Synthesis of new chiral 1,4-morpholin-2,5-dione derivatives and evaluation as α-glucosidase inhibitors. Part 3

Article abstract of DOI:10.1016/j.tetasy.2007.02.013

A series of chiral 1,4-morpholin-2,5-dione derivatives were synthesized starting from chiral synthons 1 and 2, monolactim ethers derived from l-valine, and the absolute configurations of the new stereocentres were assigned. The substrates investigation be

Full text of DOI:10.1016/j.tetasy.2007.02.013

Tetrahedron: Asymmetry 18 (2007) 562–568
Synthesis of new chiral 1,4-morpholin-2,5-dione derivatives
and evaluation as a-glucosidase inhibitors. Part 3I
Antonio Arcelli,a,* Daniele Balducci,a Sonia de Fatima Estevao Neto,b
Gianni Porzia,* and Monica Sandric
a
`
Dipartimento di Chimica ‘G.Ciamician’, Universita di Bologna, Via Selmi 2, 40126 Bologna, Italy
bDepartamento de Quimica e Bioquimica de Faculdade de Ciencias, Campo Grande, 1749-016 Lisboa, Portugal
cISTEC-CNR, Via Granarolo 64, 48018 Faenza, Italy
Received 23 January 2007; accepted 14 February 2007
Available online 13 March 2007
Dedicated to Professor Sergio Sandri on occasion of his retirement
Abstract—A series of chiral 1,4-morpholin-2,5-dione derivatives were synthesized starting from chiral synthons 1 and 2, monolactim
ethers derived from L-valine, and the absolute configurations of the new stereocentres were assigned. The substrates investigated behave
as noncompetitive inhibitors against a-glucosidases and are inactive towards b-glucosidase, a-mannosidase and a-galactosidase. Three of
these substrates show very good and specific inhibition abilities towards a-glucosidase.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
good glycosidase inhibitors.6,7 Thus, we thought it would
be interesting to synthesize new 1,4-morpholin-2,5-dione
derivatives (making use of the experience previously
acquired for analogous compounds) in order to test their
inhibitory activity towards the a-glucosidase in connection
with the derivatives previously investigated.1,2
In continuation of our studies directed towards producing
biologically active compounds,1,2 we have focused our
attention on the asymmetric synthesis of new, optically
active derivatives of 1,4-morpholin-2,5-dione. We believe
that the heterocyclic 1,4-morpholin-2,5-dione is an effective
skeleton to help further investigation and design substrates
with specific inhibition abilities towards the a-glucosidase.
In a previous paper2 we observed that the inhibitory effect
towards this enzyme is totally lost if the lactone function is
not present in these substrates. Although it is not a mimetic
of natural products, such a heterocyclic structure reveals
interesting perspectives; in fact, some nonglycosidic inhibi-
tors represent a new class of compounds with promising
therapeutic potential.3 It is possible that the skeleton of
the morpholin-2,5-dione mimics the transition state of D-
glucolactone, a good competitive inhibitor of glycosidases.4
From energy calculations the heterocyclic ring of mor-
It is important to underline that glucosidase inhibitors are
still under careful consideration owing to the potential
therapeutic application in the treatment of a variety of
diseases.3,7
2. Results and discussion
Herein, we report the results of enzymatic kinetic studies
performed on several enantiomerically pure 1,4-mor-
pholin-2,5-dione derivatives 7–10 and 19–22, prepared, by
following the procedures outlined in Schemes 1 and 2,
respectively.
`
pholin-2,5-dione in the preferred geometry adopts a sofa
conformation5 that is similar to the flattened half chair
conformation which has been shown to be important for
The synthetic path employed is based on the use of (6S)-1
and (6R)-2, 4-N-[(S)-1-phenethyl]-6-methyl-1,4-morpholin-
2,5-dione, a chiral synthon we have already used.5,8 The
alkylation of 1 and 2 occurred with practically total 1,4-
trans induction with respect to the methyl group at C-6,
as previously observed.2,5,8 (Z)-Derivatives 3 and 15 were
q Refs. 1 and 2 are considered to be Part 1 and 2, respectively.
Corresponding authors. E-mail addresses: antonio.arcelli@unibo.it;
*
gianni.porzi@unibo.it
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.02.013

A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
563
O
O
N
Ph
CH3
O
BnO
CH3
i
ii
1
O
O
O
R
BnO
R
O
O
3
3
H
H
6
S
6
S
N
N
CH3
CH3
*C
*C
O
3
4
iii
iii
BnO
H
O
O
H
H
O
O
H
O
BnO
O
O
H
H
N
N
CH3
*C
CH3
*C
O
6
trans-epoxides
5
cis-epoxides
iv
iv
O
O
O
O
OH
OH
OH
OH
H
H
H
H
3'S
3'R
3'R
3'S
O
O
O
O
BnO
R
BnO
R
2'R
OH
R
BnO
BnO
R
2'S
2'R
2'S
N
N
N
N
OH
OH
OH
CH3
CH3
CH3
CH3
*C
*C
*C
*C
O
O
O
O
9
10
7
8
v,vi
v,vi
H
v,vi
v,vi
BnO
BnO
BnO
BnO
H
H
H
R
S
S
R
O
O
O
O
S
R
S
R
HO
HO
HO
HO
R
R
R
R
O
O
O
O
H
H
NH
H
H
NH
NH
NH
*C
*C
*C
*C
3,5-trans-11
3,5-cis-12
3,5-trans-13
3,5-cis-14
C* = (S)-phenethyl
Scheme 1. Reagents and conditions: (i) 1 M LHMDS/THF, then (Z)-4-(benzyloxy)buten-2-ylmethansulfonate; (ii) 1 M LHMDS/THF, then (E)-iodo-4-
benzyloxy-2-butene; (iii) MCPBA/CH2Cl2; (iv) 1 M H2SO4/THF; (v) NH3/isopropanol at rt; (vi) reflux in toluene/ethanol 9:1.
