Synthesis of readily available fluorophenylalanine derivatives and investigation of their biological activity

Accepted Manuscript Synthesis of Readily Available Fluorophenylalanine Derivatives and Investigation of Their Biological Activity Martin Krátký, Š árka Štěpánková, Katarína Vorč áková, Lucie Navrátilová, František Trejtnar, Jiřina Stolař íková, Jarmila Vinšová PII: DOI: Reference:

S0045-2068(16)30409-6 http://dx.doi.org/10.1016/j.bioorg.2017.02.010 YBIOO 2020

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Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

12 December 2016 10 February 2017 18 February 2017

Please cite this article as: M. Krátký, S. Štěpánková, K. Vorč áková, L. Navrátilová, F. Trejtnar, J. Stolař íková, J. Vinšová, Synthesis of Readily Available Fluorophenylalanine Derivatives and Investigation of Their Biological Activity, Bioorganic Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bioorg.2017.02.010

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Synthesis of Readily Available Fluorophenylalanine Derivatives and Investigation of Their Biological Activity Martin Krátký a,*, Šárka Štěpánková b, Katarína Vorčáková b, Lucie Navrátilová c, František Trejtnar c, Jiřina Stolaříková d, Jarmila Vinšová a a

Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic b Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic c Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic d Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava, Partyzánské námĕstí 7, 702 00 Ostrava, Czech Republic *Corresponding author. Heyrovského 1203, 500 05 Hradec Králové, Czech Republic. E-mail address: [email protected], tel.: +420-495067343, fax: +420-495067166. Abstract A series of thirty novel N-acetylated fluorophenylalanine-based aromatic amides and esters was synthesized using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide or phosphorus trichloride in pyridine. They were characterized by spectral methods and screened against various microbes (Mycobacterium tuberculosis, non-tuberculous mycobacteria, other bacteria, fungi), for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and cytotoxicity. All amino acids derivatives revealed a moderate inhibition of both cholinesterases with IC 50 values for AChE and BChE of 57.88-130.75 µM and 8.25 to 289.0 µM, respectively. Some derivatives were comparable or superior to rivastigmine, an established drug. Phenyl 2-acetamido-3-(4fluorophenyl)propanoate was identified as the selective and most potent inhibitor of BChE. The esterification and amidation of parent acids led to an improved BChE inhibition. The esters are better inhibitors of BChE than the amides. The introduction of NO2 and CH3 groups into aniline ring and CF3 moiety in phenol is translated into lower IC50 values. Seven compounds showed selectivity index higher than 10 for at least one cholinesterase. Especially the esters exhibited a mild activity against Gram-positive bacteria, mycobacteria and several fungal strains with minimum inhibitory concentrations starting from 125 µM. The highest susceptibility was recorded for Trichophyton mentagrophytes fungus. Graphical Abstract

Keywords

Amides; antimicrobial activity; cholinesterases; cytotoxicity; fluorophenylalanines; in vitro activity; unnatural amino acids*

*

enzyme

inhibition;

esters;

Abbreviations: ACh: acetylcholine; AChE: acetylcholinesterase; BAC: bacitracin; BChE: butyrylcholinesterase; ChE: cholinesterases; DMAP: 4 (dimethylamino)pyridine; EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; ESBL: extended-spectrum β-lactamase; FLU: fluconazole; m-FPhe: 2-amino-3-(3-fluorophenyl)propanoic acid, meta-fluorophenylalanine; o-FPhe: 2-amino-3-(2-fluorophenyl)propanoic acid, ortho-fluorophenylalanine; p-FPhe: 2-amino-3-(4-fluorophenyl)propanoic acid, para-fluorophenylalanine; HepG2: human hepatocellular carcinoma cells; HOBt: 1-hydroxybenzotriazole; INH: isoniazid; MIC: minimum inhibitory concentration; MRSA: methicillin-resistant Staphylococcus aureus; SI: selectivity index.

1. Introduction Unnatural amino acids are important building blocks in medicinal chemistry. Replacement of the hydrogen atom with a fluorine atom with different physicochemical behaviour (lipophilicity, electronic and steric parameters etc.) often results in significant changes in the properties of the parent molecule. The introduction of a fluorine changes proteinogenic α-amino acids to unnatural (non-proteinogenic) species, thus influencing their biological behaviour [1,2], e.g., acting as antimetabolites. Phenylalanines with a fluorine atom in the positions 2, 3 or 4 of the phenyl ring have affected activity of a wide range of proteins and enzymes with many potential medicinal applications including antiviral [3] or anti-cancer agents [4]. Fluorophenylalanines-based modification of beauvericin led to increased cytotoxicity when compared to parent compound [5]. A N-Boc-3fluorophenylalanine derivative showed an antimycobacterial activity. In this case, both removal of N-protected m-FPhe and fluorine led to a decreased activity [6]. m-FPhe as an inhibitor of mycoside C biosynthesis which causes the disruption of the bacterial outer layer was able to enhance the antimycobacterial activity of an isoniazid (INH) derivative in Mycobacterium avium complex [7]. Para-fluorophenylalanine (2-amino-3-(4-fluorophenyl)propanoic acid; p-FPhe) was found to inhibit the incorporation of phenylalanine into proteins in the cell-free system. However, its MIC (minimal inhibitory concentration) activity against Escherichia coli was >250 µg/mL [8]. Laske et al. [9] identified DL-ortho/meta/para-fluorophenylalanines mimicking Phe as inhibitors of aminoacyltRNA synthetase. In this study, racemic fluorinated phenylalanines exhibited MIC values of 87-350 µM against E. coli in minimal medium but no growth inhibition in Müller-Hinton broth containing physiological amino acids. Additionally, there was no direct correlation between enzyme inhibition and antibacterial activity. DL-Meta-fluorophenylalanine (2-amino-3-(3-fluorophenyl)propanoic acid; m-FPhe) was described to be a quorum-sensing systems inhibitor from Pseudomonas aeruginosa interfering with communication of bacterial cells and their virulence [10]. p-FPhe analogues of membrane-active trichogin GA IV were active selectively against Gram-positive bacteria [11]. Di- and tripeptides containing L-meta-fluorophenylalanine were able to inhibit the growth of Candida albicans with MICs ranging from 0.5 to 63 µg/mL, while parent L-m-FPhe and peptides containing D-m-FPhe were inactive. These unnatural peptides enter the cells via active peptide transport, followed by peptidase-mediated intracellular release of L-m-FPhe. In fact, the short peptides serve as temporary carriers for L-m-FPhe, which may act by multiple proposed mechanisms of action. Importantly, the antifungal activity is antagonized in the presence of L-Phe and small proteinogenic peptides competing for uptake mediated by permeases [12]. In 2008, MICs of DL-m-FPhe for five strain of Candida albicans and Saccharomyces cerevisiae were found to be within the range of 128-256 µg/mL and 16-32 µg/mL, respectively. Peptides Leu-m-FPhe and MetMet-m-FPhe showed a higher in vitro efficacy (MIC values of 8-128 µg/mL) and they were also active against S. cerevisiae (4-256 µg/mL). The intracellular cleavage is required to release an active DL-m-FPhe and produce antifungal action. However, their cellular uptake appears to be the rate-limiting step determining their potency [13]. Inspired by the above-mentioned data, we designed and synthesized a novel series of DL-p-FPhe, DL-m-FPhe and DL-o-FPhe aromatic amides and esters. They were intended as simple more lipophilic transport forms (i.e., prodrugs) of unnatural amino acids to improve permeability through biological barriers reflecting the fact that free acid is inactive [12]. These lipophilic derivatives may reach intracellular target sites by passive diffusion, not only by active transport mediated by permeases. Primarily, novel derivatives were prepared for screening their antimicrobial activity.

Based on literature search, an additional set of experiments was performed (cytotoxicity and cholinesterases inhibition properties). Fluorophenylalanines seem to be a versatile scaffold. The choice of these assays is justified by the facts that various fluorine-containing compounds have showed also cytotoxic [14,15], antibacterial, antimycobacterial [15] and antifungal [15,16,17] properties. Moreover, several L-phenylalanine-based amides have been reported as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [18,19]. 2. Results and discussion 2.1 Chemistry N-Acetyl-DL-o/m/p-FPhe amides were synthesized by two ways. Method A employed N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) in the presence of 1hydroxybenzotriazole (HOBt; Scheme 1). This EDC-mediated amidation was used for aniline, ptoluidine and 4-chloroaniline as starting materials providing satisfactory yields within the range of 72-84%. Due to strong electron-withdrawing properties of the CF3 and NO2 moieties, 4(trifluoromethyl)aniline and 4-nitroaniline were converted to amides via PCl3 in dry pyridine (Method B; Scheme 2). This coupling is proved also for very weak nucleophiles. Yields were excellent in the case of 4-CF3-aniline derivatives (87-99 %), while 4-nitroanilides provided moderate yields of 44-58 %.

Scheme 1. Synthesis of amides 1a-3b and 1e-3e (Method A; EDC = N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride; HOBt = 1-hydroxybenzotriazole; Et 3N = triethylamine; DCM = dichloromethane; R = H, Cl, CH3).

Scheme 2. Synthesis of 4-nitro- and 4-(trifluoromethyl)anilides (Method B; R = NO2 (1c-3c), CF3 (1d-3d) Esters 1f-3j were synthesized via carbodiimide (EDC)-mediated esterification employing also DMAP and triethylamine (Scheme 3). The general yields were satisfactory ranging from 80 % up to 98 % with an exception of esterification by 4-nitrophenol. 4-Nitrophenyl derivatives h were obtained in comparatively poorer yields of 37-51 %.

Scheme 3. Synthesis of esters 1f-3j (EDC = N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; DMAP = 4-(dimethylamino)pyridine; Et 3N = triethylamine; DCM = dichloromethane; R = H, Cl, CH3, NO2, CF3). We have chosen substituents with different electronic effects as a substitution pattern for aniline and phenol used for the derivatization of the commercially available N-acetyl-o/m/pfluorophenylalanines 1-3 (Table 1): chlorine (-I and +M effects; amides a and esters f), methyl (+I effect; amides b and esters g), NO2 group (-I and -M effects; amides c and esters h), CF3 (-I effect; amides d and esters i) and H (no effect; amides e and esters j). All compounds were characterized by NMR, IR spectra and melting points. Their purity was checked by TLC and elemental analysis. 1H NMR spectra of N-acetyl-FPhe derivatives 1a-3j measured in DMSO contain doublets of the amidic hydrogen (CH3CONH) in the ranges of 8.258.43 ppm and 8.50-8.65 ppm for amides and esters, respectively. When applied CDCl3 as a solvent, this signal is shifted to ~6 ppm (2h, 3h). Methyl group in N-acetyl moiety produced a sharp singlet in the range of 1.78-1.86 ppm. The split signal at 4.58-4.76 ppm is attributed to aliphatic chiral CH. Diastereotopic hydrogens from “benzylic” CH2 group of amidic derivatives 1a-3e showed two signals in the regions of 2.99-3.20 ppm and 2.81-2.98 ppm; in the case of esters 1f-3j, their chemical shifts were within the range of 3.13-3.23 ppm and 3.05-3.14 ppm. 1H NMR spectra of anilides 1a-3e contain additional singlets attributed to the Ph-NH protons in the ranges of 9.9310.26 ppm (1a-3b, 1e-3e) and 10.41-10.80 ppm in the case of amides substituted by the electronwithdrawing substituents (10.77-10.80 ppm for 4-nitroaniline-based compounds 1c-3c and 10.4110.47 ppm for 4-(trifluoromethyl)anilides 1d and 3d). 4-(Trifluoromethyl)aniline derivative 2d was measured in deuterated acetone thus moving the signal to 9.67 ppm. 13 C NMR spectra contain signals of two C=O carbon atoms (CH3CONH and CONH-Ph in the structure of amides 1a-3e; CH3CONH and COO in the case of esters 1f-3j) in the downfield region around δ 169-172 ppm. The chemical shift of aromatic carbon in the neighbourhood of fluorine was observed in the range of 160-163 ppm. This signal is detected as a doublet with coupling constant values around 243 Hz. Signals of benzene aromatic carbons appear at 113-146 ppm. Methyl group from N-acetyl moiety produced singlet at ~22.5 ppm, while benzylic CH2 was present in a comparatively broader range of ~30-38 ppm; esters have a predominant tendency to lower values from this span. The signals of tertiary CH carbon appear in the region with chemical shift around 52-56 ppm. In the IR spectra of amides 1a-3e, most of the derivatives displayed two visible N-H stretch bands attributed to acetamide and N-phenylcarboxamide moieties. First of them appears at 3336-3356 cm– 1 for 4-nitroanilides c and 3290-3332 cm–1 for remaining derivatives, the second one at 3261-3290 cm–1. Amide I bands were observed at 1630-1652 cm–1, while amide bands II at 1533-1564 cm–1. In many cases, peaks of both carboxamide group overlapped and merged. Nitro groups from all of the derivatives showed typical two very intense peaks at 1506-1522 cm−1 (υ asym.) and 1342-1347 cm−1 (υ sym.). In the IR spectra of esters 1f-3j, an additional sharp and strong band appears in the

range of 1742-1762 cm−1 (C=O ester). The stretching frequency of N-H (CH3CONH) was recorded at 3295-3334 cm−1. Absorptions of amide I and amide II were found between 1638-1658 cm−1 and 1533-1544 cm−1, respectively. Table 1. An overview of fluorophenylalanine derivatives 1-3j Code 1 2 3

