Design, synthesis, anticonvulsant and analgesic studies of new pyrazole analogues: a Knoevenagel reaction approach

Abstract

The present work involves the design and synthesis of a number of new compounds starting from 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde. The compounds were synthesized by adopting the Knoevenagel condensation reaction to meet the structural prerequisite required for anticonvulsant and analgesic activities. The reaction of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde with substituted thiazolidine, pyrazolone, thiazolo[3,2-a]pyrimidine, Meldrum's acid and barbituric acid yielded a variety of heterocycles bearing the pyrazole moiety. The newly synthesized compounds were characterized by elemental and spectroscopic analysis; in addition, the structure of compound 1a has been elucidated by single crystal X-ray diffraction technique. The synthesized molecules were evaluated for their in vivo anticonvulsant activity using maximal electroshock seizure (MES) test, while their analgesic activity was investigated by tail flick method. Further, rotarod toxicity method was used to study the toxicity profile of the selected compounds. Among the synthesized compounds, 1a, 4a and 7a possessed potent anticonvulsant activity and 1b, 5a, 5b, 7a and 7b showed the highest analgesic activity without displaying any toxicity. Efforts were also made to establish structure–activity relationships among the tested compounds.

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1. Introduction

Epilepsy is a major neurological disorder, characterized by periodic and unpredictable occurrence of seizures.¹ A seizure is an abnormal discharge of neurons in the central nervous system specifically in the cerebral cortex of the brain. It is a major health problem that affects approximately 1% of the world population.² The behavioural symptoms may present themselves as loss of consciousness, staring, a short period of confusion, a recurring thought, peculiar feeling, emotional outburst, strange sensation, odd posture or uncontrolled movements.³ The currently available epileptic drugs (AEDs) are associated with various adverse side effects such as ataxia, hyperplasia and anaemia.^4,5 An ideal AED should prevent different types of seizures without producing side effects that adversely affect the patient's quality of life. On the other hand, pain is a pervasive public health problem. Current analgesics fall into two major classes, namely opioid agents and non-steroidal anti-inflammatory drugs (NSAIDs).⁶ However, their use is limited in both general classes of agents because of serious side effects including tolerance, physical dependence, respiratory depression and constipation.⁷ Numerous studies have also demonstrated analgesic activity of other anticonvulsant agents such as brivaracetam and lacosamide in animal models of pain and some are currently being evaluated in clinical studies.⁸ The development of novel antiepileptic agents endowed with both anticonvulsant and analgesic activities have produced a great deal of interest. In a highly saturated epilepsy market, this approach has been proven successful by providing expanded financial and marketing incentives that do not independently exist in the field of epilepsy.

Design of new synthetic compounds with appropriate therapeutic importance is a major challenge for the medicinal chemistry researchers. Molecular modification could be a productive source for new biologically active molecules.⁹ Pyrazole have attracted intense interest in recent years because of their diverse pharmacological properties particularly to reduce tissue edema and inflammation.^10,11 Pyrazole derivatives such as dipyrone, aminopyrine, isopropylantipyrine, phenyl butazone, oxyphenbutazone, celecoxib and deracoxib are potent anti-inflammatory and analgesic agents.¹² In view of the previous reports certain 1,3-diaryl pyrazoles identified targets for new anti-inflammatory and analgesic drugs.^13,14,42 Consequently, several pyrazole derivatives that exhibited anticonvulsant activity were reported.^15,16 The maximal electroshock (MES) screening for evaluating the anticonvulsant activity was found to be potency in the halogens substituted aryl ring.^17,18 Thus it was suggested that the halo substituted 4-formyl pyrazole one of the important core fragments might be capable for identifying analgesic property with a novel spectrum of anticonvulsant action.

To get a deeper insight into the structure–activity relationship of epileptic drugs (AEDs) like flupirtine, felbamate, levetiracetam, gabapentin, pregabalin, tiagabine, oxcarbazepine, phenobarbital, phenytoin, ralitoline, trimethadione, etc., groups such as cyclic imide, C–CO– and N–CO– play an important role in their anticonvulsant activities as pharmacophores (Fig. 1).^19,20 Similar kind of heterocyclic analogues of Meldrum's acid got attracted due to its cardiotonic and HIV integrase inhibitory activities.²¹ In addition, barbituric acid and its derivatives are a chemical class of compounds known for their anticonvulsant property.²² The literature review reveals that, some new substituted pyrazolone,^15,23,24 thiazolidin,^25,26 and thiazolopyrimidine^27,28 ring systems have shown appreciable anticonvulsant and analgesic property (Fig. 1).

Fig. 1 Chemical class and examples of anticonvulsant drugs representing –C–CO– and –N–CO– moiety.

In the recent years these facts have directed the search for novel antiepileptic compounds towards identification and development of potent agents endowed with both anticonvulsant and analgesic activity. Against this background, in view of these general requirements for activity, it has been planned to design and synthesize new series of halo substituted 1,3-diaryl pyrazole incorporated with above mentioned various biologically active heterocyclic compounds. Presently ongoing research mainly focuses on investigation of new anticonvulsant and analgesic agents through conventional screening and structural modifications. Taking into consideration all the compounds have been synthesized preferentially via well known Knoevenagel condensation²⁹ with less reaction time and high yield.

2. Results and discussion

2.1. Chemistry

The synthetic strategy to prepare the target compounds is depicted in Scheme 1. The required key intermediate, 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II), was constructed by following the Vilsmeier–Haack reaction, according to the previous reports of our research group.^30,31,42 Because the formyl group present on the pyrazole ring opens up the possibility of carrying out a diverse range of transformations and is an important protocol for the construction of new heterocyclic compounds. Intermediate compounds like simple and substituted thiazolidine A–C,^32–34 pyrazolone D (ref. 35) and dihydropyrimidinone E (ref. 31) were synthesized according to the reported procedure. Commercially available Meldrum's acid (F) and barbituric acid (G) were used without further purification. Importantly, all the target compounds were achieved through the well-known Knoevenagel condensation reaction, which is used for the preparation of a broad spectrum of substituted alkenes. In present study, Knoevenagel products were obtained by the reaction of pyrazole aldehyde (I-II) with the reactive methylene group containing different heterocyclic analogues (A–G) in the presence of basic catalysts. The applied Knoevenagel condensation method has several advantages such as high yield, less reaction time, simple separation and purification. The reaction time, percentage of yield and melting points of target compounds are given in Table 1. The structures of all the target compounds were further established by IR, ¹H NMR, ¹³C NMR, mass spectral and elemental analysis. The mass spectra of all the final derivatives showed comparable molecular ion peak with respect to molecular formula. The synthesized compounds gave satisfactory spectral data and were submitted for the anticonvulsant activity and analgesic property.

Scheme 1 Synthetic route for the preparation of compounds 1–7 derivatives.

