Chiral 1,4-morpholine-2,5-diones. Synthesis and evaluation as glucosidase inhibitors

Article abstract of DOI:10.1007/s00706-004-0195-5

A series of chiral 1,4-morpholine-2,5-dione derivatives were synthesized. These substrates behave as non competitive inhibitors against α-glucosidase showing a moderate inhibition (Ki < 0.8 mM) and were found inactive towards β-glucosidase. Springer-Verla

Full text of DOI:10.1007/s00706-004-0195-5

Monatshefte f€ur Chemie 135, 951–958 (2004)
DOI 10.1007/s00706-004-0195-5
Chiral 1,4-Morpholine-2,5-diones. Synthesis
and Evaluation as Glucosidase Inhibitors
ꢀ
Monica Sandri, and Sergio Sandri
Antonio Arcelli , Daniele Balducci, Alessandro Grandi, Gianni Porzi,
ꢀ
ꢀ
Universita degli Studi di Bologna, Dipartimento di Chimica ‘‘G. Ciamician’’,
40126 Bologna, Italy
Received February 3, 2004; accepted March 16, 2004
Published online June 24, 2004 # Springer-Verlag 2004
Summary. A series of chiral 1,4-morpholine-2,5-dione derivatives were synthesized. These substrates
behave as non competitive inhibitors against ꢀ-glucosidase showing a moderate inhibition (Ki<
0.8 mM) and were found inactive towards ꢁ-glucosidase.
Keywords. 1,4-Morpholine-2,5-diones; ꢀ-Glucosidase; Glycosidase inhibitors.
Introduction
Glycosidases are an important class of carbohydrate and glycoconjugate modifying
enzymes and are involved in a wide range of important biological processes [1–3].
Synthetic and naturally occurring glycosidase inhibitors play an important role not
only in elucidating the glycoside hydrolysis mechanism, but also in promising ther-
apeutic applications (antiviral [4], anticancer [5], anti-HIV [4], etc.). In fact, many
efforts to design and synthesize competitive inhibitors of glycosidases have been made
over the last years. Continuing our studies on the asymmetric synthesis of biologically
active compounds, we have focused our attention on a new family of 1,4-morpholine-
2,5-dione derivatives as potential glucosidase inhibitors. Our interest in these deriva-
tives isdue tothe factthatthegeometry of morpholine-2,5-dione (with the heterocyclic
ꢀ
ring in the preferred sofa conformation [6]) could be considered analogous to the
transition state of D-gluconolactone [2b], a good competitive inhibitor of glycosidases,
which unfortunately has limited application owing to its instability [2b, 7].
Results and Discussion
In this paper we wish to report the results of enzymatic kinetic studies performed
on several chiral derivatives of 1,4-morpholine-2,5-dione prepared enantiomeri-
ꢀ
Corresponding author. E-mail: antonio.arcelli@unibo.it

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A. Arcelli et al.
Table 1. Inhibition constants (Ki) towards ꢀ-glucosidasea
Substrates
1
3b
3c
3d
2
4b
7b
7c
10a
10b
Ki values=mM
4.80
0.70
0.80
2.38
0.30
0.70
23.70
n.i.b
3.35
0.86c
a
b
c
In HEPES buffer (pH ¼ 6.85) at 37ꢁC; no inhibition; Ki ¼ 0.3 towards ꢁ-glucosidase
Scheme 1. i) 1 M LiHMDS=THF; ii) R-Br; iii) H2 (3 atm), 10% Pd=C in CH3OH
cally pure in good yields. The products 3b–3d and 4b reported in Table 1 were
obtained by following a procedure already employed starting from (S)-phenylethyl-
amine, ethyl bromoacetate and (R,S)-2-chloropropionyl chloride [6]. After separation
by silica gel chromatography, the diastereomers (10S,6R) 1 [6] and (10S,6S)-6-
methyl-4-N-(10-phenethyl)-1,4-morpholin-2,5-dione (2) [6] were converted into the
derivatives 3b–3d and 4b, respectively (Scheme 1). As previously observed [6], the
alkylation occurred with a practically total 1,4-trans induction with respect to (C-
6)–CH3. However, the configuration of the C-3 stereocenter was confirmed by
means of the shielding effect induced by the phenyl ring of the (S)-phenylethyla-
mine group, as described in a previous paper [6], and by the NOE observed
between (C-6)–CH3 and (C-3)–H, the configuration of the C-6 stereocenter being
note. The intermediates 3a and 4a were then submitted to hydrogenolysis and the
substrates 3b and 4b were obtained in very good yields.
