Abstract
Aiming to develop novel anticonvulsant agents a new series of novel cycloalkanecarboxamide parabanic acid hybrids series 8, 9 and 10 possessing the essential structure requirements for anticonvulsant activity was synthesized starting from cycloalkanones. All final target compounds were primary screened for chemically and electrically induced seizures using pentylenetetrazole “scPTZ” and maximal electroshock seizure “MES” models. In phase I anticonvulsant evaluation compounds 8b and 10b exhibited the highest potency among all the target compounds with 100% protection towards chemically induced seizures. Results of phase II anticonvulsant screening showed that compounds 8b and 10b are more potent than standard drug ethosuximide by about 11 and 9 fold, respectively. Regarding MES test, compounds 8b and 9a-d exhibited 100% protection with ED50 values ranged between 0.107–0.177 mmol/Kg. All final compounds did not display any signs of motor impairment in the neurotoxicity screening test. Also, compounds 8a, 9a-d and 10b were devoid of hepatotoxicity as shown by measurement of serum levels of liver enzymes, albumin as well as total protein. Moreover, the cyclohexyl derivative 10b produced a significant increase of Gamma-aminobutyric acid “GABA” brain’s content of mice compared to control group confirmed its GABAergic modulating activity. Molecular docking, physicochemical and pharmacokinetic properties were carried out for all compounds as well. These outcomes support that cycloalkanecarboxamide parabanic acid hybrid is a promising scaffold to pave the way towards further development of novel class of antiepileptic drugs.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Epilepsy is a neural disorder characterized by repeated uninduced seizures due to unusual increase or synchronous nerve cell activity in the brain [1, 2]. Epilepsy could occur as a result of many causes as brain injury, tumors, infection, immune disorder or congenital anomaly. However, for many patients the major reasons are undisclosed [3, 4]. Generalized seizures and focused seizures are the two basic types of epileptic seizures. In contrast to focal seizures, which are limited to a single part of the brain, generalized seizures start with an electrical neuronal discharge that affects the entire brain [5]. The statistics of the WHO declare that epilepsy affects almost 50,000,000 individuals across the world and around 80% of cases inhabit low- and middle-income countries [6, 7]. Even though the development of novel antiepileptic drugs (AEDs), current medications fail in managing seizures in around 30% of cases which suffer from drug-resistant epilepsy (DRE) [8, 9]. According to the International League Against Epilepsy, DRE is defined as “failure of a patient’s seizures to respond to at least two antiepileptic medications that are appropriately chosen and used for an adequate period to achieve and maintain seizure freedom” [10]. Additionally, many side effects occur during the use of the available AEDs drugs like megaloblastic anemia and liver toxicity [11, 12]. Consequently, there is a great requirement to develop new antiepileptics with less complications and higher efficacy. The mechanism of FDA approved AEDs are divided to: (i) Improvement the effect of neuromediator γ-aminobutyric acid (GABA) which is the chief inhibitory mediator in the brain either by suppression of GABA reuptake, Effects on allosteric activity of GABA receptor type A or decreasing GABA metabolism through suppression of GABA aminotransferase enzyme (GABA-AT), (ii) Direct working on calcium and sodium gated channels and (iii) Decrease synaptic activation that is regulated by glutamate receptors [13, 14]. As a result of the shortage in information of pathophysiology of epilepsy along with the complicated mode of action for the majority of AEDs; the use of mechanism-driven designs in the discovery of new AEDs is not common. Therefore, the development of more anticonvulsant entities is mostly attained by either by screening investigations or the usage of hybrid pharmacophoric approach [15]. This approach counts on utilizing two or more scaffolding pharmacophores having significant pharmacological activity to be combined in a solitary molecule to achieve novel candidate with superior activity [16, 17]. The antiepileptic activity of lactams as well as cyclic imides has been reported for over 60 years. This scaffold is found in the structure of old AEDs as ethosuximide, the drug of choice in the management of petit mal seizures, and phenytoin along with the new AEDs as levetiracetam and brivaracetam (Fig. 1A) [18]. Literature survey revealed that there is a scarceness of studying the anticonvulsant effect of parabanic acids and their derivatives that can be considered as cyclic imides [19, 20]. In addition, parabanic acid is the lower homolog of barbituric acid that represents the main scaffold of phenobarbital (Fig. 1B) which suppresses seizures through improvement of γ-aminobutyric acid (GABA) action.
A Some AEDs having cyclic imides and lactams. B Homology between parabanic acid and barbituric acid. C Gabapentin structure and other anticonvulsants containing the cyclohexanecarboxamide moiety. D Parabanic acids having anticonvulsant activity
On the other hand, a lot of disubstituted cycloalkanes were reported to exhibit anticonvulsant activity such as the antiepileptic drug gabapentin and the cyclohexanecarboxamides I-III (Fig. 1C) which displayed anticonvulsant activity against electric and chemical induced seizures in animal models [21,22,23].
Based on the preceding considerations, motivated by the promising results that we had previously obtained regarding the parabanic acids IV and V as anticonvulsants [24] (Fig. 1D) and as a part of our research goal to develop new efficient anticonvulsant small molecules [24,25,26,27], a new series of parabanic acid cycloalkanecarboxamide hybrids 8a-d, 9a-d and 10a-d were constructed as potential anticonvulsants. It is worth mentioning that this is the first study which describes the synthesis and the anticonvulsant activity of parabanic acid cycloalkanecarboxamide hybrids. These newly synthesized hybrids fulfill the required pharmacophoric elements for anticonvulsants which are lipophilic aryl ring (A), hydrogen donor/acceptor unit (HAD) and electron donor atom (D) [28, 29] (Fig. 2). The anticonvulsant effect of the target compounds were assessed by using mouse models of chemically as well as electrically induced seizures. Furthermore, the neurotoxic and hepatotoxic adverse effects were also studied. Moreover, the most effective derivatives in subcutaneous pentylenetetrazole test were further investigated to determine their influence on the brain GABA level. Additionally, in silico study was conducted for these derivatives at the binding site of GABA aminotransferase (GABA-AT) to explore their GABA modulation effect.
Essential pharmacophoric elements needed for anticonvulsant activity in some AEDs and final derivatives 8a-d, 9a-d and 10a-d
Results and discussion
Chemistry
Scheme 1 illustrates the preparation of the final derivatives 8a-d, 9a-d and 10a-d. Cycloalkanones were reacted with the appropriate anilines in presence of potassium cyanide according to Strecker’s synthesis in glacial acetic acid to produce the phenyl amino cycloalkane carbonitriles 1a-c. The nitrile group was converted to corresponding amide derivatives 2a-c by partial hydrolysis with sulfuric acid at room temperature. The secondary amino group of the latter compounds was acetylated to give the respective chloroacetamide derivatives 3a-c. The hydrazines 4a-c were prepared by reflux of the chloroacetamides 3a-c with hydrazine hydrate. The penultimate semicarbazides 5a-d, 6a-d and 7a-d were achieved via reaction of appropriate isocyanate with compounds 4a-c in 1,2-dichloroethane. Subsequent cyclization of semicarbazides yielded the parabanic acids 8a-d, 9a-d and 10a-d which obtained by cyclization of the previous semicarbazides using oxalyl chloride. The chemical structures of the target compounds 8a-d, 9a-d as well as 10a-d were proved in “1H NMR” by existence of merely single signal of NH and disappearance of two NH signals of urea and in “13C NMR” by presence of 3C = O for trioxoimidazolidine at 148-159 ppm. The chemical components of the constructed products were additionally elucidated via “elemental analyses and other spectral data”.
Synthesis of the aimed derivatives 8a-d, 9a-d and 10a-d. Chemicals and conditions: (i) Potassium cyanide, glacial CH3COOH, 25 °C, 24 h. (ii) H2SO4, 25 °C, two days. (iii) ClCH2COCl, 25 °C, 24 h. (iv) 98% Hydrazine hydrate, ethanol, reflux, 8 h. (v) ClCH2CH2Cl, reflux, 3 h. (vi) Benzene, Oxalyl chloride, reflux, 3 h
Biological evaluation
Anticonvulsant effect
The anticonvulsant effect was determined following the standard protocol of the National Institute of Neurological Disorders and Stroke (NINIDS), Epilepsy section, Antiepileptic Drug Development (ADD) program. The primary anticonvulsant evaluation in phase I for the newly synthesized hybrids was performed to determine the most active compounds using subcutaneous pentylenetetrazole (scPTZ) along with the maximal electroshock seizure (MES) tests, besides assessment of the neurotoxicity using the rotarod test. In phase II antiepileptic assessment the most active candidates were subjected to median effective dose (ED50) determination.
Phase I anticonvulsant determination
Compounds were given intraperitoneally to male albino mice at a dose of 100 mg/kg. This acute dose was selected based on preliminary research that was completed before the core experiment. The outcomes of stage 1 screening are presented in Table 1. The compounds under investigations showed wide range of activity towards scPTZ–induced absence seizures that varied from 0 to 100% protection at 100 mg/kg. Compounds 8b, and 10b had the highest protection (100%) in this model of chemically stimulated seizures. On the other hand, all the compounds showed protection against MES-induced grand mal seizures ranging from 33.3 to 100% except the pentyl derivative 8c that was completely inactive. Compounds 8b and 9a-d were the most actives in this test with percent protection 100% indicating their capability to fully terminate and limit the spread of generalized tonic-clonic seizures [30, 31]. Moreover, all the tested compounds were subjected to neurotoxicological investigation using the rotarod apparatus to assess motor coordination and it was found that all of the test compounds were devoid of neurotoxicity (Table 1).
Phase II quantitative anticonvulsant evaluation
Compounds 8b and 10b that made complete protection in the scPTZ test were selected to enter phase II anticonvulsant evaluation to calculate their median effective doses (ED50) (Table 2). Similarly, Compounds 8b and 9a-9d that achieved full protection against MES induced seziures were further examined to determine their ED50 values (Table 3). Compounds 8b and 10b showed ED50 of 34.41 mg/kg (0.075 mmol/kg) and 44.01 mg/kg (0.099 mmol/kg) in the scPTZ test, respectively. Besides, Compounds 8b and 9a-d displayed ED50 of 44.49 mg/kg (0.0.107 mmol/kg), 67.61 mg/kg (0.157 mmol/kg), 77.84 mg/kg (0.181 mmol/kg), 78.64 mg/kg (0.177 mmol/kg) and 64.15 mg/kg (0.134 mmol/kg) in the MES, respectively. Notably, compound 8b possessed the lowest ED50 in both tests indicating its potential activity in protecting against grand mal and petite mal seizures.
