U.S. patent application number 13/454510 was filed with the patent office on 2013-01-24 for gyrase inhibitors and uses thereof.
This patent application is currently assigned to VERTEX PHARMACEUTICALS INCORPORATED. The applicant listed for this patent is Paul S. Charifson, David D. Deininger, Joseph Drumm, Anne-Laure Grillot, Arnaud Le Tiran, Yusheng Liao, Emanuele Perola, Steven Ronkin, Dean Stamos, Tiansheng Wang. Invention is credited to Paul S. Charifson, David D. Deininger, Joseph Drumm, Anne-Laure Grillot, Arnaud Le Tiran, Yusheng Liao, Emanuele Perola, Steven Ronkin, Dean Stamos, Tiansheng Wang.
Application Number | 20130023525 13/454510 |
Document ID | / |
Family ID | 34115255 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130023525 |
Kind Code |
A1 |
Charifson; Paul S. ; et
al. |
January 24, 2013 |
GYRASE INHIBITORS AND USES THEREOF
Abstract
The present invention relates to compounds which inhibit
bacterial gyrase and/or Topo IV and pharmaceutically acceptable
compositions comprising said compounds. These compounds, and
compositions thereof, are useful in treating bacterial infection.
Accordingly, the present invention also relates to methods for
treating bacterial infections in mammals.
Inventors: |
Charifson; Paul S.;
(Framingham, MA) ; Deininger; David D.; (Waltham,
MA) ; Grillot; Anne-Laure; (Milton, MA) ;
Liao; Yusheng; (Lexington, MA) ; Ronkin; Steven;
(Watertown, MA) ; Stamos; Dean; (Carlsbad, CA)
; Perola; Emanuele; (Brookline, MA) ; Wang;
Tiansheng; (Concord, MA) ; Le Tiran; Arnaud;
(Croissy sur Seine, FR) ; Drumm; Joseph; (Walnut
Creek, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Charifson; Paul S.
Deininger; David D.
Grillot; Anne-Laure
Liao; Yusheng
Ronkin; Steven
Stamos; Dean
Perola; Emanuele
Wang; Tiansheng
Le Tiran; Arnaud
Drumm; Joseph |
Framingham
Waltham
Milton
Lexington
Watertown
Carlsbad
Brookline
Concord
Croissy sur Seine
Walnut Creek |
MA
MA
MA
MA
MA
CA
MA
MA
CA |
US
US
US
US
US
US
US
US
FR
US |
|
|
Assignee: |
VERTEX PHARMACEUTICALS
INCORPORATED
Cambridge
MA
|
Family ID: |
34115255 |
Appl. No.: |
13/454510 |
Filed: |
April 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12246778 |
Oct 7, 2008 |
8188095 |
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13454510 |
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10767638 |
Jan 29, 2004 |
7495014 |
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12246778 |
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60443917 |
Jan 31, 2003 |
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Current U.S.
Class: |
514/221 ;
514/234.5; 514/256; 514/274; 514/275; 514/302; 514/318; 514/338;
514/395; 540/504; 544/131; 544/316; 544/331; 544/333; 546/115;
546/194; 546/269.1; 546/271.4; 546/272.4; 546/273.4; 548/305.1;
548/306.1 |
Current CPC
Class: |
C07D 403/14 20130101;
C07D 405/04 20130101; A61P 11/00 20180101; A61P 31/04 20180101;
A61P 17/00 20180101; C07D 417/14 20130101; C07D 471/04 20130101;
C07D 487/04 20130101; C07D 491/04 20130101; C07D 403/04 20130101;
A61P 31/06 20180101; C07D 405/14 20130101; A61P 43/00 20180101;
A61P 31/00 20180101; A61P 13/08 20180101; C07D 401/14 20130101;
A61P 13/02 20180101; C07D 413/14 20130101 |
Class at
Publication: |
514/221 ;
548/306.1; 514/395; 546/273.4; 514/338; 544/333; 514/256;
548/305.1; 544/316; 514/274; 544/331; 514/275; 546/194; 514/318;
546/272.4; 546/115; 514/302; 546/269.1; 546/271.4; 544/131;
514/234.5; 540/504 |
International
Class: |
A61K 31/4184 20060101
A61K031/4184; C07D 401/14 20060101 C07D401/14; A61K 31/4439
20060101 A61K031/4439; A61K 31/506 20060101 A61K031/506; C07D
405/14 20060101 C07D405/14; A61P 31/04 20060101 A61P031/04; A61K
31/4545 20060101 A61K031/4545; C07D 491/056 20060101 C07D491/056;
A61K 31/4355 20060101 A61K031/4355; C07D 413/14 20060101
C07D413/14; A61K 31/5377 20060101 A61K031/5377; A61K 31/5513
20060101 A61K031/5513; C07D 403/14 20060101 C07D403/14; C07D 403/10
20060101 C07D403/10 |
Claims
1. A compound of formula I: ##STR00353## or a pharmaceutically
acceptable salt thereof, wherein: W is selected from nitrogen, CH,
or CF; X is selected from CH or CF; Z is O or NH; R.sup.1 is phenyl
or a 5-6 membered heteroaryl ring having 1-3 heteroatoms
independently selected from oxygen, nitrogen, or sulfur, wherein:
R.sup.1 is substituted with 0-3 groups independently selected from
-(T).sub.y-Ar, R', oxo, C(O)R', CO.sub.2R', OR', N(R').sub.2, SR',
NO.sub.2, halogen, CN, C(O)N(R').sub.2, NR'C(O)R', SO.sub.2R',
SO.sub.2N(R).sub.2, or NR'SO.sub.2R'; y is 0 or 1; T is a straight
or branched C.sub.1-4 alkylidene chain, wherein one methylene unit
of T is optionally replaced by --O--, --NH--, or --S--; each R' is
independently selected from hydrogen, C.sub.1-4 aliphatic, or a 5-6
membered saturated, unsaturated, or aryl ring having 0-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, wherein: R' is substituted with 0-3 groups independently
selected from halogen, oxo, R.sup.o, N(R.sup.o).sub.2, OR.sup.o,
CO.sub.2R.sup.o, NR.sup.oC(O)R.sup.o, C(O)N(R.sup.o).sub.2,
SO.sub.2R.sup.o, SO.sub.2N(R.sup.o).sub.2, or NR.sup.o,
SO.sub.2R.sup.o, wherein: each R.sup.o is independently selected
from hydrogen, C.sub.1-4 aliphatic, or a 5-6 membered saturated,
unsaturated, or aryl ring having 0-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, and wherein: two
substituents on adjacent positions of R.sup.1 may be taken together
to form a 5-7 membered saturated, partially unsaturated, or aryl
ring having 0-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; Ar is a 3-8 membered saturated, unsaturated, or
aryl ring, a 3-7 membered heterocyclic ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, or a 5-6
membered heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, wherein: Ar is
substituted with 0-3 groups independently selected from R', oxo,
CO.sub.2R', OR', N(R').sub.2, SR', NO.sub.2, halogen, CN,
C(O)N(R').sub.2, NR'C(O)R', SO.sub.2R', C(O)R',
SO.sub.2N(R').sub.2, or NR'SO.sub.2R'; R.sup.2 is selected from
hydrogen or a C.sub.1-3 aliphatic group; and Ring A is a 5-6
membered heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, provided that said ring
has a hydrogen-bond acceptor in the position adjacent to the point
of attachment to Ring B, wherein: Ring A is substituted with 0-3
groups independently selected from R', oxo, CO.sub.2R', OR',
N(R').sub.2, SR', NO.sub.2, halogen, CN, C(O)N(R').sub.2,
NR'C(O)R', SO.sub.2R', SO.sub.2N(R').sub.2, or NR'SO.sub.2R', and
wherein: two substituents on adjacent positions of Ring A may be
taken together to form a 5-7 membered saturated, partially
unsaturated, or aryl ring having 0-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
2. The compound according to claim 1, wherein Ring A is selected
from the following optionally substituted rings: ##STR00354##
##STR00355## ##STR00356##
3. The compound according to claim 2, wherein Ring A is an
optionally substituted ring selected from rings a, f, l, s, w, y,
or z: ##STR00357##
4. The compound according to claim 1, wherein: R.sup.1 is selected
from an optionally substituted phenyl or 5-6 membered heteroaryl
ring having 1-2 nitrogens.
5. The compound according to claim 4, wherein R.sup.1 is an
optionally substituted ring selected from pyrid-2-yl, pyrid-3-yl,
pyrid-4-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl,
pyrimidin-6-yl, imidazol-1-yl, imidazol-2-yl, imidazol-4-yl, or
imidazol-5-yl.
6. The compound according to claim 5, wherein R.sup.1 is
substituted with 0-2 groups independently selected from halogen,
oxo, R', CO.sub.2R', OR', N(R').sub.2, SR', C(O)N(R).sub.2,
NR'C(O)R', SO.sub.2R', SO.sub.2N(R').sub.2, or NR'SO.sub.2R'.
7. The compound according to claim 6, wherein R.sup.2 is selected
from methyl, ethyl, isopropyl, or cyclopropyl.
8-16. (canceled)
17. The compound according to claim 1, wherein said compound is of
formula ##STR00358## or a pharmaceutically acceptable salt
thereof.
18. (canceled)
19. The compound according to claim 1 wherein R.sup.2 is ethyl.
20. A compound selected from the group consisting of: ##STR00359##
##STR00360## ##STR00361## ##STR00362## ##STR00363## ##STR00364##
##STR00365## ##STR00366## ##STR00367## ##STR00368## ##STR00369##
##STR00370## ##STR00371## ##STR00372## ##STR00373## ##STR00374##
##STR00375## ##STR00376## ##STR00377## ##STR00378## ##STR00379##
##STR00380## ##STR00381## ##STR00382## ##STR00383## ##STR00384##
##STR00385## ##STR00386## ##STR00387## ##STR00388## ##STR00389##
##STR00390## ##STR00391## ##STR00392## ##STR00393## ##STR00394##
##STR00395## ##STR00396## ##STR00397## ##STR00398## ##STR00399##
##STR00400## ##STR00401## ##STR00402## ##STR00403## ##STR00404##
##STR00405## ##STR00406## ##STR00407## ##STR00408## ##STR00409##
##STR00410## ##STR00411## ##STR00412## ##STR00413## ##STR00414##
##STR00415## ##STR00416## ##STR00417## ##STR00418## ##STR00419##
##STR00420## ##STR00421## ##STR00422## ##STR00423## ##STR00424##
##STR00425## ##STR00426## ##STR00427## ##STR00428## ##STR00429##
##STR00430## ##STR00431## ##STR00432## ##STR00433## ##STR00434##
##STR00435## ##STR00436## ##STR00437## ##STR00438## ##STR00439##
##STR00440## ##STR00441## ##STR00442## ##STR00443## ##STR00444##
##STR00445## ##STR00446## ##STR00447## ##STR00448## and I-295.
21. A composition comprising a compound according to claim 1, and a
pharmaceutically acceptable carrier, adjuvant, or vehicle.
22-24. (canceled)
25. A method of inhibiting gyrase and TopoIV activity in a patient,
comprising the step of contacting said patient with a composition
according to claim 21.
26. A method of decreasing bacterial quantity in a patient,
comprising the step of administering to said patient a composition
according to claim 21.
27. A method of treating, preventing, or lessening the severity of,
a bacterial infection in a patient, comprising the step of
administering to said patient a composition according to claim
21.
28. The method according to claim 27, wherein the bacterial
infection to be treated is characterized by the presence of one or
more of the following: Streptococcus pneumoniae, Streptococcus
pyogenes, Enterococcus faecalis, Enterococcus faecium, Klebsiella
pneumoniae, Enterobacter sps. Proteus sps. Pseudomonas aeruginosa,
E. coli, Serratia marcesens, Staphylococcus aureus, Coag. Neg.
Staph, Haemophilus influenzae, Bacillus anthracis, Mycoplasma
pneumoniae, Moraxella catarralis, Chlamydia pneumoniae, Legionella
pneumophila, Staphylococcus epidermidis, Mycobacterium
tuberculosis, or Helcoibacter pylori.
29. The method according to claim 28, wherein the bacterial
infection to be treated is selected from one or more of the
following: a urinary tract infection, a respiratory infection,
pneumonia, prostatitis, a skin or soft tissue infection, an
intra-abdominal infection, a blood stream infection, or an
infection of febrile neutropenic patients.
30. The method according to claim 29, further comprising the step
of administering to said patient an additional therapeutic agent
either as part of a multiple dosage form together with said
compound or as a separate dosage form.
31. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application 60/443,917 filed Jan. 31, 2003, the contents of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of medicinal chemistry and
relates to compounds, and pharmaceutical compositions thereof, that
inhibit bacterial gyrase and Topo IV. The compounds are useful as
inhibitors of bacterial gyrase and Topo IV activity. The present
invention also relates to methods for treating bacterial infections
in mammals and to methods for decreasing bacterial quantity in a
biological sample.
BACKGROUND OF THE INVENTION
[0003] Bacterial resistance to antibiotics has long been
recognized, and it is today considered to be a serious worldwide
health problem. As a result of resistance, some bacterial
infections are either difficult to treat with antibiotics or even
untreatable. This problem has become especially serious with the
recent development of multiple drug resistance in certain strains
of bacteria, such as Streptococcus pneumoniae (SP), Mycobacterium
tuberculosis, and Enterococcus. The appearance of vancomycin
resistant enterococcus was particularly alarming because vancomycin
was formerly the only effective antibiotic for treating this
infection, and had been considered for many infections to be the
drug of "last resort". While many other drug-resistant bacteria do
not cause life-threatening disease, such as enterococci, there is
the fear that the genes which induce resistance might spread to
more deadly organisms such as Staphylococcus aureus, where
methicillin resistance is already prevalent (De Clerq, et al.,
Current Opinion in Anti-infective Investigational Drugs, 1999, 1,
1; Levy, "The Challenge of Antibiotic Resistance", Scientific
American, March, 1998).
[0004] Another concern is how quickly antibiotic resistance can
spread. For example, until the 1960's SP was universally sensitive
to penicillin, and in 1987 only 0.02% of the SP strains in the U.S.
were resistant. However, by 1995 it was reported that SP resistance
to penicillin was about seven percent and as high as 30% in some
parts of the U.S. (Lewis, FDA Consumer magazine (September, 1995);
Gershman in The Medical Reporter, 1997).
[0005] Hospitals, in particular, serve as centers for the formation
and transmission of drug-resistant organisms. Infections occurring
in hospitals, known as nosocomial infections, are becoming an
increasingly serious problem. Of the two million Americans infected
in hospitals each year, more than half of these infections resist
at least one antibiotic. The Center for Disease Control reported
that in 1992, over 13,000 hospital patients died of bacterial
infections that were resistant to antibiotic treatment (Lewis, "The
Rise of Antibiotic-Resistant Infections", FDA Consumer magazine,
September, 1995).
[0006] As a result of the need to combat drug-resistant bacteria
and the increasing failure of the available drugs, there has been a
resurgent interest in discovering new antibiotics. One attractive
strategy for developing new antibiotics is to inhibit DNA gyrase, a
bacterial enzyme necessary for DNA replication, and therefore,
necessary for bacterial cell growth and division. Gyrase activity
is also associated with events in DNA transcription, repair and
recombination.
[0007] Gyrase is one of the topoisomerases, a group of enzymes
which catalyze the interconversion of topological isomers of DNA
(see generally, Kornberg and Baker, DNA Replication, 2d Ed.,
Chapter 12, 1992, W.H. Freeman and Co.; Drlica, Molecular
Microbiology, 1992, 6, 425; Drlica and Zhao, Microbiology and
Molecular Biology Reviews, 1997, 61, 377). Gyrase itself controls
DNA supercoiling and relieves topological stress that occurs when
the DNA strands of a parental duplex are untwisted during the
replication process. Gyrase also catalyzes the conversion of
relaxed, closed circular duplex DNA to a negatively superhelical
form which is more favorable for recombination. The mechanism of
the supercoiling reaction involves the wrapping of gyrase around a
region of the DNA, double strand breaking in that region, passing a
second region of the DNA through the break, and rejoining the
broken strands. Such a cleavage mechanism is characteristic of a
type II topoisomerase. The supercoiling reaction is driven by the
binding of ATP to gyrase. The ATP is then hydrolyzed during the
reaction. This ATP binding and subsequent hydrolysis cause
conformational changes in the DNA-bound gyrase that are necessary
for its activity. It has also been found that the level of DNA
supercoiling (or relaxation) is dependent on the ATP/ADP ratio. In
the absence of ATP, gyrase is only capable of relaxing supercoiled
DNA.
[0008] Bacterial DNA gyrase is a 400 kilodalton protein tetramer
consisting of two A (GyrA) and two B subunits (GyrB). Binding and
cleavage of the DNA is associated with GyrA, whereas ATP is bound
and hydrolyzed by the GyrB protein. GyrB consists of an
amino-terminal domain which has the ATPase activity, and a
carboxy-terminal domain which interacts with GyrA and DNA. By
contrast, eukaryotic type II topoisomerases are homodimers that can
relax negative and positive supercoils, but cannot introduce
negative supercoils. Ideally, an antibiotic based on the inhibition
of bacterial DNA gyrase would be selective for this enzyme and be
relatively inactive against the eukaryotic type II
topoisomerases.
[0009] The widely used quinolone antibiotics inhibit bacterial DNA
gyrase. Examples of the quinolones include the early compounds such
as nalidixic acid and oxolinic acid, as well as the later, more
potent fluoroquinolones such as norfloxacin, ciprofloxacin, and
trovafloxacin. These compounds bind to GyrA and stabilize the
cleaved complex, thus inhibiting overall gyrase function, leading
to cell death. However, drug resistance has also been recognized as
a problem for this class of compounds (WHO Report, "Use of
Quinolones in Food Animals and Potential Impact on Human Health",
1998). With the quinolones, as with other classes of antibiotics,
bacteria exposed to earlier compounds often quickly develop
cross-resistance to more potent compounds in the same class.
[0010] There are fewer known inhibitors that bind to GyrB. Examples
include the coumarins, novobiocin and coumermycin A1,
cyclothialidine, cinodine, and clerocidin. The coumarins have been
shown to bind to GyrB very tightly. For example, novobiocin makes a
network of hydrogen bonds with the protein and several hydrophobic
contacts. While novobiocin and ATP do appear to bind within the ATP
binding site, there is minimal overlap in the bound orientation of
the two compounds. The overlapping portions are the sugar unit of
novobiocin and the ATP adenine (Maxwell, Trends in Microbiology,
1997, 5, 102).
[0011] For coumarin-resistant bacteria, the most prevalent point
mutation is at a surface arginine residue that binds to the
carbonyl of the coumarin ring (Arg136 in E. coli GyrB). While
enzymes with this mutation show lower supercoiling and ATPase
activity, they are also less sensitive to inhibition by coumarin
drugs (Maxwell, Mol. Microbiol., 1993, 9, 681).
[0012] Despite being potent inhibitors of gyrase supercoiling, the
coumarins have not been widely used as antibiotics. They are
generally not suitable due to their low permeability in bacteria,
eukaryotic toxicity, and poor water solubility (Maxwell, Trends in
Microbiology, 1997, 5, 102). It would be desirable to have a new,
effective GyrB inhibitor that overcomes these drawbacks. Such an
inhibitor would be an attractive antibiotic candidate, without a
history of resistance problems that plague other classes of
antibiotics.
[0013] Replication fork movement along circular DNA can generate
topological changes both ahead of the replication complex as well
as behind in the already replicated regions (Champoux, J. J., Annu.
