Synthesis and biological evaluation of carvacrol-based derivatives as dual inhibitors of H. Pylori strains and ags cell proliferation

Article abstract of DOI:10.3390/ph13110405

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,¹ H/¹³ C/¹⁹ F 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.

Full text of DOI:10.3390/ph13110405

pharmaceuticals
Article
Synthesis and Biological Evaluation of
Carvacrol-Based Derivatives as Dual Inhibitors of
H. pylori Strains and AGS Cell Proliferation
1
2,
3
2
Francesca Sisto , Simone Carradori * , Paolo Guglielmi , Carmen Beatrice Traversi ,
3
6
4
3
5
Mattia Spano
, Anatoly P. Sobolev
, Daniela Secci , Maria Carmela Di Marcantonio
,
2
and Gabriella Mincione 5
Entela Haloci , Rossella Grande
1
Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy;
francesca.sisto@unimi.it
Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara,
Via dei Vestini 31, 66100 Chieti, Italy; cbtraversi@gmail.com (C.B.T.); rossella.grande@unich.it (R.G.)
Department of Chemistry and Technology of Drugs, Sapienza University of Rome, P.le A. Moro 5,
2
3
0
0185 Rome, Italy; paolo.guglielmi@uniroma1.it (P.G.); mattia.spano@uniroma1.it (M.S.);
daniela.secci@uniroma1.it (D.S.)
Institute for Biological Systems, “Annalaura Segre” Magnetic Resonance Laboratory,
CNR, 00015 Monterotondo (Rome), Italy; anatoly.sobolev@cnr.it
4
5
6
Department of Medical, Oral, and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-Pescara,
6100 Chieti, Italy; dimarcantonio@unich.it (M.C.D.M.); gabriella.mincione@unich.it (G.M.)
6
Department of Pharmacy, University of Medicine, Tirana, Rr. Dibres 369, 1001 Tirana, Albania;
entela.haloci@umed.edu.al
*
Correspondence: simone.carradori@unich.it
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 22 October 2020; Accepted: 17 November 2020; Published: 19 November 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: 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
1
13 19
analysis, H/ C/ F NMR spectra and crystallographic studies. The structure-activity relationships
evidenced that among ether derivatives the substitution with specific electron-withdrawing groups
(
CF and NO ) especially in the para position of the benzyl ring led to an improvement of the
3 2
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 g/mL). Ester derivatives
coumarin-carvacrol hybrids) displayed a slight enhancement of the inhibitory activity up to MIC
µ
(
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 g/mL against all the evaluated strains) were also assayed for their ability to
µ
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.
Keywords: carvacrol; Helicobacter pylori; AGS cells; semi-synthesis; drug resistance; dual agent; coumarin
1
. 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 [ ]. More in
1,2
Pharmaceuticals 2020, 13, 405; doi:10.3390/ph13110405
www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2020, 13, 405
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detail, carvacrol and its derivatives were shown to exert an interesting antimicrobial and antibiofilm
effects against a large panel of Gram-positive and Gram-negative bacteria and fungi [ ]. Its mechanism
3–5
of action, albeit not yet fully elucidated, could involve structural and functional alterations of the
membrane, dysregulation of nucleic acids, altered metabolism and ATP production.
Carvacrol and carvacrol-producing plants (Satureja spp., Thymus spp., and Origanum spp.) were
also studied for their ability to inhibit Helicobacter pylori (H. pylori) growth, evidencing the possibility to
introduce chemical modifications of this lead compound to improve this biological activity and/or to
enhance its poor pharmacokinetic profile [6].
H. pylori, a microaerophilic Gram-negative bacterium, colonizes about the 50% of the world’s
population representing the causative agent of the development of chronic gastritis, peptic ulcer
and gastric cancer [
Health Organization. Gastric cancer represents the third most common cancer worldwide [
epidemiological studies demonstrated that the eradication of H. pylori induces a decrease of incidence of
such malignancy [ ]. The triple therapy, consisting of a proton-pump inhibitor (PPI) and two different
antimicrobial drugs, represented the anti-H. pylori standard therapy for the last 20 years. The failure of
the above-mentioned therapy may be due to an increased antibiotic resistance to clarithromycin [10
7]; for the latter, it has been recognized as a class I carcinogen by the World
7,8] and
9
]
and levofloxacin, two of the antimicrobials used in the triple therapy. In particular, levofloxacin
was introduced a decade ago as an alternative to clarithromycin [11]. Recently, a bismuth-based
quadruple therapy consisting of PPI plus a standardized three-in-one capsule, bismuth subcitrate
potassium, metronidazole, and tetracycline has been recommended [12,13] as the first-line treatment
of multidrug-resistant H. pylori strains, in particular in areas of high clarithromycin resistance [14].
The increase of the failure rates of the triple therapy in many countries such as in Europe, Korea, Japan,
and China, [15] induced the scientific community to evaluate new therapeutical approaches in order to
decrease the development of the antibiotic resistance phenomenon. Therefore, the study of the potential
anti-H. pylori activity of carvacrol and its derivatives could be a starting point useful to assess the
therapeutic efficacy of alternative compounds inspired by natural scaffolds [16]. Moreover, carvacrol
was shown to exert anti-inflammatory (COX inhibition), antinociceptive and antiulcer activities in vitro
and in vivo [17,18], which are useful to reduce damages correlated to H. pylori colonization of the
human gastric mucosa and associated pathogenesis, whereas the potential of plant essential oils in
anticancer treatment has recently obtained many research efforts to overcome drug resistance and
multiple side effects. For these reasons, several authors also evaluated the antiproliferative efficacy of
carvacrol against human gastric adenocarcinoma (AGS) cells in vitro and in Wistar rats in vivo [19].
Starting from these premises and keeping in mind that the development of drugs from plant
secondary metabolites is a topic of the most recent ongoing research and engages large-scale
pharmacological screenings of extracts and active compounds, we aimed at designing a large library
of carvacrol-based derivatives possessing multiple tuneable functional groups for their chemical
modulation to desired properties and assuring the broadest chemical diversity. Indeed, natural product
derivatives could shed light on new therapeutic agents against human diseases due to the modulation of
the physical-chemical, toxicological and drug-like characteristics of their natural parent compound [20].
This is very important when addressing pathologies such as gastric cancer where a pluralism of
causative factors must be faced by a feasible research strategy which can evolve a multi-targeted
perspective (one molecule acting on separate targets of the disease). Moreover, this approach can
overcome issue related to combination therapy and the possibility of drug-drug interactions.
2
. Results and Discussion
2
.1. Chemistry
For the synthesis of the compounds 1–46 we followed the synthetic approach reported in the Schemes 1
and 2, taking advantage of the hydroxyl portion of carvacrol in order to synthesize ethers and esters
with modified hydrophilic/hydrophobic parameters. Ether derivatives 12 14 34 36 37 and 40 42
1
–
,
–
,
,
–

Pharmaceuticals 2020, 13, 405
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(
Scheme 1A) have been synthesized by reacting the carvacrol with the proper bromide; these reactions
were performed in N,N’-dimethylformamide (DMF), in the presence of potassium carbonate (K CO )
2
3
and under nitrogen (N ) atmosphere. Some of the products obtained in this step were further
2
employed for additional structural modifications obtained through ester hydrolysis, nitro reduction
and sulfur oxidation.
Scheme 1. Synthesis of ether compounds
1–42 and 46. (A) compounds obtained by direct modification
of the hydroxy group of carvacrol; ( ) alkaline hydrolysis of compound 12 and modification of
B
compound 13 to hydrazido compound 46; (
oxidation to compounds 38 and 39.
C) nitro reduction to amine compound 35; (D) sulfur

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Scheme 2. Synthesis of ester compounds 43–45 (coumarin-carvacrol hybrids).
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 group, located at the para position of the benzyl moiety of compound 34, was reduced
2
with sodium dithionite (Na S O ), leading to the p-NH derivative 35 (Scheme 1C). Derivative
2
2
4
2
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
solution and afforded the carboxylic acid derivatives A1
proper coumarin-3-carboxylic acid, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
-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
A
–
C
[22]. The removal of the ester function through hydrolysis was performed using 10% NaOH
–C1. Finally, coupling of carvacrol with the
1
–
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
1
13
19
confirmed by spectral studies ( H, C, and 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.
2
.2. 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).

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Figure 1. (A): ORTEP structure of compound 34; (B) Crystal structure of carvacrol-based compound 34.
2
.3. 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.
2
.4. 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.
2
.4.1. 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
3
2–64 µg/mL, respectively. All the chemical modifications can be grouped into four classes to define
robust structure-activity relationships (SARs):

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Table 1. MIC and MBC values for carvacrol (parent compound) and its semi-synthetic derivatives (1–46) against nine strains of H. pylori. Antibiotic susceptibility is
reported for each H. pylori strain.
H. pylori Strains
MIC/MBC (µg/mL)
NCTC
F4
190
23
110 R
F1
F34/497
F40/499
F40/442
1
1637
Carvacrol
64/64
128/128
64/64
64/128
128/128
64/64
16/32
32/32
64/64
8/8
128/128
128/128
64/64
>128/>128
64/64
64/128
8/16
32/32
64/64
>128/>128
64/128
64/64
128/128
64/64
64/64
>128/>128
>128/>128
128/>128
128/128
64/64
32/64
64/128
64/64
64/64
128/128
64/64
64/64
128/128
64/64
64/128
64/128
64/128
32/32
128/128
128/128
64/64
64/64
>128/>128
128/>128
64/64
128/128
128/128
64/64
64/64
128/128
64/64
64/64
128/128
64/64
32/32
32/32
64/64
16/16
128/128
128/128
64/64
>128/>128
64/64
32/64
4/8
16/16
16/32
32/32
32/32
32/32
128/>128
64/64
32/32
64/64
16/16
32/64
8/8
64/64
16/32
128/128
32/64
32/64
128/128
64/64
32/32
32/32
64/64
8/8
128/128
128/128
64/64
128/128
64/64
32/32
2/2
8/32
16/16
32/64
64/64
32/64
64/64
128/128
64/64
16/16
64/64
64/64
16/16
128/128
128/128
32/64
128/128
64/64
32/32
8/8
16/16
16/16
64/128
64/64
32/32
>128/>128
32/32
32/32
32/64
16/16
32/32
8/8
32/64
64/64
64/64
64/64
128/128
64/64
32/32
64/64
64/64
16/16
128/128
128/128
64/64
128/128
64/64
32/32
4/8
16/16
16/16
64/128
64/64
32/32
>128/>128
32/64
32/32
32/64
16/16
32/32
8/16
1
2
3
4
5
6
7
8
9
32/32
32/64
64/64
32/32
64/64
64/64
32/32
32/64
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
128/128
128/128
32/32
>128/>128
64/64
128/128
128/128
128/128
>128/>128
64/64
128/128
128/128
64/64
>128/>128
64/64
>128/>128
64/64
64/64
8/16
32/32
32/64
32/32
32/32
16/16
32/32
64/64
8/8
32/32
64/64
32/32
32/32
32/64
32/32
32/64
16/32
16/32
64/128
>128/>128
128/>128
32/64
>128/>128
32/64
>128/>128
128/128
32/32
>128/>128
32/32
64/128
32/32
32/32
>128/>128
32/64
32/64
>128/>128
64/64
128/128
64/64
32/64
>128/>128
64/64
64/128
64/64
16/32
16/32
16/32
>128/>128
64/64
>128/>128
64/64
128/128
32/64
32/32
64/64
32/32
32/64
16/16
16/64
32/32
64/64
32/32
64/64
32/64
64/64
64/64
64/64
32/32
32/32
16/32
64/64
32/64
64/64
16/32
64/64
32/64
32/32
32/64
16/32
32/32
16/16
32/64
64/64
16/32

