Synthesis and Biological Evaluation of Several Dephosphonated Analogues of CMP-Neu5Ac as Inhibitors of GM3-Synthase

Article abstract of DOI:10.1002/chem.201501770

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Full text of DOI:10.1002/chem.201501770

DOI: 10.1002/chem.201501770
Full Paper
&
Inhibitors
Synthesis and Biological Evaluation of Several Dephosphonated
Analogues of CMP-Neu5Ac as Inhibitors of GM3-Synthase**
[
c]
[b]
[b]
[c]
[b]
Paola Rota, Federica Cirillo, Marco Piccoli, Antonio Gregorio, Guido Tettamanti,
[
c]
[a, b]
Pietro Allevi, and Luigi Anastasia*
Abstract: Previous studies demonstrated that reducing the
GM3 content in myoblasts increased the cell resistance to
hypoxic stress, suggesting that a pharmacological inhibition
of the GM3 synthesis could be instrumental for the develop-
ment of new treatments for ischemic diseases. Herein, the
synthesis of several dephosphonated CMP-Neu5Ac conge-
ners and their anti-GM3-synthase activity is reported. Biologi-
cal activity testes revealed that some inhibitors almost com-
pletely blocked the GM3-synthase activity in vitro and re-
duced the GM3 content in living embryonic kidney 293A
cells, eventually activating the epidermal growth factor re-
ceptor (EGFR) signaling cascade.
[
6]
Introduction
tially mimic the effects of NEU3 overexpression. Nevertheless,
we speculated that chemically reducing the GM3 cell content
with a specific inhibitor of its synthase could result in a more
robust activation of the cell response to hypoxia. However, to
the best of our knowledge, the dephosphonate derivative 3a
(Figure 1) is the only GM3-synthase inhibitor described to date,
but its specificity and its activity in vitro on living cells has not
Sialic acid (Neu5Ac (1), Figure 1) is transferred to the terminal
portion of glycoconjugates through a process known as sialyla-
tion, which is mediated by sialyltransferases (STs), a class of en-
zymes that are localized in the Golgi apparatus and use cyti-
dine 5’-monophospho-N-acetylneuraminic acid [CMP-Neu5Ac
[1]
[7]
(
2)] as the sialyl donor. Of particular relevance is the sialyla-
been reported yet. Actually, as a CMP-Neu5Ac mimetic, its
tion of the terminal galactose residue of lactosylceramide
specificity for GM3-synthase could be modest. Along this line,
several fluorescent mimetics of CMP-Neu5Ac have been recent-
ly reported to be highly potent in inhibiting commercially
[
2]
mediated by a ST often referred to as the GM3-synthase, as it
leads to the formation of ganglioside GM3, a key precursor of
[3]
[8]
more complex ganglioside species. Gangliosides are known
to be involved in the regulation of fundamental biological pro-
cesses (including cell proliferation and migration, apoptosis,
cancer formation, and metastasis), and efforts have been made
available eukaryotic and bacterial sialyl transferases. Actually,
the use of fluorescent probes could be instrumental for an ef-
fective screening of combinatorial libraries of compounds to
identify new and more specific inhibitors.
[
4]
to adjust their bioavailability by regulating their biosynthesis.
Moreover, the reported GM3-synthase inhibitor 3a is not
commercially available. Therefore, although we approached its
synthesis, we developed a new synthetic route that allowed
the synthesis of several congeners of compound 3a, including
Along this line, we reported that sialidase NEU3 overexpression
can reduce the GM3 content on the cell surface of skeletal my-
[
5]
oblasts, causing an activation of the epidermal growth factor
receptor (EGFR) signaling cascade, ultimately upregulating HIF-
[6]
1
a and protecting cells from hypoxic stress. We also showed
that chemical inhibition of all ganglioside syntheses with 1-
phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP) could par-
[
a] Prof. L. Anastasia
Department of Biomedical Sciences for Health
Institution University of Milan, Segrate Milan (Italy)
E-mail: luigi.anastasia@unimi.it
[
b] Dr. F. Cirillo, Dr. M. Piccoli, Prof. G. Tettamanti, Prof. L. Anastasia
Laboratory of Stem Cells for Tissue Engineering
IRCCS Policlinico San Donato, San Donato Milanese, Milan (Italy)
[
c] Dr. P. Rota, Dr. A. Gregorio, Prof. P. Allevi
Department of Biomedical, Surgical and Dental Sciences
Institution University of Milan, Via Saldini 50, 20133 Milan (Italy)
[
**] CMP-Neu5Ac=cytidine 5’-monophospho-N-acetylneuraminic acid.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501770.
Figure 1. Sialic acid and target sialosides.
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the glycosides 3b and 3c and the 1,7-lactones 4a–4c
Figure 1). We speculated that the presence of the perfluoroa-
acetamidic or N-glycosidic bonds. Moreover, the aldehydes 7
and 8 partially decomposed during the chromatographic pu-
rification step on silica. Therefore, they were both used in their
crude form during the successive synthetic steps, limiting their
purification to small aliquots used for their chemical characteri-
zation. Under these synthetic conditions, the alcohol 6 was iso-
lated, in pure form, in 45% yield from the aldehyde 5. In paral-
(
cylamido groups and the inner esterification of the polar car-
boxylic moiety with the 7-hydroxyl group in the lactones 4a–
4
c could modulate their lipophilicity enhancing both the in-
hibitory activity and the cellular uptake. Finally, the anti-GM3-
synthase activity of these glycosides was tested in vitro on cell
extracts and on living cultures of embryonic kidney 293A cells.
[
13]
lel, the 2-chlorosialo donor 9a and the new perfluorinated
analogs 9b and 9c, which are needed for the successive glyco-
sidation step, were synthesized by treating the appropriate
Results and Discussion
[9b,14]
peracetylated neuraminic acid methyl esters 10a–10c
The glycosides 3a–3c and their corresponding 1,7-lactones
with acetyl chloride/methanol (Scheme 1). Then, glycosidation
of the alcohol 6 with the 2-chlorosialo donors 9a–9c, under
Koening–Knorr-like conditions, afforded the desired b-glyco-
sides 11 a–11 c in satisfactory yields (39, 36, and 27% respec-
tively), accompanied by variable, but always minor, amounts of
their 2a-epimers 12a–12c (Scheme 1). The non-fluorinated b-
glycoside 11 a was accompanied by trace amounts of the a-
epimer 12a (<1%). On the contrary, the fluorinated analogous
were accompanied by appreciable amounts of their 2a-epi-
mers 12b and 12c (20 and 24%, respectively). This confirmed
that the presence of a perfluorinated acylamido group at the
C-5 in the sialosyl donor increases the a-stereoselectivity of
4
a–4c were synthesized taking advantage of some synthetic
[9]
procedures for the N-transacylation of amides and the 1,7-
[10]
lactonization of Neu5Ac (1), which were developed in our
laboratory. The key step of the synthesis of all inhibitors was
the formation of an O-glycosidic linkage between the alcoholic
nucleoside 6 (obtained through the elongation of the 5’
carbon chain of cytidine) and each different sialic derivative,
properly protected and suitably activated at their anomeric
function (Scheme 1). To this purpose, the known cytidine alde-
[
11]
hyde 5 was converted into the alcohol 6, that is, into the
aglyconic portion of the required glycosides 3a–3c, with
[
7]
[15]
a shorter synthetic route than the one previously reported.
the glycosidation, as previously observed. The obtained gly-
cosides 11 a–11 c as well as 12b and 12c showed physico-
chemical properties, which are in agreement with their struc-
ture. However, the NMR data did not allow the direct assign-
ment of their a or b geometry that was initially attributed on
the basis of the empirical rule reported for anomeric assign-
[
16]
ments in sialosides. We assigned the b configuration to the
anomers showing a relatively smaller coupling constant be-
tween the H-7 and H-8 sialic protons (J=1.6–4.4 Hz, in contrast
to J=8.7–8.9 Hz in the a-anomers) and a relatively larger
chemical shift difference between the two vicinal protons at
the C-9 sialic carbon atom (Dd>0.85 ppm) in respect to the a-
anomers (Dd<0.2 ppm), similarly to what was previously re-
[
7]
ported in the synthesis of compound 3a. Successively, we
unequivocally confirmed the assignment by a chemical ap-
proach, showing that only those sialosides deriving from the
deprotection of the sialosides 11 b and 11 c, of assigned b ge-
ometry, were able to form the corresponding 1,7-lactones.
Then, after some unsuccessful attempts to improve the yields
of the glycosylation reaction, we decided to experiment the
preparation of the glycoside 11 a–11 c in two steps, performing
the glycosylation of the alcohol 6 with the dibromosialosyl
Scheme 1. Synthesis of the sialosyl acceptor 6, the sialosyl donors 9a–9c,
and the protected sialosides 11 a–11 c and 12a–12c. i) (Ph) P=CHCHO,
CH Cl , RT, overnight; ii) H , Pd/C, MeOH, RT, 2 h; iii) NaBH
, MeOH, À208C,
5 min; iv) AcCl (Ac=acetyl), MeOH, À108C to RT, overnight; v) AgOTf
OTf=triflate), toluene/CH NO , RT, 3 h.
3
[
17]
2
2
2
4
donors 13a–13c, prepared by simple addition of bromine to
1
(
[9c]
the glycals 14a–14c. We were confident that, as first report-
3
2
[
16a]
ed by Goto et al.,
a dibrominated sialosyl donor would pref-
erentially give the b-anomer, as a consequence of the steric
hindrance on the a side, due to the bromine atom at the 3-po-
sition. If this effect would have overbore the opposite influ-
Initially, the aldehyde 5 underwent a Wittig reaction fol-
lowed by the catalytic hydrogenation of the formed a,b-unsa-
turated aldehyde 7 into the aldehyde 8, which was then re-
[
15]
ence of the perfluorinated acylamido groups at the C-5,
duced to the alcohol 6 with NaBH . The direct transformation
a more significant selectivity in favor of the b-anomers 15a–
15c could have been observed. These intermediates should
have been converted into the desired b-glycoside 11 a–11 c by
4
of the aldehyde 7 into the alcohol 6 was also attempted by
[12]
using various chemical or catalytic methods that, however,
afforded unsatisfactory results due to some concurrent reac-
tions, such as cytidine ring hydrogenation and the cleavage of
reductive elimination with (nBu) SnH of the remaining 3-bro-
3
mine atom (Scheme 2). Actually, the glycosidation performed
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at 238C occurred quickly, affording exclusively the expected b-
glycoside 15a–15c that, by successive debromination, gave
the glycosides 11 a–11 c, showing the same physicochemical
characteristics of those previously obtained. However, the over-
all yields of this two-step process resulted in the same range
of those observed in the one-step preparation of the b-glyco-
side 11 a–11 c. For this reason, we did not consider this second
procedure as an attractive alternative and, in the successive
preparations, we used the first protocol herein reported, which
eventually allowed the obtainment of both a- and b-anomers
useful for an NMR data comparison.
1D and 2D NMR experiments, clearly supported their b-anome-
ric structure. In fact, a clear signal shift to relatively high fields
1
(d=2.37–2.43 ppm) could be observed in the H NMR spectra
for the sialic 3-equatorial hydrogen atoms, diagnostic for a b-
[
16b]
sialosidic bond in the molecules.
The shifts toward relatively
high fields were evident when the spectra were compared
with that observed for the a-glycosides 20b and 20c (d=
2.68–2.76 ppm for the corresponding protons), that were ob-
tained by regeneration of the protected functions of the a-gly-
cosides 12b and 12c with the same reaction sequences set-up
for the corresponding b-anomers (Scheme 3).
Scheme 2. Synthesis and O-sialylation of the dibromosialosyl donors 13a–
Scheme 3. Synthesis of the free sialyl glycosides 3a–3c as well as 20b and
1
3c. i) Br
2 2 2 3 2
, CH Cl , 08C to RT, 30 min; ii) AgOTf, toluene/CH NO , RT, 3h; iii)
2
0c. i) moist TFA, CH
iii) for compound 17a: NaOH, MeOH, RT, 40 min; for compounds 17b and
9b: Et N, MeOH/H O, RT, 12 h; for compounds 17c and 19c: K CO , H O/
MeOH, RT, 12 h.
2 2
Cl , heating to reflux, 1 h; ii) NaOMe, MeOH, RT, 1 h;
(
Bu) SnH, 2,2’-azobisisobutyronitrile (AIBN), THF, heating to reflux, 3 h.
3
1
3
2
2
3
2
The obtained glycosides 11 a–11 c were then sequentially
deprotected to give the desired acids 3a–3c. The acetonide
group was removed by moist trifluoroacetic acid (TFA) treat-
ment affording the diols 16a–16c that were transformed in
the esters 17a–17c by transacetylation with MeONa in MeOH.
Finally, the regeneration of the carboxyl group required the
study of the appropriate conditions for selective hydrolysis of
the ester function while retaining the perfluorinated amido
group, known to be sensitive to basic environments. Hydrolysis
of the esters 17a and 17c proceeded easily (although we
The correct assignment of the anomeric configurations have
been confirmed by chemical evidence from the successive
transformation of the b-anomers 3a–3c into the correspond-
ing 1,7-lactones 4a–4c, and from the inability of the a-isomers
20b and 20c to undergo the same transformation.
With the appropriated hydroxyl acidic glycosides 3a–3c in
hand, we attempted their direct 1,7-lactonization by using
a protocol that we recently set up for the preparation of the
[
10]
1,7-lactone of Neu5Ac (1) (Scheme 4). The reaction afforded
the desired 1,7-lactones 4a–4c with different yields, which
were good for compounds 4b and 4c (70% yield) whereas the
yield was unsatisfactory for compound 4a. No lactone was ob-
tained in the analogous reactions of the a-glycosides 20b or
20c.
found that NaHCO did not work, in contrast with a previous
3
[
7]
report ), as compound 17a carries an acetamido group that
allowed the use of methanolic sodium hydroxide, as expected,
whereas compound 17c has a heptafluorobutyric amido group
that tolerated K CO in moist methanol.
2
3
On the contrary, the hydrolysis was critical for the ester 17b,
having a base-labile trifluoroacetyl amido group, and it could
take place in a suitable way only in the presence of triethyla-
mine in aqueous methanol under strictly controlled conditions.
The obtained target sialosides 3a–3c showed physicochemi-
cal properties (mass spectra, NMR spectra), which are in agree-
Moreover, as we anticipated that the unsatisfactory yields in
the lactonization reaction of 3a were due to the lower solubili-
ty of this glycoside in the reaction solvent, we decided to at-
tempt the reaction on the corresponding acetonide 21
(Scheme 4). In this case, the reaction afforded the protected
1,7-lactone 22 in good yields (70%).
Unfortunately, the regeneration of the glycol system of the
lactone 4a, although it was attempted under various reaction
conditions with different acids, was not completely selective
and difficult to be controlled. Thus, we accepted as the best
1
ment with the assigned structures (the H NMR data of com-
[7]
pound 3a reported in the literature contained some mistakes
and erroneous signal attributions if compared with our data).
In particular an NMR analysis with complete proton and
carbon resonance assignments, achieved by combination of
conditions those when we use aqueous CF COOH (90%, heat-
3
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1
mm concentration, revealing that compounds 3a, 3b, and
0c almost completely inhibit the formation of GM3 ganglio-
2
side from lactosylceramide (Figure 2A). Then, the best inhibi-
tors (i.e., compounds 3a, 3b, and 20c) were tested at lower
concentrations, revealing that the formation of GM3 could be
inhibited roughly to half (IC50) at a concentration of 10 mm
with all three compounds (Figure 2B). Successively, we tested
their inhibitory effect on living cells. Most importantly, we
wanted to test if they could penetrate the cell membrane, as
this would be necessary for their potential use in vivo.
Scheme 4. Synthesis of the lactones 4a–4c. i) CbzCl (Cbz=carbobenzyloxy),
Et N, THF/DMF, 0 8C to RT, 30 min; ii) NaOMe, MeOH, RT, 1 h; iii) NaOH,
MeOH, RT, 40 min; iv) moist TFA, CH Cl , heating to reflux, 1 h.
3
2
2
ing to reflux, RT) affording a mixture of the desired final lac-
tone 4a (65%) and of its parent acid 3a (10%). Purification by
reverse-phase preparative column chromatography and lyophi-
lization afforded the pure lactone 4a.
Successively, in order to facilitate the penetration of the
highly hydrophilic compounds 3a–3c through the membrane
of alive cells, peracetylated derivatives of the inhibitors (i.e.,
compounds 23a–23c) were prepared, by direct acetylation of
intermediates 16a–16c (Scheme 5). In fact, the possibility of
administering the peracetylated sialosyl derivatives to cell cul-
tures has been previously reported, as they become permeable
to the membrane and are hydrolyzed to the corresponding
Figure 2. HPTLC and relative densitometric quantification of GM3-synthase
activity with [3- H] lactosylceramide (LacCer) in the presence of A) 1 mm, or
B) 10 mm solutions of the GM3-synthase inhibitors. Statistical differences
were determined by one-way analysis of variance (ANOVA). ***: p<0.001.
Ctrl=control.
3
[18]
free compounds once inside the cell. Moreover, considering
that the previous donor-based inhibitors possessed some toler-
ance of the ST enzymes to structural modifications at the
anomeric site, we decided to test also the a-anomer 20c and
so we prepared, similarly to the corresponding b-anomer, the
peracetylated derivative 22c.
As anticipated, all inhibitors were too hydrophilic to be able
to enter the cell and initial attempts were unsuccessful. On the
other hand, the peracetylated inhibitors could penetrate the
cell membrane. In fact, embryonic kidney 293A cells were cul-
tured for 3 h in the presence of 10 mm GM3-synthase peracety-
lated inhibitors, whereas the negative controls were cultured
in the presence of 0.01% DMSO, which was used to dissolve
the inhibitors. Then, the cells were subjected to metabolic radi-
3
olabeling with a pulse of 2 h with [3- H]sphingosine, always in
the presence of 10 mm inhibitors or DMSO. After a chase of
7
2 h, the sphingolipids were extracted and subjected to
HPTLC, as described in the Experimental Section. Results re-
vealed a significant decrease in the GM3 content of 18, 28, and
1
3% for compounds 23a, 23b, and 24c, respectively (Figure-
2
Scheme 5. Peracetylated sialosides 23a–23c and 24c. i) Ac O, pyridine (Py),
RT, 3 h.
s 3A and B), causing a significant increase in the lactosyl ce-
ramide and gb3 content in the organic phase (Figures 3C and
D).
Finally, we tested whether treatment with GM3-synthase in-
hibitors would activate the EGFR signaling pathway. In fact, the
GM3 ganglioside is known to bind to the EGFR and to inhibit
Inhibitory activity tests
The GM3-synthase inhibitory activity of the newly synthesized
compounds 3a–3c, 20c, 4b, and 4c was tested in vitro on
human embryonic kidney 293A cell homogenates, by using ra-
diolabeled lactosylceramide as the substrate (Figure 2). Initially,
all compounds were screened for their inhibitory activity at
[
19]
its activation through autophosphorilation (Figure 4).
This
pathway is crucial for cell survival, and we demonstrated that
[
6]
it plays an important role in myoblast survival under hypoxia.
In particular, treatment with GM3-synthase inhibitors could
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Figure 5. Western blot analysis of the EGFR pathway activation. Total pro-
teins from Hek293 cells, cultured in the presence of 10 mm GM3-synthase in-
hibitors, were extracted and analyzed by a p-EGFR and total EGFR Western
blot (A) and relative densitometric quantification (B), by a p-ERK1/2 and
total ERK Western blot (C) and relative densitometric quantification (D), by
a p-p38 and total p38 Western blot (E) and relative densitometric quantifica-
tion (F). Statistical differences were determined by one-way ANOVA. *:
p<0.05, **: p<0.01, and ***: p<0.001.
3
Figure 3. Effects of the GM3-synthase inhibitors on [3- H]sphingosine radio-
labeled sphingolipids pattern. HEK293a cells were cultured in the presence
3
of 10 mm inhibitors and metabolically labeled with [3- H]sphingosine. Radio-
chromatoscanning images of the HPTLC separation of the gangliosides con-
tained in the aqueous phase (A) and gangliosides distribution (B). Radiochro-
matoscanning images of the HPTLC separation of the sphingolipids con-
tained in the organic phase (C) and sphingolipid distribution (D). Statistical
differences were determined by one-way ANOVA. *: p<0.05, **: p<0.01,
and ***: p<0.001.
Conclusion
mimic the effects of sialidase NEU3 activation, that is, reduce
the GM3 cell content, thereby activating the EGFR signaling
cascade (Figure 4).
