Divergent synthesis of 4-epi-fagomine, 3,4-dihydroxypipecolic acid, and a dihydroxyindolizidine and their β-galactosidase inhibitory and immunomodulatory activities

Article abstract of DOI:10.1021/jo400448p

A divergent asymmetric synthesis of the titled iminosugars has been formulated starting from a chiral homoallyl alcohol as the versatile intermediate. The homoallyl alcohol was prepared by a highly diastereoselective Barbier reaction on a d-glucose-derived aldehyde. The protection of its hydroxyl function followed by reductive ozonolysis of the olefin and a subsequent one-pot three-step protocol involving a Staudinger reaction, reductive amination, and benzyloxy carbonyl protection yielded an important bicyclic furanopiperidine derivative. This was converted to the target compounds by following standard reactions. Among the synthesized compounds, 4-epi-fagomine (2b) was the best β-galactosidase inhibitor, and it also prevented LPS-mediated activation of Raw 264.7 macrophage cells. Its congener, 3,4-dihydroxypipecolic acid (4b) also showed similar trends in its cytokine- and enzyme-inhibitory properties at a low concentration (10 μM) but was proinflammatory at higher concentrations. The bicyclic compound dihydroxyindolizidine (21) reduced the proinflammatory cytokine (IL-1β and TNF-α) levels in the LPS-activated Raw 264.7 cells without showing any enzyme-inhibition activity.

Full text of DOI:10.1021/jo400448p

Article
pubs.acs.org/joc
Divergent Synthesis of 4-epi-Fagomine, 3,4-Dihydroxypipecolic Acid,
and a Dihydroxyindolizidine and Their β‑Galactosidase Inhibitory
and Immunomodulatory Activities
K. S. Ajish Kumar, J. S. Rathee, M. Subramanian, and S. Chattopadhyay*
Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400085, India
S
* Supporting Information
ABSTRACT: A divergent asymmetric synthesis of the titled
iminosugars has been formulated starting from a chiral
homoallyl alcohol as the versatile intermediate. The homoallyl
alcohol was prepared by a highly diastereoselective Barbier
reaction on a D-glucose-derived aldehyde. The protection of its
hydroxyl function followed by reductive ozonolysis of the
olefin and a subsequent one-pot three-step protocol involving
a Staudinger reaction, reductive amination, and benzyloxy
carbonyl protection yielded an important bicyclic furanopiper-
idine derivative. This was converted to the target compounds
by following standard reactions. Among the synthesized
compounds, 4-epi-fagomine (2b) was the best β-galactosidase inhibitor, and it also prevented LPS-mediated activation of
Raw 264.7 macrophage cells. Its congener, 3,4-dihydroxypipecolic acid (4b) also showed similar trends in its cytokine- and
enzyme-inhibitory properties at a low concentration (10 μM) but was proinflammatory at higher concentrations. The bicyclic
compound dihydroxyindolizidine (21) reduced the proinflammatory cytokine (IL-1β and TNF-α) levels in the LPS-activated
Raw 264.7 cells without showing any enzyme-inhibition activity.
INTRODUCTION
■
Aliphatic nitrogen-containing piperidines and pyrrolidines with
polyhydroxy pendants, also known as iminosugars, have
attracted significant interest among chemists and biologists.1
Because of their structural similarity with sugars, polyhydroxy-
lated heterocycles can act as potent substrate mimics for a
variety of glycosidases; hence, they have found importance as
diagnostic and therapeutic agents. Some of the well-known
naturally occurring iminosugars include 1-deoxynojirimycin
(1),2a fagomine (2a),2a isofagomine (3),2a−c and hydroxypipe-
colic acids (4a and 4b)2d as well as their alkyl derivatives such
as compound 5,2e,f castanospermine (6),2g swainsonine (7),2h
and so forth (Figure 1).
Figure 1. Chemical structures of some iminoalditols, bicyclic alkaloids,
and pipecolic acids.
Various stereomers of nojirimycin and its analogues are
naturally occurring. Its trihydroxy congener D-fagomine 2a has
been isolated from the seeds of the Japanese buckwheat
Fagopyrum esculentum austral Moench as well as Castanosper-
mum australe (Leguminosae), whereas two other stereomers 2c
and 2d occur in Xanthocersis zambesiaca leaves and roots.2i The
number and stereochemical disposition of their hydroxyl
groups largely decides their selectivity in inhibiting different
glycosidases as well as their therapeutic potential.3a−d For
example, compounds 6 and 7 and their derivatives possess
antiviral, antitumor, and immunomodulatory activities.4a−d
Likewise, compounds 1, 2a, and 3 show inhibitory activity
against human α-glucosidase, mammalian α-glucosidase/β-
galactosidase, and liver glycogen phoshorylase.2a Interestingly,
non-natural structural isomer 4-epi-fagomine 2b is a good
inhibitor of lysosomal α-galactosidase A in Fabry lympho-
blasts.4e,f Pipecolic acid and its derivatives possess anesthetic,5a
NMDA antagonist,5b anticoagulant,5c and glycosidase inhibitory
activities.5d Additionally, the unnatural, nonproteinogenic
amino acids are potential water-soluble auxiliaries for use in
native chemical ligation (NCL).6 Furthermore, the pipecolic
acid unit is often used as a ring-expanded homologue of proline
for conformational and ligand-binding studies in biologically
active peptides and foldamers.7
Received: September 20, 2012
© XXXX American Chemical Society
A
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Scheme 1. Diversity-Oriented Approach to the Syntheses of Iminosugars
All of these properties make them interesting synthesis
targets. Because of their limited natural occurrence, increasing
the availability of these molecules with different substitution
patterns and stereochemistry would help to establish the
structure−activity relationships that are important for their
biological effects. Hence, a number of different hydroxyl-
substituted and ring-expanded iminosugar analogues were
synthesized over the past decades using carbohydrate and
noncarbohydrate substrates as well as biocatalytic routes.8
However, these target-specific syntheses may not meet the
demand of skeletal and stereochemical diversities required for
discovery of new drugs. More flexible, universal, and efficient
methods leading to several targets with shorter synthesis routes
are desired. Designing a common strategy for the synthesis of
various bioactive molecules always has a vital significance in
organic synthesis. To this end, diversity-oriented synthesis
(DOS)-based protocols that use the same precursor to produce
compounds with both structural and stereochemical diversities
are appealing.9 As a part of our interest in the polyhydroxy
piperidines,10 presently we have attempted to devise such a
DOS strategy for this class of iminosugars.
