U.S. patent application number 11/951214 was filed with the patent office on 2008-04-17 for heterocyclic compounds and uses thereof as d-alanyl-d-alanine ligase inhibitors.
This patent application is currently assigned to PLIVA D.D.. Invention is credited to Paul J. Ala, Janid A. Ali, Jacob J. Clement, Patrick R. Connelly, Carlos H. Faerman, Christopher Faraday, John V. Gazzaniga, Andrew S. Magee, Salvatore A. Marchese, Scott T. Moe, Manuel A. Navia, Emanuele Perola, Paul Will.
Application Number | 20080090847 11/951214 |
Document ID | / |
Family ID | 23164426 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090847 |
Kind Code |
A1 |
Moe; Scott T. ; et
al. |
April 17, 2008 |
HETEROCYCLIC COMPOUNDS AND USES THEREOF AS D-ALANYL-D-ALANINE
LIGASE INHIBITORS
Abstract
The invention is based on the discovery of a new class of
heterocyclic compounds having, for example, antibacterial
properties. The D-Ala-D-Ala ligase enzyme is a critical pathway
enzyme in the bacterial cell-wall synthesis. The compounds can bind
to and inhibit the enzyme D-Ala-D-Ala ligase. The new compounds'
activity combined with their ability to cross bacterial cell
membranes makes them suitable for use as antibacterial drugs or
other antibacterial applications.
Inventors: |
Moe; Scott T.; (Marlborough,
MA) ; Ala; Paul J.; (Newark, DE) ; Perola;
Emanuele; (Cambridge, MA) ; Faerman; Carlos H.;
(Acton, MA) ; Clement; Jacob J.; (Vancouver,
CA) ; Ali; Janid A.; (Waltham, MA) ; Will;
Paul; (Stoneham, MA) ; Marchese; Salvatore A.;
(Malden, MA) ; Magee; Andrew S.; (Maynard, MA)
; Gazzaniga; John V.; (Worcester, MA) ; Faraday;
Christopher; (San Francisco, CA) ; Navia; Manuel
A.; (Lexington, MA) ; Connelly; Patrick R.;
(Harvard, MA) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770
Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
PLIVA D.D.
Zagreb
HR
10000
|
Family ID: |
23164426 |
Appl. No.: |
11/951214 |
Filed: |
December 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10185059 |
Jun 28, 2002 |
|
|
|
11951214 |
Dec 5, 2007 |
|
|
|
60301685 |
Jun 28, 2001 |
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Current U.S.
Class: |
514/262.1 ;
435/184; 544/260 |
Current CPC
Class: |
A61P 33/00 20180101;
C07D 401/12 20130101; C07D 475/08 20130101; C07D 487/04 20130101;
C07D 471/04 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/262.1 ;
435/184; 544/260 |
International
Class: |
A61K 31/495 20060101
A61K031/495; A61P 33/00 20060101 A61P033/00; C07D 475/04 20060101
C07D475/04; C12N 9/99 20060101 C12N009/99 |
Claims
1. A compound comprising the formula: ##STR86## wherein A and B are
independently selected from the group consisting of N and CR.sup.7,
provided that A and B are not both N, wherein R.sup.7 is hydrogen
or a carbon-, nitrogen-, sulfur-, halogen-, and/or
oxygen-containing function group; R.sup.1 and R.sup.2 are identical
or different --NR.sup.5R.sup.6 groups, wherein each R.sup.5 and
R.sup.6 is independently hydrogen or a carbon-containing functional
group; R.sup.3 is selected from the group consisting of hydrogen,
alkyl, amino, hydroxy, alkoxy, and alkylamino; R.sup.4 is a
carbon-, nitrogen-, sulfur-, halogen-, and/or oxygen-containing
functional group, provided that, if A is CH, B is nitrogen, and
R.sup.5 and R.sup.6 are both hydrogen, then R.sup.4 is not methyl,
isobutyl, phenyl, 4-methylphenyl, 4-chlorophenyl, 4-bromophenyl,
2-(2,5-dimethoxyphenyl)-ethyl, or --CH(OCH.sub.3).sub.2; and if A
and B are both CH groups, then R.sup.4 is an amino group other than
--NH.sub.2, (3,4-dichlorophenyl)methylamino, or
(3,4-dichlorophenyl)methyleneimino.
2. The compound of claim 1, wherein R.sup.4 is a substituted or
unsubstituted, linear, branched, or cyclic, alkyl, alkenyl,
alkynyl, aryl, aralkyl, or alkaryl group.
3. The compound of claim 1, wherein R.sup.5 and R.sup.6 are both
hydrogen.
4. The compound of claim 1, wherein R.sup.4 includes at least one
aryl group.
5. The compound of claim 4, wherein R.sup.4 is selected from the
group consisting of: ##STR87##
6. The compound of claim 4, wherein R.sup.4 is: ##STR88## wherein
R.sup.8-12 are independently selected from the group consisting of
hydrogen and carbon-, nitrogen-, sulfur-halogen- and/or
oxygen-containing functional groups.
7-11. (canceled)
12. The compound of claim 4, wherein R.sup.4 is selected from the
group consisting of --CH.sub.2O-aryl, --CH.sub.2S-aryl,
--CH.dbd.CH-aryl, and --NH(CH.sub.2-aryl).
13. (canceled)
14. The compound of claim 1, wherein R.sup.4 is selected from the
group consisting of --N(CH.sub.3)R.sup.21,
--N(CH.sub.2CH.sub.3)R.sup.21, --N(CH(CH.sub.3).sub.2)e.sup.21, and
--N(benzyl)R.sup.21, where R.sup.21 is a carbon-, nitrogen-,
sulfur-, halogen-, and/or oxygen-containing functional group.
15. The compound of claim 1, wherein R.sup.4 is selected from the
group consisting of --CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NR.sup.22R.sup.23.sub.7 and
--CH.sub.2NHC(.dbd.O)R.sup.22, wherein R.sup.22, and R.sup.23 are
independently selected from the group consisting of hydrogen and
carbon-, nitrogen-, sulfur halogen- and/or oxygen-containing
functional groups.
16. The compound of claim 15, wherein R.sup.22 is hydrogen and
R.sup.23 is --C(.dbd.O)R.sup.24, where R.sup.24 is hydrogen or a
carbon-, nitrogen-, sulfur-halogen- and/or oxygen-containing
functional group.
17-22. (canceled)
23. A compound comprising the formula: ##STR89## wherein R.sup.1
and R.sup.2 are identical or different --NR.sup.5R.sup.6 groups,
wherein each R.sup.5 and R.sup.6 is independently hydrogen or a
carbon-containing functional group; and R.sup.4 is an amino group
other than --NH.sub.2 or ##STR90##
24. A method of inhibiting D-Ala-D-Ala ligase, the method
comprising exposing D-Ala-D-Ala ligase to a compound of formula I
or formula II: ##STR91## wherein A and B are independently selected
from the group consisting of N and CR.sup.7, provided that A and B
in formula II are not both N. wherein R.sup.7 is hydrogen or a
carbon-, nitrogen-, sulfur-, halogen-, and/or oxygen-containing
function group; R.sup.1 and R.sup.2 are identical or different
--NR.sup.5R.sup.6 groups, wherein each R.sup.5 and R.sup.6 is
independently hydrogen or a carbon-containing functional group; and
R.sup.3 and R.sup.4 are independently selected from the group
consisting of hydrogen and carbon-, nitrogen-, sulfur-, halogen-,
and/or oxygen-containing functional groups; provided that R.sup.3
and R.sup.4 are not both hydrogen.
25. The method of claim 24, wherein R.sup.1 and R.sup.2 are both
--NH.sub.2.
26. The method of claim 24, wherein R.sup.3 is hydrogen.
27. The method of claim 24, wherein R.sup.3 and R.sup.4 are
independently selected from the group consisting of -branched and
straight-chain alkyl, --O-alkyl, --O-alkyl-COOH,
--O-alkyl-NR.sup.7R.sup.8, --O-alkyl-OH, --NR.sup.7R.sup.8,
--NR.sup.7-alkyl-NR.sup.8R.sup.9, --NR.sup.7-alkyl-COOH,
--NR.sup.7-alkyl-OH; --CONR.sup.7R.sup.8,
--CONR.sup.7-alkyl-NR.sup.8R.sup.9, CONR.sup.7-alkyl-COOH,
--CONR.sup.7-alkyl-OH, --S-alkyl, --S-alkyl-COOH, --S-alkyl-NR7R',
-alkyl-OH, --O-aryl, --O-aryl-COOH, --O-aryl-NR.sup.7R.sup.8,
--O-aryl-OH, --S-aryl, --S-aryl-COOH, --S-aryl-NR.sup.7R.sup.8,
--S-aryl-OH, --NR.sup.7-aryl-NR.sup.8R.sup.9, --NR.sup.7-aryl-COOH,
--NR.sup.7-aryl-OH; --CONR.sup.7-aryl-NR.sup.8R.sup.9,
--CONR.sup.7-aryl-COOH, --CONR-aryl-OH,
--CH.sub.2NR.sup.5C.sub.6H.sub.4COOH; provided that R.sup.3 and
R.sup.4 cannot simultaneously be identical branched or straight
chain alkyl groups; R.sup.1 and R.sup.2 are independently selected
from the group consisting of hydrogen, --NH.sub.2, and
--NR.sup.11R.sup.12, wherein at least one of R.sup.1 and R.sup.2 is
--NH.sub.2; R.sup.5 is lower alkyl, --H, or
--CH.sub.2NR.sup.10C.sub.6H.sub.4CONHR.sup.6; wherein R.sup.6 is
selected from the group consisting of -alkyl, -alkyl-COOH,
-alkyl-NH.sub.2, and -alkyl-OH; R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 are independently selected from
the group consisting of straight-chain alkyl, branched alkyl, aryl,
and acyl groups, optionally substituted with one or more oxygen,
nitrogen, sulfur, or halogen-based functional groups; and A is
selected from the group consisting of N and CH.
28. A method of treating a patient, the method comprising
administering to the patient an effective amount of a compound of
formula I or formula II: ##STR92## wherein A and B are
independently selected from the group consisting of N and CR.sup.7,
provided that A and B in formula II are not both N, wherein R.sup.7
is hydrogen or a carbon-, nitrogen-, sulfur-halogen- and/or
oxygen-containing function group; R.sup.1 and R.sup.2 are identical
or different --NR.sup.5R.sup.6 groups, wherein each R.sup.5 and
R.sup.6 is independently hydrogen or a carbon-containing functional
group; and R.sup.3 and R.sup.4 are independently selected from the
group consisting of hydrogen and carbon-, nitrogen-, sulfur-,
halogen-, and/or oxygen-containing functional groups; provided that
R.sup.3 and R.sup.4 are not both hydrogen.
29. The method of claim 28, wherein R.sup.1 and R.sup.2 are both
--NH.sub.2.
30. The method of claim 28, wherein R.sup.3 is hydrogen.
31. The method of claim 28, wherein R.sup.3 and R.sup.4 are
independently selected from the group consisting of: -branched and
straight-chain alkyl, --O-alkyl, --O-alkyl-COOH,
--O-alkyl-NR.sup.7R.sup.8, --O-alkyl-OH, --NR.sup.7R.sup.8,
--NR.sup.7-alkyl-NR.sup.8R.sup.9, --NR.sup.7-alkyl-COOH,
--NR.sup.7-alkyl-OH; --CONR.sup.7R.sup.8,
--CONR.sup.7-alkyl-NR.sup.8R.sup.9, --CONR.sup.7-alkyl-COOH,
--CONR.sup.7-alkyl-OH, --S-alkyl, --S-alkyl-COOH,
--S-alkyl-NR.sup.7R.sup.8, -S-alkyl-OH, --O-aryl, --O-aryl-COOH,
--O-aryl-NR.sup.7R.sup.8, --O-aryl-OH, --S-aryl, --S-aryl-COOH,
-Saryl-NR.sup.7R.sup.8, --S-aryl-OH,
--NR.sup.7-aryl-NR.sup.8R.sup.9, --NR.sup.7-aryl-COOH,
--NR.sup.7-aryl-OH; --CONR.sup.7-aryl-NR.sup.8R.sup.9,
--CONR.sup.7-aryl-COOH, --CONR.sup.7-aryl-OH,
--CH.sub.2NR.sup.5C.sub.6H.sub.4COOH; provided that R.sup.3 and
R.sup.4 cannot simultaneously be identical branched or straight
chain alkyl groups; R.sup.1 and R.sup.2 are independently selected
from the group consisting of hydrogen, --NH.sub.2, and
--NR.sup.11R.sup.12, wherein at least one of R.sup.1 and R.sup.2 is
--NH.sub.2; R.sup.5 is lower alkyl, --H, or
--CF.sup.2NR.sup.10C.sub.6H.sub.4CONHR.sup.6; wherein R.sup.6 is
selected from the group consisting of alkyl, -alkyl-COOH,
-alkyl-NH.sub.2, and -alkyl-OH; wherein R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 are independently selected from
the group consisting of straight-chain alkyl, branched alkyl, aryl,
and acyl groups, optionally substituted with one or more oxygen,
nitrogen, sulfur, or halogen-based functional groups; and A is
selected from the group consisting of N and CH.
32. A formulation comprising the compound of claim 1 combined with
an excipient suitable for administration to a subject.
33. A method of treating a subject having a microbial infection,
the method comprising administering to the subject an effective
amount of the formulation of claim 32.
34. The method of claim 33, wherein the subject is an animal.
35. A method of inhibiting bacteria growth in a non-living system,
the method comprising contacting the system with an effective
amount of the compound claim 1 to inhibit bacterial growth.
36. The method of claim 24, wherein the D-Ala-D-Ala ligase
comprises a sequence at least 90% identical to the sequence of a
D-Ala-D-Ala ligase from a species selected from the group
consisting of Escherichia coli, Chlamydia pneumoniae, Chlamydia
trachomatis, Yersinia pestis, Haemophilus influenzae, Haemophilus
ducreyi, Pseudomonas aeruginosa, Pseudomonasputida,
Xylellafastidiosa, Bordetellapertussis, Thiobacillusferrooxidans,
Neisseriameningitidis, Neisseria gonorrhoeae, Buchn era aphidicola,
Bacillus halodurans, Geobactersulfurreducens, Rickettsia
prowazekii, Zymomonas mobilis, Aquifex aeolicus thermophile,
Thermotoga maritima, Clostridium difficile, Enterococcus faecium,
Streptomyces toyocaensis, Amycolatopsis orientalis, Enterococcus
gallinarum, Enterococcushirae, EnterococcusJaecium, Enterococcus
faecalis, Streptococcus pneumoniae, Streptococcus pyogenes,
Staphylococcus aureus, Bacillus subtilis, Bacillus
stearothermophilus, Deinococcus radiodurans, Synechocystis sp.,
Salmonella typhimurium, Mycobacterium tuberculosis, Mycobacterium
avium, Mycohacterium smegmatis, Legionella pneumophila, Leuconostoc
mesenteroides, Borrelia burgdorferi, Treponema pallidum,
Vibriocholerae, and Helicobacter pylori.
