Synthesis and evaluation of α-, β-glucosidase inhibition of 1-N-carboxamide-1-azafagomines and 5-epi-1-azafagomines

Article abstract of DOI:10.1016/j.carres.2014.06.015

1-N-Carboxamide 1-azafagomines and 5-epi-1-azafagomines were obtained from 1-azafagomine and 5-epi-1-azafagomine. The hydroxyl groups and the N-2 pyridazine position were protected prior to reaction with different isocyanates to form ureas. Protective gro

Full text of DOI:10.1016/j.carres.2014.06.015

Carbohydrate Research 395 (2014) 52–57
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and evaluation of a-, b-glucosidase inhibition
of 1-N-carboxamide-1-azafagomines and 5-epi-1-azafagomines
⇑
Raquel Mendes, Vera C. M. Duarte, António Gil Fortes, Maria J. Alves
Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2014
Received in revised form 12 June 2014
Accepted 14 June 2014
Available online 28 June 2014
1-N-Carboxamide 1-azafagomines and 5-epi-1-azafagomines were obtained from 1-azafagomine and 5-
epi-1-azafagomine. The hydroxyl groups and the N-2 pyridazine position were protected prior to reaction
with different isocyanates to form ureas. Protective groups were removed leading to the target com-
pounds in 18–23% global yields. Final compounds were tested towards a- and b-glucosidases.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
1-Azafagomines
Glucosidases inhibition
Aza-Diels–Alder
1. Introduction
4-phenyl-1,2,4-triazole-3,5-dione (PTAD)1 and to diethyl azodicar-
boxylate (DEAD) leading to ( )-1-azafagomine1 (3) and ( )-5-epi-1-
azafagomine1 (6). These compounds are precursors of the target
carboxamide derivatives. ( )-1-Azafagomine (3) was obtained first
by combination of 2,4-pentadienol (2) to PTAD.1 DEAD was subse-
quently used due to its lower price compared to PTAD; the result-
ing cycloadduct 5 was obtained in 99% yield, a better yield than the
equivalent cycloadduct 4 (88%). It was found that cycloadduct 5
could be cis-dihydroxylated with OsO4 in good yield and total
selectivity, but not trans-dihydroxylated; in this case the selectiv-
ity dropped to zero. Hydrazinolysis of the urethane group fur-
nished 6 in 81% yield (Scheme 1).
1-Azafagomine and its derivatives are very good as
a- and b-
glucosidase inhibitors.1,2 The introduction of a carboxamide group
at N-1 was recently found to hugely increase the
inhibition so the compound became almost as potent as deoxynoj-
irimycin (Ki = 0.44
a-glucosidase
l
M).3,4 The Ki value obtained for (ꢀ)-1-azafago-
mine N-phenyl carboxamide (1) was Ki = 3.36 lM, slightly better
than the racemic 1-azafagomine (Ki = 3.9 l
M).2 It is also known
that an alkyl group at the same N-position greatly enhanced the
b-glucosidase inhibition, with the best result belonging to 3-phen-
ylpropyl (Ki = 0.032
-glucosidase inhibitory activity of the laevo- and the dextrorotatory
l
M).2 It was a surprising finding that the
a
1H NMR spectrum of compound 5 showed two species in 1:1
ratio, found to be rotamers. In some regions of the 1H NMR spec-
trum the duplication of peaks is clearly individualized signals. To
assign the rotamers a 1D NOE difference experiment was per-
formed in CDCl3. Irradiation at dH = 3.38 ppm (due to Ha of CH2OH
group) in one rotamer inverts Ha of this rotamer, and Ha of the other
rotamer at dH = 3.48 ppm. This fact was recently demonstrated by
Dennis to occur when the protons are under significant chemical
exchange on the saturation time scale.7 Simultaneously a slight
enhancement in intensity (8.2%) of Hb in both rotamers are
observed due to nuclear Overhauser effect at dH = 3.69 ppm (Hb of
both rotamers are coincident). 13C NMR spectrum shows either
broad peaks or duplication of signals for all carbons. The same trend
was observed for triol intermediate 7, either in 13C or 1H NMR spec-
tra. These phenomena are probably related to the difficulty of nitro-
gen’s inversion due to the two bulky groups attached to them.
1-Azafagomine (3) and 5-epi-1-azafagomine (6) were conve-
niently protected in order to keep N-1 as the only nucleophilic
enantiomers of 1-azafagomine N-phenyl carboxamide (1) were of
the same order of magnitude;4 In contrast Bols found the opposite
trend with the two enantiomers of 1-azafagomine.5 In this paper
two new 1-N-carboxamide-1-azafagomines, and two 1-N-carbox-
amide-5-epi-1-azafagomines bearing different carboxamide moie-
ties were synthetized, and its activity measured against
a- and
b-glucosidase. These compounds belong to N-sp2-iminosugars
classes known to be conformationally locked compounds display-
ing excellent selectivities.6
2. Results and discussion
The adopted synthetic methodology to these compounds
relies on Diels–Alder cycloaddition of 2,4-pentadienol (2) to
⇑
Corresponding author. Tel.: +351 253 604370; fax: +351 253604382.
E-mail address: mja@quimica.uminho.pt (M.J. Alves).
http://dx.doi.org/10.1016/j.carres.2014.06.015
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

R. Mendes et al. / Carbohydrate Research 395 (2014) 52–57
53
HO
HO
HO
OH
i)
HO
HO
HO
HO
HO
O
O
O
O
O
NH
NH
PTAD
N
N
N
N
OEt
OEt
N
N
OEt iii)
OEt
ii)
NH
NH
N
Ph
HO
HO
HO
O
5
6
3
2
7
4
Scheme 1. Synthesis of 1-azafagomine (3) and 5-epi-1-azafagomine (6). Reagents and conditions: (i) DEAD, toluene, 24 h, rt, 99%; (ii) OsO4, NMO, acetone/H2O, 9:1, 24 h, rt,
96%; (iii) NH2NH2ꢂH2O, 18 h, 100 °C, 81%.
