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10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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336
Scheme 10.18 Flavone synthesis as described by Marta etal.[106–108] Reagents/solvent
and yield: (a) acetone, K
aromatic aldehyde; (c) pyridine, I
, ClCH2OCH3, 52%; (b) 1,4-dioxane, aq. NaOH 50% (w/v),
2CO3
, then TsOH, MeOH.
2
yields bigger Aβ1–42 aggregates instead of small, toxic oligomers. These results suggest a remodeling effect by the action of chrysin (142) and related molecules, serving as the basis for the generation of glycosylated flavones with neuroprotective
potential, which shall be discussed in Section10.8.
Importantly, chrysin was synthesized by Matos etal.[106] by a new methodology
also used for the generation of similar flavones thereafter (Scheme10.18)[107, 108].
Selective protection of two hydroxyl groups of acetophloroglucinol (145) with methoxymethyl ether (MOM), sodium hydroxide-promoted Claisen–Schmidt aldol condensation of 146 with the appropriate aromatic aldehyde gave the intermediate
chalcone. In the synthesis of chrysin (142, R=H), the corresponding chalcone was
obtained by reaction with benzaldehyde in 95% yield. Then, iodine and pyridine
under reflux conditions were used for the oxidative cyclization step for the first time
in the literature, affording the monoprotected flavone (as detected by LCMS). After
work-up, acid-promoted deprotection of the MOM groups led to the synthesis of the
desired product. This final one-pot reaction step afforded chrysin 142 in 81% yield.
The individual synthesis of other flavones according to this general method will be
mentioned throughout this chapter, along with the reaction yields obtained for the
aldehyde used in each particular case.
In the context of strategy (ii) (Figure10.10), a small library of nature-inspired
flavones and their C-glucosyl derivatives was synthesized[108], from which the pair
of N-methyl piperazinyl analogs 143 and 144 (Figure 10.11) showed therapeutic
potential as protein–protein interaction inhibitors. In the initial Saturation Transfer
Difference (STD) NMR screening assays that aimed to assess the binding potential
of all synthesized molecules against Aβo, compound 143 stood out as the one presenting the highest potential for further investigation, with visible interactions with
C
Aβo at 2 μM. Later on, both 143 and 144 inhibited Aβo-PrP
binding in HEK cells,
where the C-glucosyl derivative 144 decreased the binding between these two
10.7 Amyloid Protein–Protein Interaction Inhibitors
337
proteins by 41% (p< 0.001 vs. Aβo-PrPC controls, with dose–dependent effects), and
C
143 by 26% (p< 0.01 vs. Aβo-PrP
found to enhance the extent of Aβo-PrP
controls). Interestingly, the C-glucosyl moiety was
C
disruption, thus highlighting the importance of the C-linked sugar moiety for this type of therapeutic application (p< 0.05
for compound 143 vs. compound 144) [108]. With no relevant cytotoxic effects
observed invitro, these compounds are therefore promising candidates for further
studies, namely BBB permeation assays and invivo tests aiming to prove the physi-
C
ological effects of tackling the interaction between Aβo-PrP
in AD animal models.
Interestingly, compound 133 (2,3,4,6-tetra-O-methylglucosyl)phloroglucinol
C
(Figure10.9) was able to inhibit the binding between Aβo and PrP
by 26%, while its
analogs 134–136 (Figure10.9) inhibited by 16%, 11%, and 17%, respectively[102]. As
earlier discussed, some of these compounds are Fyn kinase inhibitors (134), inhibit
Aβo-induced Fyn activation (133, 135, and 136), and all inhibited Aβo-induced Tau
hyperphosphorylation to a significant extent.
The synthesis of 143[108] was conducted on the basis of the previously described
method for the synthesis of chrysin (142) (Scheme 10.18), with a slight variation
regarding the phenol-protecting groups. Indeed, ethoxymethyl ether (EOM) was
used instead of MOM ether, allowing the optimization of the protection step, which
afforded the corresponding EOM-diprotected acetophloroglucinol derivative in
91% yield. Aldol condensation was carried out with 4-[(4-methylpiperazin-1-yl)
methyl]benzaldehyde to give the intermediate chalcone in 99% yield. Oxidative cyclization and deprotection were conducted as earlier described[106] (Scheme10.18),
giving 143 in 90% yield. A similar synthetic route was employed for the preparation
of the C-glucosyl analog 144 (Scheme 10.19) and all other C-glucosyl flavones
Scheme 10.19 Synthesis of C-glucosyl flavones as described by Matos etal.[107, 108].
