N-(4-Substituted Aryl) amino acids as potential antibacterial agents

doi:10.21203/rs.3.rs-2757757/v1

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N-(4-Substituted Aryl) amino acids as potential antibacterial agents

https://doi.org/10.21203/rs.3.rs-2757757/v1

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Resistance of bacteria to current antibiotic drugs and the reoccurrence of different ailments after several therapeutic protocols have continued to be a cause of concern. Arylated amino acids are vital synthons to many compounds, they serve as essential building blocks in the synthesis of nitrogen heterocycles with various biological activities. This research reports the synthesis of some N-aryl amino acids and evaluates their antibacterial activities. The N-aryl amino acids 3a-3j were obtained by reacting different 4-substituted fluorobenzene 1a-1d with different amino acids 2a-2g via a metal-free, base-induced aryl amination reaction of aryl halides. The antibacterial activities of the synthesised compounds were evaluated against eight bacterial strains (Four gram-positive: Bacillus subtilis (ATCC 6633), Streptococcus pneumonia (ATCC 33400), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990) and four gram-negative: Enterobacter cloacae (ATCC 43560), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 43071) and Klebsiella oxytoca (ATCC 13182) using the agar well diffusion method with streptomycin as a reference drug. The biological screening indicates the synthesised compounds 3a, 3e, and 3j have promising broad-spectrum antibacterial potential as the N-aryl amino acid displayed comparable to better activity with the standard drug against Streptococcus pneumonia, Escherichia coli, and Proteus mirabilis.

Organic Chemistry

N-aryl amino acid

prolinamides

antibacterial

drug development

Alpha-Amino acids are the building blocks of life and are therefore of immense utility. Natural amino acid derivatives are of particular interest in drug discovery and organic syntheses as well as in nutraceuticals, pharmaceuticals, agrochemicals, and materials science (Barrett, 1999). The N-arylation of amino acids is of interest because N-aryl amino acids are not only essential building blocks in the synthesis of Fused nitrogen heterocycles with distinctive biological activities (Odusami, et al., 2019) but are also important motifs in many systems of physiological importance (Kitajima, et al., 2002). Most importantly, N-aryl amino acids are desirable compounds in the incorporation of functionalized amino acids in peptides and proteins for advances in chemical biology, because they aid the development of novel procedures in studying protein structure and functions (Dougherty, 2000, King and Buchwald, 2016). N-arylation reactions have been exploited in the introduction of diversity into bioactive molecules and synthesis of anti-cancer agents with improved potencies, for example, prolinamides (Fisher and Koenig, 2011)

Antimicrobial resistance (AMR) has become a health and development threat not just in the developing world but globally. It is one of the prevailing public threats with which humanity is confronted (WHO, 2021). Due to genetic changes, misuse/overuse of antimicrobials, sanitation and hygiene amongst others, AMR has continued to prevail and as a result, bacteria of the likes of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species have been designated as pathogens of priority (WHO, 2017; De Oliveira et al., 2020). A number of them have become resistant to antibiotics such as ciprofloxacin and even carbapenem-a last-resort treatment commonly employed in the treatment of severe or high-risk bacterial infections (Vargas-Casanova et al., 2019; WHO, 2021).

Additionally, streptomycin and tetracyclines for example have an increasing incidence of resistance from Salmonella spp. of human and animal origins worldwide. These multidrug resistance usually results in increased morbidity as well as mortality (Pezzella et al., 2004; Partridge et al., 2018). Drug-resistant parasites, as well as Fungi, are not left out hence posing a significant threat to the public health systems of countries around the world due to the persistence and spread of infectious diseases (Tanwar et al., 2014; Dadgostar, 2019). As a result, there is a need for the development of new drugs to combat the effect of the resistance which has become a major challenge in society.

