Toward the Efficient Synthesis of New Phosphopantothenate Derivatives by Using Chlorophosphate Reagents

Article abstract of DOI:10.1055/s-0035-1562437

Seven new masked analogues of phosphopantothenate, suitable for an evaluation of their potential ability to restore intracellular coenzyme A (CoA) levels, were successfully synthesized in good to moderate yields. The preparation of derivatives masked with (acyloxy)alkyl groups was studied particularly in terms of synthetic efficiency, and the possibility of applying different chlorophosphate reagents for the preparation of a variety of masked derivatives was also investigated. Using this methodology, we have successfully synthesized a new chlorophosphate reagent, suitable for the introduction of the (isobutyryloxy)methyl functionality into the prodrug. Additionally, special attention was given to finding mild and economical reaction conditions for phosphorylation of biological molecules.

Full text of DOI:10.1055/s-0035-1562437

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2015, 47, A–J
paper
A
J. Pahor et al.
Paper
Synthesis
Toward the Efficient Synthesis of New Phosphopantothenate
Derivatives by Using Chlorophosphate Reagents
Jerca Pahora,b
O
Stojan Stavber*a,b,c
Alen Čusakd,e
Gregor Kosecd
Hrvoje Petkovićd
Ajda Podgoršek Berkeb,d
RO
P
Cl
OR
OH
OH
H
O
H
DIPEA, DMPA
CH2Cl2
N
OEt
N
OEt
RO
P
O
HO
OR
O
O
O
O
0 °C to r.t.
2–12 h
7 examples
up to 80% yield
a Laboratory of Organic and Bioorganic Chemistry, Department
of Physical and Organic Chemistry, Jožef Stefan Institute,
Jamova 39, 1000 Ljubljana, Slovenia
R = Me, Et, i-Pr, Ph, Tol
t-BuCO2CH2, i-PrCO2CH2
stojan.stavber@ijs.si
b Jožef Stefan International Postgraduate Schoo, Jamova 39,
1000 Ljubljana, Slovenia
c Centre of Excellence for Integrated Approaches in Chemistry
and Biology of Proteins, Jamova 39, 1000 Ljubljana, Slovenia
d Acies Bio d.o.o., Tehnološki Park 21, 1000 Ljubljana, Slovenia
e Alkemika Ltd., Kukovčeva 1, 3000 Celje, Slovenia
Received: 15.04.2016
Accepted after revision: 09.05.2016
Published online: 22.06.2016
CoA biosynthesis, pantothenate kinase.3a Currently there is
no therapeutic option available to prevent or delay progress
of PKAN,4 which provides an impetus for the development
of new compounds that could effectively restore CoA bio-
synthesis. PKAN is caused by inactivation of pantothenate
kinase, which catalyzes phosphorylation of pantothenic
acid to phosphopantothenate, the first and tightly regulated
step of CoA biosynthesis. Compounds that bypass this
blocked enzymatic step and enter the biosynthetic process
downstream in biosynthesis of CoA have been suggested as
potential therapeutic agents.3c Phosphorylated derivatives
of pantothenic acid could therefore serve as product re-
placement therapeutics, enabling a viable alternative bio-
synthetic route to CoA. In a collaborative effort we have re-
cently published that 4′-phosphopantetheine, another bio-
synthetic intermediate of CoA, can enter cells and be
converted into CoA, hence restoring intracellular CoA lev-
els.5 In a subsequent study, the feasibility of using phospho-
pantothenate bypass therapy to restore CoA levels has been
also investigated by the independent research group of
Zano et al.6 These encouraging results suggest that a re-
placement therapy is possible and hold promise for a thera-
peutic intervention which could dramatically improve the
quality of life of PKAN patients.
DOI: 10.1055/s-0035-1562437; Art ID: ss-2016-t0252-op
Abstract Seven new masked analogues of phosphopantothenate,
suitable for an evaluation of their potential ability to restore intracellu-
lar coenzyme A (CoA) levels, were successfully synthesized in good to
moderate yields. The preparation of derivatives masked with (acyl-
oxy)alkyl groups was studied particularly in terms of synthetic efficien-
cy, and the possibility of applying different chlorophosphate reagents
for the preparation of a variety of masked derivatives was also investi-
gated. Using this methodology, we have successfully synthesized a new
chlorophosphate reagent, suitable for the introduction of the (isobutyr-
yloxy)methyl functionality into the prodrug. Additionally, special atten-
tion was given to finding mild and economical reaction conditions for
phosphorylation of biological molecules.
Key words phosphopantothenate, phosphorylation, chlorophosphate
reagents, vitamins, phosphates
Pantothenic acid, also known as vitamin B5, is a water-
soluble B-complex vitamin, essential for animal and human
nutrition. Biologically, it represents a precursor for the bio-
synthesis of coenzyme A (CoA) (Figure 1), an essential co-
factor that participates in over 100 different reactions in in-
termediary metabolism.1 Beside its renowned function in
cellular metabolism, recent research revealed a broader
functionality of CoA and acyl-CoA species and their role in
regulation of many important non-metabolic processes as
well.2 Reduced levels of CoA were recently found to be in-
volved in the pathophysiology of rare but catastrophic he-
reditary neurological disorders, termed neurodegeneration
with brain iron accumulation (NBIA).3 Up to 50% of the total
reported NBIA cases are attributed to pantothenate kinase-
associated neurodegeneration (PKAN), which is caused by
mutations in the PANK2 gene encoding the key enzyme of
NH2
OH
H
H
O
O
N
N
N
N
N
HS
O
P
O–
O
P
O–
O
N
O
O
pantothenic acid
O
OH
O
HO
P
O–
phosphopantothenate
Figure 1 Coenzyme A (CoA)
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J

B
J. Pahor et al.
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Synthesis
The potential of phosphorylated derivatives of panto-
thenic acid in medicinal chemistry is severely limited due
to the lack of well-defined and universal preparation and
purification procedures. The majority of the research on
their synthesis has been published before the 1990s with a
frequently questionable formation of products and insuffi-
cient descriptions of preparative procedures. Phosphoryla-
tion has been achieved by using different phosphorylating
reagents, such as diphenyl chlorophosphate,7 dibenzyl chlo-
rophosphate,8 cyanoethyl phosphate,9 dibenzyl-N,N-diiso-
propylphosphoroamidite,10 and a few others. Complex mul-
tistep synthesis, poor yields, and low efficiency and selec-
tivity are frequent drawbacks of the existing synthetic
procedures. The quest for appropriate synthetic methods is
a key step toward unlocking the full pharmaceutical poten-
tial of this group of compounds. Consequently, the aim of
our study was to prepare novel phosphorylated derivatives
of pantothenic acid while applying innovative approaches
in the synthesis.
hemicalcium salt (1) in one step, followed by coupling with
different chlorophosphate reagents 3, as described in
Scheme 1. Since our preferred strategy was to prepare ethyl
D-pantothenate (2) in a single step, it was prepared by the
Fischer esterification protocol by treatment of commercial-
ly available D-pantothenic acid hemicalcium salt (1) with
sulfuric acid and an excess of ethanol, according to a
known, yet inadequately described literature procedure.13
After optimization, this simple and low-cost method pro-
vided ethyl D-pantothenate (2) in 35% yield.