Amides 1a 2a 3a 1b 2b 3b 1c 2c 3c 1d 2d 3d 1e 2e 3e

Structure N-acetyl-DL-o-FPhe N-acetyl-DL-m-FPhe N-acetyl-DL-p-FPhe

X NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH

R Cl Cl Cl CH3 CH3 CH3 NO2 NO2 NO2 CF3 CF3 CF3 H H H

F 234234234234234-

Chemical name 2-acetamido-3-(2-fluorophenyl)propanoic acid 2-acetamido-3-(3-fluorophenyl)propanoic acid 2-acetamido-3-(4-fluorophenyl)propanoic acid

Esters 1f 2f 3f 1g 2g 3g 1h 2h 3h 1i 2i 3i 1j 2j 3j

X O O O O O O O O O O O O O O O

R Cl Cl Cl CH3 CH3 CH3 NO2 NO2 NO2 CF3 CF3 CF3 H H H

F 234234234234234-

2.2 Biology 2.2.1 Inhibition of acetylcholinesterase and butyrylcholinesterase Fluorophenylalanine-based compounds 1-3j were investigated for their inhibitory potency against AChE from electric eel and BChE from equine serum using modified Ellman’s method. The results (Table 2) were compared with those obtained for two drugs, rivastigmine and galantamine. Carbamate rivastigmine acts as an acylating pseudoirreversible inhibitor, while galantamine is as a non-acylating competitive reversible inhibitor of ChE. Table 2. Inhibition of cholinesterases, cytotoxicity and selectivity data of FPhe 1-3j Code 1 2 3 1a 2a 3a 1b 2b 3b 1c 2c 3c

AChE IC50 [µM] 64.25±0.37 63.61±2.53 63.68±0.05 76.52±2.48 70.69±0.78 97.03±0.01 69.03±1.04 68.70±1.08 68.97±0.88 64.44±0.42 70.57±1.10 66.00±0.38

BChE IC50 [µM] 217.01±6.07 210.30±6.88 226.99±14.39 227.43±10.57 169.57±0.48 235.41±11.65 113.04±1.06 142.79±5.65 154.28±2.94 154.23±2.51 119.52±0.80 161.90±1.13

AChE/ BChE ratio 0.29 0.30 0.28 0.34 0.42 0.41 0.61 0.48 0.45 0.42 0.59 0.41

SI for AChE 64.9 57.8 56.4 >1.3 >1.4 >1.0 >1.4 >1.5 >0.7 3.4 >10.6 >0.8

SI for BChE 19.2 17.5 15.8 >0.4 >0.6 >0.4 >0.9 >0.7 >0.3 1.4 >6.3 >0.3

IC50 [µM] 4,167 3,674 3,589 >100* >100** >100* >100** >100** >50** 217.9 >750** >50**

Tested range of concentrations [µM] 10-10,000 1-10,000 10-10,000 1-100* 1-1,000 1-100* 10-2,500 10-2,500 10-2,500 1-2,000 10-2,000 1-1,000

73.42±0.61 217.55±6.05 0.34 >1.4 >0.5 >100** 1-1,000 1d 79.38±0.54 163.71±1.50 0.48 >3.1 >1.5 >250** 1-1,000 2d 84.14±0.22 289.00±6.79 0.29 >1.2 >0.3 >100* 1-100* 3d 86.25±4.82 121.06±3.52 0.71 >5.8 >4.1 >500** 10-2,500 1e 86.51±5.46 156.95±0.97 0.55 >5.8 >3.2 >500** 10-5,000 2e 99.83±3.05 89.79±2.86 1.11 >2.5 >2.8 >250** 10-5,000 3e 103.51±8.15 82.19±1.01 1.26 5.7 7.1 586.5 10-10,000 1f 114.59±9.25 52.70±0.83 2.17 4.5 9.7 513.8 10-10,000 2f 130.75±0.50 59.11±0.20 2.21 1.8 3.9 233.2 10-5,000 3f 85.36±1.26 0.68 8.7 5.9 503.4 10-5,000 1g 57.88±0.21 68.52±1.26 90.93±1.42 0.75 9.2 7.0 633.0 10-5,000 2g 73.33±2.16 83.27±0.16 0.88 5.8 5.1 425.8 10-5,000 3g 76.72±0.23 77.47±1.23 0.99 3.0 2.9 226.5 10-5,000 1h 74.28±0.34 142.41±6.44 0.52 5.0 2.6 369.1 10-5,000 2h 126.45±0.40 0.47 5.1 2.4 303.7 10-1,000 3h 59.97±0.39 67.97±0.27 37.42±0.02 1.82 5.2 9.5 356.5 10-7,500 1i 62.21±0.29 33.97±1.58 1.83 4.2 7.6 258.4 10-7,500 2i 62.86±0.53 59.77±0.42 1.05 5.2 5.5 329.8 10-7,500 3i 79.22±1.66 2.64 922.2 10-5,000 1j 29.97±0.19 11.6 30.8 87.82±3.33 53.67±0.67 1.64 9.4 826.3 10-5,000 2j 15.4 98.34±0.73 >1.0 >100* 1-100* 3j 8.25±0.22 11.92 >12.1 56.10±1.41 38.40±1.97 0.7 rivastigmine 1.54±0.02 2.77±0.15 1.8 galantamine Selectivity indexes higher than 10 and the lowest IC50 values for each enzyme are shown in bold. *testing of cytotoxicity at higher concentrations was unable due to the limited solubility of the tested compound in DMSO **measurement at higher concentration was unable due to the precipitation of the tested compound in the medium

All of the derivatives showed a considerable inhibition of both acetylcholine hydrolysing enzymes (Table 2) with IC50 values for AChE and BChE of 57.88-130.75 µM and 8.25 to 289.0 µM, respectively. Obviously, the IC50 values for butyrylcholinesterase are in a comparatively broader range than for AChE. These IC50 values are comparable or superior to several groups of recently reported cholinesterases inhibitors [20,21]. 4-Methylphenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1g was found to be the most effective inhibitor of AChE, whereas 4-chlorophenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3f exhibited the lowest inhibition of this enzyme. In the case of BChE, phenyl 2-acetamido-3-(4fluorophenyl)propanoate 3j produced the most powerful inhibition (IC50 = 8.25 µM). On the other hand, inhibitory potency of 2-acetamido-3-(4-fluorophenyl)-N-[4(trifluoromethyl)phenyl]propanamide 3d was more than thirty-five times weaker (289.0 µM). The activities of FPhe derivatives 1-3j were compared with IC50 values of rivastigmine and galantamine, two drugs used clinically for the treatment of Alzheimerʼs disease and other dementias. Four derivatives (i.e., esters 1g, 3h, 2i, 3i) were comparable to rivastigmine in AChE inhibition in vitro. Focusing on BChE, two derivatives (1j, 3j) produced a higher activity than this drug. Additionally, two esters (1i, 2i) caused a comparable enzyme inhibition. Galantamine is a more efficient inhibitor of both enzymes than any of the tested compounds. The parent amino acid 1-3 showed almost identical inhibition of AChE (IC50 ~64 µM) as well as BChE (210-227 µM). Their modification did not offer significantly improved activity towards AChE, while the esters and the amides share mostly an enhanced inhibition of BChE with only four exceptions (anilides 1a, 1d, 3a, 3d), even up to 27.5 times (3 vs. 3j). With respect to positional isomerism of the fluorine atom, isomers were comparable towards AChE or no clear tendency was observed in their activity towards BChE.

The amides caused a more uniform inhibition of AChE than the corresponding esters (64.4-99.8 µM vs. 57.9-130.8 µM). For BChE, the esters seem to be better inhibitors than the amides (IC 50 values of 8.3-142.4 µM vs. 89.8-289.0 µM). Just one amide (3e; 2-acetamido-3-(4-fluorophenyl)-Nphenylpropanamide) exhibited IC50 lower than 100 µM. The explanation of different activity of amides and esters may consist in, e.g., missing hydrogen and thus a different interaction with cholinesterases, altered lipophilicity or changes in steric and/or electronic parameters. The structure-activity relationships for the substitution of the phenol/aniline benzene are reported in Table 3. In general, the introduction of NO2 and CH3 groups into aniline ring and CF3 moiety into phenol decreased IC50 values, while chlorine did not offer any desired improvement of the activity. The presence of CH3 and NO2 is also favourable for the inhibition of AChE and hydrogen enhances activity for BChE. Table 3. SAR for the 4-substitution of aniline/phenol ring

Enzyme AChE BChE

Improving activity X = NH X=O CH3, NO2 CH3, NO2, CF3 CH3, NO2, H H, CF3

Decreasing activity X = NH X=O H Cl Cl, CF3 NO2

Considering the selectivity for AChE versus BChE (Table 2), fourteen derivatives led to a stronger inhibition of AChE (i.e., AChE/BChE IC50 values ratio lower than 0.5; parent acids 1-3, amides 1a3a, 2b, 3b, 1c, 3c, 1d-3d, and one ester, 3f). Fifteen compounds showed a balanced inhibition of both cholinesterases (AChE/BChE ratio within the range of 0.5-2.0; amides 1b, 2c, 1e-3e and esters 1g, 1g-2h, 1i-3i, 2j). Only four esters (2f, 3f, 1j, and 3j) were identified as significantly more potent inhibitors of BChE (AChE/BChE ratio >2). Phenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3j produced about 12-fold stronger inhibition of BChE thus exceeding the value of 10 which is considered to be borderline for the selectivity. In general, the amides are more selective to AChE than the corresponding esters. 2.2.2 Cytotoxicity and selectivity We also investigated cytotoxicity of the fluorophenylalanine derivatives in human hepatocellular carcinoma (HepG2) cell model (Table 2). Obviously, the esters 1f-3j and the free acids 1-3 were more soluble than the amides 1a-3e. Limited solubility of the amides in the testing medium complicated this assessment, therefore exact IC50 value was determined only for one amide (1c). IC50 values of the esters ranged from 226.5 up to 922.2 µM (compound 1a and 1j, respectively). In sum, the esterification of the starting amino acids led to the compounds with an increased cytotoxicity. The esters of 4-nitrophenol h and 4-(trifluoromethyl)phenol i share a more pronounced cytotoxicity than those derived from the other phenols. The unsubstituted phenol provided the esters with the lowest toxicity (1j, 2j). Regarding enzyme inhibition and cytotoxicity for HepG2, selectivity indexes (SI) were calculated for both cholinesterases (SI = IC50 (HepG2)/IC50 (enzyme)). Due to a substantially low cytotoxicity, the parent acids 1-3 share a sufficient selectivity for both AChE (SI of 56.4-64.9) and BChE (15.8-

