C-Triazolyl β-d-furanosides as LpxC inhibitors: Stereoselective synthesis and biological evaluation

Article abstract of DOI:10.1016/j.tet.2014.07.016

C-Triazolyl β-d-furanosides 10a-f were synthesized in a stereocontrolled way, starting from d-mannose. In the key steps of the synthesis a diastereoselective reduction of hemiketal 14 and a Cu(I) catalyzed [3+2]-cycloaddition of central building block 18

Full text of DOI:10.1016/j.tet.2014.07.016

Tetrahedron 70 (2014) 6569e6577
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
C-Triazolyl
b-
D-furanosides as LpxC inhibitors: stereoselective
synthesis and biological evaluation
a
,
b
a
c
a,
*
Sunit Kumar Jana , Marius L o€ ppenberg , Constantin G. Daniliuc , Ralph Holl
a
Institut f u€ r Pharmazeutische und Medizinische Chemie der Westf a€ lischen Wilhelms-Universit a€ t M u€ nster, Corrensstr. 48, D-48149 M u€ nster, Germany
NRW Graduate School of Chemistry, M u€ nster, Germany
Organisch-Chemisches Institut der Westf a€ lischen Wilhelms-Universit a€ t M u€ nster, Corrensstr. 40, D-48149 M u€ nster, Germany
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2014
Received in revised form 27 June 2014
Accepted 2 July 2014
Available online 8 July 2014
C-Triazolyl
b-D-furanosides 10aef were synthesized in a stereocontrolled way, starting from D-mannose.
In the key steps of the synthesis a diastereoselective reduction of hemiketal 14 and a Cu(I) catalyzed
[
3þ2]-cycloaddition of central building block 18 with various azides were performed. The synthesized
2þ
hydroxamic acids were tested for their inhibitory activity against LpxC, a Zn -dependent deacetylase
playing an important role in the biosynthesis of lipid A and therefore representing an interesting target
for the development of novel antibiotics against Gram-negative bacteria. The C-triazolyl glycosides 10aef
did not exhibit antibiotic activity. However, the described synthesis is a versatile way to access C-triazolyl
Keywords:
C-Triazolyl furanosides
LpxC inhibitors
Chiral pool synthesis
Click chemistry
b
-D
-furanosides bearing all of their substituents at the same side of the tetrahydrofuran ring.
Ó 2014 Elsevier Ltd. All rights reserved.
Stereoselective synthesis
1. Introduction
The 1,2,3-triazole moiety is a prominent structural element in
medicinal chemistry. It possesses desirable properties like meta-
1
bolic stability and the capability of forming hydrogen bonds. The
1
,2,3-triazole moiety is often used as mimetic of the peptide bond,2
but it has also been reported to bioisosterically replace trans-ole-
3
4
5
finic moieties, adenine and pyrazole rings. 1,2,3-Triazoles can be
easily accessed by a copper(I)-catalyzed [3þ2]-cycloaddition be-
tween azides and terminal alkynes, a reaction, which exhibits high
6
selectivity and functional group tolerance. Using combinatorial
synthesis, this reaction allows the generation of vast libraries of
compounds for high-throughput screening in the search for new
7
pharmacologically active compounds. In fragment-based drug
8
discovery triazoles are used for fragment linkage.
Various biologically active compounds containing a 1,2,3-
triazole moiety have been described in the literature, amongst
Fig. 1. 1,2,3-Triazole-containing bioactive compounds.
them, the
b-lactam antibiotic cefatrizine (1), the b-lactamase in-
[
(R)-3-hydroxymyristoyl]-N-acetylglucosamine (5) representing
hibitor tazobactam (2) and sugar-based 1,2,3-triazoles such as the
the first irreversible step in the biosynthesis of lipid A (Fig. 2), the
membrane anchor of lipopolysaccharide (LPS). LPS is the main
9
,10
FimH antagonist 3 and the antitumour agent 4 (Fig. 1).
Recently we have reported the stereoselective synthesis of
hydroxamic acids 7 possessing a C-triazolyl -furanosidic scaffold,
which should serve as LpxC inhibitors (Fig. 3). LpxC is a Zn -de-
pendent enzyme, which catalyzes the deacetylation of UDP-3-O-
12
component of the outer layer of the outer membrane of Gram-
negative bacteria shielding the bacteria from detergents and cer-
tain antibiotics. As the inhibition of lipid A biosynthesis is lethal to
these bacteria, LpxC inhibitors represent a class of novel antibacte-
rial agents. These inhibitors have the potential to cure even in-
fections caused by multiresistant Gram-negative bacteria, as they
exhibit their antibacterial activity via a mechanism of action, which
a
11
2þ
13
*
Corresponding author. Tel.: þ49 251 8333372; fax: þ49 251 8332144; e-mail
address: hollr@uni-muenster.de (R. Holl).
http://dx.doi.org/10.1016/j.tet.2014.07.016
0
040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

6570
S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
not exhibit any antibiotic activity at all. As two properties were
changed at the same time, it is hard to say whether the altered ste-
reochemistry or the replacement of the proximal phenyl ring of 9 by
a triazole moiety abolished the antibiotic activity of triazoles 7.
Therefore, in order to solely determine the influence of the in-
troduction of a triazole-containing lipophilic side chain, C-triazolyl
b
-furanosides 10 having the same configuration as 9 should be
synthesized.
In this paper we wish to report the synthesis of C-triazolyl b-D-
furanosides 10 and their biological evaluation as LpxC inhibitors.
With the all-cis orientation of their substituents at the furanose
ring, these compounds represent a class of C-triazolyl glycosides,
which has not been described in the literature before.
Fig. 2. LpxC catalyzed deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetyl-
glucosamine (5).
Fig. 3. Chemical structures of reported and planned LpxC inhibitors.
has not been exploited in antibiotic therapy so far. Some LpxC in-
hibitors have already been described in the literature, like the
2. Synthesis
14,15
diphenylacetylene derivative CHIR-090 (8, Fig. 3).
Derived from
In order to get access to the designed triazole derivatives, at first,
C-ethynyl furanoside 18 should be prepared, which represents the
key intermediate of the synthesis (Scheme 1). As we have already
successfully accomplished the synthesis of the corresponding C-
this hydroxamic acid, which exhibits antibacterial activity against
Escherichia coli and Pseudomonas aeruginosa being comparable to
that of ciprofloxacin, we developed C-glycosidic compounds such as
16
9
, which shows weak inhibitory activity against LpxC. In this
aryl
b-D-furanosides, our previously reported protocol for the
compound, the amide group of CHIR-090 and the hydroxyethyl
moiety of its threonyl group are replaced by a C-furanosidic scaffold.
The central dihydroxytetrahydrofuran moiety causes conforma-
tional restriction of the compound, leading to a defined relative
orientation of the hydroxamate moiety, which should complex the
Zn -ion in the active site, and the lipophilic side chain, which is
supposed to bind to the hydrophobic substrate binding tunnel of
LpxC. In order to find the optimal spatial orientation of these phar-
macophoric groups and thereby improve the biological activity of
the compounds, we began to vary the stereochemistry of the C-
preparation of these C-glycosides should also be applied for the
17
synthesis of 18. Therefore,
g-lactone 13 was prepared in a chiral
pool synthesis by reacting -mannose with acetone in the presence
D
18
of catalytic amounts of iodine, followed by a Swern oxidation of
the resulting lactol 12. A subsequent nucleophilic attack of the
acetylide, formed by deprotonation of trimethylsilyl acetylene with
2
þ
1
n-butyllithium, to lactone 13 gave hemiketal 14. In the H NMR
3
spectrum of 14 in CDCl two sets of signals can be observed in
a ratio of 3:2, indicating that in solution an equilibrium exists be-
tween the two possible anomers. However, the X-ray crystal
structure of 14 shows that the compound crystallizes solely as the
glycosides. However, the synthesized C-triazolyl a-furanosides 7 did
ꢁ
ꢁ
Scheme 1. Reagents and conditions: (a) acetone, I
2
, rt, 51%; (b) oxalyl chloride, DMSO, ꢀ78 C, then NEt
, MeOH, rt, 2. NaClO
3
, rt, 91%; (c) trimethylsilyl acetylene, n-BuLi, THF, ꢀ78 C, 98%; (d) 1.
ꢁ
BF
3
$OEt
2 3
, Et SiH, H
3
CCN, ꢀ40 C, 2. p-TsOH, MeOH, rt, 43%; (e) 1. NaIO
4
2
, 2-methylbut-2-ene, NaH
2
PO
4
, t-BuOH/H
2
O (5:2), rt, 82%; (f) MeI, K
2
CO
3
, DMF, rt, 76%;
(
g) Er(OTf)
3
, MeOH, microwave irradiation, 42%.

S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
6571
(
S)-configured hemiketal (Fig. 4). The Flack parameter for com-
additionally, a partial cleavage of the monocyclic acetonide oc-
curred during the reaction. Therefore, in order to transform the
remaining bisacetonide into diol 15, the crude product of the re-
duction was treated with catalytic amounts of p-toluenesulfonic
acid in methanol, giving diol 15 in 43% yield over the two reaction
steps.
pound 14 was refined to the value ꢀ0.1(1) and confirms the ex-
pected configuration of this compound. Hemiketal 14 contains
three heterocyclic rings: one furan ring and two dioxolane rings.