37. A method of making a compound of claim 1, comprising taking any
one of the intermediate compounds described herein and reacting it
with one or chemical reagents in one or more steps to produce a
compound of claim 1.
38. A product made by the method of claim 37.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/301,685, filed Jun. 28, 2001, which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to heterocyclic compounds and to
their use, for example, in the prophylaxis and or medical treatment
of bacterial infections and their use, for example, as antiseptics,
sterilizants, or disinfectants.
BACKGROUND OF THE INVENTION
[0003] The pathogenic processes by which microorganisms elicit
their adverse effects on subjects are generally complex and require
a defined sequence of events that implicate multiple microbial
components. If left unchecked, the proliferation of organisms can
impair the subject, resulting in chronic infection, or even death.
It is frequently necessary to bolster host defense mechanisms with
exogenous factors such as antibiotics to aid clearance of the
infecting organism from the subject.
[0004] Over time, and due in part to injudicious use of existing
antibiotic treatment regimens, organisms are becoming increasingly
resistant to the various exogenous factors available. For example,
resistance of bacteria to fluoroquinolones and beta-lactams has
been reported and will most probably increase over the next decade.
Fluoroquinolone resistance isolates from around of the world in
community-acquired pneumonia have also been increasingly described.
Further, there is a serious decrease in susceptibility of E. Coli
strains to the beta-lactams (e.g., amoxicillin), due to the
presence of R-TEM enzymes, to cotrimoxazole and trimethoprim. These
reports exemplify the necessity and continued need for the
discovery and development of new antimicrobial therapeutics in
order to provide alternative and more powerful treatment regimens
against increasingly resistant microorganisms.
SUMMARY OF THE INVENTION
[0005] The invention relates to heterocyclic compounds,
compositions comprising the compounds, and methods of using the
compounds and compound compositions. The compounds and compositions
comprising them are useful for treating disease or disease
symptoms. The invention also provides for methods of making the
compounds and methods for identifying compounds with desired
biological activity.
[0006] The invention is based on the discovery that certain
heterocyclic compounds have potent antibacterial activity, and more
specifically, activity against the enzyme D-alanyl-D-alanine ligase
("D-Ala-D-Ala ligase"; E.C. 6.3.2.4). As shown in the schematic
below, D-Ala-D-Ala ligase is believed to play a critical role in
bacterial cell growth by catalyzing assembly of D-alanyl-D-alanine
("D-Ala-D-Ala"), one of the building blocks used for peptidoglycan
crosslinking essential for bacterial cell wall biosynthesis. It is
thought that the enzyme establishes a peptide linkage that
ultimately provides the site of transacylation when the
peptidoglycan framework is crosslinked (Ellsworth et al., Chemistry
& Biology, 3:37-44, 1996). Without intending to be bound by any
theory as to the mechanism of action of the new compounds, the new
compounds are believed to bind to the adenosine triphosphate-(ATP-)
binding site of D-Ala-D-Ala ligase, and not to the D-Ala-binding
site, making the compounds competitive versus ATP. The compounds
therefore differ in this regard from other D-Ala-D-Ala ligase
inhibitors such as cycloserine and dipeptide phosphonate analogs,
which are competitive inhibitors for D-alanine and are believed to
bind to the D-alanine-binding site of the enzyme. ##STR1##
[0007] In general, the invention features compounds of the
following general structures: ##STR2## and uses thereof.
[0008] In particular, in one embodiment, the invention features a
compound having the formula: ##STR3##
[0009] where A and B can independently be either N or CR.sup.7,
where R.sup.7 is hydrogen or a carbon-, ED nitrogen-,
sulfur-halogen- and/or oxygen-containing function group (e.g.,
hydrogen, or a substituted or unsubstituted, linear, branched, or
cyclic, alkyl, alkenyl, alkynyl, aryl, aralkyl, or alkaryl group,
or a derivative of one or more of these groups where heteroatoms
are substituted for one or more of the carbon and/or hydrogen atoms
(e.g., amino groups, alkylamino groups, hydroxyl and alkoxyl
groups, thiol groups, halogens, nitro groups, phenolic groups, or
other substituted aromatic or aliphatic groups)). R.sup.1 and
R.sup.2 are identical or different --NR.sup.5R.sup.6 groups, where
each R.sup.5 and R.sup.6 can independently be hydrogen or a
carbon-containing functional group. R.sup.3 is hydrogen or an
alkyl, amino, hydroxy, alkoxy, or alkylamino group. R.sup.4 is a
carbon-, nitrogen-, sulfur-, halogen-, and/or oxygen-containing
functional group, provided that,
[0010] (1) if A and B are both nitrogen and R.sup.5 and R.sup.6 are
both hydrogen, then R.sup.4 is not --NH.sub.2, --N(H)(methyl),
--N(H)(butyl), --N(H)(hexyl), --N(H)(phenyl), --N(H)(benzyl),
--N(H)(NH.sub.2), --N(H)(CH.sub.2CH.sub.2OH),
--N(CH.sub.2CH.sub.2OH).sub.2, phenyl, N-piperadinyl, or
--S(ethyl);
[0011] (2) if A is CH, B is nitrogen, and R.sup.5 and R.sup.6 are
both hydrogen, then R.sup.4 is not methyl, isobutyl, phenyl,
4-methylphenyl, 4-chlorophenyl, 4-bromophenyl,
2-(2,5-dimethoxyphenyl)-ethyl, or --CH(OCH.sub.3).sub.2; and
[0012] (3) if A and B are both CH groups, then R.sup.4 is an amino
group other than --NH.sub.2, (3,4-dichlorophenyl)methylamino, or
(3,4-dichlorophenyl)methyleneimino.
[0013] In some cases R.sup.5 and R.sup.6 are both hydrogen.
[0014] R.sup.4 can be, for example, a substituted or unsubstituted,
linear, branched, or cyclic, alkyl, alkenyl, alkynyl, aryl,
aralkyl, or alkaryl group. In some cases, R.sup.4 includes at least
one aryl group. For example, R.sup.4 can be one of the following
groups: ##STR4##
[0015] , where R.sup.8-12 can independently be hydrogen or a
carbon-, nitrogen-, sulfur-halogen- and/or oxygen-containing
functional group (e.g., a linear or branched alkyl).
[0016] In another example where R.sup.4 includes an aryl group, the
compound has the structure: ##STR5## where R.sup.8-16 can
independently be hydrogen or a carbon-, nitrogen-, sulfur-halogen-
and/or oxygen-containing functional groups. For example, R.sup.9
can be hydrogen or methyl. In some cases, the compound has the
structure: ##STR6## where n is 1, 2, 3, or 4; R.sup.17 is
--NR.sup.18R.sup.19, where R.sup.18 and R.sup.19 can independently
be hydrogen or a carbon-, nitrogen-, sulfur-halogen- and/or
oxygen-containing functional group. In some cases, for example,
R.sup.17 can be one of the following moieties: ##STR7##
##STR8##
[0017] R.sup.8 can alternatively be, for example,
--(CH.sub.2).sub.nNR.sup.18R.sup.19, where n is 1, 2, 3, or 4; and
R.sup.18 and R.sup.19 can independently be hydrogen or a carbon-,
nitrogen-, sulfur-halogen- and/or oxygen-containing functional
group.
[0018] R.sup.4 can, alternatively, be --CH.sub.2O-aryl,
--CH.sub.2S-aryl, --CH.dbd.CH-aryl, or --NH(CH.sub.2-aryl), where
the aryl group can be any aromatic moiety, whether comprising
carbon and hydrogen only (e.g., phenyl, naphthyl, toluoyl) or
including other atoms (e.g., pyrrolyl, pyridyl, oxazolyl,
chlorophenyl, bromonaphthyl).
[0019] R.sup.4 can, alternatively, be --N(CH.sub.3)R.sup.21,
--N(CH.sub.2CH.sub.3)R.sup.21, --N(CH(CH.sub.3).sub.2)R.sup.21,
--N(benzyl)R.sup.21, --CH.sub.2NH.sub.2,
--NHCH.sub.2CH.sub.2NR.sup.22R.sup.23, or
--CH.sub.2NHC(.dbd.O)R.sup.22, wherein R.sup.21-23 can
independently be hydrogen or a carbon-, nitrogen-, sulfur-halogen-
and/or oxygen-containing functional group. In specific cases,
R.sup.22 is hydrogen and R.sup.23 is --C(.dbd.O)R.sup.24, where
R.sup.24 is hydrogen or a carbon-, nitrogen-, sulfur-halogen-
and/or oxygen-containing functional group.
[0020] In still other cases, R.sup.4 can be such that the compound
has the following structure: ##STR9## where R.sup.20 is hydrogen or
a carbon-, nitrogen-, sulfur-halogen- and/or oxygen-containing
functional group.
[0021] In other cases, R.sup.4 can be such that the compound has
the structure: ##STR10## where R.sup.25 and R.sup.26 can
independently be hydrogen or a carbon-, nitrogen-, sulfur-halogen-
and/or oxygen-containing functional group. For example, R.sup.25
can be hydrogen, alkyl, hydroxyalkyl, or aralkyl, and R.sup.26 can
be haloalkyl, hydroxyl, C(.dbd.O)OR.sup.27, or NR.sup.27R.sup.28,
where R.sup.27 and R.sup.28 can independently be hydrogen or a
carbon-, nitrogen-, sulfur-halogen- and/or oxygen-containing
functional group. R.sup.26 can alternatively be
--C(H)(aryl)(R.sup.29), where R.sup.29 is hydrogen or a carbon-,
nitrogen-, sulfur-halogen- and/or oxygen-containing functional
group. In some cases, R.sup.29 includes at least one acyl group
(e.g., a carboxylate, ketone, aldehyde, ester, amide, or thioester
group). In these or other cases, the aryl group can be a naphthyl
or benzothiophenyl group. R.sup.29 can include an alpha-hydroxy
carboxylate group (e.g., a --C(R)(OH)(COOR') group, where R and R'
can be hydrogen or a carbon-, nitrogen-, sulfur-halogen- and/or
oxygen-containing functional group).
[0022] In another embodiment, the invention features a compound
having the formula: ##STR11## where R.sup.1 and R.sup.2 are
identical or different --NR.sup.5R.sup.6 groups, where each R.sup.5
and R.sup.6 can independently be hydrogen or a carbon-containing
functional group; and R.sup.4 is an amino group other than
--NH.sub.2 or ##STR12##
[0023] In still another embodiment, the invention features a method
of inhibiting D-alanyl-D-alanine (D-Ala-D-Ala) ligase. The method
includes the step of exposing D-Ala-D-Ala ligase to a compound of
formula I or formula II: ##STR13## where
[0024] A and B can independently be N or CR.sup.7, where R.sup.7 is
hydrogen or a carbon-, nitrogen-, sulfur-halogen- and/or
oxygen-containing function group; R.sup.1 and R.sup.2 are identical
or different --NR.sup.5R.sup.6 groups, where each R.sup.5 and
R.sup.6 can independently be hydrogen or a carbon-containing
functional group; R.sup.3 and R.sup.4 can independently be hydrogen
or a carbon-, nitrogen-, sulfur-, halogen-, and/or
oxygen-containing functional group; provided that R.sup.3 and
R.sup.4 are not both hydrogen.
[0025] For example, R.sup.1 and R.sup.2 can both be --NH.sub.2.
[0026] In some cases, R.sup.3 and R.sup.4 can independently be
-branched or straight-chain alkyl, --O-alkyl, --O-alkyl-COOH,
--O-alkyl-NR.sup.7R.sup.8, --O-alkyl-OH, --NR.sup.7R.sup.8,
--NR.sup.7-alkyl-NR.sup.8R.sup.9, --NR.sup.7-alkyl-COOH,
--NR.sup.7-alkyl-OH; --CONR.sup.7R.sup.8,
--CONR.sup.7-alkyl-NR.sup.8R.sup.9, --CONR.sup.7-alkyl-COOH,
--CONR.sup.7-alkyl-OH, --S-alkyl, --S-alkyl-COOH,
--S-alkyl-NR.sup.7R.sup.8, --S-alkyl-OH, --O-aryl, --O-aryl-COOH,
--O-aryl-NR.sup.7R.sup.8, --O-aryl-OH, --S-aryl, --S-aryl-COOH,
--S-aryl-NR.sup.7R.sup.8, --S-aryl-OH,
--NR.sup.7-aryl-NR.sup.8R.sup.9, --NR.sup.7-aryl-COOH,
--NR.sup.7-aryl-OH; --CONR.sup.7-aryl-NR.sup.8R.sup.9,
--CONR.sup.7-aryl-COOH, --CONR.sup.7-aryl-OH, or
--CH.sub.2NR.sup.5C.sub.6H.sub.4COOH; provided that R.sup.3 and
R.sup.4 cannot simultaneously be identical branched or straight
chain alkyl groups; R.sup.1 and R.sup.2 can independently be
hydrogen, --NH.sub.2, or --NR.sup.11R.sup.12, where at least one of
R.sup.1 and R.sup.2 is --NH.sub.2; R.sup.5 can be lower alkyl
(i.e., C.sub.1-6 alkyl), hydrogen, or
--CH.sub.2NR.sup.10C.sub.6H.sub.4CONHR.sup.6; where R.sup.6 is
-alkyl, -alkyl-COOH, -alkyl-NH.sub.2, or -alkyl-OH; R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 can
independently be straight-chain alkyl, branched alkyl, aryl, or
acyl groups, optionally substituted with one or more oxygen,
nitrogen, sulfur, or halogen-based functional groups; and A can be
N and CH.
[0027] The D-Ala-D-Ala ligase inhibited can, for example, include a
sequence at least 90% identical to the sequence of a D-Ala-D-Ala
ligase from a species selected, for example, from the group
consisting of Escherichia coli, Chlamydophila pneumoniae, Chlamydia
trachomatis, Yersinia pestis, Haemophilus influenzae, Haemophilus
ducreyi, Pseudomonas aeruginosa, Pseudomonas putida, Xylella
fastidiosa, Bordetella pertussis, Thiobacillus ferrooxidans,
Neisseria meningitidis, Neisseria gonorrhoeae, Buchnera aphidicola,
Bacillus halodurans, Geobacter sulfurreducens, Rickettsia
prowazekii, Zymomonas mobilis, Aquifex aeolicus thermophile,
Thermotoga maritima, Clostridium difficile, Enterococcus faecium,
Streptomyces toyocaensis, Amycolatopsis orientalis, Enterococcus
gallinarum, Enterococcus hirae, Enterococcus faecium, Enterococcus
faecalis, Streptococcus pneumoniae, Streptococcus pyogenes,
Staphylococcus aureus, Bacillus subtilis, Bacillus
stearothermophilus, Deinococcus radiodurans, Synechocystis sp.,
Salmonella typhimurium, Mycobacterium tuberculosis, Mycobacterium
avium, Mycobacterium smegmatis, Legionella pneumophila, Leuconostoc
mesenteroides, Borrelia burgdorferi, Treponema pallidum, Vibrio
cholerae, and Helicobacter pylori.