19.2) with N-acetyl-o-FPhe superiority. In general, exact selectivity indexes could not be calculated for the majority of amide derivatives due to their limited solubility during cytotoxicity testing. 2Acetamido-3-(3-fluorophenyl)-N-(4-nitrophenyl)propanamide 2c showed SI of >10.6 for AChE. Most SI values of the esters were poor, i.e., they did not reach breakpoint of 10. Only derivatives of unsubstituted phenol 1j-3j exhibited a satisfactory selectivity for BChE (>12.1) and in the case of phenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1j also for AChE (11.6), thus indicating a comparative safety. Antimicrobial activity Fluorinated N-acetylphenylalanine-based amides and esters were evaluated for their in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, eight fungal species (Table 4) and both tuberculous and non-tuberculous mycobacteria (Table 5). Only susceptible strains are reported. Bacteria Enterococcus sp., E. coli, Klebsiella pneumoniae including an ESBLpositive strain, Pseudomonas aeruginosa and fungi Candida albicans, C. krusei, C. glabrata and Absidia corymbifera share a complete resistance. Among bacteria, only Staphylococci were susceptible to two derivatives (2h, 3g) with comparatively high MIC values of 250-500 µM. Focusing on fungal species, C. tropicalis and A. fumigatus were inhibited each one by only one ester (3g and 1h, respectively) at 500 µM. Derivatives 3f and 3g showed also a weak effect against T. asahii (MIC ≥ 250 µM). Trichophyton mentagrophytes was identified as the most susceptible strains (eight active derivatives with MIC values ≥ 125 µM). Obviously, neither antibacterial nor antifungal effect was detected for any amidic derivative 1a-3e, the activity was exhibited only by esters, although it is rather weak. In general, esters synthesized from N-acetyl-o-FPhe 1 and N-acetyl-p-FPhe 3 produced more frequently in vitro antimicrobial activity when compared to N-acetyl-m-FPhe esters 2. Derivatives of unsubstituted aniline e or phenol j were completely inactive indicating the importance of 4-substitution of the phenyl ring. Table 4. Antibacterial and antifungal activity of fluorophenylalanine derivatives Code 1f 3f 1g 3g 1h 2h 3h 1i FLU BAC

MIC [µM] MRSA SE CT TA AF TM 24 h 48 h 24 h 48 h 24 h 24 h 24 h 48 h 24 h 48 h 24 h 48 h 72 h 120 h >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 250 250 >250 >250 >250 >250 >250 >250 >250 >250 >250 >250 >250 250 250 250 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 250 250 >500 >500 >500 >500 500 500 500 500 250 500 500 500 125 125 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 500 500 500 500 >500 >500 >500 >500 >500 >500 250 250 250 250 250 250 250 250 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 250 250 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 125 125 >500 >500 250 250 >500 >500 7.81 125 7.81 15.62 15.62 15.62 15.62 31.25 SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; CT: Candida tropicalis 156; TA: Trichosporon asahii 1188; AF: Aspergillus fumigatus 231; TM: Trichophyton mentagrophytes 445. FLU: fluconazole, BAC: bacitracin. SA

With respect to the antimycobacterial activity (Table 5), majority of the amides 1a-3e showed no inhibition of all mycobacterial strains. Only two amidic derivatives of o-FPhe (1d, 1e) were identified as potential inhibitors with MIC value of 250 µM. Contrarily, compounds 1h and 1j from total fifteen esters were inactive. 2-Acetamido-3-(2-fluorophenyl)-N-phenylpropanamide 1e was the

most active antimycobacterial agent within this series with a uniform MIC of 250 µM against all of the mycobacteria. M. tuberculosis and both strains of M. kansasii exhibited similar susceptibility (MIC within the range of 250-1000 µM), whereas M. avium was inhibited at a higher concentration of 1000 µM and only by five esters (1f, 2f, 3f, 2h, and 3h). In general, esters of 4-chlorophenol (1f3f) were superior to other phenols involved in this study. The presence of any 4-substituent (Cl, CH3, NO2, CF3) on phenol enhances antimycobacterial activity when compared to hydrogen (esters 1j-3j). There is no significant difference between derivatives of particular isomeric Nacetylfluorophenylalanines. Table 5. Antimycobacterial activity of N-acetylfluorophenylalanine esters and amides MIC [µM] M. tuberculosis M. avium 330/88 M. kansasii 235/80 M. kansasii 6509/96 Code 331/88 14 d 21 d 14 d 21 d 7d 14 d 21 d 7d 14 d 21 d 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 1a 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 2a 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 3a 500* 500* 500* 500* 500* 500* 500* 500* 500* 500* 1b 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 2b 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 3b 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 1c 500* 500* 500* 500* 500* 500* 500* 500* 500* 500* 2c 125* 125* 125* 125* 125* 125* 125* 125* 125* 125* 3c 500* 500* 500* 500* 1d 250 250 250 250 250 250 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 2d 250* 250* 250* 250* 250* 250* 250* 250* 250* 250* 3d 1e 250 250 250 250 250 250 250 250 250 250 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 2e >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 3e 1f 500 1000 1000 1000 250 500 1000 250 500 1000 >1000 2f 500 1000 1000 500 500 1000 250 500 1000 >1000 3f 500 1000 1000 250 500 1000 250 500 1000 >1000 >1000 >1000 >1000 >1000 1g 1000 1000 1000 500 1000 >1000 >1000 >1000 >1000 >1000 2g 1000 1000 1000 500 1000 >1000 >1000 >1000 >1000 >1000 3g 1000 1000 1000 500 1000 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 1h >1000 2h 1000 1000 1000 500 1000 1000 500 1000 1000 >1000 3h 1000 1000 1000 500 1000 1000 500 1000 1000 >1000 >1000 1i 500 500 1000 1000 1000 1000 1000 1000 >1000 >1000 2i 500 500 500 1000 1000 1000 1000 1000 >1000 >1000 3i 500 500 500 1000 1000 1000 1000 1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 1j >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 2j 500 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 3j 500 0.5 1 >250 >250 >250 >250 >250 8 8 8 INH INH: isoniazid. *: at presented concentration grow of strain was observed, at duplex concentration there was present precipitate and/or turbidity; the determination of exact MIC value was not possible.

Parent acids 1-3 avoided any antimycobacterial activity (MIC >1000 µM). Analogous derivatives of N-acetyl-DL-Phe, N-acetyl-L-Phe and N-acetyl-D-Phe were also inactive in antimycobacterial, antibacterial and antifungal assays (data not shown). These findings establish a clear evidence that both modification of the free carboxyl and the presence of fluorine are crucial for the antimicrobial properties. Although newly synthesized derivatives 1a-3j share generally improved MIC values when compared to parent acids 1-3, their activity remains from moderate to negligible in spite of

increased lipophilicity (e.g., calculated logP value for N-acetyl-p-FPhe 3 is 0.87, for its Nphenylamide 3e of 2.12 and for phenylester 3j of even 2.8). An increased lipophilicity facilitates passive transport through biological barriers. A possible reason of the observed discrepancy was described previously – an antagonism of the antibacterial action of DL-fluorophenylalanines caused by the presence of natural amino acids in testing medium [9]. Based on previous reports, the proposed mechanism of action may consist in an antimetabolite action of released fluorophenylalanines affecting multiple biochemical pathways [9,12]. The esters 1f-3j caused uniformly a more effective growth inhibition of fungal and bacterial pathogens than their corresponding amides 1a-3e. The explanation may consist in a different interaction with intracellular target(s) due to missing hydrogen, higher lipophilicity of esters, changes in other physicochemical parameters, or in their combination. According to Wakiec et al. [13], the intracellular hydrolysis of the DL-m-FPhe derivatives is necessary for antifungal activity. From this point of view, aromatic esters may be hydrolysed more rapidly than amides intracellularly. However, IC50 values for hepatocytes are similar to MIC values obtained for fungi and bacteria including mycobacteria. All of the fluorinated derivatives were handled as racemates. 3. Conclusions Thirty N-acetyl-fluorophenylalanine-based compounds were synthesized using PCl3 or carbodiimide-mediated coupling with satisfactory yields. Subsequently, they underwent a screening of their potential biological activity. Importantly, all of the amino acid derivatives showed from weak to moderate inhibition of both acetylcholinesterase and butyrylcholinesterase. The modification of parent acids offers more active inhibitors of BChE but not AChE. IC50 values for BChE were within a broader concentration range when compared to AChE. One derivative produced a selective inhibition of BChE; otherwise, the inhibition is non-selective. Several derivatives were comparable or superior to rivastigmine. With respect to the substitution of ester/amidic benzene nuclei, the introduction of NO2 and CH3 groups into aniline ring and CF3 moiety into phenol enhanced activity. The expected antimicrobial activity of novel aromatic esters and amides was not found in in vitro assays. In general, the amidic derivatives avoided any significant antibacterial, antimycobacterial and antifungal properties. Drawing a comparison, the majority of the esters exhibited a mild antimycobacterial activity against both tuberculous and non-tuberculous mycobacteria and several of them also inhibited the growth of Gram-positive bacteria and Trichophyton mentagrophytes. Focusing on cytotoxicity, IC50 values for eukaryotic cell line was not sharply different from MIC obtained for prokaryotes. Contrarily, a range of investigated derivatives provided a sufficient selectivity for cholinesterases indicating a comparative safety. 4. Material and Methods 4.1 Chemistry 4.1.1 General All of the reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) or Penta Chemicals (Prague, Czech Republic), and they were used as received. The progress of the reactions and the purity of the products were monitored by thin layer chromatography (TLC) with a mixture of toluene with ethyl acetate (4:1, v/v) as eluent; plates were coated with 0.2 mm Merck 60 F254 silica gel and were visualised by UV irradiation (254 nm). Melting points were determined on

