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Article

Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones

by
Yi Hua
1,†,
Mingxia Song
1,†,
Qiaoyue Guo
1,
Yiqin Luo
1,
Xianqing Deng
1,* and
Yushan Huang
2,*
1
Health Science Center, Jinggangshan University, Ji’an 343009, China
2
Center for Evidence Based Medical and Clinical Research, First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2023, 28(8), 3408; https://doi.org/10.3390/molecules28083408
Submission received: 19 March 2023 / Revised: 10 April 2023 / Accepted: 10 April 2023 / Published: 12 April 2023
(This article belongs to the Special Issue Design, Synthesis, and Evaluations of Antidepressant/Anticonvulsant/Anti-Alzheimer Agents)
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Abstract

:
H3R is becoming an attractive and promising target for epilepsy treatment as well as the discovery of antiepileptics. In this work, a series of 6-aminoalkoxy-3,4-dihydroquinolin-2(1H)-ones was prepared to screen their H3R antagonistic activities and antiseizure effects. The majority of the target compounds displayed a potent H3R antagonistic activity. Among them, compounds 2a, 2c, 2h, and 4a showed submicromolar H3R antagonistic activity with an IC50 of 0.52, 0.47, 0.12, and 0.37 μM, respectively. The maximal electroshock seizure (MES) model screened out three compounds (2h, 4a, and 4b) with antiseizure activity. Meanwhile, the pentylenetetrazole (PTZ)-induced seizure test gave a result that no compound can resist the seizures induced by PTZ. Additionally, the anti-MES action of compound 4a fully vanished when it was administrated combined with an H3R agonist (RAMH). These results showed that the antiseizure role of compound 4a might be achieved by antagonizing the H3R receptor. The molecular docking of 2h, 4a, and PIT with the H3R protein predicted their possible binding patterns and gave a presentation that 2h, 4a, and PIT had a similar binding model with H3R.

1. Introduction

Histamine, an endogenous biological amine, mediates various physiological and pathological processes by acting on four histamine receptors in the G protein-coupled receptor (GPCR) family [1]. Among them, histamine H1 receptors (H1R) and histamine H2 receptors (H2R) are related to anaphylactic reaction and the secretion of gastric acid, respectively. Histamine H4 receptors (H4R) are mainly involved in the human body’s immune response. Histamine H3 receptors (H3R) are mainly expressed in the central nervous system (CNS). As one of the auto-receptors, it negatively regulates the synthesis and secretion of histamine in the CNS [2]. Recently it was reported as relating to the physiological processes of sleep, awakening, appetite, and learning and memory [3,4]. H3R has become a potentially important target for the diseases of narcolepsy, Alzheimer’s, schizophrenia, learning and memory disorders, and epilepsy [5,6].
In particular, as an inhibitory heteroreceptor, H3R can affect the level of some neurotransmitters, such as serotonin, norepinephrine, γ-aminobutyric acid, and glutamate in the CNS related to epilepsy [7,8,9]. It has been proved that H3R antagonists can treat epilepsy by promoting the synthesis and secretion of histamine [10,11,12]. In addition, some studies have reported that H3R antagonists have obvious protective effects on NMDA-induced neuronal damage and cell death [13,14,15]. Therefore, increasing attention has been focused on H3R in epilepsy treatment.
In the last two decades, increasing antagonists or inverse agonists of H3R have been prepared and identified with antiseizure activity in several kinds of epileptic animal models [16,17,18]. Pitolisant (PIT), an H3R antagonist and inverse agonist, was approved in the EU for narcolepsy treatment. Meanwhile, it also has been subjected to clinical Phase II trials to treat photosensitive epilepsy [19]. The results showed that PIT can inhibit photosensitive epilepsy and gave a profitable electroencephalogram (EEG) performance when taken at the dose of 30 mg or 60 mg [20]. In addition, PIT displayed a powerful antiseizure activity in the electrical kindling model of epilepsy [21].
In our previous work, dozens of 2-methyl-4-phenyloxazoles were designed and synthesized as new H3R antagonists [22]. All these compounds showed micromolar to submicromolar H3R antagonistic activities. In addition, some of them displayed an antiseizure activity in the maximal electroshock seizure (MES) test. It is interesting to note that the antiseizure activity of the representative compound I (Figure 1, I) completely vanished when it was administrated combined with an H3R agonist R-(α)-methylhistamine (RAMH), which confirmed the correlation between H3R inhibition and antiepileptic activity.
To continue the program of developing antiseizure drugs from an H3R antagonist, herein, the 2-methyl-4-phenyloxazole moiety of the compound I was interchanged by 3,4-dihydroquinolin-2(1H)-one to obtain a potential H3R antagonist (Figure 1, compound 2h). The 3,4-dihydroquinolin-2(1H)-one is an eminent pharmaceutical skeleton, which has been utilized to obtain great amounts of compounds with an antiseizure activity [23,24,25]. The hybridization of compounds I and II is expected to obtain new H3R antagonists with better antiseizure activity. Apart from compound 2h, analogs (2a-2g, 2i) using various kinds of amines replacing the piperidine group in compound 2h, and derivatives (3a-3c, 4a-4b) via adjusting the length of the link and introducing the substituents at the N atom of amide, were also synthesized.
All the titled compounds were synthesized smoothly and structurally confirmed by 1NMR, 13NMR, and HR-MS. Luciferase assay based on the cAMP-response element (CRE) was used to screen the H3R antagonism activity. Two widely used epileptic animal models, the MES model and pentylenetetrazole (PTZ)-induced seizure model, were applied to evaluate the antiseizure activity. In addition, molecular docking was carried out to understand the molecular basis of the H3R antagonistic activity of the prepared compounds. We hope that through this study, a new H3R antagonistic skeleton can be investigated to accelerate the discovery of new antiseizure drugs.

