Intramolecular ion-pair prodrugs of zanamivir and guanidino-oseltamivir

Article abstract of DOI:10.1016/j.bmc.2011.06.080

Zanamivir (ZA) is a potent anti-influenza drug, but it cannot be administrated orally because of the hydrophilic carboxylate and guanidinium groups. Guanidino-oseltamivir (GO) is another effective neuraminidase inhibitor with polar guanidinium group under

Full text of DOI:10.1016/j.bmc.2011.06.080

Bioorganic & Medicinal Chemistry 19 (2011) 4796–4802
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Intramolecular ion-pair prodrugs of zanamivir and guanidino-oseltamivir
Kung-Cheng Liu a, Pei-Shan Lee a, Shi-Yun Wang b, Yih-Shyun E. Cheng b, Jim-Min Fang a,b,
,
⇑
Chi-Huey Wong b
a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
b The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Zanamivir (ZA) is a potent anti-influenza drug, but it cannot be administrated orally because of the
hydrophilic carboxylate and guanidinium groups. Guanidino-oseltamivir (GO) is another effective neur-
aminidase inhibitor with polar guanidinium group under physiological conditions. The ester prodrugs
ZA–HNAP (5) and GO–HNAP (6) were prepared to incorporate a 1-hydroxy-2-naphthoic (HNAP) moiety
to attain good lipophilicity in the intramolecular ion-pairing forms. ZA–HNAP resumed high anti-
influenza activity (EC50 = 48 nM), in cell-based anti-influenza assays, by releasing zanamivir along with
nontoxic HNAP. Under similar conditions, the hydrolysis of the GO–HNAP ester was too sluggish to show
the desired anti-influenza activity.
Received 27 April 2011
Revised 26 June 2011
Accepted 27 June 2011
Available online 1 July 2011
Keywords:
Influenza
Neuraminidase inhibitor
Intramolecular ion-pair
Prodrug
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
to attain good oral bioavailability is required to advance their
therapeutic uses.
Influenza is a respiratory infection that causes severe health
problem. The worldwide spread of the H5N1 avian flu and the out-
break of the new type H1N1 human flu in 2009 have increased
public awareness of the potential for global influenza pandemics.
Zanamivir (ZA, 1a) is a potent neuraminidase (NA) inhibitor against
influenza virus, known as Relenza™ on market.1 However, ZA con-
taining carboxylate and guanidinium groups of high hydrophilicity,
with calculated distribution coefficient c log P = ꢀ4.13, greatly re-
duces its oral bioavailability (estimated to be 5% in humans).2 As
a result, ZA is administered intranasally, which increases risk of
bronchospasms in patients having asthma and chronic obstructive
pulmonary disease.3 Oseltamivir carboxylic acid (OC, 2a) is another
potent neuraminidase inhibitor against both influenza A and B
viruses.4 Oseltamivir is the ethyl ester (OC–Et, 2b); its phosphate
salt is an orally administered prodrug known as Tamiflu™.5,6 How-
ever, the extensive use of Tamiflu in treatment of influenza has
caused serious problem due to the rapidly developed oseltami-
vir-resistant virus strains.7,8 The resistance of influenza virus to
ZA is relatively rare in comparison with Tamiflu.7,9 By replacement
of the amino group at C-5 position with a more basic guanidine
group, the guanidino oseltamivir carboxylic acid (GOA, 3a) exerts
stronger electrostatic interactions with the residues of Glu119,
Asp151, and Glu227 in the active site of neuraminidase to show
better inhibitory activity than OC.10 Modification of ZA and GOA
Like Tamiflu, many ester-containing prodrugs have been uti-
lized to improve the intestinal permeability and oral bioavailabil-
ity.11 The ester groups in prodrugs would undergo enzymatic
hydrolysis in vivo to release the corresponding carboxylic acids
as the active drug. The oral treatment of a ZA derivative containing
a lipophilic alkoxyalkyl ester, in lieu of the carboxylic acid group,
did show significant protective effect in the mice infected by influ-
enza virus.12
The prodrugs of ZA and GOA with modification at the guanidi-
nium groups are not yet available. In another approach, 1-hydro-
xy-2-naphthoic acid (HNAP, 4) is used as a counterion of the
guanidinium group in zanamivir heptyl ester (ZA–Hept, 1c) and
guanidino oseltamivir (GO–Et, 3b) to enhance the lipophilicity of
these antiviral agents.13 This ion-pairing approach is successful
to enhance the effective permeability of ZA–Hept by the addition
of counterion HNAP in a concentration-dependent manner, pre-
sumably because the ion-pair of ZA–Hept and HNAP remains intact
during membrane permeation. However, the addition of HNAP
failed to increase the effective permeability of GO–Et, presumably
due to ion exchange with the competing endogenous anions.
In this study, we devised compounds 5 (ZA–HNAP) and 6 (GO–
HNAP) as possible ester prodrugs containing the 1-hydroxy-2-
naphthoic moiety to increase lipophilicity. Figure 1 demonstrates
this strategy comprising two concepts of ester prodrug and intra-
molecular ion-pairing. HNAP is a FDA-approved compound for
the formation of salts as active pharmaceutical ingredients.14,15
In addition, the phenol group in HNAP is considered to be
negatively charged in physiologic condition to neutralize the
⇑
Corresponding author. Tel.: +886 2 33661663; fax: +886 2 23637812.
E-mail address: jmfang@ntu.edu.tw (J.-M. Fang).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.080

K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
4797
OH
OH
O
O
O
H
O
O
O
OR
OR
OR
OH
AcHN
AcHN
AcHN
HN
NH2
R = H
HN
NH
NH
H2N
2a (OC)
H2N
2b (OC−Et) R = C2H5
1a (ZA)
R = H
3a (GOA) R = H
1b (ZA−Et) R = C2H5
1c (ZA−Hept) R = C7H15
3b (GO−Et) R = C2H5
OH OH
O
OH OH
O
H
O
H
O
O
O
O
O
O
OH
AcHN
OH
AcHN
O
O
HN
HN
NH2
HO
NH
H2N
H2N
5 (ZA−HNAP)
ZA−HNAP in ion-pair form
OH OH
H
O
Esterase cat.
hydrolysis
O
OH
O
OH
OH
AcHN
+
+
HO
HO
OH
HN
NH
4 (HNAP)
1 (ZA)
H2N
Figure 1. A strategy of using the zanamivir prodrug ZA–HNAP (5) that may exist as an intramolecular ion-pair to improve lipophilicity and cell membrane permeability.
