Structure-based design and synthesis of C-1- and C-4-modified analogs of zanamivir as neuraminidase inhibitors

Article abstract of DOI:10.1021/jm3009713

In order to exploit the 430-cavity in the active sites of neuraminidases, 22 zanamivir analogs with C-1 and C-4 modification were synthesized, and their inhibitory activities against both group-1 (H5N1, H1N1) and group-2 neuraminidases (H3N2) were determined. Compound 9f exerts the most potency, with IC₅₀ value of 0.013, 0.001, and 0.09 μM against H3N2, H5N1, and H1N1, which is similar to that of zanamivir (H3N2 IC₅₀ = 0.0014 μM, H5N1 IC₅₀ = 0.012 μM, H1N1 IC₅₀ = 0.001 μM). Pharmacokinetic studies of compound 9f in rats showed a much longer plasma half-life (t_1/2) than that of zanamivir following administration (po dose). Molecular modeling provided information about the binding model between the new inhibitors and neuraminidase, with the elongated groups at the C-1-position being projected toward the 430-loop region. This study may represent a novel starting point for the future development of improved antiflu agents.

Full text of DOI:10.1021/jm3009713

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Article
Structure-Based Design, Synthesis of C-1 and C-4 Modified
Analogs of Zanamivir as Neuraminidase Inhibitors
Enguang Feng, Woo-Jin Shin, Xuelian Zhu, Jian Li, Deju Ye, Jiang Wang, Mingyue Zheng, Jian-Ping
Zuo, Kyoung Tai No, Xian Liu, Weiliang Zhu, Wei Tang, Baik-Lin Seong, Hualiang Jiang, and Hong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3009713 • Publication Date (Web): 18 Jan 2013
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Structure-Based Design, Synthesis of C-1 and C-4 Modified Analogs
of Zanamivir as Neuraminidase Inhibitors
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Enguang Feng, WooꢀJin Shin, Xuelian Zhu, Jian Li, Deju Ye, Jiang Wang,
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Mingyue Zheng, JianꢀPing Zuo, Kyoung Tai No, Xian Liu, Weiliang Zhu, Wei
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Tang, BaikꢀLin Seong, Hualiang Jiang, and Hong Liu,
†
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu chong zhi Road, Shanghai 201203, China
‡
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei
University, Seoul 120ꢀ749, South Korea
║
These authors contributed equally to this work
Eꢀmail: hliu@mail.shcnc.ac.cn(H. Liu), blseong@yonsei.ac.kr(B.ꢀL. Seong),
tangwei@mail.shcnc.ac.cn (W. Tang), wlzhu@mail.shcnc.ac.cn (W. Zhu)
RECEIVED DATE (to be automatically inserted after your manuscript is
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Abstract
In order to exploit the 430ꢀcavity in the active sites of neuraminidases, 22 zanamivir
analogs with Cꢀ1 and Cꢀ4ꢀmodification were synthesized, and their inhibitory
activities against both groupꢀ1 (H5N1, H1N1) and groupꢀ2 neuraminidases (H3N2)
were determined. Compound 9f exerts the most potency, with IC50 value of 0.013,
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.001 and 0.09 ꢁM against H3N2, H5N1 and H1N1, which is similar to that of
zanamivir (H3N2: IC50 0.0014 ꢁM, H5N1: IC50 0.012 ꢁM, H1N1: IC50 0.001 ꢁM).
Pharmacokinetic studies of compound 9f in rats showed a much longer plasma
halfꢀlife (t ) than that of zanamivir following administration (po dose). Molecular
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modeling provided information about the binding model between the new inhibitors
and neuraminidase, with the elongated groups at the Cꢀ1 position being projected
towards the 430ꢀloop region. This study may represent a novel starting point for the
future development of improved antiꢀflu agents.
Introduction
In the face of the persistent threat of human influenza infections, and the potential
of new human or avian influenza variants to unleash a pandemic, there is much
concern about the shortage in both the number and supply of effective antiꢀinfluenza
1
virus agents. Accordingly, the discovery of effective drugs, which are not susceptible
to mutation, is an urgent need. Among the antiviral targets, neuraminidase (NA) has
been extensively investigated for drug design. The first NA inhibitors of Neu5Ac2en
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(
DANA) (1) and 4ꢀaminoꢀNeu5Ac2en (2) were designed based on the principle of the
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, 3
‘transition state analogs’ . Further optimization of the structures lead to two potent
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NA inhibitors, zanamivir (compound 3) (4ꢀguanidinoꢀNeu5Ac2en, GG167) and
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oseltamivir (compound 4) (GS4071 ethyl ester, GS4104, Ro64ꢀ0796) (Figure 1),
both are currently marketed for the treatment (and prophylaxis) of influenza A and B
virus infections.
However, the low oral bioavailability and rapid renal elimination of compound 3,
and the rapid emergence of compound 4ꢀresistant influenza viruses, have prompted
the further development of more potent and longer duration therapeutic drugs to
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combat potential human influenza pandemics.
Very recently, a longꢀacting NA
inhibitor, laninamivir (compound 5), has been approved in Japan. This drug is
effective against compound 4ꢀresistant strains and also effective when using just a
single inhaled dose via its octanoate prodrug (laninamivir octanoate, CSꢀ8958),
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demonstrating superior antiꢀinfluenza virus activity.
Figure 1
The nine known NAs of influenza A viruses can be divided phylogenetically into
two distinct groups: groupꢀ1 contains N1, N4, N5 and N8 subtypes, and groupꢀ2
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contains N2, N3, N6, N7 and N9. Groupꢀ1 NAs have a flexible ‘150ꢀloop’ providing
a novel, large cavity (150ꢀcavity) adjacent to the active site, which is absent in
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groupꢀ2 NAs. Based on this knowledge, a Cꢀ4ꢀmodified analog of compound 3 and
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a Cꢀ3ꢀmodified sialic acid derivative were recently reported to inhibit groupꢀ1 NAs
at micromolar level. Further molecular modeling studies and Xꢀray crystallography
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indicated that these two inhibitors occupied the 150ꢀcavity in the open form of
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groupꢀ1 NAs
. We have also previously reported a series of Cꢀ4ꢀtriazoleꢀmodified
analogs of compound 3 as potential antiꢀavian influenza virus (AIV) agents, and one
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compound exerts promising inhibitory activity, shown ~61% protection against AIV
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(H5N1) infection, while compound 3 was ~86% protective in the same assay.
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However, our group recently found that the 2009 H1N1 influenza pandemic NA
(
p09N1) is an atypical groupꢀ1 NA with some groupꢀ2 like features in its active site
lack of a 150ꢀcavity), implying that neuraminidase inhibitors targeted at the
(
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50ꢀcavity will probably be less effective against the subtype of groupꢀ1 variants
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containing Ile149, as argued in a recent review. Furthermore, it has been reported
that certain compound 4ꢀresistant amino acid substitutions in the NA active site are
groupꢀ1 specific. Using molecular dynamics (MD) simulations of influenza NA,
Amaro et al. observed extended conformational shifts of the 150ꢀloop and 430ꢀloop
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(Figure 2).
It was demonstrated that the binding site of NAs can open to a much
larger extent than that anticipated from prior Xꢀray structure analyses, which can be
systematically explored for the development of more potent, chemically diverse
inhibitors. Recently, a novel smallꢀmolecule inhibitor (NSC89853, 6) has been
reported by An and coꢀworkers, and the predicted binding mode of compound 6 to the
known H5N1 NA was different to that of compound 3 or compound 4. They also
suggested that the existence of another binding site within the pocket, formed by
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430ꢀloop, could be accessed by the new inhibitors. In addition, investigating the
seed extract of Alpinia katsumadain, Grienke et al. found one of the most promising
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constituent, katsumadain A, inhibited the NA of H1N1 swine influenza viruses, with
3
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IC50 values between 0.9 and 1.64 ꢁM. Due to its large molecular volume, the
extended conformation of the 430ꢀloop and 245ꢀloop appeared to be essential for the
accommodation of the ligand into the active site of NA.