Structure activity relationship
Regarding the outcomes of scPTZ test in stage I anticonvulsant assessment it was observed that the compound 8b that is swapped by branched aliphatic side chain (isopropyl) is the most active one among cyclopentyl parabanic acid hybrids 8a-d with 100% protection. Meanwhile, the activity of this compound decreased to its one-sixth and half values when the parabanic acid is substituted with normal chains of either propyl or butyl as in compounds 8a and 8c respectively. Also, the replacement of isopropyl with aralkyl side chain as compound 8d reduced the activity to its one-third value. Surprisingly, in the cyclohexyl parabanic acid hybrids 9a-d the branched isopropyl derivative 9b was completely inactive in scPTZ test. Meanwhile, compound 9c that has long chain aliphatic substitution (n-butyl) is the most active derivative with 83% protection while its shorter analog 9a substituted with n-propyl has only 33% protection. It is notable that the later compound is about 2 fold more active than its cyclopentyl analog 8a. It is worth mentioning that the aralkyl derivative 9d is more active than its corresponding cyclopentyl derivative 8d by about 1.6 fold. Concerning the cyclohexyl derivatives having tolyl substituent 10a-d it was found, as in cyclopentyl series, the most active one is the branched isopropyl derivative 10b with protection 100%. This activity was dropped to its half value upon substitution with normal chains as in compounds 10a and 10c. Unfortunately, the benzyl derivative 10d was completely devoid of activity against scPTZ induced seizures. It is remarkable that the cyclohexyl tolyl derivatives substituted with either n-propyl 10a or isopropyl 10b are more active than their unsubstituted phenyl congeners 9a and 9b. Meanwhile, the butyl derivative 9c bearing unsubstituted phenyl moiety is about 1.6 fold more active than its tolyl analog 10c. In phase II anticonvulsant evaluation the isopropyl substituted compounds 8b and 10b was more potent than the standard drug ethosuximide by 11.2 and 9.2 fold, respectively while phenobarabital was slightly more active than both the tested compounds by 1.4 and 1.7 fold, respectively. It is to be noted that the cyclopentyl derivative 8b is lightly more active than the cyclohexyl derivative 10b.
With respect to MES test, the members of cyclohexyl parabanic acid conjugates bearing unsubstituted phenyl ring 9a-c achieved 100% protection. Meanwhile, their tolyl analogs 10a-c made only 83% protection indicating that hydrophobic substitution is not favored for the activity. In the cyclopentyl parabanic acid hybrids the isopropyl derivative 8b was the most potent one with 100% protection while its normal propyl counterpart 8a displayed only 83% activity. In this series the activity was completely abolished upon introducing one carbon atom in the side chain of trioxoimidazolidine ring as observed in the butyl derivative 8c. The results of phase II anticonvulsant determination revealed that the cyclopentyl derivative 8b is generally more potent than the cyclohexyls 9a-d and it is 1.7 fold more active than its cyclohexyl analog 9b. Anticonvulsant effectiveness against MES was found to be almost same across normal chain, branched chain, and aralkyl substituents in series 9a-d of cyclohexyls, indicating that the parabanic acid substitution pattern had little effect.
Acute toxicity testing and the determination of the median lethal dosage (LD50)
All of the parabanic acids under investigations were subjected to for testing for acute toxicity and an estimate for LD50 was specified. Acute toxicity was not observed in any of the mice at doses up to 500 mg/kg. Accordingly, the LD50 might be put at greater than 500 mg/kg.
GABA level determination
In the scPTZ test, the protection against seizures may refer to GABA-mediated mechanisms, where fits are mainly repressed by drugs that aiming Gamma-aminobutyric acid receptor as benzodiazepines and barbiturates [12]. Accordingly, measurement of GABA level in the brain of animals treated with derivatives 8b and 10b, the most actives in scPTZ test, was performed to check the probable way of anticonvulsant action against PTZ that produces seizures through inhibiting GABA neurotransmitter.
As represented in Fig. 3 the level of GABA in the whole brain of control mice was 1.79 ± 0.052 μg/g brain tissue, while the cyclohexyl derivative 10b markedly increased brain GABA level (2.71 ± 0.262 μg/g brain tissue) more than normal control at p < 0.05. On the other hand, compound 8b showed comparable GABA level (1.88 ± 0.24 μg/g brain tissue) to the control group. Consequently, The principal inhibitory neuromediator accountable for the hyperpolarization of every single neuron in the frontal lobe, Gamma-aminobutyric acid, may have been involved in the antiepileptic activity of compound 10b. Potentiation of the Gamma-aminobutyric acid action could be ascribed to one of succeeding: (1) binds directly to GABA-A receptors which leads to increment the chloride ions inflow that results in excess negative membrane potential at rest and consequently reducing the of excitation of the membranes located after synapses, (2) increasing Gamma-aminobutyric acid production via glutamic acid decarboxylase enzyme modulation which is responsible for GABA synthesis by decarboxylation of glutamate, (3) inhibition of GABA catabolism through GABA transaminase enzyme suppression or (4) blockage of presynaptic GABA re-uptake [32,33,34].
Changes in GABA levels as a result of exposure to 8b and 10b, data are illustrated as barchart ± SEM. * versus control group; <0.05
Hepatotoxicity
As abovementioned liver toxicity is considered as one of most the dangerous side effects for the marketed AEDs [12]. Particular signs of liver dysfunction include increased hepatic enzyme levels. Accordingly, the most powerful compounds in both MES and scPTZ tests 8b, 9a-d and 10b were examined for their potential hepatotoxic effect. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), total protein and albumin are expressed in Table 4 and Fig. S3 Supplementary file. From these results it was concluded that all of the tested derivatives did not exhibit appreciable alternation in the content of total protein, albumin, or enzymes in the blood in comparison to negative control. This showed that all of these compounds are free from hepatotoxicity side effect.
In-Silico studies
Pharmacophore model Generation
The pharmacophore model was built to investigate the anticonvulsant activity of derivatives 8a-d, 9a-d and 10a-d. This was accomplished utilizing “common feature pharmacophore model protocol in Discovery studio software 4.1 (Accelrys, Inc, San Diego, USA)”.
Literature survey illustrated that the crucial features required for activity in antiepileptics and numerous anticonvulsant agents include the presence of an element that accepts hydrogen bonds, hydrogen bond donor and hydrophobic moiety. To build the pharmacophore, a training set of compounds (Fig. S4 Supplementary file) that have potent anticonvulsant activity and contain the fundamental structural features needed for anticonvulsant effect was used [15, 25, 35,36,37,38,39]. Ten hypotheses were obtained and the hypothesis 10 was selected as the valid best one. Validation of this selected hypothesis was depending on certain parameters as it presented complete plotting of all its features without steric clashes and having good fitting values with the training set, database search study inspecting the affinity of such hypothesis with the molecular structures of “MiniMaybridge databases” disclosed that only 15 hits have been obtained from the databases which gives indication on selectivity of this hypothesis which includes hydrogen bonding acceptor (HBA), hydrogen bonding donor (HBD), and hydrophobic property. Also, the simulated fit values for test set compounds 8a-e, 9a-e, and 10 a-e for the above hypothesis were more reliable with the experimental results compared to the other hypotheses. Table 5 and Fig. S5 Supplementary file show the measured distances and angles between the model’s structural parts (HBA, HBD, HY). In Fig. 4, it was observed how the two most effective anticonvulsants (8b, 10b) and phenobarbital fit into the parameters of the model that was developed.
Fitting of the generated pharmacophore anticonvulsant model with A 8b, B 10b, C phenobarbital
Docking study
GABA-AT is responsible for GABA degradation and certain AEDs as vigabatrin suppresses this enzyme in order to raise the concentration of Gamma-aminobutyric acid level in the central neurons [40, 41]. Consequently, molecular docking was performed to explain the effect of compounds 8b and 10b on GABA brain level. Literature survey revealed that key amino acids involved in the binding site of “GABA-AT are of His44A, Tyr69A, Ile72A, Gly136A, Ser137A, Phe189A, Arg192A, Lys203A, Ile205A, His206A, Glu265A, Ser269A, Glu270A, Glu298A, Val300A, Lys329A, Arg422A, Asn423A, Ile426A, Gly438A, Gly440A” [24, 41,42,43].
We initially examined the attaching of the co-crystallized ligand vigabatrin at the binding site of the enzyme. The protocol applied for docking was validated by re-docking of the co-crystallized ligand in the active site with RMSD of 0.9632 Å (Fig. S6 Supplementary file). Also, NH group of docking pose was capable of reproducing the important hydrogen bonds found between the co-crystallized ligand and the key amino acids Glu 270 and Lys 329, carbonyl group formed H-bond with Arg 192. Moreover, it made van der Waal interaction with Gly 191, Phe 189, Gln 301 and Ile 72 (Fig. 5A). The validation outputs indicated the appropriateness of the applied protocol for the molecular docking study of the test compounds in the active site of GABA-AT.
2D and 3D illustration of the binding of A vigabatrin B 8b C 10b to the GABA-AT active site (PDB code: 1OHW)
Regarding compound 8b, it exhibited hydrogen bonds with essential amino acids Arg 192, Lys 329, Glu 270 and additional bonds with Val 300, His 206 and Tyr 69 through carbonyl and NH groups. Trioxoimidazolidine ring made pi-alkyl interaction with Ile 72 in addition to van der Waal interaction with Gly 438, Arg 445, Gln 301, Ser 74, Gln 71, Cys 135, Ser 137, Gly 136, Gly 191 and Phe 189 (Fig. 5B).
Compound 10b exhibited the best binding interaction with the receptor; it showed several hydrogen bond interactions with crucial amino acids as Ile 72, Lys 329 through amino group, Tyr 69, Arg 192 and Arg 445 through carbonyl groups. Additionally, both Trioxoimidazolidine ring and methyl group induced pi-alkyl interaction with Ile72 and Val300, respectively as well as pi-pi interaction with Ph 189 through phenyl ring (Fig. 5C).
In Silico prediction of ADMET
The physiochemical parameters are one of the fundamentals in the design of CNS acting agents. Calculation of the molecular properties is considered as one of principle parameters in the development of new drug candidates to accomplish good bioavailability [15, 44]. Thought Lipinski rule which is known as Role of Five (RO5) [15, 45, 46] and determination of polar surface area (PSA) or topological polar surface area (TPSA) are crucial factors in estimation of drug transport characters. TPSA value less than 140 indicates the good drug transport feature through the cell membranes [22] Table 6. All the tested derivatives 8a-d, 9a-d and 10a-d displayed TPSA values within satisfactory range (Table 6).
Moreover, most of the synthesized candidates showed high GIT absorption and negative substrates for P-glycoprotein (Pgp-) transporter and so are not exposed to its efflux action. Last but not the least; they exhibited zero alerts in the pan-assay interference structure (PAINS) Table 7.
