Structure-based design, synthesis and antitumoral evaluation of enulosides

Article abstract of DOI:10.1016/j.ejmech.2017.01.036

Enulosides, carbohydrate derivatives containing an α,β-unsaturated carbonyl unit, were designed and obtained in high yields and isomeric purity. All synthesized compounds exhibited antitumoral activity in micromolar range against four tested tumor cells lines, being the best results observed for HL-60?cells. These compounds open new possibilities to prepare an array of more active, site-specific or selective antitumor agents. 2016 Elsevier Ltd. All rights reserved.

Full text of DOI:10.1016/j.ejmech.2017.01.036

European Journal of Medicinal Chemistry 128 (2017) 192e201
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-based design, synthesis and antitumoral evaluation of
enulosides
,
,
,
Jonh A.M. Santos a b, Cosme S. Santos a b, Claudia L.A. Almeida a b, Thiago D.S. Silva c,
a
c
d
b
~
~
Joao R. Freitas Filho , Gardenia C.G. Militao , Teresinha G. da Silva , Carlos H.B. da Cruz ,
Juliano C.R. Freitas a **, Paulo H. Menezes b
,
e
,
, *
a Departamento de Química, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n, 52171-900, Recife, PE, Brazil
b Departamento de Química, Universidade Federal de Pernambuco, Av. Jornalista Anibal Fernandes s/n, 50670-901, Recife, PE, Brazil
c Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901, Recife, PE, Brazil
d
ꢀ
Departamento de Antibioticos, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901, Recife, PE, Brazil
Centro de Educaçao e Saúde, Universidade Federal de Campina Grande, Acesso Professora Maria Anita Furtado Coelho, s/n, 58175-000, Cuite, PB, Brazil
e
~
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Enulosides, carbohydrate derivatives containing an a,b-unsaturated carbonyl unit, were designed and
Received 5 November 2016
Received in revised form
13 January 2017
Accepted 21 January 2017
Available online 24 January 2017
obtained in high yields and isomeric purity. All synthesized compounds exhibited antitumoral activity in
micromolar range against four tested tumor cells lines, being the best results observed for HL-60 cells.
These compounds open new possibilities to prepare an array of more active, site-specific or selective
antitumor agents. 2016 Elsevier Ltd. All rights reserved.
© 2017 Elsevier Masson SAS. All rights reserved.
Keywords:
Pseudoglycosides
Enulosides
Antitumoral
CRM-1
1. Introduction
toxicity observed [7].
Despite of the presence of an a,b-unsaturated d-lactone motif in
Several natural products that have an
a
,
b
-unsaturated
d
-lactone
several natural products with biological activity, few reports of
naturally occurring enone-containing sugars (enulosides) are
described. Examples are Microthecin [8], and Ascopyrone P [9],
both exhibiting antibacterial activity (Fig. 2).
The general strategy for the synthesis of enulosides is based on
the use of glycals as starting materials [10] and they are versatile
building blocks that can be used in a variety of transformations [11].
One example is the synthesis of enantiopure 2,4-disubstituted 6-
as structural motif are known to exhibit various biological activities
[1e3]. Among these, leptomycin B, a natural product first isolated
from Streptomyces sp. ATS1287 [4] has a prominent position (Fig. 1).
The cellular target of leptomycin B, a nuclear export inhibitor,
has been identified as CRM1, an evolutionarily conserved receptor
for the nuclear export signal of proteins [5]. Leptomycin B and
parent compounds can act as a Michael-type acceptor for biological
nucleophiles, in special the sulfhydryl groups of cysteines. Specif-
ically, the region containing Cys-529 from CRM1 forms a pocket
into which leptomycin B is readily incorporated, just like an enzy-
meesubstrate interaction [6]. However, the clinical development of
leptomycin B was subsequently discontinued due to the significant
hydroxy-1,6-dihydro-2H-pyridin-3-ones
using
an
aza-
Achmatowicz type chemistry [12].
From the pharmacological point of view, some synthetic enu-
losides exhibited activity against Mycobacterium tuberculosis H37Rv
[13], and they can also act as glycosidase inhibitors [14]. Moreover,
they exhibited excellent in vitro activities against different Gram-
positive, Gram-negative as well as resistant bacterial strains and
fungi [15]. Synthetic enolosides containing hydrophobic chains also
exhibited antifungal and antibacterial activities and a preliminary
screening indicated that some of the synthesized compounds
exhibited low toxicity to eukaryotic cells [16].
* Corresponding author.
** Corresponding author. Departamento de Química, Universidade Federal Rural
de Pernambuco, Rua Dom Manoel de Medeiros s/n, 52171-900, Recife, PE, Brazil.
E-mail addresses: julianocrufino@yahoo.com.br (J.C.R. Freitas), paulo.menezes@
pq.cnpq.br (P.H. Menezes).
http://dx.doi.org/10.1016/j.ejmech.2017.01.036
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
193
Table 1
Docking interaction energy for model compounds 2e4 against CRM1.
Entry
1
Compound
DG (Kcal/mol)
6.78
6.75
7.60
6.80
6.85
7.72
7.93
Fig. 1. Leptomycin B.
2
3
4
5
6
7
Fig. 2. Examples of natural occurring enulosides.
Thus, the synthesis of simpler and less toxic derivatives that
could act as nuclear export inhibitors for further application as
anticancer drugs would be of the great interest. In this context, the
higher electrophilic character of enulosides compared to
a,b-un-
saturated -lactones and the lower toxicity to eukaryotic cells make
d
these compounds interesting synthetic targets.
2. Results and discussion
In this work, a simple and straightforward sequence for the
synthesis of enulosides and the evaluation of their antitumoral
activity are described. The general strategy for the synthesis of the
target compounds is outlined on Fig. 3. Starting from an a-pseu-
doglycoside, 2 the sequential removal of acetyl groups would lead
to 3, which by chemoselective oxidation would give the desired
compounds, 4 (Fig. 3).
8
9
7.64
5.64
2.1. Docking analysis
As Leptomycin B plays an essential role as major tumor sup-
pressor proteins (TSPs), the predicted binding energies for some of
the envisioned compounds into the CRM1 active site were calcu-
lated (Table 1).
The energy value observed for the starting pseuglycoside
was ꢀ6.78 kcal/mol (Table 1, entry 1) and the removal of acetyl
groups resulted in a discreet increment in the binding energy to
CRM1 (Table 1, entries 1 and 2). However, a decrease in the energy
value was observed for the corresponding enuloside (Table 1, entry
3).
The increment in the bulkiness on the side chain of enulosides
resulted in a higher energy values (Table 1, entries 4 and 5) and the
presence of an unsaturated side chain proved to have a small effect
in the binding energy (Table 1, entries 6 and 7). The same behavior
was observed by the replacement of an alkyl side chain by an ar-
omatic side chain (Table 1, entry 8). These results support the
hypothesis that the side chain plays an important role in the mo-
lecular recognition.
Recently, our group described the synthesis and antitumoral
activities of (ꢀ)-massoilactone and analogous compounds [17]. By
comparison, the energy value calculated for the structurally related
a
,
b
-unsaturated
energies calculated for all other compounds (Table 1, entry 9). As
cited earlier, -unsaturated -lactones can act as a Michael-type
d-lactone was much higher when compared to the
a
,b
d
acceptor for biological nucleophiles, and as enulosides are more
electrophilic when compared to the corresponding lactones, better
activities could be observed.
2.2. Synthesis
The synthesis of required a-pseudoglycosides 2 started from the
reaction of commercially available 3,4,6-tri-O-acetyl-D-glucal, 1
and different alcohols catalyzed by TsOH under sonication [18]. In
all cases, the corresponding pseudoglycosides 2 were obtained in
good yields and anomeric selectivities (Table 2). Compounds 2a-j
were then purified by chromatographic column to yield pure
a-
anomer and the stereochemistry was supported by a NOESY
experiment [19].
Fig. 3. General strategy for the synthesis of enulosides, 4.
Despite being a simple and known method, the hydrolysis of

194
J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
Table 2
acetyl groups of stereochemically pure a-pseudoglycosides using
Synthesis of pseudoglycosides catalyzed by TsOH and promoted by ultrasound.a,b
K2CO3 in MeOH was revisited in order to find the best condition for
the reaction [19]. In all cases, the reaction was complete after five
minutes and the corresponding diols 3a-j were obtained in good
yields without any observable epimerization (Table 3).
Manganese dioxide is a widely used standard oxidant for the
synthesis of aldehydes and ketones from allylic alcohols under mild
conditions [20]. The oxidation is usually carried out at room tem-
perature and work-up is very simple, involving just the filtration of
suspended solid and elimination of solvent. The use of MnO2 [21] or
DMSO/Ac2O [16] for the synthesis of some hydroxyl enulosides was
already described, however, the oxidation required longer reaction
times to completion. Thus, in order to improve the feasibility of the
oxidation reaction using MnO2, it was studied using 3a as substrate
to determine the optimum reaction conditions (Table 4). When 0.5
equivalent of MnO2 was used, lower yields were observed (Table 4,
entry 1). By increasing the amount of MnO2 from 2.0 to 5.0
equivalents the desired product was obtained in similar yields,
however, by increasing the amount of the oxidant, a decrease in the
reaction time was observed (Table 4, entries 2 and 3). The optimum
condition for the oxidation was observed using 6.5 equivalents of
MnO2 were the oxidized product 4a was obtained in 84% yield after
only 3 h (Table 4, entry 4). Further increment in the amount of
MnO2 to 8 equivalents showed no significant changes neither in the
yield nor in the reaction time (Table 4, entry 5).
