2-Aryl-1-hydroxyimidazoles possessing antiviral activity against a wide range of orthopoxviruses, including the variola virus

Abstract

Scientific interest in orthopoxvirus infections and search for new highly effective compounds possessing antiviral activity against orthopoxviruses have significantly increased as a result of worldwide mpox outbreak in 2022. The present work deals with the synthesis of new 2-arylimidazoles exhibiting in vitro activity not only against the vaccinia virus, cowpox virus and ectromelia (mousepox) virus but also against the variola virus. Among the imidazole derivatives under consideration (1-hydroxyimidazoles, 1-methoxyimidazoles, 1-benzyloxyimidazoles, and imidazole N-oxides), the most promising antiviral activity is demonstrated by 1-hydroxyimidazoles, which may exist as two prototropic tautomers. Both of these tautomers may be manifested in different crystal structures of these compounds, according to single-crystal X-ray diffraction analysis, while predominantly one of them (N-hydroxy-tautomeric form) is present in DMSO-d₆ solutions and in the gaseous state, as shown by NMR spectroscopy and quantum-chemical calculations. The leader compound 1-hydroxy-2-(4-nitrophenyl)imidazole 4a demonstrated the highest selectivity indices against the vaccinia virus (SI = 1072) and the variola virus (SI = 373).

Newly synthesized 2-aryl-1-hydroxyimidazoles display promising antiviral activity against orthopoxviruses, including the variola virus.

1. Introduction

Poxviruses are the representatives of DNA-containing viruses that may cause diseases in both humans and animals. Currently, their most significant genus orthopoxvirus contains 12 species,¹ including the vaccinia virus (VACV), cowpox virus (CPXV), monkeypox virus (MPXV) and variola virus (VARV), that are contagious for humans. The latter is the most dangerous one, being the cause of smallpox (or variola major). Due to global vaccination, smallpox was totally eradicated.² It should be noted that this global immunization of population provided cross immunity against monkeypox (mpox) with approximately 85% efficacy.³ However, after 1980, when total planned immunization against smallpox had been terminated, immunity against orthopoxviruses in younger generations predictably decreased. For example, the outbreak of mpox has previously occurred only in Africa.⁴ However, at present, 40 years after the Smallpox Eradication Programme, the pandemic of zoonotic mpox infection outside the borders of Africa led to an increased scientific interest in the diseases caused by orthopoxviruses and the means of their treatment. There is also a concern that VARV may be used as a biological weapon⁵ or that natural mutations in zoonotic orthopoxviruses may lead to new dangerous strains.⁶ These are the reasons that in as early as 2010, the World Health Organization (WHO) published the report “Scientific Review of Variola Virus Research, 1999–2010”⁷ and comments of the WHO Advisory Group of Independent Experts on Smallpox Programme Review (AGIES).⁸ In these documents, it was particularly noted that VARV was capable of developing resistance to existing drugs, and that fact determined the need for further investigations, including the search for new anti-smallpox compounds with different mechanisms of action. As the smallpox disease is officially considered eradicated worldwide since 1980, the WHO strictly controls the storage of the stocks of VARV and complete viral genomes, and all the investigations are carried out solely with the approval of the WHO.⁹ For example, there are only two institutions in the world that are authorized to work with the variola virus (VARV)—they are the Center for Disease Control and Prevention (CDC) in Atlanta, USA, and the State Research Center of Virology and Biotechnology VECTOR of Rospotrebnadzor in Koltsovo (Novosibirsk region, Russia). As a result, the studies on the variola virus are quite rare,^10–14 whereas studies on the vaccinia virus and other orthopoxviruses are more common.¹⁵

There are four drugs approved for the treatment of orthopoxvirus infections (Fig. 1). They are cidofovir (CDV, Vistide®),¹⁶ brincidofovir (CMX001, an analogue of cidofovir)¹⁷ and currently the most effective tecovirimat (ST-246, TPOXX®)¹⁸ developed by SIGA Technologies Inc. (New York, USA). Anti-smallpox compound NIOCH-14 is the nearest analogue of ST-246.¹⁹ It also successfully passed clinical trials (NCT05976100, phase 1) and was registered in Russia.^13,19

Fig. 1. Structures of the approved anti-orthopoxvirus drugs.

Molecules containing an imidazole moiety play an important role in medicinal chemistry due to their wide range of biological activities.²⁰ Imidazole-based derivatives have demonstrated antiviral activities against ZIKV (Zika virus), HIV (human immunodeficiency virus), HPV (human papilloma virus), SARS CoV-2, influenza, dengue, etc. In the course of the development of significant antivirals, the structure–activity relationships of various imidazole derivatives against different types of viruses were reported.²¹

Previously, it has been established that 1-hydroxyimidazole derivatives exhibited antiviral activities against the vaccinia virus (VACV)^22–24 and other orthopoxviruses such as the cowpox virus (CPXV) and the mousepox (ectromelia) virus (ECTV).²⁴ It was demonstrated that the highest virus-inhibiting activity was exhibited by 2-aryl-1-hydroxyimidazole derivatives containing a phenyl moiety and electron-withdrawing groups (EWGs) in the para-position to the imidazole ring.²⁴ As a continuation of our study, in the present work, we discuss the influence of the substituents in positions 1, 3, 4, and 5 of the imidazole ring on cytotoxicity and virus-inhibiting activity against orthopoxviruses (VACV, CPXV and ECTV) including the most dangerous Variola virus (VARV).

2. Results and discussion

2.1. Synthesis

The condensation of commercially available benzaldehydes 1a–e with triketone monooximes 2 and 3 and ammonium acetate in glacial acetic acid resulted in 1-hydroxyimidazole derivatives 4a–c and 5a–c (Scheme 1) containing electron-withdrawing 4-nitro-, 4-cyano- and 4-(trifluoromethyl)phenyl moieties in position 2 of imidazole. 1-Hydroxyimidazoles 5d, e with electron-donating (methoxy- and dimethylamino-) groups in the phenyl substituent were also obtained for the comparison of the activities. Starting oximes 2 and 3 were synthesized by nitrosation of either dimedone or pentane-2,4-dione by known techniques.²⁵

Scheme 1. Synthesis of 1-hydroxyimidazoles 4a–c and 5a–e.

It is known that methylation of such 1-hydroxyimidazole derivatives proceeds selectively as an O-methylation process.^26–29 Compounds 4a, b and 5a–e were easily methylated by methyl iodide in DMF in the presence of potassium hydroxide at room temperature resulting in 1-methoxyimidazoles 6a, b, 7a–e (Scheme 2).

Scheme 2. Synthesis of 1-methoxyimidazoles 6a, b and 7a–e.

The condensation of starting oximes 2, 3 with the Schiff bases 8a–c yielded 1-methylimidazole 3-oxides 9a, c and 10a, b (Scheme 3).

Scheme 3. Synthesis of 1-methylimidazole 3-oxides 9a, c and 10a, b.

Selective substitution of the hydrogen atom of N-hydroxy group occurs not only in the course of methylation, but also in the case of introduction of bulkier substituents. Thus, alkylation of 1-hydroxyimidazole 4a by benzyl halides in the presence of potassium carbonate and potassium iodide in boiling acetonitrile led solely to benzyloxyimidazoles 11a–e (Scheme 4).

Scheme 4. Synthesis of 1-benzyimidazoles 11a–e.

Moreover, by condensation of the Schiff base 12 with corresponding oxime 2 in glacial acetic acid, 1-benzylimidazole 3-oxide 13 was synthesized (Scheme 5).

Scheme 5. Synthesis of 1-benzyimidazole 3-oxide 13.

It should be mentioned that the difference in the values of chemical shifts of ¹³C nuclei of N-methyl groups in the ¹³C NMR spectra of 1-methylimidazole 3-oxides 9a, c and 10a, b (ranging from 32.3 ppm to 33.6 ppm) and the values of chemical shifts of ¹³C carbon atoms of the supposed methoxy groups of compounds 6a, b and 7a–e (ranging from 66.1 ppm to 67.7 ppm) confirmed the course of O-methylation (Scheme 2). A similar difference in chemical shifts was observed in the case of alkylation (Scheme 4). The signal of ¹³C nuclei of benzyl CH₂-group of N-oxide 13 was observed at 48.3 ppm, while the signals of ¹³C nuclei of benzyl CH₂ groups in the case of compounds 11a–e were deshielded and observed in the range from 68.0 ppm to 81.4 ppm confirming the O-alkylation process.

2.2. Study of tautomeric forms. NMR studies in DMSO-d₆ solutions

There are two possible prototropic tautomers for 1-hydroxyimidazoles. These derivatives may exist either as an N-hydroxy-tautomer or as an N-oxide one (Scheme 6).³⁰

Scheme 6. Prototropic tautomerism of 1-hydroxyimidazoles.

It is supposed that the predominant tautomeric form may have an impact on the biological activity of small molecules.³¹ Therefore, it seems reasonable to discuss the shift of tautomeric equilibrium of the structures under consideration.

Imidazole derivatives containing a nitro group in the 2-aryl moiety were chosen for the study of prototropic tautomerism of 1-hydroxyimidazoles under consideration. The geometric parameters for two possible tautomers of 1-hydroxyimidazoles 4a and 5a were determined (Scheme 7).

Scheme 7. Difference in energy parameters (Gibbs free energies, ΔG) for the pairs of tautomers under study: a) OH- and NO-tautomers of compound 4a and b) OH- and NO-tautomers of compound 5a. A value of 0.0 corresponding to an energetically favorable tautomer of the pair, data in brackets were obtained via calculations in the presence of solvent (PCM, DMSO).

According to the quantum-chemical calculations, it was determined that compounds 4a and 5a predominantly existed in N-hydroxy-tautomeric form both in the gaseous phase and in the solvent (DMSO). For compound 4a, Gibbs energy difference between N-oxide and N-hydroxy-tautomers was 52.92 kJ mol⁻¹ in the gaseous phase and 3.51 kJ mol⁻¹ in the solvent. For 5-acetyl substituted derivative 5a, the Gibbs energy difference between tautomers was 34.53 kJ mol⁻¹ in the gas and 8.10 kJ mol⁻¹ in the solvent. The influence of DMSO decreased the energy difference between tautomers. It should also be mentioned that for the annulated derivative 4a, ΔG was lower than the one for 5-acetyl-substituted imidazole 5a.

Previously,^26,27 it has been demonstrated that the most convenient way to establish the predominant tautomeric form of 1-hydroxyimidazole in the solution was to compare the ¹H and ¹³C NMR spectra of the compounds under consideration and model structures. Similarly, substituted 1-methoxyimidazoles are commonly used as model compounds for the N-hydroxy-tautomer, while 1-methylimidazole 3-oxides stand for the N-oxide tautomer model (Scheme 6).

