Synthesis and inhibitory activities of novel C-3 substituted azafagomines: A new type of selective inhibitors of α-l-fucosidases

Article abstract of DOI:10.1016/j.bmc.2010.05.026

The synthesis of a novel aminomethyl C-3 substituted l-fuco-azafagomine and of its C-6 epimer from d-lyxose is reported. The key step of the synthesis is the introduction of the biimino (-NH-NH-) moiety by reductive hydrazination of a 1-deoxy-ketohexose with tert-butyl carbazate. The 3-aminomethyl-azafagomine derivatives were used as lead compounds in the generation of libraries of novel types of derivatives by attaching different hydrophobic groups on the aminomethyl substituent through amide linkages. These polyhydroxylated hexahydropyridazines can be viewed as a new type of diaza-C-glycoside analogues having a biimino (-NH-NH-) moiety. The conformational analysis and the glycosidase inhibitory properties of all the new C-3 substituted azafagomines synthesized are also reported. Those having l-fuco configuration have shown a selective inhibition of α-l-fucosidases.

Full text of DOI:10.1016/j.bmc.2010.05.026

Bioorganic & Medicinal Chemistry 18 (2010) 4648–4660
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and inhibitory activities of novel C-3 substituted azafagomines: A
new type of selective inhibitors of
a-L-fucosidases
*
Elena Moreno-Clavijo, Ana T. Carmona , Antonio J. Moreno-Vargas, Miguel A. Rodríguez-Carvajal,
*
Inmaculada Robina
Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Profesor García González 1, E-41012 Seville, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel aminomethyl C-3 substituted L-fuco-azafagomine and of its C-6 epimer from D-
Received 29 March 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 11 May 2010
lyxose is reported. The key step of the synthesis is the introduction of the biimino (–NH–NH–) moiety by
reductive hydrazination of a 1-deoxy-ketohexose with tert-butyl carbazate. The 3-aminomethyl-azafag-
omine derivatives were used as lead compounds in the generation of libraries of novel types of deriva-
tives by attaching different hydrophobic groups on the aminomethyl substituent through amide
linkages. These polyhydroxylated hexahydropyridazines can be viewed as a new type of diaza-C-glyco-
side analogues having a biimino (–NH–NH–) moiety. The conformational analysis and the glycosidase
inhibitory properties of all the new C-3 substituted azafagomines synthesized are also reported. Those
Keywords:
Reductive hydrazination
Hexahydropyridazines
Azafagomines
having
L
-fuco configuration have shown a selective inhibition of
a-L-fucosidases.
Ó 2010 Elsevier Ltd. All rights reserved.
a-L-Fucosidase inhibitors
1. Introduction
agomine’ (Fig. 1) that showed interesting inhibitory properties to-
wards Although several hydroxylated
-fucosidases.15
hexahydropyridazines have been reported to inhibit galactosidases
a-L
a-
L-Fucosidase is an exoglycosidase involved in the trimming of
non-reducing terminal -fucose units during the biosynthetic pro-
L
cessing of fucose-containing glycoconjugates.1 This enzyme, in
common with other glycosidases, has glycosyl transfer activity
and, therefore can be also used in the synthesis of fucosyl glycans.2
Fucosidases are associated with many disorders including inflam-
mation,3 cancer,4 cystic fibrosis,5 and fucosidosis.6 This enzyme is
considered an early biomarker for detecting hepatocellular and
H
N
OH
H
N
OH
OH
OH
NH
Me
HO
Me
1
2
HO
Ki = 1 nM
Ki = 0.81 μM
(α-
L
-fucosidases)10
(α-L
-fucosidases)15
colorectal carcinomas.7
a-L-Fucosidases have been found in human
R2
H
seminal plasma and in the membranes of human sperm cells and
facilitate sperm transport and sperm egg-interactions. Inhibitors
of these enzymes can have anticonceptive properties.8 Inhibitors
NH
N
R1
R3
OH
HO
of
effect of HIV and reduce infection.9
Among the most powerful -fucosidase inhibitors are
hydroxylated piperidines such as
-fuconojirimycin (FNJ) 110
a-L-fucosidases have been also found to inhibit the cytophatic
3a, R1 = Me, R2 = H, R3 = OH
3b, R1 = Me, R2 = H, R3 = NH2
a-
L
L-fuco
4a, R1 = H, R2 = Me, R3 = OH
L
4b, R1 = H, R2 = Me, R3 = NH2
(Fig. 1), although the intrinsic instability caused by the lability of
the N,O-acetal function prevents its biological use. The 1-hydroxy-
methyl-FNJ,11 and 1-aminomethyl-FNJ12 having a C–C bond at C-1
are stable C-glycosyl analogues13 that have been used to generate
5, R1 = Me, R2 = H, R3 = NHCOAryl, NHCOAlkyl
6, R1 = H, R2 = Me, R3 = NHCOAryl, NHCOAlkyl
Alkyl
Aryl
potent inhibitors of
a-L
-fucosidases.14 On the other hand, Bols
NH2
(CH2)10CH3
NH2
and co-workers have described the preparation of (3S,4R,5S)-3-
methylhexahydropyridazine-4,5-diol (2), the so-called ‘fuco-azaf-
Ph
NH2
* Corresponding authors. Tel.: +34 954559997; fax: +34 954624960.
E-mail addresses: anatere@us.es (A.T. Carmona), robina@us.es (I. Robina).
Figure 1.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.026

E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4649
and glucosidases,16 to the best of our knowledge, 2 is the only re-
ported example of a -fuco polyhydroxylated hexahydropyridazine.
In a preliminary communication,17 we have reported the first
synthesis and biological evaluation of -fuco-3-hydroxymethyl
(and aminomethyl) hexahydropyridazines 3a and 3b. Compound
3a was a selective and competitive inhibitor of -fucosidase from
bovine kidney. The hydroxylated pyridazine moiety can be used as
core structure to mimic the fucosyl cation generated in the enzy-
matic hydrolysis, and the hydroxymethyl or aminomethyl group
at C-3 to be further functionalized giving a library of derivatives
in a diversity-oriented synthesis. Although the interactions be-
tween some enzyme active sites and the corresponding inhibitors
have been characterized by structural and mechanistic methods,18
only limited information is available for the identification of
important factors that contribute to high inhibition potency as well
as to the dynamic motion of the glycosidase–inhibitor interaction
changing from low to high binding affinity.
We describe herein the details of the synthesis of compounds
3a and 3b and their corresponding epimers at C-6, 4a and 4b
(Fig. 1). These compounds can be viewed as a new type of diaza-
C-glycoside analogues having a biimino (–NH–NH–) moiety. We
also present the derivatization of 3b and 4b by linking different
groups to the aminomethyl side chain with the generation of small
libraries of enzymatic inhibitors, compounds 5 and 6, having aryl/
alkyl moieties. All the new compounds were tested for their inhib-
itory activity towards 12 commercially available glycosidases.
1. 2,2-DMP, PTSA
DMF
L
2. TBDPSCl, Et3N
DMAP, CH2Cl2
O
OH
TBDPSO
D
-lyxose
L
61%
O
O
7
a-L
Swern oxidation
93%
O
O
TBDPSO
O
O
8
91%
OH
Me
O
MeLi
THF, -78 oC
TBDPSO
O
9
O
NH2NHBoc, NaBH3CN
MeOH, AcOH, 65 oC
82%
Me
HO
NHBoc
N
H
TBDPSO
O
O
10
1. CbzCl, NaOH/EtOH
2. MsCl, CH2Cl2/Py
3. TMSOTf, 2,6-lutidine
CH2Cl2
71%
4. Et3N, MeOH reflux
5. Chromatographic
separation
Some of the compounds that present
L-fuco configuration consti-
tute new selective inhibitors of
range.
a-L-fucosidase in the micromolar
H
N
5
H
N
1'
3
RO
NCbz
RO
NCbz
Me
In this regard, these compounds could contribute to a better
understanding of the inhibition mechanisms of fucosidases in
terms of structural and conformational changes, charge dislocation
and protonation as it has been made with azafagomine analogues
in the case of glucosidases.19
4
O
6
Me
O
O
O
11/12 = 2.1
12, R = TBDPS
14, R = H
11, R = TBDPS
13, R = H
90%
TBAF/THF
(94% for 13)
(91% for 14)
2. Results and discussion
91%
H
TsCl, Py
TsO
2.1. Synthesis of C-3 substituted azafagomines
H
N
N
NCbz
TsO
NCbz
The retrosynthetic analysis starting from D-lyxose is indicated
in Scheme 1. The key steps of the synthesis imply cyclization of
2-methyl glycosylhydrazines, which were obtained by reductive
O
Me
O
Me
O
O
16
15
TBAN3
90%
97%
hydrazination of
-lyxose.
Thus, starting from
a
1-deoxy-ketohexose easily prepared from
THF, 50 oC
D
H
H
N
D
-lyxose, tri-O-protected -lyxonolactone 8
D
N
R
NCbz
R
NCbz
Me
could be easily obtained following a similar procedure to that de-
scribed in the literature20 (Scheme 2). Addition of MeLi21 to 8 affor-
ded the protected 1-deoxy-ketohexose 9 as a 3:1 mixture of
O
Me
O
O
O
18, R = N3
17, R = N3
SH2/Py-H2O
(94% for 19)
(93% for 20)
20, R = NH2
19, R = NH2
R2
H
NH
O
Scheme 2.
N
R1
3 + 4
N
Aryl(Alkyl)
OH
H
HO
5, R1 = Me, R2 = H
6, R1 = H, R2 = Me
anomers. Reductive hydrazination of 9 using tert-butyl carbazate,
NaBH3CN and acetic acid in MeOH at 65 °C afforded the mixture
of hydrazines 10 in 82% yield. Reaction of 10 with benzyl chlorofor-
mate, followed by mesylation and selective Boc deprotection gave
crude monohydrazides that were treated with Et3N in MeOH at re-
flux to afford cyclic derivatives 11 and 12 as a 2.1:1 mixture of
diasteroisomers. This sequence could be efficiently carried out
without intermediate purification, affording the corresponding
hexahydropyridazines in 71% overall yield after chromatographic
separation. Deprotection of these compounds by treatment with
TBAF in THF afforded the corresponding alcohols 13 and 14.
Me NHBoc
NH
O
HO
O
TBDPSO
OH
Me
O
TBDPSO
D
-Lyxose
O
O
Scheme 1. Retrosynthetic analysis for compounds 5 and 6.

4650
E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
Compounds 13 and 14 were fully characterized by their spec-
H
N
H
N
troscopic data but the configuration of C-6 was difficult to assign
through NOE experiments. Fortunately, tosylation of 13 afforded
crystalline tosyl derivative 15, the structure of which was unam-
biguously confirmed by X-ray crystallography (Fig. 2).17 Reaction
of 15 with TBAN3 (tetrabutylammonium azide) followed by reduc-
tion of the azido group with H2S, afforded protected aminomethyl
derivative 19. The same reaction sequence but starting from alco-
hol 14 gave amine 20.
Compounds 19 and 20 were used in the preparation of new
azafagomine derivatives by linking different groups through the
amine function (Scheme 3). Thus, coupling reaction of amines 19
and 20 with commercial aromatic and aliphatic carboxylic acids,
including amino acid derivatives, were efficiently carried out by
standard peptide synthesis using PyBOP/DIPEA as coupling reagent
to give 21c–f and 22c–e in good yield. For the synthesis of com-
pounds 21e,f and 22e, a further Fmoc deprotection step was per-
formed after the coupling.
H2N
NCbz
Me
20
H2N
NCbz
Me
19
O
O
O
O
(i)
(i)
O
H
O
H
N
N
R
N
NCbz
Me
R
N
NCbz
Me
H
H
O
O
O
O
21c-f
22c-e
NHR'
Ph
c
e', R' = Fmoc
e, R' = H
(ii)
R =
Deprotection of the acetonide group (and Boc group) in alcohol
13, amine 19, amides 21 and in their corresponding C-6 epimers
(14, 20, and 22) under acidic conditions afforded N-Cbz protected
hexahydropyridazines 23 and 24 in good yields. Final hydrogenol-
ysis of these compounds under acidic conditions gave unprotected
hexahydropyridazines 3, 4, 5, and 6 in quantitative yields as the
corresponding hydrochloride salts (Scheme 4).
NHR'
(CH2)10CH3
d
NHBoc
f', R' = Fmoc
f, R' = H
(ii)
Scheme 3. Reagents and conditions: (i) RCOOH, PyBOP/DIPEA, DMF (90–100%); (ii)
20% piperidine in DMF (71–92%).
The acidic conditions in the final deprotection step are critical
to avoid undesired oxidations. In the absence of acidic conditions,
hydrogenation of compound 11 afforded hydrazone 25 in good
yield as major compound (Scheme 5). The formation of this prod-
uct can be explained by oxidation of the hydrazine to the corre-
sponding azo-compound, followed by isomerization of the double
bond to the more stable hydrazone. Similar results have been pre-
viously reported in the hydrogenolysis of N-protected hexahydro-
pyridazine derivatives.19a,22
14 in DMSO-d6 shows a large coupling constant (J = 9.1 Hz) be-
tween H-3 and H-4 whereas no coupling constant between H-5
and H-6 is observed. These results, together with a coupling con-
stant of 5.0 Hz between H-4 and H-5, indicate that 14 adopts a
chair conformation (2C5, Fig. 3a) with the C-3 substituent in equa-
torial and the Me-6 in axial positions. The results of the molecular
dynamics are in agreement with the experimental observations.
In the case of the 1H NMR spectrum of major alcohol 13 (C-6 epi-
mer of 14), the values for the coupling constants H-3/H-4, H-4/H-5,
and H-5/H-6 are J3,4 = 4.2 Hz, J4,5 = 5.8 Hz, and J5,6 = 5.1 Hz. In base
of these data, the presence of only one chair conformation is dis-
carded. Additionally, the observation of medium NOEs between
Me-acetonide/H-3, H-10/H-5, and H-10/H-6 indicates that this com-
pound adopts a mixture of conformations in solution of DMSO-d6.
When the minimized structurewas obtained by molecular modeling
using the experimental coupling constants, the result was a boat
2.2. Conformational analysis
A conformational analysis of alcohols 13 and 14 based on NMR
data and molecular modeling using MacroModel23 and the force
field OPLS200124 has been carried out. The 1H NMR spectrum of
(
2,5B, Fig. 3c) which remains unchanged in a further minimization
without any restraints. However, in this structure the observation
of a NOE between Me-acetonide/H-3 is not expected. This fact indi-
cates that the compound can also adopt a chair conformation (2C5,
Fig. 3b). Thus, the experimental coupling constants are in agreement
with the presence of 2C5 and 2,5B conformations in solution in a ratio
40:60, respectively. In the case of tosyl derivative 15, although the X-
ray structure (Fig. 2) indicated a chair conformation, its 1H NMR
spectrum showed coupling constant values of 4.5 Hz for J3,4 and
5.5 Hz for J4,5 and J5,6. These values were previously observed for
compound 13, which indicates that the same conformational equi-
librium takes place in solution in this case.
According to the 1H NMR data (Table 1) of (3S,4S,5R,6S)-N,O-
protected hexahydropyridazine derivatives 13, 15, 17, 19, 21c,d,
and 21e0,f0, a correlation between coupling constant values is ob-
served. Thus, the same conformational equilibrium described be-
fore for 13 can be predicted for these compounds. In the case of
N-protected-O-unprotected derivatives 23a–c and 23e,f, the values
for the coupling constants (Table 2) obtained in a CD3OD solution
indicate also the presence of a conformational equilibrium. How-
ever, in the case of completely unprotected derivatives 3a,b and
5c,d,f (Table 3), important changes were observed for J3,4, J4,5
,
Figure 2. X-ray structure for compound 15.
and J5,6 values, indicating a conformational change in CD3OD