The tetrahydrofuran ring (O1/C2/C3/C4/C5 atoms) adopts an en-
velope conformation, with O1 atom lying well out of the plane (by
ꢀ
19
0
.598 A). The puckering parameters for this tetrahydrofuran de-
The desired C-ethynyl furanoside 18 should finally be obtained
by transforming the glycol moiety of 15 into an ester group. At first,
2
0
ꢀ
rivative ring calculated with PLATON are Q¼0.406(2) A and
ꢁ
4
¼357.3(1) . The dioxolane ring (O3/C3/C4/O4/C11 atoms) attached
the method, which successfully yielded the epimeric C-ethynyl a-
11
to the furan ring adopts a similar envelope conformation, with C11
atom lying well out of the plane (by 0.411 A). The puckering pa-
furanoside 19 was employed. This method includes a glycol
cleavage to obtain an intermediate aldehyde, a subsequent Jones
oxidation to yield the corresponding acid and a final esterification
ꢀ
ꢀ
ꢁ
rameters are Q¼0.269(8) A and 4¼132.0(2) . The second dioxolane
ring is best described as having the twisted conformation about the
using methyl iodide in the presence of K
the C-ethynyl -furanoside 15 only very poor yields of ester 17 were
obtained. Investigation of the three steps identified the oxidation of
2 3
CO . However, in case of
ꢀ
ꢁ
O6eC15 bond (with Q¼0.331(8) A and 4¼349.5(2) ). The absolute
configurations at atoms C2, C3, C4, C5 and C14 are S, S, S, R and R,
respectively.
b
the aldehyde as the crucial step. Therefore, instead of using CrO
3
in
þ
acidic medium, other methods like the Ag -mediated Tollens oxi-
dation, which was performed under basic conditions, were tested.
However, these attempts were unsuccessful as well. A possible
explanation for the failure of these metal-mediated oxidations
might be the formation of a complex between the metal ion and the
tetrahydrofuran derivative, which bears all of its substituents at the
same side of the tetrahydrofuran ring. Thus, to obtain the desired
carboxylic acid 16 an oxidation method, which is not metal-
mediated should be employed. The Pinnick oxidation with NaClO
2
21
in the presence of 2-methylbut-2-ene proved to be applicable. So
starting with the NaIO -mediated glycol cleavage of diol 15, the
4
intermediately formed aldehyde was successfully subjected to
a Pinnick oxidation to give carboxylic acid 16, which was obtained
in 82% yield over two reaction steps. Treatment of 16 with iodo-
methane in the presence of potassium carbonate led to the esteri-
fication of the carboxylic acid moiety. As additionally the
trimethylsilyl protective group was cleaved in the course of the
reaction, the transformation yielded terminal alkyne 17. The key
intermediate 18 could finally be obtained by cleaving the remaining
Fig. 4. Crystal structure of hemiketal 14.
In order to transform hemiketal 14 into a C-ethynyl glycoside,
the compound was treated with triethylsilane and boron trifluoride
etherate. This reduction proceeded in a diastereoselective manner,
yielding the corresponding C-ethynyl
b-
D-mannofuranoside, as the
acetonide of 17 with Er(OTf)
3
under microwave irradiation. In
hydride of triethylsilane was transferred to the less hindered Re-
face of the intermediately formed oxocarbenium ion. However, due
to the presence of the Lewis acid boron trifluoride etherate,
comparison to its anomer 19, whose stereochemistry was con-
firmed by X-ray crystallography, the H NMR spectrum of 18
shows clear differences (Fig. 5). Whilst the chemical shift of 2-H is
11
1
Fig. 5. Comparison of the 1H NMR spectra of the anomers 18 and 19.

6572
S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
decreased in the spectrum of 18, the signal of 5-H is shifted
downfield due to the altered location of the two protons within the
magnetic anisotropy shielding/deshielding cones of the ethynyl
group and the carboxyl moiety, respectively.
In the disc diffusion assays, E. coli BL21 (DE3) and the defective E.
coli D22 strain were grown on agar plates in the presence of filter
paper plates containing hydroxamic acids 10aef as well as CHIR-
090 and DMSO serving as positive and negative control, re-
spectively. Neither against E. coli BL21 (DE3) nor against E. coli D22,
being more sensitive towards LpxC inhibition, the triazole de-
rivatives 10aef showed inhibition of bacterial growth beyond the
6 mm filter discs.
[
3þ2]-Cycloadditions of terminal alkyne 18 with several azides
(
20aef) were carried out in the presence of copper(II) sulfate and
sodium ascorbate in a 1:1 mixture of water and tert-butanol to give
6
a set of triazole derivatives (21aef) (Scheme 2). The benzyl de-
rivative 21b was crystallized from methanol, yielding crystals
suitable for X-ray crystal structure analysis. The obtained crystal
structure confirmed the (S)-configuration at the anomeric center as
well as the substitution pattern of the triazole ring (Fig. 6). The
tetrahydrofuran ring adopts an envelope conformation, with C4
The outcome of the disc diffusion assays matches the results of
the LpxC enzyme assay, as none of the C-triazolyl
b-furanosides
10aef was able to inhibit the LpxC catalyzed deacetylation of the
enzyme’s natural substrate 5 even at concentrations up to 200
mM.
ꢀ
atom lying well out of the plane (by 0.581 A). The puckering pa-
4
. Conclusion
ꢀ
rameters calculated with PLATON program are Q¼0.375(2) A and
4
¼278.0(3) for the tetrahydrofuran derivative ring O1/C2/C3/C4/
In a chiral pool synthesis starting from
D
-mannose, C-triazolyl b-
C5. The absolute configurations at atoms C2, C3, C4 and C5 of the
five membered ring are S, S, R and S, respectively.
D
-furanosides 10aef were synthesized in a stereocontrolled man-
ner. The -configuration at the anomeric position could be estab-
b
Scheme 2. Reagents and conditions: (a) sodium ascorbate, CuSO
rt, 10a 44%, 10b 41%, 10c 58%, 10d 70%, 10e 53%, 10f 40%.
4
$5H
2
O, t-BuOH/H
2
2
O¼1:1, rt, 21a 78%, 21b 64%, 21c 66%, 21d 60%, 21e 69%, 21f 61%; (b) H NOH$HCl, NaOMe, MeOH,
lished by a stereoselective reduction of hemiketal 14 with
triethylsilane and boron trifluoride etherate. Triazole rings with
different substituents were built up by performing [3þ2]-
cycloadditions of key intermediate 18 with several azides.
The synthesized hydroxamic acids 10aef did not inhibit the
growth of E. coli in disc diffusion assays and inhibitory activity
against LpxC could not be observed. Apparently, the replacement of
the proximal phenyl ring of 9 by a triazole moiety is detrimental for
the antibacterial activity of the C-glycosides. Compared to the para-
substituted phenyl ring of 9, the polar nature of the heterocycle and
the slightly bent alignment of its substituents might not be optimal
for the binding of the side chains of hydroxamates 10aef to the
hydrophobic tunnel of LpxC.
Fig. 6. Crystal structure of triazole derivative 21b.
The obtained C-triazolyl b-furanosides 21aef were reacted with
hydroxylamine hydrochloride and sodium methanolate in metha-
nol. This final aminolysis yielded the desired hydroxamic acids
However, the described reactions offer a convenient route to
access diverse C-triazolyl
b-D-furanosides 10aef bearing four cis-
oriented substituents at their tetrahydrofuran ring.
10aef.
5
5
. Experimental section
3
. Biological evaluation
.1. Chemistry, general
In order to characterize the biological activities of C-triazolyl b-
furanosides 10aef, an LpxC inhibition assay and disc diffusion as-
says were performed (Table 1).
