Synthesis and biological evaluation of flexible and conformationally constrained LpxC inhibitors

Article abstract of DOI:10.1039/c3ob41082j

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent promising candidates for the development of antibiotics possessing a so far unexploited mechanism of action. In a chiral pool synthesis, starting from the d-mannose derived mannonolactone 4, conformationally constrained C-glycosidic as well as open chained hydroxamic acids with a defined stereochemistry were prepared. Diversity was introduced by performing C-C coupling reactions like the Sonogashira and Suzuki cross-coupling reactions. The biological evaluation of the synthesized compounds revealed that in the case of the C-glycosides a long, linear and rigid hydrophobic side chain is required for antibiotic activity against E. coli. The open chain derivatives show higher biological activity than the conformationally constrained C-glycosides. The morpholinomethyl substituted open chain derivative 43, being the most potent compound presented in this paper, inhibits LpxC with a K _i value of 0.35 μM and represents a promising lead structure. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob41082j

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Synthesis and biological evaluation of flexible and
conformationally constrained LpxC inhibitors†
Cite this: Org. Biomol. Chem., 2013, 11,
6056
Marius Löppenberg,‡ Hannes Müller,‡ Carla Pulina, Alberto Oddo, Mark Teese,
Joachim Jose and Ralph Holl*
Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent
promising candidates for the development of antibiotics possessing a so far unexploited mechanism of
action. In a chiral pool synthesis, starting from the D-mannose derived mannonolactone 4, conformation-
ally constrained C-glycosidic as well as open chained hydroxamic acids with a defined stereochemistry
were prepared. Diversity was introduced by performing C–C coupling reactions like the Sonogashira and
Suzuki cross-coupling reactions. The biological evaluation of the synthesized compounds revealed that in
the case of the C-glycosides a long, linear and rigid hydrophobic side chain is required for antibiotic
activity against E. coli. The open chain derivatives show higher biological activity than the conformation-
ally constrained C-glycosides. The morpholinomethyl substituted open chain derivative 43, being the
most potent compound presented in this paper, inhibits LpxC with a Ki value of 0.35 μM and represents a
promising lead structure.
Received 24th May 2013,
Accepted 18th July 2013
DOI: 10.1039/c3ob41082j
www.rsc.org/obc
Introduction
After most of the main classes of antibiotics used today had
been introduced to the clinic until the 1960s, for several
decades the known antibiotic scaffolds were varied in order to
find new antibiotics.1,2 However, since the 1960s, new struc-
tural classes of antibiotics were only rarely introduced into the
market. As many pharmaceutical companies have abandoned
antibiotics research, the antibacterial development pipeline is
almost empty.3 This development contrasts the growing
medical need for novel antibiotics due to the increasing
number of multidrug resistant pathogens.4 Therefore, there is
a great need for new approaches towards antibacterial anti-
biotic therapy, especially against Gram-negative bacteria, as
pathogens like E. coli and P. aeruginosa have become increas-
ingly difficult to treat with the available antibiotics.5
Gram-negative bacteria rely on lipopolysaccharide (LPS),
which serves as a diffusion barrier for many antibiotics. The
biosynthesis of LPS, which forms the outer leaflet of the outer
membrane of Gram-negative bacteria, represents an attractive
target for the development of antibiotics with a novel mechan-
ism of action.6,7 The deacetylase LpxC is an essential enzyme
Fig. 1 LpxC catalyzed deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-
N-acetylglucosamine (1).
in the biosynthesis of lipid A, which is the membrane anchor
of lipopolysaccharide. LpxC is a Zn2+-dependent enzyme,
which catalyzes the irreversible deacetylation of N-acetylgluco-
samine 1 (Fig. 1) and is conserved in virtually all Gram-nega-
tive bacteria.8,9 Since the inhibition of lipid A biosynthesis is
lethal to Gram-negative bacteria, inhibitors of LpxC represent
promising new antibiotics.
A few LpxC inhibitors have been reported so far.10–13 One of
them is the potent inhibitor CHIR-090 (Fig. 2). The N-aroyl-L-
threonine hydroxamic acid was reported to inhibit E. coli LpxC
with a Ki value of 4 nM and shows antibiotic activity against
E. coli and Pseudomonas aeruginosa in bacterial disk diffusion
assays which is comparable to that of ciprofloxacin.14 Due to
its structure, CHIR-090 is able to bind to conserved regions of
LpxC, forming a tight-binding complex.15 With its hydroxa-
mate group CHIR-090 forms a complex with the catalytic Zn2+-
ion, whilst its diphenylacetylene moiety inserts into the hydro-
phobic channel of the enzyme, which binds the natural sub-
strate’s fatty acid chain.
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-
Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
E-mail: hollr@uni-muenster.de; Fax: +49-251-8332144; Tel: +49-251-8333372
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob41082j
‡These authors contributed equally to this work.
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Fig. 2 Structures of CHIR-090 and 3a.
Recently, we have described the synthesis of the CHIR-090 and 3-bromo-substituted C-aryl β-D-mannofuranosides 7b–d
analogue 3a (Fig. 2).16 The glycosidic scaffold of this com- were obtained via a nucleophilic attack of the corresponding
pound leads to a conformational constriction keeping its halogen-substituted phenyllithium species to the D-mannose
hydroxamate moiety and the lipophilic side chain in a defined derived D-mannono-1,4-lactone 4.16,17 The resulting hemiketals
relative orientation, whilst the hydroxy groups of 3a replace the 5b–d were then reduced with Et3SiH in the presence of
one of CHIR-090’s threonyl moiety. In this paper we report on BF3·OEt2. As the hydride of triethylsilane was transferred to
the biological activity of C-glycoside 3a. Additionally, the syn- the intermediately formed oxocarbenium ions from the less
thesis and biological evaluation of further C-glycosidic LpxC hindered Re-face, the corresponding C-aryl β-D-mannofurano-
inhibitors are described. These compounds differ from 3a in sides were formed in a stereocontrolled manner. Since the
the nature of the lipophilic side chain. The variations which presence of the Lewis acid BF3·OEt2 led to a partial cleavage of
were carried out comprise differences in the length, geometry the exocyclic acetonide, in addition to the bisacetonides 6b–d,
and flexibility of this lipophilic moiety. Furthermore, in order the diols 7b–d were obtained. Treatment of the bisacetonides
to investigate the influence of the conformational restriction 6b–d with catalytic amounts of p-toluenesulfonic acid in
of the C-glycosidic scaffold on the biological activity, open- methanol led to the desired diols 7b–d via a selective cleavage
chain derivatives should be synthesized and tested for their of the less hindered exocyclic ketals. The diols 7b–d could also
antibacterial properties.
be obtained by a direct treatment of the crude products of the
reductions with p-toluenesulfonic acid in methanol, supersed-
ing the intermediate purification of the bisacetonides 6b–d.
In analogy to the synthesis of esters 8a and 9a, the vicinal
diols 7b–e were reacted with a solution containing H5IO6 and
Synthesis
First, in order to get access to the desired C-aryl glycosidic CrO3 in wet acetonitrile.16,18 The resulting carboxylic acids
scaffold, the diols 7a–e were synthesized (Scheme 1). The ethyl- were directly transformed into the esters by heating the crude
ene glycol moiety of these central building blocks can be trans- products in methanol in the presence of chlorotrimethylsilane
formed into
a hydroxamate moiety and the halogen or catalytic amounts of p-toluenesulfonic acid. As these reac-
substituents in positions 3 and 4 of the phenyl ring allow C–C tion conditions also caused the cleavage of the remaining
coupling reactions. As already described for the 4-iodophenyl ketal, in addition to the acetonide-protected esters 8b–e, the
and the phenyl derivatives 7a and 7e, the 3-iodo-, 4-bromo- diol-containing esters 9b–e were obtained. The deprotection of
Scheme 1 Reagents and conditions: (a) THF, −78 °C, 5a 71%, 5b 90%, 5c 88%, 5d 96%, 5e 95%; (b) BF3·OEt2, Et3SiH, H3CCN, −40 °C, 6a 39% + 7a 25%, 6b 27%
+ 7b 39%, 6c 30% + 7c 25%, 6d 30% + 7d 47%, 6e 24% + 7e 46%; (c) p-TsOH, MeOH, rt, 7a 65%, 7b 45%, 7c 87%, 7d 32%, 7e 73%; (d) 1. CrO3/H5IO6, H3CCN,
rt, 2. H+, MeOH, Δ, 8a 40% + 9a 37%, 8b 49% + 9b 32%, 8c 29% + 9c 25%, 8d 12% + 9d 17%, 8e 42% + 9e 15%; (e) p-TsOH, MeOH, Δ, 9a 39%, 9b 31%, 9c
42%, 9d 35%, 9e 25%; (f) H2NOH·HCl, NaOMe, MeOH, rt, 10a 10%, 10b 29%, 10c 15%, 10d 30%, 10e 17%.
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Scheme 2 Reagents and conditions: (a) phenylboronic acid, NaOCH3, 1,2-dimethoxyethane, rt, then Pd(dppf )Cl2, rt, then 8, 80 °C, 11a 42%, 11b 54%; (b) p-TsOH,
ethylene glycol, MeOH, Δ, 12a 62%, 12b 56%; (c) H2NOH·HCl, NaOMe, MeOH, rt, 13a 20%, 13b 15%; (d) trans-β-styreneboronic acid, NaOCH3, 1,2-dimethoxy-
ethane, rt, then Pd(PPh3)4, rt, then 8, 80 °C, 14a 48%, 14b 64%; (e) p-TsOH, ethylene glycol, MeOH, Δ, 15a 65%, 15b 50%; (f) H2NOH·HCl, NaOMe, MeOH, rt, 16a
37%, 16b 31%; (g) phenylacetylene, Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 17a 97%, 17b 94%; (h) H2, 1 bar, Lindlar catalyst, ethanol–ethyl acetate (3/1), rt, 70%;
(i) p-TsOH, MeOH, Δ, 75%; ( j) H2NOH·HCl, NaOMe, MeOH, rt, 40%; (k) H2, 1 bar, Pd/C, rt, THF (21a) or MeOH (21b), 21a 58%, 21b 92%; (l) p-TsOH, MeOH, Δ, 22a
80%, 22b 90%; (f) H2NOH·HCl, NaOMe, MeOH, rt, 23a 27%, 23b 49%.
the acetonides 8b–e could be achieved under the same reac-
tion conditions, yielding esters 9b–e.
For the synthesis of the (Z)-stilbene derivative 20a as well as
the phenethyl derivatives 23a and 23b, a Pd(PPh3)4-catalyzed
Finally, the aminolysis of the esters 9a–e with hydroxyl- Sonogashira coupling of C-aryl glycosides 8a and 8b with
amine gave the desired hydroxamic acids 10a–e which possess phenylacetylene was performed to give diphenylacetylene
a relatively short lipophilic side chain compared to 3a.
derivatives 17a and 17b. When the alkyne 17a was hydrogen-
In order to introduce longer side chains, C–C coupling reac- ated in the presence of a Lindlar catalyst, the (Z)-stilbene 18a
tions were performed. As bromide proved to be a worse leaving was obtained. The deprotection of acetonide 18a and the sub-
group, the iodine-substituted C-aryl glycosides 8a, b and 9a, b sequent aminolysis of the resulting diol 19a gave the desired
were used in the cross coupling reactions.
hydroxamic acid 20a. When palladium on carbon was used as
The biphenyl derivatives 13a and 13b could be obtained via a catalyst for the hydrogenation of alkynes 17a and 17b, the
a Suzuki reaction (Scheme 2).19 The Pd(dppf)Cl2-catalyzed triple bonds were hydrogenated completely to yield the phen-
cross coupling with phenylboronic acid using 1,2-dimethoxy- ethyl derivatives 21a and 21b. The use of methanol as solvent
ethane as solvent and NaOCH3 as base was performed with the was more favorable compared to THF, as the hydrogenation of
acetonides 8a and 8b, to avoid the formation of boronic esters 17b in methanol afforded 21b in 92% yield, whereas the analo-
between glycols 9a and 9b and the boronic acid. The obtained gous reaction of 17a in THF gave 21a in only 58% yield. The
biphenyls 11a and 11b were hydrolyzed by heating the com- phenethyl derivatives 21a and 21b compounds were then
pounds in methanol in the presence of a catalytic amount of deprotected and the resulting diols 22a and 22b were trans-
p-toluenesulfonic acid and the resulting diols 12a and 12b formed into the hydroxamates 23a and 23b.
were transformed into the hydroxamic acids 13a and 13b by
The envisaged Pd(PPh3)4-catalyzed Sonogashira cross
aminolysis with hydroxylamine. The (E)-stilbene derivatives coupling reactions could also be directly performed with the
16a and 16b were synthesized in the same manner by employ- glycols 9a and 9b (Scheme 3). The couplings of 9a and 9b
ing trans-β-styreneboronic acid in the Pd(PPh3)4-catalyzed with 1-hexyne yielded the hex-1-yn-1-ylphenyl derivatives 24a
Suzuki reaction.
and 24b, whilst the reactions with phenylacetylene gave
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Scheme 3 Reagents and conditions: (a) 1-hexyne, Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 24a 92%, 24b 80%; (b) H2NOH·HCl, NaOMe, MeOH, rt, 28a 18%, 28b 35%;
(c) phenylacetylene, Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 25a 97%, 25b 94%; (d) H2NOH·HCl, NaOMe, MeOH, rt, 29a 9%, 29b 22%; (e) Pd(PPh3)4, CuI, NEt3, H3CCN, rt,
27a 26%, 27b 83%; (f ) H2NOH·HCl, NaOMe, MeOH, rt, 3a 29%, 3b 48%.
