Note: Descriptions are shown in the official language in which they were submitted.
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INHIBITORS OF CELLULAR EFFLUX PUMPS OF MICROBES
This invention relates to compounds with efflux pump inhibitor properties, and
which are
therefore compounds which inhibit cellular efflux pumps of bacteria or other
microbes. Such
efflux pump inhibitors are useful, for example, against antibiotic-resistant
microbial pathogens,
for treating microbial infections by reducing the export of a co-administered
antimicrobial agent
or by preventing the export of a compound synthesized by microbes (e.g.
bacteria, fungi) to allow
or improve their growth. Thus, this invention also provides processes for
preparation of such
efflux pump inhibitors, compositions which include such efflux pump
inhibitors, and the use of the
compounds and compositions in methods for treatment of microbial infections.
Background of the Invention
Microbes are known to have developed the ability to evolve different
mechanisms of self defense
against antimicrobial agents. In particular, bacterial and fungal pathogens
have developed
mechanisms of resistance to antibiotics and antimicrobial agents used to
inhibit their growth, or to
treat infections by them in humans, animals and tissue cultures. As a result,
treatment regimens
can be adversely affected or, in some cases rendered ineffective.
One of the most frequently employed resistance strategies in both prokaryotes
and eukaryotes is
the transmembrane-protein-catalyst extrusion of drugs from the cell, with
these proteins acting
like bilge pumps, reducing the intracellular drug concentration to subtoxic
levels. (M. Ines
Vorges-Walmsley et al, Trends in Microbiology, 2001, 9:71-79).
Recent developments in the efflux area include the discovery of new monodrug
efflux systems, as
well as the realisation of the importance of multidrug efflux systems (l3.
Nikaido and H.I.
Zgurskaya, Current Opinion in Infectious Diseases, 1999, 12:529). In gram-
negative bacteria, for
example, single component efflux pumps extrude their substrates into the
periplasmic space as is
3 0 done by the transposon-encoded tetracycline- and chloramphenicol-specific
pumps, TetA and
CmIA, respectively, and the MDR pump MdfA encoded in the chromosome of
Esclzerichia coli.
Bacterial genomes sequenced to date almost invariably contain genes apparently
coding for
multidrug efflux pumps. Multidrug efflux as a major cause of intrinsic drug
resistance in many
3 5 microorganisms, or overproduction of intrinsic pumps, or acquisition of
pump genes from
external sources, all play a prominent role, often resulting in high levels of
resistance. Examples
of multicomponent efflux pumps, belonging mainly to the resistance-nodulation-
division (RND)
family members, found mostly in gram-negative bacteria, include the MDR pumps
AcrAB-ToIC
1
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WO 02/09758 PCT/INO1/00139
and MexAB-OprM from E. coli and Pseudonzouas aerugizzosa. Interplay between
efflux pumps
may provide either additive or multiplicative effects on drug resistance (A.
Lee et al, J. of
Bacteriology, 2000, 182:3142). MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM,
AcrAB-ToIC, AcrEF, MarA, SoxS, or/and Tet pumps are known to be present in
Gram negative
organisms such as P. aerugizzosa and E. coli and are reviewed in recent
publications and papers,
such as K. K. Y. Wong et al, J. of Bacteriology, 2001,183:367-374; K. Poole,
Antimicrobial Agents
and Chemother,, 2000, 44:2233-2241; R Srikumar et al, Abstracts of the 40t"
Interscience
Conference on Antimicrobial Agents and Chemother., 2000, Abstr. 441:74; Xian
Zhi Li et al,
Journal of Antimicrob. Chemother., 2000, 45:433-436; Koronakis et.al., Nature,
2000, 405:914-
919; M. Oethinger et al, Antimicrob. Agents and Chemother., 2000, 44:10-13;
W.V. Kern, et al,
Antimicrob. Agents and Chemother., 2000, 44:814-820; O..Lomovskaya, et al,
Antimicrob.
Agents and Chemother., 1999, 43:1340-1346. Active efflux has been shown to be
the primary or
molecular mechanism of a fluoroquinolone resistance in Salzzzozzella euterica
Serovar
Typhimurium (E Giraud et al, Antimicrob. Agents and Chemother., 2000, 44:1223-
1228) and in
P. arugizzosa isolates from cystic fibrosis patients (S Jalal et al,
Antimicrob. Agents and
Chemother., 2000, 44:710-712). Efflux mediated aminoglycoside and macrolide
resistance in
Buzklzolderia pseudoozallei, the causative agent of meliodosis has been
described in R. A. Moore et
al, Antimicrob. Agents and Chemother.,1999, 43:465-470.
2 0 Efflux transporters are also among different mechanisms responsible for
the resistance to
antibiotics displayed by gram-positive bacteria and mycobacteria, particularly
aerobic gram-
positive cocci. The multidrug transporter NorA, belonging to the major
facilitator superfamily
(MFS) transporters, contributes to the resistance of Staphylococcus aureus to
fluoroquinolone
antibiotics. A minireview describes effllux-mediated resistance to
fluoroquinolones in Gram-
positive bacteria and the mycobacteria (K. Poole, Antimicro. Agents and
Chemother., 2000, 44:
2595-2599). To the MFS transporters also belong the Bmr and QacA/QacB efflux
pumps of
Gram-positive bacteria, and EmrB of E. coli (H. Nikaido, Current Opinion in
Microbiology, 1:
516-523). A structure-based mechanism for drug binding by multidrug
transporters is recently
proposed using the BmrR protein from Bacillus subtilis and the multidrug
transporter MdfA from
3 0 E coli (E. E. Zheleznova et al, Trends in Biological Sciences 2000, 25:39-
43), which mechanism is
also putatively considered for the QacA/QacB efflux system of S aureus. New
MFS pumps include
PmrA (a homolog of NorA) of Streptococcus pzzeuzzzozziae (J. Broskey et al,
Journal of Antimicrob.
Chemother., 2000, 4S:Suppl. S1, 95-99; M.J. Gill etal, Antimicrob. Agents and
Chemother., 1999,
43:187-189) and Tap of Mycobacterium fortuituzzz and M. tuberculosis.
Recently, NorM, which
pumps out fluoroquinolones and some cationic agents was found to be outside
the MFS
transporter class. The role of membrane-fusion protein (MFP) structural
homologues recently
identified in low G + C Gram positive bacteria that lack an outer membrane, as
essential
2
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WO 02/09758 PCT/INO1/00139
transport accessory proteins with transporters of the ATP-Binding Cassette
(ABC) superfamily
has been described (K T Harley et al, Molecular Microbiol., 2000, 38:516-517).
The Mef efflux system mediates large fractions of erythromycin-resistant
clinical isolates of S.
pzzeuznoniae (N. J. Johnston et al, Antimicrob. Agents and Chemother., 1998,
42:2425; T.
Nishijima et al, J. Antimicrob. Chemother., 1999, 43:637). Beta-haemolytic
streptococci and
pneumococci resistant to erythromycin due to the prsence of MefA efflux pumps
in Streptococcus
pyogetzes, Mef E pumps in S. pneumotziae, and an M phenotype bearing S,
agalactiae possessing
Mef A or Mef E pumps are found to be emergent and prevalent in Europe (C.
Arpin et al, J.
Antimicrob. Chemother., 1999, 44:133-138; E. Giovanetti et al, Antimicrob.
Agents and
Chemother.,1999, 43:1935-1940).
Efflux and drug resistance in fungi and protozoa have also been described (T.
G. White et al., Clin
Microbiol. Rev. 1998, 11:382; D. Sanglard, Drug Resistance Updates 1998,
1:255; B.
Papadopoulou et al, Drug Resistance Updates 1998, 1: 266; E. Orozao et al,
Drug Resistance
Updates,1999, 2:188).
In summary, the above discussion indicates that cellular factors affecting
transport (both active
and passive transport) of antibiotics into bacterial cells are important
components of antibiotic
2 0 resistance for many microbial species.
One strategy to target resistance mechanisms of nnicrobial self defense is to
find inhibitors of
microbial efflux pumps and, in particular of bacterial and fungal efflux
pumps.
2 5 There is disclosed in PCT Patent publication WO 96/33285 and US Patent
application 5,989,832
with priority in US Application 08/427,088 and PCT/LTS96/05469, methods for
screening for
inhibitors of microbial efflux pumps, efflux pump inhibitors, compositions
containing such efflux
pump inhibitors, and methods for treating microbial infections using those
compositions, but
unlike the efflux pump inhibitors of the present invention, the efflux pump
inhibitors disclosed
3 0 are dipeptide amides specifically inhibiting a Pseudomouas aeruginosa-type
efflux pump.
There is disclosed in PCT Patent publication WO/001714, with priority in US
Application
09/108,906, compounds which have efflux pump inhibitor activity, methods of
using such efflux
pump inhibitors and pharmaceutical compositions which include such compounds,
but unlike the
3 5 efflux pump inhibitors of the present invention, the efflux pump
inhibitors disclosed are dipeptide
amide derivatives demonstrating pump inhibitory activity against P. aeruginosa
strains containing
singular efflux pumps and multiple efflux pumps.
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Patent WO 99/17791, with US priority in 60/060,898, discloses a method for
inhibiting the
selection or propagation of a bacterial mutant that overexpresses an efflux
pump wherein the
inhibitor disclosed is the dipeptide amide, L-phenylaIanyl-L-arginyl-beta-
naphthylamide (MC'-
207,110), which is unlike the efflux pump inhibitor compounds of the present
invention.
US Patent 6,245,746, US Patent 6,114,310 and WO 9937667 all with US priority
in US application
09/012,363 discloses methods of using efflux pump inhibitors which increase
the susceptibility of
microbes, in particular P. aerugiuosa strains, to antimicrobial agents and
pharmaceutical
compositions including such compounds which are unlike the efflux inhibitor
compounds of the
present invention.
US Patent 6,204,729 describes peptidomimetic, secondary amide containing
benzoxazole
derivatives as efflux pump inhibitors, methods of using such efflux pump
inhibitor compounds
and pharmaceutical compositions including such compounds which are unlike the
efflux inhibitor
compounds of the present invention.
Patent WO 00/32196, with US priority in 60/110,841, discloses inhibitors of
multidrug transport
proteins which may be used in combination with existing antibacterial agents
and/or antifungal
agents, wherein the inhibitor is an indole or a urea or an aromatic amide or a
quinoIine, all of
2 0 which inhibitors are unlike the efflux pump inhibitor compounds of the
present invention. In
addition, the inhibitors disclosed in patent WO 00/32196 are specifically
inhibitors of bacteria
expressing a norA pump, or a fungus expressing a multidrug transport protein.
Novel inhibitors of the NorA multidrug transporter of S. aureus having
structurally diverse
chemical structures were also described by P. N. Markham et al, (Antimicrob.
Agents and
Chemother., 1999, 43:2404), among which the more active compounds include (a)
those
containing an indole moiety like the previously known inhibitor, reserpine, '
(b) biphenyl urea
derivatives, (c) a substituted pyrimidinone derivative and (d) compounds INF
240 and INF 277,
but they are all unlike the efflux pump inhibitor compounds of the present
invention.
Another inhibitor of the NorA MDR .pump in a pathogenic S aureus strain is 5'-
methoxyhydnocarpin (F. R. Stermitz et al., Proc. New York Acad. of Sci., 2000,
97:1433), which
has a structure unlike the efflux pump inhibitor compounds of the present
invention.
3 5 Nocardamin, a cyclopeptide, was found to be a general antagonist of a
tetracycline efflux pump
from S. aureus. It has a structure unlike the efflux pump inhibitor compounds
of the present
invention.
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Minocycline and 1,1-dimethyl-5-(1-hydroxypropyl)-4,6,7-trimethylindan (Ro 07-
3149) inhibit the
active tetracycline efflux pump in S. aureus 743 (T.Hirata et al., Biol Pharm
Bull, 1998, 21:678).
Both the compounds have a structure unlike the efflux pump inhibitor compounds
of the present
invention.
(f)-3-(2-chloro, 5-bromophenyl) ethyl-4-fluoropiperidine as an example of 3-
arylpiperidines has
recently been described as potentiator of existing antibacterial agents
against E. coli (A.
Thorarensen et al, Bioorg, and Medicinal Chem. Letters, 2001, 11:1903-1096).
The 3-
arylpiperidine compounds have a structure unlike the efflux pump inhibitor
compounds of the
present invention.
To our knowledge no drug-like organic molecule has been identified, described
or proposed as an
inhibitor of a Mef efflux pump.
Deficiencies abound in the efflux pump inhibitors disclosed in the art prior
to our present
invention.
Reserpine is not a usable compound for therapy because of its neurotoxicity at
the concentration
2 0 required for efflux pump inhibition.
The inhibitors of single drug and multidrug transporters such as the dipeptide
amide, MC'-
207,110, are broad in specificity, inhibiting all three RND systems of P.
aerugiuosa involved in
fluoroquinolone efflux, but have not been shown to be effective against pumps
of other strains, for
instance a NorA pump. Moreover, the methods employed to demonstrate their
efflux pump
inhibitory properties are mainly ifz vitro methods. For demonstration of iu
vivo activity, recourse
has had to be taken to parenteral administration ( cf. T.E.Renau et al,
J.Med.Chem.,1999, 42,
4928), rather than oral administration, as it is generally known that oral
bioavailability is poor for
compounds of a peptidic nature.
The series of inhibitors described in WO 00/32196 and by Markham et al (vide
infra) have been
minimally and incompletely profiled in terms of their efficacy, safety and
tolerability, which has
yet to be demonstrated in itz vivo models.
Potent inhibitors of microbial efflux pumps is thus an important goal for the
improved control of
infectious diseases, allowing a renaissance for drugs that are no longer
effective owing to their
efflux (K. Poole, Journal of Pharmacy and Pharmacology, 2001, 53:283-294). The
current
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WO 02/09758 PCT/INO1/00139
inventors have synthesised, screened and identified novel inhibitors of
cellular efflux pumps of
microbes. Distinctive structural features characterise the different sets of
efflux pump inhibitors
for different microorganisms as will be described in the following
description.
6
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Summary of the Invention
The present invention describes compounds of the formula I as novel microbial
efflux pump
inhibitors, Rs R4 R4a
Rs RZ
~I ~I
R' ," i
~y, R~
wherein,
Formula I
Rl = H, Cl_6 alkyl, C3_6 cycloalkyl or optionally substituted aryl, aralkyl,
arylaminoalkyl,
aryloxyalkyl or arylS(O)talkyl, where t=0,1 or 2,
or when X is C and the nitrogen atom to which Rl is linked forms an optionally
substituted
4-, S-, 6- or 7-membered ring with X of the adjacent ring, the ring optionally
containing
one or more hetero atoms selected from nitrogen, oxygen or sulfur atoms, said
2 0 heteroatom(s) represented by Y, preferably Rl is CHZCHZ-, CHZY-, CHZCHZCHZ-
,
CHZCHZY-, CHZCHZCHZCHZ- and CHZCHZCHaY- where Y represents NH, O, or S. If the
ring is substituted, the substituent is Cl~ alkyl group.
R2= H, CHO, COORS, or CONHRIa,
2 5 where R13 = H or the NHR13 of CONHR13 is the residue of one of the 20
naturally
occurring amino acids: alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine,
proline, serine, threonine, tryptophan, tyrosine or valine or the optically
active isomers
thereof or the racemic mixtures thereof, or combinations of these amino acids
to give
3 0 dipeptidyl or tripeptidyl or polypeptidyl residues.
R3 = H, Cl_6 alkyl, C3_6 cycloalkyl, aryl, aralkyl, arylaminoalkyl,
aryloxyalkyl,
arylS(O)talkyl, where t=0,1 or 2,
(CHZ)"CH(R14) OC(=O)R~s, (CHZ)"CH(R14) C(=O)ORIS wherein n = 0-6, R14 = H, or
CH3;
3 5 and Rls = CZHS or C(CH3)s
or R3 is -(CH~)p-B ~Z
~( )a
wherein B=CH or N, and when B=CH, Z=NH or NCH3, and when B=N, Z=CH, O, NH, S
or
4 0 NCH3; p=0-2; q=0-2,
R~ = H, R4a = H, or R4 and R4a taken together are oxo (=O), or thio (=S).
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Rs= H, CI_5 alkyl, amino, alkylamino, or acylamino.
R6 = H, Cl_6 alkyl, halo such as F, Cl, Br, or I, amino, or hydroxy;
R~ = OH, halo such as F, Cl, Br, or I, or
NR9 Rlo wherein R9 and Rlo are the same or different and represent H, Cl_6
alkyl or
(CHZ)"OA,
or R9 is H and Rlo is a 4-membered, 5-membered, 6-membered, or 7-membered
carboeyclic,
mono or bicyclie ring, or mono or bicyclic heterocyclic ring linked to the
nitrogen of NR9Rlo
through an atom of the heterocycle other than the heterocyclic atom, or R9 and
Rlo taken together
with the nitrogen atom to which they are attached form part of a heterocycle
which heterocycle is
monocyclic or bicyclic.
The carbocycle and the heterocycle are optionally substituted at any position
of the carbocyclic or
heterocyclic group with COORS CONHR13, OA, Cl_6 alkyl, C3-C6 cycloalkyl,
aralkyl,
trifluoroalkyl, substituted Cl_6 alkyl, or N R16R1~.
Substituents of the alkyl group are selected from OA, NRl6Rm, or a halogen
atom. Rlg and Rl~ are
the same or different and represent H or Cl_6 alkyl, or where one of R16 and
Rl~ is hydrogen and
the other of RI6 and RIB is C3-C6 cycloalkyl, or substituted Cl_~ alkyl, or
R16 and Rl~ taken together
with the nitrogen atom form a heterocycle. When R16 or Rl~ is a substituted
alkyl group, the
substituent is selected from NRl6Ri~, alkanoyl or aminoalkanoyl,
2 0 or R~ = NHOA, NHCOORII, _ or NH(CHZ)"NR9Rlo ;
orR~= /~
-N~Z-~. where n=1, 2 or 3, Z=CH or N, and when Z=CH, W=NH or
( )n when Z=N, W is absent.
or -N cH2> Nhi- where n=0,1, or 2
3 0 such that this R~ moiety is linked either to 2 core molecules of the
Formula I to form a bis
compound or the R~ moiety has one of its link bonds linked to the core formula
of
Formula I and the second of its fink bonds is linked to a phenyl carboxylic
acid or ester
moiety thereof, the phenyl carboxylic acid or ester being optionally
substituted by the
usual aromatic substituents, such as Cl-C3 alkyl linear or branched, aralkyl
such as
3 5 benzyl, amino, alkylamino, alkanoylamino, x-aminoalkanoylamino, hydroxy,
alkoxy,
alkanoyloxy, oc-aminoalkanoyloxy, or halogen atoms, such as fluoro, chloro,
bromo.
A = H, Cl_6 alkyl, glycosyl, aralkyl, Cl_6 alkanoyl or aminoalkanoyl. The
anninoalkanoyl group
may be an aminoacid residue derived from one of the one of the 20 naturally
occurring amino
4 o acids or the optically active isomers thereof, or the racemic mixtures
thereof. The amino residue
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WO 02/09758 PCT/INO1/00139
is derived from alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine,
tryptophan, tyrosine or valine.
A may also be CgH11~6a SOsH, or PO3H2.
Rll = H, Cl_6 alkyl, C3_6 cycloalkyl, or heterocyclic group,
X = CH, C-F, C-Cl, C-CH3, C-CF3, C-OCH3, C-OCHF2, C-OCF3, N or when X is equal
to C it
forms together with the nitrogen atom of the adjacent ring an optionally
substituted 4-membered
ring, 5-membered ring, 6-membered ring, 7-membered ring, containing carbon
atoms and
optionally one or more Y atoms representing one or more nitrogen, oxygen or
sulfur atoms, such
ring being further optionally substituted by a CI_6 alkyl group;
and their pharmaceutically acceptable salts, hydrates, polymorphs and
pseudopolymorphs.
Where there are centres of asymmetry, the absolute stereochemistry can be
either R- or S-
configuration and any combination of configurations. Even racemic materials
and diastereomers
fulfil the structural generic descriptions.
Another object of the invention is to provide a process for preparing the
novel efflux pump
inhibitors of the formula I of the invention.
A further object of the invention is to provide pharmaceutical compositions
comprising the
compounds of the invention. A composition contains one or more compounds of
formula I or salt,
hydrate, polymorph or pseudopolymorph therefore. In addition to one or more
compounds of
formula I or a salt, hydrate, poIymorph or pseudopolymorph thereof, a
composition of this
invention may also include another antibiotic or antimicrobial compound.
3 0 Yet another object of the invention relates to method of treatment of
infections using the said
compounds of the invention or compositions comprising them. Treatment
comprises oral,
parenteral administration and/or topical application of an effective amount of
the compound of
the invention or its compositions, whether single or in combination with an
antibiotic or
antimicrobial agent or two or more compounds of this invention.
Yet a further object of the invention includes a method of suppressing growth
of a bacterium or
fungus expressing an efflux pump, comprising contacting said bacterium or
fungus with an efflux
pump inhibitor in the presence of a concentration of antibacterial or
antifungal agent below the
minimum inhibitory concentration (MIC) of said bacterium or fungus.
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Yet a further object of the invention includes methods for treating the
infections in humans and
animals, caused by sensitive and resistant microbial strains using an
antimicrobial agent and an
efflux pump inhibitor in an amount sufficient to reduce efflux pump activity,
wherein said efflux
pump inhibitor increases the susceptibility of said microbe to said
antimicrobial agent.
Yet a further object of the invention includes a method for prophylactic
treatment of a human or
animal, comprising administering to said human or animal at risk of a
microbial infection an
efflux pump inhibitor, wherein said efflux pump inhibitor decreases the
pathogenicity of a
microbe in said human or animal.
Yet a further object of the invention includes a method for prophylactic
treatment of human or
animal, comprising administering to said human or animal at risk of a
microbial infection an
antimicrobial agent and an efflux pump inhibitor, wherein said efflux pump
inhibitor increases
the susceptibility of a microbe to said antimicrobial agent.
Yet a further object of the invention includes a method of treatment using the
efflux pump
inhibitor compounds of the invention by administering, systemically or
topically, optically pure
compounds of the invention or stereochemically pure forms of the invention or
their salts,
2 0 hydrates, polymorphs, or pseudopolymorphs thereof to the affected human or
animal, thereby
avoiding the toxic effects associated with racemic mixtures of the compounds
of the invention.
Yet a further object of the invention is to enhance the antimicrobial activity
of an antimicrobial
agent against a microbe by contacting the microbe with an antimicrobial agent
and an efflux
2 5 pump inhibitor.
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Detailed Description of the Invention
The present invention describes heterocyclic compounds of the formula I as
novel microbial efflux
pump inhibitors
Rs R4 R4a
R6 R2
R \X N
:::, R~
Formula I
wherein,
Rl = H, Cl_6 alkyl, C3_6 cycloalkyl or optionally substituted aryl, aralkyl,
arylaminoalkyl,
aryloxyalkyl or arylS(O)talkyl, where t=0,1 or 2,
or when X is C and the nitrogen atom to which Rl is linked forms an optionally
substituted
4-, 5-, 6- or 7-membered ring with X of the adjacent ring, the ring optionally
containing
one or more hetero atoms selected from nitrogen , oxygen or sulfur atoms, said
heteroatom(s) represented by Y, prererably Ri is - CHZCHZ-, CHZY-, CHZCHZCHZ-,
2 5 CHZCHZY-, CHZCHZCHZCHZ- and CHZCHZCHZY- where Y represents NH, O, or S. If
the
ring is substituted, the substituent is Cl_6 alkyl group.
RZ = H, CHO, COORS, or CONHRIS,
where R13 = H or the NHR13 Of CONHR13 is the residue of one of the 20
naturally
3 0 occurring amino acids: alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine,
glutamic acid, glycine, histidine, isoIeucine, Ieucine, lysine, methionine,
phenylalanine,
proline, serine, threonine, tryptophan, tyrosine or valine or the optically
active isomers
thereof or the racemic mixtures thereof, or combinations of these amino acids
to give
dipeptidyl or tripeptidyl or polypeptidyl residues.
R3 = H, CI_g alkyl, C3_6 cycloalkyl, aryl, aralkyl, arylaminoalkyl,
aryloxyalkyl,
arylS(O)talkyl, where t=0,1 or 2,
(CH2)n~g~l4) OC(=O)R,S, (CHZ)"CH(R,4) C(=O)OR~S wherein n = 0-6, R14 = H or
CH3;
and RIS = CZHS, or C(CH3)s
-(CH2)p-B Z
or Rs is
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wherein B=CH or N, and when B=CH, Z=NH or NCH3, and when B=N, Z=CH, O, NH, S
or
NCH3~ p=0-2a q=0-2a
R4= H, Raa = H, or R4 and Raa taken together are oxo (=O), or thio (=S).
RS= H, Cl_5 alkyl, amino, alkylamino, or acylamino.
R6 = H, Cl_6 alkyl, halo such as F, Cl, Br, or I, amino, or hydroxy;
R~ = OH, halo such as F, CI, Br, or I, or
NR9 Rlo wherein R9 and Rlo are the same or different and represent H, Cl_6
alkyl or
(CHZ)~OA,
or R9 is H and Rlo is a 4-membered, 5-membered, 6-membered, or 7-membered
carbocyclic,
mono or ,bicyclic ring, or mono or bicyclic heterocyclic ring linked to the
nitrogen of NR9Rlo
through an atom of the heterocycle other than the heterocyclic atom, or R9 and
Rlo taken together
with the nitrogen atom to which they are attached form part of a heterocycle
which heterocycle is
monocyclic, or bicyclic.
When R9 is H and R1o is a 4-membered ring, it is preferred that the ring is a
ring such as 2-
azetidinyl, when Rlo is a 5-membered ring, it is preferred that the ring is a
ring such as 2- (or 3-)
pyrrolidinyl, or 2- (or 3-) furyl, or 2-(or 3-) thienyl, or 2-(or 4-
)imidazolyl, or oxazolyl or pyrazolyl,
2 0 or thiazolyl, or when Rlo is a 6-membered ring, it is preferred that the
ring is a ring such as 2-(or
3-, or 4-) piperidinyl, or 2-(or 3-) piperazinyl, or 2-(or 3-) morphoIinyl or
2-(or 4-)pyrimidinyl, and
when Rlo is a 7-membered heterocycle it is preferred that the heterocycle is a
heterocycle such as
azepinyl, oxazinyl, or thiazinyl, or a bicyclic heterocycle such as
tetrahydroisoquinolinyl,
quinucfidinyl, or 3-azabicyclol-[3.1.0] hex-yl.