These results, although preliminary, support the notion that
GM3-synthase inhibitors may be used to activate the cell re-
sponse to hypoxic stress, similar to what we reported for siali-
dase NEU3 overexpression. Although we cannot assure, at this
stage, the specificity of the newly synthesized inhibitors for the
GM3-synthase, the chemical approach has several advantages
over gene overexpresison, as it can be finely regulated and
suspended at any time. Although preliminary, these results
support the feasibility of the hypothesis of chemically activat-
ing the cell defense machinery as a possible new approach for
treating ischemic pathologies affecting the human heart and
the nervous system. Further studies to test the effects of se-
lected GM3-synthase inhibitors on these cell types are current-
ly undergoing in our laboratories.
Figure 4. Representation of the EGFR signaling pathway activated by GM3-
synthase inhibitors. Mechanism of the GM3-synthase inhibitors activation of
the prosurvival signaling cascade, mediated by GM3 reduction and conse-
quent phosphorilation of the EGFR.
To this purpose, Hek293A cells were incubated with 10 mm
GM3 synthase peracetlyated inhibitors for 3 h and then sub-
jected to protein extraction and Western blot analysis. Results
revealed a significant increase in the EGFR and p-EGFR (Figure-
s 5 A and B) and in its downstream targets ERK1/2, pERK1/2,
p38 MAPK and p-p38 MAPK (Figures 5C–F), confirming that
the GM3-synthase inhibitors can mimic NEU3 overexpression in
activating cell anti-apoptotic and prosurvival machinery.
Experimental Section
General information
Chemicals: All chemicals and solvents used were of analytical
grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
Water was prepared by filtering deionized water on a Milli-Q Sim-
plicity 185 filtration system from Millipore (Bedford, MA, USA). Sol-
vents were dried by using standard methods and distilled before
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use. The progress of all reactions was monitored by thin layer chro-
matography (TLC) carried out on 0.25 mm E. Merck silica gel plates
characterization by flash chromatography (AcOEt/MeOH, 95:5, v/v).
1
H NMR (500 MHz, CDCl
=7.5 Hz, 1H; H-6), 7.43 (d, J
6’ =15.8, 1H; H-5’), 6.23 (ddd, J6’
5’ =15.8 Hz, 1H; H-6’), 5.57 (brs,1H; H-1’), 5.25 (brd,
3’ =6.3 Hz, 1H; H-2’), 5.07 (dd, J3’ 2’ =6.3 Hz, 1H; H-3’),
): d=9.56 (d, J7’,
6’ =7.8 Hz, 1H; H-7’), 8.95
=7.5 Hz, 1H;
4’ =1.3,
3
(
60 F254) by using UV light, anisaldehyde/H SO /EtOH solution, or
(s, 1H; NH), 7.60 (d, J
H-5), 7.01 (dd, J5’
6
,
5
,
5 6
2
4
0
.2% ninhydrin in ethanol and heat as developing agent. All flash
,
4’ =6.2, J5’
,
,
chromatography separations were performed with normal phase
silica gel (E. Merck 230–400 mesh silica gel), following the general
J
J
6’
,
,
7’ =7.8, J6’,
,
4’ =3.9, J3’,
2’
[20]
4
.87–4.83 (m, 1H; H-4’), 2.25 (s, 3H; NHCOCH ), 1.58 (s, 3H;
3
13
protocol of Still. Melting points were measured on a SMP m.p.
3
C(CH ) ), 1.36 ppm (s, 3H; C(CH ) ); C NMR (125 MHz, CDCl ): d
apparatus (Stuart Scientific, USA) and are not corrected. Nuclear
magnetic resonance spectra were recorded at 303 K on a Bruker
AM-500 spectrometer equipped with a 5 mm inverse-geometry
3 2
3 2
3
=
193.2 (C-7’), 170.1 (NHCOCH ), 163.3 (C-4), 154.6 (C-2), 153.0 (C-
3
5
’), 147.9 (C-6), 132.3 (C-6’), 114.3 (C(CH ) ), 98.9 (C-1’), 96.8 (C-5),
3 2
1
89.0 (C-4’), 85.2 (C-3’), 85.0 (C-2’), 27.0 (C(CH ) ), 25.2 (C(CH ) ),
3 2 3 2
+
broadband probe and operating at 500.13 MHz for H and
1
3
25.0 ppm (NHCOCH ); MS (ESI positive): m/z: 372.1 [M+Na] . The
1
25.76 MHz for C. Chemical shifts are reported in parts per million
3
1
crude aldehyde 7, dissolved in MeOH (100 mL), was hydrogenated
in the presence of 10% Pd on carbon (160 mg) for 2 h. At this
time, the catalyst was filtered and the solvent was removed under
reduced pressure to afford the crude compound 8, which was
used in the next step without further purification. A small amount
of the crude aldehyde 8 was purified for the analytical characteriza-
tion by flash chromatography (AcOEt/MeOH, 95:5, v/v). M.p. 1858C
and are referenced for H spectra, to a solvent residue proton
signal (d=7.26, 3.31,and 2.50 ppm, respectively, for CDCl , CD OD,
3
3
1
3
and (CD ) SO) and for C spectra, to solvent carbon signal (central
3
3
line at d=77.0, 49.05, and 39.43 ppm, respectively, for CDCl3,
CD OD, and (CD ) SO). The chemical shifts obtained for D O solu-
3
3 3
2
tion are referenced to the internal (CH ) COH signal d=1.24 ppm
3
3
1
13
1
13
for H spectra and d=30.29 ppm for C spectra. The H and
C
2
D
0
(
from CH Cl /diisopropyl ether 7:3, v/v); [a] = +23.4 (c=1 in
1
1
1
13
2 2
resonances were assigned by H– H (COSY) and H– C (HSQC and
HMBC) correlation 2D experiments. The H NMR data are tabulated
1
chloroform); H NMR (500 MHz, CDCl ): d=9.75 (brs, 1H; H-7’), 9.23
1
3
(
s, 1H; NH), 7.63 (d, J , =7.4 Hz, 1H; H-6), 7.42 (d, J , =7.4 Hz, 1H;
6 5 5 6
as follows: s=singlet, d=doublet, brs=broad singlet, m=multip-
let, coupling constant(s) are given in hertz ([Hz]). The carbon nu-
meration used in the NMR spectra is given below.
H-5), 5.60 (brs, 1H; H-1’), 5.04 (brd, J , =6.5 Hz, 1H; H-2’), 4.71–
2’ 3’
4
2
2
.67 (m, 1H; H-3’), 4.14–4.08 (m, 1H; H-4’), 2.62–2.56 (overlapping,
H; H-6a’, H-6b’), 2.26 (s, 3H; NHCOCH ), 2.19–2.03 (overlapping,
3
H; H-5a’, H-5b’), 1.55 (s, 3H; C(CH ) ), 1.33 ppm (s, 3H; C(CH ) );
3
2
3 2
1
3
C NMR (125 MHz, CDCl ): d =201.2 (C-7’), 170.4 (NHCOCH ), 163.1
3
3
(
C-4), 154.5 (C-2), 146.8 (C-6), 114.5 (C(CH ) ), 96.7 (C-5), 96.0, (C-1’),
3 2
8
5
3
7.2 (C-4’), 84.9 (C-2’), 83.8 (C-3’), 40.0 (C-6’), 27.2 (C(CH ) ), 25.5 (C-
3 2
’), 25.3 (C(CH ) ), 25.0 ppm (NHCOCH ); MS (ESI positive): m/z:
3
2
3
+
74.6 [M+Na] . The obtained crude aldehyde 8 was dissolved in
MeOH (20 mL) and the solution was cooled at À208C. NaBH
4
(
158 mg, 2.05 mmol) was added and after 15 min the reaction was
stopped with the addition of acetone (4 mL). The reaction mixture
+
was neutralized with Amberlite resin IRC-50 (H ), filtered, and the
Optical rotations were taken on a Perkin–Elmer 241 polarimeter
solvent was removed under reduced pressure. The residue was pu-
rified by flash chromatography (AcOEt/MeOH, 90:10, v/v), to afford
equipped with a 1 dm tube. The [a] values are given in
D
À1
2
À1
À1
[
10 degcm g ] and the concentration are given in [g100mL ].
[7]
compound 6 (590 mg, 45% from compound 5), as a white solid.
Mass spectrometry was performed by using a Finnigan LCQDeca
quadrupole ion-trap mass spectrometer equipped with an ESI ion
source (Finnigan ThermoQuest, San Jose, CA, USA). The spectra
were collected in a continuous flow mode by connecting the infu-
sion pump directly to the ESI source. Solutions of the compounds
2
D
0
M.p. 97–988C (from CH Cl /diisopropyl ether 7:3, v/v); [a] = +
2
2
[7]
3
1.2 (c=1.0 in methanol) (lit. [a] = +48.6(c=1.4 in methanol));
H NMR (500 MHz, CDCl ): d=9.76 (s, 1H; NH), 7.73 (d, J , =7.5 Hz,
D
1
3
6 5
1
H; H-6), 7.42 (d, J , =7.5 Hz, 1H; H-5), 5.69 (d, J , =1.6 Hz, 1H;
5 6 1’ 2’
À1
H-1’), 4.97 (dd, J2’,1’ =1.6, J2’,3’ =6.4 Hz, 1H; H-2’), 4.62 (dd, J3’,2’ =6.4,
were infused at a flow rate of 5 mLmin , the spray voltage was
J , =4.5 Hz, 1H; H-3’), 4.19–4.16 (m, 1H; H-4’), 3.70–3.64 (overlap-
3’ 4’
set at 5.0 kV in the positive and at 4.5 kV in the negative ion mode
with a capillary temperature of 2208C. Full-scan mass spectra were
recorded by scanning a m/z range of 100–2000.
ping, 2H; H-7a’, H-7b’), 2.26 (s, 3H; NHCOCH ), 1.89–1.74 (overlap-
3
ping, 2H; H-5a’, H-5b’), 1.73–1.63 (overlapping, 2H; H-6a’, H-6b’),
13
1
.56 (s, 3H; C(CH ) ), 1.33 ppm (s, 3H; C(CH ) ); C NMR (125 MHz,
3
2
3
2
Biological: Human embryonic kidney 293A cells (Hek293A) were
CDCl ): d =170.9 (NHCOCH ), 163.2 (C-4), 154.8 (C-2), 146.0 (C-6),
3
3
obtained from Invitrogen and cultured in Dulbecco’s-modified
1
14.3 (C(CH ) ), 96.7 (C-5), 95.3 (C-1’), 87.9 (C-4’), 85.2 (C-2’), 83.9 (C-
À1
3 2
Eagle’s medium (DMEM) with high glucose content (4.5 gL ),
3
2
’), 62.2 (C-7’), 30.0 (C-5’), 28.9 (C-6’), 27.2 (C(CH ) ), 25.3 (C(CH ) ),
3
2
3 2
(
(
Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum
+
4.9 ppm (NHCOCH ); MS (ESI positive): m/z: 376.2 [M+Na] , 729.1
À1
3
FBS, Sigma-Aldrich), 4 mm l-glutamine (Gibco), 100 unitsmL
+
[
2M+Na] ; elemental analysis calcd (%) for C H N O : C 54.38, H
16 23 3 6
À1
penicillin, and 100 mgmL streptomycin (Euroclone). All cell cul-
tures were performed at 378C in a humidified incubator with 5%
CO2.
6
.56, N 11.89; found: C 54.42, H 6.60, N 11.82.
Synthesis of the chloro derivatives 9a–9c
Synthetic procedure affording alcohol 6
General procedure: To a solution of the appropriated compound
10a–10c (0.30 mmol), dissolved in acetyl chloride (5 mL) and
cooled to À108C under an argon atmosphere, anhydrous MeOH
(0.8 mL) was added. The solution was stirred overnight at RT then
the solvent was removed under reduced pressure to give a syrup,
which was crystallized from hexane/ethyl acetate (6:4, v/v) afford-
ing the derivatives 9a–9c.
A solution of aldehyde 5 (1.20 g, 3.71 mmol) and (triphenylphos-
phoranylidene)acetaldehyde (1.36 g, 4.46 mmol) in CH Cl (60 mL)
was stirred overnight at RT. Then, the orange solution was concen-
trated under reduced pressure to give the crude aldehyde 7, which
was used in the next step without further purification. A small
amount of the crude aldehyde 7 was purified for the analytical
2
2
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14619
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[
14]
Compound 9a: Starting from compound 10a
(160 mg,
which was crystallized from hexane/ethyl acetate (6:4, v/v) to
afford the 2,3-dibromo derivative 13a–13c.
[13]
0
.3 mmol) the chloro derivative 9a (118 mg, 77%) was obtained
as only stereoisomer as a white solid. H NMR (500 MHz, CDCl3):
d=5.47 (dd, J7, =2.4, J7,8 =7.0 Hz, 1H; H-7), 5.44 (d, JNH,5 =
0.2 Hz, 1H; NH), 5.39 (ddd, J , =4.8, J , =10.4, J4,3b =11.0 Hz,
H; H-4), 5.17 (ddd, J8,9a =2.7, J8,9b =5.8, J , =7.0 Hz, 1H; H-8),
.42 (dd, J , =2.7, J , =12.5 Hz, 1H; H-9a), 4.35 (dd, J , =2.4,
1
Compound 13a: Starting from the glycal 14a (284 mg,
[17]
6
0.60 mmol), the 2,3-dibromo derivative 13a (353 mg, 93%) was
1
1
4
20
4
3a
4 5
obtained as a white needles. M.p. 155–1578C; [a] =À58.1 (c=
D
[17]
21
D
8 7
1 in chloroform) (lit.
m.p. 156–1578C; [a] =À57.7 (c=1 in
1
9
a 8
9a 9b
6 7
chloroform)); H NMR (500 MHz, CDCl ): d=5.77 (dd, J , =3.5, J ,
3 4 3 4 5
J , =11.3 Hz, 1H; H-6), 4.20 (ddd, J5,NH =10.2, J , =10.4, J ,
6
5
5 4
5 6
=10.5 Hz, 1H; H-4), 5.44 (d, JNH,5 =9.1 Hz, 1H; NH), 5.42 (dd, J , =
7 6
=
11.3 Hz, 1H; H-5), 4.06 (dd, J , =5.8, J , =12.5 Hz, 1H; H-9b),
9b 8 9b 9a
.87 (s, 3H; COOCH ), 2.78 (dd, J , =4.8, J , =13.9 Hz, 1H; H-
3 3a 4 3a 3b
a), 2.28 (dd, J3 , =11.2, J , =13.9 Hz, 1H; H-3b), 2.12 (s, 3H;
OCOCH ), 2.07 (s, 3H; OCOCH ), 2.04 (s, 3H; OCOCH ), 2.03 (s, 3H;
1.7, J7,8 =7.3 Hz, 1H; H-7), 5.26 (ddd, J , =2.6, J , =5.5, J , =
8 9a
8 9b
8 7
3
3
7.3 Hz, 1H; H-8), 5.05 (d, J , =3.5 Hz, 1H; H-3), 4.54–4.42 (overlap-
3 4
ping, 3H; H-6, H-9a, H-5), 4.14 (dd, J , =5.5, J , =12.6 Hz, 1H; H-
9a 8 9a 9b
9b), 3.90 (s, 3H; COOCH ), 2.16 (s, 3H; OCOCH ), 2.12 (s, 3H;
3 3
OCOCH ), 2.08 (s, 3H; OCOCH ), 2.06 (s, 3H; OCOCH ), 1.96 ppm (s,
3 3 3
b 3a
3a 3b
3
3
3
OCOCH ), 1.91 ppm (s, 3H; NHCOCH ); MS (ESI positive): m/z: 532.9
3
3
+
[
M+Na] ; elemental analysis calcd (%) for C H ClNO : C 47.11, H
20 28 12
3H; NHCOCH ). All other physico-chemical properties practically
3
5
.53, N 2.75; found: C 47.18, H 5.61, N 2.81. Other physico-chemical
properties were identical to those reported in literature.
[17]
superimposable to those previously reported.
[13]
[9c]
Compound 13b: Starting from the glycal 14b
(316 mg,
[9c]
Compound 9b: Starting from the trifluoro derivative 10b
176 mg, 0.30 mmol) the chloro derivative 9b (137 mg, 81%) was
obtained as only stereoisomer as a white solid. M.p. 1328C; [a] =
0
.60 mmol), the 2,3-dibromo derivative 13b (391 mg, 95%) was
(
20
obtained as white needles. [a] =À30.1 (c=1 in chloroform);
D
20
1
D
H NMR (500 MHz, CDCl ): d=7.00 (d, JNH, =9.1 Hz, 1H; NH), 5.84
3 5
1
+
25.1 (c=1 in chloroform); H NMR (500 MHz, CDCl ): d=6.53 (d,
3
(dd, J , =3.2, J , =10.4 Hz, 1H; H-4), 5.40 (dd, J , =1.7, J ,
4 3 4 5 7 6 7 8
J
, =10.1 Hz, 1H; NH), 5.53 (ddd, J , =4.9, J , =10.2, J4,3b
4 3a 4 5
NH 5
=6.6 Hz, 1H; H-7), 5.27–5.23 (m, 1H; H-8), 5.06 (d, J3,4 =3.2 Hz,
1H; H-3), 4.59 (dd, J , =1.7, J , =10.8 Hz, 1H; H-6), 4.52–4.45
=
11.3 Hz, 1H; H-4), 5.44 (dd, J , =2.3, J , =7.2 Hz, 1H; H-7), 5.19
7 6
7 8
=2.6, J8,9b =5.3, J8,7 =7.2 Hz, 1H; H-8), 4.51 (dd, J6,7
6
7
6 5
(
ddd, J8,
9a
(overlapping, 2H; H-5, H-9a), 4.19 (dd, J9b,8 =5.5, J , =12.6 Hz,
9b 9a
=
2.3, J , =10.7 Hz, 1H; H-6), 4.40 (dd, J , =2.6, J , =12.6 Hz,
6 5 9a 8 9a 9b
H; H-9a), 4.13 (ddd, J5,NH =10.1, J , =10.2, J , =10.7 Hz, 1H; H-5),
5 4 5 6
.08 (dd, J9 , =5.3, J , =12.6 Hz, 1H; H-9b), 3.89 (s, 3H;
COOCH ), 2.83 (dd, J , =4.9, J , =14.0 Hz, 1H; H-3a), 2.31 (dd,
J , =11.3, J , =14.0 Hz, 1H; H-3b), 2.13 (s, 3H; OCOCH ), 2.09
s, 3H; OCOCH ), 2.05 ppm (s, 6H; 2”OCOCH ); C NMR (125 MHz,
3 3
CDCl ): d=170.8, 170.6, 170.4, 169.5 (4”OCOCH ), 165.3 (C-1),
57.6 (q, J , =38.0 Hz, COCF ), 115.3 (J , =288 Hz, COCF ), 96.0 (C-
), 73.1 (C-6), 70.7 (C-8), 68.1 (C-4), 66.9 (C-7), 62.0 (C-9), 53.7
COOCH ), 49.0 (C-5), 40.4 (C-3), 20.7, 20.5, 20.4, 20.3 ppm (4”
OCOCH ); MS (ESI positive): m/z: 586.2 [M+Na] ; elemental analysis
calcd (%) for C H ClF NO : C 42.60, H 4.47, N 2.48; found: C
2.52, H 4.48, N 2.63.
