Design and Synthesis of Novel Piperidine Urea Derivatives with Neuroprotective Properties

Keywords

ischemic stroke - Fenazinel - piperidine urea derivatives - neuroprotection

Introduction

Stroke is a severe neurological disorder resulting from circulatory disorders in the brain and is the leading cause of disability and the second leading cause of death worldwide.[1] Ischemic stroke accounts for 85% of all strokes and is characterized by high incidence, disability, mortality, and recurrence rates.[2] Currently, two main methods are used to treat ischemic stroke: thrombolytic therapy and neuroprotective therapy. However, thrombolytic therapy typically has a narrow window and bleeding risk.[3] In the early prevention and treatment of ischemic stroke, neuroprotective therapy can alleviate stroke-induced brain infarction and has several therapeutic advantages, including fewer complications.[4] However, only butylphthalide and edaravone are currently applied in clinical treatment as neuroprotective agents, and they cannot meet the clinical needs.[5] [6] Therefore, there is an urgent need to explore novel neuroprotective agents for ischemic stroke with high efficiency.

In previous work, we found that Fenazinel ([Fig. 1]) exhibited good neuroprotection in PC12 cells and in vivo rat focal cerebral ischemic animal model, and in 2006, Fenazinel entered phase I clinical trials in China as a novel neuroprotective agent.[7] [8] [9] [10] However, some side effects were observed during its clinical trials, including elevated serum creatine phosphokinase (CPK) and potential premature atrial. To determine the cause of these side effects, we performed a comprehensive evaluation of the pharmacological profile of Fenazinel, which indicated that Fenazinel had weak cardiotoxicity (hERG IC₅₀ = 8.64 μmol/L), but its major metabolite in the human body, compound M1, exhibited high cardiotoxicity (hERG IC₅₀ = 0.43 μmol/L), suggesting that M1 might contribute to drug-induced QT prolongation or drug-related cardiac toxicity ([Fig. 1]). Further studies have been conducted to decrease the metabolic toxicity of Fenazinel; for instance, the introduction of cinnamic acid and its analogs and the substitution of metabolites in Fenazinel have been shown to significantly reduce cardiotoxicity and showed potential neuroprotective activity.[11] [12]

Fig. 1 The design of novel piperidine urea derivatives (compounds A and B are reported to have neuroprotective activity with the urea group).

Through literature review, we found that the compounds containing urea groups are increasingly used in drug design.[13] Some of these compounds have demonstrated good neuroprotective activity, such as the 6-hydroxybenzothiazole urea derivative A which could protect SH-SY5Y neuroblastoma cells against α-syn-induced cytotoxicity and exhibit superior neuroprotective effect in vitro.[14] Azam et al designed and synthesized a series of thiazolyl urea derivatives, among which compound B improved the pharmacological profile in haloperidol-induced catalepsy and oxidative stress in mice, showing potential to treat Parkinson's disease ([Fig. 1]).[15] Based on the toxic metabolic position of Fenazinel and the properties of the piperidine urea groups, the pharmacophore fusion strategies were taken into account. Herein we designed a series of compounds by replacing the metabolized carbonyl group with neuroprotective urea groups. With this idea, we tried to avoid converting the carbonyl group to the hydroxyl group of Fenazinel while maintaining or enhancing the neuroprotective activity of the designed compounds. We detail below our efforts to identify novel Fenazinel derivatives with higher potency but lower cardiotoxicity.

Results and Discussion

Synthesis

The synthesis route of compound IM-4, a key intermediate for obtaining A1–A13, is outlined in [Scheme 1]. The aniline (IM-1) underwent a nucleophilic addition reaction with chloroacetyl chloride to generate IM-2, followed by a substitution reaction with 4-Boc-aminopiperidine to form IM-3, whose protecting group (Boc) was cleaved with HCl in ethyl acetate to obtain IM-4.

Scheme 1 Synthesis of the key intermediate IM-4. Reagents and conditions: (i) chloroacetyl chloride, TEA, MeCN, 0–15°C; (ii) 4-Boc-aminopiperidine, K₂CO₃, KI, MeCN, 6 hours; (iii) HCl in ethyl acetate.

