Rational Design, Synthesis, Characterization, and Anti-Bacterial Activity of Urea Derivatives Bearing 1,2,4-Triazoles as Molecular Hybrid Scaffolds

Introduction

Despite the rising demand for medicinally potent molecules for drug development, molecular hybridization techniques have rapidly arisen and strengthened over the years. A molecular hybridization is a technique in which two separate active pharmacophores are combined with or without the assistance of a linker to form a new hybrid scaffold.^1,2 The novel hybrid scaffold is expected to have higher effectiveness and selectivity profiles than the parent medications, and it may help reduce the possibility of multiple drug resistance.³ As a result of the ever-increasing need for medicinal molecules, heterocyclic compounds have long been an intriguing subject. Because of their moderate dipole character, stiffness, and durability in vivo, hydrogen bonding capabilities, the triazoles (1H-1,2,3-triazole, 1H-1,2,4-triazole) are good surrogates in bioactive compounds with a wide range of pharmacological activities.^4-6 These motifs have potential uses in HIV, anticancer, anti-protozoal, antibacterial, anti-proliferative, ß-lactamase inhibitory, anti-inflammatory, agrochemical, and material research. Some of the most potent antifungal compounds are those containing a triazole nucleus, such as itraconazole, fluconazole, and the new generation of triazoles posaconazole, voriconazole, and ravuconazole.^7-11 Among the triazoles, 1,2,4-triazole has been shown to have excellent pharmacological properties. For example, 1H-1,2,4-triazole carrying compounds are being explored as 5-lipoxygenase inhibitors, antiepileptics, anticancer drugs, and Pt (II) complexes containing 1H-1,2,4-triazoles exhibit activity like cis-platin.^12-14 Aside from pharmaceutical uses, various 1,2,4-triazole-based agro-fungicides with 1H-1,2,4-triazole as a core component are also available, such as Triadimefon, Triadimenol, Diniconazole, Flusilazole, and Difenoconazole. ^15-17 (Fig. 1).

Figure 1: Structure of bioactive compounds containing triazole moiety. Click here to View figure

Urea derivatives (R₂N(CO)NR₂), on the other hand, are used as solvents, medications, and fertilisers.^18,19 Some urea derivatives are antituberculosis, antibacterial, and anticonvulsant agents.^20-24 Given the biological relevance of these two pharmacophoric units, 1,2,4 triazole and urea, it was intriguing to create a new class of heterocyclics by molecular hybridization, in which the powerful 1,2,4-triazole moiety relates to urea derivatives. Since Bladin described the first synthetic mehtod of 1H-1,2,4-triazole in 1885,²⁵ many protocols to improve yield have been investigated, including the reaction of formyl-hydrazine with formamide (K. T. Potts, 1961),²⁶ condensation of hydrazine sulphate with formamide (M. Sekiya and S. Ishikawa, 1958),^14,27 and the reaction of N, N-Diformylhydrazine with excess ammonia (C. Ainsworth and R. G. Jones, 1955).²⁸ Most of the synthetic techniques based on this procedure have low yield formation. As a result, considerable attempts have been made in recent years to enhance the yield of 1,2,4-triazoles. Thus, in concern, production of 1,2,4-triazoles with high yield, followed by hybridization of the 1,2,4-triazoles with urea moiety to develop important pharmacophores. We began by preparing carbamates of 4-amino-1,2,4-triazoles using a multistep technique, and then we performed reactions of created carbamates of 4-amino-1,2,4-triazoles (1a-g) with amines. We also tested the hybrid scaffolds for antibacterial activity against Enterococcus feacalis, Pseudomonas, E. coli, Klebsiella pneumonia, Candida albicans, Bacillus anthracis, Proteus mirabilis, and Staphylococcus aureus.

