A short synthetic pathway via three-component coupling reaction to tamiphosphor possessing anti-influenza activity

Article abstract of DOI:10.1016/j.tet.2014.11.062

Three-component coupling reaction of (pent-3-oxy)acetaldehyde, (Z)-N-(2-nitrovinyl)acetamide, and tetraethyl 1,1-diylbis(phosphonate) is performed in a one-pot operation, followed by reduction of the nitro group and hydrolysis of the phosphonate ester, to afford 8.7% overall yield of tamiphosphor as a potent neuraminidase inhibitor with IC₅₀ and EC₅₀ values of 2.5 and 31.5 nM against wild-type H1N1 influenza virus. The tamiphosphor (5R)-epimer is a less active anti-influenza agent with IC₅₀ and EC₅₀ values of 39 and 117 nM.

Full text of DOI:10.1016/j.tet.2014.11.062

Tetrahedron 71 (2015) 266e270
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A short synthetic pathway via three-component coupling reaction to
tamiphosphor possessing anti-influenza activity
,b,
Yi-Wei Lo a, Jim-Min Fang a
*
a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
b The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2014
Received in revised form 17 November 2014
Accepted 23 November 2014
Available online 28 November 2014
Three-component coupling reaction of (pent-3-oxy)acetaldehyde, (Z)-N-(2-nitrovinyl)acetamide, and
tetraethyl 1,1-diylbis(phosphonate) is performed in a one-pot operation, followed by reduction of the
nitro group and hydrolysis of the phosphonate ester, to afford 8.7% overall yield of tamiphosphor as
a potent neuraminidase inhibitor with IC50 and EC50 values of 2.5 and 31.5 nM against wild-type H1N1
influenza virus. The tamiphosphor (5R)-epimer is a less active anti-influenza agent with IC50 and EC50
values of 39 and 117 nM.
Keywords:
Influenza
Ó 2014 Published by Elsevier Ltd.
Neuraminidase inhibitor
Michael reaction
Wittig reaction
Organocatalyst
1. Introduction
possess remarkable anti-influenza activities based on the enzy-
matic, cell, and animal assays.18,20e23 Moreover, the guanidine an-
Seasonal influenza is a contagious respiratory illness, and new
types of influenza viruses may emerge to cause global infections.
The threat of influenza is even more serious due to the cross-species
transmission of avian influenza viruses, such as H5N1 and H7N9, to
humans in the recent years. Influenza neuraminidase (NA) is a viral
surface glycoprotein that is responsible for releasing progeny in-
fluenza viruses by cleaving the linkage between the viral hemag-
glutinin and the sialo-receptor on the host cell.1,2 Inhibition of NA
has been utilized as averyeffective strategy for development of anti-
influenza drugs.3 TamifluÔ, as the phosphate salt of oseltamivir (OS,
1a),4e7 is an orally available anti-influenza drug, which is hydro-
lyzed by endogenous esterases to generate the active ingredient
oseltamivir carboxylic acid (OC, 1b) as a potent NA inhibitor.6,8e11
However, tamiflu-resistant influenza viruses such as the clinically
relevant H275Y strain of H1N1 virus have emerged over past
years.12e16 Thus, many scientists have exerted great effort to develop
new anti-influenza drugs that are also active to H275Y virus.17e19
We have previously explored tamiphosphor (TP, 2),17 a phos-
phonate congener of OC, as a potent NA inhibitor against various
avian and human influenza viruses. Strecher’s and our research
teams have further demonstrated that the TP monoesters also
alogs of TP and its monoesters are potent NA inhibitors against
H275Y virus with the IC50 values in nanomolar range.17,18
As delineated in Fig. 1, only a few methods have been utilized in
the syntheses of TP and its derivatives. In the first synthesis of TP
(path A),17
D-xylose (3) is utilized as a starting material. After
transformation of the 3 -OH group to 3 -NH2 group, a diphos-
b
a
phorylmethyl substituent is implanted to the C-5 position for the
subsequent intramolecular Wittig reaction (in the Hor-
nereWadswortheEmmons (HWE) variant)24,25 to establish the core
structure of polysubstituted cyclohexene ring. The pent-3-oxy and
amino substituents are then introduced to furnish w10% yield of the
final product of TP in overall 19 synthetic steps. The synthesis of TP
was also achieved by using N-acetyl-D-glucosamine as another
chiral-pool molecule with a preset acetamido group in the desired
absolute configuration.26 In path B,20,27,28 the N-Boc-protected OC
(1c) is subjected to photochemical HunsdieckereBarton hal-
odecarboxylation.29 A palladium-catalyzed phosphonylation of the
halocyclohexene product 6 with diethyl phosphite is carried out to
provide TP, afterconcomitant removal of the ethyl and Boc groups by
treatment with Me3SiBr. The pivotal intermediate 6 can also be
prepared from (1S,2S)-3-bromocyclohex-3,5-diene-1,2-diol (7),
a fermentation product of bromobenzene,30,31 by regio- and ster-
eoselective introduction of pent-3-oxy and acetamide groups along
with proper manipulation of the desired functional groups (path
C).27 Though the synthesis via paths B and C can provide TP in fewer
* Corresponding author. Tel.: þ886 2 33661663; fax: þ886 2 23637812; e-mail
address: jmfang@ntu.edu.tw (J.-M. Fang).