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Figure 2
It is revealed that the active site of NAs could be empirically divided into five major
subꢀsites (S1ꢀS5) which are essential for the interactions with different NA inhibitors
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(
Figure 3). The 1ꢀcarboxyl group of compound 3 was shown to form strong
hydrogen bonds with three arginine residues (Arg118, Arg292 and Arg371) in the S1
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subsite, which is very important for the NA inhibitory activity. So far, little work
has been done to synthesize Cꢀ1ꢀmodified sialic acid derivatives with the exception of
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some ester prodrugs. Very recently, with the replacement of 1ꢀcarboxyl group of
compound 3 with phosphonate group, Wong et al. discovered two phosphono anologs
of compound 3 which are more potent than compound 3 against the NAs of avian and
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human influenza viruses, including the compound 4ꢀresistant strains. Meanwhile, as
shown in Figure 2, the flexible 430ꢀloop in both groupꢀ1 and groupꢀ2 NAs is next to
the subsite S1, which can provide a large cavity (430ꢀloop) adjacent to the active site.
This suggested new opportunities to design highly effective NA inhibitors that target
both 430ꢀcavity and the known active sites (Figure 2 and 3). Based on this knowledge,
here we designed and synthesized a series of compound 3 analogs (7a-g, 8a-e and
9a-j, Table 1) with different substitutions at Cꢀ4 and Cꢀ1 positions to explore the
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30ꢀcavity. Their antiꢀinfluenza virus activities against both groupꢀ1 (H5N1 and
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H1N1) and groupꢀ2 (H3N2) NAs were tested. Meanwhile, taking compounds 7d, 8a,
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c, 9f, 9g and 9i as examples, the interaction models between inhibitors and NAs
were also studied.
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Results and discussion
Designing analogs of zanamivir. Based on the structural feature of compound 3
32
and its binding models with groupꢀ1 and groupꢀ2 NAs (Figure. 3A and Figure. 3B) ,
2 analogs (7a-g, 8a-e and 9a-j, Table 1) were designed and synthesized. In order to
explore additional interactions with 430ꢀloop as revealed in groupꢀ1 and groupꢀ2 NAs
Figure 2), a range of substitutions at Cꢀ1 and Cꢀ4 modified analogs were introduced.
2
(
In particular, we were interested in using aliphatic acids to modify the 1ꢀcarboxyl
group to retain the strong hydrogen bonds with three arginine residues (Arg118,
Arg292 and Arg371). Initially, by replacing the Cꢀ4 position of compound 3 with a
hydroxyl group as well as introducing different substituted amides at Cꢀ1 carboxyl,
we obtained analogs 7a-g. Further replacement of Cꢀ4 hydroxyl group with an amino
group led to compounds 8a-e. In an effort to gain more potency, we subsequently
introduced the guanidinium group at the Cꢀ4 position as in compound 3 to obtain a
series of derivatives 9a-e. Because the 430ꢀcavity was reported to favor aromatic
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ringꢀbased compounds, compounds 9f-j were also obtained by replacing amino acid
moieties at the Cꢀ1 position with corresponding benzylamine substituents.
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Synthesis of target compounds. The synthesis of 7a-g, 8a-e and 9a-j is shown in
Scheme 1. Compound 9 (Nꢀacetyl neuraminic acid, NANA) was esterified with
benzyl bromide to protect the 1ꢀcarboxyl group. Compound 10 was obtained using
acetic anhydride in pyridine with catalytic 4ꢀ(dimethyamino)ꢀpyridine (DMAP), then
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o
treated with trimethylsiyltrifluoromethanesulfonate (TMSOTf) in acetonitrile at 0 C
to give compound 12. The key intermediate 13 was obtained by reducing compound
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2, which reacted with different amines, then hydrolyzed with NaOH in methanol to
give compounds 7a-g. Compound 11 was treated with TMSOTf in ethyl acetate at 52
o
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C using a modification of the procedure described by Chandler et al. to give 14 in
high yield. Compound 14 was hydrolyzed, then reacted with diphenylphosphoryl
azide (DPPA) and 1,8ꢀdiazabicyclo[5,4,0]undecenꢀ7ꢀene (DBU) to yield the inverted
2
azide 16. The key compound 17 was obtained by reduction of 16 using H S (gas) in
pyridine. Reaction of 17 with Bocꢀanhydride in methanol produced intermediate 18,
which was hydrogenated over Pd/C (10%) to give compound 19. Compound 19 was
reacted with a range of different amines, deprotected with trifluoroacetic acid (TFA)
and then hydrolyzed with NaOH to give the target compounds 8a-f. Compounds 9a-j
were prepared similarly from compound 22 which was accessed from compound 21
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by the method described by Konrad F. el at.
Scheme 1
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Inhibitory activities against NAs and binding models. All 22 target compounds
were evaluated for their NA inhibition using 2’ꢀ(4ꢀmethylumbelliferyl)
aꢀ
uniform activity, we simultaneously selected N1 (H5N1, H1N1) from groupꢀ1 and N2
H3N2) from groupꢀ2 as representatives for testing, and the results are summarized in
D
ꢀ ꢀNꢀacetylneuraminic acid (MUNANA) as the substrate. In the hope of achieving
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Table 1. Although general structure-activity relationships (SAR) of these compounds
is not evident, the bioassay results provide some information for further structural
modifications. When the guanidinium group of compound 3 is replaced with a
hydroxyl group, only week NA inhibitory activities were observed for compounds
4
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a-g, which is similar to that of Neu5Ac2en (DANA, A/Mississippi/1/85(H3N2):
IC50 52 ꢁM, H5N1: IC50 0.012 ꢁM, A/Brazilll/78(H1N1): IC50 240 ꢁM). However, as
revealed by von Itzstein et al. the acidic residues Glu119, Asp151 and Glu227 in the
active sites of NA are favorable of a basic substituent at the Cꢀ4 position of the
40, 41
scaffold.
Thus, compounds 8a-e which contain an amino group had significantly
improved NA inhibition comparing with compounds 7a-g. Further replacing the Cꢀ4
amino group with a stronger base of guanidine should increase the electrostatic
interactions with the acidic residues in the NA active site. Indeed, compounds 9a-j
with a 4ꢀguanidino group showed an enhanced NA inhibition in comparison with 7a-g
and 8a-e. Generally, the introduction of the amino acid substituent to the carboxyl
group at the Cꢀ1 position of compound 3 via amide bond did not translate into better
potency as shown in compounds 9a-e compared to compound 3. Gratefully,
compound 9f bearing a 3ꢀfluorobenzyl amine exerts the greatest potency, with IC50
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value of 0.013, 0.001 and 0.09 ꢁM against H3N2, H5N1 and H1N1, which is
comparable to that of compound 3 (H3N2: IC50 0.0014 ꢁM, H5N1: IC50 0.012 ꢁM,
H1N1: IC50 0.001 ꢁM). The higher NA inhibitory potency may be ascribe to the
elongation of the aromatic ring of the molecule to the 430ꢀcavity to form hydrogen
bond and strong hydrophobic interactions with NAs. However, further conversion of
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ꢀfluorobenzyl amine into corresponding 2ꢀfluorobenzy amine, 4ꢀfluorobenzy amine,
ꢀchlorobenzy amine or 4ꢀmethylbenzyl amine did not show improved inhibition. The
incorporation of fluorine imparts special characteristics that enhance therapeutic
efficacy and improved pharmacological properties. As the second smallest substituent,
fluorine not only closely mimics hydrogen with respect to steric requirements at
enzyme receptor sites, but also leads to increased lipid solubility and hydrophobic
interaction with NAs.
