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Abstract

PDE4 is a potential target for treating mental diseases including depression, Alzheimer’s disease and schizophrenia involving in the hydrolysis of cAMP. Curcumin is well-known for its potential as a neuroprotective compound against depression. It was able to inhibit PDE4 with IC₅₀ value in the range of 10 to 20 μM with low selectivity. However, curcumin itself cannot be used clinically because of its poor drug-like properties. Herein, a series of new curcumin derivatives were designed and synthesized. Their PDE4 inhibitory activities and antidepressant effects in vivo were also evaluated. Consequently, most of these curcumin derivatives demonstrated good inhibitory activities against PDE4. Furthermore, compound 4e exhibited the most potent activity at nanomolar level (IC₅₀ value: 23 nM). It also could improve depression-like behaviors induced by chronic stress including sugar forced swimming and water consumption tests in vivo.

Keywords

behavior tests curcumin depression diarylpyrazoles PDE4 inhibitors

A series of novel curcumin derivatives were synthesized and subjected to pharmacological evaluation as PDE4 inhibitors. Consequently, compound 4e exhibited high inhibitory activity against PDE4 and good selectivities over PDE1, 2, 5, 9, and 10 as well as antidepressant-like activities in behavioral tests in vivo. Therefore, curcumin may be a valuable lead compound for discovering good PDE4 inhibitors for treating depression.

Introduction

Depression is a severe disorder by high rates of suicide and its clinical manifestations include mainly anhedonia, lack of sleep and motivation, or mental torture.^1,2 Statistics shows that the mortality rate of depression ranks among the top in various diseases, and it also is the second largest contributor to medical burden all over the world.^3
–5 The unknown etiopathology of depression partly cause to its complexity in pharmacotherapy. Presently, clinical antidepressants are developed based on the theoretical hypothesis of serotonin and noradrenaline function in the brain. However, only a small portion of patients could be relieved in response to these drugs.⁶ Novel antidepressants with different mechanisms may provide a novel perspective for new drug discovery. Therefore, the development of anti-depression agents with novel mechanism is still urgent clinically.

As a second messenger for mediating cellular responses to neurotransmitters in the brain, cAMP has been shown to be a vital target for the treatment of mental diseases.^7
–9 Phosphodiesterase 4 (PDE4), an enzyme catalyzing specifically the hydrolysis of cAMP, can regulate the cAMP/PKA/CREB pathway. Besides, PDE4 also plays a key role in modulating cAMP-mediated dopamine signaling in the postsynaptic region.¹⁰ In fact, some PDE4 inhibitors including rolipram and roflumilast have been explored for treating neuropsychiatric disorders.^11
–16 An early developed PDE4 inhibitor, rolipram, could increase cAMP signaling and exhibit antidepressant- and anxiolytic-like effects in vivo.¹⁷

It has been known that natural compounds are promising resources for depression therapy.¹⁸ Curcumin (Figure 1) is the active chemical constituent in turmeric (Curcuma longa), has been used as a common traditional Chinese medicine and food additive. It also has been well known for its various biological activities such as anti-inflammatory and antioxidant properties.¹⁹ Previous studies demonstrated that curcumin exhibits significant effects in depression-like behaviors induced by stress, but for non-induced depression, its effect is unknown.^20
–22 Interestingly, curcumin was proved to be a nonselective PDE inhibitor, inhibiting PDE1-4 with IC₅₀ values between 10 and 20 μM, and PDE5 with an IC₅₀ value of 35 μM.²³ For example, curcumin exhibited anti-angiogenic antitumour activities in vitro and in vivo through PDE2/4 inhibition. Abusnina et al. have reported that curcumin could inhibit melanoma cell proliferation by targeting PDE1. However, currently, there are no reports about curcumin derivatives as PDE4 inhibitors and their antidepressant effects.^24,25 Furthermore, the clinical application of curcumin is limited because of its poor drug-like properties such as low water-solubility and bioavailability. In order to overcome this, many of its derivatives have been reported until now, of which pyrazole derivatives of curcumin demonstrated many interesting bioactivity such as enhanced memory, antioxidant, anti-mycobacterial activities.^26
–28 Therefore, we wonder if pyrazole derivatives of curcumin can be attractive leading compounds as PDE4 inhibitors for treating depression. Herein, we constructed a series of curcumin analogue by introducing a pyrazole group in its structure and evaluated their antidepressant activities in animal models.

