CARBAPENEM ANTIBACTERIAL COMPOSITIONS AND METHODS OF TREATMENT

COMPOSITIONS ANTIBACTERIENNES CARBAPENEMS ET METHODES DE TRAITEMENT ASSOCIEES

English Abstract

The present invention relates to novel 2-(naphthosultamyl)methyl-carbapenem
antibacterial agents or pharmaceutically acceptable salts thereof in
combination with other .beta.-lactams, which are useful in treating and
preventing enterococcal infections. The combinations have anti-PBP5 activity
as well as activity against the critical PBPs of sensitive isolates. The
antibacterial compositions of the present invention thus comprise an important
contribution to therapy for treating infections caused by these difficult to
control pathogens. This combination is also useful against gram positive
microorganisms, especially methicillin resistant Staphylococcus aureus (MRSA),
methicillin resistant Staphylococcus epidermidis (MRSE), and methicillin
resistant coagulase negative Staphylococci (MRCNS).

French Abstract

L'invention concerne la combinaison de nouveaux agents antibactériens 2-(naphtosultamyl)méthyl-carbapenems ou leurs sels pharmaceutiquement acceptables et d'autres .beta.-lactames. Cette combinaison convient au traitement et à la prévention des infections aux entérocoques et exerce une activité anti-PBP5 ainsi qu'une activité contre les PBP (protéines fixatrices de pénicilline) critiques des isolats sensibles. Par conséquent, ces compositions antibactériennes constituent une contribution importante au traitement des infections dues à ces pathogènes difficiles à éradiquer. Cette combinaison convient également à la lutte contre les micro-organismes Gram positifs, notamment le Staphylococcus aureus résistant à la méthicilline (MRSA), le Staphylococcus epidermidis résistant à la méthicilline (MRSE) et les staphylocoques coagulase-négatifs résistants à la méthicilline (MRCNS).

Claims

WHAT IS CLAIMED IS:
1. An antibiotic composition comprising a pharmaceutically acceptable
carrier, a 2-(naphthosultamyl)methyl-carbapenem of structural formula I:
<IMG>
or a pharmaceutically acceptable salt thereof, wherein:
R1 represents H or methyl;
CO2M represents a carboxylic acid, a carboxylate anion, a
pharmaceutically acceptable ester group or a carboxylic acid protected by a
protecting
group;
P represents hydrogen, hydroxyl, F or hydroxyl protected by a
hydroxyl-protecting group;
each R is independently selected from: -R*; -Q; hydrogen; halo; -
CN; -NO2; -NRaRb; -ORc; -SRc; -C(O)NRaRb; -C(O)ORh; -S(O)Rc; -SO2Rc; -
SO2NRaRb; -NRaSO2Rb; -C(O)Ra; -OC(O)Ra; -OC(O)NRaRb; -NRaC(O)NRbRc; -
NRaCO2Rh; -OCO2Rh; -NRaC(O)Rb; -C1-6 straight- or branched-chain alkyl,
unsubstituted or substituted with one to four Rd groups; and -C3-7 cycloalkyl,
unsubstituted or substituted with one to four Rd groups;
each Ra, Rb and Rc independently represents hydrogen, -R*, -C1-6
straight- or branched-chain alkyl, unsubstituted or substituted with one to
four Rd
groups, or -C3-7 cycloalkyl, unsubstituted or substituted with one to four Rd
groups;
or Ra and Rb taken together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one or more of O, S, NRc,
with Rc
-27-

as defined above, or -C(O)-, said ring being unsubstituted or substituted with
one to
four Ri groups;
or Rb and Rc taken together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one to three of O, S, NRa,
with Ra
as defined above, or -C(O)-, said ring being unsubstituted or substituted with
one to
four Ri groups;
each Rd independently represents halo; -CN; -NO2;
-NReRf -ORg; -SRg; -CONReRf -COORg; -SORg; -SO2Rg; -SO2NReRf;-
NReSO2Rf;-CORe; -NRe CORf;-OCORe; -OCONReRf;-NReCONRfRg; -
NReCO2Rh; -OCO2Rh; -C(NRe)NRfRg; -NReC(NH)NRfRg; -NReC(NRf)Rg; -R* or -
Q;
Re, Rf and Rg represent hydrogen; -R*; -C1-6 straight- or branched-
chain alkyl unsubstituted or substituted with one to four Ri groups;
or Re and Rf taken together with any intervening atoms represent a 4-6-
membered saturated ring optionally interrupted by one to three of O, S, -C(O)-
or NRg
with Rg as defined above, said ring being unsubstituted or substituted with
one to four
Ri groups;
each Ri independently represents halo; -CN; -NO2;
phenyl; -NHSO2Rh; -ORh, -SRh; -N(Rh)2; -N+(Rh)3; -C(O)N(Rh)2; -SO2N(Rh)2;
heteroaryl; heteroarylium; -CO2Rh; -C(O)Rh; -OCORh; -NHCORh; guanidinyl;
carbamimidoyl or ureido;
each Rh independently represents hydrogen, a -C16
straight or branched-chain alkyl group, a -C3-C6 cycloalkyl group or phenyl,
or when
two Rh groups are present, said Rh groups may be taken in combination and
represent
a 4-G membered saturated ring, optionally interrupted by one or two of O, S,
SO2, -
C(O)-, NH and NCH3;
-28-

Q is selected from the group consisting of:
<IMGS>
wherein:
a and b are 1, 2 or 3;
L- is a pharmaceutically acceptable counterion;
.alpha. represents O, S or NRs;
.beta.,.delta.,.lambda.,µ, and .sigma. represent CRt, N or N+Rs, provided
that no more
than one of .beta.,.delta.,.lambda.,µ,and .sigma. is N+Rs;
R* is selected from the group consisting of:
<IMGS>
wherein:
d represents O, S or NRk;
e, g, x, y and z represent CRm, N or N+Rk , provided that no more than
one of e, g, x, y and z in any given structure represents N+Rk;
Rk represents hydrogen; -C1-6 straight- or branched-chain alkyl,
unsubstituted or substituted with one to four Ri groups; or -(CH2)n Q where n
= 1, 2 or
3 and Q is as previously defined;
each Rm independently represents a member selected from the group
consisting of hydrogen; halo; -CN; -NO2; -NRnRo; -ORn; -SRn; -CONRnRo; -
COORh; -SORn; -SO2Rn; -SO2NRnRo; -NRnSO2Ro; -CORn; -NRnCORo; -OCORn; -
OCONRnRo; -NRnCO2Rh; -NRnCONRoRh; -OCO2Rh; -CNRnNRoRh;-
NRnCNHNRoRh; -NRnC(NRo)Rh; -C1-6 straight- or branched-chain alkyl,
unsubstituted or substituted with one to four Ri groups; -C3-7 cycloalkyl,
unsubstituted or substituted with one to four Ri groups; and -(CH2)n Q where n
and Q
are as defined above;
Rn and Ro represent hydrogen, phenyl; -C1-6 straight- or branched-
chain alkyl unsubstituted or substituted with one to four Ri groups;
-29-

each R s independently represents hydrogen; phenyl or -C1-6 straight-
or branched-chain alkyl, unsubstituted or substituted with one to four R i
groups;
each R t independently represents hydrogen; hale; phenyl; -CN; -NO2;-
NR u R v; -OR u; -SR u; -CONR u R v; -COOR h; -SOR u; -SO2R u; -SO2NR u R v;-
NR u SO2R v; -COR u; -NR u COR v;- OCOR u; -OCONR u R v; -NR u CO2R v; -
NR u CONR v R w; -OCO2R v; -C1-6 straight- or branched-chain alkyl,
unsubstituted or
substituted with one to four R1 groups;
R u and R v represent hydrogen or -C1-6 straight- or branched-chain
alkyl, unsubstituted or substituted with one to four R i groups;
or R u and R v together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one or more of O, S, NR w or
-
C(O)-, said ring being unsubstituted or substituted with one to four R i
groups;
each R w independently represents hydrogen; -C1-6 straight- or
branched-chain alkyl, unsubstituted or substituted with one to four R i
groups; C3-6
cycloalkyl optionally substituted with one to four R i groups; phenyl
optionally
substituted with one to four R i groups, or heteroaryl optionally substituted
with 1-4 R i
groups;
or R h and R w taken together with any intervening atoms represent a 5-
6 membered saturated ring, optionally interrupted by one or two of O, S, SO2,
NH or
NCH3;
R x represents hydrogen or a C1-8 straight- or branched- chain alkyl,
optionally interrupted by one or two of O, S, SO, SO2, NR w, N+R h R w, or -
C(O)-,
said chain being unsubstituted or substituted with one to four of halo, CN,
NO2,
OR w, SR w, SOR w, SO2R w, NR h R w, N+(R h)2R w, -C(O)-R w, C(O)NR h R w,
SO2NR h R w, CO2R w, OC(O)R w, OC(O)NR h R w, NR h C(O)R w, NR h C(O)NR h R w,
or a phenyl or heteroaryl group which is in turn optionally substituted with
from one
to four R i groups or with one to two C1-3 straight- or branched- chain alkyl
groups,
said alkyl groups being unsubstituted or substituted with one to four R i
groups;
R y and R z represent hydrogen; phenyl; -C1-6 straight or branched
chain alkyl, unsubstituted or substituted with one to four R i groups, and
optionally
interrupted by O, S, NR w, N+R h R w or -C(O)-;
or R x and R y together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by O, S, SO2, NR w, N+R h R w
or -
C(O)-, unsubstituted or substituted with 1-4 R i groups,
-30-