Compounds A1–A10 were synthesized from Route 1 ([Scheme 2]) and A11–A13 from Route 2 ([Scheme 2]). The anilines (1a–1j) underwent nucleophilic addition reaction with phenyl chloroformate to produce intermediates 2a–2j, which were then reacted with IM-4 to produce 3a–3j. After purification, 3a–3j were salted with HCl in ethyl acetate, generating A1–A10. The anilines (1k–1m) were reacted with CDI to form active intermediates, followed by the addition of IM-4, and an appropriate amount of triethylamine to yield compounds 2k–2m in a one-pot fashion, and finally, the target compounds A11–A13 were obtained by salting with HCl in ethyl acetate.

Scheme 2 Synthesis of compounds A1–A13. Reagents and conditions: (i) phenyl chloroformate, pyridine, DMF, 5°C, 6 hours; (ii) IM-4, pyridine, DMF, reflux, 6 hours; (iii) HCl in ethyl acetate; (iv) CDI, DMF; (v) IM-4, DMF, r.t.

Biological Activity

L-Glutamic acid–induced injury model in SH-SY5Y cells was established to evaluate the neuroprotective effect of target compounds A1–A13 at different concentrations. The preliminary results are shown in [Table 1]. Compounds A7 and A10 showed better neuroprotective activity with a dose-dependent tendency when compared with Fenazinel, while compounds A1, A5, and A6 exhibited moderate neuroprotective activity. When the R substituent was 3-pyridine (compound A8), the compound demonstrated strong neuroprotective activity at low concentrations. However, its neuroprotective activity showed a downward trend as the concentration increased, indicating potential cytotoxicity. Generally, when R substituents are aromatic heterocyclic rings containing the S atom, the compounds exhibit good neuroprotective activity. When a substituent is introduced to the phenyl group, the neuroprotective activity of the compounds may be decreased (A1 > A2–A4).

Table 1 Preliminary neuroprotective activity of the compounds A1–A13 assessed in SH-SY5Y cells^a

Compound	R	Protective activity against SH-SY5Y cells, survival rate (%)
		0.1 μmol/L	1 μmol/L	10 μmol/L
A1		50.08 ± 5.33	54.65 ± 2.66	55.17 ± 1.41
A2		40.05 ± 6.23	48.86 ± 2.79	49.35 ± 0.97
A3		49.68 ± 2.55	51.05 ± 1.61	49.64 ± 0.47
A4		50.05 ± 0.33	46.36 ± 0.75	44.60 ± 1.52
A5		49.01 ± 0.51	52.00 ± 3.89	54.12 ± 1.77
A6		50.26 ± 2.04	49.53 ± 2.30	53.09 ± 0.71
A7		49.67 ± 13.04	55.48 ± 4.27	59.48 ± 3.47
A8		64.82 ± 1.09	57.69 ± 3.51	48.33 ± 1.08
A9		49.93 ± 0.90	48.25 ± 0.67	52.36 ± 0.87
A10		53.37 ± 1.37	57.48 ± 0.79	61.54 ± 1.89
A11		52.76 ± 3.06	51.43 ± 1.76	47.35 ± 0.56
A12		51.90 ± 3.34	54.16 ± 0.09	45.23 ± 1.66
A13		48.36 ± 1.61	47.27 ± 1.45	50.64 ± 1.75
Fenazinel	–	51.35 ± 1.77	56.27 ± 0.86	56.11 ± 1.01
L-Glutamic acid (damage model)		50.58 ± 1.13 (100 μmol/L)

Note: ^aAll data were presented as mean ± standard deviation. For each batch of experiments, only the mean and standard deviation of each compound hole under the same treatment condition were listed, without Fenazinel comparison.

The concentration of clinically used neuroprotective agents needs to be strictly controlled, as high concentrations may cause cytotoxicity.[16] [17] [18] To obtain neuroprotective agents with higher safety profiles, an thiazolyl blue (MTT) assay was conducted to test the neuroprotective ability of compounds A7 and A10 at high doses (10, 20, 50, and 100 μmol/L), using Fenazinel as a positive control group. As shown in [Table 2], all the compounds caused certain damage to SH-SY5Y cells in a dose-dependent manner. Among them, compound A10 exhibited similar damage levels to Fenazinel at different concentrations. Thus, compound A10 was chosen for the following study.

Table 2 The antiproliferative activity of the compounds A7 and A10 against SH-SY5Y nerve cells at high concentrations

Treatment	Dose (μmol/L)	Cell survival (%)
Vehicle	PBS	100.00 ± 3.29
Model (L-Glutamic acid)	PBS	57.64 ± 0.71
A7	10	53.64 ± 2.68
	20	43.86 ± 2.75
	50	41.66 ± 0.81
	100	32.61 ± 6.05
A10	10	64.08 ± 6.07
	20	59.24 ± 7.62
	50	52.75 ± 2.00
	100	48.40 ± 3.08
Fenazinel	10	62.91 ± 7.13
	20	62.14 ± 5.05
	50	52.25 ± 9.68
	100	54.66 ± 2.45

Abbreviation: PBS, phosphate buffer saline.