Unless otherwise mentioned, THF was dried with sodium/ben-
zophenone and was freshly distilled before use. Thin layer chro-
matography (tlc): Silica gel 60 254 plates (Merck). Flash
chromatography (fc): Silica gel 60, 40e64 m (MachereyeNagel);
parentheses include: diameter of the column, length of the column,
fraction size, eluent, R value. Melting point (mp): Melting point
apparatus SMP 3 (Stuart Scientific), uncorrected. Optical rotation
[deg] was determined with a Polarimeter 341 (PerkineElmer);
path length 1 dm, wavelength 589 nm (sodium D line); the unit of
F
Table 1
m
Results of the agar diffusion clearance assay and the LpxC assay
Compound
R
Zone of inhibition [mm] Enzyme assay
f
E. coli
E. coli
IC50
[
mM]
i
K [mM]
BL21 (DE3)
D22
a
CHIR-090
9
24.5
30
9
<6
<6
<6
<6
0.06 0.008
p-Phenylbutadiynyl <6
Phenyl
Benzyl
4-Fluorobenzyl
4-(Trifluoromethyl) <6
benzyl
2-Phenylethyl
3-Phenylpropyl
32
4.4
d
d
d
d
20
ꢀ1 ꢀ1
] is omitted; the con-
the specific rotation [
a
]
D
[deg mL dm
g
10a
10b
10c
10d
<6
<6
<6
>200
>200
>200
>200
ꢀ1
centration of the sample c [mg mL ] and the solvent used are given
1
13
in brackets. H NMR (400 MHz), C NMR (100 MHz): Mercury plus
400 spectrometer (Varian); in parts per million (ppm) related to
tetramethylsilane; coupling constants are given with 0.5 Hz reso-
d
1
1
0e
0f
<6
<6
<6
<6
>200
>200
d
d
1
lution. Where necessary, the assignment of the signals in the
H
13
1
1
1
13
NMR and C NMR spectra was performed using He H and He
C

S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
Major
6573
Major
3
), 26.4 (0.4C, CH
Minor
COSY NMR spectra as well as NOE (nuclear Overhauser effect) dif-
ference spectroscopy. IR: IR Prestige-21 (Shimadzu). HRMS:
MicrOTOF-QII (Bruker). HPLC methods for the determination of
product purity: Method 1: Merck Hitachi Equipment; UV detector:
25.1 (0.6C, CH
3
), 25.3 (0.6C, CH
), 25.4 (0.4C, CH
3
), 25.6
), 27.1 (0.6C,
O), 67.4 (0.6C,
O), 73.1 (0.4C, OCHCH O), 77.4 (0.6C,
Minor
Major
Minor
(0.4C, CH
3
), 26.0 (0.6C, CH
3
3
Major
Minor
CH
3
), 27.2 (0.4C, CH
3
), 67.2 (0.4C, OCHCH
2
OCHCH
2
O), 72.8 (0.6C, OCHCH
2
2
Major
Major
Major
Minor
L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
C-6
6a
), 79.6 (0.6C, C-6a
), 84.0 (0.6C, C-3a
), 79.8 (0.4C, C-6
), 86.9 (0.4C, C-3a
), 80.4 (0.4C, C-
), 92.1 (0.6C,
column: LiChrospherÒ 60 RP-select B (5
m
m); LiCroCART 250-
Ò
Minor
Minor
Major
Minor
Major
4
mm cartridge; flow rate: 1.00 mL/min; injection volume: 5.0
mL;
C^CSi
(0.4C, C-4
), 92.2 (0.4C, C^CSi
), 97.1 (0.6C, C-4
), 99.8
Minor
Minor
Major
detection at
l
¼210 nm for 30 min; solvents: A: water with 0.05% (v/
), 99.9 (0.4C, C^CSi
), 100.8 (0.6C, C^CSi
),
Minor
Major
v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoro-
acetic acid: gradient elution: (A%): 0e4 min: 90%, 4e29 min: gra-
dient from 90% to 0%, 29e31 min: 0%, 31e31.5 min: gradient from
109.5 (0.4C, C(CH
3
)
2
), 109.6 (0.6C, C(CH
3
)
2
), 113.8 (0.4C,
), ratio of anomers: 3 (Major) :
[cm ]¼3321, 1369, 1246, 1211, 1165, 1111,
Minor
Major
C(CH
3
)
2
), 113.9 (0.6C, C(CH
3
)
2
ꢀ1
2 (Minor); IR (neat): ~
n
þ
0
% to 90%, 31.5e40 min: 90%. Method 2: Merck Hitachi Equipment;
UV detector: L-7400; pump: L-6200A; column: phenomenex
1076, 1042, 1026, 841, 802, 760; HRMS (m/z): [MþH] calcd for
17 29 6
C H O Si, 357.1728; found, 357.1681; X-ray crystal structure
Ò
ꢀ
Gemini
.00 mL/min; injection volume: 5.0
min; solvents: A: acetonitrile/10 mM ammonium for-
5
m
m C6-Phenyl 110 A; LC Column 250ꢂ4.6 mm; flow rate:
analysis: recrystallization of 14 from cyclohexane gave crystals,
which were suitable for X-ray crystal structure analysis. Formula
1
m
L; detection at
l
¼254 nm for
2
0
C
17
28
H O
6
Si$C
6
H
12
,
M¼440.64,
colourless
crystal,
ꢀ
mate¼10:90 with 0.1% formic acid; B: acetonitrile/10 mM ammo-
0.15ꢂ0.12ꢂ0.02 mm, a¼6.1119(6), b¼11.6007(11), c¼18.6748(15) A,
ꢁ
ꢀ
3
ꢀ3
ꢀ1
nium formate¼90:10 with 0.1% formic acid; gradient elution: (A%):
b
¼92.469(7) , V¼1322.9(2) A ,
r
calcd¼1.106 g cm
,
m
¼1.041 mm
,
0
0
e5 min: 100%, 5e15 min: gradient from 100% to 0%, 15e20 min:
%, 20e22 min: gradient from 0% to 100%, 22e30 min: 100%. X-ray
crystal structure analyses: Datasets were collected with a Nonius
KappaCCD diffractometer. Programs used: data collection, COLLECT
empirical absorption correction (0.859ꢃTꢃ0.979), Z¼2, monoclinic,
ꢀ
space group P2
9048 reflections collected (ꢄh, ꢄk, ꢄl), [(sin
independent (Rint¼0.122) and 2137 observed reflections [I>2
1
(No. 4),
l
¼1.54178 A, T¼223(2) K,
)/
]¼0.60 A , 3674
(I)],
u and 4 scans,
ꢀ
ꢀ1
q l
s
2
(
R. W. W. Hooft, Bruker AXS, 2008, Delft, The Netherlands); data
359 refined parameters, R¼0.104, wR ¼0.312, max. (min.) residual
2
2
23
ꢀ
ꢀ3
reduction Denzo-SMN; absorption correction, Denzo; structure
electron density 0.33 (ꢀ0.25) e A , the hydrogen atoms were
calculated and refined as riding atoms. Flack parameter: ꢀ0.1(1).
solution SHELXS-97;24 structure refinement SHELXL-97
25
and
graphics, XP (BrukerAXS, 2000). Thermals ellipsoids are shown
with 15% probability for compound 14 and with 30% probability for
compound 21b, R-values are given for observed reflections, and
5.2.2. (R)-1-{(3aS,4R,6S,6aR)-2,2-Dimethyl-6-[(trimethylsilyl)ethy-
nyl]tetrahydrofuro[3,4-d][1,3]dioxol-4-yl}ethane-1,2-diol
Under N atmosphere triethylsilane (2.1 mL, 13 mmol) and BF
(15).
$OEt
2
wR values are given for all reflections. Exceptions and special fea-
2
3
2
tures: In compound 14 one SiMe
3
group and one cyclohexane sol-
(1.5 mL, 12 mmol) were added to a solution of 14 (3.6 g, 10 mmol) in
ꢁ
vent molecule were found disordered over two positions. Several
restraints (SADI, SAME, ISOR and SIMU) were used in order to im-
prove refinement stability. CCDC 1001429 (for 14) and CCDC
dry acetonitrile (50 mL) at ꢀ40 C and the reaction mixture was
stirred at this temperature for 1 h. Then a saturated aqueous solution
2 3
of K CO (5 mL) was added and the mixture was stirred for 1 h at
1001430 (for 21b) contain the supplementary crystallographic data
room temperature. The reaction mixture was diluted with water
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
(20 mL) and extracted with ethyl acetate (3ꢂ). The combined organic
2 4
phases were driedwith Na SO , filteredandthesolvent wasremoved
in vacuo. The crude product was dissolved in methanol (100 mL) and
p-toluenesulfonic acid monohydrate (380 mg, 2.0 mmol) was added.
The reaction mixture was stirred at room temperature overnight.
5
.2. Synthetic procedures
Then a saturated aqueous solution of NaHCO
mixturewas extractedwith ethyl acetate (3ꢂ). The combinedorganic
phases were driedwith Na SO , filteredandthesolvent wasremoved
in vacuo. The crude product was purified by flash column chroma-
tography (4 cm, 15 cm, 30 mL, cyclohexane/ethyl acetate¼1:1,
3
(15 mL) was added and
5
.2.1. (3aS,6R,6aS)-6-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-2,2-
dimethyl-4-[(trimethylsilyl)ethynyl]tetrahydrofuro[3,4-d][1,3]dioxol-
-ol (14). Under nitrogen atmosphere a 2.5 M solution of n-BuLi
2
4
4
(
9.7 mL, 24 mmol) in toluene was added to a solution of trime-
ꢁ
thylsilyl acetylene (3.1 mL, 22 mmol) in dry THF (20 mL) at ꢀ78 C.
R
f
¼0.26) to give 15 as colourless crystalline solid (1.3 g, 4.3 mmol, 43%
ꢁ
ꢁ
20
1
The reaction mixture was stirred at ꢀ78 C for 30 min. Then a so-
yield). Mp¼106 C; [
[ppm]¼0.18 (s, 9H, Si(CH
(br s, 1H, CH
3.6 Hz, 1H, 4-H), 3.73 (dd, J¼11.4/5.3 Hz, 1H, OCHCH
J¼11.4/2.5 Hz, 1H, OCHCH O), 4.03e4.10 (m, 1H, OCHCH
J¼4.1 Hz, 1H, 6-H), 4.71 (dd, J¼6.0/4.1 Hz, 1H, 6a-H), 4.81 (dd, J¼6.0/
a]
D
þ85.0 (c 2.6, MeOH); H NMR (CDCl
3
):
lution of 13 (5.2 g, 20 mmol) in dry THF (20 mL) was added slowly
d
3 3 3 3
) ),1.37 (s, 3H, CH ),1.52 (s, 3H, CH ), 2.36
ꢁ
over a period of 30 min. The reaction mixture was stirred at ꢀ78 C
2
OH), 2.98 (br d, J¼4.9 Hz, 1H, CHOH), 3.50 (dd, J¼8.2/
O), 3.87 (dd,
O), 4.24 (d,
for additional 20 min and then quenched with a saturated aqueous
2
solution of NH
extracted with ethyl acetate (3ꢂ). The combined organic phases
were dried with Na SO , filtered and the solvent was removed in
4
Cl. The mixture was diluted with water and
2
2
13
2
4
3.6 Hz, 1H, 3a-H); C NMR (CDCl
25.5(1C, CH ), 26.2 (1C, CH ), 64.4 (1C, OCHCH
CH O), 73.3 (1C, C-6), 80.8 (1C, C-4), 81.2 (1C, C-3a), 81.6 (1C, C-6a),
3
):
d
[ppm]¼ꢀ0.1 (3C, Si(CH
3 3
) ),
vacuo. The crude product was purified by flash column chroma-
tography (4 cm, 15 cm, 30 mL, cyclohexane/ethyl acetate¼10:1,
3
3
2
O), 69.9 (1C, OCH-
2
R
9
d
f
¼0.23) to give 14 as colourless crystalline solid (7.0 g, 19.6 mmol,
93.8 (1C, C^CSi), 98.1 (1C, C^CSi), 113.4 (1C, C(CH
)
3 2
); IR (neat): ~
n
ꢁ
20
1
ꢀ1
8% yield). Mp¼122 C; [
a]
D
þ68.3 (c 2.4, MeOH); H NMR (CDCl
3
):
[cm ]¼3375, 2955, 1416, 1373, 1250, 1211, 1080, 1015, 841, 760;
Minor
þ
[ppm]¼0.20 (s, 9H, Si(CH
3
)
3
), 1.36e1.37 (m, 6ꢂ0.4H, CH
3
), 1.38
HRMS (m/z): [MþH] calcd for C14
25 5
H O Si, 301.1466; found,
Major
Major
(
CH
s, 3ꢂ0.6H, CH
3
), 1.39 (s, 3ꢂ0.6H, CH
3
), 1.45 (s, 3ꢂ0.4H,
301.1491; HPLC (method 1): t
R
¼16.2 min, purity 99.9%.
