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Synthesis, in silico Study and Antimicrobial Evaluation of New Diesters Derived from Phthaloylglycine

Abstract

New diesters derived from phthaloylglycine (7a-7i) were synthesized and their structures characterized by infrared, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The compounds were evaluated in an in silico study, which demonstrated positive features indicating a possible drug candidate. The diesters showed antifungal activity ranging from moderate to strong against strains of Candida. Compounds 7a, 7b, 7c, 7e and 7i had a moderate minimum inhibitory concentration (MIC) of 1024 µg mL−1 against all fungal strains, while 7h showed a very good MIC of 256 µg mL−1 against Candida albicans, Candida parapsilosis and Candida krusei and 64 µg mL−1 against Candida tropicalis. However, only 7h and 7i were able to inhibit bacterial growth of strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli with an MIC of 1024 µg mL−1.

Keywords:
phthalimide; phthaloylglycine; antibacterial activity; antifungal activity


Introduction

Multidrug-resistance is posing a great threat to health care services worldwide, where infections caused by resistant bacteria and/or fungi are very difficult to treat, usually leading to therapeutic failure with high mortality rates. The development of new drugs is a prominent alternative in the control of these infections, aiming to prevent or decrease pathogen resistance to achieve better treatment outcomes.11 Silveira, G. P.; Nome, F.; Gesser, J. C.; Sá, M. M.; Terenzi, H.; Quim. Nova 2006, 29, 844.,22 Murray, P. R.; Rosenthal, K. S.; Pfaller, M. A.; Medical Microbiology, 8th ed.; Elsevier: Philadelphia, 2016. Several heterocyclic compounds possess antimicrobial properties and have been studied and evaluated as potential drug candidates. Among such compounds is phthalimide, with a distinct and valuable structure for the design and development of new varieties of drugs.

Phthalimides have an imide ring, which is responsible for their biological activity.33 Filho, V. C.; Campos, F.; Corrêa, R.; Yunes, R. A.; Quim. Nova 2003, 26, 230. These molecules have drawn attention because of their versatile range of biological applications including antibacterial, antifungal, analgesic, anti-inflammatory, antiviral, antitumor and anticonvulsant.44 Reddy, C. U. M.; Jayakar, B.; Srinivasan, R.; Int. J. Pharma Bio Sci. 2010, 1, 86. It is widely reported that phthalimide is an important biologically active pharmacophore and its derivatives have great antimicrobial activities.55 Ramesh, M.; Sabastiyan, A.; Chem. Sin. 2012, 3, 1297.

6 Fhid, O.; Doma, A. M.; Zeglam, T. H.; Baki, J.; Zitouni, M.; Sdera, W.; Pharma Chem. 2015, 7, 240.
-77 Santos, J. L.; Yamasaki, P. R.; Chin, C. M.; Takashi, C. H.; Pavan, F. R.; Leite, C. Q. F.; Bioorg. Med. Chem. 2009, 17, 3795.

To counter the mechanisms of microbial resistance already known, it is necessary to employ molecular modification strategies such as molecular lipophilicity control, which influences the biological activity of new drug candidates.88 de Almeida, C. G.; Garbois, G. D.; Amaral, L. M.; Diniz, C. C.; Le Hyaric, M.; Biomed. Pharmacother. 2010, 64, 287. This is achieved by altering the number of carbons in the alkyl chain of an ester, for example.

Due to these merits, nine diester compounds derived from phthalimide were synthesized as potential new drug candidates. The compounds initially went through a design stage and in silico evaluation, and they were then taken to the organic synthesis stage, and finally tested for antimicrobial activity.

Results and Discussion

Chemical

The synthesis of the target molecules 7a-7i involved four synthetic stages, which are described in Scheme 1.

Scheme 1
Synthetic route to obtain the target molecules. Reagents and conditions: (i) HO−R, H2SO4, reflux, 8 h, 85%; (ii) glacial acetic acid, 130 °C, 6 h, 80%; (iii) EtOH, KOH, room temperature, 2 h, 90%; (iv) DMF, NaI (1%), 100 °C, 24 h, 38-75%.

The first step was the preparation of the 2-chloroacetate esters (2a-2i) via an esterification reaction between chloroacetic acid and the selected alcohols using Fisher’s method, obtaining yields of 60-70%.99 Gupta, R.; Kumar, P.; Narasimhan, B.; Arabian J. Chem. 2017, 10, S909. In the second step, phthaloylglycine (5) was prepared by the condensation reaction of the phthalic anhydride (4) with glycine (3) in glacial acetic acid as solvent.1010 Gera, A.; Mohan, C.; Arora, S.; Curr. Org. Synth. 2018, 15, 839. Potassium phthaloylglycinate (6) was obtained by an acid-base reaction in an ethanolic solution of potassium hydroxide. The final diester products (7a-7i) were prepared from the nucleophilic substitution reaction SN2 of the 2-chloroacetate esters (2a-2i) with potassium phthaloylglycinate (6) using dimethylformamide (DMF) as solvent, catalyzed by 1% sodium iodine in reflux, with yields ranging 38-75% at this stage.

The structures of the diesters were confirmed using infrared (IR) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, including the two-dimensional techniques 1H-1H-COSY (correlation spectroscopy) and 1H-13C HSQC (heteronuclear single quantum correlation) and HMBC (heteronuclear multiple bond correlation). In the NMR spectrum of compound 7b, there were signals in the aromatic hydrogen region δH 7.88-7.74 ppm, referring to the 4 aromatic hydrogens. In the two-dimensional direct correlation spectrum (1H-13C HSQC), correlations were observed between the signal at δH 4.56 ppm, referring to the methylene hydrogen H-5, and the C-5 signal at δC 38.71 ppm and between the signal δH 4.68 ppm, referring to the methylene hydrogen H-7, and the carbon signal at δC 61.70 ppm (C-7).