Table 1
To better understand the discrepancy of activity among compounds 7, 8, 9 and
compound 3, we compared the 3D binding models of compound 3 with NA to that of
our different inhibitors to NA derived from docking simulation. Compounds with
different substituents at the Cꢀ4 position exhibit varying activity against NAs under
experimental conditions. In order to explain the difference in their activities,
compounds 7d, 8a and 9c were selected since they are identical with the exception of
Cꢀ4 substitution. The hydroxyl group of 7d and the amino group of 8a are embedded
within subsite S2 and form two hydrogen bonds with Glu119 and Asp151 of N2
(Figure 4. A, B, E and F). However, the guanidinium group of 9c forms five hydrogen
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bonds with Glu119, Asp151, Trp178 and Glu227 (Figure 4. C, D and G, H), which is
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consistent with previous findings.
In addition to the hydrogen bond, the
chargeꢀcharge interaction also plays an important role in increasing the activity of 9c.
The subsite S2 has negative charge properties (Figure 4. E, F and G), which
preferentially binds to the positiveꢀcharged substituent. The protonated guanidinium
group has the greatest positive charge among these three functional groups, followed
by protonable amino group, whereas the hydroxyl group has no positive character.
Therefore, 9c is predicted to be the most potent compound, followed by 8a, and
finally 7d, which is confirmed by the experimental test.
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Figure 4
Based on the docking results, the elongated amide groups at the Cꢀ1 position were
projected towards the 430ꢀloop region (Figure 5. A and C) to form hydrogen bonding
and/or hydrophobic interactions with NAs, while the guanidinium group establishes
hydrogen bonds and electrostatic interactions within S2 subsite similar to that of
compound 3. This explains the generally higher inhibitory activity of this series
against NAs. Among all the synthesized compounds, 9f has the most potent antiviral
activity. The elongated group at the Cꢀ1 position in 9f was projected towards the
4
30ꢀloop region to form extensive hydrophobic interactions with Val149, Ile427,
Pro431 and Thr439, which can partially compensate for weaker activity caused by the
absence of electrostatic interactions. Therefore, 9f still retains inhibitory activity
against Influenza neuraminidases, but weaker than compound 3. When compared 9g,
9
i with 9f, it seems the position of the fluorine on the phenyl ring is also very
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important, which may be ascribe to the large electronic effect on the molecule’s
dipole moment and the hydrophobic interaction with the 430ꢀcavity. Figure 5 (B and
D) showed that the fluorine substituted phenyl ring of 9g and 9i could not form
similar hydrophobic interactions as 9f.
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Figure 5
Pharmacokinetic (PK) profiles of the selected anti- influenza agent. It is
reported that compound 3 has poor bioavailability and rapidly eliminated (short
8,9
plasma halfꢀlife t1/2) following oral administration. To gain an initial understanding
about the pharmacokinetic parameters of our series, we selected compound 9f which
had the most potent antiviral activity as representative for further investigation
following intravenous (iv) and oral (po) dosing in rats. For comparison, we also listed
42
the parameters of compound 3 and compound 4 as reported in the literature in Table
2. The results showed that 9f has similar bioavailability following oral dosing to that
of compound 3 (1.41% vs 2.7%). However, a much longer plasma t1/2 for 9f was
observed after po dosing compared to that of compound 3 (2.96 h vs 1.8 h).
Meanwhile, the maximum concentration (Cmax) of 9f in plasma after oral
administration was 116 ng/mL (~0.26 ꢁM), which was much higher than compound 3
or compound 4. These results indicated that 9f may serve as a possible lead compound
for the development of antiꢀinfluenza drugs with longer duration of action.
Table 2
Conclusion
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In summary, we designed and synthesized 22 analogs of zanamivir with
modification at the Cꢀ1 position to retain the strong hydrogen bonds with three
arginine residues (Arg118, Arg292 and Arg371). To increase the structural diversity
of the analogs, we selected Cꢀ4ꢀsubstituted Neu5Ac2en with a hydroxy, amino and
guanidinium group. Compound 9f exerts the most potency, with IC50 value of 0.013,
0
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9
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0
0
.001 and 0.09 ꢁM against H3N2, H5N1 and H1N1, which is very similar to that of
compound 3 (H3N2: IC50 0.0014 ꢁM, H5N1: IC50 0.012 ꢁM, H1N1: IC50 0.001 ꢁM).
Moreover, compound 9f was shown to have a prolonged plasma halfꢀlife following
oral dosing in rat. Molecular modeling results revealed information about the binding
models between inhibitors and NAs, illustrating that the elongated amide groups at
Cꢀ1 position were projected towards the 430ꢀloop, which may aid future development
of improved inhibitors against both groupꢀ1 and groupꢀ2 NAs.
Experimental section
General information on synthesis. The reagents (chemicals) were purchased
from Lancaster, Sigma, Acros and Shanghai Chemical Reagent Company, and used
without further purification. Analytical thinꢀlayer chromatography (TLC) was
performed on HSGF 254 (150ꢀ200 ꢂm thickness, Yantai Huiyou Company, China).
Yields were not optimized. Column chromatography was performed with
CombiFlash® Companion system (Teledyne Isco, Inc.). Nuclear magnetic resonance
(NMR) spectra were performed on a Brucker AMXꢀ400 and AMXꢀ300 NMR (IS as
TMS). Chemical shifts were reported in parts per million (ppm, δ) downfield from
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tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad (br). LCꢀMS analysis were conducted
on Agilent 1100 Series HPLC with an Agilent Zorbax Eclipse XDBꢀC18 (4.6 × 50
mm, 5 ꢁm) reversed phase column. Compounds 7a-g, 8a-e and 9a-j were confirmed
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≥
95% purity (Supporting Information, Table S1). The details for purity analyses of
compounds 7a-g, 8a-e and 9a-j are described in the Supporting Information.
Neuraminidase enzyme inhibitory assay. Influenza virus A/Sydney/5/97 (H3N2),
A/Guangdong/376/2001 (H1N1) and recombinant virus containing the HA and NA
genes from A/Indonesia/5/2005 (H5N1) virus in the background of Xꢀ31 ca virus
25,26
were propagated in 11ꢀdayꢀold embryonated chicken eggs as described elsewhere.
Influenza
′ꢀ(4ꢀmethylumbelliferyl)ꢀαꢀ
Co., St. Louis, Mo.) as a substrate, as described previously with a little modification.
neuraminidase
activity
was
determined
by
using
2
D
ꢀNꢀacetylneuraminic acid (MUNANA; Sigma Chemical
19
The NA activity of each virus was determined before it was used in NA inhibition
tests. Briefly, 50 ꢁl of virus was serially diluted in enzyme buffer [sodium acetate
buffer 150 mM (pH 7.0) containing 1mM calcium chloride] and 50 ꢁl of substrate in
enzyme buffer to give a final MUNANA concentration of 100 ꢁM. The reaction
mixtures were incubated at 37 °C for 30 min. The reactions were then stopped by
addition of 100 ꢁl of 0.014 N NaOH in 83% ethanol to each well. The fluorescence of
the released 4ꢀmethylumbelliferone was quantified using
a
fluorescence
spectrophotometer (excitation wavelength, 355 nm; emission wavelength, 460 nm).
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NA inhibition was assayed by determining the drug concentration required to
reduce NA activity to 50% of control NA activity (IC50). Fiveꢀfold dilutions ranging
from 10 ꢁM to 128 nM were made of the appropriate compound, and 25 ꢁL of each
dilution was incubated with 25 ꢁL of virusꢀcontaining allantoic fluid at a standard
amount of NA activity (150 relative fluorescence units). The mixture was incubated at
0
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0
3
7 °C for 1 h, protected from light to allow interaction between the drug and virus.
The enzymatic reaction was initiated by adding 50 ꢁL of substrate in enzyme buffer at
a final concentration of 100 ꢁM. The reaction was stopped after 30 min of incubation
at 37 °C. Standard curves were constructed by plotting the percentage of fluorescence
inhibition relative to the activity of controls against the logs of inhibitor
concentration. The IC50s were obtained by interpolation of the data.