Rev. Biochem., 2001, 70, 369-413). While DNA gyrase can introduce
negative supercoils to compensate for the topological stresses
ahead of the replication fork, some overwinding can diffuse back
into the already replicated region of DNA resulting in
precatenanes. If not removed, the presence of the precatenanes can
result in interlinked (catenated) daughter molecules at the end of
replication. TopoIV is responsible for separating the catenated
daughter plasmids as well as removal of precatenanes formed during
replication ultimately allowing for segragation of the daughter
molecules into daughter cells. Topo IV is composed of two ParC and
2 parE subunits as a C2E2 tetramer (where the C and E monomers are
homologuous to the A and B monomers of gyrase, respectively) that
requires ATP hydrolysis (at the N-terminus of the E subunit) to
reset the enzyme to re-enter the catalytic cycle. Topo IV is highly
conserved among bacteria and is essential for bacterial replication
(Drlica and Zhao, Microbiol. Mol. Biol. Rev., 1997, 61, 377).
[0014] While little attention has been paid to inhibitors that
target ParE of TopoIV, the action of the newer quinolones on the
ParC region has been widely studied (Hooper, D. C., Clin. Infect.
Dis., 2000, 31(Suppl 2): S24-28). It has been demonstrated that
moxifloxacin and gatifloxacin have more balanced activities against
Gyrase and TopoIV resulting in expanded Gram positive coverage as
well as lower levels of resistance caused primary-target mutation.
In those cases, susceptibility is limited by the sensitivity of the
second target to the antibacterial agent. Thus, agents that can
effectively inhibit multiple essential targets can result in an
expanded spectrum of potencies, improved antibacterial potencies,
improved potency against single target mutants, and/or lower
spontaneous rates of resistance.
[0015] As bacterial resistance to antibiotics has become an
important public health problem, there is a continuing need to
develop newer and more potent antibiotics. More particularly, there
is a need for antibiotics that represent a new class of compounds
not previously used to treat bacterial infection. Such compounds
would be particularly useful in treating nosocomial infections in
hospitals where the formation and transmission of resistant
bacteria are becoming increasingly prevalent.
SUMMARY OF THE INVENTION
[0016] It has now been found that compounds of this invention, and
pharmaceutically acceptable compositions thereof, are effective as
inhibitors of gyrase and/or Topo IV. These compounds have the
general formula I:
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, W, X, Z, and Ring A are as defined below.
[0017] These compounds, and pharmaceutically acceptable
compositions thereof, are useful for treating or lessening the
severity of bacterial infections. In particular, the compounds of
the present invention are useful in treating or lessening the
severity of urinary tract infections, pneumonia, prostatitis, skin
and soft tissue infections, intra-abdominal infections, blood
stream infections, or infections of febrile neutropenic
patients
DESCRIPTION OF THE INVENTION
[0018] The present invention relates to a compound of formula
I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein: [0019] W is
selected from nitrogen, CH, or CF; [0020] X is selected from CH or
CF; [0021] Z is O or NH; [0022] R.sup.1 is phenyl or a 5-6 membered
heteroaryl ring having 1-3 heteroatoms independently selected from
oxygen, nitrogen, or sulfur, wherein: [0023] R.sup.1 is substituted
with 0-3 groups independently selected from -(T).sub.y-Ar, R', oxo,
C(O)R', CO.sub.2R', OR', N(R').sub.2, SR', NO.sub.2, halogen, CN,
C(O)N(R').sub.2, NR'C(O)R', SO.sub.2R', SO.sub.2N(R').sub.2, or
NR'SO.sub.2R'; [0024] y is 0 or 1; [0025] T is a straight or
branched C.sub.1-4 alkylidene chain, wherein one methylene unit of
T is optionally replaced by --O--, --NH--, or --S--; [0026] each R'
is independently selected from hydrogen, C.sub.1-4 aliphatic, or a
5-6 membered saturated, unsaturated, or aryl ring having 0-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, wherein: [0027] R' is substituted with 0-3 groups
independently selected from halogen, oxo, R.sup.o,
N(R.sup.o).sub.2, OR.sup.o, CO.sub.2R.sup.o, NR.sup.oC(O)R.sup.o,
C(O)N(R.sup.o).sub.2, SO.sub.2R.sup.o, SO.sub.2N(R.sup.o).sub.2, or
NR.sup.oSO.sub.2R.sup.o, wherein: [0028] each R.sup.o is
independently selected from hydrogen, C.sub.1-4 aliphatic, or a 5-6
membered saturated, unsaturated, or aryl ring having 0-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, and wherein: [0029] two substituents on adjacent positions
of R.sup.1 may be taken together to form a 5-7 membered saturated,
partially unsaturated, or aryl ring having 0-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; [0030] Ar
is a 3-8 membered saturated, unsaturated, or aryl ring, a 3-7
membered heterocyclic ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or a 5-6 membered
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, wherein: [0031] Ar is substituted with
0-3 groups independently selected from R', oxo, CO.sub.2R', OR',
N(R').sub.2, SR', NO.sub.2, halogen, CN, C(O)N(R').sub.2,
NR'C(O)R', SO.sub.2R', C(O)R', SO.sub.2N(R').sub.2, or
NR'SO.sub.2R'; [0032] R.sup.2 is selected from hydrogen or a
C.sub.1-3 aliphatic group; and [0033] Ring A is a 5-6 membered
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, provided that said ring has a
hydrogen-bond acceptor in the position adjacent to the point of
attachment to Ring B, wherein: [0034] Ring A is substituted with
0-3 groups independently selected from R', oxo, CO.sub.2R', OR',
N(R').sub.2, SR', NO.sub.2, halogen, CN, C(O)N(R').sub.2,
NR'C(O)R', SO.sub.2R', SO.sub.2N(R').sub.2, or NR'SO.sub.2R', and
wherein: [0035] two substituents on adjacent positions of Ring A
may be taken together to form a 5-7 membered saturated, partially
unsaturated, or aryl ring having 0-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0036] As used herein, the following definitions shall apply unless
otherwise indicated.
[0037] The phrase "optionally substituted" is used interchangeably
with the phrase "substituted or unsubstituted." Unless otherwise
indicated, an optionally substituted group may have a substituent
at each substitutable position of the group, and each substitution
is independent of the other.
[0038] The term "aliphatic" or "aliphatic group", as used herein,
means a straight-chain or branched C.sub.1-C.sub.8 hydrocarbon
chain that is completely saturated or that contains one or more
units of unsaturation, or a monocyclic C.sub.3-C.sub.8 hydrocarbon
or bicyclic C.sub.8-C.sub.12 hydrocarbon that is completely
saturated or that contains one or more units of unsaturation, but
which is not aromatic (also referred to herein as "carbocycle" or
"cycloalkyl"), that has a single point of attachment to the rest of
the molecule wherein any individual ring in said bicyclic ring
system has 3-7 members. For example, suitable aliphatic groups
include, but are not limited to, linear or branched or alkyl,
alkenyl, alkynyl groups and hybrids thereof such as
(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
[0039] The terms "alkyl", "alkoxy", "hydroxyalkyl", "alkoxyalkyl",
and "alkoxycarbonyl", used alone or as part of a larger moiety
include both straight and branched chains containing one to twelve
carbon atoms. The terms "alkenyl" and "alkynyl" used alone or as
part of a larger moiety shall include both straight and branched
chains containing two to twelve carbon atoms.
[0040] The term "heteroatom" means nitrogen, oxygen, or sulfur and
includes any oxidized form of nitrogen and sulfur, and the
quaternized form of any basic nitrogen. Also the term "nitrogen"
includes a substitutable nitrogen of a heterocyclic ring. As an
example, in a saturated or partially unsaturated ring having 0-3
heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen
may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl)
or NR.sup.+ (as in N-substituted pyrrolidinyl).
[0041] The term "unsaturated", as used herein, means that a moiety
has one or more units of unsaturation, and includes aryl rings.
[0042] The term "aryl" used alone or as part of a larger moiety as
in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic,
bicyclic and tricyclic ring systems having a total of five to
fourteen ring members, wherein at least one ring in the system is
aromatic and wherein each ring in the system contains 3 to 7 ring
members. The term "aryl" may be used interchangeably with the term
"aryl ring". The term "aryl" also refers to heteroaryl ring systems
as defined hereinbelow.
[0043] The term "heterocycle", "heterocyclyl", or "heterocyclic" as
used herein means non-aromatic, monocyclic, bicyclic or tricyclic
ring systems having five to fourteen ring members in which one or
more ring members is a heteroatom, wherein each ring in the system
contains 3 to 7 ring members.
[0044] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroaralkyl" or "heteroarylalkoxy", refers to
monocyclic, bicyclic and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, at least one ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
[0045] The term "hydrogen bond acceptor", as used herein, means an
atom capable of accepting a hydrogen bond. A typical hydrogen bond
acceptor is a sulfur, oxygen, or nitrogen atom, especially a
nitrogen that is sp.sup.2-hybridized, an ether oxygen, or a
thioether sulfur. A preferred hydrogen bond acceptor is a nitrogen
that is sp.sup.2-hybridized.
[0046] A combination of substituents or variables is permissible
only if such a combination results in a stable or chemically
feasible compound. A stable compound or chemically feasible
compound is one that is not substantially altered when kept at a
temperature of 40.degree. C. or less, in the absence of moisture or
other chemically reactive conditions, for at least a week.
[0047] It will be apparent to one skilled in the art that certain
compounds of this invention may exist in tautomeric forms, all such
tautomeric forms of the compounds being within the scope of the
invention.
[0048] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the invention. Unless otherwise stated, structures
depicted herein are also meant to include compounds that differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by a .sup.13C-- or .sup.14C-enriched carbon
are within the scope of this invention. Such compounds are useful,
for example, as analytical tools or probes in biological
assays.
[0049] Examples of suitable Ring A moieties are set forth in Table
1 below.
TABLE-US-00001 TABLE 1 ##STR00003## a ##STR00004## b ##STR00005## c
##STR00006## d ##STR00007## e ##STR00008## f ##STR00009## g
##STR00010## h ##STR00011## i ##STR00012## j ##STR00013## k
##STR00014## l ##STR00015## m ##STR00016## n ##STR00017## o
##STR00018## p ##STR00019## q ##STR00020## r ##STR00021## s
##STR00022## t ##STR00023## u ##STR00024## v ##STR00025## w
##STR00026## x ##STR00027## y ##STR00028## z ##STR00029## aa
##STR00030## bb ##STR00031## cc ##STR00032## dd ##STR00033## ee
##STR00034## ff ##STR00035## gg ##STR00036## hh
wherein each Ring A is optionally substituted as defined above.
[0050] According to one embodiment, Ring A of formula I is a
5-membered heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, provided that said ring
has a hydrogen-bond acceptor in the position adjacent to the point
of attachment to Ring B, wherein said Ring A is optionally
substituted as defined herein supra.
[0051] According to another embodiment, Ring A of formula I is a
6-membered heteroaryl ring having 1-3 nitrogens, provided that said
ring has a nitrogen atom in the position adjacent to the point of
attachment to Ring B, wherein said Ring A is optionally substituted
as defined herein supra.
[0052] In certain embodiments, Ring A moieties of formula I are
selected from rings a, b, c, d, e, f, g, h, i, j, k, l, m, p, q, r,
s, t, v, w, x, y, z, aa, bb, cc, dd, and ee, wherein each Ring A is
optionally substituted as defined above.
[0053] In other embodiments, the Ring A moieties of formula I are
selected from rings a, f, l, s, w, y, and z, wherein each Ring A is
optionally substituted as defined above.
[0054] When Ring A of formula I is a bicyclic heteroaryl ring,
preferred bicyclic Ring A moieties include benzothiazole,
benzimidazole, benzoxazole, and quinoline.
[0055] According to one embodiment, substituents on Ring A of
formula I, if present, are selected from oxo, N(R').sub.2,
C(O)N(R').sub.2, CO.sub.2R', halogen, N(R')SO.sub.2R', C(O)R', OR',
or R'. According to another embodiment, R' substituents on Ring A
of formula I include methyl, ethyl, propyl, piperazinyl,
piperidinyl, or morpholinyl, wherein said R' groups are optionally
substituted with R.sup.o, N(R.sup.o).sub.2 or OR.sup.o.
[0056] According to one embodiment, the R.sup.1 group of formula I
is optionally substituted phenyl.
[0057] According to another embodiment, the R.sup.1 group of
formula I is an optionally substituted 5-membered heteroaryl ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0058] According to another embodiment, the R.sup.1 group of
formula I is an optionally substituted 5-membered heteroaryl ring
having 1-3 nitrogens.
[0059] Yet another embodiment of the present invention relates to a
compound of formula I wherein R.sup.1 is an optionally substituted
6-membered heteroaryl ring having 1-2 nitrogens.
[0060] In certain embodiments, the R.sup.1 group of formula I is
selected from an optionally substituted phenyl or 5-6 membered
heteroaryl ring having 1-2 nitrogens. In other embodiments, the
R.sup.1 group of formula I is selected from an optionally
substituted pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, pyridone,
pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyrimidin-6-yl,
imidazol-1-yl, imidazol-2-yl, imidazol-4-yl, or imidazol-5-yl ring.
According to yet another embodiment, the R.sup.1 group of formula I
is an optionally substituted ring selected from pyrid-3-yl,
pyrid-4-yl, pyridone, pyrimidin-5-yl, or imidazol-1-yl.
[0061] In certain embodiments, substituents on the R.sup.1 group of
formula I, when present, are selected from halogen, oxo,
-(T).sub.y-Ar, R', CO.sub.2R', OR', N(R').sub.2, SR',
C(O)N(R').sub.2, NR'C(O)R', SO.sub.2R', SO.sub.2N(R').sub.2, or
NR'SO.sub.2R'. According to other embodiments, substituents on the
R.sup.1 group of formula I, when present, are selected from oxo,
fluoro, chloro, N(CH.sub.3).sub.2, NHCH.sub.2CH.sub.3,
NH-cyclopropyl, NH.sub.2, NHC(O)CH.sub.3, C(O)NHcyclopropyl,
methyl, ethyl, t-butyl, isobutyl, cyclopropyl, isopropyl,
CH.sub.2-phenyl, CH.sub.2pyridin-3-yl, OH, OCH.sub.3,
OCH.sub.2CH.sub.3, OCH.sub.2-phenyl, OCH.sub.2pyridin-3-yl,
CH.sub.2piperidinyl, CH.sub.2cyclopropyl, or
CH.sub.2CH.sub.2OCH.sub.3.
[0062] According to one embodiment, R.sup.1 is substituted with
-(T).sub.y-Ar wherein T is a straight or branched C.sub.1-3
alkylidene chain wherein one methylene unit of T is optionally
replaced by --O--, --NH--, or --S--. According to another
embodiment, T is a straight or branched C.sub.1-3 alkylidene chain
wherein one methylene unit of T is replaced by --O--, --NH--, or
--S--. Yet another embodiment of the present invention relates to a
compound of formula I wherein R.sup.1 is substituted with
-(T).sub.y-Ar and Ar is an optionally substituted 5-6 membered
saturated ring having 1-2 heteroatoms independently selected from
oxygen, nitrogen, or sulfur. According to another embodiment, the
Ar group of formula I is an optionally substituted 5-membered
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. According to yet another embodiment,
the Ar group of formula I is an optionally substituted 6-membered
heteroaryl ring having 1-3 nitrogens. Yet another embodiment
relates to a compound of formula I wherein Ar is optionally
substituted phenyl.
[0063] When the R.sup.1 group of formula I is substituted with
-(T).sub.y-Ar, examples of substituents on Ar include halogen, OR',
R', CO.sub.2R', SO.sub.2R', oxo, and C(O)R'.
[0064] According to one embodiment, when two substituents on
adjacent positions of R.sup.1 of formula I are taken together to
form an optionally substituted ring fused to R.sup.1, rings formed
thereby include 5-6 membered saturated, partially unsaturated, or
aryl rings having 0-2 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. According to another embodiment, said
ring fused to R.sup.1 is selected from a 5-membered saturated ring
having two oxygens or a 6-membered saturated ring having two
oxygens. Examples of substituents on said ring fused to R.sup.1
include halogen, such as fluorine.
[0065] One embodiment of the present invention relates to a
compound of formula I wherein R.sup.2 is selected from methyl,
ethyl, isopropyl, or cyclopropyl. According to another embodiment,
R.sup.2 is methyl or ethyl. According to yet another embodiment,
R.sup.2 of formula I is ethyl.
[0066] According to one embodiment, the present invention relates
to a compound of formula I wherein Z is NH.
[0067] According to another embodiment, the present invention
relates to a compound of formula I wherein Z is O.
[0068] Compounds of the present invention fall within the genus of
compounds described in PCT/US01/48855. However, applicants have
discovered that the presence of the Ring A moiety, as defined
above, imparts surprising and unexpectedly increased gyrase
inhibitory, TopoIV activity, and antimicrobial potency.
[0069] According to one embodiment, the present invention relates
to a compound of formula II:
##STR00037##
or a pharmaceutically acceptable salt thereof, wherein Z, R.sup.2
and Ring A are as defined above and the imidazole ring depicted is
optionally substituted in the 4-position with C(O)N(R').sub.2
and/or substituted in the 2-position with R'. Accordingly, another
embodiment of the present invention relates to a compound of
formula II-a:
##STR00038##
or a pharmaceutically acceptable salt thereof, wherein Z, R.sup.2,
R', and Ring A are as defined above.
[0070] Other embodiments describing R.sup.2 and Ring A groups of
formula II-a are those described for formula I above.
[0071] Other embodiments describing R' groups of formula II-a are
selected from hydrogen or C.sub.1-4 aliphatic.
[0072] According to one embodiment, the present invention relates
to a compound of formula II or II-a wherein Z is NH.
[0073] According to another embodiment, the present invention
relates to a compound of formula II or II-a wherein Z is O.
[0074] According to another embodiment, the present invention
relates to a compound of formula III:
##STR00039##
or a pharmaceutically acceptable salt thereof, wherein Z, R.sup.2
and Ring A are as defined above, and the pyridone ring depicted is
substituted with 0-2 groups independently selected from
--(CH.sub.2).sub.y--Ar, halogen, oxo, R', CO.sub.2R', OR',
N(R').sub.2, SR', C(O)N(R').sub.2, NR'C(O)R', SO.sub.2R',
SO.sub.2N(R').sub.2, or NR'SO.sub.2R'.
[0075] Other embodiments describing R.sup.2 and Ring A groups of
formula III are those described for formula I above.
[0076] Other embodiments describing substituents on the pyridone
ring of formula III are those described above as preferred
substituents on R.sup.1 of formula I.
[0077] According to one embodiment, the present invention relates
to a compound of formula III wherein Z is NH.
[0078] According to another embodiment, the present invention
relates to a compound of formula III wherein Z is O.
[0079] According to another embodiment, the present invention
relates to a compound of formula III-a:
##STR00040##
or a pharmaceutically acceptable salt thereof, wherein Z, R',
R.sup.2 and Ring A are as defined above.
[0080] Other embodiments describing R.sup.2 groups of formula III-a
are those described for R.sup.2 groups of formula I above.
[0081] Other embodiments describing Ring A groups of formula III-a
are those described for Ring A groups of formula I above.
[0082] In certain embodiments, the R' substituents on the pyridone
ring of formula III-a are selected from hydrogen or C.sub.1-4
aliphatic wherein R' is optionally substituted with phenyl or
pyridyl. In other embodiments, the R' substituents on the pyridone
ring of formula III-a are selected from methyl, ethyl, t-butyl,
isobutyl, cyclopropyl, isopropyl, CH.sub.2-phenyl,
CH.sub.2pyridin-3-yl, CH.sub.2piperidinyl, CH.sub.2cyclopropyl, or
CH.sub.2CH.sub.2OCH.sub.3.
[0083] According to one embodiment, the present invention relates
to a compound of formula III-a wherein Z is NH.
[0084] According to another embodiment, the present invention
relates to a compound of formula III-a wherein Z is O.
[0085] Yet another embodiment of the present invention relates to a
compound of formula IV:
##STR00041##
or a pharmaceutically acceptable salt thereof, wherein y, Z, T, Ar,
R.sup.2 and Ring A are as defined above.
[0086] Other embodiments describing Ring A and R.sup.2 groups of
formula IV are those set forth for those Ring A and R.sup.2 groups
of formula I, supra.