by a reported procedure involving six steps with chromato-
graphic purification in all of the steps.15 We are routinely
synthesizing 8 on the ∼25 g scale. As shown in Scheme 2, a Zn-
Scheme 2. Synthesis of 4-epi-Fagomine and a Pipecolic Acid
a
Analogue
We envisioned an entity of type A (Scheme 1) as a versatile
intermediate for easy access to these compounds. This design
was surmised while keeping in mind the general substitution
patterns of azasugars. Cleavage of the alkene function of A
would generate azido aldehyde B, which, as such or after
converting to a ketone, can be subjected to reductive amination
to furnish various polyhydroxy piperidine compounds C.
However, the aldehydes derived from the acetonide moiety
can be homologated and subsequently cyclized to afford
bicyclic alkaloid skeleton D. To illustrate the synthesis strategy,
we formulated a diastereoselective synthesis of the type A
intermediate and used it for the concise total syntheses of 4-epi-
fagomine 2b, pipecolic acid analogue 4b, and bicyclic
iminoalditol 18. Among these, pipecolic acid and its derivatives
are attractive targets.11 The glycosidase inhibitory activity of
pipecolic acid derivatives can be tuned by altering the
substitutions, as reflected in the moderate activity of a synthetic
alkyl derivative of the acid.12 Hence, we have synthesized 4b as
a representative compound of this class because very few
syntheses of a dihydroxypipecolic acid are reported.13 Bicyclic
indolizidine alkaloid 18 has been designed as a hybrid of
naturally occurring lentiginosine and swainsonine for studying
its glycosidase inhibitory property.
a
(a) Allyl bromide, Zn, THF, 0 to 20°C, and 7 h. (b) BnBr, NaH,
THF, 0 to 25 °C, and 4 h. (c) (i) O3, CH2Cl2, −78 °C, then PPh3,
−78 to 25 °C, 12 h; (ii) NaBH3CN, MeOH, and AcOH (cat.); (iii)
CbzCl, NaHCO3, MeOH, 0 to 25 °C, and 4 h. (d) O3, CH2Cl2, −78
°C, then Me2S, −78 to 25 °C, and 24 h. (e) (i) PPh3, MeOH, 0 to 25
°C, and 1 h; (ii) NaBH3CN, MeOH, AcOH (cat.), 0 °C, and 2 h, then
25 °C, and 8 h; (iii) CbzCl, NaHCO3, MeOH, 0 to 25 °C, and 6 h. (f)
(i) TFA−H2O (3:2), 0 °C, and 1 h; (ii) NalO4, acetone−water (4:1),
0 °C, and 30 min. (g) NaBH4, MeOH−H2O (9:1), 0 °C, and 20 min.
(h) NaH2PO4, NaClO2, 30% H2O2, CH3CN, 0 to 25 °C, and 7 h. (i)
H2 (80 psi), 10% Pd/C, MeOH, 25 °C, and 12 h.
mediated Barbier-type reaction of aldehyde 8 with allyl bromide
in moist THF afforded homoallylic alcohol 9 as the major
isomer in a 95:5 diastereomeric ratio (determined from the 1H
NMR of the reaction mixture).16 The observed selectivity in
favor of 9 could be due to the weak chelating effect of allyl zinc
with the furanose ring oxygen and carbonyl group of 8.
Danishefsky et al. have reported that the reactive conformation
in a xylose derivative similar to 8 exhibits a dihedral angle of
157.4° between the carbonyl group and the C−O bond, which
makes α-face attack the most favorable in the absence of any
strong chelation.17 This was also validated by the fact that the
RESULTS AND DISCUSSION
■
During the past several years, our group has used the furanose
form of D-glucose as the template in asymmetric synthe-
ses.8g,10,14 The present synthesis started with known azido
aldehyde 8, prepared in 50−57% overall yield from D-glucose,
B
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reaction of the Grignard reagent/allyl trimethyl silyl−BF3 with
a xylose derivative yielded the 5S-isomer predominantly.
Reaction of 9 with BnBr in THF in the presence of NaH as
the base afforded 5-O-benzyl derivative 10 in 76% yield. 5-O-
Benzyl homoallyl substrate 10 was transformed to key bicyclic
compound 11 in a one-pot four-step process in moderate
(25%) yield. This involves the ozonolysis of 10 followed by
reduction of the resultant malozonide and azide functionalities
with PPh3 to an intermediate, which undergoes reductive
amination with NaBH3CN followed by protection with benzyl
chloroformate. To improve the yield of 11, we carried out the
ozonolysis of 10 and reduced the ozonide with Me2S to isolate
aldehyde 12 in 87% yield. Subsequent reduction of the azide
functionality of 12 followed by in situ reductive amino
cyclization and Cbz protection afforded 11 in 65% yield over
three steps. Besides improving the yield of 11, the second
strategy was more advantageous for the following reason. We
found considerable degradation of compounds 9 and 10 on
storage even at a low temperature, which is possibly due to
their azide and olefin functionalities. In contrast to this,
aldehyde 12 is stable and can be stored for >1 month at 0 °C.
The 2D-ROESY study of 11 showed a long-range coupling of
H-5 with H-4 and H-3, revealing its 5R configuration (Figure
S11). This also confirmed the assigned configuration of the
newly generated center of 9.
Catalytic hydrogenation of ester 16 with HCOONH4 over 10%
Pd/C in MeOH under reflux directly furnished amide 17 via
the simultaneous deprotection of the alcohol and amine
functions, saturation of the alkene moiety, and reductive
amino cyclization. Reduction of amide 17 with LiAlH4
proceeded uneventfully to give amine 18. This was further
acetylated to furnish diacetate 19 in 96% yield.
