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ADMET and DMPK, Vol. 13 No. 1, 2025.

Original scientific paper

https://doi.org/10.5599/admet.2642

Antimicrobial and ADME properties of methoxylated, methylated and nitrated 2-hydroxynaphthalene-1 carboxanilides

Lucia Vrablova orcid id orcid.org/0009-0008-4701-5265 ; Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovicova 6, 842 15 Bratislava, Slovakia
Tomas Gonec orcid id orcid.org/0000-0003-3712-8641 ; Department of Chemical Drugs, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 00 Brno, Czech Republic
Tereza Kauerova ; Department of Pharmacology and Toxicology, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 00 Brno, Czech Republic
Michal Oravec ; Global Change Research Institute CAS, Belidla 986/4a, 603 00 Brno, Czech Republic
Izabela Jendrzejewska ; Institute of Chemistry, University of Silesia, Bankowa 12, 40007 Katowice, Poland
Peter Kollar orcid id orcid.org/0000-0003-2265-1528 ; Department of Pharmacology and Toxicology, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 00 Brno, Czech Republic
Alois Cizek ; Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic
Josef Jampilek orcid id orcid.org/0000-0003-2003-9052 ; Department of Chemical Biology, Faculty of Science, Palacky University Olomouc, Slechtitelu 27, 779 00 Olomouc, Czech Republic

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Abstract

Background and purpose
Many new compounds are being prepared to overcome the problem of increasing microbial resistance and the increasing number of infections.
Experimental approach
This study includes a series of twenty-seven mono-, di- and trisubstituted 2-hydroxynaphthalene-1-carboxanilides designed as multitarget agents. The compounds are substituted with methoxy, methyl, and nitro groups, as well as additionally with chlorine, bromine, and trifluoromethyl at various positions. All the compounds were evaluated for antibacterial activities against Gram-positive and Gram-negative bacteria and mycobacteria. Cytotoxicity on human cells was also tested.
Key results
Three compounds showed activity comparable to clinically used drugs. N-(3,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (13) showed only antistaphylococcal activity (minimum inhibitory concentration (MIC) = 54.9 μM); 2-hydroxy-N-[2-methyl-5-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (22) and 2-hydroxy-N-[4-nitro-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (27) were active across the entire spectrum of tested bacteria/mycobacteria, both against the sensitive set and against resistant isolates (MICs range 0.3 to 92.6 μM). Compound 22 was even active against E. coli (MIC = 23.2 μM). The active agents showed no in vitro cytotoxicity up to a concentration of 30 μM.
Conclusion
Compounds with trifluoromethyl in the meta-anilide position, experimental lipophilicity expressed as log k (logarithm of the capacity factor) in the range of 0.31 to 0.34 and calculated electron σ parameter for the anilide substituent higher than 0.59 were effective. The investigated compounds meet the definition of Michael acceptors. Based on ADME screening, the investigated compounds 13, 22 and 27 should have suitable physicochemical parameters for good bioavailability in the organism. Therefore, these are promising agents for further study.

Keywords

Lipophilicity; antibacterial activity; antimycobacterial activity; cytotoxicity

Hrčak ID:

329608

URI

https://hrcak.srce.hr/329608

Publication date:

30.3.2025.

Visits: 867 *

Copyright statement: Copyright © 2025 by the authors.

Copyright: 2025

License (open-access, http://creativecommons.org/licenses/by/4.0/):

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

Date received: 14 January 2025

Date revision received: 04 February 2025

Publication date (electronic): 08 February 2025

Publication date (collection): 2025

Volume: 13

Issue: 1

Electronic Location Identifier: 2642

DOI: 10.5599/admet.2642

Article Information (continued)

Categories:

Subject: Original scientific paper

Keywords:

Keywords

Keyword: Lipophilicity

Keyword: antibacterial activity

Keyword: antimycobacterial activity

Keyword: cytotoxicity

Counts:

Figures: 5

Tables: 4

Equations: 0

References: 93

Pages: 21

Antimicrobial and ADME properties of methoxylated, methylated and nitrated 2-hydroxynaphthalene-1 carboxanilides

Lucia Vrablova[1]

Tomas Gonec[2][*]

Tereza Kauerova[3]

Michal Oravec[4]

Izabela Jendrzejewska[5]

Peter Kollar[3]

Alois Cizek[6]

Josef Jampilek[1][7][*]

[1] Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovicova 6, 842 15 Bratislava, Slovakia

[2] Department of Chemical Drugs, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 00 Brno, Czech Republic

[3] Department of Pharmacology and Toxicology, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 612 00 Brno, Czech Republic

[4] Global Change Research Institute CAS, Belidla 986/4a, 603 00 Brno, Czech Republic

[5] Institute of Chemistry, University of Silesia, Bankowa 12, 40007 Katowice, Poland

[6] Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic

[7] Department of Chemical Biology, Faculty of Science, Palacky University Olomouc, Slechtitelu 27, 779 00 Olomouc, Czech Republic

Author notes:

Correspondence to: Corresponding Authors: E-mails: *t.gonec@seznam.cz

Correspondence to: *josef.jampilek@gmail.com

Abstract

Background and purpose

Many new compounds are being prepared to overcome the problem of increasing microbial resistance and the increasing number of infections.

Experimental approach

This study includes a series of twenty-seven mono-, di- and trisubstituted 2-hydroxynaphthalene-1-carboxanilides designed as multitarget agents. The compounds are substituted with methoxy, methyl, and nitro groups, as well as additionally with chlorine, bromine, and trifluoromethyl at various positions. All the compounds were evaluated for antibacterial activities against Gram-positive and Gram-negative bacteria and mycobacteria. Cytotoxicity on human cells was also tested.

Key results

Three compounds showed activity comparable to clinically used drugs. N-(3,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (13) showed only antistaphylococcal activity (minimum inhibitory concentration (MIC) = 54.9 μM); 2-hydroxy-N-[2-methyl-5-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (22) and 2-hydroxy-N-[4-nitro-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (27) were active across the entire spectrum of tested bacteria/mycobacteria, both against the sensitive set and against resistant isolates (MICs range 0.3 to 92.6 μM). Compound 22 was even active against E. coli (MIC = 23.2 μM). The active agents showed no in vitro cytotoxicity up to a concentration of 30 μM.

Conclusion

Compounds with trifluoromethyl in the meta-anilide position, experimental lipophilicity expressed as log k (logarithm of the capacity factor) in the range of 0.31 to 0.34 and calculated electron σ parameter for the anilide substituent higher than 0.59 were effective. The investigated compounds meet the definition of Michael acceptors. Based on ADME screening, the investigated compounds 13, 22 and 27 should have suitable physicochemical parameters for good bioavailability in the organism. Therefore, these are promising agents for further study.

Introduction

An increasingly common problem is the increasing number of infections caused by a wide range of microorganisms. The increase in antimicrobial resistance across the spectrum of bacteria plays a major role [1-3]. The most effective tool to counter this unfortunate trend is the effort to discover new bioactive compounds or at least innovated structures of existing drugs with a new/innovative mechanism of action [4-7]. In addition to the development of the most valuable molecules – structurally novel anti-infectives targeting new (single or multiple) targets [8,9], interesting strategies include the development of lantibiotics and bacteriocins [10,11], antimicrobial peptides and bacterial cell membrane disruptors [12-15], chemosensitizers, inhibitors of quorum sensing, virulence and biofilm formation, phage or monoclonal antibody-based therapies [16-18], drug repurposing [19,20], or nanoparticle-based strategies [21,22].

As mentioned, the current trend remains the design of so-called multitarget compounds, which are able to act on many different targets and thus interfere with the bacterial microorganism at different points of metabolism or reproduction [18,23-27]. Salicylanilides represent a promising type of multitarget compounds [28-36]. Inspired by salicylanilides, deeper research into their cyclic analogues – hydroxynaphthalenecarboxanilides – was initiated. These compounds are characterized not only by antimicrobial [37] but also by antiparasitic [38] and anticancer [39] activity. In these compounds, the essential role of the hydroxyl group has been identified, which must be free [40,41] or substituted by a group (e.g. carbamate [42]) capable of forming bonds with biomolecules. The connecting amide bridge between aromatic systems is also an important part. The position of the phenyl group is important because it manifests various physicochemical (e.g. solubility and lipophilicity), but also biological properties [37-39,43-45]. Overall, it can be said that the mentioned molecules can be considered as Michael acceptors [46-51].