The substrates 7b and 7c were synthesized following the procedure reported in
Scheme 2. The intermediate 5, obtained by alkylating p-methoxybenzylamine with
ethyl bromoacetate, was acylated with (S)-2-acetoxy-propanoyl chloride, and the
crude reaction product was submitted to alkaline hydrolysis. The 1,4-morpholine-
2,5-dione derivative 6 was then recovered after induced acid cyclization. In anal-
ogy to that observed for 1 and 2, the alkylation of intermediate 6 with benzyl
bromoacetate or with methyl 3-bromopropionate occurred with a practically total
1,4-trans induction giving 7a and 7c. The configuration of the new C-3 stereocentre
was assigned by means of a NOE between (C-6)–CH3 and (C-3)–H, the (S) con-
figuration of the C-6 stereocentre being note. By submitting the intermediate 7a to
hydrogenolysis the substrate 7b was easily obtained in very good yield.

Chiral 1,4-Morpholine-2,5-diones
953
Scheme 2. i) BrCH2COOEt=Et3N, CH2Cl2, 0ꢁC; ii) (S)-CH3CH(OAc)COCl=Et3N, CH2Cl2, 0ꢁC; iii)
NaOH=H2O, EtOH, then HCl; iv) 1 M LiHMDS=THF, R-Br; v) H2 (3 atm), 10% Pd=C in CH3OH
Scheme 3. i) p-methoxybenzylchloride, pyridine=acetone, 60ꢁC; ii) (S)-CH3CH(OAc)COCl=Et3N,
CH2Cl2; iii) NaOH in H2O=EtOH, then HCl; iv) PhCH2Br, Et3N, acetone, rt
The substrates 10a and 10b were synthesized starting from (L)-aspartic acid
dibenzyl ester p-toluenesulfonate and following the procedure reported in Scheme 3.
The derivative 8, obtained by alkylating (L)-dibenzyl aspartate with p-methoxy-
benzyl chloride, was acylated with (S)-2-acetoxypropanoyl chloride to afford the
intermediate 9. After alkaline hydrolysis, followed by acid induced cyclization, the
cis-1,4-morpholine-2,5-dione derivative 10a was obtained, which was then con-
verted into the corresponding benzyl ester 10b.
The various substrates, synthesized in optically pure form, were submitted to
the inhibitory activities screening against both ꢀ- and ꢁ-glucosidases from bakers
yeast and almonds, respectively, and the kinetic results are summarized in Table 1.
The inhibition kinetic curves showed that the compounds behave as non-competi-
tive inhibitors.
The substrates differ for the substituents at C-3, at N-4, and at C-6 positions of the
heterocyclic ring, and for the absolute configuration at C-3 and C-6 stereocenters.
Some substrates were found to be weak inhibitors of ꢀ-glucosidase, whereas all are
inactive towards ꢁ-glucosidase, except 10b, which does, however, show a weak
inhibitory activity. Better inhibition potency (Kiꢂ0.8 mM) towards ꢀ-glucosidase
was displayed by 2, 3b, 3c, 4b, and 10b (Table 1). By comparing Ki values of 1 and 2,
it appears that the absolute configuration at C-6 plays an important role. An analo-
gous behaviour can be observed for the C-3 stereocenter when R is the p-methoxy-
benzyl group (compare 7b and 10a), whereas the Ki value is not affected by the C-3

954
A. Arcelli et al.
configuration when R ¼ (S)–C6H5–CH(CH3)– (compare 3b and 4b). Thus, the chiral
(S)-phenethyl group appears more effective towards the inhibitory activity in com-
parison to the p-methoxybenzyl substituent (compare 3b with 10a and 4b with 7b).
