Design and synthesis of novel PDE4 inhibitors as potential candidates for antidepressant agents

Abstract

PDE4 inhibitors exhibit anti-stress and antidepressant-like abilities by catalyzing the hydrolysis of cAMP, a primary regulator for intracellular communication in the brain. Herein, novel diarylpyrazole derivatives are synthesized and investigated for their ability to inhibit PDE4. In vitro studies indicate that most of the synthesized compounds show significant potency for the inhibition of PDE4. Specifically, N-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)-2-morpholinoethan-1-amine exhibits the most potent PDE4 inhibition, with an IC₅₀ value of ca. 0.09 μM. It also produces antidepressant-like activities in sugar water consumption and in forced swimming tests in vivo.

A series of novel resveratrol derivatives were synthesized and evaluated as PDE4 inhibitors against depression. Among the synthesized compounds, 6e exhibits the potent ability to PDE4 and good selectivities over other PDEs. It also produced antidepressant-like activities in sugar water consumption and forced swimming tests in vivo. These results indicated that compound 6e is an effective and promising candidate against depression.

Introduction

Depression is a serious psychiatric illness characterized by anxiety, irritable moods, cognitive sleep disorders, and so on.^1,2 According to the World Health Organization, it is one of the leading causes of disability and the second largest contributor to the global burden of disease.^{3 –5} However, the etiopathology of depression presently remains unknown due to the complexity of its pharmacotherapy.^6,7 Based on classic serotonin or noradrenaline hypotheses, several antidepressants such as chlorbipram hydrochloride, fluoxetine (Flu), and venlafaxine have been developed for the treatment of depression. However, all these drugs are slower to take effect and symptoms are only improved after several weeks of treatment.^{8 –12} Furthermore, according to previous reports, at least 40% of patients do not respond to these drugs. Therefore, the development of new and effective agents with fewer adverse effects is still needed. It has been shown that cAMP, a primary regulator of intracellular communication, plays an important role in the modulation of moods.^{13 –15} Its elevation can protect dopaminergic neurons from the toxic effects of MPP⁺ (1-methyl-4-phenylpyridinium) in vitro. ¹⁶ PDE4, a cAMP-specific hydrolyzing enzyme, is a potential target for the treatment of depression.^{17 –19} In fact, several PDE4 inhibitors, such as roflumilast, rolipram, and MK-0952, display antidepressant-like effects and cognitive enhancement.^{20 –22} However, there are still no PDE4 inhibitors available for the treatment of depression, although rolipram has been evaluated in a number of preclinical and clinical studies.

Resveratrol (Figure 1), a natural polyphenol in many plant-based foods, can alleviate depression-like symptoms induced by stress in rats by functioning as a PDE4 inhibitor. Furthermore, resveratrol can increase cAMP levels both in vitro and in vivo.^23,24 Considering its chemical instability and low bioavailability, many resveratrol derivatives have been reported to overcome these problems.^{25 –27} Recently, we reported a novel resveratrol analogue (RES003) (Figure 1) via the introduction of a pyrazole group. It functions as a PDE4 inhibitor and ameliorates chronic-stress-induced depression-like behaviors. ²⁸ Encouraged by these results, herein we report our continued studies on the synthesis of such resveratrol derivatives and the evaluation of their antidepressant potential both in vitro and in vivo.

Figure 1.

The structures of resveratrol and RES003.

Results and discussion

The synthesis of compounds 6a–f is illustrated according to the previously reported method in Scheme 1. Aldol condensation between 3,5-dimethoxybenzaldehyde (1) and 4′-(diethoxymethyl)acetophenone ²⁹ (2) under standard basic conditions led to chalcone 3 in good yield. Chalcone 3 was then cyclized to give 4 in the presence of 4-toluenesulfonyl hydrazide and iodine, according to the previously reported method.^30,31 Standard acidic acetal deprotection of 4 yielded 5 in excellent yield. In the next step, condensation and reaction of compound 5 with different amines followed by reduction with NaBH₄ afforded the target compounds 6a–f in a one-pot operation. Compounds 10a–g can be obtained by a similar process using various substituted acetophenones instead of 2 (Scheme 2). All the synthesized compounds are novel apart from compounds 8a, 8c, 8e, and 8g. The structures of the products were verified by ¹H NMR, ¹³C NMR, and HRMS. Tautomers of 3,5-disubstituted pyrazoles are known to equilibrate rapidly. The tautomers of the pyrazoles shown in Schemes 1 and 2 follow those reported in the literature. ³¹

Scheme 1.

Synthesis of compounds 6a–f. Reagents and conditions: (a) NaOH, CH₃OH, rt, 12 h, 73%; (b) p-toluenesulfonyl hydrazide, I₂, K₂CO₃, C₂H₅OH, 75 °C, 2 h, 83%; (c) HCl, THF, rt, 2 h, 93%; and (d) R¹NH₂, NaBH₄, C₂H₅OH, rt, 2 h.