HO
HO
BnO
BnO
O
O
1
HO
HO
BnO
BnO
ii)
38%
i)
83%
iii)
iv)
N
NHR
NH
N
N
N
NHR
NH
N
2
NH
HO
HO
Boc
BnO
Boc
BnO
Boc
8
10
14a
12a
,R=C 6H4-p-NH2,72%
,R=C 6H4-p-NO2,91%
14b
12b
,R=C 4H9,85%
,R=C 4H9,81%
HO
O
HO
BnO
BnO
BnO
O
HO
HO
BnO
ii)
29%
i)
86%
iii)
iv)
N
NHR
NH
N
N
N
NHR
NH
N
6
NH
HO
HO
Boc
BnO
Boc
BnO
Boc
9
11
15a,R=C 6H4-p-NH2,72%
15b
13a,R=C 6H4-p-NO2,quant.
13b
,R=C 4H9,quant.
,R=C 4H9,91%
Scheme 2. Synthesis of and N-1-carboxamide 1-azafagomines (14a, b) and N-1-carboxamide 5-epi-1-azafagomines (15a, b). Reagents and conditions: (i) Boc2O, ethanol,
NaHCO3; (ii) BnBr (3.5 equiv), NaH (in paraffin), DMF (dry); (iii) isocyanate, toluene (dry), reflux; (iv) (a) Pd/C, H2, HCl 3 M (1): ethanol (5), rt, 24 h, (b) Dowex 1ꢁ2–OH.
Table 1
Ki values (lM) for the Inhibition of a- and b-glucosidases by 1-azafagomine and 5-epi-azafagomine derivatives
Compound
a
-Glucosidase (baker0s yeast)
b-Glucosidase (almonds)
( )-3
(ꢀ)-3
3.9a
0.65a
6.9a
0.3a
(+)-3
2900a
>10,000a
( )-6
(-)-1
>1000a
3.4b
137a
14.7b
(+)-1
10.6b
25.2b
( )-14a
( )-15a
( )-14b
( )-15b
>100b
12.7b
n.d.c
17.2b
14.0b
20.8b
9.0b
91.6b
a
b
c
pH 6.8.
pH 7.0.
n.d., not determined.

54
R. Mendes et al. / Carbohydrate Research 395 (2014) 52–57
centre in the molecules, ready to react with isocyanates. N-2 was
protected by reaction with Boc anhydride giving products in 83%
(8) and 86% (9) yields. The hydroxyl groups were all protected by
reaction with benzyl bromide in the presence of NaH, furnishing
the hydroxyl-protected compounds in 38% yield (10) and 29% yield
(11). Unfortunately simultaneous benzylation of N-1 occurred
lowering the yields of target compounds 10 and 11. The isolated
compounds were reacted with p-nitrophenyl and butyl isocya-
nates; ureas 12a, b and 13a, b were formed in excellent yields
(81%-quant.). Deprotection of these compounds occur by hydroge-
nation under H2, Pd/C in HCl 3 M (1): ethanol (5) at rt to give the
final compounds 14a, b and 15a, b in 72%-quant. yields (Scheme 2).
1H NMR spectra of compounds 12a, b, 13a, b showed complex
patterns due to the existence of rotamers, as referred to com-
pounds 5 and 7. The simpler 1H NMR spectrum belongs to com-
pound 13a showing broad signals for all peaks which means that
a slow interconversion between rotamers occurs in the spectrum
acquisition time.
In conclusion N-carboxamide 5-epi-1-azafagomine derivatives
display comparable -inhibition potency to N-carboxamide 1-azaf-
a
agomine derivatives in contrast to the free 5-epi-1-azafagomine
which, unlike 1-azafagomine, is a very poor inhibitor. With respect
to b-glucosidase inhibition the N-carboxamide derivatives of either
1-azafagomine or 5-epi-1-azafagomine exhibit the same potency.
In general the carboxamide functionality is exerting a negative
effect on the recognition by b-glucosidase of the compounds. The
effect of the group attached to the carboxamide function is impor-
tant in the a-glycosidase inhibition but not in b-glycosidase.
3. Experimental
3.1. General
Solvents were distilled under anhydrous conditions. 1-azafago-
mine (3) was obtained according to Bols’s protocol;5 5-epi-1-azaf-
agomine (6) was obtained by a modification of this protocol, by
changing the dienophile/diethylazodicarboxylate (DEAD) instead
of 4-phenyl-1,2,4-triazole-3,5-dione (PTAD). The synthetic
sequence of 5-epi-1-azafagomine (6) followed the same steps:
oxidation of the C@C double bond, and cleavage of the groups
attached to the six-membered ring nitrogen atoms. 2,4-Pentadie-
nol (2) was obtained by repeating the literature procedure.8 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, <0063 mm and water
pump vacuum or by flash-chromatography using silica 60 Å 230–
400 mesh as stationary phases. TLC plates (silica gel 60 F254) were
visualized either at a UV lamp or in I2. 1H NMR and 13C NMR were
run on a Varian Unity Plus 300, or Bruker Avance III 400 or Bruker
BioSpin GmbH spectrometers. Infrared spectra were recorded on a
Bomem MB 104. Samples were run as nujol mulls and oils as thin
films. Melting points are uncorrected. MS spectra were recorded on
a VG Autospec M. spectrometer.
1H and 13C NMR spectra of compounds 14a, b and 15a, b
showed of course simpler patterns. In these cases only the
hydroxymethylene and the carbamate groups are neighbours. In
general 1H and 13C NMR spectra of compounds 15a, b showed a
single set of peaks. There is an exception to the 13C spectrum of
compound 15a: the aromatic region and carbonyl group show peak
duplication. On the other hand, compound 14a showed duplication
of peaks in the aromatic region in both 1H and 13C NMR spectra,
however 14b showed two sets of peaks for all protons and carbons
in the structure.