and when Rx and Ry together represent a 4-6 membered ring as
defined above, Rz is as defined above or Rz represents an additional saturated
4-6
membered ring fused to the ring represented by Rx and Ry taken together,
optionally
interrupted by O, S, NRw or -C(O)-, said rings being unsubstituted or
substituted with
one to four Ri groups;
and another antibiotic including .beta.-lactams such as carbapenems,
cephalosporins
(ceftriaxone), penicillins; aminoglycosides (as exemplified by gentamicin and
including but not limited to amikacin, dibekacin, streptomycin, neomycin,
kanamycin,
spectinomycin, kasugamycin); fluoroquinolones {as exemplified by ciprofloxacin
and
including but not limited to trovafloxacin, sparfloxacin, gatifloxacin,
grepafloxacin,
ofloxacin, norfloxacin, floxin, levofloxacin) and related quinolones and
naphthyridines with activity against topoisomerases; chloramphenicol; as well
as
macrolides, ketolides, azalides, SynercidR, tetracyclines {including
glycylcyclines),
glycopeptides (including vancomycin, teicoplainin, LY-333328), novobiocin (and
coumermycin) and oxazolidinones or a combination thereof.
2. A composition according to claim 1 wherein the
compounds of structural formula I are represented by formula Ie:
<IMG>
or a pharmaceutically acceptable salt thereof, wherein:
R contains a positively charged moiety selected from the group
consisting of -R*, Q, and a C1-6 straight or branched alkyl chain substituted
with
one Rd group;
Rd is independently selected -R* or Q;
Q is selected from the group consisting of:
-31-

<IMGS>
wherein L-, a and b are as originally defined, and
R x represents a member selected from the group consisting of hydrogen or a C1-
8 straight- or branched- chain alkyl, optionally interrupted by one or two of
O, S,
SO, SO2, NR w, N+R h R w,
or -C(O)-, said chain being unsubstituted or substituted with one
to four of halo, CN, NO2, OR w, SR w, SOR w, SO2R w, NR h R w, N+(Rh)2R w, -
C(O)-R w, C(O)NR h R w, SO2NR h R w, CO2R w, OC(O)R w, OC(O)NR h R w,
NR h C(O)R w, NR h C(O)NR h R w, or a phenyl or heteroaryl group which is in
turn
optionally substituted with from one to four R i groups or with one to two C1-
3
straight- or branched- chain alkyl groups, said alkyl groups being
unsubstituted or
substituted with one to four Ri groups;
R* is selected from:
<IMGS>
wherein d represents NR k; R k represents -C1-6 straight or branched chain
alkyl; and
e, g, x and y represent CR m or N+R k, with R k as defined above and R m
representing
hydrogen.
3. A composition according to claim 1 wherein the compounds of
formula I are:
(1S,5R,6S)-2-(5-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
(1S,5R,6S)-2-(5-(((3-hydroxyprop-1-yl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-
em-3-carboxylate chloride;
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(1S,5R,6S)-2-(S-(( 1-methylimidazol-3-ium)methyl)(1,8-naphthosultam)methyl)-6-
[1(R)-hydroxyethyl]-1 -methylcarbapen-2-em-3-carboxylate;
(1S,5R,6S)-2-(5-(((2-( 1-methylimidazol-3-ium)-ethyl)(1,8-
naphthosultam)methyl)-6-
[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate;
(1S,5R,6S)-2-(4-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
(1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl})-
ethyl)(1,8-naphthosultam)methyl)-6-[1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-
3-
carboxylate chloride;
(1S,5R,6S)-2-{4-(2-(((3-hydroxyprop-1-yl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-
ethyl)( 1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-
3-
carboxylate chloride; or
(1S,5R,6S)-2-(4-(2-{(1 -methylimidazol-3-ium))-ethyl)(1,8-
naphthosultam)methyl}-6-
[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate.
4. A composition according to claim 3 wherein the compounds of
formula I are:
(1S,5R,6S)-2-(5-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
(1S,5R,6S)-2-(4-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct- 1 -
yl)methyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride; or
(1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-
ethyl)( 1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]- 1 -methylcarbapen-2-
em-3-
carboxylate chloride.
5. A composition according to claim 1 wherein the other
antibiotic is selected from the group consisting of cephalosporins,
penicillins,
gentamicin, ciprofloxacin, imipenem, meropenem, chloramphenicol, vancomycin,
and teicoplainin.
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6. An antibiotic composition comprising a pharmaceutically
acceptable carrier, a therapeutically effective amount of a 2-
(naphthosultamyl)methyl-
carbapenem selected from
(1S,5R,6S)-2-(5-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)( 1,8-
naphthosultam)methyl)-6-[1 (R)-hydroxyethyl]-1 -methylcarbapen-2-em-3-
carboxylate
chloride;
(1S,5R,6S)-2-(4-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride; and
(1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct- 1 -yl))-
ethyl)(1,8-naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]- 1 -methylcarbapen-2-
em-3-
carboxylate chloride; and a therapeutically effective amount of another
antibiotic
selected from carbapenems, cephalosporins, penicillins; aminoglycosides such
as
gentamicin, amikacin, dibekacin, streptomycin, neomycin, kanamycin,
spectinomycin,
kasugamycin; fluoroquinolones such as ciprofloxacin, trovafloxacin,
sparfloxacin,
gatifloxacin, grepafloxacin, ofloxacin, norfloxacin, floxin, levofloxacin;
quinolones, .
naphthyridines, chloramphenicol, macrolides, ketolides, azalides, SynercidR,
tetracyclines, glycylcyclines, glycopeptides such as vancomycin, teicoplainin,
LY-
333328; novobiocin, coumermycin, and oxazolidinones or a combination thereof.
7. A composition according to claim 6 wherein the 2-
(naphthosultamyl)methyl-carbapenem is (1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-
1,4-
diazoniabicyclo[2.2.2]oct- 1 -yl))-ethyl)( 1,8-naphthosultam)methyl)-6-[ 1 (R)-
hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate chloride; and the other
antibiotic
is selected from cephalosporins, penicillins, gentamicin, ciprofloxacin,
meropenem,
imipenem, (4R, 5S, 6S)-3-[(3S, 5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-
ylthio]-6-(1 R)-1 -hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-
2-
carboxylic acid, chloramphenicol, vancomycin, and teicoplainin.
8. An antibacterial composition according to claim 1 wherein the
other antibiotic is a carbapenem.
9. A composition according to claim 8 wherein the carbapenem is
imipenem, (4R, 5S, 6S)-3-[(3S, 5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-
ylthio]-6-(1R)-1 -hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-
-34-

carboxylic acid, or meropenem, from about 1 to about 100 mg/kg, and the 2-
(naphthosultamyl)methyl-carbapenem is (1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-
1,4-
diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)(1,8-naphthosultam)methyl)-6-[1(R)-
hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate chloride, from about 0.1 to
about 50 mg/kg.
10. A composition according to claim 9 wherein the amount of
imipenem, (4R, 5S, 6S)-3-[(3S, 5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-
ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-
carboxylic acid, or meropenem is about 2 to about 50 mg/kg and the amount of
(1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-
ethyl)(1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate chloride is about 0.5 to about 20 mg/kg.
11. An antibacterial composition comprising a pharmaceutically
acceptable carrier, about 2 to about 50 mg/kg of imipenem, a pharmaceutically
acceptable amount of cilastatin and about 0.5 to about 20 mg/kg of (1S,5R,6S)-
2-(4-
(2-(((carbamoylmethyl)-1,4-diazoniabicyclo-[2.2.2]oct-1-yl))-ethyl)(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride.
12. A composition according to claim 11 wherein the imipenem
and cilastatin are administered as Primaxin R.
13. An antibacterial composition according to claim 1 wherein the
other antibiotic is a fluoroquinolone, aminoglycoside, chloramphenicol or a
combination thereof.
14. A composition according to claim 13 wherein the
fluoroquinolone is ciprofloxacin, from about 0.1 to about 40 mg/kg, the
aminoglycoside is gentamicin from about 0.5 to about 12 mg/kg, and the 2-
(naphthosultamyl)methyl-carbapenem is (1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-
1,4-
diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)(1,8-naphthosuitam)methyl)-6-[1(R)-
hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate chloride, from about 0.1 to
about 50 mg/kg,
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15. A composition according to claim 14 wherein the amount of
ciprofloxacin is about 0.5 to about 20 mg/kg and the amount of {1S,5R,6S)-2-(4-
(2-
({(carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)( 1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride is about 0.5 to about 20 mg/kg.
16. An antibacterial composition comprising a pharmaceutically
acceptable carrier, about 0.5 to about 20 mg/kg of ciprofloxacin or about 0.5
to about
12 mg/kg of gentamicin and about 0.5 to about 20 mg/kg of (1S,5R,6S)-2-(4-(2-
(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)(1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride.
17. A composition according to claim 1 comprising 2-
(naphthosultamyl)methyl-carbapenem antibacterial agents or ophthamologically
acceptable salts thereof, in combination with a imipenem, (4R, 5S, 6S)-3-[(3S,
5S)-5-
(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-
7-
oxo-1-azabicyclo[3.2.0]kept-2-ene-2-carboxylic acid, or meropenem and another
antibiotic selected from cephalosporins, penicillins, gentamicin,
ciprofloxacin,
chloramphenicol, vancomycin, or teicoplainin.
18. A method of treating or preventing a bacterial infection in a
mammalian patient in need thereof, comprising administering to said patient an
effective amount of a composition of claim 1.
19. A method of treating or preventing enterococcal or MRSA
infections which comprises administering to a patient in need of such
treatment of a
unit dose of the composition of Claim 1.
20. A method of treating or preventing enterococcal or MRSA
infections which comprises administering to a patient in need of such
treatment of a
unit dose of the composition of Claim 6.
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21. A method of treating or preventing enterococcal or MRSA
infections which comprises administering to a patient in need of such
treatment of a
unit dose of the composition of Claim 11.
22. A method of treating or preventing enterococcal or MRSA
infections which comprises administering to a patient in need of such
treatment of a
unit dose of the composition of Claim 14.
23. A method of treating or preventing enterococcal or MRSA
infections which comprises administering to a patient in need of such
treatment of a
unit dose of the composition of Claim 16.
-37-

