Oseltamivir analogues bearing N-substituted guanidines as potent neuraminidase inhibitors

Article abstract of DOI:10.1021/jm401977j

A series of oseltamivir analogues bearing an N-substituted guanidine unit were prepared and evaluated as inhibitors of neuraminidases from four strains of influenza the two most potent analogues identified contain relatively small N-guanidine substituents (N-methyl and N-hydroxyl) and display enhanced inhibition with IC₅₀ values in the low nanomolar range against neuraminidases from wild-type and oseltamivir-resistant strains. Potential advantages of including the N-hydroxyguanidine moiety in neuraminidase inhibitors are also discussed.

Full text of DOI:10.1021/jm401977j

Brief Article
pubs.acs.org/jmc
Oseltamivir Analogues Bearing N‑Substituted Guanidines as Potent
Neuraminidase Inhibitors
Caitlin A. Mooney,†,§ Stuart A. Johnson,†,§ Peter ’t Hart,† Linda Quarles van Ufford,†
Cornelis A. M. de Haan,‡ Ed E. Moret,† and Nathaniel I. Martin*,†
†Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
‡Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL
Utrecht, The Netherlands
S
* Supporting Information
ABSTRACT: A series of oseltamivir analogues bearing an N-
substituted guanidine unit were prepared and evaluated as
inhibitors of neuraminidases from four strains of influenza. The
two most potent analogues identified contain relatively small
N-guanidine substituents (N-methyl and N-hydroxyl) and
display enhanced inhibition with IC50 values in the low
nanomolar range against neuraminidases from wild-type and
oseltamivir-resistant strains. Potential advantages of including
the N-hydroxyguanidine moiety in neuraminidase inhibitors
are also discussed.
as effective treatment of influenza A and B infections.2b,c The
INTRODUCTION
■
most successful NAIs are illustrated in Figure 1 and include
Influenza viruses infect millions of people annually and can lead
to life threatening respiratory illness. The pandemic nature of
the virus and its ability to rapidly mutate present a serious
oseltamivir (1),8 a prodrug of the corresponding oseltamivir-
carboxylate, zanamivir (2),9 laninamivir (3),10 and peramivir
(4)11 which is currently under development.
public health concern and underscore the importance of
Among the clinically used NAIs, oseltamivir has risen to
prominence largely because of its convenient oral admin-
developing effective anti-influenza agents.1 Strategies for
developing new compounds to combat influenza have generally
targeted mechanisms essential for viral replication.2 The earliest
istration compared with zanamivir and laninamivir, both of
which are delivered via inhalation, and peramivir which is
such agents to be used clinically were the adamantanes
administered by injection.2a Despite its success in recent years,
including amantadine3 and rimantadine,4 which operate by
resistance to oseltamivir is a growing concern.12 Oseltamivir
blocking the M2 ion channel required for disassembly of the
resistance is generally conferred via a single amino acid
viral particle after entry into the host cell. Adamantanes were
mutation (H274Y) in strains possessing the N1 subtype of the
found to be effective against influenza A virus infection but not
against influenza viruses of other genera. Despite early success,
the vast majority of influenza A virus strains now exhibit
neuraminidase enzyme.13 By comparison, this mutation has
little effect on the antiviral activity of the other NAIs used to
treat influenza infection.14 As shown in Figure 1, oseltamivir
resistance to these drugs and their use has largely been
discontinued.5 In response, the search for alternative anti-
(1) differs from the other NAIs in that it lacks an exocyclic
guanidine moiety. Upon the basis of available cocrystal
structures, the guanidine unit present in zanamivir (2),
influenza strategies led to the identification of the viral
neuraminidase enzyme as a promising alternative target.2 The
laninamivir (3), and peramivir (4) provides active site
neuraminidase enzyme (NA) is essential to the influenza virion
hydrogen-bonding interactions that are not present in the
and functions by cleaving terminal sialic acid residues from
glycoconjugates that are otherwise bound by the hemagglutinin
receptor-binding protein. In doing so, neuraminidases play an
essential role in the release of newly formed virions from
infected cells and facilitate propagation of the virus.6 As early as
the 1970s,7 neuraminidase inhibitors were proposed as a means
of combating influenza, and to date, a number of clinically
relevant neuraminidase inhibitors (NAIs) have been developed
oseltamivir−neuraminidase complex.14,15 These additional
interactions may offer a partial explanation for the observation
that oseltamivir resistant strains of influenza are generally
sensitive to NAIs that contain an exocyclic guanidine group.16
Received: January 7, 2014
Published: March 20, 2014
© 2014 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
fold lower than the value measured for 5 in the same inhibition
assay.17
In the present study we describe a series of new oseltamivir
analogues that incorporate various NG-substituted guanidines in
place of the amino group at the C-5 position and examine the
effect that such modifications have on neuraminidase inhibition.
