Substituted-Amidine Functionalized Monocyclic β -Lactams: Synthesis and In Vitro Antibacterial Profile

Background . Owing to the intrinsic stability against common β -lactamases and metallo-lactamases, monobactams gathered special attention in antibiotic drug development. However, so far, aztreonam is the only monobactam approved by FDA for clinical use. We designed new derivatives of aztreonam to enhance its antibacterial eﬃcacy. Methods . We synthesized a series of monocyclic β -lactams by modifying mainly at the C3 position of azetidinone ring. NH 2 group at C3 of azetidinone was attached to thiazole and thiadiazole which in turn was linked to nitrogenous heterocyclic rings via amidine moieties. We then investigated the in vitro antibacterial activities of synthesized compounds against ten bacterial strains of clinical interest in comparison to aztreonam and ceftazidime. Results . All compounds showed improved antibacterial activities against tested strains compared to reference drugs. Compounds 14d and 14e were most potent and showed the highest potency against all bacterial strains, with MIC values ranging from 0.25 µ g/mL to 8 µ g/mL, as compared to aztreonam (MIC 16 µ g/mL to > 64 µ g/mL) and ceftazidime (MIC > 64 µ g/mL). These compounds ( 14d and 14e ) may be valuable lead targets against multidrug-resistant Gram-negative bacteria.


Introduction
Continuous increase of β-lactamases [1], in association with variable resistance mechanisms [2,3], has managed the generation of multidrug-resistant (MDR) bacteria [4] over the years. Of these lactamases, extended-spectrum β-lactamases (ESBLs) are the prime cause of resistance in MDR bacteria that compromises the effectiveness of antibiotic therapy. e fact that present antibacterial drugs may be ineffective in the future requires continuous development of new antibiotics capable of combating bacterial resistance over time. To this end, different classes of β-lactams [5] are still growing areas of research despite the increasing number (more than 1000) [6] of β-lactamases.
β-Lactams are the broad spectrum and most widely used antibiotics for the treatment of serious bacterial infections. Among various classes of β-lactams including cephalosporins, carbapenems, and penicillins [5], monocyclic-β-lactams [7,8] are inherently resistant to common β-lactamases and metallolactamases (MBLs). Since the discovery of nocardicins and other monobactams from Nocardia uniformis and Pseudomonas strains, fine-tuning of the azetidinone ring is the common phenomenon to acquire the optimal potency. As a result, a vast variety of synthetic monobactam derivatives have been evaluated for antibacterial activity. e data gathered around the years revealed that the sulfonic acid group on the N1 position of the lactam ring is essential for the activation of the carbonyl group, whereas substitution at C3 and C4 maintains the stability of the molecule and plays part toward the antibacterial activity [8].
Nonetheless, despite enormous efforts, only one monobactam has been approved for clinical applications so far. Aztreonam (Figure 1) as the single FDA-approved monobactam shows a broad spectrum of activities against aerobic and anaerobic Gram-negative bacteria [9]. Although resistant to MBLs, aztreonam is susceptible to most of the ESBLs, AmpCs, and KPCs coproduced by the MBL producing Enterobacteriaceae strains [10][11][12].
erefore, many researchers are trying to improve the efficacy of aztreonam by introducing variable substituents at the C3 and C4 positions of the azetidinone ring. Recently, Reck et al. synthesized a number of compounds with variable substituents at C3 and C4 positions of azetidinone and identified a compound (LYS228) (Figure 1) that is resistant to all classes of β-lactamases. It also showed potent activity against carbapenemresistant Enterobacteriaceae (CRE) [13]. BAL30072 (Figure 1), another monobactam in phase-I clinical trials in Switzerland, demonstrated stability against MBLs, class C, and some of the class A and D β-lactamases [14].
As part of our ongoing efforts on the synthesis of monocyclic β-lactams, we previously reported the synthesis and in vitro antibacterial activity of a series of monobactams containing urea moiety. Although a few of the compounds exhibited improved antibacterial activity, none of them could achieve the acceptable MIC value against all tested strains [15]. We, therefore, decided to replace the urea moiety with amidine based on our own experience, and literature report on the previous amidine substituted monocyclic β-lactams [16]. Inspired from the literature and our previous data, the following substitutions were emphasized in the designing of final target molecules: (i) N1 position of the azetidinone ring was substituted with OSO 3 H or SO 3 H, (ii) NH 2 at C3 of the azetidinone was attached to thiazole or thiadiazole moiety which was further linked to heterocyclic (four to six membered nitrogenous) ring via a linker containing amidine and carboxylic groups, and (iii) C4 of the azetidinone ring was substituted with methyl or geminal dimethyl groups. us, designed molecules 14a-e, 14g, 18a, 18b, and 18d-e (Figure 2) were recognized as our synthetic targets. Herein, we describe their synthesis and antimicrobial activities in vitro against ten bacterial strains containing variable β-lactamases.

