Hybrid antibacterials. DNA polymerase-topoisomerase inhibitors

Article abstract of DOI:10.1021/jm0510023

Novel Gram-positive (Gram+) antibacterial compounds consisting of a DNA polymerase IIIC (pol IIIC) inhibitor covalently connected to a topoisomerase/gyrase inhibitor are described. Specifically, 3-substituted 6-(3-ethyl-4-methylanilino)uracils (EMAUs) in which the 3-substituent is a fluoroquinolone moiety (FQ) connected by various linkers were synthesized. The resulting "AU-FQ" hybrid compounds were significantly more potent than the parent EMAU compounds as inhibitors of pol IIIC and were up to 64-fold more potent as antibacterials in vitro against Gram+ bacteria. The hybrids inhibited the FQ targets, topoisomerase IV and gyrase, with potencies similar to norfloxacin but 10-fold lower than newer agents, for example, ciprofloxacin and sparfloxacin. Representative hybrids protected mice from lethal Staphylococcus aureus infection after intravenous dosing, and one compound showed protective effect against several antibiotic-sensitive and -resistant Gram+ infections in mice. The AU-FQ hybrids are a promising new family of antibacterials for treatment of antibiotic-resistant Gram+ infections.

Full text of DOI:10.1021/jm0510023

NIH Public Access
Author Manuscript
J Med Chem. Author manuscript; available in PMC 2008 October 13.
Published in final edited form as:
J Med Chem. 2006 February 23; 49(4): 1455–1465. doi:10.1021/jm0510023.
Hybrid Antibacterials. DNA polymerase:topoisomerase inhibitors
1
1
1
1
1
1
Chengxin Zhi , Zheng-yu Long , Andrzej Manikowski , Jeanne Comstock , Wei-Chu Xu ,
1
1
1
Neal C. Brown , Paul M. Tarantino Jr. , Karsten A. Holm , Edward J. Dix , George E.
1,*
2
2
2
Wright , Marjorie H. Barnes , Michelle M. Butler , Kimberly A. Foster , William A.
LaMarr , Benoit Bachand , Richard Bethell , Caroline Cadilhac , Sylvie Charron , Serge
Lamothe , Irina Motorina , and Richard Storer
2
3
3,§
3
3
3
3
3,¶
1 GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605
2 Microbiotix Inc., One Innovation Drive, Worcester, MA 01605
3 Shire BioChem Inc., Laval, Quebec, Canada
Abstract
Novel Gram-positive (Gram+) antibacterial compounds consisting of a DNA polymerase IIIC (pol
IIIC) inhibitor covalently connected to a topoisomerase/gyrase inhibitor are described. Specifically,
3-substituted-6-(3-ethyl-4-methylanilino)uracils (EMAUs) in which the 3-substituent is a
fluoroquinolone moiety (FQ) connected by various linkers were synthesized. The resulting “AU-
FQ” hybrid compounds were significantly more potent than the parent EMAU compounds as
inhibitors of pol IIIC, and were up to 64-fold more potent as antibacterials in vitro against Gram+
bacteria. The hybrids inhibited the FQ targets, topoisomerase IV and gyrase, with potencies similar
to norfloxacin, but tenfold lower than newer agents, e.g. ciprofloxacin and sparfloxacin.
Representative hybrids protected mice from lethal S. aureus infection after intravenous dosing, and
one compound showed protective effect against several antibiotic sensitive and resistant Gram+
infections in mice. The AU-FQ hybrids are a promising new family of antibacterials for treatment
of antibiotic-resistant Gram+ infections.
Introduction
The emergence of antibiotic-resistant Gram+ bacterial infections, notably with Staphylococcus
aureus, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae, has
prompted development of new chemotherapeutic agents that selectively attack new bacterial
targets. One new target which has been validated recently in Gram+ organisms is DNA
polymerase IIIC (pol IIIC), a DNA-dependent DNA polymerase which is specifically required
for replicative DNA synthesis in these organisms. Interference with pol IIIC function prevents
1,2
the replication of the Gram+ host chromosome, thus killing the host.
We previously reported that optimally substituted 3-substituted 6-anilinouracils, specifically
derivatives of 6-(3-ethyl-4-methylanilino)uracil “EMAU”, were potent inhibitors of DNA
polymerase IIIC from the Gram+ bacterium Bacillus subtilis, and had potent antibacterial
3,4
activity against a panel of Gram+ organisms. Several 3-substituted EMAU derivatives such
as HB-EMAU (1; see structure) showed a protective effect given intraperitoneally to S.
aureus infected mice, and one derivative was active given subcutaneously to S. aureus infected
*Author for correspondence: George E. Wright, Ph.D., GLSynthesis Inc., One Innovation, Drive, Worcester, MA 01605, Phone 508
754-6700, FAX 508, 754-7075, Email: george.wright@glsynthesis.com.
§
¶
Present address: Boehringer-Ingelheim (Canada) Ltd. Laval, Quebec, Canada
Present address: Laboratoires Idenix, 1682 Rue de la Valsière, 34189 Montpellier, France

Zhi et al.
Page 2
4
mice. A related compound developed by another group was reported to have efficacy after
5
intravenous dosing in the same animal model.
Not all derivatives of EMAU that were potent enzyme inhibitors had significant antibacterial
3
activity. Factors that may limit antibacterial activity could include lack of penetration of the
cell wall or membrane, removal of compound by active efflux mechanisms, and alteration of
the sensitivity of the target enzyme in its “biophase” in the bacterium. In order to further explore
the space available at the 3 position of EMAU and, thus, maximize binding to the pol IIIC
target, we undertook additional synthesis in this class of compounds. Specifically, we have
prepared derivatives of EMAU containing a variety of fluoroquinolones of known antibacterial
activity (see typical structures) linked via their secondary amino groups, which we call “AU-
FQ hybrids” (see structure). Figure 1 summarizes the structures and considerable documented
6,7
8
structure-activity relationships regarding efficacy and toxicity of the fluoroquinolones.
Based on this information we chose substituents known to impart high antibacterial potency
and low incidence of side effects to the parent “FQs” for coupling with EMAU and related pol
IIIC inhibitors. We report that these hybrid compounds have high potency against normal and
antibiotic-resistant Gram+ bacteria in culture and against relevant infections in mice, and that
they inhibit both bacterial targets, DNA polymerase IIIC and topoisomerase/gyrase.