Many anilides have been prepared, especially with pronounced lipophilic and electron-withdrawing (F/Cl/Br, CF3) substituents, which were expected to be antimicrobially active, i.e. the molecules would approach the properties of Michael acceptors [37,38,40,41,43-45]. Only a few works have dealt with alkoxy or methyl substituents [52-54]. Thus, it is a follow-up work of previous research, where anilides containing various combinations of predominantly polar and electron-donating/less electron-accepting substituents on the 2-hydroxynaphthalene-1-carboxanilide scaffold have now been synthesized and all the prepared compounds have been investigated on a wide battery of bacterial and mycobacterial species.

Experimental

General

All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA). Microwave-assisted reactions were performed using a StartSYNTH microwave lab station (Milestone, Sorisole BG, Italy). The melting points were determined on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and were uncorrected. Infrared (IR) spectra were recorded on an ATR diamond iD7 for Nicolet™ Impact 410 Fourier-transform IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA). The spectra were obtained by accumulating 64 scans with a 2 cm–1 resolution in the region of 4000-650 cm–1. All 1H- and 13C-NMR spectra were recorded on a JEOL ECZR 400 MHz NMR spectrometer (400 MHz for 1H and 100 MHz for 13C, Jeol, Tokyo, Japan) in dimethyl sulfoxide-d6 (DMSO-d6). 1H and 13C chemical shifts (ẟ) are reported in ppm. High-resolution mass spectra were measured using a high-performance liquid chromatograph Dionex UltiMate® 3000 (Thermo Scientific, West Palm Beach, FL, USA) coupled with an LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo Scientific) equipped with a HESI II (heated electrospray ionization) source in the positive mode.

General procedure for synthesis of N-(substituted phenyl)-2-hydroxynaphthalene-1-carboxamides 1-27.

2-Hydroxynaphthalene-1-carboxylic acid (5.3 mmol) and the corresponding substituted aniline (5.3 mmol) were suspended in 30 mL of dry chlorobenzene. Phosphorous trichloride (2.65 mmol) was added dropwise, and the reacting mixture was heated in the microwave reactor for 15 min at 130 °C and maximal allowed power 500 W using infrared flask-surface control of temperature. The solvent was evaporated under reduced pressure, the solid residue was washed with 2 M HCl, and the crude product was recrystallized from aqueous ethanol. All the studied compounds are presented inTable 1.

Table 1. Structure of ring-substituted 2-hydroxynaphthalene-1-carboxanilides 1-27; experimentally determined logarithm of the capacity factor (log k), logarithm of distribution coefficients at pH 6.5 (log D6.5) and pH 7.4 (log D7.4), predicted lipophilicity (log P) values and electronic σ(Ar) parameters of anilide ring of investigated compounds
ADMET-13-2642-g001.jpg
Comp.Rlog klog D6.5log D7.4log P1σ(Ar)1
1H0.03400.00110.01074.490.60
22-OCH30.31810.30150.30694.540.01
33-OCH30.17510.16430.17174.510.66
44-OCH3-0.0380-0.0491-0.03824.300.36
52,5-OCH30.33530.32630.33104.700.08
63,5-OCH30.03880.04110.04904.280.93
73,4,5-OCH3-0.1150-0.1053-0.09484.220.69
82-CH30.08580.07450.08384.830.59
93-CH30.17620.16440.17274.830.48
104-CH30.16580.15530.16324.830.46
112,5-CH30.24860.24550.25144.990.59
122,6-CH30.07850.08380.09234.990.58
133,5-CH30.33670.34290.34804.990.59
142,4,6-CH30.25390.26290.26914.840.44
152-OCH3-5-CH30.49320.49740.50124.770.01
162-OCH3-6-CH30.01240.02840.03824.770.01
172-CH3-5-OCH30.08920.10470.11134.950.76
182-Cl-5-OCH30.46350.46070.45935.151.13
192-OCH3-5-Br0.58450.58360.58355.580.12
202-OCH3-5-CF30.53350.53230.53055.770.11
213-CF3-4-OCH30.16350.17040.17455.640.58
222-CH3-5-CF30.33730.34440.34405.710.82
233-CF3-4-CH30.46050.46210.46365.710.68
242-NO20.30330.30770.31034.451.12
253-NO20.48820.34620.30584.501.09
264-NO20.09840.08620.08794.591.14
273-CF3-4-NO20.31240.32820.32205.471.36

[i] 1calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012) [55].

2-Hydroxy-N-phenylnaphthalene-1-carboxamide (1), 2-hydroxy-N-(2-methoxyphenyl)naphthalene-1-carboxamide (2), 2-hydroxy-N-(3-methoxyphenyl)naphthalene-1-carboxamide (3), 2-hydroxy-N-(4-methoxyphenyl)naphthalene-1-carboxamide (4), 2-hydroxy-N-(2-methylphenyl)naphthalene-1-carboxamide (8), 2-hydroxy-N-(3--methylphenyl)naphthalene-1-carboxamide (9), 2-hydroxy-N-(4-methylphenyl)naphthalene-1-carboxamide (10), 2-hydroxy-N-(2-nitrophenyl)naphthalene-1-carboxamide (24), 2-hydroxy-N-(3-nitrophenyl)naphthalene-1-carboxamide (25), 2-hydroxy-N-(4-nitrophenyl)naphthalene-1-carboxamide (26) were described by Gonec et al. [43].

N-(2,5-Dimethoxyphenyl)-2-hydroxynaphthalene-1-carboxamide (5)

Yield 77 %; mp 196-198 °C; IR (cm–1): 2934, 2829, 1625, 1583, 1531, 1513, 1486, 1463, 1434, 1423, 1353, 1278, 1265, 1239, 1219, 1175, 1148, 1033, 970, 959, 895, 847, 815, 792, 741, 717, 702; 1H-NMR (DMSO-d6), ẟ: 10.50 (br. s, 1H), 9.32 (s, 1H), 8.06 (d, 1H, J=2.7 Hz), 8.02 (d, 1H, J=8.7 Hz), 7.88 (d, 1H, J=9.1 Hz), 7.84 (d, 1H, J=7.8 Hz), 7.48 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.32-7.36 (m, 1H), 7.25 (d, 1H, J=9.1 Hz), 7.00 (d, 1H, J=8.7 Hz), 6.68 (dd, 1H, J=8.7 Hz, J=3.2 Hz), 3.77 (s, 3H), 3.76 (s, 3H) (see Figure S1 in Supplementary Materials); 13C-NMR (DMSO-d6), ẟ: 165.31, 153.13, 152.34, 143.23, 131.76, 131.13, 128.52, 128.04, 127.72, 127.07, 123.96, 123.12, 118.27, 116.77, 111.99, 108.05, 107.47, 56.40, 55.44 (Figure S2); HR-MS: [M+H]+ calculated 324.123034 m/z, found 324.12305 m/z.

N-(3,5-Dimethoxyphenyl)-2-hydroxynaphthalene-1-carboxamide (6)

Yield 64 %; mp 151-153 °C; IR (cm–1): 3266, 2999, 2934, 2833, 2540, 1614, 1595, 1549, 1514, 1470, 1453, 1423, 1332, 1296, 1257, 1227, 1194, 1154, 1064, 985, 846, 813, 739, 711; 1H-NMR (DMSO-d6), ẟ: 10.31 (s, 1H), 10.12 (s, 1H), 7.86 (d, 2H, J=8.4 Hz), 7.67 (d, 1H, J=8.4 Hz), 7.47 (td, 1H, J=7.3 Hz, J=1.1 Hz), 7.33 (td, 1H, J=7.3 Hz, J=1.1 Hz), 7.25 (d, 1H, J=8.8 Hz), 7.09 (s, 2H), 6.27 (s, 1H), 3.74 (s, 6H) (Figure S3); 13C-NMR (DMSO-d6), ẟ: 165.83, 160.51, 151.60, 141.30, 131.37, 130.14, 127.96, 127.38, 126.97, 123.38, 123.00, 118.61, 118.34, 97.66, 95.38, 55.08 (Figure S4); HR-MS: [M-H]+ calculated 322.10738 m/z, found 322.10788 m/z.

