Bicyclic 1-Azafagomine Derivatives: Synthesis and Glycosidase Inhibitory Testing

Article abstract of DOI:10.1055/s-0039-1690019

The synthesis of a series of 1-azafagomine derivatives that are tethered with five- and six-membered 1,2-annulated ring systems is described. These compounds were used in order to explore whether a hydrogen-bond acceptor group on the carbon atom corresponding to the glycosidic oxygen is able to interact with the catalytic acidic residue of β-glucosidase. The hydrogen-bond acceptor group was installed at various positions on the annulated ring system making it possible to study the effect of altering the position of this group.

Full text of DOI:10.1055/s-0039-1690019

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2019, 30, A–L
paper
A
en
T. C. S. Evangelista et al.
Paper
Synthesis
Bicyclic 1-Azafagomine Derivatives: Synthesis and Glycosidase
Inhibitory Testing
Tereza C. Santos Evangelistaa,b
Potent b-glucosidase inhibitor?
Poor b-glucosidase inhibitor?
Óscar Lopéz*c
O
Magne O. Sydnesa
HO
HO
HO
HO
HO
HO
n
n
N
N
José G. Fernández-Bolañosc
Sabrina Baptista Ferreirab
Emil Lindbäck*a
Hydrogen-bond
acceptor group
N
N
O
HO
HO
O
O
H
H
Catalytic residue of
β-glucosidase
O
O
O
O
O
O
a Department of Chemistry, Bioscience and Environmental Engineering,
Faculty of Science and Technology, University of Stavanger, 4036
Stavanger, Norway
emil.lindback@uis.no
b Department of Organic Chemistry, Chemistry Institute, Federal Univer-
sity of Rio de Janeiro, UFRJ, 21949-900 Rio de Janeiro, RJ, Brazil
c Departamento de Química Orgánica, Facultad de Química, Universidad
de Sevilla, c/Profesor García González 1, 41012 Seville, Spain
osc-lopez@us.es
Received: 02.06.2019
Accepted after revision: 12.07.2019
Published online: 14.08.2019
development of iminosugars as pharmaceuticals is to make
them selective for the glycosidase involved in the disease
they are targeted to combat.10 Despite this apparent draw-
back, three iminosugars, namely, N-(2-hydroxy)ethyl-DNJ
(miglitol; Glyset®),11 N-butyl-DNJ (miglustat; Zavesca®),12
and 1-deoxygalactonojirimycin (DGJ) (Galafold®)13 have
reached the commercial market as pharmaceuticals for
treatment of type 2 diabetes, Gaucher disease, and Fabry
disease, respectively. In addition, imino- and azasugars also
serve as useful tools to explore the structure and the reac-
tion mechanisms of glycosidases.14 Thence, the application
of imino- and azasugars for mapping the mechanism of
such hydrolases and their pharmaceutical properties have
triggered extensive synthesis of these sugar mimetics.15
Glycosidase catalysis has been found to proceed through
an oxocarbenium like transition state (Figure 1a) in which
the positive charge is distributed between the endocyclic
oxygen atom and the anomeric carbon atom.16 DNJ is be-
lieved to behave as a potent glycosidase inhibitor because,
in its acidic form, it resembles the positive charge on the
endocyclic oxygen atom in the transition state of glycosi-
dase catalysis.17 Similarly, isofagomine (2) was also expect-
ed to behave as a potent glycosidase inhibitor in particular
for -glucosidase, because in its acidic form, it resembles
the positive charge on the anomeric carbon atom in the
transition state.15a,17,18 In this context, perhaps, an unex-
pected observation was made when it was found that fluo-
rescence labeled derivatives of isofagomine19 and DNJ20
were able to bind -glucosidase in their neutral forms. On
the other hand, X-ray crystallographic analysis of a cello-
bio-derived isofagomine in complex with Ca15a has unam-
biguously shown that the cellobio-derived isofagomine
binds the enzyme in its acidic form.21 In order to incorpo-
rate the glycosidase inhibitory properties of both DNJ (1)
and isofagomine (2) into one single species, a hybrid
DOI: 10.1055/s-0039-1690019; Art ID: ss-2019-t0308-op
Abstract The synthesis of a series of 1-azafagomine derivatives that
are tethered with five- and six-membered 1,2-annulated ring systems is
described. These compounds were used in order to explore whether a
hydrogen-bond acceptor group on the carbon atom corresponding to
the glycosidic oxygen is able to interact with the catalytic acidic residue
of -glucosidase. The hydrogen-bond acceptor group was installed at
various positions on the annulated ring system making it possible to
study the effect of altering the position of this group.
Key words iminosugars, glycosidases, glycosidase inhibitor, hydrogen
bond, hydrogen bond acceptor, catalytic acidic residue
Glycosidases are a widespread group of enzymes in liv-
ing organisms, responsible for the cleavage of glycosidic
bonds during the degradation of oligo- and polysaccharides
and glycoconjugates.1 The ubiquitous occurrence of glycosi-
dases has marked glycosidase inhibitors as attractive phar-
maceutical targets.2 Iminosugars [such as 1-deoxynojirimy-
cin (DNJ), 1] (Figure 1a) are carbohydrate mimetics that
contain a nitrogen atom in place of the endocyclic oxygen
atom. Azasugars [such as isofagomine (2)] are a similar type
of carbohydrate mimetics, which contain a nitrogen atom in
place of a carbon atom. Aza- and iminosugars constitute to-
gether the most explored group of glycosidase inhibitors.3
This group of compounds mimic carbohydrates sufficiently
in order to interact with carbohydrate handling enzymes
but are still sufficiently stable to avoid undergoing metabo-
lization by such enzymes.4 The metabolic stability and gly-
cosidase inhibitory properties of imino- and azasugars have
attracted interest towards elucidating their potential as
treatment for diseases such as diabetes,5 cancer,6 AIDS,7 in-
fluenza,8 and lysosomal storage disorders, for example,
Gaucher and Fabry diseases.9 However, one challenge in the
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
T. C. S. Evangelista et al.
Paper
Synthesis
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
OH
NH
HO
N
NH
O
N
NH
HO
HO
HO
OH
OH
OH
DGJ (Galafold®)
HO
OH
transition state
OH
1
2
(a)
miglitol (Glyset®)
miglustat (Zavesca®)
HO
HO
HO
HO
HO
HO
HO
H
N
HO
HO
HO
HO
HO
N
N
HO
N
HO
HO
HO
HO
NH
NH
N
N
N
N
N
N
1
2
2 NH
N
NH
N
N
N
N
1
NH HO
Ph HO
HO
HO
HO
O
O
OH
OH
OH
8
OH
OH
10
3
4
5
6
7
9
weak β-glucosidase
Ki = 0.32 μM22b
Ki = 13 μM23
Ki = 150 μM25
this work
HO
Ki = 19 μM26
Ki > 8000 μM26
Ki = 0.1 μM25
Ki = 23 μM28
inhibitor
HO
HO
HO
HO
O
O
O
HO
HO
N
HO
HO
HO
HO
HO
HO
HO
HO
N
N
N
N
N
N
N
N
HO
N
N
N
HO
HO
HO
O
HO
O
O
11
12
13
14
O
O
NOH
O
O
H
O
H
O
O
O
O
(b)
(c)
O
HO
HO
HO
HO
HO
HO
Potent β-glucosidase inhibitor?
Poor β-glucosidase inhibitor?
HO
HO
HO
HO
N
N
N
N
N
N
N
N
HO
HO
15
17
18
16
Figure 1 (a) Known imino- and azasugars and their Ki values for the inhibition of sweet almond -glucosidase, (b) bicyclic iminosugars addressed in this
study as potential glycosidase inhibitors, and (c) interaction mode we want to investigate between our proposed glycosidase inhibitors and -glucosi-
dase.
structure between the two, namely, 1-azafagomine (3) (Fig-
ure 1a) was synthesized and concluded to behave as a po-
tent - and -glucosidase inhibitor.22 Subsequent work by
Vasella and co-workers led to the hydrazide analogue 4 of
1-azafagomine (3), which was found to behave as a micro-
molar -glucosidase inhibitor.23 It was suggested that the
C=O group of 4 provided interactions with the catalytic
acidic acid residue of the enzyme. However, Davies and co-
workers did not share this view.14b In addition, it was found
that N-alkyl analogues of 4, such as 5, provided very poor -
glucosidase inhibition, which was interpreted in such a way
that the NH group in 2-position of 4 might provide interac-
tion with the catalytic nucleophilic residue of the enzyme.23
Gluco-azoles 6,24 7,25 8,26 9,27 and 1028 (Figure 1a) have been
found to behave as sweet almond -glucosidase inhibitors
with inhibitory constant Ki values ranging from 0.1 M to
above 8000 M.25,26,28 Mechanistic studies with the gluco-
azoles have concluded that a nitrogen atom in the position
corresponding to the glycosidic oxygen atom is essential for
their inhibition of -glucosidase.29 Indeed, the lone pair
electrons in this glycosidic nitrogen atom establishes a hy-
drogen bond with the catalytic acidic group in the plane of
the sugar ring for -glycosidases.
Therefore, 1,2-annulation of 1-azafagomine (3) with a
five- or six-membered ring including a hydrogen bond ac-
ceptor group (oximo or oxo groups), prepared herein are at-
tractive compounds, as they might display very different
glycosidase inhibitory properties depending on the position
of the hydrogen bond acceptor group. Thus, we hypothe-
sized that compounds 11–14 (Figure 1b) with a hydrogen
bond acceptor group on the carbon corresponding to the
glycosidic oxygen atom would have the potential to form a
hydrogen bond with the catalytic acidic residue of -gluco-
sidase (Figure 1c) in a similar way as azoles 6, 7, 9, 10 and
perhaps hydrazide 4. Compounds 15 and 16 (Figure 1b) on
the other hand, include the hydrogen-acceptor oxo group
remote from the carbon corresponding to the glycosidic ox-
ygen atom and should therefore behave as weaker -gluco-
sidase inhibitors (Figure 1c) than 11–14 according to our
speculation. For comparison, we also included compounds
17 and 18 in our study, which lack a hydrogen-bond accep-
tor group in the 1,2-annulated ring system. However, 1-
azafagomine (3) displays a pKa of 5.4 (in its acidic form)30
whereas that of 4 is presumably less than 3,23 therefore, in
our design approach it could not be excluded that 17 and
18, in their acidic form, are able to form an ionic interaction
with enzymes whereas such an interaction is less likely for
11–13, 15 and 16.
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

C
T. C. S. Evangelista et al.
Paper
Synthesis
The synthesis of 15-TFA, 16, and 13 commenced from L-
xylose (19), which was converted into the known furanose
2031 in 53% yield over three steps with only one purification
(Scheme 1). By using a protocol developed by Bols and co-
workers,22b 20 was treated with Boc-hydrazide in the pres-
ence of sodium cyanoborohydride to provide hydrazide 21,
which underwent N-Alloc protection upon treatment with
chloroformate to provide 22 in 84% yield. In order to ring-
close 22 into hydrazide 23, a protocol previously used by
Bols22b and Jensen32 for similar transformations was used,
including three consecutive steps: mesylation, TFA promot-
ed Boc-deprotection, and finally heating in nitromethane in
the presence of diisopropylethylamine (DIPEA, Hünig’s
base) to provide hydrazide 23 in 72% yield, a reaction se-
quence that only required one purification. The obtained
hydrazide 23 served as a common intermediate leading to
target compounds 15-TFA, 16, and 13. Thus, in order to pre-
pare 15-TFA and 16, compound 23 was treated with 3-bro-
mopropionyl chloride and 4-bromobutyryl chloride to pro-
vide 24 and 25, respectively, where 25 was contaminated
with traces of 4-bromobutanoic acid. In addition, to obtain
24, it was essential to carry out the acylation in the absence
of base in order to avoid elimination of the bromo substitu-
ent. Subsequent palladium-catalyzed Alloc-deprotection of
24 and 25 promoted the cyclization into bicyclic compound
26 and 27, respectively. The benzyl groups of 26 were re-
moved by palladium-catalyzed hydrogenolysis in ethanol
and TFA to provide the target compound 15 in its acidic
form, 15-TFA. Interestingly, when the same de-O-benzyla-
tion protocol was used for 27, several side products were
formed, which could not be removed by silica gel column
chromatography. However, when a mixture of t-BuOH and
water was used as solvent, instead of ethanol, target com-
pound 16 was smoothly obtained. In order to reach the
third target hydrazide 13, the common intermediate 23 un-
derwent Alloc-deprotection utilizing palladium catalysis.