HNAP (1-hydroxy-2-naphthoic acid) is a FDA-approved compound for the formation of salts as active pharmaceutical ingredients. The anti-influenza drug zanamivir (1) will
be released through esterase catalyzed hydrolysis.
positively charged guanidinium group, so that compounds 5 and 6
can form intramolecular ion-pairs to improve intestinal membrane
permeation.
was protected by treatment with allyl bromide in the presence of
anhydrous K2CO3, followed by saponification to give acid 10. The
protected ZA derivative 7 was prepared from sialic acid according
to the previously reported method.16 The protecting acetyl groups
in 7 were removed by EtONa in EtOH, and the subsequent treat-
ment with 2,2-dimethoxypropane selectively protected the 8,9-
dihydroxy groups, giving acetonide 8. After saponification, the
potassium salt of carboxylate was reacted with 3-iodo-1-propanol
to provide ester 9. The coupling reaction of 9 and 10 culminated in
ZA–HNAP (5) after removal of the protecting groups. GO–HNAP
conjugate 6 was similarly synthesized from ethyl ester 1217 by a
sequence of saponification with KOH, alkylation with 3-iodo-1-
propanol, coupling with allyl HNAP, and removal of the allyl group
(Scheme 2).
2. Results and discussion
The synthesis of zanamivir–hydroxynaphthoate conjugate 5 is
illustrated in Scheme 1. The free aromatic hydroxyl group in HNAP
OAc
OH
OAc
H
H
O
CO2Et
NBoc
O
CO2Et
O
a
b
OAc
AcHN
O
AcHN
HN
The structures of ZA–HNAP and GO–HNAP conjugates were
fully characterized by their physical and spectroscopic properties
HN
NBoc
O
8
7
BocHN
BocHN
(mp, [a
]D, UV–vis, IR, HRMS, 1H and 13C NMR). No trifluoroacetic
OH
acid could be found in these samples as shown by the absence of
any fluorine signal in their 19F NMR spectra. The elemental analysis
of ZA–HNAP fits a molecular formula C26H32N4O10ꢁH2O for its
monohydrate. For comparison, the ethyl esters ZA–Et (1b)16,18
and GO–Et (3b)17,19 were also prepared by slightly modified proce-
dures as those reported previously. The presence of CF3CO2H in 1b
and 3b was confirmed by their 19F and 13C NMR spectra, showing
the fluorine signal at dF ꢂ77 as well as the carbon signals at dC
ꢂ160 and ꢂ116. Thus, ZA–Et and GO–Et existed as the salts of tri-
fluoroacetic acid, whereas ZA–HNAP and GO–HNAP conjugates
might exist as the form of intramolecular ion-pairs.
Lipophilicity is a key determinant of the pharmacokinetic
behavior of drugs. The partition coefficient (P) between octanol
and water is usually taken as a suitable measure of lipophilicity.
For an ionized compound, the distribution coefficient (log D) is
appropriate to represent the partition of drug between octanol
and PBS buffer (pH 7.4). In general speaking, most oral drugs have
the log P values between ꢀ1 and 5.20 For example, oseltamivir
H
O
O
O
O
O
c
+
HO
O
HO
NBoc
AcHN
HN
9
10
BocHN
OH
O
H
O
d
O
O
O
5 (ZA−HNAP)
O
AcHN
O
HN
O
NBoc
BocHN
11
Scheme 1. Synthesis of zanamivir–hydroxynaphthoate conjugate ZA–HNAP (5).
Reagents and conditions: (a) NaOEt, EtOH, rt, 1 h; then 2,2-dimethoxypropane, p-
TsOH, acetone, rt, 12 h; 66%; (b) KOH(aq), MeOH, rt, 0.5 h; then 3-iodo-1-propanol,
DMF, 50 °C, 4 h; 57%; (c) EDCI, DMAP, CH2Cl2, rt, 1.5 h; 68%; (d) Pd(PPh3)4,
morpholine, THF, rt, 4 h; then CF3CO2H, CH2Cl2, rt, 3 h; 75%.

4798
K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
Table 2
O
Neuraminidase inhibition and anti-influenza activities of zanamivir and guanidino-
oseltamivir derivatives
O
CO2Et
NBoc
O
a
10
O
OH
b
Entry Compound
NA inhibition IC50a (nM) Anti-influenza EC50
(nM)
AcHN
AcHN
b
HN
HN
NBoc
1
2
3
4
5
6
7
ZA
ZA–Et
ZA–Et + HNAP 1761
ZA–HNAP
GOA
2.7
1952
34
BocHN
BocHN
106
241
48
8.0
1170
1626
12
13
590
1.4
5158
4876
O
O
O
O
GO–Et
GO–
O
O
O
c
O
O
Et + HNAP
GO–HNAP
HNAP
O
O
AcHN
AcHN
8
9
23,367
>105
1465
>105
HN
HN
O
NBoc
NH2
BocHN
H2N
a
Neuraminidase inhibition against influenza virus A/WSN/1933 (H1N1).
Concentration of NA inhibitors for 50% protection of the cytopathic effects due
b
6
14
to influenza (A/WSN/1933) infection.
GO−HNAP in ion-pair form
Scheme 2. Synthesis of (guanidino-oseltamivir)–hydroxynaphthoate conjugate
GO–HNAP (6). Reagents and conditions: (a) KOH(aq), THF, rt, 0.5 h; then 3-iodo-
1-propanol, DMF, rt, 5 h; 55%; (b) EDCI, DMAP, CH2Cl2, rt, 12 h; 60%; (c) Pd(PPh3)4,
morpholine, THF, rt, 12 h; then CF3CO2H, CH2Cl2, rt, 4 h; 56%.
prevention assays are summarized in Table 2. In comparison with
the active zanamivir (entry 1) and guanidino oseltamivir carbox-
ylic acid (entry 5), the esters ZA–Et, ZA–HNAP, GO–Et and GO–
HNAP all showed inferior NA inhibition (entries 2, 4, 6 and 8) pre-
sumably due to loss of the electrostatic interactions with the three
arginine residues (Arg118, Arg292 and Arg371) in the active site of
NA. Though the anti-influenza activity of GO–HNAP conjugate
carboxylic acid (2a) exhibits a high polarity with log D value of
–1.50 at pH 7.4 (entry 1 in Table 1). Having the carboxylic group
changed to ethyl ester, oseltamivir (2b) becomes an oral drug with
an improved lipophilicity with log D value of 0.36 (entry 6).21
In order to test our hypothesis, a series of zanamivir and guani-
dino oseltamivir derivatives were also subjected to log P calcula-
tion and log D determination (Table 1). The parent carboxylic
acids ZA (1a) and GOA (3a) exhibited high polarities with log D val-
ues of ꢀ1.00 and ꢀ0.13 at pH 7.4, respectively (entries 1 and 7).