obtained from chiral synthon 1 or 2, respectively, by using
(Z)-4-(benzyloxy)buten-2-ylmethansulfonate, while (E)-
derivatives 4 and 16 were synthesized by employing (E)-
iodo-4-benzyloxy-2-butene, as an electrophile. The treat-
ment of (Z)-4-(benzyloxy)buten-2-ylmethansulfonate with
NaI does not give the corresponding (Z)-iodo-derivative,
but instead provided the isomer (E)-iodo-4-benzyloxy-2-
butene.

564
A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
O
O
Ph
N
CH3
O
CH3
BnO
2
i
ii
O
O
O
S
BnO
S
3
O
3
O
H
6
H
R
6
R
N
N
CH3
CH3
*C
*C
O
16
15
iii
iii
BnO
H
O
O
H
H
H
O
O
BnO
O
O
H
H
N
N
CH3
CH3
*C
*C
O
O
17 cis-epoxides
18 trans-epoxides
iv
iv
O
O
O
O
O
O
O
OH
OH
H
OH
H
OH
H
H
3'S
3'R
3'R
3'S
O
O
O
O
BnO
BnO
S
S
BnO
S
BnO
2'S
2'S
S
2'R
OH
2'R
N
N
N
OH
OH
N
OH
CH3
CH3
CH3
CH3
*C
*C
*C
*C
O
21
19
22
20
v,vi
v,vi
v,vi
v,vi
BnO
BnO
BnO
BnO
H
H
H
H
R
S
S
R
O
O
O
O
S
R
S
R
HO
HO
HO
HO
S
S
S
S
O
O
O
O
H
H
H
H
NH
NH
NH
NH
*C
3,5-cis-23
*C
*C
*C
3,5-trans-24
3,5-cis-25
3,5-trans-26
C* = (S)-phenethyl
Scheme 2. Reagents and conditions: (i) 1 M LHMDS/THF, then (Z)-4-(benzyloxy)buten-2-ylmethansulfonate; (ii) 1 M LHMDS/THF, then (E)-iodo-4-
benzyloxy-2-butene; (iii) MCPBA/CH2Cl2; (iv) 1 M H2SO4/THF; (v) NH3/isopropanol at rt; (vi) reflux in toluene/ethanol 9:1.
Intermediates 3 and 15 were then converted into diastereo-
meric mixture of cis-epoxides 5 and 17, respectively. Simi-
larly, intermediates 4 and 16 when submitted to the
epoxidation gave the diastereomeric mixture of trans-epox-
ides 6 and 18, respectively.
The diastereomeric mixtures of both cis- and trans-epox-
ides were not separable by silica gel chromatography and
were submitted to acid cleavage. Hence, from the diastereo-
meric mixture of cis-epoxides 5 or 17 we obtained the dia-
stereomeric mixture of diols 7 and 8 or 19 and 20,

A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
565
respectively, while from the diastereomeric mixture of
trans-epoxides 6 or 18 the diastereomeric mixture of diols
9 and 10 or 21 and 22 were recovered, respectively (see
Schemes 1 and 2). Despite the Rf being rather similar, it
was possible to separate the diastereomeric diols by silica
gel chromatography although with some difficulty. How-
ever, while diols 7 and 8 could be isolated in a pure form,
diols 10 and 19 were recovered in 80% and 90% diastereo-
meric excess, respectively. The purities of diols 9, 20, 21
and 22, as determined by HPLC–MS analysis, were greater
than 95%.
behaved as noncompetitive inhibitors. It is interesting to
note that all substrates showed inhibition activity exclu-
sively towards a-glucosidase. In fact, at 500 lM concentra-
tion, b-glucosidase (from almonds), a-mannosidase (from
jack beans) and a-galactosidase (from green coffee beans)
did not suffer inhibition.
3. Conclusion
From the values of the inhibition constants reported in
Table 1 some considerations can be inferred. First of all,
the compounds investigated are generally more effective
and selective than those previously reported.2 As a result,
we believe that the extension of the side chain at C-3, by
the introduction of a CH2OBn group, increases the biolog-
ical activity. The data collected in Table 1 show that com-
pounds 9, 10 and 21 are the most active inhibitors towards
both the a-glucosidases tested with substrate 21 in particu-
lar exhibiting very good inhibition ability. Since both 9 and
21 have the same configurations at C-20 and C-30, their ste-
reochemistry appears more important than that of the C-3
and C-6 stereocentres. In order to determine the influence
of the benzyl group on the biological activity, we per-
formed the debenzylation of 21, through hydrogenolysis
in the presence of Pd/C, to obtain the corresponding triol,
which showed an inhibitory activity more than 100-fold
smaller than substrate 21, the Ki being 1500 lM towards
a-glucosidase from baker’s yeast. Such a result suggests
that the benzyl group at the C-30 position of the side chain
probably favours the binding to an enzyme hydrophobic
zone, more than the hydroxyl group, which is only able
to form hydrogen bonds.
