Lipophosphonoxins: New modular molecular structures with significant antibacterial properties

Article abstract of DOI:10.1021/jm2009343

Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (li

Full text of DOI:10.1021/jm2009343

Article
pubs.acs.org/jmc
Lipophosphonoxins: New Modular Molecular Structures with
Significant Antibacterial Properties
Dominik Rejman,*,† Alzbeta Rabatinova,‡ Antonio R. Pombinho,§ Sona Kovackova,† Radek Pohl,†
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†
∥,⊥
⊥
#
‡
∥
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Eva Zbornıkova, Milan Kolar, Katerina Bogdanova, Otakar Nyc, Hana Sanderova, Tomas Latal,
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Petr Bartunek,§ and Libor Krasny*,‡
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†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo nam
Prague 6, Czech Republic
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. 2, 166 10
‡
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Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Vıden
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ska
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1083, 142 20 Prague 4, Czech Republic
§Center for Chemical Genetics and CZ-OPENSCREEN, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic
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v.v.i., Vıden
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ska
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1083, 142 20 Prague 4, Czech Republic
∥TRIOS, Ltd., Zakouri
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lova 142, Prague 4, 149 00, Prague, Czech Republic
⊥Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University Olomouc, 779 00 Olomouc, Czech Republic
#Department of Medical Microbiology, Teaching Hospital Motol and Charles University in Prague, Second Faculty of Medicine, V
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Uvalu 84, 150 06, Prague 5, Czech Republic
S
* Supporting Information
ABSTRACT: Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The
general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a
hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i−iii.
Lipophosphonoxins displayed significant antibacterial properties against a panel of Gram-positive species, including multiresistant
strains. The minimum inhibitory concentration (MIC) values of the best inhibitors were in the 1−12 μg/mL range, while their
cytotoxic concentrations against human cell lines were significantly above this range. The modular nature of this artificial scaffold
offers a large number of possibilities for further modifications/exploitation of these compounds.
noxin did not specifically inhibit cyst formation, it potently
inhibited vegetative growth.4
Phosphonoxin bears structural similarities with several types
of nucleoside antimicrobials: (i) polyoxins (3), (ii) muraymy-
cins (4), and (iii) caprazamycins (5) (Figure 1).
The polyoxins of general formula 3 are a new group of
antifungal antibiotics isolated from Streptomyces cacaoi that
inhibit the growth of a number of mycelial fungi5,6 by
interfering with chitin synthesis.7,8 Polyoxin D is a strong
competitive inhibitor of chitin synthase in Neurospora crassa.
Polyoxin D shares a gross structural similarity with uridine
diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc 2), in agree-
ment with its role as a competitive inhibitor.
INTRODUCTION
■
The use of antibiotics has been generally beneficial for public
health. However, very few new antibiotics have been marketed
in the last 40 years.1 Moreover, the advantages offered by
antibiotics in the treatment of infectious diseases are
endangered due to the increase in the number of antibiotic-
resistant bacterial strains. This reduces the efficiency of
antibiotic treatments and poses a serious health and economic
problem. Currently, the need for novel antibiotics is becoming
increasingly apparent.2,3
We previously synthesized an effective inhibitor of Giardia
trophozoite growth termed phosphonoxin (1) (Figure 1) with
an activity that rivaled existing therapeutics. Phosphonoxin was
designed as a transition-state inhibitor of a glycosyl transferase,
cyst wall synthase (CWS). CWS catalyzes synthesis of the
chitin-like poly β-1−3-linked N-acetylgalactosamine [poly-
(GalNAc)] that comprises about 63% of the giardia cyst wall
utilizing UDP-GalNAc (2) as a substrate. Although phospho-
The caprazamycins (CPZs) (4) were isolated from a culture
broth of the actinomycete strain Streptomyces spp. MK730−
Received: July 14, 2011
Published: October 18, 2011
© 2011 American Chemical Society
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Figure 1. Structures of phosphonoxin, UDP-GlcNAc, and structurally related natural antimicrobials and their derivatives.
Table 1. Antibacterial Activity of Muraymycin Analogues
MIC (μg/mL)
S. aureus ATCC 29213
S. aureus SR3637
E. feacalis ATCC
E. feacalis SR7914
E. faecium ATCC
E. faecium SR7917
compd
(MSSA)
(MRSA)
29212
(VRE)
19434
(VRE)
6a
7a
6b
7b
>64
>64
2
>64
>64
4
>64
>64
4
>64
>64
4
>64
>64
4
>64
>64
2
2
4
2
4
0.5
0.5
0.25
>64
vancomycin
1
1
1
>64
62F2 in 20039,10 and represent the most recent members of the
class of naturally occurring 6′-N-alkyl-5′-β-O-aminoribosyl-C-
glycyluridine antibiotics that include the liposidomycins (5)
(LPMs). The CPZs have shown excellent antimycobacterial
activity in vitro not only against drug-susceptible (MIC = 3.13
μg/mL) but also multidrug-resistant Mycobacterium tuberculosis
strains (MIC = 3.13 μg/mL) and exhibit no significant toxicity
in mice. The biological target of the 6′-N-alkyl-5′-β-O-
aminoribosyl-C-glycyluridine class of antibiotics is believed to
be MraY translocase (IC50 = 0.05 μg/mL for LPMs).11
The muraymycins (6a), isolated from a culture broth of
Streptomyces species,12 are members of a class of naturally
occurring 6′-N-alkyl-5′-β-O-aminorybosyl-C-glycyluridine anti-
biotics. Members of this family showed antibacterial activity
against Staphylococcus aureus and Enterococci and were able to
protect mice against S. aureus infection (ED50 = 1.1 mg/kg).
The muraymycins inhibit the formation of lipid II and
peptidoglycan and are believed to be inhibitors of phospho-
MurNAc-pentapeptide translocase (MraY), which is respon-
sible for the formation of lipid I in the peptidoglycan
biosynthesis pathway.13−17 Tanino recently described the
synthesis of lipophilic muraymycin analogues 6b and 7b that
differ in the stereochemistry at the carbon atom, where the
lipophilic moiety is attached. These compounds exhibited
significant activity against methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant Enterococci (VRE)
(Table 1).18
At the beginning of this study, we tested phosphonoxin for
potential antibacterial properties. However, no significant
activity was observed. We reasoned that the negative charge
on the phosphonate moiety may hamper cellular uptake. Thus,
to facilitate the cell entry, we introduced the lipophilic
hexadecyloxypropyl ester group19 to the structure and
synthesized several variants based on this structure. The
obtained structures were collectively termed lipophosphonoxins
(LPPOs), and they showed promising activities against several
Gram-positive bacterial species and low to moderate cytotox-
icities. LPPOs represent an artificial modular structure that
could be easily synthesized in few simple reaction steps.
Here we present synthesis, antibacterial activity, and
cytotoxicity of several members of the lipophosphonoxin
family.
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RESULTS AND DISCUSSION
Table 2. Vinylphosphonates 10a−h
■
General Structure of Lipophosphonoxins. LPPOs are
modular molecules consisting of these parts: a nucleoside
module (NM), a linker module (LM), an iminosugar module
(IM), and a hydrophobic module (HM) (Figure 2).
compd
B
R
10a
10b
10c
10d
10e
10f
U
PivSCH2CH2
CH3CH2
C14H29
U
U
U
C16H33
U
C18H37
U
C20H41
10g
10h
U
CBz
C16H33OCH2CH2CH2
C16H33OCH2CH2CH2
Finally, Michael addition of pyrrolidine derivatives 11a−
d23,24 or piperidine derivatives 11e−f24,25 to vinylphosphonates
10a−d was carried out, followed by deprotection, resulting in
moderate to good yields of final lipophosphonoxins 12a−l
(Scheme 3, Tables 3 and 4). The deprotection consisted of the
Figure 2. General structure of lipophosphonoxins.
a
Scheme 3
The parent phosphonoxin (1) contained uridine as the NM.
Cytidine was subsequently evaluated as an NM alternative.
LPPOs containing hexadecyloxypropyl, ethyl, pivaloylthioethyl
(SATE),20 tetradecyl, hexadecyl, octadecyl, or eicosanyl ester
groups as the HM were synthesized. Several hydroxylated
pyrrolidines and piperidines were used as the IM. The LM was
in all cases ethylphosphonic acid.
Chemistry. Vinylphosphonate synthons 9a−b were pre-
pared from nucleobase protected 2′,3′-isopropylidene nucleo-
sides 8a−b (Scheme 1). The phosphonate function was
a
(a) R1R2NH (11a−f), nBuOH, 90 °C; (b) MeNH2/MeOH (B =
CBz); (c) 0.2M HCl/MeOH.
removal of the nucleobase protecting group (in the case of
cytosine derivative) with ethanolic methylamine and the
subsequent cleavage of isopropylidene group with 0.2 M
methanolic HCl. The final products were obtained by flash
column chromatography on silica gel or using preparative
reversed phase HPLC.
a
Scheme 1
Antimicrobial Activity. The prepared compounds were
tested against a panel of selected reference bacterial strains
(Table 5). The minimal inhibitory concentration (MIC) values
obtained by the standard microdilution method26,27 were used
to evaluate the antibacterial properties of the compounds.
The compound 12a, consisting of the uridine NM,
hexadecyloxypropyl HM, and (3R,4R)-3,4-dihydroxypyrrolidine
IM, exhibited significant antibacterial properties against several
Gram-positive bacteria, such as Enterococcus faecalis (12.50 μg/
mL), Bacillus subtilis (6.25 μg/mL), and Streptococcus agalactiae
(3.13 μg/mL), respectively (Table 5). The compound 12b,
wherein the uridine NM was replaced with cytidine, exhibited a
slightly improved antibacterial activity: E. faecalis (6.25 μg/mL),
B. subtilis (3.13 μg/mL), S. agalactiae (3.13 μg/mL), and S.
aureus (25.00 μg/mL).
The replacement of the IM ((3R,4R)-3,4-dihydroxypyrroli-
dine) of 12a with its enantiomeric (3S,4S)-3,4-dihydroxypyrro-
lidine (in the case of 12c) did not change the activity. However,
when (3R,4S,5S)-3,4-trihydroxypiperidine (in the case of 12d)
or (3R,5R)-3,4-dihydroxypiperidine (in the case of 12e) were
used as the IM, the antibacterial activity was almost abolished
except for S. agalactiae (12.50 μg/mL).
a
(a) CH2CHP(O)(OMe)OH, MeIm, TPSCI, DCM/MeCN; (b)
60% aq pyridine.
introduced by esterification of monomethyl vinylphospho-
nate21,22 using triisopropylbenzensulfonylchloride (TPSCl) as a
condensing agent (Scheme 1). Obtained methyl phosphonates
were partially de-esterified by heating with 60% aqueous
pyridine to remove the methyl ester group, affording
nucleosidyl vinylphosphonates 9a−b.
Next, esterification was performed with the appropriate
alcohol using TPSCl as a condensing agent, yielding synthons
10a−h (Scheme 2; Table 2).
a
Scheme 2
The hexadecyloxypropyl HM of 12a was replaced with either
ethyl (in the case of 12g), tetradecyl (in the case of 12h),
hexadecyl (in the case of 12i), octadecyl (in the case of 12j), or
icosanyl (in the case of 12k) ester group, respectively. The
LPPO 12g with ethyl ester HM did not exhibit any antibacterial
activity. Compound 12k exhibited the lowest activity; 12h and
12j exhibited activities comparable to their parent structure,
12a. The LPPO 12i with hexadecyl ester HM appeared to be
a
(a) ROH, MeIM, TPSCI, DCM/MeCN.
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Table 3. Amines (Pyrrolidines and Piperidines) 11a−f
the most active compound of the tested series: E. faecalis (6.25
μg/mL), B. subtilis (3.13 μg/mL), S. agalactiae (1.56 μg/mL)
and S. aureus (12.50−6.25 μg/mL). In the case of the LPPO
12f with pivaloylthioethyl ester HM, no activity was observed.
None of the tested compounds exhibited MIC below 200
μg/mL against Gram-negative Escherichia coli or Pseudomonas
aeruginosa. Possible explanations included (i) problems of these
compounds with cell entry, (ii) their rapid degradation while in
the cell, (iii) their rapid export from the cell, or (iv) the lack of
molecular target(s) in Gram-negative bacteria. To better
characterize these compounds, it would be useful to distinguish
between (i) versus (ii−iv). To test the inhibitory potential of
these compounds on a Gram-negative bacterium, we had to use
permeabilized E. coli cells. Such cells have an enhanced ability
to uptake molecules from their environment. Permeabilized E.
coli cells were incubated with/without the tested compounds
(see Experimental Section for details). If (i) were correct, at
least some compound(s) would adversely affect the cell
survival. If (ii), (iii), or (iv) were correct, no difference relative
to the control would be observed.
Compounds 1, 12a, 12i, and 12k were tested on
permeabilized E. coli cells. Compound 1 had no antibacterial
effect on these cells. On the other hand, compounds 12a and
12i killed 90% of cells at 10 μM and 12k at 25 μM (Table 6).
These results suggest that the inefficiency of lipohposphonoxins
against Gram-negative bacteria is likely due to their inability to
cross the two plasmatic membranes of these species, and future
modification may be attempted to allow their entry into the
Gram-negative cell.
More importantly, these results imply that the HM may be
required for the interaction of the compounds with their
molecular target(s) because compound 1, which lacks the HM,
exhibited no activity. In other words, the HM may not be only
an auxiliary module attached to the active molecule to create its
prodrug form that is cleaved off upon the cell entry but an
integral part of the active compound.
The most active compounds were further tested against
clinically relevant multiresistant bacterial strains (Table 7).
Compounds 12a, 12b, 12c, 12i, and 12l showed significant
antibacterial activities (6.25−3.13 μg/mL) against Enterococcus
feacium VanA419/ana and Staphylococcus epidermidis 8700/B.
Compound 12h exhibited good activity against methicillin-
resistant S. aureus (MRSA) 4591 (25.00 μg/mL), Staphylococcus
haemolyticus 16568 (12.50 μg/mL), E. faecium VanA419/ana
(6.25 μg/mL), and S. epidermidis 8700/B (6.25 μg/mL).
Compound 12i exhibited slightly stronger activity than 12h
against MRSA 4591 (12.50 μg/mL), S. haemolyticus 16568
(25.00−12.50 μg/mL), E. faecium VanA419/ana (3.13 μg/mL),
and S. epidermidis 8700/B (3.13 μg/mL).
(caspase 3/7 activation), using erythroid progenitor cells
derived from human umbilical cord blood. Cell viability was
measured 48 h after exposure, while cytotoxicity and apoptosis
were measured 4 h after exposure to the compounds.
Compounds 12a, 12b and 12i, shown to have the best
antibacterial activity, had no detectable activity on normal
primary cell viability and toxicity at the MIC concentrations
(Figure 3). According to the cell viability results, these
compounds showed to be toxic to these cells at concentrations
that were well above their MIC values for most of the bacterial
strains tested (Table 8). Finally, these compounds induced only
a low level of apoptosis. Other tested compounds showed
either higher toxicity to the primary cells or lower activity/
selectivity against the selected bacterial strains (Supporting
Information Figure 1), but this still does not exclude their
potential antibacterial use, such as in topical applications.