Reagents/solvent and yield: (a) CH
64%; (c) 1,4-dioxane, aq. NaOH 50% (w/v), aromatic aldehyde; (d) pyridine, I
BBr
, DCM.
3
CN/) DCM, drierite, TMSOTf, 57%; (b) DMF, K2CO3, BnBr,
3
; then
2
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
(a)
(b)
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338
described in this chapter. Succinctly, the TMSOTf-promoted C-glucosylation of acetophloroglucinol 145 with the commercially available glucosyl donor 147 at low temperature afforded intermediate 148 in 57% yield. Subsequently, after selective
benzylation of two hydroxy groups, the base-catalyzed aldol condensation with the
appropriate aldehyde, (4-[(4-methylpiperazin-1-yl)methyl]benzaldehyde in the case
of compound 144), led to the generation of the intermediate C-glucosyl chalcone
which, in the synthesis of 144, was accomplished in 58% yield. Finally, oxidative
cyclization promoted by iodine in pyridine, followed by deprotection of all benzyl
groups gave the desired C-glucosyl flavone. This final one-pot reaction step afforded
144 (R=4-methylpiperazin-1-yl) in 84% yield.
10.8 Inhibitors of Aβo and/or Oxidative
Stress-Induced Neurotoxicity
In 2019, Rauter’s group synthesized resveratrol deoxyglycosides 150 and 151
(Scheme10.20)[109], and compared their effects with the corresponding aglycones
regarding their ability to inhibit hydrogen peroxide-induced neurotoxicity in
Scheme 10.20 Synthesis of resveratrol 2-deoxyglycosides with neuroprotective
activity[109]. Reagents/solvent and yield: (a) TPHB, THF,156, 21%; 158, 8%; (b) NaOMe,
MeOH, 150, 92%; 151, 100%.
33910.8 Inhibitors of Aβo and/or Oxidative Stress-Induced Neurotoxicity
Figure10.12 Polyphenol structures: resveratrol glycosides 150, 151, glucosyl resveratrol
152, and flavones 153, 154, two promising compounds able to inhibit Aβo and/or oxidative
stress-induced neurotoxicity.
SHSY-5Y cells. The resveratrol 2-deoxy-α--arabino-hexopyranoside 150 and the
2,6-dideoxy-α--arabino-hexopyranoside 151 were generated in a simple two-step
process (Scheme 10.20a,b), by reaction of resveratrol with the adequate acetylprotected glycals 155 or 157 in the presence of triphenylphosphane hydrobromide,
followed by deprotection with sodium methoxide[109].
Both compounds were able to significantly reduce the damage caused by hydrogen peroxide when added to the neuronal cells at 50 μM, as observed in an MTT cell
viability assay (p< 0.05 vs. hydrogen peroxide controls)[109]. Resveratrol was also
able to show some effect (p< 0.05); however, it is important to note that resveratrol
is a well-known Pan-Assay INterference compound (PAIN), and capable of interfering with biological membranes, altering their structure, fluidity, and the function of
transmembrane proteins that are at the very beginning of cell signaling pathways[110, 111]. Thus, even though resveratrol is a powerful antioxidant, it is not the
ideal candidate for optimization due to nonspecific effects that can be triggered by
its action.
With this in mind, the potential of C-glycosylation as a tool for preventing the
PAINS-type behavior exhibited by some polyphenolic compounds found in nature,
including resveratrol, was further investigated[112]. It should be noted that though
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340
Scheme 10.21 The first synthesis of glucosylresveratrol 152[112]. Reagents/solvent and
yield: (a) PivCl, pyridine, 62% yield; (b) TMSOTf, drierite, DCM/CH
yield; (c) K
yield; (f) tert-BuOK, DMF, 42% isolated yield for the (E)-isomer 164; (g) DCM, BCl
, BnBr, DMF, 85% yield; (d) LiOH, MeOH/H2O, 68% yield; (e) PCC, DCM, 81%
2CO3
CN, 162, 24% isolated
3
, 35% yield.
