Synthesis and Antibacterial Activity of Amino Acid and Dipeptide Prodrugs of IMB-070593, a Fluoroquinolone Candidate

A series of amino acid and dipeptide prodrugs of IMB-070593, a fluoroquinolone candidate discovered in our lab, were synthesized and evaluated for their water solubility and then antibacterial activity. Our results reveal that four amino acid prodrugs 4a,b,e,f and two dipeptide prodrugs 4k,l have much greater solubility (>85 mg/mL) than IMB-070593 mesylate (22.5 mg/mL). Compounds 4a and 4k show good in vivo efficacy against MSSA 12-1 (p.o./i.v., 5.32–7.68 mg/kg) and S. pneumoniae12-10 (p.o., 18.39–23.13 mg/kg) which is 1.19–1.50 fold more active than the parent drug.


Introduction
Fluoroquinolone (FQ) antibacterial agents which target two type II bacterial topoisomerase enzymes, DNA gyrase and/or topoisomerase IV, are among the most attractive drugs in the antiinfective chemotherapy field [1].

OPEN ACCESS
The structure activity relationship (SAR) studies of FQs show that the basic substituent at the C-7 position, the most amenable site for chemical changes, greatly influences their antibacterial potency, spectrum and safety [2]. The presence of five-or six-membered nitrogen heterocycle including pyrrolidine, piperazine and piperidine at this position is a particularly favorable structural feature of important FQs on the market [3]. Moreover, methyloxime-functionalized pyrrolidines as novel C-7 substituents have also been proved to be of importance with respect to biological activity and led to the discovery of some new FQ agents, such as gemifloxacin, zabofloxacin and DW286 [4][5][6].
Recently, as a part of an ongoing program to optimize FQs against bacterial pathogens and mycobacterium tuberculosis (MTB), we have focused our attention on exploring the effect of introducing an oxime group into azetidine, pyrrolidine or piperidine side chains at the C-7 position of FQs, and some of them were found to have considerable biological activity [7][8][9][10]. For example, IMB-070593 ( Figure 1), a piperidinyl-based FQ candidate discovered in our lab and in late pre-clinical stage of development currently, possesses potent antibacterial and anti-MTB activity [9,11] as well as extremely low phototoxicity, hepatotoxicity and cardiac toxicity (unpublished data). However, IMB-070593 has low water solubility (0.4 mg/mL) and its mesylate salt still exhibits poor solubility (22.5 mg/mL). Since water solubility at physiological pH is important for preclinical testing, in vivo efficacy, and parenteral formulation [12], we decided to improve the solubility of IMB-070593 by employing a prodrug strategy. One of the best strategies for increasing solubility of a drug containing a hydroxyl, carboxyl or an amino moiety is, in pharmaceutical field, to transform it into an amino acid prodrug, as exemplified by valaciclovir (a valine prodrug of acyclovir) [13]. We had previously reported that some of the amino acid prodrugs of tosufloxacin and moxifloxacin (MXFX) have better solubility and in vivo activity against clinically pathogens than the parent drugs [14,15]. Inspired by the above research results, it was decided to introduce common amino acids onto the nitrogen of the 3-amino-4-(methoxyimino)piperidine side chain of IMB-070593 by covalent binding. Thus, a series of amino acid and dipeptide prodrugs of IMB-070593 were designed, synthesized and evaluated for their water solubility and then antibacterial activity in this study. Our primary objective was to improve the solubility of IMB-070593, followed by optimizing its in vivo potency so as to finally develop the parenteral formulation.

Chemistry
Synthetic pathways to the prodrugs 4a-l of IMB-070593 are depicted in Scheme 1. According to published procedures [14,15], treatment of various amino acids 1a-j with di-tert-butyl dicarbonate (Boc 2 O) in methanol gave N-Boc-protected acids 2a-j, which were coupled with IMB-070593 in the presence of dicyclohexylcarbodiimide (DCC) to yield the condensation products 3a-j. The N-Boc protecting group on 3a-j was removed with trifluoroacetic acid (TFA) to provide the amino acid prodrugs 4a-j. Similarly, Gly-Gly-amino prodrug 4k or L-Ala-L-Ala-amino prodrug 4l could be conveniently obtained from the corresponding mono-Gly-amino prodrug 4a and N-Boc-protected glycine 2a or mono-L-Ala-amino prodrug 4b and N-Boc-protected L-alanine 2b according to the same procedures used for preparation of 4a-j. Scheme 1. Synthesis of the prodrugs 4a-l of IMB-070593. Because the methyloxime group of all the prodrugs 4a-l may be present in the E-or Zconfiguration, it was necessary to determine their geometries. Although we were unable to prepare X-ray quality single crystals of any methyloxime intermediate or product in this study, we had previously obtained the ingle crystals of 4-(methoxyimino)-3-methylaminopiperidine dihydrochloride, a N-methylated analogue of the side chain at C-7 position of IMB-070593, in which the piperidine ring adopts a chair conformation and the methyloxime geometry exists in an E-configuration [9]. Accordingly, we can speculate that the methyloxime group of the prodrugs in this study should have the same E-configuration.

