Synthesis and anticonvulsant activity of some 2-pyrazolines derived from chalcones

Nagihan Beyhan; Bedia Kocyigit-Kaymakcioglu; Salih Gümrü; Feyza Aricioglu

doi:10.1016/j.arabjc.2013.07.037

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Original Article

10 (

2_suppl

); S2073-S2081

doi:

10.1016/j.arabjc.2013.07.037

Synthesis and anticonvulsant activity of some 2-pyrazolines derived from chalcones

Nagihan Beyhan^{^a}, Bedia Kocyigit-Kaymakcioglu^{^a,⁎}, Salih Gümrü^{^b}, Feyza Aricioglu^{^b}

a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Istanbul, Turkey

b Department of Pharmacology and Psychopharmacology Research Unit, Faculty of Pharmacy, Marmara University, Istanbul, Turkey

⁎Corresponding author. Address: Marmara University Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpasa 34668, Istanbul, Turkey. Tel.: +90 212 4142962; fax: +90 212 3452952. bedia.kaymakcioglu@gmail.com (Bedia Kocyigit-Kaymakcioglu)

Received: 2012-10-3, Accepted: 2013-7-20, Published: 2013-05-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Synthesis of chalcones (1,3-diarylprop-2-en-1-ones) and 2-pyrazoline derivatives has been an active field of research due to their established pharmacological effects. In this study, a series of chalcones were prepared with methyl aryl ketones and substituted aldehydes in the presence of sodium hydroxide and methanol through Claisen-Schmidt condensation. 3,5-Disubstituted-4,5-dihydro-1H-pyrazole-1-carbothioamides were synthesized by refluxing selected chalcones and thiosemicarbazide in alkaline medium. Similarly N-3,5-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides were synthesized by refluxing selected chalcones with N-(4-chlorophenyl)semicarbazide in alkaline medium. Structures of the synthesized compounds were confirmed by elemental analysis and spectral (UV, IR, ¹H NMR, ¹³C NMR, and mass) data, which were in line with the proposed structures.

All compounds were tested for their anticonvulsant activity using pentylenetetrazole induced seizure (PTZ) and maximal electroshock seizure (MES) tests in mice at a dose level of 50 mg/kg. Among the 2-pyrazoline-1-carbothioamide derivatives, 5-(2,6-dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (2e) reduced grade-5 seizure activity and also increased survival rate in PTZ test. In MES test, 5-(4-methoxyphenyl)-3-[4-(methylsulphonyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide(2g) has not only decreased seizure severity, but also increased survival rate. Among the 2-pyrazoline-1-carboxamide derivatives, 3-(5-bromothiophen-2-yl)-N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3d) having 5-bromothiophen and 2,6-dichlorophenyl moieties and N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3e) having 5-chlorothiophen and 2,6-dichlorophenyl moieties showed remarkable activities in PTZ test. Among all tested derivatives, compound 3d was found to be the most active one and reduced grade-5 seizure severity and also increased survival rate.

Keywords

Chalcones

2-Pyrazolines

Carbothioamide

Carboxyamide

Anticonvulsant activity

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1 Introduction

Epilepsy is a central nervous system disorder characterized by unprovoked, recurrent seizures that may affect physical, mental, or behavioral functioning (Trimble and Dodson, 1994). Not only its medical influence, but also extensive psychological and economic outcomes reduce epilepsy patients’ quality of life. Successful seizure control is the key parameter in decreasing the psychosocial and economic costs of this disorder (Honarmand et al., 2011). The clinical need of refractory epilepsy remains unmet and drugs in use are not effective enough in epilepsy. Older antiepileptic drugs (AEDs), which have advantages like lower cost, wide availability and long-term experience are still in use today, but have a greater risk of serious toxicity compared to new generation AEDs, which differ in terms of mechanism of action and pharmacokinetic properties, and are also better tolerated (Eddy et al., 2011). Therefore researchers in medicinal chemistry are still proceeding to study on potential compounds (Stefan and Feuerstein, 2007; Siddiqui and Ahsan, 2010; Guan et al., 2010 ).References and further reading may be available for this article. To view references and further reading you must purchase this article. Among these compounds, 2-pyrazoline is a promising structure as a anticonvulsant agent (Küçükgüzel et al., 2000; Özdemir et al., 2007a,b ). N–CO moiety is well recognized as pharmacophoric requirements in some of the well-known anticonvulsant agents with varied mechanisms of action (Nikhil and Kishore, 2010).

During the past 20 years, the following fifteen new AEDs have been approved: eslicarbazepine acetate, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, pregabalin, retigabine, rufinamide, stiripentol, tiagabine, topiramate, vigabatrin, and zonisamide (Bialer, 2012). These new generation AEDs also affect some common mechanisms which are underlying causes of other CNS disorders. Therefore, future design of new AEDs may also have therapeutic potential in non-epileptic CNS disorders, such as neuropathic pain, migraine prophylaxis and bipolar disorder or fibromyalgia. This piquant financial situation attracts pharmaceutical industry a lot.

Recently, our group has been investigating the possible pharmacological potential of new molecules that contain a pyrazole scaffold (Koçyiğit-Kaymakçıoğlu et al., 2005, 2011). Among these series N-ethyl-N′-(3,5-dimethylpyrazole-4-yl)thiourea and N-(2-ethoxyphenyl)-N′-(3,5-dimethylpyrazole-4-yl)urea were found to show better anticonvulsant activity in MES test. In the MES test, these compounds exhibited median effective doses (ED₅₀) of 17.14 and 17.46 mg/kg, respectively. In view of these results and as a part of our enduring studies in the area of anticonvulsant agents, it was conceived to combine two pharmacophoric groups: pyrazoline ring and N–CO moiety. Pyrazoles and their reduced forms, pyrazolines, are well known nitrogen containing heterocylic compounds and various methods have been reported for their synthesis (Abid et al., 2009). In the present study, as starting compounds, a series of chalcones were prepared via Claisen Schmidt condensation and 2-pyrazoline derivatives 2a–i and 3a–e were synthesized by the reaction of chalcones with thiosemicarbazide and N-(4-chlorophenyl)semicarbazide. 2-Pyrazolines were tested for their potential anticonvulsant activity in pentylenetetrazole induced seizure (PTZ) and maximal electroshock seizure (MES) tests.

2 Results and discussion

2.1

2.1 Chemistry

The synthetic route to the target compounds is outlined in Scheme 1. The intermediated chalcones (1a–i) were prepared by reacting equimolar aldehyde and ketone in the presence of a base by conventional Claisen-Schmidt condensation. 3,5-disubstituted-4,5-dihydro-1H-pyrazole-1-carbothioamides (2a–i) were synthesized by refluxing compounds1a–i and thiosemicarbazide in the presence of alkaline medium and similiarly, N,3,5-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides (3a–e) were synthesized by refluxing selected chalcones with N-(4-chlorophenyl)semicarbazide in alkaline medium. The chalcone derivatives 1a, 1c, and 1d were original compounds. The chalcone derivatives 1b, 1e and 2-pyrazoline derivative2g have CAS numbers in SciFinder Scholar, but our study, for the first time, determined their structures.

Synthetic protocol of 1a–i, 2a–i and 3a–e.

All the compounds were isolated in satisfactory yields (35–91%) and purified by recrystallization from ethanol. The purity of the compounds was established by thin layer chromatography (TLC) and elemental analysis.

Structures of the novel synthesized compounds were confirmed by elemental analysis and spectral (UV, IR, ¹H NMR, ¹³C NMR, and mass) data, which were in line with the proposed structures.

