Development of oseltamivir phosphonate congeners as anti-influenza agents

Article abstract of DOI:10.1021/jm3008486

Oseltamivir phosphonic acid (tamiphosphor, 3a), its monoethyl ester (3c), guanidino-tamiphosphor (4a), and its monoethyl ester (4c) are potent inhibitors of influenza neuraminidases. They inhibit the replication of influenza viruses, including the oseltamivir-resistant H275Y strain, at low nanomolar to picomolar levels, and significantly protect mice from infection with lethal doses of influenza viruses when orally administered with 1 mg/kg or higher doses. These compounds are stable in simulated gastric fluid, liver microsomes, and human blood and are largely free from binding to plasma proteins. Pharmacokinetic properties of these inhibitors are thoroughly studied in dogs, rats, and mice. The absolute oral bioavailability of these compounds was lower than 12%. No conversion of monoester 4c to phosphonic acid 4a was observed in rats after intravenous administration, but partial conversion of 4c was observed with oral administration. Advanced formulation may be investigated to develop these new anti-influenza agents for better therapeutic use.

Full text of DOI:10.1021/jm3008486

Article
pubs.acs.org/jmc
Development of Oseltamivir Phosphonate Congeners as Anti-
influenza Agents
Ting-Jen R. Cheng,† Steven We∥inheimer,‡ E. Bart∥Tarbet,§ Jia-Tsron∥g Jan,† Yih-Shyun E. Cheng,†
Jiun-Jie Shie,† Chun-Lin Chen, Chih-An Chen, Wei-Che Hsieh, Pei-Wei Huang,⊥ Wen-Hao Lin,⊥
Shi-Yun Wang,† Jim-Min Fang,*,†, ∥ Oliver Yoa-Pu Hu,*,⊥ and Chi-Huey Wong*,†
†The Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei 11529, Taiwan
‡TaiMed Biologics, 5251 California Avenue, Suite 230, Irvine, California 92617, United States
§Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah 84322,
United States
∥Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan
⊥School of Pharmacy, National Defense Medical Center, No. 161, Sec. 6, Minquan E. Road, Taipei 114, Taiwan
S
* Supporting Information
ABSTRACT: Oseltamivir phosphonic acid (tamiphosphor,
3a), its monoethyl ester (3c), guanidino-tamiphosphor (4a),
and its monoethyl ester (4c) are potent inhibitors of influenza
neuraminidases. They inhibit the replication of influenza
viruses, including the oseltamivir-resistant H275Y strain, at low
nanomolar to picomolar levels, and significantly protect mice
from infection with lethal doses of influenza viruses when
orally administered with 1 mg/kg or higher doses. These
compounds are stable in simulated gastric fluid, liver
microsomes, and human blood and are largely free from binding to plasma proteins. Pharmacokinetic properties of these
inhibitors are thoroughly studied in dogs, rats, and mice. The absolute oral bioavailability of these compounds was lower than
12%. No conversion of monoester 4c to phosphonic acid 4a was observed in rats after intravenous administration, but partial
conversion of 4c was observed with oral administration. Advanced formulation may be investigated to develop these new anti-
influenza agents for better therapeutic use.
of Glu119, Asp151, and Glu 227 in the active site of influenza
NA.6 However, compounds 2a (GOC) and 2b (GOS) have not
been developed for therapeutic use. Two other anti-NA drugs,
peramivir8,9 and laninamivir,10,11 were recently approved for use
as intravenous and inhalation anti-influenza drugs, respectively.
Due to the extensive use of oseltamivir in influenza therapy,
oseltamivir-resistant viruses have emerged over the years. New
anti-influenza drugs that can also inhibit oseltamivir-resistant
strains, such as the H275Y mutant, are urgently needed for our
battle against the threat of pandemic influenza.
We have recently discovered tamiphosphor (TP, 3a in Figure
1),12 an oseltamivir phosphonate congener, which is highly active
for the inhibition of influenza neuraminidase and influenza virus
replication. Replacement of the carboxylate group with the
phosphonate group has resulted in a better binding to the
neuraminidase and thus has become effective against the
oseltamivir-resistant mutants. Moreover, guanidino-tamiphos-
phor (TPG, 4a), derived by replacing the amino group at the C-5
position with a more basic guanidino group, can furthermore
effectively inhibit the H275Y mutant of influenza neuraminidase.
INTRODUCTION
■
Influenza viruses infect humans every year through seasonal and
pandemic infections due to the appearance of new influenza
strains. For the prevention and treatment of influenza infections,
vaccines and anti-influenza drugs are available. Though vaccines
have been important for the protection of seasonal and pandemic
influenza infections, the production needs to be started months
before the onset of influenza infection. Furthermore, accurate
prediction of the incoming influenza strains remains a major
challenge. In the absence of an effective vaccine or for the
treatment of influenza infections in unprotected individuals, anti-
influenza drugs are needed.
Currently, the most effective anti-influenza drugs are
neuraminidase (NA) inhibitors.1−3 Zanamivir (ZA) is the first
marketed anti-influenza drug that is administered by inhalation
or intranasal spray.4,5 The phosphate salt of oseltamivir (OS, 1b
in Figure 1), is the most popular orally available drug for
influenza treatment.6,7 Oseltamivir is converted by endogenous
esterase to oseltamivir carboxylate (OC, 1a), which is the active
inhibitor of influenza neuraminidase. Replacement of the amine
group at the C-5 position with a more basic guanidino group (2a)
shows better inhibitory activity than that of OC, presumably due
to the stronger electrostatic interactions with the acidic residues
Received: June 15, 2012
© XXXX American Chemical Society
A
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oseltamivir carboxylate (OC, 1a), by strong electrostatic
interactions of the carboxylate group with the three arginine
residues (Arg118, Arg292, and Arg371) in the active site of NA.
The phosphonate group was used as a bioisostere of carboxylate
in drug design,15−17 because the phosphonate ion exhibits a
stronger electrostatic interaction with the guanidinium ion.18
Consistent with this rationale, tamiphosphor (3a), containing a
phosphonate group, was found to exhibit higher inhibitory
activity than OC against various influenza viruses, including A/
H1N1 (wild-type and H275Y mutant), A/H5N1, A/H3N2, and
type B viruses (Table 1). As an ester, oseltamivir is inactive to
NA. In contrast, TP monoester 3c is active in NA inhibition
because it still retains a negative charge at the phosphonate
monoester group to provide sufficient electrostatic interactions
with the three arginine residues in NA. Consistent with this
rationale, the NA inhibitory activity of TP diethyl ester (3b)
decreased dramatically (IC50 > 3 μM) against A/WSN/33
(H1N1) virus (data not shown).
The presence of an amino group at the C-5 position enhanced
the affinity of OC to the residues of Glu119, Asp151, and Glu227
in the active site of NA.1−3 Replacing the C-5 amino group with a
stronger base of guanidine should increase the electrostatic
interactions with the acidic residues in the NA active site. Indeed,
TPG (4a) showed an enhanced NA inhibitory activity in
comparison with OC and ZA. Notably, TPG and its monoester
4c also showed remarkable inhibitory activities against the
H275Y oseltamivir-resistant strain with IC50 values of 0.4 and 25
nM, respectively.
Figure 1. Chemical structures of anti-influenza agents.
Inhibition of Viruses in Cell Culture. The anti-influenza
activities of phosphonate compounds 3a, 3c, 4a, and 4c were
examined using a variety of influenza strains (Table 2). All four
compounds are very potent anti-influenza agents with EC50
values ranging from low nanomolar to picomolar levels.
Comparable anti-influenza activities were noted between the
phosphonic acids and phosphonate monoesters (3a vs 3c, and 4a
vs 4c), even though the phosphonic acids were more potent NA
inhibitors than the monoesters (Table 1). This result may be due
to the improved lipophilicity of monoesters with enhanced
intracellular uptake. We also noted that guanidino-tamiphosphor
(4a) and its monoester 4c were effective against the oseltamivir-
resistant influenza strains in the cell-based assays. The anti-
As a continuing study on the development of TP and TPG as
promising anti-influenza drugs, we report herein the pharmaco-
logical, including pharmacokinetic, properties of these com-
pounds and their monoethyl esters TP1Et (3c) and TPG1Et
(4c).
RESULTS
■
Synthesis of Oseltamivir Phosphonate Congeners. We
have previously established methods for the synthesis of
oseltamivir phosphonate congeners using D-xylose or bromo-
benzene as the starting material (Scheme 1).12,13 In brief, D-
xylose was modified to intermediate B, bearing a diphosphona-
tomethyl substituent at the C-5 position for the subsequent
intramolecular Horner−Wadsworth−Emmons reaction to con-
struct the scaffold of cyclohexenephosphonate (C). In another
approach, microbial oxidation of bromobenzene provided the
enantiopure bromoarene cis-1,2-dihydrodiol (D), which was
elaborated by a series of functional group transformations to give
an intermediate F. Substitution of the bromine atom in F with a
phosphonate group was realized by a palladium-catalyzed
coupling reaction to give compound C. Reduction of the azido
group in C afforded TP diethyl ester (3b). Treatment of 3b with
bromotrimethylsilane gave the phosphonic acid 3a, whereas
treatment with sodium ethoxide afforded the monoester 3c. By a
similar procedure, the N2,N3-bis(tert-butoxycarbonyl) derivative
of TPG diethyl ester (4b) was treated with bromotrimethylsilane
to give TPG (4a) by concurrent removal of two diethyl groups
and two tert-butoxycarbonyl groups. TPG monoester 4c was
obtained by consecutive treatments of 4b with sodium ethoxide
and acidic resin (Amberlite IR-120).
influenza cell protection activity of TPG monoester 4c (EC50
=
0.9−1.0 μM) against oseltamivir-resistant influenza was more
potent than oseltamivir (EC50 >10 μM) by over 10-fold. With
regard to toxicity, the potent anti-influenza agents 3a, 3c, 4a, and
4c were nontoxic to MDCK cells at the highest testing
concentrations (>100 μM).
