Tamiphosphor monoesters as effective anti-influenza agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.082

Oseltamivir is a potent neuraminidase inhibitor for influenza treatment. By replacing the carboxylate group in oseltamivir with phosphonate monoalkyl ester, a series of tamiphosphor derivatives were synthesized and shown to exhibit high inhibitory activities against influenza viruses. Our molecular modeling experiments revealed that influenza virus neuraminidase contains a 371-cavity near the S1-site to accommodate the alkyl substituents of tamiphosphor monoesters to render appreciable hydrophobic interactions for enhanced affinity. Furthermore, guanidino-tamiphosphor (TPG) monoesters are active to the oseltamivir-resistant mutant. TPG monohexyl ester 4e having a more lipophilic alkyl substituent showed better cell permeability and intestinal absorption than the corresponding monoethyl ester 4c, but both compounds showed similar bioavailability. Intranasal administration of TPG monoesters at low dose greatly improved the survival rate of mice infected with lethal dose of H1N1 influenza virus, whereas 4c provided better protection of the infected mice than oseltamivir and other phosphonate congeners by oral administration.

Full text of DOI:10.1016/j.ejmech.2014.04.082

European Journal of Medicinal Chemistry 81 (2014) 106e118
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tamiphosphor monoesters as effective anti-influenza agents
Chun-Lin Chen a, Tzu-Chen Lin a, Shi-Yun Wang b, Jiun-Jie Shie b, Keng-Chang Tsai c,
,
a,b,
*
Yih-Shyun E. Cheng b, Jia-Tsrong Jan b, Chun-Jung Lin d , Jim-Min Fang
,
**
,
Chi-Huey Wong b
**
a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, ROC
b The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, ROC
c National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC
d School of Pharmacy, National Taiwan University, Taipei 100, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Oseltamivir is a potent neuraminidase inhibitor for influenza treatment. By replacing the carboxylate
group in oseltamivir with phosphonate monoalkyl ester, a series of tamiphosphor derivatives were
synthesized and shown to exhibit high inhibitory activities against influenza viruses. Our molecular
modeling experiments revealed that influenza virus neuraminidase contains a 371-cavity near the S1-
site to accommodate the alkyl substituents of tamiphosphor monoesters to render appreciable hydro-
phobic interactions for enhanced affinity. Furthermore, guanidino-tamiphosphor (TPG) monoesters are
active to the oseltamivir-resistant mutant. TPG monohexyl ester 4e having a more lipophilic alkyl sub-
stituent showed better cell permeability and intestinal absorption than the corresponding monoethyl
ester 4c, but both compounds showed similar bioavailability. Intranasal administration of TPG mono-
esters at low dose greatly improved the survival rate of mice infected with lethal dose of H1N1 influenza
virus, whereas 4c provided better protection of the infected mice than oseltamivir and other phos-
phonate congeners by oral administration.
Received 31 March 2014
Received in revised form
28 April 2014
Accepted 30 April 2014
Available online 2 May 2014
Keywords:
Influenza
Virus
Neuraminidase
Inhibitor
Pharmacokinetics
Molecular modeling
Ó 2014 Elsevier Masson SAS. All rights reserved.
InChIKey:
BQLKEXSYWRBWQK-AFMQGRBTSA-N
1. Introduction
the family Orthomyxoviridae. The 8 RNA gene segments replicate at
least 11 proteins [1,2]. Hemagglutinin (HA) and neuraminidase (NA)
Influenza is a highly contagious disease that occurs in seasonal
epidemics and also emerges periodically to global pandemics.
Influenza viruses are negative-sense single-stranded RNA viruses of
are the most significant surface glycoproteins for influenza viru-
lence. Depending on the specific strain of influenza virus, HA
selectively binds to human or avian respiratory epithelial cells.
Influenza NA catalyzes the hydrolysis of the terminal sialic acid
residue from the sialo-receptors of host cells to facilitate the release
of progeny viruses for propagation and infectivity [3].
NA is a good drug target in view of its rather conserved active
site [4e6]. Four NA inhibitors zanamivir (RelenzaÔ) [7,8], oselta-
mivir (TamifluÔ as the phosphate salt) [9,10], peramivir (Rap-
iactaÔ) [11,12] and laninamivir (InavirÔ) [13,14] have been
approved for use as anti-influenza drugs. The phosphate salt of
oseltamivir (OS, 1b), is currently the most popular oral medication
for influenza therapy. Oseltamivir is converted by endogenous
esterase to oseltamivir carboxylate (OC, 1a) which is the active
component for NA inhibition [3,15,16]. The carboxylic acid in OC
provides multiple electrostatic interactions with the three arginine
residues (R118, R292 and R371) in NA [4e6]. However, oseltamivir-
resistant viruses have occurred over the years. New anti-influenza
drugs that can also inhibit oseltamivir-resistant strains, such as
Abbreviations: AUC, area under the concentration-versus-time curve; CC50, 50%
cytotoxicity concentration; CL, clearance; CPE, cytopathic effect; D, distribution
coefficient; DMEM, Dulbecco’s modified Eagle medium; EC50, half maximal effec-
tive concentration; EDTA, ethylenediaminetetraacetic acid; F (%), oral bioavailability
(fraction absorbed); GOC, guanidino-oseltamivir carboxylic acid; GOS, guanidino-
oseltamivir; HBTU, o-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate; IC50, half maximal inhibitory concentration; i.p., intraperitoneal;
i.v., intravenous; LD50, median lethal dose; MDCK, MadineDarby canine kidney;
MUNANA, 2-(4-methylumbelliferyl)-
dase; OC, oseltamivir carboxylic acid; OS, oseltamivir; P, partition coefficient;
TCID50 50% cell culture infectious dose; TP, tamiphosphor; TPG, guanidino-
a-D-N-acetylneuraminic acid; NA, neuramini-
,
tamiphosphor.
* Corresponding author. Department of Chemistry, National Taiwan University,
Taipei, 106, Taiwan, ROC.
** Corresponding authors.
E-mail addresses: clementumich@ntu.edu.tw (C.-J. Lin), jmfang@ntu.edu.tw
(J.-M. Fang), chwong@gate.sinica.edu.tw (C.-H. Wong).
http://dx.doi.org/10.1016/j.ejmech.2014.04.082
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
107
the clinically relevant H275Y mutant, are in urgent need for our
fight against the threat of pandemic influenza. Compound 2a (GOC)
shows higher inhibitory activity than OC by replacing the C-5
amino group with a more basic guanidino group, which is consid-
ered to exert stronger interactions with the acidic residues (E119,
D151, and E227) in the active site of influenza NA [9]. However,
compound 2a and its ethyl ester (2b, GOS) have not been developed
for therapeutic use.
In another aspect, a flexible 150-loop adjacent to the S2 active
site is found from the X-ray crystallographic studies of the apo and
inhibitor-bound structures of NAs [6,24]. The A-type influenza virus
NAs are divided into two categories: group-1 including N1, N4, N5
and N8 subtypes, and group-2 comprising N2, N3, N6, N7 and N9
subtypes. It is proposed that the 150-loop of group-2 NA always
exists in the closed form, whereas that of group-1 NA would change
from the open conformation to the closed form on binding with
substrate or inhibitor. In contrast, molecular dynamics simulations
suggest that both group-1 and group-2 NAs are able to adopt an
open 150-cavity within their solution-phase structural ensemble
[25]. Molecular dynamics simulations of influenza NAs further
indicate the existence of a 430-loop comprising the residues R430e
T439 adjacent to the S1 active site [26,27]. The conformational
change of 150-loop may be coupled with motion of the neighboring
430-loop to form a much larger binding pocket than that shown in
the static crystal structures. In this study, we carried out molecular
modeling experiments to reveal that influenza virus also contains a
hydrophobic 371-cavity near the NA active site that may provide
additional interactions with the alkyl groups in our designed in-
hibitors of phosphonate monoesters.
2. Results and discussion
2.1. Molecular modeling
The structure-based design of NA inhibitors has been a suc-
cessful strategy in discovery of anti-influenza drugs [4]. The phos-
phonate group has been used as a bioisostere of carboxylate in drug
design [29e31]. In comparison with carboxylate ion, a phosphonate
ion exhibits stronger electrostatic interactions with guanidinium
ion [17]. Formation of phosphonateeguanidinium complex is
thermodynamically favored [17]. Consistent with this rationale,
compound 3a containing a phosphonic acid binds strongly with the
three arginine residues in the NA active site, thus showed higher
inhibitory activity than the corresponding carboxylate 1a against
various human and avian influenza viruses, including A/H1N1
(wild-type and H275Y mutant), A/H5N1, A/H3N2 and type B viruses
[19]. Using the known N1 crystal structure (PDB code: 2HU4) [6],
our molecular docking experiments indicated that the phosphonate
group of 3a formed 8-pair hydrogen bonds with three arginine
residues (Fig. 1A), greater than the carboxyl group of 1a (6 pairs of
hydrogen bonds). The monoalkyl phosphonate ion, e.g. 3f, exhibi-
ted 7 pairs of hydrogen bonds with the three arginine residues in
the S1 site of NA (Fig.1B), in addition to the appreciable interactions
of the C3-pentoxy, C4-acetamido and C5-amino groups in the S2eS5
sites [5]. Our molecular modeling revealed that the 3-phenylpropyl
moiety in 3f also exhibited considerable interactions with I427,
P431 and K432 residues in the 430-loop. This result is in agreement
with the prediction by ensemble-based virtual screening [32],
which shows that the 430-cavity of NA favors to accept aromatic
rings and hydrophobic substituents.
Formation of phosphonateeguanidinium complex is thermo-
dynamically favored [17]. Tamiphosphor (TP, 3a), a phosphonate
congener of OC, is thus designed to have strong electrostatic in-
teractions with the three arginine residues (R118, R292 and R371)
in the active site of NA [18,19]. The phosphonate ion in 3a is also
topologically complementary to bind with the three arginine resi-
dues. Compound 3a has high NA inhibitory activities to suppress
the replication of human and avian influenza viruses. Our previous
study [19] indicates that phosphonate monoethyl ester 3c is also
very active to protect human 293T and canine MDCK cells from
infection by influenza viruses because 3c still contains a negative
charge at the phosphonate group to render the necessary electro-
static interactions with the three arginine residues in NA. Mono-
ester 3c is a drug, instead of a prodrug, because it does not
metabolize to the free phosphonic acid 3a in animal experiments
[19]. In another approach, Streicher and coworkers have demon-
strated that the phospha-oseltamivir monoesters carrying various
functional groups can be of versatile uses [20e23]. For example, the
phospha-oseltamivir monoesters equipped with aglycone mi-
metics or linked to gold nanoparticles are good for treatment and
detection of influenza viruses.
We have also prepared guanidino-tamiphosphor (TPG, 4a) and
its monoethyl ester 4c to show that they possess high inhibitory
activities against the oseltamivir-resistant mutant of influenza vi-
rus. In contrast, phosphonate diethyl esters 3b and 4b are inactive
to influenza viruses. The phosphonate compounds 3a, 3c, 4a and 4c
have similar pharmacokinetic properties in mice, rats and dogs.