The moderate inhibition activity found for these substrates can probably be
attributed to their inability to interact with the active site of the enzyme by hydro-
gen bonds and=or electrostatic interactions. In fact, competitive inhibitors, such as
nojirimycin and 1-deoxynojirimycin, which is the reference compound for the
biological activity against glycosidases, have hydroxyl groups, which may interact
with the active site of enzyme by hydrogen bond [8].
In conclusion, from these preliminary results the heterocyclic structure of the 1,4-
morpholine-2,5-dione appears to be a promising skeleton to design other substrates
with a better inhibition ability towards glycosidases. Finally, it is interesting to under-
line that our synthetic pathway to the enantiomerically pure 1,4-morpholine-2,5-
dione derivatives is rather simple and suitable for preparing other similar substrates.
Experimental
1H and 13C NMR spectra were recorded on a Gemini spectrometer at 300 MHz using CDCl3 as the
solvent. The chemical shifts are reported in ppm relative to CDCl3. The coupling constants (J) are in
Hz. UV-Vis: Cary 100 spectrophotometer. Cary software for enzyme kinetics. Optical rotation values
were measured at 25ꢁC on a Perkin-Elmer 343 polarimeter. Dry THF was distilled from sodium
benzophenone ketyl. Elemental analyses for 3b, 3c, 3d, 4b, 7b, 10a, and 10b agreed favourably with
the calculated values.
General Procedure for the Alkylation of 1 and 2
LiHMDS in THF (1M, 22 cm3) was added to a stirred solution of 5 g of 1 or 2 (21.7mmol) in 100cm3
of dry THF cooled at ꢃ78ꢁC. After about 1 h 25 mmol of alkylating reagent were added and the
reaction was monitored by TLC. When the reaction was complete, the mixture was allowed to warm to
room temperature with stirring. Diluted HCl and ethyl acetate were added and after separation the
organic solution was evaporated in vacuo. The residue was purified by silica gel chromatography
eluting with cyclohexane=ethyl acetate.
Benzyl (3S,6R)-[6-methyl-2,5-dioxo-N-((S)-1-phenethyl)-1,4-morpholin-
3-yl]acetate (3a, C22H23NO5)
Obtained in 87% yield by alkylating 1 with benzyl bromoacetate. The benzyl ester was separated by
silica gel chromatography. 1H NMR: ꢂ ¼ 1.58 (d, 3H, J ¼ 7.2 Hz), 1.69 (d, 3H, J ¼ 6.9 Hz), 2.9 (dd, 1H,
J ¼ 5.7, 17.1Hz), 3.02 (dd, 1H, J ¼ 3.6, 17.1Hz), 4.15 (m, 1H), 5.16 (m, 2H), 5.22 (q, 1H, J ¼ 6.9 Hz),
5.71 (q, 1H, J ¼ 7.2Hz), 7.27–7.45 (m, 10H) ppm; 13C NMR: ꢂ ¼ 17.1, 17.4, 37.7, 52.9, 53.1, 67.4,
74.4, 127.4, 128.3, 128.4, 128.5, 128.9, 134.6, 137.1, 166.5, 167, 169.1ppm; the product was not
obtained in a sufficiently pure form to measure its specific rotation.
(3S,6R)-[6-Methyl-2,5-dioxo-N-((S)-1-phenethyl)-1,4-morpholin-3-yl]acetic
acid (3b, C15H17NO5)
The benzyl ester 3a (0.76 g, 2 mmol) in 20 cm3 of methanol was cleaved by hydrogenolysis in the
presence of 10% Pd=C. The reaction performed at room temperature in a Parr hydrogenator under
36psi (3atm) of H2, was complete after 1 h. The catalyst was filtered off on Celite and the alcoholic

Chiral 1,4-Morpholine-2,5-diones
955
solution was evaporated under vacuum to dryness. The oily product was recovered pure in 95% yield
1
after purification by silica gel chromatography. H NMR: ꢂ ¼ 1.64 (d, 3H, J ¼ 6.8Hz), 1.69 (d, 3H,
J ¼ 6.8 Hz), 2.87 (dd, 1H, J ¼ 5.4, 17.6Hz), 3.05 (dd, 1H, J ¼ 3.2, 17.6Hz), 4.05 (dd, 1H, J ¼ 3.2,
5.4 Hz), 5.48 (q, 1H, J ¼ 6.8 Hz), 5.76 (q, 1H, J ¼ 6.8 Hz), 7.38 (m, 5ArH) ppm; 13C NMR: ꢂ ¼ 17.2,
18, 37.3, 52.8, 53.5, 75.1, 127.9, 128.9, 129.4, 136.4, 167.5, 168.5, 172.2ppm; [ꢀ]D ¼
86.2ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1, CH3OH).