Conformational Studies. It is known that 1-deoxy-
8
castanospermine adopts a C5 (I) conformation, whereas its
8a-epi-isomer takes an 5C8 (II) conformation (Figure 2.).
Figure 2. Conformation of indolizidines and piperidines.
With important precursor 11 in hand, we completed the
synthesis of 4-epi-fagomine 2b. Thus, the 1,2-acetonide
functionality in 11 was carefully deprotected using trifluoro-
acetic acetic acid (TFA)−water (3:2), and the intermediate diol
was subjected to oxidative cleavage with NaIO4 to afford N-
protected amino aldehyde 13, which is a common intermediate
for the synthesis of both 2b and 4b. In view of its instability,
without purification crude amino aldehyde 13 was subjected to
NaBH4 reduction in MeOH−water (9:1) to yield 14 in 40%
yield over three steps from 11. In the final step, catalytic
hydrogenation of 14 under pressure (80 psi) over 10% Pd/C in
MeOH at room temperature afforded 4-epi-fagomine 2b as a
semisolid in 92% yield. The analytical and spectral data of 2b
were found to be in agreement with the reported data.18 For
the synthesis of pipecolic acid derivative 4b, the aldehyde group
in 13 was oxidized with NaClO2, 30% H2O2, and NaH2PO4 to
afford corresponding acid 15 in 67% yield over three steps from
11. Catalytic hydrogenation of 15 as above gave new dihydroxy
pipecolic acid analogue 4b (Scheme 2).12
Hence, it was of interest to establish the conformation of new
indolizidine 18 in solution. As a hybrid of natural lentiginosine
and swainsonine, compound 18 can exist in conformation III or
1
IV. Because the H NMR spectroscopic pattern of 18 did not
provide much conformational information, it was converted to
acetate 19 to analyze its conformation using 2D-COSY
spectroscopy. The ddd at δ 4.85 with J = 12.0, 4.9, and 3.2
1
Hz in the H NMR spectrum of 19 was assigned to its H7
proton. The large coupling constant of 12.0 Hz suggested a
diaxial orientation of the H7a and H6a protons. The small
coupling constant of 4.9 Hz was attributed to an axial−
equatorial coupling between H7a and H6e, whereas the
smallest coupling constant of 3.2 Hz pointed to an axial−
equatorial relation between H7a and H8e protons. Taken
together, the 2D-COSY results revealed that new indolizi-
8
dine(s) 19 (and 18) adopt(s) the C5 conformation (III),
which is a favored conformation of bicyclic-D-iminosugars.
4
From this, the most common C1 conformation was assigned
for 2b and 4b.
The synthesis (Scheme 3) of bicyclic iminoalditol 18
commenced from aldehyde 13, which upon a Wittig−Horner
reaction with triethyl phosphonoacetate in the presence of NaH
in THF afforded E-ester 16 as the only isolated product.19
Although of no consequence to the synthesis, the E geometry
of the ester was confirmed from the 1H NMR spectrum.
Biological Activities. None of compounds 2b, 4b, and 18
(10, 50, 100, 250, and 500 μM) inhibited the proliferation of
the human non-small-cell lung cancer (A549), osteosarcoma
(U2-OS), and histiocytic lymphoma (U937) cells significantly,
as revealed by the MTT reduction assay.20 At a much higher
concentration (5 mM), only 4b showed a 27% inhibition of the
proliferation of A549 cells. Insulin resistance, a major
characteristic of numerous metabolic disorders such as viral
or bacterial infections, diabetes, and cancer, is induced resulting
from alterations in several glycoproteins. Earlier, increased
serum levels of β-galactosidase and β-hexosaminidase were
observed in diabetic rats,21a and these levels were reduced by
insulin.21b These findings ascertained the key roles of these
enzymes in diabetes that was also confirmed in diabetic
patients.21c Hence, we assessed the β-D-galactosidase inhibitory
property of compounds 2b, 4b, and 18 at different
concentrations (1.7, 3.4, 5.1, 6.8, and 8.5 mg/mL), and the
results are summarized in Table 1.
a
Scheme 3. Synthesis of Dihydroxy Indolizidine
a
(a) (Eto)2P(O)CH2CO2Et, NaH, THF, −40 °C, 10 min, −20 °C,
and 5 h. (b) HCOONH4, 10% Pd−C, MeOH, reflux, and 4 h. (c)
LiAlH4, THF, 0 °C to reflux, and 2 h. (d) Ac2O, Py, DMAP, 0 to 30
°C, and 6 h.
C
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almost inactive. In general, monocyclic azasugars resembling D-
galactose are good inhibitors of β-galactosidase.22 Consistent
with this, compounds 2b and 4b showed moderate-to-good β-
galactosidase inhibitory property, whereas bicyclic iminosugar
18 was ineffective. Our results with 4-epi-fagomine 2b and its
oxidized analogue 4b are noteworthy given the reported weak
β-galactosidase inhibition by D-3,4-epi-fagomine and D-3-epi-
fagomine analogues.23a The poor glycosidase inhibition by
bicyclic azasugars was reported previously.23b,c To the best of
our knowledge, the biphasic β-galactosidase inhibitory property
of compounds 4b and 18 is unprecedented among the
azasugars. Only some aliphatic alcohols and the phytoestrogen
genistein were reported to show similar behavior.24a,b
Table 1. Concentration-Dependent β-D-Galactosidase
Inhibitory Property of 2b, 4b, and 18
a
enzyme inhibition concentration (mg/mL)
compound
1.7
3.4
5.1
6.8
8.5
b
b
b
b
b
b
2b
4b
18
33.0
0
32.3
4.9
41.0
24.4
49.3
50.8
b
−8.3
−2.2
−22.1
−5.4
5.9
3.9
−0.4
a
Enzyme inhibition is expressed in percentage, considering the control
value as zero. The data (mean SEM) of three independent
experiments, each repeated three times were analyzed by paired t test
b
and one-way analysis of variance (ANOVA). p < 0.001 compared to
control.