Animal Experiment against A/WSN/33 (H1N1) Virus.
We then examined if the phosphonate compounds can protect
mice from virus-induced mortality and weight loss. When
compounds 3a, 3c, 4a, and 4c were administered orally to virus
A/WSN/33-infected mice at 10 mg/kg/day, the mice survived
and the weight recovered gradually 8 days after infection (Figure
2A and 2D). No significant protection was observed on
treatment with the phosphonate diester 3b, consistent with its
low NA inhibitory activity due to the lack of an ionic phosphate
moiety to interact with the three arginine residues in NA. Unlike
OS which is readily subject to enzymatic hydrolysis to give active
OC, there is no corresponding endogenous phosphotriesterase
to cleave the diethyl groups in 3b. When the infected mice were
treated with test compounds at 1 mg/kg/day, TPG monoester 4c
showed better protection activity in mice survival than other
compounds (Figure 2B and 2E). The phosphonate monoesters
Inhibition of Influenza Neuraminidases. The structure-
based design of NA inhibitors has been a successful strategy in
discovery of anti-influenza drugs.14 According to the structural
analysis, influenza NA interacts with the inhibitor, e.g.,
B
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Scheme 1. Outline for the Syntheses of Phosphonate Compounds 3a−3c and 4a−4c Using D-Xylose or Bromobenzene As the
a
Starting Material (3a and 4a: R1 = R2 = H; 3b and 4b: R1 = R2 = Et; 3c and 4c: R1 = H, R2 = Et)
a
Reagents and conditions:12,13 (i) Me2CO, H2SO4, CuSO4, 25 °C, 24 h; (ii) 0.05 M HCl, 50 °C, 1 h; 95% (two steps); (iii) Me3CCOCl, pyridine, 0
°C, 8 h; 89%; (iv) pyridinium dichromate, Ac2O, reflux, 1.5 h; HONH2−HCl, pyridine, 60 °C, 24 h; 82%; (v) LiAlH4, THF, 0 °C, then reflux 1.5 h;
88%; (vi) Ac2O, pyridine, 25 °C, 3 h; HCl/1,4-dioxane (4 M), BnOH, toluene, 0−25 °C, 24 h; 85%; (vii) 2,2′-dimethoxypropane, toluene, cat. p-
TsOH, 80 °C, 4 h; 90%; (viii) (CF3SO2)2O, pyridine, CH2Cl2, −15 °C, 2 h; (ix) H2C[PO(OEt)2]2, NaH, cat. 15-crown-5, DMF, 25 °C, 24 h; 73%;
(x) H2, Pd/C, EtOH, 25 °C, 24 h; (xi) EtONa, EtOH, 25 °C, 5 h, 80%; (xii) (PhO)2PON3, (i-Pr)NCN(i-Pr), PPh3, THF, 25 °C, 48 h; (xiii)
HCl, EtOH, reflux, 1 h; 74%; (xiv) (CF3SO2)2O, pyridine, CH2Cl2, −15 to −10 °C, 2 h; (xv) KNO2, 18-crown-6, DMF, 40 °C, 24 h; 71%; (xvi)
Cl3CC(NH)OCHEt2, CF3SO3H, CH2Cl2, 25 °C, 24 h; 82%; (xvii) H2, Lindlar catalyst, EtOH, 25 °C, 16 h; giving 3b (85%); (xviii) treating 3b or
4b with TMSBr, CHCl3, 25 °C, 24 h; giving 3a (85%) or 4a (75%); (xix) treating 3b or 4b with EtONa, EtOH, 25 °C, 16 h; giving 3c (82%) or 4c
(75%); (xx) treating 3b with N,N′-bis(tert-butoxycarbonyl)thiourea, HgCl2, Et3N, DMF, 0−25 °C, 10−16 h; 58%; (xxi) CF3CO2H, CH2Cl2, 0 °C, 1
h; giving 4b (72%); (xxii) E. coli JM109 (pDTG601), 37 °C, 3.5 h; 65%; (xxiii) 2,2′-dimethoxypropane, cat. H+, acetone, 0−25 °C, 0.5 h; (xxiv) N-
bromoacetamide, cat. SnBr4, H2O, CH3CN, 0 °C, 8 h; 75% (two steps); (xxv) (Me3Si)2NLi, THF, −10 to 0 °C, 0.5 h; (xxvi) 3-pentanol, BF3·OEt2,
−10 to 0 °C, 6 h; 73% (two steps); (xxvii) conc. HCl, MeOH, 50 °C, 6 h; 94%; (xxviii) AcOCMe2COBr, THF, 0−25 °C, 3.5 h; (xxix) LiBHEt3,
THF, 0−25 °C, 2 h, 82% (two steps); (xxx) (PhO)2PON3, (i-Pr)NCN(i-Pr), PPh3, THF, 40 °C, 24 h; 84%; (xxxi) diethyl phosphite, cat.
Pd(PPh3)4, 1,4-diazabicyclo[2.2.2]octane, toluene, 90 °C, 12 h; giving compound C (83%).
a
Table 1. IC50 Values (nM) of Influenza Neuraminidase Inhibition
virus
OC
ZA
3a
3c
4a
4c
A/WSN/33 (H1N1)
2.6 1.4
477 181
0.73 0.15
3.1 0.9
2.9 0.4
36 6.9
2.9 1.4
1.6 0.4
4.8 0.2
13.8 2.7
22.9 7.8
32.1 8.4
2.4 0.8
993 666
0.52 0.26
1.8 0.06
1.6 0.6
3.0 2.0
1679 43
2.2 0.6
0.09 0.05
0.4 0.1
1.1 0.1
25.1 6.6
b
A/WSN/33/H275Y (H1N1)
NIBRG14 (H5N1)
0.04 0.01
0.3 0.06
0.35 0.05
2.1 0.7
17.4 8.6
A/Taiwan/3446/2002 (H3N2)
A/Udorn/1972 (H3N2)
B/Taiwan/70641/2004
5.9 0.1
131.7 16.6
114 20.3
124.8 32.4
3.8 1.1
73.6 14
80.9 12.7
a
A fluorescent substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA), was used to determine the IC50 values that are
compound concentrations causing 50% inhibition of different influenza neuraminidase enzymes. Data are shown as mean SD of three experiments.
b
A/WSN H275Y virus by oseltamivir selection.
3c and 4c at 0.1 mg/kg/day can still exert partial protection
(Figure 2C and 2F), indicating that these two compounds may
have superior efficacy in the mouse infection model.
Figure 3 shows the survival results (A, B, and C) and weight
changes (D, E, and F) following nasal virus challenge. The
survivals of the groups treated with 10 and 1 mg/kg/day were
significantly different from the control group (Figure 3A and 3B).
However, the body weight recovery was slow and slight in mice
treated with 4a or 4c at 10 mg/kg, while the body weight
recovery was not observed in mice treated with either compound
at 1 mg/kg (Figure 3D and 3E). In fact, low body weights
persisted through day 20, and a number of mice died late in the
study on days 20 and 21 (data not shown). For the group treated
with compounds at 0.1 mg/kg/day, no significant protection was
observed except a single mouse that rapidly recovered in the
group treated with 4a (Figure 3C and 3F).
Animal Experiment against A/CA/04/2009 (H1N1,
pandemic) Virus. Compounds 4a and 4c were further tested
for their activities against pandemic influenza A (H1N1) virus A/
CA/04/2009 infection in BALB/c mice. Both compounds were
orally administered twice daily using 0.1, 1.0, and 10 mg/kg/day
doses and were compared to groups of mice receiving water
under the same treatment regimen. Mice were treated for 5 days
beginning 4 h before virus exposure and observed for weight loss
and mortality following virus challenge.
C
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a
Table 2. EC50 Values (μM) against Different Influenza Viruses Determined by Cytopathic Effect Inhibition Assays
virus
3a
3c
4a
4c
A/WSN/33 (H1N1)
0.084 0.016
0.235 0.16
7.4 3.7
0.047 0.004
0.165 0.007
0.49 0.47
0.44 0.09
>10
0.36 0.283
3.6 0.5
3.2 0.6
4.6 2.7
>10
0.18 0.014
1.4 0.283
0.9 0.2
1.0 0.22
>10
A/CA/07/2009 (pandemic H1N1)
b
A/WSN/33/H275Y (H1N1)
A/Hong Kong/2369/09/H275Y (pandemic H1N1)
A/Brisbane/10/2007 (H3N2)
A/Victoria/3/75 (H3N2)
6.55 0.36
>10
0.043 0.04
0.008 0.001
0.007 0.0003
2.0 0.28
0.041 0.05
0.002 0.005
0.005 0.0006
0.035 0.04
0.007 0.0001
0.475 0.049
>10
0.034 0.04
0.014 0.003
0.2 0.004
>10
A/Panama/2007/99 (H3N2)
A/Duck/MN/1525/81 (H5N1)
B/Florida/4/2006
1.1
0
B/Sichuan/379/99
4.25 0.64
0.695 0.31
>10
>10
a
The anti-influenza activities against different influenza strains were measured as EC50 values that are the compound concentrations for 50%
b
protection of the cytopathic effects due to the infection by different influenza strains. Data are shown as mean SD of three experiments. A/WSN
reassortant H275Y virus.
