Total Synthesis of Emmyguyacins A and B, Potential Fusion Inhibitors of Influenza Virus

Article abstract of DOI:10.1021/acs.orglett.8b03073

Fungal glycolipids emmyguyacins A and B inhibit the pH-dependent conformational change of hemaglutinin A during replication of the Influenza virus. Herein, we report the first total synthesis and structure confirmation of emmyguyacins A and B. Our efficient route, which involves regioselective functionalization of trehalose, allows rapid access to adequate amounts of chemically pure emmyguyacin analogues including the desoxylate derivatives for SAR studies.

Full text of DOI:10.1021/acs.orglett.8b03073

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis of Emmyguyacins A and B, Potential Fusion
Inhibitors of Influenza Virus
Santanu Jana, Vikram A. Sarpe, and Suvarn S. Kulkarni*
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India
S
* Supporting Information
ABSTRACT: Fungal glycolipids emmyguyacins A and B inhibit the pH-dependent
conformational change of hemaglutinin A during replication of the Influenza virus.
Herein, we report the first total synthesis and structure confirmation of
emmyguyacins A and B. Our efficient route, which involves regioselective
functionalization of trehalose, allows rapid access to adequate amounts of chemically
pure emmyguyacin analogues including the desoxylate derivatives for SAR studies.
lthough a hundred years have passed since the first major
outbreak of pandemic influenza, known as “Spanish Flu”,
A
which caused tens of millions of deaths across the globe in
1918, influenza still continues to threaten human life.1 Every
few decades, a new strain of potentially deadly influenza virus
appears and spreads all over the world. The 1957 outbreak
which started in China and spread globally took one million
lives around the world. In 1968, another outbreak caused 1 to
3 million deaths. In 2003, A(H5N1) referred to as Avian
Influenza emerged, which could pass from animals to humans,
and in 2009 the world witnessed the “Swine flu” A(H1N1)
pandemic, which started in Mexico and spread to over 214
countries. To combat the unpredictable outbreak of flu, which
involves airborne transmission and viral mutations, researchers
are always looking for novel and effective therapeutics and
vaccines against the flu.2
In 2002, Christie Boros and her group isolated two
structurally related, trehalose based novel glycolipids, emmy-
guyacin A (1) and emmyguyacin B (2) in 3:1 proportion
(Figure 1), from a potato dextrose agar fermentation of a
sterile fungus species OSI 55538.3 Structurally, these two
molecules are a pair of C4′-epimers wherein compound 1 has a
gluco-galacto trehalose core and 2 is a gluco−gluco
diastereomer. Both glycolipids contain a novel acid chain at
the 3-position, 17-oxalyloxydocosanoic acid, which was also
isolated for the first time. Despite all the attempts by Boros and
co-workers, the stereochemistry of the hydroxyl group of the
fatty acid at C17 could not be determined, perhaps due to its
distant position from the point of attachment to trehalose.
However, they could confirm that the natural products
contained both R and S enantiomers of 17-oxalyloxydocosa-
noic acid in undefined proportions. A mixture of compound 1
and 2 in a ratio of 95:5 was used for the biological studies and
was shown to inhibit replication of influenza A virus (A/X31)
in MDCK cells by inhibiting the pH dependent conformational
change of the viral glycoprotein hemaglutinin A (HA), at a
Figure 1. Structures of emmyguyacin A and emmyguyacin B.
concentrations of 9 μM. Further studies revealed that the
compounds show similar biological activity in the absence of
the oxalate ester.3 Identification of compounds that would
inhibit the conformational change of HA at low pH is a viable
approach for the development of antiviral drugs.4 In this
regard, emmyguyacins can be looked upon as important leads.
Emmyguyacin B was found to be inseparable from
emmyguyacin A even upon HPLC column chromatography.
So, it remains unclear as to whether the major component is
responsible for the inhibition of influenza or the minor
component. In order to confirm the origin of biological activity
of the mixture, and to make these compounds available in pure
form and ample amounts for SAR studies, it was necessary to
Received: September 27, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03073
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Scheme 1. Synthesis of the Side Chain of Emmyguyacins
develop a short and efficient synthetic strategy for the synthesis
of these compounds. It was envisaged that a total synthesis
using the optically pure R and S enantiomers of 17-
oxalyloxydocosanoic acid would provide access to optically
pure materials. Accordingly, we set out to synthesize R and S
fatty acid containing emmyguyacins A and B as well as their
corresponding four desoxylate derivatives 3a, 3b and 4a, 4b.