In order to confirm the predominant tautomeric form of 2-(4-nitrophenyl)substituted 1-hydroxyimidazoles 4a and 5a, the ¹H and ¹³C NMR spectra of the compounds 4a, 5a, 6a, 7a, 9a, and 10a in polar aprotic DMSO-d₆ were considered. Chemical shift values for 1-hydroxyimidazoles 4a and 5a and model structures 6a, 7a, 9a, and 10a are given in Tables 1 and 2. Correlation indices for the calculated and experimental chemical shifts do not exceed 0.99 units for all of the compounds under consideration (Fig. 2). According to both quantum-chemical calculations and experimental data (Tables 1 and 2), it is obvious that the ¹H NMR spectra were not informative enough for the determination of the prevailing tautomer as those spectra were much alike for both tautomers and model compounds.

¹³C and ¹H NMR chemical shift values (ppm) obtained experimentally and theoretically (B3LYP/6-311++G(d,p)/CSGT) for compounds 4a, 6a, and 9a^a.

δ C (DMSO-d6)a ppm
NMR 13C	Exp	Calc	Exp	Calc	Exp	Calc
	4a		6a		9a
C1′	133.8	142.2	133.0	143.2	129.8	139.9
C2′; C6′	128.5	133.8	128.5	134.3	130.4	134.9
C3′; C5′	123.9	130.2	124.2	130.2	123.4	129.3
C4′ (C–NO2)	147.4	152.8	147.8	153.1	147.1	151.1
C2	142.1	145.6	141.4	149.4	133.6	143.6
C4	149.9	160.1	150.7	161.6	139.9	151.1
C5	38.6	42.5	38.3	43.5	33.8	39.4
C6	35.4	48.7	35.4	27.0	34.2	45.2
C7	52.5	54.8	52.2	57.9	52.2	58.0
C8 (C O)	186.3	203.5	186.0	197.4	184.8	195.9
C9	123.8	125.8	122.2	129.7	123.8	131.3
2CH3	27.9	30.4	27.9	30.0	27.9	30.1
OCH3/NCH3	—	—	67.2	71.1	33.1	36.5

NMR 1H	Exp	Calc	Exp	Calc	Exp	Calc
	4a		6a		9a
N–OH	12.8 (br. s)	10.2	—	—	—	—
H-2′; H-6′	8.3 (s)b	7.5	8.3 (d, J = 8.7 Hz)	7.7	8.2 (d, J = 8.9 Hz)	7.4
H-3′; H-5′			8.4 (d, J = 8.7 Hz)	7.4	8.4 (d, J = 8.9 Hz)
OCH3/NCH3	—	—	4.1 (s)	3.7	3.6 (s)	3.0
–COCH2–	2.4(s)	2.0	2.5 (s)	1.9	2.4 (s)	1.8
2CH3	1.1 (s)	0.7	1.1 (s)	0.7	1.1 (s)	0.7
–CH2–	2.7	2.2	2.7 (s)	2.3	2.9 (s)	2.3

^aFor the convenience, the numeration of the atoms in N-oxide cycle is the same as in N-methoxy- and N-hydroxy derivatives.

^bIn this case the signals of aromatic protons are a singlet.

¹³C and ¹H NMR chemical shift values (ppm) obtained experimentally and theoretically (B3LYP/6-311++G(d,p)/CSGT) for compounds 5a, 7a, and 10a^a.

δ C (DMSO-d6) ppm
NMR 13C	Exp	Calc	Exp	Calc	Exp	Calc
	5a		7a		10a
C1′	124.4	142.1	133.1	143.2	130.9	140.0
C2′; C6′	128.5	133.9	128.9	134.3	131.9	135.4
C3′; C5′	124.3	130.1	124.7	130.3	123.4	129.3
C4′ (C–NO2)	147.6	152.6	147.7	153.0	147.2	151.3
C2	134.0	143.5	139.1	146.7	130.0	141.4
C4	142.3	156.4	143.4	154.8	133.4	145.5
C5	127.9	128.5	125.3	132.6	128.1	135.3
C6 (C O)	188.5	204.5	186.9	198.0	190.6	201.5
C7	30.3	32.7	29.8	32.1	30.4	35.5
C8	16.3	31.2	16.8	21.3	10.1	12.5
OCH3/NCH3	—	—	67.6	72.2	32.5	36.1

NMR 1H	Exp	Calc	Exp	Calc	Exp	Calc
	5a		7a		10a
N–OH	12.6 (br. s)	12.9	—	—	—	—
H-2′; H-6′	8.3 (d, J = 9.0 Hz)	7.7	8.3 (d, J = 9.5 Hz)	7.7	8.1 (d, J = 8.9 Hz)	7.4
H-3′; H-5′	8.4 (d, J = 9.0 Hz)	7.4	8.4 (d, J = 9.5 Hz)	7.4	8.4 (d, J = 8.9 Hz)	7.4
OCH3/NCH3	—	—	4.0 (s)	3.6	3.6 (s)	3.0
COCH3	2.5 (s)	2.0	2.5 (s)	1.9	2.7 (s)	2.0
CH3	2.4 (s)	2.0	2.5 (s)	1.8	2.5 (s)	2.0

^aFor the convenience, the numeration of the atoms in N-oxide cycle is the same as in N-methoxy- and N-hydroxy derivatives.

Fig. 2. Correlation of chemical shift values in the NMR spectra obtained experimentally and theoretically (QC) a) for compounds 4a and 5a, tautomeric forms; b) 1-methoxyimidazoles 6a and 7a; and c) imidazole N-oxides 9a and 10a.

While comparing the ¹³C NMR spectra of 1-hydroxyimidazole 4a, 1-methoxyimidazole 6a and imidazole 3-oxide 9a registered in DMSO-d₆ (Table 1), one can observe that the signals of ¹³C nuclei in positions 2 and 4 of the imidazole ring as well as the signal of the CH₂ group in position 5 were the most sensitive to the change in the nature of heteroatoms (Fig. 3).

Fig. 3. Comparison of the ¹³C NMR spectra of 1-hydroxyimidazole 4a, 1-methoxyimidazole 6a and imidazole 3-oxide 9a registered in DMSO-d₆ (ranging from 20 ppm to 190 ppm).

In the case of 1-methoxy-derivative 6a, the signals of ¹³C nuclei in positions 2 and 4 of the imidazole ring were observed at 141.4 ppm and 150.7 ppm, respectively, while the signal of CH₂-group in position 5 was found at 38.3 ppm. Transition from 1-methoxyimidazole 6a to 1-methylimidazole 3-oxide 9a was accompanied by the transformation of a pyridine-type nitrogen atom in the heterocycle into a pyrrole-type one. As a result, the signals of ¹³C nuclei of the carbon atoms neighboring to this changing nitrogen or conjugated to it were shifted upfield and were observed at 133.6 ppm (C2), 139.9 ppm (C4) and 33.8 ppm (C5), respectively. In the ¹³C NMR spectrum of 1-hydroxyimidazole 4a, the signals of the ¹³C nuclei in positions 2 and 4 of the imidazole ring were situated at 142.1 ppm and 149.9 ppm, respectively, while the signal of ¹³C nuclei of the CH₂ group in position 5 was observed at 38.6 ppm. These data were the evidence that the tautomer simulated by 1-methoxyimidazole (i.e., N-hydroxy-tautomeric form) prevailed in the solution in DMSO-d₆.

For 1-hydroxyimidazoles 4b, c the easily determined signals of the ¹³C nuclei of the CH₂ group in position 5 were also observed at 38.4 ppm. It means that these derivatives also existed predominantly in the N-hydroxy-tautomeric form in DMSO-d₆ solutions.

In the molecules of 5-acetylimidazoles 5a, 7a, and 10a, the lack of rigid fixation of the structure due to the absence of annulated saturated cycle led to similar differences in the positions of the signals of ¹³C nuclei of the methyl group on position 4 of the imidazole ring (Table 2).

In the ¹³C NMR spectrum of imidazole N-oxide 10a, the signal of the CH₃-group in position 4 of the imidazole ring was observed at 10.6 ppm, while in the case of N-methoxyimidazole 7a, it was deshielded and was situated at 16.8 ppm (Table 2). As for the ¹³C NMR spectrum of N-hydroxyimidazole 5a, the signal of the CH₃ group in position 4 was found at 16.0 ppm, which was the evidence of prevailing N-hydroxy-tautomers. The spectra of 1-hydroxyimidazoles 5b–e were similar.

Therefore, in the DMSO-d₆ solution, all the 1-hydroxyimidazoles under consideration predominantly existed as N-hydroxy-tautomers.

2.3. X-ray analysis data

On proceeding from the solution to the crystal state, the situation with the prevailing tautomer somehow differs.

1-Hydroxyimidazoles 4b, c with a fixed carbonyl group in position 5 of the imidazole ring in the crystal state existed in N-oxide tautomeric form (Fig. 4(a) and (b)), which was in contrast to their predominant tautomeric form in DMSO-d₆ and gaseous phase. These compounds bear different substituents in phenyl moieties but are in fact isostructural in the crystal state. The molecules of 4b, c formed infinite chains via intermolecular hydrogen bonds that involved the N-oxide function of one molecule and N–H proton of the neighboring one, as shown in Fig. 4(c) for 4b (N–H⋯O type with a distance of 2.671(2) Å). The carbonyl group in position 5 of imidazole did not participate in any type of H-bonding.

Fig. 4. Crystal structures of 4b (a) and 4c (b). The fluorine atoms in (b) are disordered. (c) Fragment of the crystal structure of 4b presenting the hydrogen-bonded chains. The dotted lines indicate the direction of hydrogen bonds.

1-Hydroxyimidazoles 5a, b with a flexible acetyl group in position 5 of the imidazole ring in crystal state existed as N-hydroxy-tautomers stabilized by short intramolecular hydrogen bonds between N-hydroxy and carbonyl groups (O–H⋯O type with a distance of 2.551(2) Å for 5a and 2.548(2) Å for 5b) (Fig. 5(a) and (b)). The flexibility of acetyl group appears to be responsible for the adjustment of molecular conformation in order to form an intramolecular hydrogen bond, what provides additional stabilization and is only possible in the case of N-hydroxy-tautomeric form. One can also emphasize the presence of strong stacking interactions between the molecules of 5b oriented antiparallel to each other (Fig. 5(c)).

Fig. 5. Molecules of 5a (a) and 5b (b) in the corresponding crystal structures. (c) Fragment of the crystal structure of 5b presenting the stacking interactions of molecules oriented antiparallel to each other. The dotted lines indicate the presence of intramolecular hydrogen bonds.

For 1-hydroxyimidazole 5c, X-ray analysis data were obtained for both the anhydrous molecule and its semihydrate (Fig. 6).

Fig. 6. Fragment of the crystal structure ((5c)₂·(H₂O)).

