Design, synthesis, and anticonvulsant effects evaluation of nonimidazole histamine H₃ receptor antagonists/inverse agonists containing triazole moiety

Abstract

Histamine H₃ receptors (H₃R) antagonists/inverse agonists are becoming a promising therapeutic approach for epilepsy. In this article, novel nonimidazole H₃R antagonists/inverse agonists have been designed and synthesised via hybriding the H₃R pharmacophore (aliphatic amine with propyloxy chain) with the 1,2,4-triazole moiety as anticonvulsant drugs. The majority of antagonists/inverse agonists prepared here exerted moderate to robust activities in cAMP-response element (CRE) luciferase screening assay. 1-(3-(4-(3-Phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3l) and 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3m) displayed the highest H₃R antagonistic activities, with IC₅₀ values of 7.81 and 5.92 nM, respectively. Meanwhile, the compounds with higher H₃R antagonistic activities exhibited protection for mice in maximal electroshock seizure (MES)-induced convulsant model. Moreover, the protection of 3m against the MES induced seizures was fully abrogated when mice were co-treated with RAMH, a CNS-penetrant H₃R agonist, which suggested that the potential therapeutic effect of 3m was through H₃R. These results indicate that the attempt to find new anticonvulsant among H₃R antagonists/inverse agonists is practicable.

Keywords:

• Epilepsy
• anticonvulsant
• H₃ receptor antagonists/inverse agonists
• hybrid
• 1,2,4-triazole

1. Introduction

Epilepsy, a very common neurologic disorder, affects about around 1% of world population^1,2. Presently, antiepileptic drugs (AEDs) are the main strategy of therapy. However, the AEDs available in the clinic such as phenytoin, carbamazepine, sodium valproate, topiramate, and oxcarbamazepine are only effective in approximately 70% of the patients with epilepsy. Moreover, their use is long-term and often accompanied with severely side effects, including naupathia, headache, and ataxia, even threaten the life of patients^3–5. Investigations for more effective and safer AEDs are still a formidable and urgent task for medicinal chemists.

The role of central histaminergic system being concerned in epilepsy have been demonstrated in many experimental and epidemiological studies, in which histamine regulated seizure susceptibility as an anticonvulsant neurotransmitter^6–8. For example, H₁-antagonists such as pyrilamine, ketotifen that decrease brain histamine levels increased the duration of convulsive phase in electrically-induced convulsions model⁹. Histidine, as the precursor of histamine, showed protection against chemically-induced convulsions in rats, via activating the histamine H₁ receptors¹⁰.

Histamine H₃ receptors (H₃R) as a G-protein coupled receptor (GPCR) binding to histamine like other histamine receptors, is expressed mainly in the central nervous system, where it acts as an auto-receptor in histaminergic neurons, and negatively regulates the synthesis and release of histamine¹¹. What is more, as a inhibitory heteroreceptor, H₃R also regulates the release of other neurotransmitters including dopamine, acetylcholine, serotonin, norepinephrine, γ-aminobutyric acid, and glutamate. These neurotransmitters, especially γ-aminobutyric acid and glutamate, are related to epilepsy inextricably^12,13. Therefore, more attention has been focussed on H₃R as an attractive therapeutic target for epilepsy treatment¹⁴.

A large number of experimental studies involved in acute and chronic models of epilepsy confirmed the anticonvulsive potential of H₃R antagonists/inverse agonists. They showed the protection against experimental seizures by feedback increase of histamine release and binding with H₁ receptors^15,16. Besides, other mechanisms might be involved in their anticonvulsive action, such as facilitating of GABA release^17–19, increasing histidine decarboxylase (HDC) activity^20,21 and synergism with AEDs^17,18,22.

Early, anticonvulsant activity of some imidazole H₃R antagonists such as thioperamide and clobenpropit was confirmed in models of epilepsy (Figure 1)^16,19,20,23. Recently, a large number of non-imidazole H₃R antagonists such as DL77 (Figure 1) prepared by a group/team of Kiec-Kononowicz exhibited excellent anticonvulsant activity in the electrically-induced seizures model and subcutaneously pentylenetetrazole (PTZ)-induced seizure model at dose-dependent, and the therapeutic action was proved through H₃R^24–28. Sadek et al. synthesised some histamine H₃R ligands (Figure 1, I) incorporating different antiepileptic structural motifs to investigate if the H₃R pharmacophore could be combined to some antiepileptic molecules, and give some new anticonvulsants by the multiple-target approaches. The results were encouraging, which indicated that the H₃R pharmacophore successfully combined to the antiepileptic molecules, maintaining the H₃R affinity and anticonvulsant activity, although the anticonvulsant activity decreased compared to the prototypal antiepileptic molecules (Figure 1)^29,30.

Figure 1. Structures of histamine H₃ receptor ligands with anticonvulsant activity, triazole derivatives with anticonvulsant activity and target compounds 3a-3q designed.

Pitolisant (PIT), a H₃R antagonist/inverse agonist, has been subjected into clinical Phase III for the treatment of epilepsy³¹. When used alone or in combination with other AEDs in the human photosensitivity model at dose ranges of 30–60 mg, PIT showed a favourable EEG profile in a dose-dependent manner³².

Supported by the above results, in this work, we designed and synthesised some novel H₃R antagonists/inverse agonists by hybriding the H₃R pharmacophore (aliphatic amine with propyloxy chain) with the 1,2,4-triazole, the latter have been identified as an important and effective anticonvulsive fragment in recent years (Figure 1, II and III)^33–37. According to Quan’s reports, the 1,2,4-triazole derivatives were likely to have several mechanisms of action such as inhibiting voltage-gated sodium ions channel and modulating GABAergic activity^38–40. And a group of Plech illustrated the anticonvulsive effects of 4-alkyl-5-aryl-1,2,4-triazole-3-thione derivatives and suggested that the influence on the voltage-gated Na⁺ channels was involved in them at least^41,42. Therefore, in this work, our strategy was to design molecules combining pharmacophores of H₃R antagonists and another anticonvulsant active pharmacophore (e.g. 1,2,4-triazole moiety) into one skeleton, and then produced a synergism for anticonvulsant active.