Compound 9c: Starting from the heptafluoro derivative 10c
206 mg, 0.30 mmol) the chloro derivative 9c (160 mg, 80%) was
1H; H-9b), 3.92 (s, 3H; COOCH ), 2.17 (s, 3H; OCOCH ), 2.11 (s, 3H;
3
3
1
4
OCOCH ), 2.10 (s, 3H; OCOCH ), 2.05 ppm (s, 3H; OCOCH );
3 3 3
13
b 8
9b 9a
C NMR (125 MHz, CDCl ): d=170.7, 170.6, 170.3, 170.1 (4”
3
3
3a 4
3a 3b
OCOCH ), 163.8 (C-1), 157.6 (J , =38.0 Hz, COCF ), 115.3 (J , =
3 C F 3 C F
3
b 3a
3a 3b
3
287.9 Hz, COCF ), 91.0 (C-2), 75.3 (C-6), 70.6 (C-8), 68.1 (C-4), 66.9
3
1
3
(
(C-7), 61.9 (C-9), 54.0 (COOCH ), 52.6 (C-3), 46.1 (C-5), 20.9, 20.6,
3
+
3
3
20.4 ppm (4”OCOCH ); MS (ESI positive): m/z (%): 710.3 [M+Na]
(100), 712.7 [M+Na] (51), 708.5 [M+Na] (53); elemental analysis
3
1
2
+
+
C F
3
C F
3
calcd (%) for C H Br F NO : C 34.96, H 3.52, N 2.04; found: C 34.8
20
24
2
3
12
(
[9c]
3
Compound 13c: Starting from the glycal 14c
(376 mg,
+
3
0
.60 mmol), the 2,3-dibromo derivative 13c (439 mg, 93%) was as
2
0
25
3
12
20
1
white needles. [a] =À18.5 (c=1 in chloroform); H NMR
D
4
(
500 MHz, CDCl ): d=7.29 (d, J , =8.8 Hz, 1H; NH), 5.89 (dd, J , =
3 NH 5 4 3
[9c]
3
7
.5, J , =10.6 Hz, 1H; H-4), 5.37 (dd, J , =1.8, J , =6.3 Hz,1H; H-
4 5 7 6 7 8
(
), 5.25 (ddd, J8,9a =2.3, J8,9b =5.7, J , =6.3 Hz, 1H; H-8), 5.08 (d,
8 7
20
obtained as only stereoisomer as a white solid. M.p. 1288C; [a] =
D
J
3
,
4
=3.5 Hz, 1H; H-3), 4.64 (dd, J
, =1.8, J , =10.8 Hz, 1H; H-6),
6 7 6 5
1
+
12.5 (c=1 in chloroform); H NMR (500 MHz, CDCl ): d=7.32 (d,
3
4.52 (dd, J , =2.3, J , =12.6 Hz, 1H; H-9a), 4.43 (ddd, J5,NH =8.8,
9a 8
9a 9b
J
, =9.7 Hz, 1H; NH), 5.55 (ddd, J , =4.9, J , =10.3, J4,3b
4 3a 4 5
11.2 Hz, 1H; H-4), 5.43 (dd, J , =2.2, J , =5.7 Hz, 1H; H-7), 5.15
7 6 7 8
NH 5
J , =10.6, J , =10.8 Hz, 1H; H-5), 4.22 (dd, J9b,8 =5.7, J , =
5 4 5 6 9b 9a
=
(
12.6 Hz, 1H; H-9b), 3.91 (s, 3H; COOCH ), 2.18 (s, 3H; OCOCH ),
3
3
ddd, J8,9a =2.5, J8,7 =5.7, J8,9b =6.4 Hz, 1H; H-8), 4.57 (dd, J6,7
2.2, J , =10.7 Hz, 1H; H-6), 4.50 (dd, J , =2.5, J , =12.5 Hz,
13
2.09 (s, 6H; 2”OCOCH ), 2.04 ppm (s, 3H; OCOCH );
C NMR
3
3
=
6
5
9a 8
9a 9b
(125 MHz, CHCl ): d=170.6, 170.5, 170.4, 169.8 (4”OCOCH ), 163.8
3
3
1
4
H; H-9a), 4.18 (ddd, J5,NH =9.7, J , =10.3, J , =10.7 Hz, 1H; H-5),
5 4 5 6
.10 (dd, J9 , =6.4, J , =12.5 Hz, 1H; H-9b), 3.88 (s, 3H;
COOCH ), 2.83 (dd, J , =4.9, J , =13.9 Hz, 1H; H-3a), 2.24 (dd,
J , =11.2, J , =13.9 Hz, 1H; H-3b), 2.13 (s, 3H; OCOCH ), 2.10
s, 3H; OCOCH ), 2.04 (s, 3H; OCOCH ), 2.03 ppm (s, 3H; OCOCH );
C NMR (125 MHz, CD OD): d=170.6, 169.7 (4”OCOCH ), 165.4 (C-
), 158.1 (J , =27 Hz, COCF CF CF ), 124.0–110.0 (COCF CF CF ),
C F 2 2 3 2 2 3
5.9 (C-2), 73.1 (C-6), 70.8 (C-4), 67.8 (C-8), 67.0 (C-7), 62.0 (C-9),
(C-1),
158.0
(J , =26.0 Hz,
C F
COCF CF CF ),
125.0–110.0
2
2
3
b 8
9b 9a
(COCF CF CF ), 90.6 (C-2), 74.7 (C-6), 70.4 (C-8), 67.5 (C-4), 67.0 (C-
2 2 3
3
3a 4
3a 3b
7), 61.8 (C-9), 54.0 (COOCH ), 52.8 (C-3), 46.6 (C-5), 20.8, 20.6, 20.5,
3
+
3
b 3a
3a 3b
3
20.3 ppm (4”OCOCH ); MS (ESI positive): m/z (%): 810.4 [M+Na]
(100), 812.6 [M+Na] (49), 808.7 [M+Na] (50); elemental analysis
3
(
+
+
3
3
3
1
3
3
3
calcd (%) for C H Br F NO : C 33.57, H 3.07, N 1.78; found: C
22
24
2
7
12
1
9
5
33.49, H 2.98, N 1.79.
3.9 (COOCH ), 49.5 (C-5), 40.6 (C-3), 20.8, 20.6, 20.5, 20.4 ppm (4”
CH COO); MS (ESI positive): m/z: 686.3 [M+Na] ; elemental analysis
3
+
3
Glycosylation with the chloro derivatives 9a–9c
calcd (%) for C H ClF NO : C 39.80, H 3.80, N 2.11; found: C
2
2
25
7
12
General procedure: Silver trifluoromethanesulfonate (284 mg,
3
9.72, H 3.78, N 2.15.
1
.10 mmol) in anhydrous toluene-MeNO2 (4 mL, 1:1, v/v) was
added to a stirred mixture of acceptor 6 (265 mg, 0.75 mmol), the
appropriate donor 9a–9c (0.95 mmol), and 4 Š molecular sieves
Synthsis of the dibromo derivatives 13a–13c
(
300 mg) in anhydrous toluene/MeNO (8 mL, 1:1, v/v), under an
2
General procedure: Bromine (0.80 mmol) was added to a solution
argon atmosphere at RT. The reaction mixture was stirred at RT for
3 h and then filtered through a pad of Celite and concentrated
under reduced pressure. Column chromatography of the residue
gave the b-anomer 11 a–11 c, respectively, as first compound
of the appropriate glycal 14a–14c (0.60 mmol) in CH Cl2 (5 mL)
2
under an argon atmosphere at 08C. After stirring for 30 min at RT,
the solvent was removed under reduced pressure to give a syrup,
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14620
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
eluted, and then the corresponding a-anomer 12a–12c, respec-
tively.
Further elution afforded compound 12b as a white solid (167 mg,
20
1
20%). M.p. 1208C; [a] =À4.70 (c=1 in chloroform); H NMR
D
(
500 MHz, CDCl ): d=9.30 (s, 1H; NH at C-4), 7.72 (d, J , =7.5 Hz,
3 6 5
Compounds 11 a and 12a: Starting from compound 9a (484 mg,
1
H; H-6), 7.42 (d, J , =7.5 Hz, 1H; H-5), 6.92 (d, J , =9.7 Hz, 1H;
5 6 NH 5’’
0
.95 mmol), column chromatography (AcOEt/MeOH, 98:2, v/v to
NH at C-5’’), 5.66 (brs, 1H; H-1’), 5.41 (ddd, J8’’,9a’’ =2.9, J , =4.6,
8’’ 9b’’
AcOEt/MeOH, 80:20, v/v) first afforded compound 11 a as a white
J , =8.7 Hz, 1H; H-8’’), 5.33 (dd, J7’’,6’’ =1.9, J7’’,8’’ =8.7 Hz, 1H; H-
8’’ 7’’
2
D
0
1
solid (306 mg, 39%). [a] =À7.2 (c=1 in methanol); H NMR
7’’), 5.09 (ddd, J4’’,3a’’ =4.6, J4’’,5’’ =10.7, J , =12.0 Hz, 1H; H-4’’),
4’’ 3b’’
(
500 MHz, CDCl ): d=9.19 (s, 1H; NH at C-4), 7.86 (d, J , =9.9 Hz,
3 NH 5”
5
1
.03 (d, J2’,3’ =6.1 Hz, 1H; H-2’), 4.64 (dd, J3’,4’ =4.7, J3’,2’ =6.1 Hz,
H; H-3’), 4.28 (dd, J6’’,7’’ =1.9, J6’’,5’’ =10.7 Hz, 1H; H-6’’), 4.26–4.19
1
7
H; NH at C-5’’), 7.58 (d, J6,5 =7.4 Hz, 1H; H-6), 7.40 (d, J , =
.4 Hz, 1H; 5-H), 5.84 (dd, J7”,8” =1.6, J7”,6” =3.0 Hz, 1H; H-7”), 5.64
5 6
(
overlapping, 2H; H-9a’’, H-9b’’), 4.16–4.04 (m, 1H; H-4’), 4.00 (ddd,
(
dd, J , =1.4, J , =6.3 Hz, 1H; H-2’), 5.40 (d, J , =1.0 Hz,1H; H-
2’ 1’’ 2’ 3’ 1’ 2
J , =9.7, J , =J , =10.7 Hz, 1H; H-5’’), 3.83–3.77 (m, 1H; H-
5’’ NH
5’’ 4’’
5’’ 6’’
1
5
4
=
(
’), 5.30 (ddd, J4’’,3a’’ =4.9, J4’’,5’’ =10.5, J , =11.1 Hz, 1H; H-4’’),
4’’ 3b’’
7
a’), 3.75 (s, 3H; COOCH ), 3.31–3.24 (m, 1H; H-7b’), 2.65 (dd, J ,
3 3a’’ 4’’
.07 (ddd, J8’’,7’’ =1.6, J , =2.0, J , =9.5 Hz, 1H; H-8’’), 4.96–
8’’ 9a’’
8’’ 9b’’
=
4.6, J3
,
=12.9 Hz, 1H; H-3a’’), 2.24 (s, 3H; NHCOCH at C-4),
a
’
’
3
b
’
’
3
.90 (overlapping, 2H; H-3’and H-9a’’), 4.50 (dd, J6’’,5’’ =10.6, J6’’,7’’
3.0 Hz, 1H; H-6’’), 4.24–4.14 (overlapping, 2H; H-4’and H-5’’), 4.07
dd, J9 =12.0, J =9.5 Hz, 1H; H-9b’’), 3.76 (s, 3H;
2
2
.13 (s, 3H; OCOCH ), 2.11 (s, 3H; OCOCH ), 2.04 (s, 3H; OCOCH ),
3
3
3
.02 (s, 3H; OCOCH ), 1.91 (dd, J
,
=12.1, J , =12.9 Hz, 1H;
3a’’ 3b’’
3
3b’’ 4’’
,
,
9b’’ 9a’’
b’’ 9a’’
H-3b’’), 1.83–1.76 (overlapping, 2H; H-5a’, H-5b’), 1.71–1.58 (over-
lapping, 2H; H-6a’, H-6b’), 1.56 (s, 3H; C(CH ) ), 1.34 ppm (s, 3H;
COOCH ), 3.55–3.50 (overlapping, 2H; H-7a’, H-7b’), 2.56 (dd, J
,
3
3a’’ 3b’’
3
2
=
12.7, J3’’,4’’ =4.9 Hz, 1H; H-3a’’), 2.21 (s, 3H; NHCOCH at C-4),
13
3
C(CH ) ); C NMR (125 MHz, CD OD): d=173.0 (NHCOCH at C-4),
3
2
3
3
2
9
.19 (s, 3H; OCOCH ), 2.01 (s, 3H; OCOCH ), 1.97–1.75 (overlapping,
3
3
1
1
2
8
8
3
72.5, 171.8, 171.6, 171.4 (4”OCOCH ), 169.6 (C-1’’), 164.7 (C-4),
3
H; 2”OCOCH , H-3b’’, H-5a’, H-5b’, NHCOCH at C-5’’), 1.74–1.55
3
3
59.3 (q, J , =37 Hz, COCF ), 157.7 (C-2), 147.6 (C-6), 117.2 (q, J , =
C F
3
C F
(
3
5
overlapping, 2H; H-6a’, H-6b’), 1.53 (s, 3H; C(CH ) ), 1.38 ppm (s,
3 2
13
87 Hz, COCF ), 115.4 (C(CH ) ), 100.1 (C-2’’), 98.1 (C-5), 95.5 (C-1’),
3
3 2
H; C(CH ) ); C NMR (125 MHz, CD OD): d=173.5 (NHCOCH at C-
3 2 3 3
8.5 (C-4’), 86.6 (C-2’), 85.3 (C-3’), 72.5 (C-6’’), 70.2 (C-4’’), 69.4 (C-
’’), 172.9 (NHCOCH3 at C-4), 172.4 (CH COO at C-9’’), 172.0
3
’’), 68.4 (C-7’’), 65.6 (C-7’), 63.4 (C-9’’), 53.4 (COOCH ), 50.8 (C-5’’),
3
(
CH COO at C-4’’ and at C-8’’), 171.9 (CH COO at C-7’’), 169.1 (C-1’’),
3 3
9.1 (C-3’’), 31.1 (C-5’), 27.6 (C(CH ) ), 27.1 (C-6’), 25.7 (C(CH ) ), 24.6
3
2
3 2
1
9
7
64.7 (C-4), 157.7 (C-2), 148.6 (C-6), 115.3 (C(CH ) ), 100.0 (C-2’’),
8.1 (C-5), 96.9 (C-1’), 88.8 (C-4’), 86.3 (C-2’), 85.6 (C-3’), 72.9 (C-8’’),
2.4 (C-6’’), 70.7 (C-4’’), 70.3 (C-7’’), 64.7 (C-7’), 63.7 (C-9’), 53.2
3 2
(
NHCOCH at C-4), 21.3, 20.8, 20.7, 20.6 ppm (4”CH COO); MS (ESI
3 3
+
positive): m/z: 903.4 [M+Na] ; elemental analysis calcd (%) for
C H F N O : C 49.09, H 5.38, N 6.36; found: C 49.15, H 5.41, N
36
47
3
4
18
(
COOCH ), 50.3 (C-5’’), 38.5 (C-3’’), 31.5 (C-5’), 27.6 (C(CH ) ), 26.7 (C-
3 3 2
6
.30.
6
2
8
’), 25.6 (C(CH ) ), 24.6 (NHCOCH at C-4), 22.9 (NHCOCH at C-5’’),
3
2
3
3
Compounds 11 c and 12c: Starting from compound 9c (631 mg,
1.0, 20.8, 20.8, 20.7 ppm (4”CH COO); MS (ESI positive): m/z:
3
+
+
0
.95 mmol) column chromatography (hexane/AcOEt, 70:30, v/v to
27.0 [M+H] , 849.3 [M+Na] ; elemental analysis calcd (%) for
AcOEt/MeOH, 95:5, v/v) first afforded compound 11 c as a white
solid (252 mg, 27%). M.p. 128–1308C; [a] = +3.9 (c=1.0 in metha-
C H N O : C 52.30, H 6.10, N 6.10; found: C 52.41, H 6.18, N 6.09.
36
50
4
18
’’
1
Further elution afforded compound 12a (2.3 mg, 0.3%). MS (ESI
nol); H NMR (500 MHz, CDCl ): d=9.18 (d, J , =9.7 Hz, 1H; NH
3
NH 5’’
+
+
positive): n/z: 827.3 [M+H] , 849.0 [M+Na] .
at C-5’’), 9.08 (s, 1H; NH at C-4), 7.58 (d, J , =7.4 Hz, 1H; H-6), 7.42
6
5
(
5
1
d, J , =7.4 Hz, 1H; H-5), 5.96 (brs, J , =1.8, J7’’,6’’ =3.2 1H; H-7’’),
5 6 7’’ 8’’
Compounds 11 b and 12b: Starting from compound 9b (536 mg,
.68 (dd, J2’,1’ =1.2, J2’,3’ =6.0 Hz, 1H; H-2’), 5.39 (d, J , =1.2 Hz,
1’ 2’
0
.95 mmol) column chromatography (hexane/AcOEt, 70:30, v/v to
H; H-1’), 5.35 (ddd, J4’’,3a’’ =5.0, J4’’,5’’ =10.4, J , =11.0 Hz, 1H;
4’’ 3b’’
AcOEt/MeOH, 95:5, v/v) first afforded compound 11 b as a white
H-4’’), 5.11 (ddd, J8’’,7’’ =1.8, J , =2.4, J , =9.4 Hz, 1H; H-8’’),
4
=
=
J9
lapping, 2H; H-7a’, H-7b’), 2.68 (dd, J3
1
2
ping, 4H; OCOCH , H-5b’), 1.78 (s, 3H; OCOCH ), 1.77–1.73 (m, 1H;
H-3b”), 1.64–1.57 (overlapping, 2H; H-6a’, H-6b’), 1.55 (s, 3H;
C(CH ) ), 1.40 ppm (s, 3H; C(CH ) ); C NMR (125 MHz, CD OD): d=
1
1
(
(
(
2
0
8’’ 9a’’
8’’ 9b’’
solid (301 mg, 36%). M.p. 1348C; [a] =À44.5 (c=1 in chloro-
D
.96–4.90 (overlapping, 2H; H-9a’’, H-3’), 4.72 (dd, J6’’,7’’ =3.2, J6’’,5’’
2
D
0
1
form); [a] =À12.2 (c=1 in methanol); H NMR (500 MHz, CDCl ):
3
10.7 Hz, 1H; H-6’’), 4.32 (ddd,
J , =9.7, J , =10.4, J ,
NH 5’’ 5’’ 4’’ 5’’ 6’’
d=9.16 (d, J , =9.8 Hz, 1H; NH at C-5’’), 9.02 (s, 1H; NH at C-4),
NH 5’’
10.7 Hz, 1H; H-5’’), 4.27–4.23 (m, 1H; H-4’), 4.07 (dd, J9
,
=9.4,
b’’ 8’’
7
.58 (d, J , =7.4 Hz, 1H; H-6), 7.42 (d, J , =7.4 Hz, 1H; H-5), 5.90
6
5
5
6
,
=12.1 Hz, 1H; H-9b’’), 3.79 (s, 3H; COOCH ), 3.60–3.52 (over-
b’’ 9a’’
3
(
dd, J , =1.8, J7’’,6’’ =3.0 Hz 1H; H-7’’), 5.67 (d, J , =6.1 Hz, 1H;
7
’
’
8
’
’
2
’
1
’
,
=5.1, J
,
=12.8 Hz,
a’’ 4’’
3a’’ 3b’’
H-1’), 5.42–5.35 (overlapping, 2H; H-1’ and H-4’’), 5.09 (ddd, J8’’,7’’
H; H-3a’’), 2.22 (s, 3H; NHCOCH at C-4), 2.21 (s, 3H; OCOCH ),
3
3
=
1.8, J , =2.1, J , =9.1 Hz, 1H; H-8’’), 4.94 (dd, J9a’’,8’’ =2.1,
8’’ 9a’’ 8’’ 9b’’
.18–2.07 (m, 1H, H-5a’), 1.99 (s, 3H; OCOCH ), 1.97–1.89 (overlap-
3
J9
,
a’’ 9b’’
=12.1 Hz, 1H; H-9a’’), 4.91–4.89 (m, 1H; H-3’), 4.71 (dd, J6’’,7’’
3
3
=
3.0, J , =10.5 Hz, 1H; H-6’’), 4.28–4.21 (overlapping, 2H; H-4’
6
’’ 5’’
and H-5’’), 4.08 (dd, J9
,
=9.1, J
, =12.1 Hz, 1H; H-9b’’), 3.78
9b’’ 9a’’
b’’ 8’’
13
3
2
3 2
3
(
(
s, 3H; COOCH ), 3.58–3.52 (overlapping, 2H; H-7a’ and H-7b’), 2.65
3
72.9 (NHCOCH3 at C-4), 172.4, 172.0, 171.6, 171.5 (4”OCOCH3),
68.9 (C-1’’), 164.7 (C-4), 159.5 (t, J , =26 Hz, COCF CF CF ), 157.6
dd, J3
,
=4.9, J
,
=12.8 Hz, 1H; H-3a’’), 2.20 (s, 6H; NHCOCH3
a’’ 4’’
3a’’ 3b’’
C F
2
2
3
at C-4, OCOCH ), 2.17–2.12 (m, 1H; H-5a’), 2.01 (s, 3H; OCOCH ),
3
3
C-2), 148.2 (C-6), 124.0–110.0 (COCF CF CF ), 115.4 (C(CH ) ), 100.0
2 2 3 3 2
C-2”), 98.2 (C-5), 96.0 (C-1’), 88.4 (C-4’), 86.4 (C-2’), 85.3 (C-3’), 72.6
C-8’’), 71.5 (C-6’’), 70.0 (C-4’’), 69.9 (C-7’’), 64.8 (C-7’), 63.5 (C-9’’),
1
.95 (s, 3H; OCOCH ), 1.93–1.86 (m, 1H; H-5b’), 1.82–1.75 (overlap-
3
ping, 4H; OCOCH , H-3b’’), 1.74–1.60 (overlapping, 2H; H-6a’, H-
3
13
6
b’), 1.55 (s, 3H; C(CH ) ), 1.40 ppm (s, 3H; C(CH ) ); C NMR
3
2
3
2
5
2
3.3 (COOCH ), 50.9 (C-5’’), 38.4 (C-3’’), 31.2 (C-5’), 27.6 (C(CH ) ),
3
3
2
(
125 MHz, CD OD): d=172.9 (NHCOCH at C-4), 172.4, 171.9, 171.7
3 3
6.9 (C-6’), 25.6 (C(CH ) ), 24.6 (NHCOCH at C-4), 21.0, 20.7 ppm
3
2
3
(
4”OCOCH ), 169.0 (C-1’’), 164.7 (C-4), 159.3 (q, J , =37 Hz, COCF ),
3 C F 3
+
(
4”CH COO); MS (ESI positive): m/z (%): 1003.0 [M+Na] (100),
3
1
57.7 (C-2), 148.4 (C-6), 117.0 (q, J , =287 Hz, COCF ), 115.4
C F 3
+
1
004.1 [M+Na]
(55); elemental analysis calcd (%) for
(
C(CH ) ), 100.0 (C-2’’), 98.2 (C-5), 96.4 (C-1’), 88.6 (C-4’), 86.3 (C-2’),
3 2
C H F N O : C 46.53, H 4.83, N 5.71; found: C 46.42, H 4.78, N
5
38
47
7
4
18
8
5.5 (C-3’), 72.6 (C-8’’), 71.5 (C-6’’), 70.0 (C-4’’, C-7’’), 64.8 (C-7’), 63.5
.89.