In recent years a number of groups have reported various
approaches to modifying the structures of oseltamivir and
zanamivir at the C-5 and C-6 positions (oseltamivir numbering;
see Figure 1). In most cases the rationale for doing so was
based on an attempt to identify analogues bearing exocyclic
substituents capable of occupying the so-called “150-cavity”
adjacent to the active site of certain neuraminidase subtypes
(N1, N4, and N8).18 Recently, the groups of Pinto and Cairo
employed click chemistry based strategies to generate
oseltamivir analogues bearing substituted triazoles at the C-5
and C-6 positions.19 While some of the analogues prepared in
this manner did show neuraminidase inhibition, they were
generally less potent than oseltamivir and zanamivir.19,20 In
another approach, von Itzstein and co-workers developed novel
sialic acid based compounds also bearing extended exocylic
substituents that were shown by crystallography to occupy the
150-cavity.21 These compound were found to effectively inhibit
the influenza virus including drug-resistant strains.21 In an
alternative approach, the groups of Lin and Fang independently
described the incorporation of various substituents at the
guanidine moiety of zanamivir (2) to produce analogues with a
range of activities.22,23 Most notable among the compounds
described by Lin and co-workers are a subset of N-
acylguanidine analogues that exhibited IC50 as low as 20 nM
against the H1N1 neuraminidase.22
Figure 1. Oseltamivir (1) with atom numbering indicated and
structures of the other known NAIs used clinically or under
development.
Further support for the inhibition-enhancing effect of the
guanidine moiety is provided by the known oseltamivir
analogue 6 wherein the C-5 amine is transformed into a
guanidine group (Figure 2). The Gilead team involved in the
RESULTS AND DISCUSSION
■
Our rationale for pursuing oseltamivir analogues with NG-
substituted guanidines at the C-5 position was twofold: first,
introduction of the guanidine moiety at the C-5 position of
oseltamivir (6) is known to enhance the neuraminidase
inhibitory activity17 and, second, the NG-substituents intro-
duced might lead to beneficial binding interactions should they
be able to access the 150-cavity adjacent to the neuraminidase
site. In designing the series of oseltamivir analogues to be
prepared, we chose to incorporate a range of small NG-
Figure 2. Structures of oseltamivir carboxylate (5) and the guanidine
analogue of oseltamivir carboxylate (6).
original development of oseltamivir found that analogue 6 was a
more potent neuraminidase inhibitor with an IC50 of 0.2 nM, 5-
Scheme 1. Synthetic Route Employed in Preparing Oseltamivir Analogues with NG-Substituted Guanidine Moiety at C-5
Position
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Journal of Medicinal Chemistry
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substituents containing three carbon atoms or less. The
synthetic approach employed in preparing these analogues is
outlined in Scheme 1. Briefly, starting from shikimic acid,
oseltamivir (1) was prepared on multigram scale according to
the routes independently described by Karpf and Trussardi at
Roche24 and the Shi group.25 Oseltamivir was then treated with
CbzNCS26 to yield thiourea 7 which served as a common
intermediate in the preparation of the various NG-modified
oseltamivir analogues prepared. Treatment of thiourea 7 with
EDCI, NEt3, and the amine of choice led to clean formation of
the protected guanidine species 8−15 as previously described
Table 1. Inhibitory Activities against Wild-Type and Mutant
Influenza Neuraminidases
a
neuraminidase inhibition, IC50 (nM)
H1N1
wild-type
H1N1
mutant
H5N1
wild-type
H5N1
e
b
c
d
compd
mutant
5
2.1 0.1
250 20
1.7 0.2
4.2 0.5
250 30
330 20
1.9 0.4
6
0.87 0.04
1.73 0.09
3.7 0.5
1.32 0.09
3.6 0.3
16
23
4
1
43
4
33
2
38
3
a
b
IC50 values reported based on triplicate measurement. Neuramini-
c
for the synthesis of other NG-substituted guanidines.26−28
A
dase from influenza virus A/California/04/2009 (H1N1). Neur-
two-step deprotection strategy29 was then utilized to yield 16−
23 which were subsequently purified by RP-HPLC. Oseltamivir
carboxylate (5) and the unsubstituted guanidine analogue (6)
were also prepared as shown in Scheme 2.
