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Molecules 2014, 19(5), 6822-6837; doi:10.3390/molecules19056822

Article
Synthesis and Antibacterial Activity of Amino Acid and Dipeptide Prodrugs of IMB-070593, a Fluoroquinolone Candidate
Tingting Zhang 1,2,, Jinwei Wu 2,, Shihong Chen 2, Kaixiang Liu 3, Yabin Lin 4, Huiyuan Guo 1 and Mingliang Liu 1,*
1
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2
Zhejiang Starry Pharmaceutical Co. Ltd., Xianju 317300, China
3
China Resources Double-Crane Pharmaceutical Co. Ltd., Beijing 100102, China
4
Harbin Stomatological Hospital, Harbin 150010, China
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: lmllyx@126.com; Tel./Fax: +86-10-6303-6965.
Received: 14 April 2014; in revised form: 20 May 2014 / Accepted: 21 May 2014 /
Published: 23 May 2014

Abstract

: 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.
Keywords:
IMB-070593; prodrugs; synthesis; water solubility; antibacterial activity

1. 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 anti-infective chemotherapy field [1].

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.

Molecules 19 06822 g001 1024
Figure 1. Structure of IMB-070593.

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Figure 1. Structure of IMB-070593.
Molecules 19 06822 g001 1024

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.

2. Results and Discussion

2.1. Chemistry

Synthetic pathways to the prodrugs 4al of IMB-070593 are depicted in Scheme 1. According to published procedures [14,15], treatment of various amino acids 1aj with di-tert-butyl dicarbonate (Boc2O) in methanol gave N-Boc-protected acids 2aj, which were coupled with IMB-070593 in the presence of dicyclohexylcarbodiimide (DCC) to yield the condensation products 3aj. The N-Boc protecting group on 3aj was removed with trifluoroacetic acid (TFA) to provide the amino acid prodrugs 4aj. 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 4aj.

Molecules 19 06822 g002 1024
Scheme 1. Synthesis of the prodrugs 4al of IMB-070593.

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Scheme 1. Synthesis of the prodrugs 4al of IMB-070593.
Molecules 19 06822 g002 1024

Reagents and Conditions: (i) (1) (Boc)2O, (C2H5)3N, CH3OH, (2) 2 N HCl; (ii) IMB-070593, DCC, CH2Cl2; (iii)/(v) (1) TFA, (2) NH4OH; (iv) 2a or 2b, DCC, CH2Cl2.

Because the methyloxime group of all the prodrugs 4al may be present in the E- or Z- configuration, 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.

2.2. Water Solubility

The prodrugs 4al 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 4al along with the parent IMB-070593 and IMB-070593 mesylate for comparison is presented in Table 1. The data reveal that all of 4al 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.

Table Table 1. Structures, physical data and solubility of compounds 4al.

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Table 1. Structures, physical data and solubility of compounds 4al.
Compd.R1R2m.p.[°C] a[α] Molecules 19 06822 i001(c, CH3OH)Water Solubility(mg/mL)
4aHH136–1370° (0.50)112.0
4bHCH3133–134−2.80°(0.50)97.0
4cH(CH3)2CH154–156−24.30°(0.50)15.6
4dH(CH3)2CHCH2109–110−20.37°(0.11)5.3
4e(CH2)3157–158−19.17°(0.24)795.1
4fH(S)-CH3CH2CH(CH3)136–138−26.68°(0.40)87.4
4gHC6H5CH2123–125−13.32°(0.77)1.0
4hHCH2OH114–116−9.01° (0.44)50.2
4iH4-(OH)C6H4CH2138–140−14.14°(0.50)0.6
4jH(R)-CH3CH(OH)111–113−15.25°(0.12)72.3
4kHH121–1230° (0.13)235.0
4lHCH3120–121−27.19°(0.23)281.5
IMB 0.4
IMB mesylate 22.5

a Melting points are uncorrected; IMB: IMB-070593.

2.3. Antibacterial Activity

2.3.1. In Vitro Activity

The prodrugs 4al 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 4al against Gram-positive and Gram-negative strains along with IMB-070593 mesylate, MXFX and levofloxacin (LVFX) for comparison, are listed in Table 2.

Table Table 2. In vitro activity of compounds 4al against Gram-positive and Gram-negative strains.

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Table 2. In vitro activity of compounds 4al against Gram-positive and Gram-negative strains.
Compd.Strains MIC (μg/mL)
S.a.MSSAMRSAMSSEMRSES.p.E.coliE.co.1E.co.2E.co.3K.p.1K.p.2P.a.1P.a.2P.a.3
4a0.250.5321320. 5416128>128832321664
4b0.1250.25641321416128>128864641664
4c0.250.25642641416>128>1281632323264
4d0.250.25644642416>128>128161281286464
4e0.252644642432>128>128166464128128
4f0.50.5642641832>128>128161281283264
4g0.1250.256416411664>128>12864>12812864128
4h1164264116128>128>12832>12812832128
4i22>128864216128>128>12832>12812832128
4j2112826421664>128>1283264643264
4k48>1281664816128>128>12864>128>128>128128
4l44>1283264832>128>128>12864>128128>128128
IMB mesylate<0.008<0.00820.060.1250.030.060.516160.060.54216
MXFX<0.008<0.00880.1250.250.06<0.0080.516160.030.52168
LVFX0.060.06640.50.030.125<0.0080.254160.030.50.5328