The lipophilicity of ethyl esters 1b and 3b increased, showing log D
values of ꢀ0.28 and 0.31 at pH 7.4, respectively (entries 2 and 8). In
mild acidic condition (pH 6.5), the lipophilicity of ZA ester would
be decreased, as shown in the low log D value (ꢀ1.31) for ZA heptyl
ester (1c in entry 3), presumably due to protonation of its guani-
dine moiety. HNAP had an aromatic moiety to impart high lipo-
philicity to the ZA–HNAP and GO–HNAP conjugates as supported
by their relatively high log D values of 0.75 and 0.82 (entries 4
and 9). The intramolecular pairing effect of phenoxide with guan-
idinium might also partially contribute to the high lipophilicity of
ZA–HNAP and GO–HNAP with good log D values required for the
development of oral drugs.
(EC50 = 1.5 lM) was still inferior to its parental carboxylic acid
(GOA), we found that the ZA–HNAP conjugate resumed a high
activity (EC50 = 48 nM) in protection of MDCK cells from infection
by H1N1 influenza virus. This result suggested the potential use
of ZA–HNAP as a prodrug of zanamivir.
The ester bonds could be hydrolyzed by esterases in the blood,
liver and kidney.11 To demonstrate the esterase catalyzed hydroly-
sis, compound 5 (ZA–HNAP) was incubated in rat plasma at 37 °C
for 24 h. The mixture was extracted by methanol and analyzed
by MALDI–TOF mass spectrometry (Fig. S1 in Supplementary data).
The protonated ions [M+H]+ for the degradative products ZA (m/z
333) and HNAP (m/z 189) were observed along with disappearance
of the parental ZA–HNAP. This result was also confirmed by LC–
QTOF MS analysis (Fig. S2 in Supplementary data). The anti-influ-
enza activity of ZA–HNAP was thus attributable to the efficient
release of the active ZA drug during incubation in the cells. The
hydrolysis of GO–HNAP was slow as shown by the MALDI–TOF
and LC–QTOF mass analyses (Figs. S3 and S4 in Supplementary
data). After 24 h incubation, a considerable content of GO–HNAP
still remained along with the degradative products of GOA and
HNAP. This phenomenon might partially explain why the anti-
influenza activity of GO–HNAP was weak under the indicated incu-
bation conditions.
The potency of the new ZA–HNAP and GO–HNAP conjugates
against the N1 neuraminidases from influenza A/WSN/1933
(H1N1) virus and their anti-influenza activity using cytopathic
Table 1
3. Conclusion
c log P and log D of zanamivir and oseltamivir derivatives
Entry
1
2
3
4
5
6
7
8
Compound
1a, ZA
1b, ZA–Et
1c, ZA–Hept
5, ZA–HNAP
2a, OC
2b, OC–Et
3a, GOA
3b, GO–Et
6, GO–HNAP
4, HNAP
c Log Pa
ꢀ4.13
ꢀ3.83
0.69
Log D (pH 7.4)b
ꢀ1.00 0.23
ꢀ0.28 0.01
NDc (–1.31)d
0.75 0.08
Zanamivir is a potent anti-influenza drug with poor oral bio-
availability due to its low lipophilicity caused by the polar carbox-
ylic acid and guanidine groups. In search of a way to circumvent
this problem, a hydroxynaphthoate conjugate ZA–HNAP (5) was
considered to act as an ester prodrug having an additional advan-
tage by intramolecular ion-pairing between the phenoxide and
guanidinium groups to increase the lipophilicity. ZA–HNAP was
prepared and characterized by physical and spectral methods. Un-
like the corresponding ethyl ester (ZA–Et, 1b) prepared as the TFA
salt, ZA–HNAP existed as an intramolecular ion-pair without com-
plexation with external acid. Our preliminary study showed that
the ZA–HNAP conjugate underwent esterase catalyzed hydrolysis
to release nontoxic HNAP compound and the active anti-influenza
drug zanamivir. The ZA–HNAP conjugate appeared to have a suit-
able lipophilicity (log D = 0.75) for oral drug development.
ꢀ0.59
0.45
NDc (–1.50)e
NDc (0.36)e
1.49
ꢀ0.22
ꢀ0.13 0.05
0.31 0.05 (–1.17)d
0.82 0.13
0.83
9
10
4.07
3.29
0.16 0.03 (0.25)f
a
Calculated values using Advanced Chemistry Development (ACD/Labs) Software
V12.01.
b
Octanol–water partition coefficient of five repeated experiments.
Not determined in this study.
Log D at pH 6.5 (adapted from Ref. 13).
Log D at pH 7.4 (adapted from Ref. 26).
Log D at pH 6.5 (adapted from Ref. 27).
c
d
e
f

K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
4799
and 1 M HCl. The organic layer was washed with saturated NaHCO3
and brine, dried over MgSO4, filtered, and concentrated by rotary
evaporation under reduced pressure. The crude material in MeOH
(10 mL) was added 1 M NaOH (1 mL), stirred for 4 h at 60 °C, and
then concentrated under reduced pressure. The mixture was di-
luted with water (20 mL), washed with ether, acidified with 1 M
HCl, and then extracted with CH2Cl2. The organic layer was dried
over MgSO4, filtered, and concentrated by rotary evaporation un-
der reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc/hexane = 3:7) to yield the allyl ether 10
4. Experimental section
4.1. General
Melting points were recorded on a Yanaco or Electrothermal
MEL-TEMP 1101D apparatus in open capillaries and are not cor-
rected. Optical rotations were measured on digital polarimeter of
Japan JASCO Co. DIP-1000; [
a]
values are given in units of 10
D
ꢀ1 deg cm2 gꢀ1. Infrared (IR) spectra were recorded on Nicolet
Magna 550-II or Thermo Nicolet 380 FT-IR spectrometers. UV–vis
spectra were measured on a Perkin Elmer Lambda 35 spectropho-
(942 mg, 75%).
(EtOAc/hexane = 3:7) Rf = 0.18; IR mmax (neat) 3455, 1738, 1365,
1232, 1228 cmꢀ1 1H NMR (400 MHz, CDCl3) d 8.21 (1H, d,
C14H12O3; yellow solid, mp 99–101 °C; TLC
tometer; extinction coefficients (e .