Cytokines such as tumor necrosis factor (TNF)-α and
interleukin (IL)-6 may modulate glucose homeostasis and alter
insulin resistance.25 Hence, the effect of compounds 2b, 4b, and
18 on cytokine production was examined using lipopolysac-
charide (LPS)-treated RAW 264.7 macrophage cells as the
model system. Endotoxins such as LPS induce cytokine
production via macrophage activation to contribute to the
Compound 2b showed a dose-dependent enzyme inhibition
at up to 6.8 mg/mL; beyond this concentration it reached a
plateau. Even at a low concentration (1.7 mg/mL), it showed
good (33%) inhibition that increased to ∼50% at 6.8 mg/mL
(46.3 mM). Compound 4b showed moderate activity (24.4%)
at 5.1 mg/mL (31.7 mM), whereas bicyclic iminoalditol 18 was
Figure 3. Concentration-dependent effect of the test compounds on LPS-stimulated production of Th1 and Th2 cytokines in RAW 264.7
macrophage cells. The cells (untreated or pretreated with different concentrations of test compounds for 1 h) were incubated with LPS (200 ng/mL)
for 16 or 24 h, and the cytokines were analyzed by ELISA. The values are the mean SEM of three independent experiments. #p < 0.001 compared
to normal and *p < 0.05, **p < 0.01, and ***p < 0.001 compared to LPS treatment. The cells that were not treated with LPS or the compounds are
designated as Cntrl.
D
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inflammatory process.26a Under in vivo conditions, LPS
stimulates macrophages that play a central role in immune
response.26b Macrophages express Th1 cytokines TNF-α,
interferon (INF)-γ, and the ILs (IL-6, IL-2, and IL-1β) as
well as Th2 cytokines IL-4 and IL-10. The balance between
Th1 and Th2 cytokines defines the nature of the immune
response.
CONCLUSIONS
■
Overall, chiral homoallylic alcohol 9 was conceived as an
important DOS intermediate and synthesized via a highly
diastereoselective allylation of a D-glucose-derived aldehyde. Its
versatility was established by converting it to two monocyclic
iminosugars (4-epi-fagomine and 4,5-dihydroxypipecolic acid)
and a new bicyclic iminosugar (dihydroxyindolizidine) in 16,
8
Initially we carried out time-dependent experiments to
determine the time point at which the maximum concentration
of the individual cytokines after the LPS treatment is present
(complete data not shown). The optimum time points were 16
h for TNF-α and IL-1β and 24 h for IL-6 and IL-10. LPS
treatment increased the levels of TNF-α (8.8-fold), IL-1β (2-
fold), IL-6 (14.3-fold), and IL-10 (5.2-fold) significantly
compared to the levels produced by the control cells.
Compounds 2b and 18 suppressed an increase in the TNF-α
level by ∼20−22% at all of the doses tested. Compound 4b (10
μM) suppressed an increase in the TNF-α level by 33.7% but
was much less effective at higher concentrations. Compound 2b
showed a dose-dependent suppression of the IL-1β level, with
the maximum reduction (35.2%) at 100 μM. Compound 4b
suppressed the IL-1β level by 33.9% at all of the tested
concentrations. However, compound 18 showed a biphasic
behavior, suppressing the IL-1β level by 63% at 10 μM, but its
efficacy was reduced thereafter. With regard to IL-6, compound
2b showed a dose-dependent suppression (12−47%), whereas
18 was effective only at 100 μM and reduced the cytokine level
by 40%. Compound 4b suppressed the IL-6 level by 57% at 10
μM, augmenting it thereafter. Both compounds 2b and 4b
suppressed the IL-10 level by ∼30−33% at all of the tested
concentrations, but 18 was much less effective (10−12%
suppression). The results are shown in Figure 3a−d. In separate
experiments, the treatment of the cells with LPS (200 ng/mL),
with the compounds alone (10, 50, and 100 μM), or with
combinations of these did not alter the cell viability, as revealed
by an MTT assay (data not shown). This indicated that the
suppression of the cytokine levels by the compounds was
unrelated to their cytotoxicity. Although cytokine imbalance is
associated with diabetes, the precise roles of the Th1 and Th2
cytokines is far from clear.27 Circulating levels of IL-6 are
elevated in type-2 diabetic patients, are correlated with insulin
sensitivity, and may predict the development of diabetes.28ac
However, IL-10 accelerated the development of diabetes in
nonobese diabetic (NOD) mice in some studies29a but
prevented diabetes in others.29b
27, and 16% overall yields, respectively. The C51conformation
of the dihydroxyindolizidine was confirmed by H NMR and
2D-COSY experiments. Previously, we found a poor glyco-
sidase inhibitory property for the L-iminosugars.14 Hence,
presently we synthesized the D-iminosugars using the β-azido
epimer of 9. However, the other stereomers of alcohol 9 can be
easily synthesized using our approach. We also demonstrated
that 2b can suppress β-galactosidase activity and the level of
cytokines that are associated with diabetes. Interestingly,
oxidation of its −CH2OH group to −CO2H, as in 4b, reduced
its efficacy and even reversed its pharmacological behavior at
higher concentrations. Further in vivo studies are required to
establish the antidiabetic properties of 2b.
EXPERIMENTAL SECTION
■
(1R)-1-[(3aR,5S,6S,6aR)-6-Azido-2,2-dimethyltetrahydrofuro-
[2,3-d][1,3]dioxol-5-yl]but-3-en-1-ol (9). To a stirred solution of
azido aldehyde 8 (5.0 g, 23.4 mmol) in THF (60 mL) were added allyl
bromide (4.46 mL, 51.6 mmol) and aqueous saturated NH4Cl (3 mL)
at 0 °C. The reaction mixture was brought to 20 °C and stirred for 7 h.
After the reaction was complete (indicated by TLC), the reaction
mixture was filtered and evaporated in vacuo to give a residue that was
purified by column chromatography (silica gel, 0−10% EtOAc/
hexane) to afford 9 (3.65 g, 61%, colorless thick liquid) as the major
product. Rf = 0.48 (15% EtOAc/hexane). [α]2D5 −14.1 (c 3.33, CHCl3).