Chemistry
Scheme 1 illustrates the multiple approaches available to synthesize AU-FQ hybrid
compounds. The simplest approach utilized pre-existing 3-(iodoalkyl)EMAUs and 7-
piperazinylfluoroquinolones (Scheme 2). Direct reaction between IB-EMAU and norfloxacin
or ciprofloxacin was an obvious choice, but we were concerned that contamination of the
product with even a small amount of the potent FQ itself could compromise the antibacterial
results. Therefore, we compared the properties of compound 2 synthesized both by direct
coupling between IB-EMAU and norfloxacin in DMF, and by treatment of the allyl ester of
norfloxacin with IB-EMAU in DMF, followed by purification of the intermediate AU-FQ ester,
and hydrolysis (LiOH). In both cases 2 was obtained in good yields, and the inhibitory
properties were identical (data not shown). However, for all subsequent syntheses, esters of
the FQs were used for reaction with iodoalkyl-EMAUs, followed by hydrolysis, to afford the
hybrids in Scheme 2 in good yields. Compounds with characteristic small alkyl groups (Et,
cPr, t-Bu) and fluorophenyl groups at the 1 position and various substituents at position 8 (halo,
methoxy) were made in this way. However, in FQs halogenation of the 8-position can result
in compounds with severe phototoxicity, and fluorophenyl groups at the 1-position can be
9
antigenic. In another strategy, 3-(4-piperazinylbutyl)EMAU reacted directly with a 6,7,8-
trifluoroquinolone ester, yielding compound 4 (Scheme 3). A third strategy required
displacement of the 7-fluoro group of polyfluoroquinolone esters by substituted piperazines,
followed by reaction with 3-(iodobutyl)EMAU and hydrolysis (Scheme 4). In this manner,
several 3-substituted-piperazinyl AU-FQ hybrids and their enantiomers were prepared. Several
derivatives bearing bicyclic diamine substituents in the FQ portion were prepared via the same
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Zhi et al.
Page 3
strategy (Scheme 5). Some potent antibacterial fluoroquinolones, e.g. trovafloxacin, contain
6,7
bicyclic diamines at position 7.
Hybrids with linkers connecting the EMAU and 7-piperazinylfluoroquinolone portions other
than butyl, including those with pentyl, heptyl and ethoxyethyl groups, were prepared by
methods analogous to the above. Except for the pentyl derivatives, e.g. 6, none was comparable
in activity to those described in Table 1 (results not shown).
Results
3-Substituted-EMAU derivatives where the 3-substituent was a “FQ” were potent inhibitors
of pol IIIC. The results presented in Table 1 show the potent inhibition (K range = 0.004–0.04
i
μM) of B. subtilis pol IIIC by all EMAU-FQ compounds. Significantly, an analog based on
10
the less pol IIIC-inhibitory 3,4-dimethylanilino compound “DMAU” (18) retained good
activity against pol IIIC (K = 0.1 μM), but a derivative bearing the essentially inactive 6-anilino
i
group (19) was much less potent (K = 29 μM). The AU-FQ hybrid compounds had significantly
i
lower MIC values against the screening set of Gram+ bacteria than typical EMAU derivatives,
such as HB-EMAU (1) (Table 1). MIC values for AU-FQ compounds were ca. 2–4-fold lower
than those for 1, although not as potent as the FQ standards norfloxacin, ciprofloxacin, and
sparfloxacin against the corresponding FQ-sensitive organisms. Among the piperazinyl and
substitutedpiperazinyl derivatives, most had similar potencies against individual strains,
varying about ±2-fold in MIC values. Derivatives with bicyclic diamines (15–17) were
generally less active, and had variable and weak activity against MRSA 1094 and the
enterococci. The hybrid derived from DMAU (18) was nearly as active as its EMAU
counterpart 5, but the unsubstituted 6- anilinouracil hybrid 19 was less potent, especially
against the MRSA and enterococcal strains (Table 1).
The Gram+ antibacterial potency of the AU-FQ compounds was similar to that of the recently
approved drug linezolid (Table 1). As with linezolid, the strains that were relatively resistant
to FQs, e.g. MRSA 1094, E. faecium 19434 and VRE 700802, remained highly sensitive to
the hybrids. Hybrids containing the 3-methylpiperazinyl moiety (12 and 13) were marginally
more potent than other hybrids, consistent with the higher potency of the corresponding FQs
11
compared with ciprofloxacin. In the case of 13, the R enantiomer was slightly, but probably
not significantly, more potent (Table 1). The enantiomers of the related 3-methylpiperazinyl
FQ temafloxacin (see structure) showed little difference in MIC vs a large panel of Gram+ and
Gram- bacteria, although the S isomer was 2–4-fold more potent in mouse protection tests than
11
the R isomer.
The Gram- organism E. coli was resistant to most compounds at the highest concentrations
tested, although several compounds showed moderate activity (Table 1), consistent with the
probability that these compounds may inhibit topo/gyrase in Gram- bacteria. No AU
compounds, e.g. 1 (Table 1), show activity against Gram- bacteria, because they lack the Gram
3
+-specific target, pol IIIC.
The effect of the hybrid compounds on the fluoroquinolone targets topoisomerase IV (topo)
and gyrase targets was tested by assaying several hybrid compounds and FQ standards against
12
the enzymes isolated from B. subtilis. Compounds 5, 13 and its enantiomers 13S and 13R,
inhibited the decatenation reaction by topo and gyrase with potencies similar to or better than
those of nalidixic acid, the prototype of the topoisomerase inhibitors, but considerably less than
the effective drugs ciprofloxacin and sparfloxacin (Table 2). That the hybrid compounds exert
action on both targets in sensitive bacteria has been demonstrated by experiments with S.
13
aureus mutants resistant to one or both AU and FQ components. For example, S. aureus
strains resistant to HB-EMAU or to ciprofloxacin were fully sensitive to compound 13, but a
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Zhi et al.
Page 4
doubly-resistant mutant was insensitive to 13. Further details of target potencies and
mechanism of action of the representative hybrid 13 are the subjects of a separate manuscript
13
submitted for publication.
Studies in mice
Compound 5, which is effectively the combination of HB-EMAU and ciprofloxacin, was
selected as a representative compound with which to explore the properties of the AU-FQ
hybrids in vivo. At acidic or alkaline pH, 5 and other hybrids were only slightly soluble in
physiological saline. To achieve concentrations required for pharmacokinetic analysis and in
vivo antibacterial testing at an acceptable dose volume, a solubility of 10–20 mg/ml was
required. As a result, the solubility of 5 in saline was tested in the presence of several cosolvents.
Solubility of 20 mg/ml was achieved with a formulation consisting of 10% N,N-
dimethylacetamide (DMA) and 10% Cremophor EL in phosphate buffered saline, adjusted to
pH 9.5. This “DCP” vehicle was used as vehicle for intravenous (iv) administration of 5 and
related compounds via the tail vein in mice.
Pharmacokinetics of 5
Disposition studies were carried out with 5 dissolved in DCP via tail vein injection to mice at
100 mg/kg. (Higher doses caused agitation and discoloration of the tail.) Mice were dosed and
blood was collected from groups of 3 animals at various times post- injection for quantitation
by HPLC (see Experimental Section for details). Figure 2 shows the resulting plasma
concentrations of 5. Peak concentration was 213 μg/mL at 5 minutes, and the concentration
declined biexponentially thereafter. Non-compartmental analysis of the data by WinNonlin
software gave an elimination half-life of 46 minutes. The mean MIC of 5 for the organism used
in antibacterial screening in mice, S. aureus (Smith), is 0.41 ±0.15 μg/mL. Therefore, the
plasma concentration of 5 was expected to exceed MIC for at least six hours following a iv
dose of 100 mg/kg; the plasma concentration at 6 hours was 0.62 ±0.22 μg/mL.
HPLC analysis of the plasma samples from the above 5-treated mice revealed a faster eluting
peak whose concentration was time-dependent. The plasma profile of this putative metabolite
in mouse plasma is shown in Figure 2. This peak was isolated and subjected to LC-MS analysis.