S.a.: S. aureus ATCC 25923. MSSA: Methicillin-sensitive S. aureus 12-4. MRSA: Methicillin-resistant S. aureus 12-1. MSSE: Methicillin-sensitive S.epidermidis 12-3. MRSE: Methicillin-resistant S. epidermidis 12-1. S.p.: S. pneumoniae ATCC 49619. E.coli: E. coli ATCC 25922. E.co.1: E. coli 12-6. E.co.2: E. coli 12-11. E.co.3: Extended-spectrum β-lactamase-producing (ESBL+) E. coli 12-14. K.p.1: K. pneumoniae 12-4. K.p.2: ESBL+ K. pneumoniae 12-7. P.a.1: P. aeruginosa 12-12. P.a.2: P. aeruginosa 12-14. P.a.3: P. aeruginosa 12-20. IMB mesylate: IMB-070593 mesylate. MXFX: Moxifloxacin. LVFX: Levofloxacin.

The prodrugs 4al show significantly less activity than the parent IMB-070593, MXFX and LVFX which is consistent with the biological character of common prodrugs. Moreover, 4al 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.

2.3.2. 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 4al 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).

The results illustrate that all of the six prodrugs have better oral activity against MSSA 12-1 (6.44–9.39 mg/kg) than IMB-070593 mesylate (9.40 mg/kg), but weaker against E. coli 12-1. Furthermore, 4a and 4k show stronger efficacy than the parent drug against S. pneumonia 12-10 (p.o., 18.39 and 23.13 mg/kg, respectively) and MSSA 12-1 (i.v., 5.71 and 5.32 mg/kg, respectively), but weaker oral activity against MRSE 12-1, MRSA 12-5 and K. pneumonia 12-1. In a word, the prodrugs seem to be more active against drug-sensitive Gram-positive strains (p.o. or i.v., such as MSSA12-1 and S. pneumonia 12-10) than IMB-070593 mesylate. Conversely, they are much weaker against drug-resistant Gram-positive strains (p.o., such as MRSE 12-1and MRSA 12-5) and Gram-negative strains (p.o., such as E. Coli 12-1 and K. pneumonia 12-1), compared with the parent drug.

Table Table 3. In vivo efficacy of selected compounds against systemic infections in mice.

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Table 3. In vivo efficacy of selected compounds against systemic infections in mice.
Infected strain(cfu/mL)CompdMIC(μg/mL)ED50 (mg/kg)a [95% confidence limit (mg/kg)]
MSSA 12-1
(3.0 × 104)
4a17.16 (10.13–4.97) b5.71 (7.97–4.11) c
4b0.59.30 (13.43–6.41)NT d
4e28.74 (12.17–6.27)NT
4f16.44 (9.75–3.83)NT
4k87.68 (10.76–5.42)5.32 (7.58–3.74)
4l89.39 (13.85–6.31)NT
IMB mesylate<0.0089.40 (13.36–6.60)7.15 (10.73–4.95)
E. coli 12-1
(6.0 × 105)
4a1617.53 (25.25–12.04)12.51 (19.41–8.37)
4b1625.17 (37.89–17.12)NT
4e3221.44 (31.18–14.83)NT
4f3217.53 (25.25–12.04)NT
4k12818.80 (26.72–13.20)14.30 (21.46–9.91)
4l>12823.43 (36.23–15.57)NT
IMB mesylate0.58.76 (12.63–6.02)7.67 (11.83–5.27)
MRSE 12-1
(4.5 × 106)
4a3250.55 (39.57–66.50)NT
4k6454.03 (40.89–77.35)NT
IMB mesylate215.33 (11.47–20.06)NT
MRSA 12-5
(4.5 × 106)
4a3272.80 (55.03–123.15)NT
4k>12886.06 (67.10–152.78)NT
IMB mesylate225.36 (20.10–32.68)NT
S.pneumonia12-10
(5.2 × 108)
4a118.39 (14.00–23.60)NT
4k223.13 (17.30–31.81)NT
IMB mesylate0.0627.57 (21.85–36.07)NT
K.pneumoniae 12-1
(4.5 × 106)
4a433.91 (26.82–44.56)NT
4k1633.30(25.16–47.78)NT
IMB mesylate0.0618.56(14.55–23.97)NT

a ED50: 50% effective dose [95% confidence limit (mg/kg)]; b Antimicrobial agents were orally administrated twice at 0 h and 6 h after infection.; c Antimicrobial agents were intravenous injected once at 0 h after infection; d NT: not tested. MRSE: MRSE: Methicillin-resistant S. epidermidis. MRSA: Methicillin-resistant S. aureus.

3. Experimental

3.1. General Information

Melting points were determined in open capillaries and are uncorrected. 1H-NMR spectra were determined on a Varian Mercury-400/500/600 spectrometer in DMSO-d6 or CDCl3 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).

