Pyrazolo[5,1-c][1,2,4]triazole: A Promising Emerging Biologically Active Scaffold in Medicinal Chemistry

Abstract

Nitrogen-containing heterocycles are essential compounds in nature, and their structural and functional diversity inspired the synthesis of a wide range of derivatives with diverse applications as pharmaceuticals, agrochemicals, dyes, polymers, cosmetics, etc. Among them, N-fused heterocycles represent an important category, due to their high potential as biologically active agents. Pyrazolo[5,1-c][1,2,4]triazoles, a class of nitrogen heterobicycles, have multiple applications as dyes and pigments. Also, a number of compounds containing this structure have been investigated for their biological activities. All the main experimental results published in the literature (both articles and patents) regarding the latter are summarized in this review.

1. Introduction

Most pharmaceuticals currently in use are small organic molecules, and, among them, nitrogen-containing heterocyclic scaffolds have shown very useful biological activities against various diseases [1]. Moreover, N-heterocyclic structures are widely distributed in nature, including in amino acids, peptides and proteins; nucleotides, nucleosides and nucleic acids; vitamins and many secondary metabolites [2]. Almost one-third of the best-selling therapeutics contain fused heterocyclic structures, justifying the high scientific interest and research effort towards these compounds. N-Fused heterocyclic compounds are basic components of several common pharmaceuticals, agrochemicals, plastics, and dyes [3,4].

Pyrazolo[5,1-c][1,2,4]triazoles are a class of N-containing biheterocyclic compounds mainly known for their use in dyes and pigments in a wide variety of fields. The pyrazolo[5,1-c][1,2,4]triazole system can theoretically exist in four tautomeric forms, namely 1H, 3H, 5H, and 7H (Figure 1); the most widely encountered is the 1H form, while the 5H form has been reported only in a few compounds, some of which are actually 5-substituted. The remaining two, not fully aromatic forms 3H and 7H, have been seen almost exclusively in 3,3- and 7,7-disubstituted compounds, respectively.

Figure 1.

The tautomeric forms of pyrazolo[5,1-c][1,2,4]triazole.

The numerous methods reported in the literature for the synthesis of the pyrazolo[5,1-c][1,2,4]triazole core have been recently reviewed [5,6].

The pyrazolo[5,1-c][1,2,4]triazole system combines two frequently used biologically active scaffolds, those of pyrazole and 1,2,4-triazole. Also, as a 5-5 bicyclic system, it is similar in size to other 5-5, 5-6, and 6-6 scaffolds. Not surprisingly, then, a number of biological activity studies have included pyrazolo[5,1-c][1,2,4]triazole derivatives, including several annulated analogs. The aim of the present review is to report all the relevant findings of these studies, in order to highlight the biological potential of this scaffold. Principally, the Reaxys and SciFinder databases were used for the literature search, covering the period between 1988 and 2025, targeting structures containing the pyrazolo[5,1-c][1,2,4]triazole core and the biological activity of these compounds.

Note: This present work is organized based on the biological activity the compounds were tested for, as described below:

1. Acetylcholinesterase inhibition activity

2. Anti-inflammatory activity

3. Analgesic activity

4. Antidiabetic activity

5. Antibacterial activity

6. Antifungal activity

7. Antiviral activity

8. Antiprotozoal activity

9. Anticancer activity

10. C3a receptor binding activity

11. Miscellaneous biological activities

12. Cytotoxicity

13. Ulcerogenic activity

2. Acetylcholinesterase Inhibition Activity

Compounds 1–5 (Figure 2), 6-amino-1,2-dihydro-3H-pyrazolo[5,1-c][1,2,4]triazole-3-thione 1, 4-amino-1-thia-3a,6,7,7b-tetraazacyclopenta[cd]inden-3(2H)-one 2, 1,7-diamino-N-(5-methyl-1,3-thiazol-2-yl)-3-thia-4,5,7a,7b-tetraazacyclopenta[cd]indene-2-carboxamide 3, ethyl 4-amino-2H-1-thia-5,7,8,8b,8c-pentaazacyclopenta[bc]acenaphthylene-3-carboxylate 4 and phenyl(4-phenyl-2H-1-thia-5,7,8,8b,8c-pentaazacyclopenta[bc]acenaphthylen-3-yl)methanone 5, were assayed for acetylcholinesterase inhibition activity [7]. Donepezil was used as reference drug. The results are displayed in Table 1. Compound 1 showed strong acetylcholinesterase inhibition activity, compounds 4 and 5 were moderate acetylcholinesterase inhibitors, while compounds 2 and 3 were weak inhibitors. (Note: This paper is of poor quality; therefore, the information in it should be taken cautiously.)

Figure 2.

Chemical structures of compounds 1–5.

Table 1.

The acetylcholinesterase inhibition activity of compounds 1–5.

Compound	IC50, μg/mL
1	14.58 ± 0.45
2	97.67 ± 2.13
3	231.94 ± 4.09
4	59.69 ± 1.18
5	52.06 ± 0.97
Donepezil	2.88 ± 0.16

3. Anti-Inflammatory Activity

[1,2,4]Triazolo[4′,3′:1,5]pyrazolo[3,4-c]dibenzo[f,h]cinnolines 6–8 (Figure 3), N-phenyl-11H-dibenzo[f,h][1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-c]cinnolin-13-amine 6, 13-phenyl-11H-dibenzo[f,h][1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-c]cinnoline 7 and 11,14-dihydro-13H-dibenzo[f,h][1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-c]cinnolin-13-one 8, were tested for anti-inflammatory activity in Wistar and Sprague Dawley rats (the animals are described as mice in the Experimental section, obviously an error) [8]. Indomethacin was used as reference drug. The compounds were administered orally at a dose of 5 mg/kg body mass. The results are listed in Table 2. Compound 6 possesses weak anti-inflammatory activity, compound 8 shows intermediary activity, while compound 7 is almost as active as Indomethacin. The high anti-inflammatory activity of compound 7 was attributed to the presence of a secondary amine of triazole [triazine in the original paper] fragment.

Figure 3.

Chemical structures of compounds 6–8.

Table 2.

The anti-inflammatory activity of compounds 6–8 in rats.

Compound	Edema Inhibition, %
	1 h	2 h	3 h
6	14.5 ± 2.4	16.7 ± 1.8	22.1 ± 1.4
7	41.9 ± 1.5	50.4 ± 2.1	61.4 ± 1.8
8	30.3 ± 1.6	39.6 ± 1.2	45.3 ± 1.1
Indomethacin	45.6 ± 1.7	52.4 ± 2.1	63.2 ± 1.8

The acute toxicity of compound 7 was determined in vivo in mice [8]. The compounds were administered by intraperitoneal injection. Indomethacin was used as standard drug. The results are displayed in Table 3. Compound 7 is moderately toxic, but less toxic than Indomethacin.

Table 3.

The cytotoxicity of compound 7 on rats.