1-N-Phenyl carboxamide derivatives of 1-azafagomine, in either
enantiomeric form was found to be potent a-glycosidase inhibitors
in a previous work.4 As an extension of this work we tested now
four new compounds: two having the 1-azafagomine backbone
14a, b, and two the 5-epi-1-azafagomine structure 15a, b. We
became interested in the 5-epi-1-azafagomine structure because
according to our previous results with 1-azafagomine N-phenyl
carboxamide derivative (1) the two enantiomers display compara-
ble inhibition, either towards
a
- and b-glucosidases.4 In fact we
3.2. Synthesis of 5-epi-1-azafagomine (6)
now acknowledge that the configuration change at C-5 of 1-azafag-
omine nucleus showed a small difference in inhibition activity
towards a-glucosidase, baker’s yeast source (15a, Ki = 12.7 lM) rel-
3.2.1. Diels–Alder cycloaddition of 2,4-pentadienol to
diethyldiazodicarboxylate
atively to (+)-1 and (ꢀ)-1, and interestingly one order of magnitude
better than 14a. However compound 15b is one order of magni-
tude less potent (Table 1). The efficiency of the recognition mech-
anism between the inhibitor and the enzyme seems to be largely
dependent on the group attached to the carboxamide functionality.
To a solution of 2,4-pentadienol (1.33 g; 16.0 mmol) in dry
toluene (10 mL) was added diethyldiazodicarboxylate (40% in
toluene, 7.30 mL, 16.0 mmol) under magnetic stirring at rt. The
stirring was continued for 24 h and the solvent removed to give
cycloadduct 5 as an orange oil (4.32 g; 99%).
mmax (neat) 3483, 1707 cmꢀ1. dH (400 MHz, CDCl3) 1.23–1.30
(12H, m, 4 ꢁ CH3, A+B), 2.58 (1H, br s, OH), 3.35 (1H, dd, J 12.3,
9.5 Hz, H-30, A), 3.45 (1H, dd, J 12.0, 9.8 Hz, H-30, B), 3.56–3.69 (2H,
m, 2 ꢁ H-30, A+B), 3.77 (1H, dd, J 13.5, 4.3 Hz, H-6, A), 3.91 (1H, br
s, H-6, B), 4.11–4.26 (8H, m, 4 ꢁ CH2, A+B), 4.30 (1H, tdd, J 6.0, 3.9,
2.2 Hz, H-6, B), 4.34–4.44 (1H, m, H-6, A), 4.72 (2H, br s, H-3, A+B),
5.66–5.88 (4H, m, H-4+H-5, A+B) ppm. dC (100 MHz, CDCl3)à 14.3
(CH3, A), 14.4 (CH3, B), 42.2 (C-6, A), 43.6 (C-6, B), 55.9 (C-3, A),
56.9 (C-3, B), 61.9 (C-30, A+B), 62.6, 62.7, 62.8, 62.9 (CH2, A+B),
123.4, 124.2, 124,6, 125.2 (C-4 or C-5, A+B), 154.9, 155.7, 156.2,
Bols have found that free N-1 is essential for the
activity of 1-azafagomines. The Ki values of alkylated compounds
towards -glucosidases were found to be similar to that of isofag-
omine (Ki = 86
M), a structure in which a carbon replaces N-1.2
a-inhibition
a
l
Possibly the good inhibition activity of 15a as well as (ꢀ)-1 and
(+)-1 can be explained by the existence of a carbonyl that is inter-
acting with the enzyme in the same way as the free N-1 does.
A rapid inspection of Table 1 shows the same kind of Ki values to
all carboxamide compounds (Ki = 9–25 lM) towards b-glucosidase,
almond source. In this case the nature of the carboxamide group
attached to N-1 is not expressed in the inhibition activity of
1-azafagomines. On the other hand free ( )-1-azafagomine (3)
shows to be 1.5 order of magnitude more potent than carboxamide
derivatives. The inhibitory activity is due exclusively to the laev-
156.3 (C@O, A+B) ppm. HRMS (ESI): calcd for
C11H18N2NaO5:
281.1108; found: 281.1109.
3.2.2. cis-Dihydroxylation of compound 5
Cycloadduct 5 (1.85 g; 7.17 mmol) was dissolved in acetone
(18.4 mL) and water (2.05 mL), N-methylmorpholine N-oxide
orotatory enantiomer (Ki = 0.3
lM); the Ki of the dextrorotatory
enantiomer is >10,000 M. Although this work deals with racemic
l
mixtures, considering that a small gap of activity exists between
both enantiomers 1, and that similar Ki values were obtained for
all carboxamide derivatives, it is likely to suppose a similar
inhibitory potential for either dextro- and laevo-enantiomers of
carboxamide derivatives.
1H NMR analysis showed a 1:1 mixture of rotamers A and B, due to inversion of
the nitrogens lone pairs/rotation of the amide bond.
à
13C NMR also showed peak duplication.

R. Mendes et al. / Carbohydrate Research 395 (2014) 52–57
55
(1.25 g; 9.30 mmol) was added followed by a solution of OsO4 in
water (4%; 0.76 mL; 0.12 mmol). The mixture was stirred for 1 d
at rt. The reaction was quenched by addition of 5% aq soln Na2S2O3
(10 mL). The acetone was removed by evaporation and the residue
redissolved in AcOEt, filtered through a pad of silica, washed with
AcOEt, and concentrated to give a yellow oil (7; 2.00 g; 96%).
mmax (neat) 3419, 2983, 2937, 1698 cmꢀ1. dH (400 MHz, CDCl3)
1.23–1.32 (12H, m, 4 ꢁ CH3, A+B), 3.12 (1H, br d, J 10.8 Hz, H-6, A),
3.21 (1H, br d, J 10.8 Hz, H-6, B), 3.50 (2H, t, J 11.6 Hz, H-30, A), 3.62
(1H, d, J 8.0 Hz, H-30, B), 3.65–3.95 (4H, br s, H-4+H-5, A+B), 4.01
(2H, dd, J 13.2, 5.6 Hz, H-6, A), 4.10–4.25 (9H, m, H-6, B+4 ꢁ CH2,
A+B), 4.52–4.64 (2H, br s, H-3, A+B) ppm. dC (100 MHz, CDCl3)à
14.3, 14.4 (CH3, A,B), 44.0, 45.7 (C-6, A,B), 58.4, 59.0 (C-3, A,B),
61.9, 62.8 (C-30, A,B), 63.0, 63.1 (2 ꢁ CH2, A or B), 63.2, 63.3
(2 ꢁ CH2, A or B), 64.0, 64.3 (C-4 or C-5, A,B), 66.4, 66.7 (C-4 or
C-5, A,B), 155.2, 156.5 (C@O, A,B), 157.1, 157.3 (C@O, A,B) ppm.
