Synthesis and Antimicrobial Activity of Sulfenimines Based on Pinane Hydroxythiols

Abstract

The widespread presence of multidrug-resistant pathogenic microorganisms challenges the development of novel chemotype antimicrobials, insensitive to microbial tools of resistance. To date, various monoterpenoids have been shown as potential antimicrobials. Among many classes of molecules with antimicrobial activity, terpenes and terpenoids are an attractive basis for the design of antimicrobials because of their low toxicity and availability for various modifications. In this work, we report on the synthesis of sulfenimines from chiral trifluoromethylated and non-fluorinated pinane-type thiols. Final compounds were obtained with yields of up to 81%. Among the 13 sulfenimines obtained, 3 compounds were able to repress the growth of both bacteria (S. aureus, both MSSA and MRSA; P. aeruginosa) and fungi (C. albicans) with an MIC of 8–32 µg/mL. Although compounds exhibited relatively high cytotoxicity (the therapeutic index of 3), their chemotype can be used as a starter point for the development of disinfectants and antiseptics for targeting multidrug-resistant pathogens.

1. Introduction

The widespread presence of multidrug-resistant bacteria and fungi challenges the development of antimicrobials of a novel chemotype, as they are insensitive to microbial tools of resistance. The acquisition of genes encoding efflux systems and enzymes that hydrolyze antimicrobials and increase biofilm formation, as well as changes in target molecules and cell wall structure, reduces the effectiveness of conventional antibiotics [1].

Among various classes of molecules able to repress the growth of pathogenic bacteria and fungi, monoterpene derivatives have a wide spectrum of antimicrobial activity [2,3,4]. Thus, the repression of the growth of both various bacteria and fungi has been reported [5,6,7,8,9,10]. The combination of terpenes with conventional antimicrobials increases the activity of the latter [11,12]. Furthermore, the fusion in one molecule of a biologically active terpene fragment and sulfur-containing functional groups, which are part of many substances with bactericidal and fungicidal activity, leads to an increase in the efficiency of the resulting thioterpenoids [2,5,13,14]. The mechanism of these synergistic effects can be a consequence of targeting the membrane itself or membrane-related proteins with terpenes. Thus, the binding site for cyclic hydrocarbons, including terpene ones [15], has been reported to be located in the cell membrane of pathogenic microorganisms. The limonene, α- and β-pinenes, and γ-terpinene are able to inhibit respiration and other energy-dependent processes localized in cell membranes of some fungi and bacteria [3,16,17,18,19]. Additionally, some derivatives of terpenes were shown to interact with membranes of eukaryotic cells [20,21].

Carane sulfenimines, sulfinimines, and N-substituted fluorine-containing sulfinamides, as well as pinane thiosulfonates obtained on the basis of monoterpene thiols, showed selective antimicrobial activity against yeasts Candida albicans and Cryptococcus neoformans, as well as the bacteria Staphylococcus aureus and Acinetobacter baumannii [22,23]. The introduction of a sulfenimine fragment in the structure of cephalosporin sulfoxides enhanced their inhibitory activity against cephalosporinase C. The activity was significantly affected by substituents in the sulfenimine moiety [24]. Substituted salicylic and nitrobenzylidene imines have been shown to be subjected as new chemotypes of antimicrobial drug candidates [25,26,27,28,29].

Nowadays, a third of the newly synthesized antimicrobials carry fluorine atoms [30], since the introduction of fluorine-containing groups enhances the membrane permeability and increases the resistance to biodegradation relative to their nonfluorinated analogs [31]. These modifications can lead to significant changes in interaction mechanisms of target with the drug and consequent shifts in biological activity of the latter, compared to hydrocarbon analogs [32,33]. Previously, CF₃-containing pinane-type monoterpene hydroxythiols for further functionalization were synthesized [34].

In this work, based on 10-hydroxyisopinocampheyl thiol 1 [35], of which its CF₃-containing analogs (10S)-2 and (10R)-3 were obtained earlier by our group [34], and nitrobenzaldehydes or substituted salicylic aldehydes, a series of sulfenimines were synthesized, and their antibacterial and antifungal activities, cytotoxicity, and mutagenicity were evaluated.

2. Results and Discussion

2.1. Synthesis of Sulfenimines from Pinane Hydroxythiols

Thiols 1–3 were treated with N-chlorosuccinimide (NCS) in liquid ammonia according to a known procedure [23,36,37,38] to form unstable sulfenamides 4–6, which entered into a condensation reaction in situ with 3,5-diiodosalicylic aldehyde (a), 4-nitrobenzaldehyde (b), 3-nitrobenzaldehyde (c), 2-nitrobenzaldehyde (d), 5-nitrosalicylic aldehyde (e) and 5-bromosalicylic aldehyde (f) to provide the corresponding sulfenimines 7a–f, 8a–f and 9a in 16–81% yields without isolation of sulfenamide intermediates (Scheme 1). Since the CF₃-containing thiol (10R)-3 is synthetically less accessible than the thiol (10S)-2 [34], only sulfenimine 9a with the 3,5-diiodosalicylic moiety was synthesized on its basis.

