Multicomponent diversity oriented synthesis of multivalent glycomimetics containing hexafluorovaline

Article abstract of DOI:10.1016/j.tet.2015.07.070

The chemoselective introduction of fluorinated moieties into biologically relevant scaffolds is now an established strategy to modulate and to study the properties of molecular leads. In this article we propose, for the first time, the diversity oriented

Full text of DOI:10.1016/j.tet.2015.07.070

Accepted Manuscript
Multicomponent Diversity Oriented Synthesis of Multivalent Glycomimetics Containing
Hexafluorovaline
M.C. Bellucci, A. Sganappa, M. Sani, A. Volonterio
PII:
S0040-4020(15)01147-3
10.1016/j.tet.2015.07.070
TET 27015
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 10 June 2015
Revised Date: 14 July 2015
Accepted Date: 27 July 2015
Please cite this article as: Bellucci MC, Sganappa A, Sani M, Volonterio A, Multicomponent Diversity
Oriented Synthesis of Multivalent Glycomimetics Containing Hexafluorovaline, Tetrahedron (2015), doi:
10.1016/j.tet.2015.07.070.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Multicomponent Diversity Oriented Synthesis of
Multivalent Glycomimetics Containing
Hexafluorovaline
a
b
c
b,c,
M.C. Bellucci, A. Sganappa, M. Sani, and A. Volonterio *
a
Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, via
Celoria 2, 20133 Milano, Italy.
b
Department of Chemistry, Materials, and Chemical Engineer “G. Natta”, Politecnico di Milano, via
Mancinelli 7, 20131 Milano, Italy
c
C.N.R. Istituto del Riconoscimento Molecolare, via Mancinelli 7, 20131 Milano, Italy.
alessandro.volonterio@polimi.it
Abstract
The chemoselective introduction of fluorinated moieties into biologically relevant scaffolds is now an
established strategy to modulate and to study the properties of molecular leads. In this article we
propose, for the first time, the diversity oriented synthesis of multivalent glycomimetics incorporating
hexafluorovaline through a straightforward multicomponent sequential process which occurs with high
yield and in mild conditions. The same process has been successfully applied to the chemistry of
1

ACCEPTED MANUSCRIPT
aminoglycosides producing a neomycin-hexafluorovaline-galactose conjugate and providing a general,
efficient strategy to functionalized aminoglycosides with sugar-hexafluorovaline tags.
Keywords: Diversity Oriented Synthesis, Multicomponent Synthesis, Multivalent Glycomimetic,
Fluorine, Aminoglycoside.
1. Introduction
The introduction of a fluorine atom or perfluorinated groups into chemical scaffolds is now a
standard strategy for modulating the chemical and biophysical properties of molecular leads and
for studying the biological aspects associated with the fluorine substitution/introduction, such as
1
metabolism, excretion properties, and fluorine-containing ligand binding interactions. This is due
to the unique properties of the fluorine atom, its strongest inductive effect compared to other
elements, its small size, and low polarizability. For instance, the highly fluorinated amino acids
hexafluorovaline (hfVal) and hexafluoroleucine (hfLeu), being often well tolerated, have been
used to modulate such properties of peptides and proteins like their hydrophobicity,
2
19
acidity/basicity, folding and stability. Moreover, F is the most stable and abundant fluorine
isotope, thus fluorinated peptides and proteins can be easily tracked with NMR and/or MRI
3
spectroscopy, even in living cells. Thus, since fluorine containing organic molecules are almost
4
absent in nature, there is great interest in developing new synthetic strategies to introduce
selectively fluorinated moieties into chemical scaffolds.
5
Multivalent glycomimetics are a class of very intriguing compounds. Indeed, there is a growing
interest in glycosidase inhibition, and in elucidating fundamental biological pathways through the
synthesis of glycomimetics. Multivalency can increase dramatically the binding potency of the
6
corresponding monovalent ligands as already demonstrated. However, despite such interest,
compared to monovalent glycomimetics, there are relatively few example in the literature of
multivalent glycosidase inhibitors, and no fluorinated multivalent glycosidase inhibitors, probably
2

ACCEPTED MANUSCRIPT
6
because they are not synthetically accessible in an easy way. One strategy to overcome such a
deficiency could be the development of straightforward methodologies for the diversity oriented
7
synthesis (DOS) of these compounds. Indeed, DOS, often combined with multicomponent
reactions (MCRs), is an indispensable tool for modern pharmaceutical and drug discovery
research programs because they offer the possibility to access molecular complexity with a
minimum of effort, in addition to atom economy, operational simplicity, and bond-forming
8
efficiency. This is well evidenced by the growing application of such strategies for the synthesis
of libraries of different collections of molecules. However, to the best of our knowledge, there are
9
no examples of DOS of multivalent, selectively fluorinated glycomimetics.
Very recently, in the frame of a research program dealing with the discovery of novel MCRs for the
1
0
11
synthesis of heterocycles and glycoconjugates, we have reported for the first time the MC
combinatorial synthesis of diversity oriented multifunctional glycomimetics 1-4, where the sugar
moieties were tethered through artificial linkers such as aspartic acid, a hydantoin ring, or urea
1
2
frameworks (Figure 1). Herein we wish to report an extension of such process, using commercially
available 4,4,4-trifluoro-3-trifluoromethyl(Tfm)-crotonic acid in order to prepare multifunctional
glycomimetics where the Asp moiety of 1-4 is substituted by hfVal.
R2 R1
R2 R1
R2 R1
Sugar
O
O
O
O
N
H
N
H
H
O
Sugar
N
Sugar
N
N
N
Sugar
N
N
N
AA-OX'
N
N
Sugar O
Sugar
H
Sugar
H
H
Sugar O
Sugar O
R1
R2
1
2
3
4
1
2
previous work R = H, R = COOEt or COOBn
1
2
this work
R = R = CF
3
Figure 1. Multivalent glycomimetics containing hfVal
We propose that the introduction of hfVal in the multivalent glycomimetic scaffolds can impart
interesting new properties to this intriguing class of compounds such as, for instance, higher metabolic
3

ACCEPTED MANUSCRIPT
1
9
stability and lipophilicity and would facilitate the investigation of target-ligand interactions through F
NMR spectroscopy.
2. Results and Discussion
4,4,4-Trifluoro-3-Tfm-crotonic acid is a commercially available fluorinated building block that, due to
the high electronegativity of the trifluoromethyl groups, has been used for the synthesis of hfVal amino
1
3
acid derivatives through anti-Michael addition of N-nucleophiles. Recently, our group has exploited
the reactivity of 4,4,4-trifluoro-3-Tfm-crotonic acid 8 for the synthesis of glycoconjugates containing
1
0,11
hfVal 10 and 11 through the MC sequential domino process depicted in Scheme 1.
The reaction of 8
with in situ generated N-glycosyl, N’-tert-butyl carbodiimides 7 produces intermediate A which
undergoes intramolecular anti-aza-Michael reaction affording a second highly reactive intermediate B.
The latter step is totally regioselective when the steric hindrance of the two N-substituents in the
carbodiimide framework is very different, namely a primary versus a tertiary alkyl group. In the absence
of a N-nucleophile, 9 intermediate B rearranges through an O→N acyl migration process affording
1
4
hydantoin scaffold 10 containing a hfVal moiety (path A, Scheme 1). Instead, in the presence of N-
nucleophile 9, such as amines, amino esters, or peptides, intermediate B undergoes nucleophilic attack
leading to the formation of urea-hfVal-glycopeptides 11 (path B, Scheme 1).
O
N
O
N
Sugar
F
3
C
N
O
CF
3
COOH
acyl migration
3
F C
Sugar-N
3
CF
3
8
Sugar
10
5
N
path A
path A
aza-Michael
Sugar
+
Sugar
N
C
N
F
3
C
O
N
N
O
H
7
or
CF3
O
N
3
F C
path B
NCO
F
3
C
CF3
2
O H N-R
6
A
COOH, R-NH
2
F3C CF3
CF
3
8
9
B
O
H
N
N
N
R
path B
H
Sugar
O
1
1
Scheme 1. Mechanism of the domino MC process
Since this process is high yielding, totally regioselective and operationally very simple (MC
domino process which does not require high temperatures, dry solvents, difficult purification
steps), we wished to exploit it for the synthesis of multivalent glycomimetics containing hfVal by
4

ACCEPTED MANUSCRIPT
using N-glycosyl amines as
N-nuclophiles, and/or by introducing a new sugar moiety in the
carbodiimide framework.
Firstly, in order to be able to prepare divalent glycomimetics 1, we checked if the MC component
process works efficiently also by using N-glycosyl amines as nucleophiles. Thus we reacted O-protected
amino-galactose 9a with acid 8 and DIC 12 under the standard conditions (CH CN as solvent, 0 °C) and
3
we were delighted to recover glycomimetic 13 in very good yield as an equimolar mixture of epimers at
1
5
the hexafluorovaline stereocentre (Scheme 2).
O
NH2
O
O
F3C CF3
O
O
CF3
TMP, CH CN
O
3
HOOC
N C N
+
+
O
NH
CF3
N
N
O
0 °C
O
8
O
H
1
2
O
9
a
O
1
3 (92%)
OMe
OH
NH2
O
OH
HO
F3C CF3
TMP, CH CN
co-solvent
CF3
3
O
O
HOOC
N C N
+
+
NH
CF3
N
N
HO
0 °C
OH
16 %
OMe
H
8
1
2
O
9
b
HO
co-solvent = none
1
4 co-solvent = MeOH 33 %
co-solvent = tert-butyl 42 %
co-solvent = DMF
51 %
Scheme 2. MC process with glycosylamine as nucleophile
We explored also the possibility of running the process with the corresponding deprotected
amino-galactose 9b. However, probably because of its low solubility in the reaction medium
(
CH CN, 0°C), the reaction produced the desired product 14 in very low yield (16%) along with
3
the formation of the undesired hydantoin in 76% yield (see Scheme 1, path A). We tried to raise
the yield of the process by adding a co-solvent in order to increase the solubility of the
nucleophile. Indeed, in the presence of 10% MeOH, the yields were raised only to a 33% due to
the concomitant formation of the corresponding urea-hfVal-OMe derivative arising from the
competitive nucleophilic attack of MeOH which is present in large excess (Scheme 1, path B).
Thus we tried with less nucleophilic tert-BuOH but without a real improvement (42% yield).
5