HRMS (ESI): calcd for C11H20N2NaO7: 315.1163; found: 315.1163.
added portionwise NaH (80% in paraffin; 3.5 equiv) under mag-
netic stirring at 0 °C. The mixture was kept stirring for 30 min at
0 °C before addition of benzyl bromide (3.5 equiv), added dropwise
at 0 °C. The reaction mixture was stirred further for 18 h at room
temperature. The reaction was quenched, by pouring the reaction
mixture onto ice, washed with water and brine. The organic layer
was dried with MgSO4 and concentrated under vacuum to give a
crude product that was subjected to dry-flash chromatography
(petroleum ether/ethyl acetate). The benzylated products were
obtained as yellow oils: N-Boc-tri-O-benzyl-1-azafagomine (10;
0.41 mmol; 38%) and N-Boc-tri-O-benzyl-5-epi-1-azafagomine
(11, 0.31 mmol; 29%).
3.4.1. N-Boc-tri-O-benzyl-1-azafagomine (10)
mmax (neat): 3471 (N–H), 3087, 3063, 3029, 3005 (Ar), 1690
(C@O) cmꢀ1. dH (400 MHz, DMSO) 1.46 (9H, s, C(CH3)3) 2.90–2.96
(2H, m, H-3+H-6), 3.49 (1H, d, J 8.4 Hz, H-4), 3.54 (1H, dd,
J 9.6, 3.2 Hz, H-30), 3.56 (1H, d, J 6.0 Hz, H-5), 3.71 (1H, dd,
J 9.6, 3.2 Hz, H-30), 4.27–4.33 (1H, br s, H-6), 4.43 (1H, d, J 11.6 Hz,
OCH2-30), 4.52 (1H, d, J 11.6 Hz, OCH2-30), 4.56 (1H, d, J 11.2 Hz,
3.2.3. Cleavage of ethyl carbamate groups in compound 7
Triol 7 (1.40 g; 4.80 mmol) was dissolved in hydrazine hydrate
(82 mL) and heated at 100 °C for 18 h. The solution was concen-
trated and the crude product was subjected to dry-flash chroma-
tography in EtOH/NH4OH (25%) 99:1 giving pure 6 (0.57 g; 81%).
dH (300 MHz, D2O) 3.00 (1H, d, J 14.4 Hz, H-6), 3.07 (1H, ddd, J
9.6, 6.4 e 3.2 Hz, H-3), 3.10 (1H, d, J 14.4 e 2.8 Hz, H-6), 3.64 (1H,
dd, J 12.0 e 3.2 Hz, H-30), 3.67 (1H, d, J 6.4 Hz, H-4), 3.84 (1H, dd,
J 12.0 e 2.8 Hz, H-30), 4.00 (1H, d, J 1.6 Hz, H-5) ppm.1
OCH2-4), 4.67 (1H, d,
J 12.0 Hz, OCH2-5), 4.72 (1H, d, J
12.0 Hz, OCH2-5), 4.88 (1H, d, J 11.2 Hz, OCH2-4), 7.23–7.39 (15H,
m, Ph) ppm. dC (100.6 MHz, DMSO) 28.2 (C(CH3)3), 47.5 (C-6),
60.1 (C-3), 68.2 (C-30), 72.2 (OCH2-30), 73.2 (OCH2-5), 74.9 (OCH2-4),
78.7 (C-5), 78.9 (C-5), 80.9 (C(CH3)3), 127.6–128.7 (C-H, Ph), 137.9,
138.1, 138.2 (Cq, Ph), 154.9 (C@O) ppm. HRMS (ESI): calcd for
C
31H38N2O5 [M+H]+ 519.2853; found: [M+H]+ 519.2844.
3.3. General procedure for N-1 protection of 1-azafagomine (3)
and 5-epi-1-azafagomine (6)
3.4.2. N-Boc-tri-O-benzyl-5-epi-1-azafagomine (11)
mmax (neat): 3322–3359 (N–H), 3088, 3063, 3030 (Ar), 1692
(C@O) cmꢀ1. dH (400 MHz, CDCl3) 1.47 (9H, s, C(CH3)3), 3.03 (1H,
br d, J 12.8 Hz, H-6), 3.46–3.52 (2H, m, H-3 and H-4), 3.60 (1H,
dd, J 9.2, 6.0 Hz, H-30), 3.75 (1H, s, H-5), 3.77 (1H, dd, J 9.2,
6.0 Hz, H-30), 4.46 (1H, d, J 12.8 Hz, H-6), 4.32 (1H, d, J 12.0 Hz,
OCH2-4 or OCH2-5), 4.51–4.58 (4H, m, OCH2-4 or OCH2-5 and
OCH2-30), 7.25–7.40 (15H, m, Ph) ppm. dC (100.6 MHz, CDCl3)
28.2 (C(CH3)3), 46.1 (C-6), 55.1 (C-3), 68.8 (C-30), 69.6 (C-5), 70.4,
70.6 (OCH2-4 and OCH2-5), 73.2 (OCH2-30), 76.1 (C-4), 80.6
(C(CH3)3), 125.5–127.8 (C-H, Ph), 137.9, 138.0, 138.1 (Cq, Ph),
155.9 (C@O) ppm. HRMS (ESI): calcd for C31H38N2O5 [M+Na]+
519.2673; found: [M+Na]+ 541.2653.
