2.1. 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).
1H and 13C spectra were recorded on Bruker Avance II+ spectrometer (14.09 T magnet), operating at 600.11 MHz 1H 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.
2.2. 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 CH
2Cl
2 (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.AA-memantine derivatives were treated with ammonia solution.
Described compounds 3a–3d were characterized recently by Stankova et al. (Amino acids in press).
Boc-Gly-Thz-memantine: 1H (DMSO-d6) δ (ppm): 0.84 (s, 6H, CH3), 1.14 (m, 2H), 1.28 (d, 2H, J = 12.2 Hz), 1.36 (d, 2H, J = 12.4 Hz,), 1.41 (s, 9H), 1.69 (m, 2H), 1.88 (m, 2H), 2.12 (m, 1H), 4.39 (d, 2H, J = 6Hz), 7.25 (s, 1H, NH), 7.81 (t, J = 6 Hz, 1H), 8.06 (s, 1H). 13C (DMSO-d6) δ (ppm): 171.9 (CO-amide), 159.9 (CO-BOC), 156.2 (Cquat-Thz), 150.6 (Cquat-Thz), 123.6 (CH-Thz), 79.9 (Cquat), 53.1 (Cquat-BOC), 50.6 (CH2), 47.4 (CH2), 42.7 (CH2), 42.4 (CH2), 32.4 (Cquat), 30.5 (2xCH3), 30.0 (CH), 28.6 (2xCH3-BOC). Yield: 65%. ESI-MS: Molecular formula: C22H33N3O3S; Mexact: 419.59, Mfound [M + H] 420.63.
Fmoc-Thz-Thz-Mem: 1H (DMSO-d6) δ (ppm): 0.84 (s, 6H, CH3), 1.16 (m, 2H), 1.29 (d, 2H, J = 12.2 Hz), 1.37 (d, 2H, J = 12.4 Hz), 1.79 (m, d, 2H, J = 11.6 Hz), 1.68 (m, d, 2H, J = 11.6 Hz), 1.93 (m, 2H), 2.12 (m, 1H), 4.26 (t, 1H, J = 6.5 Hz), 4.42 (d, 2H, J = 6.2 Hz), 4.53 (d, 2H, J = 6.7 Hz), 7.34 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.45 (s, 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.91 (d, J = 7.7 Hz, 2H), 8.20 (s, 1H), 8.35 (s, 1H). Yield: 65%. ESI–MS: Molecular formula: C34H34N4O3S2; Mexact: 610.80, Mfound [M + H] 611.76.
2.4. Biological Methods
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.