Description

CA 02341650 2001-02-26
WO 00/12048 PCT/US99/20052
TITLE OF THE INVENTION
CARBAPENEM ANTIBACTERIAL COMPOSITIONS
AND METHODS OF TREATMENT
BACKGROUND OF THE INVENTION
Within the last decade, enterococcus has become the second most
common pathogen isolated in nosocomial infections. The ability of recent
enterococcal isolates (i.e., Errterococcus (E.) faecium, Enterococcus (E.)
faecalis) to
rapidly acquire multidrug resistance to all available antimicrobials and to
disseminate
10 resistance determinants through the exchange of genetic elements has
resulted in a
serious therapeutic dilemma. Antibiotic resistance of enterococcus is
characterized
according to its susceptibility to two glycopeptides - vancomycin (VANCO) and
teicoplanin (TEICO) and is accordingly divided into three major classes of
VANCO
resistant enterococci (VRE) (phenotypes VanA, VanB, VanC and perhaps even a
new
15 class). Enterococcal VanA isolates (E. faeciurn and E. faecalis) are
resistant to high
levels of VANCO (MIC Z64 ~tg/mL) and to TEICO (MIC Z16 pg/mL) and its ...
plasmid transferable resistance is inducible by VANCO or TEICO, while class
VanB
isolates demonstrate moderate levels of resistance to VANCO ( ~-64 pg/mL and
retain susceptibility to TEICO (MIC < 2 pg/mL). Resistance to VanB isolates is
20 induced by VANCO but is not thought to be transferable. Phenotypic class
VanC
VANCO resistance is found in Enterococcus (E.) gallinarum and Enterococcus
(E.)
casseliJlavus, which possess low-level resistance to VANCO (MIC >4 and <r.32
p.g/mL) but retain susceptibility to TEICO (MIC Sl pg/mL).
VanA resistant E. jaecium (VREF) is intrinsically resistant to most (3-
25 lactam (cell-wall active) antibiotics by virtue of the relatively low
affinity of their
penicillin-binding proteins (PBPs), which are important enzymes in cell wall
biosynthesis. (3-lactam antibiotics inhibit bacterial growth through their
binding of
PBPs. Enterococci contain a normally low affinity PBP (PBPS) which in some
recent
isolates appears to be over produced and, in some cases, genetically modified.
30 Emergence of strains with resistance to most (3-lactam, aminoglycoside
and quinolone antibiotics when used as monotherapy have left only
glycopeptides
alone and in combination with aminogiycosides as the first line of defense
against
isolates resistant to (3-lactam agents. For example, treatment of serious
enterococcal
infections usually includes combinations of VANCO with a number of moderately
35 active ~i-lactams or aminoglycosides. However, this final therapeutic
option is