2. Results and discussion

2.1. Chemistry

According to Schemes 1 and 2, the target compounds (3a-3q) were synthesised smoothly. In brief, formyl hydrazine reacted with 4-aminophenol in dimethoxyl-N,N-dimethyl formamide (DMF-DMA) to give the 4-(4H-1,2,4-triazol-4-yl)phenol (1a). Compound 1a underwent a nucleophilic substitution with 1-bromo-3-chloropropane to get 4-(4-(3-chloropropoxy)phenyl)-4H-1,2,4-triazole (2a). The reaction was conducted in the presence of potassium hydroxide in dimethyl sulfoxide (DMSO) at room temperature to ensure the formation of single-substituted derivatives. Finally, proper amines reacted with compound 2a in the presence of K₂CO₃ and KI in the solvent of CH₃CN to give the desired compounds 3a-3j. To enrich the structure–activity relationship, we also prepared the derivatives of 3h via introducing the substituents at the triazole ring and adjusting the length of the link. The reaction conditions used to prepare these compounds (3k-3q) were the same as above. Compounds (3a, 3b, 3c, 3d, 3e, 3 g, 3k, 3o, and 3p) obtained as the form of oil were transformed to hydrochlorate. Their structures were characterised and confirmed by¹H-NMR, ¹³C-NMR, and HR-MS.

Scheme 1. The synthesis route and conditions for the preparation of compounds 3a-3j.

Scheme 2. The synthesis route and conditions for the preparation of compounds 3k-3q.

2.2. Biological activities

2.2.1. Evaluation of H₃R antagonistic activity

cAMP-response element (CRE) reporter gene assay has been extensively used to evaluate the efficacy of GPCR antagonists or agonists. In this work, the H₃R antagonistic activities of the prepared 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine derivatives have been screened by CRE-driven luciferase assay, in which the HEK-293 cells expressing the human H₃R and a reporter gene consisting of the firefly luciferase coding region were used^43,44. Ciproxifan (CXP) and Pitolisant (PIT) were employed as the positive controls. Initially, compounds and positive controls were tested at two concentrations (100 nM and 1 µM) to obtain the preliminary investigation of their H₃R antagonistic activities. In the assays, the antagonistic activities was positively correlated with the rise of the fluorescence value and indicated by the % antagonism. For the prominent compounds IC₅₀ values were determined at additional assays.

As seen in Table 1, majority of the synthesised compounds displayed gentle to robust H₃R antagonistic activities and eight of them exhibited micromolar inhibitory activity. The antagonistic activities of all compounds depended on the concentration treated. It is worth mentioning that compounds 3l (IC₅₀ = 7.81 nM) and 3m (IC₅₀ = 5.92 nM) displayed the most potent H₃R antagonistic activities, with the much stronger potency than that of CXP (IC₅₀ = 0.082 µM) and PIT (IC₅₀ = 0.5 µM) in the CRE reporter gene assay.

Table 1. H₃R antagonistic activity of compounds 3a-3q.

Compounds	R	n	% Antagonism^a		H3R antagonistic activity (IC50, μM)
			100 nM	1 μM
3a			49.85 ± 9.73	223.76 ± 7.56	2.99
3b			13.24 ± 4.83	15.02 ± 4.03	NT^b
3c			32.23 ± 5.69	143.98 ± 16.16	0.553
3d			11.33 ± 5.86	79.4 ± 4.50	NT
3e			9.86 ± 0.87	21.78 ± 3.04	NT
3f			9.96 ± 10.9	24.41 ± 6.30	NT
3g			−0.76 ± 4.87	20.24 ± 7.56	NT
3h			10.56 ± 7.94	239.79 ± 3.17	0.127
3i			5.19 ± 2.06	16.45 ± 1.90	NT
3j			−4.2 ± 3.11	6.12 ± 2.05	NT
3k	Me	1	133.91 ± 8.91	183.54 ± 14.41	0.021
3l	C6H5	1	148.62 ± 4.43	169.04 ± 15.04	0.00781
3m	C6H4(p-Cl)	1	201.12 ± 12.63	203.92 ± 8.44	0.00592
3n	Biphenyl	1	1.03 ± 7.79	3.36 ± 1.27	NT
3o	H	0	45.04 ± 3.61	94.20 ± 2.60	0.25
3p	H	2	25.00 ± 3.95	90.52 ± 5.71	3.44
3q	H	3	1.88 ± 12.38	2.39 ± 13.85	NT
CPX^c			33.96 ± 8.59	63.05 ± 9.33	0.082
PIT^d			54.16 ± 7.33	246.29 ± 21.48	0.51

^a% Antagonism, value represented as mean ± standard deviation of three independent experiments.

^bNT, IC₅₀ was not tested.

^cCPX, an antagonist of H₃R ciproxifan.

^dPIT, an antagonist/inverse agonist of H₃R pitolisant.

Surprisingly, antagonism percent of some compounds as well PIT were above 100%. It is well known that H₃R is a GPCR coupled with Gαi. When the ligand (histamine) binds to H₃R, the dissociated Gαi inhibits the activity of adenylate cyclase (AC) and down-regulates the level of intracellular cAMP. In this CREs driven luciferase assay, when cells were pre-treated by H₃R antagonists, the downregulation of cAMP would be inhibited, and the level of intracellular cAMP would regain to the initial level. However, in some cases, the cAMP levels raised above the initial level, giving the % antagonism greater than 100%. Our first speculation was that these compounds might stimulate the AC directly and up-regulate the level of cAMP. However, an additional assay indicated that these compounds didn’t have effects on the level of cAMP when pre-treated alone. As shown in Figure 2, forskolin (2 µM) treated group gave more than 200 times rise for the cAMP level when compared to the control group. While Histamine, ciproxifan, and compounds 3a, 3c, 3h, 3k, 3l, and 3m have no significant effects on the level of cAMP when carried out comparisons by ANOVA followed by Dunnett’s test.

Figure 2. Effects of compounds 3a, 3c, 3h, 3k, 3l, and 3m on the level of intracellular cAMP administrated alone.

Another explanation is that these compounds may be inverse agonists when binding to H₃R, which not only antagonise the function of histamine, but also give the inverse agonistic performance. Actually, PIT is a well-known H₃R antagonist and reverse agonist. So, the above results make sense. Based on the above, we further assessed the H₃R inverse agonist activity of compound 3m and PIT by using CRE-luciferase assay. In this experiment, transfected HEK-293 cells were stimulated with 10 µM forskolin or 10 µM forskolin plus different concentrations of compound 3m. The raise of luciferase activity after adding compound represented the inverse agonistic activity. The EC₅₀ was calculated by seven concentrations. Cytotoxicity appeared at 100 µM. As shown in Figure 3, PIT and compound 3m showed effective H₃R inverse agonistic activity with an EC₅₀ value of 403 and 129 nM, respectively.