[0028] In yet another embodiment, the invention features a method
of treating a patient. The method includes the step of
administering to the patient an effective amount of a compound of
formula I or formula II: ##STR14## where A, B, and R.sup.1-4 are
defined as above.
[0029] The invention also features a formulation that includes any
of the above compounds combined with an excipient suitable for
administration to a subject.
[0030] The invention also features a method of treating a subject
having a bacterial microbial infection. The method includes
administering to the subject an effective amount of a formulation
as described above. The subject can be, for example, an animal such
as a mammal (for example, a human, a horse, a lamb, a dog, a cat, a
rabbit, a mouse, a rat, a cow, a bull, a pig), a bird (for example,
a chicken, a goose, a turkey, a duck, a fowl), a fish (for example,
a salmon, a trout, a catfish, a goldfish), or other farm,
companion, or ornamental animal.
[0031] In yet another embodiment, the invention features a method
of treating a patient. The method includes the step of
administering to the patient an effective amount of any of the
above compounds, optionally with a suitable carrier.
[0032] In still another embodiment, the invention features a method
of inhibiting bacteria growth in a non-living system (e.g.,
sterilizing, disinfecting, killing bacteria in vitro). The method
includes the step of contacting the system (e.g., a medium, a
medical device, a kitchen or bathroom surface, an operating
theater), with an effective amount of any of the above compounds,
to inhibit bacterial growth.
[0033] Several parameters can be used in the selection of compounds
for use in the new methods. The parameters include, but are not
limited to, in vitro antibacterial potency and spectrum of
activity; physical-chemical properties such as lipophilicity and
solubility. The pharmacokinetic performance of the compounds of the
invention, as well as their in vitro antibacterial activity,
indicates that the compounds of the invention are useful both in
the prophylaxis and medical treatment of subjects that have
bacterial infections and as antiseptics, sterilizants, or
disinfectants.
[0034] The terms "halo" and "halogen" refer to any radical of
fluorine, chlorine, bromine or iodine.
[0035] The terms "alkyl", "alkenyl" and "alkynyl" refer to
hydrocarbon chains that may be straight-chain or branched-chain,
containing the indicated number of carbon atoms, if specified. For
example, "C.sub.1-10" or "C1-C10" indicates the group may have from
1 to 10 (inclusive) carbon atoms in it, or may by cyclic (e.g.,
including one or more rings). The terms "ring" and "ring system"
refer to a ring comprising the delineated number of atoms, said
atoms being carbon or, where indicated, a heteroatom such as
nitrogen, oxygen or sulfur (e.g., including a heterocyclyl group).
The ring itself, as well as any substituents thereon, may be
attached at any atom that allows a stable compound to be
formed.
[0036] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2 or 3 atoms of each
ring may be substituted by a substituent. Examples of aryl groups
include phenyl, naphthyl and the like, as well as heteroaryl
groups.
[0037] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered 13 bicyclic, or 11-14 membered tricyclic
ring system comprising 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S, wherein 0, 1, 2 or 3 atoms of
each ring may be substituted by a substituent. Examples of
heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,
benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl,
indolyl, thiazolyl, and the like.
[0038] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system comprising 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S, wherein 0, 1, 2 or 3 atoms of
each ring may be substituted by a substituent. Examples of
heterocyclyl groups include piperizinyl, pyrrolidinyl, dioxanyl,
morpholinyl, tetrahydrofuranyl, and the like.
[0039] Combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds. The term "stable", as used herein, refers to
compounds that possess stability sufficient to allow manufacture
and that maintains the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein (e.g.,
therapeutic or prophylactic administration to a subject or patient,
or antiseptic, wound dressing impregnation, sterilizant, or
disinfectant applications).
[0040] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the present specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0041] The invention can provide several advantages over the
existing methods of treatment. For example, the compounds of the
invention can bind to the ATP-binding site of the D-Ala-D-Ala
ligase enzyme with high specificity and are shown to be competitive
with ATP in biochemical assays. Some compounds described in this
invention have their protein-interacting functional groups situated
so as to be able to also bind to one or both of the D-alanine
binding sites of D-Ala-D-Ala ligase. These types of new compounds
(bisubstrate analogs) may have further enhanced selectivity and
potency directly associated with the ability to bridge the ATP site
and D-Ala site.
[0042] Some of the compounds of the invention may also be less
toxic than many existing antibiotics. The new compounds bind
specifically to D-Ala-D-Ala ligase, an enzyme found in bacteria but
not in human or other eukaryotic cells, so the new compounds
generally do not interfere with biological systems in patients.
Since peptidoglycans are present only in bacteria, and are absent
from mammalian cells, specific inhibition of D-Ala-D-Ala ligase can
result in highly selective antibacterial activity.
[0043] Moreover, some compounds of the invention may have several
chemical and pharmacological advantages over existing compounds
used in treating bacterial infections. These advantages may include
both chemical stability and pharmacological stability, as well as
potency, different resistance profiles, different selectivity
profiles, and decreased side-effects. The new compounds' activity
and ability to cross bacterial cell membranes also makes them
suitable for use as antibiotic drugs. The invention also envisions
veterinary uses for the treatment of infections in fish, fowl,
livestock, other food animals, sports animals, and companion
animals.
[0044] The compounds of the invention have displayed potent broad
spectrum activity against a representative panel of microorganisms,
including E. coli, S. aureus, S. pneumoniae, H. influenzae, and
others. Broad spectrum activity is also inferred from the close
sequence homology in the D-Ala-D-Ala ligases of fifty-one
representative, but evolutionarily diverse, microorganisms
representative of all bacteria. Nonetheless, individual compounds
do have greater activity against specific bacteria, creating
opportunities for the development of selective and specific
narrow-spectrum agents as well.
[0045] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The invention relates to the specific compounds exemplified
herein. Thus one embodiment of the invention is any compound
specifically delineated herein, including the compounds listed
below: ##STR15##
[0047] where A and B of structures I or II is independently either
--N--, --CH--, or --CR.sup.7--. R.sup.1, R.sup.2, R.sup.3, R.sup.4,
and R.sup.7 are independently selected.
[0048] The compounds of this invention can be synthesized using
conventional techniques. Advantageously, these compounds are
conveniently synthesized from readily available starting materials.
In general, the compounds of the formulae described herein are
conveniently obtained via methods illustrated in the schemes and
the Examples herein.
[0049] Thus, one embodiment relates to a method of making a
compound of the formulae described herein, comprising synthesizing
any one or more intermediates illustrated in the synthetic schemes
herein and then converting that intermediate(s) to a compound of
the formulae described herein. Another embodiment relates to a
method of making a compound of the formulae described herein,
comprising synthesizing any one or more intermediates illustrated
in the examples herein and then converting that intermediate(s) to
a compound of the formulae described herein. Another embodiment
relates to a method of making a compound of the formulae described
herein, comprising synthesizing any one or more intermediates
illustrated in the synthetic schemes herein and then converting
that intermediate(s) to a compound of the formulae described herein
utilizing one or more of the chemical reactions described in the
synthetic schemes or examples herein. Nucleophilic agents are known
in the art and are described in the chemical texts and treatises
referred to herein. The chemicals used in the aforementioned
methods can include, for example, solvents, reagents, catalysts,
protecting group and deprotecting group reagents, and the like. The
methods described above can also additionally comprise steps,
either before or after the steps described specifically herein, to
add or remove suitable protecting groups to ultimately allow
synthesis of the compound of the formulae described herein.
[0050] As can be appreciated by the skilled artisan, the synthetic
schemes herein are not intended to comprise a comprehensive list of
all means by which the compounds described and claimed in this
application can be synthesized. Additionally, the various synthetic
steps described above can be performed in an alternate sequence or
order to give the desired compounds. Synthetic chemistry
transformations and protecting group methodologies (protection and
deprotection) useful in synthesizing the compounds described herein
are known in the art and include, for example, those such as
described in R. Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective
Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991);
L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic
Synthesis, John Wiley and Sons (1994); M. B. Smith and J. March,
March's Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure, 5th Ed., Wiley Interscience (2001); and L. Paquette,
ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and
Sons (1995), and subsequent editions thereof.
[0051] In a typical method, compounds can be screened for
antibacterial activity against a plurality of different bacterial
strains. Compounds are assayed for potency and breadth of activity
against several strains in order to identify potential lead
compounds. The compounds can be screened for bacteriostatic
activity (i.e., prevention of bacterial growth) and/or
bacteriocidal activity (i.e., killing of bacteria). The lead
compounds can be optimized, for example, by varying substituents to
produce derivative compounds. The derivatives can be produced one
at a time or can be prepared using parallel or combinatorial
synthetic methods. In either case, the derivatives can be assayed
to generate structure-activity relationship (SAR) data, which can
then be used to further optimize the leads.
Design, Synthesis, and Biochemical Evaluation of Ligase
Inhibitors
[0052] Analogs were designed using a variety of approaches
including, traditional medicinal chemistry, systematic analoging
(e.g., systematically testing analogs with varying alkyl chain
lengths, isosteric functional groups, various aromatic
substituents), residue targeting using X-ray crystal structure
analysis, molecular modeling and computer active-site docking
experiments, computational diversity analysis, and iterative
feedback using the results from biochemical experiments. The
analogs were synthesized using a variety of synthetic methodologies
previously described in the literature by skilled artisans of the
craft. The rendered analogs were then analyzed using the described
biochemical methods herein to generate potency data. A diverse
sample of some of the analogs is described below.
[0053] We have identified a variety of substituents on the 6 and 7
positions of quinazolines, pteridines, pyridopyrimidines and
pyrimidinopyrimdines that have potent ligase inhibitory activity.
Table 1 below shows the diversity of substituents on the 6 and 7
positions capable of inhibiting the ligase enzyme (hydrogen atoms
necessary to complete the valence of nitrogen, oxygen, and carbon
atoms in the compounds are not shown, but would be understood to be
present by one of ordinary skill in the art). TABLE-US-00001 TABLE
1 Ki (E. coli ligase)/ Structure .mu.M ##STR16## 73 ##STR17## 31
##STR18## 37 ##STR19## 1 ##STR20## 55 ##STR21## 20 ##STR22## 12
##STR23## 20 ##STR24## 29 ##STR25## 14 ##STR26## 16 ##STR27## 3
##STR28## 9 ##STR29## 13 ##STR30## 12 ##STR31## 2 ##STR32## 12
##STR33## 92 ##STR34## 14 ##STR35## 0.59 ##STR36## 0.50 ##STR37##
2.9 ##STR38## 1.00 ##STR39## 0.50 ##STR40## 8.7 ##STR41## 0.13
N7-Substitution Using Lower Alkyl Groups
[0054] Various analogs of the N7-nitrogen (R1 in Table 2 below) can
be made to increase potency. Methyl, ethyl, allyl, and cyclopropyl
methyl were the most potent lower alkyl and cycloalkyl
substitutents identified. Substitution at the alpha-position (R2 in
Table 2) was allowed, in some cases CH.sub.3 and C.sub.2H.sub.5
dramatically improved activity. It would not be surprising if other
substituents at the R2 position would increase the potency of
ligase inhibition as well. Substitution of the naphthyl ring in the
4-position increased potency using --H, --CH.sub.3, or -halogen.
Replacement of the naphthyl ring with various heterocycles (for
example, benzothiophene) was found to yield analogs having potent
in vitro enzymatic activity. In this series of analogs, the
R-alpha-methyl stereoisomers were significantly more potent than
the racemates. The S-alpha-methyl stereoconfiguration provided
analogs with broader spectrum ligase potency, at the cost of high
intrinsic activity against the E. coli ligase. TABLE-US-00002 TABLE
2 E. coli Staph ligase ligase N--R1 R2 R2 chirality R3 (.mu.M)
(.mu.M) --CH3 CH3 R/S H 0.718 9.28 --CH3 C2H5 R/S H 0.452 4.60
--CH3 CH3 R H 0.326 26.80 --CH3 CH3 R/S CH3 0.277 12.25 --CH3 CH3
R/S 4,5-butylene 0.240 12.00 --CH3 CH3 R/S Cl 0.201 4.90 CH3 CH3 R
F 0.104 27.00 --CH2CH3 CH3 R/S F 0.257 19.00 --CH2CH3 CH3 R/S CH3
0.210 38.50 --CH2CH3 CH3 R CH3 0.102 71.00 --CH2CH.dbd.CH2 CH3 R/S
H 0.258 30.50 --CH2CH(CH2)2 CH3 R/S H 0.345 17.00 --CH2CH(CH2)2 CH3
R/S Br 0.279 25.00 --CH2CH(CH2)2 CH3 R/S CH3 0.211 37.00
--CH2CH(CH2)2 CH3 R/S Cl 0.179 29.00 --CH2CH(CH2)2 CH3 R CH3 0.174
62.00
Beta-Alanine Amides Analogs
[0055] Amides synthesized using the beta-alanine linker had
broad-spectrum activity. The most potent, broad-spectrum inhibitor
identified from this series was an ethylenediamine amide derivative
(i.e., the first compound in Table 3 below), which had Ki's for E.
coli and H. influenzae ligases of less than one micromolar; S.
aureus and S. pneumoniae ligase activity were single digit
micromolar (see Table 3). The meta-aminomethylbenzylamine analog
(i.e., the third compound in Table 3) was found to have Ki's less
then 1 .mu.M against 3 of the 4 ligases in the panel. In a similar
manner, the last compound in Table 3 was potent against three
ligase species. The fourth compound in Table 3, in which the
primary amine was substituted with an amidine, showed activity
similar to that of the first compound. TABLE-US-00003 TABLE 3 E.
coli Staph Strep ligase H. Influenzae ligase ligase Ki/ATP ligase
Ki/ATP Ki/ATP Ki/ATP MOLSTRUCTURE (uM) avg (uM) avg (uM) avg (uM)
avg ##STR42## 0.7 1.0 4.7 1.9 ##STR43## 9.6 0.267 9.5 3.1 ##STR44##
5.3 0.4 0.9 0.9 ##STR45## 1.0 0.6 6.3 1.4 ##STR46## 1.9 1.3 3.8 4.1
##STR47## 8.7 1.6 1.2 1.6
Carboxylic Acids and Alpha-Hydroxy Carboxylic Acids
[0056] The first compound in Table 4 was submitted as an HPLC
purified mixture of at least two stereoisomers. Protein-ligand
crystal structure analysis of this compound showed two
stereoisomers, the stereochemistry of which was determined to be
(2-R, 1'-S) and (2-S, 11'-R). The number 2 refers to the
alpha-hydroxy position, and the number 1' refers to the
alpha-methyl position. The two observed isomers are enantiomeric,
i.e., they are mirror images. The E. coli ligase potency of the
last compound in Table 4 was found to be 143 mM. The compound has a
relatively broad spectrum of activity against H. influenzae (566
nM), Staph (4.1 .mu.M), and Strep (2.6 .mu.M). TABLE-US-00004 TABLE
4 E. coli Staph Strep ligase H. Influenzae ligase ligase Ki/ATP
ligase Ki/ATP Ki/ATP Ki/ATP MOLSTRUCTURE (uM) avg (uM) avg (uM) avg
(uM) avg ##STR48## 0.611 -- 7.8 -- ##STR49## 5.3 3.7 6.7 2.1
##STR50## 4.2 5.7 10 14.6 ##STR51## 1.4 3.5 4.3 4.3 ##STR52## 0.143
0.566 4.1 2.6
N7-Primary Butyl Amine SAR
[0057] Maximum in vitro potency in this series was identified as
that having a butyl amine chain off the N7-nitrogen (i.e., the
third compound in Table 5). The alpha-methyl naphthyl chiral center
can be replaced with the achiral 2-ethoxynaphthyl substituent (see
the fourth compound in Table 5) and still maintain potent ligase
activity. The achiral molecule has a broader spectrum of ligase
activity, and was found to have a Ki of 102 nM against the ligase
isolated from E. coli. TABLE-US-00005 TABLE 5 E. coli Staph Strep
ligase H. Influenzae ligase ligase Ki/ATP ligase Ki/ATP Ki/ATP
Ki/ATP MOLSTRUCTURE (uM) avg (uM) avg (uM) avg (uM) avg ##STR53## 2
2.7 58 439 ##STR54## 0.457 5.5 46 81.2 ##STR55## 0.135 4.2 32 23.1
##STR56## 0.105 0.52 15 4.5
Additional Analogs
[0058] ##STR57## ##STR58## ##STR59## ##STR60## General Synthetic
Methodologies used in the Preparation of Analogs.