) are given in units of Mꢀ1 cmꢀ1
;
Nuclear magnetic resonance (NMR) spectra were obtained on Bru-
ker Advance-400 (400 MHz) spectrometer. Chemical shifts (d) are
given in parts per million (ppm) relative to 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, or dH 2.49/dC 39.5 for DMSO-d6. The splitting patterns are
reported as s (singlet), d (doublet), t (triplet), q (quartet), m (mul-
tiplet), dd (double of doublets) and br (broad). Coupling constants
(J) are given in Hz. Distortionless enhancement polarization trans-
fer (DEPT) spectra were taken to determine the types of carbon sig-
J = 9.6 Hz), 8.03 (1H, d, J = 8.4 Hz), 7.83 (1H, dd, J = 8.4, 1.2 Hz),
7.63 (1H, d, J = 8.8 Hz), 7.60–7.52 (2H, m), 6.28–6.17 (1H, m),
5.50 (1H, dd, J = 17.2, 1.0 Hz), 5.37 (1H, dd, J = 10.4, 1.0 Hz), 4.71
(2H, d, J = 6.0 Hz); 13C NMR (100 MHz, CDCl3) d 169.2, 157.1,
137.2, 132.5, 128.8, 128.0, 127.8, 126.7, 126.6, 124.3, 123.3,
119.2, 118.2, 77.4; ESI–HRMS (negative mode) calcd for
C
14H11O3: 227.0708, found: m/z 227.0715 [MꢀH]ꢀ.
4.3.2. Ethyl ester of zanamivir as the salt of trifluoroacetic acid
(1b)
nals. The ESI-MS experiments were conducted on
a Bruker
Daltonics BioTOF III high-resolution mass spectrometer. The MAL-
DI-TOFMS measurements were performed on a Bruker Daltonics
Ultraflez II MALDI-TOF/TOF 2000 mass spectrometer. The MALDI
matrix 2,5-dihydroxybenzoic acid (DHB) was photoionized at dif-
ferent irradiances of a UV laser with kmax at 337 or 355 nm. The
LC–QTOF MS analyses were performed using a nanoACQUITY
UltraPerformance LC system coupled to a Q-TOF Premier (both
from Waters Corp., Milford, MA). Element analysis data were ac-
quired using HERAEUS VarioEL-III.z
Ethyl ester 7 was prepared from sialic acid by a slightly modi-
fied procedure as that reported for the corresponding methyl es-
ter.16,22,23 A solution of ester 7 (33 mg, 0.06 mmol) and NaOEt
(2 mg, 0.03 mmol) in anhydrous ethanol (3 mL) was stirred at
room temperature for 1 h to remove the acetyl protecting groups.
The mixture was neutralized by Dowex 50W ꢃ 8 (H+), filtered,
and concentrated under reduced pressure to afford a crude prod-
uct. The yellow solid crude product was dissolved in CH2Cl2
(2 mL) and treated with TFA (2 mL). The mixture was stirred at
room temperature for 1 h, and then evaporated under reduced
pressure. The residue was triturated with Et2O, and the precipitate
was collected by centrifugation to give 1b (18 mg, 91%).
4.2. Materials
All reagents were reagent grade and used without further puri-
fication unless otherwise specified. All solvents were anhydrous
grade unless indicated otherwise. CH2Cl2 was distilled from CaH2.
All non-aqueous reactions were performed in oven-dried glass-
ware under a slight positive pressure of argon unless otherwise
noted. Reactions were magnetically stirred and monitored by
thin-layer chromatography on silica gel using aqueous p-anisalde-
hyde as visualizing agent. Silica gel (0.040–0.063 mm particle
sizes) and LiChroprep RP-18 (0.040–0.063 mm particle sizes) were
used for column chromatography. Flash chromatography was per-
C
14H24N4O7; colorless solid, mp 130–132 °C; UV–vis (MeOH) kmax
260 nm ( = 30,990); UV–vis (PBS) kmax 220 nm ( = 7320); IR mmax
(neat) 3405, 1673, 1377, 1280, 1202, 1138 cmꢀ1 1H NMR
(400 MHz, CD3OD) 5.86 (1H, d, J = 2.0 Hz), 4.51 (1H, d,
e
e
;
d
J = 8.4 Hz), 4.41 (1H, d, J = 10.0 Hz), 4.28–4.17 (3H, m), 3.87–3.81
(2H, m), 3.70–3.66 (2H, m), 2.01 (3H, s), 1.30 (3H, t, J = 5.6 Hz);
13C NMR (100 MHz, CD3OD) d 173.5, 162.5, 159.9 (CO2 of TFA, q,
J = 36.2 Hz), 157.9, 145.6, 116.1 (CF3 of TFA, q, J = 285.8 Hz),
107.7, 77.0, 70.3, 69.2, 63.7, 61.8, 50.4, 47.0, 21.6, 13.4; 19F NMR
(400 MHz, CD3OD) d ꢀ77.5; ESI-HRMS calcd for C14H25N4O7:
361.1723, found: m/z 361.1725 [M+H]+.
formed on silica gel of 60–200 lm particle size.
Influenza A/WSN/1933 (H1N1) virus was obtained from Dr.
Shin-Ru Shih at Chang Gung University in Taiwan. All Viruses were
cultured in the allantoic cavities of 10-day-old embryonated chick-
en eggs for 72 h, and purified by sucrose gradient centrifugation.
Madin-Darby canine kidney (MDCK) cells were obtained from
American Type Culture Collection (Manassas, Va), and were grown
in DMEM (Dulbecco’s modified Eagle medium, GibcoBRL) contain-
ing 10% fetal bovine serum (GibcoBRL) and penicillin-streptomycin
(GibcoBRL) at 37 °C under 5% CO2. Sprague DawleyÒ rat plasma
was obtained from the Development Center for Biotechnology in
Taiwan.
4.3.3. 5-Acetylamino-4-[N2,N3-bis(tert-butoxycarbonyl)
guanidino]-6-[(2,2-dimethyl-1,3-dioxolan-4-yl)-hydroxy-
methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid ethyl ester (8)
To a solution of 7 (1100 mg, 1.6 mmol) in anhydrous ethanol
(10 mL) was added NaOEt (54.4 mg, 0.8 mmol). The mixture was
stirred at room temperature for 1 h, neutralized by Dowex
50Wꢃ8 (H+), filtered, and concentrated under reduced pressure.
The residue was dissolved in anhydrous acetone (10 mL), and
p-toluenesulfonic acid (28 mg, 0.16 mmol) and 2,2-dimethoxypro-
pane (10 mL) were added. The mixture was stirred at room
temperature for 12 h. After concentration, the residue was purified
by silica gel column chromatography (EtOAc/hexane = 2:3) to af-
ford the product 8 (635 mg, 66%). C27H44N4O11; white solid, mp
4.3. Synthetic procedure and compound characterization
118–120 °C; TLC (EtOAc/hexane = 2:3) Rf = 0.22; ½a D20
ꢀ33.4 (c
ꢄ
4.3.1. 1-(2-Propenoxy)-2-naphthoic acid (10)
To a solution of 1-hydroxy-2-napthoic acid 4 (1.04 g, 5.5 mmol)
and allyl bromide (1.4 mL, 16.5 mmol) in anhydrous acetone
(12 mL) was added finely powdered K2CO3 (2.28 g, 16.5 mmol).