Semihydrate of 5c ((5c)₂·(H₂O)) was characterized by 2 molecules of 5c and one molecule of water in the asymmetric unit. Single crystals of the semihydrate of 5c were obtained from the following solvents: nitromethane, benzene, dioxane, acetone, and acetonitrile. The starting material was subjected to additional drying, and dried toluene was used as a solvent during crystallization to obtain the crystal form of compound 5c. The crystal structure of semihydrate was formed due to the protonation of one of the oxygen substituents of the imidazole ring, followed by the removal of water into the outer coordination sphere. As a result of this interaction, prototropic tautomerism was observed directly in one crystal structure, i.e., 2 tautomeric forms were present in one unit cell: one protonated as an N-hydroxy-tautomer and the other one as an N-oxide tautomer. In the molecules of N-hydroxy-tautomeric form, the intramolecular hydrogen bonds of the O–H⋯O type (distances ranged from 2.54 to 2.57 Å) were present, and this is apparently a common pattern for N-hydroxy-tautomers in crystals.

Thus, it seems that in structures under consideration, tautomeric equilibrium may easily be shifted due to any source of external influence, for example, conditions of crystallization or the presence of water molecules. As experiments on biological activity testing involve more complicated media than “just water”, one cannot unambiguously determine in what tautomeric form do the 1-hydroxyimidazoles under consideration interact with viral proteins.

The methylation of –OH group (structure 6a) led to the exclusion of the possibility of forming both intra- and intermolecular hydrogen bonds of O–H⋯O type. In the crystal structure corresponding to the molecule 6a, strong stacking interactions were observed between the aryl aromatic fragments of nearby molecules located parallel to each other, at a distance of approximately 4.06(2) Å (Fig. 7(a)). Similarly, the alkylation of –OH group by –CH₂(2-BrC₆H₄) in the crystal structure of 11b led to the exclusion of the possibility of forming both intra- and intermolecular hydrogen bonds of O–H⋯O type. However, there is a possibility of the formation of halogen intermolecular interactions (Fig. 7(b)).

Fig. 7. Fragment of the crystal structure 6a (a) and 11b (b).

2.4. Antiviral activity against orthopoxviruses in Vero cell cultures

All the synthesized 2-arylimidazoles were tested for cytotoxicity and antiviral activity against VACV in the Vero cell culture (Tables 3 and S3†).

Cytotoxicity and antiviral activity of 2-arylimidazoles 4a–c, 5a–e, 6a, b, 7a–e, 9a, c, 10a, b, 11a–e, and 13 against the vaccinia virus (VACV, Copenhagen strain) in the Vero cell culture.

No.	R1	R2	CC50, μM (M ± SD, n = 3)	IC50 (VACV), μM (M ± SD, n = 3)	SI
4a	NO2	H	142.38 ± 48.45	0.133 ± 0.03	1072
4b	CN	H	174.89 ± 41.94	0.498 ± 0.14	351
4c	CF3	H	46.25 ± 8.63	0.154 ± 0.03	300
5a	NO2	H	28.33 ± 6.89	0.651 ± 0.19	44
5b	CN	H	84.56 ± 20.45	1.12 ± 0.34	76
5c	CF3	H	3.87 ± 1.76	0.07 ± 0.03	55
5d	N(CH3)2	H	298.87 ± 67.75	12.92 ± 2.84	23
5e	OCH3	H	614.37 ± 146.44	17.91 ± 2.72	34
6a	NO2	CH3	1154.34 ± 272.41	3.96 ± 0.22	291
6b	CN	CH3	757.43 ± 97.85	28.51 ± 7.12	27
7a	NO2	CH3	443.58 ± 102.02	4.76 ± 0.11	93
7b	CN	CH3	1057.66 ± 281.26	228.02 ± 47.88	<8
7c	CF3	CH3	176.02 ± 35.20	5.90 ± 1.77	30
7d	N(CH3)2	CH3	197.19 ± 49.32	62.08 ± 14.65	<8
7e	OCH3	CH3	356.91 ± 90.28	207.35 ± 30.50	<8
9a	NO2	—	2440.93 ± 610.23	183.39 ± 42.17	13
9c	CF3	—	2080.80 ± 520.22	14.25 ± 4.13	146
10a	NO2	—	2979.00 ± 655.38	N/A	—
10b	CN	—	1508.14 ± 346.87	N/A	—
11a	NO2	CH2C6H5	1162.40 ± 180.11	6.39 ± 1.59	182
11b	NO2	CH2(2-BrC6H4)	100.15 ± 18.71	2.91 ± 0.79	34
11c	NO2	CH2(3,4-Cl2C6H3)	108.62 ± 37.15	0.61 ± 0.30	178
11d	NO2	CH2(2,6-F2C6H3)	51.71 ± 10.34	1.22 ± 0.75	43
11e	NO2	CH2(2,5-(CH3)2C6H3)	104.89 ± 22.17	1.34 ± 0.43	79
13	NO2	—	359.45 ± 97.08	27.67 ± 3.09	13
Cidofovir			1111.5 ± 163.44	32.47 ± 2.26	34
NIOCH-14			1193.4 ± 228.68	0.008 ± 0.003	149 175

As it was supposed, among the compounds under consideration, the most promising virus-inhibiting activity was displayed by 2-aryl-1-hydroxyimidazoles 4a–c and 5a–c with electron-withdrawing substituents in the aryl moiety. Both in the series of 1-hydroxyimidazoles 4a–c with annulated cycle in positions 4, 5 of imidazole and in the series of 5-acetyl-1-hydroxyimidazoles 5a–c, the least perspective were the molecules 4b and 5b with the weakest electron acceptor, i.e., cyan group (IC₅₀ = 0.49 μM for 4b and IC₅₀ = 1.12 μM for 5b). Interestingly, for 1-hydroxyimidazoles 4a, c, containing a rigidly fixed carbonyl group in position 5 of the imidazole, the values of activities for the compounds with nitro group 4a and trifluoromethyl group 4c were comparable (IC₅₀ = 0.13 μM and IC₅₀ = 0.15 μM, respectively). For 5-acetyl-1-hydroxy-4-methyl-(2-(4-trifluoromethyl)phenyl)imidazole 5c, virus-inhibiting concentration was significantly lower (IC₅₀ = 0.07 μM) than similarly substituted 2-(4-nitrophenyl)imidazole 5a (IC₅₀ = 0.65 μM). Unfortunately, all these derivatives, 4a–c and 5a–c, were comparatively cytotoxic, and the most active compound is 5c, which is also the most cytotoxic (CC₅₀ = 3.87 μM).

5-Acetyl-2-aryl-1-hydroxyimidazoles 5d, e with electron-donating groups in the aryl moiety were less cytotoxic, but their virus-inhibiting activities were rather poor.

For 2-aryl-1-hydroxyimidazoles with electron-withdrawing substituents (4a–c, 5a–c), the methylation products both by the oxygen atom (methoxyimidazoles 6a, b, 7a–c) and by the nitrogen atom (imidazole 3-oxides 9a, c, 10a, b) tended to be less cytotoxic in the Vero cell culture, especially in the case of imidazole 3-oxides. However, the decrease in cytotoxicity was also accompanied by a significant decrease in the inhibitory activity. Thus, for the most active in this series is 1-methoxyimidazole 6a with IC₅₀ = 3.96 μM. The benzylation of the leader compound 1-hydroxyimidazole 4a both at the oxygen atom (see compound 11a) and at the nitrogen atom (see compound 13) also resulted in a decrease in the cytotoxicity of the compounds – but only if there were no additional substituents in the benzyl moiety. The presence of the halogen atoms (structures 11b–d) or methyl groups (11e) in the benzyloxy moiety led to compounds that were more cytotoxic than the starting 1-hydroxyimidazole 4a. Counter-intuitively, these compounds are more active than the similar ones substituted at positions 2, 4, and 5 of heterocycle 1-methoxyimidazole 6a.

For the most active representative of this series 11c, the virus-inhibiting concentration was IC₅₀ = 0.61 μM, which was comparable to the activities of 1-hydroxyimidazoles under consideration.

It should be mentioned that imidazole 3-oxides 9a, c, 10a, b, and 13 did not reveal any significant activity.

Thus, the screening of antiviral activity of 2-arylimidazole derivatives against VACV allows deriving the following SAR (Fig. 8). The variation of the substituents in the phenyl moiety in position 2 of heterocycle may lead to the conclusion that the most effective 1-hydroxyimidazole derivatives possess electron-withdrawing groups (R¹ = NO₂, CN, and CF₃) in the para-position. The fixation of the carbonyl group in position 5 of imidazole by involvement in the aliphatic cycle results in a decrease in the cytotoxicity of the compounds and to the improvement of virus-inhibiting activity.

Fig. 8. SAR hypothesis for the structures under consideration.

As it has been previously mentioned,^26,32 for 1-hydroxyimidazoles, the presence of a carbonyl group in position 5 of the imidazole ring and a hydroxy group at the nitrogen atom allowed the formation of inter- and intramolecular H-bonds, and may also have an impact on the prevailing tautomer. In the present case, 1-hydroxyimidazoles with a free hydroxy group at the nitrogen atom (R² = H) proved to be the most active compounds, which seems to be determined by their capability of H-bond formation, while these structures were significantly cytotoxic. If the protecting groups were present (R² – methyl or benzyl), then the activity decreased, as well as the cytotoxicity of the compounds. However, if the same groups were situated at the nitrogen atom (imidazole 3-oxides), then in general the structures were not active and not cytotoxic. The influence of the substituents in positions 4 and 5 of the imidazole ring (R³ and R⁴) determined the changes in the cytotoxicity of the compounds: rigid fixation of the carbonyl group was connected to the decrease in the cytotoxicity of 2-arylimidazole derivatives.

Compounds 4a–c, 5a, 6a, 7a, and 11a–c that revealed the highest activity and moderate cytotoxicity in the experiments with VACV were also tested for the antiviral activity against such zoonotic orthopoxviruses as the cowpox virus (CPXV) and the mousepox (ectromelia) virus (ECTV) (Tables 4 and S4†).

Cytotoxicity and antiviral activity of 2-arylimidazoles 4a–c, 5a, 6a, 7a, and 11a–e against the cowpox virus (CPXV, Grishak strain) and the ectromelia virus (ECTV, K-1 strain) in the Vero cell culture.