E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4651
R
.
HCl
Cbz
N
6 H
N
Me
13,19
21c-f
(ii)
(ii)
Me
HO
(i)
(i)
NH
NH
-NH2
b
-OH
a
3
HO5
R
R
R
4
1'
OH
3a,b, 5c-f
OH
23a-f
O
H
N
N
H
.
10
HCl
H
N
Cbz
Me
HO
O
d
c
Me
HO
N
NH
14, 20
22c-e
NH
R
NH2
NH2
H
N
H
N
OH
4a,b, 6c-e
Ph
OH
24a-e
NH2
O
O
f
e
Scheme 4. Reagents and conditions: (i) HCl (1 M)/THF (1:1), 74–100%; (ii) H2, Pd–C, MeOH–HCl (5 M), quant.
Table 1
N
Me
O
H2, Pd/C
MeOH
NH
Coupling constants (J, Hz) in DMSO-d6 for protons of the hexahydropyridazine ring in
N,O-protected derivatives
OTBDPS
11
73%
O
25
Compound
J3,4
J4,5
J5,6
13
15
17
19
21c
21d
21e0
21f0
4.2
4.5
4.2
2.0
3.1
3.0
2.5
3.0
5.8
5.5
5.9
6.0
5.8
5.9
5.9
6.0
5.1
5.5
5.2
6.0
5.4
5.5
5.9
5.5
Scheme 5.
solution for these derivatives in relation to the N-protected-O-
unprotected compounds. A large coupling constant between H-3
and H-4 (10.0–10.3 Hz) and a small J5,6 value (1.2–1.3 Hz) indi-
cated that completely unprotected derivatives adopt a major chair
conformation in CD3OD solution.
2.3. Enzymatic assays
Table 2
Compounds 3a,b, 4a,b, 5c–f, 6c–e, 23, and 24 have been analyzed
for their inhibitory activities towards twelve commercially available
glycosidases.25 Compounds 23c and 24d could not be tested because
of their low solubilities under the assay conditions. Table 4 shows
Coupling constants (J, Hz) in CD3OD for protons of the hexahydropyridazine ring in N-
protected-O-unprotected derivatives
Compound
J3,4
J4,5
J5,6
23a
23b
23c
23e
23f
4.2
3.8
2.8
3.2
n.d.
3.1
3.0
2.8
3.0
3.0
5.3
5.6
n.d.
5.5
5.5
the results for the inhibition analysis towards a-L-fucosidase from
bovine kidney, b-galactosidase from Escherichia coli, amyloglucosi-
dase from Aspergillus niger, maltase from yeast and b-glucosidase
from almonds. These compounds did not inhibit any of the other
enzymes assayed:
from Aspergillus orizae,
a
-galactosidase from coffe beans, b-galactosidase
-glucosidase from rice, -mannosidase
n.d.: not determined.
a
a
from Jack beans, b-mannosidase from snail, b-xylosidase from
A. niger and b-N-acetylglucosaminidase from Jack beans.
In general, (3S,4S,5R,6S)-hexahydropyridazine derivatives
Table 3
showed specific inhibitory activity towards
a-L-fucosidase, pre-
Coupling constants (J, Hz) in CD3OD for protons of the hexahydropyridazine ring in
completely unprotected derivatives
senting only some of them a very weak activity towards other en-
zymes. Compounds 3a, 5c–f, that share the same absolute
Compound
J3,4
J4,5
J5,6
configuration than C-(2,3,4,5) of
higher inhibitory activity towards
sponding C-6 epimers, probably due to a lower similarity with the
fucopyranosyl cation generated in the transition state of the enzy-
matic hydrolysis and so indicating the importance of the S-config-
uration at Me-6 to bind the enzyme. Hydroxymethyl derivative 3a
a
a
-
L
-fucopyranosides, showed a
-L-fucosidase than their corre-
3a
3b
5c
5d
5f
10.3
10.2
10.2
10.0
10.1
2.8
2.6
2.6
2.6
2.4
1.2
n.d.
—
1.3
—
n.d.: not determined.
was a selective and competitive inhibitor of
a-L-fucosidases from
Figure 3. (a) Minimized chair conformation for compound 14. (b) Minimized chair conformation for 13. (c) Minimized twist boat conformation for 13.

4652
E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
Table 4
Inhibitory activities of C-3 substituted azafagominesa,b
Compound
a-L-Fucosidase (bovine kidney) b-Galactosidase (Escherichia coli) Amyloglucosidase (Aspergillus niger) Maltase (yeast) b-Glucosidase (almonds)
3a
97%
n.i.
n.i.
n.i.
n.i.
(IC50 = 30 lM)
Ki = 4.2
lM
3b
4a
4b
5c
44%
17%
n.i.
n.i.
n.i.
n.i.c
n.i.
n.i
n.i.
n.i
n.i.
23%
n.i.
53%
n.i.
n.i.
85%c
21%c
n.i.c
n.i.c
(IC50 = 8.6 lM)
Ki = 1.0
lM
5d
5e
70%
82%
n.i.
n.i.
n.i.
n.i.
51%
n.i.
n.i.
n.i.
(IC50 = 283
77%
lM)
5f
n.i.
n.i.
n.i.
n.i.
(IC50 = 535
53%
40%
n.i.
35%
lM)
6c
6d
6e
23a
23b
n.i.
n.i.
n.i.
n.i.
32%
n.i.
24%
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
33%
n.i.
n.i
n.i.
n.i.
92%
(IC50 = 117
lM)
23d
23e
23f
24a
24b
24c
24e
5%c
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.c
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.c
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.c
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.c
n.i.
65%
77%
27%
54%
n.i.c
16%
Percentage of inhibition at 1 mM, IC50 and Ki in lM, when measured. Optimal pH, 37 °C.
n.i.: no inhibition at 1 mM.
a
For measurement conditions see Ref. 25.
Competitive mode of inhibition for given Ki.
The percentage of inhibition was determined at 0.1 mM.
b
c
bovine kidney (97% at 1 mM concentration, Ki = 4.2
substitution of the hydroxyl group on 3a for an amino group,
compound 3b, was detrimental for the inhibition towards
l
M). The
of bearing an aromatic group that could provide 5c the appropriate
characteristics for permeability through the membranes, due to the
lipophilic nature of this group.
a-L-fucosidase (only 44% at 1 mM concentration), suggesting that
a compound with a hydroxymethyl group can establish more effec-
tive hydrogen bond interactions in the active site of the enzyme
than an aminomethyl group. However the attachment of hydro-
phobic groups to compound 3b via amide linkages, compounds
5c–f, clearly improves their inhibitory activity towards a-L-fucosi-
dase through unspecific contributions to the binding to the en-
3. Conclusions
In summary, the synthesis of new C-3 substituted
rated hydroxylated hexahydropyridazines and of their C-6 epimers
from -lyxose is reported. They can be considered the first examples
L-fuco configu-
D
of diaza-C-glycosides having a biimino (–NH–NH–) moiety and, in
the case of fuco configurated compounds, constitute interesting lead
compounds for the search of new
zyme. Of special interest is the case of biphenyl derivative 5c
that showed competitive inhibition towards
a-L-fucosidase with
a-L-fucosidase inhibitors. Pro-
a Ki = 1.0 M (85% at 0.1 mM concentration), being four times bet-
l
tected C-3 aminomethyl -fuco-azafagomine 19 and its C-6 epimer
L
ter inhibitor than alcohol 3a. The efficiency in the inhibitory activ-
ity of biphenyl derivatives was precedent26 having found that
molecular motifs containing a biphenyl moiety are high affinity-li-
gands for proteins.27 Thus, the incorporation of aromatic moieties
have been used to generate libraries of derivatives by attachment
of hydrophobic groups through an amide linkage. All the new com-
pounds have been evaluated as glycosidase inhibitors. The best re-
sult has been observed for
incorporates a biphenyl group on C-3, showing selective and com-
petitive inhibition towards -fucosidase from bovine kidney
(Ki = 1.0 M). This result shows that the incorporation of aromatic
moieties on the C-3 substituent of these hexahydropyridazine deriv-
atives increases the inhibitory activity towards -fucosidases
L-fuco-azafagomine 5c, which
to the L-fuco-hexahydropyridazine increases the inhibitory activity
remarkably as it has been previously observed in piperidine- and
a-L
pyrrolidine-based iminosugars.28 The N-Cbz protected pyridazines
l
23b,e,f showed moderate-to-high inhibition of a-L-fucosidase, 92%,
65%, and 77% at 1 mM concentration, respectively.
a
-L
The inhibition potencies of the prepared compounds are rather
remarkably, what is in agreement with previous observations in
more conventional piperidine- and pyrrolidine-based fucosidase
inhibitors.
low in comparison with other described inhibitors of
a-L-fucosi-
dase such as polyhydroxylated piperidines12,14 which display inhi-
bition constants in the low nanomolar range. This decrease in the
inhibitory activity can be attributed to the supplementary nitrogen
atom that diminishes the basicity of the N atoms and hence the
ability of resembling the fucopyranosyl cation and/or to the pres-
ence of an extra carbon chain at C-3 instead of a hydroxyl group.
However, it is worth noting that although the polyhydroxylated
4. Experimental part
4.1. General methods
Optical rotations were measured in a 1.0 cm or 1.0 dm tube
with a Perkin–Elmer 241MC spectropolarimeter. 1H and 13C NMR
spectra were obtained for solutions in CDCl3, DMSO-d6, and
derivative 3a (Ki = 4.2
present values of inhibition in the same order of magnitude as
biphenyl derivative 5c (Ki = 1.0 M), the latter has the advantage
lM) and L-fuco-azafagomine 2 (Ki = 0.81 lM)
l

E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4653
CD3OD; J values are given in Hz and d in ppm. All the assignments
were confirmed by two-dimensional NMR experiments. The FAB
mass spectra were obtained using glycerol or 3-nitrobenzyl alcohol
as the matrix. NMR and Mass spectra were registered in CITIUS
(University of Seville). TLC was performed on silica gel HF254
(Merck), with detection by UV light charring with ninhydrine
or with Pancaldi reagent [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O].
Silica gel 60 (Merck, 230 mesh) was used for preparative chroma-
tography. Glycosidases and the corresponding p-nitrophenyl-O-
glycoside substrates for inhibition assays were purchased in
Sigma–Aldrich. Molecular mechanics and molecular dynamics
studies were carried out using the Macromodel23 program (version
9.0) and the OPLS 200124 force field. The energies were minimized
by using the PR conjugate gradient method. The starting coordi-
nates for dynamics calculations where those obtained after energy
minimizations. Simulations were carried out at 350 K.
(C-6), 55.9 (C-2), 28.2 (C(CH3)3 of Boc), 26.9 (C(CH3)3 of TBDPS),
26.6, 24.8 (C(CH3)2), 19.4 (C(CH3)3), 14.3 (Me-2). FABMS m/z 581
[100%, (M+Na)+] 559 [10%, (M+H)+]. FABHRMS m/z found
581.3032, calcd for C30H46N2O6NaSi (M+Na)+: 581.3023. Data for
10b: 1H NMR (300 MHz, CDCl3, d ppm, J Hz) d 7.72–7.64 (m, 4H,
H-arom.), 7.45–7.34 (m, 6H, H-arom.), 6.40 (br s, 1H, NHBoc),
4.31 (d, 1H, J4,3 = 7.2, H-4), 4.09 (dd, 1H, J3,2 = 5.6, H-3), 3.80 (m,
1H, H-5), 3.76–3.72 (m, 2H, H-6a, H-6b), 3.34 (m, 1H, H-2), 1.51,
1.37 (2s, 3H each, C(CH3)2), 1.46 (s, 9H, C(CH3)3 of Boc), 1.20 (d,
3H, JMe-2,2 = 6.4, Me-2), 1.06 (s, 9H, C(CH3)3 of TBDPS). 13C NMR
(75.4 MHz, CDCl3, d ppm) d 156.6 (C@O of Boc), 135.7, 133.6,
129.8, 127.8 (C-arom.), 108.4 (C(CH3)2), 81.2 (C(CH3)3), 79.9 (C-3),
75.6 (C-4), 69.2 (C-5), 65.0 (C-6), 54.9 (C-2), 28.4 (C(CH3)3 of
Boc), 27.0 (C(CH3)3 of TBDPS), 26.6, 24.7 (C(CH3)2), 19.4 (C(CH3)3),
16.3 (Me-2). FABMS m/z 581 [100%, (M+Na)+], 559 [10%, (M+H)+].
FABHRMS m/z found 581.3032, calcd for C30H46N2O6NaSi
(M+Na)+: 581.3023.
4.1.1. 6-O-tert-Butyldiphenylsilyl-1-deoxy-3,4-O-isopropylidene-
D
-tagatofuranose (9)
To a stirred cooled (ꢀ78 °C) solution of 8 (1.24 g, 2.91 mmol) in
4.1.3. (3S,4S,5R,6S) and (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-
O-tert-butyldiphenylsilyl-4,5-O-isopropylidene-3-hydroxy-
methyl-6-methylhexahydropyridazine-4,5-diol (11 and 12)
To a solution of 10 (1.40 g, 2.51 mmol) in EtOH/H2O (1.5:1,
40 mL), was added NaHCO3 (0.37 g, 4.40 mmol) and CbzCl
dry THF (8 mL), MeLi (3.2 mL, 1 M in THF) was added under argon.
After 2.5 h, H2O (9 mL) was added and the mixture stirred at rt for
20 min. Then, the mixture was extracted with AcOEt and the or-
ganic phases were washed with brine, dried (Na2SO4), filtered,
and concentrated. Purification by chromatography column on silica
gel (AcOEt/petroleum ether 1:3) afforded 9 (1.17 g, 2.65 mmol,
91%) as a 1:3 mixture of distereoisomers. 1H NMR (300 MHz, CDCl3,
d ppm, J Hz) d 7.74–7.67 (m, 8H, 4H-arom.-minor., 4H-arom.-ma-
jor.), 7.44–7.34 (m, 12H, 6H-arom.-minor., 6H-arom.-major.), 4.79
(dd, 1H, J3,2 = 5.8, J3,4 = 3.9, H-4-major.), 4.76 (dd, 1H, J = 6.0,
J = 3.6, H-4-minor.), 4.43 (d, 1H, H-3-major.), 4.28–4.22 (m, 2H,
H-5-major., H-3-minor.), 4.02–3.93 (m, 2H, H-6a-major., H-6a-
minor.), 3.89–3.83 (m, 2H, H-6b-major., H-6b-minor.), 3.74 (ddd,
1H, J = 7.4, J = 5.4, J = 3.5, H-5-minor.), 1.48, 1.45 (2s, 3H each,
Me-mayor., Me-minor.), 1.37 (s, 3H, Me-minor.), 1.35 (br s, 6H,
Me-major., Me-minor.), 1.29 (s, 3H, Me-minor.), 1.06 (s, 18H,
C(CH3)3 de TBDPS-major., C(CH3)3 de TBDPS-minor.). 13C NMR
(75.4 MHz, CDCl3, d ppm) d 135.9, 135.8, 133.9, 129.7, 127.8,
127.7, 127.6 (C-arom.), 112.7, 112.4 (C(CH3)2 major., C(CH3)2 min-
or.), 105.3 (C-2 major., C-2 minor.), 85.5 (C-3 major.), 82.3 (C-3
minor.), 80.8 (C-4 major.), 80.0 (C-4 minor.), 79.7 (C-5 major.),
76.7 (C-5 minor.), 61.9 (C-6 major.), 61.5 (C-6 minor.), 27.0
(C(CH3)3 of TBDPS-major., C(CH3)3 of TBDPS-minor.), 26.3, 26.1,
25.2, 25.0, 22.8, 22.2 (3 Me major, 3 Me minor.), 19.2 (C(CH3)3 ma-
jor., C(CH3)3 minor). FABMS m/z 465 [40%, (M+Na)+]. FABHRMS m/z
found 465.2097, calcd for C25H34O5NaSi (M+Na)+: 465.2073.
(421 lL, 2.94 mmol) and the mixture stirred at rt for 7 h. Then, a
satd aq soln of NaHCO3 was added, the mixture extracted with
AcOEt (4 ꢁ 40 mL) and the organic phases dried, filtered and con-
centrated. The residue thus obtained was dissolved in dry CH2Cl2
and cooled to 0 °C. MsCl (0.58 mL, 7.5 mmol) in dry pyridine
(4.6 mL) was slowly added and the mixture was stirred at rt over-
night. Then, water was added at 0 °C and after stirring for 15 min.
at rt, the solvent was evaporated. The residue was dissolved in
CH2Cl2 and washed with H2O and brine. The organic phases were
then dried, filtered, and concentrated. The obtained residue was
then dissolved in dry CH2Cl2 and 2,6-lutidine (2.5 mL) and TMSOTf
(2.3 mL) were added under argon at 0 °C. After stirring at rt for 8 h,
a 10% aq soln of Na2CO3 was added. After extraction with CH2Cl2,
the organic phases were dried, filtered, and concentrated. The
crude product was subsequently dissolved in MeOH, Et3N was
added and the mixture heated at reflux for 72 h. After evaporation,
the residue was purified by chromatography column on silica gel
(AcOEt/petroleum ether 1:8) affording 12 (0.327 g, 0.57 mmol,
23%) and 11 (0.702 g, 1.22 mmol, 49%), as oils. Data for 12: ½a D26
ꢂ
ꢀ64.5 (c 0.82, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz)
d 7.63–7.61 (m, 4H, H-arom.), 7.49–7.30 (m, 11H, H-arom.), 5.18
(br s, 2H, CH2 of Cbz), 5.09 (br s, 1H, NH), 4.56 (q, 1H, J6,Me-
6 = 7.2, H-6), 3.99 (d, 1H, J5,4 = 5.0, H-5), 3.80 (dd, 1H, J4,3 = 9.4, H-
2
4), 3.77 (dd, 1H, J1 a,1 b = 10.7, J1 a,3 = 3.0, H-10a), 3.59 (dd, 1H,
0
0
0
0
4.1.2. 6-O-tert-Butyldiphenylsilyl-2-(tert-butoxycarbonyl)-
J1 b,3 = 9.0, H-10b), 2.89 (m, 1H, H-3), 1.28 (d, 3H, Me-6), 1.20 (s,
hydrazino-1,2-dideoxy-3,4-O-isopropylidene-
To a solution of 9 (1.36 g, 3.08 mmol) in dry MeOH (13 mL), tert-
butylcarbazate (1.22 g, 9.24 mmol), NaBH3CN (0.813 g,
12.32 mmol) and glacial AcOH (315 L) were added. After heating
D
-talitol (10)
6H, C(CH3)2), 0.98 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz, DMSO-
d6, d ppm) d 155.2 (C@O of Cbz), 136.9, 135.0, 132.6, 129.9,
128.2, 127.9, 127.8, 127.7, 127.1 (C-arom.), 108.0 (C(CH3)2), 75.0
(C-5), 70.0 (C-4), 66.3 (CH2 of Cbz), 63.3 (C-10), 59.9 (C-3), 49.1
(C-6), 27.8, 26.3 (C(CH3)2), 26.5 (C(CH3)3), 18.7 (C(CH3)3), 16.1
(Me-6). FABMS m/z 597 [50%, (M+Na)+], 575 [10%, (M+H)+]. FAB-
HRMS m/z found 597.2802, calcd for C33H42N2O5NaSi (M+Na)+:
l
for 36 h at 65 °C, a satd aq soln of NaHCO3 was added and the mix-
ture extracted with CH2Cl2. The organic phases were washed with
brine, dried, filtered and concentrated. Purification by chromatog-
raphy column on silica gel (AcOEt/petroleum ether 1:4) afforded
10 (1.41 g, 2.53 mmol, 82%) as an inseparable mixture of stereoiso-
mers 10a and 10b. Data for 10a: 1H NMR (300 MHz, CDCl3, d ppm, J
Hz) d 7.70–7.64 (m, 4H, H-arom.), 7.45–7.34 (m, 6H, H-arom.), 6.45
(br s, 1H, NHBoc), 4.38 (dd, 1H, J4,3 = 6.7, J4,5 = 1.1, H-4), 4.11 (t, 1H,
J3,2 = 6.4, H-3), 4.04 (br t, 1H, H-5), 3.78 (d, 2H, J6,5 = 6.6, H-6a, H-
6b), 3.53 (m, 1H, H-2), 1.49, 1.35 (2s, 3H each, C(CH3)2), 1.46 (s,
9H, C(CH3)3 of Boc), 1.24 (d, 3H, JMe-2,2 = 6.8, Me-2), 1.06 (s, 9H,
C(CH3)3 of TBDPS). 13C NMR (75.4 MHz, CDCl3, d ppm) d 156.4
(C@O of Boc), 135.6, 133.5, 129.7, 127.7 (C arom.), 107.8
(C(CH3)2), 81.6 (C(CH3)3), 78.4 (C-3), 76.0 (C-4), 68.9 (C-5), 64.9
597.2761. Data for 11: ½a D25
ꢂ
ꢀ18.2 (c 1.19, CH2Cl2). 1H NMR
(500 MHz, DMSO-d6, d ppm, J Hz) d 7.61–7.58 (m, 4H, H-arom.),
7.48–7.38 (m, 6H, H-arom.), 7.28–7.26 (m, 5H, H-arom.), 5.18 (br
2
d, 1H, JNH,3 = 3.2, NH), 5.05 (d, 1H, JH,H = 12.7, CH2 of Cbz), 5.02
(d, 1H, CH2 of Cbz), 4.08 (dd, 1H, J5,4 = 5.5, J5,6 = 4.8, H-5), 3.97
(qd, 1H, J6,Me-6 = 7.2, H-6), 3.96 (t, 1H, J4,3 = 5.5, H-4), 3.65 (d, 2H,
J1 ,3 = 6.7, H-10), 3.05 (m, 1H, H-3), 1.34 (d, 3H, Me-6), 1.33, 1.24
0
(2s, 3H each, C(CH3)2), 0.99 (s, 9H, C(CH3)3). 13C NMR
(125.7 MHz, DMSO-d6, d ppm) 156.0 (C@O of Cbz), 136.7, 135.0,
132.7, 129.9, 128.2, 127.9, 127.8, 127.6, 127.3 (C-arom.), 108.0
(C(CH3)2), 72.6 (C-5), 70.8 (C-4), 66.2 (CH2 of Cbz), 63.7 (C-10),

