4,5-Dihydro-1H-pyrazole: an indispensable scaffold

Abstract

Pyrazoles, categorized as nitrogen-containing heterocycles, are well known for their interminable participation in the field of perpetual research and development of therapeutical active agents. As a consequence pyrazoles became an inevitable core of numerous drugs having diverse activities. The broad spectrum of activities portrayed by the pyrazoles instigated the researchers to modify the pyrazole ring as 4,5-dihydro-1H-pyrazoles commonly known as 2-pyrazolines. The present review is a concerted effort to retrace compounds covered from 2009-till date which owe diverse biological activities to the 2-pyrazoline scaffold and also condenses the retro-synthetic approaches employed for their synthesis. This endeavor culminated in revelation that inhibitory potential varied when the substituents in particular N-substituents of 2-pyrazolines were altered.

• 4,5-Dihydro-1H-pyrazole
• biological activities
• synthesis

Introduction

Even though “pyrazoles” had been known since the 19th century when Ludwig Knorr coined the term, the first natural pyrazole β-(1-pyrazolyl) alanine, was isolated after 75 years in 1959 from the seeds of Citurllus lanatus. Pyrazoles (Figure 1) although are resistant to oxidizing and reducing agents, are known to undergo catalytic hydrogenation to pyrazolines (2-pyrazoline) in particular 4,5-dihydro-1H-pyrazole (Figure 1)1.

Figure 1. Pyrazole and 4,5-dihydro-1H-pyrazole.

Pyrazolines considered as cyclic hydrazine moiety2 possess an endocyclic double bond3,4. In comparison to pyrazoles, pyrazolines are stronger bases, less stable and behaving more like unsaturated compounds. These nitrogen containing heterocycles5 are colorless liquid which have their boiling point in the range of 120–150 °C1. Among various derivatives of pyrazolines, 4,5-dihydro-1H-pyrazole or 2-pyrazoline have been observed to be the most common derivatives6 found to be insoluble in water but owing to its lipophilic character7 soluble in propylene glycol. These electron rich nitrogen heterocycles8 can be subjected to reduction or oxidation9. On reduction 2-pyrazolines either yield pyrazolidines or undergo ring cleavage; and when oxidized they form blue or red coloring matter1. The conjugated part of the ring (–N1–N2–C3–) includes an electron donating and electron withdrawing moieties within it. As observed from the X-ray analysis, all the atoms but C₅ of the pyrazoline ring adopt a planar system10 and this deviated atom is known to play a crucial role in the development of theory in heterocyclic chemistry11,12. 2-Pyrazolines absorb light in the range 300–400 nm and emit blue fluorescence owing to the two nitrogen atoms in the heterocyclic ring13,14 and also as a consequence of the double bond hindering which occur as a result of cyclization15,16. 2- Pyrazolines owing to these properties17 have been exploited in the synthesis of synthetic fibers, fluorescent probes, in electro photography and electroluminescence.

The pyrazolines find their application as alkaloids, vitamins, pigments, and so on18. Antipyrine (2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) was the first pyrazoline derivative used in the management of inflammation and pain19. The presence of this moiety in several therapeutically active compounds encouraged the researchers in the direction of design and synthesis of novel pyrazoline derivatives possessing myriad activities. Some of the activities that pyrazolines exhibit are anticancer20,21, antitumor22, anti-androgenic23, antioxidant24, antimicrobial25–27, antiviral28, antitubercular29,30, antimalarial31, anti-amoebic32,33, COX-II34,35, monoamine oxidase36,37, xanthine oxidase38and amine oxidase39 inhibitory, and so on. With an incentive to improve the existing activities several modifications are being done on this scaffold and many of which are proved to be successful. The most extensive modification in 2-pyrazoline was the substitution of diaryl/hetroaryl groups mainly at -3, 5 position40 since it was observed that heterocycles with functional groups greatly increased solubility in water41,42. The aryl substituted pyrazolines were detected to combine the activity of pyrazoline moiety with activity of heteroarene thus proving their usefulness43. The perpetual research carried out revealed the indispensable role N-substituents of pyrazoline play in exhibiting the biological activity. As the substituent changes, a particular activity is altered either completely or to a certain extent. Substitution on the carbon of pyrazoline also modified the biological activity but activity altered significantly with the variation in Y which may be acetyl, amide, phenyl, and so on. R_1, R₂ and R may be any alkyl, aromatic or hetero-aromatic substituent (Figure 2).

Figure 2. Various substitutions on pyrazoline ring.

An exclusive review put forward by Suresh Kumar et al.44 in the year 2009 encompassed the 2-pyrazoline derivatives synthesized from 2005 to 2009 giving a lucid picture of their biological activities. In 2010, Rahman et al.11 comprehended the biological activities of 2-pyrazoline derivatives from 2000 to 2008. In 2011, M. Yusuf et al.18 compiled together the biological activities and different synthetic strategies of the pyrazoline derivatives from 2007 to 2011. Recently, Dipankar Bardalai et al.1 congregated the information on the pyrazoline derivatives synthesized (1993–2011) as anti-inflammatory and analgesic agents. The present review highpoints the pyrazoline derivatives synthesized from 2009 till date with an inducement to fathom the crucial role that substituents in particular N-substituents play in determining the biological activity. It also summarizes some of the retro-synthetic approaches employed for the synthesis of pyrazolines. In this manuscript, with the aid of SAR studies and inhibitory potential of the compounds, we have sought to establish the relationship between the N-substituents and the biological activities.