Note: Data are presented as mean ± standard error of the mean with each experiment repeated at least twice.

In vitro experiments showed that compound A10 possessed good neuroprotective activity (10 μmol/L, 61.54% survival rate) and low cytotoxicity (100 μmol/L, 48.40% cell survival) ([Tables 1] and [2]). We further performed a hERG assay to evaluate the cardiotoxicity of compound A10. The result showed that the cardiotoxicity of compound A10 was weaker (hERG IC₅₀ > 40 μmol/L) than that of Fenazinel (hERG IC₅₀ = 8 μmol/L) ([Table 3]), indicating that the cardiotoxicity of Fenazinel was effectively decreased by introducing the piperidine urea group to its structure. The result of an in vivo hypoxia tolerance assay showed that both A10 and Fenazinel were effective in prolonging the survival time of mice under experimental conditions (iv, 6 mg/kg), with compound A10 being more effective than Fenazinel ([Table 3]).

Table 3 Results of the hERG inhibition assay and hypoxia tolerance assay

Compound	hERG inhibition ratio (%) at 40 μmol/L	hERG IC50 (μmol/L)	Hypoxia tolerance assay, survival time (s)a
			6 mg/kg
A10	25.98	> 40	2,970.0 ± 119.3
Fenazinel	64.16	8.64	2,761.1 ± 409.6
Control	–	–	2,355.6 ± 356.7

Note: ^aData were expressed as mean ± standard deviation.

To further determine the neuroprotective effect of compound A10 in vivo, we established a rat model of middle cerebral artery occlusion (MCAO), and the percentage of cerebral infarction area was assessed with additional treatment of compound A10. The result ([Fig. 2]) showed significant cerebral infarction in the model group, which was improved with additional compound A10 treatment. Compound A10 exhibited a dose-dependent effect (the percentage of cerebral infarction: model: 51.06%; low concentration: 47.66%; medium concentration: 39.82%; high concentration: 32.2%). These results indicated that compound A10 has a significant anticerebral infarction effect and neuroprotective effect in MCAO rats.

Fig. 2 Compound A10 alleviated cerebral infarction in middle cerebral artery occlusion (MCAO) rats. The MCAO rats were treated with different concentrations of compound A10 (1.0, 3.0, 5.0 mg/kg). After 24 hours, the percentage of cerebral infarction was assessed using Image J software. Data are presented as mean ± standard error of the mean, and compared with a t-test. *p < 0.05 versus model group was considered significant.

Conclusion

To reduce the potential cardiotoxicity of Fenazinel, we designed and synthesized a class of novel piperidine urea derivatives and evaluated their in vitro and in vivo activities. The results showed that some of the compounds had potent neuroprotective activities in vitro. Among them, compound A10 (0.1, 1, 10 μmol/L) exhibited slightly better protective activity against L-glutamic acid–induced injury in SH-SY5Y cells than the control drug (Fenazinel). Additionally, it exhibited lower cytotoxicity at high concentrations. Moreover, compound A10 possessed weaker cardiotoxicity than Fenazinel, and was effective in prolonging the survival time of mice. Besides, compound A10 could significantly reduce the percentage of cerebral infarction in male MCAO models, and showed better anti-stroke activity with a dose-dependent tendency. In conclusion, the potential cardiotoxicity of Fenazinel could be effectively evaded by reasonable modification of its structure. Compound A10 showed comparable or better neuroprotective activity than Fenazinel both in vivo and in vitro, and is expected to be further studied as a novel neuroprotective agent.

Experimental Section

Chemistry

All the reagents and solvents were commercially available with analytical reagents. The reaction processes were monitored by thin-layer chromatography (TLC) under a 254-nm UV lamp on a silica gel plate GF254 (Qingdao Haiyang Chemical, Qingdao, China). Nuclear magnetic resonance spectra (¹H NMR and ¹³C NMR) were recorded on a Bruker Avance spectrometer (Bruker, Germany) using dimethyl sulfoxide-d ₆ (DMSO-d ₆) as a solvent. Chemical shifts were reported in parts per million (ppm, δ) with tetramethylsilane as an internal standard. ESI-MS data were recorded on an Agilent 6210 MS spectrometer. Melting point (mp) was obtained on a WRS-2A microcomputer melting point meter (Shanghai INESA Physico-Optical Instrument Co., Ltd., Shanghai, China).