OH
OH
H
N
H
N
O–
OEt
HO
HO
O
O
O
O
0.5 Ca2+
2
1
O
P
RO
Cl
OR
3
Importantly, the medicinal application of phosphorylat-
ed compounds has often been found to be ineffective, due
to limited bioavailability and high hydrophilicity related to
the charged phosphate group. In order to address the drug
delivery issues associated with biologically active phos-
phate esters, several prodrug approaches have been devel-
oped.11 These methods mainly include a preparation of a
prodrug molecule by masking the phosphate group and
providing various phosphate esters. Since the need for
chemical substitutions on phosphopantothenate in order to
achieve the desired biological activity has already been
shown,6 the preparation of new derivatives of phosphopan-
tothenate with masked phosphate groups and methods for
their preparation have been chosen as the focus of our
study.
Herein, we describe the synthesis of several novel deriv-
atives of phosphopantothenate, suitable for an evaluation of
their potential biological activity. Compounds described
with general formula 4 in Scheme 1 represent analogues of
phosphopantothenate with three masked functionalities:
the ethyl ester, utilized to mask the carboxyl group, and two
variable groups utilized to mask the charged phosphate
group. On the basis of previous successful literature exam-
ples,12 the ethyl ester group was selected for masking the
carboxylic acid terminus of phosphopantothenic acid. For
the phosphate group, two identical masking functionalities
were selected for generating a non-charged species, to
avoid the formation of an asymmetrically modified phos-
phorus atom which results from applying mixed protection
strategies such as aryl S-acyl-2-thioethyl or aryl phosphora-
midate. The synthesis of the selected compounds was ini-
tially planned according to the commercial availability of
chemicals, which prompted us to use pantothenic acid as a
starting material. All masked phosphate analogues were
synthesized from the same intermediate: ethyl D-panto-
thenate (2), which was prepared from pantothenic acid
OH
H
N
O
OEt
RO
P
O
OR
O
O
4
Scheme 1 The synthesis of proposed masked phosphopantothenate
derivatives with general formula 4
Subsequently, we turned our attention to the phosphor-
ylation of ethyl D-pantothenate (2). We carried out the
phosphorylation of 2 by using different chlorophosphates 3,
starting with commercially available reagents. In our search
toward optimal reaction conditions, a simplified phosphor-
ylation model system was selected; benzyl alcohol (5a) was
selected as the nucleophilic component for a phosphoryla-
tion with diethyl chlorophosphate (3a) (Supporting Infor-
mation, Table 1). Screening of different bases and solvents,
commonly used in phosphorylation of alcohols with chlo-
rophosphate reagents, was initially performed (Supporting
Information, Table 1), followed by further optimization ex-
periments carried out in order to optimize the equivalents
of reagents and to determine an optimal reaction time. The
highest yield was achieved when using N,N-diisopropyldi-
ethylamine with a substoichiometric amount of 4-(di-
methylamino)pyridine (DMAP) (Supporting Information,
Table 1, entry 12). According to literature data, this combi-
nation of bases is also particularly convenient for the phos-
phorylation of biological molecules with various chloro-
phosphate reagents.14 Dichloromethane was used as the
solvent of choice and the reaction was complete at ambient
temperature within two hours with a corresponding 93%
yield. In the following step, we transferred the resulting re-
action conditions from the model substrate benzyl alcohol
(5a) to the phosphorylation of ethyl D-pantothenate (2), us-
ing the same phosphorylating reagent 3a.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J

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J. Pahor et al.
Paper
Synthesis
Initially, our attempts to selectively phosphorylate the
primary hydroxyl group of substrate 2 according to our
model study were not successful. When experiments were
carried out in the same manner as with benzyl alcohol (5a),
the diphosphorylated ester of pantothenic acid 4a′ also
formed in addition to the desired product 4a (Scheme 2).
Although only 10% of this side product 4a′ was observed in
the mixture, its formation significantly hampered the puri-
fication of the desired product 4. Chromatographic separa-
tion of the products was extremely difficult, leading to sig-
nificant yield reduction during purification. The key im-
provement necessary for the existing method was related to
the manner of adding the chlorophosphate reagent. For se-
lective transformation it was critical that the phosphoryla-
tion agent was added dropwise, as a solution in CH2Cl2, to
the reaction mixture at 0 °C. To our delight, only traces of
diphosphorylated derivative 4a′ were formed.
Although alkyl and aryl masking groups used in deriva-
tives 4a–e (Table 1) are very likely to be too stable to be
used in successful prodrug strategies,11a these newly syn-
thesized derivatives represent an interesting collection of
new masked derivatives of phosphopantothenate. Their
preparation contributes to the additional knowledge in the
field of synthesis of phosphorylated derivatives of panto-
thenic acid. Since simple alkyl esters of phosphates are typ-
ically metabolically stable,11a we turned our attention to
the synthesis of phosphopantothenate derivatives masked
with functional groups that are most commonly used
among prodrug strategies.
One of the most common prodrug strategies for phos-
phates and phosphonates includes the masking of the free
phosphate with (acyloxy)alkyl groups.11a Within this series,
(pivaloyloxy)methyl (POM) protection appears to be partic-
ularly effective,11,15 with individual examples already being
FDA approved drugs.16 While sometimes successful, in
many cases the preparation of these prodrugs tends to be
very challenging, due to a complex multistep synthesis and
formation of complex mixtures with corresponding poor
yields of desired products.15h,17 In our search for the most
promising protocol for the introduction of POM groups,
three general methods have been identified in the litera-
ture. The first method includes the phosphorylation of the
precursor’s hydroxyl group to form the drug molecule with
an unmasked phosphate group, followed by double alkyla-
tion with (pivaloyloxy)methyl iodide in the presence of
DIPEA.18 The second approach involves direct condensation
of the hydroxyl compound with bis[(pivaloyloxy)methyl]
phosphate in the presence of triphenylphosphine and di-
ethyl azodicarboxylate under Mitsunobu conditions.15c Fi-
nally, the third method is the one-pot reaction between
bis[(pivaloyloxy)methyl] chlorophosphate and the hydroxyl
group of a precursor, which directly affords the desired
products.17b
O
O
P
Cl
OH
O
H
N
O
P
OH
OEt
H
N
O
O
3a
OEt
HO
O
O
O
O
O
4a
2
+
O
P
O
O
O
H
O
P
N
OEt
O
O
O
O
O
4a′
Scheme 2 Phosphorylation of ethyl D-pantothenate (2) with diethyl
chlorophosphate (3a). Reagents and conditions: DIPEA, DMPA, CH2Cl2,
r.t., 2 h.
