (2q). Following the same

procedure as 2c, treatment of 7 (250.0 mg, 0.73 mmol) with 1,3-

dimethyl-1H-pyrazole-5-carboxylic acid (122.3 mg, 0.87 mmol)

aﬀorded a mixture of diastereomers (61:39*) of the title compound

(28 mg, 8%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃, 300 K) δ

7.01 and 6.89* (bs, 1H), 6.76 and 6.72* (s, 1H), 5.98−5.85 (m, 2H),

4.79 and 4.59* (d and s*, J = 1.7 Hz, 1H), 4.35 and 4.18* (dd and d*,

J = 3.5, 11.4 Hz, J* = 11.4 Hz, 1H), 4.16 and 4.13* (s, 3H), 3.91 and

3.85* (dd, J = 11.8, 22.8 Hz, 1H), 3.71* and 3.68 (s, 3H), 3.62−

3.39* and 3.54−3.51 (m, 2H), 2.58−2.51 (m, 2H), 2.29 and 2.28* (s,

3H), 1.20 and 1.13* (s, 3H), 1.12* and 1.10 (s, 3H); ³¹P NMR (162

MHz, CDCl₃, 300 K) δ −6.33, −10.87*; UPLC t_R1.21* and 1.25;

MS (ES⁺) m/z 448 [M + H]⁺.

1

88%) as a colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 7.18

(bs, 1H), 4.03 (s, 1H), 3.60−3.49 (m, 4H), 2.51−2.48 (t, J = 5.8 Hz,

2H), 1.47 (s, 9H), 1.04 (s, 3H), 0.93 (s, 3H); UPLC t_R1.17 min; MS

(ES⁺) m/z 276 [M + H]⁺.

(((4R)-4-((3-(tert-Butoxy)-3-oxopropyl)carbamoyl)-5,5-di-

methyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Piva-

late (9b). Following the procedure described for 5, treatment of 8

(80.7 g, 293 mmol) with POCl₃(24.8 mL, 258.8 mmol) furnished

after ﬂash chromatography on SiO₂(petroleum ether/EtOAc) a

mixture of two diastereomers (59:41*) of tert-butyl 3-((4R)-2-

(benzyloxy)-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane-4-carboxa-

(((4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl 3-Ethyl-1-

methyl-1H-pyrazole-5-carboxylate (2r). Following the same

procedure as 2c, treatment of 7 (200.0 mg, 0.58 mmol) with 5-

ethyl-2-methyl-pyrazole-3-carboxylic acid (107.6 mg, 0.70 mmol)

1

mido) propanoate (110.0 g, 49%) as a white solid. H NMR (400

MHz, CDCl₃, 300 K) δ 7.47−7.39 (m, 5H), 6.91* and 6.81 (bs, 1H),

5.26−5.24 and 5.16−5.13* (d, J = 9.8 and 9.4 Hz*, 2H), 4.77 and

4.42* (m, 1H), 4.39−4.35 and 4.06−4.03* (m, 1H), 4.17−4.12 and

M

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3.86−3.73* (m, 1H), 3.60−3.53 (m, 1H), 3.51−3.53 (m, 1H), 2.51−

2.42 (m, 2H), 1.43* and 1.45 (s, 9H), 1.19 and 1.11* (s, 3H), 1.10

and 1.09* (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −4.59,

−8.67*; UPLC t_R1.72*, 1.80 min; MS (ES⁺) m/z 428 [M + H]⁺.

Treatment of this material (1.82 g, 4.26 mmol) as described for

compound 6 furnished tert-butyl 3-((4R)-2-hydroxy-5,5-dimethyl-2-

oxido-1,3,2-dioxaphosphinane-4-carboxamido)propanoate (1.40 g,

97%) as a colorless oil. ³¹P NMR (162 MHz, DMSO-d₆, 300 K) δ

−3.36; UPLC t_R0.89 min; MS (ES⁺) m/z 338 [M + H]⁺. Following

the general procedure for compound 2a, treatment of the preceding

material (1.3 g, 3.85 mmol) aﬀorded a mixture of diastereomers

(5*:95) of the title compound (2.3 g, 58%) as a white solid. ¹H NMR

(400 MHz, CDCl₃, 300 K) δ 7.02 (bs, 1H), 5.73−5.70 (d, J = 13.2

Hz, 2H), 4.82* and 4.59 (s, 1H), 4.4−4.36* and 4.21−4.18 (d, J =

15.6 Hz* and J = 11.1 Hz, 1H), 3.91−3.82 (dd, J = 11.2 Hz, 24.8 Hz,

1H), 3.64−3.56 (m, 1H), 3.52−3.44 (m, 1H), 2.51−2.47 (m, 2H),

1.48 (s, 9H), 1.28* and 1.25 (s, 9H), 1.21* and 1.15 (s, 3H), 1.14 and

1.12*(s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −5.50* and

−9.36; UPLC t_R2.14 and 2.20* min; MS (ES⁺) m/z 452 [M + H]⁺;

M.P. 101 °C.

