4-aminobenzoic acid derivatives: Converting folate precursor to antimicrobial and cytotoxic agents

biomolecules

Article

4-Aminobenzoic Acid Derivatives: Converting Folate

Precursor to Antimicrobial and Cytotoxic Agents

Martin Krátký ^1,

*

, Klára Konecˇná ², Jirˇí Janoušek ³, Michaela Brablíková ⁴,

Ondrˇej Jand’ourek ², František Trejtnar ³, Jirˇina Stolarˇíková ⁵and Jarmila Vinšová ¹

1

Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles

University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; vinsova@faf.cuni.cz

Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University,

2

Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; konecna@faf.cuni.cz (K.K.);

jando6aa@faf.cuni.cz (O.J.)

Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University,

Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; janousj2@faf.cuni.cz (J.J.);

trejtnarf@faf.cuni.cz (F.T.)

Unipetrol Centre of Research and Education, 436 70 Litvínov-Záluží 1, Czech Republic;

brablikova.michaela@gmail.com

Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava,

Partyzánské náme˘stí 7, 702 00 Ostrava, Czech Republic; Jirina.Stolarikova@zuova.cz

Correspondence: martin.kratky@faf.cuni.cz; Tel.: +420-495-067-343; Fax: +420-495-067-166

3

4

5

*

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Received: 5 November 2019; Accepted: 17 December 2019; Published: 19 December 2019

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: 4-aminobenzoic acid (PABA), an essential nutrient for many human pathogens, but

dispensable for humans, and its derivatives have exhibited various biological activities. In this study,

we combined two pharmacophores using a molecular hybridization approach: this vitamin-like

molecule and various aromatic aldehydes, including salicylaldehydes and 5-nitrofurfural, via imine

bond in one-step reaction. Resulting Schiﬀ bases were screened as potential antimicrobial and

cytotoxic agents. The simple chemical modiﬁcation of non-toxic PABA resulted in constitution of

antibacterial activity including inhibition of methicillin-resistant Staphylococcus aureus (minimum

inhibitory concentrations, MIC, from 15.62

and potent broad-spectrum antifungal properties (MIC of

exhibited notable cytotoxicity for cancer HepG2 cell line (IC₅₀

for the derivatization of PABA, it is possible to tune up the particular activities and obtain derivatives

with promising bioactivities.

µ

M), moderate antimycobacterial activity (MIC

7.81 M). Some of the Schiﬀ bases also

15.0 M). Regarding aldehyde used

≥

62.5 µM)

≥

µ

≥

µ

Keywords: 4-aminobenzoic acid; antibacterial activity; antifungal activity; cytotoxicity; Schiﬀ bases;

synthesis; vitamin

1. Introduction

Para-aminobenzoic acid (4-aminobenzoic acid, p-aminobenzoic acid; PABA; 1) is a well-known

amino acid compound in biochemistry, medicinal chemistry and with many industrial applications.

PABA is synthesized and utilized as a substrate for the synthesis of folic acid in many bacterial

species, yeasts and plants, i.e., playing a crucial role in this metabolic pathway. In addition to folate

metabolism, PABA was identiﬁed as a coenzyme Q precursor and one of the factors required for

microbial virulence. From traditional point of view, this compound belongs to the vitamins B group

(vitamin B_xor B₁₀). However, PABA is neither essential nor biosynthesized in mammals. It is produced

by symbiotic bacteria, especially Escherichia coli and absorbed from dietary intake [1–5].

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In contrast to its essentiality for some bacteria, when combined with several antibiotics, PABA

applies a synergistic antibacterial potency for diﬀerent bacterial strain including Staphylococcus

aureus or Pseudomonas aeruginosa. PABA alone showed a direct antibacterial activity against Listeria

monocytogenes, Salmonella enteritidis and E. coli. Low pH values potentiate this action. Analogous

conclusions about adjunctive/synergistic activity were obtained for antivirotics [1–3,6]. It has also

exhibited multiple antiviral and antioxidant actions. PABA and its derivatives have been utilized

as active constituents in sunscreens to protect against UVB irradiation, i.e., scavengers of reactive

oxygen species [

anticoagulants [ ]. PABA-based diagnostic tests of gastrointestinal tract state have been developed [

The potassium 4-aminobenzoate is used for the treatment of mainly dermatologic disorders such

as scleroderma, dermatomyositis and Peyronie’s disease [ ]. In agriculture, PABA has displayed

1

–

3]. PABA demonstrated also properties of cytoprotective agents [

7] and direct

2

4,8

].

3

the promotion of resistance against plant bacterial and viral pathogens and plant growth modulator

properties [4].

Nevertheless, absorption of PABA in the gastrointestinal tract represents an unfavourable

feature [3]. The acid is absorbed through a passive process and it undergoes phase II biotransformation.

PABA can be metabolized by either N-acetylation or in conjugation with glycine in the liver to produce

4-aminohippuric acid. Both these metabolic processes can take place to synthesize 4-acetamidohippuric

acid. Then, PABA and its metabolites are almost completely excreted in the urine. PABA is

generally considered non-toxic and well-tolerated. Side eﬀects of systemic exposure are predominantly

non-speciﬁc (nausea and skin rash), although hepatotoxicity may occur [8,9].

As mentioned above, the organisms biosynthesizing PABA obtain it from chorismate by a reaction

catalysed by 4-amino-4-deoxychorismate synthase. This enzyme replaces the hydroxyl group at

C4 by an amino group from chorismate, and it removes the pyruvic moiety subsequently to form

4-aminobenzoate [10]. The structure of PABA is highly speciﬁc and minimal structural alterations

lead to inhibition of enzymes utilizing PABA or involved in subsequent metabolic steps. Illustratively,

sulfonamides used for the treatment of many bacterial and protozoal infections act as structural

analogues of PABA inhibiting dihydropteroate synthase competitively [11]. A natural metabolite of

PABA, 4-acetamidobenzoic acid (acedoben, Figure 1 , I), is also able to interfere with the function of

dihydropteroate synthase [12]. This molecule is one of the components of immunomodulating and

antiviral drug inosine pranobex (Isoprinosine; Figure 1, II) [13]. Antitubercular drug 4-aminosalicylic

acid (Figure 1 , III) is utilized by dihydropteroate synthase instead of PABA and incorporated into the

folate pathway to generate a hydroxyl dihydrofolate antimetabolite, which interferes with dihydrofolate

reductase activity leading to the disruption of the biosynthesis of purines and thymidylate [11,14].

The 4-aminoaryl group is required for the biological functions and essentiality of PABA. Its chemical

modiﬁcation prevents its vitamin action and utilization.

In medicinal chemistry, a PABA-building block is used in design of drugs frequently and this

scaﬀold can be found in many drugs with various therapeutic eﬀects (Figure 1). We can distinguish

“simple” PABA-derived drugs substituted at carboxyl and/or amino group basically, or more complex

molecules with an additional substitution of benzene ring. Focusing on the ﬁrst group mentioned,

the carboxyl can be free, in salt form, or modiﬁed through ester or amide bond formation. The amino

group is usually free, mono- and di-alkylated or incorporated into amide. For example, PABA-based

drugs summarized in Figure 1 involve anticancer and cytotoxic folate antagonists (e.g., aminopterin

IV, methotrexate

V), alkylating agents, local anaesthetics (benzocaine VI, tetracaine VII, procaine

VIII, etc.), antiarrhythmics (procainamide IX), sunscreens (Padimate-O

(balsalazide XI) [12].

X) or anti-inﬂammatory drugs

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Figure 1. Examples of drugs containing the 4-aminobenzoic acid motif.

PABA hybrid compounds have been reported as potential antimicrobial agents. PABA has been

linked with other bioactive scaﬀolds involving NH₂group via amide bond [15], as a secondary amine [

or ureido moiety [16]. The derivatives of PABA showed improved radical scavenging properties and

also enhanced toxicity for cancer cells [ ]. PABA-ambazone salt exhibited improved antibacterial eﬀect

1]

5

when compared to parent drugs [17]. Schiﬀ bases with isatin (Figure 2, XII) exhibited activity against

various cancer cell lines [18]. Thus, the chemical modiﬁcation of PABA provides a viable concept for

modulation of its properties to obtain both antimicrobial and cytotoxic compounds.

Figure 2.

Examples of bioactive PABA (blue) and salicylaldehyde-derived (green) imines

and hydrazones.