Synthesis of IM-4

Chloroacetyl chloride (0.25 mol, 1.25 equiv.) and triethylamine (0.4 mol, 2.0 equiv.) were dissolved in 80 mL acetonitrile. Aniline (0.2 mol, 1.0 equiv.) was slowly added at 0 to 15°C. After the completion of the reaction monitored by TLC, the mixture was concentrated to give a residue, followed by recrystallization and purification to obtain IM-2 (18.4 g, 51%) as a white solid. ESI-MS (m/z) calcd for C₉H₁₁ClNO⁺[M + H]⁺, 184.0521; found 184.64. To a solution of 4-Boc-aminopiperidine (0.1 mol, 1.0 equiv.) in acetonitrile (40 mL) IM-2 (0.1 mol, 1.0 equiv.), K₂CO₃ (0.2 mol, 2.0 equiv.), and KI (5 mmol, 0.05 equiv.) were successively added at room temperature. The mixture was heated to 40°C. After the completion of the reaction monitored by TLC, the mixture was filtered. The filtrate was concentrated. The crude product was washed with ether to get IM-3 (32.5 g, 93.6%), which was dissolved in ethyl acetate (80 mL). HCl/ethyl acetate solution was added to adjust pH to 3 to 4. After stirring for 1 hour, a large amount of solid was precipitated and filtered to obtain the product IM-4 (27.9 g, 93.1%) as a white solid.

Synthesis of A1–A13

The arylamines (1a–1j, 5.0 mmol, 1.0 equiv.) and pyridine (8.75 mmol, 1.75 equiv.) were dissolved in N,N-dimethylformamide (DMF, 30 mL), then phenyl chloroformate (6.4 mmol, 1.28 equiv.) was slowly added at 0 to 5°C. After the completion of the reaction monitored by TLC, ice water (50 mL) was poured into the reaction solution to precipitate a white solid, which was filtered and washed with ether to yield 2a–2j.

Pyridine (2 drops) was added to a solution of IM-4 (2.26 mmol, 1.0 equiv.) in DMF (20 mL). The suspension was stirred at room temperature for 1.5 hours, and then heated to reflux. After the completion of the reaction monitored by TLC, the mixture was cooled down and concentrated to give a residue, with pH being adjusted to 9 to 10 with a saturated sodium bicarbonate solution. The solution was extracted with dichloromethane (20 mL × 2). The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 3a–3j as a yellow solid.

Arylamine (1k–1m, 5.0 mmol, 1.0 equiv.) and CDI (6.0 mmol, 1.2 equiv.) were dissolved in an appropriate amount of DMF and stirred for 2 hours. The active intermediate was obtained, then IM-4 (5.0 mmol, 1.0 equiv.) and triethylamine (15.0 mmol, 3.0 equiv.) were added. The mixture was stirred at room temperature overnight. After the completion of the reaction monitored by TLC, water was added. The mixture was extracted with ethyl acetate. The combined organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous Na₂SO₄, filtered, and concentrated to give a residue (yellow oil), followed by recrystallization with MeOH to yield intermediates 2k–2m as a white or pale yellow powder.

A1–A13 were salted from 3a–3j and 2k–2m using an HCl/ethyl acetate solution and obtained according to the method described for IM-4.

N-Benzyl-2-(4-(3-phenylureido)piperidin-1-yl)acetamide hydrochloride (A1): yield: 59.3%. mp: 80.2–82.2°C. ESI-MS (m/z): calcd for C₂₁H₂₇N₄O₂ ⁺ [M + H]⁺ 367.2129; found 367.24. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.94 (s, 1H), 9.24-9.18 (m, 1H), 7.58 (s, 3H), 7.37-7.26 (m, 5H), 6.88 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 4.0 Hz, 1H), 4.37 (d, J = 8.0 Hz, 2H), 4.05-3.98 (m, 2H), 3.74-3.67 (m, 1H), 3.49 (d, J = 8.0 Hz, 2H), 3.17 (q, J = 8.0 Hz, 1H), 2.09-1.99 (m, 2H), 1.91 (s, 1H), 1.83-1.70 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 172.12, 156.87, 139.61, 139.18, 130.02, 129.58, 128.07, 127.84, 123.53, 119.61, 63.31, 50.91, 46.66, 45.26, 34.37.