For the synthesis of glycolipids 1−4 (Figure 1), a short and
efficient synthesis of both R and S isomers of 17-
hydroxydocosanoic acid derivatives was necessary. Our syn-
thesis of the protected unsaturated (C22:1) fatty acids is
shown in Scheme 1. Both the chiral acids were synthesized
from commercially available chiral synthon (S)-epichlorohy-
drin 8 via the successive Grignard addition−epoxidation−
Grignard addition sequence followed by a Julia−Kocienski
olefination. The key sulfone fragment 75 required for
olefination was prepared from 11-bromoundecanoic acid in
three steps (Scheme 1a). First, methyl ester formation from
acid 5 using acetyl chloride6 and methanol gave compound 6
in 95% yield and was followed by displacement of the bromide
in 6 by 1-phenyl-1H-tetrazole-5-thiol (PTSH) employing
K2CO3 as a base (97%). Subsequent oxidation of the
intermediate thioether with meta-chloroperoxybenzoic acid
(92%) afforded sulfone 7.
this was followed by TBS deprotection using TBAF and
oxidation of the free primary hydroxyl group using PCC to
obtain aldehyde 12a in 70% yield over three steps. Next,
sulfone 7 was coupled with aldehyde 12a, employing
KHMDS9 as a base at −60 °C to afford 13a in 71% yield.
Methyl ester hydrolysis by LiOH produced the desired side
chain R acid derivative 14a (82%).
The enantiomeric S-acid 14b was also synthesized from 8 by
simply changing the sequence of addition of the Grignard
reagent (Scheme 1c). First, Grignard reaction with n-
butylmagnesium chloride in the presence of cuprous cyanide
at −78 °C furnished compound 1510 in 95% yield. Next,
epoxide formation to arrive at 1611 using sodium hydroxide
followed by a second Grignard reaction with (4-((tert-
butyldimethylsilyl)oxy)butyl)magnesium bromide gave the S-
alcohol 11b in 86% yield over two steps. After obtaining the S
alcohol 11b, using the same procedure employed in Scheme
1b, the S acid 14b was synthesized through the intermediacy of
aldehyde 12b (77%, 3 steps) and olefin 13b (75%).
Keeping in mind the observed equal activity of emmyguya-
cins A and B in the absence of oxalate ester,3 we proceeded to
synthesize emmyguyacins A, B and their desoxylate derivatives
3 and 4 (Figure 1). Scheme 2 outlines our strategy for the
synthesis of a suitably protected gluco-galacto trehalose core
for the synthesis of emmyguyacins A 1 and emmyguyacin A
desoxylate derivatives 3. Following the literature reported
procedure, we prepared 2,3,4,6-tetra-O-benzoyl-α-D-glucopyr-
anosyl-(1 → 1′)-2′,3′,6′-tri-O-benzoyl-α-D-galactopyranoside
18 in three steps starting from trehalose 17.12 First, hepta-
benzoylation of trehalose 17 by controlling the temperature
and stoichiometry of reagent gave 2,3,4,6- tetra-O-benzoyl-α-D-
glucopyranosyl-(1 → 1)-2′,3′,6′- tri-O-benzoyl-α-D-glucopyr-
Scheme 1b delineates the synthesis of R acid 14a. Grignard
reaction of (S)-epichlorohydrin 8 with (4-((tert-butyldimethyl-
silyl)oxy)butyl)magnesium bromide7 in the presence of
cuprous cyanide at −78 °C furnished compound 9 in 87%
yield.8 Epoxidation using sodium hydroxide followed by a
second Grignard reaction with n-butylmagnesium chloride
furnished the R-alcohol 11a in 95% yield over two steps. The
free hydroxyl group in 11a was protected as a benzyl ether, and
B
DOI: 10.1021/acs.orglett.8b03073
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Scheme 2. Synthesis of Emmyguyacin A (1) and Derivative 3
anoside (62%). Earlier, Richardson and co-workers have
reported a hepta-pivaloylation of trehalose under similar
conditions and yield.13 Next, the free C4-hydroxyl group of
the corresponding hepta-benzoate was converted into triflate
using triflic anhydride in pyridine and subsequently displaced
pTSA afforded the 4,6,3′,4′-di-O-isopropylidene derivative 19
(72%, 2 steps). The primary hydroxyl group of 19 was
protected as a TBS ether to obtain triol 20 (93%). Next, the
C2 and C2′ hydroxyl groups of triol 20 were to be selectively
masked with a suitable orthogonal protecting group which
could be removed in the presence of C3 ester at a later stage of
synthesis. For this purpose, we opted to use levulinic esters
which can be selectively cleaved by using hydrazine hydrate.16
By controlling the equivalents of levulinic acid, we could obtain
14
with NaNO2 in an SN2 manner15 to obtain compound 18
with inversion of stereochemistry at the C-4 position in 70%
yield over 2 steps. Debenzoylation of 18 followed by
isopropylidene protection using 2,2-dimethoxypropane and
C
DOI: 10.1021/acs.orglett.8b03073