Figure 3. The H₃R inverse agonistic activity (EC₅₀, μM) of Pitolisant and compound 3m.

Simple structure–activity relationships (SARs) could be obtained from Table 1. In the series of 3a-3j, the different tertiary amines significantly influenced the H₃R antagonistic activities. The N-ethyl derivative 3a showed an IC₅₀ of 2.9 µM, while the activity declined sharply for the N-propyl derivative 3b. Interestingly, compounds containing piperazine or morpholine (3d-3g) exhibited weaker activities than those with piperidine or pyrrolidine (3c and 3h). This probably attributed to the increase of the molecular polarity. The introduction of phenyl or amide group on the piperidine ring of compound 3h, gave the compounds 3i and 3j, which also decreased the H₃R antagonistic activities when compared to compound 3h. Based on the facts above, it could be concluded that the N,N-diethyl group, pyrrolidine and piperidine were more of benefit to the H₃R antagonistic activities of the 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine skeleton, and piperidine derivative (3h) was the best one with the IC₅₀ of 0.127 µM.

To enrich the structure–activity relationships, we prepared the derivatives of 3h via introducing the substituents at the triazole ring and adjusting the length of the link.

Compounds 3k, 3l, 3m, and 3n were substituted on 3-position of 1,2,4-triazole ring with methyl, phenyl, para-chlorophenyl, and biphenyl, respectively. Encouragingly, the introduction of methyl, phenyl, and para-chlorophenyl groups significantly increased the H₃R antagonistic activities, giving the two prominent compounds 3l and 3m with nanomolar IC₅₀ values. While the biphenyl substituted compound 3n showed weaker activity when compared to 3h. Replacing the three-carbon link in the compound 3h with two-carbon, four-carbon and five-carbon links, gave the compounds 3o, 3p, and 3q, respectively. It could be seen that the length of the link had a direct impact on H₃ receptor antagonistic activities of the 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine derivatives. The activity order of the link length of carbon was 3 > 2 > 4 ≫ 5.

To investigate the molecular determinants that manage the antagonistic activities of the tested compounds, molecular docking studies of PIT, 3h, and 3m with the H₃R homology model were carried out. The homology model was constructed from the crystal structure of the H₁ receptor (PDB ID: 3RZE)⁴⁵. The docking results are shown in Figure 4.

Figure 4. The predicted configurations for PIT (A), 3h (B) and 3m (C) binding with H₃R, and their overlying pattern (D).

As shown in Figure 4(A), PIT bound to H₃R through two critical H-bond interactions with Tyr115 and Glu206, and other interactions with amino acid residues Arg381, Phe193, Met378 and so on. Figure 4(B) revealed that compound 3h had a similar binding pattern to PIT, interacting with the same amino acid residues Glu206, Tyr115, Arg381, and Met378. Surprisingly, the compound 3m with the highest H₃R antagonistic activity showed a different binding pattern to PIT (as seen in Figure 4(C)). The overlying pattern of PIT, 3h, and 3m was shown in Figure 4(D). The piperidine group of 3m was not involved in the formation of the salt bridge or hydrogen-bond interactions with Glu206, which was generally considered as the critical residue of H₃R^46,47. The unexpected binding pattern of 3m might be due to the phenyl group on the triazole ring, which did not fit into the hydrophobic cavity in TMs 3-5-6 region of H₃R, even though the compound 3m showed a forceful binding with H₃R via another mode. The triazole nitrogen established an ionic bond with Glu206, and a hydrogen bond was observed between piperidine nitrogen and Tyr115. π-π shaped, and alkyl interactions with Trp371, Tyr343, Arg381, His187, Leu199, and ALA202 were observed to support the forceful binding with H₃R.

2.2.2. Anticonvulsant activity evaluation

To investigate the anticonvulsive effects, all the target compounds (3a-3q) were screened in the MES-induced and PTZ-induced convulsion models in mice. Compounds were administered intraperitoneally (i.p.) to mice at dosage of 10 mg/kg in the both models. PIT and valproic sodium (VPA) were used as positive controls in the tests.

2.2.2.1. Protective effects of H₃R antagonists/inverse agonists 3a-3q on MES-induced convulsions

Protection for the mice was defined as the reduction or abolition of the tonic hind limb extension (THLE) in the MES model in mice. As seen in Figure 5, compounds 3a, 3 g, 3h, 3l, 3m, and 3o showed moderate protection for the electro-stimulated mice with significant difference from that of the control group (p < 0.05, p < 0.01, or p < 0.001). Mice pre-treated with PIT (10 mg/kg, i.p.) and VPA (300 mg/kg, i.p.) were moderately or potently protected, respectively. Generally, the anticonvulsant activities of these compounds in MES model correlated directly to their H₃R antagonistic activities. For example, the antiepileptic activity obtained of compound 3m was the highest, and in vitro H₃R antagonistic activity measured for 3m with IC₅₀ of 5.92 nM was also the highest. Compounds 3a, 3h, 3l, and 3o, showing anticonvulsant activity in the MES model, also showed good H₃R antagonistic activities. Compound 3c, 3k, and 3p reduced the average duration of THLE, although they did not achieve a significant difference from the control group.

Figure 5. Effects of H₃R antagonists/inverse agonists 3a-3q (10 mg/kg, i.p.), PIT (10 mg/kg, i.p.) and anticonvulsant drug VPA (300 mg/kg, i.p.) against MES-induced convulsions. Protection for mice was defined as the reduction or abolition of the tonic hind limb extension (THLE) in MES model. Results are showed as mean ± SEM with seven animals in each group. Values are considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to saline-treated group.

2.2.2.2. Protective effects of H₃R antagonists/inverse agonists 3a-3q on PTZ-induced convulsions

Some experiments indicated that H₃R antagonists/inverse agonists could protect animals in PTZ-induced convulsions model^26,29. So the compounds 3a-3q, PIT, and VPA were also screened in the PTZ model in mice. Unfortunately, all compounds tested at the dose of 10 mg/kg (i.p.) did not show any protection against the seizures induced by PTZ. PIT also failed to protect the PTZ-treated mice as well at the same conditions. By contrast, anticonvulsant agent VPA showed full protection against the PTZ-induced convulsions (Figure 6).

Figure 6. Effects of compounds 3a-3q (10 mg/kg, i.p.), and reference drug PIT (10 mg/kg, i.p.) and VPA (300 mg/kg, i.p.) against PTZ-induced convulsions. Results are showed as mean ± SEM of seven mice in each group. & represent full protection.