CA 02341650 2001-02-26
WO 00/12048 PCT/US99/20052
waning to the more recent isolation of enterococcal strains with high-level
resistance
to glycopeptides. Due to the isolation of enterococccal organisms that produce
(3-
lactamases in addition to the discovery of VRE isolates with high resistance
to nearly
all (3-lactam antibiotics, etc. (which usually occurs following monotherapy)
an
increased intensity in the search to discover new and innovative anti-
enterococcal
drugs is ever pressing.
SUMMARY OF THE INVENTION
The present invention relates to novel 2-(naphthosultamyl)methyl-
10 carbapenem antibacterial agents or pharmaceutically acceptable salts
thereof in
combination with other antibiotics such as (3-lactams, aminoglycosides,
fluoroquinolones, related quinolones and naphthyridines, chloramphenicol,
macrolides, ketolides, azalides, Synercid~, tetracyclines, glycopeptides,
novobiocin , oxazolidinones and the like or a combination thereof. The
15 combination is useful against gram positive microorganisms, especially
methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant
Staphylococcus epidermidis {MRSE), and methicillin resistant coagulase
negative
Staphylococci (MRCNS).
The present invention also relates to novel 2-
20 (naphthosultamyl)methyl-carbapenem antibacterial agents or pharmaceutically
acceptable salts thereof in combination with other ~3-lactams, which are
useful in
treating and preventing enterococcal infections. The combinations have anti-
PBPS activity as well as activity against the critical PBPs of sensitive
isolates.
The antibacterial compositions of the present invention thus comprise an
25 important contribution to therapy for treating infections caused by these
difficult
to control pathogens.
Another aspect of the invention is concerned with the use of the novel
antibiotic compositions in the treatment of bacterial infections.
30 DESCRIPTION OF THE DRAWIhtGS
Figure 1 shows the effect of Compound A (described below), at
various concentrations, alone and in combination with imipenem (20 mg/kg) in
treating VanA-resistant E. faecium CL5053 using the Mouse Thigh Model.
Bacterial
clearance is expressed in logo reductions in CFUs.
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Figure 2. shows the rate of kill of VanRGentsAmps E. faecalis CL5244
by Compound A (described below), at various concentrations, alone and in
combination with 12 ug/mL of gentamicin.
5 Figure 3. shows the rate of kill of VanRGentsAmps E. faecalis CL4877
by Compound A (described below), at various concentrations, alone and in
combination with 1.0 pg/mL of ciprofloxacin.
Figure 4. shows the effect of Compound A ( 10 mg/kg) and varying
10 amounts of irnipenem and meropenem as well as Compound A (10 mg/kg) in
combination with varying amounts of imipenem or meropenem in treating VanA-
resistant E. faecium CL5053.
DETAILED DESCRIPTION OF THE INVENTION
15 The present invention relates to novel 2-(naphthosultamyl)methyl-
carbapenem antibacterial agents (such as those disclosed in US Patent
No.5,756,725,
herein incorporated by reference) in combination with other ~3-lactams,
aminoglycosides, fluoroquinolones and the like. It is known that the response
to a
given combination may be strain specific and is not solely related to the
level of
20 sensitivity/resistance to the specific members of the combination. Thus,
the
combinations of the present invention are intended to be useful on all
bacterial strains
including those not mentioned herein.
One embodiment of the invention relates to compositions comprising a
pharnnaceutically acceptable carrier, a 2-(naphthosultamyl)methyl-carbapenem
of
25 structural formula I:
~R)s
P.H N R~
H3C CH -N \
O I C02M O=S \ ' ~~R)3
O
or a pharmaceutically acceptable salt thereof, wherein:
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RI represents H or methyl;
COZM represents a carboxylic acid, a carboxylate anion, a
pharmaceutically acceptable ester group or a carboxylic acid protected by a
protecting
5 group;
P represents hydrogen, hydroxyl, F or hydroxyl protected by a
hydroxyl-protecting group;
each R is independently selected from: -R*; -Q; hydrogen; halo; -
CN; -N02; -NRaRb; -ORS; -SRS; -C(O)NRaRb; -C(O)ORh; -S(O)RB; -SOZR~; -
10 S02NRaRb; -NRaS02Rb; -C(O)Ra; -OC(O)Ra; -OC(O)NRaRb; -NRaC(O)NRbR~; -
NRaCO2Rh; -OC02Rh; -NRaC(O)Rb; -C 1 _6 straight- or branched-chain alkyl,
unsubstituted or substituted with one to four Rd groups; and -C3_~ cycloalkyl,
unsubstituted or substituted with one to four Rd groups;
each Ra, Rb and R~ independently represents hydrogen, -R*, -CI-6
15 straight- or branched-chain alkyl, unsubstituted or substituted with one to
four Rd
groups, or -C3_~ cycloalkyl, unsubstituted or substituted with one to four Rd
groups;
or Ra and Rb taken together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one or more of O, S, NR~,
with Rc
as defined above, or -C(O)-, said ring being unsubstituted or substituted with
one to
20 four Ri groups;
or Rb and Rc taken together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one to three of O, S, NRa,
with Ra
as defined above, or -C(O)-, said ring being unsubstituted or substituted with
one to
four Rl groups;
25 each Rd independently represents halo; -CN; -N02;
-NReRf; -ORB; -SRg; -CONReRf; -COORg; -SORg; -S02Rg; -S02NReRf _
NReSOzRf -CORe; -NRe CORf; -OCORe; -OCONReRf -NReCONRfRB; _
NReC02Rh; -OC02Rh; -C(NRe)NRfRg; -NReC(NH)NRfRg; -NReC(NRf)R8; -R* or -
Q
30
Re, Rf and Rg represent hydrogen; -R*; -C 1 _6 straight- or branched-
chain alkyl unsubstituted or substituted with one to four Ri groups;
or Re and Rf taken together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one to three of O, S, -C(O)-
or NRg
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with Rg as defined above, said ring being unsubstituted or substituted with
one to four
R' groups;
each R' independently represents halo; -CN; -N02;
phenyl; -NHS02Rh; -ORh, -SRh; -N(Rh)2; -N+(Rh)3; -C(O)N(Rh)2~ -S02N(Rh)2~
5 heteroaryl; heteroarylium; -C02Rh; -C(O)Rh; -OCORh; -NHCORh; guanidinyl;
carbamimidoyl or ureido;
each Rh independently represents hydrogen, a -C1-6
straight or branched-chain alkyl group, a -C3-C( cycloalkyl group or phenyl,
or when
two Rh groups are present, said Rh groups may be taken in combination and
represent
10 a 4-G membered saturated ring, optionally interrupted by one or two of O,
S, 502,
C(O)-, NH and NCH3;
Q is selected from the group consisting of
(CH 2)b
s, 0s, 0s
N' a , ~-N I! , ~--N ~ a , ~--N 0 ~N_Rx ° x
°' '' a ~ and NR R R
v ~~ .v i,
p a p,-~, ~ ~ .,
L~ ~CH 2)a
wherein:
15 a and b are 1, 2 or 3;
L- is a pharmaceutically acceptable counterion;
a represents O, S or NRs;
(3, b, ~., p and a represent CRt, N or N+Rs, provided that no more
than one of (3, S, ~., p and a is N+Rs;
20
R* is selected from the group consisting of:
e,x x~d x-y
I I , ~~ I and -~--C~ ~ z
day e~g e-g
wherein:
25 d represents O, S or NRk;
e, g, x, y and z represent CRm, N or N+Rk , provided that no more than
one of e, g, x, y and z in any given structure represents N+Rk;
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Rk represents hydrogen; -C 1 _G straight- or branched-chain alkyl,
unsubstituted or substituted with one to four R~ groups; or -(CH2)"Q where n =
l, 2 or
3 and Q is as previously defined;
each Rm independently represents a member selected from the group
S consisting of hydrogen; halo; -CN; -N02; -NRnR°; -ORn; -SRn; -
CONRnR°; -
COORh; -SORn; -S02Rn; -S02NRnR°; -NRnS02R°; -CORi ; -
NRnCOR°; -OCORn;
OCONR°R°; -NRnC02Rh; -NRnCONR°Rh; -OC02Rh; -
CNRnNR°Rh;
NRnCNHNR°Rh; -NR°C(NR°)Rh; -C1_( straight- or
branched-chain alkyl,
unsubstituted or substituted with one to four R' groups; -C3-7 cycloalkyl,
10 unsubstituted or substituted with one to four R~ groups; and -(CH2)nQ where
n and Q
are as defined above;
Rn and R° represent hydrogen, phenyl; -C1-( straight- or branched-
chain alkyl unsubstituted or substituted with one to four R~ groups;
each RS independently represents hydrogen; phenyl or -C1_G straight-
15 or branched-chain alkyl, unsubstituted or substituted with one to four R'
groups;
each Rt independently represents hydrogen; halo; phenyl; -CN; -N02;
NRuR~; -ORu; -SR°; -CONRuR~; -COORh; -SORu; -S02Ru; -S02NRuR~; -
NR~S02R"; -CORu; -NRuCOR~; -OCOR~; -OCONRuR"; -NR~C02R~; -
NRuCONR~RW; -OC02R~; -Cl-G straight- or branched-chain alkyl, unsubstituted or
20 substituted with one to four R' groups;
R° and R" represent hydrogen or -C 1 _G straight- or branched-
chain
alkyl, unsubstituted or substituted with one to four R~ groups;
or Ru and R" together with any intervening atoms represent a 4-6
membered saturated ring optionally interrupted by one or more of O, S, NRW or -
25 C(O)-, said ring being unsubstituted or substituted with one to four R'
groups;
each RW independently represents hydrogen; -C 1 _G straight- or
branched-chain alkyl, unsubstituted or substituted with one to four R' groups;
C3-6
cycloalkyl optionally substituted with one to four R' groups; phenyl
optionally
substituted with one to four Ri groups, or heteroaryl optionally substituted
with 1-4 Rl
30 groups;
or Rh and Rw taken together with any intervening atoms represent a 5-
G membered saturated ring, optionally interrupted by one or two of O, S, 502,
NH or
NCH3;
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CA 02341650 2001-02-26
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Rx represents hydrogen or a C1-g straight- or branched- chain alkyl,
optionally interrupted by one or two of O, S, SO, S02, NRw, N+RhRw, or -C(O)-,
said chain being unsubstituted or substituted with one to four of halo, CN,
N02,
ORw, SRw, SOR"~', S02Rv~', NRhRw, N+(Rh)2Rw, -C(O)-R"'r, C(O)NRhRw,
5 S02NRhRw, C02Rw, OC(O)Rw, OC(O)NRhRw, NRhC(O)Rw, NRhC(O)NRhRw,
or a phenyl or heteroaryl group which is in turn optionally substituted with
from one
to four R' groups or with one to two C1_3 straight- or branched- chain alkyl
groups,
said alkyl groups being unsubstituted or substituted with one to four Rl
groups;
RY and Rz represent hydrogen; phenyl; -C 1 _G straight or branched
10 chain alkyl, unsubstituted or substituted with one to four Rl groups, and
optionally
interrupted by O, S, NRw, N+RhRw or -C(O)-;
or RX and Ry together with any intervening atoms represent a 4-G
membered saturated ring optionally interrupted by O, S, S02, NRW , N+RhRw or -
C(O)-, unsubstituted or substituted with 1 - 4 Rl groups,
15 and when Rx and RY together represent a 4-6 membered ring as
defined above, RZ is as defined above or RZ represents an additional saturated
4-6 ~~
membered ring fused to the ring represented by Rx and RY taken together,
optionally
interrupted by O, S, NR'y or -C(O)-, said rings being unsubstituted or
substituted with
one to four Rl groups;
20 and another antibiotic including ~i-lactams such as carbapenems,
cephalosporins
(ceftriaxone), penicillins; aminoglycosides (as exemplified by gentamicin and
including but not limited to amikacin, dibekacin, streptomycin, neomycin,
kanamycin,
spectinomycin, kasugamycin); fluoroquinolones {as exemplified by ciprofloxacin
and
including but not limited to trovafloxacin, sparfloxacin, gatifloxacin,
grepafloxacin,
25 ofloxacin, norfloxacin, floxin, levofloxacin) and related quinolones and
naphthyridines with activity against topoisomerases; chloramphenicol; as well
as
macrolides, ketolides, azalides (and other related polyketides), Synercid~,
tetracyclines (including glycylcyclines), glycopeptides (including, but not
limited to,
vancomycin, teicoplainin, LY-333328), novobiocin (and coumermycin) and
30 oxazolidinones or a combination thereof.
_7_