Figure 1.

The structures of curcumin.

Results and discussion

The preparation of curcumin derivatives 3a–3i and 4a–4i was prepared easily as depicted in scheme 1. Pyrazole derivatives of curcumin (3a–3i) were prepared by heating curcumin for 8 h with phenylhydrazine, acetylhydrazine, hydroxyethyl hydrazine, ethyl hydrazinecarboxylate and various substituted phenylhydrazine in acetic acid in good yield. Their hydrogenated analogues 4a–4i were synthesized by two steps. First, the two olefinic bonds of curcumin were selectively hydrogenated catalyzed by palladium carbon. Next, compound 3 were reacted with corresponding hydrazines to form the compounds 4a–4i. Most of these curcumin derivatives are first reported except for compounds 3a, 3c, 3e, and 3g.

Scheme 1.

Synthesis of 3a–3i and 4a–4i. Reagents and conditions: (a) H₂, 10% Pd/C, C₂H₅OH, rt, 12 h, 62.9%; (b) RNHNH₂, NaHCO₃, C₂H₅OH, reflux, 12 h.

All of the curcumin derivatives were evaluated for the activities of inhibiting PDE4, and the results are shown in Table 1. Curcumin and rolipram (a commercially available PDE4 inhibitor) were applied as standard compounds for comparison. PDE4 screening and other PDE evaluations were carried out using an activity assay based on the hydrolysis of cyclic AMP and cyclic GMP. We initially focused on introducing various substituted pyrazol rings in the middle of curcumin molecule while other structural parts keep unchanged. The results showed that most of this series of compounds demonstrated better inhibitory activities than curcumin except for 3b and 3d. The data showed that the substituent at pyrazol ring has an obvious influence on their inhibitory activity against PDE4. Especially, when R group is substituted with a benzene ring, the compounds exhibited more potent inhibitory activities. For example, compounds 3e-3i exhibited potent activities (IC₅₀ < 10 μM). In contrast, the products 3a-3d had lower PDE4 inhibitory activities. To improve the molecular flexibility, the corresponding hydrogenated products (4a-4i) were obtained, most of which demonstrated better IC₅₀ values compared to the parent compound and therefore this modification seems to be favorable for their activities. Interestingly, for this series of derivatives increased activities were also observed when the pyrazol ring was substituted by various aromatic groups (4a and 4e-4f). In contrast, the compounds 4b-4d showed lower inhibitory activities toward PDE4 (IC₅₀ > 5 μM). Furthermore, the best activity is obtained when the aromatic group is methoxyphenyl substituted benzene ring (4e and 4f: 23 and 97 nM, respectively), and both meta or para positions are tolerated. Then, we further investigated the inhibitory activities of compound 4e against other PDE families, including PDE1, 2, 5, 9, and 10 to evaluate its PDE selectivity (Table 2). The results showed compound 4e display > 4000-fold selectivity with IC₅₀ > 100 μM against PDE1, PDE2, and PDE9. This compound also inhibited moderately PDE5 and PDE10 with IC50 values of 21.33 and 18.42 μM, respectively. These data revealed that compound 4e, as a PDE4 inhibitor, exhibited good selectivity against other main PDEs.

Table 1.

PDE4 inhibitory activities of curcumin derivatives.

Compound	Yield	IC50 (μM)^a
3a	71.6%	16.36 ± 1.85
3b	78.7%	23.31 ± 3.06
3c	53.1%	13.17 ± 1.08
3d	86.5%	35.67 ± 3.79
3e	80.3%	3.67 ± 0.25
3f	73.8%	8.76 ± 0.92
3g	59.6%	0.73 ± 0.08
3h	76.3%	7.56 ± 0.62
3i	75.7%	0.93 ± 0.82
4a	87.6%	2.35 ± 0.37
4b	91.2%	8.13 ± 0.92
4c	56.6%	7.55 ± 0.67
4d	62.3%	5.21 ±0.06
4e	71.6%	0.023 ± 0.003
4f	65.5%	0.097 ± 0.008
4g	75.2%	0.66 ± 0.05
4h	82.3%	1.21 ±0.16
4i	66.2%	0.19 ± 0.02
Curcumin		19.82 ± 2.31
Rolipram		0.23 ± 0.01

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

Table 2.