−1
The observed M = 806 was consistent with a glucuronide of 5. The elimination half-life of
the metabolite was 57 minutes, and the estimated AUC(0−t) ratio (metabolite/5) was 3.7. The
appearance of a glucuronide of 5 is consistent with the formation of carboxyl glucuronides of
14
several fluoroquinolones, although not of ciprofloxacin itself, which is excreted largely
15
unchanged in human patients. No metabolite that could represent the breakage of the EMAU
and FQ components was observed under these conditions.
Acute toxicity of 5 in mice
Intravenous doses of 5 in DCP via the tail vein were well tolerated by mice (n = 5) at 100 mg/
kg (dose volume of 5 mL/kg), both as a single dose or as two 50 mg/kg doses separated by 1
hour. At 200 mg/kg the mice showed a transient, seizure-like reaction to a single injection after
ca. 10 minutes, but all animals appeared normal after 16 hours.
Efficacy of 5 in the S. aureus (Smith) ip infection model
Using pharmacokinetic and modeling data, a series of experiments was designed to test the
effect of 5 in the standard murine peritonitis model of S. aureus following iv injection. The
objective was to determine antibacterial efficacy in vivo from a site distinct from the infection
8
site. Swiss-Webster mice were infected intraperitoneally (ip) with 10 colony forming units
(CFU) of S. aureus (Smith), and survival was monitored for 72 hours. Typically animals die
within 10–18 hours of the infection, and the positive control drug vancomycin, given ip at 10
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Zhi et al.
Page 5
mg/kg or iv at 30 mg/kg, protects all animals for at least 72 hours. Compound 5 was partially
effective in protecting mice from ip S. aureus (Smith) infection when administered in a single
dose of 50 mg/kg and completely effective at 75 and 100 mg/kg (Table 3). Vancomycin as
positive control was fully protective at 30 mg/kg.
Comparison of iv efficacy of AU-FQ compounds against S. aureus (Smith) infections
Representative hybrid compounds were screened, by iv dosing in DCP, for protective activity
against S. aureus (Smith) infection in mice. Single doses of all hybrids protected animals for
72 hours, and their ED values are summarized in Table 4. Compound 13 was somewhat more
50
potent than 5, and its enantiomers 13R and 13S were among the most potent hybrids in these
experiments.
Spectrum of in vivo activity of 13
The significant potency of compound 13 in the S. aureus infection model in mice prompted
detailed study of this compound against infections caused by additional bacterial strains. Ip
infections in mice were established with E. faecalis and the drug resistant isolates MRSA 1094
and VRE 700802. However, these organisms are less virulent in mice than S. aureus, and
required higher CFUs and 5% mucin as adjuvant to cause lethal infections. Even then, the mean
survival times from these infections were longer than for S. aureus (Smith), proving a challenge
for bolus iv dosing with AU-FQ compounds. The results of Table 5, based on cumulative results
for several experiments, show that single iv doses of 13 had little protective effect, except for
E. faecalis where 80% protection was seen at 75 mg/kg. Double doses of 75 mg/kg, separated
by 120 minutes, gave statistically significant, although not complete, protection from MRSA
and VRE infections. In the VRE infection, significant increases in mean survival times were
noted from single and double doses of 13 (data not shown).
Discussion
The properties of AU-FQ hybrid compounds described in this paper and its companion
12
paper show that these are unique, dual action antibacterials with potential for treatment of
antibiotic-resistant, Gram+ bacterial infections. The SAR for active hybrid compounds is
relatively “flat”, i.e. there is little variation in antibacterial potency against individual strains
except for those compounds containing AU substituents other than “3-ethyl-4-methyl”,
compounds with longer linkers, or bicyclic diamines. There have been several reports of hybrid
antibiotics consisting of fluoroquinolones covalently connected to penicillins, cephalosporins
16
and carbapenems; the linkages in these compounds may cleave, hydrolytically or
enzymatically, to give the individual components. The limited observations for hybrid 5 in
mice suggest that glucuronidation is a major pathway of metabolism. Cleavage to the respective
pol IIIC and topoisomerase/gyrase inhibitor moieties, although not observed, cannot be ruled
out, however.
The AU-FQ hybrids were fully protective in protecting mice from lethal infection by S.
aureus (Smith) at nontoxic doses (Tables 3 and 4), and a representative compound (13) gave
statistically significant protection of mice with lethal infections by other Gram+ bacteria (Table
5). The significant antibacterial activity of 13, both in vitro and in vivo, have warranted further
study of this compound. A major challenge is its low water solubility, consistent with both the
high molecular weight and likely zwitterionic property of the compound. The preparation and
study of acid and base salts of 13 are underway. In addition, potential differences in the
disposition and in vivo potencies of the enantiomers 13R and 13S have prompted more
extensive comparisons of them with the racemate.
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Zhi et al.
Page 6
Experimental Section
Materials
Reagent chemicals, solvents and chromatographic media were obtained from commercial
sources. 3-(4-Iodobutyl)-6-anilinouracils, 3-(4-piperazinylbutyl)EMAU, and 1 were
4
synthesized as described. 2-(Hydroxymethyl)piperazine was synthesized by a literature
17
method. Norfloxacin and ciprofloxacin hydrochloride were obtained from Sigma and
Mediatech Inc., respectively. Esters of polyhaloquinolone-3-carboxylic acids were prepared
by methods cited individually below.
Analytical HPLC was performed with a Hitachi LC using a Waters Symmetry C8 column (3.5
μm, 4.6 × 50 mm) and an elution gradient from 0 to 50% MeCN:H O in 20 min (unless indicated
2
otherwise). Preparative HPLC was performed on a Waters Delta Prep 400 system using a
Waters Symmetry Prep C8 column (7 μm, 19 × 150 mm) and a gradient from 25 to 50%
MeCN:H O. Plasma samples were analyzed with a Varian Prostar System using a Microsorb
2
C18 column (5 m, 4.6 × 150 mm), a mobile phase of MeCN:H O:NEt :AcOH
(25:74.7:0.2:0.1), and a detection wavelength of 282 nm, as described previously.
2
3
4
Melting points were determined on a Mel-temp apparatus and are uncorrected. Unless
otherwise noted, NMR spectra were obtained in Me SO-d solution with a Bruker Avance 300
2
6
or a Varian INOVA 400 spectrometer. Chemical shifts (δ) are in ppm from internal TMS. Mass
spectra were measured on a ThermoFinnigan LCQ Advantage ion trap instrument by APC
ionization. All new compounds were characterized for identity by HR-MS with a FAB source
on a Kratos MS50TCTA spectrometer equipped with a peak matching unit, or by EI on a JEOL
JMS-700 MStation double focusing sector spectrometer (Mass Spectrometry Facility,
University of Massachusetts, Amherst). All new compounds were at least 98% by reverse phase
HPLC in two solvent systems, except 15–17 for which one solvent system was used
(Supporting Information).
