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10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration

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Table10.2 (Continued)

311

O-GlcNAc protein

Compound Nr/Structure hOGA Ki (nM)

7.9 2.06 2.17 0.219 0.088 1.84

a) Fold increase in O-GlcNAc protein in treated rats compared to vehicle-dosed rats; b) Brain/plasma ratio=brain concentration (nmol g1)/plasma concentration (μM). The lower limit values 18.7, 7.9, and 20.5 result from plasma exposure below the limit of detection. Source: Adapted from Selnick etal.[67].

8 h 24 h 8 h 24 h 8 h 24 h

Brain exposure (nmol g−1)

Brain/Plasma

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 (Scheme10.8). This procedure led to the generation of potent inhibitors, some of them with a remarkable selectivity of hOGA over β-hexosaminidase (Table10.3). Indeed, the most selective inhibitors are 62c (NButGT) and 62d with their thiazoline ring containing a propyl group and a butyl group, respectively (Scheme10.8, Table10.3), but NButGT is the most active and selective inhibitor. The activity and selectivity of this series were compared to that of PUGNAc (Figure10.4), 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

=36 nM[39, 60].

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

10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease

312

Scheme 10.8 Synthesis of thiazoline-based OGA inhibitors 62a–62g as reported by

Macauley etal.[39] Reagents/solvent: (a) RCOCl, NEt

, DCM (b) Lawesson’s reagent, toluene

compound 59with ethyl isothiocyanate to afford the thiourea derivative 63in very high yield. After titanium tetrachloride promoted cyclization, the protected bicyclic compound 64was obtained in 90% isolated yield (Scheme10.9)[61]. Acetyl group cleavage catalyzed by potassium carbonate gave Thiamet-G 7in 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

Michaelis–Menten method [43]. It crosses the blood-brain barrier and is orally available. In addition, the authors demonstrated that this inhibitor blocks Tau

Table10.3 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 etal.[39].

10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration

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Scheme 10.9 Synthesis of Thiamet-G developed by Yuzwa etal.[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 invivo, 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 etal.[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 (Scheme10.10), 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 (Scheme10.9). 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

10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease

314

Scheme 10.10 Preparation of Thiamet-G-based thiazoline inhibitors 65 and 66a–66i as

described by Cekic etal.[40] Reagents/solvent: (a) 1. NEt SnCl

, py, NEt3; (c) 1. NaOMe, MeOH; 2. AcOH; 3. Piperidine, DMF; (d) allyl-NCS, NEt3, MeCN;

(e) TFA, DCM; (f) K

, MeOH; (g) NHR1R2·HCl, NEt3, CH3CN.

2CO3

, DCM; 2. Fmoc-NCS, py, NEt3; (b)

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

inhibition of hOGA and for that of human lysosomal hexosaminidases (Table10.4), 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

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Table10.4 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.

315

Inhibitor

Fraction protonated at pH 7.4

hOGA K (nM)

hHexB K

(μM)

hHexB/

hOGA

2.1 ± 0.3 740 ± 60[40] 350 000 7.68 0.66

4.7 ± 0.3 5.0 ± 0.6

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

3300

2.0 ± 0.2 3700 ± 670 1 850 000

350 ± 90d4800 ± 763 13 700

15 ± 5

180 ± 44 12 000 6.92 0.2

(Continued)

10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease

66h

66i

316

Table10.4 (Continued)

Inhibitor

hOGA K (nM)

60 ± 10

hHexB K

(μM)

hHexB/

hOGA

150 ± 50 2500 6.18 0.06

Fraction protonated at pH 7.4

1000 ± 200d4200 ± 1525 4200 5.33 0.01

a) Determined with the Morrison Ki fit if values are below 5 mM; b) Determined using Dixon plot analysis; c) Selectivity ratios indicating favored selectivity for hOGA over hHexB; d) Determined using Michaelis–Menten inhibition analysis. Source: Adapted from Cekic etal.[40].