PDE selectivity of compound 4e.

Compound	PDE IC₅₀ (μM)^a
Compound	PDE4	PDE1	PDE2	PDE5	PDE9	PDE10
6e	0.023 ± 0.003	> 100	> 100	21.33 ± 3.7	> 100	18.42 ± 2.16

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

Compound 4e was docked into the binding pocket of PDE4 (PDB ID: 4WCU) using Discovery Studio 2019 (Figure 2). The docking results showed that one 4-hydroxy-3-methoxyphenethyl moiety of 4e was accommodated by the hydrophobic pocket consisting of Phe372 and Ile336/Phe340, and the oxygen atom of the hydroxyl formed an H-bond with residue Gln433, and the hydrogen atom formed another H-bond with residue Met357. In addition, its benzene ring interacts with the phenyl ring of Phe372 by an interaction of π-π stacking. The CH₃O group of other 4-hydroxy-3-methoxyphenethyl moiety forms moderate hydrophobic interaction with the His160 and His204. Notably, the 4-CH₃O substituted benzene group on the pyrazole ring also exhibited hydrophobic interaction with Ile336 and Phe372, indicating this kind of N-substitution may be favorable for inhibitory activity. Unlike this binding model, curcumin only formed one hydrogen bond between its oxygen atom of carbonyl and Gln369, which may explain the much better inhibitory of compound 4e compared to that of curcumin.

Figure 2.

Binding modes of compound 4e (yellow) and curcumin (red) with PDE4 (PDB number: 4WCU). Hydrogen bonds are represented by dashed lines (green).

Encouraged by its potent PDE4 inhibition activity, compound 4e was further evaluated antidepressant activity in vivo using sucrose preference and forced swimming tests in chronic-stress-induced mice (CUS). A classic antidepressant, fluoxetine, was used as the positive control. The results in Figure 3(a) showed that the consumption of sugar water in the model group was obviously lower than that in the blank group (p < 0.001). And the consumption of sugar water increased significantly in both the large dose 4e group (12 mg/kg) and Flu (5 mg/kg) group [F_4,32 = 16.73, p < 0.01]. Medium consumption increase of sugar water was observed in 4e group (6 mg/kg), but it did not show statistical significance. In forced swimming test, a significant increase in immobility time was induced by chronic unpredictable stress (p < 0.01), as indicated in Figure 3(b). This increase was reversed by administering 4e at a dose of 6 and 12 mg/kg [F_4,40 = 13.76, p < 0.01]. The positive drug fluoxetine also could exhibit this effect (p < 0.05). On the other hand, the results in Figure 3(c) showed that the locomotor activity was not affected after compound 4e treatment at various doses. Obviously, this result indicated the antidepressant effects of compound 4e were not attributed to central stimulation and inhibition. Therefore, compound 4e demonstrated significant antidepressant activities in depression-like behavior tests.

Figure 3.

The antidepressant-like effects of compound 4e in mice. Mice were treated with vehicle, compound 4e and fluoxetine 30 minutes before (a) SPT and (b) FST. (c) Mice were treated with compound 4e 30 minutes before LA activity test. 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; D3: 3 mg/kg compound 4e; D6: 6 mg/kg compound 4e; D12: 12 mg/kg compound 4e; Flu: fluoxetine.

In summary, a series of curcumin-based PDE4 inhibitors have been designed and synthesized. The in vitro study showed that most of these compounds demonstrated better inhibitory activities than curcumin itself. Of all the curcumin derivatives, compound 4e demonstrated the best inhibitory activity with an IC₅₀ value of 23 nM and good selectivity upon PDE4. Besides, the compound can also ameliorate depression-like behaviors induced by chronic stress in two behavioral tests including SPT and FST. In conclusion, these studies showed that curcumin may be a valuable lead compound for discovering good PDE4 inhibitors to treat depression. However, further optimization and investigation of this series of curcumin derivatives are expected to obtain more drug-like PDE4 inhibitors for treating depression.