ACCEPTED MANUSCRIPT
Disappointingly, even in the presence of highly polar DMF the yields were raised only to 51%
(
Scheme 2).
Once proved that the reaction works efficiently even with O-protected N-glycosyl amines as
nucleophiles in the standard conditions, we applied the process for the four-component sequential
synthesis of a library of divalent glycoconjugates containing hfVal 1 starting from different N-tert-butyl,
N’-glycosyl carbodiimides 7, which are formed in situ by Staudinger reaction between sugar azides 5
and tert-butyl isocyanate 6, and N-glycosyl nucleophiles 9 (Table 1).
Table 1. MC combinatorial synthesis of multivalent glycomimetics 1.
CF3
HOOC
F3C
CF3
CF3
O
Ph
3
P
H
8
Sugar-N3
+
NCO
Sugar
N
C
N
N
N
N
Sugar
CH CN
Sugar-NH 9
H
5
6
3
2
7
Sugar
O
TMP, CH CN, 0°C
1
3
a
Sugar-N3
Carbodiimide
Sugar-NH2
Product
Yield (%)
Entry
1
b
N
3
O
F
3
C
CF
3
O
8
1
OMe
NH
2
O
N
C
N
O
OMe
H
N
O
O
O
O
N
N
5
a
H
O
O
7a
O
O
O
O
O
O
O
9
a
O
O
O
1
a
O
C
O
N
3
OAc
OAc
b,d
2
OMe
F
3
CF
N
3
53
78
O
N
C
N
OMe
O
O
O
O
AcO
AcO
H
AcO
AcO
N
NH
2
OAc
N
5
a
H
O
O
7a
OAc
O
O
C
O
9c
O
1
b
O
O
O
c
O
H
2
N
O
3
4
N
3
O
OMe
N
N
O
F
3
C
CF
N
3
O
O
O
O
O
H
O
O
N
O
9
d
N
H
O
O
O
O
7b
O
O
O
5
b
O
O
1c
O
O
O
b
N
3
H
2
N
O
OMe
6
8
N
C
O
N
O
F
3
C
CF
3
PivO
PivO
O
O
O
PivO
PivO
O
H
N
9d
N
N
O
O
O
AcHN
BnO
H
5
c
O
AcHN
BnO
7
c
O
OPiv
OPiv
1
d
NHAc
OBn
6

ACCEPTED MANUSCRIPT
H
2
N
AcO
OAc
O
b
AcO
AcO
83
5
6
OAc
O
OAc
O
OMe
O
AcO
AcO
HN
O
AcO
AcO
AcO
AcO
O
O
N
9
d
NH
CF3
O
O
O
O
N3
O
N
N
C
C
N
N
O
F3C
5
d
7d
1
e
O
O
O
O
b
AcO
H
2
N
F C
CF3
O
N
3
AcO
OMe
3
76
O
O
H
O
N
O
AcO
AcO
O
N
N
N
N
AcO
AcO
H
AcO
N
N
N
9d
O
AcO
N
O
O
OAc
OAc
O
OAc
AcO
1f
5
e
7
e
N
N
N
a
b
c
Isolated yields. An almost 3:1 diastereoisomeric mixture. An almost 1.5:1 diastereoisomeric
d
mixture. A 30% of the corresponding hydantoin was recovered.
Indeed, the reaction between glycosyl azides 5a-e and commercially available tert-butyl
isocyanate
6 lead to the formation of the corresponding carbodiimides 7a-e, respectively, upon
treatment with Ph P in CH CN at rt (Staudinger reaction). The resulting carbodiimides 7a-e can
3
3
also be isolated by quick flash chromatography and characterized. However, compounds 7a-e can
be used in situ in order to achieve a four component sequential process. In this case, once the
carbodiimide is formed (TLC monitoring), the temperature is cooled to 0°C and 2,4,6-
trimethylpyridine (TMP) followed by nucleophile
9 and, finally, acid 8 are added. The process
was very general, working efficiently either starting from sugar azides (where the functional
group is attached to primary carbon 6 in hexoses or carbon 5 in pentoses) leading to the formation
of multivalent glycomimetics having enzymatically stable “CH
2
NH
glycine (entries 1-4, Table 1), or attached to the anomeric carbon through a suitable linker
entries 5 and 6, Table 1). In all cases the reaction was completely regioselective leading to the
formation of regioisomers 1a-f as a mixture of diasteroisomers, which arose from the nucleophilic
attack of the less sterically congested primary -glycosyl moiety in the intramolecular anti-aza-
2
” backbone mimicking
16
(
N
17
Michael step (Scheme 1, path B). The yields were generally high except when glycosylamine 9c
was used as nucleophile (entry 2, Table 1). This result was due to the poor nucleophilicity of the
1
1d
hemiacetalic amino group and scarce solubility of 9c as already stated in previous works. In
this way we were able to obtain a small library of six divalent glycomimetics where the sugar
moieties are connected through a urea-hfVal linker. The reaction is very general, and any kind of
7

ACCEPTED MANUSCRIPT
appropriately functionalized carbohydrates could be used, such as ribose, galactose, glucose,
glucosamine, and mannose.
In order to obtain other multivalent glycomimetics containing hfVal, where the sugar moieties are
connected with different scaffolds (DOS), we tested the same reaction starting with carbodiimides
having two N-sugar substituents. In this way, considering the synthetic pathway depicted in Scheme 1,
we would be able to obtain three new multivalent glycomimetics, i.e. divalent glycomimetics 2, 3 where
the carbohydrates are connected through a hydantoin scaffold or a urea linker, respectively, and trivalent
glycomimetic 4 where the sugar moieties are tethered through a urea-hfVal scaffold. N,N’-Disugar
carbodiimides 16 could be prepared starting from the corresponding sugar azides 5 and sugar
isothiocyanates 15 (Table 2).
Table 2. MC combinatorial synthesis of multivalent glycomimetics 2-4.
Sugar
O
N
8
O
N
Sugar
TMP, CH CN
3
CF3
F3C
2
F3C CF3
O
Ph
3
P
8, XO-AA -AA -NH 17
1 2 2
Sugar-N3
+
Sugar-NCS
Sugar
N
C
N Sugar
Sugar
NH-AA -AA -OX
2 1
TMP, CH CN, 0°C
N
N
CH CN
3
5
15
3
16
H
Sugar
O
3
F3C CF3
O
H
8
, Sugar-NH2 9
Sugar
N
N
H
N
Sugar
TMP, CH CN, 0°C
3
Sugar
O
4
Sugar-N3
Sugar-NCS
Carbodiimide
Sugar-NH2
Product
Yield
Entr
y
a
(
%)
b
N
3
SCN
O
/
78
1
OMe
O
O
O
O
O
O
O
O
1
5a
O O
O
O
5
a
O
O
O
O
O
N
C
N
O
N
O
O
N
1
6a
O
O
2a
O
O
CF
3
3
F C
c
O
O
/
82
2
N
3
O
O
NCS
O
O
O
O
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
O
O
N
C
N
5
b
15b
O
O
O
O
O
O
O
CF
3
O
2
b
3
F C
1
6b
O
8