3.2. Synthesis

3.2.1. General Procedure for the Synthesis of 3aj

To a solution of amino acids 1aj (20 mmol) in methanol (90 mL) was added di-tert-butyl carbonate (8.73 g, 40 mmol) and triethylamine (11.1 mL, 80 mmol). The reaction mixture was heated to refluxing and stirred for 3 h at the same temperature, and concentrated under reduced pressure. The residue was diluted with water (40 mL), adjusted to pH 2.0–3.0 with 2 N HCl at 0–5 °C, and then extracted with ethyl acetate (50 mL × 3). The combined extracts were washed with saturated brine (30 mL), dried over anhydrous Na2SO4, and then concentrated under reduced pressure to provide 2aj as white solids [18,19,20,21,22,23] (64.8%–91.4%). A mixture of IMB-070593 (2.08 g, 4.97 mmol), 2aj (5.71 mmol), dicyclohexylcarbodiimide (1.18 g, 5.71 mmol) and dry dichloromethane (42 mL) was stirred at room temperature for 1 h and filtered. The filtrate was concentrated under reduced pressure, and the residue was treated with diethyl ether (20 mL), and then filtered. The solid was purified by column chromatography (silica gel) eluted with dichloromethane and methanol (v:v = 55:1) to afford the title compounds 3aj (36%–79%, from 1aj) as white or yellow solids.

7-(3-(2-(tert-Butoxycarbonylamino)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3a). Obtained from 2a and IMB-070593 as a white solid (86.1%), m.p.: 139–140 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.68 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.90 (d, J = 11.7 Hz, 1H, C5-H), 7.12 (d, J = 5.4 Hz, 1H), 5.09 (s, 1H), 4.70–4.59 (m, 1H), 4.18–4.14 (m, 1H), 4.04–4.01 (m, 1H), 3.92 (s, 3H, CH3O-N), 3.85 (d, J = 5.4 Hz, 1H), 3.79 (s, 3H, CH3O-C), 3.72 (q, J = 6.9 Hz, 1H), 3.62 (d, J = 9.0 Hz, 1H), 3.34 (d, J = 14.2 Hz, 1H), 3.26 (t, J = 11.9 Hz, 1H), 3.04 (t, J = 10.3 Hz, 1H), 2.34–2.32 (m, 1H), 1.47 (s, 9H, Boc), 1.30–1.19 (m, 2H, cyclopropyl CH2), 1.12–0.91 (m, 2H, cyclopropyl CH2). MS-ESI (m/z): 576.31(M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3b). Obtained from 2b as a off-white solid (84.6%), m.p.: 150–152 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.68 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.91 (d, J = 11.6 Hz, 1H, C5-H), 7.27–7.07 (m, 1H), 4.98 (s, 1H), 4.63 (s, 1H), 4.21 (s, 1H), 4.22–4.10 (m, 1H), 4.03 (s, 1H), 3.92 (d, J = 2.6 Hz, 3H, CH3O-N), 3.81 (s, 3H, CH3O-C), 3.61 (d, J = 7.7 Hz, 1H), 3.36–3.24 (m, 2H), 3.03 (t, J = 10.8 Hz, 1H), 2.32 (s, 1H), 1.46 (d, J = 6.8 Hz, 9H, Boc), 1.38–1.26 (m, 3H, CH3), 1.25–1.13 (m, 2H, cyclopropyl CH2), 1.01 (s, 2H, cyclopropyl CH2). MS-ESI (m/z): 590.32(M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)-3-methylbutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3c). Obtained from 2c as a white solid (78.6%), m.p.: 130–131 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.69 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.91 (d, J = 11.6 Hz, 1H, C5-H), 7.01–6.89 (m, 1H), 5.06 (s, 1H), 4.73–4.58 (m, 1H), 4.19–4.08 (m, 1H), 4.07–4.01 (m, 1H), 3.92 (d, J = 8.3 Hz, 3H, CH3O-N), 3.80 (s, 3H, CH3O-C), 3.61 (s, 1H), 3.48 (q, J = 7.0 Hz, 1H), 3.39–3.20 (m, 2H), 3.06–2.99 (m, 1H), 2.31 (s, 1H), 2.21–2.08 (m, 1H, CH(CH3)2), 1.45 (d, J = 2.3 Hz, 9H, Boc), 1.23–1.21(m, 2H, cyclopropyl CH2), 1.09–0.88 (m, 8H, cyclopropyl CH2, CH(CH3)2). MS-ESI (m/z): 618.65 (M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)-4-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3d). Obtained from 2d as a yellow solid (58.2%), m.p.: 142–143 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 14.67 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.91 (d, J = 11.6 Hz, 1H, C5-H), 7.26–7.02 (m, 1H), 4.88 (d, J = 19.8 Hz, 1H), 4.64 (s, 1H), 4.14 (d, J = 17.1 Hz, 1H), 4.06–4.00 (m, 1H), 3.92 (d, J = 9.4 Hz, 3H, CH3O-N), 3.80 (d, J = 2.6 Hz, 3H, CH3O-C), 3.61 (s, 1H), 3.46 (d, J = 10.3 Hz, 1H), 3.39–3.21 (m, 2H), 3.03 (t, J = 10.5 Hz, 1H), 2.38–2.24 (m, 1H), 1.94 (d, J = 9.8 Hz, 1H), 1.70 (s, 2H), 1.45 (d, J = 6.4 Hz, 9H, Boc), 1.38–1.30 (m, 1H), 1.27 (d, J = 10.0 Hz, 1H), 1.21–1.13 (m, 2H, cyclopropyl CH2), 1.04–1.00 (m, 1H), 0.98–0.95 (m, 5H). MS-ESI (m/z): 632.63 (M+H)+.