Experimental

Materials and reagents

All chemicals and reagents used (Shanghai Aladdin Bio-Chem Technology Co., Ltd) were of analytical grade. Thin-layer and column chromatography was performed using silica gel (230-400 mesh). ¹H and ¹³C NMR spectra were conducted on Bruker spectrometers and chemical shifts were reported in ppm (δ). MS measurements were performed on a Thermo Velos Pro Orbitrap Elite Hybrid Mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Measurement of melting points was carried out by a micro apparatus (Boetius).

Synthesis of 3,5-diarylpyrazoles 3a–j

1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (2): Curcumin (1) (1.20 g, 3.24 mmol) were dissolved in 30 mL of C₂H₅OH and added to 600 mg of 10% Pd/C. Under an atmosphere of hydrogen, the mixture was stirred at 25 °C for 12 h. Then, the solvent was evaporated under reduced pressure and after further purification by column chromatography compound 2 was obtained as a yellow solid (0.80 g, 62.9%; m.p. 93.1-94.6°C).

¹H NMR (400 MHz, DMSO-d6): δ = 9.97 (s, 1H), 9.46 (s, 1H), 6.85 (m,2H), 6.67 (m, 4H), 5.53 (br s, 2H), 5.42 (s, 1H), 3.71 (s, 6H), 2.85-2.73 (m, 4H), 2.55 (m, 4H). ¹³C NMR (100 MHz, DMSO-d6): δ = 202.47, 194.22, 146.31, 143.56, 132.46, 120.71, 113.97, 110.43, 109.72, 99.81, 57.32, 55.46, 45.37, 41.25, 31.53, 29.12. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₂₅O₆: 373.1651; found: 373.1669.

Synthesis of compounds 3a–j. Compound 2 (1 equivalent) was dissolved in ethanol, NaHCO₃ (1.5 equivalent), and the corresponding hydrazine (RNHNH₂, 1.5 equivalent) was added. The mixture was heated to 75 °C and stirred for 6-8 h under air. After evaporation, the resulting residue was partitioned between water and ethyl acetate. The ethyl acetate layer was then dried over MgSO₄ and the pure products 3 was obtained after column chromatography.

4,4'-((1-phenyl-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3a): White solid, 71.6% yield; m.p. 78.1-79.7 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.86 (s, 1H), 9.37 (s, 1H), 7.63-7.55 (m, 3H), 7.22 (m, 2H), 6.93 (m, 2H), 6.73-6.61 (m, 5H), 3.87 (s, 3H), 3.81 (s, 3H), 3.17 (m, 4H), 2.85 (m, 4H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.35, 148.37, 147.05, 146.21, 143.79, 140.18, 129.01, 128.31, 127.16 (2), 126.53, 125.19, 122.31, 121.76, 118.43, 116.55 (2C), 114.18, 112.01, 110.69, 100.36, 56.08, 55.82, 35.67, 34.22, 28.97, 27.22. HRMS (ESI): m/z [M + H]+ calcd for C₂₇H₂₉N₂O₄: 445.2127; found: 445.2141.

1-(3,5-bis(4-hydroxy-3-methoxyphenethyl)-1H-pyrazol-1-yl)ethan-1-one (3b): White solid, 78.7% yield; m.p. 103.2-104.5 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.55 (s, 1H), 9.31 (s, 1H), 7.12 (m, 2H), 7.03-6.91 (m, 4H), 6.63 (s, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.22 (m, 4H), 2.87 (m, 4H), 2.15 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 169.21, 150.67, 149.35, 147.86, 145.61, 138.01, 127.93, 126.21, 124.36, 123.19, 121.69, 116.32, 115.01, 111.17, 109.46, 101.25, 56.32, 55.76, 35.69, 34.21, 29.32, 27.25, 26.35. HRMS (ESI): m/z [M + H]+ calcd for C₂₃H₂₇N₂O₅: 411.1920; found: 411.1936.