4654
E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
58.7 (C-3), 50.9 (C-6), 26.8, 25.7 (C(CH3)2), 26.5 (C(CH3)3), 18.7
(C(CH3)3), 15.9 (Me-6). FABMS m/z 597 [40%, (M+Na)+], 575 [8%,
(M+H)+] FABHRMS m/z found 597.2780, calcd for C33H42N2O5NaSi
(M+Na)+: 597.2761.
353 K, d ppm) d 156.1 (C@O of Cbz), 145.1, 136.7, 132.0, 130.2,
128.3, 127.8, 127.6, 127.5 (C-arom.), 108.1 (C(CH3)2), 72.2 (C-5),
70.0, 69.9 (C-4, C-10), 66.3 (CH2 of Cbz), 56.1 (C-3), 50.1 (C-6),
26.6, 25.6 (C(CH3)2), 21.1 (Me of Ts), 15.7 (Me-6). FABMS m/z 513
[100%, (M+Na)+], 491 [10%, (M+H)+]. FABHRMS m/z found
513.1678, calcd for C24H30N2O7SNa (M+Na)+: 513.1671. Anal. Calcd
for C24H30N2O7S: C, 58.76; H, 6.16; N, 5.71; S, 6.54. Found: C, 58.75;
H, 6.11; N, 5.82; S, 6.50.
4.1.4. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-4,5-O-isopropylidene-
3-hydroxymethyl-6-methylhexahydropyridazine-4,5-diol (13)
To a solution of 11 (575.4 mg, 1.0 mmol) in THF (6 mL), TBAF
(1 M in THF, 1.0 mL) was added. After stirring at rt for 1.5 h, the sol-
vent was evaporated and the residue purified by chromatography
column on silica gel (AcOEt/petroleum ether 2:1) to afford 13
4.1.7. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-4,5-O-isopropylidene-
3-O-tosyl-3-hydroxymethyl-6-methylhexahydropyridazine-4,5-
diol (16)
(317 mg, 0.94 mmol, 94%) as an oil. ½a D25
ꢂ
+29.0 (c 1.04, CH2Cl2).
1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.37–7.29 (m, 5H, H-
Following the same procedure as for the preparation of 15, and
starting from 14, compound 16 (751 mg, 1.53 mmol, 91%) was ob-
2
arom.), 5.09 (d, 1H, JH,H = 12.6, CH2 of Cbz), 5.07 (m, 1H, NH),
5.05 (d, 1H, CH2 of Cbz), 4.56 (dd, 1H, JOH,1 b = 6.8, JOH,1 a = 4.6,
OH), 4.19 (dd, 1H, J5,4 = 5.8, J5,6 = 5.1, H-5), 4.00 (qd, 1H, J6,Me-
tained as an oil. ½a D27
ꢂ
ꢀ66.1 (c 0.97, CH2Cl2). 1H NMR (500 MHz,
0
0
3
DMSO-d6, d ppm, J Hz) d 7.79 (d, 2H, JH,H = 8.0, H-Ts), 7.44 (d,
2H, H-Ts), 7.36–7.29 (m, 5H, H-arom.), 5.13 (br s, 2H, CH2 of
Cbz), 5.02 (br s, 1H, NH), 4.50 (q, 1H, J6,Me-6 = 7.2, H-6), 4.06 (dd,
1H, J4,3 = 10.8, J4,5 = 2.6, H-4), 4.02–3.98 (m, 2H, H-5, H-10a), 3.80
(m, 1H, H-10b), 2.80 (m, 1H, H-3), 2.40 (s, 3H, CH3 of Ts), 1.22 (d,
3H, Me-6), 1.21, 1.16 (2s, 3H each, C(CH3)2). 13C NMR
(125.7 MHz, DMSO-d6, 353 K, d ppm) d 155.5 (C@O of Cbz),
145.0, 136.9, 132.1, 130.1, 128.3, 127.9, 127.6, 127.0, 125.4 (C-
arom.), 108.2 (C(CH3)2), 74.8 (C-5), 68.9, 68.8 (C-4, C-10), 66.2
(CH2 of Cbz), 57.3 (C-3), 49.1 (C-6), 27.8, 26.2 (C(CH3)2), 21.0 (Me
of Ts), 16.0 (Me-6). FABMS m/z 513 [40%, (M+Na)+], 491 [15%,
(M+H)+]. FABHRMS m/z found 513.1691, calcd for C24H30N2O7SNa
(M+Na)+: 513.1671.
6 = 7.1, H-6), 3.91 (dd, 1H, J4,3 = 4.2, H-4), 3.41 (ddd, 1H,
2
J1 b,1 a = 11.2, J1 a,3 = 6.3, H-10a), 3.33 (ddd, 1H, J1 b,3 = 7.4, H-10b),
2.90 (m, 1H, H-3), 1.38, 1.27 (2s, 3H each, C(CH3)2), 1.32 (d, 3H,
Me-6). 13C NMR (125.7 MHz, DMSO-d6, 353 K, d ppm) d 156.2
(C@O of Cbz), 136.8, 128.3, 127.6, 127.4 (C-arom.), 107.8
(C(CH3)2), 72.3 (C-5), 70.8 (C-4), 66.2 (CH2 of Cbz), 60.5 (C-10),
59.0 (C-3), 50.3 (C-6), 26.8, 25.6 (C(CH3)2), 15.9 (Me-6). CIMS m/z
337 [90%, (M+H)+]. CIHRMS m/z found 337.1768, calcd for
C17H25N2O5 (M+H)+: 337.1763. Anal. Calcd for C17H24N2O5: C,
60.70; H, 7.19; N, 8.33. Found: C, 60.66; H, 7.06; N, 8.30.
0
0
0
0
4.1.5. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-4,5-O-isopropylidene-
3-hydroxymethyl-6-methylhexahydropyridazine-4,5-diol (14)
To a solution of 12 (825 mg, 1.44 mmol) in THF (9 mL), TBAF
(1 M in THF, 1.46 mL) was added. After stirring at rt for 1.5 h, the
solvent was evaporated and the residue purified by chromatogra-
phy column on silica gel (AcOEt/petroleum ether 2:1) to afford
4.1.8. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-azidomethyl-4,5-
O-isopropylidene-6-methylhexahydropyridazine-4,5-diol (17)
To a solution of 15 (1.18 g, 2.40 mmol) in THF (25 mL), TBAN3
(2.0 g, 7.07 mmol) was added. After heating at 50 °C for 3.5 h, the
solvent was evaporated and the residue purified by chromatogra-
phy column on silica gel (AcOEt/petroleum ether 1:3) to afford
14 (440 mg, 1.31 mmol, 91%) as an oil. ½a D25
ꢀ102.1 (c 1.3, CH2Cl2).
ꢂ
1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.36–7.28 (m, 5H, H-
arom.), 5.17 (d, 1H, JH,H = 13.0, CH2 of Cbz), 5.11 (d, 1H, CH2 of
17 (780 mg, 2.16 mmol, 90%) as an oil. ½a D25
ꢀ13.8 (c 0.94, CH2Cl2).
ꢂ
2
Cbz), 4.93 (br s, 1H, NH), 4.81 (t, 1H, JOH,H = 5.5, OH), 4.53 (q, 1H,
IR (c
cmꢀ1) 3422, 3054, 2303, 2105, 1263, 895, 737, 704. 1H NMR
J6,Me-6 = 7.2, H-6), 4.00 (d, 1H, J5,4 = 4.9, H-5), 3.82 (dd, 1H,
(500 MHz, DMSO-d6, d ppm, J Hz) d 7.39–7.28 (m, 5H, H-arom.),
5.15 (br s, 1H, NH), 5.10 (d, 1H, JH,H = 12.6, CH2 of Cbz), 5.06 (d,
1H, CH2 of Cbz), 4.23 (dd, 1H, J5,4 = 5.9, J5,6 = 5.2, H-5), 4.04 (qd,
1H, J6,Me-6 = 7.1, H-6), 3.89 (dd, 1H, J4,3 = 4.2, H-4), 3.35 (dd, 1H,
2
2
J4,3 = 9.1, H-4), 3.60 (ddd, 1H, J1 a,1 b = 11.3, J1 a,3 = 2.7, H-10a), 3.27
(m, 1H, H-10b), 2.71 (m, 1H, H-3), 1.32, 1.25 (2s, 3H each,
C(CH3)2), 1.24 (d, 3H, Me-6). 13C NMR (125.7 MHz, DMSO-d6,
353 K, d ppm) d 155.2 (C@O of Cbz), 137.0, 128.3, 127.6, 127.0
(C-arom.), 107.9 (C(CH3)2), 75.1 (C-5), 70.1 (C-4), 66.2 (CH2 of
Cbz), 60.5 (C-10), 60.2 (C-3), 49.2 (C-6), 28.0, 26.4 (C(CH3)2), 16.2
(Me-6). CIMS m/z 337 [50%, (M+H)+]. CIHRMS m/z found
337.1754, calcd for C17H25N2O5 (M+H)+: 337.1763.
0
0
0
2
J1 a,1 b = 12.8, J1 a,3 = 5.0, H-10a), 3.32 (dd, 1H, J1 b,3 = 7.7, H-10b),
3.02 (m, 1H, H-3), 1.37, 1.27 (2s, 3H each, C(CH3)2), 1.31 (d, 3H,
Me-6). 13C NMR (125.7 MHz, DMSO-d6, 353 K, d ppm) d 156.2
(C@O of Cbz), 136.8, 128.2, 127.7, 127.5 (C-arom.), 108.0
(C(CH3)2), 72.3 (C-5), 71.2 (C-4), 66.2 (CH2 of Cbz), 56.9 (C-3),
50.8 (C-10), 50.2 (C-6), 26.7, 25.6 (C(CH3)2), 15.7 (Me-6). CIMS m/z
362 [10%, (M+H)+]. CIHRMS m/z found 362.1807, calcd for
C17H24N5O4 (M+H)+: 362.1828.
0
0
0
0
4.1.6. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-4,5-O-isopropylidene-
3-O-tosyl-3-hydroxymethyl-6-methylhexahydropyridazine-4,5-
diol (15)
To a 0 °C solution of 13 (957 mg, 2.84 mmol) in dry pyridine
(8 mL) was slowly added TsCl (1.38 g, 7.21 mmol). After stirring
at rt overnight, the mixture was cooled to 0 °C, water was added
(0.5 mL), and the mixture was allowed to warm to rt. Solvent
was then removed, and the residue was diluted with CH2Cl2,
washed with water and brine, dried, filtered, and concentrated.
Purification by chromatography column (AcOEt/petroleum ether
1:2) afforded 15 (1.25 g, 2.55 mmol, 90%) as a white solid. M.p.
4.1.9. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-azidomethyl-4,5-
O-isopropylidene-6-methylhexahydropyridazine-4,5-diol (18)
Following the same procedure as for the preparation of 17, and
starting from 16, compound 18 (480 mg, 1.33 mmol, 97%) was ob-
tained as an oil. ½a D24
ꢂ
ꢀ99.5 (c 0.85, CH2Cl2). 1H NMR (500 MHz,
DMSO-d6, d ppm, J Hz) d 7.38–7.28 (m, 5H, H-arom.), 5.13 (s, 2H,
CH2 of Cbz), 5.04 (br d, 1H, JNH,3 = 6.8, NH), 4.54 (br q, 1H, J6,Me-
6 = 7.3, H-6), 4.05 (d, 1H, J5,4 = 4.9, J5,6 = 0.9, H-5), 3.85 (dd, 1H,
130–132 °C (AcOEt/petroleum ether). ½a D23
ꢂ
ꢀ6.4 (c 0.45, CH2Cl2).
J4,3 = 9.0, H-4), 3.46 (br d, 1H, H-10a), 3.28 (dd, 1H, J1 b,1 a = 13.1,
0
2
0
0
1H NMR (500 MHz, DMSO-d6,
d
ppm,
J
Hz)
d
7.76 (d, 2H,
J1 b,3 = 8.2, H-10b), 2.81 (m, 1H, H-3), 1.31, 1.26 (2s, 3H each,
3JH,H = 8.0, H-Ts), 7.46 (d, 2H, H-Ts), 7.37–7.30 (m, 5H, H-arom.),
5.17 (br s, 1H, NH), 5.04 (d, 1H, JH,H = 12.7, CH2 of Cbz), 5.01 (d,
C(CH3)2), 1.26 (d, 3H, Me-6). 13C NMR (125.7 MHz, DMSO-d6,
353 K, d ppm) d 155.6 (C@O of Cbz), 137.0, 128.2, 127.6, 127.0
(C-arom.), 108.1 (C(CH3)2), 75.1 (C-5), 70.4 (C-4), 66.2 (CH2 of
Cbz), 58.0 (C-3), 50.0 (C-10), 49.2 (C-6), 28.0, 26.3 (C(CH3)2), 16.0
(Me-6). CIMS m/z 362 [15%, (M+H)+]. CIHRMS m/z found
362.1812, calcd for C17H24N5O4 (M+H)+: 362.1828. Anal. Calcd for
2
1H, CH2 of Cbz), 4.16 (t, 1H, J5,4 = J5,6 = 5.5, H-5), 4.04–3.97 (m,
3H, H-6, H-10a, H-10b), 3.83 (dd, 1H, J4,3 = 4.5, H-4), 3.10 (m, 1H,
H-3), 2.41 (s, 3H, CH3 of Ts), 1.28 (d, 3H, J6,Me-6 = 7.0, Me-6), 1.31,
1.23 (2s, 3H each, C(CH3)2). 13C NMR (125.7 MHz, DMSO-d6,