The C-3 configuration of diastereomers 3, 4, 15 and 16 was
established through the shielding effect on (C-3)–H induced
by the phenyl ring of the phenethyl group at N-4,2,5 the
configuration of the C-6 stereocentre being known from
the starting material.
The absolute configurations of the new stereocentres C-20
and C30 in 7–10 and 19–22 was assigned by following the
same procedure previously used2 and summarized in
Schemes 1 and 2. c-Lactones 11–14 and 23–26 were ob-
tained by treating diols 7–10 and 19–22, respectively, with
ammonia in isopropanol (through the assisted opening of
the morpholinone ring as described in our previous
papers2,8,9) and then by refluxing in toluene/ethanol 9:1.
As previously observed,2 the formation of c-lactones was
ascertained by the IR spectra which showed a characteristic
carbonyl absorption at shorter wavelengths (i.e., at
m = 1795–1760 cmꢀ1) than d-lactones.10 Thus, it was possi-
ble to establish the absolute configuration of the C-5 ste-
reocentre of the c-lactones by NOE experiments (the
configuration of C-3 stereocentre being known) and conse-
quently, the absolute configuration of the C-20 stereocentre
of diols could be deduced.2 Finally, because the acid cata-
lyzed opening of the epoxides occurs in a regio- and stereo-
controlled fashion, the absolute configuration at the C-30
stereocentre was established.
4. Experimental
4.1. General
The diols synthesized were then submitted to the kinetic
tests of inhibitory activities against a-glucosidases (from
both baker’s yeast and Bacillus stearothermophilus) and
the results are summarized in Table 1. The inhibition
kinetic curves showed that all compounds investigated
1H and 13C NMR spectra were recorded on a Gemini spec-
trometer at 300 MHz using CDCl3 as the solvent. Chemical
shifts are reported in ppm relative to CDCl3 and the cou-
pling constants (J) are in Hz. IR spectra were recorded
on a Nicolet 210 spectrometer. The products isolated,
which were not sufficiently pure for elemental analysis
and to measure the specific rotation, were submitted to
HPLC–MS analysis on a Hewlett-Packard Model 1100
liquid chromatograph-single-quadrupole mass-selective
detector system, with an atmospheric pressure chemical
ionization-electrospray interface. Optical rotation values
were measured at 25 °C on a Perkin–Elmer 343 polari-
meter. The enzyme kinetics were followed by Cary100
UV spectrophotometer and Cary software was employed
for calculating the inhibition constants. Dry THF was
distilled from sodium benzophenone ketyl. For the synthe-
sis and spectroscopic data of 1 and 2 (see Refs. 5 and 8).
Table 1. Inhibition constants against a-glucosidases
Substrates
C-3
C-6
C-20
C-30
Ki (lM)
a
b
7
8
(R)
(R)
(R)
(R)
(S)
(S)
(S)
(S)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(S)
n.i.c
n.i.c
40
300
804
831
400
33
9
10d
19e
20
21
22
(S)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(S)
370
520
12
194
696
4
1030
250
a From baker’s yeast.
4.2. Alkylation of 1 and 2
b From Bacillus stearothermophilus.
c No inhibition at 500 lM.
LHMDS (1 M, 10 mL) in dry THF (10 mmol) was dropped
into a solution of 10 mm of 1 or 2 in dry THF (100 mL),
cooled at ꢀ40 °C under an inert atmosphere. After about
d de = 80% (see text and Section 4).
e de = 90% (see text and Section 4).

566
A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
1 h, the bath was cooled at ꢀ78 °C and the alkylating
reagent (10 mmol) added. After about 5 h, the cooling bath
was removed allowing the reaction mixture to warm up
to rt, then 10 mL of 1 M HCl were added and the mixture
extracted with ethyl acetate. The organic extract was dried,
evaporated in vacuo and the residue purified by silica gel
chromatography eluting with hexane/ethyl acetate.
The product was not isolated in sufficiently pure form for
elemental analysis or to measure the specific rotation.
4.3. General procedure for the formation of diols 7, 8, 9, 10,
19, 20, 21 and 22
MCPBA (77%, 7 g, 21.9 mmol) was added to a solution of
3 or 4 or 15 or 16 (18.3 mmol) in 50 mL of CH2Cl2. The
reaction mixture was stirred at room temperature and
monitored by TLC. When the starting material had com-
pletely reacted, meta-chlorobenzoic acid was filtered off,
the organic phase extracted with 10% aqueous solution
of Na2CO3 and then dried on CaCl2. After evaporation of
the organic solvent under vacuum, the diastereomeric mix-
ture of epoxides was obtained in practically quantitative
yield. To the epoxide (0.7 g, 2.4 mmol), dissolved in
15 mL of THF, 1 M H2SO4 (0.5 mL) was added. The reac-
tion was monitored by TLC and stirred at room tempera-
ture for 24 h. After neutralization with 1 M NaOH, the
reaction mixture was concentrated in vacuo at about
40 °C. The crude reaction product was dissolved in ethyl
acetate and the Na2SO4 filtered off. The organic solution
was evaporated under vacuum and the residue submitted
to silica gel chromatographic separation eluting with ethyl
acetate. The reaction products were isolated in at least 85%
yield.