3-((2S,4R)-5,5-Dimethyl-2-oxido-2-((pivaloyloxy)methoxy)-

1,3,2-dioxaphosphinane-4-carboxamido)propanoic Acid (9a).

Compound 9b (13.0 g, 28.8 mmol) was dissolved in DCM (140 mL);

then, TFA (60 mL) was added dropwise. The mixture was stirred for

45 min, then the solvent was evaporated, and the residue was puriﬁed

by ﬂash chromatography on C₁₈silica eluting with H₂O/CH₃CN to

give the title compound (8.0 g, 87%) as a white solid. ¹H NMR (400

MHz, DMSO-d₆, 300 K) δ 12.27 (bs, 1H), 8.13 (bs, 1H), 5.68−5.60

(m, 2H), 4.54 (s, 1H), 4.11−4.08 (d, J = 10.1 Hz, 1H), 4.02−3.92

(dd, J = 11.2, 24.4 Hz, 1H), 3.31 (m, 2H), 2.41 (m, 2H), 1.17 (s,

9H), 1.02 (s, 3H), 0.95 (s, 3H); ³¹P NMR (162 MHz, DMSO-d₆, 300

K) δ −10.33; UPLC t_R1.24 min; MS (ES⁺) m/z 418 [M + Na]⁺;

M.P. 74 °C.

1.12 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −10.67; UPLC

t_R1.09 min; MS (ES⁺) m/z 501 [M + H]⁺.

(((2S,4R)-5,5-Dimethyl-4-((3-(2-(methylsulfonyl)ethoxy)-3-

oxopropyl)carbamoyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)-

oxy)methyl Pivalate (9f). Following the same procedure as 9c,

treatment of 9a (90.0 mg, 0.23 mmol) with 2-methylsulfonylethanol

(33.9 mg, 0.27 mmol) aﬀorded the title compound (26 mg, 23%) as a

1

colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 7.37 (bs, 1H),

5.79−5.62 (m, 2H), 4.67−4.60 (m, 2H), 4.58 (s, 1H), 4.18 (d, J =

11.2 Hz, 1H), 3.85 (dd, J = 11.3, 24.7 Hz, 1H), 3.66−3.52 (m, 2H),

3.49−3.41 (m, 1H), 3.39−3.31 (m, 1H), 3.09 (s, 3H), 2.60 (t, J = 6.1

Hz, 2H), 1.24 (s, 9H), 1.15 (s, 3H), 1.13 (s, 3H); ³¹P NMR (162

MHz, CDCl₃, 300 K) δ −9.16; UPLC t_R1.34 min; MS (ES⁺) m/z

524 [M + Na]⁺.

(((2S,4R)-4-((3-((1-Benzylpyrrolidin-3-yl)oxy)-3-oxopropyl)-

carbamoyl)-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinan-2-

yl)oxy)methyl Pivalate (9g). Following the same procedure as 9c,

treatment of 9a (90.0 mg, 0.23 mmol) with 1-benzylpyrrolidin-3-ol

(48.4 mg, 0.27 mmol) aﬀorded the title compound (8.1 mg, 29%) as a

pale oil. ¹H NMR (400 MHz, CDCl₃+ TFA, 300 K) δ 9.02−8.88 (bs,

1H), 7.94 (m, 1H), 7.51−7.47 (m, 3H), 7.42−7.40 (m, 2H), 5.84−

5.77 (m, 2H), 5.56−5.49 (m, 1H), 4.90−4.88 (m, 1H), 4.50−4.44

(m, 2H), 4.36−4.32 (m, 1H), 4.15−3.95 (m, 2H), 3.89−3.86 (m,

1H), 3.77−3.63 (m, 2H), 3.48−3.31 (m, 2H), 2.80−2.77 (m, 1H),

2.73−2.69 (m, 1H), 2.65−2.43 (m, 1H), 2.35−2.28 (m, 1H), 1.29−

1.28 (m, 9H), 1.19−1.14 (m, 6H); ³¹P NMR (162 MHz, CDCl₃+

TFA, 300 K) δ −9.48; UPLC t_R1.14 min; MS (ES⁺) m/z 555 [M +

H]⁺.