The structures and composition of sulfenimines have been proven by NMR and IR spectroscopy, and by elemental and X-ray diffraction analysis. The IR spectra of sulfenimines 7a–f, 8a–f and 9a contain absorption bands characteristic of the C=N bond in the region of 1614–1574 cm⁻¹. The ¹H and ¹³C NMR spectra contain signals from both terpene and aromatic fragments. In the ¹H NMR spectra of 7a–f, 8a–f and 9a, in comparison with the starting thiols 1–3, the proton signals of the SH groups disappear, whereas proton signals of the C¹′H=N group can be observed in the region of 8.34–8.95 ppm. In the ¹³C NMR spectra, there are signals characteristic of the C¹′=N group in the downfield region (152.0–159.9 ppm). The ¹³C NMR spectra of CF₃-containing sulfenimines 8a–f and 9a contain quartets of the C¹¹F₃ group in the range of 124.9–125.8 ppm (J_F = 283.0–284.2 Hz), as well as quartets of the C¹⁰ atom in the region of 71.1–72.6 ppm (J_F = 28.8–29.2 Hz); in contrast, in sulfenimines 7a–f, the signals of the C¹⁰ atom are in the region of 65.9–66.4 ppm.

The structure and configuration of single-crystal 8b were confirmed by X-ray diffraction analysis (Figure 1). This compound crystallizes in the chiral space group P2₁ of the monoclinic system. There are two independent molecules (A and B) of 8b in the asymmetric unit cell. They have the same molecular structure. The root-mean-square deviation of the nonhydrogen atomic positions of the A and B molecules is 0.213 Å. The greatest difference is the slight rotation of the SNCHR group. The dihedral angle between the corresponding planes when the molecules are superimposed is 11.28°. All atoms of the NO₂ group, as well as the S(1), N(1) and C(12) atoms, lie in the plane of the C(13)–C(18) phenyl ring. The N-O distances in the NO₂ group are largely aligned. This is typical for NO₂ groups in similar compounds [23,39]. In general, the main geometric characteristics for compound 8b are in good agreement with previously published related compounds [23,39].

Compound 8b has an intramolecular hydrogen bond O(1)-H(1)…N(1). The data set does not allow us to refine the position of the hydrogen atom without constraints. However, the distance between the oxygen and nitrogen atoms (2.830(5) and 2.852(5) Å in molecules A and B, respectively) indicates the implementation of the H...N interaction [40].

The resulting sulfenimines 7a–f, 8a–f and 9a were further subjected to an antibacterial and antifungal activity test, and an assessment of mutagenicity and cytotoxicity.

2.2. Antimicrobial Activity

The activity of terpenes has been shown to vary against microorganisms with various cell wall structures [2,3,4,15,16,20]. Therefore, the antimicrobial activity of the newly synthesized sulfenimine derivatives was assessed against Gram-positive bacteria (Staphylococcus aureus ATCC 29213 (MSSA) and a clinical isolate of Staphylococcus aureus resistant to methicillin (MRSA)), Gram-negative bacterium P. aeruginosa ATCC 27853, and a fluconazole-sensitive clinical isolate of Candida albicans 703. These microorganisms cause diseases of the skin, various mucosa and the respiratory tract and are characterized by a high frequency of occurrence of resistant isolates.

As could be seen from

Table 1. Antibacterial and antifungal activities, and cytotoxicity and mutagenicity of sulfenimines.

Compound	MIC, µg/mL				IC50 EBL, μg/mL	Mutagenicity in the Ames Test
	S. aureus ATCC 29213 (MSSA)	S. aureus Clinical Isolate (MRSA)	P. aeruginosa ATCC 27853	C. albicans 703 Clinical Isolate
7a	8	8	8	64	21 ± 2.7	NF *
8a	16	16	32	32	23 ± 3.8	NF
9a	256	8	1024	8	14 ± 3.5	NF
7b	64	>1024	>1024	64	20 ± 2.8	NF
8b	>1024	>1024	>1024	>1024	ND	ND
7c	32	32	32	32	45 ± 10.1	NF
8c	512	>1024	512	>1024	ND	ND
7d	32	32	>1024	512	18 ± 4.5	TA 102
8d	>1024	>1024	>1024	1024	ND	ND
7e	32	64	32	>1024	14 ± 4.6	NF
8e	16	32	32	16	35 ± 9.1	NF
7f	32	32	1024	64	9 ± 1.9	TA 102
8f	256	64	1024	16	14 ± 3.1	NF
Amikacin	4	4	4	ND	ND	ND
Ampicillin	0.5	>1024	>1024	ND	ND	ND
Ciprofloxacin	2	4	4	ND	ND	ND
Fluconazole	ND *	ND	ND	16	ND	ND
Benzalkonium chloride	1	1	4	0.5	1 ± 0.3	ND

* ND—not determined; NF—not found.

, compounds 7a, 8a, 7c and 8e repressed the growth of all test microorganisms, although the activity was moderate and MIC values were generally significantly higher than those of reference antimicrobials. Notably, trifluoromethylated sulfenimines with salicylic moiety 9a and 8f were active only against methicillin-resistant S. aureus and C. albicans; while 9a was even superior in activity to fluconazole.

Furthermore, 7d, 7f and, partially, 7b were active only on S. aureus and C. albicans; 7d and 7f in the Ames test showed mutagenicity on the Salmonella typhimurium TA102 strain, causing point mutations and reversions [41]. Sulfenimines containing the CF₃ group did not show mutagenicity in this test. Compound 7e was active only on bacteria. In general, a decrease in antibacterial properties upon the introduction of a CF₃ group in the terpene fragment of sulfenimines could be observed. Moreover, sulfenimines with a CF₃ group in the terpene moiety and a salicylaldehyde moiety, 8a, 9a, 8c and 8f, exhibit greater antifungal activity (MIC 8–32), in contrast to the nonfluorinated analogues 7a, 7e and 7f (MIC ≥ 64). Compounds 8b, 8c and 8d were inactive against all test strains, suggesting that the presence of nitrobenzylidene substituents abrogates their antimicrobial activity; neither cytotoxicity nor mutagenicity was tested.