a Büchi Melting Point Machine B-540 apparatus (BÜCHI, Flawil, Switzerland) using open capillaries, and the reported values are uncorrected. Elemental analysis (C, H, N) was performed on an automatic microanalyser CHNS-O CE instrument (FISONS EA 1110, Milano, Italy). Infrared spectra (ATR) were recorded on FT-IR spectrometer Nicolet 6700 FT-IR in the range of 400 to 4,000 cm-1. The NMR spectra were measured in CDCl3, DMSO-d6 or in acetone-d6 at ambient temperature on a Varian VNMR S500 instrument (500 MHz for 1H and 125 MHz for 13C; Varian Comp. Palo Alto, CA, USA) or a Varian Mercury-Vxbb 300 (300 MHz for 1H and 75.5 MHz for 13C; Varian, Inc., Palo Alto, CA, USA). The chemical shifts, δ, are given in ppm, with respect to tetramethylsilane as an internal standard. The coupling constants (J) are reported in Hz. The calculated logP values that are the logarithms of the partition coefficients for octan-1-ol/water, were determined using the program CS ChemOffice Ultra version 15.0 (CambridgeSoft, Cambridge, MA, USA). 4.1.2 Synthesis 4.1.2.1 Synthesis of 2-acetamido-3-(2/3/4-fluorophenyl)-N-phenylpropanamides Method A An equivalent of N-acetyl-2/3/4-fluorophenylalanine (1, 2 or 3) together with 1hydroxybenzotriazole (HOBt; 1.1 of equivalents of HOBt hydrate) and 3 equivalents of triethylamine (Et 3N) were dissolved in dichloromethane (DCM; 8 mL), followed by an addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; 1.5 of equivalents) in one portion. After 5 minutes, an equivalent of appropriate aniline was added. The reaction was monitored using TLC. The mixture was stirred at room temperature overnight. Then, the solution was evaporated till dryness, ethyl acetate was added. Resulting suspension was washed twice with 0.1 M aq HCl, 5% aq sodium bicarbonate, followed by brine. The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under reduced pressure and it was added n-hexane to start crystallization. After 24 hours at +4 °C the suspension was filtered off to give afforded amides 2. If necessary, they were recrystallized from ethyl acetate. Method B A solution of the amino acid 1, 2 or 3 (1 mmol) and 4-nitroaniline or 4-(trifluoromethyl)aniline (1 mmol) in dry pyridine (6 mL) was cooled to -10 °C, and phosphorus trichloride (1 mmol) was added dropwise under vigorous stirring. The reaction mixture was stirred for 2 h, then it was evaporated untill dryness and DCM was added. Resulting mixture was washed successively twice with 0.1 M aq HCl, 5% aq sodium bicarbonate, followed by brine. The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under reduced pressure and it was added n-hexane to start crystallization. It was performed for 24 hours at +4 °C. Then, the precipitate was filtered off to give appropriate anilides. 2-Acetamido-N-(4-chlorophenyl)-3-(2-fluorophenyl)propanamide 1a (method A). White solid; yield 77%; mp 209-212.5 °C. IR (ATR): 3317 (NH), 3261 (NH), 2932, 2851, 1689, 1647 (C=O, amide I), 1623, 1549 (C=O, amide II), 1493, 1434, 1403, 1379, 1312, 1283, 1244, 1232, 1179, 1118, 1092, 1001, 824, 794, 778, 755, 725, 666 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.16 (1H, s, NH-Ph), 8.29 (1H, d, J = 8.2 Hz, NH-Ac), 7.57 (2H, d, J = 8.8 Hz, H2´, H6´), 7.33 (2H, d, J = 8.8 Hz, H3´, H5´), 7.30-7.21 (2H, m, H4, H6), 7.14-7.06 (2H, m, H3, H5), 4.69 (1H, td, J = 8.2 Hz, J = 6.3 Hz, CH), 3.05 (1H, dd, J = 13.9 Hz, J = 6.3 Hz, CH2), 2.90 (1H, dd, J = 13.9 Hz, J = 8.3 Hz,

1H), 1.80 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.15, 169.42, 160.88 (d, J = 244.2 Hz), 137.79, 131.64 (d, J = 4.6 Hz), 128.78 (d, J = 8.1 Hz), 128.72, 127.22, 124.22 (d, J = 15.6 Hz), 124.20 (d, J = 3.4 Hz), 121.22, 115.17 (d, J = 21.9 Hz), 53.54, 31.13 (d, J = 2.1 Hz), 22.55. Anal. Calcd. for C17H16ClFN2O2 (334.77): C, 60.99; H, 4.82; N, 8.37. Found: C, 60.77; H, 5.04; N, 8.39. 2-Acetamido-N-(4-chlorophenyl)-3-(3-fluorophenyl)propanamide 2a (method A). White solid; yield 84%; mp 256.5-258.5 °C. IR (ATR): 3301 (NH), 3261 (NH), 3194, 3127, 3078, 2930, 2852, 1686, 1643 (C=O, amide I), 1612, 1549 (C=O, amide II), 1491, 1449, 1403, 1380, 1309, 1271, 1253, 1177, 1146, 1115, 1090, 1012, 943, 892, 825, 779, 742, 690, 669 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 10.24 (1H, s, NH-Ph), 8.34 (1H, d, J = 8.1 Hz, NH-Ac), 7.64-7.56 (2H, m H2´, H6´), 7.39-7.25 (3H, m, H5, H3´, H5´), 7.15-6.96 (3H, m, H2, H4, H6), 4.63 (1H, td, J = 9.3 Hz, J = 5.2 Hz, CH), 3.03 (1H, dd, J = 13.7 Hz, J = 5.2 Hz, CH2), 2.85 (dd, J = 13.6 Hz, J = 9.6 Hz, CH2), 1.79 (3H, s, CH3). 13C NMR (75 MHz, DMSO-d6): δ 170.75, 169.79, 162.39 (d, J = 242.9 Hz), 141.01 (d, J = 7.5 Hz), 138.13, 130.37 (d, J = 8.5 Hz), 129.09, 127.43, 125.73 (d, J = 2.7 Hz), 121.27, 116.29 (d, J = 21.2 Hz), 113.66 (d, J = 20.7 Hz), 55.15, 33.80, 22.80. Anal. Calcd. for C17H16ClFN2O2 (334.77): C, 60.99; H, 4.82; N, 8.37. Found: C, 61.07; H, 4.74; N, 8.16. 2-Acetamido-N-(4-chlorophenyl)-3-(4-fluorophenyl)propanamide 3a (method A). White solid; yield 77%; mp 231.5-234 °C. IR (ATR): 3320 (NH), 3290 (NH), 3190, 3136, 3077, 2930, 2851, 1680, 1630 (C=O, amide I), 1613, 1573, 1549 (C=O, amide II), 1510, 1493, 1436, 1405, 1380, 1311, 1273, 1244, 1224, 1181, 1159, 1116, 1090, 1047, 1012, 893, 879, 826, 803, 764, 730, 667 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.26 (1H, s, NH-Ph), 8.35 (1H, d, J = 8.1 Hz, NH-Ac), 7.60 (2H, d, J = 8.4 Hz, H2´, H6´), 7.35 (2H, d, J = 8.5 Hz, H3´, H5´), 7.29 (2H, dd, J = 8.3 Hz, J = 5.6 Hz, H2, H6), 7.08 (2H, t, J = 8.7 Hz, H3, H5), 4.59 (1H, td, J = 8.9 Hz, J = 5.3 Hz, CH), 2.99 (1H, dd, J = 13.7 Hz, J = 5.2 Hz, CH2), 2.82 (1H, dd, J = 13.7 Hz, J = 9.5 Hz, CH2), 1.79 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.59, 169.49, 161.16 (d, J = 241.8 Hz), 137.90, 133.96 (d, J = 2.9 Hz), 131.12 (d, J = 8.1 Hz), 128.78, 127.13, 121.03, 114.96 (d, J = 21.0 Hz), 55.18, 33.52, 22.53. Anal. Calcd. for C17H16ClFN2O2 (334.77): C, 60.99; H, 4.82; N, 8.37. Found: C, 60.89; H, 5.00; N, 8.49. 2-Acetamido-3-(2-fluorophenyl)-N-(4-methylphenyl)propanamide 1b (method A). White solid; yield 79%; mp 228-229 °C. IR (ATR): 3315 (NH), 3267 (NH), 3203, 3135, 1681, 1645 (C=O, amide I), 1612, 1546 (C=O, amide II), 1514, 1494, 1454, 1432, 1408, 1380, 1316, 1282, 1246, 1232, 1208, 1184, 1176, 1118, 1092, 1038, 953, 933, 877, 848, 816, 801, 777, 753, 724 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 9.93 (1H, s, NH-Ph), 8.25 (1H, d, J = 8.3 Hz, NH-Ac), 7.42 (2H, d, J = 8.4 Hz, H2´, H6´), 7.32-7.22 (2H, m, H4, H6), 7.14-7.05 (4H, m, H3, H5, H3´, H5´), 4.71 (1H, td, J = 8.3 Hz, J = 6.2 Hz, CH), 3.05 (1H, dd, J = 13.9 Hz, J = 6.2 Hz, CH2), 2.90 (1H, dd, J = 13.9 Hz, J = 8.4 Hz, 1H), 2.23 (3H, s, CH3-Ph), 1.80 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 169.67, 169.31, 160.88 (d, J = 244.2 Hz), 136.31, 132.53, 131.64 (d, J = 4.6 Hz), 129.15, 128.69 (d, J = 8.1 Hz), 124.36 (d, J = 15.7 Hz), 124.16 (d, J = 3.4 Hz), 119.72, 115.14 (d, J = 21.9 Hz), 53.42, 31.22 (d, J = 2.2 Hz), 22.57, 20.60. Anal. Calcd. for C18H19FN2O2 (314.35): C, 68.77; H, 6.09; N, 8.91. Found: C, 68.50; H, 6.12; N, 8.84. 2-Acetamido-3-(3-fluorophenyl)-N-(4-methylphenyl)propanamide 2b (method A). White solid; yield 84%; mp 240.5-242 °C. IR (ATR): 3290 (NH), 3265 (NH), 3204, 3140, 3080, 1676, 1640 (C=O, amide I), 1613, 1551 (C=O, amide II), 1511, 1438, 1410, 1381, 1316, 1283, 1249, 1223,

1176, 1160, 1146, 1114, 1023, 996, 953, 941, 880, 832, 813, 773, 756, 722, 690 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 9.98 (1H, s, NH-Ph), 8.26 (1H, d, J = 8.3 Hz, NH-Ac), 7.47 (2H, d, J = 8.4 Hz, H2´, H6´), 7.33-7.28 (1H, m, H5), 7.13-6.99 (5H, m, H2, H4, H6, H3´, H5´), 4.76 (1H, td, J = 8.5 Hz, J = 6.2 Hz, CH), 3.20 (1H, dd, J = 14.8 Hz, J = 5.9 Hz, CH2), 2.98 (1H, dd, J = 14.2 Hz, J = 8.4 Hz, CH2), 2.23 (3H, s, CH3-Ph), 1.78 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 169.99, 169.42, 162.12 (d, J = 242.7 Hz), 140.86 (d, J = 7.8 Hz), 136.39, 132.51, 130.04 (d, J = 8.3 Hz), 129.24, 125.47 (d, J = 2.6 Hz), 119.52, 116.01 (d, J = 21.0 Hz), 113.31 (d, J = 20.7 Hz), 54.74, 37.61, 22.55, 20.61. Anal. Calcd. for C18H19FN2O2 (314.35): C, 68.77; H, 6.09; N, 8.91. Found: C, 68.60; H, 5.94; N, 8.88. 2-Acetamido-3-(4-fluorophenyl)-N-(4-methylphenyl)propanamide 3b (method A). White solid; yield 80%; mp 264-265 °C. IR (ATR): 3294 (NH), 3269 (NH), 3206, 3140, 3084, 2987, 2940, 1676, 1640 (C=O, amide I), 1612, 1549 (C=O, amide II), 1510, 1437, 1410, 1381, 1317, 1287, 1248, 1223, 1176, 1160, 1114, 1023, 953, 936, 879, 834, 812, 755, 723, 696 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.00 (1H, s, NH-Ph), 8.26 (1H, d, J = 8.3 Hz, NH-Ac), 7.45 (2H, d, J = 8.4 Hz, H2´, H6´), 7.32-7.28 (2H, m, H2, H6), 7.12-7.05 (4H, m, H3, H5, H3´, H5´), 4.61 (1H, td, J = 9.2 Hz, J = 5.2 Hz, CH), 2.99 (1H, dd, J = 13.7 Hz, J = 5.2 Hz, CH2), 2.81 (1H, dd, J = 13.7 Hz, J = 9.5 Hz, CH2), 2.23 (3H, s, CH3-Ph), 1.78 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.10, 169.38, 161.13 (d, J = 241.9 Hz), 136.43, 134.07 (d, J = 3.2 Hz), 132.46, 131.13 (d, J = 8.1 Hz), 129.22, 119.51, 114.91 (d, J = 21.1 Hz), 55.01, 37.15, 22.57, 20.61. Anal. Calcd. for C18H19FN2O2 (314.35): C, 68.77; H, 6.09; N, 8.91. Found: C, 68.88; H, 6.01; N, 8.94. 2-Acetamido-3-(2-fluorophenyl)-N-(4-nitrophenyl)propanamide 1c (method B). Yellow solid; yield 47%; mp 259.5-262 °C. IR (ATR): 3343 (NH), 3266 (NH), 3224, 3163, 3096, 1691, 1650 (C=O, amide I), 1620, 1599, 1564 (C=O, amide II), 1539, 1506 (NO2), 1496, 1456, 1412, 1380, 1345 (NO2), 1331, 1302, 1282, 1253, 1232, 1209, 1176, 1113, 1038, 955, 864, 854, 794, 757, 749, 712, 689 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.77 (1H, s, NH-Ph), 8.43 (1H, d, J = 7.9 Hz, NHAc), 8.19 (2H, d, J = 8.9 Hz, H3´, H5´), 7.83 (2H, d, J = 8.8 Hz, H2´, H6´), 7.33-7.05 (4H, m, H3, H4, H5, H6), 4.72 (1H, q, J = 7.7 Hz, CH), 3.08 (1H, dd, J = 14.0 Hz, J = 6.4 Hz, CH2), 2.96 (1H, dd, J = 14.0 Hz, J = 8.4 Hz, 1H), 1.81 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 171.44, 169.85, 161.15 (d, J = 244.3 Hz), 145.38, 142.80, 131.93 (d, J = 4.5 Hz), 129.15 (d, J = 8.1 Hz), 125.31, 124.54 (d, J = 3.4 Hz), 124.32 (d, J = 15.7 Hz), 119.59, 115.48 (d, J = 21.9 Hz), 54.21, 31.20, 22.80. Anal. Calcd. for C17H16FN3O4 (345.33): C, 59.13; H, 4.67; N, 12.17. Found: C, 59.40; H, 4.81; N, 11.94. 2-Acetamido-3-(3-fluorophenyl)-N-(4-nitrophenyl)propanamide 2c (method B). Yellow solid; yield 44%; mp 205.5-208 °C. IR (ATR): 3356 (NH), 3270 (NH), 3078, 2938, 2842, 1693, 1645 (C=O, amide I), 1622, 1593, 1564 (C=O, amide II), 1512 (NO2), 1495, 1445, 1413, 1381, 1342 (NO2), 1291, 1259, 1215, 1180, 1145, 1113, 1000, 942, 888, 868, 851, 787, 751, 692, 685 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.79 (1H, s, NH-Ph), 8.41 (1H, d, J = 7.9 Hz, NH-Ac), 8.21 (2H, d, J = 8.8 Hz, H3´, H5´), 7.84 (2H, d, J = 8.9 Hz, H2´, H6´), 7.32-7.28 (1H, m, H5), 7.16-6.98 (3H, m, H2, H4, H6), 4.76 (1H, td, J = 9.0 Hz, J = 5.4 Hz, CH), 3.07 (1H, dd, J = 13.7 Hz, J = 5.4 Hz, CH2), 2.88 (dd, J = 13.8 Hz, J = 8.5 Hz, CH2), 1.80 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 171.44, 169.68, 162.14 (d, J = 241.9 Hz), 145.10, 142.52, 140.56 (d, J = 7.5 Hz), 130.12 (d, J = 8.5 Hz), 125.45, 125.13, 119.18, 116.05 (d, J = 21.1 Hz), 113.46 (d, J = 21.0 Hz), 55.18, 37.13, 22.47.