The mixture was stirred for 4 h at 60 °C, and then concentrated un-
der reduced pressure. The residue was partitioned between EtOAc
1.0, CHCl3); IR mmax (neat) 3309, 2981, 1725, 1647, 1609, 1559,
1369, 1251, 1151 cmꢀ1 1H NMR (400 MHz, CDCl3) d 11.30 (1H,
;
s), 8.54 (1H, d, J = 7.2 Hz), 7.91 (1H, d, J = 4.4 Hz), 5.71 (1H, d,
J = 2.4 Hz), 5.21 (1H, br s), 5.08–5.05 (1H, m), 4.33–4.30 (1H, m),
4.19–4.13 (2H, m), 4.11–4.00 (2H, m), 3.98–3.88 (1H, m), 3.45

4800
K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
(1H, d, J = 3.6 Hz), 1.93 (3H, s), 1.44 (9H, s), 1.41 (9H, s), 1.34 (3H, s),
1.28–1.22 (6H, m); 13C NMR (100 MHz, CDCl3) d 173.8, 162.2,
161.3, 157.5, 152.5, 147.0, 108.9, 106.3, 84.2, 79.9, 78.5, 74.1,
69.6, 67.3, 61.4, 51.9, 48.3, 28.1 (3ꢃ), 27.9 (3ꢃ), 26.9, 25.1, 22.8,
organic phase was dried over MgSO4, concentrated under reduced
pressure, and purified by flash chromatography on a silica gel col-
umn (EtOAc/hexane = 1:4 to 1:1). The intermediate product was
dissolved in CH2Cl2 (2 mL) and stirred with TFA (2 mL) at room
temperature for 3 h. The mixture was evaporated under reduced
pressure. The residue was triturated with Et2O, and the precipitate
was collected by centrifuge to give the ZA–HNAP conjugate 5
(33 mg, 75%). C26H32N4O10; pale yellow solid, mp 120–122 °C;
13.9; ESI–HRMS (negative mode) calcd for
C27H43N4O11:
599.2928, found: m/z 599.2926 [MꢀH]ꢀ.
4.3.4. 5-Acetylamino-4-[N2,N3-bis(tert-butoxycarbonyl)
guanidino]-6-[(2,2-dimethyl-1,3-dioxolan-4-yl)-hydroxy-
methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid 3-
hydroxypropyl ester (9)
To a solution of ester 8 (480 mg, 0.8 mmol) in MeOH (5 mL) was
added KOH (90 mg, 1.6 mmol). The mixture was stirred at room
temperature for 30 min, and then concentrated under reduced
pressure to give a potassium salt which was then dissolved in
½
a 2D1
ꢄ
+19.3 (c 1.0, MeOH); UV–vis (MeOH) kmax 316 nm (
e
e
= 5340),
= 4180),
260 nm
(
e
= 28,990); UV–vis (PBS) kmax 316 nm
(
260 nm (
e
= 4420); IR mmax (neat) 3422, 1719, 1663, 1638, 1253,
1203, 1139, 1090 cmꢀ1
;
1H NMR (400 MHz, CD3OD) d 8.35 (1H, d,
J = 8.4 Hz), 7.81 (1H, d, J = 8.4 Hz), 7.77 (1H, d, J = 8.8 Hz), 7.63
(1H, m), 7.53 (1H, m), 7.33 (1H, d, J = 8.8 Hz), 5.89 (1H, d,
J = 2.8 Hz), 4.54–4.51 (2H, m), 4.46–4.41 (4H, m), 4.23–4.18 (1H,
m), 3.89–3.81 (2H, m), 3.72–3.67 (2H, m), 2.26–2.20 (2H, m),
2.00 (3H, s); 13C NMR (100 MHz, CD3OD) d 173.5, 171.3, 162.4,
161.0, 158.0, 145.6, 137.8, 129.8, 127.8, 126.1, 125.0, 124.3,
123.6, 119.0, 108.0, 105.7, 77.1, 70.5, 69.3, 63.8, 62.5, 62.3, 50.4,
47.9, 28.1, 21.7; ESI–HRMS calcd for C26H33N4O10: 561.2197,
found: m/z 561.2194 [M+H]+. A sample of ZA–HNAP conjugate
was recrystallized from EtOH/hexane for elemental analysis. Anal.
Calcd for C26H32N4O10ꢁH2O: C, 53.97; H, 5.92, N, 9.68. Found: C,
54.25; H, 5.77; N, 9.67.
DMF (5 mL) and treated with 3-iodo-1-propanol (93 lL, 1.0 mmol).
After stirring at 50 °C for 4 h, the mixture was evaporated under re-
duced pressure. The residue was dissolved in CH2Cl2 (20 mL), and
extracted with 1 M HCl and brine, dried over MgSO4, concentrated
under reduced pressure, and purified by flash chromatography on a
silica gel column (EtOAc/hexane = 1:1 to 4:1) to afford ester 9
(287 mg, 57%). C28H46N4O12; colorless solid, mp 86–88 °C; TLC
(EtOAc/hexane = 4:1) Rf = 0.48; ½a D21
ꢄ
ꢀ23.1 (c 1.0, CHCl3); IR mmax
(neat) 3275, 2931, 1727, 1646, 1611, 1558, 1150 cmꢀ1
;
1H NMR
(400 MHz, CDCl3) d 11.32 (1H, s), 8.58 (1H, d, J = 7.6 Hz), 7.96
(1H, d, J = 0.8 Hz), 5.76 (1H, d, J = 2.0 Hz), 5.26 (1H, d, J = 4.4 Hz),
5.12–5.08 (1H, m), 4.34–4.28 (2H, m), 4.14–4.10 (1H, m), 4.07–
3.91 (3H, m), 3.68 (2H, t, J = 6.0 Hz), 3.47–3.44 (1H, m), 1.96 (3H,
s), 1.90–1.87 (2H, m), 1.46 (9H, s), 1.44 (9H, s), 1.37 (3H, s), 1.31
(3H, s); 13C NMR (100 MHz, CDCl3) d 173.9, 162.2, 161.6, 157.5,
152.6, 146.7, 109.1, 107.0, 84.3, 80.0, 78.5, 74.0, 69.7, 67.4, 62.7,
59.1, 51.7, 48.3, 31.4, 28.1 (3ꢃ), 27.9 (3ꢃ), 26.9, 25.1, 22.9; ESI-
HRMS calcd for C28H47N4O12: 631.3190, found: m/z 631.3187
[M+H]+.