Scheme 4 Reagents and conditions: (a) trimethylsilylacetylene, Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 97%; (b) AgNO3, H2O, acetone, 74%; (c) (bromoethynyl)benzene,
CuCl, HONH3Cl, nBuNH2–H2O, 0 °C, 46%; (d) p-TsOH, MeOH, Δ, 78%; (e) H2NOH·HCl, NaOMe, MeOH, rt, 38%.
diphenylacetylene derivatives 25a and 25b. Finally, the Sonoga- butadiyne derivative 32a.21 Deprotection of the acetonide 32a
shira coupling of the 3-iodo-substituted C-phenyl glycoside 9b gave diol 33a, which was transformed into hydroxamic acid
with the morpholinomethyl-substituted phenylacetylene 34a by an aminolysis with hydroxylamine.
derivative 26 yielded ester 27b which bears the side chain of
Finally, the open chain derivatives 42 and 43 were syn-
CHIR-090. The corresponding hydroxamic acids 28a, 28b, 29a, thesized (Scheme 5). Starting from diol 7a, a glycol cleavage
29b and 3b could be obtained by reacting the esters 24a, 24b, with NaIO4 was performed. The intermediate aldehyde was
25a, 25b and 27b with hydroxylamine hydrochloride and directly transformed into the corresponding dimethyl acetal 35
NaOCH3 in methanol.
with methanol in the presence of p-toluenesulfonic acid. As
For the synthesis of diphenyldiacetylene derivative 34a, the already observed for the formation of esters 8a–e, these reac-
isopropylidene protected ester 8a was subjected to a Pd(PPh3)4- tion conditions led to a partial cleavage of the isopropylidene
catalyzed Sonogashira coupling with trimethylsilylacetylene to protective group resulting in a mixture of acetonide 35 and
give compound 30a (Scheme 4). A AgNO3-catalyzed protodesily- diol 36 in a ratio of 10 : 1. In order to transform the remaining
lation of trimethylsilylacetylene derivative 30a in water contain- ketal 35 into glycol 36, it was again heated in methanol under
ing acetone yielded the terminal alkyne 31a,20 which was acidic conditions. The tetrahydrofuran ring of 36 was then
subsequently reacted with (bromoethynyl)benzene in a Cu(I)- cleaved with NaIO4. The resulting dialdehyde was subsequently
catalyzed Cadiot–Chodkiewicz cross-coupling to yield the reduced with NaBH4 to give the open chain diol 37. The (S,S)-
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Scheme 5 Reagents and conditions: (a) 1. NaIO4, MeOH, rt, 2. pTsOH, MeOH, Δ, 35 77% + 36 8%; (b) pTsOH, MeOH, Δ, 35%; (c) 1. NaIO4, MeOH, rt, 2. NaBH4,
MeOH, rt, 77%; (d) HCl–THF, Δ, 81%, (e) Br2, NaHCO3, MeOH–H2O, rt, 83%; (f) Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 40 98%, 41 81%; (g) H2NOH·HCl, NaOMe, MeOH, rt,
42 52%, 43 21%.
configuration of 37 was derived from the stereochemistry of
When tested against E. coli BL21 (DE3), none of the C-glyco-
the starting material, as only one diastereomer was found and sidic compounds showed inhibition of bacterial growth
a full conversion of one of the stereocenters during the reac- beyond the 6 mm filter discs containing the solutions of the
tion sequence appeared very unlikely to occur. In order to get substances. In the case of the open chain derivatives, a zone of
access to the corresponding aldehyde, a hydrolytic cleavage of inhibition with a diameter of 9.6 mm was observed surround-
dimethyl acetal 37 was performed. However, the spectroscopic ing the disc containing the morpholinomethyl substituted
data of the isolated product 38 indicated that the compound diphenylacetylene 43, whereas no detectable inhibition of
predominantly exists as a lactol. Hemiacetal 38 was then oxi- growth was observed with the unsubstituted diphenylacetylene
dized with Br2 in a NaHCO3 containing mixture of methanol derivative 42. When the disc diffusion assays were performed
and water (9/1) to yield methyl ester 39.22 Sonogashira reac- with the more sensitive E. coli D22 strain, the C-glycosides
tions of the iodophenyl derivative 39 with phenylacetylene and with a relatively short side chain, like the phenyl derivative
the morpholinomethyl-substituted phenylacetylene derivative 10e, the bromophenyl derivatives 10c and 10d, the iodophenyl
26 gave the diphenylacetylene derivatives 40 and 41, respect- derivatives 10a and 10b, and the biphenyl derivatives 13a and
ively. Both esters could be transformed into the corresponding 13b showed no antibacterial activity. Also in the case of the
hydroxamic acids 42 and 43 by aminolysis with hydroxyl- phenethyl and hexynyl substituted compounds 23a, 23b, 28a
amine.
and 28b, which possess a relatively flexible side chain, as well
as the compounds with a relatively bent side chain like the
styryl derivatives 16a, 16b, and 20a and the meta-substituted
diphenylacetylene derivatives 29b and 3b no inhibition of bac-
terial growth beyond the filter disc was observed. However,
when hydroxamic acids with a linear side chain like the para-
substituted diphenylacetylene derivatives 29a and 3a as well as
the diphenyldiacetylene derivate 34a were tested, promising
zones of inhibition were observed. The open chain derivatives
42 and 43 showed even larger zones of inhibition with dia-
meters of 16.2 mm and 20.4 mm, respectively.
In order to correlate the antibiotic activity of the com-
pounds with their ability to inhibit the enzymatic activity of
the deacetylase LpxC, the LpxC enzyme assay was performed.
For that purpose, the inhibition of the deacetylation of the
enzyme’s natural substrate was measured by transforming the
resulting primary amine into a fluorescent isoindole with
Biological evaluation
The synthesized hydroxamic acids were tested for their biologi-
cal activity in a LpxC enzyme assay as well as in disc diffusion
assays (Table 1), after their purity had been determined by
HPLC to be >95%.
In the disc diffusion assays, the compounds were tested
against E. coli BL21 (DE3) and the E. coli D22 strain which is
more sensitive towards LpxC inhibition. After spreading a cell
suspension of the bacteria onto an agar plate, filter discs con-
taining 15 μL of a 10 mM solution of the compounds in DMSO
were placed on the surface of the agar and the plates were
incubated overnight at 37 °C. CHIR-090 and DMSO were used
as positive and negative controls, respectively.
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Table 1 Inhibitory activity against LpxC and antibacterial activity in disc diffusion assays of the synthesized hydroxamic acids
Zone of inhibition [mm]
Enzyme assay
Compound
Substituent
E. coli BL21(DE3)
E. coli D22
IC50 [μM]
Ki [μM]
Ciprofloxacin
CHIR-090
10e
10c
10d
10a
10b
13a
13b
16a
16b
20a
23a
23b
28a
28b
29a
29b
38.5
24.5
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
9.6
29
30
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
6.5
<6
13.2
<6
9
0.06
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
32
0.008
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
H
p-Br
m-Br
p-I
m-I
p-Phenyl
m-Phenyl
p-(E)-Styryl
m-(E)-Styryl
p-(Z)-Styryl
p-Phenethyl
m-Phenethyl
p-Hexynyl
m-Hexynyl
p-Phenylethynyl
m-Phenylethynyl
p-Morpholinomethylphenylethynyl
m-Morpholinomethylphenylethynyl
p-Phenylbutadiynyl
p-Phenylethynyl
p-Morpholinomethylphenylethynyl
3a
3b
34a
42
—
—
4.4
27.6
0.352
16.2
20.4
200
2.6
43
phthalaldehyde and 2-mercaptoethanol. The compounds were introducing a phenethyl and a hexynyl residue to the phenyl ring
assayed over a range from 0.2 nM up to 200 μM. At these con- of the C-aryl glycosides. Furthermore, the C-glycosides can give
centrations no solubility problems of the substances in the access to ethylene glycol ethers with a defined stereochemistry
assay buffer were observed.
Except the diphenyldiacetylene derivative 34a, which shows cosidic scaffold and a subsequent reduction in the key step.
an IC50 value of 32 μM resulting in a Ki value of 4.4 μM, the The biological evaluation shows that the C-glycosidic hydro-
by performing a glycol cleavage with the vicinal diol of the C-gly-
C-glycosides were not able to inhibit the enzyme at concen- xamic acids with a relatively short, bent or flexible side chain
trations up to 200 μM. This also includes the phenylacetylene do not exhibit antibacterial activity against E. coli. In contrast,
derivatives 29a and 3a, which were active against E. coli D22. compounds with a long and linear side chain, which is rela-
By contrast, the open chain derivatives 42 and 43 show inhi- tively rigid, like the diphenyldiacetylene derivative 34a, show
bitory activity against LpxC, with the morpholinomethyl antibiotic activity against E. coli.
derivative 43 possessing a Ki value of 0.35 μM.
The synthesized open chain derivatives, especially the mor-
pholinomethyl substituted diphenylacetylene derivative 43,
show higher inhibitory activity in the disc diffusion test and
the LpxC enzyme assay in comparison to their conformation-
ally constrained counterparts. Apparently, the conformational
restriction realized for the presented C-aryl glycosides is not
beneficial for the biological activity of the compounds.
However, the open chain derivative 43 with its promising bio-
logical activity and Ki value represents an interesting starting
point for further variations and optimizations of its phenyl
ethylene glycol ether scaffold.
Conclusions
Applying our previously described method for the stereocon-
trolled synthesis of C-aryl glycosides, we were able to syn-
thesize hydroxamic acids with various lipophilic side chains.
In the key step, Sonogashira and Suzuki coupling reactions
were performed with the iodine substituted C-aryl glycosides.
These C–C coupling reactions allow the introduction of diverse
substituents to the aromatic moiety, which can be further
varied e.g. by hydrogenation. So starting from the C-phenyl gly-
coside 10e, the side chain was successively elongated by pre-
paring the bromophenyl, iodophenyl, biphenyl, diphenyl-
acetylene and diphenyldiacetylene derivatives. In order to vary
the geometry of the side chain, besides the 4-substituted
Experimental section
Chemistry
General. Unless otherwise mentioned, THF was dried with
C-phenyl glycosides, the corresponding 3-substituted com- sodium/benzophenone and was freshly distilled before use.
pounds were synthesized. In addition to the linear diphenylace- Thin layer chromatography (tlc): silica gel 60 F254 plates
tylene derivatives, the rather bent stilbene derivatives were (Merck). Flash chromatography (fc): silica gel 60, 40–64 μm
prepared. The flexibility of the side chain was increased by (Macherey-Nagel); parentheses include: diameter of the
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column, fraction size, eluent, Rf value. Melting point (m.p.): C(CH3)2), 126.9 (2C, Carom.), 127.3 (2C, Carom.), 127.4 (1C,
melting point apparatus SMP 3 (Stuart Scientific), uncorrected. arom.), 128.2 (2C, Carom.), 128.8 (2C, Carom.), 133.8 (1C, Carom.),
C
Optical rotation α [°] was determined with a Polarimeter 341 141.0 (1C, Carom.), 141.1 (1C, Carom.), 167.8 (1C, CvO); IR (neat):
(Perkin Elmer); path length 1 dm, wavelength 589 nm (sodium ν [cm−1] = 2986, 1761, 1488, 1437, 1383, 1206, 1096, 744; HRMS
D line); the unit of the specific rotation [α]D20 [deg mL dm−1 (m/z): [M + H]+ calcd for C21H23O5, 355.1540; found, 355.1561;
g−1] is omitted; the concentration of sample c [mg mL−1] and HPLC (method 1): tR = 20.8 min, purity 90.9%.
the solvent used are given in brackets. 1H NMR (400 MHz),
(2S,3R,4S,5S)-Methyl 5-([1,1′-biphenyl]-4-yl)-3,4-dihydroxytetra-
hydrofuran-2-carboxylate (12a). p-Toluenesulfonic acid mono-
13C NMR (100 MHz): Mercury plus 400 spectrometer (Varian);
hydrate (6 mg, 0.03 mmol) and ethylene glycol (2 drops) were
added to a solution of 11a (100 mg, 0.28 mmol) in methanol
(20 mL). The mixture was heated to reflux for 16 h. Then the
solvent was removed in vacuo and the residue was purified by
flash column chromatography (2 cm, 10 mL, cyclohexane–
ethyl acetate = 1/1, Rf = 0.38) to give 12a as a colorless solid
(55 mg, 0.17 mmol, 62%). M.p.: 153 °C; [α]2D0 = +65.1 (2.7;
δ in ppm is related to tetramethylsilane; coupling constants
are given with 0.5 Hz resolution. Where necessary, the assign-
1
ment of the signals in the H NMR and 13C NMR spectra was
performed using 1H–1H and 1H–13C COSEY NMR spectra as
well as NOE (nuclear Overhauser effect) difference spec-
troscopy. IR: IR Prestige-21(Shimadzu). HRMS: MicrOTOF-QII
(Bruker). HPLC methods for the determination of product
purity: Method 1: Merck Hitachi Equipment; UV detector:
L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
column: LiChrospher® 60 RP-select B (5 μm); LiCroCART®
250–4 mm cartridge; flow rate: 1.00 mL min−1; injection
volume: 5.0 μL; detection at λ = 210 nm for 30 min; solvents: A:
water with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with
0.05% (v/v) trifluoroacetic acid: gradient elution: (A%):
0–4 min: 90%, 4–29 min: gradient from 90% to 0%,
29–31 min: 0%, 31–31.5 min: gradient from 0% to 90%,
31.5–40 min: 90%. Method 2: Merck Hitachi Equipment; UV
detector: L-7400; pump: L-6200A; column: Phenomenex
Gemini® 5 μm C6-Phenyl 110 Å; LC column 250 × 4.6 mm;
flow rate: 1.00 mL min−1; injection volume: 5.0 μL; detection at
λ = 254 nm for 20 min; solvents: A: acetonitrile–10 mM
ammonium formate = 10 : 90 with 0.1% formic acid; B: aceto-
nitrile–10 mM ammonium formate = 90 : 10 with 0.1% formic
acid; gradient elution: (A%): 0–5 min: 100%, 5–15 min: gradi-
ent from 100% to 0%, 15–20 min: 0%, 20–22 min: gradient
from 0% to 100%, 22–30 min: 100%.