aminidase (H274Y) from influenza virus A/California/04/2009
d
(H1N1). Neuraminidase from influenza virus A/Anhui/1/2005
e
(H5N1). Neuraminidase (H274Y) from influenza virus A/Anhui/1/
2005 (H5N1).
Scheme 2. Preparation of Oseltamivir Carboxylate (5) and
Unsubstituted Guanidine Analogue (6)
The results obtained with NG-substituted analogues 16−23
indicate that the size of the substituent that can be appended to
the guanidine moiety while maintaining activity is rather
limited. Incorporation of NG-substituents larger than N-methyl
or N-hydroxyl resulted in dramatically reduced neuraminidase
inhibition, suggesting that the larger substituents here evaluated
are not able to enhance binding by accessing the 150-cavity. By
comparison, 16 and 23 showed strong inhibition of all
neuraminidases tested, including those from oseltamivir-
resistant strains. The activity of N-methyl analogue 16 was
found to be very similar to that of known unsubstituted
analogue 6 with IC50 values in the low nanomolar range against
all four neuraminidases tested. The N-hydroxy analogue (23)
was slightly less potent with IC50 values approximately 10-fold
higher than those measured for 16 (Table 1).
Docking studies were next performed to compare the
binding modes of 16 and 23 with that of oseltamivir in the
neuraminidase active site (Figure 3). Figure 3A shows
oseltamivir docked into the H274Y mutant H1N1 neuramini-
dase, according to the crystal structure published by Collins and
co-workers14 (PDB code 3CL0 with oseltamivir and PDB code
3CKZ with zanamivir, the sequence of this neuraminidase is
100% identical to the sequence of the H1N1 mutant enzyme
used in our inhibition assays). Figure 3B illustrates the
hydrogen-bonding interactions found between oseltamivir’s
exocyclic amine and the carboxylate side chains of Glu119 and
Asp 151 of the neuraminidase active site. Shown in Figure 3C
and Figure 3D, are the possible new hydrogen bonding
interactions resulting from introduction of the guanidine
moiety in analogues 16 and 23. In addition, the N-methyl
substituent of 16 appears to introduce beneficial nonpolar
interactions, while the oxygen of the N-hydroxy moiety in 23
functions as a hydrogen bond acceptor. Taken together, the
results of these docking experiments indicate that addition of an
exocyclic guanidine moiety to the oseltamivir core can lead to
beneficial hydrogen bonding interactions and restore affinity for
the H274Y mutant.
The enhanced binding suggested by the docking analysis and
the potent inhibitory activity measured for NG-hydroxy
analogue 23 with oseltamivir-sensitive and -resistant neurami-
nidases is of particular note. The pKa for a typical NG-
hydroxyguanidine moiety is generally between 8.0 and 8.5,
significantly lower than that of the corresponding guanidine
(pKa ≳ 12.5).31 Guanidines are very strong bases and are
completely ionized in serum, limiting their bioavailability and
restricting their modes of action to peripheral tissues.31a One
Compounds 16−23 were first evaluated as inhibitors of the
neuraminidase from the H1N1 wild-type influenza virus using
an established activity assay employing the fluorescent substrate
2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MU-
NANA). In addition, oseltamivir carboxylate (5) and the
unsubstituted guanidine analogue (6)17,30 were included as
positive controls. As expected, oseltamivir carboxylate (5)
effectively inhibited the wild-type H1N1 neuraminidase with an
IC50 of 2.06 0.14 nM while against the H5N1 neuraminidase
and the H274Y mutants of the H1N1 and H5N1 enzymes, the
IC50 values measured were more than 100-fold higher. By
comparison, oseltamivir analogue 6, containing the unsub-
stituted guanidine group, was found to be a potent inhibitor of
all four neuraminidases tested with IC50 values in the low
nanomolar range. An initial evaluation of analogues 16−23 was
performed by measuring each compound’s ability to inhibit the
wild-type H1N1 neuraminidase at 10, 100, and 1000 nM. This
revealed 16 and 23 to be potent inhibitors while 17−22 were
found to display little or no inhibition at the highest
concentration tested (1000 nM). Compounds 16 and 23
where therefore carried forward and more extensively evaluated
as inhibitors of the neuraminidases from wild-type and
oseltamivir-resistant H1N1 and H5N1 influenza strains
(Table 1).