Based on the analysis of compound 7b, the other diesters (7a, 7c and 7d-7i) showed a methylene hydrogen singlet (−NCH2CO2/H-5) referring to phthaloylglycine with displacement at δH 4.71-4.66 ppm and a singlet of a methylene hydrogen (−CO2CH2CO2−/H-7) referring to the part of the ester at δH 4.57-4.55 ppm. The signals of the aromatic ring hydrogens appeared at δH 7.92-7.67 ppm. The spectrum of compound 7a displayed a singlet for 3 hydrogens with terminal CH3 connected to the ester oxygen at δH 3.75 ppm (−CO2CH3). Compound 7c showed a triplet for 2 methylene hydrogens (−CO2CH2−) at δH 4.13 ppm, multiplet for 2 methylene hydrogens (-CO2CH2CH2−) at δH 1.67 ppm and a triplet for 3 methyl hydrogens (-CO2CH2CH2CH3) at δH 0.94 ppm. Compounds 7e and 7h showed a triplet for 2 methylene hydrogens (−CO2CH2−) at δH 4.13-4.18 ppm, one multiplet for 2 methylene hydrogens (−CO2CH2CH2−) at δH 1.71-1.59 ppm, a multiplet for 2 methylene hydrogens (−CO2CH2CH2CH2−) at δH 1.42-1.29 ppm and a triplet for 3 methylic hydrogens at δH 1.26-0.90 ppm. Compound 7d showed a multiplet for 1 methinic hydrogen at δH 5.09 ppm and one doublet for 6 methylene hydrogens at δH 1.26 ppm. Compound 7f showed a multiplet for 1 methylene hydrogen at δH 1.95 ppm, a doublet for 2 methylene hydrogens at δH 3.96 ppm and one doublet for 6 methylic hydrogens at δH 0.93 ppm. Compound 7g displayed a multiplet for 1 methinic hydrogen at δH 4.98-4.89 ppm, a multiplet for 2 methylene hydrogens at δH 1.67-1.53 ppm, a doublet for 3 methylic hydrogens at δH 1.23 ppm and a triplet for 3 methylic hydrogens at δH 0.88 ppm. Finally, compound 7i showed a triplet for 2 methylene hydrogens at δH 4.20 ppm, a double doublet for 1 methinic hydrogen at δH 4.02 ppm, a quartet for 2 methylene hydrogens at δH 1.54 ppm and one doublet for 6 methylic hydrogens at δH 0.92 ppm.

All nine 7a-7i diesters had three characteristic signals attributed to carbonyl C-4 and C-4’, C-6 and C-8 at δC 167.54-166.45 ppm. In the two-dimensional spectrum (HMBC) analysis of compound 7b, it was possible to allocate the displacement of the carbonyl groups referring to the compound from the couplings between 13C and 1H distant 2 and 3 connections. −CO2CH2CH3 methylene hydrogens at δC 4.22 ppm showed coupling with a carbon in −CO2CH2CH3 at δC 14.18 ppm and with carbonyl carbon C-8 at δC 166.99 ppm. Methylene hydrogen H-7 at δC 4.68 ppm coupled with carbonyl carbons C-6 and C-8 at δC 167.00 and 166.99 ppm, respectively. Methylene hydrogen H-5 at δC 4.56 ppm showed coupling with carbonyl carbons C-4 and C-4’ and C-6 at δC 167.34 and 167.00 ppm, respectively.

Based on the analysis of compound 7b, the carbonyl compounds 7a, 7c and 7d-7i can be seen in Table 1. The compounds showed two more characteristic signals referring to the methylene carbons (C-5 and C-7) at δC 38.61-38.73 and δC 61.56-61.83 ppm, respectively (Table 1). In all compounds, the signals attributed to the aromatic carbons were found at δC 123.67-134.45 ppm.

Table 1
Data of 13C NMR (101 and 126 MHz) of the diesters 7a-7i, phthaloylglycine derivatives, in CDCl3

In the spectrum of compound 7a, a signal was observed for the methyl group in the aliphatic region at δC 52.56 ppm. For compound 7c, three signals were observed for the propyl group at δC 67.20, 21.85, 10.24 ppm. For compound 7e, four signals were observed for the butyl group at δC 65.52, 30.44, 19.00 and 13.64 ppm. For compound 7h, five signals were observed for the pentyl group at δC 65.80, 28.14, 27.89, 22.25 and 13.91 ppm. For compound 7d, two signals were observed, one for the −CO2CH− carbon at δC 69.62 ppm and another for the isopropyl group methyls at δC 21.67 ppm. For compound 7f, three signals were observed, two for the −CO2CH2CH− carbons at δC 71.59 and 27.63 ppm, and one for the isopropyl group methyls at δC 18.94 ppm. For compound 7i, four signals were observed, three for the -CO2CH2CH2CH− carbons at δC 64.33, 37.09 and 24.98 ppm and one for the isopropyl group methyls at δC 22.38 ppm. For compound 7g, four signals were observed, one for −CO2CH− at δC 74.13 ppm, one for a methyl at δC 28.65 ppm and two signals for an ethyl at δC 19.30 and 9.56 ppm.

In the IR spectrum, a stretch band referring to C=O was observed, a notable feature in the structures of diesters. All 7a-7i compounds showed absorption bands ascribed to the functional group −NCH2COO− closest to the phthalimide between 1755 and 1728 cm−1, functional group −OCH2COO− referring to the terminal ester between 1776 and 1747 cm−1 and functional group −N(CO)2 related to phthalimide between 1720 and 1706 cm−1. For all compounds, the stretches of the aromatic hydrogens of phthalimide ranged from 3111 to 3043 cm−1. Two stretching bands of C−O, a strong and weak one in the range of 1193-1107 cm−1, were also observed.