4,4'-((1-(2-hydroxyethyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3c): White solid, 53.1% yield; m.p. 167.3-168.6 °C. ¹H NMR (400 MHz, DMSO-d6): δ = 9.89 (s, 1H), 9.11 (s, 1H), 7.23 (m, 2H), 7.09-6.93 (m, 4H), 6.61 (s, 1H), 4.61 (m, 1H), 4.31 (m, 2H), 3.86 (s, 3H), 3.72 (s, 3H), 3.66 (m, 2H), 3.19 (m, 4H), 2.85 m, 4H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.33, 148.91, 147.25, 146.92, 145.07, 135.92, 131.71, 128.33, 123.92, 121.55, 118.73, 113.46, 110.35, 108.73, 100.33, 62.35, 57.67, 56.09, 53.72, 35.09, 34.15, 28.15, 26.67. HRMS (ESI): m/z [M + H]+ calcd for C₂₃H₂₉N₂O₅: 413.2076; found: 413.2061.

Ethyl 3,5-bis(4-hydroxy-3-methoxyphenethyl)-1H-pyrazole-1-carboxylate (3d): White solid, 86.5% yield; m.p. 76.9-78.2 °C. ¹H NMR (400 MHz, DMSO-d6): δ = 9.57 (s, 1H), 9.23 (s, 1H), 7.39 (m, 2H), 7.13-6.92 (m, 4H), 6.67 (s, 1H), 4.03 (q, J = 7.2 Hz, 2H), 3.86 (s, 3H), 3.81 (s, 3H), 3.29 (m, 4H), 2.91 (m, 4H), 1.25 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 160.46, 150.35, 148.32, 146.95, 146.02, 145.37, 132.15, 130.76, 126.53, 125.76, 121.37, 118.09, 112.75, 110.39, 109.13, 100.55, 62.79, 56.55, 55.03, 35.57, 33.19, 29.56, 27.21, 23.08. HRMS (ESI): m/z [M + H]+ calcd for C₂₄H₂₉N₂O₆: 441.2026; found: 441.2043.

4,4'-((1-(4-methoxyphenyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3e): Pale solid, 80.3% yield; m.p. 129.1-130.6 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.93 (s, 1H), 9.21 (s, 1H), 7.56-7.43 (m, 4H), 7.17 (m, 2H), 7.05–6.93 (m, 4H), 6.73 (m, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.76 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.98, 149.25, 148.01, 146.79, 143.52, 134.23, 132.06, 131.77, 130.95, 128.51, 127.13, 126.09, 123.47 (2C), 119.16, 117.23, 115.33, 111.32 (2C), 110.56, 101.77, 57.03, 56.72, 56.03, 35.13, 32.29, 29.08, 26.33. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₃₁N₂O₅: 475.2233; found: 475.2249.

4,4'-((1-(3-methoxyphenyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3f): White solid, 73.8% yield; m.p. 136.8-137.9 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.65 (s, 1H), 9.22 (s, 1H), 7.23 (m, 2H), 7.19 (m, 3H), 7.05–6.93 (m, 5H), 6.71 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.71 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.33, 149.68, 147.23, 146.09, 143.35, 140.02, 137.52, 135.13, 132.35, 131.62, 130.59, 128.32, 127.03, 125.87, 123.21, 122.35, 120.51, 119.12, 115.32, 111.25, 100.23, 57.62, 56.87, 56.09, 35.71, 32.56, 29.33, 27.09. HRMS (ESI): m/z [M + H]+ calcd for C₂₈H₃₁N₂O₅: 475.2233; found: 475.2219.

4,4'-((1-(4-chlorophenyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3g): Brown solid, 59.6% yield; m.p. 175.2-175.6 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.32 (s, 1H), 9.09 (s, 1H), 7.62 (m, 2H), 7.26–7.13 (m, 5H), 6.89 (m, 3H), 6.67 (s, 1H), 3.87 (s, 3H), 3.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.37, 148.03, 145.09, 142.35, 140.43, 139.42, 137.56, 135.11, 132.87, 128.35 (2C), 127.03, 125.68, 122.56, 120.76, 118.52, 116.87 (2C), 111.55, 109.23, 101.25, 56.83, 56.07, 36.07, 32.11, 28.63, 27.35. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₇H₂₈ClN₂O₄: 479.1738; found: 479.1723.