Synthetic strategies

Research over the years has led to the development and introduction of several synthetic routes for the efficient synthesis of pyrazolines. Mostly the starting materials required to prepare pyrazoline compounds are obtained either via Claisen-Schmidt condensation or through synthesis of 1,3-dicarbonyl compounds. Some of the retro-synthetic approaches for the construction of pyrazolines have been sketched in Figure 3. One of the strategies involved is the parallel solution phase synthesis of 1 which involves the use of polymer bound bases A and B as depicted in route A45. Although routes B and G use the same reactants 3, route B follows microwave irradiation46 unlike route G in which the reactants are subjected to heating at 120 °C. Route B employed an efficient, rapid, and green synthesis under solvent-free conditions in the presence of scandium triflate [Sc(OTf)₃] resulting pyrazoline (1) in yield ranging from 74 to 92% in 5 min and silica chloride (route G) catalyzed one pot cyclocondensation47 afforded yield of 80% in 2 h. Suzuki-Miyaura reaction48 exploiting Pd (OAc)₂ (5 mol %) as a catalyst in the presence of SPhos (10 mol %) in an aqueous solution of K₂CO₃ (2 M) (route C) afforded pyrazoline in 60–66% yield in 8 h. Yet another strategy employed included solvent-free synthesis49 as in route D which used equivimolar concentration of methanesulphonic acid and 5 at 80 °C giving a yield of 95% in 45 min. Route E followed asymmetric synthesis50 through an enantioselective phase transfer organocatalytic addition of N-Boc hydrazine to 2 followed by a transprotection sequence allowing N-Boc transformation into N-Ac or other functional groups resulting in 1. Route F followed a simple yet highly efficient and environment friendly one-pot condensation reaction51 of 6 with tosylhydrazide in water yielding 74–92%. Route H followed a region-selective synthesis of 152 by acylation of N-Boc-N-methylhydrazones followed by TFA giving a yield of 95–98%.

Figure 3. Retrosynthetic approach for the synthesis of pyrazoline derivative.

Classification of pyrazolines

We have categorized various 2-pyrazolines into three classes on the basis of the nature of substituents (Y) at N1 position of pyrazoline as shown in Figure 4. An effort has been made to put forward certain examples in this review portraying the modification in bioactivity as a consequence of alteration of the substituents.

Figure 4. Various N-substitutions on the pyrazoline ring.

Class I

The compounds pertaining to this class are shown to exhibit the following activities:

Anticancer activity

Zeinab H.I. and her group53 synthesized and evaluated 3,5-diaryl-Δ^2-pyrazoline derivatives for their anticancer activity against the human colon (HCT-116) and breast (MCF-7) cancer cell lines. The structure–activity relationship studies of these compounds (Table 1) threw light on how substituents affect the change in biological activity. On one hand where the phenyl substitution on N1 decreased the anticancer activity (5C1, 5C2), the N-acetyl substitution (5B1) showed promising results for HCT-116 cell line. In the absence of substitution on N1 of the pyrazoline ring, these compounds owed their anticancer activity to the phenyl ring and its substitution placed at C5 of the pyrazoline ring. If the presence of an electron withdrawing group (5A1) on the phenyl ring increased the activity towards MCF-7 cancer cell line, the electron donating group (5A2) shifted the spectrum of activity towards HCT-116 cell line.

Table 1. Structural modification on 2-pyrazolines.

				IC50 (µM)
Compound	R1	R2	R3	HCT-116	MCF-7
5A1	H	F		–	3.43
5A2				7.09	–
5B1		o-CI		6.8	12
5C1		F		–	16.5
5C2		NO2		10.3	–

A.H. Banday et al.54 synthesized 17-pyrazolinyl derivatives of pregnenolone and evaluated the same against a panel of cancer cell lines - HT-29, HCT-15, 502713, HOP-62, A-545, MCF-7, SF-295. The structure–activity relationship studies along with the data obtained through IC₅₀ values (Table 2) indicated m-fluro (6C) substitution to be significant for the anticancer activity against HT-29, HCT-15, 502713, HOP-62, o-chloro (6E) substitution to be promising against HT-29 and HCT-15 and similarly good activity against 502713, MCF-7 and SF-295 was attained when the phenyl ring at C5 of the core moiety was left unsubstituted (6A). An increase in IC₅₀ values resulted as a consequence of substituted heterocyclic ring at C5 (6B) or with p-OMe (6D) thus leading to decrease in anticancer activity.

Table 2. Structural modification on steroidal based 2-pyrazolines.

		IC50 (µM)
Compound	R	HT-29	HCT-15	502713	HOP-62	A-545	MCF-7	SF-295
6A		6.35	2.31	0.56	1.46	11.0	0.43	0.63
6B		11.8	23.4	44.2	50.0	48.1	ND	50.6
6C		0.44	0.53	0.32	0.50	1.42	1.60	3.79
6D		2.37	21.8	28.5	32.9	29.2	23.1	49.5
6E		0.24	0.25	37.0	50.0	32.2	17.5	30.2

T. Liu et al.55 synthesized a series of cis-restricted 4,5-diaryl-3-aminopyrazole and tested these compounds for their anticancer potential against five human cancer cell lines namely K562, ECA-109, A-549, SMMC-7721 and PC-3 (Table 3). Substitution of trimethoxy group on ring A (7A) led to a greater increase in anticancer activity than when present on ring B (7B). Replacement of 3,4,5-trimethoxy phenyl ring (Ring B) with 4-chloro phenyl ring (7C) resulted in a substantial increase in the activity as exhibited by the IC₅₀ values. The substituted phenyl ring proved to be indispensable for activity because when ring B was substituted with a thiophene ring the activity was reduced considerably.