TPG monoester 4c in saline solution shows better oral bioavail-
ability (F ¼ 12%) than TPG 4a (F ¼ 7%) in mice [19]. We therefore aim
to investigate whether the bioavailability and pharmacokinetic
properties can be further improved in the TP and TPG
monoalkyl esters bearing more lipophilic alkyl groups, such as 3de
3g and 4de4f.
Moreover, we found a hydrophobic cleft, namely 371-cavity
(Fig. 1D), was enclosed by the R371, P431, I427, K432 and W403
residues located between the 430- and 371-loops near the S1 site.
The 3-phenylpropyl substituent of 3f extended to the 371-cavity, so
that the phenyl group was disposed in a manner to exert a signif-
icant
pecation interaction with R371, and a T-shape interaction
with the indole ring of the W403 residue in an edge-to-face
configuration [33]. In a similar fashion, the (indol-3-yl)propyl
substituent of 3g was located in the region of 430- and 371-loops to
attain additional hydrophobic,
pep and pecation interactions
(Fig. 1C). Thus, phosphonate monoesters 3f and 3g were predicted
to possess high affinity to influenza virus NAs.

108
C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
Fig. 1. Molecular models of tamiphosphor 3a (A) and phosphonate monoalkyl esters 3f (B) and 3g (C) in the active site of influenza virus neuraminidase (N1 subtype, PDB code:
2HU4). The phenylpropyl group of 3f embedded in a 371-cavity near S1 site is shown (D). The phosphonic acid moiety of 3a exhibits extensive hydrogen bonding interactions (8
pairs ligandeNA H-bonds) with three arginine residues (R118, R292 and R371) in the NA active site. In the 3feNA or 3geNA complex, there are 7 pairs H-bonds formed by the
phosphonate monoester group with R118, R292 and R371. However, the phenylpropyl substituent in 3f and (indol-3-yl)propyl substituent in 3g extend to the 371-cavity to gain
appreciable hydrophobic,
pep and pecation interactions.
2.2. Chemical synthesis
and water, and oral drugs are often found to have log P values
between ꢀ1 and 5 [34]. In lieu of log P, the distribution coefficient
(log D) is generally used to represent the partition of an ionic
compound between octanol and PBS buffer under specific pH value.
Despite having double negative charges on the phosphonate group,
3a (log D ¼ ꢀ1.04) appeared to be less hydrophilic than OC (log
We have previously synthesized phosphonic acids 3a and 4a
using -xylose as an inexpensive starting material via an intra-
D
molecular HornereWadswortheEmmons reaction with tetraethyl
methylenediphosphonate (H2C[PO(OEt)2]2) to construct the
desired cyclohexenephosphonate scaffold [18]. In another
approach, Streicher [20], Gunasekera [28] and our research teams
[29] have provided an effective synthesis of oseltamivir phospho-
nate congeners using a palladium-catalyzed phosphonylation of 1-
halocyclohexene as the key reaction. We applied this method to
synthesize a series of phosphonate monoesters 3ce3g and 4ce4f as
shown in Scheme 1. The N-Boc protected oseltamivir (5) [29] was
hydrolyzed to give the corresponding carboxylic acid 6 [28], which
was further converted to Barton ester [30,31] for the subsequent
photolysis, giving the iodocyclohexene derivative 7 in the presence
of CF3CH2I [20,28]. On the other hand, the phosphite reagents 8ce
8g were readily prepared from phosphorus trichloride with 2 equiv
of appropriate alcohols, including ethanol, butanol, hexanol, 3-
phenylpropanol and 3-(indol-3-yl)propanol. Thus, the iodo com-
pound 7 was subject to a Pd-catalyzed phosphonylation with dia-
lkyl phosphites 8ce8g to afford the phosphonate compounds 9ce
9g. The Boc protecting group in 9ce9g was removed by trifluoro-
acetic acid (TFA), and the phosphonate dialkyl esters were treated
with aqueous KOH to cleave one alkyl group, giving the desired
phosphonate monoalkyl esters 3ce3g. Alternatively, the amine
intermediate was reacted with N,N0-bis-(tert-butoxycarbonyl)-S-
methylisothiourea in the presence of HgCl2 and Et3N to give 10ce
10f, which were converted to TPG monoalkyl esters 4ce4f by
sequential treatments with TFA and KOH(aq)
.
2.3. Lipophilicity of phosphonate monoesters
Scheme 1. Synthesis of tamiphosphor and guanidino-tamiphosphor monoesters. Re-
agents and conditions: (a) 1 M KOH(aq), MeOH, 25 ꢁC, 2 h; 90%. (b) 3-hydroxy-4-
methyl-2(3H)-thiazolethione, HBTU, Et3N, CH2Cl2, 25 ꢁC, 2 h; then CF3CH2I, CH2Cl2,
In the design of an orally available drug, lipophilicity is an
important pharmacokinetic factor that must be considered. With
appropriate lipophilicity, drugs can diffuse through cell membrane
and thus having better bioavailability. The partition coefficient (P) is
a measure of lipophilicity for nonionic compound between octanol
hn
, 30 min; 54% overall yield. (c) Pd(PPh3)4, toluene, Et3N, 90 ꢁC, 2e12 h; 64e86%. (d)
CF3COOH, CH2Cl2, 25 ꢁC,
1
h; then N,N0-bis-(tert-butoxycarbonyl)-S-methyl-iso-
thiourea, HgCl2, Et3N, CH2Cl2, 25 ꢁC, 2e3 h; 60e75%. (e) CF3COOH, CH2Cl2, 25 ꢁC, 2 h;
KOH(aq), 1,4-dioxane, 25e40 ꢁC, 24e120 h; 43e68%. HBTU ¼ o-Benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium hexafluorophosphate.

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
109
Table 2
D ¼ ꢀ1.69) that carries a single negative charge [19]. Table 1 shows
the calculated and measured values of log D for a series of phos-
phonate derivatives. All the phosphonate monoesters had higher
log D values than their parental acids 3a and 4a (log D ¼ ꢀ0.98). The
lipophilicity of phosphonate monoesters increased as the aliphatic
chain elongated. The log D value increased about 1 unit when hexyl
monoester compound 3e (log D ¼ 0.30) is compared to ethyl
monoester 3c (log D ¼ ꢀ0.75) [19]. Monoesters 3f and 4f having 3-
phenylpropyl substituent showed the lipophilicity similar to the
monohexyl esters 3e and 4e. Interestingly, we also found that
guanidino-phosphonates 4ce4f were more lipophilic than their
corresponding amino compounds 3ce3f. This result was in agree-
ment with the trend of calculation (c log D). The improved lipo-
philic property of guanidino compounds might be related to their
zwitterionic structures [35,36], in which the C-5 guanidinium could
form ion-pair with the phosphonate group more favorably (in
comparison, guanidinium pKa z 14 versus aminium pKa z 10)
[17,37,38]. Without having zwitterionic structure, phosphonate
diethyl ester 4b (log D ¼ ꢀ0.10) bearing a more basic guanidine
substituent was less lipophilic than 3b (log D ¼ 0.22) having an
amine substituent at the C-5 position [19].
Inhibitory activities of tamiphosphor monoalkyl esters against wild-type H1N1 and
oseltamivir-resistant influenza viruses.a
Compoundb
Wt (WSN)a
IC50 (nM)c
H275Ya
EC50 (nM)d
IC50 (nM)c
EC50 (nM)d
3ce
3d
3e
3f
3g
4ce
4d
4e
4f
3.0 ꢂ 2.0
6.9 ꢂ 2.0
2.2 ꢂ 0.5
1.8 ꢂ 0.7
1.7 ꢂ 0.1
1.1 ꢂ 0.1
6.7 ꢂ 3.1
4.1 ꢂ 0.8
3.2 ꢂ 0.8
47 ꢂ 4
1679 ꢂ 43
1443 ꢂ 14
1719 ꢂ 56
1283 ꢂ 12
NDf
25.1 ꢂ 6.6
10.9 ꢂ 1.3
89.2 ꢂ 9.5
24.6 ꢂ 1.3
490 ꢂ 470
9154 ꢂ 32
>10,000e
>10,000e
NDf
58.6 ꢂ 6.2
18.3 ꢂ 0.4
13.1 ꢂ 6.9
13.1 ꢂ 9.5
180 ꢂ 14
7.6 ꢂ 3.5
9.3 ꢂ 8.7
3.3 ꢂ 2.1
900 ꢂ 200
97 ꢂ 18
262 ꢂ 11
224 ꢂ 16
a
Influenza viruses A/WSN/1933 (H1N1) and the H274Y mutant were used as
bioassay materials for neuraminidase inhibition and anti-influenza assays.
b
The test compounds 3ce3g and 4ce4f were nontoxic to human 293T and canine
MDCK cells at the highest testing concentrations (>10
mM).
c
A fluorescent substrate, 2-(4-methylumbelliferyl)-
a-D-N-acetylneuraminic acid
(MUNANA), was used to determine the IC50 values that are compound concentra-
tions causing 50% inhibition of different influenza neuraminidase enzymes. Data are
shown as mean ꢂ SD of three experiments.
d
The anti-influenza activities against different influenza strains were measured
as EC50 values that are the compound concentrations for 50% protection of the
cytopathic effects due to the infection by different influenza strains. Data are shown
as mean ꢂ SD of three experiments.
2.4. Neuraminidase inhibition and anti-influenza activity
e
The data are adapted from literature [19].
ND: Not determined.
f
The NA inhibitory activity was measured using a fluorogenic
substrate 2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid
(MUNANA). The anti-influenza activities were measured by pro-
tection of MadineDarby canine kidney (MDCK) cells from the viral
infection. OS (1b) as an ester is inactive to NA while its carboxylic
acid 1a is a nanomolar NA inhibitor of influenza virus. The phos-
phonic acids 3a and 4a also effectively inhibited the H1N1 virus NA
with IC50 z 2 nM [19]. Table 2 shows the NA inhibition (IC50) and
anti-influenza activities (EC50) of TP and TPG monoalkyl esters
against wild-type H1N1 virus (A/WSN/1933) and OS-resistant
H275Y mutant. Unlike OS, the phosphonate monoalkyl esters 3ce
g and 4cef all showed high NA inhibitory activities against wild-
type H1N1 virus (IC50 ¼ 1.6e6.9 nM). This result is in agreement
with our molecular docking experiments. Phosphonate monoester
has a negative charge to render sufficient electrostatic interactions
with R118, R292 and R371, and the alkyl substituent may also
provide appreciable hydrophobic interactions to increase the af-
finity with NA. Though TP monoesters were inactive to H275Y
mutant, TPG monoesters 4ce4f still showed strong inhibition to the
2.5. Cellular uptake
The MDCK cellular uptake study (Fig. 2) showed that phos-
phonate monoester 4e (R ¼ hexyl) was much more permeable than
4c (R ¼ ethyl) and 4f (R ¼ 3-phenylpropyl). In the first 10 min, the
cell permeability of 4e increased more than 10 fold than 4c and 4f.
Furthermore, the concentration of 4e reached 13,500 ng/mg pro-
tein in 120 min, whereas 4c and 4f were still at the concentrations
lower than 2000 ng/mg protein.