Conclusion
In this study a new series cycloalkanecarboxamide parabanic acid hybrids was designed and synthesized to obtain effective anticonvulsant agents. In vivo outcomes displayed that all of the examined derivatives are active at least in one model of chemically or electrically induced-seizures. The cyclopentyl derivative 8b achieved 100% protection in the two models indicating its ability to control both generalized tonic clonic and absence seizures. This compound attained the best ED50 values among the compounds under investigation (ED50 = 0.082 mmol/kg in scPTZ and 0.107 mmol/kg in MES). It is more active than the standard drug ethosuximide by about 11 fold in scPTZ test. Also, the cyclohexyl derivative 10d was active in scPTZ assay more than the standard drug by 9 fold. In addition the latter compound 10b caused a significant increase of GABA level in brain of the tested animals in comparison to control group which indicating its GABAergic modulating effect. The molecular docking study illustrated that binding with GABA-AT is the most possible mechanism of action for compounds 8b and 10d. All the target compounds in this work were devoid of neurotoxicity and the most potent derivatives exhibited no signs of hepatic dysfunction as denoted by measurement of levels of liver enzymes, albumin and total protein in serum. In silico ADMET study of the target compounds illustrated that they have acceptable drug-like properties. The achieved results in this work could open the gate towards further development of novel class of antiepileptics.
Experimental
Chemistry
All melting points were recoded using “Electrothermal Capillary apparatus” and all were uncorrected. IR spectra were determined as KBr pellets, values are characterized in cm−1. Mass spectral data were recoded using electron impact ionization technique at 70 eV. “1H NMR (500 MHz) and 13C NMR” (100 MHz) bands were achieved on “Jeol ECA 500 MHz spectrometer” and chemical shifts were presented as ppm. “Elemental microanalyses were done on Microanalytical Units in Cairo University and the National Research Center”. All final compounds exhibited purity above 95% as detected by HPLC. Analysis was carried out using Agilent 1100 series apparatus. Detection method was using diode array (DAD) ultraviolet (UV) detector was used at 254 nm. Column used was C18 column Zorbax ODS (4.6 ×150 mm i.d.,5 mm) and eluent was water: acetonitrile (30:70), flow rate = 1 mL/min, temperature 25 °C.
Preparation of 1a-c, 2a-c, 3b and 4b was achieved as per to stated methods [25, 47, 48].
Synthesis of 1- (2-chloro – N - arylacetamido) cyclolkane-1-carboxamides 3a and 3c
Chloroacetyl chloride (1.317 g, 0.0116 mol) was added to 0.0097 mol of 2a or 2c in chloroform (30 mL). The mixture was stirred at rt for 24 h followed by adding aqueous solution 10% NaOH (2 ×30 mL) and separation of the 3a-c.
1-(2-Chloro- N- phenylacetamido) cyclopentane-1-carboxamide (3a)
Pale yellow oil, yield 89.2%, 1H NMR (CDCl3) 1.64–1.81 (m, 6H, 3CH2, cyclopentyl), 2.20–2.40 (m, 2H, CH2, cyclopentyl), 3.64 (s, 2H, CH2-Cl), 6.99 (s, 2H, NH2), 7.26 (s, NH2, 2H), 7.35–7.51 (m, 5H, Har.); IR (KBr, cm−1) 3454, 3361 (NH2), 1732, 1662 (2 C = O); “13C NMR” (CDCl3) δ ppm 23.95, 35.32 (2CH2, cyclopentyl), 45.32 (CH2-Cl), 66.60 (Cq), 129.41, 129.76, 128.81, 137.78 (aromatic carbons), 168.2 (CO-CH2), 172.45 (CO-NH2); MS (EI) m/z (%): 280.75 (M+, 9.5%). Anal. calcd. for C14H17ClN2O2: C, 59.89; H, 6.10; N, 9.98. Found: C, 59.85; H, 6.14; N, 9.94.
1-(2-Chloro-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (3c)
Pale yellow oil, yield 84%, 1H NMR (CDCl3) 1.72–1.80 (m, 10H, 5CH2, cyclohexyl), 2.43 (s, 3H, CH3), 3.80 (s, 2H, CH2-Cl), 7.28 (s, 2H, NH2), 7.45 (d, 2H, J = 4 Hz, Har.), 7.47 (d, 2H, J = 4, Har.); “13C NMR” (CDCl3) δ ppm 222.41 (CH3), 22.69, 24.77, 38.68 (3CH2, cyclohexyl), 42.99 (CO-CH2), 62.69 (Cq), 129.83, 129.86, 129.88, 138.28 (arenes carbons), 176.1, 173.03 (2 C = O); IR (KBr, cm−1) 3458, 3392 (NH2), 1743, 1680 (2 C = O); MS (EI) m/z (%): 308.81 (M+, 6.2%), Anal. calcd. for C16H21ClN2O2:: C, 62.23; H, 6.85; N, 9.07. Found: C, 62.90; H, 6.95; N, 9.13.
Synthesis of 1-(2-hydrazineyl-N-arylacetamido)cycloalkane-1-carboxamides 4a and 4c
A.02 mol of hydrazine hydrate 99% was added to 0.0105 mol of 3a or 3c in absolute ethanol. For 8 hours, the reaction mixture was heated to a simmer. In order to get 4a and 4c, the produced precipitate was filtered out and refined by crystallization from ethanol.
1-(2-Hydrazineyl-N- phenylacetamido) cyclopentane -1-carboxamide (4a)
Yellowish white solid, m.p 108–110 °C, yield 85%, IR (KBr, cm−1) 3446, 3309, 3282, 3198 (NH2, NH), 1687, 1656 (C = O). “1HNMR” (CDCl3) 1.44–1.72 (m, 2H, CH2, cyclopentyl), 1.80–1.83 (m, 2H, CH2, cyclopentyl), 1.91–2.36 (m, 4H, 2CH2, cyclopentyl), 4.37 (s, 2H, CH2-CO), 7.12 (m, 2H, Har.), 7.21 (br.s, 1H, NH), 7.39–7.46 (m, 3H, Har.). (EI) m/z (%): 276.34 (M+, 15%), Anal. calcd. for C14H20N4O2: C, 60.85; H, 7.30; N, 20.28. Found: C, 60.90; H, 7.35; N, 20.35.
1-(2-Hydrazineyl-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (4c)
Yellowish brown oil, yield 83%, IR (KBr, cm−1) 3311, 3186, 3055 (NH2, NH), 1682, 1602 (C = O). “1HNMR” (CDCl3) 1.68–1.90 (m, 10H, 5CH2, cyclohexyl), 2.30 (s, 3H, CH3), 3.92 (br.s, 1H, NH), 4.35 (s, 2H, CO─CH2), 5.06 (br.s, 1H, NH), 7.28 (s, 2H, NH2), 7.36–7.46 (m, 4H, Har.). (EI) m/z (%): 304.39 (M+, 12%), Anal. calcd. for C16H24N4O2: C, 63.13; H, 7.95; N, 18.41. Found: C, 62.15; H, 7.00; N, 18.45.
Synthesis of 2-(2-((1-carbamoylcycloalkyl)(aralkyl)amino)-2-oxoethyl)-N-alkyl/aralkylhydrazine-1-carboxamides 5a-d, 6a-d and 7a-d
A 0.01 mol of the suitable isocyante was added to 0.01 mol of hydrazine 4a-c in dichloroethane and the mixture was for 3 h. The mixture was filtered off and the residue was washed with diethyl ether to get 5a-d, 6a-d and 7a-d.
2-(2-((1-Carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)-N-propylhydrazine-1-carboxamide (5a)
Yield 84%, “1HNMR” (CDCl3 d6, δ, ppm): 0.93 (t, 3H, J = 7.5 Hz, CH3-CH2), 1.46–1.50 (m, 2H, CH2-CH3), 1.60–1.67 (m, 4H, 2CH2-cyclopentyl), 1.90–1.93 (m, 2H, CH2-cyclopentyl), 2.46–2.51 (m, 2H, CH2-cyclopentyl), 3.72 (s, 2H, CH2-CH2-CH3), 3.82–3.86 (m, 2H, CH2-NH), 5.91 (br.s, 1H, NH), 7.14–7.16 (m, 2H, Har.), 7.28–7.45 (m, 3H, Har.), 7.95 (s, 2H, NH2). 13CNMR (CDCl3 d6, δ, ppm): 11.38 (CH3), 22.75 (CH2-CH3),, 23.20, 34.18 (CH2-cyclopentyl), 42.17 (CH2-CH2-CH3), 55.33 (CH2-CO) 63.22 (Cq), 128.76, 129.48, 130.14, 137.06 (aromatic carbons), 157.54, 166.98, 170.65 (3C = O), IR (KBr, cm−1) 3240, 3120, 3096, 2950 (3NH, NH2), 1701, 1664 (C = O). MS (EI) m/z (%): 361.45 (M+, 10%), Anal. calcd. for C18H27N5O3: C, 59.81; H, 7.53; N, 19.39. Found: C, 59.50; H, 7.33; N, 19.50.
2-(2-((1-Carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)-N-isopropylhydrazine-1-carboxamide (5b)
Yellowish brown oil, yield 88%,; “1HNMR” (CDCl3 d6, δ, ppm): 1.07 (d, 3H, J = 6.5 Hz, CH3-CH), 1.11–1.15 (m, 2H, CH2-cyclopentyl), 1.52–1.97 (m, 2H, 2CH2-cyclopentyl), 2.33–2.42 (m, 2H, CH2-cyclopentyl), 3.82 (s, 2H, CH2-NH), 4.38 (s, 1H, (CH3)2), 5.60 (br.s, 1H, NH), 7.02–7.14 (m, 2H, Har.), 7.23 (s, 2H, NH2), 7.40–7.44 (m, 3H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 23.30 (2CH3), 25.37, 37.22 (CH2-cyclopentyl), 41.63 (CH-CH3), 45.0 (CH2-NH), 70.8 (Cq), 128.70, 129.39, 129.51, 137.59 (aromatic carbons), 165.97, 170.08, 171.67 (3C = O), IR (KBr, cm−1) 3321, 3232, 3086, 2974 (3NH, NH2), 1666 (C = O), MS (EI) m/z (%): 361.45 (M+, 10%), Anal. calcd. for C18H27N5O3: C, 59.81; H, 7.53; N, 19.38. Found: C, 59.50; H, 7.32; N, 19.50.
