Development of novel potent orally bioavailable oseltamivir derivatives active against resistant influenza A

Article abstract of DOI:10.1021/jm401492x

With the emergence of oseltamivir-resistant influenza viruses and in view of a highly pathogenic flu pandemic, it is important to develop new anti-influenza agents. Here, the development of neuraminidase (NA) inhibitors that were designed to overcome resistance mechanisms along with unfavorable pharmacokinetic (PK) properties is described. Several 5-guanidino- and 5-amidino-based oseltamivir derivatives were synthesized and profiled for their anti-influenza activity and in vitro and in vivo PK properties. Amidine 6 and guanidine 7 were comparably effective against a panel of different A/H1N1 and A/H3N2 strains and also inhibited mutant A/H1N1 neuraminidase. Among different prodrug strategies pursued, a simple amidoxime ethyl ester (9) exhibited a superior PK profile with an oral bioavailability of 31% (rats), which is comparable to oseltamivir (36%). Thus, bioisosteric replacement of the 5-guanidine with an acetamidine - in the form of its N-hydroxy prodrug - successfully tackled the two key limitations of currently used NA inhibitors, as exemplified with oseltamivir.

Full text of DOI:10.1021/jm401492x

Article
pubs.acs.org/jmc
Development of Novel Potent Orally Bioavailable Oseltamivir
Derivatives Active against Resistant Influenza A
Dennis Schade,*,†,∥ Joscha Kotthaus,† Lukas Riebling,† Jurke Kotthaus,† Helge Muller-Fielitz,‡
̈
̈
Walter Raasch,‡ Oliver Koch,∥ Nora Seidel,§ Michaela Schmidtke,§ and Bernd Clement†
†Department of Pharmaceutical Chemistry, Pharmaceutical Institute, Christian-Albrechts University of Kiel, Gutenbergstrasse 76,
24118 Kiel, Germany
‡Institute of Experimental & Clinical Pharmacology & Toxicology, University of Lubeck, Ratzeburger Allee 160, 23538 Lubeck,
̈
̈
Germany
§Department of Virology and Antiviral Therapy, Friedrich Schiller University, Hans-Knoell-Strasse 2, 07745 Jena, Germany
∥Department of Chemistry & Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
S
* Supporting Information
ABSTRACT: With the emergence of oseltamivir-resistant influenza viruses and in view of a highly pathogenic flu pandemic, it is
important to develop new anti-influenza agents. Here, the development of neuraminidase (NA) inhibitors that were designed to
overcome resistance mechanisms along with unfavorable pharmacokinetic (PK) properties is described. Several 5-guanidino- and
5-amidino-based oseltamivir derivatives were synthesized and profiled for their anti-influenza activity and in vitro and in vivo PK
properties. Amidine 6 and guanidine 7 were comparably effective against a panel of different A/H1N1 and A/H3N2 strains and
also inhibited mutant A/H1N1 neuraminidase. Among different prodrug strategies pursued, a simple amidoxime ethyl ester (9)
exhibited a superior PK profile with an oral bioavailability of 31% (rats), which is comparable to oseltamivir (36%). Thus,
bioisosteric replacement of the 5-guanidine with an acetamidinein the form of its N-hydroxy prodrugsuccessfully tackled the
two key limitations of currently used NA inhibitors, as exemplified with oseltamivir.
classes of adamantane-based M2 ion channel protein inhibitors
and inhibitors of the viral surface protein neuraminidase (NA).
The former agents are only effective against influenza A and
suffer from several side effects and drug resistance.6,7 Today,
the classic strategy for treatment and prophylaxis of influenza is
the inhibition of viral NAs, which are key enzymes in the life
cycle of influenza viruses as their action allows virus replication
and spread, thereby significantly contributing to its pathoge-
nicity.6,7 Oseltamivir (1) represents the first orally available NA
inhibitor (NAI) that was marketed in 2000 (Figure 1).8
Primarily attributed to its approval for prophylaxis treatment
and its oral applicability, 1 represents the first choice NAI and
has been stockpiled by most countries for the case of a new
INTRODUCTION
■
According to the World Health Organization (WHO), seasonal
influenza virus infections are responsible for diseases of 3−5
million people and 250 000 to 500 000 deaths worldwide that
are either caused directly by the virus or by secondary
infections.1 People at higher risk are typically the young (age <
2), the elderly (age > 65), and patients with certain chronic
diseases or weakened immune systems.1 The last pandemic
occurring in 2009 (H1N1, “swine flu”) was more harmless than
anticipated but also caused fatal infections in pregnant
women.2,3 In recent years, highly pathogenic avian influenza
viruses of subtype H5N1 (i.e., “bird flu”, 2005) and H7N9 have
been alarming as they can be transmitted from animals to
humans.4,5
Aside from vaccination, at present, available drugs for the
treatment or prophylaxis of influenza infections belong to the
Received: September 26, 2013
© XXXX American Chemical Society
A
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zanamivir and peramivir. Therefore, many investigators in the
field made efforts to chemically modify 1 (and of NAIs in
general) to overcome resistance mechanisms of influenza
NAs.6,20 Among these efforts, the 5-position of 1 has been
explored. In analogy to zanamivir, replacement of the 5-amino
with a 5-guanidino group led to a highly potent NAI that is also
active against oseltamivir-resistant influenza strains.8,10 Ob-
viously, the guanidine moiety enables several more H-bonds as
well as strong ionic interactions that improve binding affinity.
However, such guanidine derivatives have not been further
pursued and brought to the clinic, most likely due to their main
restriction: the lack of oral bioavailability, which, indeed, has
been reported to be 4% for oseltamivir guanidine.21 The groups
of Fang and Wong made an interesting effort to further increase
binding affinity of 1 through bioisosteric replacement of the
carboxylic acid with a phosphonic acid.22 As expected, oral
bioavailability of these compounds was also very limited, with
reported values between 1 and 3% in rats and 2−12% in mice.23
Moreover, several distinct prodrug approaches have been
attempted to overcome pharmacokinetic (PK) issues of NAIs,
including intra- and intermolecular ion pairing strategies or
targeting of active transporters with specific amino acid
conjugates.24,25 In particular, the latter conceptthat has
been proposed by the Amidon groupshowed some success as
the oral bioavailability of oseltamivir guanidine could be
increased to 23% (mice, fed state) and 48% (mice, fasted
state) with a carrier-mediated L-valinate prodrug.26
Here, we describe a straightforward approach to overcome
the PK-related limitations of the potent oseltamivir guanidine
(7) by bioisosteric replacement with an acetamidine group (6),
while keeping activity against oseltamivir-resistant influenza
strains (Figure 2). Two prodrug candidates have been explored
along these lines with the simple amidine ethyl ester (8) and
the respective amidoxime analogue (9) (Figure 2). Moreover,
we demonstrate that analogous N-hydroxylated prodrugs of
oseltamivir guanidine (10, 11) are clearly inferior to the
amidine-based derivatives regarding their PK properties. The
presented studies eventually illustrate a successful balancing act
Figure 1. Chemical structures of clinically used NAIs.
