Design and synthesis of 2-phenyl-1H-benzo[d]imidazole derivatives as 17β-HSD10 inhibitors for the treatment of Alzheimer's disease

Abstract

It has been reported that 17β-HSD10 plays a key role in Alzheimer's disease. Here, a total of 44 2-phenyl-1H-benzo[d]imidazole derivatives were designed and synthesized as novel 17β-HSD10 inhibitors based on rational design and SAR studies. Among them, compound 33 (N-(4-(1,4,6-trimethyl-1H-benzo[d] imidazol-2-yl)phenyl)cyclohexanecarboxamide) showed high inhibitory efficacy (17β-HSD10 IC₅₀ = 1.65 ± 0.55 μM) and low toxicity (HepaRG IC₅₀ >100 μM). The Morris water maze experiment revealed that compound 33 could alleviate cognitive impairment induced by scopolamine in mice. This study facilitates the further development of more potent 17β-HSD10 inhibitors for the treatment of Alzheimer's disease.

It has been reported that 17β-HSD10 plays a key role in Alzheimer's disease.

1. Introduction

Alzheimer's disease (AD) is the most common form of dementia, a fatal neurodegenerative disorder primarily characterized by impairments in memory, behavior, and reasoning.^1,2 AD is considered one of the “four major killers” threatening human health.^3,4 Researchers have invested considerable energy and resources in this field, continuously advancing the study of pathological mechanisms, therapeutic methods, and early diagnostic techniques.⁵ Recent research has concentrated on understanding the underlying mechanisms of AD to identify potential therapeutic targets.⁶ Human 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is an enzyme of the short-chain dehydrogenase/reductase (SDR) superfamily.⁷ 17β-HSD10 is a mitochondrial enzyme known for its potential role in AD.⁸ 17β-HSD10 plays a crucial role in AD by binding to amyloid beta (Aβ) protein and participating in the transmission of neurotoxins associated with the disease.⁹ Therefore, clarifying the role of 17β-HSD10 in AD is of great significance for the development of AD drugs.

The human 17β-HSD10 enzyme is one of the known interacting partners of the Aβ peptide, a key peptide involved in the pathogenesis of AD.¹⁰ The generation and deposition of Aβ into extracellular insoluble plaques are key morphological features of AD and contribute to the death of numerous neurons in the human brain.^11,12 Aβ interacts with 17β-HSD10 via several amino acids in the d-loop region, which is a unique sequence insertion absent in other members of the SDR family.^13,14 This interaction has been observed in the brains of Alzheimer's disease patients and in experimental transgenic mice overexpressing amyloid precursor protein along with 17β-HSD10.¹⁵ Research has found that 17β-HSD10 is an interacting partner of Aβ and its expression has been found to be elevated in the AD brain and inhibiting 17β-HSD10 can significantly improve AD symptoms in mice.¹⁵

Current research has also found that overexpression of 17β-HSD10 disrupts the homeostasis of neurosteroid metabolism, and may alter the levels of neuroprotective steroids in different regions of the brain, such as 17β-estradiol (E2) and isoprogesterone (ALLOP).¹⁶ These two neurosteroids have significant effects on mitochondrial function, bioenergetics, antioxidant processes, and the accumulation of AD related pathological markers (Aβ and hyperphosphorylated tau protein).¹⁷ Inhibiting the activity of 17β-HSD10 enzyme provides a feasible therapeutic strategy for AD treatment, as inhibiting this enzyme can restore the disrupted homeostasis of E2 and ALLOP neurosteroids.^18,19 So far, there are few reported 17β-HSD10 inhibitors, mainly pyrazoles, steroids and benzo thiazolylurea derivatives.^14,20–30 Compounds C1, C2, and C8 all contain diarylurea/thiourea, which are commonly found in most kinase type II inhibitors, leading to selectivity issues with the compounds; it has been reported that the pyrazolo [3,4-d]pyrimidin-4(1H)-thione skeleton of compound C3 occupied the substrate binding pocket and formed a covalent adduct with NAD + cofactor at the active site of ABAD, exhibiting strong activity (IC₅₀ = 92 nM). Therefore, this skeleton of compound C3 may occupy the kinase hinge region and inhibit other kinase targets, resulting in side effects; the IC₅₀ value of compound C4 for 17β-HSD10 is 52.7 μM, indicating low activity and poor drug resistance; compounds C5–C7 contain steroid structures, which may have side effects caused by steroids; compound C9 contains multiple phenolic hydroxyl groups and is easily oxidized to quinoids, leading to toxicity. Therefore, it is very meaningful to develop novel 17β-HSD10 inhibitors with potent activity and low toxicity inhibitors (Fig. 1).

Fig. 1. Chemical structure of 17β-HSD10 inhibitors.

2. Results and discussion

2.1. Design and optimization

The co-crystal complex (PDB: 1U7T) of ABAD/HSD10 with a bound inhibitor has been reported by Kissinger, C. R. who provided a receptor structure and theoretical basis for the rational design of 17β-HSD10 inhibitors.³¹ Laura Aitken and Ondrej Benek et al. discovered novel 17β-HSD10 inhibitors with a diarylurea structure based on the co-crystal complex.^32,33 In addition, based on CADD and HTS, L. Aitken et al. discovered that benzimidazole fragments have inhibitory activity against 17β-HSD10.³⁴ These reports proved that the co-crystal complex (PDB: 1U7T) could be used as the receptor for the design of 17β-HSD10 inhibitors.

In the preliminary work, we have established a compound library that included derivatives of 4-chloropyrrolopyrimidine, indole, 7-azaindole, benzofuran, and benzimidazole. In this work, based on the work reported by Aitken et al., the previously constructed compound library was screened and compound H1 was discovered with good inhibitory activity against 17β-HSD10 (IR (%) at 5 μM = 54.85 ± 5.71).

In order to obtain 17β-HSD10 inhibitors with better properties, the binding mode of compound H1 was analyzed (Fig. 2). By analyzing the docking of H1 with 17β-HSD10, we noticed that 2-phenyl-1H-benzo [d] imidazole occupied the active pocket in a straight line posture and interacts with the amino group of Gln165 through hydrogen bonding. Importantly, we found that there was a hydrophobic cavity composed of Leu22, Thr153, Pro198 and Phe201, closely adjacent to the amino group, which showed that hydrophobic fragments could be introduced onto the amino group of compound H1. In addition, the hydrogen atom of imidazole was close to the isopropyl of Leu217 with a length distance of 3.3 Å and 4.2 Å, which shows that the molecular stacking interaction with isopropyl groups may be increased by introducing some small fragments (Fig. 2).

Fig. 2. Binding mode of hit compound H1 with the active site of 17β-HSD10 (PDB: 1U7T). 17β-HSD10 is shown in gray ribbons with selected residues colored green. Hydrogen bonds are drawn as yellow dashed lines. Compound H1 is shown with blue sticks. The illustration was generated using PyMOL.

Therefore, based on the above analysis, we believed that H1 could be reasonably optimized through two strategies to obtain an efficient 17β-HSD10 inhibitor (Fig. 2 and 3). On the one hand, hydrophobic fragments such as alkyl and aryl groups could be introduced by linking the amino group to occupy the hydrophobic cavity; on the other hand, the interaction with Leu217 could be enhanced by methylating the imidazole or altering the electron cloud distribution by changing the substituents of benzimidazole to improve the activity.

Fig. 3. The design ideas for target compounds.

2.2. Chemistry

The preparation of title compounds (1–44) is described in Schemes 1–7. As shown in Scheme 1, compounds 1–7 were obtained through a cyclization reaction. As shown in Scheme 2, compounds 8–16 were synthesized. First, the key intermediate H1 was synthesized by a nitro reduction reaction, then, intermediate H1 reacted with isocyanates to obtain a series of urea derivatives 8–16. Compounds 17–32 were obtained through an amide condensation reaction, and the synthetic route is described in Scheme 3. Compounds 33–44 were synthesized according to Schemes 4–7 through the same synthetic method as compound 17.

Scheme 1. Synthesis of compounds 1–7. Reagents and conditions: A. AcOH, DCM, 85 °C, 6 h.

Scheme 2. Synthesis of compounds 8–16. Reagents and conditions: B. Zn, NH₄Cl, MeOH, H₂O, rt, 4 h; C. DIPEA, DMAP, DMF, rt, 6 h.

Scheme 3. Synthesis of compounds 17–32. Reagents and conditions: D. DIPEA, HATU, DMF, rt, 6 h.

Scheme 4. Synthesis of compounds 33–34. Reagents and conditions: B. Zn, NH₄Cl, MeOH, H₂O, rt, 4 h; D. DIPEA, HATU, DMF, rt, 6 h; E. CH₃I, NaH, DMF, rt, 6 h.

Scheme 5. Synthesis of compounds 35–36. Reagents and conditions: B. Zn, NH4Cl, MeOH, H₂O, rt, 4 h; D. DIPEA, HATU, DMF, rt, 6 h; F. Pd (OAc)₂, K₂S₂O₈, TfOH, AcOH, MF, 100 °C, 24 h.

Scheme 6. Synthesis of compounds 37–38. Reagents and conditions: A. AcOH, DCM, 85 °C, 6 h; B. Zn, NH₄Cl, MeOH, H₂O, rt, 4 h; D. DIPEA, HATU, DMF, rt, 6 h; E. CH₃I, NaH, DMF, rt, 6 h.