Scheme 2.

Synthesis of compounds 10a–g. Reagents and conditions: (a) R²Br, K₂CO₃, DMF, 80 °C, 5–6 h; (b) 3,5-dimethoxybenzaldehyde, H₂SO₄, CH₃OH, reflux, 5 h; and (c) p-toluenesulfonyl hydrazide, I₂, K₂CO₃, C₂H₅OH, 75 °C, 2 h.

All the compounds were initially screened for their binding potency against PDE4 using rolipram as a positive control. The inhibitory activities are presented as IC₅₀ (μM) values and the results are shown in Table 1. All of the derivatives showed better inhibitory activities against PDE4 than resveratrol. Our exploration was focused on introducing different substituents at the 4′-position of one benzene ring while keeping the remaining structures fixed. The results showed that the introduction of a substituent at the 4′-position had a significant influence on the inhibitory activity. According to the screening data, we found that compounds with linear or branched-chain substitution showed poor inhibitory activity against PDE4. For example, compounds 10a–e exhibited lower activities (IC₅₀ >6.00 μM). In contrast, all of the products with (cycloalkyl)amino substitution had remarkably increased PDE4 inhibitory activities. For example, compounds 6a, 6b, and 6e showed potent inhibitory activities against PDE4 (IC₅₀ <1.00 μM). It is noticeable that compound 10f also exhibited potent activity. The most potent product was 6e possessing an aminoethyl morpholine group, which showed a nanomolar IC₅₀ values toward PDE4 (IC₅₀: 90 nM). Therefore, compound 6e was selected to evaluate the PDE4 inhibitory selectivity over other PDEs, including PDE1, 2, 5, 9, and 10 (Table 2). This compound displayed >5000-fold selectivity over PDE1 (>500 μM), PDE5 (>500 μM), and PDE10 (>500 μM) and 400-fold selectivity over PDE2 (>37.2 μM) and PDE9 (>46.1 μM), revealing that it was poorly active against other PDE family members.

Table 1.

Structures and inhibitory activities toward PDE4 by the prepared resveratrol derivatives.

Compound	Structure	Yield	IC50 (μM) a
6a		60%	0.19 ± 0.02
6b		64%	0.13 ± 0.01
6c		55%	2.21 ± 0.37
6d		60%	3.26 ± 0.22
6e		71%	0.09 ± 0.01
6f		71%	6.45 ± 0.57
10a		77%	13.51 ± 1.67
10b		62%	15.03 ± 1.41
10c		70%	10.22 ± 1.07
10d		80%	6.32 ± 0.57
10e		77%	7.27 ± 0.65
10f		58%	0.11 ± 0.01
10g		85%	2.26 ± 0.29
Rolipram			0.19 ± 0.01

a

Results are expressed as the mean of at least three experiments.

Table 2.

PDE selectivity of compound 6e.

Compound	PDE IC50 (μM) a
	PDE4	PDE1	PDE2	PDE5	PDE9	PDE10
6e	0.09 ± 0.01	>500	37.2 ± 3.1	>500	41.6 ± 3.9	>500

a

Results are expressed as the mean of at least three experiments.

In addition, a molecular docking simulation was performed to explore the interaction mode of compound 6e with PDE 4 (PDB ID: 4WCU) (Figure 2). This study showed that compound 6e can interact with PDE4 strongly via hydrophobic interactions, face-to-face π-π stacking interactions, and hydrogen bonds. Specifically, the 3,5-dimethoxybenzene fragment of 6e interacts with the phenyl ring of Phe340 and Phe372 through face-to-face π-π stacking interactions and the CH₃O group forms a hydrogen bond with GLN369. Moreover, the NH groups on the pyrazole ring and the side chain of 6e were also found to form hydrogen bonds with TYR159 and GLU230, respectively.

Figure 2.

Binding mode of compound 6e (yellow) within the active pocket of PDE4 (PDB ID: 4WCU). Hydrogen bonds are represented by dashed lines (green).