Pharmacokinetic studies. Six male SpragueꢀDawley (SD) rats (~250 g; Shanghai
Laboratory Animals Co., Shanghai, China) were housed in two rat cages (three rats
for one group) and maintained in an airꢀconditioned rat room, which were used to
determine kinetic profile after oral and iv dosing with compound 9f. In the first group,
the tested compound was administered orally at a dose of 10 mg/kg; in the second
group, the tested compound was administered intravenously at a dose of 2 mg/kg.
Serial specimens (0.5 mL) were collected via the retrobulbar vein at various time
points after the compound was given (0, 5, 15, 30 min and 1, 2, 5, 8, 24 h), and
quantification was performed by LC/MS/MS (Agilent 1100 PLC and Thermo Finigan
TSQQuantum). Pharmacokinetic parameters were calculated from the mean plasma
concentration by noncompartmental analysis.
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Molecular docking. Molecular docking was performed using Glide (gridꢀbased
ligand docking with energetics) program in the Schrödinger suite of software
(
Maestro, version 7.5.112). Glide uses the position and size of the coꢀcrystallized
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ligand to determine the binding site. Specifically, the default center of this site was the
centroid of the ligand and the default size was defined similar to the ligand. The
crystal structures of neuraminidase were retrieved from the Protein Data Bank (PDB
entry 3CKZ and 3TIC) for subtype N1 (groupꢀ1) and N2 (groupꢀ2). It remains
controversial whether water molecules should be kept during molecular docking. Two
points need to be noticed: First, it is difficult to determine exactly whether a water
molecule is conserved; second, the orientation of hydrogen bonds formed between
water molecules and different ligands is variable. In consideration of explicit water
molecules, current docking programs usually fix the orientation of the water
molecules and treat them as part of the receptor environment, which are not beneficial
to the assessment of ligandꢀreceptor interaction in many cases. In contrast, Zheng et al.
reported a fair correlation between the predicted binding free energies and the
experimental inhibitory potencies based on the reasonable binding conformations of
126 NA inhibitors obtained from molecular docking with removing all crystal water
43
molecules. Therefore, all waters and unessential ligands in the structures were
deleted for molecular docking in this study. Then the synthesized compounds were
docked to the protein in standardꢀprecision (SP) mode, with up to thirty poses saved
per compound and other default parameters.
Experimental Procedure
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General methods
General procedure for the synthesis of compounds 7a-7g
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9
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9
0
To a solution of acid 13 (0.2 mmol), EDCI (0.3 mmol), Et
3
N (0.3 mmol) in 5 mL dry
CH Cl was added an equimolar amount of the appropriate amide. The mixture was
2
2
kept stirring at room temperature for 24 h. After TLC detection to show no starting
materials, the reaction mixture was concentrated under reduced pressure and purified
by flash column chromatography to obtain intermediates. 200 ꢂL 5% NaOH aqueous
solution was added into a solution of intermediates in 5 mL methanol. After the
reaction mixture was stirred at r.t. overnight, the pH value of solution was adjusted to
5
ꢀ6 with positive ionꢀexchange resin (Dowex 50w x 4ꢀ400). The mixture was filtered,
and the solvent was removed under vacuum. The residue was lyophilized to obtain the
final products 7a-7g.
(2R,3R,4S)-3-acetamido-4-hydroxy-N-(4-methoxybenzyl)-2-((1R,2R)-1,2,3-trihyd
roxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (7a) The product was obtained
1
as white foam after lyophilization. Yield 50%. H NMR (400 MHz, CD
3
OD, ppm) δ
7
.23 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.81 (d, J = 2.4 Hz, 1H), 4.46ꢀ4.37
m, 3H), 4.18 (d, J = 10.8 Hz, 1H), 3.96 (dd, J = 10.7, 8.8 Hz, 1H), 3.83ꢀ3.77 (m, 2H),
.77 (d, J = 2.6 Hz, 3H), 3.68ꢀ3.61 (m, 1H), 3.58 (d, J = 9.4 Hz, 1H), 3.34 (d, J = 4.8
(
3
13
Hz, 1H), 2.04 (s, 3H); C NMR (126 MHz, MeOD) δ 178.62, 162.20, 158.63, 145.82,
1
29.79, 128.18, 113.07, 107.57, 76.30, 69.06, 67.88, 66.06, 62.82, 53.85, 50.19,
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+
+
4
1.63, 22.41. LCꢀMS m/z 433 [M+Na] ; HRMS (ESI) [M+Na] found m/z 433.1569,
26 2 8
calcd for C19H N O Na 433.1587.
(
S)-2-((2R,3R,4S)-3-acetamido-4-hydroxy-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4
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11
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-dihydro-2H-pyran-6-carboxamido)-4-methylpentanoic acid (7b) The product was
1
obtained as white foam after lyophilization. Yield 34%. H NMR (400 MHz, CD
3
OD,
ppm) δ 7.28 (dd, J = 13.1, 7.0 Hz, 1H), 5.84 (d, J = 2.5 Hz, 1H), 4.48ꢀ4.39 (m, 1H),
4
3
1
.35ꢀ4.25 (m, 1H), 4.18 (d, J = 10.9 Hz, 1H), 4.02 (dd, J = 10.8, 8.7 Hz, 1H),
.93ꢀ3.77 (m, 2H), 3.65 (dd, J = 11.8, 5.6 Hz, 1H), 3.61ꢀ3.56 (m, 1H), 2.04 (d, J =
1.3 Hz, 3H), 1.64ꢀ1.46 (m, 1H), 1.27ꢀ1.09 (m, 1H), 0.93 (dd, J = 14.3, 7.1 Hz, 6H);
C NMR (126 MHz, MeOD) δ 178.60, 173.42, 161.02, 145.71, 107.64, 76.83, 69.11,
7.99, 66.27, 63.03, 58.94, 50.32, 37.63, 24.20, 22.37, 20.90, 20.73. LCꢀMS m/z 403
1
3
6
ꢀ
+
[
MꢀH] ; HRMS (ESI) [M+2NaꢀH] found m/z 449.1492, calcd for C17
27 2 9 2
H N O Na
4
49.1512.
(2S,3R)-2-((2R,3R,4S)-3-acetamido-4-hydroxy-2-((1R,2R)-1,2,3-trihydroxypropyl
)-3,4-dihydro-2H-pyran-6-carboxamido)-3-methylpentanoic acid (7c) The product
1
was obtained as white foam after lyophilization. Yield 51%. H NMR (400 MHz,
CD
1H), 4.01 (dd, J = 10.8, 8.7 Hz, 1H), 3.93ꢀ3.79 (m, 2H), 3.66 (dd, J = 11.3, 5.2 Hz,
H), 3.61ꢀ3.52 (m, 1H), 2.05 (s, 3H), 1.72ꢀ1.62 (m, 2H), 1.64ꢀ1.48 (m, 1H), 0.95 (dd,
3
OD, ppm) δ 5.83 (d, J = 2.5 Hz, 1H), 4.41 (dt, J = 6.4, 3.5 Hz, 2H), 4.26ꢀ4.12 (m,
1
13
J = 7.5, 6.2 Hz, 6H); C NMR (126 MHz, MeOD) δ 178.53, 173.36, 161.01, 145.74,
1
07.53, 76.73, 69.00, 67.93, 66.29, 63.00, 52.81, 50.28, 41.82, 24.32, 22.32, 21.91,
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ꢀ
+
2
0.53. LCꢀMS m/z 403 [MꢀH] ; HRMS (ESI) [M+Na] found m/z 427.1709, calcd for
17 28 2 9
C H N O
Na 427.1693.