Figure 2. Percentage of survival (A, B, C) and average body weight (D, E, F) of mice orally administered with test compounds at indicated
concentrations twice per day and challenged with 10× LD50 of A/WSN/33 (H1N1) influenza virus. The number of mice and the average weight of the
mice at day 0 (10 mice per group) were defined as 100%, respectively. ***: P < 0.0001. **: P < 0.001. *: P < 0.05.
Animal Experiment against Oseltamivir-Resistant
H275Y H1N1 Virus. We further examined if these four
phosphonate compounds, 3a, 3c, 4a, and 4c, could protect
mice against a challenge infection with an oseltamivir-resistant
influenza A (H1N1) virus A/WSN/33/H275Y in 10× LD50. All
four compounds and oseltamivir completely protected mice from
death as well as weight loss at the 10 mg/kg/day dose (Figure 4A
and 4D). At the dose of 1 mg/kg/day, significant protection to
D
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Figure 3. Percentage of survival (A, B, C) and average body weight (D, E, F) of mice orally administered with various compounds at indicated
concentrations and challenged with 1 × 105 (3× LD50) cell culture infectious doses (TCID50) of influenza A/CA/04/2009 (H1N1) virus. Mice were
orally administered with test compounds 4 h prior to virus challenge and every day at a 12 h interval as indicated. The number of mice and the average
weight of the mice at day 0 (10 mice per group) were defined as 100%, respectively. ***: P < 0.0001. **: P < 0.001.
improve mice survival was observed in the groups treated with
phosphonates (3a, 3c, 4a, or 4c) compared to oseltamivir or
water-treated mice (Figure 4B and 4E). No significant protection
was noted by any compound treated at the dose of 0.1 mg/kg/
day (Figure 4C and 4F). These data indicate that the
phosphonate congeners are superior to oseltamivir in improving
the survival of infected mice when challenged with oseltamivir-
resistant influenza A (H1N1) viruses.
Animal Experiment against Avian Influenza H5N1
Virus. We also examined the dose−response effect of the
compounds against a challenge infection with reassortant virus
NIBRG-14, which contains HA and NA genes from the A/Viet
Nam/1194/2004 (H5N1). At the dose of 10 mg/kg/day,
significant protections were noted in mice treated with all tested
oseltamivir phosphonate congeners compared to the mice
treated with water (Figure 5A and 5D). No statistically significant
differences were observed among these four treatment groups,
though the phosphonate congeners showed an effect somewhat
more beneficial than that of oseltamivir. At the dose of 1 mg/kg/
day compounds, the treated groups provided partial protection
against virus challenge relative to the mice treated with water
(Figure 5B and 5E). At the dose of 0.1 mg/kg/day, no treatment
groups showed statistically significant protection (Figure 5C and
5F).
In summary, the mouse experiments demonstrated the
antiviral efficacy of oseltamivir phosphonate congeners,
especially the guanidino compounds 4a and 4c, by improving
mouse survival at the dose of 10 or 1 mg/kg/day following
challenge with A/WSN/33 (H1N1), A/CA/04/2009 (H1N1),
A/WSN/33/H275Y (oseltamivir-resistant strain) or avian
H5N1 influenza viruses.
Partition of Anti-influenza Compounds between
Octanol and Phosphate Buffer. Lipophilicity is an important
factor for the pharmacokinetic behavior of drugs. A measure of
lipophilicity can be deduced from the partition coefficient (P) of
a compound between octanol and water. Most oral drugs are
E
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Figure 4. Survival (A, B, C) and body weight (D, E, F) of mice challenged with 10× LD50 of oseltamivir-resistant influenza H275Y (A/WSN reassortant)
virus. Mice were orally administered with test compounds 4 h prior to virus challenge and every day at a 12 h interval as indicated. ***: P < 0.0001. *: P <
0.05.
found to have log P values between −1 and 5.19 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. OC exhibits a high polarity with log D value of −1.50 at
pH 7.4,20 whereas OS is an oral drug with an improved
lipophilicity (log D = 0.36) by changing the carboxylic group in
OC to the ethyl ester. Table 3 shows the calculated and measured
values of log D for a series of oseltamivir and phosphonate
derivatives. All the diethyl and monoethyl ester derivatives with
increased lipophilicity had log D values higher than those of their
parent acids. Despite having double negative charges on the
phosphonate group, tamiphosphor 3a (log D = −1.04) appeared
to be less hydrophilic than OC (log D = −1.69), which carries a
single negative charge. Interestingly, we also found that
guanidino compounds 2a (log D = −1.41) and 4c (log D =
−0.37) were more lipophilic than their corresponding amino
compounds 1a (log D = −1.69) and 3c (log D = −0.75). This
result was in agreement with the trend of calculation (clogD).
The improved lipophilic property of 2a and 4c might be related
to their zwitterionic structures, in which the C-5 guanidinium
could pair intramolecularly with the carboxylate or phosphonate
(single-charged) groups.21,22
Stability in Simulated Gastric Fluid. Compound 4c was
stable in simulated gastric fluid (SGF, without enzymes), and
87% of 4c remained after incubation for 30 min at room
temperature (data not shown). No apparent precipitation was
observed. According to the LC/MS/MS analysis, there was no
statistically significant difference by incubation in the presence or
absence of SGF.
Metabolic Stability in Human, Dog, and Rat Whole
Blood. TPG monoester 4c was found to be relatively stable in
human, rat, and dog whole blood (Figure s1 in Supporting
Information). No significant levels of free phosphonate
metabolite 4a (m/z 363.4 [M + H]+) were found by using
LC/MS/MS analysis.
Metabolic Stability in Human, Dog, and Rat Liver
Microsomes. The metabolic stability of compound 4c was
further tested in pooled human, male rat, and male dog liver
microsomes (Table s1 in Supporting Information). No
significant levels of the metabolite 4a (m/z 363.4 [M + H]+)
were found by using LC/MS/MS analysis. The intrinsic
F
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Figure 5. Survival (A, B, C) and average body weight (D, E, F) of mice challenged with 10× LD50 of NIBRG-14 A/Vietnam/1194/2004 (H5N1)
influenza virus. Mice were orally administered with test compounds at indicated concentrations per day. ***: P < 0.0001. **: P < 0.001. *: P < 0.05.
Table 3. Distribution Coefficients of Anti-influenza
Compounds between Octanol and PBS Buffer
clearance (CL) of 4c was very low (<2 μL/min/mg proteins),
and the in vitro half-life (t1/2) was long (>12 h). In contrast, the
control compounds (testosterone and midazolam) were readily
metabolized (65−100% in 1 h) in human, male rat, and male dog
liver microsomes.
a
b
c
entry
compound
clogP
clogD
log D
d
1
2
1a (OC)
0.45
1.49
−1.84
−0.72
−1.55
0.73
−1.69 0.16 (−1.50)
(0.36)
d
1b (OS)
Metabolic Stability in Fresh Human Liver Microsomes.
Metabolic stabilities of the phosphonate compounds 3a, 3c, 4a,
and 4c were tested in five fresh, pooled human liver microsome
preparations using the cytochrome P450 (CYP) system (with
cofactor NADPH) or the uridine 5′-diphospho-glucuronosyl-
transferase (UGT) system (with cofactor UDPGA). The
remaining levels of intact compounds 3a, 3c, 4a, and 4c were
107 8%, 92 3%, 92 7%, and 99 5% in the CYP system at
the end of the tests (1, 0.75, 1, and 0.5 h incubation, respectively),
indicating no significant metabolism of any of the compounds by
CYP enzymes. Compounds 4a and 4c were also stable in the
UGT system, with 90 6% and 91 3% remaining of intact
compounds, respectively, after 1 and 0.5 h incubation. The
stabilities of compounds 3a and 3c in the UGT system were not
tested.
3
2a (GOC)
2b (GOS)
3a (TP)
−0.22
0.83
−1.41 0.24
0.31 0.05
e
4
5
−1.25
2.18
−2.42
−1.39
−1.52
−2.17
−1.93
−1.27
−1.04 0.14
0.22 0.04
6
3b (TP2Et)
3c (TP1Et)
4a (TPG)
4b (TPG2Et)
4c (TPG1Et)
7
−0.14
−1.92
1.51
−0.75 0.10
−0.98 0.18
−0.10 0.04
−0.37 0.02
8
9
10
−0.81
a
Calculated values using Advanced Chemistry Development (ACD/
b
Laboratories) Software V12.01. Calculated values using MarvinSketch
c
(http://www.chemaxon.com/marvin/sketch/index.html). Octanol−
water partition coefficient determined at pH 7.4 from five repeated
d
e
experiments. Log D value at pH 7.4 (adapted from ref 20). Log D
value at pH 7.4 (adapted from ref 22).