1
IR (film): 2104, 1640 cm−1. H NMR (300 MHz, CDCl3): δ 1.31 (s,
3H), 1.49 (s, 3H), 1.98 (broad s, 1H, D2O exchangeable), 2.21−2.29
(m, 1H), 2.65 (m, 1H), 3.86 (m, 1H), 4.03 (dd, J = 3.3, 8.7 Hz, 1H),
4.11 (d, J = 3.3 Hz, 1H), 4.62 (d, J = 3.9 Hz, 1H), 5.18 (m, 1H), 5.24
(m, 1H), 5.78−5.93 (m, 1H), 5.86 (d, J = 3.9 Hz, 1H). 13C NMR (75
MHz, CDCl3): δ 25.9, 26.2, 38.9, 65.8, 67.9, 71.1, 83.0, 104.5, 111.8,
118.6, 133.5. Anal. Calcd for C11H17N3O4: C, 51.76; H, 6.71; N, 16.46.
Found: C, 51.62; H, 6.75; N, 16.63.
(3aR,5S,6S,6aR)-6-Azido-5-[(1R)-1-(benzyloxy)but-3-en-1-yl]-
2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (10). To a cooled
(0 °C) hexane-washed suspension of NaH (1.51 g, 31.4 mmol, 60%
suspension in oil) was dropwise added 9 (3.65 g, 14.3 mmol) in THF
(50 mL). After bringing the mixture to room temperature and stirring
for 10 min, the mixture was cooled to 0 °C, and BnBr (1.87 mL, 15.7
mmol) and Bu4NI (cat.) were added. The mixture was brought to
room temperature and stirred for an additional 4 h. The reaction was
quenched with aqueous saturated NH4Cl (3 mL), and the mixture was
concentrated in vacuo and extracted with EtOAc (3 × 15 mL). The
organic extract was washed with H2O (2 × 10 mL) and brine (1 × 5
mL) and dried. Solvent removal followed by column chromatography
(silica gel, 5% EtOAc/hexane) of the residue furnished 10 (3.71 g,
76%) as a colorless thick liquid. Rf = 0.59 (EtOAc/hexane, 1:9). [α]D25
It was observed that compound 2b reduced the Th1 and Th2
cytokines, suggesting a suppression of LPS-induced macro-
phage activation. This may reduce the immune cell-mediated
beta cell destruction that plays the most vital role in type 2
diabetes. On the basis of these results along with its β-
galactosidase inhibitory property, compound 2b may be a
potential antidiabetic agent. Compound 4b showed similar
activity at a low concentration (10 μM) but induced several
pro-inflammatory cytokines at the higher concentrations.
However, compound 18 showed poor inhibition of glycosidase
activity and IL-10 level but reduced the levels of the
proinflammatory cytokines IL-1β and TNF-α. This property
may be advantageous in managing lysosomal storage diseases
by chaperone-mediated therapy, an inexpensive alternative to
enzyme replacement therapy (ERT).30
1
−19.3 (c 1.55, CHCl3). IR (film): 2104, 1641 cm−1. H NMR (300
MHz, CDCl3): δ 1.31 (s, 3H), 1.46 (s, 3H), 2.36−2.45 (m, 1H), 2.68−
2.77 (m, 1H), 3.79 (dt, J = 4.5, 9.0 Hz, 1H), 4.07 (d, J = 3.0 Hz, 1H),
4.19 (dd, J = 3.0, 9.3 Hz, 1H), 4.52 (d, J = 11.5 Hz, 1H), 4.63 (d, J =
3.6 Hz, 1H), 4.72 (d, J = 11.5 Hz, 1H), 5.21 (m, 2H), 5.86 (d, J = 3.6
Hz, 1H), 5.87−6.01 (m, 1H), 7.28−7.38 (m, 5H). 13C NMR (75
MHz, CDCl3): δ 26.3, 26.5, 34.7, 66.1, 71.4, 75.5, 79.3, 83.2, 104.4,
112.1, 118.2, 127.7, 128.0, 129.3, 133.2, 138.0. Anal. Calcd for
C18H23N3O4: C, 62.59; H, 6.71; N, 12.17. Found: C, 62.70; H, 6.53;
N, 12.34.
(3R)-3-[(3aR,5S,6S,6aR)-6-Azido-2,2-dimethyltetrahydrofuro-
[2,3-d][1,3]dioxol-5-yl]-3-(benzyloxy)propanal (12). Ozone was
bubbled through a cooled (−78 °C) solution of 10 (3.71, 10.7 mmol)
E
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in CH2Cl2 (150 mL) until the mixture attained a persistent blue color.
After removing the excess ozone by Ar purging, Me2S (3.96 mL, 53.61
mmol) was added to the reaction mixture, and the mixture was stirred
at room temperature for 24 h. After concentration in vacuo, the
residue was purified by column chromatography (silica gel, 5%
EtOAc/hexane) to afford aldehyde 12 (3.21 g, 87%) as a colorless
thick liquid. Rf = 0.59 (10% EtOAc/hexane). [α]2D5 −4.6 (c 1.05,
CHCl3). IR (film): 2102, 1720 cm−1. 1H NMR (500 MHz, CDCl3): δ
1.30 (s, 3H), 1.48 (s, 3H), 2.76−2.82 (m, 1H), 2.89−2.96 (m, 1H),
4.07 (d, J = 2.9 Hz, 1H), 4.21 (dd, J = 3.0, 9.3 Hz, 1H), 4.24−4.27 (m,
1H), 4.60 (ABq, J = 11.5 Hz, 2H), 4.65 (d, J = 4.0 Hz, 1H), 5.84 (d, J
= 3.6 Hz, 1H), 7.26−7.38 (m, 5H), 9.82 (m, 1H). 13C NMR (125
MHz, CDCl3): δ 26.1, 26.4, 46.3, 65.8, 72.9, 76.7, 80.3, 83.3, 104.3,
112.2, 127.7, 127.9, 128.4, 137.4, 200.1. Anal. Calcd for C17H21N3O5:
C, 58.78; H, 6.09; N, 12.10. Found: C, 58.89; H, 5.93; N, 12.34. ESI−
MS: [M + Na]+ calcd for C17H21N3O5, 370.14 Da; found, 370.03 Da.