Table 1 Characterization data of compounds 1–7 series

Compounds	Reaction time (h)	Yield (%)	Melting point (°C)
1a	2.0	82	227–230
1b	2.5	70	246–247
2a	4.5	71	125–127
2b	5.0	67	145–147
3a	2.5	90	266–267
3b	2.5	84	255–257
3c	4.0	68	186–189
3d	4.0	71	203–207
4a	5.5	93	244–246
4b	6.0	79	234–237
5a	1.5	89	90–92
5b	2.0	79	99–102
6a	2.0	60	160–162
6b	1.5	42	172–175
7a	0.5	99	285–288
7b	1.5	96	263–265

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2.2. X-ray diffraction analysis

Compound 1a (deposition #CCDC 1046740†) was recrystallized from ethanol to give pale yellow crystals suitable for single crystal X-ray diffraction (crystal size 0.25 × 0.25 × 0.25 mm) with the following crystallographic parameters: a = 8.5087(8) Å, b = 10.5551(10) Å, c = 10.9292(11) Å, α = 101.832(5)°, β = 90.563(5)°, γ = 111.879(5)°, μ = 4.573 mm⁻¹, V = 887.47(15) ų, Z = 4. D_c = 1.558 Mg m⁻³, F₍₀₀₀₎ = 424, T = 293(2) K, 4.15° ≤ θ ≤ 64.78°, R = 0.0593, wR = 0.1497. The crystal structure of 1a is triclinic with a space group of P1̄ and contains the following four ring subunits viz., phenyl, 3,4-dihalophenyl, pyrazole and thiazolidine-2,4-dione. The ORTEP diagram of the molecule with thermal ellipsoids drawn at 50% probability is shown in Fig. 2(a).

Fig. 2 (a) ORTEP of the molecule 1a with thermal ellipsoids drawn at 50% probability. (b) Packing of the molecules when viewed along the b axis (dotted lines represent inter-molecular hydrogen bonds linking the molecules into chains).

The bond lengths and bond angles are in good agreement with the standard values. The dihedral angle between the central pyrazole ring and the pendant thiazolidine-2,4-dione ring and phenyl rings are 7.02(16)° and 12.12(12)° indicating that they are nearly coplanar. The dihedral angle between the pyrazole ring and the dichlorophenyl ring is 51.90(16)° indicating that dichlorophenyl ring is out of the plane of the pyrazole ring. The pyrazole ring is planar within the experimental limits with the atom C5 deviating 0.005(3)° from its mean plane. The structure exhibits both inter and intra molecular hydrogen bonds of the type N–H⋯O and C–H⋯O. The intermolecular hydrogen bonds N10⋯H10⋯O12, C15⋯H15⋯O13 and C25⋯H25⋯O13 have a length of 2.845(3) Å, 3.346(4) Å, 3.464(3) Å and an angle of 167°, 161° and 163° with symmetry codes (1−x, 2−y, 1−z), (−x, 1−y, 1−z) and (1−x, 1−y, 1−z) respectively. The packing of the molecules when viewed down from the b axis indicates that the molecules are interconnected by these hydrogen bonds to form a three dimensional chain (Fig. 2(b)).

2.3. Pharmacological screening

2.3.1. Anticonvulsant activity. The newly synthesized compounds were tested in vivo in order to evaluate their anticonvulsant activity, with the help of the MES test.^36,37 All the compounds were active at a dose level of 25 mg kg⁻¹, which is indicative of their ability to prevent the spread of convulsions. The results of the anticonvulsant activity expressed as mean extensor phase duration in seconds followed by percentage (%) protection in comparison with the standard phenytoin (25 mg kg⁻¹) are shown in Table 2 using the following equation:³⁸

% protection = {1 − (MEPD_sample/MEPD_nc)} × 100

where, MEPD_nc is the mean extensor phase duration of normal control in seconds and MEPD_sample is the mean extensor phase duration of sample in seconds.

Table 2 Anticonvulsant and toxicity screening results of target compounds

Compounds	MES test		Toxicity studies^b
	Duration of extension phase (s)^a	% protection	1 h	4 h
a Results are expressed as mean ± SEM; (n = 6). The mice were examined 2 h post i.p. injection of test samples.b Toxicity study was carried out by rotarod method at a 25, 50 and 100 mg kg^−1 doses. (—) indicates the absence of toxicity, while (x) means not tested. NT = not tested.
1a	2.58 ± 0.44	76.58	—	—
1b	2.89 ± 0.34	73.77	—	—
2a	5.35 ± 0.64	51.45	x	x
2b	5.18 ± 0.28	52.99	x	x
3a	3.05 ± 0.24	72.32	—	—
3b	3.68 ± 0.24	66.60	—	100
3c	4.49 ± 0.18	59.25	—	—
3d	4.06 ± 0.35	63.15	—	100
4a	2.23 ± 0.30	79.76	—	—
4b	3.07 ± 0.32	72.14	—	—
5a	5.54 ± 0.24	49.72	—	—
5b	5.63 ± 0.37	48.91	—	—
6a	6.48 ± 0.28	41.19	x	x
6b	4.01 ± 0.25	63.61	—	—
7a	2.32 ± 0.18	78.94	—	—
7b	3.26 ± 0.32	70.41	—	—
Phenytoin	2.01 ± 0.25	81.76	—	—
Control	11.02 ± 0.33	—	x	x

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A substance is known to possess anticonvulsant property, if it reduces or abolishes the extensor phase of MES-convulsion. The MES anticonvulsant results (Table 2) revealed that seven compounds (1a, 1b, 3a, 4a, 4b, 7a and 7b) showed excellent activity and the remaining compounds showed moderate to good anticonvulsant activity. However these compounds 1a, 4a and 7a, were found to be comparable with the reference drug phenytoin. Compounds 2(a-b), 5(a-b) and 6a emerged as the least attractive compounds in this series showing poor anticonvulsant activity as evidenced by high extensor time than phenytoin.

Attempts were made to understand the structure–activity relationship (SAR) of the molecules starting with 3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl bridged simple and substituted thiazolidine, pyrazolone, thiazolopyrimidine, Meldrum's acid and barbituric acid. The thiazolidine and its substituted compounds 1(a-b), 2(a-b) and 3(a-d), compound 1(a-b) was found to exhibit higher potency (76.58, 73.77%) than the substituted analogues. The replacement by 3-benzyl-2-(phenylimino)thiazolidin-4-one 2(a-b) (51.45, 52.99%) and by simple and substituted 2-(phenylamino)thiazol-4(5H)-one 3(a-d) (72.32, 66.60, 59.25, 63.15%) in the thiazolidine-2,4-dione 1(a-b) decreases the anticonvulsant potency, indicating the importance of substitution at different positions on thiazolidine. It is clear that the presence of more and more substituents on thiazolidine may decrease the activity gradually. Among 3(a-d), trifluoromethyl phenyl substituent (3a, 3b) showed higher percentage of protection compared to the unsubstituted phenyl rings (3c, 3d) may be due to high lipid solubility and enhancement of transport mechanism of trifluoromethyl substituent. All the synthesized compounds, 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one containing dichloro substituted 4-formyl pyrazole moiety 4a shows the highest percentage of protection (∼79.7%) and potency as compared to the standard phenytoin (∼81.7%). On the other hand, the presence of thiazolo[3,2-a]pyrimidine 5(a-b) and Meldrum's acid analogue 6(a-b) dramatically reduces the anticonvulsant activity. The presence of barbituric acid on the pyrazole ring 7(a-b) (78.94, 70.41%) renders the molecule to be more active. The results of this study demonstrated that all dichloro substituted patterns led to the significant improvement in potency than those of difluoro substituted ones.