Encouraged by these promising results, we envisioned
the method to be applicable to the phosphorylation of the
chosen substrate 2 when using different phosphorylation
agents, so we continued our synthetic efforts toward
masked derivatives of phosphopantothenate. In a subse-
quent series of experiments, the reaction of ethyl D-panto-
thenate (2) with different commercially available chloro-
phosphate reagents was investigated, affording five new de-
rivatives as described in Table 1. In all five cases, selective
phosphorylation was achieved and we were able to isolate
and purify the desired products 4a–e in good to excellent
yields (63–80%). However, our attempts to phosphorylate
pantothenic acid ester 2 with two cyclic chlorophosphate
reagents, namely 2-chloro-1,3,2-dioxaphospholane 2-oxide
(3f) and o-phenylene chlorophosphate (3g) failed (Table 1,
entries 6 and 7). Under the described reaction conditions,
extensive decomposition of the reagents with ring opening
was observed and only traces of the desired products 4f and
4g were detected in the complex reaction mixtures.
We have selected the third approach for several reasons,
as described below. Reported yields for the first and second
method are generally very low, while the yields reported
for the third approach are generally higher.15h In particular,
POM functionalization of pantothenate moiety by methods
1 and 2 provided the desired product only in marginal yield
(<10%).17 These observations are also consistent with our
own experience in the field of phosphopantothenate chem-
istry.19 Finally, our above reported successful preparation of
phoshopantothenate derivatives 4a–e by using different
chlorophosphate reagents strongly indicated that this may
be the most appropriate method for the preparation of oth-
er masked derivatives as well. Therefore, commercially un-
available bis[(pivaloyloxy)methyl] chlorophosphate was
prepared according to a procedure reported by Cole and
Hwang.17b The synthetic route is outlined in Table 2, entry
1. The reaction commenced with the trans-esterification of
trimethyl phosphate with chloromethyl pivalate to form
tris[(pivaloyloxy)methyl] phosphate 7h, with subsequent
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J

D
J. Pahor et al.
Paper
Synthesis
hydrolysis of one group with piperidine to yield the diester
bis[(pivaloyloxy)methyl] phosphate 8h. In the final step,
bis[(pivaloyloxy)methyl] phosphate 8h reacted with oxalyl
chloride to form bis[(pivaloyloxy)methyl] chlorophosphate
(3h), which was directly used in the phosphorylation of our
substrate 2. The previously optimized reaction conditions
for the phosphorylation were used, which provided the de-
sired product 4h in 35% yield (Table 3, entry 1). Our yield
was slightly lower than those reported in the literature, al-
beit synthetically useful. The application of this reagent of-
ten requires uneconomical and harsh conditions. In the lit-
erature examples,17b,20 5 equivalents bis[(pivaloyloxy)meth-
yl] chlorophosphate (3h) and 7–13 equivalents base were
used to isolate the desired compounds in 41–50% yield. In
our case, a satisfactory synthesis of product 4h (35% yield)
was accomplished by using only 1.5 equivalents bis[(piv-
aloyloxy)methyl] chlorophosphate (3h), 1.5 equivalents
DIPEA, and 0.1 equivalents DMAP (Table 3, entry 1).
Although the POM prodrug strategy is very promising
and already used in several approved drugs in the mar-
ket,16,21 it is worth mentioning the concerns about its po-
tential toxicity in long-term use, arising from its decompo-
sition products.11a The preparation of other (acyloxy)alkyl
prodrugs that do not generate pivalic acid during its bio-
Table 1 Synthesis of Masked Derivatives of Phosphopantothenate by Using Commercially Available Chlorophosphate Reagentsa
OH
O
H
O
P
N
OEt
RO
P
O
+
RO
Cl
2
OR
3
OR
O
O
4
Entry
1
Reagent 3
Reaction time
2 h
Product 4
Yieldb (%)
74
OH
O
O
H
N
OEt
EtO
P
Cl
EtO
P
O
EtO
EtO
O
O
3a
4a
OH
O
P
O
P
H
N
OEt
MeO
Cl
MeO
O
2
3
4
2 h
80
64
63
MeO
MeO
O
O
3b
4b
OH
O
O
H
N
OEt
OEt
i-PrO
P
Cl
Cl
i-PrO
P
O
O
2 h
i-PrO
i-PrO
O
O
O
3c
4c,
OH
O
H
N
O
PhO
P
PhO
P
12 h
PhO
O
PhO
3d
4d
OH
O
H
N
O
OEt
TolO
P
Cl
TolO
P
O
5
6
12 h
77
TolO
O
O
TolO
3e
4e,
OH
O
P
H
O
P
N
OEt
O
Cl
O
O
2 h
2 h
tracec
O
O
O
O
3f
4f
OH
O
P
H
N
O
P
OEt
O
Cl
O
O
7
tracec
O
O
O
O
3g
4g
a Reaction conditions: 2 (1 mmol), 3 (1.3 equiv), DIPEA (1.5 equiv), DMAP (0.1 equiv), CH2Cl2 (6 mL), under argon, 0°C, then r.t., 2–12 h.
b Yield of isolated and purified product.
c Detected by LC-MS of crude reaction mixtures.
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Synthesis
Table 2 Synthesis of (Acyloxy)alkyl and {[(Alkyloxy)carbonyl]oxy}alkyl Chlorophosphate Reagentsa
R1
R1
O
O
R1
R1
R1
O
O
O
P
O
O
P
O
P
O
i
ii
iii
R2
O
O
O
O
R2
MeO
OMe
+
R2
O
O
OH
R2
O
O
P
Cl
R2
O
Cl
MeO
R1
O
R1
R1
O
O
3.3 equiv
7
O
O
3
8
O
O
O
O
R2
R2
R2
Entry
R1
R2
7
Yieldb (%)
8
Yieldb (%)
3
Yieldc (%)
Overall yieldd (%)
1
2
3
4
H
t-Bu
i-Pr
Me
7h
7i
7j
–
74
65
18
–
8h
8i
–
81
57
–
3h
85
80
–
51
30
–
H
3i
–
H
Me
OEt
–
–
–
–
–
a Reaction conditions: (i) NaI, MeCN, reflux, 72 h; (ii) 1. piperidine, r.t., 12 h, 2. Dowex, r.t., 1 h; (iii) oxalyl chloride, DMF, CH2Cl2, r.t., 2 h.
b Yield of isolated and purified product.
c Conversions into 3h and 3i in the last step were determined by 1H NMR spectroscopy of crude reaction mixtures.
d Overall yield of three-step reaction.
conversion is therefore highly desired. Additionally, the
chemical stabilities of different (acyloxy)alkyl groups can
differ,22 and since evaluating stability is an important as-
pect of optimizing prodrug properties, the preparation of
various analogues is highly attractive from the pharmaceu-
tical standpoint. Although the preparation of other (acyl-
oxy)alkyl prodrugs is not uncommon in the literature, the
synthetic strategies used for their preparation seem less di-
verse and effective. Our experience with the preparation of
POM derivatives showed the use of bis[(pivaloyloxy)meth-
yl] chlorophosphate to be the most successful approach,
and therefore we studied the possibility of applying this
method in the preparation of other related masked ana-
logues. Thus, we tried to use the previously described effi-
cient procedure for the synthesis of bis[(pivaloyloxy)meth-
yl] chlorophophate published by Hwang and Cole17b for the
Table 3 Synthesis of Phosphopantothenate Derivatives Masked with (Acyloxy)alkyl Groupsa
O
OH
O
H
N
O
P
O
P
OEt
R
O
O
Cl
R
O
O
O
2
+
O
O
O
O
3
4
O
O
O
O
R
R
Entry
Reagent 3
Product 4
Yieldb (%)
O
OH
O
H
N
OEt
O
O
P
O
O
O
O
1
3h
35
O
O
4h
O
OH
O
P
H
N
OEt
O
O
O
O
O
O
2
3i
25
O
O
4i
a Reaction conditions: 2 (1 mmol), 3h or 3i (1.5 equiv), DIPEA (1.5 equiv), DMAP (0.1 equiv), CH2Cl2, under argon, 0 °C, then r.t., 2 h.
b Yield of isolated and purified product.