To a stirred solution of 3 (0.43 mmol)/6 (1.05 mmol) in ethanol,
respectively 1.1 mL and 2.6 mL, was added sat. soln NaHCO3
[1.08 mL (3); 1.29 mL (6)] and Boc2O [0.43 mmol (3); 1.04 mmol
(6)] at rt. The reaction mixture was stirred overnight, filtered
through a pad of silica, and washed thoroughly with ethanol. The
filtrate was concentrated to give N-Boc-1-azafagomine (8) and
N-Boc-5-epi-1-azafagomine (9) as beige oils (8; 0.71 mmol; 83%)
and (9, 0.91 mmol; 86%).
3.3.1. N-Boc-1-azafagomine (8)
mmax (neat): 3300 (O–H), 1694 (C@O) cmꢀ1. dH (400 MHz, D2O)
1.51 (9H, s, C(CH3)3), 2.73 (1H, ddd, J 9.2, 6.0, 2.8 Hz, H-3), 2.91
(1H, dd, J 13.2 e 10.8 Hz, H-6), 3.41 (1H, t, J 9,2 Hz, H-4), 3.60
(1H, ddd, J 10.8, 9.2 ,5.2 Hz, H-5), 3.71 (1H, dd, J 12.0, 6.0 Hz,
H-30), 3.90 (1H, dd, J 12.0, 2.4 Hz H-30), 4.27 (1H, dd, J 13.2,
5.2 Hz, H-6) ppm. dC (100.6 MHz, D2O) 27.6 (C(CH3)3), 49.3 (C-6),
59.2 (C-30), 61.9 (C-3), 69.8 (C-5), 71.6 (C-4), 83.3 (C(CH3)3),
156.5 (C@O) ppm. HRMS (ESI): calcd for C10H20N2O5 [M+Na]+
271.1264; found: [M+Na]+ 271.1267.
3.5. General procedure for reaction of compounds 10/11 with
isocyanates to form compounds 12a, b and 13a, b
To a solution of protected 1-azafagomine (10)/5-epi-1-azafago-
mine (11) (0.11–0.16 mmol) in dry toluene (2 mL), kept under
nitrogen and magnetic stirring, was added p-nitrophenyl/butyl iso-
cyanate (1.0–3.0 equiv) dropwise at 0 °C. The solution mixture was
transferred to an oil bath and refluxed for 4–17 h30 m, then evap-
orated till dryness, and re-dissolved in ether. The etherate was
washed with 3 M aq HCl, the organic phase was dried over MgSO4,
the filtrate evaporated to give a brownish oil in 81–92% yield.
3.3.2. N-Boc-5-epi-1-azafagomine (9)
mmax (neat): 3300 (O–H), 1637 (C@O) cmꢀ1. dH (400 MHz, D2O)
1.49 (9H, s, C(CH3)3), 3.08 (1H, ddd, J 9.2, 6.4, 3.2 Hz H-3), 3.35
(1H, br d, J 13.2 Hz, H-6), 3.66–3.71 (1H, m, H-4), 3.68 (1H, dd, J
12.0, 6.4 Hz, H-30), 3,85 (1H, dd, J 12.0, 3.2 Hz, H-30), 3.98–4.03
(1H, m, H-5), 4.05–4.10 (1H, m, H-6) ppm. dC (100.6 MHz, D2O)
29.6 (C(CH3)3), 49.7 (C-6), 59.7 (C-30), 57.6 (C-3), 66.6 (C-5), 67.4
(C-4), 82.8 (C(CH3)3), 157.4 (C@O) ppm. HRMS (ESI) calcd for
C10H20N2O5 [M+Na]+ 271.1264; found: [M+Na]+ 271.1263.
3.5.1. N-Boc-tri-O-benzyl-1-azafagomine-N-p-
nitrophenylcarboxamide 12a
Compound 10 (0.06 g; 0.12 mmol); p-nitrophenyl isocyanate:
1.5 equiv; reflux for 6h30 min. Obtained 12a, yellow oil
(0.11 mmol; 91%). mmax (neat): 3352 (N–H), 3030, 3064 (Ar), 1722
(C@O), 1690 (C@O), 1534, 1504, (NO2) cmꢀ1
. dH (400 MHz,
C3D6O)§ 1.35 (9H, s, C(CH3)3, M), 1.44 (9H, s, C(CH3)3, m), 3.42 (1H,
dd, J 14.0, 0.8 Hz, H-3, M), 3.57 (1H, br d, J 14.8 Hz, H-3, m), 3.70
(1H, s, H-4, m), 3.76–3.84 (3H, m, 2 ꢁ H-60, M, m+H-4, M), 3.89
3.4. General procedure for protection of the hydroxyl groups in
N-Boc-1-azafagomine (8) and N-Boc-5-epi-1-azafagomine (9)
To a solution of the N-Boc-1-azafagomine (8, 1.16 mmol)/
N-Boc-5-epi-1-azafagomine (9, 1.08 mmol) in DMF (6.0 mL) was
§
M, major rotamer; m, minor rotamer.