4,4'-((1-(3-chlorophenyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3h): White solid, 76.3% yield; m.p. 177.7-179.3 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.43 (s, 1H), 9.11 (s, 1H), 7.51 (m, 3H), 7.23-6.92 (m, 5H), 6.86 (m, 2H), 6.71 (s, 1H), 3.86 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.63, 148.73, 147.32, 146.02, 143.21, 141.39, 138.27, 134.29, 133.51, 132.79, 128.46, 127.08, 124.83, 123.67, 121.35, 120.01, 119.08, 115.22, 113.26, 101.56, 100.07, 56.23, 56.06, 36.55, 32.73, 28.79, 26.21. HRMS (ESI): m/z [M + H]+ calcd for C₂₇H₂₈ClN₂O₄: 479.1738; found: 479.1452.

4,4'-((1-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxyphenol) (3i): White solid, 75.7% yield; m.p. 102.2-103.5 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.47 (s, 1H), 9.12 (s, 1H), 7.49-7.43 (m, 3H), 7.11-6.95 (m, 4H), 6.83 (m, 2H), 6.75 (m, 2H), 3.82 (s, 3H), 3.71 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.79, 148.32, 146.01, 145.23, 143.51, 141.83, 140.11, 135.76, 131.27, 130.97, 128.61, 127.35 (2C), 125.43, 124.12, 123.21, 121.76, 117.33, 112.56, 111.73, 110.48, 100.03, 56.79, 56.07, 37.23, 32.55, 28.03, 26.39. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₂₈F₃N₂O₄: 513.2001; found: 513.2017.

Synthesis of compounds 4a–j. Curcumin (1 equivalent) was dissolved in ethanol, and added NaHCO₃ (1.5 equivalent) and various hydrazine (RNHNH₂, 1.5 equivalent). After refluxing for 12–15 h, the solvent was evaporated. And then the residue was dissolved with ethyl acetate and extracted by water and brine solution. After being dried by anhydrous Na₂SO₄, the ethyl acetate layer evaporated under vacuum to afford the crude product. The pure products 4 was obtained by further column chromatography.

4,4'-((1E,1'E)-(1-phenyl-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4a): Pale solid, 87.6% yield; m.p. 89.1-91.3 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.25 (s, 1H), 9.10 (s, 1H), 7.57-7.48 (m, 4H), 7.42 (m, 1H), 7.23-7.13 (m, 3H), 7.03-6.92 (m, 5H), 6.75 (m, 3H), 3.82 (s, 3H), 3.75 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.12, 148.93, 148.17, 147.09, 146.22, 142.35, 139.26, 133.12, 131.13, 129.67 (2C), 128.36, 127.66, 127.09, 125.13, 120.46, 117.52, 116.73, 115.98 (2C), 112.76, 111.13, 110.62 (2C), 101.55, 56.72, 56.03. HRMS (ESI): m/z [M + H]+ calcd for C₂₇H₂₅N₂O₄: 441.1814; found: 441.1821.

1-(3,5-Bis((E)-4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl)ethan-1-one (4b): White solid, 91.2% yield; m.p. 112.6-113.7 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.29 (s, 1H), 9.03 (s, 1H), 7.56 (d, J = 13.1 Hz, 2H), 7.12-6.93 (m, 6H), 6.61 (s, 1H), 6.49 (d, J = 13.1 Hz, 2H), 3.82 (s, 3H), 3.75 (s, 3H), 2.12 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 169.39, 151.13, 149.12, 148.08, 147.35, 146.21, 139.25, 133.79, 131.12, 128.56, 126.99, 125.18, 120.39, 118.23, 116.97, 112.09, 111.15, 110.71 (2C), 100.09, 56.17, 56.05, 27.16. HRMS (ESI): m/z [M + H]+ calcd for C₂₃H₂₃N₂O₅: 407.1607; found: 407.1619.

