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10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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306
Figure10.6 Structure of PUGNAc, nagstatin, and GlcNAcstatins designed and tested by
van Aalten and coworkers[52–54].
protonation of the exocyclic nitrogen atom of the imidazole ring would improve
OGA inhibitory properties by mimicking the charge distribution in the oxocarbenium ion, as previously reported for the inhibition of β-glycosidases[56]. Indeed,
this small molecule GlcNAcstatin is a potent inhibitor of a bacterial OGA isolated
from Clostridium perfringens, which shares high sequence similarity with that of
hOGA, showing a K
= 4.6 ± 0.1 pM[52]. By structural analysis studies, the authors
i
could confirm a tight interaction between the catalytic site and the presumably protonated imidazole[52].
By combining structural data gathered from the OGA-PUGNAc complex, e.g.
2
the (Z)-oxime stereochemistry required for activity[57], and sp
the C1 carbon, helping PUGNAc pyranose ring to assume a
hybridization of
4
E envelope conformation to mimic the transition state[58], van Aalten’s group designed and synthesized new derivatives looking for a highly specific inhibition of OGA against
hexosaminidases, by varying the N-acyl substituents and those of the imidazole
ring position2[53, 54, 59].
The first synthesis of GlcNAcstatin[52] is illustrated in Scheme10.6. It was
achieved by an elegant but very long synthetic approach with 15 reaction steps
starting from dibenzylated -xylose 24. Reduction with sodium borohydride and
protection of the free hydroxy groups with tert-butyldimethylsilyl group afforded
the open-chain sugar 25, which was selectively deprotected by acid hydrolysis to
afford the primary alcohol 26. Swern oxidation gave aldehyde 27, which reacted
with the anion of N-tritylimidazole, generated by reaction with butyl lithium, to
give a mixture of imidazoles embodying a -gulo chain in 28 and a -ido chain in
29, isolated in 53% and 18% yield, respectively. Acid detritylation of 28 with
triethylsilane was followed by benzoylation of the free hydroxy group leading
to the formation of 30. Reaction with N-iodosuccinimide gave the 4,5diiodoimidazole derivative 31, submitted to acid hydrolysis to obtain compound
32 with a free hydroxy group. Reaction with trifluoroacetic anhydride to afford
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
307
Scheme 10.6 The first synthesis of GlcNAcstatin[52]. Reagents/solvent and yield: (a) 1.
4
NaBH
, MeOH; 2. TBSCl, ImH, DMF, 95% over the two steps; (b) TFA, H2O, CHCl3, 65%; (c)
(COCl)
, DMSO, Et3N, DCM, 93%; (d) N-tritylimidazole, BuLi, THF, 57, 53% 58, 18%; (e) 1. TFA,
2
DCM, then Et
CH
CN, 90%; (g) HCl,1,4-dioxane, quant.; (h) Tf2O, Py, DCM, 89%; (i) 1. MeONa, MeOH, DCM; 2.
3
TBSOTf, iPr
Pd(PPh
PPh
, THF-H2O, then (iPrCO)2O, Et3N, 95%; (o) Pd(OH)2, H2 14.5psi, AcOH, 60%.
3
SiH; 2. BzCl, Py, DMAP cat., 91% over the two steps; (f) N-iodosuccinimide,
3
NEt, DCM, 91% over the two steps; (j) EtMgBr, THF, 93%; (k) C6H5C≡CH,
2
, CuI, Et3N, DMF, 96%; (l) TBAF, THF, 83%; (m) DPPA, DBU, toluene-THF, 90%; (n)
3)4
the triflate was followed by intramolecular cyclization to give the bicyclic derivative 33. Benzoate hydrolysis was followed by protection with the tertbutyldimethylsilyl group to give derivative 35, which was then submitted to
the Sonogashira coupling with the phenylalkyne to give 36. Reaction with
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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308
tert-butylammonium fluoride afforded the alcohol 37, which was converted into
the azide 38 with diphenylphosphoryl azide. Staudinger reduction was followed
by amine acylation with isobutyric anhydride to afford precursor 39, the hydrogenation of which gave GlcNAcstatin in 7.7% overall yield from the starting
material 24. With the promising biological results obtained for GlcNAcstatin,
van Aalten, and coworkers used this compound as scaffold, and synthesized
GlcNAcstatins A-H (Figure10.6)[53, 54, 59] to further explore the ability of this
compound family to inhibit hOGA and to be selective for hOGA against hexosaminidases. As hOGA active site has a cysteine residue (Cys215), which could
react irreversibly with the inhibitor, van Aalten and coworkers designed
GlcNAcstatins F and G comprising thiol-reactive groups in the acyl side chain
(Scheme10.7)[54]. The synthetic strategy reported in 2010[59] for GlcNAcstatin
and analogs differed from the one developed for the initial GlcNAcstatin[52],
although some reactions were also applied, as shown in Scheme10.7. The starting material used was methyl -mannopyranoside 40, which reacted with
butane-2,3-dione and trimethyl orthoformate in the presence of catalytic camphorsulfonic acid to afford the diacetal protected mannoside 41. Selective protection of the secondary alcohol with the p-methoxybenzyl group gave 42 in 54%
yield, separated from the diprotected compound, obtained in 25% yield. Reaction
of OH-6in 42 with triphenylphosphane and iodine gave the iodo derivative 43.