2-Hydroxy-N-(3,4,5-trimethoxyphenyl)naphthalene-1-carboxamide (7)

Yield 43 %; mp 223-225 °C; IR (cm–1): 3335, 2943, 2834, 1636, 1586, 1541, 1505, 1448, 1406, 1347, 1299, 1281, 1128, 1030, 1000, 980, 969, 891, 817, 774, 745, 686; 1H-NMR (DMSO-d6), ẟ: 10.29 (s, 1H), 10.11 (s, 1H), 7.86 (d, 1H, J=8.7 Hz), 7.85 (d, 1H, J=8.2 Hz), 7.68 (d, 1H, J=8.2 Hz), 7.46 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.31-7.35 (m, 1H), 7.25 (s, 2H), 7.25 (d, 1H, J=8.7 Hz), 3.76 (s, 6H), 3.65 (s, 3H) (Figure S5); 13C-NMR (DMSO-d6), ẟ: 165.58, 152.74, 151.62, 135.95, 133.39, 131.41, 130.15, 127.98, 127.38, 126.97, 123.47, 123.02, 118.65, 118.33, 96.86, 60.17, 55.70 (Figure S6); HR-MS: [M+H]+ calculated 354.133599 m/z, found 354.13345 m/z.

N-(2,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (11)

Yield 71 %; mp 162-165 °C; IR (cm–1): 2914, 1646, 1576, 1515, 1434, 1406, 1319, 1261, 1141, 1033, 962, 902, 873, 803, 742, 678, 661; 1H-NMR (DMSO-d6), ẟ: 10.13 (br. s, 1H), 9.71 (s, 1H), 7.83-7.87 (m, 3H), 7.50 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=1.1 Hz), 7.41 (s, 1H), 7.33 (t, 1H, J=7.5 Hz), 7.24 (d, 1H, J=9.1 Hz), 7.14 (d, 1H, J=7.8 Hz), 6.96 (dd, 1H, J=7.8 Hz, J=1.1 Hz), 2.32 (s, 3H), 2.28 (s, 3H) (Figure S7); 13C-NMR (DMSO-d6), ẟ: 165.56, 151.75, 136.22, 134.83, 131.66, 130.09, 130.03, 129.36, 127.94, 127.47, 126.85, 126.29, 126.05, 123.65, 122.92, 118.42, 118.34, 20.63, 17.62 (Figure S8); HR-MS: [M+H]+ calculated 292.133205 m/z, found 292.13290 m/z.

N-(2,6-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (12)

Yield 84 %; mp 195-197 °C; IR (cm–1): 3352, 2939, 2834, 1623, 1604, 1574, 1514, 1458, 1319, 1139, 1033, 970, 906, 821, 796, 756, 726, 717, 702, 661; 1H-NMR (DMSO-d6), ẟ: 10.17 (br.s, 1H), 9.67 (s, 1H), 7.89 (d, 1H, J=8.2 Hz), 7.84-7.87 (m, 2H), 7.50 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.33 (t, 1H, J=7.3 Hz), 7.25 (d, 1H, J=9.1 Hz), 7.12 (s, 3H), 2.39 (s, 6H) (Figure S9); 13C-NMR (DMSO-d6), ẟ: 165.44, 151.83, 135.59, 135.27, 131.77, 129.93, 128.01, 127.68, 127.48, 126.81, 126.42, 123.61, 122.88, 118.60, 118.29, 18.66 (Figure S10); HR-MS: [M+H]+ calculated 292.133205 m/z, found 292.13300 m/z.

N-(3,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (13)

Yield 70 %; mp 181-184 °C; IR (cm–1): 3339, 3217, 1612, 1577, 1531, 1514, 1435, 1351, 1310, 1275, 1244, 1231, 1210, 1146, 968, 842, 819, 753, 743, 685; 1H-NMR (DMSO-d6), ẟ: 10.22 (s, 1H), 10.07 (s, 1H), 7.84 (d, 2H, J=8.4 Hz), 7.67 (d, 1H, J=8.4 Hz), 7.49 (td, 1H, J=7.0 Hz, J=1.1 Hz), 7.34 (s, 2H), 7.32 (td, 1H, J=7.0 Hz, J=1.1 Hz), 7.26 (td, 1H, J=9.1 Hz, J=1.3 Hz), 7.34 (s, 1H), 2.27 (s, 6H) (Figure S11); 13C-NMR (DMSO-d6), ẟ: 165.62, 151.55, 139.48, 137.58, 131.45, 129.99, 127.93, 127.40, 126.88, 124.80, 123.44, 122.94, 118.76, 118.36, 117.13, 21.17 (Figure S12); HR-MS: [M-H]+ calculated 290.11756 m/z, found 290.11829 m/z.

2-Hydroxy-N-(2,4,6-trimethylphenyl)naphthalene-1-carboxamide (14)

Yield 72 %; mp 175-177 °C; IR (cm–1): 3338, 2944, 2831, 1623, 1602, 1577, 1512, 1460, 1396, 1369, 1319, 1223, 1206, 1155, 1030, 971, 906, 847, 821, 796, 772, 754, 724, 713, 690, 672; 1H-NMR (DMSO-d6), ẟ: 10.13 (br.s, 1H), 9.56 (s, 1H), 7.87 (d, 1H, J=8.7 Hz), 7.84 (d, 1H, J=8.7 Hz), 7.84 (d, 1H, J=9.1 Hz), 7.49 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=1.4 Hz), 7.33 (ddd, 1H, J=8.1 Hz, J=7.0 Hz, J=0.9 Hz), 7.24 (d, 1H, J=9.1 Hz), 6.93 (s, 2H), 2.34 (s, 6H), 2.26 (s, 3H) (Figure S13); 13C-NMR (DMSO-d6), ẟ: 165.52, 151.79, 135.32, 135.27, 132.65, 131.77, 129.86, 128.26, 127.99, 127.47, 126.76, 123.64, 122.85, 118.68, 118.29, 20.53, 18.55 (Figure S14); HR-MS: [M+H]+ calculated 306.148855 m/z, found 306.14862 m/z.

2-Hydroxy-N-(2-methoxy-5-methylphenyl)naphthalene-1-carboxamide (15)

Yield 73 %; mp 203-205 °C; IR (cm–1): 3342, 2945, 2833, 1633, 1585, 1530, 1514, 1482, 1462, 1434, 1370, 1353, 1321, 1300, 1272, 1257, 1210, 1148, 1123, 1030, 969, 896, 868, 817, 799, 750, 719, 687; 1H-NMR (DMSO-d6), ẟ: 10.41 (br.s, 1H), 9.26 (s, 1H), 8.14 (s, 1H), 8.01 (d, 1H, J=8.7 Hz), 7.87 (d, 1H, J=9.1 Hz), 7.84 (d, 1H, J=8.2 Hz), 7.48 (ddd, 1H, J=8.7 Hz, J=6.9 Hz, J=1.4 Hz), 7.33 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.24 (d, 1H, J=8.7 Hz), 6.92-6.98 (m, 2H), 3.78 (s, 3H), 2.31 (s, 3H) (Figure S15); 13C-NMR (DMSO-d6), ẟ: 165.22, 152.17, 147.34, 131.76, 130.89, 129.22, 128.00, 127.68, 127.39, 126.98, 124.54, 123.96, 123.06, 121.89, 118.28, 117.12, 111.12, 55.92, 20.58 (Figure S16); HR-MS: [M+H]+ calculated 308.12812 m/z, found 308.12820 m/z.