Considering the importance of behavioral studies, two behavioral models, that is, the forced swim test (FST) and the sucrose preference test (SPT), were used to evaluate the antidepressant activities of compound 6e using chronic-stress-induced mice (CUS). In the forced swimming test, the mouse was subjected to a short-term and inevitable pressure to imitate the condition of despair, and this has been shown to have a good prediction effect. The effect of immobility in the forced swim test results are shown in Figure 3(a). Chronic unpredictable stress induced a significant increase in immobility time in forced swimming tests (p < 0.01). Significant decreases in immobility time were observed on administration of compound 6e (5 mg kg⁻¹ and 10 mg kg⁻¹) compared to vehicle-treated stressed groups [F_4,32 = 15.36, p < 0.01]. Mice treated with Flu also exhibited significant antidepressant-like effects (p < 0.05). In the sucrose preference test (Figure 3(b)), the consumption of sugar water was significantly reduced in the model group (p < 0.001) compared with the blank group. The large and medium dose compound 6e groups significantly increased their consumption of sugar water in contrast to the model group [F_4,36 = 11.72, p < 0.01]. The Flu group exhibited similar effects at doses of 5 mg kg⁻¹. Figure 3(c) shows that compound 6e did not affect the locomotor activity at all doses that produced antidepressant-like effects, indicating its specific antidepressant-like effects. Overall, compound 6e was effective in both sucrose preference and forced swimming tests. Since the hydrolysis of cAMP is involved in the antidepressant and anxiolytic activities of PDE4 inhibitors, the effects of compound 6e on the accumulation of cAMP in the hippocampus of the groups that produced antidepressant-like effects were further evaluated by using an enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the cAMP levels were significantly decreased in the hippocampus of the model group (Figure 3(d)). However, the level of cAMP in the drug groups was restored at the doses of 5 and 10 mg kg⁻¹. As a positive drug, similar effects were observed on treatment with rolipram at 10 mg kg⁻¹ (p < 0.05). The present results indicate that compound 6e produces antidepressant-like activities by activation of cAMP-dependent signaling.

Figure 3.

The antidepressant-like effects of compound 6e in mice. Mice were treated with vehicle, compound 6e, fluoxetine, and rolipram 30 min before being subjected to (a) SPT and (b) FST. (c) Mice were treated with various doses of compound 6e 30 min before the locomotor activity test. (d) The effects of compound 6e on hippocampal cAMP reduced levels. Results are expressed as the mean ± SEM (n = 9-10). ^##p < 0.01 and ^###p < 0.001 versus vehicle-treated control group. *p < 0.05 and **p < 0.01 versus the model group. K: Vehicle; M: CUS model; B2.5: 2.5 mg kg⁻¹ compound 6e; B5: 5 mg kg⁻¹ compound 6e; B10: 10 mg kg⁻¹ compound 6e; Flu: fluoxetine; Rol: rolipram.

Conclusion

In summary, a series of resveratrol derivatives have been synthesized, with most of the products being potent inhibitors of PDE4. Compound 6e showed the most potent inhibitory activity with an IC₅₀ value of 0.09 μM and good selectivities over PDE1, 2, 5, 9, and 10. Furthermore, an in vivo study suggested that compound 6e exhibited antidepressant-like activities in sugar water consumption and forced swimming tests, which may be relevant to the activation of cAMP-dependent signaling. Thus, our studies showed that a PDE4 inhibitor derived from resveratrol may be useful for the treatment of depression and subsequent efforts on further optimization of compound 6e are expected to lead to more potent and selective PDE4 inhibitors with antidepressant activities.

Experimental

Materials and reagents

All chemical reagents (analytical grade) were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China) and were used as received without any purification. Reactions were monitored by flash column chromatography on silica gel (Merck Kieselgel 60, 230-400 mesh).

¹ H NMR and ¹³C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on a Bruker spectrometer in CDCl₃ or DMSO-d₆. MS spectra were acquired on a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Melting points were obtained using a Boetius micro melting point apparatus.

Synthesis of 3,5-diarylpyrazoles 6a–f

(E)-1-(4-(Diethoxymethyl)phenyl)-3-(3,5-dimethoxyphenyl)prop-2-en-1-one (3)

3,5-Dimethoxybenzaldehyde (1) (2.22 g, 10 mmol) and 4′-(diethoxymethyl)acetophenone (2) (1.67 g, 10 mmol) were dissolved in 30 mL of CH₃OH and treated with 4.67 mL of NaOH solution (3 M) at 0 °C. The mixture was then stirred for 12 h at room temperature. The reaction mixture was cooled and allowed to stay overnight, during which time a solid precipitated and was filtered. The crude product was used directly in the following step. To obtain a pure sample, following completion of the reaction, the pH of the mixture was adjusted to 7.0 with HCl (5 M). The aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic phases were dried (MgSO₄) and purified by column chromatography to afford compound 3 as a white solid (2.69 g, 73%; m.p. 129.1–130.6 °C).

¹ H NMR (400 MHz, CDCl₃): δ = 7.53 (d, J = 8.6 Hz, 2H), 7.36 (m, 3H), 6.93–6.82 (m, 3H), 6.78 (m, 1H), 6.11 (s, 1H), 3.92 (s, 6H), 3.81 (q, J = 7.2 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 188.16, 159.87 (2C), 148.23, 142.65, 139.43, 135.36, 129.72 (2C), 125.37 (2C), 122.89, 106.67 (2C), 109.32, 100.27, 61.13 (2C), 56.35 (2C), 19.57 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₇O₅: 371.1858; found: 371.1863.