The reductive opening of the pyranoside ring was carried out with activated zinc
in aqueous THF. The intermediate aldehyde formed was treated with a glyoxal
solution in methanolic ammonia to afford the imidazole 44. Its catalytic dihydroxylation with osmium tetraoxide was highly stereoselective but the configuration of the newly formed chiral center was not the required one. After silylation
of the primary alcohol, the inversion of this configuration resulted from the
introduction of two additional reaction steps, namely Swern oxidation to the
ketone and sodium borohydride reduction, giving compound 46 with the desired
-gulo stereochemistry. After cyclization to give compound 47, the strategy followed to introduce the alkyne moiety was similar to that used in the first synthesis of GlcNAcstatin[52]. Oxidative removal of the p-methoxybenzyl group with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone, azidation, azide reduction, acylation, hydrogenation, and final deprotection with aqueous TFA for 36
the target GlcNAcstatin and GlcNacstatins B, D, G, and H in 3.6%, 4.7%, 4.0%,
2.9%, and 4.0% overall yield, respectively, from compound 40. For GlcNAcstatin
F, the sulfanyl group was then deacetylated, providing this GlcNAcstatin derivative with 2.0% overall yield.
The synthesized compounds were tested to evaluate their potency for OGA inhibition and selectivity for hOGA over human hexosaminidases (Table10.2)[53, 54].
Most of them presented nanomolar K
values, with the exception of GlcNAcstatin B
i
showing a picomolar inhibition constant, the most potent hOGA GlcNAcstatin
inhibitor, and GlcNacstatin E presenting the lowest inhibition (K
Interestingly, GlcNAcstatin G, incorporating a penta-2,4-dien-1-yl group, is the most
selective inhibitor reported so far, with a selectivity over 900 000, eventually resulting from the expected irreversible reaction with the hOGA active site cysteine residue (Cys215)[54]. GlcNAcstatin G also penetrates live cells inducing cellular hyper
O-GlcNAcylation with EC
= 20 nM[54].
50
hours gave
= 8.5 μM).
i
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
309
Scheme 10.7 Preparation of GlcNAcstatin and analogs[59]. Reagents/solvent and yield:
(a) CH
COCOCH3, CH(OCH3)3(3equiv.), cat. CSA, MeOH, 95%; (b) PMBCl, NaH, n-Bu4NI, DMF,
3
54%; (c) I
NH
TIPSCl, Py, 77%; (f) 1. (COCl)
C
2H4Cl2
CuI, Et
H
2
78% or PyBOP, DIPEA, RCO
R=C
R=C
, ImH, PPh3, toluene, 78%; (d) 1. Zn, THF/H2O (10:1); 2. 40% aqueous glyoxal, 7M
2
/MeOH, 70%; (e) 1. K2(OsO4)/K3[Fe(CN)6], K2CO3, CH3SO2NH2, tert-BuOH/THF/water; 2.
3
, DMSO, DCM, then Et3N; 2. NaBH4, EtOH, 81%; (g) Tf2O, P y,
2
, 97%; (h) NIS, DMF, 73% or NIS, MeCN, PPTS, 62%; (i) EtMgBr, THF, 84%; (j) PhC≡CH,
N, Pd(PPh3)4, DMF, 93%; (k) DDQ, DCM/H2O; (l) DPPA, DBU, toluene, 85%; (m) 1.
3
,Pd/C, MeOH or EtOAc, 1 h; 2. (RCO)2O, Et3N, DCM for R=CH3, 93%; R=C2H5, 83%; i-C3H7,
H, DCM, for R=C4H9, 90%; for R=C4H5, 93%, and for
SAc, 74%; (n) TFA/H2O (95:5), R=CH3, 77%; R=C2H5, 73%; R=iC3H7, 70%;
2H4
, 68%; R=C4H5, 48%; R=C2H4SAc, 75%; (o) DMF, MeONa/MeOH, DTT, 56%.
4H9
2
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
65 (Thiamet-G)
76
79
80
81
84
85
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310
Table10.2 Pharmacodynamic of brain O-GlcNac protein in rats treated with the inhibitor
−1
mg kg
(3
, oral dose).