Methyl 3-((2R,4R)-2-(4-Fluorophenoxy)-5,5-dimethyl-2-

oxido-1,3,2-dioxaphosphinane-4-carboxamido)propanoate

(3a) and Methyl 3-((2S,4R)-2-(4-Fluorophenoxy)-5,5-dimethyl-

2-oxido-1,3,2-dioxaphosphinane-4-carboxamido)propanoate

(3b). Compound 4 (10.0 g, 42.9 mmol) was dissolved in THF (112

mL) and treated with a solution of TEA (12.6 mL, 90 mmol) in THF

(15 mL). This mixture was cooled to −78 °C and treated dropwise

with a solution of POCl₃(4.41 mL, 47.2 mmol) in THF (10 mL).

After stirring for 0.5 h, the mixture was warmed to 20 °C over 1 h.

The mixture was ﬁltered through a pad of solka ﬂoc, and the volatiles

were evaporated to give methyl 3-((4R)-2-chloro-5,5-dimethyl-2-

oxido-1,3,2-dioxaphosphinane-4-carboxamido)propanoate (13.3 g). A

portion of this material (355.0 mg, 1.13 mmol) was dissolved in DCM

(6 mL) and cooled to 0 °C. 4-Fluorophenol (103.3 mg, 0.93 mmol)

and a solution of TEA (0.55 mL, 4.09 mmol) in Et₂O (3 mL) were

added, and the mixture was stirred for 16 h at 20 °C. The mixture was

diluted with H₂O, then the organic layer was separated, and washed

with 5% aqueous citric acid, NaOH (1 N), and brine. The organic

phase was dried, ﬁltered, and concentrated to give a residue that was

puriﬁed by ﬂash chromatography on SiO₂(50−100% EtOAc/DCM)

to aﬀord a mixture of diastereomers as the title compounds. RP-

HPLC puriﬁcation of this material aﬀorded 3a (64.0 mg, 14%) as a

(((2S,4R)-4-((3-Ethoxy-3-oxopropyl)carbamoyl)-5,5-dimeth-

yl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl Pivalate

(9c). Compound 9a (300.0 mg, 0.76 mmol) and TBTU (365.5 mg,

1.14 mmol) were dissolved in MeCN (2.50 mL) and stirred for 20

min. Ethyl alcohol (0.05 mL, 0.91 mmol) and DIEA (0.26 mL, 1.52

mmol) were added and stirring was continued for 16 h. The mixture

was then ﬁltered through a Si carbonate SPE cartridge eluting with

DCM. The volatiles were evaporated, and the residue was puriﬁed by

RP-HPLC to aﬀord the title compound as a white solid (131.2 mg,

1

41%). H NMR (400 MHz, CDCl₃, 300 K) δ 6.96 (bs, 1H), 5.74−

5.69 (m, 2H), 4.57 (s, 1H), 4.20−4.15 (m, 3H), 3.85 (dd, J = 11.4,

25.2 Hz, 1H), 3.64−3.58 (m, 1H), 3.56−3.50 (m, 1H), 2.58−2.54

(m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.24 (s, 9H), 1.14 (s, 3H), 1.13 (s,

3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −10.62; UPLC t_R1.56

min; MS (ES⁺) m/z 424 [M + H]⁺; M.P. 59 °C.