Effect of the Diastereomeric Configuration at the
Phosphorus Atom on Biological Activity. The compound
12i, exhibiting the highest antibacterial activity, was separated
into both diastereomers differing in the configuration at the
phosphorus atom, yielding two pure diastereomers 12i-P1 and
12i-P2. Figure 4 shows analytical HPLC records of the mixture
and the two separated diastereomers. Diastereomer 12i-P1 with
tR = 8.20 min showed 31P NMR shift at 31.27, while
diastereomer 12i-P2 with tR = 8.69 min showed 31P NMR
shift at 32.03.
Subsequently, the antibacterial activities of both diaster-
eomers were evaluated and both diastereomers displayed
almost the same MIC values, identical with MIC values of
the original mixed compound 12i. This suggests that the
configuration at the phosphorus atom does not play an
important role in the antibacterial activity. This is reminiscent
of muraymycin analogues 6b and 7b (see Introduction) where
the chirality at the atom of the lipophilic chain attachment did
not significantly affect their antibacterial activity.18 Never-
theless, in our case, the 12i-P1 diastereomer was slightly less
toxic to erythroid progenitor cells (Figure 5).
Effect of LPPOs on Bacterial RNAP. An initial concern
had been that LPPOs, due to their amphiphilic character, might
function as nonspecific, detergent-like compounds. This
concern was dispelled by the cytotoxicity tests: the used cell
lines are highly sensitive to their environment and compounds
acting in a detergent-like manner would severely compromise
their viability, which was not the case. To further test whether
LPPOs function in a nonspecific way, the effect of several
LPPOs on the enzymatic activity of a key bacterial protein,
RNA polymerase (RNAP), was evaluated (see Experimental
Section for details). Multiple-round transcriptions with purified
components were carried out in the absence or presence of
high, 1 mM (∼700 μg/mL) concentration of selected LPPOs
(12a, 12c, 12d, and 12e). None of these compounds
significantly inhibited the enzymatic activity of RNAP, while
two control detergents, cetyltrimethylammonium bromide and
Viability, Cytotoxicity, and Apoptosis of Human
Progenitor Cells. The effects of the newly synthesized
lipophosphonoxins were tested on cell viability (metabolic
activity), cytotoxicity (membrane integrity), and apoptosis
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Table 4. Structures of Lipophosphonoxines 12a−l
cetylpyridinium bromide, completely abolished transcription at
0.2 mM (∼80 μg/mL) concentration.
played good activities against a panel of Gram-positive bacteria
strains at subcytotoxic concentrations. Importantly, the most
active compounds also exhibited antibacterial activities against
several multiresistant strains.
The molecular target(s) of LPPOs are unknown. On the
basis of a gross structural resemblance (nucleobase, long alkyl
chain), they may act in a similar way as, e.g., lipisidomycins or
caprazamycins (Figure 1). In comparison with these com-
CONCLUSIONS
■
We describe here a novel molecular scaffold leading to
structures exhibiting significant antibacterial activities. The
compounds based on this scaffold were collectively termed
lipophosphonoxins (LPPOs). The most active LPPOs dis-
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Table 5. Antibacterial Activity (against Reference Bacterial
Strains)
compd
MIC μg/mL
E. faecalis
CCM 4224
S.aureus CCM
4223
B. subtilis
S.agalactiae
a
a
12a
12.50
6.25
6.25
3.13
6.25
3.13
3.13
12b
25.00
a
12c
12d
12.50
6.25
a
12.50
6.25
a
12e
200
100
a
12f
a
a
12g
12h
12.50
6.25
6.25
100
25.00−12.50
12.50−6.25
25.00−12.50
6.25
3.13
6.25
1.56
3.13
6.25
3.13
2
a
12i
12j
a
12.50−6.25
a
12k
a
12l
6.25
100
3.13
16
2
b
CMP
c
TET
1
a
b
c
In all cases MIC = MBC. CMP = chloramphenicol. TET =
tetracycline.
Table 6. Effect of Selected Compounds on Permeabilized E.
coli
a
compd [10 μM]
killing power (log reduction )
b
1
(4.4 μg/mL)
0
1
1
Figure 3. Cell viability, cytotoxicity, and apoptosis of erythroid
progenitor cells after exposure to selected compounds (12a, 12b, and
12i). For comparison, the antibacterial activities (MIC values) of these
compounds against selected bacterial species are indicated with vertical
lines in the graphs (bacterial strains with the same MIC value are
inside the boxes).
12a
12i
12k
(7.2 μg/mL)
(6.6 μg/mL)
(7.2 μg/mL)
0.7
a
b
One log is 90% reduction in colony forming units. This compound
had no effect also at 1 mM concentration.
Table 8. Erythroid Progenitor Cells Viability
Table 7. Antibacterial Activity (against Multiresistant
Bacterial Strains)
a
compd
IC50 (μg/mL)
maximum safe concentration
MIC μg/mL
12a
12b
12c
12d
12e
12f
12g
12h
12i
91.00
67.00
47.00
149.00
53.00
>1000
nd
62.50
31.25
31.25
62.50
31.25
>1000
nd
S.
S. aureus
haemolyticus
16568
E. faecium
VanA419/ana
S. epidermidis
compd
MRSA 4591
8700/B
12a
12b
12c
12h
12i
12j
12l
6.25
6.25
6.25
12.50
3.13
6.25
6.25
3.13
6.25
6.25
12.50
3.13
6.25
6.25
25.00
12.50
200
12.5
58.00
56.00
36.00
31.00
116.00
31.25
31.25
15.60
15.60
62.50
25−12.5
12j
12k
12l
a
In all cases MIC = MBC; multiresistant bacterial strains isolated from
clinical isolates of patients from Teaching Hospital Olomouc: MRSA
(methicilin-resistant S. aureus 4591); S. haemolyticus (fluoroquinolone-
resistant strain 16568); E. faecium (vancomycin-resistant strain
VanA419/ana); S. epidermidis (methicilin-resistant strain 8700/B).
negative bacteria. Results from these experiments as well as the
research into the molecular mechanism of LPPOs will be
reported in due course.
pounds, however, LPPOs are significantly easier to synthesize.
In summary, LPPOs appear to be specific antibacterial
compounds and the molecular basis of their antibacterial
activity has yet to be elucidated experimentally. Experiments to
this effect are under way in our laboratory.
The modular composition of the scaffold and its simple
synthesis will allow further optimization of its structure to
enhance its antibacterial efficiency while decreasing its
cytotoxicity. We will also focus on modifications of lip-
ophosphonoxins to enhance their efficiency against Gram-
EXPERIMENTAL SECTION
■
Antibacterial Activity. Antimicrobial activity was assessed using
the standard microdilution method determining the minimum
inhibitory concentration (MIC) of tested samples leading to inhibition
of bacterial growth.26,27 Disposable microtitration plates were used for
the tests. The samples were diluted in brain heart infusion broth
(Himedia) to yield a concentration range between 200 and 1.56 μg/
mL (in some cases, the lower concentration range was extended to
0.78 μg/mL). The plates were inoculated with a standard amount of
the tested microbe; the inoculum density in each well was equal to
105−6 CFU/ml. The MIC was read after 24/48 h of incubation at 37
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Figure 4. The analytical HPLC chromatograms of 12i, 12i-P1, and 12i-P2. HPLC analysis was performed on Waters AutoPurification system with
2545 quarternary gradient module and 3100 single quadrupole mass detector using LUNA C18, column (Phenomenex, 100 mm × 4.6 mm, 3 μm) at
flow rate 1 mL/min using gradient, A→B/15 min, (A, 50 mM NH4HCO3 in 50% aq CH3CN; C, CH3CN). AU, absorption units.
incubated for 1 h at 37 °C by vigorous shaking. Then the mixture was
plated on LB agarose plates containing ampicillin at 100 μg/mL and
incubated at 37 °C overnight. The number of colonies obtained from
transformation with a tested compound was compared to the number
of colonies obtained after transformation without inhibitor (positive
control).
Cell Viability, Cytotoxicity, And Apoptosis. Culture and
expansion of normal human erythroid progenitor cells was described
earlier.29 Cells were plated at 25000 cells/well/20 μL in 384-well
plates immediately before compound addition. Cell viability and
cytotoxicity have a fluorescent readout, and cells were plated into black
384-well plates (Corning, Cat. no. 3571), while apoptosis has a
luminescent readout and cells were plated into white 384-well plates
(Corning, Cat. no. 3570). Compounds were prediluted in the
appropriate culture media and 5 μL of five times concentrated
solution of compounds were added to the cell supernatant.
Cytotoxicity and apoptosis were measured 4 h after compound
addition using the CytoTox-ONE homogeneous membrane integrity
assay (Promega, Cat. no. G7892) and the Caspase-Glo 3/7 assay
(Promega, Cat. no. G8091). Cell viability was measured using the
CellTiter-Blue cell viability assay (Promega, Cat. no. G8082) 48 h after
compound addition. All assays were measured using the EnVision
plate reader (PerkinElmer). Data were analyzed using GraphPad Prism
Figure 5. Cell viability, cytotoxicity, and apoptosis of erythroid
progenitor cells after exposure to diastereomers 12i-P1 and 12i-P2.
Vertical lines in the graphs represent the MIC values for the respective
bacterial strains.
5.0 statistical software, and the data were normalized to the values of
untreated control cells that were set as 100%.
Tests on Purified RNA Polymerase. Bacillus subtilis RNAP and
σ A were purified, and the holoenzyme was reconstituted as
described.30 As the template for transcriptions, we used supercoiled
°C as the minimum inhibitory concentration of the tested substance
plasmid DNA bearing a fragment containing the B. subtilis rrnB P1
promoter (pLK7).31 Multiple-round transcriptions were carried out in
that inhibited the growth of the bacterial strains.
The minimum bactericidal concentration (MBC) is characterized as
the minimum concentration of the sample required to achieve
10 μL reactions containing 6nM RNAP, 4 ng/μL supercoiled plasmid
template, 40 mM Tris−HCl pH 8.0, 10 mM MgCl2, 1 mM DTT, 0.1
irreversible inhibition, i.e. killing the bacterium after a defined period
of incubation. The MBC was examined by the inoculation method.
With an applicator, 10 μL were transferred from microplate wells with
defined concentrations of the tested sample and inoculated onto the
surface of blood agar (Trios, Czech Republic). The MBC was
determined as the lowest concentration that inhibited the visible
growth of the used bacterium.
Standard reference bacterial strains (E. faecalis CCM 4224, S. aureus
CCM 4223) from the Czech Collection of Microorganisms (CCM),
Faculty of Science, Masaryk University Brno, and S. agalactiae, B.
subtilis, methicilin-resistant S. aureus 4591, fluoroquinolone-resistant S.
haemolyticus 16568, vancomycin-resistant E. faecium VanA419/ana,
and methicilin-resistant S. epidermidis 8700/B strains obtained from
the Teaching Hospital Olomouc were used. All tested microorganisms
were stored in cryotubes (ITEST plus, Czech Republic) at −80 °C.
Antibacterial Activity against Competent E. coli. We mixed
together 20 μL of E. coli (strain DH5α) competent cells (i.e.,
permeabilized, with an increased ability to uptake molecules from the
outside),28 pUC18 plasmid DNA (5 ng) (bearing ampR as the selective
marker after transformation) and a serially diluted tested compound/
equal volume of “empty“ buffer. The mixture was incubated for 30 min
on ice followed by a heat shock performed at 42 °C for 90 s. The
mixture was then allowed to sit on ice for 5 min. Subsequently, 1 mL
of LB medium without antibiotics was added and the mixture was
mg/mL BSA, and 150 mM KCl. ATP, CTP, and GTP were 200 μM
each. UTP was 10 μM plus 2 μM [α-32P]-UTP purchased from
Institute of Isotopes Co., Ltd. The transcriptions were carried out in
the absence or presence (1 mM) of the tested compound. As a control,
cetyltrimethylammonium bromide and cetylpyridinium bromide were
used (0.2 mM).
Each sample was preincubated at 30 °C for 5 min followed by
initiation with RNAP. The reaction was stopped after 15 min at 30 °C
by 10 μL of formamide loading buffer (95% formamide, 20 mM
EDTA pH 8.0) and briefly vortexed. Samples were loaded onto 7 M
UREA 7% polyacrylamide gels and separated by electrophoresis. The
dried gels were scanned with Molecular Imager_ FX (BIO-RAD). The
amounts of the 145-nt-long transcript that originated from the rrnB P1
promoter were quantitated with Quantity One (BioRad).
Chemistry. Unless stated otherwise, all used solvents were
anhydrous. Dimethyl vinylphosphonate, tetradecanol, hexadecanol,
octadecanol, and eicosanol were purchased from Sigma Aldrich
(Czech Republic). Protected nucleosides were prepared according to
standard procedures. Hexadecyloxypropanol was prepared according
to Hostetler et al.,19 pivaloylthioethanol was prepared according to
Lefebvre et al.,23 dihydroxypyrrolidines 11a−c were prepared
according to Rejman et al.,23 piperidine derivatives 11d and 11e
were prepared according to Rejman et al.25 and Kovackova et al.24
̌
́
respectively. All reactions were performed under an inert atmosphere
7890
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Journal of Medicinal Chemistry
Article
of dry Ar or N2. TLC was performed on silica gel precoated aluminum
plates Silica gel/TLC-cards, UV 254 (Fluka), and compounds were
detected by UV light (254 nm), by spraying with 1% ethanolic
solution of ninhydrine to visualize amines, and by spraying with 1%
solution of 4-(4-nitrobenzyl)pyridine in ethanol followed by heating
and treating with gaseous ammonia (blue color, detection of alkylating
agents, e.g. mesylderivatives, phosphonate esters). The purity of the
final compounds was greater than 95%. Purity of prepared compounds
was determined by LC-MS performed on Waters AutoPurification
System with 2545 Quarternary Gradient Module and 3100 Single
Quadrupole Mass Detector using LUNA C18, column (Phenomenex,
100 mm × 4.6 mm, 3 μm) at flow rate 1 mL/min. Typical conditions:
mobile phase, A, 50 mM NH4HCO3; B, 50 mM NH4HCO3 in 50% aq
CH3CN; C, CH3CN; A→B/10 min, B→C/10 min, C/5 min.
Preparative RP HPLC was performed on LC5000 liquid chromato-
graph (INGOS-PIKRON, CR) using Luna C18 (2) column (250 mm
× 21.2 mm, 5 μm) at flow rate of 10 mL/min by a gradient elution of
methanol in 0.1 M TEAB pH 7.5 (A = 0.1 M TEAB; B = 0.1 M TEAB
in 50% aq methanol; C = methanol) or without buffer. All final
compounds were lyophilized from water. IR spectra were recorded on
a FTIR spectrometer (Bruker Equinox 55, Germany). Mass spectra
were recorded on LTQ Orbitrap XL (Thermo Fisher Scientific) using
ESI ionization. NMR spectra were measured as DMSO-d6 or D2O
solutions on Bruker AVANCE 400 (1H at 400.0 MHz, 13C at 100.6
MHz, 31P at 162.0 MHz) and/or Bruker AVANCE 500 (1H at 500.0
MHz, 13C at 125.7 MHz, 31P at 202.3 MHz) spectrometers. Chemical
shifts (in ppm, δ scale) were referenced to the residual DMSO-d6
signal (2.5 ppm for 1H and 39.7 ppm for 13C) or to 1,4-dioxane signal
product of esterification was obtained by flash chromatography on
silica gel using linear gradient of ethanol in chloroform.