Ki values are comparable to the enzyme concentration being studied, and reached

some remarkable results. The first one is related to Thiamet-G, in which K

value is

2.1 nM instead of the 21 nM given previously when evaluated by the Michaelis– Menten method[43]. The first picomolar hOGA inhibitor discovered is LSO, exhibit-

= 510 ± 50 pM and a selectivity of 3300 for hOGA over β-hexosaminidase. The

ing K

size of the chain is also important for binding. Compounds embodying N-methyl, N-ethyl, N-propyl, or N-allyl groups have K

compound with the N-butyl group suffered 100-fold decrease of activity (K

in the range of 2.0–3.2 nM, while the

= 350 nM).

The side chains with two and three carbon atoms are, indeed, the most important to increase selectivity for hOGA inhibition over β-hexosaminidase inhibition, as clearly deduced from selectivity values given in Table10.5. Interestingly, compounds 8a

Table10.5 Inhibition data of hOGA and lysosomal hexosaminidases (HexA/B) by GlcNAcstatins A-G, PUGNAC, Thiamet-G, and selectivity for hOGA[53, 54].

Compound hOGA (Ki nM) HexA/B(GH20) (Ki) Selectivity GH20/hOGA References

GlcNAcstatin 4.4 ± 0.1 550 ± 10 nM 164 [58] GlcNAcstatin A 4.3 ± 0.2 0.55 ± 0.05 nM Not selective [58] GlcNAcstatin B 0.42 ± 0.06 0.17 ± 0.05 nM Not selective [58] GlcNAcstatin D 0.74 ± 0.09 2.7 ± 0.4 nM 4 [58] GlcNAcstatin E 8500 ± 300 1100 ± 100 nM Not selective [58] GlcNAcstatin F 11.2 ± 1.4 11.0 ± 0.6 μM 1000 [59] GlcNAcstatin G 4.1 ± 0.7 >3700 >900 000 [59] GlcNAcstatin H 2.6 ± 0.3 100±30 35 000 [59] PUGNAc 35 ± 6 25 ± 2.5 Not selective [59] Thiamet-G 21 750 35 000 [43, 59]

10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration

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and 8d have some selectivity for hOGA, which is not the case for NAG-thiazoline 4a, a molecule with a similar size[39, 40]. This result reinforces the key role of the amino group in improving inhibitor selectivity over human β-hexosaminidase.

The contribution of inhibitor pK the results obtained for the fluorinated inhibitors. pK

determined by

C nuclear magnetic resonance (NMR) titration[40, 66]. The correla-

tion obtained by plotting pK

for the potency of the inhibition stood out from

of their conjugate acids was

with the corresponding logKi value suggested that pKa

dominates the effect of inhibitor binding to the active site, as compared to the steric effect resulting from increased fluor substitution. The key role of pK

in binding may

be due to optimization of hydrogen bond strength or favoring inhibitor protonated form. Quantitative methods carried out by Cekic et al. [40] demonstrated that, through their formal positive charge at physiological pH, these compounds have favorable interactions with the active site, only partly carried out within the transition state for the natural 2-acyl substrates, showing that Thiamet-G and analogs embodying an alkylamino group are tight-binding transition state analogs for hOGA. This work gave, in 2016, a new insight into the catalytic mechanism of hOGA and provided a new picomolar inhibitor representative of this compound series[40].

More recently, in 2019, further studies on this family of compounds were reported, inspired by Thiamet-G, which is well tolerated over extended treatment periods. However, it was found that it has a high polar surface (105 Å), resulting in a low diffusion rate into and out of the CNS from systemic circulation[67]. Aiming to obtain better clinical candidates, the collaboration of the companies Alectos Therapeutics Inc, Merck, and Pharmaron Beijing resulted in the generation of MK8719 (86, see Table10.6, Figure10.7), the Thiamet G analog, which is a highly potent (K

= 7.9 nM

for hOGA) and selective OGA inhibitor with excellent CNS penetration, and has been advanced to phase I clinical trials[68]. It stood out of a small library of 49 Thiamet G analogs generated by hypothesizing that modification of molecule polar substituents would result in a reduced topological polar surface area (TPSA) and consequently in a greater and faster distribution of the compound into CNS[67].