The IR spectra of the compounds 2a–i afforded pyrazoline C⚌N stretching (1589–1573 cm⁻¹), thiocarbamoyl group N–H stretching (3462–3232 cm⁻¹) and C⚌S stretching (1373–1348 cm⁻¹) bands. The CH₂ protons of the pyrazoline ring resonated as a pair of doublets of doublets at 3.10–3.22 ppm (H_A), 3.86–4.00 ppm (H_B). The CH (H_X) proton appeared as a doublet of doublets at 5.84–6.32 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring (J_AB: 17.65–18.40 Hz, J_AX: 2.90–8.01 Hz, J_BX: 11.07–13.28 Hz). The same protons for compounds 3a–e were resonated as a pair of doublets of doublets at 3.13–3.22 ppm (H_A), 3.84–3.98 ppm (H_B). Also the CH (H_X) proton appeared as a doublet of doublets at 5.43–6.42 ppm (J_AB = 17.76–18.40, J_AX = 5.05–5.50 Hz, J_BX = 11.78-13.02 Hz). N–H protons of the thiocarbamoyl group for compounds 2a–i were seen at 7.41–8.08 and 7.90–8.16 ppm as two singlets. N–H protons of the carboxamide group for compounds 3a–e were seen at 9.10–9.37 ppm as singlet. The protons belonging to the aromatic ring and the other aliphatic groups are observed with the expected chemical shift and integral values. Mass spectra (ES-MS) of compounds showed a [MH]⁺ peak, in line with their molecular formula.

2.2

2.2 Pharmacology

The anticonvulsant activity of the new compounds was determined by using PTZ (Sigma) and MES tests. The use of intact rodents, makes it easier to detect possible anticonvulsant effect regardless of the mechanisms of action. MES and PTZ tests can be used in high throughput screening, as shown by the National Institutes of Health Anticonvulsant Screening Program. Furthermore, these models can provide insight into pharmacokinetic–pharmacodynamic relations.

All compounds were suspended in 0.5% methyl cellulose and administered intraperitoneally at a dose of 50 mg/kg 30 min prior the tests. The anticonvulsant potential of these compounds was investigated by both PTZ and MES tests and results are shown in Table 1. None of the compounds tested showed anticonvulsant effect in the MES test. The PTZ model basically simulates petit mal seizures. Among the 2-pyrazoline-1-carbothioamide derivatives, compound 2e carrying thiophen and 2,6-dichlorophenyl ring in 2-pyrazoline moiety decreased grade-5 seizures and also increased survival rate in PTZ test. Compound 2c having 5-bromothiophen and 2,6-dichlorophenyl ring in 2-pyrazoline moiety increased survival rate in PTZ test. In MES test, compound 2g having 4-methylsulfonylphenyl and 4-methoxyphenyl group in 2-pyrazoline moiety not only decreased seizures but also increased survival rate. That is why it is considered as the most active compound in MES test.

Table 1 Anticonvulsant activity results of compounds 2a–i and 3a–e.

Comp.	Ar₁	Ar₂	Dose, mg/kg	PTZ test (%)		MES test (%)
Comp.	Ar₁	Ar₂	Dose, mg/kg	Grade 5	Survival	Seizure	Survival
2a	4-Trifluoromethyl	4-Methoxyphenyl	50	72^⁎⁎⁎	38^⁎⁎⁎	80^⁎⁎⁎	10^⁎⁎⁎
2b	5-Chlorothiophen-2-yl	2,6-Dichlorophenyl	50	70^⁎⁎⁎	63^⁎⁎⁎	98^⁎⁎⁎	12^⁎⁎⁎
2c	5-Bromothiophen-2-yl	2,6-Dichlorophenyl	50	75^⁎⁎⁎	75^⁎⁎⁎	75^⁎⁎⁎	10^⁎⁎⁎
2d	4-Methylsulfonylphenyl	2,6-Dichlorophenyl	50	81^⁎⁎⁎	28^⁎⁎⁎	82^⁎⁎⁎	10^⁎⁎⁎
2e	Thiophen-2-yl	2,6-Dichlorophenyl	50	41^⁎⁎⁎	48^⁎⁎⁎	50^⁎⁎⁎	60^⁎⁎⁎
2f	Phenyl	2,6-Dichlorophenyl	50	44^⁎⁎⁎	52^⁎⁎⁎	50^⁎⁎⁎	60^⁎⁎⁎
2 g	4-Methylsulfonylphenyl	4-Metoxyphenyl	50	52^⁎⁎⁎	50^⁎⁎⁎	23^⁎⁎⁎	73^⁎⁎⁎
2 h	Thiophen-2yl	3,4-Dimetoxyphenyl	50	55^⁎⁎⁎	47^⁎⁎⁎	82^⁎⁎⁎	25^⁎⁎⁎
2i	Thiophen-2yl	4-Dimethylaminophenyl	50	42^⁎⁎⁎	48^⁎⁎⁎	54^⁎⁎⁎	60^⁎⁎⁎
3a	Phenyl	2,6-Dichlorophenyl	50	80^⁎⁎⁎	22^⁎⁎⁎	70^⁎⁎⁎	38^⁎⁎⁎
3b	4- Methylsulfonylphenyl	2,6-Dichlorophenyl	50	74^⁎⁎⁎	46^⁎⁎⁎	62^⁎⁎⁎	55^⁎⁎⁎
3c	Thiophen-2-yl	2,6-Dichlorophenyl	50	40^⁎⁎⁎	50^⁎⁎⁎	60^⁎⁎⁎	60^⁎⁎⁎
3d	5-Bromothiophen-2-yl	2,6-Dichlorophenyl	50	10^⁎⁎⁎	100^⁎⁎⁎	47^⁎⁎⁎	50^⁎⁎⁎
3e	5-Chlorothiophen-2-yl	2,6-Dichlorophenyl	50	10^⁎⁎⁎	95^⁎⁎⁎	52^⁎⁎⁎	55^⁎⁎⁎
Control			0	58^⁎⁎⁎	52^⁎⁎⁎	54^⁎⁎⁎	69^⁎⁎⁎

Each group consists of 6–10 mice. Compounds were compared to control group and statistical significance.

⁎⁎⁎ p < 0.001.

Among the 2-pyrazoline-1-carboxamide derivatives, compound 3d having 5-bromothiophen and 2,6-dichlorophenyl moiety and compound 3e having 5-chlorothiophen and 2,6-dichlorophenyl moiety were found much more potent in PTZ test because of the bulk and lipophilicity. The compounds 3d and3e reduced convulsion frequency in all types of grades (from grade 1–5). In other words, it can be said that, they increased the convulsive threshold. They also prolonged onset time from 1.20 to 2.10. and 1.20 to 3.58 s. and increased survival rate nearly by twofold from 50% to 95% and 50% to 100%, respectively (Table 2). Therefore, compounds 3d and3e have anticonvulsant drug potential to treat petit mal seizures.

Table 2 Anticonvulsant properties of compounds 3d and 3e in PTZ test.

Parameter	Onset time (min)	Grade 1%	Grade 2%	Grade 3 %	Grade 4 %	Grade 5%	Survival %
PTZ	1.20	99^⁎⁎⁎	80^⁎⁎⁎	75^⁎⁎⁎	71^⁎⁎⁎	52^⁎⁎⁎	50^⁎⁎⁎
3d	2.10	67^⁎⁎⁎	38^⁎⁎⁎	30^⁎⁎⁎	18^⁎⁎⁎	10^⁎⁎⁎	100^⁎⁎⁎
3e	2.58	64^⁎⁎⁎	42^⁎⁎⁎	35^⁎⁎⁎	21^⁎⁎⁎	10^⁎⁎⁎	95^⁎⁎⁎

Each group consists of 6–10 mice. Compounds were compared to control group and statistical significance.

⁎⁎⁎ p < 0.001.

3 Conclusion

The anticonvulsant drug design was based on the presumption (Pandeya et al., 1998) that the activity in maximal electroshock (MES) evaluation requires at least one phenyl or similar aromatic group in close proximity to two electron donor atoms and the activity in the pentylenetetrazole (PTZ) evaluation requires an alkyl group substituted close to two electron donor atoms. It has been hypothesized that, ureas and thioureas displaying anticonvulsant activity interact at locations on the putative binding site designated as aryl binding site, a hydrogen bonding domain and an auxiliary aryl or other hydrophobic binding site (Dimmock et al., 2000). Carboxamide derivatives (3a–e) were found to be more potent than carbotioamide derivatives due to their ability to form strong hydrogen bonds and their hydrophobic nature. Furthermore, the carboxamide derivatives of 3d and 3e were much more potent (90% protection) than the other carboxamides in PTZ test because of the bulk they own and their lipophilicity.