However, 28 was problematic to isolate in its basic form
presumably due to oxidation, which was solved by convert-
ing it into its acidic form upon treatment with methanolic
hydrochloric acid prior to purification. In the subsequent
step, the hydrazinium chloride 28 underwent 1,2-annula-
tion into hydrazide 29 upon treatment with succinyl di-
chloride. Subsequent de-O-benzylation of 29 using palladi-
um-catalyzed hydrogenation gave target compound 13.
The synthesis of target compounds 11, 14, and 17 start-
ed from Boc-hydrazine 21 (Scheme 2), which underwent an
acylation–cyclization sequence to form hydrazide 30 in 71%
yield. The following mesylation–deprotection–cyclization
sequence resulted in the formation of the bicyclic com-
pound 31, which served as a common intermediate for tar-
get compounds 11, 14, and 17. The first target 11 was com-
pleted when 31 was subjected to palladium-catalyzed de-
O-benzylation under hydrogen atmosphere. The second tar-
get compound 17 was obtained when 31 was subjected to a
similar protocol that has been described by Viuff and Jen-
sen,32 which included lithium aluminum hydride-promoted
reduction followed by palladium-catalyzed hydrogenation.
Worth mentioning, efforts to isolate the intermediate after
reduction with lithium aluminum hydride failed. However,
hydrazine 17 did not show any tendency for decomposition
after one week in D2O at room temperature. In order to
reach the third target, hydrazide 31 was treated with
Lawesson’s reagent (LR) to provide thiohydrazide 32, which
turned out to be unstable and was therefore immediately
treated with hydroxylamine hydrochloride in the presence
of sodium bicarbonate to give amidoxime 33 in 71% yield
over two steps. In order to remove the benzyl groups
from 33, a protocol reported by Zhao and co-workers for
OBn
OBn
OH
OBn
NHBoc
NH
NHBoc
NAlloc
NH2NHBoc, NaCNBH3
AcOH, MeOH
1) HCl, MeOH, RT
2) BnBr, NaH, DMF, 0 °C
OH
OH
O
O
AllocCl, aq NaHCO3/THF
OH
OH
60 °C
77%
0 °C
84%
3) aq 4 M HCl/1,4-dioxane
BnO
BnO
HO
60–65 °C
53%
OBn
BnO
OBn
OH
OBn
19
20
21
22
1) MsCl, NEt3, CH2Cl2, 0 °C
2) TFA, CH2Cl2
3) DIPEA, MeNO2, 100 °C
O
BnO
BnO
BnO
BnO
BnO
Pd(PPh3)4, Bu3SnH
AcOH, CH2Cl2, RT
O
O
Cl
Br
n
BnO
NH
N
N
N
Br
n
72%
CH2Cl2, 0 °C
NAlloc
n
NAlloc
BnO
BnO
BnO
23
n = 1; 26, 85% Pd/C, H2, t-BuOH–H2O
n = 1; 24, 80%
n = 2; 25, 85%
1) Pd(PPh3)4, Bu3SnH, AcOH
CH2Cl2, RT
n = 2; 27, 44%
50%
Pd/C, H2, TFA, EtOH, 74%
2) methanolic HCl
93%
HO
O
BnO
BnO
O
HO
BnO
BnO
HO
HO
Cl
O
O
HO
N
O
O
Cl
HO
HO
NH
NH2
Cl
Pd/C, H2, TFA
O
N
N
N
N
N
N
O
HO
H
EtOH
84%
N
NEt3, CH2Cl2
0 °C
BnO
BnO
HO
O
CF3
15-TFA
16
O
41%
13
29
28
Scheme 1 Synthesis of hydrazides 15-TFA, 16, and 13
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

D
T. C. S. Evangelista et al.
Paper
Synthesis
BnO
BnO
HO
Boc
OBn
BnO
OBn
NHBoc
NH
1) MsCl, NEt3, CH2Cl2, 0 °C
2) TFA, CH2Cl2, RT
1) BrCH2CH2COCl
CH2Cl2, 0 °C
N
N
Pd/C, H2
TFA, EtOH
OH
HO
HO
OH
N
N
N
N
3) DIPEA, MeNO2
2) NaHCO3, EtOH
74%
O
BnO
80 °C
75%
70 °C
71%
OBn
BnO
OBn
O
O
21
30
31
11
HO
BnO
BnO
HO
HO
BnO
1) LiAlH4, TFA, RT
LR, toluene 2) Pd/C, H2, TFA, EtOH
BCl3
CH2Cl2
HO
BnO
NH4OH•HCl, NaHCO3
N
N
N
N
N
N
N
N
RT 25%
MeOH, 45 °C
71% (from 31)
–78 °C
41%
HO
BnO
HO
BnO
S
NOH
NOH
14
33
32
17
Scheme 2 Synthesis of compounds 11, 14, and 17
de-O-benzylation of similar compounds with boron trichlo-
ride was used,33 which gave the target amidoxime 14 in 41%
yield.
The tests showed that our compounds did not display any
significant inhibition for any of the enzymes in the panel.
The most significant inhibition (29%) of -glucosidase was
observed for amidoxime 14 and hydrazide 16, which con-
tain a hydrogen-bond acceptor group on the carbon corre-
sponding to the glycosidic oxygen atom and on a remote
carbon atom, respectively. Even though we speculated that
a hydrogen bond acceptor group on the carbon correspond-
ing to the glycosidic oxygen atom would implement hydro-
gen-bond interactions with the catalytic acidic residue, the
inhibition of -glucosidase by 14 is too weak for assuming
any such interaction to be significant. In addition, the very
low or no inhibition of -glucosidase by compounds 11–13
further indicates that a hydrogen-bond acceptor group on
the carbon corresponding to the glycosidic oxygen atom is
unable to interact with the catalytic acidic residue. Indeed,
the inhibition profile for compounds 11–13 is in agreement
with the observation made by Ramana and Vasella who
concluded that an N-alkyl group in 2-position of hydrazide
4, such as for 5, had a strong retarding effect on the inhibi-
tion.23 Another interesting finding in our study was that
both 1-azafagomine derivatives 17 and 18-TFA behaved as
poor glycosidase inhibitors even though 1-azafagomine (4)
itself is a very potent inhibitor of sweet almond -glucosi-
dase (Ki 0.23 M).22b In addition, 1-azafagomine derivatives
tethered with alkyl groups in 2-position have been found to
behave as even more potent -glucosidase inhibitors than
1-azafagomine.35 Therefore, it cannot be excluded that the
lack of a hydrogen atom in 1-position of 17 and 18-TFA
eliminates potential interactions with the catalytic nucleo-
philic residue at least if they ‘try’ to interact with the en-
zyme in their basic forms.
The synthesis of target compounds 12 and 18-TFA also
commenced from Boc-hydrazine 21 (Scheme 3), which un-
derwent cyclization into hydrazide 34 when it first was ac-
ylated with 4-bromobutyryl chloride and then treated by
sodium hydride. Subsequent cyclization of 34 into 35 was
achieved by mesylation, Boc-deprotection, and finally treat-
ment with DIPEA at elevated temperature in nitromethane.
Compound 35 served as the common intermediate for both
target compounds 12 and 18-TFA. The first target 12 was
obtained when the benzyl groups were removed by palladi-
um-catalyzed hydrogenation. Hydrazinium salt 18-TFA was
obtained when 35 was subjected to reduction with lithium
aluminum hydride followed by removal of the benzyl
groups by palladium-catalyzed hydrogenation in the pres-
ence of trifluoroacetic acid (TFA).
Boc
OBn
OBn
N
N
1) BrCH2CH2CH2COCl
NEt3, 0 °C
NHBoc
NH
OH
OH
2) NaH, DMF
0 °C to RT
68%
OBn
BnO
BnO
OBn
21
34
BnO
1) MsCl, NEt3, CH2Cl2, 0 °C
2) TFA, CH2Cl2, RT
Pd/C, H2
TFA, EtOH
BnO
BnO
N
N
3) DIPEA, MeNO2
80 °C
68%
70%
HO
O
35
HO
HO
HO
N
N
1) LiAlH4, THF
RT
HO
HO
N
N
2) Pd/C, TFA
EtOH
O
12
O
41%
H
In conclusion, the arming of 1-azafagomine (3) with an
1,2-annulated five- and six-membered ring containing a
hydrogen-bond acceptor group on the carbon correspond-
ing to the glycosidic oxygen atom did not implement any
favorable hydrogen-bond interactions with catalytic acidic
residue of -glucosidase. Indeed, the inhibition data were in
agreement with Vasella’s and Ramana’s finding that the
substitution of the hydrogen atom of hydrazide 4 in 2-posi-
tion with an alkyl group has a negative effect on the inhibi-
tion. The importance of such hydrogen atom was further
F3C
O
18-TFA
Scheme 3 Synthesis of compounds 12 and 18-TFA
Our compounds were tested as glycosidase inhibitors on
a panel consisting of six glycosidases (Table 1). Even though
the enzymes in the panel are not mammalian enzymes,
many of the enzymes have previously been used in the ear-
ly stage identification of relevant glycosidase inhibitors.34
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

E
T. C. S. Evangelista et al.
Paper
Synthesis
Table 1 Percent Inhibition of Compounds 11, 12, 13, 14, 15-TFA, 16, 17, and 18-TFA against a Panel of Six Glycosidasesa
Enzyme
11
N.I.
12
N.I
13
N.I.
14
15%
15-TFA
16
N.I.
17
N.I
18-TFA
-glucosidase (Saccharomyces cerivisiae)
-glucosidase (almonds)
N.I.
18%
11%
N.I.
N.I
N.I.
23%
N.I.
N.I
N.I
N.I.
12%
N.I.
14%
N.I.
12%
10%
N.I
29%
N.I.
N.I.
N.I.
N.I.
29%
N.I.
N.I
N.I.
12%
N.I
-mannosidase (Jack bean)
N.I.
N.I.
N.I.
N.I.
-galactosidase (Green coffee beans)
-galactosidase (E. coli)
N.I.
N.I.
N.I.
N.I.
N.I.
N.I.
N.I
-galactosidase (Asp. oryzae)
N.I.
N.I.
a [I] = 100 M; [S] = KM for glycosidases; pH 6.8 for glycosidases (except for -mannosidase, pH 5.6). N.I.: No inhibition.
supported by the inhibition data of compounds 17 and TFA-
18, which behaved as much weaker glycosidase inhibitors
than 1-azafagomine and its derivatives containing an alkyl
group in 2-position.
column chromatography (PE–EtOAc, 19:1 → 17:3 → 3:1) to provide
the title compound as a 1:0.6 mixture of anomers; colorless syrup;
yield: 4.49 g (53%); Rf = 0.25 (PE–EtOAc, 3:1); []D26 –12 (c 1.2, EtOAc).