5-(4-(Diethoxymethyl)phenyl)-3-(3,5-dimethoxyphenyl)-1H-pyrazole (4)

A solution of compound 3 (3.70 g, 10 mmol), p-toluenesulfonyl hydrazide (1.86 g, 10 mmol), and iodine (0.05 g, 0.2 mmol) in 40 mL of anhydrous CH₂OH was heated to 75 °C for 10 min and then treated with anhydrous K₂CO₃ (0.69 g, 0.5 mmol). The resulting mixture was stirred for 2 h under air. The solvent was removed under vacuum, and the residue was partitioned between EtOAc and water. The organic layer was then dried over MgSO₄ and concentrated under reduced pressure. Flash chromatography (EtOAc/petroleum ether) furnished compound 4 as a white solid (3.17 g, 83%; m.p. 263.5–264.3 °C).

¹ H NMR (400 MHz, DMSO-d₆): δ = 7.55 (d, J = 8.7 Hz, 2H), 7.23 (m, 2H), 7.13 (d, J = 8.7 Hz, 2H), 6.92 (m, 1H), 6.78 (m, 1H), 6.12 (s, 1H), 3.91 (s, 6H), 3.83 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 158.97 (2C), 147.65, 145.71, 137.25, 135.13, 132.96, 129.12, 127.71 (2C), 106.82 (2C), 109.35, 100.68, 99.29, 58.63, 53.49 (2C), 37.86 (2C), 21.71 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₇N₂O₄: 383.1971; found: 383.1987.

4-(3-(3,5-Dimethoxyphenyl)-1H-pyrazol-5-yl)benzaldehyde (5)

Compound 4 (3.82 g, 10 mmol) was added to THF (30 mL) in a round-bottom flask, followed by the addition of 5 mL of HCl (5 M). The mixture was stirred at room temperature for 2 h and then partitioned between EtOAc and water. The organic layer was dried over MgSO₄ and concentrated under reduced pressure. Flash chromatography (EtOAc/petroleum ether) furnished compound 5 as a yellow solid (2.85 g, 93%: m.p. 271.3–271.9 °C).

¹ H NMR (400 MHz, DMSO-d₆): δ = 9.92 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.13-7.11 (m, 2H), 6.97 (m, 1H), 6.85 (m, 1H), 3.89 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 191.32, 158.68 (2C), 147.12, 145.39, 139.16, 137.56, 135.13, 130.92 (2C), 127.13 (2C), 106.65 (2C), 100.68, 99.32, 53.16 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₇N₂O₃: 309.1239; found: 309.1226.

Synthesis of compounds 6a–f

Compound 5 (10 mmol) was dissolved in 50 mL of ethanol, and the corresponding amine (R¹NH₂) was added. The reaction mixture was refluxed for 30 min and then cooled to room temperature. NaBH₄ (5 mmol) was then added to the solution in portions, with stirring at room temperature for 2 h. The mixture was evaporated under vacuum and the residue was dissolved in EtOAc (50 mL). The solution was washed with saturated NaCl solution and water, dried over anhydrous sodium sulfate, and evaporated. Purification by silica gel column chromatography afforded pure products 6.

N-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)cyclopentanamine (6a): White powder, 59% yield; m.p. 251.1–251.7 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 7.52 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 16.2 Hz, 1H), 7.1l (d, J = 16.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 2H), 6.81 (s, 1H), 6.47 (s, 1H), 3.92 (s, 3H), 3.80 (s, 3H), 3.77 (s, 2H), 3.06 (m, 1H), 1.71 (m, 2H), 1.61 (m, 2H), 1.45 (m, 2H), 1.38 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 158.23 (2C), 149.31, 145.76, 140.82, 135.23, 131.17, 128.87 (2C), 126.11 (2C), 106.98 (2C), 100.27, 98.21, 58.63, 53.49 (2C), 51.36, 37.86 (2C), 21.71 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈N₃O₂: 378.2182; found: 378.2173.

N-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)cyclopentanamine (6b): White powder, 64% yield; m.p. 256.7–257.5 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 7.51 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 16.3 Hz, 1H), 7.11 (d, J = 16.3 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 6.80 (m, 1H), 6.47(m, 1H), 3.80 (s, 3H), 3.77(m, 3H), 3.76(s, 2H), 2.42 (m, 1H), 1.84 (m, 2H), 1.63(m, 2H), 1.05–1.22 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 159.08 (2C), 149.77, 144.36, 140.09, 136.21, 131.19, 129.65 (2C), 126.82 (2C), 106.05 (2C), 101.18, 99.06, 59.27, 53.35 (2C), 51.72, 32.12 (2C), 22.33 (2C), 20.15. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₀N₃O₂: 392.2338; found: 392.2351.