(
C-9’’), 53.3 (COOCH ), 51.0 (C-5’’), 38.4 (C-3’’), 31.4 (C-5’), 27.6
3
(
2
C(CH ) ), 26.9 (C-6’), 25.6 (C(CH ) ), 24.6 (NHCOCH at C-4), 21.0,
Further elution afforded compound 12c as a white solid (224 mg,
3
2
3 2
3
+
20
1
0.7, 20.6 ppm (4”CH COO); MS (ESI positive): m/z: 903.1 [M+Na] ;
24%). M.p. 132–1368C; [a] =À26.1 (c=1 in methanol); H NMR
3
D
elemental analysis calcd (%) for C H F N O : C 49.09, H 5.38, N
(500 MHz, CDCl ): d=8.77 (s, 1H; NH at C-4), 7.71 (d, J , =7.5 Hz,
3
6
47
3
4
18
3
6 5
6
.36; found: C 49.17, H 5.35, N 6.32.
1H; H-6), 7.40 (d, J , =7.5 Hz, 1H; H-5), 6.90 (d, J , =10.0 Hz, 1H;
5 6 NH 5’’
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14621
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
79
+
81
+
NH at C-5’’), 5.66 (d, J1’’,2’’ =1.6 Hz, 1H; H-1’), 5.40 (ddd, J , =2.7,
[ BrM+Na] (100), 929.1 [ BrM+Na] (94); elemental analysis
8’’ 9a’’
J , =4.4, J8’’,7’’ =8.8 Hz, 1H; H-8’’), 5.28 (dd, J7’’,6’’ =1.9, J7’’,8’’
8’’ 9b’’
calcd (%) for C H BrN O : C 47.74, H 5.45, N 6.19; found: C 47.66,
36 49
4
18
=
8.8 Hz, 1H; H-7’’), 5.15 (ddd, J4’’,3a’’ =4.6, J4’’,5’’ =10.5, J ,
H 5.56, N 6.09.
4’’ 3b’’
=
12.1 Hz, 1H; H-4’’), 5.02 (dd, J2’’,1’’ =1.6, J2’’,3’’ =6.5 Hz,1H; H-2’),
4
.64 (dd, J3’,4’ =4.4, J3’,2’ =6.5 Hz, 1H; H-3’), 4.32 (dd, J6’’,7’’ =1.9,
Compound 15b: Starting from the dibromide 13b (756 mg,
J , =10.7 Hz, 1H; H-6’’), 4.30–4.21 (overlapping, 2H; H-9a’’, H-
6
’’ 5’’
1
7
.10 mmol), purification by flash chromatography (hexane/AcOEt,
0:30,v/v to AcOEt/MeOH, 95:5, v/v) afforded compound 15b as
9
b’’), 4.16–4.11 (m, 1H; H-4’), 3.94 (ddd, J5’’,NH =10.0, J , =10.5,
5’’ 4’’
J , =10.7 Hz, 1H; H-5’’), 3.83–3.75 (overlapping, 2H; H-7a’,
5
’’ 6’’
a white solid (507 mg, 48%). M.p. 134–1368C decomp. (from
COOCH ), 3.33–3.27 (m, 1H; H-7b’), 2.68 (dd, J
=
OCOCH ), 2.10 (s, 3H; OCOCH ), 2.03 (s, 3H; OCOCH ), 2.00 (s, 3H;
OCOCH ), 1.87 (dd, J
1
6
,
=4.6, J ,
3
3a’’ 4’’ 3a’’ 3b’’
20
D
CH Cl /diisopropyl ether 8:2, v/v); [a] = +65.0 (c=1.0 in CH OH);
2
2
3
12.8 Hz, 1H; H-3a’’), 2.23 (s, 3H; NHCOCH at C-4), 2.14 (s, 3H;
3
1
H NMR (500 MHz, CDCl ): d=9.23 (d, J , =9.1 Hz, 1H; NH at C-
3
NH 5’’
3
3
3
5
’’), 9.04 (s, 1H; NH at C-4), 7.58 (d, J , =7.4 Hz, 1H; H-6), 7.43 (d,
6 5
,
=12.1, J , =12.9 Hz, 1H; H-3b’’), 1.83–
3a’’ 3b’’
3
3b’’ 4’’
J , =7.4 Hz, 1H; H-5), 5.91 (brs, 1H; H-7’’), 5.63 (dd, J , =1.3, J ,
5
6
2’ 1’
2’ 3’
.77 (overlapping, 2H; H-5a’, H-5b’), 1.73–1.63 (overlapping, 2H; H-
=
6.3 Hz, 1H; H-2’), 5.39 (d, J , =1.3 Hz, 1H; H-1’), 5.31 (dd, J ,
13
1’ 2’ 4’’ 3’’
a’, H-6b’), 1.56 (s, 3H; C(CH ) ), 1.34 ppm (s, 3H; C(CH ) ); C NMR
3
2
3 2
=
3.7, J , =10.0 Hz, 1H; H-4’’), 5.12 (ddd, J , =2.1, J , =2.4,
4
’’ 5’’
8’’ 7’’
8’’ 9a’’
(
(
125 MHz, CD OD): d=173.0 (NHCOCH at C-4), 172.5, 171.8, 171.4
3 3
4”OCOCH3), 169.5 (C-1’’), 164.7 (C-4), 159.6 (t, JC,F =27 Hz,
J , =9.5 Hz, 1H; H-8’’), 5.04 (dd, J
,
=2.4, J , =12.1 Hz, 1H;
8
’’ 9b’’
9a’’ 8’’
9a’’ 9b’’
H-9a’’), 4.89 (dd, J3’,4’ =1.8, J3’,2’ =6.3 Hz, 1H; H-3’), 4.76 (d, J3’’,4’’
3.6 Hz, 1H; H-3’’), 4.75–4.67 (overlapping, 2H; H-5’’, H-6’’), 4.26–
COCF CF CF ), 157.7 (C-2), 147.6 (C-6), 124.0–110.0 (COCF CF CF ),
2
2
3
2
2
3
=
115.4 (C(CH ) ), 100.0 (C-2’’), 98.1 (C-5), 95.5 (C-1’), 88.6 (C-4’), 86.6
3 2
4
.21 (m, 1H; H-4’), 4.14–4.09 (m, 1H; H-9b’’), 3.82 (s, 3H; COOCH3),
(
6
C-2’), 85.3 (C-3’), 72.4 (C-6’’), 70.2 (C-4’’), 69.5 (C-8’’), 68.5 (C-7’’),
5.6 (C-7’), 63.5 (C-9’’), 53.3 (COOCH ), 50.7 (C-5’’), 39.2 (C-3’’), 31.1
3
.64–3.55 (overlapping, 2H; H-7a’, H-7b’), 2.23 (s, 3H; NHCOCH at
3
3
C-4), 2.21 (s, 3H; OCOCH ), 2.07 (s, 3H; OCOCH ), 1.94 (s, 3H;
3
3
(
C-5’), 27.6 (C(CH ) ), 27.1 (C-6’), 25.7 (C(CH ) ), 24.6 (NHCOCH at C-
3 2 3 2 3
OCOCH ), 1.78 (s, 3H; OCOCH ), 1.77–1.55 (overlapping, 4H; H-5a’,
3
3
4
1
4
), 21.3, 20.8, 20.7, 20.6 ppm (4”OCOCH ); MS (ESI positive): m/z:
3
H-5b’, H-6a’, H-6b’), 1.54 (s, 3H; C(CH ) ), 1.39 ppm (s, 3H; C(CH ) );
+
3 2
3 2
003.1 [M+Na] ; elemental analysis calcd (%) for C H F N O : C
3
8
47
7
4
18
13
C NMR (125 MHz, CD OD): d=172.9 (NHCOCH3 at C-4), 172.5,
3
6.53, H 4.83, N 5.71; found: C 46.42, H 4.78, N 5.89.
1
71.9, 171.8, 171.3 (4”OCOCH ), 167.0 (C-1’’), 164.7 (C-4), 159.3 (q,
3
J , =37 Hz, COCF ), 157.7 (C-2), 148.6 (C-6), 117.1 (q, J , =287 Hz,
C F
3
C F
Glycosylation with the dibromo derivatives 13a–13c
General procedure: Silver trifluoromethanesulfonate (308 mg,
COCF ), 115.4 (C(CH ) ), 101.5 (C-2’’), 98.2 (C-5), 96.7 (C-1’), 88.6 (C-
3
3 2
4
’), 86.2 (C-2’), 85.5 (C-3’), 72.4 (C-8’’), 71.9 (C-6’’), 70.0 (C-4’’), 69.8
(
C-7’’), 66.4 (C-7’), 63.6 (C-9’’), 53.5 (COOCH ), 52.6 (C-3’’), 47.1 (C-
3
1
.20 mmol) in anhydrous toluene-MeNO2 (4 mL, 1:1, v/v) was
5
’’), 31.2 (C-5’), 27.6 (C(CH ) ), 26.7 (C-6’), 25.6 (C(CH ) ), 24.6
3 2 3 2
added to a stirred mixture of acceptor 6 (265 mg, 0.75 mmol), the
appropriate donor 13a–13c (1.10 mmol), and 4 Š molecular sieves
(
NHCOCH at C-4), 21.0, 20.7, 20.7, 20.5 ppm (4”OCOCH ); MS (ESI
3 3
79
+
81
+
positive): m/z (%): 981.1 [ BrM+Na] (100), 983.1 [ BrM+Na]
(
300 mg) in anhydrous toluene/MeNO2 (8 mL, 1:1, v/v) under an
argon atmosphere at RT. The reaction mixture was stirred at RT for
h and then filtered through a pad of Celite and concentrated
(
94); elemental analysis calcd (%) for C H BrF N O : C 45.06, H
36 46 3 4 18
4
.83, N 5.84; found: C 45.06, H 4.83, N 5.94.
3
under reduced pressure. The residue was purified by flash column
chromatography to afford the compound 15a–15c, respectively.
Compound 15c: Starting from the dibromide 13c (866 mg,
1.10 mmol), purification by flash chromatography (hexane/AcOEt
70:30, v/v to AcOEt/MeOH, 80:20, v/v) afforded compound 15c as
Compound 15a: Starting from the dibromide 13a (697 mg,
1
9
.10 mmol), purification by flash chromatography (AcOEt/MeOH,
8:2, v/v) afforded compound 15a as a white solid (508 mg, 51%).
20
a white solid (373 mg, 32%). M.p. 138–1408C; [a] = +8.5 (c=1 in
D
1
M.p. 154–1568C decomp. (from CH Cl /diisopropyl ether 8:2, v/v;
methanol); H NMR (500 MHz, CDCl ): d=9.23 (d, J , =8.9 Hz,
2
2
3
NH 5’’
2
D
0
[7]
[
a] =À20.2 (c=0.5 in chloroform) (lit. [a] =À47.0 (c=0.5 in
1H; NH at C-5’’), 9.04 (s, 1H; NH at C-4), 7.58 (d, J , =7.4 Hz, 1H;
D
6 5
1
methanol)); H NMR (500 MHz, CDCl ): d=9.23 (s, 1H; NH at C-4),
H-6), 7.43 (d, J , =7.4 Hz, 1H; H-5), 5.91 (dd, J , =J , =1.7 1H;
3
5 6
7’’ 8’’
7’’ 6’’
7
6
1
.89 (d, J , =9.9 Hz, 1H; NH at C-5’’), 7.59 (d, J , =7.3 Hz, 1H; H-
H-7’’), 5.63 (dd, J , =1.1, J , =6.3 Hz, 1H; H-2’), 5.39 (d, J , =
NH 5’’
6 5
2’ 1’ 2’ 3’ 1’ 2’
), 7.42 (d, J , =7.3 Hz, 1H; H-5), 5.87 (dd, J , =1.6, J , =2.8 Hz,
1.1 Hz, 1H; H-2’), 5.32 (dd, J4’’,3’’ =3.7, J , =9.9 Hz, 1H; H-4’’), 5.12
5
6
7’’ 8”
7” 6”
4’’ 5’’
H; H-7’’), 5.63 (d, J , =6.3 Hz, 1H; H-2’), 5.41 (brs, 1H; H-1’), 5.25
(ddd, J8’’,7’’ =1.7, J , =2.4, J , =9.4 Hz, 1H; H-8’’), 5.04 (dd,
2’ 3’
8’’ 9a’’
8’’ 9b’’
(
dd, J , =3.7, J4’’,5’’ =10.4 Hz, 1H; H-4’’), 5.12 (ddd, J8’’,7’’ =1.6,
J
,
=2.4, J
,
=12.1 Hz, 1H; H-9a’’), 4.89 (dd, J3’,4’ =1.7, J3’,2’
4’’ 3’’
9a’’ 8’’
9a’’ 9b’’
J , =2.1, J , =9.5 Hz, 1H; H-8’’), 5.05 (dd, J
,
=2.1, J ,
=6.3 Hz, 1H; H-3’), 4.76 (d, J3’’,4’’ =3.7 Hz, 1H; H-3’’), 4.75–4.67
(overlapping, 2H; H-5’’ and H-6’’), 4.26–4.22 (m, 1H; H-4’), 4.14–
8’’ 9a’’
8’’ 9b’’
9a’’ 8’’
9a’’ 9b’’
=
12.1 Hz, 1H; H-9a’’), 4.91 (dd, J3’,4’ =1.8, J3’,2’ =6.3 Hz, 1H; H-
3
’),4.71 (d, J3’’,4’’ =3.7 Hz, 1H; H-3’’, 4.67 (ddd, J , =9.9, J5’’,4’’
=
4.06 (m, 1H; H-9b’’), 3.82 (s, 3H; COOCH ), 3.64–3.54 (overlapping,
5’’ NH
3
J , =10.3 Hz, 1H; H-5’’), (dd, J6’’,5’’ =10.3, J6’’7’’ =2.8 Hz, 1H; H-6’’),
2H; H-7a’, H-7b’), 2.23 (s, 3H; NHCOCH3 at C-4), 2.21 (s, 3H;
OCOCH ), 2.19–2.10 (m, 1H; H-5a’), 2.08 (s, 3H; OCOCH ), 1.95 (s,
5’’ 6’’
4
.25–4.20 (m, 1H; H-4’), 4.13 (dd, J9
,
=9.5, J
,
=12.1 Hz, 1H;
b’’ 8’’
9b’’ 9a’’
3
3
H-9b’’), 3.80 (s, 3H; COOCH ), 3.63–3.53 (overlapping, 2H; H-7a’, H-
3H; OCOCH ), 1.93–1.86 (m, 1H; H-5b’), 1.78 (s, 3H; OCOCH ),
3
3
3
7
1
b’), 2.22 (s, 6H; NHCOCH at C-4, OCOCH ), 2.09 (s, 3H; OCOCH ),
1.77–1.57 (overlapping, 2H; H-6a’, H-6b’), 1.54 (s, 3H; C(CH ) ),
3
3
3
3 2
13
.94 (s, 3H; OCOCH ), 1.89 (s, 3H; OCOCH ), 1.79 (s, 3H; NHCOCH
1.39 ppm (s, 3H; C(CH3)2); C NMR (125 MHz, CD OD): d=173.0
3
3
3
3
at C-5’’), 1.70–1.58 (overlapping, 4H; H-5a’, H-5b’, H-6a’, H-6b’),
(NHCOCH at C-4), 172.5, 172.0, 171.6, 171.2 (4”OCOCH ), 167.0 (C-
1’’), 164.7 (C-4), 159.6 (t, J , =26 Hz, COCF CF CF ), 157.7 (C-2),
C F 2 2 3
3
3
1
3
1
.54 (s, 3H; C(CH ) ), 1.39 ppm (s, 3H; C(CH ) ); C NMR (125 MHz,
3
2
3 2
CD OD): d=173.5 (NHCOCH3 at C-5’’), 172.9 (NHCOCH3 at C-4),
148.3 (C-6), 124.0–110.0 (COCF CF CF ), 115.5 (C(CH ) ), 101.5 (C-2’’),
3
2 2 3 3 2
1
1
72.5, 172.0, 171.9, 171.6 (4”OCOCH ), 167.2 (C-1’’), 164.7 (C-4),
98.2 (C-5), 96.2 (C-1’), 88.3 (C-4’), 86.3 (C-2’), 85.4 (C-3’), 72.5 (C-8’’),
71.8 (C-6’’), 69.9 (C-4’’), 69.6 (C-7’’), 66.4 (C-7’), 63.5 (C-9’’), 53.6
(COOCH ), 52.6 (C-3’’), 47.2 (C-5’’), 31.1 (C-5’), 27.6 (C(CH ) ), 26.7 (C-
3
57.7 (C-2), 148.9 (C-6), 115.3 (C(CH ) ), 101.5 (C-2”), 98.2 (C-5), 97.2
3
2
(
(
C-1’), 88.8 (C-4’), 86.2 (C-2’), 85.7 (C-3’), 72.7, 72.6 (C-6’’, C-8’’), 70.6
C-4’’), 70.1 (C-7’’), 66.3 (C-7’), 63.8 (C-9’’), 53.4 (COOCH ), 52.9 (C-
3
3 2
6’), 25.6 (C(CH ) ), 24.6 (NHCOCH at C-4), 21.0, 20.7, 20.7, 20.5 ppm
3 2 3
+
3
3
2
2
’’), 46.4 (C-5’’), 31.3 (C-5’), 27.6 (C(CH ) ), 26.6 (C-6’), 25.6 (C(CH ) ),
(4”OCOCH ); MS (ESI positive): m/z: 1082.6 [M+Na] ; elemental
3
2
3 2
3
4.6 (NHCOCH at C-4), 22.9 (NHCOCH at C-5’’), 21.0, 20.8, 20.7,
analysis calcd (%) for C H BrF N O : C 43.07, H 4.38, N 5.29;
3
3
38 46
7
4
18
0.6 ppm (4”OCOCH3); MS (ESI positive): m/z (%): 927.1
found: C 43.02, H 4.38, N 5.35.