1H NMR (CDCl3, 400.13 MHz):  = 7.40–7.27 (m, 15 H, ArH), 5.52 (dd,
J1,2 = 4.2 Hz, J1,OH = 9.6 Hz, 0.4 H, 1-Hminor), 5.29 (d, J1,OH = 11.8 Hz, 0.6 H,
1-Hmajor), 4.68–4.49 (m, 6 H, 6 × CHPhmajor, 6 × CHPhminor), 4.46–4.41
(m, 1 H, 4-Hmajor, 4-Hminor), 4.14 (dd, J3,2 = 3.0 Hz, J3,4 = 5.4 Hz, 0.6 H, 3-
CH2Cl2 was dried over 4 Å molecular sieves (oven dried). For petro-
leum ether (PE), the 40–65 °C fraction was used. All reactions were
carried out under a N2 or argon atmosphere, unless otherwise speci-
fied. TLC analyses were performed on Merck silica gel 60 F254 plates
using UV light for detection. Silica gel NORMASIL 60® 40–63 m was
used for flash column chromatography. NMR spectra were recorded
on a Bruker Avance NMR spectrometer; 1H NMR spectra were record-
ed at 400.13 MHz, 13C NMR spectra were recorded at 100.61 MHz, and
19F spectra were recorded at 376.46 MHz in CDCl3, CD3OD or D2O.
Chemical shifts are reported in ppm relative to an internal standard of
residual chloroform ( = 7.26 for 1H NMR;  = 77.00 for 13C NMR), re-
sidual methanol ( = 3.31 for 1H NMR;  = 49.00 for 13C NMR). High-
resolution mass spectra (HRMS) were recorded from MeOH solutions
on a JMS-T100LC AccuTOFTM in positive electrospray ionization (ESI)
mode.
H
major), 4.08 (dd, J3,2 = 2.3 Hz, J3,4 = 4.4 Hz, 0.4 H, 3-Hminor), 4.05–4.04
(m, 0.6 H, 2-Hmajor), 4.00–3.97 (m, 1 H, 2-Hminor, OHmajor), 3.90 (d, JOH,1
9.6 Hz, 0.4 H, OHminor), 3.82–3.69 (m, 2 H, 5-Hamajor, 5-Hbmajor, 5-Haminor
5-Hbminor).
13C NMR (CDCl3, 100.61 MHz):  = 138.3 (Arminor), 137.8 (Arminor), 137.7
(Armajor), 137.6 (Armajor), 137.5 (Armajor), 136.9 (Arminor), 128.8–127.7
(Armajor, Arminor), 101.8 (1-Cmajor), 96.3 (1-Cminor), 86.7 (2-Cmajor), 81.4
(2-Cminor, 3-Cmajor), 81.1 (3-Cminor), 79.9 (4-Cmajor), 77.4 (4-Cminor, over-
lap of the solvent signal), 73.8 (CH2,major), 73.6 (CH2,minor), 73.1 (CH2,minor),
72.8 (CH2,major), 72.4 (CH2,minor), 72.0 (CH2,major), 68.8 (5-Cmajor), 68.4 (5-
=
,
C
minor).
HRMS (ESI): m/z [M + Na]+ calcd for C26H28O5Na: 443.1829; found:
443.1824.
1-(2-tert-Butyloxycarbonyl)hydrazino-1-deoxy-2,3,5-tri-O-ben-
zyl-L-xylitol (21)22b
Methyl 2,3,5-Tri-O-benzyl-L-xylofuranoside (20)
To a solution of furanose 20 (5.53 g, 13.2 mmol, 1.0 equiv), AcOH
(2.64 mL, 46.2 mmol, 3.5 equiv), and tert-butyl carbazate (3.83 g, 29.0
mmol, 2.2 equiv) in MeOH (35 mL) was added NaCNBH3 (3.32 g, 52.8
mmol, 4.0 equiv) at RT under an argon atmosphere. The mixture was
kept stirring at 60 °C overnight. The mixture was then cooled to RT
before removal of the volatiles under reduced pressure. The residue
was dissolved in sat. aq NaHCO3 (60 mL) and EtOAc (60 mL) and the
phases were separated. The aqueous layer was extracted with EtOAc
(60 mL) and the combined organic fractions were concentrated under
reduced pressure. The residue underwent purification by silica gel
flash column chromatography (PE–EtOAc, 4:1 → 3:1) to provide the
title compound as a light yellow oil; yield: 5.38 g (77%); Rf = 0.43 (PE–
EtOAc, 1:1); []D28 +4.6 (c 1.3, CHCl3) {Lit.22b []D +3.7 (c 1.0, CHCl3)}.
1H NMR (CDCl3, 400.13 MHz):  = 7.38–7.25 (m, 15 H, ArH), 6.27 (br s,
1 H, NH), 4.67 (d, J = 11.5 Hz, 2 H, 2 × CHPh), 4.59–4.52 (m, 3 H, 3 ×
CHPh), 4.47 (d, J = 12.0 Hz, 1 H, CHPh), 4.07 (t, J = 6.4 Hz, 1 H, 4-H),
4.02 (br s, 2 H, NH, OH), 3.77 (br s, 2 H, 2-H, 3-H), 3.57 (dd, J5a,4 = 6.0
Hz, J5a,5b = 9.6 Hz, 1 H, 5a-H), 3.50 (dd, J5b,4 = 6.8 Hz, J5b,5a = 9.6 Hz, 1 H,
5b-H), 3.26–3.22 (m, 1 H, 1a-H), 3.14–3.11 (m, 1 H, 1b-H), 1.50 [s, 9 H,
C(CH3)3].
AcCl (0.29 mL, 4.0 mmol, 0.2 equiv) was added dropwise to MeOH (40
mL) at RT. Then, L-xylose (19; 3.0 g, 20.0 mmol, 1.0 equiv) was added
and the obtained mixture was kept stirring at RT for 5.5 h. The tem-
perature was then decreased to 0 °C before adjusting pH (8–9) upon
dropwise addition of aq NaOH (1 M). The solvent was removed under
reduced pressure and the resulting concentrate was co-evaporated
three times from toluene. After co-evaporation, the residue was dis-
solved in anhyd DMF (64 mL). To this solution was added portionwise
NaH (60% dispersion in mineral oil, 4.24 g, 106 mmol, 5.3 equiv) and
the resulting suspension was kept stirring at RT under N2 atmosphere
for 10 min. The temperature was adjusted to 0 °C before dropwise ad-
dition of BnBr (9.8 mL, 100 mmol, 5.0 equiv). After addition, the mix-
ture was kept stirring overnight at RT. The temperature was adjusted
to 0 °C and H2O (100 mL) was added dropwise. The aqueous mixture
was extracted with EtOAc (2 × 100 mL) and the combined organic
fractions were dried (MgSO4), filtered, and concentrated under re-
duced pressure. The residue was dissolved in 1,4-dioxane (60 mL) and
aq HCl (60 mL, 4 M). The mixture was kept stirring at 60–65 °C for 2
days. After this time, the aqueous mixture was extracted with EtOAc
(2 × 75 mL) and the combined organic fractions were concentrated
under reduced pressure. The residue was purified by silica gel flash
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

F
T. C. S. Evangelista et al.
Paper
Synthesis
13C NMR (CDCl3, 100 MHz):  = 156.3 (C=O Boc), 138.1 (Ar), 138.1 (Ar),
128.3–127.7 (Ar), 80.8 [C(CH3)3], 77.5 (3-C), 76.4 (2-C), 74.1 (CH2),
73.2 (CH2), 72.1 (CH2), 71.1 (5-C), 68.1 (4-C), 50.2 (2-C), 28.3
[C(CH3)3].
1H NMR (CDCl3, 400.13 MHz):  = 7.26–7.12 (m, 15 H, ArH), 5.89–5.79
(m, 1 H, CH-vinyl), 5.22 (dd, J = 1.3, 17.3 Hz, 1 H, CHa-vinyl), 5.13 (dd,
J = 1.2, 10.4 Hz, 1 H, CHb-vinyl), 4.78 (d, J = 11.3 Hz, 1 H, CHPh), 4.63
(d, J =11.7 Hz, 1 H, CHPh), 4.58–4.53 (m, 3 H, CHa-allyl, CHb-allyl,
CHPh), 4.45 (d, J = 11.1 Hz, 1 H, CHPh), 4.41 (d, J = 11.9 Hz, 1 H, CHPh),
4.31–4.28 (m, 2 H, 6a-H, CHPh), 3.64–3.48 (m, 4 H, 3a′-H, 3b′-H, 4-H,
5-H), 2.91–2.84 (m, 2 H, 3-H, 6b-H).
13C NMR (CDCl3, 100.16 MHz):  = 155.5 (C=O Alloc), 138.2, 138.0
137.8 (Ar), 132.6 (CH-vinyl), 128.4–127.7 (Ar), 117.9 (CH2-vinyl), 78.4,
78.1 (4-C, 5-C), 74.8 (CH2), 73.2 (CH2), 72.1 (CH2), 67.6 (3′-C), 66.6
(CH2-allyl), 60.1 (3-C), 47.7 (6-C).
The NMR data were was in agreement with that reported for 21.22b
1-(1-Allyloxycarbonyl-2-tert-butyloxycarbonyl)hydrazine-1-de-
oxy-2,3,5-tri-O-benzyl-L-xylitol (22)
To a vigorously stirred solution of hydrazide 21 (645 mg, 1.20 mmol,
1.0 equiv) in sat. aq NaHCO3–THF (2:1; 12 mL) at 0 °C was added
dropwise allyl chloroformate (0.19 mL, 1.80 mmol, 1.5 equiv). The re-
sulting mixture was kept stirring for 1.5 h at 0 °C. After this time, aq
NaOH (1 M, 3 mL) was added and the mixture was kept stirring for 10
min. The aqueous mixture was extracted with EtOAc (20 mL) and
then the collected organic fraction was washed with H2O (20 mL) and
concentrated under reduced pressure. The concentrate was purified
by silica flash gel column chromatography (PE–EtOAc, 4:1 → 3:1 →
3:2) to provide the title compound as a colorless syrup; yield: 627 mg
(84%); Rf = 0.27 (PE–EtOAc, 3:2); []D26 +10 (c 3.3, EtOAc).
1H NMR (CDCl3, 400.13 MHz):  = 7.34–7.27 (m, 15 H, ArH), 6.21 (br s,
1 H, NH), 5.92–5.84 (m, 1 H, CH-vinyl), 5.31 (d, J = 17.4 Hz, 1 H, CHa-
vinyl), 5.20 (d, J = 10.1 Hz, 1 H CHb-vinyl), 4.73–4.44 (m, 8 H, 6 ×
CHPh, CHa-allyl, CHb-allyl), 4.02–3.82 (m, 4 H, 1a-H, 1b-H, 2-H, 4-H),
3.69–3.67 (m, 1 H, 3-H), 3.51–3.41 (m, 2 H, 5a-H, 5b-H), 2.51 (br s, 1
H, OH), 1.45 [s, 9 H, C(CH3)3].