1

(((2S,4R)-5,5-Dimethyl-2-oxido-4-((3-oxo-3-(thiazol-5-

ylmethoxy)propyl)carbamoyl)-1,3,2-dioxaphosphinan-2-yl)-

oxy)methyl Pivalate (9d). Following the same procedure as 9c,

treatment of 9a (90.0 mg, 0.23 mmol) with thiazol-5-ylmethanol

(31.4 mg, 0.27 mmol) aﬀorded the title compound (24 mg, 21%) as a

colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 7.30−7.13 (m,

2H), 7.13−7.07 (m, 2H), 6.98 (br s, 1H), 4.81 (d, J = 1.0 Hz, 1 H),

4.40 (dd, J = 2.6, 11.6 Hz, 1 H), 3.87 (dd, J = 11.6, 23.5 Hz, 1H), 3.74

(s, 3H), 3.66−3.47 (m, 2H), 2.58−2.53 (m, 2H), 1.16 (s, 3H), 0.85

(s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −10.84; UPLC t_R

1.46 min; MS (ES⁺) m/z 390 [M + H]⁺; and 3b (24.0 mg, 5%) as a

1

white solid. H NMR (400 MHz, CDCl₃, 300 K) δ 9.0 (s, 1H), 7.97

(s, 1H), 6.94 (bs, 1H), 5.75−5.69 (m, 2H), 5.39 (s, 2H), 4.56 (s,

1H), 4.17 (d, J = 10.9 Hz, 1H), 3.85 (dd, J = 10.9, 25.5 Hz, 1H),

3.67−3.52 (m, 2H), 2.65−2.61 (m, 2H), 1.25 (s, 9H), 1.14 (s, 3H),

1.06 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −9.33; UPLC t_R

1.46 min; MS (ES⁺) m/z 493 [M + H]⁺; M.P. 57 °C.

1

colorless oil. H NMR (400 MHz, CDCl₃, 300 K) δ 7.25−7.18 (m,

2H), 7.09−7.01 (m, 2H), 6.97 (br s, 1H), 4.72 (s, 1 H), 4.29 (d, J =

11.4 Hz, 1H), 3.92 (d, J = 11.4 Hz, 1H), 3.97 (dd, J = 11.4, 25.4 Hz,

1H), 3.72 (s, 3H), 3.59−3.69 (m, 1H), 3.58−3.49 (m, 1H), 2.63−

2.55 (m, 2H), 1.20 (s, 3H), 1.15 (s, 3H); ³¹P NMR (162 MHz,

CDCl₃, 300 K) δ −14.84; UPLC t_R1.43 min; MS (ES⁺) m/z 390 [M

+ H]⁺.

(((2S,4R)-5,5-Dimethyl-2-oxido-4-((3-oxo-3-(2-(pyridin-2-yl)-

ethoxy)propyl)carbamoyl)-1,3,2-dioxaphosphinan-2-yl)oxy)-

methyl Pivalate (9e). Following the same procedure as 9c,

treatment of 9a (125.0 mg, 0.32 mmol) with 2-(2-pyridyl)ethanol

(41.4 mg, 0.38 mmol) aﬀorded the title compound (70 mg, 61%) as a

Methyl 3-((2R,4R)-2-(4-Cyanophenoxy)-5,5-dimethyl-2-

oxido-1,3,2-dioxaphosphinane-4-carboxamido)propanoate

(3c). A solution of 4-hydroxybenzonitrile (162.3 mg, 1.36 mmol) in

Et₂O (2 mL) was stirred under nitrogen and cooled to −78 °C before

a solution of TEA (0.55 mL, 4.09 mmol) in Et₂O (3 mL) and POCl₃

(0.13 mL, 1.36 mmol) were slowly added. The mixture was stirred for

15 min at −78 °C and then warmed to 20 °C over 1 h. The mixture

was ﬁltered through a pad of solka ﬂoc, and the ﬁltrate was

1

pale oil. H NMR (400 MHz, CDCl₃, 300 K) δ 8.88 (d, J = 6.1 Hz,

1H), 8.34 (t, J = 8.5 Hz, 1H), 7.81−7.76 (m, 2H), 6.99 (bs, 1H),

5.77−5.68 (m, 2H), 4.60−4.58 (m, 3H), 4.18 (d, J = 10.9 Hz, 1H),

3.87 (dd, J = 25.5 Hz, 12.2 Hz, 1H), 3.53−3.48 (m, 2H), 3.58−3.55

(m, 2H), 2.58−2.56 (t, J = 6.1 Hz, 2H), 1.25 (s, 9H), 1.14 (s, 3H),

N

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evaporated. The residue was dissolved in THF (4 mL) and cooled to

−78 °C. A solution of compound 4 (317.8 mg, 1.36 mmol) in THF

(1 mL) and TEA (0.75 mL, 4.9 mmol) were added. After 10 min, the

cooling bath was removed and the mixture was stirred for 30 min,

then eluted through a pad of solka ﬂoc with DCM. The solution was

washed with 5% aqueous citric acid, NaOH (1 N), and brine, and

then dried and ﬁltered. Removal of the volatiles gave a residue that

was puriﬁed by ﬂash chromatography on SiO₂(50−100% EtOAc/

DCM) to aﬀord the title compound (100 mg, 18%) as a colorless oil.