2-Hydroxy-N-(2-methoxy-6-methylphenyl)naphthalene-1-carboxamide (16)

Yield 77 %; mp 142-145 °C; IR (cm–1): 3325, 2944, 2833, 1625, 1581, 1514, 1472, 1437, 1354, 1305, 1285, 1139, 1120, 1081, 1031, 970, 912, 818, 761, 741, 712; 1H-NMR (DMSO-d6), ẟ: 10.04 (br.s, 1H), 9.46 (s, 1H), 8.09 (d, 1H, J=7.8 Hz), 7.83 (d, 1H, J=9.1 Hz), 7.83 (d, 1H, J=7.8 Hz), 7.52 (ddd, 1H, J=8.6 Hz, J=7.0 Hz, J=1.4 Hz), 7.33 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.23 (d, 1H, J=8.7 Hz), 7.20 (t, 1H, J=7.8 Hz), 6.94 (d, 1H, J=7.8 Hz), 6.89 (d, 1H, J=7.3 Hz), 3.86 (s, 3H), 2.35 (s, 3H) (Figure S17); 13C-NMR (DMSO-d6), ẟ: 165.77, 155.34, 151.71, 137.06, 131.81, 129.74, 127.74, 127.42, 127.19, 126.58, 125.14, 124.30, 122.84, 121.92, 118.83, 118.29, 109.26, 55.71, 18.04 (Figure S18); HR-MS: [M+H]+ calculated 308.12812 m/z, found 308.12823 m/z.

2-Hydroxy-N-(5-methoxy-2-methylphenyl)naphthalene-1-carboxamide (17)

Yield 76 %; mp 140-143 °C; IR (cm–1): 2945, 2833, 1642, 1585, 1513, 1450, 1437, 1350, 1291, 1278, 1146, 1030, 969, 896, 845, 816, 800, 768, 746, 719, 691, 679; 1H-NMR (DMSO-d6), ẟ: 10.17 (br.s, 1H), 9.71 (s, 1H), 7.83-7.89 (m, 3H), 7.50 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=0.9 Hz), 7.31-7.36 (m, 1H), 7.26 (s, 1H), 7.25 (d, 1H, J=11.4 Hz), 7.16 (d, 1H, J=8.2 Hz), 6.74 (dd, 1H, J=8.2 Hz, J=2.7 Hz), 3.77 (s, 3H), 2.25 (s, 3H) (Figure S19); 13C-NMR (DMSO-d6), ẟ: 165.56, 157.35, 151.83, 137.20, 131.67, 130.75, 130.17, 127.95, 127.50, 126.90, 123.84, 123.69, 122.95, 118.35, 118.21, 111.14, 110.60, 55.17, 17.18 (Figure S20); HR-MS: [M+H]+ calculated 308.12812 m/z, found 308.12811 m/z.

N-(2-Chloro-5-methoxyphenyl)-2-hydroxynaphthalene-1-carboxamide (18)

Yield 87 %; mp 129-132 °C; IR (cm–1): 1635, 1583, 1512, 1456, 1435, 1413, 1353, 1273, 1231, 1213, 1190, 1168, 1150, 1126, 999, 968, 892, 860, 842, 812, 789, 743, 728, 677; 1H-NMR (DMSO-d6), ẟ: 10.48 (br.s, 1H), 9.87 (s, 1H), 8.04 (d, 1H, J=8.7 Hz), 7.89 (d, 1H, J=9.1 Hz), 7.85 (d, 1H, J=7.8 Hz), 7.70 (d, 1H, J=2.5 Hz), 7.50 (t, 1H, J=7.3 Hz), 7.44 (d, 1H, J=8.7 Hz), 7.34 (t, 1H, J=7.1 Hz), 7.25 (d, 1H, J=8.7 Hz), 6.84 (dd, 1H, J=8.9 Hz, J=2.5 Hz), 3.82 (s, 3H) (Figure S21); 13C-NMR (DMSO-d6), ẟ: 165.71, 158.22, 152.46, 135.82, 131.71, 131.08, 129.83, 128.03, 127.63, 127.05, 123.89, 123.09, 118.27, 117.29, 116.62, 111.55, 110.89, 55.56 (Figure S22); HR-MS: [M+H]+ calculated 328.073497 m/z, found 328.07379 m/z.

N-(5-Bromo-2-methoxyphenyl)-2-hydroxynaphthalene-1-carboxamide (19)

Yield 78 %; mp 231-234 °C; IR (cm–1): 1635, 1582, 1517, 1476, 1457, 1430, 1408, 1370, 1352, 1319, 1273, 1253, 1240, 1223, 1204, 1174, 1146, 1126, 1022, 966, 911, 879, 867, 816, 797, 752, 719, 700; 1H-NMR (DMSO-d6), ẟ: 10.51 (s, 1H), 9.57 (s, 1H), 8.54 (d, 1H, J=2.7 Hz), 8.00 (d, 1H, J=8.7 Hz), 7.88 (d, 1H, J=9.1 Hz), 7.84 (d, 1H, J=7.8 Hz), 7.48 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=1.4 Hz), 7.32-7.36 (m, 1H), 7.30 (dd, 1H, J=8.7 Hz, J=2.7 Hz), 7.24 (d, 1H, J=9.1 Hz), 7.06 (d, 1H, J=8.7 Hz), 3.82 (s, 3H) (Figure S23); 13C-NMR (DMSO-d6), ẟ: 165.65, 152.41, 148.49, 131.71, 131.20, 129.22, 128.03, 127.67, 127.11, 126.49, 123.88, 123.12, 123.09, 118.25, 116.53, 113.16, 111.68, 56.15 (Figure S24); HR-MS: [M+H]+ calculated 372.022975 m/z, found 372.02365 m/z.

2-Hydroxy-N-[2-methoxy-5-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (20)

Yield 75 %; mp 205-208 °C; IR (cm–1): 3425, 3194, 1641, 1616, 1583, 1533, 1510, 1484, 1463, 1438, 1341, 1323, 1267, 1232, 1207, 1164, 1124, 1108, 1074, 1018, 969, 928, 895, 812, 792, 754, 711; 1H-NMR (DMSO-d6), ẟ: 10.53 (s, 1H), 9.72 (s, 1H), 8.73 (d, 1H, J=1.8 Hz), 8.01 (d, 1H, J=8.2 Hz), 7.89 (d, 1H, J=9.1 Hz), 7.85 (d, 1H, J=7.8 Hz), 7.46-7.53 (m, 2H), 7.34 (t, 1H, J=7.3 Hz), 7.27 (d, 1H, J=8.7 Hz), 7.25 (d, 1H, J=8.7 Hz), 3.91 (s, 3H) (Figure S25); 13C-NMR (DMSO-d6), ẟ: 165.94, 152.47, 151.88, 131.73, 131.24, 128.24, 128.06, 127.68, 127.15, 124.55 (q, J=271.7 Hz), 123.89, 123.15, 121.47 (q, J=4.8 Hz), 120.89 (q, J=31.8 Hz), 118.29, 117.22 (q, J=3.9 Hz), 116.55, 111.45, 56.28 (Figure S26); HR-MS: [M+H]+ calculated 362.099854 m/z, found 362.10028 m/z.

2-Hydroxy-N-[4-methoxy-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (21)

Yield 65 %; mp 178-181 °C; IR (cm–1): 3404, 3172, 1645, 1623, 1584, 1538, 1516, 1499, 1462, 1436, 1423, 1323, 1271, 1233, 1207, 1142, 1112, 1056, 1022, 972, 897, 815, 743, 661; 1H-NMR (DMSO-d6), ẟ: 11.52 (s, 1H), 10.15 (br.s, 1H), 8.22 (d, 1H, J=2.7 Hz), 7.96 (dd, 1H, J=8.9 Hz, J=2.5 Hz), 7.84-7.88 (m, 2H), 7.69 (d, 1H, J=8.7 Hz), 7.46 (ddd, 1H, J=8.3 Hz, J=7.0 Hz, J=1.1 Hz), 7.31-7.35 (m, 1H), 7.29 (d, 1H, J=9.1 Hz), 7.26 (d, 1H, J=9.1 Hz), 3.89 (s, 3H) (Figure S27); 13C-NMR (DMSO-d6), ẟ: 165.67, 152.79 (q, J=1.9 Hz), 151.71, 132.58, 131.37, 130.31, 127.99, 127.39, 127.06, 124.63, 123.65 (q, J=271.7 Hz), 123.40, 123.06, 118.34, 118.23, 117.61 (q, J=5.8 Hz), 116.61 (q, J=29.9 Hz), 113.41, 56.28 (Figure S28); HR-MS: [M+H]+ calculated 362.099854 m/z, found 362.10037 m/z.

