Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5625_Библиотеки_им_академика_М_И_Перельмана
.pdf
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
state analogs[40]. This substrate-assisted catalytic mechanism is followed by both
hOGA and human lysosomal β-hexosaminidases, which are hydrolases of family 20
(GH20) [43], cleaving GlcNAc from terminal glycoproteins/glycosphingolipids.
Nonetheless, Vocadlo’s group experiments, in 2005[39], showed that the active site
of hOGA tolerates bulkier acetamido substituents than the one of lysosomal
β-hexosaminidase, which encouraged them to design new structures aiming at
selectivity for hOGA inhibition.
10.2.2.1 PUGNAc
In the search for OGA inhibitors, Vasella, and coworkers, in 1990, were inspired by
aldonolactones, known as potent glycosidase inhibitors, and particularly by 2acetamido-2-deoxyglucono-1,5-lactone, reported to inhibit OGA, although with a μM
. As both glucono-1,5-lactone oxime and its phenylcarbamoyl derivative were better
K
i
inhibitors of β-glucosidase than the corresponding lactone, Vasella synthesized for the
first time 2-acetamido-2-deoxy--glucono-1,5-lactone O-(phenylcarbamoyl)oxime
(PUGNAc) (Scheme 10.5)[45] to explore its ability to inhibit OGA. Starting from
oxime 1 oxidized with MnO
, they prepared the cyclized benzylidene protected (Z)-2
2
in 50% yield, with starting material recovered in 35% yield. Debenzylidenation with
Na/NH
and acetylation was followed by partial deacetylation with CH3NH2 remain-
3
ing the sugar hydroxy groups acetylated (compound 3). Reaction with phenyl isocyanate gave carbamate 5, which was deprotected with ammonia in methanol to afford
PUGNAc in 16.8% overall yield starting from oxime 1 (Scheme10.1).
Vasella and coworkers have then tested PUGNAc for the inhibition of OGA from
animal, fungal, and plant origin. They found out that PUGNAc was very effective,
particularly for the inhibition of the fungal enzyme, exhibiting K
= 40 nM[47]. This
i
potent OGA inhibitor was also the best of any glycosidase inhibitors described at
that time. However, the first synthesis has severe problems with the scale-up,
301
Scheme 10.1 The first synthesis of PUGNAc by Beer etal.[45, 46]. Reagents/solvent and
yield: (a) Activated MnO
CH
OH, 71%, (e) PhNCO, Et3N, THF, 94%; (f) saturated NH3, MeOH, 61%.
3
, MeOH, 50%; (b) Na, NH3, MeOH, 86%; (c) Ac2O, Py, 96%; (d) CH3NH2,
2
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
302
Scheme 10.2 Improved approach to prepare PUGNAc by Vasella and Mohan[48].
Reactions/solvent and yield: (a) (NH
(c) DBU, NCS/DCM, 59%; (d) PhNCO, Et
, THF/MeOH, 74%; (b) NH2OH·HCl, py/MeOH, 75%;
4)2CO3
N, DCM, 70%; (e) Aq. NH3, 73%.
3
particularly the oxidation step with MnO2, which encouraged Vasella group to
improve the synthetic approach for PUGNAc (Scheme 10.2)[48]. Starting from
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α--glucopyranose 6, the anomeric deacetylation was carried out by ammonium carbonate to give 7 in 74% yield from 100 g
starting material. The open chain oxime 8 was obtained by treatment of 7 with
hydroxylammonium chloride and pyridine in MeOH under reflux. Reaction with
N-chlorosuccinimide in DBU afforded compound 9, which reacted with PhNCO in
THF to yield phenyl carbamate 10. De-O-acetylation with saturated aqueous NH
afforded PUGNAc in 16.7% overall yield.