N-Butyl-2-(2-((1-carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (5c)
Yield 89%, “1HNMR” (CDCl3 d6, δ, ppm): 0.83(t, 3H, J = 7.65 Hz, CH3), 1.22–1.46 (m, 8H, 4CH2-cyclopentyl), 1.62–2.05 (m, 4H, (CH2)2-CH3), 2.99 (t, 2H, J = 6.7 Hz, CH2-(CH2)2-CH3), 3.40 (s, 2H,CH2-CO), 4.29 (br.s, 1H, NH), 7.01–7.15 (m, 2H, Har.), 7.10 (s, 2H, NH2), 7.11–7.40 (m, 5H, Har.), 8.29 (br.s, 1H, NH). 13CNMR (CDCl3 d6, δ, ppm): 13.82 (CH3-CH2), 20.09 (CH3-CH2), 24.81, 25.00 (2CH2-cyclopentyl), 32.06 (CH3-CH2-CH2), 40.50 (CH2-(CH2)2-CH3), 55.08 (CH2-CO), 67.91 (Cq), 115.23, 116.56, 129.26, 129.37 (aromatic carbons), 158.89, 175.99, 192.75 (3 C = O); IR (KBr, cm−1) 3458, 3358, 3302 (3NH, NH2), 16604 (C = O); MS (EI) m/z (%): 375.47 (M+, 10%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.77; N, 18.62.
N-Benzyl-2-(2-((1-carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (5d)
Pale yellow oil, yield 89%, “1HNMR” (CDCl3 d6, δ, ppm): 1.40–1.71 (m, 8H, CH2-cyclopentyl), 2.13–2.16 (m, 3H, CH2-cyclohexyl), 4.42 (s, 2H, CH2-CO), 4.46 (br.s, 1H, NH), 7.05 (br.s, 1H, NH), 7.12 (br.s, 1H, NH), 7.24 (s, 2H, NH2), 7.26–7.36 (m, 7H, Har.), 7.39–7.42 (m, 3H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 22.71, 25.4, 32.41 (2CH2-cyclopentyl), 44.23 (CH2-C6H5), 57.4 (CH2-CO), 68.51 (Cq), 119.72, 121.9, 122.7, 126.8, 127.5, 128.4, 134.59, 136.31 (aromatic carbons), 157.78, 164.7, 178.0 (C = O), IR (KBr, cm−1) 3369, 3288, 3030, 2937 (3NH, NH2), 1703, 1691, 1650 (3C = O);MS (EI) m/z (%): 409.21 (M+, 18%), Anal. calcd. for C22H27N5O3: C, 64.53; H, 6.65; N, 17.10. Found: C, 64.50; H, 6.68; N, 17.15.
2-(2-((1-Carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)- N- propyl hydrazine -1 -carboxamide (6a)
White solid, m.p. 200 °C, yield 85%, “1HNMR” (CDCl3 d6, δ, ppm): 0.83 (t, 3H, J = 8 Hz, CH3), 1.38–1.81 (m, 12H, 5CH2-cyclohexyl, 2H, CH2-CH3), 2.99 (t, 2H, J = 8 Hz, CH2-CH2-CH3), 3.37 (s, 2H, CH2-CO), 4.31 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.41–7.45 (m, 10H, Har.), 8.364.31 (br.s, 1H, NH). 13CNMR (CDCl3 d6, δ, ppm): 11.76 (CH3), 21.08 (CH2-CH3), 22.70, 23.68, 33.97 (3CH2-cyclohexyl), 41.50 (CH2-CH2-CH3), 55.54 (CH2-CO), 62.45 (Cq), 127.93, 129.09, 130.51, 137.45 (aromatic carbons), 157.85, 166.58, 168.76 (3C = O), IR (KBr, cm−1) 3371, 3300, 3223 (3NH, NH2), 1701, 1687, 1678 (C = O); MS (EI) m/z (%): 375.47 (M+, 22%). Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.76; N, 18.62.
2-(2-((1-Carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)-N-isopropylhydrazine-1-carboxamide (6b)
Yellowish white oil, yield 86%, “1HNMR” (CDCl3 d6, δ, ppm): 1.20 (d, 3H, J = 6.5 Hz, CH3), 1.84 (s, 4H, 2CH2-cyclohexyl), 1.96 (s, 6H, 3 CH2-cyclohexyl), 3.64 (s, 2H, CH2-CO), 3.96–4.03 (m, 1H, CH-CH3)2, 4.39 (br.s, 1H, NH), 7.29 (s, 2H, NH2), 7.39–7.48 (m, 5H, Har.), 13CNMR(CDCl3 d6, δ, ppm): 22.90 (CH3), 23.26, 24.51, 30.91 (3CH2-cyclopentyl), 41.63 (CH-(CH3)2), 51.93 (CH2-CO), 64.32 (Cq), 129.73, 129.99, 130.02, 137.02 (aromatic carbons), 163.94, 166.73, 175.60 (3C = O), IR (KBr, cm−1) 3429, 3421, 3196, 2981 (3NH, NH2), 1747, 1708, 1685 (C = O);MS (EI) m/z (%): 375.47 (M+, 15%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.78; H, 7.81; N, 18.67.
N-Butyl-2-(2-((1-carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (6c)
Yield 87%, “1HNMR” (CDCl3 d6, δ, ppm): 0.86 (t, 3H, J = 4 Hz, CH3), 1.27–1.40 (m, 10H, 5CH2-cyclohexyl), 1.48–1.81 (m, 4H, (CH2)2-CH3), 3.01 (t, 2H, J = 8 Hz, CH2-(CH2)2-CH3), 3.35 (s, 2H,CH2-CO), 4.29 (br.s, 1H, NH), 7.03–7.16 (m, 2H, Har.), 7.27 (s, 2H, NH2), 7.40–7.48 (m, 3H, Har.), 8.26 (br.s, 1H, NH). 13CNMR (CDCl3 d6, δ, ppm): 13.73 (CH3-CH2), 20.08 (CH3-CH2), 22.72, 24.43, 32.06 (3 CH2-cyclohexyl), 34.17 (CH3-CH2-CH2), 42.89 (CH2-(CH2)2-CH3), 58.28 (CH2-CO), 63.22 (Cq), 129.42, 130.16, 137.12, 149.12 (aromatic carbons), 157.67, 167.19, 170.49 (3C = O), IR (KBr, cm−1) 3369, 3300, 3226, 2956 (3NH, NH2), 1703, 1691, 1619 (C = O); MS (EI) m/z (%): 389.59 (M+, 15%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.76; N, 18.62.
N-Benzyl-2-(2-((1-carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (6d)
White solid, m.p. 210–212 °C, 84.5%, “1HNMR”(CDCl3 d6, δ, ppm): 1.40–1.55 (m, 5H, CH2-cyclohexyl), 1.72–1.82 (m, 2H, CH2-cyclohexyl), 2.13–2.16 (m, 3H, CH2-cyclohexyl), 4.40 (s, 2H, CH2-CO), 4.48 (br.s, 1H, NH), 7.09 (br.s, 1H, NH), 7.11 (br.s, 1H, NH), 7.24 (s, 2H, NH2), 7.26–7.34 (m, 7H, Har.), 7.39–7.45 (m, 3H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 22.71, 25.4, 32.41 (3CH2-cyclohexyl), 44.23 (CH2-C6H5), 57.4 (CH2-CO), 68.55 (Cq), 119.70, 121.7, 122.7, 126.6, 127.6, 128.3, 134.59, 136.31 (aromatic carbons), 156.79, 164.7, 178.0 (3C = O), IR (KBr, cm−1) 3369, 3288, 3203 (3NH, NH2), 1705, 1687, 1672 (C = O);MS (EI) m/z (%): 423.52 (M+, 5%), Anal. calcd. for C23H29N5O3: C, 65.23; H, 6.90; N, 16.54. Found: C, 65.25; H, 6.93; N, 16.55.
2-(2-((1-Carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)-N-propylhydrazine-1-carboxamide (7a)
Pale yellow oil, 87%, “1HNMR” (CDCl3 d6, δ, ppm): 0.95 (t, 3H, J = 4 Hz, CH3-CH2), 1.64–1.79 (m, 10H, 5CH2-cyclohexyl), 1.80 (s, 3H, CH3), 1.93–1.96 (m, 2H, CH2-CH2-CH3), 2.32 (s, 3H, CH3), 2.70 (br.s, 1H, NH), 3.73 (br.s, 1H, NH), 3.84 (t, 3H, J = 8, CH2-CH2-CH3), 4.38 (s, 2H, CH2-CO), 2.70 (s, 1H, NH), 7.22 (d, 2H, J = 4 Hz, Har.), 7.24 (d, 2H, J = 4 Hz, Har.), 7.28 (s, 2H, NH2). 13CNMR (CDCl3 d6, δ, ppm): 11.19 (CH3-CH2), 22.45, 23.34, 37.21 (3CH2, cyclohexyl), 23.41 (CH2-CH3), 44.43 (CH2-CH2-CH3), 53.94 (CH2-CO), 70.83 (Cq), 129.08, 129.22, 137.58, 140.49 (aromatic carbons), 149.06, 166.00, 171.69 (3C = O), IR (KBr, cm−1) 3290, 3215, 3196 (3NH, NH2), 1662 (C = O); MS (EI) m/z (%): 389.50 (M+, 10%), Anal. calcd. for C20H31N5O3: C, 61.67; H, 8.02; N, 17.98. Found: C, 61.65; H, 8.05; N, 17.97.
2-(2-((1-Carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)-Nisopropylhydrazine-1-carboxamide (7b)
Pale yellow oil, 90%, “1HNMR” (CDCl3 d6, δ, ppm): 1.08–1.13 (m, 4H, 2CH2-cyclohexyl), 1.15 (d, 2H, J = 8 Hz, CH3-CH), 1.17–1.27 (m, 6H, 3CH2-cyclohexyl), 1.83 (s, 3H, CH3), 2.77 (br.s, 1H, NH), 3.75 (s, 2H, CH2-CO), 4.40 (s, 1H, CH-CH3)2, 4.67 (br.s, 1H, NH), 6.70 (br.s, 1H, NH), 7.15 (d, 2H, J = 8 Hz, Har.), 7.25 (d, 2H, J = 8 Hz, Har.), 7.28 (s, 2H, NH2). 13CNMR (CDCl3 d6, δ, ppm): 23.77 (CH3), 23.97 (CH-(CH3)2), 24.21, 29.69, 37.22 (3CH2-cyclohexyl), 54.15 (CH2-CO), 70.84 (Cq), 129.20, 129.39, 129.46, 137.60 (aromatic carbons), 165.96, 170.08, 178.78 (3C = O), IR (KBr, cm−1) 3313, 3234, 3084 (3NH, NH2), 1689, 1680, 1656 (C = O); MS (EI) m/z (%): 389.50 (M+, 15%), Anal. calcd. for C20H31N5O3: C, 61.67; H, 8.02; N, 17.98. Found: C, 61.65; H, 8.03; N, 17.95.