3-{4-[1-(1-Ethyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3-ethyl-4-
methylanilino)uracil, 2
Method a—A mixture of 3-(4-iodobutyl)-6-(3-ethyl-4-methylanilino)uracil (IB-EMAU)
(241 mg, 0.56 mmol), K CO (130 mg, 0.94 mmol), and norfloxacin (150 mg, 0.47mmol) in
2
3
DMF (20 ml) was stirred at room temperature overnight. The mixture was concentrated under
reduced pressure, and the residue was purified by chromatography on silica gel using a gradient
1
of MeOH in CHCl as eluent to give 180 mg (62%) of product as a colorless solid. H NMR:
3
15.31 (s, 1H, COOH), 10.37 (s, 1H, NH), 8.92 (s, 1H, FQ-C -H), 8.07 (s, 1H, NH), 7.91 (d,
2
1H, FQ-C -H), 6.90–7.23 (m, 4H, Ar-H and FQ-C -H), 4.71 (s, 1H, C -H), 4.57 (q, 2H,
5
8
5
NCH ), 3.72 (t, 2H, NCH ), 3.30 (m, 4H, 2×CH N), 2.57 (m, 6H, 2×CH N and ArCH ), 2.37
2
2
2
2
2
(m, 2H, CH N), 2.21 (s, 3H, ArCH ), 1.37–1.58 (m, 7H, 2×CH and CH ), 1.12 (t, 3H,
2
3
2
3
ArCH CH ). HRMS: calcd for C H FN O (M + 1) 619.2966; found 619.3083.
2
3
33 40
6 5
Method b—A mixture of allyl 1-ethyl-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate
18
hydrochloride (0.85 g, 2.1 mmol), NaHCO (0.56 g, 6.7 mmol), and IB-EMAU (1.1 g, 2.6
3
mmol) in DMF (60 mL) was stirred at room temperature overnight. Water was added, and the
mixture was extracted with CHCl , and the organic extracts were dried over Na SO . After
3
2
4
removal of solvents, the residue was purified by chromatography on silica gel using 10–15%
MeOH:CHCl as eluent to give 877 mg of ester (62%) as a colorless solid. This ester (600 mg)
3
was dissolved in 80 mL of a 4:1mixture of MeOH and water. Solid LiOH (53 mg) was added
to the solution, and the mixture was stirred at room temperature overnight. The mixture was
acidified with AcOH to pH 5–6. The solvent was evaporated to dryness, and a small amount
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Zhi et al.
Page 7
of water was added to the residue. The suspension was filtered and dried in vacuo to give 557
mg (99%) of product as an off-white solid, identical with the sample made by method a.
3-{4-[1-(1-Ethyl-3-carboxy-4-oxo-6-fluoro-8-aza-7-quinolyl)-4-piperazinyl]butyl}-6-(3-ethyl-4-
methylanilino)uracil, 3
19
Ethyl 1-ethyl-6-fluoro-7-chloro-8-aza-4-quinolone-3-carboxylate was treated with IB-
EMAU and K CO in DMF as above. Hydrolysis of the resulting ester with aqueous LiOH as
2
3
1
above gave the product in 70% yield. H NMR: 15.20 (s, 1H, COOH), 10.38 (s, 1H, NH), 8.86
(s, 1H, FQ-C -H), 8.06 (s, 2H), 6.85–7.14 (m, 3H, Ar-H), 4.72 (s, 1H, C -H), 4.55 (m, 2H,
2
5
CH ), 3.88 (m, 4H, 2 × NCH ), 3.50 (m, 2H, CH N), 3.30 (m, 2H, CH N), 2.55 (q, 2H,
2
2
2
2
CH ), 2.34 (m, 4H, 2 × CH N), 2.15 (s, 3H, ArCH ), 1.56 (m, 7H, 2 × CH and CH ), 1.10 (t,
2
2
3
2
3
3H, CH ). HRMS: calcd. for C H FN O 620.2996; found 620.2995.
3
32 39
7 5
3-{4-[1-(1-Ethyl-3-carboxy-4-oxo-6,8-difluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3-ethyl-4-
methylanilino)uracil, 4
A solution of 3-[4-(1-piperazinyl)butyl]-6-(3-ethyl-4-methylanilino)uracil dihydrochloride
20
(1.2 eq), ethyl 1-ethyl-6,7,8-trifluoro-4-quinolone-3-carboxylate (1 eq), and K CO (4.0 eq)
2
3
in MeCN was heated at reflux for 16 h. The solvent was evaporated, and the residue was
chromatographed on silica gel with CHCl :MeOH as eluent, giving ca. 40% of the ester
3
intermediate. The ester was stirred in a solution of LiOH in aqueous MeOH at room temperature
overnight. After evaporation of MeOH, the solution was acidified with glacial AcOH. The
1
colorless precipitate was filtered and washed with water. Yield: 90%. H NMR: 14.82 (s, 1H,
COOH), 10.40 (s, 1H, NH), 8.90 (s, 1H, FQ-C -H), 8.02 (s, 1H, NH), 7.89 (d, 1H, FQ-C -H),
2
5
6.85–7.14 (m, 3H, Ar-H), 4.72 (s, 1H, C -H), 4.61 (m, 2H, CH ), 3.83 (m, 2H, NCH ), 3.42
5
2
2
(m, 6H, 3 × CH N), 2.57 (q, 2H, CH ), 2.33 (m, 4H, 2 × CH N), 2.17 (s, 3H, ArCH ), 1.54
2
2
2
3
(m, 7H, 2 × CH and CH ), 1.10 (t, 3H, CH ). HRMS: calcd. for C H F N O 637.2950;
2
3
3
33 39 2 6 5
found 637.2951.
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3-
ethyl-4-methylanilino)uracil, 5
A mixture of ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate (100
mg, 0.28 mmol), NaHCO (74 mg, 0.88 mmol), and IB-EMAU (184 mg, 0.43 mmol) in 30
3
mL of DMF was stirred at room temperature overnight. Water was added, and the mixture was
extracted with CHCl . The organic extracts were dried over Na SO , and, after removal of
3
2
4
solvents, the residue was purified by chromatography on silica gel using a gradient of 7–15%
1
MeOH:CHCl as eluent to give 139 mg (76%) of ester as a colorless solid. H NMR: 10.42 (s,
3
1H, NH), 8.30 (s, 1H, FQ-C -H), 8.12 (s, 1H, NH), 7.78 (d, 1H, FQ-C -H), 7.44 (d, 1H, FQ-
2
5
C -H), 6.90–7.15 (m, 3H, Ar-H), 4.75 (s, 1H, C -H), 4.20 (q, 2H, CH O), 3.72 (m, 2H,
8
5
2
NCH ), 3.65 (m, 1H, CH), 3.22 (m, 4H, 2 × CH N), 2.50–2.63 (m, 6H, 2 × CH N and
2
2
2
ArCH ), 2.35 (m, 2H, NCH ), 2.21 (s, 3H, ArCH ), 1.38–1.60 (m, 4H, 2 × CH ), 1.20–1.30
2
2
3
2
(m, 5H, CH and CH ), 1.05–1.17 (m, 5H, CH and ArCH CH ). This ester (100 mg) was
2
3
2
2
3
dissolved in 50 mL of 4:1 MeOH:H O. Solid LiOH (40 mg) was added to the solution, and the
2
mixture was stirred at room temperature overnight. After acidification with glacial AcOH to
pH 5–6, the solvent was evaporated to dryness, and a small amount of water was added. The
suspension was filtered and the solid dried in vacuo to give 87 mg (91%) of product as an off-
1
white solid. H NMR: 15.18 (s, 1H, COOH), 10.37 (s, 1H, NH), 8.65 (s, 1H, FQ-C -H), 8.05
2
(s, 1H, NH), 7.90 (d, 1H, FQ-C -H), 7.56 (s, 1H, FQ-C -H), 6.90–7.17 (m, 3H, Ar-H), 4.71
5
8
(s, 1H, C -H), 3.83 (m, 1H, CH), 3.72 (m, 2H, NCH ), 3.30 (m, 4H, 2 × CH N), 2.57 (m, 6H,
5
2
2
2 × CH N and ArCH ), 2.37 (m, 2H, CH N), 2.21 (s, 3H, ArCH ), 1.40–1.61 (m, 4H, 2 ×
2
2
2
3
CH ), 1.32 (m, 2H, CH ), 1.10–1.28 (m, 5H, CH and CH ). HRMS: calcd for
2
2
2
3
C H FN O (M + 1) 631.2966; found 631.3029.