E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4655
C17H23N5O4: C, 56.50; H, 6.41; N, 19.38. Found: C, 56.55; H, 6.47; N,
19.07.
4.28 (dd, 1H, J5,4 = 5.8, J5,6 = 5.4, H-5), 4.09 (qd, 1H, J6,Me-6 = 7.0,
H-6), 3.95 (dd, 1H, J4,3 = 3.1, H-4), 3.54 (m, 1H, H-10a), 3.16–3.11
(m, 2H, H-3, H-10b), 1.41, 1.28 (2s, 3H each, C(CH3)2), 1.33 (d, 3H,
Me-6). 13C NMR (125.7 MHz, DMSO-d6, d ppm) d 165.9 (CONH),
156.4 (C@O of Cbz), 142.7, 139.1, 136.7, 133.1, 129.0, 128.3,
128.0, 127.7, 127.6, 127.5, 126.8, 126.5 (C-arom.), 107.8
(C(CH3)2), 72.1 (C-5), 71.4 (C-4), 66.4 (CH2 of Cbz), 57.2 (C-3),
49.9 (C-6), 39.3 (C-10), 26.6, 25.5 (C(CH3)2), 15.8 (Me-6). CIMS m/
z 516 [80%, (M+H)+]. CIHRMS m/z found 516.2481, calcd for
C30H34N3O5 (M+H)+: 516.2498.
4.1.10. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-aminomethyl-
4,5-O-isopropylidene-6-methylhexahydropyridazine-4,5-diol
(19)
To a solution of 17 (750 mg, 2.076 mmol) in pyridine/H2O 1:1
(40 mL), H2S was bubbled for 1 h. Alter stirring at rt for 3 h, the sol-
vent was evaporated and the residue purified by chromatography
column on silica gel (CH2Cl2/MeOH, 6:1) to afford 19 (654 mg,
1.95 mmol, 94%) as an oil. ½a D25
ꢂ
+26.2 (c 0.92, CH2Cl2). 1H NMR
(500 MHz, DMSO-d6, d ppm, J Hz) d 7.39–7.29 (m, 5H, H-arom.),
5.10 (d, 1H, JNH,3 = 1.8, NH), 5.07 (s, 2H, CH2 of Cbz), 4.22 (t, 1H,
4.2.2. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-(p-phenylbenzoy-
laminomethyl)-4,5-O-isopropylidene-6-
J5,4 = J5,6 = 6.0, H-5), 4.05 (m, 1H, H-6), 3.84 (dd, 1H, J4,3 = 2.0, H-
methylhexahydropyridazine-4,5-diol (22c)
2
4), 2.89 (m, 1H, H-3), 2.60 (dd, 1H, J1 a,1 b = 13.0, J1 a,3 = 4.7, H-
The coupling reaction of 20 (74.8 mg, 0.223 mmol) with (1,10-
biphenyl)-4-carboxylic acid following the general procedure affor-
ded after chromatographic purification (ether/petroleum ether
10:1) derivative 22c (103.4 mg, 0.20 mmol, 90% yield) as a white
0
0
0
10a), 2.47 (dd, 1H, J1 b,3 = 10.4, H-10b), 1.40, 1.27 (2s, 3H each,
0
C(CH3)2), 1.26 (d, 3H, JMe-6,6 = 7.3, Me-6). 13C NMR (125.7 MHz,
DMSO-d6, 353 K, d ppm) d 156.3 (C@O of Cbz), 136.7, 130.4,
128.4, 127.7 (C-arom.), 107.6 (C(CH3)2), 71.3 (C-4, C-5), 66.5 (CH2
of Cbz), 58.8 (C-3), 48.9 (C-6), 41.2 (C-10), 26.4, 25.5 (C(CH3)2),
15.6 (Me-6). CIMS m/z 336 [80%, (M+H)+], 305 [45%, (M-CH2NH2)+].
CIHRMS m/z found 336.1914, calcd for C17H26N3O4 (M+H)+:
336.1923.
solid. ½a 2D5
ꢂ
ꢀ31.0 (c 0.91, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d
0
0
ppm, J Hz) d 8.60 (br t, 1H, JNH,1 a = JNH,1 b = 5.6, CONH), 7.92 (br d,
2H, J = 8.3, H-arom, biphenyl), 7.75 (br d, 2H, J = 8.2, H-arom,
biphenyl), 7.73 (br d, 2H, J = 7.4, H-arom, biphenyl), 7.50 (m, 2H,
H-arom, biphenyl), 7.41 (br t, 1H, J = 7.4, H-arom, biphenyl),
7.27–7.24 (m, 5H, H-arom.), 5.13–5.05 (m, 3H, NH, CH2 of Cbz),
4.1.11. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-aminomethyl-
4,5-O-isopropylidene-6-methylhexahydropyridazine-4,5-diol
(20)
4.52 (q, 1H, J6,Me-6 = 7.2, H-6), 4.02 (br d, 1H, J5,4 = 4.8, H-5), 3.90
2
0
0
0
(dd, 1H, J4,3 = 9.2, H-4), 3.55 (ddd, 1H, J1 a,1 b = 13.8, J1 a,3 = 3.2, H-
10a), 3.24 (ddd, 1H, J1 b,3 = 8.5, H-10b), 2.89 (m, 1H, H-3), 1.36,
0
Following the same procedure as for the preparation of 19, and
starting from 18, compound 20 (389 mg, 1.16 mmol, 93%) was ob-
1.28 (2s, 3H each, C(CH3)2), 1.29 (d, 3H, Me-6). 13C NMR
(125.7 MHz, DMSO-d6, d ppm) d 166.4 (CONH), 155.3 (C@O of
Cbz), 142.7, 139.2, 136.8, 133.3, 129.0, 128.2, 128.0, 127.8, 127.5,
126.9, 126.8, 126.4 (C-arom.), 107.9 (C(CH3)2), 75.3 (C-5), 71.8 (C-
4), 66.2 (CH2 of Cbz), 59.0 (C-3), 49.4 (C-6), 40.0–39.0 (C-10), 28.0,
26.4 (C(CH3)2), 16.3 (Me-6). CIMS m/z 516 [100%, (M+H)+]. CIHRMS
m/z found 516.2484, calcd for C30H34N3O5 (M+H)+: 516.2498.
tained as an oil. ½a D25
ꢂ
ꢀ100.8 (c 0.72, CH2Cl2). 1H NMR (500 MHz,
DMSO-d6, d ppm, J Hz) d 7.37–7.28 (m, 5H, H-arom.), 5.15 (d, 1H,
2JH,H = 13.0, CH2 of Cbz), 5.11 (d, 1H, CH2 of Cbz), 5.08 (br s, 1H,
NH), 4.52 (br q, 1H, J6,Me-6 = 7.2, H-6), 3.98 (dd, 1H, J5,4 = 5.0,
J5,6 = 0.6, H-5), 3.81 (dd, 1H, J4,3 = 9.2, H-4), 2.82 (dd, 1H,
2
J1 a,1 b = 13.0, J1 a,3 = 3.0, H-10a), 2.60 (m, 1H, H-3), 3.46 (dd, 1H,
0
0
0
0
J1 b,3 = 8.4, H-10b), 1.31, 1.26 (2s, 3H each, C(CH3)2), 1.26 (d, 3H,
4.2.3. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-dodecanoylamino-
methyl-4,5-O-isopropylidene-6-methylhexahydropyridazine-
4,5-diol (21d)
Me-6). 13C NMR (125.7 MHz, DMSO-d6, d ppm) d 155.3 (C@O of
Cbz), 137.0, 128.2, 127.6, 127.0 (C-arom.), 108.1 (C(CH3)2), 75.2
(C-5), 71.5 (C-4), 66.1 (CH2 of Cbz), 60.0 (C-3), 49.3 (C-6), 41.5
(C-10), 28.0, 26.4 (C(CH3)2), 16.2 (Me-6). CIMS m/z 336 [40%,
(M+H)+], 305 [30%, (M-CH2NH2)+]. CIHRMS m/z found 336.1929,
calcd for C17H26N3O4 (M+H)+: 336.1923.
The coupling reaction of 19 (85.2 mg, 0.254 mmol) with
dodecanoic acid following the general procedure afforded after
chromatographic purification (CH2Cl2/MeOH 50:1) derivative 21d
(127 mg, 0.246 mmol, 97% yield) as a white solid. ½a D24
ꢀ27.1 (c
ꢂ
0.54, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.57
(m, 1H, CONH), 7.38–7.29 (m, 5H, H-arom.), 5.08 (d, 1H,
JH,H = 12.6, CH2 of Cbz), 5.07 (br s, 1H, NH), 5.05 (d, 1H, CH2 of
Cbz), 4.21 (dd, 1H, J5,4 = 5.9, J5,6 = 5.5, H-5), 4.03 (qd, 1H,
J6,Me-6 = 7.1, H-6), 3.84 (dd, 1H, J4,3 = 3.0, H-4), 3.25 (m, 1H,
H-10a), 2.92–2.85 (m, 2H, H-10b, H-3), 1.95 (t, 2H, JH,H = 7.4,
CH2CONH), 1.43 (m, 2H, CH2), 1.38 (s, 3H, C(CH3)2), 1.29 (d, 3H,
Me-6), 1.26 (s, 3H, C(CH3)2), 1.23 (m, 16H, 8CH2), 0.85 (t, 3H,
JH,H = 6.9, CH3). 13C NMR (125.7 MHz, DMSO-d6, d ppm) d 172.2
(CONH), 156.2 (C@O of Cbz), 136.7, 128.3, 127.8, 127.6 (C-arom.),
107.7 (C(CH3)2), 71.9 (C-5), 71.2 (C-4), 66.3 (CH2 of Cbz), 57.2 (C-
3), 49.7 (C-6), 38.7 (C-10), 35.5 (CH2CONH), 31.3, 28.9, 28.8, 28.7,
28.6, 28.5, 26.6, 25.4, 25.1, 22.0 (9 CH2, C(CH3)2), 15.4 (Me-6),
13.9 (CH3). FABMS m/z 540 [55%, (M+Na)+], 518 [25%, (M+H)+].
FABHRMS m/z found 540.3393, calcd for C29H47N3O5Na (M+Na)+:
540.3413.
4.2. General procedure for the synthesis of amide derivatives
To a solution of the hexahydropyridazine 19 or 20 (0.1 mmol) in
the minimum amount of DMF the corresponding carboxylic acid
(0.1 mmol), DIPEA (0.2 mmol), and PyBOP (0.11 mmol) were
added. The reaction mixture was stirred at rt for 3 h and then evap-
orated. The obtained residue was dissolved in CH2Cl2 and washed
with water and brine. The organic phase was dried, filtered, con-
centrated, and purified by chromatography column on silica gel.
4.2.1. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-(p-
phenylbenzoylaminomethyl)-4,5-O-isopropylidene-6-
methylhexahydropyridazine-4,5-diol (21c)
The coupling reaction of 19 (82 mg, 0.245 mmol) with (1,10-
biphenyl)-4-carboxylic acid following the general procedure affor-
ded after chromatographic purification (ether/petroleum ether
10:1) derivative 21c (126 mg, 0.245 mmol, 100% yield) as a white
4.2.4. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-dodecanoylamino-
methyl-4,5-O-isopropylidene-6-methylhexahydropyridazine-
4,5-diol (22d)
solid. ½a 2D6
ꢂ
ꢀ95.1 (c 1.08, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d
0
0
ppm, J Hz) d 8.60 (dd, 1H, JNH,1 = 7.3, JNH,1 = 4.5, CONH), 7.89 (m,
2H, H-arom., biphenyl), 7.76–7.72 (m, 4H, H-arom., biphenyl),
7.50 (m, 2H, H-arom., biphenyl), 7.41 (tt, 1H, J = 7.3, J = 1.1, H-
arom., biphenyl), 7.30–7.23 (m, 5H, H-arom.), 5.20 (br s, 1H, NH),
The coupling reaction of 20 (77.1 mg, 0.230 mmol) with
dodecanoic acid following the general procedure afforded after
chromatographic purification (CH2Cl2/MeOH 50:1) derivative 22d
2
5.09 (d, 1H, JH,H = 12.7, CH2 of Cbz), 5.06 (d, 1H, CH2 of Cbz),
(112 mg, 0.217 mmol, 94% yield) as a white solid. ½a D23
ꢀ35.3 (c
ꢂ