1
CH2Cl2); H NMR (CDCl3): δ 3.87 (s, 3H, OCH3), 4.30 (t, J = 3.7
Hz 1H, 4-H), 4.73–4.79 (m, 2H, 2-H, 3-H), 5.19 (d, J = 3.9 Hz,
1H, 5-H), 7.33–7.38 (m, 1H, Harom.), 7.42–7.47 (m, 2H, Harom.),
7.54–7.64 (m, 6H, Harom.); 13C NMR (CDCl3): δ 52.8 (1C, OCH3),
73.8 (1C, C-4), 74.2 (1C, C-3), 79.0 (1C, C-2), 83.5 (1C, C-5),
127.2 (2C, Carom.), 127.3 (2C, Carom.), 127.4 (2C, Carom.), 127.5
(1C, Carom.), 128.9 (2C, Carom.), 134.9 (1C, Carom.), 140.9 (1C,
C
arom.), 141.2 (1C, Carom.), 172.7 (1C, CvO); IR (neat): ν [cm−1
]
= 3435, 1742, 1485, 1440, 1410, 1220, 1095, 761, 701; HRMS
(m/z): [M + H]+ calcd for C18H19O5, 315.1227; found, 315.1258;
HPLC (method 1): tR = 17.4 min, purity 97.8%.
(2S,3R,4S,5S)-5-([1,1′-Biphenyl]-4-yl)-N,3,4-trihydroxytetrahydro-
furan-2-carboxamide
(13a). Hydroxylamine
hydrochloride
(83 mg, 1.2 mmol) and sodium methoxide (65 mg, 1.2 mmol)
were added to a solution of 12a (55 mg, 0.17 mmol) in metha-
nol (20 mL) and the mixture was stirred at ambient tempera-
ture for 48 h. Then the solvent was evaporated. The residue
was dissolved in ethyl acetate and extracted with HCl (1 M).
The aqueous phase was then extracted with ethyl acetate (3×)
and the combined organic layers were dried (Na2SO4), filtered
and evaporated. The residue was purified by flash column
chromatography (1 cm, 5 mL, CH2Cl2–methanol = 9.5/0.5 +
0.1% TFA). The fractions containing 13a were collected and
evaporated. The residue was dissolved in ethyl acetate and
extracted with HCl (1 M). The aqueous phase was then
extracted with ethyl acetate (3×) and the combined organic
layers were dried (Na2SO4), filtered and evaporated to give 13a
as a colorless solid (11.3 mg, 0.04 mmol, 20%). M.p.: 156 °C;
TLC (CH2Cl2–methanol = 9/1): Rf = 0.42; [α]2D0 = +60.3 (0.7;
methanol); 1H NMR (D3COD): δ 4.21 (t, J = 4.4 Hz, 1H, 4-H),
4.49 (d, J = 7.2 Hz, 1H, 2-H), 4.74 (dd, J = 7.2/4.7 Hz, 1H, 3-H),
5.09 (d, J = 4.0 Hz, 1H, 5-H), 7.30–7.34 (m, 1H, Harom.),
7.40–7.45 (m, 2H, Harom.), 7.52–7.55 (m, 2H, Harom.), 7.58–7.63
(m, 4H, Harom.); 13C NMR (D3COD): δ 74.4 (1C, C-3), 74.9 (1C,
C-4), 80.2 (1C, C-2), 85.4 (1C, C-5), 127.4 (2C, Carom.), 127.9 (2C,
(3aR,4S,6S,6aR)-Methyl
6-([1,1′-biphenyl]-4-yl)-2,2-dimethyl-
tetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate (11a). Under
a
N2 atmosphere sodium methoxide (62 mg, 1.14 mmol) and
phenylboronic acid (105 mg, 0.86 mmol) were dissolved in 1,2-
dimethoxyethane (30 mL) and the mixture was stirred at room
temperature for 30 min. Then (1,1′-bis(diphenylphosphino)-
ferrocene)dichloropalladium(II) (65 mg, 0.09 mmol) was
added. After stirring the mixture for 10 min at room tempera-
ture a solution of 8a (230 mg, 0.57 mmol) in 1,2-dimethoxy-
ethane (15 mL) was added. Then the reaction mixture was
heated to 80 °C for 16 h, cooled to room temperature and
evaporated. The crude residue was purified by flash column
chromatography (2 cm, 10 mL, cyclohexane–ethyl acetate = 8/2,
Rf = 0.17) to give 11a as a colorless solid (85 mg, 0.24 mmol,
42%). M.p.: 154 °C; [α]2D0 = +53.2 (0.8; CH2Cl2); 1H NMR
(CDCl3): δ 1.29 (s, 3H, CH3), 1.47 (s, 3H, CH3), 3.87 (s, 3H,
OCH3), 4.42 (d, J = 4.6 Hz, 1H, 4-H), 4.72 (d, J = 3.5 Hz, 1H,
6-H), 4.87 (dd, J = 5.9/3.5 Hz, 1H, 6a-H), 5.11 (dd, J = 5.9/4.6
Hz, 1H, 3a-H), 7.31–7.37 (m, 1H, Harom.), 7.40–7.46 (m, 2H,
Carom.), 128.3 (1C, Carom.), 129.1 (2C, Carom.), 129.8 (2C, Carom.),
137.7 (1C, Carom.), 141.7 (1C, Carom.), 142.3 (1C, Carom.), 171.2
(1C, CvO); IR (neat): ν [cm−1] = 3186, 2916, 1651, 1485, 1261,
1138, 1042, 791, 756; HRMS (m/z): [M + H]+ calcd for
H
arom.), 7.53–7.62 (m, 6H, Harom.); 13C NMR (CDCl3): δ 25.1
C17H18NO5, 316.1179; found, 316.1164; HPLC (method 2): tR
=
(1C, C(CH3)2), 25.9 (1C, C(CH3)2), 52.3 (1C, OCH3), 80.8 (1C,
15.0 min, purity 98.0%.
C-4), 81.9 (1C, C-6a), 82.1 (1C, C-3a), 83.4 (1C, C-6), 113.6 (1C,
6062 | Org. Biomol. Chem., 2013, 11, 6056–6070
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(3aR,4S,6S,6aR)-Methyl 2,2-dimethyl-6-(4-((E)-styryl)phenyl)tetra- the mixture was stirred at ambient temperature for 16 h. Then
hydrofuro[3,4-d][1,3]dioxole-4-carboxylate (14a). Under N2
a
the solvent was evaporated. The residue was dissolved in ethyl
acetate and extracted with HCl (1 M). The aqueous phase was
then extracted with ethyl acetate (3×) and the combined
organic layers were dried (Na2SO4), filtered and evaporated.
The residue was purified by flash column chromatography
(1 cm, 5 mL, CH2Cl2–methanol = 9.5/0.5 + 0.1% TFA). The frac-
tions containing 16a were collected and evaporated. The
residue was dissolved in ethyl acetate and extracted with HCl
(1 M). The aqueous phase was then extracted with ethyl acetate
(3×) and the combined organic layers were dried (Na2SO4), fil-
tered and evaporated to give 16a as a colorless solid (37 mg,
0.11 mmol, 37%). M.p.: 179 °C; TLC (CH2Cl2–methanol = 9/1):
Rf = 0.36; [α]2D0 = +65.9 (1.4; methanol); 1H NMR (D3COD): δ
4.19 (t, J = 4.4 Hz, 1H, 4-H), 4.47 (d, J = 7.3 Hz, 1H, 2-H), 4.72
(dd, J = 7.3/4.8 Hz, 1H, 3-H), 5.04 (d, J = 4.1 Hz, 1H, 5-H), 7.18
(s, 2H, Ar-CHvCH-Ar), 7.21–7.26 (m, 1H, Harom.), 7.32–7.37 (m,
2H, Harom.), 7.43–7.47 (m, 2H, Harom.), 7.53–7.57 (m, 4H,
atmosphere sodium methoxide (11 mg, 0.20 mmol) and trans-
β-styreneboronic acid (22 mg, 0.15 mmol) were dissolved in
1,2-dimethoxyethane (10 mL) and the mixture was stirred at
room temperature for 30 min. Then tetrakis(triphenylpho-
sphine)palladium(0) (11.4 mg, 0.01 mmol) was added. After
stirring the mixture for 10 min at room temperature a solution
of 8a (40 mg, 0.10 mmol) in 1,2-dimethoxyethane (5 mL) was
added. Then the reaction mixture was heated to 80 °C for 16 h,
cooled to room temperature and evaporated. The crude
residue was purified by flash column chromatography (1 cm,
5 mL, cyclohexane–ethyl acetate = 8/2, Rf = 0.16) to give 14a as
a colorless solid (18 mg, 0.05 mmol, 48%). M.p.: 168 °C; [α]D20
=
1
+56.5 (2.8; CH2Cl2); H NMR (CDCl3): δ 1.28 (s, 3H, CH3), 1.45
(s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.40 (d, J = 4.6 Hz, 1H, 4-H),
4.68 (d, J = 3.6 Hz, 1H, 6-H), 4.84 (dd, J = 5.8/3.6 Hz, 1H, 6a-H),
5.09 (dd, J = 5.8/4.6 Hz, 1H, 3a-H), 7.11 (s, 2H, CHvCH),
7.23–7.29 (m, 1H, Harom.), 7.33–7.38 (m, 2H, Harom.), 7.45–7.54
(m, 6H, Harom.); 13C NMR (CDCl3): δ 25.1 (1C, C(CH3)2), 25.8
(1C, C(CH3)2), 52.3 (1C, OCH3), 80.8 (1C, C-4), 81.9 (1C, C-6a),
82.0 (1C, C-3a), 83.4 (1C, C-6), 113.5 (1C, C(CH3)2), 126.3 (2C,
H
arom.); 13C NMR (D3COD): δ 74.4 (1C, C-3), 74.9 (1C, C-4), 80.1
(1C, C-2), 85.4 (1C, C-5), 127.0 (2C, Carom.), 127.5 (2C, Carom.),
128.5 (1C, Carom.), 129.0 (2C, Carom.), 129.45 (1C, CHvCH),
129.53 (1C, CHvCH), 129.7 (2C, Carom.), 138.0 (1C, Carom.),
138.1 (1C, Carom.), 138.9 (1C, Carom.), 169.4 (1C, CvO); IR
(neat): ν [cm−1] = 3418, 3024, 2878, 1647, 1447, 1315, 1261,
1107, 1038, 968, 783, 691; HRMS (m/z): [M + H]+ calcd for
C
arom.), 126.6 (2C, Carom.), 127.7 (1C, Carom.), 128.0 (2C, Carom.),
128.6 (1C, CHvCH), 128.8 (2C, Carom.), 128.9 (1C, CHvCH),
134.2 (1C, Carom.), 137.3 (1C, Carom.), 137.5 (1C, Carom.), 167.7
(1C, CvO); IR (neat): ν [cm−1] = 2990, 2955, 2888, 1759, 1441,
1385, 1223, 1127, 1096, 1029, 818, 757, 692; HRMS (m/z):
[M + H]+ calcd for C23H25O5, 381.1697; found, 381.1747; HPLC
(method 1): tR = 21.8 min, purity 98.1%.
C19H20NO5, 342.1336; found, 342.1346; HPLC (method 1): tR
=
17.9 min, purity 95%.
(3aR,4S,6S,6aR)-Methyl
2,2-dimethyl-6-(4-(phenylethynyl)-
phenyl)tetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate
(17a).
(2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-(4-((E)-styryl)phenyl)- Under a N2 atmosphere triethylamine (0.26 mL, 1.90 mmol),
tetrahydrofuran-2-carboxylate (15a). p-Toluenesulfonic acid
monohydrate (14 mg, 0.07 mmol) and ethylene glycol (2 drops)
were added to a solution of 14a (140 mg, 0.37 mmol) in metha-
nol (30 mL). The mixture was heated to reflux for 16 h. Then
the solvent was removed in vacuo and the residue was purified
by flash column chromatography (2 cm, 10 mL, cyclohexane–
ethyl acetate = 1/1, Rf = 0.23) to give 15a as a colorless solid
(82 mg, 0.24 mmol, 65%). M.p.: 201 °C; [α]2D0 = +39.4 (1.3;
copper(I) iodide (10 mg, 0.05 mmol) and tetrakis(triphenylpho-
sphine)palladium(0) (31 mg, 0.027 mmol) were added to a
solution of 8a (110 mg, 0.27 mmol) in acetonitrile (5 mL).