[0087] According to one embodiment, the Ar group of formula IV is
an optionally substituted 5-6 membered saturated ring having 1-2
heteroatoms independently selected from oxygen, nitrogen, or
sulfur.
[0088] According to another embodiment, the Ar group of formula IV
is an optionally substituted 5-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0089] According to another embodiment, the Ar group of formula IV
is an optionally substituted 6-membered heteroaryl ring having 1-3
nitrogens.
[0090] Yet another embodiment relates to a compound of formula IV
wherein Ar is optionally substituted phenyl.
[0091] According to one embodiment, the present invention relates
to a compound of formula IV wherein Z is NH.
[0092] Examples of substituents on the Ar group of formula IV
include halogen, OR', R', CO.sub.2R', SO.sub.2R', oxo, and
C(O)R'.
[0093] According to another embodiment, the present invention
relates to a compound of formula IV wherein Z is O.
[0094] Yet another embodiment of the present invention relates to a
compound of formula V:
##STR00042##
or a pharmaceutically acceptable salt thereof, wherein y, Z,
R.sup.2 and R.sup.1 are as defined above.
[0095] Other embodiments describing R.sup.1 and R.sup.2 groups of
formula V are those set forth for those R.sup.1 and R.sup.2 groups
of formula I, supra.
[0096] According to one embodiment, the present invention relates
to a compound of formula V wherein Z is NH.
[0097] Examples of substituents on the Ar group of formula IV
include halogen, OR', R', CO.sub.2R', SO.sub.2R', oxo, and
C(O)R'.
[0098] According to another embodiment, the present invention
relates to a compound of formula V wherein Z is O.
[0099] According to another embodiment of the present invention
relates to a compound of formula VI:
##STR00043##
or a pharmaceutically acceptable salt thereof, wherein y, Z, T, Ar,
and R.sup.2 are as defined above.
[0100] Other embodiments describing the R.sup.2 group of formula VI
are those set forth for the R.sup.2 group of formula I, supra.
[0101] According to one embodiment, the Ar group of formula VI is
an optionally substituted 5-6 membered saturated ring having 1-2
heteroatoms independently selected from oxygen, nitrogen, or
sulfur.
[0102] According to another embodiment, the Ar group of formula VI
is an optionally substituted 5-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0103] According to another embodiment, the Ar group of formula VI
is an optionally substituted 6-membered heteroaryl ring having 1-3
nitrogens.
[0104] Yet another embodiment relates to a compound of formula VI
wherein Ar is optionally substituted phenyl.
[0105] According to one embodiment, the present invention relates
to a compound of formula VI wherein Z is NH.
[0106] Examples of substituents on the Ar group of formula VI
include halogen, OR', R', CO.sub.2R', SO.sub.2R', oxo, and
C(O)R'.
[0107] According to another embodiment, the present invention
relates to a compound of formula VI wherein Z is O.
[0108] Exemplary structures of formula I are set forth in Table 2
below.
TABLE-US-00002 TABLE 2 ##STR00044## I-1 ##STR00045## I-2
##STR00046## I-3 ##STR00047## I-4 ##STR00048## I-5 ##STR00049## I-6
##STR00050## I-7 ##STR00051## I-8 ##STR00052## I-9 ##STR00053##
I-10 ##STR00054## I-11 ##STR00055## I-12 ##STR00056## I-13
##STR00057## I-14 ##STR00058## I-15 ##STR00059## I-16 ##STR00060##
I-17 ##STR00061## I-18 ##STR00062## I-19 ##STR00063## I-20
##STR00064## I-21 ##STR00065## I-22 ##STR00066## I-23 ##STR00067##
I-24 ##STR00068## I-25 ##STR00069## I-26 ##STR00070## I-28
##STR00071## I-29 ##STR00072## I-30 ##STR00073## I-31 ##STR00074##
I-32 ##STR00075## I-35 ##STR00076## I-37 ##STR00077## I-38
##STR00078## I-39 ##STR00079## I-40 ##STR00080## I-41 ##STR00081##
I-42 ##STR00082## I-43 ##STR00083## I-44 ##STR00084## I-45
##STR00085## I-46 ##STR00086## I-47 ##STR00087## I-48 ##STR00088##
I-49 ##STR00089## I-50 ##STR00090## I-51 ##STR00091## I-52
##STR00092## I-53 ##STR00093## I-54 ##STR00094## I-55 ##STR00095##
I-56 ##STR00096## I-57 ##STR00097## I-60 ##STR00098## I-61
##STR00099## I-62 ##STR00100## I-63 ##STR00101## I-64 ##STR00102##
I-65 ##STR00103## I-66 ##STR00104## I-67 ##STR00105## I-68
##STR00106## I-69 ##STR00107## I-70 ##STR00108## I-71 ##STR00109##
I-72 ##STR00110## I-73 ##STR00111## I-74 ##STR00112## I-75
##STR00113## I-76 ##STR00114## I-77 ##STR00115## I-78 ##STR00116##
I-79 ##STR00117## I-80 ##STR00118## I-81 ##STR00119## I-82
##STR00120## I-83 ##STR00121## I-84 ##STR00122## I-85 ##STR00123##
I-86 ##STR00124## I-87 ##STR00125## I-88 ##STR00126## I-89
##STR00127## I-90 ##STR00128## I-91 ##STR00129## I-92 ##STR00130##
I-93 ##STR00131## I-94 ##STR00132## I-95 ##STR00133## I-96
##STR00134## I-97 ##STR00135## I-98 ##STR00136## I-99 ##STR00137##
I-100 ##STR00138## I-101 ##STR00139## I-102 ##STR00140## I-103
##STR00141## I-104 ##STR00142## I-105 ##STR00143## I-106
##STR00144## I-107 ##STR00145## I-108 ##STR00146## I-109
##STR00147## I-110 ##STR00148## I-111 ##STR00149## I-112
##STR00150## I-113 ##STR00151## I-114 ##STR00152## I-115
##STR00153## I-116 ##STR00154## I-117 ##STR00155## I-118
##STR00156## I-119 ##STR00157## I-120 ##STR00158## I-121
##STR00159## I-122 ##STR00160## I-123 ##STR00161## I-124
##STR00162## I-125 ##STR00163## I-126 ##STR00164## I-127
##STR00165## I-128 ##STR00166## I-129 ##STR00167## I-130
##STR00168## I-131 ##STR00169## I-132 ##STR00170## I-133
##STR00171## I-134 ##STR00172## I-135 ##STR00173## I-136
##STR00174## I-137 ##STR00175## I-138 ##STR00176## I-139
##STR00177## I-140 ##STR00178## I-141 ##STR00179## I-142
##STR00180## I-143 ##STR00181## I-144 ##STR00182## I-145
##STR00183## I-146 ##STR00184## I-147 ##STR00185## I-148
##STR00186## I-149 ##STR00187## I-150 ##STR00188## I-151
##STR00189## I-152 ##STR00190## I-153 ##STR00191## I-154
##STR00192## I-155 ##STR00193## I-156 ##STR00194## I-157
##STR00195## I-158 ##STR00196## I-159 ##STR00197## I-160
##STR00198## I-161 ##STR00199## I-162 ##STR00200## I-163
##STR00201## I-164 ##STR00202## I-165 ##STR00203## I-166
##STR00204## I-167 ##STR00205## I-168 ##STR00206## I-169
##STR00207## I-170 ##STR00208## I-171 ##STR00209## I-172
##STR00210## I-173 ##STR00211## I-174 ##STR00212## I-175
##STR00213## I-176 ##STR00214## I-177 ##STR00215## I-178
##STR00216## I-179 ##STR00217## I-180 ##STR00218## I-181
##STR00219## I-182 ##STR00220## I-183 ##STR00221## I-184
##STR00222## I-185 ##STR00223## I-186 ##STR00224## I-187
##STR00225## I-188 ##STR00226## I-189 ##STR00227## I-190
##STR00228## I-191 ##STR00229## I-192 ##STR00230## I-193
##STR00231## I-194 ##STR00232## I-195 ##STR00233## I-196
##STR00234## I-197 ##STR00235## I-198 ##STR00236## I-199
##STR00237## I-200 ##STR00238## I-201 ##STR00239## I-202
##STR00240## I-203 ##STR00241## I-204 ##STR00242## I-205
##STR00243## I-206 ##STR00244## I-207 ##STR00245## I-208
##STR00246## I-209 ##STR00247## I-210 ##STR00248## I-211
##STR00249## I-212 ##STR00250## I-213 ##STR00251## I-214
##STR00252## I-215 ##STR00253## I-216 ##STR00254## I-217
##STR00255## I-218 ##STR00256## I-219 ##STR00257## I-220
##STR00258## I-221 ##STR00259## I-222 ##STR00260## I-223
##STR00261## I-224 ##STR00262## I-225 ##STR00263## I-226
##STR00264## I-227 ##STR00265## I-228 ##STR00266## I-229
##STR00267## I-230 ##STR00268## I-231 ##STR00269## I-232
##STR00270## I-233 ##STR00271## I-234 ##STR00272## I-235
##STR00273## I-236 ##STR00274## I-237 ##STR00275## I-238
##STR00276## I-239 ##STR00277## I-240 ##STR00278## I-241
##STR00279## I-242 ##STR00280## I-243 ##STR00281## I-244
##STR00282## I-245 ##STR00283## I-246 ##STR00284## I-247
##STR00285## I-248 ##STR00286## I-249 ##STR00287## I-250
##STR00288## I-251 ##STR00289## I-252 ##STR00290## I-253
##STR00291## I-254 ##STR00292## I-255 ##STR00293## I-256
##STR00294## I-257 ##STR00295## I-258 ##STR00296## I-259
##STR00297## I-260 ##STR00298## I-261 ##STR00299## I-262
##STR00300## I-263 ##STR00301## I-264 ##STR00302## I-265
##STR00303## I-266 ##STR00304## I-267 ##STR00305## I-268
##STR00306## I-269 ##STR00307## I-270 ##STR00308## I-271
##STR00309## I-272 ##STR00310## I-273 ##STR00311## I-274
##STR00312## I-275 ##STR00313## I-276 ##STR00314## I-277
##STR00315## I-278 ##STR00316## I-279 ##STR00317## I-280
##STR00318## I-281 ##STR00319## I-282 ##STR00320## I-283
##STR00321## I-284 ##STR00322## I-285 ##STR00323## I-286
##STR00324## I-287 ##STR00325## I-288 ##STR00326## I-289
##STR00327## I-290 ##STR00328## I-291 ##STR00329## I-292
##STR00330## I-293 ##STR00331## I-294 ##STR00332## I-295
[0109] The compounds of this invention may be prepared in general
by methods known to those skilled in the art for analogous
compounds, as illustrated by the general Schemes I, II, III, and IV
shown below and the Examples set forth infra.
##STR00333##
[0110] Scheme I above shows a general method for preparing
N'-alkyl-N-cyanoureas (3) useful in the preparation of the
compounds of the present invention wherein Z is NH. At step (a),
cyanamide (2) is treated with an alkyl isocyanate in aqueous sodium
hydroxide to afford, after acidification, compound 3. One of skill
in the art would recognize that a variety of alkyl isocyanates
would be amenable to the reaction conditions of Scheme Ito form a
variety of N'-alkyl-N-cyanoureas.
##STR00334##
[0111] Scheme II above shows a general method for preparing the
benzimidazole compounds of the present invention wherein Z is NH or
O. The bromo-aniline (4) is treated with sodium perborate and
acetic acid to form the difluoro-nitro compound (5). Compound 5 is
treated with Ring A in the presence of sodium hydride to afford the
bi-aryl compound 6. The remaining fluoro group of compound 6 is
displaced with ammonia to form the amino compound (7). The
2-nitro-5-bromoaniline (7) is then coupled to an aryl boronic acid,
at step (d), in the presence of palladium to form the tri-aryl
compound (8). The nitro group of compound 8 is reduced to form a
diamino compound which is treated with an N'-alkyl-N-cyanourea (3)
to form benzimidazole compound of formula I wherein Z is NH
(9).
[0112] Alternatively, intermediate 8 may be used to form compounds
of formula I wherein Z is O. Compound 10 is formed by treating 8,
after reduction to the diamino compound, with
2-methyl-2-thiopseudourea and R.sup.2-chloroformate according to
the method described by L. I. Kruse et al, J. Med. Chem. 1989, 32,
409-417. One of ordinary skill in the art would recognize that the
reactions depicted in Scheme II above are amenable to a variety of
R.sup.1 and Ring A groups of the present invention.
[0113] In an alternative method, intermediate 8 is treated with
either N,N-diethlycarboxy-2-methyl-2-thiopseudourea or
N,N-diethlyureamido-2-methyl-2-thiopseudourea to form compounds 10
and 9, respectively. The syntheses of both
N,N-diethlycarboxy-2-methyl-2-thiopseudourea and
N,N-diethlyureamido-2-methyl-2-thiopseudourea are described in the
Examples set forth infra.
##STR00335##
[0114] Scheme III above shows a general method for preparing
compounds of formula II-a using methods substantially similar to
those described by Kiyomori, A.; Marcoux, J.-F.; Buchwald, S. L.,
Tetrahedron Letters, vol. 40, (1999) 2657-2660. Compound 7 is
treated with diboranic ester in the presence of Pd(dppf)/potassium
acetate in DMSO at 80.degree. C. to afford intermediate 11.
Compound II is treated with 4-C(O)N(R').sub.2-imidazole in the
presence of copper acetate to form the
4-C(O)N(R').sub.2-imidazol-1-yl compound 12. Compounds of formula
II-a are prepared from compound 12 as described in Scheme II, steps
(e), (f), and (g).
[0115] Although 4-C(O)N(R').sub.2-imidazole is used to exemplify,
one of ordinary skill in the art would recognize that a variety of
R.sup.1 groups are amenable to the displacement reaction at step
(c) to form a variety of compounds of the present invention.
Generally, the boronate intermediate 11 may be treated with a
variety of R.sup.1-halides or R.sup.1-triflates, using methods well
known to one of ordinary skill in the art, to form intermediate
compounds 12' as shown below. Using the methods recited herein and
those known to one of ordinary skill in the art, compounds 12' are
useful for preparing compounds 9 and 10 of the present invention as
depicted above at Scheme II.
##STR00336## ##STR00337##
[0116] Scheme IV above shows an alternate method for preparing
compounds of formula II-a. Compound 13 is nitrated to form 14.
Compound 14 is treated with ammonium hydroxide to form the amino
compound 15. The bromo group of compound 15 is treated with the
BrZn-Ring A reagent in the presence of Pd(PPh.sub.3).sub.4 in THF
to form compound 16. Compound 16 is treated with the
4-C(O)N(R').sub.2-imidazole in the presence of sodium carbonate to
form the 4-C(O)N(R').sub.2-imidazol-1-yl compound 18. Compounds of
formula II-a are then prepared from compound 18 as described in
Scheme II, steps (e), (f), and (g).
[0117] One of skill in the art would recognize that a variety of
compounds of the present invention may be prepared according to the
general method of Schemes I, II, III, and IV, methods known in the
art, and the synthetic Examples set forth below.
[0118] The compounds of this invention are potent inhibitors of
gyrase and Topo IV as determined by enzymatic assay. These
compounds have also been shown to have antimicrobial activity in an
antimicrobial susceptibility assay. The activity of a compound
utilized in this invention as an inhibitor of gyrase or Topo IV may
be assayed in vitro, in vivo or in a cell line according to methods
known in the art. The details of the conditions used for both the
enzymatic and the antimicrobial susceptibility assays are set forth
in the Examples below.
[0119] According to another embodiment, the invention provides a
composition comprising a compound of this invention or a
pharmaceutically acceptable salt thereof and a pharmaceutically
acceptable carrier, adjuvant, or vehicle. The amount of compound in
the compositions of this invention is such that is effective to
detectably inhibit gyrase, Topo IV, or to measurably decrease
bacterial quantity, in a biological sample or in a patient.
Preferably the composition of this invention is formulated for
administration to a patient in need of such composition. Most
preferably, the composition of this invention is formulated for
oral administration to a patient.
[0120] The term "biological sample", as used herein, includes,
without limitation, cell cultures or extracts thereof; biopsied
material obtained from a mammal or extracts thereof; and blood,
saliva, urine, feces, semen, tears, or other body fluids or
extracts thereof.
[0121] Inhibition of gyrase and/or Topo IV activity in a biological
sample is useful for a variety of purposes that are known to one of
skill in the art. Examples of such purposes include, but are not
limited to, blood transfusion, organ-transplantation, biological
specimen storage, and biological assays.
[0122] The term "patient", as used herein, means an animal,
preferably a mammal, and most preferably a human.
[0123] The term "pharmaceutically acceptable carrier, adjuvant, or
vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that
does not destroy the pharmacological activity of the compound with
which it is formulated. Pharmaceutically acceptable carriers,
adjuvants or vehicles that may be used in the compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, 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.
[0124] The term "detectably inhibit", as used herein means a
measurable change in gyrase, or Topo IV, activity between a sample
comprising said composition and gyrase, or Topo IV, and an
equivalent sample comprising gyrase, or Topo IV in the absence of
said composition.
[0125] As used herein, the term "measurably decrease bacterial
quantity", as used herein means a measurable change in the number
of bacteria between a sample containing said composition and a
sample containing only bacteria.
[0126] A "pharmaceutically acceptable salt" means any non-toxic
salt of a compound of this invention that, upon administration to a
recipient, is capable of providing, either directly or indirectly,
a compound of this invention or an inhibitorily active metabolite
or residue thereof. As used herein, the term "inhibitorily active
metabolite or residue thereof" means that a metabolite or residue
thereof is also an inhibitor of gyrase and/or Topo IV.
[0127] 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,
oxalate, 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, may be employed in the preparation of salts useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
[0128] Salts derived from appropriate bases include alkali metal
(e.g., sodium and potassium), alkaline earth metal (e.g.,
magnesium), ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 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.
[0129] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the
compositions of this invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents 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 water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium.
[0130] For this purpose, any bland fixed oil may be employed
including synthetic mono- or di-glycerides. 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, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0131] The pharmaceutically acceptable compositions of this
invention may be orally administered in any orally acceptable
dosage form including, but not limited to, capsules, tablets,
aqueous suspensions or solutions. In the case of tablets for oral
use, carriers 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 are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0132] Alternatively, the pharmaceutically acceptable compositions
of this invention may be administered in the form of suppositories
for rectal administration. These can be prepared by mixing the
agent with a suitable non-irritating excipient that is solid at
room temperature but liquid at rectal temperature and therefore
will melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0133] The pharmaceutically acceptable compositions of this
invention may also be administered topically, especially when the
target of treatment includes areas or organs readily accessible by
topical application, including diseases of the eye, the skin, or
the lower intestinal tract. Suitable topical formulations are
readily prepared for each of these areas or organs.
[0134] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0135] For topical applications, the pharmaceutically acceptable
compositions may be formulated in a suitable ointment containing
the active component suspended or dissolved in one or more
carriers. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutically acceptable compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. 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.
[0136] For ophthalmic use, the pharmaceutically acceptable
compositions may be formulated as micronized suspensions in
isotonic, pH adjusted sterile saline, or, preferably, as solutions
in isotonic, pH adjusted sterile saline, either with or without a
preservative such as benzylalkonium chloride. Alternatively, for
ophthalmic uses, the pharmaceutically acceptable compositions may
be formulated in an ointment such as petrolatum.
[0137] The pharmaceutically acceptable compositions of this
invention may also 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 conventional solubilizing or dispersing
agents.
[0138] Most preferably, the pharmaceutically acceptable
compositions of this invention are formulated for oral
administration.
[0139] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, preferably between 0.5 and about 75 mg/kg body
weight per day and most preferably between about 1 and 50 mg/kg
body weight per day of the active ingredient compound are useful in
a monotherapy for the prevention and treatment of bacterial
infections caused by bacteria such as Streptococcus pneumoniae,
Streptococcus pyogenes, Enterococcus faecalis, Enterococcus
faecium, Klebsiella pneumoniae, Enterobacter sps. Proteus sps.