(
S)-2-((2R,3R,4S)-3-acetamido-4-hydroxy-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
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4
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8
9
0
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2
3
4
5
6
7
8
9
0
-dihydro-2H-pyran-6-carboxamido)-3-methylbutanoic acid (7d) The product was
1
obtained as white foam after lyophilization. Yield 82%. H NMR (400 MHz, CD
3
OD,
ppm) δ 7.38ꢀ7.16 (m, 1H), 5.85 (d, J = 2.5 Hz, 1H), 4.41 (dd, J = 8.6, 2.5 Hz, 1H),
.29ꢀ4.23 (m, 1H), 4.18 (d, J = 10.9 Hz, 1H), 4.03 (dd, J = 10.8, 8.7 Hz, 1H), 3.86
ddd, J = 16.3, 8.9, 2.7 Hz, 2H), 3.68ꢀ3.57 (m, 2H), 2.26ꢀ2.15 (m, 1H), 2.05 (s, 3H),
4
(
13
0
1
2
.95 (dd, J = 13.4, 6.9 Hz, 6H); C NMR (126 MHz, MeOD) δ 178.53, 173.44,
61.16, 145.73, 107.65, 76.85, 69.09, 68.02, 66.28, 63.08, 59.35, 50.34, 30.98, 22.32,
ꢀ
+
0.88, 20.72. LCꢀMS m/z 389 [MꢀH] ; HRMS (ESI) [M+Na] found m/z 413.1546,
26 2 9
calcd for C16H N O
Na 413.1536.
(
S)-2-((2R,3R,4S)-3-acetamido-4-hydroxy-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4
-dihydro-2H-pyran-6-carboxamido)-2-phenylacetic acid (7e) The product was
1
obtained as yellow foam after lyophilization. Yield 75%. H NMR (400 MHz,
CD
3
OD, ppm) δ 7.45 (dd, J = 8.2, 7.4 Hz, 2H), 7.35ꢀ7.16 (m, 3H), 5.80 (dd, J = 14.3,
2
.5 Hz, 1H), 5.21 (d, J = 16.9 Hz, 1H), 4.48ꢀ4.33 (m, 1H), 4.31ꢀ4.12 (m, 1H),
4.12ꢀ3.98 (m, 1H), 3.97ꢀ3.81 (m, 2H), 3.69 (dd, J = 11.7, 6.6 Hz, 1H), 3.63ꢀ3.54 (m,
ꢀ
1
H), 2.04 (s, 3H); LCꢀMS m/z 423 [MꢀH] .
N-((2R,3R,4S)-4-hydroxy-6-(morpholine-4-carbonyl)-2-((1R,2R)-1,2,3-trihydrox
ypropyl)-3,4-dihydro-2H-pyran-3-yl)acetamide (7f) The product was obtained as
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yellow foam after lyophilization. Yield 38%. H NMR (400 MHz, CD
3
OD, ppm) δ
5
4
2
7
.28 (d, J = 2.3 Hz, 1H), 4.38 (dd, J = 8.7, 2.3 Hz, 1H), 4.18 (d, J = 10.8 Hz, 1H),
.03ꢀ3.96 (m, 1H), 3.82ꢀ3.77 (m, 1H), 3.73ꢀ3.60 (m, 9H), 3.56 (dd, J = 17.4, 8.0 Hz,
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46
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48
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13
H), 2.04 (s, 3H); C NMR (126 MHz, MeOD) δ 173.43, 163.91, 146.96, 106.93,
ꢀ
6.48, 69.20, 68.03, 65.76, 63.05, 50.24, 22.27, 20.81. LCꢀMS m/z 395 [M+Cl] ;
+
HRMS (ESI) [M+Na] found m/z 383.1412, calcd for C15
H
24
N
2
O
8
Na 383.1430.
(
2R,3R,4S)-3-acetamido-N-benzyl-4-hydroxy-2-((1R,2R)-1,2,3-trihydroxypropyl)
-
3,4-dihydro-2H-pyran-6-carboxamide (7g) The product was obtained as white
1
foam after lyophilization. Yield 68%. H NMR (400 MHz, CD OD, ppm) δ 7.39ꢀ7.15
3
(
m, 5H), 5.83 (d, J = 2.3 Hz, 1H), 4.50ꢀ4.37 (m, 3H), 4.20 (d, J = 10.8 Hz, 1H), 3.98
(
dd, J = 10.7, 8.7 Hz, 1H), 3.87ꢀ3.74 (m, 2H), 3.70ꢀ3.53 (m, 2H), 3.34ꢀ3.12 (m, 1H),
13
2
1
4
.03 (d, J = 6.1 Hz, 3H); C NMR (126 MHz, MeOD) δ 173.37, 162.31, 145.83,
37.79, 127.72, 126.79, 126.46, 107.62, 76.37, 69.03, 67.92, 66.10, 62.89, 50.28,
+
+
2.16, 20.85. LCꢀMS m/z 403 [M+Na] ; HRMS (ESI) [M+Na] found m/z 403.1466,
24 2 7
calcd for C18H N O
Na 403.1481.
General procedure for the synthesis of compounds 8a-8e
To a solution of acid 19 (0.2 mmol), EDCI (0.3 mmol), Et
3
N (0.3 mmol) in 5 mL dry
CH Cl was added an equimolar amount of the appropriate amide. The mixture was
2
2
kept stirring at room temperature for 24 h. After TLC detection to show no starting
materials, the reaction mixture was concentrated under reduced pressure and purified
by flash column chromatography to obtain intermediates 20a-e.
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% NaOH aqueous solution (200 ꢂL) was added into a solution of intermediates in
5
mL methanol. After the reaction mixture was stirred at r.t. overnight, the pH value
of solution was adjusted to 5ꢀ6 with positive ionꢀexchange resin
Dowex50WX4ꢀ400). The mixture was filtered, and the solvent was removed under
0
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2
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0
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8
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4
5
6
7
8
9
0
(
vacuum. Then trifluoroacetic acid (50 % in DCM) was added to the porducts above
abtained, and stirred at r.t. overnight. After the reaction was completed, the solvent
was evaporated to give the product 8a-e as a salt of trifluoroacetic acid, which was
+
further purified by a column of Dowex50WX8ꢀ200 (H ) resin (2.0 g).
(
S)-2-((2R,3R,4S)-3-acetamido-4-amino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-d
ihydro-2H-pyran-6-carboxamido)-3-methylbutanoic acid (8a) The product was
1
obtained as wight foam after lyophilization. Yield 80 %. H NMR (400 MHz,
CD
H), 3.71 (dd, J = 23.8, 9.0 Hz, 2H), 2.24 (s, 1H), 2.05 (s, 4H), 1.05ꢀ0.87 (m, 6H); C
NMR (126 MHz, MeOD) δ 173.02, 172.43, 160.89, 148.46, 99.87, 76.31, 69.57,
7.19, 62.73, 57.22, 49.38, 45.84, 30.13, 21.01, 17.52, 16.51. LCꢀMS m/z 340
3
OD, ppm) δ 5.81 (s, 1H), 4.69 (t, J = 11.5 Hz, 2H), 4.56ꢀ4.29 (m, 5H), 4.11 (s,
1
3
3
6
+
+
[
M+H] ; HRMS (ESI) [M+Na] found m/z 412.1683, calcd for C16
27 3 8
H N O Na
4
12.1696.
