Design, Synthesis, and Biological Evaluation of Chiral Urea and Thiourea Derivatives as Multitarget‐Directed Anti‐Alzheimer Agents

ABSTRACT

A series of novel chiral urea and thiourea compounds were designed as multitarget‐directed ligands for Alzheimer's disease and evaluated against hAChE, hBChE, and hMAO‐B, with exploratory assessment of HSD10. These chiral compounds were synthesized and fully characterized, and their enantiopurity was confirmed via HPLC. Biological screening revealed that several of the 38 single‐enantiomer derivatives exhibited potent triple inhibition of hAChE, hBChE, and hMAO‐B. Biological screening revealed that a significant number of the 38 synthesized single‐enantiomer urea and thiourea derivatives potent inhibition across the primary target panel. Among them, compounds 14 S (hAChE: 0.08 μM, hBChE: 0.93 μM, hMAO‐B: 0.02 μM), 23 R (hAChE: 0.06 μM, hBChE: 0.74 μM, hMAO‐B: 0.08 μM), 6 S (hAChE: 0.08 μM, hBChE: 0.65 μM, hMAO‐B: 0.08 μM), and 30 S (hAChE: 0.04 μM, hBChE: 0.74 μM, hMAO‐B: 0.19 μM) showed the most promising multitarget profiles. Compounds 6 S and 23 R also displayed anti‐Aβ aggregation activity, whereas 12 S combined dual hMAO‐B/hAChE inhibition with antioxidant activity. Compounds 3 R and 11 R were found to be highly active against hMAO‐B (0.03 μM and 0.06 μM, respectively) and hBChE (0.68 and 0.72 μM). HSD10 inhibition was limited overall, with only compound 26 S showing moderate activity (52.2%) at higher micromolar concentration. Molecular docking and molecular dynamics simulations supported the proposed ligand‐target interactions for the most active compounds. In addition, selected derivatives showed no acute cytotoxicity under the tested conditions in HepG2 cells and high permeability potential in the PAMPA‐BBB assay. Based on their in vitro profiles, seven compounds (3 R , 6 S , 11 R , 12 S , 14 S , 23 R , and 30 S ) were advanced to in vivo evaluation, and compounds 6 S , 12 S , and 23 R , 30 S produced significant improvements in the Morris Water Maze model. Collectively, these findings identify chiral urea and thiourea derivatives as promising lead structures for further anti‐Alzheimer drug discovery.

1. Introduction

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder that is characterized by a gradual decline of cognitive abilities. Several factors contribute to the progression of AD, including β‐amyloid (Aβ) aggregation (J. Hardy and Selkoe ²⁰⁰²), tau protein phosphorylation, increased oxidative stress (Su et al. 2008), low levels of acetylcholine (Bajda et al. 2013), elevated Monoamine oxidase B (MAO‐B) activity (Bolea et al. 2011; Weinreb et al. ²⁰¹¹), neuroinflammation of the central nervous system (Holmes 2013). Recognizing AD as a multifactorial condition—and acknowledging that current medications only alleviate its symptoms—has reinforced the development of multitarget‐directed ligand (MTDL) strategies for its treatment.

Studies have shown a correlation between the disruption of the brain's cholinergic system and memory impairment, revealing a decrease in acetylcholine concentration within cholinergic neurons in both AD and Parkinson's disease (PD). In this context, although reversible and irreversible cholinesterase inhibitors are effective in managing these diseases, they provide only symptomatic benefits without achieving palliative therapeutic effects.

Monoamine oxidases (MAOs), particularly MAO‐B, have been increasingly implicated in pathological processes associated with Alzheimer's disease (AD) (Zhong et al. 2023). Increased MAO‐B activity has been linked to oxidative stress, neuroinflammation, cholinergic neuronal damage, and amyloid‐related pathology (Behl et al. 2021; Riederer ²⁰⁰⁴). Mechanistically, MAO‐B‐catalyzed oxidative deamination generates hydrogen peroxide and reactive aldehydes, which may enhance reactive oxygen species (ROS) production and contribute to neuronal injury (Behl et al. 2021; Chatzipieris et al. ²⁰²⁵; C. Yao et al. ²⁰²²). In addition, several studies have suggested a relationship between MAO‐B activity and amyloidogenic APP processing through modulation of β‐ and γ‐secretase‐related pathways (Schedin‐Weiss et al. ²⁰¹⁷). Therefore, MAO‐B inhibition has attracted attention as part of multitarget therapeutic strategies for AD, especially in the design of compounds combining antioxidant, antiamyloid, and enzyme‐inhibitory properties (Berk et al. 2011; Christen ²⁰⁰⁰; Erol Gunal et al. ²⁰¹⁸; Jiang et al. ²⁰²⁰; Manzoor and Hoda ²⁰²⁰).

Another widely accepted hypothesis in AD pathology is the toxic production and accumulation of Aβ. Mitochondrial Aβ accumulation, which is associated with mitochondrial dysfunction and impaired synaptic trafficking, leads directly to neuronal damage (Bayer 2010; Du et al. ²⁰¹⁰; J. A. Hardy and Higgins ¹⁹⁹²; Manczak et al. ²⁰⁰⁶). Consequently, inhibiting the interaction between Aβ and mitochondrial proteins has been proposed as a potential therapeutic target for cellular protection. Aβ accumulation correlates with the production of hydrogen peroxide and nitric oxide, as well as elevated oxidative modifications of proteins and lipids (Manczak et al. 2006; Zhao and Zhao ²⁰¹³), which together promote oxidative stress and mitochondrial dysfunction (Aran and Singh 2023). Oxidative stress damages cellular functions—especially in neurons—through the interaction of reactive species with DNA, RNA, amino acids, carbohydrates, lipids, and proteins (Olufunmilayo et al. 2023). Antioxidants play a critical role in mitigating this stress by acting as electron donors and balancing excess free radicals (Srivastava et al. 2019), thus holding significant importance in the treatment of neurological diseases like AD (Guo et al. 2025).

Moreover, 17β‐hydroxysteroid dehydrogenase type 10 (17β‐HSD10 or HSD10) is a mitochondrial protein reported to interact with Aβ and to contribute to mitochondrial dysfunction, oxidative stress, and synaptic impairment in Alzheimer's disease (Du Yan et al. 1997; Yao et al. ²⁰¹¹)). Accordingly, modulation of HSD10 and/or disruption of the Aβ–HSD10 interaction has emerged as a potential complementary therapeutic strategy (Takuma et al. 2005; Yao et al. ²⁰¹¹). In this context, frentizole‐related analogues have been explored as HSD10‐targeting ligands, further supporting the relevance of this target in anti‐Alzheimer drug discovery.

Urea and thiourea derivatives hold a central role in drug development due to their capability to form multiple stable hydrogen bonds with protein and receptor targets. These drug‐target interactions govern specific biological activities and drug properties. Consequently, urea functionalities are frequently incorporated to modulate drug selectivity or improve pharmacokinetic properties in various medicinal applications, including anticancer, antibacterial, anticonvulsive, anti‐HIV, antidiabetic, and anti‐inflammatory agents (Amato et al. 2013; Gallou ²⁰⁰⁷; Prakash and Raja ²⁰¹¹; Regan et al. ²⁰⁰²; Siddiqui et al. ²⁰¹¹; Xu et al. ²⁰¹⁶). In particular, the urea related motif in MAO‐B inhibitors such as safinamide exemplifies how this scaffold contributes to central nervous system activity by forming stable hydrogen‐bonding networks within enzyme active sites, thereby enhancing both binding affinity and target selectivity (Binda et al. 2007; Murugan et al. ²⁰¹⁹). Recent studies have also demonstrated that urea‐ and thiourea‐based derivatives can inhibit AChE, BChE, Aβ plaque (Kurt et al. 2015; Teke Tuncel et al. ²⁰²⁵) and HSD10 (Figure 1) (Ganegamage et al. 2024; Hroch et al. ²⁰¹⁷; Mahmood et al. ²⁰²⁴; Saeed et al. ²⁰¹⁵; Ujan et al. ²⁰²¹; Ullah et al. ²⁰²⁴). With advances in protein structure resolution, there is growing interest in urea‐based derivatives for rational drug design (Evyapan et al. 2023; Ghosh and Gemma ²⁰¹⁴; Karaküçük‐İyidoğan et al. ²⁰²⁵).

Figure 1.

A large number of urea/thiourea derivatives showing anti‐AD activities.

Given the multifactorial pathogenesis of Alzheimer's disease (AD), the development of single chemical entities capable of modulating multiple disease‐relevant targets remains an attractive strategy in medicinal chemistry. Despite substantial progress in the pharmacological modulation of cholinesterases, monoamine oxidase B (MAO‐B), amyloid‐β (Aβ) aggregation, oxidative stress, and HSD10‐related pathways, the identification of ligands combining multitarget activity with appropriate selectivity and drug‐like properties remains challenging. In this context, the multitarget‐directed ligand (MTDL) approach has emerged as a rational framework for the design of anti‐Alzheimer agents acting on mechanistically interconnected pathological pathways. Ladostigil, generated by combining structural features of rivastigmine and rasagiline, represents a notable example of this concept (J. A. Hardy and Higgins ¹⁹⁹²) (Figure 2), while safinamide further highlights the value of urea‐related pharmacophoric elements in central nervous system drug design. Encouraged by previous reports showing that urea‐ and thiourea‐based scaffolds can engage several AD‐relevant targets, we designed a novel series of chiral derivatives incorporating these privileged motifs to explore their multitarget anti‐Alzheimer potential.

Figure 2.

Multitarget design concept of chiral urea/thiourea derivatives (the colors emphasize key pharmacophoric moieties).

Herein, we report the design, synthesis, and full characterization of a library of 38 chiral urea and thiourea derivatives. The compounds were primarily evaluated for their inhibitory activities against human acetylcholinesterase (hAChE), human butyrylcholinesterase (hBChE), and human monoamine oxidase B (hMAO‐B), while HSD10 was included as an exploratory complementary target. Several members of the series exhibited potent inhibition across this primary target panel, and selected compounds also showed anti‐Aβ aggregation and antioxidant activity. The preparation of single enantiomers enabled a detailed structure‐activity relationship analysis, revealing a marked influence of absolute configuration and arylethyl substitution pattern on potency and target selectivity. In addition, molecular docking and molecular dynamics studies were performed to support the proposed binding modes and to provide further insight into ligand–target recognition and binding stability. Selected compounds also showed high permeability potential in the PAMPA‐BBB assay and favorable effects in the Morris Water Maze model, supporting their further investigation as anti‐Alzheimer lead candidates.

2. Results and Discussion

2.1. Chemistry and Structural Elucidation

New chiral thioureas and ureas (1‐38; Scheme 1) were synthesized from the reaction of R or S‐naphthyl or phenyl ethylamine with substituted‐phenyl isocyanate/isothiocyanate. Their enantiomeric purities were evaluated by the HPLC method on optically active column materials.

Scheme 1.

Synthetic pathway for chiral thioureas and ureas (1‐38).

The synthesized single enantiomers were characterized by ¹H, ¹³C NMR, FTIR and mass spectrometry. There are two different NH groups in all molecules. In the ¹H NMR spectra of thiourea derivatives, the NH proton attached to the o‐, m‐, p‐substituted phenyl ring gave signals as singlets at around 9.5 ppm. In urea derivatives, the same NH appeared at around 8.0‐8.5 ppm as a singlet. The NH proton of thiourea derivatives attached to the naphthylethyl or phenylethyl group gave a doublet signal in the range of 8.0‐8.5 ppm interacting with the neighboring CH proton. The NH proton of urea derivatives gave a doublet signal in the aromatic region. Aromatic protons gave signals between 6.0 and 8.0 ppm and the CH proton attached to the naphthyl/phenyl ring interacting with neighboring CH₃ and NH protons, appeared as a doublet of a quartet in the range of 6.0–6.5 ppm. The CH protons of compounds 7 R , 8 S , 11 R , 12 S , 13 R , 14 S , 19 R ,20 S , 27 R , 28 S , 31 R , 32 S , 35 R , 36 S , 37 R , 38 S were seen as a broad peak. The reason for this was considered to be the E/Z transition that can be observed in ureas and thioureas (Tuncel et al. 2019). The CH₃ group adjacent to the CH group gave a doublet in the range of 1.5–1.8 ppm.

The NH protons of the synthesized thiourea and urea derivatives (1‐38) appeared in the lower field in the ¹H NMR spectra because they are expected to make hydrogen bonds with the NMR solvent DMSO‐d₆. From the literature, the oxygen atoms of polar aprotic solvents are very often involved in strong hydrogen bonding interactions with the acidic protons of solute molecules, leading to downfield shift of such protons (Gunal et al. 2016; Tuncel et al. ²⁰²³; Żuchowski and Zborowski ²⁰⁰⁶). As a matter of fact, when the ¹H NMR spectrum of 35 R was taken in CDCl₃, the NH protons shifted to the higher field (7.50 ppm and 6.40 ppm) (see Supporting information). This result was taken as a proof of the hydrogen bonding of the NH protons with DMSO‐d₆.

In the ¹³C NMR spectrum of thiourea derivatives, the C=S group gave signals around 180.0 ppm. The aromatic carbons gave signals in the range of 110.0‐160.0 ppm. The signals appearing around 50.0 ppm and 20.0 ppm were assigned to CH carbon and CH₃ carbon, respectively. Urea derivatives gave signals at around 155.0 ppm for the C=O group, 110.0‐160.0 ppm for aromatic carbons, 45.0 ppm for CH carbon, and 20.0 ppm for CH₃ carbon. In IR spectra of chiral thioureas, the observation of the stretching bands in range of 1258‐1207 cm⁻¹ for thiocarbonyl group (C=S) and 3402‐3122 cm⁻¹ for thioamide N–H bond proved the formation of thioureas. Also, in IR spectra of chiral ureas, the stretching bands in range of 1637‐1624 cm⁻¹ and 3337‐3077 cm⁻¹ confirmed the presence of carbonyl (C=O) and amide N–H bonds, respectively. The structures of the compounds were also validated by HRMS analysis. Polarimetry and HPLC analyses confirmed the absence of partial racemization, indicating that the synthesized compounds were obtained as single enantiomers.

2.2. Biological Activity Evaluation

2.2.1. Determination of Cholinesterase (ChE) Inhibitory Activities

The inhibitory ability of all urea and thiourea derivatives against human acetylcholinesterase (hAChE) and hBChE were determined using Ellman's method, with donepezil, galantamine, and tacrine as reference substances. The IC₅₀ values and theoretical K _i values are given in Table 1.

Table 1.

Inhibitory activity of compounds with human AChE, BChE and human recombinant MAO isoforms ^a and Selectivity Index (SI).