General Method C, Michael Addition and Deprotection:
Preparation of Compounds 12a−l. The mixture of alkyl
nucleosidyl mixed ester of vinylphosphonic acid (1 mmol) and
amine (1.5 mmol) in nBuOH (10 mL/mmol) was stirred at 100 °C
overnight. The mixture was concentrated in vacuo, and the protected
intermediate was obtained by flash chromatography on silica gel using
linear gradient of solvent system H1 (ethyl acetate:acetone:ethanol:-
water 4:1:1:1) in ethyl acetate. In the case of N-4-Bz-Cyt, the
intermediate was dissolved in 33% ethanolic methylamine, stirred at rt
overnight, and concentrated in vacuo (this step is skipped in the case
of uridine derivative). The intermediate was dissolved in 0.2 M
methanolic HCl (10 mL/mmol) and stirred overnight. The reaction
mixture was applied on a silica gel column. The final product was
obtained by flash chromatography using linear gradient of solvent
system H1 (ethyl acetate:acetone:ethanol:water 4:1:1:1) in ethyl
acetate and lyophilized from water.
2, 3′-Isopropylideneuridin-5′-yl Vinylphosphonate (9a). The
title compound was prepared according to general method A from
2′,3′-isopropylideneuridine (14 g, 50 mmol) in 48% yield (11.38 g,
23.93 mmol) as a yellowish thick oil. NMR of UrdPhN has shown free
acid (not Et3NH+ salt). 1H NMR (499.8 MHz, CD3OD): 1.35, 1.54 (2
× q, 2 × 3H, 4J = 0.5, (CH3)2C); 3.98 (dd, 2H, JH,P = 5.3, J5′,4′ = 3.4, H-
5′); 4.35 (m, 1H, H-4′); 4.90 (m, 2H, H-2′,3′); 5.74 (d, 1H, J5,6 = 8.1,
H-5); 5.86 (ddd, 1H, JH,P = 46.0, Jcis = 12.3, Jgem = 3.1, CHcisHtrans

CHP); 5.95 (d, 1H, J1′,2′ = 2.5, H-1′); 6.00 (ddd, 1H, JH,P = 22.8, Jtrans
18.7, Jgem = 3.1, CHcisHtransCHP); 6.12 (ddd, 1H, JH,P = 19.7, Jtrans
=
=
18.7, Jcis = 12.3, CHP); 7.86 (d, 1H, J6,5 = 8.1, H-6). 13C NMR
(125.7 MHz, CD3OD): 25.53, 27.55 ((CH3)2C); 65.31 (d, JC,P = 4.8,
CH2-5′); 82.49 (CH-3′); 85.67 (CH-2′); 86.56 (d, JC,P = 8.0, CH-4′);
93.40 (CH-1′); 102.98 (CH-5); 114.99 (C(CH3)2); 130.74 (CH2
CHP); 132.70 (d, JC,P = 174.3, CHP); 143.42 (CH-6); 152.16 (C-
2); 166.15 (C-4). 31P NMR (202.3 MHz, CD3OD): 13.16. HR-ESI:
C14H18O8N2P (M − H)− calcd 373.0806; found 373.0809
1
(3.75 ppm for H and 69.3 ppm for 13C) as internal standard in D2O.
31P NMR spectra were referenced to H3PO4 (0 ppm) as an external
standard. Coupling constants (J) are given in Hz. Complete
assignment of protons and carbons was done by analysis of correlated
homonuclear H,H-COSY and heteronuclear H,C-HSQC and H,C-
HMBC spectra. Relative configuration was checked using DPFGSE-
NOE and 2D-ROESY techniques.
4 N-Benzoyl-2′,3′-isopropylidenecytidin-5′-yl Vinylphosph-
onate (9b). The title compound was prepared according to general
method A from 4-N-benzoyl-2′,3′-isopropylidenecytidine (3.05 g, 7.87
mmol) in 52% yield (1.95 g, 4.1 mmol) as a yellowish thick oil. NMR
General Method A, Phosphonylation: Preparation of
Compounds 9a−b. Dimethyl vinylphosphonate (0.53 mL, 1.5
mmol) was treated with 60% aqueous pyridine (10 mL/mmol) at
60 °C overnight to remove one methyl ester group. The reaction
mixture was concentrated in vacuo and coevaporated with EtOH (2 ×
50 mL/mmol). The residue was dissolved in the mixture of H2O/
EtOH 1:1 (10 mL/mmol) and passed through a column of Dowex 50
in H+ form (10 mL/mmol). The resin was washed with additional
mixture of H2O/EtOH 1:1 (2 × 10 mL/mmol). The liquid was
concentrated in vacuo. The obtained monomethyl vinylphosphonate
was coevaporated with EtOH (2 × 10 mL/mmol) and toluene (2 × 10
mL/mmol). To the mixture of the monomethyl phosphonate,
protected nucleoside (1 mmol) and methylimidazole (3 mmol) in
DCM (10 mL/mmol), TPSCl (3 mmol) was added. The reaction
mixture was stirred at rt overnight. The mixture was washed with a
saturated aq NaHCO3 (10−20 mL/mmol) followed by washing with
3% aq citric acid (10−20 mL/mmol) and dried over Na2SO4. Organic
phase was concentrated in vacuo and monomethyl ester of
nucleosidevinylphosphonic acid was obtained by flash chromatography
on silica gel using linear gradient of ethanol in chloroform. The
monomethyl ester intermediate in 60% aqueous pyridine (10 mL/
mmol) was stirred at 60 °C overnight. The reaction mixture was
concentrated in vacuo, coevaporated in EtOH (2 × 10 mL/mmol),
and dissolved in the same solvent (10 mL/mmol). Dowex 50 in Et3N
form (10 mL/mmol) was added, and the suspension was stirred for 10
min. The resin was removed by filtration, and the filtrate was
concentrated in vacuo.
1
9b has shown Et3NH+ salt (not free acid). H NMR (600.1 MHz,
CD3OD): 1.31 (t, 9H, Jvic = 7.3, CH3CH2N); 1.37, 1.57 (2 × q, 2 ×
3H, 4J = 0.7, (CH3)2C); 3.20 (q, 6H, Jvic = 7.3, CH3CH2N); 4.02 (ddd,
1H, Jgem = 11.5, JH,P = 6.0, J5′b,4′ = 3.4, H-5′b); 4.06 (ddd, 1H, Jgem
=
11.5, JH,P = 4.5, J5′a,4′ = 3.0, H-5′a); 4.50 (m, 1H, H-4′); 4.91 (dd, 1H,
J3′,2′ = 6.0, J3′,4′ = 2.2, H-3′); 4.93 (dd, 1H, J2′,3′ = 6.0, J2′,1′ = 2.3, H-2′);
5.86 (ddd, 1H, JH,P = 46.0, Jcis = 12.4, Jgem = 3.1, CHcisHtransCHP);
6.00 (d, 1H, J1′,2′ = 2.3, H-1′); 6.01 (ddd, 1H, JH,P = 22.9, Jtrans = 18.7,
J
gem = 3.1, CHcisHtransCHP); 6.11 (ddd, 1H, JH,P = 19.6, Jtrans = 18.7,
Jcis = 12.4, CHP); 7.55 (m, 2H, H-m-Ph); 7.62 (bd, 1H, J5,6 = 7.5,
H-5) 7.64 (m, 1H, H-p-Ph); 7.98 (m, 2H, H-o-Ph); 8.35 (d, 1H, J6,5
=
7.5, H-6). 13C NMR (150.9 MHz, CD3OD): 9.18 (CH3CH2N); 25.45,
27.47 ((CH3)2C); 47.73 (CH3CH2N); 65.09 (d, JC,P = 4.8, CH2-5′);
82.47 (CH-3′); 87.16 (CH-2′); 87.91 (d, JC,P = 8.1, CH-4′); 95.59
(CH-1′); 98.54 (CH-5); 114.69 (C(CH3)2); 129.16 (CH-o-Ph);
129.82 (CH-m-Ph); 130.80 (CH2CHP); 132.71 (d, JC,P = 174.8, 
CHP); 134.05 (CH-p-Ph); 134.78 (C-i-Ph); 147.24 (CH-6); 157.90
(C-2); 164.99 (C-4); 169.02 (CO). 31P NMR (202.3 MHz, CD3OD):
13.02. HR-ESI: C21H25O8N3P (M + H)+ calcd 478.13738; found
478.13719.
2 Pivaloylthioethyl 2′,3′-Isopropylideneuridin-5′-yl Vinyl-
phosphonate (10a). The title compound was prepared according to
general method B from 9a (0.36 g, 1.2 mmol) and pivaloylthioethanol
(0.39 g, 2.4 mmol) in 92% yield (0.57 g, 1.1 mmol) as a colorless oil. A
General Method B, Esterification: Preparation of Com-
pounds 10a−h. TPSCl (3 mmol) was added to the mixture of
triethylamonium salt of nucleosidyl ester of vinylphosphonic acid (1
mmol), alcohol (2 mmol), and methylimidazole (3 mmol) in DCM
(10 mL/mmol). The reaction mixture was stirred at rt overnight. The
mixture was washed with saturated aq NaHCO3 (10−20 mL/mmol),
followed by washing with 3% aq citric acid (10−20 mL/mmol) and
dried over Na2SO4. Organic phase was concentrated in vacuo and the
1
mixture of diastereomers ∼1:1. H NMR (600.1 MHz, CDCl3): 1.15
(m, 18H, (CH3)3C); 1.27, 1.28, 1.49 (3 × s, 12H, (CH3)2C); 3.05,
3.06 (2 × t, 2 × 2H, Jvic = 6.8, SCH2CH2O); 3.97−4.09 (m, 4H,
SCH2CH2O); 4.17−4.25 (m, 4H, H-5′); 4.29 (m, 2H, H-4′); 4.80,
4.81 (2 × dd, 2 × 1H, J3′,2′ = 6.5, J3′,4′ = 3.7, H-3′); 4.88 (dd, 2H, J2′,3′
=
6.5, J2′,1′ = 2.3, H-2′); 5.655, 5.657 (2 × d, 2 × 1H, J5,6 = 8.1, H-5);
5.70, 5.73 (2 × d, 2 × 1H, J1′,2′ = 2.3, H-1′); 5.99, 6.00 (2 × ddd, 2 ×
7891
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Journal of Medicinal Chemistry
Article
1H, JH,P = 23.1, Jtrans = 18.5, Jcis = 12.7, CHP); 6.12, 6.14 (2 × ddd, 2
× 1H, JH,P = 52.4, Jcis = 12.7, Jgem = 1.8, CHcisHtransCHP); 6.28, 6.30
method B from 9a (1.7 g, 6 mmol) and hexadecanol (2.9 g, 12 mmol)
in 38% yield (1.38 g, 2.3 mmol) as a colorless thick oil. A mixture of
1
(2 × ddd, 2 × 1H, JH,P = 25.2, Jtrans = 18.5, Jgem = 1.8, CHcisHtrans

diastereomers ∼6:5. H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H,
CHP); 7.34, 7.38 (2 × d, 2 × 1H, J6,5 = 8.1, H-6); 10.34 (bs, 2H, NH).
13C NMR (150.9 MHz, CDCl3): 24.97, 24.99, 26.81, 26.82
((CH3)2C); 27.00 ((CH3)3C); 28.43 (d, JC,P = 6.6, SCH2CH2O);
46.19 (C(CH3)2); 64.19, 64.21 (d, JC,P = 5.0, OCH2CH2S); 64.98,
65.06 (d, JC,P = 5.4, CH2-5′); 80.39, 80.49 (CH-3′); 84.14, 84.22 (CH-
2′); 85.16, 85.43 (d, JC,P = 6.9, CH-4′); 93.41, 93.82 (CH-1′); 102.25,
CH3(CH2)15); 1.22−1.35 (m, 52H, CH3(CH2)13CH2CH2O); 1.350,
1.354, 1.573, 1.577 (4 × q, 4 × 3H, 4J = 0.7, (CH3)2C); 1.67 (m, 4H,
CH3(CH2)13CH2CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, JH,P = 7.3, Jvic
=
6.7, CH3(CH2)13CH2CH2O); 4.19−4.30 (m, 4H, H-5′); 4.35−4.39
(m, 2H, H-4′); 4.85, 4.86 (2 × dd, 2 × 1H, J3′,2′ = 6.4, J3′,4′ = 3.6, H-3′);
4.878, 4.8984 (2 × dd, 2 × 1H, J2′,3′ = 6.4, J2′,1′ = 2.3, H-2′); 5.71, 5.72
(2 × dd, 2 × 1H, J5,6 = 8.1, J5,NH = 2.2, H-5); 5.78, 5.81 (2 × d, 2 × 1H,
J1′,2′ = 2.3, H-1′); 6.03, 6.05 (2 × ddd, 2 × 1H, JH,P = 22.8, Jtrans = 18.6,
102.34 (CH-5); 114.16, 114.22 (C(CH3)2); 124.31, 124.36 (d, JC,P
=
184.6, CHP); 136.85 (CH2CHP); 141.69, 141.81 (CH-6);
150.03, 150.05 (C-2); 163.55, 163.57 (C-4); 205.41, 205.43 (CO).
31P NMR (162.0 MHz, CDCl3): 18.75, 18.88. HR-ESI: C21H32O9N2PS
Jcis = 12.8, CHP); 6.12, 6.13 (2 × ddd, 2 × 1H, JH,P = 51.7, Jcis
=
12.8, Jgem = 2.0, CHcisHtransCHP); 6.33, 6.34 (2 × ddd, 2 × 1H, JH,P
= 24.8, Jtrans = 18.6, Jgem = 2.0, CHcisHtransCHP); 7.38, 7.43 (2 × d, 2
× 1H, J6,5 = 8.1, H-6); 9.15, 9.18 (2 × bs, 2 × 2H, NH). 13C NMR
(125.7 MHz, CDCl3 ): 14.08 (CH3 (CH2 )1 5 ); 22.65
(CH3(CH2)13CH2CH2O); 25.23, 25.26 ((CH3)2C); 25.43
(CH3(CH2)13CH2CH2O); 27.08, 27.10 ((CH3)2C); 29.11, 29.32,
29.47, 29.53, 29.60, 29.61, 29.63, 29.65 (CH3(CH2)13CH2CH2O);
(M + H)+ calcd 519.1561; found 519.1562.
Ethyl 2′,3′-Isopropylideneuridin-5′-yl Vinylphosphonate
(10b). The title compound was prepared according to general
method B from 9a (1.86 g, 6.2 mmol) and ethanol (1 g, 30 mmol)
in 80% yield (2 g, 4.97 mmol) as a colorless oil. A mixture of
1
diastereomers ∼6:5. H NMR (499.8 MHz, CDCl3): 1.33, 1.34 (2 ×
30.40 (d, JC, P
= 6.3, CH3 (CH2 )13 CH2 CH2 O); 31.88
td, 2 × 3H, Jvic = 7.1, JH,P = 0.3, CH3CH2O); 1.35, 1.57 (2 × s, 2 × 6H,
(CH3)2C); 4.09−4.17 (m, 4H, CH3CH2O); 4.21−4.30 (m, 4H, H-5′);
(CH3(CH2)13CH2CH2O); 64.93, 65.00 (d, JC,P = 5.5, CH2-5′);
66.47, 66.48 (d, JC,P = 5.7, CH3(CH2)13CH2CH2O); 80.57, 80.66
(CH-3′); 84.46, 84.54 (CH-2′); 85.25, 85.55 (d, JC,P = 7.1, CH-4′);
93.40, 93.80 (CH-1′); 102.49, 102.57 (CH-5); 114.55, 114.60
(C(CH3)2); 124.99, 125.04 (d, JC,P = 184.2, CHP); 136.62,
136.66 (d, JC,P = 2.0, CH2CHP); 141.46, 141.55 (CH-6); 149.92,
149.95 (C-2); 163.05, 163.07 (C-4). 31P NMR (202.3 MHz, CDCl3):
18.59, 18.73. HR-ESI: C30H51O8N2PNa (M + Na)+ calcd 621.32752;
found 621.32778.
