INTRODUCTION

Resistance of bacterial strains to the existing antibiotics causes many problems in the therapy of diseases of bacterial aetiology (Ventola, 2015); the situation can be highly dangerous in the case of such pathogens as Pseudomonas aeruginosa (Khan et al., 2020; Pang et al., 2019; Yakout and Abdelwahab, 2022). It is known that the composition of antibiotics (Jones et al., 2022; Olsson et al., 2020) or their protection from deactivating enzymes using corresponding inhibitors (Brown and Wobst, 2022) is the possible way to solve this problem. The selection of more stable molecules in the existing groups of antibiotics is also a promising strategy to improve the therapy; Plazomicin (Wagenlehner et al., 2019), dalbavancin (Cercenado, 2017), oritavancin (Mitra et al., 2015), and meropenem (El-Gamal, 2011) serve as good examples of such molecules.

One more strategy of antimicrobial drug development, with some problems and limitations (Tommasi et al., 2015), is the development of inhibitors of bacterial protein targets, which could even form a new class of antibiotic or antibacterial agents. One of these attractive targets is bacterial TrmD, which is crucially different from its ortholog of eukaryotes and archaea (Goto-Ito et al., 2017). Blockage of this enzyme leads to +1 frameshifting at the ribosomal translation stage of protein synthesis (Gamper et al., 2015). The disruption of protein synthesis in its turn negatively influences the production of bacterial membrane proteins, such as efflux drugs pumps (Masuda et al., 2019), which leads to the misfunctioning of the outer membrane of Gram-negative bacterial cells (Zhang et al., 2013), can favorize the accumulation of antibiotics and increases their activity (Richter et al., 2017).

Small molecules draw the attention of researchers as candidates for bacterial TrmD inhibitors. Among the most promising heterocyclic systems which are present in the structure of highly active inhibitors, the assemblies of pyrazole with indole were reported (Whitehouse et al., 2019). Compounds with pyrazole heterocyclic pattern modified with 3-indolyl or 2-thienylcaboxamide substituents were identified as leaders in HTS assays aiming at the development of TrmD P. aeruginosa inhibitors (Zhong et al., 2019a). The derivatives of thieno[2,3-d]pyrimidine are one the effective classes of the well-known TrmD inhibitors (Hill et al., 2013), which allowed to disclose differences in inhibitors’ binding mechanisms between Gram-negative tRNA (Guanine37-N¹)-methyltransferases from mycobacterial and also their Gram-positive counterparts. The study of thienopyrimidine derivatives complexes with different TrmDs also helped to get a deeper insight into the molecular mechanism of their action (Zhong et al., 2019b). One of the newest results concerning libraries potential of thieno[2,3-d]pyrimidines as TrmD inhibitors is the study that revealed the derivative of 3,5,6,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidine modified at position three with 3-indolylthioacetamide substituent as the most active compound (Malasala et al., 2022). Besides, the activity of thienopyrimidines as TrmD inhibitors has also been reported (Badiger et al., 2015; Triloknadh et al., 2021). It is also interesting that thieno[2,3-d]pyrimidin-6-carboxylic acids derivatives are inhibitors of N-acetyltransferases of bacterial polysaccharides, which showed nanomolar inhibitory activity and were active against Campylobacter jejuni PglD (De Schutter et al., 2017).

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Although some compounds with 4-O-alkylthieno[2,3-d]pyrimidine core were reported to be inactive as TrmD inhibitors, the most active compound I with oxo group at position four has been reported in the same article (Zhong et al., 2019b) and 4-N-alkyl derivatives showed good activity as inhibitors to the acetyltransferase of bacterial amino-sugar II (De Schutter et al., 2017). Antimicrobial properties were also revealed earlier for thieno[2,3-d]pyrimidine-4-carboxamides, which are also the compounds with aromatic pyrimidine ring, and notably benzylamide III was the most active against P. aeruginosa (Vlasova et al., 2019). The problem of the aromatic pyrimidine ring to antibacterial activity remained unclear. We found it interesting to obtain the derivatives with a small alkyl substituent at position four of thienopyrimidine attached to the heterocyclic system without any additional atoms linker O or N atoms.