ACCEPTED MANUSCRIPT
c
O
O
75
72
3
4
N
3
O
NCS
O
O
BnOOC
17a
NH
2
O
O
F
3
C
CF
3
O
O
O
O
O
O
H
N
O
COOBn
O
O
N
H
N
O
N
C
N
O
O
5
b
15b
O
O
O
O
O
O
O
O
O
1
6b
3a
O
O
O
O
b
N
3
SCN
O
F
3
C
CF3
OMe
O
O
O
O
O
H
O
N
COOBn
1
5a
O
O
N
N
O
O
5
a
O
O
H
O
O
N
C
N
BnOOC
NH
2
O
O
O
1
6a
O
1
7b
O
O
O
O
3
b
O
O
b
N
3
SCN
O
O
7
6
6
2
5
6
OMe
F3C
O
O
CF
O
3
O
O
O
O
O
O
H
N
1
5a
NH
2
COOBn
O
O
BnOOC
N
H
O
O
N
H
N
N
H
5
a
O
O
O
O
N
C
N
O
1
6a
17c
O
O
O
O
O
3
c
O
O
N
3
SCN
O
O
NH2
O
O
O
b
OMe
O
O
F3C
O
O
O
CF
O
3
O
O
O
O
O
H
N
1
5a
O
O
5
a
O
O
O
O
O
N
H
N
O
O
N
C
N
9a
O
O
O
1
6a
O
O
O
O
O
4
a
O
O
AcO
c
O
O
OAc
O
AcO OAc
O
O
64
7
N
3
O
NCS
O
AcO
AcO
O
AcO
F
C
CF
3
3
O
AcO
O
O
O
O
O
O
H
O
N
O
NH2
O
O
N
O
9d
N
N
H
O
O
O
O
N
C
O
5
b
15b
O
O
4b
O
O
O
O
O
O
O
O
1
6b
O
a
b
c
Isolated yields. An almost 1.5:1 diastereoisomeric mixture. An almost 3:1 diastereoisomeric
mixture.
Accordingly, Staudinger reaction between ribose azide 5a and isothiocyanate 15a lead to the
clean formation of carbodiimide 16a along with Ph PS (TLC monitoring). By adding in situ TMP
3
followed by acid
mixture of diasteroisomers, where the sugar moieties are connected through a hydantoin ring
entry 1, Table 2). Under the same conditions we obtained the formation of diglycohydantoin 2b
in very high yields starting with galactose azide 5b and isothiocyanate 15b (entry 2, Table 2).
Interestingly, the reaction carried out in the presence of -amino esters nucleophiles such as H-
8, we obtained the formation of divalent glycomimetic 2a, as an almost 1.5:1
(
α
Ala-OBn 17a and H-Leu-OBn 17b furnished divalent glycodipeptides 3a,b, respectively (entry 3
9

ACCEPTED MANUSCRIPT
and 4, Table 2). The latter reaction is particularly promising because it permits the unprecedented
multicomponent synthesis of a class of very intriguing scaffolds, i.e. divalent glycopeptides
incorporating the unnatural amino acid hfVal. Moreover, the process works very efficiently also
starting from dipeptide nucleophiles such as H-Phe-Leu-OBn 17c leading to the one pot
formation of divalent glycotripeptides containing hfVal 3c in high yields (entry 5, Table 2).
Finally, in order to demonstrate that this process could also be exploited for the preparation of
more complicated trivalent glycomimetics, we tested the reaction starting with in situ generated
symmetric N,N’-diglyco carbodiimide 16a,b and sugar nucleophile 9a,d, respectively. We were
delighted that in this case the reaction also works effectively giving rise to the formation of
trivalent glycomimetics 4a,b in good yields (entries 6 and 7, Table 2). It is worth noting that, in
order to increase complexity, the synthesis of glycomimetics 2-4 could be also achieved starting
with asymmetric N,N’-diglyco carbodiimides. However, even if high yielding, due to the
similarity on the steric hindrance of the
N-substituents, the process lead to the formation of
mixtures of regioisomers that are difficult to separate (data not shown).
Beside the synthesis of multivalent glycomimetics 1-4, this process could be exploited as a general
procedure for the introduction of sugar-hfVal tags into biologically relevant molecules in a simple and
convenient way. With this in mind, we sought to functionalize aminoglycoside antibiotics.
1
8
Aminoglycosides are a class of polyaminosugars which selectively bind RNA. The selective
functionalization of such scaffolds has been exploited for the synthesis of conjugates with better
characteristics, such as, for instance, activity, selectivity, and ability to fight against aminoglycosides
1
9
resistance. In this sense, the introduction of fluorinated probes in the aminoglycoside scaffolds could
1
9
20
facilitate the investigation of RNA-ligand interaction through F NMR spectroscopy. However,
besides the interest in the development of new practical ways to selectively functionalize
aminoglycosides, there are only a few examples that use a multicomponent one-pot procedure, and, to
2
1
the best of our knowledge, none of them are used for tethering aminoglycosides with fluorinated tags.
Thus, the successful application of our process to aminoglycoside chemistry would provide a general
1
0

ACCEPTED MANUSCRIPT
way to selectively functionalize such molecules with other scaffolds (in this case carbohydrates) along
with a fluorinated tag (hfVal) with a practical one pot multicomponent procedure. Accordingly, we
decided to use neomycin derivative 18 as nucleophile in our multicomponent process. To our delight,
reacting carbodiimide 6b in the presence of acid 8 and neomycin derivative 18 under the same
conditions described above, i.e. TMP, CH CN at 0 °C, we obtained the clean regiospecific formation of
3
2
2
conjugate 19 in very good yield, as a mixture of diastereoisomers (Scheme 3).
CF3
OH NHBoc
COOH
HO
F3C
O
N
CF
3
8
HN
CF3
O
BocHN
NHBoc
1
8, TMP
O
NH
O
+
CH CN, 0°C
O
3
O
O
S
O
O
NHBoc
N
O
C
N
O
(
76%)
BocHN
HO
O
OH
O
O
O
OH
6
b
O
O
19
O
HO
NHBoc
OH NHBoc
HO
O
BocHN
NHBoc
H N
O
2
S
O
O
NHBoc
BocHN
HO
OH
O
O
OH
1
8
HO
NHBoc
Scheme 3. Synthesis of neomycin-hfVal-galactose conjugate 19
The application of this procedure for the combinatorial synthesis of different aminoglycoside
conjugates bearing a hfVal probe will be studied more in detail and reported in a forthcoming paper.
3. Conclusions
In conclusion, we have demonstrated that the MC sequential process recently developed by us can also
be for the DOS of multivalent glycomimetics containing hfVal amino acid. Indeed, the reaction between
in situ generated N-glycosyl, N’-tert-butyl carbodiimides or N, N’-diglycosyl carbodiimides with 4,4,4-
trifluoro-3-Tfm-crotonic in the presence or absence of N-nucleophiles, such as α-aminoesters, peptides
1
1

ACCEPTED MANUSCRIPT
or glycosyl amines, gave rise to the clean formation of di- or trivalent glycomimetics containing hfVal
where the glycosyl moieties are tethered trough different scaffolds, such as urea, hydantoin ring, and
urea-peptide linkers. The MC sequential domino process is completely regioselective, high yielding,
occurs under mild conditions, and is very general, working efficiently with different sugars and linkers.
This process could also be exploited as a favourable tool to conjugate biological important compounds
with a sugar-hfVal tag. For instance, the reaction performed using an appropriately functionalized
neomycin derivative such as N-nucleophile allowed us to obtain the synthesis of a neomycin-hfVal
conjugate in a very efficient way. The combinatorial synthesis of different aminoglycoside-hfVal-sugar
1
9
conjugates and the determination of their antibacterial activity and RNA interaction trough F NMR
spectroscopy is ongoing in our laboratories and will be reported in a forthcoming paper.
4
. Experimental Section
4
.1 General Methods: Commercially available reagent-grade solvents were employed without
1
1
purification. Primary glycosylazides
5
were prepared following reported procedures.
2
3
Glycosylisothiocyanate 15 were prepared by aza-Wittig reaction with CS2. Glycosylamines 9 were
prepared by catalytic hydrogenation of the corresponding azides. Neomycin derivative 18 was obtained
1
as reported in ref. 19a. H NMR spectra were run on spectrometers operating at 400 or 500 MHz.
1
Chemical shifts are expressed in ppm (δ), using tetramethylsilane (TMS) as internal standard for H and
1
3
C nuclei (δ and δ = 0.00). ESIMS was performed with an Esquire 3000 plus ion-trap mass
H
C
spectrometer equipped with an ESI source. The IR spectra were obtained by a Varian 640 high-
performance FTIR spectrometer. Elemental analysis were obtained on FlashEA 1112 NC Analyzers.
TLC was run on silica gel 60 F254 Merck. Flash chromatography (FC) was performed with silica gel 60
(
60–200 mm, Merck).
.2 Synthesis of divalent glycomimetics 1: general procedure. To a solution of glycosylazide
1 equiv.) in CH CN (0.1 M) tert-butyl isocyanate (1.05 equiv.) followed by Ph P (1.05 equiv.)
4
5
(
3
6
3
1
2