Table 3. Structural modification on 3-amino based pyrazole derivatives.

			Cytotoxicity (IC50, µM)
Compound	Ring A	Ring B	K562	ECA-109	A549	SMMC-7721	PC-3
7A	3,4,5-(OCH3)3	4-OCH3	0.08 ± 0.04	12.01 ± 2.45	1.38 ± 0.94	12.07 ± 2.66	6.95 ± 0.74
7B	4-OCH3	3,4,5-OCH3	ND	25.04 ± 6.25	40.38 ± 3.21	ND	7.68 ± 5.27
7C	3,4,5-(OCH3)3	4-Cl	0.31 ± 0.16	8.00 ± 1.35	6.03 ± 2.51	13.79 ± 5.24	0.61 ± 0.53

C. Congiu et al.56 synthesized and put efforts to test a series of novel 4, 5-dihydro pyrazole based combretastatin analogs for their anticancer potential. The structural–activity relationship and IC₅₀ values (Table 4) helped in concluding that 3,4,5-trimethoxy group at the C5 (8B) showed better activity than when it was present at C3 of the dihydropyrazole core (8A), that is, when the moiety is in the same plane as the N-acetyl group. Thus, the modifications were carried out on ring A keeping the substituent on ring B constant. Some of these modifications led to increase the activity whereas others shifted the spectrum of activity in the opposite direction. Substitutions involving the amino group (8B3, 8B4, 8B7), methyl group (8B5, 8B2) and halogens (8B6) showed promising activity. The derivatives with methoxy substituent (8B1) were found to be devoid of any activity. Hydroxyl substitution at C4 of ring A (8B9) furnished the most active compound of the series.

Table 4. Structural modifications on N₁-acetyl derivative of pyrazolines and their anticancer activity against NCIH-460 cancer cell line.

Compound	R	NCIH-460 IC50 (µM)
8B1	4-MeO	>20
8B2	4-MeS	8.0 ± 0.1
8B3	4-NH2	2.6 ± 0.1
8B4	4-Me2N	1.8 ± 0.06
8B5	4-Me	1.3 ± 0.05
8B6	3-Br	0.35 ± 0.01
8B7	3-NH2	0.74 ± 0.33
8B8	3-OH	2.3 ± 0.06
8B9	3-MeO-4-OH	0.21 ± 0.005

FabH inhibitory activity

Fifty-six analogues of 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole were synthesized by P.C. Lv et al.57 and evaluated for their potential to be used as FabH inhibitors. The minimum inhibitory concentration (MIC) values and the SAR studies (Table 5) highlighted electron releasing group on ring A and electron donating groups on the ring B essential for FabH inhibitory activity of E. coli (9A and 9B) and MIC value decreased if these substitutions were interchanged (9C). Dihalogen substitution on ring A (9D) showed significant FabH inhibition of Bacillus subtilis. These observations were further confirmed by carrying out molecular docking of the potent inhibitor into the active site of FabH of E.coli.

Table 5. Antimicrobial activities of N₁-acetyl pyrazolines.

			MIC (µg/mL)
			Gram positive			Gram negative
Compound	R1	R2	B. subtilis ATCC 6633	S. aureus ATCC 6538	S. faecalis ATCC 9790	P. aerug-inosa ATCC 13525	E. coli ATCC 35218	E. cloacae ATCC 13047
9A	4-OCH3	4-F	6.25	6.25	1.562	12.5	0.39	12.5
9B	4-OCH3	4-Cl	6.25	25	6.25	25	0.78	25
9C	4-Cl	4-OCH3	12.5	>50	25	25	25	>50
9D	3,4-Cl	4-Cl	0.78	25	6.25	25	25	>50

COX-II inhibitory activity

R. Fioravanti et al.58 synthesized 18 analogous of 1-N-substituted-3,5-diphenyl-2-pyrazoline derivatives and evaluated the cyclooxygenase activity of these compounds. They synthesized compounds in which the N1 of pyrazoline was substituted with either acetyl moiety or thiocarbamoyl moiety. The SAR studies and the IC₅₀ values (Table 6) revealed that compounds with N-acetyl group were more potent than those having thiocarbomyl group (10A and 10B). The molecular docking studies conducted highlighted the importance of 4-methanesulphonyl group on the phenyl ring at C5 for the COX-II inhibitory activity.

Table 6. COX-II inhibitory activity of some N₁-substituted pyrazoline.

Compound	R	X	IC50 (µM)
10A	2-OCH2Ph	COCH3	3.20 ± 0.24
		CSNH2	Inactive at a conc. 25µM
10B	4-CH3	COCH3	6.77 ± 0.48
		CSNH2	31.85 ± 2.57

Anti-inflammatory activity

B.P. Bandgar et al.59 synthesized 3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines and screened these compounds for their antioxidant and anti-inflammatory potential (Table 7). The selectivity of these compounds for COX-II and commendable activity shown by these compounds were attributed to the presence of carbazole moiety. Substitution of electron withdrawing group on the aryl ring enhanced the antioxidant activity resulting via the DPPH radical scavenging (11D and 11E) while the presence of electron donating group resulted in an increase of the anti-inflammatory effect (11A and 11B). These studies also outlined the fact COX-II inhibitory activity increased tremendously in substituting the aryl ring at C5 with a thiophene (11C) or pyridine ring. Molecular docking studies accompanied by the team proved the credibility of these observations.

Table 7. Anti-inflammatory & antioxidant activities of 2-pyrazolines.