Experimental Section 

Without additional purification, commercially available reagents were utilized. Column chromatography was used to manufacture and purify carbamates. Freshly dried and distilled solvents TLC was used to monitor all the reactions on pre-coated silica gel plates. Before usage, silica gel (60-80 Mesh) and anhydrous sodium sulphate were activated in a muffle furnace. The melting points were calculated using a Tempo device and results have not been modified. Bruker-DPX-300 (300 MHz) spectrometer was used to record NMR spectra. In nuclear magnetic resonance, the operational frequency of ¹H NMR is 300 MHz, whereas ¹³C NMR at 100 MHz frequency (using TMS as the internal reference). The FLASH Ea 1112 series CHN analyzer was utilized to carry out the elemental analysis procedures for C, H, and N. The Water’s Xevo G2-S Q Tof mass spectrometer was used to acquire the mass spectra.

Generalized synthesis procedure and characterization of urea derivatives (3a-3i and 5a-c):

Carbamate derivatives (1.0 equiv) were dissolved in DMF (20 vol. eq.) in a round bottom flask of 100 mL. This solution was treated with triethyl amine (2.5 eq.) and amine (1.5 eq.) before being stirred 60°C for 15 hours. TLC (thin layer chromatography) was used to track the development of the reaction (solvent: n-hexane: EtOAc: 30:70 v/v%). Upon reaction finalization, 45 mL of distilled water was used to “quench” the reaction kinetics. Following three separate 50 mL ethyl acetate extractions, the product extracted with 100 mL of brine. Thus far the received organic layer was blended and dried, over anhydrous Na₂SO₄. An off-white solid was obtained after under low pressure solvent evaporation and the residue was flushed through silica gel (200-300 mesh) column for purification using ethyl acetate/n-hexane as the eluent.

3a derivative

Off-white solid, Yield: 32.0 %; m.p. 170-174°C; ¹H NMR: δ 10.11 (s, 1H), 9.76 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.86 (dd, J = 6.8, 2.9 Hz, 4H), 7.63 – 7.45 (m, 7H), 7.31 (q, J = 8.7 Hz, 4H) ppm; m/z calcd: 389.20. Found: 390.8, 392.6 (3:1) (M⁺¹).

3b derivative

Off-white solid, Yield: 44.6 %; m.p. 162-164°C; ¹H NMR : δ 9.93 (s, 1H), 7.88 – 7.79 (m, 4H), 7.56 (q, J = 3.0 Hz, 6H), 7.29 (d, J = 2.6 Hz, 1H), 2.40 (s, 1H), 0.61 (s, 2H), 0.31 (s, 2H) ppm; m/z calcd: 319.14. Found: 320.4 (M⁺¹).

3c derivative

Off-white solid, Yield: 33.6 %; m.p. 172-174°C; ¹H NMR : δ 10.15 (s, 1H), 9.81 (s, 1H), 8.03 (s, 1H), 7.87 (s, 2H), 7.80 – 7.46 (m, 5H), 7.46 – 7.18 (m, 5H), 6.31 (d, J = 15.9 Hz, 1H) ppm; m/z calcd: 407.09. Found: 407.9, 409.2 (3:1) (M⁺¹).

3d derivative

Off-white solid, m.p. 156-160°C; Yield: 51.8 %; ¹H NMR : δ 9.99 (s, 1H), 7.83 (dd, J = 6.9, 3.1 Hz, 2H), 7.75 – 7.52 (m, 6H), 7.48 – 7.35 (m, 2H), 2.39 (d, J = 9.3 Hz, 1H), 0.61 (s, 2H), 0.31 (s, 2H) ppm; m/z calcd: 337.13. Found: 338.6 (M⁺¹).

3e derivative

Off-white solid, m.p. 168-170°C; Yield: 33.7 %; ¹H NMR : δ 10.07 (s, 1H), 9.76 (s, 1H), 7.86 (dd, J = 7.0, 2.8 Hz, 2H), 7.62 – 7.49 (m, 5H), 7.31 – 7.03 (m, 6H), 2.32 (s, 6H) ppm; m/z calcd: 417.14. Found: 418.0, 420.2 (3:1) (M⁺¹).

3f derivative

Off-white solid, m.p. 181-183°C; Yield: 52.3 %; ¹H NMR : δ 9.92 (s, 1H), 7.88 – 7.78 (m, 2H), 7.59 – 7.51 (m, 3H), 7.44 (s, 2H), 7.28 (s, 1H), 7.18 (s, 1H), 2.41 (s, 1H), 2.35 (s, 6H), 0.63 (s, 2H), 0.32 (s, 2H) ppm; m/z calcd: 347.17. Found: 348.1(M⁺¹).