N-Benzyl-2-(4-(3-(4-(trifluoromethyl)phenyl)ureido)piperidin-1-yl)acetamide hydrochloride (A2): yield: 55.4%. mp: 117.6–118.8°C. ESI-MS (m/z) calcd for C₂₂H₂₆F₃N₄O₂ ⁺ [M + H]⁺ 435.2002; found 435.21. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.83 (s, 1H), 9.24-9.19 (m, 1H), 8.92 (s, 1H), 7.40-7.25 (m, 7H), 7.05 (t, J = 8.0 Hz, 2H), 4.36 (d, J = 4.0 Hz, 2H), 4.06-3.98 (m, 2H), 3.72-3.65 (m, 1H), 3.48 (d, J = 12.0 Hz, 2H), 3.22-3.14 (m, 1H), 2.09-1.99 (m, 2H), 1.91 (s, 1H), 1.79-1.68 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 171.12, 157.09, 141.59, 139.31, 128.50, 127.70, 127.64, 126.08, 125.24, 123.90, 119.23, 60.31, 51.61, 46.76, 43.76, 31.10.

N-Benzyl-2-(4-(3-(4-fluorophenyl)ureido)piperidin-1-yl)acetamide hydrochloride (A3): yield: 67.0%. mp: 74.8–75.5°C. ESI-MS (m/z) calcd for C₂₁H₂₆FN₄O₂ ⁺ [M + H]⁺ 385.2034; found 385.19. ¹H NMR (400 MHz, DMSO-d ₆) δ 7.38-7.25 (m, 7H), 7.21 (t, J = 8.0 Hz, 2H), 6.88 (t, J = 8.0 Hz, 1H), 4.36 (d, J = 4.0 Hz, 2H), 4.05-4.02 (m, 1H), 3.98 (d, J = 4.0 Hz, 2H), 3.49 (d, J = 12.0 Hz, 2H), 3.39 (s, 1H), 3.18 (q, J = 12.0 Hz, 1H), 2.04-1.99 (m, 2H), 1.79-1.69 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 172.02, 164.06, 154.84, 138.43, 136.87, 128.42, 127.43, 127.11, 118.96, 115.03, 56.27, 51.72, 44.02, 42.25, 29.16

N-Benzyl-2-(4-(3-(4-methoxyphenyl)ureido)piperidin-1-yl)acetamide hydrochloride (A4): yield: 65.1%. mp: 74.8–75.5°C. ESI-MS (m/z) calcd for C₂₂H₂₉N₄O₃ ⁺ [M + H]⁺, 397.2234; found 397.21. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.94 (s, 1H), 9.24-9.18 (m, 1H), 8.81 (d, J = 28.0 Hz, 1H), 8.56 (s, 1H), 7.37-7.26 (m, 7H), 6.86-6.79 (m, 2H), 4.36 (d, J = 4.0 Hz, 2H), 4.05-3.98 (m, 2H), 3.70 (d, J = 8.0 Hz, 4H), 3.50-3.39 (m, 3H), 3.17 (q, J = 12.0 Hz, 1H), 2.03-1.99 (m, 2H), 1.78-1.65 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 164.06, 154.96, 153.89, 138.42, 133.22, 128.42, 127.44, 127.12, 119.55, 113.96, 56.27, 55.14, 51.76, 48.45, 44.03, 29.23.

N-Benzyl-2-(4-(3-cyclohexylureido)piperidin-1-yl)acetamide hydrochloride (A5): yield: 67.9%. mp: 87.3–88.5°C. ESI-MS (m/z) calcd for C₂₁H₃₃N₄O₂ ⁺ [M + H]⁺ 373.2598; found 373.28. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.97 (s, 1H), 9.24 (d, J = 8.0 Hz, 1H), 7.36-7.25 (m, 5H), 4.35 (d, J = 8.0 Hz, 3H), 4.05-3.98 (m, 3H), 3.63-3.53 (m, 1H), 3.47-3.41 (m, 2H), 3.40-3.34 (m, 3H), 3.30-3.26 (m, 1H), 3.14 (q, J = 12.0 Hz, 1H), 2.01-1.95 (m, 2H), 1.72-1.61 (m, 4H), 1.52-1.49 (m, 1H), 1.29-1.04 (m, 3H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 164.06, 156.74, 138.44, 128.41, 127.42, 127.09, 63.07, 56.23, 51.79, 44.06, 42.23, 33.22, 29.42, 25.31, 24.39.