2.6. Intestinal permeability
In addition to cellular uptake in renal epithelial MDCK cells,
intestinal permeability of compounds 4c and 4e were further
examined in Caco-2 cells, a model resembling human intestinal
epithelial cells. The apical-to-basolateral flux (i.e., absorption) of
compound 4c was comparable to its flux in basolateral-to-apical
direction (i.e., secretion), suggesting the absorption of compound
OS-resistant H275Y virus with IC50 < 90 nM and EC50 < 1 mM. The
cell-based assays indicated that the anti-influenza agents 3ce3g
and 4ce4f were nontoxic to human 293T and canine MDCK cells at
the highest testing concentrations (>10 mM).
Table 1
Distribution coefficients of tamiphosphor monoalkyl esters.
,d
Compound
c log Pa
c log Db
log Dc
3c
3d
3e
3f
4c
4d
4e
4f
ꢀ0.14 (ꢀ0.40)
0.93 (0.51)
1.99 (1.35)
1.95 (1.67)
ꢀ0.81 (ꢀ0.34)
0.26 (0.57)
1.32 (1.41)
1.29 (1.73)
ꢀ1.53
ꢀ0.61
0.23
ꢀ0.75 ꢂ 0.10
ꢀ0.29 ꢂ 0.04
0.30 ꢂ 0.06
0.28 ꢂ 0.02
ꢀ0.37 ꢂ 0.02
ꢀ0.16 ꢂ 0.06
0.63 ꢂ 0.08
0.66 ꢂ 0.04
0.54
ꢀ1.27
ꢀ0.36
0.48
0.80
a
Calculated values using Advanced Chemistry Development (ACD/Laboratories)
Software V12.01. Data in parenthesis were calculated using MarvinSketch (http://
www.chemaxon.com/marvin/sketch/index.html).
b
Calculated values using MarvinSketch.
Octanolewater partition coefficient determined at pH 7.4 from five repeated
c
experiments.
Fig. 2. MDCK cell uptake of phosphonate monoesters 4c (R ¼ ethyl), 4e (R ¼ hexyl) and
4f (R ¼ 3-phenylpropyl).
d
Log D value at pH 7.4 (adapted from literature [39]).

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C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
Fig. 3. Intestinal permeability of phosphonate monoesters 4c (R ¼ ethyl) and 4e (R ¼ hexyl) in Caco-2 cells.
4c is limited (Fig. 3A). In contrast, compound 4e show significantly
higher flux in the direction of apical-to-basolateral, showing its
higher intestinal absorption (Fig. 3B).
0.12 mmol/kg/day, zanamivir only provided partial protection, and
all mice died at day 9. In comparison, TPG monoesters 4c, 4e and 4f
were superior to zanamivir in improving the survival rate of
infected mice (Fig. 4B). By oral administration of test compound at a
dose of 2.6
m
mol/kg/day (Fig. 4C), compound 4c (R ¼ ethyl)
2.7. Bioavailability
exhibited much better protection of the virus infected mice than
oseltamivir, 4e and 4f. This study indicated that monoester 4e
(R ¼ hexyl) only exhibited a marginal mice protection effect by oral
administration even it showed low EC50 value in the cell-based
assay and similar bioavailability to 4c.
The pharmacokinetic parameters for compounds 4c and 4e in
male mice are listed in Table 3. The half-life of 4e was 0.36 h after
intravenous (i.v.) administration, and 1.17 h after oral administra-
tion. In comparison with 4c had half-lives of 0.71 and 2.57 h for i.v.
and oral administration. Compound 4e had shorter half-lives than
4c, presumably because 4e was a more lipophilic compound sus-
ceptible to clearance by metabolism in mice [40]. Indeed, 4e had 2e
3 fold clearance rates than 4c in i.v. and oral administrations. The
absolute oral bioavailability (F value) of compound 4e was 12.6% in
mice, similar to 4c (12.1%) but better than their parental phos-
phonic acid 4a (7%) [19]. Even though the hexyl monoester 4e
exhibited higher cellular uptake and permeability in MDCK and
Caco-2 cells than the ethyl ester 4c, no better bioavailability of 4e in
mice was obtained presumably due to the counteract of
metabolism.
3. Conclusion
In order to develop effective anti-influenza drugs, we have
prepared a series of oseltamivir phosphonate congeners, especially
focusing on the phosphonate monoesters bearing hydrophobic
alkyl substituents. Using N-Boc protected oseltamivir carboxylic
acid as a starting material, the halodecarboxylation reaction was
carried out via photolysis of the Barton ester derivative in the
presence of CF3CH2I. The Pd-catalyzed phosphonylation of the
iodocyclohexene derivative 7 with dialkyl phosphites afforded the
corresponding phosphonates, which were then elaborated to the
desired tamiphosphor and guanidino-tamiphosphor monoesters
(Scheme 1). The lipophilicity of phosphonate monoesters increased
as the aliphatic chain elongated, and the guanidino-phosphonates
(4ce4f) were more lipophilic than their corresponding amino
compounds (3ce3f). The phosphonate monoesters showed potent
NA inhibitory activities with IC50 values in low nanomolar range
against influenza viruses. Our molecular modeling experiments
2.8. Mice protection experiments against A/WSN/33 (H1N1) virus
In one experiment, 1.2 mmol/kg/day of test compound, a dose
equivalent to that of zanamivir for human therapy, was intranasally
administered to mice twice-daily on days 1e4, and day 0 before
infection with lethal doses (10 LD50) of H1N1 influenza virus (A/
WSN/1933). All mice survived at day 14 (Fig. 4A). At low dosage of
Table 3
Pharmacokinetic parameters of compounds 4c and 4e after single intravenous bolus or oral administration to male mice.
PK parameter (unit)
4ca
4e
IV, 0.25 mg/kg (N ¼ 6)
Oral, 10 mg/kg (N ¼ 6)
IV, 0.25 mg/kg (N ¼ 5)
Oral, 10 mg/kg (N ¼ 3)
k (1/h)
1.37
529
541
0.71
0.47
100
e
0.36
2575
2629
2.57
4.75
12.1
935
1.92
169b
173
0.36
1.45
100
e
0.60
361c
867
1.07
11.05
12.6
261
AUC0/t (h*ng/mL)
AUC0/N (h*ng/mL)
T1/2 (h)
CL/F (L/h/kg)
F (%)
Cmax (ng/mL)
Tmax (h)
e
1.39
e
1.00
a
The pharmacokinetic parameters of compound 4c are the mean values adapted from literature [19].
b
c
t ¼ 2.
t ¼ 12.

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
111
Fig. 4. Percentage of survival of mice by intranasal inoculation (A and B) or oral gavage (C) with test compounds at the indicated dosages (1.2, 0.12 and 2.6
mmol/kg/day). Mice were
administered twice per day. Four hours after the first dose of test compound, mice were challenged with 10 LD50 of A/WSN/33 (H1N1) influenza virus. The number of mice at day
0 (10 mice per group) were defined as 100%, respectively. ***P < 0.0001. **P < 0.001.
reveal that phosphonate monoalkyl ester contains a negative
charge on the phosphonate moiety to render sufficient electrostatic
interactions with the three arginine residues (R118, R292 and R371)
in the NA active site, and the alkyl substituent is located in the 371-
cavity near the S1-site to attain extra hydrophobic interactions the
enhanced affinity with NA. The cell-based assays showed that
tamiphosphor, guanidino-tamiphosphor and their monoesters all
positive pressure of argon unless otherwise noted. Reactions were
magnetically stirred and monitored by thin-layer chromatography
on silica gel. Flash chromatography was performed on silica gel
(60e200 mm particle size) and LiChroprep RP-18 (40e63 mm par-
ticle size). The pH values were measured with Mettler-Toledo MP-
120 pH meter. Melting points were recorded on Yanaco melting
point apparatus and are not corrected. Optical rotations were
exhibited
tamiphosphor monoesters were active to oseltamivir-resistant
H275Y mutant with EC50 < 1 M. Even though the phosphonate
potent
anti-influenza
activities.
Guanidino-
recorded on digital polarimeter of Japan JASCO Co. DIP-1000; [
a]
D
values are given in units of 10ꢀ1 deg cm2 ꢀ1. NMR spectra were
g
m
recorded on Bruker Avance-400 (400 MHz), Bruker400 AVIII
(400 MHz) and Varian Unity Plus-400 (400 MHz) spectrometer.
monohexyl ester 4e has much better cell permeability and intes-
tinal absorption than the monoethyl ester 4c, both compounds
have similar absolute bioavailability (F z 12.5%) because 4e also
has higher metabolism rate. Intranasal administration of TPG
monoesters (4c, 4e or 4f) at low dose (e.g. one tenth of zanamivir-
equivalent dose) greatly improved the survival rate of mice infected
with lethal dose of H1N1 influenza virus. Nevertheless, compound
4c is still the choice for oral administration to provide effective
protection of the virus infected mice (Fig. 4C).
Chemical shifts are given in
d values relative to tetramethylsilane
(TMS); coupling constants J are given in Hz. Internal standards were
dH 7.24/dC 77.0 (central line of t) for CHCl3/CDCl3, dH 4.80 for H2O/
D2O, dH 3.31/dC 48.2 for CD3OD, and H3PO4 in D2O (dP ¼ 0) for 31P
NMR spectra. The splitting patterns are reported as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) and dd
(double of doublets). IR spectra were recorded on a Nicolet Magna
500-II. High resolution ESI mass spectra were recorded on a Bruck
Daltonics BioTOF III high-resolution mass spectrometer. High-
performance liquid chromatography (HPLC) was performed on
Agilent 1100 series equipped with a degasser, Quat pump, and UV
detector.
4. Experimental section
4.1. General
All the reagents were commercially available and used without
further purification unless indicated otherwise. All solvents were
anhydrous grade unless indicated otherwise. All non-aqueous re-
actions were carried out in oven-dried glassware under a slight
4.2. Viruses
Influenza A/WSN/1933 (H1N1) virus was obtained from Dr.
Shin-Ru Shih at Chang Gung University in Taiwan. WSN 275Y was

112
C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
selected with tamiflu from influenza A/WSN/1933 (H1N1) in our
laboratory. All viruses were 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
penicillin-streptomycin (GibcoBRL) at 37 ꢁC under 5% CO2.
49.3, 31.2, 28.6 (3ꢄ), 26.5, 26.0, 23.7, 14.6, 10.0, 9.7; HRMS calcd for
C
21H37N2O6: 413.2652, found: m/z 413.2658 [M þ H]þ.