Focusing on the carbohydrate moiety hydroxy groups and the thiazolinesubstituted amino group, a series of analogs with structure type A (Figure10.7) was synthesized and tested[67]. Regioselective methylation of hydroxy groups or its replacement by hydrogen or by fluor atoms afforded compounds with lower TPSA. However, the potency of hOGA inhibition varied according to inhibitor structure and stereochemistry (Table 10.3). No inhibition occurred when OH-4′ was absent, as shown for compounds 70, 74, and 77, indicating that this group is important for activity. Nonetheless, some structural changes are tolerated without complete loss of activity, e.g. the replacement of the primary hydroxyl group by hydrogen (compound 73) or by fluor (compound 76), and that of OH-3′ by fluor (compounds 78 and 79). The absolute configuration was shown to be important for hOGA inhibition, together with the replacement of the N-ethyl group by the N-methyl group. By combining monofluorination of the primary alcohol with a 7-fluoro substituent in compound 82, the pharmacokinetic properties were improved when compared to the monofluorinated compound 81, as well as the selectivity over hexosaminidase inhibition, the TPSA value and the apparent permeability.

317

10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease

65 (Thiamet-G)

318

Table10.6 Data obtained in assays to determine the inhibition of hOGA, the concentration required for EC

values for elevation of all protein O-GlcNAc levels, the

selectivity over hexosaminidases, calculated TPSA, and apparent permeability.

Compound Nr/Structure

hOGA Ki (nM)

rOGA cell EC50 (nM)

hHEX Ki (nM) TPSA (Å)

Papp (10−6 cm s−1)

0.41 13.5 >10 000 105 <1.0

190 — — 94 —

>3000 — — 91 —

270 — >10 000 91 —

5.5 36.7 3600 93 1.1

69 — — 84 —

>3000 — — 86 7.0

44 364 — 84 2.6

20 176 — 84 6.1

(Continued)

10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration

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Table10.6 (Continued)

319

Compound Nr/Structure

hOGA Ki (nM)

rOGA cell EC50 (nM)

hHEX Ki (nM) TPSA (Å)

Papp (10−6 cm s−1)

>3000 — >10 000 84 —

29 443 >10 000 84 2.7

0.53 10.6 >10 000 84 —

9.0 177 1600 69 6.8

0.55 34.4 790 83 5.3

35 328 >10 000 61 26

>3000 — >10 000 47 31

28 — >10 000 47 31

(Continued)

10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease

86 (MK8719)

(a) (b)

320

Table10.6 (Continued)

Compound Nr/Structure

hOGA Ki (nM)

rOGA cell EC50 (nM)

hHEX Ki (nM) TPSA (Å)

Papp (10−6 cm s−1)

5.3 — >10 000 61 24

7.9 52.7 >10 000 80 6.4

a) Absolute configuration at position 6was not assigned. Source: Adapted from Selnick etal.[67].

The high permeability found for the difluorinated compound 82 inspired further research with structures type B (Figure10.7) and this scaffold was investigated for modifications of the amino group substituent. Compound 83, bearing a dimethylamino group, does not inhibit hOGA, while compounds 84–86, with a monosubstituted amino group, have restored this activity. Nonetheless, compound 86 (MK8719) is the one showing a good balance of hOGA potency, apparent permeability, and selectivity vs. hHEX.

The collection of Papp data for 49 compounds tested was compared to the calculated TPSA values and a reasonable correlation was found, corroborating the hypothesis based on reducing TPSA to obtain compounds with higher permeability

Figure10.7 Structure of 5-substituted methyl tetrahydropyranothiazole compounds type (a) and of 5-difluoromethyl tetrahydropyranothiazoles type (b) studied by Selnick etal.[67] to illustrate TPSA/permeability and structure/activity relationships. Source: Adapted from Selnick etal.[67].

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