pandemic.9 Zanamivir (2) was the first ever marketed NAI
(1999) and is approved for inhalative and intravenous
administration.10 As a further development of zanamivir,
laninamivir was introduced and very recently approved in
Japan.11 In the form of its octanoate prodrug (CS-8958),
laninamivir represents a long-acting NAI that requires only a
single daily inhaled dose underlining the awareness and need
for patient-compliant formulations of antiflu agents.12 The last
clinically relevant NAI is peramivir (4), which has been
approved in 2009 and 2010 for in-patient emergency treatment
of severe forms of influenza infections.13−15
In addition to the occurrence of highly pathogenic and
transmittable influenza strains, one major concern is the high
mutagenic rate of the viruses, particularly regarding the viral
surface proteins hemagglutinin and neuraminidase, which
contributes to the rapid development of resistances. In fact,
flu season 2007/2008 generated an oseltamivir-resistant H1N1
virus that spread globally and carries a H274Y mutation.16 The
matters were further complicated by sporadic emergence of
virus strains with resistance-conferring mutations of NAs, which
highlights the urgent need for new therapeutic agents.17
As indicated above, all clinically relevant NAIs suffer from
distinct drawbacks. In contrast to 1 that is orally bioavailable,
the treatment with zanamivir (or laninamivir) and peramivir is
restricted to inhalative or intravenously application, respec-
tively.18,19 On the other hand, influenza NAs already began to
develop resistances against 1 but are still susceptible to
Figure 2. Chemical structures of amidine- and guanidine-based prodrugs of oseltamivir.
B
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a
Scheme 1. Synthesis of Amidine and Guanidine Derivatives of Oseltamivir
a
Reagents and conditions: (i) S-(2-naphthylmethyl)thioacetimidate hydrobromide (1.1 equiv), EtOH, 0 °C-r.t., 2 h, 85%; (ii) KOH (3 equiv),
MeOH, 40 °C, 1 h, 88%; (iii) acetohydroximoylchloride (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C-r.t., 4 h, 70%; (iv) cyanogenbromide (1.5
equiv), NaOAc (2 equiv), EtOH, r.t., 4 h, 93%; (v) hydroxylamine (free base, 1 equiv), 1,4-dioxane, r.t., 1 h, 99%.
Figure 3. Binding mode analysis of neuraminidase X-ray structures and docking poses. (a) Overlay of oseltamivir carboxylate (5) X-ray structure
(green: pdb 3ti6) with oseltamivir amidine (6) docking pose (light blue) and oseltamivir guanidine (7) docking pose (orange); (b) overlay of
zanamivir (2) X-ray structure (green: pdb 3ti5) and oseltamivir guanidine (7) docking pose; (c) analysis of oseltamivir H-bonding within X-ray
structure (pdb 3ti6); (d) analysis of H-bonding interactions of 6 docking pose; (e) analysis of H-bonding interaction of 7 docking pose (residue
numbering based on pdb 3ti6, purple lines: H-bonding interaction).
C
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a
Table 1. Neuraminidase (NA) Inhibition of Oseltamivir Analogues in Chemiluminescence-Based Assay
Influenza Virus of Subtype H1N1
IC50 values (nM)
b
c
compd
A/Jena/5258/09
A/Jena/5555/09
A/HH/1580/09
anti H1N1 activity
A/Berlin/55/08
resistance factor
amidine 6
guanidine 7
amine 5
0.45 0.29
0.19 0.04
0.12 0.08
0.03 0.01
0.44 0.37
0.31 0.11
0.10 0.07
0.04 0.03
0.30 0.16
0.15 0.02
0.14 0.07
0.05 0.03
0.40 0.08
0.22 0.08
0.12 0.02
0.04 0.01
14.5 3.8
3.4 0.6
>100
36
15
>830
7
zanamivir 2
0.29 0.17
Influenza Virus of Subtype H3N2
IC50 values (nM)
compd
A/Hong Kong/68
A/Sachsen/6/02
2.30 1.25
A/Berlin/10/04
A/Rheinland-Pfalz/3911/03
anti H3N2 activity
amidine 6
guanidine 7
amine 5
1.87 0.81
0.83 0.15
0.44 0.38
0.66 0.56
1.30 0.16
0.75 0.30
0.15 0.05
0.45 0.07
1.79 0.88
0.63 0.13
0.17 0.10
0.20 0.00
1.82 0.41
0.80 0.15
0.24 0.14
0.45 0.19
0.99 0.36
0.18 0.09
0.49 0.16
zanamivir 2
a
b
c
Mean IC50 SD of at least three independent experiments. Oseltamivir-resistant influenza A, mutant H1N1 (H274Y). Ratio IC50 wild type
H1N1s (mean)/IC50 mutant H1N1 (A/Berlin/55/08).
between drug-likeness and antiviral activity of oseltamivir-
derived NAIs.
synthesis of amidoxime-type of prodrugs, we used freshly
prepared acetohydroximoyl chloride as a reagent,32 and
amidoxime 9 was readily accessible in good yields (70%).
Binding Mode Prediction. Docking of oseltamivir amidine
6 and guanidine 7 into H1N1 neuraminidase resulted in
reasonable high scoring docking poses. A comparison of both
molecules to oseltamivir carboxylate 5 shows an identical
binding mode (Figure 3a), and a comparison of the zanamivir
(2) complex structure and the predicted oseltamivir guanidine
(7) binding mode exhibited a similar binding of the guanidine
moiety. The binding pocket in oseltamivir H1N1 neuramini-
dase X-ray structure 3ti6 contains five water molecules, whereas
three of them are directly involved in oseltamivir binding (i.e.,
H2O840, H2O510, H2O488, Figure 3c). During docking, water
molecules H2O840 and H2O510 were replaced by the amidine
(Figure 3d) and guanidine moiety (Figure 3e). Instead,
H2O488 was not replaced and kept hydrogen bonding
interaction to amidine and guanidine. Both moieties revealed
an additional H-bonding interaction to Glu227, and guanidine
shows hydrogen bonding interactions to the backbone carbonyl
groups of Asp151 and Trp176. To summarize, 6 and 7
putatively replaced water molecules, showed additional H-
bonding interactions to the protein, and exhibited similar
binding modes as 5 and 2.