7-(3-((S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3e). The title compound 3e was obtained from 2e as a off-white solid (83.6%), m.p.: 186–187 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.67 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.91 (d, J = 11.5 Hz, 1H, C5-H), 7.00 (s, 1H), 4.63 (s, 1H), 4.22 (s, 1H), 4.10 (d, J = 15.4 Hz, 1H), 4.03 (d, J = 3.9 Hz, 1H), 3.91 (d, J = 2.6 Hz, 3H, CH3O-N), 3.81 (d, J = 4.8 Hz, 3H, CH3O-C), 3.70–3.17 (m, 6H), 3.03 (s, 1H), 2.47–2.10 (m, 2H), 2.00–1.84 (m, 2H), 1.46 (d, J = 10.9 Hz, 9H, Boc), 1.29–1.20 (m, 2H, cyclopropyl CH2), 1.00 (d, J = 4.4 Hz, 2H, cyclopropyl CH2). MS-ESI (m/z): 616.67 (M+H)+.

7-(3-((2S,3S)-2-(tert-Butoxycarbonylamino)-3-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3f). Obtained from 2f as a white solid (96.5%), m.p. : 126–127 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.68 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.91 (d, J = 11.6 Hz, 1H, C5-H), 6.99 (d, J = 5.9 Hz, 1H), 5.08 (s, 1H), 4.67 (d, J = 4.1 Hz, 1H), 4.21–3.98 (m, 3H), 3.92 (d, J = 8.5 Hz, 3H, CH3O-N), 3.80 (s, 3H, CH3O-C), 3.61 (s, 1H), 3.35 (d, J = 14.1 Hz, 1H), 3.26 (s, 1H), 3.03 (d, J = 9.6 Hz, 1H), 2.30 (s, 1H), 1.90–1.82 (m, 2H), 1.46 (s, 9H, Boc), 1.29–1.16 (m, 3H), 1.01 (s, 2H), 0.95–0.91 (m, 6H). MS-ESI (m/z): 632.35 (M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)-3-phenylpropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3g). Obtained from 2g as a white solid (79.1%), m.p.: 149–150 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.68 (s, 1H, COOH), 8.84 (s, 1H, C2-H), 7.92 (d, J = 11.6 Hz, 1H, C5-H), 7.40–7.22 (m, 5H, ph-H), 6.90–6.77 (m, 1H), 5.07–5.01 (m, 1H), 4.56 (s, 1H), 4.40 (s, 1H), 4.16–3.97 (m, 2H), 3.85 (d, J = 10.0 Hz, 3H, CH3O-N), 3.79 (s, 3H, CH3O-C), 3.56 (s, 1H), 3.31–2.75 (m, 5H), 2.28 (t, J = 12.4 Hz, 1H), 1.43 (s, 9H, Boc), 1.28–1.20 (m, 2H, cyclopropyl CH2), 1.03 (d, J = 11.3 Hz, 2H, cyclopropyl CH2). MS-ESI (m/z): 666.31 (M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)-3-hydroxypropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3h). Obtained from 2h as a off-white solid (83.3%), m.p. : 133–135 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.64 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.92 (d, J = 11.6 Hz, 1H, C5-H), 7.15 (s, 1H), 5.48 (s, 1H), 4.84–4.61 (m, 1H), 4.23 (s, 1H), 4.17–3.97 (m, 3H), 3.93 (d, J = 11.7 Hz, 3H, CH3O-N), 3.80 (d, J = 2.6 Hz, 3H, CH3O-C), 3.70–3.68 (m, 1H), 3.64–3.43 (m, 1H), 3.35–3.25 (m, 2H), 3.12 (t, J = 10.3 Hz, 1H), 2.37–2.31 (m, 1H), 1.91 (brs, 1H, OH), 1.47 (d, J = 10.2 Hz, 9H, Boc), 1.29–1.19 (m, 2H, cyclopropyl CH2), 1.03 (t, J = 7.7 Hz, 2H, cyclopropyl CH2). MS-ESI (m/z): 606.24 (M+H)+.

7-(3-((S)-2-(tert-Butoxycarbonylamino)-3-(4-hydroxyphenyl)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3i). Obtained from 2i as a off-white solid (43.9%), m.p.: 151–153 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.73 (s, 1H, COOH), 8.82 (s, 1H, C2-H), 7.89–7.84 (m, 1H, C5-H), 7.02 (d, J = 8.1 Hz, 2H), 6.98–6.76 (m, 1H), 6.72 (d, J = 8.3 Hz, 2H), 5.14 (d, J = 35.9 Hz, 1H), 4.56 (s, 1H), 4.37 (s, 1H), 4.06 (d, J = 12.0 Hz, 2H), 3.87 (d, J = 10.9 Hz, 3H, CH3O-N), 3.80 (d, J = 4.7 Hz, 3H, CH3O-C), 3.76–3.46 (m, 2H), 3.22 (d, J = 11.9 Hz, 2H), 3.03–2.89 (m, 2H), 2.38–2.23 (m, 1H), 1.44 (s, 9H, Boc), 1.26–1.19 (m, 2H, cyclopropyl CH2), 1.12–0.94 (m, 2H, cyclopropyl CH2). MS-ESI (m/z): 682.22 (M+H)+.