[0059] Synthesis of Pyrimidopyrmidines ##STR61##
[0060] Pyrimidopyrimidine compounds of the invention can be
prepared using a variety of synthetic strategies. The
pyrimidopyrimidine ring system can be synthesized in a multi-step
reaction sequence starting from an appropriately substituted
amidine (R7-C.dbd.NHNH2) and an R5-substituted
alkoxylmethylenemalonitrile. The resulting cyanoaminopyrimidine can
be condensed with guanidine to form the pyrimidopyrimine ring
system. In the case where R7 in the cyanoaminopyrimidine is either
--Cl, --Br, --S-lower alkyl, these leaving groups can be
substituted with substituted nitrogen or oxygen nucleuophiles to
provide R7-N(or O)-substituted alkyl or aryl intermediates. These
intermediates can be cyclized to their corresponding
pyrimidopyrimidines with appropriately substituted at the
7-position. ##STR62##
[0061] Another method to synthesize 7-aminosubstituted
pyrimidopyrimidines is through the nucleophilic attack of amines on
6-amino-2-bromopyrimidine-5-carbonitrile (chloro or thiomethyl, or
thioethyl can also be used as leaving groups at the 2-position),
and subsequent cyclization of the resulting, appropriately
substituted cyanoaminopyrimidine with guanidine.
[0062] If the attacking, appropriately substituted nucleophilic
amine is not commercially available, then it can be prepared using
standard methods in organic chemistry. One such standard method
used in preparing compounds in this application is by
reductive-amination. ##STR63##
[0063] In this well-known procedure, an aldehyde or ketone is
condensed with an appropriately substituted amine in the presence
of a mild reducing agent such as sodium cyanoborohydride or zinc
cyanoborohydride.
[0064] Beta-Alanine Amides and Alpha-Hydroxy-Beta-Alanine Amides
##STR64##
[0065] Amides can be synthesized by reaction of their corresponding
carboxylic acids with various primary and secondary amines.
Addition of numerous reagents described in the literature are
useful to facilitate the amide coupling process. Included in these
reagents are carbonyldiimidazole (CDI), dicyclohexylcarbodiimide
(DCC), HBTU, diethylphosphorocyanidate, and BroP.
[0066] Butylamine Analogs ##STR65##
[0067] The butylamine analogs were synthesized using a multi-step
synthetic pathway. In general, a mono-boc protected butanediamine
was reacted under reductive amination conditions to produce an
appropriately substituted mono-boc-protected naphthylmethylamine.
The secondary amine was reacted with
6-amino-5-cyano-2-halopyrimidine and the resulting intermediate
cyclized with quanidine, followed by boc-cleavage under acidic
conditions to provide the deprotected butane diamine analogs.
[0068] Direct Heterocyclic Substitution Methods ##STR66##
[0069] Compounds of the invention can also be prepared by the
aromatic nucleophilic displacement of leaving groups on the
7-position of 2,4-diaminopyrimidopyrimidine. These leaving groups
[LG] include, but are not limited to: --Cl, --Br, --SCH.sub.3,
--SC.sub.2H.sub.5, and --N(CH.sub.3).sub.3. The nucleophile used in
the displacement reaction can be --N-alkyl, --N-aryl, or
substituted alkyl or aryl amines, or --O-alkyl, --O-aryl, or
substituted alcohols or phenols.
Synthesis of Pterin Analogs
[0070] In general either 6- or 7-substituted pterin analogs can be
prepared by the reaction of an activated reagent such as 6- or
7-chloromethyl pterin with nucleophiles such as amines, and by
various other methods described in the literature other functional
groups at the 6- and 7-position of pterin such as bromomethyl,
iodomethyl, hydroxymethyl, activated hydrodroxymethyl, carbonyl,
activated carbonyl, hydroxy, chloro, bromo, or methyl can be used
as synthetic reagents for the preparation of 6- or 7-substituted
analogs. ##STR67##
[0071] The above general reaction pathway can be used to synthesize
a broad range of 7-substituted pteridine analogs. In a general
procedure, 7-chloromethyl and an appropriate amine are reacted in
an appropriate solvent such as DMF or 2-methoxyethanol for as long
as needed as determined by analysis of the reaction mixture by
HPLC, TLC, or NMR. The solvent is then removed and the product
purified by an appropriate method, usually in the form of
precipitation, recrystallization, re-precipitation of the salt by
base, or through chromatography.
Formulations, Salt Forms, and Prodrugs
[0072] As used herein, the compounds of this invention, including
the compounds of formulae described herein, are defined to include
pharmaceutically acceptable derivatives or prodrugs thereof. A
"pharmaceutically acceptable derivative or prodrug" means any
pharmaceutically acceptable salt, ester, salt of an ester, or other
derivative of a compound of this invention that, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or that enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species. Preferred prodrugs include derivatives where a
group that enhances aqueous solubility or active transport through
the gut membrane is appended to the structure of formulae described
herein. In particular, classical examples of ester prodrugs to
assist in the absorption and cell membrane penetration of analogs
containing free carboxylic acid functional groups can be
prepared.
[0073] The compounds of this invention can be modified by appending
appropriate functionalities to enhance selective biological
properties. Such modifications are known in the art and include
those that increase biological penetration into a given biological
compartment (e.g., blood, lymphatic system, central nervous
system), increase oral availability, increase solubility to allow
administration by injection, alter metabolism and alter rate of
excretion.
[0074] Pharmaceutically acceptable salts of the compounds of this
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate,
picrate, pivalate, propionate, salicylate, succinate, sulfate,
tartrate, thiocyanate, tosylate and undecanoate. Other acids, such
as oxalic, while not in themselves pharmaceutically acceptable, can
be employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts.
[0075] Salts derived from appropriate bases include alkali metal
(e.g., sodium), alkaline earth metal (e.g., magnesium), amimonium,
and N(alkyl).sub.4.sup.+salts. This invention also envisions the
quaternization of any basic nitrogen-containing groups of the
compounds disclosed herein. Water or oil-soluble or dispersible
products may be obtained by such quaternization.
[0076] Assays for Inhibition of D-Ala-D-Ala Ligase
[0077] Inhibition of D-Ala-D-Ala ligase can be assayed for using
the pyruvate kinase/lactate dehydrogenase (PK/LDH) assay described
in Example 2, and as described in the literature (e.g., in Sarthy
et al., Anal. Biochem., 254:288-290, 1997). In the bacterial cell
wall synthesis process, the ligase catalyzes the conversion of
adenosine triphosphate (ATP) to adenosine diphosphate (ADP)
concurrent with the ligation of two D-alanine residues to form
D-alanyl-D-alanine. PK then regenerates ATP from the ADP thus
created simultaneously with the conversion of phosphopyruvate to
pyruvate. LDH catalyzes the reduction of pyruvate to lactate by
converting NADH to NAD.sup.+. By monitoring the production rate of
NAD.sup.+ (e.g., using UV/Vis spectroscopy), D-Ala-D-Ala ligase
activity can be ascertained.
[0078] Compounds can be screened for % inhibition as described in
Example 3.
[0079] The inhibition constant Ki and mode of action can be
obtained as described in Example 4.
[0080] The protein sequence for the enzyme D-ala-D-ala ligase has
been determined in a variety of different bacterial species using
standard techniques in biochemistry (see Table 5). The protein
sequence from any species can be overexpressed in an appropriate
host organism such as E. coli using standard molecular biology
techniques. The ligase enzyme can be harvested, purified, and used
in the above described assay for the determination of inhibitory
activity.
[0081] In Vitro Assays for Antibacterial Activity
[0082] The compounds can be screened for antibacterial activity
using standard methods.
[0083] In one example, illustrated in Example 5 below, broth
microdilution techniques are used to measure in vitro activity of
the compounds against a given bacterial culture, to yield minimum
inhibitory concentration (MIC) data.
[0084] Microdilution Antimicrobial Susceptibility Test Assay
[0085] Stock solutions of tested compounds are prepared in
N,N-dimethylformamide (DMF) at .alpha.-concentration of 5 mg/ml.
Working solutions of the tested compounds were then prepared from
the stock solutions, in Mueller-Hinton broth (MHB) with a starting
concentration of 64 .mu.g/ml.
[0086] Bacterial inocula were prepared from overnight culture
(i.e., one fresh colony from agar plate in 5 ml MHB; H. influenzae
was grown in MHB with the addition of yeast extract, haematin, and
NAD), centrifuged 2.times.5 min/3000 rpm (for S. pneumoniae and H.
influenzae, 2.times.10 min/3000 rpm), and dispensed in 5 ml of
fresh MHB each time, such that the bacterial suspension is diluted
to obtain 100 colony forming units (cfu) in a microplate well (100
.mu.l total volume).
[0087] Microplate wells were filled with two-fold dilutions of test
compound (50 .mu.l), starting with 64 .mu.g/ml. Wells were then
filled with 50 .mu.l of bacterial inoculum (final volume: 100
.mu.l/well). The plates were incubated at 37.degree. C. for 18-24
hours (S. pneumoniae was grown in a CO.sub.2-enriched
atmosphere).
[0088] The optical density of each well at 590 nm (OD.sub.590) was
then measured with a TECAN SpectroFluor Plus.RTM., and minimum
inhibitory concentration (MIC) was defined as the concentration
that showed 90% inhibition of growth. In one example, illustrated
in Example 5, broth microdilution techniques are used to measure in
vitro activity of the compounds against a given bacterial culture,
to yield minimum inhibitory concentration (MIC) data.
[0089] Antimicrobial Agar Dilution Test
[0090] This assessment is performed essentially as described in
known literature. [See, e.g., NCCLS. Methods for Antimicrobial
Susceptibility Testing of Anaerobic Bacteria; Approved
Standard-Fourth Edition. NCCLS document M 11-A4. NCCLS: Wayne,
Pennsylvania; 1997.]
[0091] Agar medium: Brucella blood agar supplemented with hemin (5
.mu.g/ml), 5% sheep blood, and vitamin K.sub.1 (1 .mu.g/ml).
[0092] Antimicrobial Agents: Standard antimicrobial powders (e.g.,
azithromycin, chloramphenicol, nitrofurantoin, piperacillin,
clindamycin, penicillin, imipenem) and test compound, are prepared
as stock solutions [5120 .mu.g/ml in DMF (dimethylformamide)] and
diluted as indicated in Table 3 of the NCCLS Methods for
Antimicrobial Susceptibility Testing of Anaerobic bacteria;
Approved Standard-Fourth Edition 1997; M11-A4, Vol. 17 No 22.
[0093] Inoculum Preparation: The test anaerobic strains are
selected from enriched Brucella blood agar. Portions of five
colonies are directly suspended into Brucella broth medium to
achieve a turbidity equivalent to a 0.5 McFarland standard.
[0094] Procedure: The medium is prepared according to the
manufacturer's directions and distributed into screw-cap tubes. On
the day of the test, blood supplement and 2 ml of each
concentration of the antimicrobial agent are added to the
appropriate tubes of cooled (50.degree. C.) agar. The mixture of
media and antimicrobial agent is poured into standard (15.times.100
mm) round petri dishes and allowed to solidify. A
turbidity-adjusted culture of each anaerobic strain is inoculated
to each plate by a replicating device (approximately 2 .mu.l per
spot). The inoculated plates are incubated at 35.degree. C. in an
anaerobic jar. Results are recorded after 48 hours of incubation
and expressed as minimum inhibitory concentration (MIC) values.
[0095] In Vivo Assays for Antibacterial Activity
[0096] The compounds can also be tested for antibacterial efficacy
in laboratory animals. These in vivo studies include, but are not
limited to, systemic and topical models of infection, urinary tract
infection models, sepsis, antibiotic mediated colitis and wound
care. The compounds of the invention can also be evaluated in
animals to assess their pharmacokinetic profiles, such as oral
bioavailability, oral absorption, chemical half-life,
identification of metabolites, serum levels at various times, and
rate of excretion, for example.
[0097] Systemic Bacterial Infection Animal Models
[0098] Systemic models of infection are described in the
literature. The following conditions can be used to assay the
compounds in this application. Bacteria are grown in Mueller-Hinton
agar at 37.degree. C. during 24 h. For each experiment, a bacterial
suspension is prepared by inoculating 4-5 bacterial colonies onto
Mueller-Hinton broth (MHB) and by incubating at 37.degree. C. for
24 hours to yield approximately 10.sup.9 CFU/ml. BalbC female mice
are supplied by Charles River. Animals are infected by a single
administration of an LD.sub.100 dose of bacterial culture
suspension (1.times.10.sup.8 CFU/100 .mu.l per animal) in the tail
vein. A careful clinical examination is made several times a day,
and obvious clinical symptoms and mortality are recorded. Animals
survival is observed for a period of 6 days. Azithromycin is
dissolved in 0.5% methocel in saline solution and administered
orally. Test compounds are micronized with mortar and pestle and
then dissolved in methocel saline solution with 3% of DMF. The
first dose is administered 30 minutes after infection, with
following doses every 12 hours for 3 days.
[0099] Assays for Biochemical and Physical-Chemical Properties
[0100] The heterocyclic compounds of this invention can be
optimized for their in vitro "antibacterial" activity according to
the results of two types of methods, structural methodology and
physical-chemical methodology. The chemical structure can be
modified using combinations of substituents to provide compounds
that satisfy some or all of the following criteria: 1) a compound
in which the calculated or experimentally determined lipophilicity
(logP) is in the range of 0 to 2 logP units; 2) a compound that is
a substrate for any D-Ala-D-Ala ligase enzyme; 3) a compound in
which its aqueous solubility is greater than 1 .mu.g/ml. These
physical-chemical and biochemical properties are factors in the
antimicrobial effects seen in subjects (e.g., animals).