56
R. Mendes et al. / Carbohydrate Research 395 (2014) 52–57
(1H, t, J 9.8 Hz, H-60, M), 3.99 (1H, t, J 9.6 Hz H-60, m), 4.02 (1H, s, H-5,
M), 4.08 (1H, s, H-5, m), 4.34 (1H, d, J 14.8 Hz, H-3, m), 4.45–4.53
(1H, m, OCH2-4), 4.46 (1H, d, J 12.0 Hz, OCH2-60), 4.50 (1H, d, J
5.6 Hz, H-60), 3.82 (1H, ddd, J 10.8, 4.8, 2.4 Hz, H-4), 4.08–4.11
(1H, m, H-3M), 4.14 (1H, br s, H-5), 4.52–4.61 (4H, m, OCH2-4,
OCH2-60), 4.81 (1H, d, J 12.0 Hz, OCH2-5), 4.79 (1H, d, J 12.0 Hz,
OCH2-5), 5.24–5.29 (1H, br s, H-6), 7.28–7.37 (13H, m, Ph), 7.39
(2H, J 8.4 Hz, Ph) ppm. dC (100.6 MHz, C3D6O) 14.1 (CH3, M), 14.0
(CH3, m), 20.4 (CH2 ,m), 20.7 (CH2, M), 28.2, 28.3 (C(CH3), M+m),
32.6, 33.2 (CH2, M, m), 40.5 (NCH2, M), 40.7 (NCH2, m), 41.7 (H-
3, m), 42.3 (H-3, M), 54.0 (C-6), 69.0 (C-60), 70.7 (OCH2-4), 71.7
(C-4), 71.8 (OCH2-5), 72.7 (C-5), 73.7 (OCH2-60), 81.5 (C(CH3)3),
128.0–129.1 (C-H, Ph), 139.2, 139.6, 139.9 (Cq, Ph), 157.1 (C@O),
157.9 (C@O) ppm. HRMS (ESI): calcd for C36H47N3O6 [M+H]+
618.3538; found: [M+H]+ 618.3527.
14.0 Hz, H-3, M), 4.52 (1H, d,
J
12.0 Hz, OCH2-60), 4.61
(1H, d, J 11.6 Hz, OCH2-5), 4.75 (1H, d, J 11.6 Hz, OCH2-5), 4.83
(1H, d, J 11.6 Hz, OCH2-4), 5.13 (1H, t, J 7.2 Hz, H-6, M), 5.19 (1H, t,
J 7.2 Hz, H-6, m), 7.28–7.39 (15H, m, 3xPh), 7.87 (2H, d, J 8.4 Hz,
Ho, m), 7.95 (2H, d, J 8.8 Hz, Ho, M), 8.19 (2H, d, J 8.8 Hz, Hm, m),
8.20 (2H, d, J 8.4 Hz, Hm, M) ppm. dC (100.6 MHz, C3D6O) 28.1
(C(CH3)3, m), 28.2 (C(CH3)3, M), 42.8 (C-3, M), 45.7 (C-3, m), 52.9
(C-6, M+m), 68.5 (C-60, M), 68.8 (C-60, m), 71.1 (C-5, m) 71.5
(OCH2-4), 71.68 (OCH2-5), 71.7 (C-5, M), 73.5 (OCH2-60), 73.6 (C-4,
m), 73.8 (C-4, M), 81.2 (C(CH3)3, M), 81.3 (C(CH3)3, m), 119.4,
119.5 (Co, M+m), 124.3 (Cm, M+m), 127.9–129.1 (C-H, Ph), 139.2
139.4, 139.6 (Cq, Ph), 147.0 (Cq, urea) 147.3 (Cq, C-NO2) 154.9
(C@O, Boc) 156.2 (C@O, urea) ppm. HRMS (ESI): calcd for
3.6. Cleavage of protective groups to give final products (14a, b/
15a, b)
C
38H42N4O8 [M+Na]+ 705.2895; found: [M+Na]+ 705.2881.
To a solution of N-carboxamido derivatives 12a, b/13a, b
(0.06–0.11 mmol) in EtOH (5–2.5 mL) was added aq HCl sol.
(1 M, 1–0.5 mL), and Pd/C 10% (0.01–0.03 g). The reaction mixture
was stirred under H2 for 24 h at rt. After reaction completion the
mixture was filtered through a pad of Celite, washed with ethyl ace-
tate and the filtrate concentrated. The oil residue was re-dissolved
in water (20 mL), and washed with ether (20 mL). The aqueous
solution was neutralized with a Dowex (OHꢀ) basic resin and the
aqueous solution concentrated to dryness to give the final products.
3.5.2. N-Boc-tri-O-benzyl-1-azafagomine-N-butylcarboxamide
12b
Compound 10 (0.09 g; 0.16 mmol); butyl isocyanate: 2.0 equiv;
14 h reflux. Obtained 12b, brownish oil (0.13 mmol; 81%). mmax
(neat): 3365 (N–H), 3031, 3063, 3087 (Ar), 1712 (C@O), 1692
(C@O) cmꢀ1. dH (400 MHz, C3D6O)§ 0.90–1.60 (16H, m, CH3–CH2–
CH2, C(CH3), M+m), 3.15–5.15 (15H, m, NCH2, 2 ꢁ H-3, H-4, H-5,
H-6, 2 ꢁ H-60, OCH2-4, OCH2-5, OCH2-60, M+m), 7.32–7.39 (15H,
m, 3 ꢁ Ph) ppm. dC (100.6 MHz, C3D6O) 13.9, 14.0, 14.1, 14.2 (CH3,
M+m), 20.4, 20.5, 20.6, 20.7 (CH2, M+m), 28.2, 28.7 (C(CH3),
M+m), 31.7, 32.6, 33.3, 33.4 (CH2, M+m), 40.5, 40.6 (NCH2, M+m),
41.7, 42.7 (H-3, M+m), 51.9 (C-6, M), 52.3 (C-6, m), 69.3, 71.5,
73.1, 73.2 (H-60, OCH2-4, OCH2-5, OCH2-60), 71.7, 74.4 (C-4+C-5,
M), 71.3, 74.3 (C-4+C-5, m), 81.3, 81.8 (C(CH3)3), 128.1–129.4
(C-H, Ph), 139.3, 139.4, 139.5, 139.6, 139.7, 139.9 (Cq, M+m),
155.7, 156.7, 157.0, 157.7 (C@O Boc, C@O urea) ppm. HRMS (ESI):
calcd for C36H47N3O6 [M+H]+ 618.3538; found: [M+H]+ 618.3524.