Pharmaceuticals 2020, 13, 405
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Table 1. Cont.
H. pylori Strains
MIC/MBC (µg/mL)
NCTC
F4
190
23
110 R
F1
F34/497
F40/499
F40/442
1
1637
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
32/64
32/32
16/32
8/8
8/8
4/8
8/16
32/64
8/8
2/2
8/8
64/128
16/32
64/64
4/4
4/4
16/16
4/4
64/64
4/4
4/4
64/128
32/64
64/64
8/8
4/4
32/64
8/8
64/64
8/8
2/2
64/128
16/16
64/64
4/4
4/8
16/16
8/8
64/64
4/4
4/4
64/128
64/64
32/64
4/4
4/4
32/64
8/8
64/64
8/8
2/2
64/64
16/32
32/32
8/8
4/4
4/4
8/8
32/64
4/4
2/2
4/4
32/32
64/64
16/16
8/8
4/4
2/2
8/8
32/32
2/2
2/2
4/4
32/64
8/16
32/64
8/8
4/4
16/16
8/8
64/64
8/8
2/2
32/64
16/32
16/32
8/8
4/4
16/16
8/8
32/64
8/8
2/2
8/16
8/16
8/16
8/16
4/4
4/4
>128/>128
16/16
32/32
32/32
32/32
64/128
>128/>128
16/32
64/128
32/32
32/128
32/64
>128/>128
32/32
128/>128
32/32
64/>128
32/64
>128/>128
32/32
128/>128
32/32
32/128
32/64
>128/>128
32/32
128/>128
32/32
64/128
32/64
>128/>128
32/32
32/32
32/32
32/32
64/128
>128/>128
16/32
64/128
32/32
32/32
64/>128
>128/>128
32/32
32/32
32/32
32/32
32/64
>128/>128
16/16
32/32
32/32
32/32
64/128
MTZ+
CLR+
AMX−
MTZ−
CLR−
AMX−
MTZ−
CLR−
AMX−
MTZ+
CLR−
AMX−
MTZ+
CLR−
AMX−
MTZ−
CLR+
AMX−
MTZ+
CLR+
AMX−
MTZ+
CLR+
AMX−
MTZ+
CLR−
AMX−
Antibiotic
susceptibility
MTZ+ = metronidazole resistant (MIC > 8
µ
g/mL); MTZ
−
= metronidazole susceptible (MIC
≤
8
µ
g/mL); CLR+ = clarithromycin resistant (MIC > 0.25
µ
g/mL); CLR
−
= clarithromycin
susceptible (MIC
≤
0.25
µ
g/mL); AMX+ = amoxicillin resistant (MIC > 0.125
activity assays against AGS cells are highlighted in grey.
µ
g/mL); AMX
−
= amoxicillin susceptible (MIC
≤
0.125 µg/mL). Compounds selected for in vitro anti-proliferative

Pharmaceuticals 2020, 13, 405
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(
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 (OPr) and (OBu)
slightly presented MIC values comparable to carvacrol against two strains (F34/497 and F40/499),
whereas compounds (O-crotyl) and (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 , 2-Cl-4-OCH , 2-Br, and 4-Br were detrimental or
(
2
3
6
9
(
–
3
3
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 , Ph, CN, NO and NH were promising
3
2
2
to show improvements. Indeed, CF could act as a bioisostere of the NO group and in both
3
2
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 CF3
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 CH > ArSOCH > ArSCH ). Unfortunately, the introduction of bromine atoms
2
3
3
3
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.
2
.4.2. 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 (IC50 = 300
after treatment. Out of 17 carvacrol derivatives, only five (16
±
6.5
,
µ
M) in a dose-dependent manner
21 35 38 and 39) demonstrated a
,
,
dose-dependent inhibitory effect on cell viability inferior to carvacrol. All of them possessed an IC50
value higher than the reference drug (5-Fluorouracil, IC50 = 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
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-CH , 4-SOCH and 4-SO CH brought
6 and 9) or coumarin rings (compounds 43–45) directly connected to the carvacrol
3
3
3
2
3
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.

Pharmaceuticals 2020, 13, 405
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Table 2. IC50 values are expressed as mean
quintuplicate determinations.
±
standard deviation (SD) of three experiments with
Compound
IC50 (µM) a
Carvacrol
300 ± 6.5
530 ± 16
527 ± 8.0
209 ± 9.4
366 ± 13
na
6
9
16
17
20
21
29
32
33
34
35
38
39
42
43
44
45
179 ± 9.0
na
382 ± 13
615 ± 9.6
na
283 ± 12
217 ± 7.6
209 ± 10
722 ± 8.5
na
na
na
5
-Fluorouracil (5-FU)
82.3 ± 5.6
a
Data are expressed as mean ± SD, n = 3; na: not active at the maximum concentration tested (800 µM).
3
. Materials and Methods
3
.1. 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
◦
1
13
19
C). Structural analysis consisted of elemental analysis, H-/ C-/ F NMR spectra and crystallographic
1
13
studies. H and 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 and DMSO-d , as the solvents at room
temperature. Conversely, 19F spectra were recorded on a Bruker AVANCE 600 spectrometer at
3
6
5
64.7 MHz, using CDCl as the solvent. All the compounds were studied at the final concentration
3
13
1
of ~25 mg/mL. H and C chemical shifts are expressed as
solvent signal, whereas 19F chemical shifts are expressed as
δ
units (parts per millions) relative to the
units relative to an external standard
(spectrometer frequency, solvent):
δ
1
(
CF COOH, δ −76.55 ppm). H spectra are described as follows:
δ
H
3
chemical shift/ppm (multiplicity, J-coupling constant(s) in Hertz (Hz), number of protons, assignment).
1
3
C spectra are described as follows:
δ (spectrometer frequency, solvent): chemical shift/ppm
C
19
(assignment) and are fully proton decoupled. F spectra are described as follows:
δ (spectrometer
F
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, NH2)
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

Pharmaceuticals 2020, 13, 405
10 of 21
by ChemBioDraw Ultra 14.0 following IUPAC conventions. Microanalyses were performed with a
Perkin-Elmer 260 elemental analyzer (PerkinElmer, Inc., Waltham, MA, USA) for C, H and N and the
results were within
reported in the Supplementary Materials.
±
0.4% of the theoretical values. NMR spectra of all new compounds have been
3
.2. Synthesis of Carvacrol Derivatives
3
.2.1. General Procedure for the Synthesis of Compounds 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 CO , 1.2 equiv.). The suspension was stirred for 30 min at room
2
3
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 SO ) to remove water. The salt was filtered and washed three times
(
×
2
4
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 ) and proper mixtures of n-hexane/ethyl acetate.
2
3
.2.2. 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.
3
.2.3. Synthesis of Compound 46
To a stirring solution of ethyl 2-(5-isopropyl-2-methylphenoxy)acetic acid (13, 1.0 equiv.) in 10 mL
of THF was added triethyl amine (3.0 equiv.) and ethyl chloroformate (1.2 equiv.). After 30 min,
3
3
-nitrophenylhydrazine (1.3 equiv.) was added and the reaction stirred at room temperature for
h. Once the reaction was completed, the mixture was poured on ice-cold water (100 mL) and the
precipitate collected by filtration. Purification through column chromatography (SiO , n-hexane:ethyl
2
acetate 2:1) afforded the title compound 46.
3
.2.4. 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 , n-hexane:ethyl acetate 5:1)
2
to afford the amino derivative 35 as an orange viscous oil.
3
.2.5. 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