Materials and Methods
All 1 H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE NEO 400 NMR operating at 400 MHz for 1 H and 100 MHz for 13 C, respectively, and signals for NMR data are described as chemical shifts. All NMR spectra were recorded in deuterated solvents such as CDCl 3 , CD 3 OD, or DMSO-d 6 containing tetramethylsilane (TMS) an internal standard. Chemical shift (δ) values are denoted in parts per million (ppm), whereas coupling constant (J) values are provided in Hertz (Hz). Signal multiplicities are reported as follows: s, for singlet; br s, for broad singlet; d, for doublet; t, for triplet; and m, for multiplet. Final compounds were purified by Preparative HPLC using Agilent 1260 Infinity II System equipped with Agilent 10 prep-C18 250 × 21.2 mm column. Acetonitrile/water containing 0.1% trifluoroacetic acid or acetonitrile/water containing 0.1% formic acid was used as a solvent system for gradient elution at 22°C. LC-MS spectra were recorded on Agilent 1260 Infinity II System using either negative (ES − ) or positive (ES + ) ionization modes. HRMS spectra were performed on a Waters Xevo G2-XS QTof using either ES − or ES + ionization modes. Column chromatographic separation and purification were performed using glass columns filled with Qingdao Inc. Silica Gel: CC Grade (230-400 Mesh). Commercially available dry solvents were used in all synthesis experiments whereas commercial reagents were purchased from suppliers and used without purification.

Synthesis of Compound 2.
A mixture of a Co(III)catalyst (7.0 g, 8.4 mmol) and 4Å molecular sieves (10 g) in tert-butyl methyl ether (50 mL) was treated with methyl (R)oxirane-2-carboxylate (45.0 g, 441 mmol) and 4-hydroxybenzonitrile (1) (26.3 g, 220 mmol). e resulting mixture was stirred at room temperature for 48 hours, and the precipitates formed were filtered through celite pad. e filter cake was washed with ether, and the filtrate was evaporated under reduced pressure to afford a dark brown residue. e crude product was purified by column chromatography to yield the title compound 2 (79.0 g, 81.1% yield) as a brown oil. 1      7a: to a solution of compound 6a (0.97 g, 2.37 mmol) in MeOH (2 mL), a solution of diazo(diphenyl)methane (Ph 2 CN 2 , 0.42 g, 3.56 mmol) in CH 2 Cl 2 (1.5 mL) was slowly added. e resulting mixture was stirred at rt overnight and the solvent was concentrated to dryness. e crude product was purified by column chromatography to give product 7a (1.1 g, 80% yield in two steps) as a white solid. 1  8a: to a solution of compound 7a (1.2 g, 2.1 mmol), Nhydroxyphthalimide (1.03 g, 6.3 mmol ), and triphenylphosphine (1.7 g, 6.3 mmol) in anhydrous THF (5 mL), a solution of diethyl azodicarboxylate (1.09 g, 6.3 mmol) was added dropwise at 0°C. e reaction mixture was stirred at rt overnight followed by the evaporation of THF to furnish the crude product, which was purified by column chromatography to afford compound 8a (1.0 g, 66.7% yield) as white solid. 9a: to the solution of compound 8a (0.33 g, 0.45 mmol) in anhydrous ethanol (5 mL), hydrazine monohydrate (23 µL, 0.52 mmol) was added at 0°C. e resulting mixture was stirred at rt for 3.5 hours, filtered off and washed with ethanol (2 × 5 mL). e filtrate was evaporated to dryness and the residue was suspended in CH 2 Cl 2 (10 mL), filtered off and rinsed with CH 2 Cl 2 (2 × 3 mL). e filtrate was concentrated to give compound 9a (0.26 g, quantitative yield) as a pale yellow foam. e crude product was used for the next step without purification. 1 10.63 (br s, 1H). 13

Synthesis of Compound 14g. Synthesis of compound
14g was carried out according to Scheme 3. Compounds 11c and 12b were coupled according to the procedure described for 13a from the compound 13g. Boc deprotection in 13g was accomplished using analogous procedure described for compound 14a to afford the final compound 14g in 11% yield. 1 18a, 18b, and 18d-f.  Compounds 18a, 18b, and 18d-f were synthesized according to Scheme 4 using general procedures described for a representative compound for each step as follows:

Antibacterial Assay. Synthesized compounds 14 and
18 were analyzed for their antibacterial activity using the broth microdilution method, and the results are presented as minimum inhibitory concentrations (MICs, in µg/mL) for each compound and references.
e MIC values were measured according to the guidelines of the Clinical Laboratories and Standards Institute [18]. As a typical example, 14a was dissolved in DMSO and diluted, twofold in serial, with microbial growth medium (Mueller Hinton Broth II, cation adjusted) to reach the final concentration in a range of 0.063-64 µg/mL. e final DMSO concentration was kept at less than 0.5% in each sample. At this stage, bacteria (5 × 10 5 colony-forming units/mL (CFU/mL)) were added to each