(3aR,4aS,5R,8aS,8bR)-N-Benzyloxycarbonyl-5-(benzyloxy)-
2,2-dimethyloctahydro[1,3]dioxolo[4,5]furo[3,2-b]pyridine
(11). To a cooled (0 °C) and stirred solution of 12 (3.21 g, 9.33
mmol) in MeOH (140 mL) was added Ph3P (2.50 g, 9.33 mmol). The
reaction mixture was gradually brought to room temperature and
stirred for 1 h. The reaction was cooled to −20 °C, acetic acid (cat.)
was added, the reaction was stirred for 40 min, and NaCNBH3 (0.70 g,
11.10 mmol) was added in portions (10 min). After stirring at same
temperature for 2 h followed by stirring at room temperature for 8 h,
the reaction mixture was cooled to 0 °C, and NaHCO3 (2.35 g, 27.90
mmol) in H2O (15 mL) and benzyl chloroformate (0.19 mL) were
added successively. The reaction was stirred at room temperature for 6
h, concentrated in vacuo, and extracted with CHCl3 (3 × 15 mL). The
combined organic extracts were washed with H2O (3 × 10 mL) and
brine (1 × 5 mL) and dried. Solvent removal followed by column
chromatography (silica gel, 5% EtOAc/hexane) of the residue
furnished 11 (2.71 g, 65% in three steps) as a colorless thick liquid.
Rf = 0.39 (20% EtOAc/hexane). [α]2D5 −54.5 (c 2.15, CHCl3). IR
(film): 1697, 1454 cm−1. 1H NMR (500 MHz, CDCl3): δ 1.32 (s, 3H),
1.52 (s, 3H), 1.90 (m, 2H), 3.30 (broad s, 1H), 3.64 (m, 1H), 3.72
(broad s, 1H), 4.35 (broad d, J = 5.2 Hz, 1H), 4.59−4.64 (m, 4H),
5.14 (ABq, J = 12.3 Hz, 2H), 5.92 (d, J = 3.4 Hz, 1H), 7.28−7.40 (m,
10H). 13C NMR (125 MHz, CDCl3): δ 24.3, 26.6, 27.1, 38.4, 61.2,
67.4, 70.6, 72.0, 74.9, 85.2, 105.1, 112.0, 127.6, 127.7, 127.9, 128.0,
128.4, 136.2, 137.8, 155.8. Anal. Calcd for C25H29NO6: C, 68.32; H,
6.65; N, 3.19. Found: C, 68.16; H, 6.77; N, 3.05. ESI−MS: [M + Na]+
calcd for C25H29NO6, 462.19 Da; found, 462.17 Da.
60.3, 67.4, 69.5, 71.8, 75.5, 127.5, 127.8, 127.9, 128.0, 128.5, 128.6,
136.4, 137.7, 156.0. Anal. Calcd for C21H25NO5: C, 67.91; H, 6.78; N,
3.77. Found: C, 67.83; H, 6.61; N, 3.88. ESI−MS: [M + Na]+ calcd for
C21H25NO5, 394.16 Da; found, 394.09 Da.
(2R,3S,4R)-2-(Hydroxymethyl)piperidine-3,4-diol (4-epi-Fag-
omine) (2b). A solution of 14 (0.150 g, 0.40 mmol) and 10% Pd/C
(0.025 g) in MeOH (8 mL) was stirred under an H2 atmosphere at 80
psi for 12 h at 25 °C. The catalyst was filtered through a pad of Celite
545. Solvent evaporation afforded 2b (0.055 g, 92%) as a semisolid. Rf
= 0.51 (40% MeOH/CHCl3). [α]2D5 +11.7 (c 1.8, H2O); lit.18a [α]D22
+10.2 (c 1.4, H2O); lit.18b [α]D25 +10.4 (c 1.4, H2O). IR (film): 3350,
1
1708 cm−1. H NMR (700 MHz, D2O): δ 1.80−1.85 (m, 2H), 2.75−
2.79 (m, 1H), 2.93 (t, J = 6.6 Hz, 1H), 3.20−3.24 (m, 1H), 3.70−3.79
(m, 2H), 3.82−3.87 (m, 1H), 4.01 (s, 1H). 13C NMR (125 MHz,
D2O): δ 26.6, 42.6, 59.1, 61.1, 67.2, 69.3. Anal. Calcd for C6H13NO3:
C, 48.97; H, 8.90; N, 9.52. Found: C, 48.73; H, 8.97; N, 9.70. ESI−
MS: [M + H]+ calcd for C6H13NO3, 148.09 Da; found, 148.13 Da.
(2S,3S,4R)-N-Benzyloxycarbonyl-4-(benzyloxy)-3-hydroxypi-
peridine-2-carboxylic Acid (15). To a stirred solution of amino
aldehyde 13 (obtained from 11 (0.59 g, 1.4 mmol)) in CH3CN (10
mL) was added a solution of NaH2PO4 (0.04 g, 0.27 mmol) in H2O (2
mL) and 30% H2O2 (0.21 mL, 1.55 mmol). The mixture was cooled to
0 °C, and NaClO2 (0.21 g, 2.18 mmol) in H2O (4.5 mL) was added
dropwise over a period of 0.5 h. The reaction mixture was stirred at 15
°C and monitored for the evolution of gas with a bubbler connected to
the apparatus. After 6 h, the reaction was decomposed by the addition
of a small amount of Na2SO4 (0.25 g) and extracted with EtOAc (3 ×
10 mL). Evaporation of the solvent followed by column chromatog-
raphy (silica gel, 5% MeOH/CHCl3) of the residue gave 15 (0.23 g,
67% from 11) as a sticky gum. Rf = 0.60 (20% MeOH/CHCl3). [α]D25
−2.5 (c 1.1, CHCl3). IR (film): 3352, 1702 cm−1. 1H NMR (500 MHz,
CDCl3): δ 1.76 (m, 1H), 1.91 (m, 1H), 3.50−3.66 (m, 1H), 3.88
(broad s, 1H), 3.90−4.02 (m, 2H, one proton D2O exchangeable),
4.61 (ABq, J = 12.1 Hz, 2H), 5.10−5.25 (m, 4H), 7.28−7.42 (m,
10H), 8.10 (s, 1H, D2O exchangeable). 13C NMR (125 MHz, CHCl3):
δ 28.2, 36.6, 53.4, 67.9, 70.9, 71.8, 72.3, 127.2, 127.6, 127.9, 128.1,
128.4, 128.5, 136.1, 137.8, 168.1, 173.1. Anal. Calcd for C21H23NO6:
C, 65.44; H, 6.02; N, 3.63. Found: C, 65.59; H, 6.28; N, 3.83. ESI−
MS: [M + Na]+ calcd for C21H23NO6, 408.14 Da; found, 408.05 Da.