(2S)-2-((3R,4S)-3-acetamido-4-amino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dih
ydro-2H-pyran-6-carboxamido)-4-methylpentanoic acid (8b) The product was
1
obtained as white foam after lyophilization. Yield 78 %. H NMR (400 MHz,
CD
3
OD, ppm) δ 5.80 (d, J = 2.0 Hz, 1H), 4.69 (d, J = 9.5 Hz, 1H), 4.62ꢀ4.48 (m, 2H),
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.39 (dt, J = 19.6, 10.7 Hz, 2H), 4.23ꢀ4.14 (m, 1H), 4.11 (d, J = 6.9 Hz, 1H), 3.72 (d,
J = 9.8 Hz, 1H), 3.48 (dd, J = 14.1, 7.1 Hz, 1H), 2.05 (s, 3H), 1.71 (d, J = 6.3 Hz,
13
3
H), 1.27 (d, J = 5.5 Hz, 1H), 0.95 (dd, J = 8.9, 5.9 Hz, 6H); C NMR (126 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MeOD) δ 173.66, 172.99, 160.98, 148.58, 99.68, 76.16, 69.32, 67.20, 62.66, 50.24,
4
9.32, 47.67, 47.50, 47.33, 47.16, 46.99, 46.82, 46.65, 45.81, 39.85, 24.17, 21.44,
+
+
2
1.01, 20.07. LCꢀMS m/z 404 [M+H] ; HRMS (ESI) [M+Na] found m/z 426.1869,
29 3 8
calcd for C17H N O
Na 426.1852.
1
-((2R,3R,4S)-3-acetamido-4-amino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihy
dro-2H-pyran-6-carbonyl)piperidine-3-carboxylic acid (8c) The product was
1
obtained as white foam after lyophilization. Yield 50%. H NMR (400 MHz, CD
3
OD,
ppm) δ 5.20 (s, 1H), 4.66 (d, J = 9.8 Hz, 1H), 4.53ꢀ4.22 (m, 3H), 4.18ꢀ3.95 (m, 2H),
3
3
1
7
.95ꢀ3.77 (m, 1H), 3.76ꢀ3.62 (m, 1H), 3.51 (dt, J = 14.0, 10.5 Hz, 1H), 3.30 (s, 3H),
.18 (dd, J = 31.8, 12.1 Hz, 1H), 2.48 (s, 1H), 2.16ꢀ1.95 (m, 4H), 1.77 (s, 2H), 1.54 (s,
13
H); C NMR (126 MHz, MeOD) δ 174.48, 172.95, 162.99, 150.79, 98.11, 76.06,
1.89, 69.25, 67.31, 62.89, 49.24, 45.68, 43.41, 40.25, 26.44, 24.13, 20.99. LCꢀMS
+
+
m/z 424 [M+Na] ; HRMS (ESI) [M+Na] found m/z 424.1684, calcd for
17 27 3 8
C H N O
Na 424.1696.
2-((3R,4S)-3-acetamido-4-amino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro
-2H-pyran-6-carboxamido)acetic acid (8d) The product was obtained as white foam
1
after lyophilization. Yield 81%. H NMR (400 MHz, CD
3
OD, ppm) δ 5.83 (s, 1H),
4
.68 (dd, J = 11.3, 2.0 Hz, 1H), 4.52 (dd, J = 11.3, 5.7 Hz, 1H), 4.46ꢀ4.33 (m, 2H),
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
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4
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.29ꢀ4.20 (m, 1H), 4.09 (dd, J = 13.5, 6.8 Hz, 1H), 4.01 (s, 2H), 3.95ꢀ3.78 (m, 1H),
1
3
.70 (d, J = 9.6 Hz, 1H), 3.30 (dd, J = 3.1, 1.5 Hz, 3H), 2.05 (s, 3H); C NMR (126
MHz, MeOD) δ 172.97, 170.88, 161.36, 148.31, 99.71, 76.16, 69.17, 67.34, 62.77,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
4
9.29, 45.76, 39.94, 21.04. LCꢀMS m/z 348 [M+H] ; HRMS (ESI) [M+Na] found
m/z 370.1222, calcd for C13 Na 370.1226.
21 3 8
H N O
1
-((2R,3R,4S)-3-acetamido-4-amino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihy
dro-2H-pyran-6-carbonyl)piperidine-4-carboxylic acid (8e) The product was
1
obtained as white foam after lyophilization. Yield 78%. H NMR (400 MHz, CD
3
OD,
ppm) δ 5.20 (s, 1H), 4.66 (d, J = 9.4 Hz, 1H), 4.52ꢀ4.45 (m, 1H), 4.38 (dd, J = 21.0,
.3 Hz, 2H), 4.24 (d, J = 14.7 Hz, 1H), 4.15ꢀ3.97 (m, 3H), 3.90ꢀ3.75 (m, 1H), 3.69 (d,
J = 10.7 Hz, 2H), 3.54ꢀ3.43 (m, 1H), 3.27ꢀ3.16 (m, 1H), 2.96 (s, 1H), 2.63 (s, 1H),
6
1
3
2
1
2
.00 (dd, J = 21.0, 8.8 Hz, 6H), 1.63 (s, 2H); C NMR (126 MHz, MeOD) δ 179.63,
73.14, 162.75, 150.64, 98.10, 76.04, 69.29, 67.23, 62.90, 49.21, 45.71, 41.02, 39.81,
+
+
7.05, 21.07. LCꢀMS m/z 402 [M+H] ; HRMS (ESI) [M+Na] found m/z 424.1709,
Na 424.1696.
27 3 8
calcd for C17H N O
General procedure for the synthesis of compounds 9a-j
NaOH aqueous solution (5 %, 200 ꢂL) was added into a solution of intermediates 23
in 5 mL methanol. After the reaction mixture was stirred at r.t. overnight, the pH
value of solution was adjusted to 5ꢀ6 with positive ionꢀexchange resin (Dowex
50WX4ꢀ400). The mixture was filtered, and the solvent was removed under vacuum.
Then trifluoroacetic acid (50 % in DCM) was added to the porducts above abtained,
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and stirred at r.t. overnight. After the reaction was completed, the solvent was
evaporated to give the product 9a-j as a salt of trifluoroacetic acid, which was further
+
purified by a column of Dowex50WX8ꢀ200 (H ) resin (2.0 g).
10
11
12
13
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49
50
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59
60
(S)-2-((2R,3R,4S)-3-acetamido-4-guanidino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3
,
4-dihydro-2H-pyran-6-carboxamido)pentanedioic acid (9a) The product was
1
obtained as light yellow foam after lyophilization. Yield 92 %. H NMR (300 MHz,
2
D O, ppm) δ 7.95 (m, 1H), 5.85 (s, 1H), 4.57 (m, 1H), 4.40ꢀ4.13 (m, 3H), 4.08ꢀ3.85
13
(m, 2H), 3.81 – 3.64 (m, 1H), 2.38ꢀ2.21 (m, 2H), 2.11 (m, 2H), 1.96 (s, 3H);
C
NMR (126 MHz, D O) δ 181.04, 173.97, 161.96, 156.54, 146.02, 103.90, 75.80,
2
6
9.33, 67.41, 62.57, 54.89, 50.23, 47.25, 33.52, 27.58, 22.80, 21.48. LCꢀMS m/z 462
+
+
[
M+H] ; HRMS (ESI) [M+Na] found m/z 484.1673, calcd for C17
27 5
H N O10Na
4
84.1656.
(S)-2-((2R,3R,4S)-3-acetamido-4-guanidino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3
,4-dihydro-2H-pyran-6-carboxamido)-4-methylpentanoic acid (9b) The product
1
was obtained as light yellow foam after lyophilization. Yield 80 %. H NMR (400
2
MHz, D O, ppm) δ 5.85 (s, 1H), 4.55 (d, J = 9.9 Hz, 2H), 4.34ꢀ4.31 (m, 2H),
4
.03ꢀ3.90 (m, 2H), 3.80 (d, J = 9.3 Hz, 1H), 3.72ꢀ3.67 (m, 1H), 2.08 (s, 3H),
13
1.69ꢀ1.58 (m, 3H), 1.05 – 0.84 (s, 6H); C NMR (126 MHz, D
2
O) δ 178.84, 173.97,
1
61.85, 156.53, 146.18, 104.39, 75.99, 69.38, 67.11, 62.45, 53.37, 50.33, 47.25,
+
40.39, 39.89, 24.12, 21.90, 21.43, 20.50. LCꢀMS m/z 446 [M+H] ; HRMS (ESI)
+
[
M+Na] found m/z 468.2052, calcd for C18
H
31
5
N O
8
Na 468.2070.