In conclusion, a series of 2-pyrazoline derivatives have been synthesized and screened for their anticonvulsant activity. The anticonvulsant screening indicated that among the tested compounds, 2-pyrazoline-1-carboxamide derivatives carrying 5-bromothiophen, 5-chlorothiophen and 2,6-dichlorophenyl groups exhibited noteworthy activity in PTZ test. These data may be useful for future molecular modifications leading to compounds with greater favorable pharmacological properties.

4 Experimental

4.1

4.1 Materials and methods

Chemicals were procured from Aldrich Chemical Co. The reactions were monitored on Merck pre-coated aluminum TLC plates 60F-254 and the products were visualized by UV-light using chloroform and methanol as solvent system. Melting points were determined on a Kleinfield SMP II apparatus and are uncorrected. The IR spectra were recorded on a Schimadzu FTIR 8400S spectrometry. ¹H NMR spectra were recorded on Bruker (400 MHz) spectrometer instruments, in DMSO. Chemical shifts were recorded in parts per million downfield from tetramethylsilane. The splitting patterns of ¹H NMR were designed as follows: s: singlet, bs: broad singlet, d: doublet, t: triplet, q: quartet, m: multiplet. The mass spectrometry was recorded on LC-MS-Agilent 1100 series in the electrospray mode. Elemental analysis was performed on Leco CHNS-932 analyzer.

4.2

4.2 General procedure for the preparation of chalcones 1a–i

5 mmol substituted aldehyde and 5 mmol substituted/nonsubstituted ketone were dissolved in methanol (15 ml). To that mixture, 50% (w/v) NaOH (3 ml) was added and the reaction mixture was stirred for about 16 h at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice and the precipitate was filtered, dried and crystallized from suitable solvent (If the precipitate has not been obtained, the reaction mixture was neutralized with 1 N HCI).

4.2.1

4.2.1 3-(4-Methoxyphenyl)-1-[4-(trifluoromethyl)phenyl]prop-2-ene-1-one 1a

C₁₇H₁₃F₃O₂ (M.W.: 306.279), Yield 78%, mp 112–114 °C (EtOH), UV (EtOH), λ_max (log ε: 205 (4.40), 248 (4.29), 350 (4.38), IR cm⁻¹. (cm⁻¹): 3010, 2939, 2843 (CH), 1658 (C = O), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.52 (3H, 2s, OCH₃), 6.54 (1H, d, J = 8.64 Hz, COCH = CH), 6.65 (1H, d, J = 8.64 Hz, COCH = CH), 7.15-7.80 (8H, m, Ar–H), Elemental Analysis: Found, %: C 66.02; H 4.22, Calculated, %: C 66.67; H 4.28.

4.2.2

4.2.2 1-(5-Chlorothiophen-2-yl)-3-(2,6-dichlorophenyl)prop-2-ene-1-one 1b

C₁₃H₇Cl₃OS (M.W.: 317.618 g/mol), Yield 89%, mp 128–129 °C (EtOH), UV (EtOH), λ_max (log ε: 207 (4.34), 239 (4.10), 319 (4.15), IR cm⁻¹ (cm⁻¹): 3105 (CH), 1649 (C⚌O), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 7.41 (1H, d, J = 4.13 Hz, thiophen H3); 7.48 (1H, dd, J₁ = 7.65 Hz, J₂ = 7.63 Hz 2,6-dichlorophenyl H4); 7.60-7.73 (4H, m, COCH⚌CH ve 2,6-dichlorophenyl H3, H5); 8.13 (1H, d, J = 4.16 Hz, thiophen H4), Elemental Analysis: Found, %: C 48.96; H 2.29; S 10.00, Calculated, %: C 49.16; H 2.22; S 10.10.

4.2.3

4.2.3 1-(5-Bromothiophen-2-yl)-3-(2,6-dichlorophenyl)prop-2-ene-1-one 1c

C₁₃H₇BrCl₂OS (M.W.: 362.069), Yield 89%, mp 125–126 °C (EtOH), UV (EtOH), λ_max (log ε): 209 (3.98), 238 (4.10), 323 (4.20), IR cm⁻¹ (cm⁻¹): 3113 (C-H), 1647 (C⚌O), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 7.45 (1H, d, J = 8.39 Hz, 2,6-dichlorophenyl H4); 7.49 (1H, d, J = 4.21 Hz, thiophen H3); 7.60–7.73 (4H, m, COCH⚌CH ve 2,6-dichlorophenyl H3, H5); 8.06 (1H, d, J = 4.10 Hz, thiophen H4), Elemental Analysis: Found, %: C 42.97; H 2.09; S 8.68, Calculated, %: C 43.12; H 1.95; S 8.86.

4.2.4

4.2.4 3-(2,6-Dichlorophenyl)-1-[4-(methylsulphonyl)phenyl]prop-2-ene-1-one1d

C₁₆H₁₂Cl₂O₃S (M.W.: 355.235), Yield 90%, mp 191–193 °C (EtOH), UV (EtOH), λ_max (log ε): 206 (4.25), 248 (4.12), 294 (4.20). IR cm⁻¹ (cm⁻¹): 3066, 2931 (CH), 1670 (C⚌O), 1296, 1145 (SO₂), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.28 (3H, s, –SO₂CH₃); 7.44 (1H, d, J = 8.05 Hz, 2,6-dichlorophenyl H4); 7.60–7.85 (4H, m, COCH⚌CH ve 2,6-dichloro phenyl H3, H5); 8.13 (2H, d, J = 8.28 Hz, 4-methylsulphonylphenyl H2, H6); 8.28 (2H, d, J = 8.31 Hz, 4-methylsulphonylphenyl H3, H5), Elemental Analysis: Found, %: C 52.97; H 3.53; S 8.79, Calculated, %: C 54.10; H 3.40; S 9.03.

4.2.5

4.2.5 3-(2,6-Dichlorophenyl)-1-(thiophen-2-yl)prop-2-ene-1-one 1e

C₁₃H₈Cl₂OS (M.W.: 283.173), Yield 91%, mp 93–94 °C (EtOH), UV (EtOH), λ_max (log ε): 206 (4.10), 244 (4.20), 311 (4.00), IR cm⁻¹ (cm⁻¹): 3086, 3072 (CH), 1653 (C⚌O), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 7.31 (1H, dd, J₁ = 4.00 Hz, J₂ = 4.00 Hz, thiophen H3); 7.45 (1H, dd, J₁ = 8.00 Hz, J₂ = 8.00 Hz 2,6-dichlorophenyl H4); 7.58-7.80 (4H, m, COCH⚌CH ve 2,6-dichlorophenyl H3, H5); 8.15 (1H, dd, J₁ = 1.20 Hz, J₂ = 1.20 Hz, thiophen H4); 8.21 (1H, dd, J₁ = 1.20 Hz, J₂ = 1.20 Hz, thiophen H5), Elemental Analysis: Found, %: C 54.88; H 2.89; S 11.32, Calculated, %: C 55.14; H 2.85; S 11.32.

4.2.6

4.2.6 3-(2,6-Dichlorophenyl)-1-phenylprop-2-ene-1-one1f

Yield 90%, mp 84–85 °C (EtOH), (Reference mp. 84–85 °C, Jun et al., 2007), UV (EtOH), λ_max (log ε: 207 (3.85), 288 (4.10), IR cm⁻¹ (cm⁻¹): 3084, 3024 (CH), 1668 (C⚌O).

