2009-0628

· 860 · 药学学报 Acta Pharmaceutica Sinica 2010, 45 (7): 860−868

Synthesis and in vitro antibacterial activity of 7-(4-alkoxyimino-3-methyl-3-methylaminopiperidin-1-yl)quinolones

WAN Zhi-long, CHAI Yun, LIU Ming-liang*, GUO Hui-yuan, SUN Lan-ying

(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China)

Abstract: To explore new agents of quinolone derivatives with high antibacterial activity, 7-(4-alkoxyimino- 3-methyl-3-methylaminopiperidin-1-yl)quinolones were designed and synthesized, and their activity against gram-positive and gram-negative strains was tested in vitro. Sixteen target compounds were obtained. Their structures were established by 1H NMR, HRMS and X-ray crystallographic analysis. Compounds 14k and 14m−14o show good antibacterial activity against the tested five gram-positive strains and five gram-negative strains (MIC: 0.25−16 μg·mL−1), of which the most active compound 14o is 8-fold more potent than levofloxacin against S. pneumoniae (MIC: 4 μg·mL−1), and comparable to levofloxacin against S. aureus, S. epidermidis, E. faecalis and E. coli (MIC: 0.25−1 μg·mL−1), but generally less potent than gemifloxacin.

Key words: fluoroquinolone; chemical synthesis; in vitro antibacterial activity CLC number: R916 Document code: A Article ID: 0513-4870 (2010) 07-0860-09

7-(3-甲基-3-甲胺基-4-烷氧亚胺基-1-哌啶基)喹诺酮类化合物的合成与体外抗菌作用

万志龙, 柴芸, 刘明亮*, 郭慧元, 孙兰英

(中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050)

摘要: 为寻找新的喹诺酮类抗菌药, 设计合成了 16个 7-(3-甲基-3-甲胺基-4-烷氧亚胺基-1-哌啶基) 喹诺酮类

化合物, 并测定其体外抗菌活性。目标化合物结构经 1H NMR和 HRMS得到确证, 并用单晶 X-衍射分析确定其双键构型。化合物 14k和 14m～14o对所试验的 5 株革兰阳性菌和 5株革兰阴性菌表现出良好的广谱抗菌活性 (MIC: 0.25～16 μg·mL−1), 其中活性最强的化合物 14o对金葡菌、表葡菌、粪肠球菌和大肠埃希菌的活性 (MIC: 0.25～1 μg·mL−1) 与左氧氟沙星相当, 对肺炎链球菌的活性 (MIC: 4 μg·mL−1) 是左氧氟沙星的 8倍, 但总体上弱于吉米沙星。

关键词: 氟喹诺酮; 化学合成; 体外抗菌活性

The quinolones were evolved from agents used only for the treatment of urinary tract infections (UTIs) to the latest fluoroquinolones with a remarkably broad spectrum of activity and excellent pharmacokinetics allowing once-daily dosing and thus improving patient Received 2009-10-30. Project supported by the Center Commonweal Basic Scientific Research

Operation Foundation (No. IMBF-20060404). *Corresponding author Tel / Fax: 86-10-63036965,

E-mail: [email protected]

compliance[1]. New quinolones, such as gatifloxacin, moxifloxacin and gemifloxacin (GMFX), have been prescribed extensively for the treatment of respiratory tract infections, UTIs, sexually transmitted diseases and infections of the skin and soft tissue. However, as use of fluoroquinolones has increased, the continuous increase in the resistance among bacteria is generally recognized[2]. Thus, recent efforts have been directed toward the synthesis of new quinolones that can

WAN Zhi-long, et al: Synthesis and in vitro antibacterial activity of 7-(4-alkoxyimino-3-methyl-3- methylaminopiperidin-1-yl)quinolones · 861 ·

provide improved antibacterial activity against gram- positive cocci and anaerobes and against fluoroqui-nolone-resistant strains, while retaining the good gram- negative activity of early fluoroquinolones, such as ciprofloxacin and ofloxacin.

The structure-activity relationship (SAR) study of quinolone antibacterial agents showed that the basic group at the C-7 position is the most adaptable site for chemical change and an area that greatly influences their potency, spectrum and safety[3]. In general, 5- and 6-membered nitrogen heterocycles including piperazinyl, pyrrolidinyl and piperidinyl type side chains have been proven to be the optimal substituents. However, quinolone antibacterial agents possessing piperidine substitution reported in the literature were significantly fewer than that of piperazinyl- and pyrrolidinyl-based derivatives.

As part of an ongoing program to find potent new quinolones displaying strong gram-positive activity, we have focused our attention on introducing new functional groups to the piperidine ring[4−6]. Methyl group was introduced into 3-position of pyrrolidine ring for the purpose of increasing gram-positive antibacterial activity[7]. For example, as an analogue of GMFX, DW286 (Figure 1) possessing an additional methyl group at 3-position of pyrrolidine ring displays far more potent antibacterial activity than GMFX against important gram-positive organisms, methicillin- resistant staphylococcus aureus (MRSA) and ofloxacin resistant organisms, and with excellent pharmacokinetic profiles[8]. In this study, structural modifications were made on the basis of 7-(4-alkoxyimino-3-methyl-aminopiperidin-1-yl)-1, 8-naphthyridones (DZH, Figure 1) exhibiting significant in vitro antibacterial activity against gram-positive organisms, which is more potent than GMFX, linezolid and vancomycin[9]. New piperidine derivatives and a series of fluoroquinolone compounds derived from these amines at the C-7 position were designed and synthesized. These piperidine

derivatives are structurally novel, having a methylamino and a methyl group at 3-position and an alkoxyimino group at 4-position of the piperidine ring. Our primary objective was to optimize the potency of these compounds against gram-positive and gram-negative organisms.

