Synthesis and Biological Evaluation of Carvacrol-Based Derivatives as Dual Inhibitors of H. pylori Strains and AGS Cell Proliferation

This study reports on the synthesis, structural assessment, microbiological screening against several strains of H. pylori and antiproliferative activity against human gastric adenocarcinoma (AGS) cells of a large series of carvacrol-based compounds. Structural analyses consisted of elemental analysis, 1H/13C/19F NMR spectra and crystallographic studies. The structure-activity relationships evidenced that among ether derivatives the substitution with specific electron-withdrawing groups (CF3 and NO2) especially in the para position of the benzyl ring led to an improvement of the antimicrobial activity, whereas electron-donating groups on the benzyl ring and ethereal alkyl chains were not tolerated with respect to the parent compound (MIC/MBC = 64/64 µg/mL). Ester derivatives (coumarin-carvacrol hybrids) displayed a slight enhancement of the inhibitory activity up to MIC values of 8–16 µg/mL. The most interesting compounds exhibiting the lowest MIC/MBC activity against H. pylori (among others, compounds 16 and 39 endowed with MIC/MBC values ranging between 2/2 to 32/32 µg/mL against all the evaluated strains) were also assayed for their ability to reduce AGS cell growth with respect to 5-Fluorouracil. Some derivatives can be regarded as new lead compounds able to reduce H. pylori growth and to counteract the proliferation of AGS cells, both contributing to the occurrence of gastric cancer.


Introduction
Carvacrol is a naturally occurring monoterpene phenol abundant in several medicinal plants (especially within the Labiatae and Apiaceae families) which, besides its odoriferous and flavoring function, exhibits antimicrobial, food preserving, antioxidant and anticancer activities [1,2]. More in In particular, compound 12 was involved in a multistep synthesis. Firstly, it was hydrolyzed in mild conditions using lithium hydroxide (LiOH), in a mixture of water and methanol (in the ratio 50:50, v:v) at room temperature (RT), to provide the carboxylic acid derivative 13. Secondly, it was reacted with 3-nitrophenylhydrazine in ethanol to achieve the corresponding acetohydrazide 46 (Scheme 1B).
The NO 2 group, located at the para position of the benzyl moiety of compound 34, was reduced with sodium dithionite (Na 2 S 2 O 4 ), leading to the p-NH 2 derivative 35 (Scheme 1C). Derivative 37, obtained from the reaction between carvacrol and (4-(bromomethyl)phenyl)(methyl)sulfane, was treated with m-chloroperbenzoic acid (mCPBA) in dichloromethane (DCM). This reaction led to the two oxidized forms of sulfane (sulfoxide and sulfone, respectively the compounds 38 and 39) in the same step, by modulating the amount of the oxidant agent (mCPBA) added [21]. For the synthesis of the ester compounds 43-45, we synthesized the coumarin-3-carboxylic acids at first, which were then used in a condensation reaction with carvacrol (Scheme 2). Through the Knoevenagel condensation between the properly substituted 2-hydroxybenzaldehyde and the diethyl malonate, we obtained the esters A-C [22]. The removal of the ester function through hydrolysis was performed using 10% NaOH solution and afforded the carboxylic acid derivatives A1-C1. Finally, coupling of carvacrol with the proper coumarin-3-carboxylic acid, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) as condensing agents in 4-methylmorpholine (NMM), gave the title compounds 43-45. The selection of this nucleus and its substituents was suggested by the good results obtained in the evaluation of H. pylori strains previously published by some of us [23].
The compounds were stable in their solid state at room temperature. The structures were confirmed by spectral studies ( 1 H, 13 C, and 19 F NMR), whereas the purity of these compounds was confirmed by combustion analysis, X-ray diffraction studies (for compound 34), TLC parameters and melting point evaluation.

X-ray Diffraction Analysis
Crystals of compound 34 ( Figure 1) were obtained by slow evaporation from an ethyl acetate/n-hexane mixture. Information about the crystal data, experimental collection conditions and refinement as well as the structural geometric parameters are available in the Cambridge Crystallographic Data Centre in CIF format and in the Supporting information (Tables S1-S4).