Our present approach is focused on the synthesis of Schiﬀ bases of PABA and aldehydes, mainly

salicylaldehydes, and their evaluation as potential antimicrobial and cytotoxic agents. These fused

compounds combine two pharmacophores through the imine bond. Previously, we have applied

molecular hybridization approach successfully. Illustratively, salicylaldehyde-based imines and

hydrazones derived from known bioactive scaﬀolds (Figure 2, XIII

antimicrobial activity [19 21]. We have described that a potent antimicrobial activity is associated

only with the salicylidene moiety and its isomers are substantially less active [21 22]. That is why we

–XIV) have exhibited a signiﬁcant

–

,

designed predominantly 2-substituted benzylidene scaﬀold for this study. The modiﬁcation of the

amino group can result in altered vitamin function in susceptible strains and novel derivatives with an

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enhanced lipophilicity may promote limited absorption and intracellular accumulation of PABA via

passive diﬀusion.

2. Materials and Methods

2.1. Chemistry

2.1.1. General

All the reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) or Penta

Chemicals (Prague, Czech Republic) and they were used as received. The reactions and the purity

of the products were monitored by thin-layer chromatography (TLC) using a mixture with a ratio

of dichloromethane (DCM) to methanol (MeOH) of 4:1 (v/v) as the eluent. TLC plates were coated

with 0.2 mm Merck 60 F254 silica gel (Merck Millipore, Darmstadt, Germany) with UV detection

(254 nm). The melting points were determined on a Büchi Melting Point B-540 apparatus (BÜCHI,

Flawil, Switzerland) using open capillaries. The reported values are uncorrected. Infrared spectra were

recorded on a FT-IR spectrometer using ATR-Ge method (Nicolet 6700 FT-IR, Thermo Fisher Scientiﬁc,

Waltham, MA, USA) in the range of 650–4000 cm⁻¹. The NMR spectra were measured in DMSO-d₆at

ambient temperature using a Varian V NMR S500 instrument (500 MHz for ¹H and 126 MHz for ¹³C;

Varian Comp. Palo Alto, CA, USA). The chemical shifts δ are given in ppm and were referred indirectly

1

to tetramethylsilane via signals of DMSO-d₆(2.49 for H and 39.7 for ¹³C spectra). The coupling

constants (J) are reported in Hz. Elemental analysis was performed on Vario MICRO Cube Element

Analyzer (Elementar Analysensysteme, Hanau, Germany). Both calculated and found values are given

as percentages.

2.1.2. Synthesis of PABA-Derived Schiﬀ Bases 1a–1t

4-aminobenzoic acid (137.1 mg; 1 mmol) was dissolved in 7 mL of MeOH and then appropriate

aldehyde (1.1 mmol) was added in one portion. The reaction mixture was heated under reﬂux for 3 h,

then let stir at room temperature for 12 h. After this time, the reaction mixture was stored at

20 ^◦C

−

for 1 h. The resulted precipitate was ﬁltered oﬀ and washed by cold diethyl ether (DEE) thoroughly.

The products were crystallized from MeOH to obtain pure Schiﬀ bases if necessary. Alternatively,

Schiﬀ bases can be prepared using DEE as a solvent instead of MeOH. In this case, the reaction mixture

was stirred for 72 h at room temperature. The practical yields of both methods were comparable.

Toluene-4-sulfonic acid (as monohydrate; 19.0 mg; 0.1 of equivalents) was applied as an acidic

catalyst in the synthesis of two products (1f, 1t), when the uncatalyzed reaction provided low or almost

no yields.

The identity of the known compounds was established using NMR and IR spectroscopy by the

comparison with previously reported data. Additionally, their purity was checked by melting points

measurement and elemental analysis. The compounds were considered pure if they agree within

0.4% with theoretical values.

±

4-[(2-Hydroxybenzylidene)amino]benzoic acid (1a). Yellow solid; yield 92%; mp 265.5–268 ^◦C (lit. [23

]

1

276–278 ^◦C). IR: 1690 (COOH), 1608 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.95 (1H,

s, COOH), 12.71 (1H, s, OH), 8.99 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.69 (1H, dd, J = 7.6

Hz, 1.8 Hz, H6⁰), 7.51–7.42 (3H, m, H3, H5, H4⁰), 7.02–6.96 (2H, m, H3⁰, H5⁰). ¹³C-NMR (126 MHz,

DMSO-d₆):

δ 167.06, 164.99, 160.49, 152.34, 134.02, 132.80, 130.92, 128.11, 121.71, 121.22, 119.50, 115.88.

Anal. Calcd. for C₁₄H₁₁NO₃(241.25): C, 69.70; H, 4.60; N, 5.81. Found: C, 69.79; H, 4.74; N, 5.70.

4-[(5-Fluoro-2-hydroxybenzylidene)amino]benzoic acid (1b). Orange solid; yield 90%; mp 277–278.5

1

^◦C. IR: 1678 (COOH), 1570 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.92 (1H, s, COOH),

12.28 (1H, s, OH), 8.94 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.54 (1H, dd, J = 8.9 Hz, 3.2 Hz, H6⁰),

7.48–7.43 (2H, m, H3, H5), 7.30 (ddd, J = 9.1 Hz, 8.3 Hz, 3.2 Hz, H4⁰), 7.00 (1H, dd, J = 9.1 Hz, 4.5 Hz,

H3⁰). ¹³C-NMR (126 MHz, DMSO-d₆): δ 167.05, 163.30 (d, J = 2.7 Hz), 156.64, 155.20 (d, J = 234.9 Hz),

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152.46, 130.94, 129.15, 121.70, 120.97 (d, J = 23.5 Hz), 119.94 (d, J = 7.5 Hz), 118.34 (d, J = 7.6 Hz), 116.90

(d, J = 23.4 Hz). Anal. Calcd. for C₁₄H₁₀FNO₃(259.24): C, 64.87; H, 3.89; N, 5.40. Found: C, 64.69; H,

3.71; N, 5.64.

4-[(5-Chloro-2-hydroxybenzylidene)amino]benzoic acid (1c). Yellow solid; yield 91%; mp 302.5–304

1

^◦C (lit. [24] 230–232 ^◦C). IR: 1681 (COOH), 1599 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ

12.97 (1H, s, COOH), 12.60 (1H, s, OH), 8.95 (1H, s, CH=N), 8.04–7.99 (2H, m, H2, H6), 7.78 (1H, d,

J = 2.7 Hz, H6⁰), 7.48–7.43 (3H, m, H3, H5, H4⁰), 7.01 (1H, d, J = 8.8 Hz, H3⁰). ¹³C-NMR (126 MHz,

DMSO-d₆):

δ 167.02, 163.24, 159.05, 152.27, 133.42, 131.01, 130.93, 129.21, 122.93, 121.73, 120.89, 118.93.

Anal. Calcd. for C₁₄H₁₀ClNO₃(275.69): C, 60.99; H, 3.66; N, 5.08. Found: C, 61.11; H, 3.72; N, 5.16.

4-[(5-Bromo-2-hydroxybenzylidene)amino]benzoic acid (1d). Orange solid; yield 87%; mp 281.5–284

1

^◦C (lit. [25] 282 ^◦C). IR: 1681 (COOH), 1596 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.92

(1H, s, COOH), 12.62 (1H, s, OH), 8.94 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.90 (1H, d, J = 2.6 Hz

H6⁰), 7.57 (1H, dd, J = 8.8 Hz, 2.6 Hz, H4⁰), 7.48–7.44 (2H, m, H3, H5), 6.96 (1H, d, J = 8.8 Hz, H3⁰).

¹³C-NMR (126 MHz, DMSO-d₆):

167.04, 163.18, 159.47, 152.27, 136.20, 134.00, 130.95, 129.22, 121.75,

,

δ

121.52, 119.36, 110.31. Anal. Calcd. for C₁₄H₁₀BrNO₃(320.14): C, 52.52; H, 3.15; N, 4.38. Found: C,

52.25; H, 3.04; N, 4.44.

4-[(2-Hydroxy-5-iodobenzylidene)amino]benzoic acid (1e). Orange solid; yield 96%; mp 302–304 ^◦C.

1

IR: 1680 (COOH), 1593 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.96 (1H, s, COOH), 12.64

(1H, s, OH), 8.92 (1H, s, CH=N), 8.04 (1H, d, J = 2.3 Hz, H6⁰), 8.03–7.99 (2H, m, H2, H6), 7.70 (1H, dd, J

= 8.7 Hz, 2.3 Hz, H4⁰), 7.48–7.44 (2H, m, H3, H5), 6.84 (1H, d, J = 8.7 Hz, H3⁰). ¹³C-NMR (126 MHz,

DMSO-d₆):

δ 167.02, 163.20, 160.00, 152.26, 141.81, 140.04, 130.93, 129.16, 122.12, 121.74, 119.64, 81.08.