Minor
Major
Minor
3
), 1.46 (s, 3ꢂ0.6H, CH
3
), 1.51 (s, 3ꢂ0.4H, CH
3
), 1.54 (s,
Major
Major
3
ꢂ0.6H, CH
3
), 3.69 (dd, J¼8.4/3.3 Hz, 0.6H, 6-H
), 4.01e4.07
5.2.3. (3aR,4S,6S,6aR)-2,2-Dimethyl-6-[(trimethylsilyl)ethynyl]tetra-
Minor
(
m, 0.4Hþ1H, 6-H
, OCHCH
2
O (1H)), 4.10 (dd, J¼8.8/6.1 Hz, 1H,
hydrofuro[3,4-d][1,3]dioxole-4-carboxylic acid (16). Under N at-
mosphere NaIO (710 mg, 3.3 mmol) was added to a solution of 15
2
Major
OCHCH
2
O), 4.38 (ddd, J¼8.4/6.0/4.3 Hz, 0.6H, OCHCH
2
O
), 4.43
), 4.58 (d, J¼5.7 Hz, 0.4H,
Major
4
Minor
(
3
ddd, J¼8.4/6.1/4.2 Hz, 0.4H, OCHCH
2
O
(450 mg,1.5 mmol) in methanol (15 mL) and stirred for 4 h until TLC
showed complete conversion. Then the reaction mixture was di-
luted with water (20 mL) and extracted with dichloromethane
Minor
a-H
), 4.60 (d, J¼5.8 Hz, 0.6H, 3a-H
), 4.82e4.85 (m,1H, 6a-
13
3
H), ratio of anomers: 3 (Major): 2 (Minor); C NMR (CDCl ):
d
Minor
Major
[ppm]¼ꢀ0.20 (3ꢂ0.4C, Si(CH
3
)
3
), ꢀ0.18 (3ꢂ0.6C, Si(CH
3
)
3
),
(3ꢂ). The combined organic phases were dried with Na
2 4
SO ,

6574
S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
filtered and the solvent was removed in vacuo. The crude aldehyde
was dissolved in a mixture of tert-butanol and water (5:2, 10 mL).
(110 mg, 0.96 mmol) was added to a solution of 18 (90 mg,
0.48 mmol) in a 1:1 mixture of t-BuOH and H O (15 mL), followed
2
To this solution NaH
2
PO
4
(900 mg, 7.5 mmol), 2-methylbut-2-ene
by sodium ascorbate (19 mg, 0.096 mmol) and copper sulfate
pentahydrate (12 mg, 0.048 mmol). The reaction mixture was
stirred at room temperature for 24 h. The reaction was quenched
(
0.80 mL, 7.5 mmol) and sodium chlorite 80% (680 mg, 6.0 mmol)
were added. The reaction mixture was stirred at room temperature
for 30 min and then the reaction was quenched by the addition of
a saturated aqueous solution of Na SO (2 mL). Then the reaction
2 3
with a saturated aqueous solution of NaHCO
ethyl acetate (3ꢂ). The combined organic phases were dried
(Na SO ), filtered and evaporated. The residue was purified by flash
column chromatography (2 cm, 15 cm, 10 mL, dichloromethane/
3
and extracted with
mixture was diluted with water (10 mL) and extracted with ethyl
2
4
acetate (3ꢂ). The combined organic phases were dried with
Na
2
SO
4
, filtered and the solvent was removed in vacuo to give 16 as
methanol¼20:1, R
0.38 mmol, 78% yield). Mp¼142 C; [
NMR (CD OD):
[ppm]¼3.80 (s, 3H, CO
f
¼0.34) to give 21a as colourless solid (120 mg,
ꢁ
20
1
colourless crystalline solid (350 mg, 1.2 mmol, 82%
a
]
D
ꢀ31.0 (c 1.4, MeOH); H
ꢁ
20
1
yield). Mp¼136 C; [
a
]
D
þ25.0 (c 1.1, MeOH); H NMR (CDCl
3
):
),
3
d
2
CH
3
), 4.53 (dd, J¼6.1/
d
[ppm]¼0.20 (s, 9H, Si(CH
3
)
3
), 1.36 (s, 3H, CH
3
), 1.53 (s, 3H, CH
3
5.0 Hz, 1H, 4-H), 4.59e4.62 (m, 1H, 3-H), 4.69 (d, J¼5.5 Hz, 1H, 2-H),
0
0
4
.25 (d, J¼4.0 Hz, 1H, 4-H), 4.38 (d, J¼4.2 Hz, 1H, 6-H), 4.77 (dd,
5.30 (d, J¼6.1 Hz, 1H, 5-H), 7.46e7.51 (m, 1H, 4 -Hphenyl), 7.55e7.60
13
00
00
00
J¼5.9/4.2 Hz, 1H, 6a-H), 5.00 (dd, J¼5.9/4.0 Hz, 1H, 3a-H); C NMR
(m, 2H, 3 -Hphenyl, 5 -Hphenyl), 7.81e7.85 (m, 2H, 2 -Hphenyl
,
0
0
0
13
(
CDCl
3
):
d
[ppm]¼ꢀ0.2 (3C, Si(CH
3
)
3
), 25.6 (1C, CH
3
), 26.2 (1C, CH
3
),
6 -Hphenyl), 8.56 (s, 1H, 5 -Htriazol); C NMR (CD
3
OD):
d
[ppm]¼52.5
7
3.6 (1C, C-6), 80.1 (1C, C-4), 81.2 (1C, C-6a), 81.4 (1C, C-3a), 95.1
(1C, CO CH ), 73.3 (1C, C-4), 74.3 (1C, C-3), 78.0 (1C, C-5), 81.4 (1C,
2
3
00 00 0
(
1C, C^CSi(CH
3
)
3
), 96.9 (1C, C^CSi(CH
3
)
3
) 114.3 (1C, C(CH
3
)
2
),
C-2), 121.6 (2C, C-2 phenyl, C-6 phenyl), 124.8 (1C, C-5 triazol), 130.0
(1C, C-4 phenyl), 130.9 (2C, C-3 phenyl
C-1 phenyl), 147.2 (1C, C-4 triazol), 172.1 (1C, CO CH
ꢀ
1
00
00
00
1
69.0 (1C, CO
2
H); IR (neat): ~
n
[cm ]¼2955, 1728, 1381, 1246, 1211,
,
C-5 phenyl), 138.5 (1C,
þ
00
ꢀ
0
1
C
115, 1080, 1030, 841, 760; HRMS (m/z): [MþH] calcd for
2
3
); IR (neat): ~
n
1
13 21
H O
5
Si, 285.1153; found, 285.1164.
[cm ]¼3402, 1751, 1597, 1501, 1439, 1215, 1080, 1042, 760, 691;
þ
HRMS (m/z): [MþH] calcd for C14
16 3 5
H N O , 306.1084; found,
5
[
K
.2.4. (3aR,4S,6S,6aR)-Methyl 6-ethynyl-2,2-dimethyltetrahydrofuro
3,4-d][1,3]dioxole-4-carboxylate (17). Under atmosphere
(690 mg, 5.0 mmol) and methyl iodide (0.31 mL, 5.0 mmol)
306.1103; HPLC (method 1): t
R
¼10.4 min, purity 98.2%.