Pharmaceuticals 2020, 13, 405
11 of 21
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.
3
.2.6. 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.
3
.2.7. Synthesis of Compounds 43–45
To a stirring solution of the proper coumarin-3-carboxylic acid (A1–C1, 1.0 equiv.) in
4-methylmorpholine (NMM, 10 mL) under nitrogen atmosphere, were added portionwise 1-ethyl-3-
(
3-dimethylaminopropyl)carbodiimide (EDC, 1.2 equiv.) and 1-hydroxybenzotriazole (HOBt, 1.2 equiv.).
After 1 h, carvacrol (1.0 equiv.) was added and the reaction stirred for further 24 h. At the reaction
completion (by TLC), the mixture was poured on ice-cold water. The precipitate was collected by
filtration and washed with petroleum ether and n-hexane to afford title compounds 43–45 in good
yield and purity.
3
.3. Characterization Data for Carvacrol Derivatives
-ethoxy-4-isopropyl-1-methylbenzene ( ). Colourless oil, 66% yield. H NMR (400 MHz, CDCl ):
1.29 (d, J = 6.9 Hz, 6H, 2 CH ), 1.46 (t, J = 7.0 Hz, 3H, CH CH ), 2.23 (s, 3H, ArCH ), 2.87–2.94
1
2
1
3
δ
×
3
2
3
3
(
1
m, 1H, CH), 4.06–4.11 (m, 2H, OCH CH ), 6.74 (s, 1H, Ar), 6.76–6.78 (m, 1H, Ar), 7.10 (d, J = 7.5 Hz,
2
3
H, Ar). 13C NMR (101 MHz, CDCl3):
δ
15.1 (CH ), 15.8 (CH ), 24.2 (2
×
CH ), 34.2 (CH), 63.5 (OCH ),
3 2
3
3
109.6 (Ar), 117.9 (Ar), 124.2 (Ar), 130.4, 147.8 (Ar), 157.1 (Ar). Anal. Calcd for C H O: C, 80.85;
12
18
H, 10.18. Found: C, 81.12; H, 10.14.
◦
1
4
-isopropyl-1-methyl-2-propoxybenzene (
400 MHz, CDCl3):
.84–1.93 (m, 2H, CH CH CH ), 2.26 (s, 3H, ArCH ), 2.89–2.96 (m, 1H, CH), 4.00 (t, J = 6.4 Hz 2H,
2
). Colourless oil, 56% yield, mp 117–121 C. H NMR
(
1
δ
1.12 (t, J = 7.4 Hz, 3H, CH CH CH ), 1.31 (d, J = 6.9 Hz, 6H, 2
×
CH3),
2
2
3
2
2
3
3
13
OCH CH CH ), 6.75–6.79 (m, 2H, Ar), 7.11 (d, J = 7.5 Hz, 1H, Ar). C NMR (101 MHz, CDCl3):
δ
10.7
2
2
3
(
1
CH ), 15.8 (CH ), 22.9 (CH ), 24.2 (2
30.4 (Ar), 147.9 (Ar), 157.2 (Ar). Anal. Calcd for C H O: C, 81.20; H, 10.48. Found: C, 81.33; H, 10.51.
13 20
×
CH ), 34.2 (CH), 69.4 (OCH ), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar),
3
3
2
3
2
1
2
-(allyloxy)-4-isopropyl-1-methylbenzene (
CDCl3): 1.37 (s, 3H, CH ), 1.41 (s, 3H, CH ), 2.36 (s, 3H, ArCH ), 2.94–3.01 (m, 1H, CH), 4.66–4.69
m, 2H, CH2), 5.37–5.60 (m, 2H, OCH ), 6.14–6.27 (m, 1H, =CH), 6.83 (s, 1H, Ar), 6.86-6.89 (m, 1H,
3). Yellow oil, 70% yield. H NMR (300 MHz,
δ
3
3
3
(
2
Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar). 13C NMR (75 MHz, CDCl3):
δ
16.0 (CH ), 24.3 (2
×
CH ), 34.3 (CH ),
3 3
3
6
8.8 (OCH ), 110.0 (Ar), 116.9 (Ar), 118.3 (=CH ), 124.3 (Ar), 130.6 (Ar), 133.9 (=CH), 147.9 (Ar),
2
2
1
56.8 (Ar). Anal. Calcd for C H O: C, 82.06; H, 9.54. Found: C, 81.87; H, 9.58.
13 18
1
4
-isopropyl-1-methyl-2-(prop-2-yn-1-yloxy)benzene (
CDCl3): 1.35 (d, J = 6.9 Hz, 6H, 2
m, 1H, CH), 4.80 (d, J = 2.4 Hz, 1H, OCH ), 6.89 (d, J = 7.5 Hz, 1H, Ar), 6.93 (s, 1H, Ar), 7.17 (d,
4). Yellow oil, 74% yield. H NMR (300 MHz,
δ
×
CH ), 2.32 (s, 3H, ArCH ), 2.58 (t, J = 2.4 Hz, 1H, CH), 2.9–3.01
≡
3
3
(
2
J = 7.8 Hz, 1H, Ar). 13C NMR (75 MHz, CDCl3):
δ
15.9 (CH ), 24.2 (2
×
CH ), 34.2 (CH), 56.01 (OCH ),
3 2
3

Pharmaceuticals 2020, 13, 405
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75.26 (Csp), 79.2 (CspH), 110.4 (Ar), 119.2 (Ar), 124.6 (Ar), 130.8 (Ar), 147.9 (Ar), 155.8 (Ar). Anal.
Calcd for C H O: C, 82.94; H, 8.57. Found: C, 83.17; H, 8.54.
13
16
1
2
-butoxy-4-isopropyl-1-methylbenzene (
5). Colourless oil, 78% yield. H NMR (400 MHz, CDCl ):
3
δ
1.07 (t, J = 7.4 Hz, 3H, CH CH CH CH ), 1.32 (d, J = 6.9 Hz, 6H, 2
×
CH ), 1.56–1.65 (m, 2H,
3
2
2
2
3
CH CH CH CH ), 1.83–1.90 (m, 2H, CH CH CH CH ), 2.27 (s, 3H, ArCH ), 2.90–2.97 (m, 1H, CH),
4
2
2
2
3
2
2
2
3
3
13
.05 (t, J = 6.3 Hz 2H, OCH CH CH ), 6.77–6.80 (m, 2H, Ar), 7.12 (d, J = 7.5 Hz, 1H, 1Ar). C NMR
2 2 3
(
6
101 MHz, CDCl3):
δ
14.0 (CH ), 15.9 (CH ), 19.5 (CH ), 24.2 (2
×
CH ), 31.6 (CH ), 34.2 (CH),
3 2
3
3
2
7.6 (OCH ), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar), 130.4 (Ar) 147.8 (Ar), 157.2 (Ar). Anal. Calcd for
2
C H O: C, 81.50; H, 10.75. Found: C, 81.63; H, 10.77.
14
22
1
2
-(but-2-en-1-yloxy)-4-isopropyl-1-methylbenzene (
MHz, CDCl3): 1.55–1.58 (m, 6H, 2 CH ), 2.05 (d, J = 4.8 Hz, 3H, CH ), 2.54 (s, 3H, ArCH ), 3.12–3.19
m, 1H, CH), 4.76 (d, J = 5.1 Hz, 2H, OCH ), 6.01–6.18 (m, 2H, 2
6). Pale yellow oil, 69% yield. H NMR (300
δ
×
3
3
3
(
×
=CH), 7.01 (s, 1H, Ar), 7.03 (d,
2
13
J = 7.8 Hz, 1H, Ar), 7.35 (d, J = 7.5 Hz, 1H, Ar). C NMR (75 MHz, CDCl ): 16.2 (CH ), 18.1 (CH ),
δ
3
3
3
2
4.4 (2 × CH ), 34.5 (CH), 68.8 (OCH ), 110.0 (Ar), 118.3 (Ar), 124.4 (Ar), 127.1 (=CH), 129.4 (=CH),
3
2
1
30.7 Ar), 147.9 (Ar), 157.2 (Ar). Mixture of E/Z isomers with 5.2:1 ratio. For sake of clarity, we reported
only the signals related to the major isomer. Anal. Calcd for C H O: C, 82.30; H, 9.87. Found:
14
20
C, 82.33; H, 9.88.
1
4
-isopropyl-1-methyl-2-((3-methylbut-2-en-1-yl)oxy)benzene (
7
). Yellow oil, 71% yield. H NMR
(
300 MHz, CDCl3):
δ
1.29–1.32 (m, 6H, 2
×
CH ), 1.81–1.85 (m, 6H, 2 CH ), 2.26 (s, 3H, ArCH ),
×
3
3
3
2
6
2
.87–2.96 (m, 1H, CH), 4.95 (d, J = 6.6 Hz, 2H, OCH ), 5.54–5.58 (m, 1H, =CH), 6.77 (s, 1H, Ar),
2
.79–6.80 (m, 1H, Ar), 7.11 (d, J = 7.5 Hz, 1H, Ar). 13C NMR (75 MHz, CDCl3):
4.2 (2 × CH ), 25.9 (CH ), 34.2 (CH), 65.0 (OCH ), 110.0 (Ar), 118.0 (-CH=), 120.5 (Ar), 124.4 (Ar),
δ 16.0 (CH ), 18.3 (CH ),
3 3
3
3
2
1
30.4 (Ar), 137.0 (=C), 147.8 (Ar), 157.0 (Ar). Anal. Calcd for C H O: C, 82.52; H, 10.16. Found:
15 22
C, 82.61; H, 10.13.
1
4-isopropyl-1-methyl-2-(pentyloxy)benzene (
8
). Colourless oil, 78% yield. H NMR (400 MHz,
CDCl3):
δ
1.01–1.06 (m, 3H, CH CH CH CH2CH3), 1.35 (d, J = 6.0 Hz, 6H, 2 CH ), 1.45–1.61
CH , CH CH CH CH2CH ), 1.86–1.97 (m, 2H, CH2CH2CH CH CH ), 2.29 (s, 3H, ArCH ),
2 2 2 2 3 2 2 3 3
×
2
2
2
3
(
m, 4H, 2
×
2
.92–2.99 (m, 1H, CH), 4.06 (t, J = 6.4 Hz 2H, OCH2CH CH CH CH ), 6.78–6.82 (m, 2H, Ar), 7.14 (d,
2
2
2
3
J = 7.5 Hz, 1H, 1Ar). 13C NMR (101 MHz, CDCl3):
δ
14.1 (CH ), 15.9 (CH ), 22.6 (CH ), 24.2 (2
×
CH3),
3
3
2
2
8.5 (CH ), 29.2 (CH ), 34.2 (CH), 67.9(OCH ), 109.5 (Ar), 117.8 (Ar), 124.2 (Ar), 130.4 (Ar) 147.9 (Ar),
2 2 2
1
57.3 (Ar). Anal. Calcd for C H O: C, 81.76; H, 10.98. Found: C, 81.88; H, 11.01.
15 24
2
-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-4-isopropyl-1-methylbenzene (
H NMR (300 MHz, CDCl3): 1.29–1.31 (m, 6H, 2 CH ), 1.67 (s, 3H, CH ), 1.74 (s, 3H, CH ), 1.80 (s,
CH ), 2.26 (s, 3H, ArCH ), 2.89–2.94 (m, 1H, CH), 4.62 (d, J = 6.3 Hz
9). Yellow oil, 74% yield.
1
δ
×
3
3
3
3
H, CH ), 2.10–2.20 (m, 4H, 2
H, OCH ), 5.16–5.17 (m, 1H, =CH), 5.55–5.56 (m, 1H, =CH), 6.77 (s, 1H, Ar), 6.77–6.79 (m, 1H,
Ar), 7.10 (d, J = 7.5 Hz, 1H, Ar). 13C NMR (75 MHz, CDCl3):
×
,
3
2
3
2
2
δ 16.0 (CH ), 16.7 (CH ), 17.7 (CH ),
3 3 3
2
4.2 (2 × CH ), 25.7 (CH ), 26.4 (CH ), 34.2 (CH), 39.6 (CH ), 65.0 (OCH ), 109.9 (Ar), 118.0 (Ar),
3
3
2
2
2
1
20.4 (=CH), 123.9 (Ar), 124.3 (=CH), 130.4 (Ar), 131.7 (=C), 140.1 (=C), 147.7 (Ar), 157.0 (Ar). Mixture
of E/Z isomers with 2:1 ratio. For sake of clarity, we reported only the signals related to the major
isomer. Anal. Calcd for C H O: C, 83.86; H, 10.56. Found: C, 84.01; H, 10.51.
20
30
1
2
-(5-isopropyl-2-methylphenoxy)acetonitrile (10). Colourless oil, 79% yield. H NMR (300 MHz,
CDCl3): 1.30 (d, J = 7.2 Hz, 6H, 2 CH ), 2.33 (s, 3H, ArCH ), 2.97–3.06 (m, 1H, CH), 4.79 (s, 2H,
OCH ), 6.89 (s, 1H, Ar), 6.97–7.00 (m, 1H, Ar), 7.21 (d, J = 7.8 Hz, 1H, Ar). C NMR (75 MHz, CDCl3):
δ
×
3
3
13
2
δ
15.7 (CH ), 24.1 (2
×
CH ), 34.1 (CH), 53.9 (OCH ), 110.5 (Ar), 115.8 (CN), 120.8 (Ar), 124.9 (Ar),
3 2
3
1
31.3 (Ar), 148.4 (Ar), 154.9 (Ar). Anal. Calcd for C H NO: C, 76.16; H, 7.99; N, 7.40. Found: C, 76.27;
12 15
H, 8.00; N, 7.38.
1
1
-(5-isopropyl-2-methylphenoxy)propan-2-one (11). Yellow oil, 80% yield. H NMR (300 MHz,
CDCl3): 1.27 (d, J = 6.9 Hz, 6H, 2 CH ), 2.31 (s, 3H, CH ), 2.35 (s, 3H, CH ), 2.84–2.94 (m, 1H,
CH), 4.55 (s, 2H, OCH ), 6.60 (s, 1H, Ar), 6.82–6.84 (m, 1H, Ar), 7.13 (d, J = 7.8 Hz, 1H, Ar). C NMR
δ
×
3
3
3
13
2
(
75 MHz, CDCl3):
δ
15.9 (CH ), 24.1 (2
×
CH ), 26.7 (CH ), 34.1 (CH), 73.2 (OCH ), 109.3 (Ar), 119.1 (Ar),
3 3 2
3