Entry ROH
Time (min)
10
2
Yield(%)c
(a:b)
d
1
2
3
4
EtOH
90 (88:12)
91 (90:10)
83 (86:14)
95 (86:14)
2a
nPrOH
iPrOH
5
5
5
2b
2c
With the optimized reaction conditions, the oxidation was
extended to compounds 3b-j and the results are described on
Scheme 1. In all cases the desired compounds were obtained in
good yields without any observable isomerization. In addition, the
oxidation reaction seems not to be sensitive to the type of func-
tional group present in 3a-j since compounds containing alkyl,
alkenyl, alkynyl and benzyl groups were oxidized to give the cor-
responding enulosides in good yields.
2d
5
6
7
5
5
5
80 (89:11)
83 (88:12)
94 (88:12)
2.3. Antiproliferative activity
2e
2f
Compounds 2a, 3a and 4a were then submitted to a preliminary
screening at 25 mg/mL against NCI-H292 (human lung carcinoma),
MCF-7 (human breast adenocarcinoma), HL-60 (human acute
promyelocytic leukemia) and HEP-2 (human epidermoid cancer
cells) tumor cells lineages using the MTTassay [22] and the samples
with growth inhibition over 90% were used to determine the IC50
values (concentration that causes 50% growth inhibition). The re-
sults are depicted on Table 5.
Compound 4a showed an excellent percentage of inhibition and
this result can be attributed to the presence of the conjugated
double bond, since compounds 2a and 3a showed lower inhibition
(Table 5). These results, when compared to the obtained energy
values from the docking analysis are in accordance (Table 1). For
example, for compound 4a, a lower value of energy and a high
2g
8
8
84 (86:14)
92 (88:12)
82 (90:10)
2h
percentage of inhibition at 25
3 and Table 5, entry 3).
mg/mL were observed (Table 1, entry
9
10
20
Given these results, all synthesized enulosides 4a-j were eval-
uated against four tumor cell lines and the observed IC50 values are
summarized on Table 6. Doxorubicin (DOX) was employed as a
positive control. All compounds exhibited high antineoplastic ac-
tivity in micromolar scale against the tested tumor cells lines being
the best results observed for HL-60 tumor cells.
2i
10
Enulosides 4a-c, derived from simpler alcohols, showed good
IC50 values (Table 6, entries 1e3), being the best result observed for
compound 4a against HL-60 and HEP-2 tumor cell lines (Table 6,
entry 1). These results are in accordance to those obtained from the
docking analysis (Table 1). For example, the calculated energy
values for 4a and 4c (Table 1, entries 3 and 4) are in agreement with
the observed cytotoxicity (Table 6, entries 1 and 3), indicating that
2j

J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
195
a
Reaction conditions: Reactions were performed with 1 (1.36 g, 5.0 mmol), the
entry 4), albeit compound 4e and 4i, which have, respectively, a six-
membered and an aromatic ring on the side chain, displayed better
cytotoxicity against NCI-H292 tumor cell line (Table 6, entries 5 and
6). These results are not in agreement with the docking analysis,
while a lower energy value was calculated for compound 4i
(Table 1, entries 5 and 8).
appropriate alcohol (6.0 mmol) and TsOH (0.17 g, 20 mol%) in MeCN (20 mL) at 25 ꢁ
for the time indicated.
C
b
The numbers were used to assign the NMR chemical shifts (see Section 4).
c
Isolated yield.
d
The anomeric ratios were obtained by 1H NMR and confirmed by gas
chromatography.
Compound 4j was designed in an attempt to improve the water
solubility of the tested compounds, a major problem on the
development and screening of new chemotherapeutic drugs [24].
However, despite its high solubility in water, 4j displayed good
cytotoxicity only against HL-60 cells (Table 6, entry 10).
Table 3
Removal of acetyl groups from
a
-pseudoglycosides 2a-j.a,b
The inhibition of cell growth by leptomycin B was shown to
occur mainly due to the inhibitory effect of this compound on
CRM1-mediated processes [25]. The binding of leptomycin B to
CMR1 occurs in its conserved central region at a critical cysteine
residue and prevents the formation of a complex between CRM1
and the nuclear export signal of cargo proteins [26]. Recent findings
suggest that leptomycin B potently induces apoptosis and this in-
duction is related with cytochrome c release, activation of caspases,
and selective down-regulation of Mcl-1 and XIAP [27]. For Gonio-
Entry
2
3
Yield(%)c
1
2
3
4
5
6
7
8
9
10
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
2b
3c
3d
3e
3f
3g
3h
3i
95
92
96
90
92
95
98
96
94
91
thalamin [28], a simpler a,b-unsaturated d-lactone analogue, is also
known that this compound can induce apoptosis by activating
caspases [29,30] and through the loss of the mitochondrial mem-
brane potential [31].
Other a,b-unsaturated compounds such as codeinone induces
apoptosis five orders higher than that of codeine [32]. It is inter-
2j
3j
a
esting to note that the structural difference between codeinone and
Reaction conditions: Reactions were performed with 2a-j (3 mmol) and K2CO3
(6 mmol) using MeOH (12 mL) and H2O (3.6 mL) at 25 ꢁC for 5 min.
codeine resides only in the
a,b-unsaturated ketone unit. However,
b
The numbers were used to assign the NMR chemical shifts (see Section 4).
Isolated yield.
these results should be interpreted with caution, while some
a,b-
c
unsaturated compounds not only can induce apoptosis but also are
highly cytotoxic and can induce DNA fragmentation [33].
All these findings suggest that the a,b-unsaturated carbonyl is
Table 4
Effect of the amount of MnO2 in the synthesis hydroxyl enulosides.a
the main structural requirement for cytotoxic activity. For the
synthesized compounds, 4a-j this is evident by the results
described on Tables 5 and 6 However, a more detailed study about
the mechanism of action of these compounds needs to be
performed.
3. Conclusion
In summary, a new class of antitumoral agents with lower
structural complexity was designed and obtained in high yields and
isomeric purity after three sequential steps. All compounds
exhibited high antitumoral activity in micromolar range against the
tested tumor cell lines being the best results observed for HL-60
tumor cells.
The synthesized compounds showed several possibilities to
prepare an array of more active, site-specific or selective antitumor
agents.
Entry
MnO2(equiv)
Time (h)
4a (%)b
1
2
3
4
5
0.5
2.0
5.0
6.5
8.0
>48
24
10
3
40
75
80
84
86
2.5
a
Reaction conditions: Reactions were performed with 3a (1.0 mmol) using
different amounts of MnO2 in CH2Cl2 (10 mL) at 25 ꢁC for the time indicated.
b
Isolated yield.
4. Experimental section
the increase in the lengthening or branching of the carbon side-
chain can result in higher IC50 values.
4.1. Material
From all the tested compounds, the best result was observed for
enuloside 4h with an IC50 of 2.5
a value comparable to cisplatin (IC50 ¼ 1.17
m
M for HL-60 cells (Table 6, entry 8)
All solvents were purified before use as described in the litera-
ture [34]. CH2Cl2 was dried by distillation from CaH2. EtOH and
MeOH were dried by distillation from metallic magnesium. All
other commercially available reagents and solvents were used as
received. Reactions were monitored by thin-layer chromatography
on 0.25 mm E. Merck silica gel 60 plates (F254) using UV light,
vanillin and p-anisaldehyde as visualizing agents. Column chro-
matographic purification was performed using Silica Gel 60
(230e400 mesh) unless indicated otherwise [35]. All compounds
purified by chromatography were sufficiently pure for use in
further experiments, unless indicated otherwise.
mM for HL-60) [23]. In
addition, compared to 4g, which also contain an unsaturation on
the side chain, enuloside 4h showed good IC50 values against HEP-2
tumor cell line (Table 6, entries 7 and 8). It is interesting to note that
compound 4g showed the lowest energy value in the docking
analysis (Table 1, entry 7).
Enulosides containing cyclic structures on the side chain were
also tested and showed divergent but interesting features. Com-
pound 4d, which has a five-member ring on its structure exhibited
good IC50 values for HL-60 and HEP-2 tumor cell lines (Table 6,

196
J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
Scheme 1. Synthesis of hydroxyl enulosides 4a-j [The numbers were used to assign the NMR chemical shifts (see Section 4)].