N
2
2
CO
3
5.2.7. (2S,3R,4S,5S)-Methyl 5-(1-benzyl-1H-1,2,3-triazol-4-yl)-3,4-
dihydroxytetrahydrofuran-2-carboxylate (21b). (Azidomethyl)ben-
zene (220 mg, 1.7 mmol) was added to a solution of 18 (150 mg,
were added to a solution of 16 (280 mg, 1.0 mmol) in dry DMF
10 mL), and the mixture was stirred overnight. Then the reaction
(
mixture was diluted with water (50 mL) and extracted with ethyl
2
0.81 mmol) in a 1:1 mixture of t-BuOH and H O (20 mL), followed
acetate (3ꢂ). The combined organic phases were dried with
by sodium ascorbate (34 mg, 0.17 mmol) and copper sulfate pen-
tahydrate (21 mg, 0.17 mmol). The reaction mixture was stirred at
room temperature for 24 h. The reaction was quenched with
2 4
Na SO , filtered and the solvent was removed in vacuo. The residue
was purified by flash column chromatography (2 cm, 15 cm, 10 mL,
cyclohexane/ethyl acetate¼2:1, R
f
¼0.33) to give 17 as colourless
a saturated aqueous solution of NaHCO
3
and extracted with ethyl
SO ),
ꢁ
crystalline solid (170 mg, 0.76 mmol, 76% yield). Mp¼87 C;
acetate (3ꢂ). The combined organic phases were dried (Na
2
4
2
0
1
[
a]
D
þ19.0 (c 1.2, MeOH); H NMR (CDCl
3
):
d
[ppm]¼1.36 (s, 3H,
filtered and evaporated. The residue was purified by flash column
CH
CO
3
), 1.52 (s, 3H, CH
CH
3
), 2.64 (d, J¼2.3 Hz, 1H, C^CH), 3.82 (s, 3H,
chromatography (2 cm, 15 cm, 10 mL, dichloromethane/meth-
2
3
), 4.21 (d, J¼4.2 Hz, 1H, 4-H), 4.30 (dd, J¼3.8/2.3 Hz, 1H,
anol¼30:1, R
0.51 mmol, 64% yield). Mp¼141 C; [
NMR (CD OD):
[ppm]¼3.75 (s, 3H, CO
f
¼0.22) to give 21b as colourless solid (160 mg,
ꢁ
20
1
6
3
5
8
1
1
-H), 4.78 (dd, J¼5.9/3.8 Hz, 1H, 6a-H), 4.99 (dd, J¼5.9/4.2 Hz, 1H,
a]
D
ꢀ28.7 (c 1.4, MeOH); H
13
a-H); C NMR (CDCl
2.5 (1C, CO CH ), 72.4 (1C, C-6), 76.3 (1C, C^CH), 77.0 (1C, C^CH),
0.8 (1C, C-4), 81.2 (1C, C-6a), 81.6 (1C, C-3a), 114.2 (1C, C(CH ),
[cm ]¼3233, 2974, 1748, 1443,
3
):
d
[ppm]¼25.7 (1C, CH
3
), 26.1 (1C, CH
3
),
3
d
2
CH
3
), 4.41 (dd, J¼5.9/
2
3
5.0 Hz, 1H, 4-H), 4.56 (dd, J¼5.6/5.0 Hz, 1H, 3-H), 4.62 (d, J¼5.6 Hz,
1H, 2-H), 5.18 (d, J¼5.9 Hz, 1H, 5-H), 5.56 (d, J¼14.9 Hz, 1H, CH Ph),
5.61 (d, J¼14.9 Hz, 1H, CH Ph), 7.29e7.41 (m, 5H, Hphenyl), 8.06 (s,
1H, 5 -Htriazol); C NMR (CD OD): CH ), 55.0
3
)
2
2
ꢀ
1
67.0 (1C, CO
2
CH
3
); IR (neat): ~
n
2
0
13
377, 1269, 1227, 1200, 1107, 1076, 1042, 976, 903, 853, 737; HRMS
3
d
[ppm]¼52.4 (1C, CO
2
3
þ
(
m/z): [MþH] calcd for C11
H
15
O
5
, 227.0914; found, 227.0920.
(1C, CH
2
2
Ph), 73.1 (1C, C-4), 74.2 (1C, C-3), 78.1 (1C, C-5), 81.3 (1C, C-
), 126.8 (1C, C-5 triazol), 129.2 (2C, Cphenyl), 129.5 (1C, Cphenyl), 130.0
0
0
5
2
.2.5. (2S,3R,4S,5S)-Methyl 5-ethynyl-3,4-dihydroxytetrahydrofuran-
-carboxylate (18). Er(OTf) (18 mg, 0.03 mmol) was added to
(2C, Cphenyl), 136.8 (1C, Cphenyl), 146.4 (1C, C-4 triazol), 172.1 (1C,
ꢀ
1
CO
2
CH
3
); IR (neat): ~
n
[cm ]¼3468, 3267, 1732, 1435, 1250, 1107,
3
þ
a solution of 17 (110 mg, 0.5 mmol) in methanol (2 mL) in a 10 mL
1030, 964, 841, 729; HRMS (m/z): [MþH] calcd for C15
18 3 5
H N O
microwave reaction vessel. The reaction mixture was stirred at
320.1241; found, 320.1248; HPLC (method 1): t
R
¼11.0 min, purity
ꢁ
1
10 C for 60 min at 200 psi and 100 W. The reaction mixture was
96.6%; X-ray crystal structure analysis: recrystallization of 21b from
diluted with water (10 mL) and extracted with ethyl acetate (3ꢂ).
methanol gave crystals, which were suitable for X-ray crystal
The combined organic phases were dried with Na SO , filtered and
2
4
structure analysis. Formula C15
H
17
N
3
O
5
, M¼319.32, colourless
the solvent was removed in vacuo. The crude product was purified
by flash column chromatography (2 cm, 15 cm, 10 mL, dichloro-
crystal, 0.22ꢂ0.16ꢂ0.13 mm, a¼5.7872(3), b¼11.5403(6),
ꢀ
ꢀ
3
ꢀ3
,
c¼22.5145(11) A, V¼1503.6(1) A ,
rcalcd¼1.411
g
cm
ꢀ
1
methane/methanol¼30:1, R
f
¼0.25) to give 18 as colourless oil
m
¼0.904 mm , empirical absorption correction (0.825ꢃTꢃ0.891),
2
0
1
ꢀ
(
39 mg, 0.21 mmol, 42% yield). [
CD OD):
CH
.52 (d, J¼5.4 Hz,1H, 2-H), 4.70 (dd, J¼6.0/2.3 Hz,1H, 5-H); C NMR
CD OD): CH ), 73.1 (1C, C-5), 73.2 (1C, C-4),
[ppm]¼52.5 (1C, CO
3.4 (1C, C-3), 78.1 (1C, C^CH), 79.8 (1C, C^CH), 81.4 (1C, C-2),
a
]
D
þ4.4 (c 1.7, MeOH); H NMR
Z¼4, orthorhombic, space group P2
T¼223(2) K, and 4 scans, 5205 reflections collected (ꢄh, ꢄk, ꢄl),
[(sin )/
]¼0.60 A , 2478 independent (Rint¼0.030) and 2414 ob-
served reflections [I>2
(I)], 217 refined parameters, R¼0.030,
1
2
1
2
1
(No. 19),
l
¼1.54178 A,
(
CO
4
3
d
[ppm]¼2.96 (d, J¼2.3 Hz, 1H, C^CH), 3.77 (s, 3H,
u
ꢀ
ꢀ1
2
3
), 4.25 (dd, J¼6.0/5.2 Hz, 1H, 4-H), 4.45 (t, J¼5.3 Hz, 1H, 3-H),
q l
13
s
2
ꢀ
ꢀ3
(
3
d
2
3
wR ¼0.079, max. (min.) residual electron density 0.11 (ꢀ0.10) e A
,
7
the hydrogen atoms at O2 and O3 were refined freely, but with OeH
distance restraints; others were calculated and refined as riding
atoms. Flack parameter: ꢀ0.1(2).
ꢀ
1
1
71.6 (1C, CO
2
CH
3
); IR (neat): ~
n
[cm ]¼3437, 3271,1740,1439,1219,
þ
1
123, 1057, 741, 660; HRMS (m/z): [MþH] calcd for C
8 11 5
H O ,
187.0601; found, 187.0606.
5
.2.8. (2S,3R,4S,5S)-Methyl 5-[1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-
yl]-3,4-dihydroxytetrahydrofuran-2-carboxylate (21c). 1-(Azido-
methyl)-4-fluorobenzene (180 mg, 1.2 mmol) was added to
5
.2.6. (2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-(1-phenyl-1H-1,2,3-
triazol-4-yl)tetrahydrofuran-2-carboxylate (21a). 1-Azidobenzene

S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
6575
ꢁ
20
ꢀ14.9 (c 1.8, MeOH); 1H
a solution of 18 (110 mg, 0.59 mmol) in a 1:1 mixture of t-BuOH and
O (15 mL), followed by sodium ascorbate (23 mg, 0.12 mmol) and
copper sulfate pentahydrate (15 mg, 0.06 mmol). The reaction
mixture was stirred at room temperature for 24 h. The reaction was
0.41 mmol, 69% yield). Mp¼117 C; [
NMR (CD OD):
CO CH
4.61e4.64 (m, 3H, CH
a]
D
H
2
3
d
[ppm]¼3.17e3.25 (m, 2H, CH
2 2
CH Ph), 3.77 (s, 3H,
2
3
), 4.42 (dd, J¼6.0/5.0 Hz, 1H, 4-H), 4.55e4.57 (m, 1H, 3-H),
CH
Ph, 2-H), 5.17 (d, J¼6.0 Hz, 1H, 5-H),
7.16e7.18 (m, 2H, 2 -Hphenyl, 5 -Hphenyl), 7.19e7.22 (m, 1H, 4 -
2
2
0
0
00
00
quenched with a saturated aqueous solution of NaHCO
extracted with ethyl acetate (3ꢂ). The combined organic phases
were dried (Na SO ), filtered and evaporated. The residue was
purified by flash column chromatography (2 cm, 15 cm, 10 mL,
dichloromethane/methanol¼20:1, R ¼0.35) to give 21c as colour-
less solid (130 mg, 0.39 mmol, 66% yield). Mp¼132 C; [
c 1.6, MeOH); 1H NMR (CD
OD):
[ppm]¼3.76 (s, 3H, CO
.42 (dd, J¼5.9/5.1 Hz, 1H, 4-H), 4.54e4.58 (m, 1H, 3-H), 4.62 (d,
Ph),
3
and
0
0
00
H
phenyl), 7.25e7.28 (m, 2H, 3 -Hphenyl, 5 -Hphenyl), 7.93 (s, 1H,
0
13
2
4
5 -Htriazol); C NMR (CD
3
OD):
d
[ppm]¼37.5 (1C, CH
2 2
CH Ph), 52.5
(1C, CO
2
CH
3
), 52.7 (1C, CH
2
CH
2
Ph), 73.2 (1C, C-4), 74.2 (1C, C-3),
0
f
78.1 (1C, C-5), 81.2 (1C, C-2), 126.7 (1C, C-5 triazol), 127.9 (1C,
ꢁ
20
00 00 00 00
a
]
D
ꢀ20.7
CH ),
C-4 phenyl), 129.7 (2C, C-3 phenyl, C-5 phenyl), 129.9 (2C, C-2 phenyl,
00 00 0
C-6 phenyl), 138.7 (1C, C-1 phenyl), 146.0 (1C, C-4 triazol), 172.1 (1C,
(
3
d
2
3
ꢀ1
4
CO
2
CH
3
); IR (neat): ~
n
[cm ]¼3441, 1751, 1439, 1404, 1219, 1146,
þ
J¼5.6 Hz, 1H, 2-H), 5.19 (d, J¼6.0 Hz, 1H, 5-H), 5.58 (s, 2H, CH
2
1115, 1057, 964, 922, 856, 733, 694; HRMS (m/z): [MþH] calcd for
0
0
00
7
.07e7.13 (m, 2H, 3 -H4-fluorophenyl, 5 -H4-fluorophenyl), 7.37e7.42 (m,
C
16
H
20
N
3
O
5, 334.1397; found, 334.1413; HPLC (method 1):
00
00
0
13
2
H, 2 -H4-fluorophenyl, 6 -H4-fluorophenyl), 8.07 (s, 1H, 5 -Htriazol);
C
R
t ¼12.3 min, purity 96.6%.