3.6.1. 1-Azafagomine-1N-(p-aminophenyl)carboxamide (14a)
Carboxamide 12a (0.08 g; 0.11 mmol); EtOH (5 mL); HCl (1 M;
1 mL); Pd/C 10% (0.03 g). Obtained 14a, yellow oil (0.08 mmol;
72%). mmax (neat): 3583 (N–H), 3369 (O–H), 1713 (C@O) cmꢀ1. dH
(400 MHz, D2O)§ 2.92 (1H, dd, J 14.2, 0.8 Hz, H-3), 3.32 (1H, dd, J
14.2, 1.6 Hz, H-3), 3.71 (1H, d, J 13.2 Hz, H-4), 3.81 (1H, dd, J
12.0, 4.8 Hz H-60), 3.90 (1H, br s, H-5), 4.09 (1H, dd, J 12.0,
9.2 Hz, H-60), 4.41–4.45 (1H, m, H-6), 6.88 (1H, t, J 9.2, 8.4 Hz Ho,
M+m), 7.19 (2H, d, J 8.8 Hz, Hm,, M+m), 7.25 (2H, d, J 8.8 Hz, Hm,
M+m) ppm. dC (100.6 MHz, D2O) 46.4 (C-3), 56.2 (C-6), 59.0
(C-60), 64.9 (C-4), 66.2 (C-5), 116.2 (Co, m), 116.8 (Co, M), 124.4,
124.6 (Cm, M, m), 129.8, 129.9 (Cq, urea), 142.8, 144.6 (Cq, C–NO2)
155.1, 156.2 (C@O, M, m) ppm. HRMS (ESI): calcd for C10H18N4O4
[M+H]+ 283.1401; found: [M+H]+ 283.1399.
3.5.3. N-Boc-tri-O-benzyl-5-epi-1-azafagomine-N-p-
nitrophenylcarboxamide 13a
Compound 11 (0.057 g; 0.11 mmol); p-nitrophenyl isocyanate:
1.0 equiv; 4 h reflux. Obtained 13a, yellow oil (0.11 mmol; 99%).
mmax (neat): 3351 (N–H), 3030 (CH, Ar), 1711 (C@O), 1691
(C@O) cmꢀ1. dH (400 MHz, C3D6O) 1.40 (9H, s, C(CH3)3), 3.25–3.28
(1H, br s, H-3), 3.50–3.59 (1H, br s, H-6a0), 3.70–3.78 (1H, br s, H-
6b0), 3.91 (1H, br d, J 8.8 Hz, H-4), 4.22 (1H, s, H-5), 4.56–4.66
(4H, m, OCH2-4, OCH2-60), 4.66 (1H, d, J 12.0 Hz, OCH2-5), 4.80
(1H, d, J 12.0 Hz, OCH2-5), 5.25–5.34 (1H, br s, H-6), 7.29 (2H, d, J
7.2 Hz, Ph), 7.30–7.40 (11H, m, Ph), 7.43 (2H, d, J 6.8 Hz, Ph),
7.93–8.00 (2H, br s, Ho), 8.19 (2H, d, J 8.8 Hz, Hm) ppm. dC
(100.6 MHz, C3D6O) 28.8 (C(CH3)3), 43.5 (C-3), 54.9 (C-6), 68.5
(C-60), 71.1 (OCH2-4 or OCH2-60), 71.6 (C-4), 72.2 (OCH2-5), 72.8
(C-5), 73.8 (OCH2-4 or OCH2-60), 84.4 (C(CH3)3), 119.6 (Co), 125.3
(Cm), 128.2–129.2 (C-H, Ph), 139.0, 139.5, 139.6 (Cq, Ph), 143.1
(Cq, urea), 147.2 (Cq,, –NO2), 155.1 (C@O), 156.2(C@O) ppm. HRMS
(ESI): calcd for C38H42N4O8 [M+Na]+ 705.2895; found: [M+Na]+
705.2882.
3.6.2. 1-Azafagomine-1N-butylcarboxamide (14b)
Carboxamide 12b (0.06 g; 0.10 mmol); EtOH (5 mL); HCl sol
(1 mL); Pd/C 10% (0.02 g). Obtained compound 14b, yellow oil
(0.08 mmol; 85%). mmax (neat): 3388 (O–H), 1645 (C@O) cmꢀ1. dH
(400 MHz, D2O)§ 0.91 (3H, t, J 7.6 Hz, CH3, m), 0.93 (3H, t, J
7.2 Hz, CH3, M), 1.31–1.40 (2H, m, CH2, M+m,), 1.46–1.55 (2H, m,
CH2, M+m), 2.78 (1H, dd, J 13.2, 10.8 Hz, H-3, m), 2.81 (1H, dt, J
14.8, 1.2 Hz, H-3, M), 3.15–3.20 [3H(M)+2H(m), m, NCH2,
M+m,+H-3, M], 3.37 (1H, dd, J 10.0, 9.2 Hz, H-5, m), 3.55–3.59
(1H, m, H-4, m), 3.65–3.67 (1H, m, H-4, M), 3.71 (1H, dd, J 12.0,
6.4 Hz, H-60, m), 3.76 (1H, dd, J 12.0, 4.2 Hz, H-60, M), 3.88–3.86
(1H, m, H-5, M), 3.92 (1H, dd, J 12.0, 2.8 Hz, H-60, m), 4.03 (1H,
dd, J 12.0, 9.0 Hz, H-60, M), 4.31–4.34 (1H, m, H-6, M), 4.37–4.40
(2H, m, H-3+H-6, m). dC (100.6 MHz, D2O) 13.0 (CH3, m), 13.0
(CH3, M), 19.3 (CH2, m), 19.4 (CH2, M), 31.5 (CH2, m), 31.6 (CH2,
M), 39.5 (CH2, m), 39.7 (CH2, M), 46.2 (C-3, M), 47.6 (C-3, m),
56.2 (C-6, M), 62.0 (C-6, m), 59.1 (C-6,0 M), 59.4 (C-60, m), 65.0
(C-5, M), 66.2 (C-4, M), 69.9 (C-5, m), 71.9 (C-4, m), 159.7 (C@O,
m), 160.9 (C@O, M) ppm. HRMS (ESI): calcd for C10H21N3O4
[M+Na]+ 270.1424; found: [M+H]+ 270.1421.