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Synthesis
preparation of other chlorophophate reagents with differ-
ent (acyloxy)alkyl groups. To the best of our knowledge,
there is no example of the preparation and application of
other (acyloxy)alkyl chlorophosphate reagents in the exist-
ing literature. We hypothesized that the proposed approach
would be suitable for the general preparation of various
chlorophosphate reagents if we could replace chloromethyl
pivalate in the first step of the synthesis with other suitable
chlorides as described in Table 2, entries 2–4.
First, we tried to prepare the bis[(pivaloyloxy)methyl]
chlorophosphate analogue with (isobutyryloxy)methyl (i-
BOM) groups, which are structurally the closest to POM (Ta-
ble 2, entry 2). The first step of the reagent preparation, a
transesterification of trimethyl phosphate to yield
tris[(isobutyryloxy)methyl] phosphate (7i) by using chloro-
methyl isobutyrate was successfully accomplished with
comparable efficiency (65%), as in the POM case. Initially,
attempts to cleave only one i-BOM group were not success-
ful, as we obtained a mixture of bis[(isobutyryloxy)methyl]
hydrogen phosphate (8i) and mono[(isobutyryloxy)methyl]
dihydrogen phosphate in a nearly 1:1 ratio.
In contrast to the selective exclusive hydrolysis of one
POM group, the hydrolysis of both groups occurred in this
case. To circumvent this problem, different approaches
were tried. The course of the reaction was slightly shifted in
favor of the desired product when piperidine was added at
0 °C, but we failed to isolate one product exclusively. To our
delight, the separation and purification of the desired prod-
uct 8i was feasible, albeit the yield was significantly lower
(57%; Table 2, entry 2) than the same reaction step during
POM reagent preparation (81%). The chlorophoshate re-
agent 3i obtained in the last step was successfully used for
the phosphorylation of ethyl D-pantothenate (2) to provide
product 4i in moderate yield (25%) as shown in Table 3, en-
try 2.
Unfortunately, further attempts to prepare additional
chlorophosphate reagents were unsuccessful. We tried to
prepare the bis[(acetyloxy)methyl] chlorophosphate re-
agent (Table 2, entry 3). Tris[(acetyloxy)methyl] phosphate
7j was obtained only in marginal yield (18%), after pre-
esterification of trimethyl phosphate in the presence of
chloromethyl acetate. Nevertheless, we attempted to carry
out the next step as well, which resulted in total decompo-
sition of the starting material 7j, and no desired product
was observed after hydrolysis. The poor yield of the first
step and decomposition during the second step are most
likely a consequence of the lower stability of this (acetyl-
oxy)methyl group. Unfortunately, we also failed at synthesis
of the chlorophosphate reagent with ‘carbonate ester’
groups (Table 2, entry 4). In this case, the first step was al-
ready an unconquerable barrier. In an alternative method,
1-chloroethyl ethyl carbonate reacted with trimethyl phos-
phate in the presence of sodium iodide, but either polymer-
ization or decomposition occurred and the isolation was
impractical. We failed to detect the product in this complex
reaction mixture via LC-MS, and hence this functional
group was abandoned. Decrease of chemical stability in
(acyloxy)alkyl phosphate esters with less bulky alkyl sub-
stituents has been already shown.22 We assume that this
approach is not suitable for the preparation of reagents
with groups that are less stable, for instance carbonate-
moiety-containing groups and less branched alkyl chains,
such as the (acetyloxy)methyl group.
Due to the promising results of the recent studies on the
ability of phosphorylated derivatives of pantothenic acid to
restore intracellular CoA levels in cells with defective CoA
synthesis, these compounds have captured the attention of
both biological and synthetic chemists. Our work has re-
sulted in the synthesis of seven new derivatives of
phosphopantothenate in good to moderate yields. Since
phosphorylated compounds are generally unable to pene-
trate cell membranes and thus not likely to be bioactive, the
synthesis of derivatives with masked phosphate groups was
the main focus of this study. Apart from several derivatives
masked with simple alkyl and aryl groups which were syn-
thesized by using commercially available chlorophosphate
reagents, special attention was given to the preparation of
(acyloxy)alkyl derivatives as the most prominent represen-
tatives among prodrug types of phosphates. Since the ma-
jority of the literature on synthetic pathways covers only
the introduction of (pivaloyloxy)methyl (POM) groups,
with the synthesis of other (acyloxy)alkyl phosphate esters
less commonly described, we were eager to develop novel
methods for their preparation. For this purpose, the possi-
bility of applying the bis[(pivaloyloxy)methyl] chlorophos-
phate procedure17b for the preparation of other related pro-
drugs was studied. We were successful in the synthesis of
one new chlorophosphate reagent, suitable for general in-
troduction of (isobutyryloxy)methyl (i-BOM) groups into
the phosphate moiety. Finally, two new phosphopantothen-
ate derivatives masked with POM and i-BOM (acyloxy)alkyl
groups were synthesized. In addition, special attention was
given to finding mild and economical reaction conditions
for phosphorylation, suitable for use with biologically ac-
tive molecules.
Since recent studies5,6 on the potential ability of phos-
phorylated derivatives of pantothenic acid to restore nor-
mal CoA levels in cells with impaired pantothenate kinase
activity are very promising, testing the biological activity of
the prepared compounds are already in progress to evaluate
our synthetic efforts.
Chloromethyl isobutyrate (CAS 61644-18-6) and chloromethyl ace-
tate (CAS 625-56-9) were purchased from AKos GmbH (Steinen, Ger-
many). All other chemicals were obtained from commercial sources
and used without further purification. Unless otherwise indicated, all
reactions were carried out under an argon atmosphere in oven-dried
glassware using anhydrous solvents. Anhydrous solvents were ob-
tained by storing the solvents over activated 4 Å molecular sieves
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J

G
J. Pahor et al.
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Synthesis
or anhydrous Na2SO4 under an atmosphere of N2. Reactions were
monitored by TLC which was performed using silica gel/TLC cards,
DC-Alufolien-Kieselgel (Fluka, Sigma-Aldrich). Visualization was car-
ried out using UV light (254 nm) and KMnO4 stain (1.5 g KMnO4, 10 g
K2CO3, 1.25 mL 10% NaOH dissolved in 200 mL H2O). Column chroma-
tography was carried out with silica gel 60 Å, 40–63 micron. NMR
spectra were obtained at ambient temperature on a 300 MHz spec-
trometer in the solvent indicated (CDCl3). Chemical shifts (δ) are giv-
en in ppm and calibrated using an internal reference (Me4Si), the sig-
nal of residual solvent (CDCl3), or an external standard (85% H3PO4)
for 1H, 13C, and 31P NMR, respectively. High-resolution mass spectra
were measured on a Q-TOF Premier instrument (Waters-Micromass).
additional 2–12 h. The reaction mixture was quenched with H2O (10
mL) and extracted with CH2Cl2 (2 × 20 mL). The organic phase was
washed with sat. aq NH4Cl, dried over Na2SO4, and concentrated un-
der reduced pressure. The product was purified by column chroma-
tography.