34 40
6 5
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Zhi et al.
Page 8
3-{5-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]pentyl}-6-(3-
ethyl-4-methylanilino)uracil, 6
Ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and 3-(5-
iodopentyl)-6-(3-ethyl-4-methylanilino)uracil (IP-EMAU) were reacted as above, followed by
1
LiOH hydrolysis of the ester intermediate. Yield: 66%. H NMR (DMSO-d ): 15.22 (s, 1H),
6
10.37 (s, 1H), 8.65 (s, 1H), 8.08 (s, 1H), 7.89 (d, 1H), 7.65 (d, 1H), 7.11 (d, 1H), 6.93 (m, 2H),
4.73 (s, 1H), 3.81 (m, 1H), 3.70 (t, 2H), 3.34 (m, 4H), 2.55–2.60 (m, 6H), 2.36 (t, 2H), 2.22
(s, 3H), 1.53 (m, 4H), 1.25–1.32 (m, 4H), 1.07–1.20 (m, 5H). HRMS: Calcd. for
C H FN O 645.3200. Found 645.3184.
35 42
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-8-chloro-7-quinolyl)-4-piperazinyl]butyl}-6-
(3-ethyl-4-methylanilino)uracil, 7
21
Ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-piperazinyl-8-chloroquinoline-3-carboxylate and
IB-EMAU were reacted as above, followed by LiOH hydrolysis of the ester intermediate.
1
Yield: 42%. H NMR: 14.78 (s, 1H, COOH), 10.40 (s, 1H, NH), 8.90 (s, 1H, FQ-C -H), 8.04
2
(s, 1H), 7.88 (d, 1H), 6.85–7.15 (m, 3H), 4.72 (s, 1H), 4.12 (m, 1H), 3.68 (m, 2H), 3.30 (m,
4H), 2.58 (m, 6H), 2.36 (m, 2H), 2.18 (s, 3H), 1.40–1.61 (m, 4H), 0.95–1.26 (m, 7H). HRMS:
calcd. for C H ClFN O 665.2654; found 665.2662.
34 39
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-8-methoxy-7-quinolyl)-4-piperazinyl]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 8
22
Ethyl 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-7-piperazinylquinoline-3-carboxylate was
23
converted to its BF complex as described. A mixture of this complex (59 mg, 144 mmol),
2
IB-EMAU (74 mg, 0.173 mmol) and NaHCO (36 mg, 0.429 mmol) in DMF (10 mL) was
3
stirred at room temperature overnight. The mixture was concentrated under reduced pressure,
and the residue was purified by chromatography on silica gel using a gradient of 5–10%
MeOH:CHCl as eluent to give the BF complex intermediate. The intermediate was dissolved
3
2
in 30 mL of 80% MeOH:H O, 1 mL trifluoroacetic acid was added, and the mixture was heated
2
at reflux for 1.5 h. The pH was adjusted to 5–6 with 2N NaOH, the solvents were evaporated
to dryness, and 30 mL of water was added. The suspension was filtered, and the solid was dried
1
in vacuo to give 5.5 mg (38%) of product. H NMR (DMSO-d ): 14.92 (s, 1H), 10.35 (s, 1H),
6
8.71 (s, 1H), 8.13 (s, 1H), 7.74 (d, 1H), 7.15 (d, 2H), 6.93 (m, 2H), 4.73 (s, 1H), 4.10–4.30 (m,
1H), 3.78–3.86 (m, 4H), 3.30 (s, 2H), 2.60 (q, 2H), 2.23 (s, 3H), 1.30–1.70 (m, 4H), 0.90–1.30
(m, 4H). HRMS: calcd. for C H FN O 661.3150; found 661.3135.
35 42
6 6
3-{4-[1-(1-t-Butyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3-ethyl-4-
methylanilino)uracil hydrochloride, 9
24
Ethyl 1-(t-butyl)-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and IB-EMAU were
reacted as above, and the ester intermediate was hydrolyzed with aqueous LiOH. Yield:
1
63%. H NMR (DMSO-d ): 15.13 (s, 1H), 10.61 (s, 1H), 10.50 (s, 1H), 8.96 (s, 1H), 8.53 (s,
6
1H), 8.05 (d, 1H), 7.48 (d, 1H), 7.14 (d, 1H), 6.95 (m, 2H), 4.75 (s, 1H), 3.74–3.85 (m, 4H),
3.61 (m, 2H), 3.20–3.43 (m, 6H), 2.58 (q, 2H), 2.24 (s, 3H), 1.89 (s, 9H), 1.23 (m, 2H), 1.58
(m, 2H), 1.19 (t, 3H). HRMS: calcd. for C H FN O 647.3357. Found 647.3343.
35 44
6 5
3-{4-[1-(1-{2,4-Difluorophenyl}-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-
(3-ethyl-4-methylanilino)uracil, 10
25
1
Ethyl 1-(2,4-difluorophenyl)-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and IB-
EMAU gave the product after LiOH hydrolysis of the ester intermediate. Yield: 72%. H NMR:
15.02 (s, 1H), 10.35 (s, 1H), 8.86 (s, 1H), 8.10 (s, 1H), 7.88 (m, 2H), 7.65 (t, 1H), 7.43 (t, 1H),
6.86–7.14 (m, 3H), 6.20 (d, 1H), 4.72 (s, 1H), 3.68 (t, 2H), 3.50 (m, 2H), 2.95 (m, 4H), 2.55
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Zhi et al.
Page 9
(q, 2H), 2.38 (m, 4H), 2.16 (s, 3H), 1.44 (m, 4H), 1.12 (t, 3H). HRMS. Calcd. for
C H F N O 703.2856; found 703.2861.