4656
E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
0.96, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.84 (m,
1H, CONH), 7.38–7.28 (m, 5H, H-arom.), 5.13 (d, 1H, JH,H = 13.2,
CH2 of Cbz), 5.10 (d, 1H, CH2 of Cbz), 4.94 (br s, 1H, NH), 4.50 (br
56.3 (C-200), 49.3 (C-6), 46.5 (CH of Fmoc), 38.8 (C-10), 37.4 (C-300),
28.0, 26.5 (C(CH3)2), 16.2 (Me-6). FABMS m/z 727 [50%, (M+Na)+],
705 [12%, (M+H)+]. FABHRMS m/z found 727.3148, calcd for
C41H44N4O7Na (M+Na)+: 727.3108.
2
q, 1H, J6,Me-6 = 7.2, H-6), 3.98 (dd, 1H, J5,4 = 4.9, J5,6 = 0.7, H-5),
2
0
0
3.79 (dd, 1H, J4,3 = 9.3, H-4), 3.26 (ddd, 1H, J1 a,1 b = 13.9, J = 5.9,
J = 3.2, H-10a), 3.00 (ddd, 1H, J = 8.3, J = 6.2, H-10b), 2.71 (m, 1H,
H-3), 2.01 (t, 2H, JH,H = 7.5, CH2CONH), 1.46 (m, 2H, CH2), 1.31 (s,
3H, C(CH3)2), 1.26 (d, 3H, Me-6), 1.25 (s, 3H, C(CH3)2), 1.22 (m,
16H, 8CH2), 0.85 (t, 3H, JH,H = 7.0, CH3). 13C NMR (125.7 MHz,
DMSO-d6, d ppm) d 172.7 (CONH), 155.3 (C@O of Cbz), 136.9,
128.2, 127.6, 127.0 (C-arom.), 107.8 (C(CH3)2), 75.2 (C-5), 71.6 (C-
4), 66.2 (CH2 of Cbz), 59.0 (C-3), 49.3 (C-6), 38.6 (C-10), 35.4
(CH2CONH), 31.2, 28.9, 28.8, 28.7, 28.6, 28.5, 28.0, 26.4, 25.2, 22.0
(9 CH2, C(CH3)2), 16.2 (Me-6), 13.9 (CH3). FABMS m/z 540 [55%,
(M+Na)+], 518 [30%, (M+H)+]. FABHRMS m/z found 540.3381, calcd
for C29H47N3O5Na (M+Na)+: 540.3413.
4.2.7. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[(2S)-(6-N-(tert-
butoxycarbonyl)amino-2-N-(9-fluorenylmethoxycarbonyl)-
amino)hexanoylaminomethyl]-4,5-O-isopropylidene-6-
methylhexahydropyridazine-4,5-diol (21f0)
The coupling reaction of 19 (91.4 mg, 0.273 mmol) with Fmoc-
Lys(Boc)-OH following the general procedure afforded after chro-
matographic purification (CH2Cl2/MeOH 50:1) derivative 21f0
(206.2 mg, 0.263 mmol, 96% yield) as a white solid. ½a D26
ꢀ16.8 (c
ꢂ
0.85, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.89
(d, 2H, J = 7.5, H-arom.), 7.83 (m, 1H, CONH), 7.72 (dd, 2H,
00
J = 10.5, J = 7.5, H-arom.), 7.47 (d, 1H, JNH,2 = 8.0, NHFmoc), 7.41
(t, 2H, J = 7.5, H-arom.), 7.33–7.27 (m, 7H, H-arom.), 6.76 (br t,
00
4.2.5. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[((2S)-2-N-(9-
fluorenylmethoxycarbonyl)amino-3-phenyl)propanoy-
laminomethyl]-4,5-O-isopropylidene-6-methylhexahydro-
pyridazine-4,5-diol (21e0)
1H, JNH,6 = 5.5, NHBoc), 5.06 (br s, 3H, CH2 of Cbz, NH-2), 4.27–
4.19 (m, 4H, H-5, CH2 and CH of Fmoc), 4.04 (qd, 1H, J6,Me-6 = 7.0,
J6,5 = 5.8, H-6), 3.86 (m, 1H, H-200), 3.84 (dd, 1H, J = 6.0, J = 3.0, H-
4), 3.31 (m, 1H, H-10a), 2.92 (m, 1H, H-3), 2.89–2.84 (m, 3H, H-
10b, H-600), 1.60 (m, 1H, H-300a), 1.51 (m, 1H, H-300b), 1.36 (s, 12H,
C(CH3)2 and C(CH3)3), 1.34 (m, 2H, H-500), 1.27 (d, 3H, Me-6), 1.25
(m, 2H, H-400), 1.24 (s, 3H, C(CH3)2). 13C NMR (125.7 MHz, DMSO-
d6, d ppm) d 172.2 (CONH), 156.3, 156.0, 155.6 (C@O of Cbz, C@O
of Fmoc and C@O of Boc), 144.0, 143.7, 140.7, 136.9, 128.3, 127.8,
127.6, 127.5, 127.0, 125.3, 120.1 (C-arom.), 107.8 (C(CH3)2), 77.3
(C(CH3)3), 71.8 (C-5), 71.1 (C-4), 66.3 (CH2 of Cbz), 65.6 (CH2 of
Fmoc), 56.9 (C-3), 54.9 (C-200), 49.6 (C-6), 46.6 (CH of Fmoc),
40.0–38.8 (C-10, C-600), 31.5 (C-300), 29.2 (C-400), 28.3 (C(CH3)3),
26.5, 25.5 (C(CH3)2), 22.9 (C-500), 15.7 (Me-6). FABMS m/z 808
[40%, (M+Na)+]. FABHRMS m/z found 808.3842, calcd for
C43H55N5O9Na (M+Na)+: 808.3897.
The coupling reaction of 19 (78.1 mg, 0.233 mmol) with Fmoc-
Phe-OH following the general procedure afforded after chromato-
graphic purification (CH2Cl2/MeOH 50:1) derivative 21e0
(147.8 mg, 0.21 mmol, 90% yield) as a white solid. ½a D21
ꢀ15.9 (c
ꢂ
0.8, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 7.97
(dd, 1H, J = 7.2, J = 4.2, CONH), 7.87 (d, 2H, J = 7.5, H-arom.), 7.63–
7.60 (m, 3H, H-arom.), 7.39 (td, 2H, J = 7.0, J = 3.2, H-arom.),
7.35–7.22 (m, 10H, H-arom.), 7.17 (br t, 1H, J = 6.7, H-arom.),
5.06 (s, 2H, CH2 of Cbz), 4.21–4.16 (m, 2H, H-5, H-200), 4.14–4.08
(m, 3H, CH2 and CH of Fmoc), 4.02 (qd, 1H, JMe-6,6 = 7.0, J6,5 = 5.4,
H-6), 3.84 (dd, 1H, J = 6.0, J = 3.0, H-4), 3.33 (m, 1H, H-10a), 3.02
2
(dd, 1H, J3 a,3 b = 13.5, J3 a,2 = 4.5, H-300a), 2.98–2.94 (m, 2H, H-
00
00
00
00
10b, H-3), 2.80 (dd, 1H, J3 b,2 = 10.2, H-50b), 1.37, 1.26 (2s, 3H each,
C(CH3)2), 1.29 (d, 3H, Me-6). 13C NMR (125.7 MHz, DMSO-d6, d
ppm) d 171.5 (CONH), 156.3, 155.8 (C@O of Cbz and C@O of Fmoc),
143.8, 143.6, 140.6, 138.1, 136.8, 129.1, 128.2, 128.0, 127.7, 127.6,
127.4, 127.0, 126.2, 125.2, 120.0 (C-arom.), 107.7 (C(CH3)2), 72.0
(C-5), 71.2 (C-4), 66.2 (CH2 of Cbz), 65.6 (CH2 of Fmoc), 57.0 (C-
3), 56.3 (C-200), 49.8 (C-6), 46.5 (CH of Fmoc), 40.0–39.0 (C-10),
37.4 (C-300), 26.6, 25.5 (C(CH3)2), 15.8 (Me-6). FABMS m/z 727
[46%, (M+Na)+], 705 [12%, (M+H)+]. FABHRMS m/z found
727.3149, calcd for C41H44N4O7Na (M+Na)+: 727.3108.
00
00
4.2.8. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[((2S)-2-amino-3-
phenyl)propanoyl-aminomethyl]-4,5-O-isopropylidene-6-
methylhexahydropyridazine-4,5-diol (21e)
A solution of 21e0 (133 mg, 0.185 mmol) in piperidine/DMF (20%,
4 mL) was stirred at rt for 15 min. Then, the mixture was concen-
trated to dryness and the residue purified by chromatographic col-
umn (CH2Cl2/MeOH 20:1) to afford 21e (74.5 mg, 0.155 mmol,
84%) as an oil. ½a D21
ꢂ
ꢀ38.5 (c 1.1, CH2Cl2). 1H NMR (500 MHz,
DMSO-d6, d ppm, J Hz) d 7.97 (br dd, 1H, J = 7.2, J = 3.7, CONH),
7.35–7.25 (m, 7H, H-arom.), 7.21–7.17 (m, 3H, H-arom.), 5.08 (d,
1H, 2JH,H = 12.7, CH2 of Cbz), 5.04 (d, 1H, CH2 of Cbz), 5.03 (br s, 1H,
NH), 4.22 (t, 1H, J5,6 = J5,4 = 5.9, H-5), 4.05 (qd, 1H, J6,Me-6 = 7.0, H-6),
4.2.6. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-[((2S)-2-N-(9-
fluorenylmethoxycarbonyl) amino-3-phenyl)propanoylamino-
methyl]-4,5-O-isopropylidene-6-methylhexahydropyridazine-
4,5-diol (22e0)
3.84 (dd, 1H, J4,3 = 2.5, H-4), 3.37–3.30 (m, 2H, H-10a, H-200), 2.94
2
(dd, 1H, J3 a,3 b = 13.5, J3 a,2 = 4.8, H-300a), 2.92–2.85 (m, 2H, H-3,
00
00
00
00
00
00
The coupling reaction of 20 (77 mg, 0.23 mmol) with Fmoc-Phe-
OH following the general procedure afforded after chromato-
graphic purification (CH2Cl2/MeOH 50:1) derivative 22e0 (161 mg,
H-10b), 2.58 (dd, 1H, J3 b,2 = 8.4, H-300b), 1.82 (br s, 2H, NH2), 1.40,
1.27 (2s, 3H each, C(CH3)2), 1.28 (d, 3H, Me-6). 13C NMR
(125.7 MHz, DMSO-d6, d ppm) d 174.2 (CONH), 156.2 (C@O of Cbz),
138.6, 136.8, 129.2, 128.3, 128.1, 127.7, 127.5, 126.1 (C-arom.),
107.6 (C(CH3)2), 71.7 (C-5), 71.1 (C-4), 66.2 (CH2 of Cbz), 57.1 (C-3),
56.2 (C-200), 49.2 (C-6), 40.8 (C-300), 38.5 (C-10), 26.5, 25.5 (C(CH3)2),
15.6 (Me-6). CIMS m/z 483 [100%, (M+H)+]. CIHRMS m/z found
483.2607, calcd for C26H35N4O5 (M+H)+: 483.2607.
0.228 mmol, 99% yield) as a white solid. ½a D21
ꢀ37.2 (c 1, CH2Cl2).
ꢂ
1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 8.18 (br t, 1H,
0
0
JNH,1 a = JNH,1 b = 5.7, CONH), 7.87 (d, 2H, J = 7.5, H-arom.), 7.64–
7.58 (m, 2H, H-arom.), 7.42–7.38 (m, 2H, H-arom.), 7.32–7.22 (m,
11H, H-arom.), 7.17 (br t, 1H, J = 7.5, H-arom.), 5.13 (br d, 1H,
2JH,H = 13.0, CH2 of Cbz), 5.04 (d, 1H, CH2 of Cbz), 4.96 (br s, 1H,
NH), 4.50 (q, 1H, J6,Me-6 = 7.2, H-6), 4.19 (m, 1H, H-200), 4.16–4.06
(m, 3H, CH2 and CH of Fmoc), 3.98 (d, 1H, J5,4 = 5.0, H-5), 3.83 (br
dd, 1H, J4,3 = 9.0, H-4), 3.42 (m, 1H, H-10a), 3.06–2.99 (m, 2H, H-
10b, H-300a), 2.80–2.75 (m, 2H, H-3, H-300b), 1.32, 1.25 (2s, 3H each,
C(CH3)2), 1.25 (d, 3H, Me-6). 13C NMR (125.7 MHz, DMSO-d6, d
ppm) d 172.0 (CONH), 155.8, 155.4 (C@O of Cbz and C@O of Fmoc),
143.8, 143.7, 140.6, 138.4, 137.0, 129.2, 128.3, 128.0, 127.6, 127.0,
126.2, 125.4, 125.3, 120.1 (C-arom.), 108.0 (C(CH3)2), 75.2 (C-5),
71.5 (C-4), 66.2, 65.6 (CH2 of Cbz and CH2 of Fmoc), 58.3 (C-3),
4.2.9. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-[((2S)-2-amino-3-
phenyl)propanoyl-aminomethyl]-4,5-O-isopropylidene-6-
methylhexahydropyridazine-4,5-diol (22e)
Following the same procedure as for the preparation of 21e, and
starting from 22e0 (128 mg, 0.181 mmol), compound 22e (80 mg,
0.165 mmol, 92%) was obtained as an oil. ½a D27
ꢂ ꢀ68.5 (c 0.91, CH2Cl2).
1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 8.60 (br t, 1H, J = 6.7,
2
CONH), 7.33–7.17 (m, 10H, H-arom.), 5.14 (d, 1H, JH,H = 13.0, CH2
of Cbz), 5.08 (d, 1H, CH2 of Cbz), 5.01 (br s, 1H, NH), 4.50 (q, 1H,