Anal. Calcd. for C17H16FN3O4 (345.33): C, 59.13; H, 4.67; N, 12.17. Found: C, 58.98; H, 4.55; N, 12.24. 2-Acetamido-3-(4-fluorophenyl)-N-(4-nitrophenyl)propanamide 3c (method B). Yellow solid; yield 58%; mp 271-273.5 °C. IR (ATR): 3336 (NH), 3273 (NH), 3161, 3098, 1693, 1644 (C=O, amide I), 1621, 1598, 1564 (C=O, amide II), 1509 (NO2), 1496, 1442, 1412, 1381, 1344 (NO2), 1332, 1302, 1277, 1255, 1217, 1178, 1161, 1115, 1019, 955, 881, 866, 852, 835, 800, 768, 752, 700, 687 cm-1. 1 H NMR (500 MHz, DMSO-d6): δ 10.80 (1H, s, NH-Ph), 8.39 (1H, d, J = 7.9 Hz, NH-Ac), 8.21 (2H, d, J = 9.2 Hz, H3´, H5´), 7.84 (2H, d, J = 9.2 Hz, H2´, H6´), 7.36-7.26 (2H, m, H2, H6), 7.117.06 (2H, m, H3, H5), 4.64 (1H, td, J = 9.3 Hz, J = 5.3 Hz, CH), 3.03 (1H, dd, J = 13.8 Hz, J = 5.2 Hz, CH2), 2.86 (1H, dd, J = 13.6 Hz, J = 9.3 Hz, CH2), 1.80 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 171.57, 169.66, 161.20 (d, J = 242.0 Hz), 145.13, 142.50, 133.78 (d, J = 3.2 Hz), 131.16 (d, J = 8.0 Hz), 125.13, 119.17, 115.01 (d, J = 21.2 Hz), 55.44, 36.70, 22.49. Anal. Calcd. for C17H16FN3O4 (345.33): C, 59.13; H, 4.67; N, 12.17. Found: C, 59.02; H, 4.57; N, 11.99. 2-Acetamido-3-(2-fluorophenyl)-N-[4-(trifluoromethyl)phenyl]propanamide 1d (method B). White solid; yield 91%; mp 251-252.5 °C. IR (ATR): 3332 (NH), 3264 (NH), 3210, 3139, 3080, 1687, 1652 (C=O, amide I), 1611, 1541 (C=O, amide II), 1493, 1457, 1415, 1381, 1331, 1284, 1252, 1232, 1189, 1161, 1149, 1111, 1069, 1038, 1019, 953, 881, 839, 798, 775, 754, 727, 713 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.41 (1H, s, NH-Ph), 8.35 (1H, d, J = 8.0 Hz, NH-Ac), 7.76 (2H, d, J = 8.5 Hz, H3´, H5´), 7.65 (2H, d, J = 8.6 Hz, H2´, H6´), 7.32-7.04 (4H, m, H3, H4, H5, H6), 4.72 (1H, td, J = 8.2 Hz, J = 6.4 Hz, CH), 3.07 (1H, dd, J = 13.9 Hz, J = 6.4 Hz, CH2), 2.93 (1H, dd, J = 13.9 Hz, J = 8.2 Hz, 1H), 1.81 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.72, 169.50, 160.89 (d, J = 244.2 Hz), 142.43, 131.64 (d, J = 4.4 Hz), 128.84 (d, J = 8.1 Hz), 126.15 (q, J = 3.8 Hz), 124.51 (q, J = 271.5 Hz), 124.24 (d, J = 3.3 Hz), 124.14 (d, J = 17.4 Hz), 123.64 (q, J = 31.9 Hz), 119.56, 115.20 (d, J = 21.8 Hz), 53.69, 31.04, 22.53. Anal. Calcd. for C18H16F4N2O2 (368.33): C, 58.70; H, 4.38; N, 7.61. Found: C, 58.55; H, 4.47; N, 7.89. 2-Acetamido-3-(3-fluorophenyl)-N-[4-(trifluoromethyl)phenyl]propanamide 2d (method B). White solid; yield 87%; mp 268-270 °C. IR (ATR): IR (ATR): 3316 (NH), 3271 (NH), 1686, 1647 (C=O, amide I), 1612, 1553 (C=O, amide II), 1491, 1416, 1383, 1333, 1259, 1232, 1188, 1166, 1106, 1070, 1019, 943, 840, 783, 753, 691, 669 cm-1. 1H NMR (500 MHz, acetone-d6): δ 9.67 (1H, s, NHPh), 8.30 (1H, d, J = 8.0 Hz, NH-Ac), 7.80 (2H, d, J = 8.5 Hz, H3´, H5´), 7.62 (2H, d, J = 8.5 Hz, H2´, H6´), 7.32-7.26 (1H, m, H5), 7.12-6.92 (3H, m, H2, H4, H6), 4.82 (1H, td, J = 8.4 Hz, J = 6.1 Hz, CH), 3.23 (1H, dd, J = 13.9 Hz, J = 6.1 Hz, CH2), 3.01 (dd, J = 13.8 Hz, J = 8.4 Hz, CH2), 1.81 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.21, 169.69, 162.68 (d, J = 243.6 Hz), 142.27, 140.42 (d, J = 7.6 Hz), 129.94 (d, J = 8.5 Hz), 125.92 (q, J = 3.8 Hz), 125.25 (d, J = 2.7 Hz), 124.49 (q, J = 271.3 Hz), 124.67 (q, J = 32.4 Hz), 119.35, 115.96 (d, J = 21.4 Hz), 113.23 (d, J = 21.2 Hz), 55.24, 37.29, 21.81. Anal. Calcd. for C18H16F4N2O2 (368.33): C, 58.70; H, 4.38; N, 7.61. Found: C, 58.85; H, 4.29; N, 7.55. 2-Acetamido-3-(4-fluorophenyl)-N-[4-(trifluoromethyl)phenyl]propanamide 3d (method B). White solid; yield 99%; mp 273-275 °C. IR (ATR): IR (ATR): 3318 (NH), 3266 (NH), 1683, 1644 (C=O, amide I), 1612, 1549 (C=O, amide II), 1510, 1489, 1436, 1417, 1381, 1334, 1277, 1253, 1225, 1190, 1163, 1104, 1069, 1020, 953, 881, 838, 823, 804, 755, 714, 681 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.47 (1H, s, NH-Ph), 8.34 (1H, d, J = 8.1 Hz, NH-Ac), 7.78 (2H, d, J = 8.6 Hz, H3´,