4-(2-((4-(3-(3,5-Dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)amino)ethyl)phenol (6c): White powder, 55% yield; m.p. 306.3–307.1 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.32 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 16.2 Hz, 1H), 7.10 (d, J = 16.2 Hz, 1H), 6.98–6.95 (m, 3H), 6.83–6.78 (m, 2H), 6.61 (d, J = 8.6 Hz, 2H), 6.45 (m, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.75 (s, 2H), 2.71 (m, 2H), 2.59 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 158.76 (2C), 156.33, 149.01, 144.12, 141.27, 140.19, 136.82, 134.76 (2C), 131.01, 129.32 (2C), 125.69 (2C), 112.95 (2C), 106.76 (2C), 101.53, 98.22, 59.07, 52.28 (2C), 47.22, 36.91. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₂₈N₃O₃: 430.2131; found: 430.2147.

N-benzyl-1-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)phenyl)methanamine (6d): White powder, 60% yield; m.p. 281.2–281.9 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 7.52–7.58 (m, 4H), 7.45–7.48 (m, 3H), 7.18 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 6.89 (m, 1H), 6.55 (m, 1H), 4.23 (m, 2H), 4.11 (m, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.77 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.01 (2C), 156.67, 149.32, 145.11, 140.76, 139.92, 134.79 (2C), 130.29, 128.35 (2C), 127.22 (2C), 125.36, 113.37 (2C), 106.91 (2C), 101.32, 99.16, 59.25, 58.46, 51.78 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₅H₂₆N₃O₂: 400.2025; found: 400.2013.

N-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)-2-morpholinoethan-1-amine (6e): White powder, 71% yield; m.p. 269.5–270.2 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 7.55 (d, J = 8.7 Hz, 2H),7.32 (m, 1H), 7.13 (m, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.82 (m, 1H), 6.48 (m, 1H), 3.80 (s, 3H), 3.81 (s, 3H), 3.77 (m, 2H), 3.40 (m, 4H), 2.62 (m, 2H), 2.35 (m, 2H), 2.23 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 159.73 (2C), 150.12, 143.31, 140.07, 136.53, 130.86, 129.82 (2C), 127.37 (2C), 106.43 (2C), 101.29, 99.05, 62.12 (2C), 59.76, 54.17 (2C), 52.03 (2C), 51.12, 49.57. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₁N₄O₃: 423.2396; found: 423.2381.

N-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)benzyl)-1-(furan-2-yl)methanamine (6f): White powder, 71% yield; m.p. 276.2–276.8 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 7.58 (m, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.25 (m, 1H), 7.09 (m, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.82 (m, 1H), 6.48 (m, 1H), 6.52 (m, 1H), 6.28 (m, 1H), 3.80 (s, 6H), 3.73(m, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.33 (2C), 157.89, 149.71, 147.08, 145.39, 142.66, 138.17, 134.05 (2C), 131.22 (2C), 125.31, 112.95, 110.67, 106.33 (2C), 101.68, 98.35, 59.51, 57.32, 52.03 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₄N₃O₃: 390.1818; found: 390.1829.

Synthesis of compounds 10a–g

Synthesis of intermediates 8a–g

A solution of p-hydroxyacetophenone (1.36 g, 10 mmol), the corresponding bromide (R₂Br) (15 mmol), and K₂CO₃ (0.69 g, 5 mmol) in 50 mL of dry DMF was stirred at 80 °C for 5–6 h. After completion of reaction, the solution was added to a mixture of ice and H₂O, and the resulting yellow solution was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with H₂O, dried (Na₂SO₄), filtered, evaporated, and recrystallized from EtOAc.

1-(4-Ethoxyphenyl)ethan-1-one (8a): ³² White powder, 89% yield; m.p. 37.7–38.5 °C. ¹H NMR (400 MHz, CDCl₃, TMS): δ = 7.79 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H), 3.94 (q, J = 7.2 Hz, 2H), 2.41 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). ESI-MS: 165.3 [M+1]⁺.

1-(4-(1-Hydroxy-2-methylpropoxy)phenyl)ethan-1-one (8b): White solid, 76% yield; m.p. 82.1–82.6 °C. ¹H NMR (400 MHz, CDCl₃, TMS): δ = 7.73 (d, J = 8.7 Hz, 2H), 6.55 (d, J = 8.7 Hz, 2H), 4.45 (m, 1H), 1.81 (m, 1H), 1.76 (m, 1H), 2.43 (s, 3H), 1.12 (d, J = 6.5 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 196.31, 162.52, 130.36 (2C), 127.87, 115.36 (2C), 107.55, 36.27, 26.87, 19.12 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₂H₁₇O₃: 209.1178; found: 209.1173.

1-(4-(2-Methoxyethoxy)phenyl)ethan-1-one (8c): ³³ White solid, 92% yield; m.p. 63.7–64.2 °C. ¹H NMR (CDCl₃, 400 MHz): δ = (pm): 7.92 (d, 2H, J = 9.2 Hz), 6.95 (d, 2H, J = 8.8 Hz), 4.17 (t, 2H, J = 4.4 Hz), 3.76 (t, 2H, J = 4.4 Hz), 3.44 (s, 3H), 2.54 (s, 3H). ESI-MS: 195.1 [M+1]⁺.