Compound Nr/Structure hOGA Ki (nM)
0.41 1.85 1.75 0.185 0.051 0.94 >2.7
65 (Thiamet-G)
20 1.84 1.74 0.228 0.047 1.49
76
0.53 2.13 1.45 0.099 0.074 1.67
79
9 2.33 1.77 0.222 0.038 0.90 1.5
O-GlcNAc
protein
a
Brain exposure
(nmol g−1)
Brain/Plasma
b
ratio
8 h 24 h 8 h 24 h 8 h 24 h
>18.7
>29.5
80
0.55 2.44 1.65 0.068 0.025 0.60
81
28 1.80 — 0.289 — 2.22 —
84
5.3 1.81 — 0.193 — 8.81 —
85
>7.9
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
86
Table10.2 (Continued)
311
O-GlcNAc
protein
Compound Nr/Structure hOGA Ki (nM)
7.9 2.06 2.17 0.219 0.088 1.84
86
a) Fold increase in O-GlcNAc protein in treated rats compared to vehicle-dosed rats;
b) Brain/plasma ratio=brain concentration (nmol g
limit values 18.7, 7.9, and 20.5 result from plasma exposure below the limit of detection. Source:
Adapted from Selnick etal.[67].
8 h 24 h 8 h 24 h 8 h 24 h
1
)/plasma concentration (μM). The lower
a
Brain exposure
(nmol g−1)
Brain/Plasma
b
ratio
>20.5
In summary, some GlcNAcstatins are, indeed, promising candidates for further
studies toward hOGA inhibitor’s therapeutics. Nonetheless, the challenge seems to
also involve the search for synthetic approaches with less reaction steps to easily
access this family of compounds, facilitating an eventual industrial production.
10.2.2.3 Thiazoline Inhibitors
The discovered mechanism of hOGA inhibition inspired Vocadlo’s group to develop
new transition state mimics by replacing the oxazoline by a thiazoline ring containing a side chain. Linear side chains varying in size and branched chains were investigated, aiming at selectivity for hOGA over lysosomal β-hexosaminidase[39]. For
the purpose, a facile synthetic route was envisioned to access compounds 62a–62g
in good overall yields, in only three steps: N-acylation with RCOCl of peracetylated
2-amino-2-deoxy-β--glucopyranose hydrochloride 59; treatment with Lawesson’s
reagent to afford the thiazoline-fused ring; final deprotection of the acetyl groups
with sodium methoxide in methanol, and neutralization with glacial acetic acid in
methanol (Scheme10.8). This procedure led to the generation of potent inhibitors,
some of them with a remarkable selectivity of hOGA over β-hexosaminidase
(Table10.3). Indeed, the most selective inhibitors are 62c (NButGT) and 62d with
their thiazoline ring containing a propyl group and a butyl group, respectively
(Scheme10.8, Table10.3), but NButGT is the most active and selective inhibitor.
The activity and selectivity of this series were compared to that of PUGNAc
(Figure10.4), a natural product and one of the first cell-permeable hOGA inhibitors
with K
β-hexosaminidase with K
= 46 nM. Unfortunately, PUGNAc also inhibited the human lysosomal
i
=36 nM[39, 60].
i
The disadvantage of these thiazoline-based inhibitors is their limited chemical
stability in solution over periods of days to weeks. Aiming to overcome this issue,
Vocadlo’s group designed and synthesized Thiamet-G starting by N-acylation of
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312
Scheme 10.8 Synthesis of thiazoline-based OGA inhibitors 62a–62g as reported by
Macauley etal.[39] Reagents/solvent: (a) RCOCl, NEt
, DCM (b) Lawesson’s reagent, toluene
3
(c) 1. NaOMe, MeOH; 2. AcOH, MeOH. Source: Adapted from Macauley etal.[39].
compound 59with ethyl isothiocyanate to afford the thiourea derivative 63in very
high yield. After titanium tetrachloride promoted cyclization, the protected bicyclic
compound 64was obtained in 90% isolated yield (Scheme10.9)[61]. Acetyl group
cleavage catalyzed by potassium carbonate gave Thiamet-G 7in 74% overall yield.
Thiamet-G is highly selective for hOGA being able to remove GlcNAc from
O-GlcNAc-modified proteins with K
= 21 nM for hOGA as determined using the
i
Michaelis–Menten method [43]. It crosses the blood-brain barrier and is orally
available. In addition, the authors demonstrated that this inhibitor blocks Tau
Table10.3 Inhibition constants of compounds 62a–62g for hOGA and lysosomal
β-hexosaminidase, and selectivity.