No.	CC50, μM (M ± SD, n = 3)	IC50 (CPXV), μM (M ± SD, n = 3)	SI (CPXV)	IC50 (ECTV), μM (M ± SD, n = 3)	SI (ECTV)
4a	142.38 ± 48.45	1.16 ± 0.36	123	0.39 ± 0.16	358
4b	174.89 ± 41.94	17.13 ± 4.28	10	2.49 ± 0.85	70
4c	46.25 ± 8.63	2.41 ± 0.28	19	0.49 ± 0.03	94
5a	28.33 ± 6.89	5.90 ± 1.41	<8	1.30 ± 0.38	22
6a	1154.35 ± 272.41	17.92 ± 4.88	65	12.53 ± 0.57	92
7a	443.58 ± 102.02	22.27 ± 4.89	20	13.88 ± 1.13	32
11a	1162.41 ± 180.11	78.18 ± 19.54	15	30.86 ± 2.63	38
11b	100.15 ± 18.71	29.07 ± 2.99	<8	28.73 ± 2.81	<8
11c	108.62 ± 37.15	7.04 ± 1.55	15	1.80 ± 0.59	60
11d	51.71 ± 10.34	7.39 ± 1.63	<8	4.49 ± 1.34	12
11e	104.89 ± 22.17	32.33 ± 2.24	<8	14.42 ± 4.96	<8
Cidofovir	1111.5 ± 163.44	48.24 ± 4.43	23	39.44 ± 3.23	28
NIOCH-14	1193.4 ± 228.68	0.009 ± 0.004	132 600	0.008 ± 0.003	149 175

As was in the case of VACV, the most perspective virus-inhibiting activity against ECTV was exhibited by compounds 4a and 4c (IC^ECTV₅₀ = 0.39 μM and IC^ECTV₅₀ = 0.49 μM, respectively). The same compounds displayed the most interesting results against CPXV (IC^CPXV₅₀ = 1.16 μM for 4a and IC^CPXV₅₀ = 2.41 μM for 4c). Due to the already mentioned high cytotoxicity of the derivative 4c, 1-hydroxyimidazole 4a with a nitro group on the aryl moiety appeared to be the most promising one (SI^CPXV = 123; SI^ECTV = 358). In general, the activity of 2-arylimidazoles against CPXV and ECTV was lower than the one against VACV. These data correlated with our previously published results.²⁴

Compounds 4a–c and 6a with the highest selectivity indices against VACV and ECTV were also tested for the antiviral activity against the Variola virus (VARV) in the Vero cell culture (Tables 5 and S5†).

Cytotoxicity and antiviral activity of 2-arylimidazoles 4a–c and 6a against the variola virus (VARV, India3a strain) in the Vero cell culture.

No.	CC50, μM (M ± SD, n = 3)	IC50 (VARV), μM (M ± SD, n = 3)	SI (VARV)
4a	142.38 ± 48.45	0.38 ± 0.09	373
4b	174.89 ± 41.94	1.45 ± 0.36	120
4c	46.25 ± 8.63	0.18 ± 0.03	257
6a	1154.34 ± 272.41	3.96 ± 0.22	291
Cidofovir	1111.5 ± 163.44	43.15 ± 5.48	26
NIOCH-14	1193.4 ± 228.68	0.008 ± 0.003	149 175

All the studied compounds (4a–c, 6a) were active against VARV. It should be mentioned that in the molecule of 2-arylimidazole 4c with the best virus-inhibiting concentration (IC^VARV₅₀ = 0.18 μM, SI^VARV = 25), a trifluoromethyl substituent was present in the structure of the registered comparative drug NIOCH-14. However, the highest selectivity index was displayed by compound 4a with a nitro group in the aryl moiety (SI^VARV = 373).

All the structures under consideration were more effective than cidofovir. The lowest virus inhibition was observed for 2-(4-cyanophenyl)-1-hydroxyimidazole 4b (IC^VARV₅₀ = 1.45 μM) and 1-methoxy-2-(4-nitrophenyl)imidazole 6a (IC^VARV₅₀ = 3.96 μM).

3. Conclusions

In the course of the present work, novel 2-arylimidazole derivatives were synthesized and tested for anti-orthopoxvirus activity. Prototropic tautomerism of 2-aryl-1-hydroxyimidazoles was considered. By means of NMR spectroscopy, it was confirmed that all the obtained 2-aryl-1-hydroxyimidazoles 4a–c and 5a–e in the DMSO-d₆ solution existed predominantly in the N-hydroxy-tautomeric form. According to the X-ray analysis data, in the crystal state, 1-hydroxyimidazoles 5a–c also existed as the N-hydroxy-tautomer, while compounds 4b, c existed as the N-oxide tautomer. For compound 5c, both tautomeric forms were observed in the crystal simultaneously (C₁₃H₁₁F₃N₂O₂·0.5H₂O), presumably due to the influence of the water molecule.

All the new 2-arylimidazole derivatives 4a–c, 5a–e, 6a, b, 7a–e, 9a, c, 10a, b, 11a, e, and 13 were tested for the antiviral activity against VACV. Compounds with the most perspective virus-inhibiting concentrations had a 2-(4-nitrophenyl)- (4a, IC₅₀ = 0.13 μM) or a 2-(4-(trifluoromethyl)phenyl)- (5c, IC₅₀ = 0.07 μM) moieties in position 2 of the imidazole ring, while the compounds with electron-donating groups in the aryl substituent (5d, e) were less active. It should also be mentioned that derivatives with a trifluoromethyl group were more cytotoxic than those with the nitro group. Proceeding from a free acetyl group in position 5 to a fixed carbonyl group in the annulated saturated cycle led to a decrease of cytotoxicity.

2-Arylimidazole derivatives under consideration appeared to be less active against zoonotic orthopoxviruses (CPXV and ECTV) than against VACV. The leader compound 4a had the following selectivity indices: SI^VACV = 1072; SI^CPXV = 123; SI^ECTV = 358.

It was also revealed that the most perspective 2-arylimidazole derivatives were also active against the Variola virus, the leader compound in the series being 1-hydroxyimidazole 4a (IC^VARV₅₀ = 0.38 μM; SI^VARV = 373).

4. Experimental

4.1. Chemistry

4.1.1. General information

The chemicals used in the experiment were purchased from commercial sources and used without further purification.

¹H and ¹³C NMR spectra were recorded using Avance™-400 (Bruker) and Inova 400 (Agilent) spectrometers operating at 400.02 MHz (for ¹H) and 100.59 MHz (for ¹³C) respectively. The chemical shifts were determined with an accuracy of 0.01 ppm relative to residual solvent signals and translated to the internal standard (TMS), coupling constants were measured with an accuracy of 0.1 Hz. The assignment of ¹H and ¹³C signals is based on 2D NMR experiments (HMBC and HSQC), which were performed using standard pulse sequences from the Agilent library.

The elemental composition of compounds was determined from the high-resolution mass spectra (HRMS) recorded using a DFS Thermo Scientific spectrometer in the full scan mode (0–500 m/z, 70 eV electron impact ionization, direct sample injection). Moreover, high-resolution mass spectra (HRMS) were recorded using a Bruker micrOTOF II instrument with electrospray ionization (ESI).

4.1.2. General procedure for 1-hydroxyimidazoles 4a–c and 5a–e

A mixture of starting aldehyde (1 equiv.), 2-(hydroxyimino)-5,5-dimethylcyclohexane-1,3-dione 2 or 3-(hydroxyimino)pentane-2,4-dione 3 (1 equiv.) and ammonium acetate (1.02–1.25 equiv.) in glacial acetic acid was stirred at room temperature for 1–6 h and allowed to stand overnight. The reaction mixture was poured into water and the precipitate was filtered off and then purified by recrystallization or refluxing in an appropriate solvent.

3-Hydroxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (4a)

According to the general procedure, the reaction of 4-nitrobenzaldehyde 1a (4.53 g, 0.0300 mol), oxime 2 (3.87 g, 0.0300 mol) and ammonium acetate (2.53 g, 0.0325 mol) in glacial acetic acid (25 mL) afforded 5.26 g (69%) of product 4a as a yellow solid (purified by washing with diethyl ether (15 mL) and refluxing in n-hexane (20 mL)). M.p. 130–132 °C. ¹H NMR (DMSO-d₆) δ 12.81 (br. s, 1H, OH); 8.34 (s, 4H, H–Ar); 2.68 (s, 2H, CH₂); 2.39 (s, 2H, CH₂); 1.06 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.4, 149.9, 147.5, 142.2, 133.9, 128.5, 123.9, 123.8, 52.5, 38.4, 35.4, 27.9 ppm. HRMS (ESI) m/z: calcd. for C₁₅H₁₅N₃O₄ [M + H]⁺ 302.1135, found 302.1131.

4-(1-Hydroxy-5,5-dimethyl-7-oxo-4,5,6,7-tetrahydro-1H-benzo[d]imidazol-2-yl)benzonitrile (4b)

According to the general procedure, the reaction of 4-cyanobenzaldehyde 1b (2.620 g, 0.020 mol), oxime 2 (3.380 g, 0.020 mol) and ammonium acetate (1.925 g, 0.025 mol) in glacial acetic acid (20 mL) afforded 2.21 g (54%) of product 4b as a beige solid (purified by refluxing in water (25 mL)). M.p. 224–226 °C. ¹H NMR (DMSO-d₆) δ 12.52 (br. s, 1H, OH); 8.26 (d, J = 8.4 Hz, 2H, H–Ar); 7.96 (d, J = 8.4 Hz, 2H, H–Ar); 2.69 (s, 2H, CH₂); 2.40 (s, 2H, CH₂); 1.07 (s, 6H, 2CH₃) ppm. ¹H NMR (CDCl₃) δ = 11.89 (s, 1H, OH); 8.20 (d, J = 8.4 Hz, 2H, H–Ar); 7.60 (d, J = 8.4 Hz, 2H, H–Ar); 2.62 (s, 2H, CH₂); 2.31 (s, 2H, CH₂); 1.03 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.2, 149.8, 142.5, 132.6, 132.1, 128.0, 123.6, 118.5, 111.7, 52.5, 38.4, 35.3, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₆H₁₅N₃O₂ [M]⁺ 281.1159, found 281.1161.

3-Hydroxy-6,6-dimethyl-2-(4-(trifluoromethyl)phenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (4c)

According to the general procedure, the reaction of 4-(trifluoromethyl)benzaldehyde 1c (3.48 g, 0.020 mol), oxime 2 (3.380 g, 0.020 mol) and ammonium acetate (1.92 g, 0.025 mol) in glacial acetic acid (20 mL) afforded 4.43 g (68%) of product 4c as a yellow solid (purified by washing with 1% solution of sodium bicarbonate (10 mL) and further refluxing in n-hexane (20 mL)). M.p. 198–200 °C. ¹H NMR (DMSO-d₆) δ 12.57 (br. s, 1H, OH); 8.30 (d, J = 8.2 Hz, 2H, H–Ar); 7.87 (d, J = 8.2 Hz, 2H, H–Ar); 2.68 (s, 2H, CH₂); 2.39 (s, 2H, CH₂); 1.07 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.1, 149.7, 142.8, 131.8, 129.4 (d, J^CF = 31.9 Hz), 129.3, 128.2, 125.5 (d, J^CF = 4.2 Hz), 124.0 (d, J^CF = 272 Hz, C̲F₃), 123.6, 52.5, 38.4, 35.3, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₆H₁₅F₃N₂O₂ [M]⁺ 324.1080, found 324.1081.