2.2.2.3. Effects of compound 3m on MES-induced convulsions in dose dependent manner

In a further experiment, compound 3m, as the most active one in the MES-induced seizure model, was chosen to verify its protective effect in different doses. Encouragingly, the 3m-provided protections were observed and were dose dependent. The standard antagonist PIT also displayed anticonvulsive activity dose-dependently at the same condition. Notably, when pre-treated with 20 mg/kg dose, PIT could fully abrogate the tonic hind limb extension induced by electro-stimulation, showing its potential anticonvulsant activity (Figure 7). To exclude the possibility that the anticonvulsant activity of 3m was connected with sedative effect, we carried out a rotarod test for 3m. The result showed that compound 3m had no neurotoxicity at the maximum dose of 10 and 20 mg/kg (the details could be seen in Support Table 1).

Figure 7. Protective effects of compound 3m and reference drug PIT against MES-induced convulsions in different doses. Protection in the test was defined as the reduction or abolition of the THLE in mice. Results were showed as mean ± SEM with seven animals in each group. Values are considered significant at *p < 0.05, **p < 0.01, ***p < 0.001 when compared to saline-treated group. ^&PIT, at 20 mg/kg dose, fully abrogate the THLE for all the tested mice.

2.2.2.4. Effects of RAMH pre-treatment on the compound 3m-provided protection in MES-induced seizure model

To investigate the correlation between the anticonvulsant activity and H₃R antagonistic activity of compound 3m, the protection provided by compound 3m against MES-induced seizure was reassessed after the administration of RAMH (10 mg/kg, i.p.), a CNS penetrant histamine H₃R agonist. The results indicated that when co-administration with RAMH, compound 3m lost its original protective effect (Figure 8). Administration of RAMH alone also did not affect the duration of THLE of mice with p > 0.05 for saline versus RAMH. The above findings suggested that H₃R antagonism was the main contributor for the anticonvulsant activity of compound 3m in MES model. When the H₃R was blocked by H₃R antagonist 3m, histamine or other neurotransmitter such as GABA in the CNS increased, finally leading to anticonvulsive effects.

Figure 8. Protective effects of compound 3m (10 mg/kg, i.p.) against MES-induced convulsions when pre-treatment of RAMH (10 mg/kg, i.p.). Protection in the test was defined as the reduction or abolition of the THLE in mice. Results are showed as mean ± SEM with seven animals in each group. Values are considered significant at *p < 0.01 as compared to saline-treated group, and ^#p < 0.01 as compared to 3m + RAMH treated group.

3. Conclusion

To identify novel H₃R antagonists/inverse agonists with potential anticonvulsant activities, a series of 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine derivatives were designed through combining pharmacophore of H₃R antagonists and another anticonvulsant active pharmacophore (1,2,4-triazole moiety) into one molecule. The majority of those prepared compounds displayed moderate to robust H₃R antagonistic activities. The SAR analysis revealed that piperidine and triazolephenol linked by three-carbon chain was benefit for the H₃R antagonistic activity, and substitution by aromatic nucleus on the 3-position of 1,2,4-triazole further increased the H₃R antagonistic activities. The most potent H₃R antagonists/inverse agonists 3l and 3m exhibited nanomolar H₃R antagonistic activities with IC₅₀ of 7.81 nM and 5.92 nM, respectively. Molecular docking analysis demonstrated that 3m strongly bound to H₃R via interactions with Tyr115, Glu206, Trp371, Tyr343, and so on, although its binding mode was not similar to PIT. The anticonvulsive screens in vivo indicated that compounds with higher H₃R antagonistic activities showed more protection in the MES-induced convulsant model in mice, while no one was observed protective effect in PTZ-induced convulsant model. Moreover, the protection of 3m in the seizure model was fully abrogated when mice were co-treated with a H₃R agonist RAMH, which suggested that its potential therapeutic effect was through H₃R.

4. Experimental section

4.1. Synthesis

All the chemical solvents and reagents were purchased from supplier and used as received. Unless otherwise specified, reactions were monitored by thin-layer chromatography (TLC). All NMR spectrum was carried out on an AV-300 spectrometer with 300 MHz. High resolution mass spectra were measured on an MALDI-TOF/TOF mass spectrometer.

4.1.1. Synthesis of compounds 1a-1e

Taking compound 1a as an example: dimethoxyl-N, N-dimethyl formamide (DMF-DMA, 1.31 g, 11 mmol) and formyl hydrazine (0.65 g, 11 mmol) were added into a flask containing 30 ml of acetonitrile. The mixture was heated up to 60 °C for 30 min, then 4-aminophenol (0.60 g, 5.5 mmol) and acetic acid (3 mL) were added and heated up to 120 °C for 9 h. The mixture was cooled, filtered and washed by acetonitrile to give the product 1a. Chemical formula: C₈H₇N₃O (MW = 161.16). m.p. 270–272 °C, yield 73%. ¹H-NMR (300 MHz, DMSO-d₆): δ 6.90 (d, 2H, J = 8.9 Hz, Ph-H), 7.46 (d, 2H, J = 8.9 Hz, Ph-H), 8.94 (s, 2H, N = CH), 9.88 (s, 1H, OH). ¹³C-NMR (DMSO-d₆, 75 MHz): 157.75, 142.14, 126.18, 123.65, 116.60. The compounds 1 b-1e were obtained according to the above method using the other hydrazides.

4.1.2. Synthesis of compounds 2a-2h

Taking compound 2a as an example: compound 1a (0.50 g, 3.1 mmol) and KOH (0.35 g, 6.2 mmol) were put into a flask with 5 mL of DMSO. The mixture was stirred for 5 min at 20 °C. Then added 1-bromo-3-chloropropane (0.98 g, 6.2 mmol) into the mixture and continued the reaction. After completion of the reaction indicated by the TLC (developing agent ratio: CH₂Cl₂/CH₃OH = 15/1), the mixture was poured into 30 mL of water. The solution was extracted with dichloromethane three times. The organic layers were combined, washed with saturated salt water, dried over MgSO₄, filtered, and concentrated. Purification by column chromatography (silica gel, 0-5% methanol in CH₂Cl₂) gave the compound 2a. Chemical formula: C₁₁H₁₂ClN₃O (MW = 237.69). m.p. 102–104 °C, yield 81%. ¹H-NMR (300 MHz, DMSO-d₆): δ 2.15–2.23 (m, 2H, OCH₂CH₂), 3.80 (t, 2H, J = 6.5 Hz, ClCH₂), 4.16 (t, 2H, J = 6.1 Hz, OCH₂), 7.13 (d, 2H, J = 9.0 Hz, Ph-H), 7.60 (d, 2H, J = 9.0 Hz, Ph-H), 9.00 (s, 2H, N=CH). ¹³C-NMR (75 MHz, DMSO-d₆): δ 158.40, 142.03, 127.75, 123.40, 116.03, 65.22, 42.35, 32.05. As mentioned above, replaced the reactant 1a by the alternative 4-(3-substituted-4H-1,2,4-triazol-4-yl)phenols (1b-1e) to give the compounds 2b-2e. Compounds 2f, 2g, 2h were obtained by the same method as above just replacing 1-bromo-3-chloropropane by 1-bromo-2-chloroethane, 1-bromo-4-chlorobutane, 1-bromo-2-chloropentane, respectively.