Clinical Uses of the Heterocyclic Compounds
[0101] The compounds claimed in this invention can be used
therapeutically or prophylactically for treatment or prevention of
bacterial infections and/or diseases.
[0102] The invention also relates to methods, for example, of
disrupting the internal regulation of microbial growth, in a
subject, comprising the step of administering to said subject a
compound of any of the formulae described herein or a composition
comprising a compound of any of the formulae described herein. In
one embodiment, the invention relates to a method of inhibiting
microbial or bacterial activity in a subject comprising the step of
administering a compound to the subject, or a composition
comprising a compound, of any one of the formulae described herein.
Preferably, the subject is a human being or animal.
[0103] In an alternate embodiment, this invention relates to a
method of treating disease or disease symptoms in a subject
comprising the step of administering to said subject a compound, or
a composition comprising a compound, of any of the formulae
described herein. Preferably, the subject is a human being or
animal.
[0104] Infections and infectious diseases are caused from a variety
of microorganisms. The compounds of the invention may find use in
the medical treatment of infectious diseases from bacterial
sources.
[0105] Compounds that kill or limit the growth of microorganisms
may find use in the treatment of infections and infectious
diseases. Specific bacterial microorganisms are known to be
associated with the type of infection or infectious disease. Some
examples of bacterial infections and their most common causative
pathogens are given below.
[0106] Upper and lower respiratory tract infections include, but
are not limited to: bronchitis, sinusitis, pneumonia, sore throat,
chronic streptococcal infections, diphtheria, acute epiglottitis,
influenza, chronic bronchitis, middle ear infections (otitis
media), pneumonia, bronchopneumonia, Legionnaire's disease,
atypical pneumonia, whooping cough, and tuberculosis.
[0107] Bacterial microorganisms causing respiratory tract
infections include but are not limited to: S. pyogenes, S.
pneumoniae, S. aureus, H. influenzae, M. catarrhalis, N.
meningitidis, B. pertussis, Enterobacteriaceae, anaerobes,
Nocardia, Pseudomonas, C. psittaci, and C. diphtheriae.
[0108] Urinary tract infections include, but are not limited to:
urethritis, cystitis, pyelonephritis (kidney infection),
asymptomatic bacteruria, interstitial cystitis, acute urethral
syndrome, and recurrent urinary tract infections.
[0109] Bacterial microorganisms causing urinary tract infections
include but are not limited to: E. coli, Proteus, Providentia,
Pseudomonas, Klebsiella, Enterobacter, Serratia, Coag. neg.
Staphylococci, Enterococci, and C. trachomatis.
[0110] Skin and wound infections include, but are not limited to:
erythrasma, panaritium, impetigo, folliculitis, erysipelas,
cellulitis, and necrotizing fasciitis.
[0111] Bacterial microorganisms causing skin and wound infections
include but are not limited to: Streptococci, Staphylococci, P.
aeruginosa, P. acnes, Clostridia, anaerobes, and B. fragilis.
[0112] Bacterial microorganisms causing systemic infections
(bacteremia) include but are not limited to: Streptococci,
Staphylococci, Enterobacteriaceae, Pseudomonas, Bacteroides sp.,
Neisseria, H. influenzae, Brucella, Listeria, and S. typhi.
[0113] Sexually transmitted diseases of bacterial origin include,
but are not limited to: adnexitis, cervicitis, chanchroid,
urethritis, balanitis, gonorrhea, lymphogranuloma venereum,
syphilis, and granuloma inguinale.
[0114] Bacterial microorganisms causing sexually transmitted
infections include but are not limited to: Chlamydia, N.
gonorrhoeae, U. urealyticum, T pallidium, G. vaginalis, H. ducreyi,
C. granulomatis, Streptococci, Staphylococci, and
Enterobacteriae.
[0115] Gastrointestinal infections of bacterial origin include but
are not limited to: food borne infections, colitis, enteritis,
gastric ulcers, duodenal ulcers, pancreatitis, gall bladder
infections, cholera, and thyphus.
[0116] Bacterial microorganisms causing gastrointestinal infections
include but are not limited to: H. pylori, C. pylori, C. duodeni,
S. typhi, S. paratyphi, V. cholerae, anaerobes, Enterobacteriaceae,
Staphylococci, and Streptococci.
Methods of Treating Patients
[0117] E The heterocyclic compounds of the formulae delineated
herein can be administered to a patient, for example, in order to
treat an infection such as a bacterial infection. The heterocyclic
compounds can, for example, be administered in a pharmaceutically
acceptable carrier such as physiological saline, in combination
with other drugs, and/or together with appropriate excipients. The
heterocyclic compounds of the formulae herein can, for example, be
administered by injection, intravenously, intraarterially,
subdermally, intraperitoneally, intramuscularly, or subcutaneously;
or orally, buccally, nasally, transmucosally, topically, in an
ophthalmic or otic preparation, or by inhalation, with a dosage
ranging from about 0.001 to about 100 mg/kg of body weight,
preferably dosages between 10 mg and 5000 mg/dose, every 4 to 120
hours, or according to the requirements of the particular drug.
[0118] As the skilled artisan will appreciate, lower or higher
doses than those recited above may be required. Specific dosage and
treatment regimens for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health status, sex, diet,
time of administration, rate of excretion, drug combination, the
severity and course of the disease, condition or symptoms, the
patient's disposition to the disease, condition or symptoms, and
the judgment of the treating physician.
[0119] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0120] In an alternate embodiment, this invention provides methods
of treating, preventing, or relieving symptoms of disease in a
mammal comprising the step of administrating to said mammal any of
the pharmaceutical compositions and combinations described above.
Preferably, the mammal is a human. If the pharmaceutical
composition only comprises the compound of this invention as the
active component, such methods may additionally comprise the step
of administering to said mammal an additional therapeutic agent
such as, for example, macrolide antibiotics (e.g., clarithromycin),
proton pump inhibitors (e.g., omeprazole), rifamycins (e.g.,
rifampin), aminoglycosides (e.g., streptomycin, gentamycin,
tobramycin), penicillins (e.g., penicillin G, penicillin V,
ticarcillin), .beta.-lactamase inhibitors, cephalosporins (e.g.,
cefazolin, cefaclor, ceftazidime), and antimycobacterial agents
(e.g., isoniazid, ethambutol). Other suitable m agents are
delineated in infectious disease texts and publications, including
for example, Principles and Practice of Infectious Diseases, G. L.
Mandell et al. eds., 3.sup.rd ed., Churchhill Livingstone, New
York, (1990). Such additional(s) agent may be administered to the
mammal prior to, concurrently with, or following the administration
of the composition having a compound of any of the formulae
herein.
[0121] Pharmaceutical compositions of this invention comprise a
compound of the formulae described herein or a pharmaceutically
acceptable salt thereof; an additional agent selected from an
anticancer agent, an antiviral agent, antifungal agent, antibiotic,
and any pharmaceutically acceptable carrier, adjuvant or vehicle.
Alternate compositions of this invention comprise a compound of the
formulae described herein or a pharmaceutically acceptable salt
thereof; and a pharmaceutically acceptable carrier, adjuvant or
vehicle. Such compositions can optionally also comprise additional
therapeutic agents, including, for example an additional agent
selected from an anticancer agent, an antimicrobial agent, an
antiviral agent, antifungal agent, proton pump inhibitor, or
antibiotic. The compositions delineated herein include the
compounds of the formulae delineated herein, as well as additional
therapeutic agents if present, in amounts effective for achieving a
modulation of microbial or bacterial levels.
[0122] The term "pharmaceutically acceptable carrier or adjuvant"
refers to a carrier or adjuvant that can be administered to a
patient, together with a compound of this invention, and that does
not destroy the pharmacological activity thereof and is nontoxic
when administered in doses sufficient to deliver a therapeutic
amount of the compound.
[0123] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, self-emulsifying drug delivery systems
(SEDDS) such as d-.alpha.-tocopherol polyethyleneglycol 1000
succinate, surfactants used in pharmaceutical dosage forms such as
Tweens or other similar polymeric delivery matrices, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-.beta.-cyclodextrins, or other solubilized
derivatives may also be advantageously used to enhance delivery of
compounds of the formulae described herein.
[0124] The pharmaceutical compositions of this invention can be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir, preferably by oral administration or administration by
injection. The compositions can be derived from crystalline or
non-crystalline forms of the compounds. The pharmaceutical
compositions of this invention may contain any conventional
non-toxic pharmaceutically-acceptable carriers, adjuvants or
vehicles. In some cases, the pH of the formulation may be adjusted
with pharmaceutically acceptable acids, bases or buffers to enhance
the stability of the formulated compound or its delivery form. The
term "parenteral" as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion
techniques.
[0125] The pharmaceutical compositions can be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween 80) and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural pharmaceutically
acceptable oils, such as olive oil or castor oil, especially in
their polyoxyethylated versions. These oil solutions or suspensions
may also contain a long-chain alcohol diluent or dispersant, or
carboxymethyl cellulose or similar dispersing agents that are
commonly used in the formulation of pharmaceutically acceptable
dosage forms such as emulsions and or suspensions. Other commonly
used surfactants such as Tweens or Spans and/or other similar
emulsifying agents or bioavailability enhancers that are commonly
used in the manufacture of pharmaceutically acceptable solid,
liquid, or other dosage forms may also be used for the purposes of
formulation.
[0126] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, emulsions and aqueous
suspensions, dispersions and solutions. In the case of tablets for
oral use, carriers that are commonly used include lactose and corn
starch. Lubricating agents, such as magnesium stearate, are also
typically added. For oral administration in a capsule form, useful
diluents include lactose and dried cornstarch. When aqueous
suspensions and/or emulsions are administered orally, the active
ingredient may be suspended or dissolved in an oily phase is
combined with emulsifying and/or suspending agents. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0127] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
that is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0128] Topical administration of the pharmaceutical compositions of
this invention is especially useful when the desired treatment
involves areas or organs readily accessible by topical application.
For application topically to the skin, the pharmaceutical
composition should be formulated with a suitable ointment
containing the active components suspended or dissolved in a
carrier. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical composition can be formulated
with a suitable lotion or cream containing the active compound
suspended or dissolved in a carrier with suitable emulsifying
agents. Suitable carriers include, but are not limited to, mineral
oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,
cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The
pharmaceutical compositions of this invention may also be topically
applied to the lower intestinal tract by rectal suppository
formulation or in a suitable enema formulation. Topically
transdermal patches are also included in this invention.
[0129] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0130] The compounds and compositions of this invention are useful
as sterilizants, antiseptics, adjuvants in wound dressings (e.g.,
bandages), and adjuvants in wound cleansing methods (swipes,
gavage, etc.).
[0131] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, alternatively between about 0.5 and about 75 mg/kg
body weight per day (e.g., between about 10 mg and 5000 mg/dose) of
the antimicrobial compounds described herein are useful in a
monotherapy and/or in combination therapy for the prevention and
treatment of microbial mediated disease. Typically, the
pharmaceutical compositions of this invention will be administered
from about 1 to about 6 times per day or alternatively, as a
continuous infusion. Such administration can be used as a chronic
or acute therapy. The amount of active ingredient that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the subject treated and the particular
mode of administration. A typical preparation will contain from
about 5% to about 95% active compound (w/w). Alternatively, such
preparations contain from about 20% to about 80% active compound.
When the compositions of this invention comprise a combination of a
compound of the formulae described herein and one or more
additional therapeutic or prophylactic agents, both the compound
and the additional agent should be present, typically, at dosage
levels of between about 10 to 100%, and more preferably between
about 10 to 80% of the dosage normally administered in a
monotherapy regimen. The additional agents can be administered
separately, as part of a multiple dose regimen, from the compounds
of this invention. Alternatively, those agents may be part of a
single dosage form, mixed together with the compounds of this
invention in a single composition.
Other Uses
[0132] In an alternate embodiment, the inhibitory compounds
described herein may be used as platforms or scaffolds that can be
utilized in combinatorial chemistry techniques for preparation of
derivatives and/or chemical libraries of compounds. Such
derivatives and libraries of compounds have antimicrobial activity
and are useful for identifying and designing compounds possessing
antimicrobial activity. Combinatorial techniques suitable for
utilizing the compounds described herein are known in the art as
exemplified by Obrecht, D. and Villalgrodo, J. M., Solid-Supported
Combinatorial and Parallel Synthesis of Small-Molecular-Weight
Compound Libraries, Pergamon-Elsevier Science Limited (1998), and
include those such as the "split and pool" or "parallel" synthesis
techniques, solid-phase and solution-phase techniques, and encoding
techniques (see, e.g., Czarnik, A. W., Curr. Opin. Chem. Bio.,
(1997) 1, 60). Thus, one embodiment relates to a method of using
the compounds described in the formulae herein for generating
derivatives or chemical libraries comprising: 1) providing a body
comprising a plurality of wells; 2) providing one or more compounds
of the formulae described herein in each well; 3) providing an
additional one or more chemicals in each well; 4) isolating the
resulting one or more products from each well. An alternate
embodiment relates to a method of using the compounds described in
the formulae herein for generating derivatives or chemical
libraries comprising: 1) providing one or more compounds of the
formulae described herein attached to a solid support; 2) treating
the one or more compounds of the formulae described herein attached
to a solid support with one or more additional chemicals; 3)
isolating the resulting one or more products from the solid
support. In the methods described above, "tags" or identifier or
labeling moieties may be attached to and/or detached from the
compounds of the formulae herein or their derivatives, to
facilitate tracking, identification or isolation of the desired
products or their intermediates. Such moieties are known in the
art. The chemicals used in the aforementioned methods may include,
for example, solvents, reagents, catalysts, protecting group and
deprotecting group reagents and the like. Examples of such
chemicals are those that appear in the various synthetic and
protecting group chemistry texts and treatises referenced
herein.
[0133] The compounds of this invention may contain one or more
asymmetric centers and thus occur as racemates and racemic
mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are expressly included in the present invention. The compounds of
this invention may also be represented in multiple tautomeric
forms; in such instances, the invention expressly includes all
tautomeric forms of the compounds described herein (e.g.,
alkylation of a ring system may result in alkyation at multiple id
sites, the invention expressly includes all such reaction
products). The compounds may also occur in cis- or trans- or E- or
Z-double bond isomeric forms. All such isomeric forms of such
compounds are expressly included in the present invention. All
crystal forms of the compounds described herein are expressly
included in the present invention.
[0134] The invention will be further described in the following
examples. It should be understood that these examples are for
illustrative purposes only and are not to be construed as limiting
this invention in any manner.