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Table 4. Pharmacokinetic Parameters of Test Compounds after Single iv Bolus or Oral Administration to Male Rats
a
3a
3c
4a
4c
PK parameter
(unit)
iv, 1 mg/kg
(N = 6)
oral, 1 mg/kg
(N = 6)
iv, 1 mg/kg
(N = 6)
iv, 0.5 mg/kg
(N = 6)
oral, 3 mg/kg
(N = 6)
iv, 0.3 mg/kg
(N = 6)
oral, 5 mg/kg
(N = 6)
k (1/h)
0.213 0.041
2620 671
0.265 0.127
51.04 28.95
0.428 0.138
1267 336
0.902 0.251
666 171
0.485 0.090
116.7 54.5
1.285 0.037
389 35
0.669 0.126
69.6 10.2
AUC0→t (h*ng/
mL)
AUC0→∞ (h·ng/
mL)
2634 672
55.69 25.51
1286 344
669 173
128.4 59.4
390 36
77.9 10.3
t1/2 (h)
3.36 0.63
0.40 0.10
1.87 0.19
1.47 0.25
100
3.05 1.17
22.10 9.48
106.76 73.43
4.40 0.64
1.80 0.72
0.83 0.25
2.29 1.58
1.44 0.79
100
0.83 0.25
0.79 0.21
0.91 0.21
0.66 0.06
100
1.47 0.24
28.3 13.4
59.6 27.3
2.40 0.57
3.20 1.48
73.1 81.7
0.88 0.21
0.54 0.02
0.77 0.08
0.60 0.06
0.53 0.03
100
1.07 0.21
65.1 8.3
CL/F (L/h/kg)
Vd/F (L/kg)
MRT (h)
F (%)
100.9 26.4
2.05 0.27
1.20 0.16
25.8 4.4
2.33 1.21
C
max (ng/mL)
max (h)
−
−
12.92 7.60
1.46 1.36
−
−
−
−
−
−
T
1.38 0.59
a
The pharmacokinetic parameters for compound 3c in oral administration could not be deduced because its plasma concentrations were lower than
the detection limit.
Table 5. Pharmacokinetic Parameters of Compounds 4a and 4c after Single iv Bolus or Oral Administration to Male Mice
4a
4c
PK parameter (unit)
iv, 0.25 mg/kg (N = 6)
oral, 10 mg/kg (N = 6)
iv, 0.25 mg/kg (N = 6)
oral, 10 mg/kg (N = 6)
k (1/h)
1.564 0.729
354 73
373 72
0.53 0.25
0.69 0.12
0.51 0.18
0.58 0.20
100
0.300 0.073
939 346
1.368 0.625
529 103
541 108
0.71 0.57
0.47 0.08
0.44 0.24
0.63 0.22
100
0.364 0.208
2575 1441
2629 1449
2.57 1.54
4.75 2.12
20.00 18.59
3.14 0.60
12.1 6.7
AUC0→t (h·ng/mL)
AUC0→∞ (h·ng/mL)
1050 355
2.44 0.61
10.30 2.88
36.04 15.00
3.61 0.81
7.0 2.4
t1/2 (h)
CL/F (L/h/kg)
Vd/F (L/kg)
MRT (h)
F (%)
C
max (ng/mL)
max (h)
−
−
337 224
−
−
935 536
T
1.33 0.52
1.38 0.59
Table 6. Pharmacokinetic Studies in Rats and Dogs with Compound 4c in 20% HP-β-CD Aqueous Solution (iv administration) or
in Microcrystalline Cellulose (oral administration)
rat
dog
PK parameter (unit)
iv, 2 mg/kg (N = 4)
oral, 50 mg/kg (N = 4)
iv, 5 mg/kg (N = 4)
oral, 20 mg/kg (N = 4)
k (1/h)
0.16 0.01
3168 492
3170 492
4.3 0.28
0.64 0.09
0.33 0.03
0.5 0.03
100
0.31 0.01
4763 1080
4610 1053
2.2 0.08
−
0.13 0.03
24516 1918
24641 1936
5.65 1.30
3.40 0.27
0.341 0.015
1.52 0.139
100
0.13 0.03
AUC0→t (h·ng/mL)
AUC0→∞ (h·ng/mL)
11019 2975
11561 3210
5.50 1.20
t1/2 (h)
CL/F (L/h/kg)
Vd/F (L/kg)
MRT (h)
−
5.6 0.23
6.0 1.4
482 153
3.7 3.9
5.12 0.694
11.2 2.73
2423 591
1.50 0.577
F (%)
C
max (ng/mL)
max (h)
−
−
−
−
T
Protein Binding Measurements in Human, Rat, and
Dog Plasma. Plasma protein binding of TPG monoester 4c in
human, rat, and dog plasma was determined by rapid equilibrium
dialysis followed by LC/MS/MS analysis. Compound 4c showed
a high free fraction (86−91%) in plasma from all three species
(Table s2 in Supporting Information). This result was consistent
with the studies using microsome systems, which showed no
significant conversion of the monoester 4c to the phosphonic
acid metabolite 4a.
Pharmacokinetic Studies in Rats with Compounds
Dissolved in Normal Saline. The pharmacokinetic parameters
for compounds 3a, 3c, 4a, and 4c in Sprague−Dawley rats are
listed in Table 4. After intravenous (iv) administration, the half-
lives of four compounds ranged from 0.54 to 3.36 h. The CL/F
values ranged from 0.40 to 0.83 L/h/kg. After oral admin-
istration, the half-lives of compounds 3a, 4a, and 4c ranged from
1.07 to 3.05 h. The CL/F values of the three compounds ranged
from 22.1 to 65.1 L/h/kg. The absolute oral bioavailabilities (F
values) of compounds 3a, 4a and 4c were 2.33 1.21%, 3.20
1.48%, and 1.20
0.16%, respectively. The plasma concen-
trations of compound 3c after oral administration were below the
detection limit (Figure s2 in Supporting Information), so the
corresponding pharmacokinetic parameters could not be
deduced. This study indicated that the absolute oral bioavail-
ability of all four compounds in normal saline was very low with
relatively short half-lives in rats.
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Pharmacokinetic Studies in Mice with Compounds in
Normal Saline. Table 5 shows the pharmacokinetic parameters
in mice. After iv administration, the half-lives of compounds 4a
and 4c were 0.53 0.25 and 0.71 0.57 h, respectively. The CL/
lesions were observed upon necropsy of sacrificed animals after
15 days of study.
DISCUSSION
■
F values were 0.69
0.12 and 0.47
0.08 L/h/kg for
Tamiphosphor (3a), its monoester 3c, guanidino-tamiphosphor
(4a), and its monoester 4c were synthesized and found to be
potent inhibitors for influenza neuraminidases (Table 1).
Phosphonates 3a and 4a are significantly more potent than
their carboxylate congeners 1a (OC) and 2a (GOC) presumably
due to the stronger binding of phosphonate with the three
arginine residues (Arg118, Arg292, and Arg371) in the active site
of NA. Unlike oseltamivir (1b), TP monoester 3c and TPG
monoester 4c still possess effective NA inhibitory activities
because the negative charge at the phosphonate monoester
group provides the necessary electrostatic interactions with the
three arginine residues in NA. Compounds 3a, 3c, 4a, and 4c
inhibited the replication of different strains of influenza viruses at
low nanomolar to picomolar levels (Table 2). In general, the
phosphonate compounds 4a and 4c, bearing a guanidino group
at the C-5 position, are more potent than the C-5 amino
analogues 3a and 3c in inhibiting NA activity and virus
replication of the oseltamivir-resistant viruses.
compounds 4a and 4c, respectively. After oral administration, the
half-lives of compounds 4a and 4c were 2.44 0.61 and 2.57
1.54 h, respectively. The CL/F values were 10.30 2.88 and 4.75
2.12 L/h/kg for compounds 4a and 4c, respectively. The oral
bioavailability of compounds 4a (7.0 2.4%) and 4c (12.1
6.7%) in mice were appreciably higher than that in rats.
According to the plasma concentration−time curves for iv and
oral administrations of compound 4a to mice (Figure s3 in
Supporting Information), the average concentration of 4a in
plasma at 8 h after oral administration was 31.4 ng/mL (∼87
nM), which was higher than all EC50 values of the tested viruses.
The average concentration of compound 4c at 12 h was 16.8 ng/
mL (∼43 nM), which was also higher than the EC50 values of the
tested viruses. These results might account for the high survival
rates in the influenza-infected mice even though compounds 4a
and 4c had only modest absolute bioavailability.
Pharmacokinetic Studies in Rats and Dogs with
Compound 4c in 20% HP-β-CD Aqueous Solution or in
Microcrystalline Cellulose. To investigate whether bioavail-
ability of the test compound can be improved by adding
pharmaceutical excipients, compound 4c was dissolved in a 20%
2-hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution
for iv administration or in microcrystalline cellulose for oral
administration. A summary of the pharmacokinetic parameters
determined in rats and dogs is shown in Table 6. After iv
administration, the half-lives of compound 4c in rat and dog were
4.3 and 5.65 h, respectively, with corresponding CL values of 10.7
and 3.4 mL/min/kg. After oral administration, the half-lives of
compound 4c in rat and dog were 2.2 and 5.5 h, respectively. The
oral bioavailabilities of compound 4c in rat and dog were 6.0%
and 11.2%, respectively. No clear evidence of bioconversion of
monoester 4c to its free phosphonic acid 4a was observed.