1-(1-Hydroxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethan-1-one (5a)

According to the general procedure, the reaction of 4-nitrobenzaldehyde 1a (4.53 g, 0.0300 mol), oxime 3 (3.87 g, 0.0300 mol) and ammonium acetate (2.53 g, 0.0325 mol) in glacial acetic acid (25 mL) afforded 5.06 g (65%) of product 5a as a yellow solid (purified by washing with diethyl ether (15 mL) and refluxing in n-hexane (200 mL)). M.p. 182–184 °C. ¹H NMR (DMSO-d₆) δ 12.62 (br. s, 1H, OH); 8.38 (d, J = 9.0 Hz, 2H, H–Ar); 8.34 (d, J = 9.0 Hz, 2H, H–Ar); 2.54 (s, 3H, CH₃); 2.41 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 188.5, 147.6, 142.3, 134.0, 128.5, 127.9, 124.4, 124.3, 30.3, 16.3 ppm. HRMS (EI) m/z: calcd. for C₁₂H₁₁N₃O₄ [M]⁺ 261.0744, found 261.0743.

4-(5-Acetyl-1-hydroxy-4-methyl-1H-imidazol-2-yl)benzonitrile (5b)

According to the general procedure, the reaction of 4-cyanobenzaldehyde 1b (0.51 g, 0.0039 mol), oxime 3 (0.50 g, 0.0039 mol) and ammonium acetate (0.31 g, 0.0040 mol) in glacial acetic acid (20 mL) afforded 0.36 g (38%) of product 5b as a beige solid (purified by washing with water (10 mL)). M.p. 188–190 °C. ¹H NMR (DMSO-d₆) δ 12.52 (s, 1H); 8.28 (d, J = 8.2 Hz, 2H, H–Ar); 7.97 (d, J = 8.2 Hz, 2H, H–Ar); 2.53 (s, 3H, CH₃); 2.41 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 187.9, 132.5, 132.1, 131.9, 127.8, 127.3, 118.5, 111.4, 29.8, 16.1 ppm. HRMS (EI) m/z: calcd. for C₁₃H₁₁N₃O₂ [M]⁺ 241.0846, found 241.0849.

1-(1-Hydroxy-4-methyl-2-(4-(trifluoromethyl)phenyl)-1H-imidazol-5-yl)ethan-1-one (5c)

According to the general procedure, the reaction of 4-(trifluoromethyl)benzaldehyde 1c (3.48 g, 0.02 mol), oxime 3 (2.58 g, 0.02 mol) and ammonium acetate (1.93 g, 0.025 mol) in glacial acetic acid (25 mL) afforded 3.69 g (65%) of product 5c as a beige solid (purified by washing with water (20 mL) and refluxing in n-hexane (20 mL)). M.p. 98–100 °C. ¹H NMR (DMSO-d₆) δ 12.52 (br. s, 1H, OH); 8.31 (d, J = 8.2 Hz, 2H, H–Ar); 7.87 (d, J = 8.2 Hz, 2H, H–Ar); 2.53 (s, 3H, CH₃); 2.40 (s, 3H, CH₃) ppm. ¹H NMR (CDCl₃) δ 8.37 (d, J = 8.2 Hz, 2H, H–Ar); 7.74 (d, J = 8.2 Hz, 2H, H–Ar); 2.63 (s, 3H, CH₃); 2.62 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 187.9, 131.8, 129.1 (d, J^CF = 32 Hz), 127.9, 127.1, 125.5, 124.0 (d, J^CF = 272 Hz), 121.3, 29.8, 16.0 ppm. HRMS (EI) m/z: calcd. for C₁₃H₁₁F₃N₂O₂ [M]⁺ 284.0767, found 284.0764. Anal. C₁₃H₁₁N₂O₂F₃·0.5H₂O (293.08): calc. C 53.23, H 4.09, N 9.55; found C 53.22, H 4.25, N 9.50.

1-(2-(4-(Dimethylamino)phenyl)-1-hydroxy-4-methyl-1H-imidazol-5-yl)ethan-1-one (5d)

According to the general procedure, the reaction of 4-(dimethylamino)benzaldehyde 1d (4.47 g, 0.0300 mol), oxime 3 (3.87 g, 0.0300 mol) and ammonium acetate (2.50 g, 0.0325 mol) in glacial acetic acid (30 mL) afforded 1.58 g (20%) of product 5d as a yellow solid (purified by column chromatography (silica gel, eluent: chloroform)). M.p. 166–168 °C. ¹H NMR (CDCl₃) δ 8.10 (d, J = 8.8 Hz, 2H, H–Ar); 6.75 (d, J = 8.8 Hz, 2H, H–Ar); 3.03 (s, 6H, 2CH₃); 2.56 (s, 3H, CH₃); 2.53 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 12.24 (s, 1H, OH); 7.94 (d, J = 9.0 Hz, 2H, H–Ar); 6.77 (d, J = 9.0 Hz, 2H, H–Ar); 2.97 (s, 6H, 2CH₃); 2.47 (s, 3H, CH₃); 2.36 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 187.1, 150.6, 142.4128.4, 125.8, 119.9, 114.9, 111.3, 39.9, 29.3, 16.1 ppm. HRMS (EI) m/z: calcd. for C₁₄H₁₇N₃O₂ [M]⁺ 259.1315, found 259.1317.

1-(1-Hydroxy-2-(4-methoxyphenyl)-4-methyl-1H-imidazol-5-yl)ethan-1-one (5e)

According to the general procedure, the reaction of 4-methoxybenzaldehyde 1e (4.08 g, 0.0300 mol), oxime 3 (3.87 g, 0.0300 mol) and ammonium acetate (2.50 g, 0.0325 mol) in glacial acetic acid (30 mL) afforded 1.65 g (22%) of product 5e as a white solid (purified by recrystallization from water (30 mL)). M.p. 88–90 °C. ¹H NMR (CDCl₃) δ 13.86 (s, 1H, OH); 8.16 (d, J = 8.9 Hz, 2H, H–Ar); 6.97 (d, J = 8.9 Hz, 2H, H–Ar); 3.85 (s, 3H, CH₃); 2.56 (s, 3H, CH₃); 2.55 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 12.20 (br. s, 1H); 8.04 (d, J = 8.4 Hz, 2H, H–Ar); 7.05 (d, J = 8.4 Hz, 2H, H–Ar); 3.82 (s, 3H, OCH₃); 2.49 (s, 3H, CH₃); 2.38 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 187.4, 160.1, 136.9, 135.4, 129.3, 129.1, 126.2, 114.0, 55.2, 29.6, 16.3 ppm. HRMS (EI) m/z: calcd. for C₁₃H₁₄N₂O₃ [M]⁺ 246.0999, found 246.0997.

4.1.3. General procedure for 1-methoxyimidazoles 6a, b and 7a–e

A mixture of starting 1-hydroxyimidazole (1 equiv.) and potassium hydroxide (1–1.5 equiv.) in DMF was cooled to 4 °C and then methyl iodide (1 equiv.) was added dropwise while stirring. The reaction mixture was stirred at room temperature for at least 4 h and then poured into water. The obtained precipitate was filtered off, washed with water and purified if needed.

3-Methoxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (6a)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a (2.10 g, 0.007 mol), potassium hydroxide (0.56 g, 0.010 mol) and methyl iodide (0.4 mL, 0.99 g, 0.007 mol) in DMF (20 mL) afforded 1.00 g (45%) of product 6a as a yellow solid (purified by refluxing in water (20 mL) and filtering hot). M.p. 148–150 °C. ¹H NMR (DMSO-d₆) δ 8.38 (d, J = 8.7 Hz, 2H, H–Ar); 8.33 (d, J = 8.7 Hz, 4H, H–Ar); 4.08 (s, 3H, OCH₃); 2.72 (s, 2H, CH₂); 2.45 (s, 2H, CH₂); 1.08 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) 186.0, 150.8, 147.8, 141.4, 133.0, 128.6, 124.2, 122.3, 67.2, 52.2, 38.3, 35.4, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₆H₁₇N₃O₄ [M]⁺ 315.1214, found 315.1208.

4-(1-Methoxy-5,5-dimethyl-7-oxo-4,5,6,7-tetrahydro-1H-benzo[d]imidazol-2-yl)benzonitrile (6b)

According to the general procedure, the reaction of 1-hydroxyimidazole 4b (0.562 g, 0.002 mol), potassium hydroxide (0.112 g, 0.002 mol) and methyl iodide (0.12 mL, 0.284 g, 0.002 mol) in DMF (20 mL) afforded 0.140 g (24%) of chromatographically pure product 6b as a beige solid. M.p. 160–162 °C. ¹H NMR (DMSO-d₆) δ 8.24 (d, J = 8.3 Hz, 2H, H–Ar); 8.01 (d, J = 8.3 Hz, 2H, H–Ar); 4.06 (s, 3H, OCH₃); 2.71 (s, 2H, CH₂); 2.44 (s, 2H, CH₂); 1.08 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 185.8, 150.6, 141.7, 132.9, 131.3, 128.0, 122.1, 118.3, 112.3, 67.1, 52.2, 38.3, 35.3, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₇H₁₇N₃O₂ [M]⁺ 295.1315, found 295.1314.

1-(1-Methoxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethan-1-one (7a)

According to the general procedure, the reaction of 1-hydroxyimidazole 5a (2.00 g, 0.008 mol), potassium hydroxide (0.50 g, 0.009 mol) and methyl iodide (0.5 mL, 1.14 g, 0.008 mol) in DMF (20 mL) afforded 1.56 g (71%) of product 7a as a yellow solid (purified by refluxing in n-hexane (20 mL)). M.p. 122–124 °C. ¹H NMR (DMSO-d₆) δ 8.36 (d, J = 9.5 Hz, 2H, H–Ar); 8.30 (d, J = 9.5 Hz, 2H, H–Ar); 3.95 (s, 3H, CH₃); 2.54 (s, 3H, CH₃); 2.46 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.9, 147.7, 143.4, 139.1, 133.1, 128.4, 125.3, 124.2, 67.6, 29.8, 16.8 ppm. HRMS (ESI) m/z: calcd. for C₁₃H₁₃N₃O₄ [M + H]⁺ 276.0979, found 276.0979.

