Oseltamivir hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.10.005

Tamiflu_, the ethyl ester form of oseltamivir carboxylic acid (OC), is the first orally available anti-influenza drug for the front-line therapeutic option. In this study, the OC-hydroxamates, OC-sulfonamides and their guanidino congeners (GOC)

Full text of DOI:10.1016/j.bmc.2014.10.005

Bioorganic & Medicinal Chemistry 22 (2014) 6647–6654
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oseltamivir hydroxamate and acyl sulfonamide derivatives
as influenza neuraminidase inhibitors
c
b
b
Bei-Tao Hong a, Chun-Lin Chen a, Jim-Min Fang a,b, , Keng-Chang Tsai , Shi-Yun Wang , Wen-I Huang ,
⇑
Yih-Shyun E. Cheng 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
c National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Tamiflu, the ethyl ester form of oseltamivir carboxylic acid (OC), is the first orally available anti-influenza
drug for the front-line therapeutic option. In this study, the OC-hydroxamates, OC-sulfonamides and their
guanidino congeners (GOC) were synthesized. Among them, an OC-hydroxamate 7d bearing an
O-(2-indolyl)propyl substituent showed potent NA inhibition (IC50 = 6.4 nM) and good anti-influenza
activity (EC50 = 60.1 nM) against the wild-type H1N1 virus. Two GOC-hydroxamates (9b and 9d) and
Received 11 June 2014
Revised 30 September 2014
Accepted 5 October 2014
Available online 25 October 2014
one GOC-sulfonamide (12a) were active to the tamiflu-resistant H275Y virus (EC50 = 2.3–6.9 lM).
Keywords:
Influenza
Ó 2014 Elsevier Ltd. All rights reserved.
Neuraminidase inhibitor
Oseltamivir
Bioisosteres
Hydroxamate
Acyl sulfonamide
1. Introduction
sialo-receptor on host cells. The NA-catalyzed hydrolysis of the ter-
minal sialyl residue involves a cationic intermediate in oxonium-
like configuration.10–12 Oseltamivir carboxylic acid (OC, 1a) is an
NA inhibitor designed to have the cyclohexene scaffold to mimic
the sialosyl cationic intermediate.13,14 Tamiflu™, the phosphate
salt of oseltamivir (OS, 1b), is the first orally available anti-influ-
enza drug. As an ethyl ester, OS is readily hydrolyzed by endoge-
nous esterases to OC as the real active NA inhibitor. The
carboxylate group of OC provides strong electrostatic interactions
with the three arginine residues (Arg118, Arg292 and Arg371) in
the S1 site of influenza NA. OC contains a 3-pentoxy substituent,
in lieu of the glycerol side chain in sialic acid, to render hydropho-
bic interactions with the Glu276, Ala246, Arg224, and Ile222
residues in the NA active site. However, the clinically relevant
H275Y OS-resistant virus has evolved over the years because
replacement of the histine residue by a bulkier tyrosine will cause
collapse of the salt bridge between Glu276 and Arg224 to reduce
the affinity with the hydrophobic 3-pentyl moiety in OS.15,16
Many OC analogs have been synthesized to test the anti-
influenza activities, for example, using lipophilic alkyl and aryl
groups other than the 3-pentyl group,13 using different amido
groups at the C-4 position,17 or relocating the C–C double bond
in the cyclohexene scaffold.18 However, such structural
modifications have not led to better anti-influenza activity than
OC. In contrast, GOC (2) having the amino group at the C-5 position
Influenza often occurs in winter seasons and occasionally
causes pandemics due to emergence of virus mutants and unex-
pected transmission to humans. Influenza has been a long-stand-
ing threat to humans’ health over centuries. Spanish flu in 1918
has caused more than 20 million deaths because no vaccine or
anti-influenza drugs were available.1 The outbreaks of new type
H1N1 pandemic flu2 in 2009 as well as H5N1 and H7N9 avian
flus3,4 further raise the public concern of possible influenza
pandemics and cross-species transmission to humans.
There are two classes of anti-influenza drugs approved to target
the virus M2 ion channel and neuraminidase (NA). However, the
M2 ion channel inhibitors amantadine5 and rimantadine6 are no
longer recommended for treatment of influenza infection due to
their severe side effects and drug resistance. NA is a viral surface
glycoprotein responsible for cutting the connection between the
hemagglutinin (HA) and the host cell,7 an essential function to
release the progeny viruses for infection to the surrounding cells.
Inhibition of viral NA is a very effective method for influenza
therapy because the active site of NA is highly conserved across
the influenza A and B viral strains.8,9 Influenza viral HAs bind the
⇑
Corresponding author. Tel.: +886 2 33661663; fax: +886 2 27325090.
E-mail address: jmfang@ntu.edu.tw (J.-M. Fang).
http://dx.doi.org/10.1016/j.bmc.2014.10.005
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6648
B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
in OC replaced by a more basic gaunidino group does upgrade the
NA inhibitory activity.14 The guanidinium ion is expected to
increase the electrostatic interactions with the acidic residues
(Glu119, Asp151, and Glu227) in the active site of influenza NA.
However, GOC and its ethyl ester have not been developed for
therapeutic use because of their poor pharmacokinetic properties.
The recent study indicates that N-hydroxyguanidine19 and amidox-
ime20 can be utilized as surrogates of the guanidino group to
improve bioavailability. The 5-acetamidine analog of OC exhibits
high NA inhibition and anti-influenza activities against wild-type
and resistant strains.20 Schade and his co-workers also show that
the acetamidoxime analog of OS can act as a prodrug to attain good
pharmacokinetic profile with 31% oral bioavailability in rats while
the N-hydroxyguanidine analog lacks of oral bioavailability
(F = 1.5%).20 In another approach, GOC esters carrying valine or
phenylalanine are designed to render better permeability that is
facilitated by the peptide transporter–cellular activation.21
contain more electronegative atoms and hydrophobic alkyl substit-
uents that may exert additional interactions with influenza NA in
the S1 site. Compounds 9b–9d and 12a–12d were also prepared
by guanidination of 8b–8d and 10a–10d, respectively, for compar-
ison of the bioactivities.
2. Results and discussion
For the synthesis of OC hydroxamate and acyl sulfonamide
derivatives, we consider using the Boc-protecting OC (5)28,29 as
the starting material to conduct the amide bond forming reactions
(Scheme 1). Thus, the commercially available O-alkyl hydroxylam-
ines (CH3ONH2 and PhCH2ONH2 as the hydrochloride salts) were
subjected to coupling reactions with 5 using diisopropylethylamine
(DIPEA), 4-dimethylaminopyridine (DMAP) and o-benzotriazol-1-
yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU) as
the combined promoting agents to give 6b and 6c, which were
subsequently treated with trifluoroacetic acid (TFA) to afford
OC-hydroxamates 7b and 7c. Attempts for direct coupling reaction
of acid 5 with NH2OH failed, even using various promoting agents
such as HBTU, thionyl chloride, ethyl chloroformate, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDCI) and benzotriazol-
1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).
Alternatively, NH2OH was protected as the tetrahydropyran acetal
(NH2OTHP)30 and proceeded smoothly with an amide forming reac-
tion with 5 in the presence of HBTU, DIPEA and DMAP to give 6a.