http://dx.doi.org/10.1016/j.tet.2014.11.062
0040-4020/Ó 2014 Published by Elsevier Ltd.

Y.-W. Lo, J.-M. Fang / Tetrahedron 71 (2015) 266e270
267
Fig. 1. Methods for the synthesis of tamiphosphor (2). (Path A) Carbohydrate molecule, e.g., D-xylose (3), is utilized as a chiral pool to construct the core structure of polysubstituted
cyclohexene ring via an intramolecular HornereWadswortheEmmons reaction. (Path B) The carboxylic acid in the oseltamivir derivative 1c undergoes a photochemical Hunds-
diekereBarton halodecarboxylation to give a halogen compound 6, which is converted to the phosphonate ester via a palladium-catalyzed coupling reaction. (Path C) The fer-
mentation product 7 of bromobenzene is sequentially processed to the common intermediate 6. (Path D) In this study, a one-pot three-component coupling reaction of 2-(pent-3-
oxy)acetaldehyde (9), (Z)-N-(2-nitrovinyl)acetamide (12), and 1,1-diphosphonylethene (14) is devised to construct the polysubstituted cyclohexene-1-phosphonate (15) for elab-
oration to tamiphosphor.
steps, some problems such as the photochemical of 1c and microbial
preparation of chiral diol 7 in large scales remain to be solved.
To develop TP and its derivatives for the therapeutic applications,
it is needed to devise practical and large-scale synthetic methods.
Inspired by the expedient synthesis of oseltamivir that uses one-pot
multiple-component coupling reactions to construct the key in-
termediate of polysubstituted cyclohexene-1-carboxylate,32e36 we
aimed to investigate whether such straightforward methodology
could be applied to synthesize TP? Path D shows our synthetic de-
sign to prepare polysubstituted cyclohexene-1-phosphonate 15
in one-pot operation by a sequence of reactions comprising an
organocatalytic condensation of 2-(pent-3-oxy)acetaldehyde (9)
with (Z)-N-(2-nitrovinyl)acetamide (12), a HenryeMichael reaction
totrap the anion of the nitro intermediate 13 with tetraethyl ethene-
1,1-diylbis(phosphonate) (14), and an intramolecular HWE reaction.
In comparison with ethyl 2-(diethoxyphosphoryl)acrylate, H2C]
C(CO2Et)PO(OEt)2, used in oseltamivir synthesis,32e36 1,1-
diphosphorylethene 14 would be a less reactive electrophile in Mi-
chael reaction because the sp2-hybridized CeC double bond is not
fully conjugated with the phosphoryl groups in the tetrahedral
configuration.