4.4.3. 4-Acetamido-5-(tert-butoxycarbonyl)amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxylic acid (6)
Compound 5 (1.0 g, 2.4 mmol) was stirred with 1.0 M aqueous
KOH (3.5 mL) in MeOH (6.6 mL) at room temperature for 2 h, and
then neutralized with Dowex 50W ꢄ 8. The mixture was filtered,
washed with MeOH, and concentrated under reduced pressure to
give carboxylic acid 6 (0.63 g, 90%). C19H32N2O6; white solid, mp
195e197 ꢁC; TLC (EtOAc/hexane, 2:1) Rf ¼ 0.10; 1H NMR (400 MHz,
4.3. Computer modeling
The model of a specific compound in complex with the NA was
constructed through docking this compound to the crystallographic
structure of N1 neuraminidase (PDB code: 2HU4) [6]. The 3D
structure of compound (3a, 3f or 3g) was built by modifying the 3D
structure of oseltamivir acid (1a, from 2HU4) with SYBYL 8.0 pro-
gram (Tripos Associates, St. Louis, MO). GOLD 4.0.115 was used to
dock the compound onto the protein with flexible docking option
turned on [41,42]. Kollmann-all atom charges [43] were assigned to
the protein atoms, and GasteigereHückel charges [44] were
assigned to ligand atoms using the SYBYL 8.0 program. Initial 1000
independent genetic algorithm cycles of computation were carried
out with ligand torsion angles varying between ꢀ180 and 180ꢁ. The
search efficiency was set at 200% to ensure the most exhaustive
search for the docking conformational space. All other parameters
were kept the same as the default settings. The docking processes
were distributed to a 40-processor Linux cluster with Intel(R)
Xeon(TM) CPU 3.00 GHz CPUs. The resultant ligandeprotein com-
plex structures were ranked with the GOLDSCORE scoring function
to determine the top 1000 hits. Visual inspection of the top con-
formations confirmed that a consensus structure as shown in
Fig. 1A was evident. The molecular models were displayed with the
PyMOL software (DeLano Scientific, San Carlos, CA).
CD3OD)
d
6.79 (1H, s), 4.11 (1H, d, J ¼ 8.4 Hz), 3.88e3.83 (1H, m),
3.75e3.68 (1H, m), 3.41 (1H, t, J ¼ 5.6 Hz), 2.71e2.66 (1H, m), 2.28e
2.19 (1H, m), 1.96 (3H, s), 1.56e1.49 (4H, m), 1.44 (9H, s), 0.90e0.85
(6H, m); 13C NMR (100 MHz, CDCl3)
d 169.5, 168.1, 155.3, 135.0,
131.6, 81.0, 77.7, 75.4, 54.5, 49.3, 31.0, 28.4 (3ꢄ), 26.0, 25.4, 23.0, 9.6,
9.2; HRMS calcd for C19H33N2O6: 385.2339, found: m/z 385.2337
[M þ H]þ.
4.4.4. 4-Acetamido-5-(tert-butoxycarbonyl)amino-3-(1-
ethylpropoxy)-1-iodo-1-cyclohexene (7)
To a solution of carboxylic acid 6 (0.25 g, 0.65 mmol) in anhy-
drous CH2Cl2 (8 mL) were added Et3N (0.14 mL, 0.98 mmol), HBTU
(0.37 g, 0.98 mmol), and DMAP (catalytic amount). The mixture was
stirred at room temperature for 30 min, and 3-hydroxy-4-methyl
thiazolethione (0.13 g, 0.86 mmol) was added. The mixture was
stirred for another 3 h at room temperature, and concentrated
under reduced pressure. The residue was purified by flash chro-
matography on a silica gel column (EtOAc/hexane, 1:1e2:1) to
afford an intermediate Barton ester (0.3 g, 90%).
A solution containing the intermediate Barton ester (0.3 g,
0.58 mmol) and trifluoroethyl iodide (2 mL,1.5 mmol) in anhydrous
CH2Cl2 (0.3 M, 2 mL) was irradiated by 500 W lamp at room tem-
perature for 0.5 h. The mixture was concentrated under reduced
pressure, and purified by flash chromatography on a silica gel col-
umn (EtOAc/hexane, 1:3 to 1:1) to afford compound 7 (164 mg,
0.35 mmol, 54% overall yield). C18H31IN2O4; pale yellow solid, mp
161e163 ꢁC; TLC (EtOAc/hexane, 1:1) Rf ¼ 0.5; 1H NMR (400 MHz,
4.4. Synthesis and compound characterization
4.4.1. Compound characterization
New compounds were characterized by their physical and
spectroscopic properties (mp, TLC, [a
], IR, ESIeMS, 1H, 13C and 31P
NMR). Purity of synthetic compounds was assessed to be ꢃ95% by
CDCl3)
d
6.31 (1H, s), 5.61 (1H, d, J ¼ 16.4 Hz), 5.26 (1H, d, J ¼ 8.8 Hz),
HPLC analysis (Agilent HP-1100) on an HC-C18 column
4.08 (1H, q, J ¼ 9.6 Hz), 3.84e3.75 (2H, m), 3.28 (1H, t, J ¼ 5.6 Hz),
2.86 (1H, dd, J ¼ 4.4, 5.8 Hz), 2.60 (1H, dd, J ¼ 1.7, 4.08 Hz), 1.98 (3H,
s), 1.54e1.41 (13H, m), 0.90e0.85 (6H, m); 13C NMR (100 MHz,
(250 mm ꢄ 4.6 mm i.d., 5
mm particle size) using eluents at a flow
rate of 1.0 mL/min with detection at 214-nm wavelength. Com-
pounds 5 [29], 6 [28], 7 [29], 8f [45], 9c [29], 10c [29], 3c [29], 3e
[20] and 4c [29] were identified by comparison with the physical
CDCl3)
d 170.7, 155.8, 138.0, 94.5, 82.0, 79.6, 77.5, 53.3, 50.5, 45.1,
28.4 (3ꢄ), 26.2, 25.8, 23.4, 9.7, 9.4; HRMS calcd for C18H32IN2O4:
a
], IR, ESIeMS, 1H, 13C and
467.1407, found: m/z 467.1403 [M þ H]þ.
and spectroscopic properties (mp, TLC, [
31P NMR) as that reported previously.
4.4.2. Ethyl 4-acetamido-5-(tert-butoxycarbonyl)amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxylate (5)
4.4.5. Di[3-(1H-indol-3-yl)propyl] phosphite (8g)
A mixture of 3-(1H-indol-3-yl)propan-1-ol (0.64 g, 3.6 mmol)
and pyridine (0.43 g, 5.5 mmol) was added dropwise to a solution of
phosphorus trichloride (0.25 g, 1.8 mmol) in CH2Cl2 (12.0 mL) at
To a mixture of oseltamivir (0.75 g, 2.4 mmol) and NaHCO3
(0.77 g, 9.14 mmol) in THF/H2O (10 mL/10 mL) was added (Boc)2O
(0.52 g, 2.38 mmol). The mixture was stirred for 2 h at room tem-
perature, and concentrated under reduced pressure. The residue
was diluted with CH2Cl2, and washed with water. The aqueous
phase was extracted with CH2Cl2 (3ꢄ). The combined organic phase
was dried over MgSO4, filtered, and concentrated under reduced
pressure to give compound 5 (1.0 g, 2.4 mmol). C21H36N2O6;
colorless solid, mp 142e144 ꢁC; TLC (EtOAc/hexane, 1:3) Rf ¼ 0.16;
0
ꢁC. The mixture was stirred for 2 h, filtered, and rinsed with
cyclohexane. The filtrate was concentrated, dissolved in EtOAc, and
washed twice with brine. The organic layer was dried over MgSO4,
and concentrated under reduced pressure to give phosphite 8g
(0.44 g, 61% yield). C22H25N2O3P; pale yellow oil; TLC (EtOAc/hex-
ane; 1:1) Rf ¼ 0.33; 1H NMR (400 MHz, CDCl3)
d 7.94 (2H, br), 7.49
(2H, d, J ¼ 7.6 Hz), 7.25 (2H, d, J ¼ 7.2 Hz), 7.10 (2H, t, J ¼ 7.6 Hz),
7.00e7.05 (2H, m), 6.86 (2H, d, J ¼ 1.6 Hz), 6.75 (1H, d, J ¼ 692.4 Hz),
4.01e4.04 (4H, m), 2.79 (4H, t, J ¼ 7.2 Hz), 2.00 (4H, q, J ¼ 6.8 Hz);
1H NMR (400 MHz, CDCl3)
d
6.77 (1H, s), 5.82 (1H, d, J ¼ 8.8 Hz), 5.10
(1H, d, J ¼ 9.2 Hz), 4.23e4.15 (2H, m), 4.08e4.01 (1H, m), 4.06e3.95
(1H, m), 3.82e3.73 (1H, m), 3.36e3.31 (1H, m), 2.96 (1H, dd, J ¼ 1.8,
4.8 Hz), 2.31e2.24 (1H, m), 1.97 (3H, s), 1.57e1.45 (4H, m), 1.41 (9H,
s), 1.28 (3H, t, J ¼ 7.2 Hz), 0.91e0.85 (6H, m); 13C NMR (100 MHz,
13C NMR (100 MHz, CDCl3)
d
136.2 (2ꢄ), 127.2 (2ꢄ),121.8 (2ꢄ), 121.6
(2ꢄ), 119.1 (2ꢄ), 118.6 (2ꢄ), 114.6 (2ꢄ), 111.1 (2ꢄ), 65.2 (2ꢄ, d, 2JCe
¼ 6.1 Hz), 30.7 (2ꢄ, d, JCeP ¼ 6.8 Hz), 21.0 (2ꢄ); 31P NMR
3
P
CDCl3)
d
170.1, 165.2, 155.7, 137.2, 128.8, 82.1, 79.5, 75.8, 61.0, 54.6,
(162 MHz, CDCl3) d 9.1.

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
113
23
4.4.6. General procedure for the preparation of dialkyl
tamiphosphor 9ce9g
A mixture of iodocyclohexene 7 (130 mg, 0.28 mmol), phosphite
diester (8ce8g, 0.28 mmol) and Et3N (1.2 mL, 0.84 mmol) in
anhydrous toluene (2.0 mL) was deoxygenated by bubbling with
nitrogen for 10 min, and then added to tetrakis(-
114 ꢁC; TLC (EtOAc/hexane, 1:1) Rf ¼ 0.47; [
a]
ꢀ79.6 (c ¼ 1,
D
CH2Cl2); IR (film) 3283, 2963, 2934, 1686, 1657, 1566, 1533, 1454,
1367, 1297, 1253, 1013 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d 7.15e7.28
(10H, m), 6.59 (1H, d, J ¼ 22.0 Hz), 5.79 (1H, d, J ¼ 9.2 Hz), 5.01 (1H,
d, J ¼ 8.8 Hz), 3.95e4.08 (6H, m), 3.88e3.90 (1H, m), 3.76e3.80 (1H,
m), 3.29e3.32 (1H, m), 2.59 (1H, dt, J ¼ 17.6, 6.4 Hz), 2.13e2.20 (1H,
m), 1.93e2.04 (8H, m), 1.44e1.52 (6H, m), 1.41 (9H, s), 0.84e0.88
triphenylphosphine)palladium(0) (1 mg, 0.86
mmol) that was
placed in a round-bottomed flask under nitrogen atmosphere. The
resulting solution was gradually heated to 90 ꢁC and maintained at
this temperature for 3 h. The reaction mixture was filtered through
a pad of Celite, and the filtrate was concentrated under reduced
pressure to give yellow foam, which was purified by flash chro-
matography on a silica gel column (EtOAc/hexane ¼ 1:1) to afford
the desired phosphonate product (9ce9g).