Though the bioavailability of 4c is not yet optimal for convenient
oral dosing, this study indicates the possible improvement by
formulation enhancements.
Owing to the intrinsic high anti-influenza activities, all four
phosphonate compounds (3a, 3c, 4a, and 4c) can significantly
protect mice infected with several tested influenza strains
(human H1N1, pandemic H1N1, H275Y H1N1 mutant, and
avian H5N1) when administered at doses of 1 mg/kg/day or
higher (Figures 2−5). Consistent with in vitro virus replication
tests, the phosphonate congeners did show good protection
against the challenge with oseltamivir-resistant A/WSN/33/
H275Y virus.
The potent anti-influenza activity of tamiphosphor mono-
esters (3c and 4c), nearly equal to that of the free phosphonic
acids (3a and 4a), may be surprising given that there was little
evidence of bioconversion of the monoesters to the free
phosphonic acids in metabolism studies. Perhaps the higher
lipophilicity of monoester compounds resulted in higher
intracellular concentrations of the compounds which, in turn,
resulted in greater than expected anti-influenza activity. It has
been shown that GOS (2b) having a guanidino group in lieu of
the amino group in OS is no longer available to esterase
digestion.23 It is conceivable that guanidino-tamiphosphor
monoethyl ester (4c) cannot be converted to the free acid 4a;
however, little bioconversion of 3c is not expected. As the
strategy used for laninamivir24 and phospho-sulindac,25 addition
of a long aliphatic chain to phosphonate compounds 3a, 3c, 4a,
and 4c may provide more efficient bioconversion, which is
required to achieve higher levels of antiviral activity against
oseltamivir-resistant influenza viruses.
Excretion Study. After oral administration of compound 4c
(5 mg/kg) to rats, the recovery of phosphonate monoester 4c
from urine and feces was 1.5
0.4% and 43.2
12.0%,
respectively (Table s3 in Supporting Information). In the mean
time, the free phosphonic acid 4a, presumably a degradation
product of 4c, was also obtained from urine and feces at 0.4
0.2% and 27.0
3.0%, respectively. The total recovery of
compounds 4c plus 4a was estimated to be 1.9 0.5% from urine
and 70.2 12.1% from feces.
Study of Acute Toxicity in Mice. Acute toxicity was studied
in female Crl:CD1 (ICR) mice (20−30 g and 6−7 weeks old).
Tamiphosphor (3a) dissolved in water-for-injection (WIF), with
solubility up to 50 mg/mL, was administered by a single iv
injection. TP monoester 3c was suspended in carboxymethyl-
cellulose (CMC) aqueous solution (0.5%, w/v) up to 30 mg/mL
and applied by a single intraperitoneal (ip) injection. Acute
toxicity was assessed by major clinical signs including tremor,
convulsion, body jerks, hypoactivity, hunched posture, and
piloerection (Table s4 in Supporting Information). Mortality in
the treated animals occurred by iv dosing of tamiphosphor 3a
above 750 mg/kg or by ip dosing of tamiphosphor monoester 3c
at 2000 mg/kg. Clinical signs of acute toxicity which occurred in
survivors were reversed within 2 days after dosing. No gross
The results presented in this study indicate that phosphonate
compounds 3a, 3c, 4a, and 4c have similar pharmacokinetic
properties (Tables 4−6). GOC (2a) was reported to have longer
half-life (20.1 h) than OC (10.6 h) in rats.23 In contrast, slight
decrease in half-life of guanidino-tamiphosphor (4a) was found
by replacement of the amino group in TP (3a) with a guanidino
group. The compound half-lives were shorter than 4.5 h in rats
and mice but slightly longer in dogs at about 5.5 h. The absolute
oral bioavailability of all four compounds (as saline solutions) in
rats was low (≤3.20 1.48%). However, the oral bioavailability
of compounds 4a and 4c in mice was higher (up to 12.1 6.7%)
than in rats. Our study also indicates that formulation of 4c in
microcrystalline cellulose can improve its absolute oral
bioavailability to 6% in rats and 11.2 2.73% in dogs. Despite
I
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low bioavailability, phosphonates 3a, 3c, 4a, and 4c can generally
maintain the plasma concentrations in mice above the
concentration required to inhibit the influenza viruses (Table
s2 in Supporting Information), consistent with the observed
protection against lethal infection in mice. As the level of plasma
protein binding was low, the circulating compound would be
almost entirely free of bound protein and available to exert
antiviral activity in vivo.
On the basis of the in vitro metabolism studies, none of the
phosphonate compounds are metabolized extensively by CYP or
UGT enzymes in vivo nor is there evidence of bioconversion of
phosphonate monoesters to free phosphonic acids in liver
microsomes or whole blood. These findings are consistent with
the observation in pharmacokinetic studies, which showed
negligible accumulation of phosphonic acid 4a after admin-
istration of monoester 4c. After oral administration, in fact, most
of 4c (43%) and its degradation product 4a (27%) were excreted
in feces. Thus, compound 4c might be biotransformed to 4a in
gastrointestinal tract extensively. The low oral bioavailability of
4c may be due to incomplete absorption as well as partial
biotransformation to 4a in gastrointestinal tract.
As shown in this study, substitution of the carboxylate in OC
(1a) by phosphonate, giving tamiphosphor 3a, and further
substitution of the amine by guanidine, giving TPG 4a, all
provide better affinity to influenza neuraminidases, and thus
enhance the potency against influenza viruses. However,
incorporation of the charged phosphate and guanidinium ions
in 3a and 4a also causes problem in pharmacokinetics. Poor
absolute oral bioavailability (<5%) has also been encountered in
the anti-influenza agents OC (4.3%), guanidino-OC (2a, 4.0%)
and zanamivir (3.7%).23 The problem in OC has been solved by
preparation of its ethyl ester (OS) as a prodrug with good oral
bioavailability (35%) and high peak concentration (Cmax = 0.47
μg/mL) in plasma.26 Formulation of the OS phosphate salt with
appropriate filler materials further improves its bioavailability to
79% for marketing as the tamiflu capsule. The half-life and
bioavailability of compounds 3a and 4a are comparable to that of
ZA (1.8 h and 3.7%).23 To develop 3a and 4a with nonoral
administration is possible.27
As the first approach to improve the pharmacokinetic
properties of tamiphosphor 3a and TPG 4a, we synthesized
the monoethyl ester derivatives 3c and 4c to investigate their
pharmaceutical properties. Compound 4c (as the saline
solution) did show better bioavailability (F = 12%) than 4a (F
= 7%) in mice but not in rats. Using microcrystalline cellulose as
the excipient, the bioavailability of 4c was appreciably improved
in rats and was nearly 12% in dogs. It seems promising to design
other tamiphosphor prodrugs using varied monoesters to attain
better bioconversion and biopharmaceutical properties.28 In
contrast, to develop positively charged guanidine-containing
compounds into orally available drugs is still a challenging task.29
Even so, considerable progress has been made to improve the
permeability and possible oral bioavailability of ZA, for example,
by encapsulation with liposome,30 by ion-pairing with 2-
hydroxynaphthoic acid,21 and by a prodrug moiety targeting
intestinal membrane carriers.31 These new strategies and
mechanisms of advanced formulation and structural modification
for enhancing oral bioavailability of phosphate- and guanidine-
containing compounds may be applied to develop highly potent
anti-influenza agents 4a and 4c for practical therapeutic use.
EXPERIMENTAL SECTION
■
Materials and Methods. Nicotinamide adenine dinucleotide
phosphate (NADP+), glucose-6-phosphate (G6P), glucose-6-phosphate
dehydrogenase (G6PD), magnesium chloride, uridine 5′-diphospho-
glucuronic acid (UDPGA), and alamethicin were purchased from
Sigma. Phosphate buffer was prepared from potassium dihydrogen-
phosphate (KH2PO4) purchased from Nacalai Tesque Incorporation.
Acetonitrile and methanol were purchased from J. T. Baker, and formic
acid was from Sigma-Aldrich. The solution of NH4OH (5%) in MeOH
was purchased from Fluka. The reagents for cell culture including
DMEM (Dulbecco’s modified Eagle medium), fetal bovine serum, and
penicillin−streptomycin were purchased from Invitrogen (Carlsbad,
CA).
All the reagents were commercially available and used without further
purification unless indicated otherwise. All solvents were anhydrous
grade unless indicated otherwise. All nonaqueous reactions were carried
out in oven-dried glassware under a slight 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 of 60−200 μm particle
size. Melting points were recorded on an Electrothermal MEL-TEMP
1101D melting point apparatus and were not corrected. NMR spectra
were recorded on Bruker AVANCE 600 and 400 spectrometers.
Chemical shifts are given in δ values relative to tetramethylsilane
(TMS); coupling constants J are given in hertz. Internal standards were
CDCl3 (δH = 7.24), MeOH-d4 (δH = 3.31), or D2O (δH = 4.79) for 1H
NMR spectra, CDCl3 (δc = 77.0) or MeOH-d4 (δc = 49.15) for 13C
NMR spectra, and H3PO4 in D2O (δP = 0.00) 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 Thermo Nicolet 380 FT-IR spectrometer.
Optical rotations were recorded on a Perkin-Elmer Model 341
polarimeter. High resolution ESI mass spectra were recorded on a
Bruker Daltonics spectrometer.