2. Results and Discussion

2.1. Chemistry

The synthetic route of compounds 2a-2i through a two-step route is shown in Scheme 1. Compounds 3a-3c and 4a-4b were prepared smoothly as shown in Scheme 2. Briefly, compounds 1a-1d were synthesized by the reaction of 6-hydroxyquinolinone with dihaloalkane in the presence of K2CO3. The substitution reaction of compound 1a with appropriate secondary amines gave compounds 2a-2i. The substitution reaction of compounds 1b-1d with piperidine provided compound 3a-3c. Finally, compounds 4a and 4b were achieved by an alkylation of compound 2h.

2.2. Pharmacology

2.2.1. Screen for H3R Antagonistic Activity

CRE luciferase reporter assays are widely applied for the high-throughput screening of GPCR agonists and antagonists [26,27], which was used to screen the H3R antagonistic activities of the titled compounds (2a-2i, 3a-3c, and 4a-4b) in this work. PIT was used as a positive drug.
The results of targets 2a-2i, 3a-3c, and 4a-4b as H3R antagonists are summarized in Table 1. The majority of the synthesized compounds displayed outstanding H3R antagonistic activities. Among them, compounds 2a (IC50 = 0.52 μM), 2c (IC50 = 0.47 μM), 2h (IC50 = 0.12 μM), and 4a (IC50 = 0.37 μM) displayed antagonistic activities at a submicromolar level. PIT showed H3R antagonistic activity with IC50 of 0.69 μM at the same condition. When the diethyl in compound 2a was replaced by dipropyl, the H3R antagonistic activity decreased significantly with the IC50 of compound 2b higher than 50 μM. When a phenyl group was introduced onto the piperidine of compound 2h, the H3R antagonistic activity of compound 2i declined dozens of times. This suggested that substituted tertiary amine with appropriate volume is required for the H3R antagonistic activity. Altering the link length of compound 2h to obtain compounds 3a, 3b, and 3c. It can be seen that the best chain length of the carbons is three, which gave the compound 2h an IC50 of 0.12 μM. The H3R antagonistic activity decreased when the chain length became longer or shorter. Among the cyclic amine compounds, the pyrrole and piperidine substituted compounds (2c and 2h) showed higher antagonistic activities than the compounds coupled with piperazine and morpholine (2d-2g). This may be the result of the lipophilic binding of the tertiary amine with the receptor [28]

2.2.2. Evaluation of the Antiseizure Activity

To find new antiseizure candidates, the antiseizure activities of the titled compounds were evaluated via two seizure models in mice, while using PIT and antiepileptic valproic sodium (VPA) as positive controls. One of the two test models is the MES seizure model, another is the PTZ-induced seizure model. In our previous work, we found that the synthetic H3R antagonists and PIT showed an antiseizure activity at 10 mg/kg [22]. In addition, the administration of this dosage can minimize the impact of other possible side effects such as central inhibition and cardiac toxicity. The dose of the H3R antagonists 2a-2i, 3a-3c, 4a-4b, and PIT was chosen as 10 mg/kg. In addition, the tested dose of VPA was 300 mg/kg.
It could be seen that all animals in the control group experienced hindlimb stiffness after the electrical stimulation in the MES model. In the treated group, the definition of protection was the reduction or vanishing of the tonic hind limb extension (THLE) in mice. As shown in Figure 2, compounds 2h, 4a, and 4b as well as PIT and VPA decreased the duration of THLE significantly (p < 0.05, or p < 0.001), and showed good protection for mice. The majority of the compounds showing H3R antagonistic activity in Table 1 did not exhibit antiseizure activity in the MES model. However, compounds 2a, 2c, and 2h hold higher H3R antagonistic activity and displayed a descending effect for the duration of THLE, although the effect of compounds 2a and 2c has no significant difference. Compounds 4a and 4b showed an antiseizure activity in the MES with a significant decrease in the THLE in mice. The two compounds were substituted by an alkyl group on the quinolinone, which increased their ClogP value (as seen in Table 1). This may be an important contributor to their in vivo antiseizure activity in the MES test, because marketed antiepileptics usually have a high ClogP to assure their penetration through the blood–brain barrier and run up to the site of action.
The PTZ model was usually used to screen the antiepileptic candidates for the absence of seizures. In this work, compounds 2a-2i, 3a-3c, and 4a-4b were also evaluated for their antiseizure action in the PTZ model in mice, while using VPA as the positive drug. As shown in Figure 3, no compound relieved the seizures induced by PTZ at the dose of 10 mg/kg (i.p.). PIT also did not exhibit protection in mice at the same dose. While VPA fully inhibited the seizure induced by PTZ when pretreated with the dosage of 300 mg/kg. The failure of synthesized compounds in the PTZ model is expected because our previously reported H3R inhibitors were also ineffective in the PTZ model [22]. The failure of the PIT in the PTZ model also occurred in Sadek‘s study [29]. These contradictory effects observed for the synthesized H3R inhibitors and PIT in the MES and PTZ models might relate to the different levels of histamine release resulting from the seizures in different seizure models [17] A considerable increase in histamine levels was found in the brain after MES-induced seizures, whereas a tendency toward a decrease in histamine levels was observed after PTZ-kindled convulsions [30].
Based on the better performance of compounds 2h, 4a, and 4b in the MES seizure model, they were selected for further studies. To obtain the accurate effective dose of compounds 2h, 4a, and 4b in the MES model, different dosages of three compounds were applied in the MES test. Inspiringly, the protective effect was obtained in a dose-dependent manner, although the effects at the lowest dosage (3 mg/kg) have no significant difference. As shown in Figure 4, the H3R antagonist PIT also exhibited a dose-dependent antiseizure activity. In particular, PIT fully abolished the THLE induced by MES at 30 mg/kg, confirming its potential antiseizure activity.
A rotarod test was carried out to estimate the neurological safety of compounds 2h, 4a, and 4b. As described in Table 2, no compound showed neurotoxicity at the dose of 10 mg/kg and 30 mg/kg. Compound 2h showed neurotoxicity with one mouse in three at 100 mg/kg, while compounds 4a, 4b and PIT showed no neurotoxicity at 100 mg/kg. At the dose of 300 mg/kg, all animals treated with compound 2h and two-thirds of mice treated with compounds 4a and 4b were dead, which indicated that these compounds showed toxicity at the higher dose.
Up to now, we have screened out some antiepileptic compounds from the H3R antagonists belonging to 3,4-dihydroquinolin-2(1H)-ones, especially from the molecules with a strong H3R inhibitory activity. However, whether their antiepileptic activity comes from their antihistamine activity is unknown. To make this clear, the ability of compound 4a to decrease the duration of THLE in the MES model was re-evaluated in the mice pretreated by RAMH (10 mg/kg, i.p.). RAMH, as a CNS-penetrant histamine H3R agonist, can vanish or weaken the antihistamine effects of histamine antagonists. As shown in Figure 5, when co-injected with RAMH, compound 4a cannot decrease the duration of THLE (p > 0.05). This result confirmed that the H3R antagonism of 4a was an important mechanism for its antiseizure activity.