E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4657
J6,Me-6 = 7.2, H-6), 3.97(brd, 1H, J5,4 = 5.5, H-5), 3.77 (dd, 1H, J4,3 = 9.2,
(C-6), 14.3 (Me-6). CIMS m/z 297 [30%, (M+H)+]. CIHRMS m/z found
H-4), 3.37 (dd, 1H, J2 ,3 b = 8.5, J2 ,3 a = 4.5, H-200), 3.30 (m, 1H, H-10a),
297.1452, calcd for C14H21N2O5 (M+H)+: 297.1450.
00 00
00 00
2
3.08 (m, 1H, H-10b), 2.95 (dd, 1H, J3 a,3 b = 13.5, H-300a), 2.72 (m, 1H,
H-3), 2.60(dd, 1H, H-300b), 1.69 (br s, 2H, NH2), 1.31, 1.26 (2s, 3Heach,
C(CH3)2), 1.24 (d, 3H, Me-6). 13C NMR (125.7 MHz, DMSO-d6, d ppm)
d 174.6(CONH), 155.4(C@O ofCbz), 138.7, 136.9, 129.2, 128.2, 128.0,
127.6, 127.0, 126.0 (C-arom.), 107.8 (C(CH3)2), 75.2 (C-5), 71.4 (C-4),
66.1 (CH2 of Cbz), 58.4 (C-3), 56.2 (C-200), 49.3 (C-6), 40.8 (C-300), 38.6
(C-10), 28.0, 26.4 (C(CH3)2), 16.2 (Me-6). CIMS m/z 483 [50%, (M+H)+].
CIHRMS m/z found 483.2615, calcd for C26H35N4O5 (M+H)+:
483.2607.
00
00
4.2.13. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-aminomethyl-6-
methylhexahydropyridazine-4,5-diol (23b)
Compound 19 (88.4 mg, 0.264 mmol) was stirred in HCl (1 M)/
THF 1:1 (7 mL) at rt for 2 h. Solvent was then evaporated and the
residue was purified using a Dowex 50WX8 column, eluting with
MeOH (30 mL), H2O (30 mL) and NH4OH 10% (100 mL). Compound
23b (73.5 mg, 0.249 mmol, 94%) was obtained as a foam. ½a D24
ꢂ
+10.0
(c 0.81, MeOH). 1H NMR (500 MHz, CD3OD d ppm, J Hz) d 7.39–7.30
(m, 5H, H-arom.), 5.14 (d, 1H, 2JH,H = 12.4, CH2 of Cbz), 5.12 (d, 1H,
CH2 of Cbz), 4.21 (m, 1H, H-6), 3.70 (dd, 1H, J5,6 = 5.6, J5,4 = 3.0, H-5),
4.2.10. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[((2S)-6-N-(tert-
butoxycarbonyl)amino-2-amino)hexanoylaminomethyl]-4,5-O-
isopropylidene-6-methylhexahydropyridazine-4,5-diol (21f)
Following the same procedure as for the preparation of 21e, and
starting from 21f0 (189.7 mg, 0.242 mmol), compound 21f
0
0
3.48 (m, 1H, H-4), 2.89 (dt, 1H, J3,1 b = 10.8, J3,1 a = J3,4 = 3.8, H-3),
2
2.63 (dd, 1H, J1 a,1 b = 13.0, H-10a), 2.57 (dd, 1H, H-10b), 1.32 (d,
3H, JMe-6,6 = 7.0, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d
158.5 (C@O of Cbz), 137.9, 129.6, 129.3, 129.1 (C-arom.), 70.4
(C-4), 68.7 (CH2 of Cbz), 68.4 (C-5), 64.1 (C-3), 54.1 (C-6), 41.2
(C-10), 13.2 (Me-6). CIMS m/z 296 [80%, (M+H)+], 265 [40%,
(M-CH2NH2)+]. CIHRMS m/z found 296.1606, calcd for C14H22
N3O4 (M+H)+: 296.1610.
0
0
(97.1 mg, 0.172 mmol, 71%) was obtained as an oil. ½a D22
ꢀ22.1 (c
ꢂ
0.74, CH2Cl2). 1H NMR (500 MHz, DMSO-d6, d ppm, J Hz) d 8.03
(br dd, 1H, J = 7.4, J = 4.4, NHCO), 7.36–7.30 (m, 5H, H-arom.),
00
6.71 (br t, 1H, JNH,6 = 5.5, NHBoc), 5.09 (s, 2H, CH2 of Cbz), 5.06
(d, 1H, JNH,3 = 3.0, NH), 4.23 (dd, 1H, J5,4 = 6.0, J5,6 = 5.5, H-5), 4.06
(qd, 1H, J6,Me-6 = 7.0, H-6), 3.86 (dd, 1H, J4,3 = 3.0, H-4), 3.34 (m,
1H, H-10a), 3.17 (dd, 1H, J = 7.2, J = 5.2, H-200), 2.94 (m, 1H, H-3),
2.90–2.86 (m, 2H, H-10b, H-600), 1.56 (m, 1H, H-300a), 1.39 (s, 3H,
C(CH3)2), 1.37 (s, 9H, C(CH3)3), 1.34 (m, 1H, H-300b), 1.28 (d, 3H,
Me-6), 1.26 (s, 3H, C(CH3)2), 1.29–1.19 (m, 4H, H-400, H-500). 13C
NMR (125.7 MHz, DMSO-d6, d ppm) d 173.7 (CONH), 156.3, 155.5
(C@O of Cbz and C@O of Boc), 136.8, 128.3, 127.8, 127.6 (C-arom.),
107.7 (C(CH3)2), 77.3 (C(CH3)3), 71.8 (C-5), 71.1 (C-4), 66.3 (CH2 of
Cbz), 57.0 (C-3), 54.1 (C-200), 49.4 (C-6), 40.0–39.0 (C-600), 38.6 (C-
10), 33.7 (C-300), 29.3 (CH2), 28.2 (C(CH3)3), 26.5, 25.5 (C(CH3)2),
22.3 (CH2), 15.6 (Me-6). FABMS m/z 586 [40%, (M+Na)+], 564
[30%, (M+H)+]. FABHRMS m/z found 586.3195, calcd for
C28H45N5O7Na (M+Na)+: 586.3217.
4.2.14. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-aminomethyl-6-
methylhexahydropyridazine-4,5-diol (24b)
Deprotection of 20 (103 mg, 0.307 mmol) following the same
procedure as for the preparation of 23b, afforded 24b (79.3 mg,
0.269 mmol, 88%) as a foam. ½a D21
ꢂ
ꢀ66.0 (c 0.8, MeOH). 1H NMR
(500 MHz, CD3OD d ppm, J Hz) d 7.38–7.28 (m, 5H, H-arom.),
2
5.17 (d, 1H, JH,H = 12.5, CH2 of Cbz), 5.14 (d, 1H, CH2 of Cbz),
4.48 (qd, 1H, J6,Me-6 = 7.3, J6,5 = 2.2, H-6), 3.70 (m, 1H, H-5), 3.50
(dd, 1H, J4,3 = 10.1, J4,5 = 3.0, H-4), 3.05–2.98 (m, 2H, H-3, H-10a),
2
2.61 (dd, 1H, J1 b,1 a = 13.0, J1 b,3 = 8.5, H-10b), 1.24 (d, 3H, Me-6).
13C NMR (125.7 MHz, CD3OD, d ppm) d 158.6 (C@O of Cbz),
138.1, 129.5, 129.0, 128.8 (C-arom.), 72.1 (C-5), 68.5 (C-4), 68.3
(CH2 of Cbz), 59.3 (C-3), 57.0 (C-6), 42.1 (C-10), 14.3 (Me-6). CIMS
m/z 296 [100%, (M+H)+], 265 [45%, (M-CH2NH2)+]. CIHRMS m/z
found 296.1615, calcd for C14H22N3O4 (M+H)+: 296.1610.
0
0
0
4.2.11. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-hydroxymethyl-
6-methylhexahydropyridazine-4,5-diol (23a)
Compound 13 (65 mg, 0.193 mmol) was stirred in HCl (1 M)/
THF 1:1 (5 mL) at rt for 2 h. Solvent was then evaporated and the
residue purified by chromatography column (CH2Cl2/MeOH 15:1)
4.2.15. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-(p-
phenylbenzoylaminomethyl)-6-methylhexahydropyridazine-
4,5-diol (23c)
to afford 23a (55 mg, 0.186 mmol, 96%) as a white solid. ½a D26
ꢂ
Compound 21c (110 mg, 0.214 mmol) was stirred in HCl (1 M)/
THF 1:1 (5 mL) at rt for 2 h. Solvent was then evaporated and the
residue purified by chromatography column (CH2Cl2/MeOH 20:1)
+16.4 (c 0.65, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
2
7.39–7.29 (m, 5H, H-arom.), 5.16 (d, 1H, JH,H = 12.4, CH2 of Cbz),
5.12 (d, 1H, CH2 of Cbz), 4.15 (qd, 1H, J6,Me-6 = 7.0, J6,5 = 5.3, H-6),
3.72 (dd, 1H, J5,4 = 3.1, H-5), 3.57 (dd, 1H, J4,3 = 4.2, H-4), 3.52–
3.50 (m, 2H, H-10a, H-10b), 3.00 (m, 1H, H-3), 1.34 (d, 3H, Me-6).
13C NMR (125.7 MHz, CD3OD, d ppm) d 158.6 (C@O of Cbz),
138.0, 129.5, 129.2, 129.0 (C-arom.), 69.1 (C-4), 68.8 (C-5), 68.6
(CH2 of Cbz), 63.6 (C-3), 61.4 (C-10), 54.7 (C-6), 13.5 (Me-6). CIMS
m/z 297 [15%, (M+H)+]. CIHRMS m/z found 297.1454, calcd for
C14H21N2O5 (M+H)+: 297.1450.
to afford 23c (100.3 mg, 0.211 mmol, 98%) as a white solid. ½a D26
ꢂ
ꢀ75.4 (c 0.42, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
7.84 (br d, 2H, H-arom, biphenyl), 7.73–7.65 (m, 4H, H-arom,
biphenyl), 7.50–7.45 (m, 2H, H-arom, biphenyl), 7.37 (tt, 1H,
J = 5.5, J = 1.2, H-arom, biphenyl), 7.33–7.22 (m, 5H, H-arom.),
2
5.16 (d, 1H, JH,H = 12.3, CH2 of Cbz), 5.12 (d, 1H, CH2 of Cbz),
4.27 (m, 1H, H-6), 3.86–3.80 (m, 2H, H-5, H-10a), 3.63 (br t, 1H,
J4,3 = J4,5 = 2.8, H-4), 3.15–3.09 (m, 2H, H-3, H-10b), 1.36 (d, 3H,
JMe-6,6 = 7.0, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 169.4
(CONH), 158.6 (C@O of Cbz), 145.7, 141.3, 137.8, 134.1, 130.0,
129.6, 129.2, 129.1, 129.0, 128.8, 128.1, 128.0 (C-arom.), 70.0 (C-
4), 68.7 (CH2 of Cbz), 68.2 (C-5), 62.0 (C-3), 54.4 (C-6), 39.9 (C-
10), 13.2 (Me-6). CIMS m/z 476 [25%, (M+H)+]. CIHRMS m/z found
476.2193, calcd for C27H30N3O5 (M+H)+: 476.2185.
4.2.12. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-hydroxymethyl-
6-methylhexahydropyridazine-4,5-diol (24a)
Deprotection of 14 (80 mg, 0.238 mmol) following the same
procedure as for the preparation of 23a, afforded 24a (64 mg,
0.216 mmol, 91%) as a white solid. ½a D26
ꢂ
ꢀ76.3 (c 0.72, MeOH). 1H
NMR (500 MHz, CD3OD, d ppm, J Hz) d 7.40–7.28 (m, 5H, H-arom.),
2
5.17 (d, 1H, JH,H = 12.5, CH2 of Cbz), 5.14 (d, 1H, CH2 of Cbz), 4.47
4.2.16. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-(p-phenylbenzoyl-
aminomethyl)-6-methylhexahydropyridazine-4,5-diol (24c)
Deprotection of 22c (69.2 mg, 0.134 mmol) following the same
procedure as for the preparation of 23c, afforded 24c (63.7 mg,
2
0
0
(qd, 1H, J6,Me = 7.2, J6,5 = 2.0, H-6), 3.84 (dd, 1H, J1 a,1 b = 11.3,
J1 a,3 = 2.6, H-10a), 3.71 (m, 1H, H-5), 3.65 (dd, 1H, J4,3 = 10.3,
0
J4,5 = 3.0, H-4), 3.62 (dd, 1H, J1 b,3 = 6.4, H-10b), 3.06 (ddd, 1H, H-
0
3), 1.25 (d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d
158.4 (C@O of Cbz), 138.1, 129.5, 129.0, 128.8 (C-arom.), 72.1
(C-5), 68.5 (CH2 of Cbz), 66.4 (C-4), 61.6 (C-10), 59.2 (C-3), 56.6
0.134 mmol, 100% yield) as a white solid. ½a D25
ꢀ10.6 (c 0.84, MeOH).
ꢂ
1H NMR (500 MHz, CD3OD, d ppm, J Hz) 7.90 (m, 2H, H-arom, biphe-
nyl), 7.69 (br d, 2H, J = 8.0, H-arom, biphenyl), 7.66 (m, 2H, H-arom,