ACCEPTED MANUSCRIPT
were added at rt. The solution was stirred until complete formation of the corresponding
carbodiimide
1 equiv.), a solution of glycosylamine
a solution of 4,4,4-trifluoro-3-Tfm-crotonic acid
7
was achieved (TLC monitoring). The temperature was lowered to 0 °C and TMP
(
9
(1 equiv.) in a minimum amount of CH CN followed by
3
8
(1 equiv.) in a minimum amount of CH CN
3
were added. The temperature was slowly left to reach rt and the reaction, when finished (TLC
monitoring, ca. 3 h), was quenched with a 1M solution of HCl. The mixture was extracted with
AcOEt, the organic phases collected, dried over Na SO , filtered, the solvent removed under
2
4
reduced pressure and the crude purified by flash chromatography.
-1 1
1
a: Major diastereoisomer: R = 0.29 (hexane/ AcOEt 80:20); FTIR (neat) ν 1779, 1756, 1726 cm ; H
f
NMR (400 MHz, CDCl
3
), δ: 8.44 (br s, 1H), 5.49-5.46 (m, 2H), 5.11 (s, 1H), 4.63 (d, J = 5.6 Hz, 1H),
4
.59 (dd, J = 8.0 and 2.4 Hz, 2H), 4.53 (d, J = 5.6 Hz, 1H), 4.38-4.36 (m, 1H), 4.29 (dd, J = 5.2 and 2.4
Hz, 1H), 3.98-3.96 (m, 1H), 3.89-3.86 (m, 1H), 3.47 (s, 3H), 3.33-3.31 (m, 1H), 2.90-2.88 (m, 1H), 1.49
1
3
(
s, 3H), 1.49 (s, 3H), 1.47 (s, 3H), 1.41 (s, 3H), 1.35 (s, 3H), 1.32 (s, 3H), 1.30 (s, 12H); C NMR
(
100.6 MHz, CDCl ) δ: 168.3, 154.9, 123.1 (q, J = 281.7 Hz, CF ), 112.6, 111.5, 109.4, 108.5, 96.2,
3
3
8
2
7.0, 84.4, 71.4, 70.8,70.5, 70.4, 56.0, 51.4, 45.9 (septet, J = 25.8 Hz ), 40.4, 29.0, 26.3, 25.9, 25.7,
+
4.9, 24.7; ESI (m/z) 774.7 [M +Na, (100)]; Anal. calcd. for C H F N O : C 49.53, H 6.30, N 5.59;
3
1
47
6
3
11
found: C 49.53, H 6.32, N 5.60. Minor diastereoisomer: R = 0.22 (hexane/ AcOEt 80:20); FTIR (neat)
f
-
1 1
ν 1781, 1754, 1722 cm ; H NMR (400 MHz, CDCl
Hz, 2H), 5.11 (s, 1H), 4.98 (s, 1H), 4.60-4.56 (m, 2H), 4.41-4.39 (m, 1H), 4.32-4.28 (m, 2H), 4.15 (dd, J
8.0 and 2.0 Hz ,1H), 3.97-3.94 (m, 1H), 3.47-3.44 (m, 3H), 3.41 (s, 3H), 3.24 (dd, J = 16.4 and 5.2 Hz
3
), δ: 7.12 (br s, 1H), 5.77 (s, 1H), 5.49 (d, J = 5.2
=
1
3
1
H), 1.49 (s, 3H), 1.49 (s, 3H), 1.47 (s, 3H), 1.45 (s, 3H), 1.35 (s, 9H), 1.34 (s, 3H), 1.32 (s, 3H); C
NMR (100.6 MHz, CDCl ) δ: 168.0, 157.0, 123.3 (q, J = 281.5 Hz, CF ), 112.9, 110.3, 109.4, 108.7,
3
3
9
2
6
6.3, 85.8, 84.5, 82.1, 71.3, 70.9, 70.5, 65.4, 55.5, 51.5, 46.9 (s, J = 26.1 Hz ), 40.2, 29.0, 26.5, 26.0,
+
5.9, 25.1, 24.9. 24.4; ESI (m/z) 774.6 [M +Na, (100)]; Anal. calcd. for C H F N O : C 49.53, H
3
1
47
6
3
11
.30, N 5.59; found: C 49.55, H 6.31, N 5.58.
1
3

ACCEPTED MANUSCRIPT
4.3 Synthesis of divalent glycomimetics 2: general procedure. To a solution of glycosylazide 5
(
3
1 equiv.) in CH CN (0.1 M) a solution of glycosylisothiocyanante 15 (1.05 equiv.) in a minimum
amount of CH CN followed by Ph P (1.05 equiv.) were added at rt. The solution was stirred until
3
3
complete formation of the corresponding carbodiimide 16 was achieved (TLC monitoring). A
solution of 4,4,4-trifluoro-3-Tfm-crotonic acid (1 equiv.) in a minimum amount of CH CN was
added at rt and the resulting solution stirred until the reaction was finished (TLC monitoring, ca.
h). The reaction was quenched with a 1M solution of HCl. The mixture was extracted with
AcOEt, the organic phases collected, dried over Na SO , filtered, the solvent removed under
8
3
3
2
4
reduced pressure and the crude purified by flash chromatography.
2
1
1
1
3
4
3
a
:
Mixture of two diastereoisomers: Rf = 0.24 (hexane/ AcOEt 80:20); FTIR (neat)
ν
1777,
), major diastereoisomer δ: 4.96 (s, 2H), 4.75 (s,
H), 4.63-4.61 (m, 4H), 4.34-4.32 (m, 2H), 4.09 (dd, = 12.0 and 4.8 Hz, 1H), 3.95-3.92 (m,
H), 3.71-3.68 (m, 2H), 3.36 (s, 3H), 3.35 (s, 3H), 3.12 (dd, = 12.0 and 6.4 Hz, 1H), 1.44 (s,
-
1 1
754, 1728 cm ; H NMR (400 MHz, CDCl
3
J
J
H), 1.43 (s, 3H), 1.31 (s, 3H), 1.29 (s, 3H); minor diastereoisomer δ: 4.95 (s, 2H), 4.75 (s, 1H),
.63-4.61 (m, 4H), 4.34-4.32 (m, 1H), 4.31-4.29 (m, 1H), 3.95-3.92 (m, 2H), 3.71-3.68 (m, 2H),
.40-3.37 (m, 1H), 3.37 (s, 3H), 3.32 (s, 3H), 1.48 (s, 3H), 1.47 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H);
13
C NMR (100.6 MHz, CDCl ) δ: 169.0, 156.5, 156.0, 113.1, 112.5, 110.5, 110.2, 110.0, 109.8,
3
85.2, 85.0, 84.6, 83.8, 83.4, 83.3, 82.3, 82.2, 82.1, 81.9, 56.0, 55.5, 55.4, 55.3, 55.1, 48.8 (septet,
J
= 28.2 Hz), 45.9, 44.7, 42.9, 42.7, 29.6, 26.6, 26.5, 26.4, 26.3, 25.1, 25.0, 24.9, 24.8 ; ESI (m/z)
+
6
45.2 [M +Na, (100)]; Anal. calcd. for C H F N O : C 46.31, H 5.18, N 4.50; found: C 46.33,
24
32
6
2
10
H 5.20, N 4.53.
4.4 Synthesis of divalent glycomimetics 2: general procedure. To a solution of glycosylazide
5
(
1 equiv.) in CH CN (0.1 M) a solution of glycosylisothiocyanante 15 (1.05 equiv.) in a minimum
3
amount of CH CN followed by Ph P (1.05 equiv.) were added at rt. The solution was stirred until
3
3
1
4

ACCEPTED MANUSCRIPT
complete formation of the corresponding carbodiimide 16 was achieved (TLC monitoring). The
temperature was lowered to 0 °C and TMP (2 equiv.), solid
α
-aminoester hydrochloride 17 (1
(1 equiv.) in a minimum
equiv.) followed by a solution of 4,4,4-trifluoro-3-Tfm-crotonic acid
8
amount of CH CN were added. The temperature was slowly left to reach rt and the reaction, when
3
finished (TLC monitoring, ca. 3 h), was quenched with a 1M solution of HCl. The mixture was
extracted with AcOEt, the organic phases collected, dried over Na SO , filtered, the solvent
2
4
removed under reduced pressure and the crude purified by flash chromatography.
3a:
f
Major diastereoisomer: R = 0.34 (hexane/ AcOEt 70:30); FTIR (neat) ν 1769, 1753, 1723
-
1 1
cm ; H NMR (400 MHz, CDCl ), δ: 8.11 (br s, 1H), 7.38-7.35 (m, 5H), 6.57 (s, 1H), 5.48 (d,
J
3
=
4.3 Hz, 1H), 5.44 (d,
br s, 1H), 4.65-4.56 (m, 3H), 4.31-4-28 (m, 2H), 4.25-4.22 (m, 2H), 4.11 (q,
.40-3.38 (m, 4H), 1.47 (s, 6H), 1.45 (s, 3H), 1.43 (s, 3H), 1.36 (s, 6H), 1.33 (s, 3H), 1.30-1.26
J
= 4.2 Hz, 1H), 5.18 (d,
J
= 12.1 Hz, 1H), 5.11 (d,
J
= 12.1 Hz, 1H), 4.78
(
J
= 7.0 Hz, 1H),
3
1
3
(
(
m, 6H); C NMR (100.6 MHz, CDCl ) δ: 172.5, 167.5, 160.0, 136.0, 128.9, 128.6, 128.4, 123.7
3
q,
J
= 282.2 Hz), 123.2 (q,
J
= 282.2 Hz), 110.0, 109.6, 109.4, 109.0, 96.6, 71.8, 71.3, 71.2, 70.9,
= 26.5 Hz), 41.6, 26.4, 26.3, 26.2, 26.0, 25.3, 25.2,
4.9, 24.6, 17.9; ESI (m/z) 936.3 [M +Na, (100)]; Anal. calcd. for C H F N O : C 52.57, H
7
2
5
7
7
0.7, 67.2, 66.4, 65.4, 48.8, 47.4 (septet,
J
+
40
53
6
3
14
.85, N 4.60; found: C 52.58, H 5.82, N 4.62. Minor diastereoisomer: Rf = 0.30 (hexane/ AcOEt
-
1 1
0:30); FTIR (neat)
.38-7.35 (m, 5H), 6.34 (br s, 1H), 5.49 (d,
12.1 Hz, 1H), 5.11 (d, = 12.1 Hz, 1H), 4.56-4.54 (m, 2H), 5.45 (septet, J
ν
1770, 1761, 1721 cm ; H NMR (400 MHz, CDCl
= 5.08 Hz, 1H), 5.47 (d, = 5.03 Hz, 1H), 5.18 (d,
= 7.0 Hz, 1H), 4.30-
3
), δ: 7.65 (br s, 1H),
J
J
J
=
J
4
.26 (m, 2H), 4.22-4.19 (m, 2H), 3.97-3.94 (m, 1H), 3.85-3.83 (m, 1H), 3.55-3.54 (m, 1H), 3.23-
.21 (m, 2H), 1.44 (s, 6H), 1.42 (s, 3H), 1.36 (s, 6H), 1.33 (s, 3H), 1.13 (s, 6H), 1.29 (s, 3H), 1.20
3
13
(
s, 3H); C NMR (100.6 MHz, CDCl ) δ: 171.9, 168.0, 135.9, 128.9, 128.7, 128.5, 123.5 (q, J =
3
2
81.0 Hz), 123.3 (q,
J
= 282.2 Hz), 109.8, 109.6, 109.5, 108.9, 96.7, 96.6, 96.5, 72.2, 71.9, 71.3,
= 26.7 Hz), 41.5, 26.4, 26.3, 26.1, 25.3, 24.8,
70.94, 70.91, 69.2, 67.3, 67.2, 49.4, 46.6 (septet,
J
1
5