Scheme 7. Synthesis of compounds 39–44. Reagents and conditions: A. AcOH, DCM, 85 °C, 6 h; B. Zn, NH₄Cl, MeOH, H₂O, rt, 4 h; D. DIPEA, HATU, DMF, rt, 6 h; E. CH₃I, NaH, DMF, rt, 6 h.

The double bond interconversion of benzimidazole leads to the coexistence of isomers. In order to determine the absolute position of the methyl group, the crystal structures of compounds M9 and 34 were determined by X-ray crystallography (Fig. 4), indirectly proving the absolute position of imidazole methylation in these two series of derivatives. Compound M9: C₁₆H₁₅N₃O₂, orthorhombic, space group: p2₁2₁2₁; a = 7.1923(7), b = 12.7507(11), c = 15.1600(14) (Å); α = 90, β = 90, γ = 90 (°), V = 1390.3(2) nm³, T = 223.0 K, Z = 4, Dc = 1.344 g cm⁻³, F(000) = 592, reflections collected = 9310 (R_int = 0.0342), Indep. reflns = 2441, Refns obs. [I > 2σ(I)] = 2232, data/restr./paras = 2441/0/194, goodness-of-fit on F² = 1.059, R₁, wR₂ (all data) = 0.0365/0.0842, R₁, wR₂ [I > 2σ(I)] = 0.0325/0.0813, large peak/hole (e Å) = 0.154/−0.110, CCDC. No: 2385735. Compound 34: C₂₃H₂₅F2N₃O(H₂O), triclinic, space group: P1̄; a = 7.1699(13), b = 10.430(2), c = 15.039(3) (Å); α = 80.809(8), β = 89.290(7), γ = 78.757(8) (°), V = 1088.7(4) nm³, T = 223 K, Z = 2, Dc = 1.267 g cm⁻³, F(000) = 2440, reflections collected = 28 915 (R_int = 0.0737), Indep. reflns = 4943, Refns obs. [I > 2σ(I)] = 3532, data/restr./paras = 4943/0/277, goodness-of-fit on F² = 1.035, R₁, wR₂ (all data) = 0.0711/ 0.1373, R₁, wR₂ [I > 2σ(I)] = 0.0479/0.1155, large peak/hole (e Å) = 0.266/−0.247, CCDC. No: 2385734.

Fig. 4. ORTEP drawing of compounds M9 (A) and 34 (B).

2.3. SAR study

A total of 44 compounds were designed, synthesized and screened by the 17β-HSD10 inhibition rate at 5 μM and HepaRG MTT IC₅₀; 17β-HSD10-IN-1 was selected as a positive control.

Firstly, to verify the accuracy of virtual filtering, H1 and its 7 analogues (1–7) were designed and synthesized. As shown in Table 1, except for compound 4, all other compounds showed good activity, indicating that 2-phenyl-1H-benzo[d]imidazole was an advantageous skeleton for designing 17β-HSD10 inhibitors.

Table 1. The 17β-HSD10 inhibition rate and toxicity of compounds 1–7.

Compounds	R1	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
H1		54.85 ± 5.71	48.44 ± 4.33
1		44.54 ± 4.61	58.44 ± 3.15
2		50.57 ± 5.01	63.05 ± 6.78
3		24.05 ± 4.38	>100
4		61.87 ± 5.01	>100
5		62.80 ± 4.66	>100
6		65.12 ± 3.70	>100
7		54.85 ± 5.71	>100
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

It is worth noting that compound H1 exhibited the best activity; therefore focusing on compound H1, the amino group was derivatized to obtain compounds 8–16 based on reasonable design. As shown in Table 2, all the compounds exhibited good activity, and the activity increases with the increase of substituent volume, indicating that hydrophobic substituents had a significant impact on activity which was also consistent with molecular docking. Importantly, we have noticed that compounds 8–16 exhibited significant toxicity, and believed that the structure of diarylurea, as a common type II kinase inhibitor fragment, was the main factor leading to the toxicity of compounds.

Table 2. The 17β-HSD10 inhibition rate and toxicity of compounds 10–16.

Compounds	R2	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
8		42.20 ± 4.31	3.63 ± 1.01
9		57.17 ± 4.19	5.06 ± 1.25
10		64.97 ± 5.01	6.28 ± 0.68
11		54.25 ± 4.61	22.15 ± 2.35
12		77.01 ± 6.41	19.66 ± 3.21
13		66.17 ± 6.48	8.90 ± 1.25
14		62.03 ± 5.01	11.45 ± 2.15
15		78.95 ± 4.40	1.91 ± 0.98
16		75.90 ± 5.64	5.91 ± 2.15
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

In order to discover better advantageous segments in part A, based on the above results, the urea group was replaced by the amide group, and different substituents were introduced to obtain compounds 17–32. As shown in Table 3, there were significant differences in activity between compounds. Compounds 17 and 20 showed a significant decrease in activity compared to compounds 18–19, indicating the importance of the position of the benzene ring. The cyclopropyl group of compound 25 was replaced by substituents with greater steric hindrance to obtain compounds 26–32, and their activity has significantly increased, indicating that hydrophobic cavities could accommodate larger functional groups.

Table 3. The 17β-HSD10 inhibition rate and toxicity of compounds 17–32.

Compounds	R3	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
17		12.05 ± 5.44	>100
18		63.62 ± 6.71	19.92 ± 4.26
19		64.39 ± 4.82	56.21 ± 5.15
20		11.16 ± 1.85	>100
21		30.26 ± 4.31	>100
22		43.46 ± 2.91	60.36 ± 3.26
23		59.17 ± 5.73	>100
24		62.01 ± 7.14	38.99 ± 6.12
25		44.38 ± 4.40	>100
26		80.13 ± 5.03	>100
27		79.74 ± 5.73	>100
28		75.88 ± 4.19	73.67 ± 4.56
29		74.86 ± 4.53	>100
30		49.98 ± 2.91	>100
31		67.10 ± 4.66	>100
32		70.91 ± 5.03	>100
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

According to the above results, the cyclohexene ring and 1,1-difluorocyclohexane of compounds 26–27 were retained, and in order to increase the interaction with isopropyl of Leu217, imidazole was methylated to obtain compounds 33–34, respectively. As shown in Table 4, the activity of compound 33 was significantly improved, but the activity of compound 34 decreased. We believed that the reason for this difference in activity was due to the methylation of imidazole, which affected the spatial orientation of the molecule. Importantly, when NH was replaced by oxygen atoms to obtain compounds 35–36, respectively, the activity of compounds 35–36 significantly decreased. The activity of compounds 33, 26 and 35 decreased with the volume reduction of NCH₃, NH and O, indicating that it was very important to interact with the isopropyl group of Leu217.

Table 4. The 17β-HSD10 inhibition rate and toxicity of compounds 33–36.

Compounds	R4	X	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
33		–NCH3	89.76 ± 5.64	>100
34		–NCH3	38.59 ± 4.38	>100
35		O	22.12 ± 2.91	>100
36		O	9.07 ± 4.40	>100
17β-HSD10-IN-1			88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

To compare with compounds 33–34, compounds 37–38 were designed and synthesized. However, their activity was lower than that of compounds 33–34 (Table 5). Considering that single bonds were rotatable, we believed that the substituents on the benzene ring of benzimidazole had a significant impact on its activity. Therefore, to clarify the influence of substituents on the activity of the benzene ring, compounds 39–42 were designed and synthesized. As shown in Table 6, their activity has decreased, indicating the importance of benzene ring substituents. In addition, compounds 43–44 were designed and synthesized, and their activity was lower than compound 34 (Table 7), proving the importance of substituents.

Table 5. The 17β-HSD10 inhibition rate and toxicity of compounds 37–38.

Compounds	R5	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
37		36.82 ± 4.72	>100
38		45.82 ± 4.40	>100
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

Table 6. The 17β-HSD10 inhibition rate and toxicity of compounds 39–42.

Compounds	R6	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
39		65.25 ± 3.79	>100
40		56.43 ± 4.82	>100
41		26.58 ± 3.35	>100
42		16.76 ± 4.24	>100
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

Table 7. The 17β-HSD10 inhibition rate and toxicity of compounds 43–44.

Compounds	R7	IRa (%) at 5 μM	HepaRG MTT IC50b (μM)
43	H	57.72 ± 5.52	>100
44	Cl	44.85 ± 3.74	>100
17β-HSD10-IN-1		88.25 ± 6.34	75.52 ± 4.36

^aThe inhibition rate of compounds against 17β-HSD10 at 5 μM was determined.

^bDetecting the cytotoxicity of compounds using HepaRG cells. All experimental results were repeated three times, and the values were displayed as means ± SD.

And then, the reason why compound 33 exhibited potent activity against 17β-HSD10 was analyzed by docking study (Fig. 5). We noticed that compound 33 and H1 had high steric overlap. The big difference was that compound 33 formed one more hydrogen bond with the hydroxyl of Tyr168, and cyclohexane could better occupy the hydrophobic cavity. In addition, the introduction of methyl was also very important. It could be summed up that an appropriate substituent to occupy the hydrophobic cavity and interaction with the isopropyl of Leu217 were crucial to enhance the activity. In order to further determine the target of compound 33, we found through cellular thermal shift assay (CETSA) experiments that compound 33 can inhibit the degradation of 17β-HSD10 protein when co-incubated with cell lysate (Fig. 5B). In summary, compound 33 may target the 17β-HSD10 protein to exert biological activity.

Fig. 5. Binding mode of hit compound 33 with the active site of 17β-HSD10 (PDB: 1U7T). (A) 17β-HSD10 is shown in gray ribbons with selected residues colored green. Hydrogen bonds are drawn as yellow dashed lines. Compound 33 is shown with magenta sticks. The illustration was generated using PyMOL. (B) The CETSA assay used to detect the binding of 33 with the 17β-HSD10 protein in PC12 cell lysates.