As shown by Vocadlo and coworkers[39], PUGNAc is a potent inhibitor of hOGA
= 46 nM) but unfortunately, it is also a potent inhibitor of β-hexosaminidase
(K
i
= 36 nM). The dysfunction of this enzyme results in the accumulation of ganglio-
(K
i
sides and other glycoconjugates in the lysosome, causing Tay Sachs and Sandhoff
neurodegenerative diseases, as the enzyme is localized in the lysosome. This lack of
selectivity encouraged the modification of PUGNAc structure by varying the 2-N-acyl
chain and Stubbs etal.[49] reported the synthesis of PUGNAc and analogs starting
from glucosamine hydrochloride to prepare the 2-N-Boc protected precursor 11, submitted to the reaction conditions previously reported by Mohan and Vasella[48] to
prepare 12 (Scheme10.3)[49]. Treatment with phenyl isocyanate yields the carbamate 13, whose Boc deprotection is achieved with trifluoroacetic acid (TFA). Further
reaction with acyl chloride in pyridine and acetyl deprotection to release the sugar
hydroxy groups with NH
and its desired analogs. Unfortunately, they showed K
/MeOH afforded PUGNAc in 5.7% overall yield from 11
3
for hOGA and for human
i
hexosaminidase in the μM range and their selectivity was very poor[49].
The crucial step in both the synthetic approaches followed by Vasella group[48]
and Vocadlo group[49] is the N-chlorosuccinimide-mediated oxidative ring closure
of the oxime precursor in relatively low yields, as a secondary product with a fivemembered ring resulting from acetyl migration is formed. Moreover, the separation
3
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
Scheme 10.3 Synthesis of PUGNAc and N-acylated derivatives as reported by Stubbs
etal.[49] in 2006. Reagents/solvent and yield: (a) i. NH
DCM, 52% over the two steps; (b) PhNCO, Et
(48%); (e) NH
, MeOH, PUGNAc (32%), 16a (32%), 16b (26%), 16c (23%), 16d (26%), 16e
3
N, THF, 71%; (c) CF3COOH, DCM; (d) RCOCl, py, 5
3
OH·HCl, py, MeOH; ii. DBU, NCS,
2
(29%) 16f (23%) yield over two steps.
of both products was found to be quite difficult[48, 49]. Aiming to overcome this
problem, Goddard–Borger and Stubbs[50] developed a new approach, starting from
the peracetylated 2-azido precursor 17 (Scheme10.4), obtained from glucosamine in
two steps and good overall yield[48, 50]. This azido sugar reacted with benzylamine
to deacetylate the anomeric position. Synthesis of the open chain oxime succeeded in
very high yield (97%) and ring closure with DBU and N-chlorosuccinimide gave 19 in
93% yield with the desired (Z)-stereochemistry. The reductive acylation of the azido
303
Scheme 10.4 PUGNAc synthesis developed by Goddard-Borger and Stubbs[50]. Reagents/
solvent and yield: (a) NH
THP, 91%; (d) 1. PMe
OH·HCl, py, MeOH, 97%; (b) DBU, NCS, DCM, 93%; (c) PhNCO, Et3N,
2
, AcOH, 2-(PhSe)2, CH2Cl2, toluene, water, 89%; 2. NH3/MeOH, 61%.
3
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
304
group was carried out by reaction of 19 with acetic acid, trimethylphosphine, and a
catalytic amount of 2,2′-diphenyldiselenide to afford PUGNAc in 89% yield. This
was, indeed, the most fruitful and high-yielding methodology to synthesize PUGNAc,
whose access in reasonable quantities has been required for research, due to its broad
spectrum of inhibitory activity acting on enzymes interfering in a wide number of
biological processes. Interestingly, it was used for the synthesis of PUGNAc analog
with galacto configuration, termed Gal-PUGNAc[50], which is a potent and selective
inhibitor of lysosomal exo-N-acetyl--glucosaminidases[51].