ACCEPTED MANUSCRIPT
+
2
4.5; ESI (m/z) 936.2 [M +Na, (100)]; Anal. calcd. for C40
53 6 3
H F N O14: C 52.57, H 5.85, N 4.60;
found: C 52.59, H 5.86, N 4.58.
4.5 Synthesis of trivalent glycomimetics 4: general procedure. To a solution of glycosylazide
5
(1 equiv.) in CH CN (0.1 M) a solution of glycosylisothiocyanante 15 (1.05 equiv.) in a
3
minimum amount of CH CN followed by Ph P (1.05 equiv.) were added at rt. The solution was
3
3
stirred until complete formation of the corresponding carbodiimide 16 was achieved (TLC
monitoring). The temperature was lowered to 0 °C and TMP (2 equiv.), a solution of
glycosylamine
9 (1 equiv.) followed by a solution of 4,4,4-trifluoro-3-Tfm-crotonic acid 8 (1
equiv.) in a minimum amount of CH CN were added. The temperature was slowly left to reach rt
3
and the reaction, when finished (TLC monitoring, ca. 3 h), was quenched with a 1M solution of
HCl. The mixture was extracted with AcOEt, the organic phases collected, dried over Na SO ,
2
4
filtered, the solvent removed under reduced pressure and the crude purified by flash
chromatography.
4
f
a: Mixture of two diastereoisomers: R = 0.21 (hexane/ AcOEt 20:80); FTIR (neat) ν 1778, 1755,
-
1 1
1
1
1
3
721 cm ; H NMR (400 MHz, CDCl ), major diastereoisomer δ: 6.01 (br s, 1H), 5.46 (d, J = 5.1 Hz,
3
H), 5.14 (s, 1H), 4.97 (s, 1H), 4.62-4.54 (m, 6H), 4.18-4.15 (m, 3H), 4.14-4.11 (m, 2H), 3.91-3.90 (m,
H), 3.84 (d, J = 9.2 Hz, 1H), 3.78-3.76 (m, 1H), 3.53-3.50 (m, 2H), 3.42 (s, 3H), 3.37 (s, 3H), 3.25-
.22 (m, 2H), 3.08-3.05 (m, 1H), 1.48 (s, 3H), 1.45 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H), 1.30
(s, 3H), 1.29 (s, 3H), 1.28 (s, 3H); minor diastereoisomer δ: 6.38 (br s, 1H), 5.49 (d, J = 5.2 Hz, 1H),
5
3
2
1
2
.14 (s, 1H), 4.96 (s, 1H), 4.62-4.54 (m, 6H), 4.18-4.15 (m, 3H), 4.14-4.11 (m, 2H), 3.91-3.90 (m, 1H),
.84 (d, J = 9.2 Hz, 1H), 3.78-3.76 (m, 1H), 3.53-3.50 (m, 2H), 3.48 (s, 3H), 3.41 (s, 3H), 3.25-3.22 (m,
H), 3.08-3.05 (m, 1H), 1.47 (s, 3H), 1.44 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H), 1.30 (s, 3H),
1
3
.29 (s, 3H), 1.28 (s, 3H); C NMR (100.6 MHz, CDCl
3
) δ: 168.1, 167.6, 158.2, 156.0, 123.2 (q, J =
80.7 Hz), 123.0 (q, J = 281.4), 113.1, 112.8, 112.4, 112.3, 111.4, 110.3, 110.2, 110.0, 109.4, 109.3,
1
6

ACCEPTED MANUSCRIPT
1
6
4
08.6, 108.5, 96.3, 96.2, 86.5, 86.2, 85.6, 85.4, 85.3, 84.5, 82.0, 81.9, 81.7, 71.5, 71.4, 70.9, 70.5, 66.8,
5.6, 60.3, 57.9, 56.8, 55.8, 55.2, 55.1, 53.8, 51.4, 47.0, 46.7, 46.5, 46.3, 46.0, 45.8, 44.2, 43.7, 40.6,
0.3, 26.5, 26.4, 26.35, 26.31, 25.9, 25.8, 25.7, 25.0, 24.9, 24.8, 24.7, 24.6, 24.4, 24.3; ESI (m/z) 904.7
+
[
M +Na, (100)]; Anal. calcd. for C H F N O : C 49.03, H 6.06, N 4.77; found: C 49.00, H 6.08, N
3
6
53
6
3
15
4
.76.
Acknowledgments. Politecnico di Milano and ICRM-CNR are gratefully acknowledged for economic
support.
Supporting Information Available: characterization of compounds 1b-f, 2b, 3b,c, 4b, 13 and 19.
1
13
Copies of H, C NMR, and ESI-MS spectra of all new compounds.
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1
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1
2 Bellucci, M. C.; Sani, M.; Sganappa, A.; Volonterio, A. ACS Comb. Chem. 2014
3 a) Hsieh, K.-h.; Needleman, P.; Marshall, G. R. J. Med. Chem. 1987 30, 1097; b) Eberle, M.
K.; Keese, R.; Stoekli-Evans, H. Helv. Chim. Acta 1998 81, 182; c) Eberle, M. K.; Keese, R.
Helv. Chim. Acta. 2010 93, 1583.
4 For a DFT study on the mechanism of this process, see: Marcelli, T.; Olimpieri, F.;
Volonterio, A. Org. Biomol. Chem. 2011 , 5156.
, 16, 711.
1
,
,
,
1
1
1
1
,
9
5 The chiral aminosugar 9a steps in the reaction once the new stereogenic center at the hfVal
moiety is already formed.
6 Samantaray, S.; Marathe, U.; Dasgupta, S.; Nandicoori, V. K.; Roy, R. P.; J. Am. Chem. Soc.
2
008
7 In these cases we did not obtained an equimolar ratio of diastereoisomers because the
intramolecular aza-Michael nucleophilic attack arose from the chiral -sugar moiety of the
, 130, 2132.
N
carbodiimides. However, due to the distance between the reactive electronpoor C=C form
the stereogenic centers of the sugars, the diastereoselections are low, form ca. 3:1 in the case
of galactose to ca. 1.5:1 in the case of the other sugars. Since the mechanism and the
stereochimical behaviour of this process is related to that described in a previous paper (see
ref. 11a), the stereochemistry of the new formed stereocenter of the major diastereoisomer
was assessed to be (
conjugate incorporating hexafluorovaline which was determined by X-ray diffraction
8 a) Tor, Y.; Hermann, T.; Westholf, E. Chem. Biol. 1998 , R277; b) Tor, Y. ChemBioChem
003 , 998; c) Houghton, J. L.; Green, K. D.; Chen, W.; Garneau-Tsodikova, S.
R) on the basis of the structure of the corresponding peptide-sugar
1
, 5
2
, 4
1
9

ACCEPTED MANUSCRIPT
ChemBioChem. 2010
RNA 2011 , 209.
9 a) Luedtke, N. W.; Liu, Q.; Tor, Y. Biochemistry 2003
Belakhov, V.; Yaron, S.; Baasov, T.; Org. Lett. 2003 , 3575; c) Blount, K. F.; Tor, Y.
ChemBioChem. 2006 , 1612; d) Jeon, J.; Lee, K. H.; Rao, J. Chem. Commun. 2012 48
0034; e) Kumar, S.; Xue, L.; Arya, D. P. J. Am. Chem. Soc. 2010 133, 7361.
0 Lombès, T.; Moumné, R.; Larue, V.; Prost, E.; Catala, M.; Lecourt, T.; Dardel, F.; Micouin,
L.; Tisné, C. Angew. Chem. Int. Ed. 2012 51, 9530.
1 a) Park, W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem. Soc. 1996
b) Nunns, C. L.; Spence, L. A.; Slater, M. J.; Berrisford, D. J. Tetrahedron Lett. 1999
341.
,
11, 880; d) McCoy, L. S.; Xie, Y.; Tor, Y. Wiley Interdiscip. Rev.
,
2
1
, 42, 11391; b) Fridman, M.;
,
5
,
7
,
,
1
,
2
2
,
,
118, 10150;
40
,
,
9
3
22 Neomycin derivative 18 is very soluble in CH CN at 0°C, thus the protection of the
hydroxyls was not necessary to obtain high yield.
23 Garcia-Fernandez, J. M.; Ortiz Mellet, C.; Fuentes, J. J. Org. Chem. 1993, 58, 5192.
2
0

ACCEPTED MANUSCRIPT
Supporting Information
Multicomponent Diversity Oriented Synthesis of
Multivalent Glycomimetics Containing
Hexafluorovaline
Maria Cristina Bellucci, Aurora Sganappa, Monica Sani, and Alessandro Volonterio,*
Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, via
Celoria 2, 20133 Milano, Italy; C.N.R. Istituto di Chimica del Riconoscimento Molecolare, via
Mancinelli 7, 20131 Milano, Italy; Department of Chemistry, Materials, and Chemical Engineer
“
G. Natta”, Politecnico di Milano, via Mancinelli 7, 20131 Milano, Italy
alessandro.volonterio@polimi.it
Table of Contents:
Pages S2-S15
Characterization of all new compounds.
1
13
Pages S15-S91 H NMR, C NMR, and MS spectra of all new compounds.