Antiviral Activity. Among many subtypes of influenza A
viruses, currently influenza A/H1N1 and A/H3N2 subtypes are
circulating among humans, which is why we focused on these
strains in our studies, including a H1N1 virus strain that has
been reported to be oseltamivir-resistant due to a H274Y (N2
numbering) amino acid exchange in NA.33 Inhibition of NAs
from a panel of different H1N1 and H3N2 influenza A viruses
was assessed using a chemiluminescence-based assay.34,35 As
shown in Table 1, 2 served as reference compound and
potently inhibited different H3N2 NAs (0.20−0.66 nM, IC50),
wild type pandemic H1N1 (0.03−0.05 nM, IC50) as well as a
mutant H1N1 NA (0.29 nM, IC50). Oseltamivir carboxylate (5,
0.10−0.14 nM, IC50) and the novel amidine 6 (0.30−0.45 nM,
IC50) and guanidine 7 (0.15−0.31 nM, IC50) inhibited H1N1
NAs in a similar fashion. Also, no big differences in inhibition
potency between amidine 6 and guanidine 7 were obvious
against H3N2 virus NAs. However, 5 appeared to be about 3−
8-fold more potent than 6 and 7 against H3N2 and even 2-fold
more potent than 2. Regarding oseltamivir-resistant influenza
RESULTS AND DISCUSSION
■
Chemistry. Guanidine analogues of 1 have been described
early in the history of the rational NAI design and represent
potent and valuable inhibitors.8,10 Therefore, we also aimed at
overcoming PK disadvantages associated with guanidine 7, that
is, the lack of sufficient oral bioavailability.21,23 An established
prodrug concept for such highly basic functional groups is the
use of a less basic N-hydroxylated derivative that would be
bioactivated in vivo by N-reduction. Although applications of
N-hydroxyguanidines as prodrugs of guanidines have not been
extensively explored in the field, we have reported on the
underlying metabolism and could demonstrate scope and
limitations of such prodrug concepts.27−30 For the synthesis of
the desired guanidine and N-hydroxyguanidine derivatives, two
distinct synthetic approaches were realized, respectively. The
N-hydroxyguanidine moiety was built up via initial preparation
of cyanamide 12 followed by reaction with hydroxylamine to
afford the N-hydroxyguanidine prodrug of oseltamivir (11) in
excellent yields (92%, overall) (Scheme 1). In contrast,
guanidine 7 and its corresponding ethyl ester prodrug 10
were synthesized following literature protocols that employ
N,N-bis(tert-butoxycarbonyl)thiourea and HgCl2 as a desulfur-
izing reagent.8,22
Bioisosteric replacement of the guanidine group in 7 by an
amidine would be conceivable in two different ways: First, by
removing a distal NH2 group generating an acetamidine (6). A
second option would be replacing the NH group in the 5-
position by a CH2 group affording a 5-(2-amino-2-iminoethyl)
functionality. A detailed analysis of zanamivir in available X-ray
structures (data not shown) showed hydrogen bonding
interactions of both groups, and it is hard to predict which
H-bonds would be most expendable, either those from a 5-NH
group (= acetamidine) or those from a distal NH/NH2 group
(= “terminal amidine”). We ultimately argued against the latter
“terminal amidine” for a practical reason which was the difficult
synthetic accessibility of such a compound.
Starting from 1, the acetamidine group was installed using S-
(2-naphtylmethyl)thioacetimidate and afforded the amidine
ester prodrug 8 in 85% yield (Scheme 1).31 Alkaline hydrolysis
of 8 furnished the amidine carboxylate drug (6, 88%). For the
D
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A, it is known that guanidine analogue 7 exerts potent
inhibition activity (0.5−4.1 nM, IC50).8,21,22 We confirmed this
finding in our experiments (3.4 nM, IC50) and calculated a
“resistance factor” of 15 that represents a minor degree of lost
activity. Amidine 6 inhibited the oseltamivir-resistant NA from
A/Berlin/55/08 at an IC50 of 12.6 nM, which implies a
“resistance factor” of 36 (Table 1). In comparison, with an IC50
of >100 nM, we determined a “resistance factor” beyond 830
for 5. These data are in good agreement with literature-
reported increases in IC50 values (450−1000-fold, NA assays)
for seasonal and pandemic variants that carry a H274Y
mutation.36 As expected, mutant H1N1 (A/Berlin/55/08)
was still most susceptible to zanamivir (0.29 nM, IC50).
Although no meaningful difference in NA binding affinity
(i.e., similar IC50’s in NA assay) between amine 5, amidine 6,
and guanidine 7 was observed, the situation is most likely
different in mutant virus strains. Docking pose analysis of 6 and
7 indicated a similar binding mode as zanamivir (2) with the 5-
guanidino/amidino moieties occupying the same position in
NA’s active site (Figure 3). Considering that most influenza A
strains are still susceptible to 2, data presented in this study
underline the importance of this ligand binding pocket for
activity against resistant virus strains.
mutant NA as the 5-guanidino congener, although forfeiting
some potency.
In Vitro Pharmacokinetics. Solubility needs to be
evaluated in any lead optimization campaign. Low solubility
can be detrimental in many respects as it can potentially impair
oral bioavailability or formulation of a suitable dosage form. In
consideration of the high polarity of this compound class, it was
not surprising that solubility turned out to not be an issue for
the different drug candidates (6, 7) as well as the simple ethyl
ester prodrugs (9, 10). But this fact was not readily predictable
for many of the other prodrug candidates. Our results showed,
however, solubility of >50 mM (in PBS) for all oseltamivir
derivatives presented herein (Table S2, Supporting Informa-
tion). The only exceptions were amidoxime ester 9 that
exhibited a solubility of 23 mM at pH 7.4 and 9.0 and guanidine
drug 7 with a solubility of 26 mM at at pH 7.4 and 9.0. Still,
solubility values in that range are far beyond doses required to
exert their pharmacological effect, that is, inhibition of viral
NAs.
Next, we examined protein binding of all compounds, a
feature that can be clinically relevant in the context of possible
drug−drug interactions or in view of the “therapeutic window”.
As shown in Table 3, our results suggest that all compounds do
In addition, we examined all NAIs in a cell-based assay that
addresses the cytopathic effect (CPE) of influenza virus
infections (Table 2).34,35 Results from this assay paralleled
Table 3. Protein Binding of Amidine- and Guanidine-Based
Oseltamivir Derivatives
protein binding
albumin (%)
protein binding human
plasma (%)
Table 2. Cytopathic Effect (CPE) Inhibition Assay in MDCK
Cells
compd
amidine 6
a
2.2 1.5
34.8 8.8
26.7 2.5
0.4 2.8
3.7 1.4
a
amidine ester 8
n.t.
EC50 values (μM) against influenza virus
amidoxime ester 9
guanidine drug 7
guanidine ester 10
n.t.
b
compd
A/Jena/5258/2009
A/Berlin/55/08
8.6 3.0
n.t.
amidine 6
1.90 1.35
3.12 3.45
5.21 5.36
6.56 7.50
4.82 2.94
3.31 3.81
30.1 2.3
37.5 10.1
guanidine 7
oseltamivir 5
zanamivir 2
N-hydroxyguanidine
ester 11
n.t.
c
>100
1.22 1.41
a
Not tested (n.t.).
a
Mean EC50
SD of at least three independent experiments.
c
b
Oseltamivir-resistant influenza A strain. Highest tested concen-
tration.
not or only to a small extent bind to albumin (0−38%). As a
general trend, protein binding appears to increase with higher
lipophilicity. Not surprisingly, data obtained with human
plasma (only available for amidine 6, guanidine 7) reveals
higher protein binding due to the presence of several other
plasma proteins (i.e., α1-acidic glycoprotein). In the literature,
protein binding of 5 is stated with 3% and 1 with 42%.37 Thus,
with regard to protein binding, the herein presented oseltamivir
derivatives exhibit an almost identical profile as the approved
drug 1.