Here, we report the synthesis and antibacterial evaluation of N-aryl amino acids against eight bacterial strains: Bacillus subtilis (ATCC 6633), Streptococcus pneumoniae (ATCC 33400), Staphylococcus aureus (ATCC 25923), and Staphylococcus epidermidis (ATCC 14990) Enterobacter cloacae (ATCC 43560), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 43071) and Klebsiella oxytoca (ATCC 13182). The N-aryl amino acids were obtained from different 4-substituted fluorobenzene and different amino acids (Scheme 1) using a modified Ullman type coupling reaction. (Synthesis Report previously published https://doi.org/10.1002/slct.202002446).

2.1. Materials and methods

Commercially available analytical grade reagents were purchased from Sigma and Merck and used without further purification. Nuclear Magnetic Resonance Spectroscopy, High-Resolution Mass Spectrometric analysis, and cytotoxic activities of synthesised compounds were carried out at Soochow University, China, while Infra-Red Spectroscopy and antibacterial analysis were done at the University of Lagos and the University of Ibadan respectively.

Glass wares were flame-dried, and reactions were carried out under an inert (dry nitrogen gas) atmosphere. Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60 F254 precoated plates using an ethyl acetate/petroleum ether (1:2) solvent system and visualized under a UV lamp (254 nm). Column chromatography was performed with silica gel (300–400 mesh) and solvents. Melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected. Infrared spectra were recorded on a Perkin Elmer Universal (ATR Spectrum 100) FT-IR spectrometer. 1H-NMR (400 MHz & 600 MHz) and 13C-NMR (150 MHz) spectra were recorded on a Varian-Inova (400 MHz or 600 MHz) spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) are reported in parts per million (ppm), downfield from TMS. High-resolution mass spectra (m/z) were recorded on a micro TOF–QIII (ESI) spectrometer. Organic solutions were dried over anhydrous sodium sulphate (Na₂SO4) or magnesium sulphate (MgSO₄) and concentrated with a rotary evaporator at reduced pressure.

For the antibacterial assay, 8 bacterial strains: Bacillus subtilis (ATCC 6633), Streptococcus pneumoniae (ATCC 33400), Staphylococcus aureus (ATCC 25923), and Staphylococcus epidermidis (ATCC 14990) Enterobacter cloacae (ATCC 43560), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 43071) and Klebsiella oxytoca (ATCC 13182) were purchased from culture collection centers at the Nigerian Institute of Medical Research (NIMR).

2.2. General procedure for the preparation of 3a–3j

To a clean round-bottomed flask, charged with a condenser, was added amino acid 2a-g (12 mmol) in C₂H₅OH/H₂O (1:1) (20 mL) and K₂CO₃ (3.5 equiv.) The mixture was then refluxed, with stirring for 10 minutes before p-substituted-fluorobenzene 1a-d (10 mmol) was carefully added, stirred and reflux for 3 h. The reaction medium was cooled to ambient temperature and concentrated to reduce solvent volume. The concentrate was then diluted with CH₂Cl₂ (10 mL) and acidified with 6M HCl (10 mL). The aqueous layer was extracted into CH₂Cl₂ (20 mL x 3) and the combined organic layer was washed with saturated brine solution, dried with anhydrous Na₂SO₄, filtered, and concentrated in vacuo to obtain the corresponding N-(p-substituted phenyl)-amino acid adducts 3a–j in 2–90% yield upon recrystallization in petroleum ether. Some of the products were purified by column chromatography on silica gel (petroleum ether/ethyl acetate; 2:1).

2.3 Evaluation of antimicrobial activities

The in-vitro antibacterial activity of the synthesized compounds 3a-3j was carried out against 8 bacteria strains which include gram-positive bacteria: Bacillus subtilis (ATCC 6633), Streptococcus pneumoniae (ATCC 33400), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990) and gram-negative bacteria: Enterobacter cloacae (ATCC 43560), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 43071) and Klebsiella oxytoca (ATCC 13182). The in-vitro antibacterial activity was achieved using the agar well diffusion method. Streptomycin was used as positive control while Dimethysulfoxide was the negative control. The bacteria were maintained on nutrient agar.