2.3.2. Analgesic activity. The analgesic activity of the above mentioned derivatives was also evaluated by applying the tail flick method using pentazocine as a standard reference.^39,41 The test compounds and standard pentazocin were administered orally at a dose level of 10 mg kg⁻¹ by intragastric tube. The readings were recorded at regular intervals of 30 min, 60 min, 90 min and 120 min after the administration of the compounds.

The results of the analgesic activity (Table 3) clearly showed that, the newly synthesized compounds 1b, 5a, 5b, 7a and 7b exhibited good activity. When we correlate the structures of the test samples with their activity, it appears that the presence of thiazolidine, thiazolo[3,2-a]pyrimidine and barbituric acid was responsible for the enhanced analgesic activity. It was also observed that chloro substituted thiazolo[3,2-a]pyrimidine 5a and barbituric acid 7a analogues were quite active. However the presence of dichloro substituent on these heterocyclic systems are resulted in improved activity, whereas slightly less activity was observed for those possessing difluoro substituent groups (5b and 7b). Further, significant influence on the activity is observed when thiazolidine-2,4-dione 1(a-b) substituents are present on these heterocyclic ring systems. However, the presence of phenyl substitution at position 2 and 3 on thiazolidine-2,4-dione 2(a-b), 3(a-d) reduces the activity to a considerable extent. Moreover, phenyl methyl pyrazolone 4(a-b) and 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid) 6(a-b) exhibited a lower degree of analgesic activity.

Table 3 Analgesic activity of the compounds^a

Compounds	Dose mg kg^−1	Tail flick latency (s)
		Reaction time (min)
		0	30	60	90	120
a Results were expressed in mean ± SEM. (n = 6) significance levels *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the respective control.
1a	10	2.5 ± 0.1	4.8 ± 0.1***	5.5 ± 0.1***	5.7 ± 0.3***	5.5 ± 0.1***
1b	10	2.5 ± 0.4	5.2 ± 0.1***	5.6 ± 0.1***	5.7 ± 0.2***	5.9 ± 0.1***
2a	10	2.4 ± 0.1	2.8 ± 0.1	2.4 ± 0.1	2.9 ± 0.1	3.4 ± 0.8*
2b	10	2.4 ± 0.2	3.3 ± 0.1*	2.8 ± 0.9	3.1 ± 0.1	2.9 ± 0.7
3a	10	2.3 ± 0.8	3.5 ± 0.9**	3.5 ± 0.1**	3.6 ± 0.1***	4.3 ± 0.8***
3b	10	2.3 ± 0.4	2.3 ± 0.5	2.3 ± 0.13	2.3 ± 0.1	2.4 ± 0.8
3c	10	2.4 ± 0.6	2.8 ± 0.4**	3.5 ± 0.1**	4.3 ± 0.3**	4.2 ± 0.5***
3d	10	2.6 ± 0.2	3.2 ± 0.5	3.5 ± 0.1	3.9 ± 0.2	3.9 ± 0.3
4a	10	2.4 ± 0.1	2.9 ± 0.9	2.7 ± 0.1	2.5 ± 0.7	2.7 ± 0.5
4b	10	2.5 ± 0.2	3.5 ± 0.9**	3.6 ± 0.1**	3.6 ± 0.4**	3.2 ± 0.9**
5a	10	2.1 ± 0.2	6.2 ± 0.2***	6.7 ± 0.1***	6.4 ± 0.2***	6.5 ± 0.28***
5b	10	2.4 ± 0.7	5.1 ± 0.1***	5.1 ± 0.1***	5.7 ± 0.14***	5.9 ± 0.2***
6a	10	2.3 ± 0.4	4.9 ± 0.5***	5.2 ± 0.8***	4.7 ± 0.8***	4.8 ± 0.1***
6b	10	2.4 ± 0.7	5.2 ± 0.8***	5.5 ± 0.1***	4.7 ± 0.7***	5.0 ± 0.4***
7a	10	2.4 ± 0.2	6.4 ± 0.2***	5.8 ± 0.1***	6.3 ± 0.1***	6.2 ± 0.2***
7b	10	2.6 ± 0.1	4.1 ± 0.2***	5.4 ± 0.1***	5.6 ± 0.1***	5.8 ± 0.1***
Pentazocin	10	2.5 ± 0.5	7.9 ± 0.1***	7.4 ± 0.1***	6.8 ± 0.3***	6.8 ± 0.1***
Control	—	2.5 ± 0.8	2.7 ± 0.8	2.6 ± 0.1	2.5 ± 0.3	2.4 ± 0.2

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2.3.3. Toxicity study. The toxicity study was carried out for most of the compounds using the rotarod method.⁴⁰ The study was performed by observing the mice after 1 h and 4 h of test sample administration. The rotarod toxicity studies are summarized in Table 2. Except compound 3b and 3d (which showed toxicity at a high dose of 100 mg kg⁻¹), other tested samples have not shown toxicity at both the intervals. The compound 3b and 3d remained non-toxic at 1 h interval, indicating the slow onset of toxicity of the compound.

3. Conclusion

In conclusion, a new series of 4-formyl pyrazole carrying various heterocyclic systems was conveniently designed and synthesized by Knoevenagel condensation reaction with the prospect of improved analgesic and anticonvulsant properties. All the Knoevenagel reaction products were obtained with several advantages viz., lower reaction times, high yield and simple purification. Single X-ray crystals of compound 1a were developed and their crystal parameters were evaluated. The final compounds were screened for their in vivo analgesic activity and anticonvulsant activities following MES methodologies. The, anticonvulsant screening results for series of 1–7 showed that compounds 1a, 1b, 3a, 4a, 4b, 7a and 7b were good among the newly synthesized derivatives. Compounds 1a, 4a and 7a were comparable with the standard drug phenytoin. The anticonvulsant results revealed that, substituted pyrazolone, barbituric acid, simple and C-2 substituted thiazolidine-2,4-dione integrated bioactive pyrazole scaffolds emerged as potent molecules. Interestingly, the decrease in activity coincided with the increased substitution on thiazolidine. The compounds 1b, 5a, 5b, 7a and 7b exhibited prominent analgesic activity, in particularly, the compounds possessing thiazolidine-2,4-dione, thiazolo[3,2-a]pyrimidine and barbituric ring attached pyrazole moiety. Additionally, the results of the toxicity study showed that, the compounds were found to be non-toxic at all the tested doses. We conclude that further structural modifications of these molecules might lead to the development of molecules with improved potent anticonvulsant and analgesic properties.