4.2.7

4.2.7 3-(4-Methoxyphenyl)-1-[4-(methylsulphonyl)phenyl]prop-2-ene-1-one 1g

Yield 82%, mp 159–161 °C (EtOH) (Reference mp 165–166 °C, Yarishkin et al., 2008), UV (EtOH), λ_max (log ε): 205 (4.00), 249 (4.10), 353 (4.25), IR cm⁻¹ (cm⁻¹): 3009, 2972 (CH), 1658 (C⚌O), 1288, 1145 (SO₂).

4.2.8

4.2.8 3-(3,4-Dimethoxyphenyl)-1-(thiophen-2-yl)prop-2-ene-1-one1h

Yield 87%, mp 102–103 °C (EtOH) (Reference mp 106 °C, Dos Santos et al., 2008), UV (EtOH), λ_max (log ε): 208 (4.15), 266 (4.20), 364 (4.24), IR cm⁻¹. (cm⁻¹): 3107, 3075 (CH), 1637 (C⚌O).

4.2.9

4.2.9 3-[4-(Dimethylamino)phenyl]-1-(thiophen-2-yl)prop-2-ene-1-one1i

Yield 73%, mp 114–115 °C (ethanol) (Reference mp 114 °C, Navarini et al., 2009), UV (EtOH), λ_max (log ε): 205 (4.10), 277 (4.18), 427 (4.20), IR cm⁻¹ (cm⁻¹): 3089, 2904 (CH), 1629 (C⚌O).

4.3

4.3 General procedure for the preparation of 3,5-disubstituted-4,5-dihydro-1H-pyrazole-1-carbothioamides 2a–i

2 mmol chalcone was heated in ethanol (10 ml). To that mixture, the thiosemicarbazide (2.4 mmol) was added. Finally, a solution of 5 mmol NaOH in 1 ml was added and refluxed on a water bath for 8–12 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured onto ice and the precipitate was filtered, dried and crystallized from suitable solvent.

4.3.1

4.3.1 5-(4-Methoxyphenyl)-3-[4-(trifluoromethyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide2a

C₁₈H₁₆F₃N₃OS (M.W.: 379.399), Yield 72%, mp 200–202 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (4.00), 226 (4.05), 332 (4.12), IR cm⁻¹ (cm⁻¹): 3454, 3323 (NH), 1579 (C⚌N), 1363 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.17 (1H, dd, H_A, J_AB = 18.13 Hz, J_AX = 3.45 Hz); 3.72 (3H, s, –OCH₃); 3.91 (1H, dd, H_B, J_BA = 18.14 Hz, J_BX = 11.45 Hz); 5.90 (1H, dd, H_X, J_XA = 3.44 Hz, J_XB = 11.45 Hz); 6.86 (2H, d, J = 8.74 Hz, 4-methoxyphenyl H3, H5); 7.07 (2H, d, J = 8.71 Hz, 4-methoxyphenyl H2, H6); 7.81 (2H, d, J = 8.32 Hz, 4-trifluoromethylphenyl H2, H6); 8.03 (1H, bs, NH); 8.07-8.16 (2H, d, J = 8.16 Hz, 4-trifluoromethylphenyl H3, H5) 8.18 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 42.93 (pyrazole C4); 55.84 (OCH₃); 63.54 (pyrazole C5); 115.00, 123.89 126.62, 126.75, 126.79, 127.93, 128.99, 131.14, 131.46, 136.20, 136.29, 159.68 (aromatic C ve CF₃); 154.83 (pyrazole C3); 178.02 (C⚌S), Elemental Analysis: Found, %: C 56.83; H 4.12; N 11.07; S 7.67, Calculated, %: C 56.98; H 4.25; N11.08; S 8.45, ES-MS: m/z 380.399 [MH]⁺.

4.3.2

4.3.2 3-(5-Chlorothiophen-2-yl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2b

C₁₄H₁₀Cl₃N₃S₂ (M.W.: 390.738), Yield 45%, mp 256–257 °C (EtOH), UV (EtOH), λ_max (log ε): 203 (4.12), 248 (4.08), 347 (4.20), IR cm⁻¹ (cm⁻¹): 3437, 3250 (NH), 1585 (C⚌N), 1371 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.17 (1H, dd, H_A, J_AB: 18.07 Hz, J_AX: 7.70 Hz); 3.96 (1H, dd, H_B, J_BA: 18.06 Hz, J_BX: 12.97 Hz); 6.31 (1H, dd, H_X, J_XA: 7.68 Hz, J_XB: 12.95 Hz); 7.22 (1H, d, J: 3.97 Hz, thiophen H3); 7.26 (1H, t, J₁: 8.03 Hz, J₂: 8.01 Hz, 2,6-dichlorophenyl H4); 7.35 (1H, d, J: 3.93 Hz, thiophen H4); 7.39 (1H, d, J: 7.21 Hz, 2,6-dichlorophenyl H3); 7.45 (1H, d, J: 7.33 Hz, 2,6-dichlorophenyl H5); 7.57 (1H, bs, NH); 8.03 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 40.34 (pyrazole C4); 60.0 (pyrazole C5); 129.28, 129.73, 131.17, 131.68, 133.09, 133.91, 134.01, 136.21, 137.62 (aromatic C); 150.80 (pyrazole C3); 177.10 (C⚌S), Elemental Analysis: Found, %: C 43.12; H 2.42; N 10.66; S 17.03, Calculated, %: C 43.03; H 2.58; N 10.75; S 16.41, ES-MS: m/z 390.738 [MH]⁺.

4.3.3

4.3.3 3-(5-Bromothiophen-2-yl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2c

C₁₄H₁₀BrCl₂N₃S₂ (M.W.: 435.189), Yield 52%, mp 259–260 °C (EtOH), UV (EtOH), λ_max (log ε): 202 (4.10), 221 (4.09), 250 (4.00), 348 (4.25). IR cm⁻¹ (cm⁻¹): 3412, 3246 (NH), 1587 (C⚌N), 1373(C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.13 (1H, dd, H_A, J_AB = 18.06 Hz, J_AX = 7.68 Hz); 3.97 (1H, dd, H_B, J_BA = 18.08 Hz, J_BX = 13.02 Hz); 6.29 (1H, dd, H_X, J_XA = 7.67 Hz, J_XB = 13.01 Hz); 7.30 (3H, m, 2,6-dichlorophenyl H4 and thiophen H3, H4); 7.39 (1H, d, J = 7.99 Hz, 2,6-dichlorophenyl H3); 7.43 (1H, d, J = 7.97 Hz, 2,6-dichlorophenyl H5); 7.55(1H, bs, NH); 8.00 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 60.49 (pyrazole C5); 116.37, 129.15, 129.91, 130.59, 131.89, 132.16, 133.31, 135.59, 136.06, 137.02 (aromatic C); 149.98 (pyrazole C3); 176.19 (C⚌S), Elemental Analysis: Found, %: C 38.06; H 2.34; N 8.43; S 14.89, Calculated, %: C 38.64; H 2.32; N 9.66; S 14.74, ES-MS: m/z 436.189 [MH]⁺.

4.3.4

4.3.4 5-(2,6-Dichlorophenyl)-3-[4-(methylsulphonyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide 2d

C₁₇H₁₅Cl₂N₃O₂S₂ (M.W.: 428.356), Yield 39%, mp 264–265 °C (EtOH), UV (EtOH), λ_max (log ε): 205 (4.16), 232 (4.06), 317 (4.11). IR cm⁻¹ (cm⁻¹): 3450, 3331 (NH), 1581 (C⚌N), 1357 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.22 (1H, dd, H_A, J_AB = 18.36 Hz, J_AX = 7.43 Hz); 3.25 (3H, s, –SO₂CH₃); 3.98 (1H, dd, H_B, J_BA = 18.36 Hz, J_BX = 12.90 Hz); 6.32 (1H, dd, H_X, J_XA = 7.81 Hz, J_XB = 13.28 Hz); 7.23 (1H, t, J₁ = 7.82 Hz, J₂ = 8.20 Hz, 2,6-dichlorophenyl H4); 7.36 (1H, d, J = 8.21 Hz, 2,6-dichlorophenyl H3); 7.43 (1H, d, J = 8.21 Hz, 2,6-dichlorophenyl H5); 7.97 (2H, d, J = 8.60 Hz, 4-methylsulphonylphenyl H2, H6); 8.12 (2H, d, J = 8.20 Hz, 4-methyl sulphonylphenyl H3, H5); 8.0 (1H, bs, NH); 8.14 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 44.02 (SO₂CH₃); 60.72 (pyrazole C5); 127.96, 128.31, 129.15, 129.91, 130.59, 133.40, 135.61, 136.29, 137.12, 142.39 (aromatic C); 153.19 (pyrazole C3); 176.85 (C⚌S), Elemental Analysis: Found, %: C 47.76; H 3.72; N 9.68; S 15.09, Calculated, %: C 47.67; H 3.53; N 9.81; S 14.97, ES-MS: m/z 429.356 [MH]⁺.