4.3.7. Guanidino-oseltamivir as the salt of trifluoroacetic acid
(3b)13,19
Compound 12 was prepared according to the previously de-
scribed method.17 A solution of compound 12 (50 mg, 0.09 mmol)
in CH2Cl2 (3 mL) was stirred with TFA (0.1 mL) at room tempera-
ture for 4 h, was and then evaporated under reduced pressure to
give guanidino-oseltamivir 3b (40 mg, 99 %). C17H30N4O4; white
solid; mp 187–189 °C; UV–vis (MeOH) kmax 260 nm (
e = 50,630);
UV–vis (PBS) kmax 230 nm (
e
= 8370); 1H NMR (400 MHz, D2O) d
6.83 (1H, s); 4.36–4.34 (1H, d, J = 8.0 Hz); 4.26 (2H, q, J = 7.2 Hz),
3.96–3.91(1H, m), 3.87–3.80 (1H, m), 3.55 (1H, t, J = 5.2 Hz), 2.89
(1H, dd, J = 17.2, 4.8 Hz), 2.43 (1H, dd, J = 18.0, 10 Hz), 2.05 (3H,
s), 1.62–1.43 (4H, m), 1.31 (3H, t, J = 6.8 Hz), 0.93–0.84 (6H, m);
13C NMR (100 MHz, CD3OD) d 174.7, 167.7, 160.9 (CO2 of TFA, q,
J = 41.2 Hz), 159.0, 139.3, 130.0, 117.3 (CF3 of TFA, q,
J = 285.3 Hz), 84.3, 76.5, 62.6, 56.2, 52.0, 31.7, 27.6, 27.3, 23.2,
14.9, 10.2, 10.1; 19F NMR (400 MHz, CD3OD) d ꢀ76.9; ESI-HRMS
calcd for C17H30N4O4: 355.2344, found: m/z 355.2345 [M+H]+.
4.3.5. Compound 11
To a solution of ester 9 (117 mg, 0.19 mmol) in CH2Cl2 (3 mL)
was added acid 10 (43 mg, 0.19 mmol), EDCI (36 mg, 0.19 mmol)
and 4-dimethylaminopyridine (21 mg, 0.19 mmol). The mixture
was stirred at room temperature for 1.5 h, was and extracted with
1 M HCl, saturated NaHCO3 and brine. The organic phase was dried
over MgSO4, concentrated under reduced pressure, and purified by
flash chromatography on a silica gel column (EtOAc/hexane = 1:4
to 2:3) to afford ester 11 (105 mg, 68%). C42H56N4O14; pale yellow
foam; TLC (EtOAc/hexane = 2:3) Rf = 0.22; ½a D219
ꢄ
ꢀ25.5 (c 1.0,
4.3.8. 3-Hydroxypropyl 4-acetamido-5-[N2,N3-bis(tert-butoxy
carbonyl)guanidino]-3-(1-ethylpropoxy)-1-cyclohexene-1-
carboxylate (13)
CHCl3); IR mmax (neat) 2980, 2930, 1726, 1647, 1607, 1565, 1369,
1250, 1152, 1129 cmꢀ1 1H NMR (400 MHz, CDCl3) d 11.32 (1H,
;
s), 8.57 (1H, d, J = 7.6 Hz), 8.22 (1H, d, J = 8.0 Hz), 7.94 (1H, d,
J = 4.8 Hz), 7.81 (1H, d, J = 8.8 Hz), 7.58–7.47 (3H, m), 6.21–6.11
(1H, m), 5.75 (1H, d, J = 2.0 Hz), 5.45 (1H, dd, J = 17.2, 1.2 Hz),
5.27 (2H, d, J = 11.6 Hz), 5.09–5.04 (1H, m), 4.61 (1H, d,
J = 5.6 Hz), 4.45–4.42 (2H, m), 4.38–4.33 (3H, m), 4.16–4.01 (3H,
m), 3.97–3.91 (2H, m), 3.47–3.44 (1H, m), 2.18–2.14 (2H, m),
1.97 (3H, s), 1.46 (9H, s), 1.45 (9H, s), 1.37 (3H, s), 1.31 (3H, s);
13C NMR (100 MHz, CDCl3) d 173.9, 165.9, 162.2, 161.2, 157.5,
156.9, 152.6, 146.7, 136.6, 133.6, 128.7, 128.3, 127.8, 126.5,
126.4, 123.6, 119.5, 117.6, 109.0, 106.8, 84.2, 80.0, 78.5, 76.5,
73.9, 69.7, 67.4, 62.3, 61.5, 60.3, 51.9, 48.3, 28.1 (3ꢃ), 27.9 (3ꢃ),
26.9, 25.0, 22.9, 14.1; ESI-HRMS calcd for C42H57N4O14: 841.3871,
found: m/z 841.3907 [M+H]+.
To a solution of 12 (221 mg, 0.42 mmol) in MeOH (5 mL) was
added KOH (26 mg, 0.46 mmol). The mixture was stirred at room
temperature for 30 min, and then concentrated under reduced
pressure to give a potassium salt, which was dissolved in DMF
(5 mL) and treated with 3-iodo-1-propanol (52 lL, 0.55 mmol).
After stirring at room temperature for 5 h, the mixture was evapo-
rated under reduced pressure. The residue was dissolved in CH2Cl2
(20 mL) and extracted with 1 M HCl and brine. The organic phase
was dried over MgSO4, concentrated under reduced pressure, and
purified by flash chromatography on a silica gel column (EtOAc/
hexane = 1:1) to afford ester 13 (137 mg, 55%). C28H48N4O9; color-
less solid, mp 95–97 °C; TLC (EtOAc/hexane = 3:1) Rf = 0.28; ½a D20
ꢄ
ꢀ68.0 (c 1.4, CHCl3); IR mmax (neat) 3278, 2970, 2932, 1724, 1643,
1610, 1252, 1144, 1055 cmꢀ1 1H NMR (400 MHz, CDCl3) d 11.33
;
4.3.6. ZA–HNAP conjugate 5
(1H, s), 8.58 (1H, d, J = 8.4 Hz), 6.75 (1H, s), 6.51 (1H, d,
J = 8.8 Hz), 4.34–4.28 (1H, m), 4.26–4.21 (2H, m), 4.07 (1H, m),
3.97 (1H, d, J = 7.6 Hz), 3.64 (2H, t, J = 6.4 Hz), 3.29 (1H, t,
J = 5.6 Hz), 2.73–2.68 (1H, m), 2.33 (1H, dd, J = 17.6, 9.2 Hz),
1.88–1.82 (5H, m), 1.50–1.43 (22H, m), 0.86–0.78 (6H, m); 13C
To a solution of compound 11 (65 mg, 0.08 mmol) in anhydrous
THF (4 mL) were added Pd(PPh3)4 (12 mg, 0.01 mmol) and
morpholine (0.1 mL, 1.4 mmol). The mixture was stirred at room
temperature for 4 h and extracted with 1 M HCl and brine. The

K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
4801
NMR (100 MHz, CDCl3) d 170.0, 166.0, 162.9, 156.7, 152.3, 138.4,
128.2, 83.5, 82.7, 79.5, 76.1, 61.8, 58.9, 54.3, 48.2, 31.8, 30.7, 28.4
(3ꢃ), 28.1 (3ꢃ), 26.1, 25.8, 23.4, 9.7, 9.4; ESI–HRMS calcd for
determination were carried out to assess reproducibility. The
octanol and aqueous phases were then separated by centrifuga-
tion at 3000 rpm for 15 min. Each sample (300 lL) of aqueous
layer was diluted with PBS to 3 mL, and organic layer was di-
luted with MeOH to 3 mL. The concentration of drug was mea-
sured using UV spectrophotometry at kmax (MeOH) 290 nm
C
28H48N4O9: 585.3496, found: m/z 585.3500 [M+H]+.