Table 5
Preliminary screening at 25
tumor cells lineages.
and the samples were prepared as thin films on salt plates or as KBr
pellets. The melting points (mp) were obtained using a Ele-
trothermal 9100 melting point apparatus and are not corrected.
mg/mL for the compounds 2a, 3a and 4a against three
Entry
Compound
NCI-H292
(%)
MCF-7
(%)
HL60
(%)
HEP-2
(%)
4.3. Docking
1
2
3
2a
3a
4a
2.1 1.1
17.4 1.5
100 2.8
5.1 0.5
20.4 2.1
97.2 0.5
12.3 0.5
31.4 3.1
100 1.2
9.3 2.1
12.5 0.8
100 0.4
The crystallographic structure of CRM1-RAN in complex with
inhibitors were taken from the Research Collaboratory of Structural
Bioinformatics Protein Data Bank (PDB 4HAT) [36]. The ligands and
water molecules were extracted from the PDB file. All of the cal-
culations were performed with the package docking program
Autodock Tools 4.2 [37]. Initially, the structure was built using the
Gauss View 4.1 [38] and optimized using the semi-empiric model
AM1 [39] using the atomic charge units (Gasteiger model) [40]
were marked and the flexibility of both receptor and ligand were
determined using the standard program parameters, in which it
was permitted to twist the lactone side chain. The electrostatic
energy maps, atom-specific affinity and desolvatation were calcu-
lated using Autogrid 4.2 with the centre grid at ꢀ39.174, 72.955,
29.907; with 19.5 ꢂ 15.75 ꢂ 15.75 Å dimensions, and 0.375 Å
spacing. Fifty populations each with 2.500.000 fits were evaluated
using the Generic Algorithm of energy minimization with the
Autodock 4.2 program.
Table 6
Cytotoxicity (IC50) of synthesized compounds against tumor cell lines.
Entry
4
NCI-H292
MCF-7
HL60
HEP-2
a
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
18.0 ( 1.7)
15.6 ( 2.1)
24.2 ( 2.7)
19.8 ( 4.0)
2.6 ( 0.4)
10.3 ( 6.0)
6.6 ( 0.5)
12.7 ( 1.0)
3.8 ( 0.4)
35.5 ( 5.5)
0.6 ( 0.5)
-
3.5 ( 0.6)
10.7 ( 2.7)
9.7 ( 1.1)
4.2 ( 0.9)
13.3 ( 1.3)
4.9 ( 3.2)
9.3 ( 1.6)
2.5 ( 0.05)
27.3 ( 3.8)
7.9 ( 1.4)
0.20 ( 0.1)
5.8 ( 1.7)
13.4 ( 3.8)
34.9 ( 7.0)
8.0 ( 2.3)
7.5 ( 1.3)
26.1 ( 3.2)
15.4 ( 4.9)
7.1 ( 1.5)
8.5 ( 1.7)
32.1 ( 7.9)
1.3 ( 1.0)
18.3 ( 8.6)
36.0 ( 8.6)
20.7 ( 5.2)
11.0 ( 2.2)
16.3( 4.9)
10.4 ( 2.2)
a
-
9
10
11
11.1 ( 2.6)
a
4j
-
b
-
0.6 ( 0.03)
a
Not tested.
Doxorubicin was used as positive control.
b
4.4. Typical procedures
4.2. Instrumentation
4.4.1. General procedure for the synthesis of
pseudoglycosides:[18]
a-
1H NMR data were recorded at 300 or 400 MHz using a Varian
UNITY PLUS spectrometer. 1H NMR chemical shifts are reported as
To a 50 mL flask containing a solution of the 3,4,6-tri-O-acetyl-
D-glucal 1 (1.36 g, 5.0 mmol) and of the appropriate alcohol
(6.0 mmol) in acetonitrile (20 mL) was added TsOH (172.2 mg,
20 mol%). The mixture was placed in an ultrasound bath for the
time indicated on Table 3 and then diluted with EtOAc (200 mL) and
washed with a saturated solution of NaHCO3 (3 ꢂ 50 mL) and brine
(100 mL). The combined organic phases were dried over MgSO4 and
the solvents were removed under reduced pressure followed by
purification by a flash column chromatography [hexanes/EtOAc
(95:5)] to yield the corresponding 2,3-unsaturated O-glycopyr-
anosides 2a-j.
delta (
d) units in parts per million (ppm) relative to residual CDCl3
(7.26 ppm). Coupling constants (J) were reported in Hertz (Hz). 13
C
NMR data were recorded at 75 or 100 MHz using a Varian UNITY
PLUS spectrometer. 13C NMR chemical shifts were reported as delta
(d) units in parts per million (ppm) relative to the central line of
CDCl3 (77.0 ppm). High resolution mass spectra were obtained by
~
the Sao Paulo University, Chemistry Institute mass spectrometry
facility, in the electron spray ionization time of flying (ESI-TOF)
mode on a Micro TOF, Bruker Daltonics mass spectrometer. Infra-
red spectra were recorded using FT/IR spectrometer Bruker IFS 66

J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
197
4.4.1.1. Ethyl 4,6-Di-O-acetyl-2,3-dideoxy-
a
-D-erythro-hex-2-
CDCl3)
d
170.6 (C¼O), 170.2 (C¼O), 128.6 (C-3), 128.4 (C-2), 92.6 (C-
enopyranoside (2a):[18]. Colorless oil; yield 90% (1.16 g);
1), 76.6 (OCH(CH2)5), 66.6 (C-5), 65.2 (C-4), 63.0 (C-6), 33.6 (CH2),
32.0 (CH2), 25.4 (CH2), 24.3 (CH2), 24.0 (CH2), 20.9 (COCH3), 20.6
(COCH3).
20
[
a]
þ78.7 (c 1.00, MeOH); IR (thin film) nmax 2978, 2895, 1745,
D
1446, 1373, 1235, 1185, 1108 cmꢀ1; 1H NMR (300 MHz, CDCl3)
d 5.86
(dd, J ¼ 10.5 and 1.2 Hz, 1H, H-3), 5.81 (ddd, J ¼ 10.5, 2.4 and 1.8 Hz,
1H, H-2), 5.31e5.28 (m, 1H, H-4), 5.03 (br s, 1H, H-1), 4.23 (dd,
J ¼ 12.0 and 5.4 Hz, 1H, H-6 or H-60), 4.14 (dd, J ¼ 12.3 and 2.4 Hz,
1H, H-6 or H-60), 4.11e4.06 (m, 1H, H-5), 3.86e3.75 (m, 1H,
OCH2CH3), 3.60e3.50 (m, 1H, OCH2CH3), 2.07 (s, 3H, OAc), 2.05 (s,
3H, OAc),1.22 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (75 MHz, CDCl3)
4.4.1.6. Prop-2-en-1-yl 4,6-Di-O-acetyl-2,3-dideoxy-
a-D-erythro-
hex-2-enopyranoside (2f):[18]. Colorless oil; yield 83% (1.12 g);
20
[a
]
þ99.1 (c 1.00, MeOH); IR (thin film) nmax 2920, 1743, 1642,
D
1371, 1235, 1039 cmꢀ1; 1H NMR (300 MHz, CDCl3)
d 5.99e5.92 (m,
1H, OCH2CH¼CH2), 5.88 (br d, J ¼ 9.9 Hz, 1H, H-3), 5.85e5.81 (m,
1H, H-2), 5.32e5.17 (m, 3H, H-4 and OCH2CH¼CH2), 5.07 (br s, 1H,
H-1), 4.28e4.03 (m, 5H, H-5, H-6, H-60 and OCH2CH¼CH2), 2.09 (s,
d
170.3 (C¼O), 169.8 (C¼O), 128.5 (C-3), 127.4 (C-2), 93.7 (C-1), 66.2
(C-5), 64.7 (C-4), 63.8 (C-6), 62.5 (OCH2CH3), 20.5 (COCH3), 20.3
(COCH3), 14.8 (OCH2CH3).
3H, OAc), 2.07 (s, 3H, OAc); 13C NMR (75 MHz, CDCl3)
d
170.7 (C¼O),
170.2 (C¼O), 134.1 (OCH2CH¼CH2), 129.2 (C-3), 127.7 (C-2), 117.5
(OCH2CH¼CH2), 93.6 (C-1), 69.2 (OCH2CH¼CH2), 66.9 (C-5), 65.2
(C-4), 62.9 (C-6), 20.9 (COCH3), 20.7 (COCH3).