Then a solution of phenylacetylene (0.25 mL, 2.25 mmol) in
acetonitrile (3 mL) was added dropwise over a period of 2 h.
After evaporation of the solvent the residue was purified by
flash column chromatography (2 cm, 10 mL, cyclohexane–
ethyl acetate = 8/2, Rf = 0.16) to give 18a as a colorless solid
(100 mg, 0.26 mmol, 97%). M.p.: 173 °C; [α]2D0 = +63.2 (2.6;
CH2Cl2); 1H NMR (CDCl3): δ 1.27 (s, 3H, CH3), 1.43 (s, 3H,
CH3), 3.86 (s, 3H, OCH3), 4.41 (d, J = 4.6 Hz, 1H, 4-H), 4.69 (d,
J = 3.6 Hz, 1H, 6-H), 4.84 (dd, J = 5.9/3.6 Hz, 1H, 6a-H), 5.09
(dd, J = 5.9/4.6 Hz, 1H, 3a-H), 7.32–7.38 (m, 3H, Harom.),
7.44–7.48 (m, 2H, Harom.), 7.51–7.55 (m, 4H, Harom.); 13C NMR
(CDCl3): δ 25.0 (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 52.3 (1C,
OCH3), 80.8 (1C, C-4), 81.8 (1C, C-6a), 82.0 (1C, C-3a), 83.3 (1C,
C-6), 89.5 (1C, CuC), 89.6 (1C, CuC), 113.6 (1C, C(CH3)2),
123.1 (1C, Carom.), 123.4 (1C, Carom.), 127.6 (2C, Carom.), 128.4
(1C, Carom.), 128.5 (2C, Carom.), 131.4 (2C, Carom.), 131.8 (2C,
1
methanol); H NMR (CDCl3): δ 3.87 (s, 3H, OCH3), 4.27 (t, J =
3.9 Hz, 1H, 4-H), 4.72–4.76 (m, 2H, 2-H, 3-H), 5.16 (d, J = 3.9
Hz, 1H, 5-H), 7.12 (s, 2H, CHvCH), 7.24–7.29 (m, 1H, Harom.),
7.34–7.39 (m, 2H, Harom.), 7.46–7.57 (m, 6H, Harom.); 13C NMR
(CDCl3): δ 52.8 (1C, OCH3), 73.8 (1C, C-4), 74.2 (1C, C-3), 79.0
(1C, C-2), 83.5 (1C, C-5), 126.7 (2C, Carom.), 126.8 (2C, Carom.),
127.3 (2C, Carom.), 127.8 (1C, Carom.), 128.4 (1C, CHvCH),
128.8 (2C, Carom.), 129.1 (1C, CHvCH), 135.2 (1C, Carom.),
137.4 (1C, Carom.), 137.5 (1C, Carom.), 172.6 (1C, CvO); IR
(neat): ν [cm−1] = 3483, 3024, 2909, 1709, 1512, 1443, 1342,
1269, 1096, 1053, 895, 775, 691; HRMS (m/z): [M + H]+ calcd
for C20H21O5, 341.1384; found, 341.1367; HPLC (method 1): tR
= 19.0 min, purity 99.4%.
C
arom.), 135.1 (1C, Carom.), 167.7 (1C, CvO); IR (neat): ν [cm−1] =
2931, 1750, 1516, 1439, 1216, 1103, 750, 693; HRMS (m/z): [M +
H]+ calcd for C23H23O5, 379.1540; found, 379.1518; HPLC
(method 1): tR = 22.1 min, purity 95.0%.
(2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(4-((E)-styryl)phenyl)tetrahydro-
furan-2-carboxamide (16a). A 2.5
M solution of sodium
methoxide in methanol (0.35 mL, 0.87 mmol) was added to a
solution of 15a (100 mg, 0.29 mmol) and hydroxylamine
hydrochloride (40 mg, 0.58 mmol) in methanol (20 mL) and
(3aR,4S,6S,6aR)-Methyl 2,2-dimethyl-6-(4-((Z)-styryl)phenyl)-
tetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate
(18a). 17a
(200 mg, 0.53 mmol) was dissolved in an ethanol–ethyl acetate
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1
(3 : 1) mixture (30 mL) and Lindlar catalyst (20 mg) was added. +66.9 (3.6; methanol); H NMR (D3COD): δ 4.15 (t, J = 4.3 Hz,
The mixture was stirred under hydrogen (balloon) for 4 h at 1H, 4-H), 4.45 (d, J = 7.3 Hz, 1H, 2-H), 4.70 (dd, J = 7.3/4.7 Hz,
room temperature. Then the suspension was filtered through 1H, 3-H), 4.98 (d, J = 3.9 Hz, 1H, 5-H), 6.60 (s, 2H, Ar-CHvCH-
Celite® and the filtrate was concentrated in vacuo. The residue Ar), 7.15–7.25 (m, 7H, Harom.), 7.29–7.32 (m, 1H, Harom.); 13C
was purified by flash column chromatography (2 cm, 10 mL, NMR (D3COD): δ 74.4 (1C, C-3), 74.8 (1C, C-4), 80.0 (1C, C-2),
cyclohexane–ethyl acetate = 8/2, Rf = 0.28) to give 18a as a 85.4 (1C, C-5), 128.1 (1C, Carom.), 128.5 (2C, Carom.), 129.3 (2C,
colorless solid (140 mg, 0.37 mmol, 70%). M.p.: 107 °C; [α]D20
+24.2 (2.6; CH2Cl2); H NMR (CDCl3): δ 1.26 (s, 3H, CH3), 1.42 CHvCH), 131.2 (1C, CHvCH), 137.5 (1C, Carom.), 137.9 (1C,
=
Carom.), 129.4 (2C, Carom.), 129.9 (2C, Carom.), 131.1 (1C,
1
(s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.37 (d, J = 4.6 Hz, 1H, 4-H),
C
arom.), 138.8 (1C, Carom.), 169.4 (1C, CvO); IR (neat): ν [cm−1] =
4.62 (d, J = 3.6 Hz, 1H, 6-H), 4.79 (dd, J = 5.9/3.6 Hz, 1H, 6a-H), 3310, 2901, 1639, 1512, 1423, 1138, 1049, 1030, 984, 833, 772,
5.07 (dd, J = 5.9/4.6 Hz, 1H, 3a-H), 6.58 (s, 2H, CHvCH), 698; HRMS (m/z): [M + H]+ calcd for C19H20NO5, 342.1336;
7.17–7.27 (m, 7H, Harom.), 7.32–7.35 (m, 2H, Harom.); 13C NMR found, 342.1375; HPLC (method 1): tR = 18.5 min, purity
(CDCl3): δ 25.0 (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 52.3 (1C, 99.7%.
(3aR,4S,6S,6aR)-Methyl
tetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate
2,2-dimethyl-6-(4-phenethylphenyl)-
(21a). 17a
OCH3), 80.7 (1C, C-4), 81.7 (1C, C-6a), 82.0 (1C, C-3a), 83.5 (1C,
C-6), 113.5 (1C, C(CH3)2), 127.2 (1C, Carom.), 127.7 (2C, Carom.),
128.3 (2C, Carom.), 128.7 (2C, Carom.), 129.0 (2C, Carom.), 130.1
(1C, CHvCH), 130.4 (1C, CHvCH), 133.5 (1C, Carom.), 137.2
(1C, Carom.), 137.3 (1C, Carom.), 167.8 (1C, CvO); IR (neat):
ν [cm−1] = 2986, 1748, 1439, 1369, 1215, 1103, 1076, 1034, 914,
868, 829, 702; HRMS (m/z): [M + H]+ calcd for C23H25O5,
381.1697; found, 381.1703; HPLC (method 1): tR = 21.9 min,
purity 98.3%.
(85 mg, 0.22 mmol) was dissolved in THF (20 mL) and 10%
Pd/C (9 mg) was added. The mixture was stirred under hydro-
gen (balloon) for 16 h at room temperature. Then the suspen-
sion was filtered through Celite® and the filtrate was
concentrated in vacuo. The residue was purified by flash
column chromatography (1 cm, 5 mL, cyclohexane–ethyl
acetate = 8/2, Rf = 0.24) to give 21a as a colorless solid (50 mg,
0.13 mmol, 58%). M.p.: 151 °C; [α]2D0 = +31.9 (2.6; CH2Cl2);
(2S,3R,4S,5S)-Methyl
3,4-dihydroxy-5-(4-((Z)-styryl)phenyl)-
tetrahydrofuran-2-carboxylate (19a). p-Toluenesulfonic acid 1H NMR (CDCl3): δ 1.27 (s, 3H, CH3), 1.45 (s, 3H, CH3), 2.92 (s,
monohydrate (10 mg, 0.05 mmol) was added to a solution of 4H, ArCH2CH2Ar), 3.85 (s, 3H, OCH3), 4.38 (d, J = 4.6 Hz, 1H,
18a (90 mg, 0.24 mmol) in methanol (30 mL). The mixture was 4-H), 4.64 (d, J = 3.5 Hz, 1H, 6-H), 4.81 (dd, J = 5.8/3.5 Hz, 1H,
heated to reflux for 16 h. Then the solvent was removed in 6a-H), 5.05 (dd, J = 5.8/4.6 Hz, 1H, 3a-H), 7.16–7.21 (m, 5H,
vacuo and the residue was purified by flash column chromato-
Harom.), 7.25–7.30 (m, 2H, Harom.), 7.37–7.41 (m, 2H, Harom.);
graphy (1 cm, 5 mL, cyclohexane–ethyl acetate = 1/1, Rf = 0.31) 13C NMR (CDCl3): δ 25.1 (1C, C(CH3)2), 25.9 (1C, C(CH3)2), 37.9
to give 19a as a colorless solid (60 mg, 0.18 mmol, 75%). M.p.: (2C, ArCH2CH2Ar), 52.3 (1C, OCH3), 80.8 (1C, C-4), 81.8 (1C,
1
92 °C; [α]2D0 = +61.5 (2.1; CH2Cl2); H NMR (CDCl3): δ 3.84 (s, C-6a), 82.1 (1C, C-3a), 83.5 (1C, C-6), 113.5 (1C, C(CH3)2), 126.0
3H, OCH3), 4.22 (t, J = 3.9 Hz, 1H, 4-H), 4.69 (d, J = 7.6 Hz, 1H, (1C, Carom.), 127.8 (2C, Carom.), 128.3 (2C, Carom.), 128.5 (2C,
2-H), 4.73 (dd, J = 7.6/4.0 Hz, 1H, 3-H), 5.09 (d, J = 3.9 Hz, 1H,
Carom.), 128.6 (2C, Carom.), 132.3 (1C, Carom.), 141.8 (1C, Carom.),
5-H), 6.58 (d, J = 12.3 Hz, 1H, CHvCH), 6.62 (d, J = 12.3 Hz, 142.0 (1C, Carom.), 167.9 (1C, CvO); IR (neat): ν [cm−1] = 2937,
1H, CHvCH), 7.16–7.36 (m, 9H, Harom.); 13C NMR (CDCl3): δ 1754, 1517, 1435, 1383, 1218, 1114, 820, 753, 702; HRMS (m/z):
52.7 (1C, OCH3), 73.7 (1C, C-4), 74.2 (1C, C-3), 78.9 (1C, C-2), [M + H]+ calcd for C23H27O5, 383.1853; found, 383.1898; HPLC
83.6 (1C, C-5), 126.9 (2C, Carom.), 127.3 (1C, Carom.), 128.4 (2C, (method 1): tR = 22.0 min, purity 97.4%.
(2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-(4-phenethylphenyl)tetra-
hydrofuran-2-carboxylate (22a). p-Toluenesulfonic acid mono-
hydrate (6 mg, 0.03 mmol) was added to a solution of 21a
(60 mg, 0.16 mmol) in methanol (20 mL). The mixture was
heated to reflux for 16 h. Then the solvent was removed
in vacuo and the residue was purified by flash column chrom-
atography (1 cm, 5 mL, cyclohexane–ethyl acetate = 1/1, Rf =
0.33) to give 22a as a colorless solid (43 mg, 0.13 mmol, 80%).
C
arom.), 128.9 (2C, Carom.), 129.1 (2C, Carom.), 129.9 (1C,
CHvCH), 130.7 (1C, CHvCH), 134.6 (1C, Carom.), 137.2 (1C,
arom.), 137.3 (1C, Carom.), 172.5 (1C, CvO); IR (neat): ν [cm−1] =
C
3441, 2959, 1717, 1439, 1373, 1234, 1092, 1045, 876, 783,
694; HRMS (m/z): [M + H]+ calcd for C20H21O5, 341.1384;
found, 341.1358; HPLC (method 1): tR = 18.8 min, purity
98.6%.