Agar well diffusion technique as described by Adeniyi et al., (1996) was used to determine the antibacterial activity of the synthesized compounds. Sensitivity test, nutrient agar plates were seeded with 0.1 ml of an overnight culture of each bacterial strain (equivalent to 10⁷ – 10⁸ CFU mL). The seeded plates were allowed to set and a standard cork borer of 7 mm diameter was used to cut uniform wells on the surface of the agar. Exactly 0.3 mL of the test compounds already dissolved in DMSO at a concentration of 20 mg/mL was then introduced into the wells and set in the incubator at 30 ^oC for 24 h. After 24 h, the diameter of the zone of inhibition (in mm) which is the clear area around each well was measured.

2.4. Minimum Inhibitory Concentration (MIC) of the synthesized compounds

Minimum inhibitory concentrations (MICs) of the acids, prolinamides, and streptomycin were determined by a modification of standard agar dilution method procedures as previously described by Adeniyi et al. (2009). MICs were carried out for only selected compound(s) which displayed activity during the sensitivity test. Different dilutions of the compounds were prepared first at 20 mg / mL to give final concentrations in the range of 20, 10, 5, 2.5, and 1.25 mg / mL. Two milliliters (2 ml) of each dilution were introduced onto the Mueller Hinton agar (MHA, Difco, France) poured into Petri dishes and allowed to set. The agar was then streaked with the cultured bacterial strains incubated overnight (24 h) after which the plates were examined for the presence or absence of bacterial growth. The minimum concentration that inhibited microbial growth was regarded as the minimum inhibitory concentration (MIC) of the respective compounds.

The metal-free C–N coupling of para-substituted halobenzene and amino acids resulted in the formation of different 4-substituted N-aryl amino acids with yields ranging from 2–90% (Table 1)

Table 1

Metal-free C–N coupling of *para*-substituted fluorobenzene and amino acids
Entry	R₁-Ar-X		Amino acid	Product	Yield
	R₁	X			(%)
1.	NO₂	F	2a	N-(4-Nitrophenyl)-L-proline (3a)	90
2.	NO₂	F	2b	N-(4-Nitrophenyl)-D, L-pipecolinic acid (3b)	70^a
3.	CN	F	2a	N-(4-Cyanophenyl)-L-proline (3c)	69
4.	CHO	F	2a	N-(4-Formylphenyl)-L-proline (3d)	65
5.	NO₂	F	2d	N-(4-Nitrophenyl)-L-hydroxyproline (3e)	89
6.	CH₃CO	F	2a	N-(4-Acetylphenyl)-L-proline (3f)	54
7.	NO₂	F	2c	N-(4-Nitrophenyl)-L-thioproline (3g)	35^b
8.	NO₂	F	2h	N-(4-Nitrophenyl)-L-phenylalanine (3h)	89
9.	NO₂	F	2e	N-(4-Nitrophenyl)-L-alanine (3i)	25^b
10.	NO₂	F	2f	N-(4-Nitrophenyl)-L-valine (3j)	50
^{* a}12 h, ^b24 h