Anti-inflammatory activity
		% Inhibition		COX-2 enzyme inhibition assay
Compounds	Ar	COX-I	COX-II	In vitro % inhibition (100 µM)	In vivo % inhibition
Indomethacin		63.87	28.43	28.43	47.0 ± 1.1
11A		24.15	88.43	88.43	60.3 ± 4.5
11B		23.49	86.85	86.85	54.3 ± 1.1
11C		18.50	48.43	48.43	42.2 ± 11.8

Antioxidant activity
		% Radical scavenging activity
Compound	Ar			DPPH radical	SOR radical	OH radical
Ascorbic acid			91.52	51.95	88.34
11D		24.15	40.53	92.08	81.67	69.34
11E		8.85	10.15	91.74	86.26	78.64

Antidepressant and anticonvulsant activity

N-substituted thiocarbamoyl-3-(2-furyl)-5-phenyl/(2-furyl)-2- pyrazoline derivative synthesized by Z. Ozdemir and his team60 proved their potential as antidepressants and anticonvulsants by Porsolt’s behavioral despair and maximal electroshock (MES) & subcutaneous pentylenetetrazole (scMet), respectively. The compounds in the series owed their antidepressant and anticonvulsant activity to the thiocarbamoyl substitutent at N1 of the pyrazoline. Furthermore, anticonvulsant activity was enhanced several folds by substituting 2-furyl ring at C5 of pyrazoline with (12B and 12C) as represented in Table 8.

Table 8. Anticonvulsant and antidepressant activities of N1-thiourea derivatives of pyrazoline.

Antidepressant
Compound	R	Ar	Antidepressant activity (Duration of immobility) (mean ± S.E.M.)
12A	C2H5	Phenyl	139 ± 12
12B	C3H5	2-furyl	144 ± 21.4
Anticonvulsant
			MES		scMET
Compound	R	Ar	Time (hrs)	Conc (mg/kg)	Time (hrs)	Conc (mg/kg)
12C	Phenyl	2-furyl	1/2	100 & 300	No activity
12D	2-furyl	C2H5	4	300	1/2	30,100,300

Xanthine oxide inhibitory activity

Our research group40,61 synthesized 53 analogues of 1-acetyl-3,5-diaryl-4,5-dihydro (1H) pyrazoles and evaluated their xanthine oxidase activity using bovine milk xanthine oxidase enzymatic assay. Structure–activity relationships study and IC₅₀ values (Table 9) observed and confirmed by the molecular docking studies revealed that the replacement of phenyl (Ring A, 13A) with naphthalene ring (13B) increased the activity, which on replacement with 2-furyl ring resulted in further enhancement of XO inhibitory activity (13C). Replacement of ring B with 1-naphthyl (13F) resulted in decrease in activity. Replacement of phenyl with anthranyl ring resulted in complete loss of activity. Moreover, substituents on ring B also influenced the extent of activity. Deactivating groups such as nitro (13E) and halogens increased the xanthine oxidase inhibitory activity whereas activating group such as methoxy (13D) or N, N-dimethyl resulted in decrease in activity. Absence of N-acetyl substitution led to loss of the biological activity of the compound, thus making the importance of this moiety irrefutable.

Table 9. XO inhibitory activity of N₁-acetyl pyrazolines.

Compound	Ring A	Ring B	R	XO inhibitory activity IC50 (µM)
13A			H	61.4
13B			H	41.3
13C			H	26.4
13D			R2 = –OCH3	21.3
13E			R2 = NO2	13.1
13F			–	91.4

MAO inhibitory activity

N-substituted-3-[(2′-hydroxy-4′-prenyloxy)-phenyl]-5-phenyl-4,5-dihydro-(1H)-pyrazolines in particular pyrazoline N-substituted with either thiocarbamoyl or acetyl group, synthesized and evaluated by R. Fioravanti et al.62 for their potential to inhibit monoamine oxidase (Table 10). Compounds bearing acetyl substitution at N1 exhibited better activity (14A and 14B) than with thiocarbamoyl group (14C and 14D). SAR studies highlighted that irrespective of the N1 substituent; benzyloxy substitution enhanced the MAO inhibition (14A and 14C) whereas methyl or methoxy group led to a complete loss of activity (14B and 14D).

Table 10. MAO inhibitory activity of some pyrazolines.

			MAO inhibitory activity (pIC50)
Compound	X	R	hMAO-A	hMAO-B
Clorgyline (MAO-A inhibitor)			8.35	4.21
Deprenyl (MAO-B inhibitor)			4.17	7.70
14A		OCH2Ph	6.50	6.76
14B		CH3	–	–
14C		OCH2Ph	–	6.57
14D		CH3	–	–

Class II

The compounds bearing these substitutions exhibited activity against various microorganisms such as bacteria, fungi, viruses and parasites such as Plasmodium species in addition to possessing analgesic activity.

Antimicrobial activity

B.C. Revanasiddappa et al.63 synthesized and determined the pyrazoline derivatives for their in-vitro antimicrobial activity against different Gram-negative and Gram-positive bacterial strains as well as fungal strains. Although these compounds showed comparable antibacterial and antifungal activity, the activity (Table 12) is seen mainly due to the presence of electron withdrawing groups (Table 11) at R₁ on the C2 phenyl ring. Substitution of a thiophene ring at R on the phenyl ring at C5 along with an electron withdrawing group at R₁ on the phenyl ring at C3 led to an increase in the antibacterial activity especially against Bacillus subtilis and Psuedomonas aerogenosa (15A) but the substitution of a deactivating group at R and R₁ (15C) shifted the spectrum of activity towards antifungal activity. Co-relation of SAR studies and the data retrieved from the antimicrobial evaluation also featured that the presence of an activating group at R and deactivating group at R₁ led to a decrease in both antibacterial as well as antifungal activity.