Anal. Calcd. for C₁₄H₁₀INO₃(367.14): C, 45.80; H, 2.75; N, 3.82. Found: C, 46.02; H, 3.00; N, 3.55.

4-[(2-Hydroxy-5-nitrobenzylidene)amino]benzoic acid (1f). Yellowish orange solid; yield 81%; mp

1

311–313 ^◦C (decomp.). IR: 1693 (COOH), 1597 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ

13.78 (1H, s, COOH), 12.87 (1H, s, OH), 9.14 (1H, s, CH=N), 8.69 (1H, d, J = 2.9 Hz, H6⁰), 8.28 (1H, dd,

J = 9.2 Hz, 2.9 Hz, H4⁰), 8.05–8.01 (2H, m, H2, H6), 7.54–7.50 (3H, m, H3, H5), 7.14 (1H, d, J = 9.2 Hz,

H3⁰). ¹³C-NMR (126 MHz, DMSO-d₆):

δ 166.95, 166.31, 162.66, 151.24, 139.63, 130.95, 129.56, 128.90,

128.16, 121.74, 119.21, 118.36. Anal. Calcd. for C₁₄H₁₀N₂O₅(286.24): C, 58.75; H, 3.52; N, 9.79. Found:

C, 58.86; H, 3.33; N, 10.02.

4-[(2-Hydroxy-5-methylbenzylidene)amino]benzoic acid (1g). Yellow solid; yield 92%; mp 271–273

1

^◦C (lit. [26] 258 ^◦C). IR: 1681 (COOH), 1574 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.94

(1H, s, COOH), 12.41 (1H, s, OH), 8.91 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.50–7.43 (3H, m,

H3, H5, H6⁰), 7.25 (1H, dd, J = 8.4 Hz, 2.3 Hz, H4⁰), 6.88 (1H, d, J = 8.3 Hz, H3⁰), 2.27 (3H, s, CH₃).

¹³C-NMR (126 MHz, DMSO-d₆):

δ 167.05, 164.74, 158.33, 152.52, 134.78, 132.42, 130.90, 128.87, 128.04,

121.63, 119.18, 116.72, 20.09. Anal. Calcd. for C₁₅H₁₃NO₃(255.27): C, 70.58; H, 5.13; N, 5.49. Found: C,

70.81; H, 5.01; N, 5.57.

4-[(2-Hydroxy-5-methoxybenzylidene)amino]benzoic acid (1h) [27]. Brownish yellow solid; yield 92%;

1

mp 247.5–249.5 ^◦C (decomp.). IR: 1677 (COOH), 1569 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ

12.93 (1H, s, COOH), 12.04 (1H, s, OH), 8.94 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.46–7.43 (2H,

m, H3, H5), 7.28 (1H, d, J = 3.2 Hz, H6⁰), 7.06 (1H, dd, J = 9.0 Hz, 3.2 Hz, H4⁰), 6.92 (1H, d, J = 8.9 Hz

H3⁰), 3.75 (3H, s, OCH₃). ¹³C-NMR (126 MHz, DMSO-d₆):

167.06, 164.31, 154.58, 152.70, 152.15,

,

δ

130.92, 128.89, 121.63, 121.36, 119.44, 117.81, 115.01, 55.73. Anal. Calcd. for C₁₅H₁₃NO₄(271.27): C,

66.41; H, 4.83; N, 5.16. Found: C, 66.35; H, 4.69; N, 5.01.

4-[(2,5-Dihydroxybenzylidene)amino]benzoic acid (1i) [28]. Orange solid; yield 81%; mp 278–280

1

^◦C (decomp.). IR: 1679 (COOH), 1574 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.89 (1H,

s, COOH), 11.87 (1H, s, 2-OH), 9.13 (1H, s, 5-OH), 8.87 (1H, s, CH=N), 8.02–7.98 (2H, m, H2, H6),

7.46–7.43 (2H, m, H3, H5), 7.08 (1H, d, J = 3.0 Hz, H6⁰), 6.89 (1H, dd, J = 8.8 Hz, 3.0 Hz, H4⁰), 6.81 (1H,

d, J = 8.8 Hz, H3⁰). ¹³C-NMR (126 MHz, DMSO-d₆):

δ 167.08, 164.50, 153.35, 152.78, 149.92, 130.88,

128.78, 121.95, 121.66, 119.54, 117.52, 116.89. Anal. Calcd. for C₁₄H₁₁NO₄(257.07): C, 65.37; H, 4.31; N,

5.45. Found: C, 65.55; H, 4.40; N, 5.62.

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4-{[5-(Tert-butyl)-2-hydroxybenzylidene]amino}benzoic acid (1j). Orange solid; yield 85%; mp 270–272

1

^◦C. IR: 1682 (COOH), 1569 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.94 (1H, s, COOH),

12.51 (1H, s, OH), 8.99 (1H, s, CH=N), 8.03–7.99 (2H, m, H2, H6), 7.70 (1H, d, J = 2.6 Hz, H6⁰), 7.50–7.46

(3H, m, H3, H5, H4⁰), 6.92 (1H, d, J = 8.7 Hz, H3⁰), 1.28 (9H, s, CH₃). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

167.08, 165.40, 158.35, 152.52, 141.66, 131.38, 130.92, 129.04, 128.86, 121.68, 118.75, 116.51, 34.01, 31.35.

Anal. Calcd. for C₁₈H₁₉NO₃(297.35): C, 72.71; H, 6.44; N, 4.71. Found: C, 72.57; H, 6.58; N, 4.50.

4-[(2-Chloro-6-hydroxybenzylidene)amino]benzoic acid (1k). Orange solid; yield 94%; mp 279–281

1

^◦C. IR: 1681 (COOH), 1596 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 14.02 (1H, s, COOH),

13.03 (1H, s, OH), 9.17 (1H, s, CH=N), 8.05–8.01 (2H, m, H2, H6), 7.57–7.53 (2H, m, H3, H5), 7.45 (1H, t,

J = 8.2 Hz, H4⁰), 7.07 (1H, dd, J = 7.9 Hz, 1.1 Hz, H3⁰), 6.98 (1H, dd, J = 8.4 Hz, 1.0 Hz, H5⁰). ¹³C-NMR

(126 MHz, DMSO-d₆):

δ 166.95, 162.79, 161.81, 151.04, 135.83, 134.98, 130.99, 129.65, 121.93, 120.46,

116.83, 115.66. Anal. Calcd. for C₁₄H₁₀ClNO₃(275.69): C, 60.99; H, 3.66; N, 5.08. Found: C, 60.84; H,

3.74; N, 5.01.

4-[(4-Chloro-2-hydroxybenzylidene)amino]benzoic acid (1l). Yellowish orange solid; yield 90%; mp

1

304–307 ^◦C (decomp.). IR: 1674 (COOH), 1596 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 13.05

(1H, s, COOH), 12.72 (1H, s, OH), 8.99 (1H, s, CH=N), 8.04–7.99 (2H, m, H2, H6), 7.72 (1H, d, J = 8.1 Hz

,

H6⁰), 7.48–7.43 (2H, m, H3, H5), 7.07–7.03 (2H, m, H3⁰, H5⁰). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

167.01,

163.73, 161.30, 152.01, 138.18, 133.89, 130.90, 129.13, 121.70, 119.79, 118.64, 116.76. Anal. Calcd. for

C₁₄H₁₀ClNO₃(275.69): C, 60.99; H, 3.66; N, 5.08. Found: C, 60.77; H, 3.65; N, 5.20.

4-[(3-Chloro-2-hydroxybenzylidene)amino]benzoic acid (1m). Yellow solid; yield 85%; mp 296–298 ^◦C.