(6H, m); 13C NMR (100 MHz, CDCl3)
d 170.7, 156.1, 141.7, 140.7, 132.0,
1
131.9, 131.85, 131.8, 128.4 (2ꢄ), 128.3 (2ꢄ), 127.2 (C-1, d, JCe
2
¼ 216.6 Hz), 125.9 (2ꢄ), 82.2, 79.7, 76.3, 76.1, 65.3 (d, JCe
P
2
3
¼ 5.4 Hz), 65.2, 54.5, 49.2 (d, JCeP ¼ 15.2 Hz), 32.1(d, JCe
¼ 6.8 Hz), 31.8, 31.4, 31.3, 29.7, 28.4 (3ꢄ), 26.2, 25.6, 23.4, 9.7, 9.3;
P
P
31P NMR (162 MHz, CDCl3)
d 18.1; HRMS calcd for C36H52N2O7P:
655.3512, found: m/z 655.3518 [M ꢀ H]ꢀ.
4.4.10. Di[3-(1H-indol-3-yl)propyl] (3R,4R,5S)-4-acetamido-5-tert-
butoxycarbonylamino-3-(1-ethylpropoxy)-1-cyclohexene-1-
phosphonate (9g)
4.4.7. Dibutyl (3R,4R,5S)-4-acetamido-5-tert-
butoxycarbonylamino-3-(1-ethyl- propoxy)-1-cyclohexene-1-
phosphonate (9d)
According to the general procedure, iodocyclohexene 7 (130 mg,
0.28 mmol) in anhydrous toluene (2.0 mL) was treated with di[3-
(1H-indol-3-yl)propyl] phosphite 8g (110 mg, 0.28 mmol), Et3N
According to the general procedure, iodocyclohexene 7 (100 mg,
0.21 mmol) in anhydrous toluene (2.1 mL) was treated with dibutyl
phosphite 8d (60 mg, 0.32 mmol), Et3N (0.66 mg, 0.64 mmol) and
(1.2 mL, 0.84 mmol) and (Ph3P)4Pd (1 mg, 0.86 m
mol) at 90 ꢁC for 3 h
(Ph3P)4Pd (10 mg, 8.6 m
mol) at 90 ꢁC for 12 h to afford phosphonate
to afford phosphonate 9g (122 mg, 72% yield) after flash chroma-
tography on a silica gel column by elution with EtOAc/hexane (1:1)
9d (73 mg, 64% yield). C26H49N2O7P; pale yellow solid, mp 159e
24
161 ꢁC; TLC (EtOAc/hexane, 1:1) Rf ¼ 0.11; [
a
]
ꢀ68.7 (c ¼ 1,
D
CH2Cl2); IR (film) 3349, 2961, 1683, 1567, 1297 cmꢀ1 1H NMR
;
and CH2Cl2/MeOH (10:1). C40H55N4O7P; pale yellow oil; TLC (EtOAc/
24
hexane, 1:1) Rf ¼ 0.12; [
a]
ꢀ79.9 (c ¼ 1, CH2Cl2); IR (film) 3285,
(400 MHz, CDCl3)
d
6.52 (1H, d, J ¼ 21.6 Hz), 6.15 (1H, d, J ¼ 9.2 Hz),
D
2965, 2930, 1687, 1530, 1456, 1367, 1258, 1230, 1168, 1012 cmꢀ1; 1H
NMR (400 MHz, CDCl3) 8.83 (1H, s), 8.59 (1H, s), 7.53e7.56 (2H,
5.22 (1H, d, J ¼ 9.2 Hz), 3.87e4.04 (6H, m), 3.72e3.77 (1H, m), 3.28e
3.31 (1H, m), 2.51e2.58 (1H, m), 2.13e2.21 (1H, m), 1.92 (3H. s),
1.55e1.63 (8H, m), 1,29e1.47 (13H, m), 0.78e0.90 (12H, m); 13C
d
m), 7.33 (2H, t, J ¼ 8.4 Hz), 7.05e7.17 (4H, m), 6.90e6.96 (2H, m),
6.52 (1H, d, J ¼ 21.6 Hz), 5.81 (1H, d, J ¼ 9.2 Hz), 4.57 (1H, d,
J ¼ 9.6 Hz), 4.11 (1H, q, J ¼ 6.8 Hz), 3.92e4.06 (4H, m), 3.75e3.82
(1H, m), 3.70 (1H, br), 3.58 (1H, dd, J ¼ 10.0, 5.2 Hz), 3.20e3.25 (1H,
m), 2.75e2.90 (4H, m), 2.34 (1H, dt, J ¼ 19.6, 6.4 Hz), 2.04 (3H, s),
1.98 (4H, t, J ¼ 8.0 Hz), 1.43e1.47 (9H, m), 1.23e1.33 (4H, m), 0.82e
NMR (100 MHz, CDCl3)
d
170.6, 155.9, 141.1 (d, 2JCeP ¼ 6.8 Hz), 127.1
1
3
(C-1, d, JCeP ¼ 181 Hz), 82.0, 79.4, 75.9, 75.7, 65.7, 65.6 (d, JCe
¼ 7.6 Hz), 54.1, 49.0 (d, 2JCeP ¼ 14.4 Hz), 32.48, 32.46, 32.42, 31.0
P
(d, 3JCeP ¼ 6.9 Hz), 28.3 (3ꢄ), 26.1, 26.0, 23.3, 18.8, 13.6, 9.5, 9.2; 31
P
NMR (162 MHz, CDCl3)
d 16.9; HRMS calcd for C26H49N2NaO7P:
0.88 (6H, m); 13C NMR (100 MHz, CDCl3)
d 170.8, 156.3, 142.3, 136.3,
555.3175, found: m/z 555.3138 [M þ Na]þ.
136.2, 131.9, 131.8, 127.9 (C-1, d, 1JCeP ¼ 84.4 Hz), 127.2, 127.1, 121.7,
4.4.8. Dihexyl (3R,4R,5S)-4-acetamido-5-tert-
butoxycarbonylamino-3-(1-ethyl- propoxy)-1-cyclohexene-1-
phosphonate (9e)
121.6, 118.6, 118.56, 114.3, 114.1, 111.4, 111.2, 82.2, 79.7, 76.4, 65.3 (d,
2
3
2JCeP ¼ 5.4 Hz), 60.4, 55.1, 48.7 (d, JCeP ¼ 15.2 Hz), 30.3 (d, JCe
¼ 6.8 Hz), 29.9, 29.8, 28.4 (3ꢄ), 26.1, 25.5, 23.5, 21.1, 21.0, 20.8,
P
14.3, 9.7, 9.2; 31P NMR (162 MHz, CDCl3)
d 18.1. HRMS (negative
According to the general procedure, iodocyclohexene 7 (80 mg,
0.17 mmol) in anhydrous toluene (1.7 mL) was treated with dihexyl
mode) calcd for C40H54N4O7P: 733.3730, found: m/z 733.3745
[M ꢀ H]e.
phosphite 8e (51 mg, 0.20 mmol), Et3N (70
(Ph3P)4Pd (10 mg, 8.6
mol) at 90 ꢁC for 12 h to afford phosphonate
9e (71 mg, 70% yield). C30H57N2O7P; yellow oil; TLC (EtOAc/hexane,
mL, 0.51 mmol) and
m
4.4.11. General procedure for the preparation of guanidino
tamiphosphor dialkyl esters 10ce10f
23
1:1) Rf ¼ 0.38e0.43; [
2959, 1687, 1655, 1561, 1366, 1297, 1253, 1173 cmꢀ1
(400 MHz, CDCl3)
a
]
ꢀ66.5 (c ¼ 1, CH2Cl2); IR (film) 3287,
D
;
1H NMR
A solution of phosphonate compound (9ce9f, 0.073 mmol) in
anhydrous CH2Cl2 (1.0 mL) was cooled to 0 ꢁC in ice-bath, and TFA
(0.055 mL, 0.73 mmol) was added. The mixture was stirred for 2 h
at room temperature, and concentrated under reduced pressure to
give brown liquid of intermediate amine, which was treated with,
d
6.59 (1H, d, J ¼ 8.8 Hz), 6.44 (1H, d, J ¼ 21.6 Hz),
5.35 (1H, d, J ¼ 9.2 Hz), 3.79e3.97 (6H, m), 3.64e3.74 (1H, m), 3.24e
3.29 (1H, m), 2.47e2.51 (1H, m), 2.11e2.17 (1H, m), 1.86 (3H, s),
1.10e1.58 (29H, m), 0.74e0.79 (12H, m); 13C NMR (100 MHz, CDCl3)
170.5, 155.8, 141.0 (d, 2JCeP ¼ 7.6 Hz), 126.9 (C-1, d, 1JCeP ¼ 180 Hz),
N,N0-bis-(tert-butoxycarbonyl)-S-methylisothiourea
(14
mg,
d
81.9, 79.1, 75.6, 75.4, 65.9, 65.8 (d, 2JCeP ¼ 3.1 Hz), 54.0, 49.0, 48.9 (d,
0.049 mmol), HgCl2 (13.3 mg, 0.049 mmol) and triethylamine
(0.018 mL, 0.14 mmol) in CH2Cl2 (1 mL). The mixture was stirred at
room temperature for 2 h, added 1 M HCl (5 mL), and extracted
with CH2Cl2 (5 mL ꢄ 3). The organic phase was dried over MgSO4,
filtered, and concentrated under reduced pressure. The residual oil
was purified by flash column chromatography (CH2Cl2/
MeOH ¼ 20:1) to yield the desired guanidino-tamiphosphor dialkyl
ester (10ce10f).
2JCeP ¼ 14.4 Hz), 31.2, 30.8 (d, JCeP ¼ 9.1 Hz), 30.34, 30.31, 30.28,
3
30.25, 28.2 (3ꢄ), 26.0, 25.5, 25.1, 23.1, 22.4, 13.9, 9.4, 9.1; 31P NMR
(162 MHz, CDCl3)
d 17.9; HRMS calcd for C30H58N2O7P: 587.3825,
found: m/z 587.3824 [M þ H]þ.
4.4.9. Di(3-phenylprop-1-yl) (3R,4R,5S)-4-acetamido-5-tert-
butoxycarbonylamino-3-(1-ethylpropoxy)-1-cyclohexene-1-
phosphonate (9f)
According to the general procedure, iodocyclohexene 7 (100 mg,
0.21 mmol) in anhydrous toluene (2.1 mL) was treated with dioctyl
4.4.12. Dibutyl (3R,4R,5S)-4-acetamido-5-[N2,N3-bis(tert-
butoxycarbonyl)guanidino]-3-(1-ethylpropoxy)-1-cyclohexene-1-
phosphonate (10d)
According to the general procedure, compound 9d (150 mg,
0.28 mmol) was treated with TFA (0.32 mL, 4.2 mmol) in CH2Cl2
phosphite 8f (75 mg, 0.24 mmol), Et3N (100
(Ph3P)4Pd (1.0 mg, 0.86
mol) at 90 ꢁC for 3 h to afford phosphonate
9f (120 mg, 86% yield). C36H53N2O7P; pale yellow solid, mp 112e
mL, 0.64 mmol) and
m

114
C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
(0.3 mL) at room temperature for 1 h to remove the t-Boc protecting
groups. The amine product in CH2Cl2 (1 mL) was treated with N,N0-
bis-(tert-butoxycarbonyl)-S-methylisothiourea (98 mg, 0.33 mmol),
HgCl2 (92 mg, 0.32 mmol) and Et3N (0.12 mL, 0.84 mmol) at room
temperature for 3 h to yield compound 10d (136 mg, 72% yield).