2.4. Activity screening of compounds and their protective effects on cells

17β-HSD10 plays a crucial role in the pathology of Alzheimer's disease; it interacts with Aβ protein in AD patients and is involved in the transmission of neurotoxins associated with the disease. It has also been observed that there is significant neuronal cell death in AD patients. A preliminary screening of the inhibitory activity of the synthesized compounds against 17β-HSD10 was conducted at a concentration of 5 μM (Tables 1–7). The experimental results indicated that most compounds exhibited good biological activity (e.g., 12, 15, 16, 28, 29, 33) (Fig. 6). To further confirm the biological activity of the compounds, several representative compounds (12, 23, 26, 27, 33) were selected for testing their effects on 17β-HSD10 activity at different concentrations. Compound 33 exhibited the most prominent activity with an IC₅₀ of 1.65 μM (Table 8).

Fig. 6. Compound activity screening. 293T cells were transfected with the 17β-HSD10 probe to construct an in vitro screening model. Screening compounds for their inhibitory activity against 17β-HSD10 at a concentration of 5 μM (A and B). Data were obtained by at least three independent experiments, and each was performed in triplicate.

Table 8. The 17β-HSD10 IC₅₀ of high inhibitory compounds.

Compounds	17β-HSD10 IC50a (μM)	Compounds	17β-HSD10 IC50 (μM)
H1	5.24 ± 1.15	33	1.65 ± 0.55
26	2.35 ± 1.02	39	4.13 ± 1.14
27	2.58 ± 0.85	17β-HSD10-IN-1	1.32 ± 0.62

^aThe inhibition rate of compounds against 17β-HSD10 at 10 μM, 2 μM, 0.4 μM, 0.08 μM and 0.016 μM was determined. All experimental results were repeated three times, and the values were displayed as means ± SD.

Considering that the cytotoxicity of the compounds may lead to false-positive results, we further assessed the cytotoxicity of the compounds using HEPARG cells. The results showed that most compounds exhibited low cytotoxicity, suggesting their potential for further research (12, 23, 26, 27, 33) (Tables 1–7). High expression of 17β-HSD10 can inhibit cell proliferation. To further validate the biological activity of the compounds, we examined their protective effects on cell proliferation. The experimental results demonstrated that most compounds promoted cell proliferation, with compounds 4, 5, 12, 23, 26, 27, and 33 showing particularly notable activity, especially compound 33 (Fig. 7). Based on the above experimental results, compound 33 is considered to have significant potential for further investigation.

Fig. 7. The protective effect of compounds on cells. Screening the protective effect of compounds with a concentration of 5 μM on PC12 cells. Data were obtained by at least three independent experiments, and each was performed in triplicate (A and B).

2.5. In vivo pharmacokinetic evaluation of compound 33

Obtaining the in vivo metabolic data of compound 33 is crucial for evaluating its pharmacological properties. Therefore, further pharmacokinetic evaluation of compound 33 was conducted in rats through oral and intravenous injection. After oral administration of compound 33 at a dose of 10 mg kg⁻¹, the maximum concentration of the compound in plasma reached 498 μg L⁻¹ within 0.98 h, and decreased to half within 1.75 h. The area under the concentration time curve (AUC 0–∞) was 650 μg L⁻¹ h⁻¹, and the bioavailability (F%) was 22.3%, indicating that compound 33 has good oral efficacy. On the other hand, after intravenous injection of 5 mg kg⁻¹ of compound 33, the maximum blood drug concentration (C_max) was 616 μg L⁻¹, and the half-life (t_1/2) was 1.23 h. Meanwhile, we obtained a plasma protein binding (PPB) value of 87.5% for compound 33 at 20 μM concentration, which indicated that the unbound drug dose was about 12.5% of the administered dose, and the effective exposure dose could be achieved. In summary, compound 33 has good pharmacological properties (Table 9).

Table 9. In vivo PK properties of compound 33.

Dose/routes	t 1/2 (h)	T max (h)	MRT (h)	C max (μg L−1)	AUC0–∞ (μg L−1 h−1)	CL (L h−1 kg−1)	F (%)
10 mg kg−1 (po)	1.75	0.98	2.35	498	650	11.4	22.3
5 mg kg−1 (iv)	1.23		1.95	616	1460	114

2.6. Morris water maze experiment

To assess the in vivo activity of compound 33, we induced a cognitive impairment model in mice using scopolamine and evaluated behavioral changes through the Morris water maze experiment. After a continuous 10 day treatment to establish the model, we conducted 5 days of learning and memory training, followed by an exploratory test on day 6 to evaluate behavior. The results are shown in Fig. 8. Compared to the blank control group, the mice treated with scopolamine (3 mg kg⁻¹) exhibited significant memory deficits, as evidenced by disorganized movement patterns and difficulty in locating the platform, indicating the successful establishment of the Alzheimer's disease mouse model. The donepezil (10 mg kg⁻¹) positive control group demonstrated that treatment with donepezil helped simplify the mice's movement trajectories and reduced the time taken to reach the platform. We found that the experimental group of mice treated with a low dose of compound 33 (10 mg kg⁻¹) exhibited behavioral improvements. In the high-dose group (20 mg kg⁻¹), this behavioral improvement even slightly surpassed that of the donepezil positive control group. Additionally, we observed that the body weight of the treated mice did not change, indicating that compound 33 also has good safety. Taken together, these experiments demonstrate that compound 33 can improve cognitive function and memory impairments in mice.

Fig. 8. Compound 33 can alleviate cognitive impairment induced by scopolamine in mice. (A) The effect of compound 33 on mouse body weight. (B) During the experiment, mice searched for platform representative trajectory maps.

3. Conclusion

In order to discover novel 17β-HSD10 inhibitors with high activity and low toxicity, in-depth structure-based design was used for further exploration in this study. On the basis of early hit compound H1, through a series of structural modification and optimization, a total of 44 new 2-phenyl-1H-benzo[d]imidazole derivatives were designed and synthesized, of which the most promising compound 33 (N-(4-(1,4,6-trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide) exhibited lower toxicity and higher activity. In vitro experiments have found that this compound 33 could alleviate cognitive impairment induced by scopolamine in mice. Collectively, the results support further development of this compound as a promising lead 17β-HSD10 inhibitor and explore its therapeutic potential for AD.

4. Experimental section

4.1. General methods for chemistry

Reactions were monitored by thin layer chromatography (TLC) on pre-coated silica GF₂₅₄ plates. ¹H and ¹³C NMR spectra of all the compounds (H1, 1–44) were obtained using a Bruker AM-300 (¹H, 400 MHz; ¹³C, 100 MHz) spectrometer with DMSO-d₆ as the solvent and TMS as the internal standard.

General procedure for synthesis of compound H1, 1–7

3,5-Dimethyl-1,2-phenylenediamine (2 g, 14.68 mmol), DMF (20 mL), AcOH (1.76 g, 29.37 mmol) and p-nitrobenzaldehyde (2.44 g, 16.15 mmol) were added into the flask (100 mL), then the mixture was stirred at 85 °C for 6 h. It was cooled to room temperature and ethyl acetate was added, then washed with saturated salt solution. The organic layer was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 3 : 1) with a yield of 51%.

Nitro raw materials (200 mg, 748.26 μmol), NH4Cl (800.47 mg, 14.97 mmol), methanol (5 mL) and water (5 mL) were added into the flask, then zinc powder (489.21 mg, 7.48 mmol) was added. It was stirred at room temperature for 4 h. Ethyl acetate was added and stirred for 5 minutes, and then the reaction mixture was filtered. The filtrate was separated and the organic phase was obtained and dried with anhydrous Na₂SO₄, then purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1 : 1) to obtain 100 mg of the product (H1) with a yield of 56%. Compounds 1–7 were obtained according to similar procedures.

4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)aniline (H1)

¹H NMR (400 MHz, DMSO-d₆) δ 12.18 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.07 (s, 1H), 6.74 (s, 1H), 6.66 (d, J = 8.2 Hz, 2H), 5.55 (s, 2H), 2.49 (s, 3H), 2.36 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 150.72, 130.62, 128.13, 123.72, 118.16, 113.95, 21.76, 17.31. HR-MS (ESI): calcd for C₁₅H₁₅N₃ [M + H]⁺, 238.1339, found 238.1342.

2-(4-Fluorophenyl)-4,6-dimethyl-1H-benzo[d]imidazole (1)

¹H NMR (400 MHz, DMSO-d₆) δ 12.58 (d, J = 83.0 Hz, 1H), 8.48–8.00 (m, 2H), 7.39 (t, J = 8.9 Hz, 2H), 7.28–7.00 (m, 1H), 6.83 (s, 1H), 2.51 (q, J = 1.8 Hz, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.55, 162.10, 132.10, 129.05, 127.57, 127.54, 116.44, 116.22, 40.55, 40.39, 40.34, 40.18, 40.13, 39.93, 39.72, 39.51, 39.30, 21.76, 17.17. HR-MS (ESI): calcd for C₁₅H₁₃FN₂ [M + H]⁺, 241.1136, found 241.1139.

