Design and synthesis of neuroprotective methylthiazoles and modification as NO-chimeras for neurodegenerative therapy

Abstract

Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.

Figure 1

Inhibition by picrotoxin of neuroprotection elicited by MZ derivatives in primary neuronal cultures subject to excitotoxic insult. Primary cortical cultures (10–11 DIV) were subjected to 24 h of glutamate (Glut; 100 μM) toxicity with co-treatment of muscimol (Musc) or MZ derivatives (50 μM). Picrotoxin (Picro; 100 μM) was added, where indicated, 1 h before glutamate treatment to provide blockade of GABA_A signaling. Survival was measured with the MTT assay and normalized to vehicle treated controls. Data shows mean and SEM from at least six individual experiments analyzed by ANOVA with post hoc Dunnett’s MCT comparing to non-glutamate treated vehicle control, or comparing the efficacy of picrotoxin in blocking each drug treatment effect with a two-tailed t-test: * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 2

Representative kinetic traces for hydrolysis of 36 and 37 (10 μM) in phosphate buffer (100 mM, pH 7.4). Incubations were conducted at room temperature and monitored by HPLC with UV detection at 254nm and fit to a pseudo-first order rate equation. A putative mechanism is shown for rate acceleration by anchimeric stabilization of the anionic transition state by the nitrate group acting as intramolecular Lewis acid.

Figure 3

Restoration of CA1-LTP impairment in 3-month-old APP/PS1 mouse hippocampal slices. (A) NO-chimera 32a rescued the LTP impairment in APP/PS1 mice (F_(1,16) = 4.129, p = 0.05, compared with vehicle; 50–80 min analysis); (B) NO-chimera 42 showed a tendency towards reversal of LTP impairment without reaching significance (F_(1,12) = 2.537, p = 0.13, compared with vehicle).

Figure 4

Procognitive effects of NO-chimeras evaluated in the scopolamine-induced memory deficit model using the STPA task. Scopolamine was administered 30 min and drug was administered 20 min prior to the start of training and memory was tested 24 h after training and drug treatment. NO-chimeras, 28, 29, 32a, 32b, 42 and 43 (***, p<0.01, compared with vehicle), and 40 (**, p<0.05, compared with vehicle) significantly reversed the scopolamine induced memory deficit. Data were obtained from at least 5 mice per group.

Figure 5

MRM LC-MS/MS chromatograms of extracted mouse brain samples 20 min after i.p. delivery of each drug at a dosage that was equimolar with 1 (4.5 μmol/kg): A) 26 with 8 used as internal standard; B) 28 with 8 used as internal standard; C) 22a with 22b used as internal standard; D) 32a with 22b used as internal standard.

Scheme 1

Design concept for novel nomethiazole NO-chimeras based upon neuroprotective MZ scaffolds.

Scheme 2

Reagents and conditions: a) Grignard reagent, THF, 0°C to r.t.; b) MsCl, NEt₃ then NaN₃, CH₃CN, r.t.; c) alkyne, CuSO₄·5H₂O, sodium ascorbate, tBuOH/H₂O (1:1, v/v); d) LiAlH₄, THF, reflux; e) MsCl, NEt₃ then NaN₃, CH₃CN, reflux; f) MOM-Br, K₂CO₃, THF; g) ethynyltrimethylsilane, CuI, Pd(Ph₃P)₂Cl₂, NEt₃, THF; h) 3, CuSO₄·5H₂O, sodium ascorbate, K₂CO₃, MeOH-H₂O (2:1); i) HCl/i-PrOH (1.5 M), 70°C; j) 4-nitrooxy-butan-1-ol (for 18) or 1-acetoxy-4-hydroxybutane (for 17), Ph₃P, DIAD, 0°C to r.t ; then K₂CO₃, MeOH (for 17).

Scheme 3

Reagents and conditions: (a) NBS, CH₃CO₂H, dioxane-H₂O; (b) K₂CO₃, MeOH; (c) Grignard reagent, THF, 0°C to r.t.; (d) NaN₃, CH₃CN/H₂O (1:1, v/v), reflux; (e) NaH, DMSO, 90°C, 5h; (f) 3-ethynyl pyridine, CuSO₄·5H₂O, sodium ascorbate, tBuOH/H₂O (1:1, v/v); (g) fuming HNO₃, Ac₂O, CH₂Cl₂, 0°C; (h) 3-ethynyl pyridine, toluene, reflux, 48h; (i) 2-mercaptoethanol, Na₂CO₃, CH₃CN/H₂O (2:1); (j) 3-pyridineboronic acid, (2-biphenyl)di-tert-butylphosphine, Pd(OAc)₂, KF, DMF, 120°C.

Scheme 4

Reagents and conditions: (a) fuming HNO₃, Ac₂O, CH₂Cl₂, 0°C; (b) TEMPO, PhI(OAc)₂, DCM; (c) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, tBuOH; (d) 35 (for 36, 44) or 1-Methylcyclopropane-1-carboxylic acid (for 37), EDCI, DIPEA, HOBT, DCM; (e) NaH, CH₃I, DMF; (f) bis(2-bromomethyl)ether, NaH, DMF; (g) AgNO₃, CH₃CN, reflux; (h) LiAlH₄, THF, reflux; (i) triphosgene, AcOEt, reflux; (j) 34 (for 42) or 4-nitrooxy-butan-1-ol (for 43), NEt₃, THF.