Pursuing their research to control PUGNAc selectivity, Stubbs and coworkers[44], in 2016, synthesized new PUGNAc analogs, where the carbamoyl moiety is
varied in size, hydrophobicity, and shape. For the purpose, they envisioned three
series of compounds. In one of them, the PUGNAc phenyl group is substituted in
the para-position with methyl or methoxy groups, or with a bromine atom, or
replaced by a benzyl group. Another series has a cyclic or acyclic aliphatic structure
replacing the phenyl group and the third one has NHPh of the carbamate replaced
by an amino acid derivative. To access PUGNAc and the designed 63 compounds,
the authors developed a new methodology, in which a colorogenic intermediate carbonate 21 is formed in situ by reaction of 9 with 4-nitrophenyl chloroformate, and
then transformed into carbamates in good overall yield by reaction with anilines or
amines, in a one-pot reaction (Scheme 10.5). The inhibition of OGA and of
Scheme 10.5 Synthesis of PUGNAc and representative analogs with modified carbamate
moiety as developed by Stubbs and coworkers[44]. Reagents/solvent: (a) 1. 4-nitrophenyl
chloroformate, DIPEA, THF; 2. Amine or aniline-based compound, DIPEA; (b) NH
, MeOH.
3
O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
10.2
Table10.1 Inhibition constant of compounds 23a–23i over OGA and HEXB.
K
(μM)
i
305
Inhibitor
PUGNAc 0.036[34] 0.046[34]
23a 0.021
23b 0.111
23c 0.047
23d 14
23e 1.3
23f 45
23g 17
23h 161
23i 0.77
Source: Adapted from Hattie etal.[44].
HEXB OGA
± 0.005 0.028±0.007
± 0.019 0.178 ± 0.042
± 0.002 0.056 ± 0.016
± 3.5 230 ± 78
± 0.17 78 ± 26
± 16 420 ± 140
± 4 220 ± 75
± 8 188 ± 72
± 0.031 11 ± 4.8
β-hexosaminidase B found for this small library of compounds and the Ki values of
the most representative compounds (Table10.1) showed that the aryl derivatives
have, in general, a similar potency as PUGNAc, although the electron-donating
methoxy group decreased potency as compared to PUGNAc. All the aliphatic derivatives were less potent than PUGNAc for both OGA and HEXB, which is the product of the lysosomal hexosaminidase gene HEXB. Nonetheless, the results obtained
demonstrate that the envisioned structural changes were efficient in tuning selectivity to HEXB over OGA, leading to the discovery of potent and selective inhibitors
of HEXB.
In conclusion, all efforts to achieve selectivity of PUGNAc for OGA failed, either
by changing the carbamate moiety, or the sugar N-acylation. Also, the Gal-PUGNAc
analog, in which sugar position 4has the opposite configuration of that exhibited by
PUGNAc, is a selective inhibitor of the human lysosomal β-hexosaminidases,
changing GM2ganglioside levels in cultured cells, but is not able to affect O-GlcNAc
levels [51]. Nonetheless, these selective HEXB inhibitors may become powerful
small molecules for defining the role played by GM2in biological processes at the
molecular level in diseases with abnormal accumulation of GM2, and for creating
models to follow up disease stages.
10.2.2.2 GlcNAcstatins
In 2006, van Aalten and coworkers[52] presented the first rationally designed glucoimidazole GlcNAcstatin, inspired by the structure of nagstatin (Figure 10.6), a
natural product and potent inhibitor of hexosaminidase[55]. GlcNAcstatin has a
molecular architecture similar to that of nagstatin, bearing an isopropylamido
group on position 8 and a phenethyl group at position 2. The authors expected that
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
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
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 hours gave
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
= 8.5 μM).
i
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
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
310
Table10.2 Pharmacodynamic of brain O-GlcNac protein in rats treated with the inhibitor
−1
(3 mg kg
, 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
81
84
85
0.55 2.44 1.65 0.068 0.025 0.60
>7.9
28 1.80 — 0.289 — 2.22 —
5.3 1.81 — 0.193 — 8.81 —
Соседние файлы в папке Библиотека им академика М.И. Перельмана