In silico assessment of the metabolism and its safety significance of multitarget propargylamine ASS234

Physiopathological disturbances associated to Alzheimer disease (AD) seem to result from a complex interaction of multiple environmental and genetic factors. Until now, the scientific community has not found an efficient treatment to mitigate the natural course of the disease. However, the design and synthesis of multitarget small molecules,1 able to interact in two or more targets, may open an avenue to explore new treatment options. In this context, N‐((5‐(3‐(1‐benzylpiperidin‐4‐yl) propoxy)‐1‐methyl‐1H‐indol‐2‐yl)methyl)‐N‐methylprop‐2‐yn‐1‐amine (ASS234) (Figure 1) has emerged as a new multitarget small compound for the potential treatment and prevention of AD. Among its reported actions, ASS234 acts simultaneously as both a reversible inhibitor of human acetyl and butyrylcholinesterase (AChE/BuChE), as an irreversible inhibitor of human monoamine oxidase A and B (MAO A/B) and has the ability to inhibit both Αβ_1‐42 and Αβ_1‐40 self‐aggregation and to penetrate into the brain.2, 3 Furthermore, the mechanism of action of ASS234 has been explored both in vitro, playing an interesting role as a modulator of the apoptotic process,3 of the antioxidant system,4 and wingless‐type MMTV integration site family (Wnt) signaling pathway,5 and in vivo, enhancing monoaminergic system 6 and lowering Aβ plaques and gliosis in transgenic mouse model (APPswe/PS1∆E9) of AD.7

Figure 1.

Meteor Nexus V3.1.0 (KB 2018 1.0.0, certified by Lhasa Limited, Leeds, Yorkshire, UK) prediction for ASS234 (first and second generation, plausible reasoning threshold). Supporting evidence likelihood of a single biotransformation is represented as yellow for plausible and orange for probable metabolites. Biotransformation route name, chemical formula, and exact/average molecular mass is displayed for each compound

Collectively, these evidences need additional toxicological information before extrapolating these data to preclinical studies. The European Union legislation and the OCDE Guidance promotes the use of alternative nonanimal methods to obtain toxicological information for a wide variety of chemicals and its derivatives.8, 9 In this way, the present study aimed to assess the ASS234 metabolism and carcinogenic risk. The International Conference on Harmonisation (ICH) of technical requirements for registration of pharmaceuticals for human use has developed a guideline for the assessment and control of mutagenic impurities to limit potential carcinogenic risk, ICH M7.8 This guideline purpose is to provide a framework to identify mutagenic alerts with computational toxicology assessment, this analysis need to be performed using two complementary (Q)SAR methodologies. To reach this objective, we have used an in silico prediction system from Lhasa ltd. (Leeds, UK), Derek Nexus V3.2.0 (expert rule‐based methodology) and Sarah Nexus V3.0.0 (statistical‐based methodology) to obtain a classification.9 From this ICH M7 guideline, regulatory agencies accept results from (Q)SAR methodologies instead of in vitro testing. Nevertheless, application of expert knowledge is necessary to support or overturn any (Q)SAR prediction.10

On the basis of this framework, we firstly work out a likely Phase I metabolism fate of ASS234, predicted by Meteor Nexus version 3.1.0. (KB 2018 1.0.0) (Figure 1). Meteor Nexus predicted first and second generation of metabolites by an Absolute/Relative reasoning, with a minimum likelihood established level of “plausible”. Each predicted metabolite was selected to evaluate its mutagenic potential under the ICH M7 guideline (Table 1).

Table 1.

Metabolites predicted by Meteor Nexus V3.1.0 (KB 2018 1.0.0, certified by Lhasa Limited, Leeds, Yorkshire, UK)