4.3.9. Compound 14
A mixture of compound 13 (177 mg, 0.30 mmol) acid 10 (90 mg,
0.39 mmol), EDCI (114 mg, 0.45 mmol) and 4-dimethylaminopyri-
dine (34 mg, 0.30 mmol) in CH2Cl2 (5 mL) was stirred at room tem-
perature for 12 h. The mixture was extracted with 1 M HCl,
saturated NaHCO3 and brine. The organic phase was dried over
MgSO4, concentrated under reduced pressure, and purified by flash
chromatography on a silica gel column (EtOAc/hexane = 1:3 to 1:1)
to afford compound 14 (144 mg, 60%). C42H58N4O11; colorless solid,
(e = 14,440) and kmax (PBS) 290 nm (e = 4610) at pH 7.4 for the
chromophore of 1-hydroxy-2-naphthoic acid. 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.
4.5. Incubation of conjugates in plasma and mass spectrometric
analyses
mp 75–77 °C; TLC (EtOAc/hexane = 2:1) Rf = 0.54; ½a D20
ꢀ60.6 (c 1.1,
ꢄ
CHCl3); IR mmax (neat) 3278, 2973, 2933, 1723, 1644, 1614, 1416,
1336, 1232, 1141, 1054 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d 11.36
Sprague DawleyÒ rat plasma (550
lL) and 50 lL of analyte were
(1H, s), 8.61 (1H, d, J = 8.0 Hz), 8.24 (1H, d, J = 8.0 Hz), 7.84–7.81
(2H, m), 7.62–7.51 (3H, m), 6.79 (1H, s), 6.26 (1H, d, J = 9.2 Hz),
6.24–6.14 (1H, m), 5.47 (1H, d, J = 17.2 Hz), 5.29 (1H, d,
J = 10.4 Hz), 4.64 (2H, d, J = 5.6 Hz), 4.45 (2H, t, J = 5.6 Hz), 4.40–
4.32 (3H, m), 4.13–4.07 (1H, m), 3.90 (1H, d, J = 6.8 Hz), 3.31–
3.25 (1H, m), 2.77 (1H, dd, J = 19.6, 5.2 Hz), 2.38 (1H, dd, J = 17.2,
9.6 Hz), 2.20–2.35 (2H, m), 1.90 (3H, s), 1.47–1.48 (22H, m),
0.94–0.79 (6H, m); 13C NMR (100 MHz, CDCl3) d 170.0, 165.8,
165.5, 162.9, 156.7, 156.6, 152.3, 138.3, 136.6, 133.4, 128.6,
128.2, 128.1, 127.7, 126.4, 123.7, 123.6, 119.4, 117.6 (2ꢃ), 83.5,
82.6, 79.5, 77.3, 76.1, 61.9, 61.8, 54.4, 48.1, 30.6, 28.4, 28.2 (3ꢃ),
28.1 (3ꢃ), 26.1, 25.8, 23.4, 9.7, 9.4; ESI-HRMS calcd for
mixed to yield a final concentration of 1 mM. The mixture was
incubated at 37 °C for 24 h, extracted with methanol (3.6 mL) by
vortex mixing at 4 °C for 2 h, and then subjected to centrifugation
at 10,000 rpm for 20 min. The precipitate was removed by filtra-
tion. The filtrate was concentrated under reduced pressure, and
the content of enzymatic cleavage products was analyzed by MAL-
DI-TOFMS.
Alternatively, the mixture resulted from incubation was sub-
jected to LC–QTOF MS analysis. The sample was loaded onto a
Sep-PakÒ Plus tC18 cartridge (Waters Corp.), and flushed with
deionized water (2 ꢃ 2 mL) to remove excess salts and proteins,
followed by elution with MeOH/H2O (1:1, 4 ꢃ 2 mL) to collect ana-
lyte. The analyte was evaporated to dryness, and re-dissolved in
C
42H58N4O11: 795.4180, found: m/z 795.4178 [M+H]+.
CH3CN/H2O (1:9, 100
Acquity UPLC BEH C18 column (1.7
Corp.). The injection volume was 2–5
50 min. A mixture of 0.1% formic acid in water (mobile phase A)
and 0.1% formic acid in acetonitrile (mobile phase B) was used with
l
L) for LC–QTOF MS analysis on a nano
m, 75
m ꢃ 250 mm, Waters
L and the total run time was
4.3.10. GO–HNAP conjugate 6
l
l
l
To a solution of 4 (74 mg, 0.09 mmol) in anhydrous THF (5 mL)
was added Pd(PPh3)4 (35 mg, 0.03 mmol) and morpholine (0.1 mL,
1.4 mmol). The mixture was stirred at room temperature for 12 h,
and then extracted with 1 M HCl and brine. The organic phase was
dried over MgSO4, concentrated under reduced pressure, and puri-
fied by flash chromatography on a silica gel column (EtOAc/hex-
ane = 1:2). The intermediate product was dissolved in CH2Cl2
(3 mL) and treated with TFA (0.1 mL). After stirring at room tem-
perature for 4 h, the mixture was evaporated under reduced pres-
sure. The residue was triturated with Et2O, and the precipitate was
collected by centrifugation to give conjugate 6 (26 mg, 56 %).
a gradient program at a flow rate of 0.3 lL/min at ambient temper-
ature. To analyze the product mixture formed by incubation of ZA–
HNAP in plasma, the gradient program of mobile phase was set: a
linear increase from 10:90 to 85:15 B/A in 25 min, and kept at
85:15 B/A for 10 min. The column was washed for 15 min before
returning to original conditions. To analyze the product mixture
formed by incubation of GO–HNAP in plasma, the gradient pro-
gram of mobile phase was set: a linear increase from 8:92 to
70:30 B/A in 25 min, from 70:30 to 85:15 B/A in 5 min, and kept
at 85:15 B/A for 5 min.