4.3.6

4.3.6 5-(2,6-Dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2e

C₁₄H₁₁Cl₂N₃S₂ (M.W.: 356.293), Yield 42%, mp 202–203 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (4.10), 224 (4.22), 338 (4.00), IR cm⁻¹ (cm⁻¹): 3410, 3248 (N–H), 1589 (C⚌N), 1361 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.20 (1H, dd, H_A, J_AB = 18.40 Hz, J_AX = 7.60 Hz); 4.00 (1H, dd, H_B, J_BA = 18.40 Hz, J_BX = 12.80 Hz); 6.30 (1H, dd, H_X, J_XA = 7.60 Hz, J_XB = 12.80 Hz); 7.17 (1H, dd, J₁ = 3.60 Hz, J₂ = 3.60 Hz, thiophen H4); 7.24-7.46 (3H, m, 2,6-dichlorophenyl H-3, H4, H5); 7.47-7.52 (2H, d, J: 3.20 Hz, thiophen H3 ve NH); 7.78 (1H, d, J = 4.80 Hz, thiophen H5); 8.00 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 40.93 (pyrazole C4); 60.34 (pyrazole C5); 128.76, 129.14, 129.85, 130.58, 130.75, 131.35, 133.30, 134.26, 135.61, 137.14 (aromatic C); 150.92 (pyrazole C3); 176.10 (C⚌S), Elemental Analysis: Found, %: C 47.52; H 3.08; N 10.88; S 18.38, Calculated, %: C 47.19; H 3.11; N 11.79; S 18.00, ES-MS: m/z 357.293 [MH]⁺.

4.3.7

4.3.7 5-(2,6-Dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide2f

C₁₆H₁₃Cl₂N₃S (M.W.: 350.265), Yield 43%, mp 209–210 °C (EtOH), UV (EtOH), λ_max (log ε): 203 (4.05), 222 (4.15), 321 (4.18), IR cm⁻¹ (cm⁻¹): 3435, 3232 (NH), 1587 (C⚌N), 1348 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.20 (1H, dd, H_A, J_AB = 18.36 Hz, J_AX = 7.81 Hz); 3.97 (1H, dd, H_B, J_BA = 17.97 Hz, J_BX = 12.90 Hz); 6.30 (1H, dd, H_X, J_XA = 7.81 Hz, J_XB = 12.90 Hz); 7.24–7.46 (3H, m, 2,6-dichlorophenyl H3, H4, H5); 7.46–7.51 (3H, m, phenyl H3, H4, H5); 7.82 (1H, bs, NH); 7.85-7.90 (2H, m, phenyl H2, H6); 8.10 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 60.34 (pyrazole C5); 127.68, 129.11, 129.36, 129.79, 130.56, 131.10, 131.46, 133.37, 135.64, 137.31 (aromatic C); 154.77 (pyrazole C3); 176.48 (C⚌S), Elemental Analysis: Found, %: C 54.78; H 3.87; N 11.94; S 9.11, Calculated, %: C 54.86; H 3.74; N 12.00; S 9.15, ES-MS: m/z 351.265 [MH]⁺.

4.3.8

4.3.8 5-(4-Methoxyphenyl)-3-[4-(methylsulphonyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide2g

C₁₈H₁₉N₃O₃S₂ (M.W.: 389.491), Yield 39%, mp 226–227 °C (EtOH), UV (EtOH), λ_max (log ε): 201 (4.10), 226 (4.08) 334 (4.12), IR cm⁻¹ (cm⁻¹): 3462, 3346 (NH), 1583 (C⚌N), 1400 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.17 (1H, dd, H_A, J_AB = 18.03 Hz, J_AX = 7.56 Hz); 3.25 (3H, s, –SO₂CH₃); 3.72 (3H, s, –OCH₃); 3.93 (1H, dd, H_B, J_BA = 18.04 Hz, J_BX = 11.45 Hz); 5.90 (1H, dd, H_X, J_XA = 8.01 Hz, J_XB = 11.46 Hz); 6.87 (2H, d, J = 8.75 Hz, 4-methoxyphenyl H3, H5); 7.07 (2H, d, J = 8.72 Hz, 4-methoxyphenyl H2, H6); 7.98 (2H, d, J = 8.64 Hz, 4-methylsulphonylphenyl H2, H6); 8.15 (2H, d, J = 8.53 Hz, 4-methylsulphonylphenyl H3, H5); 8.08 (1H, bs, NH); 8.11 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 42.86 (pyrazole C4); 44.02 (SO₂CH₃); 55.72 (OCH₃); 63.45 (pyrazole C5); 110.00, 114.52, 127.36, 127.93, 128.44, 135.56, 136.47, 142.40, 158.92 (aromatic C); 153.91 (pyrazole C3); 177.14 (C⚌S), Elemental Analysis: Found,%: C 55.20; H 4.90; N 11.11; S 16.74, Calculated,%: C 55.51; H 4.92; N 10.79; S 16.47, ES-MS: m/z 390.491 [MH]⁺.

4.3.8

4.3.8 5-(3,4-Dimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2h

C₁₆H₁₇N₃O₂S₂ (M.W.: 347.455), Yield 32%. mp 183–184 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (4.12), 222 (4.20), 277 (4.09), 341 (4.10), IR cm⁻¹ (cm⁻¹): 3444, 3323 (NH), 1573 (C⚌N), 1348 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.12 (1H, dd, H_A, J_AB = 17.75 Hz, J_AX = 3.07 Hz); 3.71 (6H, s, –OCH₃); 3.87 (1H, dd, H_B, J_BA = 17.75 Hz, J_BX = 11.20 Hz); 5.85 (1H, dd, H_X, J_XA = 3.01 Hz, J_XB = 11.16 Hz); 6.58 (1H, d, J = 8.29 Hz, 3,4-dimethoxyphenyl H5); 6.79 (1H, s, 3,4-dimethoxyphenyl H2); 6.88 (1H, d, J = 8.31 Hz, 3,4-dimethoxyphenyl H6); 7.15 (1H, dd, J₁ = 4.82 Hz, J₂ = 3.84 Hz, thiophen H4); 7.48 (1H, d, J = 3.49 Hz, thiophen H3); 7.59 (1H, bs, NH); 7.78 (1H, d, J = 4.95 Hz, thiophen H5); 7.96 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 43.87 (pyrazole C4); 56.13, 56.18 (3-OCH₃ and 4-OCH₃); 63.30 (pyrazole C5); 110.29, 112.52, 117.53, 128.80, 131.02, 131.74, 134.55, 135.80, 148.47, 149.35 (aromatic C); 151.86 (pyrazole C3); 176.48 (C⚌S), Elemental Analysis: Found, %: C 54.77; H 5.07; N 11.82; S 18.21, Calculated, %: C 55.31; H 4.93; N 12.09; S 18.46, ES-MS: m/z 348.455 [MH]⁺.