4.4.1.2. n-Propyl 4,6-Di-O-acetyl-2,3-dideoxy-
a-D-erythro-hex-2-
enopyranoside (2b):[18]. Colorless oil; yield 91% (1.24 g);
20
[
a]
þ97.4 (c 1.00, MeOH); IR (thin film) nmax 2964, 2881, 1747,
D
1450, 1371, 1234, 1182, 1105 cmꢀ1
;
1H NMR (300 MHz, CDCl3)
4.4.1.7. Prop-2-yn-1-yl 4,6-Di-O-acetyl-2,3-dideoxy-
a-D-erythro-
d
5.95e5.81 (m, 2H, H-3 and H-2), 5.32e5.29 (m, 1H, H-4), 5.02 (br
hex-2-enopyranoside (2g):[41]. White solid; yield 94% (1.26 g); mp
20
s, 1H, H-1), 4.25e4.17 (m, 2H, H-6 and H-60), 4.13e4.06 (m, 1H, H-5),
3.70 (dt, J ¼ 9.6 and 6.6 Hz, 1H, OCH2CH2CH3), 3.46 (dt, J ¼ 9.6 and
6.6 Hz, 1H, OCH2CH2CH3), 2.08 (s, 3H, OAc), 2.07 (s, 3H, OAc), 1.61
(sex, J ¼ 7.5 Hz, 2H, OCH2CH2CH3), 0.92 (t, J ¼ 7.5 Hz, 3H,
58e59 ꢁC; [
2935, 2893, 2129, 1742, 1375, 1239, 1030 cmꢀ1; 1H NMR (300 MHz,
CDCl3)
a]
þ138.6 (c 1.00; MeOH); IR (KBr pellet)nmax 3281,
D
d
5.92 (d, J ¼ 10.2 Hz, 1H, H-3), 5.83 (dt, J ¼ 10.2 and 1.5 Hz,
1H, H-2), 5.35 (ddd, J ¼ 9.6, 3.0 and 1,5 Hz, 1H, H-4), 5.22 (br s, 1H,
H-1), 4.31 (d, J ¼ 2.4 Hz, 2H, OCH2C≡CH), 4.25 (dd, J ¼ 12.4 and
5.4 Hz, 1H, H-6 or H-60), 4.16 (dd, J ¼ 12.4 and 2.4 Hz, 1H, H-6 or H-
60), 4.09 (ddd, J ¼ 9.6, 5.4 and 2.4 Hz, 1H, H-5), 2.07 (t, J ¼ 2.4 Hz, 1H,
OCH2C≡CH), 2.09 (s, 3H, OAc), 2.07 (s, 3H, OAc); 13C NMR (75 MHz,
OCH2CH2CH3); 13C NMR (75 MHz, CDCl3)
d
170.7 (C¼O), 170.2
(C¼O), 128.9 (C-3), 127.8 (C-2), 94.3 (C-1), 70.5 (C-5), 66.7
(OCH2CH2CH3), 65.2 (C-4), 62.9 (C-6), 22.8 (OCH2CH2CH3), 20.9
(COCH3), 20.7 (COCH3), 10.6 (OCH2CH2CH3).
CDCl3)
d
170.7 (C¼O), 170.2 (C¼O), 129.7 (C-3), 127.1 (C-2), 92.7 (C-
4.4.1.3. i-Propyl 4,6-Di-O-acetyl-2,3-dideoxy-
a
-D-erythro-hex-2-
1), 78.9 (OCH2C≡CH), 74.8 (OCH2C≡CH), 67.1 (C-5), 65.0 (C-4), 62.7
(C-6), 55.0 (OCH2C≡CH), 20.9 (COCH3), 20.7 (COCH3).
enopyranoside (2c):[18]. Colorless oil; yield 83% (1.13 g);
20
[
a]
þ95.4 (c 1.00, MeOH); IR (thin film) nmax 2971, 2902, 1745,
D
1450, 1372, 1317, 1233, 1184, 1127 cmꢀ1; 1H NMR (300 MHz, CDCl3)
4.4.1.8. But-3-yn-1-yl 4,6-Di-O-acetyl-2,3-dideoxy-
hex-2-enopyranoside (2h):[41]. Colorless oil; yield 84% (1.18 g);
þ91,3 (c 1,00; MeOH); IR (KBr pellet) nmax 3280, 2917, 1742,
a-D-erythro-
d
5.77 (br d, J ¼ 11.7 Hz, 1H, H-3), 5.70 (dt, J ¼ 11.7 and 1.8 Hz, 1H, H-
2), 5.21e5.17 (m, 1H, H-4), 5.03 (br s, 1H, H-1), 4.17e4.01 (m, 3H, H-
5, H-6 and H-60), 3.96e3.84 (m, 1H, OCH(CH3)2), 1.99 (s, 3H, OAc),
1.98 (s, 3H, OAc), 1.15 (d, J ¼ 6.3 Hz, 3H, OCH(CH3)2), 1.08 (d,
[a]
D
1371, 1230, 1043, 974 cmꢀ1 1H NMR (300 MHz, CDCl3)
; d 5.88 (d,
J ¼ 10.2 Hz,1H, H-3), 5.82 (ddd, J ¼ 9.6, 2.7 and 1.5 Hz,1H, H-2), 5.29
(ddd, J ¼ 9.6, 2.7 and 1.8 Hz, 1H, H-4), 5.06 (br s, 1H, H-1), 4.27e4.19
(m, 2H, H-6 and H-60), 4.12 (ddd, J ¼ 9.6, 5.4 and 3.0, 1H, H-5), 3.85
(dt, J ¼ 16.5 and 6.6 Hz, 1H, OCH2CH2C≡CH), 3.67 (dt, J ¼ 16.5 and
6.6, 1H, OCH2CH2C≡CH), 2.51 (td, J ¼ 6.6 and 2.7 Hz, 2H,
OCH2CH2C≡CH), 2.08 (s, 3H, OAc), 2.06 (s, 3H, OAc), 1.97 (t,
J ¼ 6.0 Hz, 3H, OCH(CH3)2); 13C NMR (75 MHz, CDCl3)
170.8 (C¼O),
d
170.3 (C¼O), 128.8 (C-3), 128.4 (C-2), 92.8 (C-1), 70.7 (OCH(CH3)2),
66.7 (C-5), 65.4 (C-4), 63.1 (C-6), 23.5 (OCH(CH3)2), 21.9
(OCH(CH3)2), 20.9 (COCH3), 20.7 (COCH3).
4.4.1.4. Cyclopentyl
hex-2-enopyranoside (2d):[18]. Colorless oil; yield 95% (1.42 g);
4,6-Di-O-acetyl-2,3-dideoxy-
a
-D-erythro-
J ¼ 2.7 Hz, 1H, OCH2CH2C≡CH); 13C NMR (75 MHz, CDCl3)
d 170.7
(C¼O), 170.2 (C¼O), 129.3 (C-3), 127.4 (C-2), 94.5 (C-1), 80.9
(OCH2CH2C≡CH), 69.4 (OCH2CH2C≡CH), 66.9 (OCH2CH2C≡CH), 66.8
(C-5), 65.1 (C-4), 62.8 (C-6), 20.9 (COCH3), 20.7 (COCH3), 20.0
(OCH2CH2C≡CH).
20
[
a
]
þ82.2 (c 1.00, MeOH); IR (thin film) nmax 2940, 2861, 2673,
D
2137, 1746, 1456, 1373, 1234, 1039 cmꢀ1; 1H NMR (300 MHz, CDCl3)
d
5.81 (br d, J ¼ 10.5 Hz, 1H, H-3), 5.74 (dt, J ¼ 10.5 and 2.4 Hz,1H, H-
2), 5.23 (dd, J ¼ 9.6 Hz and 1.2 Hz, 1H, H-4), 5.08 (br s, 1H, H-1), 4.23
(dd, J ¼ 12.0 and 5.4 Hz, 1H, H-6 or H-60), 4.12 (dd, J ¼ 12.0 and
2.4 Hz, 1H, H-6 or H-60), 4.06 (ddd, J ¼ 9.6, 5.4 and 2.4 Hz, 1H, H-5),
2.04 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.76e1.47 (m, 9H, OCH(CH2)4 and
4.4.1.9. Benzyl 4,6-Di-O-acetyl-2,3-dideoxy-
a-D-erythro-hex-2-
enopyranoside (2i):[18]. Colorless oil; yield 92% (1.47 g);
20
[a
]
þ61.4 (c 1,00; MeOH); IR (thin film) nmax 3030, 2899, 1745,
D
OCH(CH2)4); 13C NMR (75 MHz, CDCl3)
d
170.8 (C¼O), 170.3 (C¼O),
1656, 1490, 1451, 1371, 1234, 1042, 696 cmꢀ1
;
1H NMR (300 MHz,
128.6 (C-3), 128.4 (C-2), 93.6 (C-1), 80.6 (OCH(CH2)4), 66.8 (C-5),
65.2 (C-4), 63.0 (C-6), 33.4 (CH2), 32.3 (CH2), 23.4 (CH2), 23.1 (CH2),
20.9 (COCH3), 20.7 (COCH3).