4-(5-Acetyl-1-methoxy-4-methyl-1H-imidazol-2-yl)benzonitrile (7b)

According to the general procedure, the reaction of 1-hydroxyimidazole 5b (0.145 g, 0.6 mmol), potassium hydroxide (0.034 g, 0.6 mmol) and methyl iodide (0.037 mL, 0.085 g, 0.6 mmol) in DMF (5 mL) afforded 0.040 g (27%) of chromatographically pure product 7b as a beige solid. M.p. 188–190 °C. ¹H NMR (DMSO-d₆) δ 8.22 (d, J = 8.2 Hz, 2H, H–Ar); 8.01 (d, J = 8.2 Hz, 2H, H–Ar); 3.93 (s, 3H, OCH₃); 2.53 (s, 3H, CH₃); 2.46 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 188.0, 132.9, 132.6, 131.9, 127.9, 127.8, 127.3, 118.5, 111.4, 67.4, 29.8, 16.0 ppm. HRMS (EI) m/z: calcd. for C₁₄H₁₃N₃O₂ [M]⁺ 255.1002, found 255.1002.

1-(1-Methoxy-4-methyl-2-(4-(trifluoromethyl)phenyl)-1H-imidazol-5-yl)ethan-1-one (7c)

According to the general procedure, the reaction of 1-hydroxyimidazole 5c (0.71 g, 0.0025 mol), potassium hydroxide (0.16 g, 0.0030 mol) and methyl iodide (0.16 mL, 0.36 g, 0.0025 mol) in DMF (20 mL) afforded 0.55 g (74%) of chromatographically pure product 7c as a beige solid. M.p. 68–70 °C. ¹H NMR (CDCl₃) δ 8.28 (d, J = 8.1 Hz, 2H, H–Ar); 7.79 (d, J = 8.1 Hz, 2H, H–Ar); 3.95 (s, 3H, OCH₃); 2.63 (s, 3H, CH₃); 2.61 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 8.26 (d, J = 8.1 Hz, 2H, H–Ar); 7.91 (d, J = 8.1 Hz, 2H, H–Ar); 3.92 (s, 3H, OCH₃); 2.53 (s, 3H, CH₃); 2.46 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.7, 143.1, 139.8, 131.1, 129.8, 129.6, 128.0, 125.9, 125.9, 125.0, 124.8, 123.0, 67.3, 29.6, 16.7 ppm. HRMS (ESI) m/z: calcd. for C₁₄H₁₃F₃N₂O₃ [M + H]⁺ 299.1002, found 299.0998.

1-(2-(4-(Dimethylamino)phenyl)-1-methoxy-4-methyl-1H-imidazol-5-yl)ethan-1-one (7d)

According to the general procedure, the reaction of 1-hydroxyimidazole 5d (0.78 g, 0.003 mol), potassium hydroxide (0.22 g, 0.004 mol) and methyl iodide (0.19 mL, 0.43 g, 0.003 mol) in DMF (15 mL) afforded 0.48 g (59%) of chromatographically pure product 7d as a yellow solid. M.p. 98–100 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.8 Hz, 2H, H–Ar); 6.74 (d, J = 8.8 Hz, 2H, H–Ar); 3.84 (s, 3H, CH₃); 3.02 (s, 6H, 2CH₃); 3.53 (s, 3H, CH₃); 3.52 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 7.90 (d, J = 9.0 Hz, 2H, H–Ar); 6.81 (d, J = 9.0 Hz, 2H, H–Ar); 3.84 (s, 3H, CH₃); 2.99 (s, 6H, 2CH₃); 2.47 (s, 3H, CH₃); 2.41 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 185.8, 151.1, 143.3, 142.6, 128.5, 123.8, 114.2, 111.7, 66.1, 39.6, 29.4, 16.8 ppm. HRMS (EI) m/z: calcd. for C₁₅H₁₉N₃O₂ [M]⁺ 273.1472, found 273.1470.

1-(1-Methoxy-2-(4-methoxyphenyl)-4-methyl-1H-imidazol-5-yl)ethan-1-one (7e)

According to the general procedure, the reaction of 1-hydroxyimidazole 5e (0.50 g, 0.002 mol), potassium hydroxide (0.168 g, 0.003 mol) and methyl iodide (0.12 mL, 0.28 g, 0.002 mol) in DMF (1 mL) afforded 0.35 g (67%) of product 7e as a beige solid (purified by recrystallizing from water (50 mL)). M.p. 110–112 °C. ¹H NMR (DMSO-d₆) δ 7.99 (d, J = 8.9 Hz, 2H, H–Ar); 7.09 (d, J = 8.9 Hz, 2H, H–Ar); 3.85 (s, 3H, OCH₃); 3.83 (s, 3H, CH₃); 2.49 (s, 3H, CH₃); 2.42 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.2, 160.6, 143.1, 141.6, 129.0, 124.1, 119.7, 114.4, 66.5, 55.3, 29.4, 16.7 ppm. HRMS (EI) m/z: calcd. for C₁₄H₁₆N₂O₃ [M]⁺ 260.1155, found 260.1156.

4.1.4. (E)-N-Methyl-1-(4-nitrophenyl)methanimine (8a)

To 4-nitrobenzaldehyde 1a (1.51 g, 0.01 mol) while stirring at room temperature, a 40% aqueous solution (0.78 g) containing 0.31 g (0.01 mol) of methyl amine was added dropwise. The reaction mixture was stirred at room temperature for 5 h. The product was extracted with ether (20 mL × 2) and the extract was dried over anhydrous potassium carbonate. The solvent was removed under reduced pressure, yielding 1.24 g (76%) of chromatographically pure product 8a as a beige solid. M.p. 106–108 °C ¹H NMR (CDCl₃) δ 8.37 (s, 1H, N CH); 8.27 (d, J = 8.5 Hz, 2H, Ar–H); 7.87 (d, J = 8.5 Hz, 2H, Ar–H); 3.59 (s, 3H, CH₃) ppm.

4.1.5. (E)-4-((Methylimino)methyl)benzonitrile (8b)

4-Cyanobenzaldehyde 1b (3.93 g, 0.03 mol) was dissolved in water (30 mL) with heating and stirring. Then, a 40% aqueous solution (2.33 g) containing 0.93 g (0.03 mol) of methyl amine was added dropwise. The reaction mixture was stirred at 50–55 °C for 5 h and cooled to room temperature, and then the precipitate was filtered off and purified by refluxing in n-hexane (20 mL) and filtering hot. Product 8b (3.84 g, 89%) was obtained as white needles. M.p. 60–62 °C. ¹H NMR (CDCl₃) δ 8.30 (s, 1H, N CH); 7.79 (d, J = 8.3 Hz, 2H, Ar–H); 7.68 (d, J = 8.3 Hz, 2H, Ar–H); 3.55 (s, 3H, CH₃) ppm.

4.1.6. (E)-N-Methyl-1-(4-(trifluoromethyl)phenyl)methanimine (8c)

To 4-(trifluoromethyl)benzaldehyde 1c (5.22 g, 0.03 mol) under stirring at room temperature, a 40% aqueous solution (2.33 g) containing 0.93 g (0.03 mol) of methyl amine was added dropwise. The reaction mixture was stirred at room temperature for 6 h and allowed to stand overnight. Then, the solvent was removed under reduced pressure yielding 4.70 g (84%) of chromatographically pure product 8c as a light yellow oil. ¹H NMR (CDCl₃) δ 8.31 (s, 1H, N CH); 7.81 (d, J = 7.9 Hz, 2H, Ar–H); 7.66 (d, J = 7.9 Hz, 2H, Ar–H); 3.55 (s, 3H, CH₃) ppm.

4.1.7. General procedure for 1-methylimidazole 3-oxides 9a, c and 10a, b

A mixture of starting azomethine (1 equiv.) and the appropriate oxime (1 equiv.) in glacial acetic acid was stirred at room temperature for 2 h and allowed to stand overnight. The reaction mixture was poured into water and the product was extracted with chloroform. The extract was sequentially washed with potassium carbonate solution (3%) and water and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was triturated with diethyl ether in order to obtain the chromatographically pure desired product.

1,6,6-Trimethyl-2-(4-nitrophenyl)-4-oxo-4,5,6,7-tetrahydro-1H-benzo[d]imidazole 3-oxide (9a)

According to the general procedure, the reaction of azomethine 8a (0.49 g, 0.003 mol) and oxime 2 (0.51 g, 0.003 mol) in glacial acetic acid (23 mL) afforded 0.24 g (25%) of product 9a as a yellow solid. M.p. 228–230 °C. ¹H NMR (CDCl₃) δ 8.38 (d, J = 8.9 Hz, 2H; H–Ar); 8.08 (d, J = 8.9 Hz, 2H, H–Ar); 3.69 (s, 3H, NCH₃); 2.77 (s, 2H, CH₂); 2.49 (s, 2H, CH₂); 1.22 (s, 6H, 2CH₃) ppm. ¹H NMR (DMSO-d₆) δ 8.38 (d, J = 8.9 Hz, 2H, H–Ar); 8.18 (d, J = 8.9 Hz, 2H, H–Ar); 3.64 (s, 3H, CH₃); 2.85 (s, 2H, CH₂); 2.38 (s, 2H, CH₂); 1.11 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 184.9, 147.1, 139.9, 133.7, 130.4, 129.9, 123.8, 123.5, 52.3, 34.2, 33.8, 33.2, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₆H₁₇N₃O₄ [M]⁺ 315.1214, found 315.1210.

1,6,6-Trimethyl-4-oxo-2-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazole 3-oxide (9c)

Mixture of azomethine 8c (1.87 g, 0.01 mol) and oxime 2 (1.69 g, 0.01 mol) in glacial acetic acid (20 mL) was stirred at room temperature for 2 h. The reaction mixture was poured into water (50 mL), and the precipitate was filtered off and refluxed in water yielding 1.77 g (52%) of product 9c as a beige solid. M.p. 210–212 °C. ¹H NMR (CDCl₃ and DMSO-d₆) δ 7.60 (d, J = 8.1 Hz, 2H; H–Ar); 7.40 (d, J = 8.1 Hz, 2H, H–Ar); 3.26 (s, 3H, NCH₃); 2.41 (s, 2H, CH₂); 2.05 (s, 2H, CH₂); 0.81 (s, 6H, 2CH₃) ppm. ¹H NMR (DMSO-d₆) δ 8.08 (d, J = 8.5 Hz, 2H; H–Ar); 7.92 (d, J = 8.5 Hz, 2H, H–Ar); 3.61 (s, 3H, NCH₃); 2.84 (s, 2H, CH₂); 2.37 (s, 2H, CH₂); 1.11 (s, 6H, 2CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 184.7, 147.4, 143.1, 141.0, 139.3, 138.4, 130.3, 130.2, 129.4, 125.2, 52.2, 34.2, 33.7, 32.8, 27.9 ppm. HRMS (EI) m/z: calcd. for C₁₇H₁₆N₂O₂F₃ [M]⁺ 337.1158, found 337.1153.