7-(3-((2S,3R)-2-(tert-Butoxycarbonylamino)-3-hydroxybutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3j). Obtained from 2j as a white solid (47.7%), m.p.: 134–136 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.64 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.92 (d, J = 11.6 Hz, 1H, C5-H), 7.33 (s, 1H), 5.42 (s, 1H), 4.74–4.65 (m, 1H), 4.42–4.33 (m, 1H), 4.17–4.03 (m, 3H), 3.92 (t, J = 4.3 Hz, 3H, CH3O-N), 3.80 (s, 3H, CH3O-C), 3.61 (s, 1H), 3.29 (dd, J = 22.8, 13.8 Hz, 2H), 3.11 (t, J = 11.4 Hz, 1H), 2.43–2.26 (m, 1H), 1.80 (brs, 1H, OH), 1.46 (d, J = 11.6 Hz, 9H, Boc), 1.26–1.20 (m, 5H, cyclopropyl CH2, CH3CH), 1.01 (s, 2H, cyclopropyl CH2). MS-ESI (m/z): 620.21 (M+H)+.

3.2.2. General Procedure for the Synthesis of 4aj

To a stirring solution of trifluoroacetic acid (8.0 mL) was added 3aj (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 Na2SO4, and then concentrated under reduced pressure to provide the title compounds 4aj.

7-(3-(2-Aminoacetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4a). Obtained from 3a as a yellow solid (71.8%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.83 (s, 1H, C2-H), 8.14 (s, 1H), 7.90 (d, J = 8.9 Hz, 1H, C5-H), 4.71 (s, 1H), 4.03–3.81 (m, 6H), 3.62 (s, 1H), 3.49–3.12 (m, 6H), 2.41 (s, 2H), 1.25–1.21 (m, 2H, cyclopropyl CH2), 1.02 (d, J = 17.5 Hz, 2H, cyclopropyl CH2). 13C-NMR (150 MHz, DMSO-d6) δ 176.33, 172.48, 165.59, 156.28, 154.37, 150.61, 146.23, 138.59(d, J = 12.2 Hz), 134.06, 121.49, 106.69, 106.46, 63.24, 61.33, 55.74, 49.15, 44.50, 40.73, 24.85, 18.55, 9.03, 8.86. MS-ESI (m/z): 476.23(M+H)+. HRMS-ESI (m/z): C22H27O6N5F, Calcd: 476.193899(M+H)+; Found: 476.19389(M+H)+.

7-(3-((S)-2-Aminopropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4b). Obtained from 3b as a yellow solid (88.3%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.81 (s, 1H, C2-H), 8.38–8.00 (m, 1H), 7.87 (d, J = 11.7 Hz, 1H, C5-H), 4.67 (s, 1H), 4.06 (s, 2H), 3.93 (s, 3H, CH3O-N), 3.82 (s, 3H, CH3O-C), 3.57 (d, J = 13.5 Hz, 2H), 3.31 (d, J = 11.1 Hz, 2H), 3.11 (s, 1H), 2.55–2.30 (m, 1H), 1.38–1.33 (m, 2H), 1.26–1.19 (m, 3H), 1.03 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 177.15, 175.65, 166.78, 157.70, 153.47, 150.12, 146.20, 139.02 (d, J = 12 Hz), 133.91, 122.77, 108.40, 107.96, 62.84, 62.06, 56.65, 51.00, 50.64, 50.24, 40.70, 25.67, 21.77, 9.86, 9.53. MS-ESI (m/z): 490.25 (M+H)+.HRMS-ESI (m/z): C23H29O6N5F, Calcd: 490.20964 (M+H)+; Found: 490.20968 (M+H)+.

7-(3-((S)-2-Amino-3-methylbutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4c). Obtained from 3c as a light yellow solid (70.0%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.70 (s, 1H, C2-H), 8.29–8.24 (m, 1H), 7.77 (d, J = 10.6 Hz, 1H, C5-H), 4.59–4.55 (m, 1H), 4.16 (s, 1H), 3.83 (s, 3H, CH3O-N), 3.76 (d, J = 5.2 Hz, 3H, CH3O-N), 3.73–3.58 (m, 1H), 3.56–3.41 (m, 1H), 3.28–3.16 (m, 1H), 3.03 (d, J = 3.5 Hz, 2H), 2.60–2.50 (m, 1H), 1.98–1.94 (m, 1H), 1.91–1.72 (m, 1H), 1.21–0.91 (m, 4H, 2 × cyclopropyl CH2), 0.87 (d, J = 6.8 Hz, 1H), 0.80 (d, J = 6.8 Hz, 2H), 0.74–0.70 (m, 3H). 13C-NMR (100 MHz, DMSO-d6) δ 176.30, 174.16, 165.61, 156.74, 154.45, 150.62, 146.08(d, J = 18 Hz), 138.60, 134.04, 121.33, 106.65, 106.42, 63.11, 61.27, 59.66, 55.47, 49.25, 40.71, 30.30, 24.76, 19.29, 16.65, 16.53, 8.98, 8.88. MS-ESI (m/z): 518.35 (M+H)+.HRMS-ESI (m/z): C25H33O6N5F, Calcd: 518.24094 (M+H)+; Found: 518.24080 (M+H)+.