2.3. Molecular Docking

To make clear the molecular binding mode of the titled compounds with H3R and understand the molecular basis of their H3R antagonistic activity, the molecular docking of 2h, 4a, and PIT with the H3R protein was carried out. In this docking study, the 3D structure of H3R was constructed from the structure-known H1R protein (PDB ID: 3RZE) by homology modeling [31].
As shown in Figure 6, compounds 2h, 4a, and PIT bound to H3R in the same binding pocket and had similar binding patterns. The piperidine group in the compounds 2h and 4a formed a Pi–Pi interaction with the amino acid residue Pro184 of H3R. The phenyl group on the quinoline ring bound to the amino acid residues Tyr115 and Met378 with Pi–Pi force and alkyl interaction, respectively. The amide group in compound 2h formed a critical H-bond interaction with Glu206 and Ser203, while this interaction vanished in the docking of compound 4a because of the hinder of the N-substitution. This may be the important reason for the lower H3R antagonistic activity of 4a than 2h. However, the propyl group in compound 4a formed hydrophobic interactions with Tyr374 and Met378. PIT and H3R interacted through the similar amino acid residues Tyr115, Pro184, His187, Ala190, Ala202, Glu206, Trp371, Met378, and so on. The overlying pattern of compounds 2h, 4a, and PIT was shown in Figure 7, which vividly presented that 2h, 4a, and PIT had a similar binding model with H3R. The docking score for compound 2h and H3R protein binding pocket was obtained to be 115.14, which is higher than that of compound 4a and PIT with a score of 109 and 103.19, respectively. The docking score is consistent with their antihistamine effects. These results supported the suggestion that compounds 2h and 4a play their antiseizure effects by binding and inhibiting the H3R receptor, with a similar binding model to PIT.

3. Materials and Methods

3.1. Chemical Part

Unless otherwise specified, the reagents used in this work were bought from Macklin Inc. All the reactions were monitored by thin-layer chromatography (TLC). After purification, the products were sent to the analysis center for a structure confirmation. The NMR spectrums were measured on a Bruker AV-300 spectrometer. The HR-MS of compounds was measured on a Xevo G2-XS QTOF mass spectrometer.

3.1.1. Synthesis Procedure of 6-(Chloroalkoxy)-3,4-dihydroquinolin-2(1H)-one (1a-1d)

Taking compound 1a as an example: 6-hydroxyquinolinone (1.63 g, 10 mmol), 1-bromo-3-chloropropane (1.87 g, 12 mmol), and potassium carbonate (2.76 g, 20 mmol) were added into a round-bottomed flask with 20 mL acetonitrile. After refluxing the mixture for 24 h, the finish of the reaction was identified by the TLC monitoring with 25% ethyl acetate in petroleum ether. Then the solvent was removed and the leavings were purified using silica gel column chromatography (1% methanol in DCM) to obtain the compound 1a. The compounds 1b-1d were obtained according to the above method using the other haloalkanes. The characterization for the four compounds is listed below.
6-(3-Chloropropoxy)-3,4-dihydroquinolin-2(1H)-one (1a) Mp 99–102 °C, yield 79%. 1H-NMR (CDCl3, 300 MHz): δ 2.20–2.27 (m, 2H, OCH2CH2), 2.63 (t, 2H, J = 8.5 Hz, CH2), 2.95 (t, 2H, J = 8.5 Hz, CH2), 3.75 (t, 2H, J = 6.3 Hz, ClCH2), 4.09 (t, 2H, J = 6.3 Hz, OCH2), 6.72–6.81 (m, 3H, Ph-H), 9.32 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 171.9, 154.7, 131.1, 125.0, 116.4, 114.5, 113.2, 64.7, 41.5, 32.3, 30.6, 25.7. HR-MS (ESI) calcd for C12H15ClNO2+ ([M + H]+): 240.0786; found: 240.0791.
6-(2-Chloroethoxy)-3,4-dihydroquinolin-2(1H)-one (1b) Mp 151–152 °C, yield 81%. 1H-NMR (CDCl3, 300 MHz): δ 2.63 (t, 2H, J = 8.6 Hz, CH2), 2.96 (t, 2H, J = 8.5 Hz, CH2), 3.81 (t, 2H, J = 5.8 Hz, ClCH2), 4.21 (t, 2H, J = 5.8 Hz, OCH2), 6.76–6.78 (m, 3H, Ph-H), 8.76 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 171.5, 154.1, 131.5, 125.2, 116.2, 115.0, 113.5, 68.6, 41.9, 30.6, 25.7. HR-MS (ESI) calcd for C11H13ClNO2+ ([M + H]+): 226.0629; found: 226.0628.
6-(4-Chlorobutoxy)-3,4-dihydroquinolin-2(1H)-one (1c) Mp 146–148 °C, yield 71%. 1H-NMR (CDCl3, 300 MHz): δ 1.91–1.99 (m, 4H, OCH2(CH2)2), 2.61 (t, 2H, J = 7.6 Hz, CH2), 2.93 (t, 2H, J = 7.7 Hz, CH2), 3.62 (t, 6H, J = 5.8 Hz, N-CH2), 3.96 (t, 2H, J = 6.2 Hz, O-CH2), 6.69–6.75 (m, 3H, Ph-H), 8.85 (s, 1H, CONH). 13C-NMR (CDCl3, 75 MHz): δ 171.6, 154.9, 130.9, 125.0, 116.3, 114.5, 113.1, 67.4, 44.7, 30.6, 29.3, 26.7, 25.7. HR-MS (ESI) calcd for C13H17ClNO2+ ([M + H]+): 254.0942; found: 254.0947.
6-((5-Chloropentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (1d) Mp 128–130 °C, yield 72%. 1H-NMR (CDCl3, 300 MHz): δ 1.59–1.93 (m, 6H, OCH2(CH2)3), 2.62 (t, 2H, J = 7.6 Hz, COCH2), 2.93 (t, 2H, J = 7.6 Hz, PhCH2), 3.56 (t, 2H, J = 5.9 Hz, ClCH2), 3.93 (t, 2H, J = 6.2 Hz, OCH2), 6.69–6.81 (m, 3H, Ph-H), 9.51 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 172.0, 154.9, 130.9, 124.9, 116.4, 114.4, 113.1, 68.0, 44.9, 32.3, 30.6, 28.6, 25.7, 23.5. HR-MS (ESI) calcd for C14H19ClNO2+ ([M + H]+): 268.1099; found: 268.1106.