Comp	IC50 AChE (μM)	K i(app) b AChE (μM)	IC50 BChE (μM)	K i(app) bBChE(μM)	IC50 MAO‐A(μM)	IC50 MAO‐B(μM)	SIIC50 MAO‐A/IC50MAO‐B	K i MAO‐A(μM)	K i MAO‐B(μM)	SIK i MAO‐A/K i MAO‐B
1 R	0.106 ± 0.036	0.026	0.754±0.023	0.290	> 1000	0.37 ± 0.02	> 2700.00	> 1000	0.09 ± 0.01	> 11,111.00
2 S	0.133 ± 0.081	0.032	1.231±0.024	0.486	40.00 ± 2.07	0.18 ± 0.09	222.22	9.33 ± 0.22	0.03 ± 0.002	311.00
3 R	0.103 ± 0.036	0.025	0.681±0.012	0.259	> 1000	0.03 ± 0.003	> 37,000.00	> 1000	0.01 ± 0.001	> 142,000.00
4 S	0.108 ± 0.015	0.026	0.646±0.014	0.245	> 1000	0.60 ± 0.03	> 1600	> 1000	0.12 ± 0.01	> 8000.00
5 R	0.101 ± 0.001	0.024	0.753±0.014	0.289	14.90 ± 1.15	0.06 ± 0.002	248.33	3.04 ± 0.22	0.02 ± 0.001	202.66
6 S	0.076 ± 0.011	0.018	0.653±0.011	0.248	2.80 ± 0.16	0.08 ± 0.004	34.57	0.72 ± 0.05	0.02 ± 0.001	50.00
7 R	0.094 ± 0.009	0.022	5.684±0.119	2.320	68.45 ± 4.39	0.20 ± 0.02	342.25	16.50 ± 1.30	0.03 ± 0.01	550.00
8S	0.088 ± 0.009	0.021	0.941 ± 0.021	0.366	8.01 ± 0.46	0.24 ± 0.02	33.38	2.00 ± 0.15	0.06 ± 0.04	33.33
9 R	0.132 ± 0.003	0.032	1.919 ± 0.055	0.769	4.55 ± 0.27	0.09 ± 0.004	50.55	1.33 ± 0.10	0.01 ± 0.001	95.00
10 S	0.099 ± 0.048	0.023	0.786 ± 0.013	0.303	9.02 ± 0.55	0.11 ± 0.01	82.00	2.39 ± 0.14	0.01 ± 0.001	183.85
11 R	0.116 ± 0.569	0.028	0.724 ± 0.013	0.277	3.24 ± 1.09	0.06 ± 0.021	54.00	3.40 ± 0.21	0.08 ± 0.004	44.74
12 S	0.090 ± 0.006	0.021	1.416 ± 0.031	0.562	8.44 ± 0.49	0.09 ± 0.003	93.77	2.13 ± 0.15	0.02 ± 0.001	106.50
13 R	0.127 ± 0.005	0.031	0.671 ± 0.014	0.255	2.16 ± 0.11	0.08 ± 0.003	27.00	0.50 ± 0.03	0.02 ± 0.001	23.81
14 S	0.079 ± 0.040	0.019	0.931 ± 0.023	0.363	3.90 ± 0.19	0.02 ± 0.001	229.41	0.88 ± 0.05	0.004 ± 0.001	220.00
15 R	0.075 ± 0.030	0.018	0.791 ± 0.016	0.305	5.29 ± 0.36	0.18 ± 0.001	29.39	1.02 ± 0.009	0.04 ± 0.002	27.57
16 S	0.141 ± 0.042	0.034	0.814 ± 0.016	0.315	5.05 ± 0.31	0.30 ± 0.02	16.83	1.34 ± 0.11	0.06 ± 0.002	22.71
17 R	0.150 ± 0.019	0.036	0.483 ± 0.009	0.178	2.59 ± 0.14	0.09 ± 0.01	30.12	0.70 ± 0.044	0.02 ± 0.001	35.00
18 S	0.105 ± 0.013	0.025	3.864 ± 0.075	1.571	4.80 ± 0.18	0.04 ± 0.002	120.00	1.55 ± 0.10	0.01 ± 0.001	172.22
19 R	0.176 ± 0.008	0.043	0.563 ± 0.009	0.211	4.86 ± 0.19	0.13 ± 0.009	37.38	1.55 ± 0.11	0.04 ± 0.002	40.79
20 S	0.107 ± 0.096	0.026	1.239 ± 0.021	0.489	5.00 ± 0.20	0.31 ± 0.014	16.66	1.98 ± 0.12	0.08 ± 0.004	25.38
21 R	0.102 ± 0.029	0.024	3.481 ± 0.109	1.412	4.81 ± 0.20	0.05 ± 0.001	96.20	1.24 ± 0.10	0.02 ± 0.001	65.26
22 S	0.178 ± 0.007	0.043	0.512 ± 0.024	0.191	5.69 0.23	0.06 ± 0.003	94.83	1.77 ± 0.11	0.01 ± 0.001	177.00
23 R	0.059 ± 0.042	0.014	0.741 ± 0.014	0.284	2.50 ± 0.19	0.08 ± 0.003	31.25	0.80 ± 0.02	0.02 ± 0.009	47.05
24 S	0.403 ± 0.428	0.098	0.997 ± 0.019	0.391	5.07 ± 0.26	0.21 ± 0.01	24.14	1.55 ± 0.10	0.05 ± 0.002	29.81
25 R	0.121 ± 0.022	0.029	1.404 ± 0.026	0.557	1.24 ± 0.10	0.04 ± 0.001	31.00	0.29 ± 0.01	0.02 ± 0.001	14.50
26 S	0.114 ± 0.016	0.028	0.854 ± 0.017	0.331	5.25 ± 0.28	0.05 ± 0.002	105.00	1.46 ± 0.12	0.01 ± 0.001	132.73
27 R	0.104 ± 0.229	0.025	0.791 ± 0.018	0.305	34.00 ± 1.90	0.09 ± 0.003	377.78	8.77 ± 0.26	0.02 ± 0.001	438.50
28 S	0.104 ± 0.196	0.025	1.308 ± 0.035	0.518	8.90 ± 0.42	0.21 ± 0.01	42.38	2.00 ± 0.11	0.05 ± 0.002	40.00
29 R	0.180 ± 0.360	0.043	1.197 ± 0.035	0.472	14.55 ± 1.06	0.07 ± 0.002	207.86	3.48 ± 0.15	0.02 ± 0.001	174.00
30 S	0.044 ± 0.061	0.011	0.744 ± 0.026	0.286	18.56 ± 1.13	0.19 ± 0.01	97.68	5.50 ± 0.23	0.04 ± 0.001	137.50
31 R	0.165 ± 0.003	0.040	0.728 ± 0.013	0.279	12.00 ± 1.07	5.20 ± 0.22	2.31	4.01 ± 0.13	1.55 ± 0.11	2.59
32 S	0.123 ± 0.048	0.029	1.088 ± 0.026	0.427	12.90 ± 1.07	4.88 ± 0.13	2.64	4.00 ± 0.11	1.29 ± 0.09	3.10
33 R	0.149 ± 0.014	0.036	1.501 ± 0.029	0.598	15.50 ± 1.03	1.18 ± 0.09	13.14	4.88 ± 0.21	0.52 ± 0.01	9.38
34 S	0.131 ± 0.037	0.031	0.704 ± 0.015	0.269	27.00 ± 1.32	1.29 ± 0.16	20.93	7.00 ± 0.24	0.43 ± 0.01	16.28
35 R	0.149 ± 0.042	0.036	0.593 ± 0.011	0.223	3.99 ± 0.16	4.11 ± 0.19	0.97	1.03 ± 0.09	0.97 ± 0.02	1.06
36 S	0.051 ± 0.020	0.012	0.649 ± 0.013	0.246	4.33 ± 0.17	4.28 ± 0.24	1.01	0.61 ± 0.02	0.58 ± 0.01	1.05
37 R	0.081 ± 0.028	0.019	1.891 ± 0.043	0.758	5.02 ± 0.18	1.20 ± 0.09	4.18	1.90 ± 0.10	0.32 ± 0.01	5.94
38 S	0.097 ± 0.019	0.023	0.649 ± 0.021	0.246	5.05 ± 0.18	1.36 ± 0.09	4.04	1.01 ± 0.08	0.24 ± 0.01	4.21
Donepezil	0.01 ± 0.001	0.002	1.7 ± 0.03	0.70
Galantamine			6.15 ± 0.14	1.94
Harmine								0.09 ± 0.001	1300.00	7.69 × 10−4
Lazabemide					> 1000	0.05	20,000.00	> 1000	0.01 ± 0.001	> 125,000.00
Tacrine c			0.01 ± 0.003

^a All synthesized compounds are competitive and reversible inhibitors of hMAO‐B; majority of them are hMAO‐B selective.

^b Ki values were calculated from experimentally determined IC₅₀ values using the IC50‐to‐Ki converter of Cer et al. (2009).

^c Available from literature (Turgutalp et al. 2022).

2.2.1.1. AchE Inhibitory Activity of Compounds

All compounds exhibited remarkably strong hAChE inhibitory activity, with IC₅₀ values ranging from 0.04 to 0.40 μM. The most potent inhibitor was the urea derivative 30 S , which features a naphthalen‐1‐ylethyl group on one urea nitrogen and a 4‐chlorophenyl group on the other. Based on IC₅₀ values, compound 30 S was approximately four times more potent than its R‐enantiomer, a finding that aligns with the calculated Kᵢ values from molecular docking studies. The second most active compound, 36 S , contains a phenethyl group and a 2‐fluorophenyl group on the thiourea moiety and was found to be three times more potent than its R‐enantiomer, 35 R . The third most active compound, 23 R , also bears a naphthylethyl substituent on the thiourea, similar to 30 S . The structure–activity relationship (SAR) of these chiral urea and thiourea derivatives as hAChE inhibitors is illustrated in Figure 3. Notably, 9 of the top 10 most active compounds, with the exception of 30 S , contain a thiourea moiety, suggesting that the presence of sulfur may enhance inhibitory potency compared to oxygen in the urea analogs. In general, compounds bearing a naphthylethyl group displayed higher hAChE inhibitory activity than their phenethyl analogs, with the exception of compounds 32 S , 36 S , and 38 S , which bear 2‐methoxy, 2‐fluoro, and 3‐fluoro substituents, respectively, on the phenyl ring.

Figure 3.

SAR of chiral thioureas and ureas as hAChE inhibitors.

2.2.1.2. HBChE Inhibitory Activity of Compounds

All compounds exhibited stronger hBChE inhibitory activity than reference compound galantamine (IC₅₀ 6.15 μM), but weaker activity than tacrine (IC₅₀ 0.01 μM). Most of the compounds investigated in this study exhibited significantly stronger hBChE inhibitory activity compared to the reference inhibitor donepezil (IC₅₀ = 1.7 μM). The most active compound of the series was compound 17 R (IC₅₀ 0.48 μM), which carries a naphthalen‐1‐ylethyl and 3‐fluorophenyl group on the urea. The second most active compound was compound 22 S (IC₅₀ 0.51 μM), which carries naphthalen‐1‐ylethyl and 4‐fluorophenyl groups on the urea nitrogen. The third most active derivative was compound 19 R , which carries the substituents of compound 22 S on a thiourea core instead of a urea core (IC₅₀ 0.56 μM). Figure 4 shows the structure–activity relationships of chiral thiourea and urea derivatives as hBChE inhibitors. Contrary to hAChE assay, in general, compounds bearing phenethyl group are more active than their counterparts bearing naphthylethyl group except compounds 33 R , 34 S and 37 R with R = 3‐MeO, and 3‐F. The presence of fluoro substituents on the phenyl ring generally tends to improve activity relative to methoxy and chloro analogs. Majority of the R enantiomers display higher activity compared to the S enantiomers. Among the thiourea derivatives bearing a phenylethyl group (compounds 31–38) instead of naphthalen‐1‐ylethyl, and a 2‐substituted phenyl ring (e.g., 31 R , 32 S , 35 R , 36 S ), compounds with an absolute R configuration exhibited greater hBChE inhibitory activity compared to their S‐configured counterparts. Interestingly, within the 3‐substituted phenyl derivatives (33 R , 34 S , 37 R , 38 S ), the trend was reversed, and compounds with the S configuration demonstrated superior biological activity.

Figure 4.

SAR of chiral thioureas and ureas as hBChE inhibitors.

When both anticholinesterase activities were evaluated together, it was observed that compounds 30 S , 36 S , 6 S and 2 3R are the most active dual inhibitors.

In order to provide insight into in vitro findings and to explain binding mechanisms, all compounds were docked into the active sites of the target enzymes using Autodock Vina software(Trott and Olson 2010) following the previously reported docking protocols and validations (Minovski et al. 2019; Yelekçi and Erdem ²⁰²³). The most potent hAChE inhibitor 30 S displayed one of the strongest binding affinities in the list (ΔG −11.5 kcal/mol, K _icalc 0.004 μM (in vitro calculated K _i 0.01 μM)). The most potent hBChE inhibitors 17 R and 22 S were among the best binding inhibitors with ΔG −10.9 and −10.5 kcal/mol (K _icalc 0.01 and 0.02 µM). Binding pose of the most potent hAChE and hBChE inhibitors (30 S and 17 R ) are given in Figure 5. hAChE active site is composed of several cavities known as acyl binding site, choline binding site, catalytic triad, oxyanion hole and peripheral anionic site (Rosenberry et al. 2017). 30 S is well accommodated in the pocket of hAChE, orienting its pharmacophore groups towards three sites. In the peripheral anionic site, it binds to the aromatic rings of Trp286 and Tyr341 via π‐π stacking interactions with its naphthyl group. Its phenyl group interacts with Gly120‐Gly121 in the oxyanion hole and with Trp86 in the choline binding site via π‐π stacking.

Figure 5.

Binding poses of the most potent hAChE and hBChE inhibitors, 30S (green) and 17R (pink), in their complexes with hAChE and hBChE enzymes including the K _icalc values and binding energies. Green dashed lines represent H‐bonding while pink lines represent hydrophobic interactions.

hBChE active site is similar to hAChE but six aromatic amino acid residues are replaced by aliphatic amino acids. The most potent hBChE inhibitor 17 R fitted well into the cavities formed by the acyl binding site, catalytic triad and choline binding site, as observed in the most of the effective hBChE inhibitors in the literature (Rosenberry et al. 2017; Teke Tuncel et al. ²⁰²⁵). In the acyl binding site, its fluorophenyl group was well located in the pocket surrounded by Trp231, Phe329 and Leu286 which provided several π‐π and π‐alkyl interactions with these residues and two H‐bonding interactions of fluorine atom with the backbones of Leu286 and Pro285. Naphthyl group displayed π‐π interactions with Trp82 in the choline binding site and with His438 in the catalytic triad.

2.2.2. hMAO Inhibitory Activities of Compounds

The hMAO inhibitory ability of the synthesized compounds was determined using recombinant hMAO isoforms. The concentration of p‐tyramine, used as a substrate, was maintained between 0.05 and 0.50 mM. Harmine was chosen as the selective reference inhibitor for hMAO‐A, and lazabemide as the selective reference inhibitor for hMAO‐B. Both compounds reversibly and competitively inhibited the hMAO isoforms. The specific enzyme activities of the recombinant hMAO isoforms were calculated as 0.16 nmol/mg/min for hMAO‐A and 0.14 nmol/mg/min for hMAO‐B. Comparative results are provided in Table 1. The kinetic properties of hMAO inhibition were determined using Lineweaver‐Burk plots. The K _i values for the inhibition of the hMAO isoforms by the new compounds indicated that all compounds inhibited at least one isoform of hMAO (Table 1). The K _i values were consistent with the findings from the IC₅₀ results.

All compounds exhibited competitive and reversible inhibition of hMAO‐B, with IC₅₀ values ranging from 0.02 to 5.20 μM. Notably, compounds 14 S , 3 R , 18 S , and 25 R demonstrated stronger inhibitory activity than the reference inhibitor lazabemide, while compounds 21 R and 26 S showed comparable potency to that of lazabemide.

When the IC₅₀ values of phenylethyl thioureas (31 R −38 S ) were compared to the corresponding naphthylethyl thioureas in the group of 1 R −30 S , an apparent decline (4.5‐fold to 68‐fold, depending on different substituents) in the activity of 31 R −38 S was observed. Similarly, the compounds with high hMAO‐B selectivity carried a naphthalen‐1‐ylethyl group on the thiourea nitrogen, while the nonselective compounds were derivatives carrying a phenylethyl group on the thiourea nitrogen. Except for the compounds 31 R −38 S , the remaining compounds selectively inhibited the hMAO‐B. Figure 6 illustrates the structure–activity relationships of chiral thiourea and urea derivatives as hMAO‐B inhibitors. For compounds 1 R −30 S , which carry the naphthylethyl group, urea derivatives generally display stronger hMAO‐B inhibitory activities than the corresponding thiourea derivatives. The structure‐activity relationship highlighted the higher inhibitory ability of naphthylethyl fragment and carbonyl oxygen with respect to the phenylethyl and the thiocarbonyl moiety. It is expected that naphthyl group leads to the enhancement of hydrophobic interactions with the aromatic cage of the hMAO‐B active site. Stronger activity of compounds with Y = O than Y = S can be attributed to the better H‐bonding ability of the urea fragment.