(2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(4-((Z)-styryl)phenyl)tetrahydro-
furan-2-carboxamide (20a). A 2.5
M
solution of sodium M.p.: 136 °C; [α]2D0 = +54.9 (3.1; CH2Cl2); 1H NMR (CDCl3):
methoxide in methanol (0.12 mL, 0.29 mmol) was added to a δ 2.90–2.96 (m, 4H, ArCH2CH2Ar), 3.85 (s, 3H, OCH3), 4.24 (t,
solution of 19a (40 mg, 0.12 mmol) and hydroxylamine hydro- J = 3.9 Hz, 1H, 4-H), 4.70–4.76 (m, 2H, 2-H, 3-H), 5.13 (d, J =
chloride (18 mg, 0.26 mmol) in methanol (20 mL) and the 3.9 Hz, 1H, 5-H), 7.17–7.24 (m, 5H, Harom.), 7.25–7.31 (m, 2H,
mixture was stirred at ambient temperature for 16 h. Then the
H
arom.), 7.38–7.41 (m, 2H, Harom.); 13C NMR (CDCl3): δ 37.8
solvent was evaporated. After the addition of HCl (1 M), the (1C, ArCH2CH2Ar), 37.9 (1C, ArCH2CH2Ar), 52.7 (1C, OCH3),
mixture was extracted with ethyl acetate (3×). The combined 73.7 (1C, C-4), 74.2 (1C, C-3), 78.9 (1C, C-2), 83.6 (1C, C-5),
organic layers were dried (Na2SO4), filtered and evaporated. 126.1 (1C, Carom.), 127.0 (2C, Carom.), 128.5 (2C, Carom.), 128.6
The residue was purified by flash column chromatography (2C, Carom.), 128.8 (2C, Carom.), 133.3 (1C, Carom.), 141.8 (1C,
(1 cm, 5 mL, CH2Cl2–methanol = 9/1, Rf = 0.43) to give 20a as a
C
arom.), 142.0 (1C, Carom.), 172.6 (1C, CvO); IR (neat): ν [cm−1] =
3332, 3028, 2949, 2866, 1714, 1440, 1227, 1081, 887, 781,
colorless solid (16 mg, 0.047 mmol, 40%). M.p.: 145 °C; [α]D20
=
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704; HRMS (m/z): [M + H]+ calcd for C20H23O5, 343.1540; (1C, C-1′phenyl), 172.6 (1C, CvO); IR (neat): ν [cm−1] = 33791,
found, 343.1533; HPLC (method 1): tR = 19.41 min, purity 2932, 2874, 1745, 1506, 1437, 1221, 1090, 817, 742; HRMS
98.9%.
(2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(4-phenethylphenyl)tetrahydro-
furan-2-carboxamide (23a). Hydroxylamine hydrochloride
(m/z): [M + H]+ calcd for C18H23O5, 319.1540; found, 319.1571;
HPLC (method 1): tR = 19.3 min, purity 99.2%.
(2S,3R,4S,5S)-Methyl
3,4-dihydroxy-5-(4-(2-phenylethynyl)-
(43 mg, 0.61 mmol) and sodium methoxide (44 mg, phenyl)-tetrahydrofuran-2-carboxylate (25a). Under a N2 atmos-
0.82 mmol) were added to a solution of 22a (70 mg, phere triethylamine (0.54 mL, 3.85 mmol), copper(I) iodide
0.20 mmol) in methanol (20 mL) and the mixture was stirred (21 mg, 0.11 mmol) and tetrakis(triphenylphosphine)palla-
at ambient temperature for 48 h. Then the solvent was evapo- dium(0) (63 mg, 0.055 mmol) were added to a solution of 9a
rated. The residue was dissolved in ethyl acetate and extracted (200 mg, 0.55 mmol) in acetonitrile (10 mL). Then a solution
with HCl (1 M). The aqueous phase was then extracted with of phenylacetylene (0.5 mL, 4.55 mmol) in acetonitrile (5 mL)
ethyl acetate (3×) and the combined organic layers were dried was added dropwise over a period of 2 h. After evaporation of
(Na2SO4), filtered and evaporated. The residue was purified by the solvent the residue was purified by flash column chromato-
flash column chromatography (1 cm, 5 mL, CH2Cl2–methanol = graphy (2 cm, 10 mL, n-hexane–ethyl acetate = 1/1, Rf = 0.22) to
9.5/0.5 + 0.1% TFA). The fractions containing 23a were col- give 25a as a colorless solid (180 mg, 0.53 mmol, 97%). M.p.:
1
lected and evaporated. The residue was dissolved in ethyl 147 °C; [α]2D0 = +67.8 (1.8; CH2Cl2); H NMR (CDCl3): δ 2.85 (s
acetate and extracted with HCl (1 M). The aqueous phase was br, 1H, OH), 3.18 (s br, 1H, OH), 3.86 (s, 3H, OCH3), 4.25–4.29
then extracted with ethyl acetate (3×) and the combined (m, 1H, 4-H), 4.71–4.76 (m, 2H, 2-H, 3-H), 5.15 (d, J = 4.0 Hz,
organic layers were dried (Na2SO4), filtered and evaporated to 1H, 5-H), 7.32–7.38 (m, 3H, Harom.), 7.45–7.49 (m, 2H, Harom.),
give 23a as a colorless solid (19 mg, 0.06 mmol, 27%). M.p.: 7.52–7.58 (m, 4H, Harom.); 13C NMR (CDCl3): δ 52.8 (1C, OCH3),
161 °C; TLC (CH2Cl2–methanol = 9/1): Rf = 0.24; [α]2D0 = +51.1 73.8 (1C, C-4), 74.1 (1C, C-3), 78.9 (1C, C-2), 83.4 (1C, C-5), 89.3
(2.0, methanol); 1H NMR (D3COD): δ 2.90 (s, 4H, ArCH2CH2Ar), (1C, CuC), 89.8 (1C, CuC), 123.2 (1C, Carom.), 123.3 (1C,
4.14 (t, J = 4.3 Hz, 1H, 4-H), 4.45 (d, J = 7.3 Hz, 1H, 2-H), 4.71
Carom.), 127.0 (2C, Carom.), 128.4 (1C, Carom.), 128.5 (2C, Carom.),
(dd, J = 7.3/4.7 Hz, 1H, 3-H), 4.99 (d, J = 3.9 Hz, 1H, 5-H), 131.7 (2C, Carom.), 131.8 (2C, Carom.), 136.2 (1C, Carom.), 172.6
7.12–7.19 (m, 5H, Harom.), 7.21–7.26 (m, 2H, Harom.), 7.33–7.36 (1C, CvO); IR (neat): ν [cm−1] = 3443, 3060, 2957, 2892, 1741,
(m, 2H, Harom.); 13C NMR (D3COD): δ 38.9 (1C, ArCH2CH2Ar), 1514, 1440, 1408, 1221, 1092, 765, 692; HRMS (m/z): [M + Na]+
39.1 (1C, ArCH2CH2Ar), 74.3 (1C, C-3), 74.8 (1C, C-4), 80.0 (1C, calcd for C20H18O5Na, 361.1046; found, 361.1046; HPLC
C-2), 85.5 (1C, C-5), 126.9 (1C, Carom.), 128.6 (2C, Carom.), 129.0 (method 1): tR = 19.2 min, purity 99.5%.
(2S,3R,4S,5S)-5-(4-(Hex-1-yn-1-yl)phenyl)-N,3,4-trihydroxytetra-
hydrofuran-2-carboxamide (28a). Hydroxylamine hydrochloride
(118 mg, 1.7 mmol) and sodium methoxide (129 mg,
2.38 mmol) were added to a solution of 24a (107 mg,
0.34 mmol) in methanol (20 mL) and the mixture was stirred
at ambient temperature for 48 h. Then the solvent was evapo-
rated. The residue was dissolved in ethyl acetate and extracted
(2C, Carom.), 129.3 (2C, Carom.), 129.5 (2C, Carom.), 135.9 (1C,
arom.), 142.4 (1C, Carom.), 143.1 (1C, Carom.), 169.4 (1C, CvO);
IR (neat): ν [cm−1] = 3393, 3306, 3027, 2922, 1654, 1452, 1139,
1053, 827, 780, 697; HRMS (m/z): [M
H]+ calcd for
C
+
C19H22NO5, 344.1492; found, 344.1527; HPLC (method 2): tR
=
15.5 min, purity 99.4%.
(2S,3R,4S,5S)-Methyl 5-(4-(hex-1-yn-1-yl)phenyl)-3,4-dihydroxy-
tetrahydrofuran-2-carboxylate (24a). Under a N2 atmosphere tri- with HCl (1 M). The aqueous phase was then extracted with
ethylamine (0.4 mL, 2.9 mmol), copper(I) iodide (16 mg, ethyl acetate (3×) and the combined organic layers were dried
0.08 mmol) and tetrakis(triphenylphosphine)palladium(0) (Na2SO4), filtered and evaporated. The residue was purified by
(48 mg, 0.04 mmol) were added to a solution of 9a (150 mg, flash column chromatography (1 cm, 5 mL, CH2Cl2–methanol =
0.41 mmol) in acetonitrile (10 mL). Then a solution of 1- 9.5/0.5 + 0.1% TFA). The fractions containing 28a were col-
hexyne (0.4 mL, 3.4 mmol) in acetonitrile (5 mL) was added lected and evaporated. The residue was dissolved in ethyl
dropwise over a period of 2 h. After evaporation of the solvent acetate and extracted with HCl (1 M). The aqueous phase was
the residue was purified by flash column chromatography then extracted with ethyl acetate (3×) and the combined
(2 cm, 10 mL, cyclohexane–ethyl acetate = 1/1, Rf = 0.27) to give organic layers were dried (Na2SO4), filtered and evaporated to
24a as a colorless solid (121 mg, 0.38 mmol, 92%). M.p.: 82 °C; give 28a as a colorless solid (20 mg, 0.06 mmol, 18%). M.p.:
1
[α]2D0 = +77.3 (2.5; CH2Cl2); H NMR (CDCl3): δ 0.90 (t, J = 7.3 127 °C; TLC (CH2Cl2–methanol = 9/1): Rf = 0.50; [α]2D0 = +56.3
Hz, 3H, CH2CH2CH2CH3), 1.42–1.53 (m, 2H, CH2CH2CH2CH3), (2.8; methanol); 1H NMR (D3COD): δ 0.97 (t, J = 7.2 Hz, 3H,
1.54–1.63 (m, 2H, CH2CH2CH2CH3), 2.41 (t, J = 7.0 Hz, 2H, CH2CH2CH2CH3), 1.45–1.62 (m, 4H, CH2CH2CH2CH3), 2.41 (t,
CH2CH2CH2CH3), 3.84 (s, 3H, OCH3), 4.23 (t, J = 3.8 Hz 1H, J = 6.9 Hz, 2H, CH2CH2CH2CH3), 4.17 (t, J = 4.4 Hz, 1H, 4-H),
4-H), 4.68–4.74 (m, 2H, 2-H, 3-H), 5.11 (d, J = 3.8 Hz, 1H, 5-H), 4.46 (d, J = 7.1 Hz, 1H, 2-H), 4.70 (dd, J = 7.1/4.8 Hz, 1H, 3-H),
7.37–7.43 (m, 4H, Harom.); 13C NMR (CDCl3): δ 13.8 (1C, 5.01 (d, J = 4.1 Hz, 1H, 5-H), 7.30–7.34 (m, 2H, Harom.),
CH2CH2CH2CH3), 19.3 (1C, CH2CH2CH2CH3), 22.2 (1C, 7.36–7.40 (m, 2H, Harom.); 13C NMR (D3COD): δ 14.0 (1C,
CH2CH2CH2CH3), 31.0 (1C, CH2CH2CH2CH3), 52.8 (1C, OCH3), CH2CH2CH2CH3), 19.7 (1C, CH2CH2CH2CH3), 23.0 (1C,
73.8 (1C, C-4), 74.1 (1C, C-3), 78.9 (1C, C-2), 80.4 (1C, Ar-CuC), CH2CH2CH2CH3), 32.1 (1C, CH2CH2CH2CH3), 74.3 (1C, C-3),
83.4 (1C, C-5), 90.9 (1C, Ar-CuC), 124.1 (1C, C-4′phenyl), 126.8 74.8 (1C, C-4), 80.1 (1C, C-2), 81.5 (1C, Ar-CuC), 85.3 (1C, C-5),
(2C, C-2′phenyl, C-6′phenyl), 131.7 (2C, C-3′phenyl, C-5′phenyl), 135.2 90.7 (1C, Ar-CuC), 124.6 (1C, C-4′phenyl), 128.6 (2C, C-2′phenyl
,
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C-6′phenyl), 131.9 (2C, C-3′phenyl
,
C-5′phenyl), 138.1 (1C, 135.3 (1C, C-1phenyl), 167.7 (1C, COOCH3); IR (neat): ν [cm−1] =
C-1′phenyl), 169.3 (1C, CvO); IR (neat): ν [cm−1] = 3246, 2930, 2982, 2959, 2160, 1759, 1381, 1211, 1099, 864, 841, 752; HRMS
1638, 1510, 1131, 1038, 779; HRMS (m/z): [M + H]+ calcd for (m/z): [M + H]+ calcd for C20H27O5Si: 375.1622; found:
C17H22NO5, 320.1493; found, 320.1502; HPLC (method 2): tR
16.0 min, purity 96.0%.
(2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(4-(2-phenylethynyl)phenyl)-
=
375.1631; HPLC (method 1): tR = 22.1 min, purity 97.2%.