4.34−4.39 (m, 2H, H-4′); 4.86, 4.87 (2 × dd, 2 × 1H, J3′,2′ = 6.4, J3′,4′
=
3.7, H-3′); 4.92 (dd, 2H, J2′,3′ = 6.5, J2′,1′ = 2.4, H-2′); 5.71, 5.73 (2 × d,
2 × 1H, J5,6 = 8.1, H-5); 5.78, 5.805 (2 × d, 2 × 1H, J1′,2′ = 2.4, H-1′);
6.05, 6.06 (2 × ddd, 2 × 1H, JH,P = 22.9, Jtrans = 18.6, Jcis = 12.8, 
CHP); 6.12, 6.14 (2 × ddd, 2 × 1H, JH,P = 51.6, Jcis = 12.8, Jgem = 2.0,
CHcisHtransCHP); 6.34, 6.35 (2 × ddd, 2 × 1H, JH,P = 24.7, Jtrans
=
=
18.6, Jgem = 2.0, CHcisHtransCHP); 7.40, 7.44 (2 × d, 2 × 1H, J6,5
8.1, H-6); 10.15 (bs, 2H, NH). 13C NMR (125.7 MHz, CDCl3): 16.18
(d, JC,P = 6.2, CH3CH2O); 25.10, 25.12, 26.95, 26.96 ((CH3)2C);
62.30 (d, JC,P = 5.5, CH3CH2O); 64.94, 64.99 (d, JC,P = 5.8, CH2-5′);
80.54, 80.60 (CH-3′); 84.31, 84.39 (CH-2′); 85.32, 85.52 (d, JC,P = 7.1,
CH-4′); 93.51, 93.78 (CH-1′); 102.36, 102.44 (CH-5); 114.34, 114.38
(C(CH3)2); 124.91, 124.95 (d, JC,P = 184.2, CHP); 136.51, 136.53
(d, JC,P = 2.3, CH2CHP); 141.67, 141.73 (CH-6); 150.08, 150.11
(C-2); 163.58 (C-4). 31P NMR (202.3 MHz, CDCl3): 18.49, 18.66.
HR-ESI: C16H24O8N2P (M + H)+ calcd 403.12648; found 403.12632.
Tetradecyl 2′,3′-Isopropylideneuridin-5′-yl Vinylphospho-
nate (10c). The title compound was prepared according to general
method B from 9a (2.45 g, 5.15 mmol) and tetradecanol (2.2 g, 10.31
mmol) in 40% yield (1.17 g, 2.05 mmol) as a colorless thick oil. A
Octadecyl 2′,3′-Isopropylideneuridin-5′-yl Vinylphospho-
nate (10e). The title compound was prepared according to general
method B from 9a (2.18 g, 4.58 mmol) and octadecanol (2.48 g, 9.17
mmol) in 43% yield (1.23 g, 1.96 mmol) as a colorless thick oil. A
1
mixture of diastereomers ∼6:5. H NMR (499.8 MHz, CDCl3): 0.88
(m, 6H, CH3(CH2)17); 1.20−1.40 (m, 60H, CH3(CH2)15CH2CH2O);
4
1.350, 1.354, 1.573, 1.576 (4 × q, 4 × 3H, J = 0.7, (CH3)2C); 1.66
(m, 4H, CH3(CH2)15CH2CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, JH,P
=
7.3, Jvic = 6.7, CH3(CH2)15CH2CH2O); 4.19−4.29 (m, 4H, H-5′);
4.34−4.39 (m, 2H, H-4′); 4.85, 4.86 (2 × dd, 2 × 1H, J3′,2′ = 6.4, J3′,4′
3.7, H-3′); 4.88, 4.89 (2 × dd, 2 × 1H, J2′,3′ = 6.4, J2′,1′ = 2.3, H-2′); 5.71,
=
5.72 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.77, 5.81 (2 × d, 2 × 1H, J1′,2′
2.3, H-1′); 6.03, 6.04 (2 × ddd, 2 × 1H, JH,P = 22.9, Jtrans = 18.6, Jcis
=
=
1
mixture of diastereomers ∼6:5. H NMR (499.8 MHz, CDCl3): 0.88
12.7, CHP); 6.16, 6.19 (2 × ddd, 2 × 1H, JH,P = 51.7, Jcis = 12.7, Jgem
(m, 6H, CH3(CH2)13); 1.20−1.35 (m, 44H, CH3(CH2)11CH2CH2O);
4
= 2.0, CHcisHtransCHP); 6.33, 6.34 (2 × ddd, 2 × 1H, JH,P = 25.6,
1.350, 1.354, 1.573, 1.576 (4 × q, 4 × 3H, J = 0.7, (CH3)2C); 1.67
J
trans = 18.6, Jgem = 2.0, CHcisHtransCHP); 7.38, 7.43 (2 × d, 2 × 1H,
(m, 4H, CH3(CH2)11CH2CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, JH,P
7.3, Jvic = 6.7, CH3(CH2)11CH2CH2O); 4.19−4.29 (m, 4H, H-5′);
4.35−4.39 (m, 2H, H-4′); 4.85, 4.86 (2 × dd, 2 × 1H, J3′,2′ = 6.4, J3′,4′
3.6, H-3′); 4.88, 4.89 (2 × dd, 2 × 1H, J2′,3′ = 6.4, J2′,1′ = 2.3, H-2′); 5.71,
=
J6,5 = 8.1, H-6); 9.37 (bs, 2H, NH). 13C NMR (125.7 MHz, CDCl3):
14.08 (CH3(CH2)17); 22.64 (CH3(CH2)15CH2CH2O); 25.22, 25.25
((CH3)2C); 25.43 (CH3(CH2)15CH2CH2O); 27.07, 27.09
((CH3)2C); 29.10, 29.31, 29.46, 29.53, 29.60, 29.61, 29.63, 29.65
=
5.72 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.77, 5.81 (2 × d, 2 × 1H, J1′,2′
2.3, H-1′); 6.03, 6.04 (2 × ddd, 2 × 1H, JH,P = 22.9, Jtrans = 18.6, Jcis
=
=
( C H 3 ( C H 2 ) 1 5 C H 2 C H 2 O ) ; 3 0 . 3 9 ( d , J C , P
=
6 . 2 ,
CH3(CH2)15CH2CH2O); 31.87 (CH3(CH2)15CH2CH2O); 64.93,
65.01 (d, JC,P = 5.5, CH2-5′); 66.46, 66.47 (d, JC,P = 5.7,
CH3(CH2)15CH2CH2O); 80.57, 80.66 (CH-3′); 84.45, 84.53 (CH-
2′); 85.27, 85.56 (d, JC,P = 7.0, CH-4′); 93.44, 93.83 (CH-1′); 102.49,
12.7, CHP); 6.16, 6.19 (2 × ddd, 2 × 1H, JH,P = 51.6, Jcis = 12.7, Jgem
= 2.0, CHcisHtransCHP); 6.33, 6.34 (2 × ddd, 2 × 1H, JH,P = 25.7,
J
trans = 18.7, Jgem = 2.0, CHcisHtransCHP); 7.38, 7.43 (2 × d, 2 × 1H,
J6,5 = 8.1, H-6); 9.34 (bs, 2H, NH). 13C NMR (125.7 MHz, CDCl3):
14.08 (CH3(CH2)13); 22.64 (CH3(CH2)11CH2CH2O); 25.23, 25.25
((CH3)2C); 25.43 (CH3(CH2)11CH2CH2O); 27.08, 27.09
((CH3)2C); 29.10, 29.30, 29.46, 29.52, 29.59, 29.60, 29.62, 29.64
102.58 (CH-5); 114.53, 114.58 (C(CH3)2); 124.97, 125.01 (d, JC,P
=
184.2, CHP); 136.62, 136.66 (d, JC,P = 2.0, CH2CHP); 141.47,
141.56 (CH-6); 149.97, 150.00 (C-2); 163.14, 163.16 (C-4). 31P
NMR (202.3 MHz, CDCl3): 18.59, 18.74. HR-ESI: C32 H56 O8 N2 P
[M + H]+ calcd 627.37688, found 627.37738.
( C H 3 ( C H 2 ) 1 1 C H 2 C H 2 O ) ; 3 0 . 3 9 ( d , J C , P
=
6 . 2 ,
CH3(CH2)11CH2CH2O); 31.87 (CH3(CH2)11CH2CH2O); 64.93,
65.01 (d, JC,P = 5.5, CH2-5′); 66.47, 66.48 (d, JC,P = 5.7,
CH3(CH2)11CH2CH2O); 80.57, 80.66 (CH-3′); 84.45, 84.53 (CH-
2′); 85.26, 85.55 (d, JC,P = 7.0, CH-4′); 93.43, 93.83 (CH-1′); 102.49,
Icosanyl 2′,3′-Isopropylideneuridin-5′-yl Vinylphosphonate
(10f). The title compound was prepared according to general method
B from 9a (2.35 g, 4.94 mmol) and icosanol (2.95 g, 9.9 mmol) in 34%
yield (1.1 g, 1.68 mmol) as a colorless white wax. A mixture of
102.58 (CH-5); 114.54, 114.59 (C(CH3)2); 124.97, 125.02 (d, JC,P
=
1
diastereomers ∼6:5. H NMR (499.8 MHz, CDCl3): 0.88 (m, 6H,
184.3, CHP); 136.60, 136.62 (CH2CHP); 141.47, 141.55 (CH-
6); 149.96, 149.99 (C-2); 163.11 (C-4). 31P NMR (202.3 MHz,
CDCl3): 18.59, 18.74. HR-ESI: C28H48O8N2P [M + H]+ calcd
571.31428, found 571.31445, C28H47O8N2NaP [M + Na]+ calcd
593.29622, found 593.29616.
CH3(CH2)19); 1.20−1.36 (m, 68H, CH3(CH2)17CH2CH2O); 1.351,
1.354, 1.574, 1.577 (4 × q, 4 × 3H, 4J = 0.7, (CH3)2C); 1.66 (m, 4H,
CH3(CH2)17CH2CH2O); 4.03, 4.04 (2 × dt, 2 × 2H, JH,P = 7.3, Jvic
=
6.7, CH3(CH2)17CH2CH2O); 4.19−4.29 (m, 4H, H-5′); 4.35−4.39
(m, 2H, H-4′); 4.84, 4.86 (2 × dd, 2 × 1H, J3′,2′ = 6.4, J3′,4′ = 3.4, H-3′);
4.87, 4.89 (2 × dd, 2 × 1H, J2′,3′ = 6.4, J2′,1′ = 2.3, H-2′); 5.706, 5.714 (2
Hexadecyl 2′,3′-Isopropylideneuridin-5′-yl Vinylphospho-
nate (10d). The title compound was prepared according to general
7892
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Journal of Medicinal Chemistry
Article
× d, 2 × 1H, J5,6 = 8.1, H-5); 5.77, 5.80 (2 × d, 2 × 1H, J1′,2′ = 2.3, H-
1′); 6.03, 6.06 (2 × ddd, 2 × 1H, JH,P = 22.8, Jtrans = 18.6, Jcis = 12.7, 
CHP); 6.17, 6.19 (2 × ddd, 2 × 1H, JH,P = 51.7, Jcis = 12.7, Jgem = 2.0,
H-1′); 6.145, 6.17 (2 × ddd, 2 × 1H, JH,P = 24.0, Jtrans = 18.4, Jcis = 12.8,
CHP); 6.257, 6.261 (2 × ddd, 2 × 1H, JH,P = 52.0, Jcis = 12.8, Jgem
=
2.1, CHcisHtransCHP); 6.30, 6.31 (2 × ddd, 2 × 1H, JH,P = 26.0, Jtrans
= 18.4, Jgem = 2.1, CHcisHtransCHP); 7.54 (m, 4H, H-m-Bz); 7.59 (d,
2H, J5,6 = 7.7, H-5); 7.64 (m, 2H, H-p-Bz); 7.99 (m, 4H, H-o-Bz);
8.16, 8.17 (2 × d, 2 × 1H, J6,5 = 7.7, H-5). 13C NMR (150.9 MHz,
CD3OD): 14.48 (CH3(CH2)15); 23.75 (CH3(CH2)13CH2CH2O);
25.46, 25.48 ((CH3)2C); 27.27 (CH3(CH2)14CH2O); 27.43
((CH3)2C); 30.49, 30.61, 30.78, 30.80, 30.81 (CH3(CH2)14CH2O);
CHcisHtransCHP); 6.33, 6.34 (2 × ddd, 2 × 1H, JH,P = 25.6, Jtrans
=
=
18.6, Jgem = 2.0, CHcisHtransCHP); 7.37, 7.43 (2 × d, 2 × 1H, J6,5
8.1, H-6); 9.08 (bs, 2H, NH). 13C NMR (125.7 MHz, CDCl3): 14.10
(CH3(CH2)19); 22.66 (CH3(CH2)17CH2CH2O); 25.24, 25.26
((CH3)2C); 25.44 (CH3(CH2)17CH2CH2O); 27.09, 27.11
((CH3)2C); 29.12, 29.33, 29.48, 29.55, 29.62, 29.65, 29.67
( C H 3 ( C H 2 ) 1 7 C H 2 C H 2 O ) ; 3 0 . 4 1 ( d , J C , P
=
6 . 3 ,
31.70 (d, JC, P
(CH3(CH2)13CH2CH2O); 64.96, 64.98 (d, JC,P
OCH2CH2CH2OC16H33); 66.97, 67.04 (d, JC,P = 5.6, CH2-5′);
67.42, 67.43 (OCH2CH2CH2OC16H33); 72.10 (CH3(CH2)14CH2O);
82.64, 82.635, 82.644 (CH-3′); 86.57, 86.58 (CH-2′); 88.23, 88.30 (d,
=
6.5, OCH2CH2CH2OC16H33); 33.09
5.7,
CH3(CH2)17CH2CH2O); 31.89 (CH3(CH2)17CH2CH2O); 64.92,
64.99 (d, JC,P = 5.5, CH2-5′); 66.48, 66.50 (d, JC,P = 5.7,
CH3(CH2)17CH2CH2O); 80.57, 80.66 (CH-3′); 84.47, 84.56 (CH-
2′); 85.25, 85.54 (d, JC,P = 7.0, CH-4′); 93.44, 93.83 (CH-1′); 102.50,
=
102.59 (CH-5); 114.56, 114.62 (C(CH3)2); 124.98, 125.02 (d, JC,P
=
J
C,P = 7.2, CH-4′); 97.60, 97.71 (CH-1′); 98.51, 98.54 (CH-5); 115.10,
184.2, CHP); 136.65, 136.70 (d, JC,P = 1.9, CH2CHP); 141.41,
141.50 (CH-6); 149.90, 149.93 (C-2); 162.92, 162.95 (C-4). 31P
NMR (202.3 MHz, CDCl3): 18.59, 18.74. HR-ESI: C34H60O8N2P [M
+ H] calcd 655.40818, found 655.40857; C34H59O8N2NaP [M + Na]
calcd 677.39012, found 677.39032.