Both the Boc and THP protecting groups in 6a were concomitantly
removed by treatment with TFA to give the desired hydroxamic
O
O
O
O
OR
OH
AcHN
AcHN
NH2
HN
NH
NH2
1a
1b
(OC) R = H
(OS) R = Et
2
(GOC)
O
P
O
P
OR
OH
OR
OH
O
O
acid 7a. We also synthesized hydroxamate 7d containing
a
AcHN
AcHN
3-(indol-2-yl)propyl substituent to test whether it would provide
good NA inhibitory activity. The aralkyl group in 7d may reside in
the hydrophobic 371-cavity that is enclosed by the
Arg371, Pro431, Ile427, Lys432 and Trp403 residues near the S1 site
NH2
HN
NH
NH2
3a
3b
3c
(TP) R = H
R = Et
R = n-Hex
4a
4b
4c
(TPG) R = H
R = Et
R = n-Hex
to attain additional hydrophobic, p–p and p
–cation interactions.31
For guanidination, 7b–7d were reacted respectively with
1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine in the presence
of Et3N to give the Boc-protecting compounds 8b–8d, which
were subsequently treated with TFA to give the desired products
9b–9d.
By the similar procedure, the coupling reactions of acid 5 with a
series of sulfonamides were also carried out, followed by removal
of the Boc protecting group with TFA, to afford acyl sulfonamides
10a–10d. The amino group in 10a–10d was further elaborated to
guanidino group by treatment with 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine to afford GOC-sulfonamides 12a–12d after
removal of the Boc groups.
Bioisosteres are the functional groups through rational modifica-
tions of an active composition to mimic the structure with similar
chemical, physical, electronic, conformational and biological prop-
erties.22 Many medicinal chemists have taken the advantages of
bioisosterism to optimize the pharmacokinetic properties of lead
compounds to develop safer and more clinically effective agents.
As carboxylic acid is an important functional group in drugs, the
bioisosteres of carboxylic acid have been extensively investigated.23
By replacing the carboxylic group in OC, the phosphonate congener
3a (tamiphosphor, TP) shows remarkable neuraminidase inhibition
and anti-influenza activities in the cell-based and animal experi-
ments.24,25 The monoesters of TP,25–28 for example, 3b and 3c, are
also effective anti-influenza drugs because the singly-charged phos-
phonate group still retains substantial electrostatic interactions
with the three arginine residues (Arg118, Arg292 and Arg371) in
the active site of neuraminidase. As expected, guanidino TP (TPG,
4a) and its monoesters (e.g., 4b and 4c) exhibit even better inhibi-
tory activities against avian and human influenza viruses including
the drug-resistant H275Y strain.24,25
According to molecular modeling experiments, we have previ-
ously proposed a 371-cavity near the S1-site of influenza NA to
accommodate appropriate alkyl substituents.28 As a continuing
research, we report herein the synthesis and anti-influenza activi-
ties of two series of OC bioisosteres: hydroxamates 7a–7d and acyl
sulfonamides 10a–10d (Scheme 1). Hydroxamic acid (pKa ꢀ 8.5) is
weaker than carboxylic acid (pKa ꢀ 4.7), whereas acyl sulfonamide
(pKa ꢀ 5–6) has the acidity close to that of carboxylic acid. Com-
pared with the carboxylic acid group in OC, the hydroxamate
groups in 7a–7d and the acyl sulfonamide groups in 10a–10d
The enzymatic assays for NA inhibition were conducted using a
fluorescent substrate, 2-(4-methylumbelliferyl)-a-D-N-acetylneu-
raminic acid (MUNANA), whereas the anti-influenza activities
were measured by the cytopathic effect of Madin–Darby canine
kidney (MDCK) cells due to infection of influenza viruses. Table 1
shows the IC50 and EC50 values of OC-hydroxamates 7a–7d, GOC-
hydroxamates 9b–9d, OC-sulfonamides 10a–10d and GOC-sulfon-
amides 12a–12d. In comparison with OC, the NA inhibitory activity
of 7a was much lower, presumably because the hydroxamic acid in
7a was less acidic to attain strong interactions with the three argi-
nine residues in the S1 site of influenza NA. By incorporation of
alkyl substituents at the O-atom of hydroxamate moiety, com-
pounds 7b–7d displayed better NA inhibitory and anti-influenza
activities than 7a. In this series, hydroxamate 7d bearing an
(indol-2-yl)propyl substituent was the best inhibitor, showing
the IC50 value of 6.4 nM and the EC50 value of 60.1 nM, close to
the activity of OC against the wild-type H1N1 virus.
The molecular modeling of 7d in the active site of influenza
virus NA (Fig. 1) indicates that the hydroxamate moiety exhibits
extensive hydrogen bonding interactions (4 pairs ligand–NA

B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
6649
O
O
O
O
O
O
O
O
O
O
O
RONH2
a
N
H
R
b
N
H
R
d
N
H
R
OH
AcHN
AcHN
AcHN
AcHN
NHBoc
NHBoc
NH2
HN
NBoc
5
6a
6b
6c
6d
R = 2-THP
R = Me
R = CH2Ph
R = (CH2)3-(2-indole)
7a
7b
7c
7d
R = H
R = Me
R = CH2Ph
R = (CH2)3-(2-indole)
NHBoc
8b
8c
8d
R = Me
c
RSO2NH2
R = CH2Ph
R = (CH2)3-(2-indole)
b
b
O
O
S
O
O
O
S
O
O
O
S
O
O
O
O
O
N
H
R
d
N
H
R
N
H
R
O
O
b
N
H
R
AcHN
AcHN
AcHN
AcHN
NH2
HN
NBoc
HN
NH
NH2
HN
NH
NH2
R = CH2Ph
10a
10b
10c
10d
R = Me
NHBoc
R = Me
R = CF3
R = n-Bu
R = Ph
R = CF3
R = n-Bu
R = Ph
11a
11b
11c
11d
12a
12b
12c
12d
R = Me
R = CF3
R = n-Bu
R = Ph
9b
9c
9d
R = Me
R = (CH2)3-(2-indole)
Scheme 1. Synthesis of oseltamivir hydroxamate and acyl sulfonamide derivatives. Reagents and reaction conditions: (a) i-Pr2NEt, DMAP, HBTU, CH2Cl2, rt, 10–12 h. (b)
CF3CO2H, CH2Cl2, rt, 1 h. (c) Et3N, DMAP, HBTU, CH2Cl2, rt, 10 h. (d) (BocNH)(TfNH)C@NBoc, Et3N, CH2Cl2, rt, 10–12 h. Overall yields: 7a (47%), 7b (51%), 7c (54%), 7d (42%), 9b
(41% from 7b), 9c (42% from 7c), and 9d (37% from 7d), 10a (47%), 10b (51%), 10c (49%), and 10d (43%), 12a (32% from 10a), 12b (39% from 10b), 12c (36% from 10c), and 12d
(28% from 10d). Boc = tert-butoxycarbonyl; HBTU = o-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate; DMAP = 4-dimethylaminopyridine; THP =
tetrahydropyran; Tf = trifluorosulfonyl.
In the series of OC-sulfonamides, 10a bearing a methyl substitu-
ent in the moiety of acyl sulfonamide showed modest NA inhibi-
tory activity against the wild-type and H275Y H1N1 viruses with
IC50 values of 67.5 and 401 nM, respectively. However, compound
10b having an electronegative trifluoromethyl substituent
decreased the NA inhibitory activity by 5–17 folds relative to
10a. Compounds 10c and 10d having bulkier butyl and phenyl sub-
stituents also caused reduced binding to influenza NA. Though the
acidity of acyl sulfonamides is similar to carboxylic acid, OC-sul-
fonamides 10a–10d did not display good NA inhibition. This result
suggested that the acidity of bioisosteres was not a single factor
responsible for good anti-influenza activity.
