Synthesis and antimicrobial activity of 1H-1,2,3-triazole and carboxylate analogues of metronidazole

Introduction

Metronidazole (1) is an important antimicrobial agent which has been clinically used successfully for a long time. It was originally used for the treatment of infections caused by Trichomonas varginalis and later it was applied to treat various other infections [1]. For the last 45 years metronidazole (1) is in extensive use for the management of anaerobic infections. The compound possesses a broad spectrum of activity against various Gram-positive as well as Gram-negative organisms [2]. It is also a cost-effective drug. Due to its impressive antimicrobial activity and limited adverse effect metronidazole (1) has been considered as a “Gold Standard” antibiotic (Figure 1).

However, to avoid the problem related to clinical resistance to this antimicrobial agent some novel and improved analogues of this compounds are required. In this regard we suggested the modification of the alcohol tail of metronidazole by incorporating an N-heterocyclic moiety.

Nitrogen-containing heterocycles play a vital role in agrochemicals and pharmaceuticals [3]. Among these heterocyclic systems, the 1H-1,2,3-triazoles are very important in organic chemistry due to their broad spectrum of applications in biochemical, biomedicinal, pharmaceuticals, and materials sciences [4]. Their chemistry underwent a substantial growth over the past decades [5]. They are widely used in industrial applications such as photographic materials, dyes, agrochemicals, photostabilizers, and corrosion inhibitors (copper alloys) [6]. Incorporation of the 1H-1,2,3-triazole moiety is well known to impact on the physical, chemical and biological potential properties of organic molecules. Due to this reason, many efforts have been exerted to develop new synthetic methodologies toward the 1H-1,2,3-triazole group containing organic entities.

However, earlier methods of the synthesis of aliphatic and aromatic esters of metronidazole are associated with different drawbacks such as long conversion times, low yields and preparation of their respective acid chlorides by using thionyl chlorides and these acid chlorides were then made to react with the -OH functionality of metronidazole to get different esters [7]. Here we report a convenient method for the synthesis of aliphatic and aromatic esters of metronidazole.

Furthermore, derivatives of metronidazole scaffolds are known to have a large range of biological activities including tumorhypxia agents [8], antiprotozoal activity [9], antimicrobial [10], antitumour [11], carbonic anhydrase IX inhibitors [12], trichomonas vaginalis activity [13], antileishmanial agents [14] (Figure 2). We have recently synthesized several 1H-1,2,3-triazole-containing molecules with impressive biological activities [15].

In continuation of our research work on 1H-1,2,3-triazole derivatives [16], we have synthesized a series of new 1H-1,2,3-triazole and carboxylate derivatives of metronidazole (5a–i and 7a–e). The choice of 1H-1,2,3-triazole was based on its known activities and its broad range of applications in biochemical, pharmaceutical, biomedicinal and materials sciences [4,5].

Results and Discussion

Chemistry: synthesis of 1H-1,2,3-triazole analogues of metronidazole

Metronidazole (1) has a free primary hydroxy group. The first step was initiated by the protection of the primary hydroxy group of metronidazole (1) with p-toluenesulfonyl chloride in dry DCM in the presence of triethylamine at 0 °C to room temperature. The reaction afforded the desired metronidazole tosylate 2 in high yield (96%) [17]. In the next step, the metronidazole tosylate 2 under treatment with NaN₃ in DMF at 70 °C afforded the corresponding metronidazide 3 in 88% yield [18].

The ¹H NMR spectrum of metronidazide 3 showed a singlet at δ 7.93 for the 1H-imidazole proton. Two triplet signals at δ 4.40 and δ 3.74 were assigned to four methylene protons of –N–CH₂-CH₂–N₃. A singlet peak at δ 2.50 was due to methyl protons on the imidazole ring. The high-resolution mass spectrometric data at 197.0737 (M + H)⁺ confirmed the structure of metronidazide 3.

Single crystals of metroazide compound 3 were grown from slow evaporation of DCM solution. The structure of metronidazide 3 was unambiguously confirmed by single crystal X-ray analysis (Figure 3).

The next step was carried out by using “click” chemistry involving the 1,3-dipolar cycloaddition reaction between metronidazide 3 and alkyne derivative 4a in the presence of CuI and Hünig’s base with MeCN as a solvent. The reaction furnished the desired product metronidazole 1H-1,2,3-triazole derivative 5a as a pale yellow solid in 85% yield [19,20].

Similarly, using the same reaction conditions and procedure described for the synthesis of the 1H-1,2,3-triazole derivative of metronidazole 5a, analogues 5b–i were obtained in 86–94% yield using the different alkyne derivatives 4b–i. The synthesis of the new 1H-1,2,3-triazole derivatives of metronidazole is summarized in Scheme 1 and Table 1.

Their chemical structures (5a–i) were confirmed by spectroscopic techniques (¹H NMR, ¹³C NMR) and HRMS.

The ¹H NMR spectrum of 1H-1,2,3-triazole compound 5c showed two singlet signals at δ 8.13 and 7.99 corresponding to the 1H-imidazole and 1H-1,2,3-triazole protons, respectively. The four aromatic protons appeared in the region of δ 7.67–7.05 ppm. A doublet signal at δ 4.77 is due to the four methylene protons of –N–CH₂-CH₂–Ph. A singlet peak at δ 1.86 is attributed to methyl protons on the imidazole ring. The ¹⁹F NMR spectrum of 1H-1,2,3-triazole compound 5c showed a singlet at δ −113.61 corresponding to one fluorine atom of the phenyl ring. The high-resolution mass spectrometric data at 317.1141 (M + H)⁺ supported the structure of 1H-1,2,3-triazole compound 5c.