N-Butyl-2-(2-((1-carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (7c)
Pale yellow, 89%, “1HNMR” (CDCl3 d6, δ, ppm): 0.87 (t, 3H, J = 7.5 Hz, CH3-CH2), 1.15 (t, 4H, J = 7 Hz, CH2, CH2-CH3), 1.95–1.1.98 (m, 10H, 5CH2-cyclohexyl), 3.62 (s, 2H,CH2-CO), 3.79 (t, 2H, J = 7.5 Hz, CH2-CH2-CH2-CH3), 4.31 (br.s, 1H, NH), 7.07 (d, 2H, J = 5.8 Hz, Har.), 7.16 (d, 2H, J = 5.8 Hz, Har.), 7.20 (s, 2H, NH2), 7.98 (br.s, 1H, NH), 8.91 (br.s, 1H, NH), 13CNMR (CDCl3 d6, δ, ppm): 13.7 (CH3-CH2), 18.2 (CH3-CH2), 20.8 (CH3), 22.4, 25.3 (2CH2-cyclohexyl), 31.8 (CH2-cyclohexyl), 36.81 (CH3-CH2-CH2), 42.8 (CH2-(CH2)2-CH3), 58.2 (CH2-CO), 71.0 (Cq), 129.0, 129.2, 137.5, 140.4 (aromatic carbons), 159.1, 166.7, 180.2 (3 C = O), IR (KBr, cm−1) 3330, 3214, 3050, 2937 (3NH, NH2), 1730, 1688, 1672 (C = O); MS (EI) m/z (%): 403.53 (M+, 7%), Anal. calcd. for C21H33N5O3: C, 62.51; H, 8.24; N, 17.36. Found: C, 62.54; H, 8.25; N, 17.35.
N-Benzyl-2-(2-((1-carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (7d)
Oil, 85%, “1HNMR” (CDCl3 d6, δ, ppm): 1.68–2.00 (m, 10H, 5CH2-cyclohexyl), 2.34 (s, 3H, CH3), 4.28 (s, 2H, CH2-CO), 5.23 (s, 2H, CH2-C6H5), 7.10 (s, 1H, NH), 7.22–7.29 (m, 9H, Har.), 8.99 (br.s, 1H, NH). 13CNMR (CDCl3 d6, δ, ppm): 21.02 (CH3), 22.21, 24.25, 30.21 (3CH2-cyclohexyl), 43.86 (CH2-C6H5), 52.43 (CH2-CO), 70.55 (Cq), 127.15, 127.34, 128.49, 129.69, 129.91 131.21, 134.21, 137.52 (aromatic carbons), 158.21, 166.82, 180.10 (3C = O), IR (KBr, cm−1) 3400, 3332, 3294, (3NH, NH2), 1693, 1662 (3C = O); MS (EI) m/z (%): 437.54 (M+, 30%), Anal. calcd. for C24H31N5O3: C, 65.88; H, 7.14; N, 16.01. Found: C, 65.89; H, 7.15; N, 16.03.
Synthesis of substituted (2,4,5-trioxoimidazolidin-1-yl)amino-N-phenylacetamido)cycloalkane-1-carboxamides 8a-d, 9a-d and 10a-d
To a 0.001 mol of 5a-d, 6a-d or 7a-d in dry benzene a 0.002 mol of oxalyl chloride was supplemented. The mix was heated for 3 h then benzene was allowed to evaporate. The residual substance was subjected to column chromatography using mixture of CHCl2 and CH3OH as mobile phase to obtain 8a-d, 9a-d and 10a-d.
1-(N- Phenyl-2-((2,4,5 - trioxo-3-propyl imidazolidin-1-yl)amino)acetamido) cyclopentane-1-carboxamide (8a)
White solid, m.p. 110–112 °C, yield 80%, HPLC: purity 98.7%, Rt = 3.98 min. “1HNMR” (CDCl3 d6, δ, ppm): 0.91–1.01 (m, 3H, CH3-CH2), 1.61–1.91 (m, 10H, 4CH2-cyclopentyl, CH2-CH3), 3.63 (t, 2H, J = 8, CH2-CH2-CH3), 3.73 (s, 2H, CH2-CO), 4.59 (br.s, 1H, NH), 7.22 (s, 2H, NH2), 7.38–7.54 (m, 5H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 11.13 (CH3), 20.94 (CH2-CH3), 23.92, 36.14 (2 CH2-cyclopentyl), 41.67 (CH2-CH2-CH3), 44.47 (CH2-CO), 71.09 (Cq), 115.56, 128.35, 130.10, 135.70 (aromatic carbons), 149.09, 152.55, 154.54 (3C = O, imidazolidine), 166.5, 169.30 (2C = O); IR (KBr, cm−1) 3365, 3251 (NH, NH2), 1755, 1689, 1631 (C = O);MS (EI) m/z (%): 415.45 (M+, 4%), Anal. calcd. for C20H25N5O5: C, 57.82; H, 6.07; N, 16.86. Found: C, 57.81; H, 6.15; N, 16.75.
1-(2-((3 – Isopropyl - 2,4,5-trioxo imidazolidin-1-yl)amino) – N -phenylacetamido)cyclopentane-1-carboxamide (8b)
Yellowish brown oil, yield 75%, HPLC: purity 99.4%, Rt = 4.30 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.07–1.11 (m, 2H, CH2-cyclopentyl), 1.14 (d, 6H, J = 6.5, (CH3)2), 1.22–1.26 (m, 2H, CH2-cyclopentyl), 1.39–1.50 (m, 4H, 2CH2-cyclopentyl) 2.07 (br.s, 1H, NH), 3.71 (s, 2H, CH2-CO), 4.51 (m, 1H, CH-(CH3)2), 7.26 (s, 2H, NH2), 7.36–7.45 (m, 5H, Har.). 13C NMR (CDCl3, δ, ppm): 20.43 (CH3), 23.00, 37.00 (2CH2-cyclopentyl), 42.47 (CH2-CO), 51.16 (CH-(CH3)2), 63.85 (Cq), 129.65, 129.77, 130.12, 137.57 (aromatic carbons), 148.78, 151.81, 159.06 (3 C = O, imidazolidine), 165.06, 177.20 (2 C = O), IR (KBr, cm−1) 3304, 3234, 3219 (NH, NH2), 1662, 1610, 1593 (C = O); MS (EI) m/z (%): 415.45 (M+, 25%), Anal. calcd. for C20H25N5O5: C, 57.82; H, 6.07; N, 16.86. Found: C, 57.72; H, 6.15; N, 16.69.
1-(2-((3-Butyl-2,4,5-trioxoimidazolidin-1-yl)amino)-N-phenylacetamido)cyclopentane-1-carboxamide (8c)
Yellowish brown oil, yield 80 %, HPLC: purity 96.5%, Rt = 3.39 min. “1HNMR” (CDCl3 d6, δ, ppm): 0.87 (t, 3H, J = 8 Hz, CH3), 1.13–1.18 (m, 2H, CH3-CH2), 1.23–1.27 (m, 2H, CH2-CH2-CH3), 1.29–1.68 (m, 8H, 4CH2-cyclopentyl), 3.50 (s, 2H, CH2-CO), 3.80 (t, 2H, J = 8 Hz, CH2-CH2-CH2-CH3), 4.50 (br.s, 1H, NH), 7.22 (br.s, 2H, NH2), 7.27–7.38 (m, 5H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 13.70 (CH3), 18.18 (CH2-CH3), 24.14, 36.64 (2 CH2-cyclopentyl), 29.38 (CH2-CH2- CH3), 42.88 (CH2-CH2- CH2-CH3), 52.98 (CH2-CO), 72.38 (Cq), 119.31, 128.65, 129.02 (3CHar.), 137.38 (Car.), 148.45, 155.07, 159.22 (3C = O, imidazolidine), 163.07, 177.28 (2C = O). IR (KBr, cm−1) 3444, 3419, 3360 (NH, NH2), 1755, 1747, 1734 (C = O); MS (EI) m/z (%): 429.48 (M+, 20%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.80; H, 6.31; N, 16.35.
1-(2- ((3 -Benzyl- 2,4,5- trioxo imidazolidin-1-yl) amino)- N- phenylacetamido) cyclopentane-1-carboxamide (8d)
Pale yellow viscous oil, yield 71%, HPLC: purity 98%, Rt = 7.18 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.14–1.27 (m, 4H, 2CH2-cyclopentyl), 1.94–2.08 (m, 4H, 2CH2-cyclopentyl), 4.71 (s, 2H, CH2-C6H5), 4. 76 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.16–7.29 (m, 10H, Har.), 13CNMR (CDCl3 d6, δ, ppm): 25.01, 35.55 (2 CH2-cyclopentyl), 42.93 (CH2-C6H5), 43.95 (CH2-CO), 67.90 (Cq), 128.96, 128.98, 129.01 129.07, 129.08, 129.12, 136.90, 137.25 (aromatic carbons), 148.56, 151.68, 153.43 (3C = O, imidazolidine), 166.92, 172.75 (2C = O). IR (KBr, cm−1) 34195, 3273, 3186, 3089 (NH, NH2), 1759, 1720, 1680, 1665, 1631 (5 C = O); MS (EI) m/z (%): 463.39 (M+, 10%), Anal. calcd. for C24H25N5O5: C, 62.19; H, 5.44; N, 15.11. Found: C, 62.21; H, 5.45; N, 15.15.
1-(N- Phenyl-2-((2,4,5 - trioxo- 3-propyl imidazolidin-1-yl)amino) acetamido) cyclohexane-1-carboxamide (9a)
White solid, m.p. 120 °C, yield 75%, HPLC: purity 97.6%, Rt = 5.05 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.10–1.23 (m, 3H, CH3-CH2), 1.41–1.91 (m, 12H, 5CH2-cyclohexyl, CH2-CH3), 3.61 (t, 2H, J = 8, CH2-CH2-CH3), 3.75 (s, 2H, CH2-CO), 4.59 (br.s, 1H, NH), 7.22 (s, 2H, NH2), 7.38–7.54 (m, 5H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 11.14 (CH3), 20.84 (CH2-CH3), 23.92, 36.14 (2 CH2-cyclopentyl), 41.61 (CH2-CH2-CH3), 44.47 (CH2-CO), 69.12 (Cq), 115.56, 128.35, 130.10 (3CHar.), 135.91 (Car.), 149.09, 152.55, 154.54 (3C = O, imidazolidine), 166.2, 169.30 (2C = O), IR (KBr, cm−1) 3313, 3294, 3142 (NH, NH2), 1690, 1685, 1630 (C = O); MS (EI) m/z (%): 429.48 (M+, 25%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.71; H, 6.38; N, 16.29.