2-(4-Chlorophenyl)-4,6-dimethyl-1H-benzo[d]imidazole (2)

¹H NMR (400 MHz, DMSO-d₆) δ 12.65 (d, J = 84.4 Hz, 1H), 8.30–8.07 (m, 2H), 7.62 (dd, J = 8.5, 6.1 Hz, 2H), 7.13 (s, 1H), 6.83 (s, 1H), 2.52–2.49 (m, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.25, 141.81, 134.52, 129.78, 129.40, 128.40, 124.21, 109.03, 40.56, 40.35, 40.14, 39.93, 39.82, 39.72, 39.60, 39.51, 39.30, 21.78, 17.09. HR-MS (ESI): calcd for C₁₅H₁₃ClN₂ [M + H]⁺, 257.0841, found 257.0844.

4,6-Dimethyl-2-(p-tolyl)-1H-benzo[d]imidazole (3)

¹H NMR (400 MHz, DMSO-d₆) δ 12.45 (d, J = 81.8 Hz, 1H), 8.04 (dd, J = 28.1, 8.2 Hz, 2H), 7.31 (dd, J = 8.1, 3.7 Hz, 2H), 7.13 (d, J = 54.7 Hz, 1H), 6.77 (d, J = 4.0 Hz, 1H), 2.47 (d, J = 2.0 Hz, 3H), 2.41–2.26 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 151.62, 150.47, 144.37, 141.83, 139.68, 139.52, 135.22, 133.10, 131.88, 130.91, 129.89, 129.80, 128.23, 128.20, 128.13, 126.98, 126.64, 124.99, 123.93, 121.07, 116.34, 108.88, 40.57, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 21.84, 21.69, 21.42, 17.57, 17.11. HR-MS (ESI): calcd for C₁₆H₁₆N₂ [M + H]⁺, 237.1387, found 237.1382.

4,6-Dimethyl-2-(4-nitrophenyl)-1H-benzo[d]imidazole (4)

¹H NMR (400 MHz, DMSO-d₆) δ 12.95 (d, J = 88.6 Hz, 1H), 8.51–8.38 (m, 4H), 7.25 (d, J = 59.5 Hz, 1H), 6.89 (d, J = 11.7 Hz, 1H), 3.36 (s, 4H), 2.55 (d, J = 8.8 Hz, 3H), 2.40 (d, J = 9.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.13, 148.07, 147.88, 144.43, 142.07, 136.78, 136.76, 135.60, 133.53, 133.49, 131.86, 130.57, 129.00, 127.86, 127.74, 127.51, 126.24, 124.71, 124.69, 124.61, 124.54, 124.38, 121.73, 116.88, 109.29, 40.56, 40.36, 40.15, 40.04, 39.94, 39.73, 39.52, 39.31, 21.88, 21.67, 21.53, 17.53, 17.04. HR-MS (ESI): calcd for C₁₅H₁₃N₃O₂ [M + H]⁺, 268.1081, found 268.1086.

4,6-Dimethyl-2-(3-nitrophenyl)-1H-benzo[d]imidazole (5)

¹H NMR (400 MHz, DMSO-d₆) δ 12.96 (d, J = 82.3 Hz, 1H), 9.25–8.86 (m, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.31 (s, 1H), 7.92–7.78 (m, 1H), 7.24 (d, J = 55.8 Hz, 1H), 6.88 (d, J = 10.3 Hz, 1H), 2.56 (s, 3H), 2.41 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 148.75, 132.87, 132.44, 130.99, 124.31, 121.08, 40.56, 40.35, 40.14, 39.93, 39.72, 39.52, 39.31, 21.77, 17.26. HR-MS (ESI): calcd for C₁₅H₁₃N₃O₂ [M + H]⁺, 268.1081, found 268.1086.

Methyl-4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)benzoate 11 (6)

¹H NMR (400 MHz, DMSO-d₆) δ 12.80 (d, J = 85.2 Hz, 1H), 8.33 (dd, J = 29.9, 8.5 Hz, 2H), 8.12 (dd, J = 8.6, 2.4 Hz, 2H), 7.23 (d, J = 58.2 Hz, 1H), 6.87 (d, J = 9.3 Hz, 1H), 3.90 (d, J = 1.2 Hz, 3H), 2.55 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.32, 150.16, 149.06, 144.38, 141.95, 135.44, 135.09, 133.36, 132.91, 131.50, 130.42, 130.29, 130.23, 130.12, 128.72, 127.12, 126.78, 125.79, 124.41, 121.53, 116.71, 109.17, 52.72, 40.55, 40.40, 40.35, 40.14, 39.93, 39.72, 39.51, 39.30, 21.86, 21.68, 17.54, 17.07. HR-MS (ESI): calcd for C₁₇H₁₆N₂O₂ [M + H]⁺, 281.1285, found 281.1286.

Methyl-2-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)acetate (7)

¹H NMR (400 MHz, DMSO-d₆) δ 12.54 (d, J = 82.7 Hz, 1H), 8.22–8.06 (m, 2H), 7.43 (dd, J = 8.2, 3.5 Hz, 2H), 7.18 (d, J = 55.4 Hz, 1H), 6.82 (d, J = 5.5 Hz, 1H), 3.78 (d, J = 2.0 Hz, 2H), 3.64 (s, 3H), 2.53 (s, 3H), 2.38 (d, J = 8.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.92, 150.19, 141.87, 136.23, 135.29, 132.11, 130.34, 130.24, 129.57, 129.36, 128.28, 127.01, 126.73, 126.38, 124.06, 108.95, 52.26, 21.78, 17.12. HR-MS (ESI): calcd for C₁₈H₁₈N₂O₂ [M + H]⁺, 295.1442, found 295.1445.

General procedure for synthesis of compounds 8–16

H1 (100 mg, 0.4 mmol), DMAP (15 mg), DIPEA (54 mg, 0.42 mmol) and DMF (5 mL) were added into the flask. After 3 min, isocyanate (97 mg, 0.6 mmol) was added. It was stirred at room temperature for 2 h. Ethyl acetate and water were added into the mixture. The mixture was left to stand and the liquid to separate, then the organic phase was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2 : 1) to obtain 48 mg of product (8) with a yield of 29%. Compounds 9–16 were obtained according to similar procedures.

1-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-ethylurea (8)

¹H NMR (400 MHz, DMSO-d₆) δ 12.35 (d, J = 82.0 Hz, 1H), 8.70 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.61–7.48 (m, 2H), 7.07 (s, 1H), 6.78 (s, 1H), 6.22 (s, 1H), 3.13 (dd, J = 7.2, 5.6 Hz, 2H), 2.51 (d, J = 1.8 Hz, 3H), 2.38 (s, 3H), 1.07 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 155.38, 152.84, 140.91, 130.43, 130.16, 129.85, 124.70, 123.48, 122.41, 118.75, 117.82, 114.68, 60.23, 40.56, 40.40, 40.35, 40.15, 39.94, 39.73, 39.52, 39.31, 15.91. HR-MS (ESI): calcd for C₁₈H₂₀N₄O [M + H]⁺, 309.1710, found 309.1705.

1-Butyl-3-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)urea (9)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (s, 1H), 8.67 (s, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.58–7.49 (m, 2H), 7.12 (s, 1H), 6.79 (s, 1H), 6.24 (t, J = 5.7 Hz, 1H), 3.15–3.06 (m, 2H), 2.51 (d, J = 2.0 Hz, 3H), 2.37 (s, 3H), 1.49–1.39 (m, 2H), 1.36–1.26 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 155.44, 142.40, 127.52, 123.41, 117.76, 40.61, 40.56, 40.40, 40.35, 40.19, 40.14, 39.93, 39.73, 39.52, 39.31, 39.17, 32.31, 21.78, 20.00, 17.31, 14.18. HR-MS (ESI): calcd for C₂₀H₂₄N₄O [M + H]⁺, 337.2023, found 337.2018.

1-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(p-tolyl)urea (10)

¹H NMR (400 MHz, DMSO-d₆) δ 12.41 (d, J = 73.1 Hz, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.2 Hz, 3H), 6.79 (s, 1H), 2.52 (s, 3H), 2.38 (s, 3H), 2.25 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 152.86, 141.55, 137.41, 131.31, 129.69, 127.59, 124.24, 118.84, 118.36, 40.56, 40.35, 40.14, 39.93, 39.72, 39.52, 39.31, 21.79, 20.82. HR-MS (ESI): calcd for C₂₃H₂₂N₄O [M + H]⁺, 371.1867, found 371.1872.

1-(4-Chlorophenyl)-3-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)urea (11)

¹H NMR (400 MHz, DMSO-d₆) δ 12.53 (s, 1H), 8.95 (d, J = 19.5 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 7.09 (s, 1H), 6.80 (s, 1H), 2.52 (d, J = 1.9 Hz, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 152.75, 139.02, 129.14, 125.98, 124.52, 120.30, 118.58, 40.56, 40.35, 40.14, 39.94, 39.73, 39.52, 39.31, 21.80. HR-MS (ESI): calcd for C₂₂H₁₉ClN₄O [M+H]⁺, 391.1321, found 391.1316.

1-(4-Bromophenyl)-3-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)urea (12)

¹H NMR (400 MHz, DMSO-d₆) δ 12.42 (d, J = 81.6 Hz, 1H), 8.95 (d, J = 19.3 Hz, 2H), 8.17–8.04 (m, 2H), 7.65–7.57 (m, 2H), 7.47 (s, 4H), 7.15 (d, J = 51.5 Hz, 1H), 6.80 (s, 1H), 2.52 (d, J = 1.7 Hz, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 152.71, 141.25, 139.45, 132.03, 124.53, 120.69, 118.57, 113.86, 40.57, 40.41, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 21.79. HR-MS (ESI): calcd for C₂₂H₁₉BrN₄O [M + H]⁺, 435.0815, found 435.0797.

