Chain-Branched polyhydroxylated Octahydro-1H-Indoles as potential leads against lysosomal storage diseases

Article abstract of DOI:10.3390/ph12020047

Here, the synthesis and glycosidase inhibition properties of the two first known 3-ethyloctahydro-1H-indole-4,5,6-triols are reported. This study shows the transformation of D-glucose into polyhydroxylated 1-(2-nitrocyclohexane) acetaldehydes, followed by a protocol involving the formation of the azacyclopentane ring. Results of inhibitory potency assays and docking calculations show that at least one of them could be a lead for optimization in the search for compounds that behave like folding chaperones in lysosomal storage diseases.

Full text of DOI:10.3390/ph12020047

pharmaceuticals
Article
Chain-Branched Polyhydroxylated
Octahydro-1H-Indoles as Potential Leads against
Lysosomal Storage Diseases
Juan C. Estévez 1 , Marcos A. González , M. Carmen Villaverde , Yuki Hirokami ,
,2
1,2
2
3
3
1
1,2
1,2,
Atsushi Kato , Fredy Sussman , David Reza
and Ramón J. Estévez
*
1
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares, Universidade de Santiago
de Compostela, 15782 Santiago de Compostela, Spain; juancarlos.estevez@usc.es (J.C.E.);
mar.gonzalez.castro@gmail.com (M.A.G.); fredy.sussman@usc.es (F.S.); david.reza@gmail.com (D.R.)
Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago de
Compostela, Spain; mc.villaverde@usc.es
2
3
Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan;
yuhi@saitama-med.ac.jp (Y.H.); kato@med.u-toyama.ac.jp (A.K.)
*
Correspondence: ramon.estevez@usc.es; Tel.: +34-881815731
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 13 February 2019; Accepted: 19 March 2019; Published: 29 March 2019
Abstract: Here, the synthesis and glycosidase inhibition properties of the two first known
-ethyloctahydro-1H-indole-4,5,6-triols are reported. This study shows the transformation of
3
D-glucose into polyhydroxylated 1-(2-nitrocyclohexane) acetaldehydes, followed by a protocol
involving the formation of the azacyclopentane ring. Results of inhibitory potency assays and
docking calculations show that at least one of them could be a lead for optimization in the search for
compounds that behave like folding chaperones in lysosomal storage diseases.
Keywords: sugars; iminosugars; glycosidase inhibition
1
. Introduction
Iminosugars [1–4] and aminocarbasugars [5–7] are sugar mimics that inhibit a variety of enzymes
of therapeutic interest, including glycosidases and glycosyltransferases. They have been shown
to be lead molecules for the treatment of diseases such as diabetes, viral infections, or lysosomal
storage disorders. Some representative examples are included in Figure 1. Thus, the N-alkylated
®
1
-deoxynojirimycin miglitol (II) (Glyset ) [
8
,
9
] and the aminocarbasugar voglibose (IV) [10
,
11] have
®
been approved for the treatment of type II diabetes [12
for the treatment of Gaucher disease [14
polyhydroxylated indolizidine alkaloid [
,
13], and miglustat (III) (Zavesca ) is prescribed
,
15]. The bicyclic iminosugar castanospermine (
V) [16,17] is a
3
,
18] that can be considered as a conformational restricted
analogue of miglitol. This compound and its derivatives have received considerable attention as
potential antineoplastic and immunosuppressive agents, but, unfortunately, they have shown toxicity
to human cells [19–22]. This led to the study of analogues of these leads aimed at altering their activity
and toxicity profile. Specifically, the polyhydroxylated octahydroindole (VI) and its N-hydroxyalkyl
derivatives were studied as castanospermine analogues where the position of the N-atom was changed,
allowing them to be considered as conformational restricted carbasugars [23,24].
Pharmaceuticals 2019, 12, 47; doi:10.3390/ph12020047
www.mdpi.com/journal/pharmaceuticals

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Figure 1. Selection of iminosugars and aminocarbasugars.
As a contribution to the search for new castanospermine analogues, and as a part of ongoing
project on synthetic application of nitro sugars and related compounds [25], we report herein
the synthesis and biological evaluation of compounds 10 and 13 as novel polyhydroxylated
octahydroindoles bearing an alkyl substituent at the C-3 position. Results of the inhibitory potency
assays and docking calculations have shown that at least one of them could be a lead for optimization
in the search for compounds that behave like folding chaperones, useful as drugs against lysosomal
storage diseases like GM1 gangliosidosis and Morquio B disease [26].
2
. Results
2
.1. Chemical Synthesis
Michael addition of carbonyl synthetic equivalents to electron-deficient nitro alkenes has proven
to be a promising method for the enantioselective synthesis of γ−nitro acids [27
,28]. In this regard,
carbasugar nitro olefins are suitable scaffolds for this and other synthetic purposes [29], although they
are practically unexplored. They include in their structure a preformed carbocyclic ring bearing several
hydroxy substituents in a well-defined spatial orientation and a nitroethylene subunit suitable for
Michael addition of nucleophiles.
In order to apply this methodology, the key six-membered nitroolefin 4b was obtained from
the known nitro sugar
1 [30], according to a protocol developed by us for the synthesis of the first
polyhydroxylated 1-nitrocyclohexane [31].
Removal of the isopropylidene protecting group of 1 under acidic conditions [trifluoracetic acid
(
TFA)/H O] was followed by a potassium carbonate-promoted intramolecular Henry reaction of the
2
resulting ε-nitro aldehyde 3 (the open form of the tricomponent mixture 2 + 3), which was directly
reacted with potassium carbonate in methanol followed by acetic anhydride and a catalytic amount of
dimethoxyaminopyridine (DMAP) in order to promote its dehydration aimed at obtaining the cyclic
nitroolefin 4b, via 4a (Scheme 1).
Scheme 1. Conditions: (
i) TFA/H O, rt, 5 h. (ii) 2% K CO /MeOH, rt, 4 h. (iii) Ac O, DMAP (cat),
2 2 3 2
Et O, rt, 6 h (77%, three steps).
2
Michael addition of butyraldehyde to 4b, catalyzed by pyrrolidine, resulted in an epimeric
mixture of -nitro aldehydes and , which were isolated by column chromatography (Scheme 2).
γ
5
6
The stereochemical outcome of this reaction, which is controlled by the substrate, can be rationalized
on the basis of the Felkin–Ahn rule.

