Syntheses and Antibiotic Evaluation of 2-{[(2R,4R)-4-Carboxy-2-hydroxypyrrolidin-1-yl]carbonyl}benzene-1,5-dicarboxylic Acids and 2-Carbamoylbenzene-1,5-dicarboxylic Acid Analogues

Our search for new antibiotics led to the syntheses and biological evaluation of new classes of dicarboxylic acid analogues. The syntheses involve nucleophilic addition of different substituted benzylamine, aniline, alkylamine, and 4-hydroxyl-L-proline with carbamoylbenzoic acid. The results of the antimicrobial activity as indicated by the zone of inhibition (ZOI) showed that Z 10 is the most active against Pseudomonas aeruginosa (32 mm) and least active against Candida stellatoidea (27 mm) and Vancomycin Resistant Enterococci (VRE) (27 mm), while Z 7 shows the least zone of inhibition (22 mm) against Methicillin Resistant Staphylococcus aureus (MRSA). The minimum inhibition concentration (MIC) determination reveals that Z 10 inhibits the growth of tested microbes at a low concentration of 6.25 μg/mL, while Z 9 and Z 12 inhibits the growth of most microbes at a concentration of 12.5 μg/mL, recording the least MIC. The Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) results revealed that Z 10 has the highest bactericidal/fungicidal effect on the test microbes, at a concentration of 12.5 μg/mL, with the exception of Candida stellatoidea and Vancomycin Resistant Enterococci (VRE) with MBC/MFC of 25 μg/mL. The result of this investigation reveals the potential of the target compounds (Z 1–3,5,7–12) in the search for new antimicrobial agents.


Introduction
Development of novel bioactive drugs in chemical warfare against bacteria, fungi, and other infectious diseases has become an important and challenging task for the synthetic and medicinal chemists. Many research programs are tailored towards the design and synthesis of new drugs, for their chemotherapeutic application. The emergence of antimicrobial resistance threatens the effective prevention and treatment of an ever increasing range of infections caused by bacteria, parasites, virus, and fungi. New resistance mechanisms are emerging and spreading globally; the appearance and widespread use of fake and substandard drugs have further compounded the problem [1]. The HIV epidemic around the world has led to an increase in the number of immunocompromised patients, which in turn has led to an increase in the number of systemic bacterial and fungal infections [2]. Compounds containing carboxylic acid functional groups are playing a major role in the field of medicine. Generally, they play an active and critical role in the biochemistry of human or animal physiology. They have been involved in studies as antibacterial [3], anti-inflammatory [4], antiplatelet [5], antimicrobial [6], anticancer [7], antifungal [8], and analgesic and antiseptic [9].
Currently, there are more than 450 clinically approved drugs containing a free carboxylic acid group [10]. To the best of our knowledge no biological studies and syntheses of these compounds have been reported. We present here the syntheses of different substituted carboxylic and dicarboxylic acid analogues, with electron withdrawing and donating groups (OH, OCH 3 , CH 3 , NO 2 , and F) and explored their potentials as antibacterial and antifungal drugs.

General Procedure for the Synthesis of Z 1-3,5,7-12 .
A solution of 1,3-dioxo-2-benzofuran-5-carbonyl chloride (5 g, 23.8 mmol) was weighed into a round bottom flask containing 100 mL of KOH aq (5%) and then stirred for 30 min; the solid product (1,3-dioxo-2-benzofuran-5-carboxylic acid) formed was then filtered under suction. The products were pure enough for further reaction without further purification; this acid (0.3 g, 1.56 mmol) was further transferred to a 10 mL round bottom flask containing dichloromethane (7 mL) and a magnetic stirrer and 1.5 equiv. of different classes of amine were each added individually to the respective flasks and the mixture refluxed for at least two hours. The heat was then removed and the reaction stirred for a further 30 min. The reaction mixtures were allowed to stand at room temperature for a further 30 min; the solid precipitates formed were then filtered under suction and washed thoroughly with dichloromethane to remove any excess amines (Scheme 1). After drying, they were recrystallized from dichloromethane to give the purified products (Table 1).

2-[(3-Methoxypropyl)carbamoyl]benzene-1,5-dicarboxylic
Acid (Z 9 ). White solid powder (24% yield) was prepared according to the general procedure from 1,3-dioxo-2benzofuran-5-carboxylic acid 2 (0.     [11] was used to determine the antimicrobial activity of the test compounds. Pure cultures of the organism were inoculated on to Mueller Hinton Agar (MERCK) and incubated for 24 h at 38 ∘ C for bacteria and 48 h at 34 ∘ C for fungi. About 5 discrete colonies were aseptically transferred using sterile wire loops into tubes containing sterile normal saline (0.85% NaCl) and were adjusted to a turbidity of 0.5 McFarland Standard. The suspensions were then inoculated on the surface of sterile Mueller-Hinton Agar plates using sterile cotton swabs. A sterile 6 mm diameter Cork borer was used to make holes (wells) into the set of inoculated Mueller-Hinton Agar. The wells were filled with different concentration of the test compounds. The plates were then incubated; all the tests were performed in triplicate and the antimicrobial activities were determined as mean diameter of inhibition zone (mm) produced by the test compounds.

Minimum Inhibition Concentration (MIC).
The MIC was determined for the compounds using microbroth dilution method in accordance with National Committee for Clinical Laboratory Standard [12]. Serial dilution of the least concentration of the compounds that showed activity was prepared using test tubes containing 9 mL of double strength nutrient broth (OXOID). The test tubes were inoculated with the suspension of the standardized inoculum and incubated at 38 ∘ C for 24 h. MICs were recorded as the lowest concentration of the compounds showing no visible growth (turbidity) in the broth.

Chemistry.
Our synthetic approach involved two steps (Scheme 1). In the first step, the acid chloride is hydrolysed by the aqueous KOH to form the corresponding carboxylic acid. The reaction takes place at room temperature under stirring condition for 30 min. The second step involves nucleophilic addition of the substituted amine, which attack the anhydride ring, breaking it open to form the second carboxylic acid group and an amide. This reaction was carried out in DCM under reflux condition for at least 2 h. The overall yields are given in the experimental section and they ranges between 35 and 99%. The structures of the compounds were confirmed by the use of 1 H and 13 C NMR with application of 2D NMR where necessary.

Biological Results
. The synthesised compounds were tested against eleven bacteria including two resistance bacteria, Methicillin Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE), and four fungi. The test compounds had significant zones of inhibitions against all tested organism as compared to the standard drug. Compound Z 10 had the best activity among the compounds tested, with zone of inhibition ranging from 32 to 27 mm on the test microbes, but was not able to inhibit five bacteria (S. dysenteriae, P. aeruginosa, P. vulgaris, E. coli, and S. pyogenes) ( Table 2). This was the compound without any substitution on the benzyl ring. Compounds Z 7 and Z 11 had zones ranging from 30 to 22 mm, while Z 1 , Z 2 , and Z 5 were in the range between 29 and 24 mm. The zone of inhibition of Z 3 , Z 9 , and Z 12 was in the range between 28 and 20 mm, as compared to the zone of the standard drugs (32 to 40 mm).
Minimum inhibitory concentration (MIC) results (Table 3) reveal that a low concentration of 6.25 g/mL of the test compounds (Z 1−3 , Z 5,7−12 ) inhibited the growth of