Compound	LD50 (i.p.), mg/kg
7	155
Indomethacin	50

2-[(6-Amino-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl)methyl]-4-(2,4,6-trimethylphenyl)phthalazin-1(2H)-one 9 (Figure 4) was tested for anti-inflammatory activity in rats [9]. The control group (each group included 6 rats) was injected with 2% w/v acacia mucilage, while a second group was treated with Indomethacin as reference drug. The other seven groups were injected with the compound studied. The compounds were administered intraperitoneally at a dose of 1.5 mg/kg body mass. Then the rat paw oedema was induced with 0.1 mL w/v carrageenan subcutaneously. The results are listed in Table 4. Compound 9 possesses moderate anti-inflammatory activity.

Figure 4.

Chemical structure of compound 9.

Table 4.

The anti-inflammatory activity of compound 9 in rats.

Compound	Paw Oedema Volume Mean + S.D. (mL)	Percentage Inhibition of Paw Oedema	Dose (mg kg−1 p.o.)
2% Gum acacia (control)	0.62 ± 0.029	–	10 mL kg−1
9	0.46 ± 0.019	25.80	50
Indomethacin	0.25 ± 0.012	59.67	1.5

4. Analgesic Activity

Compounds 6–8 (Figure 3) were tested for analgesic activity in Webster mice, with Valdecoxib as reference drug [8]. The compounds were administered subcutaneously at a dose of 5 mg/kg body mass. The results are listed in Table 5. Compound 6 showed intermediate analgesic activity, compound 7 possessed only weak activity, while compound 8 was very active, with its activity after 30 min becoming practically equal to that of Valdecoxib. The high analgesic activity of compound 8 was attributed to the presence of the secondary amine of the triazole fragment in its structure.

Table 5.

The analgesic activity of compounds 6–8 in mice.

Compound	Relative Potency
	10 min	30 min	60 min
6	0.68 ± 0.03	0.73 ± 0.02	0.81 ± 0.04
7	0.22 ± 0.01	0.24 ± 0.01	0.27 ± 0.02
8	0.85 ± 0.01	0.93 ± 0.03	0.99 ± 0.01
Valdecoxib	1.00	1.00	1.00

5. Antidiabetic Activity

Compounds 10–14 (Figure 5), 1-(6-methyl-1H-pyrazolo[5,1-c][1,2,4]triazol-1-yl)ethan-1-one 10, 1-(6-methyl-7-thioxo-7,8-dihydro-1H,5H-azeto[3′,2′:4,5]pyrazolo[5,1-c][1,2,4]triazol-1-yl)ethan-1-one 11, 1-acetyl-6-methyl-1H-pyrazolo[5,1-c][1,2,4]triazole-7-carbothioamide 12, 6-methyl-1H-pyrazolo[5,1-c][1,2,4]triazole-7-carbonitrile 13, and 6-methyl-1H-pyrazolo[5,1-c][1,2,4]triazole 14 (Figure 5) were tested for antidiabetic activity (α-glucosidase and α-amylase inhibitory activities) [10]. The antidiabetic drug Acarbose was used as reference. The results are shown in Table 6. It can be observed that compound 12 is the most potent inhibitor of α-glucosidase, while compounds 14 and 11 are potent inhibitors of α-amylase. Molecular docking studies were also reported in this paper.

Figure 5.

Chemical structures of compounds 10–14.

Table 6.

The antidiabetic inhibitory activity of compounds 10–14.

Compound	Antidiabetic Activity (IC50 in μM)
	α-Glucosidase	α-Amylase
10	595.04 ± 43.42	260.40 ± 0.27
11	503.43 ± 35.11	121.15 ± 0.65
12	216.22 ± 17.32	764.56 ± 0.10
13	433.32 ± 88.31	219.89 ± 0.08
14	300.45 ± 47.87	109.43 ± 6.12
Acarbose	309.11 ± 22.32	618.87 ± 0.76

6. Antibacterial Activity

2-[5-(5,6-Diphenyl-1,2,4-triazin-3-yl)-6-phenyl-5H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]acetonitrile 15 (Figure 6) was tested in vitro for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris and was found to be inactive in all cases [11].

Figure 6.

Chemical structure of compound 15.

In another study, three chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazoles 16–18 (Figure 7), chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole-3(2H)-thione 16, 2-methyl-5,6,7,8-tetrahydro-2′H-spiro[chromene-4,3′-chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole] 17, and 3′,6′-dimethyl-1′-phenyl-1′H,2H-spiro[chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole-3,4′-pyrano[2,3-c]pyrazole] 18 were tested in vitro for antibacterial activity against two Gram-positive bacteria, Bacillus cereus and Staphylococcus albus, and two Gram-negative bacteria species, Pseudomonas aeruginosa and E. coli [12]. The results are displayed in Table 7. As can be seen, these compounds showed variable activity against bacteria.

Figure 7.

Chemical structures of compounds 16–18.

Table 7.

The antibacterial activity of compounds 16–18 against Gram-positive and Gram-negative bacteria (inhibition zone diameter, mm).

Compound	B. cereus	S. albus	P. aeruginosa	E. coli
16	4	3	2	2
17	2	4	3	3
18	2	2	2	2

The cytotoxicity of compounds 16–18 (Figure 7) was determined on brine shrimp (Artemia salina) larvae [12]. The results are shown in Table 8. Compound 17 has low toxicity, while compounds 16 and 18 are non-toxic.

Table 8.

The cytotoxicity of compounds 16–18 against Artemia salina.

Compound	Artemia salina a
16	D
17	C
18	D

^a Artemia salina (brine shrimp) test. A, high toxicity, 75–100% dead larvae; B, moderate toxicity, 50–75% dead larvae; C, low toxicity, 25–50% dead larvae; D, non-toxic, less than 25% dead larvae.

Pyrazolo[5,1-c][1,2,4]triazoles 19–21 (Figure 8), 4-{[6-(1,1-dimethylethyl)-3-(pyridin-2-yl)-7H-pyrazolo[5,1-c][1,2,4]triazol-7-ylidene]methyl}-N,N-diethyl-3-methylaniline 19, 4-{[3-butyl-6-(1,1-dimethylethyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl]imino}-2,6-dichlorocyclohexa-2,5-dien-1-one 20, and tetrabutylammonium 4-{[3-butyl-6-(1,1-dimethylethyl)-7H-pyrazolo[5,1-c][1,2,4]triazol-7-ylidene]amino}-2,6-dichlorophenolate 21 were tested for antibacterial activity against Salmonella typhimurium TA98 and TA100 [13]. H-1 [Kathon biocide; a 3:1 mixture of 5-chloro-2-methylisothiazol-3(2H)-one and 2-methylisothiazol-3(2H)-one)] was used as reference. The results are shown in Table 9 and Table 10. It can be seen that compounds 19–21 have antibacterial activity, while, at the same time, they are less toxic than the reference antibacterial agent (no increase in the number of revertant colonies was observed).

Figure 8.

Chemical structures of compounds 19–21.

Table 9.

