Synthesis and In-Silico Analysis of Novel Tetrahydroquinolines and Their Antioxidant Activity ^†

Abstract

Within the area of study of neurodegenerative diseases, particularly Alzheimer’s disease (AD), this research focused on the synthesis and evaluation of novel tetrahydroquinoline (THQ) derivatives with potential antioxidant activity. The toluidine N-propargylation synthesis protocol was optimized, achieving a significant increase in yield by using sodium carbonate and reaction temperature variation. Subsequently, four THQ compounds with alkene variation were successfully synthesized, including some which had not been previously reported in the literature. The synthesized compounds were characterized by nuclear magnetic resonance (NMR), mass spectrometry, and infrared spectroscopy (IR), which confirmed their structures and purity. In silico analyses performed with SwissADME and OSIRIS Property Explorer indicated that most of the compounds exhibited excellent drug-like characteristics and favorable pharmacokinetic profiles. Antioxidant evaluation was performed using DPPH and ABTS assays, in which all compounds demonstrated excellent antioxidant capacity, with EC50 values below 10 μg/mL in the ABTS assay, significantly outperforming the ascorbic acid control (EC50 = 35 μg/mL). The results suggest that the predominant radical-scavenging mechanism is single electron transfer (SET). This study provides a solid foundation for further investigations into the potential of THQs’ derivatives as antioxidants, and potential cholinesterase inhibitors in the context of neurodegenerative diseases such as Alzheimer’s. As a future projection, an enzymatic evaluation, including regarding mechanisms of action and the exploration of a hybrid synthesis of THQ/triazole, is proposed based on these promising results.

1. Introduction

Tetrahydroquinolines (THQs) are nitrogen-containing heterocyclic organic compounds that exhibit a wide range of biological activities, including antioxidant and cholinesterase inhibitory effects [1]. Furthermore, research suggests that these compounds may play a role in cancer treatment [2,3] and exhibit antibacterial activity [4].

One practical way to obtain THQs is by organic synthesis. Different methods have been described for obtaining these compounds [5], with the Povarov or hetero Dields–Alder reactions being two of the most widely used [6]. These reactions have evolved, generating different options based on the reagents and catalyst used [7]. In general terms, the Povarov reaction involves the condensation of three components: an aniline, an aldehyde and an electron-rich alkene. The reaction requires a Lewis acid as a catalyst and results in a THQ [8].

Because THQs are compounds with bioactive properties, there is growing interest in further improving such capabilities. Organic synthesis allows the design and obtaining of specific organic compounds, in this case THQs. Consequently, a rational design of these compounds can be carried out, seeking to improve their properties, whether antioxidant, inhibitory, antibacterial, or otherwise.

As an objective of this study, we propose the synthesis of novel THQs with variations in the types of alkene used in the reaction, in order to synthetize novel THQ with alkenes like indene and vinyl caprolactam, which have not been reported in the literature. The compounds tested are shown in

Table 1. Compound abbreviations, nomenclature, reaction yields and the structures of synthesized tetrahydroquinolines.

Compound Abbreviation	Nomenclature	Molecular Weight (g/mol)	Reaction Yield %	Chemical Structure
A	1-methyl-2-(N-formylamino)-1,2,3,4-tetrahydroquinoline	218.29	47.84
B	1-methyl-2-(2,3-dihydroindene)-1,2,3,4-tetrahydroquinoline	259.34	43.75
C	1-methyl-2-(N-caprolactamyl)-1,2,3,4-tetrahydroquinoline	254.33	44.52
D	1-methyl-2-(N-pyrrolidonyl)-1,2,3,4-tetrahydroquinoline	282.38	41.38

.

2. Methods

2.1. General Information

All reagents were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and used without further purification. The obtained products were characterized by spectral data (IR, MS, ¹H-NMR, ¹³C-NMR). The progress of the reactions was monitored by thin layer chromatography on aluminum TLC plates. Column chromatography was performed using silica gel (60–120 mesh) and the solvents used were of analytical grade.