3.2. Antibacterial activity of compounds 3a -j

Table 1 reveals the zones of inhibition (ZI) of compounds 3a-j at 20 mg/mL, the higher the ZI, the better the activity. At lower concentrations (10 mg/mL, 5 mg/mL, 2.5 mg/mL, and 1.25 mg/mL) only two (N-(4-Nitrophenyl)-L-proline 3a and N-(4-Nitrophenyl)-L-valine3j) of the compounds were able to sufficiently inhibit the growth of the selected bacteria. Compounds 3a and 3j displayed the best antibacterial activity against all the bacterial strains screened except against Bacillus subtilis which was resistant to the test compounds. Interestingly, both compounds 3a and 3j had higher zones of inhibition than streptomycin against four out of the eight bacterial strains screened: Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Proteus mirabilis. And amongst the four bacteria, Compound 3a displayed the highest zone of inhibition against Staphylococcus aureus. Furthermore, it was observed that 3a, 3d, 3i, and 3j with ZI of 12 mm, 16 mm, 18 mm, and 12 mm respectively were more potent against Proteus mirabilis and Streptococcus pneumoniae (ZI = 16, 16, 14, 18 mm respectively) than streptomycin (8 and 8 mm respectively). However, none of the compounds were able to inhibit the growth of Bacillus subtilis asides from the standard drug. The best activity across the N-aryl amino acid was observed against Proteus mirabilis and Streptococcus pneumoniae where four of the amino acids displayed higher zones of inhibition (> 8mm; > 16mm respectively) than streptomycin (ZI = 8 mm).

Table 1

Zones of Inhibition (ZI) of synthesised N-Aryl amino acids at 20 mg/mL
Bacterial strains	3a R₁ = NO₂ n = 3	3b R₁ = NO₂ n = 4	3c R₁ = CN n = 3	3d R₁ = CHO n = 3	3e R₁ = NO₂ R₂ = OH n = 3	3f R₁ = CH₃CO n = 3	3g R₁ = NO_2, thioproline n = 3	3h R₁ = NO₂ n = 0	3i R₁ = NO₂ n = 0	3j R₁ = NO₂ n = 0	Strep
a) Gram-positive
Bacillus subtilis	-	-	-	-	-	-	-	-	-	-	40
Streptococcus pneumoniae	16	10	-	16	6	8	10	6	14	18	10
Staphylococcus aureus	22	10	16	12	14	12	-	12	10	20	16
Staphylococcus epidermidis	8	10	16	16	14	12	-	10	12	18	30
b) Gram-negative
Enterobacter cloacae	8	4	-	8	4	2	-	4	4	4	16
Escherichia coli	12	6	8	6	6	8	6	6	8	20	8
Proteus mirabilis	12	6	8	16	6	8	6	4	12	18	8
Klebsiella oxytoca	10	8	-	12	6	4	10	6	10	12	12

Streptomycin (Strep) - Standard drug.

Surprisingly, the only N-aryl amino acid with sulfur embedded within its ring 3g was not able to inhibit the growth of as many bacterial strains compared to other compounds despite the biological significance of sulfur-containing molecules. The aforementioned was only able to inhibit the growth of four bacteria with lower zones of inhibition compared to other N-aryl amino acids in the series (Table 1). The least activity was recorded against Enterobacter cloacae across all compounds tested. The activity of these compounds can be attributed to the presence of the electron-withdrawing group, amine functionality, N-alkyl chain substituents, rigid proline ring, and hydroxyl functional group, the majority of which are present within the structure of the standard drug - Streptomycin. The exceptional activities observed with compounds 3a and 3j can be attributed to the presence of the nitro functional group common to the two while 3c and 3d possess the cyano and the hydroxy functions (both of which are also present in Streptomycin) respectively (Fig. 1) (Atsushi, et al., 2020; Osinubi et al., 2020).

Having obtained results from the sensitivity test, the Minimum Inhibitory Concentration (Table 2) of the most active compounds was determined with concentrations of 20, 10, 5, 2.5, and 1.25 mg/mL. It was observed that 3j displayed the best antibacterial activity against Escherichia coli at a concentration of 1.25 mg/mL while compounds 3a and 3c were both active at 2.5 mg/mL. Additionally, 3j was the most active against Klebsiella oxytoca at a concentration of 2.5 mg/mL while other compounds were only active between 10–20 mg/mL, it is worthy to state that compound 3a sufficiently inhibited the growth of seven out of the eight bacteria screened with a MIC of 5 mg/mL, this observation correlates with the work of Odusami et al., 2019 who reported that N-(2-nitrophenyl) pyrrolidine-2-carboxylic acid displayed better antibacterial activity against all the gram-negative bacterial strains they tested except for K. oxytoca. As for the 4-Cyano substituted moiety, it is probable that the presence of the cyano group in N-(4-Cyanophenyl)-L-proline 3c enhanced its activity towards Escherichia coli (2.5 mg/mL) among other bacterial strains screened (Table 2) hence the reason for the significant activity.