Table 11. Various of N₁-pyridamide pyrazoles.

Compound	R	R1
15A	2-Thiophene	p-NO2
15B	p-(CH3)N	p-Br
15C	p-Cl	p-Cl
15D	p-OCH3	p-Cl

Table 12. Antimicrobial activity of various of N₁-pyridamide pyrazoles.

	Diameter of the zone of inhibition (mm) at 10 µg/mL concentration
	Anti-bacterial activity				Anti-fungal activity
Compound	S. aureus	B. subtilis	E. coli	P. aeruginosa	C. albicans	A. niger
15A	12	16	13	16	15	14
15B	13	13	16	14	15	14
15C	14	14	15	12	16	16
15D	14	14	14	13	15	15
Streptomycin	24	21	22	22	–	–
Griseofulvin	–	–	–	–	23	22
Control (DMF)	–	–	–	–	–	–

Anti-tubercular activity

M.A. Ali et al.64 synthesized pyrazoline derivatives and tested their anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv. Compound with 2,6-dichloro group substitution [16A, anilino-3-(4-hydroxy-3-methylphenyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-1-pyrazolylmethanethione] (Table 13) produced highest efficacy and exhibited >90% inhibition at very low concentration. 2,6-Dichloro substituted derivatives exhibited relatively higher inhibitory activity although the presence of electron rich groups such as, 4-chloro, 2-chloro, and 3-nitro substituted analogue (16B) led to significant decrease in inhibitory activity against M. tuberculosis H37Rv. Pyrazoline analogues with 3,4-dimethoxy phenyl substitution (16C) and 3,4,5-trimethoxy phenyl substitution (16D) showed relatively moderate anti-tubercular activity. Compounds having R = CH₃ (16E) and R₁ = 3,4,5-trimethoxy phenyl presented relatively low inhibitory activity against M. tuberculosis H37Rv.

Table 13. MIC values for antituberculer activity.

Compound	R	R1	Antituberculer activity MIC (µg/mL)
16A	2-OCH3	2,6-Dichloro phenyl-	1.66
16B	2-OCH3	3-Nitro phenyl-	–
16C	2-OCH3	3,4-Dimethoxy phenyl-	5.67
16D	2-OCH3	3,4,5-Trimethoxy phenyl-	–
16E	2-CH3	3,4,5-Trimethoxy phenyl-	–

Anti-amoebic activity

A team of A.R. Bhat32 synthesized thiocarbamoyl bis-pyrazoline derivatives and assessed these compounds for their potential as anti-amoebic against HM1: IMSS strain of Entamoeba histolytica via the microdilution technique. The SAR studies (Table 14) carried out helped in inferring that the aromatic ring on N-thiocarbamoyl (17A and 17B) proved to have better activity than the cyclic groups (17C and 17D). The electron withdrawing substituent on the aromatic ring also contributed to the activity. Among the compounds containing the cyclic group, increase in activity was proportional to the ring size.

Table 14. Anti-amoebic activity of bispyrazolines.

Compound	R	Anti-amoebic activity IC50 (µM)
17A		0.42
17B		0.62
17C		0.92
17D		1.16
Metronidazole		1.8

M.Y. Wani et al. (65) synthesized and subjected 1,3,5-trisubstituted pyrazoline derivatives to in-vitro anti-amoebic screening to assess their potential against growth of Entamoeba histolytica. The information drawn from SAR studies (Table 15) revealed that compounds having methyl groups at R₁ and R₂ possessed the highest activity (18C). Replacing the methyl group at R₂ with methoxy group ensued in a small decrease in activity (18D). Activity was further decreased when methyl group from 18D was removed. Compounds with electron withdrawing group at R₁ (18A and 18B) resulted in higher IC₅₀ values thus indicating lower activity. The team also determined the safety profile for the synthesized compounds which held an inverse relation to the IC₅₀ values measured.

Table 15. In-vitro anti-amoebic activity of compounds against HM1: IMSS strain of Entamoeba histolytica and their toxicity profile.

			Anti-amoebic activity
Compound	R1	R2	IC50 (µM)	Safety Profile (SI)
18A	Cl	H	3.15	28.76
18B	Cl	OCH3	2.63	>38.02
18C	CH3	CH3	0.86	>116.27
18D	CH3	OCH3	1.08	>92.59
Metronidazole			1.80	>55.55

Anti HIV activity

Mohamed A. Ali and his group66 synthesized and assessed nicotinoyl substituted pyrazolines for their activity against HIV strains IIIB and ROD (Table 16). Among the series, the compound having a phenyl ring bearing electron donating groups at C5 of the pyrazoline (19A) showed the highest activity. Derivatives with heteroaromatic ring (19B) showed moderate activity. In addition, the SAR studies signified that presence of electron withdrawing groups resulted in fall of activity and this was found to be inversely proportional to the number of substitutions, that is dihalogen substitution showed better activity than monohalogen (19C and 19D).

Table 16. Anti-HIV activity of N₁-pyridamide pyrazoles.

Compound	R	IC50 (µM)
19A	4-Dimethylaminophenyl-	5.7
19B	Furyl-	6.8
19C	4-Chlorophenyl-	>36.1
19D	2,6-Dichlorophenyl-	>13.83

Anti-malarial activity

B.N. Acharya et al.67 synthesized and evaluated a series of nicotinoyl substituted pyrazolines for their potential as antimalarials against chloroquine sensitive (MRC-02) and chloroquine resistant (RKL 9) strains of Plasmodium falciparum. Among these derivatives, the SAR studies (Table 17) assisted in inferring that the compounds possessing electron withdrawing groups at either ortho or para (20B) or both (20A) showed commendable activity against both the strains. Substitution involving electron releasing group resulted in abolishing of the activity to a small extent for chloroquine sensitive strains although it retained good activity for chloroquine resistant strains.