4.2.1. (3R,6S)-3-(40-Benzyloxy-(20Z)-butenyl)-6-methyl-4-
[(S)-phenethyl]-morpholine-2,5-dione 3. The compound
was obtained in 90% yield by alkylating 1 with (Z)-4-(benz-
yloxy)buten-2-ylmethansulfonate prepared from the (Z)-
4-(benzyloxy)-2-buten-1-ol. 1H NMR: d 1.55 (d, 3H,
J = 6.6); 1.6 (d, 3H, J = 6.9); 1.75 (m, 1H); 2.15 (m, 1H);
3.72 (m, 2H); 4.1 (dd, 1H, J = 4.8, 10.5); 4.25 (qAB, 2H,
J = 10); 4.94 (q, 1H, J = 6.6); 5.29 (m, 1H); 5.67 (m, 1H);
5.96 (q, 1H, J = 6.9), 7.4 (m, 10ArH). 13C NMR: d 15.4,
15.9, 29.3, 51, 55.1, 64.8, 71.6, 73.1, 124.8, 127.1, 127.5,
127.8, 127.9, 128, 128.4, 129.9, 137.6, 138.3, 165.6, 166.2.
HPLC–MS: 394.2 [M+1]+, 416.2 [M+Na]+. The product
was not isolated in sufficiently pure form for elemental
analysis or to measure the specific rotation.
4.2.2. (3R,6S)-3-(40-Benzyloxy-(20E)-butenyl)-6-methyl-4-
[(S)-phenethyl]-morpholine-2,5-dione 4. The compound
was obtained in 90% yield by alkylating 1 with (E)-iodo-
4-benzyloxy-2-butene prepared from (Z)-4-(benzyl-
1
oxy)buten-2-ylmethansulfonate. H NMR: d 1.59 (d, 3H,
4.3.1. (3R,6S,20S,30S)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
J = 7.2); 1.65 (d, 3H, J = 6.9); 1.76 (m, 1H); 2.01 (m,
1H); 3.89 (m, 2H); 4.15 (dd, 1H, J = 4.5, 9.6); 4.49 (s,
2H); 5.02 (q, 1H, J = 6.9); 5.39 (m, 2H); 6.1 (q, 1H,
J = 7.2); 7.4 (m, 10ArH). 13C NMR: d 15.8, 16.4, 34.7,
51.4, 55.9, 69.5, 71.9, 73.5, 125.4, 127.4, 127.5, 127.8,
128.2, 128.4, 128.7, 131.7, 137.9, 138.6, 165.9, 166.8.
[a]D = ꢀ207 (c 0.4, CHCl3). Anal. Calcd for C23H27NO4:
C, 72; H, 7.13; N, 3.67. Found: C, 72.1; H, 7.15; N, 3.68.
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione
7. The
1
product was obtained from 3 in 40% yield. H NMR: d
1.45 (m, 1H); 1.52 (d, 3H, J = 6.9); 1.78 (d, 3H, J = 6.9);
1.78 (d, 3H, J = 6.9); 2.35 (m, 1H); 3.57 (m, 2H); 3.66
(m, 1H); 3.98 (dd, 1H, J = 8.1, 10.2); 4.54 (s, 2H); 4.65 (m,
1H); 4.79, (q, 1H, J = 6.9); 5.12 (q, 1H, J = 6.9); 7.4 (m,
10ArH). 13C NMR: d 16.8, 21.2, 28.8, 51.2, 54.1, 64.6,
70.7, 71.9, 73.2, 77, 127, 127.6, 127.7, 128.2, 128.3, 128.8,
137.4, 138.6, 173.7, 174.3. [a]D = +13.5 (c 2, CHCl3). Anal.
Calcd for C24H29NO6: C, 67.43; H, 6.84; N, 3.28. Found:
C, 67.12; H, 6.86; N, 3.22.
4.2.3. (3S,6R)-3-(40-Benzyloxy-(20Z)-butenyl)-6-methyl-4-
[(S)-phenethyl]-morpholine-2,5-dione 15. The compound
was obtained in 90% yield by alkylating 1 with (Z)-4-(benz-
yloxy)buten-2-ylmethansulfonate prepared from the (Z)-
4-(benzyloxy)-2-buten-1-ol. 1H NMR: d 1.62 (d, 3H,
J = 6.6); 1.65 (d, 3H, J = 7.2); 2.70 (m, 2H); 3.87 (dd,
1H, J = 5.4, 8.7); 4.04 (m, 2H); 4.53 (m, 2H); 4.97 (q,
1H, J = 6.6); 5.62 (m, 1H); 5.88 (m, 2H); 7.4 (m, 10ArH).
13C NMR: d 16.3, 17.2, 31.1, 52.1, 55.8, 65.2, 72.2, 73.4,
125.4, 126.8, 127.6, 128.1, 128.2, 128.8, 130.5, 137.6,
138.0, 166.3, 166.5. [a]D = +84.8 (c 0.9, CHCl3). Anal.
Calcd for C23H27NO4: C, 72; H, 7.13; N, 3.67. Found: C,
71.84; H, 7.12; N, 3.65.