Pan Assay Interference Compounds (PAINS) Evaluation
All designed inhibitors have been analyzed by means of three different theoretical tools, such as ZINC PAINS Pattern Identifier [24], False Positive Remover [25], and FAF-Drug4 [26]. Our compounds were not reported as potential PAINS or covalent inhibitors by none of the considered algorithms.

Biological Assay
After a proper purification and characterization, the compounds were subjected to in vitro biological experiments to assess their inhibitory activity against H. pylori growth and AGS cells aiming at discovering the structural requirements to achieve a dual agent.

In Vitro Inhibitory Activity against H. pylori Strains
Disposing of all envisioned products, the in vitro inhibitory activity against nine strains of H. pylori (one reference strain and eight clinical isolates) characterized by a different antibiotic susceptibility pattern was evaluated and the data reported in Table 1. The susceptibility pattern followed the breakpoints as classified in the international EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines for H. pylori strains.
The parent compound, carvacrol, displayed MIC and MBC values in the range 16-64 and 32-64 μg/mL, respectively. All the chemical modifications can be grouped into four classes to define robust structure-activity relationships (SARs): 1) Alkyloxy derivatives 1-13: In general, these derivatives were characterized by an increasing alkyl chain, linear or branched, saturated or unsaturated, functionalized with additional moieties (cyano, ketone, ester, carboxylic acid). None of these modifications led to an improvement of the inhibitory activity with respect to the parent compound. Only derivatives 2 (OPr) and 3 (OBu) slightly presented MIC values comparable to carvacrol against two strains (F34/497 and F40/499), whereas compounds 6 (O-crotyl) and 9 (O-geranyl) were endowed with inferior MIC and MBC values up to 16 and 8 μg/mL, respectively, toward all the strains; 2) Benzyloxy derivatives 14-40 and 46: The simplest representative of this class (14, O-benzyl) had an anti-H. pylori activity comparable to carvacrol, whereas other substitutions on the aryl ring such as 3,4-diCl, 2,6-diF, 3-F, 3-OCH3, 2-Cl-4-OCH3, 2-Br, and 4-Br were detrimental or didn't produce a strong increment of the antimicrobial activity. Conversely, some substituents, especially in the para position of the aryl ring, such as CF3, Ph, CN, NO2 and NH2 were promising to show improvements. Indeed, CF3 could act as a bioisostere of the NO2 group and in both series we can highlight the following activity order: p > m > o

Pan Assay Interference Compounds (PAINS) Evaluation
All designed inhibitors have been analyzed by means of three different theoretical tools, such as ZINC PAINS Pattern Identifier [24], False Positive Remover [25], and FAF-Drug4 [26]. Our compounds were not reported as potential PAINS or covalent inhibitors by none of the considered algorithms.

Biological Assay
After a proper purification and characterization, the compounds were subjected to in vitro biological experiments to assess their inhibitory activity against H. pylori growth and AGS cells aiming at discovering the structural requirements to achieve a dual agent.