Table 17. Antimalarial activity of some N₁-pyridamide pyrazoles.

					Antimalarial activity
Compound	R1	R2	R3	R4	MRC-02	RKL9
20A	Cl	H	Cl	H	0.0375 ± 0.005	0.0680 ± 0.019
20B	Br	H	H	H	0.0272 ± 0.005	0.0640 ± 0.003

Analgesic activity

R. S. Joshi et al.68 synthesized a series of 3,2-(4,5-dihydro-5-(4-morphilinophenyl)-1H-pyarazol-3-yl) phenols and its N-phenylpyrazol-1-carbothioamide and evaluated them for their analgesic activity by acetic acid induced writhing response in mice and anti-inflammatory activity against carrageenan induced rat paw edema model in rats. The compounds (Table 18) having the N-phenylpyrazol-1-carbothioamide moiety (21D) disclosed better activity than the pyrazoline compound. Although the presence of a halogen (Br) (21B) resulted in improvement of the activity but there was no direct correlation between enhancement of the anti-inflammatory activity and the presence of electron donating and electron withdrawing group. This might be owing to the fact that substitution of a dihalogen doesn’t produce any significant activity when compared to monohalogen substitution.

Table 18. Anlgesic and anti-inflammatory activities of morpholine containing pyrazolines.

						Increase in paw volume (mL) after carageenan administration
Compound	X	R1	R2	R3	Number of writhings	2 h	3 h	4 h	24 h
Control					71 ± 3	1.49 ± 0.20	1.64 ± 0.15	1.57 ± 14	1.92 ± 0.12
Diclofenac					08 ± 2	0.18 ± 0.03	0.18 ± 0.015	0.18 ± 0.02	0.03 ± 0.01
21A	H	H	H	Cl	53 ± 4	1.52 ± 0.12	1.60 ± 0.18	1.61 ± 0.12	1.92 ± 0.18
21B		H	H	Br	15 ± 4	0.86 ± 0.15	0.52 ± 0.14	0.64 ± 0.10	0.98 ± 0.12
21C		H	H	Cl	09 ± 2	0.64 ± 0.10	0.72 ± 0.14	0.72 ± 0.12	0.86 ± 0.12
21D		H	H	Br	13 ± 3	0.87 ± 0.12	0.69 ± 0.13	0.91 ± 0.17	0.92 ± 0.16

Class III

The compounds bearing these substituents at the pyrazoline N1 showed antimicrobial activity mainly against bacteria and fungi in addition to anti-inflammatory, anti-infective and anticancer activity.

Antimicrobial activity

Zeinab H. I. et al.53 synthesized pyrazoline derivatives (Table 19) and tested them for their in-vitro antimicrobial activity against Gram-positive bacteria Staphylococcus aureus, Bacillis subtilis, the Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and fungi Aspergillus fumigatus, Penicillium italicum, Syncephalastrum racemosum and Candida albicans

The data obtained from the SAR studies projected that dinitrophenyl ring substitution on the N1 of pyrazoline indicated more commendable activity than an unsubstituted phenyl ring (Table 20). Phenyl ring bearing electron withdrawing groups (22A, 22B, 22C) at C3 and C5 of the pyrazoline ring displayed excellent activity against Gram-positive bacteria and also E. coli whereas phenyl ring with electron donating groups (22C) displayed only moderate activity against the Gram-positive bacteria. Derivatives in which the pyrazoline ring is substituted with thiophene ring (22D) at the C3 of the pyrazoline ring were seen to act against only Escherichia coli. Moreover, it was observed that the compounds that possessed significant antibacterial activity also displayed commendable antifungal activity (Table 21). Akin to the antibacterial activity, phenyl rings bearing electron withdrawing groups (22A) at C3 and C5 of the pyrazoline ring demonstrated excellent antifungal activity.

Table 19. Structural modification of some N-substituted pyrazolines.

Compound	Ar	R	R′	R″
22A	C6H4 Br-4	H	N(CH3)2
22B	C6H4 Br-4	Cl	H	Ph
22C		–OCH3	H
22D		H	Cl	Ph
22E	C6H5	H	H

Table 20. Antibacterial activity of 22A–22E.

	Gram-positive bacteria						Gram-negative bacteria
	S. aureus			B. subtilis			E. coli			P. aeruginosa
Compound	1.25	2.5	5.0	1.25	2.5	5.0	1.25	2.5	5.0	1.25	2.5	5.0
22A	+	+	++	+	++	++	+	+	++	0	0	+
22B	0	0	+	0	0	+	+	+	+	0	0	0
22C	0	+	+	0	+	+	0	+	+	0	0	0
22D	0	0	0	0	0	0	0	+	+	0	0	0
22E	+	+	++	+	++	++	+	++	++	0	0	+

Chloramphenicol is the standard antibacterial control.

Inhibition values = 0.1–0.5 cm beyond control = +; Inhibition values = 0.6–1.0 cm beyond control = ++; and 0 = not detected.

Table 21. Antifungal activity of 22A and 22B.