Moreover, assessment of chemical stability is key in the
process of lead optimization and was done with the oseltamivir
drug candidates (6, 7) and their prodrugs (8−11) as
summarized in detail in Supporting Information (Figures S1−
S3). Such data is important for nomination of preferred drug
and prodrug candidates as well as for proper interpretation of in
vitro and in vivo PK studies as described in the following
sections. Briefly, all compounds showed excellent chemical
stability in PBS within the pH range of 2.0−9.0 (Figures S2 and
S3, Supporting Information). These data underline the high
stability of the ethyl ester under acidic and alkaline conditions,
which is relevant in the stomach and intestine after oral
administration. Moreover, incubations with human and murine
plasma resulted in rapid enzymatic hydrolysis of ethyl ester,
which is, in fact, desired for prodrug activation. Along these
the trend observed for NA inhibition as discussed above.
However, 6 and 7 were even more effective than 2 and 5.
Oseltamivir carboxylate was least active against the mutant
influenza A strain (i.e., not active at highest tested dose of 100
μM), while the amidine and guanidine analogues still
performed well. Notably, no cytotoxicity was detected in
MDCK cells at inhibitor concentrations up to 200 μg/mL (i.e.,
6 = 0.53 mM, 7 = 0.55 mM). Moreover, cellular data was
further confirmed in a virus yield reduction assay where again
both oseltamivir amidine and guanidine were more potent than
2 and 5 reference compounds (Table S1, Supporting
Information).
In summary, amidine 6 performs similarly potent toward
wild-type NAs compared to its guanidine analogue 7 and is
about 4-fold less potent against mutant (oseltamivir-resistant)
influenza A. Amidine 6 is about 7-fold more potent against
mutant influenza A than its 5-amino congener (5). From these
results we conclude thatamong all oseltamivir derivatives
evaluated in this studya 5-guanidino moiety still leads to
highest binding affinity to NAs. This effect becomes most
prominent for the herein tested mutant NA (H274Y).
However, an amidine group in 5-position is completely
tolerated in wild-type NAs and leads to similar efficacy against
E
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lines, more thorough studies on in vitro bioactivation using
distinct liver enzyme preparations were required to test if
activation of all prodrugs occurs in sufficient rates to justify
further in vivo studies in rats. As shown in Table 4, all prodrugs
In Vivo Bioavailability. The ultimate goal of our studies
was to show oral bioavailability of the newly developed
oseltamivir derivatives considering this general problematic
nature of NAIs. Therefore, in vivo PK studies were done with
all herein reported oseltamivir prodrugs. Oral application of all
amidine-based derivatives resulted in measurable plasma
concentrations of the active from (i.e., amidine 6) over a
time period of 360 min (Figure 4). Highest plasma levels were
detected after application of the amidoxime ester prodrug 9
with a bioavailability of 31.4% and plasma half-life of 111 min
(Table 5). Similarly performed studies in rats with the
marketed drug 1 revealed a comparable oral bioavailability (F
= 36%) with a reduced plasma half-life (44 min).21,38
Significantly lower plasma concentrations of amidine 6 were
obtained after oral administration of the amidine drug itself (6,
F = 3.9%) as well as the amidine ester (8, F = 0.8%). An
interesting finding was the identification of oseltamivir
amidoxime carboxylic acid as a major metabolite (plasma)
after oral application of the amidoxime prodrug 9 (data not
shown). This metabolite provides insight into the mode of
bioactivation as it suggests that in vivo N-reduction is indeed
the rate-determining step, which is eventually in good
agreement with our in vitro bioactivation data. These results
are also in accordance to our previous observations with similar
prodrugs.39,40
Table 4. In vitro Enzymatic Bioactivation of Oseltamivir
a
Prodrugs to Oseltamivir Carboxylate
conversion rate (nmol/min/mg protein)
N-hydroxy-
enzyme
source
amidine
ester 8
amidoxime
ester 9
guanidine
ester 10
guanidine
ester 11
PL Ms
0.30 0.11
0.15 0.01
0.42 0.10
0.66 0.05
0.64 0.01
0.56 0.04
0.20 0.02
0.15 0.01
0.83 0.14
7.51 0.66
0.51 0.06
5.34 0.15
0.40 0.03
2.30 0.20
0.29 0.01
0.56 0.06
0.13 0.02
n.t.
0.22 0.01
0.21 0.01
0.22 0.01
n.d.
4.77 0.13
0.67 0.02
1.53 0.14
n.d.
PL Mt
PL 9000g
PL Cyt
HL Ms
HL Mt
HL 9000g
HL Cyt
PL CE
1.06 0.02
0.79 0.04
0.32 0.03
0.50 0.08
1.35 0.15
1.76 0.05
1.74 0.06
0.76 0.09
0.28 0.01
n.t.
a
Liver enzyme sources: pig liver (PL), human liver (HL), microsomes
(Ms), mitochondria (Mt), cytosol (Cyt), 9000g supernatant (9000g),
carboxyl esterase (CE), not detected (n.d.), not tested (n.t.).
A disappointing outcome of the herein presented in vivo
studies was the lack of oral bioavailability of the N-hydroxy
prodrug 11 of oseltamivir guanidine (F = 1.5%, Table 6, Figure
4) despite its promising in vitro PK profile. The simple
guanidine ester prodrug 10 and 7 revealed similarly low oral
bioavailabilities (i.e., 1.3% and 1.9%), which are in good
agreement with literature-reported values in rats (i.e., 10 = 2.1%
and 7 = 4.0%).21 In a recently published study with a very
interesting class of oseltamivir phosphonates, the authors also
struggled with a lack of bioavailability of 5-amino, 5-guanidino,
and distinct guanidino phosphonate esters of 1 (F = 1−3% in
rats, F = 2−12% in mice).23 At this point, it can only be
speculated that guanidine-based oseltamivir derivatives are
either insufficiently absorbed in the intestine and/or rapidly
excreted after uptake, processes that can obviously not be
avoided with an N-hydroxy prodrug as has been shown here.
Currently, only the L-valine carrier-mediated prodrug for
oseltamivir guanidine concept reported by the Amidon group
seems to mask some of the detrimental PK features of the
guanidine moiety through targeting of active transporters.26
were activated to their corresponding drugs at moderate to high
turnover rates. Interestingly, the N-hydroxylated compounds
(9, 11) are more efficiently activated than the simple ester
prodrugs (8, 10). These results suggest that N-hydroxylated
oseltamivir prodrugs might be better substrates for esterases
than the highly basic and positively charged amidine 6 or
guanidine 7, respectively. However, this phenomenon was
much less pronounced in incubations with human and murine
plasma, which clearly revealed a good enzymatic activation of
the ethyl esters (Figures S2 and S3, Supporting Information).