Results and discussion 1 Chemistry

The detailed synthetic pathways to 4-alkoxyimino- 3-methyl-3-methylaminopiperidine dihydrochlorides 11a, 11b starting from 1-tert-butoxycarbonyl-4-piperidone (1) and novel fluoroquinolones 14a−14p were depicted in Schemes 1 and 2, respectively.

Sixteen target compounds 14a−14p were synthe-sized in our study, and their structures were established by 1H NMR and HRMS spectra. Their physical constants and spectral data were depicted in Table 1.

It is obvious that the target compounds 14a−14p and intermediates 11a, 11b are all racemes. However, it was necessary to determine the geometry of the oximino group because it could exist in the E or Z configuration. Although we were not successful in preparing X-ray quality single crystals of compounds 14a−14p, we were able to obtain X-ray data for the intermediate 10a. As expected, the six-membered piperidine ring adopts a chair conformation and the methyloxime geometry of 10a was confirmed to have the E-configuration [10]. 2 Pharmacology

The synthesized fluoroquinolones 14a−14p were evaluated for their in vitro antibacterial activity against representative gram-positive and gram-negative strains using standard techniques. The minimum inhibitory concentration (MIC) values of the compounds against the ten strains are presented in Table 2. The antibac-terial activity of the reference compounds GMFX and levofloxacin (LVFX) are also included.

Figure 1 Chemical structures of GMFX, DW286 and DZH


(a) (CH3O)2CO, 70% NaH, C6H5CH3, 80 ℃; (b) CH3I, K2CO3, (CH3)2CO, 40 ℃; (c) RONH2·HCl, Et3N, EtOH; 55−60 ℃; (d) NaOH, MeOH, 50 ℃, and then HOAc; (e) ClCOOBu-i, Et3N, −14 to −12 ℃; (f) NH3 gas, 0 − 5 ℃; (g) NaBrO, CH3CN, 5 ℃; (h) Boc2O, EtOH, rt; (i) CH3I, 70% NaH, THF, rt; (j) HCl gas, CH2Cl2, rt.

Scheme 1 Synthesis of 4-alkoxyimino-3-methyl-3-methylaminopiperidine dihydrochlorides 11a, 11b

Compd. A R1 R Compd. A R1 R

14a N CH3 14b N C2H5

14c N 2,4-F2-C6H3 CH3 14d N 2,4-F2-C6H3 C2H5

14e COCHF2 CH3 14f COCHF2 C2H5

14g CF C2H4F CH3 14h CF C2H4F C2H5

14i CF C2H5 CH3 14j CF C2H5 C2H5

14k CF CH3 14l CF C2H5

14m COCH3 CH3 14n COCH3 C2H5

14o CH3 14p C2H5

Scheme 2 Synthesis of target compounds 14a−14p


Table 1 Physical constants and spectral data of compounds 14a−14p

Compd. Yield

/% mp/˚C 1H NMR / δ (400 MHz, CDCl3)

HR-FAB-MS Calcd (Found)

14a 76.7 216−217 1.07−1.11 (m, 2H, cyclopropyl), 1.24−1.30 (m, 5H, cyclopropyl, CH3), 2.25 (s, 3H, NCH3), 2.52−2.59 (m, 1H, piperidine), 3.05−3.12 (m, 1H, piperidine), 3.43−3.53 (m, 2H, piperidine), 3.61−3.67 (m, 1H, cyclopropyl), 3.90 (s, 3H, OCH3), 4.28−4.31 (m, 1H, piperidine), 4.40−4.45 (m, 1H, piperidine), 8.07−8.10 (d, J = 13.2 Hz, 1H, H-5 quinolone), 8.73 (s, 1H, H-2 quinolone)

[C20H25FN5O4 +H]+

418.189 1 (418.187 0)

14b 66.3 199−200 1.08−1.09 (m, 2H, cyclopropyl), 1.26−1.34 (m, 8H, cyclopropyl, OCH2CH3, CH3), 2.28 (s, 3H, NCH3), 2.60−2.61 (m, 1H, piperidine), 3.07−3.11 (m, 1H, piperidine), 3.51−3.58 (m, 2H, piperidine), 3.66−3.68 (m, 1H, cyclopropyl), 4.13−4.18 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.31−4.38 (m, 2H, piperidine), 8.07−8.10 (d, J = 13.2 Hz, 1H, H-5 quinolone), 8.73 (s, 1H, H-2 quinolone)

[C21H27FN5O4+H]+

432.204 7 (432.205 4)