	A. fumigatus			P. italicum			S. racemosun			C. albicans
Compound	1.25	2.5	5.0	1.25	2.5	5.0	1.25	2.5	5.0	1.25	2.5	5.0
22A	+	+	++	+	+	0	+	++	++	0	+	+
22B	0	0	0	0	+	+	0	0	0	0	+	+

Shamsuzzaman and his coworkers69 synthesized and screened 2′-(2″, 4″-dinitrophenyl)-5a-cholestano [5, 7-c d] (Table 22) to assess their antimicrobial activity against different bacterial strains (Corynebacterium xerosis, Staphylococcus epidermidis and Escherichia coli) method and fungal strains (Mucor azygosporus, Claviceps purpurea and Aspergillus niger) employing the broth dilution and agar diffusion method, respectively. The promising antifungal and antibacterial activity shown by the compound was attributed to the dinitrophenyl ring substituted at the N1 of the pyrazoline ring.

Table 22. Antimicrobial activity of steroidal pyrazolines.

Compound	MIC (Antibacterial)
2′-(2″,4″-dinitrophenyl)-5a-cholestano [5,7-c d] pyrazolines	E. coli	C. xerosis	S. epidermidis
	3.125	0.781	0.195
	MIC (Antifungal)
	M. azygosporus	C. purpurea	A. niger
	3.125	6.250	1.562

S. P. Shaktinathan and his research group43 synthesized a series of novel 2-naphthyl pyrazolines and also evaluated these compounds for their antimicrobial activity against bacterial strains (Gram-positive Micrococcous luteus, Bacillus substilis, Staphylococcus aureus and Gram-negative Escherichia coli and Klebsiella) and against fungal strains (Aspergillus niger and Trichoderma viride) by utilising the well-known disc diffusion technique (Table 23). The activity was confirmed by measuring the diameter of the zone of inhibition. Among the electron withdrawing groups substituted, chlorine (23C) showed better activity against Gram-negative strains especially against E. coli and fluorine substitution (23A) exhibited moderate antibacterial activity against E. coli and B. subtilis. The electron releasing groups (23D) showed no activity against Gram-positive strains but showed the highest activity against Gram-negative E. coli strain. Antifungal activity more or less followed the same trend; chlorine substitution rendered the compound active against T. viride but not against A. niger.

Table 23. Antimicrobial activity of 23A–23D.

Antibacterial activity (mm)
		Gram-positive bacteria			Gram-negative bacteria
Compound	X	B. subtilis	M. luteus	S. aureus	E. coli	K. pneumoniae
23A	4-F	9	7	6	9	7
23B	4-Br	8	8	7	7	7
23C	4-Cl	8	7	–	10	7
23D	4-CH3	6	–	–	12	7
Ampicillin		20	18	18	18	12

Antifungal activity (mm)
Compound	X	A. niger	T. viride
23A	2-Cl	–	10
23B	3-Cl	–	10
23C	4-F	9	7
Miconazole		14	16

Antibacterial activity

Mamta Rani and her research group70,71 synthesized three series of pyrazoline derivatives with the same basic framework but different substituent and assessed their antibacterial activity against strains of Aeromonas hydrophila, Yersinia enterocolitica, Listeria monocytogenes and Staphylococcus aureus with the help of Halo Zone Test72 and also compared their activity by measuring the zone of inhibition (Table 24). Derivatives having furan ring at C5 of the pyrazoline ring were observed to possess better activity than the derivatives with thiophene ring.

Table 24. Antimicrobial activity of 24A1–24B3.

			Halo zone test (unit, mm)
			Microorganisms
Compound	Ar	X	A. hydrophila	Y. enterocolitica	L. monocytogenes	S. aureus
Gentamycin			21	–	–	17
24A1			21.5	24.4	21.3	22.8
24B1			25.3	18.6	20.8	24.5
24A2			22.3	21.4	24.3	22.8
24B2			21.7	25.2	19.4	23.6
24A3			20.5	22.4	24.3	22.8
24B3			21.7	18.4	21.2	19.4

Anti-inflammatory activity

S. Bano et al.73 synthesized 2-pyrazolines bearing benzene sulphonamides (Table 25) and evaluated these compounds for their anti-inflammatory activity. The benzene sulphonamides substituted at the N1 of the pyrazoline ring attributed to these compounds their activity. p-chloro substitution on the C5 phenyl ring increased the anti-inflammatory effect as compared to o-hydroxy substitution on the C5 phenyl ring.

Table 25. Anti-inflammatory activity of benzene sulphonamide derivative of 2-pyrazolines.

	Carrageenan-induced rat paw edema assay
	3 h		5 h
Compound	Increase in the paw volume (ml)	% Inhibition	Increase in the paw volume (ml)	% Inhibition
Celecoxib	0.12 ± 0.0041	79.59	0.09 ± 0.0031	82
	0.12 ± 0.004	80	0.09 ± 0.003	81.82
	0.125 ± 0.005	78.95	0.125 ± 0.004	75

S. Khode and his group74 synthesized 5-(substituted) aryl-3-(3-coumarinyl)-1-phenyl-2-pyrazolines and evaluated both their in vitro as well as in-vivo anti-inflammatory activity (Table 26). The IC₅₀ activity exhibited by these compounds revealed that activity increased with the electron withdrawing group with fluro-substitution (25B) showing better activity than the chloro (25C) substitution although activity was also dependent on the number of groups (25A) substituted. Compound 25C and 25D showed good inhibitory activity at second and fourth hour, respectively, while the most active compounds 25A and 25B exhibited excellent inhibitory activity at second and fourth hour, respectively.

Table 26. Anti-inflammatory activity of coumarin based 2-pyrazolines.