¹H NMR (400 MHz, DMSO-d₆, 300 K) δ 8.34 (br t, J = 5.6 Hz, 1H),

7.92 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 4.73 (d, J = 11.4

Hz, 1H), 4.52 (app t, J = 11.0 Hz, 1H), 4.15 (dd, J = 11.0, 13.6 Hz,

1H), 3.58 (s, 3H), 3.40−3.27 (m, 2H), 2.48−2.42 (m, 2H), 1.14 (s,

3H), 0.76−0.84 (m, 3H); ³¹P NMR (162 MHz, DMSO-d₆, 300 K) δ

−15.08; UPLC t_R1.34 min; MS (ES⁺) m/z 397 [M + H]⁺.

Methyl 3-((2R, 4R)-5, 5-Dimethyl-2-oxido-2-(4-

(triﬂuoromethyl)phenoxy)-1,3,2-dioxaphosphinane-4-

carboxamido)propanoate (3d). Following the same procedure as

3c, treatment of 4 (431.7 mg, 1.85 mmol) with 4-triﬂuoromethyl-

phenol (300.0 mg, 1.85 mmol) aﬀorded the title compound (22 mg,

3%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.70 (d,

J = 8.8 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.05 (br s, 1H), 4.85 (d, J =

2.0 Hz, 1H), 4.42 (dd, J = 3.3, 11.6 Hz, 1H), 3.93 (dd, J = 11.4, 23.2,

1H), 3.72 (s, 3H), 3.67−3.57 (m, 1H), 3.57−3.48 (m, 1H), 2.61−

2.53 (m, 2H), 1.19 (s, 3H), 0.91 (s, 3H); ³¹P NMR (162 MHz,

CDCl₃, 300 K) δ −11.47; UPLC t_R1.70 min; MS (ES⁺) m/z 440 [M

+ H]⁺.

Methyl 3-((2R,4R)-5,5-Dimethyl-2-oxido-2-(4-(pentaﬂuoro-

l6-sulfanyl)phenoxy)-1,3,2-dioxaphosphinane-4-

carboxamido)propanoate (3e). Following the same procedure as

3c, treatment of 4 (317.85 mg, 1.36 mmol) with 4-(pentaﬂuorothio)-

phenol (300.0 mg, 1.36 mmol) aﬀorded the title compound (59 mg,

8%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.84 (d,

J = 8.7 Hz, 2H), 7.42 (d, J = 8.7 Hz, 2H), 7.06 (bs, 1H), 4.84 (d, J =

2.2 Hz, 1H), 4.42 (dd, J = 3.9, 11.4 Hz, 1H), 3.97 (dd, J = 11.4, 22.8

Hz, 1H), 3.73 (s, 3H), 3.68−3.51 (m, 2H), 5.58 (t, J = 5.7 Hz, 2H),

1.22 (s, 3H), 0.97 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ

−11.35; UPLC t_R1.79 min; MS (ES⁺) m/z 498 [M + H]⁺.

Methyl 2-(((2R,4R)-4-((3-Methoxy-3-oxopropyl)carbamoyl)-

5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)-

benzoate (3f). Following the same procedure as 3c, treatment of 4

(317.85 mg, 1.36 mmol) with methyl 2-hydroxybenzoate (207.3 mg,

1.36 mmol) aﬀorded the title compound (120 mg, 20%) as a colorless

oil. ¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.94−7.92 (m, 1H), 7.59−

7.55 (m, 1H), 7.46 (bs, 1H), 7.40−7.38 (m, 1H), 7.33−7.29 (m, 1H),

4.81 (d, J = 2.8, 1H), 4.40 (dd, J = 3.1, 11.6 Hz, 1H), 3.94 (dd, J =

11.6, 23.9 Hz, 1H), 3.95 (s, 3H), 3.68 (s, 3H), 3.62−3.57 (m, 2H),

2.6 (t, J = 6.5 Hz, 2H), 1.19 (s, 3H), 0.96 (s, 3H); ³¹P NMR (162

MHz, CDCl₃, 300 K) δ −12.57; UPLC t_R1.40 min; MS (ES⁺) m/z

430 [M + H]⁺.

methanol (2.0 mL) at 0 °C. The reaction was allowed to warm to

RT and then heated to reﬂux (T = 68 °C) for 2 h. The solution was

concentrated in vacuo, treated with tert-butyl methyl ether, and the

resulting crystals were removed by ﬁltration to aﬀord the title

1

compound (590.0 mg, 99%). H NMR (methanol-d₄, 400 MHz) δ

3.7−3.8 (m, 3H), 3.3−3.5 (m, 1H), 3.0−3.1 (m, 1H), 2.9−3.0 (m,

1H), 2.5−2.6 (m, 1H)