Pseudomonas aeruginosa, E. coli, Serratia marcesens, Staphylococcus
aureus, Coag. Neg. Staph, Haemophilus influenzae, Bacillus
anthracis, Mycoplasma pneumoniae, Moraxella catarralis, Chlamydia
pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis,
Staphylococcus epidermidis, or Helicobacter pylori.
[0140] Typically, the pharmaceutical compositions of this invention
will be administered from about 1 to 5 times per day or
alternatively, as a continuous infusion. Or, alternatively, the
compositions of the present invention may be administered in a
pulsatile formulation. 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 host treated and the particular mode
of administration. A typical preparation will contain from about 5%
to about 95% active compound (w/w). Preferably, such preparations
contain from about 20% to about 80% active compound.
[0141] When the compositions of this invention comprise a
combination of a compound of formula I and one or more additional
therapeutic or prophylactic agents, both the compound and the
additional agent should be present at dosage levels of between
about 10% to 80% of the dosage normally administered in a
monotherapy regime.
[0142] 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 or
disease symptoms.
[0143] 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, and the patient's disposition
to the disease and the judgment of the treating physician.
[0144] Depending upon the particular condition, or disease, to be
treated or prevented, additional therapeutic agents, which are
normally administered to treat or prevent that condition, may also
be present in the compositions of this invention. As used herein,
additional therapeutic agents that are normally administered to
treat or prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated". Such
agents include, but are not limited to, an antibiotic, an
anti-inflammatory agent, a matrix metalloprotease inhibitor, a
lipoxygenase inhibitor, a cytokine antagonist, an
immunosuppressant, an anti-cancer agent, an anti-viral agent, a
cytokine, a growth factor, an immunomodulator, a prostaglandin, an
anti-vascular hyperproliferation compound, or an agent which
increases the susceptibility of bacterial organisms to
antibiotics.
[0145] Agents which increase the susceptibility of bacterial
organisms to antibiotics are known. For example, U.S. Pat. No.
5,523,288, U.S. Pat. No. 5,783,561 and U.S. Pat. No. 6,140,306
describe methods of using bactericidal/permeability-increasing
protein (BPI) for increasing antibiotic susceptibility of
gram-positive and gram-negative bacteria. Agents that increase the
permeability of the outer membrane of bacterial organisms have been
described by Vaara, M. in Microbiological Reviews (1992) pp.
395-411, and the sensitization of gram-negative bacteria has been
described by Tsubery, H., et al, in J. Med. Chem. (2000) pp.
3085-3092.
[0146] According to another embodiment, the invention provides a
method for treating or lessening the severity of a bacterial
infection in a patient comprising the step of administering to said
patient a composition according to the present invention.
[0147] According to another embodiment, the invention provides a
method of inhibiting gyrase in a biological sample.
[0148] According to another embodiment, the invention provides a
method of inhibiting Topo IV in a biological sample.
[0149] According to another embodiment, the invention provides a
method of decreasing bacterial quantity in a biological sample.
[0150] According to another embodiment, the invention provides a
method of decreasing bacterial quantity in a biological sample, but
further comprising the step of contacting said biological sample
with an agent which increases the susceptibility of bacterial
organisms to antibiotics.
[0151] The pharmaceutical compositions and methods of this
invention will be useful generally for controlling bacterial
infections in vivo. Examples of bacterial organisms that may be
controlled by the compositions and methods of this invention
include, but are not limited to, the following organisms:
Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus
faecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter
sps. Proteus sps. Pseudomonas aeruginosa, E. coli, Serratia
marcesens, Staphylococcus aureus, Coag. Neg. Staph, Haemophilus
infuenzae, Bacillus anthracis, Mycoplasma pneumoniae, Moraxella
catarralis, H. influenzae, Chlamydia pneumoniae, Legionella
pneumophila, Mycobacterium tuberculosis, Helicobacter pylori,
Staphylococcus epidermidis. Chlamydia pneumoniae, Legionella
pneumophila, Mycobacterium tuberculosis, or Helibacter pylori.
[0152] The compositions and methods will therefore be useful for
controlling, treating or reducing the advancement, severity or
effects of nosocomial or non-nosocomial infections. Examples of
nosocomial uses include, but are not limited to, urinary tract
infections, respiratory infections such as pneumonia, surgical
wound infections, and blood stream infections (also known as
bacteremia). Examples of non-nosocomial uses include but are not
limited to urinary tract infections, pneumonia, prostatitis, skin
and soft tissue infections, intra-abdominal infections, and therapy
for febrile neutropenic patients.
[0153] The term "pharmaceutically effective amount" refers to an
amount effective in treating or ameliorating a bacterial infection
in a patient. The term "prophylactically effective amount" refers
to an amount effective in preventing or substantially lessening a
bacterial infection in a patient.
[0154] The compounds of this invention may be employed in a
conventional manner for controlling bacterial infections levels in
vivo and for treating diseases or reducing the advancement or
severity of effects which are mediated by bacteria. Such methods of
treatment, their dosage levels and requirements may be selected by
those of ordinary skill in the art from available methods and
techniques.
[0155] For example, a compound of this invention may be combined
with a pharmaceutically acceptable adjuvant for administration to a
patient suffering from a bacterial infection or disease in a
pharmaceutically acceptable manner and in an amount effective to
lessen the severity of that infection or disease.
[0156] Alternatively, the compounds of this invention may be used
in compositions and methods for treating or protecting individuals
against bacterial infections or diseases over extended periods of
time. The compounds may be employed in such compositions either
alone or together with other compounds of this invention in a
manner consistent with the conventional utilization of enzyme
inhibitors in pharmaceutical compositions. For example, a compound
of this invention may be combined with pharmaceutically acceptable
adjuvants conventionally employed in vaccines and administered in
prophylactically effective amounts to protect individuals over an
extended period of time against bacterial infections or
diseases.
[0157] The compounds of formula I may also be co-administered with
other antibiotics to increase the effect of therapy or prophylaxis
against various bacterial infections. When the compounds of this
invention are administered in combination therapies with other
agents, they may be administered sequentially or concurrently to
the patient. Alternatively, pharmaceutical or prophylactic
compositions according to this invention comprise a combination of
a compound of formula I and another therapeutic or prophylactic
agent.
[0158] The additional therapeutic agents described above may be
administered separately, as part of a multiple dosage regimen, from
the inhibitor-containing composition. Alternatively, these agents
may be part of a single dosage form, mixed together with the
inhibitor in a single composition.
[0159] In order that this invention be more fully understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
EXAMPLES
Example 1
5-Bromo-1,3-difluoro-2-nitro-benzene
[0160] To a suspension of sodium perborate tetrahydrate (1.04 g, 5
mmol) in acetic acid (20 mL), stirred at 55.degree. C., was added a
solution of 4-bromo-2,6-difluoroaniline in acetic acid (10 mL) over
1 hour in a dropwise fashion. After stirring at 55.degree. C. for
an additional 3 hours, the solution was allowed to cool to room
temperature and filtered. The filtrate was poured into ice, and
extracted twice with ethyl acetate. The combined organic extracts
were washed successively with 5.times.100-mL portions of water,
brine, dried (MgSO.sub.4), and concentrated in vacuo. The resulting
residue was purified by column chromatography over silica gel
eluted with ethyl acetate:hexanes (1:20) to afford 780 mg of the
title compound as a tan solid. .sup.1H NMR (CDCl.sub.3) .delta.
7.32 (dt, 2H).
Example 2
1-(5-Bromo-3-fluoro-2-nitro-phenyl)-1H-pyrazole
[0161] To a suspension of sodium hydride (44 mg, 1.1 mmol, 60% oil
dispersion) in THF (4 mL), stirred at 0.degree. C., was added a
solution of pyrazole (72 mg, 1.05 mmol) in THF (1 mL). The
resulting mixture was stirred at 0.degree. C. for 5 minutes and a
solution of 5-bromo-1,3-difluoro-2-nitro-benzene (238 mg, 1 mmol)
in THF (1 mL) was added. The mixture was stirred at room
temperature for 1 hour, quenched by addition of water (1 mL), then
partitioned between water (20 mL) and ethyl acetate (50 mL). The
organic layer was washed with brine, dried (MgSO.sub.4), and
concentrated in vacuo. The residue was purified by column
chromatography over silica gel eluted with ethyl acetate:hexanes
(1:6), to afford 240 mg (86%) of the title compound. .sup.1H NMR
(CDCl.sub.3) .delta. 6.55 (t, 1H), 7.45 (d, 1H), 7.60 (s, 1H), 7.80
(m, 2H). MS M+1 287, M+1+2 289.
Example 3
5-Bromo-2-nitro-3-pyrazol-1-yl-phenylamine
[0162] To a solution of
1-(5-bromo-3-fluoro-2-nitro-phenyl)-1H-pyrazole (240 mg, 0.84 mmol)
in ethanol (3 mL) was added ammonia (3 mL, 2N in methanol. The
resulting mixture was heated in a sealed tube at 80.degree. C. for
16 hours then concentrated in vacuo. The residue was purified by
column chromatography over silica gel eluted with ethyl
acetate:hexanes (1:3) to afford 205 mg (86%) of the title compound
as a yellow solid. .sup.1H NMR (CDCl.sub.3) .delta. 5.20 (br s,
2H), 6.50 (t, 1H), 6.9 (d, 1H), 7.1 (d, 1H), 7.7 (d, 1H), 7.8 (d,
1H). MS M+1 283, M+1+2 285.
Example 4
2-Nitro-3-pyrazol-1-yl-5-pyridin-3-yl-phenylamine
[0163] To a solution of 5-bromo-2-nitro-3-pyrazol-1-yl-phenylamine
(200 mg, 0.71 mmol) in THF (8 mL) was added, successively,
3-pyridyl-diethyl borane (157 mg), (tetrakistriphenylphosphine)
palladium(0) (84 mg), and sodium carbonate (1.1 mL, 2.2 mmom of 2M
aqueous). The resulting mixture was stirred at 70.degree. C.
overnight then cooled to room temperature. The reaction mixture was
diluted with ethyl acetate (100 mL) and washed with water (50 mL),
brine (50 mL), dried (MgSO.sub.4) then concentrated in vacuo. The
resulting residue was purified by column chromatography over silica
gel eluted with a gradient of ethyl acetate:hexanes (1:3, 1:2, 1:0,
2:1, 4:1, 8:1), to afford 120 mg (60%) of the title compound as a
yellow solid. .sup.1H NMR (DMSO-d.sub.6) .delta. 6.45 (br, s 2H),
6.55 (t, 1H), 7.1 (s, 1H), 7.25 (s, 1H), 7.55 (m, 1H), 7.7 (s, 1H),
8.1 (dt, 1H), 8.3 (d, 1H), 8.7 (d, 1H), 8.9 (s, 1H).
Example 5
1-Ethyl-3-(7-pyrazol-1-yl-5-pyridin-3-yl-1H-benzoimidazol-2-yl)-urea
(1-2)
[0164] A suspension of
2-nitro-3-pyrazol-1-yl-5-pyridin-3-yl-phenylamine (120 mg, 0.40
mmol) and 10% palladium on carbon (12 mg) in ethyl acetate (10 mL)
was placed in a Parr hydrogenator under a hydrogen pressure of 45
psi. The mixture was shaken for 16 hours, filtered and the filtrate
concentrated in vacuo. The resulting residue was diluted with
H.sub.2SO.sub.4 (1.6 mL or 1N), and N'-ethyl-N-cyanourea (0.8 mL,
1M) was added. The mixture was heated at 95.degree. C. for 4 hours
then concentrated in vacuo. The residue was purified by preparative
HPLC to afford 75 mg of the title compound as the bis-TFA salt
which was converted to the free base to afford the title compound.
.sup.1H NMR (DMSO-d.sub.6) .delta. 1.1 (t, 3H), 3.2 (m, 2H), 7.0
(m, 1H), 7.3 (d, 1H), 7.5 (m, 1H), 7.55 (s, 1H), 8.0 (d, 1H), 8.55
(dd, 1H), 8.85 (s, 1H), 10.1 (s, 1H), 12.0 (s, 1H). LC/MS one peak,
M+1 348.23, M-1 346.18.
Example 6
N'-Ethyl-N-cyanourea
[0165] To a 20.degree. C. solution of sodium hydroxide (1.5 M
aqueous, 50 mL, 75.02 mmol) was added cyanamide (8.5 g, 202.25
mmol) then ethyl isocyanate (4 mL, 50.56 mmol) was added in a
dropwise fashion over 10 minutes. After stirring for 30 minutes,
additional sodium hydroxide (3M, 25 mL. 75.02 mmol) and ethyl
isocyanate (4 mL, 50.56 mmol) were added. The resulting solution
was then aged for a minimum of 30 minutes before using directly
without isolation.
Example 7
4-(Pyridin-3-yl)-2-nitroaniline
[0166] To a solution of 4-bromo-2-nitroaniline (4.8 g, 22 mmol) in
DME (100 mL) was added pyridine-3-boronic acid 1,3-propanediol
cyclic ester (4 g, 24 mmol), sodium bicarbonate (45 mL, 1M), and
tetrakis(triphenylphosphine)palladium (0.05 eq). The resulting
mixture was heated at 90.degree. C. for 8 hours then cooled to room
temperature. The solids were collected, washed with water, 5% EtOAc
in Hexane and dried to afford the title compound (5 g). .sup.1H NMR
(CDCl.sub.3) .delta. 8.8 (d, 1H), 8.55 (m, 1H), 8.35 (d, 1H), 7.85
(dd, 1H), 7.65 (dd, 1H), 7.35 (m, 1H), 6.95 (d, 1H), 6.25 (br s,
2H).
Example 8
2-Bromo-6-nitro-4-pyridin-3-yl-phenylamine
[0167] To a solution of 4-(pyridin-3-yl)-2-nitroaniline (1.3 g, 9
mmol) in HOAc (25 mL) was added bromine (1.58 g, 9.9 mmol) in HOAc
(5 mL). The resulting mixture was stirred at room temperature for
one hour and then quenched with ice-water. The solids were
collected, washed with water and dried. The solids in EtOAc was
then washed with NaOH (2N; 20 mL), water, brine and concentrated in
vacuo. The concentrate was purified by chromatography [Silica Gel,
ethyl acetate:hexanes (1:1)] to afford the title compound (0.8 g).
.sup.1H NMR (CDCl.sub.3) .delta. 8.83 (d, 1H), 8.55 (m, 1H), 8.41
(d, 1H), 8.15 (d, 1H), 7.96 (m, 1H), 7.41 (m, 1H), 6.80 (br s, 2H).
(M+1) 294.
Example 9
2-Nitro-6-pyridin-2-yl-4-pyridin-3-yl-phenylamine
[0168] A mixture of 2-bromo-6-nitro-4-pyridin-3-yl-phenylamine (100
mg, 1 eq), 2-pyridylznic bromide (6 eq) and
tetrakis(triphenylphosphine)palladium (0.1 eq) in THF (10 mL) was
heated at 100.degree. C. for 18 hours. The reaction was quenched
with water (2 mL). The product was extracted with EtOAc
(20.times.3). The combined organic layer was then concentrated in
vacuo and the residue was purified by chromatography (Silica Gel,
EtOAC) to afford the title compound (75 mg) as a yellow solid.
(M+1) 293.
Example 10
3-Pyridin-2-yl-5-pyridin-3-yl-benzene-1,2-diamine
[0169] To a solution of
2-nitro-6-pyridin-2-yl-4-pyridin-3-yl-phenylamine (75 mg, 0.26
mmol) in ethyl acetate (20 mL) was added 10% palladium on carbon
(50 mg). The resulting suspension was placed in a Parr
hydrogenation apparatus under 40 psi hydrogen gas while shaking at
ambient temperature for one hour. The catalyst was removed by
filtration and the filtrate concentrated in vacuo to afford
compound the title compound (50 mg, 0.19 mmol).
Example 11
1-Ethyl-3-(7-pyridin-2-yl-5-pyridin-3-yl-1H-benzoimidazol-2-yl)-urea
(1-31)
[0170] To a solution
3-pyridin-2-yl-5-pyridin-3-yl-benzene-1,2-diamine (50 mg, 0.19
mmol) and sulfuric acid (0.76 mL, 1 N) in water (1 mL) was added
N'-ethyl-N-cyanourea (0.76 mL, 1 M). Enough sulfuric acid was added
dropwise to achieve pH 3. The resulting mixture was heated at
100.degree. C. for 8 hours. The reaction mixture was then cooled to
ambient temperature. The solids were collected, washed with water
and dried. The solids were purified by chromatography (Silica Gel,
EtOAc, then 10% MeOH in EtOAc) to afford compound 5 (27 mg).
.sup.1H NMR (CDCl.sub.3) .delta. 8.92 (d, 1H), 8.80 (m, 1H), 8.52
(m, 1H), 8.30 (m, 1H), 8.21 (d, 1H), 8.04 (s, 1H), 7.94 (m, 1H),
7.75 (s, 1H), 7.56 (d, 1H), 7.37 (m, 2H), 3.36 (q, 2H), 1.24 (t,
3H). (M+1) 359.
Example 12
##STR00338##
[0171] 2,2-Dimethyl-N-(2-pyrimidin-2-yl-phenyl)-propionamide
[0172] A 5 L flask was charged with the above depicted boronic acid
as a tetrahydrate (281.4 grams, 960 mmoles), 2-chloropyrimidine
(100 g, 874 mmoles), NaHCO.sub.3 (146.8 grams, 1.746 moles), and
Pd(PPh.sub.3).sub.4 (10.0 grams, 8.72 mmoles). Water (1 L) and
dimethoxyethane (1 L) were added, and the mixture was heated slowly
to 83.degree. C. (internal temperature) over a 1 hour period with
overhead stirring. After .about.2 hours all solids had dissolved.
The reaction was allowed to stir for 8 hours. The mixture was
cooled to room temperature and stirred overnight after which time a
thick precipitate had formed. The crude mixture was diluted with
water (2 L) and stirred for an additional 2 hours after which time
the mixture was filtered and the solids were washed sequentially
with water, 0.1 N NaOH, and water again. The solids were then dried
under high vacuum at 50.degree. C. to afford the title compound
(.about.233 grams) as a tan powder.
Example 13
##STR00339##
[0173]
N-(4-Bromo-2-pyrimidin-2-yl-phenyl)-2,2-dimethyl-propionamide
[0174] To a room temperature suspension of
2,2-dimethyl-N-(2-pyrimidin-2-yl-phenyl)-propionamide (.about.117
grams, 437 mmoles) in acetic acid (1 L) was added bromine (67 mL,
1.31 moles) as a solution in 100 mL of acetic acid over a 1 hour
period. The heterogenous mixture was stirred at room temperature
for 5 hours over which time a thick precipitate formed. The mixture
was then poured over ice, diluted with 1N Na.sub.2S.sub.2O.sub.3 (2
L), and stirred for 1 hour. The solids were filtered, resuspended
in water (2 L), stirred for 1 hour, then filtered and washed with
water again. The resulting solids were pumped to dryness at
50.degree. C., resuspended in HOAc (1 L), and treated with bromine
(22 mL, 430 mmoles) in acetic acid solution (20 mL) over a 20
minute period. The resulting heterogenous mixture was stirred for 5
hours, then quenched and treated as described above. The resulting
solids were vacuum dried at 50.degree. C. to afford the title
compound (165 grams) as a tan powder.
Example 14
##STR00340##
[0175]
N-(4-Bromo-2-nitro-6-pyrimidin-2-yl-phenyl)-2,2-dimethyl-propionami-
de
[0176] To a 5.degree. C. suspension of
N-(4-bromo-2-pyrimidin-2-yl-phenyl)-2,2-dimethyl-propionamide (32.6
grams, 97.5 mmoles) in TFA (400 mL) was added 90% nitric acid (70
mL, 1.46 mmoles) over a 30 minute period. The mixture was then
allowed to warm to room temperature and stir for a total of 2
hours. The crude reaction (now homogenous) was poured into ice
producing a pasty mass. The mixture was diluted to 2 L total volume
with water, treated with 500 mL of methanol, and vigorously stirred
for 12 hours. The resulting solids were filtered, washed with
copious amounts of water, then vacuum dried at 50.degree. C. to
afford the title compound (29.9 grams, 81% yield) as a tan
powder.