3.1.2. The Synthesis Procedure of 6-(3-(Substituted-aminopropoxy)-3,4-dihydroquinolin-2(1H)-one (2a-2i) and Its Analogues (3a-3c)

Taking compound 2a as an example: compounds 1a (1.20 g, 5 mmol), diethylamine (0.73g, 10 mmol), K2CO3 (1.38 g, 10 mmol), and KI (1.0 g, 6 mmol) were added to a round-bottomed flask with 20 mL acetonitrile. After refluxing the mixture for 24 h, the finish of the reaction was confirmed by the TLC monitoring with 50% ethyl acetate in a petroleum ether. After cooling to room temperature, the mixture was filtered, and the filtrate was evaporated to obtain a crude product, which was purified on silica gel column chromatography (1.5% MeOH in DCM) to obtain compound 2a. Compounds 2b-2i were prepared according to the above method with the compound 1a and other secondary amines. The compounds 3a (3b, 3c) were prepared according to the above method with the compounds 1b (1c, 1d) and piperidine. The characterization for these compounds is listed below.
6-(3-(Diethylamino)propoxy)-3,4-dihydroquinolin-2(1H)-one hydrochioride (2a) Mp 68–70 °C, yield 76%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.23 (t, 6H, J = 7.6 Hz, CH3), 2.08–2.15 (m, 2H, CH2), 2.38 (t, 2H, J = 7.5 Hz, CH2), 2.82, (t, 2H, J = 7.5 Hz, CH2), 3.05–3.17 (m, 6H, N-CH2), 4.00 (t, 2H, J = 6.0 Hz, O-CH2), 6.72–6.81 (m, 3H, Ph-H), 9.93 (s, 1H, CONH), 10.81 (s, 1H, HCl). 13C-NMR (DMSO-d6, 75 MHz): δ 170.21, 153.36, 132.46, 125.34, 116.25, 114.65, 113.53, 65.64, 46.57, 43.13, 30.79, 25.53, 23.54, 8.87. HR-MS (ESI) calcd for C16H25N2O2+ ([M + H]+): 277.1911; found: 277.1912.
6-(3-(Dipropylamino)propoxy)-3,4-dihydroquinolin-2(1H)-one hydrochloride (2b) Mp 57–59 °C, yield 71%. 1H-NMR (DMSO-d6, 300 MHz): δ 0.96 (t, 6H, J = 7.5 Hz, CH3), 1.71–1.84 (m, 4H, CH2), 2.17–2.22 (m, 2H, CH2), 2.43 (t, 2H, J = 7.5 Hz, CH2), 2.84 (t, 2H, J = 7.5 Hz, CH2), 2.98–3.05 (m, 4H, N-CH2), 3.20–3.26 (m, 2H, N-CH2), 3.99 (t, 2H, J = 5.6 Hz, O-CH2), 6.61–6.79 (m, 3H, Ph-H), 9.84 (s, 1H, CONH), 10.73 (s, 1H, HCl). 13C-NMR (DMSO-d6, 75 MHz): δ 175.10, 158.50, 137.05, 129.70, 121.09, 119.07, 118.00, 70.02, 59.02, 54.72, 35.56, 30.48, 28.39, 21.78, 16.03. HR-MS (ESI) calcd for C18H29N2O2+ ([M + H]+): 305.2224; found: 305.2223.
6-(3-(Pyrrolidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one hydrochioride (2c) Mp 120–122 °C, yield 83%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.86–2.07 (m, 6H, CH2), 2.40 (t, 2H, J = 7.5 Hz, CH2), 2.84 (t, 2H, J = 7.5 Hz, CH2), 3.05 (s, 2H, N-CH2), 3.33 (s, 2H, N-CH2), 3.59 (s, 2H, N-CH2), 4.00 (s, 2H, J = 6.0 Hz, O-CH2), 6.3–6.80 (m, 3H, Ph-H), 9.44 (s, 1H, CONH), 9.90 (s, 1H, HCl). 13C-NMR (DMSO-d6, 75 MHz): δ 170.22, 153.79, 132.50, 125.34, 116.24, 114.60, 113.59, 65.41, 53.89, 51.94, 30.80, 25.89, 25.55, 23.06. HR-MS (ESI) calcd for C16H23N2O2+ ([M + H]+): 275.1754; found: 275.1755.
6-(3-(Piperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (2d) Mp 150–151 °C, yield 78%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.77–1.86 (m, 2H, CH2), 2.37–2.51 (m, 12H, CH2), 2.83 (t, 2H, J = 7.2 Hz, N-CH2), 3.92 (t, 2H, J = 6.1 Hz, O-CH2), 6.69–6.76 (m, 3H, Ph-H), 9.86 (s, 1H, N-H). 13C-NMR (DMSO-d6, 75 MHz): δ 170.19, 154.28, 132.15, 125.28, 116.23, 114.48, 113.45, 66.53, 55.15, 53.11, 46.11, 30.84, 26.76, 25.57, HR-MS (ESI) calcd for C16H24N3O2+ ([M + H]+): 290.1863; found: 290.1864.
6-(3-(4-Methylpiperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (2e) Mp 159–160 °C, yield 79%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.49–1.67 (m, 7H, CH2, NCH3), 1.95–2.05 (m, 2H, CH2), 2.40 (t, 2H, J = 7.6 Hz, N-CH2), 2.81–2.89 (m, 8H, N-CH2), 3.97 (t, 2H, J = 6.2 Hz, O-CH2), 6.70–6.79 (m, 3H, Ph-H), 9.89 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 170.19, 153.98, 132.39, 125.32, 116.24, 114.57, 113.56, 65.98, 54.54, 53.35, 30.82, 25.56, 25.01, 24.13, 22.75. HR-MS (ESI) calcd for C17H26N3O2+ ([M + H]+): 304.2020; found: 304.2015.