Methyl (R)-3-(2,4-Dihydroxy-3,3-dimethylbutanamido-¹⁵N)-

propanoate-1,2,3-¹³C₃(11). To a stirred solution of (R)-

pantolactone (3127.7 mg, 24.3 mmol) in methanol (11.9 mL) were

added (3-methoxy-3-oxopropyl-1,2,3-¹³C₃)amide-¹⁵N HCl salt (10)

and TEA (2.3 mL, 16.4 mmol). The mixture was stirred at reﬂux

overnight. After cooling to RT, the solvent was evaporated and the

resulting residue was puriﬁed by column chromatography on SiO₂

(0−90% EtOAc in PE) to aﬀord the title compound (653.0 mg, 67%).

¹H NMR (chloroform-d, 400 MHz, 300 K) δ 7.2−7.3 and 7.1−7.0

(m, 1H), 4.04 (s, 1H), 3.8−3.8 (m, 1H), 3.7−3.7 (m, 3H), 3.5−3.6

(m, 2H), 3.4−3.5 (m, 1H), 2.7−2.8 (m, 1H), 2.4−2. 5 (m, 1H), 2.07

(s, 1H), 1.06 (s, 3H), 0.94 (s, 3H).

Methyl 3-((4R)-2-(Benzyloxy)-5,5-dimethyl-2-(oxido-¹⁸O)-

1,3,2-dioxaphosphinane-4-carboxamido-¹⁵N)propanoate-

1,2,3-¹³C₃(12). Following the same procedure as 5, using 11 (4.4 g,

18.5 mmol) and P¹⁸OCl₃aﬀorded a mixture of diastereomers (50:50)

of the title compound (5.0 g, 69%). ³¹P NMR (162 MHz, CDCl₃, 300

K) δ −3.13 and −7.31; UPLC t_R1.33 and 1.39 min; MS (ES⁺) m/z

392 (M + H)⁺.

Methyl 3-((4R)-2-Hydroxy-5,5-dimethyl-2-(oxido-¹⁸O)-1,3,2-

dioxaphosphinane-4-carboxamido-¹⁵N)propanoate-1,2,3-¹³C₃

(13). Following the same procedure as 6, using 12 (2.0 g, 5.1 mmol)

1

aﬀorded the title compound (1.5 g, 99%). H NMR (DMSO-d₆, 400

MHz, 300 K) δ 8.1−8.2 and 8.0−7.9 (m, 1H), 4.43 (s, 1H), 3.9−4.1

(m, 1H), 3.7−3.9 (m, 1H), 3.6−3.6 (m, 3H), 3.4−3.5 (m, 1H), 3.1−

3.3 (m, 1H), 2.5−2.7 (m, 1H), 2.3−2.5 (m, 1H), 0.98 (s, 3H), 0.94

(s, 3H); ³¹P NMR (162 MHz, DMSO-d₆, 300 K) δ −8.11.

(((4R)-4-((3-Methoxy-3-oxopropyl-1, 2, 3-^{1 3}C₃)-

carbamoyl-¹⁵N)-5,5-dimethyl-2-(oxido-¹⁸O)-1,3,2-dioxaphos-

phinan-2-yl)oxy)methyl Pivalate (2a +6 amu). Following the

same procedure as 2a, treatment of 13 (1.5 g, 5.1 mmol) with

chloromethyl-2,2-dimethylpropanoate (1.3 mL, 8.7 mmol) aﬀorded a

mixture of diastereomers (5*:95) of the title compound (700 mg,

33%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃, 300 K) δ 7.08−7.02 and 6.84−6.78

(br s, 1H), 5.81−5.65 (m, 2H), 4.78−4.56 (m, 1H), 4.35−4.16 (m,

1H), 3.91−3.77 (m, 2H), 3.71 (s, 3H), 3.48−3.30 (m, 1H), 2.79−

2.67 (m, 1H), 2.47−2.33 (m, 1H), 1.23 (s, 9H), 1.13 (s, 3H), 1.11 (s,

3H); ³¹P NMR (162 MHz, CDCl₃, 300 K) δ −5.38* and −9.27;

UPLC t_R1.47 and 1.52* min; MS (ES⁺) m/z 416 [M + H]⁺.