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org 14622
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Reductive debromination
=4.9 Hz, 1H; H-2’), 4.12 (dd, J9b’’
b’’), 4.05–3.96 (overlapping, 2H; H-4’, H-5’’), 3.87–3.78 (overlap-
ping, 4H; H-3’, COOCH ), 3.67–3.59 (m, 1H; H-7a’), 3.48–3.42 (m,
,8’’ =7.4, J9a’’,9b’’ =12.4 Hz, 1H; H-
9
General procedure: Tri-n-butyltin hydride (0.05 mL, 0.20 mmol)
and AIBN (10 mg, 0.061 mmol) were added to a solution of the ap-
propriate compound 15a–15c (0.10 mmol) in THF (5 mL) and the
mixture was heated to reflux for 3 h. Then the reaction mixture
was concentrated under reduced pressure and the residue was tri-
tured with diisopropyl ether/hexane (8:2, v/v) to afford compound
3
1
H; H-7b’), 2.51 (dd, J3
,
=4.9, J
,
=12.9 Hz, 1H; H-3a’’), 2.18
a’’ 4’’
3a’’ 3b’’
(
s, 3H; NHCOCH3 at C-4), 2.12 (s, 3H; OCOCH ), 2.03 (s, 3H;
3
OCOCH ), 2.02 (s, 3H; OCOCH ), 1.97 (s, 3H; OCOCH ), 1.94–
3
3
3
1
.77 ppm (overlapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b”);
13
C NMR (125 MHz, CD OD): d=173.0 (NHCOCH3 at C-4), 172.5,
3
11a–11 c, respectively, with all the physico-chemical properties
1
72.2, 171.7, 171.5 (4”OCOCH ), 169.0 (C-1’’), 164.4 (C-4), 159.3 (q,
3
identical to those previously reported.
J , =38 Hz, COCF ), 158.0 (C-2), 146.1 (C-6), 117.1 (q, J , =287 Hz,
C F
3
C F
Compounds 11 a: Starting from compound 15a (90 mg,
COCF ), 100.0 (C-2’’), 98.3 (C-5), 94.1 (C-1’), 84.4 (C-4’), 76.2 (C-2’),
3
0
(
.10 mmol) the glycoside 11 a was obtained as a white solid
67 mg, 81%).
Compounds 11 b: Starting from compound 15b (96 mg,
.10 mmol) the glycoside 11 b was obtained as a white solid
73 mg, 83%).
Compounds 11 c: Starting from compound 15c (105 mg,
75.0 (C-3’), 72.7 (C-8”), 71.6 (C-6”), 70.0 (C-4”), 69.9 (C-7’’), 65.0 (C-
7’), 63.5 (C-9’’), 53.3 (COOCH ), 51.0 (C-5’’), 38.5 (C-3’’), 31.1 (C-5’or
3
C-6’), 27.2 (C-6’ or C-5’), 24.6 (NHCOCH
20.6 ppm (4”OCOCH ); MS (ESI positive): m/z: 863.1 [M+Na] ; ele-
at C-4), 21.0, 20.7,
3
+
0
(
3
mental analysis calcd (%) for C33
found: C 47.22, H 5.28, N 6.50.
H F N O18: C 47.15, H 5.16, N 6.66;
43 3 4
0
8
.10 mmol) the glycoside 11 c was obtained a white solid (85 mg,
7%).
Compound 16c: Starting from compound 11 c (392 mg,
0.40 mmol), purification by flash chromatography (AcOEt/MeOH,
9
7
0:10, v/v) afforded compound 16c as a white solid (297 mg,
Acetonide deprotection to afford compounds 16a–16c as well
as 18b and 18c: A solution of the appropriate compound 11 a–
20
1
9%). M.p. 130–1328C; [a] = +19.4 (c=1.0 in CH OH); H NMR
D
3
(
500 MHz, CD OD): d=8.00 (d, J , =7.5 Hz, 1H; H-6), 7.47 (d, J , =
3 6 5 5 6
11 c or 12b and 12c (0.40 mmol) in dichloromethane (5 mL) was
7
.5 Hz, 1H; H-5), 5.81 (d, 1’,2’ =2.2 Hz, 1H; H-1’), 5.38–5.28 (overlap-
=2.3, J , =12.4 Hz,
added to CF COOH (45 mL) containing 5% H O. The mixture was
3
2
ping, 3H; H-7’’, H-4’’, H-8’’), 4.77 (dd, J
1
,
heated to reflux and stirred for one hour. Then the solution was
neutralized with a weak basic resin (IRA 67), filtered, and evaporat-
ed under reduced pressure to give a syrup, which was purified by
flash column chromatography to afford compound 16a–16c or
9a’’ 8’’
9a’’ 8’’
H; H-9a’’), 4.32 (d, J6’’,5’’ =10.4 Hz, 1H; H-6’’), 4.17 (dd, J2’,1’ =2.2,
J2’,3’ =5.2 Hz, 1H; H-2’), 4.15–4.00 (overlapping, 3H; H-4’, H-5’’ H-
9
b’’), 3.84 (dd, J3’,2’ =5.2, J3’,4’ =7.0 Hz, 1H; H-3’), 3.81 (s, 3H;
COOCH ), 3.67–3.60 (m, 1H; H-7a’), 3.48–3.41 (m, 1H; H-7b’), 2.53
1
8b and 18c, respectively.
3
(
dd, J3
,
=5.0, J
,
=12.9 Hz, 1H; H-3a’’), 2.18 (s, 3H; NHCOCH3
a’’ 4’’
3a’’ 3b’’
Compound 16a: Starting from compound 11 a (331 mg,
at C-4), 2.12 (s, 3H; OCOCH ), 2.04 (s, 3H; OCOCH ), 2.02 (s, 3H;
OCOCH ), 1.98–1.76 ppm (overlapping, 8H; OCOCH , H-5a’, H-5b’,
H-6a’, H-6b’, H-3b’’);
3
3
0
9
7
.40 mmol), purification by flash chromatography (AcOEt/MeOH,
3
3
0:10, v/v) afforded compound 16a as a white solid (236 mg,
13
2
D
5
1
C NMR (125 MHz, CD
3
OD): d=173.0
5%). M.p. 118–1218C; [a] = +14.2 (c=1 in methanol); H NMR
(
NHCOCH at C-4), 172.5, 172.2, 171.6, 171.4 (4”OCOCH ), 169.0 (C-
3 3
(
500 MHz, CD OD): d=8.01 (d, J , =7.5 Hz, 1H; H-6), 7.47 (d, J , =
3 6 5 5 6
1
1
’’), 164.4 (C-4), 159.6 (t, J , =26 Hz,COCF CF CF ), 158.0 (C-2),
C F 2 2 3
7
.5 Hz, 1H; H-5), 5.81 (d, J , =2.3 Hz, 1H; H-1’), 5.41 (dd, J ,
1’ 2’
7’’ 6’’
46.1 (C-6), 121.0–106.0 (COCF CF CF ), 99.9 (C-2’’), 98.3 (C-5), 94.0
2
2
3
=
2.2, J , =4.1 Hz, 1H; H-7’’), 5.30 (ddd, J , =2.5, J8’’,7’’ =4.1,
7’’ 8’’
8’’ 9a’’
(
6
C-1’), 84.3 (C-4’), 76.2 (C-2’), 75.0 (C-3’), 72.7 (C-8’’), 71.6 (C-6’’),
9.9, 69.8 (C-4’’, C-7’’), 65.0 (C-7’), 63.5 (C-9’’), 53.3 (COOCH ), 50.9
J , =7.6 Hz, 1H; H-8’’), 5.23 (ddd, J , =4.9, J4’’,5’’ =10.4, J ,
8’’ 9b’’
4’’ 3a’’
4’’ 3b’’
3
=
11.0 Hz, 1H; H-4’’), 4.82 (dd, J9
,
=2.5, J , =12.4 Hz, 1H; H-
a’’ 8’’ 9a’’ 8’’
(
C-5’’), 38.5 (C-3’’), 31.1 (C-5’), 27.2 (C-6’), 24.6 (NHCOCH at C-4),
3
9
a’’), 4.19 (dd, J2’,1’ =2.3, J2’,3’ =5.2 Hz, 1H; H-2’), 4.15—4.07 (over-
2
1.0, 20.7, 20.6 ppm (4”OCOCH ); MS (ESI positive): m/z: 963.1
3
lapping, 2H; H-6’’, H-9b’’), 4.06–4.04 (m, 1H; H-4’), 3.95 (dd, J , =
5’’ 4’’
+
[M+Na] ; elemental analysis calcd (%) for C H F N O : C 44.69, H
35 43 7 4 18
J , =10.4 Hz, 1H; H-5’’), 3.84 (dd, J3’,2’ =5.3, J3’,4’ =7.2 Hz, 1H; H-
5’’ 6’’
4
.61, N 5.96; found: C 44.52, H 4.70, N 5.93.
3
1
’), 3.80 (s, 3H; COOCH ), 3.67–3.61 (m, 1H; H-7a’), 3.48–3.42 (m,
3
H; H-7b’), 2.47 (dd, J3
,
=4.9, J
, =13.0 Hz, 1H; H-3a’’), 2.18
3a’’ 3b’’
Compound 18b: Starting from compound 12b (352 mg,
0.40 mmol), purification by flash chromatography (AcOEt/MeOH,
85:15, v/v) afforded compound 18b (255 mg, 76%) as a white
a’’ 4’’
(
s, 3H; NHCOCH at C-4), 2.12 (s, 3H; OCOCH ), 2.04–2.02 (overlap-
3 3
ping, 6H; 3”OCOCH3), 2.00–1.76 ppm (overlapping, 11 H;
2
D
0
1
NHCOCH at C-5’’, OCOCH , H-5a’, H-5b’, H-6a’, H-6b’, H-3b’’);
solid. M.p. 132–1348C; [a]
(500 MHz, CD OD): d=8.08 (d, J
7.4 Hz, 1H; H-5), 5.85 (d, J1’
H-8’’), 5.33 (d, J7’’ 8’’ =8.7 Hz, 1H; H-7’’), 4.96–4.84 (m, 1H; H-4’’),
4.36–4.27 (overlapping, 2H; H-6’’, H-9a’’), 4.18–4.15 (m, 1H; H-2’),
4.14–4.03 (overlapping, 3H; H-3’, H-4’, H-9b’’), 4.02 (dd, J5’’
6’’ =10.7 Hz, 1H; H-5’’), 3.90–3.80 (overlapping, 4H; H-7a’,
COOCH ), 3.44–3.38 (m, 1H; H-7b’), 2.71 (dd, J3a’’
=12.5 Hz, 1H; H-3a’’), 2.20 (s, 3H; NHCOCH ), 2.17 (s, 3H;
CH COO), 2.13 (s, 3H; CH COO), 2.01 (s, 3H; CH COO), 1.99 (s, 3H;
CH COO), 1.94–1.72 ppm (overlapping, 5H; H-5a’, H-5b’, H-6a’, H-
=À24.1 (c=1 in MeOH); H NMR
3
3
13
C NMR (125 MHz, CD OD): d=173.5 (NHCOCH3 at C-5’’), 173.0
3
, =7.4 Hz, 1H; H-6), 7.49 (d, J , =
6 5 5 6
3
(
NHCOCH at C-4), 172.6, 172.3, 172.0, 171.7 (4”OCOCH ), 169.2 (C-
,2’ =1.4 Hz, 1H; H-1’), 5.47–5.41 (m, 1H;
3
3
1
’’), 164.4 (C-4), 158.0 (C-2), 146.2 (C-6), 100.0 (C-2’’), 98.3 (C-5), 94.2
,
(
(
C-1’), 84.4 (C-4’), 76.2 (C-2’), 75.0 (C-3’), 73.1 (C-8’’), 72.4 (C-6’’), 70.6
C-4’’), 70.1 (C-7’’), 64.9 (C-7’), 63.7 (C-9’’), 53.3 (COOCH ), 50.3 (C-
, =
4’’
3
5
’’), 38.5 (C-3’’), 31.3 (C-5’), 27.3 (C-6’), 24.6 (NHCOCH at C-4), 22.8
J ,
5’’
3
(
NHCOCH at C-5’’), 21.0, 20.8 ppm (4”OCOCH ); MS (ESI positive):
3
,4’’ =4.6, J3a’’,
3b’’
3
3
+
m/z: 809.3 [M+Na] ; elemental analysis calcd (%) for C H N O :
3
3
3
46
4
18
C 50.38, H 5.89, N 7.12; found: C 50.46, H 5.80, N 7.03.
3
3
3
3
Compound 16b: Starting from compound 11 b (352 mg,
+
6
b’, H-3b’’); MS (ESI positive): m/z: 863.6 [M+Na] ; elemental anal-
0
9
7
.40 mmol), purification by flash chromatography (AcOEt/MeOH,
ysis calcd (%) for C H F N O : C 47.15, H 5.16, N 6.66; found C
47.66, H 5.20, N 6.40.
33
43
3
4
18
0:10, v/v) afforded compound 16b as white a solid (259 mg,
2
D
0
1
7%). M.p. 128–1308C; [a] = +21.5 (c=1 in methanol); H NMR
(
7
=
500 MHz, CD OD): d=8.01 (d, J , =7.5 Hz, 1H; H-6), 7.47 (d, J , =
Compound 18c: Starting from compound 12c (392 mg,
0.40 mmol), purification by flash chromatography (AcOEt/MeOH,
85:15, v/v) afforded compound 18c (271 mg, 72%) as a white
3
6 5
5 6
.5 Hz, 1H; H-5), 5.81 (d, J , =2.0 Hz, 1H; H-1’), 5.39 (dd, J ,
1’ 2’
7’’ 6’’
2.0, J , =4.2 Hz, 1H; H-7’’), 5.38–5.29 (overlapping, 2H; H-4’’,
7
’’ 8’’
20
1
H-8’’), 4.78 (dd, J9
J , =2.0, J6’’,5’’ =9.5 Hz, 1H; H-6’’), 4.18 (dd, J2’,1’ =2.0, J2’,3’
,
=2.1, J
,
=12.4 Hz, 1H; H-9a’’), 4.28 (dd,
solid. M.p. 122–1248C; [a] =À14.9 (c=1 in methanol); H NMR
a’’ 8’’
9a’’ 9b’’
D
(500 MHz, CD OD): d=8.06 (d, J , =7.5 Hz, 1H; H-6), 7.46 (d, J , =
6’’ 7’’
3
6 5
5 6
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14623
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
1
7
.5 Hz, 1H; H-5), 5.83 (d, J , =2.5 Hz, 1H; H-1’), 5.41 (ddd, J ,
nol); H NMR (500 MHz, CD OD): d=7.60 (d, J , =7.4 Hz, 1H; H-6),
3 6 5
1
’ 2’
8’’ 9a’’
=
2.7, J , =5.2, J8’’,7’’ =8.9 Hz, 1H; H-8’’), 5.29 (dd, J7’’,6’’ =2.1,
5.93 (d, J , =7.4 Hz, 1H; H-5), 5.77 (d, J , =3.2 Hz, 1H; H-1’), 4.20–
5 6 1’ 2’
8’’ 9b’’
J , =8.9 Hz, 1H; H-7’’), 4.96–4.88 (ddd, J , =4.7, J4’’,5’’ =10.4,
4.09 (overlapping, 3H; H-2’, H-4’’, H-6’’), 4.00 (dd, J , =J , =
7’’ 8’’
4’’ 3a’’
5’’ 4’’ 5’’ 6’’
J , =12.0 Hz, 1H; H-4’’), 4.34 (dd, J6’’,7’’ =2.1, J , =10.7 Hz, 1H;
10.3 Hz, 1H; H-5’’), 3.96–3.90 (m, 1H; H-4’), 3.86–3.75 (overlapping,
7H; H-3’, H-7a’, H-8’’, H-9a’’, COOCH ), 3.67 (dd, J =5.2,
4’’ 3b’’
6’’ 5’’
H-6’’), 4.29 (dd, J9a’’ 8’’
,
=2.7, J , =12.5 Hz, 1H; H-9a’’), 4.15 (dd,
,
9b’’ 8’’
9a’’ 8’’
3
J , =2.5, J , =5.2 Hz, 1H; H-2’), 4.09–4.00 (overlapping, 3H; H-4’,
J
,
=11.3 Hz, 1H; H-9b’’), 3.46 (d, J7’’,8’’ =9.4 Hz, 1H; H-7’’), 3.30–
2’ 1’
2’ 3’
9a’’ 9b’’
H-5’’, H-9b’’), 3.88–3.78 (overlapping, 5H; H-3’, H-7a’, COOCH3),
.42–3.36 (m, 1H; H-7b’), 2.70 (dd, J3 =4.7, J =12.7 Hz, 1H;
3.24 (m, 1H; H-7b’), 2.39 (dd, J
3a’’), 1.94–1.71 (overlapping, 4H; H-5a’, H-5b’, H-6a’, H-6b’),
,
=4.9, J
,
=12.9 Hz, 1H; H-
3a’’ 4’’
3a’’ 3b’’
3
,
,
3a’’ 3b’’
a’’ 4’’
13
H-3a’’), 2.18 (s, 3H; NHCOCH ), 2.15 (s, 3H; CH COO), 2.11 (s, 3H;
1.66 ppm (dd, J
,
=11.5, J
,
=12.9 Hz, 1H; H-3b’’); C NMR
3
3
3b’’ 4’’
3a’’ 3b’’
CH COO), 1.99 (s, 3H; CH COO), 1.96 (s, 3H; CH COO), 1.91–
(125 MHz, CD OD): d=171.5 (C-1’’), 167.7 (C-4), 159.7 (q, J , =
3 C F
3
3
3
1
.69 ppm (overlapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b);
37 Hz, COCF ), 158.5 (C-2), 142.8 (C-6), 117.6 (q, J , =287 Hz,
3 C F
1
3
C NMR (125 MHz, CD OD): d=173.0 (NHCOCH3 at C-4), 172.5,
COCF ), 100.1 (C-2’’), 96.3 (C-5), 93.1 (C-1’), 84.3 (C-4’), 75.8 (C-2’),
3
3
1
71.9, 171.5, 171.4 (4”CH COO), 169.5 (C-1’’), 164.4 (C-4), 159.6 (t,
75.0 (C-3’), 71.7 (C-8’’), 71.4 (C-6’’), 70.1 (C-7’’), 67.3 (C-4’’), 65.2 (C-
3
J , =27 Hz, COCF CF CF ), 158.1 (C-2), 146.1 (C-6), 124.0–110.0
9’’), 64.4 (C-7’), 54.3 (C-5’’), 53.4 (COOCH ), 41.9 (C-3’’), 31.1 (C-5’),
C F
2
2
3
3
+
(
(
COCF CF CF ), 100.1 (C-2’’), 98.2 (C-5), 93.8 (C-1’), 84.7 (C-4’), 76.4
C-2’), 75.0 (C-3’), 72.4 (C-6’’), 70.2 (C-4’’), 69.5 (C-8’’), 68.6 (C-7’’),
5.7 (C-7’), 63.5 (C-9’), 53.3 (COOCH ), 50.7 (C-5’’), 39.2 (C-3’’), 30.7
27.0 ppm (C-6’); MS (ESI positive): m/z: 653.0 [M+Na] ; MS (ESI
2
2
3
À
negative): m/z: 629.1 [MÀH] ; elemental analysis calcd (%) for
6
C H F N O : C 43.81, H 5.28, N 9.04; found: C 43.62, H 5.10, N
3
23 33
3
4
13
(
(
C-5’), 27.5 (C-6’), 24.6 (NHCOCH at C-4), 21.3, 20.8, 20.7, 20.6 ppm
9.30.
Compound 17c: Starting from compound 16c (188 mg,
.20 mmol), purification of the crude product by flash chromatog-
raphy (AcOEt/MeOH, 80:20, v/v) afforded compound 17c (130 mg,
3
+
4”CH COO); MS (ESI positive): m/z: 963.1 [M+Na] ; elemental
3
analysis calcd (%) for C H F N O : C 44.69, H 4.61, N 5.96; found:
C 44.66, H 4.73, N 6.03.