4.1.3. Synthesis of compounds 3a-3q

Taking compound 3a as an example: in a 100 mL round-bottom flask with 15 mL of acetonitrile, compound 2a (0.40 g, 1.68 mmol), diethylamine (0.245 g, 3.36 mmol), K₂CO₃ (0.46 g, 3.36 mmol) and potassium iodide (0.56 g, 3.36 mmol) were added one by one. The mixture was heated up to 110 °C for 12–16 h. After cooing the mixture to 40 °C, it was filtered and dried by vacuum to obtain a residue. Purification by column chromatography (silica gel, 0–20% methanol in CH₂Cl₂) gave the compound 3a. The same conditions were used to prepare the compounds 3b-3q. Compounds 3a, 3c, 3d, 3e, 3 g, 3k, 3o, and 3p obtained as oils were transformed into the corresponding hydrochloride by hydrogen chloride in CH₂Cl₂.

4.1.4. Characterisation for the target compounds

4.1.4.1. 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-N,N-diethylpropan-1-amine hydrochloride (3a)

Chemical formula: C₁₅H₂₂N₄O × HCl (MW = 310.83). m.p. 105–106 °C, yield 82%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.25 (t, 6H, J = 7.2 Hz, CH₃), 2.18 (t, 2H, J = 9.0 Hz, OCH₂CH₂), 3.08–3.16 (m, 6H, N(CH₂)₃), 4.17 (t, 2H, J = 6.0 Hz, OCH₂), 7.19 (d, 2H, J = 8.8 Hz, Ph-H), 7.75 (d, 2H, J = 8.8 Hz, Ph-H), 9.84 (s, 2H, N=CH), 10.96 (s, 1H, HCl). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.34, 142.39, 126.44, 124.35, 116.08, 66.00, 48.04, 46.59, 23.40, 8.87. ESI-HRMS calculated for C₁₅H₂₃N₄O⁺ ([M-Cl]⁺): 275.1866; found: 275.1860.

4.1.4.2. 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-N,N-dipropylpropan-1-amine hydrochloride (3b)

Chemical formula: C₁₇H₂₆N₄O × HCl (MW = 338.88). m.p. 180–183 °C, yield 62%. ¹H-NMR (300 MHz, CDCl₃,): δ 1.05 (t, 6H, J = 7.3 Hz, CH₃), 1.77–1.90 (m, 4H, N(CH₂CH₂)₂), 2.27–2.37 (m, 2H, OCH₂CH₂), 3.15 (t, 4H, J = 8.0 Hz, N(CH₂)₂), 3.40 (t, 2H, J = 8.3 Hz, NCH₂), 4.18 (t, 2H, J = 5.6 Hz, OCH₂), 7.05 (d, 2H, J = 8.9 Hz, Ph-H), 7.43 (d, 2H, J = 8.9 Hz, Ph-H), 8.62 (s, 2H, CH=N)., 9.51 (s, 1H, HCl). ¹³C-NMR (75 MHz, CDCl₃): δ 163.08, 146.48, 132.18, 128.41, 120.58, 69.93, 59.42, 55.13, 28.54, 21.98, 15.92. ESI-HRMS calculated for C₁₇H₂₇N₄O⁺ ([M-Cl]⁺): 303.2179; found: 303.2178.

4.1.4.3. 4-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-4H-1,2,4-triazole hydrochloride (3c)

Chemical formula: C₁₅H₂₀N₄O × HCl (MW = 308.81). m.p. 120–122 °C, yield 56%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.92–2.00 (m, 4H, NCH₂CH₂), 2.19–2.22 (m, 2H, OCH₂CH₂), 3.00–3.53 (m, 6H, N(CH₂)₃), 4.17 (t, 2H, J = 5.7 Hz, OCH₂), 7.17 (d, 2H, J = 8.7 Hz, Ph-H), 7.72 (d, 2H, J = 8.7 Hz, Ph-H), 9.75 (s, 2H, N=CH), 11.45 (s, 1H, HCl). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.25, 142.36, 126.57, 124.25, 116.07, 66.03, 53.21, 51.46, 25.53, 23.24. ESI-HRMS calculated for C₁₅H₂₁ClN₄O⁺ ([M-Cl]⁺): 273.1710; found: 273.1711.

4.1.4.4. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperazine dihydrochloride (3d)

Chemical formula: C₁₅H₂₁N₅O × 2HCl (MW = 323.83). m.p. 146–148 °C, yield 52%. ¹H-NMR (300 MHz, CDCl₃): δ 2.20–2.30 (m, 2H, J = 7.0 Hz, OCH₂CH₂), 3.32 (t, 2H, J = 7.8 Hz, NCH₂), 3.37–3.57 (m, 8H, Piperazine-H), 4.17 (t, 2H, J = 5.8 Hz, OCH₂), 7.16 (d, 2H, J = 8.9 Hz, Ph-H), 7.68 (d, 2H, J = 8.9 Hz, Ph-H), 9.44 (s, 2H, CH=N), 10.04 (s, 2H, HCl). ¹³C-NMR (75 MHz, DMSO-d₆): δ 158.91, 142.29, 127.01, 123.96, 116.10, 65.89, 53.41, 48.17, 45.78, 23.50. ESI-HRMS calculated for C₁₅H₂₂N₅O⁺ ([M-2HCl + H]⁺): 288.1819; found: 288.1820.