6-(3-(4-Phenylpiperazin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (2f) Mp 199–201 °C, yield 80%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.89 (t, 2H, J = 6.2 Hz, CH2), 2.37–2.51 (m, 8H, CH2), 2.83 (t, 2H, J = 7.3 Hz, CH2), 3.12 (s, 4H, CH2), 3.96 (t, 2H, J = 6.2 Hz, O-CH2), 6.75–6.79 (m, 4H, Ph-H), 6.92 (d, 2H, J = 7.2 Hz, Ph-H), 7.21 (t, 2H, J = 8.6 Hz, Ph-H), 9.89 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 170.20, 154.28, 151.51, 132.17, 129.36, 125.29, 119.20, 116.23, 115.77, 114.50, 113.46, 66.54, 54.95, 53.27, 48.67, 30.84, 26.76, 25.57. HR-MS (ESI) calcd for C22H28N3O2+ ([M + H]+): 366.2176; found: 366.2177.
6-(3-(Morpholino)propoxy)-3,4-dihydroquinolin-2(1H)-one (2g) Mp 146–147 °C, yield 79%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.83–1.92 (m, 2H, CH2), 2.40–2.46 (m, 8H, CH2), 2.86 (t, 2H, J = 7.3 Hz, N-CH2), 3.62 (t, 4H, J = 4.5 Hz, N-CH2), 3.94 (t, 2H, J = 6.2 Hz, O-CH2), 6.64–6.79 (m, 3H, Ph-H), 9.85 (s, 1H, N-H). 13C-NMR (DMSO-d6, 75 MHz): δ 170.19, 154.26, 132.15, 125.28, 116.22, 114.46, 113.43, 66.67, 66.45, 55.36, 53.85, 30.83, 26.42, 25.56. HR-MS (ESI) calcd for C16H23N2O3+ ([M + H]+): 291.1703; found: 291.1702.
6-(3-(Piperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (2h) Mp 138–140 °C, yield 79%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.78–1.87 (t, 2H, CH2), 2.38 (t, 2H, J = 7.4 Hz, CH2), 2.47–2.60 (m, 6H, CH2), 2.60 (s, 4H, CH2), 2.82 (t, 2H, J = 7.4 Hz, CH2), 3.09 (s, 4H, N-CH2), 3.24 (s, 2H, N-CH2), 3.93 (t, 2H, J = 6.2 Hz, O-CH2), 6.69–6.76 (m, 3H, Ph-H), 9.89 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 170.20, 154.19, 132.18, 125.29, 116.23, 114.51, 113.49, 66.29, 54.48, 49.76, 43.43, 41.15, 30.83, 26.40, 25.56. HR-MS (ESI) calcd for C17H25N2O2+ ([M + H]+): 289.1911; found: 289.1912.
6-(3-(4-Phenylpiperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (2i) Mp 174–176 °C, yield 78%. 1H-NMR (DMSO-d6, 300 MHz): δ 1.27 (s, 1H, CH), 1.79–2.03 (m, 6H, CH2), 2.30 (s, 2H, J = 6.2 Hz, CH2), 2.49–2.68 (m, 4H, N-CH2), 2.89 (t, 2H, J = 6.2 Hz, N-CH2), 3.16 (d, 2H, J = 7.9 Hz, N-CH2), 4.00 (s, 2H, J = 6.2 Hz, O-CH2), 6.66–6.81 (m, 3H, Ph-H), 7.18–7.31 (m, 5H, Ph-H), 9.67 (s, 1H, CONH). 13C-NMR (DMSO-d6, 75 MHz): δ 170.4, 154.2, 145.7, 131.7, 128.4, 126.7, 126.1, 124.7, 116.1, 114.1, 113.1, 66.2, 55.0, 53.9, 41.8, 32.8, 30.7, 26.3, 25.7. HR-MS (ESI) calcd for C23H29N2O2+ ([M + H]+): 365.2224; found: 365.2223.
6-(2-(Piperidin-1-yl)ethoxy)-3,4-dihydroquinolin-2(1H)-one (3a) Mp 147–149 °C, yield 77%. 1H-NMR (CDCl3, 300 MHz): δ 1.66–1.97 (m, 8H, CH2), 2.50 (t, 2H, J = 7.3 Hz, CH2), 2.89 (t, 2H, J = 7.3 Hz, CH2), 3.10 (t, 2H, J = 6.0 Hz, CH2), 3.58 (t, 2H, J = 4.7 Hz, CH2), 4.38 (t, 2H, J = 4.7 Hz, O-CH2), 6.70–6.86 (m, 3H, Ph-H), 9.64 (s, 1H, CONH). 13C-NMR (CDCl3, 75 MHz): δ 175.40, 157.54, 137.55, 129.85, 121.19, 119.28, 118.24, 67.72, 60.80, 58.61, 49.32, 35.43, 30.44, 27.69. HR-MS (ESI) calcd for C16H23N2O2+ ([M + H]+): 275.1754; found: 275.1756.
6-(4-(Piperidin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (3b) Mp 150–151 °C, yield 78%. 1H-NMR (CDCl3, 300 MHz): δ 1.58–1.68 (m, 2H, CH2), 1.82–2.01 (m, 8H, CH2), 2.62 (t, 2H, J = 7.5 Hz, CH2), 2.75–2.85 (m, 6H, CH2), 2.96 (t, 2H, J = 7.5 Hz, N-CH2), 3.99 (t, 2H, J = 6.1 Hz, O-CH2), 6.67–6.74 (m, 3H, Ph-H), 7.70 (s, 1H, CONH). 13C-NMR (CDCl3, 75 MHz): δ 170.83, 154.74, 130.82, 125.16, 115.97, 114.69, 113.20, 67.70, 58.00, 53.80, 30.64, 27.00, 25.74, 23.91, 23.06, 21.99. HR-MS (ESI) calcd for C18H27N2O2+ ([M + H]+): 303.2067; found: 303.2068.
6-((5-(Piperidin-1-yl)pentyl)oxy)-3,4-dihydroquinolin-2(1H)-one (3c) Mp 150–151 °C, yield 79%. 1H-NMR (CDCl3, 300 MHz): δ 1.53–1.63 (m, 4H, CH2), 1.80–1.89 (q, 4H, CH2), 2.00–2.11 (m, 6H, CH2), 2.62 (t, 2H, J = 7.4 Hz, CH2), 2.93–3.01 (m, 6H, -NCH2), 3.97 (t, 2H, J = 6.2 Hz, 0-CH2), 6.66–6.76 (m, 3H, Ph-H), 7.61 (s, 1H, N-H). 13C-NMR (CDCl3, 75 MHz): δ 170.76, 164.81, 130.76, 125.17, 116.93, 114.67, 113.23, 67.66, 57.39, 53.26, 30.66, 28.66, 25.74, 23.67, 23.34, 22.36, 22.02. HR-MS (ESI) calcd for C19H29N2O2+ ([M + H]+): 317.2224; found: 317.2222.