By replacing the amino group in 10a–10d with a more basic gua-
nidino group, compounds 12a–12d did exert much higher inhibi-
tory activities against the wild-type H1N1 virus. However, only
compound 12a showed somewhat better activity than OC against
the H275Y mutant of H1N1 virus with IC50 value of 19.5 nM and
Table 1
Neuraminidase inhibition and anti-influenza activity
a
b
Compd
IC50 (nM)
EC50 (nM)
A/WSN/33c
H275Yc
A/WSN/33c
H275Yc
OC
7a
7b
7c
7d
9b
9c
9d
10a
10b
10c
10d
12a
12b
12c
12d
1.78 0.55
384 14
45.4 11.6
31.1 6.7
6.4 2.0
6.82 1.03
8.49 1.61
151.2 11.8e
67.5 30.6
1203 367
656 262
574 105
20.3 1.9
94.0 15.9
41.5 8.7
37.6 9.0
260 21
28.2 3.6
359 107
11,026 1541
No effectd
No effectd
No effectd
No effectd
2770 1579
16143 11502
2268 442
No effectd
No effectd
No effectd
No effectd
6902 766
No effectd
No effectd
No effectd
No effectd
6648 322
1010 54
1108 89
781 45
127
4
72.4 34.9
60.1 2.7
71.7 3.9
23.8 11.6
90.7 49.8
401 162
2223 472
1977 23
7487 2500
59.1 0.6
6946 1971
1227 51
19,085 4681
No effectd
No effectd
No effectd
19.5 2.2
832 524
1108 427
13,399 228
484
4
261 119
593 34
EC50 value of 6.9 lM. The guanidino groups in 12a–12d were con-
sidered to render stronger interactions with the acidic residues
acidic residues of Glu119, Asp151 and Glu227 in the S2 site of
NA.14 The tendency of inhibitory activity in the two acyl-sulfon-
amide series was similar, i.e. 10a/12a (R = CH3) > 10d/12d
(R = Ph) ꢀ 10c/12c (R = n-Bu) > 10b/12b (R = CF3).
a
A fluorescent substrate, 2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid
(MUNANA), was used to determine the IC50 values that are compound concentra-
tions causing 50% inhibition of different influenza neuraminidases. Data are shown
as mean SD of three experiments.
b
The anti-influenza activities against different influenza viruses were measured
as EC50 values that are the compound concentrations for 50% protection of the
cytopathic effects due to the infection by different influenza viruses. Data are
shown as mean SD of three experiments.
The effects of guanidino group in GOC-hydroxamates 9b–9d are
discrepant. The O-methyl GOC-hydroxamate 9b showed much bet-
ter activities than the corresponding OC-hydroxamate 7b in NA
inhibition and cell protection against H1N1 and mutant influenza
viruses. In comparison with 7c, the O-benzyl GOC-hydroxamte 9c
also showed better cell protection against H1N1 virus and the
H275Y mutant. Compounds 9d and 7d showed similar NA inhibi-
tion against H275Y mutant, but 9d exhibited a higher IC50 value
(151 nM) than 7d (6.4 nM) against wild-type H1N1 virus for
unclear reason. Nonetheless, both compounds displayed equal inhi-
c
A/WSN/33 is human H1N1 virus, and H275Y is an oseltamivir-resistant mutant.
d
The IC50 or EC50 values were over 100
lM.
e
Data for two repeated triplicate experiments.
H-bonds) with three arginine residues (R118, R292 and R371) and
the phenol group of Y347 in the NA active site. The oxygen of the
acetamido group in 7d forms a hydrogen bond with the guanidi-
nium group of R152. In addition, the amine group in 7d forms ionic
interactions with residues D151, E119 and E227. The (2-indo-
lyl)propyl substituent in 7d extends to the 371-cavity to obtain
bition activity against the H275Y strain (IC50 ꢀ 1.2
noted that GOC-hydroxamates 9b and 9d were effective to protect
cells against the infection of H275Y mutant (EC50 = 2.3–2.8 M).
Compounds 9b, 9d and 12a were nontoxic to MDCK and human
293T cells (CC50 > 100 M). Based on the CC50 data of the
lM). It was
l
appreciable
H-bond,
hydrophobic,
p–p
and
p–cation
interactions.28,31
l

6650
B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
Figure 1. Molecular model of 7d in the active site of influenza virus neuraminidase (N1 subtype, PDB id: 2HU4). The hydrogen bonds and ionic interactions are shown in
green and yellow dotted lines, respectively.
anti-influenza agents, unspecific effects (from cytotoxicity) could
be excluded for the entire series of OC derivatives (Table 1).
(doublet), t (triplet), q (quartet), m (multiplet), br (broad) and dd
(doublet of doublets). IR spectra were recorded on Varian 640 or
Nicolet Magna 500-II. Optical rotations were recorded on digital
polarimeter of Japan JASCO Co. DIP-1000. [a] values were given
D
3. Conclusion
in units of 10–1 deg cm2 g–1. ESI mass spectra were recorded on a
high-resolution mass spectrometer.
Oseltamivir carboxylic acid (OC) is a potent neuraminidase
inhibitor, and the ethyl ester OS is developed as an effective orally
available anti-influenza drug. In this study, we first synthesized
two series of OC bioisosteres, that is, OC-hydroxamates 7a–7d
and OC-sulfonamides 10a–10d, as well as the guanidino congeners
9b–9d and 12a–12d. In the series of OC-hydroxamates, 7d bearing
an O-(2-indolyl)propyl substituent showed the best NA inhibitory
activity, presumably because the substituent can locate in the
371-cavity near the S1 site of NA to attain additional hydrophobic,
4.2. Materials
Influenza A/WSN/1933 (H1N1) was from Dr. Shin-Ru Shih at
Chang Gung University in Taiwan. All viruses were cultured in
the allantoic cavities of 10-day-old embryonated chicken 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) containing 10%
fetal bovine serum (GibcoBRL) and penicillin–streptomycin
(GibcoBRL) at 37 °C under 5% CO2.
p–p and p–cation interactions. In the series of GOC-sulfonamides,
12a is an effective NA inhibitor with the EC50 value slightly lower
than OC against the drug-resistant H275Y virus. The GOC-hydroxa-
mates 9b and 9d were even better anti-influenza agents against
The oseltamivir-resistant A/WSN H275Y mutant was created
using a 12-plasmid system that was 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. An H275Y
mutation was introduced in the NA gene, and the sequence was
confirmed to generate the A/WSN H275Y virus.
the H275Y mutant (EC50 ꢀ 2.5
lM). Our study indicates that the
NA inhibitory activity cannot simply be accounted on to the acidity
and the numbers of electron-negative atoms in the carboxylate
isosteres. So far, only OC phosphonate congeners have been dem-
onstrated to possess enhanced NA inhibition by having higher
acidity with complementary topology to interact with the three
arginine residues (Arg 181, Arg292 and Arg371) in the NA active
site.24,26
4.3. Synthetic procedures and compound characterization
4. Experimental section
4.1. General
Compound 5 was prepared according to the previously reported
method.28,29
All the reagents were commercially available and used without
further purification unless indicated otherwise. All solvents were
anhydrous grade unless indicated otherwise. Reactions were mag-
netically stirred and monitored by thin-layer chromatography on
silica gel. Flash chromatography was performed on silica gel (40–
4.3.1. N-Hydroxy (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (7a)
To a solution of the Boc protected oseltamivir acid (5, 78 mg,
0.2 mmol) in CH2Cl2 (2 mL) were added (tetrahydro-2H-pyran-2-
oxy)amine30 (28 mg, 0.24 mmol), HBTU (91 mg, 0.24 mmol), DIPEA
(0.1 mL, 0.6 mmol) and DMAP (7.4 mg, 0.02 mmol). The mixture
was stirred at room temperature for 10 h, and then concentrated
under reduced pressure. The residue was dissolved in CH2Cl2,
washed with 1.0 M HCl, saturated NaHCO3 and brine. The organic
layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give 6a as pale yellow oil,
which was used in the next step without further purification.