4.1.4.5. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-methylpiperazine hydrochloride (3e)

Chemical formula: C₁₆H₂₃N₅O × HCl (MW = 337.85). m.p. 220–223 °C, yield 65%. ¹H-NMR (300 MHz, CDCl₃): δ 2.26 (s, 2H, OCH₂CH₂), 2.84 (s, 3H, CH₃), 3.35-3.66 (m, 10H, N(CH₂)₅), 4.17 (t, 2H, J = 5.2 Hz, OCH₂), 7.16 (d, 2H, J = 8.7 Hz, Ph-H), 7.68 (d, 2H, J = 8.7 Hz, Ph-H), 9.46 (s, 2H, CH=N), 10.57 (s, 1H, HCl). ¹³C-NMR (75 MHz, DMSO-d₆): δ 158.93, 142.30, 126.96, 123.99, 116.10, 65.90, 54.53, 48.75, 45.75, 23.60, 15.53. ESI-HRMS calculated for C₁₆H₂₄N₅O⁺ ([M-Cl]⁺): 302.1975; found: 302.1976.

4.1.4.6. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-phenylpiperazine (3f)

Chemical formula: C₂₁H₂₅N₅O (MW = 363.47). m.p. 172–174 °C, yield 64%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.91–1.97 (m, 2H, OCH₂CH₂), 2.47–2.55 (m, 6H, N(CH₂)₃), 3.12–3.15 (m, 4H, N(CH₂)₂), 4.09 (t, 2H, J = 6.2 Hz, OCH₂), 6.76 (t, 1H, J = 7.2 Hz, Ph-H), 6.92 (d, 2H, J = 8.0 Hz, Ph-H), 7.10 (d, 2H, J = 8.9 Hz, Ph-H), 7.2 (t, 2H, J = 7.9 Hz, Ph-H), 7.58 (d, 2H, J = 8.9 Hz, Ph-H), 9.00 (s, 2H, CH=N). ¹³C-NMR (75 MHz, DMSO-d₆): δ 158.70, 151.50, 142.03, 129.36, 127.52, 123.37, 119.21, 116.00, 115.77, 66.81, 54.82, 53.26, 48.68, 26.60. ESI-HRMS calculated for C₂₁H₂₆N₅O⁺ ([M + H]⁺): 364.2132; found: 364.2133.

4.1.4.7. 4-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)morpholine hydrochloride (3g)

Chemical formula: C₁₅H₂₀N₄O₂ × HCl (MW = 324.81). m.p. 237–239 °C, yield 51%. ¹H-NMR (300 MHz, CDCl₃): δ 2.27 (q, 2H, J = 6.0 Hz, OCH₂CH₂), 3.12-3.48 (m, 6H, N(CH₂)₃), 3.92 (t, 4H, J = 7.7 Hz, OCH₂), 4.16 (t, 2H, J = 6.0 Hz, OCH₂), 7.14 (d, 2H, J = 8.9 Hz, Ph-H), 7.67 (d, 2H, J = 8.9 Hz, Ph-H), 9.52 (s, 2H, N=CH), 11.44 (s, 1H, HCl). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.02, 142.22, 126.81, 123.93, 116.01, 65.94, 63.63, 53.95, 51.55, 23.37. ESI-HRMS calculated for C₁₅H₂₁N₄O₂⁺ ([M - Cl]⁺): 289.1659; found: 289.1658.

4.1.4.8. 1–(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3h)

Chemical formula: C₁₆H₂₂N₄O (MW = 286.38). m.p. 249–252 °C, yield 67%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.38–1.91 (m, 6H, NCH₂CH₂(CH₂)₂), 2.20–2.30 (m, 2H, OCH₂CH₂), 2.83–3.44 (m, 6H, N(CH₂)₃), 4.15 (t, 2H, J = 6.0 Hz, OCH₂), 7.17 (d, 2H, J = 8.9 Hz, Ph-H), 7.70 (d, 2H, J = 8.9 Hz, Ph-H), 9.62 (s, 2H, CH=N). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.09, 142.30, 126.78, 124.09, 116.07, 66.20, 53.67, 52.40, 23.59, 22.71, 21.90. ESI-HRMS calculated for C₁₆H₂₃N₄O⁺ ([M + H]⁺): 287.1866; found: 287.1867.

4.1.4.9. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-phenylpiperidine (3i)

Chemical formula: C₂₂H₂₆N₄O (MW = 362.48). m.p. 124–126 °C, yield 50%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.88–2.10 (m, 6H, NCH₂CH₂), 2.17–2.22 (m, 2H, NCH₂), 2.56 (t, 1H, J = 6.0 Hz, NCH₂CH₂CH), 2.64 (t, 2H, J = 7.0 Hz, NCH₂), 3.14 (t, 2H, J = 6.4 Hz, NCH₂), 4.11 (t, 2H, J = 6.0 Hz, OCH₂), 7.03-7.32 (m, 9H, Ph-H), 8.47 (s, 2H, CH=N). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.49, 146.04, 141.92, 128.46, 126.83, 126.71, 126.24, 123.98, 115.85, 66.88, 55.36, 54.42, 42.51, 33.23, 26.68. ESI-HRMS calculated for C₂₂H₂₇N₄O⁺ ([M + H]⁺): 363.2179; found: 363.2178.

4.1.4.10. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine-4-carboxamide (3j)

Chemical formula: C₁₇H₂₃N₅O₂ (MW = 329.40). m.p. 196–198 °C, yield 68%. ¹H-NMR (300 MHz, CDCl₃): δ 1.74–2.06 (m, 7H, NCH₂(CH₂)₃CH), 2.14–3.00 (m, 6H, N(CH₂)₃), 4.07 (d, 2H, J = 6.5 Hz, OCH₂), 5.70 (s, 2H, NH₂), 7.02 (d, 2H, J = 8.9 Hz, Ph-H), 7.28 (d, 2H, J = 8.9 Hz, Ph-H), 8.39 (s, 2H, CH=N). ¹³C-NMR (75 MHz, CDCl₃): δ 177.53, 159.53, 141.87,126.30, 123.98, 115.81, 66.76, 54.96, 53.23, 42.71, 28.94, 26.71. ESI-HRMS calculated for C₁₇H₂₄N₅O₂⁺ ([M + H]⁺): 330.1925; found: 330.1926.

