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

,b New diesters derived from phthaloylglycine ( 7a - 7i ) were synthesized and their structures characterized by infrared, 1 H and 13 C 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 .


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. 1,2 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. 3 These molecules have drawn attention because of their versatile range of biological applications including antibacterial, antifungal, analgesic, anti-inflammatory, antiviral, antitumor and anticonvulsant. 4 It is widely reported that phthalimide is an important biologically active pharmacophore and its derivatives have great antimicrobial activities. [5][6][7] 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. 8 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.
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%. 9 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. 10 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 S N 2 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 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy, including the two-dimensional techniques 1 H-1 H-COSY (correlation spectroscopy) and 1 H- 13  All nine 7a-7i diesters had three characteristic signals attributed to carbonyl C-4 and C-4', C-6 and C-8 at d C 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 13 C and 1 H distant 2 and 3 connections. −CO 2 CH 2 CH 3 methylene hydrogens at d C 4.22 ppm showed coupling with a carbon in −CO 2 CH 2 CH 3 at d C 14.18 ppm and with carbonyl carbon C-8 at d C 166.99 ppm. Methylene hydrogen H-7 at d C 4.68 ppm coupled with carbonyl carbons C-6 and C-8 at d C 167.00 and 166.99 ppm, respectively. Methylene hydrogen H-5 at d C 4.56 ppm showed coupling with carbonyl carbons C-4 and C-4' and C-6 at d C 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 d C 38.61-38.73 and d C 61.56-61.83 ppm, respectively (Table 1). In all compounds, the signals attributed to the aromatic carbons were found at d C 123.67-134.45 ppm.
In the spectrum of compound 7a, a signal was observed for the methyl group in the aliphatic region at d C 52.56 ppm. For compound 7c, three signals were observed for the propyl group at d C 67. 20 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 −NCH 2 COO− closest to the phthalimide between 1755 and 1728 cm −1 , functional group −OCH 2 COO− 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 authors 11,12 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. 13 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 Explorer 14 and Swiss ADME 15 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. 16 The values determined in this study are shown in Table 2.
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 Å 2 , 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 Å 2 , a high probability of good oral bioavailability. 11 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. 14 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 druglikeness 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
( 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. [17][18][19] 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. [20][21][22] 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.
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, 20,23,24 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, 1 H and 13 C 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.

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). 1 H and 13 C NMR spectra were obtained on two different instruments: a Bruker Avance Ultrashield TM (400 MHz for 1 H and 101 MHz for 13 C) and Bruker Avance Ultrashield TM (500 MHz for 1 H and 126 MHz for 13 C). Deuterated chloroform (CDCl 3 ) and deuterated dimethyl sulfoxide (DMSO-d 6 ) 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 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 MgSO 4 . The ethyl ether was evaporated in a rotary evaporator, yielding the respective ester derivatives. 1 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. 29 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.