CA 02341650 2001-02-26
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The invention is described herein in detail using the terms defined
below unless otherwise specified.
Carboxylate anion refers to a negatively charged group -COO'.
The term "alkyl" refers to a monovalent alkane (hydrocarbon)
S derived radical containing from 1 to 10 carbon atoms unless otherwise
defined. It
may be straight, branched or cyclic. Preferred alkyl groups include methyl,
ethyl,
propyl, isopropyl, butyl, t-butyl, cyclopentyl and cyclohexyl. When
substituted,
alkyl groups may be substituted with up to four substituent groups, selected
from
Rd and Rl, as defined, at any available point of attachment. When the alkyl
group
10 is said to be substituted with an alkyl group, this is used interchangeably
with
"branched alkyl group".
Cycloalkyl is a specie of alkyl containing from 3 to I S carbon
atoms, without alternating or resonating double bonds between carbon atoms. It
may contain from 1 to 4 rings which are fused.
15 The term "alkenyl" refers to a hydrocarbon radical straight,
branched or cyclic containing from 2 to 10 carbon atoms and at least one
carbon to ...
carbon double bond. Preferred alkenyl groups include ethenyl, propenyl,
butenyl
and cyclohexenyl.
The term "alkynyl" refers to a hydrocarbon radical straight or
20 branched, containing from 2 to 10 carbon atoms and at least one carbon to
carbon
triple bond. Preferred alkynyl groups include ethynyl, propynyl and butynyl.
Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and
the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl and
the
like. An aryl group thus contains at least one ring having at least 6 atoms,
with up
25 to five such rings being present, containing up to 22 atoms therein, with
alternating (resonating) double bonds between adjacent carbon atoms or
suitable
heteroatoms. The preferred aryl groups are phenyl, naphthyl and phenanthrenyl.
Aryl groups may likewise be substituted as defined. Preferred substituted
aryls
include phenyl and naphthyl.
30 The term "heteroaryl" refers to a monocyclic aromatic hydrocarbon
group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10
atoms,
containing at least one heteroatom, O, S or N, in which a carbon or nitrogen
atom
is the point of attachment, and in which one or two additional carbon atoms is
optionally replaced by a heteroatom selected from O or S, and in which from
_g_