7-(3-((S)-2-Amino-4-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4d). Obtained from 3d as a yellow solid (71.5%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.71 (s, 1H, C2-H), 8.46–8.23 (m, 1H), 7.77 (d, J = 10.6 Hz, 1H, C5-H), 4.55–4.49 (m, 1H), 4.17 (s, 1H), 3.83 (s, 3H, CH3O-N), 3.76 (d, J = 8.3 Hz, 3H, CH3O-C), 3.69 (s, 1H), 3.48–3.21 (m, 3H), 3.03 (t, J = 16.1 Hz, 1H), 2.64 (d, J = 6.4 Hz, 1H), 1.72 (s, 1H), 1.56 (s, 1H), 1.41 (s, 1H), 1.35–0.96 (m, 5H), 0.86–0.81 (m, 3H), 0.77–0.73 (m, 3H). 13C-NMR (100 MHz, DMSO-d6) δ 176.31, 175.04, 165.60, 156.72, 154.44, 150.60, 146.02 (d, J = 33 Hz), 138.66, 134.03, 121.43, 106.65, 106.42, 63.13, 61.27, 55.51, 52.99, 49.16, 44.07, 40.70, 24.87, 23.97, 23.12, 23.03, 21.85, 8.97, 8.93. MS-ESI (m/z): 532.40 (M+H)+. HRMS-ESI (m/z): C26H35O6N5F, Calcd: 532.25659 (M+H)+; Found: 532.25671 (M+H)+.

7-(4-(methoxyimino)-3-((S)-pyrrolidine-2-carboxamido)piperidin-1-yl)-1-Cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4e). Obtained from 3e as a yellow solid (66.8%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.81 (s, 1H, C2-H), 8.45–8.39 (m, 1H), 7.87 (d, J = 11.7 Hz, 1H, C5-H), 4.67 (s, 1H), 4.04–3.98 (m, 2H), 3.92 (d, J = 7.8 Hz, 3H, CH3O-N), 3.81 (s, 4H), 3.57 (d, J = 12.5 Hz, 1H), 3.31–3.27 (m, 2H), 3.15 (t, J = 10.7 Hz, 1H), 3.05–2.93 (m, 2H), 2.51–2.31 (m, 1H), 2.15–2.10 (m, 1H), 2.00–1.95 (m, 1H), 1.88–1.56 (m, 2H), 1.24–1.19 (m, 2H, cyclopropyl CH2), 1.02 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 177.12, 175.17, 166.76, 157.61, 153.61, 150.11, 146.10, 138.97 (d, J = 12 Hz), 133.91, 122.64, 108.41, 107.97, 62.81, 61.96, 60.82, 56.59, 50.52, 49.88, 47.37, 40.68, 30.92, 26.21, 25.61, 9.86, 9.53. MS-ESI (m/z): 516.36 (M+H)+.HRMS-ESI (m/z): C25H31O6N5F, Calcd: 516.22529 (M+H)+; Found: 516.22545 (M+H)+.

7-(3-((2S,3S)-2-Amino-3-methylpentanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4f). Obtained from 3f as a yellow solid (88.3%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.73 (d, J = 3.5 Hz, 1H, C2-H), 7.91–7.57 (m, 1H, C5-H), 7.46 (s, 1H), 4.71 (s, 1H), 4.13–4.04 (m, 3H), 3.90 (d, J = 6.9 Hz, 3H, CH3O-N), 3.79 (s, 3H, CH3O-C), 3.57–3.47 (m, 1H), 3.34–3.19 (m, 2H), 2.41 (s, 1H), 2.13 (s, 1H), 1.63–1.58 (m, 1H), 1.22 (d, J = 6.4 Hz, 4H), 1.06–0.97 (m, 6H), 0.92 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 176.74, 175.34, 166.81, 154.83, 153.05, 150.25, 145.93(d, J = 12 Hz), 138.91, 134.05, 122.08, 107.94, 107.61, 63.12, 62.15, 58.19, 55.96, 50.94, 50.48, 40.87, 37.01, 25.38, 14.86, 11.75, 9.76. MS-ESI (m/z): 532.33 (M+H)+.HRMS-ESI (m/z): C26H35O6N5F, Calcd: 532.25659 (M+H)+; Found: 532.25653 (M+H)+.

7-(3-((S)-2-Amino-3-phenylpropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4g). Obtained from 3g as a yellow solid (82.4%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.72 (s, 1H, C2-H), 8.28 (d, J = 4.6 Hz, 1H), 7.90–7.70 (m, 1H, C5-H), 7.40–6.90 (m, 5H, ph-H), 4.54 (s, 1H), 4.17 (s, 1H), 3.83 (d, J = 8.0 Hz, 3H, CH3O-N), 3.76–3.66 (m, 4H), 3.47 (d, J = 5.7 Hz, 2H), 3.31 (s, 2H), 3.23–3.07 (m, 1H), 2.96–2.91 (m, 2H), 2.74–2.51 (m, 1H), 1.26–0.83 (m, 4H, 2 × cyclopropyl CH2). 13C-NMR (100 MHz, DMSO-d6) δ 176.29, 173.76, 165.66, 156.71, 154.33, 150.64, 146.24, 138.35(d, J = 29 Hz), 134.00, 129.24, 128.02, 126.01, 121.57, 106.69, 106.46, 63.14, 61.30, 56.00, 55.68, 49.25, 40.68, 40.45, 24.70, 16.37, 8.95. MS-ESI (m/z): 566.40 (M+H)+. HRMS-ESI (m/z): C29H33O6N5F, Calcd: 566.24094 (M+H)+; Found: 566.24108 (M+H)+.