For the further preparation of attractive antimicrobial substances, we additionally considered two possible main ways of modifying the carboxylic function at position six of the thienopyrimidine cycle: one of them is the preparation of amides and the other one is the use of heterocyclization methods which showed their effectiveness for construction of thieno[2,3-d]pyrimidines bearing 6-heteryl substituent with antimicrobial properties (Hill et al., 2013; Vlasov et al., 2021; Zhong et al., 2019b).

The examples of 4-alkylthieno[2,3-d]pyrimidines are quite rare, although recently some 4-methylthieno[2,3-d]pyrimidines with different heterocycles at position six were reported as ones with antiproliferative dose-dependent activity according to NCI-H460 xenograft model and their safety profile was found acceptable (Lin et al., 2018). The building blocks for these structures were obtained by reaction of 4-chlorothieno[2,3-d]pyrimidine with methylboronic acid. We found this approach promising and decided to test an opportunity of synthesis of 4,5-dimethylthieno[2,3-d]pyrimidine-6-carboxylic acid as a perfect building block for the synthesis of amides and the construction of benzimidazole substituent at position six of the core heterocyclic system.

The first preparation of 4-alkyl derivatives of thieno[2,3-d]pyrimidine was reported in 1971 (Manhas and Sharma, 1971). The authors used 4-chloride of 2-phenyl-5,6,7,8-tetrahydro[1]-benzothieno[2,3-d]pyrimidine as the starting compound for the preparation of 4-methylsulphonylmethyl-2-phenyl-5,6,7,8-tetrahydro[1]-benzothieno[2,3-d]pyrimidine, which was further reduced with aluminum amalgam in aqueous tetrahydrofuran. The example of incidental preparation of 4-alkyl thieno[2,3-d]pyrimidine occurred during the experimental selection of metalation reagent (Therkelsen et al., 2004). Preparation of 4-methyl derivatives by cyclization of 4-chloro-5-(trimethylsilylethynyl)pyrimidines with the addition of sodium hydrosulfide was also suggested (Sakamoto et al., 1986). Preparation of 4-alkylthieno[2,3-d]pyrimidines by Suzuki coupling was first reported for the preparation of 4-butyl derivative as the result of the interaction of Pd(PPh₃)₄-mediated reaction of the corresponding chloride with butylboronic acid (Fernandez-Mato et al., 2011); similar transformations for methylboronic acid were reported recently (Lin et al., 2018).

Figure 1. The derivatives of thieno[2,3-d]pyrimidine with antibacterial properties. [Click here to view]

In view of the high effectiveness of methylation in the reaction of different aryl and hetaryl halides in reaction with methylboronic acid (Falb et al., 2017; Karroum et al., 2018), we studied the Pd-catalyzed reaction of previously reported ethyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (Triloknadh et al., 2018) with methylboronic acid (Fig. 2). It is interesting that the reaction appeared very sensitive to the quality of the starting chloride. For the crude chloride in the reaction with boronic acid, we saw the traces of the by-product with quasimolecular ion peak 459 (m/z), which probably resulted from Pd-catalyzed side N-arylation reaction of the chloride 1 with its oxo-derivative, which might have been as admixture. Therefore, for the scaling up of the reaction, the starting chloride 1 was purified chromatographically.

The reaction of pure compound 1 with methylboronic acid catalyzed by PdCl₂ dppf allowed the preparation of 4-methyl derivative 2 in satisfactory yield (Fig. 2). At the next step, the ester 2 was hydrolyzed in alkaline conditions to give acid 3 with an excellent yield. Synthesis of amides 4 was performed by reaction of the acid 3 with the series of primary benzyl amines and morpholine in DMF, which was promoted by CDI. All of the amides 4 were obtained with good yields 43%–92%. In the ¹H NMR spectra of the compounds 4 two signals of methyl groups at positions 4 and 5 of thienopyrimidine are observed at 2.54–2.75 and 2.90–2.92 ppm; the position of the down-field signal is not much influenced by the amide substituent which confirms that this is the signal of CH₃ group at more distant form carboxamide substituent position four. For all of the compound 4, the signal of a proton of thieno[2,3-d]pyrimidine at position two is observed as a singlet at 8.90–8.98 ppm. For benzylamides 4a-4e the doublet signals of the methylene group are observed at 4.43–4.49 ppm while the broaden signal of NH is obviously seen at 8.86–9.10 ppm; the signals of aromatic protons are observed at 6.92–7.40 ppm. For morpholyl amide 4f the signals of methylene groups are at 3.19–3.69 ppm. ¹³C NMR spectra of the synthesized derivatives 4 have signals of both CH₃ groups of thieno[2,3-d]pyrimidine at 16.1–16.6 ppm and 24.0–24.6 ppm.