4.3.2. (3R,6S,20R,30R)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione
8. The
1
product was obtained from 3 in 40% yield. H NMR: d
1.52 (d, 3H, J = 6.6); 1.6 (m, 1H); 1.79 (d, 3H, J = 6.9);
2.4 (m, 1H); 3.61 (m, 2H); 3.78 (t, 1H, J = 10.2); 3.85 (m,
1H); 4.51 (qAB, 2H, J = 12); 4.52 (m, 1H); 4.79 (q, 1H,
J = 6.6); 5.17 (q, 1H, J = 6.9); 7.4 (m, 10ArH). 13C
NMR: d 16.7, 21.2, 27.3, 53.4, 54.1, 64.7, 70.1, 71.3, 76.6,
127, 127.5, 128.2, 128.9, 137.7, 138.3, 172.9, 173.9. [a]D =
ꢀ10.6 (c 0.5, CHCl3). Anal. Calcd for C24H29NO6: C,
67.43; H, 6.84; N, 3.28. Found: C, 67.66; H, 6.85; N, 3.25.
4.2.4. (3S,6R)-3-(40-Benzyloxy-(20E)-butenyl)-6-methyl-4-
[(S)-phenethyl]-morpholine-2,5-dione 16. The compound
was obtained in 90% yield by alkylating 1 with (E)-iodo-
4-benzyloxy-2-butene prepared from the (Z)-4-(benzyl-
4.3.3. (3R,6S,20S,30R)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione
9. The
1
product was obtained from 4 in 30% yield. H NMR: d
1.51 (d, 3H, J = 6.6); 1.60 (m, 1H); 1.76 (d, 3H, J = 7);
2.15 (m, 1H); 3.40 (m, 2H); 3.86 (dd, 1H, J = 7.8, 10.6);
3.94 (m, 1H); 4.50 (m, 2H); 4.68 (m, 1H); 4.79 (q, 1H,
J = 6.6); 5.12 (q, 1H, J = 7); 7.4 (m, 10ArH). 13C NMR:
d 16.9, 21.3, 27.3, 53.4, 54.3, 64.8, 70.2, 71.2, 73.5, 77.1,
127.1, 127.7, 127.8, 128.4, 128.5, 129.0, 137.6, 138.3,
173.1, 174.1. HPLC–MS: 410.1 [M+1ꢀH2O]+, 428.1
1
oxy)buten-2-ylmethansulfonate. H NMR: d 1.64 (d, 3H,
J = 6.6); 1.68 (d, 3H, J = 6.6); 2.68 (m, 2H); 3.91 (t, H,
J = 6.6); 3.99 (m, 2H); 4.51 (s, 2H); 5.06 (q, 1H, J = 6.6);
5.74 (m, 2H); 5.86 (q, 1H, J = 6.6 Hz); 7.4 (m, 10ArH).
13C NMR d 16.6, 17.6, 36.3, 52.5, 56.3, 69.5, 72.2, 73.6,
125.2, 127.0, 127.6, 128.3, 129.0, 132.3, 137.9, 138.1,
166.5, 166.8. HPLC–MS: 394.2 [M+1]+, 416.2 [M+Na]+.

A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
567
[M+1]+, 450.1 [M+Na]+, 877.2 [2M+Na]. The product
was not isolated in sufficiently pure form for elemental
analysis or to measure the specific rotation.
4.3.8. (3S,6R,20R,30S)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione 22. The
product was obtained from 16 in 40% yield. H NMR: d
1
1.41 (d, 3H, J = 6.6); 1.66 (d, 3H, J = 7); 2.45 (m,2H);
3.52 (m,2H); 3.82 (dd, 1H, J = 8.4, 9.8); 3.92 (m, 1H);
4.53 (qAB, 2H, J = 11.6); 4.52 (m, 1H); 4.76 (m, 1H); 5.13
(q, 1H, J = 6.6); 7.4 (m, 10ArH). 13C NMR: d 19.7; 21.2;
28.2; 52.0; 54.3; 64.7; 70.1; 70.6; 73.2; 77.8; 126.5; 127.5;
127.6; 128.2; 128.7; 137.3; 138.3; 173.3; 174.6. HPLC–
MS: 410.1 [M+1ꢀH2O]+, 428.1 [M+1]+, 450.1 [M+Na]+,
877.2 [2M+Na]+. The product was not isolated in suffi-
ciently pure form for elemental analysis or to measure
the specific rotation.
4.3.4. (3R,6S,20R,30S)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione 10. The
product, obtained from 4, was recovered in 40% yield as
a wax in a 90:10 diastereomeric mixture with 9, respec-
1
tively. H NMR: d 1.50 (d, 3H, J = 6.6); 1.60 (m, 1H);
1.77 (d, 3H, J = 7); 2.42 (m, 1H); 3.58 (m, 2H); 3.74 (t,
1H, J = 10); 4.15 (m, 1H); 4.38 (m, 1H); 4.52 (s, 2H);
4.79 (q, 1H, J = 7); 5.18 (q, 1H, J = 6.6); 7.4 (m,
10ArH). 13C NMR: d 17.0, 21.4, 26.9, 52.5, 54.4, 64.7,
70.1, 71.0, 73.4, 78.3, 127.0, 127.7, 127.9, 128.4, 128.5,
128.9,
137.3,
138.5,
174.2.