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(
S)-2-((2R,3R,4S)-3-acetamido-4-guanidino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3
,
4-dihydro-2H-pyran-6-carboxamido)-3-methylbutanoic acid (9c) The product
1
was obtained as light yellow foam after lyophilization. Yield 95 %. H NMR (400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, D O, ppm) δ 5.87 (s, 1H), 4.56 (d, J = 10.2 Hz, 2H), 4.35 (t, J = 9.9 Hz, 1H),
2
4
.20 (d, J = 5.2 Hz, 1H), 3.96 (dd, J = 15.4, 8.3 Hz, 2H), 3.81 (d, J = 9.3 Hz, 1H),
.71 (dd, J = 10.7, 4.6 Hz, 1H), 2.26ꢀ2.17 (m, 1H), 2.10 (d, J = 10.6 Hz, 3H), 0.97
3
13
(dd, J = 14.2, 6.9 Hz, 6H); C NMR (126 MHz, D
2
O) δ 177.39, 173.97, 161.86,
1
1
56.53, 146.08, 104.47, 76.10, 69.52, 67.05, 62.45, 59.93, 50.38, 47.27, 30.26, 21.43,
+
+
8.29, 16.62. LCꢀMS m/z 432 [M+Na] ; HRMS (ESI) [M+Na] found m/z 454.1900,
2 5 8
calcd for C17H N O
Na 454.1914.
(S)-2-((2R,3R,4S)-3-acetamido-4-guanidino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3
,
4-dihydro-2H-pyran-6-carboxamido)propanoic acid (9d) The product was
1
obtained as light yellow foam after lyophilization. Yield 89 %. HꢀNMR (400 MHz,
D O, ppm): δ 5.87 (d, J = 2.0 Hz, 1H), 4.57 (d, 2H), 4.37ꢀ4.29 (m, 2H), 4.02ꢀ3.96 (m,
2
2
H), 3.81 (d, J = 9.6 Hz, 1H), 3.75 (q, J = 6.8 Hz, 1H), 2.78 (s, 1H), 2.11 (s, 3H), 1.45
13
(d, J = 6.8 Hz, 3H); C NMR (126 MHz, D
2
O) δ 181.04, 179.22, 174.08, 161.62,
1
56.58, 104.14, 75.87, 69.44, 67.25, 62.54, 50.37, 50.26, 47.27, 22.86, 21.49, 16.97.
+
+
LCꢀMS m/z 404[M+H] ; HRMS (ESI) [M+Na] found m/z 426.1613, calcd for
C H N O Na 426.1601.
15 25
5
8
3
-((2R,3R,4S)-3-acetamido-4-guanidino-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-d
ihydro-2H-pyran-6-carboxamido)propanoic acid (9e) The product was obtained as
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light yellow foam after lyophilization. Yield 85 %. H NMR (300 MHz, CD
3
OD,
ppm) δ 7.99 (d, J = 10.7 Hz, 1H), 5.72 (s, 1H), 4.57ꢀ4.35 (m, 2H), 4.20 (t, J = 9.1 Hz,
H), 4.06ꢀ3.91 (m, 1H), 3.75 (dd, J = 41.2, 9.2 Hz, 3H), 3.53 (d, J = 4.7 Hz, 2H), 2.55
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
(
d, J = 6.3 Hz, 2H), 2.01 (s, 3H); C NMR (126 MHz, MeOD) δ 173.56, 172.66,
1
3
61.55, 157.05, 146.72, 102.96, 76.45, 69.33, 67.81, 62.78, 49.60, 35.15, 34.41,
ꢀ
+
2.46, 20.80. LCꢀMS m/z 402 [MꢀH] ; HRMS (ESI) [M+H] found m/z 404.1797,
404.1781.
26 5 8
calcd for C15H N O
(
2R,3R,4S)-3-acetamido-N-(3-fluorobenzyl)-4-guanidino-2-((1R,2R)-1,2,3-trihydr
oxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (9f) The product was obtained
1
as light yellow foam after lyophilization. Yield 85 %. HꢀNMR (400 MHz, D
2
O, ppm)
δ 7.33ꢀ7.30 (m, 1H), 7.13ꢀ6.97 (m, 3H), 5.77ꢀ5.74 (m, 1H), 4.51ꢀ4.42 (m, 4H),
1
3
4
1
1
6
.24ꢀ4.08 (m, 2H), 3.82ꢀ3.68 (m, 2H), 2.01 (s, 3H); C NMR (126 MHz, MeOD) δ
62.89 (J = 248 Hz), 162.23, 161.92, 157.37, 147.27, 141.09, 129.83 (J = 7 Hz),
22.90 (J = 8 Hz), 113.77 (J = 22 Hz), 113.55 (J = 21 Hz), 103.56, 76.82, 70.65,
+
9.70, 68.18, 63.07, 49.96, 42.05, 21.34. LCꢀMS m/z 440[M+H] ; HRMS (ESI)
+
[
M+H] found m/z 440.1934, calcd for C19
H
27
5
N O
6
F 440.1945.
(
2R,3R,4S)-3-acetamido-N-(2-fluorobenzyl)-4-guanidino-2-((1R,2R)-1,2,3-trihydr
oxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (9g) The product was obtained
1
as light yellow foam after lyophilization. Yield 92 %. HꢀNMR (400 MHz, D O, ppm)
2
δ 7.35ꢀ7.28 (m, 2H), 7.15ꢀ7.08 (m, 2H), 5.73 (d, J = 2.1 Hz, 1H), 4.53 (s, 2H),
4
.46ꢀ4.41 (m, 2H), 4.24ꢀ4.18 (m, 1H), 3.83ꢀ3.78 (m, 2H), 3.71ꢀ3.67 (m, 2H), 2.01 (s,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
3
1
7
2
H); C NMR (126 MHz, D O) δ 174.36, 162.24 (J = 217.98 Hz), 159.42, 156.87,
46.64, 129.47 (J = 8 Hz), 129.30 (J = 8 Hz), 123.96, 115.22 (J = 22 Hz), 104.59,
+
6.19, 69.60, 67.45, 62.72, 50.52, 47.58, 37.16, 21.76. LCꢀMS m/z 440[M+H] ;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
HRMS (ESI) [M+H] found m/z 440.1956, calcd for C H N O F 440.1945.
1
9
27
5
6
(
2R,3R,4S)-3-acetamido-4-guanidino-N-(4-methylbenzyl)-2-((1R,2R)-1,2,3-trihyd
roxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (9h) The product was obtained
1
as light yellow foam after lyophilization. Yield 85 %. HꢀNMR (400 MHz, D
2
O, ppm)
δ 8.34 (s, 1H), 7.18 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 5.74 (d, J = 2.4 Hz,
1
3
1
H), 4.40ꢀ4.64 (m, 5H), 4.08ꢀ4.24 (m, 3H), 3.65ꢀ3.69 (m, 1H), 2.30 (s, 3H), 2.01 (s,
1
3
H); C NMR (126 MHz, D
2
O) δ 174.36, 163.00, 156.85, 146.72, 137.56, 134.34,
29.20, 127.19, 104.50, 76.19, 69.59, 67.47, 62.73, 50.51, 47.59, 42.54, 21.76, 19.96.
+
+
LCꢀMS m/z 436 [M+H] ; HRMS (ESI) [M+H] found m/z 436.2213, calcd for
20 30 5 6
C H N O
436.2196.