4.3.19

4.3.19 5-[4-(Dimethylamino)phenyl]-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide2i

C₁₆H₁₈N₄S₂ (M.W.: 330.470), Yield 39%, mp 222–224 °C (EtOH), UV (EtOH), λ_max (log ε: 207 (4.10), 262 (4.09), 341 (4.20), IR cm⁻¹ (cm⁻¹): 3439, 3240 (NH), 1563 (C⚌N), 1366 (C⚌S), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 2.85 (6H, s, N(CH₃)₂); 3.10 (1H, dd, H_A, J_AB = 17.65 Hz, J_AX = 2.98 Hz); 3.86 (1H, dd, H_B, J_BA = 17.65 Hz, J_BX = 11.08 Hz); 5.84 (1H, dd, H_X, J_XA = 2.90 Hz, J_XB = 11.07 Hz); 6.67 (2H, d, J = 8.81 Hz, 4-dimethylaminophenyl H3, H5); 6.94 (2H, d, J = 8.75 Hz, 4-dimethylaminophenyl H2, H6); 7.15 (1H, dd, J₁ = 3.65 Hz, J₂ = 3.65 Hz, thiophen H4); 7.41-7.53 (2H, d, J = 3.65 Hz, thiophen H3 and NH); 7.76 (1H, d, J = 5.04 Hz, thiophen H5); 7.90 (1H, bs, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 43.86 (pyrazole C4); 63.20 (pyrazole C5); 113.10, 126.84, 128.84, 130.97, 131.68, 134.70, 150.30 (aromatic C); 151.93 (pyrazole C3); 176.29 (C⚌S), Elemental Analysis: Found,%: C 58.18; H 5.47; N 16.82; S 19.38, Calculated,%: C 58.15; H 5.49; N 16.95; S 19.4,. ES-MS: m/z 331.71 [MH]⁺.

4.4

4.4 General synthesis methods of N,3,5-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides 3a-e

5 mmol 4-chlorophenylisocyanate was dissolved in diethyl ether. To the stirred mixture, 5 mmol hydrazine was added dropwise and stirred for 15 min. The obtained 4-chlorophenylsemicarbazides were filtered, dried and washed with petrol ether. 2 mmol chalcone was dissolved in ethanol (10 ml) and then, the semicarbazide was added. Finally, a solution of 5 mmol NaOH in 1 ml water was added. After the completion of the reaction by TLC, the reaction mixture was poured onto ice and the precipitate was filtered, dried and crystallized from suitable solvent.

4.4.1

4.4.1 N-(4-chlorophenyl)semicarbazide

Yield 98%, mp 275–277 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (3.98), 247 (4.10), IR cm⁻¹. (cm⁻¹): 3330, 3292, 3218, 3098, 2980, 2924, 1667, 1592, 1530 1491, 1455, 1405, 1093.

4.4.2

4.4.2 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide3a

C₂₂H₁₆Cl₃N₃O (M.W.: 445.740), Yield 35%, mp 183–185 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (4.00), 225 (4.12), 269 (4.22), 323 (4.25), IR cm⁻¹ (cm⁻¹): 3382 (NH), 1678 (C⚌O), 1579 (C⚌N), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.22 (1H, dd, H_A, J_AB = 17.94 Hz, J_AX = 5.50 Hz); 3.72 (3H, s, OCH₃); 3.90 (1H, dd, H_B, J_BA = 17.94 Hz, J_BX = 12.04 Hz); 5.50 (1H, dd, H_X, J_XA = 5.50 Hz, J_XB = 12.01 Hz); 7.19–7.66 (12H, m, Ar–H) 9.19 (1H, s, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 42.55 (pyrazole C4); 60.73 (pyrazole C5); 130.11, 130.43, 131.32, 135.53, 135.97, 137.57, 151.14 (aromatic C); 151.83 (pyrazole C3); 159.16 (CC⚌O), Elemental Analysis: Found, %: C 59.36; H 3.55; N 9.62, Calculated, %: C 59.41; H 3.63; N 9.45, ES-MS: m/z 351.265 [MH]⁺.

4.4.3

4.4.3 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(4-methylsulfonylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3b

C₂₃H₁₈Cl₃N₃OS (M.W.: 522.832), Yield 53%, mp 237–239 °C (EtOH), UV (EtOH), λ_max (log ε): 205 (4.10), 232 (4.22), 317 (4.15), IR cm⁻¹ (cm⁻¹): 3440, 3325 (NH), 1695 (C⚌O), 1595 (C⚌N), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.22 (1H, dd, H_A, J_AB = 18.36 Hz, J_AX = 7.43 Hz); 3.25 (3H, s, –SO₂CH₃); 3.98 (1H, dd, H_B, J_BA = 18.36 Hz, J_BX = 12.90 Hz); 6.32 (1H, dd, H_X, J_XA = 7.81 Hz, J_XB = 13.28 Hz); 7.23 (1H, t, J₁ = 7.82 Hz, J₂ = 8.20 Hz, 2,6-dichlorophenyl H4); 7.30 (2H, d, J = 8.86 Hz, 4-chlorophenyl H3, H5); 7.39 (1H, d, J = 7.99 Hz, 2,6-dichlorophenyl H3); 7.43 (1H, d, J = 7.97 Hz, 2,6-dichlorophenyl H5); 7.66 (2H, d, J = 8.91 Hz, 4-chlorophenyl H2, H6); 7.97 (2H, d, J = 8.60 Hz, 4-methylsulphonylphenyl H2, H6); 8.12 (2H, d, J = 8.20 Hz, 4-methylsulphonylphenyl H3, H5); 9.15 (1H, s, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 44.02 (SO₂CH₃); 60.72 (pyrazole C5); 127.96, 128.31, 129.15, 129.91, 130.59, 133.40, 135.61, 136.29, 137.12, 142.39 (aromatic C); 153.19 (pyrazole C3); 161.82 (C⚌O), Elemental Analysis: Found, %: C 52.10; H 3.42; N 8.60; S 6.50, Calculated, %: C 52.84; H 3.47; N 8.04; S 6.13, ES-MS: m/z 523.832 [M + H].

4.4.4

4.4.4 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3c

C₂₀H₁₄Cl₃N₃OS (M.W.: 450.769), Yield 45%, mp 166–168 °C (EtOH), UV (EtOH), λ_max (log ε): 204 (4.10), 224 (4.12), 338 (4.20), IR cm⁻¹ (cm⁻¹): 3360, 3248 (NH), 1670 (C⚌O), 1590 (C⚌N), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.20 (1H, dd, H_A, J_AB = 18.40 Hz, J_AX = 7.60 Hz); 4.00 (1H, dd, H_B, J_BA = 18.40 Hz, J_BX = 12.80 Hz); 6.30 (1H, dd, H_X, J_XA = 7.60 Hz, J_XB = 12.80 Hz); 7.17 (1H, dd, J₁ = 3.60 Hz, J₂ = 3.60 Hz, thiophen H4); 7.24–7.46 (5H, m, 2,6-dichlorophenyl H-3, H4, H5 ve 4-chlorophenyl H3, H5); 7.47–7.52 (1H, d, J = 3.20 Hz, thiophen H3); 7.62 (2H, d, J = 8.87 Hz, 4-chlorophenyl H2, H6); 7.78 (1H, d, J = 4.80 Hz, thiophen H5); 9.25 (1H, s, NH),¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 40.93 (pyrazole C4); 60.34 (pyrazole C5); 128.76, 129.14, 129.85, 130.58, 130.75, 131.35, 133.30, 134.26, 135.61, 137.14 (aromatic C); 150.92 (pyrazole C3); 166.10 (C⚌O): Elemental Analysis: Found, %: C 47.52; H 3.08; N 10.88; S 18.38, Calculated, %: C 53.29; H 3.13; N 9.32; S 7.11, ES-MS: m/z 451.768 [MH]⁺.