product 13, predominantly in the threo-isomer (Scheme 1).35 The
threo-isomer showed the CHO group at dH 9.61, whereas the erythro
isomer displayed the aldehyde proton at dH 9.57. We then carried on
the reaction of 13 (as a mixture of threo and erythro isomers) with
bisphosphorylethene 14 in the presence of Cs2CO3. The 1H NMR
spectrum of the crude product showed a vinyl proton (H-2) at dH
6.60 with a large coupling constant (JHeP¼21.6 Hz),17 indicating that
cyclohexene-1-phosphonate 15 was formed by tandem Hen-
ryeMichael reaction and intramolecular HWE reaction. The elimi-
nation species of phosphoric acid diethyl ester, (OEt)2P(O)(OH)
generated from HWE reaction, exhibited the phosphorus(V) signal
at dP¼0 in the 31P NMR spectrum. However, an appreciable amount
of the side product 16, showing a sodiated molecular ion [MþNa]þ
at m/z 729.2616, was also observed. Compound 16 was presumably
derived from a second HenryeMichael reaction of 14 with 15.33 The
NOESY correlation between H-4 (at dH 4.58) and the methylene
protons (H-10 at dH 2.69) of C5 substituent was consistent with the
attack of 14 at the 15-anion from the less hindered face. There are
three phosphorus signals occurring at dP 17.2, 23.0, and 23.4 in
compound 16. The characteristic vinyl proton (H-2) appeared at dH
6.60 (JHeP¼22 Hz), and the methine proton (H-20) adjacent to the
phosphoryl groups exhibited at dH 2.57 with a large HeP coupling
constant of 25.4 Hz. The NOESY correlation between H-3 (at dH
3.84) and NH (at dH 6.31) of C4 acetamide group revealed the trans-
relationship at C3 and C4.
2. Results and discussion
We first validated the asymmetric Michael reaction of aldehyde
9 with nitroalkene 12 using (S)-diphenylprolinol trimethylsilyl
ether (11a) as the chiral organocatalyst to furnish the addition
Our attempts to reduce the formation of 16, e.g., by conducting
the reaction at low temperature (ꢀ30 ꢁC) or using less amounts of

268
Y.-W. Lo, J.-M. Fang / Tetrahedron 71 (2015) 266e270
amine product 17 was obtained by acidebase extraction, and iso-
lated as the Boc-protected compound 18 by subsequent treatment
with Boc2O. The two epimers were then successfully separated by
flash chromatography to give (5S)-18 and (5R)-18, respectively, in
10.1% and 13.6% isolated yields (calculated from nitroalkene 12).
According to our previously reported procedure,27 (5S)-18 was
treated with bromotrimethylsilane to afford tamiphosphor 2 hav-
ing the (5S)-configuration in 86% yield by concomitant hydrolysis of
the phosphonate group and removal of Boc group. The epimer (5R)-
2 was obtained from (5R)-18 by a similar procedure.
The NA inhibitory activities were measured by a fluorescence
assay using 2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid
(MUNANA) as the substrate, while the anti-influenza activities
were evaluated by the cytopathic effect of H1N1 virus (A/WSN/
1933) on MadineDarby canine kidney (MDCK) cells. Tamiphosphor
was a potent anti-influenza agent (IC50¼2.5 nM and EC50¼31.5 nM),
whereas its epimer (5R)-2 showed inferior activities with the IC50
and EC50 values of 38.7 and 116.6 nM, respectively. This result was
in agreement with the molecular model of NAetamiphosphor
complex,17 in which the 5ꢀNHþ3 substituent of tamiphosphor
exhibited considerable electrostatic interactions with the acidic
residues of Glu119, Asp151, and Glu227 in the S2 site of NA. This
study thus provides direct evidence that the C-5 aminium group in
(S)-chirality is appropriate to bind with influenza NA. In compari-
son, a recent study revealed that the (4S)-epimer of OC completely
lost anti-influenza activity.37 The influence of wrong stereo-
disposition in the C4-acetamido group appeared to be more drastic
than that in the C5-amino group for anti-influenza activity.
3. Conclusion
Starting from the chiral amine-promoted Michael reaction of
aldehyde 9 with nitroenamide 12, the sequential reactions com-
prising a second Michael addition with 1,1-diphosphorylethene 14,
an HWE reaction and an reversion of the side product 16 were
carried out by one-pot operation to give the (3,4,5-trisubstituted)
cyclohexene-1-phosphonate 15. After nitro reduction and pro-
tection of the resulting amine, the two diastereomers of 18 were
separated by flash chromatography, and then individually treated
with bromotrimethylsilane to afford tamiphosphor and its (5R)-
epimer. This synthetic protocol could be performed in open system
without rigorous exclusion of oxygen or moisture. The whole pro-
cess involved only one chromatographic separation of (5S)-18 and
(5R)-18. Thus, 8.7% overall yield (based on nitroenamide 12) of
tamiphosphor was obtained by a straightforward manner, and
upgraded to gram-scale synthesis. The starting materials 9, 12 and
14 were readily prepared from inexpensive reagents by known
methods. The chiral prolinol derivative was employed as an eco-
friendly organocatalyst to establish the desired absolute configu-
rations at the C-3 and C-4 positions. The side product 16 was ef-
fectively reverted by sodium ethoxide to the key intermediate 15.