14c 74.8 203−205 1.16 (s, 3H, CH3), 2.14−2.16 (d, J = 10.4 Hz, 3H, NCH3), 2.33−2.39 (m, 1H, piperidine), 2.80−2.90 (m, 1H, piperidine), 3.18−3.35 (m, 2H, piperidine), 3.86 (s, 3H, OCH3), 3.95−4.03 (m, 2H, piperidine), 7.07−7.09 (m, 2H, ArH), 7.39−7.44 (1H, m, ArH), 8.10−8.14 (d, J = 13.2 Hz, 1H, H-5 quinolone), 8.67 (s, 1H, H-2 quinolone)

[C23H23F3N5O4+H]+ 490.170 2

(490.169 6)

14d 72.5 210−212 1.20 (s, 3H, CH3), 1.23−1.26 (t, J = 7.2 Hz, 3H, OCH2CH3), 2.14−2.17 (d, J = 10.0 Hz, 3H, NCH3), 2.36−2.37 (m, 1H, piperidine), 2.82−2.89 (m, 1H, piperidine), 3.19−3.36 (m, 2H, piperidine), 3.96− 4.01 (m, 2H, piperidine), 4.08−4.13 (q, J = 7.2 Hz, 2H, OCH2CH3), 7.10−7.12 (m, 2H, ArH), 7.40− 7.44 (1H, m, ArH), 8.10−8.14 (d, J = 13.2 Hz, 1H, H-5 quinolone), 8.67 (s, 1H, H-2 quinolone)

[C24H25F3N5O4+H]+ 504.185 9

(504.187 7)

14e 57.6 206−207 0.87−0.92 (m, 1H, cyclopropyl), 1.04−1.11 (m, 1H, cyclopropyl), 1.19−1.39 (m, 5H, cyclopropyl and CH3), 2.28 (s, 3H, NCH3), 2.40−2.48 (m, 1H, piperidine), 3.17−3.24 (m, 2H, piperidine), 3.36−3.42 (m, 2H, piperidine), 3.53−3.56 (m, 1H, piperidine), 3.91 (s, 3H, OCH3), 4.07−4.12 (m, 1H, cyclopropyl), 6.57−6.94 (t, J = 74.8 Hz, 1H, OCHF2), 8.03−8.06 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.85 (s, 1H, H-2 quinolone)

[C22H26F3N4O5+H]+ 483.185 5

(483.182 5)

14f 54.8 186−189 0.83−1.08 (m, 3H, cyclopropyl), 1.25−1.57 (m, 7H, cyclopropyl, CH3 and OCH2CH3), 2.31 (s, 3H, NCH3), 2.40−2.48 (m, 1H, piperidine), 3.21−3.24 (m, 2H, piperidine), 3.43−3.52 (m, 3H, piperidine), 4.07−4.12(m, 1H, cyclopropyl), 4.08−4.13 (q, J = 6.8 Hz, 2H, OCH2CH3), 6.71−7.18 (t, J = 74.8 Hz, 1H, OCHF2), 8.04−8.07 (d, J = 12.0 Hz, 1H, H-5 quinolone), 8.85 (s, 1H, H-2 quinolone)

[C23H28F3N4O5+H]+ 497.201 2

(497.201 7)

14g 65.6 201−204 1.27 (s, 3H, CH3), 2.27 (s, 3H, NCH3), 2.31-2.34 (m, 1H, piperidine), 3.21−3.43 (m, 5H, piperidine), 3.91 (s, 3H, OCH3), 4.76−4.88 (m, 4H, CH2CH2F), 7.99−8.02 (d, J = 11.2 Hz, 1H, H-5 quinolone), 8.63 (s, 1H, H-2 quinolone)

[C20H24F3N4O4+H]+

441.175 0 (441.175 6)

14h 63.2 177−179 1.26−1.29 (t, J = 6.8 Hz, 3H, OCH2CH3), 1.39 (s, 3H, CH3), 2.30 (s, 3H, NCH3), 2.72−3.00 (m, 1H, piperidine), 3.24−3.42 (m, 5H, piperidine), 4.10−4.17 (q, J = 6.8 Hz, 2H, OCH2CH3), 4.71−4.88 (m, 4H, CH2CH2F), 7.99−8.02 (d, J = 10.8 Hz, 1H, H-5 quinolone), 8.62 (s, 1H, H-2 quinolone)

[C21H26F3N4O4+H]+ 455.190 6

(455.190 2) 14i 69.8 204−206 1.28 (s, 3H, CH3), 1.54−1.58 (t, J = 7.0 Hz, 3H, NCH2CH3), 2.32 (s, 3H, NCH3), 2.35−2.37 (m, 1H,

piperidine), 3.20−3.42 (m, 5H, piperidine), 3.91 (s, 3H, OCH3), 4.45−4.52 (m, 2H, NCH2), 7.98−8.01 (d, J = 11.2 Hz, 1H, H-5 quinolone), 8.64 (s, 1H, H-2 quinolone)

[C20H25F2N4O4+H]+ 423.184 4

(423.183 6) 14j 62.7 185−187 1.26−1.30 (m, 6H, CH3 and OCH2CH3), 1.54−1.58 (t, 3H, NCH2CH3), 2.29 (s, 3H, NCH3), 2.34−2.38