3.1.3. Synthesis Procedure of 1-Alkyl-6-(3-(piperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (4a-4b)

Sodium hydroxide (0.12 g, 3 mmol), compound 2h (0.6 g, 2 mmol), and bromopropane (0.37 g, 3 mmol) were placed into a flask with 20 mL of acetonitrile, and the mixture was refluxed for 24 h. Then the brine was added after evaporating the solvent of the mixture. The combined organic layer was dried over anhydrous MgSO4 after the extraction with DCM 3 times (30 mL × 3). A crude product was obtained after filtration and evaporation. The residue was purified by silica gel column chromatography with 1% methanol in DCM to obtain the compound 4a. The bromopropane was replaced by pentane bromide to give the compound 4b. The characterization for the two compounds is listed below.
6-(3-(Piperidin-1-yl)propoxy)-1-propyl-3,4-dihydroquinolin-2(1H)-one (4a) oil, yield 78%. 1H-NMR (CDCl3, 300 MHz): δ 0.87 (t, 3H, J = 7.5 Hz, CH3), 1.51–1.63 (m, 4H, CH2), 1.77–1.85 (m, 4H, CH2), 2.12–2.21 (m, 2H, CH2), 2.52 (t, 2H, J = 7.4 Hz, CH2), 2.75–2.85 (m, 8H, CH2), 3.78 (t, 2H, J = 7.5 Hz, N-CH2), 3.96 (t, 2H, J = 6.1 Hz, O-CH2), 6.65–6.83 (m, 3H, Ph-H). 13C-NMR (CDCl3, 75 MHz): δ 169.63, 154.01, 133.25, 128.12, 115.77, 114.49, 112.61, 65.92, 55.49, 53.91, 43.56, 31.34, 25.76, 25.14, 23.97, 22.97, 20.34, 11,14. HR-MS (ESI) calcd for C20H31N2O2+ ([M + H]+): 331.2380; found: 331.2381.
1-Pentyl-6-(3-(piperidin-1-yl)propoxy)-3,4-dihydroquinolin-2(1H)-one (4b) oil, yield 77%. 1H-NMR (CDCl3, 300 MHz): δ 0.90 (t 3H, J = 7.1 Hz, CH3), 1.29–1.72 (m, 10H, CH2), 2.13 (s, 4H, CH2), 2.44–2.63 (m, 4H, CH2), 2.84 (t, 2H, J = 6.2 Hz, N-CH2), 3.18–3.23 (m, 4H, N-CH2), 3.88 (t, 2H, J = 7.5 Hz, N-CH2), 4.07 (t, 2H, J = 4.8 Hz, O-CH2), 6.70–6.91 (m, 3H, Ph-H). 13C-NMR (CDCl3, 75 MHz): δ 169.61, 153.66, 133.67, 128.31, 115.83, 114.51, 112.58, 65.35, 55.40, 53.57, 42.17, 31.85, 29.03, 26.88, 25.83, 23.99, 22.55, 22.43, 22.07, 14.01. HR-MS (ESI) calcd for C22H35N2O2+ ([M + H]+): 359.2693; found: 359.2694.