In general, the synthesized sulfenimines exhibited high cytotoxicity on the embryonic bovine lung (EBL) cells: CC₅₀ values were the least toxic, whereas active sulfenimines 7c and 8e exceeded only the corresponding MIC by 2–3-fold. This fact makes sulfenimines suitable only as antiseptics, which is similar to benzalkonium chloride due to a similar therapeutic index (CC₅₀/MIC).

3. Conclusions

Thus, new monoterpene sulfenimines based on monoterpene pinane thiols, including those containing a CF₃ group, have been synthesized for the first time. The described sulfenimines, generally, have moderate antibacterial and antifungal activity and high cytotoxicity in vitro, which limits their direct application. By contrast, the revealed effects of monoterpene and aromatic moieties on the antimicrobial activity of sulfenimines allows for further modeling of compounds with given selectivity for pathogenic microorganisms.

4. Materials and Methods

4.1. General Information

IR spectra were registered on a Shimadzu IR Prestige 21 infrared Fourier spectrometer in a thin layer or in KBr pellets. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance 300 spectrometer (300 and 75 MHz) in CDCl₃ using the signal of the indicated solvent as an internal standard (See Supplementary Materials). ¹³C NMR spectra were registered in the J-modulation mode. The complete assignment of ¹H and ¹³C signals was performed using 2D homo- (¹H–¹H COSY, ¹H–¹H NOESY) and heteronuclear (¹H–¹³C HSQC, ¹H–¹³C HMBC) experiments. ¹⁹F NMR spectra were recorded on a Bruker Avance 300 spectrometer (282 MHz) and on a Spinsolve 60 HF Ultra spectrometer (58 MHz) in CDCl₃ using the signal of CF₃COOH as an external standard. For easier interpretation of the NMR spectra, the carbon atoms of structures 7a–f, 8a–f and 9a were numbered, in some cases, contrary to the recommendations of IUPAC. Elemental analysis was carried out on an EA 1110 CHNS-O automatic analyzer. Melting points were determined on a Sanyo Gallenkamp MPD350BM3.5 instrument and were not corrected. Optical rotation was measured on an automated digital polarimeter, the Optical Activity PolAAr 3001 (UK). Sorbfil plates were used for thin-layer chromatography; the visualizing agent was a solution of phosphoromolybdic acid in ethanol. Alfa Aesar silica gel (0.06–0.2 mm) was used for column chromatography. The commercially available N-chlorosuccinimide, 98% (Alfa Aesar); 3,5-diiodosalicylaldehyde, 98+% (Alfa Aesar); 4-nitrobenzaldehyde, 99% (Alfa Aesar); 3-nitrobenzaldehyde, 99% (Alfa Aesar); 2-nitrobenzaldehyde, 98+% (Alfa Aesar); 5-nitrosalicylaldehyde, 98% (Alfa Aesar); and 5-bromosalicylaldehyde, 98% (Alfa Aesar) were used without additional purification.

The diffraction data for compound 8b were collected on an Oxford Xcalibur Eos diffractometer (Mo-K_α radiation, ω-scan technique, λ = 0.71073 Å). The intensity data were integrated by the CrysAlisPro [42] program. The SCALE3 ABSPACK algorithm [42] was used to perform absorption corrections. The structure was solved by dual methods [43] and was refined on ${\mathrm{F}}_{\mathrm{hkl}}^2$ using the SHELXTL package [44]. All nonhydrogen atoms were refined anisotropically. All H atoms, with the exception of hydrogens of the hydroxyl groups, were placed in calculated positions and were refined using a riding model (U_iso(H) = 1.5U_eq(C) for CH₃ groups and U_iso(H) = 1.2U_eq(C) for other groups). The H(1A) and H(1B) atoms in 8b were located on the differential Fourier map and were refined isotropically with AFIX 147. The PhNO₂ fragment in molecule B was disordered over two positions. To refine the disordered atoms, the AFIX 66, SAME, SADI, FLAT and ISOR instructions were used. CCDC 2193190 contains the supplementary crystallographic data. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures (accessed on 3 November 2022).

((1S,2R,3S,5R)-3-Mercapto-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)methanol (1) [35], (S)-2,2,2-trifluoro-1-((1S,2R,3S,5R)-3-mercapto-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)ethan-1-ol (2) and (R)-2,2,2-trifluoro-1-((1S,2R,3S,5R)-3-mercapto-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)ethan-1-ol (3) [34] were synthesized according to known procedures.

4.2. General Procedure for the Synthesis of Sulfenimines

The procedure is based on methods for the synthesis of sulfenimines [23,36,37,38].