The antibacterial activity of compounds 19–21 against Salmonella typhimurium TA100 ^a.

Compound	Dose (μg/Plate)											Obs.
	5000	1250	313	78	20	10	5	1.25	0.5	0.31	0.2
H-1	–	–	–	–	–	–	–	K	K	K	K	control
19	K	K	K	K	K	K	○	○	–	–	–
20	K	K	K	K	○	○	○	–	–	–	–
21	K	K	K	K	K	–	○	○	○	–	–

^a (K) indicates that Salmonella has been killed, (–) indicates that it has not been measured, and (○) indicates that no increase in the number of revertant colonies was observed.

Table 10.

The antibacterial activity of compounds 19–21 against Salmonella typhimurium TA98.

Compound	Dose (μg/Plate)											Obs.
	5000	1250	313	78	20	10	5	1.25	0.5	0.31	0.2
H-1	–	–	–	–	–	–	–	K	K	–	●	control
19	K	K	K	K	○	K	○	○	–	–	–
20	K	K	K	○	○	–	–	–	–	–	–
21	K	K	K	○	○	–	○	○	–	–	–

(K) indicates that Salmonella has been killed, (–) indicates that it has not been measured, (●) indicates that the number of revertant colonies was more than twice the number of spontaneous revertant colonies in the solvent control group, and (○) indicates that no increase in the number of revertant colonies was observed.

Pyrazolo[5,1-c][1,2,4]triazol-6(5H)-ones 22–24 (Figure 9), 3-(2-methyl-1H-indol-3-yl)-7-(phenyldiazenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 22, 3-(2-methyl-1H-indol-3-yl)-7-(4-methylphenyldiazenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 23 and 7-(4-chlorophenyldiazenyl)-3-(2-methyl-1H-indol-3-yl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 24, were also tested in vitro for antibacterial activity against four bacteria, S. aureus, P. aeruginosa, B. subtilis and E. coli [14]. Chloramphenicol was used as reference under the same conditions. The results are displayed in Table 11. Compound 23 exhibited the highest degree of inhibition against P. aeruginosa and E. coli, while compound 22 had a high inhibition effect against P. aeruginosa. Compound 24 showed a high degree of inhibition against E. coli. However, the activities of the tested compounds are much lower than those of the standard antibacterial agent used.

Figure 9.

Chemical structures of compounds 22–24. 22: R = H; 23: R = Me; 24: R = Cl.

Table 11.

The biological activities of compounds 22–24 against bacteria at 5 mg/mL concentration (inhibition zone diameter, cm) ^a.

Compound	S. aureus	P. aeruginosa	B. subtilis	E. coli
22	+	++	-	-
23	+	++	-	++
24	+	-	-	++
Chloramphenicol	1.0	2.8	2.6	1.0

^a Inhibition zone diameter beyond control/(sign): 1.1–1.5 cm/(+++); 0.6–1.0 cm/(++); 0.1–0.5 cm/(+); 0 cm/(-).

5-(5,6-Diphenyl-1,2,4-triazin-3-yl)-7-methyl-3-phenyl-5H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-d]pyrimidin-9(8H)-one 25 (Figure 10) was tested in vitro for antibacterial activity against three bacteria, S. aureus (MTCCB 737), Staphylococcus epidermidis (MTCCB 1824), and E. coli (MTCCB 1652) [15]. Tetracycline was used as standard drug against bacterial strains at 30 μg/mL concentration. The results are shown in Table 12. It can be noted that compound 25 showed good inhibition activities against all species of bacterial strains with respect to Tetracycline. The good antibacterial activity was attributed to the presence of the pyrazolo[3,4-b]pyrimidine scaffold fused with the bioactive heterocyclic moiety of 1,2,4-triazole. The minimum inhibitory concentrations (MIC, μg/mL) of compound 25 against S. aureus (MTCCB 737) and E. coli (MTCCB 1652) are shown in Table 13.

Figure 10.

Chemical structure of compound 25.

Table 12.

The antimicrobial activity of compound 25 at 100 μg/mL concentration (inhibition zone diameter, mm) ^a.

Compound	S. aureus (MTCCB 737)	S. epidermidis (MTCCB 1824)	E. coli (MTCCB 1652)
25	27	27	26
Tetracycline	30	25	28

^a 12 mm or less: resistant or no inhibition, 13–17 mm: moderate inhibition, 18 mm or more: maximum inhibition.

Table 13.

The minimum inhibitory concentration (MIC, μg/mL) of compound 25 against Staphylococcus aureus and Escherichia coli, respectively.

Compound	S. aureus (MTCCB 737)	E. coli (MTCCB 1652)
25	50	25
Tetracycline	6.25	12.5

Compound 25 was also tested for cytotoxicity against A. salina larvae [15]. Bleomycin and gallic acid were used as standards. As can be seen in Table 14, compound 25 showed low toxicity against A. salina larvae.

Table 14.

The cytotoxicity of compound 25 against Artemia salina.

Compound	95% Confidence Limit ppm			Regression Equation	X2 (df)
	LC50	Lower	Upper
25	3.54	2.08	6.02	y = 3.98 + 1.85x	3.38 (2)
Bleomycin	0.41	0.27	0.62	y = 3.16 + 2.98x	0.62 (2)
Gallic acid	4.53	3.33	6.15	y = 3.93 + 1.62x	1.25 (2)

In another study, compounds 6 and 8 (Figure 3) were tested in vitro for antibacterial activity against the same three bacteria, S. aureus, S. epidermidis, and E. coli [8]. The same standard as above was used (Tetracycline at 30 μg/mL concentration). The results are shown in Table 15. It can be seen that compound 8 showed good inhibition against all the species of bacteria, while compound 6 showed weak inhibition.

Table 15.

The antimicrobial activity of compounds 6 and 8 at 100 μg/mL concentration (inhibition zone diameter, mm) ^a.

Compound	S. aureus	S. epidermidis	E. coli
6	10	9	5
8	25	23	20
Tetracycline	30	25	28

^a 15 mm or less: resistant or no inhibition, 16–20 mm: moderate inhibition, 20 mm or more: maximum inhibition.

Compound 9 (Figure 4) and its analog 2-[(6-methyl-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl)methyl]-4-(2,4,6-trimethylphenyl)phthalazin-1(2H)-one 26 (Figure 11) were tested in vitro for antibacterial activity against four bacterial strains, E. coli, S. aureus, B. subtilis and Salmonella typhi [9,16]. Amoxicillin was used as standard drug. The observed activities are given in Table 16. The results indicate that compound 9 exhibited good antibacterial activity against S. aureus, B. subtilis, and S. typhi and lower activity against E. coli, while compound 26 showed weak activity against all four bacterial species.

Figure 11.

Chemical structure of compound 26.

Table 16.

The biological activities of compound 26 against bacteria (minimum inhibitory concentration (MIC), μg/mL).