3.5.4. N-Boc-tri-O-benzyl-5-epi-1-azafagomine-N-
butylcarboxamide 13b
Compound 11 (0.024 g; 0.046 mmol); butyl isocyanate:
3.0 equiv; 17h30 min, reflux. Obtained 13b as
a yellow oil,
(0.04 mmol; 91%). mmax (neat): 3365 (N–H), 3087, 3063, 3030
(Ar), 1711 (C@O), 1691 (C@O) cmꢀ1. dH (400 MHz, C3D6O)§ 0.89–
0.96 (3H, m, CH3, M+m), 1.32–1.37 (2H, m, CH2, M+m), 1.39, 1.40
(9H, s, C(CH3)3, M+m), 1.44–1.50 (2H, m, CH2, M+m), 3.11–3.27
(5H, m, NCH2 m, 2 ꢁ H-3m, H-3M), 3.25 (2H, td, J 7.2, 5.6 Hz,
NCH2, M), 3.47 (1H, br d, J 9.6 Hz, H-60), 3.67 (1H, dd, J 9.6,
3.6.3. 5-epi-1-Azafagomine-1N-(p-aminophenyl)carboxamide
(15a)
Carboxamide 13a (0.07 g; 0.09 mmol); EtOH (7 mL); HCl sol.
(2 mL); Pd/C 10% (0.03 g). Obtained compound 15a as a yellow

R. Mendes et al. / Carbohydrate Research 395 (2014) 52–57
57
oil (0.07 mmol; 73%). mmax (neat): 3446 (OH and NH), 1640
(C@O) cmꢀ1
dH (400 MHz, D2O) 2.89–2.92 (1H, m, H-6),
absorbance into product concentrations. Initial velocities were cal-
culated from the slopes of the Abs versus time graphs for each con-
centration of substrate, and these were used to construct
Michaelis–Menten plots. The kinetic parameters KM, KMapp and
Vmax were determined by fitting the experimental results to a rect-
angular hyperbole using the Origin 8 Graph Pad. The inhibition
type was established as competitive for all enzymes and inhibitors
tested, using 4 different concentrations of inhibitors (in triplicate).
Individual Ki values were obtained from the linear regression plot
of KM/KMapp as a function of the inhibitor concentration, [I], where
the slope corresponds to 1/Ki. Reported Ki values are expressed as
average of 3 independent Ki determinations.
.
2.96–2.99 (2H, m, H-3), 3.78–3.87 (1H, m, H-60), 3.89–3.95 (1H,
m, H-4), 4.08 (1H, t, J 2.6 Hz, H-5), 4.65 (1H, ddd, J 14.4, 5.6,
2.4 Hz, H-60), 7.12 (1H, d, J 8.0 Hz, Ho), 7.35 (1H, d, J 8.0 Hz, Hm)
ppm. dC (100.6 MHz, D2O) 42.0 (C-6), 45.1 (C-3), 58.3 (C-60), 64.4
(C-4), 66.5 (C-5), 117.2 (Co), 123.4 (Cm), 123.39, 131.80 (Cq),
146.12 (C@O) ppm. HRMS (ESI): calcd for
C10H18N4O4
[M-C7H7N2O]+ 148.0770 ; found: [M-C7H7N2O.]+ 148.0788.
3.6.4. 5-epi-1-Azafagomine-1N-butylcarboxamide (15b)
Carboxamide 13b (0.04 g; 0.06 mmol); EtOH (2.5 mL); HCl sol
(1 M; 0.5 mL); Pd/C (0.01 g). Obtained compound 15b, yellow oil
(0.06 mmol; quant.). mmax (neat): 3388 (O-H), 1645 (C@O) cmꢀ1
.
Acknowledgments
dH (400 MHz, D2O) 0.94 (3H, t, J 7.4 Hz, CH3) 1.34–1.36 (2H, m,
CH2), 1.48–1.52 (2H, m, CH2), 2.86–2.89 (2H, m, H-3), 3.01–3.20
(2H, m, NCH2), 3.75 (1H, dd, J 12.0, 6.4 Hz, H-60), 3.81 (1H, dd, J
12.0, 9.6 Hz, H-60), 3.86 (1H, ddd, J 9.6, 6.8, 2.8 Hz, H-4), 4.04 (1H,
t, J 2.8 Hz, H-5), 4.57 (1H, ddd, J 8.4, 6.4, 2.4 Hz H-6) ppm. dC
(100.6 MHz, D2O) 13.1 (CH3), 19.4 (CH2), 31.6 (CH2), 39.5 (NCH2),
44.9 (C-3), 58.3 (C-6), 58.4 (C-60), 64.5 (C-4), 66.5 (C-5), 161.0
(C@O) ppm. HRMS (ESI): calcd for C10H21N3O4 [M+Na]+ 270.1424;
found: [M+Na]+ 270.1423.
We thank FCT for project funding PTDC/QUI/67407/2006 and
PTDC/QEQ-MED/1671/2012; FCT and FEDER for funding NMR
spectrometer Bruker Avance III 400 as part of the National NMR
Network. V.C.M.D. also thanks for PhD Grant SFRH/BD/61290/2009.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.06.
015.
3.7. Measurement of glycosidase inhibition
a
-Glucosidase from bakers’ yeast (EC 3.2.1.20, Sigma G-5003)
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lL, was tested using 1 unit/mL of b-glucosidase or 0.21 units/
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a
-glucosidase, in the absence and in the presence of inhibitor
(14a, b and 15a, b: 6.66 ꢁ 10ꢀ6 to 50 ꢁ 10ꢀ6 M). Blanks were set
containing all reaction components but enzyme. All assays were
performed in triplicate.
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
same conditions used for the enzyme assays, was used to convert
e
l = 570,000 Mꢀ1 (pH 7.0), determined in the