Example 15
##STR00341##
[0177] 4-Bromo-2-nitro-6-pyrimidin-2-yl-phenylamine
[0178] A suspension of
N-(4-bromo-2-nitro-6-pyrimidin-2-yl-phenyl)-2,2-dimethyl-propionamide
(29.9 grams, 78.8 mmoles) in conc. HCl (200 mL) was refluxed for 8
hours. The partially homogeneous crude reaction was then cooled to
room temperature, diluted with water (500 mL), and the resulting
precipitate was stirred for 1 hour. The solids were then filtered,
washed with water, and vacuum dried at 50.degree. C. to afford the
title compound (21.1 grams, 91% yield) as an orange powder.
Example 16
##STR00342##
[0179]
2-Nitro-6-pyrimidin-2-yl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-
-2-yl)-phenylamine
[0180] A mixture of 4-bromo-2-nitro-6-pyrimidin-2-yl-phenylamine
(1.82 g, 6.2 mmol), bis(pinacolato)diboron (3.144 g, 12.4 mmol),
PdCl.sub.2dppf.sub.2 (453 mg, 0.6 mmol) and KOAc (3.03 g, 31 mmol)
in dioxane (60 ml) was heated at 105.degree. C. for 2.5 hours. The
reaction was filtered and washed with dichloromethane. The combined
filtrates were concentrated under vacuum and water (100 ml) was
added to the residue. Extraction with dichloromethane (3.times.50
ml), drying and concentration gave a residue, which was washed with
ether-hexane to afford the title compound (2.07 g, 98%).
Example 17
##STR00343##
[0181]
N-[2-(3-Fluoro-pyridin-2-yl)-phenyl]-2,2-dimethyl-propionamide
[0182] A 3 L flask was charged with the above depicted boronic acid
as a tetrahydrate (92.1 grams, 314 mmoles), chlorofluoropyridine
(37.6 g, 286 mmoles), NaHCO.sub.3 (48.0 grams, 572 mmoles), and
Pd(PPh.sub.3).sub.4 (3.3 grams, 2.86 mmoles). Water (300 mL) and
dimethoxyethane (300 mL) were added, and the mixture was heated
slowly to 83.degree. C. (internal temperature) over a 1 hour period
with overhead stirring. After .about.2 hours all solids had
dissolved. The reaction was allowed to stir for 10 hours. The
mixture was cooled to room temperature and stirred overnight after
which time a thick gum had formed. The crude mixture was diluted
with water (2 L) and stirred for an additional 2 hours. The mixture
was then allowed to rest without stirring until the gum had settled
to the bottom of the flask. The liquid phase was removed via
vacuum, then replaced with 0.1 N NaOH and stirred for 15 minutes.
The gum was allowed to settle and the liquid removed via vacuum.
The gum was then similarly washed three times with water, then
transferred to a 1 neck flask as an acetone solution. The mixture
was concentrated in vacuo and azeotroped five times with ethyl
acetate.
Example 18
##STR00344##
[0183]
N-[4-Bromo-2-(3-fluoro-pyridin-2-yl)-phenyl]-2,2-dimethyl-propionam-
ide
[0184] To a room temperature suspension of
N-[2-(3-fluoro-pyridin-2-yl)-phenyl]-2,2-dimethyl-propionamide
(.about.77 mmoles) in acetic acid (300 mL) was added bromine (12
mL, 228 mmoles) as a solution in 50 mL of acetic acid over a 1 hour
period. The heterogenous mixture was stirred at room temperature
for 5 hours over which time a thick precipitate formed. The mixture
was then poured over ice, diluted with 1N Na.sub.2S.sub.2O.sub.3
(500 mL), and stirred for 1 hour. The solids were filtered,
re-suspended in water (2 L), stirred for 1 hour, then filtered and
washed with water again. The resulting solids were pumped to
dryness at 50.degree. C., re-suspended in HOAc (400 mL), and
treated with bromine (4 mL, 76 mmoles) in acetic acid solution (20
mL) over a 20 minute period. The resulting heterogenous mixture was
stirred for 5 hours, then quenched and treated as described above.
The resulting solids were vacuum dried at 50.degree. C. to afford
the title compound (19.1 grams, 72%) as a tan powder.
Example 19
##STR00345##
[0185]
N-[4-Bromo-2-(3-fluoro-pyridin-2-yl)-6-nitro-phenyl]-2,2-dimethyl-p-
ropionamide
[0186] To a suspension of
N-[4-bromo-2-(3-fluoro-pyridin-2-yl)-phenyl]-2,2-dimethyl-propionamide
(6.45 grams, 18.4 mmoles) in TFA (100 mL) and TFAA (25.5 mL, 183.6
mmole), at 0.degree. C., was added a TFA solution (30 mL) of 90%
fuming nitric acid (2.46 mL, 55.1 mmoles) over a 45 minute period.
The mixture was then stirred at 0.degree. C. for a total of 4
hours. The crude reaction (now homogenous) was poured into ice
producing a pasty mass. The mixture was diluted to 500 mL total
volume with water, treated with 50 mL of methanol, and vigorously
stirred for 12 hours. The resulting solids were filtered, washed
with copious amounts of water, then dried in vacuo at 50.degree. C.
to afford the title compound (6.1 grams, 82% yield) as a tan
powder.
Example 20
##STR00346##
[0187] 2-(3,5-Difluoro-phenyl)-pyrimidine
[0188] A solution of the difluoroboronic acid (5.4 g, 34.1 mmoles)
and 2-chloropyrimidine (3.0 g, 26.2 mmoles) in ethanol (50 mL) was
treated with Na.sub.2CO.sub.3 (3.6 g, 34.1 mmoles) and
Pd(PPh.sub.3).sub.4 (1.5 g, 1.31 mmoles) then heated at reflux for
3 days. The resulting mixture was then diluted with EtOAc, Silica
gel added, and the resulting slurry stirred for 3 hours at room
temperature. The crude mixture was then filtered through a silica
gel pad with EtOAc, concentrated in vacuo, and flash
chromatographed (silica gel, 19/1-14/1-9/1-7/1 hexanes/EtOAc
gradient) to afford the title compound (1.38 g, 27%) as a white
solid. .sup.1H NMR (dmso-d.sub.6, 500 MHz): 8.95 (d, 2H); 7.98 (m,
2H); 7.57 (dd, 1H); 7.48 (m, 1H).
Example 21
##STR00347##
[0189] 2-(3,5-Difluoro-2-nitro-phenyl)-pyrimidine
[0190] To a room temperature solution of
2-(3,5-difluoro-phenyl)-pyrimidine (1.2 g, 6.24 mmole) in
H.sub.2SO.sub.4 (3 mL) was added 90% HNO.sub.3 (0.375 mL, 9.37
mmoles) over 10 seconds via syringe. The resulting mixture was
stirred at room temperature for 1 hour then poured into ice. The
resulting heterogeneous mixture was then diluted with water, warmed
to room temperature, and filtered. The solids were washed with
water and dried in vacuo to afford the title compound (1.53 g,
100%) as a tan solid. .sup.1H NMR (dmso-d.sub.6, 500 MHz): 8.92 (d,
2H); 8.67 (m, 1H); 7.94 (m, 1H); 7.65 (dd, 1H).
Example 22
##STR00348##
[0191] 5-Fluoro-2-nitro-3-pyrimidin-2-yl-phenylamine
[0192] To a solution of 2-(3,5-difluoro-2-nitro-phenyl)-pyrimidine
(1.5 g, 6.32 mmoles) in dioxane (10 mL) was added tBuNH.sub.2 (6.6
mL, 63.24 mmoles) at room temperature. The mixture was heated to
100.degree. C. in a sealed tube for 10 hours. The mixture was then
cooled to room temperature, poured into water, and the solids
stirred for 1 hour. The mixture was filtered, solids washed with
water until filtrate was clear. The crude product was then diluted
in MeOH, 6N HCl added, and the resulting mixture heated at reflux
for 3 hours. The reaction was cooled to room temperature and poured
into ice. The resulting heterogeneous mixture was warmed to room
temperature, filtered, solids washed with water until filtrate ran
clear, and dried in vacuo to afford the title compound (1.33 g,
90%) as an orange powder. .sup.1H NMR (dmso-d.sub.6, 500 MHz): 8.87
(d, 2H); 7.52 (dd, 1H); 7.08 (dd, 1H); 6.86 (dd, 1H); 6.60 (s,
2H).
Example 23
##STR00349##
[0193]
1-(3-Amino-4-nitro-5-pyrimidin-2-yl-phenyl)-1H-imidazole-4-carboxyl-
ic acid cyclopropylamide
[0194] To a mixture of
5-fluoro-2-nitro-3-pyrimidin-2-yl-phenylamine (650 mg, 2.77 mmole)
in DMF (5 mL) was added 17 (545 mg, 3.6 mmoles) and
Na.sub.2CO.sub.3 (381 mg, 3.60 mmoles) at room temperature. The
resulting mixture was heated to 125.degree. C. for 6 hours, then
cooled to room temperature. The resulting mixture was diluted with
water and the yellow precipitate was stirred for 1 hour. The crude
reaction was filtered and the solids washed with water until the
filtrate ran clear. The washed solids were then dried in vacuo to
afford the title compound (960 mg, 95%) as a yellow powder. .sup.1H
NMR (dmso-d.sub.6, 500 MHz): 8.91 (d, 1H); 8.42 (s, 1H); 8.29 (s,
1H); 8.08 (d, 1H); 7.52 (dd, 1H); 7.36 (d, 1H); 7.29 (d, 1H); 6.59
(s, 2H); 2.89 (m, 1H); 0.072 (m, 2H); 0.64 (m, 2H).
Example 24
##STR00350##
[0195] N,N-Diethlycarboxy-2-methyl-2-thiopseudourea
[0196] To a mixture of 2-methyl-2-thiopseudourea sulfate (22.8 g,
81.9 mmol) in methylene chloride (200 mL) was added triethylamine
(34.5 mL, 245.7 mmol) and ethyl chloroformate (20.65 g, 245 mmol).
After stirring over night the mixture was washed with water, brine
then dried over sodium sulfate, filtered and concentrated in vacuo
to a pungent oil which was flash chromatographed (10% ethyl
acetate/hexanes) to provide the title compound (16.68 g, 86.9% Y)
as a colorless oil which solidified on standing. .sup.1H NMR (500
Mhz, CDCl.sub.3) .differential.1.3 (q, 6H), 2.41 (s, 3H), 4.22 (m,
4H).
Example 25
##STR00351##
[0197] N,N-Diethlyureamido-2-methyl-2-thiopseudourea
[0198] To a mixture of 2-methyl-2-thiopseudourea sulfate (2.0 g,
7.18 mmol) in water (3 mL) was added ethyl isocyanate (1.137 mL,
14.37 mmol) followed by dropwise 6N NaOH to a stable pH 8. After 1
hour at pH8 the biphasic solution was diluted with aqueous
saturated sodium bicarbonate and extracted into ethyl acetate
(3.times.100 mL). The combined organic layers were washed with
brine and dried over sodium sulfate, filtered then concentrated in
vacuo to afford the title compound as a pungent oil (1.54 g,
92.7%). TLC (50% Ethyl acetate/methylene chloride) and .sup.1H NMR
suggests that the material is a mixture of mono and diacyl
pseudourea. .sup.1H NMR (500 Mhz, CDCl.sub.3) .differential.1.18
(m2, 6H), 2.31 and 2.41 (2s, 3H), 3.28 (m, 4H).
Example 26
##STR00352##
[0199]
[5-(4-Cyclopropylcarbamoyl-imidazol-1-yl)-7-pyrimidin-2-yl-1H-benzo-
imidazol-2-yl]-carbamic acid ethyl ester
[0200] To a solution of
1-(3-amino-4-nitro-5-pyrimidin-2-yl-phenyl)-1H-imidazole-4-carboxylic
acid cyclopropylamide (65 mg, 0.178 mmoles) in MeOH (10 mL) was
added Ra--Ni (2 drops of water slurry, catalytic) and the resulting
suspension was placed under 45 psi of H.sub.2 (Parr shaker) for 2
hours. The resulting mixture was then filtered, concentrated,
diluted with 3 mL of pH=3.5 buffer (made from 1M H.sub.2SO.sub.4
with enough NaOAc to raise pH to 3.5), and treated with
N,N-diethlycarboxy-2-methyl-2-thiopseudourea (0.267 mL of a 1M
solution of N,N-diethlycarboxy-2-methyl-2-thiopseudourea in
dioxane) at room temperature. The resulting mixture was refluxed
for 5 hours resulting in a heterogeneous suspension. The reaction
was cooled to room temperature, diluted with water and enough
NH.sub.4OH to raise the pH to .about.6.0. The solids were then
filtered and washed sequentially with water, 2/1 water/ethanol,
EtOAc, and then hexanes. The resulting solids were suspended in
MeOH, 2 equivalents of methanesulfonic acid was added, and
concentrated in vacuo to afford the title compound (75, 70%) as an
off-white solid. .sup.1H NMR (dmso-d.sub.6, 500 MHz): 9.28 (s, 1H);
9.08 (d, 1H); 8.8-7.4 (v. broad s, 4H); 8.67 (s, 1H); 8.53 (s, 1H);
8.46 (d, 1H); 8.05 (d, 1H); 7.59 (dd, 1H); 4.33 (q, 2H); 2.88 (m,
1H); 2.35 (s, 6H); 1.34 (t, 3H); 0.76 (m, 2H); 0.61 (m, 2H).
Example 27
[0201] We have prepared other compounds of formula I by methods
substantially similar to those described in Schemes I through IV,
Examples 1 through 26, and by methods known in the art. The
characterization data for these compounds is summarized in Table 3
below and includes LC/MS (observed) and .sup.1H NMR data.
[0202] .sup.1H NMR data is summarized in Table 3 below wherein
.sup.1H NMR data was obtained at 500 MHz in deuterated DMSO, unless
otherwise indicated, and was found to be consistent with structure.
Compound numbers correspond to the compound numbers listed in Table
2.