CDCl3) 7.38e7.29 (m, 5H, Haryl), 5.93e5.83 (m, 2H, H-3 and H-2),
d
5.36e5.31 (m, 1H, H-4), 5.14 (br s, 1H, H-1), 4.81 (d, J ¼ 11.7 Hz, 1H,
OCH2), 4.59 (d, J ¼ 11.7 Hz, 1H, OCH2), 4.25 (dd, J ¼ 11.4 and 5.4 Hz,
1H, H-6 or H-60), 4.19e4.13 (m, 2H, H-5 and H-6), 2.11 (s, 3H, OAc),
4.4.1.5. Cyclohexyl 4,6-Di-O-acetyl-2,3-dideoxy-
2-enopyranoside (2e):[18]. Colorless oil; yield 80% (1.23 g);
a-D-erythro-hex-
2.09 (s, 3H, OAc); 13C NMR (75 MHz, CDCl3)
(C¼O), 137.5 (Caryl), 129.2 (Caryl), 128.4 (C-3), 127.9 (C-2), 127.8
(Caryl), 127.7 (Caryl), 93.6 (C-1), 70.2 (OCH2), 66.9 (C-5), 65.2 (C-4),
62.8 (C-6), 20.9 (COCH3), 20.8 (COCH3).
d
170.8 (C¼O), 170.2
20
[
a
]
þ85.9 (c 1.00, MeOH); IR (thin film) nmax 2933, 2858, 2659,
D
2134, 1747, 1450, 1370, 1233, 1187, 1036 cmꢀ1
CDCl3)
;
1H NMR (300 MHz,
d
5.87 (br d, J ¼ 10.5 Hz,1H, H-3), 5.77 (dt, J ¼ 10.5 and 1.5 Hz,
1H, H-2), 5.25 (dd, J ¼ 9.3 and 1.2 Hz, 1H, H-4), 5.13 (br s, 1H, H-1),
4.23e4.09 (m, 3H, H-5, H-6 and H-60), 3.65e3.56 (m, 1H,
OCH(CH2)5), 2.05 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.93e1.83 (m, 2H,
OCH(CH2)5), 1.71e1.69 (m, 2H, OCH(CH2)5), 1.52e1.49 (m, 1H,
OCH(CH2)5), 1.38e1.16 (m, 5H, OCH(CH2)5); 13C NMR (75 MHz,
4.4.1.10. 2-(2-(2-Methoxyethoxy)ethoxy)ethanyl 4,6-Di-O-acetyl-
2,3-dideoxy-
a
-D-erythro-hex-2-enopyranoside
(2j):[18].
Colorless oil; yield 82% (1.53 g); [
a]
20 þ75.7 (c 1.00; MeOH); IR (thin
D
film) nmax 2878, 1743, 1453, 1371, 1237, 1048 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) d 5.87 (br s, 2H, H-3), 5.32 (br d, 1H, H-2), 5.08 (br

198
J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
s, 1H, H-1), 4.25 (dd, J ¼ 12.0 and 5.1 Hz, 1H, H-6 or H-60), 4.16 (dd,
J ¼ 12.0 and 2.1 Hz, 1H, H-6 or H-60), 4.11 (ddd, J ¼ 9.6, 5.1 Hz and
2.1 Hz, 1H, H-5), 3.75e3.62 (m, 10H, OCH2), 3.56e3.53 (m, 2H,
OCH2), 3.38 (s, 3H, OCH3), 2.09 (s, 3H, OAc), 2.07 (s, 3H, OAc); 13C
(75 MHz, CDCl3) d 133.1 (C-3), 126.7 (C-2), 93.3 (C-1), 80.2
(OCH(CH2)4), 71.3 (C-5), 64.1 (C-4), 62.6 (C-6), 33.5 (CH2), 32.2
(CH2), 23.5 (CH2), 23.2 (CH2).
NMR (75 MHz, CDCl3)
d
170.8 (C¼O), 170.3 (C¼O), 129.1 (C-3), 127.7
4.4.2.5. Cyclohexyl 2,3-dideoxy-
a-D-erythro-hex-2-
(C-2), 94.6 (C-1), 71.9 (CH2), 70.6 (OCH2), 70.5 (OCH2), 70.5 (OCH2),
70.4 (OCH2), 67.8 (OCH2), 66.8 (C-5), 65.2 (C-4), 62.9 (C-6), 59.0
(OCH3), 20.9 (COCH3), 20.8 (COCH3).
enopyranoside (3e):[45]. Colorless oil; yield 92% (0.51 g);
20
[
a
]
þ 57,6 (c 1.0, MeOH); IR (thin film) nmax 3425, 2928, 1450,
D
1099, 765 cmꢀ1
;
1H NMR (300 Hz, CDCl3):
d
5.93 (br d, J ¼ 9.9 Hz,
1H, H-3), 5.71 (dt, J ¼ 10.5 and 2.4 Hz, 1H, H-2), 5.11 (br s, 1H, H-1),
4.19 (br d, J ¼ 5.1 Hz, 1H, H-4), 3.84 (d, J ¼ 4.2 Hz, 2H, H-6 and H-60),
3.72 (dd, J ¼ 4.8 and 4.2 Hz, 1H, H-5), 3.61 (s, 1H, OCH(CH2)5), 2.22
(br s, 2H, OH), 1.93e1.14 (m, 10H, OCH(CH2)5); 13C NMR (75 MHz,
4.4.2. General procedure for the hydrolysis
a-pseudoglycosides 2a-j
To a 25 mL flask containing the appropriate
a
-pseudoglycosides
2a-j (3.0 mmol) in MeOH (12 mL) were added distilled water
(3.6 mL) and K2CO3 (0.83 g; 6.0 mmol). The mixture was stirred at
room temperature for 5 min and the solvent was removed in vacuo.
The product was then diluted with EtOAc (50 mL) and washed with
a saturated solution of NH4Cl (3 ꢂ 50 mL) and brine (50 mL). The
combined organic phases were dried over MgSO4 and the solvents
were removed under reduced pressure followed by purification by
flash column chromatography [hexanes/EtOAc (1:1)] to yield the
desired compounds 3a-j.
CDCl3)
d 133.1 (C-3), 126.8 (C-2), 92.5 (C-1), 76.6 (OCH(CH2)5), 71.3
(C-5), 64.2 (C-4), 62.7 (C-2), 33.9 (CH2), 32.1 (CH2), 25.5 (CH2), 24.4
(CH2), 24.2 (CH2)
4.4.2.6. Prop-2-en-1-yl 2,3-dideoxy-a-D-erythro-hex-2-
enopyranoside (3f):[46]. White solid; yield 95% (0.52 g);
20
[a
]
þ 57,6 (c 1.0, MeOH); mp 68e69 ꢁC IR (thin film) nmax 3401,
D
2896,1660,1450,1044, 698 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d 5.97
(d, J ¼ 10.8 Hz, 1H, OCH2CH¼CH2), 5.94e5.88 (m, 1H, H-3), 5.75 (dt,
J ¼ 10.8 and 2.4 Hz, 1H, H-2), 5.30 (dd, J ¼ 17.2 and 1.6 Hz, 1H,
OCH2CH¼CH2), 5.20 (d, J ¼ 10.0 Hz, 1H, OCH2CH¼CH2), 5.03 (br s,
1H, H-1), 4.27e4.19 (m, 2H, H-6 and H-60), 4.07 (dd, J ¼ 9.6 and
6.4 Hz, 1H, H-4), 3.87e3.84 (m, 2H, OCH2CH¼CH2), 3.74e3.68 (m,
1H, H-5), 2.82 (d, J ¼ 7.6 Hz, 1H), 2.49 (br s, 1H, OH), 2.02 (br s, 1H,
4.4.2.1. Ethyl 2,3-dideoxy-a-D-erythro-hex-2-enopyranoside
20
(3a):[42]. White solid; yield 95% (0.49 g); [
a
]
þ146.4 (c 1.00,
D
25
MeOH) [lit.40
[a
]
þ75.7 (c 1.00; MeOH); mp 88e89 ꢁC; IR (thin
D
film) nmax 3320, 2934, 1476, 1385, 1109, 787 cmꢀ1 1H NMR
;
(300 MHz, CDCl3)
d
5.9 (br d, J ¼ 10.2 Hz, 1H, H-3), 5.75 (br d,
J ¼ 9.9 Hz, 1H, H-2), 4.99 (br s, 1H, H-1), 4.19 (br s, 1H, H-4),
3.90e3.80 (m, 3H, H-5, H-6 and H-60), 3.74e3.68 (m, 1H, OCH2CH3),
3.61e3.53 (m, 1H, OCH2CH3), 2.92 (br s, 1H, OH), 2.57 (br s, 1H, OH),
OH); 13C NMR (75 MHz, CDCl3)
d
134.2 (OCH2CH¼CH2), 133.6 (C-3),
126.1 (C-2), 117.5 (OCH2CH¼CH2), 93.5 (C-1), 71.5 (C-5), 69.3 (C-4),
64.2 (OCH2CH¼CH2), 62.6 (C-6).