4658
E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
(dd, 1H, J1 b,3 = 10.0, H-10b), 2.79 (m, 1H, H-3), 2.74 (dd, J3 b,2 = 7.4,
H-300b), 1.31 (d, 3H, JMe-6,6 = 7.0, Me-6). 13C NMR (125.7 MHz, CD3OD,
d ppm) d 176.5 (CONH), 158.3 (C@O of Cbz), 138.9, 137.9, 130.4,
129.6, 129.58, 129.3, 129.1, 127.7 (C-arom.), 68.9 (C-4), 68.6 (CH2
of Cbz), 68.3 (C-5), 61.6 (C-3), 57.8 (C-200), 54.3 (C-6), 42.3 (C-300),
39.3 (C-10), 13.2 (Me-6). CIMS m/z 443 [65%, (M+H)+]. CIHRMS m/z
found 443.2289, calcd for C23H31N4O5 (M+H)+: 443.2294.
0
00
00
biphenyl), 7.48–7.45 (m, 2H, H-arom, biphenyl), 7.41 (tt, 1H, J = 7.5,
J = 1.5, H-arom, biphenyl), 7.34 (m, 2H, H-arom.), 7.25 (br s, 3H, H-
arom.), 5.16 (d, 1H, JH,H = 12.5, CH2 of Cbz), 5.12 (d, 1H, CH2 of
2
Cbz), 4.50 (qd, 1H, J6,Me-6 = 7.2, J6,5 = 2.2, H-6), 3.96 (br d, 1H,
2
J1 a,1 b = 13.9, H-10a), 3.75 (m, 1H, H-5), 3.55 (dd, 1H, J4,3 = 10.0,
0
0
J4,5 = 3.1, H-4), 3.30 (m, 1H, H-10b), 3.22 (td, 1H, J3,1 b = 9.5,
0
J3,1 a = 2.5, H-3), 1.27 (d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD, d
0
ppm) d 170.6 (CONH), 158.6 (C@O of Cbz), 145.7, 141.3, 138.0,
134.3, 130.0, 129.5, 129.1, 129.0, 128.7, 128.1, 128.0 (C-arom.),
72.2 (C-5), 68.6 (CH2 of Cbz), 68.2 (C-4), 58.3 (C-3), 56.9 (C-6), 41.1
(C-10), 14.4 (Me-6). CIMS m/z 476 [30%, (M+H)+]. CIHRMS m/z found
476.2184, calcd for C27H30N3O5 (M+H)+: 476.2185.
4.2.20. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-[((S)-2-amino-3-
phenyl)propanoyl-aminomethyl]-6-methylhexahydropy-
ridazine-4,5-diol (24e)
Compound 22e (62.8 mg, 0.130 mmol) was stirred in HCl
(1 M):THF 1:1 (3.4 mL) at rt for 5 h. Solvent was then evaporated
and the residue was purified using a Dowex 50WX8 column, elut-
ing with MeOH (30 mL), H2O (30 mL), and NH4OH 10% (150 mL).
Compound 24e (51.8 mg, 0.117 mmol, 90%) was obtained as an
4.2.17. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-dodecanoyl-
aminomethyl-6-methylhexahydropyridazine-4,5-diol (23d)
Deprotection of 21d (99 mg, 0.191 mmol) following the same
procedure as for the preparation of 23c, afforded 23d (83.6 mg,
oil. ½a 2D5
ꢂ
ꢀ26.9 (c 0.68, MeOH). 1H NMR (500 MHz, CD3OD, d
0.175 mmol, 92%) as an oil. ½a D27
ꢂ
ꢀ22.3 (c 0.64, MeOH). 1H NMR
ppm, J Hz) d 7.38–7.36 (m, 2H, H-arom.), 7.33 (t, 2H, J = 7.5, H-
(300 MHz, CD3OD, d ppm, J Hz) d 7.40–7.28 (m, 5H, H-arom.), 5.18
arom.), 7.29–7.26 (m, 3H, H-arom.), 7.22–7.18 (m, 3H, H-arom.),
2
2
(d, 1H, JH,H = 12.3, CH2 of Cbz), 5.11 (d, 1H, CH2 of Cbz), 4.22 (m,
5.17 (d, 1H, JH,H = 13.0, CH2 of Cbz), 5.13 (d, 1H, CH2 of Cbz),
1H, H-6), 3.77 (dd, 1H, J = 5.6, J = 3.0, H-5), 3.58–3.53 (m, 2H, H-4,
H-10a), 2.97–2.85 (m, 2H, H-10b, H-3), 2.04 (br t, 2H, JH,H = 7.5,
CH2CONH), 1.53 (m, 2H, CH2), 1.33 (d, 3H, JMe-6,6 = 7.0, Me-6), 1.29
(m, 16H, 8CH2), 0.90 (t, 3H, JH,H = 6.9, CH3). 13C NMR (75.4 MHz,
CD3OD, d ppm) d 176.0 (CONH), 158.5 (C@O of Cbz), 137.9, 129.6,
129.3, 129.1 (C-arom.), 69.9 (C-4), 68.7 (CH2 of Cbz), 68.2 (C-5),
61.9 (C-3), 54.4 (C-6), 39.4 (C-10), 37.2 (CH2CONH), 33.1, 30.7,
30.6, 30.5, 30.4, 30.3, 26.9, 23.7 (9 CH2), 14.4 (CH3), 13.2 (Me-6).
CIMS m/z 478 [100%, (M+H)+]. CIHRMS m/z found 478.3293, calcd
for C26H44N3O5 (M+H)+: 478.3281. Anal. Calcd for C26H43N3O5: C,
65.38; H, 9.07; N, 8.80. Found: C, 64.82; H, 8.66; N, 8.86.
4.46 (qd, 1H, J6,Me-6 = 7.3, J6,5 = 2.2, H-6), 3.70 (m, 1H, H-5), 3.62
2
(dd, 1H, J1 a,1 b = 14.0, J1 a,3 = 2.7, H-10a), 3.58 (m, 1H, H-200), 3.44
0
0
0
(dd, 1H, J4,3 = 10.5, J4,5 = 3.1, H-4), 3.08 (dd, 1H, J1 b,3 = 9.0, H-10b),
0
3.02–2.97 (m, 2H, H-300a, H-3), 2.81 (m, 1H, H-300b), 1.22 (d, 3H,
Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 176.8 (CONH),
158.6 (C@O of Cbz), 138.7, 138.1, 130.4, 129.6, 129.5, 129.0,
128.8, 127.8 (C-arom.), 72.1 (C-5), 68.6 (CH2 of Cbz), 68.0 (C-4),
57.8 (C-3), 57.6 (C-200), 56.9 (C-6), 42.2 (C-300), 40.3 (C-10), 14.4
(Me-6). CIMS m/z 443 [95%, (M+H)+]. CIHRMS m/z found
443.2296, calcd for C23H31N4O5 (M+H)+: 443.2294.
4.2.21. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[((S)-2,6-diamino)-
hexanoylaminomethyl]-6-methylhexahydropyridazine-4,5-diol
(23f)
4.2.18. (3S,4S,5R,6R)-1-N-Benzyloxycarbonyl-3-dodecanoyl-
aminomethyl-6-methylhexahydropyridazine-4,5-diol (24d)
Deprotection of 22d (86.2 mg, 0.167 mmol) following the same
procedure as for the preparation of 23c, afforded 24d (77.4 mg,
Deprotection of 21f (74.1 mg, 0.131 mmol) following the same
procedure as for the preparation of 24e, afforded 23f (41 mg,
0.162 mmol, 97%) as an oil. ½a D22
ꢂ
ꢀ40.2 (c 0.79, MeOH). 1H NMR
0.097 mmol, 74%) as an oil. ½a D22
ꢂ
ꢀ22.8 (c 0.55, MeOH). 1H NMR
(300 MHz, DMSO-d6, 363 K, d ppm, J Hz) d 7.42 (br s, 1H, CONH),
7.38–7.28 (m, 5H, H-arom.), 5.09 (s, 2H, CH2 of Cbz), 4.75 (br d,
1H, JNH,3 = 6.0, NH), 4.34–4.26 (m, 3H, H-6, OH-4, OH-5), 3.58 (m,
1H, H-5), 3.45–3.38 (m, 2H, H-4, H-10a), 3.11 (m, 1H, H-10b), 2.95
(m, 1H, H-3), 2.08 (t, 2H, JH,H = 7.3, CH2CONH), 1.51 (m, 2H, CH2),
1.27 (m, 16H, 8CH2), 1.18 (d, 3H, JMe-6,6 = 7.2, Me-6), 0.87 (t, 3H,
JH,H = 6.6, CH3). 13C NMR (125.7 MHz, DMSO-d6, 363 K, d ppm) d
172.4 (CONH), 155.1 (C@O of Cbz), 136.7, 127.7, 127.0, 126.0 (C-
arom.), 70.1 (C-5), 66.4 (C-4), 65.7 (CH2 of Cbz), 56.8 (C-3), 54.2
(C-6), 38.4 (C-10), 35.1 (CH2CONH), 30.7, 28.4, 28.38, 28.3, 28.2,
28.1, 28.0, 24.7, 21.4 (9 CH2), 13.7 (Me-6), 13.2 (CH3). CIMS m/z
478 [80%, (M+H)+]. CIHRMS m/z found 478.3261, calcd for
C26H44N3O5 (M+H)+: 478.3281.
(500 MHz, CD3OD, d ppm, J Hz) d 7.36–7.31 (m, 5H, H-arom.),
2
5.18 (d, 1H, JH,H = 12.3, CH2 of Cbz), 5.13 (d, 1H, CH2 of Cbz),
4.06 (m, 1H, H-6), 3.78 (dd, 1H, J5,6 = 5.5, J5,4 = 3.0, H-5), 3.58–
3.55 (m, 2H, H-4, H-10a), 3.15 (m, 1H, H-200), 3.01–2.94 (m, 2H, H-
3, H-10b), 2.71 (t, 2H, JH,H = 7.2, H-600), 1.65 (m, 1H, H-300a), 1.55–
1.45 (m, 3H, H-500, H-300b), 1.36 (m, 2H, H-400), 1.33 (d, 3H,
J6,Me-6 = 7.0, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 177.5
(CONH), 158.5 (C@O of Cbz), 138.0, 129.3, 129.1, 129.0 (C-arom.),
70.0 (C-4), 68.7 (CH2 of Cbz), 68.3 (C-5), 61.7 (C-3), 56.1 (C-200),
54.5 (C-6), 41.8 (C-600), 39.4 (C-10), 35.9 (C-300), 32.1 (C-500), 23.9
(C-400), 13.2 (Me-6). CIMS m/z 424 [5%, (M+H)+]. CIHRMS m/z found
424.2560, calcd for C20H34N5O5 (M+H)+: 424.2560.
4.3. Hydrogenation of Cbz. General procedure
4.2.19. (3S,4S,5R,6S)-1-N-Benzyloxycarbonyl-3-[((S)-2-amino-3-
phenyl)propanoyl-aminomethyl]-6-methylhexahydropy-
ridazine-4,5-diol (23e)
To
a
solution of the N-protected hexahydropyridazine
(0.1 mmol) in MeOH (4 mL), Pd/C (10%) and HCl (5 M, 0.4 mmol)
were added. The mixture was hydrogenated at 1 atm for 2–4 h.
The mixture was then diluted with MeOH, filtered through Celite,
and evaporated, affording the corresponding unprotected deriva-
tives in quantitative yields.
Compound 21e (78.5 mg, 0.163 mmol) was stirred in HCl (1 M)/
THF 1:1 (8.5 mL) at rt for 5 h. Solvent was then evaporated and the
residuewasdissolvedinwater(1.5 mL)andtreatedwith25%NH4OH
(1.3 mL). After 1.5 h at rt, the solvent was evaporated and the residue
purified by chromatography column (CH2Cl2/MeOH/NH4OH
10:1:0.1) to afford 23e (64 mg, 0.145 mmol, 89%) as an oil. ½a D22
ꢂ
4.3.1. (3S,4S,5R,6S)-3-Hydroxymethyl-6-methylhexahy-
dropyridazine-4,5-diol hydrochloride (3a)
ꢀ8.5 (c 1.33, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
7.39–7.26 (m, 7H, H-arom.), 7.23–7.16 (m, 3H, H-arom.), 5.12 (d,
Hydrogenation of 23a (32.3 mg, 0.109 mmol) following the gen-
1H, JH,H = 12.3, CH2 of Cbz), 5.09 (d, 1H, CH2 of Cbz), 4.18 (m, 1H,
eral procedure afforded 3a (21.6 mg, 0.109 mmol) as an oil. ½a D22
ꢂ
2
H-6), 3.74 (dd, 1H, J5,6 = 5.5, J5,4 = 3.0, H-5), 3.53 (dd, 1H,
ꢀ44.6 (c 0.6, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz)
2
2
J1 a,1 b = 13.5, J1 a,3 = 4.7, H-10a), 3.48 (br t, 1H, J4,3 = 3.2, H-4), 3.37
3.85–3.82 (m, 2H, H-5, H-10a), 3.74 (dd, 1H, J1 b,1 a = 11.6,
0
0
0
0
0
2
(m, 1H, H-200), 2.94 (dd, 1H, J3 a,3 b = 13.4, J3 a,2 = 6.4, H-300a), 2.87
J1 b,3 = 5.5, H-10b), 3.63 (dd, 1H, J4,3 = 10.3, J4,5 = 2.8, H-4), 3.35 (qd,
00
00
00
00
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E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
4659
0
1H, J6,Me-6 = 6.8, J6,5 = 1.2, H-6), 3.24 (ddd, 1H, J3,1 a = 2.9, H-3), 1.28
(d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 69.6 (C-5),
68.1 (C-4), 60.1 (C-10), 58.0 (C-6), 57.5 (C-3), 14.0 (Me-6). CIMS
m/z 163 [40%, (M+H)+]. CIHRMS m/z found 163.1083, calcd for
C6H15N2O3 (M+H)+: 163.1083.
7.95 (br d, 2H, J = 7.5, H-arom.), 7.74 (br d, 2H, J = 8.5, H-arom.),
7.67–7.65 (m, 2H, H-arom.), 7.46 (br t, 2H, J = 7.5, H-arom.), 7.38
(tt, 1H, J = 7.5, J = 1.2, H-arom.), 3.82–3.76 (m, 3H, H-4, H-5, H-
10a), 3.67 (m, 1H, H-10b), 3.60–3.52 (m, 2H, H-3, H-6), 1.40 (d,
3H, JMe-6,6 = 6.8, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d
170.8 (CONH), 146.0, 141.1, 133.6, 130.0, 129.2, 129.0, 128.1 (C-
arom.), 69.8, 66.4 (C-5, C-4), 59.3, 57.1 (C-3, C-6), 39.9 (C-10),
12.9 (Me-6). CIMS m/z 342 [5%, (M+H)+]. CIHRMS m/z found
342.1806, calcd for C19H24N3O3 (M+H)+: 342.1818.
4.3.2. (3S,4S,5R,6R)-3-Hydroxymethyl-6-methylhexahydro-
pyridazine-4,5-diol hydrochloride (4a)
Hydrogenation of 24a (27.3 mg, 0.092 mmol) following the gen-
eral procedure afforded 4a (18.2 mg, 0.092 mmol) as an oil. ½a D22
ꢂ
ꢀ12.3 (c 0.85, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz)
4.3.7. (3S,4S,5R,6S)-3-Dodecanoylaminomethyl-6-
2
3.85 (dd, 1H, J1 a,1 b = 11.5, J1 a,3 = 3.7, H-10a), 3.81 (dd, 1H,
methylhexahydropyridazine-4,5-diol hydrochloride (5d)
Hydrogenation of 23d (45.3 mg, 0.095 mmol) following the gen-
0
0
0
J4,3 = 7.4, J4,5 = 2.9, H-4), 3.77 (dd, 1H, J1 b,3 = 6.9, H-10b), 3.71 (m,
0
1H, H-5), 3.51 (qd, 1H, J6,Me-6 = 6.9, J6,5 = 5.4, H-6), 3.36 (td, 1H,
H-3), 1.34 (d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d
70.1 (C-5), 65.4 (C-4), 60.5 (C-3), 60.2 (C-10), 56.6 (C-6), 13.2
(Me-6). CIMS m/z 163 [65%, (M+H)+]. CIHRMS m/z found
163.1087, calcd for C6H15N2O3 (M+H)+: 163.1083.
eral procedure afforded 5d (36 mg, 0.095 mmol) as a solid. ½a D22
ꢂ
ꢀ20.4 (c 0.64, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
3.86 (dd, 1H, J5,4 = 2.6, J5,6 = 1.3, H-5), 3.59 (m, 1H, H-10a), 3.41
(dd, 1H, J4,3 = 10.0, H-4), 3.37 (qd, 1H, J6,Me-6 = 6.7, H-6), 3.33–
3.26 (m, 2H, H-10b, H-3), 2.24 (t, 2H, JH,H = 7.5, CH2CONH), 1.61
(m, 2H, CH2), 1.32–1.29 (m, 19H, 8CH2, Me-6), 0.90 (t, 3H,
JH,H = 7.0, CH3). 13C NMR (125.7 MHz, CD3OD, d ppm) d 177.4
(CONH), 70.0 (C-4), 69.5 (C-5), 58.7 (C-6), 56.6 (C-3), 39.7 (C-10),
37.0 (CH2CONH), 33.1, 30.7, 30.6, 30.5, 30.4, 26.9, 23.7 (9 CH2),
14.4 (CH3), 13.7 (Me-6). CIMS m/z 344 [100%, (M+H)+]. CIHRMS
m/z found 344.2905, calcd for C18H38N3O3 (M+H)+: 344.2913.
4.3.3. (3S,4S,5R,6S)-3-Aminomethyl-6-methylhexahydro-
pyridazine-4,5-diol dihydrochloride (3b)
Hydrogenation of 23b (43.2 mg, 0.146 mmol) following the gen-
eral procedure afforded 3b (34 mg, 0.146 mmol) as an oil. ½a D22
ꢂ
ꢀ34.9 (c 0.74, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
3.92 (m, 1H, H-5), 3.57 (dd, 1H, J4,3 = 10.2, J4,5 = 2.6, H-4), 3.52
2
0
0
(m, 1H, H-6), 3.50 (m, 1H, H-3), 3.38 (dd, 1H, J1 a,1 b = 13.5,
4.3.8. (3S,4S,5R,6R)-3-Dodecanoylaminomethyl-6-
J1 a,3 = 4.0, H-10a), 2.98 (dd, 1H, J1 b,3 = 8.9, H-10b), 1.34 (d, 3H,
J6,Me-6 = 6.7, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 70.2
(C-4), 69.3 (C-5), 59.3 (C-6), 53.9 (C-3), 40.4 (C-10), 13.6 (Me-6).
CIMS m/z 162 [50%, (M+H)+]. CIHRMS m/z found 162.1246, calcd
for C6H16N3O2 (M+H)+: 162.1243.
methylhexahydropyridazine-4,5-diol hydrochloride (6d)
Hydrogenation of 24d (51.5 mg, 0.108 mmol) following the gen-
0
0
eral procedure afforded 6d (41 mg, 0.108 mmol) as a solid. ½a D22
ꢂ
ꢀ4.5 (c 0.75, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
3.76 (m, 1H, H-5), 3.77 (br dd, 1H, J4,3 = 7.3, J4,5 = 2.5, H-4), 3.56–
2
3.50 (m, 1H, H-6), 3.52 (dd, 1H, J1 a,1 b = 14.4, J1 a,3 = 4.2, H-10a),
0
0
0
0
4.3.4. (3S,4S,5R,6R)-3-Aminomethyl-6-methylhexahydro-
pyridazine-4,5-diol dihydrochloride (4b)
3.43 (dd, 1H, J1 b,3 = 7.2, H-10b), 3.36 (m, 1H, H-3), 2.24 (t, 2H,
JH,H = 7.5, CH2CONH), 1.61 (m, 2H, CH2), 1.37 (d, 3H, JMe-6,6 = 7.0,
Me-6), 1.32–1.29 (m, 16H, 8CH2), 0.90 (t, 3H, JH,H = 7.0, CH3). 13C
NMR (125.7 MHz, CD3OD, d ppm) d 177.6 (CONH), 69.8 (C-5),
66.2 (C-4), 59.2 (C-3), 56.9 (C-6), 39.2 (C-10), 37.0 (CH2CONH),
33.0, 30.73, 30.72, 30.6, 30.5, 30.4, 30.3, 26.9, 23.7 (9 CH2), 14.4
(CH3), 12.9 (Me-6). CIMS m/z 344 [100%, (M+H)+]. CIHRMS m/z
found 344.2903, calcd for C18H38N3O3 (M+H)+: 344.2913.
Hydrogenation of 24b (41 mg, 0.138 mmol) following the gen-
eral procedure afforded 4b (32.6 mg, 0.138 mmol) as an oil. ½a D22
ꢂ
ꢀ11.6 (c 0.33, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
3.86 (m, 1H, H-5), 3.72 (dd, 1H, J4,3 = 8.9, J4,5 = 2.7, H-4), 3.67 (qd,
0
0
1H, J6,Me-6 = 7.2, J6,5 = 3.7, H-6), 3.54 (td, 1H, J3,1 b = 8.8, J3,1 a = 4.5,
2
H-3), 3.33 (m, 1H, H-10a), 3.16 (dd, 1H, J1 a,1 b = 13.2, H-10b), 1.45
(d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 69.6 (C-5),
66.1 (C-4), 58.3 (C-6), 55.3 (C-3), 40.0 (C-10), 12.0 (Me-6). CIMS
m/z 162 [40%, (M+H)+]. CIHRMS m/z found 162.1247, calcd for
C6H16N3O2 (M+H)+: 162.1243.
0
0
4.3.9. (3S,4S,5R,6S)-3-[((2S)-2-Amino-6-phenyl)propanoyl-
aminomethyl]-6-methylhexahydropyridazine-4,5-diol
dihydrochloride (5e)
Hydrogenation of 23e (25.3 mg, 0.057 mmol) following the gen-
4.3.5. (3S,4S,5R,6S)-3-(p-Phenylbenzoylaminomethyl)-6-
methylhexahydropyridazine-4,5-diol hydrochloride (5c)
Hydrogenation of 23c (50.5 mg, 0.106 mmol) following the gen-
eral procedure afforded 5e (22 mg, 0.057 mmol) as a solid. ½a D22
ꢂ
+5.7 (c 0.67, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
7.38–7.35 (m, 2H, H-arom.), 7.32–7.29 (m, 3H, H-arom.), 4.16
eral procedure afforded 5c (40.5 mg, 0.106 mmol) as a solid. ½a D22
ꢂ
(dd, 1H, J2 ,3 b = 8.0, J2 ,3 a = 7.0, H-200), 3.85 (br s, 1H, H-5), 3.52
00 00
00 00
ꢀ13.7 (c 0.88, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
7.95 (d, 2H, J = 8.5, H-arom.), 7.72 (d, 2H, J = 8.5, H-arom.), 7.66
(dd, 1H, J1 a,1 b = 14.0, J1 a,3 = 3.0, H-10a), 3.47–3.44 (m, 2H, H-4,
2
0
0
0
H-6), 3.41 (dd, 1H, J1 b,3 = 7.0, H-10b), 3.28 (m, 1H, H-3), 3.23 (dd,
0
2
1H, J3 a,3 b = 14.0, H-300a), 3.04 (dd, 1H, H-300b), 1.30 (d, 3H, JMe-
6,6 = 7.0, Me-6). 13C NMR (125.7 MHz, CD3OD, d ppm) d 170.6
(CONH), 135.7, 130.5, 130.1, 128.9 (C-arom.), 69.6 (C-4), 69.5 (C-
5), 58.8 (C-6), 56.2 (C-3), 55.9 (C-200), 39.8 (C-10), 38.6 (C-300), 13.6
(Me-6). CIMS m/z 309 [10%, (M+H)+]. CIHRMS m/z found
309.1924, calcd for C15H25N4O3 (M+H)+: 309.1927.
00
00
(m, 2H, H-arom.), 7.46 (m, 2H, H-arom.), 7.38 (tt, 1H, J = 7.0,
2
0
0
J = 1.5, H-arom.), 3.90 (m, 1H, H-5), 3.82 (dd, 1H, J1 a,1 b = 14.4,
J1 a,3 = 3.0, H-10a), 3.59 (dd, 1H, J1 b,3 = 7.3, H-10b), 3.53 (dd, 1H,
J4,3 = 10.2, J4,5 = 2.6, H-4), 3.47 (m, 1H, H-3), 3.43 (br q, 1H,
J6,Me-6 = 6.7, H-6), 1.31 (d, 3H, Me-6). 13C NMR (125.7 MHz, CD3OD,
d ppm) d 170.8 (CONH), 145.9, 141.2, 133.8, 129.1, 129.0, 128.1,
128.0 (C-arom.), 70.2 (C-4), 69.6 (C-5), 58.6 (C-6), 56.9 (C-3), 40.4
(C-10), 13.7 (Me-6). CIMS m/z 342 [30%, (M+H)+]. CIHRMS m/z
found 342.1813, calcd for C19H24N3O3 (M+H)+: 342.1818.
0
0
4.3.10. (3S,4S,5R,6R)-3-[((2S)-2-Amino-3-phenyl)propanoyl-
aminomethyl]-6-methylhexahydropyridazine-4,5-diol
dihydrochloride (6e)
4.3.6. (3S,4S,5R,6R)-3-(p-Phenylbenzoylaminomethyl)-6-
methylhexahydropyridazine-4,5-diol hydrochloride (6c)
Hydrogenation of 24c (40.1 mg, 0.084 mmol) following the gen-
Hydrogenation of 24e (32 mg, 0.072 mmol) following the gen-
eral procedure afforded 6e (27.4 mg, 0.072 mmol) as a solid. ½a D22
ꢂ
+21.4 (c 0.76, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
eral procedure afforded 6c (32 mg, 0.084 mmol) as a solid. ½a D25
ꢂ
7.38–7.36 (m, 2H, H-arom.), 7.32–7.30 (m, 3H, H-arom.), 4.16
ꢀ5.5 (c 1.24, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
(dd, 1H, J2 ,3 b = 8.2, J2 ,3 a = 6.5, H-200), 3.77 (br s, 1H, H-5), 3.66
00 00
00 00