63 lm particle size) and LiChroprep RP-18 (40–63 lm particle
size). Yields were reported for spectroscopically pure compounds.
Melting points were recorded on a Yanaco melting point apparatus.
1H and 13C NMR spectra were recorded on Varian Unity Plus-400
(400 MHz) spectrometer. 19F NMR spectra were recorded on Varian
Unity Plus-400 (400 MHz) spectrometer. Chemical shifts were
given in d values relative to tetramethylsilane (TMS); coupling
constants J were given in Hz. Internal standards were CDCl3
(dH = 7.24), CD3OD (dH = 3.31) or D2O (dH = 4.79) for 1H NMR
spectra; and CDCl3 (dC = 77.0) or CD3OD (dC = 49.15) were for 13C
NMR spectra. The splitting patterns are reported as s (singlet), d
A
solution of the above-prepared compound 6a in
anhydrous CH2Cl2 (1 mL) was cooled to 0 °C in an ice-bath, and

B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
6651
trifluoroacetic acid (0.24 mL, 3.22 mmol) was added. The mixture
was stirred and allowed to warm up to room temperature for 1 h,
and then concentrated under reduced pressure. The residue was
reaction with N-hydroxyphthalimide in the presence of Ph3P and
diisopropyl azodicarboxylate (DIAD) to afford 3-(1H-indol-2-yl)
propoxylamine after cleaving the phthalimide moiety with
hydrazine.
purified on
a reversed-phase RP-18 column (MeOH/H2O = 1:
9–1:4) to give the desired product 7a (28 mg, 47%). C14H25N3O4;
To a solution of the Boc protected oseltamivir acid (5, 78 mg,
0.2 mmol) in CH2Cl2 (2 mL) were added 3-(1H-indol-2-yl)prop-
oxylamine (as the hydrochloric salt, 137 mg, 0.72 mmol), DIPEA
(0.1 mL, 0.6 mmol), HBTU (91 mg, 0.24 mmol) and DMAP (7.4 mg,
0.02 mmol). The mixture was stirred at room temperature for
12 h, and then concentrated under reduced pressure. The residue
was dissolved in CH2Cl2, washed with 1.0 M HCl, saturated NaHCO3
and brine. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (EtOAc/hexane = 3:1) to give
6d (71 mg, 63%). C30H44N4O6; pale yellow oil; 1H NMR (400 MHz,
CDCl3) d 9.84 (1H, br), 9.71 (1H, br), 7.49 (1H, d, J = 7.6 Hz), 7.34
(1H, d, J = 7.6 Hz), 7.06 (1H, t, J = 7.6 Hz), 7.01 (1H, t, J = 7.6 Hz),
6.69 (1H, br d, J = 6.8 Hz), 6.27 (1H, s), 6.15 (1H, s), 5.52 (1H, d,
J = 9.2 Hz), 4.08–3.99 (2H, m), 3.86–3.81 (2H, m), 3.32 (1H, t,
J = 5.6 Hz), 2.85 (2H, t, J = 6.6 Hz), 2.69 (1H, dd, J = 17.2, 5.4 Hz),
2.31 (1H, dd, J = 17.2, 10.2 Hz), 2.03 (3H, s), 1.84 (2H, t,
J = 6.0 Hz), 1.72–1.13 (14H, m), 0.93–0.82 (6H, m); 13C NMR
(100 MHz, CDCl3) d 171.2, 166.9, 156.3, 138.8, 136.1, 132.8,
120.3, 128.5, 120.4, 119.3, 119.0, 110.8, 99.0, 82.9, 79.6, 75.8,
74.8, 55.1, 49.8, 31.0, 28.6, 28.4 (3ꢂ), 26.2, 25.7, 24.0, 23.3, 9.8,
20
white solid; mp 224–226 °C; [
a
]
D
ꢁ11.5 (c 0.5, MeOH); IR (film)
3272, 2968, 2939, 2866, 1673, 1539, 1202, 1139 cm–1
;
1H NMR
(400 MHz, D2O) d 6.50 (1H, t, J = 2.0 Hz), 4.40 (1H, t, J = 8.8 Hz),
4.18 (1H, dd, J = 11.6, 8.8 Hz), 3.76–3.68 (1H, m), 3.67–3.62 (1H,
m), 3.00–2.95 (1H, m), 2.71–2.63 (1H, m), 2.20 (3H, s),
1.71–1.54 (4H, m), 1.01–0.94 (6H, m); 13C NMR (100 MHz, D2O)
d 175.1, 173.9, 133.1, 132.4, 84.2, 75.7, 53.2, 49.7, 29.8, 25.5,
25.2, 22.3, 8.5, 8.4; HRMS calcd for C14H26N3O4:300.1923, found:
m/z 300.1931 [M+H]+.
4.3.2. N-Methoxy (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (7b)
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with O-methyl hydroxylamine
hydrochloride (17 mg, 0.24 mmol) was conducted in the presence
of HBTU (91 mg, 0.24 mmol), DIPEA (0.1 mL, 0.6 mmol) and DMAP
(7.4 mg, 0.02 mmol) to give compound 6b, which was subse-
quently treated with trifluoroacetic acid (0.24 mL, 3.22 mmol) to
give 7b (32 mg, 51%). The purity of product 7b was 98.7% as shown
by HPLC on an HC-C18 column (Agilent, 4.6 ꢂ 250 mm, 5
lm
porosity), tR = 15.2 min (MeOH/H2O = 3:7). C15H27N3O4; hygro-
9.3; HRMS calcd for
579.3149 [M+Na]+.
C30H44N4NaO6:579.3159, found: m/z
22
scopic solid; [
2940, 2882, 1672, 1548, 1440, 1375, 1303, 1202, 1137, 1078,
1021, 943, 844, 800, 723 cm–1 1H NMR (400 MHz, CDCl3) d 6.40
a]
ꢁ57.5 (c 1, MeOH); IR (film) 3475, 3273, 2969,
D
A
solution of the above-prepared compound 6d (71 mg,
;
0.12 mmol) in anhydrous CH2Cl2 (1 mL) was cooled to 0 °C in an
ice-bath, and trifluoroacetic acid (0.24 mL, 3.22 mmol) was added.