Incubations with carboxyl esterases showed turnover rates of
0.83 0.14 nmol/min/mg (8) and 1.35 0.15 nmol/min/mg
protein (10), additionally stressing sufficient bioactivation of
these ethyl esters (Table 4).
In summary, oseltamivir amidine 6 and guanidine 7 as well as
all prodrugs exhibit excellent in vitro PK properties as they are
stable under various physiologically relevant conditions, do not
excessively bind to plasma proteins, and are sufficiently
bioactivated.
Figure 4. Plasma concentration curves of oseltamivir amidine drug 6 (left) and oseltamivir guanidine drug 7 (right) in rats after oral administration
of prodrug candidates. Drugs and prodrugs 6−11 were administered orally at 50 mg/kg as suspensions (10% gum arabic in H2O). Blood samples
from different time points were quantified for either 6 or 7 by HPLC analysis. Each data point represents mean SD from n = 3−6 rats.
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Table 5. In Vivo Pharmacokinetic Parameters of Amidine-Based Oseltamivir Prodrugs
a
PK parameters
application
amidine 6
amidine ester 8
amidoxime ester 9
oral
iv
oral
oral
5
rats (n)
5
3
6
b
MW (g/mol)
dose (mg/kg)
AUC (μg min/mL)
tmax (min)
325
10
325
50
434
369
50
50
578 149
113 48
44 15
17 13
797 193
82 20
31
6
Cmax (μg/mL)
MRT (min)
t1/2 (min)
0.6 0.2
126 16
0.2 0.1
97 29
3.1 0.7
206 61
32
34
5
9
c
c
d
68 29
42 23
111 56
c
c
e
F (%)
100 25.8
3.9 1.7
0.8 0.6
31.4 7.6
a
b
c
Molecular weight (MW), area under the curve (AUC), mean residence time (MRT), bioavailability (F). HBr salt. No significant differences.
d
e
Significantly different from i.v. (p < 0.05, one-way ANOVA with Dunnett’s post test). Significantly different (p < 0.001, one-way ANOVA with
Tukey’s post test).
primarily because of its oral formulation (i.e., capsules). In fact,
all remaining clinically relevant NAIs cannot be administered
orally but either by inhalation (i.e., 2) or by parenteral injection
(2, 3).
Table 6. In Vivo Pharmacokinetic Parameters of Guanidine-
Based Oseltamivir Prodrugs
N-hydroxy-
PK
guanidine
guanidine
ester 11
a
parameters
guanidine 7
ester 10
With oseltamivir amidine 6 and its bifunctional prodrug
amidoxime ethyl ester 9, we introduced an antiflu strategy that
tackles both major problems associated with currently available
NAIs. First, the 5-amidine analogue of oseltamivir (i.e., 6) is
comparably potent against different wild-type influenza A
strains as 1 but additionally showed efficacy (IC50 = 14.5 nM)
against oseltamivir-resistant influenza (A/Berlin/55/08, H274Y
mutant). Docking experiments revealed that additional
interactions in the NA active site occur, to a similar extent as
guanidine-containing NAIs such as oseltamivir guanidine 7 or
zanamivir (2). Second, amidoxime prodrug 9 exhibits an in vivo
PK profile that is competitive with 1 with comparable oral
bioavailabilities (31% versus 36%). A possible asset of 9 can be
seen in its prolonged plasma half-life (111 min versus 44 min
for 1) that means a retardation effect that could be beneficial
for the required dosing regimen. In general, the amidine-based
oseltamivir derivatives described here exhibit excellent in vitro
PK properties as they are stable under various physiologically
relevant conditions, appear to not excessively bind to plasma
proteins, and are sufficiently bioactivated. Moreover, extension
of the 5-amino to a 5-acetamidino group is a minor change of
the molecule minimizing the risk for unforeseen toxicity or
undesired side effects. In addition, with 9 no complex double-
prodrug or carrier-mediated prodrug concepts have been
implemented ensuring intestinal uptake by simple mechanisms,
that is, passive diffusion. Further assets of the herein presented
amidine-based oseltamivir derivatives are their straightforward
synthetic accessibility, their high physicochemical stability, and
the crystalline state of 9, features that will facilitate formulation
technologies.
application
iv
oral
3
oral
5
oral
4
rats (n)
5
b
MW
(g/mol)
326
326
468
370
dose
(mg/kg)
10
50
50
50
AUC (μg
min/mL)
288 161
27 13
13
8
19 11
tmax (min)
103 29
0.3 0.2
60 26
91 54
Cmax
(μg/mL)
0.1 0.1
0.1 0.1
MRT (min)
t1/2 (min)
F (%)
59 24
132
9
c
157 45
168 77
c
c
c
94 36
73 10
87 32
69 38
d
d
d
100.0 56
1.9 0.9
1.3 0.7
1.5 0.9
a
Molecular weight (MW), area under the curve (AUC), mean
b
c
residence time (MRT), bioavailability (F). TFA salt. No significant
differences. No significant differences.
d
The authors recently reported on an oral biovailability of their
oseltamivir guanidine prodrug of 23% under fed and 48% under
fasted conditions (mice).
In summary, the results presented here emphasize the high
efficacy of an amidoxime prodrug strategy, a concept that
unifies improved intestinal uptake (i.e., absorption) and
adequate bioactivation. Amidoxime prodrug 9 exhibits an in
vivo PK profile that can be viewed as equivalent to 1 with
comparable oral bioavailabilities (31% versus 36%). A possible
advantage of amidoxime 9 over 1 could be its prolonged plasma
half-life (111 min versus 44 min) that means a retardation effect
that in turn could be beneficial for the required dosing regimen.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
1
In view of the threat of a highly pathogenic flu pandemic and of
emerging drug resistance, it is of critical importance to identify
and develop novel anti-influenza agents. A general issue with
antiflu therapy is the high mutagenic rate of the viruses,
especially concerning their surface proteins hemagglutinin and
neuraminidase, which contributes to the rapid development of
resistance against currently approved drugs. In this regard,
particularly alarming is the occurrence of oseltamivir-resistant
influenza strains. In the case of a flu pandemic, 1 is currently the
drug of choice and has been stockpiled by most countries,
Chemistry. General. H (300 MHz) and 13C (75 MHz) NMR
spectra were obtained on a Bruker ARX 300 spectrometer at 300 K.