4. Experimental section

4.1. Materials and methods

All the reagents were purchased from commercial suppliers Sigma-Aldrich, Spectrochem India and used without further purification. Melting points were determined in an open capillary tube and were uncorrected. The progress of each reaction was monitored by ascending thin layer chromatography (TLC) on silica gel G (Merck 1.05570.0001), visualized by UV light. The IR spectra (in KBr pellets) were recorded on a Shimadzu-FTIR spectrometer and the wave numbers were given in cm⁻¹. The ¹H NMR and ¹³C NMR spectra were recorded (CDCl₃/DMSO-d₆ mixture) on a Bruker AMX-400 NMR spectrometer with 5 mm PABBO BB-1H TUBES with TMS as internal standard. The X-ray intensity data were collected at a temperature of 296 K on a Bruker Proteum2 CCD diffractometer equipped with an X-ray generator operating at 45 kV and 10 mA, using CuK_α radiation. Mass spectra were recorded in Agilent Technology LC-mass spectrometer. Elemental analyses were carried out using VARIO EL-III (Elementar Analysensysteme GmBH).

4.1.1. General procedure for the preparation of 4-formylpyrazole (I-II). (3,4-Dichlorophenyl)ethanone phenylhydrazone (0.01 mol) was added to a mixture of the Vilsmeier–Haack reagent (prepared by dropwise addition of 1.2 mL POCl₃ in ice cooled 5 mL DMF) and refluxed for 6 h. The reaction mixture was poured into crushed ice followed by neutralization using sodium bicarbonate. The product obtained was filtered, washed with water and recrystallized from ethanol.³¹
3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I). M.p. 150–152 °C; FT-IR (KBr) ν_max (cm⁻¹): 3041 (Ar C–H), 1678 (C=O), 1571 (C=N), 1489 (C=C), 813 (C–Cl); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.42–7.83 (m, 9H, Ar-CH), 9.97 (s, 1H, CHO); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 119.79, 122.75, 128.43, 129.10, 130.24, 130.61, 131.21, 131.77, 132.34, 137.15, 138.88, 149.98 (Ar-C), 184.87 (aldehyde C=O); LCMS (m/z): 317.12 [M + H] (³⁵Cl₂), 319.14 [M + H + 2] (³⁵Cl³⁷Cl), 321.24 [M + H + 4] (³⁷Cl₂); anal. calcd for C₁₆H₁₀Cl₂N₂O: C, 60.59; H, 3.18; N, 8.83. Found: C, 60.56; H, 3.13; N, 8.86.
3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (II). M.p. 95–98 °C; FT-IR (KBr) ν_max (cm⁻¹): 3056 (Ar-C–H), 1675 (C=O), 1578 (C=N), 1453 (C=C), 1086 (C–F); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.44–9.37 (m, 9H, Ar-CH), 9.96 (s, 1H, CHO); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 117.99, 119.71, 122.58, 126.04, 128.33, 129.18, 130.19, 136.83, 138.88, 148.48, 149.19, 150.43, 150.91, 151.65 (Ar-C), 184.87 (aldehyde C=O); LCMS (m/z): 285.2 [M + H]; anal. calcd for C₁₆H₁₀F₂N₂O: C, 67.60; H, 3.55; N, 9.85. Found: C, 67.62; H, 3.53; N, 9.86.

4.1.2. Procedure for the synthesis of (Z)-5-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiazolidine-2,4-dione 1(a-b). A well stirred solution of 1,3-thiazolidine-2,4-dione (A) (0.01 mol) in 10 mL of acetic acid was buffered with sodium acetate (0.02 mol) and added with the 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II) (0.015 mol).³⁴ The solution was refluxed for 2–3 h and then poured into ice-cold water. The yellow precipitate was filtered and washed with water. The resulting crude product was recrystallized from ethanol to afford title compounds.
(Z)-5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiazolidine-2,4-dione (1a). M.p. 227–230 °C; FT-IR (KBr) ν_max (cm⁻¹): 3392 (NH), 2960 (Ar-H), 1668 (C=O), 1602 (C=N), 1529 (C=C), 758 (C–Cl); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.39–8.01 (m, 9H, Ar-H), 8.70 (s, 1H, pyrazole-CH), 12.54 (s, 1H, thiazolidine-NH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 116.23, 119.63, 121.99, 123.53, 127.73, 127.86, 128.18, 129.72, 130.41, 130.90, 131.94, 132.81, 132.84, 139.00, 151.66 (Ar-C), 167.12, 167.23 (thiazolidine-2,4-dione C=O); LCMS (m/z): 416.2 [M + H] (³⁵Cl₂), 418.0 [M + H + 2] (³⁵Cl³⁷Cl), 420.2 [M + H + 4] (³⁷Cl₂); anal. calcd for C₁₉H₁₁Cl₂N₃O₂S: C, 54.82; H, 2.66; N, 10.09; S, 7.70. Found: C, 54.86; H, 2.60; N, 10.07; S, 7.67.
(Z)-5-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiazolidine-2,4-dione (1b). M.p. 246–247 °C; FT-IR (KBr) ν_max (cm⁻¹): 3293 (NH), 2958 (Ar-H), 1678 (C=O), 1591 (C=N), 1509 (C=C), 1078 (C–F); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.19–7.99 (m, 9H, Ar-H), 8.89 (s, 1H, pyrazole-CH), 12.03 (s, 1H, thiazolidine-NH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 115.26, 118.51, 120.11, 122.46, 125.72, 127.84, 128.35, 129.64, 129.44, 130.56, 131.53, 132.56, 133.01, 134.78, 140.06, 152.48 (Ar-C), 167.78, 167.84 (thiazolidine-2,4-dione C=O); LCMS (m/z): 384.2 [M + H]; anal. calcd for C₁₉H₁₁F₂N₃O₂S: C, 59.53; H, 2.89; N, 10.96; S, 8.36. Found: C, 59.51; H, 2.89; N, 10.98; S, 8.39.

4.1.3. Procedure for the synthesis of (2Z, 5Z)-3-benzyl-5-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(phenylimino)thiazolidin-4-one 2(a-b). A mixture of (Z)-3-benzyl-2-(phenylimino)thiazolidin-4-one (B) (0.01 mol), 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II) (0.01 mol) and sodium acetate (0.015 mol) in glacial acetic acid was refluxed for 4–5 h.³³ After completion of the reaction (TLC check), cold water was added to reaction mixture, separated solid was filtered and the product was recrystallized using ethanol.
(2Z,5Z)-3-Benzyl-5-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(phenylimino)thiazolidin-4-one (2a). M.p. 125–127 °C; FT-IR (KBr) ν_max (cm⁻¹): 2973 (Ar-H), 1759 (C=O), 1665 (C=N), 1563 (C=C), 751 (C–Cl); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 5.21 (s, 2H, N–CH₂), 7.04–7.92 (m, 19H, Ar-H), 8.14 (s, 1H, pyrazole-CH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 46.52 (–N–CH₂–C–), 116.45, 119.76, 121.13, 124.94, 127.76, 127.99, 128.55, 129.13, 129.48, 129.62, 129.79, 130.53, 130.80, 131.75, 132.76, 133.17, 135.96, 139.10, 147.98, 148.79, 151.90 (Ar-C), 161.20 (thiazolidin-4-one, C=N), 166.10 (thiazolidin-4-one, C=O); LCMS (m/z): 581.0 [M + H] (³⁵Cl₂), 583.0 [M + H + 2] (³⁵Cl³⁷Cl), 585.0 [M + H + 4] (³⁷Cl₂); anal. calcd for C₃₂H₂₂Cl₂N₄OS: C, 66.09; H, 3.81; N, 9.63; S, 5.51. Found: C, 66.07; H, 3.85; N, 9.67; S, 5.48.
(2Z,5Z)-3-Benzyl-5-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(phenylimino)thiazolidin-4-one (2b). M.p. 145–147 °C; FT-IR (KBr) ν_max (cm⁻¹): 2982 (Ar-H), 1769 (C=O), 1675 (C=N), 1568 (C=C), 1053 (C–F); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 5.16 (s, 2H, N–CH₂), 6.99–7.81 (m, 19H, Ar-H), 8.02 (s, 1H, pyrazole-CH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 44.51 (–N–CH₂–C–), 113.05, 117.53, 119.51, 121.38, 125.75, 126.78, 127.59, 128.67, 129.08, 129.63, 129.99, 130.29, 130.87, 131.55, 132.15, 132.88, 133.59, 135.34, 137.54, 139.89, 146.59, 148.88, 150.98 (Ar-C), 160.23 (thiazolidin-4-one, C=N), 165.20 (thiazolidin-4-one, C=O); LCMS (m/z): 549.2 [M + H]; anal. calcd for C₃₂H₂₂F₂N₄OS: C, 70.06; H, 4.04; N, 10.21; S, 5.84. Found: C, 70.05; H, 4.07; N, 10.17; S, 5.85.