13C NMR (CDCl3, 100.61 MHz):  = 156.0 (C=O Alloc), 154.7 (C=O Boc),
138.0 (3 × Ar), 132.3 (CH-vinyl), 128.5–127.7 (Ar), 118.1 (CH2-vinyl),
117.6 (CH2-vinyl-rotamer), 81.4 [C(CH3)3], 77.8 (3-C), 76.5 (2-C), 74.4
(CH2), 73.3 (CH2), 72.7 (CH2), 71.3 (5-C), 69.0 (4-C), 66.8 (CH2-allyl),
51.2 (1-C), 50.6 (1-C-rotamer), 28.2 [C(CH3)3].
HRMS (ESI): m/z [M + Na]+ calcd for C35H44N2O8Na: 643.2990; found:
643.2980.
HRMS (ESI): m/z [M + H]+ calcd for C30H35N2O5: 503.2540; found:
503.2534.
(3R,4R,5R)-1-N-Allyloxycarbonyl-2-N-(3′-bromopropionyl)-4,5-
dibenzyloxy-3-benzyloxymethylhexahydropyridazine (24)
Compound 23 (0.28 g, 0.55 mmol, 1.0 equiv) was dissolved in anhyd
CH2Cl2 (5 mL) and cooled to 0 °C. 3-Bromopropionyl chloride (0.07
mL, 0.66 mmol, 1.2 equiv) was added dropwise under an argon atmo-
sphere and the resulting mixture was kept stirring at 0 °C. After 10
min, the reaction was quenched with H2O (5 mL) and diluted with
CH2Cl2 (15 mL). The phases were separated and the organic layer was
washed with sat. aq NaHCO3 (15 mL), dried (MgSO4), and concentrat-
ed under reduced pressure. The residue was purified by silica gel flash
column chromatography (PE–EtOAc, 8:2 → 7:3) to provide the title
compound as a colorless syrup; yield: 283 mg (80%); Rf = 0.50 (PE–
EtOAc, 4:1); []D26 –48 (c 0.7, EtOAc).
Compound 24 appeared as a mixture of rotamers (major/minor′ 3:2)
on the NMR time scale.
1H NMR (CDCl3, 400.13 MHz):  = 7.35–7.21 (m, 15 H, ArH), 5.89–5.75
(m, 1 H, CH-vinyl, CH′-vinyl), 5.33–5.17 (m, 3 H, 3-H, 3′-H, CH2-vinyl,
CH2′-vinyl), 4.75–4.69 (m, 1.6 H, 2 × CHPh, CHPh′), 4.65–4.37 (m, 7 H,
6a-H, 4 × CHPh, 5 × CHPh′, CH2-allyl, CH2′-allyl), 4.30 (dd, J6a′,5′ = 2.4
Hz, J6a′,6b′ = 14.7 Hz, 0.4 H, 6a′-H), 3.99–3.98 (m, 0.4 H, 4′-H), 3.88–3.87
(m, 0.6 H, 4-H), 3.85 (d, J3a′′,3b′′ = 9.4 Hz, 0.4 H, 3a′′-H), 3.77–3.51 (m, 5
(3R,4R,5R)-1-N-Allyloxycarbonyl-4,5-dibenzyloxy-3-benzyl-
oxymethylhexahydropyridazine (23)
H, 3a′-H, 3b′-H, 3b′′-H, 5-H, 5′-H, 6b′-H, CH2, CH2′), 3.70 (0.6 H, J6b,5
1.5 Hz, J6b,6a = 14.1 Hz, 6b-H), 3.07–2.86 (2 H, m, CH2, CH2′).
=
To a solution of hydrazide 22 (626 mg, 1.01 mmol, 1.0 equiv) and NEt3
(0.21 mL, 1.31 mmol, 1.3 equiv) in CH2Cl2 (8 mL) at 0 °C was added
dropwise MsCl (0.10 mL, 1.31 mmol, 1.3 equiv) under an argon atmo-
sphere. The mixture was kept stirring at 0 ° for 5 min. After this time,
sat. aq NaHCO3 (10 mL) and CHCl3 (10 mL) were added. The phases
were separated and the aqueous layer was extracted with CHCl3 (10
mL). The combined organic fractions were dried (MgSO4), filtered,
and concentrated under reduced pressure. The residue was dissolved
in toluene (3 × 10 mL) and concentrated under reduced pressure. The
residue was dissolved in methylene chloride (3.6 mL) and TFA (2.4
mL) was then added (2.4 mL). The resulting mixture was kept stirring
at RT under an argon atmosphere for 40 min. The volatiles were then
removed under reduced pressure. The residue was dissolved in a mix-
ture of CHCl3 (15 mL) and sat. aq NaHCO3 (15 mL). The phases were
separated and the aqueous layer was extracted with CHCl3 (15 mL).
The combined organic extracts were dried (MgSO4), filtered, and con-
centrated under reduced pressure. The residue was dissolved in tolu-
ene (3 × 10 mL) and concentrated under reduced pressure. The resi-
due was dissolved in anhyd MeNO2 (9 mL) and DIPEA was added (0.52
mL, 3.03 mmol, 3.0 equiv). The resulting mixture was kept stirring at
100 °C under an argon atmosphere for 2 h. After this time, the vola-
tiles were removed under reduced pressure and the residue under-
went purification by silica gel flash column chromatography (PE–EtOAc,
4:1 → 3:1) to provide the title compound as a yellow oil; yield: 363
mg (72%); Rf = 0.63 (PE–EtOAc, 3:2); []D28 +29 (c 0.7, EtOAc).
13C NMR (CDCl3, 100.61 MHz):  = 172.3 (C=O), 172.0 (C=O′), 156.6
(C=O Alloc′), 156.2 (C=O Alloc), 138.4, 138.1, 137.6 (Ar), 131.8 (CH′-
vinyl), 131.5 (CH-vinyl), 128.4–126.9 (Ar), 119.6 (CH2-vinyl), 118.7
(CH2′-vinyl), 73.0 (CH2, CH2′), 72.9 (5′-C), 72.6 (5-C), 71.2 (CH2, CH2′),
71.0 (CH2′), 70.8 (CH2), 70.7 (4-C), 69.7 (4′-C), 67.4 (CH2-allyl, CH2′-al-
lyl), 67.3 (3′′-C), 67.2 (3′-C), 51.5 (3-C), 51.4 (3′-C), 45.4 (6′-C), 43.6 (6-
C), 35.4 (CH2, CH2′), 26.6 (CH2′), 26.4 (CH2).
HRMS (ESI): m/z [M + Na]+ calcd for C33H37BrN2O6Na: 659.1727 and
661.1707; found: 659.1713 and 661.1695.
(3R,4R,5R)-1-N-Allyloxycarbonyl-2-N-(4′-bromobutyryl)-4,5-
dibenzyloxy-3-benzyloxymethylhexahydropyridazine (25)
Compound 25 was synthesized in the same way as compound 24.
Compound 23 (0.21 g, 0.42 mmol, 1.0 equiv) was reacted with 4-bro-
mobutyryl chloride (0.06 mL, 0.50 mmol, 1.2 equiv) to give 25 as a
colorless syrup (the product contained traces of 4-bromobutanoic ac-
id); yield: 232 mg (85%). Purified by silica gel flash column chroma-
tography (PE–EtOAc, 8:2); Rf = 0.55 (PE–EtOAc, 4:1).
Compound 25 appeared as a mixture of rotamers (major/minor′ =
3:2) on the NMR time scale.
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

G
T. C. S. Evangelista et al.
Paper
Synthesis
1H NMR (CDCl3, 400.13 MHz):  = 7.35–7.22 (m, 15 H, ArH), 5.92–5.77
(m, 1 H, CH-vinyl, CH′-vinyl), 5.33–5.17 (m, 3 H, 3-H, 3′-H, CH2-vinyl,
CH2′-vinyl), 4.75–4.38 (m, 8.6 H, 6a-H, CH2-allyl, CH2′-allyl, 6 × CHPh,
6 × CHPh′), 4.32 (dd, J6a′,5′ = 2.2 Hz, J6a′,6b′ =14.5 Hz, 0.4 H, 6a′-H), 3.99–
3.97 (m, 0.4 H, 4′-H), 3.98–3.58 (m, 1 H, 4-H, 3a′′-H), 3.78–3.71 (m, 1
H, 3a′-H, 3b′′-H), 3.66 (dd, J3b′,3′ = 6.6 Hz, J3b′,3a′ = 9.8 Hz, 0.6 H, 3b′-H),
3.61–3.60 (m, 0.6 H, 5-H), 3.55–3.42 (m, 2.8 H, 5′-H, 6b′-H, CH2Br,
CH2Br′), 3.39 (dd, J6b,5 = 1.6 Hz, J6b,6a = 14.2 Hz, 0.6 H, 6b-H), 2.73–2.61
[m, 1 H CHa–C(O)N, CH′a–C(O)N], 2.53–2.43 [m, 1 H, CHb–C(O)N,
CHb′–C(O)N], 2.32–2.14 (m, 2 H, CH2, CH2′).
13C NMR (CDCl3, 100.61 MHz):  = 174.1 (C=O), 173.8 (C=O′), 156.5
(C=O Alloc′), 156.2 (C=O Alloc), 138.4, 138.2, 137.7, 137.6 (Ar), 131.9
(CH′-vinyl), 131.6 (CH-vinyl), 128.4–127.0 (Ar), 119.4 (CH2-vinyl),
118.6 (CH2′-vinyl), 73.1 (CH2, CH2′), 72.9 (5-C′), 72.6 (5-C), 71.1 (CH2,
CH2′), 70.9 (4-C), 70.8 (CH2, CH2′), 69.9 (4′-C), 67.4, 67.3 (CH2-allyl,
CH2′-allyl, 3′-C, 3′′-C), 51.6 (3′-C), 51.4 (3-C), 45.3 (6′-C), 43.2 (6-C),
33.8 (CH2′-Br), 33.5 (CH2-Br), 29.7 [CH2′–C(O)N], 29.6 [CH2–C(O)N],
27.5 (CH2′), 27.3 (CH2).
2.4 Hz, J6a,6b = 12.0 Hz, 1 H, 6a-H), 3.25 (ddd, J = 2.6, 12.7, 13.7 Hz, 1 H,
4a-H), 3.03–2.98 (m, 1 H, 4b-H), 2.75 (dd, J6b,7 = 1.9 Hz, J6b,6a = 12.0 Hz,
1 H, 6b-H), 2.60–2.45 (m, 2 H, 2a-H, 2b-H), 2.20–2.08 (m, 1 H, 3a-H),
1.73–1.66 (m, 1 H, 3b-H).
13C NMR (CDCl3, 100.61 MHz):  = 167.0 (1-C), 138.6, 138.0, 137.9
(Ar), 128.4–127.4 (Ar), 74.1 (7-C), 72.8 (CH2), 71.2 (CH2), 70.9 (CH2),
70.3 (8-C), 67.7 (9′-C), 54.2 (6-C), 51.3, 51.1 (4-C, 9-C), 30.2 (2-C), 16.6
(3-C).
HRMS (ESI): m/z [M + H]+ calcd for C30H35N2O: 487.2591; found:
487.2585.
(6R,7R,8R)-6,7-Dihydroxy-8-hydroxymethylhexahydro-1H-pyra-
zolo[1,2-a]pyridazin-1-one Trifluoroacetate (15-TFA)
A mixture of 26 (32 mg, 0.067 mmol), 10% Pd/C (32 mg), and TFA (3
drops) in EtOH (10 mL) was degassed under an argon atmosphere and
H2 (1 atm) was introduced. The hydrogenation was left overnight and
at the end, volatiles were removed, and the reaction mixture was pu-
rified by silica gel flash column chromatography (CH2Cl2–MeOH, 95:5
→ 90:10) to afford the desired product 15-TFA as a colorless syrup;
HRMS (ESI): m/z [M + Na]+ calcd for C34H39BrN2O6Na: 673.1884 and
675.1863; found: 673.1872 and 675.1852.