Hexadecyloxypropyl 2′,3′-Isopropylideneuridin-5′-yl Vinyl-
phosphonate (10g). The title compound was prepared according to
general method B from 9a (1.8 g, 6.1 mmol) and hexadecylox-
ypropanol (1.45 g, 3.05 mmol) in 71% yield (1.42 g, 2.16 mmol) as a
colorless white wax. A mixture of diastereomers ∼1:1. 1H NMR (500.0
MHz, CDCl3): 0.88 (m, 6H, CH3(CH2)15); 1.23−1.33 (m, 52H,
CH3(CH2)13CH2CH2O); 1.348, 1.352 (2 × q, 2 × 3H, 4J = 0.6,
(CH3)2C); 1.54 (m, 4H, CH3(CH2)13CH2CH2O); 1.57 (s, 6H,
115.11 (C(CH3)2); 125.63, 125.67 (d, JC,P = 183.9, CHP); 129.25
(CH-o-Bz); 129.83 (CH-m-Bz); 134.14 (CH-p-Bz); 134.70 (C-i-Bz);
138.01, 138.04 (d, JC,P = 1.9, CH2CHP); 148.26, 148.34 (CH-6);
157.58, 157.60 (C-2); 165.58 (C-4); 169.19 (CO-Bz). 31P NMR
(202.3 MHz, CD3OD): 19.62, 19.72. HR-EI: C40H62N3O9P (M + H)+
calcd 759.4224; found 759.4225.
Hexadecyloxypropyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxy-
pyrrolidin-1-N-yl)ethylphosphonate (12a). The mixture of dihy-
droxypyrrolidine 11a (0.12 g, 1.18 mmol) and phosphonate 10g (0.4
g, 0.61 mmol) in nBuOH (15 mL) was stirred at 105 °C overnight.
The solvent was removed, and the isopropylidene intermediate was
obtained by flash chromatography on silica gel using linear gradient of
H1 in ethyl acetate. This compound was without further character-
ization dissolved in 0.5 M methanolic HCl (30 mL), and the mixture
was stirred at rt for 4 h. The desired product was obtained after
evaporation of solvent in 34% overall yield (152 mg, 0.205 mmol) as a
(CH3)2C); 1.92, 1.94 (2
× p, 2 × 2H, Jvic = 6.1,
OCH2CH2CH2OC16H33); 3.38, 3.39 (2 × t, 2 × 2H, Jvic = 6.7,
CH3(CH2)13CH2CH2O); 3.48, 3.49 (2 × t, 2 × 2H, Jvic = 6.1,
1
OCH2CH2CH2OC16H33); 4.13, 4.15 (2 × td, 2 × 2H, Jvic = 6.1, JH,P
=
white amorphous solid. A mixture of diastereomers ∼6:5. H NMR
4.8, OCH2CH2CH2OC16H33); 4.20−4.30 (m, 4H, H-5′); 4.34−4.38
(m, 2H, H-4′); 4.85, 4.86 (2 × dd, 2 × 1H, J3′,2′ = 6.5, J3′,4′ = 3.6, H-3′);
4.92 (dd, 2H, J2′,3′ = 6.5, J2′,1′ = 2.3, H-2′); 5.700, 5.704 (2 × d, 2 × 1H,
(499.8 MHz, CD3OD): 0.90 (m, 6H, CH3(CH2)15); 1.25−1.38 (m,
5 2 H , C H 3 ( C H 2 ) 1 3 C H 2 C H 2 O ) ; 1 . 5 5 ( m , 4 H ,
CH3(CH2)13CH2CH2O); 1.93 (m, 4H, OCH2CH2CH2OC16H33);
2.12−2.25 (m, 4H, CH2P); 2.71 (dd, 4H, Jgem = 10.6, Jvic = 3.0, H-
2b,5b-pyrr); 2.86−3.00 (m, 4H, CH2N); 3.116, 3.118 (2 × dd, 2 × 2H,
J
5,6 = 8.1 H-5); 5.74, 5.76 (2 × d, 2 × 1H, J1′,2′ = 2.3, H-1′); 6.03, 6.05
(2 × ddd, 2 × 1H, JH,P = 22.9, Jtrans = 18.4, Jcis = 12.7, CHP); 6.16,
6.18 (2 × ddd, 2 × 1H, JH,P = 51.8, Jcis = 12.7, Jgem = 1.9, CHcisHtrans

Jgem = 10.6, Jvic = 5.0, H-2a,5a-pyrr); 3.42, 3.43 (2 × t, 2 × 2H, Jvic
=
CHP); 6.33, 6.35 (2 × ddd, 2 × 1H, JH,P = 25.5, Jtrans = 18.4, Jgem = 1.9,
CHcisHtransCHP); 7.34, 7.39 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C
NMR (125.7 MHz, CDCl3): 14.07 (CH3(CH2)15); 22.62
(CH3(CH2)13CH2CH2O); 25.19, 25.21 ((CH3)2C); 26.08
(CH3(CH2)14CH2O); 27.04, 27.06 ((CH3)2C); 29.29, 29.45, 29.55,
29.57, 29.58, 29.63 (CH3(CH2)14CH2O); 30.66, 30.68 (d, JC,P = 6.4,
OCH2CH2CH2OC16H33); 31.85 (CH3(CH2)13CH2CH2O); 63.56
63.57 (d, JC,P = 5.5, OCH2CH2CH2OC16H33); 64.92, 65.01 (d, JC,P
= 5.5, CH2-5′); 66.32, 66.36 (OCH2CH2CH2OC16H33); 71.15, 71.16
(CH3(CH2)14CH2O); 80.60, 80.67 (CH-3′); 84.44, 84.53 (CH-2′);
85.38, 85.62 (d, JC,P = 7.1, CH-4′); 93.82, 94.12 (CH-1′); 102.57,
6.6, CH3(CH2)13CH2CH2O); 3.515, 3.522 (2 × t, 2 × 2H, Jvic = 6.1,
OCH2CH2CH2OC16H33); 4.08 (m, 4H, OCH2CH2CH2OC16H33);
4.11−4.21 (m, 8H, H-3′,4′, H-3,4-pyrr); 4.216, 4.218 (2 × dd, 2 × 1H,
J2′,3′ = 5.1, J2′,1′ = 4.1, H-2′); 4.26 (ddd, 1H, Jgem = 11.6, JH,P = 6.8, J5′b,4′
= 4.5, H-5′b); 4.30−4.34 (m, 2H, H-5′); 4.36 (ddd, 1H, Jgem = 11.6,
J
H,P = 6.6, J5′a,4′ = 2.9, H-5′a); 5.751, 5.753 (2 × d, 2 × 1H, J5,6 = 8.1, H-
5); 5.845 (d, 2H, J1′,2′ = 4.1, H-1′); 7.70, 7.73 (2 × d, 2 × 1H, J6,5 = 8.1,
H-6). 13C NMR (125.7 MHz, CD3OD): 14.47 (CH3(CH2)15); 23.73
(CH3(CH2)14CH2O); 24.75, 24.81 (d, JC,P = 139.6, CH2P); 27.28,
30.47, 30.63, 30.76, 30.79 (CH3(CH2)14CH2O); 31.75, 31.78 (d, JC,P
6.2, OCH2CH2CH2OC16H33); 33.06 (CH3(CH2)14CH2O); 50.81,
50.85 (CH2N); 60.95, 60.98 (CH2-2,5-pyrr); 64.86, 64.93 (d, JC,P
=
102.64 (CH-5); 114.42, 114.46 (C(CH3)2); 124.75, 124.80 (d, JC,P
=
=
184.0, CHP); 136.75, 136.77 (d, JC,P = 1.9, CH2CHP); 141.43,
141.48 (CH-6); 150.16 (C-2); 163.29, 163.32 (C-4). 31P NMR (202.3
MHz, CDCl3): 18.64, 18.80. HR-ESI: C33H58O9N2P (M + H)+ calcd
657.3874; found 657.3876.
Hexadecyloxypropyl 4-N-Benzoyl-2′,3′-isopropylidenecyti-
din-5′-yl Vinylphosphonate (10h). The title compound was
prepared according to general method B from 9b (0.65 g, 1.36
mmol) and hexadecyloxypropanol (0.81 g, 2.7 mmol) in 78% yield
(0.81 g, 1.06 mmol) as a white foam. A mixture of diastereomers ∼1:1.
1H NMR (600.1 MHz, CD3OD): 0.895 (m, 6H, CH3(CH2)15); 1.24−
1.35 (m, 52H, CH3(CH2)13CH2CH2O); 1.368, 1.371 (2 × q, 2 × 3H,
4J = 0.6, (CH3)2C); 1.51 (m, 4H, CH3(CH2)13CH2CH2O); 1.57 (s,
6H, (CH3)2C); 1.89, 1.91 (2 × p, 2 × 2H, Jvic = 6.1,
OCH2CH2CH2OC16H33); 3.38, 3.39 (2 × t, 2 × 2H, Jvic = 6.7,
CH3(CH2)13CH2CH2O); 3.47 (td, 2H, Jvic = 6.1, JH,P = 1.2,
6.7, OCH2CH2CH2OC16H33); 66.52, 66.54 (d, JC,P = 6.2, CH2-5′);
67.49, 67.53 (OCH2CH2CH2OC16H33); 70.79, 70.86 (CH-3′); 72.14
(CH3(CH2)14CH2O); 74.87, 74.93 (CH-2′); 78.12, 78.19 (CH-3,4-
pyrr); 83.57, 83.58 (d, JC,P = 6.6, CH-4′); 91.75, 91.85 (CH-1′); 103.02
(CH-5); 142.58, 142.64 (CH-6); 152.18, 152.20 (C-2); 165.99 (C-4).
31P NMR (202.3 MHz, CD3OD): 31.37, 31.64. HR-ESI:
C34H63O11N3P (M + H)+ calcd 720.41947; found 720.41939.
Hexadecyloxypropyl Cytidin-5′-yl 2-([3R,4R]-3,4-Dihydroxy-
pyrrolidin-1-N-yl)ethylphosphonate (12b). The mixture of dihy-
droxypyrrolidine 11a (0.05 g, 0.49 mmol) and benzoylcytosine
vinylphosphonate 10h (0.34 g, 0.45 mmol) in nBuOH (10 mL) was
stirred at 100 °C for 12 h. The solvent was removed and the protected
intermediate was purified by flash chromatography on silica gel using
linear gradient of H1 in ethyl acetate. White foam obtained after
evaporation of solvents was without further characterization (only LC-
MS confirmation) dissolved in 8 M ethanolic methylamine (10 mL)
and left aside at rt overnight. The mixture was concentrated in vacuo,
coevaporated with ethanol (2 × 20 mL), dissolved in 0.2 M
methanolic HCl (10 mL), and the reaction mixture was left aside at
rt overnight. After concentration in vacuo (note: temperature in the
OCH2 CH2 CH2 OC1 6 H3 3 ); 3. 49 (t, 2H, Jv i c
= 6.1,
O C H 2 C H 2 C H 2 O C 1 6 H 3 3 ) ; 4 . 0 9 − 4 . 1 6 ( m , 4 H ,
OCH2CH2CH2OC16H33); 4.27−4.36 (m, 4H, H-5′); 4.48 (m, 2H,
H-4′); 4.91 (dd, 2H, J3′,2′ = 6.2, J3′,4′ = 3.6, H-3′); 5.06, 507 (2 × dd, 2 ×
1H, J2′,3′ = 6.2, J2′,1′ = 1.8, H-2′); 5.87, 5.88 (2 × d, 2 × 1H, J1′,2′ = 1.8,
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bath did not exceed 40 °C) and coevaporation with ethanol (3 × 20
mL), the final product was obtained by preparative HPLC in 27%
overall yield (90.6 mg, 120 μM) as a white amorphous solid. A mixture
of diastereomers ∼8:7. 1H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H,
CH3(CH2)15); 1.25−1.38 (m, 52H, CH3(CH2)13CH2CH2O); 1.55
( m , 4 H , C H 3 ( C H 2 ) 1 3 C H 2 C H 2 O ) ; 1 . 9 3 ( m , 4 H ,
OCH2CH2CH2OC16H33); 2.10−2.18 (m, 4H, CH2P); 2.56 (bm,
4H, H-2b,5b-pyrr); 2.75−2.87 (bm, 4H, CH2N); 3.00 (bm, 4H, H-
The solvent was removed and the isopropylidene intermediate was
obtained by flash chromatography on silica gel using linear gradient of
ethanol in chloroform. This compound was without characterization
dissolved in 0.5 M methanolic HCl (20 mL), and the mixture was
stirred at rt for 2 h. After concentration in vacuo (note: temperature in
the bath did not exceeded 40 °C) and coevaporation with ethanol (3 ×
20 mL), the desired product was obtained by flash chromatography on
silica gel using linear gradient of H1 in ethyl acetate in 68% overall
yield (80 mg, 0.11 mmol) as a white amorphous solid. A mixture of
2a,5a-pyrr); 3.41, 3.43 (2
× t, 2 × 2H, Jvic = 6.7,
1
diastereomers ∼7:3. H NMR (499.8 MHz, CD3OD): 0.90 (m, 6H,
CH3(CH2)13CH2CH2O); 3.51, 3.53 (2 × t, 2 × 2H, Jvic = 6.0,
OCH2CH2CH2OC16H33); 4.04 (m, 4H, H-3,4-pyrr); 4.08−4.23 (m,
10H, H-2′,3′,4′-pyrr, OCH2CH2CH2OC16H33); 4.25−4.42 (m, 4H, H-
CH3(CH2)15); 1.25−1.38 (m, 52H, CH3(CH2)13CH2CH2O); 1.55
( m , 4 H , C H 3 ( C H 2 ) 1 3 C H 2 C H 2 O ) ; 1 . 9 3 ( m , 4 H ,
OCH2CH2CH2OC16H33); 2.10−2.18 (m, 4H, CH2P); 2.43, 2.54 (2
× bm, 2 × 4H, H-2,6-pip); 2.75 (m, 4H, CH2N); 3.42, 3.43 (2 × t, 2 ×
2H, Jvic = 6.6, CH3(CH2)13CH2CH2O); 3.52, 3.53 (2 × t, 2 × 2H, Jvic
= 6.1, OCH2CH2CH2OC16H33); 3.68 (bm, 4H, H-3,5-pip); 3.81 (bm,
2 H , H - 4 - p i p ) ; 4 . 1 1 − 4 . 2 2 ( m , 1 0 H , H - 2 ′ , 3 ′ , 4 ′ ,
OCH2CH2CH2OC16H33); 4.23−4.38 (m, 4H, H-5′); 5.746, 5.749 (2
× d, 2 × 1H, J5,6 = 8.1, H-5); 5.84 (d, 2H, J1′,2′ = 4.1, H-1′); 7.71, 7.73
(2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR (125.7 MHz, CD3OD):
14.45 (CH3(CH2)15); 23.55, 23.65 (d, JC,P = 138.5, CH2P); 23.75
(CH3(CH2)14CH2O); 27.30, 30.49, 30.63, 30.77, 30.80
5′); 5.83, 5.84 (d, 2H, J1′,2′ = 3.0, H-1′); 5.93, 5.94 (2 × d, 2 × 1H, J5,6
=
7.6, H-5); 7.77, 7.79 (2 × d, 2 × 1H, J6,5 = 7.6, H-6). 13C NMR (150.9
MHz, CD3OD): 14.47 (CH3(CH2)15); 23.75 (CH3(CH2)14CH2O);
25.09, 25.14 (d, JC,P = 139.1, CH2P); 27.29, 27.30, 30.49, 30.64, 30.77,
30.80 (CH3(CH2)14CH2O); 31.76, 31.79 (d, JC,P = 6.3,
OCH2CH2CH2OC16H33); 33.08 (CH3(CH2)14CH2O); 50.55
(CH2N); 60.99, 61.02 (CH2-2,5-pyrr); 64.71, 64.79 (d, JC,P = 6.6,
OCH2CH2CH2OC16H33); 66.24, 66.31 (d, JC,P = 6.4, CH2-5′); 67.51,
67.55 (OCH2CH2CH2OC16H33); 70.52, 70.55 (CH-3′); 72.15
(CH3(CH2)14CH2O); 75.68, 75.70 (CH-2′); 78.61, 78.66 (CH-3,4-
pyrr); 83.13, 83.16 (d, JC,P = 6.6, CH-4′); 92.89, 92.90 (CH-1′); 96.17,
96.18 (C-5); 142.56, 142.64 (CH-6); 158.26, 158.27 (C-2); 167.646,
167.653 (C-4). 31P NMR (202.3 MHz, CD3OD): 32.67, 32.99. HR-
ESI: C34H64O10N4P (M + H)+ calcd 719.4355; found 719.4357.