CA 02341650 2001-02-26
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1 to 3 additional carbon atoms are optionally replaced by nitrogen
heteroatoms,
said heteroaryl group being optionally substituted as described herein.
Examples
of this type are pyrrole, pyridine, oxazole, thiazole and oxazine. Additional
nitrogen atoms may be present together with the first nitrogen and oxygen or
5 sulfur, giving, e.g., thiadiazote. Examples include the following:
~ NH N~NH NHS
pyrrole (pyrrolyl) imidazole {imidazolyi) thiazole {thiazolyl)
N~ w O w S
oxazole (oxazolyl) furan (furyl) thiophene (thienyl)
N=~
N~NH .~ NH ~ ,O
N N
triazole (triazolyl) pyrazole (pyrazolyl) isoxazole (isoxazolyl)
I.
N
N N
isothiazole {isothiazolyl) pyridine {pyridinyl) pyrazine
(pyrazinyl)
I N I ~~N
N N
pyridazine (pyridazinyl) pyrimidine (pyrimidinyl)
l~N
~NJ
triazine (triazinyl)
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CA 02341650 2001-02-26
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Heteroarylium refers to heteroaryl groups bearing a quaternary
nitrogen atom and thus a positive charge. . Examples include the following:
~-NON-CHs .,-NHS
,N + +
--NCO ~ N-CH3
+ i + ;+
~N~N-CH i \
N~, 3 ~ O
+ N N+
CH3 CH3
~N/ ~ ~N
~ J +~
N+ N
\ N~N'CH3
~N'N ~N~
CH3
When a charge is shown on a particular nitrogen atom in a ring
which contains one or more additional nitrogen atoms, it is understood that
the
charge may reside on a different nitrogen atom in the ring by virtue of charge
resonance that occurs.
+/
\N~N-CHa ~ \N-~N ~ CHs
N~/ N.~/
and
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~N~N CH3 ~ ~N ~ CH3
+ iN~
The term "heterocycloalkyl" refers to a cycloalkyl group
(nonaromatic) in which one of the carbon atoms in the ring is replaced by a
heteroatom selected from O, S or N, and in which up to three additional carbon
atoms may be replaced by hetero atoms.
The terms "quaternary nitrogen" and "positive charge" refer to
tetravalent, positively charged nitrogen atoms including, e.g., the positively
charged nitrogen in a tetraalkylammonium group (e. g. tetramethylammonium),
10 heteroarylium, (e.g., N-methyl-pyridinium), basic nitrogens which are
protonated
at physiological pH, and the like. Cationic groups thus encompass positively
charged nitrogen-containing groups, as well as basic nitrogens which are
protonated at physiologic pH.
The term "heteroatom" means O, S or N, selected on an
15 independent basis.
Halogen and "halo" refer to bromine, chlorine, fluorine and iodine.
Alkoxy refers to C1-C4 alkyl-O-, with the alkyl group optionally
substituted as described herein.
When a group is termed "substituted", unless otherwise indicated,
20 this means that the group contains from 1 to 4 substituents thereon. With
respect
to R, Ra, Rb and Rc, the substituents available on alkyl groups are selected
from
the values of Rd. Many of the variable groups are optionally substituted with
up
to four R' groups. With respect to Re, Rf and Rg, when these variables
represent
substituted alkyl, the substituents available thereon are selected from the
values of
25 Rl.
When a functional group is termed "protected", this means that the
group is in modified form to preclude undesired side reactions at the
protected
site. Suitable protecting groups for the compounds of the present invention
will
be recognized from the present application taking into account the level of
skill in
30 the art, and with reference to standard textbooks, such as Greene, T. W. et
al.
Protective Groups in Organic Synthesis Wiley, New York (1991). Examples of
suitable protecting groups are contained throughout the specification.
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CA 02341650 2001-02-26
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In some of the carbapenem compounds of the present invention, M
is a readily removable carboxyl protecting group, and/or P represents a
hydroxyl
which is protected by a hydroxyl-protecting group. Such conventional
protecting
groups consist of known groups which are used to protectively block the
hydroxyl
5 or carboxyl group during the synthesis procedures described herein. These
conventional blocking groups are readily removable, i.e., they can be removed,
if
desired, by procedures which will not cause cleavage or other disruption of
the
remaining portions of the molecule. Such procedures include chemical and
enzymatic hydrolysis, treatment with chemical reducing or oxidizing agents
under
10 mild conditions, treatment with a transition metal catalyst and a
nucleophile and
catalytic hydrogenation.
Examples of carboxyl protecting groups include
allyl, benzhydryl, 2-naphthylmethyl, benzyl, silyl such as t-
butyldimethylsilyl
(TBDMS), phenacyl, p-methoxybenzyl, o-nitrobenzyl, p-methoxyphenyl, p-
15 nitrobenzyl, 4-pyridylmethyl and t-butyl.
Examples of suitable C-6 hydroxyethyl protecting groups include
triethylsilyl, t-butyldimethylsilyl, o-nitrobenzyloxycarbonyl, p-
nitrobenzyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, t-
butyloxycarbonyl,
2,2,2-trichloroethyioxyearbonyl and the like.
20 The carbapenems of this invention, other than the novel 2-
(naphthosultamyl)methyl-carbapenem described above, includes imipenem (plus or
minus cilastatin), meropenem, biapenem, panipenem, (4R, SS, 6S)-3-[3S,SS)-5-(3-
carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-( 1 R)-1-hydroxyethyl]-4-methyl-
7-
oxo-1-azabicyclo[3.2.0]kept-2-ene-2-carboxylic acid, an enantiomer,
diastereomer or
25 pharmaceutically acceptable salt thereof.
The (i-lactams included in this invention include carbapenems such as
imipenem (plus or minus cilastatin; with cilastatin = Primaxin~), meropenem,
biapenem, panipenem, (4R, SS, 6S)-3-[3S,SS)-5-(3-
carboxyphenylcarbamoyl}pyn olidin-3-ylthio]-6-( 1 R)-1-hydroxyethylJ-4-methyl-
'7-
30 oxo-1-azabicyclo[3.2.OJhept-2-ene-2-carboxylic acid, an enantiomer,
diastereomer or
pharmaceutically acceptable salt thereof and the like, cephalosporins and
cephamycins
such as cefoperazone, cefotiam, cefpirome, cefmetazole, cafazolin, cephalexin,
cefuroxime, cefaclor, cefotaxime, ceftriaxone, moxalactam, cefixime and the
like and
peniciliins such as ampicillin, amoxicillin, piperacillin, nafcillin,
oxacillin, cloxacillin
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CA 02341650 2001-02-26
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and combinations of penicillins with a beta-lactamase inhibitor such as
sulbactam,
clavulanic acid, tazobactam, and the like.
The carbapenem compositions of the present invention are useful
per se and in their pharmaceutically acceptable salt and ester forms for the
treatment of bacterial infections in animal and human subjects. The term
"pharmaceutically acceptable ester, salt or hydrate," refers to those salts,
esters and
hydrated forms of the compounds of the present invention which would be
apparent to the pharmaceutical chemist. i.e., those which are substantially
non-
toxic and which may favorably affect the pharmacokinetic properties of said
10 compounds, such as palatability, absorption, distribution, metabolism and
excretion. Other factors, more practical in nature, which are also important
in the
selection, are cost of the raw materials, ease of crystallization, yield,
stability,
solubility, hygroscopicity and flowability of the resulting bulk drug.
Conveniently, pharmaceutical compositions may be prepared from the active
1 S ingredients in combination with pharmaceutically acceptable carriers.
Thus, the
present invention is also concerned with pharmaceutical compositions and
methods of treating bacterial infections utilizing as an active ingredient the
novel
carbapenem compounds.
With respect to -C02M, which is attached to the carbapenem
20 nucleus at position 3, this represents a carboxylic acid goup (M represents
H), a
carboxylate anion (M represents a negative charge), a pharmaceutically
acceptable
ester (M represents an ester forming group) or a carboxylic acid protected by
a
protecting group (M represents a carboxyl protecting group). The
pharmaceutically acceptable salts referred to above may take the form -COOM,
25 where M is a negative charge, which is balanced by a counterion, e.g., an
alkali
metal cation such as sodium or potassium. Other pharmaceutically acceptable
counterions may be calcium, magnesium, zinc, ammonium, or alkyiammonium
cations such as tetramethylammonium, tetrabutylammonium, choline,
triethylhydroammonium, rneglumine, triethanolhydroammonium, etc.
30 The pharmaceutically acceptable salts referred to above also
include acid addition salts. Thus, the Formula I compounds can be used in the
form of salts derived from inorganic or organic acids. Included among such
salts
are the following: acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
35 cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
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CA 02341650 2001-02-26
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glucoheptanoate, giycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate,
maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate,
pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate,
tartrate, thiocyanate, tosylate and undecanoate.
The pharmaceutically acceptable esters are such as would be
readily apparent to a medicinal chemist, and include, for example, those
described
in detail in U.S. Pat. No. 4,309,438. Included within such pharmaceutically
acceptable esters are those which are hydrolyzed under physiological
conditions,
such as pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and
methoxymethyl, and others described in detail in U.S. Pat. No. 4,479,947.
These
are also referred to as "biolabile esters".
Biolabile esters are biologically hydrolizable, and may be suitable
for oral administration, due to good absorption through the stomach or
intestinal
15 mucosa, resistance to gastric acid degradation and other factors. Examples
of
biolabile esters include compounds in which M represents an alkoxyalkyl,
alkylcarbonyIoxyalkyl, alkoxycarbonyloxyalkyl, cycloalkoxyalkyl,
alkenyloxyalkyl, aryloxyalkyl, alkoxyaryl, alkyIthioalkyl,
cycloalkylthioalkyl,
alkenylthioalkyl, arylthioalkyl or alkylthioaryl group. These groups can be
20 substituted in the alkyl or aryl portions thereof with acyl or halo groups.
The
following M species are examples of biolabile ester forming moieties.:
acetoxymethyl, 1-acetoxyethyl, 1-acetoxypropyl, pivaloyloxymethyl,
1-isopropyloxycarbonyloxyethyl, 1-cyclohexyloxycarbonyloxyethyl, phthalidyl
and (2-oxo-S-methyl-1,3-dioxolen-4-yl)methyl.
25 L- can be present or absent as necessary to maintain
the appropriate charge balance. When present, L- represents a pharmaceutically
acceptable counterion. Most anions derived from inorganic or organic acids are
suitable. Representative examples of such counterions are the following:
acetate,
adipate, aminosalicylate, anhydromethylenecitrate, ascorbate, aspartate,
benzoate,
30 benzenesuIfonate, bromide, citrate, camphorate, camphorsulfonate, chloride,
estolate, ethanesulfonate, fumarate, glucoheptanoate, gluconate, glutamate,
lactobionate, malate, maleate, mandelate, methanesulfonate, pantothenate,
pectinate, phosphate/diphosphate, polygalacturonate, propionate, salicylate,
stearate, succinate, sulfate, tartrate and tosylate. Other suitable anionic
species
35 will be apparent to the ordinarily skilled chemist.
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CA 02341650 2001-02-26
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Likewise, when L- represents a species with more than one
negative charge, such as malonate, tartrate or ethylenediamine-tetraacetate
(EDTA), an appropriate number of carbapenem molecules can be found in
association therewith to maintain the overall charge balance and neutrality.
It is preferred that least one of the R groups attached to the
naphtho-sultam platform contains a positively charged moiety. Thus, it can
include -R* or Q, or a moiety which in turn contains a positively charged