(3aR,4S,6S,6aR)-Methyl 6-(4-ethynylphenyl)-2,2-dimethyltetra-
hydrofuro[3,4-d][1,3]dioxole-4-carboxylate (31a). Silver nitrate
tetrahydrofuran-2-carboxamide (29a). Hydroxylamine hydro- (20 mg, 0.12 mmol) and water (2.11 mL, 0.117 mol) were
chloride (63 mg, 0.90 mmol) and sodium methoxide (73 mg, added to a solution of 30a (439 mg, 1.17 mmol) in acetone
1.35 mmol) were added to a solution of 25a (100 mg, (20 mL). The mixture was stirred for 24 h at room temperature
0.30 mmol) in methanol (20 mL) and the mixture was stirred in the dark. Then, water was added and mixture was extracted
at ambient temperature for 16 h. Then the solvent was evapor- with CH2Cl2 (3×). The combined organic layers were dried
ated. The residue was dissolved in ethyl acetate and extracted (Na2SO4) and filtered and the solvent was removed in vacuo.
with HCl (1 M). The aqueous phase was then extracted with The residue was purified by flash column chromatography
ethyl acetate (3×) and the combined organic layers were dried (2 cm, 10 mL, cyclohexane–EtOAc = 3/1, Rf = 0.23) to give 31a
(Na2SO4), filtered and evaporated. The residue was purified by as a colorless solid (261 mg, 0.86 mmol, 74%). M.p.: 144 °C;
1
flash column chromatography (1 cm, 5 mL, CH2Cl2–methanol = [α]2D0 = +53.8 (1.3; CH2Cl2); H NMR (CDCl3): δ 1.26 (s, 3H, C
9.5/0.5 + 0.1% TFA). The fractions containing 29a were col- (CH3)2), 1.41 (s, 3H, C(CH3)2), 3.06 (s, 1H, PhCuCH), 3.85 (s,
lected and evaporated. The residue was dissolved in ethyl 3H, COOCH3), 4.39 (d, J = 4.5 Hz, 1H, 4-H), 4.67 (d, J = 3.6 Hz,
acetate and extracted with HCl (1 M). The aqueous phase was 1H, 6-H), 4.82 (dd, J = 5.9/3.6 Hz, 1H, 6a-H), 5.08 (dd, J = 5.9/
then extracted with ethyl acetate (3×) and the combined 4.5 Hz, 1H, 3a-H), 7.41–7.51 (m, 4H, Harom.); 13C NMR (CDCl3):
organic layers were dried (Na2SO4), filtered and evaporated to δ 25.0 (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 52.3 (1C, COOCH3),
give 29a as a colorless solid (10 mg, 0.03 mmol, 9%). M.p.: 77.5 (1C, PhCuCH), 80.8 (1C, C-4), 81.8 (1C, C-6a), 82.0
115 °C; TLC (CH2Cl2–methanol = 9/1): Rf = 0.44; [α]2D0 = +37.4 (1C, C-3a), 83.2 (1C, C-6), 83.7 (1C, PhCuCH), 113.6 (1C, C
(1.5; methanol); 1H NMR (D3COD): δ 4.22 (t, J = 4.4 Hz, 1H, (CH3)2), 121.9 (1C, C-4phenyl), 127.5 (2C, C-2phenyl, C-6phenyl),
4-H), 4.48 (d, J = 7.0 Hz, 1H, 2-H), 4.72 (dd, J = 7.0/4.8 Hz, 1H, 131.9 (2C, C-3phenyl, C-5phenyl), 135.7 (1C, C-1phenyl), 167.7 (1C,
3-H), 5.06 (d, J = 4.1 Hz, 1H, 5-H), 7.34–7.40 (m, 3H, Harom.), COOCH3); IR (neat): ν [cm−1] = 3267, 2986, 1751, 1439, 1300,
7.46–7.53 (m, 6H, Harom.); 13C NMR (D3COD): δ 74.3 (1C, C-3), 1211, 1099, 822, 748, 679; HRMS (m/z): [M + H]+ calcd for
74.8 (1C, C-4), 80.2 (1C, C-2), 85.3 (1C, C-5), 90.0 (1C, CuC), C17H19O5: 303.1227; found: 303.1179; HPLC (method 1): tR
90.2 (1C, CuC), 123.6 (1C, Carom.), 124.7 (1C, Carom.), 128.8 18.0 min, purity 95.9%.
=
(3aR,4S,6S,6aR)-Methyl
2,2-dimethyl-6-(4-(phenylbuta-1,3-
(2C, Carom.), 129.4 (1C, Carom.), 129.6 (2C, Carom.), 132.0 (2C,
diyn-1-yl)phenyl)tetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate
(32a). Copper(I) chloride (3 mg, 0.03 mmol) was added to an
aqueous solution (30% v/v) of n-butylamine (10 mL) at
ambient temperature. The resulting blue color was discharged
by addition of a few crystals of hydroxylamine hydrochloride.
Addition of 31a (180 mg, 0.60 mmol) at ambient temperature
C
arom.), 132.5 (2C, Carom.), 139.2 (1C, Carom.), 168.0 (1C, CvO);
IR (neat): ν [cm−1] = 3229, 2925, 1669, 1201, 1134, 1027, 755;
HRMS (m/z): [M + H]+ calcd for C19H18NO5, 340.1179; found,
340.1200; HPLC (method 2): tR = 15.8 min, purity 95.2%.
(3aR,4S,6S,6aR)-Methyl
2,2-dimethyl-6-(4-((trimethylsilyl)-
[1,3]dioxole-4-carboxylate
ethynyl)phenyl)tetrahydrofuro[3,4-d]
(30a). Under a nitrogen atmosphere copper(I) iodide (5 mg, led to a yellow acetylide suspension that was immediately
0.03 mmol), tetrakis(triphenylphosphine)palladium(0) (31 mg, cooled by a water-ice bath. Then, (bromoethynyl)benzene
0.03 mmol) and triethylamine (0.26 mL, 1.90 mmol) were (0.09 mL, 0.71 mmol) was added at once. Then the water-ice
added to a solution of 8a (110 mg, 0.27 mmol) in dry aceto- bath was removed and Et2O (5 mL) was added. The reaction
nitrile (20 mL). Then a solution of trimethylsilylacetylene mixture was stirred for 30 min. During this time hydroxyl-
(0.06 mL, 0.41 mmol) in dry acetonitrile (5 mL) was added amine hydrochloride was added when the mixture turned
dropwise over a period of 3 h. Then, the solvent was removed green or blue. When the color of the suspension turned rusty,
in vacuo. The residue was purified by flash column chromato- the reaction was terminated. Then, water was added and the
graphy (1 cm, 5 mL, cyclohexane–EtOAc = 3/1, Rf = 0.29) to give mixture was extracted with Et2O (3×). The combined organic
30a as a colorless solid (99 mg, 0.26 mmol, 97%). M.p.: 167 °C; layers were dried (Na2SO4) and filtered and the solvent was
[α]2D0 = +32.7 (1.2; CH2Cl2); 1H NMR (CDCl3): δ 0.24 (s, 9H, removed in vacuo. The residue was purified by flash column
Si(CH3)3), 1.25 (s, 3H, C(CH3)2), 1.39 (s, 3H, C(CH3)2), 3.85 (s, chromatography (1 cm, 5 mL, cyclohexane–EtOAc = 3/1, Rf =
3H, COOCH3), 4.39 (d, J = 4.5 Hz, 1H, 4-H), 4.67 (d, J = 3.6 Hz, 0.23) to give 32a as a colorless solid (110 mg, 0.27 mmol,
1H, 6-H), 4.81 (dd, J = 5.8/3.6 Hz, 1H, 6a-H), 5.08 (dd, J = 5.8/ 46%). M.p.: 177 °C; [α]2D0 = +64.7 (3.7; CH2Cl2); 1H NMR
4.5 Hz, 1H, 3a-H), 7.38–7.48 (m, 4H, Harom.); 13C NMR (CDCl3): (CDCl3): δ 1.27 (s, 3H, C(CH3)2), 1.42 (s, 3H, C(CH3)2), 3.85 (s,
δ 0.13 (3C, Si(CH3)3), 25.1 (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 3H, COOCH3), 4.40 (d, J = 4.5 Hz, 1H, 4-H), 4.68 (d, J = 3.6 Hz,
52.3 (1C, COOCH3), 80.8 (1C, C-4), 81.8 (1C, C-6a), 82.0 (1C, 1H, 6-H), 4.83 (dd, J = 5.9/3.6 Hz, 1H, 6a-H), 5.09 (dd, J = 5.9/
C-3a), 83.3 (1C, C-6), 94.3 (1C, PhCuCSi(CH3)3), 105.2 (1C, 4.5 Hz, 1H, 3a-H), 7.31–7.40 (m, 3H, Harom.), 7.43–7.47 (m, 2H,
PhCuCSi(CH3)3), 113.6 (1C, C(CH3)2), 122.9 (1C, C-4phenyl),
127.4 (2C, C-2phenyl, C-6phenyl), 131.8 (2C, C-3phenyl, C-5phenyl), (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 52.3 (1C, COOCH3), 74.1 (2C,
H
arom.), 7.51–7.55 (m, 4H, Harom.); 13C NMR (CDCl3): δ 25.0
6066 | Org. Biomol. Chem., 2013, 11, 6056–6070
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PhCuCCuCPh), 80.8 (1C, C-4), 81.7 (2C, PhCuCCuCPh), ν [cm−1] = 3310, 2955, 2897, 1639, 1481, 1315, 1142, 1049, 787,
81.8 (1C, C-6a), 82.0 (1C, C-3a), 83.2 (1C, C-6), 113.7 (1C, 748; HRMS (m/z): [M + H]+ calcd for C21H18NO5: 364.1179;
C(CH3)2), 121.6 (1C, Carom.), 121.9 (1C, Carom.), 127.7 (2C, found: 364.1191; HPLC (method 2): tR = 16.2 min, purity
Carom.), 128.6 (2C, Carom.), 129.3 (1C, Carom.), 132.3 (2C, Carom.), 95.1%.
(3aR,4S,6S,6aR)-4-(Dimethoxymethyl)-6-(4-iodophenyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxole (35). Sodium meta-
periodate (885 mg, 4.14 mmol) was added to a solution of 7a
(1.12 g, 2.76 mmol) in methanol (30 mL). The reaction was
stirred at ambient temperature for 3 h. Then, water was added
and the mixture was extracted with ethyl acetate (3×). The com-
132.6 (2C, Carom.), 136.2 (1C, Carom.), 167.7 (1C, COOCH3); IR
(neat): ν [cm−1] = 2982, 2940, 1755, 1435, 1373, 1211, 1099,
829, 752, 687; HRMS (m/z): [M + H]+ calcd for C25H23O5:
403.1540; found: 403.1562; HPLC (method 1): tR = 23.3 min,
purity 97.6%.
(2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-(4-(phenylbuta-1,3-diyn-
1-yl)phenyl)tetrahydrofuran-2-carboxylate (33a). p-Toluenesulfo- bined organic layers were dried (Na2SO4) and filtered and the
nic acid monohydrate (14 mg, 0.07 mmol) was added to a solu- solvent was removed in vacuo. The residue was dissolved in
tion of 32a (150 mg, 0.37 mmol) in methanol (20 mL). The methanol (30 mL) and p-toluenesulfonic acid monohydrate
mixture was heated to reflux for 48 h. Then the solvent was (105 mg, 0.55 mmol) was added. The reaction was heated to
removed in vacuo. Then, methanol (20 mL) was added and the reflux overnight. Then water was added and the mixture was
mixture was heated to reflux for an additional 48 h. Then the extracted with ethyl acetate (3×). The combined organic layers
solvent was removed in vacuo and the residue was purified by were dried (Na2SO4) and filtered and the solvent was removed
flash column chromatography (1 cm, 5 mL, cyclohexane– in vacuo. The residue was purified by flash column chromato-
EtOAc = 1/1, Rf = 0.21) to give 33a as a yellowish solid (105 mg, graphy (3 cm, 20 mL, cyclohexane–ethyl acetate = 8/2 → 1/2) to
0.29 mmol, 78%). M.p.: 157 °C; [α]2D0 = +72.6 (4.4; CH2Cl2); give 35 (cyclohexane–ethyl acetate = 8/2, Rf = 0.25) as a color-
1H NMR (CDCl3): δ 2.90 (d, J = 9.6 Hz, 1H, OH), 3.13–3.20 (m, less solid (890 mg, 2.12 mmol, 77%) and 36 (cyclohexane–ethyl
1H, OH), 3.85 (s, 3H, COOCH3), 4.27 (dt, J = 9.6/3.9 Hz, 1H, acetate = 1/2, Rf = 0.32) as a colorless solid (80 mg, 0.21 mmol,
4-H), 4.70–4.75 (m, 2H, 2-H, 3-H), 5.14 (d, J = 3.9 Hz, 1H, 5-H), 8%).