We also studied the reaction of acid 3 in conditions similar to amides 4 synthesis using o-phenylenediamine as an amine. It was found that the reaction at 120°C–130°C during 2–3 hours results in the product of benzimidazole ring closure with the formation of 6-(1H-benzimidazol-2-yl)-4,5-dimethylthieno[2,3-d]pyrimidine 5. In the ¹H NMR spectrum of the derivative 6, the signals of thienopyrimidine CH3 groups are observed as two singlets at 2.94 ?? 2.96 ppm; the proton at position two of the core heterocyclic system shows its signal at 8.90 ppm. The signals of the benzimidazole system in the spectrum are observed as two groups at 7.25 (br. s, 2?, ? Ar) and 7.64 (br. s, 2?, ? Ar), while the signal of benzimidazole NH is at 12.91 ppm.

Figure 2. Preparation of 4-methylthieno[2,3-d]pyrimidine derivatives 4a-f, 5, and 6a-c. [Click here to view]

Compound 5 with its benzimidazole fragment was found suitable for alkylation with chloroacetamides. The alkylation was performed in dimethylformamide using potassium carbonate as the base. 2-[2-(4,5-dimethylthieno[2,3-d]pyrimidin-6-yl)-1H-benzimidazol-1-yl]-N-arylacetamides 6?-6? were isolated with good yields and crystallized from ethanol. ¹H NMR spectra of the compounds 6 in comparison with the compound 5 have additional signals of protons of the CH₂ group of acetamide in the region 5.10–5.13 ppm, while the signals of NH groups are observed in the region 10.26–10.34 ppm. The ¹³C NMR spectra of 6?-6? differ from the spectrum of 5 by additional signals of CH₂ at 47.7–47.8 ppm and C=O signals at 165.1–165.5 ppm.

The results of antimicrobial activity screening, which was performed by agar “well” method for all of the synthesized compounds, showed that all of the compounds with the fragment of 4-methylthieno[2,3-d]pyrimidine were active against most bacteria and Candida fungi (Table 1). Among the Gram-positive bacteria B. subtilis ???? 6633 was the most sensitive; the compounds also most effectively inhibited the growth of P. aeruginosa ???? 27853 among the Gram-negative strains. Ester 2 has been found more active than the corresponding acid 3. The introduction of the benzylamide fragment also increased the activity of the compounds 4c, 4d, and 4e correspondingly 4-Cl, 4-F, and 4-OCH₃ against B. subtilis and P. aeruginosa. Amide 4f with morpholine fragment did not display high antimicrobial activity regardless of its better solubility. The results of the antimicrobial activity study also showed the introduction of a benzimidazole ring instead of a carboxyl group. The antimicrobial activity of the compounds synthesized was found to be a little less than the activity of the reference drug Streptomycin, which was selected as the reference drug which is an aminoglycoside antibiotic with a wide spectrum of activity against both Gram-positive and Gram-negative bacteria; it is also active against Mycobacterium tuberculosis; this drug is also widely used in clinics (Ahmed et al., 2013; Jansen et al., 2016; Tereshko et al., 2003). Unfortunately, not of the TrmD inhibitors, for now, has been approved for medical use.

To suggest the possible mechanism of antibacterial activity for all the compounds which were studied for the antimicrobial activity we performed the docking study to the active site of TrmD selective inhibitors. The TrmD which was isolated from P. aeruginosa (Zhong et al., 2019b) was used as a protein model. The validation of docking methodology and RMSD calculation of the reference ligand pose to the native ligand were reported earlier (Vlasov et al., 2021, 2022). According to the docking studies, the obtained compounds showed different affinity to the active site TrmD inhibitor (Table 2).

The lowest level of affinity was predicted for both ester 2 and acid 3 (−7.4 kcal/mole against −8.2 kcal/mole affinity for the native ligand). Transformation of the carboxyl group to the benzimidazole cycle improves the binding energy for derivative 5 (−8.0 kcal/mole). Alkylation of the benzimidazole cycle increases the affinity; the scoring function for arylacetamides 6?-? reaches −8.7 kcal/mole value. The affinity results were predicted for amides 4, especially benzylamides 4a-e, which showed the values of scoring function from −9.3 to −9.9 kcal/mole. The calculated scoring function values are in good correlation with in vitro experimental results.