In a U-shaped tube, while cooling to −70 °C in an acetone bath, 7–10 mL of liquid NH₃ (dry) was condensed. While maintaining the bath temperature, NCS (143 mg, 1.069 mmol) was carefully added. The mixture was stirred for 5 min, then thiol 1 (0.823 mmol, either 2 or 3) dissolved in 2 mL of CH₂Cl₂ was introduced into the tube with a syringe. The reaction mixture was stirred for one hour, gradually increasing the temperature to −30 °C until unstable sulfenamide 2 was formed. After complete conversion of the thiol (monitored by TLC), the corresponding aldehyde (2.1 mmol, a–f) was added to the reaction mixture. As ammonia evaporated, CH₂Cl₂ was added. After complete evaporation of ammonia, the reaction mixture was heated to room temperature. After 12 h, the reaction mixture was filtered off on a Schott filter under reduced pressure. The organic phase was treated with a NaCl solution and extracted with CH₂Cl₂ (3 × 20 mL). The combined organic phases were dried over Na₂SO₄. The solvent was then distilled off under reduced pressure. The resulting mixture was separated by silica gel column chromatography using the same eluent systems as for TLC.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-3,5-diiodobenzylidene)-2-(hydroxymethyl)-6,6-dimethylbicyclo[3.1.1]heptane-3-sulfenamide (7a). Yield: 55.5%; yellow-orange powder; m.p.: 64.1 °C; ${\left[\alpha \right]}_D^{25}$ +56.6 (c 1.71, CHCl₃); R_f 0.43 (petr. ether:EtOAc, 2:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.04 (3H, s, CH₃-8), 1.14 (1H, d, J = 9.8, H-7α), 1.27 (3H, s, CH₃-9), 2.04–2.11 (2H, m, H-5, C-10-OH), 2.18–2.29 (3H, m, H-1, H-4α, H-2), 2.50 (1H, dtd, J = 9.5, 6.6, 2.2, H-7β), 2.60–2.72 (1H, m, H-4β), 3.64 (1H, dt, J = 9.5, 6.6, H-3), 3.69–3.84 (2H, m, H-10), 7.43 (1H, d, J = 2.0, H-7′), 7.99 (1H, d, J = 2.0, H-5′), 8.35 (1H, s, H-1′), 12.45 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.5 (C-8), 27.5 (C-9), 32.4 (C-7), 35.5 (C-4), 38.8 (C-6), 41.7 (C-5), 42.5 (C-3), 42.7 (C-1), 49.7 (C-2), 66.0 (C-10), 80.7 (C-6′), 86.5 (C-4′), 121.7 (C-2′), 138.7 (C-7′), 147.7 (C-5′), 157.6 (C-3′), 158.8 (C-1′). IR spectrum (KBr, ν, cm⁻¹): 3404 (Ar-OH), 3383 (OH), 1574 (C=N). Elemental analysis calcd. (%) for C₁₇H₂₁I₂NO₂S: C 36.64, H 3.80, N 2.51; found: C 37.03, H 4.17, N 2.64.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-3,5-diiodobenzylidene)-6,6-dimethyl-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8a). Yield: 69%; yellow powder; m.p.: 98.5 °C; ${\left[\alpha \right]}_D^{25}$ +0.2 (c 0.85, CHCl₃); R_f 0.53 (CHCl₃). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.06 (3H, s, CH₃-8), 1.25 (1H, d, J = 9.5, H-7α), 1.27 (3H, s, CH₃-9), 2.05–2.11 (1H, m, H-5), 2.20–2.21 (1H, m, H-1), 2.29–2.38 (2H, m, H-2, H-4α), 2.40–2.50 (1H, m, H-7β), 2.59–2.79 (2H, m, H-4β, C-10-OH), 4.05–4-21 (2H, H-3, H-10), 7.44 (1H, d, J = 2.0, H-7′), 8.00 (1H, d, J = 2.0, H-5′), 8.34 (1H, s, H-1′), 12.20 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.1 (C-8), 26.9 (C-9), 30.4 (C-7), 35.1 (C-4), 38.1 (C-6), 40.7 (C-5), 40.8 (C-3), 42.4 (C-1), 46.4 (C-2), 72.6 (q, J_F = 29.2, C-10), 80.7 (C-6′), 86.6 (C-4′), 121.6 (C-2′), 124.9 (q, J_F = 283.0, C-11), 138.8 (C-7′), 147.9 (C-5′), 157.4 (C-3′), 158.4 (C-1′). ¹⁹F NMR (282 MHz, CDCl₃, δ, ppm): −76.20 (3F, s, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3460 (OH), 1576 (C=N), 1273, 1151, 1123 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₀F₃I₂NO₂S: C 34.58, H 3.22, N 2.24; found: C 34.95, H 3.60, N 2.08.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-3,5-diiodobenzylidene)-6,6-dimethyl-2-((R)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (9a). Yield: 16%; light-yellow gummy oil; ${\left[\alpha \right]}_D^{26}$ +30.8 (c 0.4, CHCl₃); R_f 0.37 (CHCl₃). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.13 (3H, s, CH₃-8), 1.14 (1H, d, J = 9.5, H-7α), 1.30 (3H, s, CH₃-9), 2.05–2.13 (1H, m, H-5), 2.18 (1H, br.s, C-10-OH), 2.36–2.47 (3H, m, H-1, H-2, H-4α), 2.56–2.74 (2H, m, H-2, H-7β, H-4β), 3.87 (1H, td, J = 9.2, 6.6, H-3), 4.19–4-29 (1H, m, H-10), 7.45 (1H, d, J = 1.5, H-7′), 8.02 (1H, d, J = 1.5, H-5′), 8.40 (1H, s, H-1′), 12.32 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 24.3 (C-8), 27.9 (C-9), 33.8 (C-7), 35.6 (C-4), 38.4 (C-6), 41.7 (C-5), 42.0 (C-1), 43.4 (C-3), 46.3 (C-2), 71.2 (q, J_F = 29.9, C-10), 80.7 (C-6′), 88.5 (C-4′), 121.6 (C-2′), 125.1 (q, J_F = 283.1, C-11), 138.9 (C-7′), 148.0 (C-5′), 157.6 (C-3′), 159.4 (C-1′). ¹⁹F NMR (58 MHz, CDCl₃, δ, ppm): −74.43 (3F, d, J = 7.4, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3466 (OH), 1576 (C=N), 1128, 1552, 1123 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₀F₃I₂NO₂S: C 34.58, H 3.22, N 2.24; found: C 34.58, H 3.14, N 2.63.