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Figure 3. Oseltamivir carboxylate (1) and guanidine-modified analogues 16 and 23 docked into the active site of H274Y mutant H1N1
neuraminidase. (A) MSMS molecular surface representation32 of the neuraminidase active site with oseltamivir carboxylate (1) bound and the 150-
cavity indicated. Entry to the cavity appears to be via a relatively small channel, and the cavity itself is quite polar. (B) Hydrogen-bonding interactions
between oseltamivir’s exocyclic amine and the carboxylate side chains of Glu119 and Asp 151. (C) Binding of NG-methylguanidine containing
analogue 16 is predicted to be facilitated by hydrogen-bonding with Trp178, Glu227, and Glu 277. (D) NG-Hydroxyguanidine moiety in analogue 23
is predicted to function as a hydrogen-bond donor with Glu119, Asp151, and Glu227 and as a hydrogen-bond acceptor with Arg156.
approach to overcoming these limitations is to “tailor the
basicity” of guanidine compounds by instead using the
corresponding NG-hydroxy analogue. The reduced basicity of
the NG-hydroxyguanidine moiety has been exploited to
generate analogues that are less ionized at physiological pH
and show altered physiological distributions relative to the
corresponding guanidine.31a
CONCLUSION
■
The oseltamivir analogues described in this study provide new
insights into the opportunities and limitations for enhancing
neuraminidase inhibition via introduction of N-substituted
guanidine groups into the parent compound. Among the
analogues here prepared, those bearing NG-substituents larger
than methyl and hydroxyl groups displayed dramatically
reduced activity. These findings suggest that access to the
150-cavity is not possible for substituents appended to the
terminal nitrogen atoms of the exocyclic guanidine moiety. By
comparison, N-methyl and N-hydroxy analogues 16 and 23
exhibit potent inhibition of influenza neuraminidases, including
those from oseltamivir-resistant strains. The activity of the NG-
hydroxy analogue 23 is particularly noteworthy given that NG-
hydroxy guanidines can show improved bioavailability relative
to the corresponding unsubstituted guanidine species. These
findings also point toward a possible approach for altering the
bioavailability of the other major NAIs (Figure 1). In contrast
to oseltamivir, the poor oral bioavailability observed with
zanamivir (2) and the other major NAIs is largely attributable
to the presence of a free carboxylate and an exocyclic guanidine.
While the carboxylate moiety in these compounds can be
masked as an ester prodrug (as in oseltamivir), attenuating the
highly basic guanidine group provides an additional challenge.
On the basis of the findings here presented, it may be of
interest to prepare the corresponding NG-hydroxyguanidine
analogues of zanamivir (2), laninamivir (3), and peramivir (4)
and evaluate their inhibitory activity and (oral) bioavailability.
Such investigations are ongoing in our laboratories, the results
of which will be presented in due course.
As described above, oseltamivir’s oral bioavailability is largely
responsible for its popularity compared with the other NAIs. A
significant contribution to oseltamivir’s bioavailability as a
prodrug is attributable to the ethyl ester that is hydrolyzed in
vivo. In addition, the fact that oseltamivir contains an exocyclic
primary amine rather than a guanidine moiety may further
enhance its oral bioavailability. We here show that introduction
of a guanidine moiety into the oseltamivir carboxylate structure
as in 6, 16, and 23 leads to an enhanced inhibitory activity
against oseltamivir-resistant neuraminidases in vitro. In a recent
report, Schade and Clement described the in vivo activity of the
ethyl ester prodrugs of structurally similar compounds.33 Most
notable is their finding that an oseltamivir analogue bearing an
exocyclic amidine moiety at C-5 maintains the bioavailability of
the parent compound and displays enhanced antiviral activity
against an oseltamivir resistant H1N1 influenza strain. Taken
together with our own results, these findings indicate that the
inclusion of modified amidine and guanidine groups at the C-5
position provides a means by which oseltamivir resistance can
be overcome.