65.3 (d, 2JCeP ¼ 6.1 Hz), 54.4, 48.2 (d, 2JCeP ¼ 14.4 Hz), 32.0 (d, 3JCe
¼ 6.8 Hz), 31.7, 30.9, 30.8, 28.3 (3ꢄ), 28.2, 28.01 (3ꢄ), 27.96, 26.1,
P
25.6, 23.3, 9.7, 9.2; 31P NMR (162 MHz, CDCl3)
d
18.1; HRMS calcd for
C
42H64N4O9P: 799.4411, found: m/z 799.4413 [M þ H]þ.
C
32H59N4O9P; colorless oil; TLC (CH2Cl2/MeOH, 20:1) Rf ¼ 0.63;
4.4.15. General procedure for the preparation of tamiphosphor
monoesters 3ce3g and guanidino-tamiphosphor monoesters 4ce4f
A solution of tamiphosphor dialkyl ester (9ce9g, 0.18 mmol) in
CH2Cl2 (0.13 mL) was treated with TFA (0.14 mL, 19 mmol) at room
temperature for 2 h. The mixture was concentrated under reduced
pressure, dissolved in 1,4-dioxane (2.6 mL), and added 1 M KOH(aq)
(1.8 mL). The mixture was stirred at 40 ꢁC for 24e60 h (monitored
by 1H NMR), and Dowex 50W ꢄ 8 resin was added to neutralize the
solution in MeOH for 5 min. The mixture was filtered, and the
filtrate was concentrated under reduced pressure. The crude
product was purified by DE-50 anion exchange resin and C-18 gel
columns. The product was dissolved in aqueous NH4OH (2 mL,
16 M)/MeOH (5 mL), stirred at room temperature for 0.5 h, and then
lyophilized to afford tamiphosphor monoester 3ce3g. Compounds
4ce4f were similarly prepared from guanidino-tamiphosphor
dialkyl esters 10ce10f (0.13 mmol) by the procedure similar to
that for 3ce3g.
23
[
a
]
ꢀ57.6 (c ¼ 1, CH2Cl2); IR (film) 3272, 2963, 1730, 1639, 1416,
D
1367, 1307, 1249 cmꢀ1 1H NMR (400 MHz, CDCl3)
; d 11.35 (1H, s),
8.56 (1H, d, J ¼ 8.0 Hz), 6.59 (1H, d, J ¼ 21.6 Hz), 6.34 (1H, d,
J ¼ 9.2 Hz), 4.34e4.38 (1H, m), 4.12 (1H, q, J ¼ 8.4 Hz), 3.92e4.03
(5H, m), 3.30e3.34 (1H, m), 2.59e2.66 (1H, m), 2,25e2.32 (1H, m),
1.90 (3H, s), 1.23e1.66 (30H, m), 0.82e0.94 (12H, m); 13C NMR
2
(100 MHz, CDCl3)
d
170.1, 162.8, 156.6, 152.3, 141.8 (d, JCe
¼ 6.8 Hz), 126.3 (C-1, d, 1JCeP ¼ 181 Hz), 83.5, 82.5, 79.5, 76.5, 76.2,
P
2
2
65.8, 65.75, 65.6 (d, JCeP ¼ 6.1 Hz), 54.4, 48.2 (d, JCeP ¼ 14.4 Hz),
32.51, 32.45, 30.9 (d, 3JCeP ¼ 9.2 Hz), 28.3 (3ꢄ), 28.1 (3ꢄ), 26.1, 25.6,
23.3, 18.8, 13.7, 9.7, 9.3; 31P NMR (162 MHz, CDCl3)
d 17.9; HRMS
calcd for C32H58N4O9P: 673.3941, found: m/z 673.3951 [M ꢀ H]ꢀ.
4.4.13. Dihexyl (3R,4R,5S)-4-acetamido-5-[N2,N3-bis(tert-
butoxycarbonyl)guanidino]-3-(1-ethylpropoxy)-1-cyclohexene-1-
phosphonate (10e)
According to the general procedure, compound 9e (85 mg,
0.14 mmol) was treated with TFA (0.11 mL, 1.4 mmol) in CH2Cl2
(1.0 mL) at room temperature for 1 h to remove the t-Boc protecting
groups. The amine product in CH2Cl2 (0.5 mL) was treated with
4.4.16. Monobutyl (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-phosphonate (3d)
According to the general procedure, compound 9d (100 mg,
0.18 mmol) in CH2Cl2 (0.13 mL) was treated with TFA (0.14 mL,
19 mmol) to give crude amino product, which was dissolved in 1,4-
dioxane (2.6 mL) and treated with 1 M aqueous KOH (1.8 mL) at
40 ꢁC for 36 h to afford compound 3d (47 mg, 66% yield) as the
ammonium salt. The purity of product 3d was 95.2% as shown by
N,N0-bis-(tert-butoxycarbonyl)-S-methylisothiourea
(46
mg,
0.16 mmol), HgCl2 (43 mg, 0.16 mmol) and Et3N (0.06 mL,
0.43 mmol) at room temperature for 3 h to yield compound 10e
(63 mg, 60% yield). C36H67N4O9P; colorless oil; TLC (EtOAc/hexane,
22
1:1) Rf ¼ 0.32; [
a
]
e59.6 (c ¼ 1, CH2Cl2); IR (film) 3449, 3277,
D
2960, 2930, 1730, 1639, 1611, 1561, 1418, 1368, 1153 cmꢀ1; 1H NMR
(400 MHz, CDCl3)
HPLC on an HC-C18 column (Agilent, 4.6 ꢄ 250 mm, 5
mm) with
elution of MeOH/H2O (1:1), tR ¼ 6.2 min (UV detection at 214 nm
d
11.36 (1H, s), 8.58 (1H, d, J ¼ 8.0 Hz), 6.59 (1H, d,
wavelength). C17H33N2O5P; white solid; mp 227e229 ꢁC; TLC (2-
J ¼ 21.6 Hz), 6.42 (1H, d, J ¼ 9.2 Hz), 434e4.40 (1H, m), 4.11e4.16
(1H, m), 3.91e4.09 (5H, m), 3.29e3.34 (1H, m), 2.60e2.67 (1H, m),
2.25e2.33 (1H, m),1.90 (3H, s), 1.22e1.65 (38H, m), 0.80e0.89 (12H,
22
propanol/H2O/NH4OH, 10:2:3) Rf ¼ 0.61; [
a]
ꢀ9.8 (c ¼ 0.55,
D
MeOH); IR (film) 3484, 3315, 2962, 2875, 1636, 1557, 1459, 1397,
1299, 1182, 1059, 1032 cmꢀ1; 1H NMR (400 MHz, CD3OD)
d
8.19 (1H,
m); 13C NMR (100 MHz, CDCl3)
d 170.1, 162.8, 156.7, 152.4, 141.9 (d,
2JCeP ¼ 7.6 Hz), 126.2 (C-1, d, 1JCeP ¼ 182 Hz), 83.5, 82.5, 79.5, 76.5,
d, J ¼ 8.4 Hz), 6.39 (1H, d, J ¼ 18.4 Hz), 4,10 (1H, d, J ¼ 7.6 Hz), 3.96
(2H, q, J ¼ 6.4 Hz), 3.79 (2H, t, J ¼ 6.4 Hz), 3.36e3.45 (1H, m), 2.78
(1H, dt, J ¼ 16.8, 6.8 Hz), 2.35e2.42 (1H, m), 2.03 (3H,s), 1.38e1.64
2
2
76.3, 66.2, 66.1, 66.0 (d, JCeP ¼ 6.1 Hz), 54.5, 48.3 (d, JCe
¼ 14.4 Hz), 31.4, 31.0 (d, 3JCeP ¼ 9.9 Hz), 30.5, 30.4, 29.7, 28.3 (3ꢄ),
P
(8H, m), 0.87e0.97 (9H, m); 13C NMR (100 MHz, CD3OD)
d 174.8,
28.1 (3ꢄ), 26.1, 25.7, 25.28, 25.26, 23.3, 22.6, 14.1, 9.7, 9.3; 31P NMR
136.4 (d, 2JCeP ¼ 7.2 Hz), 132.6 (C-1, d, 1JCeP ¼ 171 Hz), 83.4, 76.6 (d,
(162 MHz, CDCl3)
d 17.9; HRMS calcd for C36H68N4O9P: 731.4724,
2
3
3JCeP ¼ 5.7 Hz), 65.5 (d, JCeP ¼ 171 Hz), 54.8, 51.6 (d, JCe
found: m/z 731.4734 [M þ H]þ.
¼ 13.5 Hz), 34.3 (d, 2JCeP ¼ 6.9 Hz), 30.8 (d, 3JCeP ¼ 10.7 Hz), 27.5,
P
4.4.14. Di(3-phenylprop-1-yl) (3R,4R,5S)-4-acetamido-5-[N2,N3-
bis(tert-butoxycarbonyl)guanidino]-3-(1-ethylpropoxy)-1-
cyclohexene-1-phosphonate (10f)
26.8, 23.3, 20.3, 14.3, 10.0, 9.7; 31P NMR (162 MHz, CD3OD)
d
12.2;
HRMS calcd for C17H32N2O5P: 375.2049, found: m/z 375.2051
[M þ H]þ.