7.30–7.41 (m, 3H, Harom.), 7.42–7.50 (m, 2H, Harom.), 7.50–7.60
35: m.p.: 154 °C; [α]2D0 = +39.0 (c = 2.0; CH2Cl2); 1H NMR
(m, 4H, Harom.); 13C NMR (CDCl3): δ 52.8 (1C, COOCH3), 73.9 (CDCl3): δ 1.27 (s, 3H, C(CH3)2), 1.43 (s, 3H, C(CH3)2), 3.48 (s,
(1C, C-4), 74.0 (1C, PhCuCCuCPh), 74.1 (1C, C-3), 74.3 (1C, 3H, OCH3), 3.51 (s, 3H, OCH3), 3.71 (dd, J = 7.7/3.8 Hz, 1H,
PhCuCCuCPh), 78.9 (1C, C-2), 81.5 (1C, PhCuCCuCPh), 4-H), 4.55 (d, J = 3.8 Hz, 1H, 6-H), 4.74 (d, J = 7.7 Hz, 1H,
81.8 (1C, PhCuCCuCPh), 83.4 (1C, C-5), 121.6 (1C, Carom.), (H3CO)2CH), 4.76 (dd, J = 6.0/3.8 Hz, 1H, 6a-H), 4.83 (dd, J =
121.9 (1C, Carom.), 127.1 (2C, Carom.), 128.6 (2C, Carom.), 129.4 6.0/3.8 Hz, 1H, 3a-H), 7.11–7.16 (m, 2H, 2′-H4-iodophenyl
,
(1C, Carom.), 132.7 (4C, Carom.), 137.4 (1C, Carom.), 172.7 (1C, 6′-H4-iodophenyl), 7.63–7.68 (m, 2H, 3′-H4-iodophenyl, 5′-H4-iodophenyl);
COOCH3); IR (neat): ν [cm−1] = 3480, 3426, 2951, 1759, 1740, 13C NMR (CDCl3): δ 24.6 (1C, C(CH3)2), 25.8 (1C, C(CH3)2), 54.0
1439, 1227, 1076, 752, 687; HRMS (m/z): [M + H]+ calcd for (1C, OCH3), 55.5 (1C, OCH3), 80.6 (1C, C-4), 81.2 (1C, C-3a),
C22H19O5: 363.1227; found: 363.1257; HPLC (method 1): tR
20.4 min, purity 95.2%.
=
82.1 (1C, C-6a), 83.4 (1C, C-6), 93.7 (1C, C-4′4-iodophenyl), 102.5
(1C, (H3CO)2CH), 112.6 (1C, C(CH3)2), 129.6 (2C, C-2′4-iodophenyl
,
(2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(4-(phenylbuta-1,3-diyn-1-yl)-
phenyl)tetrahydrofuran-2-carboxamide (34a). Hydroxylamine
hydrochloride (41 mg, 0.59 mmol) and a 2 M solution of
sodium methanolate in methanol (0.29 mL, 0.59 mmol) were
added to a solution of 33a (85 mg, 0.23 mmol) in dry methanol
(20 mL) and the mixture was stirred at ambient temperature
C-6′4-iodophenyl), 135.3 (1C, C-1′4-iodophenyl), 137.0 (2C,
C-3′4-iodophenyl, C-5′4-iodophenyl); IR (neat): ν [cm−1] = 2978, 2940,
1481, 1381, 1269, 1200, 1092, 1072, 976, 814; HRMS (m/z):
[M + H]+ calcd for C16H22IO5: 421.0506; found: 421.0529;
HPLC (method 1): tR = 19.9 min, purity 95.1%.
(2S,3R,4S,5S)-2-(Dimethoxymethyl)-5-(4-iodophenyl)tetrahydro-
for 16 h. Then water was added and the mixture was extracted furan-3,4-diol (36). p-Toluenesulfonic acid monohydrate
with ethyl acetate (3×). The combined organic layers were dried (105 mg, 0.55 mmol) was added to a solution of 35 (1.16 g,
(Na2SO4) and filtered and the solvent was removed in vacuo. 2.76 mmol) in methanol (20 mL). The mixture was heated to
The residue was purified by flash column chromatography reflux for 64 h. Then, water was added and the mixture was
(1 cm, 5 mL, CH2Cl2–methanol = 9/1, Rf = 0.27) to give 34a as a extracted with ethyl acetate (3×). The combined organic layers
yellowish solid (32 mg, 0.09 mmol, 38%). M.p.: 178 °C were dried (Na2SO4) and filtered and the solvent was removed
(decomposition); [α]D20 = +100.1 (1.0; methanol); 1H NMR in vacuo. The residue was purified by flash column chromato-
(D3COD): δ 4.21–4.28 (m, 1H, 4-H), 4.47–4.53 (m, 1H, 2-H), graphy (3 cm, 20 mL, cyclohexane–ethyl acetate = 1/2, Rf =
4.69–4.75 (m, 1H, 3-H), 5.05–5.10 (m, 1H, 5-H), 7.34–7.45 (m, 0.32) to give 36 as a colorless solid (365 mg, 0.96 mmol, 35%).
1
3H, Harom.), 7.45–7.59 (m, 6H, Harom.); 13C NMR (D3COD): δ M.p.: 48 °C; [α]2D0 = +65.8 (c = 3.7; CH2Cl2); H NMR (CDCl3): δ
74.1 (1C, C-3), 74.3 (1C, PhCuCCuCPh), 74.4 (1C, 2.77–2.90 (d br, J = 6.5 Hz, 1H, PhCHCHOH), 3.23–3.32 (m br,
PhCuCCuCPh), 74.6 (1C, C-4), 80.0 (1C, C-2), 82.0 (1C, 1H, (H3CO)2CHCHCHOH), 3.54 (s, 3H, OCH3), 3.56 (s, 3H,
PhCuCCuCPh), 82.2 (1C, PhCuCCuCPh), 85.0 (1C, C-5), OCH3), 4.13 (dd, J = 6.5/4.6 Hz, 1H, 2-H), 4.24–4.30 (m, 1H,
121.8 (1C, Carom.), 122.8 (1C, Carom.), 128.7 (2C, Carom.), 129.5 4-H), 4.52–4.58 (m, 1H, 3-H), 4.71 (d, J = 4.6 Hz, 1H,
(2C, Carom.), 130.3 (1C, Carom.), 132.8 (2C, Carom.), 133.3 (2C, (H3CO)2CH), 4.90 (d, J = 4.7 Hz, 1H, 5-H), 7.12–7.16 (m,
Carom.), 140.1 (1C, Carom.), 169.0 (1C, CONHOH); IR (neat): 2H, 2′-H4-iodophenyl
,
6′-H4-iodophenyl), 7.66–7.70 (m, 2H, 3′-
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H4-iodophenyl, 5′-H4-iodophenyl); 13C NMR (CDCl3): δ 55.7 (1C, (1H), CH2OHE1 (0.7H), 2-HE1 (0.7H)), 4.82 (dd, J = 5.2/2.3 Hz,
OCH3), 55.8 (1C, OCH3), 73.2 (1C, C-3), 73.5 (1C, C-4), 78.6 (1C, 0.3H, 2-HE2), 6.55 (d, J = 5.9 Hz, 0.7H, OCHOHE1), 6.70 (d, J =
C-2), 82.1 (1C, C-5), 93.5 (1C, C-4′4-iodophenyl), 103.9 (1C, 5.2 Hz, 0.3H, OCHOHE2), 7.20–7.28 (m, 2H, 2′-H4-iodophenyl
,
(H3CO)2CH), 129.1 (2C, C-2′4-iodophenyl, C-6′4-iodophenyl), 136.7 6′-H4-iodophenyl), 7.69–7.74 (m, 2H, 3′-H4-iodophenyl, 5′-H4-iodophenyl),
(1C, C-1′4-iodophenyl), 137.3 (1C, C-3′4-iodophenyl, C-5′4-iodophenyl); ratio of epimers E1 : E2 = 70 : 30; 13C NMR (DMSO-D6): δ 57.6
IR (neat): ν [cm−1] = 3468, 3348, 2932, 2835, 1481, 1327, 1200, (0.3C, CH2OHE2), 59.6 (0.7C, CH2OHE1), 63.0 (0.7C, C-6E1), 64.1
1096, 1072, 748; HRMS (m/z): [M + H]+ calcd for C13H18IO5: (0.3C, C-6E2), 69.3 (0.3C, C-5E2), 70.0 (0.7C, C-5E1), 73.9 (0.3C,
381.0193; found: 381.0204; HPLC (method 1): tR = 15.1 min, C-3E2), 75.1 (0.7C, C-3E1), 89.7 (0.7C, C-2E1), 90.9 (0.3C, C-2E2),
E1
purity 95.1%.
93.5 (1C, C-4′4-iodophenylE1+E2), 129.0 (1.4C, C-2′4-iodophenyl
,
C-6′4-iodophenylE1), 129.3 (0.6C, C-2′4-iodophenylE2, C-6′4-iodophenylE2),
(S)-2-((S)-2-Hydroxy-1-(4-iodophenyl)ethoxy)-3,3-dimethoxypropan-
1-ol (37). Sodium metaperiodate (1.26 g, 5.89 mmol) was
added to a solution of 36 (746 mg, 1.96 mmol) in methanol
(30 mL). The reaction was stirred at ambient temperature for
16 h. Then, water was added and the mixture was extracted
with ethyl acetate (3×). The combined organic layers were dried
(Na2SO4) and filtered and the solvent was removed in vacuo.
The residue was dissolved in methanol (20 mL) and sodium
E2
136.9 (0.6C, C-3′4-iodophenyl
,
C-5′4-iodophenylE2), 137.0
E1
(0.6C, C-3′4-iodophenyl
,
C-5′4-iodophenylE1), 138.5 (0.3C,
C-1′4-iodophenylE2), 139.1 (0.7C, C-1′4-iodophenylE1), ratio of
epimers E1 : E2 = 70 : 30; IR (neat): ν [cm−1] = 3298, 3156, 2967,
2889, 1485, 1312, 1138, 999, 972, 818; HPLC (method 1): tR
=
14.1 min, purity 96.4%.
(S)-Methyl 3-hydroxy-2-((S)-2-hydroxy-1-(4-iodophenyl)ethoxy)-
borohydride (371 mg, 9.81 mmol) was added. The reaction was propanoate (39). Sodium bicarbonate (375 mg, 4.46 mmol) was
stirred at ambient temperature for 30 min. Then hydrochloric added to a solution of H26 (75 mg, 0.22 mmol) in methanol–
acid (1 M) was added and the mixture was extracted with ethyl water (9/1, 10 mL). Then a 1 M solution of Br2 in methanol–
acetate (3×). The combined organic layers were dried (Na2SO4) water (9/1, 0.45 mL, 0.45 mmol) was added. The reaction was
and filtered and the solvent was removed in vacuo. The residue stirred at ambient temperature overnight. Then, sodium thio-
was purified by flash column chromatography (3 cm, 20 mL, sulfate and water were added and the mixture was extracted
cyclohexane–ethyl acetate = 1/2, Rf = 0.18) to give 37 as a color- with ethyl acetate (3×). The combined organic layers were dried
less oil (580 mg, 1.52 mmol, 77%). [α]2D0 = +54.9 (c = 13.5; (Na2SO4) and filtered and the solvent was removed in vacuo.
CH2Cl2); 1H NMR (CDCl3): δ 3.21 (s, 3H, OCH3), 3.39 (s, 3H, The residue was purified by flash column chromatography
OCH3), 3.51–3.57 (m, 1H, (H3CO)2CHCHCH2OH), 3.59–3.73 (1 cm, 5 mL, cyclohexane–ethyl acetate = 1/2, Rf = 0.14) to give
(m, 2H, PhCHCH2OH), 3.79 (dd,
(H3CO)2CHCHCH2OH), 3.88 (dd,
J
=
11.9/5.4 Hz, 1H, 39 as a colorless oil (68 mg, 0.19 mmol, 83%). [α]2D0 = +32.7 (c =
=
11.9/3.9 Hz, 1H, 2.9; CH2Cl2); 1H NMR (CDCl3): δ 3.62 (s, 3H, COOCH3), 3.67
J
(H3CO)2CHCHCH2OH), 4.26 (d, J = 5.3 Hz, 1H, (H3CO)2CHCH- (dd, J = 12.1/2.8 Hz, 1H, PhCHCH2OH), 3.78 (dd, J = 12.1/9.0
CH2OH), 4.60 (dd, J = 8.0/3.9 Hz, 1H, PhCHCH2OH), 7.09–7.16 Hz, 1H, PhCHCH2OH), 3.85 (dd,
J
=
=
12.2/5.6 Hz, 1H,
12.2/2.8 Hz, 1H,
(m, 2H, 2′-H4-iodophenyl, 6′-H4-iodophenyl), 7.65–7.72 (m, 2H, H3COOCCHCH2OH), 3.98 (dd,
J
3′-H4-iodophenyl, 5′-H4-iodophenyl); 13C NMR (CDCl3): δ 55.5 (1C, H3COOCCHCH2OH), 4.04 (dd, J = 5.6/2.8 Hz, 1H, H3COOCCH-
OCH3), 56.1 (1C, OCH3), 61.1 (1C, CH2OH), 67.6 (1C, CH2OH), CH2OH), 4.48 (s br, 1H, OH), 4.60 (s br, 1H, OH), 4.61 (dd, J =
79.2 (1C, (H3CO)2CHCHCH2OH), 82.7 (1C, PhCHCH2OH), 93.7 9.0/2.8 Hz, 1H, PhCHCH2OH), 7.07–7.13 (m, 2H,
(1C, C-4′4-iodophenyl), 105.9 (1C, (H3CO)2CH), 128.8 (2C, 2′-H4-iodophenyl, 6′-H4-iodophenyl), 7.65–7.70 (m, 2H, 3′-H4-iodophenyl
,
C-2′4-iodophenyl
,
C-6′4-iodophenyl), 137.6 (1C, C-3′4-iodophenyl
,
5′-H4-iodophenyl); 13C NMR (CDCl3): δ 52.4 (1C, COOCH3), 62.3
C-5′4-iodophenyl), 138.9 (1C, C-1′4-iodophenyl); IR (neat): ν [cm−1] = (1C, CH2OH), 66.9 (1C, CH2OH), 78.7 (1C, H3COOCCH-
3356, 2928, 2832, 1589, 1481, 1400, 1192, 1061, 976, 814; CH2OH), 83.1 (1C, PhCHCH2OH), 94.3 (1C, C-4′4-iodophenyl),
HRMS (m/z): [M + H]+ calcd for C13H20IO5: 383.0350; found: 129.1 (2C, C-2′4-iodophenyl, C-6′4-iodophenyl), 137.3 (1C, C-1′4-iodophenyl),
383.0365; HPLC (method 1): tR = 16.3 min, purity 95.3%.