(3S,6R)-3-(E)-But-2-enyl-6-methyl-N-[(S)-1-phenethyl]-1,4-morpholine-2,5-dione
(3c, C17H21NO3)
Obtained in 85% yield by alkylating 1 with (E)-1-bromobut-2-ene. The reaction was worked up after
8 h and the product was isolated pure as an oil after silica gel chromatography. 1H NMR: ꢂ ¼ 1.65 (d,
3H, J ¼ 6.8 Hz), 1.66 (d, 3H, J ¼ 7.1 Hz), 1.68 (d, 3H, J ¼ 7.1 Hz), 2.6 (m, 2H), 3.85 (t, 1H, J ¼ 6.5 Hz),
5 (q, 1H, J ¼ 6.8Hz), 5.4 (m, 1H), 5.6 (m, 1H), 5.85 (q, 1H, J ¼ 7.1 Hz), 7.3 (m, 5H) ppm; 13C NMR:
ꢂ ¼ 16.6, 17.2, 17.4, 17.7, 52.2, 56.4, 73.4, 123.3, 126.9, 128, 128.8, 131.1, 138.8, 166.4, 166.9ppm;
[ꢀ]D ¼ 94.1ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.8, CHCl3).
(3S,6R)-N-[(S)-1-Phenethyl]-3-[3-methylbutyl]-6-methyl-1,4-morpholine-2,5-dione
(3d, C18H23NO3)
Obtained in 86% yield by alkylating 1 with 1-bromo-3-methylbut-2-ene. The reaction was worked up
after 4 h and the product was isolated pure as an oil after silica gel chromatography. 1H NMR: ꢂ ¼ 1.62
(d, 3H, J ¼ 7.2 Hz), 1.64 (d, 3H, J ¼ 6.8 Hz), 1.67 (s, 3H), 1.7 (s, 3H), 2.6 (m, 2H), 3.83 (dd, 1H,
J ¼ 4.9, 8.3Hz), 5.05 (q, 1H, J ¼ 6.8 Hz), 5.15 (m, 1H), 5.85 (q, 1H, J ¼ 7.2Hz), 7.3 (m, 5H) ppm; 13C
NMR: ꢂ ¼ 16.9, 17.5, 18, 25.9, 32, 52.5, 56.4, 73.8, 116.7, 127.2, 128.1, 128.4, 137.5, 138.3, 166.8,
168 ppm; [ꢀ]D ¼ 108.7ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.4, CHCl3).
Benzyl (3R,6S)-[6-methyl-2,5-dioxo-N-((S)-1-phenethyl)-1,4-morpholin-3-yl]acetate
(4a, C22H23NO5)
Obtained in 88% yield by alkylating 2 with benzyl bromoacetate. The benzyl ester was submitted to
1
silica gel chromatography. H NMR: ꢂ ¼ 1.67 (m, 6H), 2.33 (dd, 1H, J ¼ 6.2, 17.2Hz), 2.5 (dd, 1H,
J ¼ 4, 17.2 Hz), 4.62 (dd, 1H, J ¼ 4, 6.2 Hz), 4.95 (m, 2H), 5.24 (q, 1H, J ¼ 6.6 Hz), 5.98 (q, 1H,
J ¼ 7.2 Hz), 7.31 (m, 10H) ppm; 13C NMR: ꢂ ¼ 15.4, 17.2, 36.6, 50.8, 52.2, 67.1, 74.4, 127.2, 128.2,
128.5, 128.6, 128.9, 134.8, 139.5, 166.7, 167.2, 168.9 ppm; the product was not obtained in a suffi-
ciently pure form to measure the specific rotation.