1

IR: 1689 (COOH), 1596 (-CH=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 13.99 (1H, s, COOH), 13.01

(1H, s, OH), 9.08 (1H, s, CH=N), 8.05–8.01 (2H, m, H2, H6), 7.65 (1H, dd, J = 7.8 Hz, 1.6 Hz, H6⁰), 7.61

(1H, dd, J = 7.9 Hz, 1.5 Hz, H4⁰), 7.57–7.54 (2H, m, H3, H5), 7.01 (1H, t, J = 7.8 Hz, H5⁰). ¹³C-NMR (126

MHz, DMSO-d₆):

δ 166.95, 165.17, 156.69, 150.75, 133.77, 132.22, 130.92, 129.52, 121.87, 120.56, 120.19,

119.85. Anal. Calcd. for C₁₄H₁₀ClNO₃(275.69): C, 60.99; H, 3.66; N, 5.08. Found: C, 61.09; H, 3.79; N,

5.30.

4-[(3,5-Dichloro-2-hydroxybenzylidene)amino]benzoic acid (1n). Orange solid; yield 89%; mp 317.5–319

1

^◦C (lit. [29] 300 ^◦C). IR: 1689 (COOH), 1597 (-CH=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 14.02

(1H, s, COOH), 13.03 (1H, s, OH), 9.04 (1H, s, CH=N), 8.05-8.01 (2H, m, H2, H6), 7.75–7.73 (2H, m,

H4⁰, H6⁰), 7.56–7.53 (2H, m, H3, H5). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

166.90, 164.07, 155.94, 150.30,

132.83, 131.06, 130.96, 129.83, 122.47, 121.93, 121.88, 120.66. Anal. Calcd. for C₁₄H₉Cl₂NO₃(309.00): C,

54.22; H, 2.93; N, 4.52. Found: C, 54.10; H, 3.11; N, 4.36.

4-[(3-Bromo-5-chloro-2-hydroxybenzylidene)amino]benzoic acid (1o). Orangish solid; yield 92%; mp >

1

300 ^◦C (decomp.). IR: 1685 (COOH), 1595 (-CH=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 14.05 (1H,

s, COOH), 13.03 (1H, s, OH), 9.02 (1H, s, CH=N), 8.05–8.02 (2H, m, H2, H6), 7.86 (1H, d, J = 2.5 Hz

,

H4⁰), 7.77 (1H, d, J = 2.6 Hz, H6⁰), 7.57–7.53 (2H, m, H3, H5). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

166.88,

164.08, 156.90, 150.16, 135.60, 131.76, 130.95, 129.82, 122.84, 121.90, 120.38, 111.36. Anal. Calcd. for

C₁₄H₉BrClNO₃(352.95): C, 47.42; H, 2.56; N, 3.95. Found: C, 47.55; H, 2.70; N, 4.13.

4-[(5-Chloro-2-hydroxy-3-iodobenzylidene)amino]benzoic acid (1p). Reddish orange solid; yield 84%;

1

mp 290 ^◦C (decomp). IR: 1671 (COOH), 1589 (-CH=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 14.04

(1H, s, COOH), 13.03 (1H, s, OH), 8.96 (1H, s, CH=N), 8.05–8.02 (2H, m, H2, H6), 7.97 (1H, d, J = 2.6

Hz, H4⁰), 7.78 (1H, d, J = 2.6 Hz, H6⁰), 7.58–7.53 (2H, m, H3, H5). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

166.90, 164.09, 159.36, 150.17, 141.24, 132.50, 130.96, 129.77, 123.32, 121.94, 119.23, 87.53. Anal. Calcd.

for C₁₄H₉ClINO₃(400.93): C, 41.87; H, 2.26; N, 3.49. Found: C, 42.11; H, 2.39; N, 3.31.

4-[(2-Hydroxy-3,5-diiodobenzylidene)amino]benzoic acid (1q). Red solid; yield 95%; mp 289–291 ^◦C

1

(decomp.). IR: 1667 (COOH), 1583 (-CH=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 14.08 (1H, s,

COOH), 13.02 (1H, s, OH), 8.94 (1H, s, CH=N), 8.16 (1H, d, J = 2.1 Hz, H4⁰), 8.05–8.00 (3H, m, H2,

H6, H6⁰), 7.57–7.53 (2H, m, H3, H5). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

166.90, 163.95, 160.22, 150.14,

148.93, 141.47, 130.95, 129.72, 121.91, 120.77, 88.51, 81.51. Anal. Calcd. for C₁₄H₉I₂NO₃(492.87): C,

34.11; H, 1.84; N, 2.84. Found: C, 33.87; H, 2.00; N, 2.87.

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4-{[(5-Nitrofuran-2-yl)methylene]amino}benzoic acid (1r). Yellow solid; yield 80%; mp 233–235 ^◦C

1

(decomp.) (lit. [30] 234 ^◦C decomp.). IR: 1673 (COOH), 1591 (-CH=N-) cm⁻¹. H-NMR (500 MHz,

DMSO-d₆):

Hz, furan H4⁰), 7.48 (1H, d, J = 3.9 Hz, furan H3⁰), 7.43–7.39 (2H, m, H3, H5). ¹³C-NMR (126 MHz,

DMSO-d₆): 167.00, 153.92, 152.74, 152.47, 149.99, 130.84, 129.37, 121.53, 119.15, 114.20. Anal. Calcd.

δ 12.95 (1H, s, COOH), 8.64 (1H, s, CH=N), 8.03–7.98 (2H, m, H2, H6), 7.83 (1H, d, J = 3.9

δ

for C₁₂H₈N₂O₅(260.04): C, 55.39; H, 3.10; N, 10.77. Found: C, 55.60; H, 2.94; N, 10.89.

4-[(2-Methoxybenzylidene)amino]benzoic acid (1s). White solid; yield 95%; mp 231.5–234 ^◦C (lit. [24

]

1

220–221 ^◦C). IR: 1683 (COOH), 1593 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 12.85 (1H, s,

COOH), 8.84 (1H, s, CH=N), 8.02 (1H, dd, J = 7.7 Hz, 1.8 Hz, H6⁰), 7.99–7.95 (2H, m, H2, H6), 7.54 (1H,

ddd, J = 8.8 Hz, 7.2 Hz, 1.8 Hz, H4⁰), 7.28–7.23 (2H, m, H3, H5), 7.17 (1H, d, J = 8.4 Hz, H3⁰), 7.07 (1H, t,

J = 7.5 Hz, H5⁰). ¹³C-NMR (126 MHz, DMSO-d₆):

δ 167.21, 159.72, 157.26, 156.24, 133.92, 130.84, 127.99,

127.21, 123.68, 121.09, 120.91, 112.30, 56.01. Anal. Calcd. for C₁₅H₁₃NO₃(255.09): C, 70.58; H, 5.13; N,

5.49. Found: C, 70.44; H, 5.02; N, 5.60.

4-[(4-Hydroxybenzylidene)amino]benzoic acid (1t). Yellowish brown solid; yield 80%; mp 251–253

1

^◦C (lit. [31] 254–255 ^◦C). IR: 1681 (COOH), 1599 (-HC=N-) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ

10.45 (1H, s, COOH), 9.79 (1H, s, OH), 8.65 (1H, s, CH=N), 8.00–7.97 (2H, m, H2, H6), 7.79–7.74 (2H, m,

H2⁰, H6⁰), 7.38–7.34 (2H, m, H3, H5). 6.96–6.93 (2H, m, H3⁰, H5⁰). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

167.59, 162.02, 161.41, 156.39, 131.39, 130.86, 128.03, 127.65, 121.12, 116.27. Anal. Calcd. for C₁₄H₁₁NO₃

(241.25): C, 69.70; H, 4.60; N, 5.81. Found: C, 69.66; H, 4.51; N, 5.97.

2.1.3. Synthesis of 1u

4-aminobenzoic acid (137.1 mg; 1 mmol) was dissolved in 7 mL of MeOH, followed by addition of

salicylaldehyde (117

µL; 1.1 mmol), sodium cyanoborohydride (62.8 mg; 1 mmol) and ﬁnally glacial

acetic acid (100 L). The mixture was stirred for 24 h at room temperature. After this time, the reaction

µ

mixture was diluted with distilled water and stirred for additional 1 h. The resulted precipitate was

ﬁltered oﬀ and washed by water. The product was crystallized from ethyl acetate/n-hexane to obtain

pure 1u.