ACCEPTED MANUSCRIPT
1
1
3: Mixture of two diastereoisomers: R = 0.38 (hexane/ AcOEt 60:40); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 8.25 (br s, 1H), 5.48 (t, J = 4.8 Hz, 1H), 4.67 (m, 2H), 4.57 (d, J
3
=
2.0 Hz, 1H), 4.36 (m, 1H), 4.27 (m, 1H), 4.13 (m, 1H),3.97 (m, 1H), 3.82 (m, 2H), 3.57 (m, 2H),
3
4
.35 (m, 1H), 1.44 (m, 6H), 1.28 (m, 18H); minor diastereoisomer δ: 8.25 (br s, 1H), 5.48 (t, J =
.8 Hz, 1H), 4.67 (m, 2H), 4.55 (d, J = 2.4 Hz, 1H), 4.36 (m, 1H), 4.27 (m, 1H), 4.13 (m, 1H),3.97
1
3
(m, 1H), 3.82 (m, 2H), 3.57 (m, 2H), 3.23 (m, 1H), 1.44 (m, 6H), 1.28 (m, 18H); C NMR (100.6
MHz, CDCl ) δ: 169.3, 158.1, 123.0 (q, J = 281.2 Hz, CF ), 122.8 (q, J = 284.1 Hz, CF ), 109.3,
3
3
3
1
4
08.4, 108.3, 96.3, 96. 2,71.2, 70.8, 70.6, 65.9, 65.8, 56.2, 55.9, 50.2, 50.0, 47.0 (s, J = 25.9 Hz ),
0.0, 39.8, 25.9, 25.8, 25.7, 24.8, 24.3, 23.0, 21.2, 20.7, 20.5 ; ESI (m/z) 616.3 [M +Na, (100)];
+
Anal. calcd. for C H F N O : C 48.56, H 6.28, N 7.08; found: C 48.58, H 6.30, N 7.09.
2
4
37
6
3
7
1
1
b: Mixture of two diastereoisomers: R = 0.27 (hexane/ AcOEt 60:40); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 8.44 (br s, 1H), 5.47 (s, 1H), 5.26-5.19 (m, 2H), 5.07-54.92 (m,
3
2H), 4.60-4.53 (m, 4H), 4.30 (m, 1H), 4.22 (m, 1H), 4.05-4.00 (m, 2H), 3.78 (m, 1H), 3.45 (m, 1H),
3.40 (s, 3H), 3.20 (dd, J = 16.0 and 3.6 Hz, 1H), 2.03-1.94 (four s, 12H), 1.47 (s, 3H), 1.31 (s, 3H),
1.30 (s, 9H); minor diastereoisomer δ: 8.42 (br s, 1H), 5.47 (s, 1H), 5.26-5.19 (m, 2H), 5.07-54.92
(
1
m, 2H), 4.60-4.53 (m, 4H), 4.30 (m, 1H), 4.22 (m, 1H), 4.05-4.00 (m, 2H), 3.78 (m, 1H), 3.45 (m,
H), 3.38 (s, 3H), 3.00 (m, 1H), 2.03-1.94 (four s, 12H), 1.53 (s, 3H), 1.31 (s, 3H), 1.28 (s, 9H); C
1
3
NMR (100.6 MHz, CDCl ) δ: 170.45, 170.41, 169.9, 169.8, 169.7, 169.6, 169.4, 168.3, 122.9 (q, J
3
=
8
(
279.7 Hz), 122.5 (q, J = 278.5 Hz), 113.4, 112.8, 110.7, 110.3, 87.0, 86.6, 85.2, 84.1, 84.0, 83.7,
2.0, 78.2, 73.8, 73.3, 73.2, 73.1, 70.5, 70.4, 68.3, 68.2, 61.8, 55.8, 55.3, 54.3, 51.6, 51.5, 47.0
septet, J = 26.1 Hz), 45.8 (septet, J = 28.2 Hz), 29.4, 28.9, 28.8, 26.5, 26.3, 24.9, 24.7, 20.5, 20.4,
+
2
5
0.3, 20.1, ; ESI (m/z) 862.6 [M +Na, (100)]; Anal. calcd. for C H F N O : C 47.20, H 5.64, N
.00; found: C 47.23, H 5.62, N 4.99.
3
3
47
6
3
15
1
1
c: Mixture of two diastereoisomers: R = 0.24 (hexane/ AcOEt 80:20); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 5.53 (d, J = 4.8 Hz, 1H), 4.96 (s, 1H), 4.60 (m, 4H), 4.32 (m,
3
1
2
H), 4.24 (m, 2H), 3.98 (m, 1H), 3.68 (m, 2H), 3.50 (m, 1H), 3.37 (s, 3H), 3.36 (m, 1H), 3.10 (m,
H), 1.46-1.45 (three s, 9H), 1.35-1.33 (three s, 9H), 1.32 (s, 9H); minor diastereoisomer δ: 5.52
(d, J = 4.6 Hz, 1H), 4.95 (s, 1H), 4.60 (m, 4H), 4.32 (m, 1H), 4.24 (m, 2H), 4.01 (m, 1H), 3.71 (m,
2
H), 3.50 (m, 1H), 3.37 (s, 3H), 3.38 (m, 1H), 3.10 (m, 2H), 1.46-1.45 (three s, 9H), 1.35-1.33
1
3
(three s, 9H), 1.29 (s, 9H); C NMR (100.6 MHz, CDCl ) δ: 168.6, 167.7, 162.6, 158.3, 156.2,
3
1
8
4
2
4
23.0 (q, J = 283.7 Hz), 112.35, 112.30, 109.8, 109.7, 109.6, 109.5, 109.4, 109.2, 96.3, 96.2, 96.1,
5.5, 85.4, 85.3, 85.2, 82.2, 71.6, 71.5, 70.8, 70.7, 70.6, 70.5, 70.4, 70.3, 68.6, 55.1, 51.25, 51.22,
6.4 (septet, J = 26.1 Hz), 42.7, 36.4, 29.4, 28.9, 28.8, 28.3, 26.4, 26.3, 26.0, 25.9, 25.8, 25.7, 25.6,
+
5.0, 24.8, 24.7, 24.3, 24.1; ESI (m/z) 774.6 [M +Na, (100)]; Anal. calcd. for C H F N O : C
3
1
47
6
3
11
9.53, H 6.30, N 5.59; found: C 49.55, H 6.31, N 5.60.
1
1
d: Mixture of two diastereoisomers: R = 0.58 (hexane/ AcOEt 50:50); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 7.27 (m, 6H), 6.27 (br s, 1H), 5.63 (d, J = 10.4 Hz, 1H), 5.32 (m,
3
1
H), 4.94 (m, 2H), 4.86 (m, 1H), 4.75 (s, 1H), 4.56-4.28 (m, 6H), 4.19 (m, 1H), 3.52 (m, 2H), 3.47
(s, 3H), 3.21 (m, 2H), 1.84 (s, 3H), 1.46 (s, 3H), 1.35-1.12 (three s, 30H); minor diastereoisomer δ:
7
4
3
1
.27 (m, 6H), 5.98 (br s, 1H), 5.60 (d, J = 10.4 Hz, 1H), 5.32 (m, 1H), 4.94 (m, 2H), 4.86 (m, 1H),
.78 (s, 1H), 4.56-4.28 (m, 6H), 4.08 (m, 1H), 3.52 (m, 2H), 3.32 (s, 3H), 3.21 (m, 2H), 1.83 (s,
1
3
H), 1.42 (s, 3H), 1.35-1.12 (three s, 30H); C NMR (100.6 MHz, CDCl ) δ: 178.6, 178.5, 177.0,
3
76.9, 169.7, 167.6, 167.3, 162.5, 158.7, 135.9, 128.8, 128.7, 128.6, 128.5, 128.2, 128.0, 127.9,