N-Benzyl-2-(4-(3-(1,3,4-thiadiazol-2-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A6): yield: 45.7%. mp: 137.6–138.7°C. ESI-MS (m/z) calcd for C₁₇H₂₃N₆O₂S⁺ [M + H]⁺ 375.1598; found 375.16. ¹H NMR (400 MHz, DMSO-d ₆) δ 10.58 (s, 1H), 8.97 (s, 1H), 8.26 (dt, J = 20.0 Hz, 4.0 Hz, 1H), 7.33-7.30 (m, 2H), 7.26-7.22 (m, 3H), 6.82 (s, 1H), 4.30 (t, J = 8.0 Hz, 2H), 3.51 (s, 1H), 2.98-2.94 (m, 2H), 2.75-2.67 (m, 2H), 2.19 (dt, J = 28.0 Hz, 8.0 Hz, 2H), 1.81 (d, J = 8.0 Hz, 1H), 1.71 (d, J = 4.0 Hz, 1H), 1.51 (td, J = 12.0 Hz, 4.0 Hz, 1H), 1.35 (td, J = 8.0 Hz, 4.0 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 171.12, 166.69, 158.29, 146.78, 139.31, 128.50, 127.70, 127.64, 60.31, 51.61, 46.82, 43.76, 31.08.

N-Benzyl-2-(4-(3-(5-methylthiazol-2-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A7): yield: 61.5%. mp: 142.0–144.8°C. ESI-MS (m/z) calcd for C₁₉H₂₆N₅O₂S⁺ [M + H]⁺ 388.1802; found 388.25. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.96 (s, 1H), 8.27 (s, 1H), 7.32-7.23 (m, 5H), 6.95 (s, 1H), 6.54 (s, 1H), 4.31 (d, J = 4.0 Hz, 2H), 3.50 (s, 1H), 2.98 (s, 2H), 2.71 (s, 2H), 2.28-2.22 (m, 5H), 1.81 (d, J = 4.0 Hz, 2H), 1.47 (d, J = 4.0 Hz, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 164.03, 159.70, 152.70, 152.51, 138.45, 128.41, 127.43, 127.10, 125.22, 56.24, 51.46, 44.62, 42.23, 28.73, 11.23.

N-Benzyl-2-(4-(3-(pyridin-3-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A8): yield: 55.8%. mp: 82.8–83.9°C. ESI-MS (m/z) calcd for C₂₀H₂₆N₅O₂ ⁺ [M + H]⁺ 368.2081; found 368.16. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.52 (s, 1H), 8.50 (d, J = 4.0 Hz, 1H), 8.25 (t, J = 4.0 Hz, 1H), 8.10 (dd, J = 4.0 Hz, 3.6 Hz, 1H), 7.87 (dq, J = 8.0 Hz, 4.0 Hz, 1H), 7.32-7.30 (m, 2H), 7.25-7.22 (m, 4H), 6.29 (d, J = 8.0 Hz, 1H), 4.30 (t, J = 4.0 Hz, 2H), 3.51-3.48 (m, 1H), 2.97 (s, 1H), 2.94 (d, J = 4.0 Hz, 1H), 2.75-2.71 (m, 2H), 2.21 (t, J = 4.0 Hz, 1H), 2.15 (t, J = 4.0 Hz, 1H), 1.80 (dd, J = 8.0 Hz, 4.0 Hz, 2H), 1.46 (td, J = 8.0 Hz, 4.0 Hz, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 171.12, 156.89, 144.28, 143.03, 139.31, 136.19, 128.50, 127.70, 127.64, 126.25, 125.03, 60.31, 51.61, 46.76, 43.76, 31.10.

N-Benzyl-2-(4-(3-(1H-pyrazol-3-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A9): yield: 47.5%. mp: 105.9–107.4°C. ESI-MS (m/z) calcd for C₁₈H₂₅N₆O₂ ⁺ [M + H]⁺ 375.2034; found 357.26. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.22 (s, 1H, CONH), 7.88 (d, J = 4.0 Hz, 2H, CONH), 7.36-7.31 (m, 3H), 7.26-7.22 (m, 3H), 5.75 (d, J = 2.0 Hz, 1H), 5.27 (s, 2H), 4.30 (d, J = 4.0 Hz, 2H), 3.57 (t, J = 4.0 Hz, 1H), 2.98 (s, 2H), 2.78 (d, J = 8.0 Hz, 2H), 2.19 (t, J = 4.0 Hz, 2H), 1.76 (d, J = 8.0 Hz, 2H), 1.69-1.64 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 164.07, 148.85, 138.50, 130.27, 128.42, 127.44, 127.40, 127.10, 100.18, 56.34, 51.67, 45.32, 42.24, 28.47.