3
5
43
7
4
18
0
20
8
9%) as a white solid. M.p. 130–1328C; [a] = +5.0 (c=1 in meth-
D
1
anol); H NMR (500 MHz, CD OD): d=7.60 (d, J , =7.5 Hz, 1H; H-
3
6 5
Deacetylation
6
4
1
), 5.93 (d, J , =7.5 Hz, 1H; H-5), 5.77 (d, J , =3.4 Hz, 1H; H-1’),
5 6 1’ 2’
.20–4.02 (overlapping, 4H; H-2’, H-4’’, H-5’’, H-6’’), 3.96–3.89 (m,
H; H-4’), 3.86–3.74 (overlapping, 7H; H-3’, H-7a’, H-8’’, H-9a’’,
General procedure: To a solution of the appropriate compound
6a–16c or 18b and 18c (0.20 mmol) in dry methanol (5 mL),
1
COOCH ), 3.63 (dd, J
,
=5.6, J
,
=11.4 Hz, 1H; H-9b’’), 3.44 (d,
a methanolic solution of sodium methoxide (0.5m, 1.5 mL) was
added. After stirring for 1 h at RT, the solution was neutralized by
addition of a weakly acid resin (Amberlite CG50), filtered, and con-
centrated under reduced pressure. The residue was purified by
flash chromatography (compounds 16b and 16c or 18b and 18c)
or by preparative RP-HPLC chromatography to afford compounds
3
9b’’ 8’’
9a’’ 9b’’
J7’’,8’’ =9.4 Hz, 1H; H-7’’), 3.29–3.24 (m, 1H; H-7b’), 2.39 (dd, J
,
3a’’ 4’’
=
4.8, J
,
=12.9 Hz, 1H; H-3a’’), 1.93–1.69 (overlapping, 4H; H-
3a’’ 3b’’
5
=
a’, H-5b’, H-6a’, H-6b’), 1.66 ppm (dd,
12.9 Hz, 1H; H-3b’’); C NMR (125 MHz, CD OD): d=171.6 (C-1’’),
J
,
=11.2,
J ,
3a’’ 3b’’
3b’’ 4’’
13
3
1
67.7 (C-4), 159.9 (t, J , =26 Hz, COCF CF CF ), 158.4 (C-2), 142.8
C F 2 2 3
(
C-6), 120.9–105.0 (COCF CF CF ), 100.1 (C-2’’), 96.3 (C-5), 93.1 (C-1’),
1
7a–17c or 19b and 19c. In all cases, the crude residue obtained
2 2 3
8
7
4
4.2 (C-4’), 75.8 (C-2’), 75.0 (C-3’), 71.7 (C-8’’), 71.4 (C-6’’), 70.2 (C-
’’), 67.3 (C-4’’), 65.3 (C-9’’), 64.4 (C-7’), 54.3 (C-5’’), 53.5 (COOCH3),
after the appropriate hydrolytic treatment was purified by prepara-
tive HPLC chromatography by using an Atlantis C-18-Preper T3
ODB (5 mm, 19”10 mm) HPLC column and starting from 100% of
2.0 (C-3’’), 31.0 (C-5’), 27.0 ppm (C-6’); MS (ESI positive): m/z: 753.1
+
À
[M+Na] ; MS (ESI negative): m/z: 729.1 [MÀH] ; elemental analysis
aqueous 0.1% (v/v) formic acid to 100% CH CN as eluent.
3
calcd (%) for C H F N O : C 41.10, H 4.55, N 7.67; found: C 41.30,
25
33
7
4
13
Compound 17a: Starting from compound 16a (157 mg,
H 4.40, N 7.82.
Compound 19b: Starting from compound 18b (168 mg,
.20 mmol), purification of the crude product by flash chromatog-
raphy (AcOEt/MeOH, 80:20, v/v) afforded compound 19b (106 mg,
0
.20 mmol), the crude product was purificated by preparative
HPLC chromatography by using an Atlantis C-18-Preper T3 ODB
5 mm, 19”10 mm) HPLC column and starting from 100% of aque-
0
(
ous 0.1% (v/v) formic acid to 100% CH CN as eluent, to afford
compound 17a as white solid (104 mg, 90%). M.p. 118–1208C;
3
2
D
0
8
4%) as a white powered. M.p. 132–1348C; [a] =À16.2 (c=1 in
1
2
D
5
1
MeOH); H NMR (500 MHz, CD OD): d=7.61 (d, J , =7.3 Hz, 1H; H-
[a] = +15.1 (c=1 in water); H NMR (500 MHz, D O): d=7.71 (d,
3
6 5
2
6
4
3
), 5.95 (d, J , =7.3 Hz, 1H; H-5), 5.79 (brd, J , =1.7 Hz, 1H; H-1’),
J , =7.6 Hz, 1H; H-6), 6.12 (d, J , =7.6 Hz, 1H; H-5), 5.88 (d, J , =
5
6
1
’
2
’
6
5
5 6
1’ 2’
.09 (brs, 1H; H-2’), 3.99–3.91 (overlapping, 2H; H-4’, H-5’’), 3.90–
.80 (overlapping, 8H; H-3’, H-6’’, H-7a’, H-8’’, H-9a’’, COOCH ), 3.71
3
4
.9 Hz, 1H; H-1’), 4.37 (dd, J , =3.9, J , =4.9 Hz, 1H; H-2’), 4.14–
.00 (overlapping, 3H; H-4’, H-4’’, H-3’), 3.96–3.92 (overlapping, 2H;
2’ 1’ 2’ 3’
3
(
ddd, J , =4.6, J , =10.2, J , =12.4 Hz, 1H; H-4’’), 3.64 (dd,
H-5’’, H-6’’), 3.88–3.81 (overlapping, 5H; COOCH , H-8’’, H-9a’’),
4’’ 3a’’ 4’’ 5’’ 4’’ 3b’’
3
J
,
=5.9, J
,
=11.9 Hz, 1H; H-9b’’), 3.50–3.42 (overlapping,
3
1
.80–3.69 (overlapping, 2H; H-7a’, H-9b’’), 3.63 (d, J7’’,8’’ =9.3 Hz,
H; H-7’’), 3.44–3.38 (m, 1H; H-7b’), 2.47 (dd, J3 =4.9, J
9b’’ 8’’
9a’’ 9b’’
2
1
H; H-7’’, H-7b’), 2.68 (dd, J
,
=4.6, J
,
=12.8 Hz, 1H; H-3a’’),
,
,
3a’’ 3b’’
3a’’ 4’’
3a’’ 3b’’
a’’ 4’’
.91–1.63 ppm (overlapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b’’);
=
13.2 Hz, 1H; H-3a’’), 2.09 (s, 3H; NHCOCH ), 1.99–1.75 ppm (over-
3
13
À
MS (ESI negative): m/z: 629.2 [MÀH] ; elemental analysis calcd (%)
lapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b’’); C NMR (125 MHz,
D O): d=176.2 (NHCOCH ), 171.9 (C-1’’), 166.6 (C-4), 157.6 (C-2),
for C H F N O : C 43.81, H 5.28, N 8.89; found C 43.94, H 5.40, N
23 33
3
4
13
2
3
8
.72.
1
2
43.4 (C-6), 100.0 (C-2’’), 97.5 (C-5), 92.0 (C-1’), 84.5 (C-4’), 75.0 (C-
’), 74.3 (C-3’), 71.9 (C-6’’), 71.2 (C-8’’), 69.3 (C-7’’), 67.7 (C-4’’), 64.7
Compound 19c: Starting from compound 18c (188 mg,
0.20 mmol), purification of the crude product by flash chromatog-
raphy (AcOEt/MeOH, 80:20, v/v) afforded compound 19c (126 mg,
(
C-9’’), 64.6 (C-7’), 54.8 (COOCH ), 53.2 (C-5’’), 40.7 (C-3’’), 30.5 (C-5’),
3
2
6.3 (C-6’), 23.4 ppm (NHCOCH ); MS (ESI positive): m/z: 599.3
3
+
[M+Na] ; elemental analysis calcd (%) for C H N O : C 47.91, H
20
2
3
36
4
13
86%) as a white solid. M.p. 125–1278C; [a] =À11.1 (c=1 in meth-
D
6
.29, N 9.72; found: C 47.87, H 6.09, N 9.83.
1
anol); H NMR (500 MHz, CD OD): d=7.61 (d, J , =7.3 Hz, 1H; H-
3
6 5
Compound 17b: Starting from compound 16b (168 mg,
.20 mmol), purification of the crude product by flash chromatog-
raphy (AcOEt/MeOH, 80:20, v/v) afforded compound 17b (115 mg,
6), 5.94 (brd, J , =7.3 Hz, 1H; H-5), 5.78 (brs, 1H; H-1’), 4.09 (brs,
5 6
0
1H; H-2’), 4.03 (dd, J , =J , =10.2 Hz, 1H; H-5’’), 3.96–3.91 (m,
5’’ 6’’ 5’’ 4’’
1H; H-4’), 3.90–3.79 (overlapping, 8H; H-3’, H-6’’, H-7a’, H-8’’, H-
9a’’, COOCH ), 3.81–3.72 (ddd, J , =4.5, J4’’,5’’ =10.4, J ,
2
0
9
1%) as white solid. M.p. 120–1218C; [a] = +18.1 (c=1 in metha-
D
3
4’’ 3a’’
4’’ 3b’’
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14624
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
1
=
11.6 Hz, 1H; H-4’’), 3.69 (dd, J9
,
=6.3, J
,
=11.6 Hz, 1H; H-
water); H NMR (500 MHz, D O): d=7.86 (d, J , =7.9 Hz, 1H; H-6),
2 6 5
b’’ 8’’
9a’’ 9b’’
9
b’’), 3.49–3.42 (overlapping, 2H; H-7b’, H-7’’), 2.68 (dd, J3
,
=4.5,
6.23 (d, J , =7.9 Hz, 1H; H-5), 5.83 (d, J , =3.8 Hz, 1H; H-1’), 4.35
a’’ 4’’
5
6
1
’
2
’
J3
a’’ 3b’’
,
=12.7 Hz, 1H; H-3a’’), 1.92–1.65 ppm (overlapping, 5H; H-
(dd, J2’,1’ =3.8, J2’,3’ =5.1 Hz, 1H; H-2’), 4.23 (ddd, J , =4.9, J4’’,5’’
4’’ 3a’’
1
3
5
a’, H-5b’, H-6a, H-6b, H-3b’’); C NMR (125 MHz, CD OD): d=170.9
=10.4, J , =11.7 Hz, 1H; H-4”), 4.13–3.97 (overlapping, 4H; H-3’,
3
4’’ 3b’’
(
1
C-1’’), 167.7 (C-4), 160.0 (t, J , =26 Hz, COCF CF CF ), 158.4 (C-2),
H-4’, H-5’’, H-6’’), 3.89–3.82 (overlapping, 2H; H-8’’, H-9a’’), 3.69–
3.62 (overlapping, 2H; H-7a’, H-9b’’), 3.53 (d app, J7’’,8’’ =9.7 Hz,
C F
2
2
3
42.6 (C-6), 124.0–110.0 (COCF CF CF ), 100.3 (C-2’’), 96.3 (C-5), 92.8
2 2 3
(
C-1’), 84.1 (C-4’), 75.9 (C-2’), 75.0 (C-3’), 73.7 (C-6’’ or C-8’’), 72.8 (C-
1H; H-7’’), 3.42–3.36 (m, 1H; H-7b’), 2.43 (dd, J3
,
=4.9, J ,
a’’ 4’’ 3a’’ 3b’’
6
5
’’ or C-8’’), 70.3 (C-7’’), 68.3 (C-4’’), 64.8 (C-7’, C-9’’), 54.1 (C-5’’),
=13.2 Hz, 1H; H-3a’’), 1.96–1.87 (m, 1H; H-5a’), 1.84–1.70 ppm
13
3.3 (COOCH ), 42.0 (C-3’’), 30.7 (C-5’), 27.2 ppm (C-6’); MS (ESI pos-
(overlapping, 4H; H-5b’, H-6a’, H-6b’, H-3b’’); C NMR (125 MHz,
D O): d=174.4 (C-1’’), 160.6–158.8 (q, J , =37 Hz, COCF , C-4),
3
+
itive): m/z: 753.6 [M+Na] ; elemental analysis calcd (%) for
2
C F
3
C H F N O : C 41.10, H 4.55, N 7.67; found: C 41.19, H 4.61, N
149.0 (C-2), 144.9 (C-6), 116.4 (q, J , =286 Hz, COCF ), 100.0 (C-2’’),
25
33
7
4
13
C F 3
7
.59.
95.8 (C-5), 91.4 (C-1’), 84.6 (C-4’), 74.4 (C-2’), 73.5 (C-3’), 70.7 (C-8’’),
7
5
6
4
0.2 (C-6’’), 68.8 (C-7’’), 66.6 (C-4’’), 64.1 (C-9’’), 63.6 (C-7’), 53.5 (C-
’’), 40.5 (C-3’’), 30.0 (C-5’), 25.8 ppm (C-6’); MS (ESI negative): m/z:
Regeneration of the carboxylic group
General procedure
À
15.2 [MÀH] ; elemental analysis calcd (%) for C H F N O : C
22
31
3
4
13
2.86, H 5.07, N 9.09; found: C 43.01, H 5.0, N 9.12.
Compound 3c: Starting from ester 17c (73 mg, 0.10 mmol), after
For acetylamido derivate 17a: An acqueous solution of NaOH
hydrolysis and HPLC purification, compound 3c (62 mg, 87%) was
(
(
1m, 0.5 mL) was added to a solution of metyl ester 17a
0.10 mmol) in MeOH (1.0 mL) and the solution was stirred for
20
obtained as a white solid. M.p. 125–1268C; [a] = + 22.1 (c=1 in
D
1
water); H NMR (500 MHz, D O): d=7.64 (d, J , =7.6 Hz, 1H; H-6),
2
6 5
4
0 min at RT. Then the reaction mixture was neutralized with
6
.05 (d, J , =7.6 Hz, 1H; H-5), 5.86 (d, J , =4.0 Hz, 1H; H-1’), 4.30
5 6 1’ 2’
weakly acid resin (Amberlite CG50), filtered, and the solvent was
removed under reduced pressure.
(
dd, J2’,1’ =4.0, J2’,3’ =4.6 Hz, 1H; H-2’), 4.20 (ddd, J , =4.9, J4’’,5’’
4’’ 3a’’
=
10.2, J4’’,3b’’ =11.2 Hz, 1H; H-4’’), 4.09–4.00 (overlapping, 4H; H-3’,
For the trifluoroacetylamido derivatives 17b and 19b: Et N
H-4’, H-5’’, H-6’’), 3.91–3.82 (overlapping, 2H; H-8’’, H-9a’’), 3.66–
3.58 (overlapping, 2H; H-7a, H-9b’’), 3.47 (d, J7’’,8’’ =9.4 Hz, 1H; H-
3
(
(
0.90 mL) was added to a solution of the appropriate methyl ester
0.10 mmol) in MeOH/H O (1.5 mL, 2:1, v/v) and the solution was
7’’), 3.37–3.31 (m, 1H; H-7b’), 2.40 (dd, J
,
=4.9, J
,
=13.2 Hz,
2
3a’’ 4’’
3a’’ 3b’’
stirred for 12 h at RT. Then the solvent was removed under re-
duced pressure and the residue was recovered with water and
1H; H-3a’’), 1.95–1.69 (overlapping, 4H; H-5a’, H-5b’, H-6a’, H-6b’),
13
1.66 ppm (dd, J3
,
=11.2, J
,
=13.2 Hz, 1H; H-3b’’); C NMR
b’’ 4’’
3a’’ 3b’’
lyophilized many times until complete elimination of Et N.
(125 MHz, D O): d=176.1 (C-1’’), 166.9 (C-4), 160.4 (t, J , =27 Hz,
2 C F
3
For the heptafluorobutirylamido derivatives 17c and 19c: A so-
COCF CF CF ), 158.3 (C-2), 142.2 (C-6), 120.9–107.0 (COCF CF CF ),
2 2 3 2 2 3
lution of the appropriate metyl ester (0.10 mmol) in MeOH/H O
100.6 (C-2’’), 97.1 (C-5), 91.0 (C-1’), 84.0 (C-4’), 74.6 (C-2’), 73.6 (C-3’),
70.8 (C-8’’), 70.0 (C-6’’), 69.1 (C-7’’), 67.0 (C-4’’), 64.3 (C-9’’), 63.2 (C-
7’), 53.8 (C-5’’), 41.0 (C-3’’), 30.1 (C-5’), 25.9 ppm (C-6’); MS (ESI neg-
2
(
1.0 mL; 2:1, v/v) saturated with K CO , was stirred for 12 h at RT.
2 3
Then the reaction mixture was neutralized with weakly acid resin
Amberlite CG50), filtered, and the solvent was removed under re-
À
À
(
ative): m/z: 715.2 [MÀH] , 737.4 [MÀ2H+Na] ; elemental analysis
duced pressure.
calcd (%) for C24
H 4.13, N 7.30.
H F N O13: C 40.23, H 4.36, N 7.82; found: C 40.60,
31 7 4
In all cases, the crude residue obtained after the appropriate hy-
drolytic treatment was purified by preparative HPLC chromatogra-
phy by using a Atlantis C-18-Preper T3 ODB (5 mm, 19”10 mm)
HPLC column and starting from 100% of aqueous 0.1% (v/v)
Compound 20b: Starting from ester 19b (63 mg, 0.10 mmol), after
hydrolysis and HPLC purification, compound 20b (53 mg, 86%)
25
was obtained as a white solid. M.p. 125–1288C; [a] =À20.5 (c=
D
1
formic acid to 100% CH CN as eluent.
1 in water); H NMR (500 MHz, D O): d=7.56 (d, J , =7.4 Hz, 1H;
3
2
6 5
H-6), 5.94 (d, J , =7.4 Hz, 1H; H-5), 5.78 (d, J , =3.7 Hz, 1H; H-1’),
Compound 3a: Starting from ester 17a (58 mg, 0.10 mmol), after
hydrolysis and HPLC purification, compound 3a (48 mg, 85%) was
5 6
1’ 2’
4
5
.22–4.16 (m, 1H; H-2’), 4.02–3.89 (overlapping, 3H; H-3’, H-4’, H-
’’), 3.86–3.70 (overlapping, 4H; H-6’’, H-7a’, H-8’’, H-9a’’), 3.70–3.61
2
5
[7]
obtained as a white solid. [a] = +13.9 (c=1 in water) (lit. [a] =
D
D
1
(
m, 1H; H-4’’), 3.53 (dd, J
,
=6.2, J
,
=11.5 Hz, 1H; H-9b’’),
À26.6 (c=0.1 in water)); H NMR (500 MHz, D O): d=7.64 (d, J ,
9b’’ 8’’
9a’’ 9b’’
2
6 5
3
.49–3.39 (overlapping, 2H; H-7b’, H-7’’), 2.68 (dd, J
,
=4.6,
=
7.5 Hz, 1H; H-6), 6.05 (d, J , =7.5 Hz, 1H; H-5), 5.86 (d, J , =
3a’’ 4’’
5
6
1’ 2’
J
5
, =12.6 Hz, 1H; H-3a’’), 1.80–1.54 ppm (overlapping, 5H; H-
3a’’ 3b’’
a’, H-5b’, H-6a’, H-6b’, H-3b’’); C NMR (125 MHz, D O): d=173.6
2
3
4
3
.9 Hz, 1H; H-1’), 4.31 (dd, J , =3.9, J , =5.4 Hz, 1H; H-2’), 4.12–
2’ 1’
2’ 3’
13
.04 (overlapping, 2H; H-4’, H-4’’), 4.02 (dd, J , =5.4 Hz, 1H; H-3’),
3’ 2’
(
(
(
C-1’’), 166.3 (C-4), 159.8 (q, J , =37 Hz, COCF ), 157.8 (C-2), 141.5
C-6), 120.0–110.0 (COCF ), 100.8 (C-2’’), 96.5 (C-5), 90.2 (C-1’), 83.4
C-4’), 74.1 (C-2’), 73.0 (C-3’), 72.1 (C-6’’ or C-8’’), 71.8 (C-6’’ or C-8’’),
.93–3.79 (overlapping, 4H; H-5’’, H-6’’, H-8’’, H-9a’’), 3.66 (dd, J9
,
C F 3
b’’ 8’’
=
5.3, J9
,
=11.7 Hz, 1H; H-9b’’), 3.62–3.55 (m, 1H; H-7a’), 3.52
3
a’’ 9b’’
(
d, J , =9.4 Hz, 1H; H-7’’), 3.37–3.30 (m, 1H; H-7b’), 2.37 (dd,
7’’ 8’’
6
3
8.4 (C-7’’), 68.0 (C-4’’), 64.5 (C-7’), 62.7 (C-9’’), 52.9 (C-5’’), 40.7 (C-
J3
1
,
=5.0, J
,
=13.1 Hz, 1H; H-3a’’), 2.04 (s, 3H; NHCOCH3),
a’’ 4’’
3a’’ 3b’’
’’), 29.3 (C-5’), 25.5 ppm (C-6’); MS (ESI negative): m/z: 615.1
.94–1.85 (m, 1H; H-5a’), 1.83–1.69 (overlapping, 3H; H-5b’, H-6a’,
13
À
[
5
MÀH] ; elemental analysis calcd (%) for C H F N O : C 42.86, H
H-6b’), 1.63 ppm (dd, J3b’’ 3a’’
,
=13.1 Hz, 1H; H-3b’’); C NMR
23 31
3
4
13
.07, N 9.09; found: C 42.79, H 5.06, N 9.06
(
125 MHz, D O): d=176.3 (C-1’’), 175.6 (NHCOCH ), 167.0 (C-4),
2
3
1
4
58.4 (C-2), 142.4 (C-6), 100.7 (C-2’’), 97.1 (C-5), 91.2 (C-1’), 84.1 (C-
’), 74.7 (C-2’), 73.8 (C-3’), 70.9 (C-6’’), 70.8 (C-8’’), 69.2 (C-7’’), 67.9
Compound 20c: Starting from ester 19c (73 mg, 0.10 mmol), after
hydrolysis and HPLC purification, compound 20c (60 mg, 84%)
(
2
C-4’’), 64.4 (C-9’’), 63.3 (C-7’), 52.9 (C-5’’), 40.8 (C-3’’), 30.3 (C-5’),
5.9 (C-6’), 22.9 ppm (NHCOCH ); MS (ESI negative): m/z: 561.3
25
was obtained as a white solid. [a] =À11.3 (c=1 in water);
D
1
3
H NMR (500 MHz, D O, MeOH as reference standard): d=7.63 (d,
2
-
À
[
MÀH] , 583.3 [MÀ2H+Na] ; elemental analysis calcd (%) for
J , =7.5 Hz, 1H; H-6), 6.05 (brd, J , =7.5 Hz, 1H; H-5), 5.86 (d,
6
5
5 6
C H N O : C 46.97, H 6.09, N 9.96; found: C 46.87, H 6.00, N 9.85.