2.2. Synthesis

2.2.1. Synthesis of N-Propargyl-Toluidine

In a round-bottom flask, 3 g of toluidine (28 mmol), 5.9756 g of Potassium Carbonate (K₂CO₃) (43 mmol), 1.1719 g of potassium iodide (KI) (7 mmol), and 10 mL of dimethylformamide were added and left for agitation for 15 min in an ice bath (0 °C), additionally, a solution of 2.4 mL of propargyl bromide (80 wt.%) in 5 mL of Dimethylformamide was prepared (21.5 mmol). After 15 min, this solution was added drop by drop into the flask, with the agitation and ice bath being maintained; when the dripping was finished, the ice bath was removed, and the product of the reaction was maintained at room temperature for 2 h, to be monitored by thin layer chromatography. The solution was extracted with 20 mL of Brinz solution and 20 mL of ethyl acetate (20 mL × 3). The organic phase was separated, and filtered with approximately 5% Sodium Sulfate. Finally, the product of interest was purified by liquid chromatography on silica gel, eluting with a mixture of ethyl acetate and petroleum ether.

2.2.2. Synthesis of Tetrahydroquinolines

In a round-bottom flask, the resulting propargyl-toluidine (approx. 1 g) was added into 10 mL of Acetonitrile; formaldehyde (70%) (5–6 mL) was added in excess, and agitated between 30 and 35 °C for 15 min. After this time the corresponding alkene (vinyl formamide, Vinyl-pyrrolidine, Indeno, Vinyl-caprolactam) was added dropwise, in slight excess (1,1 molar equivalents of the propargyl-toluidine obtained). It was kept between 30 and 35 °C, and shaking was maintained for 24 h. The solution was extracted with 20 mL of Brinz solution and 20 mL of ethyl acetate (20 mL × 3). It was filtered with approximately 5% Sodium Sulfate. Finally, the product of interest was purified by liquid chromatography on silica gel, eluting with a mixture of ethyl acetate and petroleum ether.

2.3. Biological Activity

2.3.1. Measurement of DPPH Radical-Scavenging Activity

The synthesized compounds were evaluated for their antioxidant activity by scavenging DPPH+ free radicals, following a procedure described previously [9]. Ascorbic acid was used as a reference standard, with an SC₅₀ value of 1.5 mg/mL.

2.3.2. Measurement of ABTS Radical-Scavenging Activity

The synthesized compounds were evaluated for their antioxidant activity against ABTS+ radicals, following a previously reported procedure [10]. Ascorbic acid was used as a reference standard, with an SC₅₀ value of 35 μg/mL.

2.3.3. In Silico Prediction of Pharmacokinetic Properties

To study the potential toxicological risks, tools such as SwissADME and OSIRIS were used [11,12]. SwissADME was used to perform in silico analysis of the pharmacokinetic and pharmacodynamic properties, assessing the absorption, distribution, metabolism and excretion of the THQs. OSIRIS, on the other hand, allowed the prediction of potential adverse effects by analyzing the chemical structure of the compounds and identifying fragments associated with toxicity.

3. Results and Discussion

3.1. Synthesis

In the case of the synthesis of THQ with the Povarov reaction, the results obtained were consistent with what was expected, showing uniform yields for all compounds, as observed in Table 1. On the other hand, the results of the optimization of the synthesis of N-propargyl-toluidine indicated that, when allowed to react for 2 h at room temperature, crystals are obtained, which contrasts the previously described method [13], in which the reaction was carried out on ice, and oils were obtained. Regarding the yields, the substitution of potassium carbonate with sodium carbonate was evaluated; this substitution resulted in a yield of 68.23%, while maintaining the reaction at room temperature, which represents a significant increase in yield.

All the new synthesized THQs were structurally characterized using NMR spectroscopic techniques, mass spectrometry, and IR spectroscopy. In the IR spectra, typical vibration bands of the propargyl fragment (3209–3302 cm⁻¹) were observed. All ¹H and ¹³C NMR spectra of the synthesized THQs were very similar, and were characterized by the presence of three groups of signals: aromatic, aliphatic, and those close to the heteroatom. The mass spectra correspond to the expected masses for the proposed structures, which constitutes evidence that the Diels–Alder reaction was carried out successfully.

3.2. Biological Activity

All the obtained compounds were evaluated as antioxidant agents in the presence of the stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl) at a concentration ranging from 10 to 100 μL, and were compared to ascorbic acid. The DPPH scavenging activity was poor, with EC₅₀ values higher than 100 μg/mL. On the other hand,

Table 2. Antioxidant activity in in vitro DPPH and ABTS assays of the new THQs compounds.

Compound	EC50 DPPH (μg/mL)	EC50 ABTS (μg/mL)
A	>100	1.09
B	>100	1.52
C	>100	<10
D	>100	<10
Ascorbic acid	1500	35

showed that the compounds have good activity in scavenging the ABTS radical, presenting better EC₅₀ values than those found for ascorbic acid, which was used as a reference.