3g derivative

Off-white solid, m.p. 167-169°C; Yield: 47.2 %;  ¹H NMR : δ 10.14 (br, 1H), 9.84 (br, 1H), 7.82 – 7.14 (m, 11H), 2.47 (s, 3H), 2.31 (s, 3H) ppm; m/z calcd: 435.13. Found: 436.1, 437.3 (3:1) (M⁺¹).

3h derivative

Off-white solid, m.p. 174-178°C; Yield: 53.6 %; ¹H NMR : δ 9.99 (s, 1H), 7.82-7.14 (m, 7H), 2.50 (s, 6H), 1.24 (s, 1H), 0.45 (dd, J = 177.7, 77.9 Hz, 4H) ppm; m/z calcd: 365.17. Found: 366.3 (M⁺¹).

3i derivative

Off-white solid, m.p. 169-171°C; Yield: 56.1 %; ¹H NMR: δ 9.87 (s, 1H), 7.71-7.39 (m, 4H), 7.24-7.06 (m, 2H), 6.75 (m, 1H), 2.36 (s, 6H), 1.24 (m, 1H), 0.56 (m, 2H), 0.27 (m, 2H) ppm; m/z calcd: 381.14. Found: 382.4, 384.1 (3:1), (M⁺¹).

5a derivative

Off-white solid, m.p. 190-194°C; Yield: 59.5 %; ¹H NMR : δ 9.99 (s, 1H), 9.81 (s, 1H), 7.79 (s, 2H), 7.66 – 7.24 (m, 7H), 2.65 (s, 3H). ppm; m/z calcd: 359.83. Found: 360.0, 362.6 (3:1) (M⁺¹).

5b derivative

Off-white solid, m.p. 184-187°C; Yield: 50.4 %; ¹H NMR : δ 9.97 (s, 1H), 9.50 (s, 1H), 7.43 (d, J = 62.3 Hz, 8H), 2.66 (s, 3H)  ppm; m/z calcd: 394.28. Found: 395.6, 397.4 (3:1) (M⁺¹).

5c derivative

Off-white solid, m.p. 176-180°C; Yield: 49.0 %; ¹H NMR : δ 9.97 (s, 1H), 9.61 (s, 1H), 7.75 – 7.43 (m, 2H), 7.32 (s, 1H), 7.14 (s, 1H), 6.09 (s, 1H), 2.42 (s, 3H), 1.24 (m, 1H), 0.58 (m, 2H), 0.29 (m, 2H) ppm; m/z calcd: 323.80. Found: 324.8, 326.3 (3:1) (M⁺¹).

Results and Discussion

As a result, Scheme 1 shows the final synthesis pathway for the 4H-1,2,4-triazol-4-amine (1a-g). In the first phase, ethyl benzoate was synthesized by reacting a benzoic acid solution in ethanol with sulphuric acid. The produced ethyl benzoate was then treated with hydrazine hydrate in the second step, resulting in the synthesis of benzohydrazide. Following that, in the third stage, N-benzoyl benzohydrazides were synthesized by reacting benzohydrazides with benzoic acid. Following the synthesis, N-benzoyl benzohydrazides were treated with thionyl chloride to produce 2,5-diphenyl-1,3,4-oxadiazole. Following the reaction of 2,5-diphenyl-1,3,4-oxadiazoles with hydrazine hydrate, 3,5-diphenyl-4H-1,2,4-triazole-4-amines are formed. In the last phase, phenyl (3,5-diphenyl-4H-1,2,4-triazole-4-yl) carbamates are made by reacting 3,5-diphenyl-4H-1,2,4-triazole-4-amines with phenyl formate under reflux conditions in the presence of THF and triethylamine.

Scheme 1: Synthetic pathway for the 4-amino-4H-1,2,4-triazoles (1a-g). Click here to View  scheme

The interaction of the generated 4H-1,2,4-triazol-4-amine (1a-g) with amines (2a, b) in DMF using trimethyl amine at about 60°C for 15 hours yielded urea derivatives (3a-i) in moderate yield (30 to 60%). (Scheme 2, Table 1). All the products are off-white solids that have been identified using spectral and elemental analysis.