Hexadecyloxypropyl Uridin-5′-yl 2-([3S,4S]-3,4-Dihydroxy-
pyrrolidin-1-N-yl)ethylphosphonate (12c). The mixture of dihy-
droxypyrrolidine 11b (0.26 g, 2.56 mmol) and phosphonate 10g (0.84
g, 1.28 mmol) in nBuOH (30 mL) was stirred at 105 °C overnight.
The solvent was removed, and the isopropylidene intermediate was
obtained by flash chromatography on silica gel using linear gradient of
H1 in ethyl acetate. This compound was without further character-
ization dissolved in 0.5 M methanolic HCl (40 mL), and the mixture
was stirred at rt for 4 h. After concentration in vacuo (note:
temperature in the bath did not exceeded 40 °C) and coevaporation
with ethanol (3 × 20 mL) the desired product was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate in 36% overall yield (344 mg, 0.46 mmol) as a white
(CH3 (CH2 )1 4 CH2 O); 31.79, 31.82 (d, JC , P
= 6.2,
OCH2CH2CH2OC16H33); 33.08 (CH3(CH2)14CH2O); 51.75
(CH2N); 54.40 (CH2-2,6-pip); 64.72, 64.78 (d, JC,P = 6.5,
OCH2CH2CH2OC16H33); 66.37 (d, JC,P = 6.3, CH2-5′); 67.51, 67.55
(OCH2CH2CH2OC16H33); 69.67 (CH-3,5-pip); 70.81, 70.83 (CH-
3′); 71.83 (CH-4-pip); 72.17 (CH3(CH2)14CH2O); 74.97, 75.01 (CH-
2′); 83.59, 83.63 (d, JC,P = 6.7, CH-4′); 91.84, 91.92 (CH-1′); 103.00
(CH-5); 142.56, 142.60 (CH-6); 152.18, 152.19 (C-2); 166.05 (C-4).
31P NMR (202.3 MHz, CD3OD): 32.91, 33.23. HR-ESI:
C35H65O11N3P (M + H)+ calcd 734.4347; found 734.4351.
Hexadecyloxypropyl Uridin-5′-yl 2-([3R,5R]-3,4-Dihydroxy-
piperidin-1-N-yl)ethylphosphonate (12e). The mixture of dihy-
droxypiperidine 11e (0.05 g, 0.42 mmol) and phosphonate 10g (0.25
g, 0.38 mmol) in nBuOH (5 mL) was stirred at 105 °C overnight. The
solvent was removed, and the isopropylidene intermediate was
obtained by flash chromatography on silica gel using linear gradient
of ethanol in chloroform. This compound was without characterization
dissolved in 0.5 M methanolic HCl (40 mL), and the mixture was
stirred at rt for 3 h. After concentration in vacuo (note: temperature in
the bath did not exceeded 40 °C) and coevaporation with ethanol (3 ×
20 mL), the desired product was obtained by chromatography on silica
gel using linear gradient of H1 in ethyl acetate in 76% overall yield
(230 mg, 0.29 mmol) as a white amorphous solid. A mixture of
1
amorphous solid. A mixture of diastereomers ∼6:5. H NMR (499.8
MHz, CD3OD): 0.90 (m, 6H, CH3(CH2)15); 1.25−1.39 (m, 52H,
CH3(CH2)13CH2CH2O); 1.55 (m, 4H, CH3(CH2)13CH2CH2O); 1.95
(m, 4H, OCH2CH2CH2OC16H33); 2.37−2.52 (m, 4H, CH2P); 3.16
(bm, 2H, H-2b,5b-pyrr); 3.42 (t, 2H, Jv i c
= 6.7,
CH3(CH2)13CH2CH2O); 3.425 (bm, 2H, H-2b,5b-pyrr); 3.43 (t,
2H, Jvic = 6.7, CH3(CH2)13CH2CH2O); 3.515 (m, 4H, CH2N); 3.52,
3.53 (2 × t, 2 × 2H, Jvic = 6.1, OCH2CH2CH2OC16H33); 3.59, 3.92 (2
× bm, 2 × 2H, H-2a,5a-pyrr); 4.14 (m, 2H, H-4′); 4.16 (m, 2H, H-3′);
4.18−4.27 (m, 10H, H-2′, OCH2CH2CH2OC16H33, H-3,4-pyrr); 4.31
(ddd, 1H, Jgem = 11.5, JH,P = 7.6, J5′b,4′ = 5.4, H-5′b); 4.36 (m, 2H, H-
5′); 4.40 (ddd, 1H, Jgem = 11.5, JH,P = 7.3, J5′a,4′ = 2.9, H-5′a); 5.742,
1
diastereomers ∼6:4. H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H,
CH3(CH2)15); 1.25−1.38 (m, 52H, CH3(CH2)13CH2CH2O); 1.55
(m, 4H, CH3(CH2)13CH2CH2O); 1.70 (bm, 4H, H-4-pip); 1.93 (m,
4H, OCH2CH2CH2OC16H33); 2.12−2.19 (m, 4H, CH2P); 2.36, 2.57
(2 × m, 2 × 4H, H-2,6-pip); 2.66−2.76 (m, 4H, CH2N); 3.42, 3.43 (2
× t, 2 × 2H, Jvic = 6.6, CH3(CH2)13CH2CH2O); 3.51, 3.53 (2 × t, 2 ×
2H, Jvic = 6.1, OCH2CH2CH2OC16H33); 4.01 (m, 4H, H-3,5-pip);
4.12−4.24 (m, 10H, H-2′,3′,4′, OCH2CH2CH2OC16H33); 4.24−4.40
(m, 4H, H-5′); 5.751, 5.754 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.854,
5.746 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.78, 5.80 (2 × d, 2 × 1H, J1′,2′
=
3.9, H-1′); 7.68, 7.70 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR (125.7
MHz, CD3OD): 14.45 (CH3(CH2)15); 23.32 (d, JC,P = 142.0, CH2P);
23.74 (CH3(CH2)14CH2O); 27.29, 30.48, 30.64, 30.76, 30.79
5.856 (2 × d, 2 × 1H, J1′,2′ = 4.1, H-1′); 7.71, 7.74 (2 × d, 2 × 1H, J6,5
=
8.1, H-6). 13C NMR (150.9 MHz, CD3OD): 14.49 (CH3(CH2)15);
23.49, 23.53 (d, JC,P = 138.4, CH2P); 23.75 (CH3(CH2)14CH2O);
27.29, 27.30, 30.49, 30.65, 30.78, 30.81 (CH3(CH2)14CH2O); 31.76,
(CH3 (CH2 )1 4 CH2 O); 31.70, 31.72 (d, JC , P
= 6.1,
OCH2CH2CH2OC16H33); 33.07 (CH3(CH2)14CH2O); 52.18, 52.22
(CH2N); 60.67, 60.75, 60.98, 61.01 (CH2-2,5-pyrr); 65.33, 65.58 (d,
JC,P = 6.7, OCH2CH2CH2OC16H33); 67.16 (d, JC,P = 6.5, CH2-5′);
67.44 (OCH2CH2CH2OC16H33); 67.45 (d, JC,P = 4.3, CH2-5′); 67.47
( O C H 2 C H 2 C H 2 O C 1 6 H 3 3 ) ; 7 0 . 7 7 ( C H - 3 ′ ) ; 7 2 . 1 6
(CH3(CH2)14CH2O); 74.55, 74.60 (CH-2′); 75.67, 75.68, 76.04
(CH-3,4-pyrr); 83.29 (d, JC,P = 6.2, CH-4′); 83.40 (d, JC,P = 5.8, CH-
4′); 92.83, 92.85 (CH-1′); 103.03, 103.10 (CH-5); 143.12, 143.19
(CH-6); 152.13, 152.14 (C-2); 166.00 (C-4). 31P NMR (202.3 MHz,
CD3OD): 26.79, 27.26.
31.83 (d, JC, P
(CH3(CH2)14CH2O); 40.63, 40.65 (CH2-4-pip); 52.02, 52.04 (d,
C,P = 2.0, CH2N); 60.07, 60.10 (CH2-2,6-pip); 64.63, 64.75 (d, JC,P
= 6.3, OCH2CH2CH2OC16H33); 33.08
J
=
6.6, OCH2CH2CH2OC16H33); 65.58 (CH-3,5-pip); 66.27, 66.52 (d,
JC,P = 6.2, CH2-5′); 67.51, 67.54 (OCH2CH2CH2OC16H33); 70.77,
70.84 (CH-3′); 72.13, 72.15 (CH3(CH2)14CH2O); 74.99, 75.05 (CH-
2′); 83.59, 83.60 (d, JC,P = 6.6, CH-4′); 91.65, 91.66 (CH-1′); 102.99
(CH-5); 142.45, 142.51 (CH-6); 152.16, 152.18 (C-2); 165.99 (C-4).
31P NMR (202.3 MHz, CD3OD): 33.22, 33.53. HR-ESI:
Hexadecyloxypropyl Uridin-5′-yl 2-([3R,4S,5S]-3,4-Trihy-
droxypiperidin-1-N-yl)ethylphosphonate (12d). The mixture of
trihydroxypiperidine 11e (0.04 g, 0.33 mmol) and phosphonate 10g
(0.1 g, 0.16 mmol) in nBuOH (5 mL) was stirred at 105 °C overnight.
C35H65O10N3P (M + H)+ calcd 718.4402; found 718.4402.
2 Pivaloylthioethyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyr-
rolidin-1-N-yl)ethylphosphonate (12f). The mixture of dihydrox-
7894
dx.doi.org/10.1021/jm2009343|J. Med. Chem. 2011, 54, 7884−7898

Journal of Medicinal Chemistry
Article
ypyrrolidine 11a (0.25 g, 2.4 mmol) and phosphonate 10a (0.57 g, 1.1
mmol) in nBuOH (12 mL) was stirred at 100 °C for 12 h. The solvent
was removed, and the product was obtained by flash chromatography
on silica gel using linear gradient of H1 in ethyl acetate in 85% yield
(0.58 g, 0.93 mmol) as white foam (HR-ESI: C25H39O11N3PS (M −
H)− calcd 620.2048; found 620.2049). The solution of this
intermediate (0.58 g, mmol) in 0.2 M methanolic HCl (50 mL) was
stirred at rt overnight. The mixture was concentrated in vacuo, and the
product was obtained by preparative reverse phase HPLC in 26%
overall yield (0.177 g, 0.29 mmol) as a white amorphous solid. A
mixture of diastereomers ∼5:3. 1H NMR (499.8 MHz, CD3OD): 1.23,
1.24 (2 × s, 2 × 9H, (CH3)3C); 2.46 (m, 4H, CH2P); 3.12−3.24 (m,
4H, SCH2CH2O); 3.38 (bm, 4H, H-2b,5b-pyrr); 3.51 (m, 4H,
CH2N); 3.66 (bm, 4H, H-2a,5a-pyrr); 4.11−4.22 (m, 8H, H-3′,4′,
gel using linear gradient of H1 in ethyl acetate in 66% overall yield
(862 mg, 1.36 mmol) as a white amorphous solid. A mixture of two
diastereomers ∼6:5. H NMR (600.1 MHz, CD3OD): 0.90 (m, 6H,
1
CH3(CH2)13); 1.25−1.43 (m, 44H, CH3(CH2)11CH2CH2O); 1.68
(m, 4H, CH3(CH2)11CH2CH2O); 2.06−2.19 (m, 4H, CH2P); 2.56
(m, 4H, H-2b,5b-pyrr); 2.74−2.88 (m, 4H, CH2N); 3.00 (m, 4H, H-
2a,5a-pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.06−4.13 (m, 4H,
CH3(CH2)11CH2CH2O); 4.13−4.16 (m, 4H, H-3′,4′); 4.200, 4.202
(2 × dd, 2 × 1H, J2′,3′ = 5.0, J2′,1′ = 4.0, H-2′); 4.24 (ddd, 1H, Jgem
11.6, JH,P = 6.7, J5′b,4′ = 4.3, H-5′b); 4.29 (ddd, 1H, Jgem = 11.6, JH,P
7.2, J5′b,4′ = 2.4, H-5′b); 4.31 (ddd, 1H, Jgem = 11.6, JH,P = 5.0, J5′a,4′
=
=
=
2.6, H-5′a); 4.35 (ddd, 1H, Jgem = 11.6, JH,P = 6.6, J5′a,4′ = 2.8, H-5′a);
5.736, 5.745 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H,
J1′,2′ = 4.0, H-1′); 7.71, 7.74 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR
(150.9 MHz, CD3 OD): 14.46 (CH3 (CH2 )1 3 ); 23.75
(CH3(CH2)11CH2CH2O); 25.11 (d, JC,P = 139.5, CH2P); 25.16 (d,
JC,P = 139.0, CH2P); 26.65, 30.27, 30.28, 30.50, 30.68, 30.71, 30.72,
OCH2CH2S); 4.26 (m, 6H, H-2′, H-3,4-pyrr); 4.34 (ddd, 1H, Jgem
=
11.6, JH,P = 8.0, J5′b,4′ = 5.4, H-5′b); 4.36−4.40 (m, 2H, H-5′); 4.41
(ddd, 1H, Jgem = 11.6, JH,P = 7.7, J5′a,4′ = 2.8, H-5′a); 5.746, 5.750 (2 ×
d, 2 × 1H, J5,6 = 8.1, H-5); 5.797, 5.800 (2 × d, 2 × 1H, J1′,2′ = 4.0, H-
1′); 7.68, 7.70 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR (125.7 MHz,
CD3OD): 23.43, 23.49 (d, JC,P = 142.2, CH2P); 27.66 ((CH3)3C);
29.52, 29.58 (d, JC,P = 6.6, SCH2CH2O); 47.56 (C(CH3)3); 52.13,
30.78, 30.80, 30.82 (CH3(CH2)11CH2CH2O); 31.55, 31.57 (d, JC,P
=
6.0, CH3(CH2)11CH2CH2O); 33.09 (CH3(CH2)11CH2CH2O); 50.57,
50.60 (d, JC,P = 1.0, CH2N); 61.01, 61.03 (CH2-2,5-pyrr); 66.36, 66.42
(d, JC,P
= 6.3, CH2-5′); 66.60, 67.63 (d, JC,P = 6.8,
52.17 (CH2N); 60.86, 60.89 (CH2-2,5-pyrr); 66.20, 66.34 (d, JC,P
=
CH3(CH2)11CH2CH2O); 70.81, 70.88 (CH-3′); 74.95, 75.01 (CH-
2′); 78.60, 78.65 (CH-3,4-pyrr); 83.63, 83.64 (d, JC,P = 6.6, CH-4′);
91.57, 91.75 (CH-1′); 102.95, 102.98 (CH-5); 142.56, 142.57 (CH-6);
152.18, 152.22 (C-2); 166.03 (C-4). 31P NMR (202.3 MHz, CD3OD):
32.08, 32.38. HR-ESI: C29H53O10N3P (M + H)+ calcd 634.34631,
found 634.34635.