(m, 1H, piperidine), 3.23−3.45 (m, 5H, piperidine), 4.12−4.18 (q, J = 6.8 Hz, 2H, OCH2CH3), 4.45− 4.53 (m, 2H, NCH2), 7.99−8.02 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.63 (s, 1H, H-2 quinolone)

[C21H27F2N4O4+H]+ 437.200 0

(437.200 5) 14k 72.1 207−208 1.12−1.22 (m, 2H, cyclopropyl), 1.27 (s, 3H, CH3), 1.29−1.32 (m, 2H, cyclopropyl), 2.30 (s, 3H,

NCH3), 2.32−2.40 (m, 1H, piperidine), 3.20−3.42 (m, 5H, piperidine), 3.90 (s, 3H, OCH3), 3.99−4.01 (m, 1H, cyclopropyl), 7.93−7.96 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.80 (s, 1H, H-2 quinolone)

[C21H25F2N4O4+H]+ 435.184 4

(435.185 6) 14l 75.7 194−195 1.16−1.22 (m, 2H, cyclopropyl), 1.26−1.35 (m, 8H, cyclopropyl, CH3 and OCH2CH3), 2.32 (s, 3H,

NCH3), 2.35−2.43 (m, 1H, piperidine), 3.20−3.43 (m, 5H, piperidine), 3.99−4.01 (m, 1H, cyclopropyl), 4.13−4.18 (q, J = 7.2 Hz, 2H, OCH2CH3), 7.93−7.96 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.80 (s, 1H, H-2 quinolone)

[C22H27F2N4O4+H]+ 449.200 0

(449.201 5)

14m 52.9 181−183 1.02−1.06 (m, 2H, cyclopropyl), 1.22−1.26 (m, 2H, cyclopropyl), 1.49 (s, 3H, CH3), 2.51 (s, 3H, NCH3), 2.70−2.72 (m, 1H, piperidine), 3.02−3.06 (m, 1H, piperidine), 3.35−3.37 (m, 1H, piperidine), 3.48−3.55 (m, 2H, piperidine), 3.67−3.69 (m, 1H, piperidine), 3.71 (s, 3H, OCH3-8 quinolone), 3.94 (s, 3H, OCH3), 3.96−4.02 (m, 1H, cyclopropyl), 7.86−7.89 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.83 (s, 1H, H-2 quinolone)

[C22H28FN4O5+H]+ 447.204 4

(447.205 3)

14n 63.3 159−161 1.01−1.03 (m, 2H, cyclopropyl), 1.22−1.31 (m, 5H, cyclopropyl and OCH2CH3), 1.49 (s, 3H, CH3), 2.50 (s, 3H, NCH3), 2.72−2.74 (m, 1H, piperidine), 3.03−3.06 (m, 1H, piperidine), 3.35−3.37 (m, 1H, piperidine), 3.50−3.56 (m, 2H, piperidine), 3.70−3.72 (m, 4H, piperidine and OCH3), 3.96−4.00 (m, 1H, cyclopropyl), 4.02−4.13 (q, J = 6.8 Hz, 2H, OCH2CH3), 7.86−7.89 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.83 (s, 1H, H-2 quinolone)

[C23H30FN4O5+H]+ 461.220 0

(461.224 1)

14o 48.2 159−162 1.45−1.49 (m, 6H, 2×CH3), 2.49 (s, 3H, NCH3), 2.82−2.88 (m, 2H, piperidine), 3.35−3.56 (m, 4H, piperidine), 3.90 (s, 3H, OCH3), 4.36−4.44 (m, 1H, OCH2), 4.62−4.71 (m, 1H, OCH2), 4.93−4.95 (m, 1H, NCH), 7.64−7.66 (d, J = 11.6 Hz, 1H, H-5 quinolone), 8.94 (s, 1H, H-2 quinolone)

[C21H26FN4O5+H]+ 433.188 7

(433.188 8) 14p 76.7 216−217 1.14−1.29 (m, 9H, 3×CH3), 2.12−2.22 (m, 2H, piperidine), 2.26 (s, 3H, NCH3), 3.35−3.56 (m, 4H,

piperidine), 4.11−4.16 (q, J = 6.8 Hz, 2H, OCH2CH3), 4.35−4.49 (m, 3H, OCH2CH), 7.73−7.76 (d, J = 11.2 Hz, 1H, H-5 quinolone), 8.62 (s, 1H, H-2 quinolone)

[C22H28FN4O5+H]+ 447.204 4

(447.202 8)


Table 2 In vitro antibacterial activity of compounds 14a−14p against selected strains. S. a.: Staphylococcus aureus ATCC29213; S. e.: Staphylococcus epidermidis ATCC12228; S. p.: Streptococcus pneumoniae 97100; S. py.: Streptococcus pyogenes 9619; E. f.: Enterococcus faecalis ATCC29212; E. c.: Escherichia coli ATCC26; K. p.: Klebsiella pneumoniae 7; P. a.: Pseudomonas aeruginosa 17; S. s.: Shigella sonnei 51592; E. c.: Enterobacter cloacae 45301

MIC / μg·mL−1 Compd.