ASSOCIATED CONTENT

■

sı

* Supporting Information

Methyl (S)-2-Amino-3-(4-(((2R,4R)-4-((3-methoxy-3-

oxopropyl)carbamoyl)-5,5-dimethyl-2-oxido-1,3,2-dioxaphos-

phinan-2-yl)oxy)phenyl)propanoate Hydrochloride (3g). Fol-

lowing the same procedure as 3c, treatment of 4 (500.0 mg, 1.69

mmol) with methyl 3-[[(2R)-3,3-dimethyl-2,4-bis(oxidanyl)-

butanoyl]amino]propanoate (394.9 mg, 1.69 mmol) aﬀorded (S)-2-

((tert-butoxycarbonyl)amino)-3-(4-(((2R,4R)-4-((3-methoxy-3-

oxopropyl)carbamoyl)-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinan-

2-yl)oxy)phenyl)propanoate. Treatment of this material with a

solution of HCl (0.2 M in Et₂O, 0.5 mL) gave a precipitate that

was ﬁltered and dried to aﬀord the title compound (15 mg, 23%) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆, 300 K) δ 8.38−8.33 (bs,

2H), 8.29−8.27 (bs, 1H), 7.27−7.20 (m, 4H), 4.72 (d, J = 9.0 Hz,

1H), 4.40−4.34 (m, 1H), 4.30−4.27 (m, 1H), 4.17−4.12 (m, 1H),

3.68 (s, 3H), 3.60 (s, 3H), 3.38−3.33 (m, 1H), 3.08 (m, 2H), 2-52−

2.50 (m, 2H), 1.10 (s, 3H), 0.82 (s, 3H); ³¹P NMR (162 MHz,

DMSO-d₆, 300 K) δ −13.33; UPLC t_R0.93 min; MS (ES⁺) m/z 473

[M + H]⁺; M.P. 50 °C.

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01531.

Procedure for the PBEC BBB assay, assessment of the

eﬀect of compound 2a on tubulin and histone

acetylation, ROESY spectra of compound 1, ROESY

spectra of compound 2a, X-ray crystal structure of

compound 2a, ¹H and ³¹P NMR spectra of key

compounds, and UPLC chromatograms of key com-

pounds (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

(3-Methoxy-3-oxopropyl-1,2,3-¹³C₃)amide-¹⁵N HCl Salt (10).

Thionyl chloride (4.1 mL, 56.4 mmol) was added dropwise to a

stirred solution of β-alanine-¹³C₃,¹⁵N 3-aminopropanoic acid in

Daniel Elbaum − Travere, San Diego, California 92130,

United States; orcid.org/0000-0002-6012-7965;

Email: ddelbaum@gmail.com

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01531

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(4) Schneider, S. A.; Hardy, J.; Bhatia, K. P. Syndromes of

Neurodegeneration with Brain Iron Accumulation (NBIA): An

Update on Clinical Presentations, Histological and Genetic Under-

pinnings, and Treatment Considerations. Mov. Disord. 2012, 42−53.

(5) Alfonso-Pecchio, A.; Garcia, M.; Leonardi, R.; Jackowski, S.

Compartmentalization of Mammalian Pantothenate Kinases. PLoS

One 2012, 7, No. e49509.

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Delineation of Neurodegeneration with Brain Iron Accumulation. J.

Med. Genet. 2008, 46, 73−80.

(7) Martinez, D. L.; Tsuchiya, Y.; Gout, I. Coenzyme A Biosynthetic

Machinery in Mammalian Cells. Biochem. Soc. Trans. 2014, 42, 1112−

1117.

(8) Orellana, D. I.; Santambrogio, P.; Rubio, A.; Yekhlef, L.;

Cancellieri, C.; Dusi, S.; Giannelli, S. G.; Venco, P.; Mazzara, P. G.;

Cozzi, A.; Ferrari, M.; Garavaglia, B.; Taverna, S.; Tiranti, V.;

Broccoli, V.; Levi, S. Coenzyme A Corrects Pathological Defects in

Human Neurons of PANK 2-associated Neurodegeneration. EMBO

Mol. Med. 2016, 8, 1197−1211.

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Correction of a Genetic Deficiency in Pantothenate Kinase 1 Using

Phosphopantothenate Replacement Therapy. Mol. Genet. Metab.

2015, 116, 281−288.

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Quinton, M. S.; Lyons, K. A.; Vaino, A.; Harper, S. Fosmetpantoten-

ate (RE-024), a Phosphopantothenate Replacement Therapy for

Pantothenate Kinase-Associated Neurodegeneration: Mechanism of

Action and Efficacy in Nonclinical Models. PLoS One 2018, 13,

No. e0192028.