37 38 3
6 5
3-{4-[1-(1-(4-Fluorophenyl)-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3-
ethyl-4-methylanilino)uracil, 11
25
Ethyl 1-(4-fluorophenyl)-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and IB-
1
EMAU gave the product after LiOH hydrolysis of the ester intermediate. Yield: 78%. H NMR
(DMSO-d ): 15.08 (s, 1H), 10.37 (s, 1H), 8.68 (s, 1H), 8.11 (s, 1H), 7.98 (d, 1H), 7.80 (m,
6
2H), 7.49 (m, 2H), 6.86–7.14 (m, 3H), 6.40 (d, 1H), 4.72 (s, 1H), 3.68 (t, 2H), 3.06 (m, 4H),
2.57 (m, 6H), 2.30 (m, 2H), 2.18 (s, 3H), 1.50 (m, 4H), 1.15 (t, 3H). HRMS: Calcd. for
C H F N O 685.2950; found 685.3004.
37 39 2
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 12
26
A solution of ethyl 1-cyclopropyl-6,7-difluoro-4-oxo-quinoline-3-carboxylate and 2-
methylpiperazine in DMSO was heated at 80 °C for 3 h. The resulting ethyl 1-cyclopropyl-6,7-
difluoro-4-oxo-7-(3-methylpiperazinyl)quinoline-3-carboxylate (isolated as described for the
27
8-fluoro analog ) was treated with IB-EMAU, followed by hydrolysis of the ester
1
intermediate, to give the product. Yield: 38%. H NMR: 15.10 (s, 1H), 10.37 (s, 1H), 8.78 (s,
1H), 8.05 (s, 1H), 7.88 (d, 1H), 6.88–7.17 (m, 3H), 4.72 (s, 1H), 3.83 (m, 1H), 3.70 (m, 2H),
3.50 (m, 4H), 2.30–3.30 (m, 10H), 2.18 (s, 3H), 1.10–1.65 (m, 14H). HRMS: calcd. for
C H FN O 546.3200; found 645.3196.
35 42
6 5
(R)-3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 12R
Treatment of ethyl 1-cyclopropyl-6,7-difluoro-4-oxoquinoline-3-carboxylate with (R)-2-
methylpiperazine as above gave (R)-ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-(3-
methylpiperazinyl)quinoline-3-carboxylate. Reaction with IB-EMAU and hydrolysis of the
1
ester gave the (R) enantiomer. Yield: 65%. H NMR: as for 12. HRMS: calcd. for
C H FN O 645.3200; found 645.3184.
35 42
6 5
(S)-3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl} 6-(3-ethyl-4-methylanilino)uracil, 12S
Treatment of ethyl 6,7-difluoro-4-oxoquinoline-3-carboxylate with (S)-2-methylpiperazine
gave (S)-ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-(3-methylpiperazinyl)quinoline-3-
carboxylate. Reaction with IB-EMAU and hydrolysis of the ester gave the (S) enantiomer.
1
Yield: 41%. H NMR: as for 12. HRMS: calcd. for C H FN O 645.3200; found 645.3198.
35 42
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6,8-difluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 13
A mixture of ethyl 1-cyclopropyl-6,8-difluoro-4-oxo-7-(3-methylpiperazinyl)quinoline-3-
27
carboxylate, (320 mg, 0.82 mmol), NaHCO (414 mg, 3 mmol), and IB-EMAU (580 mg,
3
1.36 mmol) in 50 mL DMF was stirred at room temperature overnight. Water was added, and
the mixture was extracted with CHCl , and then dried over Na SO . After removal of solvents,
3
2
4
the residue was purified by chromatography on silica gel using a gradient of 7–15%
1
MeOH:CHCl as eluent to give 196 mg (35%) of ester as a colorless solid. H NMR: 10.48 (s,
3
1H), 8.52 (s, 1H), 8.05 (s, 1H), 7.78 (d, 1H), 6.86–7.15 (m, 3H), 4.78 (s, 1H), 4.17 (q, 2H),
3.90(m, 1H), 3.72 (m, 3H), 3.32 (m, 4H), 2.70–2.93 (m, 2H), 2.54 (q, 2H), 2.35 (m, 2H), 2.12
(s, 3H), 1.38–1.60 (m, 4H), 0.95–1.30 (m, 13H) ppm. The ester (85 mg, 0.12 mmol) was
dissolved in 20% aqueous MeOH (30 mL), LiOH (40 mg, 0.95 mmol) was added, and the
mixture was stirred at room temperature overnight. The mixture was acidified with AcOH to
J Med Chem. Author manuscript; available in PMC 2008 October 13.

Zhi et al.
Page 10
pH 5–6, the solvent was evaporated to dryness, and a small amount of water was added to the
residue. The suspension was filtered and dried in vacuo to give 72 mg (89%) of product as an
1
off-white solid. H NMR: 15.20 (s, 1H), 10.36 (s, 1H), 8.72 (s, 1H), 8.50 (s, 1H), 7.82 (d, 1H),
6.86–7.15 (m, 3H), 4.78 (s, 1H), 4.12 (m, 1H), 3.74 (m, 2H), 3.33 (m, 4H), 2.90 (m, 2H), 2.12
(s, 3H), 1.40–1.61 (m, 4H), 0.95–1.22 (m, 10H, CH ). HRMS: calcd. for C H F N O
2
35 41 2 6 5
663.3106; found 663.3163.
(R)-3-{4-[1-(1-cyclopropyl-3-carboxy-4-oxo-6,8-difluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 13R
This compound was prepared by the same procedure as for 13, but with the use of (R)-ethyl
1-cyclopropyl-6,8-difluoro-4-oxo-7-(3-methylpiperazinyl)quinoline-3-carboxylate. Yield:
1
45% overall. H NMR: as for 13. HRMS: calcd. for C H F N O 663.3106; found 663.3108.
35 41 2
6 5
(S)-3-{4-[1-(1-cyclopropyl-3-carboxy-4-oxo-6,8-difluoro-7-quinolyl)-4-(3-methylpiperazinyl)]
butyl}-6-(3-ethyl-4-methylanilino)uracil, 13S
This compound was prepared by the same procedure as for 13, but with the use of (S)-ethyl 1-
cyclopropyl-6,8-difluoro-4-oxo-7-(3-methylpiperazinyl)quinoline-3-carboxylate. Yield: 35%
1
overall. H NMR: as for 13. HRMS: calcd. for C H F N O 663.3106; found 663.3136.
35 41 2
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6,8-difluoro-7-quinolyl)-4-(3-
hydroxymethylpiperazinyl)]butyl}-6-(3-ethyl-4-methylanilino)uracil, 14
A mixture of ethyl 1-cyclopropyl-6,8-difluoro-4-oxo-7-[3-(hydroxymethyl)piperazinyl]
28
quinoline-3- carboxylate (400 mg, 1 mmol), NaHCO (250 mg, 3 mmol), and IB-EMAU
3
(1.1g, 2.6 mmol) in DMF (80 mL) was stirred at room temperature overnight. The solvent was
removed, and water was added to the residue. The suspension was extracted with CHCl , and
3
the organic extracts were dried over Na SO . After removal of solvents, the residue was
2
4
purified by chromatography on silica gel using 7–15% MeOH:CHCl as eluent to give 320 mg
3
(46%) of ethyl ester as a colorless solid. The ester (200 mg) was dissolved in 4:1MeOH:H O
2
(80 mL), LiOH (60 mg) was added, and the solution was stirred at room temperature overnight.