ACCEPTED MANUSCRIPT
1
8
5
2
9
6
23.0 (q, J = 284.8 Hz), 122.7 (q, J = 285.7 Hz), 112.4, 109.9, 109.8, 96.4, 95.9, 95.8, 85.7, 85.5,
5.4, 85.3, 82.1, 82.0, 70.8, 70.4, 70.3, 70.2, 70.0, 69.9, 69.8, 69.7, 69.6, 69.4, 68.9, 68.5, 55.3,
5.1, 51.8, 51.7, 51.1, 50.9, 47.7 (septet, J = 25.1 Hz), 43.5, 42.7, 42.5, 38.9, 38.8, 36.4, 31.4, 29.6,
9.3, 29.1, 29.0, 28.6, 28.4, 28.3, 27.0, 26.9, 26.5, 26.4, 26.3, 24.8, 23.0, 22.9, 22.8; ESI (m/z)
+
93.8 [M +Na, (100)]; Anal. calcd. for C H F N O : C 54.43, H 6.64, N 5.77; found: C 54.45, H
4
4
64
6
4
13
.65, N 5.80.
1
1
e: Mixture of two diastereoisomers: R = 0.37 (hexane/ AcOEt 50:50); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 7.32 (br s, 1H), 5.28 (m, 2H), 5.22 (m, 1H), 4.97 (d, J = 2.8 Hz,
3
1
3
5
H), 4.80 (d, J = 2.0 Hz, 1H)4.36 (m, 1H), 4.27 (m, 1H), 4.13 (m, 1H),3.97 (m, 1H), 3.82 (m, 2H),
.57 (m, 2H), 3.35 (m, 1H), 1.44 (m, 6H), 1.28 (m, 18H); minor diastereoisomer δ: 8.25 (br s, 1H),
.48 (t, J = 4.8 Hz, 1H), 4.67 (m, 2H), 4.55 (d, J = 2.4 Hz, 1H), 4.36 (m, 1H), 4.27 (m, 1H), 4.13
1
3
(m, 1H),3.97 (m, 1H), 3.82 (m, 2H), 3.57 (m, 2H), 3.23 (m, 1H), 1.44 (m, 6H), 1.28 (m, 18H); C
NMR (100.6 MHz, CDCl ) δ: 169.3, 158.1, 123.0 (q, J = 281.2 Hz, CF ), 122.8 (q, J = 284.1 Hz,
3
3
CF ), 109.3, 108.4, 108.3, 96.3, 96. 2,71.2, 70.8, 70.6, 65.9, 65.8, 56.2, 55.9, 50.2, 50.0, 47.0 (s, J =
3
+
2
5.9 Hz ), 40.0, 39.8, 25.9, 25.8, 25.7, 24.8, 24.3, 23.0, 21.2, 20.7, 20.5 ; ESI (m/z) 616.3 [M +Na,
(
100)]; Anal. calcd. for C H F N O : C 48.16, H 5.95, N 4.68; found: C 48.17, H 5.97, N 4.67.
36 53 6 3 16
1
1
f: Major diastereoisomer: R = 0.45 (hexane/ AcOEt 50:50); H NMR (400 MHz, CDCl ), δ: 7.80
f 3
(s, 1H), 6.25 (br s, 1H), 5.83 (d, J = 9.6 Hz, 1H), 5.42 (t, J = 9.6 Hz, 1H), 5.37 (t, J = 9.6 Hz, 1H),
5
1
3
.26 (t, J = 9.6 Hz, 1H), 4.93 (s, 1H), 4.49 (m, 2H), 4.42 (br s, 1H), 4.37 (dd, J = 12.2 and 4.4 Hz,
H), 4.25 (m, 1H), 4.21 (m, 2H), 4.00 (m, 2H), 3.95 (m, 1H), 3.34 (s, 3H), 3.22 (m, 1H), 2.11 (s,
1
3
H), 2.08 (s, 3H), 2.05 (s, 3H), 1.89 (s, 3H), 1.60 (s, 3H), 1.45 (s, 3H), 1.33 (s, 9H); C NMR
(
100.6 MHz, CDCl ) δ: 170.3, 169.9, 169.2, 168.7, 167.9, 155.9, 144.8, 122.8 (q, J = 282.1 Hz),
3
1
21.6, 112.4, 109.9, 86.1, 85.9, 85.2, 85.1, 81.8, 75.4, 72.5, 70.9, 67.8, 61.2, 60.3, 55.2, 51.5, 46.6
(
[
septet, J = 27.2 Hz), 42.5, 29.6, 28.9, 28.3, 26.4, 24.8, 20.9, 20.4, 20.0, 14.2; ESI (m/z) 943.7
+
M +Na, (100)]; Anal. calcd. for C H F N O : C 46.96, H 5.47, N 9.13; found: C 46.98, H 5.48,
3
6
50
6
6
15
N 9.11.
1
Minor diastereoisomer: R = 0.38 (hexane/ AcOEt 50:50); H NMR (400 MHz, CDCl ), δ: 7.71 (s,
f
3
1
H), 5.97 (br s, 1H), 5.81 (d, J = 9.2 Hz, 1H), 5.43 (t, J = 9.2 Hz, 1H), 5.35 (t, J = 9.2 Hz, 1H), 5.25
(t, J = 9.2 Hz, 1H), 4.97 (s, 1H), 4.57 (d, J = 6.0 Hz, 1H), 4.53 (d, J = 6.0 Hz, 1H), 4.49 (br s, 1H),
4
1
1
1
8
2
5
.39 (d, J = 13.2 Hz, 1H), 4.32 (dd, J = 12.8 and 5.2 Hz, 1H), 4.21 (m, 1H), 4.18 (d, J = 13.2 Hz,
H), 4.00 (m, 2H), 3.67 (m, 1H), 3.41 (s, 3H), 3.23 (m, 1H), 2.11 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H),
1
3
.86 (s, 3H), 1.68 (s, 3H), 1.47 (s, 3H), 1.30 (s, 9H); C NMR (100.6 MHz, CDCl ) δ: 170.4, 169.9,
3
69.3, 168.5, 167.9, 155.9, 144.5, 123.0 (q, J = 282.7 Hz), 121.2, 112.4, 109.1, 86.0, 85.6, 85.5,
2.0, 75.4, 75.3, 72.5, 70.4, 67.6, 61.4, 55.5, 51.5, 46.7 (septet, J = 26.2 Hz), 42.3, 29.7, 28.9, 26.3,
+
4.7, 20.6, 20.5, 20.0; ESI (m/z) 943.7 [M +Na, (100)]; Anal. calcd. for C H F N O : C 46.96, H
3
6
50
6
6
15
.47, N 9.13; found: C 46.96, H 5.49, N 9.14.
1
2
b: Mixture of two diastereoisomers: R = 0.24 (hexane/ AcOEt 80:20); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 5.41 (d, J = 5.2 Hz, 1H), 5.38 (d, J = 4.8 Hz, 1H), 4.62 (s, 1H),
3
4
.56 (m, 2H), 4.24 (m, 2H), 4.14 (m, 4H), 3.82 (m, 2H), 3.46 (m, 1H), 3.21 (dd, J = 14.8 and 5.2
Hz, 1H), 1.44-1.37 (four s, 12H), 1.30-1.24 (four s, 12H); minor diastereoisomer δ: 5.46 (d, J = 5.2
Hz, 1H), 5.38 (d, J = 4.8 Hz, 1H), 4.91 (s, 1H), 4.56 (m, 2H), 4.24 (m, 2H), 4.14 (m, 4H), 3.82 (m,
1
3
2
H), 3.45 (m, 2H), 1.44-1.37 (four s, 12H), 1.30-1.24 (four s, 12H); C NMR (100.6 MHz, CDCl )
3