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E. Moreno-Clavijo et al. / Bioorg. Med. Chem. 18 (2010) 4648–4660
9. Robina, I.; Moreno-Vargas, A. J.; Carmona, A. T.; Vogel, P. Curr. Drug. Metab.
2004, 5, 329.
(br d, 1H, J4,3 = 6.0, H-4), 3.55–3.52 (m, 1H, H-6), 3.53 (dd, 1H,
2
J1 a,1 b = 14.4, J1 a,3 = 4.0, H-10a), 3.45 (br dd, 1H, J1 b,3 = 7.5, H-10b),
0
0
0
0
10. Dubernet, M.; Defoin, A.; Tarnus, C. Bioorg. Med. Chem. Lett. 2006, 61, 1172.
11. (a) Fleet, G. W. J.; Namgoong, S. K.; Barker, C.; Baines, S.; Jacob, G. S.;
Winchester, B. Tetrahedron Lett. 1989, 30, 4439; (b) Andrews, D. M.; Bird, M.
I.; Cunningham, M. M.; Ward, P. Bioorg. Med. Chem. Lett. 1993, 3, 2533; (c)
Beacham, A. R.; Smelt, K. H.; Biggadike, K.; Britten, C. J.; Hackett, L.;
Winchester, B. G.; Nash, R. J.; Griffiths, R. C.; Fleet, G. W. J. Tetrahedron Lett.
1998, 39, 151.
2
3.34 (m, 1H, H-3), 3.24 (dd, 1H, J3 a,3 b = 14.0, H-300a), 3.04 (dd,
1H, H-300b), 1.38 (d, 3H, JMe-6,6 = 6.9, Me-6). 13C NMR (125.7 MHz,
CD3OD, d ppm) d 170.6 (CONH), 135.6, 130.5, 130.2, 128.9, (C-
arom.), 69.8 (C-5), 66.0 (C-4), 58.3 (C-3), 57.5 (C-6), 55.9 (C-200),
39.6 (C-10), 38.6 (C-300), 12.6 (Me-6). CIMS m/z 309 [60%, (M+H)+].
CIHRMS m/z found 309.1920, calcd for C15H25N4O3 (M+H)+:
309.1927.
00
00
12. Wu, C.-Y.; Chang, C.-F.; Chen, J. S.-Y.; Wong, C.-H.; Lin, C.-H. Angew. Chem., Int.
Ed. 2003, 42, 4661.
13. (a) Davis, B. G. Tetrahedron: Asymmetry 2009, 20, 645; (b)Iminosugars: From
Synthesis to Therapeutic Applications; Compain, P., Martin, O. R., Eds.; Wiley-
VCH: Weinheim, 2007; (c) Robina, I.; Vogel, P. Synthesis 2005, 675.
14. Ho, C.-W.; Lin, Y.-N.; Chang, C.-F.; Li, S.-T.; Wu, Y.-T.; Wu, C.-Y.; Chang, C.-F.;
Liu, S.-W.; Li, Y.-K.; Lin, C.-H. Biochemistry 2006, 45, 5695; (b) Chang, C.-F.; Ho,
C.-W.; Wu, C.-Y.; Chao, T.-A.; Wong, C.-H.; Lin, C.-H. Chem. Biol. 2004, 11, 1301.
15. Jensen, H. H.; Jensen, A.; Hazzel, R. G.; Bols, M. J. Chem. Soc., Perkin Trans. 1
2002, 1190.
16. (a) Bols, M.; Hazell, R. G.; Thomsen, I. B. Chem. Eur. J. 1997, 3, 940; (b) Bols, M.;
Liang, X. J. Org. Chem. 1999, 64, 8485; (c) Lohse, A.; Jensen, K. B.; Lundgren, K.;
Bols, M. Bioorg. Med. Chem. 1999, 7, 1965; (d) Ernholt, B. V.; Thomsen, I.; Jensen,
K. B.; Bols, M. Synlett 1999, 701; (e) Ernholt, B. V.; Thomsen, I.; Lohse, A.;
Plesner, I. W.; Jensen, K. B.; Hazell, R.; Liang, X.; Jacobsen, A.; Bols, M. Chem. Eur.
J. 2000, 6, 278; (f) Jensen, H. H.; Bols, M. J. Chem. Soc., Perkin Trans. 1 2001, 905;
(g) Meloncelli, P. J.; Stick, R. V. Aust. J. Chem. 2006, 59, 827; (h) López López, O.;
Bols, M. ChemBioChem 2007, 8, 657; (i) Monbaliu, J.-C.; Marchand-Brynaert, J.
Synthesis 2009, 1876.
4.3.11. (3S,4S,5R,6S)-3-[((2S)-2,6-Diamino)hexanoyla-
minomethyl]-6-methylhexahydropyridazine-4,5-diol
hydrochloride (5f)
Hydrogenation of 23f (22 mg, 0.052 mmol) following the gen-
eral procedure afforded 5f (20 mg, 0.052 mmol) as a solid. ½a D25
ꢂ
ꢀ1.6 (c 0.5, MeOH). 1H NMR (500 MHz, CD3OD, d ppm, J Hz) d
3.97 (br t, 1H, J2 ,3 a = J2 ,3 b = 6.5, H-200), 3.89 (m, 1H, H-5), 3.66
00 00
00 00
2
(dd, 1H, J1 a,1 b = 14.2, J1 a,3 = 2.7, H-10a), 3.54 (dd, 1H, J4,3 = 10.1,
J4,5 = 2.4, H-4), 3.50 (q, 1H, J6,Me-6 = 6.6, H-6), 3.44 (dd, 1H,
0
0
0
J1 b,3 = 7.2, H-10b), 3.37 (m, 1H, H-3), 2.98 (br t, 2H, J6 ,5 = 7.5, H-
600), 1.95 (m, 1H, H-300a), 1.89 (m, 1H, H-300b), 1.75 (m, 2H, H-500),
1.54 (m, 2H, H-400), 1.32 (d, 3H, Me-6). 13C NMR (125.7 MHz,
CD3OD, d ppm) d 170.8 (CONH), 69.7 (C-4), 69.5 (C-5), 58.8 (C-6),
56.1 (C-3), 54.3 (C-200), 40.3 (C-600), 39.9 (C-10), 32.0 (C-300), 28.0
(C-500), 23.0 (C-400), 13.7 (Me-6). CIMS m/z 290 [45%, (M+H)+], 272
[100%, (M-NH3)+]. CIHRMS m/z found 290.2186, calcd for
C12H28N5O3 (M+H)+: 290.2192.
0
00 00
17. Moreno-Clavijo, E.; Carmona, A. T.; Moreno-Vargas, A. J.; Álvarez, E.; Robina, I.
Synlett 2010, 1367.
18. (a) Varrot, A.; Tarling, C. A.; Macdonald, J. M.; Stick, R. V.; Zechel, D. L.; Withers,
S. G.; Davies, G. J. J. Am. Chem. Soc. 2003, 125, 7496; (b) Kim, Y. W.; Lovering, A.
L.; Chen, H.; Kantner, T.; McIntosh, L. P.; Strynadka, N. C.; Withers, S. G. J. Am.
Chem. Soc. 2006, 128, 2202; (c) Gloster, T. M.; Meloncelli, P.; Stick, R. V.; Zechel,
D.; Vasella, A.; Davies, G. J. J. Am. Chem. Soc. 2007, 129, 2345; (d) Caines, M. E.;
Hancock, S. M.; Tarling, C. A.; Wrodnigg, T. M.; Stick, R. V.; Stutz, A. E.; Vasella,
A.; Withers, S. G.; Strynadka, N. C. Angew. Chem., Int. Ed. 2007, 46, 4474; (e)
Kuntz, D. A.; Tarling, C. A.; Withers, S. G.; Rose, D. R. Biochemistry 2008, 47,
10058; (f) Lammerts van Bueren, A.; Ardèvol, A.; Fayers-Kerr, J.; Luo, B.; Zhang,
Y.; Sollogoub, M.; Blèriot, Y.; Rovira, C.; Davies, G. J. J. Am. Chem. Soc. 2010, 132,
1804; (g) Wu, H. J.; Ho, C.-W.; Ko, T.-P.; Popat, S. D.; Lin, C.-H.; Wang, A. H.-J.
Angew. Chem., Int. Ed. 2010, 49, 337.
19. (a) Hansen, S. U.; Bols, M. J. Chem. Soc., Perkin Trans. 2 2000, 665; (b) Bülow, A.;
Plesner, I. W.; Bols, M. J. Am. Chem. Soc. 2000, 122, 8567; (c) Lohse, A.; Hardlei,
T.; Jensen, A.; Plesner, I. W.; Bols, M. Biochem. J. 2000, 349, 211; (d) Jensen, H.
H.; Lyngbye, L.; Jensen, A.; Bols, M. Chem. Eur. J. 2002, 8, 1218; (e) Sivertsen, A.
C.; Gasior, M.; Bjerring, M.; Hansen, S. U.; López López, O.; Nielsen, N. C.; Bols,
M. Eur. J. Org. Chem. 2007, 1735.
Acknowledgments
We thank the Ministerio de Ciencia e Innovación of Spain
(CTQ2008-01565/BQU) and the Junta de Andalucía (FQM 345) for
financial support. E.M.-C. thanks the Ministerio de Educación for
a FPU fellowship.
Supplementary data
20. (a) Jiang, S.; Rycroft, A. D.; Singh, G.; Wang, X.-Z.; Wu, Y.-L. Tetrahedron Lett.
1998, 39, 3809; (b) Cheng, X.; Khan, N.; Mootoo, D. R. J. Org. Chem. 2000, 65,
2544.
21. Jones, N. A.; Jenkinson, S. F.; Soengas, R.; Fanefjord, M.; Wormald, M. R.; Dwek,
R. A.; Kiran, G. P.; Devendar, R.; Takata, G.; Morimoto, K.; Izumoric, K.; Fleet, G.
W. J. Tetrahedron: Asymmetry 2007, 18, 774.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.026.
References and notes
22. Stone, K. J.; Greenberg, M. M.; Blackstock, S. C.; Berson, J. A. J. Am. Chem. Soc.
1989, 111, 3659.
1. (a) Ma, B.; Simala-Grant, J. L.; Taylor, D. E. Glycobiology 2006, 16, 158R; (b)
Vocadlo, D.; Davies, G. J. Curr. Opin. Chem. Biol. 2008, 12, 539; (c) Davies, G. J.;
Ducros, V. M.-A.; Varrot, A.; Zechel, D. L. Biochem. Soc. Trans. 2003, 31, 523; (d)
Benesova, E.; Markova, M.; Lipovova, P.; Kralova, B. Chem. Listy 2005, 9, 324.
2. (a) Berteau, O.; Bielicki, J.; Kilonda, A.; Delphine Machy, D.; Anson, D. S.; Kenne,
L. Biochemistry 2004, 43, 7881; (b) Osanjo, G.; Dion, M.; Drone, J.; Solleux, C.;
Tran, V.; Rabiller, C.; Tellier, C. Biochemistry 2007, 46, 1022.
23. Mohamadi, F.; Richards, N. G. I.; Guida, W. C.; Liskamp, R.; Canfield, C.; Chang,
G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
24. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. J. Phys. Chem. B
2001, 105, 6474.
25. (a) Saul, R.; Chambers, J. P.; Molyneux, R. J.; Elbein, A. D. Arch. Biochem. Biophys.
1983, 221, 593; (b) Brandi, A.; Cicchi, S.; Cordero, F. M.; Frignoli, R.; Goti, A.;
Picasso, S.; Vogel, P. J. Org. Chem. 1995, 60, 6806.
26. Carmona, A. T.; Popowycz, F.; Gerber-Lemaire, S.; Rodríguez-García, E.; Robina,
I.; Vogel, P. Bioorg. Med. Chem. 2003, 11, 4897.
27. Hadjuk, P. J.; Bures, M.; Praestgaard, V.; Fesik, S. W. J. Med. Chem. 2000, 43,
3443.
28. For pyrrolidine-based iminosugars, see: (a) Robina, I.; Moreno-Vargas, A. J.;
Fernández-Bolaños, J. G.; Fuentes, J.; Vogel, P. Bioorg. Med. Chem. Lett. 2001,
11, 2555; (b) Moreno-Vargas, A. J.; Carmona, A. T.; Mora, F.; Vogel, P.;
Robina, I. Chem. Commun. 2005, 4949; (c) For piperidine-based iminosugars,
see Ref. 12.
3. Hooper, L. V.; Gordon, J. I. Glycobiology 2001, 11, 1R.
4. Ayude, D.; Fernández-Rodríguez, J.; Rodríguez-Berrocal, F. J.; Martínez-Zorzano,
V. S.; De Carlos, A.; Gil, E.; Paez de la Cadena, M. Oncology 2000, 59, 310.
5. Glick, M. C.; Kothari, V. A.; Liu, A.; Stoykova, L. I.; Scanlin, T. F. Biochimie 2001,
83, 743.
6. Willems, P. J.; Gatti, R.; Darby, J. K.; Romeo, G.; Durand, P.; Dumon, J. E.; O’Brien,
J. S. Am. J. Med. Genet. 1991, 38, 111.
7. Hutchinson, W. L.; Johnson, P. J.; Du, M. Q.; Williams, R. Clin. Sci. 1991, 81, 177.
8. (a) Khunnsook, S.; Bean, B.; McGowan, S. R.; Alhadeff, J. A. Biol. Reprod. 2003, 68,
709; (b) Khunnsook, S.; Alhadeff, J. A.; Bean, B. Mol. Hum. Reprod. 2002, 8, 221.