The mixture was stirred and allowed to warm up to room temper-
ature for 1 h, and then concentrated under reduced pressure. The
residue was purified on a reversed-phase RP-18 column (MeOH/
H2O = 1:9–1:4) to give the desired product 7d (36 mg, 66%). The
purity of product 7d was 97.1% as shown by HPLC on an HC-C18
(1H, s), 4.20 (1H, d, J = 8.4 Hz), 3.97 (1H, dd, J = 11.2, 8.4 Hz), 3.72
(3H, s), 3.54–3.48 (1H, m), 3.42 (1H, quint, J = 5.2 Hz), 2.83 (1H,
dd, J = 18.2, 5.0 Hz), 2.51–2.44 (1H, m), 2.03 (3H, s), 1.59–1.44
(4H, m), 0.93–0.85 (6H, m). 13C NMR (100 MHz, CDCl3) d 174.9,
134.1, 129.6, 84.0, 75.8, 63.4, 54.6, 50.8, 34.1, 29.6, 27.3, 26.7,
23.3, 9.9, 9.7; HRMS calcd for C15H28N3O4: 314.2080, found: m/z
314.2080 [M+H]+.
column (Agilent, 4.6 ꢂ 250 mm,
(MeOH/H2O = 3:7). C25H36N4O4; hygroscopic solid; [
1.0, MeOH); IR (film) 3422, 2967, 2866, 2844, 1675, 1543, 1433,
1373, 1203, 1139, 1055, 1033, 1015 cm–1 1H NMR (400 MHz,
5 lm porosity), tR = 14.2 min
20
a]
ꢁ20.6 (c
D
4.3.3. N-Benzyloxy (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (7c)
;
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with O-benzyl hydroxylamine
hydrochloride (38 mg, 0.24 mmol) was conducted in the presence
of HBTU (152 mg, 0.24 mmol), DIPEA (0.1 mL, 0.6 mmol) and
DMAP (7.4 mg, 0.02 mmol) to give compound 6c, which was subse-
quently treated with trifluoroacetic acid (0.24 mL, 3.22 mmol) to
give 7c (82 mg, 68%). The purity of product 7c was 96.7% as shown
D2O) d 7.64 (1H, d, J = 7.6 Hz), 7.50 (1H, d, J = 8.4 Hz), 7.24 (1H, t,
J = 7.4 Hz), 7.17 (1H, t, J = 7.4 Hz), 6.40 (1H, s), 4.31 (1H, br d,
J = 7.6 Hz), 4.13–4.10 (1H, m), 4.07 (2H, t, J = 6.2 Hz), 3.68–3.60
(2H, m), 3.55 (1H, t, J = 5.4 Hz), 2.98 (2H, t, J = 7.2 Hz), 2.96–2.90
(1H, m), 2.67–2.56 (1H, m), 2.18–2.15 (5H, m), 1.66–1.51 (4H,
m), 0.99–0.91 (6H, m); 13C NMR (100 MHz, CD3OD) d 173.6,
167.4, 138.8, 137.9, 133.0, 129.3, 127.6, 121.6, 118.6, 111.0,
107.0, 82.7, 82.6, 74.5, 63.9, 50.0, 49.5, 39.0, 28.3, 26.0, 25.5, 25.4,
22.0, 8.6, 8.4; HRMS calcd for C25H37N4O4: 457.2815, found: m/z
457.2821 [M+H]+.
by HPLC on an HC-C18 column (Agilent, 4.6 ꢂ 250 mm, 5
lm
porosity), tR = 12.7 min (MeOH/H2O = 3:7). C21H31N3O4; hygro-
23
scopic solid; [
2878, 1673, 1639, 1543, 1526, 1458, 1436, 1374, 1300, 1203,
1184, 1135, 1074, 1019 cm–1 1H NMR (400 MHz, D2O)
a]
ꢁ22.6 (c 1.0, MeOH); IR (film) 2967, 2937,
D
;
d
4.3.5. N-Methoxy (3R,4R,5S)-4-acetamido-5-guanidino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (9b)
7.60–7.55 (5H, m), 6.24 (1H, t, J = 2.0 Hz), 5.05 (2H, s), 4.33 (1H,
d, J = 9.0 Hz), 4.12 (1H, dd, J = 11.8, 9.0 Hz), 3.70–3.63 (1H, m),
3.63–3.56 (1H, m), 2.88 (1H, dd, J = 17.0, 5.8 Hz), 2.62–2.55 (1H,
m), 2.18 (3H, s), 1.69–1.52 (4H, m), 0.99–0.93 (6H, m); 13C NMR
(100 MHz, D2O) d 174.9, 137.1, 133.9, 130.6 (2ꢂ), 129.8, 129.6
(4ꢂ), 84.0, 79.0, 75.9, 54.6, 50.8, 29.7, 27.3, 26.7, 23.3, 9.9, 9.7;
HRMS calcd for C21H32N3O4: 390.2393, found: m/z 390.2393
[M+H]+.
NEt3 (0.041 mL, 0.3 mmol) and 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (47 mg, 0.12 mmol) were added to a solution
of compound 7b (31 mg, 0.1 mmol) in CH2Cl2 (1 mL). The mixture
was stirred at 25 °C for 10 h, and then concentrated under reduced
pressure. The residue was dissolved in CH2Cl2, washed with 1.0 M
HCl, saturated NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash silica gel column chromatography
CH2Cl2/MeOH = 20:1) to give 8b as colorless oil.
A solution of the above-prepared compound 8b in anhydrous
CH2Cl2 (1 mL) was cooled to 0 °C in an ice-bath, and trifluoroacetic
acid (0.24 mL, 3.22 mmol) was added. The mixture was stirred at
room temperature for 1 h, and then concentrated under reduced
pressure. The residue was purified by reversed-phase RP-18
4.3.4. N-[3-(1H-Indol-2-yl)propoxy] (3R,4R,5S)-4-acetamido-5-
amino-3-(1-ethylpropoxy)-1-cyclohexenecarboxamide (7d)
The details for preparation of 3-(1H-indol-2-yl)propoxylamine
is reported in Supplementary data. In brief, 2-iodo-trifluoroaceta-
mide was reacted with 3-butyn-1-ol in the presence of Pd(OAc)2
to give 2-(3-hydroxypropyl)indole,32 which underwent Mitsunobu

6652
B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
column (MeOH/H2O = 1:9–1:4) to give the desired product 9b
(15 mg, 41%). The purity of product 9b was 99.3% as shown by
4.3.8. N-Methylsulfonyl (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (10a)
HPLC on an HC-C18 column (Agilent, 4.6 ꢂ 250 mm, 5
l
m poros-
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with methanesulfonamide
(23 mg, 0.24 mmol) was conducted in the presence of HBTU
(91 mg, 0.24 mmol), NEt3 (0.08 mL, 0.6 mmol) and DMAP (7.4 mg,
0.02 mmol). The residues was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give pale yellow oil,
which was subsequently treated with trifluoroacetic acid
(0.24 mL, 3.22 mmol) to give 10a (34 mg, 47%). The purity of prod-
uct 10a was 99.2% as shown by HPLC on an HC-C18 column (Agi-
ity), tR = 11.4 min (MeOH/H2O = 3:7). C16H29N5O4; hygroscopic
22
solid; [
a
]
D
ꢁ43.9 (c 1, MeOH); IR (film) 3416, 3179, 2771, 1643,
1447, 1325, 1268, 1117, 1025, 920 cm–1
;
1H NMR (400 MHz,
CD3OD) d 6.41 (1H, t, J = 2.4 Hz), 4.20 (1H, br d, J = 2.4 Hz), 3.89
(2H, br t, J = 3.0 Hz), 3.72 (3H, s), 3.41 (1H, quint, J = 5.6 Hz), 2.73
(1H, dd, J = 18.4 Hz, 3.2 Hz), 2.36 (1H, br dd, J = 11.2 Hz, 6.4 Hz),
1.98 (3H, s), 1.59–1.43 (4H, m), 0.93–0.86 (6H, m); 13C NMR
(100 MHz, CD3OD) d 174.4, 158.7, 134.3, 130.6, 126.2, 84.0, 76.2,
64.4, 56.2, 51.7, 31.4, 27.3, 26.9, 23.0, 9.90, 9.85; HRMS calcd for
lent, 4.6 ꢂ 250 mm,
H2O = 3:7). C15H27N3O5S; hygroscopic solid; [
MeOH); IR (film) 3274, 2965, 2937, 2878, 1658, 1550, 1463, 1373,
1324, 1242, 1100, 1064, 1023, 970, 852, 752 cm–1 1H NMR
5
lm
porosity), tR = 12.5 min (MeOH/
22
C
16H30N5O4: 356.2298, found: m/z 356.2305 [M+H]+.