Ethyl (R)-3-[4-(Diethoxyphosphoryloxy)-2-hydroxy-3,3-dimethyl-
butanamido]propanoate (4a)
Product 4a was prepared according to general procedure 1 from di-
ethyl chlorophosphate (3a; 193 μL, 1.3 mmol, 1.3 equiv). The reaction
mixture was stirred for 2 h at r.t. Purification by column chromatog-
raphy (silica gel, CH2Cl2–MeOH, 95:5) afforded the product as a pale
yellow oil.
Ethyl (R)-3-(2,4-Dihydroxy-3,3-dimethylbutanamido)propanoate
Yield: 284 mg (74%); Rf = 0.25 (CH2Cl2–MeOH, 95:5).
(2)13
1H NMR (300 MHz,): δ = 0.86 (s, 3 H), 1.12 (s, 3 H), 1.26 (t, J = 7.1 Hz, 3
H), 1.35 (td, J = 0.9, 7.1 Hz, 6 H), 2.55 (t, J = 6.4 Hz, 2 H), 3.54 (m, 2 H),
3.6 (dd, J = 7.5, 10.0 Hz, 1 H), 3.98 (s, 1 H), 4.18–4.07 (m, 7 H), 7.44
(app br t, J = 6.0 Hz, 1 H, NH).
13C NMR (76 MHz, CDCl3): δ = 14.09, 16.03 (d, J = 6.6 Hz), 18.53, 20.92,
34.13, 34.51, 39.36 (d, J = 4.9 Hz), 60.55, 64.13 (app t, J = 5.9 Hz),
73.43, 73.49 (d, J = 6.0 Hz), 171.97, 172.36.
To a stirred suspension of calcium D-pantothenate 1 (4.765 g, 20
mmol) in EtOH (120 mL), sulfuric acid (1.13 mL, 20 mmol) was added.
The reaction mixture was heated overnight at reflux temperature.
The mixture was directly flushed through a silica column with EtOAc
(200 mL) and concentrated under reduced pressure. Purification by
column chromatography (silica gel, EtOAc–hexane, 95:5) afforded the
product as a slightly yellow oil.
31P NMR (120 MHz, CDCl3): δ = 0.59 (s).
Yield: 1.826 g (35%); Rf = 0.23 (hexane–EtOAc, 95:5).
HRMS (ESI): m/z [M + H]+ calcd for C15H31NO8P: 384.1787; found:
384.1798.
1H NMR (300 MHz, CDCl3): δ = 0.91 (s, 3 H), 0.99 (s, 3 H), 1.27 (t, J = 7.1
Hz, 3 H), 2.56 (t, J = 6.2 Hz, 2 H), 3.46–3.63 (m, 4 H), 3.77 (br s, 1 H),
4.01 (s, 1 H), 4.16 (q, J = 7.1 Hz, 2 H), 4.32 (br s, 1 H), 7.32 (app br t, J =
5.7 Hz, 1 H, NH).
Ethyl (R)-3-[2,4-Bis(diethoxyphosphoryloxy)-3,3-dimethylbutan-
amido]propanoate (4a′)
13C NMR (76 MHz, CDCl3): δ = 14.27, 20.34, 21.28, 34.20, 34.76, 39.38,
60.99, 71.27, 77.57, 172.46, 173.46.
HRMS (ESI): m/z [M + H]+ calcd for C11H22NO5: 248.1498; found:
248.1493.
To a stirred solution of ethyl D-pantothenate (2) (247 mg, 1 mmol),
DIPEA (528 μL, 3 mmol), and DMAP (25 mg, 0.2 mmol) in CH2Cl2 (5
mL) at 0 °C, diethyl chlorophosphate (3a; 386 μL, 2.6 mmol) was add-
ed dropwise under argon. The reaction mixture was allowed to warm
to r.t. and stirred for an additional 4 h. The reaction mixture was
quenched with H2O (10 mL) and extracted with CH2Cl2 (2 × 20 mL).
The organic phase was washed with sat. aq NH4Cl, dried over Na2SO4,
and concentrated under reduced pressure. Purification by column
chromatography (silica gel, EtOAc–MeOH, 98:2) afforded the product
as a colorless oil.
Benzyl Diethyl Phosphate (6a)23
To a stirred solution of benzyl alcohol (5a; 108 mg, 1 mmol), DIPEA
(264 μL, 1.5 mmol), and DMAP (12 mg, 0.1 mmol) in CH2Cl2 (5 mL),
diethyl chlorophosphate (3a; 192 μL, 1.3 mmol) was added dropwise
under argon. The reaction mixture was stirred at ambient tempera-
ture for 2 h. The reaction mixture was quenched with H2O (5 mL) and
extracted with CH2Cl2 (3 × 10 mL). The organic phase was dried over
Na2SO4, and concentrated under reduced pressure. Purification by
column chromatography (silica gel, CH2Cl2–MeOH, 97:3) afforded the
product as a yellow oil.
Yield: 227 mg (44%); Rf = 0.18 (EtOAc–MeOH, 98:2).
1H NMR (300 MHz, CDCl3): δ = 1.03 (s, 3 H), 1.08 (s, 3 H), 1.27 (t, J = 7.1
Hz, 3 H), 1.35 (m, 12 H), 2.56 (t, J = 6.4 Hz, 2 H), 3.56 (m, 2 H), 3.88 (dd,
J = 4.5, 9.9 Hz, 1 H), 3.97 (dd, J = 4.9, 9.9 Hz, 1 H), 4.14 (m, 8 H), 4.56 (d,
J = 8.8 Hz, 1H), 6.95 (app br t, J = 6.0 Hz, 1 H, NH).
13C NMR (76 MHz, CDCl3): δ = 14.16, 16.12 (m), 20.39, 20.59, 33.91,
34.75, 39.10 (dd, J = 6.3, 2.2 Hz), 60.71, 63.81–64.44 (m), 71.97 (d, J =
5.7 Hz), 80.54 (d, J = 6.6 Hz), 167.82, 172.00.
Yield: 195 mg (80%); Rf = 0.20 (CH2Cl2–MeOH, 97:3).
1H NMR (300 MHz, CDCl3): δ = 1.30 (td, J = 1.0, 7.1 Hz, 6 H), 4.02–4.15
(m, 4 H), 5.07 (d, J = 8.1 Hz, 2 H), 7.30–7.42 (m, 5 H).
13C NMR (76 MHz, CDCl3): δ = 16.13 (d, J = 6.8 Hz), 63.88 (d, J = 5.9 Hz),
69.10 (d, J = 5.5 Hz), 127.91, 128.51, 128.60, 136.10 (d, J = 6.9 Hz).
31P NMR (120 MHz, CDCl3): δ = –0.46 (s).