(1S,2R,3S,5R)-2-(Hydroxymethyl)-6,6-dimethyl-N-((E)-4-nitrobenzylidene)bicyclo-[3.1.1]heptane-3-sulfenamide (7b). Yield: 81%; yellowish oil; ${\left[\alpha \right]}_D^{26}$ −82.0 (c = 0.6, CHCl₃); R_f = 0.22 (petr. ether:EtOAc, 3:1). ¹H, ¹³C NMR and IR spectral data correspond to those given in Ref. [37].

(1S,2R,3S,5R)-6,6-Dimethyl-N-((E)-4-nitrobenzylidene)-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8b). Yield: 75%; light-yellow crystal; m.p.: 48.8 °C; ${\left[\alpha \right]}_D^{25}$−1.5 (c 1.41, CHCl₃); R_f 0.28 (petr. ether:EtOAc, 5:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.09 (3H, s, CH₃-8), 1.28 (3H, s, CH₃-9), 1.28 (1H, d, J = 10.3, H-7α), 1.60–1.68 (1H, m, H-4α), 2.05–2.11 (1H, m, H-5), 2.28–2.35 (1H, m, H-1), 2.37–2.46 (1H, m, H-7β), 2.49–2.59 (1H, m, H-4β), 2.94 (1H, dt, J = 10.3, 3.3, H-2), 3.94–4.07 (1H, m, H-10), 4.37 (1H, dt, J = 10.8, 4.5, H-3), 5.58 (1H, br.d, J = 8.8, C-10-OH), 7.72 (2H, d, J = 8.8, H-3′, H-7′), 8.29 (2H, d, J = 8.8, H-4′, H-6′), 8.54 (1H, s, H-1′). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.0 (C-8), 26.9 (C-9), 29.7 (C-7), 29.9 (C-4), 38.1 (C-6), 38.7 (C-3), 40.1 (C-5), 43.3 (C-1), 51.5 (C-2), 71.1 (q, J_F = 28.8, C-10), 124.4 (C-4′, C-6′), 125.8 (q, J_F = 283.1, C-11), 127.8 (C-3′, C-7′), 140.1 (C-2′), 148.7 (C-5′), 155.3 (C-1′). ¹⁹F NMR (282 MHz, CDCl₃, δ, ppm): −76.37 (3F, s, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3238 (OH), 1601 (C=N), 1522, 1344 (NO₂), 1267, 1169, 1125 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₁F₃N₂O₃S: C 53.72, H 5.26, N 6.96; found: C 53.33, H 5.21, N 7.04. A single crystal of 8b was grown from the hexane-Et₂O system. A colorless prismatic crystal of the monoclinic system had a size of 0.77×0.39×0.13 mm, space group P2₁, a = 16.6479(3), b = 7.08470(10), c = 17.9640(4) Å, β = 111.720(2)°, V = 1968.34(7) ų, Z = 4, μ = 0.212 mm⁻¹, d_calc = 1.358 g/cm³ and F(000) = 840. A dataset of 42,548 reflections was collected at scattering angles of 2.101° < θ < 25.027°, of which 6946 were independent (R_int = 0.0357), including 5644 reflections with I > 2σ(I). The final refinement parameters were R₁ = 0.0548, wR₂ = 0.1118 (all data), R₁ = 0.0406 and wR₂ = 0.1006 [I > 2σ(I)], with GooF = 0.945. Δρ_e = 0.266/−0.171 e Å^–3; Flack parameter = −0.03(2).

(1S,2R,3S,5R)-2-(Hydroxymethyl)-6,6-dimethyl-N-((E)-3-nitrobenzylidene)bicyclo-[3.1.1]heptane-3-sulfenamide (7c). Yield: 53%; light-yellow oil; ${\left[\alpha \right]}_D^{25}$ −40.4 (c 2.18, CHCl₃); R_f 0.30 (PhH:EtOAc, 10:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.04 (3H, s, CH₃-8), 1.26 (3H, s, CH₃-9), 1.27 (1H, d, J = 10.3, H-7α), 1.89 (1H, ddd, J = 14.1, 5.7, 2.9, H-4α), 2.02–2.15 (1H, m, H-5, H-1), 2.39–2.60 (1H, m, H-7β, H-4β), 2.67 (1H, dtd, J = 8.7, 5.9, 2.2, H-2), 3.37 (1H, br.s, C-10-OH), 3.64 (1H, dd, J = 10.3, 5.9, H-10α), 3.81–3.87 (2H, m, H-3, H-10), 7.58 (1H, t, J = 8.1, H-6′), 7.96 (1H, d, J = 8.1, H-7′), 8.22 (1H, d, J = 8.1, H-5′), 8.38 (1H, s, H-3′), 8.54 (1H, s, H-1′). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.5 (C-8), 27.3 (C-9), 31.5 (C-7), 32.4 (C-4), 38.6 (C-6), 41.2 (C-5), 42.8 (C-3), 44.1 (C-1), 53.1 (C-2), 66.4 (C-10), 122.1 (C-3′), 124.4 (C-5′), 129.9 (C-6′), 131.9 (C-7′), 137.4 (C-2′), 148.7 (C-4′), 154.0 (C-1′). IR spectrum (KBr, ν, cm⁻¹): 3402 (OH), 1614 (C=N), 1530, 1350 (NO₂). Elemental analysis calcd. (%) for C₁₇H₂₂N₂O₃S: C 61.05, H 6.63, N 8.38; found: C 61.38, H 6.37, N 7.91.