4-[(2-Hydroxybenzyl)amino]benzoic acid (1u). Greyish solid; yield 74%; mp 198–199.5 ^◦C (lit. [32

]

1

203–204 ^◦C). IR: 3277, 1670 (COOH) cm⁻¹. H-NMR (500 MHz, DMSO-d₆):

δ 11.95 (1H, s, COOH), 9.56

(1H, s, OH), 7.64 (2H, d, J = 8.7 Hz, H2, H6), 7.14 (1H, dd, J = 7.6 Hz, 2.7 Hz, H6⁰), 7.05 (1H, td, J = 7.6

Hz, 1.7 Hz, H4⁰), 6.85-6.79 (2H, m, NH, H3⁰), 6.73 (1H, td, J = 7.4 Hz, 1.1 Hz, H5⁰), 6.60-6.56 (2H, m, H3,

H5), 4.24 (2H, d, J = 5.8 Hz, CH₂). ¹³C-NMR (126 MHz, DMSO-d₆):

δ 167.68, 155.21, 152.80, 131.26,

128.39, 127.93, 125.09, 119.00, 117.06, 115.11, 111.14, 41.03. Anal. Calcd. for C₁₄H₁₃NO₃(243.09): C,

69.12; H, 5.39; N, 5.76. Found: C, 69.21; H, 5.51; N, 5.49.

2.2. Pharmacology

2.2.1. Antibacterial Activity

For the purpose of screening for antibacterial activity of the derivatives four Gram-positive and

four Gram-negative strains of clinical importance were used, namely: Staphylococcus aureus ATCC 29213,

CCM 4223, methicillin-resistant Staphylococcus aureus ATCC 43300, CCM 4750 (MRSA), Staphylococcus

epidermidis H 6966/08 (clinical isolate), Enterococcus faecalis ATCC 29212, CCM 4224; Escherichia coli ATCC

25922, CCM 3954, Klebsiella pneumoniae D 11750/08 (clinical isolate), extended-spectrum β-lactamase

positive Klebsiella pneumoniae J 14368/08 (clinical isolate), and Pseudomonas aeruginosa ATCC 27853,

CCM 3955. These strains were obtained from the Czech Collection of Microorganisms (CCM, Brno,

Czech Republic) or they are clinical isolates from the Department of Clinical Microbiology, University

Hospital in Hradec Králové, Czech Republic.

The microdilution broth method was performed according to EUCAST (The European Committee

on Antimicrobial Susceptibility Testing) instructions with slight modiﬁcations [33]. Brieﬂy, the

cultivation was done in Cation-adjusted Mueller-Hinton broth (CAMHB, M-H 2 Broth, Sigma-Aldrich)

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at 35

±

2 ^◦C. Tested compounds were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) to

produce stock solutions. The ﬁnal concentration of DMSO in the testing medium did not exceed 1%

(v/v) of the total solution composition and did not aﬀect the growth of bacteria. Positive (microbe solely),

negative (cultivation medium and DMSO) controls and internal quality standards were involved in

each assay. Antibacterial activity is expressed as minimum inhibitory concentration (MIC, reported

◦

in

µ

M) after 24 and 48 h of static incubation in the dark and humidiﬁed atmosphere at 35

±

2 C.

The experiments were performed in duplicates. For the results to be valid, the diﬀerence in MIC

determined from two parallel measurements must not be greater than one step on the dilution scale.

The results were analysed by visual inspection.

Parent PABA as well as topical antibiotics bacitracin (active especially against Gram-positive species

including MRSA) and neomycin (an aminoglycoside active predominantly against Gram-negative

strains) were involved as the reference drugs. Standard antibiotics (ciproﬂoxacin, gentamicin; data not

shown) and internal quality control/reference strains are routinely included in parallel testing-basic

screening of antibacterial activity.

2.2.2. Antimycobacterial Activity

The in vitro antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv (331/88; Mtb.)

and three strains of nontuberculous mycobacteria (Mycobacterium avium 330/88 [resistant to isoniazid,

rifamycines, oﬂoxacin, and ethambutol], Mycobacterium kansasii 235/80 from the Czech National

Collection of Type Cultures, and a clinical isolate of M. kansasii 6509/96) was evaluated using a

previously reported method [34]. The micromethod for the determination of MIC was used involving

the Šula’s semisynthetic medium (SEVAC, Prague, Czech Republic). The investigated compounds

were added to the medium as solutions in DMSO; the ﬁnal volume contained 1.0% DMSO (v/v).

The following concentrations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2 and 1

were determined after incubation at 37 ^◦C for 14 and 21 days, for M. kansasii strains additionally for 7

days. MIC (in M) was the lowest concentration at which the complete inhibition of mycobacterial

µM. The MIC

µ

growth occurred. First-line oral antitubercular drug isoniazid (INH) and parent PABA were involved

as reference compounds.

2.2.3. Antifungal Activity

The antifungal properties were evaluated against ﬁve yeast strains, namely: Candida albicans

ATCC 44859, Candida tropicalis 156, Candida krusei E28, Candida glabrata 20/I, Trichosporon asahii 1188

and three strains of ﬁlamentous fungi: Aspergillus fumigatus 231, Lichtheimia corymbifera 272, and

Trichophyton interdigitale 445. A microdilution broth method was performed according to EUCAST II,

III instructions with slight modiﬁcations [35,36]. Brieﬂy, tested compounds were dissolved in DMSO

and diluted in a twofold manner with RPMI-1640 medium with l-glutamine, supplemented with 2%

glucose (w/v) and buﬀered to pH 7.0 with 3 morpholinopropane-1-sulfonic acid (all components were

purchased from Sigma-Aldrich, USA). The ﬁnal concentration of DMSO in the tested medium did not

exceed 1% (v/v) of the total solution composition, and it was conﬁrmed that this concentration did not

inhibit the fungal growth. Static incubation was performed in the dark and humidiﬁed atmosphere, at

35

±

2 ^◦C for 24 and 48 h (72 and 120 h for Trichophyton interdigitale). Positive controls consisted of

test microbe solely, while negative controls consisted of medium and DMSO. Internal quality control

was included too. Visual inspection was used for MIC endpoints evaluation. The experiments were

conducted in duplicates. For the results to be valid, the diﬀerence in MIC determined from two parallel

measurements must not be greater than one step on the dilution scale.

PABA and triazole antimycotic drug ﬂuconazole were involved as the reference compounds. MIC

values of ﬂuconazole mean MIC₅₀values.

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2.2.4. Cytotoxicity

HepG2, human hepatocellular carcinoma cells (ATCC HB-8065; passage 5–8) purchased from

Health Protection Agency Culture Collections (ECACC, Salisbury, UK) were cultured in Minimum

Essentials Eagle Medium supplemented with 10% fetal bovine serum, 1% l-glutamine solution

and non-essential amino acid solution (Sigma-Aldrich, St. Louis, MO, USA) in a humidiﬁed

atmosphere containing 5% CO₂at 37 ^◦C. For subculturing, the cells were harvested after trypsin/EDTA

(Sigma-Aldrich, St. Louis, USA) treatment at 37 ^◦C. To evaluate cytotoxicity, the cells treated with

the tested substances were used as experimental groups whereas untreated HepG2 cells served as

control groups.

The cells were seeded in a density of 10 000 cells per well in a 96-well plate. The following day

the cells were treated with each of the tested substances dissolved in DMSO (maximal incubation

concentration of DMSO was 1% v/v). The tested substances were prepared at diﬀerent incubation

concentrations in triplicates according to their solubility. Simultaneously, the controls representing

100% cell viability, 0% cell viability (the cells treated with 10% DMSO), no cell control and vehiculum

controls were also involved in triplicates. After 24 h incubation in a humidiﬁed atmosphere containing

5% CO₂at 37 ^◦C, the reagent from the kit CellTiter 96 AQueous One Solution Cell Proliferation Assay

(CellTiter 96; Promega, Fitchburg, WI, USA) was added. After 2 h incubation at 37 ^◦C absorbance of

samples was recorded at 490 nm (TECAN, Inﬁnita M200, Grödig, Austria). A standard toxicological

parameter IC₅₀was calculated by nonlinear regression from a semilogarithmic plot of incubation

concentration versus percentage of absorbance relative to untreated controls using GraphPad Prism

software (version 6; GraphPad Software, Inc., La Jolla, CA, USA). Results of the experiments are

presented as inhibitory concentration which reduces the viability of the cell population to 50% from

the maximal viability (IC₅₀).

Parent PABA and a cytotoxic/anticancer drugs tamoxifen (as tamoxifen citrate) and cisplatin were

involved as the reference drugs.