Taken together, this method appears to be more appealing to
tamiphosphor synthesis than the previously reported meth-
ods.17,20,26e28 In this study, we also compare the IC50 and EC50
values of tamiphosphor with its epimer (5R)-2 to evaluate the in-
fluence of the disposition of 5-amino group in anti-influenza ac-
tivity in a quantitative sense.
Scheme 1. Synthesis of tamiphosphor (2) and its (5R)-epimer via multiple-component
reactions. Reagents and reaction conditions: (a) ClCH2CO2H (40 mol %), CH2Cl2, 0 ꢁC,
24 h; (b) Cs2CO3, 0 ꢁC, 24 h; (c) NaOEt, EtOH, 0 ꢁC, 7.5 h; (d) Zn, 2 M HCl(aq), EtOH, 25 ꢁC,
6 h; (e) Boc2O, Et3N, CH2Cl2, 25 ꢁC, 18 h; separation of (5S)-18 and (5R)-18 by flash
chromatography; (f) Me3SiBr, CH2Cl2, 25 ꢁC, 18 h; aqueous NH4HCO3; 86% yield. The
steps (a)e(c) were conducted in one-pot operation.
bisphosphonate 14, failed. However, we found that 16 underwent
a retro-Michael reaction on treatment with a strong base (e.g.,
NaOEt) to give the desired product 15 as a less polar compound
according to the TLC analysis. Compound 15 existed as an in-
separable mixture of two 5-epimers. The desired epimer, (5S)-15,
showed the H-5 and the methine proton on the pent-3-oxy group
at dH 3.80 and 3.31, respectively, whereas the corresponding pro-
tons in (5R)-15 occurred at dH 4.02 and 3.45. The released side
product 14 was readily scavenged by NaOEt, via an oxa-Michael
reaction, to avoid re-addition to 15.
In the one-pot process, the amine-promoted reaction of 9
(2 equiv) with 12 (1 equiv) gave an intermediate 13, as a mixture of
threo- and erythro-isomers, which was subsequently treated with
bisphosphonate 14 (1.5 equiv) and Cs2CO3 at 0 ꢁC for 24 h, followed
by addition of NaOEt (5 equiv) in EtOH solution for reversion of 16,
to accomplish the synthesis of 15 without contamination of 16. The
product 15 existed as a 1:1.2 mixture of two diastereomers (5S)-15
and (5R)-15 as shown by the 1H NMR spectrum. According to the
previous reports,32e36 we also attempted to perform the thia-
Michael reaction of cyclohexene-1-phosphonate 15 with p-tolue-
nethiol, so that the cyclohexene ring would be converted to a -
cyclohexane framework for better epimerization of (5R)-15 (in 4,5-
cis configuration) to the desired (5S)-15 (in 4,5-trans configura-
tion). However, the anticipated thia-Michael reaction of 15 could
not be realized by using p-toluenethiol and other thiols under
thermal or radical conditions, presumably because the cyclo-
hexene-1-phosphonate was a relatively weak Michael acceptor.
The nitro group in 15 (as a mixture of 5S/5R epimers, 1:1.2) was
selectively reduced by zinc powder in acidic conditions. The crude
4. Experimental section
4.1. General
All the reagents and solvents were reagent grade and used
without further purification unless otherwise specified. Reactions
were magnetically stirred and monitored by thin-layer chroma-
tography on silica gel using aqueous p-anisaldehyde as visualizing

Y.-W. Lo, J.-M. Fang / Tetrahedron 71 (2015) 266e270
269
agent. Silica gel (0.040e0.063 mm particle sizes) was used for
column chromatography. Flash chromatography was performed on
1025 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
6.57 (1H, d, J¼21.6 Hz), 5.90
(1H, d, J¼8.8 Hz), 5.10 (1H, d, J¼9.2 Hz), 4.09e3.98 (5H, m),
3.89e3.87 (1H, br s), 3.80e3.75 (1H, m), 3.30 (1H, m), 2.62e2.55
(1H, m), 2.22e2.15 (1H, m), 1.95 (3H, s), 1.50e1.44 (4H, m), 1.39 (9H,
s),1.30e1.27 (6H, m), 0.87e0.79 (6H, m); 13C NMR (150 MHz, CDCl3)
silica gel of 60e200 mm particle size. Molecular sieves were acti-
vated under high vacuum at 220 ꢁC over 6 h.
