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10.6 Fyn Kinase Inhibitors
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their high-affinity partner, the PrPC. Once in complex with Aβo, PrPC interacts physically with the mGluR5 and, in the cytoplasm, Fyn kinase binds to mGluR5 and is
phosphorylated. This activated complex then results in the phosphorylation of the
NR2B subunit of the N-methyl--aspartate receptor (NMDA), which then leads to a
calcium influx that triggers the phosphorylation of downstream signaling molecules. With chronic exposure to Aβo at the neuronal surface, this cascade of events
ultimately culminates in Tau hyperphosphorylation, which is responsible for synaptic dysfunction and cognitive decline in AD patients.
Not many compounds have been identified to this point as Fyn kinase inhibitors
with proven concomitant downstream effects. In 2015, Kaufman etal.[101] investigated the brain-permeable Src family kinase inhibitor AZD0530, also known as
saracatinib (123, Scheme10.15), as a compound with potential against AD.
Saracatinib is a non-glycosylated C-5-substituted anilinoquinazoline and was
originally discovered as an anticancer agent by Hennequin etal.[103] in 2006, along
with a series of analogs investigated in structure–activity relationship (SAR) studies.
1
Biological assays showed that, when given at 5 mg kg
per day, this Fyn kinase
inhibitor prevented Aβo-induced signaling, Tau phosphorylation, and deposition,
while being able to rescue special memory and synaptic depletion in AD transgenic
mice[98]. In another study by Tang etal.[104], later in 2020, the same dose of saracatinib promoted similar improvements in phosphorylated Tau accumulation in the
331
Scheme 10.15 Synthesis of saracatinib (123)[102]. Reagents/solvent and yield: (a)
formamidine acetate, 2-methoxyethanol, DIPEA, then IPA (52%); (b) BnOH, NaH, DMA; (c)
HCl, 98%; (d) anhydrous toluene, POCl
92%; (g), Ph
(j) distillation, NaOH, 89%; (k) 2-(4-methylpiperazin-1-yl)ethanol, Ph
IPA, 86%.
P, DTAD, tetrahydropyran-4-ol, toluene; (h) HCl, dioxane, then IPA, 80%; (i) TFA;
3
, DIPEA; (e) anhydrous toluene, 69%; (f) pyridine, HCl,
3
P, DTAD, THF, then
3
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
332
hippocampus, accompanied by memory improvements in mice with transgenic and
traumatic tauopathy.
The original 100 mg-scale synthesis of saracatinib[103] included 17linear steps, 7
of which were protection/deprotection reactions. With some practical concerns
raised for a potential scale-up synthesis, four years later, an optimized route for the
kilogram manufacture of saracatinib was developed [102]. This route started from
the difluoro quinazolinone 125, accessed in 52% yield from ester 124 by using forma-
midine acetate in 2-methoxyethanol (Scheme10.15), followed by addition of isopropanol and crystallization. Then, after a double S
Ar reaction to allow the replacement
N
of both fluorine groups with benzyloxy moieties by reaction with benzyl alcohol in
the presence of sodium hydride and dimethylacetamide (DMA), intermediate 126
was chlorinated in the presence of phosphorous oxychloride and DIPEA but 127 was
not isolated and reacted with 128 to give anilinoquinazoline 129, which was obtained
as a hydrochloride salt in 69% yield. Selective debenzylation gave the 5-hydroxy intermediate 130 in 92% yield. Compound 131 was then accessed as a hydrochloride salt
in 80% yield by reaction with tetrahydropyran-4-ol under Mitsunobu conditions,
with subsequent addition of hydrochloric acid in 1,4-dioxane, followed by crystallization. After deprotection of the remaining benzyl group, followed by distillation to
dryness and neutralization with sodium hydroxide, the final Mitsunobu coupling of
intermediate 132 with 2-(4-methylpiperazin-1-yl)ethanol gave saracatinib 123 which,
after crystallization, was obtained as a difumarate in 86% yield.
To the best of our knowledge, saracatinib (123) was the first Fyn kinase inhibitor
with proven downstream effects in an AD model and with adequate pharmacokinetic properties. However, the very first sugar-based Fyn kinase inhibitors were not
published until 2020[103]. Indeed, Rauter and coworkers identified a new family of
nature-inspired glucosylpolyphenols (121–124, Figure10.9) that were shown to distinctively interfere with the Aβo-Fyn-Tau neuronal signaling cascade, with the
advantage of having a much easier, straightforward, and efficient synthesis compared to the route described for saracatinib.
Synthesis of the three per-O-methylated C-glucosyl polyphenols 133–135 was
conducted by Matos et al.[105] in only two reaction steps, in good to very good
overall yield (Scheme10.16). Briefly, after full methylation of the commercially
available methyl α--glucopyranoside (137) with sodium hydride and methyl iodide,
the TMSOTf-promoted C-glucosylation reaction of each polyphenol in the presence
of drierite gave the desired final compound. This final reaction step afforded compound 133 in 53% yield, compound 134 in 37% yield, and compound 135 in 45% yield.