H5´), 7.66 (2H, d, J = 8.7 Hz, H2´, H6´), 7.34-7.24 (2H, m, H2, H6), 7.12-7.06 (2H, m, H3, H5), 4.63 (1H, td, J = 9.3 Hz, J = 5.3 Hz, CH), 3.01 (1H, dd, J = 13.8 Hz, J = 5.2 Hz, CH2), 2.84 (1H, dd, J = 13.8 Hz, J = 9.5 Hz, CH2), 1.80 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 171.13, 169.57, 161.18 (d, J = 241.9 Hz), 142.49, 133.86 (d, J = 3.2 Hz), 131.13 (d, J = 8.0 Hz), 126.22 (q, J = 3.8 Hz), 124.51 (q, J = 271.3 Hz), 123.60 (q, J = 31.8 Hz), 119.37, 114.98 (d, J = 21.1 Hz), 55.24, 36.88, 22.50. Anal. Calcd. for C18H16F4N2O2 (368.33): C, 58.70; H, 4.38; N, 7.61. Found: C, 58.92; H, 4.34; N, 7.90. 2-Acetamido-3-(2-fluorophenyl)-N-phenylpropanamide 1e (method A). White solid; yield 72%; mp 212-214 °C. IR (ATR): 3308 (NH), 3063, 2940, 1668, 1644 (C=O, amide I), 1600, 1537 (C=O, amide II), 1494, 1444, 1369, 1321, 1305, 1287, 1231, 1212, 1183, 1175, 1111, 1080, 1048, 959, 906, 878, 848, 794, 764, 754, 722, 688 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 10.04 (1H, s, NHPh), 8.28 (1H, d, J = 8.2 Hz, NH-Ac), 7.54 (2H, d, J = 7.7 Hz, H2´, H6´), 7.34-7.19 (4H, m, H4, H6, H3´, H5´), 7.15-6.98 (3H, m, H3, H5, H4´), 4.72 (1H, td, J = 8.2 Hz, J = 6.2 Hz, CH), 3.06 (1H, dd, J = 13.9 Hz, J = 6.2 Hz, CH2), 2.90 (1H, dd, J = 13.9 Hz, J = 8.4 Hz, 1H), 1.80 (3H, s, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 170.24, 169.64, 161.17 (d, J = 244.1 Hz), 139.12, 131.92 (d, J = 4.4 Hz), 129.08, 129.02 (d, J = 8.2 Hz), 124.60 (d, J = 15.9 Hz), 124.47 (d, J = 3.6 Hz), 123.91, 119.97, 115.44 (d, J = 21.8 Hz), 53.74, 31.47, 22.85. Anal. Calcd. for C17H17FN2O2 (300.33): C, 67.99; H, 5.71; N, 9.33. Found: C, 68.17; H, 5.60; N, 9.47. 2-Acetamido-3-(3-fluorophenyl)-N-phenylpropanamide 2e (method A). White solid; yield 75%; mp 202-204 °C. IR (ATR): 3297 (NH), 3266 (NH), 3205, 3146, 3092, 2936, 1688, 1646 (C=O, amide I), 1621, 1610, 1601, 1554 (C=O, amide II), 1498, 1446, 1381, 1314, 1299, 1252, 1179, 1160, 1143, 1114, 1079, 1031, 996, 953, 942, 889, 845, 763, 752, 689 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.09 (1H, s, NH-Ph), 8.30 (1H, d, J = 8.2 Hz, NH-Ac), 7.56 (2H, d, J = 7.6 Hz, H2´, H6´), 7.337.26 (3H, m, H5, H3´, H5´), 7.14-7.10 (2H, m, H4, H6), 7.07-6.98 (2H, m, H2, H4´), 4.66 (1H, td, J = 9.0 Hz, J = 5.1 Hz, CH), 3.03 (1H, dd, J = 13.7 Hz, J = 5.1 Hz, CH2), 2.85 (dd, J = 13.7 Hz, J = 9.7 Hz, CH2), 1.79 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.26, 169.45, 162.12 (d, J = 242.8 Hz), 140.84 (d, J = 7.6 Hz), 138.91, 130.05 (d, J = 8.3 Hz), 128.87, 125.47 (d, J = 2.5 Hz), 123.59, 119.50, 116.02 (d, J = 21.0 Hz), 113.33 (d, J = 20.8 Hz), 54.79, 37.56, 22.55. Anal. Calcd. for C17H17FN2O2 (300.33): C, 67.99; H, 5.71; N, 9.33. Found: C, 67.89; H, 5.56; N, 9.44. 2-Acetamido-3-(4-fluorophenyl)-N-phenylpropanamide 3e (method A). White solid; yield 76%; mp 220.5-221.5 °C. IR (ATR): 3299 (NH), 3270 (NH), 3205, 3147, 3093, 2920, 2828, 1683, 1647 (C=O, amide I), 1620, 1609, 1601, 1533 (C=O, amide II), 1509, 1491, 1446, 1382, 1318, 1296, 1283, 1248, 1222, 1181, 1160, 1114, 1093, 1080, 1019, 955, 900, 883, 837, 827, 796, 754, 719, 700, 690 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 10.09 (1H, s, NH-Ph), 8.28 (1H, d, J = 8.3 Hz, NH-Ac), 7.59-7.55 (2H, m, H2´, H6´), 7.33-7.27 (4H, m, H3, H6, H3´, H5´), 7.12-7.02 (3H, m, H3, H5, H4´), 4.63 (1H, td, J = 8.9 Hz, J = 5.2 Hz, CH), 3.00 (1H, dd, J = 13.7 Hz, J = 5.2 Hz, CH2), 2.82 (1H, dd, J = 13.7 Hz, J = 9.6 Hz, CH2), 1.79 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.38, 169.44, 161.15 (d, J = 241.6 Hz), 138.94, 134.05 (d, J = 2.9 Hz), 131.14 (d, J = 7.9 Hz), 128.87, 123.58, 119.50, 114.95 (d, J = 21.0 Hz), 55.06, 37.11, 22.56. Anal. Calcd. for C17H17FN2O2 (300.33): C, 67.99; H, 5.71; N, 9.33. Found: C, 67.74; H, 5.87; N, 9.19. 4.1.2.2 Synthesis of phenyl 2-acetamido-3-(2/3/4-fluorophenyl)propanoates

An equivalent of N-acetyl-2/3/4-fluorophenylalanine (1, 2 or 3) together with 1.1 of equivalents of corresponding phenol, 1 equivalent of triethylamine (Et 3N) and 4-(dimethylamino)pyridine (DMAP; 0.1 eq.) were dissolved in dichloromethane (7 mL). After complete dissolution, EDC (1.5 of equivalents) was added in one portion. The reaction was monitored using TLC. It was stirred at room temperature overnight. The reaction mixture was washed three times with 0.1 M aq HCl, 10% aq sodium carbonate, followed by brine. The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under reduced pressure. Crystallization was initiated by the addition of n-hexane. It was performed for 24 hours at +4 °C and the mixture was filtered off to give esters 1f-3j. If necessary, they were recrystallized from ethyl acetate. 4-Chlorophenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1f. White solid; yield 85%; mp 136137.5 °C. IR (ATR): 3323 (NH), 3072, 2948, 1747 (OC=O), 1641 (C=O, amide I), 1587, 1543 (C=O, amide II), 1494, 1484, 1457, 1372, 1353, 1299, 1273, 1232, 1222, 1205, 1168, 1104, 1088, 1040, 1013, 977, 916, 888, 869, 842, 812, 797, 756, 706, 682 cm-1. 1H NMR (500 MHz, DMSOd6): δ 8.60 (1H, d, J = 6.9 Hz, NH), 7.49-7.43 (2H, m, H3´, H5´), 7.39-7.29 (2H, m, H4, H6), 7.217.13 (2H, m, H3, H5), 7.01-6.96 (2H, m, H2´, H6´), 4.61 (1H, dt, J = 8.4 Hz, J = 7.0 Hz, CH), 3.21 (1H, dd, J = 13.8 Hz, J = 7.0 Hz, CH2), 3.10 (1H, dd, J = 13.8 Hz, J = 8.4 Hz, CH2), 1.85 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.29, 169.87, 160.98 (d, J = 243.7 Hz), 149.16, 132.01 (d, J = 4.5 Hz), 130.27, 129.63, 129.22 (d, J = 8.2 Hz), 124.54 (d, J = 3.3 Hz), 123.82 (d, J = 15.3 Hz), 123.49, 115.35 (d, J = 21.9 Hz), 52.89, 30.16 (d, J = 2.3 Hz), 22.25. Anal. Calcd. for C17H15ClFNO3 (335.76): C, 60.81; H, 4.50; N, 4.17. Found: C, 60.80; H, 4.40; N, 4.41. 4-Chlorophenyl 2-acetamido-3-(3-fluorophenyl)propanoate 2f. White solid; yield 81%; mp 139.5140.5 °C. IR (ATR): 3309 (NH), 3071, 2939, 1745 (OC=O), 1638 (C=O, amide I), 1590, 1544 (C=O, amide II), 1484, 1448, 1372, 1352, 1301, 1275, 1244, 1220, 1203, 1171, 1142, 1087, 1030, 1013, 981, 949, 875, 842, 814, 799, 777, 741, 711, 694, 678 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.56 (1H, d, J = 6.9 Hz, NH), 7.49-7.44 (2H, m, H3´, H5´), 7.38-7.33 (1H, m, H5), 7.19-7.04 (3H, m, H2, H4, H6), 7.04-6.98 (2H, m, H2´, H6´), 4.63 (1H, dt, J = 8.9 Hz, J = 6.5 Hz, CH), 3.18 (1H, dd, J = 13.8 Hz, J = 6.2 Hz, CH2), 3.08 (1H, dd, J = 13.8 Hz, J = 9.0 Hz, CH2), 1.85 (3H, s, CH3). 13 C NMR (126 MHz, DMSO-d6): δ 170.43, 169.89, 162.24 (d, J = 243.1 Hz), 149.20, 140.04 (d, J = 7.8 Hz), 130.33 (d, J = 8.2 Hz), 130.26, 129.65, 125.56 (d, J = 2.7 Hz), 123.51, 116.17 (d, J = 21.3 Hz), 113.69 (d, J = 20.9 Hz), 53.95, 36.07, 22.26. Anal. Calcd. for C17H15ClFNO3 (335.76): C, 60.81; H, 4.50; N, 4.17. Found: C, 60.57; H, 4.35; N, 4.01. 4-Chlorophenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3f. White solid; yield 89%; mp 140141 °C. IR (ATR): 3295 (NH), 3077, 2931, 1749 (OC=O), 1640 (C=O, amide I), 1542 (C=O, amide II), 1510, 1485, 1446, 1378, 1351, 1303, 1273, 1218, 1205, 1164, 1087, 1031, 1013, 977, 911, 863, 851, 822, 814, 795, 717, 678 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 8.56 (1H, d, J = 6.8 Hz, NH), 7.51-7.41 (2H, m, H3´, H5´), 7.38-7.27 (2H, m, H2, H6), 7.19-7.09 (2H, m, H3, H5), 7.01-6.96 (2H, m, H2´, H6´), 4.58 (1H, dt, J = 8.6 Hz, J = 6.6 Hz, CH), 3.18-2.97 (2H, m, CH2), 1.84 (3H, s, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 170.57, 169.92, 161.37 (d, J = 242.4 Hz), 149.21, 133.28 (d, J = 3.0 Hz), 131.31 (d, J = 8.2 Hz), 130.29, 129.68, 123.57, 115.24 (d, J = 21.2 Hz), 54.28, 35.67, 22.30. Anal. Calcd. for C17H15ClFNO3 (335.76): C, 60.81; H, 4.50; N, 4.17. Found: C, 60.54; H, 4.67; N, 4.30.