Low resolution mass spectra were recorded on a Bruker-Esquire-LC
with electrospray ionization (ESI). Purity of synthetic products was
≥96% as determined by LC-MS. Recordings of high mass resolution
spectra (HRMS) were conducted on a Bruker 7.4 T FTICR mass
spectrometer BioApex II equipped with an ESI-ion source (Agilent);
purification of synthesized compounds was performed by column
chromatography using silica gel (particle size 40−63 μm). Flash
chromatography on a RP18 column (43 g, RediSep) was performed
with the CombiFlashRETRIEVE system. Starting materials were
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commercially available and used without further purification. All
solvents were distilled and dried according to standard procedures. 1
as a starting material was obtained from the proprietary medicinal
product Tamiflu (capsules) by several extractions as described in
Supporting Information. Oseltamivir guanidine (7, GS4116) and the
guanidine ester prodrug (10) were synthesized according to literature-
described protocols.22
The mixture was stirred 4 h at ambient temperature and evaporated in
vacuo, and the crude product was purified by column chromatography
(SiO2, CH2Cl2/MeOH, 5−10%). Yield: 1 g (93%) of a colorless oil.
TLC: Rf = 0.39 (CH2Cl2/MeOH, 9:1). 1H NMR (DMSO-d6): δ/ppm
= 0.79 (t, 3J = 7.3 Hz, 3H), 0.84 (t, 3J = 7.1 Hz, 3H), 1.22 (t, 3J = 7.1
Hz, 3H), 1.40 (mc, 4H), 1.86 (s, 3H), 2.10−2.28 (m, 1H), 2.86 (dd, 2J
3
3
= 17.2 Hz, J = 5.2 Hz, 1H), 3.23 (mc, 1H), 3.35 (quint, J = 5.7 Hz,
1H), 3.60 (td, 3J = 8.9, 11.0 Hz, 1H), 4.11 (mc, 1H), 4.16 (q, 3J = 7.1
Hz, 2H), 6.64 (mc, 1H), 6.89 (d, 3J = 7.0 Hz, 1H), 7.95 (d, 3J = 9.1 Hz,
1H). 13C NMR (DMSO-d6): δ/ppm = 8.8, 9.4, 14.0, 23.0, 25.1, 25.6,
30.6, 53.0, 55.1, 60.5, 74.5, 80.7, 116.2, 127.6, 138.4, 165.2, 169.7.
(e) (3R,4R,5S)-Ethyl 4-acetamido-5-(N-(N′-hydroxy)guanidine)-3-
(1-ethylpropoxy)cyclohex-1-enecarboxylate (11). A total of 213 mg
of cyanamide 13 (0.6 mmol) was dissolved in 5 mL of dry 1,4-dioxane,
and 20 mg of hydroxylamine (1 equiv, free base)41 was added. The
mixture was stirred for 30 min at room temperature and evaporated to
dryness. After azeotroping with CH2Cl2 as well as Et2O, a white
powder was obtained. Yield: 222 mg (100%) of a white powder. TLC:
Rf = 0.20 (EtOAc/MeOH, 6:4). 1H NMR (DMSO-d6): δ/ppm = 0.80
(t, 3J = 7.3 Hz, 3H), 0.84 (t, 3J = 7.4 Hz, 3H), 1.24 (t, 3J = 7.1 Hz, 3H),
(a) (3R,4R,5S)-Ethyl 4-acetamido-5-(N-acetimidamido)-3-(1-
ethylpropoxy)cyclohex-1-ene-carboxylate Hydrobromide (8). One
gram (3.2 mmol) of 1 was dissolved in 10 mL of EtOH and cooled to
0 °C. A total of 1.04 g of S-(naphthylmethyl)acetimidobromide (1.1
equiv)31 was added, and the mixture was stirred at room temperature
for 1 h. The mixture was dried in vacuo and resuspended in 80 mL of
water. The solution was washed with Et2O and concentrated to
dryness in vacuo. The product (85%) contained small amounts of
starting material and was further purified by column chromatography
(SiO2, CH2Cl2/MeOH, 5−10%). Yield: 960 mg (71%) of a white
1
powder. TLC: Rf = 0.65 (CH2Cl2/MeOH, 9:1). H NMR (DMSO-
d6): δ/ppm = 0.79 (t, 3J = 7.4 Hz, 3H), 0.85 (t, 3J = 7.4 Hz, 3H), 1.23
3
(t, J = 7.1 Hz, 3H), 1.44 (mc, 4H), 1.83 (s, 3H), 2.11 (s, 3H), 2.33
2
(mc, 1H), 2.67 (dd, 2J = 17.6 Hz, 3J = 4.7 Hz, 1H), 3.42 (quint, 3J = 5.6
Hz, 1H), 3.82 (mc, 1H), 4.05 (mc, 1H), 4.17 (q, 3J = 7.1 Hz, 2H), 4.35
(mc, 1H), 6.69 (mc, 1H), 8.04 (br d, 3J = 9.0 Hz, 1H), 8.63 (br s, 1H),
9.25, 9.35 (2 × br s, 1H). 13C NMR (DMSO-d6): δ/ppm = 8.8, 9.4,
14.0, 18.9, 22.7, 25.2, 25.7, 28.7, 50.9, 53.4, 60.6, 74.1, 81.1, 127.7,
138.0, 164.0, 165.1, 169.7. MS (ESI): m/z = 354 [M + H]+.
1.40 (mc, 4H), 1.83 (s, 3H), 1.99−2.07 (m, 1H), 2.86 (dd, J = 16.7
3
3
Hz, J = 2.5 Hz, 1H), 3.38 (quint, J = 5.5 Hz, 1H), 3.49 (mc, 1H),
3.80 (m, 1H), 4.01 (mc, 1H), 4.14 (q, 3J = 7.1 Hz, 2H), 4.24 (mc, 1H),
4.92 (s, 2H), 6.64 (mc, 1H), 7.72 (br s, 1H), 7.79 (d, 3J = 8.8 Hz, 1H).
13C NMR (DMSO-d6): δ/ppm = 9.0, 9.3, 14.1, 22.9, 25.2, 25.6, 30.9,
49.1, 53.1, 60.4, 75.2, 80.9, 128.8, 137.5, 154.3, 165.7, 169.8. MS (ESI):
m/z = 741 [2 M + H]+, 393 [M + Na]+, 386, 371 [M + H]+. HRMS
(ESI): m/z calc. for C17H30N4O5 [M + H]+: 371.22890, found:
371.22911.
(b) (3R, 4R, 5S)-4-Acetamido-5-(N-acetimidamido)-3-(1-
ethylpropoxy)cyclohex-1-ene-carboxylic Acid (6). Ethyl ester 8
(217 mg, 0.5 mmol) was stirred in 10 mL of MeOH and 1.5 mL of
1 M KOH (in MeOH, 3 equiv) for 1 h at 40 °C. This solution was
diluted with water and the pH was adjusted to 7−8 and concentrated
to dryness. The crude product was purified by flash-chromatography
on a RP18 column (eluent: H2O). Product containing fractions were
Computer Modeling. Protein structures of 5 (pdb 3ti6) and 2 (pdb
3ti5) in complex with H1N1 neuraminidase were retrieved from the
protein data bank (www.pdb.org). Overlay of both structures were
done using UCSF Chimera.42 Hydrogens were added to the protein
structure using MOE and Protonate 3D.43 For docking, the prepared
protein structure pdb 3ti6 and GOLD 5.0 were used with standard
settings and ChemPLP for pose creation.44 In each docking run, 100
poses were created, and water molecules H2O488, H2O510, H2O544,
H2O628, and H2O840 were kept during docking but could be replaced
by the docked ligand.
