Subnanomolar indazole-5-carboxamide inhibitors of monoamine oxidase B (MAO-B) continued: indications of iron binding, experimental evidence for optimised solubility and brain penetration

Figures & data

Figure 1. Structures of selective MAO-B inhibitors.

Table 1. MAO inhibitory activity and HYDE estimated affinities against hMAO-B.

	IC50 (nM)^a					Binding affinity (kJ/mol)^e
Compound	hMAO-A	hMAO-B	SI^b	Ki (nM)^c	Ki HYDE (nM)^d	ΔH	−TΔS	ΔG
NTZ-1006	>10,000	0.59 ± 0.09	>16959	0.26 ± 0.04	2–185	−38.0	−6.2	−44.2
NTZ-1032	>10,000	0.68 ± 0.04	14706	0.30 ± 0.02	4–400	−38.0	−4.2	−42.2
NTZ-1034	>10,000	1.59 ± 0.16	>6289	0.70 ± 0.07	5–464	−38.0	−3.8	−41.8
NTZ-1091	>10,000	0.39 ± 0.05	25,641	0.17 ± 0.02	0–37	−38.0	−9.9	−47.9
NTZ-1441	≥10,000	0.66 ± 0.06	≥15,151	0.29 ± 0.03	1–103	−38.0	−7.5	−45.5
NTZ-1471	>10,000	1.52 ± 0.18	6579	0.67 ± 0.08	1–112	−38.0	−7.3	−45.3
Selegiline	1700^f	6.59 ± 1.09^f	258	2.91 ± 0.48	8–757	0.0	−41.4	−41.4
Safinamide	45,000^f	5.18 ± 0.04^f	5000^f	2.29 ± 0.02	2–187	−21.8	−22.5	−44.3

^a The values are the mean ± SEM (n = 3).

^b SI: Selectivity Index = IC₅₀ (hMAO-A)/IC₅₀ (hMAO-B).

^c The K_i values were calculated from the experimentally measured IC₅₀ hMAO-B values according to the equation by Cheng and Prusoff²⁴: K_i =IC₅₀ (1 + [S]/K_m) with substrate concentration of p-tyramine [S] = 150 μM and Michaelis constant K_m =118.8 μM.

^d K_{i HYDE}: estimated HYDE K_i range values from the compounds’ best docking conformations within the human MAO-B (PDB: 2V5Z²⁰).

^e HYDE estimated thermodynamic binding values; ΔG: Gibbs free energy = ΔH –TΔS; ΔH: sum of interactions (enthalpy); −TΔS: sum of desolvation terms (entropy).

^f Data from Ref.¹⁵.

Figure 2. (A) SeeSAR visualisation of the binding of NTZ-1006 (blue) and NTZ-1091 (off-white) overlaid onto the crystal structure of the hMAO-B-safinamide complex (PDB: 2V5Z). HYDE visual affinity assessment: green = favourable, red = unfavourable and non-coloured = not relevant for affinity. (B) Bar diagrams representing a semi-quantitative decomposition of enthalpic (ΔH: sum of interactions) and entropic part (−TΔS: sum of desolvation terms) for all heavy atoms of the Gibbs free energy (ΔG, kJ/mol) of tested compounds in comparison to the reference MAO-B inhibitor safinamide (SAF).

Figure 3. (A) Reactivation of recombinant human MAO-B enzyme by irreversible inhibitor selegiline (30 nM), reversible inhibitor safinamide (50 nM), and representative compounds NTZ-1006 (1.0 nM) and NTZ-1091 (1.0 nM). (B) Lineweaver–Burk plot of the inhibition of hMAO-B enzyme in the absence (no inhibitor) and in the presence of different concentrations (0.5 and 1.0 nM) of NTZ-1091. The reciprocal hMAO-B inhibitory activity of the compound was plotted versus the reciprocal substrate concentration. 1/V: 1/velocity of reaction [1/(nmol p-tyramine/min/mg protein)]; 1/[S]: 1/substrate concentration (1/mM p-tyramine).

Table 2. Physicochemical, drug-like, and in vitro ADME properties of investigated and reference MAO-B inhibitors.