a]
ꢁ52.9 (c 1,
D
4.3.6. N-Benzyloxy (3R,4R,5S)-4-acetamido-5-guanidino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (9c)
;
(400 MHz, CD3OD) d 6.68 (1H, s), 4.19 (1H, br d, J = 8.0 Hz), 3.96
(1H, dd, J = 11.2 Hz, 8.0 Hz), 3.50–3.41 (2H, m), 3.04 (3H, s), 2.90
(1H, br dd, J = 17.2 Hz, 8.4 Hz), 2.45–2.39 (1H, m), 2.03 (3H, s),
1.59–1.46 (4H, m), 0.94–0.87 (6H, m); 13C NMR (100 MHz, CD3OD)
d 174.9, 174.1, 135.0, 134.4, 83.7, 76.4, 54.7, 51.4, 40.8, 30.4, 27.3,
26.6, 23.4, 10.0, 9.7; HRMS calcd for C15H28N3O5S: 362.1750, found:
m/z 362.1752 [M+H]+.
By a procedure similar to that for 9b, guanidination of com-
pound 7c (32 mg, 0.08 mmol) with 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (37 mg, 0.096 mmol) was conducted in the
presence of NEt3 (0.033 mL, 0.24 mmol) in CH2Cl2 (1 mL). The resi-
due was purified by flash silica gel column chromatography (CH2-
Cl2/MeOH = 20:1) to give 8c as pale yellow oil, which was
subsequently treated with trifluoroacetic acid (0.19 mL,
2.57 mmol) to give the desired product 9c (14 mg, 42%) after puri-
fication on a reversed-phase RP-18 column (MeOH/H2O = 1:9–1:4).
The purity of product 9c was 96.7% as shown by HPLC on an HC-C18
4.3.9. N-Trifluoroethylsulfonyl (3R,4R,5S)-4-acetamido-5-
amino-3-(1-ethylpropoxy)-1-cyclohexenecarboxamide (10b)
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with trifluoromethanesulf-
onamide (36 mg, 0.24 mmol) was conducted in the presence of
HBTU (91 mg, 0.24 mmol), NEt3 (0.08 mL, 0.6 mmol) and DMAP
(7.4 mg, 0.02 mmol). The residues was purified by flash silica gel
column chromatography (CH2Cl2/MeOH = 20:1) to give pale yellow
oil, which was subsequently treated with trifluoroacetic acid
(0.24 mL, 3.22 mmol) to give 10b (42 mg, 51%). The purity of prod-
uct 10b was 97.4% as shown by HPLC on an HC-C18 column (Agi-
column (Agilent, 4.6 ꢂ 250 mm,
(MeOH/H2O = 3:7). C22H33N5O4; hygroscopic solid; [
1, MeOH); IR (film) 3163, 2974, 2378, 1734, 1670, 1635, 1540,
1506, 1472, 1203, 1176, 1136, 1078, 722 cm–1 1H NMR
5 lm porosity), tR = 17.5 min
23
a]
ꢁ30.3 (c
D
;
(400 MHz, CD3OD) d 7.44–7.41 (2H, m), 7.39–7.32 (3H, m), 6.32
(1H, br d, J = 2.0 Hz), 4.16 (1H, br d, J = 6.0 Hz), 3.91–3.82 (2H, m),
3.41–3.34 (1H, m), 2.70 (1H, dd, J = 16.6, 3.8 Hz), 2.37–2.31 (1H,
m), 1.97 (3H, s), 1.56–1.42 (4H, m), 0.92–0.86 (6H, m); 13C NMR
(100 MHz, CD3OD) d 174.3, 158.8, 137.1, 134.0, 130.7 (2ꢂ), 130.6,
129.8 (3ꢂ), 129.6, 84.0, 79.0, 76.3, 56.1, 51.7, 31.4, 27.3, 26.9,
23.0, 9.9, 9.8; HRMS calcd for C22H34N5O4: 432.2611, found: m/z
432.2618 [M+H]+.
lent, 4.6 ꢂ 250 mm,
H2O = 3:7). C15H24F3N3O5S; hygroscopic solid; [
MeOH); IR (film) 3444, 2973, 1652, 1558, 1456, 1374, 1281,
1198, 1096, 845 cm–1 1H NMR (400 MHz, CD3OD) d 6.80 (1H, d,
5
l
m
porosity), tR = 7.8 min (MeOH/
22
a]
D
ꢁ65.9 (c 1,
;
J = 2.0 Hz), 4.19 (1H, d, J = 8.4 Hz), 4.02–3.96 (1H, m), 3.50–3.40
(2H, m), 2.93 (1H, dd, J = 17.6, 5.6 Hz), 2.45–2.37 (1H, m), 2.04
(3H, s), 1.59–1.43 (4H, m), 0.94–0.87 (6H, m); 13C NMR
4.3.7. N-[3-(1H-Indol-2-yl)propoxy] (3R,4R,5S)-4-acetamido-5-
guanidino-3-(1-ethylpropoxy)-1-cyclohexenecarboxamide (9d)
By a procedure similar to that for 9b, guanidination of com-
pound 7d (36 mg, 0.08 mmol) with 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (37 mg, 0.096 mmol) was conducted in the
presence of NEt3 (0.033 mL, 0.24 mmol) in CH2Cl2 (1 mL). The resi-
due was purified by flash silica gel column chromatography (CH2-
Cl2/MeOH = 20:1) to give 8d as pale yellow oil, which was
subsequently treated with trifluoroacetic acid (0.19 mL,
2.57 mmol) to give the desired product 9d (15 mg, 37%) after puri-
fication on a reversed-phase RP-18 column (MeOH/H2O = 1:9–1:4).
The purity of product 9d was 97.2% as shown by HPLC on an HC-C18
(100 MHz, CD3OD)
d
174.8, 173.6, 135.6, 134.6, 121.9
(q, JC–F = 321 Hz), 83.7, 76.3, 54.5, 51.5, 30.1, 27.4, 26.8, 23.3, 9.9,
9.7; 19F NMR (396 MHz, CD3OD) d ꢁ80.26 (s, CF3); HRMS calcd
for C15H25F3N3O5S: 416.1467, found: m/z 416.1468 [M+H]+.
4.3.10. N-Butylsulfonyl (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (10c)
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with n-butylsulfonamide
(33 mg, 0.24 mmol) was conducted in the presence of HBTU
(91 mg, 0.24 mmol), NEt3 (0.08 mL, 0.6 mmol) and DMAP (7.4 mg,
0.02 mmol). The residues was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give pale yellow oil,
which was subsequently treated with trifluoroacetic acid
(0.24 mL, 3.22 mmol) to give 10c (40 mg, 49%). The purity of prod-
uct 10c was 97.8% as shown by HPLC on an HC-C18 column (Agi-
column (Agilent, 4.6 ꢂ 250 mm,
(MeOH/H2O = 3:7). C26H38N6O4; hygroscopic solid; [
1, MeOH); IR (film) 3273, 3170, 2966, 2925, 2876, 1671, 1548,
1458, 1434, 1377, 1262, 1209, 1136, 1082, 943, 719 cm–1 1H
5 lm porosity), tR = 23.3 min
23
a]
ꢁ25.7 (c
D
;
NMR (400 MHz, CD3OD) d 7.39 (1H, d, J = 7.6 Hz), 7.25 (1H, t,
J = 4.0 Hz), 7.00 (1H, t, J = 7.2 Hz), 6.93 (1H, t, J = 7.2 Hz), 6.78 (1H,
s), 6.13 (1H, s), 4.24 (2H, t, J = 6.4 Hz), 4.15 (1H, d, J = 7.6 Hz),
3.90–3.76 (2H, m), 3.37 (1H, quint, J = 5.6 Hz), 2.87 (2H, t,
J = 7.4 Hz), 2.74 (1H, dd, J = 17.6, 5.2 Hz), 2.30–2.22 (1H, m), 2.12
(2H, quint, J = 6.8 Hz), 1.98 (3H, s), 1.56–1.44 (4H, m), 0.93–0.86
(6H, m); 13C NMR (100 MHz, CD3OD) d 174.4, 167.4, 158.7, 140.1,
139.1, 138.1, 130.3, 129.6, 121.6, 120.5, 120.1, 111.6, 100.0, 83.8,
76.1, 65.9, 55.9, 51.6, 31.3, 29.4, 27.4, 27.0, 26.0, 23.0, 10.0, 9.8;
HRMS calcd for C26H39N6O4: 499.3054, found: m/z 499.3051
[M+H]+.