NMR (CD
3
OD):
d
[ppm]¼52.4 (1C, CO
2 3 2
CH ), 54.1 (1C, CH Ph), 73.1
(
1C, C-4), 74.2 (1C, C-3), 78.1 (1C, C-5), 81.3 (1C, C-2), 116.7 (d,
5.2.11. (2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-[1-(3-phenylpropyl)-
1H-1,2,3-triazol-4-yl]tetrahydrofuran-2-carboxylate (21f). (3-
Azidopropyl)benzene (190 mg, 1.2 mmol) was added to a solution
2
of 18 (110 mg, 0.59 mmol) in a 1:1 mixture of t-BuOH and H O
(15 mL), followed by sodium ascorbate (23 mg, 0.12 mmol) and
copper sulfate pentahydrate (157 mg, 0.06 mmol). The reaction
mixture was stirred at room temperature for 24 h. The reaction was
0
0
00
, C-5 4-fluorophenyl), 126.7 (1C,
J¼22.0 Hz, 2C, C-3 4-fluorophenyl
0
00
00
C-5 triazol), 131.4 (d, J¼8.4 Hz, 2C, C-2 4-fluorophenyl, C-6 4-fluorophenyl),
00
0triazol
1
32.9 (d, J¼3.2 Hz, 1C, C-1 4-fluorophenyl), 146.5 (1C, C-4
), 164.2
0
0
(
[
9
3
9
d, J¼246 Hz, 1C, C-4 4-fluorophenyl), 172.1 (1C, CO
2
CH
3
); IR (neat): ~
n
ꢀ1
cm ]¼3471, 2970, 1732, 1512, 1435, 1288, 1227, 1107, 1061, 1042,
þ
68, 837, 772; HRMS (m/z): [MþH] calcd for C15
3 5,
H17FN O
38.1147; found, 338.1107; HPLC (method 1): t
R
¼11.7 min, purity
quenched with a saturated aqueous solution of NaHCO
extracted with ethyl acetate (3ꢂ). The combined organic phases
were dried (Na SO ), filtered and evaporated. The residue was
purified by flash column chromatography (2 cm, 15 cm, 10 mL,
dichloromethane/methanol¼20:1, R ¼0.38) to give 21f as colour-
less oil (130 mg, 0.36 mmol, 61% yield). [
3
and
7.3%.
2
4
5.2.9. (2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5{1-[4-(trifluoromethyl)
benzyl]-1H-1,2,3-triazol-4-yl}tetrahydrofuran-2-carboxylate
21d). 1-(Azidomethyl)-4-(trifluoromethyl)benzene (220 mg,
f
2
0
(
a
]
D
ꢀ10.6 (c 2.2, MeOH);
[ppm]¼2.20e2.26 (m, 2H, CH CH CH Ph),
2.63e2.66 (m, 2H, CH CH CH Ph), 3.78 (s, 3H, CO CH ), 4.39 (t,
CH CH
1
1.1 mmol) was added to a solution of 18 (100 mg, 0.54 mmol) in
H NMR (CD
3
OD):
d
2
2
2
a 1:1 mixture of t-BuOH and H
2
O (15 mL), followed by sodium
2
2
2
2
3
ascorbate (21 mg, 0.11 mmol) and copper sulfate pentahydrate
14 mg, 0.054 mmol). The reaction mixture was stirred at room
temperature for 24 h. The reaction was quenched with a saturated
aqueous solution of NaHCO
and extracted with ethyl acetate (3ꢂ).
The combined organic phases were dried (Na SO ), filtered and
J¼7.1 Hz, 2H, CH
2
2
2
Ph), 4.45 (dd, J¼5.9/5.1 Hz, 1H, 4-H), 4.58
(
(t, J¼5.3 Hz, 1H, 3-H), 4.65 (d, J¼5.6 Hz, 1H, 2-H), 5.21 (d, J¼6.1 Hz,
1H, 5-H), 7.16e7.20 (m, 1H, Hphenyl), 7.21e7.23 (m, 2H, Hphenyl),
0
13
3
7.26e7.30 (m, 2H, Hphenyl), 8.07 (s, 1H, 5 -Htriazol); C NMR
(CD OD): CH CH Ph), 33.4 (1C,
[ppm]¼33.0 (1C, CH
CH CH CH Ph), 50.7 (1C, CH CH CH Ph), 52.5 (1C, CO CH ), 73.2
2
4
3
d
2
2
2
evaporated. The residue was purified by flash column chromatog-
raphy (2 cm, 15 cm, 10 mL, dichloromethane/methanol¼20:1,
2
2
2
2
2
2
2
3
(1C, C-4), 74.2 (1C, C-3), 78.1 (1C, C-5), 81.3 (1C, C-2), 126.7 (1C, C-
triazol), 127.2 (1C, Cphenyl), 129.5 (4C, Cphenyl), 142.0 (1C, Cphenyl),
0
R
f
¼0.34) to give 21d as colourless solid (130 mg, 0.32 mmol, 60%
5
ꢁ
20
1
0
ꢀ1
yield). Mp¼159 C; [
a
]
D
ꢀ20.8 (c 2.1, MeOH); H NMR (CD
3
OD):
146.1 (1C, C-4 triazol), 172.1 (1C, CO
2
CH
3
); IR (neat): ~
n
[cm ]¼3406,
d
[ppm]¼3.76 (s, 3H, CO
2
CH
3
), 4.44 (dd, J¼6.0/5.0 Hz, 1H, 4-H), 4.56
2951, 1755, 1497, 1439, 1215, 1119, 1080, 745, 702; HRMS (m/z):
þ
(
(
H
dd, J¼5.6/5.0 Hz, 1H, 3-H), 4.64 (d, J¼5.6 Hz, 1H, 2-H), 5.21
[MþH] calcd for C17
H
22
N
3
O
5
348.1554; found, 348.1563; HPLC
0
0
d, J¼6.0 Hz, 1H, 5-H), 5.71 (s, 2H, CH
2
Ph), 7.49e7.52 (m, 2H, 2 -
(method 1): t
R
¼14.0 min, purity 95.6%.
0
0
00
00
phenyl, 6 -Hphenyl), 7.66e7.69 (m, 2H, 3 -Hphenyl, 5 -Hphenyl), 8.13 (s,
0
13
1
H, 5 -Htriazol); C NMR (CD
1C, CH Ph), 73.2 (1C, C-4), 74.2 (1C, C-3), 78.1 (1C, C-5), 81.3 (1C,
C-2), 125.5 (q, J¼271 Hz, 1C, CF
3
OD):
d
[ppm]¼52.4 (1C, CO
2
CH
3
), 54.2
5.2.12. (2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(1-phenyl-1H-1,2,3-triazol-
4-yl)tetrahydrofuran-2-carboxamide (10a). Hydroxylamine hydro-
chloride (120 mg, 1.8 mmol) and a 2.0 M solution of sodium
methoxide in methanol (0.88 mL, 1.8 mmol) were added to a solu-
tion of 21a (110 mg, 0.35 mmol) in dry methanol (5 mL). The
reaction mixture was stirred at ambient temperature for 20 h until
TLC showed complete conversion of the ester. The reaction mixture
was acidified with 1.0 M HCl to pH 5e6. Then the mixture was
extracted with ethyl acetate (3ꢂ). The combined organic phases
(
2
0
0
3
), 126.8 (q, J¼3.8 Hz, 2C, C-3 phenyl
,
0
0
0
00
00phenyl
C-5 phenyl), 127.1 (1C, C-5 triazol), 129.7 (2C, C-2 phenyl, C-6
),
0
0phenyl
00
1
31.5 (q, J¼32.1 Hz, 1C, C-4
), 141.3 (1C, C-1 phenyl), 146.7 (1C,
0
ꢀ1
C-4 triazol), 172.1 (1C, CO
2
CH
3
); IR (neat): ~
n
[cm ]¼3302, 2959,
2
901, 1759, 1439, 1327, 1219, 1107, 1065, 1018, 964, 926, 826, 748;
þ
HRMS (m/z): [MþH] calcd for C16
H
17
F
3
N
3
O5, 388.1115; found,
3
88.1131; HPLC (method 1): t
R
¼15.1 min, purity 97.4%.
were dried with Na
The crude mixture was purified by flash column chromatography
(1 cm, 15 cm, 5 mL, dichloromethane/methanol¼9:1, R ¼0.29) to
2 4
SO , filtered and the solvent was dried in vacuo.