(1S,2R,3S,5R)-6,6-Dimethyl-N-((E)-3-nitrobenzylidene)-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8c). Yield: 24%; white powder; m.p.: 122.7 °C; ${\left[\alpha \right]}_D^{27}$−83.3 (c 0.6, CHCl₃); R_f 0.34 (petr. ether:EtOAc, 5:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.09 (3H, s, CH₃-8), 1.28 (3H, s, CH₃-9), 1.28 (1H, d, J = 10.3, H-7α), 1.64 (1H, dt, J = 14.3, 4.0, H-4α), 2.05–2.11 (1H, m, H-5), 2.28–2.35 (1H, m, H-1), 2.37–2.46 (1H, m, H-7β), 2.54 (1H, ddt, J = 13.9, 11.3, 2.5, H-4β), 2.96 (1H, dt, J = 10.3, 3.3, H-2), 3.94–4.07 (1H, m, H-10), 4.37 (1H, dt, J = 10.8, 4.5, H-3), 5.64 (1H, br.d, J = 8.1, C-10-OH), 7.63 (1H, t, J = 7.6, H-6′), 7.97 (1H, d, J = 7.6, H-7′), 8.27 (1H, d, J = 7.6, H-5′), 8.35 (1H, s, H-3′), 8.53 (1H, s, H-1′). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.0 (C-8), 26.9 (C-9), 29.7 (C-7), 29.9 (C-4), 38.1 (C-6), 38.5 (C-3), 40.1 (C-5), 43.2 (C-1), 51.4 (C-2), 71.1 (q, J_F = 28.8, C-10), 122.8 (C-3′), 125.1 (C-5′), 125.7 (q, J_F = 283.1, C-11), 130.3 (C-6′), 131.6 (C-7′), 136.6 (C-2′), 148.7 (C-4′), 155.2 (C-1′). ¹⁹F NMR (282 MHz, CDCl₃, δ, ppm): −76.38 (3F, d, J = 7.0, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3379 (Ar-OH), 3264 (OH), 1595 (C=N), 1530, 1350 (NO₂), 1271, 1169, 1121 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₁F₃N₂O₃S: C 53.72, H 5.26, N 6.96; found: C 53.95, H 5.28, N 7.01.

(1S,2R,3S,5R)-2-(Hydroxymethyl)-6,6-dimethyl-N-((E)-2-nitrobenzylidene)bicyclo-[3.1.1]heptane-3-sulfenamide (7d). Yield: 81%; yellow oil; ${\left[\alpha \right]}_D^{25}$ −34.5 (c 1.4, CHCl₃); R_f 0.23 (PhH:EtOAc, 10:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.03 (3H, s, CH₃-8), 1.25 (3H, s, CH₃-9), 1.26 (1H, d, J = 9.8, H-7α), 1.86 (1H, ddd, J = 14.3, 5.5, 2.9, H-4α), 2.02–2.12 (2H, m, H-5, H-1), 2.39–2.59 (2H, m, H-7β, H-4β), 2.68 (1H, dtd, J = 8.7, 5.9, 2.2, H-2), 3.45 (1H, br.s, C-10-OH), 3.60 (1H, dd, J = 9.5, 6.6, H-10α), 3.78–3.90 (2H, m, H-10β, H-3), 7.52 (1H, td, J = 7.9, 1.7, H-5′), 7.66 (1H, t, J = 7.8, H-6′), 7.95 (1H, dd, J = 7.9, 1.7, H-7′), 8.00 (1H, d, J = 7.9, H-4′), 8.94 (1H, s, H-1′). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.5 (C-8), 27.2 (C-9), 31.4 (C-7), 32.0 (C-4), 38.6 (C-6), 41.2 (C-5), 42.6 (C-3), 44.2 (C-1), 53.2 (C-2), 66.4 (C-10), 124.6 (C-4′), 128.4 (C-7′), 130.3 (C-5′), 130.4 (C-2′), 133.6 (C-6′), 147.7 (C-3′), 152.0 (C-1′). IR spectrum (KBr, ν, cm⁻¹): 3395 (OH), 1605 (C=N). Elemental analysis calcd. (%) for C₁₇H₂₂N₂O₃S: C 61.05, H 6.63, N 8.38; found: C 61.29, H 7.04, N 8.19.