In Vitro Inhibitory Activity against H. pylori Strains
Disposing of all envisioned products, the in vitro inhibitory activity against nine strains of H. pylori (one reference strain and eight clinical isolates) characterized by a different antibiotic susceptibility pattern was evaluated and the data reported in Table 1. The susceptibility pattern followed the breakpoints as classified in the international EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines for H. pylori strains.
The parent compound, carvacrol, displayed MIC and MBC values in the range 16-64 and 32-64 µg/mL, respectively. All the chemical modifications can be grouped into four classes to define robust structure-activity relationships (SARs):  (1) Alkyloxy derivatives 1-13: In general, these derivatives were characterized by an increasing alkyl chain, linear or branched, saturated or unsaturated, functionalized with additional moieties (cyano, ketone, ester, carboxylic acid). None of these modifications led to an improvement of the inhibitory activity with respect to the parent compound. Only derivatives 2 (OPr) and 3 (OBu) slightly presented MIC values comparable to carvacrol against two strains (F34/497 and F40/499), whereas compounds 6 (O-crotyl) and 9 (O-geranyl) were endowed with inferior MIC and MBC values up to 16 and 8 µg/mL, respectively, toward all the strains; (2) Benzyloxy derivatives 14-40 and 46: The simplest representative of this class (14, O-benzyl) had an anti-H. pylori activity comparable to carvacrol, whereas other substitutions on the aryl ring such as 3,4-diCl, 2,6-diF, 3-F, 3-OCH 3 , 2-Cl-4-OCH 3 , 2-Br, and 4-Br were detrimental or didn't produce a strong increment of the antimicrobial activity. Conversely, some substituents, especially in the para position of the aryl ring, such as CF 3 , Ph, CN, NO 2 and NH 2 were promising to show improvements. Indeed, CF 3 could act as a bioisostere of the NO 2 group and in both series we can highlight the following activity order: p > m > o. The presence of a fluorine atom or a trifluoromethyl group into an organic scaffold can lead to changes in the physical, chemical and biological properties, often associated with an increase lipophilicity and electronegativity but a relatively small size, which can favour entry into the cell membranes. The presence of two CF 3 in compound 22 didn't synergistically contribute to an improved inhibitory action. As regards sulfur-based compounds (37-39) we highlighted a better activity with a higher sulfur oxidation state (ArSO 2 CH 3 > ArSOCH 3 > ArSCH 3 ). Unfortunately, the introduction of bromine atoms in compounds 30 and 31 reduced the inhibitory effect likely due to their low ability to act as H-bond acceptors and their higher atomic radius, both determining a negative steric constrain. Moreover, electron-donating groups were not tolerated. (3) Bicyclic and heteroaryl derivatives 41 and 42: The change of the benzyl group into a naphthalene led to a total loss of inhibitory activity, whereas phthalimide can be tolerated; (4) Coumarin ester derivatives 43-45: These compounds imparted a slight improvement of the anti-Helicobacter activity against all the strains with respect to carvacrol. These results were in accordance with those obtained with the same substitution pattern previously published by us [23,27].
Regarding the mechanism of action, it is reasonable to consider these compounds as good bactericidal inhibitors, being the value of MBC/MIC ratio between 1 and 2.

Effects of Carvacrol and Its Derivatives on Cell Viability of AGS Cell Line
As a follow-up study, the selection of the candidates for biological assays was guided by the anti-Helicobacter pylori activity (Table 1, compounds highlighted in gray). AGS cells, were incubated for 24 h with the specified molecules or with 0.1% DMSO vehicle (control). Data shown are the means ± SD of three experiments with quintuplicate determinations. Carvacrol showed cytotoxic effects by reducing cell viability of AGS cells (IC 50 = 300 ± 6.5 µM) in a dose-dependent manner after treatment. Out of 17 carvacrol derivatives, only five (16, 21, 35, 38 and 39) demonstrated a dose-dependent inhibitory effect on cell viability inferior to carvacrol. All of them possessed an IC 50 value higher than the reference drug (5-Fluorouracil, IC 50 = 82.3 ± 5.6 µM) ( Table 2).
More in detail, carvacrol was a medium potency anti-proliferative agent against AGS cells. From the results shown in Table 2, it is possible to highlight that the introduction of alkyl substituents (compounds 6 and 9) or coumarin rings (compounds 43-45) directly connected to the carvacrol oxygen led to a loss of inhibitory activity. The presence of a benzyl moiety, especially meta or para substituted, exerted some improvements. In particular, 4-CF 3 , 3-CH 3 , 4-SOCH 3 and 4-SO 2 CH 3 brought to compounds endowed with a stronger effect with respect to carvacrol. Other clear SAR trends are not observable. These small and easily accessible molecules are promising motifs in the development of dual agents able not only to reduce H. pylori growth, but also to counteract the proliferation of AGS cells at higher concentration. a Data are expressed as mean ± SD, n = 3; na: not active at the maximum concentration tested (800 µM).