Biology. Control Compounds. 5 (GS4071) was kindly provided
by Hoffmann-La Roche AG (Basel, Switzerland) and 2 (GG167) by
GlaxoSmithKline (Uxbridge, UK). For antiviral assays, compound
stocks were prepared in H2O and stored at 4 °C (stability of all
oseltamivir derivatives was confirmed as described in detail in
Supporting Information).
1
combined and lyophilized. Yield: 88% of a white powder. H NMR
(D2O, 300 MHz): δ/ppm = 0.84 (t, 3J = 7.4 Hz, 3H), 0.89 (t, 3J = 7.4
Hz, 3H), 1.38−1.63 (m, 4H), 2.03 (s, 3H), 2.23 (s, 3H), 2.43 (mc,
1H), 2.82 (dd, 2J = 17.5 Hz, 3J = 4.8 Hz, 1H), 3.53 (quint, 3J = 5.4 Hz,
1H), 3.93−4.09 (m, 2H), 4.36 (mc, 1H), 6.71 (br s, 1H). 13C NMR
(D2O): δ/ppm =11.2, 11.3, 21.8, 24.7, 27.9, 28.3, 32.0, 53.8, 56.8, 78.0,
86.8, 133.1, 138.4, 167.9, 173.8, 177.3. MS (ESI): m/z = 348 [M +
Na]+, 326 [M + H]+. HRMS (ESI): m/z calc. for C16H27N3O4 [M +
H]+: 326.20743, found: 326.20737.
(c) (3R,4R,5S)-Ethyl 4-acetamido-5-(N-(N′-hydroxy)-
acetimidamido)-3-(1-ethylpropoxy)-cyclohex1-enecarboxylate (9).
A total of 465 mg (1.49 mmol) of 1 and 290 mg of DIPEA (389
μL, 1.5 equiv) were dissolved in 5 mL of CH2Cl2 and cooled to 0 °C.
To this solution, freshly prepared acetohydroximoylchloride (209 mg,
1.5 equiv)32 was added dropwise. The mixture was stirred for 4 h at
room temperature. After addition of 15 mL of water, the mixture was
stirred for another hour. The phases were separated, and the aqueous
phase was extracted with CH2Cl2 (4×) to increase yields. The pooled
organic phases were dried (Na2SO4) and evaporated to dryness. The
product was purified by column chromatography (SiO2, CH2Cl2/
MeOH, 9:1). Yield: 70% of a crystalline colorless solid. TLC: Rf = 0.29
Cells and Viruses. MDCK cells (Friedrich-Loeffler Institute, Riems,
Germany) were grown in Eagle’s minimum essential medium
(EMEM) supplemented with 100 U/mL penicillin and 100 U/mL
streptomycin, 10% fetal bovine serum, and 2 mM L-glutamine. EMEM
medium for virus propagation, titration, antiviral tests of viruses
contained 100 U/mL penicillin and 100 U/mL streptomycin, 2 μg/mL
trypsin, and 0.1% sodium bicarbonate. The seasonal H1N1 A/Berlin/
55/08 (swab sample was published as A/342/2009),33 the H3N2 A/
Sachsen/6/02, A/Berlin/10/04, and A/Rheinland-Pfalz/3911/03 (all
kindly provided by the National Reference Centre of influenza viruses
at the Robert Koch Institute, Berlin, Germany), pandemic H1N1
influenza viruses A/Jena/5258/09, A/Jena/5555/09 (both as pub-
1
3
(CH2Cl2/MeOH, 9:1). H NMR (DMSO-d6): δ/ppm = 0.80 (t, J =
7.4 Hz, 3H), 0.85 (t, 3J = 7.4 Hz, 3H), 1.23 (t, 3J = 7.1 Hz, 3H), 1.43
(mc, 4H), 1.80 (s, 3H), 1.95 (s, 3H), 2.38 (mc, 1H), 2.62 (dd, 2J = 17.4
Hz, 3J = 5.0 Hz, 1H), 3.40 (quint, 3J = 5.6 Hz, 1H), 3.65 (mc, 1H) 3.78
(dd, 2J = 17.4 Hz, 3J = 8.7 Hz, 1H) 4.15 (q, 3J = 7.1 Hz, 2H), 4.19 (mc,
1H), 6.67 (mc, 1H), 6.81 (br d, 3J = 9.1 Hz, 1H), 7.99 (d, 3J = 8.6 Hz,
1H), 9.73 (br s, 1H). 13C NMR (DMSO-d6): δ/ppm = 9.0, 9.4, 13.9,
14.0, 22.7, 25.2, 25.6, 31.3, 50.6, 53.6, 60.4, 74.4, 81.0, 128.4, 137.7,
153.5, 165.5, 169.5. MS (ESI): m/z = 392 [M + Na]+, 370 [M + H]+.
HRMS (ESI): m/z calc. for C18H31N3O5 [M + H]+: 370.23365, found:
370.23379.
lished),33 A/Hamburg/1580/09 (kindly provided by Ralf Durrwald,
̈
IDT Biologica GmbH, Dessau, Germany), and the H3N2 A/
HongKong/68 (strain collection of the Department of Virology and
Antiviral Therapy, Jena University Hospital, Germany) were used in
the present study. Virus titers (TCID50) were determined according to
Reed and Muench.45
Neuraminidase (NA) Inhibition Assay. Inhibition of viral NA was
examined using a chemiluminescence-based assay (NA-star kit, Tropix,
Applied Biosystems, Darmstadt, Germany) as described in the
literature.16,33,34 IC50 values were calculated from at least three
independent experiments.
Cytophatic Effect (CPE) Inhibition Assay. Two-day-old MDCK cell
monolayer without or with test compounds was infected with influenza
(d) (3R,4R,5S)-Ethyl 4-acetamido-5-cyanamido-3-(1-
ethylpropoxy)cyclohex-1-enecarboxylate (12). One gram (3.2
mmol) of 1 was dissolved in 20 mL of EtOH, and 525 mg of
NaOAc (2 equiv) was added. To this solution, 500 mg of cyanogen
bromide (1.5 eq, dissolved 20 mL EtOH) were slowly added dropwise.