28
yield: 10 mg (74%); Rf = 0.16 (CHCl3–MeOH, 9:1); []D –55 (c 0.30,
MeOH).
(6R,7R,8R)-6,7-Dibenzyloxy-8-benzyloxymethylhexahydro-1H-
pyrazolo[1,2-a]pyridazin-1-one (26)
1H NMR (D2O, 400.13 MHz):  = 4.18 (br s, 1 H, 8-H), 4.01–3.96 (m, 1
H, 8a′-H), 3.83 (br s, 1 H, 6-H), 3.76 (br s, 1 H, 7-H), 3.71 (dd, J8b′,8 = 4.5
Hz, J8b′,8a′ = 11.5 Hz, 1 H, 8b′-H), 3.55–3.48 (m, 1 H, 3a-H), 3.07–2.80
(m, 4 H, 2a-H, 3b-H, 5a-H, 5b-H), 2.57–2.49 (m, 1 H, 2b-H).
13C NMR (D2O, 100.61 MHz):  = 174.2 (1-C), 163.0 (q, J = 35 Hz, C=O),
116.3 (q, J = 292 Hz, CF3), 67.2 (6-C), 65.2 (7-C), 59.1 (8-C), 58.9 (8′-C),
55.6 (5-C), 48.9 (3-C), 29.5 (2-C).
A solution of hydrazide 24 (0.17 g, 0.28 mmol, 1.0 equiv), AcOH (0.09
mL, 1.58 mmol, 5.7 equiv), and Pd(PPh3)4 (16 mg g, 0.014 mmol, 0.05
equiv) in CH2Cl2 (4 mL) was kept stirring for 5 min at RT under an ar-
gon atmosphere. After this time, Bu3SnH (0.18 mL, 0.67 mmol, 2.4
equiv) was added dropwise. After addition, the reaction mixture was
kept stirring and the reaction was complete after 5 min. The solvent
was removed under reduced pressure and the residue was purified by
silica gel flash column chromatography (PE–EtOAc, 1:1 → 2:3 → 7:13)
provided the title compound as a yellow syrup; yield: 112 mg (85%);
Rf = 0.15 (PE–EtOAc, 7:13); []D26 –62 (c 0.4, EtOAc).
19F NMR (D2O, 376.46 MHz):  = –75.6.
HRMS (ESI): m/z [M + Na]+ calcd for C8H14N2O4Na: 225.0846; found:
225.0843.
1H NMR (CDCl3, 400.13 MHz):  = 7.32–7.27 (m, 15 H, ArH), 4.71 (br s,
1 H, 8-H), 4.65 (d, J = 12.0 Hz, 1 H, CHPh), 4.56–4.40 (m, 5 H, 5 ×
CHPh), 3.95–3.60 (m, 1 H, 8a′-H), 3.81–3.77 (m, 2 H, 7-H, 8b′-H), 3.58–
3.55 (m, 2 H, 3a-H, 6-H), 3.01–2.93 (m, 2 H, 5a-H, 5b-H), 2.84–2.80
(m, 1 H, 3b-H), 2.71–2.63 (m, 1 H, 2a-H), 2.50–2.41 (m, 1 H, 2b-H).
13C NMR (CDCl3, 100.61 MHz):  = 171.0 (1-C), 138.3, 137.6 137.5 (Ar),
128.4–127.5 (Ar), 73.5 (6-C), 73.0 (CH2), 71.3 (CH2), 71.1 (CH2), 68.4
(7-C), 67.2 (8′-C), 55.3 (5-C), 52.2 (8-C), 50.3 (3-C), 30.9 (2-C).
(7R,8R,9R)-7,8-Dihydroxy-9-hydroxymethylhexahydropyridazi-
no[1,2-a]pyridazin-1(2H)-one (16)
A suspension of 27 (54 mg, 0.110 mmol) and 10% Pd/C (108 mg ) in t-
BuOH–H2O (10:1, 5.5 mL) was degassed under an argon atmosphere
and H2 (1 atm) was introduced. The hydrogenation was left for 48 h
and at the end, volatiles were removed under reduced pressure, and
the concentrate was purified by silica gel flash column chromatogra-
phy (CH2Cl2–MeOH, 95:5 → 90:10 + 0.2% NH4OH) to afford the desired
product 16 as a yellow solid; yield: 12.1 mg (50%); Rf = 0.34 (CHCl3–
MeOH, 8:2); []D26 –90.3 (c 0.6, MeOH).
HRMS (ESI): m/z [M + H]+ calcd for C29H33N2O4: 473.2254; found:
473.2429.
1H NMR (D2O, 400.13 MHz):  = 4.68–4.66 (m, 1 H, 9-H), 4.06 (dd, J9a′,9
=
9.3 Hz, J 9a′,9b′ = 11.7 Hz, 1 H, 9a′-H), 3.74–3.70 (m, 2 H, 7-H, 8-H), 3.68
(dd, J9b′,9 = 4.4 Hz, J 9b′,9a′ = 11.7 Hz, 1 H, 9b′-H), 3.39 (dd, J6a,7 = 2.3 Hz,
J6a,6b = 12.6 Hz, 1 H, 6a-H), 3.21–3.14 (m, 1 H, 4a-H), 3.04–2.99 (m, 1
H, 4b-H), 2.67 (dd, J6b,7 = 2.3 Hz, J6b,6a = 12.6 Hz, 1 H, 6b-H), 2.46–2.42
(m, 2 H, 2a-H, 2b-H), 2.15–2.04 (m, 1 H, 3a-H), 1.70–1.62 (m, 1 H, 3b-
H).
13C NMR (D2O, 100.61 MHz):  = 171.2 (1-C), 67.3 (7-C), 66.9 (8-C),
59.1 (9-C), 58.9 (9′-C), 54.9 (6-C), 50.3 (4-C), 29.4 (2-C), 15.2 (3-C).
HRMS (ESI): m/z [M + Na]+ calcd for C9H16N2O4Na: 239.1002; found:
(7R,8R,9R)-7,8-Dibenzyloxy-9-benzyloxymethylhexahydropyri-
dazino[1,2-a]pyridazin-1(2H)-one (27)
A solution of hydrazide 25 (55.6 mg, 0.085 mmol, 1.0 equiv), AcOH
(0.03 mL, 0.53 mmol, 6.0 equiv), and Pd(PPh3)4 (0.005 g, 0.0043
mmol, 0.05 equiv) in CH2Cl2 (2 mL) was kept stirring for 5 min at RT
under an argon atmosphere. After this time, Bu3SnH (0.06 mL, 0.22
mmol, 2.6 equiv) was added dropwise. After addition, the reaction
mixture was kept stirring and the reaction was complete after 5 min.
The solvent was removed under reduced pressure and the residue was
purified by silica gel flash column chromatography (PE–EtOAc, 1:1 →
2:3) provided the title compound 27 as a yellow syrup; yield: 18.2 mg
(44%); Rf = 0.18 (PE–EtOAc, 2:3); []D26 –61 (c 0.8, EtOAc).
239.1001.
(3R,4R,5R)-4,5-Dibenzyloxy-3-benzyloxymethylhexahydropyri-
dazine Hydrochloride (28)
1H NMR (CDCl3, 400.13 MHz):  = 7.35–7.26 (m, 15 H, ArH), 5.44–5.41
(m, 1 H, 9-H), 4.75 (d, J = 11.9 Hz, 1 H, CHPh), 4.56–4.53 (m, 3 H, 3 ×
CHPh), 4.46 (d, J = 12.0 Hz, 2 H, 2 × CHPh), 3.98–3.89 (m, 2 H, 9a′-H,
A solution of hydrazide 23 (105 mg, 0.209 mmol, 1.0 equiv), AcOH (63
L, 1.1 mol, 5.0 equiv), and Pd(PPh3)4 (12 mg, 0.010 mmol, 0.05 equiv)
in CH2Cl2 (2 mL) was kept stirring for 5 min at RT under an argon at-
9b′-H), 3.74 (br s, 1 H, 8-H), 3.57–3.56 (m, 1 H, 7-H), 3.34 (dd, J6a,7
=
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

H
T. C. S. Evangelista et al.
Paper
Synthesis
mosphere. Bu3SnH (110 L, 0.418 mmol, 2.0 equiv) was added drop-
wise and the mixture was kept stirring for 5 min. Methanolic HCl (1.5
mL, 0.5 M) was added dropwise. Then, the volatiles were removed un-
der reduced pressure. Purification of the residue by silica gel flash col-
umn chromatography [CH2Cl2–methanolic HCl (0.1 M), 195:5 →
192.5:7.5] provided the title compound 6 as a white foam; yield: 88
mg (93%); Rf = 0.63 (PE–EtOAc, 3:2); []D26 –3 (c 0.7, MeOH).
1H NMR (CD3OD, 400.13 MHz):  = 4.75–4.72 (m, 1 H, 6-H), 4.42 (dd,
J9a,8 = 1.8 Hz, J9a,9b = 13.6 Hz, 1 H, 9a-H), 3.93 (dd, J6a′,6 = 8.5 Hz, J6a′,6b′
=
11.8 Hz, 1 H, 6a′-H), 3.79–3.75 (m, 2 H, H-7, H-8), 3.70 (dd, J6b′,6 = 4.8
Hz, J6b′,6a′ = 11.8 Hz, 1 H, 6b′-H), 3.36 (dd, J9b,8 = 2.2 Hz, J9b,9a = 13.6 Hz,
1 H, 9b-H), 2.90–2.75 (m, 2 H), 2.55–2.46 (m, 2 H).
13C NMR (CD3OD, 100.61 MHz):  =172.7, 172.0 (1-C, 4-C), 67.8, 67.6
(7-C, 8-C), 61.0 (6-C), 60.9 (6′-C), 47.7 (9-C), 30.0, 29.4 (2-C, 3-C).
1H NMR (CD3OD, 400.13 MHz):  = 7.36–7.19 (m, 15 H, ArH), 4.81 (d,
J = 11.2 Hz, 1 H, CHPh), 4.72 (d, J = 11.7 Hz, 1 H, CHPh), 4.65 (d, J = 11.7
Hz, 1 H, CHPh), 4.55 (d, J = 11.2 Hz, 1 H, CHPh), 4.53 (d, J = 11.8 Hz, 1
H, CHPh), 4.47 (d, J = 11.8 Hz, 1 H, CHPh), 3.79–3.78 (m, 1 H, 5-H), 3.72
(dd, J3a′,3 = 4.4 Hz, J3a′,3b′ = 10.3 Hz, 3a′-H), 3.68–3.64 (m, 2 H, 3b′-H, 4-
H), 3.56 (dd, J6a,5 = 3.8 Hz, J6a,6b = 13.1 Hz, 1 H, 6a-H), 3.23 (br s, 1 H, 3-
H), 2.97 (dd, J6b,5 = 9.6 Hz, J6b,6a = 13.1 Hz, 1 H, 6a-H).
13C NMR (CD3OD, 100.61 MHz):  =139.3 (2 Ar), 138.9 (Ar), 129.7–
129.1 (Ar), 77.2 (5-C), 76.3 (4-C), 75.9 (CH2), 74.5 (CH2), 73.6 (CH2),
66.8 (3′-C), 60.6 (3-C), 48.4 (6-C, overlaps with the solvent signal).
HRMS (ESI): m/z [M + Na]+ calcd for C9H14N2O5Na: 253.0795; found:
253.0793.