Synthesis of compound 136 required a few additional reaction steps (Scheme10.17)
but is still simpler than that of saracatinib (123). Firstly, the C-glycosylation reaction
carried out as previously described afforded the intermediate 139 in 63% yield. It is
interesting to note that this reaction occurred via a Friedel–Crafts-type mechanism,
being the first exception to the Fries-type rearrangement ever reported in the
C-glycosylation of unprotected phenols. Compound 139 was then submitted to a
benzoylation reaction with benzoyl chloride, imidazole, and dichloromethane as a
solvent to afford its derivative 140 in 88% yield, which was deprotected to give the
MeO
OMe
MeO
OMe
MeO
135
136
OMe
OMe
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10.6 Fyn Kinase Inhibitors
333
MeO
MeO
HO
O
H
H
OH
133
OMe
OH
OHHO
OMe
O
OH
O
MeO
HO
HO
HO
O
H
OH
134
OH
OH
O
H
O
O
O
O
Figure10.9 Structure of Fyn kinase inhibitor 134 and of the inhibitors of Aβo induced Fyn
activation 133, 135, and 136.
desired compound 136 in 8% yield. The low yield of this final step was due to the
hydrolysis of the ester groups and could potentially be further optimized.
In terms of their bioactivity, the per-O-methylated C-glucosyl phloroglucinol 133
and the per-O-methylated C-glucosyl hydroquinone 134 (Figure10.9) were found to
significantly inhibit Fyn kinase activity at 10 μM (p< 0.01 vs. untreated controls) in
hiPSC-derived neural progenitor cells. On the other hand, compounds 133, 135, and
136 (Figure 10.9) were able to inhibit Aβo-induced Fyn activation at the same
Scheme 10.16 Synthesis of per-O-methylated C-glucosyl polyphenols 133–135[105]].
Reagents/solvent and yield: (a) DMF, NaH, MeI, 91%; (b) dry CH
CN, hydroquinone,
3
phloroglucinol or acetophloroglucinol, drierite, TMSOTf. Source: Adapted from Matos
etal.[105].
–
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
334
Scheme 10.17 Synthesis of per-O-methylated C-glucosyl polyphenols[103]. Reagents/
solvent and yield: (a) DMF, NaH, MeI, 91%; (b) dry CH
imidazole, BzCl, 88%; (d) DCM, BBr
.
SMe2, 8%.
3
CN, drierite, TMSOTf, 63%; (c) DCM,
3
concentration (p < 0.001 vs. untreated Aβo controls). Importantly, all four compounds (133–136) reduced Aβo-induced Tau hyperphosphorylation (at 10 μM) to
levels below controls, which once more supports Fyn kinase as a therapeutic target for AD.
Even though other analog molecules also exhibited bioactivity in the SAR study
conducted by the authors, this group of glycoconjugates displayed the adequate
physicochemical features (optimal log D and effective permeability in a PAMPA
assay) without any relevant cytotoxic effects at 50 μM[103], which makes them can-
didates for further development as CNS-active therapeutic agents.
10.7 Amyloid Protein–Protein Interaction Inhibitors
As previously discussed, the interactions between Aβo and its high-affinity partner
C
at the neuronal cell surface are crucial in triggering Fyn activation and subse-
PrP
quent events leading to tau hyperphosphorylation in AD (Figure10.10). Hence,
when thinking of protein–protein interaction inhibition strategies aiming to directly
disrupt the involvement of Aβ in the development of this signaling cascade, two
main approaches arise: (i) tackling the aggregation process that turns Aβ monomers
(Aβm) into Aβo and (ii) inhibiting the binding between Aβo and the PrP
(Figure10.10).
In the context of approach (i), a group of flavonoids was investigated for their ability to inhibit Aβ aggregation[106]. The authors highlighted the ability of catecholtype flavonoids, such as the natural compound quercetin (141, Figure 10.11), to
completely abolish Aβ aggregation by acting as Michael acceptors once oxidized
into the corresponding o-quinones. This process is characteristic of Pan Assay
C
10.7 Amyloid Protein–Protein Interaction Inhibitors
HO
143
144
OH
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335
(i)
(iI)
Aβ
Aβ
m
o
Aβ
o
Aβo + PrP
c
Figure10.10 Two major processes to be targeted by amyloid protein–protein interaction
inhibitors in the context of AD.
Interference Compounds (PAINS) with the catechol motif, which are able to indiscriminately interfere with many other molecular targets. Hence, highly promiscuous compounds such as quercetin (141) should not be regarded as ideal candidates
for further optimization. Yet, they ultimately uncovered the potential of chrysin
(142, Figure10.11), another natural compound, to act as an anti-aggregation compound. Even though ThT fluorescence assays did not reveal a significant change in
the presence of amyloid aggregates by the effects of this compound (tested in concentrations from 0.5 to 100 μM), Atomic Force Microscopy (AFM) experiments
showed that, compared with structurally similar compounds, chrysin preferably
OH
HO
OH O
141
OH O
O
OH
N
O
N
HO
HO
HO
HO
H
O
OH O
142
OH
OH
O
O
OH O
Figure10.11 Amyloid anti-aggregation agents (141, 142) and Aβo-PrPC disrupting agents
(143, 144).
N
N
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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
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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
tance of the C-linked sugar moiety for this type of therapeutic application (p<
controls). Interestingly, the C-glucosyl moiety was
C
disruption, thus highlighting the impor-
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)
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
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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
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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
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