Figure 6.

SAR of chiral thioureas and ureas as hMAO‐B inhibitors.

Among the compounds with higher selectivity, the strongest inhibition was observed for compound 3 R , which had an IC₅₀ value of 0.03 μM, and the highest selectivity index (SI) values. The IC₅₀ value of compound 3 R was superior to that of the well‐known hMAO‐B inhibitor lazabemide (IC₅₀ 0.05 μM), and it also showed greater selectivity (lazabemide IC₅₀ SI > 20,000). Further, compound 1 R emerged as the second most selective hMAO‐B inhibitor with an IC₅₀ 0.37 μM, and SI greater than 11,111. Although compound 14 S showed lower selectivity (SI 220), it was the most potent hMAO‐B inhibitor among all compounds, with IC₅₀ 0.02 μM. Generally, the R isomers were found to be more effective than the S isomers for most of the compounds exhibiting hMAO‐B inhibition.

MAO docking scores revealed that binding affinities of all compounds were stronger for hMAO‐B than for hMAO‐A confirming the hMAO‐B selectivity of these ligands. Among the most selective hMAO‐B inhibitors (1 R , 3 R and 4 S ), molecule 3 R was the most potent inhibitor (ΔG −10.8 kcal/mol, K _icalc 0.01 µM). Among all compounds, 14 S displayed the strongest binding affinity (ΔG −11.3 kcal/mol, K _icalc 0.005 µM) compromising the experimental inhibitory activity (IC₅₀ 0.02 µM, K _i 0.004 µM). Binding pose of 14 S in the active sites of hMAO‐B and its interaction with important amino acid residues are shown in Figure 7.

Figure 7.

Binding pose of 14S (red) in its complexes with hMAO‐B and hAChE enzymes including the K _icalc values and binding energies. Green dashed lines represent H‐bonding while pink lines represent hydrophobic interactions.

In the active site of hMAO‐B, 14 S occupied the entrance cavity rather than the “aromatic cage” (Akyüz et al. 2007; Li et al. ²⁰⁰⁶). Its phenyl group showed π‐alkyl interactions with Leu164 and Ile199 which acts as a gate between the entrance and substrate cavities. Urea group was involved in H‐bond with Ile199 backbone. Other π‐alkyl interactions were observed with Cys172 and Leu 171 via the naphthyl group. Thus, phenyl/naphthyl rings and urea groups were the important fragments involved in these interactions. Compound 14 S, which possesses the fluorophenyl same as in the known drug safinamide (Figure 2), displayed similar binding orientation as the fluorophenyl group of safinamide (Yelekçi and Erdem 2023).

According to both in vitro and in silico results, the most potent hMAO‐B inhibitor 14 S was also one of the best hAChE inhibitors in the study (IC₅₀ 0.08 µM, K _i 0.02 µM) revealing its dual‐activity. Therefore, binding pose of 14 S which occupies the peripheral anionic site and acyl binding pocket of hAChE is also given in Figure 7.

2.2.3. Beta‐Amyloid (Aβ) Fibril Assays

Amyloid beta protein is generated through the sequential cleavage of the transmembrane APP by secretases. In the “amyloidogenic” pathway, which leads to the formation of Aβ, APP is cleaved by gamma‐secretase at either the 40th or 42nd amino acid, resulting in the production of Aβ (1‐40) or Aβ (1‐42), respectively. Approximately 90% of the residues form Aβ (1‐40), however, the Aβ (1‐42) is more toxic and aggregates more easily. As a result, Aβ (1‐42) is the most abundant form found in amyloid plaques. In this instance, the Aβ‐related activity of the synthesized compounds was investigated to determine their capacity to interfere with this process of aggregation, which is a critical event in Alzheimer's pathology.

Table 2 provides data on the inhibition of Aβ (1‐40) and Aβ (1) fibril formation, the results are given as IC₅₀ values. Overall, compounds with an S configuration, specifically those carrying a thiourea and naphthalen‐1‐ylethyl moiety, were found to be more effective in Aβ aggregation inhibition than the R isomers, which bore the urea and phenylethyl group. The most effective derivatives in the series were compounds 6 S , 24 S , 28 S , 5 R , 8 S and 16 S .

Table 2.

Aβ (1‐40) and Aβ (1‐42) fibril inhibition of compounds. ^a

Compound	Aβ (1‐40) IC50 (μM)	Compound	Aβ (1‐40) IC50 (μM)	Aβ (1‐42) IC50 (μM)	Aβ (1‐42) IC50 (μM)
1 R	356.79	N/A	20 S	88.68	N/A
2 S	149.53	N/A	21 R	N/A	N/A
3 R	195.58	92.16	22 S	84.37	N/A
4 S	51.06	N/A	23 R	40.02	9.23
5 R	24.64	15.25	24 S	15.53	8.22
6 S	19.31	9.66	25 R	13.85	33.87
7 R	N/A	N/A	26 S	30.28	24.57
8 S	19.23	18.34	27 R	1096.05	9.21
9 R	N/A	N/A	28 S	17.22	11.54
10 S	61.71	N/A	29 R	70.18	N/A
11 R	51.39	75.56	30 S	182.53	108.13
12 S	75.05	49.33	31 R	170.73	N/A
13 R	N/A	N/A	32 S	1001.44	N/A
14 S	N/A	N/A	33 R	N/A	N/A
15 R	N/A	22.72	34 S	74.63	281.19
16 S	26.71	23.03	35 R	2150.58	N/A
17 R	42.75	N/A	36 S	154.20	N/A
18 S	N/A	N/A	37 R	N/A	N/A
19 R	N/A	19.48	38 S	N/A	409.62
Selegiline	40.30	28.72

^a In cases where compounds did not demonstrate fibril inhibition, they are indicated as “N/A” in the table.

2.2.4. In Vitro antioxidant activity

The antioxidant ability by quantifying the scavenging activity against the DPPH (1,1‐diphenyl‐2‐picrylhydrazil) radical, a well‐established stable organic nitrogen radical, was determined. The most effective derivatives in the series were 19 R , 31 R , 1 R , 35 R , 11 R and 2 S , respectively (Table 3).

Table 3.

IC₅₀ values of all compounds for DPPH free radical scavenging assay, CUPRAC and superoxide anion scavenging capacity.

Compound	DPPH IC50 (mM)	CUPRAC IC50 (μM)	Superoxide anion scavenging capacity (μM)	Compound	DPPH IC50 (mM)	CUPRAC IC50 (μM)	Superoxide anion scavenging capacity (μM)
1 R	4.84	36.10 ± 1.05	27.90 ± 0.18	21 R	> 25	> 100	> 100
2 S	5.56	37.77 ± 0.68	29.82 ± 0.69	22 S	> 25	> 100	> 100
3 R	> 25	66.45 ± 1.90	51.00 ± 0.93	23 R	> 25	> 100	> 100
4 S	> 25	97.23 ± 1.29	> 100	24 S	9.01	45.40 ± 1.18	37.63 ± 0.81
5 R	> 25	> 100	> 100	25 R	> 25	46.65 ± 1.00	35.27 ± 0.63
6 S	> 25	> 100	> 100	26 S	> 25	> 100	> 100
7 R	> 25	> 100	> 100	27 R	19.31	> 100	> 100
8S	> 25	> 100	> 100	28 S	> 25	> 100	> 100
9 R	> 25	> 100	> 100	29 R	> 25	> 100	> 100
10 S	> 25	> 100	> 100	30 S	> 25	> 100	> 100
11 R	5.45	37.11 ± 1.29	29.28 ± 0.13	31 R	4.53	35.50 ± 1.09	31.90 ± 0.29
12 S	5.49	37.88 ± 1.07	33.24 ± 0.51	32 S	> 25	> 100	> 100
13 R	> 25	90.00 ± 1.29	> 100	33 R	> 25	> 100	> 100
14 S	> 25	> 100	> 100	34 S	> 25	> 100	> 100
15 R	20.44	48.22 ± 0.91	33.20 ± 0.54	35 R	5.19	33.02 ± 0.90	28.77 ± 0.72
16 S	22.44	49.02 ± 1.00	60.45 ± 1.00	36 S	6.02	39.25 ± 1.24	49.00 ± 0.71
17 R	> 25	> 100	> 100	37 R	> 25	> 100	> 100
18 S	> 25	90.22 ± 1.08	60.16 ± 1.00	38 S	> 25	> 100	> 100
19 R	3.98	34.05 ± 0.88	26.90 ± 0.83	Trolox	0.05	28.36 ± 1.12
20 S	> 25	> 100	> 100	BHT			21.00 ± 0.98

The results from the CUPRAC antioxidant method also supported the findings from the DPPH assay. Compounds 35 R , 19 R , 31 R , 1 R , 11 R , 2 S and 12 S demonstrated stronger antioxidant activity compared to the reference compound Trolox (Table 3).

The superoxide anion scavenging capacity yielded similar results to the previous assays. Among the synthesized compounds 19 R , 1 R , 35 R , 11 R , 2 S , 31 R , 15R and 12 S demonstrated the strongest superoxide anion scavenging activity (Table 3).

According to the results of all three antioxidant tests, the compounds with the R configuration demonstrated greater efficacy. Among all the compounds, 19 R , 31 R , 1 R , 35 R , and 11 R stood out, with the 19 R compound, which carries a naphthalen‐1‐ylethyl group on one thiourea nitrogen and a 4‐fluorophenyl group on the other, showing the most potent antioxidant activity compared to the others. Among these, compounds 1 R and 31 R had a naphthalen‐1‐ylethyl and phenylethyl group on the 1‐position of the thiourea nitrogen, with a 2‐methoxyphenyl group on the other nitrogen. Similarly, compounds 11 R and 35 R have a 2‐fluorophenyl ring on the urea nitrogen. Among the S‐isomers, 2 S showed the most effective result.

An analysis of the chemical structures of the most potent compounds revealed that they contain a 2‐OCH₃ group and a fluorine substituent. It is believed that phenolic moieties enhance antioxidant activity by stabilizing free radicals, while the fluorine substituent contributes to the antioxidant effect by increasing lipophilicity and altering electron density.

2.2.5. Cytotoxicity Assay

HepG2 cells served as the selected cellular model to examine the potential effects of the synthesized compounds. The primary objective was to evaluate cytotoxicity when these compounds were administered at concentrations of 1, 5, and 25 µM during a 24‐h incubation period. Cell viability and cytotoxicity were assessed by comparing the results to a control group treated with an equivalent volume of DMSO as the compounds. Table 4 presents a comprehensive summary of the findings, displaying cell viability percentages and associated p values for each tested concentration. The data obtained from this experiment indicate that, even at the highest concentration of 25 µM, the synthesized compounds did not induce cytotoxic effects within the 24 h.

Table 4.

Percentage of cell viability and associated p values for the synthesized 1‐38 compounds at concentrations of 1, 5, and 25 µM, in comparison to the control group (DMSO).

Comp	Conc.	% Viability	p value	Comp	Conc.	% Viability	p value
1 R	1 µM	120.02 ± 8.62*	0.018	20 S	1 µM	102.41 ± 3.41	0.018
	5 µM	123.26 ± 32.02	0.246		5 µM	118.42 ± 38.02	0.246
	25 µM	129.00 ± 47.68	0.313		25 µM	99.24 ± 23.28	0.313
2 S	1 µM	148.49 ± 77.37	0.300	21 R	1 µM	144.94 ± 56.30	0.300
	5 µM	120.85 ± 19.57	0.122		5 µM	138.97 ± 11.48**	0.122
	25 µM	142.82 ± 38.71	0.111		25 µM	162.92 ± 79.27	0.111
3 R	1 µM	98.18 ± 14.04	0.931	22 S	1 µM	129.38 ± 48.73	0.931
	5 µM	102.71 ± 15.72	0.722		5 µM	124.77 ± 12.75*	0.722
	25 µM	102.01 ± 16.94	0.787		25 µM	144.86 ± 19.36*	0.787
4 S	1 µM	108.86 ± 11.42	0.272	23 R	1 µM	99.24 ± 10.04	0.272
	5 µM	113.59 ± 28.16	0.464		5 µM	116.14 ± 5.31	0.464
	25 µM	100.70 ± 16.65	0.852		25 µM	99.69 ± 8.97	0.852
5 R	1 µM	96.07 ± 8.97	0.569	24 S	1 µM	114.45 ± 15.74	0.569
	5 µM	157.50 ± 63.57	0.259		5 µM	112.83 ± 2.34	0.259
	25 µM	112.99 ± 23.88	0.516		25 µM	103.18 ± 4.29	0.516
6 S	1 µM	119.63 ± 31.17	0.479	25 R	1 µM	97.88 ± 1.28	0.479
	5 µM	116.81 ± 7.087	0.051		5 µM	96.16 ± 6.54	0.051
	25 µM	107.26 ± 7.68	0.433		25 µM	100.41 ± 7.84	0.433
7 R	1 µM	105.84 ± 13.80	0.778	26 S	1 µM	115.34 ± 3.50	0.778
	5 µM	97.43 ± 8.20	0.126		5 µM	111.07 ± 10.74	0.126
	25 µM	105.81 ± 10.87	0.609		25 µM	108.15 ± 11.53	0.609
8 S	1 µM	112.89 ± 11.56	0.795	27 R	1 µM	113.89 ± 4.27	0.795
	5 µM	115.35 ± 11.45	0.561		5 µM	107.19 ± 7.34	0.561
	25 µM	115.84 ± 11.22	0.491		25 µM	101.75 ± 0.21	0.491
9 R	1 µM	103.88 ± 3.70	0.214	28 S	1 µM	102.57 ± 3.60	0.214
	5 µM	105.74 ± 14.44	0.741		5 µM	98.27 ± 7.81	0.741
	25 µM	111.03 ± 8.44	0.775		25 µM	103.02 ± 11.11	0.775
10 S	1 µM	94.87 ± 8.13	0.603	29 R	1 µM	162.50 ± 14.58	0.603
	5 µM	106.79 ± 19.86	0.677		5 µM	172.14 ± 16.45	0.677
	25 µM	96.67 ± 20.08	0.896		25 µM	161.73 ± 15.78*	0.896
11 R	1 µM	109.14 ± 24.66	0.531	30 S	1 µM	185.17 ± 12.02	0.531
	5 µM	98.19 ± 14.50	0.845		5 µM	106.23 ± 14.04	0.845
	25 µM	95.02 ± 7.63	0.458		25 µM	121.42 ± 14.12	0.458
12 S	1 µM	146.45 ± 56.42	0.198	31 R	1 µM	120.34 ± 7.55	0.198
	5 µM	109.37 ± 20.80	0.460		5 µM	91.50 ± 11.22	0.460
	25 µM	125.38 ± 27.93	0.167		25 µM	101.83 ± 24.71	0.167
13 R	1 µM	102.41 ± 3.41	0.382	32 S	1 µM	98.37 ± 35.83	0.382
	5 µM	118.42 ± 38.02	0.601		5 µM	133.60 ± 39.05	0.601
	25 µM	99.24 ± 23.28	0.944		25 µM	87.42 ± 12.73	0.944
14 S	1 µM	99.24 ± 10.04	0.584	33 R	1 µM	103.17 ± 17.73	0.584
	5 µM	116.14 ± 5.31	0.072		5 µM	97.43 ± 10.46	0.072
	25 µM	99.69 ± 8.97	0.801		25 µM	93.65 ± 2.99	0.801
15 R	1 µM	109.45 ± 8.67	0.491	34 S	1 µM	116.60 ± 4.67	0.491
	5 µM	112.83 ± 2.34	0.059		5 µM	99.09 ± 10.25	0.059
	25 µM	108.18 ± 2.77	0.116		25 µM	95.92 ± 3.63	0.116
16 S	1 µM	97.88 ± 1.28	0.109	35 R	1 µM	87.38 ± 16.43	0.109
	5 µM	96.16 ± 6.54	0.354		5 µM	80.44 ± 7.99	0.354
	25 µM	115.40 ± 29.05	0.664		25 µM	97.97 ± 34.81	0.664
17 R	1 µM	108.157 ± 11.53	0.747	36 S	1 µM	189.58 ± 89.92	0.747
	5 µM	115.34 ± 3.50	0.057		5 µM	94.90 ± 18.04	0.057
	25 µM	111.07 ± 10.74	0.638		25 µM	82.09 ± 15.98	0.638
18 S	1 µM	101.75 ± 0.21	0.191	37 R	1 µM	120.39 ± 13.03	0.191
	5 µM	113.89 ± 4.27	0.169		5 µM	107.96 ± 6.55	0.169
	25 µM	107.19 ± 7.34	0.988		25 µM	93.74 ± 17.64	0.988
19 R	1 µM	97.87 ± 10.24	0.902	38 R	1 µM	115.41 ± 29.05	0.902
	5 µM	106.79 ± 19.86	0.677		5 µM	103.64 ± 6.85	0.677
	25 µM	96.67 ± 20.08	0.896		25 µM	101.23 ± 0.61	0.896

Note: Significance levels are indicated as follows:

^* p < 0.05 vs. DMSO

^** p < 0.01 vs. DMSO.