Oseltamivir carboxylic acid (1a, OC), oseltamivir (1b, OS, tamiflu as
the phosphate salt), guanidino-OC (2a, GOC), guanidino-OS (2b,
GOS), zanamivir (ZA, Relenza), and the oseltamivir phosphonate
congeners including tamiphosphor (3a, TP), tamiphosphor diethyl ester
(3b, TP2Et), guanidino-tamiphosphor (4a, TPG), and guanidino-
tamiphosphor diethyl ester (4b, TPG2Et) were prepared in our
laboratory according to the previously reported procedures.12,13 The
synthetic procedures for tamiphosphor monoethyl ester (3c, TP1Et)
and guanidino-tamiphosphor monoethyl ester (4c, TPG1Et) are
described as follows. Purity of these compounds was assessed to be
≥95% by HPLC analyses (Agilent 1100 series) on an HC-C18 column
(5 μm porosity, 4.6 × 250 mm) using MeOH/H2O (1:1) as the eluent
and UV detector at λ = 214 nm.
Tamiphosphor Monoethyl Ester (3c). A solution of tamiphos-
phor diethyl ester 3b (1.43 g, 3 mmol) in ethanol (50 mL) was treated
with sodium ethanoate in ethanol (4.5 mmol, 4.5 mL of 1 M solution)
under a nitrogen atmosphere. The mixture was stirred for 16 h at room
temperature and then acidified with Amberlite IR-120 (H+-form). The
heterogeneous solution was stirred at 40 °C for 2 h, filtered, and
concentrated in vacuo. The residual oil was taken up in water (15 mL)
and subjected to lyophilization. The residual colorless solids were
washed with cold acetone (20 mL × 3), dissolved in aqueous NH4HCO3
(15 mL of 0.1 M solution), stirred for 1 h at room temperature, and then
lyophilized to afford the ammonium salt of TP monoethyl ester 3c (898
mg, 82%) as white solids. The purity of product was >98% as shown by
HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm) with
elution of MeOH/H2O (50:50), tR = 7.5 min (UV detection at 214-nm
20
wavelength). C15H32N3O5P, mp 65−67 °C; [α]D = −36.2 (c = 0.7,
H2O); IR (neat) 3503, 3211, 2921, 1714, 1658, 1121 cm−1; H NMR
1
(600 MHz, D2O) δ 6.33 (1 H, d, JP‑2 = 19.2 Hz), 4.23 (1 H, d, J = 9.4 Hz),
3.99 (1 H, dd, J = 10.2, 5.1 Hz), 3.86−3.84 (2 H, m), 3.53 (1 H, br s),
3.48−3.45 (1 H, m), 2.76−2.73 (1 H, m), 2.41−2.37 (1 H, m), 2.07 (3
H, s), 1.61−1.40 (4 H, m), 1.24 (3 H, t, J = 6.8 Hz), 0.89 (3 H, t, J = 7.1
Hz), 0.84 (3 H, t, J = 7.1 Hz); 13C NMR (150 MHz, D2O) δ 175.1, 136.4,
130.3 (C-1, d, JP‑1 = 168 Hz), 84.2, 76.2, 76.1, 61.3, 53.6, 49.6, 29.9, 25.5,
J
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25.2, 22.3, 15.8, 8.5; 31P NMR (242 MHz, D2O) δ 12.89; HRMS calcd
for C15H28N2NaO5P [M + Na − NH4]+: 370.1639, found: m/z
370.1643.
min, absorbance at 490 nm was read on a plate reader. Influenza virus
TCID50 was determined using the Reed−Muench method.34,35
Determination of IC50 of Neuraminidase Inhibitors. The
neuraminidase activity was measured using either fluorogenic or
luminescence substrate. In one approach, the neuraminidase activity
was measured using diluted allantoic fluid harvested from influenza
virus-infected embryonated eggs and a fluorogenic substrate 2′-(4-
methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma).
For the 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 μM substrate. The fluorescence of the
released 4-methylumbelliferone was measured in Envision plate reader
(Perkin-Elmer, Wellesley, MA) using excitation and emission wave-
lengths of 365 and 460 nm, respectively. The inhibitor IC50 values were
determined from the dose−response curves by plotting the percent
inhibition of NA activity versus inhibitor concentrations using Prism 5
(GraphPad Software, Inc., San Diego, CA).
Alternatively, the effect of inhibitor on viral NA activity was
performed using a commercially available kit with a chemiluminescent
substrate of sialic acid 1,2-dioxetane derivative (NA-Star Influenza
Neuraminidase Inhibitor Resistance Detection Kit, Applied Biosystems,
Foster City, CA) in 96-well solid white microplates following the
manufacturer’s instructions. Inhibitor in half-log dilution increments
was incubated with virus (as the source of neuraminidase). The amount
of virus in each microwell was approximately 500 cell culture infectious
doses (CCID50). Plates were preincubated for 10 min at 37 °C prior to
addition of chemiluminescent substrate. Following addition of substrate,
plates were incubated for 30 min at 37 °C. The NA activity was evaluated
using a Centro LB 960 luminometer (Berthold Technologies) for 0.5 s
immediately after addition of NA-Star accelerator solution. Fifty percent
inhibitory concentrations (IC50 values) of viral NA activity were
determined by plotting percent chemiluminescent counts versus log10 of
the concentration of inhibitor.
Determination of EC50 of Neuraminidase Inhibitors. The anti-
influenza virus activities of NA inhibitors were measured by the EC50
values, i.e., the concentrations of the compound required for 50%
protection of the influenza virus infection-mediated cytopathic effects
(CPE). A 50−100 μL amount of diluted influenza virus (100 TCID50)
was mixed with equal volumes of NA inhibitors at varied concentrations.
The mixtures were then used to infect 100 μL of MDCK cells at 1 × 105
cells/mL in 96-well plates. After 48−72 h incubation at 37 °C under 5%
CO2, the CPE were determined 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 values were determined by fitting the curve of percent CPE versus
the concentrations of NA inhibitor using Graph Pad Prism 4.
Animal Experiments for Virus Challenges. Female BALB/c mice
(18−20 g) were obtained from Charles River Laboratories or National
Laboratory Animal Center (Taiwan). The mice were quarantined for
48−72 h before use. The mice were anesthetized by intraperitoneal (ip)
injection of zoletil (or ketamine/xylazine) and inoculated intranasally
with 25 μL of infectious influenza virus. The test compounds were
dissolved in sterile water and administered to mice at the indicated
dosages by oral gavage (po) twice daily for 5 days. Control mice received
sterile water on the same schedule. Ten mice per test group were used
throughout the studies. Four hours after the first dose of drug, mice were
inoculated with influenza virus at 3−10× mouse LD50, depending upon
the virus strain. Mice were observed daily for 14−21 days for survival and
body weight.
Guanidino-tamiphosphor Monoethyl Ester (4c). By a proce-
dure similar to that for compound 3c, TPG diethyl ester 4b (2.73 g, 4
mmol) was treated with sodium ethanoate in ethanol, followed by
workup using Amberlite IR-120 and ion exchange with NH4HCO3, to
give the ammonium salt of TPG monoethyl ester 4c (1.22 g, 75%) as
white solids. The purity of product was >98% as shown by HPLC on an
HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm) with elution of MeOH/
H2O (50:50), tR = 7.9 min (UV detection at 214-nm wavelength).
C16H34N5O5P, mp 70−72 °C; [α]D20 = −11.5 (c = 0.6, H2O); IR (neat)
3521, 1931, 1756, 1623, 1210 cm−1; 1H NMR (600 MHz, D2O) δ 6.29
(1 H, d, JP‑2 = 19.1 Hz), 4.25−4.22 (1 H, m), 3.91−3.82 (4 H, m), 3.51
(1 H, br s), 2.57−2.55 (1 H, m), 2.242.20 (1 H, m), 2.01 (3 H, s), 1.63−
1.49 (3 H, m), 1.44−1.40 (1 H, m), 1.24 (3 H, t, J = 6.9 Hz), 0.88 (3 H, t,
J = 7.0 Hz), 0.82 (3 H, t, J = 7.0 Hz); 13C NMR (150 MHz, D2O) δ
174.5, 160.3, 136.5, 131.8 (C-1, d, JP‑1 = 171 Hz), 84.2, 76.9, 76.8, 61.2,
55.6, 51.0, 31.6, 25.6, 25.3, 22.0, 15.7, 8.5; HRMS calcd for
C16H30N4NaO5P (M + Na − NH4)+: 412.1857, found: m/z 412.1859.
Viruses. Influenza A/WSN/33 (H1N1) viruses was obtained from
Dr. Shin-Ru Shih (Chang Gung University in Taiwan) or from Dr.
Kenneth Cochran (University of Michigan, Ann Arbor). A/Taiwan/
3446/2002 (H3N2) and B/Taiwan/70641/2004 were gifts from Dr.