Melting points were recorded on a Yanaco or Electrothermal
MEL-TEMP 1101D apparatus in open capillaries and are not cor-
rected. Optical rotations were measured on digital polarimeter of
d
170.9, 156.3,141.5, 127.4 (C-1, d, JCeP¼181 Hz), 82.1, 79.8, 76.0, 62.1,
62.0, 54.5, 49.1, 31.2, 28.3 (3ꢂ), 26.0, 25.5, 23.3, 16.3, 16.4, 9.5, 9.1;
Japan JASCO Co. DIP-1000. [
a]
values are given in units of
31P NMR (162 MHz, CDCl3)
d 17.99; HRMS (ESI): calcd for
D
10ꢀ1 deg cm2 ꢀ1. Infrared (IR) spectra were recorded on Nicolet
g
C
22H42N2O7P: 477.2730, found: m/z 477.2545 [MþH]þ.
Magna 550-II or Thermo Nicolet 380 FT-IR spectrometers. Nuclear
magnetic resonance (NMR) spectra were obtained on Varian Unity
Plus-400 (400 MHz) or Bruker Avance-400 (400 MHz) spectrom-
4.2.2. Compound (5R)-18. C22H41N2O7P; pale yellowoil; [
a
]
20 ꢀ68.8
D
(c 1.0, CHCl3); IR (film) 3297, 2978, 1687, 1536, 1168, 1033 cmꢀ1; 1H
eter. Chemical shifts (
d
) are given in parts per million (ppm) relative
NMR (400 MHz, CD3OD)
d
6.60 (1H, d, J¼22 Hz), 4.2 (1H, br s),
to dH 7.24/dC 77.0 (central line of t) for CHCl3/CDCl3, dH 4.80 for H2O/
D2O, or dH 3.31/dC 48.2 for MeOH-d4. The splitting patterns are re-
ported as s (singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet), dd (double of doublets), and br (broad). Coupling constants (J)
are given in hertz (Hz). The ESI-MS experiments were conducted on
a Bruker Daltonics BioTOF III high-resolution mass spectrometer.
Compound 11a was commercially available. Compounds 9,32
12,34 and 1438,39 were prepared according to the previously re-
ported methods.
4.20e4.06 (5H, m), 3.97e3.95 (1H, m), 3.48 (1H, m), 2.55e2.49 (1H,
m), 2.26e2.19 (1H, m), 1.98 (3H, s), 1.60e1.46 (4H, m), 1.44 (9H, s),
1.40e1.28 (6H, m), 0.95e0.87 (6H, m); 13C NMR (100 MHz, CD3OD)
d
174.0, 159.3, 141.2, 129.4 (C-1, d, JCeP¼180 Hz), 83.5, 80.48, 74.2,
63.9, 63.7, 52.7, 47.7, 29.3, 29.2, 28.9 (3ꢂ), 27.6, 27.59,16.9,16.8,10.4,
10.0; 31P NMR (162 MHz, CDCl3)
d 18.93; HRMS (ESI): calcd for
C
22H42N2O7P: 477.2730, found: m/z 477.2545 [MþH]þ.