In silico study

The stages of developing new drug candidates demand a high cost of resources and time. To reduce these costs, theoretical studies have been of fundamental importance in the indication of factors that qualify new chemical compounds as potential drugs. Several authors1111 Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.; J. Med. Chem. 2002, 45, 2615.,1212 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.; Adv. Drug Delivery Rev. 2012, 64, 4. highlight the importance of the pharmacokinetic parameters absorption, distribution, metabolism and excretion (ADME), which give information about the permeability and concentration of certain compounds in therapeutic targets and their consequent elimination by the body. ADME parameters can be checked by in silico studies on the basis of calculations of physicochemical properties such as lipophilicity (clog P), water solubility (log S) and molecular weight (MW).

In the 1990s, Lipinski et al.1313 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.; Adv. Drug Delivery Rev. 1997, 23, 3. presented a relationship between pharmacokinetic and physicochemical parameters, indicating that the molecules with high potential to become a drug were those that resembled existing drugs in certain measured properties. Their study resulted in “Lipinski’s rule of 5”, which has only four factors (whose values are multiples of five): molar mass ≤ 500 g mol−1, log P ≤ 5, number of hydrogen bond acceptors ≤ 10 (accounted for as a function of N or O atoms in the molecule), and number of hydrogen bond donors ≤ 5 (represented as a function of the NH or OH groups in the molecule).

In this work, the in silico study of the 7a-7i diesters was performed to determine the Lipinski parameters using OSIRIS Property Explorer1414 Sander, T.; OSIRIS Property Explorer; Idorsia Pharmaceuticals Ltd., Switzerland, 2001. Available at https://www.organic-chemistry.org/prog/peo, accessed on August 01, 2019.
https://www.organic-chemistry.org/prog/p...
and Swiss ADME1515 Daiana, A.; Michielin, O.; Zoete, V.; Sci. Rep. 2017, 7, 42717. software. In addition to these, other parameters such as rotating bonds (Rb), topological polar surface area (TPSA), absorption percentage (ABS), drug-likeness and drug score were included in the study, since they are important parameters in the design of new drug candidates. ABS was calculated using the equation ABS(%) = 109 - (0.345 × TPSA) according to Zhao et al.1616 Zhao, Y. H.; Abraham, M. H.; Le, J.; Hersey, A.; Luscombe, C. N.; Beck, G.; Sherborne, B.; Cooper, I.; Pharm. Res. 2002, 19, 1446. The values determined in this study are shown in Table 2.

Table 2
In silico studies evaluating Lipinski's rule of five, topological surface area (TPSA), solubility (log S), adsorption percentage (ABS), rotating bonds, drug-likeness and drug score of the compounds 7a-7i

The in silico results displayed in Table 2 showed that all 7a-7i diesters were in line with Lipinski’s rule of 5, indicating that these compounds may show good oral availability. The TPSA values of all the 7a-7i diesters were 89.98 Å2, indicating good permeability in the plasma membrane of cells and a moderate absorption percentage of 77.95%. The number of Rb ranged from 6 to 10 for compounds 7a-7i, which indicated, along with a TPSA below 140 Å2, a high probability of good oral bioavailability.1111 Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.; J. Med. Chem. 2002, 45, 2615.

The log S (Ali method) of the diesters 7a-7g and 7i showed values between −2.21 and −3.96, indicating that the compounds were soluble, while diester 7h showed a value of −4.07 and was described as moderately soluble. More than 80% of drugs on the market show values higher than −4.00.1414 Sander, T.; OSIRIS Property Explorer; Idorsia Pharmaceuticals Ltd., Switzerland, 2001. Available at https://www.organic-chemistry.org/prog/peo, accessed on August 01, 2019.
https://www.organic-chemistry.org/prog/p...

The drug-likeness value of the 7a-7i diesters varied between −8.1 and −17.6, where the highest value was found for 7i and the lowest for 7h. When this value is closer to being positive, the molecule contains more moieties that are often present in commercial drugs; ideally, the drug-likeness value should be positive. The drug score value combines clog P, log S, molecular weight and toxicity risks and varies from 0.0 to 1.0 which can be used to predict the overall potential of a compound to be a new drug candidate. The values obtained with this synthesis approach ranged from 0.21 for diester 7i to 0.38 for diesters 7a and 7b, suggesting that the series of diesters 7a-7i has the potential to include new drug candidates.

Biological studies

Antibacterial activity

The in vitro antibacterial activity of the compounds 7a-7i was evaluated by the microdilution method with four strains of pathogenic bacteria, Staphylococcus aureus ATCC-6538, Staphylococcus epidermidis ATCC-12228, Escherichia coli ATCC-25922 and Pseudomonas aeruginosa ATCC-9027, using gentamicin (64 µg mL−1) as the standard control drug (Table 3). The antibacterial activity of the products was interpreted and considered as active or inactive, according to the following minimum inhibitory concentration (MIC) criteria: below 600 µg mL−1 = strong/optimum activity; 600-1500 µg mL−1 = moderate activity; above 1500 µg mL−1= weak activity or inactive product.1717 Holetz, F. B.; Pessini, G. L.; Sanches, N. R.; Cortez, D. A. G.; Nakamura, C. V.; Dias Filho, B. P.; Mem. Inst. Oswaldo Cruz 2002, 97, 1027.

18 Houghton, P. J.; Howes, M. J.; Lee, C. C.; Steventon, G.; J. Ethnopharmacol. 2007, 110, 391.
-1919 Sartoratto, A.; Machado, A. L. M.; Delarmelina, C.; Figueira, G. M.; Duarte, M. C. T.; Rehder, V. L. G.; Braz. J. Microbiol. 2004, 35, 275.