group.
Yet another embodiment of this invention is realized when the
compounds of structural formula I are represented by formula Ie:
HO H N R~ /
H3C CH -IV
o N~ 2 _v I
Ie CO 2 O
O
or a pharmaceutically acceptable salt thereof, wherein:
15 R contains a positively charged moiety selected from the group
consisting of -R*, Q, and a C 1-6 straight or branched alkyl chain substituted
with
one Rd group;
Rd is independently selected -R* or Q;
Q is selected from the group consisting of:
1CH21b
0 ,s,.~
and ~-IV o ~N-Rx
(CH )a
wherein L-, a and b are as originally defined, and
RX represents a member selected from the group consisting of hydrogen or a C 1
_
g straight- or branched- chain alkyl, optionally interrupted by one or two of
O, S,
SO, S02, NRw, N+RhRw, or -C(O)-, said chain being unsubstituted or substituted
with one to four of halo, CN, N02, ORw, SRw, SORw, S02Rw, NRhRw,
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N+(Rh)2R~'v, -C(O)-Rv', C(O)NRhRw, S02NRhRw, C02Rw, OC(O)Rw,
OC(O)NRhRw, NRhC(O)Rw, NRhC(O)NRhRw, or a phenyl or heteroaryl group
which is in turn optionally substituted with from one to four R' groups or
with one
to two C 1 _3 straight- or branched- chain alkyl groups, said alkyl groups
being
unsubstituted or substituted with one to four R' groups;
R* is selected from:
e~X ~ ~ d
II and
d~y e~g .
10 wherein d represents NRk; Rk represents -C 1 _6 straight or branched chain
alkyl;
and e, g, x and y represent CRm or N+Rk, with Rk as defined above and Rm
representing hydrogen.
Still another embodiment of the invention is realized when the
compounds of formula I are:
15 (1S,SR,bS~-2-(5-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-I-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
(IS,SR,6S)-2-(5-(((3-hydroxyprop-1-yl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)( 1,8-naphthosultam)methyl)-6-[ I (R)-hydroxyethyl]-1-methylcarbapen-
2-
20 em-3-carboxylate chloride;
( 1 S,SR,6S)-2-(5-(( 1-methylimidazol-3-ium)methyl)( 1,8-naphthosultam)methyl)-
6-
[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate;
( 1 S,SR,6S)-2-(5-(((2-( 1-methylimidazol-3-ium)-ethyl)( 1,8-
naphthosultam)methyl)-6-
[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate;
25 (1S,SR,6S)-2-(4-(({carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-I-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[ 1 {R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
( I S,SR,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-
ethyl)( 1, 8-naphthosul tam)methyl)-6-[ 1 (R)-hydroxyethyl ]-1-methylcarbapen-
2-em-3-
30 carboxylate chloride;
( 1 S,SR,6S)-2-(4-(2-(((3-hydroxyprop-1-yl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl))-
ethyl)( 1,8-naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-
em-3-
carboxylate chloride; or
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( I S,SR,6S)-2-(4-(2-(( 1-methylimidazol-3-ium))-ethyl)( 1,8-
naphthosultam)methyl)-6-
[ 1 (R)-hydroxyethyl]- I -methylcarbapen-2-em-3-carboxylate.
A preferred embodiment of the invention is realized when the
compounds of formula I are:
5 (IS,SR,6S)-2-{5-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
( 1 S,SR,6S)-2-(4-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2Joct-1-
yl)methyl)( 1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethylJ-1-methylcarbapen-2-em-3-
carboxylate
chloride; or
( 1 S,SR,6S)-2-(4-(2-{((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2Joct-1-yl))-
ethyl)( I ,8-naphthosultam)methyl)-6-[ 1(R)-hydroxyethyl]-I-methylcarbapen-2-
em-3-
carboxylate chloride.
Another preferred embodiment of the invention is realized when the
15 other antibiotic employed is selected from the group consisting of
cephalosporins,
penicillins, gentamicin, ciprofloxacin, imipenem, meropenem, chloramphenicol,
vancomycin, or teicoplainin.
In yet another aspect of this invention, the novel antibacterial
compositions of this invention comprise a pharmaceuticallly acceptable
carrier, a
therapeutically effective amount of a 2-{naphthosultamyl)methyl-carbapenem
selected
from
( 1 S,SR,6S)-2-(S-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-
yl)methyl)( 1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride;
25 (1S,SR,6S)-2-(4-(((carbamoylmethyl)-1,4-diazoniabieyclo[2.2.2Joct-1-
yl)methyl)(1,8-
naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride; and
( 1 S,SR,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo(2.2.2]oct-1-yl))-
ethyl)( 1,8-naphthosultam)methyl)-6-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-
em-3-
carboxylate chloride; and a therapeutically effective amount of another
antibiotic
selected from carbapenems, cephalosporins (ceftriaxone), penicillins;
aminoglycosides (as exemplified by gentamicin and including but not limited to
amikacin, dibekacin, streptomycin, neomycin, kanamycin, spectinomycin,
kasugamycin); fluoroquinolones (as exemplified by ciprofloxacin and including
but
not limited to trovafloxacin, sparfloxacin, gatifloxacin, grepafloxacin,
ofloxacin,
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norfloxacin, floxin, levofloxacin) and related quinolones and naphthyridines
with
activity against topoisomerases; chloramphenicol; as well as macrolides,
ketolides,
azalides (and other related polyketides), Synercid~, tetracyclines (including
glycylcyclines), glycopeptides (including, but not limited to, vancomycin,
teicoplainin, LY-333328), novobiocin (and coumermycin) and oxazoiidinones or a
combination thereof.
An aspect of this invention is realized when the 2-
(naphthosultamyl)methyl-carbapenem is (1S,SR,6S)-2-(4-(2-(((carbamoylmethyl)-
1,4-diazoniabicyclo[2.2.2)oct-1-yl))-ethyl)( 1,8-naphthosultam)methyl)-6-[ 1
(R)-
10 hydroxyethyl)-1-methylcarbapen-2-em-3-carboxylate chloride; and the other
antibiotic selected is from cephalosporins, penicillins, gentamicin,
ciprofloxacin,
imipenem, meropenem, (4R, SS, 6S)-3-[(3S, SS)-5-(3-
carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-( 1 R)-1-hydroxyethylJ-4-methyl-
7-
oxo-1-azabicyclo[3.2.OJhept-2-ene-2-carboxylic acid, chloramphenicol,
I S vancomycin, and teicoplainin.
Another embodiment of this invention relates to 2-
(naphthosultamyl)methyl-carbapenem antibacterial agents or ophthamologically
acceptable salts thereof, described above, in combination with other
carbapenems.
An aspect of this invention is realized when the other carbapenem is
20 (4R, SS, 6S)-3-[(3S, SS)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-
(1R)-1-
hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid,
imipenem, or meropenem from about 1 to about 100 mg/kg, preferably about 2 to
about 50 mg/kg and the 2-(naphthosultamyl)methyl-carbapenem is (1S,SR,6S)-2-(4-
(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)( 1,8-
25 naphthosultam)methyl)-6-[1{R)-hydroxyethyI]-1-methylcarbapen-2-em-3-
carboxylate
chloride, from about 0.1 to about 50 mg/kg, preferably about 0.5 to about 20
mg/kg.
Another embodiment of this invention relates to from about novel 2-
(naphthosultamyl)methyl-carbapenem antibacterial agents or ophthamologically
acceptable salts thereof, described above, in combination with
fluoroquinolones,
30 aminoglycosides, or chloramphenicol or a combination thereof.
An aspect of this invention is realized when the fluoroquinolone is
ciprofloxacin, from about 0.1 to about 40 mg/kg, preferably about 0.5 to about
20
mg/kg of the aminoglycoside is gentamicin from about 0.5 to about 12 mg/kg,
and
the 2-(naphthosultamyl)methyl-carbapenem is (1S,SR,6S)-2-(4-(2-
35 (((carbamoyImethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-ethyl)(1,8-
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naphthosultam)methyl)-G-[ 1 (R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride, from about 0.1 to about 50 mg/kg, preferably about o.5 to about 20
mg/kg.
Still another embodiment of this invention relates to novel 2-
(naphthosultamyl)methyl-carbapenem antibacterial agents or ophthamologically
acceptable salts thereof, described above, in combination with a imipenem or
meropenem and another antibiotic selected from cephalosporins, penicillins,
gentamicin, ciprofloxacin, chloramphenicol, vancomycin, or teicoplainin.
The compositions of the present invention are valuable as
antibacterial agents active against enterococcal infections, particularly
against
10 clinical isolates of multiply resistant E. faecium. The compositions of the
present
invention are also valuable as antibacterial agents active against various
gram
positive and gram negative bacteria. Many of compositions of the present
invention are also biologically active against MRSA/MRCNS.
Suitable subjects for the administration of the formulation of the
1 S present invention include mammals, primates, man, and other animals. In
vitro
antibacterial activity is predictive of in vivo activity when the compositions
are
administered to a mammal infected with a susceptible bacterial organism.
Using standard susceptibility tests, the compositions of the
invention are determined to be active against MRSA and enterococcal
infections.
20 The compounds of the invention are formulated in pharmaceutical
compositions by combining the compounds with a pharmaceutically acceptable
carrier. Examples of such carriers are set forth below.
The compounds may be employed in powder or crystalline form, in
liquid solution, or in suspension. They may be administered by a variety of
25 means; those of principal interest include: topically, orally and
parenterally by
injection (intravenously or intramuscularly).
Compositions for injection, a preferred route of delivery, may be
prepared in unit dosage form in ampules, or in multidose containers. The
injectable compositions may take such forms as suspensions, solutions, or
30 emulsions in oily or aqueous vehicles, and may contain various formulating
agents. Alternatively, the active ingredient may be in powder (lyophilized or
non-
lyophilized) form for reconstitution at the time of delivery with a suitable
vehicle,
such as sterile water. In injectable compositions, the carrier is typically
comprised
of sterile water, saline or another injectable liquid, e.g., peanut oil for
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intramuscular injections. Also, various buffering agents, preservatives and
the
like can be included.
Topical applications may be formulated in carriers such as
hydrophobic or hydrophilic bases to form ointments, creams, lotions, in
aqueous,
oleaginous or alcoholic liquids to form paints or in dry diluents to form
powders.
Oral compositions may take such forms as tablets, capsules, oral
suspensions and oral solutions. The oral compositions may utilize carriers
such as
conventional formulating agents, and may include sustained release properties
as
well as rapid delivery forms.
10 The dosage to be administered depends to a large extent upon the
condition and size of the subject being treated, the route and frequency of
administration, the sensitivity of the pathogen to the particular compound
selected,
the virulence of the infection and other factors. Such matters, however, are
left to
the routine discretion of the physician according to principles of treatment
well
1 S known in the antibacterial arts. Another factor influencing the precise
dosage
regimen, apart from the nature of the infection and peculiar identity of the
individual being treated, is the molecular weight of the compound.
The novel antibiotic compositions of this invention for human
delivery per unit dosage, whether liquid or solid, comprise from about 0.01 %
to as
20 high as about 99% of the 2-(naphthosultamyl)methyl-carbapenem discussed
herein, the preferred range being from about 10-GO% and from about 1 % to
about
99.99% of one or more of other antibiotics such as those discussed herein,
preferably from about 40% to about 90%. The composition will generally contain
from about 125 mg to about 3.0 g of the 2-(naphthosultamyl)methyl-carbapenem
25 discussed herein; however, in general, it is preferable to employ dosage