(3R,6S)-[6-Methyl-2,5-dioxo-N-((S)-1-phenethyl)-1,4-morpholin-3-yl]acetic acid
(4b, C15H17NO5)
The benzyl ester 4a was cleaved by hydrogenolysis, following the procedure described for 3b.
After purification by silica gel chromatography, the product was recovered as an oil in 93% yield.
1H NMR: ꢂ ¼ 1.69 (m, 6H), 2.38 (dd, 1H, J ¼ 6.3, 17.1Hz), 2.47 (dd, 1H, J ¼ 4.2, 17.1Hz), 4.63 (dd,
1H, J ¼ 4.2, 6.3 Hz), 5.2 (q, 1H, J ¼ 6.9 Hz), 5.98 (q, 1H, J ¼ 6.9 Hz), 7.4 (m, 5ArH) ppm; 13C-NMR:
ꢂ ¼ 15.5, 17, 36.2, 51.7, 52.3, 74.3, 127.1, 128.3, 128.9, 138.8, 167.1, 167.3, 172.5ppm;
[ꢀ]D ¼ ꢃ144.7ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1, CH3OH).
3-N-(p-Methoxybenzyl)glycine ethylester (5, C12H17NO3)
Ethyl bromoacetate (12 cm3, 110 mmol) was dropped to a solution of 15.5cm3 of p-methoxybenzyla-
mine (110 mmol) and 15.3cm3 of triethylamine (110 mmol) in 50 cm3 of CH2Cl2 cooled at 0ꢁC. When

956
A. Arcelli et al.
the addition was complete, the cooling bath was removed allowing the reaction mixture to warm up to
rt. The reaction was then monitored by TLC and after 3 h H2O was added. The organic phase was
separated, washed with H2O, and dried (CaCl2). The organic solvent was evaporated in vacuo and the
crude reaction product was submitted to purification by silica gel chromatography. The product was
recovered in 85% yield. 1H NMR: ꢂ ¼ 1.28 (t, 3H, J ¼ 7 Hz), 2 (bs, NH), 3.4 (s, 2H), 3.82 (m, 5H) 4.2
(q, 2H, J ¼ 7 Hz), 6.9 (m, 2ArH), 7.3 (m, 2ArH) ppm; 13C-NMR: ꢂ ¼ 13.8, 49.5, 52.2, 54.7, 60.2,
113.4, 129, 131.3, 158.4, 172 ppm.
(6S)-N-(p-Methoxybenzyl)-6-methyl-1,4-morpholine-2,5-dione (6, C13H15NO4)
To a solution of 11.5 g of 5 (50 mmol) in 50cm3 of CH2Cl2 and 8.3 cm3 of triethylamine (60 mmol),
cooled at 0ꢁC, 8.25g of (S)-2-acetoxy propanoylchloride (55 mmol) was dropped under stirring. After
30min the cooling bath was removed allowing the reaction mixture to warm up to rt and the reaction
was monitored by TLC. After 1 h, diluted HCl was added, the organic phase separated and dried
(CaCl2). The organic solvent was evaporated under vacuum to dryness and the residue was dissolved in
50cm3 of ethanol. To the solution it was added under stirring 8 g of NaOH (200mmol) dissolved in
80cm3 of 50% EtOH=H2O and the reaction was monitored by TLC. After the starting material had
disappeared, the ethanol was evaporated and the aqueous solution acidified by adding concentrated
HCl and stirred at rt. The reaction product was extracted with ethyl acetate and the organic solution
evaporated in vacuo. The residue was purified by silica gel chromatography. The pure product was
1
recovered as an oil in 80% yield. H NMR: ꢂ ¼ 1.67 (d, 3H, J ¼ 7 Hz), 3.82 (s, 3H), 4 (qAB, 2H,
J ¼ 18 Hz), 4.56 (qAB, 2H, J ¼ 14.7 Hz), 4.94 (q, 1H, J ¼ 7 Hz), 6.89 (m, 2H), 7.2 (m, 2H) ppm; 13C
NMR: ꢂ ¼ 17.3, 42, 48.8, 55.2, 74.9, 114.3, 126.5, 129.6, 159.5, 165.2, 166ppm; [ꢀ]D ¼
ꢃ35ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 0.2, CHCl3).