6.6, OCH2CH2S); 67.40 (d, JC,P = 6.8, CH2-5′); 67.50 (d, JC,P = 6.4,
CH2-5′); 70.77 (CH-3′); 74.54, 74.58 (CH-2′); 75.90 (CH-3,4-pyrr);
83.29, 83.34 (d, JC,P = 6.0, CH-4′); 92.65, 92.82 (CH-1′); 103.10,
103.12 (CH-5); 143.11, 143.14 (CH-6); 152.17 (C-2); 166.01 (C-4);
207.15 (COS). 31P NMR (202.3 MHz, CD3OD): 26.92, 27.39. HR-
ESI: C22H37O11N3PS (M + H)+ calcd 582.18809; found 582.18811.
Ethyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-N-
yl)ethylphosphonate (12g). The mixture of dihydroxypyrrolidine
11a (0.24 g, 2.3 mmol) and phosphonate 10b (0.6 g, 1.5 mmol) in
nBuOH (12 mL) was stirred at 100 °C for 12 h. The solvent was
removed, and the intermediate was obtained by flash chromatography
on silica gel using linear gradient of H1 in ethyl acetate. The
intermediate (0.76 g, 1.5 mmol) was dissolved in 0.5 M methanolic
HCl (100 mL), and the mixture was stirred at rt overnight. The
mixture was concentrated in vacuo, coevaporated with diethylether (3
× 50 mL) and ethylacetate (2 × 50 mL), and the product was
obtained by crystallization from a mixture of ethanol and diethylether
(1:1) in 77% yield (0.58 g, 1.16 mmol) as a white solid. Characterized
by NMR as hydrochloride, a mixture of diastereomers ∼1:1. 1H NMR
(600.1 MHz, DMSO-d6): 1.235, 1.242 (2 × s, 2 × 3H, Jvic = 7.0,
CH3CH2); 2.38 (m, 4H, CH2P); 3.07 (bd, 2H, Jgem = 12.1, H-2b or
5b-pyrr); 3.30 (bm, 6H, CH2N, H-2b or 5b-pyrr); 3.42, 3.67 (2 × bm,
2 × 2H, H-2a,5a-pyrr); 3.95, 3.97 (2 × t, 2 × 1H, J3′,2′ = J3′,4′ = 4.9, H-
3′); 3.98 (m, 2H, H-4′); 4.05 (m, 4H, CH3CH2); 4.07 (m, 2H, H-2′);
4.09 (m, 4H, H-3,4-pyrr); 4.13, 4.20 (2 × m, 4H, H-5′); 5.38, 5.55 (2
Hexadecyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-
N-yl)ethylphosphonate (12i). The mixture of dihydroxypyrrolidine
11a (0.36 g, 3.45 mmol) and phosphonate 10d (1.38 g, 2.3 mmol) in
nBuOH (20 mL) was stirred at 100 °C overnight. The solvent was
removed and isopropylidene intermediate was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate. This compound was without further characterization dissolved
in 0.5 M methanolic HCl (50 mL), and the mixture was stirred at rt for
4 h. The reaction mixture was concentrated in vacuo and coevaporated
with ethanol (3 × 40 mL). The desired product was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate in 39% overall yield (590 mg, 0.89 mmol) as a white
amorphous solid. A mixture of two diastereomers ∼1:1. 1H NMR
(600.1 MHz, CD3OD): 0.90 (m, 6H, CH3(CH2)15); 1.24−1.43 (m,
5 2 H , C H 3 ( C H 2 ) 1 3 C H 2 C H 2 O ) ; 1 . 6 8 ( m , 4 H ,
CH3(CH2)13CH2CH2O); 2.06−2.18 (m, 4H, CH2P); 2.55 (m, 4H,
H-2b,5b-pyrr); 2.73−2.86 (m, 4H, CH2N); 2.99 (m, 4H, H-2a,5a-
pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.05−4.12 (m, 4H,
CH3(CH2)13CH2CH2O); 4.12−4.16 (m, 4H, H-3′,4′); 4.198, 4.200
(2 × dd, 2 × 1H, J2′,3′ = 5.1, J2′,1′ = 4.1, H-2′); 4.24 (ddd, 1H, Jgem
11.6, JH,P = 6.5, J5′b,4′ = 4.2, H-5′b); 4.28 (ddd, 1H, Jgem = 11.6, JH,P
7.2, J5′b,4′ = 2.4, H-5′b); 4.30 (ddd, 1H, Jgem = 11.6, JH,P = 5.2, J5′a,4′
=
=
=
× bs, 2 × 2H, OH-2′,3′); 5.67, 5.68 (2 × dd, 2 × 1H, J5,6 = 8.1, J5,NH
=
2.6, H-5); 5.76 (d, 2H, J1′,2′ = 5.2, H-1′); 5.83 (bs, 4H, OH-3,4-pyrr);
7.64, 7.66 (2 × d, 2 × 1H, J6,5 = 8.1, H-6); 10.61 (bs, 2H, NH-pyrr);
11.39 (s, 2H, NH-3). 13C NMR (150.9 MHz, DMSO-d6): 16.49 (d,
JC,P = 5.1, CH3CH2); 22.05, 22.10 (d, JC,P = 139.2, CH2P); 50.90
(CH2N); 58.89, 58.95, 59.07 (CH2-2,5-pyrr); 66.37, 66.44 (d, JC,P
6.2, CH2CH3); 65.51, 65.56 (d, JC,P = 6.5, CH2-5′); 69.67, 69.73 (CH-
3′); 72.81, 72.85 (CH-2′); 74.41, 74.57 (CH-3,4-pyrr); 82.23 (d, JC,P
2.7, H-5′a); 4.34 (ddd, 1H, Jgem = 11.6, JH,P = 6.6, J5′a,4′ = 2.8, H-5′a);
5.73, 5.74 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H,
J1′,2′ = 4.1, H-1′); 7.71, 7.74 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR
(150.9 MHz, CD3 OD): 14.46 (CH3 (CH2 )1 5 ); 23.75
(CH3(CH2)13CH2CH2O); 25.14, 25.19 (d, JC,P = 139.3, CH2P);
26.65, 30.27, 30.28, 30.50, 30.68, 30.69, 30.72, 30.73, 30.78, 30.81
=
=
5.2, CH-4′); 88.84, 88.94 (CH-1′); 102.36, 102.37 (CH-5); 141.21,
141.23 (CH-6); 150.95, 150.97 (C-2); 163.44 (C-4). 31P NMR (202.3
MHz, DMSO-d6): 27.31, 27.58. HR-ESI: C17H29O10N3P (M + H)+
calcd 466.1585; found 466.1585.
(CH3(CH2)13CH2CH2O); 31.55, 31.57 (d, JC,P = 6.0,
CH3(CH2)13CH2CH2O); 33.09 (CH3(CH2)13CH2CH2O); 50.55,
50.59 (d, JC,P = 1.0, CH2N); 61.02, 61.05 (CH2-2,5-pyrr); 66.34,
66.42 (d, JC,P = 6.3, CH2-5′); 66.60, 67.63 (d, JC,P = 6.8,
CH3(CH2)13CH2CH2O); 70.81, 70.89 (CH-3′); 74.96, 75.02 (CH-
2′); 78.65, 78.69 (CH-3,4-pyrr); 83.64, 83.66 (d, JC,P = 6.6, CH-4′);
91.58, 91.76 (CH-1′); 102.94, 102.98 (CH-5); 142.57, 142.58 (CH-6);
152.18, 152.22 (C-2); 166.04 (C-4). 31P NMR (202.3 MHz, CD3OD):
32.33, 32.63. HR-ESI: C31H57O10N3P (M + H)+ calcd 662.37761,
found 662.37759.
Hexadecyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-
N-yl)ethylphosphonate (12i-P1). The title compound was ob-
tained by preparative HPLC from 12i using Waters AutoPurification
system with 2545 quarternary gradient module and 3100 single
quadrupole mass detector using LUNA C18, column (Phenomenex,
Tetradecyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-
1-N-yl)ethylphosphonate (12h). The mixture of dihydroxypyrro-
lidine 11a (0.42 g, 4.1 mmol) and phosphonate 10c (1.17 g, 2.05
mmol) in nBuOH (30 mL) was stirred at 100 °C overnight. The
solvent was removed, and the isopropylidene intermediate was
obtained by flash chromatography on silica gel using linear gradient
of H1 in ethyl acetate. This compound was without further
characterization dissolved in 0.5 M methanolic HCl (50 mL), and
the mixture was stirred at rt for 4 h. The reaction mixture was
concentrated in vacuo and coevaporated with ethanol (3 × 40 mL).
The desired product was obtained by flash chromatography on silica
7895
dx.doi.org/10.1021/jm2009343|J. Med. Chem. 2011, 54, 7884−7898

Journal of Medicinal Chemistry
Article
250 mm × 21.2 mm, 5 μm) at flow rate 10 mL/min using gradient: A,
50 mM NH4HCO3 in 50% aq CH3CN; C, CH3CN; A→A/10 min,
A→B/50 min. 1H NMR (600.1 MHz, CD3OD): 0.90 (t, 3H, Jvic = 7.2,
CH3(CH2)15); 1.26−1.41 (m, 26H, CH3(CH2)13CH2CH2O); 1.69
(m, 2H, CH3(CH2)13CH2CH2O); 2.20 (m, 2H, CH2P); 2.80 (bdd,
2H, Jgem = 10.5, Jvic = 1.9, H-2b,5b-pyrr); 3.00 (bm, 2H, CH2N); 3.19
(bdd, 2H, Jgem = 10.9, Jvic = 4.5, H-2a,5a-pyrr); 4.05−4.16 (m, 6H, H-
3′,4′, H-3,4-pyrr, CH3(CH2)13CH2CH2O); 4.21 (dd, 1H, J2′,3′ = 5.1,
J2′,1′ = 4.2, H-2′); 4.26 (ddd, 1H, Jgem = 11.3, JH,P = 7.0, J5′b,4′ = 4.7, H-
5′b); 4.36 (ddd, 1H, Jgem = 11.3, JH,P = 6.7, J5′a,4′ = 2.6, H-5′a); 5.74 (d,
(150.9 MHz, CD3 OD): 14.47 (CH3 (CH2 )1 7 ); 23.75
(CH3(CH2)15CH2CH2O); 25.13, 25.17 (d, JC,P = 139.3, CH2P);
26.65, 30.28, 30.29, 30.49, 30.68, 30.69, 30.72, 30.73, 30.78, 30.79,
30.80 (CH3(CH2)15CH2CH2O); 31.55, 31.57 (d, JC,P = 6.0,
CH3(CH2)15CH2CH2O); 33.09 (CH3(CH2)15CH2CH2O); 50.55,
50.57 (d, JC,P = 1.1, CH2N); 61.01, 61.04 (CH2-2,5-pyrr); 66.34,
66.42 (d, JC,P = 6.3, CH2-5′); 66.60, 67.62 (d, JC,P = 6.8,
CH3(CH2)15CH2CH2O); 70.80, 70.88 (CH-3′); 74.96, 75.02 (CH-
2′); 78.63, 78.68 (CH-3,4-pyrr); 83.63, 83.65 (d, JC,P = 6.7, CH-4′);
91.56, 91.74 (CH-1′); 102.95, 102.98 (CH-5); 142.55, 142.57 (CH-6);
152.18, 152.22 (C-2); 166.03 (C-4). 31P NMR (202.3 MHz, CD3OD):
32.12, 32.43. HR-ESI: C33H61O10N3P (M + H)+ calcd 690.40891,
found 690.40891.
1H, J5,6 = 8.1, H-5); 5.83 (d, 1H, J1′,2′ = 4.2, H-1′); 7.73 (d, 1H, J6,5
=
8.1, H-6). 13C NMR (150.9 MHz, CD3OD): 14.46 (CH3(CH2)15);
23.76 (CH3(CH2)13CH2CH2O); 24.66 (d, JC,P = 140.2, CH2P); 26.64,
3 0 . 2 9 , 3 0 . 5 0 , 3 0 . 6 9 , 3 0 . 7 4 , 3 0 . 7 8 , 3 0 . 7 9 , 3 0 . 8 2
Icosanyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-
N-yl)ethylphosphonate (12k). The mixture of dihydroxypyrroli-
dine 11a (0.26 g, 2.52 mmol) and phosphonate 10f (1.1 g, 1.68 mmol)
in nBuOH (17 mL) was stirred at 100 °C overnight. The solvent was
removed, and the isopropylidene intermediate was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate. This compound was without further characterization dissolved
in 0.5 M methanolic HCl (50 mL), and the mixture was stirred at rt for
4 h. The reaction mixture was concentrated in vacuo and coevaporated
with ethanol (3 × 40 mL). The desired product was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate in 70% overall yield (840 mg, 1.17 mmol) as a white
amorphous solid. A mixture of two diastereomers ∼6:5. 1H NMR
(600.1 MHz, CD3OD): 0.90 (m, 6H, CH3(CH2)19); 1.25−1.43 (m,
6 8 H , C H 3 ( C H 2 ) 1 7 C H 2 C H 2 O ) ; 1 . 6 9 ( m , 4 H ,
CH3(CH2)17CH2CH2O); 2.06−2.23 (m, 4H, CH2P); 2.68 (dd, 4H,
( C H 3 ( C H 2 ) 1 3 C H 2 C H 2 O ) ; 3 1 . 5 4 ( d , J C , P
=
6 . 0 ,
CH3(CH2)13CH2CH2O); 33.09 (CH3(CH2)13CH2CH2O); 51.04
(CH2N); 60.99 (CH2-2,5-pyrr); 66.66 (d, JC,P = 6.4, CH2-5′); 67.84
(d, JC,P = 6.8, CH3(CH2)13CH2CH2O); 70.86 (CH-3′); 74.84 (CH-2′);
77.87 (CH-3,4-pyrr); 83.55 (d, JC,P = 6.4, CH-4′); 91.96 (CH-1′);
102.98 (CH-5); 142.74 (CH-6); 152.19 (C-2); 166.03 (C-4). 31P
NMR (202.3 MHz, CD3OD): 31.27.
Hexadecyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-
N-yl)ethylphosphonate (12i-P2). The title compound was ob-
tained by preparative HPLC from 12i using Waters AutoPurification
system with 2545 quarternary gradient module and 3100 single
quadrupole mass detector using LUNA C18, column (Phenomenex,
250 mm × 21.2 mm, 5 μm) at flow rate 10 mL/min using gradient: A,
50 mM NH4HCO3 in 50% aq CH3CN; C, CH3CN; A→A/10 min,
A→B/50 min. 1H NMR (600.1 MHz, CD3OD): 0.90 (t, 3H, Jvic = 7.2,
CH3(CH2)15); 1.26−1.43 (m, 26H, CH3(CH2)13CH2CH2O); 1.69
(m, 2H, CH3(CH2)13CH2CH2O); 2.18 (m, 2H, CH2P); 2.72 (bd, 2H,
J
gem = 10.6, Jvic = 2.9, H-2b,5b-pyrr); 2.84−2.97 (m, 4H, CH2N); 3.10
(dd, 4H, Jgem = 10.6, Jvic = 5.0, H-2a,5a-pyrr); 4.07 (m, 4H, H-3,4-
pyrr); 4.08−4.13 (m, 4H, CH3(CH2)17CH2CH2O); 4.13−4.16 (m,
4H, H-3′,4′); 4.207, 4.210 (2 × dd, 2 × 1H, J2′,3′ = 5.0, J2′,1′ = 4.2, H-2′);
4.25 (ddd, 1H, Jgem = 11.6, JH,P = 6.8, J5′b,4′ = 4.4, H-5′b); 4.31 (m, 2H,
H-5′); 4.35 (ddd, 1H, Jgem = 11.6, JH,P = 6.6, J5′a,4′ = 2.7, H-5′a); 5.735,
J
gem = 10.5, H-2b,5b-pyrr); 2.94 (bm, 2H, CH2N); 3.13 (bdd, 2H, Jgem
= 10.5, Jvic = 5.0, H-2a,5a-pyrr); 4.05−4.15 (m, 6H, H-3′,4′, H-3,4-pyrr,
CH3(CH2)13CH2CH2O);4.21 (dd, 1H, J2′,3′ = 5.0, J2′,1′ = 4.1, H-2′);
4.27−4.34 (m, 2H, H-5′); 5.73 (d, 1H, J5,6 = 8.1, H-5); 5.84 (d, 1H,
J1′,2′ = 4.1, H-1′); 7.70 (d, 1H, J6,5 = 8.1, H-6). 13C NMR (150.9 MHz,
CD3OD): 14.46 (CH3(CH2)15); 23.76 (CH3(CH2)13CH2CH2O);
24.78 (d, JC,P = 139.8, CH2P); 26.64, 30.28, 30.50, 30.68, 30.69,
5.743 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.84, 5.85 (2 × d, 2 × 1H, J1′,2′
=
4.2, H-1′); 7.71, 7.74 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR (150.9
M H z , C D 3 O D ) : 1 4 . 4 6 ( C H 3 ( C H 2 ) 1 9 ) ; 2 3 . 7 5
(CH3(CH2)17CH2CH2O); 24.85, 24.91 (d, JC,P = 139.8, CH2P);
26.64, 30.28, 30.29, 30.49, 30.69, 30.73, 30.74, 30.77, 30.79, 30.80
30.73, 30.79, 30.81, 30.82 (CH3(CH2)13CH2CH2O); 31.56 (d, JC,P
=
6.0, CH3(CH2)13CH2CH2O); 33.10 (CH3(CH2)13CH2CH2O); 50.94
(CH2N); 60.99 (CH2-2,5-pyrr); 66.59 (d, JC,P = 6.1, CH2-5′); 67.71 (d,
JC,P = 6.8, CH3(CH2)13CH2CH2O); 70.81 (CH-3′); 74.93 (CH-2′);
78.06 (CH-3,4-pyrr); 83.59 (d, JC,P = 6.6, CH-4′); 92.02 (CH-1′);
102.96 (CH-5); 142.69 (CH-6); 152.17 (C-2); 166.04 (C-4). 31P
NMR (202.3 MHz, CD3OD): 32.03.
(CH3(CH2)17CH2CH2O); 31.55, 31.57 (d, JC,P
CH3(CH2)17CH2CH2O); 33.09 (CH3(CH2)17CH2CH2O); 50.81,
50.86 (CH2N); 60.98, 61.02 (CH2-2,5-pyrr); 66.52, 66.54 (d, JC,P
= 6.0,
=
6.1, CH2-5′); 67.69, 67.73 (d, JC,P = 7.1, CH3(CH2)17CH2CH2O);
70.80, 70.87 (CH-3′); 74.90, 74.95 (CH-2′); 78.19, 78.26 (CH-3,4-
pyrr); 83.60 (d, JC,P = 6.5, CH-4′); 91.75, 91.93 (CH-1′); 102.97,
102.99 (CH-5); 142.65 (CH-6); 152.17, 152.20 (C-2); 166.03 (C-4).
31P NMR (202.3 MHz, CD3OD): 31.29, 31.57. HR-ESI:
Octadecyl Uridin-5′-yl 2-([3R,4R]-3,4-Dihydroxypyrrolidin-1-
N-yl)ethylphosphonate (12j). The mixture of dihydroxypyrrolidine
11a (0.3 g, 2.94 mmol) and phosphonate 10e (1.23 g, 1.96 mmol) in
nBuOH (20 mL) was stirred at 100 °C overnight. The solvent was
removed, and the isopropylidene intermediate was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate. This compound was without further characterization dissolved
in 0.5 M methanolic HCl (50 mL), and the mixture was stirred at rt for
4 h. The reaction mixture was concentrated in vacuo and coevaporated
with ethanol (3 × 40 mL). The desired product was obtained by flash
chromatography on silica gel using linear gradient of H1 in ethyl
acetate in 68% overall yield (920 mg, 1.33 mmol) as a white
amorphous solid. A mixture of two diastereomers ∼6:5. 1H NMR
(600.1 MHz, CD3OD): 0.90 (m, 6H, CH3(CH2)17); 1.25−1.43 (m,
6 0 H , C H 3 ( C H 2 ) 1 5 C H 2 C H 2 O ) ; 1 . 6 8 ( m , 4 H ,
CH3(CH2)15CH2CH2O); 2.06−2.19 (m, 4H, CH2P); 2.55 (m, 4H,
H-2b,5b-pyrr); 2.73−2.86 (m, 4H, CH2N); 2.99 (m, 4H, H-2a,5a-
pyrr); 4.04 (m, 4H, H-3,4-pyrr); 4.05−4.13 (m, 4H,
CH3(CH2)15CH2CH2O); 4.13−4.16 (m, 4H, H-3′,4′); 4.199, 4.201
C35H65O10N3P (M + H)+ calcd 718.44021, found 718.44030.
Hexadecyl Uridin-5′-yl 2-([3R,5S]-3-Hydroxy-5-
(hydroxymethyl)pyrrolidin-1-N-yl)ethylphosphonate (12l).
The mixture of dihydroxypyrrolidine 11d (0.12 g, 1.0 mmol) and
phosphonate 10d (0.3 g, 0.5 mmol) in nBuOH (5 mL) was stirred at
95 °C overnight. The solvent was removed, and the isopropylidene
intermediate was obtained by flash chromatography on silica gel using
linear gradient of H1 in ethyl acetate. This compound was without
further characterization dissolved in 0.5 M methanolic HCl (50 mL)
and the mixture was stirred at rt for 4 h. The reaction mixture was
concentrated in vacuo and coevaporated with ethanol (3 × 40 mL).
The desired product was obtained by flash chromatography on silica
gel using linear gradient of H1 in ethyl acetate in 60% overall yield
(200 mg, 0.3 mmol) as a white amorphous solid. A mixture of
1
diastereomers ∼6:5. H NMR (500.0 MHz, CD3OD): 0.90 (m, 6H,
CH3(CH2)15); 1.24−1.43 (m, 52H, CH3(CH2)13CH2CH2O); 1.70
(m, 4H, CH3(CH2)13CH2CH2O); 2.03, 2.17 (2 × m, 2 × 1H, H-4-
pyrr); 2.44−2.58 (m, 4H, CH2P); 3.30 (m, 2H, H-2b-pyrr); 3.53 (m,
2H, CHaHbN); 3.70 (m, 2H, H-2a-pyrr); 3.73 (m, 2H, CHaHbOH);
3.78 (m, 2H, CHaHbN); 3.93 (m, 2H, CHaHbOH); 3.96 (m, 2H, H-5-
pyrr); 4.07−4.18 (m, 8H, H-3′,4′, CH3(CH2)13CH2CH2O); 4.24 (m,
2H, H-2′); 4.28−4.42 (m, 4H, H-5′); 4.53 (m, 2H, H-3-pyrr); 5.75 (d,
(2 × dd, 2 × 1H, J2′,3′ = 4.8, J2′,1′ = 4.2, H-2′); 4.24 (ddd, 1H, Jgem
11.6, JH,P = 6.7, J5′b,4′ = 4.2, H-5′b); 4.28 (ddd, 1H, Jgem = 11.6, JH,P
7.4, J5′b,4′ = 2.5, H-5′b); 4.30 (ddd, 1H, Jgem = 11.6, JH,P = 5.2, J5′a,4′
=
=
=
2.7, H-5′a); 4.34 (ddd, 1H, Jgem = 11.6, JH,P = 6.5, J5′a,4′ = 2.7, H-5′a);
5.735, 5.745 (2 × d, 2 × 1H, J5,6 = 8.1, H-5); 5.85, 5.86 (2 × d, 2 × 1H,
J1′,2′ = 4.2, H-1′); 7.71, 7.74 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR
7896
dx.doi.org/10.1021/jm2009343|J. Med. Chem. 2011, 54, 7884−7898

Journal of Medicinal Chemistry
Article
2H, J5,6 = 8.1, H-5); 5.81, 5.83 (2 × d, 2 × 1H, J1′,2′ = 4.0, H-1′); 7.69,
7.71 (2 × d, 2 × 1H, J6,5 = 8.1, H-6). 13C NMR (125.7 MHz,
CD3OD): 14.44 (CH3(CH2)15); 23.17, 23.22 (d, JC,P = 140.5, CH2P);
23.73 (CH3(CH2)13CH2CH2O); 26.59, 30.28, 30.29, 30.47, 30.66,
30.73, 30.76, 30.79 (CH3(CH2)13CH2CH2O); 31.51 (d, JC,P = 5.9,
CH3(CH2)13CH2CH2O); 33.07 (CH3(CH2)13CH2CH2O); 37.35
(CH2-3); 52.72, 52.76 (CH2N); 60.82 (CH2OH); 62.25, 62.28
(CH2-2-pyrr); 67.18, 67.38 (d, JC,P = 6.3, CH2-5′); 68.14, 68.37 (d, JC,P
= 6.8, CH3(CH2)13CH2CH2O); 70.14 (CH-3-pyrr); 70.40, 70.49
(CH-5-pyrr); 70.75, 70.79 (CH-3′); 74.66, 74.71 (CH-2′); 83.39,
83.45 (d, JC,P = 6.4, CH-4′); 92.40, 92.46 (CH-1′); 103.08, 103.12
(CH-5); 142.94, 142.97 (CH-6); 152.17, 152.19 (C-2); 166.00 (C-4).
31P NMR (162.0 MHz, CD3OD): 27.81, 28.26. HR-ESI:
C32H59O10N3P (M + H)+ calcd 676.39326, found 676.39306.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Cell viability, cytotoxicity and apoptosis of erythroid progenitor
cells after exposure to selected compounds and IR data of
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(12) McDonald, L. A.; Barbieri, L. R.; Carter, G. T.; Lenoy, E.;
Lotvin, J.; Petersen, P. J.; Siegel, M. M.; Singh, G.; Williamson, R. T.
Structures of the muraymycins, novel peptidoglycan biosynthesis
inhibitors. J. Am. Chem. Soc. 2002, 124, 10260−10261.
(13) Bugg, T. D. H.; Lloyd, A. J.; Roper, D. I. Phospho-
MurNAcpentapeptide translocase (MraY) as a target for antibacterial
agents and antibacterial proteins. Infect. Dis. Drug Targets 2006, 6, 85−
106.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: rejman@uochb.cas.cz, Phone: (+420220183371)
(D.R.); E-mail: krasny@biomed.cas.cz , Phone:
(+420241063208) (L.K.).
(14) Kimura, K.; Bugg, T. D. H. Recent advances in antimicrobial
nucleoside antibiotics targeting cell wall biosynthesis. Nat. Prod. Rep.
2003, 20, 252−273.
(15) Bouhss, A.; Mengin-Lecreulx, D.; Le Beller, D.; Van Heijenoort,
J. Topological analysis of the MraY protein catalyzing the first
membrane step of peptidoglycan synthesis. Mol. Microbiol. 1999, 34,
576−585.
(16) Bouhss, A.; Trunkfield, A. E.; Bugg, T. D.; Mengin-Lecreulx, D.
The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS
Microbiol. Rev. 2008, 32, 208−33.
ACKNOWLEDGMENTS
■
Support by grant nos. 2B06065 (Ministry of Education, Youth
and Sports of the Czech Republic and Trios), LC06077
(Ministry of Education, Youth and Sports of the Czech
Republic), 204/09/0583 and P302/11/0855 (Czech Science
Foundation), and Institutional Research Concepts
AV0Z50200510 and Z40550506 is gratefully acknowledged.
The authors are indebted to the staff of the Department of
Organic Analysis for the measurements of HR-MS.
(17) Al-Dabbagh, B.; Henry, X.; El Ghachi, M.; Auger, G.; Blanot,
D.; Parquet, C.; Mengin-Lecreulx, D.; Bouhss, A. Active site mapping
of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine
1-phosphate transferase superfamily, catalyzing the first membrane
step of peptidoglycan biosynthesis. Biochemistry 2008, 47, 8919−8928.
(18) Tanino, T.; Ichikawa, S.; Al-Dabbagh, B.; Bouhss, A.; Oyama,
H.; Matsuda, A. Synthesis and Biological Evaluation of Muraymycin
Analogues Active against Anti-Drug-Resistant Bacteria. Med. Chem.
Lett. 2010, 1, 258−262.
(19) Hostetler, K. Y.; Beadle, J. R.; Hornbuckle, W. E.; Bellezza, C.
A.; Ilia, A.; Tochkov, I. A.; Cote, P. J.; Gerin, J. L.; Korba, B. E.;
Tennant, B. C. Antiviral Activities of Oral 1-O-Hexadecylpropanediol-
3-phosphoacyclovir and Acyclovir in Woodchucks with Chronic
Woodchuck Hepatitis Virus Infection. Antimicrob. Agents Chemother.
2000, 44 (7), 1964−1969.
(20) Lefebvre, I.; Perigaud, C.; Pompon, A.; Aubertin, A.-M.;
Girardet, J.-L.; Kirn, A.; Gosselin, G.; Imbach, J. L. Mononucleoside
phosphotriester derivatives with S-acyl-2-thioethyl bioreversible
phosphate-protecting groups: intracellular delivery of 3′-azido-2′,3′-
dideoxythymidine 5′-monophosphate. J. Med. Chem. 1995, 38, 3941−
3950.
(21) Boutevin, B.; Hervaud, Y.; Jeanmarie, T.; Boulahna, A.; Elasri,
M. Monodealkylation Des Esters Phosphoniques Synthese De
Monosels Et De Monoacides Phosphoniques. Phosphorus Sulfur
2001, 174, 1−14.
ABBREVIATIONS USED
■
C, cytosine; CBz, 4-N-benzoylcytosine; CPZ, caprazamycin;
CWS, cyst wall synthase; ED50, effective dose; HM, hydro-
phobic module; HPLC, high performance liquid chromatog-
raphy; IM, an iminosugar module; LM, linker module; LPM,
liposidomycin; LPPO, lipophosphonoxin; MBC, minimum
bactericidal concentration; MIC, minimum inhibitory concen-
tration; NM, nucleoside module; [poly(GalNAc)], poly β-1−3-
linked N-acetylgalactosamine; SATE, pivaloylthioethyl; TPSCl,
triisopropylbenzensulfonylchloride; U, uracil; UDP-GalNAc,
uridinediphosphate-N-acetyl-alpha-D-galactosamine
REFERENCES
■
(1) Winn, M.; Goss, R. J. M.; Kimurab, K.; Bugg, T. D. H.
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