4,4'-((1E,1'E)-(1-(2-hydroxyethyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4c): Pale solid, 56.6% yield; m.p. 223.6-225.1 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.33 (s, 1H), 9.12 (s, 1H), 7.59 (d, J = 13.2 Hz, 2H), 7.09-6.92 (m, 6H), 6.63 (s, 1H), 6.47 (d, J = 13.2 Hz, 2H), 4.66 (m, 1H), 4.32 (m, 2H), 3.87 (s, 3H), 3.79 (s, 3H), 3.65 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.86, 149.19, 148.87, 147.67, 145.21, 131.76, 131.03, 130.23, 126.23, 125.16, 121.55, 120.93, 118.22, 114.03, 113.02, 110.21 (2C), 108.53, 101.62, 62.79, 56.19, 56.01, 53.26. HRMS (ESI): m/z [M + H]+ calcd for C₂₃H₂₅N₂O₅: 409.1763; found: 409.1776.

Ethyl 3,5-bis((E)-4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-carboxylate (4d): White solid, 62.3% yield; m.p. 83.7-85.3 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.36 (s, 1H), 9.07 (s, 1H), 7.62 (d, J = 13.1 Hz, 2H), 7.06-6.91 (m, 6H), 6.62 (s, 1H), 6.46 (d, J = 13.1 Hz, 2H), 4.01 (q, J = 7.2 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 159.23, 151.87, 149.23, 148.36, 146.97, 145.22, 131.77, 131.01, 130.55, 127.19, 125.13, 122.01, 120.95, 118.31, 114.03, 112.65, 110.79 (2C), 108.43, 101.66, 62.85, 56.53, 56.12, 23,76. HRMS (ESI): m/z [M + H]+ calcd for C₂₄H₂₅N₂O₆: 437.1713; found: 409.1701.

4,4'-((1E,1'E)-(1-(4-methoxyphenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4e): White solid, 71.6% yield; m.p. 126.2-127.5 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.63 (s, 1H), 9.39 (s, 1H), 7.66-7.53 (m, 5H), 7.41 (m, 1H), 7.23–7.11 (m, 3H), 7.01–6.92 (m, 4H), 6.79 (m, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.37, 149.02, 148.55, 147.03, 146.59, 144.17, 142.12, 133.91, 132.19, 131.56, 129.36 (2C), 127.91, 127.03, 126.55, 125.63, 120.52, 118.07, 116.75 (2C), 115.46, 112.15, 111.03 (2C), 100.76, 57.03, 56.72, 56.03. HRMS (ESI): m/z [M + H]+ calcd for C₂₈H₂₇N₂O₅: 471.1920; found: 471.1932.

4,4'-((1E,1'E)-(1-(3-methoxyphenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4f): White solid, 65.5% yield; m.p. 132.6-133.8 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.76 (s, 1H), 9.31 (s, 1H), 7.65-7.51 (m, 4H), 7.43 (m, 2H), 7.22–7.13 (m, 3H), 7.03–6.91 (m, 5H), 6.77 (m, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.76, 149.11, 148.35, 146.83 (2C), 143.25, 142.17, 133.63, 132.11, 131.77, 130.32, 129.58, 128.42, 127.33 (2C), 126.51, 123.29, 120.51, 118.79, 116.21, 113.68, 112.11, 110.63 (2C), 100.09, 57.39, 56.58, 56.13. HRMS (ESI): m/z [M + H]+ calcd for C₂₈H₂₇N₂O₅: 471.1920; found: 471.1937.

4,4'-((1E,1'E)-(1-(4-chlorophenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4g): Brown solid, 75.2% yield; m.p. 171.1-172.3 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.32 (s, 1H), 9.09 (s, 1H), 7.43 (m, 2H), 7.15-7.01 (m, 6H), 6.73 (m, 4H), 6.63 (m, 3H), 3.87 (s, 3H), 3.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 150.23, 148.71, 147.57, 146.03, 142.15, 140.63 (2C), 137.23, 134.69, 131.21, 130.56, 128.13, 127.76, 127.13, 124.05, 123.71, 121.86, 120.53, 117.03, 116.37, 115.91, 112.37, 110.81, 109.55, 101.31, 56.31, 55.29. HRMS (ESI): m/z [M + H]+ calcd for C₂₇H₂₄ClN₂O₄: 475.1425; found: 475.1412.