Table 2

Minimum inhibitory concentration (MIC) (mg/mL) of synthesised compounds (3a, 3b, 3c, 3d, 3e, 3f, 3h, 3j)
Bacterial strains	3a	3b	3c	3d	3e	3f	3h	3j
a) Gram-positive
Bacillus subtilis	-	-	-	-	-	-	-	-
Streptococcus pneumoniae	5	20	> 20	10	> 20	20	> 20	20
Staphylococcus aureus	5	20	10	> 20	10	10	> 20	10
Staphylococcus epidermidis	5	> 20	10	10	10	10	10	10
b) Gram-negative
Enterobacter cloacae	5	> 20	> 20	> 20	> 20	20	> 20	20
Escherichia coli	2.5	5	2.5	10	5	5	10	1.25
Proteus mirabilis	5	20	10	10	10	20	> 20	5
Klebsiella oxytoca	5	10	> 20	20	> 20	20	10	2.5

Of all the bacterial strains screened, the test compounds were observed to be most active against Escherichia coli because all the compounds 3a, 3b, 3c, 3d, 3e, 3f, 3h, 3j inhibited its growth even at a concentration as low as 1.25 mg/mL (Table 2).

By comparison, it is sufficient to state that the N-aryl amino acid 3a N-(4-Nitrophenyl)-L-proline and 3j N-(4-Nitrophenyl)-L-valine (with lower inhibitory concentration and significant broad-spectrum antibacterial activity) seem better motifs in the search of compounds that can serve as drug leads for combating drug-resistant bacterial against Streptococcus pneumoniae, Escherichia coli, Proteus mirabilis, and Klebsiella oxytoca especially. These findings are also in consonance with the work of Odusami et al., 2019 who reported that N-(Nitrophenyl)cycloamino acids sufficiently inhibited the growth of Escherichia coli and Proteus mirabilis among other bacteria screened.

The findings of this study indicated that the ten N-aryl amino acids evaluated for their antibacterial activities showed significant antibacterial activity. Of all the compounds tested, N-(4-Nitrophenyl)-L-proline 3a, was more potent than streptomycin against Escherichia coli. While compounds N-(4-Nitrophenyl)-L-proline 3a, N-(4-Nitrophenyl)-D, L-pipecolinic acid 3d, N-(4-Nitrophenyl)-L-alanine 3i, and N-(4-Nitrophenyl)-L-valine 3j were more potent than streptomycin against Streptococcus pneumoniae. The significant antibacterial activities of the N-aryl amino acids reported in this research work can be attributed to the effect of amino acids which are precursors in many important bioactive molecules, thus, indicating them as lead compounds for future drug development in combating bacterial resistance.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Funding Information

This work was funded, in parts, by the University of Lagos Central Research Committee (CRC No. 2015/25), Nigerian Government TETFund IBR (CRC/TETFund/No. 2018/016)

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Adejoke Osinubi. The first draft of the manuscript was written by Olayinka Asekun and Oluwole B. Familoni and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Scheme 1 is available in the Supplementary Files section

Scheme1.png
Scheme 1: Synthesis of N-aryl amino acids 3a-j

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N-(4-Substituted Aryl) amino acids as potential antibacterial agents

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials and methods

2.2. General procedure for the preparation of 3a–3j

2.3 Evaluation of antimicrobial activities

2.4. Minimum Inhibitory Concentration (MIC) of the synthesized compounds

3. Results And Discussion

3.2. Antibacterial activity of compounds 3a -j

4. Conclusion

Declarations

References

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Supplementary Files

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