Compound	S. aureus	B. subtilis	S. typhi	E. coli
9	25	25	25	50
26	100	250	200	100
Amoxicillin	6.25	6.25	6.25	6.25

In another study, six pyrazolo[5,1-c][1,2,4]triazoles 27–32 (Figure 12), ethyl 7-[(4-chlorophenyl)diazenyl]-6-oxo-1-phenyl-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 27, ethyl 7-[(4-chlorophenyl)diazenyl]-1-(4-methylphenyl)-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 28, 3-acetyl-7-[(4-chlorophenyl)diazenyl]-1-phenyl-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 29, 3-acetyl-7-[(4-chlorophenyl)diazenyl]-1-(4-methoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 30, 3-acetyl-1-(4-methylphenyl)-7-(4-methylphenyldiazenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 31, and 3-benzoyl-7-(4-methylphenyldiazenyl)-1-phenyl-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 32 were tested in vitro for antibacterial activity against two Gram-positive bacteria, Streptococcus pneumoniae and B. subtilis, and two Gram-negative bacteria species, P. aeruginosa and E. coli [17]. Ampicillin and Gentamicin were used as standard antibacterial agents for Gram-positive and Gram-negative bacteria, respectively. The results are displayed in Table 17. With the exception of compounds 27, 29, and 30, which showed low activity against B. subtilis, and compound 28, which revealed low activity against S. pneumoniae, the compounds tested showed no antibacterial activity.

Figure 12.

Chemical structures of compounds 27–32. 27: R = H; 28: R = Me; 29–31: R¹ = Me; 29: R² = Cl, R³ = H; 30: R² = Cl, R³ = OMe; 31: R² = R³ = Me; 32: R¹ = Ph, R² = Me, R³ = H.

Table 17.

The biological activities of compounds 27–32 against Gram-positive and Gram-negative bacteria (inhibition zone diameter, mm µg⁻¹ sample).

Compound	S. pneumoniae	B. subtilis	P. aeruginosa	E. coli
27	NA a	10.2 ± 0.53	NA	NA
28	14.6 ± 0.22	NA	NA	NA
29	NA	9.3 ± 0.44	NA	NA
30	NA	8.2 ± 0.53	NA	NA
31	NA	NA	NA	NA
32	NA	NA	NA	NA
Ampicillin	23.8 ± 0.2	32.4 ± 0.3	–	–
Gentamicin	–	–	17.3 ± 01	19.9 ± 0.3

^a NA—no activity.

In another study from the same laboratory, fifteen pyrazolo[5,1-c][1,2,4]triazoles 33–47 (Figure 13), ethyl 6-oxo-1-phenyl-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 33, ethyl 1-(4-chlorophenyl)-6-oxo-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 34, ethyl 1-(4-methylphenyl)-6-oxo-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 35, ethyl 1-(4-nitrophenyl)-6-oxo-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 36, ethyl 1-(3-chlorophenyl)-6-oxo-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 37, ethyl 1-(3-nitrophenyl)-6-oxo-7-(phenyldiazenyl)-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 38, ethyl 7-[(4-nitrophenyl)diazenyl]-6-oxo-1-phenyl-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 39, ethyl 1-(4-chlorophenyl)-7-[(4-nitrophenyl)diazenyl]-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 40, ethyl 1-(4-methylphenyl)-7-[(4-nitrophenyl)diazenyl]-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 41, ethyl 1-(4-nitrophenyl)-7-[(4-nitrophenyl)diazenyl]-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 42, ethyl 1-(3-chlorophenyl)-7-[(4-nitrophenyl)diazenyl]-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 43, ethyl 1-(3-nitrophenyl)-7-[(4-nitrophenyl)diazenyl]-6-oxo-5,6-dihydro-1H-pyrazolo[5,1-c][1,2,4]triazole-3-carboxylate 44, 3-acetyl-1-phenyl-7-(phenyldiazenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 45, 3-acetyl-7-[(4-nitrophenyl)diazenyl]-1-phenyl-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 46, and 3-benzoyl-1-phenyl-7-(phenyldiazenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 47 were tested in vitro for antibacterial activity against the same four bacterial strains, using the same standards for reference [18]. The determined activities are included in Table 18. The results reveal that compounds 34, 35, 37, 39, 45, and 46 have medium to weak activity against both Gram-positive bacteria, while compound 47 shows weak activity against B. subtilis. With the exception of compound 35, which has medium activity against E. coli, the compounds tested showed no activity against Gram-negative bacteria. A molecular docking study was also included in the work.

Figure 13.

Chemical structures of compounds 33–47.

Table 18.

The antibacterial activities of compounds 33–47 against Gram-positive and Gram-negative bacteria (inhibition zone diameter, mm/µg sample).

Compound	S. pneumoniae	B. subtilis	P. aeruginosa	E. coli
33	NA a	NA	NA	NA
34	12.9 ± 0.19	15.1 ± 0.44	NA	NA
35	14.9 ± 0.22	16.2 ± 0.36	NA	12.3 ± 0.27
36	NA	NA	NA	NA
37	12.3 ± 0.36	14.1 ± 0.17	NA	NA
38	NA	NA	NA	NA
39	11.3 ± 0.19	12.2 ± 0.44	NA	NA
40	NA	NA	NA	NA
41	NA	NA	NA	NA
42	NA	NA	NA	NA
43	NA	NA	NA	NA
44	NA	NA	NA	NA
45	12.9 ± 0.12	15.1 ± 0.34	NA	NA
46	13.2 ± 0.19	15.7 ± 0.44	NA	NA
47	NA	10.8 ± 0.34	NA	NA
Ampicillin	23.8 ± 0.2	32.4 ± 0.3	–	–
Gentamicin	–	–	17.3 ± 0.1	19.9 ± 0.3

^a NA—no activity.

A Chinese patent reports the in vitro testing of chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazoles 48–50 (Figure 14), N-(2,4-difluorophenyl)chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole-1(11H)-carboxamide 48, N-ethylchromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole-1(11H)-carboxamide 49 and N-(4-ethyl-2-fluorobenzyl)chromeno[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazole-1(11H)-carboxamide 50, against S. aureus, with Penicillin as standard [19]. The results are shown in Table 19. Compounds 48 and 50, both containing a fluorine-substituted benzene ring, show good inhibition against S. aureus, while the activity of compound 49 is significantly lower.

Figure 14.

Chemical structures of compounds 48–50. 48: R = 2,4-F₂-C₆H₃; 49: R = Et; 50: R = 2-F-4-Et-C₆H₃CH₂.

Table 19.

The antibacterial activities of compounds 48–50 against S. aureus (inhibition zone diameter, mm).

Compound	Inhibition Zone Diameter, mm
48	23.70
49	13.46
50	25.94
Penicillin	22.50

7. Antifungal Activity

Compound 15 (Figure 6) was also tested in vitro for antifungal activity against Aspergillus niger and Penicillium chrysogenum (formerly known as P. notatum) and was found to be inactive in all cases [11].