5
.2.10. (2S,3R,4S,5S)-Methyl 3,4-dihydroxy-5-(1-phenethyl-1H-1,2,3-
(21e). (2-Azidoethyl)
benzene (170 mg, 1.2 mmol) was added to a solution of 18 (110 mg,
.59 mmol) in a 1:1 mixture of t-BuOH and H O (15 mL), followed
triazol-4-yl)tetrahydrofuran-2-carboxylate
f
give 10a as colourless crystalline solid (47 mg, 0.15 mmol,
ꢁ
20
1
0
2
44%). Mp¼56 C; [
a]
D
ꢀ16.9 (c 1.1, MeOH); H NMR (CD
3
OD):
by sodium ascorbate (23 mg, 0.12 mmol) and copper sulfate pen-
tahydrate (15 mg, 0.06 mmol). The reaction mixture was stirred at
room temperature for 24 h. The reaction was quenched with
d
[ppm]¼4.45 (dd, J¼5.6/4.8 Hz, 1H, 4-H), 4.53 (d, J¼5.8 Hz, 1H, 2-
H), 4.63 (dd, J¼5.8/4.8 Hz, 1H, 3-H), 5.30 (d, J¼5.6 Hz, 1H, 5-H),
0
0
00
7.47e7.52 (m, 1H, 4 -Hphenyl), 7.56e7.61 (m, 2H, 3 -Hphenyl
,
0
0
0
0
0
0
a saturated aqueous solution of NaHCO
3
and extracted with ethyl
SO ),
5 -Hphenyl), 7.83e7.87 (m, 2H, 2 -Hphenyl, 6 -Hphenyl), 8.55 (s, 1H,
0
13
acetate (3ꢂ). The combined organic phases were dried (Na
2
4
5 -Htriazol); C NMR (CD
3
OD):
d
[ppm]¼73.6 (1C, C-4), 74.2 (1C,
0
0
00
00
filtered and evaporated. The residue was purified by flash column
chromatography (ؼ2 cm, h¼15 cm, V¼10 mL, dichloromethane/
C-3), 78.2 (1C, C-5), 81.1 (1C, C-2), 121.6 (2C, C-2 phenyl, C-6 phenyl),
0
00
124.2 (1C, C-5 triazol), 130.1 (1C, C-4 phenyl), 130.9 (2C, C-3 phenyl
,
00 00 0
methanol¼20:1, R
f
¼0.38) to give 21e as colourless solid (140 mg,
C-5 phenyl), 138.5 (1C, C-1 phenyl), 146.9 (1C, C-4 triazol), 168.9 (1C,

6576
S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
ꢀ
1
00
0
00
00
CONHOH); IR (neat): ~
n
[cm ]¼3206, 2986, 2901, 1663, 1501, 1238,
6 -Hphenyl), 7.66e7.70 (m, 2H, 3 -Hphenyl, 5 -Hphenyl), 8.10 (s, 1H,
þ
13
1
3
9
134, 1053, 756, 691; HRMS (m/z): [MþH] calcd for C13
H
15
N
4
O
5
,
5 -Htriazol); C NMR (CD
3
OD):
d
[ppm]¼54.2 (1C, CH
2
Ph), 73.6 (1C,
07.1037; found, 307.1067; HPLC (method 2): t
R
¼12.6 min, purity
C-4), 74.1 (1C, C-3), 78.2 (1C, C-5), 80.9 (1C, C-2), 125.5 (q, J¼271 Hz,
0
00
6.4%.
1C, CF
3
), 126.4 (1C, C-5 triazol), 126.9 (q, J¼3.9 Hz, 2C, C-3 phenyl
,
0
0
00
00
C-5 phenyl), 129.7 (2C, C-2 phenyl, C-6 phenyl), 131.6 (q, J¼32.2 Hz, 1C,
00 00 0
5
.2.13. (2S,3R,4S,5S)-5-(1-Benzyl-1H-1,2,3-triazol-4-yl)-N,3,4-
(10b). Hydroxylamine
C-4 phenyl), 141.2 (1C, C-1 phenyl),146.4 (1C, C-4 triazol), 168.9 (1C,
ꢀ1
trihydroxytetrahydrofuran-2-carboxamide
CONHOH); IR (neat): ~
n
[cm ]¼3217, 1663, 1420, 1323, 1165, 1119,
þ
hydrochloride (87 mg, 1.3 mmol) and a 2.0 M solution of sodium
methoxide in methanol (0.63 mL, 1.3 mmol) were added to a solu-
tion of 21b (80 mg, 0.25 mmol) in dry methanol (10 mL). The re-
action mixture was stirred at ambient temperature for 16 h until
TLC showed complete conversion of the ester. The reaction mixture
was evaporated and purified by flash column chromatography
1065, 1018, 822; HRMS (m/z): [MþH] calcd for C15
16 3 4 5,
H F N O
389.1067; found, 389.1093; HPLC (method 2): t
R
¼14.5 min, purity
97.1%.
5.2.16. (2S,3R,4S,5S)-N,3,4-Trihydroxy-5-(1-phenethyl-1H-1,2,3-
triazol-4-yl)tetrahydrofuran-2-carboxamide (10e). Hydroxylamine
hydrochloride (94 mg, 1.4 mmol) and a 2.0 M solution of sodium
methoxide in methanol (0.68 mL, 1.4 mmol) were added to a solu-
tion of 21e (90 mg, 0.27 mmol) in dry methanol (10 mL). The
reaction mixture was stirred at ambient temperature for 16 h until
TLC showed complete conversion of the ester. The reaction mixture
was evaporated and purified by flash column chromatography
(
1 cm, 15 cm, 5 mL, dichloromethane/methanol¼9:1, R ¼0.26) to
f
give 10b as colourless solid (33 mg, 0.10 mmol, 41% yield).
ꢁ
20
1
Mp¼50 C; [
a]
D
ꢀ6.2 (c 1.3, MeOH); H NMR (CD
3
OD):
d
[ppm]¼
4
.33 (t, J¼5.0 Hz, 1H, 4-H), 4.46 (d, J¼6.1 Hz, 1H, 2-H), 4.60 (dd,
J¼6.0/4.8 Hz, 1H, 3-H), 5.19 (d, J¼5.3 Hz, 1H, 5-H), 5.59 (s, 2H,
0
13
CH
CD
2
Ph), 7.32e7.39 (m, 5H, Hphenyl), 8.03 (s, 1H, 5 -Htriazol); C NMR
(
3
OD):
d
[ppm]¼55.0 (1C, CH
2
Ph), 73.5 (1C, C-4), 74.1 (1C, C-3),
(1 cm, 15 cm, 5 mL, dichloromethane/methanol¼9:1, R ¼0.30) to
f
0
7
8.3 (1C, C-5), 80.8 (1C, C-2), 126.0 (1C, C-5 triazol), 129.2 (2C,
give 10e as colourless solid (48 mg, 0.14 mmol, 53%
ꢁ
20
1
C
1
3
phenyl), 129.5 (1C, Cphenyl), 130.0 (2C, Cphenyl), 136.7 (1C, Cphenyl),
46.1 (1C, C-4 triazol), 168.9 (1C, CONHOH); IR (neat): ~
202, 1659, 1454, 1323, 1227, 1123, 1057, 1026, 718; HRMS (m/z):
yield). Mp¼51 C; [
a]
D
ꢀ4.7 (c 1.0, MeOH); H NMR (CD
3
OD):
0
ꢀ1
n
[cm ]¼
d
[ppm]¼3.21 (t, J¼7.3 Hz, 2H, CH
2
CH
2
Ph), 4.33 (t, J¼5.0 Hz, 4-H),
4.46 (d, J¼6.0 Hz, 1H, 2-H), 4.58 (dd, J¼6.0/4.8 Hz, 3-H), 4.63
þ
[
(
MþH] calcd for C14
H
17
N
4
O
5
321.1193; found, 321.1200; HPLC
(t, J¼7.3 Hz, 2H, CH
2
CH
2
Ph), 5.16 (d, J¼5.4 Hz, 1H, 5-H), 7.15e7.17
0
0
00
00
method 2): t
R
¼12.6 min, purity 95.4%.
(m, 2H, 2 -Hphenyl, 6 -Hphenyl), 7.20e7.22 (m, 1H, 4 -Hphenyl),
0
0
00
0
13
7
.26e7.28 (m, 2H, 3 -Hphenyl, 5 -Hphenyl), 7.88 (s, 1H, 5 -Htriazol);
NMR (CD OD): CH Ph), 52.8 (1C, CH CH Ph),
[ppm]¼37.5 (1C, CH
73.6 (1C, C-4), 74.1 (1C, C-3), 78.2 (1C, C-5), 80.9 (1C, C-2), 126.1 (1C,
C
5
4
.2.14. (2S,3R,4S,5S)-5-[1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl]-N,3,
-trihydroxytetrahydrofuran-2-carboxamide (10c). Hydroxylamine
3
d
2
2
2
2
0 00 00 00
C-5 triazol), 127.9 (1C, C-4 phenyl), 129.7 (2C, C-3 phenyl, C-5 phenyl),
hydrochloride (100 mg, 1.5 mmol) and a 2.0 M solution of sodium
methoxide in methanol (0.74 mL, 1.5 mmol) were added to a solu-
tion of 21c (100 mg, 0.30 mmol) in dry methanol (10 mL). The re-
action mixture was stirred at ambient temperature for 16 h until
TLC showed complete conversion of the ester. The reaction mixture
was evaporated and purified by flash column chromatography
0
0
00
00
129.8 (2C, C-2 phenyl, C-6 phenyl), 138.7 (1C, C-1 phenyl), 145.7 (1C,
C-4 triazol), 169.0 (1C, CONHOH); IR (neat): ~
0
ꢀ1
n
[cm ]¼3198, 2901,
þ
1659, 1454, 1227, 1123, 1057, 1026, 748, 698; HRMS (m/z): [MþH]
calcd for C15
19 4 5
H N O , 335.1350; found, 335.1380; HPLC (method 2):
t
R
¼13.2 min, purity 95.5%.