4.1.4. Procedure for the synthesis of (Z)-5-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(3-substitutedphenylamino)thiazol-4(5H)-one 3(a–d). A well stirred solution of 2-(3-(trifluoromethyl)phenylamino)thiazol-4(5H)-one (C) (0.01 mol) in 17.5 mL of acetic acid was buffered with sodium acetate (0.012 mol) and added with the 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II) (0.015 mol).³⁴ The solution was refluxed for 2–4 h and then poured into ice-cold water. The yellow precipitate was filtered and washed with water; the resulting crude product was purified by recrystallization from acetic acid–DMF (2 : 1) to afford title compounds.
(Z)-5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(3-(trifluoromethyl)phenylamino)thiazol-4(5H)-one (3a). M.p. 266–267 °C; FT-IR (KBr) ν_max (cm⁻¹): 3285 (NH), 3023, 2916 (Ar-H), 1659 (C=O), 1601 (C=N), 1516 (C=C), 1344 (Ar-CF₃), 1025 (C–F), 742 (C–Cl); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.32–8.29 (m, 13H, Ar-CH), 8.74 (s, 1H, pyrazole-CH), 9.97 (s, 1H, NH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 116.73, 120.04, 120.97, 121.24, 125.21, 127.48, 128.04, 128.24, 128.40, 128.56, 128.83, 129.41, 129.53, 129.75, 130.07, 130.80, 131.08, 132.03, 133.04, 133.45, 136.24, 139.38, 148.26, 149.07 (Ar-C), 162.15 (thiazolone, C=N), 166.38 (thiazolone, C=O); LCMS (m/z): 558.9 [M + H] (³⁵Cl₂), 560.9 [M + H + 2] (³⁵Cl³⁷Cl), 562.9 [M + H + 4] (³⁷Cl₂); anal. calcd for C₂₆H₁₅Cl₂F₃N₄OS: C, 55.82; H, 2.70; N, 10.02; S, 5.73. Found: C, 55.80; H, 2.73; N, 10.00; S, 5.71.
(Z)-5-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(3-(trifluoromethyl)phenylamino)thiazol-4(5H)-one (3b). M.p. 255–257 °C; FT-IR (KBr) ν_max (cm⁻¹): 3278 (NH), 3002, 2968 (Ar-H), 1692 (C=O), 1606 (C=N), 1526 (C=C), 1350 (Ar-CF₃), 1018 (C–F); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.39–8.17 (m, 13H, Ar-CH), 8.89 (s, 1H, pyrazole-CH), 9.93 (s, 1H, NH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 115.75, 119.08, 120.87, 122.00, 124.47, 126.53, 127.51, 128.32, 128.56, 129.03, 129.57, 129.95, 130.19, 130.79, 131.22, 132.63, 133.09, 134.84, 136.34, 138.88, 148.06, 149.12 (Ar-C), 161.02 (thiazolone, C=N), 166.18 (thiazolone, C=O); LCMS (m/z): 527.2 [M + H]; anal. calcd for C₂₆H₁₅F₅N₄OS: C, 59.31; H, 2.87; N, 10.64; S, 6.09. Found: C, 59.30; H, 2.88; N, 10.67; S, 6.08.
(Z)-5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(phenylamino)thiazol-4(5H)-one (3c). M.p. 186–189 °C; FT-IR (KBr) ν_max (cm⁻¹): 3252 (NH), 3012, 2973 (Ar-H), 1698 (C=O), 1604 (C=N), 1526 (C=C), 750 (C–Cl); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.28–8.04 (m, 14H, Ar-CH), 8.54 (s, 1H, pyrazole-CH), 9.89 (s, 1H, NH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 107.75, 116.51, 118.78, 119.64, 126.04, 127.32, 128.53, 129.01, 129.98, 130.04, 131.86, 132.07, 133.64, 133.95, 136.53, 139.57, 142.50, 149.13, 150.34 (Ar-C), 162.02 (thiazolone, C=N), 167.12 (thiazolone, C=O); LCMS (m/z): 491.2 [M + H] (³⁵Cl₂), 493.2 [M + H + 2] (³⁵Cl³⁷Cl), 495.2 [M + H + 4] (³⁷Cl₂); anal. calcd for C₂₅H₁₆Cl₂N₄OS: C, 61.11; H, 3.28; N, 11.40; S, 6.53. Found: C, 61.14; H, 3.28; N, 11.47; S, 6.58.
(Z)-5-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-(phenylamino)thiazol-4(5H)-one (3d). M.p. 203–207 °C; FT-IR (KBr) ν_max (cm⁻¹): 3251 (NH), 3005, 2956 (Ar-H), 1679 (C=O), 1610 (C=N), 1530 (C=C), 1016 (C–F); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.22–8.22 (m, 14H, Ar-CH), 8.91 (s, 1H, pyrazole-CH), 9.94 (s, 1H, NH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 106.92, 116.54, 118.34, 118.54, 119.86, 121.54, 126.18, 127.16, 128.82, 129.54, 129.98, 130.14, 130.59, 136.52, 142.53, 149.16, 150.21, 150.56, 150.89 (Ar-C), 163.22 (thiazolone, C=N), 167.11 (thiazolone, C=O); LCMS (m/z): 458.9 [M + H]; anal. calcd for C₂₅H₁₆F₂N₄OS: C, 65.49; H, 3.52; N, 12.22; S, 6.99. Found: C, 65.50; H, 3.48; N, 12.17; S, 6.97.