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to 2:1). Yields of 8−15 ranged from 70% to quantitative. As an
example, analytical data for 8 are given below; data for all compounds
are provided in the Supporting Information.
EXPERIMENTAL SECTION
■
General Procedures. Reagents, Solvents, and Solutions.
Oseltamivir was prepared from shikimic acid based upon a
combination of recently published synthetic routes.24,25,34 Oseltamivir
carboxylate (5) and the corresponding unsubstituted guanidine
analogue (6) were prepared as previously described.30 CbzNCS was
prepared following literature protocols.26 All other reagents employed
were of American Chemical Society (ACS) grade or finer and were
used without further purification unless otherwise stated. Recombinant
influenza A neuraminidase enzymes H1N1 wild-type, H1N1(H274Y),
H5N1 wild-type, and H5N1(H274Y) were obtained from Sino
Biological Inc.
Purification Techniques. All reactions and fractions from column
chromatography were monitored by thin layer chromatography (TLC)
using plates with a UV fluorescent indicator (normal SiO2, Merck 60
F254). One or more of the following methods were used for
visualization: UV absorption by fluorescence quenching; iodine
staining; phosphomolybdic acid/ceric sulfate/sulfuric acid/H2O (10
g/1.25 g/12 mL/238 mL) spray; 50% sulfuric acid spray. Flash
chromatography was performed using Merck type 60, 230−400 mesh
silica gel. Product purities were confirmed to be ≥95% by RP-HPLC
employing an analytical C18 column and a water/acetonitrile gradient
moving from 5% to 95% MeCN (0.1% TFA) over 30 min (flow rate
1.0 mL/min).
(3R,4R,5S)-Ethyl 4-Acetamido-5-(2-(benzyloxycarbonyl)-3-
methylguanidino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxy-
late (8). Yield: 227 mg, 96%. 1H NMR (300 MHz, CDCl3) δ 8.97 (br
s, 1H), 7.41−7.23 (m, 5H), 6.79 (s, 1H), 5.85−5.64 (m, 2H), 5.10 (s,
2H), 4.38−4.25 (m, 1H), 4.20 (q, 2H, J = 7.2 Hz), 4.14−3.94 (m,
2H), 3.36 (app quin, 1H, J = 5.6 Hz), 2.86 (dd, 1H, J = 23.2, 5.3 Hz),
2.76 (d, 3H, J = 5.1 Hz), 2.34 (ddt, 1H, J = 18.0, 9.0, 2.2 Hz), 1.92 (s,
3H), 1.58−1.42 (m, 4H), 1.29 (t, 3H, J = 7.3 Hz), 0.95−0.83 (m, 6H);
13C NMR (75 MHz, CDCl3) δ 171.8, 166.0, 164.0, 160.8, 137.8, 136.5,
129.7, 128.3, 127.8, 127.6, 82.1, 75.2, 66.5, 61.0, 54.0, 49.7, 31.0, 27.5,
26.3, 25.8, 23.2, 14.2, 9.5, 9.3. HRMS (ESI, M + H) calcd for
C26H39N4O6, 503.2864; found, 503.2875.
Synthesis of N-Substituted Guanidine Compounds 16−23.
General Procedure. The Cbz-protected ethyl ester intermediate (0.4
mmol) was dissolved in 1.0 mL of MeOH (or dioxane if methanol
does not dissolve). Once fully dissolved, an aqueous solution of 2 M
KOH (0.5 mL, 1.0 mmol, 2.5 equiv of KOH) was added and the
reaction mixture stirred for 3−4 h. Once TLC confirmed complete
hydrolysis, Amberlite IR-120 resin (1 g) was added directly to acidify
the mixture. After the mixture was stirred slowly for 5 min the resin
was filtered off and rinsed with an additional 25 mL of MeOH. The
MeOH and H2O were then removed under vacuum, and the resulting
oil was used directly in the next step.
1
Instrumentation for Compound Characterization. H NMR
spectra were recorded at 300.1 MHz with chemical shifts reported in
parts per million (ppm) downfield relative to tetramethylsilane
(TMS). 1H NMR data are reported in the following order: multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sex, sextet;
sept, septet; m, multiplet), number of protons, and coupling constant
(J) in hertz (Hz). When appropriate, the multiplicity is preceded by br,
indicating that the signal was broad. 13C NMR spectra were recorded
at 75.5 MHz with chemical shifts reported relative to CDCl3 δ 77.0.