1.24 (t, J ¼ 6.9 Hz, 3H, OCH2CH3); 13C NMR (75 MHz, CDCl3)
d 133.4
(C-3), 126.2 (C-2), 94.1 (C-1), 71.4 (C-5), 64.1 (C-4), 64.1 (C-6), 62.6
4.4.2.7. Prop-2-yn-1-yl 2,3-dideoxy-a-D-erythro-hex-2-
(OCH2CH3), 15.2 (OCH2CH3).
enopyranoside (3g):[47]. White solid; yield 98% (0.54 g);
[a
]
20 þ137.7 (c 1.00; MeOH); mp 76e77 ꢁC; IR (thin film) nmax 3369,
D
4.4.2.2. nPropyl 2,3-dideoxy-
(3b):[43]. Colorless oil; yield 92% (0.52 g); [
CH2Cl2); IR (thin film) nmax 3384, 2932, 1437, 1375, 1100, 687 cmꢀ1
1H NMR (300 MHz, CDCl3)
a
-D-erythro-hex-2-enopyranoside
3252, 2910, 2124, 1654, 1453, 1093, 737 cmꢀ1
;
1H NMR (400 MHz,
20
a
]
þ77,7 (c 1,00;
CDCl3)
d
6.01 (br d, J ¼ 10.2 Hz,1H, H-3), 5.67(dd, J ¼ 10.2 and 2.8 Hz,
D
;
1H, H-2), 5.20 (br s, 1H, H-1), 4.31 (d, J ¼ 2.8 Hz, 2H, H-6 and H-60),
4.28e4.23 (m, 1H, H-4), 3.93e3.83 (m, 2H, OCH2C≡CH), 3.76e3.69
(m, 1H, H-5), 2.47 (t, J ¼ 3.2 Hz, 1H, OCH2C≡CH), 2.17 (br s, 2H, OH);
d
5.96 (br d, J ¼ 9.9 Hz, 1H, H-3), 5.76 (br
d, J ¼ 10.5 Hz, 1H, H-2), 4.98 (br s, 1H, H-1), 4,20 (dq J ¼ 9.0 Hz end
1.2 Hz, 1H, H-4), 3.87e3.85 (m, 3H, H-5, H-6 and H-60), 3.76e3.68
(m, 2H, OCH2CH2CH3), 2.42 (br s, 2H, OH), 1.63. (sext, 2H,
J ¼ 13.8 Hz, OCH2CH2CH3), 0.94 (t, J ¼ 7.2, 3H, OCH2CH2CH3); 13C
13C NMR (100 MHz, CDCl3)
d 133.9 (C-3); 125.7 (C-2); 92.8 (C-1);
79.3 (OCH2C≡CH); 74.7 (OCH2C≡CH); 71.7 (C-5); 64.2 (C-4); 62.6 (C-
6); 55.1 (OCH2C≡CH).
NMR (75 MHz, CDCl3) d 133.2 (C-3), 126.3 (C-2), 94.2 (C-1), 71.4 (C-
5), 70.5 (OCH2CH2CH3), 64.2 (C-4), 62.7 (C-6), 22.9 (OCH2CH2CH3),
10.6 (OCH2CH2CH3).
4.4.2.8. But-3-yn-1-yl 2,3-dideoxy-
enopyranoside (3h). Colorless oil; yield 96% (0.57 g); [
(c 1,00; MeOH); IR (thin film) nmax 3396, 3259, 2926, 2130, 1435,
1099, 754 cmꢀ1; 1H NMR (300 MHz, CDCl3)
a-D-erythro-hex-2-
20
a]
þ74,7
D
4.4.2.3. iPropyl 2,3-dideoxy-
a
-D-erythro-hex-2-enopyranoside
d
5.96 (br d, J ¼ 9.9 Hz,
20
(3c):[44]. Colorless oil; yield 96% (0.54 g); [
a
]
þ80.4 (c 1.00,
1H, H-3), 5.77 (dt, J ¼ 10.4 and 2.4 Hz, 1H, H-2), 5.03 (br s, 1H, H-1),
4.23 (br s, 1H, H-4), 3.90e3.82 (m, 3H, H-4, H-6 and H-60), 3.75 (dd,
J ¼ 8.7 and 4.2 Hz,1H, H-5), 3.69e3.63 (m, 2H, OCH2CH2C≡CH), 2.58
(br s, 1H, OH), 2.51 (td, J ¼ 7.2 and 2.4 Hz, 2H, OCH2CH2C≡CH), 2.35
(br s, 1H, OH), 2.00 (t, J ¼ 2.4 Hz, 1H, OCH2CH2C≡CH); 13C NMR
D
MeOH) [lit.40
[
a
]
25 þ103.4 (c 1.00; CH2Cl2); IR (thin film) nmax 3392,
D
2970, 1430, 1386, 1095, 974 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
5.94
(br d, J ¼ 10. Hz, 1H, H-3), 5.70 (dt, J ¼ 10 Hz and 2.4 Hz,1H, H-2),
5.07 (br s, 1H, H-1), 4.19 (br d, J ¼ 8.4 Hz, 1H, H-4), 3.96 (sept, 1H,
OCH(CH3)2), 3.84 (br s, 2H, H-6 and H-60), 3.75e3.70 (m, 1H, H-5),
1.23 (d, J ¼ 6.0 Hz, 3H, OCH(CH3)2), 1.17 (d, J ¼ 6.4 Hz, 3H,
(100 MHz, CDCl3)
d 133.6 (C-3), 125.9 (C-2), 94.5 (C-1), 81.1
(OCH2CH2C≡CH), 71.5 (OCH2CH2C≡CH), 69.5 (C-5), 66.7 (C-4), 64.1
(OCH2CH2C≡CH), 62.6 (C-6), 20.1 (OCH2CH2C≡CH); HRMS (ESI,
MeOH:H2O) calcd for C10H14O4Na [M þ Na]þ 221.0784, found
221.0788.
OCH(CH3)2); 13C NMR (75 MHz, CDCl3)
d 133.2 (C-3), 126.8 (C-2),
92.6 (C-1), 71.2 (OCH(CH3)2), 70.4 (C-5), 64.2 (C-4), 62.7 (C-6), 23.7
(OCH(CH3)2), 21.9 (OCH(CH3)2).
4.4.2.4. Cyclopentyl 2,3-dideoxy-
enopyranoside (3d):[45]. Colorless oil; yield 90% (0.58 g);
a
-D-erythro-hex-2-
4.4.2.9. Benzyl 2,3-dideoxy-
(3i):[48]. Colorless oil; yield 94% (0.66 g); [
MeOH); IR (thin film) nmax 3323, 3031, 2988, 1454, 1093, 690 cmꢀ1
1H NMR (300 MHz, CDCl3):
7.37e7.27 (m, 5H, Haryl), 5.94 (br d,
J ¼ 9.9 Hz, 1H, H-3), 5.76 (dt, J ¼ 10.5 and 2.4 Hz, 1H, H-2), 5.08 (br s,
1H, H-1), 4.77 (br d, J ¼ 11.7 Hz, 1H, OCH2), 4.60 (d, J ¼ 11.7 Hz, 1H,
OCH2), 4.19 (br d, J ¼ 9.2 Hz,1H, H-4), 3.79 (d, J ¼ 4.2 Hz, 2H, H-6 and
H-60), 3.73 (dd, J ¼ 9.0 Hz and 4.2 Hz, 1H, H-5), 2.40 (br s, 2H, OH);
a-D-erythro-hex-2-enopyranoside
20
a
]
þ31.7 (c 1.0,
D
[
a
]
20 þ 58,1 (c 1.0, MeOH); IR (thin film) nmax 3399, 2956,1437, 1097,
;
D
788 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
5.93 (d, J ¼ 10.4 Hz, 1H, H-
d
3), 5.71 (dd, J ¼ 10.4 and 2.4 Hz, 1H, H-2), 5.04 (br s, 1H, H-1),
4.26e4.20 (m, 3H, H-4, H-6 and H-60), 3.87e3.83 (m, 1H,
OCH(CH2)4), 3.71 (dd, J ¼ 8.8 and 4.4 Hz, 1H, H-5), 2.85 (br s, 1H,
OH), 2.54 (br s, 1H, OH) 1.84e1.52 (m, 8H, OCH(CH2)4); 13C NMR

J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
199
20
13C NMR (75 MHz, CDCl3)
128.0 (Caryl), 127.8 (C-2), 126.0 (Caryl), 93.7 (C-1), 71.6 (C-5), 70.3 (C-
4), 64.1 (OCH2), 62.5 (C-6).
d
137.7 (Caryl), 133.6 (C-3), 128.4 (Caryl),
mp 76.4e77.0 ꢁC; [
a]
- 28.1 (c 0.92, CHCl3); IR (thin film) nmax
D
3294, 2946, 1698, 1437, 1108, 787 cmꢀ1 1H NMR (300 Hz, CDCl3):
;
d
6.86 (dd, J ¼ 10.2 and 3.6 Hz, 1H, H-2), 6.10 (d, J ¼ 10.5, 1H, H-3),
5.33 (d, J ¼ 3.6 Hz, 1H, H-1), 4.50 (t, J ¼ 4.6 Hz, 1H, H-5), 4.36e4.30
(m, 1H, OCH(CH2)4), 4.02 (dd, J ¼ 11.8 and 4.6 Hz, 1H, H-6 or H-60),
3.93 (dd, J ¼ 11.8 and 4.6 Hz, 1H, H-6 or H-60), 2.24 (br s, 1H, OH),
1.86e1.70 (m, 6H, OCH(CH2)4), 1.62e1.56 (m, 2H, OCH(CH2)4); 13C
4.4.2.10. 2-(2-(2-Methoxyethoxy)ethoxy)ethanyl 2,3-dideoxy-
a-D-
erythro-hex-2-enopyranoside (3j). Colorless oil; yield 91% (0.80 g);
20
[
a]
þ23,02 (c 1,00; CH2Cl2); IR (thin film) nmax 3412, 2889, 1646,
D
1456, 1352, 1093 cmꢀ1
;
1H NMR (300 MHz, CDCl3)
d
5.96 (br d,
NMR (75 MHz, CDCl3): d 195.7 (C-4), 144.3 (C-2), 127.0 (C-3), 92.7
J ¼ 10.2 Hz, 1H, H-3), 5.76 (dt, J ¼ 9.9 and 2.4 Hz, 1H, H-2), 5.01 (br s,
1H, H-1), 4.12 (d, J ¼ 7.5 Hz,1H, H-4), 3.95e3.76 (m, 4H, H-5, H-6, H-
60 and OCH2), 3.69e3.62 (m, 9H, OCH2), 3.57e3.54 (m, 2H, OCH2),
(C-1), 80.6 (OCH(CH2)4), 73.7 (C-5), 61.3 (C-6), 32.9 (CH2), 31.8
(CH2), 23.0 (CH2), 22.2 (CH2).