S. a. S. e. S. p. S. py. E. f. E. c. K. p. P. a. S. s. E.c.

14a 8 8 32 128 64 16 8 64 16 16

14b 8 8 128 >128 16 32 32 >128 128 32

14c 2 4 32 >128 32 32 32 >128 32 32

14d 8 8 >128 >128 128 >128 >128 >128 >128 >128

14e 0.5 2 8 64 2 4 4 32 8 8

14f 1 2 64 64 8 32 16 128 32 32

14g 2 2 32 64 8 8 16 128 16 16

14h 2 64 >128 >128 >128 >128 >128 128 64 16

14i 2 2 32 64 8 8 8 128 16 16

14j 2 64 >128 >128 >128 >128 >128 >128 128 32

14k 0.25 1 8 8 2 1 1 16 4 4

14l 1 32 >128 >128 4 >128 >128 >128 16 8

14m 0.25 0.5 8 8 2 2 2 16 4 4

14n 0.25 0.5 8 16 2 8 2 16 8 8

14o 0.25 0.5 4 16 1 1 2 8 2 4

14p 0.25 2 128 128 64 2 64 64 16 4

GMFX <0.03 <0.03 2 2 0.06 0.06 0.5 0.125 0.06 0.06

LVFX 0.25 0.5 32 4 1 1 1 0.5 1 0.125

The novel fluoroquinolones 14a−14p have generally

potent antibacterial activity against the ten strains. Compounds 14k and 14m−14o showed good antibacterial activity against the tested five gram-positive strains and five gram-negative strains (MIC: 0.25−16 μg·mL−1), of which the most active compound 14o was found to be 8-fold more potent than LVFX against S. pneumoniae (MIC: 4 μg·mL−1), and comparable to LVFX against S. aureus, S. epidermidis, E. faecalis and E. coli (MIC: 0.25−1 μg·mL−1), but generally less potent than GMFX. 3 Conclusion and discussion

In summary, a series of novel 7-(4-alkoxyimino- 3-methyl-3-methylaminopiperidin-1-yl) fluoroquinolone derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity against representative gram-positive and gram-negative strains. All of the novel fluoroquinolones demonstrate potent antibacterial activity, of which compounds 14k and 14m−14o show good antibacterial activity (MIC: 0.25−16 μg·mL−1), and the most active compound 14o is 8-fold more potent than LVFX against S. pneumoniae, and comparable to LVFX against S. aureus, S. epidermidis, E. faecalis and E. coli, but they are generally less potent than GMFX against the tested gram-positive and gram-

negative strains. These results show that introduction of another

methyl group into 3-position of piperidine ring causes reduced antibacterial activity, which is contrary to the activity profiles of pyrrolidine-containing fluoroquinolones. In addition, the fluoroquinolones featuring methyloxime-incorporated piperidino-substitution at C-7 position are more active than those of the corresponding analogues containing ethyloxime.

Experimental 1 General experimental procedures

Melting points were determined on X6 precision melting-point apparatus and uncorrected. 1H NMR spectra were determined on a Varian Mercury-400 MHz spectrometer using tetramethylsilane as an internal standard. Electrospray ionization (ESI) mass spectra were obtained on a MDSSCIEX Q-Tap mass spectrometer. Fast Atom Bombardment (FAB) mass spectra and HR-MS were obtained on an AutoSpec Ultima-TOF mass spectrometer. The reference drugs GMFX and LVFX were synthesized in our lab according to the references[11−13].


2 Chemistry 2.1 Methyl 1-tert-butoxycarbonyl-4-oxopiperidine- 3-carboxylate (2) To a suspension of 70% sodium hydride (30.20 g, 0.88 mol) in dry toluene (500 mL), dimethyl carbonate (43.20 g, 0.48 mol) was added dropwise in 0.5 h at room temperature under an atmosphere of nitrogen. After addition of a few drops of methanol, a solution of 1-tert-butoxycarbonyl-4- piperidone (1, 48.00 g, 0.24 mol) dissolved in dry toluene (200 mL) was added dropwise to the reaction mixture while stirring at 80 ℃ in 1 h. The reaction mixture was stirred for 3 h at the same temperature and then cooled to 0 ℃ (ice bath), adjusted to pH 6−6.5 with acetic acid. The resulting mixture was diluted with cold distilled water (100 mL) and adjusted to pH 8 with 5% sodium hydroxide solution. The toluene layer was separated and the aqueous layer was extracted with toluene (100 mL). The combined toluenes were dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solid obtained was dried in vacuo to give the title compound 2 (54.50 g, 88.0%) as an off-white solid, mp 32−34 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.47 (s, 9H, Boc), 2.35−2.38 (t, J = 6.0 Hz, 2H, H-5), 3.54−3.57 (t, J = 6.0 Hz, 2H, H-6), 3.76 (s, 3H, OCH3), 4.05 (s, 2H, H-2). ESI-MS m/z: 258 [M+H]+. 2.2 Methyl 1-tert-butoxycarbonyl-3-methyl-4-oxopiperidine-3-carboxylate (3) To a suspension of compound 2 (53.68 g, 0.21 mol) and anhydrous potassium carbonate (100.93 g, 0.73 mol) in dry acetone (500 mL), a solution of methyl iodide (23.38 mL, 0.38 mol) dissolved in dry acetone (250 mL) was added at room temperature under an atmosphere of nitrogen. The reaction mixture was heated to 40 ℃ and stirred for 6 h, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane (300 mL) and washed with distilled water and saturated saline separately, dried over anhydrous sodium sulfate, filtered and concentrated. The yellow oily residue was treated with petroleum ether (300 mL) and filtered. The solid obtained was washed twice with petroleum ether and dried in vacuo to give the title compound 3 (45.36 g, 80.1%) as a white solid, mp 42−43 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.31 (s, 3H, CH3), 1.49 (s, 9H, Boc), 2.45−2.51 (m, 1H, H-5), 2.76 (s, 1H, H-2), 3.06−3.10 (m, 1H, H-6), 3.30−3.37 (m, 1H, H-5), 3.72 (s, 3H,