22
34
4
13
J , =3.9 Hz, 1H; H-1’), 4.31–4.24 (m, 1H; H-2’), 4.09–3.96 (overlap-
1’ 2’
Compound 3b: Starting from ester 17b (63 mg, 0.10 mmol), after
hydrolysis and HPLC purification, compound 3b (51 mg, 83%) was
ping, 3H; H-3’, H-4’, H-5’’), 3.92–3.78 (overlapping, 4H; H-6’’, H-7a’,
H-8’’, H-9a’’), 3.78–3.69 (m, 1H; H-4’’), 3.61 (dd, J9 =6.3, J
=11.5 Hz, 1H; H-9b’’), 3.54–3.48 (overlapping, 2H; H-7b’, H-7’’),
,
,
9a’’ 9b’’
b’’ 8’’
2
0
obtained as a white solid. M.p. 129–1318C; [a] = +18.9 (c=1 in
D
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14625 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
2
.76 (dd, J3
,
=4.8, J
, =12.5 Hz, 1H; H-3a’’), 1.88–1.65 ppm
3a’’ 3b’’
64.7 (C-7’), 63.6 (C-9’’), 52.8 (C-5’’), 38.0 (C-3’’), 31.0 (C-5’), 27.7 (C-
6’), 27.0 (C(CH ) ), 25.7 (C(CH ) ), 22.5 ppm (NHCOCH ); MS (ESI neg-
a’’ 4’’
13
(
(
overlapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b’’); C NMR
125 MHz, D O, MeOH as reference standard): d=173.7 (C-1’’),
3
2
3 2
À
3
ative): m/z: 583.1 [MÀH] ; elemental analysis calcd (%) for
2
1
66.3 (C-4), 159.8 (t, J , =26 Hz, COCF CF CF ), 157.7 (C-2), 141.5
C H N O : C 51.37, H 6.21, N 9.58; found: C 51.28, H 6.29, N 9.61.
C F
2
2
3
25 36
4
12
(
8
C-6), 120.0–110.0 (COCF CF CF ), 100.8 (C-2’’), 96.5 (C-5), 90.2 (C-1’),
2 2 3
3.4 (C-4’), 74.0 (C-2’), 73.0 (C-3’), 72.1 (C-6’’ or C-8’’), 71.9 (C-6’’ or
A solution of compound 22 (50 mg, 0.08 mmol) in dichlorome-
thane (8 mL) was added to CF COOH (20 mL) containing 10% of
3
C-8’’), 68.4 (C-7’’), 68.1 (C-4’’), 64.5 (C-7’), 62.6 (C-9’’), 53.0 (C-5’’),
H O. The reaction mixture was heated to reflux and stirred for one
2
4
7
4
0.6 (C-3’’), 29.3 (C-5’), 25.5 ppm (C-6’); MS (ESI negative): m/z:
hour. Then the solution was neutralized with a weak basic resin
(IRA 67), filtered, and evaporated under reduced pressure The
crude residue was purified by preparative HPLC chromatography
by using an Atlantis C-18-Preper T3 ODB (5 mm, 19”10 mm) HPLC
column and starting from 100% of aqueous 0.1% (v/v) formic acid
À
15.3 [MÀH] ; elemental analysis calcd (%) for C H F N O : C
2
4
31
7
4
13
0.23, H 4.36, N 7.82; found: C 40.39, H 4.24, N 7.85.
Synthesis of compound 21: Compound 11 a (165 mg, 0.2 mmol)
was treated according to the general procedures for deacetylation
and for regeneration of carboxylic group, to afford compound 21
to 100% CH CN as eluent to afford, after lyophilization, first the
3
2
0
(
95 mg, 79%) as a white solid. [a] = +39.1 (c=1 in water);
pure compound 4a (30 mg, 65%) and then compound 3a (5 mg,
10%).
D
1
H NMR (500 MHz, D O): d=7.56 (d, J , =7.5 Hz, 1H; H-6), 5.95 (d,
2
6 5
J , =7.5 Hz, 1H; H-5), 5.74 (d, J , =2.5 Hz, 1H; H-1’), 5.01 (dd,
20
D
1
5
6
1’ 2’
Compound 4a: [a] = +31.0 (c=1 in methanol); H NMR
J , =2.5, J , =6.5 Hz, 1H; H-2’), 4.73–4.69 (overlapping with H O,
2’ 1’
2’ 3’
2
(500 MHz, CD OD): d=7.60 (d, J , =7.5 Hz, 1H; H-6), 5.91 (d, J , =
7.5 Hz, 1H; H-5), 5.77 (d, J , =3.3 Hz, 1H; H-1’), 4.56 (brs, 1H; H-
6’’), 4.44 (d, J7’’,8’’ =7.8, 1H; H-7’’), 4.11–4.08 (m, 1H; H-2’), 4.06–4.02
(m, 1H; H-4’’), 3.96–3.97–3.89 (overlapping, 2H; H-4’, H-5’’), 3.84–
.68 (overlapping, 5H; H-3’, H-7a’, H-8’’, H-9a’’, H-9b’’), 3.50–3.43
(m, 1H; H-7b’), 2.10 (dd, J3 =3.4, J =14.1 Hz, 1H; H-3a’’),
=14.2 Hz, 1H; H-3b’’), 2.00 (s, 3H;
NHCOCH ), 1.92–1.66 ppm (overlapping, 4H; H-5a’, H-5b’, H-6a’, H-
3
6 5
5 6
1
1
H; H-3’), 4.16–4.12 (m, 1H; H-4’), 4.00 (ddd, J4 ,
=4.9, J , =
’’ 3a’’
4’’ 5’’
1’ 2’
0.4, J , =10.8 Hz, 1H; H-4’’), 3.84–3.70 (overlapping, 4H; H-5’’,
4
’’ 3b’’
H-6”, H-8’’, H-9a’’), 3.66 (dd, J9
,
b’’ 8’’
=5.6, J
,
9a’’ 9b’’
=12.1 Hz, 1H; H-
9
7
b’’), 3.42–3.51 (overlapping, 2H; H-7a’, H-7’’), 3.29–3.22 (m, 1H; H-
=4.9, J =13.1 Hz, 1H; H-3a’’), 1.97 (s, 3H;
3
b’), 2.29 (dd, J3
,
a’’ 4’’
,
3a’’ 3b’’
,
,
3a’’ 3b’’
a’’ 4’’
NHCOCH3), 1.77–1.70 (overlapping, 2H; H-5a’, H-5b’), 1.66–
2.05 (dd, J3
,
=2.1, J
,
b’’ 4’’
3b’’ 3a’’
1
3
.51 ppm (overlapping, 6H; C(CH ) , H-6a’, H-6b’’, H-3b’’), 1.32 (s,
3 2
13
3
H; C(CH ) ); C NMR (125 MHz, D O): d=176.1 (C-1’’), 175.4
+
3
2
2
6b’); MS (ESI positive): m/z: 567.1 [M+Na] ; elemental analysis
calcd (%) for C H N O : C 48.53, H 5.92, N 10.29; found: C 48.69,
(
NHCOCH ), 167.1 (C-4), 157.8 (C-2), 143.7 (C-6), 115.6 (C(CH ) ),
3 3 2
22
32
4
12
1
7
7
2
00.5 (C-1”), 96.7 (C-5), 93.5 (C-1’), 86.9 (C-4’), 85.0 (C-2’), 83.7 (C-3’),
0.8 (C-6’’), 70.7 (C-8’’), 69.0 (C-7’’), 67.8 (C-4’’), 64.2 (C-9’’), 63.0 (C-
’), 52.8 (C-5’’), 40.7 (C-3’’), 30.2 (C-5’), 26.6 (C-6’), 25.7 (C(CH ) ),
H 5.76, N 10.01.
All other physic-chemical properties of compound 3a are superim-
posable to those previously reported for the same compound.
3
2
5.1 (C(CH ) ), 22.8 ppm (NHCOCH ); MS (ESI negative): m/z: 601.4
3
2
3
À
Compound 4b: Starting from compound 3b (61 mg, 0.10 mmol),
purification by flash chromatography (AcOEt/MeOH, 85:15, v/v) af-
forded compound 4b (42 mg, 70%) as a white solid. M.p. 123–
[MÀH] ; elemental analysis calcd (%) for C H N O : C 49.83, H
2
5
38
4
13
6
.36, N 9.30; found: C 49.89, H 6.14, N 9.10.
2
D
0
1
1
248C; [a] = +19.1 (c=1 in methanol); H NMR (500 MHz,
CD OD): d=7.62 (d, J , =7.5 Hz, 1H; H-6), 5.96 (d, J , =7.5 Hz, 1H;
3
6 5
5 6
1,7-Lactonization
H-5), 5.80 (d, J , =3.5 Hz, 1H; H-1’), 4.67 (brs, 1H; H-6’’), 4.47 (d,
1’ 2’
General procedure: The appropriate starting material 3b, 3c, or
J , =7.9 Hz, 1H; H-7’’), 4.14–4.08 (overlapping, 2H; H-2’, H-4’’),
7’’ 8’’
2
1 (0.10 mmol), dissolved in DMF (1.0 mL) under stirring, was
cooled to 08C and diluted with THF (1.5 mL). Then CbzCl (0.03 mL;
.25 mmol, 2.5 equiv) in THF (0.4 mL) was added in a single por-
4.00 (brs, 1H; H-5’’), 3.95–3.90 (m, 1H; H-4’), 3.86–3.75 (overlap-
ping, 4H; H-3’, H-7a’, H-8’’, H-9a’’), 3.75–3.69 (m, 1H; H-9b’’), 3.49–
0
3.42 (m, 1H; H-7b’), 2.16 (dd, J3
,
=3.4, J
,
=14.2 Hz, 1H; H-
a’’ 4’’
3a’’ 3b’’
tion, followed by Et N (0.04 mL; 0.3 mmol) in a single addition. The
mixture was then stirred for 30 min at 238C. At this time, methanol
3a’’), 2.07 (dd, J
,
=1.5, J
,
=14.2 Hz, 1H; H-3b’’), 1.89–
3
3b’’ 4’’
3b’’ 3a’’
1
3
1.61 ppm (overlapping, 4H; H-5a’, H-5b’, H-6a’, H-6b’); C NMR
(125 MHz, CD OD): d=170.1 (C-1’’), 167.6 (C-4), 159.0 (q, J , =
(
0.3 mL) was added and the stirring was continued for 15 min.
3
C F
Evaporation of the solvent under reduced pressure (22 mmHg and
then at 10 mmHg) afforded a crude residue, which, after purifica-
38 Hz, COCF ), 158.6 (C-2), 142.5 (C-6), 117.3 (q, J , =287 Hz,
3
C
F
À1
COCF ), 96.5 (C-5), 96.1 (C-2’’), 92.5 (C-1’), 84.4 (C-4’), 79.7 (C-7’’),
3
tion by flash chromatography give the appropriate pure lactone
76.0 (C-2’), 74.9 (C-3’), 73.0 (C-8’’), 71.3 (C-6’’), 67.0 (C-4’’), 65.0 (C-
7’), 63.5 (C-9”), 53.8 (C-5’’), 38.0 (C-3’’), 30.9 (C-5’), 27.2 ppm (C-6’);
MS (ESI positive): m/z: 621.2 [M+Na] ; elemental analysis calcd (%)
4
b, 4c, or 22.
+
Compound 4a: Starting from 21 (60 mg, 0.10 mmol), purification
for C H F N O : C 44.15, H 4.88, N 9.36; found: C 44.71, H 4.65, N
by flash chromatography (AcOEt/MeOH, 80:20, v/v) afforded com-
22 29
3
4
12
2
0
9.27.
pound 22 (41 mg, 70%) as a white solid. [a] = +23.1 (c=1 in
D
1
methanol); H NMR (500 MHz, CD OD): d=7.61 (d, J6,5 =7.5 Hz,
Compound 4c: Starting from compound 3c (72 mg, 0.10 mmol),
3
1
H; H-6), 5.91 (d, J , =7.5 Hz, 1H; H-5), 5.77 (d, J , =2.3 Hz, 1H;
purification by flash chromatography (AcOEt/MeOH, 85:15, v/v) af-
5
6
1’ 2’
2
0
H-1’), 4.95 (dd, J , =2.3, J , =6.5 Hz, 1H; H-2’), 4.63 (dd, J , =4.8,
J , =6.5 Hz, 1H; H-3’), 4.58 (brs, 1H; H-6’’), 4.46 (dd, J , <1.0,
3
forded compound 4c (50 mg, 71%) as a white solid. [a] =À42.9
2
’ 1’
2’ 3’
3’ 4’
D
1
(c=1 in methanol); H NMR (500 MHz, CD OD): d=7.62 (d, J ,
’2’
7’’ 6’’
3
6 5
J , =8.0 Hz, 1H; H-7’’), 4.07–4.04 (m, 1H; H-4’’), 4.04–3.99 (m, 1H,
7’ 8’
=7.5 Hz, 1H; H-6), 5.96 (d, J , =7.5 Hz, 1H; H-5), 5.81 (d, J , =
5 6 1’ 2’
H-4’), 3.98–3.95 (m, 1H, H-5’’), 3.82–3.69 (overlapping, 4H; H-7a’, H-
’’, H-9a’’, H-9b’’), 3.48–3.42 (m, 1H; H-7b’), 2.12 (dd, J3a’’ 4’’ =3.3,
J3 =14.1 Hz, 1H; H-3a’’), 2.06 (dd, J =2.1, J =14.1 Hz,
3.4 Hz, 1H; H-1’), 4.68 (brs, 1H; H-6’’), 4.47 (d, J7’’,8’’ =7.8 Hz, 1H; H-
7’’), 4.13–4.07 (overlapping, 2H; H-2’, H-4’’), 4.04 (brs, 1H; H-5’’),
3.96–3.88 (m, 1H; H-4’), 3.87–3.76 (overlapping, 4H; H-3’, H-7a’, H-
8’’, H-9a’’), 3.75–3.70 (m, 1H; H-9b’’), 3.48–3.43 (m, 1H; H-7b’), 2.15
8
,
,
,
,
3b’’ 3a’’
a’’ 3b’’
3b’’ 4’’
1
5
H; H-3b’’), 2.02 (s, 3H; NHCOCH ), 1.84–1.78 (overlapping, 2H; H-
3
a’, H-5b’), 1.74–1.66 (overlapping, 2H; H-6a’, H-6b’), 1.55 (s, 3H;
(dd, J3
,
=3.4, J
,
=14.2 Hz, 1H; H-3a’’), 2.07 (dd, J , =2.0,
a’’ 4’’
3a’’ 3b’’ 3b’’ 4”
1
3
C(CH ) ), 1.35 ppm (s, 3H; C(CH ) ); C NMR (125 MHz, CD OD): d=
J , =14.2 Hz, 1H; H-3b’’), 1.89–1.68 ppm (overlapping, 4H; H-
3
2
3 2
3
3b’’ 3a’’
13
1
73.0 (NHCOCH ), 170.3 (C-1’’), 168.0 (C-4), 158.0 (C-2), 144.5 (C-6),
5a’, H-5b’, H-6a’, H-6b’); C NMR (125 MHz, CD OD): d=170.0 (C-
3
3
1
15.5 (C(CH ) ), 96.3 (C-5), 96.1 (C-2’’), 94.7 (C-1’), 87.6 (C-4’), 86.2
1’’), 167.6 (C-4), 159.4 (t, J , =27 Hz, COCF CF CF ), 158.5 (C-2),
3
2
C F
2
2
3
(
C-2’), 85.3 (C-3’), 79.8 (C-7’’), 73.2 (C-8’’), 72.1 (C-6’’), 67.7 (C-4’’),
142.6 (C-6), 120.1–109.3 (COCF CF CF ), 96.4 (C-5), 96.1 (C-2’’), 92.6
2 2 3
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org 14626
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(
(
3
C-1’), 84.3 (C-4’), 79.7 (C-7’’), 76.0 (C-2’), 74.9 (C-3’), 73.0 (C-8’’), 71.3
3H; OCOCH ), 2.07 (s, 3H; OCOCH ), 1.98 (s, 3H; OCOCH ), 1.96 (s,
3
3
3
C-6’’), 66.9 (C-4’’), 65.0 (C-7’), 63.5 (C-9’’), 54.1 (C-5’’), 38.0 (C-3’’),
3H; OCOCH ), 1.92–1.81 (overlapping, 5H; H-5b’, H-6a’, OCOCH ),
3
3
+
0.9 (C-5’), 27.2 ppm (C-6’); MS (ESI positive): m/z: 721.1 [M+Na] ;
1.79 (dd, J3
,
=12.8, J , =11.3 Hz, 1H; H-3a’’), 1.54–1.46 ppm
a’’ 3b’’
3’’ 4’’
13
elemental analysis calcd (%) for C H F N O : C 41.27, H 4.18, N
(m, 1H; H-6b’); C NMR (125 MHz, CDCl ): d=170.9, 170.5, 170.2,
2
4
29
7
4
12
3
8
.02; found: C 41.11, H 4.31, N 8.13.
170.2, 169.4 (NHCOCH at C-4, CH COO at C-9’’, CH COO at C-4’’,
3
3
3
CH COO at C-8’’, CH COO at C-7’’, CH COO at C-2’, CH COO at C-3’),
3
3
3
3
1
67.5 (C-1’’), 162.4 (C-4), 157.8 (q, J , =37 Hz, COCF ), 155.7 (C-2),
C F
3
Acetylation affording the derivatives 23a–23c and 24c
147.8 (C-6), 116.0 (q, J
,
C
F
=287 Hz, COCF
), 98.9 (C-2’’), 97.1 (C-5),
3
9
5.2 (C-1’), 84.0 (C-4’), 74.7 (C-3’), 72.1 (C-2’), 72.0 (C-8’’), 70.6 (2C,
General procedure: Acetic anhydride (0.5 mL) was added to a solu-
tion of the appropriate compound 16a–16c or 18c (0.20 mmol) in
pyridine (1.0 mL) and the resulting solution was stirred for 3 h at
RT. Then, MeOH (0.4 mL) was added and the solution was concen-
trated under reduced pressure. The residue was recovered with ac-
queous HCl (1m) and extracted with ethyl acetate. The organic
C-6’’, C-7’’), 69.5 (C-4’’), 63.9 (C-7’), 62.8 (C-9’), 52.6 (COOCH ), 49.4
3
(
C-5’’), 37.5 (C-3’’), 29.7 (C-5’), 26.0 (C-6’), 24.6 (NHCOCH at C-4),
3
2
9
4
0.9, 20.7, 20.6, 20.4 ppm (6”CH COO); MS (ESI positive): m/z:
3
+
47.6 [M+Na] ; elemental analysis calcd (%) for C H F N O : C
37
47
3
4
20
8.05, H 5.12, N 6.06; found C 48.18, H 5.40, N 6.38.
layers were washed with aqueous NaHCO and concentrated under
reduced pressure to give a crude residue, which was purified by
flash chromatography.
Compound 23c: Starting from compound 16c (188 mg,
0.20 mmol), purification by flash chromatography (AcOEt/MeOH,
90:10, v/v) afforded compound 23c (182 mg 89%) as a white solid.