Pharmaceuticals 2020, 13, 405
13 of 21
1
24.1 (Ar), 130.9 (Ar), 148.1 (Ar), 155.9 (Ar), 206.5 (C=O). Anal. Calcd for C H O : C, 75.69; H, 8.80.
13 18 2
Found: C, 75.84; H, 8.77.
1
Ethyl 2-(5-isopropyl-2-methylphenoxy)acetate (12). Yellow oil, 77% yield. H NMR (300 MHz,
CDCl3):
δ
1.28–1.33 (m, 3H, CH2CH3), 1.34 (s, 3H, CH ), 1.36 (s, 3H, CH ), 2.34 (s, 3H, ArCH ),
3
3
3
2
.86–2.93 (m, 1H, CH), 4.26–4.33 (m, 2H, CH2CH ), 4.68 (s, 2H, OCH ), 6.67 (s, 1H, Ar), 6.83 (d,
3 2
13
J = 7.8 Hz, 1H, Ar), 7.10 (d, J = 7.8 Hz, 1H, Ar). C NMR (75 MHz, CDCl3):
δ
14.2 (CH ), 15.8 (CH ),
3
3
2
4.1 (2 × CH ), 34.1 (CH ), 61.0 (CH2CH ), 65.7 (OCH ), 109.7 (Ar), 119.2 (Ar), 124.5 (Ar), 130.8 (Ar),
3
3
3
2
147.7 (Ar), 156.2 (Ar), 169.1 (Ar). Anal. Calcd for C H O : C, 71.16; H, 8.53. Found: C, 71.22; H, 8.51.
14 20 3
1
2
-(5-isopropyl-2-methylphenoxy)acetic acid (13). Viscous white oil, 91% yield. H NMR (300 MHz,
CDCl3): 1.23 (d, J = 6.9 Hz, 6H, 2 CH ), 2.26 (s, 3H, ArCH ), 2.85–2.95 (m, 1H, CH), 4.70 (s, 2H,
OCH ), 6.61 (s, 1H, Ar), 6.81–6.83 (m, 1H, Ar), 7.08–7.11 (m, 1H, Ar). C NMR (75 MHz, CDCl3):
δ
×
3
3
13
δ
15.7
2
(
1
CH ), 24.0 (2
×
CH ), 34.0 (CH ), 65.3 (OCH ), 110.0 (Ar), 119.8 (Ar), 124.5 (Ar), 131.0 (Ar), 148.1 (Ar),
3
3
3
2
55.5 (Ar). Anal. Calcd for C H O : C, 69.21; H, 7.74. Found: C, 69.11; H, 7.71.
12 16 3
1
2
-(benzyloxy)-4-isopropyl-1-methylbenzene (14). Yellow oil, 88% yield. H NMR (300 MHz,
CDCl3): 1.54 (m, 6H, 2 CH ), 2.57 (s, 3H, ArCH ), 3.11–3.20 (m, 1H, CH), 5.34 (s, 2H, OCH ),
.04–7.07 (m, 2H, Ar), 7.37 (d, J = 7.8 Hz, 1H, Ar), 7.54–7.67 (m, 3H, Ar), 7.73 (d, J = 6.9 Hz, 2H, Ar).
δ
×
3
3
2
7
1
3
C NMR (75 MHz, CDCl3):
δ
16.3 (CH ), 24.4 (2
×
CH ), 34.4 (CH), 70.1 (OCH ), 110.2 (Ar), 118.6 (Ar),
3 2
3
1
24.6 (Ar), 127.5 (2 Ar), 128.0 (Ar), 128.7 (2
×
×
Ar), 130.8 (Ar), 137.9 (Ar), 148.1 (Ar), 157.1 (Ar). Anal.
Calcd for C H O: C, 84.96; H, 8.39. Found: C, 85.01; H, 8.42.
17
20
1
4
-isopropyl-1-methyl-2-((2-methylbenzyl)oxy)benzene (15). Yellow oil, 86% yield. H NMR
300 MHz, CDCl3): 1.40 (d, J = 6.9 Hz, 6H, 2 CH ), 2.37 (s, 3H, ArCH ), 2.52 (s, 3H, ArCH ),
.96–3.10 (m, 1H, CH), 5.16 (s, 2H, OCH ), 6.90–6.95 (m, 2H, Ar), 7.22 (d, J = 7.5 Hz, 1H, Ar), 7.35–7.37
(
δ
×
3
3
3
2
(
3
2
m, 3H, Ar), 7.50–7.52 (m, 1H, Ar). 13C NMR (75 MHz, CDCl3):
4.3 (CH), 68.5 (OCH ), 109.9 (Ar), 118.3 (Ar), 124.5 (Ar), 126.1 (Ar), 128.1 (Ar), 128.5 (Ar), 130.4 (Ar),
δ
16.1 (CH ), 19.1 (CH ), 24.3 (2 × CH )
,
3
3
3
2
1
30.7 (Ar), 135.5 (Ar), 136.7 (Ar), 148.0 (Ar). Anal. Calcd for C H O: C, 84.99; H, 8.72. Found: C, 85.17;
18 22
H, 8.70.
1
4
-isopropyl-1-methyl-2-((3-methylbenzyl)oxy)benzene (16). Pale yellow oil, 85% yield. H NMR
300 MHz, CDCl3): 1.58 (d, J = 6.9 Hz, 6H, 2 CH ) 2.60 (s, 3H, ArCH ), 2.67 (s, 3H, ArCH ), 3.16–3.21
m, 1H, CH), 5.33 (s, 2H, OCH ), 7.07–7.10 (m, 2H, Ar), 7.38–7.43 (m, 2H, Ar), 7.56–7.58 (m, 3H, Ar).
(
(
δ
×
3
3
3
2
1
3
C NMR (75 MHz, CDCl3):
18.6 (Ar), 124.7 (Ar), 128.3 (Ar), 128.7 (Ar), 128.8 (Ar), 130.8 (Ar), 137.9 (Ar), 138.3 (Ar), 148.1 (Ar),
57.2 (Ar). Anal. Calcd for C H O: C, 84.99; H, 8.72. Found: C, 85.11; H, 8.73.
δ
16.4 (CH ), 21.7 (CH ), 24.5 (2
×
CH ), 34.5 (CH), 70.2 (OCH ), 110.2 (Ar),
3
3
3 2
1
1
1
8
22
4
-isopropyl-1-methyl-2-((4-methylbenzyl)oxy)benzene (17). Yellow oil, 73% yield. 1H NMR
(
3
300 MHz, CDCl3):
δ
1.61 (d, J = 6.9 Hz, 6H, 2
×
CH ), 2.61 (s, 3H, ArCH ), 2.67 (s, 3H, ArCH ),
3
3
3
.18–3.21 (m, 1H, CH), 5.35 (s, 2H, OCH ), 7.09–7.13 (m, 2H, Ar), 7.42 (d, J = 6.9 Hz, 2H, Ar), 7.50 (d,
2
13
J = 8.4 Hz, 2H, Ar), 7.68 (d, J = 7.5 Hz, 2H, Ar). C NMR (75 MHz, CDCl3):
δ
16.4 (CH ), 21.5 (CH ),
3
3
2
4.6 (2 × CH ), 34.5 (CH), 70.0 (OCH ), 110.1 (Ar), 118.5 (Ar), 124.6 (Ar), 127.7 (2
×
Ar), 129.5 (2
×
Ar),
3
2
130.9 (Ar), 134.9 (Ar), 137.6 (Ar), 148.1 (Ar), 157.3 (Ar). Anal. Calcd for C H O: C, 84.99; H, 8.72.
18 22
Found: C, 84.88; H, 8.69.
1
2-((3,5-dimethylbenzyl)oxy)-4-isopropyl-1-methylbenzene (18). Yellow oil, 63% yield. H NMR
(
400 MHz, CDCl ): δ 1.27–1.30 (m, 6H, 2 × CH ), 2.29–2.30 (m, 3H, ArCH ), 2.38 (bs, 6H, 2 × ArCH ),
3
3
3
3
2
.89–2.93 (m, 1H, CH), 5.05 (s, 2H, OCH ), 6.79–6.83 (m, 2H, 2
×
Ar), 7.00 (s, 1H, Ar), 7.13 (bs, 3H, 3 × Ar).
2
1
3
C NMR (101 MHz, CDCl3):
10.2 (Ar), 118.3 (Ar), 124.5 (Ar), 125.2 (2
47.9 (Ar), 157.0 (Ar). Anal. Calcd for C H O: C, 85.03; H, 9.01. Found: C, 85.23; H, 9.05.
δ
16.0 (CH ), 21.4 (2
×
CH ), 24.2 (2 CH ), 34.1 (CH), 70.1 (OCH ),
×
3
3
3
2
1
1
×
Ar), 129.4 (2
×
Ar), 130.5 (Ar), 137.5 (Ar), 138.0 (Ar),
19
24
4
-isopropyl-1-methyl-2-((2-(trifluoromethyl)benzyl)oxy)benzene (19). Colourless oil, 84% yield.
1
H NMR (300 MHz, CDCl3): 1.60 (d, J = 6.3 Hz, 6H, 2 CH ), 2.70 (s, 3H, ArCH ), 3.17–3.26 (m, 1H,
CH), 5.67 (s, 2H, OCH ), 7.13–7.17 (m, 2H, Ar), 7.46 (d, J = 7.2, 1H, Ar), 7.62–7.67 (m, 1H, Ar), 7.81–7.86
m, 1H, Ar), 8.01 (d, J = 8.1 Hz, 1H, Ar), 8.17 (d, J = 8.1 Hz, 1H, Ar). C NMR (75 MHz, CDCl3):
δ
×
3
3
2
13
(
δ 16.3
(
CH ), 24.3 (2 CH ), 34.4 (CH), 66.1 (OCH ), 110.2 (Ar), 119.0 (Ar), 123.3 (CF ), 124.5 (Ar), 126.0 (Ar),
×
3
3
2
3