Table 3
Minimum inhibitory concentration (MIC) of diesters against bacterial strains used

Only 7h and 7i, diesters with the longest alkyl chain, showed moderate antibacterial activity, with an MIC of 1024 µg mL−1 against strains of S. aureus ATCC-6538, S. epidermidis ATCC-12228, E. coli ATCC-25922 and P. aeruginosa ATCC-9027 (Table 3). Studies have shown that the activity of compounds with antibacterial properties against Gram-positive and Gram-negative bacteria is improved by increasing their lipophilicity.2020 Silva, R. H. N.; Silva, D. F.; Nóbrega, F. R.; Oliveira, A. J. M. S.; Lima, E. O.; Souza, D. P.; J. Chem. Pharm. Res. 2017, 9, 89.

21 Echeverría, J.; Opazo, J.; Mendoza, L.; Urzúra, A.; Wilkens, M.; Molecules 2017, 22, 608.
-2222 Podunavac-Kuzmanović, S. O.; Cvetković, D. D.; Barna, D. J.; J. Serb. Chem. Soc. 2008, 73, 967. However, further studies should be conducted to identify what makes 7h and 7i substances able to act against both types of bacteria. The other diester compounds, 7a, 7b, 7c, 7d, 7e, 7f and 7g, showed no inhibition on bacterial growth of the strains used.

Antifungal activity

The in vitro antifungal activity of compounds 7a-7i was evaluated by the microdilution method with eight strains of pathogenic yeasts, Candida albicans ATCC-76645 and LM-111, Candida tropicalis ATCC-13803 and LM-07, Candida parapsilosis ATCC-22019 and LM-302, Candida krusei ATCC-6258 and LM-656, using amphotericin B (32 µg mL−1) as the standard control drug. The antifungal activity of the products was interpreted and considered as active or inactive, according to the following MIC criteria: below 600 µg mL−1 = strong/optimum activity; 600-1500 µg mL−1 = moderate activity; above 1500 µg mL−1= weak activity or inactive product.1717 Holetz, F. B.; Pessini, G. L.; Sanches, N. R.; Cortez, D. A. G.; Nakamura, C. V.; Dias Filho, B. P.; Mem. Inst. Oswaldo Cruz 2002, 97, 1027.

18 Houghton, P. J.; Howes, M. J.; Lee, C. C.; Steventon, G.; J. Ethnopharmacol. 2007, 110, 391.
-1919 Sartoratto, A.; Machado, A. L. M.; Delarmelina, C.; Figueira, G. M.; Duarte, M. C. T.; Rehder, V. L. G.; Braz. J. Microbiol. 2004, 35, 275. Of the nine diesters tested, only compounds 7d, 7f, and 7g did not show any antifungal activity, while 7a, 7b, 7c, 7e, 7h and 7i exerted 100% inhibition against all Candida strains tested (Table 4).

Table 4
Minimum inhibitory concentration (MIC) of diesters against fungal strains tested

The compounds 7a, 7b, 7c, 7e and 7i presented antifungal activity with a minimum inhibitory concentration (MIC) of 1024 µg mL−1 against all Candida strains. The compound 7h showed antifungal activity with an MIC of 256 µg mL−1 against the strains C. albicans ATCC-76645, C. albicans LM-111, C. tropicalis ATCC-13803, C. parapsilosis ATCC-22019, C. parapsilosis LM-302, C. krusei ATCC-6258 and C. krusei LM-656; and 64 µg mL−1 against C. tropicalis LM-07. Comparing the experimental (biological) results with the theoretical (in silico), in relation to the increase in alkyl chain length of the terminal esters of compounds 7a-7i, we observed that MIC was inversely proportional to lipophilicity. In relation to the isomers, we observed a decrease in MIC of the compounds 7c, 7e and 7h with N-alkyl chains, in relation to the compounds 7d, 7f, 7g and 7i with branched chains. The results are in accordance with the literature,2020 Silva, R. H. N.; Silva, D. F.; Nóbrega, F. R.; Oliveira, A. J. M. S.; Lima, E. O.; Souza, D. P.; J. Chem. Pharm. Res. 2017, 9, 89.,2323 Podunavac-Kuzmanović, S.; Markov, S.; Barna, D.; J. Theor. Comput. Chem. 2007, 6, 687.,2424 Rezaee, S.; Khalaj, A.; Adibpour, N.; Saffary, M.; Daru, J. Pharm. Sci. 2009, 17, 256. which reports better activities and smaller MICs for compounds with longer chain and consequently greater lipophilicity. The results (Table 4) were considered moderate for diester compounds 7a, 7b, 7c, 7e and 7i and strong for 7h, in terms of antifungal activity.

Conclusions

Nine new diesters were synthesized and characterized using IR, 1H and 13C NMR spectroscopic techniques. The in silico study showed that all synthesized diesters were in line with Lipinski’s rule of 5, indicating good oral bioavailability with drug administration, thus being a good new drug candidate. In the antibacterial activity study, only diesters 7h and 7i showed moderate antibacterial activity (MIC of 1024 µg mL−1) against all strains tested. In the antifungal activity study, diesters 7a-7c, 7e and 7i also had moderate activity (MIC of 1024 µg mL−1) against all Candida strains, while 7h displayed strong activity (MIC of 64-256 µg mL−1) against all Candida strains. The results indicate that both the increase in the linear alkyl chain of the terminal esters and their different geometric arrangements have an influence on biological activity. Future studies involving the synthesis of new diesters with alkyl chains longer than five carbons will be carried out to determine to what extent alkyl chain length influences biological activity.

Experimental

Chemical

All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Brazil) and used without further purification. The progress of the reactions was monitored by thin layer chromatography (TLC) on silica gel plates. The compounds were purified by recrystallization in ethanol and confirmed by determining the melting point (mp) range on an MQAPF-3 brand hotplate. Fourier transform infrared (FTIR) spectra were obtained on a Shimadzu Prestige-21 spectrometer using attenuated total reflectance (ATR). 1H and 13C NMR spectra were obtained on two different instruments: a Bruker Avance UltrashieldTM (400 MHz for 1H and 101 MHz for 13C) and Bruker Avance UltrashieldTM (500 MHz for 1H and 126 MHz for 13C). Deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (DMSO-d6) were used as solvent, and tetramethylsilane (TMS) was used for the internal standard. Chemical shifts (d) were measured in parts per million (ppm), and the coupling constants (J), in hertz (Hz).