3
glycosides, such as 150 and 151 are easily synthesized, they are highly susceptible to
hydrolysis in the gut, and therefore may reach their therapeutic targets as aglycones
when administered orally. The same does not occur with the chemically and enzymatically stable C-glucosyl polyphenol derivatives, which became the focus of our
research. The first synthesis of C-glucosyl resveratrol 152 (Scheme10.21) was carried out starting by TMSOTf-catalyzed Fries-type reaction of benzylated glucopyranose 147 with the pivaloyl protected 5-(hydroxymethyl)benzene-1,3-diol 160 to
afford the C-glucoside 161, chromatographically separated from the secondary
product 162 and isolated in 24% yield. Benzylation of the free hydroxy groups was
followed by depivaloylation with lithium hydroxide in methanol/water and subsequent oxidation with pyridinium chlorochromate, obtaining the aldehyde 163 in
46.8% overall yield from 161. Horner–Wadsworth–Emmons olefination with diethyl
(4-benzyloxyphenyl)methylphosphonate in the presence of tert-BuOK in DMF
afforded the protected (E)-isomer 164, isolated in 55% yield. Full debenzylation was
possible with BCl
to give purified (E)-glucosyl resveratrol 152 in 35% yield.
3
10.9 Carbohydrate–Protein Interactions as Potential Therapeutic Targets Against AD
Compound 152 was then compared to its aglycone resveratrol concerning the
ability to alter membrane dipole potential. Indeed, resveratrol and other polyphenols, typically regarded in the literature as PAINS, were found to significantly alter
membrane dipole potential (p < 0.0001 vs. untreated controls), which could be a
mechanism underpinning the broad bioactivities they have been described to exert
when studied in cell models of disease. In contrast, the corresponding C-glucosyl
derivatives, including compound 152, did not exert the same effects (not statistically
different vs. untreated controls), suggesting that C-glucosylation may be considered
as a valuable approach to prevent the promiscuous membrane-disrupting effects of
lipophilic, planar compounds, such as resveratrol.
It is important to note that the addition of the C-glucosyl moiety does not mean
loss of bioactivity in the case of polyphenolic compounds. In fact, maintenance or
even an increase in activity is possible with C-glucosylation, and this is well illustrated in this review by the results reported for compounds 143 and 144 (Figure10.11)
as well as those given in the literature by Rauter’s group for other glucosylpolyphenols[113, 114]. Moreover, many other bioactive C-glycosides here are presented to
confirm the usefulness of C-glycosylation as a means to generate bioactive molecular entities with therapeutic potential against neurodegenerative processes.
Another great example is here given by the 4′-morpholinylflavone derivatives 153
and 154 (Figure 10.12). Synthesized by Matos etal. [108] in 2019, these two compounds used at 50 μM concentrations were able to normalize cell viability of SHSY-5Y
cells exposed to oxidative stress induced by H
-treated controls (p< 0.0001 for compound 154 and p< 0.05 for 153, vs. H2O2-
to H
2O2
, with significant differences relative
2O2
treated controls, respectively). Furthermore, both compounds significantly reduced
Aβ-induced neurotoxicity in the same cell model with a similar efficacy pattern
(p< 0.0001 vs. Aβ-treated controls for 154; p< 0.05 vs. Aβ-treated controls for 153).
Flavone 154, the best in this study, showed adequate log D and effective permeability
in a PAMPA assay, and was not cytotoxic in concentrations up to 100 μM, being,
therefore, a promising lead for future investigation against AD[109].
341
10.9 Carbohydrate–Protein Interactions as Potential
Therapeutic Targets Against AD
10.9.1 Lipid-Raft Gangliosides as Membrane Accumulation Sites for
Toxic Aβ Aggregates
Carbohydrate–protein and carbohydrate–carbohydrate interactions are key in many
physiological and pathological processes, and AD is no exception. Indeed, increasing evidence points toward membrane clusters enriched in ganglioside GM1, the
most common brain ganglioside, as seeding locations for Aβ peptides [115].
Gangliosides are glycosphingolipids carrying one or more sialic acid units in their
glycans and are usually located in the outer leaflet of the plasma membrane in the
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so-called lipid rafts, which are essential in signal transduction processes[116]. In
these microdomains, also containing sphingomyelin and cholesterol, GM1 forms a
string-like cluster with a hydrophobic sugar–lipid interface that is greatly prone to
interact with and accommodate Aβ peptides, thereby restricting their spatial rearrangements and promoting the formation of cross-β-sheets[117]. The binding affinity between ganglioside clusters and Aβ peptides seems to increase with the number
of glycolipid-decorating sugar moieties, with reports of hydrogen bond interactions
between Aβ and sugar hydroxyl groups, or hydrophobic CH-π interactions between
aromatic side chains of Aβ amino acid residues and the CH groups of the glycan
backbone as the major driving force of binding of Aβ to lipid bilayers[118].