13C NMR spectra were recorded using the attached proton test (APT)
The crude carboxylic acid species from the previous step (0.4
mmol) was treated with trifluoroacetic acid (15.0 mL, 202 mmol) and
thioanisole (2.4 mL, 20 mmol) at room temperature and the reaction
mixture stirred overnight. The next morning the TFA was removed
under vacuum to yield the crude product as an oil (also contains
residual thioanisole). The product was purified using RP-HPLC
employing a preparative C18 column and an H2O/MeCN gradient
moving from 28.5% to 50% MeCN (0.1% TFA) over 60 min (flow
rate, 18.0 mL/min). Fractions containing the desired product were
combined and lyophilized to yield the pure compounds as amorphous
white powders. As an example, analytical data for compound 16 are
given below; data for all compounds are provided in the Supporting
Information.
sequence. High resolution mass spectrometry (HRMS) analyses were
performed using a TOF LC/MS instrument. All literature compounds
had NMR and mass spectra consistent with the assigned structures.
Synthetic Procedures and Compound Characterization.
(3R,4R,5S)-Ethyl 4-Acetamido-5-(3-((benzyloxy)carbonyl)-
thioureido)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate
(Thiourea Intermediate 7). Oseltamivir freebase (2.31 g, 7.41
mmol) was dissolved in dichloromethane (100 mL) and treated with a
0.5 M solution of CbzNCS in CH2Cl2 (15 mL, 7.5 mmol) and
triethylamine (1.1 mL, 7.8 mmol). After the mixture was stirred for 1 h
at room temperature, TLC analysis indicated partial conversion to the
thiourea. An additional 3.0 mL of the 0.5 M CbzNCS solution (1.5
mmol) was added to the mixture. After the mixture was stirred for
another 0.5 h, TLC verified that the reaction had reached completion.
The CH2Cl2 was then removed under reduced pressure, and the
residue applied directly to a silica column, eluting with EtOAc/hexane
(1:2). The desired thiourea was obtained as a white solid (2.63g, 5.20
mmol). Yield: 70.0%. 1H NMR (300 MHz, CDCl3) δ 9.94 (d, 1H, J =
8.4 Hz), 8.02 (s, 1H), 7.46−7.31 (m, 5H), 6.86 (s, 1H), 5.82 (d, 1H, J
= 9.3 Hz), 5.17 (q, 2H, J = 5.1 Hz), 4.84−4.70 (m, 1H), 4.36−4.26
(m, 1H), 4.21 (q, 2H, J = 6.9 Hz), 4.07−4.00 (m, 1H), 3.37 (app quin,
1H, J = 6.0 Hz), 2.88 (dd, 1H, J = 17.7, 5.4 Hz), 2.55 (ddt, 1H, J =
18.0, 9.3, 2.7 Hz), 1.97 (s, 3H), 1.55−1.42 (m, 4H), 1.29 (t, 3H, J =
7.2 Hz), 0.93−0.80 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 175.0,
165.7, 161.0, 147.0, 132.8, 129.6, 124.2, 124.1, 124.0, 123.8, 78.0, 71.0,
63.7, 56.3, 49.1, 48.7, 24.9, 21.4, 21.0, 18.7, 18.6, 10.1, 5.4. HRMS
(ESI, M + H) calcd for C25H36N3O6S, 506.2319; found, 506.2331.
