Synthesis, Antibacterial, and Antifungal Activities of Hybrid Molecules Based on Alzheimer Disease Drugs and Bearing an Amino Acid Fragment

The objective of the current study was to develop novel compounds–memantine derivatives with antimicrobial activity designed for application in treatment of bacterial and fungal infections in patients suffering from Alzheimer’s disease. To realize this objective, a series of six memantine hybrid molecules were synthesized, characterized by 1H NMR, 13C NMR, MS, X-ray, and tested for their biological properties as anti-Alzheimer agents and their antimicrobial potential.


Introduction
There have been links between Alzheimer's disease (AD) and infections that cause a long-term activation of the immune system, a process known as chronic inflammation. Some of the infections that are thought to be linked to Alzheimer's include herpes, pneumonia, and infection with bacteria [1]. In the last few decades, neonatal sepsis and meningitis (NSM) remain a leading cause of infant mortality and morbidity, despite the use and availability of antibiotics. Bacterial E. coli is the most common Gram-negative pathogen causing NSM [2]. Regardless of the remarkable successes, there is still an unmet need for new effective therapeutic approaches to treat the disease. On the other hand, chronic viral, bacterial, and fungal infections might be causative factors for the inflammatory pathway in AD [3]. However, it is not yet clear whether these infections might trigger Alzheimer's or cause it to get worse. It could be that people with Alzheimer's are more susceptible to picking up infections. The blood-brain barrier (BBB) protects the brain by controlling what substances can pass from the blood into brain tissue. In Alzheimer's disease, the blood-brain barrier is damaged, particularly in the brain region affected by Alzheimer's. Evidences suggest that inflammation, the Alzheimer's hallmark amyloid protein and the ApoE4 gene, which are all linked to Alzheimer's disease, can contribute to the breakdown of the blood-brain barrier. Once it has been weakened, bacteria, viruses, and other harmful substances can get through the BBB into the brain more easily. This may explain why certain viruses and bacteria, such as herpes and spirochetes, are more common in the brains of people with AD [4]. Using the blood-brain barrier (BBB) and RNA-sequence models, Nie, P. et al. undertook a drug repositioning study to identify unknown antimicrobial activities for known drugs. The authors demonstrated for the first time that memantine (MEM), a drug approved by the Food and Drug Administration (FDA) for the treatment of Alzheimer's disease, can very effectively block E. coli-caused bacteremia and meningitis in a mouse model of NSM. MEM was able to synergistically improve the antibacterial activity of ampicillin in neonatal mice with bacteremia and meningitis and also significantly regulate anti-inflammatory factors.
In the present study, we report on the design, synthesis, and antimicrobial properties of new memantine derivatives functionalized with amino acids, e.g., peptidomimetics. The results of the study suggest that some of the memantine analogues reveal a potential for application as antimicrobial agents [5][6][7][8][9][10].

General
Unless otherwise stated, the starting materials, reagents, and solvents were obtained from commercial purchase and used as supplied without further purification. Analytical thin-layer chromatography (TLC) was run on Merck silica gel 60 F-254, with detection by UV light (254 nm). Memantine hydrochloride (MH) was purchased from Sigma-Aldrich (St. Louis, MO, USA), amino acids were purchased from Bachem (Berlin, Germany). 1 H and 13 C spectra were recorded on Bruker Avance II+ spectrometer (14.09 T magnet), operating at 600.11 MHz 1 H frequencies, equipped with a 5 mm BBO probe with z-gradient coil. The temperature was maintained at 293 K, using a Bruker B-VT 3000 temperature unit with airflow of 535 L/h. All chemical shifts are reported in parts per million (ppm), referenced against teramethylsylane (TMS, 0.00 ppm) or using the residual solvent signal (7.27 ppm for CDCL3 of 2.5 ppm for DMSO). Electrospray ionization-mass spectrometry (ESI-MS) experiments were acquired on Bruker Compact QTOF-MS (Bruker Daltonics, Bremen, Germany) and controlled by the Compass 1.9 Control software. The data analysis was performed and the mono-isotopic mass values were calculated using Data analysis software v 4.4 (Bruker Daltonics, Billerica, MA, USA). The analyses were conducted in the positive ion mode at a scan range from m/z 50 to 1000, and nitrogen was used as a nebulizer gas at a pressure of 4 psi and flow of 3 L/min for the dry gas. The capillary voltage and temperature were set at 4500 V and 220 °C, respectively. An external calibration for mass accuracy was carried out by using sodium formate calibration solution. The precursor ion of each compound was selected, and ESI-MS/MS analysis was performed by collision-induced dissociation (CID); nitrogen was the collision gas, and the collision energy varied from 5 to 40 eV. MSn experiments were conducted on an ion trap instrument Esquire 3000 (Bruker Daltonics, Bremen, Germany) and controlled by the Esquire Control 5.3.11 software. ESI-MS data were collected in positive-ion mode at a scan range from m/z 50 to 500. In all ESI-MS measurements, the nebulizer gas pressure was 124.1 kPa at a flow rate of 5 L min-1; the desolvation temperature was 300 °C and capillary voltage was adjusted to 4000 V. The sample solutions were delivered to the nebulizer by a syringe pump (Cole Parmer, Vernon Hills, IL, USA) at a flow rate 3 µL min −1 .