HPLC–MS:
410.1
4.4. Conversion of diols into c-lactones
[M+1ꢀH2O]+, 428.1 [M+1]+, 450.1 [M+Na]+, 877.2
[2M+Na]. The product was not isolated in sufficiently pure
form for elemental analysis or to measure the specific
rotation.
A solution of diol 9–12 (1.5 g, 5 mmol) in 100 mL of iso-
propanol was cooled at 0 °C and then saturated with
NH3 by bubbling for about 30 min. The reaction flask
was stopped and kept for 3 days at rt. After testing by
TLC, the ammonia and the organic solvent were evapo-
rated in vacuo. The residue was submitted to cyclization
by refluxing in 50 mL of toluene/ethanol = 9:1 for 24 h
and the reaction monitored by TLC. The organic solvents
were evaporated to dryness under vacuum and the residue
submitted to silica gel chromatography eluting with ethyl
acetate. The c-lactones were recovered in 70–80% yield.
4.3.5. (3S,6R,20S,30S)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione 19. The
product, obtained from 15, was recovered in 40% yield as
a wax in 95:5 diastereomeric mixture with 20, respectively.
1H NMR: d 1.42 (d, 3H, J = 6.9); 1.65 (d, 3H, J = 6.6);
2.34 (m, 1H); 2.63 (m, 1H); 3.63 (m, 2H); 3.80 (t, 1H,
J = 10.2); 3.93 (m, 1H); 4.59 (m, 4H); 5.18 (q, 1H,
J = 6.6); 7.4 (m, 10ArH). 13C NMR: d 19.7, 21.3, 28.8,
53.0, 54.5, 64.7, 70.1, 71.4, 73.3, 76.9, 126.6, 127.6, 127.9,
128.2, 128.8, 137.7, 138.1, 172.0, 174.6. HPLC–MS: 410.1
[M+1ꢀH2O]+, 428.1 [M+1]+, 450.1 [M+Na]+, 877.2
[2M+Na]. The product was not isolated in sufficiently pure
form for elemental analysis or to measure the specific
rotation.
4.4.1. (3R,5S,10S)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 11. The product
1
was obtained from 7. H NMR: d 1.42 (d, 3H, J = 6.9);
2.25 (m, 1H); 2.5 (m, 1H); 3.5 (m, 2H); 3.66 (t, 1H,
J = 8.7); 3.79 (m, 1H); 3.85 (q, 1H, J = 6.9); 4.55 (s, 2H);
4.6 (m, 1H); 7.4 (m, 10ArH). 13C NMR: d 24, 32.6, 53.4,
56.2, 70.7, 71.9, 73.4, 77.3, 126.3, 127.3, 127.7, 127.9,
128.4, 128.6, 137.4, 143.9, 178. HPLC–MS: 356.3
[M+1]+, 378.2 [M+Na]+. IR (CHCl3) m (cmꢀ1) = 3620
(OH), 1772 (C@O). The product was not isolated in suffi-
ciently pure form for elemental analysis or to measure
the specific rotation.
4.3.6. (3S,6R,20R,30R)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione 20. The
product was obtained from 15 in 35% yield. H NMR: d
1
1.41 (d, 3H, J = 6.6); 1.68 (d, 3H, J = 7); 2.4–2.7 (m,
2H); 3.6 (m, 2H); 3.8 (m, 1H); 3.97 (dd, 1H, J = 8.6, 10);
4.56 (s, 2H); 4.59 (m, 1H); 4.74 (m, 1H); 5.12 (q, 1H,
J = 7); 7.4 (m, 10ArH). 13C NMR: d 20.0, 21.6, 30.4,
52.0, 54.6, 64.7, 70.7, 72.2, 73.5, 76.6, 126.6, 127.8, 127.9,
128.4, 128.5, 128.9, 137.4, 138.3, 173.3, 174.8. HPLC–
MS: 410.1 [M+1ꢀH2O]+, 428.1 [M+1]+, 450.1 [M+Na]+,
877.2 [2M+Na]+. The product was not isolated in suffi-
ciently pure form for elemental analysis or to measure
the specific rotation.
4.4.2. (3R,5R,10R)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 12. The product
1
was obtained from 8. H NMR: d 1.45 (d, 3H, J = 6.9);
2.1 (m, 1H); 2.4 (m, 1H); 3.4 (t, 1H, J = 8.7); 3.62 (m,
2H); 3.8 (m, 2H); 4.55 (m, 1H); 4.6 (s, 2H); 7.4 (m,
10ArH). 13C NMR: d 24.1, 31.9, 54.3, 56.2, 70.3, 71.4,
73.6, 77.8, 126.4, 127.5, 127.8, 127.9, 128.5, 128.8, 137.6,
143.3, 176.5. [a]D = ꢀ65.6 (c 0.7, CHCl3). IR (CHCl3) m
(cmꢀ1) = 3615 (OH), 1774 (C@O). Anal. Calcd for
C21H25NO4: C, 70.96; H, 7.09; N, 3.94. Found: C, 71.11;
H, 7.1; N, 3.93.