2.2.6. HSD10 Inhibitory Activity of Compounds

The prepared compounds were screened for their inhibition activity towards 17β‐HSD10 at 20 and 10 μM concentration (Table 5). Compounds that showed inhibition higher than 50% (i.e., residual activity lower than 50% of uninhibited enzymatic reaction) were consequently forwarded for 1 μM screening. The selected compounds did not show inhibition higher than 50% at 1 μM (i.e., residual activity lower than 50%). Among the 11 selected urea and thiourea derivatives, which demonstrated high hMAO and cholinesterase inhibitory activity, only compound 26 S stood out in HSD10 enzyme inhibition assay. At concentrations of 20 and 10 µM, compound 26 S exhibited significantly better results compared to other derivatives in the series, with HSD10 inhibition 52.2% (Table 5). Notably, this compound shares a structural similarity with frentizole, an urea‐based molecule approved by the FDA. However, at a lower concentration of 1 µM, compound 26 S did not show significant activity.

Table 5.

Enzyme inhibition of HSD10 for selected compounds.

Comp	20 µM		10 µM		1 µM
	Enzyme Inh.	Standard deviation	Enzyme Inh.	Standard deviation	Enzyme Inh.	Standard deviation
1 R	0.0%	0.7%	0.0%	2.3%
3 R	0.0%	0.6%	0.0%	2.4%
5 R	0.0%	2.6%	16.4%	3.4%
7 R	2.0%	3.1%	13.3%	9.8%
10 S	58.6%	10.0%	21.1%	6.8%
14 S	8.6%	4.2%	0.0%	6.5%
18 S	27.1%	1.6%	8.3%	2.1%
23 R	0.0%	4.2%	0.0%	3.4%
26 S	55.8%	0.9%	52.2%	2.1%	0.0%	1.7%
27 R	0.0%	1.1%	0.0%	5.1%
30 S	8.8%	6.3%	0.0%	6.5%
K1152 **	—	—	78%	3.4%	50.5%	9.9%

^**See the reference (Benek et al. 2023).

According to molecular docking calculations, compound 26 S which showed 52.2% HSD10 inhibition at 10 µM concentration, binds to HSD10 enzyme with moderate binding affinity (ΔG −7.1 kcal/mol, K _icalc 6.24 µM). This compound also exhibits very strong activity against hMAO‐B (experimental IC₅₀ 0.05 µM), and moderate activities against hAChE, and hBChE enzymes demonstrating its multimodal activity. The enzyme‐ligand interactions of 26 S in the active sites of HSD10 and hMAO‐B are shown in Figure 8.

Figure 8.

Binding pose of 26 S (orange) in its complexes with HSD10 and hMAO‐B enzymes including the K _icalc values and binding energies. Green dashed lines represent H‐bonding while pink lines represent hydrophobic interactions.

In the Aβ‐binding site of HSD10, it exhibits π‐alkyl interactions with Ala95, Ala97, Leu205, Leu206, Leu209 via its aromatic rings and H‐bond interactions with Gln162 through urea N‐H moiety. In the hMAO‐B active site, it occupies the gate pocket through its 3‐chlorophenyl group and extends its naphthyl group toward the aromatic cage making π‐π interactions with Tyr398 and π‐alkyl interactions with Leu171. At the gate pocket, it shows π‐alkyl interactions and H‐bonding with Ile199 and carbon‐hydrogen bond with the backbone of Ile198.

The Gibbs free binding energies (ΔG) and calculated inhibition constants of the compounds that bind to hAChE, hBChE, hMAO‐A, hMAO‐B, HSD10 enzymes are given in Table S2.

2.2.7. In Vitro Blood–Brain Barrier Permeation

Compounds/drugs designed for the central nervous system must cross blood‐brain barrier (BBB) to reach their therapeutic targets. The ability of the newly synthesized derivatives to cross the BBB was determined using the PAMPA‐BBB (Parallel Artificial Membrane Permeability Assay for the Blood‐Brain Barrier) assay. Standard drugs such as testosterone, verapamil, β‐estradiol, progesterone, corticosterone, piroxicam, hydrocortisone, lomefloxacin, and dopamine were used in the method. Due to the high cost of PAMPA tests, only compounds with the highest potential for multimodal effects (1 R , 3 R , 5 R , 7 R , 10 S ,14 S , 18 S , 23 R , 26 S , 27 R , 30 S ) were selected for testing. Compounds with a P_e (10⁻⁶ cm s⁻¹) > 4.00 are rated as having better BBB permeability, and all tested compounds demonstrated high permeability potential to cross into the central nervous system (Table 6).

Table 6.

Results of the BBB permeability test (PAMPA‐BBB) for the selected compounds and standards.

Compound	Bibliography** Pe (x10−6 cm s−1)	Experimental Pe (x10−6 cm s−1)***	Permeability
1 R		10.52 ± 0.99	CNS+
3 R		6.40 ± 0.22	CNS+
5 R		7.30 ± 0.26	CNS+
7 R		7.44 ± 0.30	CNS+
10 S		9.56 ± 0.29	CNS+
14 S		6.77 ± 0.31	CNS+
18 S		8.17 ± 0.29	CNS+
23 R		9.01 ± 0.40	CNS+
26 S		8.12 ± 0.30	CNS+
27 R		6.66 ± 0.16	CNS+
30 S		10.12 ± 0.50	CNS+
Testosterone	17.20	18.11 ± 1.09	CNS+
Verapamil	16.10	16.00 ± 0.96	CNS+
β‐estradiol	11.90	11.70 ± 0.76	CNS+
Progesterone	9.10	8.99 ± 0.24	CNS+
Corticosterone	5.10	5.18 ± 0.14	CNS+
Piroxicam	2.40	2.47 ± 0.13	CNS+/‐
Hydrocortisone	1.70	1.77 ± 0.11	CNS‐
Dopamine	0.20	0.23 ± 0.01	CNS‐

Note: Compounds were dissolved in DMSO (1% w/v) to 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration was 50 µg/mL.

^**Taken from Di et al. (2003).

^***Values are expressed as mean ± standard error (n = 3).

2.2.8. In Vivo Evaluation of Selected Compounds

The cognitive effects of the selected synthesized compounds were evaluated in vivo using the Morris Water Maze (MWM) test following oral administration to mice with an AD like phenotype induced by aluminum chloride (AlCl₃).

Analysis of swimming velocity in the MWM revealed no statistically significant differences in locomotor performance either between experimental groups or across testing days. These findings suggest that neither the administration of AlCl₃ nor the tested compounds affected the general motor abilities of the mice and, therefore, did not influence their capacity to locate the hidden platform (Figure 9a).

Figure 9.

Performance of experimental groups in the Morris Water Maze task. (a) No significant variations in swimming speed were detected across testing days or between treatment groups, indicating comparable locomotor activity. (b) Progressive reduction in escape latency from Day 1 to Day 4 demonstrates the acquisition of spatial learning (*p < 0.05 between Day 1 and Day 4 within each group). (c) During the probe trial on Day 5, the proportion of time spent in the target quadrant was significantly reduced in the Alzheimer's group, whereas treatment with the test compounds increased this measure compared to both control and AD groups (n = 8 for each group; p < 0.05 vs. control; ^# p < 0.05 vs. AD).

Throughout the first 4 days of testing, mice were trained to locate the hidden platform, and learning performance was subsequently evaluated on the fifth day using the probe trial. The progressive reduction in escape latency from Day 1 to Day 4 confirmed that the animals successfully acquired the task. In all treatment groups, except for the untreated Alzheimer's model, mice demonstrated shorter latencies on Day 4 compared to Day 1, reflecting effective learning. Since cognitive deficits are a hallmark of AD, these results suggest that AlCl₃ exposure induced learning impairments consistent with Alzheimer‐like pathology, whereas administration of the tested compounds produced a partial improvement in spatial learning performance (Figure 9b).

In the probe trial, conducted after removal of the platform, the time spent by mice in the target quadrant was evaluated. The AD group and the untreated animals spent significantly less time in the platform quadrant compared to the control group, consistent with spatial and memory impairments that are characteristic of AD. Among the synthesized compounds, mice treated with 6 S , 12 S , 23 R , and 30 S spent a comparable amount of time in the target quadrant to that of the control animals, and this duration was significantly greater than that of the AD group (Figure 9c). These findings indicate that 1 week of subchronic treatment produced notable improvements in memory performance. Compound 6 S exhibited the most pronounced effect on learning and memory; however, its efficacy did not differ statistically from that of 12 S , 23 R , or 30 S . Although 3 R , 11 R , and 14 S did not produce statistically significant improvements compared with the AD group, they showed a tendency toward enhanced learning and memory performance. It is likely that extending the treatment period could lead to statistically significant effects for these compounds as well.

2.3. ADME Predictions

It is important to determine the pharmacokinetics and drug‐likeness parameters of the designed compounds to evaluate their ADME properties and minimize the adverse effects. These parameters were predicted using SwissADME webtool (Daina et al. 2014, ²⁰¹⁷) and are given in SI Table S1. All compounds showed the BBB permeability and passed Lipinski's, Ghose's, Veber's, and Egan's drug‐likeness filters (Lipinski et al. 2001). Drug‐likeness refers to the qualitative assessment of a molecule's potential as an orally bioavailable drug candidate based on its structural and physicochemical properties. In addition to these drug‐likeness filters SwissADME also describes six bioavailability criteria: Lipophilicity: −0.7 < XLOGP3 < 5.0, Size: 150 < MW < 500 g/mol, Polarity: 20 < TPSA < 130 Ų, Insolubility: −6 < Log S (ESOL) < 0, Insaturation: 0.25 < Fraction Csp³ < 1, Flexibility: 0 < Num. rotatable bonds < 9. All compounds conform with six bioavailability criteria except insaturation with Csp³ fractions ranging from 0.11 to 0.19. The most potent compound of each enzyme (hAChE 30 S , hBChE 17 R , hMAO‐B 14 S , HSD10 26 S ) were selected as representatives and, their bioavailability radar and boiled egg (Daina et al. 2014, ²⁰¹⁷) graphs are shown in SI Figure S58. Five of the six physicochemical properties related to bioavailability are within the acceptable pink zone. With only insaturation being outside the pink zone, these compounds are likely to be orally bioavailable. Boiled egg is the graphical representation of gastrointestinal absorption and BBB permeability which are associated with WLOGP (lipophilicity) and TPSA (polarity) parameters. The compounds fall into the yellow region of the boiled egg indicating that they exhibit high BBB permeation and gastrointestinal absorption. Inhibition of the CYP450 isoenzymes increases drug accumulation by decelerating metabolism, and likely to increase drug‐drug interactions. Compounds 30 S , 17 R , 14 S and 26 S were predicted as not inhibitors of metabolism enzymes CYP1A2, CYP3A4, and as inhibitors of CYP2C19, CYP2C9, CYP2D6 which might be optimized in further studies.

2.4. Molecular Dynamic Simulations

The MD simulations were employed to reveal behavior of selected compounds 14 S , 26 S or 30 S in the active sites of hAChE, hMAO‐B or HSD10. Stabilities of the selected enzyme‐ligand complexes which were obtained from molecular docking were further validated with 100 ns MD simulations in order to account for full flexibility and dynamics of these complexes. MD simulations were run with GROMACS 5.1.4 package (Abraham et al. 2015) employing GROMOS 54a7 force field (Schmid et al. 2011) for generated enzyme topology and using ATB and PRODRG 2.5 web server (Schüttelkopf and van Aalten ²⁰⁰⁴; Stroet et al. ²⁰¹⁸) for parametrization. Because of the high computational cost of MD, only the enzyme‐ligand complexes of the most potent hAChE inhibitor 30 S (hAChE‐30 S ), dual‐target compound 14 S (hMAO‐B‐14 S and hAChE‐14 S ), and HSD10 ‐26 S complex were simulated along with their holo/apo enzymes.

Analysis of MD trajectories justified that the ligands of these four enzyme‐ligand complexes remained bound to the enzymes, mostly retaining their position in the active site throughout the simulation. As shown in Figures S58–S61, the snapshots extracted from the starting, 20, 40, 60, 80, and 100 ns MD trajectories and the binding interactions at the end of simulation, confirm the structural stabilities of these enzyme‐inhibitor complexes.

Figure 10 demonstrates RMSD plots of four selected enzyme‐ligand complexes together with the corresponding holo/apo enzymes. The RMSD values of hMAO‐B‐14 S complex (Figure 10a) altered around 0.2‐0.35 nm. At the beginning of the simulation, RMSD values slightly increased up to about 20 ns. Then, fluctuations were relatively small and remained stable after 80 ns. This was caused by the slight movement of the ligand from the gate pocket towards the aromatic cage in the active site, which allowed new π‐π interactions of its naphthyl group with the aromatic residues Tyr435 and Tyr60 as illustrated in Figure S58. hAChE complexes showed more stability than others throughout the simulation. RMSD values of hAChE‐14 S and hAChE‐30 S complexes (Figure 10b) varied around 0.2–0.32 nm. The HSD10 ‐26 S complex RMSD values reached equilibrium after 10 ns simulation and balanced around RMSD 0.35 nm. In general, enzyme‐ligand complexes of 14 S , 30 S , and 26 S displayed smaller RMSD profiles than holo/apo form of the corresponding enzyme.

Figure 10.

RMSD plots of the enzyme‐ligand complexes of the most potent inhibitors along with the corresponding holo/apo enzymes (black): (a) hMAO‐B‐14S (red), (b) hAChE‐14S (red), hAChE‐30S (green), and (c) HSD10‐26S (orange).

The RMSF plots are given in SI Figure S62, which shows each residue's fluctuations over the simulation time. They demonstrated negligible fluctuations around the binding site residues. Consequently, RMSD and RMSF plots of hMAO‐B, hAChE and HSD10 complexes confirmed the stabilities of enzyme‐ligand complexes along 100 ns simulations and were consistent with our in vitro and molecular modeling data.

3. Conclusion

Despite advances in rational drug design, genomics, proteomics, and high‐throughput screening techniques, the development of novel single‐target drugs has fallen short of expectations for AD therapeutics over the past decades. In contrast, MTDLs offer a broader therapeutic scope by interacting with multiple pharmacologically relevant targets, often triggering a range of indirect, network‐based beneficial effects. Consequently, MTDLs not only increase the number of viable drug targets but also introduce new drug classes with plausibly reduced side effects and lower toxicity.