3.37 (br s, 3H, OCH3); 13C NMR (75 MHz, CDCl3)
d 133.6 (C-3), 125.9
4.4.3.5. Cyclohexyl 2,3-dideoxy-
4-ulose (4e):[45]. White solid; yield 88% (0.199 g); [
1.00, CHCl3); mp 99-98 ꢁC; IR (thin film) nmax 3368, 2929, 1693,
1437, 1056, 764 cmꢀ1 1H NMR (300 MHz, CDCl3):
6.88 (dd,
a-D-glycero-hex-2-enopyranosid-
(C-2), 94.2 (C-1), 71.8 (OCH2), 71.5 (OCH2), 70.5 (OCH2), 70.5 (OCH2),
70.3 (OCH2), 70.2 (CH2), 67.3 (C-5), 64.7 (C-4), 63.0 (C-6), 58.9
(OCH3); HRMS (ESI, MeOH:H2O) calcd for C13H24O7Na [M þ Na]þ
315.1414, found 315.1413.
20
a]
- 23,8 (c
D
;
d
J ¼ 10.6 and 3.6 Hz, 1H, H-2), 6.11 (d, J ¼ 10.0, 1H, H-3), 5.42 (d,
J ¼ 3.5 Hz, 1H, H-1), 4.53 (t, J ¼ 4.7 Hz, 1H, H-5), 4.06e3.88 (m, 2H,
H-6 and H-60), 3.77e3.68 (m, 1H, OCH(CH2)5), 2.26 (br s, 1H, OH),
1.95e1.92 (m, 2H, OCH(CH2)5), 1.77e1.70 (m, 2H, OCH(CH2)5),
1.58e1.54 (m, 1H, OCH(CH2)5), 1.43e1.90 (m, 5H, OCH(CH2)5); 13C
NMR (75 MHz, CDCl3): d 196.2 (C-4),144.9 (C-2),127.5 (C-3), 91.4 (C-
1), 77.2 (OCH(CH2)5), 74.0 (C-5), 61.7 (C-6), 33.7 (CH2), 31.9 (CH2),
25.4 (CH2), 24.2 (CH2), 24.0 (CH2).
4.4.3. General procedure for the synthesis of enulosides 4a-j
To a 25 mL flask containing the appropriate compound 3a-j
(1.0 mmol) in dichloromethane (10 mL) was added MnO2 (2.26 g;
26.0 mmol). The mixture was stirred at room temperature for the
time indicated on Scheme 1 and the solvent was filtered using celite
in vacuo. The solvents were removed under reduced pressure fol-
lowed by purification by flash column chromatography [hexanes/
EtOAc (7:3)] to yield the desired compounds 4a-j.
4.4.3.6. Prop-2-en-1-yl 2,3-dideoxy-
enopyranosid-4-ulose (4f):[50]. Colorless oil; yield 92% (0.17 g);
20 - 8.4 (c 1.00; CHCl3); IR (thin film) nmax 3352, 2901, 1697, 1422,
a-D-glycero-hex-2-
4.4.3.1. Ethyl 2,3-dideoxy-
ulose (4a):[45]. White solid; yield 84% (0.15 g); [
1.00; CHCl3); mp 85e86 ꢁC; IR (thin film) nmax 3314, 2978, 1692,
1450, 1386, 1109, 789 cmꢀ1; 1H NMR (300 MHz, CDCl3):
6.89 (dd,
a-D-glycero-hex-2-enopyranosid-4-
20
a
]
- 13.5 (c
[a]
D
D
1106, 657 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d
6.90 (dd, J ¼ 10.5 and
d
4.2 Hz, 1H, H-2), 6.14 (d, J ¼ 10.5 Hz, 1H, H-3), 6.01e5.88 (m, 1H,
OCH2CH¼CH2), 5.37e5.30 (m, 2H, OCH2CH¼CH2), 5.28e5.24 (m,
1H, H-1), 4.51 (t, J ¼ 4.6 Hz, 1H, H-5), 4.36e4.29 (m, 1H,
OCH2CH¼CH2), 4.20e4.13 (m, 1H, OCH2CH¼CH2), 4.06e4.00 (m,
1H, H-6 or H-60), 3.96e3.91 (m, 1H, H-6 or H-60), 2.23 (br s, 1H, OH);
J ¼ 10.5 and 3.6 Hz, 1H, H-2), 6.12 (d, J ¼ 9.9 Hz, 1H, H-3), 5.28 (d,
J ¼ 3.6 Hz, 1H, H-1), 4.49 (t, J ¼ 4.8 Hz, 1H, H-5), 4.02 (dd, J ¼ 12.3
and 4.8 Hz, 1H, H-6 or H-60), 3.96e3.85 (m, 2H,, H-6 or H-60, and
OCH2CH3), 3.71e3.61 (m, 1H, OCH2CH3), 2.31 (br s, 1H, OH), 1.27 (t,
J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (75 MHz, CDCl3):
d
195.8 (C-4),
13C NMR (75 MHz, CDCl3):
d 195.7 (C-4), 143.9 (C-2), 133.4
144.2 (C-2), 127.7 (C-3), 92.9 (C-1), 74.1 (C-5), 65.0 (OCH2CH3), 61.7
(C-6), 15.1 (OCH2CH3).
(OCH2CH¼CH2), 127.9 (C-3), 118.4 (OCH2CH¼CH2), 92.2 (C-1), 74.2
(C-5), 69.8 (OCH2CH¼CH2), 61.6 (C-6).
4.4.3.2. n-Propyl 2,3-dideoxy-
ulose (4b). Colorless oil; yield 86% (0.16 g); [
CHCl3); IR (thin film) nmax 3368, 2974, 1702, 1437, 1375, 1104,
764 cmꢀ1 1H NMR (400 MHz, CDCl3):
a-D-glycero-hex-2-enopyranosid-4-
4.4.3.7. Prop-2-yn-1-yl 2,3-dideoxy-
a-D-glycero-hex-2-
20
a
]
- 12.1 (c 1,00;
D
enopyranosid-4-ulose (4g). Colorless oil; yield 89% (0.162 g);
20
[a
]
þ29.8 (c 1.00; CHCl3); IR (thin film) nmax 3369, 3256, 2926,
D
;
d
6.89 (dd, J ¼ 10.4 and
2138, 1703, 1402, 1098, 757 cmꢀ1 1H NMR (300 Hz, CDCl3):
; d 6.91
3.6 Hz, 1H, H-2), 6.11 (d, J ¼ 10.4, 1H, H-3), 5.26 (d, J ¼ 3.6 Hz, 1H, H-
1), 4.48 (t, J ¼ 4.4, 1H, H-5), 4.03e3.90 (m, 2H, H-6 or H-60), 3.79 (dt,
J ¼ 9.6 and 6.8 Hz, 1H, OCH2CH2CH3), 3.54 (dt, J ¼ 9.6 Hz and 6.8 Hz,
1H, OCH2CH2CH3), 2.41 (br s, 1H, OH), 1.64 (sext, J ¼ 7,2 Hz, 2H,
OCH2CH2CH3), 0.94 (t, J ¼ 7.2 Hz, 3H, OCH2CH2CH3); 13C NMR
(dd, J ¼ 10.5 and 3.3 Hz, 1H, H-2), 6.18 (d, J ¼ 10.5 Hz, 1H, H-3), 5.50
(d, J ¼ 3.3 Hz, 1H, H-1), 4.51 (t, J ¼ 4.6 Hz, 1H, H-5), 4.41e4.39 (t,
J ¼ 2.4 Hz, 2H, OCH2C≡CH), 4.04 (dd, J ¼ 11.8 and 4.6 Hz, 1H, H-6 or
H-60), 3.95 (dd, J ¼ 11.8 and 4.6 Hz, 1H, H-6 or H-60), 2.54e2.52 (m,
1H, OCH2C≡CH), 2.20 (br s, 1H, OH); 13C NMR (75 MHz, CDCl3):
(75 MHz, CDCl3):
d 195.6 (C-4), 143.9 (C-2), 127.3 (C-3), 92.7 (C-1),
d
194.9 (C-4), 142.9 (C-2), 127.8 (C-3), 91.2 (C-1), 78.1 (OCH2C≡CH),
73.9 (C-5), 70.9 (OCH2CH2CH3), 61.3 (C-6), 22.5 (OCH2CH2CH3), 10.2
(OCH2CH2CH3); HRMS (ESI, MeOH:H2O) calcd for C9H14O4Na
[M þ Na]þ 209.0784, found 209.0785.
75.1 (OCH2C≡CH), 74.0 (C-5), 61.1 (C-6), 55.4 (OCH2C≡CH).