Compound nr.
hOGA Ki
(μM)
β-hexosaminidase Ki
(μM)
β-hexosaminidase Ki/
hOGA K
62a (NAG-thiazoline) 0.070 0.070 1
62b 0.12 32 270
62c (NbutGT) 0.23 340 1500
62d 1.5 4600 3100
62e 57 11
000 100
62f 1.6 720 700
62g 5.7 4000 190
PUGNAc 0.046 0.036 0.8
Source: Adapted from Macauley etal.[39].
i
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Scheme 10.9 Synthesis of Thiamet-G developed by Yuzwa etal.[61] Reagents/solvent: (a)
CH
N=C=S, Et3N, CH3CN (b) SnCl4, DCM (c) K2CO3, MeOH.
3CH2
phosphorylation in cultured neuron-like cells and decreases phosphorylation of Tau
invivo, thus becoming an interesting compound for further investigation of its functional role in AD pathology. In 2014, Yuzwa et al. [62] investigated the role of
O-GlcNAc on APP and β-amyloid production in mice exhibiting both Tau and
β-amyloid pathologies, using Thiamet-G to increase the global levels of O-GlcNAc
in bigenic Tau/APP mutant mice (TAPP mice)[62], and concluded that pharmacological inhibition of OGA prevents cognitive decline and amyloid plaque formation
in the studied mutant mice. They showed that Thiamet-G increases O-GlcNAc levels in TAPP mouse brain, leading to reduction of neuritic plaques and amyloidogenic β-amyloid peptides levels, and blocking the onset of cognitive impairment.
Intrigued by the role of this inhibitor, in 2016, Cekic etal.[40] explored substitution
of the amino group to promote activity and selectivity for hOGA inhibition. They
designed and synthesized a series of Thiamet-G derivatives by modifying
N-substitution, aiming to understand the role of the alkyl side chain size, the influence of altered electronic properties on the binding, and the effect of inhibitor pK
The series comprises compounds type 66 (Scheme10.10), in which the amino group
is either free or substituted with methyl, allyl, ethyl (Thiamet-G), propyl, and butyl
groups, with 2-fluoroethyl, 2,2-difluoroethyl and 2,2,2-trifluoroethyl groups. The
synthetic approach (Scheme 10.10) is inspired by that applied for Thiamet-G
(Scheme10.9). Briefly, reaction of salt 59 either with N-fluorenylmethyloxycarbonyl
(Fmoc) protected or N-allyl protected isothiocyanate afforded intermediates 67a
and 67b, respectively. Cyclization of 67a was accomplished by reaction with titanium tetrachloride, while that of the N-allyl intermediate was possible with
TFA. Deprotection to 66a was carried out as for Thiamet-G, while deacetylation of
68b succeeded with potassium carbonate in methanol. The series 66c–66i and
Thiamet-G (65) were prepared starting from the isothiocyanate 69, which reacted
313
.
a
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314
Scheme 10.10 Preparation of Thiamet-G-based thiazoline inhibitors 65 and 66a–66i as
described by Cekic etal.[40] Reagents/solvent: (a) 1. NEt
SnCl
, py, NEt3; (c) 1. NaOMe, MeOH; 2. AcOH; 3. Piperidine, DMF; (d) allyl-NCS, NEt3, MeCN;
4
(e) TFA, DCM; (f) K
, MeOH; (g) NHR1R2·HCl, NEt3, CH3CN.
2CO3
, DCM; 2. Fmoc-NCS, py, NEt3; (b)
3
with the respective ammonium chloride in acetonitrile in the presence of triethylamine to afford the thiourea intermediate. After cyclization promoted by TFA and
deacetylation with potassium carbonate in methanol, compounds 65 and 66c–66i
were obtained in good yields.
With compounds in hand, Vocadlo and coworkers determined K
values for the
i
inhibition of hOGA and for that of human lysosomal hexosaminidases (Table10.4),
which are the products of HEXA and HEXB genes. They used Michaelis–Menten
kinetics for the less potent inhibitors[40], while for the most potent ones, they
applied the Copeland modified Morrison method[63–65], which can be used when
65 Thiamet-G
66a
66b
66c
66d
66e
66f
66g
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Table10.4 Ki values for hOGA and βHexB, Ki selectivity ratios of inhibitors 65, 66a–66i for
hOGA over hHexB, and pK
values for Thiamet-G and inhibitors 66g–66i.
a
315
Inhibitor
pK
a
Fraction
protonated
at pH 7.4
hOGA K
(nM)
a
hHexB K
i
(μM)
b
hHexB/
i
hOGA
c
2.1 ± 0.3 740 ± 60[40] 350 000 7.68 0.66
4.7 ± 0.3 5.0 ± 0.6
d
1100
3.2 ± 0.4 2850 ± 570 950 000
2.4 ± 0.2 13.0 ± 3.8 5400
0.51 ± 0.05 1.70 ± 0.19
d
3300
2.0 ± 0.2 3700 ± 670 1 850 000
350 ± 90d4800 ± 763 13 700
d
15 ± 5
180 ± 44 12 000 6.92 0.2
(Continued)
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