6-Amino-1H-pyrazolo[5,1-c][1,2,4]triazole-3-thiol 51 (Figure 15) was tested in vitro for antifungal activity against four fungal species, Aspergillus ochraceus Wilhelm (AUCC-230), P. chrysogenum Thom (AUCC-530), Aspergillus flavus Link (AUCC-164), and Candida albicans (Robim) Berkho (AUCC-1720) [20]. Mycostatin (30 μg/mL) was used as reference. [This compound is actually a tautomer form of compound 1, reported as such in this paper.] The results are listed in Table 20. Compound 51 is nearly as active as Mycostatin against A. ochraceus, P. chrysogenum, and A. flavus (MIC values were 50–75 μg/mL) but less active against C. albicans.

Figure 15.

Chemical structure of compound 51.

Table 20.

The antifungal activity of compound 51 against four species of fungi (inhibition zone diameter, mm).

Compound	A. ochraceus	P. chrysogenum	A. flavus	C. albicans
51	34	36	35	24
Mycostatin	36	40	38	40

Compounds 22–24 (Figure 9) were tested in vitro for antifungal activity against four fungal species, Aspergillus fumigatus, Penicillium italicum, Syncephalastrum racemosum, and Candida albicans [14]. The fungicide Terbinafin was used as reference. The results are displayed in Table 21. Compounds 22 and 23 exhibited the highest degree of inhibition against A. fumigatus; the rest of the activities were low. However, the activities of the tested compounds are much lower than that of the standard antifungal agent used.

Table 21.

The antifungal activities of compounds 22–24 (inhibition zone diameter, cm) ^a.

Compound	A. fumigatus	P. italicum	S. racemosum	C. albicans
22	++	+	-	+
23	++	+	+	-
24	+	-	-	-
Terbinafin	3.0	3.6	3.6	3.0

^a Inhibition zone diameter beyond control/(sign): 1.1–1.5 cm/(+++); 0.6–1.0 cm/(++); 0.1–0.5 cm/(+); 0 cm/(-).

Compound 25 (Figure 10) was tested in vitro for antifungal activity against three fungi, A. fumigatus, Aspergillus niger, and Alternaria alternata [15]. Ketoconazole was used as standard drug against fungal strains at 30 μg/mL concentration. The results are shown in Table 22. It can be noted that compound 25 showed good inhibition activities against A. fumigatus with respect to Ketoconazole, and lower inhibition against the other two species. The minimum inhibitory concentrations (MIC, μg/mL) of compound 25 against A. niger and A. alternata are shown in Table 23.

Table 22.

The antifungal activity of compound 25 at 100 μg/mL concentration (inhibition zone diameter, mm) ^a.

Compound	A. fumigatus	A. niger	A. alternata
25	17	12	8
Ketoconazole	18	20	21

^a 12 mm or less: resistant or no inhibition, 13–17 mm: moderate inhibition, 18 mm or more: maximum inhibition.

Table 23.

The minimum inhibitory concentration (MIC, μg/mL) of compound 25 against Aspergillus niger and Aspergillus alternata, respectively.

Compound	A. niger	A. alternata
25	50	50
Ketoconazole	6.25	6.25

Compounds 6 and 8 (Figure 3) were tested in vitro for antifungal activity against the same three fungal species, A. fumigatus, A. niger, and A. alternata [8]. The same standard as above was used (Ketoconazole at 30 μg/mL concentration). The results are shown in Table 24. It can be seen that compound 8 shows good inhibition against all the species of fungi, while compound 6 displays weak inhibition.

Table 24.

The antifungal activities of compounds 6 and 8 at 100 μg/mL concentration (inhibition zone diameter, mm).

Compound	A. fumigatus	A. niger	A. alternata
6	12	9	10
8	21	19	19
Ketoconazole	18	20	21

15 mm or less: resistant or no inhibition, 16–20 mm: moderate inhibition, 20 mm or more: maximum inhibition.

Compound 26 (Figure 11) was tested in vitro for antifungal activity against two fungal strains, A. niger and C. albicans [16]. Again, Ketoconazole was used as standard drug (30 μg/mL concentration). The observed activities are given in Table 25. The results indicate low activities of compound 26 against both fungal strains.

Table 25.

The biological activities of compound 26 against fungi (minimum inhibitory concentration (MIC), μg/mL).

Compound	A. niger	C. albicans
26	250	250
Ketoconazole	31.25	31.25

Compound 9 (Figure 4) was subjected to the same tests as its analog 26 above [9]. The determined activities are reported in Table 26. This compound exhibited moderate activity against A. niger and low activity against C. albicans.

Table 26.

The biological activities of compound 9 against fungi (minimum inhibitory concentration (MIC), μg/mL).

Compound	A. niger	C. albicans
9	62.5	125
Ketoconazole	31.25	31.25

When compounds 27–32 (Figure 12) were tested in vitro for antifungal activity against four fungal species, A. fumigatus, S. racemosum, Geotricum candidum, and C. albicans, using Amphotericin B as standard antifungal agent, they showed no antifungal activity at all [17].

Compounds 33–47 (Figure 13), when tested under the same conditions, also showed no antifungal activity at all [18].

8. Antiviral Activity

9-[3-(4-Methoxyphenyl)oxiran-2-yl]-6b,7,11,11a-tetrahydro-5H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[4,3-c]quinolin-6(6aH)-one 52 (Figure 16) was tested in vitro for antiviral activity against infectious bursal disease virus (IBDV) in specific pathogen-free (SPF) chicken embryos, with un-inoculated SPF eggs as control of embryo [21]. Ribavirin was used as reference drug. The results are shown in Table 27. Compound 52 displayed weak activity against IBDV.

Figure 16.

Chemical structure of compound 52.

Table 27.

The in vitro antiviral activity of compound 52.

Compound	CC50 a Μl/Egg	IC50	Therapeutic Index b	Titer of the Virus c	S/P Ratio d
IBDV only	–	–	–	107 log2 EID50	–
52	>500	≤9	55.5%	106 log2 EID50	0.34
Ribavirin	>300	≤7	42.8%	102 log2 EID50	–
Negative control	–	–	–	0 log2 EID50	0.01
Vaccinated group only	–	–	–	–	0.34

^a Cytotoxicity concentration of fifty. ^b TI = CC₅₀/IC₅₀. ^c EID₅₀ (egg infective dose fifty) with a mixture of IBDV with each compound virus titer. ^d S/P ratio: sample/positive ratio.

9. Antiprotozoal Activity

3-Cyclopropyl-6-[4-methoxy-3-(pyridin-3-yl)phenyl]-7,7-dimethyl-7H-pyrazolo[5,1-c][1,2,4]triazole 53 (Figure 17) was tested for antiprotozoal activity (phenotypic activity against intracellular amastigotes) against the causative agent of Chagas disease, Trypanosoma cruzi [22]. The results are shown in Table 28. The pIC₅₀ value of 53 indicates a moderate activity compared with that of the lead compound, NPD-0227 (2-isopropyl-5-[4-methoxy-3-(pyridin-3-yl)phenyl]-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one) [23] (6.4).