1-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-methoxyphenyl)urea (13)

¹H NMR (400 MHz, DMSO-d₆) δ 12.40 (d, J = 81.4 Hz, 1H), 8.84 (s, 1H), 8.58 (s, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 7.43–7.32 (m, 2H), 7.15 (d, J = 50.6 Hz, 1H), 6.95–6.84 (m, 2H), 6.79 (s, 1H), 3.73 (s, 3H), 2.56–2.51 (m, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 155.03, 153.01, 141.66, 132.97, 127.52, 124.14, 120.59, 118.32, 114.46, 55.62, 40.56, 40.41, 40.35, 40.15, 39.94, 39.82, 39.73, 39.52, 39.31, 21.79. HR-MS (ESI): calcd for C₂₃H₂₂N₄O₂ [M + H]⁺, 387.1816, found 387.1805.

1-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (14)

¹H NMR (400 MHz, DMSO-d₆) δ 12.42 (d, J = 80.4 Hz, 1H), 8.99 (s, 2H), 8.20–8.01 (m, 2H), 7.73–7.51 (m, 4H), 7.31 (d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 6.80 (s, 1H), 2.52 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 152.79, 143.15, 141.25, 139.33, 127.57, 124.54, 122.27, 121.93, 119.96, 118.58, 40.56, 40.35, 40.15, 39.94, 39.73, 39.52, 39.31, 21.78. HR-MS (ESI): calcd for C₂₃H₁₉F₃N₄O₂ [M + H]⁺, 441.1533, found 441.1537.

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)urea (15)

¹H NMR (400 MHz, DMSO-d₆) δ 12.59 (s, 1H), 9.26 (d, J = 60.8 Hz, 2H), 8.23 (d, J = 2.3 Hz, 1H), 8.18 (d, J = 8.3 Hz, 2H), 7.82–7.60 (m, 4H), 7.27–7.10 (m, 1H), 6.89–6.84 (m, 1H), 2.59 (s, 3H), 2.44 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.77, 152.74, 140.96, 139.68, 132.49, 127.59, 127.34, 127.04, 124.79, 124.65, 124.30, 123.63, 122.93, 121.93, 118.86, 117.32, 117.27, 60.23, 40.56, 40.40, 40.35, 40.14, 39.93, 39.72, 39.51, 39.30, 36.23, 31.21, 21.77, 21.22, 17.30, 14.53. HR-MS (ESI): calcd for C₂₃H₁₈ClF₃N₄O [M + H]⁺, 459.1194, found 459.1197.

1-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (16)

¹H NMR (400 MHz, DMSO-d₆) δ 12.42 (d, J = 81.4 Hz, 1H), 9.10 (d, J = 36.8 Hz, 2H), 8.24–7.99 (m, 3H), 7.74–7.43 (m, 4H), 7.34 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 52.7 Hz, 1H), 6.80 (s, 1H), 2.52 (s, 3H), 2.38 (d, J = 6.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.83, 152.84, 141.08, 140.91, 130.43, 130.16, 129.85, 127.52, 126.04, 124.70, 123.34, 122.41, 118.74, 114.68, 60.23, 40.56, 40.41, 40.35, 40.15, 39.94, 39.73, 39.52, 39.31, 21.78, 21.23, 14.54. HR-MS (ESI): calcd for C₂₃H₁₉F₃N₄O [M + H]⁺, 425.1584, found 425.1585.

General procedure for synthesis of compounds 17–44

H1 (100 mg, 0.42 mmol), HATU (169.75 mg, 0.46 mmol), TEA (50 mg, 0.48 mol) and DMF (5 mL) were added into the flask, After 30 min, acid (83 mg, 0.5 mmol) was added and stirred at room temperature for 6 h. Ethyl acetate and water were added into the mixture. The mixture was left to stand and the liquid to separate, then the organic phase was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2 : 1) to obtain 72 mg of the product (17) with a yield of 44%. Compounds 18–44 were obtained according to similar procedures.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)benzamide (17)

¹H NMR (400 MHz, DMSO-d₆) δ 12.48 (d, J = 82.5 Hz, 1H), 10.46 (s, 1H), 8.17 (dd, J = 28.3, 8.4 Hz, 2H), 8.06–7.90 (m, 4H), 7.66–7.51 (m, 3H), 7.17 (d, J = 53.5 Hz, 1H), 6.82 (d, J = 3.5 Hz, 1H), 2.53 (d, J = 4.7 Hz, 3H), 2.38 (d, J = 7.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.13, 151.40, 150.71, 141.59, 140.87, 135.09, 131.52, 129.25, 128.82, 128.73, 127.49, 126.45, 125.42, 124.37, 121.10, 119.39, 40.56, 40.35, 40.14, 39.93, 39.73, 39.52, 39.31, 38.47, 31.21, 21.78, 17.30. HR-MS (ESI): calcd for C₂₂H₁₉N₃O [M + H]⁺, 342.1601, found 342.1608.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylacetamide (18)

¹H NMR (400 MHz, DMSO-d₆) δ 12.45 (d, J = 82.3 Hz, 1H), 10.41 (s, 1H), 8.11 (dd, J = 28.4, 8.6 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 6.6 Hz, 4H), 7.26 (s, 1H), 7.15 (d, J = 53.5 Hz, 1H), 6.80 (s, 1H), 3.68 (s, 2H), 2.51 (d, J = 1.9 Hz, 3H), 2.37 (d, J = 7.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.36, 140.98, 131.51, 127.49, 125.30, 124.36, 119.33, 56.49, 21.78, 19.03, 17.29, 15.11, 7.87. HR-MS (ESI): calcd for C₂₃H₂₁N₃O [M + H]⁺, 356.1758, found 356.1749.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-phenylpropanamide (19)

¹H NMR (400 MHz, DMSO-d₆) δ 12.63 (s, 1H), 10.16 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 2.4 Hz, 4H), 7.23–7.11 (m, 2H), 6.81 (s, 1H), 2.94 (t, J = 7.7 Hz, 2H), 2.68 (t, J = 7.7 Hz, 2H), 2.52 (s, 3H), 2.38 (s, 3H).

¹³C NMR (101 MHz, DMSO-d₆) δ 169.83, 136.31, 129.61, 128.82, 127.07, 125.73, 119.51, 43.84, 40.56, 40.36, 40.15, 39.94, 39.73, 39.61, 39.52, 39.31. HR-MS (ESI): calcd for C₂₄H₂₃N₃O [M + H]⁺, 370.1914, found 370.1908.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-4-phenylbutanamide (20)

¹H NMR (400 MHz, DMSO-d₆) δ 12.43 (d, J = 82.2 Hz, 1H), 10.12 (s, 1H), 8.10 (dd, J = 28.2, 8.6 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.25–7.17 (m, 3H), 7.14–6.67 (m, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.52 (s, 3H), 2.37 (d, J = 8.2 Hz, 5H), 1.98–1.86 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.19, 140.82, 135.24, 132.22, 128.93, 128.19, 127.31, 126.10, 120.67, 56.49, 40.54, 40.33, 40.12, 39.91, 39.70, 39.50, 39.29, 21.79, 19.03. HR-MS (ESI): calcd for C₂₅H₂₅N₃O [M + H]⁺, 384.2071, found 384.2066.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-5-phenylpentanamide (21)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (d, J = 81.4 Hz, 1H), 10.12 (s, 1H), 8.07 (d, J = 9.1 Hz, 2H), 7.79–7.71 (m, 2H), 7.27 (d, J = 7.4 Hz, 2H), 7.23–7.04 (m, 4H), 6.80 (s, 1H), 2.62 (s, 2H), 2.51 (d, J = 2.0 Hz, 3H), 2.38 (s, 5H), 1.63 (s, 4H).

¹³C NMR (101 MHz, DMSO-d₆) δ 175.03, 141.13, 127.38, 125.33, 119.41, 56.49, 45.37, 40.56, 40.40, 40.35, 40.14, 39.93, 39.72, 39.52, 39.31, 29.58, 25.86, 25.69, 21.77, 19.03, 17.29. HR-MS (ESI): calcd for C₂₆H₂₇N₃O [M + H]⁺, 398.2227, found 398.2224.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cinnamamide (22)

¹H NMR (400 MHz, DMSO) δ 12.58, 10.47, 8.16, 8.14, 7.88, 7.86, 7.67, 7.67, 7.66, 7.65, 7.65, 7.62, 7.47, 7.47, 7.46, 7.46, 7.45, 7.43, 7.16, 6.90, 6.86, 6.81, 2.53, 2.38.

¹³C NMR (101 MHz, DMSO-d₆) δ 164.16, 141.01, 140.87, 135.10, 130.39, 129.54, 128.28, 127.55, 125.86, 122.51, 119.65, 40.57, 40.41, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 21.79, 17.31. HR-MS (ESI): calcd for C₂₄H₂₁N₃O [M + H]⁺, 368.1758, found 368.1755.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)isonicotinamide (23)

¹H NMR (400 MHz, DMSO-d₆) δ 12.53 (d, J = 83.1 Hz, 1H), 10.72 (s, 1H), 8.87–8.76 (m, 2H), 8.27–8.12 (m, 2H), 8.01–7.87 (m, 4H), 7.12 (s, 1H), 6.82 (s, 1H), 2.53 (s, 3H), 2.39 (d, J = 4.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.65, 151.56, 151.10, 150.79, 150.39, 142.20, 140.51, 140.08, 135.10, 132.06, 129.32, 127.59, 125.88, 124.81, 123.22, 122.08, 121.18, 120.84, 40.61, 40.56, 40.40, 40.35, 40.14, 39.94, 39.73, 39.52, 39.31, 21.76, 17.28, 0.56. HR-MS (ESI): calcd for C₂₁H₁₈N₄O [M + H]⁺, 343.1554, found 343.1554.