Shin-Ru Shih (Chang Gung University in Taiwan). The reassortant
H5N1 virus NIBRG14 created with hemagglutinin as well as
neuraminidase genes from A/Vietnam/1194/2004, and the other
genes from PR8 were originally from National Institute for Biological
Standards and Control (Hertfordshire, UK). Influenza A/California/
07/2009 (H1N1, Pandemic), A/Brisbane/10/2007 (H3N2), and A/
Udorn/1972 (H3N2) were obtained from Centers for Disease Control
(Taiwan). The oseltamivir-resistant A/WSN H275Y mutant was
created using a 12-plasmid system that is based on cotransfection of
mammalian cells with 8 plasmids encoding virion sense RNA under the
control of a human PolI promoter and 4 plasmids encoding mRNA
encoding the RNP complex (PB1, PB2, PA, and nucleoprotein gene
products) under the control of a PolII promoter.32,33 An H275Y
mutation was introduced in the NA gene, and the sequence was
confirmed to generate the A/WSN H275Y virus. Alternatively, the
oseltamivir-resistant WSN mutant was selected by six passages in
Madin-Darby canine kidney (MDCK) cells with gradually increased OC
(oseltamivir carboxylate) concentrations. This mutant influenza grows
well in the presence of 1 μM OC and carries a single H275Y mutation at
its NA gene as confirmed by sequence analysis. Influenza A/Panama/
2007/99 (H3N2), A/Hong Kong/2369/2009 (H275Y, oseltamivir-
resistant H1N1), B/Florida/4/2006, and B/Sichuan/379/99 viruses
were obtained from the Centers for Disease Control and Prevention
(Atlanta, GA). Influenza A/Victoria/3/75 (H3N2) virus was purchased
from the American Type Culture Collection (Manassas, VA). Mouse-
adapted influenza A/California/04/2009 (H1N1) and A/Duck/MN/
1525/81 were kindly provided by Drs. Elena Govorkova and Robert
Webster (St Jude Children’s Research Hospital, Memphis, TN),
respectively. All viruses were cultured in the allantoic cavities of 10-
day-old embryonated chicken eggs for 72 h and purified by sucrose
gradient centrifugation. In another preparation, A/California/04/2009
(H1N1) virus was adapted to replication in the lungs of BALB/c mice by
nine sequential passages through mouse lungs. Virus was plaque-purified
in MDCK cells, and a virus stock was prepared by growth in
embryonated chicken eggs and then MDCK cells.
Determination of Influenza Virus TCID50. MDCK cells were
obtained from American Type Culture Collection (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 incubation of serially
diluted influenza virus in 100 μL solution with 100 μL of MDCK cells at
1 × 105 cells/mL in 96-well microplates. The infected cells were
incubated at 37 °C under 5% CO2 for 48−72 h and added to each well
with 100 μL per well of CellTiter 96 AQueous Non-Radioactive Cell
Proliferation Assay reagent (Promega). After incubation at 37 °C for 15
Statistical Analysis. Kaplan−Meier survival curves were generated
and compared by the Log-rank (Mantel−Cox) test using Prism 5.0b
(GraphPad Software Inc.). Where statistical significance was seen,
pairwise comparisons were made by the Gehan−Breslow−Wilcoxon
test. Mean body weights were analyzed by ANOVA followed by Tukey’s
multiple comparison test using Prism 5.0b. For stability studies in gastric
fluid and fresh human liver microsomes, and pharmacokinetic study with
compound in normal saline, the Microsoft Excel 2002 (e.g., mean, SD, %
CV, % diff) and SPSS 13.0 software (ANOVA and linear regression)
were used.
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Ethical Regulation of Laboratory Animals. This study was
conducted in accordance with the approval of the Institutional Animal
Care and Use Committee of Utah State University dated Sept 20, 2010
(expires Sept 19, 2013), or the Institutional Animal Care and Use
Committee, National Defense Medical Center, Taiwan (2009−2011),
or the Institutional Animal Care and Use Committee, Academia Sinica,
Taiwan. The work was done in the AAALAC-accredited Laboratory
Animal Research Center of Utah State University, or in the AAALAC-
accredited Animal Center of National Defense Medical Center, or in the
BSL-3 Laboratory of Genomics Research Center, Academia Sinica,
Taiwan. The U.S. Government (National Institutes of Health) approval
was renewed on April 7, 2010 (Animal Welfare Assurance no. A3801-
01) in accordance to the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (Revision; 2010).36
Determination of Octanol−Buffer Partition Coefficients. The
standard solutions of compound in MeOH at various concentrations of
1.0, 0.5, 0.1, 0.05, and 0.01 (mg/mL) were measured by HPLC (Agilent
1100 series) on an HC-C18 column (5 μm porosity, 4.6 × 250 mm)
using MeOH/H2O (1:1) as the eluent and UV detector at λ = 214 nm.
The integral area of the peak corresponding to the compound was used
(average of triple experiments) to establish a standard curve by
Microsoft Excel.
To test compound partitioning, the respective test compounds (∼1.0
mg) were placed in an 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 sample (25 μL) of
aqueous layer was measured by HPLC. The concentration 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 coefficient, DB = [Bt]oct/[Bt]aq, is determined, where
[Bt]oct and [Bt]aq are the concentrations of the drug in organic and
aqueous phases, respectively.
min at 13 000g, and the supernatant was transferred to a plate for LC/
MS/MS analysis. The experiment was performed in triplicates.
Metabolic Stability of Compound in Whole Blood and
Microsomes. Human, dog, and rat whole blood samples were obtained
from normal healthy individuals. Stock solutions of test compounds
were prepared in dimethyl sulfoxide (DMSO) at the concentration of 1
mM and diluted to a final concentration of 500 μM. Test compounds
were incubated with whole blood at a final concentration of 5 μM at 37
°C at 100 rpm on an orbital shaker. Aliquots were removed at 0, 15, 30,
45, 60, and 120 min, and the reaction was stopped by the addition of 5
volumes of cold methanol. After centrifugation at 20 000g for 20 min to
precipitate protein, an aliquot of 200 μL from the supernatant was used
for LC/MS/MS analysis. All experiments were performed in duplicates.
Human, male dog, and male rat pooled liver microsomes were
purchased from BD Biosciences and stored at −80 °C prior to use. A
master solution containing 250 μg of microsomes in 5.6 mM phosphate
buffer and 5.6 mM MgCl2 was prepared and mixed with test compounds
or control solution (testosterone and midazolam) at the final
concentration of 3 μM. The mixture was prewarmed at 37 °C for 2
min, and then the reaction was started at 37 °C with the addition of 50
μL of ultrapure H2O (for negative control) or 10 mM NADPH solution
at the final concentration of 1 mM. Aliquots of 50 μL were removed
from the reaction at 0, 10, 20, 30, and 60 min and stopped by the
addition of 3 volumes of cold methanol. After centrifugation at 16 000
rpm for 10 min, an aliquot (100 μL) of the supernatant was collected for
LC/MS/MS analysis. All experiments were performed in duplicate. All
calculations were carried out using Microsoft Excel 2003. The slope
value, k, was determined by linear regression of the natural logarithm of
the peak area of the parent drug vs incubation time curve. Peak areas
were determined from extracted ion chromatograms. The in vitro half-
life (in vitro t1/2) was determined from the slope value: in vitro t1/2
=
−(0.693/k). Conversion of the in vitro t1/2 (in min) into the in vitro
intrinsic clearance (in vitro CLint, in μL/min/mg proteins) was
performed using the following equation (mean of duplicate determi-
nations):
LC/MS/MS Determination. Samples were separated with a
Shimadzu liquid chromatograph separation system equipped with a
DGU-20A3 degasser, a LC-20AD solvent delivery unit, a CBM-20A
system controller, CTO-10ASVP column oven, and a CTC Analytics
HTC PAL System. The samples (10 μL) were separated on a
preguarded Phenomenex Luna C18 column (5 μ, 2.0 × 50 mm) with
acetonitrile (A) and water (B) (both containing 0.1% formic acid) in a
sequence of A/B 1:9 (0−2 min), 1:0 (2−2.3 min), and 1:9 (2.3−3.5
min) at 25 °C with an elution rate of 600 μL/min.
For stability studies in gastric fluid and fresh human liver microsomes,
and for pharmacokinetic studies with compounds in normal saline, the
respective samples of rat plasma (100 μL), mouse plasma (10 μL), and
microsomes (200 μL) were chromatographically separated with a
preguarded Waters Atlantis T3 3 μm C18 (2.1 × 100 mm) column. The
mobile phase was CH3CN/H2O/HCO2H (40:60:0.1, v/v/v).
Mass spectrometric analysis was performed using an API 3000 LC-
MS/MS from Applied Biosystems Inc. (Canada) with an electrospray
ionization (ESI) interface. Ionization was conducted in the positive
mode, and the ion source temperature was maintained at 400 °C. High
purity nitrogen gas was used as the collision-induced dissociation
(CAD) gas, curtain gas, and nebulizer gas. Positive multiple reaction
monitoring (MRM) mode was used for the quantification. The selected
transitions of m/z were 321.1 [M + H]+ → 174.2 [M − pentyloxy −
acetamide]+ for compound 3a (collision energy, 31 eV), 349.3 [M + H]+
→ 261.2 [M − pentyloxy]+ for compound 3c (collision energy, 20 eV),
363.4 [M + H]+ → 216.1 [M − pentyloxy − acetamide]+ for compound
4a (collision energy, 32 eV), and 391.3 [M + H]+ →303.3 [M −
pentyloxy]+ for compound 4c (collision energy, 27 eV).