When Rg and Rio taken together with the nitrogen atom to which they are
attached form part of a
heterocycle which heterocycle is monocyclic, it is preferred that the
heterocycle is such as
azetidine, pyrrolidine, furan, thiophene, imidazole, oxazole, pyrazole,
thiazole, piperidine,
piperazine, pyrimidine, azepine, oxazine, thiazine, or bicyclic such as
isoquinoline, quinuclidine,
3 0 amino-3-azabicyclo [3.1.0]hexane.
The carbocycle and the heterocycle are optionally substituted at any position
of the heterocyclic
group with COORS CONHR13, OA, Cx_6 alkyl, aralkyl, trifluoroalkyl, substituted
alkyl, or N
RISRi~~
Substituents of the alkyl group are selected from OA, NRISRI~, or a halogen
atom. R16 and Rm are
the same or different and represent H or Cl_6 alkyl, or where one of R16 and
Rl~ is hydrogen and
the other of RI6 and R~~ is C3-C6 cycloalkyl, or substituted alkyl, or R16 and
Rl~ taken together
12
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WO 02/09758 PCT/INO1/00139
with the nitrogen atom form a heterocycle. When R16 or RIB is a substituted
alkyl group, the
substituent is selected from NR16R1~, alkanoyl or aminoalkanoyl
or R~ = NHOA, NHCOORII, NH(CHz)"NR9Rlo
or R~ _ ~--~
-N~Z-~ where n=1, 2 or 3, Z=CH or N, and when Z=CH, W=NH or
when Z=N, W has no meaning.
or -N char NH- where n=0,1, or 2
such that this R~ moiety is linked either to 2 core molecules of the Formula I
to form a bis
compound or the R~ moiety has one of its link bonds linked to the core formula
of Formula I and
the second of its link bonds is linked to a phenyl carboxylic acid or ester
moiety thereof, optionally
substituted by the usual aromatic substituents, such as Cl-C3 alkyl linear or
branched, aralkyl
such as benzyl, amino, alkylamino, alkanoylamino, oc-aminoalkanoylamino,
hydroxy, alkoxy,
alkanoyloxy, oc-aminoalkanoyloxy, or halogen atoms, such as fluoro, chloro,
bromo.
A = H, Cl_5 alkyl, glycosyl, aralkyl, CI_6 alkanoyl or aminoalkanoyI. The
aminoalkanoyl group
may be an aminoacid residue derived from one of the one of the 20 naturally
occurring amino
acids or the optically active isomers thereof, or the racemic mixtures
thereof. The amino residue
is derived from alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid,
2 5 glycine, histidine, isoleucine, Ieucine, lysine, methionine,
phenylalanine, proline, serine threonine,
trytophan, tyrosine or valine.
A may also be CgHllOg, SO3H, or P03Hz,
3 0 Rll = H, C1.6 alkyl, C3_6 cycloalkyl, or heterocyclic group such as a 4-
membered ring, it is preferred
that the ring is a ring such 2-azetidinyl, a 5-membered ring such as 2- (or 3-
) pyrrolidinyl, or 2- (or
3-) furyl, or 2-(or 3-) thienyl, or 2-(or 4-) imidazolyl, or oxazolyl or
pyrazolyl, or thiazolyl, or a 6-
membered ring such as 2-(or 3-,or 4-) piperidinyl, or 2-(or 3-) piperazinyl,
or 2-(or 3-)
morpholinyl, or 2-(or 4-)pyrimidinyl, or a 7-membered heterocycle such as
azepinyl, or oxazinyl,
35 or thiazinyl, or a bicyclic heterocycle such as tetrahydroisoquinolinyl, or
quinuclidinyl, or 3-
azabicyclol-X3.1.0]hex-yl
X = CH, C-F, C-Cl, C-CH3, C-CF3, C-OCH3, C-OCHFz, C-OCF3, N or when X is equal
to C it
forms together with the nitrogen atom of the adjacent ring a 5-membered ring,
6-membered ring,
40 7-membered ring, optionally containing besides carbon atoms additional Y
atoms representing
13
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WO 02/09758 PCT/INO1/00139
one or more nitrogen , oxygen or sulfur atoms, such ring being further
optionally substituted by a
Cl_6 alkyl group;
and their pharmaceutically acceptable salts, hydrates, polymorphs and
pseudopolymorphs.
It is preferred that R4 and R4a are combined together to form an oxo group and
Rs is hydrogen or
fluorine.
Where there are centres of asymmetry, the absolute stereochemistry can be
either R- or S-
configuration and any combination of configuration. Even racemic materials and
diastereomers
fulfil the structural generic descriptions.
The term "alkyl" refers to a branched or unbranched Cl-C6 aliphatic
hydrocarbon group.
The term "aralkyl" refers to a Cl-C6 alkyl group substituted with an aryl
group which aryl group
is defined below. One example of an aralkyl group is a benzyl group.
The terms "arylaminoalkyl", "aryloxyalkyl", and "arylS(O)talkyl refers to an
aryl group as
defined below that is bonded respectively through an NH, oxygen , or S(O)S to
an alkyl group as
defined above.
The term "aryl" refers to an aromatic group which has at least one ring having
conjugated ~
electron system and includes both carbocyclic aryl (e.g., phenyl, naphthyl)
and heterocyclic aryl
groups (e.g. pyridyl, pyrimidyl, pyrazinyl, thienyl, furyl, pyrrolyl,
pyrazolyl, imidazolyl, thiazolyl
and oxazolyl). The aryl group is preferably 5 to 14 carbons, more preferably 5
to 10 carbons.
2 5 Aryl moieties includes monocyclic, bicyclic, and tricyclic rings, where
each ring has preferably five
or six members and are 6n (or 6 pi)-annelated ring system or substituted 6u
annelated ring
systems composed of a mix of carbocyclic and heterocyclic units (e.g. benzo,
pyrido, pyrimido,
pyrazino, thieno, furano, pyrrolo, pyrazolo, imidazolo, thiazolo and oxazolo).
The term "6n-
annelated.ring system" refers to a ring which has 6~ electron and is
considered aromatic. The
3 0 ring may have 6 items in its backbone, such as pyridine, pyrimidine,
benzene, or it may have less
than 6 items in its backbone, such as pyrrole, pyrazole, furan, thiazole,
oxazole or thiophene. The
aryl moiety may be optionally monosubstituted or disubstituted independently
with lower linear
or branched Cl-C6 alkyl or hydroxyl, alkoxy, alkylthio, halogen, haloalkyl,
mercapto, amino,
mono linear or branched Cl-C3alkylamino, di linear or branched Cl-
C3alkylamino, phenyl,
3 5 substituted phenyl or optionally substituted phenylamino wherein the
phenyl is substituted by
usual aromatic substituents, such as Cl-C3 alkyl linear or branched, aralkyI
such as benzyl, amino,
alkylamino, alkanoylamino, oc-aminoalkanoylamino, hydroxy, alkoxy,
alkanoyloxy, oc-
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WO 02/09758 PCT/INO1/00139
aminoalkanoyloxy, or halogen atoms, such as fluoro, chloro, or bromo. An
example of a bicyclic
ring system is for instance pyrazolopyridinyl optionally further substituted
e.g. by a phenyl group
or a substituted phenyl group.
"Alkylamino" is for example NHCH3, N(CH3)z, NHCzHs, N(CzHs)z, NHCsH~, N(C3H~)z
and
"Acylamino" is for example NHCOCH3, NHCOOCzHs or NHC(=O)O(CH3)3.
15
"Alkanoyl" is for example acetyl, propionyl, ethoxycarbonyl, t-butoxycarbonyl,
' (CH2)nC~l4 OC(=O)R15 is for example acetoxymethyI, pivaloxymethyl,
pivaloyloxyethyl,
pyrrolidinylethyl, piperidinylethyl, morpholinylethyl.
' (CH2)nC~l4 C(=O)OR15 is for example methoxycarbonylmethyl,
ethoxycarbonylmethyl.
-(CH~)p-B Z
is for example pyrrolidinylethyl, piperidinylethyl, morpholinylethyl
The preferred acid addition salts are those of hydrochloride, hydrobromide,
hydroiodide,
sulphate, sulfonate, sulfamate, phosphate and salts of organic acids such as
acetate, lactate,
succinate, oxalate, maleate, fumarate, malate, tartrate, citrate, ascorbate,
gluconate, benzoate,
cinnamate, methane sulphonate and p-toluene sulphonate. Preferred alkali
addition salts are
2 5 lithium, sodium, potassium salts, and alkaline earth salts are magnesium,
calcium salts, or
ammonium, or organic amines such as diethanolamine, N-methylglucamine,
guanidine or
heterocyclic amines such as choline, piperidine, N-methyl-4-hydroxypiperidine,
hydroxyethylpyrrolidine, hydroxyethylpiperidine, morpholine,
hydroxyethylmorpholine,
piperazine, N-methyl piperazine and the like or basic amino acids such as
optically pure or
3 0 racemic isomers of arginine, lysine, histidine, tryptophan and the like.
A number of compounds of this invention are disclosed in our PCT patent
application number
PCT/IN99/00016 and in our US patent applications 09/566,875, 09/640,947,
09/850,669 and
601286,291. The subject matter of PCT application PCT/IN99/00016 and of US
applications
3 5 09/566,875 09/566,875, 09/640,947, 09/850,669 and 60/286,291 are
incorporated herein by
reference.
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WO 02/09758 PCT/INO1/00139
A list of preferred compounds of the invention as novel microbial efflux pump
inhibitors is
provided below:
R~
Formula I
R1 = CzHs, C3Hs, CH(CH3)CHZCHZSC6H5, C6H4(2-CF3),
where (2-CF3) means that the CF3 group is attached at the 2 position,
C6H4(4-CF3)
where (4-CF3) means that the CF3 group is attached at the 2 position,
2 0 C6H4(4-F),
where (4-F) means that the F atom is attached at the 4 position, and
CsH3(2~4-r' a)
where (2,4-FZ) means that there are 2 fluorine atoms, one at the 2 position
and the other at the 4
position.
30
X..Y..N = C-CHZCHZCH(CH3), C-OCHZCH(CH3), C-OCHZCHZCH(CH3)
RZ = COON, COOCZHS, COOCH(CH3)Z, COO(CH2)3CH3, COOCHZC6H5, COOCHZCOOCZHS,
COO(CHZ)2-morpholino,
-C(=O)O N-CH3
3 5 COphe-lys-Ome, where phe-lys-Ome is the methylester of the dipeptide from
phenylalanine and lysine.
~~~a=O
RS = H, CH3, or NHZ,
R6= H,orF
40 R~ = F, Br, pyrrolidin-3-yl-amino, pyrrolidin-3-alkoxycarbonylamino,
piperidin-4-yl-amino,
pentaalkylpiperidin-4-yl-alkylamino, [loc,Soc,6oc]-3-azabicyclo[3.1.0]hex-6-yl-
amino,
quinuclidinyl-3-yl-amino,
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WO 02/09758 PCT/INO1/00139
3-aminopyrrolidinyl, 5-aminopyrrolidinyl, aminopyrrolidinyl optionally further
monopoly substituted with Cl-C6 alkyl, (mono/poly
aminoalkanoyl)aminopyrrolidinyl;
alkoxycarbonyl (mono/poly aminoalkanoyl) aminopyrrolidinyl;
acetamidopyrrolidinyl
optionally further monopoly substituted with CI-C6 alkyl, hydroxypyrrolidinyl,
piperidinyl optionally further monopoly substituted with Cl-C6 alkyl, or
halogen;
aminopiperidinyl optionally further monopoly substituted with Cl-C6 alkyl,
alkoxycarbonyl; aminopiperidinyl optionally further monolpoly substituted with
Cl-C6
alkyl, mono/dialkylaminopiperidinyl optionally further monopoly substituted
with CI-Cs
alkyl, acetamidopiperidin-1-yl,1-phenyl-4,5,6,7-tetrahydropyrazolo[4,3-
c]pyridin-1-yl,
mono/polyalkylpiperidinyl, carboxyamidopiperidinyl optionally further monopoly
substituted with Cl-C6 alkyl, hydroxypiperidinyl optionally further
substituted with one or
more C~-C6 alkyl or aminoCl-C6alkyl, alkoxypiperidinyl;
alkanoyloxypiperidinyl, oc-
aminoalkanoyloxypiperidinyl, alkoxycarbonyl-oc-aminoalkanoyloxypiperidinyl,
alkoxycarbonyl-oc-aminoalkanoyaminopiperidinyl;~ hydroxypiperidinyl optionally
further
monopoly substituted with Cl-C6 alkyl, pyrrolidinyl-4-piperidinyl, 4-
(piperidin-4-yl)-
aminoalkylpiperidinyl,
piperazinyl optionally further monopoly substituted with Cl-C6 alkyl,
mono/polyalkylpiperazinyl; alkanoylpiperazine optionally further monopoly
substituted
with Cl-C6 alkyl, 4-(2-oxazolidinone-1-yl)-alkylpiperazine-1-yl;
cyclopropylpiperazinyl;
2 0 morpholino, mono/polyalkylmorphoLino,
1,2,3,4-tetrahydroisoquinolin-2-yl,
6-amino-3-azabicyclo[3.1.0]hex-3-yl, 6-alkoxycarbonylamino-3-
azabicyclo[3.1.0]hex-3-yl,
3-aralkyl-3-azabicyclo[3.1,0]hex-6-yl amino or
1-aryl-4,5,6,7-tetrahydropyrazolo [4,3-c]pyridin-1-yl
Wherever stereoisomeric forms of the substituted heterocyclic moieties are
possible, they
axe also here included.
X = C-H, C-OCH3, C-F, N and when X is linked to N of the adjacent ring, it has
the
3 0 meanings as defined above for X..Y...N
Compounds within the generic description as contained in formula I can be
obtained by synthetic
chemistry methods known to those skilled in the chemical arts. The methods
described in our
PCT application PCT/IN99100016 and US applications 09/566,875 , 09/640,947,
09/850,669 and
3 5 60/286,291 are incorporated herein by reference.
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WO 02/09758 PCT/INO1/00139
The pharmaceutically acceptable acid addition salts of compounds I are
prepared in a
conventional manner by treating a solution or suspension of the free base I
with about one
chemical equivalent of a pharmaceutically acceptable acid. Conventional
concentration and
recrystallisation techniques are employed in isolating the salts. Illustrative
of suitable acids are
hydrochloric, hydrobromic, hydroiodic, sulphuric, sulfamic, sulfonic,
phosphoric, acetic, lactic,
succinic, oxalic, malefic, fumaric, malic, tartaric, citric, ascorbic,
gluconic, benzoic, cinnamic,
methanesulfonic and p-toluenesulfonic acid.
The pharmaceutically acceptable cationic salts of compounds I may be prepared
by conventional
methods from the corresponding acids e.g. by reaction with about one equimolar
amount of a
base. Examples of suitable cationic salts are those of alkali metals such as
lithium, sodium or
potassium, alkaline earth metals such as magnesium or calcium or ammonium or
organic amines
such diethanolamine, N-methylglucamine, guanidine or heterocyclic amines such
as choline,
piperidine, N-methyl-4-hydroxypiperidine, hydroxyethylpyrrolidine,
hydroxyethylpiperidine,
morpholine, hydroxyethylmorpholine, piperazine, N-methyl piperazine and the
like or basic
amino acids such as optically pure or racemic isomers of arginine, lysine,
histidine, tryptophan
and the like.
The hydrates, pseudopolymorphs and polymorphs are prepared by methods known in
the art.
2 0 Detailed descriptions of different methods to generate hydrates, solvates,
pseudopolymorphs and
polymorphs and to characterise them are described in chapters 5 and 6 of the
book entitled
"Polymorphism in Pharmaceutical Solids" edited by Harry G Brittain (Marcel
Dekker Tnc., New
York), pp. 183-278, 1999.
The following is a list of compounds that inhibit the efflux pump of different
organisms. The list
of compounds and organisms is not inclusive. There are compounds on this list
that will inhibit
the efflux pump of other organisms.
Some specific efflux puma inhibitor com,~ounds of the invention are:
Some Preferred Compounds of the Invention Disulayin~ Inhibition of the Efflux
Pumu of
Stanhvlococcus aureus 1199 B (Nor A i1
1. 1-Ethyl-6-fluoro-1, 4-dihydro -7-(1', 2',3',4'-tetrahydroisoquinolin-2-yl)-
4-oxo-
3 5 quinoline-3-carboxylic acid and its salts.
2. 1-Ethyl-6,8-fluoro-1, 4-dihydro -7-(4'-acetoxypiperidin-1-yl)-4-oxo-
quinoline-3-
carboxylic acid and its salts.
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3. 1-Ethyl-6,8-fluoro-1, 4-dihydro -7-(4'-~2'-(2'-oxazolidin-1-yl) ethyl)
piperazin-1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
4. 1- Ethyl-6, 8-difluoro-1, 4-dihydro -7-{(loc,5oc,6oc)-6-amino-3-azabicyclo
[3.1.0]-hex-3-
yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
5. 5-Amino-1- ethyl -6, 8-difluoro-1, 4-dihydro -7-(3'-amino-5'-methyl
pyrrolidin-1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
6. 5-Amino-1- ethyl-6, 8-difluoro-1, 4-dihydro -7-(4'-aminopiperidin-1-yl)-4-
oxo-
quinoline-3-carboxylic acid and its salts.
7. 5-Amino-1- ethyl -6, 8-difluoro-1, 4-dihydro -7-{4'-(acetamido) piperidin-1-
yl~-4-oxo-
quinoline-3-carboxylic acid and its salts.
8. 5-Amino-1- ethyl-6, 8-difluoro-1, 4-dihydro -7-{(loc,5oc,6oc)-6'-(t-
butoxycarbonyl
amino)-3-azabicyclo [3.1.0]-hex-3-yl)-4-oxo-quinoline-3-carboxylic acid and
its salts.
9. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7-(3'-acetamido-5'-methylpyrrolidin-1-
yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
10. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7-(3'-amino-5'-methylpyrrolidin-1-yl)-
4-oxo-
quinoline-3-carboxylic acid and its salts.
l I. 1-Cyclopropyl-6-fluoro-1,4-dihydro--7-(4'-acetoxypiperidin-1-yl)-4-oxo-
quinoline-3-
carboxylic acid and its salts.
12. 1-Cyclopropyl-6-fluoro-1,4-dihydro -7- f 4'-(dimethylamino) piperidin-1-
yl)-4-oxo-
2 0 quinoline-3-carboxylic acid and its salts.
13. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(3',5'-dimethylpiperidin-1-yl)-4-oxo-
quinoline-3-
carboxylic acid and its salts.
14. 1-Cyclopropyl-6-fluoro-1, 4-dihydro -7-(4'-hydroxy-3',5'-dimethylpiperidin-
1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
2 5 15. 1-Cyclopropyl-6-fluoro-1, 4-dihydro- -7-(3', 4', 5'-trimethyl
piperazin-1-yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
16. 1-Cyclopropyl-6-fluoro-1, 4-dihydro- 7-(3', 5'-dimethyl-4'-ethyl piperazin-
1-yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
17. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-5-methyl- 7-(4'-ethoxypiperidin-1-yl)-
4-oxo-
3 0 quinoline-3-carboxylic acid and its salts.
18. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-5-methyl- 7-(3', 3'-dimethylpiperazin-
1-yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
19. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-(dimethylamino)-3'-
methyl
piperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
3 5 20. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-hydroxy-3'-isobutyl
piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
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21. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-hydroxy-3',3'-dimethyl
piperidin-
1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
22. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-hydroxy-3',5'-dimethyl
piperidin-
1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
23.1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(3'-methylpiperazin-1-yl)-4-
oxo-
quinoline-3-carboxylic acid and its salts.
24. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(cis-4'-amino-3',5'-
dimethylpiperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
25. 1-Cyclopropyl-6,8-difiuoro-5-methyl-1,4-dihydro-7-(4'-hydroxy-3'-
aminomethyl
piperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
26. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(5' amino-2'-methyl-
pyrrolidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
27. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-{3'-(L-Ala-L-Ala)
amino
pyrrolidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
28. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7- f 4'-(di-n-
butylamino) piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts,
29. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-{4'-(t-butoxycarbonyl-
L-Ala-L-
Ala)aminopiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
30. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(4'- propionoxy
piperidin-1-yl)-4-
2 0 oxo-quinoline-3-carboxylic acid and its salts.
3I. 5-Amino-I-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(4'-hydroxy-3',3'-
dimethyl-
piperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
32. 5-Amino-1-cyclopropyl-6,8-difluoro-1, 4-dihydro -7- f 4'-(1-pyrrolidinyl)
piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
2 5 33. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-}4'-[(piperidin-4-yl)
aminomethyl]-
piperidin-1-yl,~-4-oxo-quinoline-3-carboxylic acid and its salts.
34. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-}(1,2',2', 6',6'-
pentamethyl
piperidin-4-yl)methylamino}-4-oxo-quinoline-3-carboxylic acid and its salts.
35. 5-Amino-1-cyclopropyl-6,8-difluoro-1, 4-dihydro -7-(3',5'-dimethyl
morpholin-1-yl)-4-
3 0 oxo-quinoline-3-carboxylic acid and its salts.
36. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(4'-cyclopropyl
piperazin-1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
37. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(3', 5'-dimethyl-4-
pivaloyl
piperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
3 5 38. Ethyl 6,8-difluoro-7-(4-hydroxypiperidin-1-yl)-1-(1-phenylthio-3(S~-
but-3-yl)-1,4-
dihydro-4-oxo-quinoline-3-carboxylate
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39. 1- (2'-Trifluoromethylphenyl) -6-fluoro-1, 4-dihydro- -7-(3', 3', 4'-
trimethyl piperazin-
1-yl)-.4-oxo-quinoline-3-carboxylic acid and its salts.
40. 5-Amino-1- (2'-trifluoromethylphenyl)-6,8-difluoro-1, 4-dihydro -7-
(morpholin-1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
41. 5-Amino-1- (2'-trifluoromethylphenyl) -6, 8-difluoro-1, 4-dihydro -7-
(3',S'-
dimethylmorpholin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
42. 5-Amino-1- (2'-trifluoromethylphenyl) -6, 8-difluoro-1, 4-dihydro -7-
(3',5'-dimethyl
piperazinyl-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
43. 5-Amino-1- (4'-trifluoromethylphenyl) -6, S-difluoro-1, 4-dihydro -7-(3'-
aminopyrrolidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
44. 1- (4'-Fluorophenyl) -6-fluoro-1,4-dihydro -7-{4'-ethylamino)piperidin-1-
yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
45. 1- (2',4'-Difluorophenyl) -6-fluoro-1, 4-dihydro-7-(3', 5'-dimethyl
piperidin-1-yl)-4-
oxo-quinoline-3-carboxylic acid and its salts.
46. 5-Amino-1- (2',4'-difluorophenyl) -6, 8-difluoro-1, 4-dihydro -7-(3'-
hydroxy-5'-
methylpyrrolidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
47. 5-Annino-1- (2',4'-difluorophenyl) -6, 8-difluoro-1, 4-dihydro -7-(3',3'-
dimethyl
piperazinyl-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
48. 1-Cyclopropyl-6-fluoro-1, 4-dihydro -7-{(3'-aminoethoxycarbonyl)pyrrolidin-
3-yl)-4-
2 0 oxo-1,8-naphthyridine-3-carboxylic acid and its salts.
49. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(pyrrolidin-3-yl-amino)-4-oxo-
naphthyridine-3-
carboxylic acid and its salts.
50. 1- (2',4'-Difluorophenyl) -6-fluoro-1, 4-dihydro -7-(piperidin-4-yl-amino)-
4-oxo-1,8-
naphthyridine-3-carboxylic acid and its salts.
51. Ethyl-1- (2',4'-difluorophenyl) -6-fluoro-1, 4-dihydro -7-f [loc,5oc,6oc]-
3-N-benzyl-3-
azabicyclo[3.1.0]hex-6-yl-amino-4-oxo-1,8-naphthyridine-3-carboxylate and its
salts
52. 1-(2,4-difluorophenyl) -6-fluoro-7-(1-phenyl-4,5,6,7-tetrahydropyrazolo
[4,3-c]pyridin-1-yl-1, 4-dihydro-4-oxo-1, 8-naphthyridine- 3-carboxylic acid
and its
salts.
3 0 53. (S)-(-)-9-Fluoro-6,7-dihydro-8-(4'-carboxamidopiperidin-1-yl)-5-methyl-
1-oxo-1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid and its salts.
54. (R)-(+)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid L-arginine salt
55. (S)-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxy-3',3'-dimethylpiperidin-1-yl)-5-
methyl-1-
oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid and its salts.
21
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56. (S)-(-)-N-methylpiperidin-1-yl-9-fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-
1-yl)-5-
methyl-1-oxo-1H,5H-benzo [i,j ] quinolizine-2-carboxylate.
57. (S)-(-)-Morpholinoethyl-9-fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-
5-methyl-1-
oxo-LH,SH-benzo[i,j]quinolizine-2-carboxylate and its salts
58. Ethoxycarbonylmethyl (R)-(+)- 9-fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-
1-yl)-5-
methyl-1-oxo-1H,5H-benzo [i,j ] quinolizine-2-carboxylate
Some Preferred Bis Compounds of the Invention Displayin~ Inhibition of the
Efflux Pumu of
Stanlzylococcus auz~eus 1199 B (Nor A i1
1. N-1- f 7-(1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-quinoline-3-carboxylic
acid)}-N-3-
amino-(7-(1-(2,4-difluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-
3-
carboxylic acid)}-pyrrolidine.
2. N-1-{7-(1-cyclopropyl) -6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine- 3-
carboxylic
acid)}-N-3-amino-{7-(1-cyclopropyl) -6-fluoro-1, 4-dihydro-4-oxo-1, 8-
naphthyridine-
3-carboxylic acid)}-pyrrolidine
3. N-1- j7-(1-cyclopropyl) -6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine- 3-
carboxylic
acid)}-N-3-amino-{7-(1-cyclopropyl -6,8-difluoro-5-amino-1, 4-dihydro-4-oxo-
quinofine-3-carboxylic acid)}-pyrrolidine
4. N-1- f 7-(1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine- 3-
carboxylic
2 0 acid)}-N-4- j7-(1-cycIopropyl-6,8-difluoro-5-amino-1, 4-dihydro-4-oxo-
quinoline-3-
carboxylic acid)}-piperazine
5. N-1- f 7-(1-cyclopropyl-6-fluoro-5-methyl-1,4-dihydro-4-oxo-quinolone-3-
carboxylic
acid)}-N-3-amino- f 7-( I-(2,4-difluorophenyl) -6-fluoro- I, 4-dihydro-4-oxo-
I, 8-
naphthyridine-3-carboxylic acid)}-pyrrolidine
2 5 6. N-1- {7-(1-cyclopropyl-6-fluoro-5-methyl-1, 4-dihydro-4-oxo-quinolone-3-
carboxylic
acid)}-N-4-amino f 7-(1-cyclopropyl-6,8-difluoro-5-amino-1, 4-dihydro-4-oxo-
quinoline-3-carboxylic acid)}-piperidine
7. N-I- }7-(1-cyclopropyl-6-fluoro-5-methyl-1, 4-dihydro-4-oxo-quinolone-3-
carboxylic
acid)}-N-3-amino f 7-(1-cyclopropyl-6,8-difluoro-5-amino-1, 4-dihydro-4-oxo-
quinoline-
3 0 3-carboxylic acid)}-pyrrolidine
8. N-I- (7-(1-(2,4-difluorophenyl) -6-fluoro-1, 4-dihydro-4-oxo-1, 8-
naphthyridine- 3-
carboxylic acid)}-N-4- f 7-(1-cyclopropyl-6-fluoro-I, 4-dihydro-4-oxo-
quinoline-3-
carboxylic acid)}-piperazine.