7-(3-((S)-2-Amino-3-hydroxypropanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4h). Obtained from 3h as a yellow solid (66.2%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.71 (s, 1H, C2-H), 8.36 (d, J = 5.1 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H, C5-H), 4.76 (s, 1H), 4.56–4.52 (m, 1H), 4.17 (s, 1H), 3.84 (d, J = 0.9 Hz, 3H, CH3O-N), 3.81–3.70 (m, 4H), 3.60–3.35 (m, 4H), 3.26–3.25 (m, 2H), 3.11–2.96 (m, 1H), 2.56–2.51 (m, 1H), 1.13–0.96 (m, 4H, 2 × cyclopropyl CH2). 13C-NMR (100 MHz, DMSO-d6) δ 176.36, 172.77, 165.60, 156.82, 154.37, 150.61, 146.29 (d, J = 11 Hz), 138.67, 134.05, 121.49, 106.69, 106.42, 64.90, 63.18, 61.34, 56.76, 55.65, 49.40, 40.75, 24.85, 15.16, 8.92. MS-ESI (m/z): 506.28 (M+H)+.HRMS-ESI (m/z): C23H29O7N5F, Calcd: 506.20455 (M+H)+; Found: 506.20465 (M+H)+.

7-(3-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4i). Obtained from 3i as a light yellow solid (83.4%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 9.11 (d, J = 15.9 Hz, 1H), 8.71 (d, J = 4.5 Hz, 1H, C2-H), 8.23 (t, J = 8.0 Hz, 1H), 7.79 (dd, J = 11.8, 6.1 Hz, 1H, C5-H), 6.95 (t, J = 7.6 Hz, 2H), 6.59 (dd, J = 14.6, 8.3 Hz, 2H), 4.53 (s, 1H), 4.18 (dd, J = 6.8, 3.8 Hz, 1H), 3.83 (d, J = 7.8 Hz, 3H, CH3O-N), 3.74 (d, J = 10.8 Hz, 3H, CH3O-C), 3.67 (d, J = 7.8 Hz, 1H), 3.54–3.44 (m, 1H), 3.42–3.34 (m, 2H), 3.26–3.16 (m, 1H), 3.15–2.89 (m, 2H), 2.79 (d, J = 11.0 Hz, 1H), 2.65–2.53 (m, 1H), 1.16–1.01 (m, 4H, 2 × cyclopropyl CH2). 13C-NMR (100 MHz, DMSO-d6) δ 176.40, 173.94, 165.64, 156.74, 155.78, 154.30, 150.63, 146.26, 138.72(d, J = 38 Hz), 134.07, 130.27, 130.09, 128.33, 127.95, 121.53, 114.88, 106.69, 106.48, 63.15, 61.31, 56.18, 55.80, 49.71, 49.24, 40.75, 24.87, 15.15, 9.06, 8.96. MS-ESI (m/z): 582.30 (M+H)+.HRMS-ESI (m/z): C29H33O7N5F, Calcd: 582.23585 (M+H)+; Found: 582.23593 (M+H)+.

7-(3-((2S,3R)-2-Amino-3-hydroxybutanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4J). Obtained from 3J as a yellow solid (73.3%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.71 (s, 1H, C2-H), 8.38 (d, J = 7.0 Hz, 1H), 7.78 (dd, J = 11.8, 3.1 Hz, 1H, C5-H), 4.63–4.50 (m, 1H), 4.22–4.10 (m, 1H), 3.91–3.80 (m, 4H), 3.79–3.59 (m, 4H), 3.51–3.47 (m, 1H), 3.31–3.12 (m, 2H), 3.10–3.02 (m, 1H), 2.99 (d, J = 4.1 Hz, 1H), 2.60–2.51 (m, 1H), 1.29–1.23 (m, 1H), 1.13–1.11 (m, 2H), 1.04–0.97 (m, 3H), 0.91–0.73 (m, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 176.34, 173.04, 165.60, 156.84, 154.39, 150.61, 146.10, 138.56 (d, J = 38 Hz), 134.06, 122.24, 106.66, 106.42, 67.14, 63.17, 61.32, 60.19, 55.60, 49.47, 39.94, 24.85, 20.08, 19.94, 8.94. MS-ESI (m/z): 520.28 (M+H)+. HRMS-ESI (m/z): C24H31O7N5F, Calcd: 520.22020 (M+H)+; Found: 520.22083 (M+H)+.

3.2.3. General Procedure for the Synthesis of 3kl

A mixture of 4ab (0.82 mmol), 2ab (0.82 mmol), dicyclohexylcarbodiimide (0.17 g, 0.82 mmol) and dry dichloromethane (20 mL) was stirred at room temperature for 4 h, filtered and concentrated under reduced pressure. The solid was treated with diethyl ether (3 mL) and filtered. The solid was purified by column chromatography (silica gel) eluted with dichloromethane and methanol (v:v = 60:1) to afford the title compounds 3kl.

7-(3-(2-(2-(tert-Butoxycarbonylamino)acetamido)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3k). Obtained from 4a and 2a as a light yellow solid (30.8%), m.p.: 122–124 °C.1H-NMR (400 MHz, CDCl3) δ (ppm) 14.71 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.90 (d, J = 11.6 Hz, 1H, C5-H), 6.97 (d, J = 5.6 Hz, 1H), 6.75 (s, 1H), 5.07 (s, 1H), 4.72–4.56 (m, 1H), 4.11 (d, J = 7.0 Hz, 1H), 4.06–3.95 (m, 3H), 3.93 (s, 3H, CH3O-N), 3.86 (d, J = 3.7 Hz, 2H), 3.78 (s, 3H, CH3O-C), 3.61 (d, J = 10.0 Hz, 1H), 3.34–3.24 (m, 2H), 3.07 (t, J = 10.5 Hz, 1H), 2.42–2.26 (m, 1H), 1.45 (s, 9H, Boc), 1.26–1.21 (m, 2H, cyclopropyl CH2), 1.07–0.98 (m, 2H, cyclopropyl CH2). MS-ESI (m/z): 633.17 (M+H)+.