2-Hydroxy-N-[2-methyl-5-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (22)

Yield 67 %; mp 143-146 °C; IR (cm–1): 3230, 2927, 1634, 1623, 1585, 1544, 1514, 1492, 1438, 1418, 1326, 1277, 1264, 1224, 1163, 1111, 1076, 972, 925, 883, 818, 761, 739, 710; 1H-NMR (DMSO-d6), ẟ: 10.26 (s, 1H), 10.03 (s, 1H), 8.06 (s, 1H), 7.85-7.89 (m, 3H), 7.48-7.53 (m, 3H), 7.33-7.37 (m, 1H), 7.26 (d, 1H, J=9.1 Hz), 2.42 (s, 3H) (Figure S29); 13C-NMR (DMSO-d6), ẟ: 165.96, 151.98, 137.12, 136.78, 131.57, 131.38, 130.45, 127.09, 127.48, 127.07, 126.76 (q, J=31.8 Hz), 124.29 (q, J=271.7 Hz), 123.58, 123.06, 121.54 (q, J=3.9 Hz), 121.46 (q, J=3.9 Hz), 118.33, 117.76, 18.03 (Figure S30); HR-MS: [M+H]+ calculated 346.10494 m/z, found 346.10507 m/z.

2-Hydroxy-N-[4-methyl-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (23)

Yield 60 %; mp 155-160 °C; IR (cm–1): 3053, 2674, 1634, 1598, 1584, 1539, 1513, 1502, 1436, 1419, 1328, 1273, 1241, 1207, 1167, 1140, 1123, 1107, 1053, 1042, 967, 809, 744, 682; 1H-NMR (DMSO-d6), ẟ: 10.62 (s, 1H), 10.17 (s, 1H), 8.28 (d, 1H, J=1.8 Hz), 7.84-7.90 (m, 3H), 7.68 (d, 1H, J=8.2 Hz), 7.44-7.48 (m, 1H), 7.42 (d, 1H, J=8.2 Hz), 7.31-7.35 (m, 1H), 7.26 (d, 1H, J=8.7 Hz), 2.42 (s, 3H) (Figure S31); 13C-NMR (DMSO-d6), ẟ: 166.05, 151.76, 137.85, 132.65, 131.32, 130.39, 130.28 (q, J=1.9 Hz), 128.00, 127.49 (q, J=28.9 Hz), 127.38, 127.09, 124.50 (q, J=273.6 Hz), 123.33, 123.07, 122.57, 118.33, 118.16, 116.15 (q, J=5.8 Hz), 18.23 (q, J=1.9 Hz) (Figure S32); HR-MS: [M+H]+ calculated 346.10494 m/z, found 346.10532 m/z.

2-Hydroxy-N-[4-nitro-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (27)

Yield 18 %; mp 157-160 °C; IR (cm–1): 3272, 1672, 1657, 1623, 1514, 1437, 1417, 1353, 1333, 1279, 1235, 1214, 1179, 1143, 1042, 968, 891, 834, 815, 804, 754, 744, 681; 1H-NMR (DMSO-d6), ẟ: 11.24 (s, 1H), 10.35 (br.s, 1H), 8.52 (s, 1H), 8.23-8.29 (m, 2H), 7.92 (d, 1H, J=9.1 Hz), 7.88 (d, 1H, J=8.2 Hz), 7.70 (d, 1H, J=8.7 Hz), 7.48 (ddd, 1H, J=8.6 Hz, J=7.0 Hz, J=1.4 Hz), 7.33-7.38 (m, 1H), 7.28 (d, 1H, J=9.1 Hz) (Figure S33); 13C-NMR (DMSO-d6), ẟ: 167.00, 152.14, 144.12, 141.45, 131.12, 131.03, 128.11, 127.85, 127.37, 127.35, 123.27, 123.13, 123.08 (q, J=32.8 Hz), 122.37, 122.11 (q, J=273.6 Hz), 118.27, 117.32 (q, J=6.1 Hz), 117.20 (Figure S34); HR-MS: [M+H]+ calculated 377.074368 m/z, found 377.07495 m/z.

Lipophilicity determination by HPLC

An HPLC system Agilent 1200 equipped with a DAD detector (Agilent, Santa Clara, CA, USA) was used. A chromatographic column Symmetry® C18 5 μm, 4.6×250 mm, part No. WAT054275 (Waters Corp., Milford, MA, USA) was used. The HPLC separation process was monitored and evaluated with EZChrom Elite software ver. 3.3.2 (Agilent) [56]. Isocratic elution by a mixture of MeOH p.a. (72 %) and H2O-HPLC Mili-Q grade (28 %) as a mobile phase was used for the determination of capacity factor k. Isocratic elution by a mixture of MeOH p.a. (72 %) and acetate-buffered saline (pH 7.4 and pH 6.5) (28 %) as a mobile phase was used for the determination of distribution coefficients expressed as D7.4 and D6.5. The total flow of the column was 1.0 mL min-1, the injection volume was 20 μL, the column temperature was 40 °C, and the sample temperature was 10 °C. The detection wavelength of 210 nm was chosen. A KI methanolic solution was used to determine the dead times (tD). Retention times (tR) were measured in minutes. The capacity factors k were calculated according to the formula k = (tR–tD)/tD, where tR is the retention time of the solute, and tD is the dead time obtained using an unretained analyte. The distribution coefficients DpH were calculated according to the formula DpH = (tR–tD)/tD. Each experiment was repeated three times. The experimental values of lipophilicity of individual compounds are shown inTable 1.

Antibacterial screening

In vitro antibacterial activity of the synthesized compounds was evaluated against representatives of multidrug-resistant bacteria, three clinical isolates of methicillin-resistant S. aureus: clinical isolate of animal origin, MRSA 63718 [57] (Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Czech Republic), and MRSA SA 630 and MRSA SA 3202 [57] (National Institute of Public Health, Prague, Czech Republic), both of human origin. These three clinical isolates, carrying the mecA gene [58], were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 μg mL-1 (VA2-MRSA) within the susceptible range for MRSA 63718) methicillin-resistant S. aureus (VS-MRSA) [57]. Vancomycin- and methicillin-susceptible S. aureus ATCC 29213 and vancomycin-susceptible Enterococcus faecalis ATCC 29212, obtained from the American Type Culture Collection, were used as the reference and quality control strains. Three vanA gene-carrying vancomycin-resistant isolates of E. faecalis (VRE 342B, VRE 368, VRE 725B) were provided by Oravcova et al. [59]. In addition, all the prepared compounds were tested against the Gram-negative bacteria E. coli ATCC 25922 (American Type Culture Collection).

The minimum inhibitory concentrations (MICs) were evaluated using the microtitration broth method according to the CLSI [60,61], with some modifications. The compounds were dissolved in DMSO (Sigma, St. Louis, MO, USA) to get a concentration 10 μg mL-1 and diluted in a microtitration plate in an appropriate medium, i.e. Cation Adjusted Mueller–Hinton Broth (CaMH, Oxoid, Basingstoke, UK) for staphylococci, E. coli; and Brain Heart Infusion Broth (BHI, Oxoid) for enterococci to reach the final concentration of 256 to 0.125 μg mL-1. Microtitre plates were inoculated with test microorganisms so that the final concentration of was 105 bacterial cells in a microtiter plate. Ampicillin and ciprofloxacin (Sigma) were used as reference drugs. A drug-free control and a sterility control were included. The plates were incubated for 24 h at 37 °C for all the tested bacteria. After static incubation in the darkness in an aerobic atmosphere, the MIC was visually evaluated as the lowest concentration of the tested compound, which completely inhibited the growth of the microorganism. The experiments were repeated three times. The results are summarized inTable 2.