4.1.5. Procedure for the synthesis of (Z)-4-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 4(a-b). An equimolar mixture of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II) (0.01 mol) and 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (D) (0.01 mol) in acetic acid (10 mL) and sodium acetate (0.015 mol) were refluxed for 5–6 h.³⁵ After completion of the reaction, the reaction mixture was allowed to cool and poured over the crushed ice. The solid thus separated was collected by filtration and recrystallized from acetic acid.
(Z)-4-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (4a). M.p. 244–246 °C; FT-IR (KBr) ν_max (cm⁻¹): 2994 (Ar-H), 1736 (C=O), 1680 (C=N), 1589 (C=C), 759 (C–Cl); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 2.28 (s, 3H, CH₃), 7.20–7.97 (m, 14H, Ar-CH), 10.24 (s, 1H, pyrazole CH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 28.42 (CH₃), 115.72, 118.32, 120.59, 122.23, 123.48, 127.26, 127.82, 128.70, 128.86, 129.76, 130.28, 130.31, 132.19, 134.93, 142.67, 143.52, 143.94, 145.81, 147.09 (Ar-C), 162.88 (pyrazolone C=O); LCMS (m/z): 473.4 [M + H] (³⁵Cl₂), 475.3 [M + H + 2] (³⁵Cl³⁷Cl), 477.2 [M + H + 4] (³⁷Cl₂); anal. calcd for C₂₆H₁₈Cl₂N₄O: C, 65.97; H, 3.83; N, 11.84. Found: C, 65.94; H, 3.82; N, 11.86.
(Z)-4-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (4b). M.p. 234–237 °C; FT-IR (KBr) ν_max (cm⁻¹): 2963 (Ar-H), 1732 (C=O), 1687 (C=N), 1592 (C=C), 1099 (C–F); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 2.31 (s, 3H, CH₃), 7.16–7.89 (m, 14H, Ar-CH), 10.18 (s, 1H, pyrazole CH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 116.23, 119.02, 120.75, 122.13, 124.12, 125.13, 126.89, 127.84, 128.77, 128.92, 129.66, 130.22, 130.89, 132.33, 134.89, 142.68, 143.45, 144.08, 145.86, 147.69 (Ar-C), 162.33 (pyrazolone C=O); LCMS (m/z): 441.0 [M + H]; anal. calcd for C₂₆H₁₈F₂N₄O: C, 70.90; H, 4.12; N, 12.72. Found: C, 70.93; H, 4.10; N, 12.69.

4.1.6. Procedure for the synthesis of (Z)-ethyl-2-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-5-(4-fluorophenyl)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate 5(a-b). A mixture of ethyl 4-(4-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (E) (0.01 mol), 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (I-II) (0.01 mol), monochloroacetic acid (0.015 mol) and anhydrous sodium acetate (0.015 mol) was dissolved in glacial acetic acid (12 mL)-acetic anhydride (10 mL) mixture.³⁰ The reaction mixture were refluxed for 1–2 h and cooled to room temperature and poured over crushed ice with vigorous stirring. The precipitated solid was filtered, washed with cold water and recrystallized from ethanol.
(Z)-Ethyl-2-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-5-(4-fluorophenyl)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (5a). M.p. 90–92 °C; FT-IR (KBr) ν_max (cm⁻¹): 2977, 2864 (Ar-H), 1708 (ester C=O), 1596 (cyclic C=O), 1547 (C=N), 1511 (C=C), 1159 (C–O), 1031 (C–F), 757 (C–Cl); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 1.16 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 4.09 (q, 2H, J = 5.2 Hz, –OCH₂), 2.52 (s, 3H, CH₃), 6.16 (s, 1H, pyrimidine-CH), 6.98–7.80 (m, 13H, Ar-H), 8.18 (s, 1H, pyrazole CH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 14.06 (–OCH₂CH₃), 22.78 (CH₃), 54.92 (–OCH₂), 60.60 (CH), 108.99, 115.48, 115.69, 116.23, 119.33, 119.72, 123.06, 127.24, 127.92, 128.00, 128.25, 129.55, 129.76, 129.91, 130.36, 131.39, 132.74, 133.24, 133.37, 135.86, 138.94, 152.22, 152.43, 154.98, 161.48, 163.94 (Ar-C), 164.52, 165.28 (C=O); LCMS (m/z): 633.0 [M + H] (³⁵Cl₂), 635.0 [M + H + 2] (³⁵Cl³⁷Cl), 637.0 [M + H + 4] (³⁷Cl₂); anal. calcd for C₃₂H₂₃Cl₂FN₄O₃S: C, 60.67; H, 3.66; N, 8.84; S, 5.06. Found: C, 60.69; H, 3.68; N, 8.90; S, 5.03.
(Z)-Ethyl-2-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-5-(4-fluorophenyl)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (5b). M.p. 99–102 °C; FT-IR (KBr) ν_max (cm⁻¹): 2976, 2939 (Ar-H), 1707 (ester C=O), 1603 (cyclic C=O), 1511 (C=N, C=C), 1243 (C–O), 1056 (C–F); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 1.30 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 3.78 (q, 2H, J = 5.4 Hz, –OCH₂), 2.63 (s, 3H, CH₃), 5.98 (s, 1H, pyrimidine-CH), 7.03–7.98 (m, 13H, Ar-H), 8.24 (s, 1H, pyrazole CH); ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 14.16 (–OCH₂CH₃), 22.25 (CH₃), 53.99 (–OCH₂), 59.60 (CH), 109.13, 114.89, 115.62, 116.37, 119.08, 119.86, 123.66, 127.14, 127.98, 128.11, 128.58, 129.69, 129.79, 129.98, 130.78, 131.52, 132.64, 133.53, 133.86, 135.76, 138.79, 151.32, 152.44, 154.98, 160.18, 162.44 (Ar-C), 164.58, 165.37 (C=O); LCMS (m/z): 600.9 [M + H]; anal. calcd for C₃₂H₂₃F₃N₄O₃S: C, 63.99; H, 3.86; N, 9.33; S, 5.34. Found: C, 63.96; H, 3.84; N, 9.33; S, 5.33.