N-Benzyl-2-(4-(3-(5-methyl-1,3,4-thiadiazol-2-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A10): yield: 56.4%. mp: >220°C. ESI-MS (m/z) calcd for C₁₈H₂₅N₆O₂S⁺ [M + H]⁺ 389.1754; found 440.20. ¹H NMR (600 MHz, DMSO-d ₆): δ 9.18 (s, 1H), 8.59 (s, 1H), 7.37-7.08 (m, 5H), 4.38-4.36 (d, 2H), 3.93 (s, 2H), 3.56-3.53 (m, 2H), 3.31 (m, 1H), 3.21-3.16 (m, 2H), 2.45 (s, 3H), 2.17-2.14 (m, 2H), 2.06-1.96 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 172.05, 164.07, 158.31, 153.22, 138.48, 128.44, 127.46, 127.14, 56.26, 51.54, 44.60, 42.26, 28.85, 14.77.

N-Benzyl-2-(4-(3-(5-methylisoxazol-3-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A11): yield: 68.4%. mp: 206.3–208.4°C. ESI-MS (m/z) calcd for C₁₉H₂₆N₅O₃ ⁺ [M + H]⁺ 372.2030; found 372.24. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.93 (s, 1H), 9.29 (s, 1H), 9.17 (d, J = 8.0 Hz, 1H), 7.37-7.25 (m, 5H), 6.98 (d, J = 8.0 Hz, 1H), 6.40 (s, 1H), 4.36 (d, J = 8.0 Hz, 2H), 3.98 (s, 2H), 3.69 (d, J = 8.0 Hz, 1H), 3.48 (d, J = 12.0 Hz, 2H), 3.17 (q, J = 8.0 Hz, 2H), 2.32 (s, 3H), 2.02 (d, J = 12.0 Hz, 2H), 1.76 (t, J = 8.0 Hz, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 168.71, 164.06, 158.90, 153.38, 138.42, 128.41, 127.42, 127.10, 95.45, 56.26, 51.59, 44.26, 42.25, 28.97, 12.07.

N-Benzyl-2-(4-(3-(isoxazol-3-yl)ureido)piperidin-1-yl)acetamide hydrochloride (A12): yield: 65.6%. mp: 83.4–85.3°C. ESI-MS (m/z) calcd for C₁₈H₂₄N₅O₃ ⁺ [M + H]⁺ 358.1874; found 358.19. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.95 (s, 1H), 9.46 (s, 1H), 9.18 (s, 1H), 8.67 (s, 1H), 7.37-7.25 (m, 5H), 6.72 (s, 1H), 4.35 (d, J = 4.0 Hz, 2H), 4.03 (d, J = 4.0 Hz, 2H), 3.49 (d, J = 12.0 Hz, 2H), 3.18 (d, J = 8.0 Hz, 2H), 2.03 (d, J = 12.0 Hz, 2H), 1.77 (t, J = 12.0 Hz, 2H), 1.17 (t, J = 8.0 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 169.12, 157.45, 156.89, 155.54, 140.31, 128.70, 128.55, 128.24, 96.38, 65.31, 50.75, 46.21, 44.76, 30.98.

2,2'-((Carbonylbis(azanediyl))bis(piperidine-4,1-diyl))bis(N-benzylacetamide) hydrochloride (A13): yield: 67.7%. mp: 108.1–109.3°C. ESI-MS (m/z) calcd for C₂₉H₄₁N₆O₃ ⁺ [M + H]⁺ 521.3235; found 521.37. ¹H NMR (400 MHz, DMSO-d ₆) δ 10.00 (s, 1H), 9.27 (d, J = 8.0 Hz, 1H), 7.36-7.25 (m, 5H, ArH), 4.35 (d, J = 8.0 Hz, 2H), 4.01-3.97 (m, 2H), 3.58 (d, J = 8.0 Hz, 1H), 3.45 (d, J = 12.0 Hz, 2H), 3.15 (t, J = 12.0 Hz, 2H), 1.95 (d, J = 12.0 Hz, 2H), 1.71-1.62 (m, 2H); ¹³C NMR (151 MHz, DMSO-d ₆) δ 164.06, 156.81, 138.46, 128.40, 127.42, 127.08, 56.21, 51.69, 44.10, 42.22, 29.36.