Preparation of 2-chloroacetate esters (2a-2i)

In a 100-mL round-bottomed flask equipped with a condenser, a mixture of chloroacetic acid (10 mmol), alcohol (methyl, ethyl, propyl, butyl and isopentyl) (50 mL) and concentrated sulfuric acid (1 mL) was heated under reflux conditions for 4 h. Afterwards, excess solvent was rotary evaporated and the residue poured into cold water. The residue was transferred to a separation funnel containing 250 mL of water and 50 mL of ethyl ether were then added. The organic phase was separated and washed repeatedly with 10% sodium bicarbonate to neutral pH and then dried with anhydrous MgSO4. The ethyl ether was evaporated in a rotary evaporator, yielding the respective esters, which were used in the next step of the synthesis.

Methyl 2-chloroacetate (2a)

Colorless liquid; yield: 80%; IR (ATR) ν / cm−1 2957 (CHAliph), 1753 (C=O), 1299 and 1002 (O−CAliph), 789 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.08 (s, 2H, CH2Aliph), 3.80 (s, 3H, OCH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.7, 53.0, 40.6.

Ethyl 2-chloroacetate (2b)

Colorless liquid; yield: 93%; IR (ATR) ν / cm−1 2985, 2942 (CHAliph), 1735 (C=O), 1287 and 1024 (O−CAliph), 781 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.24 (q, 2H, OCH2Aliph), 4.05 (s, 2H, CH2Aliph), 1.30 (t, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.3, 62.2, 40.9, 14.0.

Propyl 2-chloroacetate (2c)

Colorless liquid; yield: 92%; IR (ATR) ν / cm−1 2970, 2881 (CHAliph), 1737 (C=O), 1290 and 1056 (O−CAliph), 792 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.15 (t, 2H, OCH2Aliph), 4.06 (s, 2H, CH2Aliph), 1.75-1.64 (hex, 2H, CH2Aliph), 0.96 (t, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.3, 67.7, 40.9, 21.8, 10.3.

Isopropyl 2-chloroacetate (2d)

Colorless liquid; yield: 80%; IR (ATR) ν / cm−1 2983, 2939 (CHAliph), 1732 (C=O), 1375 (isopropyl), 1287 and 1024 (O−CAliph), 792 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 5.09 (m, 1H, OCHAliph), 4.02 (t, 2H, CH2Aliph), 1.28 [d, 6H, (CH3Aliph)2]; 13C NMR (101 MHz, CDCl3) δ 166.8, 70.1, 41.2, 21.6.

Butyl 2-chloroacetate (2e)

Colorless liquid; yield: 80%; IR (ATR) ν / cm−1 2960, 2936, 2873 (CHAliph), 1737 (C=O), 1288 and 1020 (O-CAliph), 785 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.20 (t, 2H, OCH2Aliph), 4.06 (s, 2H, CH2Aliph), 1.70-1.61 (m, 2H, CH2Aliph), 1.40 (sext, 2H, CH2Aliph), 0.94 (t, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.4, 66.1, 40.9, 30.4, 18.9, 13.6.

Isobutyl 2-chloroacetate (2f)

Colorless liquid; yield: 78%; IR (ATR) ν / cm−1 2962, 2875 (CHAliph), 1736 (C=O), 1378 (isobutyl), 1288 and 1174 (O−CAliph), 788 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.08 (s, 2H, CH2Aliph), 3.98 (d, 2H, OCH2Aliph), 1.98 (m, 1H, CHAliph), 0.95 [d, 6H, (CH3Aliph)2]; 13C NMR (101 MHz, CDCl3) δ 167.4, 72.1, 40.9, 27.6, 18.9.

sec-Butyl 2-chloroacetate (2g)

Colorless liquid; yield: 65%; IR (ATR) ν / cm−1 2976, 2939, 2881 (CHAliph), 1732 (C=O), 1381 (sec-butyl), 1288 and 1190 (O−CAliph), 792 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.99-4.88 (t, 1H, OCHAliph), 4.04 (s, 2H, CH2Aliph), 1.72-1.52 (m, 2H, CH2Aliph), 1.26 (d, 3H, CH3Aliph), 0.92 (t, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.01, 74.69, 41.23, 28.66, 19.31, 9.59.

Pentyl 2-chloroacetate (2h)

Colorless liquid; yield: 90%; IR (ATR) ν / cm−1 2958, 2933, 2862 (CHAliph), 1737 (C=O), 1180 and 1045 (O-CAliph), 792 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.19 (t, 2H, OCH2Aliph), 4.06 (s, 2H, CH2Aliph), 1.72-1.62 (qt, 2H, CH2Aliph), 1.40-1.30 [m, 4H, (CH2Aliph)], 0.92 (t, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.4, 66.3, 40.9, 28.1, 27.8, 22.2, 13.8.

Isopentyl 2-chloroacetate (2i)

Colorless liquid; yield: 75%; IR (ATR) ν / cm−1 2960, 2873 (CHAliph), 1736 (C=O), 1386 (isopentyl), 1184 and 1047 (O−CAliph), 784 (C−Cl); 1H NMR (400 MHz, CDCl3) δ 4.22 (s, 2H, CH2Aliph), 4.05 (t, 2H, OCH2Aliph), 1.75-1.63 (m, 1H, CHAliph), 1.56 (q, 2H, CH2Aliph), 0.93 [d, J 6.7 Hz, 6H, (CH3Aliph)2]; 13C NMR (101 MHz, CDCl3) δ 167.3, 64.9, 40.9, 37.0, 24.9, 22.3.