1-Deoxy-2,3,5-tri-O-benzyl-1-(2-(tert-butoxycarbonyl)-5-oxopyr-
azolidin-1-yl)-L-xylitol (30)
To a solution of hydrazide 21 (1.72 g, 3.20 mmol, 1.0 equiv) in CH2Cl2
(25 mL) was added dropwise 3-bromopropionyl chloride (0.34 mL,
3.37 mmol, 1.05 equiv) at 0 °C under an argon atmosphere. The re-
sulting mixture was kept stirring at 0 °C for 2 h. After this time, NaH-
CO3 (1.61 g, 19.2 mmol, 6.0 equiv) and EtOH (1 mL) were added before
the volatiles were removed under reduced pressure. The residue was
suspended in EtOH (35 mL) and kept stirring at 70 °C for 20 h. The
solvent was removed under reduced pressure and the concentrate
was purified by silica gel flash column chromatography (PE–EtOAc,
3:1 → 7:3 → 1:1) to provide the title compound as a colorless syrup;
yield: 1.35 g (71%); Rf = 0.47 (PE–EtOAc, 9:11); []D28 + 11 (c 1.1, EtO-
Ac).
1H NMR (CDCl3, 400.13 MHz):  = 7.35–7.28 (m, 15 H, ArH), 4.77 (d, J =
11.2 Hz, 1 H, CHPh), 4.65 (d, J = 11.8 Hz, 1 H, CHPh), 4.59 (d, J = 11.2
Hz, 1 H, CHPh), 4.53 (d, J = 11.8 Hz, 1 H, CHPh), 4.50 (d, J = 12.0 Hz, 1 H,
CHPh), 4.46 (d, J = 11.8 Hz, 1 H, CHPh), 4.17–4.11 (m, 1 H, 1a-H), 4.05–
3.98 (m, 3 H, 1b-H, 2-H, 4-H), 3.94–3.87 (m, 1 H, 3a′-H), 3.75 (dd, J =
2.7, 5.8 Hz, 1 H, 3-H), 3.53–3.43 (m, 3 H, 3b′-H, 5a-H, 5b-H), 2.55–2.47
(m, 2 H, OH, 4a′-H), 2.38–2.31 (m, 1 H, 4b′-H), 1.49 [s, 9 H, C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C26H31N2O3: 419.2329; found:
419.2326.
(6R,7R,8R)-7,8-Dibenzyloxy-6-benzyloxymethylhexahydropyri-
dazino[1,2-a]pyridazine-1,4-dione (29)
To a solution of hydrazine hydrochloride 28 (87 mg, 0.19 mmol, 1.0
equiv) in anhyd CH2Cl2 (1.5 mL) at –10 °C was added dropwise a solu-
tion of succinyl chloride (23 L, 0.211 mmol, 1.1 equiv) in CH2Cl2
(0.12 mL) under a N2 atmosphere. Then, a solution of NEt3 (93 L, 0.67
mmol, 3.5 equiv) in CH2Cl2 (1 mL) was added dropwise. After addition,
the mixture was kept stirring at –10 °C for 30 min. After this time, sat.
aq NaHCO3 (10 mL) and CH2Cl2 (10 mL) were added and the phases
were separated. The aqueous layer was extracted with CH2Cl2 (10 mL)
and the combined organic fractions were concentrated under reduced
pressure. Purification of the residue by silica gel flash column chro-
matography (PE–EtOAc, 13:7 → 3:2) provided the title compound 29
as a yellow syrup; yield: 39.4 mg (41%); Rf = 0.31 (PE–EtOAc, 2:3);
[]D28 –41 (c 0.4, EtOAc).
13C NMR (CDCl3, 100.61 MHz):  = 172.1 (5′-C), 157.1 (C=O Boc), 138.1
(Ar), 137.9 (Ar), 137.8 (Ar), 128.3–127.6 (Ar), 82.8 [C(CH3)3], 77.7 (3-
C), 75.3 (2-C), 74.4 (CH2), 73.2 (CH2), 72.4 (CH2), 71.2 (5-C), 69.1 (4-C),
46.9 (3′-C), 45.4 (C-1), 31.3 (4′-C), 28.0 [C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C34H43N2O7: 591.3065; found:
591.3054.
1H NMR (CDCl3, 400.13 MHz):  = 7.34–7.22 (m, 15 H, ArH), 5.22 (m, 1
H, 6-H), 4.74 (dd, J9a,8 = 1.1 Hz, J9a,9b = 13.9 Hz, 1 H, 9a-H), 4.67 (d, J =
11.9 Hz, 1 H, CHPh), 4.64 (d, J = 11.7 Hz, 1 H, CHPh), 4.52–4.48 (m, 2 H,
2 × CHPh), 4.43–4.37 (m, 2 H, 2 × CHPh), 3.89 (dd, J6a′,6 = 9.30 Hz, J6a′,6b′
=
(5R,6R,7R)-6,7-Dibenzyloxy-5-benzyloxymethylhexahydro-1H-
pyrazolo[1,2-a]pyridazin-1-one (31)
10.3 Hz, 1 H, 6a′-H), 3.67–3.62 (m, 2 H, 7-H, 8-H), 3.48 (dd, J6b′,6 = 9.30
Hz, J6b′,6a′ = 10.3 Hz, 1 H, 6b′-H), 3.24 (dd, J9b,8 = 2.0 Hz, J9b,9a = 13.9 Hz,
1 H, 9b-H), 2.72–2.43 (m, 4 H, 2a-H, 2b-H, 3a-H, 3b-H).
13C NMR (CDCl3, 100.61 MHz):  = 169.8, 169.6 (1-C, 4-C), 138.0 (Ar),
137.4 (2 × Ar), 128.7–127.7 (Ar), 72.9 (CH2), 71.7 (8-C), 71.6 (CH2),
71.1 (CH2), 70.7 (7-C), 66.9 (6′-C), 53.0 (6-C), 41.7 (9-C), 29.4, 28.6 (2-
C, 3-C).
To a solution of pyrazolidine 30 (1.33 g, 2.25 mmol, 1.0 equiv) and
NEt3 (0.47 mL, 3.38 mmol, 1.5 equiv) in anhyd CH2Cl2 (17 mL) was
added dropwise MsCl (0.23 mL, 2.93 mmol, 1.3 equiv) at 0 °C under
an argon atmosphere. The resulting mixture was kept stirring for 7
min before the addition of sat. aq NaHCO3 (15 mL). The phases were
separated and the aqueous layer was extracted with CHCl3 (15 mL).
The combined organic fractions were dried (MgSO4), filtered, and
concentrated under reduced pressure. The resulting residue was dis-
solved in toluene (20 mL) and concentrated in vacuo. After concentra-
tion, the residue was dissolved in anhyd CH2Cl2 (8.1 mL) and TFA (5.4
mL) was added. The mixture was kept stirring at RT for 45 min. After
this time, the volatiles were removed under reduced pressure. The
residue was dissolved in toluene (3 × 15 mL) and concentrated in vac-
uo. The concentrate was dissolved in MeNO2 (20 mL) and DIPEA (1.96
mL, 11.3 mmol, 5.0 equiv) was added. The mixture was kept stirring
at 80 °C under an argon atmosphere for 3 h. After this time, the mix-
ture was allowed to reach RT before removal of the volatiles under re-
duced pressure. The residue underwent purification by silica gel flash
HRMS (ESI): m/z [M + H]+ calcd for C30H33N2O5: 501.2384; found:
501.2379.
(6R,7R,8R)-7,8-Dihydroxy-6-hydroxymethylhexahydropyridazi-
no[1,2-a]pyridazine-1,4-dione (13)
A mixture of 29 (39 mg, 77.9 mol), 10% Pd/C (39 mg), and TFA (3
drops) in EtOH (10 mL) was degassed under an argon atmosphere and
H2 (1 atm) was introduced. The hydrogenation was left overnight and
at the end, volatiles were removed, and the reaction mixture was pu-
rified by silica gel flash column chromatography (CHCl3–MeOH, 95:5
→ 90:10) to afford the desired product as a colorless solid; yield: 15.0
mg (84%); Rf = 0.12 (CHCl3–MeOH, 9:1); []D25.9 +23 (c 0.4, MeOH).
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

I
T. C. S. Evangelista et al.
Paper
Synthesis
column chromatography (PE–EtOAc, 3:2 → 1:1 → 3:7) to provide the
title compound as a light yellow solid; yield: 796 mg (75%); Rf = 0.22
(PE–EtOAc, 7:13); []D28 +4 (c 0.58, EtOAc).
13C NMR (CD3OD, 100.61 MHz):  = 70.8 (7-C), 70.7 (6-C), 68.1 (5-C),
58.5 (5′-C), 56.1 (8-C), 52.1 (1-C), 50.6 (3-C), 22.2 (2-C).
HRMS (ESI): m/z [M + H]+ calcd for C8H17N2O3: 189.1234; found:
1H NMR (CDCl3, 400.13 MHz):  = 7.33–7.18 (m, 15 H, ArH), 4.95 (d, J =
10.8 Hz, 1 H, CHPh), 4.72 (d, J = 11.3 Hz, 1 H, CHPh), 4.63 (d, J = 11.3
Hz, 1 H, CHPh), 4.53–4.50 (m, 3 H, 3 × CHPh), 4.41 (dd, J8a,7 = 4.9 Hz,
J8a,8b = 12.5 Hz, 1 H, 8a-H), 3.74–3.53 (m, 5 H, 3a-H, 5a′-H, 5b′-H, 6-H,
7-H), 2.92–2.87 (m, 2 H, 3b-H, 8b-H), 2.58–2.53 (m, 2 H, 2a-H, 2b-H),
2.46-2.44 (m, 1 H, 5-H).
13C NMR (CDCl3, 100.61 MHz):  = 170.3 (1-C), 138.1 (Ar), 137.7 (Ar),
137.5 (Ar), 128.5–127.7 (Ar), 78.2 (C-7), 78.0 (6-C), 75.3 (CH2), 73.5
(CH2), 72.2 (CH2), 68.9 (5-C), 65.8 (5′-C), 48.5 (3-C), 42.8 (8-C), 30.9
(2-C).
189.1232.
(5R,6R,7R)-6,7-Dibenzyloxy-5-benzyloxymethylhexahydro-1H-
pyrazolo[1,2-a]pyridazin-1-one Oxime (33)
To a solution of 31 (200.8 mg, 0.425 mmol) in toluene (8 mL) was add-
ed Lawesson’s reagent (171.8 mg, 0.425 mmol) under an argon atmo-
sphere. The resulting mixture was kept stirring for 24 h at RT. After
this time, the solvent was removed under reduced pressure and the
crude product was purified by flash column chromatography (PE–
EtOAc, 8:2) to afford the desired product 32 as a yellow oil; yield:
185.8 mg (89%). Compound 32 was not fully characterized due to in-
stability and was immediately used in the following step.
HRMS (ESI): m/z [M + H]+ calcd for C29H33N2O4: 473.2435; found:
473.2426.