4.3.7. (3S,6R,20S,30R)-3-(40-Benzyloxy-20,30-dihydroxybutyl)-
6-methyl-4-[(S)-phenethyl]-morpholine-2,5-dione 21. The
product was obtained from 16 in 40% yield. H NMR: d
1
1.44 (d, 3H, J = 6.6); 1.68 (d, 3H, J = 7.0); 2.42 (m, 1H);
2.64 (m, 1H); 3.64 (m, 2H); 3.77 (t, 1H, J = 9.8); 4.20 (q,
1H, J = 5.8); 4.47 (m, 1H); 4.56 (s, 2H); 4.61 (m, 1H);
5.16 (q, 1H, J = 7); 7.4 (m, 10ArH). 13C NMR: d 19.9,
21.6, 28.9, 52.9, 54.6, 64.8, 70.2, 71.1, 73.5, 76.7, 126.7,
127.7, 127.8, 128.1, 128.4, 129.0, 137.6, 138.1, 172.1,
174.9. HPLC–MS: 410.1 [M+1ꢀH2O]+, 428.1 [M+1]+,
450.1 [M+Na]+, 877.2 [2M+Na]+. The product was not
isolated in sufficiently pure form for elemental analysis or
to measure the specific rotation.
4.4.3. (3R,5S,10R)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 13. The product
was obtained from 9. H NMR: d 1.43 (d, 3H, J = 6.9);
2.15 (m, 1H); 2.57 (m, 1H); 3.46–3.62 (m, 3H); 3.77 (m,
1H); 3.83 (q, 1H, J = 6.9); 4.55 (m, 1H); 4.56 (s, 2H); 7.4
(m, 10ArH). 13C NMR: d 24.1, 30.7, 53.1, 56.3, 70.3, 70.6,
73.6, 77.5, 126.4, 127.4, 127.8, 128.0, 128.5, 128.7, 137.3,
143.8, 177.5. HPLC–MS: 356.3 [M+1]+, 378.2 [M+Na]+.
IR (CHCl3) m (cmꢀ1) = 3625 (OH), 1770 (C@O). The
1

568
A. Arcelli et al. / Tetrahedron: Asymmetry 18 (2007) 562–568
product was not isolated in sufficiently pure form for ele-
mental analysis or to measure the specific rotation.
4.5. Enzyme kinetics
4.5.1. Materials. a-Glucosidase (EC 3.2.1.20) from baker’s
yeast and from B. stearothermophilus, b-glucosidase
(EC 3.2.1.2) from almonds, a-mannosidase (EC 3.2.1.24)
from Jack bean, a-galactosidase (EC 3.2.1.22) from green
coffee beans, p-nitrophenyl glucosides, 4-(2-hydroxy-
ethyl)-1-piperazinethanesulfonic acid and its potassium salt
(HEPES) were purchased from Sigma.
4.4.4. (3R,5R,10S)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 14. The product
was obtained from 10. H NMR: d 1.44 (d, 3H, J = 6.6);
1
2.09 (m, 1H); 2.48 (m, 1H); 3.37 (dd, 1H, J = 8.4, 10.2);
3.60 (m, 2H); 3.82 (q, 1H, J = 6.6); 3.97 (m, 1H); 4.39
(m, 1H); 4.56 (s, 2H); 7.4 (m, 10ArH). 13C NMR: d 24.2,
31.9, 54.5, 56.3, 70.3, 70.9, 73.6, 77.4, 126.4, 127.5, 127.8,
128.0, 128.5, 128.8, 137.5, 143.4, 176.7. HPLC–MS: 356.3
[M+1]+, 378.2 [M+Na]+. IR (CHCl3) m (cmꢀ1) = 3612
(OH), 1775 (C@O). The product was not isolated in suffi-
ciently pure form for elemental analysis or to measure
the specific rotation.
4.5.2. Kinetics. The kinetic hydrolyses of glucosides were
carried out at pH = 6.85 and were followed at k = 400 nm
by Carry 100 UV spectrophotometer at 37 0.01 °C. Ten
cells, filled with 0.1 M HEPES buffer solution containing
0.05–0.2 units of enzyme, were thermostated in the cell
holder of UV spectrophotometer. Stock solutions (10–
100 lL) of inhibitors, dissolved in ethanol, were added to
enzyme buffer solution and thermostated for about
15 min. The glucosides solutions were added to the buffered
enzyme solutions by ten Hamilton syringes previously
thermostated. Noncompetitive inhibition constants (Ki)
were calculated from the equation Ki ¼ V 0max½I0ꢁ=
ðV max ꢀ V 0maxÞ where V m0 ax and Vmax are the maximum rates
measured in the presence and in the absence of inhibitor,
respectively, and [I0] is the inhibitor concentration. The
kinetics were carried out in duplicate runs and the Ki repro-
ducibility was in the range 10–15%.
4.4.5. (3S,5S,10S)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 23. The product
1
was obtained from 19. H NMR: d 1.43 (d, 3H, J = 6.6);
1.82 (m, 1H); 2.08 (m, 1H); 3.46 (t, 1H, J = 8.4); 3.57 (m,
2H); 3.77 (m, 1H); 4.19 (q, 1H, J = 6.6); 4.42 (m, 1H);
4.55 (s, 2H); 7.4 (m, 10ArH). 13C NMR: d 24.4, 33.0,
55.6, 57.5, 70.4, 71.2, 73.5, 77.4, 127.0, 127.4, 127.7,
127.9, 128.4, 128.5, 137.6, 144.5, 177.0. [a]D = ꢀ18.7 (c
0.6, CHCl3). IR (CHCl3) m (cmꢀ1) = 3620 (OH), 1770
(C@O). Anal. Calcd for C21H25NO4: C, 70.96; H, 7.09;
N, 3.94. Found: C, 71.22; H, 7.07; N, 3.95.