Preparation of 2-(1,3-dioxoisoindolin-2-yl) acetic acid (5)

A solution of phthalic anhydride (5 mmol) and glycine (5 mmol) in glacial acetic acid was stirred under reflux for 6 h. Afterwards, the solvent was evaporated under reduced pressure, and the solid residue was washed with distilled water, filtered, dried and recrystallized. Yield: 80%; mp 189-190 °C; IR (ATR) ν / cm−1 3475 (OH), 3099-3051 (CHAr), 1612-1467 (C=CAr), 2985-2872 (CHAliph), 1770-1718 (C=O); 1H NMR (400 MHz, DMSO-d 6) δ 13.25 (s, 1H, OH), 7.92 (m, 2H), 7.89-7.85 (m, 2H), 4.32 (s, 2H, CH2Aliph); 13C NMR (101 MHz, DMSO-d 6) δ 168.92, 167.26, 134.83, 131.44, 123.42, 38.92.

Preparation of potassium acetate 2-(1,3-dioxoisoindolin-2-yl) (6)

An ethanolic solution (50 mL) of potassium hydroxide (10 mmol) was added slowly to an ethanolic solution of phthaloylglycine (10 mmol). The reaction mixture was stirred for 3 h. Afterwards, the solvent was evaporated under low pressure and the precipitate dried. Yield: 90%; mp > 250 °C; IR (ATR) ν / cm−1 3097-3041 (CHAr), 1537-1466 (C=CAr), 2987-2927 (CHAliph), 1743-1714 (C=O).

Preparation of derivatives of phthaloylglycine (7a-7i)

The potassium salt of phthaloylglycine (10 mmol) was placed in a flask with 10 mmol alkyl chloroacetate in 10 mL of DMF. The mixture was stirred under reflux for 24 h. After 24 h of reaction time, the product was allowed to cool at room temperature. After 10 min, cold distilled water was added and the mixture was then transferred to a separation funnel containing 250 mL of water, followed by the addition of 50 mL of ethyl ether. The organic phase was separated then dried with anhydrous MgSO4. The ethyl ether was evaporated in a rotary evaporator, yielding the respective ester derivatives.

2-Methoxy-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7a)

White solid; yield: 75%; mp 95-96 °C; IR (ATR) ν / cm−1 3074 (CHAr), 2995-2948 (CHAliph), 1759 (C=O), 1728 (C=O), 1708 (C=O), 1185 and 1107 (O−CAliph); 1H NMR (400 MHz, CDCl3) δ 7.92-7.83 (m, 2H, H-1 and H-1’), 7.77-7.67 (m, 2H, H-2 and H-2’), 4.69 (s, 2H, H-7), 4.55 (s, 2H, H-5), 3.75 (s, 3H, OCH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.54 (C-4 and C-4’), 167.40 (C-6), 167.03 (C-8), 134.45 (C-1 and C-1’), 132.07 (C-2 and C-2’), 123.82 (C-3 and C-3’), 61.56 (C-7), 52.56 (OCH3Aliph), 38.73 (C-5).

2-Ethoxy-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7b)

White solid; yield: 75%; mp 85-86 °C; IR (ATR) ν / cm−1 3068 (CHAr), 2987-2935 (CHAliph), 1747 (C=O), 1747 (C=O), 1718 (C=O), 1182 and 1114 (O−CAliph); 1H NMR (500 MHz, CDCl3) δ 7.87 (dd, J 5.2, 3.1 Hz, 2H, H-1 and H-1’), 7.73 (dd, J 5.2, 3.1 Hz, 2H, H-2 and H-2’), 4.67 (s, 2H, H-7), 4.55 (s, 2H, H-5), 4.21 (q, J 7.1 Hz, 2H, OCH2Aliph), 1.26 (t, J 7.1 Hz, 3H, CH3Aliph); 13C NMR (126 MHz, CDCl3) δ 167.34 (C-4 and C-4’), 167.00 (C-6), 166.99 (C-3 and C-3’), 134.38 (C-1 and C-1’), 132.05 (C-2 and C-2’), 123.76 (C-3 and C-3’), 61.76 (OCH2Aliph), 61.70 (C-7), 38.71 (C-5), 14.09 (CH3Aliph).

2-Oxo-2-propoxyethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7c)

White solid; yield: 72%; mp 76-77 °C; IR (ATR) ν / cm−1 3111-3076 (CHAr), 2980-2928 (CHAliph), 1754 (C=O), 1754 (C=O), 1714 (C=O), 1170 and 1118 (O−CAliph); 1H NMR (500 MHz, CDCl3) δ 7.89 (dd, J 5.5, 3.1 Hz, 2H, H-1 and H-1’), 7.75 (dd, J 5.5, 3.1 Hz, 2H, H-2 and H-2’), 4.69 (s, 2H, H-7), 4.56 (s, 2H, H-5), 4.13 (t, J6.7 Hz, 2H, OCH2Aliph), 1.71-1.62 (m, 2H, CH2Aliph), 0.93 (t, J 7.4 Hz, 3H, CH3Aliph); 13C NMR (126 MHz, CDCl3) δ 167.25 (C-4 and C-4’), 166.99 (C-6), 166.88 (C-8), 134.27 (C-1 and C-1’), 132.02 (C-2 and C-2’), 123.69 (C-3 and C-3’), 67.20 (OCH2Aliph), 61.60 (C-7), 38.64 (C-5), 21.85 (CH2Aliph), 10.24 (CH3Aliph).