(2R,3R,4S)-3-acetamido-4-guanidino-N-(4-fluorobenzyl)-2-((1R,2R)-1,2,3-trihydr
oxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (9i) The product was obtained
1
as light yellow foam after lyophilization. Yield 81 %. HꢀNMR (400 MHz, D
2
O, ppm)
δ 7.30ꢀ7.35 (m, 2H), 7.01ꢀ7.07 (m, 2H), 5.73 (d, 2.1Hz, 1H), 4.40ꢀ4.44 (m, 4H),
13
4.21ꢀ4.24 (m, 1H), 3.79ꢀ3.82 (m, 2H), 3.66ꢀ3.70 (m, 2H), 2.01 (s, 3H); C NMR (125
MHz, MeOD) δ 173.09, 162.54 (J = 114.6 Hz), 161.05, 157.39, 147.28, 134.17,
129.16, 129.09, 114.74, 114.57, 103.45, 76.78, 69.65, 68.14, 63.06, 49.91, 47.93,
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+
+
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1.81, 21.12. LCꢀMS m/z 440 [M+H] ; HRMS (ESI) [M+H] found m/z 440.1946,
27 5 6
calcd for C19H N O F 440.1945.
(
2R,3R,4S)-3-acetamido-4-guanidino-N-(3-chlorobenzyl)-2-((1R,2R)-1,2,3-trihyd
10
11
12
13
14
15
16
17
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60
roxypropyl)-3,4-dihydro-2H-pyran-6-carboxamide (9j) The product was obtained
1
as light yellow foam after lyophilization. Yield 83 %. HꢀNMR (400 MHz, D
2
O, ppm)
δ 7.22ꢀ7.32 (m, 4H), 5.74 (s, 1H), 4.42ꢀ4.46 (m, 4H), 4.19ꢀ4.25 (m, 1H), 3.78ꢀ3.85
13
(
m, 2H), 3.66ꢀ3.71 (m, 2H), 2.02 (s, 3H); C NMR (100 MHz, MeOD) δ 173.08,
1
7
62.30, 157.47, 147.26, 140.62, 133.93, 129.66, 127.21, 126.96, 125.63, 103.76,
+
6.89, 69.69, 68.18, 63.10, 49.95, 48.21, 42.07, 21.23. LCꢀMS m/z 456 [M+H] ;
+
HRMS (ESI) [M+H] found m/z 456.1646, calcd for C19
H
27
N
5
O
6
Cl 456.1650.
(2S,4S,5R,6R)-benzyl 5-acetamido-2,4-dihydroxy-6-((1R,2R)-1,2,3-trihydroxypro
pyl)tetrahydro-2H-pyran-1-carboxylate (10) stirred suspension of
A
Nꢀacetylneuraminic acid (10 g, 32.4 mmol) in 10 mL of water was treated with
Cs CO (5 g), and the pH value of solution was adjusted to 7ꢀ8. Then the reaction
2
3
mixture was concentrated to be a glassy solid under reduced pressure. The glassy
solid salt was dissolved in DMF (30 mL) and BnBr (6 mL) was added dropwise.
After being stirred for 24 h, the resulting mixture was filtered. Compound 10
crystallized when the filtrate was poured into DCM (1000 mL) with agitate vigorously
1
(
10.8 g). HꢀNMR (400 MHz, D O, ppm): δ 7.31ꢀ7.41 (m,5H), 5.22 (dd, J = 22.4,
2
12.8 Hz, 2H), 4.04 (m, 1H), 3.99 (dd, J = 10.4, 1.6 Hz, 1H), 3.80 (d, J = 2.8 Hz, 1H),
3
.77 (m, 1H), 3.70 (m, 1H), 3.62 (dd, J = 10.8, 5.2 Hz,1H), 3.48 (dd, J = 9.2, 1.2
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Hz,1H), 2.22 (dd, J = 12.8, 5.2 Hz, 1H), 1.91 (dd, J =12.8, 11.2Hz, 1H); LCꢀMS m/z
+
3
98 [M+H] .
(1S,2R)-1-((2R,3R,4S,6R)-3-acetamido-4,6-diacetoxy-6-(benzyloxycarbonyl)tetra
0
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0
hydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (11) suspension of
A
compound 10 (10.8 g,) in 50 mL of pyridine was treated with catalytic amount of
DMAP (350 mg) in ice bath, and then 26 mL of acetic anhydride was added dropwise.
The resulting mixture was stirred for 24 h at room temperature. After most of solvent
was evaporated under reduced pressure, 300 mL of ethyl acetate was added, and the
resulting mixture was washed sequentially by 2 M HCl (100 mL × 3), saturated
aqueous sodium bicarbonate (100 mL × 2), and saturated brine (100 mL). The organic
layer was dried by MgSO
4
, and filtered, the solvent was evaporated to give
buffꢀcolored syrup, which was further purified by flash column chromatography
(ethyl acetate) to yield the expected product 11 (7.4 g). Yield of the above two steps:
1
3
5
3
2
7.6 %. HꢀNMR (300 MHz, CDCl , ppm): δ 7.34 (s, 5H), 5.42ꢀ5.36 (m, 2H),
3
.25ꢀ5.13 (m, 3H), 5.09ꢀ5.07 (m, 1H), 4.44 (dd, J = 12.3, 2.7 Hz, 1H), 4.17ꢀ4.06 (m,
H), 2.54 (dd, J = 13.5, 4.8 Hz, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 2.04ꢀ2.03 (m, 1H),
+
.01 (s, 9H), 1.88 (s, 3H), 1.82 (br, 1H); LCꢀMS m/z 610 [M+H] 。
(1S,2R)-1-((2R,3R,4S)-3-acetamido-4-acetoxy-6-(benzyloxycarbonyl)-3,4-dihydro
2H-pyran-2-yl)propane-1,2,3-triyl triacetate (12) Compound 11 (7 g) was
-
dissolved in CH
protection of N
3
CN (30 mL) and 5 mL of TMSOTf was added dropwise under the
o
2
at 0 C, and continued to stir for 2.5 h. The reaction mixture was
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allowed to pour into a vigorously stirred mixture of iceꢀcold saturated aqueous sodium
bicarbonate (500 mL, including a lot of solid sodium bicarbonate). After 10 min, the
solution was filtered and the aqueous phase was extracted with ethyl acetate (100 mL
10
11
12
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60
×
2). The combined organic layer was concentrated, and the resulting residue was
further purified by flash column chromatography (ethyl acetate) to yield the expected
1
product 12 (3.8 g, yield 60 %). HꢀNMR(400 MHz, CD
3
OD, ppm): δ 7.35ꢀ7.12 (m,
5
H), 6.0 (d, J = 6.9 Hz, 1H), 5.49ꢀ5.43 (m, 1H), 5.35ꢀ5.32 (m, 1H), 5.20 (s, 2H), 4.55
(dd, J = 3.3, 2.0 Hz, 1H), 4.38ꢀ4.32 (m, 2H), 4.15ꢀ4.02 (m, 2H), 2.06(s, 3H), 2.04 (s,
+
3
H), 2.01 (s, 3H), 2.00 (s, 3H),1.90 (s, 3H); LCꢀMS m/z 550 [M+H] .
(
2R,3R,4S)-3-acetamido-4-acetoxy-2-((1S,2R)-1,2,3-triacetoxypropyl)-3,4-dihydr
o-2H-pyran-6-carboxylic acid (13) Compound 12 (3 g) was dissolved in ethanol (20
mL), and 10%Pd/C (300 mg) was added to the reaction mixture. A slow stream of H
gas was added to the system. After about 10 min, the H gas was stopped when all
2
2
compound 12 was consumed as evidenced by TLC. Then the reaction mixture was
filtered, and concentrated to obtain the compound 13 under reduced pressure (2.4 g,
yield 95 %).
(1S,2R)-1-((3aR,4R,7aR)-6-(benzyloxycarbonyl)-2-methyl-4,7a-dihydro-3aH-pyr
ano[3,4-d]oxazol-4-yl)propane-1,2,3-triyl triacetate (14) After compound 11 (7.4
g) was dissolved in ethyl acetate (40 mL) and 6.4 mL of TMSOTf was added
2
dropwise under the protection of N at room temperature. Then the temperature was
o
raised to 52 C and continued to stir for 2.5 h. The reaction mixture was allowed to
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