3.2. Pharmacological Assays

3.2.1. Luciferase Assay for the H3R Antagonistic Activity

Luciferase assay was taken to screen the H3R antagonistic activity of the compounds 2a-2i, 3a-3c, and 4a-4b. PIT was used as the positive control. Firstly, all the target compounds were screened at 5 μM and 50 μM. In addition, the compounds with an inhibitory rate bigger than 50% were subjected to further assay to obtain the IC50. The detailed experimental operations refer to the previous paper [22,31].

3.2.2. In Vivo Pharmacology Tests for the Antiseizure Activity

The antiseizure activity of the compounds 2a-2i, 3a-3c, and 4a-4b was examined through the MES and PTZ models. The neurological safety of the compounds 2h, 4a, and 4b was evaluated by the rotarod test. In the MES test, the definition of protection was the reduction or vanishing of the THLE in mice. In the PTZ test, the seizures were induced through the subcutaneous administration of PTZ (85 mg/kg). The antiseizure activities of the compounds in the PTZ test were evaluated by comparing the convulsion scores in mice between taking the compounds or not. VPA and PIT were used as positive drugs. To explore the anticonvulsive mechanism, compound 4a (10 mg/kg) was co-injected with RAMH (10 mg/kg). In addition, the THLE was recorded and compared with that of the mice treated by compound 4a alone. The detailed experimental operations in the MES, PTZ, and rotarod tests refer to our previous paper [22,32]. All the animal experiments were carried out on 4-week-old KunMing male mice weighing from 20 to 25 g. The mice were housed collectively in polycarbonate cages in groups of 30, where they were maintained on a 12 h light/dark cycle in a temperature controlled (25 ± 2 °C) laboratory with free access to food and water. In the MES and PTZ models, seven animals were used in each group. In the rotarod test, three animals were used in each group. Each animal was used only once. All the experiments referring to animals were applied according to the Guide for the Care and Use of Laboratory Animals and were approved by the Medical Ethics Committee of Jinggangshan University, approval number: 220811002.

3.2.3. Statistics

GraphPad Prism was used to carry out the statistical analysis. In the seizure tests, data were provided as the mean ± standard error of the mean (SEM). In addition, one-way ANOVA followed by Dunnett’s post hoc test was executed for group comparison. The statistical significance was defined when the p value < 0.05 [22].

3.2.4. Molecular Docking

The molecular docking was conducted through the Flexible Docking in Discovery Studio 2019 [22]. The 3D structure of H3R was constructed from the structure-known H1R protein (PDB ID: 3RZE) by homology modeling [30]. The 3D structures of the compounds 2h, 4a, and PIT were constructed by ChemDraw 16.0, energy minimized, and placed into the protein cavity to flexible binding with the homology model of H3R. After the docking running finished, the binding patterns were provided, and the interactions between the compounds 2h, 4a, PIT, and H3R were analyzed.

4. Conclusions

In this work, a battery of 6-aminoalkoxy-3,4-dihydroquinolin-2(1H)-one derivatives were synthesized to screen their H3R antagonistic activities and evaluate their antiseizure activities. The majority of the target compounds displayed a potent H3R antagonistic activity. Among them, the compounds 2a, 2c, 2h, and 4a showed a submicromolar H3R antagonistic activity. The MES model screened out three compounds (2h, 4a, and 4b) with antiseizure activity, while all compounds did not show any protective effect for mice against the seizures induced by PTZ. Additionally, the antiseizure mechanism of compound 4a was proved to be related to H3R antagonism. The molecular docking of 2h, 4a, and PIT with the H3R protein predicted their possible binding patterns and gave a presentation that 2h, 4a, and PIT had a similar binding model with H3R.

Author Contributions

Conceptualization, Y.H. (Yi Hua) and X.D.; Methodology, M.S.; Software, Y.H. (Yi Hua) and M.S.; Validation, Y.H. (Yushan Huang); Investigation, Y.H. (Yi Hua), Q.G. and Y.L.; Writing—original draft, M.S.; Writing—review & editing, X.D.; Visualization, X.D.; Supervision, X.D. and Y.H. (Yushan Huang); Project administration, X.D.; Funding acquisition, X.D. and Y.H. (Yushan Huang). All authors have read and agreed to the published version of the manuscript.

Funding

The foundation of this research was the National Natural Science Foundation of China (NO.22167017) and the scientific research fund of Jiangxi provincial education department (No. GJJ201004).

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee of Jinggangshan University (protocol code 220811002, date of approval: 11 August 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