		Anti-inflammatory activity
		In vitro % inhibition
Compound	Ar	2 h	4 h	In vivo % inhibition after treatment
25A	2,4-Cl2-C6H3	56.7 ± 0.030	67.5 ± 0.024	45.4 ± 1.63
25B	4-F-C6H4	44.9 ± 0.023	66.7 ± 0.011	40.5 ± 1.80
25C	4-Cl-C6H4	39.2 ± 0.026	64.7 ± 0.021	39.1 ± 0.81
25D	3-OMe-C6H4	38.3 ± 0.030	61.5 ± 0.013	20.9 ± 1.25
Diclofenac		63.7 ± 0.017	78.7 ± 0.013	53.0 ± 1.92

Anti-infective agents

P. M. Sivakumar and his team75 synthesized 1, 3, 5-triphenyl-2-pyrazolines and screened these compounds for their anti-infective activities against Mycobacterium tuberculosis H₃₇Rv, bacterial and fungal strains. It was observed (Table 27) that sulfonylmethyl (26A) substitution increased the activity towards the H₃₇Rv strain since it was observed to bring the log P value to 3 (ideal for penetration through mycobacterial cell). Compounds having thiomethyl substitution (26C) in the A-ring resulted in higher activity against the bacterial and the fungal strains. Activity possessed by the active compounds (26C, 26D and 26E) was attributed to the halogen substitution at the -p and/or -m position in the B-ring, thus, highlighting the importance of halogen substitution for antibacterial and antifungal activities.

Table 27. Antimicrobial activity of N₁-phenyl substituted pyrazolines.

Anti-tubercular activity (% reduction in rlu)
Compound	R	X	50 µg/ml						100 µg/ml
26a	SO2CH3	–	1.70						50.71
26b	SCH3	–	0.00						17.65
Antibacterial activity (mic in µm)
Compound	R	X		E. coli	P. vulgaris		E. aerogenes		S. aureus	S. typhii		B. subtilis
26a	SO2CH3	–		0.166	0.332		0.332		0.664	0.332		0.332
26c	SCH3	Cl		0.152	0.304		0.304		0.608	0.304		0.304
26d	SCH3	Br		0.148	0.295		0.295		0.591	0.295		0.295
Antifungal activity (% inhibition at 2mg/ml concentration)
Compound	R	X		F. proliferatum		C. tropicalis		A. flavus			A. niger
26a	SO2CH3	–		97.61 ± 0.47		30.64 ± 0.30		65.85 ± 1.82			47.20 ± 5.03
26c	SCH3	Cl		88.26 ± 0.90		95.58 ± 1.05		88.44 ± 1.92			97.54 ± 0.07
26e	SCH3	F		98.71 ± 0.16		97.41 ± 1.40		94.82 ± 0.16			97.28 ± 1.31

Anticancer activity

M. Shaharyar et al.76 synthesized series of benzimidazole bearing 2-pyrazolines and tested these compounds against various cancer cell lines belonging to different panels (Table 28) such as renal, breast, colon, melanoma, prostate, and so on. Most active compound of the series was found to be 2-[5-(3, 4-dimethoxyphenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1H-benzimidazole. Based on close examinations of the substituent, it was concluded that the role of electron donating group on the phenyl ring at C5 of the pyrazoline ring had a great influence on anticancer activity.

Table 28. In-vitro anticancer activity of compound NSC 748326.

Panel	Cancer cell lines	GI50 (µM)
Leukemia	CCRF-CEM	2.23
	RPMI-8226	2.76
Renal	CAKI-1	3.6
	UO-31	2.2
Prostate	PC-3	6.27
	DU-145	33.1
Colon	HCT-116	3.94
	HCT-15	4.1
Breast	MCF7	2.41
	MDA-MB-468	5.25

Conclusion

Modifications on the pyrazole ring are rightly claimed to have inextricable contribution to the multifaceted activities projected by the pyrazoline moiety. The broad spectrum of activities exhibited by the 2-pyrazolines makes them an indispensable scaffold for the synthesis of numerous pharmacologically active compounds. The review was put forward with an incentive to congregate the 2-pyrazoline derivatives synthesized from 2009-till date and classifying them in a manner that would justify the activities portrayed by these derivatives. Although, the substituents on the pyrazoline ring, whether on nitrogen (N1, N2) or on the carbon (C3, C4, and C5) proved to be significant, the decisive role was played by the N1 of the pyrazoline. The plethora of information, retrieved from the structure–activity relationship studies, IC₅₀ values, safety profiles and confirmation by the molecular docking studies, helped paint a lucid picture of the potential held by the substituents to influence the extend of inhibitory potential commendably. Amide, acetyl or thiocarbamoyl substitution on the N1 of the pyrazoline portrayed activity that varied over a wide range of activity such as anticancer activity, oxidase inhibitory activity, anti-inflammatory activity, antidepressant activity, and so on. On the contrary, aromatic or heteroaromatic substitution on the N1 contributed to synthesis of compounds which had the potential as antimicrobials, anti-HIV, anti-amoebic, antiparasitic, antitubercular and analgesic. Not many efforts have been made so far to isolate and separate the enantiomeric forms of N1-substituted 2-pyrazolines. An understanding of interactions of the resolved single isomer with the target in co-crystal structures may be beacon for offering the scope of tuning and having permutation and combination of a variety of N-substituents at 2-pyrazolines for the design of potential and specific inhibitors.

Declaration of interest

This study was financially supported by DST, New Delhi (DST project Grant NO. SR/FT/CS-71/2011).

Acknowledgements

Dr. Raj Kumar is thankful to DST, New Delhi for providing the financial aid (DST project Grant NO. SR/FT/CS-71/2011) to carry out the present work. Authors are also thankful to Dean Academic Affairs, Central University of Punjab, Bathinda for his constant encouragement.