amounts
in the range of from about 250 mg to 1000 mg and from about 200mg to about 5 g
of the other antibiotics discussed herein; preferably from about 250 mg to
about
1000 mg. In parenteral administration, the unit dosage will typically include
the
pure compound in sterile water solution or in the form of a soluble powder
30 intended for solution, which can be adjusted to neutral pH and isotonic.
The invention described herein also includes a method of treating a
bacterial infection in a mammal in need of such treatment comprising
administering to said mammal the claimed composition in an amount effective to
treat said infection.
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The preferred methods of administration of the claimed
compositions include oral and parenteral, e.g., i.v. infusion, i.v. bolus and
i.m.
injection fonmulated so that a unit dosage comprises a therapeutically
effective
amount of each active component or some submultiple thereof.
S For adults, about S-50 mg/kg of body weight, preferably about 250
mg to about 1000 mg per person of the 2-(naphthosultamyl)methyl-carbapenem
antibacterial compound and about 250 mg, to about 1000 mg per person of the
other antibiotics) given one to four times daily is prefer ed. More
specifically, for
mild infections a dose of about 250 mg two or three times daily of the 2-
10 (naphthosultamyl)methyl-carbapenem antibacterial compound and about 250 mg
two or three times daily of the other antibiotic is recommended. For moderate
infections against highly susceptible gram positive organisms a dose of about
500
mg each of the 2-(naphthosultamyl)methyl-carbapenem and the other antibiotics,
three or four times daily is recommended. For severe, life-threatening
infections
15 against organisms at the upper limits of sensitivity to the antibiotic, a
dose of
about S00-2000 mg each of the 2-(naphthosultamyl)methyl-carbapenem and the
other antibiotics, three to four times daily may be recommended.
For children, a dose of about 5-25 mg/kg of body weight given 2, 3,
or 4 times per day is preferred; a dose of 10 mg/kg is typically recommended
20 The claimed compositions contain antibiotic agents which are of
the broad class known as carbapenems. Many carbapenems are susceptible to
attack by a renal enzyme known as dehydropeptidase (DHP). This attack or
degradation may reduce the efficacy of the carbapenem antibacterial agent.
Many
of the compounds of the present invention, on the other hand, are less subject
to
25 such attack, and therefore may not require the use of a DHP inhibitor.
However,
such use is optional and contemplated to be part of the present invention.
Inhibitors of DHP and their use with carbapenems are disclosed in,
e.g.,[European
Patent Application Nos. 79102616.4, filed July 24, 1979 (Patent No. 0 007
614);
and 82107174.3, filed August 9, 1982 (Publication No. 0 072 014)].
30 The compositions of the present invention may, where DHP
inhibition is desired or necessary, be combined or used with the appropriate
DHP
inhibitor as described in the aforesaid patents and published application. The
cited European Patent Applications define the procedure for determining DHP
susceptibility of the present carbapenems and disclose suitable inhibitors,
35 combination compositions and methods of treatment. It is preferred that a
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pharmaceutically acceptable amount of cilastatin is administered with the
carbapenems. A preferred weight ratio of DHP to carbapenem: DHP inhibitor in
the combination compositions is about 1:1. The amount of DHP to employed
generally can be determined by the skilled artisan. US Patent 4539208, herein
incorporated by reference illustrates the amount of DHP that can be used with
a
carbapenem.
A preferred DHP inhibitor is 7-(L-2-amino-2-carboxy-ethylthio)-2-
(2,2-dimethylcyclopropanecarboxamide)-2-heptenoic acid or a useful salt
thereof.
Thus, another aspect of this invention is realized when the 2-
i 0 (naphthosultamyl)methyl-carbapenem antibacterial agents or
ophthamologically
acceptable salts thereof, described above, are in combination with imipenem or
meropenem containing 7-(L-2-amino-2-carboxy-ethylthio)-2-{2,2-
dimethylcyclopropanecarboxamide)-2-heptenoic acid or a useful salt thereof.
When imipenem contains cilastatin it is preferred that composition is
Primaxin~.
15 The invention is further described in connection with the following
non-limiting examples.
EXAMPLE 1
The relative in-vivo protective abilities of (1S,SR,6S)-2-(4-(2
(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl))-ethyl){ 1,8
20 naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride {Compound A) in combination with imipenem (IPM) to influence
clearance
of CL5053 from local soft tissue (thigh) was measured in a cyclophosphamide-
treated
mouse model. Multple antibiotic treatments were administered (s.c.) over a 5
day
period. Tissues were aseptically removed on day +7 and processed.
MATERIALS and METHODS
Localized Tissue Infection Model
Bacterial Culture Preparation
Stock cultures of E. faecium CL5053 (obtained from Bellevue Hospital
30 in NYC) were prepared from slants or L-tubes. A shake flask containing --75
mL of
trypticase soy broth (TSB) was inoculated with ~lmL of seed culture followed
by
shaking at 250 rpm for 10-12 hours at 35°C. The culture was then washed
(i.e.,
centrifuged in a Sorvall RC-SB at 5000 rpm ~15 min and bacterial cells
concentrated
twofold [to 109 to 1010 colony forming units (CFU/mL)] in fresh TSB containing
10% glycerol (or in PBS-10% glycerol) by reconstituting the pellet in half the
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original volume. Finally, ~1 mL aliquots of the washed stock culture was
stored in -
70°C for later use to initiate cultures for animal infections.
Prior to infection, 1 mL of the above frozen culture was thawed in cold
H20, warmed to 35°C and added to ~75 mL fresh TSB before incubating at
35°C for
5 ~10 hours as described above. The 10 hour culture was then washed as above
to
remove media containing toxins, etc. released during cell growth and bacterial
death.
The cell pellet was then reconstituted with fresh TSB in half the original
volume. 0.2
mL of the washed, concentrated culture (challenge inoculum) was injected
intramuscularly (i.m.) into the right thighs of mice. Infected mice were
further
divided into appropriate groups of S or 10 animals each for antibiotic
treatments.
Antibiotic Preparations
The antibiotics of this invention were prepared in 10 mM 3-[-
Morpholinolpropanesulfonic acid (MOPS) buffer, pH 7.1. For all carbapenems,
the
above MOPS buffer also contains cilastatin, a renal dehydropeptidase-I (DHP-1)
inhibitor, at a concentration which would yield a final dose of 40 mg/kg in a
0.5 mL
inoculum/mouse regardless of antibiotic concentration. Antibiotic dosing (s.c.
in 0.5
mL) was initiated one hour following bacterial challenge (+1 hour), followed
by two
treatments at four hour intervals ( +S and +9 hours). Over the next 5 days,
further
20 antibiotic treatments were administered once or twice daily (at ~10 to 1 2
and at ~24
hour intervals, respectively) starting at x-20 hours and continuing for a
total of either
8 or 13 treatments. Two days after the last antibiotic treatment (day +7),
animals were
sacrificed and thigh tissues were prepared for CFU determination.
Animals
Random outbred CD1 mice (Charles River Labs) were
immunocompromised by a single injection of cyclophosphamide (Cytoxan) prepared
in PBS at a final concentration of 250 mg/kg/0.5 mLJmouse -4 days prior to
i.m.
bacterial challenge with E. faecium CL5053. Immediately before or after
bacterial
30 challenge, 0.5 mL of a 0.45 pm membrane filtered ferric iron (Fe+3)/PBS
solution
was injected i.p. at 20 mg/kg. The source of Fe+3 was obtained by appropriate
PBS
solubilization of ferric ammonium citrate (consisting of 18.5% Fe+3 by weight)
followed by filtering through a 0.45 pm membrane just prior to its i.p.
administration
at the time of bacterial challenge.
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Preparation of Tissue for Detection of Colony Formin Units i(CFU~
On day +7 mice were euthanized, the lower abdomen and thighs were
flushed with 70% ethanol and the outer skin aseptically removed. Denuded thigh
tissues were aseptically collected and placed in tubes containing 4 mL of
sterile
5 PBS/10% glycerol for temporary -70°C storage or for same day
processing. On the
day of plating, frozen thighs were thawed in cold H20, ground with a Polytron
homogenizer for 5-10 seconds each and placed on ice for further dilutions.
Tenfold
serial dilutions of each sample were made in tubes containing 0.9 mL of cold
fresh
TSB prior to plating 0.1 mL from selected dilution tubes onto Enterococcus-
specific
m-Enterococcus agar plates. Plates were incubated at 35°C for 2 to 3
days and the
number of CFU's remaining per thigh determined.
Results
Figure 1 show that Compound A alone effectively protects mice
(reducing 3.3, 2.08, ~0.8 and 0.6 logs of CFU, respectively with 40, 20, 10
and S
mg/kg). However, combinations of Compound A with a fixed amount of IPM (20
mg/kg) were significantly synergistic (e.g., .20 mg/kg of IPM increased the
effective
log clearance of 5 mg/kg of Compound A from 0.6 to 2.75 logs).
EXAMPLE 2
To evaluate the combination of Compound A with gentamicin to
influence clearance of VanRGentSAmpS (where R = resistant and S=sensitive) E.
faecalis CL 5244 (obtained from Washington University School of Medicine, St.
Louis, MO) partial checkerboard synergy studies were performed followed by
time-
kill studies.
To evaluate the combination of Compound A with ciprofloxacin to
influence clearance of VanRGentSAmpS (where R = resistant and S=sensitive) E.
faecalis CL 4877 (obtained from University of Maryland Hospital, University of
Maryland Medical System, 22 South Green St., Baltimore, MD 21201 ) partial
checkerboard synergy studies were also performed followed by time-kill
studies.
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MATERIALS and METHODS
Antibiotics
Compound A stock solution was prepared in 10 mM MOPs buffer, pH
7.0, and its concentration confirmed using a spectrophotometric assay.
Gentamicin
(Sigma Chemical Co.) stock solutions were prepared in 10 mM MOPs buffer, pH
7.0
on a weight per volume basis.
Bacterial Strains
10 E. faecalis, E. faeci:m: and E. gallinanun strains were provided by the
Merck Clinical Culture Collection.
Media
Organisms maintained on Brain Heart Infusion (BHI) (Difco) agar
slants were grown overnight in BHI broth (Difco) prior to testing. The test
medium
used was BHI broth.
MIC Determinations
MICs were determined by broth microdilution. Serial twofold
20 dilutions of each antibiotic were prepared in BHI broth and inoculated with
106
CFU/mL. The MIC is defined as the lowest concentration of antibiotic that
resulted in
no visible growth after incubation at 35°C for 22 hours.
Checkerboard and Time-Kill SSmergy Studies
25 Serial twofold antibiotic dilutions were prepared in BHI broth and
inoculated with 106 CFU/mL. Follow-up time-kill studies were done in flasks.
Compound A or Compound A in combination with gentamicin (at clinically
relevant
concentrations) were added to flasks containing BHI broth (50 mL,/flask),
inoculated
with 106 CFU/mL and incubated at 35°C with shaking at 85 rpm. Viable
counts
30 were obtained at appropriate time intervals on BHI agar plates.
Results
Figure 2 shows that Compound A alone is effective against
VanRGentSAmpS E. faecalis CL 5244. However, combinations of Compound A
35 with a fixed amount of gentamicin were significantly synergistic.
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Figure 3 shows that Compound A alone is effective against
VanRGentsAmps E. faecalis CL 4877. However, combinations of Compound A
with a fixed amount of ciprofloxacin were signif cantly effective.
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Representative Drawing