4.1.4.11. 1-(3-(4–(3-methyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine hydrochloride (3k)

Chemical formula: C₁₇H₂₄ClN₄O × HCl (MW = 336.86). m.p. 240–242 °C, yield 61%. ¹H-NMR (300 MHz, CDCl₃ + DMSO-d₆): δ 1.86–2.43 (m, 8H, NCH₂CH₂CH₂), 2.64 (s, 3H, N=CCH₃), 2.94–3.55 (m, 6H, N(CH₂)₃), 4.22 (s, 2H, OCH₂), 7.13 (d, 2H, J = 5.8 Hz, Ph-H), 7.55 (d, 2H, J = 5.8 Hz, Ph-H), 9.33 (s, 1H, CH=N), 11.31 (s, 1H, HCl). ¹³C- NMR (75 MHz, CDCl₃ + DMSO-d₆): δ 164.95, 156.81, 149.03, 132.21, 129.15, 120.75, 70.66, 59.08, 57.74, 28.43, 27.48, 26.65, 15.04. ESI-HRMS calculated for C₁₇H₂₅N₄O⁺ ([M-Cl]⁺): 301.2023; found: 301.2025.

4.1.4.12. 1-(3-(4-(3-phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3l)

Chemical formula: C₂₂H₂₆N₄O (MW = 362.48). m.p. 88–90 °C, yield 59%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.68 (s, 2H, NCH₂CH₂CH₂), 1.84–1.92 (m, 4H, NCH₂(CH₂)₂, 2.18–2.30 (m, 2H, OCH₂CH₂), 3.18-3.25 (m, 6H, N(CH₂)₃), 4.13 (t, 2H, J = 6.0 Hz, OCH₂), 7.00 (d, 2H, J = 8.9 Hz, Ph-H), 7.23 (d, 2H, J = 8.9 Hz, Ph-H), 7.32-7.46 (m, 5H, Ph-H), 8.45 (s, 1H, CH=N). ¹³C-NMR (75 MHz, CDCl₃): δ 163.64, 157.72, 150.07, 134.62, 133.40, 133.26, 132.35, 132.05, 131.39, 120.31, 70.19, 59.33, 58.11, 28.99, 28.09, 26.66. ESI-HRMS calculated for C₂₂H₂₇N₄O⁺ ([M + H]⁺): 363.2179; found: 363.2178.

4.1.4.13. 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3m)

Chemical formula: C₂₂H₂₅ClN₄O (MW = 396.92). m.p. 165–167 °C, yield 75%. ¹H-NMR (300 MHz, CDCl₃): δ 1.54 (s, 2H, NCH₂CH₂CH₂), 1.72 (s, 4H, NCH₂(CH₂)₂), 2.09–2.14 (m, 2H, OCH₂CH₂), 3.08–3.33 (m, 6H, N(CH₂)₃), 4.10 (t, 2H, J = 6.0 Hz, OCH₂), 7.06 (d, 2H, J = 8.8 Hz, Ph-H), 7.34 (d, 2H, J = 8.8 Hz, Ph-H), 7.41 (d, 2H, J = 8.6 Hz, Ph-H), 7.48 (d, 2H, J = 8.6 Hz, Ph-H), 8.79 (s, 1H, CH=N). ¹³C-NMR (75 MHz, DMSO-d₆): δ 159.14, 151.96, 146.43, 135.01, 130.54, 129.21, 128.04, 127.56, 126.12, 115.85, 65.98, 54.08, 52.99, 29.45, 24.31, 23.56. ESI-HRMS calculated for C₂₂H₂₆ClN₄O⁺ ([M + H]⁺): 397.1790; found: 397.1791.

4.1.4.14. 1-(3-(4-(3-([1,1’-biphenyl]-4-yl)-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3n)

Chemical formula: C₂₈H₃₀N₄O (MW = 438.58). m.p. 66–68 °C, yield 58%. ¹H-NMR (300 MHz, DMSO-d₆): δ 1.73 (s, 2H, NCH₂CH₂CH₂), 2.01–2.09 (m, 4H, NCH₂CH₂), 2.39–2.48 (m, 2H, OCH₂CH₂), 3.12–3.20 (m, 6H, N(CH₂)₃), 4.14 (t, 2H, J = 6.0 Hz, OCH₂), 6.96 (d, 2H, J = 8.8 Hz, Ph-H), 7.20 (d, 2H, J = 8.8 Hz, Ph-H), 7.35-7.58 (m, 9H, Ph-H), 8.34 (s, 1H, CH=N). ¹³C-NMR (75 MHz, DMSO-d₆): δ 158.97, 153.04, 145.16, 142.55, 139.81, 128.89, 128.86, 127.91, 127.63, 127.63, 127.22, 127.01, 125.05, 115.61, 65.70, 55.17, 53.69, 24.32, 23.08, 22.30. ESI-HRMS calculated for C₂₈H₃₁N₄O⁺ ([M + H]⁺): 439.2492; found: 439.2494.

4.1.4.15. 1-(2-(4–(4H-1,2,4-triazol-4-yl)phenoxy)ethyl)piperidine hydrochloride (3o)

Chemical formula: C₁₅H₂₀N₄O × HCl (MW = 308.81). m.p. 85–87 °C, yield 72%. ¹H-NMR (300 MHz, CDCl₃): δ 1.63–1.96 (m, 6H, NCH₂(CH₂)₂CH₂), 2.95–3.07 (m, 2H, OCH₂CH₂), 3.48 (t, 4H, J = 5.5 Hz, N(CH₂)₂), 4.55 (t, 2H, J = 5.1 Hz, OCH₂), 7.23 (d, 2H, J = 8.8 Hz, Ph-H), 7.75 (d, 2H, J = 8.8 Hz, Ph-H), 9.69 (s, 2H, CH=N), 11.21 (s, 1H, HCl). ¹³C-NMR (75 MHz, CDCl₃): δ 158.38, 142.33, 127.14, 124.20, 116.33, 63.37, 54.89, 53.01, 22.70, 21.68. ESI-HRMS calculated for C₁₅H₂₁N₄O⁺ ([M-Cl]⁺): 273.1710; 273.1711.

4.1.4.16. 1-(4–(4-(4H-1,2,4-triazol-4-yl)phenoxy)butyl)piperidine hydrochloride (3p)

Chemical formula: C₁₇H₂₄N₄O × HCl (MW = 336.86). m.p. 80–82 °C, yield 69%. ¹H-NMR (300 MHz, CDCl₃): δ 1.88–2.21 (m, 8H, NCH₂(CH₂)₃CH₂), 2.22–2.36 (m, 2H, OCH₂CH₂), 2.82–3.73 (m, 6H, N(CH₂)₃), 4.08 (t, 2H, J = 5.8 Hz, OCH₂), 7.00 (d, 2H, J = 8.9 Hz, Ph-H), 7.30 (d, 2H, J = 8.9 Hz, Ph-H), 8.49 (s, 2H, CH=N). 10.22 (s, 1H, HCl). ¹³C-NMR (75 MHz, CDCl₃): δ 158.93, 141.86, 126.87, 123.86, 115.89, 67.27, 56.93, 53.26, 26.37, 22.50, 21.88, 20.76. ESI-HRMS calculated for C₁₇H₂₅N₄O⁺ ([M-Cl]⁺): 301.2023; found: 301.2021.