Figure 17.

Chemical structure of compound 53.

Table 28.

Phenotypic activity of compound 53 against intracellular amastigotes of T. cruzi (Tulahuen strain) and MRC-5 cells.

Compound	pIC50 a		SI b
	T. cruzi	MRC-5
53	4.8	<4.2	>4

^a All reported values are within a standard deviation of ±0.2 and the result of at least n = 2. ^b The selectivity index is calculated by dividing the cytotoxicity (IC₅₀) by the T. cruzi activity (IC₅₀).

10. Anticancer Activity

3-(Pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazoles 54–59, 1-[6-methyl-3-(pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl]ethan-1-one 54, its hydrazinium salt 55, 1,2-bis{1-[6-methyl-3-(pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl]ethylidene}hydrazine 56, 7-{1-[2-(4-chlorophenyl)hydrazinylidene]ethyl}-6-methyl-3-(pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazole 57, 1-[6-methyl-3-(pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl]ethan-1-one oxime 58, and 1-[6-methyl-3-(pyridin-4-yl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl]ethan-1-one O-thiophene-2-carbonyl oxime 59 (Figure 18) were tested in vitro for cytotoxicity against Ehrlich–Lettre ascites carcinoma (EAC) tumor cells, with Doxorubicin as reference [24]. The results are displayed in Table 29. Compounds 56 and 57 proved to be active toward the used tumor cells; compounds 54, 55, and 58 showed moderate activities, while compound 59 showed no activity, possibly because of its low solubility in the culture medium.

Figure 18.

Chemical structures of compounds 54–59.

Table 29.

The in vitro cytotoxic activity of compounds 54–59.

Compound	Nonviable Cells (%) Concentration (μg/mL)
	100	50	25
54	60%	55%	45%
55	60%	50%	35%
56	70%	60%	45%
57	90%	85%	80%
58	50%	35%	10%
59	NA a	NA	NA
Doxorubicin	100%	55%	20%

^a NA—no activity.

The activity of compounds 56 and 57 against a liver carcinoma cell line (Hep G2) was also examined, again with Doxorubicin as reference [24]. The results are shown in Table 30. The low IC₅₀ value of compound 57 is an indicator of its high inhibitory activity against Hep G2.

Table 30.

The in vitro anti-Hep G2 activity results of compounds 56 and 57.

Compound	IC50, μg/mL
56	60.27
57	8.12
Doxorubicin	43.60

The in vitro antitumor activity for five fluoroquinolones 60–64 (Figure 19), 3-[6-(3,4-dihydroxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]-6-fluoro-1-methyl-7-(piperazin-1-yl)quinolin-4(1H)-one 60, 1-cyclopropyl-3-[6-(3,4-dihydroxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one 61, 1-cyclopropyl-3-[6-(3,4-dihydroxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]-7-(4-ethylpiperazin-1-yl)-6-fluoroquinolin-4(1H)-one 62, racemic 6-[6-(3,4-dihydroxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolin-7-one 63, and its S enantiomer 64 against L1210 (murine leukemia) and CHO (Chinese hamster ovary) cell lines was evaluated via their respective IC₅₀ values [25]. The results are summarized in Table 31. While the five parent fluoroquinolone antibiotics (norfloxacin, ciprofloxacin, enrofloxacin, ofloxacin, and levofloxacin) had poor inhibitory activities against these cancer line cells (IC₅₀ > 150 μmol/L), compounds 60–64 showed IC₅₀ values < 10 μmol/L, with compounds 61 and 64 being the most active.

Figure 19.

Chemical structures of compounds 60–64. 60: R = Et, R¹ = H; 61, 62: R = cyclopropyl; 61: R¹ = H; 62: R¹ = Et.

Table 31.

The in vitro antitumor activity of compounds 60–64 (IC₅₀, μmol/L).

Compound	L1210	CHO
60	<10	<10
61	0.14	2.2
62	<10	<10
63	<10	<10
64	1.2	3.5

In another study, another five fluoroquinolones 65–69 (Figure 20), S-{1-acetyl-3-[7-(4-acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl]-6-phenyl-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl} ethanethioate 65, S-{1-acetyl-3-[7-(4-acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl]-6-(4-methoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl} ethanethioate 66, S-{1-acetyl-3-[7-(4-acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl]-6-(4-methylphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl} ethanethioate 67, S-{1-acetyl-3-[7-(C)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl]-6-(4-chlorophenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl} ethanethioate 68, and S-[1-acetyl-3-[7-(4-acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl]-6-(4-nitrophenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-7-yl} ethanethioate 69 were tested in vitro for antitumor activity against L1210 (murine leukemia), HL-60 (human leukemia) and CHO (Chinese hamster ovary) cell lines, with Ciprofloxacin as standard [26]. The results are given in Table 32. All these compounds showed IC₅₀ values in the micromolar range, with compound 69 being the most active against all three cell lines, while Ciprofloxacin showed much weaker activity. The structure–activity relationships from these two studies [25,26] show that by substituting the 3-carboxyl group in fluoroquinolones with a fused heterobicycle is conducive to antitumor activity, and that the pyrazolo[5,1-c][1,2,4]triazoles investigated have greater antitumor activity than their triazolo[3,4-b][1,3,4]thiadiazine precursors and, in the case of compounds 65–69, the latter’s 4-acetylpiperazinyl analogs.

Figure 20.

Chemical structures of compounds 65–69. 65: R = H; 66: R = OMe; 67: R = Me; 68: R = Cl; 69: R = NO₂.

Table 32.

The in vitro antitumor activity of compounds 65–69 (IC₅₀, μmol/L).

Compound	L1210	HL-60	CHO
65	7.3	5.3	7.2
66	15.4	12.7	6.7
67	8.2	5.2	5.0
68	4.7	2.8	4.2
69	2.6	1.4	1.0
Ciprofloxacin	>150	>150	>150

N-(6-Amino-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl)benzamide 70 (Figure 21) was tested in vitro for antitumor activity against a panel of four human tumor cell lines: hepatocellular carcinoma Hep G2, lung fibroblasts WI 38, kidney of a normal adult African green monkey VERO, and breast cancer MCF-7 [27]. 5-Fluorouracil was used as reference. The results are reported in Table 33. As can be observed, compound 70 showed weak inhibitory activity against all four cell lines.

Figure 21.

Chemical structure of compound 70.

Table 33.

The in vitro antitumor activity of compound 70 (IC₅₀, μmol/L).