In this study, the majority of the synthesized chiral urea and thiourea derivatives demonstrated potent multifunctional activity as potential anti‐Alzheimer's agents. These compounds showed significant inhibition of cholinesterases and hMAO‐B, attenuated self‐induced Aβ aggregation, and exhibited strong antioxidant properties. Adhering to the multitarget drug paradigm, we sought to establish a balanced interaction across several targets concurrently to tackle the multifactorial characteristics of AD. Most of the investigated compounds demonstrated multiple activities inhibiting hAChE, hBChE and hMAO‐B in nanomolar to micromolar range. Among the compounds studied, several highly bioactive MTDLs were identified. The most potent hAChE inhibitor 30 S (0.04 µM) acted as a dual cholinesterase inhibitor (hBChE 0.74 µM) while also inhibiting hMAO‐B (0.19 µM). Furthermore, compounds 14 S (hMAO‐B 0.02 µM, hAChE 0.08 µM, hBChE 0.93 µM), 23 R (hAChE 0.06 µM, hBChE 0.74 µM, hMAO‐B 0.08 µM) and 6 S (hAChE 0.08 µM, hBChE 0.65 µM, hMAO‐B 0.08 µM) displayed exceptionally strong triple inhibition. Compounds 6 S and 23 R also exhibit good inhibitory activity against Aβ fibril formation. Compound 8 S exhibited high activity against hAChE, hBChE, and Aβ, while compounds 27 R and 5 R showed strong activity against hMAO‐B, hBChE and Aβ fibrils. Compounds 3 R and 11 R were highly effective against hMAO‐B (0.03 µM and 0.06 µM) and hBChE (0.68 and 0.72 µM), whereas compound 12 S was active against hMAO‐B (0.09 µM) and hAChE (0.09 µM). The antioxidant profile of compound 12 S was also quite promising. Additionally, compounds 36 S and 15 R exhibited high activity against both hAChE and hBChE. Notably, compound 26 S showed moderate activity against HSD10 (55.8%) while maintaining high hMAO‐B (0.05 µM) and hBChE (0.85 µM) inhibitory activity. The most promising compounds showing multimodal activities are schematically summarized in Figure 11.

Figure 11.

The most promising compounds showing multimodal activities (blue and red colors emphasize thiourea and urea derivatives, respectively).

In the in vivo experiments, the tested compounds successfully crossed the blood–brain barrier and exerted pharmacological effects consistent with their in vitro profiles, demonstrating their central activity and suitability for oral administration. Behavioral findings indicated that the selected compounds improved memory and spatial learning performance, with 6 S , 12 S , 23 R , and 30 S producing statistically significant improvements. When interpreted alongside the in vitro data, the pronounced efficacy of 6 S and 23 R appears to be associated not only with their modulatory activity on the relevant target enzymes but also with their ability to reduce Aβ amyloid accumulation. Furthermore, the additional antioxidant capacity of 12 S may contribute to its greater efficacy compared to 3 R and 11 R , which exhibited milder effects on cognitive performance. It is conceivable that extending the treatment duration could allow these latter compounds to achieve statistically significant improvements as well.

Overall, these findings support the hypothesis that this newly synthesized series exerts a neuroprotective effect through multiple mechanisms—including enzymatic modulation, attenuation of amyloid pathology, and antioxidant activity—thereby contributing to the observed improvements in learning and memory. In conclusion, the presented urea and thiourea derivatives demonstrate compelling bioactivity across multiple tested targets. Their multimodal action should be explored further, and the SAR broadened to fully realize the therapeutic potential of these molecules.

4. Experimental

4.1. Chemistry

All reagents and solvents were obtained commercially (Sigma‐Aldrich, Merck, TCI) and used without further purification. Melting points were determined on Electrothermal 9100 melting point apparatus. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of all compounds were recorded on a Varian‐Mercury VX‐400 MHz‐BB. HRMS was carried out on Waters SYNAPT G1 MS analyser. Polarimetric measurements were carried out on Rudolph Research Analytical Autopol I Automatic Polarimeter. The propagation of reactions was followed by TLC using silica gel 60‐F254. IR spectra were recorded on Thermo Nicolet 380 FT‐IR spectrometer. Liquid chromatography analyses with UV detector (λ = 254 nm) were performed using CHIRALPAK‐IC (particle size: 5 mm; column size: 250 × 4.6 mm).

4.1.1. General Procedure for the Preparation of Compounds (1‐38)

To a solution of chiral amine (5 mmol) in 10 mL of dichloromethane was added the corresponding isocyanate or isothiocyanate (5 mmol). The solution was stirred for 2 h at room temperature. At the end of this the solvent was removed and the crude product was purified by crystallization using ethyl acetate‐hexane mixture (Touchard et al. 1997).

4.1.2. Compound 1R: (R−1‐(2‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 440° (0.1 g/100 mL acetone). Yield: 46%, m.p.: 74°C‐75°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphtyl)ethylamine and 2‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 8.95 (s, 1H, NH), 8.54 (d, 1H, NH, J = 7.2 Hz), 8.20 (d, 1H, Ar, J = 8.2 Hz), 8.13 (d, 1H, Ar, J = 8.0 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.89 (d, 1H, Ar, J = 8.0 Hz), 7.67‐7.50 (m, 4H, Ar), 7.09‐6.86 (m, 3H, Ar), 6.88 (t, 1H, CH, J = 6.8 Hz), 3.76 (s, 3H, OCH₃), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 179.6, 151.0, 139.6, 133.8, 131.2, 129.0, 128.6, 128.3, 127.0, 125.9, 125.6, 123.9, 123.0, 119.8, 111.74, 55.8, 48.7, 20.7 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 337.1375; Found 337.1377. ATR‐FTIR: 3254 (N–H); 1236 (C=S) cm⁻¹.

4.1.3. Compound 2S: (S−1‐(2‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+ ) 440° (0.1 g/100 mL acetone). Yield: 49%, m.p.: 74°C–75°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine ve 2‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO δ 8.95 (s, 1H, NH), 8.54 (d, 1H, NH, J = 7.2 Hz), 8.20 (d, 1H, Ar, J = 8.2 Hz), 8.13 (d, 1H, Ar, J = 8.0 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.89 (d, 1H, Ar, J = 8.0 Hz), 7.67‐7.50 (m, 4H, Ar), 7.09‐6.86 (m, 3H, Ar), 6.88 (t, 1H, CH, J = 6.8 Hz), 3.76 (s, 3H, OCH₃), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm.¹³C NMR (100 MHz, DMSO): δ 179.6, 151.0, 139.6, 133.8, 131.2, 129.0, 128.6, 128.3, 127.0, 125.9, 125.6, 123.9, 123.0, 119.8, 111.74, 55.8, 48.7, 20.7 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 337.1375; Found 337.1377. ATR‐FTIR: 3254 (N–H); 1248 (C=S) cm⁻¹.

4.1.4. Compound 3R: (R−1‐(2‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (−) 240° (0.1 g/100 mL acetone). Yield: 74%, m.p.: 212°C‐214°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 2‐methoxyphenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.17 (d, 1H, NH, J = 8.0 Hz), 8.09 (dd, 1H, Ar, J = 1.8 Hz, J = 7.6 Hz), 8.00 (s, 1H, NH), 7,97 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar J = 8.0 Hz), 7.64‐43 (m, 5H, Ar), 6.95 (dd, 1H, Ar J = 1.4 Hz, J = 8.0 Hz), 6.88‐6.79 (m, 2H, Ar), 5.63 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 3.82 (s, 3H, OCH₃), 1.53 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 154.6, 147.5, 141.3, 133.8, 130.9, 129.7, 129.2, 127.7, 126.7, 125.9, 123.4, 120.5, 117.6, 111.2, 55.8, 44.9, 22.5 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂O₂: 321.1603; Found 321.1603. ATR‐FTIR: 3337, 3288 (NH); 1626 (C=O) cm⁻¹.

4.1.5. Compound 4S: (S−1‐(2‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 240° (0.1 g/100 mL acetone). Yield: 71%, m.p.: 212°C–214°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine ve 2‐methoxyphenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.17 (d, 1H, NH, J = 8.0 Hz), 8.09 (dd, 1H, Ar, J = 1.8 Hz, J = 7.6 Hz), 8.00 (s, 1H, NH), 7,97 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar J = 8.0 Hz), 7.64‐43 (m, 5H, Ar), 6.95 (dd, 1H, Ar J = 1.4 Hz, J = 8.0 Hz), 6.88‐6.79 (m, 2H, Ar), 5.63 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 3.82 (s, 3H, OCH₃), 1.53 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 154.6, 147.5, 141.3, 133.8, 130.9, 129.7, 129.2, 127.7, 126.7, 125.9, 123.4, 120.5, 117.6, 111.2, 55.8, 44.9, 22.5 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂O₂: 321.1603; Found 321.1622. ATR‐FTIR: 3337, 3288 (NH), 1626 (C=O) cm⁻¹.

4.1.6. Compound 5R: (R−1‐(3‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 440° (0.1 g/100 mL acetone). Yield: 88%, m.p.: 152°C‐153°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 3‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.48 (s, 1H, NH), 8.24 (d, 1H, NH, J = 7.6 Hz), 8.20 (d, 1H, Ar, J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.88 (d, 1H, Ar, J = 8.0 Hz), 7.62‐7.16 (m, 6H, Ar), 6.92 (d, 1H, Ar, J = 8.0 Hz), 6.62 (dd, 1H, Ar, J = 2.2 Hz, J = 8.0 Hz), 5.63 (t, 1H, CH, J = 6.8 Hz), 3.56 (s, 3H, OCH₃), 1.63 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 179.6, 159.8, 147.9, 139.3, 134.0, 131.3, 129.8, 129.0, 128.1, 126.8, 126.2, 125.9, 123.9, 123.5, 114.8, 109.9, 108.0, 55.4, 49.3, 20.6 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 335.1218; Found 335.1215. ATR‐FTIR: 3310 (N–H); 1258 (C=S) cm⁻¹.

4.1.7. Compound 6S: (S−1‐(3‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 440° (0.1 g/100 mL acetone). Yield: 74%, m.p.: 152°C‐153°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 3‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.48 (s, 1H, NH), 8.24 (d, 1H, NH, J = 7.6 Hz), 8.20 (d, 1H, Ar, J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.88 (d, 1H, Ar, J = 8.0 Hz), 7.62‐7.16 (m, 6H, Ar), 6.92 (d, 1H, Ar, J = 8.0 Hz), 6.62 (dd, 1H, Ar, J = 2.2 Hz, J = 8.0 Hz), 5.63 (t, 1H, CH, J = 6.8 Hz), 3.56 (s, 3H, OCH₃), 1.63 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 179.6, 159.8, 147.9, 139.3, 134.0, 131.3, 129.8, 129.0, 128.1, 126.8, 126.2, 125.9, 123.9, 123.5, 114.8, 109.9, 108.0, 55.4, 49.3, 20.6 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 335.1218; Found 335.1215. ATR‐FTIR: 3310 (N–H); 1258 (C=S) 1598 cm⁻¹.

4.1.8. Compound 7R: (R−1‐(4‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (‐) 540° (0.1 g/100 mL acetone). Yield: 67%, m.p.: 137°C‐138°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.25 (s, 1H, NH), 8.21 (d, 1H, NH, J = 8.2 Hz), 7.99 (br, 1H, Ar), 7,97 (d, 1H, Ar, J = 8.0 Hz), 7.87 (d, 1H, Ar, J = 8.0 Hz), 7.65‐7.42 (m, 4H, Ar), 7.29 (d, 2H, Ar, J = 8.0 Hz), 6.87 (d, 2H, Ar, J = 8.2 Hz), 6.29 (br, 1H, CH), 3.73 (s, 3H, OCH₃), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.4, 156.7, 139.8, 133.9, 132.5, 131.1, 129.1, 127.9, 126.1, 125.8, 123.8, 123.0, 114.1, 55.4, 49.3, 21.1 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 335.1218; Found 335.1218. ATR‐FTIR: 3367 (NH); 1247 (C=S)cm⁻¹.

4.1.9. Compound 8S: (S−1‐(4‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 540° (0.1 g/100 mL acetone). Yield: 86%, m.p.: 137°C‐138°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 4‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.25 (s, 1H, NH), 8.21 (d, 1H, NH, J = 8.2 Hz), 7.99 (br, 1H, Ar), 7,97 (d, 1H, Ar, J = 8.0 Hz), 7.87 (d, 1H, Ar, J = 8.0 Hz), 7.65‐7.42 (m, 4H, Ar), 7.29 (d, 2H, Ar, J = 8.0 Hz), 6.87 (d, 2H, Ar, J = 8.2 Hz), 6.29 (br, 1H, CH), 3.73 (s, 3H, OCH₃), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ δ 180.4, 156.7, 139.8, 133.9, 132.5, 131.1, 129.1, 127.9, 126.1, 125.8, 123.8, 123.0, 114.1, 55.4, 49.3, 21.1 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂OS: 335.1218; Found 335.1191. ATR‐FTIR: 3367 (NH); 1236 (C=S) cm⁻¹.

4.1.10. Compound 9R: (R−1‐(4‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (‐) 140° (0.1 g/100 mL acetone). Yield: 82%, m.p.: 226°C‐227°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐methoxyphenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.21 (s, 1H, NH), 8.18 (d, 1H, Ar, J = 8.4 Hz), 7.96 (d, 1H, Ar, J = 7.8 Hz), 7,84 (d, 1H, J = 8 Hz), 7.67‐7.47 (m, 4H, Ar), 7.29 (d, 2H, Ar, J = 8.4 Hz), 6.81 (d, 2H, NH, J = 8.4 Hz), 6.66 (d, 1H, Ar, J = 8.0 Hz), 5.64 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 3.69 (s, 3H, OCH₃), 1.54 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 155.0, 154.2, 141.2, 134.0, 130.7, 128.9, 128.0, 126.6, 125.8, 123.26, 122.3, 119.7, 114.4, 55.4, 45.2, 22.7 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂O₂: 321.1603; Found 321.1611. ATR‐FTIR: 3297 (NH); 1624 (C=O) cm⁻¹.

4.1.11. Compound 10S: (S−1‐(4‐Methoxyphenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 140° (0.1 g/100 mL acetone). Yield: 91%, m.p.: 226°C‐227°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine ve 4‐ methoxyphenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.21 (s, 1H, NH), 8.18 (d, 1H, Ar, J = 8.4 Hz), 7.96 (d, 1H, Ar, J = 7.8 Hz), 7,84 (d, 1H, J = 8 Hz), 7.67‐7.47 (m, 4H, Ar), 7.29 (d, 2H, Ar, J = 8.4 Hz), 6.81 (d, 2H, NH, J = 8.4 Hz), 6.66 (d, 1H, Ar, J = 8.0 Hz), 5.64 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 3.69 (s, 3H, OCH₃), 1.54 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 155.0, 154.2, 141.2, 134.0, 130.7, 128.9, 128.0, 126.6, 125.8, 123.26, 122.3, 119.7, 114.4, 55.4, 45.2, 22.7 ppm. HRMS (TOF MS ES+): Calculated for C₂₀H₂₀N₂O₂: 321.1603; Found 321.1610. ATR‐FTIR: 3300 (NH); 1624 (C=O) cm⁻¹.

4.1.12. Compound 11R: (R−1‐(2‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 40° (0.1 g/100 mL acetone). Yield: 65%, m.p.: 143°C‐144°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 2‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.13 (s, 1H, NH), 8.50 (d, 1H, NH, J = 6.0 Hz), 8.18 (d, 1H, Ar, J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.89 (d, 2H, Ar, J = 8.0 Hz), 7.68–7.05 (m, 7H, Ar), 6.26 (br, 1H, CH), 1.62 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.9, 139.5, 133.9, 131.1, 129.1, 128.1, 127.6, 127.5 (d, J = 13.6 Hz) 126.8, 126.5 (d, J = 7.9 Hz), 126.2, 125.9, 124.30 (d, J = 3.6 Hz), 123.8, 123.2, 115.9, 115.7, 49.6, 21.0 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 325.1175; Found 325.1172. ATR‐FTIR: 3299 (NH); 1217 (C=S) cm⁻¹.