Table 2. In vitro activities (MICs) of investigated compounds against bacteria compared to ampicillin (AMP) and ciprofloxacin (CPX)
Comp.RMIC, μM
S. aureusMRSA 63718MRSA
SA 630		MRSA
SA 3202		E. faecalisVRE
342B		VRE
725B		VRE
368		E. coli
1H>972>972243122>972>972>972>972972
22-OCH3>873>873>873>873>873>873>873>873873
33-OCH3436436873218>873>873>873>873873
44-OCH3>873>873>873>873>873>873>873>873873
52,5-OCH3792792792792792792792792792
63,5-OCH3198396396198792792792792792
73,4,5-OCH3838838838419724724724724838
82-CH3462462462462462462462462462
93-CH3231462231231462462462462462
104-CH3231462462462462462462462462
112,5-CH3110110110110879879879879879
122,6-CH3439879439439879879879879879
133,5-CH354.954.954.954.9879879879879879
142,4,6-CH3838838838419838838838838838
152-OCH3-5-CH3833833833833833833833833833
162-OCH3-6-CH3833833833833416208833208833
172-CH3-5-OCH3104416208208833833833833833
182-Cl-5-OCH348.819548.897.6781781781781781
192-OCH3-5-Br688688688688688688688688688
202-OCH3-5-CF3709709709709709709709709709
213-CF3-4-OCH388.617788.688.688.617788.688.6709
222-CH3-5-CF32.905.792.902.9046.392.692.646.323.2
233-CF3-4-CH323.237192.746.3741741741741741
242-NO226.041510451.9830830830830830
253-NO220826.0208208830830830830830
264-NO2>830830415104415830830830830
273-CF3-4-NO20.3321.331.331.3321.342.542.521.3680
AMP–5.7245.845.845.82.8111.511.511.545.8
CPX–0.7524.224.224.21.513.021933.020.377

Antimycobacterial screening

The evaluation of in vitro antimycobacterial activity of the compounds was performed against Mycobacterium tuberculosis ATCC 25177/H37Ra, Mycobacterium kansasii DSM 44162 and Mycobacterium smegmatis ATCC 700084.

The broth dilution micro-method in Middlebrook 7H9 medium (Difco, Lawrence, KS, USA) supplemented with ADC Enrichment (Difco) was used to determine the minimum inhibitory concentration (MIC) as previously described [61]. The compounds were dissolved in DMSO (Sigma), and the final concentration of DMSO did not exceed 2.5 % of the total solution composition. The final concentrations of the evaluated compounds, ranging from 256 to 0.125 μg mL-1, were obtained by twofold serial dilution of the stock solution in a microtiter plate with a sterile medium. Bacterial inocula were prepared by transferring colonies from culture to sterile water. The plate was inoculated by tested microorganisms. The final concentration of bacterial cells was 1.5×106 for M. tuberculosis and 105 cells in a microtiter plate for other mycobacteria. Isoniazid and rifampicin (Sigma) were used as reference antimycobacterial drugs. Drug-free controls, sterility controls, and controls consisting of medium and DMSO alone were included. The plates were incubated for a defined time at an appropriate temperature (3 days at 37 °C for M. smegmatis, and 14 days at 37 °C for M. tuberculosis and M. kansasii). After incubation, the MIC was visually evaluated as the lowest concentration of the tested compound, which completely inhibited the growth of the microorganism. The MICs against M. tuberculosis were evaluated by Alamar blue (Oxoid). After incubation, 10 % of Alamar blue was added to each well, and the plate was incubated for 24 h. The MIC values were assessed as the lowest concentration of the tested compounds, which prevented changing blue resazurin to pink resorufin. The experiments were repeated three times. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration of the compound at which no visible bacterial growth was observed. The MIC value is routinely and widely used in bacterial assays and is a standard detection limit according to the CLSI [60]. The results are summarized inTable 3.

Table 3. In vitro activities (MICs) of investigated compounds against mycobacteria compared to isoniazid (INH), rifampicin (RIF), and in vitro cytotoxicity assay (LC50) of choice compounds on human monocytic leukemia THP-1 cells compared to oxaliplatin (OXP), camptothecin (CMP)
Comp.RMIC, μMLC50, μM
M. tuberculosisM. kansasiiM. smegmatis
1H48615.2486>20 [43]
22-OCH3>873>873>873–
33-OCH3218109218–
44-OCH3109218436–
52,5-OCH3396792792–
63,5-OCH339698.9396>30
73,4,5-OCH3210838210–
82-CH3231115462–
93-CH3231115231–
104-CH3231115>923–
112,5-CH3439110220–
122,6-CH3439879879–
133,5-CH3439110220>30
142,4,6-CH3210838210–
152-OCH3-5-CH3416833833–
162-OCH3-6-CH3416833416–
172-CH3-5-OCH3416833416–
182-Cl-5-OCH3391391781–
192-OCH3-5-Br344688688–
202-OCH3-5-CF3354709709–
213-CF3-4-OCH3354709177–
222-CH3-5-CF346.346.323.2>30
233-CF3-4-CH337174146.3–
242-NO210451.9208>20 [43]
253-NO2104104208>20 [43]
264-NO21044152082.5±0.1 [43]
273-CF3-4-NO285.021.321.3>30
INH–36.5233117–
RIF–9.710.15019.4–
OXP––––1.7±0.6
CMP––––0.16±0.07

Cytotoxicity assay

Cytotoxicity of the compounds was determined using an LDH assay kit (Roche Diagnostics, Mannheim, Germany) as described previously [43,54]. Human monocytic leukemia THP-1 cells (European Collection of Cell Cultures, Salisbury, UK) were exposed for 24 h at 37 °C to various compound concentrations ranging from 0.37 to 30 μM in RPMI 1640 medium. For LDH assays, cells were seeded into 96-well plates (5×104 cells/well in 100 μL culture medium) in triplicate in serum-free RPMI 1640 medium and measurements were taken 24 h after the treatment with the compounds. The maximum concentration of DMSO (Sigma) in the assays never exceeded 0.1 %. Oxaliplatin and camptothecin (Sigma) were used as reference drugs. The median lethal dose values, LC50, were deduced through the production of a dose-response curve. All data from three independent experiments were evaluated using GraphPad Prism 5.00 software (GraphPadSoftware, San Diego, CA, USA) [62]. The results are summarized inTable 3.

Results and discussion

Chemistry

The compounds were prepared by a simple (click chemistry method) but innovative microwave synthesis from commercially available building blocks (2-hydroxy-1-naphthoic acid and multisubstituted anilines) in anhydrous chlorobenzene in the presence of PCl3. The synthesis is depicted inScheme 1 and a list of all studied compounds is given inTable 1. The unsubstituted derivative 1 and monosubstituted compounds 2-4, 8-10, 24-26 have already been described by Gonec et al. [43] but are listed here for the completeness of the entire study.

Scheme 1. Synthesis of ring-substituted 2-hydroxynaphthalene-1-carboxanilides 1-27. Reagents and conditions: (a) PCl3, chlorobenzene, microwave synthesis (500 W, 130 °C, 15 min)
ADMET-13-2642-g002.jpg

Since the basis for understanding the behavior of bioactive molecules is the knowledge of their lipo-hydrophilic properties, lipophilicity parameters were determined for all compounds, namely log k (logarithm of the capacity factor) and log D (logarithm of the distribution coefficient) at physiological pH 6.5 and 7.4.

The capacity factor and distribution coefficients were measured by RP-HPLC on a C18 column with methanol as an organic modifier of the mobile phase. In addition to the experimental lipophilicities, predicted log P values were also calculated using ACD/Percepta [55], seeTable 1. In addition to lipophilicity,Table 1 also shows the predicted (ACD/Percepta [55]) electronic σ(Ar) parameters of the whole substituted anilide ring, characterizing the electron-withdrawing or donating ability of the molecular system. The values of σ(Ar) are found in a wide range from 0.01 to 1.36, so the compounds contain substituents with both electron-donating and slightly electron-withdrawing properties.

The graphs inFigure 1 show the correlations between the experimental and calculated lipophilicity values, and as can be seen from the correlation coefficients r, which range from 0.60 to 0.63 (n = 27), the agreement is small, which probably indicates a significant influence of the free phenol group, which the software cannot capture. On the other hand, the graphs inFigure 2 illustrate the relationships between log k and log D, which, according to the correlation coefficient r of ca. 0.99 (n = 27), are very good.