3
20
1
[a] = +21.3 (c=1 in CH OH); H NMR (500 MHz, CDCl ): d=9.09
D
3 3
Compound 23a: Starting from compound 16a (157 mg,
(
s, 1H; NH at C-4), 8.82 (d, JNH,5’’ =9.8 Hz, 1H; NH at C-5’’), 7.65 (d,
0
9
.20 mmol), purification by flash chromatography (AcOEt/MeOH,
J , =7.5 Hz, 1H; H-6), 7.41 (d, J , =7.5 Hz, 1H; 5-H), 5.97 (dd,
6
5
5 6
9:1, v/v) afforded compound 23a (159 mg, 91%) as a white solid.
20
1
J , =J , =5.2 Hz, 1H; H-2’), 5.71–5.67 (overlapping, 2H; H-7’’, H-
2’ 1’ 2’ 3’
[a] = +32.1 (c=1 CH OH); H NMR (500 MHz, CDCl ): d=9.16 (s,
D
3 3
1
’), 5.35–5.27 (overlapping, 2H; H-3’, H-4’’), 5.20 (ddd, J8’’,7’’ =J ,
8’’ 9a’’
1
9
H; NH at C-4), 7.63 (d, J6,5 =7.5 Hz, 1H; H-6), 7.50 (d, JNH,5’’ =
.9 Hz, 1H; NH at C-5’’), 7.41 (d, J , =7.5 Hz, 1H; 5-H), 6.05 (dd,
=
2.4, J ,
=9.0 Hz, 1H; H-8’’), 4.88 (dd, J
,
=2.4, J
,
=
8
’’ 9b’’
9a’’ 8’’
9a’’ 9b’’
5
6
1
4
1
3
2
2.1 Hz, 1H; H-9a’’), 4.38 (dd, J6’’,7’’ =2.8, J6’’,5’’ =10.5 Hz, 1H; H-6’’),
J , =4.6, J , =5.7 Hz, 1H; H-2’), 5.70 (dd, J , =J , =2.4 Hz,
2’ 1’
2’ 3’
7’’ 8’’
7’’ 6’’
.30 (ddd, J5’’,NH =9.8, J , =J , =10.5, 1H; H-5’’), 4.15–4.12 (m,
5’’ 4’’
5’’ 6’’
1
H; H-7’’), 5.58 (d, J , =4.6 Hz, 1H; H-1’), 5.36 (dd, J , =4.6, J ,
1’ 2’ 4’’ 3’’ 4’’ 5’’
H; H-4’), 4.07 (dd, J9
,
=12.1, J , =9.0 Hz, 1H; H-9b’’), 3.78 (s,
b’’ 9a’’ 9b’’ 8’’
=
=
5.7 Hz, 1H; H-3’), 5.23 (ddd, J4’’,3a’’ =5.0, J4’’,5’’ =10.5, J ,
4’’ 3b’’
H; COOCH ), 3.70–3.63 (m, 1H; H-7a’), 3.56–3.49 (m, 1H; H-7b’),
3
11.1 Hz, 1H; H-4’’), 5.14 (ddd, J , =J , =2.4, J , =9.5 Hz,
8’’ 7’’
8’’ 9a’’
8’’ 9b’’
.60 (dd, J3
,
=12.8, J3’’,4’’ =5.0 Hz, 1H; H-3a’’), 2.21 (s, 3H;
a’’ 3b’’
1
H; H-8’’), 4.89 (dd, J9
,
=2.3, J
,
=12.4 Hz, 1H; H-9a’’), 4.38
a’’ 8’’
9a’’ 9b’’
NHCOCH at C-4), 2.14–2.10 (overlapping, 4H; OCOCH , H-5a’), 2.10
3
3
(
dd, J6’’7’’ =2.4, J6’’,5’’ =10.5 Hz, 1H; H-6’’), 4.22–4.14 (overlapping,
(
(
s, 3H; OCOCH ), 2.08 (s, 3H; OCOCH ), 1.97 (s, 3H; OCOCH ), 1.96
3
3
3
2
3
2
H; H-4’, H-5’’), 4.06 (dd, J9
,
=12.4, J , =9.5 Hz, 1H; H-9b’’),
b’’ 9a’’ 9b’’ 8’’
s, 3H; OCOCH ), 1.91 (s, 3H; OCOCH ), 1.90–1.81 (overlapping, 2H;
3
3
.76 (s, 3H; COOCH ), 3.66–3.61 (overlapping, 2H; H-7a’, H-7b’),
3
H-5b’, H-6a’), 1.77 (dd, J3
,
=12.8, J , =11.4 Hz, 1H; H-3a’’),
a’’ 3b’’
3’’ 4’’
.52 (dd, J3
,
=12.7, J3’’,4’’ =5.0 Hz, 1H; H-3a’’), 2.22 (s, 3H;
a’’ 3b’’
13
1
.59–1.48 ppm (m, 1H; H-6b’); C NMR (125 MHz, CDCl ): d=170.7,
3
NHCOCH at C-4), 2.18 (s, 3H; OCOCH ), 2.13 (s, 3H; OCOCH ), 2.09
3
3
3
1
70.5, 170.3, 170.1, 169.3 (NHCOCH3 at C-4, CH COO at C-9’’,
3
(
s, 3H; OCOCH ), 2.01 (s, 3H; OCOCH ), 1.96 (s, 3H; OCOCH ), 189–
3 3 3
CH COO at C-4’’, CH COO at C-8’’, CH COO at C-7’’, CH COO at C-2’,
3
3
3
3
1
1
.85 (overlapping, 8H; OCOCH , H-5a’, H-5b’, NHCOCH at C-5’’),
3 3
13
CH COO at C-3’), 167.5 (C-1’’), 162.4 (C-4), 158.3 (t, J , =27 Hz,
3
C
F
.82–1.51 ppm (overlapping, 3H; H-3b’’, H-6a’, H-6b’); C NMR
COCF CF CF ), 155.5 (C-2),147.2 (C-6), 125.1–109.3 (COCF CF CF ),
2
2
3
2
2
3
(
125 MHz, CDCl ): d=170.3, 170.5, 170.4, 170.2, 169.5 (NHCOCH at
3 3
9
7
9
2
8.8 (C-2’’), 97.1 (C-5), 94.1 (C-1’), 83.5 (C-4’), 74.2 (C-3’), 72.6 (C-2’),
2.1 (C-8’’), 70.7 (C-6’’), 70.4 (C-7’’), 69.4 (C-4’’), 63.8 (C-7’), 62.8 (C-
C-4, NHCOCH at C-5’’, CH COO at C-9’’, CH COO at C-4’’, CH COO
3
3
3
3
at C-8’’, CH COO at C-7’’, CH COO at C-2’, CH COO at C-3’), 167.2
3
3
3
’), 52.7 (COOCH ), 49.3 (C-5’’), 37.5 (C-3’’), 29.7 (C-5’), 25.9 (C-6’),
3
(
C-1’’), 162.6 (C-4), 155.3 (C-2), 147.7 (C-6), 98.9 (C-2’’), 96.9 (C-5),
4.6 (NHCOCH3 at C-4), 20.8, 20.7, 20.6, 20.5, 20.4, ppm (6”
9
7
5.3 (C-1’), 83.8 (C-4’), 74.6 (C-3’), 72.7 (C-2’), 72.4 (C-8’’), 71.5 (C-6’’),
+
CH COO); MS (ESI positive): m/z: 1047.2 [M+Na] ; elemental analy-
3
0.6 (C-7’’), 70.0 (C-4’’), 63.6 (C-7’), 63.0 (C-9’), 52.6 (COOCH ), 48.4
3
sis calcd (%) for C H F N O : C 45.71, H 4.62, N 5.47; found C
39
47
7
4
20
(
C-5’’), 37.4 (C-3’’), 29.9 (C-5’), 26.1 (C-6’), 24.7 (NHCOCH at C-4),
3
4
5.61, H 4.75, N.5.33.
2
3.2 (NHCOCH at C-5’’), 21.1, 20.7, 20.5 ppm (6”CH COO); MS (ESI
3 3
+
positive): m/z: 893.5 [M+Na] ; elemental analysis calcd (%) for
C H F N O : C 51.03, H 5.79, N 6.43; found C 51.23, H 5.56,
Compound 24c: Starting from compound 18c (188 mg,
0.20 mmol), purification by flash chromatography (AcOEt/hexane,
37
50
7
4
20
N.6.32.
9
0:10, v/v) afforded compound 24c (180 mg 88%) as a white solid.
20
1
Compound 23b: Starting from compound 16b (168 mg,
M.p. 129–1328C; [a] =À18.3 (c=1 in MeOH); H NMR (500 MHz,
D
0
9
.20 mmol), purification by flash chromatography (AcOEt/MeOH,
CDCl ): d=9.42 (s, 1H; NH at C-4), 7.94 (d, J , =9.0 Hz, 1H; NH at
3
NH 5’’
0:10, v/v) afforded compound 23b (165 mg, 89%) as a white
C-5’’), 7.79 (d, J , =7.5 Hz, 1H; H-6), 7.49 (d, J , =7.5 Hz, 1H; 5-H),
6 5 5 6
2
D
0
1
solid. M.p. 130–1318C; [a] = +21.3 (c=1 in CH OH); H NMR
6.05 (d, J , =4.5, 1H; H-1’), 5.40 (ddd, J , =2.5, J , =6.0, J8’’,7’’
3
1’ 2’ 8’’ 9a’’ 8’’ 9b’’
(
500 MHz, CDCl ): d=9.00 (s, 1H; NH at C-4), 8.94 (d, J , =9.8 Hz,
=8.7 Hz, 1H; H-8’’), 5.35 (dd, J7’’,6’’ =2.0, J7’’,8’’ =8.7 Hz, 1H; H-7’’),
3
NH 5’’
1
7
H; NH at C-5’’), 7.60 (d, J6,5 =7.5 Hz, 1H; H-6), 7.41 (d, J , =
5.31 (dd, J2’,1’ =4.5, J2’,3’ =5.8 Hz, 1H; H-2’), 5.29 (ddd, J , =4.7,
5 6
4’’ 3a’’
.5 Hz, 1H; H-5), 6.15 (dd, J , =J , =5.5 Hz, 1H; H-2’), 5.74 (dd,
J4’’,5’’ =10.5, J , =12.0 Hz, 1H; H-4’’), 5.09 (dd, J3’,2’ =J , =5.8 Hz,
2
’ 1’
2’ 3’
4’’ 3b’’
3’ 4’
J , =J , =2.3 Hz, 1H; H-7’’), 5.58 (d, J , =5.5 Hz, 1H; H-1’),
1H; H-3’), 4.35–4.30 (overlapping, 2H; H-6’’, H-9a’’), 4.17–4.04 (over-
lapping, 3H; H-4’, H-5’’, H-9b’’), 3.82–3.77 (m, 1H; H-7a’), 3.67 (s,
7’’ 8’’
7’’ 6’’
1’ 2’
5
.38–5.30 (overlapping, 2H; H-3’, H-4’’), 5.15 (ddd, J8’’,7’’ =J ,
8’’ 9a’’
=
2.3, J8’’,9b’’ =9.1 Hz, 1H; H-8’’), 4.89 (dd, J9a’’ 8’’
,
=2.3, J
,
=
3H; COOCH ), 3.30–3.24 (m, 1H; H-7b’), 2.71 (dd, J , =4.7, J ,
9a’’ 9b’’
3 3a’’ 4’’ 3a’’ 3b’’
1
4
1
3
2
2.2 Hz, 1H; H-9a’’), 4.62 (dd, J6’’7’’ =2.3, J6’’,5’’ =10.5 Hz, 1H; H-6’’),
.22 (ddd, J5’’,NH =9.8, J , =J , =10.5, 1H; H-5’’), 4.19–4.14 (m,
=12.7 Hz, 1H; H-3a’’), 2.25 (s, 3H; NHCOCH ), 2.15 (s, 3H;
3
CH COO), 2.09 (s, 3H; CH COO), 2.08–2.07 (overlapping, 6H; 2”
5’’ 4’’
5’’ 6’’
3
3
H; H-4’), 4.06 (dd, J9
,
=12.2, J
,
=9.1 Hz, 1H; H-9b’’), 3.78 (s,
CH COO), 2.02 (s, 3H; CH COO), 2.01 (s, 3H; CH COO), 1.90–
3 3 3
b’’ 9a’’
9b’’ 8’’
H; COOCH ), 3.70–3.64 (m, 1H; H-7a’), 3.57–3.51 (m, 1H; H-7b’),
1.60 ppm (overlapping, 5H; H-5a’, H-5b’, H-6a’, H-6b’, H-3b”);
3
3
1
.59 (dd, J3
,
=12.8, J3’’,4’’ =5.0 Hz, 1H; H-3a’’), 2.30–2.22 (m, 1H;
C NMR (125 MHz, CDCl ): d=170.9, 170.7, 170.6, 170.1, 170.0,
3
a’’ 3b’’
H-5a’), 2.20 (s, 3H; NHCOCH at C-4), 2.17 (s, 3H; OCOCH ), 2.11 (s,
169.6, 169.4 (NHCOCH at C-4, CH COO at C-9’’, CH COO at C-4’’,
3 3 3
3
3
Chem. Eur. J. 2015, 21, 14614 – 14629
www.chemeurj.org
14627
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
CH COO at C-8’’, CH COO at C-7’’, CH COO at C-2’, CH COO at C-3’),
form/methanol (2:1, v/v) and 20% water. The aqueous and organic
phase obtained were counted for their radioactivity and analyzed
by HPTLC. [3- H]Sphingosine of the organic phase was separated
3
3
3
3
1
68.1 (C-1’’), 162.7 (C-4), 158.1 (t, J , =27 Hz, COCF CF CF ), 155.3
C F 2 2 3
3
(
C-2), 144.0 (C-6), 125.1–109.3 (COCF CF CF ), 98.6 (C-2’’), 97.2 (C-5),
2
2
3
8
6
8.9 (C-1’), 81.7 (C-4’), 73.9 (C-3’), 72.9 (C-2’), 71.4 (C-8’’), 68.9 (C-6’’),
by using the solvent system chloroform/methanol/water (55:20:3,
v/v/v). The solvent system chloroform/methanol/0.2% aqueous so-
8.5 (C-7’’), 67.6 (C-4’’), 64.3 (C-7’), 62.5 (C-9’), 52.6 (COOCH ), 49.8
3
(
C-5’’), 38.0 (C-3’’), 29.8 (C-5’), 25.7 (C-6’), 24.8 (NHCOCH at C-4),
lution of CaCl (60:40:9, v/v/v) was employed to analyzed the [3-
H]sphingosine of the aqueous phase. Radioactive lipids were vi-
sualized with a Beta-Imager 2000 (Biospace, Paris, France) and
identified by comparison with radiolabeled standards. The radioac-
tivity associated with the individual lipids was determined with the
specific b-Vision software (Biospace, Paris, France).
3
2
3
2
1
4
1.0, 20.7, 20.6, 20.5, 20.3 ppm (6”CH COO); MS (ESI positive): m/z:
3
+
047.1 [M+Na] ; elemental analysis calcd (%) for C H F N O : C
3
9
47
7
4
20
5.71, H 4.62, N 5.47; found C 45.53, H 4.70, N.5.39.
GM3 synthase assay
Cells cultured in 100 mm dishes, as described above, were harvest-
ed by using a plastic scraper and washed two times with phos-
phate-buffered saline (PBS, Euroclone). Cells were resuspended in
Western blot analysis
Cells were cultured for 72 h with 10% FBS DMEM in the presence
of 0.01% DMSO or 10 mm GM3 synthase inhibitors. After treatment,
the cells were rinsed twice with cold PBS, harvested by scraping,
and lysed in PBS containing aprotinin, leupeptin, and pepstatin by
sonication. Protein samples corresponding to 30 mg of cell fraction
were denatured by boiling for 5 min in sodium dodecyl sulfate
(SDS) sample buffer and subjected to sodium dodecylsulfate poly-
acrylamide gel electrophoresis (SDS/PAGE) (10% w/v gel). Subse-
quently, the proteins were transferred to a nitrocellulose mem-
brane (Thermo Scientific) by electroblotting for 2 h at 100 V.
1
50 mm sodium cacodylate/HCl buffer, pH 6.6 (20 mg of cell pro-
À1
teinmL ) with protease inhibitors (AEBSF at 104 mm, aprotinin at
8
0 mm, bestatin at 4 mm, E-64 at 1.4 mm, leupeptin at 2 mm, and
pepstatin A at 1.5 mm) (Sigma–Aldrich) and homogenized with
Dounce homogenizer (10 strokes, tight). In each tube, 10 mL of Tri-
ton CF-54 1.5% in chloroform/methanol (2:1, v/v) were mixed with
3
0
4
.5–50 nmol of [3- H(sphingosine)]LacCer (corresponding to
5 nCi), from a stock solution in chloroform/methanol (2:1, v/v)
and dried under N . To this mixture, 8 mL of 750 mm sodium caco-
2
dylate/HCl buffer (pH 6.6), 4 mL of 125 mm MgCl , 4 mL of 125 mm
2
The membranes were incubated overnight in Tris-buffered saline
2
-mercaptoethanol, 10 mL of 5 mm CMP-NeuAc, and 10 mL of the
(
TBS, 10 mm Tris-HCl, pH 7.4, 150 mm NaCl), 0.1% (v/v) Tween 20
cell homogenate (containing 200 mg of proteins) were added in
a total reaction volume of 50 mL. A negative control was performed
by using heat-inactivated cell homogenate (1008C for 3 min)
whereas a positive control was performed by using an activated
cell homogenate. The GM3 synthase inhibitors were tested at two
different concentration, that is, 1 mm and 10 mm, in the presence
of activated cell homogenate. The incubation was performed at
containing 5% (w/v) dried milk (Biorad) or 5% (w/v) bovine serum
albumin (BSA, Sigma–Aldrich) for the blocking buffer. Blots were in-
cubated with a primary antibody in the appropriate blocking solu-
tion for 3 h at room temperature. The following primary antibodies
were used: anti-phospho-EGFR Tyr-1148, 1:1000 dilution (cell sig-
naling); anti-EGFR, 1:1000 dilution (cell signaling); anti-phospho-
ERK1/2 Thr-202/Tyr-204, 1:1000 dilution (cell signaling); anti-ERK1/
3
78C for three hours with continuous shaking. The reaction was
2
, 1:1000 dilution (cell signaling); anti-phospho-p38 MAPK Thr-180/
stopped by adding 1.5 mL of chloroform/methanol (2:1, v/v). The
reaction mixture was analyzed by HPTLC by using the solvent
system chloroform/methanol/water 55:20:3 (v/v/v). Radioactive
lipids were detected and quantified by radioactivity imaging as de-
scribed below.
Tyr-182, 1:1000 dilution (cell signaling); anti-p38 MAPK, 1:1000 dilu-
tion (cell signaling); anti-calnexin, 1:10000 dilution (epitomics). The
membranes were washed four times for 10 min with T-TBS and
then incubated with the appropriate secondary antibody conjugat-
ed (anti-mouse and anti-rabbit HRP-conjugated, (Dako)) with
horseradish peroxidase for 1 h at RT, 1:2000 dilution. After four
washes in T-TBS, the protein bands were detected by using an ECL
detection kit (Amersham), as described by the manufacturer.
Metabolic labeling of cell sphingolipids
3
[
3- H]Sphingosine dissolved in ethanol was transferred into a gall
sterile tube and dried under a nitrogen stream. The residue was
then dissolved in an appropriate volume of pre-warmed 10% FBS
Acknowledgements
DMEM to obtain a final concentration of 0.25 mCi per 100 mm dish
À9
(
corresponding to 2.6”10 m). Cells were pre-treated for 72 h in
We gratefully acknowledge Professor Mario Anastasia for the
valuable suggestions and his unrelenting encouragement, and
Irene Delcarro for her skilled technical assistance and Dr. Luca
Pignataro for preparative HPLC support.
the presence of 10 mm GM3 synthase inhibitors, whereas the nega-
tive control were cultured for 72 h in the presence of 0.01%
DMSO. Then the samples were incubated with 10% FBS DMEM
3
containing [3- H]sphingosine and 10 mm solutions of the inhibitors
or DMSO. After 2 h incubation, the medium was removed and the
cells were chased for 48 h with 10% FBS DMEM always containing
Keywords: glycosides · inhibitors · sialic acids · sphingolipids
1
0 mm inhibitors or DMSO, in order to reach the metabolic steady
state. At the end of chase, the cells were washed and harvested in
ice-cold phosphate-buffered saline by scraping. Cell suspensions
were frozen and then lyophilized.
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Full Paper
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Published online on August 11, 2015
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