(
1 cm, 15 cm, 5 mL, dichloromethane/methanol¼9:1, R ¼0.26) to
f
give 10c as colourless solid (58 mg, 0.17 mmol, 58% yield).
5.2.17. (2S,3R,4S,5S)-N,3,4-Trihydroxy-5-[1-(3-phenylpropyl)-1H-
1,2,3-triazol-4-yl]tetrahydrofuran-2-carboxamide (10f). Hydroxy-
lamine hydrochloride (110 mg, 1.6 mmol) and a 2.0 M solution of
sodium methoxide in methanol (0.79 mL, 1.6 mmol) were added to
a solution of 21f (110 mg, 0.32 mmol) in dry methanol (10 mL). The
reaction mixture was stirred at ambient temperature for 16 h until
TLC showed complete conversion of the ester. The reaction mixture
was evaporated and purified by flash column chromatography
ꢁ
20
1
Mp¼77 C (decomposition); [
a]
D
ꢀ10.2 (c 1.0, MeOH); H NMR
(
CD
3
OD):
d
[ppm]¼4.33 (t, J¼5.0 Hz, 1H, 4-H), 4.46 (d, J¼6.1 Hz, 1H,
2
5
-H), 4.60 (dd, J¼6.0/4.7 Hz 1H, 3-H), 5.19 (d, J¼5.3 Hz, 1H, 5-H),
00 00
2
.58 (s, 2H, CH Ph), 7.07e7.14 (m, 2H, 3 -H4-fluorophenyl, 5 -H4-
00
00
fluorophenyl), 7.37e7.42 (m, 2H, 2 -H4-fluorophenyl, 6 -H4-fluorophenyl),
0
13
8
.04 (s, 1H, 5 -Htriazol); C NMR (CD
CH Ph), 73.5 (1C, C-4), 74.1 (1C, C-3), 78.2 (1C, C-5), 80.8 (1C, C-2),
16.7 (d, J¼22.0 Hz, 2C, C-3 4-fluorophenyl, C-5 4-fluorophenyl),126.0 (1C,
3
OD):
d
[ppm]¼54.2 (1C,
2
0
0
00
1
(1 cm, 15 cm, 5 mL, dichloromethane/methanol¼9:1, R
f
¼0.30) to
0
00
00
ꢁ
C-5 triazol), 131.4 (d, J¼8.4 Hz, 2C, C-2 4-fluorophenyl, C-6 4-fluorophenyl),
give 10f as colourless solid (44 mg, 0.13 mmol, 40% yield). Mp¼49 C;
0
0
0triazol
20
1
1
(
~
n
32.9 (d, J¼3.2 Hz, 1C, C-1 4-fluorophenyl), 146.2 (1C, C-4
), 164.2
[
a
]
D
ꢀ1.8 (c 1.6, MeOH); H NMR (CD
3
OD):
d
[ppm]¼2.20e2.28 (m,
CH CH Ph), 4.36 (t,
0
0
d, J¼246 Hz, 1C, C-4 4-fluorophenyl), 168.9 (1C, CO
2
NHOH); IR (neat):
2H, CH
2
CH
2
CH
2
Ph), 2.63e2.67 (m, 2H, CH
2
2
2
ꢀ
1
[cm ]¼3213, 1655, 1508, 1223, 1126, 1057, 1018, 822, 772; HRMS
J¼5.0 Hz, 1H, 4-H), 4.40 (t, J¼7.0 Hz, 2H, CH CH CH Ph), 4.48 (d,
2
2
2
þ
(
m/z): [MþH] calcd for C14
H
16FN
4
O5, 339.1099; found, 339.1127;
J¼6.0 Hz,1H, 2-H), 4.61 (dd, J¼6.0/4.7 Hz,1H, 3-H), 5.21 (d, J¼5.3 Hz,
HPLC (method 2): t
R
¼13.1 min, purity 96.4%.
1H, 5-H), 7.17e7.23 (m, 3H, Hphenyl), 7.25e7.30 (m, 2H, Hphenyl), 8.03
0
13
(
s, 1H, 5 -Htriazol);
CH CH CH Ph), 33.4 (1C, CH
C
NMR (CD
3
OD):
d
[ppm]¼32.9 (1C,
CH CH Ph),
5
.2.15. (2S,3R,4S,5S)-N,3,4-Trihydroxy-5-{1-[4-(trifluoromethyl)ben-
2
2
2
2
CH CH Ph), 50.8 (1C, CH
2
2
2
2
2
zyl]-1H-1,2,3-triazol-4-yl}tetrahydrofuran-2-carboxamide
10d). Hydroxylamine hydrochloride (90 mg, 1.3 mmol) and
a 2.0 M solution of sodium methoxide in methanol (0.63 mL,
.3 mmol) were added to a solution of 21d (100 mg, 0.26 mmol) in
dry methanol (4 mL). The reaction mixture was stirred at ambient
temperature for 16 h until TLC showed complete conversion of the
ester. The reaction mixture was evaporated and purified by flash
column chromatography (1 cm, 15 cm, 5 mL, dichloromethane/
73.6 (1C, C-4), 74.2 (1C, C-3), 78.3 (1C, C-5), 80.9 (1C, C-2), 126.0 (1C,
C-5 triazol), 127.2 (1C, Cphenyl), 129.5 (2C, Cphenyl), 129.6 (2C, Cphenyl),
0
(
0
142.0 (1C, Cphenyl), 145.9 (1C, C-4 triazol), 169.0 (1C, CONHOH); IR
ꢀ
1
1
(neat): ~
n
[cm ]¼3210, 2920,1659, 1497, 1454,1126, 1061, 1026, 745,
þ
698; HRMS (m/z): [MþH] calcd for C16
21 4 5
H N O 349.1506; found,
349.1539; HPLC (method 2): t
R
¼13.8 min, purity 95.5%.
5.3. Biological evaluation
methanol¼9:1, R
.18 mmol, 70% yield). Mp¼88 C; [
NMR (CD OD):
[ppm]¼4.35 (t, J¼5.0 Hz, 1H, 4-H), 4.47
f
¼0.26) to give 10d as colourless solid (70 mg,
ꢁ
20 1
0
a
]
D
ꢀ12.2 (c 1.3, MeOH);
H
5.3.1. Agar diffusion clearance assay. The antibiotic activity of the
synthesized inhibitors was determined by agar disc diffusion
clearance assays. Liquid cultures of E. coli BL21 (DE3) and the an-
3
d
(
d, J¼6.0 Hz, 1H, 2-H), 4.60 (dd, J¼6.0/4.8 Hz, 1H, 3-H), 5.21 (d,
00
26
J¼5.3 Hz, 1H, 5-H), 5.71 (s, 2H, CH
2
Ph), 7.49e7.53 (m, 2H, 2 -Hphenyl
,
tibiotic resistant strain E. coli D22 were grown overnight in LB

S.K. Jana et al. / Tetrahedron 70 (2014) 6569e6577
6577
broth27 at 37 C, 200 rpm. Overnight cell suspension (150
spread evenly onto LB agar Petri dishes. Each compound (15
10 mM in DMSO) was applied onto circular filter paper (Ø 6 mm,
ꢁ
m
L) was
L)
9. Schwardt, O.; Rabbani, S.; Hartmann, M.; Abgottspon, D.; Wittwer, M.; Kleeb, S.;
Zalewski, A.; Smiesko, M.; Cutting, B.; Ernst, B. Bioorg. Med. Chem. 2011, 19,
m
6454e6473.
(
10. Yu, J. L.; Wu, Q. P.; Zhang, Q. S.; Liu, Y. H.; Li, Y. Z.; Zhou, Z. M. Bioorg. Med. Chem.
thickness 0.75 mm, Carl Roth). Pure DMSO, serving as a negative
Lett. 2010, 20, 240e243.
11. Jana, S. K.; Loppenberg, M.; Daniliuc, C. G.; Jose, J.; Holl, R. Tetrahedron 2013, 69,
and CHIR-090,15 serving as a positive control were also spotted. The
9434e9442.
ꢁ
Petri dishes were incubated overnight at 37 C and the diameter of
1
2. Barb, A. W.; Zhou, P. Curr. Pharm. Biotechnol. 2008, 9, 9e15.
the zone of growth inhibition was measured for each compound.
13. Clements, J. M.; Coignard, F.; Johnson, I.; Chandler, S.; Palan, S.; Waller, A.;
Wijkmans, J.; Hunter, M. G. Antimicrob. Agents Chemother. 2002, 46, 1793e1799.
1
4. Barb, A. W.; McClerren, A. L.; Snehelatha, K.; Reynolds, C. M.; Zhou, P.; Raetz, C.
R. H. Biochemistry 2007, 46, 3793e3802.
5
.3.2. Protein purification and LpxC assay. Purification of LpxC and
11
the LpxC enzyme assay were performed as previously described.
15. McClerren, A. L.; Endsley, S.; Bowman, J. L.; Andersen, N. H.; Guan, Z. Q.; Ru-
dolph, J.; Raetz, C. R. H. Biochemistry 2005, 44, 16574e16583.
1
6. Loppenberg, M.; Muller, H.; Pulina, C.; Oddo, A.; Teese, M.; Jose, J.; Holl, R. Org.
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