4.1.13. Compound 12S: (S−1‐(2‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 40° (0.1 g/100 mL acetone). Yield: % 75, m.p.: 143°C–144°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 2‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.13 (s, 1H, NH), 8.50 (d, 1H, NH, J = 6.0 Hz), 8.18 (d, 1H, Ar, J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.89 (d, 2H, Ar, J = 8.0 Hz), 7.68 .05 (m, 7H, Ar), 6.26 (br, 1H, CH), 1.62 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.9, 139.5, 133.9, 131.1, 129.1, 128.1, 127.6, 127.5 (d, J = 13.6 Hz) 126.8, 126.5 (d, J = 7.9 Hz), 126.2, 125.9, 124.30 (d, J = 3.6 Hz), 123.8, 123.2, 115.9, 115.7, 49.6, 21.0 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 325.1175; Found 325.1175. ATR‐FTIR: 3306 (NH); 1216 (C=S) cm⁻¹.

4.1.14. Compound 13R: (R−1‐(2‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (−) 160° (0.1 g/100 mL acetone). Yield: 71%, m.p.: 195°C‐197°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 2‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.32 (s, 1H, NH), 8.18‐8.12 (m, 2H, Ar), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar, J = 8.0 Hz), 7.67 – 7.46 (m, 4H, Ar), 7.29 (d, 1H, NH J = 7.2 Hz), 7.18 (t, 1H, Ar, J = 8.0 Hz), 7.06 (t, 1H, Ar, J = 8.0 Hz), 6.93‐6.89 (m, 1H, Ar) 5.64 (br, 1H, CH), 1.55 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 154.4, 152.9, 150.7, 141.1, 133.7, 130.7, 129.2, 128.6, 127.7, 126.5, 125.9, 124.6, 123.6, 122.5, 122.0, 119.9, 115.3, 45.1, 22.7 ppm HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1248. ATR‐FTIR: 3300 (NH); 1633 (C=O) cm⁻¹.

4.1.15. Compound 14S: (S−1‐(2‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 160° (0.1 g/100 mL acetone). Yield: 88%, m.p.: 195°C‐197°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 2‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.32 (s, 1H, NH), 8.18‐8.12 (m, 2H, Ar), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar, J = 8.0 Hz), 7.67 – 7.46 (m, 4H, Ar), 7.29 (d, 1H, NH J = 7.2 Hz), 7.18 (t, 1H, Ar, J = 8.0 Hz), 7.06 (t, 1H, Ar, J = 8.0 Hz), 6.93‐6.89 (m, 1H, Ar) 5.64 (br, 1H, CH), 1.55 (d, 3H, CH₃, J = 6.8 Hz) ppm.¹³C NMR (100 MHz, DMSO): δ δ 154.4, 152.9, 150.7, 141.1, 133.7, 130.7, 129.2, 128.6, 127.7, 126.5, 125.9, 124.6, 123.6, 122.5, 122.0, 119.9, 115.3, 45.1, 22.7 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1195. ATR‐FTIR: 3305(NH); 1633 (C=O) cm⁻¹.

4.1.16. Compound 15R: (R−1‐(3‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 220° (0.1 g/100 mL acetone). Yield: 43%, m.p.: 69°C–72°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 3‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.55 (s, 1H, NH), 8.36 (d, 1H, NH, J = 7.6 Hz), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar, J = 8.4 Hz), 7.85 (d, 1H, Ar, J = 8.4 Hz), 7.62 (d, 1H, Ar J = 11.6 Hz), 7.60 – 7.48 (m, 4H, Ar), 7.27 (td, 1H, Ar, J = 6.8 Hz, J = 8.2 Hz), 7.14 (dd, 1H, Ar, J = 8.4 Hz, J = 1.1 Hz), 6.84 (dt, 1H, Ar, J = 8.4 Hz, J = 0.8 Hz), 6.23 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.60 (d, 3H, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO) δ 179.7, 163.4, 160.9, 141.9 (d, J = 10.8 Hz), 139.5, 133.9, 131.1, 130.4 (d, J = 9.8 Hz), 129.2, 128.2, 126.9, 126.2, 126.0, 123.8, 123.4, 118.1, 110.4 (d, J = 20.7 Hz), 49.3, 20.9 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 325.1175; Found 325.1175. ATR‐FTIR: 3402, 3200 (NH); 1216 (C=S) cm⁻¹.

4.1.17. Compound 16S: (S−1‐(3‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 220° (0.1 g/100 mL acetone). Yield: 59%, m.p.: 69°C–72°C. This compound was synthesized according to the general procedure by using S‐(‐)−1‐(1‐naphthyl)ethylamine and 3‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.55 (s, 1H, NH), 8.36 (d, 1H, NH, J = 7.6 Hz), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar, J = 8.4 Hz), 7.85 (d, 1H, Ar, J = 8.4 Hz), 7.62 (d, 1H, Ar J = 11.6 Hz), 7.60 – 7.48 (m, 4H, Ar), 7.27 (td, 1H, Ar, J = 6.8 Hz, J = 8.2 Hz), 7.14 (dd, 1H, Ar, J = 8.4 Hz, J = 1.1 Hz), 6.84 (dt, 1H, Ar, J = 8.4 Hz, J = 0.8 Hz), 6.23 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.60 (d, 3H, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO) δ 179.7, 163.4, 160.9, 141.9 (d, J = 10.8 Hz), 139.5, 133.9, 131.1, 130.4 (d, J = 9.8 Hz), 129.2, 128.2, 126.9, 126.2, 126.0, 123.8, 123.4, 118.1, 110.4 (d, J = 20.7 Hz), 49.3, 20.9 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 325.1175; Found 325.1218. ATR‐FTIR: 3402, 3205 (NH); 1216 (C=S) cm⁻¹.

4.1.18. Compound 17R: (R−1‐(3‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (‐) 60° (0.1 g/100 mL acetone). Yield: 95%, m.p.: 228°C–230°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 3‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.50 (s, 1H, NH), 8.13 (d, 1H, Ar, J = 8.4 Hz), 7.93 (dd, 1H, Ar, J = 1.6 Hz, J = 8.0 Hz), 7.82 (d, 1H, Ar, J = 8.0 Hz), 7.61–7.46 (m, 4H, Ar), 7.41 (td, 1H, Ar, J = 2.4 Hz, J = 8.0 Hz), 7.20 (td, 1H, Ar, J = 7.0 Hz, J = 8.2 Hz), 6.97 (ddd, 1H, Ar, J = 0.8 Hz, J = 2.8 Hz, J = 8.2 Hz), 6.82 (d, 1H, NH, J = 8.0 Hz), 6.66 (dt, 1H, J = 2.4 Hz, J = 8.0 Hz), 5.60 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.51 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 164.0, 161.7, 154.5, 142.7 (d, J = 11.5 Hz), 141.0, 133.9, 130.7, 130.6 (d, J = 10.0 Hz), 129.2, 127.8, 126.7, 126.1 (d, J = 13.7 Hz), 123.6, 122.6, 113.7 (d, J = 2.2 Hz), 107.8 (d, J = 21.1 Hz), 104.6 (d, J = 26.7 Hz), 45.4, 22.4. ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1247. ATR‐FTIR: 3308, 3077 (NH); 1628 (C=O) cm⁻¹.

4.1.19. Compound 18S: (S−1‐(3‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 60° (0.1 g/100 mL acetone). Yield: 98%, m.p.: 228°C–230°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 3‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.50 (s, 1H, NH), 8.13 (d, 1H, Ar, J = 8.4 Hz), 7.93 (dd, 1H, Ar, J = 1.6 Hz, J = 8.0 Hz), 7.82 (d, 1H, Ar, J = 8.0 Hz), 7.61–7.46 (m, 4H, Ar), 7.41 (td, 1H, Ar, J = 2.4 Hz, J = 8.0 Hz), 7.20 (td, 1H, Ar, J = 7.0 Hz, J = 8.2 Hz), 6.97 (ddd, 1H, Ar, J = 0.8 Hz, J = 2.8 Hz, J = 8.2 Hz), 6.82 (d, 1H, NH J = 8.0 Hz), 6.66 (dt, 1H, J = 2.4 Hz, J = 8.0 Hz), 5.60 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.51 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 164.0, 161.7, 154.5, 142.7 (d, J = 11.5 Hz), 141.0, 133.9, 130.7, 130.6 (d, J = 10.0 Hz), 129.2, 127.8, 126.7, 126.1 (d, J = 13.7 Hz), 123.6, 122.6, 113.7 (d, J = 2.2 Hz), 107.8 (d, J = 21.1 Hz), 104.6 (d, J = 26.7 Hz), 45.4, 22.4. ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1247. ATR‐FTIR: 3308, 3077 (NH); 1628 (C=O) cm⁻¹.

4.1.20. Compound 19R: (R−1‐(4‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 200° (0.1 g/100 mL acetone). Yield: 51%, m.p.: 80°C–83°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.40 (s, 1H, NH), 8.22 (d, 1H, NH, J = 7.2 Hz), 8.16 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar J = 8.0 Hz), 7.84 (d, 1H, Ar, J = 8.0 Hz), 7.60 – 7.39 (m, 6H, Ar), 7.14–7.05 (m, 2H, Ar), 6.25 (br, 1H, CH), 1.59 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.4, 139.7, 136.3, 133.9, 131.1, 129.2, 128.1, 126.8, 126.2, 126.0, 125.5, 123.9, 123.3, 115.6, 115.4, 49.4, 21.1 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 323.1018; Found 323.1014. ATR‐FTIR: 3319 (NH); 1216 (C=S) cm⁻¹.

4.1.21. Compound 20S: (S−1‐(4‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 200° (0.1 g/100 mL acetone). Yield: 36%, m.p.: 80°C–83°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 4‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.40 (s, 1H, NH), 8.22 (d, 1H, NH, J = 7.2 Hz), 8.16 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar J = 8.0 Hz), 7.84 (d, 1H, Ar, J = 8.0 Hz), 7.60 – 7.39 (m, 6H, Ar), 7.14–7.05 (m, 2H, Ar), 6.25 (br, 1H, CH), 1.59 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.4, 139.7, 136.3, 133.9, 131.1, 129.2, 128.1, 126.8, 126.2, 126.0, 125.5, 123.9, 123.3, 115.6, 115.4, 49.4, 21.1 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂S: 323.1018; Found 323.1015. ATR‐FTIR: 3319 (NH); 1211 (C=S) cm⁻¹.

4.1.22. Compound 21R: (R−1‐(4‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (−) 80° (0.1 g/100 mL acetone). Yield: 31%, m.p.: 230°C–232°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.36 (s, 1H, NH), 8.12 (d, 1H, Ar, J = 8.4 Hz), 7.92 (dd, 1H, Ar, J = 1.6 Hz, J = 8.0 Hz), 7.80 (d, 1H, J = 8.0 Hz), 7.61– 6.97 (m, 8H, Ar), 6.71 (d, 1H, NH, J = 8.0 Hz), 5.60 (dq, 1H, CH, J = 7.2 Hz, J = 6.8 Hz), 1.51 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 158.5, 156.2, 154.8, 141.1, 137.1, 133.9, 130.7, 129.1, 127.7, 126.7, 126.0, 123.5, 122.5, 119.6, 119.6, 115.7, 115.5, 45.1, 22.6 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1269. ATR‐FTIR: 3319 (NH); 1626 (C=O) cm⁻¹.

4.1.23. Compound 22S: (S−1‐(4‐Fluorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 80° (0.1 g/100 mL acetone). Yield: 42%, m.p.: 230°C–232°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 4‐fluorophenyl isocyanate. ¹H NMR (400 MHz, DMSO): δ 8.36 (s, 1H, NH), 8.12 (d, 1H, Ar, J = 8.4 Hz), 7.92 (dd, 1H, Ar, J = 1.6 Hz, J = 8.0 Hz), 7.80 (d, 1H, J = 8.0 Hz), 7.61– 6.97 (m, 8H, Ar), 6.71 (d, 1H, NH, J = 8.0 Hz), 5.60 (dq, 1H, CH, J = 7.2 Hz, J = 6.8 Hz), 1.51 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ δ 158.5, 156.2, 154.8, 141.1, 137.1, 133.9, 130.7, 129.1, 127.7, 126.7, 126.0, 123.5, 122.5, 119.6, 119.6, 115.7, 115.5, 45.1, 22.6 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇FN₂O: 307.1247; Found 307.1251. ATR‐FTIR: 3319 ((NH); 1626 (C=O) cm⁻¹.

4.1.24. Compound 23R: (R−1‐(3‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 100° (0.1 g/100 mL acetone). Yield: 27%, m.p.: 96°C‐98°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 3‐chlorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.51 (s, 1H, NH), 8.38 (d, 1H, NH J = 7.6 Hz), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar, J = 8.0 Hz), 7.76 (s, 1H, Ar), 7.54–7.08 (m, 7H, Ar), 6.23 (dq, 1H, CH, J = 6.8 Hz, J = 6.4 Hz), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 179.9, 141.7, 139.4, 133.9 133.0, 131.1, 130.5, 129.2, 128.2, 126.9, 126.2, 125.9, 123.8, 123.7, 123.4, 122.0, 121.0, 49.4, 20.9 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂S: 339.0723; Found 339.0702. ATR‐FTIR: 3401, 3203 (NH); 1217 (C=S) cm⁻¹.

4.1.25. Compound 24S: (S−1‐(3‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 100° (0.1 g/100 mL acetone). Yield: 50%, m.p.: 96°C–98°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine ve 3‐chlorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO): δ 9.51 (s, 1H, NH), 8.38 (d, 1H, NH J = 7.6 Hz), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.94 (d, 1H, Ar, J = 8.0 Hz), 7.85 (d, 1H, Ar, J = 8.0 Hz), 7.76 (s, 1H, Ar), 7.54–7.08 (m, 7H, Ar), 6.23 (dq, 1H, CH, J = 6.8 Hz, J = 6.4 Hz), 1.61 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 179.9, 141.7, 139.4, 133.9 133.0, 131.1, 130.5, 129.2, 128.2, 126.9, 126.2, 125.9, 123.8, 123.7, 123.4, 122.0, 121.0, 49.4, 20.9 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂S: 339.0723; Found 339.0722. ATR‐FTIR: 3401, 3206 (NH); 1217 (C=S) cm⁻¹.

4.1.26. Compound 25R: (R−1‐(3‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 20° (0.1 g/100 mL acetone). Yield: 92%, m.p.: 254°C−256°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 3‐chlorophenyl isocyanate. ¹H NMR (400 MHz, DMSO) δ 8.57 (s, 1H, NH), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.93 (d, 1H, Ar, J = 7.2 Hz), 7.82 (d, 1H, Ar, J = 8.0 Hz), 7.64 (t, 1H, Ar, J = 2.0 Hz), 7.61–7.46 (m, 4H, Ar), 7.20 (t, 1H, Ar, J = 8.0 Hz), 7.13 (ddd, 1H, Ar, J = 1.0 Hz, J = 2.0 Hz, J = 8.2 Hz), 6.91 (ddd, 1H, Ar, J = 1.0 Hz, J = 2.0 Hz, J = 8.0 Hz), 6.84 (d, 1H, NH, J = 7.6 Hz), 5.61 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.52 (d, 3H, CH₃, J = 7.2 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 154.5, 142.4, 141.0, 133.9, 133.6, 130.8, 129.2, 127.8, 126.7, 126.1, 125.9, 123.6, 122.6, 121.2, 117.31, 116.4, 45.2, 22.5 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂O: 323.0951; Found 323.0952. ATR‐FTIR: 3311 (NH); 1625 (C=O) cm⁻¹.