Benzyl (3R,6S)-[N-(p-methoxybenzyl)-6-methyl-2,5-dioxo-1,4-morpholin-3-yl]acetate
(7a, C22H23NO6)
The intermediate 6 was alkylated with benzyl bromoacetate following the procedure employed to
1
synthesize 3a. The benzyl ester was obtained as an oil in 85% yield. H NMR: ꢂ ¼ 1.64 (d, 3H,
J ¼ 7 Hz), 2.93 (dd, 1H, J ¼ 4.4, 17.4Hz), 3.19 (dd, 1H, J ¼ 4.4, 17.4Hz), 3.79 (s, 3H), 4.15 (d, 1H,
J ¼ 14.8Hz), 4.32 (t, 1H, J ¼ 4.4 Hz), 5.03 (d, 1H, J ¼ 14.8Hz), 5.10 (qAB, 2H, J ¼ 12.2Hz), 5.26 (q,
1H, J ¼ 7 Hz), 6.86 (m, 2H), 7.16 (m, 2H), 7.36 (m, 5H) ppm; 13C NMR: ꢂ ¼ 17.8, 35.5, 46.7, 54.5,
55.2, 67.5, 74.7, 114.4, 126.8, 128.4, 128.6, 129.3, 134.5, 159.4, 166.5, 166.8, 169.4 ppm; the product
was not obtained in a sufficiently pure form to measure the specific rotation.
(3R,6S)-[4-(p-Methoxybenzyl)-6-methyl-2,5-dioxo-1,4-morpholin-3-yl]acetic acid
(7b, C15H17NO6)
The benzyl ester 7a was cleaved by hydrogenolysis, as described for 3b. The product was recovered as
an oil in 94% yield after purification by silica gel chromatography. 1H-NMR: ꢂ ¼ 1.68 (d, 3H,
J ¼ 7 Hz), 2.95 (dd, 1H, J ¼ 4.6, 17.6Hz), 3.12 (dd, 1H, J ¼ 4, 17.6 Hz), 3.8 (s, 3H), 4 (d, 1H,
J ¼ 15 Hz), 4.26 (dd, 1H, J ¼ 4, 4.6 Hz), 5.24 (d, 1H, J ¼ 15Hz), 5.35 (q, 1H, J ¼ 7 Hz), 6.9 (m,
2H), 7.15 (m, 2H), 7.66 (bs, 1H) ppm; 13C NMR: ꢂ ¼ 18, 36.6, 45.6, 54.4, 55.2, 74.6, 114.2, 126.6,
129.4, 159.2, 166.8, 168.1, 174.1 ppm; [ꢀ]D ¼ ꢃ87.1ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 0.6, CH3OH).
Methyl (3R,6S)-3-[N-(p-methoxybenzyl)-6-methyl-2,5-dioxo-1,4-morpholin-3-yl]propionate
(7c, C17H21NO6)
It was obtained in 80% yield by alkylating the intermediate 6 with methyl 3-bromopropionate. The
product was recovered as an oil after silica gel chromatography. 1H NMR: ꢂ ¼ 1.65 (d, 3H, J ¼ 6.9 Hz),

Chiral 1,4-Morpholine-2,5-diones
957
2.1 (m, 2H), 2.46 (m, 2H), 3.68 (s, 3H), 3.8 (s, 3H), 4.04 (d, 1H, J ¼ 14.7Hz), 4.05 (m, 1H), 5.02 (q,
1H, J ¼ 6.9 Hz), 5.1 (d, 1H, J ¼ 14.7Hz), 6.86 (m, 2H), 7.2 (m, 2H) ppm; 13C NMR: ꢂ ¼ 16.6, 25.3,
28.9, 47, 51.8, 55.1, 57.3, 73.4, 114.2, 127, 129.5, 159.3, 165.8, 166.5, 172.1 ppm; the product was not
obtained in a sufficiently pure form to measure the specific rotation.