1-(2- ((3-Isopropyl -2,4,5 – trioxo imidazolidin-1-yl) amino) -N-phenyl acetamido) cyclohexane-1-carboxamide (9b)
White solid, m.p. 160 °C, yield 80%, HPLC: purity 99.4%, Rt = 4.27 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.11 (d, 6H, J = 4 Hz, (CH3)2), 1.38–1.61 (m, 10H, 5CH2-cyclohexyll), 3.65 (s, 2H, CH2-CO), 4.33–4.50 (m, 1H, CH-(CH3)2), 4.93 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.27 (s, 2H, Har.), 7.36–7.48 (m, 3H, Har.); 13C NMR (CDCl3, δ, ppm): 20.68 CH3, 22.49, 25.85, 29.68 (3 CH2-cyclohexyl), 46.79 (CH2-CO), 53.57 (CH-(CH3)2), 66.06 (Cq), 129.61, 129.75, 129.87, 134.78 (aromatic carbons), 148.23, 156.44, 157.21 (3C = O, imidazolidine), 164.89, 166.19 (2 C = O), IR (KBr, cm−1) 3209, 3184, (NH, NH2), 1762, 1728, 1697 (C = O); MS (EI) m/z (%): 429.48 (M+, 10%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.75; H, 6.34; N, 16.30.
1-(2-((3-Butyl-2,4,5-trioxo imidazolidin-1-yl) amino)- N-phenyl acetamido) cyclohexane-1-carboxamide (9c)
White solid, m.p. 170 °C, yield 75%, HPLC: purity 97.6%, Rt = 6.59 min. “1HNMR” (CDCl3 d6, δ, ppm): 0.96 (t, 3H, J = 4 Hz, CH3), 1.25 36–1.41 (m, 2H, CH2- CH3), 1.61–1.63 (m, 10H, 5CH2-cyclohexyl), 3.66 (s, 2H, CH2-CO), 3.73 (t, 2H, CH2-(CH2)2 -CH3), 4.54 (br.s, 1H, NH), 7.18 (s, 2H, Har.), 7.26 (br.s, 2H, NH2), 7.27–7.46 (m, 3H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 13.73 (CH3), 19.89 (CH2-CH3), 20.04, 22.51, 29.82 (3CH2-cyclohexyl), 29.96 (CH2-CH2- CH3), 42.90 (CH2-CH2- CH2-CH3), 53.42 (CH2-CO), 63.23 (Cq), 129.61, 129.82, 130.08, 136.56, 137.38 (aromatic carbons.), 148.78, 151.81, 153.71 (3C = O, imidazolidine), 165.06, 167.69 (2 C = O). MS (EI) m/z (%): 443.50 (M+, 10%), IR (KBr, cm−1) 3392, 3385, 3275 (NH, NH2), 1795, 1759, 1685 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.55; H, 6.62; N, 15.83.
1-(2-((3-Benzyl-2,4,5- trioxo imidazolidin-1-yl) amino)-N-phenyl acetamido) cyclohexane-1-carboxamide (9d)
White solid, m.p. 175 °C, yield 90%, HPLC: purity 97.9%, Rt = 8.25 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.25 (s, 4H, 2CH2-cyclohexyl), 1.46–1.72 (m, 6H, 3 CH2-cyclohexyl), 4.84 (s, 2H, CH2-CO), 4.87 (s, 2H, CH2-C6H5),7.25 (s, 2H, NH2), 7.35–7.37 (m, 10H, Har.) 7.50 (br.s, 1H, NH). 13CNMR (CDCl3 d6, δ, ppm): 22.89, 34.52, 34.58 (3CH2-cyclohexyl), 43.52 (CH2-C6H5), 44.03 (CH2-CO), 64.40 (Cq), 128.79, 128.97, 129.01, 129.03, 129.12, 129.18, 133.82, 134.99 (aromatic carbons), 149.94, 151.59, 153.31 (3C = O, imidazolidine), 164.89, 166.19 (2 C = O), MS (EI) m/z (%): 477.52 (M + , 54), IR (KBr, cm−1) 3419, 3273, 3186 (NH, NH2), 1759, 1680, 1625 (C = O); Anal. calcd. for C25H27N5O5: C, 62.88; H, 5.70; N, 14.67. Found: C, 62.86; H,5.68; N, 14.63.
1-(N-(p-Tolyl)-2-((2,4,5-trioxo-3-propylimidazolidin-1-yl)amino)acetamido)cyclohexane-1-carboxamide (10a)
White solid, m.p. 110–112 °C, yield 75%, HPLC: purity 97%, Rt = 3.81 min. “1HNMR” (CDCl3 d6, δ, ppm): 0.92 (t, 3H, J = 4 Hz, CH2-CH3), 1.56–1.59 (m, 2H, CH2-CH3), 1.66–1.73 (m, 10H, 5CH2-cyclohexyl), 1.79 (s, 3H, CH3), 3.85 (t, 2H, J = 8, CH2-CH2-CH3), 3.73 (s, 2H, CH2-CO), 4.53 (br.s, 1H, NH), 7.28 (s, 2H, NH2), 7.36 (d, 2H, J = 8, Har.), 7.46 (d, 2H, J = 8 Hz, Har.). 13CNMR (CDCl3 d6, δ, ppm): 11.10 (CH3-CH2), 21.14 (CH3-C6H5), 23.94, 24.17, 36.97 (3CH2-cyclohexyl), 42.39 (CH2-CH2-CH3), 44.46 (CH2-CO), 72.38 (Cq), 128.75, 129.56, 129.73, 140.71 (aromatic carbons), 149.0, 152.55, 154.54 (3C = O, imidazolidine), 172.28, 177.53 (2 C = O). MS (EI) m/z (%): 443.50 (M+, 50%), IR (KBr, cm−1) 3290, 3165 (NH, NH2), 1685, 1662 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.54; H, 6.57; N, 15.78.
1-(2- ((3-Isopropyl -2,4,5 –trioxo imidazolidin-1-yl) amino)-N- (p-tolyl) acetamido) cyclohexane-1-carboxamide (10b)
White solid, m.p. 150–152 °C, yield 83%, HPLC: purity 99%, Rt = 5.94 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.14 (d, 6H, J = 6.5 Hz, (CH3)2), 1.43–1.54 (m, 10H, 5CH2-cyclohexyl), 1.77 (s, 3H, C6H5-CH3), 3.87 (s, 2H, CH2-CO), 4.41–4.57 (m, 1H, CH-(CH3)2), 7.17 (d, 2H, J = 6.5 Hz, Har.), 7.27 (s, 2H, NH2), 7.44 (d, 2H, J = 6.5 Hz, Har.); “13C NMR” (CDCl3, δ, ppm): 20.42 (CH-(CH3)2), 21.15 (CH3-C6H5), 22.32, 25.16, 29.72 (3 CH2-cyclohexyl), 46.17 (CH2-CO), 53.52 (CH-(CH3)2), 67.12 (Cq), 129.41, 129.65, 129.81, 135.73 (aromatic carbons), 148.21, 154.42, 157.13 (3C = O, imidazolidine), 164.42, 167.16 (2 C = O), MS (EI) m/z (%): 443.50 (M+, 50%), IR (KBr, cm−1) 3155, 3091 (NH, NH2), 1745, 1708, 1680 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.57; H, 6.55; N, 15.77.
1-(2-((3-Butyl -2,4,5-trioxo imidazolidin-1-yl) amino)-N-(p-tolyl) acetamido) cyclohexane-1-carboxamide (10c)
White solid, m.p. 120 °C, yield 75%, HPLC: purity 99%, Rt = 6.07 min. “1HNMR” (CDCl3 d6, δ, ppm): 0.86 (t, 3H, J = 4.4 Hz, CH2-CH3), 1.24–1.30 (m, 2H, CH2-CH2-CH3), 1.24–1.30 (m, 2H, CH2-CH2-CH3), 1.47–1.69 (m, 10H, 5CH2-cyclohexyl), 1.97 (s, 3H, C6H4-CH3), 3.58 (s, 2H, CH2-CO), 3.80 (t, 2H, J = 7.4 Hz, CH2-(CH2)2 -CH3), 4.49 (br.s, 1H, NH), 7.20 (br.s, 2H, NH2), 7.27 (d, 2H, J = 8.4 Hz, Har.), 7.31 (d, 2H, J = 8.4 Hz, Har.). MS (EI) m/z (%): 457.53 (M+, 15%), IR (KBr, cm−1) 3385, 3311 (NH, NH2), 1755, 1687 (C = O); Anal. calcd. for C23H31N5O5: C, 60.38; H, 6.83; N, 15.31. Found: C, 60.36; H, 6.81; N, 15.30.
1-(2-((3-Benzyl - 2,4,5- trioxoimidazolidin-1-yl)amino)-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (10d)
White solid, m.p. 160 °C, yield 75%, HPLC: purity 95.9%, Rt = 6.60 min. “1HNMR” (CDCl3 d6, δ, ppm): 1.19–1.37 (m, 10H, 5CH2-cyclohexyl), 1.74 (CH3-C6H4), 3.66 (s, 2H, CH2-CO), 4.77 (s, 2H, CH2-C6H5), 4.85 (br.s, 1H, NH), 5.29 (s, 2H, NH2), 7.17–7.42 (m, 10H, Har.). 13CNMR (CDCl3 d6, δ, ppm): 20.7 (CH3), 22.4, 25.1, 36.9 (2 CH2-cyclohexyl), 44.6 (CH2-C6H5), 53.1 (CH2-CO), 70.9 (Cq), 129.06, 129.54, 129.64, 129.68, 129.77, 133.81, 137.05, 136.57 (aromatic carbons), 150.41, 154.44, 159.31 (3C = O, imidazolidine), 166.20, 177.51 (2C = O). MS (EI) m/z (%): 491.55 (M+, 30%), IR (KBr, cm−1) 3307 (NH, NH2), 1747, 1714, 1645 (C = O); Anal. calcd. for C26H29N5O5: C, 63.53; H, 5.95; N, 14.25. Found: C, 63.50; H, 5.61; N, 14.27.
Biological Evaluation
Animals
The anticonvulsant effect of the new hydrids 8a-d, 9a-d as well as 10a-d was carried on Male mature white mice range in weight from “20–25 g” that obtained from “European Reef Animal House Colony (Egypt)”. They were retained in the ambient condition for humidity, light/dark cycle and temperature. The mice were fed a regular diet and had free access to water. Prior to the start of the pharmacological experiments, mice were acclimated to laboratory conditions. Every part of the experimental procedures involving animals were conducted in agreement with the Ethics Committee of the Misr University for Science & Technology (Approval number MC-7). All of the compounds under investigation were suspended in a solution of 1 percent of Tween 80.