1-(4-(4-Methoxybutoxy)phenyl)ethan-1-one (8d): White solid, 89% yield; m.p. 78.3–78.9 °C. ¹H NMR (CDCl₃, 400 MHz): δ = (pm): 7.93 (d, 2H, J = 8.8 Hz), 6.91 (d, 2H, J = 8.8 Hz), 4.16 (t, 2H, J = 4.4 Hz), 3.77 (t, 2H, J = 4.4 Hz), 3.42 (s, 3H), 2.54 (s, 3H), 1.76 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 196.72, 162.56, 130.87 (2C), 127.13, 116.13 (2C), 68.53, 65.22, 57.12, 26.81, 23.13, 21.59. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₃H₁₉O₃: 223.1334; found: 223.1329.

1-(4-(Pent-4-en-1-yloxy)phenyl)ethan-1-one (8e): ³⁴ Colorless oil, 83% yield; ¹H NMR (400 MHz, CDCl₃): δ = 7.91 (d, 2H, J = 8.9 Hz), 6.92 (d, 2H, J = 8.9 Hz), 5.93–5.89 (m, 1H), 5.18-4.92 (m, 2H), 4.07 (t, 2H, J = 6.4 Hz), 2.55 (s, 3H), 2.41–2.17 (m, 2H), 2.09–1.77 (m, 2H). ESI-MS: 205.3 [M+1]⁺.

2-(4-Acetylphenoxy)-N-benzylacetamide (8f): Pale solid, 72% yield; m.p. 149.1–149.6 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.76 (d, 2H, J = 8.7 Hz), 7.43–7.31 (m, 3H), 6.94 (m, 3H), 6.87 (m, 1H), 4.63–4.59 (m, 4H), 2.51 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 196.67, 168.36, 157.28, 134.77, 128.31 (2C), 127.27 (2C), 126.79, 126.35 (2C), 123.17, 113.66 (2C), 68.21, 49.15, 26.19. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₇H₁₈NO₃: 284.1287; found: 284.1290.

1-(4-((3,5-Difluorobenzyl)oxy)phenyl)ethan-1-one (8g): ³⁵ White solid, 83% yield; m.p. 127.3–128.1 °C. ¹H NMR (400 MHz, CDCl₃, TMS): δ = 7.82 (d, 2H, J = 8.8 Hz), 6.93 (d, J = 8.8 Hz, 2H), 6.85-6.81 (m, 2H), 6.73 (m 1H), 4.56 (s, 2H), 2.43 (s, 3H). ESI-MS: 263.7 [M+1]⁺.

Synthesis of chalcones 9

Compound 8 (10 mmol) and 3,5-dimethoxybenzaldehyde (1.67 g, 10 mmol) were dissolved in 30 mL of CH₃OH, followed by the addition of a few drops of concentrated H₂SO₄. The mixture was then heated at reflux for 5 h. The solvent was cooled, and the mixture allowed to stay overnight. The precipitated solid was filtered and the crude product was used directly in the following step.

Synthesis of pyrazoles 10a–g

A solution of compound 9 (10 mmol), p-toluenesulfonyl hydrazide (1.86 g, 10 mmol), and iodine (0.05 g, 0.2 mmol) in 40 mL of anhydrous C₂H₅OH was heated at 75 °C for 10 min and then treated with anhydrous K₂CO₃ (0.69 g, 0.5 mmol). Then, reaction mixture was stirred for 2 h under an air atmosphere. The solvent was removed under vacuum and the residue was partitioned between EtOAc and water. The organic layer was dried over MgSO₄ and concentrated under reduced pressure. Flash column chromatography (EtOAc/petroleum ether) furnished the desired pyrazole 10a–g.

3-(3,5-Dimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-pyrazole (10a): White solid, 77% yield; m.p. 222.6–223.7 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.55–7.52 (m, 2H), 7.40–7.30 (m, 3H), 6.60 (s, 1H), 6.52–6.49 (m, 2H), 3.82 (s, 6H), 3.80 (m, 2H), 1.31 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 161.82 (2C), 150.19, 146.71, 140.32, 136.15, 130.03, 127.93 (2C), 126.15 (2C), 113.59 (2C), 101.76, 99.32, 60.03, 53.66 (2C), 20.61. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₂₁N₂O₃: 325.1552; found: 325.1563.