Water Solubility
The prodrugs 4a-l were initially evaluated for their water solubility which was determined by HPLC measurement of the concentration of a micromembrane filtered saturated solution [16]. The solubility of 4a-l along with the parent IMB-070593 and IMB-070593 mesylate for comparison is presented in Table 1. The data reveal that all of 4a-l have greater solubility (0.6-795.1 mg/mL) than that of IMB-070593 (0.4 mg/mL), and two dipeptide prodrugs 4k and 4l (235.0 and 281.5 mg/mL, respectively) are more soluble than the corresponding amino acid prodrugs 4a and 4b (97.0 and 112.0 mg/mL, respectively). It is encouraging that prodrugs 4a,b,e,f,k,l have also much greater solubility (>85 mg/mL) than the parent IMB-070593 mesylate (22.5 mg/mL) which suggest that the six prodrugs could display better in vivo efficacy than IMB-070593 mesylate.

In Vitro Activity
The prodrugs 4a-l were evaluated for their in vitro antibacterial activity against representative bacterial strains using standard techniques [17]. Minimum inhibitory concentration (MIC) is defined as the concentration of the compound required to give complete inhibition of bacterial growth, and MIC values of 4a-l against Gram-positive and Gram-negative strains along with IMB-070593 mesylate, MXFX and levofloxacin (LVFX) for comparison, are listed in Table 2. The prodrugs 4a-l show significantly less activity than the parent IMB-070593, MXFX and LVFX which is consistent with the biological character of common prodrugs. Moreover, 4a-l are generally more active against Gram-positive strains except methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) than Gram-negative ones, and they have virtually no activity against all of the tested Gram-negative strains except for few exceptions.

In Vivo Activity
Mice protection tests were used to evaluate in vivo efficacy of the prodrugs 4a,b,e,f,k,l with greater solubility than IMB-070593 mesylate, given the fact that there is no obvious difference among of 4a-l with regard to their in vitro activity. The efficacy of them was initially tested against two clinical isolate strains (methicillin-sensitive S. aureus / MSSA 12-1, E. Coli 12-1), and then 4a of the amino acid prodrugs and 4k of the dipeptide ones with better efficacy were chosen for further evaluation their protective effects in vivo (p.o.) against other four clinical isolates and that (i.v.) against the above two strains, IMB-070593 mesylate was used as the control drug ( Table 3).

General Information
Melting points were determined in open capillaries and are uncorrected. 1 H-NMR spectra were determined on a Varian Mercury-400/500/600 spectrometer in DMSO-d 6 or CDCl 3 using tetramethylsilane (TMS) as an internal standard. Electrospray ionization (ESI) mass spectra and high resolution mass spectra (HRMS) were obtained on a MDSSCIEX Q-Tap mass spectrometer and AccuTOF CS JMS-T100CS (JEOL) mass spectrometer. Unless otherwise noted, the reagents were obtained from commercial supplier and used without further purification. TLC was performed on silica gel plates (Merck, ART5554 60F254).

General Procedure for the Synthesis of 4a-j
To a stirring solution of trifluoroacetic acid (8.0 mL) was added 3a-j (1.80 mmol) in portions over a period of 0.5 h at −5-0 °C and stirred for 3 h at the same temperature, and then concentrated under reduced pressure. The residue was treated with diethyl ether (10 mL) and then filtered. The solid was dissolved in methanol (2 mL) and adjusted to pH 7.0 with the ammonia water, and then extracted with dichloromethane (30 mL × 3). The combined extracts were washed with saturated brine (10 mL) and dried over anhydrous Na 2 SO 4 , and then concentrated under reduced pressure to provide the title compounds 4a-j.

General Procedure for the Synthesis of 4k-l
To a stirring solution of trifluoroacetic acid (3.0 mL) was added 3k-l (0.47 mmol) in portions over a period of 0.5 h at −5-0 °C, stirred for 2 h at the same temperature and then concentrated under reduced pressure. The residue was treated with diethyl ether (7 mL) and filtered. The solid was dissolved in methanol (1 mL) and adjusted to pH 7.0 with the ammonia water, and then extracted with dichloromethane (50 mL × 3). The combined extracts were washed with saturated brine (10 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to provide the title compounds 4k-l.  13

MIC Determination
All compounds were screened for their in vitro antibacterial activity against representative Grampositive and Gram-negative strains, by means of standard twofold serial dilution method using agar media [17]. Minimum inhibitory concentration (MIC) is defined as the minimum concentration of the compound required to give complete inhibition of bacterial growth after incubation at 35 °C for 18-24 h.

Conclusions
In summary, a series of amino acid and dipeptide prodrugs of IMB-070593 were designed, synthesized and evaluated for their water solubility and antibacterial activity in this study. Our results reveal that the solubility (>85 mg/mL) of four amino acid prodrugs 4a,b,e,f and two dipeptide prodrugs 4k,l was much greater than that of IMB-070593 mesylate (22.5 mg/mL). Compounds 4a and 4k were found to have lower ED 50 values when administered by intravenous injection, compared with orally. Moreover, both of them show stronger efficacy against drug-sensitive Gram-positive strains than the parent drug, as opposed to Gram-negative and drug-resistant Gram-positive strains.