4-Methylphenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1g. White solid; yield 81%; mp 122.5124 °C. IR (ATR): 3333 (NH), 3072, 2945, 1742 (OC=O), 1643 (C=O, amide I), 1585, 1541 (C=O, amide II), 1506, 1493, 1454, 1441, 1372, 1352, 1298, 1275, 1227, 1198, 1169, 1157, 1122, 1103, 1040, 1019, 978, 940, 914, 886, 853, 838, 819, 793, 756, 698 cm-1. 1H NMR (500 MHz, DMSOd6): δ 8.57 (1H, d, J = 6.9 Hz, NH), 7.40-7.28 (2H, m, H4, H6), 7.21-7.14 (4H, m, H3, H5, H3´, H5´), 6.84-6.79 (2H, m, H2´, H6´), 4.63 (1H, dt, J = 8.4 Hz, J = 7.1 Hz, CH), 3.20 (1H, dd, J = 13.8 Hz, J = 7.0 Hz, CH2), 3.08 (1H, dd, J = 13.8 Hz, J = 8.4 Hz, CH2), 2.27 (3H, s, CH3-Ph), 1.84 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.54, 169.78, 161.00 (d, J = 244.0 Hz), 148.19, 135.28, 132.02 (d, J = 4.4 Hz), 129.99, 129.19 (d, J = 8.2 Hz), 124.52 (d, J = 3.3 Hz), 123.89 (d, J = 15.3 Hz), 121.24, 115.34 (d, J = 21.7 Hz), 52.81, 30.35 (d, J = 2.0 Hz), 22.31, 20.53. Anal. Calcd. for C18H18FNO3 (315.34): C, 68.56; H, 5.75; N, 4.44. Found: C, 68.41; H, 5.87; N, 4.55. 4-Methylphenyl 2-acetamido-3-(3-fluorophenyl)propanoate 2g. White solid; yield 95%; mp 121122 °C. IR (ATR): 3309 (NH), 3070, 2928, 1749 (OC=O), 1652 (C=O, amide I), 1647, 1588, 1533 (C=O, amide II), 1507, 1489, 1456, 1373, 1352, 1289, 1257, 1214, 1190, 1168, 1142, 1127, 1033, 1018, 980, 942, 882, 816, 778, 753, 725, 694, 669 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.52 (1H, d, J = 7.1 Hz, NH), 7.38-7.32 (1H, m, H5), 7.20-7.04 (3H, m, H2, H4, H6), 6.86-6.82 (2H, m, H2´, H6´), 4.64 (1H, dt, J = 8.9 Hz, J = 6.6 Hz, CH), 3.17 (1H, dd, J = 13.7 Hz, J = 6.2 Hz, CH2), 3.07 (1H, dd, J = 13.7 Hz, J = 9.0 Hz, CH2), 2.28 (3H, s, CH3-Ph), 1.84 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.65, 169.77, 162.22 (d, J = 243.1 Hz), 148.21, 140.09 (d, J = 7.6 Hz), 135.26, 130.29 (d, J = 8.4 Hz), 129.98, 125.55 (d, J = 2.6 Hz), 121.24, 116.16 (d, J = 21.1 Hz), 113.63 (d, J = 20.9 Hz), 53.86, 36.24, 22.29, 20.50. Anal. Calcd. for C18H18FNO3 (315.34): C, 68.56; H, 5.75; N, 4.44. Found: C, 68.58; H, 5.49; N, 4.32. 4-Methylphenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3g. White solid; yield 93%; mp 107.5109.5 °C. IR (ATR): 3301 (NH), 3073, 2937, 1753 (OC=O), 1657 (C=O, amide I), 1603, 1544 (C=O, amide II), 1508, 1373, 1305, 1288, 1224, 1213, 1195, 1168, 1158, 1124, 1097, 1043, 1019, 979, 854, 835, 821, 789, 730, 692 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.50 (1H, d, J = 7.0 Hz, NH), 7.37-7.31 (2H, m, H2, H6), 7.21-7.10 (4H, m, H3, H5, H3´, H5´), 6.84 (2H, d, J = 8.0 Hz, H2´, H6´), 4.60 (1H, q, J = 7.4 Hz, CH), 3.13 (1H, dd, J = 13.8 Hz, J = 6.4 Hz, CH2), 3.07 (1H, dd, J = 13.8 Hz, J = 8.8 Hz, CH2), 2.28 (3H, s, CH3-Ph), 1.84 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.75, 169.76, 161.33 (d, J = 242.2 Hz), 148.22, 135.25, 133.32 (d, J = 3.0 Hz), 131.26 (d, J = 8.0 Hz), 129.98, 121.26, 115.17 (d, J = 21.0 Hz), 54.15, 35.83, 22.31, 20.51. Anal. Calcd. for C18H18FNO3 (315.34): C, 68.56; H, 5.75; N, 4.44. Found: C, 68.80; H, 5.67; N, 4.71. 4-Nitrophenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1h. White solid; yield 51%; mp 119.5121.5 °C. IR (ATR): 3321 (NH), 3072, 2944, 1747 (OC=O), 1638 (C=O, amide I), 1615, 1591, 1542 (C=O, amide II), 1522 (NO2), 1493, 1456, 1374, 1347 (NO2), 1298, 1233, 1212, 1163, 1104, 1091, 1040, 1013, 976, 917, 895, 866, 850, 799, 758, 745, 696 cm-1. 1H NMR (500 MHz, DMSOd6): δ 8.65 (1H, d, J = 6.7 Hz, NH), 8.32-8.27 (2H, m, H3´, H5´), 7.40-7.29 (2H, m, H4, H6), 7.277.23 (2H, m, H2´, H6´), 7.21-7.14 (2H, m, H3, H5), 4.63 (1H, dt, J = 8.5 Hz, J = 6.8 Hz, CH), 3.23 (1H, dd, J = 13.9 Hz, J = 6.9 Hz, CH2), 3.14 (1H, dd, J = 13.8 Hz, J = 8.4 Hz, CH2), 1.86 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 169.99, 169.83, 160.98 (d, J = 243.9 Hz), 155.24, 145.29, 131.99 (d, J = 4.4 Hz), 129.25 (d, J = 8.3 Hz), 125.52, 124.56 (d, J = 3.4 Hz), 123.74 (d, J = 15.3 Hz), 122.93, 115.35 (d, J = 21.6 Hz), 53.04, 29.95 (d, J = 2.0 Hz), 22.18. Anal. Calcd. for C17H15FN2O5 (346.31): C, 58.96; H, 4.37; N, 8.09. Found: C, 59.04; H, 4.36; N, 8.35.

4-Nitrophenyl 2-acetamido-3-(3-fluorophenyl)propanoate 2h. White solid; yield 42%; mp 134136.5 °C. IR (ATR): 3308 (NH), 3077, 2937, 1746 (OC=O), 1638 (C=O, amide I), 1615, 1591, 1542 (C=O, amide II), 1519 (NO2), 1488, 1447, 1375, 1345 (NO2), 1297, 1275, 1248, 1212, 1156, 1145, 1111, 1099, 1032, 1011, 977, 941, 897, 881, 863, 851, 779, 745, 705, 690, 673 cm-1. 1H NMR (500 MHz, CDCl3): δ 8.28-8.22 (2H, m, H3´, H5´), 7.36-7.30 (1H, m, H5), 7.19-7.15 (2H, m, H2´, H6´), 7.04-7.92 (3H, m, H2, H4, H6), 6.02 (1H, d, J = 7.5 Hz, NH), 5.06 (1H, dt, J = 7.5 Hz, J = 6.5 Hz, CH), 3.26 (2H, d, J = 6.4 Hz, CH2), 2.06 (3H, s, CH3). 13C NMR (126 MHz, CDCl3): δ 169.99, 169.43, 162.91 (d, J = 247.4 Hz), 154.69, 145.65, 137.73 (d, J = 7.2 Hz), 130.44 (d, J = 8.3 Hz), 125.30, 124.89 (d, J = 2.9 Hz), 122.15, 116.23 (d, J = 21.4 Hz), 114.59 (d, J = 20.8 Hz), 53.42, 37.48 (d, J = 1.9 Hz), 22.98. Anal. Calcd. for C17H15FN2O5 (346.31): C, 58.96; H, 4.37; N, 8.09. Found: C, 59.11; H, 4.57; N, 8.00. 4-Nitrophenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3h. White solid; yield 37%; mp 157159.5 °C. IR (ATR): 3311 (NH), 3070, 2933, 1749 (OC=O), 1638 (C=O, amide I), 1614, 1591, 1542 (C=O, amide II), 1521 (NO2), 1509, 1488, 1377, 1346 (NO2), 1299, 1271, 1211, 1155, 1092, 1025, 1011, 974, 915, 894, 865, 848, 827, 796, 745, 717, 696, 670 cm-1. 1H NMR (500 MHz, CDCl3): δ 8.28-8.24 (2H, m, H3´, H5´), 7.22-7.01 (6H, m, H2, H3, H5, H6, H3´, H5´), 5.99 (1H, d, J = 7.5 Hz, NH), 5.04 (1H, q, J = 6.8 Hz, CH), 3.24 (2H, d, J = 6.5 Hz, CH2), 2.05 (3H, s, CH3). 13C NMR (126 MHz, CDCl3): δ 169.99, 169.59, 162.23 (d, J = 246.7 Hz), 154.73, 145.65, 130.95 (d, J = 3.4 Hz), 130.78 (d, J = 8.1 Hz), 125.31, 122.16, 115.83 (d, J = 21.4 Hz), 53.61, 37.04, 23.00. Anal. Calcd. for C17H15FN2O5 (346.31): C, 58.96; H, 4.37; N, 8.09. Found: C, 58.86; H, 4.45; N, 7.93. 4-(Trifluoromethyl)phenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1i. White solid; yield 88%; mp 124-126 °C. IR (ATR): 3330 (NH), 3071, 2942, 1751 (OC=O), 1655 (C=O, amide I), 1612, 1541 (C=O, amide II), 1514, 1494, 1372, 1351, 1329, 1298, 1233, 1210, 1169, 1153, 1118, 1101, 1065, 1040, 1019, 979, 937, 891, 874, 846, 795, 758, 682 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.65 (1H, d, J = 6.8 Hz, NH), 7.79 (2H, d, J = 8.4 Hz, H3´, H5´), 7.41-7.29 (2H, m, H4, H6), 7.227.14 (2H, m, H3, H5, H2´, H6´), 4.64 (1H, dt, J = 8.4 Hz, J = 6.9 Hz, CH), 3.23 (1H, dd, J = 13.8 Hz, J = 7.0 Hz, CH2), 3.13 (1H, dd, J = 13.9 Hz, J = 8.4 Hz, CH2), 1.86 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.12, 169.97, 161.01 (d, J = 244.0 Hz), 153.34, 132.03 (d, J = 4.4 Hz), 129.26 (d, J = 8.2 Hz), 127.16 (q, J = 3.8 Hz), 126.80 (q, J = 32.2 Hz), 124.57 (d, J = 3.4 Hz), 124.12 (q, J = 271.9 Hz), 123.80 (d, J = 15.4 Hz), 122.68, 115.37 (d, J = 21.8 Hz), 53.01, 30.09 (d, J = 2.0 Hz), 22.23. Anal. Calcd. for C18H15F4NO3 (369.31): C, 58.54; H, 4.09; N, 3.79. Found: C, 58.75; H, 4.11; N, 3.52. 4-(Trifluoromethyl)phenyl 2-acetamido-3-(3-fluorophenyl)propanoate 2i. White solid; yield 91%; mp 125-127.5 °C. IR (ATR): 3315 (NH), 3071, 2942, 1747 (OC=O), 1638 (C=O, amide I), 1613, 1591, 1542 (C=O, amide II), 1512, 1487, 1447, 1375, 1350, 1328, 1295, 1273, 1252, 1217, 1169, 1156, 1116, 1100, 1065, 1017, 979, 947, 874, 845, 778, 727, 698, 679 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.61 (1H, d, J = 6.8 Hz, NH), 7.80 (2H, d, J = 8.3 Hz, H3´, H5´), 7.39-7.33 (1H, m, H5), 7.25-7.05 (5H, m, H2, H4, H6, H2´, H6´), 4.65 (1H, dt, J = 8.9 Hz, J = 6.4 Hz, CH), 3.20 (1H, dd, J = 13.8 Hz, J = 6.2 Hz, CH2), 3.10 (1H, dd, J = 13.8 Hz, J = 9.0 Hz, CH2), 1.86 (3H, s, CH3). 13 C NMR (126 MHz, DMSO-d6): δ 170.27, 169.99, 162.27 (d, J = 243.1 Hz), 153.38, 140.02 (d, J = 7.6 Hz), 130.36 (d, J = 8.4 Hz), 127.17 (q, J = 3.7 Hz), 126.79 (q, J = 32.1 Hz), 125.58 (d, J = 2.7