4.3. (3R,4R,5S)-4-Acetamido-5-amino-3-(1-ethylpropoxy)cy-
clohex-1-ene-1-phosphonic acid (2, tamiphosphor)27
4.2. Diethyl (3R,4R,5S)-4-acetamido-5-tert-butox-
ycarbonylamino-3-(1-ethylpropoxy)cyclohex-1-ene-1-
phosphonate (18)27
Diethyl phosphonate (5S)-17 (50 mg, 0.1 mmol) was dissolved in
CH2Cl2 (1 mL) and treated with bromotrimethylsilane (160 mg,
1.05 mmol) at 0 ꢁC. The reaction mixture was warmed to room
temperature and stirred for 18 h. After which, the mixture was
concentrated under reduced pressure. The residue was taken up in
water (0.5 mL), stirred for 2 h at room temperature, and subject to
lyophilization. The residual pale yellow solids were washed with
Et2O (3ꢂ20 mL) to give white solids, which were dissolved in
aqueous NH4HCO3 (0.1 M solution, 2 mL), stirred for 1 h at room
temperature, and then lyophilization to afford tamiphosphor 2 as
ammonium salts (32 mg, 86% yield).
Freshly prepared aldehyde 9 (1.0 g, 7.7 mmol) was added to
a suspension of (S)-diphenylprolinol trimethylsilyl ether (11a,
250 mg, 0.76 mmol), nitroenamide 12 (0.50 g, 3.8 mmol), and
chloroacetic acid (152 mg,1.52 mmol) in CH2Cl2 at 0 ꢁC. The mixture
was stirred for 24 h, then bisphosphonate 14 (1.36 g, 5.76 mmol)
and Cs2CO3 (11.5 mmol, 3.76 g) were added at 0 ꢁC. After stirring for
24 h at 0 ꢁC, the mixture was concentrated under reduced pressure
at 0 ꢁC. The residue containing 15 and 16 was dissolved in absolute
alcohol (EtOH, 10 mL) as purchased without prior removal of water.
NaOEt (1.3 g, 19 mmol) was added to the solution at 0 ꢁC. The
mixture was stirred at the same temperature for 7.5 h, and then
quenched with cold 2 M HCl (10 mL). The aqueous layer was
extracted with CH2Cl2 (20 mL) for three times. The combined or-
ganic extracts were washed with saturated NaHCO3 solution
(25 mL), dried over MgSO4, and concentrated under reduced
pressure to give a crude product 15.
The above-prepared crude product was dissolved in EtOH
(10 mL) and added 2 M aqueous HCl (10 mL). Activated zinc powder
(1754 mg, 25.8 mmol) was added to the resulting solution and
stirred for 6 h at 25 ꢁC before filtration. Aqueous ammonia (5 mL,
28%) was added tothe filtrate. The aqueous layer was extracted three
times with 10% MeOH/CH2Cl2. The combined organic phase was
extracted three times with 2 M HCl. The combined aqueous extracts
were adjusted to pH 10 with 28% aqueous ammonia and extracted
three times with 10% MeOH/CH2Cl2. The combined organic extracts
were washed with brine, dried over MgSO4, and concentrated under
reduced pressure to give the crude amine product 17. The crude
amine was dissolved in CH2Cl2. To the solution was added triethyl-
amine (313 mg, 3.1 mmol, 0.4 mL), followed by addition of di-tert-
butyl dicarbonate (338 mg, 1.55 mmol). The mixture was stirred for
18 h at 25 ꢁC until the reaction was completed as shown by TLC
analysis. The mixture was concentrated under reduced pressure.
The residue was purified by flash column chromatography (SiO2,
CH2Cl2/2-propanol¼15:1) to afford(5S)-18 (183 mg,10.1% yield form
12) and (5R)-18 (247 mg, 13.5% yield form 12).
C
13H25N2O5P; white solid; mp 246e250 ꢁC (lit.27 238ꢀ240 ꢁC);
20
20
[a
]
ꢀ48.51 (c 1.0, H2O) (lit.27
[
a]
ꢀ56.7 (c 1.2, H2O)); IR (film)
D
D
3125, 3032, 2960, 1653, 1558, 1019 cmꢀ1; 1H NMR (600 MHz, D2O)
d
6.33 (1H, d, JP-1¼19.2 Hz), 4.26 (1H, d, J¼9.0 Hz), 3.94 (1H, dd,
J¼12.0, 8.7 Hz), 3.58e3.55 (2H, m), 2.86e2.81 (1H, m), 2.54e2.48
(1H, m), 2.11 (3H, s), 1.62e1.56 (3H, m), 1.51e1.46 (1H, m), 0.92 (3H,
t, J¼7.8 Hz), 0.87 (3H, t, J¼7.8 Hz); 13C NMR (150 MHz, D2O)
d 177.9,
136.9, 134.9 (C-1, d, JP-1¼172 Hz), 87.1, 78.8, 55.7, 52.2, 32.0, 28.2,
27.9, 25.1, 11.3, 11.2; 31P NMR (162 MHz, D2O)
d 10.16; HRMS calcd
for C13H26N2O5P [MþH]þ: 321.1579, found: m/z 321.1569.