Chemistry
Unless otherwise indicated, all reactions were carried out under a positive pressure of nitrogen in washed and oven-dried glassware. All the solvents and reagents were directly used as supplied by Sigma-Aldrich (Milan, Italy) without further purification. Where mixtures of solvents are specified, the stated ratios are volume:volume. All melting points were measured on a SMP1 melting point apparatus (Stuart ® , Staffordshire, UK) and are uncorrected (temperatures are reported in • C). Structural analysis consisted of elemental analysis, 1 H-/ 13 C-/ 19 F NMR spectra and crystallographic studies. 1 H and 13 CNMR spectra were mainly recorded at 300 MHz and 75 MHz (Mercury spectrometer, Varian, Santa Clara, CA, USA), while some compounds were analysed at 400 MHz and 101 MHz on a Bruker spectrometer (Milan, Italy), using CDCl 3 and DMSO-d 6 , as the solvents at room temperature. Conversely, 19 F spectra were recorded on a Bruker AVANCE 600 spectrometer at 564.7 MHz, using CDCl 3 as the solvent. All the compounds were studied at the final concentration of~25 mg/mL. 1 H and 13 C chemical shifts are expressed as δ units (parts per millions) relative to the solvent signal, whereas 19 F chemical shifts are expressed as δ units relative to an external standard (CF 3 COOH, δ −76.55 ppm). 1 H spectra are described as follows: δ H (spectrometer frequency, solvent): chemical shift/ppm (multiplicity, J-coupling constant(s) in Hertz (Hz), number of protons, assignment). 13 C spectra are described as follows: δ C (spectrometer frequency, solvent): chemical shift/ppm (assignment) and are fully proton decoupled. 19 F spectra are described as follows: δ F (spectrometer frequency, solvent): chemical shift/ppm (multiplicity, J-coupling constant(s) in Hertz, number of fluorine, assignment). Multiplets are abbreviated as follows: br-broad; s-singlet; d-doublet; t-triplet; q-quartet; td-triplet of doublets; m-multiplet. The exchangeable protons (OH, NH 2 ) were assessed by the addition of deuterium oxide. The processing and analyses of the NMR data were carried out with MestreNova. Preparative chromatography was carried out employing silica gel (high purity grade, pore size 60 Å, 230-400 mesh particle size). All the purifications and reactions were carried out by thin layer chromatography (TLC) performed on 0.2 mm thick silica gel-aluminium backed plates (60 F254). Spot visualization was performed under short-and long-wavelength (254 and 365 nm, respectively) ultra-violet irradiation. Where given, systematic compound names were generated  1-12, 14-34, 36, 37, and 40-42 To a stirring solution of carvacrol (1 equiv.) in dry DMF (10 mL) was added freshly ground and anhydrous potassium carbonate (K 2 CO 3 , 1.2 equiv.). The suspension was stirred for 30 min at room temperature; then, the proper (substituted)benzyl, diarylmethyl, heteroarylmethyl or alkyl bromide (1.0 equiv.) was added and the reaction stirred until disappearance of the starting reagents, as detected by TLC. Once the reaction was completed, the mixture was poured into ice-cold water (100 mL) and extracted with dichloromethane (DCM, 3 × 20 mL). The organics were reunited and added with anhydrous sodium sulphate (Na 2 SO 4 ) to remove water. The salt was filtered and washed three times with small amounts (5 mL) of dry DCM. The organic phase was evaporated in vacuo to afford the crude extract containing the target molecule that was recovered through column chromatography, employing silica gel (SiO 2 ) and proper mixtures of n-hexane/ethyl acetate.

Synthesis of Compound 13
To a stirring solution of ethyl 2-(5-isopropyl-2-methylphenoxy) acetate (12, 1.0 equiv.) in 10 mL of methanol was added dropwise lithium hydroxide (1.2 equiv.) dissolved in 10 mL of water. The reaction was stirred at room temperature for 24 h; then, the mixture was concentrated in vacuo to remove methanol and quenched with 3N HCl (15 mL). The precipitate was collected by filtration and washed with n-hexane to give the title compound 13, without further purification requirements.