Dibenzyl (L)-N-(p-methoxybenzyl)aspartate (8, C26H27NO5)
(L)-Aspartic acid dibenzyl ester p-toluenesulfonate (purchased from Aldrich) (4.85 g, 10mmol) dis-
solved 20cm3 of acetone was stirred at about 60ꢁC with 1.7 g of p-methoxybenzyl chloride (11 mmol)
in the presence of 1.6 cm3 of pyridine. When the TLC showed the reaction practically complete (after
2–3 days) the mixture was evaporated in vacuo and the residue dissolved in ethyl acetate. The organic
solution was washed with H2O, dried, and after removal of the organic solvent in vacuo the residue was
submitted to silica gel chromatography. The product was recovered as an oil in 50% yield. 1H NMR:
ꢂ ¼ 1.65 (bs, 1H), 2.7 (dd, 1H, J ¼ 6.9, 15.6Hz), 2.78 (dd, 1H, J ¼ 6, 15.6 Hz), 3.62 (d, 1H,
J ¼ 12.6Hz), 3.71 (dd, 1H, J ¼ 6, 6.9 Hz), 3.76 (d, 1H, J ¼ 12.6 Hz), 3.77 (s, 3H), 5.1 (m, 4H), 6.8
(m, 2H), 7.15 (m, 2H), 7.3 (m, 10H) ppm; 13C NMR: ꢂ ¼ 36, 50.8, 54.9, 55.2, 66.9, 67.8, 114, 124.8,
128.1, 128.2, 128.3, 130.7, 134.4, 134.9, 159.6, 169, 169.5 ppm; the product was not obtained in a
sufficiently pure form to measure the specific rotation.
Dibenzyl (L)-N-(p-methoxybenzyl)-N-[(2S)-acetoxypropionyl]aspartate (9, C31H33NO8)
(S)-2-Acetoxypropanoyl chloride (1.5cm3, 10 mmol) was added to a stirred solution of 4.33 g of 8
(10 mmol) in 22cm3 of CH2Cl2 and 15cm3 of triethylamine, cooled at 0ꢁC. The reaction, monitored by
TLC, after 1 h was slowly warmed to rt and acidified with diluted HCl. The organic phase was
separated, washed, dried (CaCl2), and the solvent was evaporated in vacuo. The residue was submitted
to silica gel chromatography and the product was recovered in 85% yield. 1H NMR: ꢂ ¼ 1.35 (d, 3H,
J ¼ 6.6 Hz), 2 (s, 3H), 2.81 (dd, 1H, J ¼ 7.2, 17.1Hz), 3.25 (dd, 1H, J ¼ 6.6, 17.1 Hz), 3.71 (s, 3H),
4.33 (d, 1H, J ¼ 16.2Hz), 4.48 (dd, 1H, J ¼ 6.6, 7.2 Hz), 4.57 (d, 1H, J ¼ 16.2Hz), 5.05 (m, 4H), 5.38
(q, 1H, J ¼ 6.6Hz), 6.69 (m, 2H), 7.3 (m, 12H) ppm; 13C NMR: ꢂ ¼ 16.6, 20.4, 34.1, 51.3, 54.9, 56,
66.4, 67, 67.1, 113.6, 127.9, 128.1, 128.2, 128.3, 129.2, 135.1, 135.3, 159, 168.8, 169.9, 170.3,
170.8ppm; the product was not obtained in a sufficiently pure form to measure the specific rotation.
(3S,6S)-[4-(p-Methoxybenzyl)-6-methyl-2,5-dioxo-1,4-morpholin-3-yl]acetic acid
(10a, C15H17NO6)
NaOH (0.4g, 10 mmol) dissolved in 10 cm3 of 50% EtOH=H2O was added to 5.5 g of 9 (10 mmol) in
10cm3 of ethanol under stirring at rt and the reaction was monitored by TLC. After about 2 h the
reaction mixture was concentrated in vacuo, acidified with 5 N HCl, and then extracted with ethyl
acetate. The organic solution was dried and evaporated in vacuo. The pure product was recovered in
90% yield after silica gel chromatography. 1H NMR: ꢂ ¼ 1.54 (d, 3H, J ¼ 7.2Hz), 3 (dd, 1H, J ¼ 5.4,
17.4Hz), 3.13 (dd, 1H, J ¼ 3.9, 17.4Hz), 3.82 (s, 3H), 4.12 (d, 1H, J ¼ 15Hz), 4.37 (m, 1H), 5.11 (q,
1H, J ¼ 7.2 Hz), 5.2 (d, 1H, J ¼ 15 Hz), 6.8 (m, 2H), 7.2 (m, 2H) ppm; 13C NMR: ꢂ ¼ 19.1, 35.1, 46.2,
53.2, 55.3, 75.6, 114.6, 126.4, 129.4, 159.6, 165.9, 166.3, 172.8ppm; [ꢀ]D ¼ ꢃ17.9ꢁ cm3 gꢃ1 dmꢃ1
(c ¼ 0.2, CH3OH).
Benzyl (3S,6S)-[4-(p-Methoxybenzyl)-6-methyl-2,5-dioxo-1,4-morpholin-3-yl]acetate
(10b, C22H23NO6)
To a solution of 1.6 g of 10a (5mmol) in 15 cm3 of acetone and 1.5cm3 of triethylamine was added
0.63g of benzyl chloride (5 mmol) and the reaction was stirred at rt. After 12 h acetone was evaporated

958
A. Arcelli et al.: Chiral 1,4-Morpholine-2,5-diones
in vacuo and first ethyl acetate and then diluted HCl were added to the residue. The organic phase was
separated, washed, dried, and the organic solvent was evaporated in vacuo. The residue was purified by
1
silica gel chromatography and the product was recovered in 85% yield. H NMR: ꢂ ¼ 1.84 (d, 3H,
J ¼ 7 Hz), 2.99 (dd, 1H, J ¼ 5.2, 17.2Hz), 3.12 (dd, 1H, J ¼ 4, 17.2Hz), 3.79 (s, 3H), 4.21 (d, 1H,
J ¼ 15.4Hz), 4.41 (dd, 1H, J ¼ 4, 5.2 Hz), 4.99 (d, 1H, J ¼ 15.4Hz), 5.08 (q, 1H, J ¼ 7 Hz), 5.09 (qAB
,
2H, J ¼ 12Hz), 6.87 (m, 2H), 7.17 (m, 2H), 7.35 (m, 5H) ppm; 13C NMR: ꢂ ¼ 19.2, 35.9, 46.4, 53.8,
55.2, 67.2, 75.5, 114.3, 126.9, 128.2, 128.4, 128.5, 129.1, 134.7, 159.3, 165.8, 166, 169 ppm;
[ꢀ]D ¼ 11.3ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 0.4, CHCl3).
Enzyme Kinetics
Materials: ꢀ-Glucosidase (EC 3.2.1.20) from baker yeast, ꢁ-glucosidase (EC 3.2.1.2) from almonds,
p-nitrophenyl glucosides and N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic acid potassium salt
(HEPES) were purchased from Sigma.
The kinetic hydrolyses of glucosides were carried out at pH¼ 6.85 in presence of 0.1M HEPES
buffer solution and 0.05–0.2 units of enzyme. The stock solutions of the inhibitors were prepared by
dissolving 1, 2, 3c, 3d, 7c, and 10b in EtOH and 3b, 4b, 7b, and 10a in HEPES. Stock solution (20–
60mm3) was added to buffered enzyme solutions contained in 10–12 cells placed in the multicell
holder accessory of a Cary 100 spectrophotometer and thermostated at 37 ꢄ 0.02ꢁC. The solutions
were incubated for 15min. Then, appropriate aliquots of the p-nitrophenyl glucoside (thermostated at
37 ꢄ 1ꢁC in Hamilton syringes) were added to the enzyme solutions and the initial rates were followed
at ꢃ ¼ 400 nm by monitoring the formation of p-nitrophenol. Non-competitive inhibition constants (Ki)
were calculated from the equation Ki ¼ v0max[Io]=(vmax–v0max), where v0max and vmax are the maximum
rates measured in presence and in absence of inhibitor and [Io] is the inhibitor concentration. The Ki
values were obtained by means of the Cary software (Enzyme Kinetics) and are reported in Table 1.
The reproducibility was in the range 5–12%.
Acknowledgements
Thanks are due to the University of Bologna for financial support (Ricerca Fondamentale Orientata, ex
quota 60%).
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