4.4. (3R,4R,5R)-4-Acetamido-5-amino-3-(1-ethylpropoxy)-1-
cyclohexene phosphonic acid ((5R)-2)
Diethyl phosphonate (5R)-17 (50 mg, 0.1 mmol) was dissolved in
CH2Cl2 (1 mL) and treated with bromotrimethylsilane (160 mg,
1.05 mmol) at 0 ꢁC. The reaction mixture was warmed to room
temperature and stirred for 18 h. After which, the mixture was
concentrated under reduced pressure. The residue was taken up in
water (0.5 mL), stirred for 2 h at room temperature, and subject to
lyophilization. The residual pale yellow solid residue was washed
with Et2O (3ꢂ20 mL) to give white solids, which was dissolved in
aqueous NH4HCO3 (0.1 M solution, 2 mL), stirred for 1 h at room
temperature, and then lyophilization to afford (5R)-2 as ammo-
nium salts (33 mg, 89% yield).
C
13H25N2O5P; white solid; mp 218e224 ꢁC (dec); [
a
]
20 ꢀ58.83 (c
D
1.0, H2O); IR (film) 3037, 2967, 2875,1647,1540,1070 cmꢀ1; 1H NMR
(600 MHz, D2O)
d
6.15 (1H, d, JP-2¼18.8 Hz), 4.12 (1H, d, J¼8.1 Hz),
4.2.1. Compound
(5S)-18. C22H41N2O7P; white solid; mp
3.94 (1H, dd, J¼7.2, 4.3 Hz), 3.77e3.74 (1H, m), 3.59e3.56 (1H, m),
2.79e2.73 (1H, m), 2.40e2.36 (1H, m), 2.04 (3H, s), 1.65e1.56 (3H,
m), 1.50e1.44 (1H, m), 0.95 (3H, t, J¼7.4 Hz), 0.89 (3H, t, J¼7.4 Hz);
173e175 ꢁC (lit.27 mp 167e169 ꢁC); [
a]
20 ꢀ65.5 (c 1.0, CHCl3) (lit.27
D
20
[
a]
ꢀ88.8 (c 1.14, CHCl3)); IR (film) 3389, 2933, 1695, 1565, 1172,
D

270
Y.-W. Lo, J.-M. Fang / Tetrahedron 71 (2015) 266e270
13C NMR (150 MHz, D2O)
d
178.2, 139.1 (C-1, d, JP-1¼170 Hz), 132.2,
Genomics Research Center, Academia Sinica) for helpful discussion,
as well as the Ministry of Science and Technology for financial
support (NSC 101-2325-B-002-024).
85.8, 75.5, 50.4, 49.4, 28.6, 28.0, 27.9, 24.6, 12.3, 10.9; 31P NMR
(162 MHz, D2O)
d
10.05; HRMS calcd for C13H26N2O5P [MþH]þ:
321.1579, found: m/z 321.1578.
Supplementary data
4.5. Determination of influenza virus TCID50
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2014.11.062.
Influenza A/WSN/1933 (H1N1) (from Dr. Shin-Ru Shih, Chang
Gung University, Taiwan) was cultured in the allantoic cavities of 10-
day-old embryonated chicken eggs for 72 h, and purified by sucrose
gradient centrifugation. MadineDarby canine kidney (MDCK) cells
were obtained from American Type Culture Collection (Manassas,
Va), and were grown in DMEM (Dulbecco’s modified Eagle medium,
GibcoBRL) containing 10% fetal bovine serum (GibcoBRL) and pen-
icillinestreptomycin (GibcoBRL) at 37 ꢁC under 5% CO2.
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Acknowledgements
€
We thank Dr. Yih-Shyun E. Cheng (the Genomics Research
Center, Academia Sinica) for bioassays, Dr. Jiun-Jie Shie (the
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