Figure 1. Comparison of experimentally determined values of log k (A), log D6.5 (B), and log D7.4 (C) with calculated log P (ACD/Percepta [55]) of carboxanilides 1-27
ADMET-13-2642-g003.jpg
Figure 2. Cross-correlations of experimentally determined values of log k versus log D6.5 (A), log k versus log D7.4 (B) and log D6.5 versus log D7.4 (C) of carboxanilides 1-27
ADMET-13-2642-g004.jpg

Figure 3 shows the order of the compounds according to the increasing log k value. The least lipophilic are methoxylated derivatives 7 (R = 3,4,5-OCH3) and 4 (R = 4-OCH3), while the most lipophilic are compounds 20 (R = 2-OCH3-5-CF3) and 19 (R = 2-OCH3-5-Br). The unsubstituted derivative 1 (the fourth least lipophilic compound in this series) showed the largest deviation between the log k and log D values.

Figure 3. Order of individual derivatives arranged according to increasing log k values
ADMET-13-2642-g005.jpg

Regarding all these observations, it should be summarized that for these 2-hydroxynaphthalene-1-carboxamides on the ring-multisubstituted anilide, standard commercially available lipophilicity prediction programs are unable to provide relevant data due to the high incidence of intra- and intermolecular interactions. On the other hand, the presence of an ionizable acidic phenolic group in the vicinity of the amide bond does not cause significant differences in the experimental values obtained for different mobile phase properties/compositions.

In vitro biological activities

The biological properties were assessed for in vitro antibacterial and antimycobacterial activity. In addition, all the compounds were also evaluated for their cytotoxicity against human monocytic leukemia THP-1 cells. The selection of the studied bacterial strains was adopted following the CLSI (National Committee for Clinical Laboratory Standards) international reference methodologies [63], i.e. standardization. For this purpose, universally sensitive collection strains from ATCC (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922) were selected. The second aspect of strain selection was the current state of occurrence of strains with an epidemiologically significant type of resistance, represented by clinical isolates of human and veterinary origin, i.e. different sequence types limited to human and animal populations, e.g. methicillin-resistant Staphylococcus aureus (MRSA) SA 3202, SA 630, 63718 isolates carrying the mecA gene [57]. In the case of vancomycin-resistant E. faecalis (VRE) 342B, 368, and 725B isolates carrying the vanA gene [59], these were isolates from wild birds colonized from US hospital wastewater, as confirmed. Therefore, it can be concluded that the tested strains differed in the spectrum of antibiotic resistance, genetic makeup, and probably accessory genome. Activities are expressed as minimum inhibitory concentrations (MICs), as shown inTable 2.

To obtain a comprehensive overview of the antibacterial properties of the investigated compounds, all derivatives were tested in vitro against Mycobacterium tuberculosis ATCC 25177/H37Ra, Mycobacterium kansasii DSM 44162 and Mycobacterium smegmatis ATCC 700084; activities are expressed as MICs as reported inTable 3. In order to reduce risks, a replacement of model pathogens is commonly used in basic laboratory screening. For M. tuberculosis, avirulent strain H37Ra is used, which has a similar pathology as M. tuberculosis strains infecting humans and, thus, represents a good model for testing antitubercular agents [64]. The genus Mycobacterium is a closely related group of fast- and slow-growing species. In addition to M. tuberculosis, there are a number of other so-called atypical (non-tuberculous) mycobacteria, important environmental pathogens, that cause a wide range of diseases (pulmonary diseases, lymphadenitis, skin and soft tissue diseases, gastrointestinal and skeletal infections), especially in immunocompromised patients [65-68]. These non-tuberculous strains include the fast-growing, e.g. M. smegmatis [69,70] and the slow-growing, e.g. M. kansasii [71,72].

Antimicrobial activities

Looking atTables 2 and3, it should be noted that the compounds had very limited activity. Of all the studied derivatives, only a total of 7 compounds (13, 18, 21-24 and 27) showed some activity, with 13 (R = 3,5-CH3), 22 (R = 2-CH3-5-CF3) and 27 (R = 3-CF3-4-NO2) being truly effective. Of this number, it is, of course, not possible to meaningfully discuss structure-activity relationships. On the other hand, it is important to note that despite the small number of active compounds, their efficacy, when they were active, was at the level of clinically used drugs.

Compounds 13, 22 and 27 were active against S. aureus/MRSA, two of which (22, 27) were also active against E. faecalis/VRE. Since the compounds were active against both the collection strains and resistant isolates, it is possible to speculate on a specific mechanism of action different from that of beta-lactam or quinolone antibiotics and the demonstrated resistance. The lower potency against E. faecalis/VRE compared to S. aureus/MRSA is likely due to the overall higher resistance of E. faecalis/VRE, including their ability to be facultative anaerobic bacteria [73-76]. It should be added that compound 22 was the only one that surprisingly showed activity against the Gram-negative collection strain E. coli. Several compounds also demonstrated activity against mycobacteria. Derivatives 22 and 27 were active against all three evaluated mycobacterial species. In addition, 23 (R = 3-CF3-4-CH3) showed activity against the fast-growing M. smegmatis and 24 (4-NO2) also against the slow-growing M. kansasii.

Considering the activities of previously published monosubstituted derivatives, 2-hydroxy-N-(2-nitrophenyl)naphthalene-1-carboxamide (24) was the most antimicrobially active (data seeTables 2 and3), followed by N-(4-bromophenyl)-2-hydroxynaphthalene-1-carboxamide and 2-hydroxy-N-(4-trifluoromethylphenyl)naphthalene-1-carboxamide against MRSA SA 630 and SA 3202 (MICs 47 and 94 μM, respectively) and M. kansasii (MICs 93 and 23 μM, respectively) [43]. Therefore, it can be stated that overall the previously described compounds had even more limited effects than these disubstituted derivatives.

Suppose these new observations are generalized from the point of view of the significance of substituents in the anilide part of the molecule. In that case, it is necessary to state that substitution with methoxy groups is completely disadvantageous for any antimicrobial activity. The situation changes slightly if the methoxy moiety is replaced by a methyl group; compare 5 (R = 2,5-OCH3) and 6 (R = 3,5-OCH3) with 11 (R = 2,5-CH3) and 13 (R = 3,5-CH3). Similar findings were published recently [52,54]. Disubstitution with 3,5-CH3 led to an increase in antistaphylococcal activity. The subsequent combination of methyl with a CF3 group in the meta position (compounds 23 and especially 22) resulted in a further significant increase and, above all, the extension of activity to E. faecalis/VRE, Gram-negative bacteria and mycobacteria (compare 20 (R = 2-OCH3-5-CF3 and 22 (R = 2-CH3-5-CF3)). The positive influence of the CF3 moiety on the potency and extension of antimicrobial activity was also observed in the nitrated disubstituted derivative 27 (compared with compound 26). These observations (the advantage of combining CH3 or NO2 with CF3) are completely new and have not been found in previously studied isomers [37,54].

The individual derivatives ordered by increasing electron σ(Ar) parameter are shown inFigure 4, where log k values are also given for comparison. The hatched bars in the graph indicate seven compounds (i.e. 13, 18, 21-24 and 27) demonstrating some activity. The first more significant individual effect was achieved at a log k value of 0.16 (compound 21, R = 3-CF3-4-OCH3). On the other hand, the activity disappeared at log k values of 0.46 (compounds 23 (R = 3-CF3-4-CH3), 18 (R = 2-Cl-5-OCH3)). The highest/widest activity was achieved with a log k value higher than 0.31 (27, R = 3-CF3-4-NO2) and lower than 0.34 (22, R = 2-CH3-5-CF3). So, it is evident that lipophilicity plays a secondary role.

Figure 4. Order of individual derivatives arranged according to increasing electron σ(Ar) parameter compared to log k values. Compounds with some effect are hatched in blue-red
ADMET-13-2642-g006.jpg

On the other hand, the potency and wide antimicrobial activity are much more influenced by the electron σ(Ar) parameter; it is advantageous if it is higher than 0.58 (derivative 21 (R = 3-CF3-4-OCH3), seeFigure 4). In accordance with the Michael acceptor theory, the higher the substituent causes an electron deficit, the better. Therefore, it seems that this is a substituent-dependent activity (dependent on the type and position of the substituents), where the electron-deficient state of the molecule (characterized by the magnitude of the parameter σ(Ar)) plays a significant role, similarly as described, e.g. in [29,43,77-80]. Thus, it can be said that the investigated effective compounds meet the definition of so-called Michael acceptors, where in addition to suitable lipophilicity for penetration through the bacterial wall, the overall electron deficit in the molecule is key for efficacy and binding to targets (bacterial biomolecules) appears to occur primarily through polar groups and a planar π-π system.