Synthesis
General procedure: The compounds 1a-e were synthesized with memantine according to a known procedure (Scheme 1). For the compounds 1a-e (3 mmol), DIPEA (3.1 mmol) was added to a solution of TBTU (3 mmol) in CH2Cl2 (15 mL). After stirring, the mixture was treated with memantine (3 mmol), along with DMAP (3 mmol). This mixture was stirred at RT for 3 h, and then evaporated to dryness. The residues were purified by column chromatography using chloroform/methanol (95:5). The Boc-protected group was removed by TFA (5 mL) at 0 °C for 1 h. After that, TFA.AAmemantine derivatives were treated with ammonia solution. Described

Sample Crystallization
In order to obtain single crystals with suitable quality, the dry powder substances of Beta-Alanine-Memantine (C17H27F3N2O3) and 4-F-Phenilalanine-Memantine (C23H32F4N2O4) were dissolved in ethanol, methanol, and acetone at room temperature. Large crystals (0.3 × 0.2 × 0.15 mm 3 ) suitable for single crystal X-ray studies formed within 2-3 days by slow evaporation. Crystals were obtained from the three solvents used, but the best quality crystal grew in acetone conditions.

Data Collection and Crystal Structure Refinement
A single crystal was mounted on a glass capillary and all data were collected at room temperature (290 K) on an Oxford diffraction Supernova diffractometer using Mo-Kα radiation (λ = 0.71013 Å) from micro-focus source. The determination of unit cell parameters, data integration, scaling and absorption corrections were carried out using the CrysalisPro [11]. The phases were obtained by direct methods with ShelxS-2018. The refinement of the structure involved several cycles of refinement using full-matrix least-squares on F 2 with the ShelxL-2018 package [12]. The N hydrogen atoms were positioned from difference Fourier map, while all other hydrogen atoms were placed at idealized positions. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were refined using the riding model. Visual representation of the two molecules present in the asymmetric unit were carried out using ORTEP [13], while three-dimensional packing and hydrogen bonding interactions were drawn using Mercury [14]. The data in the Crystallographic Information File (coordinates, structure factors, etc.) were verified using IUCr checkCIF/PLATON [15].

Thermal Analyses
Differential thermal analysis (DTA) and thermo gravimetric measurements (TG) were carried out simultaneously in a thermal analyzer Stanton Redcroft STA780 under the following conditions: heating rate of 5 °C/min -1 , dry argon as a carrier gas (−30 mL/min −1 ) and sample weight of 10-11 mg, in corundum crucible.

Agar Disk-Diffusion Method
Agar disk-diffusion testing, developed in 1940 [16], is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing. Nowadays, many accepted and approved standards are published by the Clinical and Laboratory Standards Institute (CLSI) for bacteria and yeasts testing [17]. In this well-known procedure, agar plates are inoculated with a standardized inoculum of the test microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test compound at a desired concentration, are placed on the agar surface. The Petri dishes are incubated under suitable conditions. Generally, the antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism, and then the diameters of inhibition growth zones are measured.

Standard Antibacterial in Vitro Metrics. Minimum inhibitory concentrations were investigated by Abedon [18].
Time-kill test for evaluation of bacteriostatic/bactericide activity [19]. Test cultures were prepared as described above with an initial concentration of 0.1, 0.3, and 0.5 McF. To each culture was added 0.5 mL of 1 mM solution of test compound. Samples were incubated at 37 °C. OD585 measurements were taken at 2, 3, 4, 5, and 6 h after incubation. In the samples, CFU/mL must be determined and compared to control data in same hour. The results are the following: • if xi > xk-the compound stimulates microorganisms to grow; • if xi = xk-the compound does not affect the microorganisms; • if xi = CFU/mL before the incubation-the compound has a bacteriostatic effect; • if xi < xk-the compound has a bactericide effect.