According to the general procedure, compound 9f (110 mg,
0.15 mmol) was treated with TFA (0.11 mL, 1.5 mmol) in CH2Cl2
(0.5 mL) at room temperature for 1 h to remove the t-Boc protecting
groups. The amine product in CH2Cl2 (1.7 mL) was treated with
4.4.17. Mono(3-phenylprop-1-yl) (3R,4R,5S)-4-acetamido-5-
amino-3-(1-ethylpropoxy)-1-cyclohexene-1-phosphonate (3f)
According to the general procedure, compound 9f (60 mg,
0.091 mmol) in CH2Cl2 (0.70 mL) was treated with TFA (0.07 mL,
1.3 mmol) to give crude amino product, which was dissolved in 1,4-
dioxane (0.1 mL) and treated with 1 M aqueous KOH (0.91 mL) at
25 ꢁC for 48 h to afford compound 3f (25 mg, 63% yield) as the
ammonium salt. The purity of product was >99% as shown by HPLC
N,N0-bis-(tert-butoxycarbonyl)-S-methylisothiourea
(48
mg,
0.17 mmol), HgCl2 (45 mg, 0.17 mmol) and Et3N (0.07 mL,
0.46 mmol) at room temperature for 3 h to yield compound 10f
(98 mg, 75% yield). C42H63N4O9P; pale yellow oil; TLC (EtOAc/hex-
ane, 1:1) Rf ¼ 0.34; [
a]
23 ꢀ43.7 (c ¼ 1, CH2Cl2); IR (film) 2973, 2934,
D
1789, 1729, 1639, 1416, 1368, 1307, 1229, 1146 cmꢀ1
;
1H NMR
on an HC-C18 column (Agilent, 4.6 ꢄ 250 mm, 5
mm) with elution of
MeOH/H2O (40:60), tR ¼ 7.9 min (UV detection at 214 nm wave-
(400 MHz, CDCl3)
d
11.43 (1H, s), 8.70 (1H, d, J ¼ 8.4 Hz), 7.22e7.35
(10H, m), 6.70 ( 1H, d, J ¼ 22.0 Hz), 6.46 (1H, d, J ¼ 9.2 Hz), 4.47e4.55
(1H, m), 4.17e4.24 (1H, m), 4.03e4.13 (5H, m), 3.39 (1H, m), 2.76
(4H, t, J ¼ 7.6 Hz), 2.68e2.72 (1H, m), 2.32e2.38 (1H, m), 2.05 (4H, t,
length). C22H35N2O5P; white solid, mp 229e231 ꢁC; TLC (2-
22
propanol/H2O/NH4OH, 10:2:3) Rf ¼ 0.74; [
a
]
ꢀ37.0 (c ¼ 0.2,
D
MeOH); IR (film) 3445, 2964, 2939, 1455, 1375, 1296, 1060,
J ¼ 7.2 Hz), 2.00 (3H, s), 1.99e1.70 (22H, m), 0.90e0.97 (6H, m); 13
C
1030 cmꢀ1 1H NMR (400 MHz, CD3OD)
;
d
8.19 (1H, d, J ¼ 8.8 Hz),
2
NMR (100 MHz, CDCl3)
d
170.8, 162.8, 157.0, 152.6, 142.3 (d, JCe
7.12e7.26 (5H, m), 6.40 (1H, d, J ¼ 18.8 Hz), 4,10 (1H, d, J ¼ 8.0 Hz),
3.93e3.99 (1H, m), 3.82 (3H, q, J ¼ 6.4 Hz), 3.36e3.44 (2H, m), 2.80
(1H, dt, J ¼ 16.8, 7.2 Hz), 2.71 (2H, t, J ¼ 8.0 Hz), 2.37e2.44 (1H, m),
¼ 7.6 Hz), 140.9, 132.2, 132.1, 128.6, 128.4, 126.1, 126.3 (C-1, d, 1JCe
¼ 182 Hz), 83.8, 82.6, 80.1, 79.7, 77.3, 76.3, 76.1, 67.1, 65.5, 65.4,
P
P

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
115
2
2
2
2.03 (3H, s), 1.90e1.92 (2H, m), 1.46e1.58 (4H, m), 0.87e0.91 (6H,
(d, JCeP ¼ 18.9 Hz), 65.9 (d, JCeP ¼ 5.5 Hz), 56.4, 52.4 (d, JCe
m); 13C NMR (100 MHz, CD3OD)
d
174.9, 143.2, 136.7 (2ꢄ), 129.62
¼ 12.8 Hz), 32.9, 32.7 (d, 3JCeP ¼ 6.1 Hz), 32.2, 31.7, 27.5, 27.0, 23.8,
P
(2ꢄ), 128.27 (C-1, d, 1JCeP ¼ 254 Hz), 83.5, 76.7, 76.5, 65.2, 54.9, 51.6
23.0, 14.6, 10.1, 9.8; 31P NMR (162 MHz, CD3OD)
d
13.0; HRMS calcd
2
3
(d, JCeP ¼ 12.9 Hz), 34.1 (d, JCeP ¼ 6.0 Hz), 33.3, 31.0, 30.9, 27.5,
for C20H38N4O5P: 445.2580, found: m/z 445.2581 [M þ H]þ.
26.8, 23.3, 10.1, 9.7; 31P NMR (162 MHz, CD3OD)
d 12.3; HRMS calcd
for C22H36N2O5P: 439.2362, found: m/z 439.2361 [M þ H]þ.
4.4.21. Mono(3-phenylpropyl) (3R,4R,5S)-4-acetamido-3-(1-
ethylpropoxy)-5-guanidino-1-cyclohexene-1-phosphonate (4f)
According to the general procedure, compound 10f (100 mg,
0.13 mmol) in CH2Cl2 (1.25 mL) was treated with TFA (0.11 mL,
1.3 mmol) to give crude guanidino product, which was dissolved in
1,4-dioxane (1.3 mL) and treated with 1 M aqueous KOH (1.3 mL) at
25 ꢁC for 48 h to afford compound 4f (60 mg, 55% yield). The purity
of product 4f was >99% as shown by HPLC on an HC-C18 column
4.4.18. Mono[(1H-3-Indol-3-yl)propyl] (3R,4R,5S)-4-acetamido-5-
amino-3-(1-ethylpropoxy)-1-cyclohexene-1-phosphonate (3g)
According to the general procedure, compound 9g (55 mg,
0.075 mmol) in CH2Cl2 (1.0 mL) was treated with TFA (0.055 mL,
0.75 mmol) to give crude amino product, which was dissolved in
1,4-dioxane (0.75 mL) and treated with 1 M aqueous KOH (0.75 mL)
at 25 ꢁC for 48 h to afford compound 3g (15 mg, 43%) as the
(Agilent, 4.6 ꢄ 250 mm, 5
mm) with elution of MeOH/H2O (6:4),
ammonium salt.
C24H36N3O5P; TLC (2-propanol/H2O/NH4OH,
tR ¼ 7.5 min (UV detection at 214 nm wavelength). C23H37N4O5P;
7:2:3) Rf ¼ 0.71; 1H NMR (400 MHz, CD3OD)
d 7.52 (1H, d,
white solid; mp 252e254 ꢁC; TLC (2-propanol/H2O/NH4OH, 10:2:3)
22 ꢀ8.98 (c ¼ 0.2, MeOH); IR (film) 3442, 2964, 1650,
J ¼ 8.0 Hz), 7.30 (1H, d, J ¼ 8.4 Hz), 6.95e7.11 (3H, m), 6.40 (1H, d,
J ¼ 19.2 Hz), 4.05 (1H, d, J ¼ 7.6 Hz), 3.85e3.96 (3H, m), 3.61e3.66
(1H, m), 3.46e3.50 (1H, m), 2.75e2.93 (3H, m), 2.34e2.41 (1H, m),
1.91e2.08 (5H, m), 1.47e1.55 (4H, m), 0.80e0.90 (6H, m); 31P NMR
Rf ¼ 0.47; [
a]
D
1506, 1455, 1388, 1188, 1061 cmꢀ1
;
1H NMR (400 MHz, CD3OD)
d
7.12e7.24 (5H, m), 6.39 (1H, d, J ¼ 19.2 Hz), 4.08 (1H, d, J ¼ 6.8 Hz),
3.93 (1H, dd, J ¼ 9.6, 7.6 Hz), 3.74e3.85 (3H, m), 3.37e3.41 (1H, m),
2.72 (2H, t, J ¼ 8.0 Hz), 2.67e2.72 (1H, m), 2.29e2.31 (1H, m), 1.98
(162 MHz, CDCl3)
d 12.4; HRMS calcd for C24H37N3O5P: 476.2308,
found: m/z 476.2308 [M ꢀ H]e.
(3H, s),1.89e1.96 (2H, m),1.47e1.56 (4H, m), 0.87e0.93 (6H, m); 13
C
NMR (100 MHz, CD3OD)
d
174.3, 158.7, 140.3, 136.3 (2ꢄ), 129.6 (2ꢄ),
128.3 (C-1, d, 1JCeP ¼ 255 Hz), 83.5, 77.1, 77.0, 65.1, 56.0, 52.4, 34.1,
4.4.19. Monobutyl (3R,4R,5S)-4-acetamido-3-(1-ethylpropoxy)-5-
guanidino-1-cyclohexene-1-phosphonate (4d)
33.3, 32.4 (2ꢄ), 27.5, 27.0, 23.0, 10.0, 9.9; 31P NMR (162 MHz,
According to the general procedure, compound 10d (100 mg,
0.15 mmol) in CH2Cl2 (0.15 mL) was treated with TFA (0.17 mL,
22 mmol) to give crude gaunidino product, which was dissolved in
EtOHeTHF (1.5 mL/1.5 mL) and treated with 1 M aqueous KOH
(1.8 mL) at 40 ꢁC for 36 h to afford compound 4d (32 mg, 52% yield)
as the ammonium salt. The purity of product 4d was >99% as
shown by HPLC on an HC-C18 column (Agilent, 4.6 ꢄ 250 mm,
CD3OD) d 13.0; HRMS calcd for C23H38N4O5P: 481.2580, found: m/z
481.2574 [M þ H]þ.
4.5. Determination of octanolebuffer partition coefficients
The standard solutions of test compound in MeOH at various
concentrations of 1.0, 0.5, 0.1, 0.05 and 0.01 mg/mL were measured
5
m
m) with elution of MeOH/H2O (1:1), tR ¼ 5.9 min (UV detection
by HPLC (Agilent 1100 series) on an HC-C18 column (5
4.6 ꢄ 250 mm) using MeOH/H2O (1:1) as the eluent and UV de-
tector at
mm porosity,
at 214 nm wavelength). C18H35N4O5P; white solid; mp 247e249 ꢁC;
TLC (2-propanol/H2O/NH4OH, 10:2:3) Rf ¼ 0.57; [
a]
22 ꢀ8.2 (c ¼ 0.2,
D
l
¼ 214 nm. The integral area of the peak corresponding to
MeOH); IR (film) 3541, 3172, 1800, 1706, 1667, 1403, 1247, 1182,
1059, 1020 cmꢀ1 1H NMR (400 MHz, CD3OD)
; d 6.42 (1H, d,
the compound was used (average of triple experiments) to estab-
lish standard curve by Microsoft Excel.
J ¼ 19.2 Hz), 4.37 (1H, d, J ¼ 8.0 Hz), 3.99e4.05 (1H, m), 3.82e3.95
(3H, m), 3.61e3.66 (1H, m), 2.76e2.84 (1H, m), 2.42e2.48 (1H, m),
2.15 (3H, s), 1.40e1.75 (8H, m), 0.99e1.04 (6H, m), 0.96 (3H, t,
To test compound partitioning, the respective test compounds
(w1.0 mg) were placed in Eppendorf tube, and octanol (0.75 mL)
and phosphate buffer saline (0.75 mL of 0.01 M solution, pH 7.4)
were added. The solution was equilibrated at 37 ꢁC using magnetic
stirring at 1200 rpm for 24 h. The octanol and aqueous phases were
then separated by centrifugation at 6000 rpm for 5 min. Each
J ¼ 7.2 Hz); 13C NMR (100 MHz, CD3OD)
d
174.3, 158.7, 136.2 (d, 2JCe
1
3
¼ 6.4 Hz), 133.7 (C-1, d, JCeP ¼ 171 Hz), 83.4, 77.0 (d, JCe
P
¼ 19.2 Hz), 65.5 (d, 2JCeP ¼ 5.0 Hz), 56.0, 52.3 (d, 3JCeP ¼ 12.8 Hz),
P
34.3 (d, 2JCeP ¼ 7.1 Hz), 32.3 (d, 3JCeP ¼ 9.2 Hz), 27.5, 27.0, 23.0, 20.3,
14.3, 10.0, 9.9; 31P NMR (162 MHz, CD3OD)
d 13.0; HRMS calcd for
sample (25 mL) of aqueous layer was measured by HPLC. The con-
C
18H36N4O5P: 419.2423, found: m/z 419.2428 [M þ H]þ.
centration of drug in the aqueous phase was deduced by calibration
with the above-established standard curve. Five replicates of each
determination were carried out to assess reproducibility. From
these data, the apparent octanol/buffer (pH 7.4) partition coeffi-
cient, DB ¼ [Bt]oct/[Bt]aq, is determined, where [Bt]oct and [Bt]aq are
the concentrations of the drug in organic and aqueous phases,
respectively.