OCH3), 4.12 (s, 1H, H-2), 4.48−4.51 (m, 1H, H-6). ESI-MS m/z: 272 [M+H]+. 2.3 Methyl 1-tert-butoxycarbonyl-4-methoxyimino- 3-methylpiperidine-3-carboxylate (4a) To a stirring solution of compound 3 (15.00 g, 55.3 mmol) dissolved in ethanol (50 mL), a solution of methoxylamine hydrochloride (5.05 g, 60.5 mmol) and triethylamine (8.37 mL, 60.5 mmol) in 80% ethanol (25 mL) was added dropwise at 55−60 ℃ in 15 min. The reaction mixture was stirred at the same temperature for 1.5 h and concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3×50 mL). The combined extracts were washed with 1 mol·L−1 HCl (2×10 mL) and saturated brine separately, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was recrystallized from petroleum ether to give the title compound 4a (14.71 g, 88.6%) as a white solid, mp 75−76 ℃ . 1H NMR (400 MHz, CDCl3) δ: 1.36 (s, 3H, CH3), 1.46 (s, 9H, Boc), 2.39− 2.40 (m, 1H, piperidine), 2.92−3.16 (m, 3H, piperidine), 3.70 (s, 3H, COOCH3), 3.86 (s, 3H, NOCH3), 3.82− 3.83 (m, 1H, piperidine), 4.32−4.36 (m, 1H, piperidine). ESI-MS m/z: 301 [M+H]+. 2.4 Methyl 1-tert-butoxycarbonyl-4-ethoxyimino-3- methylpiperidine-3-carboxylate (4b) The title compound was obtained in a similar manner as for the preparation of compound 4a. White solid (90.3%), mp 51−53 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.22− 1.26 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.36 (s, 3H, CH3), 1.46 (s, 9H, Boc), 2.42−2.44 (m, 1H, piperidine), 2.92− 3.19 (m, 3H, piperidine), 3.70 (s, 3H, OCH3), 3.79− 3.80 (m, 1H, piperidine), 4.07−4.12 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.30−4.33 (m, 1H, piperidine). ESI-MS m/z: 315 [M+H]+. 2.5 1-tert-Butoxycarbonyl-3-carbamyl-4-methoxyimino-3-methylpiperidine (7a) To a stirring solution of compound 4a (17.00 g, 56.6 mmol) in methanol (100 mL), a solution of 18% sodium hydroxide solution (25 mL) was added dropwise at room temperature. The reaction mixture was heated to 50 ℃ and stirred for 2 h at the same temperature. After removal of the methanol under reduced pressure, the reaction mixture was diluted with distilled water (30 mL), adjusted to pH 6−6.5 with acetic acid and filtered to give the acid 5a as a white solid. The solid 5a was dissolved in dichloromethane (150 mL), and to this solution


triethylamine (8.8 mL, 63.6 mmol) was added. The reaction mixture was cooled to −14 to −12 ℃ using an ice-salt bath, isobutyl chloroformate (9.0 mL, 69.2 mmol) was added and stirred for 0.5 h at the same temperature to give the ester 6a. To the reaction mixture containing the ester 6a, ammonia gas was pumped cautiously at 0−5 ℃ for 0.5 h, washed with distilled water and saturated brine, separately, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting yellow residue was recrystallized from ethyl acetate to give the title compound 7a (13.50 g, 83.6%) as a white solid, mp 126−127 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.37 (s, 3H, CH3), 1.47 (s, 9H, Boc), 2.14−2.31 (m, 1H, piperidine), 2.95−3.10 (m, 3H, piperidine), 3.72−3.80 (m, 1H, piperidine), 3.90 (s, 3H, OCH3), 4.35−4.37 (m, 1H, piperidine), 5.37 (br, 1H, CONH), 6.19, 6.78 (2s, 1H, CONH). ESI-MS m/z: 286 [M+H]+. 2.6 1-tert-Butoxycarbonyl-3-carbamyl-4-ethoxyimino- 3-methylpiperidine (7b) The title compound was obtained in a similar manner as for the preparation of compound 7a. White solid (78.6%), mp 108−109 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.24−1.27 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.38 (s, 3H, CH3), 1.52 (s, 9H, Boc), 2.20−2.35 (m, 1H, piperidine), 2.99−3.15 (m, 3H, piperidine), 3.75−3.78 (m, 1H, piperidine), 4.11− 4.16 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.33−4.35 (m, H, piperidine), 5.46 (br, 1H, CONH), 6.19, 6.66 (2s, 1H, CONH). ESI-MS m/z: 300 [M+H]+. 2.7 1-tert-Butoxycarbonyl-3-amino-4-methoxyimino- 3-methylpiperidine (8a) To a solution of compound 7a (2.00 g, 7.0 mmol) in acetonitrile (80 mL), freshly prepared sodium hypobromite solution[14] (14 mL) was added dropwise at 0−5 ℃ in 0.5 h. The reaction mixture was stirred at the room temperature for 2 h, the organic layer was separated and concentrated under reduced pressure. The residue was dissolved in distilled water (30 mL), adjusted to pH 2.5 with 2 mol·L−1 HCl, washed with ethyl acetate (3×30 mL). The water layer was adjusted to pH 12 with 15% sodium hydroxide solution, and then extracted with ethyl acetate (3×50 mL). The combined organic extracts were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to give the title compound 8a (0.91 g, 50.6%) as a colorless liquid. 1H NMR (400 MHz, CDCl3)