4.1.7. Procedure for the synthesis of 5-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione 6(a-b). A mixture of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (0.01 mol) (I-II) and Meldrum's acid (F) (0.02 mol) in presence of catalytic amount of sodium acetate(0.015 mol) in glacial acetic acid (5 mL) refluxed for 1–2 h. The reaction mixture was allowed to cool. The solid that separated was filtered, dried and recrystallized from ethanol.
5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (6a). M.p. 160–162 °C; FT-IR (KBr) ν_max (cm⁻¹): 2973, 2865 (Ar-H), 1759 (C=O), 1665 (C=N), 1563 (C=C), 1208 (C–O), 751 (C–Cl); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 1.25 (s, 6H, 2CH₃), 7.42–8.07 (m, 9H, Ar-CH), 8.54 (s, 1H, pyrazole-CH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 21.12, 21.19 (CH₃), 119.50, 121.32, 122.90, 125.07, 127.52, 128.34, 128.58, 129.61, 130.00, 130.44, 130.71, 131.15, 131.72, 132.14, 138.68, 148.46, 150.40 (Ar-C), 162.50, 162.90 (C=O); LCMS (m/z): 443.0 [M + H] (³⁵Cl₂), 445.0 [M + H + 2] (³⁵Cl³⁷Cl), 447.0 [M + H + 4] (³⁷Cl₂); anal. calcd for C₂₂H₁₆Cl₂N₂O₄: C, 59.61; H, 3.64; N, 6.32. Found: C, 59.65; H, 3.67; N, 6.31.
5-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (6b). M.p. 172–175 °C; FT-IR (KBr) ν_max (cm⁻¹): 2986, 2953 (Ar-H), 1768 (C=O), 1657 (C=N), 1548 (C=C), 1224 (C–O), 1081 (C–F); ¹H NMR (CDCl₃, 400 MHz, δ ppm): 2.41 (s, 6H, 2CH₃), 7.39–8.11 (m, 9H, Ar-CH), 8.53 (s, 1H, pyrazole-CH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 21.34, 21.38 (CH₃), 119.24, 121.56, 122.98, 125.38, 126.45, 127.98, 128.87, 129.52, 130.01, 130.54, 130.89, 131.17, 131.68, 133.08, 137.94, 147.49, 151.40 (Ar-C), 163.41, 163.97 (C=O); LCMS (m/z): 410.4 [M + H]; anal. calcd for C₂₂H₁₆F₂N₂O₄: C, 64.39; H, 3.93; N, 6.83. Found: C, 64.35; H, 3.94; N, 6.85.

4.1.8. Procedure for the synthesis of 5-((3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione 7(a-b). A mixture of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (0.01 mol) (I-II) and barbituric acid (G) (0.01 mol) in glacial acetic acid (10 mL) in presence of catalytic amount of sodium acetate (0.015 mol) refluxed for 30 min to 2 h.⁴³ The reaction mixture was allowed to cool. The separated yellow solid was filtered, dried and recrystallized from ethanol.
5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (7a). M.p. 285–288 °C; FT-IR (KBr) ν_max (cm⁻¹): 3442, 3336 (NH), 2916 (Ar-H), 1714 (C=O), 1614 (C=N), 1514 (C=C), 750 (C–Cl). ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.45–8.09 (m, 9H, Ar-CH), 9.75 (s, 1H, pyrazole-CH), 11.30 (s, 2H, thiazolidine NH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 115.59, 115.74, 120.25, 128.74, 130.05, 130.43, 131.48, 131.65, 131.94, 132.29, 132.86, 135.10, 138.94, 150.71 (Ar-C), 163.06, 163.99 (C=O); LCMS (m/z): 424.8 [M + H] (³⁵Cl₂), 426.8 [M + H + 2] (³⁵Cl³⁷Cl), 428.8 [M + H + 4] (³⁷Cl₂); anal. calcd for C₂₀H₁₂Cl₂N₄O₃: C, 56.22; H, 2.83; N, 13.11. Found: C, 56.22; H, 2.83; N, 13.11.
5-((3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (7b). M.p. 263–265 °C; FT-IR (KBr) ν_max (cm⁻¹): 3440, 3368 (NH), 2952 (Ar-H), 1718 (C=O), 1652 (C=N), 1505 (C=C), 1015 (C–F); ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 7.38–8.03 (m, 9H, Ar-CH), 9.55 (s, 1H, pyrazole-CH), 11.24 (s, 2H, thiazolidine NH); ¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 114.89, 115.92, 121.32, 127.89, 129.15, 130.68, 131.32, 131.73, 131.99, 132.19, 133.16, 135.18, 138.32, 149.68 (Ar-C), 164.18, 164.24 (C=O); LCMS (m/z): 395.1 [M + H]; anal. calcd for C₂₀H₁₂F₂N₄O₃: C, 60.92; H, 3.07; N, 14.21. Found: C, 60.91; H, 3.07; N, 14.25.

4.2. Pharmacology

4.2.1. Anticonvulsant activity. MES test:³⁶⁻³⁸ inbred male albino mice weighing between 40–50 g were used in the study. They housed under standard laboratory conditions for week before the experiments. The animals were transferred to the laboratory 1 h before starting the experiment. The experiment was carried out according to committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines and Institutional Animal Ethical Committee approved all procedures. Each mice was used only once.

Mice were divided into different groups with six mice in each group. Suspensions of the compounds/standard in 0.5% Tween-80 in saline were administered intraperitoneally (i.p.) at 25 mg kg⁻¹. The untreated group was administered the same volume of the vehicle. A drop of 0.9% saline was instilled in each eye prior to the application of electrodes (Centroniks Electroconvulsiometer, Inco Co., Ambala India). Generalized tonic clonic seizures were induced in the animals by using an electroconvulsiometer (Inco Co., Ambala, India) at 50 Hz alternating current of 40 mA intensity for 0.2 s using a pair of ear clip electrodes. Failure to extend the hind limbs to an angle with the trunk >90° is defined as protection. Phenytoin was used as standard drug at a dose level of 25 mg kg⁻¹. The results are given in Table 2. The seizure pattern was recorded at 2 h after administration of the dose.

4.2.2. Analgesic activity. The analgesic activity of the above mentioned derivatives was also evaluated by applying the tail flick method using pentazocine as a standard reference.^39,41 Wistar albino mice of either sex (150–200 g) in the groups of six animals, each one were selected by random sampling technique. The 10 mg kg⁻¹ of the test compounds were administered orally by intragastric tube. Pentazocin (10 mg kg⁻¹) was administered orally as a reference drug for comparison. The reading was recorded at regular intervals of 0 min, 30 min, 60 min and 90 min after administration of compounds. A cut off point of 10 seconds was observed to prevent the tail damage.

4.2.3. Rotarod neurotoxicity test. Rotarod test⁴⁰ was performed to detect the motor deficit in mice. Animals were divided in groups of 4 animals and trained to stay on an accelerating rotarod that rotates at 10 revolutions per minute. The rod diameter was 3.2 cm. Trained animals (able to stay on the rotarod for at least two consecutive periods of 90 s) were given an i.p. injection of the test compounds at doses of 25, 50 and 100 mg kg⁻¹ and tested after 1 and 4 h. Neurological deficit was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of the three trials. The dose at which animal fell off the rod was determined.

Acknowledgements

The authors gratefully acknowledge the UGC, SAP and DST-PURSE for the financial assistance. We are grateful to IISC Bangalore and USIC Mangalore University for providing the spectral analysis. The authors also grateful to the Institution of Excellence, Vijnana Bhavana, University of Mysore, India, for providing the single-crystal X-ray diffractometer facility.