TABLE-US-00003 TABLE 3 Characterization Data for Selected Compounds
of Formula I Compound No. I- M - 1 (obs) M + 1 (obs) .sup.1H NMR 16
347.2 349.2 1.1 (t, 3H) 3.2 (q, 2H) 6.8 (t, 1H) 7.5 (m, 1H), 7.7
(s, 1H) 7.9 (s, 1H) 8.1 (d, 1H) 8.3 (s, 1H) 8.6 (d, 1H) 8.9 (s,
1H)9.6 (s1H) 10.3 (s, 1H) 20 360.3 362.3 (CD.sub.3OD): 8.89 (dd,
1H); 8.51 (dd, 1H); 8.42-8.29 (br. s, 1H), 8.18 (ddd, 1H);
7.94-7.77 (br. s, 1H); 7.63 (br. s); 7.58 (br. s, 1H); 7.53 (dd,
1H); 3.32 (q, 2H); 2.21 (s, 3H); 1.23 (t, 3H) 24 391.3 393.3 1.13
(t, 3H), 1.3 (t, 3H) 3.24 (q, 2H), 3.37 (q, 2H), 7.82 (s, 1H), 7.82
(s1H), 7.96 (t, 1H)8.19 (s, 1H), 8.56 (s, 1H), 8.62 (d, 1H), 8.82
(d, 1H), 9.15 (s, 1H), 11.02 (s, 1H) 42 390.3 392.2 1.13 (t, 3H)
2.45 (s, 3H) 3.23 (q, 2H) 3.46 (s, 3H) 6.58 (m, 4H), 7.78 (m, 3H)
9.11 (s, 1H) 10.51 (s, 1H) 12.18 (s, 1H) 43 -- -- 1.15 (t, 3H),
3.25 (m, 2H), 3.35 (s, 3H), 4.6 (s, 2H), 7.4 (br s, 1H), 7.55 (s,
1H), 7.8 (m, 1H), 8.0 (d, 1H), 8.05 (d, 1H), 8.6 (m, 1H), 8.7 (m,
1H), 9.2 (s, 1H), 10.4 (br s, 1H) 49 -- -- 1.3 (t, 3H), 4.3 (q,
2H), 6.65 (t, 1H), 7.75 (d, 1H), 7.85 (dd, 1H), 7.9 (s, 1H), 8.05
(d, 1H), 8.5 (d, 1H), 8.75 (dd, 1H), 9.1 (s, 1H0, 11.7 (br s, 1H),
50 377.2 379.1 1.23 (t, 3H), 2.89 (s, 3H), 3.36 (q, 2H), 7.93 (d,
1H), 8.16 (d, 1H), 8.26 (d, 1H), 8.33 (d, 1H), 8.86 (d, 1H), 8.97
(d, 1H), 9.30 (d, 1H) 51 -- -- 1.1 (t, 3H), 1.25 (t, 3H), 3.25 (q,
2H), 3.37 (s, 3H), 4.05 (q, 2H), 6.6 (m, 4H), 7.65 (s, 1H), 7.9 (m,
2H) 9.1 (br s, 1H), 10.2 (br s, 1H), 11.8 (br s, 1H) 54 -- -- 0.5
(m, 2H), 0.8 (m, 2H), 2.7 (m, 1H), 6.4 (br s, 1H), 6.7 (m, 1H),
7.75 (s, 1H), 7.8 (m, 1H), 7.85 (s, 1H), 8.05 (m, 1H), 8.5 (brs,
1H), 8.7 (m, 1H), 9.05 (s, 1H), 9.15 (s, 1H), 10.2 (br s, 1H) 55 --
-- 1.15 (t, 3H), 3.25 (m, 2H), 7.25 (m, 1H), 7.5 (br s, 1H), 7.7
(m, 1H), 7.85 (s, 1H), 8.3 (s, 1H), 8.4 (m, 1H), 8.7 (m, 2H), 8.85
(s, 1H), 9.1 (s, 1H), 9.15 (dd, 1H), 10.5 (br s, 1H), 57 377.1
379.2 9.08 (d, 1H); 8.48 (br. s, 1h); 8.13, (d, 1H); 7.95 (d, 1H);
7.88 (s, 1H); 7.25 (d, 1H); 6.75 (d, 1H); 6.64 (s, 1H);, 6.62 (dd,
1H); 6.4-5.7 (br. s, 2H); 5.69 (q, 2H); 3.48 (s, 3H); 1.48 (t, 3H)
61 -- -- 1.13 (t, 3H) 2.38 (s, 3H) 3.24 (q, 2H) 5.36 (s, 2H) 6.71
(m, 2H), 6.83 (s, 1H) 7.18 (d, 2H) 7.28 (t, 1H) 7.38 (m, 2H) 7.76
(s, 1H) 7.92 (s, 2H), 8.30 (s, 1H) 9.08 (s, 1H) 11.50 (s, 1H) 62
404.3 406.3 12.15, 11.81 (s, 1H), 10.34, 9.99 (s, 1H), 9.13, 8.99
(s, 1H), 7.99-7.81 (m, 3H), 7.68 (s, 1H), 7.30-6.59 (m, 4H), 5.09
(m, 1H), 3.23 (t, 2H), 1.33 9 (d, 6H), 1.13 (t, 3H) 63 -- -- 1.15
(t, 3H) 2.44 (s, 3H) 3.25 (q, 2H) 5.44 (s, 2H) 6.70 (m, 3H), 7.40
(d, 1H) 7.49 (t, 1H) 7.75 (s, 1H) 7.85 (m, 1H) 7.97 (s, 2H) 8.12
(s, 1H), 8.60 (d, 1H) 9.09 (s, 1H) 11.21 (s, 1H) 64 -- -- 1.2 (t,
3H), 2.2 (m, 2H), 3.3 (m, 2H), 3.65 (m, 2H), 4.1 (t, 2H), 7.75 (s,
1H), 7.84 (s, 1H), 7.87 (s, 1H), 7.8 (m, 1H0, 8.5 (m, 1H), 8.65 (m,
1H), 9.0 (s, 1H) 65 423.1 425.1 (MeOH-d.sub.4 & CDCl.sub.3):
8.30-7.85 (m, 4H), 6.78 (s, 1H), 6.58 (s, 1H), 3.60 (s, 3H), 3.37
(q, 2H), 2.80 (s, 3H), 1.25 (t, 3H) 67 -- -- 1.15 (t, 6H), 3.45 (q,
4H), 6.7 (s, 1H), 7.7 (m, 2H), 7.9 (s, 1H), 8.1 (s, 1H), 8.4 (m,
1H), 8.7 (m, 1H), 9.1 (m, 2H), 10.6 (br s, 1H), 68 453.2 455.2
12.17, 11.81 (s, 1H), 10.35, 9.99 (s, 1H), 9.13, 9.00 (s, 1H), 8.52
(s, 1H), 7.99-7.69 (m, 5H), 7.32-7.27 (m, 2H), 6.93-6.59 (m, 4H),
5.23 (s, 2H), 3.22 (q, 2H), 1.13 (t, 3H) 69 407.3 409.2
(MeOH-d.sub.4, HCl salt)): 8.62 (s, 1H), 7.96-7.93 (m, 2H), 7.59
(s, 1H), 6.67 (s, 1H), 5.81 (s, 1H), 3.45, 3.39 (s, 3H), 3.36 (q,
2H), 3.28, 3.20 (s, 3H), 1.23 (t, 3H) 70 -- -- 1.15 (t, 3H), 1.35
(t, 3H), 3.25 (q, 2H), 4.3 (q, 2H), 7.1 (br s, 1H), 7.85 (s, 1H),
8.05 (m, 1H), 8.2 (s, 1H), 8.3 (s, 1H), 8.8 (m, 1H), 8.85 (d, 1H),
9.25 (s, 1H0, 9.65 (s, 1H), 10.7 (br s, 1H), 71 416.2 418.2 0.84
(m, 2H) 1.14 (m, 5H) 2.55 (s, 3H) 2.91 (m, 1H) 3.26 (q, 2H), 6.59
(s, 1H) 6.65 (s, 1H) 6.69 (s, 1H) 7.65 (s, 1H) 7.81 (m, 1H) 7.97
(s, 1H), 8.07 (s, 1H) 9.07 (s, 1H) 11.59 (s, 1H) 72 -- -- 1.1 (t,
3H), 3.2 (q, 2H), 7.1 (br s, 1H), 7.8 (s, 1H), 8.0 (m, 1H), 8.2 (s,
1H), 8.25 (s, 1H), 8.7 (m, 1H), 8.8 (m, 1H), 9.2 (s, 1H), 9.6 (s,
1H), 10.7 (br s, 1H), 73 448.2 450.2 (CD.sub.3OD): 1.18-1.26 (m,
9H), 3.27 (s, 3H), 3.36 (q, 2H), 4.21 (s, 2H), 6.65-6.68 (m, 1H),
6.90-6.94 (m, 1H), 6.98-7.01 (m, 1H), 7.78-7.84 (m, 2H), 7.93-7.96
(m, 1H), 8.09-8.11 (m, 1H), 8.74-8.76 (m, 1H) 74 434.3 436.3
(CD.sub.3OD): 1.21-1.27 (m, 9H), 3.36 (q, 2H), 4.17 (s, 2H),
6.65-6.68 (m, 1H), 6.95-6.99 (m, 1H), 7.00-7.03 (m, 1H), 7.79-7.82
(m, 1H), 7.89 (d, 1H), 7.94-7.96 (m, 1H), 8.09-8.11 (m, 1H),
8.73-8.76 (m, 1H) 75 -- -- 1.1 (t, 3H), 3.0 (br s, 3H), 3.25 (m,
5H), 7.0 (br s, 1H), 7.75 (m, 2H), 8.05 (s, 1H), 8.15 (s, 1H), 8.45
(m, 1H), 8.7 (m, 1H), 9.1 (s, 1H), 9.4 (s, 1H), 10.4 (br s, 1H), 77
448.3 450.2 (CD.sub.3OD): d 1.24 (t, 3H), 1.27 (d, 3H), 1.47 (d,
3H), 3.36 (q, 2H), 3.37 (s, 3H), 3.58-3.67 (m, 1H), 5.21-5.28 (m,
1H), 6.68-6.71 (m, 1H), 7.80 (d, 1H), 7.85 (s, 1H), 7.95-7.99 (m,
2H), 8.27 (s, 1H), 8.38 (d, 1H), 8.78-8.82 (m, 1H). 78 448.3 450.3
(CD.sub.3OD): d 1.24 (t, 3H), 1.31 (d, 3H), 1.51 (d, 3H), 3.38 (q,
2H), 3.42 (s, 3H), 3.66-3.73 (m, 1H), 5.44-5.51 (m, 1H), 6.68-6.71
(m, 1H), 7.94 (d, 1H), 7.96-7.98 (m, 1H), 8.03 (s, 1H), 8.07 (s,
1H), 8.32 (s, 1H), 8.43 (d, 1H), 8.81-8.86 (m, 1H). 79 434.3 436.2
(CD.sub.3OD): d 1.24 (t, 3H), 1.32 (d, 3H), 1.50 (d, 3H), 3.37 (q,
2H), 3.93-4.02 (m, 1H), 5.14-5.22 (m, 1H), 6.67-6.71 (m, 1H), 7.90
(d, 1H), 7.95-7.98 (m, 1H), 8.02 (s, 1H), 8.04 (s, 1H), 8.32 (s,
1H), 8.41 (d, 1H), 8.81-8.85 (m, 1H). 82 428.2 430.1 9.0 (m, 1H),
8.6 (d, 1H), 8.4 (m, 1H), 8.1-8.2 (m, 2H), 8.0 (m, 1H), 7.8 (m,
1H), 7.5 (m, 2H), 6.6 (s, 1H), 4.8 (s, 1H), 2.55 (s, 3H), 3.25 (m,
2H), 2.1 (s, 3H), 1.1 (t, 3H) 83 417.1 419.1 11.01 (br. s, 1H);
9.10 (d, 1H); 8.37, (s, 1H); 8.19 (s, 1H); 7.97 (s, 1H); 7.78 (br.
s, 1H); 7.58 (m, 1H); 7.08, (s, 1H); 6.68 (m, 1H); 3.88 (dd, 2H);
3.22 (dq, 2H); 2.99 (dd, 2H);, 1.91 (ddd, 2H); 1.85 (ddd, 2H); 1.11
(t, 3H). 84 377.2 379.2 (MeOD-d.sub.3): 8.72 (br s, 1H), 8.58 (s,
1H), 8.40 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.14 (s, 1H), 6.68
(s, 1H), 3.60 (s, 3H), 3.21 (q, 2H), 1.24 (t, 3H). 85 430.2 432.2
(MeOD-d.sub.3, salt): 8.64 (d, 1H), 8.14 (s, 1H), 8.00 (s, 1H),
7.97 (d, 1H), 7.64 (s, 1H), 6.66 (dd, 1H), 6.51 (s, 1H), 3.89 (s,
3H), 3.48 (s, 3H), 3.37 (q, 2H), 1.24 (t, 3H) 87 360.1 362.1
MeOD-d.sub.3, 1.24 (t, 3H), 2.42 (s, 3H), 3.38 (q, 2H), 6.87 (s,
1H), 7.94 (s, 1H), 8.15 (m, 2H), 8.85 (d, 1H), 8.98 (d, 1H), 9.30
(s, 1H) 88 388 390 9.27 (s, 2H). 9.20 (S, 1H), 8.27 (s, 1H). 8.10
(m, 1H), 7.94 (s, H), 7.92 (d, 1H), 6.95 (d, 1H), 4.10 (s, 3H),
3.25 (m, 2H), 1.11 (t, 3H) 89 386.9 389.2 (CD.sub.3OD) 8.92-6.96
(m, 9ArH), 3.99 (s, 3H), 3.36 (q, 2H), 1.24 (t, 3H) 90 457.1 459.2
-- 91 429.2 431.2 (CD.sub.3OD): 8.97 (s, 1H); 8.89 (d, 1H); 8.49
(d, 1H); 8.37 (m, 2H); 8.22 (ddd, 1H); 7.93 (d, 1H); 7.64 (dd, 1H);
3.38 (q, 2H); 2.91 (m, 1H); 1.25 (t, H): 0.88 (m, 2H); 0.67 (m,
2H). 92 360.13 362.19 1.15 (t, 3H), 3.25 (m, 2H), 3.9 (s, 3H), 7.15
(m, 1H), 7.65 (m, 1H), 7.8 (s, 1H), 8.0 (s, 1H), 8.2 (m, 1H), 8.3
(m, 1H), 8.65 (m, 1H), 9.0 (m, 2H), 10.3 (br s, 1H) 93 371 373 1.12
(t, 3H), 3.25 (m, 2H), 4.2 (bs, 2H), 7.0.2-7.25 (m, 1H), 7.5 (m,
1H), 7.81 (m, 1H), 8.08 (t, 1H), 8.22 (m, 1H), 8.61-8.48 (m, 2H),
8.4 (d, 1H), 8.46 (s, 1H). 94 430 432 0.92 (d, 6H), 1.12 (m, 3H),
2.23 (m.1H), 2.83 (d, 2H), 3.35 (m, 2H), 7.5 (m, 1H), 7.61 (bs,
1H), 7.89 (s, 1H), 8.1 (m, 1H), 8.4 (s, 1H), 8.58 (m, 1H), 8.78
(bs, 1H), 8.3 (d, 1H). 95 431.03 433.2 (CD.sub.3OD) 1.2 (7, 3H),
3.3 (q, 2H), 3.8 (m, 2H), 4.6 (m, 2H), 7.4 (m, 1H), 7.8 (m, 1H),
8.0 (s, 1H), 8.3 (m, 2H), 8.5 (m, 1H), 8.7 (m, 2H), 9.1 (s, 1H) 96
507.2 509.2 (CD.sub.3OD): 8.9 (d, 1H), 8.55 (d, 1H), 8.4 (s, 1H),
8.3 (m, 1H), 8.0 (s, 1H), 7.7 (m, 1H), 7.3 (t, 1H), 7.0 (s, 2H),
6.85 (d, 1H), 6.7 (d, 2H), 5.5 (s, 2H), 3.7 (s, 3H), 3.3 (q, 2H),
2.5 (s, 3H), 1.25 (t, 3H) 97 401 403 9.1 (s, 1H), 8.6 (d, 2H), 8.3
(m, 1H), 8.1 (s, 1H), 7.9 (s, 1H), 7.8 (s, 1H) 7.5 (m, 1H), 7.0 (d,
1H), 4.3 (m, 2H), 3.3 (m, 2H), 1.4 (t, 3H), 1.1 (t, 3H). 98 497.03
499.18 , 1.16 (t, 3H) 3.25 (q, 2H) 4.44 (d, 2H) 7.17 (t, 2H) 7.38
(t, 2H)7.53 (t, 1H) 7.79 (m, 1H) 7.87 (s, 1H) 8.10 (t, 1H) 8.32 (s,
1H)8.51 (s, 1H) 8.57 (d, 1H) 8.77 (s, 1H) 8.82 (d, 1H) 8.94 (t,
1H)11.10 (br s, 1H) 99 432 434 -- 100 418.25 420.15 1.1 (t, 3H),
3.2 (m, 2H), 3.95 (s, 3H), 4.1 (s, 2H), 7.2 (br s, 1H), 7.25 (brs,
1H), 7.9 (s, 1H), 8.2 (m, 1H), 8.25 (s, 1H), 8.7 (d, 1H), 9.05 (s,
2H), 10.4 (br s, 1H) 101 431 433 8.65 (d, 1H), 8.28 (s, 1H), 8.24
(s, 1H), 7.98 (d, 1H), 7.78 (d, 1H), 7.38 (m, 1H), 6.93 (s, 1H),
6.89 (dd, 1H), 4.51 (q, 2H), 3.63 (s, 3H), 3.35 (q, 2H), 1.55 (t,
3H), 1.22 (t, 3H) 102 375 377 8.86 (d, 1H), 8.69 (m, 1H), 8.52 (d,
1H), 8.16 (m, 2H), 7.79 (m, 2H), 7.56 (m, 1H), 7.50 (m, 1H), 3.55
(m, 2H)1.23, (t, 3H) 103 430 432.1 8.87 (br. s, 1H); 8.81 (d, 1H);
8.66 (br. s., 1H); 8.49 (s, 1H); 8.39 (br. s., 1H); 8.25 (d, 1H);
8.09 (ddd, 1H); 7.87 (d, 1H); 7.52 (dd, 1H); 5.3 (very br. s, 5 H);
4.29 (q, 2H); 2.85 (m, 1H); 1.31 (t, 3H); 0.72 (m, 2H); 0.63 (m,
104 417.19 419.14 1.1 (t, 3H), 3.2 (q, 2H), 3.45 (s, 3H), 4.1 (s,
3H), 6.7 (dd, 1H), 6.85 (d, 1H), 7.15 (br s, 1H), 7.35 (br s, 1H),
7.8 (d, 1H), 7.95 (s, 1H), 8.2 (s, 1H), 8.4 (br s, 1H), 8.7 (d,
1H), 10.5 (br s, 1H) 105 406 408 8.97 (br. s, 2H); 8.60 (d, 1H);
7.99 (br. s., 1H); 7.96 (m, 1H); 7.81 (br. s., 1H); 7.57 (m, 1H);
3.98 (s, 3H); 3.25 (m, 2H); 1.11 (t, 3H). 106 508.2 510.2 1.1 (t,
3H), 2.4 (s, 3H), 3.2 (q, 2H), 4.1 (s, 3H), 5.4 (s, 2H), 6.65 (s,
1H), 6.75 (s, 1H), 7.15 (br s, 1H), 7.3 (m, 2H), 7.35 (m, 1H), 7.8
(m, 1H), 7.95 (s, 1H), 8.25 (s, 1H), 8.35 (br s, 1H), 8.5 (m, 1H),
8.75 (m, 1H), 10.5 (br s, 1H) 107 495.2 497.2 8.8 (m, 1H), 8.2 (m,
1H), 7.9 (s, 1H), 7.8 (m, 1H), 7.4 (m, 2H), 7.35 (d, 2H), 7.25 (d,
2H), 6.7-6.8 (m, 2H), 5.3 (2, 2H), 4.0 (m, 1H), 3.4 (q, 2H), 2.4
(s, 3H), 1.1 (t, 3H) 108 417 419 9.12 (br. s, 1H); 8.60 (d, 1H);
8.39 (m, 1H); 8.36 (s, 1H); 8.25 (s, 1H); 7.89 (s,
1H); 7.70 (dd, 1H); 7.41 (m, 1 H); 4.13 (s, 3H); 3.98 (s, 3H); 3.26
(m, 2H); 1.15 (t, 3H). 109 358.3 360.1 (CD.sub.3OD) 9.61 (d, 1H),
9.22 (d, 1H), 8.83-8.79 (m, 3H), 8.65 (d, 1H), 8.47 (d, 1H), 8.02
(dd, 1H), 8.00 (s, 1H), 3.37 (q, 2H), 1.24 (t, 3H) 110 388.3 390.2
(CDCl.sub.3) 14.05 (br s, 1H), 12.85 (br s, 1H), 8.37 (t, 1H), 7.97
(d, 1H), 7.88 (d, 1H), 7.79-7.74 (m, 2H), 7.63 (dd, 1H), 7.46 (d,
1H), 6.62 (dd, 1H), 5.75 (br s, 1H), 3.45-3.40 (m, 2H), 1.27 (t,
3H) 111 492.3 494.1 10.3 (s, 1H), 8.9 (d, 1H), 8.6 (m, 1H), 8.5 (d,
1H), 8.2 (s, 1H), 8.05 (t, 1H), 7.8-7.9 (m, 2H), 7.5 (m, 2H), 7.4
(d, 1H), 7.3 (m, 1H), 6.7 (s, 1H), 6.6 (s, 1H), 3.3 (q, 2H), 1.9
(d, 3H), 1.1-1.2 (t, 3H). 112 475.2 477.2 10.62 (s, 1H); 8.74 (d,
1H); 8.65 (s, 1H); 8.40 (s, 1H); 8.28 (s, 1H); 8.23 (s, 1H); 7.87
(s, 1H); 7.43 (s, 1H); 7.32 (m, 2H); 5.52-4.41 (br. s, 3H); 4.10
(s, 3H); 3.24 (dt, 2H); 1.41 (s, 9H); 1.13 (t, 3H). 113 494.2 496.2
(CD.sub.3OD) 9.06 (s, 1H); 8.93 (d, 1H); 8.71 (d, 1H); 8.50 (m,
2H); 8.39 (s, 1H); 8.00 (s, 1H); 7.87 (dd, 1H); 7.42 (d, 2H); 7.35
(dd, 2H); 7.25 (dd, 1H); 5.27 (q, 1H); 4.41 (q, 2H); 1.62 (d, 3H);
1.42 (t, 3H) 114 370.2 372.2 12.09 & 12.74 (s, 1H), 10.25 &
9.94 (s, 1H), 9.12 & 8.94 (s, 1H), 8.35-6.58 (m, 9H), 3.24 (m,
2H), 1.13 (t, 3H) 115 442.1 444.3 (CD.sub.3OD) 8.65 (d, 1H), 7.94
(s, 1H), 7.91 (d, 1H), 7.72 (d, 2H), 7.66 (s, 1H), 7.40 (d, 2H),
6.63 (dd, 1H), 4.52 (s, 2H), 3.40 (t, 2H), 3.35 (q, 2H), 2.47 (t,
2H), 2.09-2.03 (m, 2H), 1.24 (t, 3H) 116 382 384 -- 117 415 417
9.17 (br. s, 1H); 9.0 (m, 1H); 8.98 (d, 1H); 8.72 (d, 1H); 8.55 (m,
1H); 8.38 (s, 1H); 7.94 (s, 1H); 7.86 (d, 1H); 7.79 (m, 1H); 3.97
(s, 3H); 3.27 (m, 2H); 1.15 (t, 3H). 118 401 403 -- 119 403.3 405.2
(CD.sub.3OD): 8.78 (s, 1H), 8.67 (d, 1H), 8.37 (d, 1H), 8.24 (d,
1H), 8.22 (s, 1H), 8.04 (d, 1H), 7.91 (s, 1H), 7.67 (dd, 1H), 7.40
(dd, 1H), 4.22 (s, 3H), 3.34 (q, 2H), 1.23 (t, 3H) 120 434.32
436.23 1.1 (t, 3H), 2.8 (s, 6H), 3.2 (m, 2H), 3.4 (s, 3H), 4.6 (s,
2H), 6.6 (d, 1H), 6.8 (s, 1H), 7.2 (br s, 1H), 7.6 (s, 1H), 7.8 (d,
1H), 7.9 (s, 1H), 7.95 (s, 1H), 10.0 (br s, 1H), 10.2 (br s, 1H)
121 414 416 (CD.sub.3OD): 9.40 (br. s, 1H); 9.07 (d, 1H); 8.98 (d,
1H); 8.91 (d, 1H); 8.80 (s, 1H); 8.60 (s, 1H); 8.24 (dd, 1H); 8.11
(d, 1H); 7.84 (dd, 1H); 3.35 (m, 2H); 3.01 (s, 3H); 1.25 (t, 3H).