4.4.3.8. But-3-yn-1-yl 2,3-dideoxy-
a-D-glycero-hex-2-
4.4.3.3. i-Propyl 2,3-dideoxy-
ulose (4c):[49]. Colorless oil; yield 86% (0.16 g); [
CHCl3); IR (thin film) nmax 3313, 2977, 1697, 1430, 1371, 1105,
762 cmꢀ1; 1H NMR (300 MHz, CDCl3):
a-D-glycero-hex-2-enopyranosid-4-
enopyranosid-4-ulose (4h). Colorless oil; yield 78% (0.15 g);
20
20
a
]
- 8.6 (c 0,87,
[a
]
- 26.1 (c 1,00; CHCl3); IR (thin film) nmax 3304, 3266, 2920,
D
D
1698, 1405, 1104, 731 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d 6.91 (dd,
d
6.87 (dd, J ¼ 9.9 and 3.3 Hz,
J ¼ 10.6 and 3.5 Hz, 1H, H-2), 6.14 (d, J ¼ 10.0 Hz, 1H, H-3), 5.34 (d,
J ¼ 3.5 Hz, 1H, H-1), 4.57 (t, J ¼ 4.7 Hz, 1H, H-5), 4.06e3.92 (m, 3H,
H-6, H-60 and OCH2CH2C≡CH), 3.81e3.73 (m, 1H, OCH2CH2C≡CH);
2.54 (td, J ¼ 7.2 and 2.8 Hz, 2H, OCH2CH2C≡CH), 2.21 (br s, 1H, OH),
2.01 (t, J ¼ 2.8 Hz, 1H, OCH2CH2C≡CH); 13C NMR (75 MHz, CDCl3):
1H, H-2), 6.11 (d, J ¼ 9.9 Hz, 1H, H-3), 5.38 (d, J ¼ 3.3 Hz, 1H, H-1),
4.52 (t, J ¼ 4.6, 1H, H-5), 4.10e3.99 (m, 2H, H-6 and H-60), 3.92 (d,
J ¼ 5.9 Hz,1H, OCH(CH3)2), 2.23 (br s, 1H, OH),1.27 (d, J ¼ 5.9 Hz, 3H,
OCH(CH3)2), 1.23 (d, J ¼ 5.9 Hz, 3H, OCH(CH3)2); 13C NMR (75 MHz,
CDCl3):
d
196.1 (C-4), 144.7 (C-2), 127.5 (C-3), 91.4 (C-1), 74.0 (C-5),
d 195.6 (C-4), 143.6 (C-2), 127.9 (C-3), 93.3 (C-1), 80.7
71.3 (OCH(CH3)2), 61.7 (C-6), 23.2 (OCH(CH3)2), 21.8 (OCH(CH3)2).
(OCH2CH2C≡CH), 74.3 (OCH2CH2C≡CH), 69.7 (C-5), 67.3
(OCH2CH2C≡CH), 61.7 (C-6), 19.9 (OCH2CH2C≡CH); HRMS (ESI,
MeOH:H2O) calcd for C10H12O4Na [M þ Na]þ 219.0627, found
219.0622.
4.4.3.4. Cyclopentyl 2,3-dideoxy-
a-D-glycero-hex-2-
enopyranosid-4-ulose (4d):[45]. White solid; yield 89% (0.19 g);

200
J.A.M. Santos et al. / European Journal of Medicinal Chemistry 128 (2017) 192e201
4.4.3.9. Benzyl 2,3-dideoxy-
a
-D-glycero-hex-2-enopyranosid-4-
References
20
ulose (4i):[51]. Colorless oil; yield 90% (0.21 g); [
1.00; CHCl3); IR (thin film) nmax 3361, 3030, 2920, 1699, 1454,
1101 cmꢀ1 1H NMR (300 MHz, CDCl3):
7.41e7.30 (m, 5H, Haryl),
a
]
þ22.1 (c
D
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J.E. Carvalho, Effect of goniothalamin on the development of Ehrlich solid
tumor in mice, Bioorg. Med. Chem. 18 (2010) 6742e6747.
;
d
6.90 (dd, J ¼ 10.6 and 3.5 Hz, 1H, H-2), 6.14 (d, J ¼ 10.6 Hz, 1H, H-3),
5.37 (d, J ¼ 3.5 Hz, 1H, H-1), 4.85 (d, J ¼ 11.7 Hz, 1H, OCH2), 4.71 (d,
J ¼ 11.7 Hz, 1H, OCH2), 4.49 (t, J ¼ 4.7 Hz, 1H, H-5), 4.02 (dd, J ¼ 12.0
and 4.7 Hz, 1H, H-6 or H-60), 3.90 (dd, J ¼ 12.0 and 4.4 Hz, 1H, H-6 or
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H-60), 2.52 (br s, 1H, OH); 13C NMR (75 MHz, CDCl3):
d 195.3 (C-4),
143.6 (C-2), 136.4 (Caryl), 128.1 (C-3), 127.8 (Caryl), 127.7 (Caryl), 127.4
(Caryl), 91.9 (C-1), 73.8 (C-5), 70.6 (OCH2), 61.1 (C-6).
4.4.3.10. 2-(2-(2-Methoxyethoxy)ethoxy)ethanyl 2,3-dideoxy-
glycero-hex-2-enopyranosid-4-ulose (4j). Colorless oil; yield 75%
(0.22 g); [
20 - 11.7 (c 1.00, CHCl3); IR (thin film) nmax 3423, 3030,
2881, 1694, 1456, 1352, 1106, 957 cmꢀ1; 1H NMR (300 MHz, CDCl3):
a-D-
a
]
D
d
6.92 (dd, J ¼ 10.6 and 3.5 Hz,1H, H-2), 6.11 (d, J ¼ 10.6 Hz,1H, H-3),
5.34 (d, J ¼ 3.5 Hz, 1H, H-1), 4.59 (t, J ¼ 4.7 Hz, 1H, H-5), 4.01e3.95
(m, 2H, H-6 and H-60), 3.86e3.79 (m, 1H, OCH2), 3.74e3.70 (m, 2H,
OCH2), 3.69e3.61 (m, 8H, OCH2), 3.57e3.52 (m, 2H, OCH2), 3.37 (s,
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synthesis of new antibiotics from sugars, Pure Appl. Chem. 84 (2012)
803e816;
3H, OCH3), 2.36 (br s, 1H, OH); 13C NMR (75 MHz, CDCl3):
d 195.8 (C-
4), 143.9 (C-2), 127.7 (C-3), 93.3 (C-1), 74.4 (OCH2), 71.8 (OCH2), 70.6
(OCH2), 70.5 (OCH2), 70.4 (OCH2), 70.4 (OCH2), 68.3 (C-5), 61.6 (C-
6), 58.9 (OCH3); HRMS (ESI, MeOH:H2O) calcd for C13H24O7Na
[M þ Na]þ 313.1257, found 313.1262.
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carbonyl systems: synthesis and their usefulness as scaffolds in carbohy-
drate chemistry, Carbohydr. Res. 343 (2008) 1523e1539.
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Dihydro-2H-Pyridin-3-ones, 2,3-disubstituted 4-iodopyridines, and enantio-
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Antimicrob. Agents Chemother. 58 (2014) 3530e3532.
4.4.4. Antiproliferative activity
The antiproliferative activities of the synthesized compounds
were evaluated in the following human cancer cells lines: NCI-
H292 (human lung carcinoma), MCF-7 (human breast adenocarci-
noma), HL-60 (human acute promyelocytic leukemia) and HEP-2
(human epidermoid cancer cells) obtained from Rio de Janeiro
Cell Bank (RJ-Brazil). All cancer cells were maintained in RPMI 1640
or DMEM medium supplemented with 10% fetal bovine serum,
2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin at
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Y.S. Prabhakar, A.K. Shaw, Synthesis of 2,3,6-trideoxy sugar triazole hybrids as
potential new broad spectrum antimicrobial agents, Eur. J. Med. Chem. 83
(2014) 474e489.
37 ꢁC with 5% CO2. The cytotoxicity of all compounds was tested
using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-
lium bromide (MTT) (Sigma Aldrich Co., St. Louis, MO/USA)
reduction assay. For all experiments, tumor cells were plated in 96-
well plates (105 cells/mL for adherent cells or 3 ꢂ 105 cells/mL for
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Y. Queneau, Synthesis of sugars embodying conjugated carbonyl systems and
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tion Antimicrobial Activity Toxic., Bioorg. Med. Chem. 19 (2011) 926e938.
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leukemias). Tested Compounds (0.1e25 mg/mL) dissolved in DMSO
0.1% were added to each well and incubated for 72 h. Control
groups received the same amount of DMSO. After 69 h of treatment
~
L.C.C. de Araújo, G.C.G. Militao, T.G. da Silvac, R.A. Oliveira, P.H. Menezes,
Synthesis and evaluation of (ꢀ)-Massoialactone and analogues as potential
anticancer and anti-inflammatory agents, Eur. J. Med. Chem. 76 (2014)
291e300.
25
mL of MTT (5 mg/mL) was added, three hours later, the MTT
formazan product was dissolved in 100
m
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