Calculation of the median lethal dose (LD50) and acute toxicity test
The mice were distributed randomly into groups (6 mice per group) Preliminary investigations were performed to detect the minimum dose that causes death of all mice (LD100). If the LD100 is not achieved, the chemical has a good safety profile, and the LD50 will be reported as greater than 0.5 g/kg. Animals were observed during the first 2 hours after receiving the maximal i.p. dosage of these chemicals for any signs of abnormal behavior or fatalities, and then once an hour for the next 4 hours. The monitoring was kept up each 2 h till the 12th h then at 24 h to inspect any late changes. The behaviors and any other toxic manifestations of the mice were monitored every day for three days and the animals were maintained under monitoring for two weeks.
Stage I: Preliminary evaluation of anticonvulsant effect
Subcutaneous pentylenetetrazole (scPTZ)-induced seizures method
A group of 6 animals serves as negative control and the test compounds or the standard antiepileptic drug were given i.p. to groups of 6 mice. Subsequently one hour of intraperitoneal injection of the substances, pentylenetetrazole (PTZ) was administrated 85 mg/kg subcutaneous injection, in the fatty fold of skin near the base of the neck [49, 50]. This dosage has been reported to produce clonic seizures that continues for at least five seconds in 97% of the mice under investigations.
Each mouse is put in a separate plastic cage for monitoring that persists for half an hour. The occurrence of tonic-clonic convulsions long-lasting for more than five seconds was detected. Lacking of a single five seconds episode of clonic convulsions through the period of monitoring (30 min) is considered as the end point in this experiment [51]. The results were expressed as percent protection.
Maximal electroshock seizure (MES) test
The experiment was carried out following the reported method described by White et al. and Krall et al. [52, 53]. Thirty min after the intraperitoneal injection of the compounds, in order to cause convulsions in mice, an electrical current was applied. Ugo Basil, ECT Unit, 57800 adjusted to a fixed 25 mA current intensity and 0.2 s stimulus duration, and at 50 Hz” via auricle electrodes. The fact that the test chemical was able to prevent tonic 180-degree hind limb flexion raises the possibility that it might slow the spread of MES-induced convulsions [54].
Neurotoxicity
Employing the motor coordination apparatus, neuronal toxicity of examined compounds was assessed for every signs of changes in motion coordination [51]. Before beginning the experiment, the mice were trained to keep their equilibrium on “a rotating 2.5 cm diameter of plastic rod (Ugo-Basile Accele. ROTA-ROD for mice, UGO Basile, 7650, Italy) at a fixed speed of 10 rpm for one minute”. Mice which fulfilling this prerequisite criteria and effectively maintain their balance on the rod for three times were selected to enter the experiment. Each test substance was injected, and then the experiment was conducted 30 minutes later. The mice were put back on the rotating rod, and their motor abilities were monitored for up to 60 seconds. The inability to keep the balance on the rod during one minute is considered as a sign of motor impairment and neurotoxicity [55].
Stage II: Determination of ED50
ED50 is defined as “the dose of the compound which protecting half of the mice in the group against scPTZ or MES seizures”. To determine the ED50, the chemical was administered into every groups of mice (n = 6) at varying dosages, resulting in a spectrum of seizure protection from 16 to 83%. The ED50 and its associated confidence interval might be deduced from the provided data plot.
Quantitative determination of GABA level in whole brain
Animals were divided into three groups and each group contains 6 mice. The first group was negative control group and was injected using a Tween 80 water suspension at concentration of 1%.The mice in the second and third groups were administered compounds 8b or 10b, correspondingly of 100 mg/ kg, intraperitoneally. The compounds were given for 7 days. One day after the final dose of the examined substance, the mice were sedated followed by brains dissection and were laid on ice and stored at a temperature of −80 degrees Celsius. Consequently, brains were crushed using “a homogenizer (Medical instruments, MPW-120, Poland), with ice-cooled saline (0.9% NaCl) to prepare 20% w/v homogenate. The homogenate was then centrifuged at 4000 rpm for 5 min at 4 °C using a cooling centrifuge to remove cell debris (Laborzentrifugen, 2k15, Sigma, Germany) then the supernatant was used for determination of brain’s γ- aminobutyric acid (GABA) content” following the previously reported method [56].
Hepatotoxicity
Animals were divided into seven groups (n = 6). The first group was the control group and was injected with 1% Tween 80 suspension; Groups (2–7) were administered substances 8b, 9a-d and 10b, correspondingly of 100 mg/ kg, intraperitoneally suspended in 1% Tween 80. The compounds were given for seven days. One day afterward the final dosing of the examined substances, the mice were sedated followed by collection of blood samples from the heart perforation then centrifugation at 4000 rpm for ten min to separate the serum. Analyses of the liver serum enzymes, total protein in addition of albumin were measured using commercially available kits “Biodiagnostic, Egypt”.
Statistical analysis
Before proceeding in the statistical analysis, data values were checked for normality using the Shapiro test and for heteroscedasticity using the Brown-Forsythe test. The data are presented as means ± S.E. Data were processed by one-way ANOVA followed by the Tukey–Kramer Post hoc test. GraphPad Prism software (version 8; GraphPad Software, Inc., San Diego, CA, USA) was employed to perform the statistical analysis and to establish the graphical representation.
In-Silico studies
Pharmacophore generation
The hypothesis was created through common feature pharmacophore model protocol in Discovery studio software 4.1 (Accelrys, Inc, San Diego, USA). Training set 1–10 was used to generate the anticonvulsant pharmacophore; they were stated to have antiepileptic action. There are different conformational models were constructed for of each structure of the test sets to generate the common feature hypotheses, whereas ten hypotheses were generated.
Docking study
Docking study was performed using Discovery studio software 4.1 (Accelrys, Inc, San Diego, USA). The crystal structure of GABA-AT (PDB: 1OHW) [57] was downloaded from RCSB protein data bank (http://www.rscb.org/pdb). In general, the docking workflow was listed as: (1) Protein was prepared using the “Protein Preparation protocol”. All water molecules were deleted from the structure of the complex. Hydrogen atoms and charges were added during a brief relaxation. After optimizing the hydrogen bond network, the crystal structure was minimized. (2) The ligand was prepared with ligand preparation protocol, including adding hydrogen atoms, then energy minimization. (3) The center of the grid box, grid position and spacing was defined with the intrinsic ligand. The top-ranking poses were determined. Ten of the most stable docking poses for each ligand were obtained and arranged according to their docking scores (-CDOCKER energy). The ideal pose which fulfills the reported key binding features was selected.
Physicochemical parameters
All physicochemical descriptors and pharmacokinetic properties were calculated using swissADME a fee online web tool
References
Nazar S, Siddiqui N, Alam O. Recent progress of 1, 3, 4‐oxadiazoles as anticonvulsants: Future horizons. Arch Pharm. 2020;353:1900342.
Zadali R, Baghery M, Abbasi M, Yavari N, Miran M, Ebrahimi SN. Anticonvulsant activity of Iranian medicinal plants and molecular docking studies of isolated phytochemicals. S Afr J Bot. 2022;149:646–57.
Toolabi M, Khoramjouy M, Aghcheli A, Ayati A, Moghimi S, Firoozpour L, et al. Synthesis and radioligand‐binding assay of 2, 5‐disubstituted thiadiazoles and evaluation of their anticonvulsant activities. Arch Pharm. 2020;353:2000066.
Emami S, Valipour M, Komishani FK, Sadati-Ashrafi F, Rasoulian M, Ghasemian M, et al. Synthesis, in silico, in vitro and in vivo evaluations of isatin aroylhydrazones as highly potent anticonvulsant agents. Bioorg Chem. 2021;112:104943.
Beghi E. The Epidemiology of Epilepsy. Neuroepidemiology. 2020;54:185–91.
(Epilepsy: World Health Organization; [updated 2 February 2022; cited 2022 12 December] Available from: https://www.who.int/news-room/fact-sheets/detail/epilepsy.
Ataollahi E, Solhjoo A, Rezaei Z, Behrouz M, Heidari R, Shahbazi MR, et al. Novel 1,4 benzothiazine 3-one derivatives as anticonvulsant agents: Design, synthesis, biological evaluation and computational studies. Comput Biol Chem. 2023;104:107870.
Meng Q, Ren X, Wang R, Han Y, Li X, Zhang Q, et al. Design, synthesis, anticonvulsant activity and structure-activity relationships of novel 7-Azaindole derivatives. Bioorg Chem. 2023;133:106430.
Dawidowski M, Król M, Szulczyk B, Chodkowski A, Podsadni P, Konopelski P, et al. Structure-activity relationship and cardiac safety of 2-aryl-2-(pyridin-2-yl)acetamides as a new class of broad-spectrum anticonvulsants derived from Disopyramide. Bioorg Chem. 2020;98:103717.
Amaye IJ, Harper T, Jackson-Ayotunde PL. Design and development of trifluoromethylated enaminone derivatives as potential anticonvulsants. J Fluor Chem. 2021;251:109886.
Aboul‐Enein MN, El‐Azzouny AAS, Saleh OA, Amin KM, Maklad YA, Hassan RM. Synthesis and anticonvulsant activity of substituted‐1, 3‐diazaspiro [4.5] decan‐4‐ones. Arch Pharm. 2015;348:575–88.
Noureldin NA, Kothayer H, Lashine ESM, Baraka MM, El‐Eraky W, Awdan SAE. Synthesis, anticonvulsant activity, and SAR study of novel 4‐quinazolinone derivatives. Arch Pharm. 2017;350:1600332.
Sahu M, Siddiqui N, Iqbal R, Sharma V, Wakode S. Design, synthesis and evaluation of newer 5, 6-dihydropyrimidine-2 (1H)-thiones as GABA-AT inhibitors for anticonvulsant potential. Bioorg Chem. 2017;74:166–78.
Hashemia SM, Emami S, Masihib PH, Shakiba A, Dehestani L, Ahangar N. Synthesis of 2-aryl-3-triazolyl-indoles from phenacyltriazole-derived hydrazones: Exploring new scaffolds for anticonvulsant activity. J Mol Struct. 2023;1276:134704.
Marzouk AA, Bass AK, Ahmed MS, Abdelhamid AA, Elshaier YA, Salman AM, et al. Design, synthesis and anticonvulsant activity of new imidazolidindione and imidazole derivatives. Bioorg Chem. 2020;101:104020.
Hassan RM, Aboutabl ME, Bozzi M, El-Behairy MF, El Kerdawy AM, Sampaolese B, et al. Discovery of 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids as novel PPARα agonists with anti-hyperlipidemic and antioxidant activities: Design, synthesis and in vitro/in vivo biological evaluation. Bioorg Chem. 2021;115:105170.
Hassan RM, Ali IH, Abdel‐Maksoud MS, Abdallah HM, El Kerdawy AM, Sciandra F, et al. Design and synthesis of novel quinazolinone‐based fibrates as PPARα agonists with antihyperlipidemic activity. Arch Pharm. 2022;355:2100399.