7-(3-((S)-2-((S)-2-(tert-Butoxycarbonylamino)propanamido)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3l). Obtained from 4b and 2b as a yellow solid (71.5%), m.p.: 126–128 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm) 14.68 (s, 1H, COOH), 8.83 (s, 1H, C2-H), 7.90 (d, J = 11.6 Hz, 1H, C5-H), 7.18–6.92 (m, 1H), 6.71 (d, J = 20.8 Hz, 1H), 4.93 (s, 1H), 4.70–4.58 (m, 1H), 4.53–4.48 (m, 1H), 4.16 (s, 1H), 4.16–4.03 (m, 2H), 3.92 (d, J = 4.0 Hz, 3H, CH3O-N), 3.79 (d, J = 1.5 Hz, 3H, CH3O-C), 3.59 (s, 1H), 3.40–3.18 (m, 2H), 3.14–2.97 (m, 1H), 2.34–2.32 (m, 1H), 1.63–1.29 (m, 15H, Boc, 2 × CH3), 1.28–1.16 (m, 2H, cyclopropyl CH2), 1.09–0.87 (m, 2H, cyclopropyl CH2). MS-ESI (m/z): 661.33 (M+H)+.

3.2.4. General Procedure for the Synthesis of 4kl

To a stirring solution of trifluoroacetic acid (3.0 mL) was added 3kl (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 Na2SO4, and concentrated under reduced pressure to provide the title compounds 4kl.

7-(3-(2-(2-Aminoacetamido)acetamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4k). Obtained from 3k as a yellow solid (73.3%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.81 (s, 1H, C2-H), 7.98–7.80 (m, 2H), 7.11 (d, J = 5.9 Hz, 1H), 4.75–4.56 (m, 1H), 4.14–4.10 (m, 1H), 4.07–4.00 (m, 2H), 3.97 (d, J = 5.6 Hz, 1H), 3.92 (s, 3H, CH3O-N), 3.79 (s, 3H, CH3O-C), 3.60 (d, J = 8.0 Hz, 1H), 3.52–3.44 (m, 1H), 3.44 (s, 1H), 3.35–3.19 (m, 2H), 3.09 (t, J = 10.7 Hz, 1H), 2.46–2.22 (m, 1H), 1.27–1.18 (m, 2H, cyclopropyl CH2), 1.13–0.95 (m, 2H, cyclopropyl CH2). 13C-NMR (100 MHz, CDCl3) δ 177.07, 173.52, 168.75, 166.74, 157.52, 152.99, 150.12, 146.05, 138.85(d, J = 12 Hz), 133.94, 122.74, 108.33, 107.96, 62.95, 62.11, 56.43, 50.57, 50.15, 44.79, 42.87, 40.70, 25.66, 9.86, 9.53. MS-ESI (m/z): 533.32 (M+H)+.HRMS-ESI (m/z): C24H30O7N6F, Calcd: 533.21545 (M+H)+; Found: 533.21660 (M+H)+.

7-(3-((S)-2-((S)-2-Aminopropanamido)propanamido)-4-(methoxyimino)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4l). Obtained from 3l as a yellow solid (34.7%). 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.81 (s, 1H, C2-H), 7.86 (dd, J = 11.6, 6.2 Hz, 1H, C5-H), 7.75 (t, J = 7.8 Hz, 1H), 7.28–7.10 (m, 1H), 4.66–4.62 (m, 1H), 4.56–4.47 (m, 1H), 4.13–4.00 (m, 2H), 3.92 (d, J = 4.6 Hz, 3H, CH3O-N), 3.80 (s, 3H, CH3O-C), 3.68–3.44 (m, 2H), 3.32–3.25 (m, 2H), 3.08 (t, J = 10.4 Hz, 1H), 2.34 (t, J = 12.7 Hz, 1H), 1.44–1.33 (m, 5H), 1.26–1.21 (m, 3H), 1.03 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 177.11, 175.83, 172.12, 166.76, 159.46, 153.12, 150.11, 146.14(d, J = 12 Hz), 138.80, 133.90, 122.82, 108.39, 107.98, 62.90, 62.07, 56.37, 50.78, 50.60, 48.68, 48.47, 40.69, 25.69, 21.66, 18.13, 9.80, 9.58. MS-ESI (m/z): 561.44 (M+H)+.HRMS-ESI (m/z): C26H34O7N6F, Calcd: 561.24675 (M+H)+; Found: 561.24691 (M+H)+.

3.3. MIC Determination

All compounds were screened for their in vitro antibacterial activity against representative Gram-positive 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.

4. 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 ED50 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.

Acknowledgments

This work was supported by the National S&T Major Special Project on Major New Drug Innovations (2012ZX09301002-001-017/023, 2014ZX09507009-003) and NSFC 81373267-003.

Author Contributions

Conceived and designed the experiments: Mingliang Liu, Huiyuan Guo. Performed the experiments: Tingting Zhang, Jinwei Wu and Kaixiang Liu. Analyzed the data: Tingting Zhang, Jinwei Wu, Shihong Chen, Kaixiang Liu and Yabin Lin. Wrote the paper: Tingting Zhang, Mingliang Liu, Huiyuan Guo. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 3bj, 3l and 4ai and 4k are available from the authors.
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