2-Isopropoxy-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7d)

White solid; yield: 38%; mp 74-75 °C; IR (ATR) ν / cm−1 3105-3078 (CHAr), 2984-2943 (CHAliph), 1768 (C=O), 1753 (C=O), 1720 (C=O), 1179 and 1115 (O-CAliph); 1H NMR (400 MHz, CDCl3) δ 7.92-7.88 (m, 2H, H-1 and H-1’), 7.77-7.74 (m, 2H, H-2 and H-2’), 5.09 (m, 1H, OCHAliph), 4.66 (s, 2H, H-7), 4.57 (s, 2H, H-5), 1.26 [(d, J 6.3 Hz, 6H, (CH3Aliph)2]; 13C NMR (101 MHz, CDCl3) δ 167.28 (C-4 and C-4’), 166.91 (C-6), 166.45 (C-8), 134.29 (C-1 and C-1’), 132.00 (C-2 and C-2’), 123.69 (C-3 and C-3’), 69.62 (CHAliph), 61.83 (C-7), 38.63 (C-5), 21.67 (CH3Aliph).

2-Butoxy-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7e)

White solid; yield: 68%; mp 58-59 °C; IR (ATR) ν / cm−1 3101-3068 (CHAr), 2964-2943 (CHAliph), 1763 (C=O), 1748 (C=O), 1716 (C=O), 1187 and 1114 (O-CAliph); 1H NMR (400 MHz, CDCl3) δ 7.91-7.88 (m, 2H, H-1 and H-1’), 7.76 (m, 2H, H-2 and H-2’), 4.70 (s, 2H, H-7), 4.57 (s, 2H, H-5), 4.18 (t, J 6.7 Hz, 2H, OCH2Aliph), 1.67-1.59 (m, 2H, CH2Aliph), 1.42-1.32 (m, 2H, CH2Aliph), 0.93 (t, J 7.4 Hz, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.26 (C-4 and C-4’), 167.02 (C-6), 166.90 (C-8), 134.30 (C-1 and C-1’), 131.99 (C-2 and C-2’), 123.69 (C-3 and C-3’), 65.52 (OCH2Aliph), 61.60 (C-7), 38.62 (C-5), 30.44 (CH2Aliph), 19.00 (CH2Aliph), 13.64 (CH3Aliph).

2-Isobutoxy-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7f)

White solid; yield: 66%; mp 69-70 °C; IR (ATR) ν / cm−1 3099-3079 (CHAr), 2965-2935 (CHAliph), 1750 (C=O), 1750 (C=O), 1707 (C=O), 1178 and 1114 (O-CAliph); 1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J 5.5, 3.0 Hz, 2H, H-1 and H-1’), 7.75 (dd, J 5.5, 3.0 Hz, 2H, H-2 and H-2’), 4.71 (s, 2H, H-7), 4.57 (s, 2H, H-5), 3.96 (d, J 6.7 Hz, 2H, OCH2Aliph), 1.95 (m, 1H, CHAliph), 0.93 [(d, J 6.7 Hz, 6H, (CH3Aliph)2]; 13C NMR (101 MHz, CDCl3) δ 167.26 (C-4 and C-4’), 167.00 (C-6), 166.90 (C-8), 134.30 (C-1 and C-1’), 131.99 (C-2 and C-2’), 123.69 (C-3 and C-3’), 71.59 (CHAliph), 61.57 (C-7), 38.61 (C-5), 27.63 (OCH2Aliph), 18.94 (CH3Aliph).

2-(sec-Butoxy)-2-oxoethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7g)

White solid; yield: 60%; mp 68-69 °C; IR (ATR) ν / cm−1 3099-3082 (CHAr), 2974-2939 (CHAliph), 1776 (C=O), 1755 (C=O), 1707 (C=O), 1179 and 1114 (O−CAliph); 1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J 5.5, 3.0 Hz, 2H, H-1 and H-1’), 7.75 (dd, J 5.5, 3.0 Hz, 2H, H-2 and H-2’), 4.98-4.89 (m, 1H, OCHAliph), 4.67 (s, 2H, H-7), 4.57 (s, 2H, H-5), 1.67-1.53 (m, 2H, CH2Aliph), 1.23 (d, J 6.3 Hz, 3H, CH3Aliph), 0.88 (t, J 7.5 Hz, 3H, CH3Aliph); 13C NMR (101 MHz, CDCl3) δ 167.26 (C-4 and C-4’), 166.91 (C-6), 166.60 (C-8), 134.29 (C-1 and C-1’), 132.00 (C-2 and C-2’), 123.68 (C-3 and C-3’), 74.13 (OCHAliph), 61.78 (C-7), 38.62 (C-5), 28.65 (CH2Aliph), 19.30 (CH3Aliph), 9.56 (CH3Aliph).

2-Oxo-2-(pentyloxy)ethyl 2-(1,3-dioxoisoindolin-2-yl)acetate (7h)

Brown solid; yield: 67%; mp 39-40 °C; IR (ATR) ν / cm−1 3100-3043 (CHAr), 2959-2929 (CHAliph), 1774 (C=O), 1752 (C=O), 1706 (C=O), 1193 and 1115 (O-CAliph); 1H NMR (500 MHz, CDCl3) δ 7.91-7.87 (m, 2H, H-1 and H-1’), 7.77-7.73 (m, 2H, H-2 and H-2’), 4.69 (s, 2H, H-7), 4.57 (s, 2H, H-5), 4.16 (t, J 6.8 Hz, 2H, OCH2Aliph), 1.68-1.61 (m, 2H, CH2Aliph), 1.36-1.29 (m, 2H, CH2Aliph), 0.90 (t, J 7.0 Hz, 3H, CH3Aliph); 13C NMR (126 MHz, CDCl3) δ 167.24 (C-4 and C-4’), 166.99 (C-6), 166.88 (C-8), 134.27 (C-1 and C-1’), 132.02 (C-2 and C-2’), 123.68 (C-3 and C-3’), 65.80 (OCH2Aliph), 61.60 (C-7), 38.64 (C-5), 28.13 (CH2Aliph), 27.89 (CH2Aliph), 22.25 (CH2Aliph), 13.91 (CH3Aliph).