3. Results and Discussion

3.1. Chemistry

We synthesized twenty PABA-derived Schiﬀ bases 1a–1t and one “reduced” compound 1u (i.e.,

1a with reduced imine double bond). The synthesis of Schiﬀ bases (Scheme 1) involved a condensation

of the amino acid with aldehydes in MeOH under reﬂux (3 h) or DEE at room temperature (72

h). Exceptionally, an acidic organic catalyst (0.1 equivalent of toluene-4-sulfonic acid monohydrate)

was used to promote the reaction (imines 1f and 1t). The yields were from good to excellent

(80–96%). Seventeen Schiﬀ bases were prepared from substituted salicylaldehydes (1a

–1q), one

(1r) from 5-nitrofuran-2-carbaldehyde (5-nitrofurfural) and three compounds are analogues of 1a

:

O-methyl derivative 1s, 4-hydroxy isomer 1t and molecule 1u without double bond. Aforementioned

4-[(2-hydroxybenzyl)amino]benzoic acid 1u was obtained by reductive amination from PABA and

salicylaldehyde using sodium cyanoborohydride and glacial acetic acid in MeOH (yield 74%; Scheme 2).

The structures of the aimed compounds are summarized in Table 1.

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Table 1. Antibacterial activity of PABA derivatives 1.

MIC [µM]

R¹

R²

Code

SA

MRSA

SE

EF

EC

KP

KP-E

24 h

PA

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

1s

1t

1u

PABA

1

H

5-F

5-Cl

5-Br

5-I

5-NO₂

5-CH₃

5-OCH₃

5-OH

5-tert-Bu

6-Cl

4-Cl

H

5-Cl

5-I

H

Cl

Br

I

>500

>250

500

>500

500

>500

125

15.62

62.5

>500

>250

500

>500

500

>500

125

15.62

62.5

>500

>250

250

>500

>250

250

>500

62.5

250

500

250

>500

500

>500

62.5

250

500

250

>500

500

>500

>250

500

>500

500

>500

62.5

>500

>250

500

>500

500

>500

62.5

>500

>250

>500

500

>500

>250

>500

500

>500

>250

>500

500

>500

500

>500

>250

>500

500

>500

500

>500

>250

>500

500

>500

500

>500

>250

>500

500

>500

500

>500

>250

>500

500

>500

500

>500

>250

>500

500

>500

500

>500

250

>500

250

>500

250

>500

250

31.25

15.62

62.5

31.25

15.62

62.5

15.62

31.25

62.5

125

>500

31.25

62.5

125

>500

62.5

500

>500

62.5

500

>500

62.5

500

>500

62.5

500

>500

125

500

>500

125

500

>500

125

500

>500

125

500

>500

I

-

62.5

>500

-

>500

BAC

NEO

-

7.81

ND

15.62

ND

15.62

ND

15.62

ND

15.62

ND

31.25

ND

15.62

ND

62.5

ND

>500

3.9

>500

3.9

>500

1.95

>500

1.95

>500

3.9

>500

3.9

>500

7.81

>500

15.62

BAC = bacitracin; NEO = neomycin; PABA = 4-aminobenzoic acid. SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus

epidermidis H 6966/08; EF: Enterococcus faecalis J 14365/08. EC: Escherichia coli CCM 4517; KP: Klebsiella pneumoniae D 11750/08; KP-E: ESBL-positive Klebsiella pneumoniae J 14368/08; PA:

Pseudomonas aeruginosa CCM 1961. ND = not determined. One or two of the best MIC value(s) for each strain are shown in bold.

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Scheme 1. Synthesis of PABA-based imines 1a–

1t (R¹= H, 5-F, 4-Cl, 5-Cl, 6-Cl, 5-Br, 5-I, 5-NO₂, 5-CH₃,

5-OCH₃, 5-OH, 5-t-Bu; R²= H, Cl, Br, I; R³= 2-OCH₃, 4-OH; TsOH = toluene-4-sulfonic acid; DEE =

diethyl ether).

Scheme 2. Synthesis of the derivative 1u via reductive amination.

New compounds were characterized by melting points, NMR and IR spectroscopy. Their purity

was checked by the TLC and elemental analysis additionally. The identity of the known compounds

was established using NMR and IR spectroscopy by the comparison with previously reported data.

Their purity was checked in an identical way as for novel derivatives.

3.2. Antibacterial Activity

PABA derivatives 1a–1u were evaluated for their antibacterial action using four Gram-positive

strains of Staphylococcus aureus ATCC 29213, CCM 4223, methicillin-resistant Staphylococcus aureus ATCC

43300, CCM 4750 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus faecalis ATCC 29212, CCM

4224 and four Gram-negative strains, Escherichia coli ATCC 25922, CCM 3954, Klebsiella pneumoniae D

11750/08, extended spectrum β-lactamase positive K. pneumoniae J 14368/08, and Pseudomonas aeruginosa

ATCC 27853, CCM 3955 (Table 1). Bacitracin, neomycin and PABA itself were used for comparison of

MIC, ciproﬂoxacin and gentamicin as internal quality standards (their MIC data are not shown).

Free PABA lacked any antibacterial properties (MIC > 500

were more susceptible (MIC from 15.62 M) than Gram-negative species (

the compounds share no activity). Illustratively, P. aeruginosa was inhibited only by two imines at

500 M (1i and 1r). On the other hand, S. epidermidis was found being the most susceptible strain

15.62 M) followed by both drug-susceptible and methicillin-resistant S. aureus. Importantly, the

µM). Generally, Gram-positive strains

µ

≥

62.5 M and majority of

µ

1

µ

(≥

µ

growth inhibition of S. aureus is independent of the presence of methicillin-resistance, thus indicating

no cross-resistance with this drug. The MIC values obtained for the most active imines are identical

to those obtained for bacitracin, an antibiotic used for the treatment of topical infections caused by

Gram-positive bacteria. Due to a lack of a signiﬁcant activity of 1a–1u against Gram-negative strains,

none of the derivatives were superior to aminoglycoside drug neomycin.

The following structure-activity relationships (SAR) were identiﬁed:

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Substituted salicylidene moiety is essential: derivatives of unsubstituted salicylaldehyde (1a) and

its reduced analogue 1u, 2-methoxybenzaldehyde (1s) and 4-hydroxybenzaldehyde (1t) avoided any

antibacterial action.

1.

2.

The presence of 5-F (1b), 5-t-Bu (1j) and chlorine at the positions 4 and 6 of the salicylic ring (1k

1l) did not improve activity of 1a.

,

Nitro (1f) and hydroxy group (1i) with opposite electron properties led to similar

results—inhibition of multiple Gram-positive strains including MRSA, but at high concentrations

(≥ 250 µM).

3.

4.

5.

6.

The 5-substitution by a single halogen (Cl 1c, Br 1d, I 1e) improves the inhibition of S. epidermidis;

in the case of 1e MRSA, too (MIC from 62.5 µM).

Electron-donation groups (CH₃1g, OCH₃1h) together with 3-Cl (1m) produced inhibition of

only S. epidermidis, but with MIC from 250 µM.

5-Nitrofurylidene PABA derivative 1r inhibited all three staphylococcal strains (MIC 62.5–125

µM).

The Schiﬀ bases derived from 3,5-dihalogenosalicylaldehydes (1n

successful modiﬁcation of PABA with MIC values of 15.62 M, especially if the particular

halogens are diﬀerent (1o 1p). These latter compounds containing one iodine atom also

–1q) represent the most

≥

µ

,

demonstrated activity against three Gram-negative strains (E. coli, K. pneumoniae).

In summary, the presence of at least one heavier halogen is essential, favouring derivatives of

5-chloro-3-iodo/bromo-salicylaldehydes. These substituents provide both wide-spectrum antibacterial

activity and comparatively low MIC values. Interestingly, similar results were obtained for sulfadiazine

Schiﬀ bases previously [20]. MIC of 5-nitrofurfural-derived Schiﬀ base 1r (62.5–125

g/mL) is fully comparable to breakpoint of its clinically used analogue nitrofurantoin for staphylococci

(32–64 µg/mL) according to EUCAST [33].

Although PABA is an essential molecule for folate biosynthesis, it was reported as an agent

inhibiting E. coli, Listeria monocytogenes and Salmonella enteritidis [ ], but at supraphysiological

concentrations. Although the strains used by Richards et al. are distinct from our study, their

reported MIC values of PABA were substantially higher (9–24 mM) [ ]. Giving together with the

µM, i.e., 16.25–32.51

µ

6

ineﬀectiveness of PABA in our assay, it is obvious that the antibacterial action depends on the presence

of imine bond resulting from the reaction with substituted salicylaldehydes or 5-nitrofurfural, i.e.,

the bioactivity is established by this simple chemical modiﬁcation of the vitamin unequivocally. The

substitution of NH₂group by carbonyl group could interfere with the vitamin function of PABA and

thus folate biosynthesis. Alternatively, PABA may be considered as a carrier for intrinsic bioactive

salicylic and nitrofuran scaﬀolds. From this point of view, PABA that has demonstrated selective

bacterial uptake (S. aureus, E. coli, M. tuberculosis) [37] should help to accumulate Schiﬀ bases and

thus salicylic and nitrofuran molecules within bacterial cells where they can disrupt cellular functions

leading to growth inhibition or cellular death.