Results and Discussion
Synthesis of novel compounds-memantine hybrid molecules, with antimicrobial activity, was designed for application in treatment of bacterial and fungal infections in patients suffering from dementia of Alzheimer's type.
The peptide bond between the memantine and amino acids and peptidomimetics was formed using the TBTU coupling reagent [20]. The protected groups were removed in CH2Cl2/TFA at 0 °C (Scheme 1).

X-ray Study
Single crystal X-ray study showed that the compounds crystallize in a centrosymmetric manner (SG P21/c, No. 14) for the Beta-Alanine-Memantine and in a non-centrosymmetric manner (SG P212121, No. 19) for the 4-F-Phenilalanine-Memantine. The asymmetric units of the two studied compounds are shown in Figures 1 and 2.
The bond lengths and angles of the two molecules are comparable (Tables 1 and 2). The aromatic ring system present in the 4-F-Phenilalanine-Memantine molecule is essentially planar with rmsd of 0.002 Å. The overlay of the two molecules shows that the memantine geometry is highly conserved.    The observed difference is related to the β-Alanine-Memantine and 4-F-Phenilalanine-Memantine orientation (Figure 3). Having in mind the similarity (bond lengths and angles), one should expect similar hydrogen bonding interactions, as the side chain difference provides the main difference between the structures. Indeed, in both structures, intermolecular N-H … O hydrogen bonds (Table 3) stabilize the three-dimensional packing of the molecules. The crystal structure of 4-F-Phenilalanine-Memantine and β-Alanine-Memantine also reveals the presence of Trifluoroacetic acid (TFA) that produces the zwitterionic structure. One solvent water molecule is present in the 4-F-Phenilalanine-Memantine.

Thermal Behavior
The thermal behavior of the F-Phenilalanine-Memantine and β-Alanine-Memantine compounds was investigated between room temperature and 250 °C using DTA-TG-DTG analysis (Figure 4).
For the β-Alanine-Memantine ( Figure 4A), DTA measurement shows a slow endothermic effect at 150-170 °C without loss of mass on the TG curve. Such kind of endo effect can be related to the melting point of the compound. A second endo effect is detected at higher temperatures of 200-250 °C. The second effect is accompanied by mass losses and can be associated with decomposition of β-Alanine-Memantine.
Similarly for the 4-F-Phenilalanine-Memantine ( Figure 4B), DTA measurement shows two endothermic effects: the first is in the range 150-170 °C, and the second, 200-250 °C. The first endo effect is accompanied by ~3% weight losses, which corresponds to a loss of a water molecule and is in agreement with the crystal structure. The second DTA effect is associated with pronounced weight losses and is the melting and decomposition of the 4-F-Phenilalanine-Memantine. After the melting, a pronounced weight loss is started due to the volatility. The two processes-melting and decomposition-cannot be clearly separated, because they seem to occur almost simultaneously.
The compound 4-F-Phe memantine shows the highest antibacterial activity against both types of Gram bacteria. Its inhibition effect is similar with that of commercially available tetracycline (30 µg/disk). Antibacterial activity of compounds Val-memantine and Thz-memantine is lower and variable, between 10 and 16 mm inhibition area. Compound β-Ala-memantine demonstrates activity only against S. enterica, while compounds Gly-memantine and Fmoc-Thz-Thz-memantine are both inactive. All tested compounds were dissolved in DMSO. This solvent, 4-F-L-Phenylalanine, tetracycline, and the memantine (Nemdatine 10 mg tablets) were included as controls.
Comparing analysis of antimicrobial activity shows that compound Val-memantine has greater activity against Gram (−) bacteria. Among Gram (+) bacteria, only Bacillus megaterium growing was affected. Table 4. Antibacterial activity of chemical derivatives of memantine (10 mM) against Gram (+) and Gram (−) bacteria. The area of inhibition are presented in mm.