Stability of Compound in Simulated Gastric Fluid. To
determine whether the test compound is likely to be stable in the
stomach, a simulated gastric fluid (SGF, without enzymes) was prepared
by dissolving 2 g of NaCl in 1 L of ddH2O and adjusting the pH to 1.2
with HCl.37 The test compound was incubated at a concentration of 2.5
mg/mL in the presence or absence of simulated gastric fluid for 30 min
at room temperature. After 30 min, the samples were centrifuged for 10
in vitro CLint = (0.693/t1/2) × [volume of incubation (μL)
/amount of proteins (mg)]
Other metabolic stability studies were conducted in five pooled fresh
human liver microsomes. An incubation mixture containing test
compound (80 ng/mL), phosphate buffer (0.1 M, pH = 7.4), MgCl2
(5 mM), and NADPH regenerating solution (1 mM NADP+, 10 mM
G6P, and 2 IU of G6PD) or UDPGA (2 mM) was vortexed well. The
reaction mixture, in a final volume of 500 μL, was preincubated for 1 min
at 37 °C, and the human liver microsomes (0.5 mg/mL),38 freshly
prepared from five human livers obtained from the Department of
Surgery, Tri-Service General Hospital (Taipei, Taiwan), were added to
start the reaction. Each reaction was stopped at 5, 10, 15, 20, 30, 45, and
60 min by adding 500 μL of cold acetonitrile. After termination of the
incubation, oseltamivir (50 μL, 40 ng/mL) was added as an internal
standard for analysis. The sample was centrifuged, and 200 μL aliquot
was taken and evaporated to dryness under reduced pressure. The dried
sample was reconstituted with 50% methanol for LC/MS/MS analysis.
All experiments were performed in triplicates.
Pharmacokinetic Studies in Rats with Compounds in Normal
Saline. All four compounds were administered as aqueous solutions in
normal saline. Male rats were purchased from BioLASCO Taiwan Co.,
Ltd. The test compound was administered to six Sprague−Dawley rats
(250−350 g) as a single intravenous (iv) dose (0.3−1 mg/kg of body
weight) or as a single oral dose (1−5 mg/kg). At predetermined time
points up to 24 h postdosing, blood samples were collected via a tail
lateral vein, placed into heparinized tubes, and processed to extract the
plasma, which was then stored at −20 °C. As an example of a
representative sampling schedule, plasma samples were collected at 0.08,
0.17, 0.33, 0.67, 1, 2, 4, 6, 8, 10, 12, 16, and 24 h after administration of
the iv dose to the rats and at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, and
12 h after administration of the oral dose to the rats. The samples were
extracted by Oasis MCX cartridge. The concentrations of compounds in
the eluate were determined by LC/MS/MS analysis.
L
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Pharmacokinetic Studies in Mice with Compounds in Normal
Saline. The compounds were administered as aqueous solutions in
normal saline. Male mice were purchased from BioLASCO Taiwan Co.,
Ltd. The compounds were administered to six mice as a single iv 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,
10, 12, and 24 h and then stored at −20 °C. Deproteinized plasma
samples were centrifuged for 10 min at 13 000g, and the supernatant was
transferred to a plate for LC/MS/MS analysis. The concentrations of
compound in the mice plasma samples were determined by LC/MS/MS
analysis.
Pharmacokinetic Studies in Rats and Dogs with Compound
Dissolved in 20% HP-β-CD in Water or in Microcrystalline
Cellulose. Compound 4c was prepared as aqueous solution in 20% 2-
hydroxypropyl-β-cyclodextrin (HP-β-CD) in water or in microcrystal-
line cellulose. A sample of 4c was administered to rats or dogs as a single
iv dose (2 mg/kg and 5 mg/kg of body weight in rats and dogs,
respectively) or as a single oral dose (50 mg/kg and 20 mg/kg in rats and
dogs, respectively). Plasma samples were collected at 0.08, 0.25, 0.5, 1, 2,
3, 4, 5, 6, 8, and 24 h after administration of the iv dose and at 0.25, 0.5, 1,
2, 3, 4, 5, 6, 8, and 24 h after administration of the oral dose. The
concentrations of test compound in the rat and dog plasma samples
were determined by LC/MS/MS analysis.
%free = (concentration in buffer chamber/
concentration in plasma chamber) × 100%
%bound = 100% − %free
Excretion Study of Compound 4c. A single oral dose (5 mg/kg)
of compound 4c was administered by gavage to six SD rats (average
weight, 270 g). The rats were kept in metabolic cages, and their urine
and feces were collected at 4, 8, 12, 24, and 48 h. The contents of
compounds were determined by LC/MS/MS analysis.
ASSOCIATED CONTENT
* Supporting Information
Supplementary figures, tables, and NMR spectra. This material is
■
S
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(J.M.F.) Tel: 8862-3366-1663. Fax: 8862-2363-7812. E-mail:
jmfang@ntu.edu.tw. (O.Y.-P.H.) Tel: 8862-8792-3100 ext
18207. Fax: 8862-8792-4859. E-mail: hyp@ndmctsgh.edu.tw.
(C.-H.W.) Tel: 8862-2789-9400. Fax 8862-2785-3852. E-mail:
chwong@gate.sinica.edu.tw.
■
Pharmacokinetic Analysis. The pharmacokinetic parameters were
obtained using a pharmacokinetic program WinNonlin, fitting data to a
noncompartmental model. The pharmacokinetic parameters including
the area under the plasma concentration-versus-time curve (AUC) to
the last sampling time (AUC0−t), to the time infinity (AUC0−∞), the
terminal-phase half-life (t1/2), the maximum concentration of
compound in plasma (Cmax), the time of Cmax (Tmax), the mean
residence time (MRT), and the first-order rate constant associated with
the terminal portion of the curve (k) were estimated via linear regression
of time vs log concentration. The total plasma clearance (CL) was
calculated as dose/AUCiv, and the apparent volume of distribution (Vd)
of drug administered iv was calculated as CL × k. The oral bioavailability
(F) of the test compound by oral administration was calculated from the
AUC0−∞ of the oral dose divided by the AUC0−∞ of the iv dose.
Protein Binding Study. Plasma protein binding was determined
using rapid equilibrium dialysis method.39 Test compounds were
prepared in DMSO at the concentration of 1 mM and diluted in series in
50% DMSO/H2O. Human, rat, or dog plasma was then spiked with the
compound(s) at a final concentration of 1 μM with the final
concentration of DMSO less than 1%. All compounds were tested in
duplicates. Ranitidine and testosterone were included as the control
compounds. A rapid equilibrium dialysis device in 96-well format with
dialysis membrane of 8000 Da cutoff (Pierce) was used according to the
manufacturer’s instructions. Briefly, the base plate was rinsed with 20%
ethanol for 10 min, followed by two rinses with ultrapure water. The
base plate was then air-dried and used immediately. After the inserts
were placed open-end-up into the wells of the base plate, 300 μL of
phosphate buffer (pH 7.4) was added to the buffer chamber and 100 μL
of spiked plasma sample was added into the sample chamber (indicated
by a red ring). The device was then covered with a sealing tape and
incubated at 37 °C at approximately 100 rpm on an orbital shaker for 4 h.
An aliquot of 50 μL from both buffer and plasma chambers were
collected into separate microcentrifuge tubes. Fifty microliters of plasma
was added to the aliquot from the buffer chamber while 50 μL of PBS
was added to the collected plasma samples. A 300 μL amount of MeOH
was then added to precipitate protein and release compound(s). After
being vortexed and incubated for 30 min, the samples were centrifuged
for 15 min at 16 000g, and the supernatant was transferred to a plate for
LC/MS/MS analysis.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Academia Sinica, the National Science Council of
Taiwan (grant NSC-98-2321-B-001-022), National Institute of
Health of United States (NIH contract N01-AI-30063 to E.B.T.),
and TaiMed Biologics for financial supports, as well as Dr.
Donald F. Smee (Utah State University) and Mr. Shi-Chiang Li
(National Defense Medical Center) for technical support.
ABBREVIATIONS USED
■
AUC, area under the concentration-versus-time curve; CL,
clearance; CPE, cytopathic effect; CYP, cytochrome P 450; D,
distribution coefficient; DMEM, Dulbecco’s modified Eagle
medium; EC50, half maximal effective concentration; F (%), oral
bioavailability (fraction absorbed); GOC, guanidino-oseltamivir
carboxylic acid; GOS, guanidino-oseltamivir; HP-β-CD, 2-
hydroxypropyl-β-cyclodextrin; HPLC, high-performance liquid
chromatography; IC50, half maximal inhibitory concentration; ip,
intraperitoneal; iv, intravenous; LD50, median leathal dose; MS,
mass spectrometry; MDCK, Madin−Darby canine kidney;
MRT, the mean residence time; NA, neuraminidase; OC,
oseltamivir carboxylic acid; OS, oseltamivir; P, partition
coefficient; SGF, simulated gastric fluid; TCID50, 50% cell
culture infectious dose; TP, tamiphosphor; TP1Et, tamiphos-
phor monoethyl ester; TP2Et, tamiphosphor diethyl ester; TPG,
guanidino-tamiphosphor; TPG1Et, guanidino-tamiphosphor
monoethyl ester; TPG2Et, guanidino-tamiphosphor diethyl
ester; UDPGA, uridine 5′-diphosphoglucuronic acid; UGT,
uridine 5′-diphospho-glucuronosyltransferase; ZA, zanamivir
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