3.3. Antimycobacterial Activity

Schiﬀ bases 1a–1t and phenol 1u were screened in vitro against Mtb. 331/88 (i.e., H₃₇Rv) and three

non-tuberculous mycobacterial strains: M. avium 330/88, M. kansasii 235/80 and M. kansasii 6509/96 (a

clinical isolate). A ﬁrst-line antitubercular drug INH and parent PABA were used as reference drugs

for comparison (Table 2).

Biomolecules 2020, 10, 9

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Table 2. Antimycobacterial activity of PABA derivatives 1.

MIC [µM]

R²

R

Code

Mtb. 331/88

M. avium 330/88

M. kansasii 235/80

M. kansasii 6509/96

14 d

21 d

14 d

21 d

7 d

14 d

21 d

7 d

14 d

21 d

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

H

5-F

5-Cl

5-Br

5-I

5-NO₂

5-CH₃

5-OCH₃

5-OH

5-tert-Bu

6-Cl

4-Cl

H

5-Cl

5-I

H

Cl

Br

I

-

>1000

500

250

500

250

500

250

500

250

>1000

500

250

500

1000

250

500

250

125

1000

>1000

1

>1000

1000

500

>1000

500

1000

250

500

125

250

500

250

>1000

500

250

500

125

500

>1000

500

250

500

1000

250

500

250

1000

500

250

500

250

500

250

125

1000

8

1000

500

250

500

1000

250

500

250

125

1000

8

1000

500

1000

500

1000

500

1000

500

250

1000

8

500

1000

>1000

1000

500

1000

500

250

>1000

1000

>1000

>250

>1000

1000

>1000

1000

500

1000

500

>1000

>250

-

62.5

1000

>1000

0.5

62.5

1000

>250

125

1s

1t

1u

1000

>1000

>250

>1000

1000

>1000

>250

PABA 1

INH

INH = isoniazid; PABA = 4-aminobenzoic acid; Mtb. = Mycobacterium tuberculosis. One or two of the lowest MIC value(s) for each strain are shown in bold.

Biomolecules 2020, 10, 9

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In general, although none of the compounds was inactive completely, antimycobacterial activity

of the derivatives 1a 1u is comparatively weak with MIC from 62.5 M. 5-nitrofurfural derivative 1r

showed the lowest MIC (62.5–250 M); only this derivative outstripped INH in the case of M. avium

–

µ

and M. kansasii 235/80 (i.e., INH-resistant strains). This ﬁnding was expected regarding previous

studies, e.g., [38]. The Schiﬀ bases derived from unsubstituted salicylaldehyde (1a), its 4-OH isomer

(

1t), 2-methoxybenzaldehyde (1s) and reduced compound 1u share only negligible antimycobacterial

activity corresponding to the parent PABA. The substitution of salicylaldehyde (compounds 1b 1q

1p and 1q) improved growth inhibition of

–

;

especially tert-butyl 1j and halogenated imines 1i, 1k, 1l,

mycobacteria, but only moderately.

3.4. Antifungal Activity

PABA derivatives 1a–1u were investigated also against eight fungal strains. The panel of human

pathogens covers ﬁve yeast strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida

krusei E28, Candida glabrata 20/I, Trichosporon asahii 1188) and three moulds (Aspergillus fumigatus 231,

Lichtheimia corymbifera 272, and Trichophyton interdigitale 445). Azole drug ﬂuconazole and parent PABA

were used for comparison of MIC (Table 3).

PABA itself showed no antifungal properties (MIC > 500

corymbifera were found being the most resistant species (MIC from 125

and T. interdigitale were inhibited from the concentration of 7.81 M. Interestingly, the inhibition of

these strains by particular derivatives were virtually identical. The PABA Schiﬀ bases were superior to

ﬂuconazole for seven strains: C. tropicalis (1c 1e 1h 1k 1m 1r), C. krusei (inherent FLU-resistant; (1e

1o 1r; other derivatives were comparable—1c 1d 1k 1m, and 1n), C. glabrata (1d 1e 1m 1o 1q), T.

asahii (1d 1e 1m 1q; others are comparable: 1f 1h 1r), A. fumigatus (1d 1e 1m 1q), L. corymbifera (1f

1m 1q) and T. interdigitale (1p 1q; additional derivatives were superior or comparable after 120 h of

treatment).

These SAR were found:

µM). In general, A. fumigatus and L.

µM). On the other hand, yeasts

µ

–

,

–

,

–

,

–

,

–

,

–

,

–

,

1.

Substituted salicylidene or 5-nitrofurfurylidene moiety is indispensable: unsubstituted

salicylaldehyde 1a), its reduced analogue 1u 2-methoxybenzaldehyde 1s and

4-hydroxybenzaldehyde (1t) share no antimycotic potency.

5-F (1b) and 5-alkyls (1g, 1j) are not translated into any antifungal action.

(

,

( )

2.

3.

The presence of NO₂(1f), OH (1i), OCH₃(1h) and 4-Cl (1l) substituents provides active compounds

but with higher MIC values (≥ 250 µM).

4.

Focusing on isomeric 6-Cl (1k), 5-Cl (1c), 4-Cl (1l) and 3-Cl (1m) derivatives, the increase of the

activity is as follows: 4-Cl (MIC

the introduction of second chlorine atom (3,5-Cl₂) did not improve the activity (1n; MIC

µM).

≥

250

µ

M) < 5-Cl

≤

6-Cl < 3-Cl (MIC

≥

31.25 µM). Interestingly,

≥

125

5.

6.

The activity of single halogen-substituted derivatives (5-F 1b, 5-Cl 1c, 5-Br 1d, 5-I 1e) is increasing

with the atomic mass/lipophilicity/atomic radius of the halogens (5-F > 500 M; 5-I 31.25 M).

3,5-Dihalogenosalicylidene imines (1n–1q) were identiﬁed as the most active derivatives providing

both low MIC ( 7.81 M) and broad-spectrum of the activity. The presence of at least one iodine

µ

≥

µ

≥

µ

atom results in the highest antifungal properties (1o and 1p).

Obviously, SAR are predominantly analogous to antibacterial action. Recently, Laborda et al. [39

]

have discovered PABA as antifungal substance of Lysobacter antibioticus supressing phytopathogenic

fungi, even though at high concentrations again (IC₅₀of 1–3 mM), i.e., being up to three orders of

magnitude less potent then our compounds. The maximal concentration of PABA investigated in

our study was lower than reported as eﬀective [39]. To the best of our knowledge, no activity of

PABA-based imines working against human pathogenic fungi has been reported and this property is

presented herein for the ﬁrst time. Again, a simple structural change of this vitamin-like molecule

constitutes new bioactivity.

Biomolecules 2020, 10, 9

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Table 3. Antifungal activity of PABA derivatives 1.