9. N-3-azabycyclo f 7-(1-(2,4-difluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-1,8-
3 5 naphthyridine-3-carboxylic acid)}-[1~,5oc,6oc]-N-6-amino- f 7-1-(2,4-
difluorophenyl)-6-
fluoro-1,4-dihydro-4-oxo-I,8-naphthyridine-3-carboxylic acid)}-[3.1.0] hexane
22
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10. N-1- {7-(1-cyclopropyl-6,8-difluoro-5-amino-1, 4-dihydro-4-oxo-quinolone-3-
carboxylic acid)}-N-4-amino-{ethyl 2,3,6-trifluorophenyl-4-carboxylate}-
piperidine
Some Preferred Compounds of the Invention Displayin~ Inhibition of the Efflux
Pumps of
Pseudomouas aeru~iuosa 23587
1. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-5-methyl- 7-(4'-methoxypiperidin -1-yl)-
4-oxo-
quinoline-3-carboxylic acid and its salts.
2. 7-Bromo-1-cyclopropyl-6-fluoro-5-methyl-1,4-dihydro-4-oxo-quinoline-3-
carboxylic
acid and its salts.
3. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-amino-3'-
methylpiperidin-1-yl)-
4-oxo-quinoline-3-carboxylic acid and its salts.
4. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-amino-3'-methyl
piperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
5. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro -7-{3,3-dimethyl-4'-
ethylamino piperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
6. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-amino-3'-3'-dimethyl
piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
7. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-{4'-(dimethylamino)piperidin-
1-yl}-
2 0 4-oxo-quinoline-3-carboxylic acid and its salts
8. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-hydroxy-4'-
methylpiperidin-1-yl)-
4-oxo-quinoline-3-carboxylic acid and its salts.
9. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(3',3'-dimethyl-4'-
hydroxypiperidin-
1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
2 5 10. I-Cyclopropyl-6,8-difluoro-5-methyl-1,4-dihydro-7-(3'-aminomethyl-4'-
hydroxypiperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
11. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(3'-aminomethyl-4'-
hydroxypiperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
12. 1-Ethyl-6,8-difluoro-1,4-dihydro-7-(3'-5'-dimethylpiperazin-1-yl)-4-oxo-
quinoline-3-
3 0 carboxylic acid and its salts.
13. 1-Cyclopropyl-6-fluoro-I,4-dihydro-7-(4'-ethyl-3'-methylpiperazin-1-yl)-4-
oxo-
quinoline-3-carboxylic acid and its salts.
14. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(3'-5'-dimethyl-4'-ethylpiperazin-1-
yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
3 5 15. 1-Ethyl-6, 8-difluoro-1, 4-dihydro -7-{(loc,5oc,6oc)-6'-amino-3'-
azabicyclo [3.1.0] hex-3'-
yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
23
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16. 5-Amino-1-(2',4'-difluorophenyl)-6,8-difluoro-1,4-dihydro-7-(3'-
aminopyrrolidin-1-yI)-
4-oxo-quinoline-3-carboxylic acid and its salts.
17. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-((3'-amimoethoxycarbonyl
pyrrolidin-3-yl}-4-oxo-quinoiine-3-carboxylic acid and its salts.
. 18. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(pyrrolidin-3'-ylamino)-4-oxo-
naphthyridine-3-
carboxylic acid and its salts.
19. 1-(Z',4'-Difluorophenyl)-6-fluoro-1,4-dihydro-7-(piperidin-4'-ylamino)-4-
oxo-
naphthyridine-3-carboxylic acid and its salts.
20. 1-Cycloprogyl-6-fluoro-1, 4-dihydro -7-(4'-amino-3'-ethylpiperidin-1-yl)-4-
ozo-
naphthyridine-3-carboxylic acid and its salts.
21. (S)-( )-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,SFI-
benzo[ij]quinolizine-2-carboxylic acid 0.2 hydrate.
22. (S)-( )-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-I-oxo-
1H,5H
benzo[i,j]quinolizine-2-carboxylic acid, choline salt.
23. (S)-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-I-oxo-
1H,5H-
benzo[ij]quinolizine-2-carboxylic acid. 1-Hydroxyethylpyrrolidine salt.
24. (S)-( )-9-Fluoro-6,7-dihydra-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
IH,SH-
benzo[ij]quinolizine-2-carboxylic acid, diethanolamine salt.
25. (S)-( )-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H
2 0 benzo[ij]quinolizine-2-carboxylate, L-histidine salt.
26. (RS)-(~)-9-Fluoro-6,7-dihydro-8-(4'-(D-phenylaIanyloxy) piperidin-1-ylj-5-
methyl-1-
oxo-1H,5H-benzojij]quinolizine-2-carboxylic acid hydrochloride.
27. (RS)-(~)-9-Fluoro-6,7-dihydro-8-f4'-(L-oc-aspartyloxy) piperidin-1-yl]-5-
methyl-1-oxo-
1H,5H-benzo[ij]quinolizine-2-carboxylic acid hydrochloride.
2 5 28. (RS)-(~)-9-Fluoro-6,7-dihydro-8-(4'-(L-leucyloxy)piperidin-1-yl}-5-
methyl 1-oxo
1H,5H-benzo[ij]quinolizine-2-carboxylic acid hydrochloride.
29. (S)-(-)-9-Fluoro-6,7-dihydro-8-{4'-(D-leucyloxy)piperidin-1-yl]-5-methyl-1-
oxo-1H,SH-
benzo[ij]quinolizine-2-carboxylic acid hydrochloride.
30. (S)-(-)-9-Fluoro-6,7-dihydro-8-k4'-(L-alanyloxy)piperidin-1-yl]-5-methyl 1-
oxo-1H,5H-
30 benzo[ij]quinolizine-2-carboxylic acid hydrochloride.
31. (S)-( ~Morpholinoethyl-9-fluoro-6.7-dihydra-8-(4'-hydroxypiperidin-1-yl)-5-
methyl-1-
oxo-1H,5H-benzo[ij]quinolizine-2-carboxylate and its salts.
32. (R)-(+)-g,9-difluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[ij]quinolizine-
2- [S-
phenylalanyl-S-lysine methyl ester]carboxamide.
3 5 33. (RS)-(~)-9-Fluoro-6,7-dihydro-8-(traps-4'-hydroxy-3'-methylpiperidin-1-
yl)-5-methyl-
1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid and its salts.
24
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34. (RS)-(t)-9-Fluoro-6,7-dihydro-8-(cis-4'-hydroxy-3'-methylpiperidin-1-yl)-5-
methyl-1-
oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
35. (S)-(-)-9-Fluoro-6,7-dihydro-8-(traps-4'-hydroxy-3'-methylpiperidin-1-yl)-
5-methyl-1-
oxo-1H,5H-benzo[i,jjquinolizine-2-carboxylic acid and its salts.
36. 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-9-ftuoro-2,3-dihydro-3-methyl-10-(4'-
hydroxy-3'-
ethylpiperidin-1'-yl)-7-oxo-6-carboxylic acid and its salts.
37. 10-Fluoro-11-[(loc,5x,6oc )-6-amino-3-azabicyclo[3.1.0]hex-3-yl]-3,4-
dihydro-4(S)-
methyl-8-oxo-2H,8H-pyrido[1,2,3-efJ-1,5-benzoxazepine-7-carboxylic acid.
hydrochloride.
Some Preferred Compounds of the Invention Displayins Inhibition of the Efflux
Pumps of
Esclzerischia coli 2051
1. 7-Bromo-1-cyclopropyl-6-fluoro-5-methyl-1,4-dihydro-4-oxo-quinoline-3-
carboxylic
acid and its salts.
2. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4' -amino-3'-
methylpiperidin-1-yl)-
4-oxo-quinoline-3-carboxylic acid and its salts.
3. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-amino-3'-3'-dimethyl
piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
4. S-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,jJquinolizine-2-carboxylic acid 0.2 hydrate.
5. S-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid, choline salt.
6. S-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid, l-hydroxyethylpyrrolidine salt.
7. S-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid, diethanolamine salt.
8. (S)-(-)-9-Fluoro-6.7-dihydro-8- f 4'-(D-leucyloxy)piperidin-1-yl}-5-methyl-
1-oxo-1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid hydrochloride.
3 0 9. (S)-(-)-Morpholinoethyl-9-fluoro-6.7-dihydro-8-(4'-hydroxypiperidin-1-
yl)-5-methyl-1-
oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylate and its salts
10. (R)-(+)-g,9-difluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-
benzo[i,j]quinolizine-2- [S-
phenylalanyl-S-lysine methyl ester] carboxamide.
11. S-(-)-9-Fluoro-6,7-dihydro-8-(traps-4'-hydroxy-3'-methylpiperidin-1-yl}-5-
methyl-1-
oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
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12. 10-Fluoro-11-[(loc,5oc,6oc )-6-amino-3-azabicyclo[3.1.0]hex-3y1]-3,4-
dihydro-4(S)
methyl-8-oxo-2H,8H-pyrido[1,2,3-efJ-1,5-benzoxazipin-5-yl-7-carboxylic acid
hydrochloride.
Some Preferred Compounds of the Invention Disulayin~ Inhibition of the Mef
Efflux
Pump of Streptococcus pueuffaohiae 3514 and Strevtococcus yyo~ehes 26-00
1. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-(4'-amino-3'-methyl
piperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
2. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-(methylamino)-
3',3'-dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
3. i-Propyl 1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro -7-(4'-amino-3',3'-
dimethyl-piperidin-1-yl}-4-oxo-quinoline-3-carboxylate and its salts.
4. n-Butyl 1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro -7-(4'-amino-3',3'-
dimethyl-
piperidin-1-yl}-4-oxo-quinoline-3-carboxylate and its salts.
5. Ethoxycarbonylmethyl 1-cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-
amino-3',3'-dimethylpiperidin-1-yl)-4-oxo-quinoline-3-carboxylate and its
salts.
6. Benzyl I-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro -7- f 4'-(t-
butoxycarbonyl
amino)-3',3'-dimethylpiperidin-1-yl)-4-oxo-quinoline-3-carboxylate and its
salts.
2 0 7. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-N-(t-
butoxycarbonyl-L-
alanyl) amino-3',3'-dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid
hydrochloride.
8. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-L-alanylamino-3',3'-
dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid hydrochloride.
2 5 9. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(3',3'-dimethyl-4'-(t-
butoxy-
carbonylvalinylamino)piperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its
salts.
10. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro -7-(3',3'-dimethyl-4'-(L)-
valyl-
aminopiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid hydrochloride.
3 0 11. 1-Cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7- f 4'-(L)-aspartylamino-
3',3'-
dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid hydrochloride
12. 1-Ethyl-6,8-difluoro-1,4-dihydro-7-(4'-ethylaminopiperidin-1'-yl)-4-oxo-
quinoline-3-carboxylic acid and its salts.
13. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(4'-amino-3'-methyl
piperidin -
3 5 1-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
26
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14. 5-Amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(quinuclidinyl-3-yl-
amino)-4-
oxo-quinoline-3-carboxylic acid and its salts.
15. 5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro-7- {(loc,5oc,6x)-6-amino-
N-
benzyl-3-azabicycIo [3.LOj hex-6-yI]-4-oxo-quinoline-3-carboxylic acid and its
salts.
16. 1-(3'-Fluorophenyl)-6-fluoro -1, 4-dihydro -7-(4'-methylpiperazin-1'-yI)-4-
oxo-
quinoline-3-carboxylic acid and its salts.
17. 1-(2,4-Difluorophenyl) -6-fluoro-1, 4-dihydro-7- (4'-ethylaminopiperidin-
1'-yI)- 4-
oxo-quinoline-3-carboxylic acid and its salts.
18. 1-(2',4'-Difluorophenyl)-6-fluoro-5-methyl-I, 4-dihydro -7-(4'-
aminopiperidin-1'-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
19. 1-(2',4'-Difluorophenyl)-6-fluoro-5-methyl-1,4-dihydro-7-(4'-methylamino
piperidin-1'-yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
20. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(3'-aminopyrrolidin-1'-yl)-4-oxo-1,8-
° naphthyridine-3-carboxylic acid and its salts.
21. 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-{(1 oc,5x,6oc)-6-amino--N-benzyl-3-
azabicyclo [3.1.0] hex-6-yl}-4-oxo-1,8-naphthyridine-3-carboxylic acid and its
salts.
22. 1-(2,4-Difluorophenyl) -6-fluoro-1, 4-dihydro-7-(3'-aminopyrrolidin-1'-yl)-
4-oxo-
1,8-naphthyridine-3-carboxylic acid and its salts.
2 0 23. 1-(2,4-Difluorophenyl) -6-fluoro-1, 4-dihydro-7-{(loca5oc,6oc)-6-amino-
N-benzyl-3-
azabicyclo [3.1.0]hex-6-yl}- 4-oxo-1,8-naphthyridine-3-carboxylic acid and its
salts.
24. 1-(2,4-Difluorophenyl)-6-fluoro-1,4-dihydro-7-(3',3'-dimethyl-4'-hydroxy
piperdin-
1'-yl)-4-oxo-1,8-naphthyridine-3-carboxylic acid and its salts.
25. (RS)-(t)-9-Fluoro-6, 7-dihydro-8-{4'-(L-a-aspartyloxy)piperidin-1-ylj-5-
methyl-1-
oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid hydrochloride.
26. 7H-Pyrido [1,2,3-de]-1,4-benzoxazine-9-fluoro-2,3-dihydro-3-methyl-10-(3'-
ethyl 4'-
hydroxypiperidin-1'-yl)-7-oxo-6-carboxylic acid and its salts.
27. 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-9-fluoro-2, 3-dihydro-3-methyl-10- (3'-
amino
methyl-4'-hydroxypiperidin-1'-yl)-7-oxo-6-carboxylic acid and its salt.
3 0 28. 1-Cyclopropyl-6, 8-difluoro-5-methyl-1, 4-dihydro -7-(3', 3'-dimethyl-
4'-ethylamino
piperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid and its salt.
29. 1-cyclopropyl-6,7,8-trifluoro-5-methyl-1,4-dihydro - 4-oxo-quinoline-3-
carboxylic
acid.
30. (S)-(-)-9-Fluoro-6,7-dihydro-8- (3', 3'-dimethyl-4'-ethylaminopiperidin-1-
yl)-5-
methyl-1-oxo-1H, 5H-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
27
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31. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7- (3'-aminomethyl-4'-hydroxypiperidin
1-yl)-
4-oxo-quinoline-3-carboxylic acid and its salts.
32. 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7- (4'-dimethylamino-3'-
methylpiperidin-1-
yl)-4-oxo-quinoline-3-carboxylic acid and its salts.
33. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-7-{4'-cyclopropyl
aminopiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts
34. 1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-(t-butoxycarbonyl (L)-
Ala-
Ala)amino-3', 3'-dimethyl piperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid
hydrochloride.
35. 5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-ethylamino-
3', 5'-
dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid and its salts.
36. Ethyl 1-(2,4-difluorophenyl) -6-fluoro -1, 4-dihydro-7- (4-amino-3-
ethylpiperidin-
1-yl)- 4-oxo-1,8-naphthyridine-3-carboxylate
37. 1-(2,4-difluorophenyl) -6-fluoro-1, 4-dihydro-7- (4-amino-3, 5-
dimethylpiperidin-1-
Z5 y1)- 4-oxo-1,8-naphthyridine-3-carboxylic acid and its salts.
38. Ethyl 1-(2,4-difluorophenyl) -6-fluoro-5-methyl-1, 4-dihydro-7- (4-amino-
3, 3-
dimethylpiperidin-1-yl)- 4-oxo-1,8-naphthyridine-3-carboxylate
39. (S)-(-)-9-fluoro-6.7-dihydro-8- (4'-hydroxy- 3'-fluoropiperidin-1-yl)-5-
methyl-1-
oxo-1H, 5H-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
2 0 40. 10-Fluoro-11- (4-aminopiperidin-1-yl)-3,4-dihydro-4 (S)-methyl-8-oxo-
2H, 8H-
pyrido[1,2,3-efJ-1,5-benzoxazipin-7-carboxylic acid and its salt.
41. (RS)-(t)-6, 7-dihydro-8- (traps-4'-hydroxy-3'-methylpiperidin-1-yl)-5-
methyl-1-
oxo-1H, SH-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
42. (RS)-(t)-6, 7-dihydro-8- (cis-4'-hydroxy-3'-methylpiperidin-1-yl)-5-methyl-
1-oxo-
25 1H, 5H-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
43. (RS)-(~)-6, 7-dihydro-8- (4'-hydroxy-3', 3'-dimethylpiperidin-1-yl)-5-
methyl-1-oxo-
1H, SH-benzo[i,j]quinolizine-2-carboxylic acid and its salts.
Particularly appropriate examples of a microbe appropriate for the use of an
efflux pump
3 0 inhibitor are pathogenic bacterial species, such as Streptococcus
pzzeumouiae, Streptococcus
pyogetzes, Pseudonzoraas aerugitzosa, Esclaerischia coli, Staplzylococcus
aureus which can be
intrinsically resistant to commonly used antibacterial agents. Exposing these
bacteria to an efflux
pump inhibitor can significantly slow the export of an antibacterial agent
from the interior of the
cell or the export of siderophores. For instance, overexpression of the norA
multidrug transporter
3 5 has been reported for strains of S aureus for fluoroquinolone resistance
both iu-vitz~o (Yoshida et.
al., 1990; Kaatz et.al., 1990) and ifz-vivo (Trucksis et.al., 1991).
Therefore, if another antibacterial
agent is administered in conjunction with the efflux pump inhibitor, the
antibacterial agent, which
28
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WO 02/09758 PCT/INO1/00139
would otherwise be maintained at a very low intracellular concentration by the
export process,
can accumulate to a concentration which will inhibit the growth of the
bacterial cells. This growth
inhibition can be due to either bacteriostatic or bactericidal activity,
depending on the specific
antibacterial agent used. While P. aerugi~zosa is an example of an appropriate
bacterium, other
bacterial and microbial species including those described above may contain
similar broad
substrate pumps, which actively export a variety of antimicrobial agents, and
thus can also be
appropriate targets.
In addition as suggested above, for some microbial, e.g., bacterial, species,
efflux pump inhibitors
can decrease the virulence of the microbe, for example, by inhibiting the
transport of factors
important for pathogenicity. Again using P. aerugiuosa as an example,
inhibition of an efflux
pump in this bacterium inhibits the uptake of iron, which is important for
pathogenicity. The
mechanism of bacterial iron transport involves molecules called siderophores,
which are
synthesised and exported by bacterial cells via efflux pumps. These
siderophores bind tightly to
iron scavenged from the host, and are then taken up by the bacteria. In this
way, the iron needed
for bacterial metabolism is obtained, and an infection can be maintained.
Therefore, illustrating the utility of efflux pump inhibitors, inhibiting the
efflux pump of
Streptococcus pheuzuozziae, Stzeptococcus pyogeues, Pseudomo~zas aerugi~zosa,
Eschezisclaia coli,
2 0 Staphylococcus aureus allows obtaining one or more of the following
biological effects:
1. Stz~eptococcus p~zeunzo~ziae, Streptococcus pyogefzes, Pseudozzzouas
aerugiuosa, Esclaerisclaia coli,
Staphylococcus auz~ercs strains will become susceptible to antibiotics that
could not be used for
treatment of the respective bacterial infections, or become more susceptible
to antibiotics
2 5 which do inhibit the respective bacterial growth.
2. Streptococcus pyieumouiae, Streptococcus pyogeszes, Pseudozrzouas
aerugizzosa, Escherischia coli,
Staphylococcus auzeus strains will become more susceptible to antibiotics
currently used for
treatment of the respective bacterial infections.
3. ~ruletzce of Streptococcus pueumouiae, Streptococcus pyogetzes,
Pseudozzzonas aerugiuosa,
Esclzezisclzia coli, Staphylococcus aureus will be attenuated because the
availability of an
essential siderephore bearing element will be hampered.
3 5 4. The inhibition of the pumps or of one of the components of the pumps
may be lethal or
prevent growth.
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Obtaining even one of these effects provides a potential therapeutic treatment
for infections by
these bacteria. Also, as previously mentioned, similar pumps are found in
other microorganisms.
Some or all of the above effects can also be obtained with those microbes, and
they are therefore
also appropriate targets for detecting or using efflux pump inhibitors. Thus,
the term "microbes"
include, for example, bacteria, fungi, yeasts, and protozoa.
As indicated, the bacterium to be inhibited through the use of an efflux pump
inhibitor can be
from other bacterial groups or species, such bacterial groups of species
including but not limited
to one of the following:
Pseudozzzoztas aeruginosa, Pseudomonas fluorescens, Pseudomozzas acidovorans,
Pseudozzzonas
alcaligezzes, Pseudonzonas putida, Stezzotroplzozzzonas nzaltophilia,
Butklzolderia capacia, Aeromonas
hydroplzilia, Esclzerichia coli, Citrobactez~ freundil, Salnzonella
ttyplzitrzuriunz, Salmonella typhi,
Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Slzigella
flexzzeri, Slzigella sottnei,
Enterobacter cloacae, Enterobacter aerogetzes, Klebsiella pneunzoniae,
Klebsiella oxytoca, Sezratia,
»zarcescetzs, Francisella tularensis, Morganella nzorganii, Pzoteus
nzizabilis, Proteus vulgaris,
Pzovidehcia alcalifaciens, Ptovidencia rettgeri, Providencia stuaroi,
Acinetobacter calcoaceticus,
Acinetobacter haezrtolyocus, Yersinia ezzter~colitica, Yezsizzia pesos,
Yersizzia pseudotnbezculosis,
Yezsinia itztertzzedia, Bordetella pertussis, Bozdetella pazapertussis,
Bordetella brotzclziseptica,
Haenzophilus influen.zae, Haenzopliilus parai~zflue~zzae, Haezzzopltilus
duczeyi, Pasteurella znultocida,
2 0 Pastenrella haemolytica, Branlzamella catarrhalis, Helicobacter pylori,
Cantpylobactez~ fetus,
Canzpylobacter jejutzi, Canzpylobacter coli, Borrelia bnrgdorferi, Tdibzio
clzolerae, T~ibrio
paralzaetzzolyticus, Legionella pneunzophila, Listeria mozzocytogenes,
Neissezia gozzoz~rhoeae,
Neisseria nzeningiodis, Kingella, Morazella, Gardenerella vagizzalis,
Bactezoides fzagilis, Bactezoides
distasonis, Bacteroides 3~S~A Itozzzology group, Bacteroides distasonis,
Bactezoides ovalus,
Bacteroides tlzetaiotaonzicron, Bacteroides utziformis, Bacter~ides
eggertlzii, Bacteroides
splanchnicus, Clostridium di, f, ficile, Mycobacterzurzt tubercucosts,
myconacterzuzzz avtum,
Mycobacterium izttracellulare, Mycobacterium lepzae, Cozy~zebactez~ium
diphtlzeziae,
Corynebacteriunz ulcerans, Stt~eptococcus pneunzozziae, Streptococcus
agalactiae, Streptococcus
pyogezzes, Enterococcns faecalis, Enterococcus faecium, Staphylococcus aureus,
Staphylococcus
3 0 epidezmidis, Staphylococcus saproplzyticus, Staphylococcus itzternzedius,
Staphylococcus lzyicus
subsp. lzyicus, Staphylococcus haemolyticus, Staphylococcus Izonzinis and
Staphylococcus
saccharolyticus.
The term "efflux pump" refers to a protein assembly which exports substrate
molecules from the
3 5 cytoplasm or periplasm of a cell, in an energy dependent fashion. Thus an
efflux pump will
typically be located in the cytoplasmic membrane of the cell (spanning the
cytoplasmic
membrane). In Gram-negative bacteria the pump may span the periplasmic space
and there may
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
also be portion of the efflux pump which spans the outer membrane. Certain
efflux pumps will
include a polypeptide which has at least 50% amino acid sequence similarity
with a polypeptide
which is part of the P. aerugitzosa mexAlmexB/oprM efflux pump or the efflux
pump
overexpressed by P.aerugiuosa strain K385, or the efflux pump overexpressed by
P. aerugiuosa
strain PA040998E. Due to the described sequence similarity of a component
polypeptide of the
efflux pump, such an efflux pump is termed a P. aeruginosa-type efflux pump.
An "efflux pump inhibitor" is a compound which specifically interferes with
the ability of an
efflux pump to export its normal substrate, or other compounds such as an
antibiotic. The
inhibitor may have intrinsic antimicrobial (e.g. antibacterial) activity of
its own, but at least a
significant portion of the relevant activity is due to the efflux pump
inhibiting activity. Of
particular interest in this izzvention, are compounds which inhibit the export
or activity of efflux
pumps which have a broad substrate range which includes antibacterial agents.
The term
Streptococcus pfzeumouiae-type efflux pump inhibitor refers to an efflux pump
inhibitor which
inhibits a Streptococcus pueunzouiae-type efflux pump. The term "Pseudomouas
aerugiuosa- type
efflux pump inhibitor" refers to an efflux pump inhibitor which inhibits a
Pseudonzonas
aerugiuosa-type efflux pump. The term "Escherisclzia coli-type efflux pump
inhibitor" refers to an
efflux pump inhibitor which inhibits an Esclzerisclaia coli -type efflux pump.
The term
"Staphylococcus aureus-type efflux pump inhibitor" refers to an efflux pump
inhibitor which
2 0 inhibits a Staphylococcus aureus -type efflux pump.
In another aspect, this invention provides a method for treating a microbial
infection, e.g., a
bacterial infection, in an animal by administering to an animal suffering from
such an infection an
efflux pump inhibitor as described above in an amount sufficient to reduce
efflux pump activity.
In a preferred embodiment, the inhibitor is one which decreases the
pathogenicity of the microbe.
Such a decrease in pathogenicity can be obtained, for example, by interfering
with essential
bacterial element acquisition by inhibiting the transport of siderophores. The
pathogenicity may
also be reduced by reducing or eliminating the microbial products which cause
tissue-damaging
3 0 effects-to the host. Other methods of reducing pathogenicity are, however,
also within this aspect.