N-(4-(4,6-dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)nicotinamide (24)

¹H NMR (400 MHz, DMSO-d₆) δ 12.52 (d, J = 83.0 Hz, 1H), 10.67 (s, 1H), 9.19–9.10 (m, 1H), 8.79 (dd, J = 4.8, 1.7 Hz, 1H), 8.33 (dt, J = 8.0, 2.0 Hz, 1H), 8.19 (dd, J = 27.7, 8.3 Hz, 2H), 8.02–7.90 (m, 2H), 7.60 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.26–7.09 (m, 1H), 6.82 (s, 1H), 2.53 (s, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.69, 152.71, 150.59, 149.21, 140.51, 136.00, 131.63, 130.93, 127.41, 126.31, 124.45, 124.01, 120.70, 56.49, 40.57, 40.41, 40.35, 40.20, 40.15, 39.94, 39.73, 39.52, 39.31, 21.79, 19.03, 17.30. HR-MS (ESI): calcd for C₂₁H₁₈N₄O [M + H]⁺, 343.1554, found 343.1556.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclopropanecarboxamide (25)

¹H NMR (400 MHz, DMSO-d₆) δ 12.48 (s, 1H), 10.42 (s, 1H), 8.10 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.15 (s, 1H), 6.80 (s, 1H), 2.52 (s, 3H), 2.38 (s, 3H), 1.82 (td, J = 7.5, 3.9 Hz, 1H), 0.84 (t, J = 5.5 Hz, 4H). ¹³C NMR (101 MHz, DMSO) δ 171.91, 142.52, 140.91, 128.77, 128.73, 127.40, 126.15, 125.48, 119.37, 40.56, 40.35, 40.14, 39.93, 39.72, 39.52, 39.31, 36.78, 35.39, 31.14, 25.22, 21.78. HR-MS (ESI): calcd for C₁₉H₁₉N₃O [M + H]⁺, 306.1601, found 306.1604.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (26)

¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.15 (s, 1H), 6.81 (s, 1H), 2.52 (s, 3H), 2.38 (s, 4H), 1.88–1.72 (m, 4H), 1.67 (d, J = 11.1 Hz, 1H), 1.50–1.37 (m, 2H), 1.35–1.14 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.69, 150.31, 142.13, 141.87, 140.87, 135.23, 131.78, 128.83, 128.80, 128.02, 127.63, 127.27, 126.30, 125.49, 123.91, 119.45, 119.34, 108.83, 40.56, 40.41, 40.35, 40.14, 39.93, 39.73, 39.52, 39.31, 36.28, 35.08, 27.18, 21.84, 17.57, 17.13. HR-MS (ESI): calcd for C₂₂H₂₅N₃O [M + H]⁺, 348.2071, found 348.2075.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-4,4-difluorocyclohexane-1-carboxamide (27)

¹H NMR (400 MHz, DMSO-d₆) δ 12.45 (d, J = 81.2 Hz, 1H), 10.21 (s, 1H), 8.23–8.02 (m, 2H), 7.86–7.66 (m, 2H), 7.15 (d, J = 51.1 Hz, 1H), 6.80 (s, 1H), 2.51 (s, 4H), 2.38 (s, 3H), 2.10 (s, 2H), 2.02–1.63 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.61, 150.25, 144.40, 141.85, 140.90, 140.77, 135.23, 133.10, 131.82, 130.90, 128.03, 127.62, 127.26, 125.65, 124.93, 119.60, 119.49, 116.25, 108.83, 42.47, 40.55, 40.39, 40.34, 40.13, 39.92, 39.71, 39.50, 39.30, 32.90, 32.67, 32.43, 26.07, 25.98, 21.83, 21.69, 17.55, 17.11, 0.56. HR-MS (ESI): calcd for C₂₂H₂₃F₂N₃O [M + H]⁺, 384.1882, found 384.1882.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-4-methylcyclohexane-1-carboxamide (28)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (d, J = 82.5 Hz, 1H), 10.03 (d, J = 26.9 Hz, 1H), 8.10 (dd, J = 28.6, 8.4 Hz, 2H), 7.76 (dd, J = 8.9, 2.5 Hz, 2H), 7.15 (d, J = 52.6 Hz, 1H), 6.80 (s, 1H), 2.52 (d, J = 1.7 Hz, 3H), 2.47 (d, J = 4.1 Hz, 1H), 2.37 (d, J = 6.9 Hz, 3H), 1.82 (s, 4H), 1.62–1.40 (m, 5H), 1.40–1.19 (m, 1H), 0.94 (d, J = 6.9 Hz, 2H), 0.89 (d, J = 6.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.09, 174.94, 141.15, 141.10, 127.35, 125.39, 125.33, 119.48, 119.42, 45.15, 43.08, 40.55, 40.34, 40.13, 39.92, 39.71, 39.62, 39.50, 39.29, 34.44, 32.06, 31.09, 29.54, 28.82, 25.51, 23.02, 21.77, 19.90, 17.29. HR-MS (ESI): calcd for C₂₃H₂₇N₃O [M + H]⁺, 362.2227, found 362.2225.

(1r,4r)-N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-4-isopropylcyclohexane-1-carboxamide (29)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (d, J = 80.1 Hz, 1H), 10.05 (s, 1H), 8.08 (s, 2H), 7.81–7.72 (m, 2H), 7.15 (d, J = 43.7 Hz, 1H), 6.80 (s, 1H), 2.51 (s, 3H), 2.37 (s, 3H), 2.34–2.24 (m, 1H), 1.89 (dd, J = 13.6, 3.5 Hz, 2H), 1.81–1.73 (m, 2H), 1.50–1.37 (m, 3H), 1.09–0.95 (m, 3H), 0.87 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.06, 141.09, 127.37, 125.39, 119.42, 45.57, 43.24, 40.61, 40.55, 40.35, 40.14, 39.93, 39.72, 39.51, 39.30, 32.81, 29.70, 28.97, 21.78, 20.12. HR-MS (ESI): calcd for C₂₅H₃₁N₃O [M + H]⁺, 390.2540, found 390.2541.

N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide (30)

¹H NMR (400 MHz, DMSO-d₆) δ 12.46 (d, J = 73.0 Hz, 1H), 10.14 (s, 1H), 8.10 (d, J = 8.2 Hz, 2H), 7.87–7.68 (m, 2H), 7.12 (s, 1H), 6.80 (s, 1H), 3.98–3.80 (m, 2H), 3.40 (d, J = 3.2 Hz, 1H), 3.34 (d, J = 3.6 Hz, 1H), 2.62 (dd, J = 10.5, 5.0 Hz, 1H), 2.51 (s, 3H), 2.38 (s, 3H), 1.82–1.53 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.73, 140.93, 127.41, 125.54, 119.52, 66.83, 42.27, 40.53, 40.32, 40.11, 39.90, 39.69, 39.59, 39.49, 39.28, 29.28, 21.77, 17.29. HR-MS (ESI): calcd for C₂₁H₂₃N₃O₂[M + H]⁺, 350.1864, found 350.1864.

(3r,5r,7r)-N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)adamantane-1-carboxamide (31)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (d, J = 80.9 Hz, 1H), 9.35 (s, 1H), 8.20–8.02 (m, 2H), 7.90–7.78 (m, 2H), 7.15 (d, J = 51.3 Hz, 1H), 6.80 (s, 1H), 2.51 (s, 3H), 2.38 (s, 3H), 2.08–2.01 (m, 3H), 1.94 (d, J = 2.9 Hz, 6H), 1.72 (t, J = 3.2 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 176.64, 141.06, 131.45, 127.13, 125.48, 124.32, 120.41, 60.24, 41.51, 40.55, 40.34, 40.13, 39.93, 39.72, 39.51, 39.30, 38.68, 36.46, 28.13, 21.77, 21.22, 17.29, HR-MS (ESI): calcd for C₂₆H₂₉N₃O [M + H]⁺, 400.2384, found 400.2383.

14.54.(1r,3R,5S,7r)-N-(4-(4,6-Dimethyl-1H-benzo[d]imidazol-2-yl)phenyl)-3,5-dimethyladamantane-1-carboxamide (32)

¹H NMR (400 MHz, DMSO-d₆) δ 12.44 (d, J = 81.6 Hz, 1H), 9.36 (s, 1H), 8.16–8.03 (m, 2H), 7.83 (dd, J = 8.8, 1.6 Hz, 2H), 7.23–7.07 (m, 1H), 6.80 (t, J = 2.4 Hz, 1H), 2.52 (s, 3H), 2.37 (d, J = 7.5 Hz, 3H), 2.12 (p, J = 3.2 Hz, 1H), 1.80–1.71 (m, 2H), 1.56 (q, J = 12.2 Hz, 4H), 1.43–1.29 (m, 4H), 1.17 (s, 2H), 0.86 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 176.37, 141.04, 127.01, 125.53, 120.41, 50.61, 44.85, 43.46, 42.70, 40.56, 40.41, 40.35, 40.14, 39.93, 39.73, 39.52, 39.31, 37.35, 31.25, 30.90, 29.33, 21.79. HR-MS (ESI): calcd for C₂₈H₃₃N₃O [M + H]⁺, 428.2697, found 428.2697.