(3S,5S)-3-(Hydroxymethyl)-5-(4-iodophenyl)-1,4-dioxan-2-ol
(38). A solution of 37 (535 mg, 1.40 mmol) in 1 M hydrochloric
acid–THF (1/1, 20 mL) was heated to reflux overnight. Then, a
saturated aqueous solution of sodium bicarbonate was added
and the mixture was extracted with ethyl acetate (3×). The com-
137.8 (1C, C-3′4-iodophenyl, C-5′4-iodophenyl), 171.2 (1C, COOCH3);
IR (neat): ν [cm−1] = 3364, 2951, 2878, 1736, 1589, 1435, 1207,
1057, 818, 729; HRMS (m/z): [M + H]+ calcd for C12H16IO5:
367.0037; found: 367.0053; HPLC (method 1): tR = 15.4 min,
purity 96.7%.
(S)-Methyl
3-hydroxy-2-((S)-2-hydroxy-1-(4-((4-(morpholino-
bined organic layers were dried (Na2SO4) and filtered and the methyl)phenyl)ethynyl)phenyl)-ethoxy)propanoate (41). Under a
solvent was removed in vacuo. The residue was purified by nitrogen atmosphere copper(I) iodide (19 mg, 0.10 mmol),
flash column chromatography (3 cm, 20 mL, cyclohexane– tetrakis(triphenylphosphine)palladium(0) (57 mg, 0.05 mmol)
ethyl acetate = 1/2, Rf = 0.26) to give 38 as a colorless solid and triethylamine (0.48 mL, 3.44 mmol) were added to a solu-
(382 mg, 1.14 mmol, 81%). M.p.: 108 °C; [α]2D0 = −8.5 (c = 4.6; tion of 39 (180 mg, 0.49 mmol) in dry acetonitrile (20 mL).
CH2Cl2); 1H NMR (DMSO-D6): δ 3.38–3.43 (m, 0.7H, 3-HE1), Then a solution of 4-(4-ethynylbenzyl)morpholine (26) (0.79 g,
3.48–3.69 (m, 3 × 0.7H + 4 × 0.3H, CH2OHE1+E2 (2H), 3-HE2 3.93 mmol) in dry acetonitrile (5 mL) was added dropwise over
(0.3H), 6-HE1+E2 (1H)), 3.96 (dd, J = 11.8/7.3 Hz, 0.7H, 6-HE1), a period of 3 h. Then, the solvent was removed in vacuo. The
4.11 (dd, J = 11.9/3.4 Hz, 0.3H, 6-HE2), 4.48 (dd, J = 6.0/5.1 Hz, residue was purified twice by flash column chromatography
0.3H, CH2OHE2), 4.71–4.76 (m, 3 × 0.7H + 1 × 0.3H, 5-HE1+E2 (1 cm, 5 mL, CH2Cl2–methanol = 19/1, Rf = 0.27) to give 41 as a
6068 | Org. Biomol. Chem., 2013, 11, 6056–6070
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yellowish solid (175 mg, 0.40 mmol, 81% yield). M.p.: 90 °C; compound (10 mM in DMSO) was applied onto circular filter
1
[α]2D0 = +43.3 (3.5; methanol); H NMR (CDCl3): δ 2.41–2.48 (m, paper (Ø 6 mm, thickness 0.75 mm, Carl Roth). Pure DMSO,
4H, N(CH2CH2)2), 3.51 (s, 2H, PhCH2N), 3.63 (s, 3H, COOCH3), serving as a negative, and CHIR-090,12 serving as a positive
3.69–3.75 (m, 5H, PhCHCH2OH (1H), N(CH2CH2)2), 3.84 (dd, control, were also spotted. The Petri dishes were incubated
J = 12.0/9.0, 1H, PhCHCH2OH), 3.88 (dd, J = 12.2/5.7, 1H, overnight at 37 °C and the diameter of the zone of growth inhi-
H3COOCCHCH2OH), 4.00 (dd, J = 12.2/3.0 Hz, 1H, H3COO- bition was measured for each compound.
CCHCH2OH), 4.09 (dd, J = 5.7/3.0 Hz, 1H, H3COOCCHCH2OH),
4.68 (dd, J = 9.0/2.7 Hz, 1H, PhCHCH2OH), 7.29–7.37 (m, 4H, LpxCC63A (pETEcLpxCC63A) was kindly provided by Carol
arom.), 7.44–7.53 (m, 4H, Harom.); 13C NMR (CDCl3): δ 52.3 Fierke.25 The C63A mutation lowers the undesired influence of
Protein purification. The plasmid for the expression of
H
(1C, COOCH3), 53.7 (2C, N(CH2CH2)2), 62.5 (1C, H3COOCCH- Zn2+ concentration on enzymatic activity. The purification of
CH2OH), 63.3 (1C, PhCH2N), 67.0 (1C, PhCHCH2OH), 67.1 (2C, LpxC was performed essentially as previously described.26
N(CH2CH2)2), 78.8 (1C, H3COOCCHCH2OH), 83.5 (1C, Weak anion exchange was performed with a column contain-
PhCHCH2OH), 88.9 (1C, PhCuCPh), 89.0 (1C, PhCuCPh), ing 30 mL diethylaminoethylcellulose (DEAE)-Sepharose fast
122.0 (1C, Carom.), 123.6 (1C, Carom.), 127.2 (2C, Carom.), 129.3 flow media (GE Healthcare). Eluted fractions containing the
(2C, Carom.), 131.7 (2C, Carom.), 131.9 (2C, Carom.), 137.7 (1C, desired enzyme were concentrated and desalted with mole-
C
arom.), 138.4 (1C, Carom.), 171.2 (1C, COOCH3); IR (neat): cular weight cut-off (MWCO) spin columns (10 kDa, PALL Cor-
ν [cm−1] = 3483, 3329, 2959, 2855, 1744, 1439, 1219, 1114, 1060, poration). Strong anion exchange was then performed with a
833; HRMS (m/z): [M + H]+ calcd for C25H30NO6: 440.2068; column containing 30 mL of quaternary ammonium-sepharose
found: 440.2056; HPLC (method 1): tR = 14.6 min, purity 97.5%. (Q-Sepharose) fast flow media (GE Healthcare). The fractions
containing LpxC (peak elution at 18.6 mS cm−1) were concen-
(S)-N,3-Dihydroxy-2-((S)-2-hydroxy-1-(4-((4-(morpholinomethyl)-
phenyl)ethynyl)phenyl)-ethoxy)propanamide
(43). Hydroxyl-
trated and desalted as above using MWCO columns. The final
step of protein purification was performed with a pre-packed
size exclusion chromatography column containing 300 mL of
Superdex 200 (GE Healthcare). LpxCC63A emerged in a peak
after 200 mL of elution buffer. The purified LpxC was concen-
trated with MWCO columns and stored in 100 μL aliquots at
80 °C in Bis/Tris buffer 50 mM, pH 6.0, containing 150 mM
NaCl. The presence of the enzyme during the purification pro-
gress was confirmed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue
staining. The purified LpxC had a purity above 95% according
to SDS-PAGE, and a concentration of 500 μg mL−1 according to
the Bradford assay (Bio-Rad protein assay).
LpxC assay. A fluorescence-based microplate assay for LpxC
activity was performed as described by Clements et al.10 The
wells in a black non-binding, 96 wells fluorescence microplate
(Greiner Bio One, Frickenhausen) were filled with 97 μL of a
40 mM sodium morpholinoethanesulfonic acid buffer (pH 6.0)
containing 25 μM UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetyl-
glucosamine, 80 μM dithiothreitol and 0.02% Brij 35. Inhibi-
tors were dissolved in DMSO and assayed over a range starting
from 0.2 nM up to 200 μM. The microplate was incubated for
30 min at 37 °C in a plate shaker. After the addition of 500 ng
purified LpxC, the biochemical reaction was stopped by
adding 40 μL of 0.625 M sodium hydroxide, further incubation
for 15 min and neutralization by adding 40 μL of 0.625 M
acetic acid. The deacetylated product UDP-3-O-[(R)-3-hydroxy-
myristoyl]glucosamine was converted into a fluorescing iso-
indole by adding 120 μL of 250 nM o-phthaldialdehyde-
2-mercaptoethanol in 0.1 M borax27 and detected by a Mithras
amine hydrochloride (117 mg, 1.68 mmol) and a 2 M solution
of sodium methanolate in methanol (0.84 mL, 1.68 mmol)
were added to a solution of 41 (123 mg, 0.28 mmol) in dry
methanol (20 mL) and the mixture was stirred at ambient
temperature for 16 h. Then water was added and the mixture
was extracted with ethyl acetate (3×). The combined organic
layers were dried (Na2SO4) and filtered and the solvent was
removed in vacuo. The residue was purified by flash column
chromatography (1 cm, 5 mL, CH2Cl2–methanol = 9/1, Rf =
0.13) to give 43 as a colorless solid (26 mg, 0.06 mmol, 21%
yield). M.p.: 86 °C; [α]D20 = +31.9 (9.0; methanol); 1H NMR
(D3COD): δ 2.41–2.49 (m, 4H, N(CH2CH2)2), 3.51–3.55 (m, 2H,
PhCH2N), 3.64–3.71 (m, 5H, PhCHCH2OH (1H), N(CH2CH2)2),
3.72–3.87 (m, 3H, PhCHCH2OH (1H), HOHNOCCHCH2OH
(1H), HOHNOCCHCH2OH), 3.88–3.94 (m, 1H, HOHNOCCH-
CH2OH), 4.69–4.75 (m, 1H, PhCHCH2OH), 7.31–7.43 (m, 4H,
H
arom.), 7.43–7.54 (m, 4H, Harom.); 13C NMR (D3COD): δ 54.6
(2C, N(CH2CH2)2), 62.6 (1C, HOHNOCCHCH2OH), 63.9 (1C,
PhCH2N), 67.3 (1C, PhCHCH2OH), 67.7 (2C, N(CH2CH2)2), 79.7
(1C, HOHNOCCHCH2OH), 83.6 (1C, PhCHCH2OH), 89.9 (1C,
PhCuCPh), 90.4 (1C, PhCuCPh), 123.5 (1C, Carom.), 124.5 (1C,
Carom.), 128.6 (2C, Carom.), 130.8 (2C, Carom.), 132.5 (2C, Carom.),
132.7 (2C, Carom.), 138.9 (1C, Carom.), 139.8 (1C, Carom.), 169.3
(1C, COONHOH); IR (neat): ν [cm−1] = 3726, 3626, 3225, 2855,
1663, 1516, 1111, 1045, 833, 667; HRMS (m/z): [M + H]+ calcd
for C24H29N2O6: 441.2020; found: 441.2045; HPLC (method 2):
tR = 10.9 min, purity 98.5%.
Biological evaluation
Agar diffusion clearance assay. The antibiotic activity of the plate reader (Berthold, Bad Wildbad) at 340 nm excitation and
synthesized inhibitors was determined by agar disc diffusion 460 nm emission wavelengths. The calculation of the IC50
clearance assays. Liquid cultures of E. coli BL21 (DE3) and the values was performed with the aid of the software GraphPad-
antibiotic resistant strain E. coli D2223 were grown overnight in Prism, which were then converted into Ki values using the
LB broth24 at 37 °C, 200 rpm. 150 μL of overnight cell suspen- Cheng–Prusoff equation. The KM value was calculated from the
sion was spread evenly onto LB agar Petri dishes. 15 μL of each Lineweaver–Burk plot. To validate the test system, the IC50
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Organic & Biomolecular Chemistry
value of CHIR-090 was measured and was found to be compar-
able to the one in the literature.
S. W. Chung, C. Desbonnet, J. Doty, M. Doroski,
J. J. Engtrakul, T. M. Harris, M. Huband, J. D. Knafels,
K. L. Leach, S. Liu, A. Marfat, A. Marra, E. McElroy,
M. Melnick, C. A. Menard, J. I. Montgomery, L. Mullins,
M. C. Noe, J. O’Donnell, J. Penzien, M. S. Plummer,
L. M. Price, V. Shanmugasundaram, C. Thoma,
D. P. Uccello, J. S. Warmus and D. G. Wishka, J. Med.
Chem., 2012, 55, 914–923.
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