4.4.20. Monohexyl (3R,4R,5S)-4-acetamido-3-(1-ethylpropoxy)-5-
guanidino-1-cyclohexene-1-phosphonate (4e)
According to the general procedure, compound 10e (180 mg,
0.23 mmol) in CH2Cl2 (0.30 mL) was treated with TFA (0.18 mL,
23 mmol) to give crude guanidino product, which was dissolved in
1,4-dioxane (7.6 mL) and treated with 1 M aqueous KOH (2.3 mL) at
25 ꢁC for 60 h to afford compound 4e (68 mg, 68% yield) as the
ammonium salt. The purity of product was >96% as shown by HPLC
4.6. Determination of influenza virus TCID50
on an HC-C18 column (Agilent, 4.6 ꢄ 250 mm, 5
m
m) with elution of
MDCK cells were obtained from American Type Culture Collec-
tion (Manassas, VA), and were grown in DMEM containing 10% fetal
bovine serum and penicillin-streptomycin at 37 ꢁC under 5% CO2.
The TCID50 (50% tissue culture infectious dose) was determined by
MeOH/H2O (70:30), tR ¼ 8.7 min (UV detection at 214 nm wave-
length). C20H39N4O5P; white solid, mp 245e247 ꢁC; TLC (2-
22
propanol/H2O/NH4OH, 10:2:3) Rf ¼ 0.63; [
a
]
ꢀ44.6 (c ¼ 1,
D
MeOH); IR(film) 3325, 2960, 2935, 2860, 1661, 1556, 1462, 1375,
incubation of serially diluted influenza virus in 100 mL solution with
1319, 1188, 1060, 1016 cmꢀ1; 1H NMR (400 MHz, CD3OD)
d
6.37 (1H,
100
m
L MDCK cells at 1 ꢄ 105 cells/mL in 96-well microplates. The
d, J ¼ 19.2 Hz), 4.08 (1H, m), 3.92 (1H, t, J ¼ 10 Hz), 3.74e3.83 (3H,
m), 3.38e3.43 (1H, m), 2.64e2.71 (1H, m), 2.02e2.31 (1H, m), 1.98
(3H, s), 1.32e1.71 (12H, m), 0.88e0.94 (9H, m); 13C NMR (100 MHz,
infected cells were incubated at 37 ꢁC under 5% CO2 for 48e72 h
and added to each wells with 100 mL per well of CellTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay reagent (Promega). After
CD3OD)
d
174.2, 158.7, 136.5, 133.6 (C-1, d, 1JCeP ¼ 171 Hz), 83.4, 77.1
incubation at 37 ꢁC for 15 min, absorbance at 490 nm was read on a

116
C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
plate reader. Influenza virus TCID50 was determined using Reede
Muench method [46,47].
supernatant was removed and was evaporated to dryness by ni-
trogen gas. The residue was reconstituted with 100 L of 50%
methanol and an aliquot of 50 L of sample was injected into a
m
m
4.7. Determination of IC50 of neuraminidase inhibitors
HPLC system. For protein assay, cells were solubilized in 1.0 mL of
1% Triton X-100, and the protein content of the cells was deter-
mined using the DC protein assay (Bio-Rad) with bovine serum
albumin as the standard.
The neuraminidase (NA) activity was measured using diluted
allantoic fluid harvested from influenza virus infected embryonated
eggs. A fluorometric assay was used to determine the NA activity
Caco-2 cells (5 ꢄ 104 cells) were seeded into millicell insert
(4.2 cm2 culture area, Millipore, Merck, USA), and the culture me-
dium was changed every 2 days. The differentiation status of the
monolayer was evaluated by measuring the transepithelial elec-
trical resistance (TEER) (Millicell ERS epithelial volt-ohm meter,
with a fluorogenic substrate 2-(4-methylumbelliferyl)-a-D-N-ace-
tylneuraminic acid (MUNANA; Sigma). For inhibition test, the
compound of interest was incubated with diluted virus-infected
allantoic fluid for 10 min at room temperature followed by the
addition of 200
m
M of substrate. The fluorescence of the released 4-
Millipore).
values > 300 U
Permeability
cm2. The experiments were conducted for time-
g/mL of test com-
was
conducted
until
TEER
methylumbelliferone was measured in Envision plate reader (Per-
kineElmer, Wellesley, MA) using excitation and emission wave-
lengths of 365 and 460 nm, respectively. Inhibitor IC50 values, i.e.,
the concentrations of the compound required for 50% inhibition of
the NA, were measured in triplicate and determined from the
doseeresponse curves by plotting the percent inhibition of NA
activity versus inhibitor concentrations using Prism 5 (GraphPad
Software, Inc., San Diego, CA).
dependency by incubating cells with 100
pound for 15e120 min. First, the cells were rinsed three times with
HBSS (1 mM CaCl2, 0.5 mM MgCl2, 5 mM KCl, 10 mM HEPES, 1 mM
Na2HPO4, 140 mM NaCl, 5 mM D-glucose, pH 7.4). HBSS was loaded
to the receiver side (1.5 mL) before incubation and the drug solution
was added to donor side (apical (AP) side or basolateral (BL) side)
(1.5 mL). An aliquot (200
(15, 30, 60,120 min) and 50
each sampling, 200 L of HBSS was supplemented to receiver side
m
mL) was collected from the receiver side
mL was directly injected into HPLC. After
4.8. Determination of EC50 and CC50 of neuraminidase inhibitors
m
to maintain a constant volume of each side.
The anti-influenza virus activities of NA inhibitors were
measured by the EC50 values, i.e., the concentrations of the com-
pound required for 50% protection of the influenza virus infection-
The HPLC system (Hitachi High-Technologies Co., Tokyo, Japan)
consisted of a model L-2130 pump, a model L-2200 Autosampler, a
model L-2400 ultraviolet detector, and a 4.6 mm ꢄ 250 mm
mediated cytopathic effects (CPE). Fifty to 100
mL of diluted influ-
Mightysil column (RP-18, 5 mm; MetaChem, CA, USA). The mobile
enza virus (100 TCID50) were mixed with equal volumes of NA in-
hibitors at varied concentrations. The mixtures were then used to
phase consisted of 50% methanol in water (for 4c) or 65% methanol
in water (for 4e and 4h). The sample was analyzed at flow rate of
1.0 mL/min and was detected by ultraviolet at a wavelength of
214 nm. The lower quantitation limits were 500 ng/mL.
infect 100
m
L of MDCK cells at 1 ꢄ 105 cells/mL in 96-wells. After
48e72 h incubation at 37 ꢁC under 5% CO2, the CPE were deter-
mined with CellTiter 96 AQueous Non-Radioactive Cell Proliferation
Assay reagent as described above, or alternatively with 0.11 final
percentage of neutral red for 2 h. Inhibitor EC50 value were
measured in triplicate and determined by fitting the curve of
percent CPE versus the concentrations of NA inhibitor using Graph
Pad Prism 4.
4.10. Pharmacokinetic study
Compound 4e was administered as aqueous solutions in normal
saline. Male ICR mice were purchased from BioLASCO Taiwan Co.,
Ltd. The compound was administered to mice as a single i.v. dose
(0.25 mg/kg of body weight) or as a single oral dose (10 mg/kg).
Plasma were prepared from tail lateral vein bleeds at 0.17, 0.33, 0.67,
1, 2, 4, 6, 8, and 12 h, and then stored at ꢀ70 ꢁC. Deproteinized
plasma samples were centrifuged for 10 min at 13,000 g and the
supernatant was transferred to a plate for LC/MS/MS analysis.
The pharmacokinetic parameters were obtained using a phar-
The CC50 values (50% cytotoxic concentrations) of NA inhibitors
to MDCK cells were determined by the procedures similar to the
EC50 determination but without virus infection.
4.9. Cellular uptake and intestinal permeability
The MDCK type II cells (or Caco-2 cells) were continuously
cultured in minimum essential medium supplemented with 0.11 g/
L of sodium pyruvate, 1.5 g/L of sodium bicarbonate, 1% antibiotics
macokinetic program WinNonlin, fitting data to
a
non-
compartmental model. The pharmacokinetic parameters including
the area under the plasma concentration-versus-time curve (AUC)
to the last sampling time, (AUC0/12), to the time infinity (AUC0/N),
the terminal-phase half-life (T1/2), the maximum concentration of
compound in plasma (Cmax), the time of Cmax (Tmax), and the first
order rate constant associated with the terminal portion of the
curve (k) were estimated via linear regression of time versus log
concentration. The total plasma clearance (CL) was calculated as
dose/AUCi.v.. The oral bioavailability (F) of the test compound by
oral administration was calculated from the AUC0eN of the oral
dose divided by the AUC0/N of the i.v. dose.
(10,000 U/mL of penicillin,10 mg/mL of streptomycin, and 25 mg/mL
of amphotericin B), and 10% (for MDCK cells) or 20% (for Caco-2
cells) fetal bovine serum. The cells were routinely maintained in
75 cm2 flasks at 37 ꢁC in a 5% CO2 and 95% humidity atmosphere.
The culture medium was changed every 2 days. Cells were passaged
after reaching 80% confluence. For subculturing, the cells were
removed enzymatically (0.25% trypsin/EDTA), and subcultured in a
T75 flask.
MDCK-II cells (5 ꢄ 104 cells) were grown on 6-well Nunclon
Multidishes (9.6 cm2 culture area, polystyrene, Nunc, Denmark)
and were used for experiments 5 days after seeding. The experi-
ments were conducted for time-dependency by incubating cells
4.11. Animal experiments for virus challenges
with 100
m
g/mL of test compound (4c, 4e or 4h) for 30e120 min.
Female BALB/c mice (18e20 g) were obtained from Charles River
Laboratories or National Laboratory Animal Center (Taiwan). The
mice were quarantined for 48e72 h before use. The mice were
anesthetized by intraperitoneal (i.p.) injection of zoletil (or keta-
The cells were then washed with 1.5 mL of ice-cold ECF buffer
(122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM
HEPES, 25 mM NaHCO3, 0.4 mM K2HPO4, 10 mM D-glucose, pH 7.4)
three times. The cells were then scrapped and sonicated in 1 mL
mine/xylazine) and inoculated intranasally with 25
influenza virus. The test compounds were dissolved in sterile water,
mL of infectious
methanol. After centrifugation (14,000 rpm, 25 ꢁC, 10 min), the

C.-L. Chen et al. / European Journal of Medicinal Chemistry 81 (2014) 106e118
117
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4.13. Ethical regulation of laboratory animals
This study was conducted in accordance with the approval of
the Institutional Animal Care and Use Committee, Academia Sinica,
Taiwan. The work was done in the BSL-3 Laboratory of Genomics
Research Center, Academia Sinica, Taiwan.
Acknowledgments
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virus which utilize selective binding to the neuraminidase, Org. Biomol. Chem.
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We thank Academia Sinica and National Science Council for
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.04.
082. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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