31P NMR (120 MHz, CDCl3): δ = –1.32 (s, 1 P), –0.73 (s, 1 P).
HRMS (ESI): m/z [M + H]+ calcd for C19H40NO11P2: 520.2077; found:
520.2075.
HRMS (ESI): m/z [M + H]+ calcd for C11H18O4P: 245.0943; found:
245.0946.
Ethyl (R)-3-[4-(Dimethoxyphosphoryloxy)-2-hydroxy-3,3-dimeth-
ylbutanamido]propanoate (4b)
Phosphorylation of Ethyl D-Pantothenate (2) with Commercially
Available Reagents 3; General Procedure 1
Product 4b was prepared according to general procedure 1 from di-
methyl chlorophosphate (3b; 146 μL, 1.3 mmol, 1.3 equiv). The reac-
tion mixture was stirred for 2 h at r.t. Purification by column chroma-
tography (silica gel, EtOAc) afforded the product as a colorless oil.
To a stirred solution of ethyl D-pantothenate (2; 247 mg, 1 mmol),
DIPEA (264 μL, 1.5 mmol), and DMAP (12 mg, 0.1 mmol) in CH2Cl2 (4
mL) at 0 °C, a solution of the appropriate chlorophosphate reagent
3a–g (1.3 mmol) in CH2Cl2 (2 mL) was added dropwise under argon.
The reaction mixture was allowed to warm to r.t. and stirred for an
Yield: 284 mg (80%); Rf = 0.29 (EtOAc).
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J. Pahor et al.
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1H NMR (300 MHz, CDCl3): δ = 0.86 (s, 3 H), 1.13 (s, 3 H), 1.26 (t, J = 7.2
Hz, 3 H), 2.55 (t, J = 6.3 Hz, 2 H), 3.55 (m, 2 H), 3.68 (dd, J = 7.6, 10.0
Hz, 1 H), 3.79 (d, J = 11.2 Hz, 3 H), 3.80 (d, J = 11.1 Hz, 3 H), 3.99 (wide
s, 1 H), 4.19–4.10 (m, 3 H), 4.67 (wide s, 1 H), 7.39 (app br t, J = 5.9 Hz,
1 H, NH).
13C NMR (76 MHz, CDCl3): δ = 14.22, 18.58, 21.01, 34.24, 34.67, 39.55
(d, J = 5.5 Hz), 54.69 (m), 60.74, 73.46, 73.86 (d, J = 5.9 Hz), 172.15,
172.34.
Yield: 391 mg (77%); Rf = 0.24 (EtOAc–heptane, 55:45).
1H NMR (300 MHz, CDCl3): δ = 0.83 (s, 3 H), 1.10 (s, 3 H), 1.24 (t, J = 7.1
Hz, 3 H), 2.32 (br s, 6 H), 2.51 (t, J = 6.3 Hz, 2 H), 3.51 (ddt, J = 2.8, 6.5,
8.9 Hz, 2 H), 3.81 (dd, J = 8.6, 10.0 Hz, 1 H), 3.87 (s, 1 H), 4.13 (q, J = 7.2
Hz, 2 H), 4.31 (dd, J = 7.7, 10.1 Hz, 1 H), 7.11 (m, 8 H), 7.28 (app br t, J =
5.9 Hz, 1 H, NH).
13C NMR (76 MHz, CDCl3): δ = 14.26, 18.45, 20.82, 21.09, 34.25, 34.69,
39.72 (d, J = 5.1 Hz), 60.75, 73.41, 75.05 (d, J = 6.4 Hz), 119.86 (app dd,
J = 4.6, 7.0 Hz), 130.39, 135.29, 135.40, 148.27 (app t, J = 7.0 Hz),
171.99, 172.13.
31P NMR (120 MHz, CDCl3): δ = 2.64 (s).
HRMS (ESI): m/z [M + H]+ calcd for C13H27NO8P: 356.1474; found:
356.1475.
31P NMR (120 MHz, CDCl3): δ = –9.52 (s).
HRMS (ESI): m/z [M + H]+ calcd for C25H35NO8P: 508.2100; found:
508.2114.
Ethyl (R)-3-[4-(Diisopropoxyphosphoryloxy)-2-hydroxy-3,3-di-
methylbutanamido]propanoate (4c)
Tris[(pivaloyloxy)methyl] Phosphate (7h)17b
Product 4c was prepared according to general procedure 1 from diiso-
propyl chlorophosphate (3c; 198 μL, 1.3 mmol, 1.3 equiv). The reac-
tion mixture was stirred for 2 h at r.t. Purification by column chroma-
tography (silica gel, CH2Cl2–MeOH, 98:2) afforded the product as a
pale yellow oil.
To a solution of trimethyl phosphate (2.8 g, 20 mmol) in anhydrous
MeCN (17 mL) were sequentially added chloromethyl pivalate (11.2
mL, 78 mmol) and NaI (8.99 g, 60 mmol). The reaction mixture was
heated at reflux (85 °C) for 72 h, cooled to ambient temperature, and
diluted with Et2O (160 mL). The organic phase was washed with H2O
(2 × 50 mL) and sat. aq Na2S2O3 solution (2 × 50 mL), dried over
Na2SO4, and concentrated. Purification by column chromatography
(silica gel, hexane–EtOAc 4:1) afforded a viscous pale yellow oil.
Yield: 263 mg (64%); Rf = 0.23 (CH2Cl2–MeOH, 98:2).
1H NMR (300 MHz, CDCl3): δ = 0.83 (s, 3 H), 1.17 (s, 3 H), 1.26 (t, J = 7.1
Hz, 3 H), 1.35 (m, 12 H), 2.55 (t, J = 6.4 Hz, 2 H,), 3.55 (m, 2 H), 3.99
(wide s, 1 H), 4.19–4.07 (m, 3 H), 4.63 (m, 2 H), 4.95 (br s, 1 H), 7.45
(app br t, J = 5.9 Hz, 1 H, NH).
Yield: 6.478 g (74 %); Rf = 0.25 (hexane–EtOAc, 4:1).
13C NMR (76 MHz, CDCl3): δ = 14.26, 18.25, 21.30, 23.70 (m), 34.33,
34.64, 39.70 (d, J = 4.7 Hz), 60.69, 73.17 (d, J = 6.4 Hz), 73.42 (d, J =
11.9 Hz), 73.42, 172.08, 172.23.
31P NMR (120 MHz, CDCl3): δ = –0.31 (s).
HRMS (ESI): m/z [M + H]+ calcd for C17H35NO8P: 412.2100; found:
1H NMR (300 MHz, CDCl3): δ = 1.24 (s, 27 H), 5.66 (d, J = 13.7 Hz, 6 H).
13C NMR (76 MHz, CDCl3): δ = 26.95, 38.89, 82.93 (d, J = 5.0 Hz),
176.73.
31P NMR (120 MHz, CDCl3): δ = –4.74 (s).
HRMS (ESI): m/z [M + H]+ calcd for C18H34O10P: 441.1890; found:
412.2107.
441.1901.