Gram (−) Bacteria Gram (+) Bacteria S. enterica E. coli St. aureus B. megaterium
St. epidermidis Gly-memantine n/a n/a n/a n/a n/a 4-F -Phe memantine  20  22  21  22  24  Val-memantine  11  11 n/a 16 n/a β-Ala-memantine 16 n/a n/a n/a n/a Thz-memantine n/a 13 10 16 n/a Fmoc-Thz-Thzmemantine n/a n/a n/a n/a n/a Memantine.HCl n/a n/a n/a n/a n/a 4-F-L-Phenylalanine n/a n/a n/a n/a n/a DMSO n/a n/a n/a n/a n/a Tetracycline 26 26 28 34 n/a Compound β-Ala-memantine shows activity against Salmonella enterica, while Thz-memantine affects mostly Gram (+) bacteria with the exception of Staphylococcus epidermidis, which growing was not influenced. It is obvious that there is selectivity of inhibition among diverse memantine analogues. The exception is 4-F-Phe memantine, which affects the growing of all tested strains.
The investigation of 1 mM compounds concentration inhibitory effect was observed only at Gram (+) bacteria St. aureus and B. megaterium by compound 4-F-Phe memantine.

Yeasts
Yeasts are widespread in nature. Often they cause mycosis in individuals with immunocompromised status. The main causes are Candida and Rhodotorula. Recent studies indicate that the incidence of mycosis caused by Rhodotorula is between 0.5% and 20.3% in the US and Europe. The investigated isolates were resistant to the antibiotic fluconazole.
The antifungal activity data is presented in Table 5. The compound 4-F-Phe memantine 2 is the only one active against all tested yeast microorganisms. Its effect is similar to the well-known antifungal medication Nystatin. Compound Gly-memantine demonstrates activity only against Sacch. cerevisiae. Experimental data shows that the memantine derivatives have an antimicrobial effect against model prokaryote microorganisms and tested eukaryote cultures as well. Table 5. Antifungal activity of chemical derivatives of memantine (10 mM) against model yeast microorganisms. The area of inhibition are presented in mm.

Candida lusitaniae
Sacch. cerevisiae Gly-memantine n/a n/a 14 4-F-Phe-memantine 26 23 21 Val-memantine n/a n/a n/a β-Ala-memantine n/a n/a n/a Thz-memantine n/a n/a n/a Fmoc-Thz-Thz-memantine n/a n/a n/a Memantine.HCl n/a n/a n/a 4-F-L-Phenylalanine n/a n/a n/a DMSO n/a n/a n/a Nystatin 26 24 n/a

Quantitative Analysis of Antimicrobial Activity: Determination of Minimum Inhibitory Concentration (MIC)
The results from the MIC test are presented in Table 6. Compound Gly-memantine is active against Salmonella enteria and B. megaterium with MIC value 1.25 mM. Regarding β-Ala-memantine, we observed low antimicrobial activity (MIC = 10 mM) against E. coli. Compound 4-F-Phe-memantine demonstrated the highest antimicrobial potential against all tested microorganisms. The MIC values are in a wide range, from 1.25 mM to 0.078 mM. Compound Thz-memantine is also active against all tested microorganism at higher concentrations, compared to compound 4-F-Phe memantine. Val-memantine shows activity, but at high concentrations.

Time-Kill Test for Evaluation of Bacteriostatic/Bactericide Activity
Time-kill test of 4-F-Phe-memantine, conducted with the most sensitive bacterial species Salmonella enterica, shows bactericidal effect and 100% inhibition of bacterial growth after 7 h of cultivation in the presence of the test substance ( Figure 5). For the same period, the control reached a cell density above 6 × 10 8 CFU/mL. 4-F-Phe-memantine showed a strong inhibitory effect on the growth of filamentous fungi (Table  7). It is in the range of 8% for Penicillium chaviforme and 62.9% for Fusarium graminearum.

Conclusions
The investigated memantine analogues with amino acids exhibit inhibitory effects on individual test microorganisms. Val-memantine shows greater efficacy against Gram-negative bacterial employees, and toward Gram-positive conditions that raise a plant only on the Bacillus megaterium. β-Ala-memantine has an effect exclusively on Salmonella enterica plants, and Gly-Thiazole has a good inhibitory effect against Gram-positive bacteria.
The 4-F-Phe-memantine was effective on all the strains required. The inhibitory effect is commensurate with this time to control the commercially available tetracycline and nystatin. The hybrid 4-F-Phe-memantine is the most promising for possible application as a new anti-infective hostdirected therapeutic agent against clinically significant, conditionally pathogenic bacteria in patients suffering from moderate-to-severe dementia of Alzheimer's type.

Conflicts of Interest:
The authors declare no conflict of interest.