Pharmaceuticals 2020, 13, 405
14 of 21
1
26.1 (Ar), 127.7 (Ar), 128.6 (Ar), 131.0 (Ar), 132.4 (Ar), 136.7 (Ar), 148.3 (Ar), 156.7 (Ar). 19F NMR
(
564.7 MHz, CDCl3): δ −58.63 (s, 3F, ArCF ). Anal. Calcd for C H F O: C, 70.12; H, 6.21. Found:
3
18 19 3
C, 69.98; H, 6.20.
4
-isopropyl-1-methyl-2-((3-(trifluoromethyl)benzyl)oxy)benzene (20). Colourless oil, 78% yield.
H NMR (300 MHz, CDCl3): 1.58 (d, J = 6.9 Hz, 6H, 2 CH ), 2.59 (s, 3H, ArCH ), 3.14–3.23 (m,
H, CH), 5.36 (s, 2H, OCH ), 7.09 (s, 1H, Ar), 7.11–7.12 (m, 1H, Ar), 7.40 (d, J = 7.5 Hz, 1H, Ar),
1
δ
×
3
3
1
7
2
1
3
.69–7.74 (m, 1H, Ar), 7.84 (d, J =7.5 Hz, 1H, Ar), 7.90 (d, J = 7.5 Hz, 1H Ar), 8.06 (s, 1H, Ar). C NMR
16.1 (CH ), 24.3 (2 CH ), 34.4 (CH), 69.2 (OCH ), 110.0 (Ar), 119.0 (Ar), 124.0 (Ar),
(
75 MHz, CDCl3):
δ
×
3
3
2
124.0 (Ar), 124.1 (Ar), 124.6 (CF ), 124.7 (Ar), 129.2 (Ar), 130.6 (Ar), 131.0 (Ar), 139.0 (Ar), 148.3 (Ar),
3
1
56.8 (Ar). 19F NMR (564.7 MHz, CDCl3):
δ –60.92 (s, 3F, ArCF ). Anal. Calcd for C H F O: C, 70.12;
3 18 19 3
H, 6.21. Found: C, 70.35; H, 6.22.
4
-isopropyl-1-methyl-2-((4-(trifluoromethyl)benzyl)oxy)benzene (21). Viscous colourless oil,
1
8
3
4% yield. H NMR (300 MHz, CDCl ):
.00–3.12 (m, 1H, CH), 5.30 (s, 2H, OCH ), 6.96 (s, 1H, Ar), 6.98–7.01 (m, 1H, Ar), 7.30 (d, J = 7.5 Hz, 1H,
Ar), 7.74 (d, J = 8.4 Hz, 2H, Ar), 7.82 (d, J = 8.4 Hz, 2H, Ar). C NMR (75 MHz, CDCl3):
4.2 (2 × CH ), 34.3 (CH), 69.0 (OCH ), 110.0 (Ar), 118.9 (Ar), 124.5 (Ar), 125.5 (2 Ar), 125.6 (Ar),
27.3 (Ar), 130.3 (CF ), 130.9 (Ar), 141.9 (Ar), 148.2 (Ar), 156.6 (Ar). F NMR (564.7 MHz, CDCl ):
δ
1.46 (d, J = 7.2 Hz, 6H, 2
×
CH ), 2.49 (s, 3H, ArCH ),
3 3
3
2
13
δ 16.1 (CH3)
,
2
1
×
3
2
19
3
3
δ −60.77 (s, 3F, ArCF ). Anal. Calcd for C H F O: C, 70.12; H, 6.21. Found: C, 70.17; H, 6.19.
3
18 19 3
2
-((3,5-bis(trifluoromethyl)benzyl)oxy)-4-isopropyl-1-methylbenzene (22). Yellow oil, 80% yield.
H NMR (400 MHz, CDCl3): 1.26–1.28 (d, J =6.8 Hz, 6H, 2 CH ), 2.30 (s, 3H, CH ), 2.87–2.94 (m,
H, CH), 5.21 (s, 2H, CH ), 6.78 (s, 1H, Ar), 6.83–6.85 (d, J = 7.6, 1H, Ar), 7.14–7.16 (d, J = 7.6, 1H,
1
δ
×
3
3
1
2
Ar), 7.88 (s, 1H, Ar), 7.97 (s, 2H, 2
3
×
Ar). 13C NMR (101 MHz, CDCl3):
δ
15.9 (CH ), 24.1 (2
×
CH3),
3
4.1 (CH), 68.5 (OCH ), 109.9 (Ar), 119.3 (Ar), 121.7 (Ar), 121.9 (Ar), 124.4 (Ar), 124.6 (Ar), 127.1 (Ar),
2
19
1
2
30.9 (2 × Ar), 132.0 (2
×
CF ), 140.3 (Ar), 148.1 (Ar), 156.1 (Ar). F (564.7 MHz, CDCl3):
δ
–66.85 (s, 6F,
3
× ArCF ). Anal. Calcd for C H F O: C, 60.64; H, 4.82. Found: C, 60.80; H, 4.81.
3
19 18 6
1
2
-((3-fluorobenzyl)oxy)-4-isopropyl-1-methylbenzene (23). Colourless oil, 82% yield. H NMR
(
300 MHz, CDCl3):
δ
1.59–1.63 (m, 6H, 2
×
CH ), 2.63 (s, 3H, ArCH ), 3.17–3.24 (m, 1H, CH), 5.34 (s,
3 3
2
H, OCH ), 7.10–7.14 (m, 2H, Ar), 7.26–7.31 (m, 1H, Ar), 7.41–7.45 (m, 1H, Ar), 7.50–7.63 (m, 3H, Ar).
2
1
3
C NMR (75 MHz, CDCl3):
δ
16.3 (CH ), 24.4 (2
×
CH ), 34.5 (CH), 69.2 (OCH ), 110.1 (Ar), 114.2 (Ar),
3 2
3
1
14.8 (Ar), 118.9 (Ar), 122.7 (Ar), 124.6 (Ar), 130.3 (Ar), 131.0 (Ar), 140.7 (Ar), 148.2 (Ar), 156.9 (Ar) 163.4
C-F
19
F-H
(
d,
J = 244.9 Hz, C-F). F NMR (564.7 MHz, CDCl3): δ −111.26 (td,
J = 9.0 Hz, 6.0 Hz, 1F, ArF).
Anal. Calcd for C H FO: C, 79.04; H, 7.41. Found: C, 78.91; H, 7.39.
17
19
1
2
-((3,5-difluorobenzyl)oxy)-4-isopropyl-1-methylbenzene (24). Colourless oil, 80% yield. H NMR
(
5
1
1
400 MHz, CDCl3):
δ
1.27–1.29 (d, J = 6.8 Hz, 6H, 2
×
3CH ), 2.31 (s, 3H, CH ), 2.87–2.94 (m, 1H, CH),
3 3
.10 (s, 2H, CH ), 6.75 (s, 1H, Ar), 6.76–6.84 (m, 2H, 2
×
Ar), 7.01–7.06 (m, 2H, Ar), 7.13–7.15 (d, J = 7.6 Hz
,
2
H, Ar). 13C NMR (101 MHz, CDCl3):
09.6 (Ar), 118.9 (Ar), 124.4 (Ar), 130.7 (Ar), 141.8 (Ar), 148.0 (Ar), 156.3 (Ar), 161.9 (Ar), 164.4 (Ar).
δ
16.0 (CH ), 24.1 (2 CH ), 34.1 (CH), 68.6 (OCH ), 102.9 (Ar),
×
3
3
2
1
9
F (564.7 MHz, CDCl3):
C, 73.94; H, 6.58.
,3-difluoro-2-((5-isopropyl-2-methylphenoxy)methyl)benzene (25). Colourless oil, 89% yield.
H NMR (300 MHz, CDCl3): 1.65 (d, J = 6.9 Hz, 6H, 2 CH ), 2.56 (s, 3H, ArCH ), 3.20–3.29 (m, 1H,
CH), 5.50 (s, 2H, OCH ), 7.13–7.19 (m, 3H, Ar), 7.30 (s, 1H, Ar), 7.41 (d, J = 7.5 Hz, 1H, Ar), 7.44–7.54 (m,
δ –113.48 (m, 2F, ArF). Anal. Calcd for C H F O: C, 73.89; H, 6.57. Found:
17 18 2
1
1
δ
×
3
3
2
1
1
1
H, Ar). 13C NMR (75 MHz, CDCl3):
δ
16.0 (CH ), 24.4 (2
×
CH ), 34.5 (CH), 58.4 (OCH ), 110.7 (Ar),
3 2
3
11.5 (Ar), 111.7 (Ar), 113.6 (Ar), 119.3 (Ar), 125.1 (Ar), 130.7 (Ar), 131.0 (Ar), 148.2 (Ar), 157.0 (Ar),
C-F
C-F
19
62.3 (d,
t,
J = 248.3 Hz, C-F), 162.3 (d,
J = 249.5 Hz, C-F). F NMR (564.7 MHz, CDCl3): δ −112.80
F-H
(
J = 6.6 Hz, 2F, ArF). Anal. Calcd for C H F O: C, 73.89; H, 6.57. Found: C, 74.00; H, 6.58.
17 18 2
1
4-isopropyl-2-((3-methoxybenzyl)oxy)-1-methylbenzene (26). Colourless oil, 80% yield. H NMR
(
3
2
400 MHz, CDCl3):
δ
1.26–1.29 (m, 6H, 2
×
CH ), 2.30 (s, 3H, ArCH ), 2.87–2.92 (m, 1H, CH), 3.86 (s,
3
3
H, OCH ), 5.10 (s, 2H, OCH ), 6.81 (bs, 2H, 2
×
Ar), 6.89–6.91 (d, J = 8.4 Hz, 1H, Ar), 7.07 (bs, 2H,
3
2
13
× Ar), 7.12–7.14 (d, J = 7.6 Hz, 1H, Ar), 7.32–7.36 (t, J = 8.2 Hz, 1H, Ar). C NMR (101 MHz, CDCl3):