C
29H38N4O7; white solid, mp 136–138 °C; ½a D20
ꢀ36.4 (c 0.4,
ꢄ
MeOH); UV–vis (MeOH) kmax 315 nm
(
e
= 29,550), 270 nm
= 13,490); IR max (neat)
3413, 2967, 1703, 1663, 1253, 1205 cmꢀ1 1H NMR (400 MHz,
(e
= 106,930); UV–vis (PBS) kmax 270 nm (
e
m
;
4.6. Determination of influenza virus TCID50
CD3OD) d 8.34 (1H, d, J = 8.0 Hz), 7.80 (1H, d, J = 8.0 Hz), 7.75 (1H,
d, J = 8.8 Hz), 7.65–7.60 (1H, m), 7.56–7.50 (1H, m), 7.33 (1H, d,
J = 8.4 Hz), 6.82 (1H, d, J = 2.0 Hz), 4.56–4.51 (2H, m), 4.40 (2H, t,
J = 6.4 Hz), 4.17 (1H, d, J = 6.4 Hz), 3.91–3.84 (2H, m), 3.35–3.30
(1H, m), 2.84 (1H, dd, J = 16.8, 4.0 Hz), 2.38–2.33 (1H, m), 2.23
(2H, m), 1.98 (3H, s), 1.51–1.40 (4H, m), 0.09–0.84 (6H, m); 13C
NMR (100 MHz, CD3OD) d 172.0, 177.0, 158.4, 139.2, 138.6,
133.1, 133.0, 130.6, 130.0, 129.3, 128.7, 126.9, 125.1, 124.5,
119.9, 106.6, 83.8, 76.1, 63.7, 63.3, 55.9, 51.7, 31.5, 29.3, 27.4,
27.0, 23.0, 10.1, 9.8; ESI–HRMS calcd for C29H38N4O7: 555.2819,
found: m/z 555.2819 [M+H]+.
The TCID50 (50% tissue culture infectious dose) was determined
by serial dilution of the influenza virus stock solution onto 100 lL
MDCK cells at 1 ꢃ 105 cells/mL in 96-well microplates. The infected
cells were incubated at 37 °C under 5.0% CO2 for 48 h and added to
each wells with 100 l
L per well of CellTiter 96Ò AQueous Non-
Radioactive Cell Proliferation Assay reagent (Promega). After incu-
bation at 37 °C for 15 min, absorbance at 490 nm was read on a
plate reader. Influenza virus TCID50 was determined using Reed-
Muench method.24,25
4.7. Determination of neuraminidase activity by a fluorescent
assay
4.4. Determination of octanol-buffer partition coefficients
Solutions of each compound (100
l
g/mL) were prepared in
The neuraminidase activity was measured using diluted allan-
toic fluid harvested from influenza A/WSN/1933 (H1N1) infected
embryonated eggs. A fluorometric assay was used to determine
the NA activity with the fluorogenic substrate 20-(4-methylumbel-
octanol saturated phosphate buffered saline (0.01 M, pH 7.4).
These aqueous solutions were then equilibrated at 37 °C with
an equivalent volume (0.5 mL) of buffer saturated octanol using
magnetic stirring at 1200 rpm for 24 h. Five replicates of each
liferyl)-
a
-D
-N-acetylneuraminic
acid
(MUNANA;
Sigma).

4802
K.-C. Liu et al. / Bioorg. Med. Chem. 19 (2011) 4796–4802
The fluorescence of the released 4-methylumbelliferone was mea-
sured in Envision plate reader (Perkin–Elmer, Wellesley, MA) using
excitation and emission wavelengths of 365 and 460 nm, respec-
tively. Neuraminidase activity was determined at 200 lM of MUN-
ANA. Enzyme activity was expressed as the fluorescence increase
References and notes
1. Ryan, D. M.; Ticehurst, J.; Dempsey, M. H.; Penn, C. R. Antimicrob. Agents
Chemother. 1994, 38, 2270.
2. Cass, L. M. R.; Efthymiopoulos, C.; Bye, A. Clin. Pharmacokinet. 1999, 36, 1.
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Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R.
C. J. Am. Chem. Soc. 1997, 119, 681.
during 15 min incubation at room temperature.
4.8. Determination of IC50 of neuraminidase inhibitor
5. Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Li, W.; Sidwell, R. W.; Huffman, J. H.;
Sweet, C.; Jakeman, K. J.; Merson, J.; Lacy, S. A.; Lew, W.; Williams, M. A.; Zhang,
L.; Chen, M. S.; Bischofberger, N.; Kim, C. U. Antimicrob. Agents Chemother. 1998,
42, 640.
Neuraminidase inhibition was determined by mixing inhibitor
and neuraminidase for 10 min at room temperature followed by
6. Sidwell, R. W.; Huffman, J. H.; Barnard, D. L.; Bailey, K. W.; Wong, M. H.;
Morrison, A.; Syndergaard, T.; Kim, C. U. Antiviral Res. 1998, 37, 107.
7. Collins, P. J.; Haire, L. F.; Lin, Y. P.; Liu, J. F.; Russell, R. J.; Walker, P. A.; Skehel, J.
J.; Martin, S. R.; Hay, A. J.; Gamblin, S. J. Nature 2008, 453, 1258.
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Med. 2009, 361, 2296.
the addition of 200 lM of substrate. Inhibitor IC50 value were
determined from the dose-response curves by plotting the percent
inhibition of NA activity versus inhibitor concentrations using
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The anti-flu activities of neuraminidase inhibitors were mea-
sured by the EC50 values that were the concentrations of NA
inhibitor for 50% protection of the H1N1 CPE activities. Fifty
microliters diluted H1N1 at 100 TCID50 were mixed with equal
volumes of NA inhibitors at varied concentrations. The mixtures
were used to infect 100
l
L of MDCK cells at 1 ꢃ 105 cells/mL in
96-wells. After 48 h incubation at 37 °C under 5.0% CO2, the cyto-
pathic effects (CPE) were determined with CellTiter 96Ò AQueous
Non-Radioactive Cell Proliferation Assay reagent as described
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Acknowledgment
We thank the National Science Council for financial support.
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Supplementary data
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A Laboratory
Supplementary data (MS, 1H, 13C, 19F NMR spectra) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.06.080.