The solution was brought to pH 5–6 with glacial AcOH, and the solvents were evaporated to
dryness, and a small amount of water was added to the residue. The suspension was filtered,
1
and the solid was dried in vacuo to give 176 mg (92%) of product as an off-white solid. H
NMR: 15.20 (s, 1H), 10.45 (s, 1H), 8.68 (s, 1H), 8.42 (s, 1H), 7.8 (d, 1H), 6.86–7.14 (m, 3H),
4.76 (s, 1H), 4.57 (s, 1H), 4.08 (m, 1H), 3.74 (m, 4H), 2.3–3.6 (m, 11H), 2.18 (s, 3H), 1.38–
1.6 (m, 4H), 1.05–1.26 (m, 7H). HRMS: calcd. for C H F N O 679.3055; found 679.3046.
35 41 2
6 6
3-{5-[1-(1-Ethyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-3-(1,3-diazabicyclononyl)]pentyl}-6-(3-
ethyl-4-methylanilino)uracil, 15
20
A mixture of 7-chloro-1-ethyl-6-fluoro-4-oxoquinoline-3-carboxylic acid (270 mg, 1
29
mmol), 2,8-diazabicyclo[4.3.0]nonane trifluoroacetate (TFA) (360 mg, 1 mmol,) and 1,4-
diazabicyclo[5.4.0]undec-7-ene (DBU) (0.45 ml, 3 mmol) in 1-methyl-2-pyrrolidinone (3 mL)
was heated at 120 °C for 18 h. The solvent was removed, and the residue was purified by
preparative HPLC with 25–50% MeCN:H O as eluent to obtain 91 mg (26 %) of intermediate.
2
A mixture of this intermediate (50 mg, 0.14 mmol), IP-EMAU (60 mg, 0.14 mmol), and
K CO (60 mg, 0.43 mmol) in DMF (2 mL) was heated at 90 °C for 2 h. After removal of
2
3
solvent, the residue was purified by preparative HPLC with 25–50% MeCN:H O as eluent to
2
1
obtain 17 mg (10%) of 15 as the TFA salt. H NMR: 10.94 (s, 1H), 10.34 (s, 1H), 9.59 (s, 1H),
9.32 (s, 1H), 8.66 (s, 1H), 8.32 (d, 1H), 7.56 (dd, 1H), 7.34–7.39 (m, 2H), 7.16 (m, 1H), 5.15
(s, 1H), 4.97 (m, 4H), 3.29–4.65 (m, 9H), 3.0 (q, 2H), 2.66 (s, 3H), 1.84–2.22 (m, 7H), 1.73
(m, 2H), 1.56 (t, 3H). HRMS: calcd for C H FN O , 673.3514; found, 673.3500.
37 46
6 5
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Zhi et al.
Page 11
3-{5-[1-(1-Cyclopropyl-3-carboxy-6-fluoro-4-oxo-7-quinolyl)-3-(1,3-diazabicyclononyl)]
pentyl}-6-(3-ethyl-4-methylanilino)uracil, 16
20
A mixture of 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-quinoline-3-carboxylic acid (360 mg,
29
1.28 mmol), 2,8-diazabicyclo[4.3.0] nonane as the TFA salt (450 mg, 1.27 mmol), and DBU
(0.6 ml, 3.84 mmol) in 1-methyl-2-pyrrolidinone (3 mL) was heated at 120 °C for 18 h. The
solvent was removed, and the residue was purified by preparative HPLC as above to obtain
161 mg (34%) of intermediate as the TFA salt. A mixture of this intermediate (55.4 mg, 0.113
mmol), IP-EMAU (50 mg, 0.113 mmol), and K CO (50 mg, 0.36 mmol) in DMF (2 mL) was
2
3
heated at 90 °C for 1 h. After removal of solvent, the residue was purified by preparative HPLC
1
as above to obtain 15.6 mg (20%) of 16 as the TFA salt. H NMR: 11.06 (s, 1H), 10.46 (s, 1H),
9.72 (s, 1H), 9.16 (s, 1H), 8.77 (s, 1H), 8.41 (d,1H), 7.71 (m, 2H), 7.49 (m, 2H), 5.27 (s, 1H),
3.44–4.79 (m, 12H), 3.13 (q, 2H), 2.79 (s, 3H), 2.35 (m, 6H), 2.09 (m, 2H), 1.86 (m, 5H), 1.68
(m, 5H). HRMS: calcd for C H FN O , 685.3514; found, 685.3521.
38 46
6 5
3-{5-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-3-(5-oxa-1,3-
diazabicyclononyl)]pentyl}-6-(3-ethyl-4-methylanilino)uracil, 17
20
A mixture of 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-quinoline-3-carboxylic acid (0.25 g,
29
0.85 mmol), cis-2-oxa-5,8-diazabicyclo[4,3-0]nonane dihydrochloride (0.18 g, 0.93 mmol),
and DBU in DMF (5 mL) was heated at 95 °C for 5 h. The solvent was removed, and the residue
was crystallized from MeOH:Et O to give 0.24 g (70%) of intermediate as a colorless solid.
2
A mixture of the intermediate (0.1 g, 0.24 mmol), IP-EMAU (0.13 g, 0.29 mmol), and
K CO (51.6 mg, 0.37 mmol) in DMF (2 mL) was heated at 90 °C for 3 h. Water was added,
2
3
the mixture was extracted with CH Cl , and the organic extracts were dried over Na SO . After
2
2
2
4
removal of solvents, the residue was purified by chromatography on silica gel using a gradient
of 2–10% MeOH:CH Cl as eluent to give 15 mg (10%) of the ester. The ester (15 mg, 0.02
2
2
mmol) was dissolved in MeOH (0.5 mL), aqueous 2N NaOH (1 ml) was added, and the mixture
was stirred at room temperature for 5 h. After evaporation of MeOH under reduced pressure,
water was added and the mixture was acidified with glacial AcOH to pH 5–6. The suspension
was filtered, and the solid was washed with water and dried in vacuo to give 12 mg of a yellow
solid. Purification by preparative HPLC in a gradient of 30–50% MeCN:H O as eluent gave
2
1
8 mg (55%) of 17 as the TFA salt. H NMR: 10.39 (s, 1H), 8.53 (s, 1H), 8.08 (s, 1H), 7.80 (d,
1H), 7.07 (m, 2H), 6.87 (m, 2H), 4.64 (s, 1H), 4.1 (m, 1H), 3.0–4.0 (m, 12H), 2.53 (q, 2H),
2.35 (m, 2H), 2.17 (s, 3H), 1.17–2.1 (m, 10H), 1.1 (t, 3H). HRMS: calcd for C H FN O ,
37 44
6 6
687.3306; found, 687.3316.