δ: 1.27 (s, 3H, CH3), 1.43 (s, 9H, Boc), 1.75 (br, 2H, NH2), 2.30−2.33 (m, 1H, piperidine), 2.90−3.00 (m, 3H, piperidine), 3.67−3.70 (m, 1H, piperidine), 3.82 (s, 3H, OCH3), 3.85−3.88 (m, 1H, piperidine). ESI-MS m/z: 258 [M+H]+. 2.8 1-tert-Butoxycarbonyl-3-amino-4-ethoxyimino- 3-methylpiperidine (8b) The title compound was obtained in a similar manner as for the preparation of compound 8a. Colorless liquid (66.0%). 1H NMR (400 MHz, CDCl3) δ: 1.22−1.26 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.28 (s, 3H, CH3), 1.46 (s, 9H, Boc), 1.79 (br, 2H, NH2), 2.28−2.36 (m, 1H, piperidine), 2.97− 3.07 (m, 3H, piperidine), 3.69−3.72 (m, 1H, piperidine), 3.90−3.93 (m, 1H, piperidine), 4.05−4.10 (q, J = 7.2 Hz, 2H, OCH2CH3). ESI-MS m/z: 272 [M+H]+. 2.9 1-tert-Butoxycarbonyl-3-(N-tert-butoxycarbonyl) amino-4-methoxyimino-3-methylpiperidine (9a) To a stirring solution of compound 8a (1.73 g, 6.7 mmol) in ethanol (30 mL), di-tert-butyl dicarbonate (1.62 g, 7.4 mmol) was added portionwise. The reaction mixture was stirred for 2 h at the room temperature and then concentrated under reduced pressure to give the title compound 9a (2.40 g, 100%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 1.43 (s, 9H, Boc), 1.48 (s, 9H, Boc), 1.52 (s, 3H, CH3), 2.35−2.38 (m, 1H, piperidine), 2.89−3.18 (m, 3H, piperidine), 3.66−3.68 (m, 1H, piperidine), 3.86 (s, 3H, OCH3), 4.29−4.30 (m, 1H, piperidine), 5.67 (s, 1H, NH). ESI-MS m/z: 358 [M+H]+. 2.10 1-tert-Butoxycarbonyl-3-(N-tert-butoxycarbonyl) amino-4-ethoxyimino-3- methylpiperidine (9b) The title compound was obtained in a similar manner as for the preparation of compound 9a. Colorless oil (98.5%). 1H NMR (400 MHz, CDCl3) δ: 1.23−1.27 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.44 (s, 9H, Boc), 1.49 (s, 9H, Boc), 1.52 (s, 3H, CH3), 2.31−2.33 (m, 1H, piperidine), 2.98−3.17 (m, 3H, piperidine), 3.95−3.96 (m, 1H, piperidine), 4.08−4.13 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.35−4.38 (m, 1H, piperidine), 5.73 (s, 1H, NH). ESI-MS m/z: 372 [M+H]+. 2.11 1-tert-Butoxycarbonyl-3-(N-tert-butoxycarbonyl) methylamino-3-methyl-4-methoxyiminopiperidine (10a) To a stirring solution of compound 9a (2.4 g, 6.7 mmol) in dry tetrahydrofuran (40 mL), 70% sodium hydride (0.46 g, 13.4 mmol) was added at 0 ℃ in 0.5 h using an ice bath, and then stirred for 0.5 h at the room temperature. After addition of methyl iodide (0.84