122 415 417 -- 123 435.28 437.26 1.3 (t, 3H), 2.8 (s, 6H), 3.5 (s,
3H), 4.2 (q, 2H), 4.6 (s, 2H), 6.6 (dd, 1H), 6.7 (s, 1H), 7.7 (s,
1H), 7.8 (d, 1H), 8.0 (m, 2H), 10.1 (br s, 1H), 11.7 (br s, 1H) 124
431.2 433.2 9.28 (s, 1H); 9.08 (d, 1H); 8.8-7.4 (v. broad s, 4H);
8.67 (s, 1H); 8.53 (s, 1H); 8.46 (d, 1H); 8.05 (d, 1H); 7.59 (dd,
1H); 4.33 (q, 2H); 2.88 (m, 1H); 2.35 (s, 6H); 1.34 (t, 3H); 0.76
(m, 2H); 0.61 (m, 2H). 125 447.4 -- 9.10 (s, 1H); 8.60 (d, 1H);
8.47 (m, 2H); 7.95 (s, 1H); 7.83 (s, 1H); 7.12 (d, 1H); 5.2-3.6
(br. m, 7H); 2.86 (M, 1H); 1.30 (t, 3H); 0.75 (m, 2H); 0.64 (m, 2H)
126 439.2 441.2 (CDCl.sub.3) 13.89 (br s, 1H), 12.89 (br s, 1H),
8.28 (d, 1H), 8.25 (d, 1H), 7.96 (d, 1H), 7.74 (s, 1H), 7.69-7.43
(m, 6H), 7.40 (s, 1H), 6.64 (dd, 1H), 6.11 (br s, 1H), 3.46-3.40
(m, 2H), 1.27 (t, 3H) 127 418.2 420.2 (CD.sub.3OD) 9.72 (d, 1H),
8.87 (d, 1H), 8.82 (d, 1H), 8.11 (d, 1H), 8.09 (d, 1H), 7.85 (d,
1H), 7.39 (d, 1H), 7.36 (d, 1H), 4.43 (q, 2H), 3.35 (q, 2H), 1.44
(t, 3H), 1.24 (t, 3H) 128 449.8 452.1 (CD.sub.3OD) 9.21 (d, 1H),
8.83 (ddd, 1H), 8.81 (dd, 1H), 8.68 (d, 1H), 8.26 (d, 1H), 8.18 (d,
1H), 8.07 (dd, 1H), 8.05 (d, 1H), 7.50 (dd, 1H), 3.36 (q, 2H), 3.34
(s, 3H), 1.23 (t, 3H) 129 -- -- 9.31 (s, 1H); 9.08 (d, 2H); 8.63
(s, 1H); 8.43 (d, 1H); 8.02 (d, 1H); 7.89 (s, 1H); 7.57 (t, 1H);
8.8-6.6 (very br. s, 4H); 4.31 (q, 2H); 2.32 (s, 6H); 1.42 (s, 9H);
1.33 (t, 3H). 130 495.4 497.2 9.07 (d, 2H); 8.73 (d, 1H); 8.56 (s,
1H); 8.44 (d, 1H); 8.03 (d, 1H); 7.57 (t, 3H); 7.44 (d, 2H); 7.36
(dd, 2H); 7.26 (dd, 1H); 6.95-5.90 (very broad s., 5H); 5.18 (dt,
1H); 4.32 (q, 2H); 2.32 (s, 6H); 1.53 (d, 3H); 1.33 (t, 3H). 131
432 434 (CD.sub.3OD) 1.08 (d, 6H), 1.43 (t, 3H), 2.33 (m, 1H), 2.91
(d, 2H), 4.45 (q, 2H), 7.53 (t, 1H), 8.08 (s, 1H), 8.79 (s,
1H)<8.94 (s, 1H), 9.05 (d, 2H), 9.5 (s, 1H). 132 514.23 516.23
8.84 (d, 1H), 8.81-8.83 (m, 1H), 8.35 (s, 1H), 8.06 (dd, 1H), 7.90
(dd, 1H), 7.80 (d, 1H), 6.80 (br s, 1H), 6.70 (d, 1H), 4.30 (q,
2H), 3.42-3.54 (m, 4H), 3.48 (s, 3H), 3.12-3.17 (m, 4H), 2.81 (d,
3H), 1.32 (t, 3H) ppm 133 501.33 503.26 -- 134 521.6 523.2
(CD.sub.3OD) 8.82 (s, 1H), m 8.46 (d, 1H), 8.30 (s, 1H), 8.13 (dd,
1H), 7.90 (s, 1H), 7.55 (dd, 1H), 7.28 (dd, 1H), 6.86-6.77 (m, 4H),
6.74 (s, 1H), 3.77 (s, 3H), 3.36 (q, 2H), 2.41 (br s, 3H), 1.97 (d,
3H), 1.24 (t, 3H) 135 470.3 472.5 (CD.sub.3OD) 8.85 (br s, 1H),
8.45 (d, 1H), 8.34 (dd, 1H), 8/26 (dd, 1H), 8.13 (ddd, 1H), 7.96
(dd, 1H), 7.56 (dd, 1H), 7.43 (dd, 1H), 7.29 & 7.20 (s, 1H),
5.49 (m, 1H), 3.66-3.25 (m, 4H), 3.38 (q, 2H), 2.94 (2s, 3H), 2.52
(m, 1H), 2.38 (m, 1H), 2.15 (m, 1H) 136 521.3 523.3 (CD.sub.3OD)
8.81 (d, 1H), 8.43 (d, 1H), 8.27 (s, 1H), 8.11 (ddd, 1H), 7.88 (s,
1H), 7.54 (dd, 1H), 7.28 (dd, 1H), 6.87 (d, 1H), 6.84 (d, 1H), 6.81
(s, 1H), 4.42 (q, 2H), 3.78 (s, 3H), 2.50 (br s, 3H), 2.00 (d, 3H),
1.41 (t, 3H) 137 522.6 524.2 (CD.sub.3OD) 8.90 (d, 1H), 8.81 (d,
1H), 8.49 (dd, 1H), 8.41 (s, 1H), 8.06 (s, 1H), 7.84 (dd, 1H), 7.29
(dd, 1H), 6.99 (br s, 1H), 6.92 (s, 1H), 6.87 (d, 1H), 6.84 (d,
1H), 6.81 (s, 1H), 4.42 (q, 2H), 3.78 (s, 3H), 2.50 (br s, 3H),
2.00 (d, 3H), 1.41 (t, 3H) 138 525 527 1.2 (t.3H). 1.5 (d, 3H),
3.73 (s, 3H), 4.3 (q, 2H), 5.13 (m, 1H), 6.8 (d, 1H), 7.0 (m, 2H),
7.24 (t, 1H), 7.53 (t, 1H), 7.93 (s, 1H), 8.24 (s, 1H), 8.32 (m,
2H), 8.4 (s, 1H), 9.08 (s, 1H), 11.79 (bs, 1H), 12.18 (s, 1H). 139
522.5 524.2 (CD.sub.3OD): 8.86 (d, 1H), 8.74 (d, 1H), 8.41 (dd,
1H), 8.35 (s, 1H), 7.99 (s, 1H), 7.78 (dd, 1H), 7.28 (dd, 1H), 6.85
(d, 1H), 6.83 (d, 1H), 6.80 (s, 2H), m 6.76 (s, 1H), 4.41 (q, 2H),
3.77 (s, 3H), 2.41 (br s, 3H), 1.97 (d, 3H), 1.41 (t, 3H) 140 499
501 -- 141 425.3 427.2 (CD.sub.3OD) 8.8 (d, 1H), 8.35 (d, 1H), 8.2
(s, 1H), 8.1 (t, 1H), 8.0 (s, 1H), 7.9 (d, 1H), 7.8 (s, 1H),
7.65-7.7 (dd, 1H), 7.5 (t, 1H), 7.49 (d, 1H), 4.6 (s, 2H), 4.45 (s,
1H), 3.4 (q, 2H), 3.2 (s, 3H), 3.15 (s, 1H), 1.2 (t, 3H) 142 426.3
428.2 (CD.sub.3OD): 8.8 (d, 1H), 8.6 (d, 1H), 8.3 (m, 2H), 8.1 (s,
1H), 8.0 (d, 1H), 7.9 (s, 1H), 7.7 (d, 1H), 4.6 (s, 2H), 4.45 (s,
1H), 4.4-4.5 (q, 2H), 3.3 (s, 3H), 3.15 (s, 1H), 1.4 (t, 3H) 143
498 500 -- 144 497 499 (CD.sub.3OD) 1.42 (t, 3H), 1.84 (d.3H), 4.49
(q, 2H), 6.43 (q, 1H). 7.58 (t, 1H), 8.03 (t, 1H), 8.08 (s, 1H0,
8.23 (d, 1H), 8.67 (t, 1H), 8.73 (s, 1H), 8.39 (d, 2H), 8.88 (d,
2H), 9.06 (s, 2H). 145 457 459 (CD.sub.3OD) 1.4 (t, 3H), 3.48 (d,
4H), 3.99 (s, 4H), 4.41 (s, 2H), 7.7 (d, 1H), 8.07 (s, 1H) 8.05 (m,
3H), 8.73 (s, 1H), 8.9 (s, 1H), 9.12 (s, 1H). 146 -- 502.21
(CD.sub.3OD) 8.89 (s, 1H), 8.49 (d, 1H), 8.45 (s, 1H), 8.22 (d,
1H), 8.11 (d, 1H), 8.06 (s, 1H), 7.47 (br d, 1H), 7.36 (br s, 1H),
3.91 (s, 3H), 3.68-3.89 (br m, 8H), 3.38 (q, 2H), 1.25 (t, 3H) ppm
147 -- 487.2 (CD.sub.3OD) 8.99 (s, 1H), 8.54 (d, 1H), 8.46 (s, 1H),
8.27 (d, 1H), 8.01 (s, 1H), 7.85 (br s, 1H), 6.97 (br s, 1H), 6.91
(br s, 1H), 4.47 (q, 2H), 3.66-3.69 (m, 5H), 3.59-3.62 (m, 2H),
2.01-2.06 (m, 2H), 1.96-2.00 (m, 2H), 1.43 (t, 3H) ppm 148 375 377
(CD.sub.3OD): 9.02 (t, 2H), 8.98 (s, 2H), 8.82 (d, 1H), 8.05 (d,
1H), 7.54 (t, 1H), 4.48 (q, 2H), 1.44 (t, 3H) 149 -- 486.22
(CD.sub.3OD) 8.98 (s, 1H), 8.44 (m, 1H), 8.38 (br s, 1H), 8.19 (br
d, 1H), 8.02 (br s, 1H), 7.98 (br s, 1H), 7.15 (br s, 1H), 7.13 (br
s, 1H), 3.79 (br s, 3H), 3.67 (m, 2H), 3.61 (m, 2H), 3.37 (q, 2H),
2.05 (m, 2H), 2.00 (m, 2H), 1.25 (t, 3H) p 150 494.3 496.2
(CD.sub.3OD) 9.0 (d, 2H), 8.8 (s, 1H), 8.6 (d, 1H), 8.2 (t, 1H),
7.95 (s, 1H), 7.8 (d, 1H), 7.65 (t, 1H), 7.5 (t, 1H), 6.9 (s, 1H),
6.6 (s, 1H), 6.1 (m, 1H), 4.4 (q, 2H), 2.7 (s, 3H), 2.05 (d, 3H),
1.4 (t, 3H) 151 493.4 495.2 (CD.sub.3OD) 8.95 (d, 2H), 8.6 (s, 1H),
8.5 (d, 1H), 8.1 (t, 1H), 7.8 (s, 1H), 7.7 (d, 1H), 7.5 (t, 1H),
7.4 (t, 1H), 6.8 (s, 1H), 6.6 (s, 1H), 6.0 (m, 1H), 3.35 (q, 2H),
2.6 (s, 3H), 2.0 (d, 3H), 1.2 (t, 3H) 152 -- 462.21 (CD.sub.3OD)
8.90 (d, 1H), 8.81 (d, 1H), 8.49 (m, 1H), 8.39 (d, 1H), 8.04 (d,
1H), 7.85 (m, 1H), 7.02 (br s, 1H), 6.90 (d, 1H), 4.82 (m, 1H,
underneath water peak), 4.42 (q, 2H), 4.29 (m, 1H), 3.72 (m, 1H),
3.35 (s, 3H), 2.65 & 2.90 (s, 3H), 1 153 504.3 506.3 12.14 (s,
1H); 11.11 (s, 1H); 9.59 (s, 1H); 9.08 (d, 2H); 8.38 (s, 1H); 8.33
(d, 1H); 8.22 (s, 1H); 7.95 (d, 1H); 7.56 (dd, 1H); 4.32 (q, 2H);
4.12 (br. s, 4H); 3.62 (br. s, 4H); 2.46 (br. s, 2H); 1.31 (dd,
2H); 1.22 (t, 3H). 154 474.3 476.3 10.31 (s, 1H); 9.09 (d, 2H);
8.79 (s, 1H); 8.44 (s, 1H); 8.42 (s, 1H); 8.00 (s, 1H); 7.68 (t,
2H); 7.7-6.6 (br. s, 3H); 4.32 (q, 2H); 3.55 (m, 4H); 3.12 (m, 4H);
2.34 (s, 6H); 1.32 (t, 3H). 155 479.3 481.2 1.33 (t, 3H) 2.36 (s,
3H) 4.35 (q, 2H) 5.60 (s, 2H) 6.84 (s, 1H) 6.88 (s, 1H) 7.68 (t,
1H)7.81 (d, 2H) 7.99 (s, 1H) 8.26 (t, 1H) 8.35 (s, 1H)8.78 (d, 1H)
8.89 (d, 3H) 11.75 (br s, 2H) 156 -- 461.22 (CD.sub.3OD) 8.83 (m,
1H), 8.44 (d, 1H), 8.25 (d, 1H), 8.12 (m, 1H), 7.88 (d, 1H), 7.54
(m, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 4.68 (m, 1H), 4.32 (m, 1H),
3.70 (m, 1H), 3.38 (q, 2H), 3.35 (s, 3H), 2.56 (s, 3H), 1.60 (d,
3H), 1.24 (t, 3H) ppm 157 486.3 488.3 (CD.sub.3OD) 1.37 (t, 3H)
1.57 (m, 4H) 2.27 (m, 1H) 2.82 (s, 3H) 3.41 (t, 2H) 3.96 (d,
2H)4.29 (d, 2H) 4.43 (q, 2H) 7.27 (s, 1H) 7.50 (s, 1H) 7.90 (t, 1H)
8.17 (s, 1H) 8.55 (s, 1H) 8.659 (t, 1H) 8.95 (d, 1H) 9.09 (d, 1H)
158 474.3 476.3 (CD.sub.3OD) 1.08 (d, 6H) 1.42 (t3H) 2.76 (s, 3H)
3.57 (m, 1H) 2.82 (t, 2H)4.42 (m, 4H) 6.98 (s, 1H) 7.05 (s, 1H)
7.86 (t, 1H) 8.09 (s, 1H) 8.41 (s, 1H)8.51 (t, 1H) 8.82 (d, 1H)
8.91 (d, 1H) 159 458 460 (CD.sub.3OD) 1.43 (t, 3H), 3.48 (bs, 24H),
3.99 (bs, 4H), 4.48 (1, 2H), 4.63 (s, 2H), 7.55 (t, 1H), 7.71 (d,
1H), 8.1 (s, 1H), 8.33 (d, 1H), 8.9 (1H), 9.03 (d, 2H), 9.13
(s.1H). 160 457 459 (CD.sub.3OD) 1.35 (t, 3H), 3.3-3.5 (m, 6H),
3.99 (bs, 4H), 4.62 (s, 2H), 7.52 (t, 1H), 7.7 (d, 1H), 8.08 (s,
1H), 8.32 (d, 1H), 8.88 (s, 1H),
9.05 (d, 2H)9.1 (s, 1H). 161 446.28 448.2 1.2 (t, 3H), 3.2 (m, 2H),
3.3 (br s, 4H), 3.9 (br s, 4H), 4.7 (s, 2H), 7.7 (s, 1H), 7.8 (br
s, 1H), 7.95 (m, 1H), 8.1 (s, 1H), 8.25 (s, 1H), 8.7 (m, 1H), 8.8
(m, 1H), 9.2 (s, 1H), 10.6 (br s, 1H) 162 550.3 552.3 12.32 (s,
1H); 11.18 (s, 1H); 9.08 (d, 2H); 8.62 (s, 1H); 8.39 (s, 1H); 8.37
(s, 1H); 7.98 (s, 1H); 7.62 (m, 2H); 7.58 (dd, 1H); 7.48 (m, 3H);
6.4-5.9 (br. s, 3H); 4.38 (s, 2H); 4.33 (q, 2H); 3.42 (m, 4H); 3.15
(m, 4H); 1.35 (t, 3H). 163 501.3 503.3 (CD.sub.3OD) 9.1 (d, 1H),
8.8 (t, 1H), 8.5 (d, 1H), 8.4 (s, 1H), 8.3 (s, 1H), 8.2 (t, 1H),
7.7 (s, 1H), 7.5 (s, 1H), 4.8 (t, 2H), 4.4 (q, 2H), 4.1 (d, 2H),
3.9 (t, 2H), 3.75 (d, 2H), 3.7 (t, 2H), 3.4 (m, 2H), 3.3 (s, 2H),
2.85 (s, 3H), 2.0 (s, 5H), 1.4 (t, 3H) 164 500.4 502.3 (CD.sub.3OD)
8.75 (d, 1H), 8.3 (d, 1H), 8.2 (s, 1H), 8.0-8.05 (t, 1H), 7.8 (s,
1H), 7.5 (t, 1H), 6.8 (s, 1H), 6.7 (s, 1H), 4.5-4.6 (t, 2H), 4.0
(brd s, 4H), 3.5-3.6 (t, 4H), 3.4 (q, 2H), 3.3 (t, 2H), 2.6 (s,
3H), 1.2-1.3 (t, 3H) 165 474 476 (CD.sub.3OD) 9.04 (d, 1H), 8.71
(d, 1H), 8.34 (d, 1H), 8.23 (dd, 1H), 7.94 (d, 1H), 7.84 (m, 1H),
7.65 (d, 1H), 7.57 (m, 1H), 4.56 (s, 2H), 3.98 (t, 4H), 3.45 (t,
4H), 3.37 (q, 2H), 1.24 (t, 3H) 166 475 477 (CD.sub.3OD): 9.07 (d,
1H), 8.72 (d, 1H), 8.49 (d, 1H), 8.28 (dd, 1H), 8.04 (d, 1H), 7.92
(m, 1H), 7.69 (d, 1H), 7.63 (m, 1H), 4.63 (s, 2H), 4.47 (q, 2H),
3.99 (m, 4H), 3.44 (m, 4H), 1.43 (t, 3H) 167 522.3 524.4
(CD.sub.3OD) 8.94 (d, 2H), 8.65 (s, 1H), 7.85 (s, 1H), 7.44 (dd,
1H), 7.27 (dd, 1H), 6.84 (d, 2H), 6.80 (s, 1H), 6.70 (s, 1H), 6.64
(s, 1H), 3.77 (s, 3H), 3.36 (q, 2H), 2.39 (br s, 3H), 1.96 (d, 3H),
1.24 (t, 3H) 168 -- 474.3 (CD.sub.3OD) 1.45 (t, 3H) 1.80 (d, 3H)
2.69 (m, 2H) 3.50 (m, 2H) 3.97 (m, 4H) 4.49 (q, 2H) 4.72 (m,
1H)7.55 (t, 1H) 7.71 (d, 1H) 1.15 (s, 1H) 8.33 (d, 1H) 8.91 (s, 1H)
9.08 (d, 2H) 9.17 (s, 1H) 169 445.44 447.24 1.3 (t, 3H), 2.9 (br s,
6H), 3.6 (brs, 2H), 4.3 (q, 2H), 4.6 (m, 2H), 7.1 (m, 1H), 7.6 (m,
1H), 7.9 (s, 1H), 8.2 (m, 1H), 8.25 (s, 1H), 8.3 (m, 1H), 8.6 (d,
1H), 8.7 (d, 1H), 9.1 (s, 1H), 9.7 (br, s, !H) 170 444.43 446.22