Synthesis of Cbz-Protected N-Substituted Guanidine Com-
pounds 8−15. General Procedure. Thiourea precursor 7 (253 mg,
0.50 mmol) was dissolved in CH2Cl2 (8 mL) and treated with the
amine of choice (2 equiv, 1.0 mmol), triethylamine (2 equiv, 0.14 mL,
1.0 mmol), and EDCI−HCl (2 equiv, 191 mg, 1.0 mmol) after which
the mixture was stirred at room temperature. After TLC confirmed
complete conversion to the substituted guanidine (typically 2−5 h)
CH2Cl2 was removed under reduced pressure and the residue applied
directly to a silica column (typical solvent system EtOAc/hexane 1:2
(3R,4R,5S)-4-Acetamido-5-((E)-2-methylguanidino)-3-(pen-
tan-3-yloxy)cyclohex-1-enecarboxylic Acid (16). Yield: 49 mg,
1
36%. H NMR (300 MHz, D2O) δ 6.72 (s, 1H), 4.21 (d, 1H, J = 8.4
Hz,), 3.80 (q, 1H, J = 9.0 Hz), 3.72−3.64 (m, 1H), 3.43 (app quin,
1H, J = 5.1 Hz), 2.73 (d, 1H, J = 5.1 Hz), 2.70 (s, 3H), 2.29 (dd, 1H, J
= 17.1, 9.9 Hz), 1.89 (s, 3H), 1.46−1.24 (m, 4H), 0.78−0.68 (m, 6H);
13C NMR (75 MHz, D2O) δ 177.2, 171.9, 159.1, 140.8, 130.3, 86.9,
78.0, 57.4, 53.1, 32.4, 30.0, 28.1, 27.7, 24.4, 11.0; HRMS (ESI, M + H)
calcd for C16H29N4O4, 341.2183; found, 341.2202.
Neuraminidase Inhibition Assay. Compounds 2, 6, and 16−23
were evaluated as neuraminidase inhibitors by following an established
protocol provided in a commercially available assay kit (NA-Fluor
influezna neuraminidase assay kit from Life Technologies). For 5, 6,
16, and 23, IC50 values were determined from the dose−response
curves by plotting percent inhibition of neuraminidase activity versus
inhibitor concentration using GraphPad Prism 4.
Docking Studies. The docking studies performed made use of the
crystal structure of the H274Y mutant H1N1 neuraminidase,
previously published by Collins and co-workers14 (PDB codes 3CL0
with oseltamivir and 3CKZ with zanamivir). Oseltamivir carboxylate
(5) and 16 and 23 were docked into the neuraminidase active site
using Autodock 4.235 as part of YASARA 12.4.1.36 For the docking
experiments the original oseltamivir carboxylate structure was
converted to those of analogues 16 and 23 in YASARA, which also
allowed for cleaning of the molecules with regard to atom types and
bond types. Initial minimization and simulated annealing of the ligand
and the side chains of the neuraminidase residues within 7 Å were
performed using the AMBER03 force field37 followed by 250 rigid
docking runs with ga_pop_size set at 15 000 (the only change to the
default YASARA macro36). The local search algorithm used was based
on the Solis−Wets method,38 and the free energy was estimated
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Journal of Medicinal Chemistry
Brief Article
according to existing methods.39 The best docking result was
minimized with the YASARA minimization experiment (steepest
decent followed by simulated annealing with the AMBER03 force
field). Explicit solvent was not included in the docking simulation or in
the free energy estimate, where a charge-based desolvation term was
computed.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and protocols for the synthesis of
oseltamivir and all new compounds; H and 13C NMR spectra
1
for 2, 6−24; analytical RP-HPLC traces for 2, 6, 16−23;
neuraminidase inhibition assay details and IC50 curves for 2, 6,
16, and 23. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: n.i.martin@uu.nl. Telephone: +31-618785274.
■
Author Contributions
§C.A.M. and S.A.J. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Javier Sastre Torano is kindly acknowledged for providing
̃
HRMS analysis results. Financial support was provided by The
Netherlands Organization for Scientific Research (VIDI grant
to N.I.M.). The Utrecht Institute for Pharmaceutical Sciences
(UIPS) and Utrecht University are also gratefully acknowl-
edged for their support.
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M. Laninamivir prodrug CS-8958, a long-acting neuraminidase
inhibitor, shows superior anti-influenza virus activity after a single
administration. Antimicrob. Agents Chemother. 2010, 54, 1256−1264.
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Hutchison, T. L.; Babu, Y. S.; Bantia, S.; Elliott, A. J.; Montgomery, J.
A. Systematic structure-based design and stereoselective synthesis of
novel multisubstituted cyclopentane derivatives with potent antiin-
fluenza activity. J. Med. Chem. 2001, 44, 4379−4392.
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defenses. N. Engl. J. Med. 2005, 353, 2633−2636. (b) Dharan, N. J.;
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C.; Marshall, S. A.; St George, K.; Epperson, S.; Brammer, L.; Klimov,
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ABBREVIATIONS USED
NA, neuraminidase; NAI, neuraminidase inhibitor; RP-HPLC,
reverse phase high performance liquid chromatography
■
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