Neuroprotection Assay Against L-Glutamic Acid–induced Cell Damage in SH-SY5Y Cells

The SH-SY5Y cells were purchased from American Type Culture Collection (ATCC), and cultured in EMEM/F12 (ATCC) with 10% fetal bovine serum (EX-CELL), 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco, Shanghai, China) at a condition of 37°C, 5% CO₂. SH-SY5Y cells were divided into normal culture group, model group, and treatment group. Fenazinel (Shanghai Institute of Pharmaceutical Industry Co., Ltd., Shanghai, China) was used as a positive control drug. For primary screening of the neuroprotective activity of the compounds, the concentration gradient was 0.1, 1, and 10 µmol/L (two multiple holes for each concentration); for further screening of the cytotoxicity of a compound, the concentration gradient was 10, 20, 50, and 100 µmol/L (three multiple holes for each concentration). SH-SY5Y cells in the treatment group were pretreated with different concentrations of the test substance for 30 minutes and then incubated with 300 µmol/L L-glutamic acid obtained from Sigma for 24 hours. Normal and L-glutamic acid–injured SY5Y cells were treated with the same volume of phosphate buffer saline (PBS). After 24 hours, an MTT assay was performed to assess cell viability using an MTT assay kit (Abcam) according to the manufacturer's instruction. Optical density (OD) was assessed through spectrophotometry with a micro-plate reader (Thermo Fisher Scientific, Shanghai, China) at 570 nm. The following formula was used: cell viability rate (%) = (OD values of the treated groups − OD values of model groups)/(OD values of the normal groups − OD values of model groups) × 100%.

Hypoxia Tolerance Assay

Male ICR mice (18–20 g, Sipu-BiKAI, SCXK2008–0016) were divided into control and treatment groups (n = 10 per group). Mice in the treatment groups were given Fenazinel or A10 at a dose of 20 mg/kg through a tail vein intravenous injection. Mice in the control group were given the same volume of DMSO. After the treatment, mice were placed in 250 mL grinding mouth bottles containing 5 g sodium lime (one mouse in a bottle). After sealing, the survival time of the mice was observed and recorded.

hERG Inhibitory Activity Assay

hERG inhibition ratio test assay was conducted in CHO-hERG cells (ATCC) according to a reported study.[19] The whole-cell recordings were conducted using Automated Qpatch. The cells were treated with different concentrations of Fenazinel and compound A10 (0.16–40 μmol/L). The degree of hERG channel inhibition was determined by the following equation: Inhibition (%) = (1 − variation in the current before and after the addition of a test substance/variation in the current before and after the addition of a medium) × 100. The experiments were performed three times in parallel. Cardiotoxicity of the compound was predicted as IC₅₀ values of the hERG channel, as well as the hERG inhibition ratio (%) of the compound at a high dose (40 μmol/L).

MCAO-Induced Cerebral Ischemia/Reperfusion Injury Model

The Sprague-Dawley (SD) rats (male, 240.0 ± 20.0 g, Beijing Charles River Co., Ltd., Beijing, China) were raised under the condition of a 12-hour light/12-hour dark cycle, 25 ± 2°C, and a relative humidity of 45 ± 5% for 7-day acclimation at the Pharmacological Evaluation and Research Animal Center. Before the operation, the rats were fasted for 12 hours. After anesthetizing the rats with 1% pentobarbital sodium (Sigma) at a dose of 40 mg/kg, the skin of the neck was incised and the tissue was bluntly separated to expose the internal carotid arteries and right common carotid artery. The middle cerebral artery was blocked by a nylon thread inserted from the common carotid artery. After 1.5 hours of ischemia, the nylon thread was removed and the incision was sutured. The successful establishment of the MCAO model could be observed as a contralateral lower limb paralysis in rats.

The MCAO rats were given a solution (5% DMSO, 40% PEG 400, 5% Tween 80, and normal saline) containing different concentrations of compound A10 (1, 3, and 5 mg/kg, iv). At the endpoint of the experiment, rats were anesthetized with 1% pentobarbital sodium (40 mg/kg) and sacrificed. The complete brain was isolated. After being cleaned with PBS and frozen at −20°C, the brains were cut into 2-mm thick slices, then dyed with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St. Louis, MO, United States) solution at 37°C for 30 minutes. The percentage of infarct area was quantified using Image J software. Each experiment was repeated five times.

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