lent, 4.6 ꢂ 250 mm,
H2O = 3:7). C18H33N3O5S; hygroscopic solid; [
MeOH); IR (film) 3439, 1643, 1556, 1453, 1377, 1322, 1225,
1094, 921 cm–1 1H NMR (400 MHz, CD3OD) d 6.67(1H, s), 4.22
5
l
m
porosity), tR = 10.5 min (MeOH/
22
a]
ꢁ52.5 (c 1,
D
;
(1H, br d, J = 8.2 Hz), 3.97 (1H, br dd, J = 10.8, 8.2 Hz), 3.54–3.50
(1H, m), 3.49–3.42 (1H, m), 3.32–3.24 (2H, m), 2.91 (1H, br dd,
J = 17.0, 4.6 Hz), 2.44 (1H, br dd, J = 17.0, 10.2 Hz), 2.04 (3H, s),
1.74 (2H, quint, J = 6.8 Hz), 1.60–1.55 (4H, m), 1.54–1.41 (2H, m),
1.00–0.87 (9 H, m); 13C NMR (100 MHz, CD3OD) d 174.9, 172.7,

B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
6653
135.0, 134.2, 83.7, 76.4, 54.6, 53.3, 51.2, 30.2, 27.4, 27.1, 26.7, 23.4,
22.8, 14.2, 10.0, 9.7; HRMS calcd for C18H34N3O5S: 404.2219,
found: m/z 404.2231 [M+H]+.
subsequently treated with trifluoroacetic acid (0.077 mL,
1.0 mmol) to give 12b (18 mg, 39%). The purity of product 12b
was 99.6% as shown by HPLC on an HC-C18 column (Agilent,
4.6 ꢂ 250 mm,
16H26F3N5O5S; hygroscopic solid; [
(film) 3416, 1640, 1453, 1372, 1293, 1199, 1073, 932 cm–1
5 lm porosity), tR = 9.4 min (MeOH/H2O = 3:7).
22
4.3.11. N-Phenylsulfonyl (3R,4R,5S)-4-acetamido-5-amino-3-(1-
ethylpropoxy)-1-cyclohexenecarboxamide (10d)
C
a]
ꢁ19.6 (c 1, MeOH); IR
D
;
1H
By a procedure similar to that for 7a, the amide bond forming
reaction of acid 5 (78 mg, 0.2 mmol) with benzenesulfonamide
(38 mg, 0.24 mmol) was conducted in the presence of HBTU
(91 mg, 0.24 mmol), NEt3 (0.08 mL, 0.6 mmol) and DMAP (7.4 mg,
0.02 mmol). The residues was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give pale yellow oil,
which was subsequently treated with trifluoroacetic acid
(0.24 mL, 3.22 mmol) to give 10d (36 mg, 43%). The purity of prod-
uct 10d was >95% as shown by HPLC on an HC-C18 column (Agilent,
NMR (400 MHz, CD3OD) d 6.79 (1H, s), 4.16 (1H, br t, J = 3.4 Hz),
3.91 (1H, dd, J = 10.0, 8.0 Hz), 3.79–3.73 (1H, m), 3.41 (1H, quint,
J = 5.6 Hz), 2.79 (1H, dd, J = 17.8, 5.6 Hz), 2.33–2.25 (1H, m), 1.97
(3H, s), 1.57–1.45(4H, m), 0.94–0.86 (6H, m); 13C NMR (100 MHz,
CD3OD)
d 174.4, 174.2, 158.6, 135.8, 135.5, 121.8 (q, JC–F
= 340 Hz)83.9, 76.6, 55.9, 52.1, 31.5, 27.5, 27.0, 23.0, 9.93, 9.91;
19F NMR (396 MHz, CD3OD) d ꢁ80.10 (s, CF3); HRMS calcd for
C16H27F3N5O5S: 458.1685, found: m/z 458.1685 [M+H]+.
4.6 ꢂ 250 mm,
C20H29N3O5S; hygroscopic solid; [
3408, 2360, 1638, 1558, 1382, 1319, 1135, 1082, 839 cm–1
NMR (400 MHz, CD3OD) d 7.92 (2H, br d, J = 7.2 Hz), 7.51–7.42
(3H, m), 6.66 (1H, s), 4.19 (1H, br), 3.96 (1H, br), 3.45–3.41 (2H,
m), 2.86 (1H, br), 2.37 (1H, br), 2.01 (3H, s), 1.57–1.44 (4H, m),
0.92–0.85 (6H, m); 13C NMR (100 MHz, CD3OD) d 174.8, 173.5,
145.1, 135.0, 134.4, 132.5, 129.4 (2ꢂ), 128.2 (2ꢂ), 83.6, 76.4, 54.5,
51.4, 30.2, 27.3, 26.7, 23.4, 10.0, 9.7; HRMS calcd for C20H30N3O5S:
424.1906, found: m/z 424.1908 [M+H]+.
5
l
m
porosity), tR = 9.0 min (MeOH/H2O = 3:7).
4.3.14. N-Butylsulfonyl (3R,4R,5S)-4-acetamido-5-guanidino-3-
(1-ethylpropoxy)-1-cyclohexenecarboxamide (12c)
22
a]
ꢁ61.1 (c 1, MeOH); IR (film)
D
;
1H
By a procedure similar to that for 12a, guanidination of com-
pound 10c (40 mg, 0.1 mmol) with 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (47 mg, 0.12 mmol) was conducted in the
presence of NEt3 (0.041 mL, 0.3 mmol) in CH2Cl2 (1 mL). The resi-
due was purified by flash silica gel column chromatography
(CH2Cl2/MeOH = 20:1) to give 11c as pale yellow oil, which was
subsequently treated with trifluoroacetic acid (0.077 mL,
1.0 mmol) to give 12c (16 mg, 36%). The purity of product 12c
was 97.1% as shown by HPLC on an HC-C18 column (Agilent,
4.3.12. N-Methylsulfonyl (3R,4R,5S)-4-acetamido-5-guanidino-
3-(1-ethylpropoxy)-1-cyclohexenecarboxamide (12a)
4.6 ꢂ 250 mm, 5
19H35N5O5S; hygroscopic solid; [
3423, 2094, 1640, 1545, 1372, 1312, 1220, 1111, 1067 cm–1 1H
;
NMR (400 MHz, CD3OD) d 6.69 (1H, s), 4.18 (1H, br), 4.00 (2H,
br), 3.44 (1H, quint, J = 5.6 Hz), 3.28–3.16 (2H, m), 2.78 (1H, d,
J = 16.0 Hz), 2.42 (1H, d, J = 16.0 Hz), 1.97 (3H, s), 1.78–1.69 (2H,
m), 1.60–1.34 (6H, m), 0.96–0.88 (9H, m); 13C NMR (100 MHz,
CD3OD) d 175.1, 174.2, 158.6, 136.0, 134.6, 83.8, 76.5, 56.5, 53.2,
52.1, 32.1, 27.5, 27.3, 26.9, 23.1, 23.0, 14.3, 10.0, 9.95; HRMS calcd
for C19H36N5O5S: 446.2437, found: m/z 446.2432 [M+H]+.
lm porosity), tR = 12.8 min (MeOH/H2O = 3:7).