(1S,2R,3S,5R)-6,6-Dimethyl-N-((E)-2-nitrobenzylidene)-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8d). Yield: 52%; yellow oil; ${\left[\alpha \right]}_D^{25}$ −58.8 (c 1.69, CHCl₃); R_f 0.35 (PhH). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.08 (3H, s, CH₃-8), 1.28 (3H, s, CH₃-9), 1.32 (1H, d, J = 10.6, H-7α), 1.60–1.68 (1H, m, H-4α), 2.05–2.11 (1H, m, H-5), 2.29–2.36 (1H, m, H-1), 2.37–2.46 (1H, m, H-7β), 2.49–2.59 (1H, m, H-4β), 2.97 (1H, dt, J = 10.3, 3.3, H-2), 3.93–4.06 (1H, m, H-10), 4.34 (1H, dt, J = 10.3, 4.4, H-3), 5.57 (1H, br.d, J = 8.1, C-10-OH), 7.58 (1H, t, J = 7.9, H-5′), 7.72 (1H, t, J = 7.9, H-6′), 7.85 (1H, d, J = 7.9, H-7′), 8.06 (1H, d, J = 7.9, H-4′), 8.95 (1H, s, H-1′). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.0 (C-8), 26.8 (C-9), 29.5 (C-7), 29.9 (C-4), 38.1 (C-6), 38.7 (C-3), 40.1 (C-5), 43.2 (C-1), 51.5 (C-2), 71.1 (q, J_F = 28.8, C-10), 124.8 (C-4′), 125.7 (q, J_F = 283.0, C-11), 128.3 (C-7′), 130.0 (C-2′), 130.9 (C-5′), 134.0 (C-6′), 147.7 (C-3′), 153.4 (C-1′). ¹⁹F NMR (282 MHz, CDCl₃, δ, ppm): −76.12 (3F, d, J = 6.6, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3364 (OH), 1605 (C=N), 1528, 1344 (NO₂), 1269, 1169, 1123 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₁F₃N₂O₃S: C 53.72, H 5.26, N 6.96; found: C 53.98, H 5.31, N 6.60.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-5-nitrobenzylidene)-2-(hydroxymethyl)-6,6-dimethylbicyclo[3.1.1]heptane-3-sulfenamide (7e). Yield: 66%; yellow powder; m.p.: 108.5 °C; ${\left[\alpha \right]}_D^{25}$ +65.4 (c 1.17, CHCl₃); R_f 0.29 (PhH:EtOAc, 10:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.05 (3H, s, CH₃-8), 1.15 (1H, d, J = 9.5, H-7α), 1.27 (3H, s, CH₃-9), 2.04–2.11 (2H, m, H-5, C-10-OH), 2.18–2.35 (3H, m, H-1, H-4α, H-2), 2.46 (1H, dtd, J = 9.5, 6.6, 2.2 H-7β), 2.58–2.68 (1H, m, H-4β), 4.36 (1H, dt, J = 10.3, 6.6, H-3), 3.73–3.86 (2H, m, H-10), 7.01 (1H, d, J = 8.8, H-4′), 8.13–8.18 (2H, m, H-5′, H-7′), 8.60 (1H, s, H-1′), 12.40 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.4 (C-8), 27.4 (C-9), 32.2 (C-7), 35.0 (C-4), 38.8 (C-6), 41.6 (C-5), 42.4 (C-3), 42.8 (C-1), 50.0 (C-2), 65.9 (C-10), 117.7 (C-4′), 119.4 (C-2′), 126.2 (C-7′), 126.9 (C-5′), 140.3 (C-6′), 158.8 (C-1′), 163.9 (C-3′). IR spectrum (KBr, ν, cm⁻¹): 3553 (Ar-OH), 3537 (OH), 1593 (C=N), 1520, 1339 (NO₂). Elemental analysis calcd. (%) for C₁₇H₂₂N₂O₄S: C 58.27, H 6.33, N 7.99; found: C 57.87, H 5.85, N 7.60.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-5-nitrobenzylidene)-6,6-dimethyl-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8e). Yield: 41%; yellow gummy oil; ${\left[\alpha \right]}_D^{25}$ +38.9 (c 1.14, CHCl₃); R_f 0.30 (petr. ether:EtOAc, 4:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.07 (3H, s, CH₃-8), 1.27 (3H, s, CH₃-9), 1.30 (1H, d, J = 9.0, H-7α), 2.05–2.13 (1H, m, H-5), 2.20–2.29 (2H, m, H-1, H-4α), 2.40–2.51 (3H, m, H-2, H-7β, C-10-OH), 2.64–2.76 (1H, m, H-4β), 4.09–4.23 (2H, m, H-3, H-10), 7.02 (1H, d, J = 8.8, H-4′), 8.14–8.19 (2H, m, H-5′, H-7′), 8.59 (1H, s, H-1′), 12.10 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.1 (C-8), 26.8 (C-9), 30.0 (C-7), 34.3 (C-4), 38.0 (C-6), 40.3 (C-5), 40.6 (C-3), 42.4 (C-1), 46.7 (C-2), 72.4 (q, J_F = 29.2, C-10), 117.8 (C-4′), 119.4 (C-2′), 125.0 (q, J_F = 283.1, C-11), 126.4 (C-7′), 127.0 (C-5′), 140.3 (C-6′), 158.7 (C-1′), 163.9 (C-3′). ¹⁹F NMR (282 MHz, CDCl₃, δ, ppm): −76.17 (1F, d, J = 6.5, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3509 (OH), 1593 (C=N), 1522, 1341 (NO₂), 1277, 1167, 1123 (CF₃). Elemental analysis calcd. (%) for C₁₈H₂₁F₃N₂O₄S: C 51.67, H 5.06, N 6.70; found: C 51.69, H 5.27, N 6.82.

(1S,2R,3S,5R)-N-((E)-5-Bromo-2-hydroxybenzylidene)-2-(hydroxymethyl)-6,6-dimethylbicyclo[3.1.1]heptane-3-sulfenamide (7f). Yield: 61%; light-yellow gummy oil; ${\left[\alpha \right]}_D^{26}$ +55.9 (c 0.2, CHCl₃); R_f 0.36 (PhH:EtOAc, 5:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.04 (3H, s, CH₃-8), 1.14 (1H, d, J = 9.9, H-7α), 1.27 (3H, s, CH₃-9), 1.63 (1H, br.s, C-10-OH), 2.03–2.09 (1H, m, H-5), 2.18–2.34 (3H, m, H-1, H-4α, H-2), 2.44–2.53 (1H, m, H-7β), 2.56–2.66 (1H, m, H-4β), 3.59 (1H, dt, J = 9.9, 6.6, H-3), 3.69–3.85 (2H, m, H-10), 6.84 (1H, d, J = 8.8, H-4′), 7.27 (1H, d, J = 2.2, H-7′), 7.35 (1H, dd, J = 8.8, 2.2, H-5′), 8.48 (1H, s, H-1′), 11.51 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.4 (C-8), 27.5 (C-9), 32.4 (C-7), 35.1 (C-4), 38.8 (C-6), 41.7 (C-5), 42.4 (C-1), 42.8 (C-3), 50.1 (C-2), 66.1 (C-10), 110.8 (C-6′), 118.9 (C-4′), 121.5 (C-2′), 132.5 (C-7′), 134.3 (C-5′), 157.8 (C-3′), 159.7 (C-1′). IR spectrum (KBr, ν, cm⁻¹): 3341 (OH), 1587 (C=N). Elemental analysis calcd. (%) for C₁₇H₂₂BrNO₂S: C 53.13, H 5.77, N 3.64; found: C 53.10, H 5.71, N 3.55.