Ethyl (R)-3-[4-(Diphenoxyphosphoryloxy)-2-hydroxy-3,3-dimeth-
ylbutanamido]propanoate (4d)
Bis[(pivaloyloxy)methyl] Hydrogen Phosphate (8h)17b
Phosphate 7h (1 g, 2.3 mmol) was dissolved in piperidine (7 mL) and
stirred at r.t. for 12 h. The solution was concentrated and further
evaporated in vacuo until a constant weight was reached (935 mg,
99% yield). The crude oil (935 mg, 2.28 mmol) was dissolved in H2O
(20 mL) and treated with Dowex W50X2 H+ form resin (19.2 g, 11.4
mmol, 0.6 mmol/g). The suspension was stirred at ambient tempera-
ture for 1 h. The resin was filtered and washed with H2O. The filtrate
was concentrated and dried in vacuo affording a white solid.
Product 4d was prepared according to general procedure 1 from di-
phenyl chlorophosphate (3d; 272 μL, 1.3 mmol, 1.3 equiv). The reac-
tion mixture was stirred for 12 h at r.t. Purification by column chro-
matography (silica gel, EtOAc–heptane, 60:40) afforded the product
as white oil.
Yield: 303 mg (63%); Rf = 0.29 (EtOAc–heptane, 6:4).
1H NMR (300 MHz, CDCl3): δ = 0.84 (s, 3 H), 1.09 (s, 3 H), 1.24 (t, J = 7.1
Hz, 3 H), 2.51 (t, J = 6.3 Hz, 2 H), 3.51 (m, 2 H), 3.86 (m, 2 H), 4.13 (q,
J = 7.1 Hz, 2 H), 4.33 (dd, J = 7.4, 9.8 Hz, 1 H), 7.17–7.28 (m, 7 H, 6 × CH
+ NH), 7.31–7.38 (m, 4 H).
13C NMR (76 MHz, CDCl3): δ = 14.25, 18.52, 21.05, 34.23, 34.70, 39.69
(d, J = 5.5 Hz), 60.77, 73.49, 75.17 (d, J = 6.4 Hz), 120.16 (m), 125.69,
125.79, 129.98, 150.42 (app t, J = 7.1 Hz), 171.99, 172.16.
Yield: 602 mg (81%).
1H NMR (300 MHz, CDCl3): δ = 1.23 (s, 18 H), 5.62 (d, J = 13.2 Hz, 4 H),
8.45 (br s, 1 H).
13C NMR (76 MHz, CDCl3): δ = 26.96, 38.85, 82.88 (d, J = 5.1 Hz),
177.02.
31P NMR (120 MHz, CDCl3): δ = –3.37 (s).
HRMS (ESI): m/z [M + H]+ calcd for C12H24O8P: 327.1209; found:
31P NMR (120 MHz, CDCl3): δ = –10.17 (s).
HRMS (ESI): m/z [M + H]+ calcd for C23H31NO8P: 480.1787; found:
327.1207.
480.1785.
Bis[(pivaloyloxy)methyl] Chlorophosphate (3h)17b
Ethyl (R)-3-{4-[bis(p-tolyloxy)phosphoryloxy]-2-hydroxy-3,3-di-
A solution of hydrogen phosphate 8h (576 mg, 1.77 mmol) and DMF
(17 μL, 0.22 mmol) in CH2Cl2 (6.5 mL) was added dropwise to a stirred
solution of oxalyl chloride (788 μL, 8.85 mmol) in CH2Cl2 (6.5 mL) un-
der argon at ambient temperature. The reaction mixture was stirred
for 2 h. The solvent was evaporated under argon to provide a crude
yellow oil (516 mg, 1.5 mmol), which was directly used in the next
step.
methylbutanamido}propanoate (4e)
Product 4e was prepared according to general procedure 1 from di-p-
tolyl chlorophosphate (3e; 386 mg, 1.3 mmol, 1.3 equiv). The reaction
mixture was stirred for 12 h at r.t. Purification by column chromatog-
raphy (silica gel, EtOAc–heptane, 55:45) afforded the product as a
white oil.
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J. Pahor et al.
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Synthesis
Ethyl (R)-3-(4-{Bis[(pivaloyloxy)methyl]phosphoryloxy}-2-hy-
droxy-3,3-dimethylbutanamido)propanoate (4h)
1H NMR (300 MHz, CDCl3): δ = 1.15 (d, J = 7.0 Hz, 12 H), 2.58 (hept, J =
7.0 Hz, 2 H), 5.53 (d, J = 10.1 Hz, 4 H).
13C NMR (76 MHz, CDCl3): δ = 18.72, 33.92, 83.08 (d, J = 4.1 Hz),
176.62.
31P NMR (120 MHz, CDCl3): δ = –3.45 (s).
HRMS (ESI): m/z [M + H]+ calcd for C10H20O8P: 299.0896; found:
To a stirred solution of ethyl D-pantothenate (2; 247 mg, 1 mmol),
DIPEA (264 μL, 1.5 mmol), and DMAP (12 mg, 0.1 mmol) in CH2Cl2
(6.5 mL) at 0 °C, a solution of chlorophosphate 3h (516 mg, 1.5 mmol)
in CH2Cl2 (6.5 mL) was added dropwise under argon. The reaction
mixture was allowed to warm to r.t. and stirred for an additional 2 h.
The reaction mixture was quenched with H2O (10 mL) and extracted
with CH2Cl2 (2 × 20 mL). The organic phase was washed with sat. aq
NH4Cl, dried over Na2SO4, and concentrated under reduced pressure.
Purification by column chromatography (silica gel, CH2Cl2–MeOH,
98:2) afforded the product as a yellow oil.
299.0893.
Bis[(isobutyryloxy)methyl] Chlorophosphate (3i)
A solution of hydrogen phosphate 7i (1.789 g, 6 mmol) and DMF (59
μL, 0.76 mmol) in CH2Cl2 (24 mL) was added dropwise to a stirred
solution of oxalyl chloride (2.6 mL, 30 mmol) in CH2Cl2 (24 mL) under
argon at ambient temperature. The reaction mixture was stirred for 2
h. The solvent was evaporated under argon to provide a crude orange
paste (1.517 g, 4.8 mmol), which was directly used in the next step.
Yield: 194 mg (35%); Rf = 0.26 (CH2Cl2–MeOH, 98:2).
1H NMR (300 MHz, CDCl3): δ = 0.86 (s, 3 H), 1.12 (s, 3 H), 1.28–1.24
(m, 18 H), 2.55 (t, J = 6.2 Hz, 2 H), 3.56 (m, 2 H), 3.72 (dd, J = 7.4, 9.9
Hz, 1 H), 4.00 (d, J = 5.0 Hz, 1 H), 4.15 (m, 3 H), 4.25 (d, J = 5.8 Hz, 1 H),
5.61–5.70 (m, 4 H), 7.31 (app br t, J = 5.9 Hz, 1 H, NH).
13C NMR (76 MHz, CDCl3): δ = 14.25, 18.44, 21.08, 26.88, 34.24, 34.69,
38.81, 39.46 (d, J = 5.9 Hz), 60.73, 73.41, 74.53 (d, J = 5.9 Hz), 82.98
(app t, J = 4.2 Hz) 172.03, 172.16, 176.83, 176.92.