32
(5R,6R,7R)-6,7-Dihydroxy-5-hydroxymethylhexahydro-1H-pyra-
zolo[1,2-a]pyridazin-1-one (11)
1H NMR (CDCl3, 400.13 MHz):  = 7.28–7.11 (m, 15 H, ArH), 5.07 (dd,
J8a,7 = 5.6 Hz, J8a,8b = 13.0 Hz, 1 H, 8a-H), 4.87 (d, J = 11.2 Hz, 1 H, CHPh),
4.69 (d, J = 11.2 Hz, 1 H, CHPh), 4.57 (d, J = 11.5 Hz, 1 H, CHPh), 4.48–
4.41 (m, 3 H, 3 × CHPh), 3.72–3.68 (m, 1 H, 6-H), 3.67–3.50 (m, 4 H,
3a-H, 5a′-H, 5b′-H, 7-H), 3.14 (ddd, J = 4.02, 9.3, 13.0 Hz, 1 H, 2a-H),
3.07–2.94 (m, 2 H, 2b-H, 8b-H), 2.89–2.82 (m, 1 H, 3b-H), 2.50 (ddd,
J = 1.9, 3.8, 9.4 Hz, 1 H, 5-H).
13C NMR (CDCl3, 100.61 MHz):  = 188.6 (1-C), 138.0 (Ar), 137.6 (Ar),
137.5 (Ar), 128.7–128.0 (Ar), 77.2 (6-C, 7-C), 75.3 (CH2), 73.7 (CH2),
72.6 (CH2), 68.5 (5-C), 66.5 (5′-C), 49.4 (3-C), 46.4 (8-C), 43.4 (2-C).
A mixture of hydrazide 31 (54.7 mg, 0.116 mmol), 10% Pd/C (54.7
mg), and TFA (3 drops) in EtOH (10 mL) was degassed under an argon
atmosphere at RT and H2 (1 atm) was introduced by means of a bal-
loon. The mixture was kept stirring overnight before removal of the
volatiles under reduced pressure. The residue underwent purification
by silica flash column chromatography (CHCl3–MeOH, 19:1 → 9:1) to
provide the title compound as a colorless syrup; yield: 17.4 mg (74%);
Rf = 0.21 (CHCl3–MeOH, 17:3); []D27 +32 (c 0.49, MeOH).
1H NMR (CD3OD, 400.13 MHz):  = 4.13 (dd, J8a,7 = 5.7 Hz, J8a,8b = 12.6
Hz, 1 H, 8a-H), 3.92 (dd, J5a′,5 = 2.3 Hz, J5a′,5b′ = 12.3 Hz, 1 H, 5a′-H), 3.82
(dd, J5a′,5 = 3.7 Hz, J5b′,5a′ = 12.3 Hz, 1 H, 5b′-H), 3.67–3.61 (m, 1 H, 3a-
H), 3.50–3.45 (m, 1 H, 6-H), 3.42–3.35 (m, 1 H, 7-H), 3.08–3.01 (m, 1
H, 3b-H), 2.72–2.61 (m, 2 H, 2a-H, 8b-H), 2.54–2.44 (m, 1 H, 2b-H),
2.28 (ddd, J5,5a′ = 2.3 Hz, J5,5a′ = 3.7 Hz, J5,6 = 9.4 Hz, 1 H, 5-H).
To a solution of compound 32 (185.8 mg, 3.81 mmol, 1.0 equiv) in
MeOH (10 mL) were added NaHCO3 (470.6 mg, 5.71 mmol) and
NH2OH·HCl (396.7 g, 5.71 mmol). The resulting mixture was kept stir-
ring at 45 °C for 15 h. After this time, the reaction mixture was treated
with CH2Cl2 (20 mL) and sat. aq NaHCO3 (20 mL) and the phases were
separated. The aqueous layer was extracted with CH2Cl2 (20 mL) and
the combined organic fractions were dried (MgSO4), filtered, and con-
centrated under reduced pressure. The crude product was purified by
flash column chromatography (PE–EtOAc, 7:3 → 6:4) to afford the de-
sired product 33 as a colorless syrup; yield: 147 mg (71%); Rf = 0.30
(PE–EtOAc, 3:2); []D28 + 34 (c 0.8, EtOAc).
13C NMR (CD3OD, 100.61 MHz):  = 172.5 (1-C), 72.7 (6-C), 72.1 (5-C),
71.2 (7-C), 59.7 (5′-C), 49.0 (3-C, overlaps with the solvent signal),
46.0 (8-C), 31.7 (2-C).
HRMS (ESI): m/z [M + H]+ calcd for C8H15N2O4: 203.1026; found:
203.1026.
(5R,6R,7R)-5-Hydroxymethylhexahydro-1H-pyrazolo[1,2-a]pyri-
dazine-6,7-diol (17)
33
1H NMR (CDCl3, 400.13 MHz):  = 7.27–7.10 (m, 15 H, ArH), 4.87 (d, J =
11.0 Hz, 1 H, CHPh), 4.64 (d, J = 11.0 Hz, 1 H, CHPh), 4.55–4.87 (d, J =
11.6 Hz, 1 H, CHPh), 4.44–4.41 (m, 3 H, 3 × CHPh), 3.89 (dd, J8a,7 = 5.0
Hz, J8a,8b = 11.7 Hz, 1 H, 8a-H), 3.65–3.51 (m, 4 H, 5a′-H, 5b′-H, 6-H, 7-
H), 3.37–3.32 (m, 1 H, 3a-H), 2.86–2.80 (m, 1 H, 2a-H), 2.71–2.62 (m,
2 H, 2b-H, 8b-H), 2.52–2.45 (m, 1 H, 3b-H), 2.38–2.34 (m, 1 H, 5-H).
13C NMR (CDCl3, 100.61 MHz):  = 161.4 (1-C) 138.5 (Ar), 138.2 (Ar),
137.8 (Ar), 128.6–127.8 (Ar), 79.1, 78.4 (6-C, 7-C), 75.3 (CH2), 73.6
(CH2), 72.3 (CH2), 68.1 (5-C), 66.5 (5′-C), 49.4 (3-C), 47.9 (8-C), 26.4
(2-C).
To a solution of compound 31 (42 mg, 88.9 mol) in anhyd THF (1 mL)
was added dropwise a suspension of LiAlH4 (20.2 mg, 0.533 mmol, 6
equiv) in THF (1.5 mL) at RT under an argon atmosphere. Immediately
after addition, sat. aq NH4Cl (5.0 mL) was added dropwise. The result-
ing aqueous mixture was extracted with EtOAc (2 × 10 mL). The com-
bined organic fractions were dried (MgSO4), filtered, and concentrat-
ed under reduced pressure. The concentrate was dissolved in EtOH–
TFA (9:1, 5 mL) and 10% Pd/C (42 mg) was added. The resulting sus-
pension was degassed and then H2 (1 atm) was introduced. The reac-
tion mixture was kept stirring overnight at RT. The solvent was re-
moved under reduced pressure and the concentrate underwent puri-
fication by silica gel flash column chromatography (CHCl3–MeOH, 9:1
+ 0.2% NH4OH → 9:1 + 0.3% NH4OH → 9:1 + 0.4% NH4OH) to provide
the title compound as a light yellow syrup; yield: 4.1 mg (25%); Rf =
0.20 (CHCl3–MeOH, 17:3 + 0.4% NH4OH); []D27 –25 (c 0.24, MeOH).
HRMS (ESI): m/z [M + H]+ calcd for C29H34N3O4: 488.2544; found:
488.2534.
(5R,6R,7R,E)-6,7-Dihydroxy-5-hydroxymethylhexahydro-1H-pyra-
zolo[1,2-a]pyridazin-1-one Oxime (14)
1H NMR (D2O, 400.13 MHz):  = 3.84–3.74 (m, 2 H, 5a′-H, 5b′-H),
3.63–3.56 (m, 1 H, 7-H), 3.40–3.35 (m, 1 H, 6-H), 3.32–3.26 (m, 2 H,
8a-H, 3a-H), 3.13–3.07 (m, 1 H, 1a-H), 2.48–2.37 (m, 2 H, 1b-H, 3b-H),
2.27–2.21 (m, 2 H, 5-H, 8b-H), 2.03–1.96 (m, 2 H, 2a-H, 2b-H).
Compound 33 (0.13 g, 0.26 mmol, 1.0 equiv) was dissolved in anhyd
CH2Cl2 at –78 °C under an argon atmosphere. After stirring for 20 min,
BCl3 (8.6 mL, 8.6 mmol, 1 M in hexane, 33.0 equiv) was added and the
resulting mixture was kept stirring at –78 °C for 3 h and then at 0 °C
overnight. After this time, the mixture was warmed up to RT and con-
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

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T. C. S. Evangelista et al.
Paper
Synthesis
centrated under reduced pressure. The crude product was purified by
column chromatography (MeCN–H2O, 185:15) to afford the target
product 14 as a colorless solid; yield: 23 mg (41%); Rf = 0.15 (MeCN–
H2O, 9:1); []D28 +51 (c 0.8, MeOH).
1H NMR (D2O, 400.13 MHz):  = 3.84 (dd, J5a′,5 = 2.3 Hz, J5a′,5b′ = 12.9 Hz,
1 H, 5a′-H), 3.79 (dd, J5b′,5 = 3.4 Hz, J5b′,5a′ = 12.9 Hz, 1 H, 5b′-H), 3.71
(dd, J8a,7 = 5.6 Hz, J8a,8b = 12.0 Hz, 1 H, 8a-H), 3.59 (ddd, J7,8a = 5.6 Hz, J7,9
= 9.0 Hz, J7,8b = 10.8 Hz, 1 H, 7-H), 3.46–3.40 (m, 2 H, 3a-H, 6-H), 2.92
(ddd, J = 2.8, 4.5, 14.1 Hz, 1 H, 2a-H), 2.74–2.61 (m, 2 H, 2b-H, 3b-H),
2.57 (dd, J8b,7 = 10.8 Hz, J8b,8a = 12.0 Hz, 1 H, 8b-H), 2.30 (ddd, J5,5a′ = 2.3
Hz, J5,5b′ = 3.4 Hz, J5,6 = 9.9 Hz, 1 H, 5-H).
RT for 1 h. After this time, the volatiles were removed under reduced
pressure and the concentrate was dissolved in toluene (3 × 15 mL) and
concentrated under reduced pressure. Then, the residue was dis-
solved in MeNO2 (27 mL) and DIPEA (2.6 mL, 14.9 mmol, 6.2 equiv)
was added. The resulting mixture was heated under an argon atmo-
sphere at 80–85 °C for 10 h. After this time, the mixture was cooled
down to RT before the volatiles were removed under reduced pres-
sure. Purification of the residue by silica gel flash column chromatog-
raphy (PE–EtOAc, 4:2 → 14:11 → 1:1) provided the title compound as
a light yellow solid; yield: 795 mg (68%); Rf = 0.29 (PE–EtOAc, 2:3).
1H NMR (CDCl3, 400.13 MHz):  = 7.37–7.17 (m, 15 H, ArH), 4.98–4.95
(m, 2 H, 9a-H, CHPh), 4.76 (d, J = 11.4 Hz, 1 H, CHPh), 4.65 (d, J = 11.4
Hz, 1 H, CHPh), 4.53–4.43 (m, 3 H, 3 × CHPh), 3.64–3.58 (m, 4 H, 6a′-H,
6b′-H, 7-H, 8-H), 3.52–3.49 (m, 1 H, 4a-H), 3.23–3.16 (m, 1 H, 4b-H),
2.95–2.93 (m, 1 H, 6-H), 2.81–2.75 (m, 1 H, 9b-H), 2.50–2.47 (m, 2 H,
2a-H, 2b-H), 2.17–2.07 (m, 1 H, 3a-H), 1.72–1.69 (m, 1 H, 3b-H).
13C NMR (D2O, 100.61 MHz):  = 162.9 (1-C), 70.6 (6-C), 69.5 (7-C),
68.7 (5-C), 57.6 (5′-C), 48.9 (8-C), 48.3 (3-C), 25.9 (2-C).