Detailed analysis of the interaction of the ligands with amino acids of the active site shows many hydrophobic interactions. Among them, there was the interaction of methyl group at position four of thieno[2,3-d]pyrimidine with the pyrrolidine cycle of proline Pro94, which can be a sign of the existence of stable ligand-enzyme conformations. All the studied ligands interact with the residues of glutamic acid Gly145, 146, and/or glutamine Gln95, which were proven to be the site of TrmD cofactor S-adenosylmethionine (SAM) binding. This determines the deep immersion of 4-methylthieno[2,3-d]pyrimidines in the cavity of the active site competing with SAM and thus inhibiting bacterial TrmDs like the one isolated from P. aeruginosa. As to the results of the docking, the best parameters of antibacterial activity via inhibition of TrmD were shown by benzyl amides 4?-? (Fig. 3).

Table 1. Antimicrobial activity screening results for 4-methylthieno[2,3-d]pyrimidine derivatives 2,3, 4a-f, 5, 6a-c. [Click here to view]

Table 2. The resuls of the docking studies of 4-methylthieno[2,3-d]pyrimidine derivatives 2, 3, 4a-f, 5, 6a-c to the active site of selective TrmD P. aeruginosa inhibito rs. [Click here to view]

Figure 3. 3D visualization of the ligand 4b and the amino acids of the active site of selective TrmD inhibitors (a) conformation with reference inhibitor (yellow structure) (Zong et al., 2019 ) in the active site of P. aeruginosa TrmD. [Click here to view]

CONCLUSION

As the result of modification of position four of thieno[2,3-d]pyrimidine using Suzuki reaction of methyl boronic acid and readily available ethyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-carboxylate the ester and 4,5-dimethylthieno[2,3-d]pyrimidine-6-carboxylic acid resulted from hydrolysis of the ester were obtained. Modification of 4,5-dimethylthieno[2,3-d]pyrimidine-6-carboxylic acid gave the series of amides and the derivative with benzimidazole fused system at position six. All of the synthesized compounds showed antimicrobial properties, especially against the strains of B. subtilis and P. aeruginosa. The most promising antimicrobial properties were determined for benzyl amides of 4,5-dimethylthieno[2,3-d]pyrimidine-6-carboxylic acid. The docking studies of the obtained compounds to the active site of selective TrmD inhibitors revealed that benzyl amides also had the best binding parameters to the tRNA (Guanine37-N¹)-methyltransferase of P. aeruginosa. The docking studies confirmed the suggested mechanism of antibacterial action as in silico results are in correlation with the in vitro experiment.

ACKNOWLEDGMENTS

All the authors and contributors are truly grateful to Enamine Ltd. Company for providing analytical data like ¹H, ¹³C NMR, and LC-MS spectra. They acknowledge the kind help of Dr. Tatyana P. Osolodchenko, Ph.D. in Biology, and her colleagues from the Mechnikov Institute of Microbiology and Immunology of the NAMS of Ukraine (Kharkiv) for the performance of antibacterial and antifungal studies.

AUTHOR CONTRIBUTIONS

Concept and design were contributed by S.V.V., V.A.G., O.D.V., and H.I.S.; data acquisition and data analysis were contributed by K.Y.K., V.S.V, P.E.S., H.I.S., O.D.V., O.V.B., and S.V.V.; preparation and submission of the manuscript were done by S.V.V., H.I.S., K.Y.K., and O.V.B.; supervision and final approval were contributed by V.A.G., S.V.V., H.I.S., and O.V.B. All the authors have made a significant contribution to the content of the submitted manuscript; they adjusted it and agree with its publication.

FINANCIAL SUPPORT

The research was funded by the Ministry of Health Care of Ukraine at the expense of the State Budget in framework # 2301020 “Scientific and scientific-technical activity in the field of health protection” on the topic “Synthesis and study of new thienopyrimidines for the detection of antimicrobial and related types of pharmacological activity” (State registration number: 0121U109472; Order of the Ministry of Health of Ukraine of November 17, 2020, ? 2651).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY

All data generated and analyzed are included in this research article.

PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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