Compound	MW	pIC50	Stability	tPSA^a	%ABS	HBA/D^a	logBB^a	logS7.4	logD7.4	LLE
NTZ-1006	306.15	9.23	Stable	57.78	89.07	4/1	0.065	n.d. (−4.56^a)	n.d. (3.73^a)	5.50
NTZ-1032	289.04	9.17	Stable	57.78	89.07	4/2	0.030	n.d. (−4.48^a)	n.d. (3.00^a)	6.17
NTZ-1034	273.24	8.80	Stable	57.78	89.07	4/2	−0.002	−4.22 (−4.61^a)	2.04 (2.40^a)	6.76
NTZ-1091	320.17	9.41	Stable	46.92	92.81	4/2	0.246	−4.75 (−4.82^a)	3.24 (3.76^a)	6.17
NTZ-1441	303.06	9.18	Stable	46.92	92.81	4/2	0.216	−3.87 (−4.76^a)	2.19 (3.15^a)	6.99
NTZ-1471	287.27	8.82	Stable	46.92	92.81	4/2	0.187	−3.67 (−4.81^a)	n.d. (2.64^a)	6.18
SEL	187.29	8.18	–	3.24	107.88	4/1	0.570	n.d. (−4.10^a)	n.d. (2.35^a)	5.83
SAF	302.35	8.29	–	64.68	86.67	4/1	−0.083	n.d. (−4.06^a)	n.d. (2.89^a)	5.40
^b,^cCNS+	≤400	>8	–	<70	≥60	≤7/≤3	≥–1	≥ −5.0	1–4	>5

MW: molecular weight; pIC₅₀ at human MAO-B; Stability: chemical stability of compounds in 10 mM phosphate buffer at pH 7.4; tPSA: topological surface area (in Ų); %ABS: % of absorption = 109–0.345×tPSA (Ref.²⁹); HBA/D: number of hydrogen bond acceptors/donors; logBB: blood-brain partition coefficient (lit. 31); logS: solubility (in mol/L) at pH 7.4 in 60 mM phosphate buffer or pure water at rt; logD: distribution coefficient at pH 7.4 in 60 mM phosphate buffer at rt; LLE: ligand-lipophilicity efficiency = pIC50–logD (Ref.³⁰); n.d.: not determined; SEL: Selegiline; SAF: Safinamide.

^a Calculated values using the StarDrop module in SeeSAR 5.5, 2017¹⁸.

^b Ref.^25–27.

^c CNS+: required ranges for compound penetration to the central nervous system (CNS).

Figure 4. (A) HPLC chromatogram of a standard 1.0 mM equimolar mixture of compounds NTZ-1471 (a, ret. time 7.52 min), NTZ-1441 (b, ret. time 8.25 min) and NTZ-1091 (c, ret. time 9.17 min) in methanol (MeOH). Conditions: 40% methanol mobile phase, 10 μL sample injection volume, and 250 μL/min flow rate. (B) Mean solubility of the compounds in pure water and 50% methanol measured by LC/ESI-MS method. (C) Distribution of BBB mean permeability of standard drugs and subclass II compounds measured in the PAMPA assay.

Table 3. Solubility and PAMPA-BBB data for selected compounds.

	Solubility in water^a		Solubility in 50% methanol^a		PAMPA-BBB^a,^b
Compound	μg/mL	μM	μg/mL	mM	Pe (×10^−6 cm/s)	−logPe
NTZ-1091	5.62 ± 0.17	17.6 ± 0.5	156 ± 19	0.49 ± 0.06	117.5 ± 6.4	3.91
NTZ-1441	42.1 ± 2.3	136 ± 5	609 ± 49	2.04 ± 0.18	144.2 ± 3.4	3.84
NTZ-1471	61.6 ± 9.6	215 ± 33	610 ± 26	2.12 ± 0.09	148.1 ± 2.4	3.83

^a The values are the mean ± SD (n ≥ 3).

^b PAMPA blood-brain barrier (BBB) values were determined under stirring at the pH value of the Prisma HT buffer (Pion).

Figure 5. UV–visible studies of Fe²⁺ or Fe³⁺ chelating ability of deferiprone (DFP) in the absence or presence of 10 mM FeSO₄ (A) and FeCl₃ (B) and compounds NTZ-1006 (black line), NTZ-1032 (red line), and NTZ-1441 (blue line) before and after addition of 40 μL of 10 mM FeSO₄ (C), or FeCl₃ (D). Superposition UV–visible spectra were obtained by measurement of absorbance of compound alone (50 μM) and FeSO₄ or FeCl₃ without compound (both 130 μM) in DMSO 50%.

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