Pharmaceuticals 2020, 13, 405
15 of 21
δ
16.0 (CH ), 24.1 (2
×
CH ), 34.1 (CH), 55.2 (OCH ), 69.8 (OCH ), 110.0 (Ar), 112.7 (Ar), 113.2 (Ar),
3
3
3
2
118.4 (Ar), 119.4 (Ar), 124.4 (Ar), 129.5 (Ar), 130.5 (Ar), 139.3 (Ar), 147.9 (Ar), 156.8 (Ar), 159.8 (Ar).
Anal. Calcd for C H O : C, 79.96; H, 8.20. Found: C, 80.11; H, 8.5.
18
22
2
1
2
-((4-chlorobenzyl)oxy)-4-isopropyl-1-methylbenzene (27). Pale yellow oil, 79% yield. H NMR
300 MHz, CDCl3): 1.52 (d, J = 6.9 Hz, 6H, 2 CH ), 2.52 (s, 3H, ArCH ), 3.11–3.20 (m, 1H, CH),
.24 (s, 2H, OCH ), 7.01–7.04 (m, 2H, Ar), 7.34 (d, J = 7.8 Hz, 1H, Ar), 7.54–7.61 (m, 4H, Ar). C NMR
(
5
δ
×
3
3
13
2
(
1
75 MHz, CDCl3):
δ
16.3 (CH ), 24.4 (2
×
CH ), 34.4 (CH), 69.2 (OCH ), 110.2 (Ar), 118.8 (Ar), 124.5 (Ar),
3 2
3
28.7 (2
×
Ar), 128.8 (2
×
Ar), 130.9 (Ar), 133.7 (Ar), 136.4 (Ar), 148.1 (Ar), 156.9 (Ar). Anal. Calcd for
C H ClO: C, 74.31; H, 6.97. Found: C, 74.43; H, 7.00.
17
19
2
-chloro-1-((5-isopropyl-2-methylphenoxy)methyl)-4-methoxybenzene (28). Colourless oil,
1
8
2% yield. H NMR (400 MHz, CDCl3):
CH ), 2.87–2.92 (m, 1H, CH), 3.84 (s, 3H, OCH ), 5.13 (s, 2H, CH ), 6.79–6.81 (m, 2H, 2
m, 1H, Ar), 6.99–7.00 (d, J = 2.4 Hz, 1H, Ar), 7.10–7.12 (d, J = 7.6 Hz, 1H, Ar), 7.50–7.52 (d, J = 8.4 Hz
δ
1.26–1.28 (d, J = 7.2 Hz, 6H, 2
×
CH ), 2.28 (s, 3H,
3
×
Ar), 6.86–6.89
3
3
2
(
2
6
,
13
H, 2 × Ar). C NMR (101 MHz, CDCl3):
δ
16.0 (CH ), 24.1 (2
×
CH ), 34.1 (CH), 55.6 (OCH ),
3
3
3
6.9 (OCH ), 110.2 (Ar), 112.9 (Ar), 114.8 (Ar), 118.5 (Ar), 124.4 (Ar), 127.3 (Ar), 129.9 (Ar), 130.5 (Ar),
2
1
33.5 (Ar), 148.0 (Ar), 156.6 (Ar), 159.7 (Ar). Anal. Calcd for C H ClO : C, 70.93; H, 6.94. Found:
18 21 2
C, 71.10; H, 6.92.
1
,2-dichloro-4-((5-isopropyl-2-methylphenoxy)methyl)benzene (29). Colourless oil, 92% yield.
H NMR (300 MHz, CDCl3): 1.59 (d, J = 6.9 Hz, 6H, 2 CH ), 2.58 (s, 3H, ArCH ), 3.16–3.21 (m,
H, CH), 5.22 (s, 2H, OCH ), 7.05 (s, 1H, Ar), 7.10 (d, J = 7.5 Hz, 1H, Ar), 7.39 (d, J = 7.5 Hz, 1H, Ar),
1
δ
×
3
3
1
7
δ
2
13
.48–7.52 (m, 1H, Ar), 7.64 (d, J = 8.1 Hz, 1H, Ar), 7.79–7.80 (m, 1H, Ar). C NMR (75 MHz, CDCl3):
16.4 (CH ), 24.5 (2 CH ), 34.5 (CH), 68.5 (OCH ), 110.0 (Ar), 119.1 (Ar), 124.5 (Ar), 126.6 (Ar),
×
3
3
2
129.2 (Ar), 130.7 (Ar), 131.1 (Ar), 131.8 (Ar), 132.8 (Ar), 138.3 (Ar), 148.2 (Ar), 156.7 (Ar). Anal. Calcd
for C H Cl O: C, 66.03; H, 5.87. Found: C, 65.94; H, 5.88.
17
18
2
◦
.
2
-((2-bromobenzyl)oxy)-4-isopropyl-1-methylbenzene (30). White powder, 61% yield, mp 51–52 C
1.27 (d, J = 6.9 Hz, 6H, 2 CH ), 2.32 (s, 3H, ArCH ), 2.83–2.94 (m,
H, CH), 5.16 (s, 2H, OCH ), 6.79–6.82 (m, 2H, Ar), 7.13 (d, J = 7.8 Hz, 1H, Ar), 7.16–7.23 (m, 1H, Ar),
1
H NMR (300 MHz, CDCl3):
δ
×
3
3
1
7
3
2
13
.34–7.40 (t, 1H, Ar), 7.60–7.65 (m, 2H, Ar). C NMR (75 MHz, CDCl3):
δ
16.1 (CH ), 24.2 (2
×
CH3),
3
4.1 (CH), 69.2 (OCH ), 110.1 (Ar), 118.5 (Ar), 122.1 (Ar), 124.4 (Ar), 127.6 (Ar), 128.7 (Ar), 129.0 (Ar),
2
1
30.6 (Ar), 132.5 (Ar), 136.9 (Ar), 148.0 (Ar), 156.4 (Ar). Anal. Calcd for C H BrO: C, 63.96; H, 6.00.
17 19
Found: C, 63.85; H, 5.99.
1
2
-((4-bromobenzyl)oxy)-4-isopropyl-1-methylbenzene (31). Colourless oil, 66% yield. H NMR
300 MHz, CDCl3): 1.55 (d, J = 6.9 Hz, 6H, 2 CH ), 2.56 (s, 3H, ArCH ), 3.12–3.18 (m, 1H, CH),
.25 (s, 2H, OCH ), 7.04–7.09 (m, 2H, Ar), 7.38 (d, J = 7.2 Hz, 1H, Ar), 7.55 (d, J = 8.1 Hz, 2H, Ar),
(
δ
×
3
3
5
7
6
2
13
.71–7.75 (m, 2H, Ar). C NMR (75 MHz, CDCl3):
δ
16.4 (CH ), 21.5 (CH ), 24.5 (2
×
CH ), 34.5 (CH),
3
Ar), 130.9 (Ar), 131.9 (2
3
3
9.3 (OCH ), 110.0 (Ar), 118.8 (Ar), 121.9 (Ar), 124.5 (Ar), 129.1 (2
×
×
Ar),
2
1
36.9 (Ar), 148.1 (Ar), 156.9 (Ar). Anal. Calcd for C H BrO: C, 63.96; H, 6.00. Found: C, 63.99; H, 6.02.
1
7
19
◦
4
-isopropyl-1-methyl-2-((2-nitrobenzyl)oxy)benzene (32). White powder, 91% yield, mp 56–57 C
.
1
H NMR (300 MHz, CDCl3):
δ
1.25 (d, J = 7.2 Hz, 6H, 2
×
CH ), 2.32 (s, 3H, ArCH ), 2.86–2.91 (m, 1H,
3
3
CH), 5.50 (s, 2H, OCH ), 6.78 (s, 1H, Ar), 6.80–6.83 (dd, J = 1.2 Hz, J = 15 Hz, 1H, Ar), 7.13 (d, J = 7.8 Hz
,
2
1
H, Ar), 7.50–7.53 (m, 1H, Ar), 7.69–7.75 (m, 1H, Ar), 7.98–8.01 (m, 1H, Ar), 8.17–8.20 (m, 1H, Ar).
13
C NMR (75 MHz, CDCl3):
24.2 (Ar), 124.9 (Ar), 128.2 (Ar), 128.5 (Ar), 130.7 (Ar), 134.0 (Ar), 134.4 (Ar), 146.9 (Ar), 148.2 (Ar),
56.1 (Ar). Anal. Calcd for C H NO : C, 71.56; H, 6.71; N, 4.91. Found: C, 71.71; H, 6.70; N, 4.92.
δ
16.1 (CH ), 24.1 (2
×
CH ), 34.1 (CH), 66.6 (OCH ), 110.0 (Ar), 118.8 (Ar),
3
3 2
1
1
17
19
3
1
4
-isopropyl-1-methyl-2-((3-nitrobenzyl)oxy)benzene (33). Yellow viscous oil, 72% yield. H NMR
400 MHz, CDCl3): 1.36 (d, J = 6.9 Hz, 6H, 2 CH ), 2.38 (s, 3H, ArCH ), 2.96–3.00 (m, 1H, CH),
.24 (s, 2H, OCH ), 6.88–6.91 (m, 2H, Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar), 7.60–7.65 (m, 1H, Ar), 7.89 (d,
(
5
δ
×
3
3
2
13
J = 7.5 Hz, 1H, Ar), 8.23 (d, J = 7.5 Hz, 1H, Ar), 8.43 (s, 1H, Ar). C NMR (101 MHz, CDCl3):
δ 16.1
(
CH ), 24.2 (2 CH ), 34.2 (CH), 68.5 (OCH ), 109.9 (Ar), 119.0 (Ar), 121.9 (Ar), 122.7 (Ar), 124.3 (Ar),
×
3
3
2