4.4.6. (3S,5R,10R)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 24. The product
was obtained from 20. H NMR: d 1.41 (d, 3H, J = 6.6);
Acknowledgement
1
1.98 (m, 2H); 2.20; 5.25 (m, 2H); 3.71 (m, 2H); 4.11 (q,
1H, J = 6.6); 4.42 (m, 1H); 4.53 (s, 2H); 7.4 (m, 10ArH).
13C NMR: d 24.5, 33.8, 54.8, 57.9, 70.7, 72.0, 73.5, 77.1,
127.0, 127.2, 127.8, 127.9, 128.4, 128.5, 137.4, 144.9, 178.3.
[a]D = ꢀ94.6 (c 0.5, CHCl3). IR (CHCl3) m (cmꢀ1) = 3624
(OH), 1772 (C@O). Anal. Calcd for C21H25NO4: C, 70.96;
H, 7.09; N, 3.94. Found: C, 71.1; H, 7.11; N, 3.94.
Thanks are due to the University of Bologna for financial
support (Ricerca Fondamentale Orientata).
References
1. Arcelli, A.; Balducci, D.; Grandi, A.; Porzi, G.; Sandri, M.;
Sandri, S. Monatsh. Chem. 2004, 135, 951.
2. Arcelli, A.; Balducci, D.; Grandi, A.; Porzi, G.; Sandri, M.;
Sandri, S. Tetrahedron: Asymmetry 2005, 16, 1495, and
references cited therein.
3. Borges de Melo, E.; da Silveira Gomes, A.; Carvalho, I.
Tetrahedron 2006, 62, 10277.
4. Stutz, A. E. Iminosugars as Glycosidase Inhibitors; Wiley-
VCH: Weinheim, 1999.
4.4.7. (3S,5S,10R)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 25. The product
1
was obtained from 21. H NMR: d 1.41 (d, 3H, J = 6.6);
1.9 (m, 1H); 2.1 (m, 1H); 3.45 (dd, 1H, J = 8.7, 9.6); 3.53
(m, 2H); 3.97 (m, 1H); 4.22 (q, 1H, J = 6.6); 4.32 (m,
1H); 4.55 (qAB, 2H, J = 10.5); 7.4 (m, 10ArH). 13C
NMR: d 24.3, 32, 55.6, 57.3, 70.1, 73.4, 77.1, 126.9,
127.2, 127.6, 127.8, 128.4, 137.4, 144.4, 177.4.
[a]D = ꢀ63.7 (c 0.4, CHCl3). IR (CHCl3) m (cmꢀ1) = 3618
(OH), 1772 (C@O). Anal. Calcd for C21H25NO4: C,
70.96; H, 7.09; N, 3.94. Found: C, 71.21; H, 7.1; N, 3.95.
5. Porzi, G.; Sandri, S. Tetrahedron: Asymmetry 1996, 7, 189,
and references cited therein.
6. (a) Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem.
Soc. 1993, 115, 11682; (b) Kajimoto, T.; Liu, K. K.-C.;
Pedersen, R. L.; Zhong, Z.; Ikikawa, Y.; Porco, J. A.; Wong,
C. H. J. Am. Chem. Soc. 1991, 113, 6187; (c) Ermert, P.;
Vasella, A. T.; Weber, M.; Rupitz, K.; Withers, S. G.
Carbohydr. Res. 1993, 250, 113; (d) Eightman, T. D.; Vasella,
A. T. Angew. Chem., Int. Ed. 1999, 38, 750.
7. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem.
Rev. 2002, 102, 515.
8. Madau, A.; Porzi, G.; Sandri, S. Tetrahedron: Asymmetry
1996, 7, 825.
9. Arcelli, A.; Porzi, G.; Sandri, S. Tetrahedron 1996, 52,
4141.
4.4.8. (3S,5R,10S)-5-(20-Benzyloxy-10-hydroxyethyl)-3-[(S)-
phenylethylamino]-dihydro-furan-2-one 26. The product
1
was obtained from 22. H NMR: d 1.42 (d, 3H, J = 6.6);
1.9 (m, 1H); 2.2 (m, 1H); 3.54 (m, 3H); 3.82 (m, 1H); 4.16
(q, 1H, J = 6.6); 4.45 (m, 1H); 4.54 (qAB, 2H, J = 11.7); 7.4
(m, 10ArH). 13C NMR: d 24.5, 31.7, 54.3, 57.4, 70.3, 70.7,
73.5, 77.4, 126.9, 127.3, 127.6, 128, 128.4, 128.5, 137.3,
144.7, 177.8. HPLC–MS: 356.3 [M+1]+, 378.2 [M+Na]+.
IR (CHCl3) m (cmꢀ1) = 3623 (OH), 1773 (C@O). The prod-
uct was not isolated in sufficiently pure form for elemental
analysis or to measure the specific rotation.
10. Silverstein, R. M.; Webster, F. X. Spectrometric Identification
of Organic Compounds, 6th ed.; John Wiley & Sons, 1999; p
98.