EXAMPLES
[0135] Liquid chromatographic data was obtained using a
Hewlett-Packard (HP) 1090 Series Liquid Chromatograph coupled to a
Diode Array Detector [Restek Allure C18 Column; particle size, 5
.mu.M; column length, 150 mm; column diameter, 4.6 mm; flow rate, 1
ml/min; Solvent program, from 95% H.sub.2O (w/0.1% TFA)/5%
CH.sub.3CN (w/0.1% TFA) to 100% CH.sub.3CN (w/0.1% TFA) in 8
minutes, then held constant for 3 minutes; detection wavelength,
254 nm]. Mass Spectral data were obtained on either an Agilent 1100
LC/MS or Thermofinigan AQA/Gilson LC/MS system. .sup.1H- and
.sup.13C-NMR spectra were obtained on a Bruker AC-300 MHz
instrument. Medium pressure flash chromatography was performed on
an Isco Inc., Combiflash Sg100c system. Thin-layer chromatography
was performed using EM Science silica gel 60 F.sub.254 plastic TLC
plates. Melting points were determined in open-air capillary tubes
in a Meltemp II apparatus. UV light was used for detecting
compounds on the TLC plates. Reagents used in reactions were
purchased from the Aldrich Chemical Co. (Milwaukee, Wis.), Sigma
Chemical Co. (Saint Louis, Mo.), Fluka Chemical Corp. (Milwaukee,
Wis.), Fisher Scientific (Pittsburgh, Pa.), TCI America (Portland,
Oreg.), Transworld Chemicals, Inc. (Rockville, Md.), Maybridge
Chemical Ltd., (London, England) or Lancaster Synthesis (Windham,
N.H.).
Example 1
Synthesis of Ligase Inhibitors
[0136] ##STR68##
N7-Methyl-N-7-(1-naphthalen-1-yl-propyl)-pyrimido[4,5-d]pydrimidine-2,4,7--
triamine
[0137] Compound 1: To a solution of 4.25 g (27.2 mmol)
naphthaldehyde in 30 ml dry ether in ice water bath was slowly
added 13 ml of ethylmagnesium bromide, 3 M in ether. The mixture
was stirred for another 30 min at room temperature and then
quenched by adding 40 ml of 1N HCl solution. The organic layer was
washed with water (20 ml), sat. sodium bicarbonate (20 ml.times.2),
brine (20 ml) and then dried over anhydrous sodium sulfate.
Evaporation of the organic solvent gave a crude product 1 which was
directly used for the next step of the reaction without further
purification.
[0138] Compound 2: The crude product 1 was dissolved in 30 ml
acetone and to the resulting mixture, bathed in an ice water bath,
was slowly added Jone's reagent until the brown color persisted.
The solution was further stirred for 15 min at room temperature and
then 5 ml of isopropanol was added. After 50 ml of ethyl acetate
was added, the resulting mixture was washed with water (30 ml),
sat. sodium bicarbonate (30 ml.times.2), sat. NaCl and then dried
over anhydrous sodium sulfate. Evaporation of the organic solvents
gave an oily residue which was then purified by silica gel column
chromatography. 4.01 g of ketone 2 was obtained.
[0139] Compound 3: To the mixture of 4.01 g ketone 2 and 16.3 ml of
methylamine in methanol, 2 M, was added 1.61 g sodium
cyanoborohydride and 160 mg of zinc chloride. The resulting mixture
was stirred overnight at 50.degree. C. Adding 1N HCl quenched the
reaction. After most of the methanol was removed in vacuo, the
solution was extracted with dichloromethane (15 ml.times.2). The pH
of the aqueous layer was adjusted to about 9 with 2 N NaOH. The
product was then extracted with dichloromethane (15 ml.times.3).
The combined organic layer was washed with sat. NaCl and then dried
over anhydrous sodium sulfate. Evaporation of the solvent gave 3.54
g of compound 3.
[0140] Compound 4: A mixture of 2.89 g (14.5 mmol) pyrimidine 11,
3.54 g (15 mmol) of compound 3 and 2.5 ml (18 mmol) triethylamine
in 25 ml of 2-methoxyethanol was stirred at 80.degree. C. for 2 h.
The resulting mixture was cooled down to room temperature and the
solvent was evaporated to give an oily residue. 30 ml of ethyl
acetate was added to dissolve the residue and the resulting
solution was washed three times with water then dried over
anhydrous sodium sulfate. Evaporation gave an oily residue, which
was then purified by silica gel column chromatography. 3.95 g of
product 4 was obtained as white powder.
N7-Methyl-N-7-(1-naphthalen-1-yl-propyl)-pyrimido[4,5-d]pyrimidine-2,4,7-t-
riamine
[0141] To a solution of 3.60 g of compound 4 in 40 ml of
2-methoxyethanol was added 24 ml of IM guanidine in methanol and 16
ml of 1.5 M CH.sub.3ONa in CH.sub.3OH. The mixture was stirred at
140.degree. C. for 12 h with an equipped Dean-Stark trap to remove
the methanol solution. The reaction mixture was cooled down and
evaporated in vacuo to give an oily residue, which was then
dissolved in 30 ml of methanol. 50 ml of water was added to
precipitate the product. The product was then purified by
recrystallization from methanol, and the recrystallized product was
then stirred in methanol three times. 1.95 g of the product was
obtained as white powder. The purity of it was greater than 99%
based on HPLC analysis. ##STR69##
N7-(1-Benzo[b]thiophen-3-yl-ethyl)-N-7-methyl-pyrimido[4,5-d]pyrimidine-2,-
4,7-triamine
[0142] A mixture of 2.89 g (14.5 mmol) pyrimidine 11, 2.87 g (15
mmol) methylamine 1a and 2.5 ml (18 mmol) triethylamine in 25 ml of
2-methoxyethanol was stirred at 80.degree. C. for 2 h. The reaction
mixture was cooled down to room temperature and the solvent was
evaporated to give a oily residue. 30 ml of ethyl acetate was added
to dissolve the residue and the resulting solution was washed three
times with water then dried over sodium sulfate. Evaporation gave
an oily residue, which was then purified via the recrystallization
from ether/hexane. 3.95 g of product 1b was obtained as white
powder.
[0143] To a solution of 3.71 g 1a in 40 ml of 2-methoxyethanol was
added 24 ml of 1M guanidine in methanol and 16 ml of 1.5 M
CH.sub.3ONa in CH.sub.3OH. The mixture was stirred at 140.degree.
C. for 12 h with an equipped Dean-Stark trap to remove the methanol
solution. The reaction mixture was cooled down and evaporated in
vacuo to give an oily residue, which was then dissolved in 30 ml of
methanol. 50 ml of water was added to precipitate the product. The
product was then purified by the recrystallization from methanol,
then stirring in methanol three times. 920 mg of product was
obtained as white powder. The purity of it was 98.52% based on HPLC
analysis. ##STR70##
N7-Methyl-N7-[1-(4-methyl-naphthalen-1-yl)-ethyl]-pyrimido[4,5-d]pyrimidin-
e-2,4,7-triamine
[0144] A similar procedures as for the preparation of compound
N7-(1-Benzo[b]thiophen-3-yl-5
ethyl)-N-7-methyl-pyrimido[4,5-d]pyrimidine-2,4,7-triamine was used
for the preparation of compound
N7-Methyl-N-7-[1-(4-methyl-naphthalen-1-yl)-ethyl]-pyrimido[4,5-d]pyrimid-
ine-2,4,7-triamine. 860 mg of final product was obtained as white
powder, which had 98.80% of HPLC purity. Synthesis of 7-amino
Substituted Pyrimidopyrimidines (structure IV) ##STR71##
[0145] The amines (structure II) were prepared by reductive
amination of 1-acetonaphthone with corresponding amines (scheme 2)
structure II. ##STR72##
(II A) Tert-butyl
N-(2-{[1-(1-naphthyl)ethyl]amino}ethyl)carbamate
[0146] To a solution of 1-acetonaphthone (152 .mu.l, 1 mmol) in
acetonitrile (2 ml) was added tert-butyl N-(2-aminoethyl)-carbamate
(189 .mu.l, 1.2 mmol), NaBH.sub.3CN (126 mg, 2 mmol) and anhydrous
ZnCl.sub.2 (136 mg, 1 mmol). The reaction was heated over night at
80.degree. C. in a screw cap vial with magnetic stirring. The
precipitate was filtered off and the solution was evaporated. The
residue was dissolved in 3 ml 0.11N HCl and extracted with
methylenchloride (2.times.5 ml). The combined organic layers were
dried (na.sub.2so.sub.4) and evaporated. The crude product was
purified by silica gel (sp) chromatography using 1.CH.sub.2Cl.sub.2
and 2. CH.sub.2Cl.sub.2/MeOH (10/0.1) as an eluent to give a white
waxen product 77%. The structure characterization of the products
was made with .sup.1HNMR; .sup.13CNMR; MS (m/z): 315
(MH.sup.+).
(II B)
Tert-butyl-N-(2-{[1-(1-naphthyl)ethyl]amino}propyl)carbamate
[0147] To a solution of 1-acetonaphthone (152 .mu.l, 1 mmol) in
acetonitrile (2 ml) was added tert-butyl
N-(3-aminopropyl)-carbamate (209 .mu.l, 1.2 mmol), NaBH.sub.3CN
(126 mg, 2 mmol) and anhydrous ZnCl.sub.2 (136 mg, 1 mmol). The
reaction was heated for 70 hours at 80.degree. C. in a screw cap
vial with magnetic stirring. The precipitate was filtered off and
the solution was evaporated. The residue was dissolved in 3 ml 0.1N
HCl and extracted with methylenechloride (1.times.5 ml). The
organic layer was dried (Na.sub.2SO.sub.4) and evaporated. The
crude product was purified by silica gel flash chromatography using
1. CHCl.sub.2/MeOH (10/0.1) and 2. CH.sub.2Cl.sub.2/MeOH (10/1), as
an eluent, to give an oily product 70%. The structure
characterization of the products was made with .sup.1HNMR;
.sup.13CNMR; MS (m/z): 329 (MH.sup.+).
(II C)
Tert-butyl-N-(2-{[1-(1-naphthyl)ethyl]amino}butyl)carbamate
[0148] To a solution of 1-acetonaphthone (152 .mu.l, 1 mmol) in
acetonitrile (2 ml) was added N-Boc-1,4-diaminobutane (229 .mu.l,
1.2 mmol), NaBH.sub.3CN (126 mg, 2 mmol) and anhydrous ZnCl.sub.2
(136 mg, 1 mmol). The reaction was heated for 55 hours at
80.degree. C. in a screw cap vial with magnetic stirring. The
precipitate was filtered off and the solution was evaporated. The
residue was dissolved in 3 ml 0.1N HCl and extracted with
methylenchloride (1.times.5 ml). The organic layer was dried
(Na.sub.2SO.sub.4) and evaporated. The crude product was purified
by silica gel flash chromatography using 1. CH.sub.2Cl.sub.2/MeOH
(10/0.1) and 2. CH.sub.2Cl.sub.2MeOH (10/1), as an eluent, to give
an oily product 80%. The structure characterization of the products
was made with .sup.1HNMR; .sup.13CNMR; MS (m/z): 343 (MH.sup.+).
##STR73##
III A
4-Amino-2-{[4-aminoethyl)[1-naphthyl)ethyl]amino}-5-pyrimidinecarbon-
itrile
[0149] 4-amino-2-bromopyrimidine-5-carbonitrile (1 mmol, 199 mg),
tert-butyl n-(2-{[1-(1-naphthyl)ethyl]amino}ethyl)carbamate (IIA)
(1.2 mmol, 377 mg), N,N-diisopropylethylamine (DIEA) (2 mmol, 342
.mu.l) and 2-methoxyethanol (2 ml) were placed in screw cap vial
and heated at 150.degree. C. for 4 hours. 2-methoxyethanol was
evaporated. The residue was dissolved in 3 ml 0.1N HCl and
extracted with methylenchloride (2.times.5 ml). The combined
organic layers were dried (Na.sub.2SO.sub.4) and evaporated. The
crude product was purified by silica gel (sp) chromatography using
CH.sub.2Cl.sub.2/MeOH (10/0.1), as an eluent, to give a white
amorphous product.
[0150] To the amorphous product was added a cold solution of 50%
trifluoroacetic acid in dichloromethane (1 ml) and the mixture
agitated for 1 hour at room temperature. The solution was
evaporated. To the crude product was added saturated solution of
na.sub.2co.sub.3 and extracted with methylenchloride (2.times.5
ml). The organic layers were dried (Na.sub.2SO.sub.4) and
evaporated. Yielded: 36% white solid. The structure
characterization of the products was made with HNMR, .sup.13CNMR;
MS (m/z): 333 (MH.sup.+).
IIIB
4-Amino-2-{[4-aminopropyl)[1-naphthyl)ethyl]amino}-5-pyrimidinecarbon-
itrile
[0151] 4-amino-2-bromopyrimidine-5-carbonitrile (1 mmol, 199 mg),
tert-butyl N-(2-{[1-(1-naphthyl)ethyl]amino}propyl)carbamate (IIB)
(1.2 mmol, 394 mg), N,N-diisopropylethylamine (diea) (2 mmol, 342
.mu.l) and 2-methoxyethanol (2 ml) was placed in screw cap vial and
heated at 150.degree. c. for 5 hours. 2-metoxyethanol was
evaporated. The residue was dissolved in 3 ml 0.1N HCl and
extracted with methylenchloride (2.times.5 ml). The combined
organic layers were dried (Na.sub.2SO.sub.4) and evaporated. The
crude product was purified by silica gel (sp) chromatography using
CH.sub.2Cl.sub.2/MeOH (10/0.1) as an eluent to give a white
amorphous product.
[0152] To the amorphous product was added a cold solution of 50%
trifluoroacetic acid in dichloromethane (2 ml) and the mixture
agitated for 1 hour at room temperature. The solution was
evaporated. To the crude product was added saturated solution of
Na.sub.2CO.sub.3 and extracted with methylenchloride (2.times.5
ml). The organic layers were dried (Na.sub.2SO.sub.4) and
evaporated. Yielded: 66% white solid. The structure
characterization of the products were made with .sup.1HNMR;
.sup.13CNMR; MS (m/z): 347 (MH.sup.+).
III
C.sub.4-Amino-2-{[4-aminobutyl)[1-naphthyl)ethyl]amino}-5-pyrimidineca-
rbonitrile
[0153] 4-amino-2-bromopyrimidine-5-carbonitrile (1 mmol, 199 mg),
tert-butyl N-(2-{[1-(1-naphthyl)ethyl]amino}butyl)carbamate (IIC)
(1.2 mmol, 410 mg), N,N-diisopropylethylamine (DIEA) (2 mmol, 342
.mu.l) and 2-methoxyethanol (2 ml) was placed in screw cap vial and
heated at 150.degree. C. for 4 hours. 2-metoxyethanol was
evaporated. The residue was dissolved in 3 ml 0.1N HCl and
extracted with methylenchloride (2.times.5 ml). The combined
organic layers were dried (Na.sub.2SO.sub.4) and evaporated. The
crude product was purified by silica gel (sp) chromatography using
CH.sub.2Cl.sub.2/MeOH (10/0.1) as an eluent to give a white
amorphous product.