Scheme 2: Synthesis of urea derivatives from 4-amino-1,2,4-triazoles based Carbamates Click here to View scheme

Table 1: Synthesis of urea derivatives from 4-amino-1,2,4-triazoles based Carbamates (Scheme 1).

Entry	Compound	R	R1	R3	Yield(%)
1	3a	C6H5	C6H5	4-CI-C6H4	32.0%
2	3b	C6H5	C6H5	c-propyl	44.6%
3	3c	3-F- C6H4	C6H5	4-CI-C6H4	33.6%
4	3d	3-F- C6H4	C6H5	c-propyl	51.8%
5	3e	C6H5	3,5-(Me)2-C6H3	4-CI-C6H4	33.7%
6	3f	C6H5	3,5-(Me)2-C6H3	c-propyl	52.3%
7	3g	3-F- C6H4	3,5-(Me)2-C6H3	4-CI-C6H4	47.2%
8	3h	3-F- C6H4	3,5-(Me)2-C6H3	c-propyl	53.6%
9	3i	2-CI- C6H4	3,5-(Me)2-C6H3	c-propyl	56.1%

 

Based on the previous results, the interaction of methylthio substituted 4H-1,2,4-triazol-4-amine-based carbamate compounds (4a-c) with 2a, b was also explored under identical reaction conditions (Scheme 3, Table 2). All the products are off-white solids that have been characterized using spectral and elemental analysis.

Scheme 3: Synthesis of urea derivatives from 3-(methylthio)-4-amino-1,2,4-triazole based Carbamates. Click here to View scheme

Table 2: Synthesis of urea derivatives from 3-(methylthio)-4-amino-1,2,4-triazole based Carbamates. (Scheme 3).

Entry	Compound	R	R3	Yieid (%)
1	5a	C6H5	4-CI- C6H5	59.5%
2	5b	2-CI- C6H5	4-CI- C6H5	50.4%
3	5c	2-CI- C6H5	c-propyl	49.0%

 

Optimization

However, as the penultimate stage in the synthesis of urea preparation via carbamates, we received a highly messy product with low yield, which we then optimized as tabulated in table 3. A reaction between 3,5-diphenyl substituted 4H-1,2,3-triazole-4-amine and phenyl formate in the organic solvents (i.e., THF, Acetonitrile, DMF, and 1,4-dioxane) and bases (i.e., K₂CO₃, Cs₂CO₃, DIPEA, pyrimidine and triethylamine) yielded phenyl substitited carbamate. Using carbamate in a refluxing mixture of THF and triethylamine with phenyl formate, we obtained excellent results at a range of temperatures.

Table 3: Optimization of reaction media for the synthesis of target compounds

Entry	Solvents	Time (h)	Yields (%)
1	THF	26 h	59%
2	Acetonitrile	25 h	46%
3	1,4-dioxane	28 h	42%
4	DMF	15 h	50%

 

Note: Each solvent system explored with K₂CO₃, Cs₂CO₃, DIPEA, pyrimidine and triethylamine base, the best result obtained with the combination of THF solvent-triethylamine base

Biological Activity

Anti-bacterial activity

The anti-bacterial activities of the synthesised urea derivatives were evaluated against the following bacterial species: Enterococcus faecalis (EF) (ATCC 29212), Klebsiella pneumoniae (KP) (ATCC 700603), Candida albicans (CA) (MTCC 3017), Escherichia coli (EC) (ATCC 25922), Pseudomonas aeruginosa (PA) (ATCC 15442), Bacillus anthracis (BA) (ATCC 14578). According to the findings, the urea derivatives (3a-i and 5b) exhibited substantial anti-bacterial action in vivo (figure 2). Table 4 displays the results of anti-bacterial efficiencies testing in-vitro. 