GM1-bound Aβ peptides exhibit an extremely elevated potential to accelerate Aβ
aggregation[117]. Furthermore, contrarily to the earlier mentioned nontoxic Aβ
fibrils formed in solution, membrane-anchored Aβ fibrils formed in such a hydrophobic context are, in fact, cytotoxic and display unique physicochemical properties[116]. Over time, such Aβ fibrils with potent toxicity accumulate in the form of
amyloid plaques, causing lipid raft disruptions that affect cellular processes relying
on the normal function of these microdomains[119]. Hence, compounds targeting
the interactions between GM1 sialic acid units and Aβ side chains may be seen as
promising therapeutic targets against AD, namely those able to suppress β-sheet
formation by stabilizing α-type helical secondary structures of Aβ on the ganglioside
clusters[117].
It is interesting to note that the formation of GM1 clusters in the first place is
highly dependent on cholesterol concentrations, as the establishment of hydrogen
bonds with GM1 and sphingomyelin assures the required cohesion between lipids
for the clustering process to occur[116]. Notably, high levels of serum cholesterol
positively correlate with an increased risk of dementia, with reports showing a
decreased prevalence of AD in subjects prescribed with cholesterol-lowering
drugs[120]. Moreover, the presence of the cholesterol transporter ApoE4 variant,
linked to abnormal cholesterol metabolism in the brain, is strongly associated with
the occurrence of late-onset AD[121]. The crucial role of cholesterol in gangliosidecontaining lipid rafts may explain why that is the case. Therefore, tackling cholesterol metabolism and/or the assembly of GM1 clusters in the brain is also an
appealing therapeutic target for drug discovery against AD.
10.9.2 The Role of Microglial Cells in Aβ Brain Clearance
Senile plaques, the extracellular deposits of Aβ aggregates typically found in the
brain of patients with AD, are enriched in a variety of other components besides the
Aβ peptide, namely sialylated glycoproteins and gangliosides. As previously
described, gangliosides are able to bind Aβ and initiate their aggregation into toxic
fibrils, thus playing a key role in the formation of these plaques[113]. It would be
expected that microglial cells, the resident macrophages of the brain, would be able
to effectively clear the amyloid aggregates observed in senile plaques by phagocytosis, but this is not the case[122, 123]. These cells express several recognition receptors at the cellular surface, including sialic acid-binding, immunoglobulin-like
10.10 Conclusion
lectin receptors known as Siglecs. Specifically, microglia express Siglec-11, which
belongs to the subfamily of CD33 proteins, known to suppress immune cell activation. CD33 are overexpressed in postmortem brain samples of AD patients and seem
to inhibit the activity of microglia by specifically binding to sialylated conjugates,
such as gangliosides, which might explain why senile plaques are not efficiently
eliminated by microglial cells through phagocytosis[123, 124].
In 2019, a study was published by Dukhinova and coworkers[125] with brainganglioside deficient 5XFAD mice lacking the gene that encodes for α2,3sialyltransferase, which is required for the synthesis of all major brain
gangliosides, including GM1. This animal model for AD overexpresses three
mutant human amyloid proteins and two presenilin PS1 genes. These animals
were found to have a significantly lower level of amyloid plaques compared to
wild-type 5XFAD animals of the same age (p< 0.0001), without neuronal loss
and with a comparable cognitive function to 5XFAD mice non-lacking the α2,3sialyltransferase gene[125]. Interestingly, animals unable to produce sialylated
brain gangliosides had lower microglia activation levels (p < 0.05), although
these cells underwent important morphological changes of activation making
them capable of phagocyting Aβ aggregates. Yet, the authors also reported that
treatment of wild-type 5XFAD mice with a sialic acid-binding lectin targeting
sialic acid units of major brain gangliosides successfully decreased amyloid deposition, inhibited neuroinflammation, boosted the expression of synaptic markers,
and improved the cognitive function of these animals (p< 0.05 vs. controls)[125].
Moreover, in a recent study by Griciuc etal.[124], APP/PS1mice, another animal
model for the study of AD, treated with microRNA targeting CD33 (miRCD33) at
an early stage, effectively displayed reduced CD33 microglial expression, and
resulted in a significantly decreased Aβ plaque burden in the brain of these animals (24.4% decrease in Aβ plaque area, p = 0.035 vs. controls). However, it
should be noted that in an invitro study, where a CD33knockout was performed
in human macrophages and microglia, despite a higher phagocytic activity
toward Aβ, CD33 deletion also resulted in oxidative damage and inflammation,
contrarily to CD33-expressing microglia[126]. Together, this evidence validates
the potential of ganglioside sialic acid masking as a therapeutic strategy to
decrease the amyloid burden in AD.