Single crystals of 1H-1,2,3-triazole compound 5c were grown from slow evaporation of MeOH. The structure of 1H-1,2,3-triazole compound 5c was unambiguously confirmed by single crystal X-ray analysis (Figure 4).

Synthesis of carboxylate analogues of metronidazole

Compound 1 reacted with different acid chlorides (6a–e) in the presence of pyridine, a catalytic amount of DMAP and in dry DCM at room temperature. The reaction proceeded smoothly to give the desired metronidazole carboxylate derivatives 7a–e in 86–93% yields [21,22]. The synthesis of the new metronidazole carboxylate derivatives is summarized in Scheme 2 and Table 2.

Their chemical structures (7a–e) were confirmed by spectroscopic techniques (¹H NMR, ¹³C NMR and HRMS).

The ¹H NMR spectrum of compound 7b showed two doublet signals at δ 8.26 and 8.07 which are due to the four aromatic protons of the phenyl ring. A singlet signal at δ 7.95 is for the 1H-imidazole proton. Two doublet signals at δ 4.73 and δ 4.71 are assigned to the four methylene protons of –N–CH₂-CH₂–Ph. A singlet peak at δ 2.48 is due to the methyl protons on the imidazole ring. The high-resolution mass spectrometric data at 321.0842 (M + H)⁺ supported the structure of compound 7b.

Single crystals of compound 7b were grown from slow evaporation of MeOH + DCM (1:1) solution. The structure of compound 7b was unambiguously confirmed by single crystal X-ray analysis (Figure 5).

In this article, chemical transformations of novel metronidazole 1H-1,2,3-triazole derivatives via “click” chemistry and carboxylate derivatives can lead to a wide range of biological applications.

Antimicrobial activity

The general structural pattern of the synthesized metronidazole derivatives is shown in Figure 6. Two pharmacophoric elements (metronidazole core and triazole moiety) were considered as rigid motif with an alkyl/aryl group attached to the triazole unit. A diverse array of functional groups in the aromatic ring influencing the antimicrobial activity of the molecules have been utilized.

Antifungal activity of compounds

The antifungal activity of all compounds were evaluated by inhibiting the growth of Didymella sp. (Figure 7 and Table 3). The fungal colony after 7 days of control treatment was noted to be 8.6 cm in diameter.

Whereas, the growth of the fungal colony was detected maximum, i.e., 8.8 ± 0.2 and 9.0 ± 0.3 cm against compound 2 and 3, respectively. However, compound 5e and 7c efficiently inhibited the fungal growth by limiting the colony diameter to 3 ± 0.3 and 3.1 ± 0.2 cm followed equally by compound 7b and compound 5b with 4.1 ± 0.3 and 4.6 ± 0.2 cm, respectively. Compared to control and metronidazole treatments, fungal growth under compound 5e and compound 7c treatment was detected 2.8, 2.7 folds and 2.5, 2.6 folds less, respectively. All of the synthesized compounds except compounds 2 and 3 showed a higher inhibition rate of fungal growth as compared to the control and metronidazole (Figure 7 and Table 3). The inhibition zones were recorded after 7 days of treatment and compared with growth area of fungi growing in control conditions.

Antibacterial activity

To determine the bacterial growth inhibiting effects of compounds, bacterial OD₆₀₀ was measured at different time points i.e., 12, 24, 36 and 48 h (Figure 8 and Table 4). The results revealed that all compounds were able to inhibit the bacterial growth by showing suppressed OD but with varied sensitivity. OD at 12 h reading was detected minimum, and an increase was detected over the time. At 2 h time point, the inhibitory effect of compound 5b was significantly higher by demonstrating minimum OD among all tested compounds, while compound 7e and metronidazole treated bacteria exhibited maximum OD. Similarly, a slight OD enhancement was recorded in bacterial growth under all tested compounds from 24–48 h. However, the trend of suppressed bacterial OD by compound 5b was maintained at all-time points, which suggest that the inhibitory effects of compound 5b could be sustained for a considerably longer period of time. However, inhibitory effects of compound 5c was noted to be enhanced over the time and exhibited same inhibitory effects as compound 4 at 48 h time point. All of the tested compounds illustrated higher inhibitory effects at 36 and 48 h time point as compared to metronidazole. Taken together, the current findings demonstrate that all compounds in particular compound 5b and 5c inhibited bacterial growth and proved to be more potent than metronidazole.

Conclusion

In summary, a series of novel metronidazole 1H-1,2,3-triazole and carboxylate derivatives (5a–i and 7a–e) were synthesized via “click” chemistry, and evaluated for their antimicrobial activity (antifungal and antibacterial) in vitro. All the synthesized compounds (except 2 and 3 for antifungal studies) showed higher inhibition rates of fungal and bacterial growths when compared to control and the parent compound, metronidazole. Amongst the tested compounds 5b, 5c, 5e, 7b and 7e displayed excellent potent antimicrobial activity. The present study has added one more step in exploring the 1H-1,2,3-triazole moiety in the medicinal field. In addition, the above-mentioned activity of all the active compounds was reported for the first time for these derivatives.