4.4.5

4.4.5 3-(5-Bromothiophen-2-yl)-N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3d

C₂₀H₁₃BrCl₃N₃OS (M.W.: 529.665), Yield 53%, mp 198–200 °C (EtOH), UV (EtOH), λ_max (log ε): 202 (4.14), 221 (4.10), 250 (4.20), 348 (4.10), IR cm⁻¹ (cm⁻¹): 3400, 3250 (NH), 1686 (C⚌O), 1580 (C⚌N), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.13 (1H, dd, H_A, J_AB = 18.06 Hz, J_AX = 7.68 Hz); 3.97 (1H, dd, H_B, J_BA = 18.08 Hz, J_BX = 13.02 Hz); 6.29 (1H, dd, H_X, J_XA = 7.67 Hz, J_XB = 13.01 Hz); 7.30 (3H, m, 2,6-dichlorophenyl H4 ve thiophen H3, H4); 7.39 (1H, d, J = 7.99 Hz, 2,6-dichlorophenyl H3); 7.43 (1H, d, J: 7.97 Hz, 2,6-dichlorophenyl H5); 9.05 (1H, s, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 60.49 (pyrazole C5); 116.37, 129.15, 129.91, 130.59, 131.89, 132.16, 133.31, 135.59, 136.06, 137.02 (aromatic C); 149.98 (pyrazole C3); 164.18 (C⚌O), Elemental Analysis: Found, %: C 45.50; H 2.45; N 7.90; S 6.00, Calculated, %: C 45.35; H 2.47; N 7.93; S 6.05, ES-MS: m/z 530.665 [MH]⁺.

4.4.6

4.4.6 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3e

C₂₀H₁₃Cl₄N₃OS (M.W.: 485.214), Yield 45%, mp 256–257 °C (EtOH), UV (EtOH), λ_max (log ε): 203 (4.05), 248 (4.15), 347 (4.20), IR cm⁻¹ (cm⁻¹): 3440, 3220 (NH), 1690 (C⚌O), 1580 (C⚌N), ¹H NMR (400 MHz, DMSO-d₆/TMS), δ, ppm (J, Hz): 3.17 (1H, dd, H_A, J_AB = 18.07 Hz, J_AX = 7.70 Hz); 3.96 (1H, dd, H_B, J_BA = 18.06 Hz, J_BX = 12.97 Hz); 6.31 (1H, dd, H_X, J_XA = 7.68 Hz, J_XB = 12.95 Hz); 7.22 (1H, d, J = 3.97 Hz, thiophen H3); 7.26 (1H, t, J₁ = 8.03 Hz, J₂ = 8.01 Hz, 2,6-dichlorophenyl H4); 7.30 (2H, d, J = 8.80 Hz, 4-chlorophenyl H3, H5); 7.35 (1H, d, J = 3.93 Hz, thiophen H4); 7.39 (1H, d, J = 7.21 Hz, 2,6-dichlorophenyl H3); 7.45 (1H, d, J = 7.33 Hz, 2,6-dichlorophenyl H5); 7.66 (2H, d, J = 8.80 Hz, 4-chlorophenyl H2, H6); 9.10 (1H, s, NH), ¹³C NMR (100 MHz), (DMSO-d₆/TMS), δ(ppm): 40.34 (pyrazole C4); 60.0 (pyrazole C5); 129.28, 129.73, 131.17, 131.68, 133.09, 133.91, 134.01, 136.21, 137.62 (aromatic C); 151.20 (pyrazole C3); 164.20 (C⚌O), Elemental Analysis: Found, %: C 49.48; H 2.68; N 8.64; S 6.58, Calculated, %: C, 49.51; H, 2.70; N, 8.66; S, 6.61, ES-MS: m/z 486.214 [MH]⁺.

4.5

4.5 Pharmacology

Male and female adult BALB/c mice weighing 20–30 g were used. The animals were housed in colongy cages, under standard laboratory conditions on a 12-h light–dark cycle (8 a.m.–8 p.m.), with free access to food and tap water. Room temperature and relative humidity were maintained at 22 ± 1 °C and 60%, respectively. All tests were conducted during the light phase of day. After the adaption period of 2 days, experimental groups were chosen randomly. Each mouse was used only once. The experimental protocols were approved by the Animal Care and Use Committee of Marmara University (16.04.2009–02.04.2009).

4.5.1

4.5.1 Anticonvulsant activity

The anticonvulsant activities of the new compounds were determined by using PTZ (Sigma) and MES tests. These rodent models are widely used as standard methods for predicting protection against generalized absence and tonic–clonic seizures in humans (Borowicz et al., 2010). All synthesized compounds were suspended in 0.5% methyl cellulose and administered at a dose of 50 mg/kg 30 min prior the tests. Effective dose 50 (ED₅₀) value for PTZ (60 mg/kg) and convulsive current 50 (CC₅₀) of animals and the 95% fiducial limits were calculated by the method of Litchfield and Wilcoxon (1949).

4.5.2

4.5.2 PTZ test

The animals of the control group received same volume of saline in PTZ test. 30 min after the administration of the test compounds, mice were injected intraperitoneally with 60 mg/kg PTZ and observed for 15 min. Motor responses were graded from 0 to 5 according to the scale of Racine, where grade 1: no movements, grade 2: head twitching and myoclonic jerks (MKJ), grade 3: clonic forelimb convulsions, grade 4: three plus change in posture, and grade 5: falling back and generalized convulsions with tonic extension (Racine, 1972).

4.5.3

4.5.3 MES test

MES test was performed 30 min after the administration of the test compounds. Electroshocks were evoked through a current transmitter producing square waves (Ari Technical ECT unit). In the MES test, seizures were elicited with a 60-Hz alternating current of 25 mA intensity in BALB/c mice. The current was applied via ear clip electrodes for 450 ms. During the shock, electrodes were attached to each animal’s ear and the animals lay on their backs, while their tails are fixed. Thus, observation of the tonic and clonic convulsions appearing during the seizure was ensured (Krall et al., 1978).

4.5.4

4.5.4 Statistics

Statistical analyses were evaluated by using analysis of variance (ANOVA) followed by unpaired Student’s t-test using Prism 3.0 (GraphPad Software, San Diego, CA, USA). ED₅₀ values, as well as their statistical evaluation, were estimated by computer probit analysis, according to Litchfield and Wilcoxon (1949).

Acknowledgement

This study was supported by the Marmara University Scientific Research Projects Commission (BAPKO, Project number SAG-A -040609-0140).

References

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Introduction

Results and discussion

Chemistry
Pharmacology

Conclusion

Experimental

Materials and methods
General procedure for the preparation of chalcones 1a–i
General procedure for the preparation of 3,5-disubstituted-4,5-dihydro-1H-pyrazole-1-carbothioamides 2a–i
General synthesis methods of N,3,5-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides 3a-e
Pharmacology

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[1] Abid M., Bhat A.R., Athar F., Azam A., . Synthesis, spectral studies and antiamoebic activity of new 1-N-substituted thiocarbamoyl-3-phenyl-2-pyrazolines. Eur. J. Med. Chem.. 2009;44:417-425.
[Google Scholar]

[2] Bialer M., . Chemical properties of antiepileptic drugs (AEDs) Adv. Drug Delivery Rev.. 2012;64:887-895.
[Google Scholar]

[3] Borowicz K.K., Furmanek-Karwowska K., Morawska M., Luszczki J.J., Czuczwar S.J., . Effect of acute and chronic treatment with milnacipran potentiates the anticonvulsant activity of conventional antiepileptic drugs in the maximal electroshock-induced seizures in mice. Psychopharmacology. 2010;207:661-669.
[Google Scholar]

[4] Dimmock J.R., Vashishtha S.C., Stables J.P., . Urelylene anticonvulsants and related compounds. Pharmazie. 2000;55:490-494.
[Google Scholar]

[5] Dos Santos L., Lima L.A., Cechinel-Filho V., Corrêa R., Buzzi F.C., Nunes R.J., . Synthesis of new 1-phenyl-3-{4-[(2E)-3- phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives with anti-nociceptive activity. Bioorg. Med. Chem.. 2008;16:8526-8534.
[Google Scholar]

[6] Eddy C.M., Rickards H.E., Cavanna A.E., . The cognitive impact of antiepileptic drugs. Ther. Adv. Neurol. Disord.. 2011;4(6):385-407.
[Google Scholar]