H
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virus in a concentration causing complete CPE within 48 h. After
incubation for 48 h at 37 °C, cells were fixed and stained with crystal
violet as described previously.46 Inhibition of virus-induced CPE was
determined photometrically on a Dynatech plate reader. Antiviral
efficacy was calculated by comparison of optical density of drug-treated
and untreated virus-infected cells. On the basis of the dose response
curve of at least two independent experiments linear interpolation was
used to calculate IC50 values. Note: None of the test compounds were
toxic to MDCK cells treated for 72 h as described previously.46
Protein Binding. Protein binding was determined in a 4% albumin
solution (or in human plasma) at three different concentrations (10,
25, 50 μM) of the test compound. After shaking for 10 min at r.t.,
samples were centrifuged at 10 000 rpm (15 min) in Vivaspin 500 (10
kDa cutoff) ultrafiltration units. Control samples without protein
confirmed that the test compounds were not retained by the
ultrafiltration unit membrane. Filtrate were analyzed by HPLC:
Waters 717 plus autosampler, Waters 600 Controller and Waters 2487
Dual λ absorbance detector (set at 210 nm); Column: LiChrospher 60
RP-select B (125 × 4 mm, 5 μm), RP-select B guard column; Isocratic
elution (1 mL/min): for quantification of oseltamivir amidine (6) and
oseltamivir guanidine (7): solvent A (0.1% TFA, 10 mM phosphate
buffer, pH 3.0) and solvent B (MeOH) at 60/40 ratio. Retention
times: 4.4 0.2 min (7), 4.5 0.2 min (6); for quantification of all
other oseltamivir derivatives: solvent A (0.1% TFA, 10 mM phosphate
buffer, pH 3.0) and solvent B (MeOH) at 50/50 ratio. Retention
times: 3.7 0.1 min (10), 4.2 0.1 min (9), 4.6 0.1 min (11), 4.8
0.1 min (8).
20, 45, 90, 150, 240, 360 min after i.v. injection. Plasma samples were
obtained by centrifugation at 10.000g for 5 min and were immediately
frozen (−80 °C). Animals were sacrificed by decapitation under
isofurane anesthesia 360 min after application of test compounds.
Plasma samples were treated with equal volumes of MeOH (0.2%
TFA). Samples were shaken for 45 min and frozen (−80 °C). After
thawing and shaking for 15 min, the samples were centrifuged at
13000g for 15 min. The supernatant was analyzed by LC/MS. LC/MS
analytic: Agilent 1100 HPLC system with a 1100 binary pump, 1100
well-plate autosampler, 1100 PDA detector, and a Bruker Esquire ESI-
MS; Column: LiChrospher 60 RP-select B (125 × 3 mm, 5 μm) and
RP-select B guard column; flow rate: 0.3 mL/min, 50 μL injection
volume, detection at 190−400 nm, quantification for m/z = [M]+ and
[M + H]+. Solvent A: H2O (0.1% TFA), solvent B: MeOH (0.1%
TFA), gradient elution: 45%-75% B (0−8 min), 75% B (8−10 min),
75%-45% B (10−11 min), 45% B (11−17 min); Retention times: 5.1
0.3 min (6), 5.1 0.3 min (7). Pharmacokinetic parameters were
calculated using PK Solutions 2.0 software (Summit Research Services,
Montrose, USA). Statistical analyses were performed with GraphPad
Prism version 4.02 for Windows (GraphPad Software, San Diego,
USA).
ASSOCIATED CONTENT
* Supporting Information
■
S
Supplementary data, figures, tables, and extended experimen-
tals. This material is available free of charge via the Internet at
http://pubs.acs.org.
In Vitro Bioactivation. Prodrugs were incubated with subcellular
enzyme preparations from human and porcine liver (i.e., microsomes,
mitochondria, 9000g supernatants, cytosol). A typical incubation
sample consisted of 500 μM prodrug, 500 μM NADH, 1 U carboxyl
esterase (pig liver), and 0.3 mg of the respective enzyme preparation in
150 μL of potassium phosphate buffer (100 mM, pH 6.3). Samples
were incubated at 37 °C for 30 min, and reactions were stopped by
addition of 150 μL of MeCN. After centrifugation (13 000 rpm, 15
min) the supernatant was analyzed by HPLC (see below).
Bioactivation of ester prodrugs (i.e., 8 and 10) was investigated
using porcine liver carboxyl esterase. Incubations were performed over
60 min and contained 200 μM prodrug and 3 U esterase in 200 μL of
potassium phosphate buffer (100 mM, pH 7.4). Samples were taken
and analyzed by HPLC every 15 min. HPLC analytic was the same as
described above but using the following isocratic elution: solvent A
(0.1% TFA, 10 mM phosphate buffer, pH 6.5) and solvent B (MeOH)
at 70/30 ratio. Retention times: 4.8 0.2 min (6), 4.7 0.2 min (7).
In Vivo Bioavailability. Oral bioavailability of all oseltamivir
derivatives was examined with male Sprague−Dawley rats (Janivier,
France). The study was conducted according to the National Institutes
of Health Guidelines for the Care and Use of Laboratory Animals and
AUTHOR INFORMATION
Corresponding Author
*E-mail: dennis.schade@tu-dortmund.de. Phone: +49-231-
7557083.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Ulrich Girreser for performing NMR and MS
experiments and gratefully acknowledge the excellent technical
assistance of Melissa Zietz, Finn Clausen, Franziska Hohm,
Clara Schoeder, and Birgit Jahn. Financial support by
Innovationsstiftung Schleswig-Holstein in the form of the
“Transferpramie 2010” as well as by the Bundesministerium fur
̈
̈
Bildung und Forschung (01KI1006J) is acknowledged.
ABBREVIATIONS
NA, neuraminidase; PK, pharmacokinetic; CPE, cytopathic
effect; MDCK cells, Madin-Darby canine kidney epithelial cells
■
authorized by the local regulatory authority (= Ministerium fur
̈
Landwirtschaft, Umwelt und landliche Raume des Bundeslandes
̈
̈
Schleswig-Holstein). The animals were kept at r.t. with a 12:12 h dark
(8 AM-8 PM)-light (8 PM-8 AM) cycle. They received a standard diet
and water ad libitum. Rats (385 9 g) were habituated to research
assistants and vice versa 2 weeks before drug treatment was initiated.
Rats were randomized into groups receiving either amidine drug (6)
i.v. (n = 5), guanidine drug (7) i.v. (n = 5), amidine drug (6) p.o. (n =
3), guanidine drug (7) p.o. (n = 3), or one of the prodrugs p.o. (each n
= 4−6). Surgery: Two days before the pharmacokinetic study, chronic
polyethylene catheters have been inserted during pentobarbitone
anesthesia (67 mg/kg) into the right femoral vein and artery.
Catheters were tunneled under the back skin, exteriorised in the region
of the cervical vertebra, and fixed at the skin. Thereafter, rats were
housed individually in cages (height × width × length: 20 × 22 × 25
cm) until the end of the study.
For oral administration, compounds were suspended in a solution of
gum arabic (10%, H2O) and administered orally by gavage at 50 mg
kg−1 body weight. Blood samples (ca. 300 μL) were taken with
microvettes (EDTA-coated) after 30, 60, 90, 120, 180, 240, and 360
min via an arterial implanted catheter. Intravenous administration of 6
and 7 were done at 10 mg kg−1. Blood samples were collected 5, 10,
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