1-(4-(3-(3,5-Dimethoxyphenyl)-1H-pyrazol-5-yl)phenoxy)-2-methylpropan-1-ol (10b): White solid, 62% yield; m.p. 268.5–269.1 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.64 (m, 2H), 7.40–7.28 (m, 3H), 6.74 (s, 1H), 6.51 (m, 1H), 6.42 (m, 1H), 4.46 (m, 1H), 3.82 (s, 6H), 1.83 (m, 1H), 1.81–1.71 (m, 1H), 0.99 (d, J = 6.5 Hz, 3H), 0.72 (d, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.52 (2C), 150.33, 145.02, 141.91, 136.63, 130.25, 127.76 (2C), 125.83 (2C), 113.72 (2C), 107.91, 101.39, 98.26, 53.17 (2C), 35.39, 19.61(2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₂₅N₂O₄: 369.1814; found: 369.1822.

3-(3,5-Dimethoxyphenyl)-5-(4-(2-methoxyethoxy)phenyl)-1H-pyrazole (10c): White solid, 70% yield; m.p. 236.2–236.8 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.46 (d, J = 8.8 Hz, 2H), 7.06 (m, 1H), 6.96–6.93 (m, 2H), 6.69–6.65 (m, 2H), 6.41 (m, 1H), 4.17 (m, 2H), 3.85 (s, 6H), 3.77 (m, 2H), 3.49 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.73 (2C), 150.11, 146.56, 141.36, 136.53, 131.57, 128.22 (2C), 126.82 (2C), 113.49 (2C), 101.27, 98.96, 69.35, 65.12, 58.21, 53.66 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₃N₂O₄: 355.1658; found: 355.1647.

3-(3,5-Dimethoxyphenyl)-5-(4-(4-methoxybutoxy)phenyl)-1H-pyrazole (10d): White solid, 80% yield; m.p. 247.6–248.9 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.46 (d, J = 8.8 Hz, 2H), 7.28 (s, 1H), 6.97–6.85 (m, 2H), 6.68–6.66 (m, 2H), 6.40 (m, 1H), 4.03 (m, 2H), 3.85 (s, 6H), 3.48 (m, 2H), 3.38 (s, 3H), 1.95–1.85 (m, 2H), 1.85–1.73 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.11 (2C), 150.75, 147.21, 142.33, 136.09, 131.36, 129.01 (2C), 127.35 (2C), 113.79 (2C), 101.16, 99.49, 68.57, 65.03, 57.19, 53.63 (2C), 23.16, 21.63. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₇N₂O₄: 383.1971; found: 383.1978.

3-(3,5-Dimethoxyphenyl)-5-(4-(pent-4-en-1-yloxy)phenyl)-1H-pyrazole (10e): White solid, 77% yield; m.p. 201.2–201.7 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.47 (d, J = 8.8 Hz, 2H), 7.08 (m, 1H), 6.98–6.89 (m, 2H), 6.69 (s, 2H), 6.42 (s, 1H), 5.91 (m, 1H), 5.09 (m, 2H), 4.02 (m, 2H), 3.86 (s, 6H), 2.29 (m, 2H), 1.97-1.87 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 160.32 (2C), 151.96, 146.17, 142.66, 136.82, 135.01, 131.79, 128.83 (2C), 127.05 (2C), 115.26, 113.71 (2C), 100.32, 98.75, 68.19, 53.63 (2C), 32.62, 22.73. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₅N₂O₃: 365.1865; found: 365.1852.

N-Benzyl-2-(4-(3-(3,5-dimethoxyphenyl)-1H-pyrazol-5-yl)phenoxy)acetamide (10f): White solid, 58% yield; m.p. 292.8–293.5 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.48 (d, J = 8.8 Hz, 2H), 7.40-7.35 (m, 3H), 7.06 (m, 1H), 6.96–6.88 (m, 4H), 6.69–6.67 (m, 2H), 6.41 (m, 1H), 4.62-4.57 (m, 4H), 3.85 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 159.68 (2C), 157.22, 149.36, 146.93, 140.01, 138.89, 134.72, 132.01, 128.35 (2C), 127.22 (2C), 125.97 (2C), 123.36, 113.31 (2C), 107.06 (2C), 101.39, 99.53, 68.53, 52.66 (2C), 49.13. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₂₆N₃O₄: 444.1923; found: 444.1931.

5-(4-((3,5-Difluorobenzyl)oxy)phenyl)-3-(3,5-dimethoxyphenyl)-1H-pyrazole (10g): White solid, 85% yield; m.p. 263.2–263.9 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.68 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.49-7.47 (m, 2H), 7.05–6.96 (m, 3H), 6.67 (m, 1H), 6.41 (m, 1H), 5.18 (s, 2H), 3.86 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ = 162.71 (2C), 160.36 (2C), 159.65, 149.36, 145.93, 141.17, 139.98, 131.19, 128.31 (2C), 126.86 (2C), 125.01, 112.13 (2C), 106.07 (2C), 101.78, 98.25, 69.32, 52.83 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₁F₂N₂O₃: 423.1520; found: 423.1533.