The host is an animal and may be, for example, chickens and turkeys, and in
certain preferred
embodiments in a mammal, e.g. a human.
In certain preferred embodiments, the microbial infection may be due to
bacteria, which may, for
example, be any of the bacterial species indicated above, but specifically
including Streptococcus
pueunzouiae, Pseudomouas aerugiuosa, Esclaerischia coli, Staplzylococcus
aureus.
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WO 02/09758 PCT/INO1/00139
In a related aspect, this invention provides a method of treating an animal
suffering from a
microbial infection by administering to the animal an efflux pump inhibitor in
an amount
sufficient to reduce efflux pump activity. In this aspect, the efflux pump
inhibitor in one which
reduces the iiz vivo viability of a microbe involved in the infection. By
reducing the i~z vivo
viability, the infected animal can more readily clear its body of the
infection, or the microbes may
even be killed. In particular embodiments the animal is a mammal. Also in
particular
embodiments, the microbe may be from one of a variety of pathogenic bacterial
species,
specifically including those listed above.
The term "izz vivo viability" refers to the ability of a microbe, e.g., a
bacterium, to survive or grow
in a host, such as an animal. Therefore, an efflux pump inhibitor which
reduces the ifz vivo
viability of a microbe may stop the growth of the microbe and/or kill the
microbe. Such efflux
pump inhibitors, therefore, are antimicrobial agents.
In a further related aspect, this invention includes a method for prophylactic
treatment of an
animal, e.g., a mammal. In this method, an efflux pump inhibitor which reduces
the pathogenicity
of a microbe is administered to a mammal at risk of a microbial infection,
e.g., a bacterial
infection.
2 0 In a related aspect, the invention provides a method for treating a
microbial infection in an
animal, specifically including in a mammal, by treating an animal suffering
from such an infection
with an antimicrobial agent and an efflux pump inhibitor which increases the
susceptibility of the
microbe for that antimicrobial agent. In this way a microbe involved in the
infection can be
treated using the antimicrobial agent in smaller quantities, or can be treated
with an antimicrobial
agent which is not therapeutically effective when used in the absence of the
efflux pump inhibitor.
Thus, this method of treatment is especially appropriate for the treatment of
infections using an
antimicrobial agent alone due to a need for high dosage levels (which can
cause undesirable side
effects), or due to lack of any clinically effective antimicrobial agents.
However, it is also
appropriate for treating infections involving microbes which are susceptible
to particular
3 0 antimicrobial agents as a way to reduce the dosage of those particular
agents. This can reduce the
risk of side effects, but can also reduce the selection effect for highly
resistant microbes resulting
from the consistent high level use of a particular antimicrobial agent. In
particular embodiment
the microbe is a bacterium, which may, for example, be from any of the groups
or species
indicated above. Also in particular embodiments various antibacterial agents
can be used. These
3 5 include quinolones, tetracyclines, glycopeptides, aminoglycosides, beta-
lactams, rifamycins,
coumermycins, macrolides, and chloramphenicol. In particular embodiments an
antibiotic of the
above classes can be, for example, one of the following:
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WO 02/09758 PCT/INO1/00139
Beta-Lactam Antibiotics
Imipenem, meropenem, saneftrinem, biapenem, cefaclor, cefadroxil, cefamandole,
cefatrizine,
cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid,
cefoperazone, ceforanide,
cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin,
ceftazidime, cefteram,
ceftezole, ceftibuten, ceftizoxime, ceftriazone, cefurozime, cefuzonam,
cephaaceterile, cephalexin,
cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine,
cefmetazole, cefoxitin,
cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin,
amoxicillin, amicillin,
azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin,
dicloxacillin, methicilloin,
mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin,
temocillin, ticarcillin,
cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-04.67, BK-
218, FK-037, DQ-
2556, FK-518, Cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000,
LY206763
Macrolides
azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin,
rosaramicin,
roxithromycin, troleandomycin, telithromycin and other ketolides.
2 0 ~uinolones
Amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine,
loMefloxacin, nalidixic
acid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin,
difloxacin, marbofloxacin,
rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin,
trovafloxacin, alatrofloxacin,
grepafloxacin, moxifloxacin, gatifloxacin, gemifloxacin, nadifloxacin,
PD131628, PD140248, (1-35,
AM-1155, NM394, T-3761, rufloxacin, OPC-17116, DU-6859a (identified in Sato,
K. et. al., 1992,
Afatimicrob AgefZts Clae»aother. 37:1491-98), DV-7751a (identified in Tanaka,
M. et. al., 1992
Antin:tcrob Agents Clzemotlzer 37:2212-18).
3 0 Tetracyclines
Chlortetracycline, demeclocyline, doxycycline, lymecycline, methacycline,
minocycline,
oxytetracycline, tetracycline,
3 5 Aminogl~cosides
Amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin,
netilmicin,
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WO 02/09758 PCT/INO1/00139
ribostanycin, sisomicin, spectinomycin, streptomycin, tobramycin, clindamycin,
lincomycin.
Oxazolidinones
Linezolid, eperezolid
Each of the above compounds have been reported in the literature. Other
antibiotic compounds
which may be identified which are effluxed by particular bacteria can also be
utilised with the
efflux pump inhibitors of this invention.
In the context of the response of a microbe, such as a bacterium, to an
antimicrobial agent, the
term "susceptibility" refers to the sensitivity of the microbe for the
presence of the antimicrobial
agent. So, to increase the susceptibility means that the microbe will be
inhibited by a lower
concentration of the antimicrobial agent in the medium surrounding the
microbial cells. This is
equivalent to saying that the microbe is more sensitive to the antimicrobial
agent. In most cases
the minimum inhibitory concentration (MIC) of that antimicrobiaI agent will
have been reduced.
As used herein, the term "treating" refers to administering a pharmaceutical
composition for
prophylactic and/or therapeutic purposes. The term "prophylactic treatment"
refers to treating
an organism such as a human patient, who is not yet infected, but who is
susceptible to, or
2 0 otherwise at risk of, a particular infection. The terms "susceptible" and
"risk" do not refer to the
status of organisms of that type generally, but rather refers to a
significantly enhanced risk. Such
risk may for example be due to a specific exposure to a particular potentially
infective agent, to a
generally weakened physical condition, or immune system deficiency.
Preferably, for humans the
enhanced risk is sufficient such that a prudent doctor familiar with the
treatment of the potential
2 5 infection would find prophylactic treatment medically warranted. The term
"microbial infection"
refers to a disease state or some adverse condition(s), such as the presence
of a pathogenic
microorganism in a body fluid like blood, urine, cerebrospinal or organ
tissue, which are
otherwise sterile or free of pathogenic microorganisms. The term "therapeutic
treatment" refers
to administering treatment to a patient already suffering from an infection.
Thus, in preferred
3 0 embodiments, treating is the administration to a mammal (either for
therapeutic or prophylactic
purposes) of therapeutically effective amounts of a potentiator and an
antibacterial (or
antimicrobial) agent in combination (either simultaneously or serially). The
term "potentiator"
refers to a compound such as an efflux pump inhibitor which has the ability to
increase the
concentration of existing antibiotics in a microbial cell.
By "therapeutically effective amount" or "pharmaceutically effective amount"
is meant an
amount of an efflux pump inhibitor, or amounts individually of an efflux pump
inhibitor and an
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CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
antimicrobial agent, as disclosed for this invention, which have a therapeutic
effect, which
generally refers to the inhibition to some extent of the normal metabolism of
microbial cells
causing or contributing to a microbial infection. The doses of efflux pump
inhibitor and
antimicrobial agent which are useful in combination as a treatment are
therapeutically effective
amounts. Thus, as used herein, a therapeutically effective amount means those
amounts of efflux
pump inhibitor and antimicrobial agent which, when used in combination,
produce the desired
therapeutic effect as judged by clinical trial results and/or model animal
infection studies. In
particular embodiments, the efflux pump inhibitor and antimicrobial agent are
combined in pre-
determined proportions and thus a therapeutically effective amount would be an
amount of the
combination. This amount and the amount of the efflux pump inhibitor and
antimicrobial agent
individually can be routinely determined by one of skill in the art, and will
vary, depending on
several factors, such as the particular microbial strain involved and the
particular efflux pump
inhibitor and antimicrobial agent used. This amount can further depend upon
the patient's
height, weight, sex, age and medical history. For prophylactic treatments, a
therapeutically
effective amount is that amount which would be effective if a microbial
infection existed.
A therapeutic effect relieves, to some extent, one or more of the symptoms of
the infection, and
includes curing an infection. "Curing" means that the symptoms of active
infections are
eliminated, including the elimination of excessive numbers of viable microbes
of those involved in
2 0 the infection. However, certain long-term or permanent effects of the
infection may exist even
after a cure is obtained (such as extensive tissue damage).
The term "microbial infection" refers to the invasion of the host mammal by
pathogenic microbes.
This includes the excessive growth of microbes which are normally present in
or on the body of a
mammal. More generally, a microbial infection can be any situation in which
the presence of a
microbial populations) is damaging to a host mammal. Thus, a mammal is
"suffering" from a
microbial infection when excessive numbers of a microbial population are
present in or on a
mammal's body, or when the effects of the presence of a microbial population
(s) is damaging the
cells or other tissue of a mammal. Specifically, this description applies to a
bacterial infection.
The term "administration" or "administering" refers to a method of giving a
dosage of an
antimicrobial pharmaceutical composition to a mammal, where the method is,
e.g., topical, oral,
intravenous, intraperitoneal, or intramuscular. The preferred method of
administration can vary
depending on various factors e.g., the components of the pharmaceutical
composition, the site of
3 5 the potential or actual bacterial infection, the microbe involved, and the
severity of an actual
microbial infection.
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
The term "mammal" is used in its usual biological sense. Thus, it specifically
includes humans,
cattle, horses, dogs, and cats, but also includes many other species.
This invention also features a method of enhancing the antimicrobial activity
of an antimicrobial
agent against a microbe, in which such a microbe is contacted with an efflux
pump inhibitor, e.g.,
a non-tetracycline specific efflux pump inhibitor, to an efflux pump in the
cell, and an
antibacterial agent. The efflux pump inhibitor is a compound as described
above. Thus, this
method makes an antimicrobial agent more effective against a cell which
expresses an efflux pump
when the cell is treated with the combination of an antimicrobial agent and
efflux pump inhibitor.
In particular embodiments the microbe is a bacterium or a fungus, such as any
of those described
above; the antibacterial agent can be selected from a number of structural
classes of antibiotics
including, e.g., beta-lactams, glycopeptides, aminoglycosides, quinolones,
tetracyclines, rifamycins,
coumermycins, macrolides, and chloramphenicol. In particular embodiments an
antibiotic of the
above classes can be as stated above.
In a further aspect this invention provides pharmaceutical compositions
effective for treatment of
an infection of an animal, e.g., a mammal, by a microbe, such as a bacterium
or a fungus. The
composition includes a pharmaceutically acceptable carrier and an efflux pump
inhibitor as
described above. In preferred embodiments, such compositions contain efflux
pump inhibitors,
2 0 which are themselves effective antimicrobial agents, even in the absence
of another antimicrobial
agent (i.e., have intrinsic antimicrobial activity). Thus, pharmaceutical
composition including
such efflux pump inhibitors can be used either alone or in conjunction with
another antimicrobial
agent. Also in preferred embodiments, the efflux pump inhibitors in
pharmaceutical compositions
of this aspect are efflux pump inhibitors which enhance the effectiveness of
an antimicrobial agent
2 5 other than the efflux pump inhibitor, so such compositions would generally
be used in
combination with such other antimicrobial agent. The invention also provides
pharmaceutical
compositions similarly effective for treatment of an infection of a mammal
which include an efflux
pump inhibitor and an antimicrobial agent. Similarly, the invention provides
antimicrobial
formulations which include an antimicrobial agent, an efflux pump inhibitor,
and a carrier. In
3 0 preferred embodiments, the antimicrobial agent is an antibacterial agent.
A "carrier" or "excipient" is a compound or material used to facilitate
administration of the
compound, for example, to increase the solubility of the compound. Solid
carriers include, e.g.,
starch, lactose, dicalcium phosphate, sucrose, and kaolin. Liquid carriers
include, e.g., sterile
3 5 water, saline, buffers, non-ionic surfactants, and edible oils such as
oil, peanut and sesame oils. In
addition, various adjuvants such as are commonly used in the art may be
included. These and
other such compounds are described in the literature, e.g., in the Merck
Ifadex, Merck & Compasay,
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CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
Rahway, NJ. Considerations for the inclusion of various components in
pharmaceutical
compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and
Gilman's; The .
Pharmacological Basis of Therapeutics, 8t" Ed., Pergamon Press.
In yet another aspect, the invention provides a method of suppressing growth
of a microbe, e.g., a
bacterium, expressing an efflux pump, e.g., a non-tetracycline-specific efflux
pump. As illustrated
by the case where the microbe is a bacterium, the method involves containing
that bacterium with
' an efflux pump inhibitor, e.g., a non-tetracycline-specific efflux pump
inhibitor, in the presence of
a concentration of antibacterial agent below the MIC of the bacterium. This
method is useful, for
example, to prevent or cure contamination of a cell culture by a bacterium
possessing an efflux
pump. However, it applies to any situation where such growth suppression is
desirable.
In another related aspect, the invention provides a method fox reducing a
population of a microbe,
e.g., a bacterial strain, involving contacting the population with an efflux
pump inhibitor which
inhibits a component of an efflux pump expressed in the microbe in that
population, which is
essential for the growth of the microbe expressing that efflux pump. In
particular embodiments,
that component is cytoplasmic membrane component. As indicated above, such
efflux pump
inhibitors may act in various ways, including, but not limited to, acting
directly on the essential
component, or acting to inhibit the expression of that component.
The term "reducing a population" means that the microbes of that population
are being killed.
This is distinguished from the action of a static agent, e.g., a
bacteriostatic agent, which prevents
the bacteria from growing and multiplying but does not kill the microbes.
Accordingly, in the
context of this aspect, an "essential component" of an efflux pump is one
which is essential to the
in vivo survival of the microbe, i.e., the survival in a host.
In yet another aspect, this invention provides a method for enhancing growth
of an animal by
administering an efflux pump inhibitor to the animal, which inhibits an efflux
pump expressed in
a bacterial strain in the animal, and which inhibits the growth of the
bacterial strain. Such a
3 0 growth enhancing effect may result from the reduced energy consumption by
the bacteria, which
increases the food energy available to the animal. This method is appropriate,
for example, for
use with cattle, swine, and fowl such as chickens and turkeys.
In an additional aspect, the invention provides novel compounds having efflux
pump activity.
3 5 These compounds have chemical structures as described above.
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As indicated above, while the present invention is presently exemplified by
activity against
bacteria, compounds of the present invention also have activity against other
microbes, for
example against yeasts and/or other fungi. Thus, the above aspects also
include embodiments in
which described compounds are active or effective against such other microbes.
In further aspect, the invention provides a method of making a pharmaceutical
composition
comprising the steps of identifying an efflux pump inhibitor having a chemical
structure of the
formula I; synthesizing said efflux pump inhibitor and preparing a
pharmaceutical composition
containing said efflux pump inhibitor. The efflux pump inhibitor may have the
chemical structure
as described above. The pharmaceutical composition may also contain one or
more antimicrobial
agents, e.g., as identified above, and one or more carriers, diluents, and
excipients. Further, in
preferred embodiments, the efflux inhibitor compound is active against a
microbe, e.g., a
bacterium, as identified above.
Identification of Efflux Pumn Inhibitors
Identification of efflux pump inhibitors having structures as described for
the present invention
was performed using screening methods known to those skilled in the art of
biological techniques
and are described in detail below. In particular, the screening method based
on inhibition of
2 0 microbial growth in the presence of a subinhibitory concentration of an
antibacterial agent which
is normally effluxed by the test microbe and a concentration of a test
compound was used for
identifying some of the active compounds disclosed herein. In this method,
inhibition of growth of
the microbe is indicative that export of the antibacterial agent is inhibited
by the test compound,
and that the test compound is therefore an efflux pump inhibitor. The mode of
action of the test
compound so identified can then be confirmed as inhibiting active efflux.
However, other
screening methods for detecting efflux pump inhibitors can also be used.
Synthesis of Derivatives of Efflux Pumu Inhibitors from Screening
3 0 The inventors have screened a library of synthetic chemicals and
identified several compounds
that effectively inhibit the respective efflux pumps of Staphylococcus aureus
1199B NorA~,
Streptoc~ccus przeumoniae 3514, Pseudoviofaas aerugitaosa 23587, Escherisclaia
coli 2051. Some of
these compounds were found to be also effective against presently unidentified
multidrug
transporters of other microorganisms.
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The library of compounds was obtained by synthesis according to methods as
described in our
copending applications PCT application PCT/IN99/OOOI6, US applications
09/566,875, 09/640,947,
and 09/850,669 and by methodologies described in a later section below.
Exemplary compounds of the present invention were synthesised by methods as
described in the
examples below. Those skilled in the art will understand how to synthesise
additional compounds
within the scope of this invention based on the described syntheses and the
knowledge of those
skilled in the art of chemical synthesis.
The following examples illustrate methods of screening, which have led to the
identification of
efflux pump inhibitors. They, however, do not Iimit the scope of methods that
can be used for
screening or the kinds of efflux pump inhibitors that can be found by such
varied methods.
Screening of Efflux Pumu Inhibitors - In-vivo
Inhibitors of the bacterial efflux pumps are generally initially characterised
in vitro. Those which
show effective inhibition of the pumps) and which show synergistic activity
with antibiotics are
selected fox evaluation in vivo. Efficacy testing will be done using standard
procedures. Primary
efficacy evaluation may be done using the murine septicemia model (M.G.
Bergeron, 1978, Scand.
2 0 J. Infect. Dis. Suppl. 14:189-206; S.D. Davis, 1975, Antimicrob. Agents
Chemother. 8:50-53). In
this model a supra-lethal dose of bacteria is used to challenge the rodents.
Treatment is initiated,
varying either or both times) of treatment and dose of antibiotic. In these
experiments both the
antibiotic and the efflux pump inhibitor doses are varied. A positive result
is indicated by
significant increase in protection from the lethal infection by the
combination of the potentiator
2 5 (the efflux pump inhibitor) and the antibiotic versus the antibiotic
alone.
A second efficacy model which is used is the mouse soft tissue infection model
(Vogelman et.al.
1988, J. Infect. Dis. 157:287-298). In this model anaesthetised mice are
infected with an
appropriate titer of bacteria in the muscle of the hind thigh. Mice are either
neutropenic
3 0 (cyclophosphamide treated at 125 mg/kg on days -4, -2, and 0) or
immunocompetent. The
infecting dose is commonly 105 - 106 colony forming units per animal.
Treatment with the
combination of the efflux pump inhibitor and/or antibiotics follows infection,
or can occur before
infection. The proliferation (or death) of the bacteria within the thigh
muscle is monitored over
time. Effective combinations show greater activity than the antibiotic alone.
Activity is defined as
3 5 reduction in growth rate of the test bacteria in the murine tissue.
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Pharmaceutical Comuositions and Modes of Administration
The particular c4mpound that is an efflux pump inhibitor can be administered
to a patient either
by itself, or in combination with an antimicrobial, e.g., antibacterial,
agent, or in pharmaceutical
compositions where it is mixed with a suitable carriers) or excipient(s) or
diluent(s). A
combination of an efflux pump inhibitor with an antimicrobial agent can be of
at least two
different types. In one, a quantity of an efflux pump inhibitor is combined
with a quantity of an
antimicrobial agent in a mixture, e.g., in a solution or powder mixture. In
such mixtures, the
relative quantities of the inhibitor and the antimicrobial agent may be varied
as appropriate for
the specific combination and expected treatment. In a second type of
combination an inhibitor
and an antimicrobial agent can be covaIently linked in such manner that the
linked molecules
can be cleaved within the cell. However, the term "in combination" can also
refer to other
possibilities, including serial administration of an inhibitor and other
antimicrobial agent. In
addition, an efflux pump inhibitor and/or another antimicrobial agent may be
administered in
pro-drug forms, i.e. the compound is administered in a form which is modified
within the cell to
produce the functional form. In treating a patient exhibiting a disorder of
interest, a
therapeutically effective amount of an agent or agents such as these is
administered. A
therapeutically effective dose refers to that amount of the compounds) that
results in
amelioration of symptoms or a prolongation of survival in a patient, and may
include elimination
2 0 of a microbial infection.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the
LDso (the dose lethal to 50% of the population) and the EDSO (the dose
therapeutically effective in
50% of the population). The dose ratio between toxic and therapeutic effects
is the therapeutic
index and it can be expressed as the ratio LDSO, EDso, Compounds which exhibit
large
therapeutic indices are preferred. The data obtained from these cell culture
assays and animal
studies can be used in formulating a range of dosage for use in human. The
dosage of such
compounds lies preferably within a range of circulating concentrations that
include the EDSo
3 0 with little or no toxicity. The dosage may vary within this range
depending upon the dosage and
dosage form employed and the route of administration utilised. It is
preferable that the
therapeutic serum concentration of an efflux pump inhibitor should be in the
range of 0.1-100
mcg/ml, more preferably 0.1 - 50 mcg/ml, even more preferably 0.1 - 20 mcg/ml,
even more
preferably 1.0- 50 mcg./ml or most preferably 1.0 - 20 mcg/ml.
For any compounds used in the method of the invention, the therapeutically
effective dose can be
estimated initially from cell culture assays. For example, a dose can be
formulated in animal
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models to achieve a circulating plasma concentration range that includes the
ICSO as determined
in cell culture. Such information can be used to more accurately determine
useful dosage in
humans. Levels in plains may be measured, e.g. by HPLC.
In particular preferred embodiments, the efflux inhibitor in a pharmaceutical
composition has a
structure as shown by the generic structures described above.
The exact formulation, route of administration and dosage can be chosen by the
individual
physician in view of the patients condition. (See e.g. Fingl et.al., in The
Pharmacological Basis of
Therapeutics, 1975, Ch.l, p.1). It should be noted that the attending
physician would know how
and when to terminate, interrupt or adjust administration due to toxicity, or
to organ
dysfunctions. Conversely, the attending physician would also know to adjust
treatment to higher
levels if the clinical response were not adequate, (precluding toxicity). The
severity of the
condition may, for example, be evaluated, in part, by standard prognostic
evaluation methods.
Further, the does and perhaps dose frequency will also vary according to the
age, body weight
and response of the individual patient. A programme comparable to that
discussed above may
be used in veterinary medicine.
Depending on the specific infection being treated, such agents may be
formulated and
2 0 administered systemically or locally. Techniques for formulation and
administration may be
found in Remington's Pharmaceutical Sciences, 19t" ed., Mack Publishing Co.,
Easton, Pa.
(1990). Suitable routes may include oral, rectal, transdermal, vaginal,
transmucosal, or intestinal
administration; parentral delivery, including intramuscular, subcutaneous,
intramedullary,
injections, as well as intrathecal, direct intraventricular, intravenous,
intraperitonial, intranesal,
or intraocular injections just to name a few.
The pharmaceutical compositions are prepared according to conventional
procedures used by
persons skilled in the art to make stable and effective compositions. In the
solid, liquid,
parenteral and topical dosage forms, an effective amount of the active
compound or the active
3 0 ingredient is any amount, which produces the desired results.
For the purpose of this invention the pharmaceutical compositions may contain
the active
compounds of the invention, their derivatives, salts or hydrates thereof, in a
form to be
administered alone, but generally in a form to be administered in admixture
with a
3 5 pharmaceutical carrier selected with regard to the intended route of
administration and
standard pharmaceutical practice. Suitable carriers which can be used are, for
example, diluents
or excipients such as fillers, extenders, binders, emollients, wetting agents,
disintegrants, surface
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active agents and lubricants which are usually employed to prepare such drugs
depending on the
type of dosage form.
Any suitable route of administration may be employed for providing the patient
with an effective
dosage of the compound of the invention, their derivatives, salts or hydrates
thereof. For
example, oral, rectal, parenteral (subcutaneous, intramuscular, intravenous),
transdermal,
topical and like forms of administration may be employed. Dosage forms include
(solutions,
suspensions, etc) tablets, pills, powders, troches, dispersions, suspensions,
emulsions, solutions,
capsules, injectable preparations, patches, ointments, creams, lotions,
shampoos and the like.
Pharmaceutical compositions of the present invention suitable for oral
administration may be
presented as discrete units such as capsules, cachets, or tablets, or aerosol
sprays, each
containing a predetermined amount of the active ingredient, as a powder or
granules, or as a
solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-
water emulsion, or
a water-in-oil liquid emulsion. Such compositions may be prepared by any of
the methods of
pharmacy, but all methods include the step of bringing into association the
active ingredient with
the carrier which constitutes one or more necessary ingredients. In general,
the compositions are
prepared by uniformly and intimately admixing the active ingredient with
liquid carriers or
finely divided solid carriers or both, and then, if necessary, shaping the
product into the desired
2 0 presentation.
The compositions of the present invention include compositions such as
suspensions, solutions,
elixirs, aerosols, and solid dosage forms. Carriers as described in general
above are commonly
used in the case of oral solid preparations (such as powders, capsules and
tablets), with the oral
solid preparations being preferred over the oral liquid preparations. The most
preferred oral
solid preparation is tablets.
Because of their ease of administration, tablets and capsules represent the
most advantageous
oral dosage unit form, in which case solid pharmaceutical carriers are
employed. Examples of
3 0 suitable carriers include excipients such as lactose, white sugar, sodium
chloride, glucose
solution, urea, starch, calcium carbonate, kaolin, crystalline cellulose and
silicic acid, binders
such as water, ethanol, propanol, simple syrup, glucose, starch solution,
gelatine solution,
carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and
polyvinyl
pyrrolidone, disintegrants such as dried starch, sodium alginate, agar powder,
laminaria powder,
3 5 sodium hydrogen carbonate, calcium carbonate, Tween (fatty acid ester of
polyoxyethylenesorbitan), sodium lauryl sulfate, stearic acid monoglyceride,
starch, and lactose,
disintegration inhibitors such as white sugar, stearic acid glyceryl ester,
cacao butter and
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hydrogenated oils, absorption promoters such as quaternary ammonium bases and
sodium
lauryl sulfate, humectants such as glycerol and starch, absorbents such as
starch, lactose, kaolin,
bentonite and colloidal silicic acid, and lubricants such as purified talc,
stearic acid salts, boric
acid powder, polyethylene glycol and solid polyethylene glycol.
The tablet, if desired, can be coated, and made into sugar-coated tablets,
gelatine-coated tablets,
enteric-coated tablets, film-coated tablets, or tablets comprising two or more
layers.
If desired, tablets may be coated by standard aqueous or nonaqueous
techniques.