(1S,2R,3S,5R)-N-((E)-2-Hydroxy-5-bromobenzylidene)-6,6-dimethyl-2-((S)-2,2,2-trifluoro-1-hydroxyethyl)bicyclo[3.1.1]heptane-3-sulfenamide (8f). Yield: 38%; yellow oil; m.p.: 122.7 °C; ${\left[\alpha \right]}_D^{26}$ +29.0 (c 1.3, CHCl₃); R_f 0.34 (PhH:CH₂Cl₂, 2:1). ¹H NMR (300 MHz, CDCl₃, δ, ppm, J/Hz): 1.05 (3H, s, CH₃-8), 1.25 (1H, d, J = 9.9, H-7α), 1.25 (3H, s, CH₃-9), 2.02–2.09 (1H, m, H-5), 2.18–2.34 (2H, m, H-1, H-4α), 2.37–2.50 (2H, m, H-7β, H-2), 2.55–2.80 (2H, m, H-4β, C-10-OH), 4.00–4.20 (2H, m, H-3, H-10), 6.83 (1H, d, J = 8.8, H-4′), 7.27 (1H, s, H-7′), 7.34 (1H, d, J = 8.8, 2.2, H-5′), 8.45 (1H, s, H-1′), 11.25 (1H, br.s, C-3′-OH). ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.0 (C-8), 26.9 (C-9), 30.3 (C-7), 34.7 (C-4), 38.1 (C-6), 40.5 (C-3), 40.8 (C-5), 42.5 (C-1), 46.6 (C-2), 72.6 (q, J_F = 29.8, C-10), 110.9 (C-6′), 118.9 (C-4′), 121.4 (C-2′), 125.0 (q, J_F = 284.2, C-11), 132.7 (C-7′), 134.5 (C-5′), 157.7 (C-3′), 159.9 (C-1′). ¹⁹F NMR (58 MHz, CDCl₃, δ, ppm): −74.11 (3F, d, J = 6.1, CF₃-11). IR spectrum (KBr, ν, cm⁻¹): 3455 (OH), 1589 (C=N), 1271, 1171, 1123 (CF₃), 1076 (CBr). Elemental analysis calcd. (%) for C₁₈H₂₁BrF₃NO₂S: C 47.80, H 4.68, N 3.10; found: C 47.53, H 4.31, N 3.07.

4.3. Antibacterial Activity

Minimum inhibitory concentrations (MICs) of compounds were determined by the broth microdilution assay in 96-well plates (Eppendorf, Hamburg, Germany) according to the EUCAST rules for antimicrobial susceptibility testing [45] in full Mueller–Hinton broth (MH). Briefly, the bacterial suspension containing 10⁸ CFUs/mL was subsequently diluted to 1:300 with MH broth in microwell plates to obtain a 10⁶ cells/mL suspension, and then incubated at 37 °C for 24 h. The stock solutions of compounds to be tested were prepared in DMSO and added to the final concentrations of compounds to be tested, which ranged from 1 to 1048 µg/mL. The MIC was determined as the lowest concentration of an antibiotic for which no visible bacterial growth could be observed after 24 h of incubation. The assessment was performed five times and the typical (median) value was considered as the MIC.

4.4. Antifungal Activity

MICs on C. albicans were determined using the broth microdilution method in 96-well plates (Eppendorf) with MH broth, as recommended in the protocol CLSI M27-A3 [46]. C. albicans was grown overnight and diluted with MH broth until a density of 10⁷ cells/mL was reached, obtaining the working solution. Then, 2-fold serial dilutions of compounds in concentrations from 1 to 1024 μg/mL were prepared in MH broth and seeded with fungi (1% v/v of working solution) with subsequent incubation at 37 °C for 24 h. The MIC was defined as the lowest concentration of the compound at which no visible growth could be seen. The assessment was performed five times and the typical (median) value was considered as the MIC.

4.5. Mutagenicity and Cytotoxicity

The mutagenicity of compounds was evaluated in the Ames test with S. typhimurium TA98, TA100 and TA102 strains, as described in [41]. The spot-test modification was applied to avoid false-negative results due to the antibacterial activity of compounds. The tested compound was considered to be mutagenic if the number of revertant colonies increased more than 2 times when close to the filter paper with the compound.

The cytotoxicity of compounds was determined using the microtetrazolium test (MTT) on BFL cells. The cells were cultured in DMEM—Dulbecco’s Modified Eagle’s Medium (Sigma Aldrich, St. Louis, MO, USA)—that was supplemented with 10% FBS, 2 mM of L-glutamine, 100 µg/mL of penicillin and 100 µg/mL of streptomycin. Cells were seeded in 96-well plates with a density of 3000 cells per well and left overnight to allow for the attachment. Next, cells were cultured at 37 °C and 5% CO₂ in the presence of compounds of interest at various concentrations from 1.25 to 160 µg/mL. After 24 h of cultivation, the cells were subjected to the MTT assay. The formazan was solubilized by DMSO and the optical density was measured on the Tecan Infinite 200Pro at 570 nm. The concentration required to inhibit cellular dehydrogenase activity by 50% (CC₅₀ value) was calculated by using the GraphPad Prism 6.0 software.