MIC [µM]

R¹

R²

Code

CA

CT

CK

CG

TA

AF

LC

TI

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

72 h

120 h

1a

1b

1c

1d

1e

H

5-F

5-Cl

5-Br

5-I

5-NO₂

5-CH₃

5-OCH₃

5-OH

5-tert-Bu

6-Cl

4-Cl

H

5-Cl

5-I

H

Cl

Br

I

>500

>125

62.5

500

>125

250

500

>500

125

250

31.25

125

31.25

7.81

250

>500

>125

62.5

500

>125

250

500

>500

125

>500

31.25

125

31.25

7.81

250

>500

125

>500

125

>500

125

>500

125

62.5

>500

>125

500

>500

250

>500

250

125

>500

125

62.5

31.25

>500

>125

250

>500

125

500

62.5

125

15.62

7.81

250

>500

125

62.5

31.25

>500

>125

250

>500

125

>500

62.5

125

15.62

7.81

250

>500

>125

125

62.5

500

>125

500

>500

125

500

125

31.25

7.81

15.62

500

>500

>125

125

62.5

500

>500

>125

250

>500

>125

250

>500

>125

>250

>125

500

>125

>500

>125

>500

500

250

>500

>125

>250

>125

500

>125

>500

>125

>500

500

250

>500

125

62.5

31.25

500

>125

>500

125

500

125

250

62.5

7.81

250

>500

125

62.5

31.25

500

>125

>500

125

>500

125

250

62.5

7.81

250

>500

125

62.5

>500

>125

500

>500

125

500

250

125

62.5

15.62

31.25

62.5

>500

125

1f

>500

>125

500

>500

62.5

500

62.5

250

62.5

31.25

15.62

250

>500

>125

500

>500

125

>500

62.5

250

62.5

31.25

15.62

250

>500

>125

>500

>125

250

500

250

125

>500

>125

>500

>125

>500

250

500

250

125

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

1s

1t

1u

PABA

1

>125

500

>500

>125

>500

125

31.25

7.81

15.62

500

>500

62.5

15.62

31.25

62.5

>500

I

-

125

-

>500

-

>500

FLU

0.24

125

250

31.25

500

250

500

7.81

125

FLU = ﬂuconazole; PABA = 4-aminobenzoic acid. CA: Candida albicans ATCC 44859; CT: Candida tropicalis; CK: Candida krusei E28; CG: Candida glabrata 20/I; TA: Trichosporon asahii

1188; AF: Aspergillus fumigatus 231; LC: Lichtheimia corymbifera 272; TI: Trichophyton interdigitale 445. One or two of the best MIC value(s) for each strain are shown in bold.

Biomolecules 2020, 10, 9

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Notably, Meir and Osherov [40] presented and highlighted vitamin biosynthesis as promising

antifungal targets including folic acid biosynthesis utilizing PABA. The deﬁcient synthesis of PABA

and withdrawal from food hampered the virulence of Aspergillus spp. completely. Moreover, PABA

inhibitors will likely be active against ongoing infections.

3.5. Cytotoxicity

The cytotoxicity of the PABA derivatives 1 were screened using the standard hepatic cancer cell

line HepG2 (Table 4) from two reasons. First, salicylidene-based compounds have been proposed

as anticancer agents supressing also HepG2 cells [41]. On the other hand, they can serve as an

in vitro model for the hepatotoxicity as the surrogate of normal liver cells during preclinical drug

discovery [42,43].

Table 4. Cytotoxicity of the compounds 1 for HepG2 cells.

Code

R¹

R²

IC₅₀[µM]

Range of Concentrations Tested

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

H

5-F

5-Cl

5-Br

5-I

5-NO₂

5-CH₃

5-CH₃O

5-OH

5-tert-Bu

6-Cl

4-Cl

H

5-Cl

5-I

-

H

Cl

Br

I

-

>1500 *

497.6

142.9

178.7

113.4

243.8

956.4

530.4

ND

1–1500

1–500

1–1500

1–500

1–1500

1–500

1–1500

80.8

279.6

263.9

247.4

98.7

85.7

53.4

57.8

15.0

1090.0

>1500 *

>500 *

19.6

1q

1r

1s

1t

1u

Tamoxifen

Cisplatin

PABA 1

21.3

>1500 *

* measurement at higher concentration was unable due to the precipitation of the compound in the medium. PABA

= 4-aminobenzoic acid; ND = not determined.

The CellTiter 96 assay used is based on the reduction of tetrazolium dye in living cells to formazan,

which is then determined colorimetrically. This reduction is related to availability of NADH or NADPH.

The decline in levels of these metabolically important compounds in the cell causes that the production

of formazan is reduced. For quantitative comparison of cytotoxicity, the parameter IC₅₀was used. IC₅₀

is deﬁned as the concentration that reduces the viability of the cells to 50% of the maximal viability.

We were able to determine the exact IC₅₀of the majority of the substances. However, IC₅₀values

of parent PABA 1, 1a, 1t and 1u were higher than tested range of concentrations. The measurement

was not possible due to the precipitation of the compound in the medium. The IC₅₀of 1i could not be

determined with the used method due to strong interaction with the testing reagent.

The eﬀective cytotoxic concentrations among the compounds diﬀer in the extent of several

orders of magnitude. We could identify both several relatively non-toxic compounds for HepG2 cells

(IC₅₀> 500

µM: 1a, 1g, 1h, 1s, 1t, 1u and PABA 1), other compounds exhibited moderate toxicity.

5-nitrofurylidene-based imine was identiﬁed as the most toxic compound (IC₅₀= 15.0 µM), followed

Biomolecules 2020, 10, 9

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by polyhalogenated derivatives and 5-tert-butyl compound 1j. Interestingly, there are not identical

SAR as for antimicrobial action: heavier/more lipophilic halogens do not result in uniformly enhanced

toxicity and, regarding particular chlorinated isomers, 5-isomer is approximately twice more toxic than

other. The IC₅₀value of the most cytotoxic compound (nitrofuran derivative 1r) was fully comparable

to those obtained for anticancer drugs tamoxifen (19.6

active compounds exhibited IC₅₀in the same order of magnitude (1j, 1n–1p).

In addition to PABA “non-imine” cytotoxic derivatives [ 12 44], Farooq et al. [18] discovered

µM) and cisplatin (21.3 µM), other the most

1,5, ,

N-alkylisatin-3-iminobenzoic acids, i.e., Schiﬀ bases of substituted isatin and PABA, as potential

cytotoxic/antileukemic agents with IC₅₀for human normal as well as cancer cell lines comparable to

our the most active molecules. Thus, the formation of Schiﬀ bases could convert non-toxic PABA to

moderate cytotoxic agents, depending on the carbonyl compound used. Seemingly negligible changes

to the structure may result in substantially higher toxicity for malignant cells (illustratively, switch of a

lipophilic and electron-donating substituent methyl 1g to tert-butyl 1j increases toxicity almost twelve

times). Since PABA as the parent compound itself is non-toxic and safe for human administration [8,9],

the cytotoxicity should be a consequence of the properties of the attached aldehyde and/or imine bond.

4. Conclusions

In this study, we synthesized twenty-one hybrid compounds of folate precursor 4-aminobenzoic

acid, an essential vitamin for many human pathogens, and bioactive aldehydes, predominantly

substituted salicylaldehydes and 5-nitrofurfural. These scaﬀolds were fused through imine bond to

form Schiﬀ bases. Advantageously, the aimed compounds are easily synthetically available.

The modiﬁcation of PABA in this way led to constitution of various bioactivities that were not

exhibited by the parent drug in our assays. SAR revealed following essential structural features:

(1) presence of imine bond; (2) (5-nitrofuran-2-yl)methylene or 2-hydroxybenzylidene (salicylidene)

scaﬀold; (3) substitution of salicylic moiety, especially by one or more heavier halogen(s). Many of

the new compounds share antibacterial action especially against Gram-positive strains including

MRSA, mild inhibition of mycobacteria and broad-spectrum antifungal properties. The most potent

agents against Gram-positive strains were halogenated, especially 3,5-dihalogenated derivatives with

MIC values from 15.62

µM. The 5-nitrofurfurylidene scaﬀold was proven to be beneﬁcial for the

inhibition of mycobacteria (MIC of

non-toxic parent amino acid can result in compounds with cytotoxic action for cancer cells, but many

derivatives remained non-toxic. The presence of heavier halogen(s), especially iodine, highly lipophilic

≥

62.5 M). Moreover, the simple chemical derivatization of

µ

tert-butyl and nitro group (IC₅₀from 15.0 µM) enhanced the cytotoxicity of the Schiﬀ bases for HepG2

line. In sum, structure-activity relationships highlighted the presence of dihalogenated salicylic and

5-nitrofurane moieties. Regarding the carbonyl compound used for the modiﬁcation of the PABA

structure, it is possible to tune up the particular activities to identify even more promising derivatives

for further development.

Author Contributions: Conceptualization: M.K. and J.V.; Methodology, M.K., K.K., F.T. and J.S.; Investigation,

M.K., K.K., J.J., M.B., O.J. and J.S.; Writing—Original Draft Preparation, M.K. and K.K.; Writing—Review and

Editing, F.T. and J.V.; Supervision, M.K. and J.V. All authors have read and agreed to the published version of

the manuscript.

Funding: This research was funded by the Czech Science Foundation, grant number 17–27514Y, and by the project

EFSA-CDN [grant No. CZ.02.1.01/0.0/0.0/16_019/0000841] co-funded by ERDF.

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

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