N-(4-(1,4,6-Trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (33)

¹H NMR (400 MHz, DMSO-d₆) δ 10.07 (s, 1H), 7.78 (q, J = 8.5 Hz, 4H), 7.17 (s, 1H), 6.87 (s, 1H), 3.80 (s, 3H), 2.42 (s, 6H), 1.80 (dd, J = 24.1, 12.4 Hz, 4H), 1.67 (d, J = 11.6 Hz, 1H), 1.44 (q, J = 12.2, 11.7 Hz, 2H), 1.36–1.00 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.11, 152.12, 140.94, 140.43, 136.87, 131.78, 130.17, 128.26, 125.13, 124.20, 119.17, 108.09, 45.41, 40.56, 40.35, 40.14, 39.93, 39.73, 39.52, 39.31, 32.14, 29.58, 25.86, 25.70, 21.94, 16.87. HR-MS (ESI): calcd for C₂₃H₂₇N₃O [M + H]⁺, 362.2227, found 362.2232.

4,4-Difluoro-N-(4-(1,4,6-trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexane-1-carboxamide (34)

¹H NMR (400 MHz, DMSO-d₆) δ 10.22 (s, 1H), 7.78 (d, J = 4.4 Hz, 4H), 7.18 (s, 1H), 6.87 (s, 1H), 3.80 (s, 3H), 2.69 (s, 1H), 2.42 (s, 3H), 2.33 (s, 1H), 2.12 (s, 3H), 2.02–1.64 (m, 8H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.68, 152.05, 140.67, 140.43, 136.88, 131.82, 130.20, 128.28, 125.39, 124.21, 119.31, 108.10, 42.51, 40.55, 40.35, 40.14, 39.93, 39.72, 39.51, 39.30, 32.92, 32.69, 32.45, 32.14, 26.80, 26.08, 25.99, 21.94, 16.86. HR-MS (ESI): calcd for C₂₃H₂₅F₂N₃O [M + H]⁺, 398.2039, found 398.2044.

N-(4-(4,6-Dimethylbenzo[d]oxazol-2-yl)phenyl)cyclohexanecarboxamide (35)

¹H NMR (400 MHz, DMSO-d₆) δ 10.19 (s, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 1.5 Hz, 1H), 7.03 (s, 1H), 2.52 (s, 3H), 2.42 (s, 3H), 2.36 (dt, J = 11.5, 3.4 Hz, 1H), 1.87–1.71 (m, 4H), 1.66 (d, J = 11.1 Hz, 1H), 1.49–1.37 (m, 2H), 1.34–1.14 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.25, 161.49, 150.56, 142.90, 139.17, 135.13, 129.26, 128.32, 126.78, 121.29, 119.54, 108.47, 45.40, 40.57, 40.36, 40.15, 39.94, 39.73, 39.53, 39.32, 29.53, 25.83, 25.66, 21.73, 21.71, 16.68, 16.65. HR-MS (ESI): calcd for C₂₂H₂₄N₂O₂ [M + H]⁺, 349.1911, found 349.1913.

N-(4-(4,6-Dimethylbenzo[d]oxazol-2-yl)phenyl)-4,4-difluorocyclohexane-1-carboxamide (36)

¹H NMR (400 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.11 (d, J = 8.8 Hz, 2H), 7.87–7.80 (m, 2H), 7.36 (d, J = 1.5 Hz, 1H), 7.03 (s, 1H), 2.53 (s, 4H), 2.42 (s, 3H), 2.18–2.06 (m, 2H), 2.00–1.78 (m, 4H), 1.77–1.65 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.83, 173.81, 161.44, 150.57, 142.62, 139.16, 135.18, 129.30, 128.35, 126.80, 121.54, 119.68, 108.49, 42.51, 40.57, 40.41, 40.36, 40.15, 39.95, 39.74, 39.53, 39.32, 32.88, 32.64, 32.41, 26.03, 25.93, 21.72, 16.66. HR-MS (ESI): calcd for C₂₂H₂₂F₂N₂O₂ [M + H]⁺, 385.1723, found 385.1719.

N-(4-(1,5,7-Trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (37)

¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.24 (s, 1H), 6.81 (s, 1H), 3.97 (s, 3H), 2.70 (s, 3H), 2.42–2.36 (m, 1H), 2.36 (s, 3H), 1.89–1.72 (m, 4H), 1.67 (d, J = 10.9 Hz, 1H), 1.49–1.37 (m, 2H), 1.36–1.13 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.12, 154.05, 143.86, 141.03, 133.63, 131.08, 130.50, 126.53, 124.99, 121.72, 119.15, 117.12, 45.41, 34.50, 29.58, 25.86, 25.69, 21.45, 18.68. HR-MS (ESI): calcd for C₂₃H₂₇N₃O [M + H]⁺, 362.2227, found 362.2228.

4,4-Difluoro-N-(4-(1,5,7-trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexane-1-carboxamide (38)

¹H NMR (400 MHz, DMSO-d₆) δ 10.24 (s, 1H), 7.85–7.75 (m, 2H), 7.74–7.62 (m, 2H), 7.24 (s, 1H), 6.81 (s, 1H), 3.97 (s, 3H), 2.70 (s, 3H), 2.60–2.52 (m, 1H), 2.36 (s, 3H), 2.19–2.06 (m, 2H), 2.02–1.64 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.71, 173.68, 153.97, 143.81, 140.76, 133.62, 131.12, 130.56, 126.57, 126.52, 125.21, 124.13, 121.75, 119.27, 117.11, 56.49, 42.51, 40.56, 40.41, 40.35, 40.14, 39.93, 39.73, 39.61, 39.52, 39.31, 34.50, 32.92, 32.68, 32.45, 26.08, 25.99, 21.45, 19.03, 18.68. HR-MS (ESI): calcd for C₂₃H₂₅F₂N₃O [M + H]⁺, 398.2039, found 398.2041.

N-(4-(1-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (39)

¹H NMR (400 MHz, DMSO-d₆) δ 10.08 (s, 1H), 7.87–7.72 (m, 4H), 7.62 (dd, J = 24.3, 7.8 Hz, 2H), 7.25 (dt, J = 19.0, 7.2 Hz, 2H), 3.87 (s, 3H), 2.38 (tt, J = 11.8, 3.7 Hz, 1H), 1.93–1.72 (m, 4H), 1.67 (d, J = 11.6 Hz, 1H), 1.51–1.37 (m, 2H), 1.37–1.15 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.16, 153.37, 142.94, 141.21, 137.07, 130.28, 124.81, 122.59, 122.29, 119.24, 119.21, 110.88, 45.42, 32.16, 29.58, 25.86, 25.69. HR-MS (ESI): calcd for C₂₁H₂₃N₃O [M + H]⁺, 334.1914, found 334.1915.

N-(4-(6-Chloro-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (40)

¹H NMR (400 MHz, DMSO-d₆) δ 10.11 (s, 1H), 7.85–7.74 (m, 5H), 7.65 (d, J = 8.5 Hz, 1H), 7.24 (dt, J = 8.6, 1.5 Hz, 1H), 3.86 (s, 3H), 2.37 (td, J = 9.8, 8.0, 5.8 Hz, 1H), 1.90–1.73 (m, 4H), 1.66 (d, J = 11.3 Hz, 1H), 1.43 (qd, J = 12.1, 2.9 Hz, 2H), 1.26 (dddt, J = 24.8, 17.0, 13.0, 6.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.21, 154.48, 141.64, 141.42, 137.86, 130.33, 27.09, 124.29, 122.62, 120.45, 119.23, 111.06, 45.41, 32.43, 29.57, 25.85, 25.68. HR-MS (ESI): calcd for C₂₁H₂₂ClN₃O [M + H]⁺, 368.1525, found 368.1527.

N-(4-(1,5,6-Trimethyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (41)

¹H NMR (400 MHz, DMSO-d₆) δ 10.07 (s, 1H), 7.84–7.71 (m, 4H), 7.38 (d, J = 24.9 Hz, 2H), 3.81 (s, 3H), 2.34 (d, J = 15.7 Hz, 7H), 1.88–1.73 (m, 4H), 1.67 (d, J = 11.0 Hz, 1H), 1.43 (qd, J = 12.1, 2.8 Hz, 2H), 1.35–1.14 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.10, 152.45, 141.57, 140.97, 135.68, 131.21, 130.51, 130.10, 125.08, 119.34, 119.18, 110.88, 45.41, 32.10, 29.59, 25.86, 25.70, 20.63, 20.40. HR-MS (ESI): calcd for C₂₃H₂₇N₃O [M + H]⁺, 362.2227, found 362.2226.

N-(4-(5,6-Dichloro-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexanecarboxamide (42)

¹H NMR (400 MHz, DMSO-d₆) δ 10.11 (s, 1H), 8.03 (s, 1H), 7.93 (s, 1H), 7.81 (s, 4H), 3.88 (s, 3H), 2.37 (tt, J = 11.9, 3.7 Hz, 1H), 1.87–1.73 (m, 4H), 1.67 (d, J = 11.6 Hz, 1H), 1.49–1.37 (m, 2H), 1.36–1.13 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.22, 155.75, 142.40, 141.67, 136.73, 130.41, 124.95, 124.79, 123.87, 120.26, 119.21, 112.84, 45.42, 32.68, 29.56, 25.84, 25.68. HR-MS (ESI): calcd for C₂₁H₂₁Cl₂N₃O [M + H]⁺, 402.1135, found 402.1137.