Structure	Likelihood	Formula	Biotransformation Name	Phase I Enzyme	Derek Prediction	Sarah Prediction	QSAR Prediction	ICH M7 Class
ASS234	‐	C29H37N30	‐	‐			Impossible/Negative	Class 5
M1	Plausible	C29H35N3O2	Lactams from Aza‐Alicyclic Compounds	CYP450			Impossible/Negative	Class 5
M6	Plausible	C29H37N3O2	Para Hydroxylation of Monosubstituted Benzene Compounds	CYP450			Impossible/Negative	Class 5
M12	Probable	C28H35N30	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M13	Probable	C22H31N3O	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M14	Probable	C26H35N3O	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M15	Probable	C4H7N	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M16	Probable	C25H30N2O3	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
			Oxidation of Primary Alcohols	ADH
M17	Probable	C3H2O2	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M18	Probable	C7H6O2	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
			Oxidation of Primary Alcohols	ADH
M19	Probable	C25H32N2O2	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M20	Probable	C3H4O	Oxidative N‐Dealkylation	CYP450			Impossible/Positive	Class 2
M21	Probable	C7H8O	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M22	Plausible	C29H39N3O2	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M23	Plausible	C29H37N3O3	Oxidative N‐Dealkylation	CYP450			Impossible/Negative	Class 5
M24	Plausible	C14H16N2O	Beta‐Oxidation of Carboxylic Acids	ACD, ECH, HCD, BKT			Impossible/Negative	Class 5
M25	Plausible	C15H23NO	Oxidative O‐Dealkylation	CYP450			Impossible/Negative	Class 5
M26	Plausible	C15H21NO2	Oxidative O‐Dealkylation	CYP450			Impossible/Negative	Class 5
			Oxidation of Primary Alcohols	ADH
M29	Plausible	C28H35N3O	Oxidative O‐Dealkylation	CYP450			Impossible/Negative	Class 5
M43	Probable	C28H33N3O2	Oxidative N‐Demethylation of Aromatic Heterocycles	CYP450			Impossible/Negative	Class 5
M65	Probable	C28H35N3O2	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M92	Probable	C21H29N3O	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M93	Probable	C25H33N3O	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M95	Probable	C28H37N3O2	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M96	Probable	C28H35N3O3	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M97	Probable	C13H14N2O	Oxidative N‐Demethylation	CYP450			Impossible/Equivocal	Inconclusive
M99	Probable	C27H33N3O	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5
M237	Plausible	C13H17NO2	Oxidative N‐Demethylation	CYP450			Impossible/Negative	Class 5

Meteor was setup for human Phase I biotransformation reactions. ICH M7 Classification outcomes predicted by Derek V3.2.0 and Sarah V3.0.0 for Mutagenicity in vitro (bacterium).

CYP450, Cytochrome P450; ADH, Alcohol dehydrogenase; ECH, Enoyl‐CoA hydratase; HCD, 3‐hidroxyacyl‐CoA dehydrogenase; BKT, Beta‐ketothiolase; ACD, Acyl‐CoA dehydrogenase.

Two complementary (Q)SAR methodologies, Derek (KB 2018 1.1) and Sarah (Model 2.0), predicted an absence of structural alerts for ASS234 and 22 of the 24 metabolites. Under ICH M7 guideline, this is sufficient to conclude that there is no mutagenic concern, and no further testing is recommended (Class 5). (Table 1) However, M97, a second generation metabolite, obtained an uncertain result, whereas Derek nexus predicted a confident negative prediction, Sarah gathered an equivocal prediction (7% negative, the equivocal level is settled at 8%), there is almost an equal weight of evidence for and against the proposition. After reviewing the prediction details and the training set molecules, the most similar compounds to M97 have a negative outcome, and the most different structures that contain well‐known mutagenic regions do not share those regions with M97. Therefore, and in light of confident Derek negative prediction, we can conclude an overall call of negative. M20, a first generation metabolite, fired a positive bacterial mutagenicity result, after finding an exact match to be 100% positive and overturning a negative result obtained by Derek. This means that further hazard assessment and/or control measures are needed (Class 2). Further metabolism studies to confirm the biotransformation route and levels of these possible metabolites should be developed.

In addition, the concept of Threshold of Toxicological Concern (TTC) has been developed to define an acceptable intake for any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effects.8 The exposure to a metabolite identified as a mutagen is not necessarily associated with an increased cancer risk, additional analytical controls should be developed to ensure that the mutagenic impurity is at or below the acceptable cancer risk level.8 In spite of the outcome detected for M20 (Class 2), bacterial mutagenicity positive but no rodent carcinogenicity data available, the existence of a potential impurity structural alert alone is considered insufficient to assess carcinogenicity risk. Considering TTC‐based acceptable intake of mutagenic impurity (1.5 μg person/day) can derive an acceptable limit for control for long‐term treatments.

In light of this structure‐based assessment, there are no bacterial mutagenicity outcome alerts for the parent compound ASS234, thereby there is no mutagenic concern. The first and second generation metabolites displayed interesting results with no mutagenic concern results. Only M20 specific impurity, displayed an alert. Nevertheless, the apparition of M20 in vivo should be verified to avoid false positives. Finally, risk assessment should be developed to derive in acceptable intakes, if needed.

CONFLICT OF INTEREST

The authors declare no conflict of interest.