In moulding the pharmaceutical composition into pills, a wide variety of
conventional carriers
known in the art can be used. Examples of suitable carriers are excipients
such as glucose,
lactose, starch, cacao butter, hardened vegetable oils, kaolin and talc,
binders such as gum arabic
powder, tragacanth powder, gelatin, and ethanol, and disintegrants such as
laminaria and agar.
In molding the pharmaceutical composition into a suppository form, a wide
variety of carriers
known in the art can be used. Examples of suitable carriers include
polyethylene glycol, cacao
butter, higher alcohols, gelatine, and semi-synthetic glycerides.
A second preferred method of administration is parenterally for intramuscular,
intravenous or
2 0 subcutaneous administration.
A third preferred route of administration is topically, for which creams,
ointments, shampoos,
lotions, dusting powders and the like are well suited.
In addition to the common dosage forms set out above, the compounds of the
present invention
may also be administered by controlled release means and/or delivery devices
such as those
described in U S Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123 and
4,008,719; the
disclosures of which are hereby incorporated by reference.
When the pliarmaceutical composition is formulated into an injectable
preparation, in
formulating the pharmaceutical composition into the form of a solution or
suspension, all
diluents customarily used in the art can be used. Examples of suitable
diluents are water, ethyl
alcohol, polypropylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene
sorbitol, and
sorbitan esters. Sodium chloride, glucose or glycerol may be incorporated into
a therapeutic
3 5 agent.
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The antimicrobial pharmaceutical composition may further contain ordinary
dissolving aids,
buffers, pain-alleviating agents, and preservatives, and optionally colouring
agents, perfumes,
flavours, sweeteners, and other drugs.
For topical application, there are employed as non-sprayable forms, viscous to
semi-solid or solid
forms comprising a carrier compatible with topical application and having a
dynamic viscosity
preferably greater than water. Suitable formulations include but are not
limited to solutions,
suspensions, emulsions, creams, ointments, powders, liniments, salves,
aerosols, etc., which are, if
desired, sterilized or mixed with auxiliary agents, e.g. preservatives,
antioxidants, stabilizers,
wetting agents, buffers or salts for influencing osmotic pressure, etc. For
topical application, also
suitable are sprayabIe aerosol preparations wherein the active ingredient
preferably in,
combination with a solid or liquid inert carrier material,
It will be apparent to those skilled in the art that many modifications, both
to materials and
methods may be practised without departing from the purpose and scope of this
invention.
Preparations of intermediates and of exemplary compounds of the invention
The compounds of the present invention may be readily prepared in accordance
with the
2 0 following synthesis schemes, as illustrated in the specific examples
provided. However, those
skilled in the art wfll recognise that other synthetic pathways for forming
the compounds of this
invention can be utilised, and that the following is provided merely by way of
example and is not
limiting to the present invention. It will be further recognised that various
protecting and
deprotecting strategies will be employed which are standard in the art (see
e.g., "Protective
Groups in Organic Synthesis", by Green and Wuts). Those skilled in the art
will recognise that
the selection of any particular protecting group (e.g. amine, hydroxy and
carboxyl protecting
groups) will depend on the stability of the protected moiety with regard to
the subsequent
reaction conditions and will understand the appropriate selection.
3 0 General Procedures for preparing, the compounds of the invention.
A 7-halo quinolone of the structure shown below
3 5 ~ R4 R4a
Rs R2
Halo
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where halo represents F, Cl, or Br, RZ represents COON, COOCH3, COOCZHs, Rz,
R4 and R4a
taken together represents a difluoroboron coordination complex shown as -
C(=O)OB(FZ)...0= or
R2, Rd and Rda taken together represents a diacetoxyboron coordination complex
shown as -
C=O(O)B(OAc)Z and Ri, R4, R4a, Rs, R6, X and Y are as hereinbefore described
is treated with an
appropriate amine of the formula R9RIONH, where R9 and Rlo have the meanings
hereinbefore
described in an organic solvent such as acetone, alcohol, acetonitrile,
dimethyl sulphoxide, N,N-
dimethylformamide preferably acetonitrile or dimethyl sulphoxide, optionally
in the presence of a
base such as triethylamine, pyridine, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN),
diazabicyclo[5.4.0]undec-7-ene (DBi~ preferably triethylamine at 50°C -
120°C, preferably 70°C -
90°C for 4- 24 hr. When RZ represents COOCH3 or COOCZHs or R2, R4 and
Rda represent -
C(=O)OB(Fz)...0= or C=O(O)B(OAc)2 or -C(=O)OB(OAc)2, the product obtained is
hydrolysed
by aqueous alkali preferably sodium hydroxide or a base preferably
triethylamine in solution in a
solvent such as ethanol.
a) The compounds I of the invention which are esters at a carboxylic acid
group may be
prepared by treating the free acid of compounds of formula I in solution in an
appropriate
solvent, preferably N,N-dimethylformamide, with the corresponding halo
compound,
2 0 preferably chloro or bromo-compound, in the presence of a base, preferably
anhydrous
potassium carbonate, at an elevated temperature, preferably 50°C for an
extended period of
time, preferably 6 hours.
b) The compounds of the invention which are esters at a carboxylic acid group
may be
prepared by treating the free acid of compound of formula I in solution in an
appropriate
solvent, preferably N,N-dimethylacetamide, with the corresponding hydroxy
compound, in
the presence of a base, preferably triethylamine, in presence of a catalyst,
preferably 4-N,N-
dimethylaminopyridine, and in the presence of a dehydrating agent, preferably
N,N-
dicyclohexylcarbodimide at an elevated temperature, preferably 100°C
for an extended
3 0 period of time, preferably 24 hours.
c) The compounds of formula I of the invention which are amides at a
carboxylic acid groups
may be prepared by coupling the free acid of compound of formula I with
ammonia or an
appropriate amine or an amino acid appropriately protected at the acid
functionality of the
3 5 amino acids with a protecting group. The -COON protecting groups for amino
acids are
known in the art. Examples of suitable -COOH protecting groups for amino acids
are
methyl, ethyl, t-butyl and benzyl groups. The -COON protecting group is
removed by
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hydrolysis or by hydrogenation. The coupling of a -COON group of compound of
formula
I with the amino group of the amino acid is also known in the art. The
reaction may be
conducted with or without a solvent at a range of temperatures in the presence
of a coupling
agent.
d) The compounds of the invention which are amides at an NH2 or an NH group
are prepared
by coupling the free amino group of a compound of formula I or by coupling the
free NH
bearing compound of formula I with an appropriate acylating agent such as an
acyl
anhydride or an acyl chloride in the presence of a condensing agent such as a
base e.g.
triethylamine or aqueous sodium hydroxide optionally in the presence of a
solvent such as
N,N-dimethyl acetamide at an elevated temperature of 50°C -
100°C for an extended period
of time upto 24 hours. For compound that contain two NH2 groups, two NH groups
or one
NH2 and one NH group, it may be necessary when so desired to use a protecting
group on
the NH2 group or NH group which is desired to remain unreactive. Protecting
groups for
,15 NH2 and NH groups axe well known to those skilled in the art.
e) The compounds of the invention which are esters of a free hydroxy group may
be prepared
by treating the free hydroxy compound of formula I with an organic acid, an
organic dibasic
acid or appropriate N-protected amino acid or polypeptide as defined above.
Nitrogen
2 0 protecting groups are known in the art. Examples of suitable nitrogen
protecting groups are
Cl-C6 acyl, CZ-C6 alkoxycarbonyl optionally substituted benzyloxycarbonyl,
aryloxycarbonyl, silyl, trityl, tetrahydropyranyl, vinyloxycarbonyl, O-
nitrophenylsulfonyl,
diphenylphosphinyl, p-toluenesulfonyI, and benzyI. The nitrogen protecting
group is
removed by methods known in the art such as hydrogenation or hydrolysis. The
ester
25 forming reaction may be conducted with or without a solvent at a range of
temperatures in
the presence of a suitable condensing agent, known to those skilled in the
art.
f) The compounds of the invention which are alkyl ethers of a free hydroxy
group may be
prepared by treating the compound bearing the free hydroxy group with an alkyl
halide in
3 0 an organic solvent in the presence of a base or .a condensing agent at
temperatures upto the
boiling point of the solvent for a period of time upto 24 hours, by methods
known to those
skilled in the art.
g) The compounds of the invention which are mono or dialkyl derivatives of a
free amino group
35 may be prepared by treating the compound bearing the free amino group with
appropritae
molar amounts of an alkylhalide in an organic solvent optionally in the
presence of a base or
a condensing agent at temperatures upto the boiling point of the solvent for a
period of time
upto 24 hours, by methods known to those skilled in the art.
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h) General Method for making aminoacid esters at the 8-(4'-hydroxypiperidine
substituent of
RS-/R-/S-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j] quinolizine-2-carboxylic acid '
RS-/R-/S-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-benzo
[i,j] quinolizine-2-carboxylic acid (lmm, 360 mg), and triethylamine ( 1 mm,
0.145 ml) was
dissolved in dimethylacetamide ( I5 ml). t-Butyloxycarbonyl aminoacid (1.2 mm)
and
dimethylaminopyridine (1.2 mm, 150 mg) were added, followed by
dicyclohexylcarbodiimide
under ice cooling. After 30 minutes at 0°C, the mixture was stirred at
room temperature
overnight. Dicyclohexylurea was filtered and the mixture was diluted with
ethylacetate,
transferred in a separating funnel, washed with 0.5 N hydrochloric acid, 1 N
sodiumbicarbonate and brine,dried over sodium sulphhate and evaporated to give
t-
butoxycarbonylaminoacyloxy derivative of RS-/R-/S-9-fluoro-6,7-dihydro-8- (4'-
hydroxypiperidin-1-yl)..5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-
carboxylic acid
Trifluoroacetic acid (10 ml) was added to the product obtained in the previous
step. After 30
mins at room temperature, the acid was evaporated and the trifluoroacetate
salt was
2 0 precipitated by addition of ether. If need be the product could be
purified by high pressure
liquid chromatography on a C8 or C18 column. Dissolving the trifluoroacetate
salt in 0.1 N
hydrochloride acid and freeze drying provided the hydrochloride salt.
i) General Method for making an amide derivative at the 2-carboxyl substituent
of RS-/R-lS-
9 Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid using an alpha amino acid
RS-/R-/S-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid (lmm, 360 mg), and triethylamine ( 1
mm, 0.145 ml)
3 0 were are dissolved in dimethylacetannide ( 15 ml) . Isobutylchloroformate
(lmm, 0.13 ml)
was added under ice cooling, After 5 min a solution of aminoacid ester
hydrochloride (2
mm) and triethylamine (Z mm, 0.28 ml) in dimethylacetamide (10 m1) was added
followed
by dimethylaminopyridine (1 mm, 125 mg) and the mixture was stirred at room
temperature overnight. The mixture was then dffuted with ethyl acetate,
transferred in a
3 5 separating funnel, washed with 0.5 N hydrochloric acid, 1 N sodium
bicarbonate and brine,
dried over sodiumsulphate and evaporated to give 'the ester of the respective
amide from RS-
/R-/S-9-Fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid.
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The ester was dissolved in methanol, 1 N sodium hydroxide (1.1 eqivalent) was
added. The
mixture was acidified, extracted with ethylacetate, washed with brine , dried
over
sodiumsulphate and evaporated to provide the title compound. If need be the
product may
be purified by high pressure liquid chromatography on a C8 or C18 column.
Optionally,
dissolving the acid in water with one equivalent sodiumbicarbonate followed by
freeze
drying provided the sodium salt of the title compound.
j) The pharmaceutically acceptable acid addition salts of compounds I are
prepared in a
conventional manner by treating a solution or suspension of the free base I
with about one
chemical equivalent of a pharmaceutically acceptable acid. Conventional
concentration and
recrystalisation techniques are employed in isolating the salts. Illustrative
of suitable acids
are hydrochloric, hydrobromic, hydroiodic, sulphuric, sulfamic, sulfonic,
phosphoric, acetic,
lactic, succinic, oxalic, malefic, fumaric, malic, tartaric, citric, ascorbic,
gluconic, benzoic,
cinnamic, methanesulfonic and p-toluenesulfonic acid.
k) The pharmaceutically acceptable cationic salts of compounds of formula I
may be prepared
by conventional methods from the corresponding acids e.g. by reaction with
about one
equimolar amount of a base. Examples of suitable cationic salts are those of
alkali metals
such as lithium, sodium or potassium, alkaline earth metals such as magnesium
or calcium
2 0 or ammonium or organic amines such diethanolamine, N-methylglucamine,
guanidine or
heterocyclic amines such as choline, piperidine, N-methyl-4-hydroxypiperidine,
hydroxyethylpyrrolidine, hydroxyethylpiperidine, morpholine,
hydroxyethylmorpholine,
piperazine, N-methyl piperazine and the like or basic amino acids such as
optically pure or
racemic isomers of arginine, lysine, histidine, tryptophan and the like.
30
The following examples describe the methods of synthesis of the compounds of
the invention.
Some Preferred Compounds of the Invention Displayin~ Inhibition of the Efflux
Pumn of
Staplzydococcus aureus 1199 B (Nor A ~'1
Example 1
1 Ethyl-6-fluoro-1, 4-dihydro -7-(1', 2', 3', 4'-tetrahydroisoauinolin-2-yl)-4-
oxo-auinoline-3-
carboxylic acid.
3 5 A mixture of 1-ethyl-6, 7-difluoro-1, 4-dihydro-4-oxo-quinoIone-3-
carboxylic acid (2.5 g, 8.3
mmole), 1,2,3,4-tetrahydroisoquinoline (1.67 g, 16.7 mmole) and triethylamine
(0.5 ml) in DMSO
(15 ml) was stirred at 140 °C for 24 hr and concentrated in vacuum and
the residue thus obtained
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was triturated with 25m1 water. The solid separated was filtered and dried to
furnish the required
product. Yield 2.0 g (62 %), m.p. 220 °C . CZ1H19FNZO3, m/z 385 (M+1).
Example 2
I-Ethyl-6,8-fluoro-1, 4-dihydro -7-(4'-acetoxypiperidin-1-yll-4-oxo-auinoline-
3-carboxylic acid
A mixture of 1-ethyl-6, 7-difluoro-1, 4-dihydro-4-oxo-quinolone-3-carboxylic
acid (2.5 g, 8.3
mmole), 1,2,3,4-tetrahydroisoquinoline (1.67 g, 16.7 mmole) and triethylamine
(0.5 ml) in
dimethylsulfoxide (15 ml) was stirred at 140 °C for 24 hr and
concentrated in vacuum and the
residue thus obtained was triturated with 25m1 water. The solid separated was
filtered and dried
to furnish the required product. Yield S6%,m.p. 220-22 °C .
CZ1H19FN203, m/z 395 (M+1).
Example 3
1-Ethyl-6,8-fluoro-1, 4-dihydro -7-(4'-~2'-(2'-oxazolidin-1yD ethyl~ piuerazin-
1 yll-4-oxo-guinoline-
3-carboxylic acid.
A mixture of 1-ethyl-6, 7-difluoro-1, 4-dihydro-4-oxo-quinolone-3-carboxylic
acid (2.5 g, 8.3
mmole), 1,2,3,4-tetrahydroisoquinoline (1.67 g, 16.7 mmole) and triethylamine
(0.5 ml) in
dimethylsulfoxide (15 ml) was stirred at 140 °C for 24 hr and
concentrated in vacuum and the
residue thus obtained was triturated with 25m1 water, The solid separated was
fltered and dried
to furnish the required product. Yield 42%, m.p.192-94 °C .
CZIHzaFaNaOs~ m/z 451 (M+1).
Example 4
1-Ethyl-6, 8-difTuoro-1, 4-dihydro -7-floc, 5oc, 6~)-6-amino-3-azabicyclo
13.1.0)-hex-3-yl}-4-oxo-
guinofine-3-carboxylic acid.
The condensation of (loc, 5~c, 6x)-6-t-b~toxycarbonylamino-3-azabicyclo
[3.1.0]-hexane with 1
ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-quinoline-3-carboxylic acid was
carried in similar
manner as described in example 1 which on hydrolysis with conc. HCl furnished
titled product.
3 0 Yield 60 %, m.p 224-26 °C, C1~H1~FZN303, m/z 350 (M+1).
Example 5
5-Amino-1- ethyl -6, 8-difluoro-1, 4-dihydro -7-(3'-amino-5'-methyl nyrrolidin-
1-yl)-4-oxo-
guinoline-3-carboxylic acid.
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The condensation of 5-amino-1-ethyl -6, 7, 8-trifluoro-1, 4-dihydro- 4-oxo-
quinoline-3-carboxylic
acid with 3-amino-5-methyl pyrrolidine in a similar manner as described in
example 1 give the
titled product. Yield 41 %, m.p 238-40 °C, CI~HIgFZNqO3, mlz 393 (M+1).
Example 6
5-Amino-1- ethyl-6, 8-difluoro-1, 4-dihydro -7-(4'-aminopiperidin-1-yl)-4-oxo-
c~uinoline-3-
carboxylic acid.
Tt was prepared according to procedure described in example 5 where 4-
aminopiperidine was
used in place of 3-amino-5-methyl pyrrolidine. Yield 67 %, m.p 204-06
°C, CZ°HZ8FZN4O3, m/z 411
(M+1).
Example 7
5 Amino-1- ethyl -6, 8-difluoro-1, 4-dihydro -7-14'-(acetamido) uineridin-1-
yl~ 4-oxo-auinoline-3-
carboxylic acid.
It was prepared in a similar manner as described in example 5 where 4-
(acetamido) piperidine
was used in place of 3-amino-5-methyl pyrrolidine. Yield (45%), m.p 280-82
°C, C,9H22F2N4~5~ ~z
425 (M+1).
Example 8
5 Amino 1 ethyl 6, 8 difluoro-1 4-dihydro -7-((loc, Soc, 6x)-6'-(N-t-
butoxycarbonyl amino)-3-
azabicyclo (3 1.Ol-hex-3-y1~4-oxo-guinoline-3-carboxylic acid.
It was prepared according to procedure described in example 5 where (lx, Soc,
6oc)-6-(N-t-
butoxycarbonyl amino)-3-azabicyclo [3.1.0]-hexane was used in place of 3-amino-
5-methyl
pyrrolidine and the t-butoxycarbonyl protecting group in the product was
removed using conc.
HCI.. Yield 92 mg (47 %), m.p 220-2Z °C, CZZHz~FzNaU4, m/z 449
(M+1).
3 0 Example 9
1-Cyclonropyl-6-fluoro-1, 4-dihydro-7- (3'-acetamido-5'-methylnyrrolidin-1-yl)-
4-oxo-auinoline-3-
carboxylic acid,
The condensation of 1-cyclopropyl-6-fluoro-7-chloro-1, 4-dihydro- 4-oxo-
quinoline-3-carboxylic
3 5 acid with 3-acetamido-5-methyl pyrrolidine in a similar manner as
described in example 1 give the
titled product. Yield (68%), m.p 270-72 °C, CzoHaaFNs4a~ ~z 388 (M+1).
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Example 10
1-Cycloprouyl-6-fluoro-1, 4-dihydro-7- (3'-amino-5'-methyluyrroIidin-I-yI)-4-
oxo-auinoIine-3-
carbox~ic acid.
It was prepared in a similar manner as described in example 9 where 3-amino-5-
methylpyrrolidin
was used in place of 3-acetamido-5-methyl pyrrolidine to furnish the titled
product. Yield (41 %),
m.p 346 °C, C18H2°FN303, m/z 346 (M+1).
Example 11
1-Cycloprouyl-6-fluoro-1, 4-dihydro- -7-(4'-acetoxypineridin -1-yl)-4-oxo-
quinoline-3-carboxylic
acid.
It was prepared in a similar manner as described in example 9 where 4-acetoxy
piperidine was
used in place of 3-acetamido-5-methyl pyrrolidine. Yield 50 %, m.p 258-60
°C, CZ~Hr~F5N403, m/z
469 (M+1).
Example 12
1-Cyclopronyl-6-fluoro-1, 4-dihydro -7-d4'-(N-dimethylamino)~iperidin-1-yll-4-
oxo-auinoline-3-
carboxylic acid.
It was prepared in a similar manner as described in example 9 where 4-(N-
dimethyl
aminopiperidine was used in place of 3-acetamido-5-methyl pyrrolidine. Yield
59 %, m.p 284-86
°Ca CzWzsI''NzCs~ ~z 403 (M+1)
2 5 Example 13
1-Cyclopropyl-6-fluoro-1, 4-dihydro- -7-(3', 5'-dimethyl uiperidin-1-yl)-4-oxo-
auinoline-3-
carboxylic acid.
It was prepared in a similar manner as described in example 9 where 3,5-
dimethyl piperidine was
3 o used in place of 3-acetamido-5-methyl pyrrolidine. Yield 23 %, m.p 212-14
°C, CZ°H23FN203, m/z
359 (M+1).
Example 14
1-Cycloprouyl-6-fluoro-1,1, 4-dihydro -7-(4'-hydroxy-3', 5'-dimethylpiperidin-
1-yll-4-oxo-auinoline-
3 5 3-carboxylic acid.
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It was prepared in a similar manner as described in example 9 where 4-hydroxy-
3,S-
dimethylpiperidine was used in place of 3-acetamido-5-methyl pyrrolidine.
Yield 38 %, m.p 178-
80 °C, Czo~zsf'NZOa, m/z 375 (M+1).
Example 15
1-Cyclouro~yl-6-fluoro-1, 4-dihydro- -7-(3', 4', 5'-trimethyl piperazin-1-yl)-
4-oxo-auinoline-3-
carboxylic acid.
It was prepared in a similar manner as described in example 9 where 3,4,5-
trimethyl piperazine
was used in place of 3-acetamido-5-methyl pyrrolidine.
Yield 48 %, m.p 200-02 °C, CZ°H24FN3O3, m/z 373 (M+1).
Example 16
1-Cyclopropyl-6-fluoro-1, 4-dihYdro- 7-(3', 5'-dimethyl-4'-ethyl piuerazin-1-
yl)-4-oxo-auinoline-3-
carboxylic acid.
It was prepared in a similar manner as described in example 9 where (3,5-
dimethyl-4-ethyl
piperazine was used in place of 3-acetamido-5-methyl pyrrolidine. Yield 79 %,
m.p 215-20 °C,
C21A26FN3C3~ ~Z 388 (M+1).
Example 17
1-Cyclouropyl-6-fluoro-1, 4-dihvdro-5-methyl- 7- (4'-ethoxypiperidin-1-vl)-4-
oxo-auinoline-3-
carboxylic acid.
2 5 The condensation of 1-cyclopropyl-6, 7-difluoro-5-methyl-1, 4-dihydro- 4-
oxo-quinoline-3-
carboxylic acid with 4-ethoxypiperidine in a similar manner as described in
example 1 give the
titled product. Yield 40 %, m.p 180-82 °C, CZ1FI25FN2C4~ ~Z 388 (M+1).
Example 18
3 0 1-Cyclopropyl-6-fluoro-1, 4-dihydro-5-methyl- 7-(3', 3'-dimethylpiperazin -
1-yl)-4-oxo-auinoline-
3-carboxylic acid.
It was prepared in a similar manner as described in example 17 where 3,3-
dimethyl piperazine
was used in place of 4-ethoxypiperidine. Yield 50 %, m.p 238-42 °C,
CZOH24FN3~3~ m/z 374 (M+1).
Example 19
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1-Cycloprobvl-6-fluoro-8-methoxy-1, 4-dihydro -7-f4' ~N-dimethylamino)-3'-
methylpiperidin-1-
yli-4-oxo-auinoline-3-carboxylic acid.
A mixture of [1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxo-
quinoline-3-carboxylate-
03, 04] difluoroboron chelate (100 mg, 0.29 mmole) and 4-(N-dimethylamino)-3-
methylpiperidine
(100 mg, 0.7 mmole) in acetonitrile (5 ml) was stirred at 80 °C for 6hr
and cooled. Solvent was
removed, borate complex was heated under reflux using triethylamine (0.1 ml)
in ethanol (5 ml),
solvent was removed and solid obtained was purified on silica column to
furnish the required
product. Yield 74 %, C22I~28FN304, m.p.180-82 °C m/z 417 (M+1).
Example 20
1-Cyclopropyl-6-fluoro-8-methoxv-1, 4-dihydro -7-(4'-hydroxy-3'-
isobutylpiperidin-1-yl)-4-oxo-
auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 19 where 4-hydroxy-
3-
isobutylpiperidine was used in place of 4-(N-dimethylamino)-3-
methylpiperidine. Yield (59%),
m.p 190-92 °C, C23H29FN2~5~ ~z 433 (M+1).
Example 21
2 0 1-CyclopropyI-6-fluoro-8-methoxy-1, 4-dihvdro -7-(4'-hvdroxy-3', 3'-
dimethylpiperidin-1-yl)-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 19 where 4-hydroxy-
3, 3
dimethylpiperidine was used in place of 4-(N-dimethylamino)-3-
methylpiperidine. Yield 29 %,
2 5 m.p 240-42 °C, CZIHzsFNzUa, m/z 405 (M+1).
Example 22
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-hydroxy-3', 5'-
dimethylpiperidin-1-yl)-4-
oxo;guinoline-3-carboxylic acid.
It was prepared according to procedure described in example 19 where 4-hydroxy-
3, 5-
dimethylpiperidine was used in place of 4-(N dimethylamino)-3-
methylpiperidine. Yield 27 %,
m.p 230-32 °C, CZIHz~F1V30a, m/z 405 (M+1).
3 5 Example 23
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(3'-methylpiperazin-1-yl)-4-
oxo-guinoline-3-
carboxylic acid.
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It was prepared in a similar manner as described in example 19 where 3-methyl
piperazine was
used in place of 4-(N-dimethylamino)-3-methylpiperidine. Yield (48 %), m.p 260-
61 °C,
Ci9HzzT' NsOd, m/z 375 (M+1).
Example 24
5-Amino-1-cvclopro~yl-6-fluoro-8-methoxy-1, 4-dihydro -7-(cis-4'-amino-3', 5'-
dimethylnineridin-
1-yl)-4-oxo-guinoline-3-carboxylic acid.
The condensation of 5-amino-1-cyclopropyl-6, 7-difluoro -1, 4-dihydro- 4-oxo-
quinoline-3
carboxylic acid with cis-4-amino-3, 5-dimethylpiperidine in a similar manner
as described in
example 1 gave the titled product. Yield 37 %, m.p 248-50 °C,
CZ1HZ~FNdOa, m/z 419 (M+1).
Example 25
1-Cyclonronyl-6,8-difluoro-5-methyl-1,4-dihydro -7-(4'-hydroxy-3'-aminomethvl
piperidin-1-yl)-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 24 where 4-hydroxy-
3-
aminomethylpiperidine was used in place of cis-4-amino-3, 5-
dimethylpiperidine. Yield 42 %, m.p
270-75 °C, CZOII23F2N3~4~ ~z 408 (M+1).