4-Acetyl-1,5-dimethyl-2-(4-nitrophenyl)-1H-imidazole 3-oxide (10a)

According to the general procedure, the reaction of azomethine 8a (1.24 g, 0.008 mol) and oxime 3 (0.98 g, 0.008 mol) in glacial acetic acid (30 mL) afforded 1.40 g (64%) of product 10a as a yellow solid. M.p. 180–182 °C. ¹H NMR (CDCl₃) δ 8.37 (d, J = 8.8 Hz, 2H; H–Ar); 8.00 (d, J = 8.8 Hz, 2H, H–Ar); 3.65 (s, 3H, NCH₃); 2.82 (s, 3H, CH₃); 2.62 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 8.37 (d, J = 8.9 Hz, 2H, H–Ar); 8.14 (d, J = 8.9 Hz, 2H, H–Ar); 3.61 (s, 3H, NCH₃); 2.70 (s, 3H, CH₃); 2.53 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 190.6, 147.2, 133.4, 131.9, 130.9, 130.0, 128.1, 123.4, 32.5, 30.4, 10.1 ppm. HRMS (ESI) m/z: calcd. for C₁₃H₁₄N₃O₄ [M + H]⁺ 276.0979, found 276.0985.

4-Acetyl-2-(4-cyanophenyl)-1,5-dimethyl-1H-imidazole 3-oxide (10b)

According to the general procedure, the reaction of azomethine 8b (1.44 g, 0.01 mol) and oxime 3 (1.29 g, 0.01 mol) in glacial acetic acid (10 mL) afforded 1.58 g (59%) of product 10b as a yellow solid. M.p. 170–172 °C. ¹H NMR (CDCl₃) δ 7.90 (d, J = 8.3 Hz, 2H, H–Ar); 7.82 (d, J = 8.3 Hz, 2H, H–Ar); 3.62 (s, 3H, N–CH₃); 2.82 (s, 3H, CH₃); 2.61 (s, 3H, CH₃) ppm. ¹H NMR (DMSO-d₆) δ 8.04 (d, J = 8.4 Hz, 2H, H–Ar); 8.01 (d, J = 8.4 Hz, 2H, H–Ar); 3.58 (s, 3H, CH₃); 2.70 (s, 3H, CH₃); 2.52 (s, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 190.5, 133.0, 132.1, 132.1, 130.4, 128.3, 128.0, 118.4, 111.7, 32.3, 30.3, 10.0 ppm. HRMS (EI) m/z: calcd. for C₁₄H₁₂N₃O₂ [M]⁺ 254.0924, found 254.0926.

4.1.8. General procedure for 1-benzyloxyimidazoles 11a–e

A mixture of 1-hydroxyimidazole 4a (0.6 g, 2.0 mmol), potassium iodide (0.03 g, 0.2 mmol), potassium carbonate (0.28 g, 2.0 mmol) and appropriate benzyl halide (2.0 mmol) in acetonitrile (15 mL) was refluxed with stirring for 24 h. The reaction mixture was cooled to room temperature and poured into water, and the precipitate was filtered off, dried on air and subjected to column chromatography (silica gel, eluent: chloroform–methanol = 15 : 1) in order to obtain individual products.

3-(Benzyloxy)-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (11a)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a with benzyl chloride (0.23 mL, 0.25 g, 2.0 mmol) afforded 0.01 g (13%) of product 11a as a beige solid. M.p. 202–205 °C. ¹H NMR (DMSO-d₆) δ 8.28 (d, J = 8.9 Hz, 2H, H–Ar); 8.07 (d, J = 8.9 Hz, 2H, H–Ar); 7.29 (td, J = 5.8, 2.7 Hz, 1H, H–Ar); 7.22 (d, J = 5.8 Hz, 4H, H–Ar); 5.22 (s, 2H, CH₂); 2.73 (s, 2H, CH₂); 2.50 (s, 2H, CH₂); 1.11 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.9, 151.5, 148.0, 143.3, 133.6, 132.6, 130.9, 130.1, 129.7, 129.4, 128.8, 124.2, 122.7, 81.8, 52.7, 38.8, 35.9, 28.4 ppm. HRMS (EI) m/z: calcd. for C₂₂H₂₁N₃O₄ [M]⁺ 391.1527, found 391.1523.

3-((2-Bromobenzyl)oxy)-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (11b)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a with 2-bromobenzyl bromide (0.50 g, 2.0 mmol) afforded 0.56 g (60%) of product 11b as a yellow solid. M.p. 145–147 °C. ¹H NMR (DMSO-d₆) δ 8.19 (d, J = 8.8 Hz, 2H, H–Ar); 7.90 (d, J = 8.8 Hz, 2H, H–Ar); 7.34 (dd, J = 7.5, 1.6 Hz, 1H, H–Ar); 7.21–7.09 (m, 3H, H–Ar); 5.32 (s, 2H, CH₂); 2.73 (s, 2H, CH₂); 2.49 (s, 2H, CH₂); 1.11 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.6, 151.2, 147.6, 143.5, 133.2, 133.0, 132.6, 131.9, 131.8, 129.3, 127.8, 125.2, 123.5, 122.3, 80.6, 52.4, 38.4, 35.5, 28.1 ppm. HRMS (EI) m/z: calcd. for C₂₂H₂₀N₃O₄⁷⁹Br [M]⁺ 469.0632, found 469.0623.

3-((3,4-Dichlorobenzyl)oxy)-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (11c)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a with 3,4-dichlorobenzyl bromide (0.48 g, 2.0 mmol) afforded 0.18 g (20%) of product 11c as a yellow solid. M.p. 185–187 °C. ¹H NMR (DMSO-d₆) δ 8.25 (d, J = 8.9 Hz, 2H, H–Ar); 7.93 (d, J = 8.9 Hz, 2H, H–Ar); 7.46–7.39 (m, 2H, H–Ar); 7.05 (dd, J = 8.2, 2.0 Hz, 1H, H–Ar); 5.20 (s, 2H, CH₂); 2.73 (s, 2H, CH₂); 2.49 (s, 2H, CH₂); 1.11 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.5, 151.2, 147.7, 143.3, 133.2, 133.0, 132.8, 132.6, 131.0, 130.6, 130.5, 129.2, 124.3, 123.6, 122.0, 79.7, 52.3, 38.4, 35.5, 28.2, 28.0 ppm. HRMS (EI) m/z: calcd. for C₂₂H₁₉N₃O₄³⁵Cl₂ [M]⁺ 459.0747, found 459.0750.

3-((2,6-Difluorobenzyl)oxy)-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (11d)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a with 2,6-difluorobenzyl chloride (0.33 g, 2.0 mmol) afforded 0.37 g (43%) of product 11d as an orange solid. M.p. 142–145 °C. ¹H NMR (DMSO-d₆) δ 8.18 (d, J = 8.8 Hz, 2H, H–Ar); 7.84 (d, J = 8.8 Hz, 2H, H–Ar); 7.29–7.19 (m, 1H, H–Ar); 6.75 (t, J = 8.3 Hz, 2H, H–Ar); 5.36 (s, 2H, CH₂); 2.71 (s, 2H, CH₂); 2.47 (s, 2H, CH₂); 1.10 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.7, 161.8 (d, J^C–F = 250.6 Hz), 151.2, 147.6, 143.4, 133.3, 132.9, 130.6, 129.1, 123.4, 122.3, 111.2 (d, J^C–F = 24.2 Hz), 67.8, 52.3, 38.4, 35.6, 27.8 ppm. HRMS (EI) m/z: calcd. for C₂₂H₁₉F₂N₃O₄ [M]⁺ 427.1338, found 427.1337.

3-((2,5-Dimethylbenzyl)oxy)-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzo[d]imidazol-4-one (11e)

According to the general procedure, the reaction of 1-hydroxyimidazole 4a with 2,5-dimethylbenzyl chloride (0.31 g, 2.0 mmol) afforded 0.27 g (32%) of product 11e as a yellow solid. M.p. 123–125 °C. ¹H NMR (DMSO-d₆) δ 8.23 (d, J = 8.8 Hz, 2H, H–Ar); 7.92 (d, J = 8.8 Hz, 2H, H–Ar); 6.93 (d, J = 7.7 Hz, 1H, H–Ar); 6.82 (d, J = 7.7 Hz, 1H, H–Ar); 6.76 (s, 1H, H–Ar); 5.18 (s, 2H, CH₂); 2.73 (s, 2H, CH₂); 2.09 (s, 3H, CH₃); 2.06 (s, 3H, CH₃); 1.12 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 186.5, 151.2, 147.6, 143.7, 135.1, 134.8, 133.3, 132.3, 130.4, 130.4, 130.1, 129.3, 128.8, 123.5, 122.4, 79.4, 52.4, 38.5, 35.5, 28.0, 20.3, 17.9 ppm. HRMS (EI) m/z: calcd. for C₂₄H₂₅N₃O₄ [M]⁺ 419.1840, found 419.1847.

4.1.9. (E)-N-Benzyl-1-(4-nitrophenyl)methanimine (12)

A mixture of benzyl amine (1.10 g, 0.01 mol) and 4-nitrobenzaldehyde 1a (1.50 g, 0.01 mol) in ethanol (30 mL) was refluxed for 3 h. Then the solvent was removed under reduced pressure yielding 2.20 g (90%) of product 12 as a yellowish solid. M.p. 54–56 °C. ¹H NMR (DMSO-d₆) δ 8.67 (s, 1H, CH); 8.31 (d, J = 7.1 Hz, 2H, H–Ar); 8.04 (d, J = 7.1 Hz, 2H, H–Ar); 7.26–7.36 (m, 5H, H–Ar); 4.84 (s, 2H, CH₂) ppm.

4.1.10. 1-Benzyl-6,6-dimethyl-2-(4-nitrophenyl)-4-oxo-4,5,6,7-tetrahydro-1H-benzo[d]imidazole 3-oxide (13)

A mixture of oxime 2 (0.85 g, 5 mmol) and azomethine 12 (1.20 g, 5 mmol) in glacial acetic acid (10 mL) was stirred at room temperature for 24 h. Then the reaction mixture was poured into water (50 mL) and extracted with chloroform (50 mL × 2). The extract was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was triturated with n-hexane, filtered off and washed with diethyl ether yielding 1.01 g (52%) of product 13 as a beige solid. M.p. 171–173 °C. ¹H NMR (DMSO-d₆) δ 8.29 (d, J = 8.4 Hz, 2H, H–Ar); 7.95 (d, J = 8.4 Hz, 2H, H–Ar); 7.36–7.22 (m, 3H, H–Ar); 7.01 (d, J = 7.1 Hz, 2H, H–Ar); 5.35 (s, 2H, CH₂); 2.77 (s, 2H, CH₂); 2.41 (s, 2H, CH₂); 1.05 (s, 6H, CH₃) ppm. ¹³C NMR (DMSO-d₆) δ 185.3, 147.5, 140.2, 135.2, 134.0, 130.8, 129.8, 129.1, 128.1, 126.2, 124.4, 123.7, 52.2, 48.5, 34.6, 34.1, 27.8 ppm. HRMS (EI) m/z: calcd. for C₂₂H₂₁N₃O₄ [M]⁺ 391.1527, found 391.1520.