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Scheme 2. Conditions: (i) pyrrolidine, CH Cl , rt, 3 h (50% for 5, 21% for 6); (ii) NaClO /NaH PO ,
2 2 2 2 4
◦
2
-methyl-2-butene, t-BuOH/H O (3:1), rt, 2 h, 88%; (iii) Zn dust, MeOH/AcOH (1:1), 0 C, 14 h (90%
2
for
8
, 85% for 11); (iv) 1 M NaOH/MeOH, rt, 4 h /69% for 9 and for 12); (v) 10% Pd/C, MeOH, H2
(1 atm) (87% for 10, 88% for 13).
The structure of compounds
5 and 6 were unambiguously identified by X-ray experiments
(Figure 2). In the latter case this was corroborated by the X-ray structure of the corresponding
carboxylic acid 7, which was obtained by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation
of 6.
(
5)
(7)
Figure 2. X-ray structures of compounds 5 and 7.
Finally, γ−nitroaldehydes
octahydroindoles 10 and 13 (Scheme 2). Reaction of compound
h directly provided the corresponding octahydroindole , by the reduction of the nitro group to amino,
followed by a spontaneous reductive amination of the resulting γ−amino aldehyde. Basic hydrolysis
of with NaOH resulted in a selective hydrolysis of its acetate subunits to give derivative . Catalytic
hydrogenation of gave the trihydroxylated octahydroindole 10. On the other hand, the epimeric
octahydroindole 13 was similarly obtained from γ−nitro aldehyde 6 via compounds 11 and 12.
5
and
6
were satisfactorily converted into the corresponding
5
with Zn dust in an acidic media for 14
8
8
9
9

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2
.2. Glycosidase Inhibition Assays
Table 1 displays results of the inhibition of a variety of glycosidases by iminosugars 10 and 13
as the inhibition percentage at 1000 M. As seen from this table, these compounds display by far the
,
µ
highest inhibition activity against bovine
levels under 50%.
β-galactosidase (amongst all enzymes assayed), albeit at
Table 1. Data on inhibition of various glycosidases by compounds 10 and 13.
Enzyme
Compound 10
Compound 13
Miglitol (IC )
50
α-Glucosidase
Yeast
a
a
16%
0%
4.1%
19.5%
70 µM
0.17 µM
>1000 µM
a
a
Rice
0%
a
a
Rat intestinal maltase
0%
β-Glucosidase
Almond
Bovine liver
a
5% a
21.7%
12%
0%
>1000 µM
>1000 µM
a
a
α-Galactosidase
Coffee beans
a
0% a
21.7%
15.9%
>1000 µM
a
a
0
%
β-Galactosidase
Bovine liver
Lactase
a
a
45.6%
0.6%
35.8%
7.4%
>1000 µM
>1000 µM
a
a
α-Mannosidase
Jack bean
0% a
0% a
0% a
2.3% a
12.9% a
0% a
β-Mannosidase
Snail
>1000 µM
>1000 µM
803
α-L-Fucosidase
Bovine kidney
7.5% a
0% a
α-L-Rhamnosidase
Penicillium decumbens
β-Glucronidase
E.coli
18.1% a
0% a
12.3% a
0% a
>1000 µM
131
α,α-Trehalase
Porcine kidney
Amyloglucosidase
Aspergillus niger
0% a
0% a
>1000 µM
a
inhibition % at 1000 µM.
2
.3. Docking Studies
To further shed some light on the differences between the inhibition activities of these compounds
for lysosomal -galactosidase ( -Gal), we performed docking simulations that included the above
β
β
mentioned compounds 10 and 13, as well as galactose, a catalytic product of this enzyme. We
included this latter compound as a point of reference, since the structure of its complex with human
β-galactosidase (with whom the bovine variant shares a highly conserved binding domain) is known.
The docking results for these compounds are summarized in Table 2. This table lists the scoring
value for the top-scoring poses obtained in three docking calculations that differ in the options used
in this work, which include “early termination search”, “diverse solutions”, and docking runs that
allow for active site side chain flexibility (see methods section for details). The table also includes the
hydrogen bond pattern between the ligands and some residues of the protein.

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Table 2. Results of the docking simulations and score values for the top pose of each run.
Fitness & Search Options a
Diverse Solutions
Ligand
Protein Residue HB
Early Termination
CHEM GOLD PLP
Side Flexible Search
CHEM GOLD
PLP
52.84
3-OH
3-OH
CHEM GOLD
PLP
PDB
-
3-OH
3-OH
6-OH
4-OH
2-OH
Galactose score
Tyr83(OH)
Ala128(N)
21.88
3-OH
54.49
3-OH
3-OH
6-OH
4-OH
2-OH
55.63
3-OH
18.57
50.17
3-OH
12.57
3-OH
3-OH
53.60
42.29
3-OH
3-OH
4-OH
6-OH
3-OH
Glu129(OE1)
Glu129(OE2)
Asn187(ND2)
Asn187(OD)
Glu188(OE1)
Glu188(OE2)
Glu268(OE1)
Glu268(OE2)
Tyr333(OH)
6-OH
4-OH
2-OH
4,6-OH 6-OH
4-OH
4-OH
2-OH
4,6-OH 4,6-OH 4-OH
4-OH
2-OH
4-OH
2-OH
4-OH
2-OH
6-OH
2-OH
Galactose
2-OH
1-OH
1-OH
1-OH
1-OH
2-OH
2-OH
6-OH
44.31
6-OH
1-OH
1-OH
1-OH
1-OH
1-OH
2-OH
2-OH
6-OH
45.78
6-OH
1-OH
1-OH
1,2-OH 1-OH
1-OH
2-OH
2-OH
6-OH
27.58
6-OH
6-OH
6-OH
N
2-OH
2-OH
6-OH
52.90
5-OH
5-OH
5-OH
2-OH
2-OH
2-OH
6-OH
2-OH
6-OH
49.82
6-OH
6-OH
6-OH
N
2-OH
6-OH
27.19
6-OH
2-OH
6-OH
44.25
5-OH
6-OH
38.17
6-OH
6-OH
6-OH
N
1
0 score
20.47
6-OH
6-OH
Tyr83(OH)
Ile126(O)
Ala128(N)
Glu129(OE1)
Glu129(OE2)
Asn187(ND2)
Asn187(OD)
Glu188(OE1)
Glu188(OE2)
Glu268(OE1)
Glu268(OE2)
Tyr333(OH)
N
N
6-OH
N
N
6-OH
4-OH
N
1
0
5-OH
5,6-OH 5-OH
5-OH
5,6-OH
5,6-OH 4-OH
5,6-OH
4,5-OH
4,5-OH 4,5-OH 4,5-OH 4,5-OH
4,5-OH 4,5-OH
4-OH
4-OH
4-OH
4-OH
4-OH
6-OH
6-OH
N
6-OH
N
1
3 score
25.37
46.53
5-OH
43.62
25.53
5,6-OH 5-OH
45.76
43.10
17.65
6-OH
56.23
39.34
6-OH
Tyr83(OH)
Ile126(O)
Ala128(N)
5-OH
6-OH
6-OH
4-OH
5-OH
Glu129(OE1)
Glu129(OE2)
Asn187(ND2)
Asn187(OD)
Glu188(OE1)
Glu188(OE2)
Glu268(OE1)
Glu268(OE2)
Tyr333(OH)
6-OH
6-OH
6-OH
6-OH
N
N
5-OH
1
3
6-OH
6-OH
5-OH
5-OH
4-OH
4-OH
4-OH
N
4-OH
4,5-OH 4-OH
5-OH
4-OH
4,5-OH 4,5-OH
4,5-OH
5-OH
6-OH
4-OH
5-OH
4,5-OH 4-OH
4-OH
6-OH
6-OH
6-OH
a
The color codes in this table are used to indicate the number of runs that share the same top exit pose. See text
below for further explanation.
In this table we have used a color code for every scoring value as well as the hydrogen bond
pattern. It is used to identify common poses obtained across different docking simulations. Compounds
whose poses share the same color in different docking runs have a similar top exit pose. For instance,
most of the scoring values in the docking results for galactose are colored with a single color (green), to
indicate that there seems to be a consensus on the pose reached by these compounds, which reproduces
the one found in the crystal structure of the β-galactosidase-galactose complex found in Protein Data
Bank (PDB) entry 3HTC (see Figure 3). This outcome serves to validate the docking protocol used for
these calculations. Docking of ligand 10 produces a similar pose in most of the cases (see Figure 4),
colored cyan in Table 2. Compound 13 displays a larger number of different poses than compound 10
,
indicating this latter ligand fits better in the binding site, a result that may underlie its higher inhibitory
activity (see Table 1). Figure 5 shows the poses that result for compound 13 using all the three scoring
functions. Most of the poses obtained for this ligand are un-colored, in order to point out that these are
single binding conformations which do not display comparable poses amongst the different docking
runs. The only pose that is reproduced in more than one run (for this compound) was obtained with
GoldScore and has the largest number of hydrogen bonds.

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(
a)
(b)
Figure 3. (a) Top galactose poses obtained in early termination runs with the various scoring functions
used, superimposed on the one observed in the crystal structure, drawn in yellow. The ChemScore
structure is drawn in red, while the GoldScore and ChemPLP are drawn in black and cyan, respectively.
(
b) Hydrogen bond interactions between the ligand and the protein residues, resulting from the
GoldScore scoring protocol run, observed as well in PDB entry 3THC [32].
(
a)
(b)
Figure 4.
functions used. The ChemScore structure is drawn in red, while the GoldScore and ChemPLP are
drawn in black and cyan, respectively. ( ) Hydrogen bond interactions between the ligand and the
protein residues, resulting from the GoldScore scoring protocol run.
(a) Top poses obtained for compound 10 in early termination runs with the various scoring
b

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(
a)
(
b)
Figure 5. (a) Top poses obtained for compound 13 in early termination runs with the various scoring
functions used. The ChemScore structure is drawn in red, while the GoldScore and ChemPLP are
drawn in black and cyan, respectively. ( ) Hydrogen bond interactions between the ligand and the
b
protein residues, resulting from the GoldScore scoring protocol run.
To further asses the uniqueness of the binding poses for the three ligands used in our studies
we have reviewed the “early termination” protocol results. In this protocol the best pose search is
terminated as soon as a specified number of runs have a similar solution, within a given Root Mean
Square Deviation (RMSD) of each other (see methods section). The results show that the number of
runs increases for the weaker binder (compound 13) as compared to compound 10, indicating a lack
of consensus for the best pose for the former. For instance, compound 13 does not find a consensus
solution with the ChemPLP scoring function in the early termination run. Finally, cluster analysis
results indicate that the latter compound present fewer clusters at the RMSD cut-off used in this study
(
results not shown), an outcome that further validates the hypothesis behind the difference in affinity.
3
. Discussion
This work presents new synthetic routes to polyhydroxylated octahydroindoles with
pharmacological chaperone (PC) therapeutic potential aimed at autosomal recessive diseases like GM1
gangliosidosis and Morquio B, a group of lysosomal storage disorders resulting from the abnormal
metabolism of macro-substances such as glycosphingolipids, glycogen, mucopolysaccharides, and
glycoproteins [33]. This abnormal metabolism may result in neurodegenerative disorders as well as
dwarfism and skeletal abnormalities [34].
These diseases are caused by mutations in the lysosomal β-galactosidase (β-Gal), which are
frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation [26].
PC therapy is a novel approach that uses small molecule ligands of the mutant enzyme to promote the
correct folding and prevent endoplasmic reticulum-associated degradation while promoting trafficking
to the lysosome [33]. The affinity of the leads for the enzyme should not be high since it is desirable that
the activity of the enzyme should be preserved. The range of affinities for these compounds usually

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span from the milli- to the micro-molar level. Some of the efficacious leads are galactose analogues. In
this family of ligands the hydroxyl groups serve to maintain the structural binding specificity. The
compounds studied here (10 and 13) are polyhydroxylated octahydroindoles and hence represent a
departure from the galactose analogue paradigm. The results indicate that these compounds display
inhibitory potency only for β-Gal at a milli-molar level, making them a good starting point for further
development as PCs. The docking calculations performed herein for these compounds have allowed us
to rationalize the small difference in binding between these compounds. As seen above, compound 10
displays a single well defined binding pose, while its epimer 13 loses this pose specificity, an outcome
that may explain the observed binding affinity order.
The β-galactosidase mutations that originate the four phenotypes of the GM1 gangliosidosis and
Morquio B diseases have been identified and have been mapped into the crystallographic structure of
this molecule. Most mutations fall outside the active site and hence will not disturb the ligand–protein
interactions. Actually, one of the phenotypes of this disease does not present any mutations in the
active site. The other three phenotypes each present a single mutation that could affect the hydrogen
bond pattern described above or ligand–protein van der Waals contacts [34]. As a conclusion, the
present work opens new venues for PC therapies other than galactose analogs. It will be desirable
in the future to carry out the binding kinetics and docking studies of our compounds with the few
known β-galactosidase active site mutants in order to advance in the lead compound generation.
4
. Materials and Methods
4
.1. Methods for the Glycosidase Inhibition Studies
The enzymes
-galactosidase (from coffee beans),
-mannosidase (from snails),
Penicillium decumbens), -glucuronidases (from Escherichia coli),
α
-glucosidase (from yeast, rice),
-galactosidase (from bovine liver),
-L-fucosidase (from bovine kidney),
-trehalase (from porcine kidney),
β
-glucosidase (from almonds, bovine liver),
-mannosidase (from jack
-L-rhamnosidase (from
α
β
α
beans),
β
α
α
β
α
,α
amyloglucosidase (from Aspergillus niger), p-nitrophenyl glycosides, and various disaccharides were
purchased from Sigma-Aldrich Co. Brush border membranes were prepared from the rat small
intestine according to the method of Kessler et al. [35], and were assayed at pH 6.8 for rat intestinal
maltase using maltose. For rat intestinal glucosidases and porcine kidney trehalase activities, the
reaction mixture (0.2 mL) contained 25 mM substrate and the appropriate amount of enzyme, and the
◦
◦
incubations were performed for 10 min at 37 C. The reaction was stopped by heating at 100 C for
min. After centrifugation (600 g; 10 min), 0.035 mL of the resulting reaction mixture were added
to 2.1 mL of the Glucose CII-test Wako (Wako Pure Chemical Ind., Osaka, Japan). The absorbance at
05 nm was measured to determine the amount of the released D-glucose. Other glycosidase activities
3
5
were determined using an appropriate p-nitrophenyl glycoside as substrate at the optimum pH of
each enzyme. The reaction mixture (0.2 mL) contained 2 mM of the substrate and the appropriate
amount of enzyme. The reaction was stopped by adding 0.4 mL of 400 mM Na CO . The released
2
3
p-nitrophenol was measured spectrometrically at 400 nm.
4
.2. Docking Protocol
We carried out the docking simulations with the suite of modules resident in the program
GOLD [36]. We scored the poses with three of the scoring resident functions (i.e., GoldScore [37
ChemScore [39–41], and ChemPLP [42]).
,38],
For the docking predictions to our target we used the X-ray structure of human
-Gal) bound to galactose (PDB entry 3THC) [34]. As a first step we selected the catalytic domain
residues 29 to 360) and we cleaned up the target structure discarding alternative conformations and
β-galactosidase
(
(
hβ
adding hydrogen atoms, using the Discovery Studio (DS) modules [43]. We defined the binding site as
including all the -Gal catalytic domain atom residues that lay at 6 Å from the ligand (i.e., -galactose).
We performed the docking simulations using three different approaches in the GOLD “Fitness Search
β
β

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Options” running setup: in the first one we used the “allow early termination” option, which instructs
GOLD to terminate docking of a ligand as soon as a specified number of runs have a similar solution,
within a given RMSD of each other. In the second one we used the “generate diverse solutions” option
in which diversity is reinforced during the ligand mapping stage. This aim is reached by comparing
the RMSD of the current solution against those that have already been generated. If the RMSD is below
the diversity threshold or the maximum of solutions per cluster has been reached, the mapping is
rejected and the process repeated until an acceptable solution is generated. In the last one we used the
“allow early termination” option but leaving flexibility of the side chains of the active site residues
Tyr83, Glu129, Asn187, Glu188, Glu268, and Tyr333. In all cases the docking conformations generated
by the genetic algorithm were evaluated by the all three scoring functions mentioned above. In the
case of galactose, we validated our methodology by searching for poses similar to the crystallographic
one. In the case of ligands 10 and 13 we performed an additional analysis, searching (across all docking
options) for hydrogen bond interactions similar to the ones observed in the galactose complex.
4
.3. Chemical Synthesis Methods
Melting points were determined using a Kofler Thermogerate apparatus and are uncorrected.
Specific rotations were recorded on a JASCO DIP-370 optical polarimeter, infrared spectra on a MIDAC
FTIR spectrophotometer, and nuclear magnetic resonance spectra on a Bruker WM-250 or a Varian
Mercury 300 apparatus. Mass spectra were obtained on a Kratos MS 50 TC mass spectrometer.
Elemental analyses were obtained from the Elemental Analysis Service at the University of Santiago
de Compostela. Thin layer chromatography (tlc) was performed using Merck GF-254 type 60 silica gel
and ethyl acetate/hexane mixtures as eluents; the tlc spots were visualized with Hanessian mixture.
Column chromatography was carried out using Merck type 9385 silica gel. Solvents were purified as
in reference [44].
4
.3.1. (1R,2S,3S)-2-(Benzyloxy)-5-nitrocyclohex-4-ene-1,3-diyl diacetate (4b)
A 2:1 TFA/H O mixture (90 mL) was added to compound (285 mg, 0.88 mmol) and the resulting
mixture was stirred at room temperature for 5 h. The solvents were evaporated under vacuum and
co-evaporated with toluene (3 10 mL). The crude was solved in methanol (20 mL), 2% aq. K CO
9 mL) were added, and the mixture was stirred at room temperature for 4 h. The reaction was then
1
2
×
2
3
(
neutralized with DOWEX 50WX4-50 (previously acidified at pH = 1), filtered and concentrated to
dryness, to provide 4a.
Ac O (1.8 mL, 19.4 mmol) and a catalytic amount of DMAP (30 mg) were added to a solution of
2
crude 4a in Et O (60 mL) and the resulting solution was stirred at room temperature for 12 h. The
2
reaction was then concentrated to dryness and the residue was solved in CH Cl (30 mL) and washed
2
2
with H O (3
×
15 mL). The organic layer was dried (anhydrous Na SO ), filtered and concentrated
2 4
2
to dryness. The residue was purified by flash column chromatography (AcOEt/Hex 1:3) to give
20
compound 4b (237 mg, 77% yield), as an amorphous white solid. [
α
]D : +69.5 (c 2.2, CHCl3).
1
H-RMN (Cl CD, 250 MHz, ppm): 1.96 (s, 3H, CH ); 1.98 (s, 3H, CH ); 2.66–2.77 (m, 1H, H-6); 2.99–3.10
3
3
3
0
m, 1H, H-6 ); 3.81 (dd, J = 6.9 Hz, J = 4.3 Hz, 1H); 4.64 (s, 2H, CH Ph); 5.15 (dt, J = 7.0, J = 5.3 Hz, 1H,
2
(
H-1); 5.51 (tt, J = 4.1 Hz, J = 1.9 Hz, 1H, H-3); 6.99 (d, J = 3.4 Hz, 1H, H-4); 7.17–7.32 (m, 5H, 5xH-Ar).
13
C-RMN (Cl CD, 62.5 MHz, ppm): 20.8 (CH ); 20.9 (CH ); 27.4 (CH ); 67.9 (CH); 68.7 (CH); 73.7
3
3
3
2
(
(
CH ); 75.1 (CH); 127.7 (CH); 127.8 (2xCHAr); 128.2 (CHAr); 128.6 (2xCHAr); 137.4 (C); 148.2 (C); 169.7
2
−
CO); 170.0 (CO). IR (ν, cm ): 1747 (s, C=O); 1528 (m, NO ), 1340 (m, NO ). HRMS (ESI ): calculated
2 2
1
+
+
for C H NNaO [M + Na] : 372.1054. Found: 372.1061.
17
19
7
4
.3.2. (1R,2S,3S,4R,5S)-2-(Benzyloxy)-5-nitro-4-((S)-1-oxobutan-2-yl)cyclohexane-1,3-diyl diacetate (5)
and (1R,2S,3S,4R,5S)-2-(benzyloxy)-5-nitro-4-((R)-1-oxobutan-2-yl)cyclohexane-1,3-diyl diacetate (6)
n-Butanal (211 L, 2.34 mmol) and pyrrolidine (99 L, 1.21 mmol) were added to a solution
µ
µ
of nitroolefin 4b (157 mg, 0.45 mmol) in CH Cl (4 mL). The resulting solution was stirred at room
2
2

Pharmaceuticals 2019, 12, 47
10 of 14
temperature for 3 h and then was concentrated to dryness. Flash column chromatography of the
residue (EtOAc/hexane 1:4) provided compound (98 mg, 50% yield, white solid) and its epimer
5
6
(21% yield, clear gum).
◦
]D20
◦
17.3 (c 1.5, CHCl ). H–RMN (Cl CD,
3 3
1
Compound
2
5
. m.p.: 112.5–114.0 C (EtOAc/Hex). [
α
=
−
50 MHz, ppm): 0.88 (t, J = 7.3 Hz, 3H, CH2CH3); 1.13–1.33 (m, 2H, CH2CH ); 1.74 (s, 3H, CH ); 1.92
s, 3H, CH ); 1.75–2.13 (m, 2H, H-6+H-6 ); 2.63 (dt, J = 4.2 Hz; J = 12.2 Hz, 1H, H-4); 2.92 (td, J = 1.9 Hz;
3
3
0
(
3
J = 11.5 Hz, 1H, H-2a); 3.54 (t, J = 9.4 Hz, 1H, H-2); 4.54 (ABq, J = 11.4 Hz, 2H, CH Ph); 4.61–4.93 (m,
2
1
3
3
6
H, H-1 + H-3 + H-5); 7.12–7.28 (m, 5H, 5xAr-H); 9.44 (d, J = 1.1 Hz, 1H, H-C=O). C-RMN (Cl CD,
3
2.5 MHz, ppm): 13.5 (CH ); 16.0 (CH ); 20.6 (CH3); 20.9 (CH ); 33.8 (CH ); 44.0 (CH); 52.3 (CH); 69.1
3
2
3
2
(
1
CH); 70.3 (CH); 75.1 (CH ); 81.5 (CH); 82.2 (CH); 127.7 (2xCH-Ar); 128.0 (CH-Ar); 128.6 (2xCH-Ar);
2
−
37.6 (C); 169.1 (CO); 169.8 (CO); 200.1 (CO). IR (ν, cm ): 1745 (s, C=O); 1556 (s, NO ), 1369 (s, NO ).
2 2
1
+
+
MS-ESI (m/z, %): 444.1 (100, [M + Na] ). EA: Calculated for C H NO : C, 59.85; H, 6.46; N, 3.32.
21
27
8
Found: C, 59.84; H, 6.54; N, 3.16.
]D20
=
−
1.9 (c 1.0, CHCl ). H-RMN (Cl CD, 250 MHz, ppm): 0.89 (t, J = 7.1 Hz, 3H,
◦
1
Compound
6. [α
3
3
-
CH2CH3); 1.15–1.52 (m, 2H, -CH2CH ); 1.90 (s, 3H, CH ); 1.91 (s, 3H, CH ); 1.74–2.07 (m, 2H, H-6 +
3 3 3
H-6 ); 2.51 (dt, J = 12.1 Hz, J = 4.41 Hz,1H, H-4); 2.75–2.85 (m, 1H, H-2a); 3.58 (t, J = 9.2 Hz, 1H, H-2);
0
4
9
.52-4.67 (m, 3H, CH Ph + H-5); 4.81 (ddd, J = 4.6 Hz; J = 9.5 Hz; J = 11.6 Hz, 1H, H-1); 5.01 (dd, J
=
3,2
2
13
.2 Hz; J3,4 = 11.1 Hz, 1H, H-3); 7.14–7.30 (m, 5H, 5xAr-H); 9.44 (s, 1H, H-C=O). C-RMN (Cl CD, 62.5
3
MHz, ppm): 13.2 (CH ); 17.4 (CH ); 20.9 (CH ); 21.0 (CH ); 33.9 (CH ); 43.5 (CH); 51.5 (CH); 70.2 (CH);
3
2
3
3
2
7
(
0.5 (CH); 75.2 (CH ); 80.6 (CH); 81.9 (CH); 127.6 (2xCH-Ar); 128.0 (CH-Ar); 128.6 (2xCH-Ar); 137.8
2
−
C); 169.8 (2xCO); 201.3 (CO). IR (ν, cm ): 1752 (s, C=O); 1718 (s, C=O), 1555 (s, NO ), 1372 (s, NO ).
2 2
1
+
+
HRMS (ESI ): Calculated for C H NNaO [M + Na] : 444.1629. Found: 444.1623.
21
27
8
4
.3.3. (R)-2-((1R,2S,3S,4R,6S)-2,4-Diacetoxy-3-(benzyloxy)-6-nitrocyclohexyl)butanoic acid (7)
-Methyl-2-butene (115
2
µ
L, 1.09 mmol), NaH PO .2H O (50 mg, 0.32 mmol) and NaClO (13.5
2
4
2
2
◦
mg, 0.15 mmol) were added to a 0 C cooled solution of aldehyde
6
(115 mg, 0.27 mmol) in a 5:2
t
t
BuOH/H O mixture (7 mL). The reaction was then stirred at room temperature for 2 h, the BuOH
2
was removed under vacuum, and the resulting mixture was extracted with EtOAc (3
×
10 mL). The
organic layers were dried (anhydrous Na SO ), filtered, and the solvents were removed under vacuum.
2
4
The residue was submitted to flash column chomathography (EtOAc/Hexane 1:2) to give compound
7
◦ ◦
105 mg, 88% yield), as a solid mp: 146.8-148.1 C (EtOAc/Hexane). [α]D = +10.8 (c 1.4, CHCl ).
3
20
(
1
H-RMN (Cl CD, 250 MHz, ppm): 0.92 (t, J = 7.2 Hz, 3H, CH CH ); 1.38–2.10 (m, 2H, CH CH ); 1.94
3
2
3
2
3
0
s, 3H, CH ); 1.98 (s, 3H, CH ); 2.18–2.24 (m, 2H, H-5 + H-5 ); 2.59 (dt, J = 4.6 Hz; J = 12.3 Hz, 1H, H-1);
3 3
(
2
.80 (td, J = 1.9 Hz; J = 11.2 Hz, 1H, H-2a); 3.61 (t, J = 9.1 Hz, 1H, H-3); 4.57–4.70 (m, 3H, CH Ph +
2
H-6); 4.92 (tdd, J = 11.5 Hz, J = 8.8 Hz, J = 5.1 Hz, 1H, H-2); 5.15 (dd, J = 9.0 Hz; J = 11.2 Hz, 1H, H-4);
13
7
.20–7.36 (m, 5H, 5xAr-H); 9.24 (sa, 1H CO H). C-RMN (Cl CD, 62.5 MHz, ppm): 13.0 (CH ); 21.0
2 3 3
(2xCH ); 21.5 (CH ); 34.3 (CH ); 45.2 (CH); 45.6 (CH); 70.3 (CH); 70.6 (CH); 74.9 (CH ); 82.0 (CH); 82.2
3 2 2 2
(
CH); 127.8 (2xCH-Ar); 127.9 (CH-Ar); 128.6 (2xCH-Ar); 137.9 (C); 169.8 (CO); 169.9 (CO); 177.5 (CO).
−
1
+
IR (ν, cm ): 1746 (s, C=O); 1711 (s, C=O); 1557 (s, NO ); 1370 (s, NO ). HRMS (ESI ): Calculated for
C H NNaO [M + Na] : 460.1578. Found: 460.1555.
2
2
+
21
27
9
4
.3.4. (3S,3aR,4S,5S,6R,7aS)-5-(Benzyloxy)-3-ethyloctahydro-1H-indole-4,6-diyl diacetate (8)
◦
Zinc dust (969 mg, 14.82 mmol) was added to a 0 C cooled solution of aldehyde
5
(250 mg, 0.59
◦
mmol) in a 1:1 MeOH/AcOH mixture (15 mL) and the resulting suspension was stirred at 0 C for 14
h. The reaction was filtered through a celite pad, which was eluted with methanol, and the filtrate was
concentrated to dryness. The residue was disolved in Cl CH (15 mL) and was washed with saturated
2
2
aq. NaHCO (2x10 mL). The organic layer was dried (anhydrous Na SO ), filtered, and concentrated
3
2
4
to dryness under vacuum. Flash column chromathography of the resulting oil (CH Cl /MeOH 9:1)
2
2
20
provided compound
8
(206 mg, 90% yield,), as a white amorphous solid. [
α
]D
=
−
10.8 (c 1.8, CHCl3).

Pharmaceuticals 2019, 12, 47
11 of 14
1
0
H-RMN (Cl CD, 250 MHz, ppm): 0.77 (t, J = 7.4 Hz, 3H, CH2CH3); 1.09–1.49 (m, 5H, H-3 + H-7 + H-7
3
+
1
CH2-CH ); 1.90 (s, 3H, CH ); 1.91 (s, 3H, CH ); 2.06 (bs, 1H, NH); 2.27 (ddd, J = 3.4 Hz, J = 5.1 Hz, J =
3 3 3
1.7 Hz, 1H, H-3a); 2.65–2.74 (m, 2H, H-2 + H-2 ); 3.21 (t, J = 10.0 Hz, 1H, H-7a); 3.53 (t, J = 9.3 Hz, 1H,
0
H-5); 4.58 (ABq, J = 11.6 Hz, 2H, CH Ph); 4.89 (ddd, J = 5.1 Hz, J = 9.5 Hz, J = 11.3 Hz, 1H, H-6); 4.99
2
13
(
dd, J = 9.1 Hz, J = 11.1 Hz, 1H, H-4); 7.16–7.28 (m, 5H, 5x-Ar-H). C-RMN (Cl CD, 62.5 MHz, ppm):
3
1
2.0 (CH ); 21.0 (2xCH ); 21.9 (CH ); 35.3 (CH ); 39.7 (CH); 51.4 (CH ); 52.0 (CH); 54.5 (CH); 71.1 (CH);
3
3
2
2
2
7
3.5 (CH); 74.7 (CH ); 84.8 (CH); 127.4 (2xCH-Ar); 127.5 (CH-Ar); 128.3 (2xCH-Ar); 138.3 (C); 169.8
2
−
CO); 169.9 (CO). IR (ν, cm ): 3205 (b, NH), 1740 (s, C=O). HRMS (ESI ): Calculated for C H NO
21 30 5
1
+
(
[
+
M + H] : 376.2118. Found: 376.2122.
4
.3.5. (3S,3aR,4S,5S,6R,7aS)-5-(Benzyloxy)-3-ethyloctahydro-1H-indole-4,6-diol (9)
A 1 M NaOH methanolic solution (1 mL, 1 mmol) was added to a solution of amine
.08 mmol) in methanol (2 mL) and the resulting solution was stirred at room temperature for 4 h.
8
(30 mg,
0
Half of the methanol was then evaporated, and the reaction was neutralized with saturated NH Cl
4
solution and extracted with ethyl acetate (3
×
10 mL). The joined organic layers were dried (anhydrous
Na SO ), filtered, and concentrated to dryness under vacuum. Purification of the resulting oil by flash
2
4
column chromathography (CH Cl /MeOH 7:1) gave compound
9
(16 mg, 69% yield), as a clear gum.
2
2
20
1
[
α]D = +3.0 (c 1.0, CH OH) H-RMN (CD OD, 250 MHz, ppm): 0.88 (t, J = 7.3 Hz, 3H, CH -CH3);
3
3
2
1
(
.19–1.58 (m, 4H); 1.92–2.07 (m, 2H); 2.18 (ddd. J = 3.4 Hz, J = 5.0 Hz, J = 11.7 Hz, 1H, H-3a); 2.68–2.88
0
m, 2H, H-2 + H-7a); 3.15 (t, J = 8.8 Hz, 1H); 3.31–3.46 (m, 1H); 3.54–3.64 (m, 1H, H-6); 4.83 (s, 2H,
13
CH Ph); 7.18–7.31 (m, 3H, 3xAr-H); 7.37–7.42 (m, 2H, 2xAr-H). C-RMN (CD OD, 62.5 MHz, ppm):
2
3
1
7
2.7 (CH ); 28.1 (CH ); 36.7 (CH ); 45.3 (CH); 52.3 (CH ); 54.8 (CH); 59.4 (CH); 72.7 (CH); 75.8 (CH);
3 2 2 2
−
1
6.6 (CH); 90.4 (CH); 128.5 (CH-Ar); 129.2 (4xCH-Ar); 140.5 (C). IR (ν, cm ): 3371 (b, NH + OH).
+
+
HRMS (ESI ): Calculated for C H NO , [M + H] : 292.1907. Found: 292.1902
17
26
3
4
.3.6. (3S,3aR,4S,5S,6R,7aS)-3-Ethyloctahydro-1H-indole-4,5,6-triol (10)
Pd/C 10% (15 mg) was added to a deoxygenated solution of amine
9 (15 mg, 0.05 mmol) in
methanol (2 mL) and the resulting suspension was stirred at room temperature for 6 h, under a
hydrogen atmosphere (P = 1 atm). The reaction was then filtered through a celite pad, eluted with
methanol, and the filtrate concentrated to dryness. Flash column chromathography of the residue
2
0
(
+
CH Cl /MeOH 5:1) allowed isolation of compound 10 (9 mg, 87% yield) as a yellow oil. [
α
]D
=
2
2
1
16.4 (c 0.9, MeOH). H-RMN (CD OD, 250 MHz, ppm): 0.91 (t, J = 7.4 Hz, 3H, CH2CH3); 1.22–1.39
3
(
3
m, 1H); 1.49–1.69 (m, 2H); 1.91–2.20 (m, 2H), 2.28 (dt, 1H, J = 11.5 Hz, J = 4.2 Hz, 1H); 2.99–3.19 (m,
H), 3.40–3.64 (m, 3H). 13C-RMN (CD OD, 62,5 MHz, ppm): 12.5 (CH ); 28.0 (CH ); 33.5 (CH ); 39.2
3
3
2
2
−
1
(CH); 42.6 (CH); 62.7 (CH ); 66.9 (CH); 72.0 (CH); 75.4 (CH); 81.2 (CH). IR (
ν
, cm ): 3405 (b, NH +
2
+
OH). HRMS (ESI+): Calculated for C H NO [M + H] : 202.1438. Found: 202.1438.
1
0
20
3
4
.3.7. (3R,3aR,4S,5S,6R,7aS)-5-(Benzyloxy)-3-ethyloctahydro-1H-indole-4,6-diyl diacetate (11)
Starting from aldehyde (100 mg, 0.24 mmol) and following the same procedure as for compound
6
20
8
, compound 11 was obtained (76 mg, 85% yield), as a white amorphous solid. [
α
]D = +9.3 (c 1.8,
1
CHCl ). H-RMN (Cl CD, 250 MHz, ppm): 0.79–0.93 (m, 4H), 0.97–1.12 (m, 1H), 1.18–1.51 (m, 4 H),
3
3
1
1
.76–1.83 (m, 1H), 1,93 (s, 3H, CH ), 1,96 (s, 3H, CH ), 2.35 (ddd, J = 11.6 Hz, J = 5.0 Hz, J = 3.7 Hz,
3 3
H), 2.80 (td, J = 11.2 Hz, J = 3.3 Hz, 1H), 3.18–3.24 (m, 1H), 3.53–3.59 (m, 1H), 4.62 (ABq, J = 11.6 Hz,
2
7
H, CH Ph), 4.93 (ddd, J = 11.4 Hz, J = 9.6 Hz, J = 4.9 Hz, 1H), 5.13 (dd, J = 11.6 Hz, J = 8.7 Hz, 1H),
2
13
.21–7.33 (m, 5H, 5xAr-H). C-RMN (Cl CD, 62.5 MHz, ppm): 12.1 (CH ); 21.1 (CH ); 21.2 (CH ); 22.1
3
3
3
3
(
(
CH ); 35.4 (CH ); 39.9 (CH); 51.5 (CH ); 52.1 (CH); 54.6 (CH); 71.3 (CH); 73.7 (CH); 74.9 (CH ); 85.0
2 2 3 2
−
1
CH); 127.6 (2xCH); 127.7 (2xCH); 128.5 (CH); 138.4 (C); 170.0 (CO); 170.1 (CO). IR (ν, cm ): 3220 (b,
+
+
NH); 1741 (s, C=O). HRMS (ESI ): Calculated for C H NO [M + H] : 376.2118. Found: 376.2122.
21
30
5

Pharmaceuticals 2019, 12, 47
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4
.3.8. (3R,3aR,4S,5S,6R,7aS)-5-(Benzyloxy)-3-ethyloctahydro-1H-indole-4,6-diol (12)
Reaction of amine 11 (54 mg, 0.14 mmol) under the same conditions as for the preparation of amine
20
1
9
gave compound 12 (29 mg, 69% yield), as a clear gum. [α]D = +18.2 (c 2.1, CH OH). H-RMN
3
(
CD OD, 250 MHz, ppm): 0.92 (t, J = 7.3 Hz, 3H, CH2CH3); 1.13 (tq, J = 13.4 Hz, J = 7.0 Hz, 1H,
3
H
-CHCH ); 1.27–1.48 (m, 1H,
H
-CHCH ); 1.61 (td, J = 11.4 Hz, J = 7.6 Hz, 1H, H-7); 1.76 (dtd, J = 14.9
3
3
0
Hz, J = 7.5 Hz, J = 3.3 Hz,1H, H-7 ); 2.11–2.25 (m, 2H, H-3 + H-3a); 2.61 (td, J = 11.6 Hz, J = 3.5 Hz, 1H,
H-7a + H-2); 2.77 (dd, J = 11.5 Hz, J = 3.2 Hz, 1H); 3.19–3.27 (m, 2H, H-2 + H-5); 3.52–3.65 (m, 2H);
13
4.87–4.91 (m, 2H, CH Ph); 7.22–7.37 (m, 3H, 3xAr-H), 7.46–7.49 (m, 2H, 2xAr-H). C-RMN (CD OD,
2 3
6
2.5 MHz, ppm): 12.4 (CH ); 23.1 (CH ); 38.7 (CH ); 41.1 (CH); 52.1 (CH ); 55.2 (CH); 56.0 (CH); 71.8
3
2
2
2
−
1
(
CH); 73.1 (CH); 76.5 (CH); 91.1 (CH ); 128.5 (2xCH); 129.2 (3xCH); 140.6 (C). IR (
ν
, cm ): 3371 (b, NH
2
+
+
OH). HRMS (ESI+): Calculated forC H NO [M + H] : 292.1907. Found: 292.1909.
17
26
3
4
.3.9. (3R,3aR,4S,5S,6R,7aS)-3-ethyloctahydro-1H-indole-4,5,6-triol (13)
Following the same procedure as for amine
9
, amine 12 (18 mg, 0.06 mmol) gave compound 13 (11
20
1
mg, 88% yield), as a yellow solid. [
α
]D = +36.4 (c 1.1, CH OH). H-RMN (CD OD, 250 MHz, ppm):
3
3
0
3
6
.90 (t, 3H, J = 7.3 Hz, CH2CH3); 1.10–1.43 (m, 3H); 1.81–1.96 (m, 2H); 2.11–2.46 (m, 4H); 3.13–3.67 (m,
13
H). C-RMN (CD OD, 62.5 MHz, ppm): 12.1 (CH ); 24.1 (CH ); 35.2 (CH); 38.3 (CH ); 52.5 (CH);
3
3
2
2
−
1
+
3.6 (CH ); 63.7 (CH); 71.5 (CH); 72.2 (CH); 82.3 (CH). IR (
ν
, cm ): 3408 (b, NH + OH). HRMS (ESI ):
2
+
Calculated for C H NO [M + H] : 202.1438. Found: 202.1438.
X-ray crystal structure for compound 5, X-ray crystal structure for compound
NMR spectra for compounds 4b, 5, 6, 7, 8, 9, 10, 11, 12, and 13 are in Supplementary Materials.
10
20
3
7 C
, 1H NMR and 13
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/12/2/47/s1.
Author Contributions: R.J.E., M.C.V. and A.K. conceived the original concept; R.J.E. and J.C.E. developed the
synthetic methodology; M.C.V. and F.S. performed the docking studies; R.J.E., A.K., F.S., and M.C.V. interpreted
the resulting data; J.C.E., M.G., and Y.H. performed experimental work; R.J.E., A.K., F.S. and M.C.V. performed
writing—original draft preparation; R.J.E., A.K., F.S., and M.C.V. performed writing—reviewing and editing; R.J.E.
and A.K. were responsible for supervision, project administration, and funding acquisition.
Funding: This work has received financial support from the Spanish Ministry of Science and Innovation
(
CTQ2009-08490), the Xunta de Galicia (Centro Singular de Investigación de Galicia, accreditation 2016–2019,
ED431B 2018/13; Project CN2011/037 and Project GRC2014/040), and the European Union (European Regional
Development Fund-ERDF). It has also received a Grant-in-Aid for Scientific Research (C) from the Japanese Society
for the Promotion of Science (JSPS KAKENHI Grant Number JP17K08362) (AK).
Conflicts of Interest: The authors declare no conflict of interest.
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