Enzymatic assay

All the enzymatic activities of PDEs were measured using ³H-cGMP or ³H-cAMP as the substrate. PDEs were first incubated with a buffer containing 50 mmol L⁻¹ of Tris-HCl, pH 7.8, 10 mmol L⁻¹ MgCl₂, and ³H-cAMP or ³H-cGMP (40,000 cpm assay⁻¹) at 24 °C for 30 min. The reaction was subsequently terminated by the addition of ZnSO₄ and Ba(OH)₂. The activity of the test compound was assessed by measuring the amount of ³H-cGMP or ³H-cAMP resulting from enzyme cleavage. The reaction product ³H-AMP or ³H-GMP was precipitated by adding BaSO₄, while unreacted ³H-cAMP or ³H-cGMP remained in the supernatant. Radioactivity in the supernatant was measured in 2.5 mL of Ultima Gold liquid scintillation. The IC₅₀ value of each inhibitor was measured at eight or more different concentrations of the inhibitor in the presence of ³H-cAMP or ³H-cGMP. Each measurement was repeated at least three times. The IC₅₀ values were calculated by a nonlinear regression method using GraphPad Prism 5.0 Software.

Animals and treatment

Male ICR mice, weighing between 22 and 25 g, were obtained from the Animal Center at Changzhou University. All experiments were carried out according to the National Institute of Health Guide for Care and Use of Laboratory Animals (1996) and were approved by the Institutional Animal Care and Use Committee of Changzhou University (NO. Y20220057). Fluoxetine and rolipram were purchased from Sigma Chemical Co. (USA). The tested compounds and rolipram were dissolved in saline containing 5% DMSO, 5% solutol, and 90% saline. The positive control drug fluoxetine was dissolved in saline. Mice were subjected to intragastric administration (compound 6e: 2.5, 5.0, and 10 mg kg⁻¹) or intraperitoneal injections (fluoxetine: 5 mg kg⁻¹ and rolipram: 10 mg kg⁻¹) based on a volume of 10 mL kg⁻¹ body weight.

The chronic unpredictable stress procedure (CUS) was carried out as described previously with minor modifications. ³⁶ First, mice were housed individually for one day and subjected to chronic unpredictable stress for 10 days. Then, the animals were exposed to two different stressors each day including 2-h restraint, damp bedding, tilted cage, 10-min forced swimming and overnight illumination. All procedures were performed in isolated rooms adjacent to the housing room. Control mice were individually housed for the same period of time and were handled daily for 30 s in the housing room without being stressed.

After the CUS procedure, the sucrose preference test was assessed on the following day and then a forced swimming test was carried out the next day. Mice were treated with vehicle, compound 6e, and fluoxetine 30 min before being subjected to SPT and FST. The drugs and vehicle were administered (i.g. or i.p.) once a day from the first day and continued until the final day. After behavioral testing, all the animals were used for biochemical analysis.

Behavioral tests

Sucrose preference test

The sucrose preference test was performed according to the previous description with minor modifications. ³⁷ Before the procedure, animals were deprived of food and water for 14 h. Each animal was first habituated to drink from two bottles each filled with tap water for 24 h. Then, they were each given free access within another 24 h to two bottles, one containing a sucrose (1%) solution and the other tap water. The position of the bottles (right or left) was reversed 12 h later to avoid any lateralization bias on the part of the animal. Mice were not deprived of food during this period. Finally, the bottles were weighed and recorded before and after the sucrose preference test. The sucrose preference was calculated as the ratio of the consumed sucrose solution relative to the total amount of liquid consumed.

Forced swimming test

According to the previous description, animals were individually placed in glass cylinders (25 cm height, 10 cm diameter) containing 10 cm depth of water at 24 ± 1 °C for 6 min, allowing for free swimming. There was a 15-min pretest followed 24 h later by a 5-min test. A mouse was determined to be immobile when there were only small movements necessary to keep its head above water. The immobility time was recorded during the 5-min testing period.

Locomotor activity test

The locomotor activity (LA) test was assessed using an open-field chamber according to a previous procedure. ³⁸ The floor of the chamber was divided into 16 identical squares. Mice were individually placed in the center of the chamber and allowed to acclimatize for 15 min. Their paws were contacted or disconnected from the active bars which can produce random configurations. The locomotor counts of mice were measured by counting the number of line crossings over 10 min.

cAMP concentration assay

The mice brain tissues were dissected and stored at −80 °C until analysis after the mice had been sacrificed. All the tissues were homogenized in ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride and centrifuged at 14,000 rpm for 20 min at 4 °C. The cAMP levels were measured using the mouse cAMP ELISA kit (ab133051, Biocompare, South San Francisco, CA) following the manufacturer’s instructions.

Docking simulations

Molecular docking of compound 6e into the three-dimensional X-ray structure of PDE4 (PDB code: 4WCU) was carried out using the CDOCKER protocol of Discovery Studio 2019.