4.1.27. Compound 26S: (S−1‐(3‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (−) 20° (0.1 g/100 mL acetone).: Yield: 87%, m.p.: 255°C–256°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 3‐chlorophenyl isocyanate. ¹H NMR (400 MHz, DMSO) δ 8.57 (s, 1H, NH), 8.14 (d, 1H, Ar, J = 8.4 Hz), 7.93 (d, 1H, Ar, J = 7.2 Hz), 7.82 (d, 1H, Ar, J = 8.0 Hz), 7.64 (t, 1H, Ar, J = 2.0 Hz), 7.61–7.46 (m, 4H, Ar), 7.20 (t, 1H, Ar, J = 8.0 Hz), 7.13 (ddd, 1H, Ar, J = 1.0 Hz, J = 2.0 Hz, J = 8.2 Hz), 6.91 (ddd, 1H, Ar, J = 1.0 Hz, J = 2.0 Hz, J = 8.0 Hz), 6.84 (d, 1H, NH, J = 7.6 Hz), 5.61 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 1.52 (d, 3H, CH₃, J = 7.2 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 154.5, 142.4, 141.0, 133.9, 133.6, 130.8, 129.2, 127.8, 126.7, 126.1, 125.9, 123.6, 122.6, 121.2, 117.31, 116.4, 45.2, 22.5 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂O: 323.0951; Found 323.0953. ATR‐FTIR: 3311(NH); 1626 (C=O) cm⁻¹.

4.1.28. Compound 27R: (R−1‐(4‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 220° (0.1 g/100 mL acetone). Yield: 78%, m.p.: 70°C–73°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐chlorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.49 (s, 1H, NH), 8.32 (d, 1H, NH J = 7.2 Hz), 8.18 (d, 1H, Ar J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.88 (d, 1H, Ar, J = 8.0 Hz), 7.62‐7.52 (m, 6H, Ar), 7.33 (d, 2H, Ar, J = 8.4 Hz), 6.26 (br, 1H, CH), 1.62 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.0, 139.6, 139.1, 133.9, 131.1, 129.2, 128.7, 128.1, 128.0, 126.9, 126.2, 125.9, 124.5, 123.8, 123.4, 49.4, 21.0 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂S: 339.0723; Found 339.0724. ATR‐FTIR: 3184 (NH); 1236 (C=S) cm⁻¹.

4.1.29. Compound 28S: (S−1‐(4‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 220° (0.1 g/100 mL acetone). Yield: 90%, m.p.: 70°C–73°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine ve 4‐chlorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.49 (s, 1H, NH), 8.32 (d, 1H, NH J = 7.2 Hz), 8.18 (d, 1H, Ar J = 8.4 Hz), 7.97 (d, 1H, Ar, J = 8.0 Hz), 7.88 (d, 1H, Ar, J = 8.0 Hz), 7.62‐7.52 (m, 6H, Ar), 7.33 (d, 2H, Ar, J = 8.4 Hz), 6.26 (br, 1H, CH), 1.62 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO):): δ 180.0, 139.6, 139.1, 133.9, 131.1, 129.2, 128.7, 128.1, 128.0, 126.9, 126.2, 125.9, 124.5, 123.8, 123.4, 49.4, 21.0 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂S: 339.0723; Found 339.0720. ATR‐FTIR: 3194 (NH); 1236 (C=S) cm⁻¹.

4.1.30. Compound 29R: (R−1‐(4‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (−) 20° (0.1 g/100 mL acetone). Yield: 43%, m.p.: 238°C–242°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐(1‐naphthyl)ethylamine and 4‐chlorophenyl isocyanate. ¹H NMR (400 MHz, DMSO) δ 8.55 (s, 1H, NH), 8.16 (d, 1H, Ar, J = 8.4 Hz), 7.96 (d, 1H, Ar, J = 8.0 Hz), 7.84 (d, 1H, Ar, J = 8.0 Hz), 7.67–7.47 (m, 4H, Ar), 7.41 (d, 2H, Ar, J = 8.8 Hz), 7.25 (d, 2H, Ar, J = 8.8 Hz), 6.82 (d, 1H, NH, J = 8.0 Hz), 5.63 (dq, 1H, CH, J = 7.2, Hz, J = 6.8 Hz), 1.54 (d, 3H, CH₃, J = 6.8 Hz). ¹³C NMR (100 MHz, DMSO): δ 154.6, 141.0, 139.7, 133.9, 130.7, 129.1, 128.9, 127.7, 126.7, 126.1, 125.9, 125.0, 123.5, 122.5, 119.5, 45.1, 22.6 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂O: 323.0951; Found 323.0978. ATR‐FTIR: 3305 (NH); 1637 (C=O) cm⁻¹.

4.1.31. Compound 30S: (S−1‐(4‐Chlorophenyl)−3‐(1‐(Naphthalen‐1‐yl)Ethyl)Urea)

[α]²⁶ ₃₆₀ = (+) 20° (0.1 g/100 mL acetone). Yield: 46%, m.p.: 238°C–242°C. This compound was synthesized according to the general procedure by using S‐(−)−1‐(1‐naphthyl)ethylamine and 4‐chlorophenyl isocyanate. ¹H NMR (400 MHz, DMSO) δ 8.55 (s, 1H, NH), 8.16 (d, 1H, Ar, J = 8.4 Hz), 7.96 (d, 1H, Ar, J = 8.0 Hz), 7.84 (d, 1H, Ar, J = 8.0 Hz), 7.67–7.47 (m, 4H, Ar), 7.41 (d, 2H, Ar, J = 8.8 Hz), 7.25 (d, 2H, Ar, J = 8.8 Hz), 6.82 (d, 1H, NH, J = 8.0 Hz), 5.63 (dq, 1H, CH, J = 7.2, Hz, J = 6.8 Hz), 1.54 (d, 3H, CH₃, J = 6.8 Hz). ¹³C NMR (100 MHz, DMSO): δ 154.6, 141.0, 139.7, 133.9, 130.7, 129.1, 128.9, 127.7, 126.7, 126.1, 125.9, 125.0, 123.5, 122.5, 119.5, 45.1, 22.6 ppm. HRMS (TOF MS ES+): Calculated for C₁₉H₁₇ClN₂O: 323.0951; Found 323.0959. ATR‐FTIR: 3302 (NH); 1637 (C=O) cm⁻¹.

4.1.32. Compound 31R: (R−1‐(2‐Methoxyphenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 260° (0.1 g/100 mL acetone). Yield: 73%, m.p.: 106°C–108°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐phenylethylamine and 2‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 8.96 (s, 1H, NH), 8.45 (d, 1H, Ar, J = 8.0 Hz), 8.02 (d, 1H, NH, J = 7.6 Hz), 7.51–6.99 (m, 7H, Ar), 6.89 (t, 1H, Ar, J = 7.6 Hz), 5.56 (br, 1H, CH), 3.81 (s, 3H, OCH₃), 1.45 (d, 3H, CH_3, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.1, 151.5, 144.4, 129.2, 128.8, 128.3, 127.2, 127.1, 126.7, 125.4, 125.1, 120.1, 111.6, 56.0, 53.0, 22.4 ppm. HRMS (TOF MS ES+): Calculated for C₁₆H₁₈N₂OS: 287.1218; Found 287.1219. ATR‐FTIR: 3239 (NH); 1249 (C=S) cm⁻¹.

4.1.33. Compound 32S: (S−1‐(2‐Methoxyphenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 260° (0.1 g/100 mL acetone). Yield: 65%, m.p.: 106°C–108°C. This compound was synthesized according to the general procedure by using S‐(‐)−1‐phenylethylamine and 2‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 8.96 (s, 1H, NH), 8.45 (d, 1H, Ar, J = 8.0 Hz), 8.02 (d, 1H, NH, J = 7.6 Hz), 7.51–6.99 (m, 7H, Ar), 6.89 (t, 1H, Ar, J = 7.6 Hz), 5.56 (br, 1H, CH), 3.81 (s, 3H, OCH₃), 1.45 (d, 3H, CH_3, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.1, 151.5, 144.4, 129.2, 128.8, 128.3, 127.2, 127.1, 126.7, 125.4, 125.1, 120.1, 111.6, 56.0, 53.0, 22.4 ppm. HRMS (TOF MS ES+): Calculated for C₁₆H₁₈N₂OS: 287.1218; Found 287.1218. ATR‐FTIR: 3238 (NH); 1249 (C=S) cm⁻¹.

4.1.34. Compound 33R: (R−1‐(3‐Methoxyphenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 300° (0.1 g/100 mL acetone). Yield: 66%, m.p.: 66°C–68°C. This compound was synthesized according to the general procedure by usingR‐(+)−1‐phenylethylamine and 3‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.44 (s, 1H, NH), 8.15 (d, 1H, NH, J = 8.0 Hz), 7.41–7.13 (m, 7H, Ar), 6.93 (d, 1H, Ar J = 8.0 Hz), 6.63 (dd, 1H, Ar, J = 8.0 Hz, J = 2.5 Hz,), 5.54 (dq, 1H, CH, J = 6.8 Hz, J = 7.2 Hz), 3.69 (s, 3H, OCH₃), 1.45 (d, 3H, CH_3, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO) δ 179.9, 159.8, 144.3, 141.1, 129.7, 128.8, 127.3, 126.8, 115.1, 109.9, 108.6, 55.5, 53.1, 22.3 ppm. HRMS (TOF MS ES+): Calculated for C₁₆H₁₈N₂OS: 287.1218; Found 287.1226. ATR‐FTIR: 3325, 3122 (NH); 1207 (C=S) cm⁻¹.

4.1.35. Compound 34S: (S−1‐(3‐ Methoxyphenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 300° (0.1 g/100 mL acetone). Yield: 73%, m.p.: 66°C–68°C. This compound was synthesized according to the general procedure by using S‐(‐)−1‐phenylethylamine and 3‐methoxyphenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.44 (s, 1H, NH), 8.15 (d, 1H, NH, J = 8.0 Hz), 7.41–7.13 (m, 7H, Ar), 6.93 (d, 1H, Ar, J = 8.0 Hz), 6.63 (dd, 1H, Ar, J = 8.0 Hz, J = 2.5 Hz,), 5.54 (dq, 1H, CH,J = 6.8 Hz, J = 7.2 Hz), 3.69 (s, 3H, OCH₃), 1.45 (d, 3H, CH_3, J = 6.8 Hz) ppm.¹³C NMR (100 MHz, DMSO) δ δ 179.9, 159.8, 144.3, 141.1, 129.7, 128.8, 127.3, 126.8, 115.1, 109.9, 108.6, 55.5, 53.1, 22.3 ppm. HRMS (TOF MS ES+): Calculated for C₁₆H₁₈N₂OS: 287.1218; Found 287.1223. ATR‐FTIR: 3326, 3122 (NH); 1207 (C=S) cm⁻¹.

4.1.36. Compound 35R: (R−1‐(2‐Fluorophenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 280° (0.1 g/100 mL chloroform). Yield: 95%, m.p.: 80°C–82°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐phenylethylamine and 2‐ fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.17 (s, 1H, NH), 8.42 (d, 1H, NH J = 6.4 Hz), 7.85–7.08 (m, 9H, Ar), 5.54 (br, 1H, CH), 1.47 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO) δ 181.0, 144.2, 128.8, 127.9, 127.7, 127.6, 127.5, 127.3 126.6, 124.3, 116.0, 53.3, 22.4 ppm. HRMS (TOF MS ES+): Calculated for C₁₅H₁₅FN₂S: 273.0862; Found 273.0864. ATR‐FTIR: 3195 (NH); 1217 (C=S) cm⁻¹.

4.1.37. Compound 36S: (S−1‐(2‐Fluorophenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 280° (0.1 g/100 mL chloroform). Yield: 85%, m.p.: 80°C–82°C. This compound was synthesized according to the general procedure by using S‐(‐)−1‐phenylethylamine and 2‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.17 (s, 1H, NH), 8.42 (d, 1H, NH J = 6.4 Hz), 7.85–7.08 (m, 9H, Ar), 5.54 (br, 1H, CH), 1.47 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO) δ 181.0, 144.2, 128.8, 127.9, 127.7, 127.6, 127.5, 127.3 126.6, 124.3, 116.0, 53.3, 22.4 ppm. HRMS (TOF MS ES+): Calculated for C₁₅H₁₅FN₂S: 273.0862; Found 273.0862. ATR‐FTIR: 3195 (NH); 1217 (C=S) cm⁻¹.

4.1.38. Compound 37R: (R−1‐(3‐Fluorophenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (−) 90° (0.1 g/100 mL acetone). Yield: 79%, m.p.: 92°C–94°C. This compound was synthesized according to the general procedure by using R‐(+)−1‐phenylethylamine and 3‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.58 (s, 1H, NH), 8.30 (d, 1H, NH, J = 7.6 Hz), 7.61 (d, 1H, Ar, J = 11.2 Hz), 7.39–7.15 (m, 7H, Ar), 6.86 (td, 1H, Ar, J = 2.4 Hz, J = 8.0 Hz), 5.51 (br, 1H, CH), 1.45 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.0, 163.4, 160.9, 144.1, 142.0, 141.9, 130.4 (d, J = 9.6 Hz), 128.8, 127.3, 126.7, 118.2, 110.5 (d, J = 21.2 Hz), 109.1 (d, J = 21.2 Hz), 53.0, 22.3 ppm. HRMS (TOF MS ES+): Calculated for C₁₅H₁₅FN₂S: 273.0862; Found 273.0863. ATR‐FTIR: 3207 (NH); 1254 (C=S) cm⁻¹.

4.1.39. Compound 38S: (S−1‐(3‐Fluorophenyl)−3‐(1‐Phenylethyl)Thiourea)

[α]²⁶ ₃₆₀ = (+) 90° (0.1 g/100 mL acetone). Yield: 90%, m.p.: 92°C–94°C. This compound was synthesized according to the general procedure by using S‐(‐)−1‐Phenylethylamine and 3‐fluorophenyl isothiocyanate. ¹H NMR (400 MHz, DMSO) δ 9.58 (s, 1H, NH), 8.30 (d, 1H, NH, J = 7.6 Hz), 7.61 (d, 1H, Ar, J = 11.2 Hz), 7.39–7.15 (m, 7H, Ar), 6.86 (td, 1H, Ar, J = 2.4 Hz, J = 8.0 Hz), 5.51 (br, 1H, CH), 1.45 (d, 3H, CH₃, J = 6.8 Hz) ppm. ¹³C NMR (100 MHz, DMSO): δ 180.0, 163.4, 160.9, 144.1, 142.0, 141.9, 130.4 (d, J = 9.6 Hz), 128.8, 127.3, 126.7, 118.2, 110.5 (d, J = 21.2 Hz), 109.1 (d, J = 21.2 Hz), 53.0, 22.3 ppm. HRMS (TOF MS ES+): Calculated for C₁₅H₁₅FN₂S: 273.0862; Found 273.0868. ATR‐FTIR: 3207 (NH); 1254 (C=S) cm⁻¹.

4.2. Biological Activity Assessments

4.2.1. Determination of Cholinesterase (ChE) Inhibitory Activities

The Ellman method was used to determine the inhibitory effects of compounds on both hAChE and hBChE enzymes (Ellman et al. 1961). hAChE and hBChE activity measurements were performed at 25°C in 500 mM KP (pH 7.4) medium containing 5 mM acetylthiocholine or 5 mM butyrylthiocholine and 0.125 mM DTNB as substrate. The reactions were initiated by adding the enzymes. FLUOstar Omega Microplate Reader (BMG LABTECH) was used to monitor the absorbance change at 412 nm. Reference compounds, donepezil and selegiline, were used for comparative analysis of the experimental results (Eyer et al. 2003). The results are given as IC₅₀ values (n = 6). The IC₅₀ from the dose−response equation was converted to an inhibitory constant (Ki) using a IC₅₀‐to‐Ki calculator (Cer et al. 2009) The IC₅₀‐to‐Ki converter computes Ki values from experimentally determined IC₅₀ values for inhibitors of enzymes that obey classic Michaelis‐Menten kinetics.