[0154] To the amorphous product was added a cold solution of 50%
trifluoroacetic acid in dichloromethane (1.5 ml) and the mixture
agitated for 1 hour at room temperature. The solution was
evaporated. To the crude product was added saturated solution of
Na.sub.2CO.sub.3 and extracted with methylenchloride (3.times.5
ml). The organic layers were dried (na.sub.2so.sub.4) and
evaporated. Yielded: 54% white solid. The structure
characterization of the products was made with .sup.1HNMR;
.sup.13CNMR; MS (m/z): 361 (MH.sup.+).
[0155] The forming 7-substituted pyrimido pyrimidines (structure
IV) was carried out by the condensation of 2-substituted
2,4-diamino-5-pyrimidinecarbonitriles (structure III) with
guanidine (scheme 4). ##STR74## Compound IVC
[0156]
4-Amino-2-{[4-aminobutyl)[1-naphthyl)ethyl]amino}-5-pyrimidinecarb-
onitrile (IIIC) (0.26 mmol, 95 mg) was dissolved in 1.2 ml of the
guanidine free base (the preparation see below) in
2-methoxyethanol. The reaction mixture was stirred in screw cap
vial at 150.degree. C. for 1.5 hours. 2-metoxyethanol was
evaporated. The water was added and the precipitate was filtered
and the crude product was purified by silica gel (sp)
chromatography using CH.sub.2Cl.sub.2/MeOH/NH.sub.3 (2/1/0.1) as an
eluent to give a white solid 42%. The structure characterization of
the products were made with .sup.1HNMR; .sup.13CNMR; MS (m/z): 403
(MH.sup.+).
Compound IVB
[0157]
4-Amino-2-{[4-aminopropyl)[1-naphthyl)ethyl]amino}-5-pyrimidinecar-
bonitrile (IIIB) (0.26 mmol, 93 mg) was dissolved in 1.3 ml of the
guanidine free base (the preparation see below) in
2-methoxyethanol. The reaction mixture was stirred in screw cap
vial at 150.degree. C. for 1.5 hours. 2-metoxyethanol was
evaporated. The water was added and the precipitate was filtered
and the crude product was purified by silica gel (sp)
chromatography using CH.sub.2Cl.sub.2/MeOH/NH.sub.3 (2/1/0.1) as an
eluent to give a white solid 38%. The structure characterization of
the products were made with .sup.1HNMR; .sup.13CNMR; MS (m/z): 389
(MH.sup.+).
Compound IVA
[0158]
4-Amino-2-{[4-aminoethyl)[1-naphthyl)ethyl]amino}-5-pyrimidinecarb-
onitrile (IIIA) (0.3 mmol, 100 mg) was dissolved in 1.4 ml the
guanidine free base (the preparation see below) in
2-methoxyethanol. The reaction mixture was stirred in screw cap
vial at 150.degree. C. for 1.5 hours.
[0159] 2-metoxyethanol was evaporated. The water was added and the
precipitate was filtered and the crude product was purified by
silica gel (sp) chromatography using CH.sub.2Cl.sub.2/MeOH/NH.sub.3
(10/2/0.2) as an eluent to give a white solid 46%. The structure
characterization of the products was made with .sup.1HNMR;
.sup.13CNMR; MS (m/z): 375 (MH.sup.+).
The preparation of the Guanidine Free Base in 2-methoxyethanol
[0160] In a separate container sodium metal (Ig, 44 mmol) was added
to 30 ml of 2-methoxyethanol, stirred under an inert atmosphere
until no sodium metal was observed in solution.
[0161] In a separate container was made a guanidine hydrochloride
(4.2 g, 44 mmol) solution in 2-methoxyethanol (30 ml). To this
solution was added the sodium methoxyethoxide solution. Upon
addition a white precipitate was formed (NaCl). The reaction was
stirred at 25.degree. C. for 30 minutes. The precipitate was
filtered and the solution was stored in refrigerator and used as a
solution of guanidine free base. ##STR75##
3-[(5,7-Diamino-pyrimido[4,5-d]pyrimidin-2-yl)-(2-ethoxy-naphthalen-1-ylme-
thyl)-amino]-2-hydroxy-propionic acid
[0162] The alpha-hydroxy carboxylic acid was synthesized in a
multi-step procedure starting from isoserine, using experimental
methods and conditions similar to those described in detail
elsewhere in this application. The enantiomeric alcohols can be
synthesized stereoselectively utilizing the reaction of
2-ethoxynaphthylmethylamine on esters of glycinic acid
(epoxide).
N-(2-Amino-ethyl)-3-[(5,7-diamino-pyrimido[-4,5-d]pyrimidin-2-yl)-(2-ethox-
y-naphthalen-1-ylmethyl)-amino]-2-hydroxy-propionamide
[0163] To
3-[(5,7-diamino-pyrimido[4,5-d]pyrimidin-2-yl)-(2-ethoxy-naphth-
alen-1-ylmethyl)-amino]-2-hydroxy-propionic acid (200 mg, 0.445
mmol) in dry DMF (2 ml) was added BroP (249 mg, 0.534 mmol). After
the mixture was stirred for 30 min, DIEA (126 mg, 0.979 mmol) was
added. After stirring for another 20 min, ethylene diamine (53.5
mg, 0.89 mmol) was added. The mixture was then stirred at room
temperature for 16 hours and was directly purified by prep HPLC to
yield 35 mg (16%) of the title compound: MS m/z (M+H) 492. General
Procedure used Reacting Various Amines with Aromatic Ketones under
Reducing Conditions ##STR76## The following procedure is based on a
literature method (J. Org. Chem. 1985, 50, 1927-1932).
[0164] At room temperature, to a solution of 1-acetylnaphthylene (1
equiv, 1 mmol, 170 mg, 151 .mu.l) in methanolic methylamine
solution (4 equiv, 4 mmol, 2 ml of 2 M in methanol) was added solid
sodium cyanoborohydride (2 equiv, 2 mmol, 126 mg) and anhydrous
zinc chloride (1 t, equiv, 1 mmol, 136 mg). The reactions were
heated to 65.degree. C. in an open tube with magnetic stirring.
[0165] The course of the reaction can be monitored by either TLC or
HPLC. At 30 min, several peaks were observed. An authentic sample
of N-methyl-1-naphthylethylamine was used for comparison. At 1
hour, the reaction was >50% complete. At 4 hours the ketone had
completely disappeared and the product was 95+% pure, contaminated
with only <5% intermediate imine. Heating the reaction longer
may have resulted in a cleaner product. We recommend the reaction
time of at least 6 hours for this specific ketone, although
reaction time may vary dependent on the nature of the ketone. These
reactions were worked up in the usual manner and the crude products
were purified using standard laboratory techniques. ##STR77##
3-[(5,7-Diamino-pyrimido[4,5-d]pyrimidin-2-yl)-(2-ethoxy-naphthalen-1-ylme-
thyl)-amino]-propionic acid
[0166] ##STR78##
{4-[(5,7-Diamino-pyrimido[4,5-d]pyrimidin-2-yl)-(2-ethoxy-naphthalen-1-ylm-
ethyl)-amino]-butyl}-carbamic acid tert-butyl ester
[0167] A suspension of the substituted-cyanoaminopyrimdine (12.37
g, 0.025 mol), guanidine hydrochloride (7.16 g, 0.075 mol), solid
sodium methoxide (5.40 g, 0.1 mol) in methoxyethanol (150 ml) was
heated to reflux for 48 hours. The reaction was determined to be
complete by monitoring by HPLC. The reaction mixture was cooled to
room temperature and poured into excess water. The solid material
was collected on a filter, dried under vacuum, to provide 13.05
grams of crude material that was used in the next step without
further purification.
N7-(4-Amino-butyl)-N-7-(2-ethoxynaphthalen-1-ylmethyl)-pyrimido[4,5-d]pyri-
midine-2,4,7-triamine
[0168] The mono-boc-protected intermediate (13 g) was added slowly
over a period of 10 min to ice cold trifluoroacetic acid (75 ml)
with rapid stirring. The reaction was complete as observed by
HPLC/MS analysis of an aliquot. The reaction mixture was poured
into an ice cold solution of sodium ethoxide (5%) to precipitate
the product. The HPLC product was collected on a filter and dried
to provide 8.0 grams of solid: HPLC Rt=2.882 min, 99% pure, MS m/z
433 (pos). ##STR79## Step 1: Reductive Amination
[0169] 20.0 g (0.12 mole) of the ketone was dissolved in 100 ml (2
eq.) of a 2M methanolic solution of methylamine. In a separate
flask cooled to 0.degree. C. was added 8.3 g (0.5 eq.) of
ZnCl.sub.2 and 7.7 g (1.0 eq.) NaCNBH.sub.3 in about 10 ml of MeOH.
The Zn(CNBH.sub.3).sub.2 was allowed to mix at 0 degrees for about
5 minutes and then added as a slurry to the ketone/amine mixture.
The reaction was brought to a gentle reflux overnight. The reaction
was allowed to cool to room temp. and rotovaped to dryness. The
material was allowed to sit under high vacuum in order to remove
any residual methylamine.
[0170] The white solid was triturated with Et.sub.2O however, a
viscous oil resulted. Better results were observed by trituration
with Et.sub.2O by adding enough MeOH to keep the material from
oiling out. The material was filtered and washed with ether and
allowed to air dry. TLC of the solid versus an authentic sample
provided by ET showed identical mobility. Based on weight however,
recovery was >100% and it was assumed that inorganic salts were
still present.
[0171] Due to the contaminating salts present, a small amount of
the material was used in the next step. No obvious problems were
observed. The amino acid was therefore used without further
purification.
Step 2: Reaction with Cl-Pyrimidine
[0172] 25 g (theoretical yield 21.8 g) of the amino acid was
dissolved in approximately 50 ml of ethoxyethanol. To this was
added 17.0 g (0.9 eq. based on ketone) of
4-amino-2-chloropyrimidine-5-carbonitrile and 42 ml (2.0 eq.) of
DIEA and the reaction was allowed to mix for about 2 hrs. at 80
degrees (temp of oil bath). TLC showed none of the chloride
remaining. The reaction was allowed to cool to room temp and the
concentrated to about 10 ml on a rotary evaporator. The resulting
slurry was diluted with about 400 ml of water and the pH was
adjusted to 5-6 (pH paper) using conc. HOAc, at which point a light
yellow solid formed. The material was allowed to sit at 0 degrees
overnight, filtered and washed with about 1 L of water and air aL
dried. Recrystallization from H.sub.2O/MeOH provided .about.25 g of
the intermediate (69%). TLC (CH.sub.2Cl.sub.2 10% MeOH)
R.sub.f.about.0.1. There did appear to be a fast moving material,
however, it was very minor and the product was used without further
purification.
Step 3: Cyclization Reaction
[0173] For the scale-up, ethoxyethanol was used as solvent in order
to increase the temperature of the reaction to about 135.degree. C.
To 16.0 g (54 mmol) of the intermediate was dissolved in .about.75
ml of ethoxyethanol. To this was added 10.2 g (2.0 eq.) of
guanidine hydrochloride and 11.6 g (4.0 eq.) of NaOMe and the
reaction was brought to a gentle reflux under argon. TLC was used
to monitor the disappearance of starting material. After about 30
hours, the reaction was cooled and an additional 5.1 g (1.0 eq.) of
guanidine hydrochloride and 2.9 g (1.0 eq.) NaOMe was added and the
mixture brought back to reflux. By TLC, the reaction appeared
complete after about 72 hours.
[0174] The reaction was allowed to cool and concentrated to about
20 ml on a rotary evaporator. The resulting slurry was diluted with
about 600 ml of water and the pH was adjusted to 5-6 with con HOAc.
The product precipitated out of the solution and was allowed to sit
overnight at 0 degrees. The product was filtered and washed with
copious amounts of water, followed by copious amounts of MeOH
(remove any unreacted starting material) and air dried. Isolate
.about.13.5 g (.about.75%) of material. HPLC analysis showed that
there was a minor polar impurity, the same one observed in the
small scale reaction. The material can be used without further
purification. ##STR80##
[0175] [2-(4-Amino-5-cyano-pyrimidin-2-ylamino)-ethyl]-carbamic
acid tert-butyl ester (5 g, 18 mmol) was dissolved in 60 ml of a
50/50 v/v dichloromethane trifluoroacetic acid solution. Vigorous
effervescence is observed upon addition of liquid to the solid. The
solution is then stirred under an inert atmosphere for 30 minutes
and then sample is taken for HPLC analysis to determine
deprotection is complete. When deprotection is complete, the
solution is concentrated to apparent dryness in vacuo.
[0176] In a separate container sodium metal (0.675 g) is added
slowly to 30 ml of 2-methoxyethanol under an inert atmosphere until
no sodium metal is observed in solution. Guanidine hydrochloride:
(2.645 g) solution in 2-methoxyethanol (30 ml) was made in a
separate container. To this solution was added the sodium
methoxyethoxide solution. Upon addition, a white precipitate of
sodium chloride formed, and the resulting solution was stirred 30
minutes. This solution was filtered in an inert atmosphere, added
to the crude residue from step one, and stirred vigorously. Within
15 minutes, a yellow precipitate was observed. The precipitate was
filtered to yield N7-(2-Amino-ethyl)-pyrimido[4,5-d
pyrimidine-2,4,7-triamine (2.1 g, 52% yield). ##STR81##
4-(N-[2,4-d]amino-6-pteridinyl-methyl]-N-methylamino)-benzoic acid
4-aminobutyl amide
[0177] To a suspension of
4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino]benzoic acid
hemihydrochloride (250 mg, 0.73 mmol) in dry DMF (20 ml) were added
N,N-diisopropylethylamine (250 .mu.l, 1.46 mmol, 2 equiv) and
diethyl cyanophosphonate (225 .mu.l, 1.46 mmol, 2 equiv).
Dissolution occurred rapidly and the reaction was stirred for 4
hours at room temperature. 1,4-Diaminobutane (367 .mu.l, 3.65 mmol,
5 equiv) and N,N-diisopropylethylamine (250 .mu.l, 1.46 mmol, 2
equiv) were then added and the solution was stirred 45 minutes at
room temperature. The completion of reaction was verified by HPLC,
and solid NaHCO.sub.3 was added., The solvent was then evaporated
under reduced pressure and the residue suspended in a small amount
of methanol. Addition of dilute aqueous NH.sub.4OH was then
followed by filtration and a rinse with water gave the product upon
drying to yield 153 mg (53%): MS m/z (M+H) 396, HPLC R.sub.t 3.80
min. ##STR82##
4-(N-[2,4-d]amino-6-pteridinyl-methyl]-N-methylamino)-benzoic acid
4-hydroxyphenyl amide
[0178] To a suspension of
4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino]benzoic acid
hemihydrochloride (100 mg, 0.29 mmol) in dry DMF (-6 ml) were added
2 equivalents of N,N-diisopropylethylamine (100 ul, 0.58 mmol) and
2 equivalents of diethyl cyanophosphonate (90 ul, 0.58 mmol).