Isopentyl 2-(2-(1,3-dioxoisoindolin-2-yl)acetoxy)acetate (7i)

Yellow liquid; yield: 64%; IR (ATR) ν / cm−1 3097-3080 (CHAr), 2958 (CHAliph), 1753 (C=O), 1753 (C=O), 1716 (C=O), 1176 and 1114 (O−CAliph); 1H NMR (500 MHz, CDCl3) δ 7.89 (dd, J 5.3, 3.1 Hz, 2H, H-1 and H-1’), 7.75 (dd, J 5.3, 3.1 Hz, 2H, H-2 and H-2’), 4.69 (s, 2H, H-7), 4.57 (s, 2H, H-5), 4.20 (t, J 6.9 Hz, 2H, OCH2Aliph), 4.02 (m, 1H, CHAliph), 1.54 (q, J 6.9 Hz, 2H, CH2Aliph), 0.92 [d, J 6.6 Hz, 6H, (CH3Aliph)2]; 13C NMR (126 MHz, CDCl3) δ 167.22 (C-4 and C-4’), 166.98 (C-6), 166.88 (C-8), 134.28 (C-1 and C-1’), 132.00 (C-2 and C-2’), 123.67 (C-3 and C-3’), 64.33 (CHAliph), 61.61 (C-7), 38.63 (C-5), 37.09 (OCH2Aliph), 24.98 (CH2Aliph), 22.38 (CH3Aliph).

Antimicrobial activity

Test substance

Solutions of the synthesized compounds 7a-7i were prepared at the time of the tests by dissolving the compound in 5% DMSO and 2% Tween 80 (Sigma-Aldrich, São Paulo, Brazil), and completing the final volume with sterile distilled water.2525 Nascimento, P. F. C.; Nascimento, A. C.; Rodrigues, C. S.; Antoniolli, A. R.; Santos, P. O.; Júnior, A. M. B.; Trindade, R. C.; Rev. Bras. Farmacogn. 2007, 17, 108.,2626 Pereira, F. O.; Mendes, J. M.; Lima, I. O.; Mota, K. S. L.; Oliveira, W. A.; Lima, E. O.; Pharm. Biol. 2015, 53, 228.

Culture media

The fungal and bacterial strains were maintained in Sabouraud dextrose agar (SDA) and brain heart infusion (BHI) medium (Difco Laboratories Ltd., USA, France), respectively. For biological activity assays, BHI broth and Roswell Park Memorial Institute (RPMI)-1640 medium with L-glutamine and no sodium bicarbonate (Difco Laboratories Ltd., USA, France and INLAB, São Paulo, Brazil) were used for tests with bacteria and fungi, respectively. The culture media were prepared according to the manufacturer’s instructions and sterilized by autoclaving at 121 °C and 1 atm for 15 min.

Microorganisms

The following strains were used for antimicrobial activity assays: Staphylococcus aureus ATCC-6538, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC-9027, Escherichia coli ATCC-25922, Candida albicans ATCC-76645 and LM-111, C. tropicalis ATCC-13803 and LM-07, C. parapsilosis ATCC-22019 and LM-302, C. krusei ATCC-6258 and LM-656. The microorganisms were obtained from the Micoteca (collection) of the Mycology Laboratory, Department of Pharmaceutical Sciences (DCF), Health Sciences Center (CCS) of the Federal University of Paraíba (UFPB), Brazil. The fungal and bacterial strains were maintained at 4 °C in SAD and BHI, respectively. For use in the assays, the fungi and bacteria were harvested in SAD and BHI, respectively, and incubated at 35 ± 2 °C for 24-48 h. The microorganism suspension was prepared according to the 0.5 McFarland scale tube and was adjusted by the use of a spectrophotometer (Leitz-Phtometer 340-800) to 90% T (530 nm), corresponding to approximately 106 colony-forming unit (CFU) mL−1 for fungi and 108CFU mL−1 for bacteria.2727 Clinical and Laboratory Standards Institute (CLSI); Document M100-S17, M7-A6: Performance Standards for Antimicrobial Susceptibility Testing; Approved Standard, 6th ed.; CLSI: Wayne, PA, 2003.,2828 Cleland, R.; Squires, E. In Antibiotics in Laboratory Medicine; Lorian, V., ed.; Lippincott Williams & Wilkins: Baltimore, 1991, p. 739.

Determination of minimum inhibitory concentration (MIC)

The determination of the MIC of the products in bacterial and fungal strains was performed using the broth microdilution method with 96-well round-bottom microplates (TPP, Switzerland) containing. Initially, 100 µL of RPMI/BHI broth were distributed in the wells of the microdilution plates. Next, 100 µL of the substances were dispensed in the first column of wells, and through a twofold serial dilution, concentrations of 1024 µg mL−1 down to 64 µg mL−1 were obtained. Finally, 10 µL of the bacterial and fungal suspensions were added to the wells. In parallel, controls were included: microorganisms (BHI + bacteria and RPMI + yeasts) and culture medium (RPMI/BHI), to assure the strains’ viability and sterility of the medium, respectively; and negative control with the antimicrobials gentamicin (100 µg mL−1) for bacteria and amphotericin B (100 µg mL−1) for fungi. The prepared plates were aseptically closed and incubated at 35 ± 2 °C for 24-48 h.

In the biological assay with bacteria, after 24 h of incubation, 20 µL of 0.01% resazurin dye indicator (INLAB), a colorimetric redox, were added.2929 Mann, C. M.; Markham, J. L.; J. Appl. Microbiol. 1998, 84, 538. A change in dye color from blue to red indicated microbial growth, and if the color remained blue, it meant the absence of microbial growth. The MIC for each product was defined as the lowest concentration capable of visually inhibiting microbial growth with no dye color change.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by the Brazilian agencies CNPq and CAPES. Dr A. Leyva (USA) provided English editing of the manuscript.

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Publication Dates

  • Publication in this collection
    30 Apr 2020
  • Date of issue
    May 2020

History

  • Received
    13 Aug 2019
  • Accepted
    19 Nov 2019
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