Kamiński K, Wiklik B, Obniska J. Synthesis and anticonvulsant activity of new N-phenyl-2-(4-phenylpiperazin-1-yl) acetamide derivatives. Med Chem Res. 2015;24:3047–61.
Zankowska-Jasińska W, Borowiec H, Golus J, Kolasa A, Zaleska B, Krzywosiński L, et al. Synthesis and pharmacological investigations of 3-(aminoalkylene)-1-aryl-2-thioxo-4, 5-imidazolidinedione and 2, 4, 5-imidazolidinetrione derivatives. Pol J Pharmacol Pharm. 1990;42:49–58.
Zankowska-Jasińska W, Borowiec H, Kolasa A, Ostrowska K, Zaleska B, Przemyk B, et al. 1, 3-disubstituted 2-thioxo-4, 5-imidazolidinediones and 2, 4, 5-imidazolidinetriones and their anticonvulsant activity. Pol J Pharmacol Pharm. 1990;42:59–68.
Aboul-Enein MN, El-Azzouny AA, Maklad YA, Ismail MA, Ismail NS, Hassan RM. Design and synthesis of certain substituted cycloalkanecarboxamides structurally related to safinamide with anticonvulsant potential. Res Chem Intermed. 2015;41:3767–91.
Aboul‐Enein MN, El‐Azzouny AA, Ragab F, Abdel‐Maksoud MS, Abd‐Allah WH, Maklad Y. Synthesis, molecular modeling, anticonvulsant and antinociceptive properties of New 1, 1‐Disubstituted Cyclohexane and 1, 3‐Diazaspiro [4.5] decane Derivatives. ChemistrySelect. 2019;4:1360–5.
Aboul-Enein MN, El- El-Azzouny AA, Amin KM, Aboutabl ME, Abo-Elmagd MI. Synthesis, molecular modeling studies, and anticonvulsant evaluation of novel 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides and their acetate esters. Arch Pharm. 2018;351:e1800269.
Aboutabl ME, Hassan RM, El-Azzouny AA-S, Aboul-Enein MN, Abd-Allah WH. Design and synthesis of novel parabanic acid derivatives as anticonvulsants. Bioorg Chem. 2020;94:103473.
Aboul-Enein MN, El-Azzouny AA, Attia MI, Maklad YA, Aboutabl ME, Ragab F, et al. Anticonvulsant profiles of certain new 6-Aryl-9-substituted-6, 9-diazaspiro-[4.5] decane-8, 10-diones and 1-Aryl-4-substituted-1, 4-diazaspiro [5.5] undecane-3, 5-diones. Inter J Mol Sci. 2014;15:16911–35.
Aboul-Enein HY, Aboul-Enein MN, El-Azzouny AA, Saleh OA, Hassan RM, Amin KM. Enantioseparation of Substituted 1, 3-Diazaspiro [4.5]Decan-4-Ones: HPLC comparative study on different polysaccharide type chiral stationary phases. J Chromatogr Sci. 2018;56:160–65.
Abd-Allah WH, Anwar MA, Mohammed ER, Elbaset MA, El Moghazy SM. Exploring new cyclohexane carboxamides based GABA agonist: Design, synthesis, biological evaluation, in silico ADME and docking studies. Bioorg Chem. 2023;136:106561.
Grover G, Pal R, Bhatia R, Yar MS, Nath R, Singh S, et al. Design, synthesis, and pharmacological evaluation of aryl oxadiazole linked 1,2,4-triazine derivatives as anticonvulsant agents. Med Chem Res. 2022;31:781–93.
Alhamzani AG, Yousef TA, Abou-Krisha MM, Raghu MS, Yogesh Kumar K, Prashanth MK, et al. Design, synthesis, molecular docking and pharmacological evaluation of novel triazine-based triazole derivatives as potential anticonvulsant agents. Bioorg Med Chem Lett. 2022;77:129042.
Löscher W, Fassbender CP, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models. Epilepsy Res. 1991;8:79–94.
Piredda S, Gale K. A crucial epileptogenic site in the deep prepiriform cortex. Nature. 1985;317:623–5.
Lukasiuk K, Lason W. Emerging molecular targets for anti-epileptogenic and epilepsy modifying drugs. Int J Mol Sci. 2023;24:2928.
Tritsch NX, Granger AJ, Sabatini BL. Mechanisms and functions of GABA co-release. Nat Rev Neurosci. 2016;17:139–45.
Aboutabl ME. Antiepileptic drugs: progress and development. Egypt Pharm J. 2018;17:129–40.
He X, Zhong M, Zhang T, Wu W, Wu Z, Xiao Y, et al. Synthesis and anticonvulsant activity of ethyl 1-(2-arylhydrazinecarboxamido)-2, 2-dimethylcyclopropanecarboxylate derivatives. Eur J Med Chem. 2012;54:542–8.
Hassan MZ, Khan SA, Amir M. Design, synthesis and evaluation of N-(substituted benzothiazol-2-yl) amides as anticonvulsant and neuroprotective. Eur J Med Chem. 2012;58:206–13.
Quan ZS, Wang J-M, Rho J-R, Kwak K-C, Kang H-C, Jun C-S, et al. Synthesis of 6-alkyloxyl-3, 4-dihydro-2 (1H)-quinoliones and their anticonvulsant activities. Bull Korean Chem Soc. 2005;26:1757–60.
Stöhr T, Kupferberg HJ, Stables JP, Choi D, Harris RH, Kohn H, et al. Lacosamide, a novel anti-convulsant drug, shows efficacy with a wide safety margin in rodent models for epilepsy. Epilepsy Res. 2007;74:147–54.
He X, Zhong M, Zhang T, Wu W, Wu Z, Yang J, et al. Synthesis and anticonvulsant activity of N-3-arylamide substituted 5, 5-cyclopropanespirohydantoin derivatives. Eur J Med Chem. 2010;45:5870–7.
Nanavati SM, Silverman RB. Design of potential anticonvulsant agents: mechanistic classification of GABA aminotransferase inactivators. J Med Chem. 1989;32:2413–21.
Abd-Allah WH, Osman EEA, Anwar MA-E-M, Attia HN, El Moghazy SM. Design, synthesis and docking studies of novel benzopyrone derivatives as anticonvulsants. Bioorg Chem. 2020;98:103738.
Partap S, Yar MS, Hassan MZ, Akhtar MJ, Siddiqui AA. Design, synthesis, and pharmacological screening of pyridazinone hybrids as anticonvulsant agents. Arch Pharm. 2017;350:1700135.
Sahu M, Siddiqui N, Naim MJ, Alam O, Yar MS, Sharma V, et al. Design, synthesis, and docking study of pyrimidine–triazine hybrids for GABA estimation in animal epilepsy models. Arch Pharm. 2017;350:1700146.
Refsgaard HH, Jensen BF, Brockhoff PB, Padkjær SB, Guldbrandt M, Christensen MS. In silico prediction of membrane permeability from calculated molecular parameters. J Med Chem. 2005;48:805–11.
Poudrel J, Hullot P, Vidal J, Girard J, Rossi J, Muller A, et al. Synthesis and pharmacological profile of new 1, 3-disubstituted cyclohexanes as leukotriene B4 receptor antagonists. Bioorg Med Chem Lett. 1996;6:2349–54.
Aboul-Enein MN, El-Azzouny A. 1-Alkyl-1, 4-diazaspiro [4.5] decane and [5.5] undecane-3, 5-diones as analgesics and anticonvulsants. Acta Pharm Suec. 1986;23:107–14.
Abd-Allah WH, Elshafie MF. Synthesis and biological evaluation of certain new cyclohexane-1-carboxamides as apoptosis inducers. Oriental J Chem. 2018;34:825.
Abd‐Allah WH, Salman A, Sabry Saad S. Anticancer activity of newly synthesized 1, 1‐disubstituted cyclohexane‐1‐carboxamides: in vitro caspases mediated apoptosis activators in human cancer cell lines and their molecular modeling. Drug Dev Res. 2019;80:933–47.
Fariello R, McArthur R, Bonsignori A, Cervini M, Maj R, Marrari P, et al. Preclinical evaluation of PNU-151774E as a novel anticonvulsant. J Pharmacol Exp Ther. 1998;285:397–403.
Nath R, Yar MS, Pathania S, Grover G, Debnath B, Akhtar J. Synthesis and anticonvulsant evaluation of indoline derivatives of functionalized aryloxadiazole amine and benzothiazole acetamide. J Mol Struct. 2021;1228:129742.
El-Ansary SL, Hassan GS, Rahman DEA, Farag NA, Hamed MI, Baset MADesign. synthesis and biological evaluation of some new succinimide, 2-iminothiazoline and oxazine derivatives based benzopyrone as anticonvulsant agents. Inter J Pharm Pharm Sci. 2016;8:222–8.
White NC, Hedenquist JW. Epithermal gold deposits: styles, characteristics and exploration. SEG Discovery. 1995;23:1–13.
Krall R, Penry J, White B, Kupferberg H, Swinyard E. Antiepileptic drug development: II. Anticonvulsant drug screening. Epilepsia. 1978;19:409–28.
Valipour M, Naderi N, Heidarli E, Shaki F, Motafeghi F, Talebpour Amiri F, et al. Design, synthesis and biological evaluation of naphthalene-derived (arylalkyl)azoles containing heterocyclic linkers as new anticonvulsants: A comprehensive in silico, in vitro, and in vivo study. Eur J Pharm Sci. 2021;166:105974.
Raveesha R, Kumar KY, Raghu MS, Benaka Prasad SB, Alsalme A, Krishnaiah P, et al. Synthesis, in silico ADME, toxicity prediction and molecular docking studies of N-substituted[1,2,4]triazolo[4,3-a]pyrazine derivatives as potential anticonvulsant agents. J Mol Struct. 2022;1255:132407.
Heinrikson RL, Meredith SC. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem. 1984;136:65–74.
Storici P, De Biase D, Bossa F, Bruno S, Mozzarelli A, Peneff C, et al. Structures of gamma-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5’-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with gamma-ethynyl-GABA and with the antiepilepsy drug vigabatrin. J Biol Chem. 2004;279:363–373.
Acknowledgements
The authors are thankful to Science, Technology, and Innovation Funding Authority (STDF), Egypt for supporting this work through Research Support Grant (RSG) No. 43691.
Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Abd-Allah, W.H., Abd El-Maksoud, M.S., Elbaset, M.A. et al. Design and synthesis of novel cycloalkanecarboxamide parabanic acid hybrids as anticonvulsants. Med Chem Res 33, 89–106 (2024). https://doi.org/10.1007/s00044-023-03166-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00044-023-03166-z