Example 26
5-Amino-1-cyclopronyl-6, 8-difluoro-1, 4-dihydro -7-(5'-amino-2'-methyl-
nyrrolidin-1-yl)-4-oxo-
guinoline-3-carboxylic acid.
2 5 The condensation of 5-amino-1-cyclopropyl-6, 7, 8-trifluoro -1, 4-dihydro-
4-oxo-quinoline-3-
carboxylic acid with 5-amino-2-methyl-pyrrolidine in a similar manner as
described in example 1
gave the titled product. Yield 40 %, m.p 224-30 °C, C18g2oF'2N4C3~ ~z
379. (M+1)
Example 27
3 0 5 Amino 1 cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-13'-(L-Ala-L-Ala
amino) pyrrolidin-1-yl}-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 3-(L-Ala-
L-Ala amino)
pyrrolidine was used in place of 5-amino-2-methyl-pyrrolidine. Yield 50 %, m.p
234-36 °C,
3 5 Cz3HZ~FZN6O5, m/z 506. (M+1)
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Example 28
5-Amino-1-cyclourouyl-6, 8-difluoro-1, 4-dihydro -7-f4'-(N-di-n-
butylamino2pineridin-1-vl)-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 4-(N-di-n-
butylamino)
piperidine was used in place of 5-amino-2-methyl-pyrrolidine. Yield (63%), m.p
>320 °C,
C26~36F2N4~3f ~Z 49I. (M+1)
Example 29
5-Amino-1-cycloprouyl-6, 8-difluoro-1, 4-dihydro -7-f4'-(-t-butoxycarbonyl
amino-L-Ala-L-Alal
aminouiperidin-1-yl~-4-oxo-auinoline-3-carboxylic acid.
A mixture of 5-amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-{4-
aminopiperidin-1-yl}-4-oxo-
quinoline-3-carboxylic acid (200 mg, 0.52 mmole), t-butoxycarbonyl amino-L-Ala-
L-Ala (134 mg,
0.52 mmole) and 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (128 mg
0.52 mmole)
in dry methylene dichloride for 2hr, washed with water (10 ml) and organic
layer was separated,
dried over sodium sulphate and concentrated to give titled product. Yield 54 %
, m.p 164-68 °C;
C29H39F2N6O7~ ~z 622 (M+1).
2 0 Example 30
5-Amino-1-eycl~~ropyl-6, 8-difluoro-1, 4-dihydro -7-(4'- nrouionoxy pineridin-
1-yll-4-oxo-
guinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 4-
propionoxy piperidine
2 5 where was used in place of 5-amino-2-methyl-pyrrolidine.
Yield 46 %, m.p 224-26 °C, CZ1H23FZN3O5, m/z 436. (M+1)
Example 31
S-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(4'-hydroxy-3', 3'-
dimethyl-pineridin-1-yl)-4-
3 0 oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 4 -
hydroxy-3, 3-
dimethylpiperidine was used in place of 5-amino-2-methyl-pyrrolidine
Yield 71 %, m.p 236-38 °C, Cz0H23F2N304~ ~z 408. (M+1)
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Example 32
5-Annino-1-cycIopropyl-6, 8-difluoro-I, 4-dihydro -7-I4'-(1-pyrrolidinyIl
piperidin-1-yl)-4-oxo-
auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 4-(1-
pyrrolidinyl)
piperidine was used in place of 5-amino-2-methyl-pyrrolidine
Yield 59%, m.p 224-26 °C, C22I~26F2N4~3~ ~z 433. (M+1)
Example 33
5-Amino-1-cyclopropvl-6, 8-difluoro-1, 4-dihydro -7-f4'-fN-(uiperidin-4-yl)
aminomethyll-
uiperidin-1-vl)-4-oxo-auinoline-3-carboxylic acid.
It was prepared in a sinnilar manner as described in example 26 where 4 -[N-
(piperidin-4-yl)
aminomethyl]piperidine was used in place of 5-amino-2-methyl-pyrrolidine.
Yield 60 %, m.p 300(d) °C, C24Ii31F2N5C3~ ~z 476. (M+1)
Example 34
5-Amino-1-cyclopropyl-6, 8-difluoro-1, 4-dihydro -7-(N-1-2',2', 6',6'-
pentamethyllpiperidin-4-
y1)methylamino-4-oxo-auinoline-3-carboxylic acid and its salt.
It was prepared in a similar manner as described in example 26 where amino- [N-
1,N-4-2,2,6,6-
hexamethyl]piperidine was used in place of 5-amino-2-methyl-pyrrolidine.
Yield 41 %, m.p 200-02 °C, C24~I32F2N403~ ~z 463. (M+1)
2 5 Example 35
5-Amino-1-cyclopropyl-6,8-difluoro-1, 4-dihydro -7-(3',5'-dimethyl morpholin-1-
yl)-4-oxo-
quinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 3,5-
dimethyl morpholine
3 0 was used in place of 5-amino-2-methyl-pyrrolidine. Yield 30 %, m.p 286-88
°C, C,9H21F2Ns~a~ ~z
4I2. (M+1)
Example 36
5-Amino-1-cyclonrouyl-6, 8-difluoro-1, 4-dihydro -7-(4'- cyclopronyl_piperazin-
1-yl)-4-oxo-
3 5 auinoline-3-carboxylic acid.
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It was prepared in a similar manner as described in example 26 where 4 -
cyclopropyl piperazine
was used in place of 5-amino-2-methyl-pyrrolidine Yield 46%, m.p 260(d)
°C, CzoHzzFzNaOs, m/z
485. (M+1)
Example 37
5-Amino-1-cyclouronyl-6, 8-difluoro-1, 4-dihydro -7-(3', 5'-dimethyl-4-
uivalovl piperazin-1-yl)-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 26 where 3,5-
dimethyl-4-pivaloyl
piperazine was used in place of 5-amino-2-methyl-pyrrolidine. Yield 30%, m.p
290-94 °C,
CzaHzsr' zNa4a~ ~z 472 (M+1).
Example 38
Ethyl 6,8-Difluoro-7-(4-hydroxyuiperidin-1-yl)-1-(1-phenylthio-3 (S1-but-3-yl)-
1,4-dihydro-4-oxo-
auinoline-3-carboxylate
A mixture of ethyl 6,7,8-trifluoro-1-(1-phenylthio-3(~-but-3-yl)-1,4-dihydro-4-
oxo-quinoline-3-
carboxylate (100 mg, 0.229 mmol), 4-hydroxypiperidine (46 mg, 0.45 mmol) and
triethylamine (46
mg, 0.45 mmol) was stirred in acetonitrile (5.0 ml) at 85 °C (bath
temperature) for 12 h. After
2 0 completion of the reaction (monitored by tlc), the solvent was evaporated
under reduced pressure.
Diluted with water (20 ml) and extracted with chloroform (3 x 30 ml) and
evaporated to give the
crude product, which on silica gel column purification using 5 % MeOH in
chloroform gave 100
mg (84 %) of the product as viscous liquid. 1H NMR (200 MHz, CDC13): 81.40 (t,
J= 7.0 Hz, 3H, -
CHZCH3), 1.60 (d, J= 6.0 Hz, 3H, -CHCH3), 1.65-1.81 (m, 4H, 2 x -CHz-),1.95-
2.15 (4H, 2 x -CHz-
2 5 ), 2.70 (quint., 1H, H-C2'), 2.90 (quint, 1H, H-C2'), 3.09-3.10 (m, 2H, -
CHZS-), 3.80-3.98 (m, 1H, -
CHOH), 4.39 (q, 2H, -CHZCH3), 5.20-5.39 (m, 1H, -CHCH3), 7.20 (s, 5H, arom),
7.95 (dd, J =12.5,
2.0 Hz, 1H, H-C5), 8.45 (s,1H, H-C2); ( ESMS txlz 517 (MII-'~,100 %),
Example 39
3 0 1-(2'-Trifluoromethylphenyl) -6-fluoro-1, 4-dihydro- -7-(3', 3',_4' -
trimethvlt~inerazin-1-yl)-4-oxo-
auinoline-3-carboxylic acid
The condensation of with 1-(2'-trifluoromethylphenyl) -6, 7-difluoro-1, 4-
dihydro-4-oxo-quinoline-
3-carboxylic acid 3,3,4 -trimethylpiperazine in a similar manner as described
in example 1 gave
35 the titled product. Yield 24%, m.p 238-42°C, CzaHzsT'aNsOz, m/z 47$.
(M+1)
Example 40
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5-Amino-1- (2'-trifluoromethylphenyl)-6,8-difluoro-1, 4-dihydro -7-(morpholin-
1-yl)-4-oxo-
guinoline-3-carboxylic acid.
The condensation of 5-amino-1-(Z'-trifluoromethylphenyl) -6, 7, 8-trifluoro-1,
4-dihydro-4-oxo-
quinoline-3-carboxylic acid with morpholine in a similar manner as described
in example 1 gave
the titled product. Yield 52 %, m.p 228-30 °C, CzlH,6F5N304, m/z 470.
(M+1)
Example 41
5-Amino-I-~2'-trifluoromethylphenyl) -6, 8-difluoro-1, 4-dihvdro -7-(3',5'-
dimethvlmorpholin-1-
yl)-4-oxo-guinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 39 where 3,5-
dimethyl morpholine
was used in place morpholine. Yield 49%, m.p 270-72 °C, Cz3HzoFsNs~a,
~z 498. (M+1)
Example 42
5-Amino-I- (2'-trifluoromethyluhenyD -6, 8-difluoro-1, 4-dihydro -7-(3',5'-
dimethyl piperizine-1-
yl)-4-oxo-guinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 39 where 3,5-
dimethyl piperazine
2 0 was used in place of morpholine. Yield 60 %, m.p 190(d) °C,
Cz3H21F5N4~3, m/z 497. (M+1)
Example 43
5-Amino-1- (4'-trifluoromethylphenyl) -6, 8-difluoro-1, 4-dihydro -7-(3'-
aminouyrrolidin-I-yl)-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 39 where 3-amino
pyrrolidine was
used in place of morpholine. Yield 50 %, m.p 258-60 °C, Cz1H17F5N4~3,
~z 469 (M+1).
Example 44
1-(4'-FluorophenvlL 6-fluoro-1, 4-dihydro -7-f4'-ethylamino) hiperidin-1-yl)-4-
oxo-auinoline-3-
carboxylic acid.
The condensation of 1-(4'-fluorophenyl) -6, 7-difluoro-1, 4-dihydro -4-oxo-
quinoline-3-carboxylic
acid with 4-ethylaminopiperidine in a similar manner as described in example 1
gave the titled
product. Yield 51 %, m.p 270-72°C, Cz3H23r'2N3~3, ~z 428. (M+1)
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Example 4S
1-(2', 4'-Difluorophenyl) -6-fluoro-1, 4-dihydro-7-( 3', 5'-dimethylpiperidin-
1-yl)-4-oxo-auinoline-
3-carboxylic acid.
The condensation of with 1-(2', 4'-difluorophenyl)-6, 7-difluoro-1, 4-dihydro-
4-oxo-quinoline-3-
carboxylic acid with 3, 5-dimethylpiperidine in a similar manner as described
in example 1 gave
the titled product. Yield 43%, m.p 224-26 °C, Cz3IIz1F3N2~3, ~z 431.
(M+1)
Example 46
5-Amino-1- (2', 4'-difluoronhenyD -6, 8-difluoro-1, 4-dihydro -7-(3'-hydroxy;
5'-methyluyrrolidin-
1-yl)-4-oxo-guinoline-3-carboxylic acid.
The condensation of 5-Amino-1- (2', 4'-difluorophenyl) -6, 7, 8-trifluoro-1, 4-
dihydro -4-oxo-
quinoline-3-carboxylic acid with 3-hydroxy-5-methylpyrrofidine in a similar
manner as described
in example 1 gave the titled product. Yield 64%, m.p °C, CzIHI~F~N3O4,
m/z 453. (M+1)
Example 47
2 0 5-Amino-1- (2',4'-difluorophenyl) -6, 8-difluoro-1, 4-dihydro -7-(3',3'-
dimethvl pinerazin-1-yD-4-
oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 45 where 3,3-
dimethyl piperazine
was used in place of 3-hydroxy-5-methylpyrrolidine. Yield 41 %, m.p 212-14
°C, CzzHzoFaNa~3~
2 5 m/z 465. (M+1)
Example 48
1-Cyclopropyl-6-fluoro-l, 4-dihydro -7-~(3'-aminoethoxycarbonyl)uvrrolidin-3-
yl)-4-oxo-
3 0 nanhthyridine-3-carboxylic acid,
1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-4-oxo-naphthyridine-3-carboxylic acid
(0.1g, 0.32 mmole)
and (3-N-ethoxycarbonyl) aminopyrrolidine (0.14g, 14 mmole) in~acetonitril
(5m1) was stirred at
80 °C for 4 hr and cooled. Solvent was concentrated to dryness. Water
(25 ml) was added to
35 reaction mixture and solid obtained was purified to give the required
product. Yield 23%,
m.p.254-56 °C, C19Hz1FN405, m/z 406. (M+1)
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Example 49
1-Cyclopropyl-6-fluoro-1, 4-dihydro -7-(pyrrolidin-3jy1-aminol-4-oxo-
l,8naphthyridine-3-
carboxylic acid.
It was prepared in a similar manner as described in example 47 where 3-
aminopyrrolidin was
used in place of (3-N-ethoxycarbonyl) aminopyrrolidine. Yield 42%, m.p 122-24
°C, CIgHI~FNqO3,
m/z 333. (M+1)
Example 50
1-(2', 4'-Difluorophenyl) -6-fluoro-1, 4-dihydro -7-(niperidin-4-yl-amino)-4-
oxo-1,8-naphthyridine-
3-carboxylic acid.
Ethyl 1- (2,4-difluorophenyl) -6,7-difluoro-1,4-dihydro-4-oxo-naphthyridine-3-
carboxylate (0.5 g,
1.3 mmole) and (3-N-ethoxycarbonyl) aminopyrrolidine (0.3 g, 1.74 mmole) in
acetonitril (5 ml)
was stirred at 80 °C for 3 hr and cooled. Solvent was concentrated to
dryness and 5% aqueous
sodium hydroxide (lOml) was added to reaction mixture and stirred for 2 hr,
acidified with conc.
hydrochloric acid solid separated was filtered and dried to furnish the
required product. Yield
2 0 74%, m.p >320 °C, CZ°I~17F3N4~3~ ~z 419. (M+1)
Example 51
Ethyl-1- (2', 4'-difluorouhenyl) -6-fluoro-1, 4-dihydro -7-~flx,5x,6oc1-3-N-
benzyl-3-azabicyclo
2 5 L3.1.01 hex-6-vl-amino-4-oxo-1,8-naphthyridine-3-carboxylate and its salt.
Ethyl 1- (2,4-difluorophenyl) -6,7-difluoro-1, 4-dihydro-4-oxo-naphthyridine-3-
carboxylate (2 g,
5.2 mmole) and 6-amino- (lx, 5oc, 6oc)-3-N-benzyl-3-azabicyclo [3.1.0] hexane
(1.16 g, 5.1 mmole)
in acetonitrile (5 ml) was stirred at 80 °C for 3 hr and cooled.
Solvent was concentrated to dryness
and added water (15m1); solid separated was filtered and dried to furnish the
required product.
3 0 Yield 36%, m.p 187-90 °C, C29~25F3N4~3~ ~z 535. (M+1)
Example 52
Ethyl 6-fluoro-7-f 1-Phenyl-4,5,6y7-tetrahvdropyrazolo f 4,3-cl pyridin-1-yll-
1-(2,4-difluorophenyl)-
3 5 4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxyIate
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A mixture of ethyl-1-(2,4-difluorophenyl)- 6,7-difluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-
carboxylate(500 nag, 1.30 mmol), 1-phenyl-4,5,6,7-tetrahydropyrazolo(4,3-
c]pyridine (200 mg, 1.30
mmol) and triethylamine (260 mg, 2.61 mmol) was stirred in acetonitrile (5.0
ml) at 85 °C (bath
temperature) for 6 hr. Solvent was concentrated to dryness. Added water (20
ml) and extracted
with chloroform (3 x 30 ml). Organic layer was separated, dried and removed to
give crude
product which was purified on silica column to give titled product as a
yellowish solid. Yield 70 %,
m.p 85-90°C, ESMS ntlZ S46 (MH+,100 %).
Example 53
(S)-(-)-9-Fluoro-6.7-dihydro-8- (4'-carboxamidopiperidin-1-yl)-5-methyl-1-oxo-
1H, 5H-benzo
fi,ilguinolizine-2-carboxylic acid.
(O-B)-diacetoxy- {S- (-)-8,9-difluoro-5-methyl-6,7-dihydro -1-oxo-1H,5H-
benzo[i,j] quinolizine-2-
carboxylic acid}borane was heated with 4-carboxamidopiperdine in acetonitrile
for 6hr solvent
was removed and borate complex was decomposed using lOml of 5% aqueous sodium
hydroxide
and stirred for 2hr, acidified with conc. hydrochloride. Solid separated was
filtered, dried and
purified on silica column to furnish required product. Yield 34 %, m.p OC,
CZOH22FN304a ~Z 388
(M+1).
2 0 Example 54
(S)-(-)-9-Fluoro-6 7-dihydro-8- f4'-hydroxyuiperidin-I-yI)-5-methyl-I-oxo-
IH,SH-benzofi,ii
guinolizine-2-carboxylic acid L-ar~inine salt.
2 5 ' To a stirred suspension of (S)-(-)- 9-fluoro-6,7-dihydro-8-(4'-
hydroxypiperidin-1-y1)-5-methyl-1-
oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid (5 g, 0.0138 m) in acetone
(50 ml) was added
finely powdered L-arginine (2.51 g, 0.0138 m) then distilled water (20 ml) was
added to it.
Reaction mixture was warmed on water bath and two times water (5 ml) was added
to it at the
interval of 10 min. Clear solution was stirred for lhr. Solvent was removed
under high vacuum
3 0 and solid was dried overnight under high vacuum to furnish title product.
Yield 96%, m.p 234-37
°C~ C25H35FN606~ m/z) 535. (M+1)
Example 55
3 5 (S)-(-)-9-Fluoro-6,7-dihydro-8-(4'-hydroxy-3',3'-dimethvluiueridin-1-yl)-5-
methyl-1-oxo-1H,5H-
benzofi,ilc,Luinolizine-2-carboxylic acid.
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It was prepared in a similar manner as described in example 51 where 4-hydroxy-
3, 3-
dimethylpiperidine was used in place 4-carboxamidopiperdine. Yield 42%, m.p
220 °C,
CziHzsT' NzOaa ~z 389. (M+1)
Example 56
L )-(-)-N-methylniperidino-9-fluoro-6,7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-
methyl-1-oxo-
1H,5H-b enzo f i,i 1 guinolizine-2-carboxylate.
A mixture of (S)-(-) -9-fluoro-6.7-dihydro-8- (4'-hydroxypiperidin-1-yl)-5-
methyl-1-oxo-1H, 5H-
benzo[i,j]quinolizine-2-carboxylic acid a (0.5 g, 1.38 mmole), N,N-
dimethylamino pyridine ( 0.203
g, 1.66 mmole), N,N-dicyclohexylcarbodiimde (0.286 g, 1.38 mmole),
triethylamine (0.2 ml, 1.9
mmole), N-methyl-4-hydroxypiperidine (0.192 g, 1.66 mmole) were dissolved in
N,N-
dimethylacetamide (20 ml) stirred at 100 °C for 24hr. Reaction mixture
was cooled to room temp.
and diluted with ethyl acetate (50 ml). Washed with 0.5N hydrochloride acid
and saturated
sodium bicarbonate, Organic layer was dried over sodium sulphate. Solvent
removed and solid
obtained was purified over silica column to furnish title product. Yield 56 %,
m.p 170-75 °C,
CzsHszT' NsOa, ~z 458. (M+1)
2 0 Example 57
Ethoxy carbonylmethyl fR)-(+)- 9-fluoro-6,7-dihydro-8-(4'-hydroxyp~eridin-1-
yl)-5-methyl-1-
oxo-1H,5H-benzo f i,j 1 guinolizine-2-carboxylate,
A mixture of (R)-(+)- 9-fluoro-6.7-dihydro-8-(4'-hydroxypiperidin-1-yl)-5-
methyl-1-oxo-1H,5H-
benzo[i,j]quinolizine-2-carboxylic acid (0.5 g, 1.388 mmole) and anhydrous
potassium carbonate
(0.192 g, 1.39 mmole) was suspended in dry N,N-dimethylformamide (20 ml)
stirred at 50 °C for
7hr. Ethylbromoacetate (0.35 g, 2.08 mmole) was added and reaction mixture was
stirred for l7hr.
Solvent was removed and extracted with chloroform (25m1 x 2). Organic layer
was dried and solid
3 0 obtained was crystallized from chloroform: hexane (2:8) to furnish the
title product. Yield 71 %,
m.p 130-35 °C, Cz3Hz~FNzOs, m/z 447. (M+1)
Example 58
(S~~-Morpholinoethyl-9-fluoro-6,7-dih~dro-8- (4'-hydroxyniueridin-1-yD-5-
methyl-1-oxo-1H,
5H-benzo (i,il q_uinolizine-2-carboxyliate.
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A mixture of (S)-(-) -9-fluoro-6.7-dihydro-8- (4'-hydroxypiperidin-1-yl)-5-
methyl-1-oxo-1H, 5H-
benzo[i,j]quinolizine-2-carboxylic acid a (0.5 g, 1.38 mmole), N,N-
dimethylamino pyridine ( 0.203
g, 1,66 mmole), N,N-dicyclohexylcarbodiimde (0.286 g, 1.38 mmole),
triethylamine (0.2 ml, 1.9
mmole), N-(2-hydroxyethyl)morpholine (0.218 g, 1.66 mmole) were dissolved in
N,N-
dimethylacetamide (20 ml) stirred at 100 °C for 24 hr. Reaction mixture
was cooled to room temp.
and diluted with ethyl acetate (50 ml). Washed with 0.5N hydrochloride acid
and saturated
sodium bicarbonate. Organic layer was dried over sodium sulphate. Solvent
removed and solid
obtained was purified over silica column to furnish title product. Yield 58 %,
m.p 245-50 °C,
C25g32FN3~5~ ~z 474. (M+1)
Some Preferred Comuounds of the Invention Disulayin~ Inhibition of the Efflux
Pumps of
Pseudonaohas aeru~itzosa 23587 and E.coli 2051
Example 1
1-Cyclopro~pvl-6-fluoro-1, 4-dihydro-5-methyl- 7-(4'-methoxypiueridin -1-yI)-4-
oxo-quinoline-3-
carboxylic acid.
A mixture of 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-5-methyl- -4-oxo-
quinoline-3-carboxylic
acid (500 mg, 1.3 mmole), 4-methoxypiperidin -1-yl (600 mg, 5 mmole) and
triethylamine (0.2 ml)
2 0 in dimethyl sulfoxide (5 ml) was stirred at 140 °C for 24 hr and
cooled. Solvent was concentrated
to dryness triturated with water (25 ml) solid thus separated was filtered and
dried to furnish the
required product. Yield 57 %, m.p 130-38 °C, CZOHa3FN204, m/z 375
(M+1).
Example 2
7-Bromo-1-cyclopropyl-6-fluoro-5-methyl-1, 4-dihydro-4-oxo-guinoline-3-
carboxylic acid and its
salts.
The preparation of the compound is described in PCT WO 89/06649.
3 0 Example 3
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-amino-3'-methylpiperidin-
1-yl)-4-oxo-
guinoline-3-carboxylic acid.
A mixture of [1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-methoxy-4-oxo-quinoline-
3-carboxylate-
03,04]difluoroboron chelate (0.1 g, 0.29 mmole) and 4-amino-3-methyl
piperidine (0.2 g, 1.75
mmole) in acetonitrile (20 ml) was refluxed for 6 hr. The reaction mixture was
concentrated to
dryness. The obtained residue was treated with triethylamine (3 ml) and
ethanol (15 m1) and
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refluxed fox 16 hr. The resulting mixture was concentrated to dryness; the
solid thus obtained was
triturated with water (10 ml), filtered, washed with water, dried and purified
by preparative
HPLC to furnish the required product. Yield 0.04 g (35%), m.p.238-40°C,
C2°H2dFN3Od, m/z 390
o (M+1), PMR (CD30D): 0.84-1.42 (7H, m, 2 X CH2 & CH3), 1.8-2.4 (3H, m, CH &
CH2), 3.02 (1H,
m, N-CH), 3.18-3.72 (4H, m, 2 X N-CH2), 3.8 (3H, S, OCH3), 4.18 (1H, m, c-CH),
7.82 (1H, d, H-5,
J=16 Hz), 8.9 (1H, s, H-2).
Example 4
5-Amino-1-cvclonropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-amino-3'-methyl
pineridin-1-yll-4-
oxo-guinoline-3-carboxylic acid.
The condensation of 5-amino-1-cyclopropyl-6, 7-difluoro-8-methoxy -1, 4-
dihydro- 4-oxo-
quinoline-3-carboxylic acid with 4-amino-3-methylpiperidine was carried out in
a similar manner
as described in example 1, gave the titled product. Yield 50 %, m.p 260-62
°C, C2°H25FN404, m/z
405 (M+1).
Example 5
5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-i3', 3'-dimethyl-4'-
ethylaminopiperidin-1-yl~-4-oxo-~uinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 4 where 4-
ethylamino-3,3-
dimethylpiperidine was used in place of 4-amino-3-methylpiperidine. Yield 54
%, m.p 205 °C,
C23H31FN404~ ~z 447 (M+1).
2 5 Example 6
1-Cyclouropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-amino-3'-3'-
dimethylpiueridin-1-yll-4-oxo-
guinoline-3-carboxylic acid.
1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-{4'-N-benzyloxycarbonyl-amino-
3',3'-
3 0 dimethylpiperidin-1-yl)-4-oxo-quinoline-3-carboxylic acid (5.5 g, 9.3
mmole) was stirred with
Conc. HCl (75 ml) for 2 hr at 30°C, concentrated to dryness, triturated
with acetone and filtered.
The obtained hydrochloride was dissolved in water-methanol, neutralized with
triethylamine (2
ml), concentrated and crystallisation from methanol furnished the required
product. Yield 3.8 g
(31 %), m.p.256-58 °C, C21H2sFN3Oa, m/z 404 (M+1).
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It was prepared in a similar manner as described in Example 3, where 4-annino-
3,3-
dimethylpiperidine was used in place of 4-amino-3-methylpiperidine. Yield 0.24
g (10%), m.p.25$-
60°C, CZ1HZ6I~'N304, m/z 404 (M+1).