This results suggest that the radical trapping mechanism used by the compounds in each assay is different. Both assays are mixed, being able to use either the HAT (hydrogen atom transfer) or SET (single electron transfer) mechanism [14].

The incubation time was different for each assay, at 5 min for DPPH and 30 min for ABTS. We propose that the radical trapping mechanism for each compound is primarily SET, since this mechanism is slower, as it occurs in two steps. Moreover, the solvents used (methanol for DPPH, and ethanol/water (1:1) for ABTS) are protic polar solvents, which favor the SET mechanism [14].

It is important to note that the proposed SET mechanism for both tests is an approximation based on the results obtained. To verify the radical trapping mechanism, it would be ideal to use a third assay specific to one type of mechanism rather than one of a mixed nature.

3.3. Analysis In Silico of the Compounds

Pharmacokinetic analysis is crucial, as it provides important information on the potential uses of these compounds as drugs. The compounds are required to cross the blood–brain barrier (BBB) and have high gastrointestinal absorption (HIA). All of the proposed compounds meet these requirements (

Table 3. Swiss ADME pharmacokinetic predictions and OSIRIS toxicological risks for new THQs. (): non-toxic; (): high toxicity.

Compound	Pharmacokinetics	PGP Substrate 1	BBB 2 Penetration	PAINS 3	MUT 4	TUM 5	IRRI 6	REP 7	DL 8
A	-	No	Yes	0
B	Inhibitor of CYP1A2, CYP2C9, CYP2D6	Yes	Yes	0
C	Inhibitor of CYP2C19, CYP2C9, CYP2D6	No	Yes	0
D	Inhibitor of CYP2C19, CYP2D6	No	Yes	0

¹ P-glycoprotein (P-gp) substrate; ² BBB: blood–brain barrier; ³ PAINS: interference compounds in assays. ⁴ MUT: mutagenic; ⁵ TUM: tumorigenic; ⁶ IRRI: irritant; ⁷ REP: reproductive effects; ⁸ DL: drug-likeness.

).

Another important parameter is that the compounds are not substrates of permeability glycoprotein (P-gp), and that they are inhibitors of the cytochrome P450 family. P-gp is responsible for eliminating molecules unknown to the human body, by regulating outflow [15]. On the other hand, the P450 family of cytochromes is responsible for eliminating molecules, such as those related to drugs, by metabolic biotransformation [16], so their inhibition is important. Of the four synthetized compounds, are not substrates for P-gp, with the exception being compound B. In general, the proposed THQs are good CYP inhibitors, inhibiting three CYPs each, with compound A being an exception (Table 3).

Drug-likeness evaluates different approaches, including those of Lipinski, Ghose, Veber, Egan, and Muegge. These are filters for molecules with potential use as oral drugs, based on chemical properties such as molecular weight, lipophilicity, hydrogen bonding, flexibility, polarity, and bioavailability [12]. Three of four compounds meet all of these standards, with the exception being Compound C, which has more than two heteroatoms and high lipophilicity (Table 3).

The “boiled egg” graph provided by SwissADME indicates the absorption of the compounds in the intestine (HIA) and their ability to cross the blood–brain barrier (BBB). The proposed compounds exhibit high gastrointestinal absorption and blood–brain barrier permeability, except for compound B (THQ indeno), which is a P-gp substrate (Figure S1).

OSIRIS Property Explorer evaluates biological risks associated with the compounds studied, such as mutagenesis, tumorigenesis, irritancy, and reproductive effects. None of the four compounds tested present these risks. It also gives an evaluation of the drug-likeness of the compounds.

4. Conclusions

This study successfully optimized the synthesis of N-propargyl-toluidine and developed two novel tetrahydroquinoline (THQ) derivatives. Key findings include:

• Improved synthesis yields using Na₂CO₃ at room temperature.
• Favorable in silico drug-like properties for most compounds, with no significant predicted toxicological risks.
• Exceptional antioxidant activity in the ABTS assay, for every compound (EC₅₀ < 10 μg/mL), outperforming ascorbic acid (EC₅₀ = 35 μg/mL).
• Evidence suggesting a primary single electron transfer (SET) mechanism for radical-scavenging.

These results provide a foundation for further exploration of THQ derivatives as potential antioxidant agents, especially in the context of neurodegenerative diseases. Future work should focus on enzymatic assays, detailed bioinformatic analyses, and investigation of the compounds’ mechanisms of action. The promising antioxidant activity and favorable predicted properties position these THQs as intriguing candidates for further development in neurodegenerative disease research.