22
C
a
]
D
ꢁ6.7 (c 1, MeOH); IR (film)
NEt3 (0.041 mL, 0.3 mmol) and 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (47 mg, 0.12 mmol) were added to a solution
of compound 10a (36 mg, 0.1 mmol) in CH2Cl2 (1 mL). The mixture
was stirred at 25 °C for 10 h, and then concentrated under reduced
pressure. The residue was dissolved in CH2Cl2, washed with 1.0 M
HCl, saturated NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was purified by flash silica gel column chromatography
CH2Cl2/MeOH = 20:1) to give 11a as pale yellow oil.
A solution of the above-prepared compound 11a in anhydrous
CH2Cl2 (1 mL) was cooled to 0 °C in an ice-bath, and trifluoroacetic
acid (0.24 mL, 3.22 mmol) was added. The mixture was stirred at
room temperature for 1 h, and then concentrated under reduced
pressure. The residue was purified by reversed-phase RP-18 col-
umn (MeOH/H2O = 1:9–1:4) to give the desired product 12a
(13 mg, 32%). The purity of product 12a was 95.3% as shown by
4.3.15. N-Phenylsulfonyl (3R,4R,5S)-4-acetamido-5-guanidino-
3-(1-ethylpropoxy)-1-cyclohexenecarboxamide (12d)
By a procedure similar to that for 12a, the guanidination of
compound 10d (42 mg, 0.1 mmol) with 1,3-di-Boc-2-(trifluoro-
methylsulfonyl)guanidine (47 mg, 0.12 mmol) was conducted in
the presence of NEt3 (0.041 mL, 0.3 mmol) in CH2Cl2 (1 mL). The
residue was purified by flash silica gel column chromatography
(CH2Cl2/MeOH = 20:1) to give 11d as pale yellow oil, which was
subsequently treated with trifluoroacetic acid (0.077 mL,
1.0 mmol) to give 12d (13 mg, 28%). The purity of product 12d
was 99.7% as shown by HPLC on an HC-C18 column (Agilent,
HPLC on an HC-C18 column (Agilent, 4.6 ꢂ 250 mm, 5
lm poros-
ity), tR = 27.4 min (MeOH/H2O = 3:7). C16H29N5O5S; hygroscopic
22
solid; [a]
ꢁ35.3 (c 0.8, MeOH); IR (film) 3416, 1652, 1437,
1H NMR (400 MHz, CD3OD) d
D
1415, 1318, 1017, 925, 720 cm–1
;
6.63 (1H, s), 4.27 (1H, br d, J = 7.2 Hz), 3.89 (1H, br t, J = 9.2 Hz),
3.77–3.71 (1H, m), 3.47 (1H, br t, J = 5.4 Hz), 3.07 (3H, s), 2.80
(1H, dd, J = 17.4 Hz, 4.6 Hz), 2.32 (1H, dd, J = 17.4 Hz, 9.4 Hz), 2.00
(3H, s), 1.58–1.43 (4H, m), 0.94–0.85 (6H, m); 13C NMR
(100 MHz, CD3OD) d 174.71, 174.65, 158.7, 135.6, 134.7, 84.4,
76.7, 56.3, 52.2, 40.9, 31.9, 27.3, 26.7, 23.1, 9.9 (2ꢂ); HRMS calcd
for C16H30N5O5S:404.1968, found: m/z 404.1971 [M+H]+.
4.6 ꢂ 250 mm, 5
C21H31N5O5S; hygroscopic solid; [
(film) 3564, 3220, 2920, 1659, 1437, 1407, 1318, 1016, 952, 903,
711 cm–1 1H NMR (400 MHz, CD3OD) d 7.91 (2H, d, J = 6.8 Hz),
7.51–7.42 (3H, m), 6.71 (1H, s), 4.25 (1H, br d, J = 7.2 Hz), 3.85
(1H, br t, J = 11.2 Hz), 3.73–3.67 (1H, m), 3.46 (1H, quint,
J = 5.6 Hz), 2.72 (1H, dd, J = 17.6, 5.4 Hz), 2.16 (1H, dd, J = 17.6,
9.8 Hz), 2.00 (3H, s), 1.59–1.43 (4H, m), 0.95–0.86 (6H, m); 13C
NMR (100 MHz, CD3OD) d 174.5, 174.0, 158.4, 145.3, 135.6,
134.9, 132.3, 129.2 (2ꢂ), 128.1 (2ꢂ), 83.8, 76.4, 56.2, 52.0, 31.6,
27.3, 26.8, 22.9, 9.8 (2ꢂ); HRMS calcd for C21H32N5O5S:466.2124,
found: m/z 466.2119 [M+H]+.
lm porosity), tR = 12.7 min (MeOH/H2O = 3:7).
22
a]
D
ꢁ14.9 (c 0.5, MeOH); IR
;
4.3.13. N-Trifluoromethylsulfonyl (3R,4R,5S)-4-acetamido-5-
guanidino-3-(1-ethylpropoxy)-1-cyclohexenecarboxamide
(12b)
By a procedure similar to that for 12a, guanidination of com-
pound 10b (42 mg, 0.1 mmol) with 1,3-di-Boc-2-(trifluoromethyl-
sulfonyl)guanidine (47 mg, 0.12 mmol) was conducted in the
presence of NEt3 (0.041 mL, 0.3 mmol) in CH2Cl2 (1 mL). The
residue was purified by flash silica gel column chromatography
(CH2Cl2/MeOH = 20:1) to give 11b as pale yellow oil, which was
4.4. Determination of influenza virus TCID50
The TCID50 (50% tissue culture infectious dose) was determined
by serial dilution of the influenza virus stock solution onto 100 lL

6654
B.-T. Hong et al. / Bioorg. Med. Chem. 22 (2014) 6647–6654
MDCK cells at 1 ꢂ 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
Supplementary data
each well with 100 l
L of CellTiter 96Ò AQueous Non-Radioactive
Supplementary data (synthetic schemes, procedures and com-
pound characterization, 1H, 13C, and 19F NMR spectra, and HPLC
diagrams) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2014.10.005. These
data include MOL files and InChiKeys of the most important com-
pounds described in this article.
Cell Proliferation Assay reagent (Promega). After the incubation
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.
4.5. Determination of neuraminidase activity by a fluorescent
assay
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The neuraminidase activity was measured by using diluted
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15 min incubation at room temperature.
4.6. Determination of IC50 of neuraminidase inhibitors
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ated CPE (cytopathic effects). 50
100 TCID50 was mixed with equal volumes of NA inhibitors at var-
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lL of diluted influenza virus at
l
MDCK cells at 1 ꢂ 105 cells/mL in 96-wells. After 48 h of incubation
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Acknowledgment
We thank the Ministry of Science and Technology (Taiwan) and
Academia Sinica (Taiwan) for financial support.