Example 7
1-Cyclonrouyl-6-fluoro-8-methoxy-1,4-dihydro-7-~4'-dimethylanninopiueridin-1-
yl~-4-oxo-
auinoline-3-carboxylic acid.
l0 The preparation of the compound is described in US 4822801
Example 8
1-Cycl~~ropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-hydroxy-4'-
methyluineridin-1-yl)-4-oxo-
auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 19 (NorA), where 4-
hydroxy-4-
methylpiperidine was used in place of 4-dimethylamino-3-methylpiperidine Yield
35 %, m.p 220-
24 °C, CZOI323FNzOs, m/z 390 (M+1).
2 0 Example 9
1-~clouroQyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(3', 3'-dimethyl-4'-hydroxy-
niperidin-1-yl)-4-
oxo-guinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 19 (NorA), where 4-
hydroxy-3, 3-
dimethylpiperidine was used in place of 4-dimethylamino-3-methylpiperidine
Yield 56 %, m.p
240-42 °C, CZ1HZSFN205, mlz 404 (M+1).
Example 10
1-Cyclopropyl-6, 8-difluoro-5-methyl-1, 4-dihydro -7-(3'-aminomethyl-4'-
hydroxy- pineridin-1-yl)-
4-oxo-auinoline-3-carboxylic acid.
The condensation of 1-cyclopropyl-6, 7, 8-trifluoro -5-methyl-1, 4-dihydro- 4-
oxo-quinoline-3-
3 5 carboxylic acid with 3-aminomethyl-4-hydroxy-piperidine was carried out in
a similar manner as
described in example 1 gave the titled product. Yield 45.9 %, m.p 270-75
°C, CZOH23FzN3~4~ ~z
408 (M+1).
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Example 11
5-Amino-1- (cyclo~~,ropyl)-6,8-difluoro-1, 4-dihydro-7- (3'-aminomethyl-4'-
hydroxy-piperidin-1-
yl)-4-oxo-auinoline-3-carboxylic acid.
The condensation of 5-amino-1-cyclopropyl-6, 7, 8-trifluoro -1, 4-dihydro- 4-
oxo-quinoline-3-
carboxylic acid with 3-aminomethyl-4-hydroxy-piperidine was carried out in a
similar manner as
described in example 1, gave the titled product. Yield 52.8 %, m.p 202-04
°C, Cl9HzzFzNaCa~ ~z
409 (M+1).
Example 12
1-Ethyl-6, 8-difluoro-1, 4-dihydro -7-(3'-5'-dimethvlpiperazin-1-vl)-4-oxo-
auinoline-3-carboxylic
acid.
The condensation of 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-quinoline-3-
carboxylic acid with
3-5-dimethylpiperazine was carried out in a similar manner as described in
example 1, gave the
titled product. Yield 58 %, m.p 230 °C (d), C1gH22F2N303~ ~z 367 (M+1).
'
2 0 Example 13
1-C~,clopronyl-6-fluoro-1 4-dihvdro-7- (4'-ethyl-3'-metl~lpinerazin-1-vl)-4-
oxo-cruinoline-3-
carboxylic acid.
The condensation of 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-4-oxo-quinoline-3-
carboxylic acid
with 4-ethyl-3-methylpiperazine was carried out in a similar manner as
described in example 1
gave the titled product. Yield 51 %, m.p 200 °C, Cz°Hz4FN303,
m/z 374 (M+1).
Example 14
1-Cyclopropyl-6-fluoro-1 4-dihydro- -7-(3'-5'-dimethyl-4'-ethvlpiperazin-1-yl)-
4-oxo-auinoIine-3-
3 0 carboxylic acid.
It was prepared in a similar manner as described in example 13, where 3-5-
dimethyl-4-
ethylpiperazine was used in place of 4-ethyl-3-methylpiperazine. Yield 79 .%,
m.p 215-20 °C,
C2IH26FN3~3~ ~z 388 (M+1).
d
Example 15
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1-Ethyl-6, 8-difluoro-1, 4-dihydro -7-floc, Sac, 6oc)-6'-amino-3'-azabicyclo
(3.1.01 hex-3-yl~ 4-oxo-
auinoline-3-carboxylic acid.
The condensation of (loc, 5°c, 6oc)-6-t-butoxycarbonylamino-3-
azabicyclo [3.1.0]-hexane with 1-
ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-quinoline-3-carboxylic acid was
carried in similar
manner as described in example 1, which on hydrolysis with cone. HCl furnished
titled product.
Yield 22.4 %, m.p 2243-26 °C, C1~H1~FZN3O3, m/z 350 (M+I).
Example 16
5-Amino-1- (2', 4'-difluorophenyD -6, 8-difluoro-1, 4-dihydro -7-(3-
aminopyrrolidine -1-yl)-4-oxo-
uinoline-3-carboxylic acid.
The condensation of 5-amino-1- (2', 4'-difluorophenyl) -6, 7, 8-trifluoro-1, 4-
dihydro-4- oxo-
quinoline-3-carboxylic acid with 3-aminopyrrolidine was carried out in a
similar manner as
described in example 1, gave the titled product. Yield 71 %, m.p 268 °C
(d), CZOH16F4N4~3~ m/z 437
(M+1).
Example 17
2 0 5-Amino-1- cyclopropyl-6, 8-difluoro-1, 4-dihydro-7- f,(3'-amino
ethoxvcarbonylpyrrolidin-3-yl)-4-
oxo-guinofine-3-carboxylic acid.
The condensation of 5-amino-1- cyclopropyl-6, 7, 8-trifluoro-1, 4-dihydro-4-
oxo-quinoline-3-
carboxylic acid was carried out with 3-amino ethoxycarbonyl pyrrolidine in a
similar manner as
described in example 1 gave the titled product. Yield 68.3 %, m.p 180-84
°C, CZOHzIFaNa~s~ ~z
437 (M+1).
Example 18
3 0 1Cyclopropyl-6-fluoro-1, 4-dihydro -7-(pyrrofidin-3'-ylamino)-4-oxo-
nanhthyridine-3-carboxylic
acid,
Prepared according to example 48 in the list of compounds for the NorA pump.
3 5 Example 19
1-(2', 4'-Difluorouhenyl)-6-fluoro-1, 4-dihydro-7- (niueridin-4'-ylaminol-4-
oxo-naphthyridine-3-
carboxylic acid.
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Prepared according to example 49 in the Iist of compounds for the NorA pump.
Example 20
l~Cyclopropyl-6-fluoro-1, 4-dihydro -7-(4'-amino-3'-ethylpiperidin-1-yl)-4-oxo-
naphthyridine-3-
carboxylic acid.
The condensation of 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-4-oxo-
naphthyridine-3-carboxylic
acid with 4-amino-3-ethylpiperidine was carried out in acetonitrile in a
similar manner as
described in example 1, gave the titled product. Yield 71 %, m.p 218-20
°C, C19H23FN4~3, m/z 375
(M+1).
Example 21
S-(-)-9-Fluoro-6, 7-dihydro-8- (4'-hvdroxynineridin-1-vll-5-methyl-1-oxo-1H,
5H-
benzofi,iloluinolizine-2-carboxylic acid 0.2 hydrate.
S- ( )-9-Fluoro-6, 7-dihydro-8- (4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-IH,
SH benzo[i,jj
quinolizine-2-carboxylic acid was crystallized from aqueous ethanol to furnish
titled product.
Yield 75 %, m.p 244-46 °C, Cl9HziFNz04, m/z 361(M+1).
Example 22
S- (-)-9-Fluoro-6, 7-dihydro-8-(4'-hydroxypiperidin-1-~)-5-methyl-1-oxo-1H,SH-
benzofi,ilguinolizine-2-carboxylic acid, choline salt.
S- (-)-9-Fluoro-6, 7-dihydro-8- (4'-hydroxypiperidin-1-yl)-5-methyl-I-oxo-1H,
5H-benzo[i,j]
quinolizine-2-carboxylic acid (0.5 g,1.388 mmole) was dissolved in methanol
(50 ml) to it was
added 45 % w/w solution of choline base (0.374 ml, L388 mmoIe) and stirred at
50 °C for S hr, and
concentrated to dryness. Yield 80 %, m.p 190-92 °C, Cz4H35FN3~6~ m/z
482 (M+1)
3 0 Example 23
S- (-)-9-Fluoro-6, 7-dihydro-8-(4'-hydroxypiueridin-1-yl)-5-methyl-1-oxo-1H,
5H-
benzofi,ilauinolizine-2-carboxylic acid, 1-hydroxyeth~uyrrolidine salt.
It was prepared according to procedure described in example 22, where 1-
hydroxy
3 5 ethylpyrrolidine (0.156 g, 1.388 mmole) was used in place of choline base.
Yield 78 %, m.p 252 °C,
C25H33FN3~5~ ~z 476 (M+1).
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Example 24
S- (-)-9-FIuoro-6, 7-dihydro-8- (4'-hydroxyniperidin-I-yl)-5-methyl-1-oxo-
1H,SH-
benzofi,ilauinolizine-2-carboxylic acid, diethanolamine salt.
It was prepared according to procedure described in example 22, where
diethanolamine (0.146 g,
1.388 mmole) was used in place of choline base. Yield 57 %, m.p 255-60
°C, Cz3HsiFNsOg, mlz 466
(M+1)
Example 25
(S)-(-)- 9-Fluoro-6.7-dihydro-8- (4'-hydroxypiperidin-1-yl)-5-methyl-1-oxo-
1H,5H-
benzofi,ilauinolizine-2-carboxylic acid (L1 histidine salt.
It was prepared according to procedure described in example 22, where L-
histidine (0.21 g, 1.38
mmole) was used in place of choline base. Yield 98 %, m.p 278-84 °C,
C31H39FN8O8, m/z 671
(M+1)
Example 26
,(RS)-(+)-9-Fuoro-6 7-dihydro-8-14'-(D-phenylalaninoxy) uiperidin-1-yl)-5-
methyl-1-oxo-1H, 5H-
benzofi,ilauinolizine-2-carboxylic acid hydrochloride.
(RS)-(t)-9-Fuoro-6, 7-dihydro-8-(4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,
5H-benzo[i,j]
quinolizine-2-carboxylic acid (360 mg, lmm), and triethylamine (0.145 ml, lmm)
are dissolved in
dimethylacetamide (15 ml. t-Butyloxycarbonyl (D)-phenylalanine (265 mg, 1 mm)
and
dimethylamino pyridine (125 mg, 1 mm) were added, followed by dicyclohexyl
carbodiimide
2 5 under ice cooling. Stirred 30 minutes at 0° C then at room
temperature overnight.
Dicyclohexylurea was filtered and the mixture diluted with ethylacetate,
transferred to a
separating funnel, washed with 0.5 N hydrochloric acid, 1N sodiumbicarbonate
and brine, dried
(sodium sulphate) and evaporated to give the t-butoxycarbonyl protected
derivative.
3 0 Tritluoroacetic acid (10 ml) was added to the derivative obtained, stirred
for 30 wins at room
temp., the acid was evaporated and the trifluoroacetate salt was precipitated
by addition of ether.
Purified by RP-HPLC on a C18 column. Dissolved the trifluoroacetate salt in
0.1 N hydrochloride
acid and freeze-dried provided the titled salt. Yield 75 %, m.p (hygroscopic)
°C, C23H26FN3O~, m/z
543 (M+I)
Example 27
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(RS)-(~)-9-Fluoro-6,7-dihvdro-8-~4'-(L-a-aspartyloxy)piperidin-1-yl~-methyl-1-
oxo-1H,SH-
benzofi,ilauinolizine-2-carboxylic acid hydrochloride.
It was prepared according to procedure described in example 26, where t-
butoxycarbonyl (L)-
asparticacid ( 133 mg, 1 mmole) was used in place t-Butyloxycarbonyl (D)-
phenylalanine Yield 90
%, m.p (hygroscopic) °C, C23H26FN3O7~ m/z 671 (M+1)
Example 28
(RS)-(~)-9-Fluoro-6,7-dihydro-8- f4'-(L-leucyloxy)pineridin-1_yl)-5-methyl-1-
oxo-1H,5H-
benzo~sjlguinolizine-2-carboxylic acid hydrochloride.
It was prepared according to procedure described in example 26, where t-
butoxycarbonyl (L)-
asparticacid ( 131 mg, 1 mmole) was used in place t-Butyloxycarbonyl (L)-
leucine. Yield 43 %,
m.p 166 °C, CZSHsaFlY305, m/z 475.5 (M+1)
Example 29
(S)-(-)-9-Fluoro-6,7-dihydro-8- i4'-(D-leucvloxy)piperidin-1-yl)-5-methyl-1-
oxo-1H,5H-
benzofi,ilauinolizine-2-carboxylic acid hydrochloride.
It was prepared according to procedure described in example 26, where t-
butoxycarbonyl (L)-
asparticacid ( 131 mg, l mmole) was used in place t-Butyloxycarbonyl (D)-
phenylalanine. Yield 94
%, m.p 220-23 °C, CZSH32FN3~5~ m/z 671 (M+1)
Example 30
(S)-(-)-9-Fluoro-6,7-dihydro-8- f4'-(L-alanyloxy) piperidin-1-y1I-5-methyl-1-
oxo-1H,5H-
benzofi.ilauinolizine-2-carboxylic acid hydrochloride.
It was prepared according to procedure described in example 26, where t-
butoxycarbonyl (L)-
aIanine ( 89 mg, I mmole) was used in place t-Butyloxycarbonyl (D)-
phenylalanine. Yield 95 %,
m.p 160-65 °C, CZZH26FN305, m/z 467.5 (M+1),
3 5 Example 31
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(Sl-(-)-Mor~pholinoethyl-9-fluoro-6.7-dihydro-8- (4'-hydroxyniperidin-1-yl)-5-
methyl-1-oxo-1H,
5H-benzo ~i,j 1 guinolizine-2-carboxylate.
Prepared according to example 55 in the list of compounds for the NorA pump.
Example 32
(Rl-(+)-9,8-fluoro-8-fluoro-6, 7-dihydro-5-methyl-1-oxo-1H, 5H-benzo ~i,il
quinolizine-2-
carboxamido uhe-lys-OMe
R-Nadifloxacin (285 mg, 1 mmole), and triethylamine (0.145 ml, lmmole) are
dissolved in
dimethylacetamide (I5 ml) . IsobutylchIoroformate (0.13 ml, 1 mmole) is added
under ice cooling,
sirred for 5 min, a solution of S-Phe-S-Lys (2- chlorobenzyloxycarbonyl 1)-
methylester
trifluoroacetate (0.6 g, 1 mmole) and triethylamine (0.14 ml, lmmole) in
dimethylacetamide (10
ml) was added followed by dimethylaminopyridene (125 mg, 1 mmole) and the
mixture was
stirred at room temperature overnight. The mixture was then diluted with ethyl
acetate,
transferred in a separating funnel, washed with 0.5 N hydrochloric acid, 1N
sodium bicarbonate
and brine, dried (sodium sulphate) and evaporated to dryness. The above
product is dissolved in
methanol- acetic acid -water (15-2-2 ml), and chlorobenzyloxycarbonyl
protecting group was
2 0 removed by bubbling hydrogen in presence of Palladium/charcoal. The
catalyst was filtered and
filtrates evaporated to gave syrup, which solidified on triturating with ether
to gave the titled
compound. The compound was purified by prep-HPLC using water-acetonitrile-0.1
trifluoroacetic acid system to get after freeze drying a white powder as
trifluoro acetate salt. Yield
74 %, m.p 80-82 °C, C3°H3~F2N405' m/z 467.5 (M+1)_
Example 33
(~~~)-9-Fluoro-6, 7-dihydro-8- (trans-4'-hvdroxy-3'-methylpiperidin-1-yD-5-
methyl-1-oxo-1H,
5H-benzofi,ilguinolizine-2-carboxylic acid.
(O-B)-diacetoxy-SRS- (t)-8,9-difluoro-5-methyl-6, 7-dihydro -1-oxo-1H, 5H-
benzo[i,j] quinolizine-
2-carboxylic acid}borane (471 mg, L15 mmole) was heated with trans-4-hydroxy-3-
methylpiperidine (400 mg, 3.47 mmole) in acetonitrile (15 ml) for 6 hr,
solvent was removed, and
borate complex was decomposed using of 5% aqueous sodium hydroxide (lOml) and
stirred for 2
3 5 hr, acidified with conc. hydrochloric acid. Solid separated was filtered,
dried and purified on silica
column to furnish titled product. Yield 66 %, m.p 150-52 °C,
C2°H23FNZO4, m/z 375 (M+1).
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Example 34
(RS)-(t)-9-Fluoro-6, 7-dihvdro-8- (cis-4'-hvdroxy-3'-methylpiperidin-1-yl)-5-
methyl-1-oxo-1H,SH-
benzofi,ilauinolizine-2-carboxylic acid.
It was prepared according to procedure described in example 33, where cis-4-
hydroxy-3-
methylpiperidine was used in place of traps-4-hydroxy-3-methylpiperidine,
Yield 48 %, m.p 146-
48 °C, CZ°HZaFNaOø, m/z 375 (M+1).
Example 35
S-(-)-9-Fluoro-6, 7-dihydro-8- (traps-4'-hydroxv-3'-methylniueridin-1-pll-5-
methyl-1-oxo-1H, 5H-
benzo[i,ilauinolizine-2-carboxylic acid.
It was prepared according to procedure described in example 33, where (O-B)-
diacetoxy-(S)-(-)-
8,9-difluoro-5-methyl-6, 7-dihydro -1-oxo-1H, 5H-benzo[ij] quinolizine-2-
carboxylic acid)borane
was used in place of OB diacetoxy was used in place of (O-B)-diacetoxy-{RS-
(~)-8,9-difluoro-5-
methyl-6, 7-dihydro -1-oxo-1H, 5H-benzo[i,j] quinolizine-2-carboxylic
acid}borane. Yield 42 %,
m.p 184-86 °C, CZ°HzsFN20a, ~z 375 (M+1).
Example 36
7H-Pyridof1,2,3-del-1,4-benzoxazine-9-fluoro-2, 3-dihydro-3-methyl-10- (4'-
hydroxy-3'-
ethyluiperidin-1'-yl)-7-oxo-6-carboxylic acid.
The condensation of 7H-pyrido[1,2,3-de]-1,4-benzoxazine-9, 10-difluoro-2, 3-
dihydro-3-methyl-7-
oxo-6-carboxylic acid with 4-hydroxy 3-ethylpiperidine was carried out in a
similar manner as
described in example 1, gave the titled product. Yield 86 %, m.p 178-80
°C, CZ°Hz3FNaOsa m/z 391
(M+1).
Example 37
10-Fluoro-11- f(lxz5oc, 6x )-6-amino-3-azabicyclof3.l,Olhex-3y11-3,4-dihydro-
4(S)-methyl-8-oxo-
2H,8H-pyrido f 1,2,3-efl-1,5-benzoxaziuin-7-carboxylic acid. HCI.
A mixture of 10,11-Difluoro-3, 4-dihydro-4 (S)-methyl-8-oxo-2H, 8H-
pyrido[1,2,3-de]-1,S-
benzoxazepine-7-carboxylic acid (300 mg, 1.017 mmol), 6-amino-3-azabicyclo
[3.1.0]hexane (201
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mg, 1.017 mmol) and triethylamine (206 mg, 2.03 mmol) was stirred in
acetonitrile (5 ml) at 85 °C
for 96 hr. After completion of the reaction (monitored by tlc), the solvent
was evaporated under
reduced pressure. Diluted with water (20 ml) and extracted with chloroform (4
x 25 ml). The
chloroform extracts were concentrated and the residue was dissolved in 2.0 N
HCl (20 ml) and
washed with ethyl acetate (2x20 ml) to remove the unreacted material if any.
The aqueous layer
was filtered and lyophilized to give titled product. Yield 48 %, m.p. 200-204
°C, C19Hx1C1FN304,
m/z 411 (M+1).
Some Preferred Compounds of the Invention Displayin~ Inhibition of the Efflux
Pumps of E.coli
2051
The compounds listed in Table 3 are common with the respective compounds in
Table 2. Their
preparation is described under the examples for the efflux pump inhibitors
described under
Pseudotraotzas aerugitaosa.
Some preferred Compounds with EPI activity against Mef pumps of Streptococcus
pheun:oitia
3514 and Streptococcus pyoe~etzes 26 00
Example 1
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihvdro -7-(4'-amino-3'-methyl piperidin-
1-yl)-4-oxo-
guinoline-3-carboxylic acid.
It was prepared as described in example 3 in the list of EPIs active against
Pseudomonas
2 5 aeruginosa.
Example 2
5-Amino-1-cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-f4'-(methylamino)-3',
3'-
3 0 dimethylpiperidin-1-yl'r-4-oxo-cruinoline-3-carboxylic acid.
The condensation of 5-amino-1-cyclopropyl-6, 7-difluoro-8-methoxy-1, 4-dihydro-
4-oxo-
quinoline-3-carboxylic acid with 4-methylamino-3, 3-dimethylpiperidine was
carried out in a
similar manner as described in example 1 (NorA), gave the titled product.
Yield 54 %, m.p 250
3 5 °C, Cz2H29FN4~4~ mJz 433 (M+1).
Example 3
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i-Propyl I-cyclopropvl-6-fluoro-8-methoxy-1,4-dihydro -7-f4'-amino-3',3'-
dimethyl- piperidin-1-
yl)-4-oxo-nuinoline-3-carboxylate.
In a dry dimethylformaide (15 ml) was taken a mixture of 1-cyclopropyl-6-
fluoro-8-methoxy-1, 4-
dihydro -7-(4'-t-butoxycarbonylamino-3', 3'-dimethylpiperidin-1-yl)-4-oxo-
quinoline-3-carboxylic
acid (1 g, 1.98 mmole), potassium carbonate (0.275 g, 1.98 mmole) was stirred
at 70 °C for 7hr.
Added 1-iodopropane (0.S g, 2.98 mmole) to reaction mixture and stirred at the
same temperature
for l6hr. Solvent was concentrated up to dryness and (15 ml) water was added
to it and extracted
with chloroform (25 ml x 2). Organic layer was separated, dried, concentrated
and the residue was
hydrolysied with trifluoroacetic acid in methylene dichloride and purified on
silica column to
furnish titled product. Yield 45 %, m.p I40-42 °C, Cz4HsaFNsO4, ~z 446
(M+1).
Example 4
n-Butyl I-cycIopropyl-6-fluoro-8-methoxy-I,4-dihydro -7-(4'-amino-3' 3'-
dimethyl-p_iperidin-1-yl)-
4-oxo-auinoline-3-carboxylate.
2 0 It was in a similar manner as described in example 3, where 1-iodobutane
was used in place of 1-
iodopropane.Yield 55 %, m.p 120-25 °C, CZSfIsaFN30a, m/z 460 (M+1).
Example 5
Ethoxvcarbony methyl I-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-
amino-3', 3'-
dimethylpiperidin-I-yl)-4-oxo-auinoline-3-carboxylate.
It was in a similar manner as described in example 3, where bromo ethyl
acetate was used in place
3 0 of 1-iodopropane.Yield 87 %, m.p 135-40 °C, CZSHszFNs06, m/z 489
(M+1).
Example 6
Benzyl 1-cyclopropvl-6-fluoro-8-methoxy-1, 4-dihydro -7-f4-N-t-butoxycarbonyl)-
amino-3', 3'-
3 5 dimethylpiperidin-1-vl)-4-oxo-guinoline-3-carboxylate.
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It was in a similar manner as described in example 3, where benzyl bromide was
used in place of
1-iodopropane. Yield 67 %, m.p 152-54 °C, C33I~OFN306, m/z 594 (M+1).
Example 7
1-Cvclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-N-(t-butoxycarbonyl-L-
alanyl)-amino-3', 3'-
dimethylpiperidin-1-yI~-4-oxo-auinoline-3-carboxylic acid hydrochloride.
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-}4'-amino-3', 3'-
dimethylpiperidin-1-yl}-4-oxo-
quinoline-3-carboxylic acid(1.0 gm, 2.48 mmole), N- t-butoxycarbonyl-(L)-
alanine (0.94 gm, 4.96
mmole) and 2-ethoxy-N-ethyoxycarbonyl dihydro quinoline (EEDQ) (061 gm, 2.48
mmole) were
dissolved in dichromethane (300 mI)and the mixture is stirred at room
temperature for 48 hr. The
mixture is washed with water, separate the layer, dried and concentrated.
Product purified by
silica gel column to furnish titled product. Yield 84 %, m.p 170-75 °C,
C29H39FN4O~, mlz 575
(M+1).
Example 8
1-Cyclopropyl-6-fluoro-8-methoxv-1, 4-dihydro -7-(4'-(L)-alaninvlamino-3', 3'-
dimethylpiperidin-
1-yl)-4-oxo-auinoline-3-carboxylic acid hydrochloride.
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-(N-t-butoxycarbonyl-L-
alanyl) amino-3', 3'-
dimethylpiperidin-1-yl}-4-oxo-quinoline-3-carboxylic acid (0.9 gm ) is
hydrolysed with 6N HCl (20
ml) at room temperature for 1 hr. The mixture is concentrated to furnish the
titled product. Yield
75 %, m.p 245-50°C, C24HsiF''N405.HC1 m/z 475 (M+1).
Example 9
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(3', 3'-dimethyl-4'-N-(t-
butoxycarbonyl-L-
valyl)-aminopiperidin-1-y11~-4-oxo-auinoline-3-carboxylic acid and its salts.
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-amino-3', 3'-
dimethylpiperidin-1-yl}-4-oxo-
quinoline-3-carboxylic acid(1.0 gm, 2.48 mmole), N- t-butoxycarbonyl -(L)-
valine (1.07 gm, 4.96
mmole) and 2-ethoxy-N-ethyoxycarbonyl dihydro quinoline (EEDQ) (0.61 gm, 2.48
mmole) were
dissolved in dichromethane (300 ml)and the mixture is stirred at room
temperature for 48 hr. The
mixture is washed with water, separate the layer, dried and concentrated.
Product purified by
silica gel column to furnish titled product. Yield 37 %, m.p 80-82 °C,
C31H43FN4O~, m/z 603
(M+1).
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Example 10
1-Cyclouropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(3',_3'-dimethyl-4'-L-valinyl
aminopiperidin-I-
yl~-4-oxo-auinoline-3-carboxylic acid hydrochloride.
I-CycIopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-(N-t-butoxycarbonyl-L-
vaIyl) amino-3', 3'-
dimethylpiperidin-1-yl~-4-oxo-quinoline-3-carboxylic acid (0.4 gm) is
hydrolysed with 6N
hydrochloride acid (20 ml) at room temperature for 1 hr. The mixture is
concentrated to furnish
the titled product. Yield 75 %, m.p 150-55°C, CZSH3sFNaOs~HCI m/z 503
(M+1).
Example 1I
1 ~Cyclouropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-(4'-(L)-aspartylamino-3~, 3'-
dimethvl piueridin-
1-yl~-4-oxo-guinoline-3-carbox~ c acid hydrochloride.