Dissolution occurred rapidly and the reaction was stirred for 3-4
hours at room temperature. For the 4-hydroxyphenyl amide, 1.05
equivalents of .rho.-aminophenol (33 mg, 0.30 mmol) and 2
equivalents of N,N-diisopropylethylamine (100 ul, 0.58 mmol) were
then added and the solution was stirred overnight at room
temperature. The completion of reaction was verified by HPLC, and
solid NaHCO.sub.3 was added. The solvent was then evaporated under
reduced pressure and the residue suspended in a small amount of
methanol. Addition of dilute aqueous NH.sub.4OH was then followed
by filtration and a rinse with either dilute aqueous acetic acid
gave the product upon drying. Yield 77 mg (0.19 mmol, 64%), MS m/z
(M-H) 415, HPLC retention time 4.66 minutes. ##STR83##
(N-benzyl[2,4-d]amino-6-pteridinyl-methyl]-N-methylamine)
[0179] Five Fold Excess of N-Methylbenzylamine (245.4 Mg, 261
.mu.L, 2.02 Mmol) was Dissolved in DMF (4 ml) in a 15 ml screw cap
vial equipped with a magnetic stirrer.
2,4-Diamino-6-chloromethylpterin (100 mg, 0.405 mmol) was added and
mixed well. The reaction mixture was stirred at 60.degree. C. for 4
h. Analytical HPLC analysis of an aliquot confirmed the absence of
pterin starting material. The solvent was removed under reduced
pressure at 60.degree. C. The resulting a mixture was washed with
EtOH (2.times.15 ml), then the solvent was removed under a stream
of nitrogen and the resulting product dried on high vacuum 18 h.
NMR and MS were obtained and confirm product structure and purity.
##STR84##
(N-(alpha-methyl-4-methylbenzy)[2,4-diamino-6-pteridinyl-methyl]-N-methyla-
mine)
[0180] A five fold excess of (S)-(+)-alpha-4-dimethylbenzylamine
(272 mg, 298 ul, 2.02 mmol) was dissolved in DMF (4 ml) in a 15 ml
screw cap vial equipped with a magnetic stirrer.
2,4-Diamino-6-chloromethylpterin (100 mg, 0.405 mmol) was added and
mixed well. The reaction mixture was stirred at 60.degree. C. for 4
h. Analytical HPLC analysis of an aliquot confirmed the absence of
pterin starting material. The solvent was removed under reduced
pressure at 60.degree. C. The resulting mixture was washed with
EtOH (2.times.15 ml), then the solvent was removed under a stream
of nitrogen and the resulting product dried on high vacuum 18 h.
Analytical HPLC, 1H-NMR, and MS were consistent with the structure
of the product and the product was of high purity.
Example 2
D-Ala-D-Ala-Ligase Ki Determination
[0181] The synthetic analogs of Example 1 were dissolved in
dimethylsulfoxide (DMSO) at a concentration of 100 mM on the day of
screening, using a vortex mixer and sonication if necessary for
dissolution. The solutions were kept at room temperature until
screening was completed.
[0182] A 10 mM NADH (Sigma) stock solution was prepared freshly on
the day of screening by dissolving 32 .mu.mol NADH in 3.2 ml
double-distilled water. The NADH solution was kept on ice. Stock
solutions containing 50 mM phosphoenolpyruvate (PEP; Sigma), 500
.mu.M HERMES, 30 mM adenosine triphosphate (ATP; Sigma), 200 mM
D-alanine (Sigma), and 4.times. core buffer (i.e., 400 mM Hepes, 40
mM magnesium chloride, and 40 mM potassium chloride), were also
stored on ice. A stock solution of pyruvate kinase/lactate
dehydrogenase (PK/LDH) was also obtained from Sigma.
[0183] Table of Final concentrations are dependent on the type of
screening: TABLE-US-00006 ANALOGS' % ANALOGS' Ki AND ANALOGS' Ki
INHIBITION MODE OF INHIBITION Type of Final concentration Final
concentration Final concentration screening in enzyme mix in enzyme
mix in enzyme mix Core buffer 1.times. 1.times. 1.times. 4.times.
NADH 500 .mu.M 500 .mu.M 500 .mu.M 10 mM PEP 50 mM 2 mM 2 mM 2 mM
PK/LDH 0.02 ml/ml 0.02 ml/ml 0.02 ml/ml mix enzyme stock enzyme
stock enzyme stock solution solution solution Hermes 200 nM 400-600
nM 200 nM 500 .mu.M
[0184] TABLE-US-00007 ANALOGS' % ANALOGS' Ki AND ANALOGS' Ki
INHIBITION MODE OF INHIBITION Final concentration Final
concentration Final concentration Type of screening in substrate
mix in substrate mix in substrate mix Solution A Screening A B C
ATP 30 mM 4 mM 20 uM 4 mM 4 mM 100 uM D-Ala 200 mM 2 mM 64 mM 2 mM
64 mM 64 mM Core buffer 1.times. 1.times. 1.times. 1.times.
1.times. 1.times.
Example 3
Determination of Ki of Analogs
[0185] For each set of test compounds, two 96-well plates were
used: an inhibitor plate and an enzyme plate. The test compounds
correspond to rows A-G of the plates. Adenosine (Sigma) dissolved
in DMSO, used as a control, corresponds to row H of each plate.
[0186] The enzyme solution was allowed to equilibrate to 25.degree.
C.
[0187] Dilutions were prepared in the inhibitor plate as follows:
50 .mu.l DMSO was added to each well of columns 1-11, rows A-G, of
the inhibitor plate. 50 .mu.l DMSO were added to each well of
columns 1-11, row H. 100 .mu.l of the 100 mM test solutions were
added to column 12, rows A-G (i.e., the first compound in row A,
the second compound in row B, and so on). 100 .mu.l of a 100 mM
Adenosine solution was added to column 12, row H.
[0188] 50 .mu.l of the test solution was transferred from column 12
in each row to column 11 of the a same row, mixing the solution
with the DMSO. 50 .mu.l of solution was then transferred from
column 11 in each row to column 10 in the same row, 50 .mu.l from
column 10 was transferred to column 9, and so on, down to column 2.
No solution was transferred to column 1. Multichannel pipettors
were used in making the serial dilution.
[0189] 120 .mu.l of the enzyme solution was added to each well of
the enzyme plate.
[0190] The substrate solutions were brought to 25.degree. C.
[0191] The analogs and enzymes were then incubated at 25.degree. C.
Since the reactions were initiated in columns, the analog addition
is also in columns. At t=0 minutes, 5 .mu.l analog was transferred
from each well of columns 1-4 of the inhibitor plate to the
corresponding well of the enzyme plate. At t=4 minutes, 5 .mu.l
analog was transferred from each well of columns 5-8 of the
inhibitor plate to the corresponding well of the enzyme plate. At
t=8 minutes, 5 .mu.l analog was transferred from each well of
columns 9-12 of the inhibitor plate to the corresponding well of
the enzyme plate. The inhibitor plate was then frozen.
[0192] At t=18-19 minutes, the substrate solution was taken from
25.degree. C. to a Spectromax.RTM. UV-vis spectrophotometer. At
t=20 minutes, within a 30 second timeframe, 125 .mu.l of substrate
solution was added to each well of columns 1-4, and the absorbance
at 340 nm was read. At t=24 minutes and t=28 minutes, respectively,
the process was repeated for columns 5-8 and 9-12.
[0193] Thus, the concentrations of the compounds in columns 1-12 in
each row were 0, 1.9 .mu.M, 3.9 .mu.M, 7.8 .mu.M, 15.6 .mu.M, 31.2
.mu.M, 62.5 .mu.M, 125 .mu.M, 250 uM, 500 .mu.M, 1 mM, and 2 mM,
respectively.
[0194] The reduction values were multiplied by -4.06 to convert
mOD/min units to nM/sec (OD=.lamda.LM; .lamda.=6220 1/Mcm; L=0.66
cm; mOD/sec=6220.times.0.66.times.(mM/sec).times.60;
(mOD/sec).times.4.06=nM/sec); multiplied by -1 since NADH
absorbance decreases as more product is generated).
[0195] Plots of reaction rates vs. inhibitor concentration were
generated using Kaleidograph.RTM., and K.sub.i values were
determined after the data was fitted to the proper equation.
[0196] Most of the stages are alternatively done using the SciClone
automated liquid handling machine. These stages are: adding the
enzyme mix to the enzyme plate, dispensing 50 ul DMSO in each well
of columns 1-11 rows A-H of the inhibitor plate, serial dilutions
of the analogs+adenosine control in the inhibitor plate, adding the
analog inhibitors from the inhibitor plate to the enzyme plate,
adding the substrate to the enzyme plate.
Example 4
Analogs % Inhibition
[0197] The assay procedure described above was repeated, except
that inhibitor plates were prepared with 5 mM solutions of the
inhibitors in the plates (rather than by serial dilutions), to
result in a final concentration of 100 .mu.M inhibitor in the final
reaction mix. Enzyme activity in the presence of DMSO was used as a
100% activity reference.
[0198] Refer to the table above for exact concentrations.
Example 5
Analogs' Ki and Mode of Inhibition
[0199] The assay procedure described above was repeated, using
three different substrate solutions, each in a different enzyme
plate. The final concentrations in the reaction mixtures were: (A)
2 mM ATP and 1 mM D-alanine; (B) 2 mM ATP and 32 mM D-alanine; and
(C) 50 .mu.M ATP and 32 mM D-alanine. The same inhibitor plate was
used with all three enzyme plates. Adenosine (Sigma) and
cycloserine (Sigma) were used as controls. Refer to the table above
for exact concentrations.
Example 6
Microdilution Antimicrobial Susceptibility Test Assay
[0200] Stock solutions of test compounds were prepared in DMF at a
concentration of 5 mg/ml. Working solutions of the tested compounds
were then prepared from the stock solutions, in Mueller-Hinton
broth (MHB) with starting concentration of 64 .mu.g/ml (i.e., 25.6
.mu.l of stock solution in 974.4 .mu.l of MHB=128 .mu.g/ml, which
was diluted with an equal volume of bacterial inoculum in the
procedure that follows).
[0201] Bacterial inocula were prepared from overnight culture
(i.e., one fresh colony from agar plate in 5 ml MHB; H. influenzae
was grown in MHB with the addition of yeast extract, haematin, and
NAD), centrifuged 2.times.5 min/3000 rpm (for S. pneumoniae and H.
influenzae, 2.times.10 min/3000 rpm), and dispensed in 5 ml of
fresh MHB each time, such that the bacterial suspension is diluted
to obtain 100 colony forming units (cfu) in a microplate well (100
.mu.l total volume).
[0202] The microplate wells were then filled with twofold dilutions
of tested compound (50 .mu.l), starting with 64 .mu.g/ml. Columns
2-12 were filled with 50 .mu.l of bacterial innoculum (final
volume: 100 .mu.l/well). The plates were incubated at 37.degree. C.
for 18-24 hours (S. pneumoniae was grown in a CO.sub.2-enriched
atmosphere).
[0203] The optical density of each well at 590 nm (OD.sub.590) was
then measured with a TECAN SpectroFluor Plus.RTM., and minimum
inhibitory concentration (MIC) was defined as the concentration
that showed 90% inhibition of growth.
Example 7
MIC Determination using Overexpressing E. coli
[0204] The procedure of Example 5 was repeated, with the following
modifications:
[0205] The media used for growing bacteria was luria broth (LB)
with added antibiotics (20 mg/l chloramphenicol for pBAD vectors,
100 mg/l ampicillin for pTAC vectors for plasmid selection) or M9
minimal media with D-mannitol as a carbon source.
[0206] The bacteria used for innoculum in LB were prepared as
follows: Overnight culture was diluted 1:50 in a fresh LB media and
incubated at 37.degree. C. on a shaker at 250 rpm. After mid-log
stage was reached (OD.sub.600=0.5-1.0, about 3 hours), operon
regulator (glucose, arabinose, or IPTG) was added, and the bacteria
were further incubated for 3 hours. After 3 hours, OD.sub.600 was
measured again to estimate the bacterial count number, and the
culture was diluted in LB media (antibiotics--chloramphenicol or
ampicillin and regulators were added in double concentrations).
Final bacterial inoculum was around 10,000 cfu/well.
[0207] The bacteria used for innoculum in M9 minimal media were
prepared as follows: Overnight culture in LB was centrifuged
2.times.5 min/3000 rpm, washed with M9 media, diluted 1:50 in M9
minimal media, left at 37.degree. C. for 14 hours
(OD.sub.600.about.0.5), operon regulator was added, and the
bacteria were further incubated for 3 hours. After 3 hours,
OD.sub.600 was measured to estimate bacteria number, and the
culture was diluted in M9 minimal media (antibiotics
-chloramphenicol or ampicillin and regulators were added in double
concentrations). The final bacterial innoculum was around 10,000
cfu/well.
[0208] Optical density was read out after 24 and 48 hours because
of the slower bacterial growth in minimal media.
Example 8
Computer Modeling Protocol used to Predict the Ligase Inhibitory
Activity of Representative Analogs
[0209] A virtual library of 7-substituted pteridines was generated
by combining the chloromethylpteridine core shown below at left
with a set of commercially available amines, according to the
following scheme: ##STR85##
[0210] A set of 1500 commercially available amines was selected
from Available Chemicals id Directory (ACD, MDL) based on the
following criteria:
[0211] MW<300;
[0212] No reactive or toxic functional groups;
[0213] General drug-like properties;
[0214] Available from known and reliable suppliers.
[0215] The corresponding pteridine derivatives were generated with
the analog builder module implemented in Cerius2 (MSI).
Conformational search was performed on the generated analogs with
Catalyst (MSI), and a total of 32,000 conformers (.about.20 per
molecule) were docked into the active site of D-Ala-D-Ala-ligase
with the EUDOC program (Mayo Clinic). The conformation of the
active site used for docking was derived from the x-ray
crystallographic structure of the complex between the enzyme, ADP
and a phosphinate inhibitor (obtained from the protein databank,
pdb code: 2dln), with rearrangement and minimization of the side
chain conformation of lysine 215.
[0216] The "best"-binding conformer of each molecule was then
extracted from the docking results. The corresponding orientations
in the active site were re-scored with a set of scoring functions
implemented in the program CSCORE (Tripos). The solutions were then
ranked on the basis of consensus scoring, using the function
Chemscore as secondary criterion. A set of 76 high-ranking
compounds were selected and re-docked with the FlexX program
(Tripos), using the same conformation of the enzyme active site.
The docking solutions were re-scored with CS CORE. The final
selection of 50 compounds was based on consensus between the
results obtained with the two docking programs. The predicted Ki's,
calculated with Chemscore on the FlexX-generated solutions, were in
the range 0.1-10 .mu.M.
[0217] The calculations were performed on an SGI Octane
(2.times.250 MHz CPU, 512 MB RAM), an SGI O2 (270 MHz CPU, 128 MB
RAM) and a cluster of ten SGI Indigo2 computers (195 MHz CPU, 512
MB RAM).
OTHER EMBODIMENTS
[0218] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *