Advances in the synthesis of homochiral (-)-1-azafagomine and (+)-5-epi-1-azafagomine. 1-N-phenyl carboxamide derivatives of both enantiomers of 1-azafagomine: Leads for the synthesis of active α-glycosidase inhibitors.

Article abstract of DOI:10.1021/jo201486q

A new expeditious preparation of homochiral ( - )-1-azafagomine and (+)-5-epi-1-azafagomine has been devised. Stoodley's diastereoselective cycloaddition of dienes bearing a 2,3,4,6-tetraacetyl glucosyl chiral auxiliary to 4-phenyl-1,2,4-triazole-3,5-dione was merged with Bols's protocol for functionalizing alkenes into molecules bearing a glucosyl framework. Homochiral (+)-5-epi-1-azafagomine was synthetized for the first time. Partial reductive cleavage of the phenyltriazolidinone moiety afforded new homochiral 1-N-phenyl carboxamide derivatives of 1-azafagomine. Both enantiomers of these derivatives were synthetized and tested, displaying a very good enzymatic inhibition toward baker's yeast α-glucosidase. The molecular recognition mechanism of the 1-N-phenyl carboxamide derivative of 1-azafagomine by α-glucosidase from baker's yeast was studied by molecular modeling. The efficient packing of the aromatic ring of the 1-N-phenyl carboxamide moiety into a hydrophobic subsite (pocket) in the enzyme's active site seems to be responsible for the improved binding affinity in relation to underivatized ( - )-1-azafagomine and (+)-1-azafagomine.

Full text of DOI:10.1021/jo201486q

Article
pubs.acs.org/joc
Advances in the Synthesis of Homochiral (−)-1-Azafagomine and
(+)-5-epi-1-Azafagomine. 1-N-Phenyl Carboxamide Derivatives of
both Enantiomers of 1-Azafagomine: Leads for the Synthesis of
Active α-Glycosidase Inhibitors.
M. Jose Alves,*,† Flora T. Costa,‡ Vera C. M. Duarte,† Antonio Gil Fortes,† Jose A. Martins,†
́
́
and Nuno M. Micaelo†
†
́
Departamento de Quimica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
cias da Saude, Universidade Fernando Pessoa, R. Carlos da Maia, 296, 4100-150 Porto, Portugal
‡Faculdade de Cien
̂
́
S
* Supporting Information
ABSTRACT: A new expeditious preparation of homochiral
(−)-1-azafagomine and (+)-5-epi-1-azafagomine has been
devised. Stoodley's diastereoselective cycloaddition of dienes
bearing a 2,3,4,6-tetraacetyl glucosyl chiral auxiliary to 4-
phenyl-1,2,4-triazole-3,5-dione was merged with Bols's proto-
col for functionalizing alkenes into molecules bearing a
glucosyl framework. Homochiral (+)-5-epi-1-azafagomine was
synthetized for the first time. Partial reductive cleavage of the
phenyltriazolidinone moiety afforded new homochiral 1-N-
phenyl carboxamide derivatives of 1-azafagomine. Both
enantiomers of these derivatives were synthetized and tested,
displaying a very good enzymatic inhibition toward baker's yeast α-glucosidase. The molecular recognition mechanism of the
1-N-phenyl carboxamide derivative of 1-azafagomine by α-glucosidase from baker's yeast was studied by molecular modeling. The
efficient packing of the aromatic ring of the 1-N-phenyl carboxamide moiety into a hydrophobic subsite (pocket) in the enzyme's
active site seems to be responsible for the improved binding affinity in relation to underivatized (−)-1-azafagomine and (+)-
1-azafagomine.
INTRODUCTION
■
The synthesis of iminosugars is receiving increasing interest
because many of these structures are biological tools and
potential therapeutics. The first iminosugar medicine registered
was miglitol (Glyset, PHARMACIA, and UPJOHN).1 The
biological properties of iminosugars arise from their interfer-
ence with glycosidases, the natural carbohydrate degrading
enzymes, and with carbohydrate-recognizing receptors spread
in all living organisms. 1-Deoxynojirimycine (1) is a natural
iminosugar resembling the structure of glucose. The biological
activity of this compound seems to be dependent on its
conjugated ammonium form mimicking the transition state for
glycoside cleavage.2 Bols and co-workers have demonstrated
that (−)-1-azafagomine (2) is a potent competitive inhibitor of
almond β-glucosidase (Ki = 0.32 μM), yeast α-glucosidase (Ki =
6.9 μM), and isomaltase (Ki= 0.27 μM).3 On the other hand,
racemic (±)-5-epi-1-azafagomine (3) was found to be a much
weaker glycosidase inhibitor of almond β-glucosidase (Ki =
137 μM) and Escherichia coli β-galactosidase (Ki = 149 μM)
(Figure 1).4 5-epi-1-Azafagomine (3), as far as we could find,
was previously unknown in any of the enantiomeric pure forms.
The synthesis of homochiral (−)-1-azafagomine (2) was
accomplished by Bols and co-workers through a synthetic
Figure 1. Structure of some known iminosugars.
sequence based on the Diels−Alder cycloaddition to 4-phenyl-
1,2,4-triazole-3,5-dione (PTAD) 4 to achiral dienes: 2,4-penta-
dienoic acid, methyl 2,4-pentadienoate, and 2,4-pentadienol
(5).4 The racemic cycloadduct 6 obtained from 2,4-pentadienol
and PTAD was resolved by lipase-mediated transesterification.
The olefin portion of each enantiomer's precursor of 2 was
oxidized to oxirane and further opened under highly acidic condi-
tions to yield the glucosyl framework of 1-azafagomine, compound
7. After hydrazinolysis, both enantiomers of 1-azafagomine
were obtained in 9% total yield5 (Scheme 1). Osmilation of the
double bond on the racemic cycloadduct 6 led to racemic diol
8, which after hydrazinolysis gave 5-epi-1-azafagomine (3).
Received: November 30, 2010
Published: October 21, 2011
© 2011 American Chemical Society
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Scheme 1
a
Scheme 2
a
(i) Et3SiH, TFA, DCM, 5 h, rt, 61%; (ii) oxone, CF3COCH3, NaHCO3, CH3CN/H2O (3.5:2), 24 h, 65%; (iii) H2O, H2SO4 (exc.), reflux, 8 h,
52%; (iv) NaBH4 (3 equiv), EtOH, 3 d, rt, 59%; (v) NH2NH2·H2O, 100 °C, 18 h, 68% (−)-2, 64% (+)-3; (vi) OsO4, NMO, acetone:H2O (2:1),
5 d, rt, 79%; (vii) NaBH4 (3 equiv), EtOH, 3 d, rt, 52%.
Bols3 also achieved the synthesis of (−)-1-azafagomine (2)
from relatively expensive L-xylose in six steps. L-2,3,5-Tribenzyl
xylofuranose was isolated as an intermediate after three steps
with no explicit yield. 1-Azafagomine was then isolated in 37%
overall yield from this intermediate.6,7
temperature for 1 day generated a 3:1 mixture of oxiranes as
reported previously by Bols for his racemic compounds.4
Selective crystallization afforded the major isomer 12 in 65%
yield. Opening of oxirane 12 was achieved with total regio- and
stereoselectivity by refluxing in aqueous H2SO4 giving 13.
Osmilation of compound 11 produced cis-diol 14 with total
stereoselectivity. Selective reduction of trans-diol 13 and cis-diol
14 with NaBH4 gave, respectively, triols 15 and 16. These
compounds were further treated with hydrazine under reflux to
produce the target compounds: (−)-1-azafagomine (2) in 14%
overall yield and (+)-5-epi-1-azafagomine (3) in 26% overall yield
from alkene (−)-11 (Scheme 2). Whereas (−)-1-azafagomine 2 is
a known compound, (+)-5-epi-1-azafagomine 3 was obtained
enantiomerically pure for the first time. Pure (+)-5-epi-1-
azafagomine displays NMR spectra compatible with the data
published for the racemic compound 3. The specific optical
rotation obtained for (+)-5-epi-1-azafagomine is αD = +65 (H2O,
c = 0.70). The specific optical rotation value measured for (−)-1-
azafagomine, αD = −20 (H2O, c = 0.85), differs from the one
reported in the literature, αD = −9.8 (H2O, c = 0.85).3
New Synthetic Sequence for Preparing Homochiral
1-N-Phenyl Carboxamide Derivatives of 1-Azafagomine
(+)-22 and (−)-22. Stoodley was able to prepare the precursors
of (−)-22 and (+)-22 from (E,E)-diene 9b and (E,Z)-diene 17,
respectively, by the method described in Scheme 3.9
(E,E)-Diene 9b was isolated in 70% yield and (E,Z)-diene 17
in 14% yield. Applying Still's olefination, the yield of the (E,Z)-
diene could be improved to 36%9 (Scheme 3). Having in mind
the shortcomings in the synthesis of compound 17,
epimerization of compound 10 obtained by Stoodley's method
was tried in various conditions: (i) triethylamine in MeOH, (ii)
NaN3 in MeOH, and (iii) triethylamine/p-chlorothiophenol in
Alternatives to the enzymatic resolution of racemic adducts
of type 6 are desirable for the production of chiral synthons for
further elaboration into homochiral compounds. Stoodley, in
the 1990s, combined 2,4-pentadienoates, bearing a tetraacetyl
glucosyl chiral auxiliary in the position 1, compound 9a, with
PTAD to obtain cycloadduct 10 in 70% yield and in a high
degree of diastereoselectivity (Scheme 2).8,9 The chemistry of
cycloadduct 10 had been pushed forward for the synthesis of
dehydropiperazic acid, a nonproteinogenic amino acid con-
stituent of antrimycins-linear heptapeptides with antitubercular
activity.10 Lately, dienes bearing oxazolidinone chiral auxiliary
were combined with PTAD to generate (S)-piperazinic acid.11
To the best of our knowledge, nobody has merged the Stoodley
cycloaddition entry into chiral alkenes of type 11 with Bols's
olefin functionalization methodology for synthetizing enantio-
pure iminosugars.
RESULTS AND DISCUSSION
■
New Synthetic Sequence for Preparing Homochiral
(−)-1-Azafagomine and (+)-5-epi-1-Azafagomine. In this
paper we report a new synthetic route for obtaining homochiral
(−)-1-azafagomine (−)-2 and (+)-5-epi-1-azafagomine (+)-3
from chiral alkene (−)-11 (Scheme 2).
Cycloadduct 10 was submitted to reductive cleavage with
triethylsilane, according to Stoodley's protocol, to generate
known compound (−)-11.9 Treatment of 11 with oxone/
trifluoroacetone in the presence of NaHCO3 at room
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Scheme 3. Stoodley’s Synthetic Sequence for Precursors Related to Compounds (−)-2, (−)-22, and (+)-22
a
Scheme 4. Epimerization of Compound 10 into Compound 18
a
Reagents: NEt3, p-chlorothiophenol, MeOH, 0 °C → rt.
MeOH (Scheme 4). When triethylamine was the sole reagent,
isolation of compound 18 was difficult because of the competing
elimination of glucosyl moiety giving the 1,3-diene compound.
The same applied for the attempt with NaN3. The mixture of
triethylamine/p-chlorothiophenol afforded after 40 min of reaction
88% yield of compound 18. This represents an important
achievement concerning the synthesis of compound 18. Extending
the reaction time, a Michael addition of p-chlorothiophenol
occurs, leading to compound (±)-19 (Scheme 4).
The structure of compound 18 was unambiguously
confirmed by X-ray crystallography (Figure 2).
Figure 3. ORTEP view of compound 20.
The Schiff salt initially formed by reduction of one of the
carbonyl groups is trapped by internal nucleophilic attack of the
alcohol function. A large excess of hydride was necessary to
cleave intermediate 20 to final product 21. Compounds 20 and
21 display a major difference in their 13C NMR spectra: a peak
at δC = 86.3 ppm, assigned to the methylene attached to the
oxygen and nitrogen atoms in compound 20, is not apparent in
the 13C NMR spectrum of compound 21.
ii. In the Synthesis of Compounds (−)-22 and (+)-22. At-
tempted epoxidation of compound 21 was unsuccessful, leading
to a complex mixture. As an alternative, compound (−)-13 was
subjected to treatment with LiAlH4 in THF to give compound
(−)-22. The synthesis of compound (+)-22 was obtained from
18 (Scheme 4) by reductive cleavage of the glucosyl moiety to
give (+)-11 followed by the functional group transformation
described in Scheme 6.
The enantiopure compounds (−)-1-N-phenyl carboxamide
1-azafagomine (−)-22 and (+)-1-N-phenyl carboxamide
1-azafagomine (+)-22 were obtained following the same
sequence of reactions in 29 and 10% overall yield starting
from compounds 10 and 18, respectively.
Figure 2. ORTEP view of compound 18.
Reduction of the Ester and Urea Groups in the
Triazolidinone Moiety. i. In the Synthesis of Compound
(−)-21. When compound (−)-11 was treated with 7 equi-
valents of freshly opened LiAlH4, a new compound formed,
1
according to H NMR spectroscopy. If the LiAlH4 was not
strictly fresh, a mixture of two compounds was observed on the
1H NMR spectrum. Further treatment with LiAlH4 converted
the mixture into the same compound observed before. The
structure of the intermediate in the reduction process was
determined by X-ray crystallography and identified as
compound 20 (Figure 3).
Knowledge of the bridged structure of compound 20 allowed
us to propose a plausible mechanism for its formation and the
formation of compound 21 (Scheme 5).
1-N-Phenyl Carboxamide Derivatives of 1-Azafagomine:
New Leads for the Synthesis of Potent α-Glycosidase
Inhibitors. A structure−activity relationship study of a series
2-N-alkylated 1-azafagomines as glycosidase inhibitors revealed
that these compounds are better β-glycosidase inhibitors than
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a
Scheme 5
a
(i) LiAIH4 (7 equiv from a bottle opened for a long time), THF, 4 h, 0 °C → rt, 86%; (ii) LiAIH4 (15 equiv from a recently opened bottle), THF,
4, 0 °C → rt, 48%.
Scheme 6
retains the nucleophile aspartate 214 and the catalytic residues
glutamate 276 and aspartate 349. Our theoretical binding
affinity estimate for (+)-22 and (−)-22 against yeast
α-glucosidase are of −7.8 and −7.5 kcal/mol, respectively.
The binding affinity free energy difference between the two
enantiomers is within the docking standard error (∼2 kcal/mol),
and consequently, it suggests low enantiomeric discrimination
of (+)-22 and (−)-22. However, our experimental Ki data
(Table 1) indicates a preferential binding of (−)-22 when
compared to (+)-22. This discrepancy can be explained by the
lack of mobility of enzyme to reorganize during the docking
experiments and, consequently, to properly recognize the most
potent enantiomeric compound, (−)-22. Despite this, the
observation of the binding pose of both enantiomers of 22
provides a structural explanation of their binding mechanism
(Figure 4) and a rational approach for their future improve-
ment. These complexes correspond to lowest binding energy
pose. They belong to the most populated cluster of docking
solutions, 7 and 8 docking poses out of 20, respectively, for
each enantiomer.
The α-glucosidase enzyme active site is characterized by
two distinct regions: a highly polar region due to the presence
of histidine 348, glutamate 276, aspartate 68, 214, and 349,
and arginine 212 and 439 and a hydrophobic pocket flaked
by phenylalanine 157 and 177, leucine 218, and alanine
278 (Figure 4). The binding affinity between the two 22
enantiomers and yeast α-glucosidase is the result of several
strong hydrogen bonding interactions between two hydroxyl
and amine groups of (+)-22 and (−)-22 with aspartate 68, 214,
and 349, arginine 212 and 439, and histidine 348 of the
α-glucosidase enzyme active site. The highlighted nonbonded
interactions of (−)-22 with histidine 348, arginine 439, and
aspartate 68 are shorter for this enantiomer when compared to
(+)-22, suggesting a stronger interaction of (−)-22 with the
enzyme. On the basis of these observations, the molecular
modeling study suggests that the binding affinity can be
improved either by (a) increasing the length of the N-phenyl-
1-carboxamide moiety or (b) introducing donor/acceptor
moieties on the p-position of the aromatic ring. These avenues
are being currently pursued in order to improve the binding
affinity and, ideally, selectivity.
α-glycosidase inhibitors. Moreover, the inhibition constant (Ki)
was found to be dependent on the chain length. The best
results have been obtained with the N-propylphenyl derivative
23 (Ki = 0.032 μM) and the N-hexyl derivative 24 (Ki =
0.055 μM)12 (Table 1).
The N-propylphenyl derivative 23 is around an order of
magnitude more effective as a β-glucosidase inhibitor than
(−)-1-azafagomine 2. On the other hand, derivative 23 is a
much weaker inhibitor of α-glucosidase than its parent com-
pound (2), making compound 23 a potent inhibitor selective
for β-glucosidase.12
The most striking results of the inhibition studies with the
1-N-phenyl carboxamide derivatives of 1-azafagomines 22 are
Ki values toward α-glucosidase substantially lower than the
N-propylphenyl derivative 23. Compound (−)-22, displaying
the same stereochemistry as (−)-1-azafagomine 2, is around
two times more active, whereas its isomer (+)-22 is slightly less
active. Both enantiomers of compound 22 display lower activity
toward β-glucosidase than their parent compound 2 and the
N-propylphenyl derivative. A moderate α/β selectivity was
observed for compounds 22. The low Ki values obtained for
compounds 22 toward α-glucosidase suggests an efficient
recognition mechanism between both enantiomers and the
enzyme. The levorotatory isomer was slightly more active than
the dextrorotatory isomer (Table 1). This observation is in
contrast with the results reported by Bols, who demonstrated
that (−)-1-azafagomine is the active enantiomer, whereas
(+)-1-azafagomine is virtually inactive toward the same
α-glucosidase that was used in this study.3
The yeast α-glucosidase enantioselective discrimination
toward (+)-22 and (−)-22 was studied using molecular
docking methodologies. This enzyme, as well as the
Saccharomyces cerevisiae enzyme used for the homology
modeling,13 belongs to the glycoside hydrolase family 13
(GH 13). This family of retaining glucosidases is characterized
by strong recognition of the a glucoside moiety of synthetic
p-nitrophenyl glucosides and heterogeneous substrates such as
sucrose, while being inactive toward hydration of D-glucal and
the hydrolysis of p-nitrophenyl α-2-deoxyglucosides.14,15 The
active site structure in Figure 4 illustrates that this enzyme
CONCLUSIONS
■
In this paper we report the preparation of homochiral
1-azafagomine (−)-2 and (+)-5-epi-1-azafagomine (+)-3. The
synthetic route devised merges Stoodley diastereoselective
Diels−Alder cycloaddition methodology with Bols protocol
for functionalizing alkenes into molecules bearing sugar-like
frameworks. Novel 1-N-phenyl carboxamide derivatives of
1-azafagomine 22 were obtained in enantiomeric pure forms.
The epimerization of cycloadduct 10 was revealed to be the key
step in the synthesis of the dextrorotatory compound 22. This
methodology represents an advantageous alternative to other
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Table 1. Ki Values (μM) for the Inhibition of α- and β-Glucosidases by Compounds 22 and Other Azasugars at Different
pH Values
a
b
c
d
pH 6.8. pH 7.0. pH 5.0. Enzyme inactive.
Figure 4. Structure of the lowest binding free energy complexes between yeast α-glucosidase binding site and the enantiomers of compound 22: (A)
(+)-22 and (B) (−)-22. The figure is rendered with Corey−Pauling−Koltun (CPK) coloring scheme. Selected side-chain residues of the yeast
α-glucosidase binding pocket are rendered in sticks and labeled with the three-letter amino acid code name and sequence residue number.
Compounds are rendered in ball-and-stick style.
more conventional approaches for obtaining enantiopure (+)-2
(not isolated in this work) and its derivatives (e.g., N-phenyl
carboxamides). Compounds 22 were tested as inhibitors against
α- and β-glucosidases. Both enantiomeric forms of 22 are
potent inhibitors of α-glucosidase, in contrast to the current
wisdom that only (−)-2 enantiomer of 1-azafagomine is active
toward α- and β-glucosidase. The low Ki value determined
toward α-glucosidase inhibition is particularly relevant in
comparison with its analogue N-propylphenyl azafagomine,
the compound in Table 1 with a closer side chain length. The
molecular recognition mechanism between the enatiomeric
compounds 22 and the α-glucosidase studied by molecular
modeling has shown that the aromatic group is accommodated
in a hydrophobic pocket of the enzyme binding site with polar
characteristics at its end. This evidence has provided further
clues for improving the binding affinity and, possibly, the α/β
selectivity by increasing the length of the N-carboxamide
moiety and the introduction of donor/acceptor hydrogen bond
groups on the aromatic ring.
The results of this study suggest that 1-N-phenyl
carboxamide derivatives of 1-azafagomine are potential new
leads for the synthesis of potent α-glycosidase inhibitors.
EXPERIMENTAL SECTION
■
General Methods. Solvents were distilled under anhydrous
conditions. The (S)-ethyl 2-phenyl-2,4,9-triazabicyclo[4.3.0]non-6-
ene-1,3-dione-5-carboxylate (−)-11 was obtained according to
Stoodley's protocol for the (S)-methyl carboxylate derivative; 2,3,4,6-
tetra-O-acetyl-α-D-glucopyranosyl bromide (ABG) was prepared
according to the literature,16 and potassium 3-hydroxypropenal17 was
combined to ABG18 followed by addition of tributylphosphorane.9
The diene obtained was subjected to 4-phenyl-1,2,4-triazole-3,5-dione
(PTAD) to obtain the adduct 10. The glucose moiety was removed
by reduction with triethylsilane.10 Compound 7 (R = CH2OH) was
obtained according to the literature.4 Functionalization of the double
bond was obtained by osmilation with OsO4 in acetone/water or with
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oxone, trifluoromethylacetone, in the presence of NaHCO3 and
aqueous acetonitrile. Reduction of the oxazolidinone was done either
with freshly opened LiAlH4 1 M in THF or with long-term opened
bottles (over a month) of LiAlH4 1 M in THF. All reagents were
purchased and used without further purification. Glassware was dried
prior to use. Compounds were purified by dry flash chromatography
using silica 60, <0,063 mm and water pump vacuum or by flash-
chromatography using silica 60A 230−400 mesh as stationary phases.
TLC plates (silica gel 60 F254) were visualized either at a UV lamp or
in I2.
(C−H, Ph), 129.1 (C−H, Ph), 131.1 (Cq, Ph), 152.3 (CO), 153.3
(CO), 166.7 (CO ester) ppm. EA Calcd for C15H15N3O4, C,
59.79%; H, 5.02%; N, 13.95%. Found, C, 59.90%; H, 4.93%; N,
13.86%.
Synthesis of (R)-Ethyl 2-Phenyl-2,4,9-triazabicyclo[4.3.0]-
non-6-ene-1,3-dione-5-carboxylate (+)-11. To a solution of the
cycloadduct 18 (1.03 g; 1.59 mmol) in DCM (25 mL) were added
triethylsilane (9.85 mL; 0.78 mol) and trifluoroacetic acid (9.85 mL;
0.17 mol). The resulting yellow solution was kept under stirring at rt
for 5 h. The solvent was removed under vacuum, and the residue was
redissolved in DCM (25 mL). The solution was washed with an
aq saturated solution of NaHCO3 (3 × 25 mL) and water (25 mL).
The combined organic layers were dried over magnesium sulfate
and filtered, and the solvent was evaporated. From the residual oil
crystallized a white solid that was washed with diethyl ether and
Synthesis of Ethyl 5-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyra-
nosyloxy)-2,4-pentadienoate 9. To a solution of ethyl tributyl
phosphorane9 (3.37 g; 11.70 mmol) in DCM (15 mL) was added
3-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosyloxy)-1-propenal17 (1.32 g;
3.42 mmol); the orange solution formed was stirred at rt for 24 h. The
solvent was evaporated, giving an oil subjected to dry-flash
chromatography (petroleum ether/diethyl ether; gradient of polarity)
20
proved to be the title compound ((+)-11, 0.292 g; 61%): [α]D
+370.0° (c = 1, CH2Cl2).
(9, 1.17 g; 63%): [α]D +27.0° (c = 1, CH2Cl2); νmax (Nujol, cm−1)
20
Synthesis of (5S,6R,7S)-Ethyl 6,7-Epoxy-2-phenyl-2,4,9-
triazabicyclo[4.3.0]nonano-1,3-dione-5-carboxylate (−)-12. To
a solution of compound (−)-11 (0.48 g; 1.59 mmol) in acetonitrile
(28.0 mL), water (16.0 mL) and 1,1,1-trifluoracetone (3.21 mL), were
added solid NaHCO3 (2.44 g; 29.02 mmol) and oxone (12.00 g;
39.04 mmol) for 20 min at 0 °C. The mixture was stirred for 18 h. A
new portion of solid NaHCO3 (2.44 g; 29.02 mmol) and oxone (12.00 g;
39.04 mmol) was added and stirred for another 4 h. Then water
(100 mL) was added to the reaction mixture, which was extracted with
CHCl3 (8 × 40 mL). The organic layers were combined and dried
with magnesium sulfate. After removal of the solvent, recrystallization
with diethyl ether gave a white solid identified as the title compound
((−)-12, 0.329 g; 65%): [α]D20 − 238.0° (c = 0.8, CH2Cl2); mp 218−
220 °C; νmax (Nujol, cm−1) 2950, 2923, 1775, 1750, 1715, 1458, 1094,
1
2955, 2934, 1736, 1698, 1635; H NMR (δH, 300 MHz, CDCl3) 1.27
(3H, t, J 7.2 Hz, CH3), 2.01, 2.03, 2.04, 2.08 (12H, 4 × s, 4 ×
CH3CO2), 3.81 (1H, ddd, J 9.0, 6.0, 3.0 Hz, H-5′), 4.13 (1H, dd, J 9.0,
3.0 Hz, H-6′), 4.18 (2H, q, J 6.0 Hz, CH2), 4.26 (1H, dd, J 12.0, 3.0
Hz, H-6′), 4.87 (1H, d, J 9.0 Hz, H-1′), 5.08−5.29 (3H, m, H-2′ + H-3′
+ H-4′), 5.78 (1H, d, J 15.0 Hz, H-2), 5.90 (1H, t, J 12.0 Hz, H-4),
6.81 (1H, d, J 12.0 Hz, H-5), 7.20 (1H, dd, J 15.0, 12.0 Hz, H-3) ppm;
13C NMR (δC, 75.5 MHz, CDCl3) 14.2 (CH3), 20.5, 20.5, 20.7
(CH3CO2), 60.1 (CH2), 61.6 (C-6′), 67.8, 70.6, 72.3 (C-2′, C-3′, C-4′),
72.4 (C-5′), 99.7 (C-1′), 110.1 (C-4), 118.7 (C-2), 140.9 (C-3), 152.6
(C-5), 167.0 (CO ester), 169.1, 169.2, 170.1, 170.5 (CH3CO2) ppm.
Synthesis of (5S,8S)-Ethyl 8-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-
glucopyranosyloxy)-2-phenyl-2,4,9-triazabicyclo[4.3.0]non-6-
eno-1,3-dione-5-carboxylate 10. To a solution of diene 9 (1.31 g;
2.86 mmol) in DCM (15 mL) was added 4-phenyl-1,2,4-triazole-3,5-
dione (0.50 g; 2.86 mmol), giving a red colored solution that quickly
lost its color. The reaction mixture was stirred for a further 30 min and
then evaporated. The residue was tritured with ethyl ether. A white
solid was formed and filtered, giving the title compound (10, 1.302 g;
70%): [α]D20 +23,8° (c = 1, CH2Cl2); νmax (Nujol, cm−1) 2954, 2853,
1743, 1620, 1635, 1218; 1H NMR (δH, 300 MHz, CDCl3) 1.35 (3H, t,
J 6.0 Hz, CH3), 2.00, 2.01, 2.02, 2.04 (12H, 4 × s, 4 × CH3CO2), 3.87
(1H, ddd, J 12.0, 6.0, 3.0 Hz, H-5′), 4.05 (1H, dd, J 12.0, 3.0 Hz, H-6′),
4.27 (1H, dd, J 15.0, 9.0 Hz, H-6′), 4.34 (2H, dq, J 7.2, 1.2 Hz, CH2),
5.01 (2H, m, H-5 + H-2′), 5.09 (1H, t, J 9.9 Hz, H-4′), 5.19−5.26 (2H,
m, H-3′ + H-1′), 6.06−6.12 (2H, m, H-8 + H-7), 6.19−6.26 (1H, m,
H-6), 7.39−7.56 (5H, m, Ph) ppm; 13C NMR (δC, 75.5 MHz, CDCl3)
14.0 (CH3), 20.5, 20.6, 20.6 (CH3CO2), 56.7 (C-5), 61.5 (C-6′), 62.9
(CH2), 67.8 (C-4′), 71.0 (C-2′), 72.1 (C-5′), 72.8 (C-3′), 74.6 (C-8),
97.2 (C-1′), 123.3 (C-7), 124.9 (C-6), 125.4 (C−H, Ph), 128.5 (C−H,
Ph), 129.2 (C−H, Ph), 130.7 (Cq, Ph), 150.2 (CO), 151.4 (C
O), 165.6 (CO ester), 169.4, 169.4, 170.2, 170.7 (CH3CO2) ppm.
HRMS (FAB): Calcd for C29H33N3O14, 648.2041. Found, 648.2041.
Synthesis of (S)-Ethyl 2-Phenyl-2,4,9-triazabicyclo[4.3.0]-
non-6-ene-1,3-dione-5-carboxylate (−)-11. To a solution of the
cycloadduct 10 (1.31 g; 2.03 mmol) in DCM (20 mL) were added
triethylsilane (12.7 mL; 0.78 mol) and trifluoroacetic acid (12.7 mL;
0.17 mol). The resulting yellow suspension was kept under stirring at
rt for 5 h. The solvent was removed under vacuum and the residue
redissolved in DCM (30 mL). The solution was washed with an aq
saturated solution of NaHCO3 (3 × 50 mL) and water (50 mL). The
combined organic layers were dried over magnesium sulfate and
filtered, and the solvent was evaporated. From the residual oil
crystallized a white solid that was washed with diethyl ether and
1
1034; H NMR (δH, 300 MHz, CDCl3) 1.32 (3H, t, J 7.1 Hz, CH3),
3.53−3.65 (2H, m, H-7 + H-8), 3.89 (1H, dd, J 5.6, 3.8 Hz, H-6),
4.19−4.40 (2H, m, CH2), 4.47 (1H, dd, J 13.6, 1.4 Hz, H-8), 5.01 (1H,
d, J 5.7 Hz, H-5), 7.33−7.51 (5H, m, Ph) ppm; 13C NMR (δC, 75.5
MHz, CDCl3) 14.1 (CH3), 43.0 (C-8), 49.0 (C-6), 50.1 (C-7), 54.8
(C-5), 62.7 (CH2), 125.6 (C−H, Ph), 128.4 (C−H, Ph), 129.1 (C−H,
Ph), 130.9 (Cq, Ph), 153.3 (CO), 153.56 (CO), 165.6 (CO
ester) ppm. HRMS (FAB): Calcd for C15H16N3O5, 318.1089. Found,
318.1087.
Synthesis of (5R,6S,7R)-Ethyl 6,7-Epoxy-2-phenyl-2,4,9-
triazabicyclo[4.3.0]nonano-1,3-dione-5-carboxylate (+)-12. To
a solution of compound (+)-11 (0.37 g; 1.21 mmol) in acetonitrile
(21.1 mL), water (12.3 mL), and 1,1,1-trifluoracetone (2.40 mL) were
added solid NaHCO3 (1.86 g; 29.02 mmol) and oxone (9.15 g;
39.04 mmol) for 20 min at 0 °C. The mixture was stirred for 18 h. A
new portion of solid NaHCO3 (1.86 g; 29.02 mmol) and oxone (9.15 g;
39.04 mmol) was added and stirred for another 4 h. Then water (60 mL)
was added to the reaction mixture, which was extracted with DCM
(10 × 40 mL). The organic layers were combined and dried with
magnesium sulfate. After removal of the solvent, recrystallization with
diethyl ether gave a white solid identified as the title compound
20
((+)-12, 0.233 g; 60%): [α]D + 232.8° (c = 0.8, CH2Cl2).
Synthesis of (5S,6R,7R)-Ethyl 6,7-Dihydroxy-2-phenyl-2,4,9-
triazabicyclo[4.3.0] Nonane-1,3-dione-5-carboxylate (−)-13. To
a solution of epoxide (−)-12 (0.20 g; 0.63 mmol) in water (30 mL)
was added concentrated H2SO4 (0,5 mL), and the mixture was
refluxed for 8 h. After this time, solid NaHCO3 (0.86 g; 10.24 mmol)
was added, and the water was evaporated until dryness. The residue
was dissolved in ethyl acetate (100 mL) and washed with NaCl (50 mL).
The organic phase was separated, and the aqueous phase was extracted
with ethyl acetate (100 mL). The organic phases were combined and
dried with magnesium sulfate, filtered, and concentrated in the rotary
evaporator. The yellowish solid obtained was washed with diethyl
ether and found to be the title compound ((−)-13, 0.110 g; 52%):
[α]D20 −22.8° (c = 2, acetone); νmax (Nujol, cm−1) 3596−3540, 2954,
20
proved to be the title compound ((−)-11, 0.313 g; 51%): [α]D
−311.0° (c = 2.15, CH2Cl2); mp 140−142 °C; νmax (Nujol, cm−1)
2954, 2923, 1742, 1714; 1H NMR (δH, 300 MHz, CDCl3) 1.29 (3H, t,
J 7.2 Hz, CH3), 3.99−4.06 (1H, dm, H-8), 4.25 (2H, q, J 7.2 Hz,
CH2), 4.38−4.45 (1H, dm, H-8), 5.09−5.12 (1H, m, H-5), 6.05−6.16
(2H, m, H-6 + H-7), 7.38−7.57 (5H, m, Ph) ppm; 13C NMR (δC,
75.5 MHz, CDCl3) 14.1 (CH3), 43.1 (C-8), 55.9 (C-5), 62.4 (CH2),
119.7 (C-6 or C-7), 123.3 (C-6 or C-7), 125.6 (C−H, Ph), 128.2
1
2923, 1729, 1698, 1122, 1088; H NMR (δH, 400 MHz, CDCl3) 1.24
(3H, m, CH3), 3.64 (1H, d, J 12.0 Hz, H-8), 3.95 (1H, bs, H-7), 3.99
(1H, d, J 12.8 Hz, H-8), 4.13−4.28 (2H, m, CH2), 4.46 (1H, t, J
2.8 Hz, H-6), 4.74 (1H, d, J 2.8 Hz, H-5), 7.38−7.50 (5H, m, Ph) ppm;
9589
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Article
13C NMR (δC, 100 MHz, CDCl3) 13.9 (CH3), 44.3 (C-8), 59.4 (C-5),
10 (0.22 g; 0.33 mmol) in methanol (5 mL) were added
4-chorothiophenol (0.10 g; 0.68 mmol) and triethylamine at 0 °C
and under magnetic stirring. After 40 min, the solvent was evaporated,
and the crude was subjected to dry-flash chromatography (petroleum
ether/ether 1:3). The product was obtained as a white solid (18;
62.6 (CH2), 65.9 (C-7), 67.4 (C-6), 125.9 (C−H, Ph), 128.6 (C−H,
Ph), 129.3 (C−H, Ph), 131.0 (Cq, Ph), 152.1 (CO), 154.1 (C
O), 167.3 (CO ester) ppm. HRMS (FAB): Calcd for C15H18N3O6,
336.1196. Found, 336.1207.
20
0.187 g; 0.30 mmol; 88%): mp 154−157 °C; [α]D + 219.7° (c = 1,
Synthesis of (5R,6S,7S)-Ethyl 6,7-Dihydroxy-2-phenyl-2,4,9-
triazabicyclo[4.3.0] Nonane-1,3-dione-5-carboxylate (+)-13. To
a solution of epoxide (+)-12 (0.23 g; 0.73 mmol) in water (35 mL)
was added concentrated H2SO4 (0.7 mL), and the mixture was
refluxed for 10 h. After this time, solid NaHCO3 (1.42 g; 16.90 mmol)
was added, and the water was evaporated until dryness. The residue
was dissolved in ethyl acetate (100 mL) and washed with NaCl (50 mL).
The organic phase was separated, and the aqueous phase was extracted
with ethyl acetate (3 × 100 mL). The organic phases were combined
and dried with magnesium sulfate, filtered, and concentrated in the
rotary evaporator. The yellowish solid obtained was washed with
diethyl ether and found to be the title compound ((+)-13, 0.121 g;
1
acetone); νmax (Nujol, cm−1) 2955, 2924, 1744, 1723, 1226; H NMR
(δH, 400 MHz, CDCl3) 1.28 (3H, t, J 7.2 Hz, CH3), 1.88, 1.98, 2.02,
2.07 (12H, 4 × s, 4 × CH3CO2), 3.74−3.79 (1H, m, H-5′), 4.15 (1H,
dd, J 12.4, 2.4 Hz, H-6′), 4.23 (2H, q, J 7.2 Hz, CH2), 4.25 (1H, dd,
J 12.0, 4.4 Hz, H-6′), 4.95 (1H, dd, J 9.6, 8.0 Hz, H-2′), 5.07 (1H, t,
J 10.0 Hz, H-4′), 5.15 (1H, dd, J 5.2, 2.0 Hz, H-5), 5.18−5.23 (2H, m,
H-1′ + H-3′), 5.96 (1H, d, J 4.8 Hz, H-8), 6.07 (1H, ddd, J 10.0, 4.4,
2.0 Hz, H-6), 6.31 (1H, ddd, J 10.0, 5.2, 0.8 Hz, H-7), 7.27−7.56 (5H,
m, Ph) ppm; 13C NMR (δC 100 MHz, CDCl3) 14.0 (CH3), 20.5, 20.5,
20.7 (CH3CO2), 56.0 (C-5), 61.6 (C-6′), 62.7 (CH2), 68.0 (C-4′), 71.1
(C-2′), 72.2 (C-5′), 72.6 (C-3′), 76.0 (C-8), 99.6 (C-1′), 123.9 (C-7),
124.1 (C-6), 125.6 (C−H, Ph), 128.6 (C−H, Ph), 129.2 (C−H, Ph),
130.8 (Cq, Ph), 151.5 (CO), 153.4 (CO), 166.0 (CO ester),
169.2, 169.4, 170.1, 170.6 (CH3CO2) ppm. EA Calcd for
C29H33N3O14, C, 53.79%; H, 5.14%; N, 6.49%. Found, C, 53.58%;
H, 5.23%; N, 6.38%.
Synthesis of (S)-N-Phenyl-3-oxa-1,9-diazabicyclo[3.3.1]non-
6-ene-9-carboxylate 20. To a solution of ester (−)-11 (0.205 g;
0.68 mmol) solubilized in dry THF (13 mL) was added LiAlH4 1 M in
THF (7 equiv, 5.2 mL), from a flask containing a white deposit, at
0 °C. The mixture was kept under stirring for 4 h at rt. The reaction
was quenched with a sequence addition of water (1 drop), aq NaOH
15% (2 drops), and water (1 drop), during which time a large amount
of H2 was released. Then a portion of water (15 mL) was added, and
the mixture was extracted with ethyl acetate (4 × 25 mL). The
combined organic layers were washed with saturated aq NaHCO3
(25 mL) and brine (25 mL) and then dried over MgSO4. After
evaporation of the ethyl acetate, a yellowish crude crystallized giving
20 (0.088 g; 0.36 mmol; 48%): [α]D20 −57.5° (c = 0.4, CHCl3); νmax
(Nujol, cm−1) 3320, 1670, 1604, 1591, 1530; 1H NMR (δH, 300 MHz,
CDCl3) 3.45 (1H, dd, J 18.3, 1.2 Hz, H-8), 3.71 (1H, d, J 10.5 Hz,
H-4), 3.90 (dd, J 2.7, 11.1 Hz, H-4), 4.00 (1H, dd, J 18.3, 1.2 Hz, H-8),
4.56 (1H, d, J 10.5 Hz, H-2), 4.66 (1H, bs, H-5), 4.69 (d, J 10.5 Hz,
H-2), 6.07 (2H, bs, H-6+ H-7), 7.04 (t, J 7.2 Hz, CH, Ph), 7.31 (2H, t,
J 7.2 Hz, CH, Ph), 7.49 (2H, d, J 7.2 Hz, CH, Ph), 8.00 (1H, bs, NH)
ppm; 13C NMR (δC, 100 MHz, CDCl3) 44.4 (C-5), 50.7 (C-8), 66.7
(C-4), 86.3 (C-2), 118.7 (CH, Ph), 122.9 (CH, Ph), 127.8 (C-7 or
C-6), 128.9 (C-6 or C-7), 128.9 (C−H, Ph), 138.5 (Cq, Ph), 153.1
(CO) ppm. HRMS (FAB): Calcd for C13H16N3O2, 246.124252.
Found, 246.124172.
20
49%): [α]D +26.9° (c = 0.5, acetone).
Synthesis of (5S,6R,7S)-Ethyl 6,7-Dihydroxy-2-phenyl-2,4,9-
triazabicyclo[4.3.0] Nonane-1,3-dione-5-carboxylate 14. To a
solution of (−)-11 (0.30 g; 1.00 mmol) in acetone (1 mL) and water
(0.5 mL) were added 4-methylmorpholine N-oxide (0.18 g; 1.49
mmol) and a solution of OsO4 in water 4% (108 mL). The mixture of
stirred for 5 days. Then an aq solution of Na2S2O3 5% (25 mL) was
added to mixture, which was stirred for 15 min. The solution was
extracted with ethyl acetate (4 × 30 mL) and the organic phases were
washed with water (10 mL). The organic phase was dried over
MgSO4, filtered, and concentrated to give a white solid (14, 0.26 g;
79%): [α]D −110.6° (c = 2.05, acetone); νmax (Nujol, cm−1) 3425,
20
1
1768, 1749, 1736, 1287, 1204; H NMR (δH, 400 MHz, CDCl3) 1.27
(3H, t, J 7.2 Hz, CH3), 3.35 (1H, d, J 10.8 Hz, H-8), 3.83 (1H, ddd,
J 10.0, 5.2, 2.8 Hz, H-7), 4.03 (1H, dd, J 11.6, 5.2 Hz, H-8), 4.24 (2H,
q, J 7.2 Hz, CH2), 4.52 (1H, t, J 2.8 Hz, H-6), 4.90 (1H, d, J 3.6 Hz,
H-5), 7.40−7.49 (5H, m, Ph) ppm; 13C NMR (δC, 100 MHz, CDCl3)
14.0 (CH3), 43.2 (C-8), 60.6 (C-5), 62.8 (CH2), 65.1 (C-7), 67.2
(C-6), 125.8 (C−H, Ph), 128.6 (C−H, Ph), 129.3 (C−H, Ph), 130.9
(Cq, Ph), 151.4 (CO), 153.8 (CO), 166.4 (CO ester) ppm.
HRMS (FAB): Calcd for C15H18N3O6, 336.1196. Found, 336.1195.
Synthesis of (5S,6R,7R)-6,7-Dihydroxy-5-hydroxymethyl-2-
phenyl-2,4,9-triazabicyclo[4.3.0] Nonane-1,3-dione 15. To a
solution of the diol (−)-13 (0.07 g; 0.22 mmol) in ethanol (3 mL) was
added NaBH4 (8 mg; 0.22 mmol) under magnetic stirring at room
temperature. After 1 h, an aliquot was quenched with HCl 0.4 M,
extracted with ethyl acetate, dried over magnesium sulfate, and
1
concentrated. H NMR spectrum showed that the reaction was not
completed, so a new amount of NaBH4 (8 mg; 0.22 mmol) was added,
and the mixture was stirred for another 4 h. The procedure was
repeated with addition of NaBH4 (8 mg; 0.22 mmol). The reaction
was quenched with aq HCl 0.4 M (4.4 mL); the mixture was stirred for
10 min and evaporated. The residue was dissolved in water (10 mL)
and extracted with ethyl acetate (8 × 15 mL). The organic phases were
combined and dried over magnesium sulfate. Evaporation of the
solvent gave a white solid identified as the title compound (15, 0.037
Synthesis of (S)-6-(Hydroxymethyl)-N-phenyl-2,3-dihydro-
pyridazine-1(6H)-carboxamide 21. To the ester (−)-11 (0.15 g;
0.5 mmol) solubilized in dry THF (10 mL) was added LiAlH4 1 M in
THF (15 equiv; 13.5 mL), freshly open, at 0 °C. The mixture was kept
under stirring for 4 h at rt. The reaction was quenched by a drop of
water followed by 2 drops of aq NaOH 15% and another drop of
water, during which time a large amount of H2 was released. Then a
portion of water (40 mL) was added, and the mixture was extracted
with ethyl acetate (5 × 40 mL). The combined organic layers were
washed with saturated aq NaHCO3 (50 mL) and brine (50 mL) and
then dried over MgSO4. After evaporation of the ethyl acetate, a
yellowish crude was obtained, from which crystallized a solid (0.10 g;
20
g; 59%): [α]D −70.4° (c = 1.2, acetone). The spectroscopic data of
the racemic mixture was reported before.4
Synthesis of (5S,6R,7S)-6,7-Dihydroxy-5-hydroxymethyl-2-
phenyl-2,4,9-triazabicyclo[4.3.0] Nonane-1,3-dione 16. To a
solution of the diol 14 (0.224 g; 0.73 mmol) in ethanol (7 mL) was
added NaBH4 (0.083 g). The mixture was stirred at room temperature
overnight. After addition of aq HCl 0.4 M (15.3 mL), the mixture was
stirred for 15 min. Then the solvent was removed under vacuum, and
the residue was dissolved in water (20 mL) and saturated aq solution
of NaHCO3 (10 mL) and extracted with ethyl acetate (14 × 25 mL).
The organic layers were combined, dried over magnesium sulfate, and
concentrated. It obtained a white solid identified as the title compound
20
0.43 mmol; 86%): [α]D −150.8° (c = 0.4, CHCl3); νmax (Nujol,
1
cm−1) 3359, 3268, 3058, 1636, 1601, 1592, 1536; H NMR (δH, 300
MHz, CDCl3) 3.30 (1H, bd, J 17.2 Hz, H-3), 3.48−3.55 (1H, m, H-3),
3.75 (1H, dd, J 10.8, 5.2 Hz, CH2OH), 3.95 (1H, dd, J 11.2, 3.2 Hz,
CH2OH), 4.20 (1H, dd, J 11.2, 2.4 Hz, OH), 4.78 (1H, bs, H-6), 5.83
(1H, dm, J 8.4 Hz, H-4 or H-5), 6.13 (1H, dm, J 8.4 Hz, H-4 or H-5),
7.02 (1H, t, J 7.6 Hz, CH, Ph), 7.30 (2H, t, J 7.6 Hz, CH, Ph), 7.47
(2H, d, J 7.6 Hz, CH, Ph), 8.60 (1H, bs, NH) ppm; 13C NMR (δC,
100 MHz, CDCl3) 45.3 (C-6), 50.7 (C-3), 65.1 (CH2OH), 122.7
(CH, Ph), 124.5 (C-4 or C-5), 128.3 (C-5 or C-4), 128.9 (CH, Ph),
138.7 (Cq, Ph), 155.0 (CO) ppm. HRMS (FAB): Calcd for
C12H16N3O2, 234.124432. Found, 244.124252.
20
(16, 0.112 g; 52%): [α]D −8.0° (c = 0.75, acetone). The spectro-
scopic data of the racemic mixture was reported before.4
Synthesis of Ethyl (5R,8S)-8-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-
glucopyranosyloxy)-2-phenyl-2,4,9-triazabiciclo[4.3.0]non-6-
ene-1,3-dione-5-carboxylate 18. To a suspension of compound
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The Journal of Organic Chemistry
Article
Synthesis of (4R,5R,6R)-4,5-Dihydroxy-6-(hydroxymethyl)-N-
phenylhexahydropyridazine-1-carboxamide (−)-22. To a sol-
ution of (−)-13 (0.06 g; 0.18 mmol) in dry THF (8 mL) was added at
0 °C a solution of LiAlH4 1 M in THF (7 equiv; 2.51 mL). The
reaction mixture stirred for 3 h at rt, and then the quenching was
followed by sequential addition of 1 drop of water, one drop of aq
NaOH 15%, and water (20 mL). The aqueous solution was extracted
with ethyl acetate (6 × 60 mL). The organic layers were combined,
dried, and evaporated, giving an oil that was submitted to PLC
(DCM/methanol 10%), giving the title compound (−)-22 (0.014 g;
except the amide bonds in both enantiomers of compound 22. The
protonation state of the amine N-1 and N-2 of the compounds was set
neutral, in agreement with previous NMR evidence.20 The grid for
probe-target energy calculations was placed with its center at the
enzyme-binding site. The docking grid size was 42 × 40 × 42 grid
points with 0.375 Å spacing. For each ligand, 20 runs using the
Lamarckian genetic algorithm with 150 individuals in each population
were carried out. The maximum number of generations was set to
27 × 103 and the maximum number of energy evaluations to 5 × 106.
The resulting docking solutions were clustered using AUTODOCK
with a structural root-mean-square deviation cutoff of 1 Å. Since no
experimental structure exists for the yeast α-glucosidase enzyme, a
theoretical structural model of this enzyme was derived using
MODELER,21 employing the crystal structure of isomaltase from
S. cerevisiae structure (PDB ID: 3A4A)13 as template. Isomaltase and
α-glucosidase from S. cerevisiae share 72% sequence similarity. Twenty
models were generated using an initial alignment between the
isomaltase and α-glucosidase enzyme sequences. The model with the
lowest objective function21 was chosen, and its quality was evaluated
on the basis of its stereochemistry given by Procheck.22 A high quality
model of the yeast α-glucosidase enzyme was obtained with no
residues in disallowed regions in the Ramachandran plot. The
protonation states of the acidic and basic residues were set to their
standard state found in aqueous solution at pH 7.
20
0.05 mmol, 29%): [α]D −54.4° (c = 0.6, methanol); νmax (neat,
cm−1) 3346, 2925, 1656, 1592, 1534; 1H NMR (δH, 400 MHz,
CDCl3) 2.92 (1H, dt, J 1.2, 14.8 Hz, H-3), 3.32 (1H, dd, J 14.8, 2.0 Hz,
H-3), 3.69−3.73 (1H, m, H-6), 3.82 (1H, dd, J 12.0, 4.8 Hz, CH2OH),
3.90−3.92 (1H, m, H-4), 4.11(1H, dd, J 12.2, 9.0 Hz, CH2OH), 4.44−
450 (1H, m, H-5), 7.19−7.43 (5H, m, Ph); 13C NMR (δC, 100 MHz,
CDCl3) 46.4 (C-3), 56.1 (C-5), 59.0 (CH2OH), 64.8 (C-6), 66.2 (C-
4), 121.5 (CH, Ph), 125.0 (CH, Ph), 129.1 (CH, Ph), 138.0 (Cq, Ph),
153.6 (CO) ppm. HRMS (FAB): Calcd for C12H18N3O4, 268.1219.
Found, 268.1222.
Synthesis of (4S,5S,6S)-4,5-Dihydroxy-6-(hydroxymethyl)-N-
phenylhexahydropyridazine-1-carboxamide (+)-22. To a sol-
ution of (+)-13 (0.12 g; 0.36 mmol) in dry THF (10 mL) was added
at 0 °C a solution of LiAlH4 1 M in THF (7 equiv; 5.03 mL). The
reaction mixture was stirred for 1.5 h at rt, and then the quenching was
followed by sequential addition of 1 drop of water, one drop of aq
NaOH 15%, and water (50 mL). The aqueous solution was extracted
with ethyl acetate (10 × 40 mL). The organic layers were combined,
dried, and evaporated, giving an oil that was submitted to PLC
(DCM/methanol 10%), giving the title compound (+)-22 (0.010 g;
ASSOCIATED CONTENT
■
S
* Supporting Information
Crystallographic data and ORTEP drawing for compounds 18
1
and 20 (CIF) and H, 13C, HMBC, and HMQC NMR spectra
20
of all compounds. This material is available free of charge via
the Internet at http://pubs.acs.org/.
0.04 mmol, 10%): [α]D +51.3° (c = 1, methanol).
Measurement of Glycosidase Inhibition. α-Glucosidase from
baker's yeast (EC 3.2.1.20, Sigma G-5003) and β-glucosidase from
almonds (EC 3.2.1.21, Sigma G-0395) were used as model
glycosidases. Enzyme assays were conducted in 96 well Nunc plates,
using 4-nitrophenyl α-D-glucopyranoside or 4-nitrophenyl β-D-
glucopyranoside as substrates, in phosphate buffer 100 mM, pH 7.0
or citrate buffer 100 mM, pH 5.0 at 25 °C. A range of substrate
concentrations from 3.3 × 10−5 M to 2.0 × 10−3 M (11 different
concentrations), in a final volume of 300 μL, was tested using 0.2
units/mL of β-glucosidase or 0.15 units/mL of α-glucosidase, in the
AUTHOR INFORMATION
Corresponding Author
*E-mail: mja@quimica.uminho.pt.
■
ACKNOWLEDGMENTS
■
We thank FCT for project funding PTDC/QUI/67407/2006
and FCT and FEDER for funding NMR spectrometer Bruker
Avance III 400 as part of the National NMR Network. M.N.M.
acknowledges the contract research program “Compromisso
absence and in the presence of inhibitor ((+)- and (−)-22, 5 × 10−6
M
and 10 × 10−6 M). Blanks were set containing all reaction components
but enzyme. All assays were performed in triplicate.
̂
com a Ciencia” Reference C2008-UMINHO-CQ-03 and access
to the Minho University GRIUM cluster.
The formation of 4-nitrophenol was monitored for 20 min at 25 °C,
measuring the absorbance (1 reading each minute) at 400 nm. A value
of εl = 787.73 M−1 (pH 7.0) or 28.29 M−1 (pH 5.0), determined in
the same conditions as used for the enzyme assays, was used to convert
absorbance into product concentration. Initial velocities were
calculated from the slopes of the absorbance vs time graphs for each
concentration of substrate and used to construct Michaelis−Menten
plots. The kinetic parameters KM and Vmax were determined by fitting
the experimental results to a rectangular hyperbole using the Origin 8
Graph Pad and by Lineweaver−Burk analysis. The inhibition type was
established as competitive for all enzymes and inhibitors tested, using
two different concentrations of inhibitors (in duplicate) and by
examining the Lineweaver−Burk plot. For each inhibitor concen-
tration, individual Ki values were obtained using the expression for
competitive inhibition (Ki= [I]/((KMapp/KM)-1)), where KM and KMapp
represent the Michaelis−Menten constant in the absence and in the
presence of inhibitor, respectively. Reported Ki values are expressed as
average of two independent Ki determinations.
Structural Molecular Modeling Studies. Structural enzyme−
compound complexes and theoretical binding free energy of (−)-2,
(+)-2, and 22 toward yeast α-glucosidase structure were done with
computational docking methodologies using AUTODOCK 4.19 The
modeling of the enzyme−compound complexes with almond β-
glucosidase was not calculated because, to the best of our knowledge,
no structure or protein sequence is available. In the docking
calculations, all possible torsions of the compounds were set flexible
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