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Scheme 3. Synthesis of Emmyguyacin B (2) and Derivative 4
the 2,2′-di-O-acylated compound 21 in 73% yield. The
remaining C3-OH group was acylated with the acyl chain
14a to obtain compound 22 in 82% yield. Simultaneous
removal of the benzyl protecting group and double bond
reduction in 22 under palladium catalyzed hydrogenation
conditions gave compound 23, which is the key intermediate
for the synthesis of emmyguyacin A 1a and emmyguyacin A
derivative 3a. Selective removal of both the levulinic ester
protecting groups in 23 using hydrazine hydrate (95%)
followed by hydrolysis of the intermediate under acidic
conditions afforded the C17 R-acid containing the desoxylate
derivative of emmyguyacin 3a in 97% yield. Next, for the
synthesis of emmyguyacin A, compound 23 was acylated with
oxalic acid monobenzyl ester 24, which could be synthesized
from oxalyl chloride.17 Finally, removal of both the levulinic
groups using hydrazine acetate (77%), followed by aqueous
TFA hydrolysis (96%), removed the TBS ether as well as the
isopropylidene acetal, and debenzylation of the intermediate
under catalytic hydrogenation conditions (84%) afforded
emmyguyacin A (1a). Scheme 2b delineates the total synthesis
of emmyguyacin A 1b and desoxylate derivative 3b using S
acid 14b, starting from intermediate 21 and following the same
procedure employed in Scheme 1a, via O3 esterification,
hydrogenolysis, oxalate ester formation (only for 1b), and
global deprotection in similar yields. The 1H and 13C NMR of
emmyguyacin A matched perfectly well with the data of
isolated emmyguyacin A reported by Boros and co-workers,
thus confirming its proposed structure (see Supporting
Information).3 Both emmyguyacins 1a and 1b show identical
1H and 13C NMR spectral data.
For the synthesis of emmyguyacin B (2) (Scheme 3) we
started with the known trehalose tricyclohexylidene acetal
D
DOI: 10.1021/acs.orglett.8b03073
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derivative 28,18 which can be conveniently prepared from
trehalose in a single step in 69% yield.19 Regioselective 2-O-
acylation of 2,3-diol 28 with levulinic acid via a DCC-mediated
coupling furnished the monoacylated compound 29 in 90%
yield. The remaining C3-OH was acylated with acid 14a in the
presence of DCC and DMAP to obtain compound 30 in 91%
yield. Palladium catalyzed hydrogenation of compound 30
gave saturated alcohol 31. Deprotection of levulinic ester
group in compound 31 with hydrazine hydrate followed by
treatment of the intermediate with aqueous TFA furnished the
desired desoxylate emmyguacin B derivative (4a) in 90% yields
over two steps. Alternatively, compound 31 could be acylated
with oxalic acid monobenzyl ester 24 to obtain compound 32
in 91% yield. Finally, removal of the levulinic group was carried
out by using hydrazine acetate (82%) followed by aqueous
TFA hydrolysis (95%) and debenzylation of the intermediate
under catalytic hydrogenation conditions to afford emmyguya-
cin B (2a) in 84% yield.
Scheme 3b delineates the total synthesis of emmyguyacin B
2b and derivative 4b using S acid 14b along similar lines
starting from intermediate 29 and following the same
procedure employed in Scheme 2a, via O3 esterification,
hydrogenolysis, oxalate ester formation (only for 2b), and
global deprotection in equally good yields.
In conclusion, we have accomplished the first total synthesis
of emmyguyacins A and B containing R and S forms of 17-
oxalyloxydocosanoic acid in an efficient manner in overall
yields of 2.7% (1a), 3.7% (1b), 12.2% (2a), and 13.1% (2b).
The synthetic route involves a total of 24 steps for
emmyguyacin A and 18 steps for emmyguyacin B with a
longest linear sequence of 15 steps each. En route we could
also synthesize the corresponding four desoxylate derivatives of
the emmyguyacins. The synthesized glycolipids which are
available in pure enantiomeric forms for the first time can now
be tested for influenza virus inhibition studies.
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1H and 13C NMR spectra for all compounds and 1H−1H
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: suvarn@chem.iitb.ac.in.
ORCID
Santanu Jana: 0000-0001-7665-5386
Suvarn S. Kulkarni: 0000-0003-2884-876X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank SERB, DST (Grant No. EMR/2014/000235), and
University Grants Commission (UGC-ISF Project F. No. 6-2/
2016(IC)) for financial support. S.J. and V.A.S. thank CSIR−
New Delhi for fellowships.
E
DOI: 10.1021/acs.orglett.8b03073
Org. Lett. XXXX, XXX, XXX−XXX