Hz), 124.13 (q, J = 272.5 Hz), 122.70, 116.19 (d, J = 21.1 Hz), 113.72 (d, J = 20.8 Hz), 54.05, 36.01, 22.24. Anal. Calcd. for C18H15F4NO3 (369.31): C, 58.54; H, 4.09; N, 3.79. Found: C, 58.59; H, 3.97; N, 3.56. 4-(Trifluoromethyl)phenyl 2-acetamido-3-(2-fluorophenyl)propanoate 3i. White solid; yield 82%; mp 132.5-134 °C. IR (ATR): 3330 (NH), 3081, 2933, 1754 (OC=O), 1658 (C=O, amide I), 1601, 1541 (C=O, amide II), 1509, 1374, 1324, 1287, 1222, 1209, 1196, 1167, 1159, 1123, 1102, 1064, 1040, 1018, 980, 878, 853, 830, 821, 799, 788, 726 cm-1. 1H NMR (500 MHz, DMSO-d6): 8.59 (1H, d, J = 6.8 Hz, NH), 7.80 (2H, d, J = 8.4 Hz, H3´, H5´), 7.38-7.32 (2H, m, H2, H6), 7.21 (2H, d, J = 8.4 Hz, H2´, H6´), 7.17-7.11 (2H, m, H3, H5), 4.61 (1H, dt, J = 8.7 Hz, J = 6.6 Hz, CH), 3.16 (1H, dd, J = 13.8 Hz, J = 6.4 Hz, CH2), 3.07 (1H, dd, J = 13.8 Hz, J = 8.8 Hz, CH2), 1.86 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.36, 169.97, 161.38 (d, J = 242.4 Hz), 153.38, 133.22 (d, J = 2.9 Hz), 131.30 (d, J = 8.1 Hz), 127.17 (q, J = 3.7 Hz), 126.78 (q, J = 32.2 Hz), 124.13 (q, J = 272.6 Hz), 122.72, 115.24 (d, J = 21.1 Hz), 54.35, 35.60, 22.25. Anal. Calcd. for C18H15F4NO3 (369.31): C, 58.54; H, 4.09; N, 3.79. Found: C, 58.32; H, 4.10; N, 3.94. Phenyl 2-acetamido-3-(2-fluorophenyl)propanoate 1j. White solid; yield 80%; mp 112.5-113.5 °C. IR (ATR): 3325 (NH), 3063, 2937, 1762 (OC=O), 1655 (C=O, amide I), 1541 (C=O, amide II), 1492, 1456, 1438, 1373, 1353, 1307, 1289, 1272, 1230, 1204, 1190, 1164, 1150, 1121, 1070, 1026, 981, 939, 909, 875, 864, 820, 767, 739, 721, 688 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.59 (1H, d, J = 7.1 Hz, NH), 7.42-7.14 (7H, m, H3, H4, H5, H6, H3´, H4´, H5´), 6.96-6.92 (2H, m, H2´, H6´), 4.65 (1H, dt, J = 8.5 Hz, J = 7.1 Hz, CH), 3.22 (1H, dd, J = 13.8 Hz, J = 7.0 Hz, CH2), 3.10 (1H, dd, J = 13.8 Hz, J = 8.4 Hz, CH2), 1.85 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.43, 169.81, 161.00 (d, J = 244.1 Hz), 150.40, 132.02 (d, J = 4.5 Hz), 129.67, 129.20 (d, J = 8.2 Hz), 126.11, 124.53 (d, J = 3.4 Hz), 123.87 (d, J = 15.6 Hz), 121.56, 115.35 (d, J = 21.8 Hz), 52.85, 30.31 (d, J = 2.3 Hz), 22.29. Anal. Calcd. for C17H16FNO3 (301.31): C, 67.76; H, 5.35; N, 4.65. Found: C, 67.88; H, 5.18; N, 4.81. Phenyl 2-acetamido-3-(3-fluorophenyl)propanoate 2j. White solid; yield 81%; mp 92-93.5 °C. IR (ATR): 3296 (NH), 3065, 2938, 1749 (OC=O), 1655 (C=O, amide I), 1590, 1543 (C=O, amide II), 1488, 1450, 1375, 1294, 1275, 1248, 1231, 1210, 1188, 1168, 1147, 1121, 1070, 1047, 986, 945, 936, 906, 885, 867, 833, 797 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 8.55 (1H, d, J = 7.0 Hz, NH), 7.43-7.33 (3H, m, H5, H3´, H5´), 7.27-7.23 (1H, m, H4´), 7.20-7.04 (3H, m, H2, H4, H6), 6.98-6.95 (2H, m, H2´, H6´), 4.65 (1H, dt, J = 8.9 Hz, J = 6.6 Hz, CH), 3.18 (1H, dd, J = 13.8 Hz, J = 6.3 Hz, CH2), 3.08 (1H, dd, J = 13.7 Hz, J = 8.9 Hz, CH2), 1.85 (3H, s, CH3). 13C NMR (126 MHz, DMSOd6): δ 170.59, 169.86, 162.26 (d, J = 243.0 Hz), 150.46, 140.10 (d, J = 7.7 Hz), 130.34 (d, J = 8.5 Hz), 129.71, 126.13, 125.59 (d, J = 2.7 Hz), 121.60, 116.20 (d, J = 21.3 Hz), 113.77 (d, J = 20.8 Hz), 53.94, 36.23, 22.31. Anal. Calcd. for C17H16FNO3 (301.31): C, 67.76; H, 5.35; N, 4.65. Found: C, 67.90; H, 5.49; N, 4.76. Phenyl 2-acetamido-3-(4-fluorophenyl)propanoate 3j. White solid; yield 98%; mp 108-110.5 °C. IR (ATR): 3334 (NH), 3075, 2922, 2849, 1746 (OC=O), 1656 (C=O, amide I), 1610, 1576, 1541 (C=O, amide II), 1508, 1492, 1465, 1374, 1350, 1311, 1282, 1252, 1230, 1197, 1162, 1154, 1097, 1070, 1042, 1024, 983, 920, 909, 857, 841, 829, 741, 725, 692 cm-1. 1H NMR (500 MHz, DMSOd6): δ 8.53 (1H, d, J = 7.0 Hz, NH), 7.40 (2H, t, J = 7.8 Hz, H3´, H5´), 7.37-7.32 (2H, m, H2, H6), 7.24 (1H, t, J = 7.4 Hz, H4´), 7.17-7.11 (2H, m, H3, H5), 6.96 (2H, d, J = 7.6 Hz, H2´, H6´), 4.61

(1H, dt, J = 8.7 Hz, J = 6.7 Hz, CH), 3.14 (1H, dd, J = 13.8 Hz, J = 6.4 Hz, CH2), 3.05 (1H, dd, J = 13.8 Hz, J = 8.8 Hz, CH2), 1.85 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): δ 170.68, 169.84, 161.36 (d, J = 242.3 Hz), 150.46, 133.32 (d, J = 3.0 Hz), 131.30 (d, J = 8.1 Hz), 129.70, 126.12, 121.61, 115.21 (d, J = 21.3 Hz), 54.22, 35.81, 22.32. Anal. Calcd. for C17H16FNO3 (301.31): C, 67.76; H, 5.35; N, 4.65. Found: C, 67.55; H, 5.14; N, 4.52. 4.2 Biology 4.2.1 Inhibition of cholinesterases The IC50 values were determined using the spectrophotometric Ellman’s method. All of the tested compounds were dissolved in 0.01 M DMSO and then diluted in demineralised water to 0.001 M and 0.0001 M. Acetylcholinesterase was obtained from electric eel (Electrophorus electricus L.) and butyrylcholinesterase was from equine serum. Rivastigmine and galantamine were involved as reference drugs. The efficacy of inhibitors is expressed as IC50 values, representing the concentration required for 50% inhibition of the enzyme [22]. All of the enzymes, galantamine and rivastigmine were purchased from Sigma-Aldrich (St. Louis, USA). 4.2.2 Cytotoxicity evaluation Cytotoxicity was determined for the human hepatocellular liver carcinoma cell line HepG2 (passage 31-42). The cells purchased from Health Protection Agency Culture Collections (ECACC, Salisbury, UK) was routinely cultured in Minimum Essential Eagle Medium MEM (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10 % fetal bovine serum (PAA), 1 % L-glutamine solution (Sigma-Aldrich) and 1 % non-essential amino acid solution (Sigma-Aldrich) in a humidified atmosphere containing 5 % CO2 at 37 °C. The cells were harvested after trypsin/EDTA (SigmaAldrich) treatment at 37 °C. To evaluate the cytotoxicity, the HepG2 cells treated with the tested substances were used as experimental groups whereas untreated HepG2 cells served as control groups. The HepG2 cells were seeded in density 1×104 cells per well on a 96-well plate. Next day they were treated with tested substances dissolved in DMSO (maximal incubation concentration of DMSO was 1 %). The tested substances were prepared according to their solubility at incubation concentrations 1–10,000 µM. The treatment was carried out in a humidified atmosphere containing 5 % CO2 at 37 °C in triplicates for 24 h. The controls representing 100% cell viability, 0% cell viability (the cells treated with 10 % DMSO), control with no cells and vehiculum controls were incubated in triplicates simultaneously. After 24h exposure the reagent from the kit CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) was added according to the recommendation of the manufacturer. After 2h incubation at 37 °C in humidified, 5 % CO2 atmosphere the absorbance was recorded at 490 nm. Inhibitory curves were constructed for each compound plotting incubation concentrations vs. percentage of absorbance relative to untreated control. The standard toxicological parameter IC50, i.e., the inhibitory concentration reducing the cell viability to 50 % of the maximal (control) viability, was calculated by nonlinear regression analysis of the inhibitory curves using GraphPad Prism software (version 6.0; GraphPad Software, San Diego, CA, USA). 4.2.3 In vitro antimicrobial evaluation The antibacterial activities were assayed against a panel of eight Gram-positive and Gram-negative strains: Staphylococcus aureus CCM 4516/08, methicillin-resistant Staphylococcus aureus H 5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08;

Escherichia coli CCM 4517, Klebsiella pneumoniae D 11750/08, extended-spectrum β-lactamase (ESBL) positive Klebsiella pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961. The microdilution broth method modified according to standard M07-A07 in Mueller-Hinton broth (HiMedia Laboratories, Mumbai, India) adjusted to pH 7.4 (±0.2) was used. The tested compounds were dissolved in DMSO to final concentrations ranging from 500 to 0.49 µM. Bacitracin (BAC) was used as a reference drug. A bacterial inoculum in sterile water was prepared to reach 0.5 on the McFarland scale (1.5 × 108 CFU/mL). The minimum inhibitory concentrations were assayed as a reduction in growth of at least 95% (IC95) compared with the control. The results were analysed visually. The MIC values were determined after 24 and 48 h of incubation in the dark at 35 °C (±0.1) in a humid atmosphere [23]. The antifungal properties were evaluated against four Candida strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, and Candida glabrata 20/I), Trichosporon asahii 1188 and three strains of filamentous fungi (Aspergillus fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445). The microdilution broth method was used according to the CLSI M27-A3 and M38-A2 guidelines in RPMI 1640 with glutamine (KlinLab, Prague, Czech Republic) buffered to pH 7.0 with 0.165 mol of 3-morpholino-propane-1-sulphonic acid (Sigma-Aldrich, Darmstadt, Germany). DMSO served as a diluent for all of the compounds. In yeast, the final size of the inoculum was 5 × 10 3 ± 0.2 CFU/mL, and in the case of the moulds, the final size of the inoculum was 0.5–5 × 104 CFU/mL. Fluconazole (FLU) was involved as a comparative drug. The MIC values for yeasts and filamentous fungi were assayed as a reduction of growth of at least 80% (IC 80) or of at least 50% (IC50) compared with the control, respectively. The results were analysed visually and/or spectrophotometrically at 540 nm. The MIC values were determined after 24 and 48 h of incubation in the dark at 35 °C (±0.1) in a humid atmosphere, but for T. mentagrophytes, the final MIC values were determined after 72 and 120 h of incubation [17]. The antimycobacterial activity against Mycobacterium tuberculosis 331/88 (H37Rv; dilution of this strain was 10−3), Mycobacterium avium 330/88 (dilution of 10−5), and two strains of Mycobacterium kansasii, namely 235/80 (dilution of 10−4) and the clinically isolated strain 6509/96 (dilution of 10−5) was evaluated using a previously described method [23]. The following concentrations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, and 1 μM. MIC is the lowest concentration at which complete inhibition of mycobacterial growth was observed. INH was chosen as a reference compound. The experiments were prepared in quadruplicates and the determination was repeated twice. Acknowledgements This work was supported by the Czech Science Foundation project No. 17-27514Y. This work was also supported by the Charles University (Project SVV 260 293). The authors wish to acknowledge financial support from the University of Pardubice, Faculty of Chemical Technology. We want to thank to Ida Dufková for the excellent performance of antibiotic susceptibility tests, the staff of the Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy, for the technical assistance and J. Urbanová, M.A., for the language help provided. Declaration of interest The authors declare no conflict of interest.

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Highlights  Aromatic esters and amides of isomeric N-acetylfluorophenylalanines were obtained.  All the compounds inhibit acetylcholinesterase and butyrylcholinesterase.  IC50 values from 8.25 µM.  Some derivatives were superior to rivastigmine and showed a high selectivity index.  Mild antimicrobial activity (bacteria, mycobacteria, fungi) of phenyl esters.