4,4-Difluoro-N-(4-(1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexane-1-carboxamide (43)

¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 7.81 (s, 4H), 7.69–7.57 (m, 2H), 7.26 (ddd, J = 11.8, 7.6, 1.3 Hz, 2H), 3.88 (s, 3H), 2.54 (s, 1H), 2.19–2.06 (m, 2H), 2.01–1.79 (m, 4H), 1.78–1.65 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.73, 173.71, 153.29, 142.89, 140.95, 137.05, 130.33, 125.03, 122.63, 122.33, 119.33, 119.24, 110.91, 42.52, 40.57, 40.41, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 32.92, 32.68, 32.45, 32.17, 26.08, 25.98. HR-MS (ESI): calcd for C₂₁H₂₁Cl₂N₃O [M + H]⁺, 370.1726, found 370.1726.

N-(4-(6-Chloro-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)-4,4-difluorocyclohexane-1-carboxamide (44)

¹H NMR (400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 7.81 (s, 4H), 7.78 (d, J = 2.1 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 3.87 (s, 3H), 2.55 (d, J = 11.1 Hz, 1H), 2.12 (d, J = 10.1 Hz, 2H), 2.00–1.79 (m, 4H), 1.77–1.65 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 173.76, 173.74, 154.42, 141.66, 141.15, 137.87, 130.37, 127.09, 124.56, 122.62, 120.48, 119.33, 111.09, 42.52, 40.57, 40.41, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 32.91, 32.68, 32.44, 26.07, 25.97. HR-MS (ESI): calcd for C₂₁H₂₁F₂N₃O [M + H]⁺, 404.1336, found 404.1335.

4.2. Cytotoxicity evaluation of compounds

The HepaRG cells in the proliferation phase were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate. After incubating in a CO₂ incubator for 24 h, the cells reached approximately 60% confluence and were then used for experiments. The cells were treated with compounds at different concentrations (0.8 μM, 4 μM, 20 μM, and 100 μM) for 24 h. Subsequently, 2 μL of MTT solution (5 mg mL⁻¹, Sigma-Aldrich) was added, and the cells were incubated for an additional 4 h. After 4 h, the cell culture supernatant was aspirated, and DMSO was added to each well to dissolve the crystals formed in the 96-well plate. The absorbance was measured at 492 nm using a microplate reader.

PC12 cells were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate and pre-cultured in a CO₂ incubator for 24 h (37 °C, 5% CO₂). A compound at a concentration of 10 μM was added to the culture plate for a 2 h pre-treatment, followed by treatment with 5 mM l-glutamate for 24 h. Subsequently, 100 μL of CCK-8 solution was added to each well and incubated for 2 h, after which the absorbance was measured at 450 nm using a microplate reader.

At least three independent experiments were conducted to determine the IC50 values, which are expressed as means ± standard deviation. All experimental data were analyzed using GraphPad Prism software.

4.3. Compound activity screening

Preparation of transfection reagents: add 24 μL of Lipofectamine 2000 and 600 μL of opti-MEM, and incubate at room temperature for 5 min. Add 9.6 μL of DNA and 600 μL of opti-MEM. After 20 min, add 4800 μL of opti-MEM and gently mix. When the 293T cells reach 85–95% confluence, digest with 0.25% trypsin and seed into a 96-well plate, using complete culture medium without antibiotics, and incubate in a 37 °C incubator for 24 h. After 24 h, discard the original culture medium from the plate, wash once with 1× PBS, and add 100 μL of the prepared transfection reagent (DNA–lipid complex) to each well. Incubate in a 37 °C incubator for 4 h. After 4 h, remove the transfection reagent from the plate, wash once with 1× PBS, and add 200 μL of complete culture medium to each well for an additional 20 h of incubation for subsequent experiments. Add complete culture medium containing 5 μM of the compound to each well and incubate for 24 h. After discarding the medium, add complete culture medium containing 10 μM of the 17β-HSD10 probe to each well and continue incubating for 30 min. Discard the culture medium, add 100 μL of PBS to each well, and then measure the fluorescence using a multifunctional microplate reader with an excitation wavelength of 435 nm and an emission wavelength of 520 nm.

4.4. The protective effect of compounds on cells

Preparation of transfection reagent: add 24 μL of Lipofectamine 2000 and 600 μL of opti-MEM, and let them react at room temperature for 5 min. Add 9.6 μL of DNA and 600 μL of opti-MEM. After 20 min, add 4800 μL of opti-MEM and mix gently. Cell transfection: when 3–4 passages of cells reach 85–95% confluence, digest with 0.25% trypsin and seed into a 96-well plate. Culture in complete medium without antibiotics in a 37 °C incubator for 24 h. After 24 h, discard the original culture medium, wash once with 1× PBS, and add 100 μL of the prepared transfection reagent (DNA–lipid complex) to each well. Incubate in a 37 °C incubator for 4 h. After 4 h, remove the transfection reagent, wash once with 1× PBS, and add 200 μL of complete medium to each well for continued culture for 20 h for subsequent experiments. Add complete medium containing 5 μM of the compound to each well and incubate for 24 h. After discarding the medium, add 10 μL of CCK-8 solution to each well and continue incubation for 2 h. Subsequently, measure the absorbance at 450 nm using a multifunctional microplate reader.

4.5. Cellular thermal shift assay

PC12 cells were cultured in cell culture dishes (100 mm) and treated or not with 10 μM compound, then incubated for 4 h. Discard the supernatant, add cell lysate, and evenly divide the cell lysate into several portions. Then, each tube was heated at a single temperature (25, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, and 78 °C) for 2 min. After that, cells were broken by ultrasonic treatment and centrifuged at 12 000 rpm for 20 min, and analyzed by western blot assay.

4.6. PK study

The experimental compound 33 was administered intravenously (5 mg kg⁻¹) and orally at a single dose (10 mg kg⁻¹) to Sprague–Dawley rats. After administration, blood samples were collected at different times and centrifuged to obtain plasma. The obtained plasma sample was precipitated with acetonitrile and analyzed for compounds using an LC–MS/MS system. PK parameters were calculated using the plasma concentration–time curve.

4.7. Plasma protein binding

Plasma doped with a concentration of 20 μM of compound and blank buffer was incubated in a CO₂ incubator at 37 °C for 4 hours. After incubation, the concentrations of compounds in plasma and buffer were quantified by LC–MS/MS. The unbound value was derived by dividing the compound concentration in the buffer chamber by the total concentration in the plasma chamber.

4.8. Research on animal behavior

All experiments and animal care procedures were approved by the Animal Resource Center of Anhui Medical University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (20230875). In this experiment, 8 week-old male Kunming mice, weighing between 18 and 22 g, were placed in the environment for one week to acclimatize. Mice were then randomly assigned to five groups: (1) control group, administered 0.5% sodium carboxymethyl cellulose (CMC-Na) solution via gavage; (2) model group; (3) high-dose compound 33 group (20 mg kg⁻¹); (4) low-dose compound 33 group (10 mg kg⁻¹); (5) positive control group with donepezil (10 mg kg⁻¹). Except for the control group, which only received saline injections, the other groups received intraperitoneal injections of scopolamine hydrobromide to establish an Alzheimer's disease model (for 10 consecutive days, with one dose per day). On day 5, the mice underwent training in the water maze experiment while receiving both gavage and intraperitoneal injections of the modeling drug, continuing until testing on day 10. The water maze apparatus primarily consists of a circular pool (diameter 150 cm × height 60 cm), an escape platform (diameter 10 cm), a data acquisition system, and an analysis system. The circular pool can be divided into four quadrants, with the escape platform consistently positioned at the center of the third quadrant. At the start of the experiment, water was added to the pool until the water level was 1 cm above the escape platform, and titanium dioxide powder (0.25 g L⁻¹) was added to make the water opaque. During the training phase, the mice were placed facing the inner wall and randomly introduced into the pool from each of the four quadrants. If a mouse located the escape platform within one minute, it was allowed to rest on the platform for 15 seconds; if unsuccessful, it was gently guided to the platform for a 15 second rest. Continuous training was conducted for five days, followed by the exploration experiment starting on day six. In the exploration experiment, mice were placed in the pool from the first quadrant and given one minute to find the platform, during which their movement trajectories and time taken to locate the platform were observed, recorded, and analyzed.

5. Statistical analyses

GraphPad Prism was utilized for statistical analysis through one-way ANOVA. The data were presented as mean ± standard deviation (SD), and statistical significance was determined at p < 0.05.

Abbreviation

AD

Alzheimer's disease

¹H NMR

Proton nuclear magnetic resonance

¹³C NMR

Carbon nuclear magnetic resonance

HR-MS

High-resolution electron impact mass spectra

DMF

N,N-Dimethylformamide

THF

Tetrahydrofuran

MeOH

Methanol

EtOAc

Ethyl acetate

DCM

Dichloromethane

DIPEA

N,N-Diisopropylethylamine

TEA

Triethylamine

IR

Inhibitory rate

IC₅₀

Half maximal inhibitory concentration

Data availability

The data supporting this study's findings are available from the corresponding author upon reasonable request.

Author contributions

Xiaohan Liu, Bin Zhou, Jinyuan Lin and Xingxing Zhang performed the synthetic experiments and analyzed the data; Yan Chen, Chenwen Shao, Banfeng Ruan and Liuzeng Chen performed the bioassays and analyzed the data; Ban-Feng Ruan, Xingxing Zhang, and Yong Qian conceived and wrote the article. All authors contributed to and approved the manuscript.

Conflicts of interest

The authors confirm that this article content has no conflict of interest.