HRMS (ESI): m/z [M + H]+ calcd for C8H16N3O4: 218.1135; found:
218.1135.
13C NMR (CDCl3, 100.61 MHz):  = 167.3 (1-C), 138.4 (Ar), 138.1 (Ar),
137.6 (Ar), 128.6–127.8 (Ar), 78.9, 78.8 (7-C, 8-C), 75.2 (CH2), 73.5
(CH2), 72.2 (CH2), 66.7 (6′-C), 64.9 (6-C), 46.5 (4-C), 44.7 (9-C), 30.0
(2-C), 17.2 (3-C).
HRMS (ESI): m/z [M + Na]+ calcd for C30H34N2O4Na: 509.2411; found:
509.2403.
1-Deoxy-2,3,5-tri-O-benzyl-1-[2-(tert-butoxycarbony)-6-oxohexa-
hydropyridazine-1-yl]-l-xylitol (34)
To a solution of hydrazide 21 (1.65 g, 3.07 mmol, 1.0 equiv) and NEt3
(0.51 mL, 3.68 mmol, 1,2 equiv) in CH2Cl2 (33 mL) was added drop-
wise 4-bromobutyryl chloride (0.37 mL, 3.23 mmol, 1.05 equiv) at 0
°C under an argon atmosphere. The resulting reaction mixture was
kept stirring at 0 °C for 20 min. After this time, sat. aq NaHCO3 (20 mL)
was added and the phases were separated. The aqueous layer was ex-
tracted with CH2Cl2 (20 mL) and the combined organic extracts were
dried (MgSO4), filtered, and concentrated under reduced pressure.
The residue was dissolved in toluene (3 × 20 mL) and concentrated
under reduced pressure. Then, the residue was dissolved in DMF (20
mL) under an argon atmosphere and NaH (60% dispersion in mineral
oil, 368.4 mg, 9.21 mmol, 3.0 equiv) was added. The resulting mixture
was kept stirring at 0 °C for 20 min and then at RT for 2.7 h before the
dropwise addition of H2O (20 mL) at 0 °C. Then, the phases were sepa-
rated and the aqueous layer was extracted with EtOAc (2 × 20 mL).
The combined organic extracts were concentrated under reduced
pressure and the concentrate underwent purification by silica gel
flash column chromatography (PE–EtOAc, 7:3 → 13:7) to obtain the
target compound 34 as a colorless syrup; yield: 1.27 g (68%); Rf = 0.33
(PE–EtOAc, 1:1); []D28 +11(c 0.75, EtOAc).
(6R,7R,8R)-7,8-Dihydroxy-6-(hydroxymethyl)hexahydropyridazi-
no[1,2-a]pyridazin-1(2H)-one (12)
Prepared in a similar way as compound 11. The hydrazide 35 (41 mg,
0.084 mmol) was subjected to Pd-catalyzed hydrogenation to provide
the title compound 12 as a white solid; yield: 13 mg (70%); Rf = 0.20
(CHCl3–MeOH, 9:1 + 0.4% NH4OH); []D26 +134 (c 0.25, MeOH).
1H NMR (CD3OD, 400.13 MHz):  = 4.65–4.60 (m, 1 H, 9a-H), 3.97 (dd,
J6a′,6 = 2.0 Hz, J6a′,6b′ = 12.3 Hz, 1 H, 6a′-H), 3.73(dd, J6b′,6 = 4.6 Hz, J6b′,6a′
=
12.3 Hz, 1 H, 6a′-H), 3.67–3.62 (m, 1 H, 4a-H), 3.41–3.34 (m, 2 H, 7-H,
8-H), 3.14 (ddd, J = 2.6, 13.0, 14.6 Hz, 1 H, 4b-H), 2.72 (ddd, J6,6a′ = 2.0
Hz, J6,6b′ = 4.6 Hz, J6,7 = 9.0 Hz, 1 H, 6-H), 2.65–2.59 (m, 1 H, 9b-H),
2.49–2.45 (m, 2 H, 2a-H, 2b-H), 2.27–2.14 (m, 1 H, 3a-H), 1.72–1.65
(m, 1 H, 3b-H).
13C NMR (CD3OD, 100.61 MHz):  = 170.0 (1-C), 73.8, 71.9 (7-C, 8-C),
68.4 (6-C), 60.0 (6′-C), 48.4 (9-C), 47.2 (4-C), 30.6 (2-C), 17.8 (3-C).
Very complicated NMR spectra due to rotamers.
HRMS (ESI): m/z [M + H]+ calcd for C9H17N2O4: 217.1183; found:
217.1182.
1H NMR (CDCl3, 400.13 MHz):  = 7.37–7.28 (m, 15 H), 4.79–4.44 (m,
7 H), 4.07–3.95 (m, 3 H), 3.71 (dd, J = 3.0, 5.4 Hz, 1 H), 3.48–3.47 (m, 3
H), 3.10–3.29 (m, 1 H), 2.34–2.14 (m, 5 H), 1.48 (s, 9 H).
13C NMR (CD3OD, 100.61 MHz):  = 172.6, 155.6 138.1, 138.0, 128.4–
127.7, 82.3, 76.2, 74.5, 73.3, 72.5, 71.9, 71.3, 69.3, 68.9, 47.9, 45.9,
44.0, 30.0, 28.2, 21.4.
(1R,2R,3R)-2,3-Dihydroxy-1-(hydroxymethyl)octahydropyridazi-
no[1,2-a]pyridazine Trifluoroacetate (18-TFA)
To a solution of 35 (151 mg, 0.310 mmol, 1.0 equiv) in anhyd THF (3.6
mL) was added dropwise a suspension of LiAlH4 (70.7 mg, 1.86 mmol,
6.0 equiv) in THF (3.6 mL) at RT under an argon atmosphere. The flask
containing the LiAlH4 suspension was rinsed with THF (1.8 mL),
which was transferred to the reaction mixture. After addition, the re-
action was immediately quenched at 0 °C by adding dropwise sat. aq
NH4Cl (10 mL). The aqueous mixture was extracted with EtOAc (2 ×
10 mL). The combined organic fractions were dried (MgSO4), filtered,
and concentrated in vacuo. The residue was dissolved in EtOH (17.1
mL) and TFA (1.9 mL), and then 10% Pd/C (151 mg) was added and H2
(1 atm) was introduced. The mixture was kept stirring overnight be-
fore removal of the volatiles under reduced pressure. The residue was
purified twice by silica gel flash column chromatography using the
eluent systems: 1) CH2Cl2–MeOH–NH4OH, 900:100:1 → 900:100:2;
2) CH2Cl2–MeOH, 9:1. The relevant fractions were concentrated and
dissolved in MeOH (5 mL) and TFA (0.4 mL) was added dropwise. Re-
HRMS (ESI): m/z [M + H]+ calcd for C35H45N2O7: 605.3221; found:
605.3211.
(6R,7R,8R)-7,8-Dibenzyloxy-9-benzyloxymethylhexahydropyri-
dazino[1,2-a]pyridazin-1(2H)-one (35)
To a solution of compound 34 (1.45 g, 2.98 mmol, 1.0 equiv) and NEt3
(0.62 mL, 4.47 mmol, 1.9 equiv) in CH2Cl2 (27 mL) was added drop-
wise MsCl (0.30 mL, 3.87 mmol, 1.6 equiv) under an argon atmo-
sphere at 0 °C. The resulting mixture was kept stirring for 15 min be-
fore sat. aq NaHCO3 (15 mL) was added. The phases were separated
and the organic layer was extracted with CHCl3 (15 mL). The com-
bined organic fractions were dried (MgSO4), filtered, and concentrat-
ed under reduced pressure. The residue was dissolved in toluene (20
mL) and concentrated under reduced pressure. The residue was dis-
solved in CH2Cl2 (10.8 mL) and TFA (7.2 mL) and was kept stirring at
© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L

K
T. C. S. Evangelista et al.
Paper
Synthesis
moval of the volatiles under reduced pressure provided the title com-
pound as a light yellow syrup; yield: 40.2 mg (41%); Rf = 0.51 (CH2Cl2–
MeOH, 4:1).
1H NMR (D2O, 400.13 MHz):  = 3.98–3.91 (m, 2 H, 1a′-H, 1b′-H),
3.79–3.72 (m, 1 H, 3-H), 3.67–3.63 (m, 1 H, m, 8a, 8a-H and 5a-H, may
be reverse), 3.63–3.58 (m, 1 H, 2-H), 3.44–3.37 (m, 2 H, 4a-H, 5a-H),
3.09–3.03 (m, 1 H, 5b-H, 5b-H and 8b-H, may be reverse), 2.94–2.88
(m, 3 H, 1-H, 4b-H, 8b-H), 1.86–1.81 (m, 2 H, 6a-H, 7a-H), 1.71–1.66
(m, 2 H, 6b-H, 7b-H).
13C NMR (D2O, 100.61 MHz):  = 163.0 (q, JC,F = 36 Hz, C=O), 116.4 (q,
JC,F = 291 Hz, CF3), 68.2 (2-C, 1-C), 67.2 (3-C), 57.2 (4-C), 56.3 (5-C, 5-C
and 8-C, may be reverse), 55.2 (1′-C), 51.9 (8-C), 20.9 (6-C, 7-C).
19F NMR (D2O, 376.46 MHz):  = –75.6.
HRMS (ESI): m/z [M + H]+ calcd for C9H19N2O3: 203.1390; found:
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0039-1690019.
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Glycosidase Inhibition Assays
Measurement of the glycosidase inhibition was carried out following
the methodology reported by Bols and co-workers22a with the follow-
ing commercially-available glycosidases: -glucosidase (Saccharomy-
ces cerivisiae), -glucosidase (almonds), -mannosidase (Canavalia
ensiformis, Jack bean), -galactosidase (green coffee beans), -galac-
tosidase (Aspergillus oryzae), -galactosidase (Escherichia coli). As
substrates, the corresponding p-nitrophenyl glycopyranosides (o-ni-
trophenyl derivatives for -galactosidases) were used.
Percentage of inhibition was determined in 1.2 mL PS cuvettes con-
taining a 0.1 M phosphate buffered medium (pH 6.8 for glucosidases
and galactosidases, and 5.6 for -mannosidase); for that purpose, a
substrate concentration equal to the expected KM value of the en-
zyme, and an inhibitor concentration of 100 M were used. Mother
inhibitor solutions were prepared in DMSO (5% fixed ratio of the assay
medium, which was proved to have no effects on enzyme functional-
ity).
Temperature of the assay was settled at 25 °C for glucosidases and ga-
lactosidases, and at 35 °C for -mannosidase. The formation of the
corresponding o- or p-nitrophenolates was monitored spectrophoto-
metrically at 400 nm (420 nm for -galactosidases) for a period of 120
s. In the case of -mannosidase, aliquots (200 L) were taken for a pe-
riod of 190 s, and added to a 1 M Na2CO3 solution; the absorbance of
the corresponding mixture was registered at 400 nm.
(14) (a) Heightman, T. D.; Vasella, A. T. Angew. Chem. Int. Ed. 1999,
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Spanish MICINN (CTQ2016-78703-P), the Junta de Andalucía
(FQM134), the European Regional Development Fund (FEDER)
(501100008530), and the University of Stavanger.
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Acknowledgment
T.C.S.E. is grateful for the provision of a three-month scholarship from
the Diku funded NorBra project enabling a research stay at the Uni-
versity of Stavanger and thanks Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior – Brasil (CAPES) for a Ph.D. fellowship.
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Paper
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© 2019. Thieme. All rights reserved. — Synthesis 2019, 30, A–L