Ethyl (R)-3-(4-{Bis[(isobutyryloxy)methyl]phosphoryloxy}-2-hy-
droxy-3,3-dimethylbutanamido)propanoate (4i)
To a stirred solution of ethyl D-pantothenate (2; 247 mg, 1 mmol),
DIPEA (264 μL, 1.5 mmol), and DMAP (12 mg, 0.1 mmol) in CH2Cl2 (9
mL) at 0 °C, a solution of chlorophosphate 3i (474 mg, 1.5 mmol) in
CH2Cl2 (9 mL) was added dropwise under argon. The reaction mixture
was allowed to warm to r.t. and stirred for an additional 2 h. The reac-
tion mixture was quenched with H2O (10 mL) and extracted with
CH2Cl2 (2 × 20 mL). The organic phase was washed with sat. aq NH4Cl,
dried over Na2SO4, and concentrated under reduced pressure. Purifi-
cation by column chromatography (silica gel, CH2Cl2–MeOH, 97:3) af-
forded the product as a pale yellow oil.
31P NMR (120 MHz, CDCl3): δ = –2.22 (s).
HRMS (ESI): m/z [M + H]+ calcd for C23H43NO12P: 556.2523; found:
556.2536.
Tris[(isobutyryloxy)methyl] Phosphate (7i)
To a solution of trimethyl phosphate (7.0 g, 50 mmol) in anhydrous
MeCN (43 mL) were sequentially added chloromethyl isobutyrate
(25.96 mL, 195 mmol) and NaI (22.48 g, 150 mmol). The reaction
mixture was heated at reflux (85 °C) for 72 h, cooled to ambient tem-
perature, and diluted with Et2O (400 mL). The organic phase was
washed with H2O (2 × 100 mL) and saturated Na2S2O3 solution (2 ×
100 mL), dried over Na2SO4, and concentrated. Purification by column
chromatography (silica gel, hexane–EtOAc, 4:6) afforded a viscous
pale yellow oil.
Yield: 133 mg (25%); Rf = 0.22 (CH2Cl2–MeOH, 97:3).
1H NMR (300 MHz, CDCl3): δ = 0.85 (s, 3 H), 1.13 (s, 3 H), 1.21 (app dd,
J = 1.5, 7.0 Hz, 12 H), 1.26 (t, J = 7.1 Hz, 3 H), 2.55 (t, J = 6.2 Hz, 2 H),
2.63 (m, 2 H), 3.56 (m, 2 H), 3.71 (dd, J = 7.7, 10.0 Hz, 1 H), 4.00 (d, J =
6.2 Hz, 1 H), 4.15 (m, 4 H), 5.65 (m, 4 H), 7.28 (app br t, J = 6.0 Hz, 1 H,
NH).
13C NMR (76 MHz, CDCl3): δ = 14.28, 18.39, 18.67 (br s), 21.10, 33.83,
34.27, 34.73, 39.53 (d, J = 6.1 Hz), 60.77, 73.41, 74.58 (d, J = 5.9 Hz),
82.83 (app dd, J = 2.5, 5.0 Hz), 171.97, 172.19, 175.41, 175.50.
Yield: 12.938 g (65%); Rf = 0.33 (hexane–EtOAc, 4:6).
1H NMR (300 MHz, CDCl3): δ = 1.21 (d, J = 7.0 Hz, 18 H), 2.63 (hept, J =
7.0 Hz, 3 H), 5.6 (d, J = 13.7 Hz, 6 H).
31P NMR (120 MHz, CDCl3): δ = –2.43 (s).
13C NMR (76 MHz, CDCl3): δ = 18.59, 33.79, 82.70 (d, J = 5.1 Hz),
HRMS (ESI): m/z [M + H]+ calcd for C21H39NO12P: 528.2210; found:
528.2223.
175.21.
31P NMR (120 MHz, CDCl3): δ = –4.76 (s).
HRMS (ESI): m/z [M + H]+ calcd for C15H28O10P: 399.1420; found:
399.1410.
Tris(acetyloxymethyl) Phosphate (7j)
To a solution of trimethyl phosphate (1.4 g, 10 mmol) in anhydrous
MeCN (8.5 mL) were sequentially added chloromethyl acetate (4.0
mL, 39 mmol) and NaI (4.50 g, 30 mmol). The reaction mixture was
heated at reflux (85 °C) for 72 h, cooled to ambient temperature, and
diluted with Et2O (50 mL). The organic phase was washed with H2O (2
× 20 mL) and saturated Na2S2O3 solution (2 × 20 mL), dried over
Na2SO4, and concentrated. Purification by column chromatography
(silica gel, EtOAc–heptane, 7:3) afforded a viscous pale yellow oil.
Bis[(isobutyryloxy)methyl] Hydrogen Phosphate (8i)
Phosphate 7i (4.90 g, 12.3 mmol) was dissolved in piperidine (30 mL)
at 0 °C. The reaction mixture was allowed to warm to r.t. and stirred
for an additional 4 h. The solution was concentrated and further
evaporated in vacuo until a constant weight was reached (4.67 g,
99.0% yield). The crude oil (4.67 mg, 12.2 mmol) was dissolved in H2O
(100 mL) and treated with Dowex W50X2 H+ form resin (102 g, 61
mmol, 0.6 mmol/g). The suspension was stirred at ambient tempera-
ture for 1 h. The resin was filtered and washed with H2O. The filtrate
was concentrated and dried in vacuo. Purification by column chroma-
tography (silica gel, EtOAc–MeOH gradiently from 9:1 to 1:9) afforded
a pale yellow oil.
Yield: 0.566 g (18%); Rf = 0.32 (hexane–EtOAc, 4:6).
1H NMR (300 MHz, CDCl3): δ = 2.16 (s, 9 H), 5.65 (d, J = 13.6 Hz, 6 H).
13C NMR (76 MHz, CDCl3): δ = 20.70, 82.67 (d, J = 5.1 Hz), 169.22.
31P NMR (120 MHz, CDCl3): δ = –5.27 (s).
HRMS (ESI): m/z [M + H]+ calcd for C9H16O10P: 315.0481; found:
315.0476.
Yield: 2.10 g (57%); Rf = 0.19 (EtOAc–MeOH, 8:2).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J

J
J. Pahor et al.
Paper
Synthesis
Acknowledgment
(f) Gao, F.; Yan, X.; Shakya, T.; Baettig, O. M.; Ait-Mohand-
Brunet, S.; Berghuis, A. M.; Wright, G. D.; Auclair, K. J. Med.
Chem. 2006, 49, 5273.
The financial support of the Slovenian Research Agency (PR-05021
and PR-0134) is greatly appreciated. We are grateful to the European
Regional Development Fund and the Government of Slovenia – Minis-
try of Education, Science and Sport for the award of the ‘research
voucher’ grant (Contract No. 3330-13-500038). This work was also
supported by the European Commission’s Seventh Framework Pro-
gramme (grant number FP7/2007-2013, HEALTH-F2-2011, grant
agreement number 277984, TIRCON to Acies Bio d.o.o.). The authors
are grateful to the Slovenian NMR Centre at the National Institute of
Chemistry, and Mass Spectroscopy Centre at the Jožef Stefan Institute.
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Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1562437.
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, A–J