(2S,3S,4R)-3,4-Dihydroxypiperidine-2-carboxylic Acid (3,4-
Dihydroxypipecolic Acid) (4b). Following the same procedure as
for 2b, compound 15 (0.18 g, 0.46 mmol) was catalytically
hydrogenated to obtain 4b (0.072 g, 93%) as a white solid. Rf =
0.46 (30% MeOH/CHCl3). mp: 245−247 °C. [α]2D5 +7.5 (c 2.1,
H2O). IR (KBr): 3374, 2934, 1048 cm−1. 1H NMR (500 MHz, D2O):
δ 1.90−2.05 (m, 2H), 3.04−3.15 (td, J = 4.3, 12.8 Hz, 1H), 3.49 (d, J
= 12.6 Hz, 1H), 3.95−4.01 (m, 1H), 4.18 (s, 1H), 4.48 (s, 1H). 13C
NMR (125 MHz, D2O): δ 23.5, 41.5, 60.3, 66.8, 67.1, 169.3. Anal.
Calcd for C6H11NO4: C, 44.72; H, 6.88; N, 8.69. Found: C, 44.65; H,
6.68; N, 8.81. ESI−MS: [M + Na]+ calcd for C6H11NO4, 184.06 Da;
found, 184.05 Da, 206.01 Da [+2Na].
(2R,3S,4R)-N-Benzyloxycarbonyl-4-(benzyloxy)-2-
(hydroxymethyl)piperidin-3-ol (14). A solution of 11 (0.24 g, 0.54
mmol) in TFA−H2O (2.50 mL, 3:2) was stirred at 0 °C for 1 h. TFA
was removed azeotropically with toluene in vacuo to afford the
hemiacetal as a thick liquid (2.53 g). To a cooled (0 °C) solution of
the crude hemiacetal in acetone/water (5.00 mL, 9:1) was added
NaIO4 (0.13 g, 0.6 mmol). After stirring for 30 min, excess NaIO4 was
decomposed with ethylene glycol (0.10 mL), the mixture was
concentrated in vacuo, and the residue was extracted with CHCl3 (3
× 10 mL) to obtain crude α-amino aldehyde 13 (0. 235 g) as a thick
liquid.
Ethyl (2E)-3-[(2R,3S,4R)-1-Benzyloxycarbonyl-4-benzyloxy-
3-hydroxypiperidin-2-yl]prop-2-enoate (16). To a cooled (−40
°C) and stirred suspension of hexane-washed NaH (0.21 g, 2.52 mmol,
60% suspension in oil) was dropwise added triethyl phosphonoacetate
(0.55 mL, 2.74 mmol). After 10 min, amino aldehyde 13 (obtained
from 11 (0.85 g, 2.29 mmol)) in THF (10 mL) was added into the
mixture. After 10 min, the mixture was gradually warmed to −20 °C
and stirred until the completion of the reaction (∼5 h, indicated by
TLC). The mixture was treated with aqueous saturated NH4Cl (2 mL)
and extracted with EtOAc (3 × 10 mL). The organic extract washed
with H2O (2 × 10 mL) and brine (1 × 5 mL) and dried. Solvent
removal in vacuo followed by column chromatography of the residue
(silica gel, 25% EtOAc/hexane) afforded pure 16 (0.48 g, 48%) as a
colorless thick liquid. Rf = 0.30 (25% EtOAc/hexane). [α]2D5 −5.9 (c
To a cooled (0 °C) and stirred solution of 13 in 90% aqueous
MeOH (10 mL) was added NaBH4 (0.02 g, 0.55 mmol). After the
completion of the reaction (20 min, indicated by TLC), aqueous
saturated NH4Cl (1 mL) was added, and the mixture was concentrated
in vacuo and extracted with CHCl3 (3 × 15 mL). The combined
organic extracts were washed with H2O (2 × 10 mL) and brine (1 × 5
mL) and dried. Solvent removal followed by column chromatography
(silica gel, 0−15% EtOAc/hexane) of the residue furnished diol 14
(40% from 11 in three steps) as a colorless thick liquid. Rf = 0.35 (40%
EtOAc/hexane). [α]2D5 −43.3 (c 2.8, CHCl3). IR (film): 1672, 1435
1
cm−1. H NMR (500 MHz, CDCl3): δ 1.58−1.67 (m, 3H, partially
1
D2O exchangeable), 1.99 (broad d, J = 13.4 Hz, 1H), 2.77 (broad s,
1H, D2O exchangeable), 3.35 (broad s, 1H), 3.83 (broad s, 1H), 3.88
(broad s, 1H), 3.98 (broad s, 2H), 4.33 (broad s, 1H), 4.53 (d, J = 11.7
Hz, 1H), 4.69 (d, J = 11.7 Hz, 1H), 5.15 (ABq, J = 12.6 Hz, 2H),
7.28−7.39 (m, 10H). 13C NMR (125 MHz, CDCl3): δ 27.3, 35.6, 56.7,
3.4, CHCl3). IR (film): 3355, 2890, 1693, 1681 cm−1. H NMR (700
MHz, CDCl3): δ 1.25 (t, J = 7.1 Hz, 3H), 1.59 (m, 2H, partially D2O
exchangeable), 1.99 (m, 1H), 3.13 (t, J = 12.2 Hz, 1H), 3.77 (broad s,
1H), 3.86 (d, J = 2.3 Hz, 1H), 3.95 (m, 1H), 4.17 (q, J = 7.1 Hz, 2H),
4.42 (d, J = 11.9 Hz, 1H), 4.65 (d, J = 11.9 Hz, 1H), 5.02 (m, 1H),
F
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5.11 (s, 2H), 5.86 (d, J = 14.9 Hz, 1H), 7.17 (dd, J = 14.8, 6.1 Hz, 1H),