References

1. N. Pessah, M. Bialer, B. Wlodarczyk, R. H. Finnell and B. Yagen, J. Med. Chem., 2009, 52, 2233–2242.
2. P. Yogeeswari, D. Sriram, J. V. Ragavendran and R. Thirumurugan, Curr. Drug Metab., 2005, 6, 127–139.
3. J. A. Cramer, S. Mintzer, J. Wheless and R. H. Mattson, Expert Rev. Neurother., 2010, 10, 885–891.
4. M. Naithani, S. Chopra, B. L. Somani and R. K. Singh, Curr. Neurobiol., 2010, 1, 117–120.
5. Z. Lin and P. K. Kadaba, Med. Res. Rev., 1997, 17, 537–572.
6. M. W. Decker and M. D. Meyer, Biochem. Pharmacol., 1999, 58, 917–923.
7. (a) G. Vangegas, C. Ripamonti, A. Sbanotto and F. de Conno, Canc. Nurs., 1998, 21, 289–297; (b) C. Ecoffey, Cah. Anesthesiol., 1991, 39, 115–119; (c) T. D. Walsh, J. Pain Symptom Manage., 1990, 5, 362–367; (d) E. A. Kowaluk, K. J. Lynch and M. F. Jarvis, Annu. Rep. Med. Chem., 2000, 35, 21–30.
8. A. M. Waszkielewicz, A. Gunia, K. Słoczyn ska and H. Marona, Curr. Med. Chem., 2011, 18, 4344.
9. J. L. Kraus, Pharmacol. Res. Commun., 1983, 15, 183–189.
10. A. F. R. Fatma, M. A. G. Nagwa, H. G. Hanan and F. S. Mona, Chem. Pharm. Bull., 2013, 61, 834–845.
11. W. B. Roland, X. Han, Z. Xiaoming, M. Kristine, G. Jennifer, F. Mike, A. Matthew, H. P. Matthew, W. Min, M. E. Michele, R. Kelly, N. V. Vellarkad, T. Philip and H. Randall, Bioorg. Med. Chem. Lett., 2006, 16, 3713–3718.
12. K. K. Sivakumar, A. Rajasekaran, P. Senthilkumar and P. P. Wattamwar, Bioorg. Med. Chem. Lett., 2014, 24, 2940–2944.
13. P. K. Sharma, S. Kumar, P. Kumar, P. Kaushik, D. Kaushik, Y. Dhingra and K. Aneja, Eur. J. Med. Chem., 2010, 45, 2650–2655.
14. F. A. Ragab, N. M. Abdel Gawad, H. H. Georgey and M. F. Said, Eur. J. Med. Chem., 2013, 63, 645–654.
15. M. Abdel-Aziz, G. E. A. Abuo-Rahma and A. A. Hassan, Eur. J. Med. Chem., 2009, 44, 3480–3487.
16. Z. Ozdemir, H. B. Kandilci, B. Gumusel, U. Calis and A. A. Bilgin, Eur. J. Med. Chem., 2007, 42, 373–379.
17. P. Yogeeswari, D. Sriram, L. R. J. Suniljit, S. S. Kumar and J. P. Stables, Eur. J. Med. Chem., 2002, 37, 231–236.
18. J. R. Dimmock, K. K. Sidhu, S. D. Tumber, S. K. Basran, M. Chen, J. W. Quail, U. Pugazhenthi, T. Lechler and J. P. Stables, Eur. J. Med. Chem., 1995, 30, 287–301.
19. G. Sharma, J. Y. Park and M. S. Park, Bioorg. Med. Chem. Lett., 2008, 18, 3188–3191.
20. K. Kaminski, S. Rzepka and J. Obniska, Bioorg. Med. Chem. Lett., 2011, 21, 5800–5803.
21. Y. Cao, Y. Zhang, S. Wu, Q. Yang, X. Sun, J. Zhao, F. Pei, Y. Guo, C. Tian, Z. Zhang, H. Wang, L. Ma, J. Liu and X. Wang, Bioorg. Med. Chem., 2015, 23, 149–159.
22. L. S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, McGraw-Hill, New Delhi, 1991, p. 358.
23. K. K. Sivakumar, A. Rajasekaran, P. Senthilkumar and P. P. Wattamwar, Bioorg. Med. Chem. Lett., 2014, 24, 2940–2944.
24. M. Amir, H. Kumar and S. A. Khan, Bioorg. Med. Chem. Lett., 2008, 18, 918–922.
25. K. M. Amin, D. E. Abdel Rahman and Y. A. Al-Eryani, Bioorg. Med. Chem., 2008, 16, 5377–5388.
26. L. J. S. Knutsen, C. J. Hobbs, C. G. Earnshaw, A. Fiumana, J. Gilbert, S. L. Mellor, F. Radford, N. J. Smith, P. J. Birch, J. R. Burley, S. D. C. Ward and I. F. James, Bioorg. Med. Chem. Lett., 2007, 17, 662–667.
27. A. A. Nehad, A. E. Amr and A. I. Alhusien, Monatsh. Chem., 2007, 138, 559–567.
28. A. A. Abu-Hashem, M. A. Gouda and F. A. Badria, Eur. J. Med. Chem., 2010, 45, 1976–1981.
29. G. W. Wang and B. Cheng, ARKIVOC, 2004, 4, 4–8.
30. S. Viveka, Dinesha, S. S. Laxmeshwar and G. K. Nagaraja, Molbank, 2012, 3, M776.
31. S. Viveka, Dinesha, L. N. Madhu and G. K. Nagaraja, Monatsh. Chem., 2015, 146, 1547–1555.
32. R. Lesyk, B. Zimenkovsky, D. Atamanyuk, F. Jensen, K. Kiec-Kononowicz and A. Gzell, Bioorg. Med. Chem., 2006, 14, 5230–5240.
33. S. G. Patil, R. R. Bagul, M. S. Swami, S. N. Hallale, V. M. Kamble, N. S. Kotharkar and K. Darade, J. Chem. Pharm. Res., 2011, 3, 69–76.
34. S. S. Gawande, S. C. Warangkar, B. P. Bandgar and C. N. Khobragade, Bioorg. Med. Chem., 2013, 21, 2772–2777.
35. A. M. Vijesh, A. M. Isloor, S. Isloor, K. N. Shivananda, P. C. Shyma and T. Arulmoli, Der Pharma Chemica, 2011, 3, 454–463.
36. M. R. Shiradkar, M. Ghodake, K. G. Bothara, S. V. Bhandari, A. Nikalje, K. C. Akula, N. C. Desai and P. J. Burange, ARKIVOC, 2007, 14, 58–64.
37. M. Babu, K. Pitchumani and P. Ramesh, Bioorg. Med. Chem. Lett., 2012, 22, 1263–1266.
38. C. S. Sharma, R. K. Nema and V. K. Sharma, Stamford J. Pharm. Sci., 2009, 2, 42–47.
39. K. Meena Kumari, M. V. Amberkar, T. Shanbhag, K. L. Bairy and S. Shenoy, Indian J. Physiol. Pharmacol., 2011, 55, 13–24.
40. N. W. Dunham and T. S. Miya, J. Am. Pharm. Assoc., Sci. Ed., 1957, 46, 208–209.
41. S. Viveka, Dinesha, P. Shama, G. K. Nagaraja, N. Deepa and M. Y. Sreenivasa, Res. Chem. Intermed., 2015 DOI:10.1007/s11164-015-2170-7.
42. S. Viveka, Dinesha, P. Shama, G. K. Nagaraja, S. Ballav and S. Kerkar, Eur. J. Med. Chem., 2015, 101, 442–451.
43. N. P. Badige, N. S. Shetty, R. S. Lamani and I. A. M. Khazi, Heterocycl. Commun., 2009, 15, 433–442.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1046740. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra17391d

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