Compound	Hep G2	WI 38	VERO	MCF-7
70	65.6	65.0	69.8	66.8
5-Fluorouracil	8.6	3.2	6.5	2.3

Pyrazolo[5,1-c][1,2,4]triazoles 27–32 (Figure 12) and 71–73 (Figure 22), 3-acetyl-1-(4-chlorophenyl)-7-[(4-chlorophenyl)diazenyl]-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 71, 3-acetyl-1-(4-chlorophenyl)-7-[(4-methylphenyl)diazenyl]-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 72, and 3-acetyl-1-(4-methoxyphenyl)-7-[(4-methylphenyl)diazenyl]-1H-pyrazolo[5,1-c][1,2,4]triazol-6(5H)-one 73 were tested in vitro for antitumor activity against hepatocellular carcinoma Hep G2 and colon cancer HCT116 cell lines, with 5-fluorouracil, Doxorubicin and Imatinib as standard drugs [17]. The results are summarized in Table 34. Some of the tested compounds showed good activity against both cancer cell lines: the most active compounds against Hep G2 hepatocellular carcinoma cells were compounds 27, 29, and 72, while compounds 28, 71, and 72 were the most active against colon cancer HCT116 cells.

Figure 22.

Chemical structures of compounds 71–73. 71: R = R¹ = Cl; 72, 73: R = Me; 72: R¹ = Cl; 73: R¹ = OMe.

Table 34.

The in vitro antitumor activity of compounds 27–32 and 71–73 (IC₅₀, μmol/L).

Compound	Hep G2	HCT116
27	19.4	38.4
28	41.8	21.3
29	21.6	39.3
30	43.9	37.5
31	80.9	65.1
32	27.6	24.3
71	23.3	21.4
72	12.4	20.9
73	94.9	82.8
Doxorubicin	0.46	0.42
Imatinib	9.7	18.9
5-Flourouracil	1.2	3.5

Compounds 74–78 (Figure 23), 6-(4-methylphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole 74, 6-(4-chlorophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole 75, 6-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole 76, 6-(4-methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole 77, and 2-methoxy-5-[3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-6-yl]aniline 78 were tested in vitro for antitumor activity against human gastric adenocarcinoma SGC-7901, human oral epithelial cancer KB, and human fibrosarcoma HT1080 cell lines, with Combretastatin A-4 and Doxorubicin as standard drugs [28]. The results are presented in Table 35. All the tested compounds showed good inhibition of all three cancer cell lines studied, with compounds 75 and 78 being the most active.

Figure 23.

Chemical structures of compounds 74–78. 74: R = 4-Me; 75: R = 4-Cl; 76: R = 4-OMe; 77: R = 3-NO₂-4-OMe-; 78: R = 3-NH₂-4-OMe.

Table 35.

The in vitro antitumor activity of compounds 74–78.

Compound	Inhibition Rate, (%, 10 μg/mL)
	SGC-7901	KB	HT1080
74	90.3	89.9	90.6
75	95.9	96.3	95.5
76	90.5	91.8	88.9
77	88.7	89.5	90.3
78	96.0	97.5	96.5
Combretastatin A-4	75.0	74.4	76.4
Doxorubicin	87.6	85.3	88.4

The acute toxicity of compounds 75 and 76 (Figure 23) was determined in vivo in mice at a dose of 500 mg/kg [28]. The compounds were administered by intraperitoneal injection. Since all the mice survived and returned to normal after the administration of this compound stopped, the LD₅₀ value for intraperitoneal administration was considered greater than 500 mg/kg.

The most active compounds in vitro, 75 and 78, were also investigated in vivo on S-180 sarcoma model mice, using 5-fluorouracil as reference [28]. The results are presented in Table 36.

Table 36.

The in vivo antitumor activity of compounds 75 and 78.

Compound	Dose mg/kg	Administration Method	Number of Animals		Body Weight g		Tumor Weight x ± SD	Inhibition Rate %
			Initial	Final	Initial	Final
none	–	–	10	10	20.2	26.9	1.80 ± 0.45	–
75	10	i.p.	10	10	20.1	27.2	0.65 ± 0.19	63.9
75	5	i.p.	10	10	20.3	26.5	0.62 ± 0.23	65.6
78	10	i.p.	10	10	20.5	26.5	0.69 ± 0.25	61.7
78	5	i.p.	10	10	20.1	27.0	0.76 ± 0.28	57.8
5-Fluorouracil	50	i.v.	10	10	20.7	23.9	0.57 ± 0.22	68.3

The same compounds 74–78 were later tested again in vitro for antiproliferative activity against human gastric adenocarcinoma SGC-7901, human lung adenocarcinoma A549, and human fibrosarcoma HT1080 cell lines [29], with two compounds with potent antiproliferative activity, SMART [30] [(2-phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone] and ABI [31] [(2-phenyl-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone] (Figure 24) as positive controls. The results are displayed in Table 37. It can be observed that all the pyrazolo[5,1-c][1,2,4]triazoles tested showed modest antiproliferative activity, especially in comparison with SMART. A number of 1H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole analogs were also tested in this study, and several of these compounds showed potent antiproliferative activity at sub-micromolar or nanomolar concentrations against the three different cancer cell lines; one derivative in particular, the analog of 78, showed activities close to those of SMART (0.022 ± 0.006, 0.029 ± 0.011, 0.027 ± 0.013, respectively).

Figure 24.

Chemical structures of compounds SMART and ABI.

Table 37.

The in vitro antiproliferative activity of compounds 74–78.

Compound	IC50 ± SD, μM
	SGC-7901	A549	HT1080
74	33.5 ± 1.9	29.3 ± 1.2	51.3 ± 2.6
75	6.12 ± 0.13	1.21 ± 0.09	6.81 ± 0.21
76	26.8 ± 1.1	26.4 ± 2.0	26.5 ± 2.2
77	48.3 ± 1.5	58.2 ± 2.9	35.6 ± 3.8
78	13.8 ± 1.6	17.4 ± 2.0	17.8 ± 1.5
SMART	0.019 ± 0.008	0.029 ± 0.009	0.028 ± 0.011
ABI	0.81 ± 0.08	0.98 ± 0.11	0.15 ± 0.05

Compounds 33–47 (Figure 13) were tested in vitro for antitumor activity against Hep G2 and HCT116 cell lines, using 5-fluorouracil, Doxorubicin, and Imatinib as standard drugs [18]. The results are shown in Table 38. These compounds showed weak to moderate activity against both cell lines, the most active being compounds 43 and 47.

Table 38.

The in vitro antitumor activity of compounds 33–47.

Compound	IC50, μg/mL
	Hep G2	HCT116
33	29	39.7
34	>100	>100
35	22.2	23.3
36	72.5	94.6
37	45.5	88.6
38	38.8	40.1
39	48.1	83.1
40	22.7	35.2
41	31.3	59.5
42	>100	>100
43	11.3	12.5
44	23.6	38.9
45	77.4	>100
46	46.3	96.5
47	11.9	18.9
Doxorubicin	0.42	0.46
Imatinib	18.9	9.7
5-Flourouracil	4.6	4.3

1H-[1,2,4]Triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridines 79–83: (7,9-dimethyl-1H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridin-3-yl)(4-fluorophenyl)methanone 79, (4-chlorophenyl)(7,9-dimethyl-1H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridin-3-yl)methanone 80, (5-bromothiophen-2-yl)(7,9-dimethyl-1H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridin-3-yl)methanone 81, 3-(7,9-dimethyl-1H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridine-3-carbonyl)-2H-chromen-2-one 82, and 1-(7,9-dimethyl-1H-[1,2,4]triazolo[4′,3′:1,5]pyrazolo[3,4-b]pyridin-3-yl)ethan-1-one 83 (Figure 25) were tested in vitro for antitumor activity against HCT116, Hep G2, HeLa (cervical cancer) and MCF-7 cell lines, with Doxorubicin as reference [32]. The results are listed in Table 39. Compound 82 exhibited very good activity against all cell lines, while all the other compounds demonstrated weak activity in all cases. The remarkable activity of compound 82 could be attributed to the presence of the coumarin fragment in its molecule.