Authors

Giulio Auciello − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Annalise Di Marco − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Odalys Gonzalez Paz − Departments of Chemistry and

Biology, IRBM Science Park, 00071 Pomezia, Rome, Italy

Savina Malancona − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy;

orcid.org/0000-0002-5388-6533

Steven Harper − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Maria Beconi − Travere, San Diego, California 92130, United

States

Ilaria Rossetti − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Alina Ciammaichella − Departments of Chemistry and

Biology, IRBM Science Park, 00071 Pomezia, Rome, Italy

Paola Fezzardi − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Andrea Vecchi − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Elena Bracacel − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Daniel Cicero − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

Edith Monteagudo − Departments of Chemistry and Biology,

IRBM Science Park, 00071 Pomezia, Rome, Italy

(11) Klopstock, T.; Escolar, M. L.; Marshall, R. D.; Perez-Duenas,

̃

B.; Tuller, S.; Videnovic, A.; Greblikas, F. The FOsmetpantotenate

Replacement Therapy (FORT) Randomized, Double-Blind, Placebo-

Controlled Pivotal Trial: Study Design and Development Method-

ology of a Novel Primary Efficacy Outcome in Patients with

Pantothenate Kinase-Associated Neurodegeneration. Clin. Trials

2019, 16, 410−418.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01531

Notes

The authors declare no competing ﬁnancial interest.

(12) Jeong, S. Y.; Hogarth, P.; Placzek, A.; Gregory, A. M.; Fox, R.;

Zhen, D.; Hamada, J.; van der Zwaag, M.; Lambrechts, R.; Jin, H.;

Nilsen, A.; Cobb, J.; Pham, T.; Gray, N.; Ralle, M.; Duffy, M.;

Schwanemann, L.; Rai, P.; Freed, A.; Wakeman, K.; Woltjer, R. L.;

Sibon, O. C. M.; Hayflick, S. J. 4 ′-Phosphopantetheine Corrects CoA,

Iron, and Dopamine Metabolic Defects in Mammalian Models of

PKAN. EMBO Mol. Med. 2019, 11, No. e10489.

ACKNOWLEDGMENTS

■

The authors thank Mabel Tse, Lachy McClean, Feriandis

Greblikas, and Bob Ternansky for a critical reading of the

manuscript, and Bill Rote for both a critical reading of the

manuscript and shepherding it through the internal Travere

publication system.

(13) Srinivasan, B.; Baratashvili, M.; van der Zwaag, M.; Kanon, B.;

Colombelli, C.; Lambrechts, R. A.; Schaap, O.; Nollen, E. A.;

DEDICATION

■

^§This paper is dedicated to the memory of Steven Harper

̌

Podgorsek, A.; Kosec, G.; Petkovic, H.; Hayflick, S.; Tiranti, V.;

Reijngoud, D.-J.; Grzeschik, N. A.; Sibon, O. C. M. Extracellular 4 ′-

Phosphopantetheine Is a Source for Intracellular Coenzyme A

Synthesis. Nat. Chem. Biol. 2015, 11, 784−792.

(1968−2019).

ABBREVIATIONS

■

(14) Sharma, L. K.; Subramanian, C.; Yun, M.-K.; Frank, M. W.;

White, S. W.; Rock, C. O.; Lee, R. E.; Jackowski, S. A Therapeutic

Approach to Pantothenate Kinase Associated Neurodegeneration.

Nat. Commun. 2018, 9, No. 4399.

DIEA, diisopropylethylamine; MRP, human multidrug resist-

ance protein; NBIA, neurodegeneration with brain iron

accumulation; PanK, pantothenate kinase; PANK, gene

encoding pantothenate kinase; PBEC, porcine brain endothe-

lial cells; PKAN, pantothenate kinase-associated neurodegen-

eration; POC, phosphonyloxycarbonate; POM, pivaloyloxy-

methyl; PPA, phosphopantothenic acid; TEA, triethylamine

(15) Freel Meyers, C. L.; Borch, R. F. Activation Mechanisms of

Nucleoside Phosphoramidate Prodrugs. J. Med. Chem. 2000, 43,

4319−4327.

(16) De Clercq, E. The Clinical Potential of the Acyclic (and Cyclic)

Nucleoside Phosphonates. The Magic of the Phosphonate Bond.

Biochem. Pharmacol. 2011, 82, 99−109.

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Cyclic Phosphopantothenic Acid Prodrugs for Treatment of Pantothenate Kinase-Associated Neurodegeneration

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