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-(3,4-
dimethylanilino)uracil, 18
Ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and 3-(4-
iodobutyl)-6-(3,4-dimethylanilino)uracil were reacted as described for 5. Yield: 56%. H NMR
1
(DMSO-d ): 15.22 (s, 1H), 10.37 (s, 1H), 8.66 (s, 1H), 8.06 (s, 1H), 7.92 (d, 1H), 7.55 (d, 1H),
6
7.13 (d, 1H), 6.83–7.06 (m, 2H), 4.72 (s, 1H), 3.59–3.89 (m, 3H), 2.59 (s, 4H), 2.38 (m, 2H),
2.19 (d, 4H), 0.94–1.64 (m, 8H). HRMS: calcd. for C H FN O 617.2887; found 617.2863,
33 38
6 5
3-{4-[1-(1-Cyclopropyl-3-carboxy-4-oxo-6-fluoro-7-quinolyl)-4-piperazinyl]butyl}-6-
anilinouracil, 19
Ethyl 1-cyclopropyl-6-fluoro-4-oxo-7-piperazinylquinoline-3-carboxylate and 3-(4-
1
iodobutyl)-6-anilinouracil were reacted as described for 5. Yield: 77%. H NMR (DMSO-
d ): 15.21 (s, 1H), 10.62 (s, 1H), 8.65 (s, 1H), 8.37 (s, 1H), 7.90 (d, 1H), 7.57 (s, 1H), 7.12–
6
7.50 (m, 5H), 4.82 (s, 1H), 3.70–3.90 (m, 3H), 3.35 (m, 4H), 2.57 (m, 4H), 2.37 (m, 2H), 1.40–
1.68 (m, 4H), 1.32 (m, 2H), 1.20 (m, 2H). HRMS: calcd. for C H FN O 589.2574; found
31 34
6 5
589.2592.
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Zhi et al.
Page 12
Enzyme Assays
DNA polymerase IIIC (pol IIIC) of B. subtilis was the homogenous recombinant protein
10
expressed and prepared as described previously. The enzyme was assayed in a 96-well plate
30
format by using activated calf thymus DNA as a substrate as described. Apparent inhibition
constants (K values) were determined in a truncated assay lacking the competitor dGTP as
i
31
11
described previously. Topoisomerase IV and gyrase from B. subtilis were prepared and
assayed as described.
Bacterial Strains
The standard panel for determination of Minimum Inhibitory Concentration (MIC) values
included S. aureus 25923, S. aureus 13709 (Smith), E. faecalis 29212, and E. faecium 19434,
all purchased from the American Type Culture Collection (ATCC, Manassas, VA).
Methicillin-resistant S. aureus (MRSA 1094) and vancomycin-resistant E. faecium (VRE
700802) are clinical isolates provided by the University of Massachusetts Medical School. B.
subtilis (BD54) is a standard laboratory strain. E. coli (J53) was provided by Prof. Martin
Marinus, University of Massachusetts Medical School.
Minimum Inhibitory Concentration (MIC)
3
Measurement of Minimum Inhibitory Concentration (MIC) was done as described, by
incubating bacteria at 37 °C for 16 to 24 h in the presence of twofold serial dilutions of test
compounds. Compounds were dissolved in and diluted from DMSO stocks, and all assays
contained 1% DMSO. MIC values are the lowest concentrations of test compounds at which
bacterial growth was not apparent (<25% of the DMSO control, based on optical density at
600 nm).
Animal studies
Pathogen-free Swiss-Webster mice (males, 20–24 g) were purchased from Taconic Farms
(Germantown, NY). The animals were housed at the University of Massachusetts Medical
School (UMMS) Animal Medicine facility. All animal experiments were approved by the
UMMS institutional animal care and use committee. Mice were allowed free access to food
and water throughout the studies.
Pharmacokinetic studies
Compound 5 was given as an iv bolus dose of 20 mg/kg in DCP to mice by tail vein injection
in a volume of 5 mL/Kg. At appropriate times mice were anesthetized with halothane, and
blood was collected by cardiac puncture and placed in heparinized tubes. Plasma was harvested,
and a 0.3 mL aliquot was mixed with an equal volume of MeCN, and samples were centrifuged
to precipitate protein. The supernatant was evaporated (SpeedVac®), and the residue was
suspended in 200 μL of 20% MeCN:H O for analysis by reverse phase HPLC. Conditions were
2
4
as previously described. The mobile phase consisted of MeCH:H O:NEt :AcOH
2
3
(25:74.7:0.2:0.1) at a flow rate of 1 mL/min and a detection wavelength of 282 nm. The
concentration of 5 was determined by comparison of peak areas with those from a standard
curve generated from plasma samples spiked with compound. The linear range was 1 to 40
μg/mL. Mass spectra of 5 and its putative metabolite were measured with a ion trap instrument
by APC ionization, and samples were introduced by infusion from solutions in water or by
HPLC in the above conditions.
Antibacterial efficacy in vivo
S. aureus (Smith) was grown at 37 °C to log-phase in Luria Broth (LB), and other organisms
were grown in brain-heart infusion broth (BHIB). The colony forming units (CFU) were
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Zhi et al.
Page 13
determined using a nomogram relating CFU to optical density at 600 nm. Bacteria were washed
in fresh cold broth, and given by ip injection to mice as a suspension in 0.5 mL of broth, and
with or without 5% mucin as indicated in Tables 3 and 5. Groups of five or ten mice were
treated at 15 min post-infection and/or various times post-infection with vehicle, test compound
in vehicle, or vancomycin hydrochloride (Vancocin®, Lilly) in saline at 30 mg/Kg. Mice were
returned to their cages and monitored for mortality for up to 72 hours.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
The authors thank Milka Yanachkova for excellent technical assistance. This work was supported by small business
grant GM060828 from the National Institutes of Health and by Shire BioChem Inc., Laval, Quebec, Canada.
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Figure 1.
Overview of SAR of fluoroquinolone antibacterial drugs. PK, pharmacokinetics.
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Figure 2.
Mean plasma concentrations of 5 (■) and its glucuronide metabolite (▲) after a 100 mg/kg iv
dose of 5 to mice (n = 3).
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Scheme 1.
Retrosynthetic analysis of AU-FQ hybrids.
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Scheme 2.
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Scheme 3.
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Scheme 4.
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Scheme 5.
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Table 2
Inhibition of B. subtilis topoisomerases by selected compounds.
*
IC50(μg/ml)
Cpd
gyrase
topo
5
13
13S
13R
nalidixic acid
ciprofloxacin
sparfloxacin
28.4
21.7
9.2
11.7
34.6
1.9
22.9
28.9
13.2
24.5
276
0.74
0.5
0.4
*
see Experimental Section for assay details.
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Table 3
Effect of 5 given iv on survival of S. aureus (Smith) ip infected mice.
a
Treatment
Dose (mg/kg)
Survivors (n = 10)
b
− (5 mL/Kg)
0
10
Vehicle
c
30
Vancomycin
5
“
“
50
75
100
7
10
10
a
b
c
8
Groups of 10 mice were infected ip with 10 CFU S. aureus (Smith) and dosed iv 15 minutes later; survivors were counted at 72 hours post infection.
DCP.
in saline.
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Table 4
Effect of single iv doses of AU-FQ hybrids on survival of mice after ip infection with S. aureus (Smith).
*
ED50 (mg/Kg)
Cpd
8
10
12
12R
12S
13
13R
13S
14
25
27
20
20
30
15
20
15
35
*
via tail vein in DCP vehicle, dosed at 5 mL/Kg, 15 min post-infection
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