Table 4: Anti-bacterial activities of carbamate extracts

Compound Name		EF	PA	EC	KP	CA	BA	PM	SAB
Standard	IZ	21, 30, 31 Mean =61.3	34,34,45 Mean =83	23,35,30 Mean=70	33,16,34 Mean=60.3	31,15,31 Mean=56.3	38,34,40 Mean=85.3	21,26,40 Mean=60.3	32,36,40 Mean=81.3
3a	IZ	14mm	11mm	17mm	16mm	12mm	11mm	15mm	8mm
	AI	0.2	0.1	0.2	0.2	0.2	0.1	0.2	0.09
3b	IZ	10mm	11mm	12mm	10mm	10mm	10mm	12mm	11mm
	AI	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
3c	IZ	12mm	15mm	9mm	17mm	16mm	11mm	12mm	10mm
	AI	0.1	0.1	0.1	0.2	0.2	0.1	0.1	0.1
3d	IZ	10mm	12mm	10mm	11mm	8mm	10mm	11mm	10mm
	AI	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
3e	IZ	9mm	10mm	11mm	6mm	7mm	8mm	10mm	16mm
	AI	0.1	0.1	0.1	0.09	0.1	0.09	0.1	0.1
3f	IZ	13mm	10mm	15mm	10mm	10mm	9mm	13mm	14mm
	AI	0.2	0.1	0.2	0.1	0.1	0.1	0.2	0.1
3g	IZ	13mm	7mm	10mm	10mm	14mm	12mm	11mm	10mm
	AI	0.2	0.08	0.1	0.1	0.2	0.1	0.1	0.1
3h	IZ	9mm	14mm	12mm	10mm	15mm	13mm	14mm	8mm
	AI	0.1	0.1	0.1	0.1	0.2	0.1	0.2	0.09
3i	IZ	11mm	14mm	13mm	10mm	13mm	10mm	12mm	11mm
	AI	0.1	0.1	0.1	0.1	0.2	0.1	0.1	0.1
5b	IZ	12mm	13mm	14mm	7mm	7mm	10mm	13mm	11mm
	AI	0.2	1.5	0.2	0.1	0.1	0.1	0.2	0.1

Figure 2: Zone of inhibition (mm) against 3(a-i) and 5b derivatives. Click here to View figure

Anti-bacterial activity

Synthesized carbamate derivatives were tested for antibacterial activity using the disc-diffusion technique against Enterococcus faecalis (EF) (ATCC 29212), Pseudomonas aeruginosa (PA) (ATCC 15442), Escherichia coli (EC) (ATCC 25922), Candida albicans (CA) (MTCC 3017), Klebsiella pneumoniae KP) (ATCC 700603), , Bacillus anthracis (BA) (ATCC 14578). By inoculating the bacterial cultures into nutritional agar, they were cultured for 24 hours at 30 0.1°C. Urea derivatives were kept dry at room temperature and solubilized in dimethyl sulfoxide (20 mg in per mL DMSO). Mueller The petri dishes were filled with Hinton Agar Media (15 cm³) and heated to 45 °C to harden the medium. Specifically, 50 µL of normal saline solution were poured onto 9 cm Petri dishes and incubated to grow the aforementioned culture medium (105-106 bacteria per mL). Carbamate (50 µL) injections were made into discs and firmly pushed onto the solid agar medium. For 24 hours, the Petri plates were kept at 37 °C. At the conclusion of the time, the inhibition zones created on the medium were measured in millimetres using a zone reader. Similarly, the standard disc zone (tetracycline) was computed.

Conclusion

A new series of molecular hybrids i.e., urea derivatives carrying 1,2,4-triazole molecular hybrid scaffolds were synthesized via reaction of carbamates of 4-amino-1,2,4-triazoles with amines in DMF for 15 hours using trimethyl amine. The approach was further expanded to produce urea derivatives of 3-(methylthio)-4-amino-1,2,4-triazoles. Antibacterial activity for all synthesized derivatives were evaluated against Enterococcus faecalis, Pseudomonas, E. Coli, Klebsiella pneumonia, Candida albicans, Bacillus anthracis, Proteus mirabilis, and Staphylococcus aureus. All the synthesized compounds found promising antibacterial activity in the test.

Acknowledgement

Thanks are due to MNIT, Jaipur (India) for instrumental facilities.

Conflict of Interest

There is no conflict of interest.

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