4.2.2. Determination of hMAO Inhibitory Activities

Inhibitory activities of the synthesized compounds on hMAO isoforms were determined with the help of recombinant hMAO‐A and ‐B isoforms. The hMAO activity was determined with the Amplex Red MAO assay kit (Molecular Probes, USA).

The recombinant enzymes were diluted in reaction buffer, pH 7.4, and specific inhibitors, namely clorgyline and pargyline, added at a 0.5‐mM concentration to 100 µL of enzyme solution and incubated at room temperature for 30 min. The positive control solution was made by dilution of the working solution of 20 mM H₂O₂ in reaction buffer to a final concentration of 10 µM. The negative control comprised the reaction buffer without H₂O₂. The reaction was started by adding 100 µL of the Amplex Red reagent to all the wells with the samples and the controls and incubated for 30 min at room temperature. The measurment was carried out by following the fluorescence, which appeared at an excitation wavelength of 530 nm and emission wavelength of 590 nm. SI was calculated as (K _i value of hMAO‐A)/(K _i value of hMAO‐B). Thus, the inhibiting effect of the compounds is increased on the hMAO‐A isoform when the probe SI value decreases, and the inhibiting effect of the compounds is increased on the hMAO‐B isoform together with increasing the SI value.

4.2.2.1. Kinetic Studies

The synthesized compounds were dissolved in DMSO at a maximum final concentration of 1% and tested within the appropriate concentration range of 0.001 µM to 5.00 mM. Selegiline (an irreversible selective hMAO‐B inhibitor), lazabemide (a reversible selective hMAO‐B inhibitor), and moclobemide (a reversible selective hMAO‐A inhibitor) were also dissolved in DMSO and used within the same concentration range. The type of enzyme inhibition was determined using Lineweaver–Burk plots, and IC₅₀ and Kᵢ values were calculated. Protein concentrations were measured using the SIGMA Total Protein Kit (Micro TP0100), according to the Bradford method (Bradford 1976).

4.2.2.2. Reversibility Studies

The reversibility of the inhibitory effects of the synthesized compounds on hMAO isoforms was assessed by dialysis. Recombinant enzymes were prepared in 0.05 M potassium phosphate buffer (pH 7.4) containing 5% sucrose and 1% DMSO, at a concentration five times the Kᵢ value, and incubated at 37°C for 15 min. Known inhibitors—selegiline, lazabemide, and moclobemide‐ were prepared and incubated using this method. These mixtures were then placed in the dialysis bags of size 25 × 16 mm and of molecular weight cut‐off of 12,000, and were dialyzed against dialysis buffer containing 100 mM potassium phosphate, pH 7.4, and 5% sucrose at 4°C for 24 h. The dialysis process was repeated twice. The remaining hMAO activity was measured after dialysis.

4.2.3. Beta‐Amyloid (Aβ) Fibril Assays

4.2.3.1. Preparation of Aβ (1‐40) and (1‐42) Fibrils

500 µM Aβ (1‐40) and 500 µM Aβ (1‐42) peptides were dissolved in 0.02% cold ammonium solution and thoroughly mixed using vortex agitation. Out of the 100 µL solution, 20 µL was aliquoted for incubation to initiate fibril formation. The remaining solution was preserved as fresh fibrils at −80°C. To prepare Aβ (1‐40) and Aβ (1‐42) fibrils, a reaction mixture containing 50 µM freshly prepared Aβ (1‐40/1‐42), 100 mM NaCl, and 50 mM phosphate buffer at pH 7.5, resulting in a total volume of 2400 µL was prepared and incubated 48 h. Fibril formation was determined through thioflavin T (ThT) fluorescence measurements (Baysal et al. 2017).

4.2.3.2. ThT Fluorescence Measurements

ThT fluorescence measurements of Aβ (1–40) and Aβ (1–42) fibrils were performed with an excitation wavelength of 448 nm and an emission wavelength of 490 nm. These measurements were conducted using a reaction mixture consisting of 5 mM ThT and 50 mM glycine–NaOH buffer at pH 8.5. ThT fluorescence measurements were performed utilizing a Shimadzu (RF‐5301PC) fluorescence spectrophotometer, following a method previously reported (Hasegawa et al. 1999).

4.2.3.3. Inhibition Studies of Aβ (1‐40) and (1‐42) Fibrils

Aβ fibril (either Aβ (1‐40) or Aβ (1‐42)) at a concentration of 50 µM was subjected to incubation in a 500 µL reaction medium comprising 100 mM NaCl and 50 mM phosphate buffer at pH 7.5. Various concentrations of the synthesized compounds were introduced into the mixture, and subsequently, ThT fluorescence intensity was measured. For inhibition experiments, the test compounds were initially formulated as concentrated stock solutions and subsequently serially diluted into the reaction medium just prior to measurement to achieve the specified final concentrations; solutions were prepared to ensure homogeneity at the working concentrations utilized for IC50 determination. The reduction in spectral values indicated the inhibition of fibril formation in the presence of these compounds (Baysal et al. 2017). The results are given as IC₅₀ values (n = 3).

4.2.4. In Vitro antioxidant activity

4.2.4.1. DPPH Free Radical Scavenging Activity

The DPPH assay was conducted with minor alterations to the previously established protocol (Fukumoto and Mazza 2000). A fresh DPPH working solution was prepared and protected from light for each experiment. Test compounds were prepared at the desired concentrations. In a 96‐well microplate, the DPPH solution was mixed with compound solutions to obtain a final DPPH concentration of 150 µM in each well. Control wells contained DPPH plus the corresponding volume of solvent without compound. To correct for any intrinsic absorbance of test compounds, compound blank wells (compound solution without DPPH) were prepared in parallel. The plate was incubated for 30 min at room temperature in the dark. After incubation, absorbance was measured at 520 nm using a FLUOstar Omega (BMG LABTECH) microplate reader.

Radical scavenging activity was calculated as percent inhibition relative to the control using blank correction, as follows:

$$\%\mathrm{inhibition}=(1-\frac{A_{\mathrm{Sample}}-A_{\mathrm{Blank}}}{A_{\mathrm{Control}}-A_{\mathrm{Blank}\left(\mathrm{control}\right)}})\times 100,$$

where A _Sample is the absorbance of DPPH + compound, A _Blank is the absorbance of compound without DPPH, A _Control is the absorbance of DPPH + control, and A _{Blank(control)} is the absorbance of control without DPPH. Trolox was included as a reference antioxidant under the same conditions. Dose‐response curves were generated from the percent inhibition values, and IC₅₀ values were determined by nonlinear regression. Each concentration was tested in six replicates (n = 6), and data are reported as IC₅₀ values.

4.2.4.2. CUPRAC Antioxidant Assay

The CUPRAC test is the most suitable test for demonstrating the effects of glutathione and thiol‐type antioxidants since the interactions of antioxidants present in the environment do not affect their absorbance properties in this test. Its suitability for redox potential, especially when working at pH levels close to physiological pH, the stability of CUPRAC reagents, and its suitability for lipophilic antioxidants make this test superior to other electron‐transfer based tests.

60 µL of copper(II) chloride (CuCl2) solution (1.0 × 102 M), 60 µL of ammonium acetate (NH4 Ac) buffer solution at pH 7.0 (1.0 M), 60 µL of neocuproine solution (7.5 × 10‐3 M), and 66 µL of compound solution at different concentrations were mixed. The mixture was incubated at room temperature for 30 min, and measurements were taken at a wavelength of 450 nm. Trolox was used as reference. Increased absorbance indicates increased reduction capability. Dose‐response curves were generated from the percent inhibition values, and IC₅₀ values were determined by nonlinear regression. Each concentration was tested in six replicates (n = 6), and data are reported as IC₅₀ values (µM) (Apak et al. 2004).

4.2.4.3. Superoxide Anion Scavenging Capacity Assay

The superoxide anion‐scavenging capacities of the synthesized compounds were determined according to the method proposed by Liu (Liu et al. 1997). Superoxide radicals were generated in the PMS‐NADH system, and their reduction of nitroblue tetrazolium was monitored. Absorbance was measured at 560 nm against the standard. A decrease in absorbance indicated an increased capacity of the compounds to scavenge superoxide anions. BHT was used as reference. Dose‐response curves were generated from the percent inhibition values, and IC₅₀ values were determined by nonlinear regression. Each concentration was tested in six replicates (n = 6), and data are reported as IC₅₀ values (µM) (Liu et al. 1997).

4.2.5. Cytotoxicity Assay

XTT‐based cell proliferation kit (Biological Industries, USA) was used to determine the effect of the synthesized compounds on the proliferation of the HepG2 cell line. 96‐well plates were seeded with approximately 4 × 10⁴ to 6 × 10⁴ cells. After reaching confluence, the compounds were administered at concentrations of 1, 5, and 25 µM, followed by incubation at 37°C in a CO₂ incubator for 24 h. Subsequently, 50 µL of XTT solution per well was added, and the cells were subjected to further incubation at 37°C for 2 h. Following incubation, the absorbance was measured using FLUOstar Omega Microplate Reader (BMG LABTECH) at 450 nm. The results were standardized against values obtained from the control group, which received an equivalent volume of DMSO as the compounds (n = 6).

4.2.6. HSD10 Inhibition

The recombinant 17β‐HSD10 was expressed in Escherichia coli BL21 (DE3) strain and purified as described previously (Schmidt et al. 2020). For the compounds' inhibitory ability and IC₅₀ determination, the SAAC method using acetoacetyl‐CoA (AAC) as a substrate and absorbance readout at 340 nm was used (Schmidt et al. 2020). In brief, 0.15 μg of recombinant 17β‐HSD10 was mixed with 320 μmol/L nicotinamide adenine dinucleotide (NADH) as an enzyme cofactor in assay buffer (10 mM Tris‐HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.001% Tween 20 and 0.01% bovine serum albumin) and the mixture was preincubated for 5 min at 37°C with the tested inhibitor (or vehicle control). The reaction was started by the addition of 320 μmol/L AAC and monitored as a change in absorbance at 340 nm for 5 min in 10‐s intervals. Compounds were dissolved in anhydrous DMSO and further diluted in the assay buffer to reach the final concentrations. All data were analyzed in GraphPad Prism 7 software using non‐linear regression.

4.2.7. In Vitro Blood–Brain Barrier Permeation (BBB) Assay

The selected derivatives were tested for their ability to cross the BBB using the PAMPA‐BBB test. The drugs used as standards in this method (testosterone, verapamil, β‐estradiol, progesterone, corticosterone, piroxicam, hydrocortisone, lomefloxacin, dopamine) and porcine brain lipid (PBL) were purchased from Merck (Germany); donor and acceptor microplates were from Millipore (USA). In brief, the compounds were dissolved in DMSO at 100 mg/mL and then diluted in PBS/EtOH 7:3 and added into donor plates. The acceptor plate was placed on top and incubated at 25°C for 16 h. Afterwards, the donor plate was removed, and the concentrations of the compounds in the wells of the acceptor plate were measured by a UV plate reader. The abilities of compounds to penetrate the BBB were assessed as follows (Di et al. 2003):

• CNS+ (very good BBB penetration): Pe (10⁻⁶ cm s⁻¹) > 4.00

• CNS± (weak BBB penetration): Pe (10⁻⁶ cm s⁻¹) 4.00‐2.00

• CNS‐ (poor BBB penetration): Pe (10⁻⁶ cm s⁻¹) < 2.00

4.2.8. In Vivo Assessment of the Spatial Learning and Memory With Morris Water Maze Test

To evaluate the in vivo effects of the compounds, they were administered orally via gavage to mice with an AD like model induced by Aluminum Chloride (AlCl₃). After 1 week of administration, the Morris Water Maze test was conducted on the mice. The Morris Water Maze test is one of the most commonly used tests to evaluate anti‐Alzheimer effect. This test assesses the spatial memory of animals and provides important information regarding the effectiveness of the treatment (Kazkayasi and Telli 2024).

4.2.8.1. Animals

A total of 72 male Balb/c albino mice (n = 8 for each group) weighing 25–30 g were used in the experiments. The mice were brought to the laboratory 1 week before the experiments began to acclimate to the environment. They were housed in rooms maintained at 22 ± 2°C with a 12‐h light/dark cycle and fed a standard pellet diet. Food and water were provided ad libitum. The experiments were approved by Hacettepe University Animal Experiments Local Ethics Committee and was held according to Directive 2010/63/EU.

4.2.8.2. Development of Alzheimer's Disease‐Like Model Induced by AlCl₃ and Administration of Compounds

Because it is a reproducible and relatively easy model, it was preferred for developing AD very common. Furthermore, it allows for the evaluation of cognitive changes associated with AD. Accordingly, 40 mg/kg of AlCl3 was administered intraperitoneally (i.p.) to male Balb/c mice for 30 days (Mansour Suliman ²⁰¹⁵). At the end of 30 days, seven compounds (3 R , 6 S , 11 R , 12 S , 14 S , 23 R , and 30 S ) which are found effective after in vitro and in silico methods, were administered for 1 week at a common screening dose of 20 mg/kg via oral gavage.

4.2.8.3. Morris Water Maze Test

One hour after the last dose of the compounds was administered, the Morris Water Maze test was performed on the mice. In this test, a black pool filled with water with a diameter of 122 cm had a black platform submerged 3 cm below the water level. The pool was filled with water at a temperature of 22°C–26°C, and cues of a size visible to the mice while swimming were hung on the walls of the room where the pool was located. Each test day, the mice were released from a different location in the pool four times, and the time taken for them to find and climb onto the submerged platform was measured. The test was repeated at the same time for four consecutive days to allow the mice to learn the location of the platform. On the fifth day, the platform was removed, and the test was conducted. The experiments were recorded using a video tracking system (Videomot2, TSE Systems, Thuringia, Germany), and learning curves were plotted based on the time taken to find the platform. The time spent in the quadrant where the platform was located on the fifth day was compared to evaluate the effectiveness of the newly synthesized compounds.

4.2.8.4. Statistical Analysis

Statistical analysis was performed using one‐way analysis of variance (ANOVA) and post hoc Tukey test. The results are presented as mean ± standard error. Two way ANOVA was performed to analyse learning curve in MWM test.

4.3. Molecular Modeling Methodology

Pharmacokinetic parameters were predicted using SwissADME (Daina et al. 2014, ²⁰¹⁷) website. Molecular docking and molecular dynamic simulations were performed using Autodock Vina (Trott and Olson 2010) and Gromacs 5.1.4 package (Abraham et al. 2015), respectively. Detailed procedures and validations are given in the Supplementary Information.

Author Contibutions

Sule Erol Gunal: conceptualization, writing – review and editing, writing – original draft, validation, visualization, methodology, investigation, formal analysis, data curation. Senel Teke Tuncel: conceptualization, writing – review and editing, writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation. İlke Demir: conceptualization, writing – review and editing, writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation, software. İpek Baysal: methodology, investigation, Gökçen Telli: methodology, investigation. Safiye Sag Erdem: conceptualization, writing – review and editing, writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation, software. Gulberk Ucar: methodology, investigation, Michaela Hanzlova: methodology, investigation. Kamil Musilek: formal analysis, data curation, writing – review and editing. İlknur Dogan: project administration, supervision, resources, conceptualization, writing – review and editing, methodology, validation, investigation, formal analysis. Nesrin Gökhan Kelekçi: project administration, supervision, conceptualization, writing – review and editing, writing – original draft, validation, investigation, formal analysis, data curation. TUBİTAK: funding acquisition.

Conflicts of Interest

Sule Erol Gunal, Senel Teke Tuncel, İlke Demir, İpek Baysal, Safiye Sag Erdem, Gulberk Ucar, İlknur Dogan, and Nesrin Gokhan Kelekci are listed as inventors on Patent No. TR 2023 016729 B related to the compounds described in this manuscript. The other authors declare no conflicts of interest. This disclosure is provided in accordance with the journal's policy on transparency and competing interests.

Supporting information

Supporting File 1

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.