ACCEPTED MANUSCRIPT
δ: 169.7, 169.6, 169.5, 157.2, 156.8, 122.8 (q, J = 281.5 Hz), 122.3 (q, J = 280.8 Hz), 109.9, 109.8,
1
7
4
2
09.7, 109.5, 109.0, 108.7, 108.7, 108.5, 96.3, 96.23, 96.20, 96.1, 71.5, 71.4, 71.3, 71.1, 70.9, 70.8,
0.7, 70.5, 70.44, 70.40, 70.3, 66.9, 64.6, 64.3, 63.6, 57.2, 57.1, 55.4, 49.2 (septet, J = 28.5 Hz),
8.9 (septet, J = 27.9 Hz), 42.2, 42.1, 42.0, 40.2, 39.9, 29.6, 26.0, 25.9, 25.8, 25.76, 25.70, 25.05,
+
5.03, 24.7, 24.6, 24.55, 24.51, 24.3 ; ESI (m/z) 757.1 [M +Na, (100)]; Anal. calcd. for
C H F N O : C 49.05, H 5.49, N 3.81; found: C 49.02, H 5.50, N 3.79.
30
40
6
2
12
1
3
b: Mixture of two diastereoisomers: R = 0.35 (hexane/ AcOEt 60:40); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 8.21 (br s, 1H), 7.34 (m, 5H), 6.60 (br s, 1H), 5.16 (d, J = 12.0
3
Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H), 5.00 (s, 1H), 4.97 (s, 1H), 4.62-4.57 (m, 7H), 4.28 (t, J = 6.0 Hz,
1H), 4.00 (m, 1H), 3.48 (m, 1H), 3.44 (s, 3H), 3.40 (s, 3H), 3.33 (m, 1H), 1.63 (m, 3H), 1.47 (s,
3H), 1.46 (s, 3H), 1.30 (s, 3H), 1.29 (s, 3H), 0.91 (m, 6H); minor diastereoisomer δ: 7.52 (br s,
1H), 7.34 (m, 5H), 6.16 (t, J = 5.6 Hz, 1H), 5.17 (d, J = 12.0 Hz, 1H), 5.11 (d, J = 12.0 Hz, 1H),
5.04 (s, 1H), 4.97 (s, 1H), 4.62-4.57 (m, 7H), 4.34 (dd, J = 8.4 and 6.0 Hz, 1H), 4.00 (m, 1H), 3.48
(
3
1
m, 1H), 3.41 (s, 3H), 3.40 (s, 3H), 3.06 (m, 1H), 1.63 (m, 3H), 1.47 (s, 3H), 1.46 (s, 3H), 1.33 (s,
1
3
H), 1.31 (s, 3H), 0.91 (m, 6H); C NMR (100.6 MHz, CDCl ) δ: 171.9, 171.6, 167.8, 167.3,
3
58.3, 156.2, 135.6, 135.5, 128.5, 128.4, 128.3, 128.28, 128.21, 128.1, 128.0, 122.7 (q, J = 289.2
Hz), 122.4 (q, J = 282.6 Hz), 113.1, 112.9, 112.3, 112.2, 111.4, 110.4, 110.3, 110.2, 87.0, 86.3,
6.2, 85.6, 85.4, 84.6, 84.4, 82.3, 82.0, 66.9, 66.8, 56.0, 55.8, 55.3, 55.2, 51.5, 51.4, 46.6 (septet, J
26.8 Hz), 46.0 (septet, J = 26.5 Hz), 44.0, 43.8, 41.2, 40.8, 26.5, 26.4, 26.3, 24.9, 24.8, 24.7, 24.6,
8
=
+
2
2.7, 21.7, 21.6; ESI (m/z) 866.1 [M +Na, (100)]; Anal. calcd. for C H F N O : C 52.67, H 6.09,
37 51 6 3 12
N 4.98; found: C 52.68, H 6.10, N 4.96.
1
3
c: Major diastereoisomer: R = 0.34 (hexane/ AcOEt 40:60); H NMR (500 MHz, CDCl ), δ: 7.58
f 3
(
4
(
3
br s, 1H), 7.37-7.22 (m, 10H), 6.12 (br s, 1H), 5.18 (d, J = 12.5 Hz, 1H), 5.14 (d, J = 12.5 Hz, 1H),
.97 (s, 1H), 4.86 (m, 1H), 4.64-4.47 (m, 8H), 4.28 (m, 1H), 3.75-3.68 (m, 2H), 3.44 (s, 3H), 3.41
m, 2H), 2.99 (m, 2H), 2.96 (m, 3H), 1.71 (m, 1H), 1.57 (m, 2H), 1.49 (s, 3H), 1.45 (s, 3H), 1.31 (s,
1
3
H), 1.26 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H); C NMR (100.6 MHz, CDCl )
3
δ: 171.8, 170.6, 167.9, 137.0, 135.8, 128.7, 128.5, 128.4, 128.1, 128.0, 126.6, 123.6 (q, J = 280.7
Hz), 123.2 (q, J = 281.7 Hz), 113.1, 112.4, 110.9, 110.8, 86.3, 85.8, 84.1, 81.9, 81.3, 66.7, 58.8,
5
2
5
5.8, 55.5, 54.2, 53.4, 51.4, 46.1 (septet, J = 26.2 Hz), 43.4, 40.8, 37.0, 29.6, 26.4, 26.2, 24.8, 24.4,
+
2.6, 22.1; ESI (m/z) 1013.4 [M +Na, (100)]; Anal. calcd. for C H F N O : C 55.75, H 6.10, N
4
6
60
6
4
13
.65; found: C 55.76, H 6.09, N 5.67.
1
Minor diastereoisomer: R = 0.28 (hexane/ AcOEt 40:60); H NMR (400 MHz, CDCl ), δ: 7.30-
f
3
7
.17 (m, 12H), 6.52 (br s, 1H), 6.02 (br s, 1H), 5.08 (s, 2H), 4.93 (s, 1H), 4.91 (s, 1H), 4.58-4.49
(m, 6H), 4.34 (q, J = 7.2 Hz, 1H), 4.23 (t, J = 6.8 Hz, 1H), 4.17 (m, 1H), 3.42 (m, 1H), 3.34 (s, 3H),
3
3
1
1
.33 (s, 3H), 3.20-3.14 (m, 4H), 1.65-1.52 (m, 5H), 1.43 (s, 3H), 1.42 (s, 3H), 1.27 (s, 3H), 1.26 (s,
13
H), 0.86 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); C NMR (100.6 MHz, CDCl ) δ: 172.3,
3
69.8, 167.3, 158.1, 137.3, 135.6, 129.1, 128.5, 128.4, 128.2, 128.0, 126.7, 123.2 (q, J = 284.7 Hz),
13.4, 112.4, 110.5, 110.1, 86.2, 85.4, 84.5, 81.9, 81.8, 66.7, 56.1, 56.0, 55.1, 51.1, 46.7 (q, J = 27.2
Hz), 44.0, 41.1, 36.3, 29.6, 26.5, 26.4, 24.9, 24.8, 24.7, 24.6, 22.7, 21.8, ; ESI (m/z) 1013.4
+
[
M +Na, (100)]; Anal. calcd. for C H F N O : C 55.75, H 6.10, N 5.65; found: C 55.75, H 6.11,
46 60 6 4 13
N 5.66.

ACCEPTED MANUSCRIPT
1
4
b: Mixture of two diastereoisomers: R = 0.18 (hexane/ AcOEt 20:80); H NMR (400 MHz,
f
CDCl ), major diastereoisomer δ: 5.58 (d, J = 5.2 Hz, 1H), 5.54 (d, J = 4.8 Hz, 1H), 5.30 (s, 1H),
3
5
.28 (s, 1H), 5.23 (m, 1H), 4.80 (d, J = 2.0 Hz, 1H), 4.59 (m, 2H), 4.32 (m, 1H), 4.29 (m, 2H), 4.26
(m, 1H), 4.11 (m, 2H), 3.98 (m, 2H), 3.84 (m, 2H), 3.74 (m, 1H), 3.49 (m, 3H), 3.21 (m, 3H), 2.16
(
3
s, 3H), 2.11 (s, 3H), 2.41 (s, 3H), 1.99 (s, 3H), 1.84 (m, 2H), 1.46 (s, 3H), 1.45 (s, 9H), 1.35 (s,
H), 1.34 (s, 6H), 1.27 (s, 3H); C NMR (100.6 MHz, CDCl ) δ: 170.6, 169.9, 169.7, 168.2, 122.9
3
1
3
(q, J = 280.7 Hz), 109.5, 109.3, 108.5, 97.5, 96.3, 96.1, 71.5, 70.9, 70.7, 70.5, 70.4, 69.6, 69.1, 68.5,
6
2
4
6.7, 66.2, 65.9, 62.5, 46.3, 46.1, 45.8, 37.2, 29.3, 28.9, 26.0, 25.9, 25.8, 25.0, 24.96, 24.89, 24.3,
+
4.1, 20.8, 20.7, 20.6, 20.5; ESI (m/z) 1123.1 [M +Na, (100)]; Anal. calcd. for C H F N O : C
4
7
67
6
3
22
9.52, H 5.92, N 3.69; found: C 49.50, H 5.94, N 3.70.
1
1
9: Mixture of two diastereoisomers: R = 0.23 (CHCl /CH OH 95:5); H NMR (400 MHz,
f
3
3
CD OD), major diastereoisomer δ: 6.36 (br s, 1H), 6.34 (br s, 1H), 5.59 (s, 1H), 5.26 (br s, 1H),
3
5
.16 (d, J = 2.8 Hz, 1H), 4.92 (s, 1H), 4.39 (m, 1H), 4.31 (m, 1H), 4.19 (m, 3H), 4.09 (m, 2H), 3.89
(m, 3H), 3.76 (m, 2H), 3.69 (m, 1H), 3.47-3.17 (m, 15H), 2.89 (m, 2H), 2.68 (m, 2H), 1.97 (m, 1H),
1
5
4
2
1
9
7
.47-1.28 (eleven s, 102H); minor diastereoisomer δ: 6.36 (br s, 1H), 6.34 (br s, 1H), 5.58 (s, 1H),
.26 (br s, 1H), 5.14 (d, J = 2.6 Hz, 1H), 4.92 (s, 1H), 4.39 (m, 1H), 4.31 (m, 1H), 4.19 (m, 3H),
.09 (m, 2H), 3.89 (m, 3H), 3.76 (m, 2H), 3.69 (m, 1H), 3.47-3.17 (m, 15H), 2.89 (m, 2H), 2.68 (m,
1
3
H), 1.97 (m, 1H), 1.47-1.28 (eleven s, 102H); C NMR (100.6 MHz, CDCl ) δ: 168.3, 168.0,
3
57.5, 157.1, 156.8, 156.3, 122.7 (q, J = 282.3 Hz), 122.3 (q, J = 282.7Hz), 109.3, 109.2, 109.0,
9.3, 98.0, 96.5, 96.4, 96.2, 85.7, 81.2, 80.1, 79.3, 79.0, 78.0, 74.3, 74.1, 74.0, 73.2, 72.1, 72.0,
1.6, 71.2, 70.8, 70.7, 70.5, 70.4, 70.2, 67.5, 66.1, 55.6, 52.9, 51.0, 50.9, 50.8, 50.4, 45.9 (septet, J
=
27.2 Hz), 41.4, 40.3, 39.6, 34.4, 34.1, 30.9, 30.8, 28.2, 28.0, 27.7, 27.5, 27.4, 27.3, 25.6, 25.2,
+
2
5.1, 24.9, 24.8, 24.0, 23.9, 23.2; ESI (m/z) 1846.0 [M +Na, (71)]; Anal. calcd. for
C H F N O S: C 50.73, H 7.13, N 6.92; found: C 50.70, H 7.15, N 6.89.
77
129
6
9
31

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F3C CF
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major diast.
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.0
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