4,4'-((1E,1'E)-(1-(3-chlorophenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4h): Brown solid, 82.3% yield; m.p. 175.7-175.2 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.28 (s, 1H), 9.15 (s, 1H), 7.65-7.51 (m, 4H), 7.25-6.91 (m, 8H), 6.81 (m, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.87, 148.32, 148.09, 147.55, 146.97, 143.08, 140.63, 134.15, 133.22, 131.37, 131.05, 128.61, 128.17, 127.19, 124.65, 123.51, 120.82, 120.07, 117.43, 116.12, 115.93, 112.36, 110.85, 110.16, 100.92, 56.23, 56.06. HRMS (ESI): m/z [M + H]+ calcd for C₂₇H₂₄ClN₂O₄: 475.1425; found: 475.1437.

4,4'-((1E,1'E)-(1-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (4i): Yellow solid, 66.2% yield; m.p. 97.3-98.9 °C. ¹ H NMR (400 MHz, DMSO-d6): δ = 9.29 (s, 1H), 9.11 (s, 1H), 7.58-7.49 (m, 4H), 7.43 (m, 1H), 7.25-7.13 (m, 3H), 7.02–6.93 (m, 4H), 6.77 (m, 3H), 3.81 (s, 3H), 3.77 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 151.32, 148.91, 148.16, 147.02, 146.35, 143.21, 142.19, 133.23, 132.55, 131.69, 128.35 (2C), 127.97, 127.01, 125.91 (2C), 125.15, 123.72, 120.51, 117.51, 116.73, 115.27, 112.77, 111.15, 110.63, 100.98, 56.19, 56.11. HRMS (ESI): m/z [M + H]+ calcd for C₂₈H₂₄F₃N₂O₄: 509.1688; found: 509.1673.

Enzymatic assay

The substrate ³H-cGMP and ³H-cAMP were used to assay PDE enzymatic activities by the phosphodiesterase scintillation proximity assay (SPA). First, PDEs were incubated with a solution of Tris–HCl (50 mmol/L, pH 7.8), MgCl₂ (10 mmol/L), and ³H-cAMP or ³H-cGMP (40 000 cpm/assay) at 24 °C for 30 minutes. After complete hydrolysis, the reaction was terminated by adding ZnSO₄ and then adding the solution of 0.2 N Ba(OH)₂ to precipitate the product ³H-GMP. Then, the radioactivity of unreacted ³H-cGMP in the supernatant was measured, and the IC₅₀ values were measured.

Animals and treatment

Male ICR mice, 22-25 g, were purchased from Nanjing University of Traditional Chinese Medicine. The animal tests were approved by the Institutional Animal Care and Use Committee of Changzhou University. Saline containing 5% DMSO was used to dissolve all of the test compounds, but for fluoxetine, the saline did not contain DMSO. Mice were administrated intragastrically (compound 4e: 3, 6, and 12 mg/kg) or intraperitoneal injections (fluoxetine: 5 mg/kg). The chronic unpredictable stress procedure (CUS) was performed according to the previous procedures.²⁹ And then, the sucrose preference test was carried out on the next day, and then on the following day, the forced swimming test was performed. Thirty minutes before the sucrose preference test (SPT) and forced swimming test (FST) mice were administered (i.g. or i.p.) and continued once a day until the final day.

Behavioral tests

SPT and locomotor activity (LA) tests were conducted according to the previous description.^30,31 For FST, mice were induced individually into a glass cylinder (20 cm diameter, 40 cm depth). And the cylinder was filled with water of 25 cm at 25 °C for 10 min pre-swimming. Then, the mice were placed in the cylinder for 6 min after 24 h. During the last 4 min, the total immobility time was recorded. And those necessary posture for the mouse to keep its head above water were excluded.

Docking simulations

Molecular docking was carried out on Discovery Studio 2019 using the CDOCKER method with the crystal structure of PDE4 (PDB number: 4WCU), obtained from protein data bank (RSC).

Footnotes

Availability of data and materials

The data sets used and analyzed during this study are available from the corresponding author upon request.

Consent for publication

Written informed consent for publication was obtained from all authors.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was sponsored by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX22_1314).

ORCID iD

Xianfeng Huang

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