4.1.4.17. 1-(5-(4-(4H-1,2,4-triazol-4-yl)phenoxy)pentyl)piperidine (3q)

Chemical formula: C₁₈H₂₆N₄O × HCl (MW = 314.43). m.p. 70–74 °C, yield 77%. ¹H-NMR (300 MHz, CDCl₃): δ 1.56–2.09 (m, 14H, NCH₂(CH₂)₃(CH₂)₂CH₂), 3.04–3.09 (m, 4H, N(CH₂)₂), 4.01 (t, 2H, J = 6.0 Hz, OCH₂), 7.00 (d, 2H, J = 8.8 Hz, Ph-H), 7.30 (d, 2H, J = 8.8 Hz, Ph-H), 8.44 (s, 2H, CH=N). ¹³C-NMR (75 MHz, CDCl₃): δ 159.25, 141.88, 126.69, 123.91, 115.86, 67.76, 57.25, 53.28, 28.32, 23.37, 22.49, 21.92, 21.88. ESI-HRMS calculated for C₁₈H₂₇N₄O⁺ ([M + H]⁺): 315.2179; found: 315.2180.

4.2. Pharmacology

4.2.1. In vitro screening

4.2.1.1. Cell culture and transfection

Human thalamus poly-A RNA (Clontech, Palo Alto, CA, USA) was used to clone the hH₃R gene by RT-PCR. DNA PCR primers were designed in the light of the reported human histamine receptor gene sequences (GenBank accession no.AF140538). HEK-293 cells were cultured and transfected for the luciferase assay. The detailed procedures were described in the previous publication⁴⁰.

4.2.1.2. CRE-driven reporter gene assay

Stable HEK-293 cells, which had been co-transfected with H₃R and pCRE-Luc, were seeded in a 96-well plate overnight, and were grown to 90–95% confluence^43,44. Then the cells were treated with various concentrations of tested compounds in serum-free DMEM and incubated for 20 min. Cells were then stimulated with 100 nM Histamine in serum-free DMEM containing 2 µM Forskolin and incubated for 4 h at 37 °C. Firefly luciferase assay kits (Ken-real, Shanghai, China) were used to determine the luciferase activity.

4.2.2. In vivo pharmacology

4.2.2.1. Drugs and animals

Valproic acid (VPA) was obtained from melongpharma, Dalian, China. Ciproxifan maleate was purchased from Shanghai Hanxiang Biotechnology Co., Ltd, China. R-(α)-methyl-histamine (RAMH), Pitolisant (PIT), and Pentylenetetrazol (PTZ) were bought from Macklin Co. KunMing mice were purchased from Changsha tianqin Biotechnology Co., Ltd, China and used with the body weight 20–25 g. Procedures involving animals were performed according to the Guide for the Care and Use of Laboratory Animals (8th Edition, National Academies Press, Washington, DC), and was approved by the local animal ethics committee (Institutional Animal Ethics Committee of Jinggangshan University, approval number: 201906018).

4.2.2.2. MES-induced seizure

Ear stimulation with alternating current (0.2 s, 60 Hz, 50 mA) was used to induce the seizures in mice. The reduction or abolition of the hind limb tonic extension (THLE) of mice was considered protective against the MES-induced seizures^29,48. Test compounds and positive drugs VPA (300 mg/kg) and PIT (10 mg/kg) were i.p. administrated half an hour prior to the electric stimulation. To investigate the mechanism of action, the most promising one 3m was chose for a further test. In one group of animals of seven mice, compound 3m (10 mg/kg) co-injected with RAMH 10 mg/kg with 5 min interval. The animals in other three groups were single treated with RAMH 10 mg/kg, compound 3m 10 mg/kg, and vehicle, respectively.

4.2.2.3. PTZ-induced seizures

PTZ (85 mg/kg) was injected subcutaneously to induce seizures. Firstly, vehicle, tested compounds 3a-3q (10 mg/kg), and positive controls (VPA 300 mg/kg, PIT 10 mg/kg) were administered i.p. After 30 min, PTZ was injected to all the animals. Animals were observed for 30 min (experiment period) for any convulsion signs, and graded scores were used to assess the seizures severity. The following are the specific meaning of graded scores: score 0 = normal, score 1 = eyelids or facial twitches, score 2 = agitation with body twisting, score 3 = myo-clonic jerks or rearing, score 4 = turn over into one side position or running violently, and score 5 = turn over into back position, limbs tonic extension, or die during the experiment period³⁰.

4.2.3. Statistics

GraphPad Prism was used for statistical analysis. All data in vivo were presented as the mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA), followed by the Dunnett’s post-test were conducted for multiple comparisons. The statistical significance was defined as p values <0.05.

4.2.4. Docking

4.2.4.1. Homology modelling

Crystal structure of the histamine H₁ receptor (PDB ID: 3RZE) was used to construct the H₃ receptor homology model⁴⁵. The hH₃R primary sequence was downloaded from the Universal Protein Resource (UniProt ID: Q9Y5N1). DS MODELLER (Discovery Studio 2019) was used to construct a 3D model of the H₃R. Then the model was assessed in accordance with the PDF Total Energy and the Profile-3D procedure. The 3D model of H₃R with the lowest PDF total energy was selected for the next docking test.

4.2.4.2. Molecular docking

To consider the conformation of the protein and its ligands, Flexible Docking (Discovery Studio 2019) was used for the docking procedure. The initial ligand and water were removed, and hydrogen atoms were added. Three-dimensional structures of compounds 3h, 3m as well as PIT were generated and then placed into the protein structure during the molecular docking procedure. Interactions of the protein with 3h, 3m, and PIT were analysed.

Acknowledgements

The School of Life Sciences, Jinggangshan University was appreciated for providing the BIOVIA Discovery Studio software in this study.

Disclosure statement

The authors declare that there is no conflict of interest.

Additional information

Funding

Thanks for the financial support provided by the National Natural Science Foundation of China [No. 21562028].