Cytotoxicity

Preliminary in vitro cytotoxicity screening of selected active compounds was performed using the THP-1 cell line. Cytotoxicity was expressed as LC50 value (lethal concentration for 50 % of the cell population), seeTable 3. Treatment with 30 μM of the new compounds did not result in a significant lethal effect on THP-1 cells (e.g. LC50 values of oxaliplatin and camptothecin were 1.7±0.64 and 0.16±0.07 μM). Among nine previously synthesized monosubstituted 2-hydroxynaphthalene-1-carboxanilides, the cytotoxicity of four of them was previously examined. A significant lethal effect was detected only in the case of compound 26 (LC50 = 2.5 μM), while compounds 1, 24, and 25 did not show significant cytotoxicity (LC50 > 20 μM) [43]. Based on these observations, it can be concluded that tested substances except 26 can be considered non-toxic substances for the subsequent design of new antimicrobial agents.

Computational ADME Properties

In the early stage of the drug discovery process, especially orally administered drugs, it is extremely important to perform at least indicative ADME profiling to provide critical information about the basic behavior of a potential drug in the body. Basic information obtained from such studies helps guide further structural optimizations to obtain molecules with more favorable pharmacokinetic parameters while maintaining the existing efficiency potential. A variety of software is advantageously used for basic ADME screening, and ADME profiling is subsequently verified for selected drug candidates in preclinical and clinical studies [81-83]. The Lipinski Rule of Five (Ro5) is one of the most accepted recommendations concerning the physicochemical parameters of biologically active compounds, and all medicinal chemists try to follow it when designing molecules [84]. Ro5 contains the limits of specific molecular descriptors (MW <500, log P <5, HBD <5, HBA <10) set based on experimentally and statistically obtained results so that a compound that meets this recommendation has a higher chance of becoming a drug. However, a good drug-like score does not make a molecule a drug, and vice versa [85,86]. In addition, information has been added about compounds related to the Veber rule, which states that a compound with 10 or fewer rotational bonds (RB) and a polar surface area (TPSA) of no higher than 1.4 nm2 (140 Å2) should have good oral bioavailability [81,87]. It is clear that ADMET-friendly properties, such as lipophilicity, polar surface area, etc, are important in the context of specific ligand-receptor interactions.

The followingTable 4 shows the predicted ADME-influencing properties of the most effective compounds (13, 22, 27). The compounds are rather rigid and expected to be nearly planar [54]. All contain a free acidic phenolic group, which is crucial for biological activity [40,41,43], meaning that these acidic compounds in plasma bind predominantly to human serum albumin [88]. In addition to Ro5 parameters, other parameters, such as intestinal absorption and permeation into the brain (the effectiveness of antibiotics in the brain is important), are listed inTable 4. All the parameters were predicted using the commercially available program ACD/Percepta [55].

Table 4. Values of parameters characterizing physicochemical properties of discussed effective 2-hydroxynaphthalene-1-carboxanilides predicted using ACD/Percepta [55]
Comp.MWlog PHBDHBARBTPSA, nm2Parachor, cm3ka / min-1log BBlog PSBrain plasma equilibrim rate
13291.344.992320.49641.030.0550.54-1.1-3.2 (brain penetration sufficient for CNS activity)
22345.325.712330.49660.590.055-0.11-1.2-3.5 (probably CNS inactive due to low brain penetration)
27376.295.472640.98678.430.055-0.241.3-3.5 (probably CNS inactive due to low brain penetration)

All the discussed agents have molecular weights (MW) significantly <500. On the other hand, the compounds have rather higher lipophilicity (log P ≥5). All the compounds meet the criteria for the number of H-bond donors (HBD) and acceptors (HBA). The number of rotatable bonds (RB) is in the narrow range of 2 to 4. The topological polar surface area (TPSA) has been recognized as a good indicator of intestinal drug absorption (TPSA <1.2-1.4 nm2 (<120-140 Å2)) and blood-brain barrier (BBB) penetration (TPSA <0.6 nm2 (<60 Å2)) [87,89,90]. The TPSA value of 0.49 nm2 (49 Å2) indicates that compounds 13 and 22 should have good intestinal absorption as well as adequate BBB penetration. On the other hand, the TPSA = 0. 98 nm2 (98 Å2) for compound 27 suggests only good intestinal absorption. The predicted value of the absorption rate in the jejunum (ka = 0.055 min-1) is the same for all derivatives. A remarkable prediction was made by ACD/Percepta [55] for blood-brain barrier (BBB) permeation. In general, log BB ≥ 0.3 is for BBB permeable drugs and log BB ≤ -0.3 is for impermeable drugs [91]. The log BB values of -0.11 (22) and -0.24 (27), respectively, indicate that both compounds are probably CNS inactive due to low brain penetration (as already suggested by the TPSA value for 27). Conversely, the log BB = 0.54 for 13 indicates good BBB penetration. More informative are the values of the permeability surface-area product (expressed as log PS) [92] in the range of -1.2 to 1.3. The brain plasma equilibrium rate predicted by ACD/Percepta [55] for compound 13 is -3.2, suggesting that this compound may achieve sufficient brain penetration for CNS activity.

In summary, after preliminary in silico ADME screening using commercially available software, it can be assumed that investigated compounds 13, 22, and 27 should have suitable physicochemical parameters for adequate bioavailability in the body. Unfortunately, for the most effective of the compounds 37, probably due to the presence of the NO2 group, BBB penetration was not predicted. Of course, much more accurate results could be obtained using other experiments [93].

Conclusions

A series of nine previously synthesized monosubstituted 2-hydroxynaphthalene-1-carboxanilides was enriched with seventeen new di- and trisubstituted 2-hydroxynaphthalene-1-carboxanilides and all the compounds were tested for their in vitro antibacterial and antimycobacterial activity. Only five compounds showed antimicrobial activity, and three of them were comparable to drugs that were clinically used. N-(3,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (13) was active only against S. aureus and MRSA isolates, 2-hydroxy-N-[2-methyl-5-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (22) and 2-hydroxy-N-[4-nitro-3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide (27) were active across the entire spectrum of tested bacteria/mycobacteria, both against susceptible and resistant isolates. Compound 22 was even active against Gram-negative E. coli. These active agents showed no in vitro cytotoxicity against THP-1 cells up to a concentration of 30 μM. Based on preliminary in silico ADME screening using commercially available software, it can be assumed that investigated compounds 13, 22 and 27 should have suitable physicochemical parameters for adequate bioavailability in the body. From the observations, it can be stated that the two most active compounds 22 and 27 are substituted with a CF3 moiety in the meta position of the anilide, in addition to the methyl or nitro group. The CF3 moiety thus proved to be a necessary prerequisite for antimicrobial activity in structures of this type. It was indirectly verified that the biological effects of this scaffold are based on the Michael acceptor theory. The CF3 moiety, primarily due to its electron-withdrawing properties, meets the definition of Michael acceptors, where the overall electron deficit in the molecule (caused by appropriate substitution) appears to be essential for the expected binding to targets in the bacterial cell, resulting in antimicrobial activity. Given the overall structure of the investigated compounds, multiple mechanisms of action can be assumed; efforts to discover them will subsequently be carried out using proteomic and molecular biological experiments.

Supplementary material

1H and 13C NMR spectra of the new discussed compounds are available from the corresponding author on request, or athttps://pub.iapchem.org/ojs/index.php/admet/article/view/2642.

Acknowledgements

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic within the CzeCOS program (Grant No. LM2023048). This work was also supported by the Slovak Research and Development Agency (project APVV-22-0133).

Notes

[2] Conflicts of interest Conflict of interest: The authors declare no conflicts of interest.

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