Pharmaceuticals 2020, 13, 405
16 of 21
1
29.6 (Ar), 130.9, 133.1 (Ar), 139.9 (Ar), 148.1 (Ar), 148.4 (Ar), 156.3 (Ar). Anal. Calcd for C H NO :
17 19 3
C, 71.56; H, 6.71; N, 4.91. Found: C, 71.43; H, 6.73; N, 4.92.
4
-isopropyl-1-methyl-2-((4-nitrobenzyl)oxy)benzene (34). Yellow amber powder, 95% yield,
◦
1
mp 88–95 C. H NMR (300 MHz, CDCl ):
.84–2.88 (m, 1H, CH), 5.19 (s, 2H, CH ), 6.71 (s, 1H, Ar), 6.80 (d, J = 7.8 Hz, 1H, Ar), 7.11 (d, J =7.5 Hz
H, Ar), 7.64 (d, J = 8.1 Hz, 2H, Ar), 8.26 (d, J = 8.7 Hz, 2H, Ar). C NMR (75 MHz, CDCl3): δ 15.9 (CH3)
4.1 (2 CH ), 34.1 (CH ), 68.5 (OCH ), 109.7 (Ar), 110.0 (Ar), 119.0 (2
27.4 (Ar), 130.8 (Ar), 145.1 (Ar), 148.1 (C=O), 156.2 (C=O). Anal. Calcd for C H NO : C, 71.56;
δ
1.23 (d, J = 6.3 Hz, 6H, 2
×
CH ), 2.28 (s, 3H, ArCH ),
3 3
3
2
1
2
1
,
,
2
13
×
× Ar), 123.8 (2 × Ar), 124.3 (Ar),
3
3
2
17
19
3
H, 6.71; N, 4.91. Found: C, 71.63; H, 6.69; N, 4.90.
1
4-((5-isopropyl-2-methylphenoxy)methyl)aniline (35). Orange viscous oil, 70% yield. H NMR
(
2
300 MHz, CDCl3):
δ
1.24–1.27 (m, 6H, 2
×
CH ), 2.23 (s, 3H, ArCH ), 2.86–2.91 (m, 1H, CH), 3.86 (bs,
3
3
H, NH , D O exch.), 4.97 (s, 2H, OCH ), 6.75–6.80 (m, 4H, Ar), 7.09 (d, J = 7.8 Hz, 1H, Ar), 7.28 (d,
2
2
2
13
J = 8.7 Hz, 2H, Ar). C NMR (75 MHz, CDCl3):
1
1
δ
16.1 (CH ), 24.2 (2
×
CH ), 34.2 (CH), 70.1 (OCH ),
3
3
2
10.1 (Ar), 115.1 (2
×
Ar), 118.2 (Ar), 124.5 (Ar), 124.6 (Ar), 128.9 (2
×
Ar), 130.4 (2
×
Ar), 130.8 (Ar),
46.0 (Ar), 147.8 (Ar), 157.1 (Ar). Anal. Calcd for C H NO: C, 79.96; H, 8.29; N, 5.49. Found: C, 80.14;
17 21
H, 8.27; N, 5.51.
◦
4
-((5-isopropyl-2-methylphenoxy)methyl)benzonitrile (36). White powder, 79% yield, mp 74–76 C.
H NMR (300 MHz, CDCl3): 1.21–124 (m, 6H, 2 CH ), 2.27 (s, 3H, ArCH ), 2.81–2.88 (m, 1H,
CH), 5.14 (s, 2H, OCH ), 6.71 (s, 1H, Ar), 6.79 (d, J = 7.8 Hz, 1H, Ar), 7.11 (d, J = 7.5 Hz, 1H, Ar),
1
δ
×
3
3
2
13
7.58 (d, J = 8.4 Hz, 2H, Ar), 7.70 (d, J = 8.1 Hz, 2H, Ar). C NMR (75 MHz, CDCl3):
δ
16.0 (CH3),
Ar),
Ar), 143.1 (Ar), 148.1 (Ar), 156.2 (Ar). Anal. Calcd for C H NO: C, 81.47; H, 7.22;
2
4.1 (2 × CH ), 34.1 (CH), 68.9 (OCH ), 109.7 (Ar), 111.4 (CN), 118.8 (Ar), 124.3 (Ar), 127.3 (2
×
3
2
1
30.7 (Ar), 132.4 (2
N, 5.28. Found: C, 71.56; H, 6.71; N, 4.91.
4-((5-isopropyl-2-methylphenoxy)methyl)phenyl)(methyl)sulfane (37). White powder, 81% yield,
×
18
19
(
◦
1
mp 61–62 C. H NMR (300 MHz, CDCl ):
δ
1.35 (d, J = 6.9 Hz, 6H, 2
×
CH ), 2.35 (s, 3H, ArCH ),
3 3
3
2.56 (s, 3H, SCH ), 2.92–2.99 (m, 1H, CH), 5.19 (s, 2H, OCH ), 6.86–6.88 (m, 2H, Ar), 7.18 (d, J = 8.1 Hz
,
3
2
1
3
1
1
1
H, Ar), 7.35–7.38 (m, 2H, Ar), 7.65 (d, J = 8.7 Hz, 2H, Ar). C NMR (75 MHz, CDCl3):
δ
15.9 (SCH3),
Ar),
Ar), 130.6 (Ar), 134.5 (Ar), 138.0 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C H OS:
6.2 (CH ), 24.4 (2 CH ), 34.3 (CH), 69.6 (OCH ), 110.0 (Ar), 118.5 (Ar), 124.4 (Ar), 126.8 (2
×
×
3
3
2
28.0 (2
×
18
22
C, 75.48; H, 7.74. Found: C, 75.37; H, 7.72.
-isopropyl-1-methyl-2-((4-(methylsulfinyl)benzyl)oxy)benzene (38). Yellow viscous oil, 51% yield.
H NMR (300 MHz, CDCl3): 1.24 (d, J = 6.3 Hz, 6H, 2 CH ), 2.27 (s, 3H, ArCH ), 2.77 (s, 3H, SCH ),
.85–2.87 (m, 1H, CH), 5.15 (s, 2H, OCH ), 6.75–6.80 (m, 2H, Ar), 7.09–7.12 (m, 1H, Ar), 7.65 (bs, 4H,
4
1
δ
×
3
3
3
2
2
13
Ar). C NMR (75 MHz, CDCl3):
δ
16.0 (CH ), 24.1 (2
×
CH ), 34.1 (CH), 44.7 (SCH ), 69.1 (OCH ),
3 3 2
3
109.9 (Ar), 118.7 (Ar), 124.1 (Ar), 124.3 (2
×
Ar), 128.0 (2
×
Ar), 130.7 (Ar), 141.2 (Ar), 145.0 (Ar),
1
48.0 (Ar), 156.4 (Ar). Anal. Calcd for C H O S: C, 71.49; H, 7.33. Found: C, 71.35; H, 7.30.
1
8
22
2
4
-isopropyl-1-methyl-2-((4-(methylsulfonyl)benzyl)oxy)benzene (39).
Yellow viscous oil,
1
2
(
5% yield. H NMR (300 MHz, CDCl )
m, 1H, CH), 3.07 (s, 3H, SO CH ); 5.17 (s, 2H, OCH ), 6.72 (s, 1H, Ar), 6.79 (d, J = 7.8 Hz, 1H, Ar),
δ
1.21–1.22 (m, 6H, 2
×
CH ), 2.27 (s, 3H, ArCH ); 2.84–2.86
3 3
3
2 3 2
7
.09–7.12 (d, J = 7.5 Hz, 1H, Ar); 7.67 (d, J = 8.4 Hz, 2H, Ar), 7.97 (d, J = 8.1 Hz, 2H, Ar). Anal. Calcd for
C H O S: C, 67.89; H, 6.96. Found: C, 68.01; H, 6.99.
18
22
3
0
◦
4
-((5-isopropyl-2-methylphenoxy)methyl)-1,1 -biphenyl (40). White solid, 90% yield, mp = 99–100 C.
H NMR (400 MHz, CDCl3): 1.29–1.31 (d, J = 6.8 Hz, 6H, 2 CH ), 2.33 (s, 3H, ArCH ), 2.89–2.96
m, 1H, CH), 5.18 (s, 2H, OCH ), 6.81–6.85 (m, 2H, 2 Ar), 7.14–7.16 (d, J = 7.6 Hz, 1H, Ar), 7.38–7.42
Ar), 7.57–7.60 (d, J = 8.4 Hz, 2H, 2
1
δ
×
3
3
(
(
×
2
m, 1H, Ar), 7.48–7.52 (m, 2H, 2
×
×
Ar), 7.65–7.68 (m, 4H, 4 × Ar).
1
3
C NMR (101 MHz, CDCl3):
δ
16.1 (CH ), 24.2 (2
×
CH ), 34.2 (CH), 69.7 (OCH ), 110.1 (2
×
Ar),
3
3
2
1
1
18.4 (2 Ar), 124.5 (Ar), 127.2 (2
×
×
Ar), 127.3 (2
×
Ar), 127.4 (2
×
Ar), 127.7 (2
×
Ar), 128.8 (Ar),
30.6 (Ar), 126.7 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C H O: C, 87.30; H, 7.64. Found: C, 87.47;
23
24
H, 7.65.