7.23−7.39 (m, 10H). 13C NMR (175 MHz, CDCl3): δ 14.2, 26.9, 33.9,
56.0, 60.3, 67.9, 69.5, 71.4, 73.1, 122.2, 127.0, 127.2, 127.9, 128.1,
128.4, 128.5, 128.6, 136.3, 137.8, 142.5, 155.3, 166.1. Anal. Calcd for
C25H29NO6: C, 68.32; H, 6.65; N, 3.19. Found: C, 68.44; H, 6.52; N,
3.31. ESI−MS: [M + Na]+ calcd for C25H29NO6, 462.18 Da; found,
462.17 Da.
(7R,8S,8aR)-7,8-Dihydroxyhexahydroindolizidine-3-(2H)-one
(17). To a solution of 16 (0.28 g, 0.63 mmol) in anhydrous MeOH
(20 mL) was added HCO2NH4 (0.20 g, 3.15 mmol) and 10% Pd/C
(30 mg). The reaction mixture was refluxed for 4 h and filtered
through Celite 540. The filtrate was concentrated in vacuo, and the
residue was purified by column chromatography (silica gel, 5%
MeOH/CHCl3/NH3 (1drop)) to afford 17 (0.10 g, 90%) as white
the mean
SEM of three independent experiments, with each
experiment repeated three times. The data were analyzed by a paired t
test and one-way analysis of variance (ANOVA).
Cytokine Estimation. The levels of TNF-α, IL-1β, IL-6, and IL-10
in the culture supernatants were measured using ELISA kits according
to the manufacturer’s instructions. Briefly, Raw 264.7 cells (1 × 106/
well) untreated or pretreated with different concentrations of the test
compounds for 1 h were incubated with LPS (200 ng/mL) for 16 h to
assay TNF-α and IL-1β levels and for 24 h to estimate IL-6 and IL-10
levels. The culture medium was collected, and the concentrations of
the cytokines in each sample were determined using standard curves.
For the estimation of TNF-α and IL-1β, the culture supernatants were
diluted 1:10 by sample diluents provided in the kit. The values are
expressed as the mean SEM of three independent experiments, with
each experiment repeated three times. The data were analyzed by a
paired t test and one-way analysis of variance (ANOVA).
crystals. Rf = 0.30 (10% MeOH/CHCl3). mp: 162−163 °C. [α]D25
+
6.1 (c 1.8, CH3OH). IR (KBr): 3357, 1658 cm−1. 1H NMR (500 MHz,
CD3OD): δ 1.58 (m, 1H), 1.74 (m, 1H), 2.06 (m, 2H), 2.29 (m, 1H),
2.41 (m, 1H), 2.73 (m, 1H), 3.67 (m, 3H), 4.00 (ddd, J = 13.2, 5.2, 1.6
Hz, 1H). 13C NMR (175 MHz, CD3OD): δ 18.3, 25.7, 30.1, 37.1, 60.1,
69.8, 70.1, 175.8. Anal. Calcd for C8H13NO3: C, 56.13; H, 7.65; N,
8.18. Found: C, 56.01; H, 7.72; N, 8.29. ESI−MS: [M + H]+ calcd for
C8H13NO3, 172.09 Da; found, 172.08 Da.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(7R,8S,8aR)-Octahydroindolizine-7,8-diol (18). To a cooled (0
°C) and stirred suspension of LiAlH4 (0.048 g, 1.27 mmol) in THF (3
mL) was added 17 (0.087 g, 0.50 mmol) in THF (15 mL). The
reaction mixture was slowly brought to room temperature and refluxed
for 2 h. The reaction was quenched with EtOAc (5 mL) and aqueous
saturated NH4Cl (1 mL), the mixture was filtered through Celite 540,
and the resultant thick liquid was purified by column chromatography
(silica gel, 5% MeOH/CHCl3) to afford 18 (73 mg, 92%) as a white
solid. Rf = 0.20 (40% MeOH/CHCl3). mp: 126−127 °C. [α]2D5 + 3.31
AUTHOR INFORMATION
Corresponding Author
*E-mail: schatt@barc.gov.in.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
A.K.S. gratefully acknowledges BRNS-DAE (Mumbai, India)
for a K. S. Krishnan Research Associateship award.
■
1
(c 3.2, H2O). IR (KBr): 3366, cm−1. H NMR (500 MHz, D2O): δ
2.0−2.25 (m, 6H), 3.06−3.12 (m, 2H), 3.43 (m, 1H), 3.62−3.67(m,
2H), 4.04 (m, 1H), 4.23 (d, J = 2.1 Hz, 1H). 13C NMR (125 MHz,
D2O): δ 20.1, 23.1, 24.9, 48.4, 52.1, 66.2, 68.0. Anal. Calcd for
C8H15NO2: C, 61.12; H, 9.62; N, 8.91. Found: C, 61.25; H, 9.54; N,
9.13. ESI−MS: [M + H]+ calcd for C8H15NO2, 158.12 Da; found,
158.16 Da.
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C12H19NO4, 242.13 Da; found, 242.13 Da.
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200, BioTek Winooski, VT). For the control sample, the test
compounds were omitted. The β-galactosidase activity was determined
using a standard plot of 4-nitrophenol, and the values are expressed as
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