[7] Guan L.P., Sui X., Deng X.Q., Quan Y.C., Quan Z.S., . Synthesis and anticonvulsant activity of a new 6-alkoxy-[1,2,4]triazolo[4,3-b]pyridazine. Eur. J. Med. Chem.. 2010;45:1746-1752.
[Google Scholar]

[8] Honarmand A., Safavi M., Zare M., . Gabapentin: an update of properties and therapeutic use in epilepsy. J. Res. Med. Sci.. 2011;16(9):1062-1069.
[Google Scholar]

[9] Jun N., Hong G., Jun K., . Synthesis and evaluation of 2′,4′,6′-trihydroxychalcones as a new class of tyrosinase inhibitors. Bioorg. Med. Chem.. 2007;15:2396-2402.
[Google Scholar]

[10] Koçyiğit-Kaymakçıoğlu B., Rollas S., Korcegez E., Aricioglu F., . Synthesis and biological evaluation of new N-substituted-N′-(3,5-di/1,3,5-trimethylpyrazole-4-yl) thiourea/urea derivatives. Eur. J. Pharm. Sci.. 2005;26:97-103.
[Google Scholar]

[11] Koçyiğit-Kaymakcioğlu B., Aker R.G., Tezcan K., Sakalli E., Ketenci S., Oruç-Emre E.E., Akin D., Gurbanova A., Terzioğlu B., Onat F., Rollas S., . Anticonvulsant activity of 3,5-dimethylpyrazole derivatives in animal models. Med. Chem. Res.. 2011;20(5):607-611.
[Google Scholar]

[12] Krall R.L., Penry J.K., White B.G., Kupferberg H.J., Swinyard E.A., . Anticonvulsant drug development: anticonvulsant drug screening. Epilepsia. 1978;19:409-428.
[Google Scholar]

[13] Küçükgüzel Ş.G., Rollas S., Erdeniz H., Kiraz M., Ekinci A.C., Vidin A., . Synthesis, characterization and pharmacological properties of some 4-arylhydrazono-2-pyrazoline-5-one derivatives obtained from heterocyclic amines. Eur. J. Med. Chem.. 2000;35:761-771.
[Google Scholar]

[14] Litchfield J.T., Wilcoxon F.A., . A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther.. 1949;96:99-113.
[Google Scholar]

[15] Navarini AL., Chiaradia LD., Mascarello A., Fritzen M., Nunes RJ., Yunes RA., Creczynski-Pasa TB., . Hydroxychalcones induce apoptosis in B16-F10 melanoma cells via GSH and ATP depletion. Eur. J. Med. Chem.. 2009;44:1630-1637.
[Google Scholar]

[16] Nikhil D., Kishore P.B., . Synthesis, anticonvulsant activity and 3D-QSAR study of some prop-2-eneamido and 1-acetyl-pyrazoline derivatives of aminobenzothiazole. Eur. J. Med. Chem.. 2010;45:149-159.
[Google Scholar]

[17] Özdemir A., Turan-Zitouni G., Kaplancıklı Z.A., Revial G., Güven K., . Synthesis and antimicrobial activity of 1-(4-aryl-2-thiazolyl)-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives. Eur. J. Med. Chem.. 2007;42:403-409.
[Google Scholar]

[18] Özdemir Z., Kandilci H.B., Gümüşel B., Çalış Ü., Bilgin A.A., . Synthesis and studies on antidepressant and anticonvulsant activities of some 3-(2-furyl)-pyrazoline derivatives. Eur. J. Med. Chem.. 2007;42:373-379.
[Google Scholar]

[19] Pandeya S.N., Mishra V., Sign P.N., Rupainwar D.C., . Anticonvulsant activity of thioureido derivatives of acetopheonone semicarbazones. Pharmacol. Res.. 1998;37:17-22.
[Google Scholar]

[20] Racine R.J., . Modification of seizure activity by electrical stimulation II. Motor seizure. Electroencephalogr. Clin. Neurophysiol.. 1972;32:281-294.
[Google Scholar]

[21] Siddiqui N., Ahsan W., . Triazole incorporated thiazoles as a new class of anticonvulsants: design, synthesis and in vivo screening. Eur. J. Med. Chem.. 2010;45:1536-1543.
[Google Scholar]

[22] Stefan H., Feuerstein T.J., . Novel anticonvulsant drugs. Pharmacology & Therapeutics. 2007;113:165-183.
[Google Scholar]

[23] Trimble M.R., Dodson W.E., eds. Epilepsy and quality of life. New York: Raven Press Ltd., New York; 1994.

[24] Yarishkin O.V., Ryu H.W., Park J.Y., Yang M.S., Hongb S.G., Park K.H., . Sulfonate chalcone as new class voltage-dependent K+ channel blocker. Bioorg. Med. Chem. Lett.. 2008;18:137-140.
[Google Scholar]

Synthesis and anticonvulsant activity of some 2-pyrazolines derived from chalcones

Abstract

Keywords

Chalcones

2-Pyrazolines

Carbothioamide

Carboxyamide

Anticonvulsant activity

1 Introduction

2 Results and discussion

2.1 Chemistry

2.2 Pharmacology

3 Conclusion

4 Experimental

4.1 Materials and methods

4.2 General procedure for the preparation of chalcones 1a–i

4.2.1 3-(4-Methoxyphenyl)-1-[4-(trifluoromethyl)phenyl]prop-2-ene-1-one 1a

4.2.2 1-(5-Chlorothiophen-2-yl)-3-(2,6-dichlorophenyl)prop-2-ene-1-one 1b

4.2.3 1-(5-Bromothiophen-2-yl)-3-(2,6-dichlorophenyl)prop-2-ene-1-one 1c

4.2.4 3-(2,6-Dichlorophenyl)-1-[4-(methylsulphonyl)phenyl]prop-2-ene-1-one1d

4.2.5 3-(2,6-Dichlorophenyl)-1-(thiophen-2-yl)prop-2-ene-1-one 1e

4.2.6 3-(2,6-Dichlorophenyl)-1-phenylprop-2-ene-1-one1f

4.2.7 3-(4-Methoxyphenyl)-1-[4-(methylsulphonyl)phenyl]prop-2-ene-1-one 1g

4.2.8 3-(3,4-Dimethoxyphenyl)-1-(thiophen-2-yl)prop-2-ene-1-one1h

4.2.9 3-[4-(Dimethylamino)phenyl]-1-(thiophen-2-yl)prop-2-ene-1-one1i

4.3 General procedure for the preparation of 3,5-disubstituted-4,5-dihydro-1H-pyrazole-1-carbothioamides 2a–i

4.3.1 5-(4-Methoxyphenyl)-3-[4-(trifluoromethyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide2a

4.3.2 3-(5-Chlorothiophen-2-yl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2b

4.3.3 3-(5-Bromothiophen-2-yl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2c

4.3.4 5-(2,6-Dichlorophenyl)-3-[4-(methylsulphonyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide 2d

4.3.6 5-(2,6-Dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2e

4.3.7 5-(2,6-Dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide2f

4.3.8 5-(4-Methoxyphenyl)-3-[4-(methylsulphonyl)phenyl]-4,5-dihydro-1H-pyrazole-1-carbothioamide2g

4.3.8 5-(3,4-Dimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide 2h

4.3.19 5-[4-(Dimethylamino)phenyl]-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide2i

4.4 General synthesis methods of N,3,5-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides 3a-e

4.4.1 N-(4-chlorophenyl)semicarbazide

4.4.2 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide3a

4.4.3 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(4-methylsulfonylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3b

4.4.4 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3c

4.4.5 3-(5-Bromothiophen-2-yl)-N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3d

4.4.6 N-(4-chlorophenyl)-5-(2,6-dichlorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide 3e

4.5 Pharmacology

4.5.1 Anticonvulsant activity

4.5.2 PTZ test

4.5.3 MES test

4.5.4 Statistics

Acknowledgement

References

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