Figure 25.

Chemical structures of compounds 79–83. 79: R = F; 80: R = Cl.

Table 39.

The in vitro antitumor activity of compounds 79–83.

Compound	IC50 ± SD, μM
	HCT116	Hep G2	HeLa	MCF-7
79	45.51 ± 2.80	38.53 ± 2.62	64.90 ± 3.36	34.33 ± 2.32
80	82.68 ± 4.21	72.26 ± 4.00	91.45 ± 4.63	65.27 ± 3.71
81	74.28 ± 3.84	68.67 ± 3.74	85.90 ± 4.20	57.26 ± 3.30
82	7.71 ± 0.62	10.84 ± 0.90	13.11 ± 1.01	9.29 ± 0.73
83	92.72 ± 4.70	84.30 ± 4.32	>100	52.51 ± 2.92
Doxorubicin	5.23 ± 0.33	4.50 ± 0.20	5.57 ± 0.46	4.17 ± 0.20

Later, in a study on the discovery of 1,2,4-triazole-based inhibitors of aromatase (CYP19A1), for the treatment of hormone receptor (HR)-positive breast cancer, compounds 79–82 were among the 78 compounds used to generate the pharmacophore model [33]. Compounds 80 and 81 were included in the training set (39 compounds), and compounds 79 and 82 in the test set (39 compounds). In the end, this study led to two 1,2,4-triazole-based structures with better estimated activity and fit value than the standard drug Letrozole.

N-(4-{[(6-Chloropyridazin-3-yl)amino]sulfonyl}phenyl)-N′-(3-sulfanyl-7H-pyrazolo[5,1-c][1,2,4]triazol-6-yl)urea 84 (Figure 26) was subjected to an in vitro cytotoxicity screening against 21 cancer cell lines, representing eight subpanels: leukemia (CCRF-CEM and SR), non-small-cell lung (EKVX, HOP-62, HOP-92 and NCI-H522), central nervous system (SF-268 and SNB-75), melanoma (UACC-62), ovarian (IGROV1 and SK-OV-3), renal (A498, CAK-1 and UO-31), prostate (PC-3), and breast (MCF7, MDA-MB231/ATCC, HS 578T, BT-549, T-47D, and MDA-MB-468), at a single dose of 10⁻⁵ M (10 μM) [34]. The results were reported as the % growth inhibition (GI) against the cell lines. With the exception of the leukemia SR cell line, against which compound 84 displayed minimal activity (GI = 10%), and renal cancer UO-31 cell line (GI = 14%), no inhibitory activities of this compound were observed (GI < 10%).

Figure 26.

Chemical structure of compound 84.

11. C3a Receptor Binding Activity

6-(3′,4′-Dimethyl-[1,1′-biphenyl]-4-yl)-3-methyl-1H-pyrazolo[5,1-c][1,2,4]triazole 85 (Figure 27) showed good C3a receptor binding activity, with an IC₅₀ value of 71 nM [35].

Figure 27.

Chemical structure of compound 85.

12. Miscellaneous Biological Activities

An in silico screen of 1.1 million compounds was performed, using three-dimensional E47-Id1 interaction mapping to identify small molecules that could potentially inhibit the E47-Id1 interaction [36]. Ethyl 6-methyl-3-(5-nitrothiophen-2-yl)-1H-pyrazolo[5,1-c][1,2,4]triazole-7-carboxylate 86 (Figure 28) was among the 364 structures identified by this screen, but not among the compounds showing the most pronounced anti-Id activity in the gel shift assay.

Figure 28.

Chemical structure of compound 86.

13. Cytotoxicity

2-{1-[6-(1,1-Dimethylethyl)-1H-pyrazolo[5,1-c][1,2,4]triazol-3-yl]ethyl}-1H-isoindole-1,3(2H)-dione hydromethanesulfonate (mesylate) 87 (Figure 29) was tested for toxicity in rats by oral administration [37]. In contrast to the corresponding hydrochloride, which caused strong skin irritation, salt 87 had an acute toxicity of 2000 mg/kg or more and caused no skin irritation, mutagenicity, or rash.

Figure 29.

Chemical structure of compound 87.

14. Ulcerogenic Activity

Compound 7 (Figure 3) was also tested for ulcerogenic activity in rats at dose levels of 10, 50, and 100 mg/kg body mass [8]. Indomethacin was used as reference drug. The compounds were administered per os. The results are shown in Table 40. It can be observed that compound 8 possesses no ulcerogenic activity.

Table 40.

The ulcerogenic activity of compound 7 in rats ^a.

Compound	Dose (mg/kg)
	10	50	100
Control	0/6	0/6	0/6
7	0/6(0)	0/6(0)	0/6(0)
Indomethacin	3/6(1.5 ± 0.2) b,c	5/6(1.9 ± 0.2) b,c	6/6(2.2 ± 0.2) b,c

^a Number of rat lesions bigger than 0.5 mm in length per total number of rats. ^b Mean ulcer lesions ± SEM (mm) (n = 6) in parentheses. ^c Significant difference at p < 0.05 compared with the control.

15. Conclusions

A number of studies on the biological activities of various pyrazolo[5,1-c][1,2,4]triazoles have been conducted and published, including several patents. Although the number of pyrazolo[5,1-c][1,2,4]triazoles studied as drug candidates so far is not very great (<100) compared with other scaffolds, this moiety is a promising motif for future drug discovery and development projects. Significantly, the pyrazolo[5,1-c][1,2,4]triazoles investigated so far have shown little or no toxicity. One obvious direction is that of synthetic nucleoside analogs, with the pyrazolo[5,1-c][1,2,4]triazole scaffold as a good isostere for purine. There are many methods developed for the synthesis and further functionalization of pyrazolo[5,1-c][1,2,4]triazoles, enabling the modification of existing structures for the generation of libraries of compounds and selection of the best drug candidates.

Author Contributions

Conceptualization, B.-N.P.; validation, V.-G.P., V.B. and F.P.; resources, B.-N.P.; data curation, B.-N.P.; writing—original draft preparation, B.-N.P.; writing—review and editing, B.-N.P. and F.P.; visualization, B.-N.P.; supervision, V.B. and F.P.; project administration, F.P.; funding acquisition, V.B. and F.P. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.