mL, 13.4 mol), the reaction mixture was stirred at 40 ℃ for 5 h and cooled to room temperature, adjusted to pH 7 with 1 mol·L−1 HCl and then concentrated under reduced pressure. The residue was diluted with ethyl acetate (50 mL), washed with distilled water (25 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, dried in vacuo to give the title compound 10a (2.37 g, 95.0%) as a white solid, mp 107−109 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.34 (s, 3H, CH3), 1.41 (s, 9H, Boc), 1.46 (s, 9H, Boc), 2.24−2.25 (m, 1H, piperidine), 2.87−2.88 (m, 1H, piperidine), 2.91 (s, 3H, NCH3), 2.95−3.08 (m, 2H, piperidine), 3.82 (s, 3H, OCH3), 3.84−3.86 (m, 1H, piperidine), 4.30−4.31 (m, 1H, piperidine). ESI-MS m/z: 372 [M+H]+. 2.12 1-tert-Butoxycarbonyl-3-(N-tert-butoxycarbonyl) methylamino-4-ethoxyimino-3-methylpiperidine (10b) The title compound was obtained in a similar manner as for the preparation of compound 10a. White solid (93.6%), mp 56−58 ℃. 1H NMR (400 MHz, CDCl3) δ: 1.20−1.24 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.34 (s, 3H, CH3), 1.41 (s, 9H, Boc), 1.46 (s, 9H, Boc), 2.28−2.30 (m, 1H, piperidine), 2.91 (s, 3H, NCH3), 2.95-3.03 (m, 3H, piperidine), 3.78−3.82 (m, 1H, piperidine), 4.03− 4.07 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.30−4.32 (m, 1H, piperidine). ESI-MS m/z: 386 [M+H]+. 2.13 4-Methoxyimino-3-methyl-3-methylaminopiperidine dihydrochloride (11a) To a stirring solution of compound 10a (2.30 g, 6.2 mmol) in dichloro-methane (50 mL), dried hydrochloride gas was pumped at 0−5 ℃ using an ice bath for 0.5 h. The reaction mixture was allowed to stir for another 0.5 h at room temperature, the resulting solid was collected by suction, and dried in vacuo to afford the title compound 11a (1.04 g, 97.8%) as a white solid, mp 224−226 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 1.59 (s, 3H, CH3), 2.51 (s, 3H, NCH3), 2.76−2.82 (m, 1H, piperidine), 2.95− 3.02 (m, 1H, piperidine), 3.11−3.22 (m, 2H, piperidine), 3.49−3.60 (m, 2H, piperidine), 3.90 (s, 3H, OCH3), 9.82 (br, 4H, 2NH2

+). ESI-MS m/z: 172 [M+H]+. 2.14 4-Ethoxyimino-3-methyl-3-methylaminopiperidine dihydrochloride (11b) The title compound was obtained in a similar manner as for the preparation of compound 11a. White solid (98.8%), mp 202−203 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 1.23−1.25 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.76 (s, 3H, CH3), 2.58 (s, 3H, NCH3), 2.95−2.97 (m, 1H, piperidine), 3.19−3.20 (m, 1H,

piperidine), 3.46−3.50 (m, 2H, piperidine), 3.86−3.88 (m, 1H, piperidine), 4.19−4.24 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.56−4.60 (m, 1H, piperidine), 9.97 (br, 2H, NH2

+), 10.53, 10.60 (2br, 2H, NH2+). ESI-MS m/z:

186 [M+H]+ . 2.15 1-Cyclopropyl-6-fluoro-7-(4-methoxyimino-3- methyl-3-methylaminopiperidin-1-yl)-1, 4-dihydro-4- oxo-1, 8-naphthyridine-3-carboxylic acid (14a) A mixture of compound 11a (0.59 g, 2.4 mmol), triethyl-amine (0.5 mL) and dry acetonitrile (10 mL) was stirred at room temperature for 10 min, 7-chloro-1-cyclopropyl- 6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine-3-carboxylic acid 12a (0.56 g, 2.0 mmol) was added to the solution and stirred for 1.5 h at the same temperature under an atmosphere of nitrogen. The resulting solid was collected by suction, washed with distilled water and ethanol, respectively, dried in vacuo to afford the title compounds.

The compounds 14b−14d, by coupling reaction of compound 11b with 12a, or compounds 11a, 11b with 12b, were synthesized in a similar manner. 2.16 1-Cyclopropyl-6-fluoro-7-(4-methoxyimino-3- methyl-3-methylaminopiperidin-1-yl)-8-difluoromethoxyl-1, 4-dihydro-4-oxo-quinoline-3-carboxylic acid (14e) A mixture of boric acid (0.25 g, 4.0 mmol) and acetic anhydride (3 mL, 19.1 mmol) was stirred at 110 ℃ for 1.5 h. Then cooled and acetic acid (6 mL) was added to the solution and stirred at 110 ℃ for another 1 h. Then cooled to 50−60 ℃, ethyl 1-cyclopropyl- 6, 7-difluoro-8-difluoromethoxyl-1, 4-dihydro-4-oxo- quinoline-3-carboxylate 13a (0.58 g, 1.6 mmol) was added and stirred at 110 ℃ for 1.5 h. Then cooled to room temperature and poured into cold water (50 mL). The resulting solid was collected by suction, and dried. The solid obtained (0.69 g, 1.5 mmol) was added to a mixture of compound 11a (0.73 g, 3 mmol), triethylamine (1.2 mL) and dry acetonitrile (15 mL), which was stirred at 60 ℃ for 6 h. After completion of the condensation, the reaction mixture was concentrated under reduced pressure. To the residue 6% sodium hydroxide solution (15 mL) was added, stirred at 40 ℃ for 1 h, and then adjusted to pH 7 with 2 mol·L−1 HCl, the solid product was collected by suction. The crude product was recrystallized from dichloromethane and ethanol to give the title compound as a white solid.

The compounds 14f−14p, by coupling reaction of compound 11b with 13a, or compounds 11a, 11b with


13b−13f, were synthesized in a similar manner. 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[15]. 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 ℃ for 18–24 h.

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