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Molecules 28 03408 g001 550
Figure 1. The structures of reported anticonvulsive compounds I, II, and target compound 2h.
Figure 1. The structures of reported anticonvulsive compounds I, II, and target compound 2h.
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Molecules 28 03408 sch001 550
Scheme 1. The synthesis of a first series of 3,4-dihydroquinolin-2(1H)-ones 2a-2i.
Scheme 1. The synthesis of a first series of 3,4-dihydroquinolin-2(1H)-ones 2a-2i.
Molecules 28 03408 sch001
Molecules 28 03408 sch002 550
Scheme 2. The synthesis of a second and third series of 3,4-dihydroquinolin-2(1H)-ones 3a-3c and 4a-4b.
Scheme 2. The synthesis of a second and third series of 3,4-dihydroquinolin-2(1H)-ones 3a-3c and 4a-4b.
Molecules 28 03408 sch002
Molecules 28 03408 g002 550
Figure 2. The protection of H3R antagonists 2a-2i, 3a-3c, 4a-4b (10 mg/kg, i.p.), PIT (10 mg/kg, i.p.), and the antiepileptic drug VPA (300 mg/kg, i.p.) in the maximal electroshock seizure model. The definition of protection was the reduction or vanishing of the tonic hind limb extension (THLE). Data are shown as mean ± SEM in the figure, which are derived from seven animals in each group. * p < 0.05, *** p < 0.001 when compared to the control group (CTL).
Figure 2. The protection of H3R antagonists 2a-2i, 3a-3c, 4a-4b (10 mg/kg, i.p.), PIT (10 mg/kg, i.p.), and the antiepileptic drug VPA (300 mg/kg, i.p.) in the maximal electroshock seizure model. The definition of protection was the reduction or vanishing of the tonic hind limb extension (THLE). Data are shown as mean ± SEM in the figure, which are derived from seven animals in each group. * p < 0.05, *** p < 0.001 when compared to the control group (CTL).
Molecules 28 03408 g002
Molecules 28 03408 g003 550
Figure 3. The performance of compounds 2a-2i, 3a-3c, and 4a-4b (10 mg/kg, i.p.), H3R antagonist PIT (10 mg/kg, i.p.), and antiepileptic VPA (300 mg/kg, i.p.) in the PTZ-induced seizure model. The seizure scores came from seven animals in each group and are shown as mean ± SEM in the figure. This represents that VPA fully inhibited the convulsive behavior induced by PTZ.
Figure 3. The performance of compounds 2a-2i, 3a-3c, and 4a-4b (10 mg/kg, i.p.), H3R antagonist PIT (10 mg/kg, i.p.), and antiepileptic VPA (300 mg/kg, i.p.) in the PTZ-induced seizure model. The seizure scores came from seven animals in each group and are shown as mean ± SEM in the figure. This represents that VPA fully inhibited the convulsive behavior induced by PTZ.
Molecules 28 03408 g003
Molecules 28 03408 g004 550
Figure 4. The protection of compounds 2h, 4a, 4b, and PIT against the maximal electroshock-induced seizures in doses of 3, 10, and 30 mg/kg. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control group (saline).
Figure 4. The protection of compounds 2h, 4a, 4b, and PIT against the maximal electroshock-induced seizures in doses of 3, 10, and 30 mg/kg. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to the control group (saline).
Molecules 28 03408 g004
Molecules 28 03408 g005 550
Figure 5. The protection of compound 4a (10 mg/kg, i.p.) against the maximal electroshock-induced seizures in mice pretreated with RAMH (10 mg/kg, i.p.). Data come from seven animals in each group and are shown as mean ± SEM in the figure. * p < 0.01 when compared to the control group (saline-treated), # p < 0.01 when compared to the combined group (4a + RAMH).
Figure 5. The protection of compound 4a (10 mg/kg, i.p.) against the maximal electroshock-induced seizures in mice pretreated with RAMH (10 mg/kg, i.p.). Data come from seven animals in each group and are shown as mean ± SEM in the figure. * p < 0.01 when compared to the control group (saline-treated), # p < 0.01 when compared to the combined group (4a + RAMH).
Molecules 28 03408 g005
Molecules 28 03408 g006 550
Figure 6. The predicted binding configurations when 2h (A), 4a (B), and PIT (C) dock with H3R.
Figure 6. The predicted binding configurations when 2h (A), 4a (B), and PIT (C) dock with H3R.
Molecules 28 03408 g006
Molecules 28 03408 g007 550
Figure 7. The overlying pattern of compound 2h (blue), 4a (green), and PIT (red) in the binding pocket of the H3R.
Figure 7. The overlying pattern of compound 2h (blue), 4a (green), and PIT (red) in the binding pocket of the H3R.
Molecules 28 03408 g007
Table
Table 1. H3R antagonistic effects of targets 2a-2i, 3a-3c, and 4a-4b.
Molecules 28 03408 i001
Table 1. H3R antagonistic effects of targets 2a-2i, 3a-3c, and 4a-4b.
Molecules 28 03408 i001
CompoundsRnR′H3R Antagonistic Activity
(IC50, μM)		ClogP a
2aMolecules 28 03408 i0021H0.522.72
2bMolecules 28 03408 i0031H>503.78
2cMolecules 28 03408 i0041H0.472.29
2dMolecules 28 03408 i0051H>501.23
2eMolecules 28 03408 i0061H38.110.74
2fMolecules 28 03408 i0071H25.643.63
2gMolecules 28 03408 i0081H46.041.64
2hMolecules 28 03408 i0091H0.122.85
2iMolecules 28 03408 i0101H38.474.26
3aMolecules 28 03408 i0110H7.492.55
3bMolecules 28 03408 i0122H2.882.96
3cMolecules 28 03408 i0133H>503.49
4aMolecules 28 03408 i0141C3H70.373.98
4bMolecules 28 03408 i0151C5H111.915.03
PIT---0.694.82
a ClogP was calculated by ChemDraw 16.0 (CambridgeSoft, Cambridge, MA, USA).
Table
Table 2. The neurotoxicity of compounds 2h, 4a, and 4b (i.p.) in the rotarod test in mice.
Table 2. The neurotoxicity of compounds 2h, 4a, and 4b (i.p.) in the rotarod test in mice.
CompoundsNeurotoxicity
10 mg/kg30 mg/kg100 mg/kg
2h0/30/31/3
4a0/30/30/3
4b0/30/30/3
PIT0/30/30/3
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MDPI and ACS Style

Hua, Y.; Song, M.; Guo, Q.; Luo, Y.; Deng, X.; Huang, Y. Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones. Molecules 2023, 28, 3408. https://doi.org/10.3390/molecules28083408

AMA Style

Hua Y, Song M, Guo Q, Luo Y, Deng X, Huang Y. Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones. Molecules. 2023; 28(8):3408. https://doi.org/10.3390/molecules28083408

Chicago/Turabian Style

Hua, Yi, Mingxia Song, Qiaoyue Guo, Yiqin Luo, Xianqing Deng, and Yushan Huang. 2023. "Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones" Molecules 28, no. 8: 3408. https://doi.org/10.3390/molecules28083408

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