1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-amino-3', 3'-
dimethyIpiperidin-I-yl}-4-oxo-
quinoline-3-carboxylic acid(1.0 gm, 2.48 mmole), N-Boc-L-aspartic acid (1.15
gm, 4.96 mmole)
and 2-ethoxy-N-ethyoxycarbonyl dihydro quinoline (EEDQ) (0.61 gm, 2.48 mmole)
were dissolved
2 0 in dichromethane (300 m1)and the mixture is stirred at room temperature
for 48 hr. The mixture
is washed with water, separate the layer, dried and concentrated. Which was
hydrolysed with 6N
hydrochloric acid and at room temperature for 1 hr and concentrate to furnish
titled product.
Yield 54 %, m.p 250-55 °C, Ca5H3iFNaO~.HC1 m/z 618 (M+1).
2 5 Example 12
I-Ethyl-6, 8-difluoro-I, 4-dihydro-7- (4'-ethylaminoniperidin-1'-yl)-4-oxo-
auinoline-3-carboxylic
acid and its salts.
3 0 It was prepared in a similar manner as described in example 5 (NorA),
where 4-ethyl
aminopiperidine was used in place of 3-amino-5-methylpyrrolidine. Yield 44 %,
m.p 260-62 °C,
C19~24F2N4~3~ ~z 395 (M+1)
Example 13
5-Amino-1- cyclopropyl-6, 8-difluoro-1, 4-dihydro-7- (4'-amino-3'-
methylpiperidin-1-yll-4-oxo-
auinoline-3-carboxylic acid.
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It was in a similar manner as described in example 26 in the list of EPIs
active against NorA,
where 4-amino-3-methylpiperidine was used in place of 5-amino-2-
methylpyrrolidine.Yield 55 %,
m.p 228-30 °C, C19H22F2N4C6a ~z 393 (M+1).
Example 14
5-Amino-1-cyclonropyl-6, 8-difluoro-1, 4-dihydro-7-(3'-auinuclidinyl-3'-yl-
amino)-4-oxo-
auinoline-3-carbolic acid and its salts.
It was prepared in a similar manner as described in example 26 in the list of
EPIs active against
NorA, where 3-aminoquinuclidine was used in place of 5-amino-2-methyl-
pyrrolidine. Yield 53 %,
m.p 280-82 °C, CZOH22F2N4C3~ ~z 404 (M+1)
Example 15
5 Amino 1-cyclourouvl-6, 8-difluoro-1, 4-dihydro-7- ~(loc, 5oc, hoc)-6-amino-N-
benzyl-3-azabicyclo
[3.1.01 hex-6-yll-4-oxo-auinoline-3-carboxylic acid and its salts.
It was prepared in a similar manner as described in example 26 in the list of
EPIs active against
NorA, where {(lxa 5oc, 6x)-6-amino-N-benzyl-3-azabicyclo [3.1.0] hexane was
used in place of 5-
amino-2-methyl-pyrrolidine. Yield 75 %, m.p 270-72 °C, CzSHzaFzNaCs, ~z
467 (M+1)
Example 16
1-(3'-Fluorophenyl)-6-fluoro -1 4-dihydro -7-(4'-methylpiperazin-1'-yl)-4-oxo-
auinoline-3-
carboxylic acid and its salts.
The condensation of 1-(3'-fluorophenyl) -6, 7-difluoro-1, 4-dihydro -4-oxo-
quinoline-3-carboxylic
acid with 4-methylpiperazine in a similar manner as described in example 1 in
the list of EPIs
3 0 active against NorA, gave the titled product. Yield 58 %, m.p 298-02
°C, Cz1H19FzNs03, mlz 400.
(1vI+1)
Example 17
1-(2', 4'-Difluoronhenyl)-6-fluoro-1, 4-dihydro -7-(4'-ethylaminonineridin-1'-
yll-4-oxo-auinoline-3-
carboxylic acid .
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It was in a similar manner as described in example 45 in the list of EPIs
active against NorA,
where 4-ethylamino piperidine was used in place of 3,3-
dimethylpiperidine.Yield 62 %, m.p 240-
42°C, C23IiZ2h3N30g, m/z 446 (M+1).
Example 18
1-~2', 4'-Difluoronhenyp-6-fluoro-5-methyl-1, 4-dihydro -7-(4'-aminopiperidin-
1'-yD-4-oxo-
c~uinofine-3-carboxylic acid and its salts.
The condensation of 1- (2, 4-difluorophenyl) -6, 7, 8-trifluoro -5-methyl-1, 4-
dihydro- 4-oxo-
quinoline-3-carboxylic acid with 4-aminopiperidine in a similar manner as
described in example 1
in the list of EPIs active against NorA, gave the titled product. Yield 58 %,
m.p 236-38 °C,
C22H20F3N3O3~ ~z 432 (M+1)
Example 19
1-(2', 4'-DifluoronhenyD-6-fluoro-5-methyl-1, 4-dihydro -7-(4'-
methylaminopiperidin-1'-yl)-4-oxo-
auinoline-3-carboxylic acid and its salts.
2 0 It was prepared in a similar manner as described in example 18, where 4-
methylamino piperidine
was used in place of 4-aminopiperidine. Yield 52%, m.p 204-06 °C,
C23H22F3N3~3, ~z 346 (M+1)
Example 20
2 5 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7- (3'-aminouyrrolidin-3'-vl)-4-oxo-
1,8-naphthyridine-3-
carboxylic acid.
It was prepared in a similar manner as described in example 47 in the list of
EPIs active against
NorA, where 3-amino pyrrolidine was used in place of 3-amino
ethoxycarbonylpyrrolidine.Yield
3 0 47 %, m.p 120-22 °C, C16g17FN4o3~ m/z 323 (M+1)
Example 21
1-Cyclopropyl-6-fluoro-1, 4-dihydro-7-1(Ioc, Soc, hoc)-6-amino-N-benzvl-3-
azabicyclo f3.1.01 hex-6-
3 5 yl~-4-oxo-1,8-naphthyridine-3-carboxylic acid ,
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It was prepared in a similar manner as described in example 47 in the list of
EPIs active against
NorA, where {(Ioc, Soc, hoc)-6-amino-N-benzyl-3-azabicyclo [3.1.0] hexane was
used in place of 3-
amino ethoxycarbonylpyrrolidine Yield 67 %, m.p 290-92 °C, C24H23FN403,
m/z 434 (M+1)
Example 22
1-(2,4-DifluorophenyI) -6-fluoro-I, 4-dihvdro-7-(3'-aminouyrrolidin-3'-yl)- 4-
oxo-1,8-
naphthyridine-3-carboxylic acid.
It was prepared in a similar manner as described in example 49 in the fist of
EPIs active against
NorA, where 3-amino pyrrolidine was used in place of 4-amino
ethoxycarbonylpiperidine. Yield
97 %, m.p 268-70 °C, C21H19F3N4~3~ ~z 433 (M+1)
Example 23
1-(2,4-Difluorouhenyl) -6-fluoro-1, 4-dihydro-7- f (1°c, 5oc, 6x)-6-
amino-N-benzyl-3-azabicyclo
(3.1.01 hex-6-yl)- 4-oxo-1,8-nauhthyridine-3-carboxylic acid.
It was prepared in a similar manner as described in example 49 in the list of
EPIs active against
NorA, where {(loc, Soc, 6oc)-6-annino-N-benzyl-3-azabicyclo [3.1.0] hexane was
used in place of 4-
2 0 amino ethoxycarbonypiperidine. Yield 74 %, m.p 236-38 °C,
CZ~H21F3Nd~3~ ~z 507 (M+1)
Example 24
1-(2,4-Difluorophenyl) -6-fluoro-1, 4-dil~dro-7- (3', 3'-dimethyl-4'-
hydroxyuiperdin-1-yl)-4-oxo-
nanhthyridine-3-carboxylic acid.
It was prepared in a similar manner as described in example 49 in the Iist of
EPIs active against
NorA, where 4-hydroxy-3', 3'-dimethyIpiperidine was used in place of 3-amino
ethoxycarbonylpyrrolidine Yield 76 %, m.p 194-98 °C, C22H20F3N3~4~ ~z
448 (M+1)
Example 25
(RS)-(t)-9-Fluoro-6, 7-dihydro-8- (4'-(L-a-aspartyloxypiueridin-1-yll-5-methyl-
1-oxo-1H, SH-
benzofi,ilauinolizine-2-carboxylic acid hydrochloride.
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Tt was prepared in a similar manner as described in example 51 in the list of
EPIs active against
NorA, where 4-(L-oc-aspartyloxy) piperidine was used in place of 4-
carboxamidopiperidine.Yield
68 %, m.p 210-12 °C, Cz3HzsFN30~, m/z 513 (M+1).
Example 26
7H-Pyridofl,2,3-del-1,4-benzoxazine-9-fluoro-2, 3-dihydro-3-methyl-10- (4'-
hydroxv-3'-
ethylpiperidin-1'-yD-7-oxo-6-carboxylic acid.
Tt was prepared as described in example 35 in the list of EPIs active against
Pseudoznozzas
aeruginosa.
Example 27
7H-Pyrido(1,2,3-de)-1,4-benzoxazine-9-fluoro-2, 3-dihydro-3-methyl-10- (3'-
amino methyl-4'-
hydroxyuiueridin-1'-yD-7-oxo-6-carboxylic acid.
It was prepared in a similar manner as described in example 26, where 3-
aminomethyl-4-
hydroxypiperidine was used in place of 4-hydroxy-3-ethylpiperidine. Yield 69
%, m.p 278-80 °C,
C19~22FN3~5~ ~z 392 (M+1).
Example 28
1-CyclouroQyl-6, 8-difluoro-5-methyl-1, 4-dihydro -7-(3', 3'-dimethyl-4'-
ethylaminouiperidin-1-yl)-
4-oxo-auinoline-3-carboxylic acid.
,
It was prepared in a similar manner as described in example 10 of
Pseudoznozzas aerugizzosa,
where 3, 3-dimethyl-4-ethyIaminopiperidine was used in place of 3-aminomethyl-
4-
hydroxypiperidine. Yield 52 %, m.p 156-58 °C, C23H30FN3C3~ ~z 415
(M+1).
3 0 Example 29
1-cvclonrouyl-6,7, 8-trifluoro-5-methyl-1, 4-dihydro - 4-oxo-auinoline-3-
carboxylic acid.
~uowzz compound (PCT WO 89/06649)
3 5 Example 30
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WO 02/09758 PCT/INO1/00139
(S)-~)-9-Fluoro-6.7-dihydro-8- (3', 3'-dimethyl-4'-ethvlaminoniperidin-1-vll-5-
methyl-1-oxo-1H,
5H-benzofi,~lguinolizine-2-carboxylic acid.
It was prepared in a similar manner as described in example 51 in the list of
EPIs active against
NorA, where 3, 3-dimethyl-4-ethylaminopiperidine was used in place 4-
carboxamidopiperidine.
Yield 51%, m.p 302-04 °C, C23H3°FN3U3, tn/z 415. (M+1)
Example 31
1-Cvcloprouyl-6-fluoro-1, 4-dihydro-7- (3'-aminomethyl-4'-hvdroxyuiperidin 1-
yl)-4-oxo-
guinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 13 in the list of
EPIs active against
Pseudomouas aerugiuosa, where 3-amino methyl-4-hydroxypiperidin was used in
place 4-ethyl-3-
methylpiperazine. Yield 63%, m.p 230-34 °C, C18HZ1FN404, m/z 378. (M+1)
Example 32
1-C,~clopropyl-6-fluoro-1 4-dihydro-7- (4'-dimethylamino-3'-methyluiperidin-1-
yl)-4-oxo-
gninoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 13 in the list of
EPIs active against
Pseudomouas aerugiuosa, where 4-dimethylamino-3- methylpiperidine was used in
place 4-ethyl-3-
methylpiperazine. Yield 41%, m.p 180-88 °C, CZOH24FN303, m/z 374. (M+1)
2 5 Example 33
5 Amino-1- cyclopronyl-6-fluoro-8-methoxy-1, 4-dihydro-7- (4'-
cyclouropyla~nogi~eridin-1-yl)-
4-oxo-guinoline-3-carboxylic acid.
3 0 It was in a similar manner as described in example 24 in the list of EPIs
active against NorA,
where 4-cyclopropylamino piperidine was used in place of 3,3-
dimethylpiperidine.Yield 65 %,
m.p 218-20°C, C23H29F2N4~4~ ~z 445 (M+1).
Example 34
1-Cyclonrowl-6-fluoro-8-method-1, 4-dihydro -7-~-4'-(t-butoxycarbonyl (L)-Ala-
Ala) amino 3',
3'-dimethylpiperidin-1-yl)-4-oxo-guinoline-3-carboxylic acid
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1-Cyclopropyl-6-fluoro-8-methoxy-1, 4-dihydro -7-{4'-amino-3', 3'-
dimethylpiperidin-1-yl}-4-oxo-
quinoline-3-carboxylic acid (1.0 gm, 2.48 mmole), 4-(N-t-butoxycarbonyl)-L-
alanine (0.94 gm, 4.96
mmole) and 2-ethoxy-N-ethyoxycarbonyl dihydroquinoline (EEDQ) (0.61 gm, 2.48
mmole) were
dissolved in dichromethane (300 ml) and the mixture is stirred at room
temperature for 48 hr. The
mixture is washed with water, separate the layer, dried and concentrated.
Product purified by
sifica gel column to furnish titled product. Yield 84 %, m.p. 170-75
°C, C29H39FN4~7a ~z 575
(M+1).
Example 35
5-Amino-1-cycloprouyl-6-fluoro-8-methoxy-1, 4-dihydro -7-f4'-ethylamino-3', 5'-
dimethylniueridin-1-yl)-4-oxo-auinoline-3-carboxylic acid.
It was prepared in a similar manner as described in example 2, where 4-
ethylamino-3, 5-
dimethylpiperidine was used in place of 4-methylamino-3, 3-dimethylpiperidine.
Yield 38 %, m.p
255-58 °C, ~23Ii31FN4~4~ m/z 447 (M+1)
Example 36
2 0 Ethyl 1-(2,4-difluoronhenyl) -6-fluoro -1, 4-dihydro-7- (4-amino-3-
ethylvpiueridin-1-yl)- 4-oxo-1,8-
nanhthyridine-3-carboxylate
It was prepared in a similar manner as described in example 49 in the list of
EPIs active against
NorA, where 4-amino-3-ethylpiperidine was used in place of 4-amino
ethoxycarbonylpiperidine.
2 5 Yield 48 %, m.p 178-80 °C, Ca4H25F3N40a, m/z 475 (M+1)
Example 37
1-(2,4-difluorophen~l) -6-fluoro-1, 4-dihydro-7- (4-amino-3, 5-
dimethyluiperidin-1-yl)- 4-oxo-1,8-
3 0 nanhthyridine-3-carboxylic acid.
It was prepared in a similar manner as described in example 49 in the list of
EPIs active against
NorA, where 4-amino-3, 5-dimethylpiperidine was used in place of 4-amino
ethoxycarbonylpiperidine. Yield 73 %, m.p 180-82 °C, C22H21F3N4~3a ~z
447 (M+1)
Example 38
82
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Ethyl 1-(2,4-difluoronhenyl)-6-fluoro-5-methyl-1, 4-dihydro-7- (4-amino-3, 3-
dimethylniperidin-1-
yl)- 4-oxo-1,8-nauhthyridine-3-carboxylate
It was prepared in a similar manner as described in example 49 in the list of
EPIs active against
NorA, where 4-amino-3,3-dimethylpiperidine was used in place of 4-amino
ethoxycarbonylpiperidine. Yield 67 %, m.p 148-52 °C, CZSHZ~F3N403, m/z
489 (M+1)
Example 39
(S)-(-)-9-Fluoro-6.7-dihydro-8-(4'-hydrox 3'-fluoroniperidin-1-yl)-5-methyl-1-
oxo-1H, 5H-
benzofi,jlguinolizine-2-carboxylic acid.
It was prepared in a similar manner as described in example 51 in the list of
EPTs active against
NorA, where 4-hydroxy-3-fluoropiperidine was used in place 4-
carboxamidopiperdine. Yield 46
%, m.p 180 °C, C19H~°F2N2O4, m/z 379 (M+1)
Example 40
10-Fluoro-11- (4-aminoniperidin-1-yl)-3,4-dihydro-4(S)-methyl-8-oxo-2H,8H-
pyridof1,2,3-ef1-1,5-
benzoxazepin-7-carboxylic acid, hydrochloride.
It was prepared in a similar manner as described in example 36 in the list of
EPIs active against
Pseudo~raostas aefugihosa, where 4-amino piperidine was used in place [(loc,
5oc, 6x)-6-amino-3-
azabicyclo [3.1.0] hexane. Yield 56 %, m.p 208-10 °C, C,9H23C1FN3O4,
m/z 413 (M+1).
2 5 Example 41
(RS)-(t)-6, 7-dihydro-8- (traps-4'-hydroxy-3'-methylpiperidin-1-yl)-5-methyl-1-
oxo-1H, 5H-
benzofi,,j]guinolizine-2-carboxylic acid.
3 0 It was prepared in a similar manner as described in example 32 in the list
of EPIs active against
Pseudonzoftas aerugifiosa, Yield 59 %, m.p 152-54 °C,
CZ°H24N2~a~ ~z 356 (M+1).
Example 42
3 5 (RS)-(f)-6, 7-dihydro-8- (cis-4'-hydroxy-3'-methylpiperidin-1-yl)-5-methyl-
1-oxo-1H, 5H-
benzo[i,jlguinolizine-2-carboxylic acid.
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It was prepared in a simitlar manner as described in example 32 in the list of
EPIs active against
Pseudonaouas aerugiuosa, Yield 62 %, m.p I62-64 °C, CzogzaNz~a, m/z 356
(M+1).
Example 43
(RS)-(f)-6, 7-dihydro-8- (4'-hydroxy-3', 3'-dimethylait~eridin-1-yl)-5-methyl-
1-oxo-1H, 5H-
benzofi,ilauinolizine-Z-carboxylic acid.
It was prepared in a similar manner as described in example 32 in the list
active against P.
aerugiuosa, Yield 43.%, m.p 258-60 °C, Cz~HzsN2O~, m/z 370 (M+1)
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Susceptibility Testing
Particular exemplary efflux pump inhibitor compounds within the generic
descriptions of the
compounds of this invention were evaluated for potentiation effect. The iu
vitro microbiological
data for antibiotic potentiation is presented in Tables 1- S.
Testing method for inhibition of the efflux pump of Staphylococcus aureus
1199B NorA+
Compounds disclosed herein were studied for potentiation effect by determining
the Fractional
l0 Inhibition Concentration (FIC) index of compounds and ciprofloxacin.
Potentiation effect is
observed by the reduction of minimal inhibitory concentration (MIC) of
ciprofloxacin in the
presence of the experimental efflux pump inhibitor. The activity of an efflux
pump inhibitor in
combination with a fluoroquinolone such as ciprofloxacin is assessed by the
checkerboard assay
and the FIC index was calculated. An FIC index value of S O.S is synergistic,
of O.S - 2.0 is additive
and > 2.0 is antagonistic (Antimicrobial Combination In Antibiotics in
Laboratory Medicine, Ed.
Victor Lorian, MD, Fourth edition 1996, pp 333-338).
In short, the assay involves broth microdilution method performed as
recommended by the
National Committee for Clinical Laboratory Standards (NCCLS) 1997 (Methods fox
Dilution of
2 0 Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,
Fourth edition; Approved
Standard, NCCLS document M7-A4, vol. 17, No.2). The test organism used for the
assay is
Staphyloeoccus aureus 1199 B NorA+.
In this assay, multiple dilution of two drugs viz. the experimental efflux
pump inhibitor and
2 5 ciprofloxacin are being tested, alone and in combination, at
concentrations equal to above and
below the respective MICs. In the case of the efflux pump inhibitors of the
invention, a number of
compounds display intrinsic antimicrobial activity, but are tested at sub-MIC
concentrations of 4
mcg - 20 mcg/ml.
3 0 Stock solutions of the efflux pump inhibitors tested are prepared in
dimethyl sulfoxide (DMSO) at
a final concentration of S mg/ml. Stock solutions are further diluted in
Mueller Hinton Broth as
per the need of the assay. Ciprofloxacin is dissolved in water,
The checkerboard assay is performed in microtiter plates. Ciprofloxacin is
diluted in the X-axis,
3 5 each column containing a single concentration of ciprofloxacin. The efflux
pump inhibitor is
diluted in the Y axis, each row containing an equal concentration of efflux
pump inhibitor. The
result of these manipulations is that each well of the microtiter plate
contains a unique
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
combination of concentrations of the two agents. Bacterial inoculum is added
at 5 x 105 CFU/ml.
Microtiter plates are incubated at 35°C for 20 hours. Reading is done
by visibly noting turbidity
and confirmed using a microtiter plate reader (Molecular Devices). The FIC
index is calculated as
(A) (B)
------------- + -=----------- - FICA + FICB = FIC Index
~CA) ~CB)
(A) is the concentration of the experimental efflux pump inhibitor in the well
that is the lowest
inhibitory concentration in the Y-axis. (MICA) is the MIC of Staphylococcus
aureus to
experimental efflux pump inhibitor alone. FICA is the fractional inhibitory
concentration of an
experimental efflux pump inhibitor. (B) is the concentration of the
ciprofloxacin in the well that is
the lowest inhibitory concentration in the X-axis. FICB is the fractional
inhibitory concentration of
ciprofloxacin. (Antimicrobial Combination In Antibiotics in Laboratory
Medicine, Ed. Victor
Lorian, MD, Fourth edition 1996, pp 333-338).
Testing method for inhibition of the efflux pumps of Psezzdonzofzas
aeru,~ijzosa 23587. E.coli 2051.
Streptococcus pszeumotziae 3514 azzd Streptococcus nyo~efzus 26-00
Antibiotic Diffusion Assay
The activity of efflux pump inhibitor was also assessed using an antibiotic
diffusion assay. For this
assay Pseudomozzas aeruginosa 23587 is used. Pseudozzzozzas aeruginosa 23587
is a clinical isolate
containing multiple efflux pumps which can be mexAB, mexCD, mexEF but riot
limited to mex
2 5 classification only. Pseudomofzas aerugizzosa are grown in tryptic Soya
broth for 20 hours at 35°C.
Two antibiotic assay plates are prepared in Mueller Hinton Agar using above
inoculum. The
bacterial count in a plate is 1 x 106 cells/ml. In one of the plates,
levofloxacin is incorporated at 5
mcg/ml, while in the control plate an equivalent amount of water is added. An
experimental
efflux pump inhibitor is evaluated by adding a series of concentrations of the
test compound
3 0 ranging from 0.025 mcg to 200 mcg./well in agar plate. The plates are
incubated at 35°C for 20
hours and the zone of inhibition was recorded in mm (table 2). The difference
in the diameter of
zone of inhibition was calculated by subtracting from the diameter of the zone
of inhibition in the
levofloxacin containing plate the diameter of the zone of inhibition in the
water containing control
plate. Example, if the diameter of the zone of inhibition in the levofloxacin
plate is X mm and the
3 5 diameter of the zone of inhibition in the control plate is Y mm, the
difference Z = X - Y and is a
measure of the activity of the efflux pump inhibitor.
The activity of an efflux pump inhibitor of E.coli ZOSI was also assessed
using an antibiotic
diffusion assay. For this assay E.coli 2051 is used. E.coli 2051 is a clinical
isolate containing
86
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
multiple efflux pumps. E.cvli 2051 are grown in tryptic soya broth for 20
hours at 35°C. Two
antibiotic assay plates are prepared in Mueller Hinton Agar using above
inoculum. The bacterial
count in a plate is 1 x 106 cellslml. In one of the plates, levofloxacin is
incorporated at 15 mcg/ml,
while in the control plate an equivalent amount of water is added. An
experimental efflux pump
inhibitor is evaluated by adding a series of concentrations of the test
compound ranging from
0.025 mcg to 200 mcg./well in agar plate. The plates are incubated at
35°C for 20 hours and the
zone of inhibition was recorded in mm (table 3) . The difference in the
diameter of zone of
inhibition was calculated by subtracting from the zone of inhibition in the
levofloxacin containing
plate the diameter of the zone of inhibition in the control plate.
To detect the macrolide efflux pump (Mef) inhibitor, Streptococcus pneumoftiae
3514 (Mef+) is
used which was obtained from the Centre for Disease Control (CDC), Atlanta. A
similar 2 plate
assay method as described above is used with small modifications.
Streptococcus pneu~rtoniae 3514
(Mef+) is grown in Columbia blood agar for 18 hours at 25°C. The growth
was suspended in
sterile todd hewitt broth and CFU were adjusted using macfarland's standard to
1 x 108 CFU/ml.
0.5 ml of this is added to the Mueller l3inton agar supplemented with 5% sheep
blood, final
bacterial count was 106 cells/ml. In one of the two plates 0.5 mcg/ml
azithromycin is incorporated
and to the control plate an equivalent amount of water is added. The plates
are incubated at 35°C
for 20 hours and the zone of inhibition was recorded in mm (table 4). The
difference in the
2 0 diameter of zone of inhibition was calculated by subtracting from the
diameter of the zone of
inhibition in the azithromycin containing plate the diameter of the zone of
inhibition in the
control plate.
To detect the macrolide efflux pump (Mef) inhibitor, Streptococcus pyogehus 26
00 is used which
was obtained from the Centre for Disease Control (CDC), Atlanta. A similar 2
plate assay method
as described above is used with small modifications. Streptococcus pyogestus
26-00 (Mef+) is grown
in Columbia blood agar for 18 hours at 25°C. The growth was suspended
in sterile todd hewitt
broth and CFU were adjusted using macfarland's standard to 1 x 10g CFU/ml. 0.5
ml of this is
added to the Mueller Hinton agar supplemented with 5% sheep blood, final
bacterial count was
3 0 106 cells/ml. In one of the two plates 0.5 mcg/ml azithromycin is
incorporated and to the control
plate an equivalent amount of water is added. The plates are incubated at
35°C for 20 hours and
the zone of inhibition was recorded in mm (table 4). The difference in the
diameter of zone of
inhibition was calculated by subtracting from the diameter of the zone of
inhibition in the
azithromycin containing plate the diameter of the zone of inhibition in the
control plate.
87
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
The results of the screening are provided in the following tables:
88
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
DC
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89
CA 02417799 2003-O1-30
WO 02/09758 PCT/INO1/00139
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required or mandatory, but that other elements are optional and may or may not
be
present. By "consisting of is meant including, and limited to, whatever
follows the
phrase "consisting of'. Thus, the phrase "consisting of indicates that the
listed elements
may be present. By "consisting essentially of ' is meant including any
elements listed after
the phrase, and limited to other elements that do not interfere with or
contribute to the
activity or action specified in the disclosure for the listed elements. Thus,
the phrase
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mandatory,
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depending upon
whether or not they affect the activity or action of the listed elements.
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