Synthesis of Compound 35
To a stirring solution of 4-isopropyl-1-methyl-2-((4-nitrobenzyl)oxy)benzene (compound 34, 1.0 equiv.) in tetrahydrofuran (THF, 15 mL) was added dropwise a freshly prepared solution of sodium dithionite (5.5 equiv.) dissolved in a basic solution made of water (15 mL) and sodium bicarbonate (5.5 equiv.). The reaction was stirred at room temperature until completion (assessed by TLC); then THF was evaporated in vacuo and the aqueous phase extracted with DCM (3 × 20 mL). The organics were reunited, dried over sodium sulphate and filtered to remove the salt. DCM was evaporated in vacuo to give the crude extract, that was purified by column chromatography (SiO 2 , n-hexane:ethyl acetate 5:1) to afford the amino derivative 35 as an orange viscous oil.

Synthesis of Compounds 38 and 39
To a stirring solution of (4-((5-isopropyl-2-methylphenoxy)methyl)phenyl)(methyl) sulfane (37, 1 equiv.) in DCM (10 mL) placed on ice/water bath (0-5 • C), was added dropwise a freshly prepared solution of 3-chloroperbenzoic acid (1 equiv.) dissolved in 5 mL of DCM in an ice-bath. The reaction was followed by TLC and after 8 h another aliquot of 3-chloroperbenzoic acid (1 equiv. in 5 mL of DCM) was added and the reaction stirred at room temperature for further 24 h. Once the reaction completion was reached (appearance on TLC of the two spots relative to sulfoxide and sulfone derivatives), the mixture was concentrated in vacuo and the two compounds separated by column chromatography on silica gel (n-hexane:ethyl acetate, 5:1) to give the title compounds 38 and 39.

Synthesis of Intermediates A/A1-C/C1
For the synthesis of the coumarin-3-carboxylic acids A1-C1 we used the synthetic procedures previously reported by our group [22]. Briefly, the Knoevenagel cyclization between the proper substituted salicylaldehydes (1 equiv.) and diethyl malonate (1 equiv.) was performed in ethanol (25 mL) with catalytic amounts of piperidine. The reaction was followed by TLC until disappearance of their starting reagents. Once the reaction completed, the mixture was poured into ice-cold water and the solid collected by filtration. The powder was washed with n-hexane to obtain the title ester compounds A-C.
The corresponding ester (A, B or C, 1 equiv.) was then dissolved in ethanol (10 mL) and hydrolyzed by using 20% NaOH solution (25 mL). After reaction completion assessed by means of TLC, the ethanol was evaporated in vacuo. The solution was quenched with 3N HCl (20 mL) leading to precipitation of the coumarin-3-carboxylic acid, that was collected by filtration and washed with n-hexane, affording the title compounds A1-C1 without further purification requirements.

Conclusions
Based on the shortlisted hits, a large series of carvacrol-based molecules were designed, synthesized and evaluated for their ability to act as dual agents (inhibitory action against the growth of H. pylori and AGS cells) along with the assessment of robust structure-activity relationships. Several hits with required balance of activities were extrapolated as a result of this study. Moreover, the most active compounds displayed antimicrobial activity with similar MIC and MBC values toward H. pylori strains with a different antibiotic susceptibility pattern, thus suggesting a mechanism of action alternative to metronidazole, amoxicillin and clarithromycin. The most important result is that the anti-Helicobacter pylori activity was not only strictly related to the presence of an OH moiety, as reported for the general antibacterial activity of the parent compound. Among the compounds evaluated, some analogues exhibited MIC/MBC values in the low µg/mL range. In this regard, the most noteworthy compounds displaying the lowest MIC/MBC activity against H. pylori, such as compounds 16 and 39, also showed good activity against AGS cells (IC 50 compound 16 = 209 µM; IC 50 compound 39 = 209 µM). Further studies might explain the potential synergistic effects of the combination of these derivatives with the currently used therapeutics. In addition, the possibility to treat drug resistant H. pylori strains would be beneficial in the clinical setting due to the high development of resistance attributed to H. pylori. Finally, whereas the development of biofilms by H. pylori as well as the ability of the microorganism to enter the Viable But Non-Culturable (VBNC) state represent two different survival strategies that induce resistance/tolerance of the microorganism or the microbial community to antimicrobial drugs [36], future studies will be carried out to evaluate the potential activity of such molecules on both H. pylori biofilm and VBNC state.

Conflicts of Interest:
The authors declare no conflict of interest.