Pharmaceuticals 2020, 13, 405
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1
-((5-isopropyl-2-methylphenoxy)methyl)naphthalene (41). Yellow-brown sticky solid, 98% yield.
1
H NMR (300 MHz, CDCl3): 1.29 (d, J = 7.2 Hz, 6H, 2 CH ), 2.21 (s, 3H, ArCH ), 2.88–2.97 (m, 1H,
CH), 5.52 (s, 2H, OCH ), 6.80–6.83 (m, 1H, Ar), 6.95–6.96 (m, 1H, Ar), 7.48 (s, 1H, Ar), 7.50 (m, 1H,
δ
×
3
3
2
Ar), 7.51–7.59 (m, 2H, Ar), 7.66 (d, J = 7.2 Hz, 1H, Ar) 7.86–7.94 (m, 2H, Ar), 8.10–8.14 (m, 1H, Ar).
1
3
C NMR (75 MHz, CDCl3):
23.9 (Ar), 124.6 (Ar), 125.3 (Ar), 125.8 (Ar), 126.2 (Ar), 126.3 (Ar), 128.6 (Ar), 128.8 (Ar), 130.6 (Ar),
31.6 (Ar), 132.9 (Ar), 133.7 (Ar), 148.0 (Ar), 156.9 (Ar). Anal. Calcd for C H O: C, 86.85; H, 7.64.
δ
16.0 (CH ), 24.2 (2
×
CH ), 34.2 (CH), 68.6 (OCH ), 110.0 (Ar), 118.4 (Ar),
3
3 2
1
1
21
22
Found: C, 86.97; H, 7.66.
2
-((5-isopropyl-2-methylphenoxy)methyl)isoindoline-1,3-dione (42). Colourless viscous oil, 88%
yield. 1H NMR (300 MHz, CDCl3):
(
7
1
δ
1.23–1.26 (m, 6H, 2 CH ), 2.18 (s, 3H, ArCH ), 2.80–2.93
×
3
3
m, 1H, CH), 5.67 (s, 2H, OCH ), 6.79–6.82 (m, 1H, Ar), 7.06–7.04 (m, 1H, Ar), 7.74–7.78 (m, 2H, Ar),
.88–7.92 (m, 2H, Ar). 13C NMR (75 MHz, CDCl3):
12.6 (Ar), 120.3 (Ar), 123.8 (2
2
δ
15.8 (CH ), 24.1 (2
×
CH ), 34.0 (CH), 65.5 (OCH ),
3
3
2
×
Ar), 125.5 (Ar), 130.8 (Ar), 131.8 (Ar), 134.5 (2
×
Ar), 148.0 (Ar),
1
54.3 (Ar), 167.2 (2 × C=O). Anal. Calcd for C H NO : C, 73.77; H, 6.19; N, 4.53. Found: C, 73.69;
19
19
3
H, 6.18; N, 4.55.
5
-isopropyl-2-methylphenyl 6-methyl-2-oxo-2H-chromene-3-carboxylate (43). White powder, 76%
◦
1
yield, mp 122–124 C. H NMR (300 MHz, CDCl ):
ArCH3 carv.), 2.44 (s, 3H, ArCH3 coum.), 2.89–2.95 (m, 1H, CH), 7.01–7.07 (m, 2H, Ar), 7.17–7.19 (m,
H, Ar), 7.28–7.31 (m, 1H, Ar), 7.44–7.51 (m, 2H, Ar), 8.69 (s, 1H, =CH). 1 C NMR (75 MHz, CDCl3):
δ
1.24 (d, J = 6.9 Hz, 6H, 2
×
CH ), 2.22 (s, 3H,
3
3
3
1
δ
16.0 (CH ), 20.8 (CH3 coum.), 23.9 (2
×
CH ), 33.6 (CH ), 116.7 (Ar), 117.4 (Ar), 117.6 (Ar), 119.8 (Ar),
3 3
3
124.5 (Ar), 127.2 (Ar), 129.4 (Ar), 131.0 (Ar), 134.8 (Ar), 136.0 (Ar), 148.2 (Ar), 149.0 (Ar), 149.9 (Ar),
1
53.6 (Ar), 156.8 (C=O), 161.6 (C=O). Anal. Calcd for C H O : C, 74.98; H, 5.99. Found: C, 75.07;
21
20
4
H, 6.00.
5
-isopropyl-2-methylphenyl 6-chloro-2-oxo-2H-chromene-3-carboxylate (44). White powder, 70%
◦
1
yield, mp 114–115 C. H NMR (300 MHz, CDCl ):
ArCH ), 2.87–2.92 (m, 1H, CH), 7.00 (s, 1H, Ar), 7.04–7.08 (m, 1H, Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar),
.34–7.37 (d, J = 8.1 Hz, 1H, Ar), 7.60–7.65 (m, 2H, Ar), 8.64 (s, 1H, =CH). C NMR (75 MHz, CDCl3):
δ
1.24 (d, J = 6.9 Hz, 6H, 2
×
CH ), 2.21 (s, 3H,
3
3
3
13
7
δ
15.9 (CH ), 23.9 (2 CH ) 33.6 (CH), 118.4 (Ar), 118.7 (Ar), 118.8 (Ar), 119.6 (Ar), 124.7 (Ar), 127.1 (Ar),
×
3
3
128.7 (Ar), 130.3 (Ar), 131.0 (Ar), 134.6 (Ar), 148.2 (Ar), 148.3 (Ar), 148.9 (Ar), 153.7 (Ar), 155.8 (C=O),
61.1 (C=O). Anal. Calcd for C H ClO : C, 67.33; H, 4.80. Found: C, 67.40; H, 4.79.
1
20
17
4
5
-Isopropyl-2-methylphenyl 6-bromo-2-oxo-2H-chromene-3-carboxylate (45). White powder, 73%
◦
1
yield, mp 134–138 C. H NMR (300 MHz, CDCl ):
ArCH ), 2.84–2.96 (m, 1H, CH), 7.00 (s, 1H, Ar), 7.05–7.08 (m, 1H, Ar), 7.19 (d, J = 7.5 Hz, 1H, Ar),
.30 (d, J = 8.7 Hz, 1H, Ar), 7.74–7.80 (m, 2H, Ar), 8.64 (s, 1H, =CH coumarin). C NMR (75 MHz,
δ
1.24 (d, J = 6.3 Hz, 6H, 2
×
CH ), 2.21 (s, 3H,
3
3
3
13
7
CDCl3): 15.9 (CH ), 23.9 (2 CH ), 33.6 (CH), 117.5 (Ar), 118.6 (Ar), 118.8 (Ar), 119.3 (Ar), 119.6 (Ar),
δ
×
3
3
1
1
24.7 (Ar), 127.1 (Ar), 131.1 (Ar), 131.7 (Ar), 137.4 (Ar), 148.2 (Ar), 148.2 (Ar), 148.9 (Ar), 154.2 (Ar),
55.8 (C=O), 161.0 (C=O). Anal. Calcd for C H BrO : C, 59.87; H, 4.27. Found: C, 59.92; H, 4.28.
-(5-isopropyl-2-methylphenoxy)-N -(3-nitrophenyl)acetohydrazide (46). White solid; 62% yield,
mp = 127–128 C. H NMR (400 MHz, CDCl ):
2
0
17
4
0
2
◦
1
δ
1.26–1.28 (d, J = 6.8 Hz, 6H, 2
×
CH ), 2.34 (s, 3H,
3
3
CH ), 2.88–2.95 (m, 1H, CH), 4.76 (s, 2H, OCH ), 6.41–6.42 (bs, 1H, NH, D O exch.), 6.73 (s, 1H, Ar),
3
2
2
6
.89–6.91 (d, J = 7.6 Hz, 1H, Ar), 7.15–7.18 (m, 2H, 2
×
Ar), 7.37–7.39 (t, J = 8.0 Hz, 1H, Ar), 7.69–7.71
13
(m, 1H, Ar), 7.77–7.79 (m, 1H, Ar), 8.33–8.34 (bs, 1H, NH, D O exch.). C NMR (101 MHz, CDCl3):
2
δ 16.1 (CH3), 24.1 (2
20.2 (Ar), 123.8 (Ar), 130.0 (Ar), 131.2 (Ar), 148.7 (Ar), 148.9 (Ar), 149.2 (Ar), 155.1 (Ar), 169.0 (C=O).
Anal. Calcd for C H N O : C, 62.96; H, 6.16; N, 12.24. Found: C, 63.05; H, 6.19; N, 12.29.
×
CH ), 34.1 (CH), 67.4 (OCH ), 108.0 (Ar), 109.9 (Ar), 116.0 (Ar), 119.1 (Ar),
3 2
1
18
21
3
4
3
.4. Crystal Structure Determination of Compound 34
C17H19NO3, M = 285.33, Monoclinic, space group P 21/c, a = 8.380(1), b = 15.416(1), c = 11.375(1)Å,
−1
β
= 95.856(7), V = 1462.6(2)Å3, Z = 4 Dc = 1.296, µ = 0.089 mm , F(000) = 608. 9670 reflections

Pharmaceuticals 2020, 13, 405
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were collected with a 4.356 <
θ
< 29.273 range with a completeness to theta 99.1%; 3395 were unique,
the parameters were 190 and the final R index was 0.0619 for reflections having I > 2σI.
A light yellow prismatic shaped crystal (0.07 0.06 0.03) was used for data collection. Hydrogen
×
×
atoms were all assigned in calculated positions and refined as isotropic. No relevant hydrogen
bonds were detected. CCDC 1980137 contains the supplementary crystallographic data for this
molecule. Data can be obtained free of charge from the Cambridge Crystallographic Data Centre [28].
Collection was carried out with a KM4 Xcalibur2 goniometer (Oxford Diffraction, Abingdon, UK)
at 100 K. Mo/K
ULTRA assembly, and an Oxford Diffraction Excalibur PX Ultra CCD were used for cells parameters
determination and data collection. The integrated intensities, measured using the scan mode,
α
radiation (40 mA/
−
40 KV), monochromated by an Oxford Diffraction Enhance
ω
were corrected for Lorentz and polarization effects [29]. Direct methods of SIR2004 [30] were used
in solving the structure and the refinement was performed using the full-matrix least squares on
F2 provided, within WinGX v.2013.3 routine [31], by SHELXL2014 [32]. Multi-scan symmetry-related
measurement was used as experimental absorption correction type.
3
.5. Anti-Helicobacter Pylori Activity
The MIC determination was performed by modified broth microdilution assay as previously
described [33]. For MBC evaluation 10
Skirrow agar plates surface and incubated for 72 h at 37 C in microaerophilic conditions. The MBC
was defined as the concentration that killed 99.9% of the initial inoculum.
µ
L of suspensions without visible growth were spotted on
◦
3
.6. Cell Lines and Treatments
The human adenocarcinoma gastric cell line (AGS), was derived from an untreated human
adenocarcinoma of the stomach and retained the same cytological characteristics of the malignant cells
obtained from Caucasian patients [34]. The AGS cells (ECACC 89090402) were obtained from CLS
Cell Lines Services GmbH (Epplheim, Germany) and were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 10% Foetal Bovine
Serum (FBS) (EuroClone S.p.A., Pero, Italy).
Working solutions of carvacrol and its derivatives (
6, 9, 16, 17, 20, 21, 29, 32–35, 38, 39, 42–45)
(600 mM) were freshly prepared in dimethyl sulfoxide (DMSO) and in DMEM according to the
experimental design by serial dilutions in complete culture medium. The final concentration of DMSO
in experiments was 0.14%. No toxicity on AGS cells was observed (data not shown). 5-Fluorouracil
was used as positive control.
3
.7. Cell Viability
Cell viability was tested by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(
4-sulfophenyl)-2H-tetrazolium)) assay (Promega, Madison, WI, USA). The concentration of carvacrol
and its derivatives for treatments was selected based on concentration—response curves constructed
in preliminary experiments (data not shown). Briefly, AGS cells were seeded in 96-well plates
3
(
(
6 × 10 cells/well) and treated for 24 h with different concentrations (50–800
µM) of specific molecules
5 replica wells for each treatment condition).
Briefly, cells were incubated with the MTS solution for at least 1 h and cell viability was determined
colorimetrically by measuring the absorbance at 490 nm using GloMax-Multi Detection System
Promega, Madison, WI, USA). Cell viability was expressed as the percentage compared with the
(
untreated cells designated as 100%. The IC50 value was calculated from the concentration-response
curves by nonlinear regression analysis [35].
3
.8. Statistical Analysis
A p value of 0.05 was considered statistically significant. IC50 values was calculated using the
GraphPad Prism 7 software.

Pharmaceuticals 2020, 13, 405
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4
. 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
noteworthy compounds displaying the lowest MIC/MBC activity against H. pylori, such as compounds
6 and 39, also showed good activity against AGS cells (IC50 compound 16 = 209 µM; IC50 compound
9 = 209 µM). Further studies might explain the potential synergistic effects of the combination of these
µg/mL range. In this regard, the most
1
3
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.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/11/405/s1.
Table S1: Crystal data and structure refinement for compound 34, Table S2: Atomic coordinates (x 104) and
equivalent isotropic displacement parameters (Å2x 103) for compound 34. U(eq) is defined as one third of the
◦
trace of the orthogonalized Uij tensor, Table S3: Bond lengths [Å] and angles [ ] for compound 34, Table S4
:
Anisotropic displacement parameters (Å2x 103) for compound 34. The anisotropic displacement factor exponent
1
13
19
takes the form: −2 2[h2a*2U11 + . . . + 2 h k a* b* U12]. H, C and F NMR spectra of new compounds.
Author Contributions: Conceptualization, S.C. and R.G.; methodology, S.C., F.S., P.G., and M.S.; formal analysis,
C.B.T., A.P.S., M.C.D.M., E.H.; D.S., S.C., and G.M.; writing and original draft preparation, S.C. and R.G. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was financed by intramural grants by Ministero Italiano dell’Universit
MIUR) FAR2019 (ex 60%), held by Simone Carradori.
à e della Ricerca
(
Conflicts of Interest: The authors declare no conflict of interest.
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