343
10.10 Conclusion
With more than 50 million people affected globally, AD is undoubtedly one of the
major public health concerns of the modern society for which effective diseasemodifying therapies are urgently needed. Yet, the complex and multifactorial
nature of this pathology makes the quest for new drug candidates a difficult challenge. As a matter of fact, reports on so far unexplored molecular players, cell–cell
interactions, and key pathophysiological pathways in neurodegeneration are
added to the literature almost everyday. While aggregation of Aβ is widely accepted
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as one of the causes of AD, other therapeutic targets are increasingly seen as alternative or additional choices when it comes to drug discovery and development.
As in many other pathological processes, carbohydrates play an important role in
the processes leading to synaptic dysfunction and neuronal death, namely imbalances in posttranslational protein modification, ganglioside-promoted amyloid
aggregation, recognition of amyloid aggregates by microglial cells via protein–
carbohydrate interactions, among others. In this context, this critical review covered several different sugar-related therapeutic targets for drug development, as
well as the most recently published carbohydrate-based drug candidates that act by
mimicking natural substrates and transition states of glycosylation reactions. Both
the synthesis of these compounds and the most important biological activity data
available in the literature are here discussed. What’s more, we have also covered a
group of very recent nature-inspired sugar-linked polyphenols with remarkably
promising results concerning Fyn inhibition, amyloid protein–protein interaction
inhibition, and prevention of amyloid-oxidative stress-induced neuronal death. As
here exemplified, in some cases the synthesis of such compounds can be much
more straightforward and efficient compared to other known small molecules
intended for the same use. Importantly, the sugar moiety can increase the desired
bioactivity when linked to an aglycone with therapeutic potential, while mitigating
undesirable PAINS-type behavior toward cell membranes.
The pool of data covered in this chapter illustrates the versatility of carbohydrates
and carbohydrate-linked molecules in terms of their synthesis and bioactivity, while
supporting their use as molecular scaffolds against neurodegenerative disorders,
particularly AD.
List ofAbbreviations
Aβ amyloid β
Aβ1–40
Aβ1–42 42-Residue Aβ
Aβm Aβ monomers
Aβo Aβ oligomers
AcCl acetyl chloride
ACh acetylcholine
AChE acetylcholinesterase
ACP 2-acetamido-6-chloropurine
AD Alzheimer’s disease
AFM atomic force microscopy
AICD APP intracellular domain
ALN alendronate
ApoE4 apolipoprotein E4
APP Aβ precursor protein
40-Residue Aβ
APP-CTFα me mbrane-bound C-terminal fragment of APP
generated by α-secretase
APP-CTFβ me mbrane-bound C-terminal fragment of APP
generated by β-secretase
AUC area under the curve
BAβACs sol uble complexes between Aβ and BChE in the
presence of ApoE4
BACE1 APP β-secretase
BChE butyrylcholinesterase
Bn benzyl
Boc tert-butyloxycarbonyl
BSA bis(trimethylsilyl)acetamide
BzCl benzoyl chloride
CD33 sialic acid binding Ig-like lectin 3
ChEs cholinesterases
CNS central nervous system
COSs chitosan oligosaccharides
CP 6-Chloropurine
CSA camphorsulfonic acid
CSPGs chondroitin sulfate proteoglycans
CTS chitosan
CTS-ALN-NPs ALN-loaded CTS nanoparticles
Cys cysteine
DAST diethylaminosulfur trifluoride
DBU 1,8-diazabicyclo(5.4.0)undec-7-ene
DCM dichloromethane
DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DFT density functional theory
-Glc -glucose
DIPEA N,N-diisopropylethylamine
DMA dimethylacetamide
-Man -mannose
DMAP 4-dimethylaminopyridine
DMF dimethylformamide
DMS dimethyl sulfide
DMSO dimethylsulfoxide
DON 6-Diazo-5-oxonrleucine
DPPA diphenylphosphoryl azide
DTAD di-tert-butyl azodicarboxylate
DTT dithiothreitol
EC50 half maximal effective concentration
EOM ethoxymethyl ether
Fmoc fluorenylmethyloxycarbonyl
345List ofAbbreviations
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