4.2. Calculation methods

The optimization of geometric parameters of systems under study with the subsequent solution of oscillation problem was performed at the DFT-theory level using the B3LYP functional^33,34 combined with the 6-311++G(d,p) basis set.^35–38 The absence of imaginary unit in the matrix of the second derivatives of the wave function was considered as evidence for the location of the stationary point on the potential energy surface. Thermodynamic parameters were calculated within the gaseous phase approximation at 298 K and atmospheric pressure and also subject to implicit solvation.³⁹ The PCM models with permittivity of the medium ε = 46.826 (corresponding to the value of permittivity of dimethyl sulfoxide (DMSO)) were used as a solvent. ¹³C and ¹H chemical shifts were calculated within the CSGT approximation^35,38 relative to tetramethyl silane (δ – 0 ppm). The calculations were carried out using the GAUSSIAN 16 software package.⁴⁰

4.3. Single-crystal X-ray analysis

Single crystals of the following compounds were obtained by a slow evaporation technique from saturated solutions of 5a (carbon tetrachloride), 5c (nitromethane), 5b (acetone), 4b (ethanol) and 11b (ethanol).

X-ray diffraction experiments with the samples of 5a, 5c, 5b, 4b and 11b were conducted using a TD-5000 single crystal diffractometer with MoKα (λ = 0.71073 Å) and a Dectris Pilatus 200k HPC detector. Data reduction was carried out using the CrysAlisPro v.171.42.49 software,⁴¹ and absorption correction was applied by the multi-scan method. The structure solution was obtained by SHELXT⁴² and the structure refinement was performed by SHELXL⁴³ using the Olex2 version 1.5 (ref. 44) software as a GUI. All non-hydrogen atoms were refined anisotropically, and the ADP of hydrogen atoms was U_iso(H) = 1.2·U_eq(atom) for non-methyl, and U_iso(H) = 1.5·U_eq(carbon) for methyl hydrogen atoms. The trifluoromethyl group of 5c was disordered, for F4a, F5A, F6A and F4B, F5B, F6B atoms DELU restrains were performed, and distances F5A–F6A, F6A–F4a, F4a–F5A, F4B–F6B, F5B–F6B and F5B–F4B were restrained by DANG instruction on 2.116 Å.

XCRD data have been deposited with the Cambridge Crystallographic Data Centre (CCDC) (see Tables S1 and S2†) and are available from the authors or at the address: https://www.ccdc.cam.ac.uk/structures (accessed on 4 March 2024).

4.4. Virology studies

Viruses

In the course of the work, the following orthopoxviruses were used: the vaccinia virus (VACV, strain Copenhagen), the Cowpox virus (CPXV, strain Grishak), the Ectromelia virus (mousepox) (ECTV, strain K-1) and Variola virus (VARV, strain India-3a). All the above-mentioned orthopoxviruses were obtained from the State Collection of Virus Infection and Rickettsiosis Agents of State Research Center of Virology and Biotechnology VECTOR of Rospotrebnadzor (Koltsovo, Novosibirsk region). The orthopoxviruses were grown in the Vero cell culture (kidney epithelial cells from an African green monkey) in DMEM (Dulbecco's modified Eagle's medium). The concentration of virus in the culture liquid was determined by a plaque technique by means of titration of virus-containing samples in the Vero cell culture, and then it was calculated and expressed in decimal logarithm (log₁₀) of plaque forming units in mL (PFU mL⁻¹).⁴⁵ The concentration of the orthopoxviruses in the samples used in the present work was from 4.5 to 6.7 log₁₀ PFU mL⁻¹. The cultured and used series of viruses with the indicated titer in work was stored at −70 °C.

4.4.1. Cell cultures

The passage cultures of Vero cells (kidney cells of an African green monkey) obtained from the Collection of Cell Cultures of State Research Center of Virology and Biotechnology VECTOR of Rospotrebnadzor (Koltsovo, Novosibirsk region, Russia) were used in the present work. A monolayer of cells was grown in 96-well plates (containing 100 μL of cell suspension with a concentration of 1 × 10⁵ cells per mL per well) in DMEM (BioloT Ltd, Russia) in the presence of 10% fetal bovine serum (FBS) (“HyClone”, USA) with the addition of penicillin (100 IU mL⁻¹), streptomycin (100 μg mL⁻¹) and 2.5 u mL⁻¹ of amphotericin B. The plates with cells were placed in a thermostat at 37 °C, with 5% CO₂ and 100% humidity for 2–3 days until a confluent cell monolayer was formed. In the course of cultivation of orthopoxviruses in Vero cells, the same medium with antibiotics with the addition of 2% FBS was used as a maintenance medium.

The evaluation of the antiviral activity of chemically synthesized compounds in vitro by a colorimetric method. The evaluation of the antiviral efficiency of the compounds was carried out using our adapted and modified technique.⁴⁶ Specimens of chemical compounds under study were preliminarily dissolved in dimethylsulfoxide (DMSO) in 20 mg mL⁻¹ concentration and stored in a freezing chamber at minus 70 °C. In the wells of 96-well plates containing a monolayer of Vero cells in 100 μL of DMEM with 2% fetal serum, first 50 μL of serial dilutions of the compounds under study were added, followed by the addition of 50 μL of orthopoxviruses in doses causing 100% cell destruction of control monolayer without compounds: 1000 PFU. Dilutions of orthopoxviruses were prepared in DMEM with 2% serum.

For the evaluation of cytotoxicity of samples, eight 3-fold dilutions of each specimen (1200, 400, 133.3, 44.4, 14.8, 4.9, 1.6 and 0.55 μg mL⁻¹) were prepared along with the control one – 0 μg mL⁻¹ in DMEM with 2% serum. For the evaluation of the antiviral activity of samples against orthopoxviruses, eight 3-fold dilutions (150, 50, 16.7, 5.5, 1.85, 0.6, 0.2 and 0.07 μg mL⁻¹) were prepared along with the control one – 0 μg mL⁻¹ in DMEM with 2% serum.

The dilutions of the specimens to be introduced to Vero cells were prepared in DMEM supplemented with 2% serum. Then every dilution was introduced into 6 wells in the volume of 50 μL. As a result, in a 96-well plate, 6 rows of wells with eight 3-fold dilutions of every specimen were obtained. Three rows out of 6 were used for the evaluation of antiviral activity – in these wells, 50 μL of virus-containing liquid per well was introduced. Three remaining rows were used for the evaluation of toxicity of the specimen under study – in these wells 50 μL of nutrient medium was introduced. Thus, the total volume of the liquid in every well was 200 μL. The starting concentration of the compound when testing their cytotoxicity was 300.0 μg mL⁻¹ and further 100.0, 33.3, 11.1, 3.7, 1.23, 0.4, 0.14 and 0 μg mL⁻¹ respectively. For certain compounds possessing antiviral activity with toxic concentrations >300 μg mL⁻¹, the cytotoxicity was additionally tested beginning with 600 μg mL⁻¹ concentration in the wells of plate. The starting concentration of compound when testing their antiviral activity was 37.5 μg mL⁻¹ and further 12.5, 4.175, 1.375, 0.46, 0.15, 0.05, 0.017 and 0 μg mL⁻¹ respectively. For certain compounds possessing antiviral activity with concentrations <0.017 μg mL⁻¹, the antiviral activity was additionally tested beginning with 1.375 μg mL⁻¹ concentration in the wells of the plate.

The toxic activity of the compounds was determined by the cell death caused by the compounds in the wells of the plate into which no virus was introduced. The monolayers of cells in the wells of the plate were used as controls – either the ones into which virus without compounds (virus control) was introduced or the monolayers of cells in wells into which neither the virus nor the compound was introduced (cell culture control).

After incubation of the plates with cells in the presence of either both virus and compounds, or only virus or compounds, or neither virus nor compound, for 4 days, the monolayers of the cells were stained with the vital dye neutral red. During 1.5 h, the dye was adsorbed by living cells that were not destroyed by the action of the virus or by the toxic action of the tested compound. After removing the dye and washing the wells from its unbound fraction by Hanks' solution, a lysis buffer was added: 0.01 M monoammonium phosphate, pH 3.5 (50%) and ethanol (50%). In this solution, the cell membranes were destroyed and the dye was transferred into the solution. The amount of the dye adsorbed by the living cells of the monolayer and transferred into the solution was evaluated by its optical density (OD), which is an indication of the number of cells in the monolayer that were undisturbed under the influence of either virus or compound, thus allowing us to evaluate the toxicity and antiviral activity of the tested compounds. The OD was measured using an Emax plate spectrophotometer (Molecular Devices, USA) at a length of 490 nm. The OD measurement results depending on the concentration of the compound were presented in semilog coordinates. Thus, concentration of the compounds was given at abscissa (x) axis at the logarithmic scale, while the optical density was given at ordinate (y) at the linear scale. The results were processed using the SoftMax 4.0 program (Molecular Devices, USA) that automatically calculated the 50% cytotoxic concentration (CC₅₀ in μg mL⁻¹) and 50% virus-inhibiting (effective) concentration (IC₅₀ in μg mL⁻¹) of the compounds. CC₅₀ is the concentration of the compound in the nutrient medium at which 50% of cells in uninfected monolayers are destroyed (loose viability). IC₅₀ is the concentration of the compound in the nutrient medium at which 50% of cells in infected monolayers are not destroyed (remain viable). The obtained CC₅₀ and IC₅₀ values of the compounds expressed in μg mL⁻¹ (Tables S3–S5†) were recalculated and presented in μM.

On the basis of CC₅₀ and IC₅₀ values, the selectivity index (SI) was calculated. The SI is a value indicating the number of times the cytotoxic concentration of the compound is greater than its virus-inhibiting concentration: SI = CC₅₀/IC₅₀. The SI value lower than 8 is considered to be unacceptable for the compounds that may be perspective as antiviral drugs.⁴⁴

Author contributions

Synthesis: E. I. B., E. A. K. X-ray crystallography data: D. S. K., N. E. B., S. G. A. Quantum-chemical calculations: S. S. B., M. G. I. Experimental data of NMR spectra: M. A. P. Biological activity data/performed the biological assays: N. I. B., O. A. S., L. N. Sh., A. S. O., D. A. O., O. V. P., A. P. A. Manuscript preparation: E. I. B., P. A. N., L. N. Sh., S. G. A., O. I. Ya. Project conception and supervision: P. A. N., V. P. P., N. F. S.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.