1.2 (t, 3H), 2.9 (br s, 6H), 3.25 (m, 2H), 3.6 (br s, 2H), 4.6 (m,
2H), 7.1 (m, 1H), 7.4 (br s, 1H), 7.6 (m, 1H), 7.9 (s, 1H), 8.1 (br
s, 1H), 8.2 (s, 1H), 8.3 (m, 1H), 8.6 (d, 1H), 8.7 (d, 1H), 9.1 (s,
1H), 9.8 (br s, 1H), 10.5 (br s, 1H) 171 460.2 462.2 (CD.sub.3OD)
8.95 (d, 1H), 8.86 (d, 1H), 8.57 (dt, 1H), 8.46 (d, 1H), 8.17 (d,
1H), 7.93 (t, 1H), 7.62 (br s 1H), 7.33 (d, 1H), 5.14 (m, 1H), 4.42
(q, 2H), 4.26 (m, 1H), 3.75 (dd, 1H), 3.36 (s, 3H), 2.81 (s, 3H),
1.42 (d, 3H), 1.30 (t, 3H) ppm 172 485 487 (CD.sub.3OD) 1.45-1.12
(m, 3H), 1.9 (m, 1H), 2.1 (m, 2H), 2.32 (m, 1H), 3.5-3.3 (m, 5H),
3.7 (m, 4H), 3.99 (bs, 1H), 4.4 (q, 2H), 4.57 (bd, 1H), 7.58 (d,
1H), 7.8 (t, 1H), 8.03 (s, 1H0, 8.32 (d, 1H), 8.42 (m, 2H), 8.75
(d, 1H), 8.88 (s, 1H) 9.1 (s, 1H). 173 484 486 (CD.sub.3OD) 1.15
(t, 3H), 1.4 (t, 3H), 3.02 (s, 2H), 3.6 (m, 2H), 3.68 (m, 2H), 3.95
(s, 2H), 4.4 (m, 2H), 4.61 (s, 2H), 7.8 (m, 2H), 8.08 (s, 1H), 8.48
(m, 3H), 8.78 (d, 1H), 8.9 (d, 1H), 9.17 (s, 1H). 174 503.2 505.2
(CDCl.sub.3) 8.94 (d, 1H), 8.87 (d, 1H), 8.15 (d, 1H), 8.11 (d,
2H), 7.95 (ddd, 1H), 7.90 (s, 1H), 7.70 (d, 1H), 7.42 (dd, 1H),
4.76 (s, 2H), 4.46 (q, 2H), 3.82 (t, 4H), 3.64 (t, 4H), 3.40 (s,
6H), 1.45 (t, 3H) 175 593.4 595.3 10.85 (s, 1H); 8.84 (s, 1H); 8.72
(s, 1H); 8.58 (d, 1H); 8.52 (s, 1H); 8.30 (d, 1H); 8.08 (s, 1H);
7.92 (d, aH); 7.44 (d, 2H); 7.34 (dd, 2H); 7.24 (t, 1H); 6.05-4.9
(br. s); 5.21 (dq, 1H); 4.78 (d, 2H); 4.31 (q, 2H); 3.58 (d, 2H);
3.52 ( 176 543.5 545.3 11.76 (s, 1H); 9.59 (s, 1H); 8.89 (d, 1H);
8.78 (s, 1H); 8.65 (s, 1H); 8.40 (s, 1H); 8.05 (d, 1H); 7.9-6.2
(br. s, 2H); 4.87 (d, 2H); 4.32 (q, 2H); 3.96 (dd, 2H); 3.68 (dd,
2H); 3.58 (m, 4H); 3.20 (m, 2H); 2.94 (d, 3H); 1.98 (m, 2H); 1.88
177 542.5 544.3 11.12 (s, 1H); 10.84 (s, 1H); 9.08 (s, 1H); 8.87
(d, 1H); 8.66 (s, 1H); 8.41 (s, 1H); 8.01 (s, 1H); 7.96 (d, 1H);
7.58 (s, 1H); 6.3-4.6 (br. s, 6H); 4.82 (d, 2H); 3.96 (dd, 2H);
3.58 (m, 6H); 3.26 (m, 2H); 3.15 (m, 2H); 2.84 (d, 3H); 1.96 ( 178
470.4 472.3 9.05 (d, 2H); 8.54 (s, 1H); 7.89 (s, 1H); 8.01 (s, 1H);
7.78 (d, 1H); 7.55 (t, 1H); 7.05 (d, 1H); 5.5-4.2 (br. s, 1H); 4.33
(q, 2H); 3.50 (dd, 2H); 3.36 (dd, 2H); 3.12 (s, 3H); 2.85 (s, 3H);
1.33 (t, 3H). 179 415.4 417.3 (CD.sub.3OD) 9.54 (d, 1H), 9.21 (m,
1H), 9.15 (s, 1H), 8.97 (d, 1H), 8.92 (d, 1H), 8.83 (d, 1H), 8.28
(dd, 1H), 8.20 (d, 1H), 7.69 (m, 1H), 4.62 (s, 2H), 4.48 (q, 2H),
3.00 (s, 6H), 1.44 (t, 3H) ppm 180 414.4 416.3 (CD.sub.3OD) 9.54
(d, 1H), 9.22 (m, 1H), 9.05 (s, 1H), 8.97 (d, 1H), 8.93 (d, 1H),
8.79 (d, 1H), 8.28 (dd, 1H), 8.17 (d, 1H), 7.62 (m, 1H), 4.59 (s,
2H), 3.39 (q, 2H), 2.99 (s, 6H), 1.25 (t, 3H) ppm 181 513 515
(CD.sub.3OD): 9.14 (d, 1H), 9.06 (d, 1H), 8.85 (t, 1H), 8.67 (t,
1H), 8.46 (d, 1H), 8.37 (d, 1H) 8.11 (m, 1H), 7.97 (d, 1H), 7.55
(m, 1H), 4.07 (brs, 2H), 3.88 (t, 4H), 3.71 (brs, 2H), 3.48 (t,
4H), 3.36 (q, 2H), 1.24 (t, 3H) 182 477.4 479.37 0.9 (d, 6H), 1.3
(t, 3H), 2.1 (m, 1H), 2.9 (s, 6H), 3.75 (d, 2H), 4.3 (q, 2H), 4.6
(s, 2H), 6.6 (dt, 1H), 6.7 (d, 1H), 7.7 (s, 1H), 7.8 (d, 1H), 7.95
(s, 1H), 8.010.2 (br s, 1H) (s, 1H), 183 476.42 478.41 0.9 (d, 6H),
1.1 (t, 3H), 2.1 (m, 1H), 2.9 (s, 6H), 3.2 (m, 2H), 3.8 (d, 2H),
4.6 (s, 2H), 6.6 (dt, 1H), 6.7 (d, 1H), 7.3 (br s, 1H), 7.7 (s,
1H), 7.8 (d, 1H), 7.95 (s, 1H), 8.0 (s, 1H), 10.2 (br s, 1H) 184
514 516 -- 185 470.4 472.2 (CD.sub.3OD) 1.25 (t, 3H) 1.78 (d, 3H)
3.30 (m, 2H) 3.35 (q, 2H) 3.50 (m, 2H) 3.93 (m, 4H)4.73 (q, 1H)
7.72 (t, 1H) 7.45 (d, 1H) 8.01 (s, 1H) 8.27 (t, 1H) 8.35 (m, 2H)
8.52 (d, 1H) 8.91 (d, 1H) 9.18 (s, 1H) 186 498.4 500.3 (CD.sub.3OD)
1.16 (m, 3H) 1.26 (m, 6H) 1.80 (d, 3H) 2.82 (t, 2H) 3.20 (d, 1H)
3.35 (q, 2H)3.72 (d, 1H) 3.91 (m, 1H) 4.04 (m, 1H) 4.68 (q, 1H)
7.65 (t, 1H) 7.71 (d, 1H) 8.01 (s, 1H)8.22 (t, 1H) 8.35 (m, 2H)
8.49 (d, 1H) 8.89 (d, 1H) 9.18 (s, 1H) 187 499.5 501.3 (CD.sub.3OD)
1.16 (m, 3H) 1.29 (d, 3H) 1.41 (t, 3H) 1.79 (d, 3H) 2.82 (t, 2H)
3.20 (d, 1H) 3.74 (d, 1H) 3.92 (m, 1H) 4.04 (m, 1H) 4.43 (q, 2H)
4.69 (q, 1H) 7.70 (d, 1H)7.87 (t, 1H) 8.11 (s, 1H) 8.35 (d, 1H)
8.46 (m, 2H) 8.75 (d, 1H) 8.92 (d, 1H) 9.34 (s, 1H) 188 486 488
(CD.sub.3OD): 8.91 (m, 1H), 8.65 (m, 1H), 8.47 (m, 1H), 8.31 (m,
2H), 8.29 (m, 1H), 7.96 (m, 1H) 7.73 (m, 1H), 7.19 (m, 1H), 4.08
(m, 2H), 3.90 (m, 2H), 3.72 (m, 4H), 3.66 (m, 4H), 3.37 (m, 2H),
1.24 (t, 3H) 189 487 489 (CD.sub.3OD): 8.89 (d, 1H), 8.69 (d, 1H),
8.61 (d, 1H), 8.41 (t, 1H), 8.32 (d, 1H), 8.20 (dd, 1H) 7.95 (d,
1H), 7.78 (t, 1H), 7.10 (d, 1H), 4.42 (q, 2H), 4.09 (m, 2H), 3.86
(m, 2H), 3.70 (m, 4H), 3.33 (m, 4H), 1.24 (t, 3H) 190 388.38 390.19
1.2 (t, 3H), 3.3 (m, 2H), 3.5 (s, 3H), 6.6 (dd, 1H), 6.7 (d, 1H),
7.6 (t, 1H), 7.8 (d, 1H), 7.95 (br s, 1H), 8.0 (s, 1H), 8.6 (s,
1H), 9.05 (d, 2H) 191 359.3 361.2 -- 192 446.4 448.3 -- 193 474.2
476.4 (CD.sub.3OD) 9.14 (s, 1H), 9.08 (d, 2H), 8.91 (s, 1H), 8.39
(d, 1H), 8.14 (s, 1H), 7.78 (s, 1H), 7.55 (dd, 1H), 4.72 (s, 2H),
4.48 (q, 2H), 3.81 (t, 2H), 3.56 (t, 2 H), 3.46 (q, 2H), 3.41 (s,
3H), 1.44 (t, 3H), 1.43 (t, 3H) 194 486.1 488.1 (CDCl.sub.3) 12.31
(br s, 1H), 8.95 (s, 1H), 8.93 (d, 2H), 8.58 (s, 1H), 8.05 (s, 1H),
8.01 (d, 1H), 7.50 (d, 1H), 7.24 (dd, 1H), 4.46 (q, 2H), 3.78 (m,
2H), 3.72 (s, 2H), 2.78 (d, 2H), 1.91 (dd, 2H), 1.41 (t, 3H), 1.17
(d, 6H) 195 -- -- 11.5 (br s, 1H), 9.19 (br s, 1H), 8.97 (s, 1H),
8.88 (d, 1H), 8.45 (m, 1H), 8.40 (s, 1H), 7.98 (d, 1H), 7.77 (d,
1H), 7.60 (d, 1H), 4.57 (s, 2H), 4.47 (br d, 2H), 4.32 (q, 2H),
3.90 (br m, 4H), 3.32 (br s, 4H), 2.80 (d, 6H), 1.34 197 460.2
462.2 (CD.sub.3OD &CDCl.sub.3): 8.98 (d, 2H), 8.85 (d, 1H),
8.60 (d, 1H), 8.16 (dd, 1H), 7.86 (d, 1H), 7.67 (d, 1H), 7.40 (dd,
1H), 4.37 (q, 2H), 3.81 (s, 2H), 3.60 (t, 2H), 3.37 (s, 3H), 2.74
(t, 2H), 2.38 (s, 3H), 1.41 (t, 3H) 198 459.3 461.2 (CD.sub.3OD):
8.95 (d, 2H), 8.83 (s, 1H), 8.54 (s, 1H), 8.15 (d, 1H), 7.82 (s,
1H), 7.65 (d, 1H), 7.39 (dd, 1H), 3.79 (s, 2H), 3.59 (t, 2H), 3.36
(q, 2H), 3.35 (s, 3H), 2.72 (t, 2H), 2.36 (s, 3H), 1.24 (t, 3H) 199
488 490 -- 200 487 489 -- 201 488 490 -- 202 487 489 -- 203 489 491
9.06 (m, 4H), 8.50 (d, 1H), 7.98 (d, 1H), 7.54 (t, 1H), 4.79 (t,
2H), 4.31 (q, 2H), 4.01 (m, 2H), 3.79 (m, 2H), 3.67 (m, 2H), 3.56
(m, 2H), 3.25 (m, 2H) 1.33 (t, 3H) 204 488 490 (CD.sub.3OD): 9.02
(m, 4H), 8.75 (s, 1H), 7.97 (d, 1H), 7.52 (m, 1H), 4.88 (m, 2H),
3.75-3.73 (m, 10H), 3.35 (m, 2H), 1.25 (t, 3H) 205 400.1 402.1 1.31
(t, 3H) 2.70 (s, 3H) 4.32 (q, 2H) 7.55 (t, 1H) 8.00 (s, 1H) 8.09
(d, 1H)8.14 (t, 1H) 8.39 (s, 1H) 8.44 (d, 1H) 8.70 (d, 1H) 8.81 (d,
1H) 9.19 (s, 1H) 206 458.1 460 (CD.sub.3OD) 9.41 (d, 2H), 8.92 (m,
1H), 8.80 (d, 1H), 8.48 (m, 2H), 8.10 (br s, 1H), 7.84 (m, 1H),
4.76 (s, 2H with water peak), 4.42 (q, 2H), 4.11 (br m, 2H), 3.95
(nr m, 2H), 3.71 (br m, 2H), 3.45 (br m, 2H), 1.42 (t, 3H) ppm. 207
457.2 459.1 (CD.sub.3OD) 9.33 (s, 2H), 8.89 (d, 1H), 8.53 (d, 1H),
8.44 (d, 1H), 8.20 (m, 1H), 8.04 (d, 1H), 7.63 (m, 1H), 4.76 (s,
2H, with water peak), 4.12 (br m, 2H), 3.94 (br m, 2H), 3.71 (br m,
2H), 3.44 (br m, 2H), 3.39 (q, 2H), 1.25 (t, 3H) ppm 208 391 393 --
209 441.1 443 (CD.sub.3OD) 1.43 (t, 3H) 4.50 (s, 2H) 4.89 (s, 2H)
7.55 (t, 1H) 8.10 (d, 1H)8.20 (s, 1H) 8.87 (d, 1H) 9.01 (s, 1H)
9.10 (d, 2H) 9.30 (s, 1H) 210 472.4 474.2 (CD.sub.3OD) 9.25 (s,
1H), 9.02 (d, 2H), 8.87 (d, 1H), 8.65 (d, 1H), 8.18 (s, 1H), 8.08
(d, 1H), 7.54 (dd, 1H), 4.88 (s, 2H), 4.49 (q, 2H), 4.27 (br s,
1H), 3.68 (m, 4H), 3.40 (s, 3H), 2.32 (m, 2H), 1.45 (t, 3H) 211
471.2 473.1 (CD.sub.3OD) 9.16 (s, 1H), 8.98 (d, 2H), 8.76 (d, 1H),
8.48 (d, 1H), 8.05 (s, 1H), 7.91 (d, 1H), 7.50 (dd, 1H), 4.81 (s,
2H), 4.27 (br s, 1H), 3.69 (m, 4H), 3.40 (s, 3H), 3.38 (q, 2H),
2.32 (m, 2H), 1.26 (t, 3H) 212 485.2 487.3 (CD.sub.3OD) 9.16 (s,
1H), 9.03 (d, 2H), 8.87 (s, 1H), 8.50 (d, 1H), 8.14 (s, 1H), 7.92
(d, 1H), 7.54 (dd, 1H), 4.75 (s, 2H), 4.49 (q, 2H), 4.06 (s, 2H),
3.74 (br s, 4H), 3.06 (s, 3H), 1.45 (t, 3H) 213 484.2 486.2
(CD.sub.3OD) 9.16 (s, 1H), 9.01 (d, 2H), 8.78 (s, 1H), 8.52 (d,
1H), 8.07 (s, 1H), 7.96 (d, 1H), 7.52 (dd, 1H), 4.77 (s, 2H), 4.07
(s, 2H), 3.75 (br s, 4H), 3.38 (q, 2H), 3.06 (s, 3H), 1.26 (t, 3H)
214 350.1 12.28 (br. s, 1H); 11.77 (br. s, 1H); 9.73 (s, 1H); 9.08
(d, 2H); 8.49 (d, 1H); 8.37 (m, 1H); 8.08 (d, 1H); 7.94 (m, 1H);
7.59 (t, 1H); 4.32 (q, 2H); 2.30 (s, 3H);
1.32 (t, 3H). 215 502.4 504.1 (CD.sub.3OD) 9.17 (s, 1H), 9.02 (d,
2H), 8.85 (s, 1H), 8.48 (d, 1H), 8.15 (s, 1H), 7.90 (d, 1H), 7.53
(dd, 1H), 4.81 (d, 1H), 4.77 (d, 1H), 4.48 (q, 2H), 4.18 (br s,
2H), 3.75 (m, 4H), 3.47 (s, 6H), 1.45 (t, 3H) 216 347.2 349.1 12.28
(br. s, 1H); 10.22 (br. s, 1H); 9.53 (s, 1H); 9.08 (d, 2H); 8.41
(d, 1H); 8.29 (m, 1H); 7.98 (d, 1H); 7.83 (m, 1H); 7.57 (t, 1H);
7.22 (m, 1H); 3.26 (dq, 2H); 2.31 (s, 3H); 1.15 (t, 3H). 217 501.4
503.1 (CD.sub.3OD) 9.15 (s, 1H), 9.03 (d, 2H), 8.85 (s, 1H), 8.43
(d, 1H), 8.10 (s, 1H), 7.83 (d, 1H), 7.53 (dd, 1H), 4.78 (d, 1H),
4.74 (d, 1H), 4.18 (br s, 2H), 3.74 (m, 4H), 3.47 (s, 6H), 3.38 (q,
2H), 1.26 (t, 3H) 218 456 458 (CD.sub.3OD) 1.24 (t, 3H), 3.38 (m,
2H), 3.57 (bs, 4H), 3.99 (bs, 4H), 4.63 (s, 2H), 7.68 (m, 1H), 7.73
(m, 1H), 8.02 (s, 1H), 8.24 (t, 1H), 8.48 (m, 1H), 8.49 (d, 1H),
8.9 (d, 1H), 9.13 (s, 1H). 219 -- -- 12.25 (s, 1H); 11.75 (s, 1H);
9.61 (d, 1H); 9.07 (d, 2H); 8.45 (s, 1H); 8.11 (s, 1H); 8.02 (d,
1H); 7.59 (t, 1H); 4.31 (q, 2H); 2.38 (s, 3H); 2.29 (s, 3H); 1.32
(t, 3H). 220 502.3 504.2 (CD.sub.3OD) 9.10 (d, 1H), 9.05 (d, 2H),
8.91 (d, 1H), 8.32 (dd, 1H), 8.11 (d, 1H), 7.69 (d, 1H), 7.55 (dd,
1H), 4.73 (d, 1H), 4.69 (d, 1H), 4.49 (q, 2H), 4.17 (br s, 2H),
3.70 (m, 4H), 3.46 (s, 6H), 1.44 (t, 3H) 221 501.3 503.2
(CD.sub.3OD) 9.09 (s, 1H), 9.05 (d, 2H), 8.86 (s, 1H), 8.29 (d,
1H), 8.09 (s, 1H), 7.67 (d, 1H), 7.54 (dd, 1H), 4.72 (d, 1H), 4.67
(d, 1H), 4.17 (br s, 2H), 3.71 (m, 4H), 3.47 (s, 6H), 3.39 (q, 2H),
1.26 (t, 3H) 222 -- 559 (CDCl.sub.3) 12.29 (br s, 1H), 8.94 (s,
1H), 8.94 (d, 2H), 8.60 (s, 1H), 8.04 (s, 1H), 8.02 (d, 1H), 7.50
(d, 1H), 7.25 (dd, 1H), 4.46 (q, 2H), 3.75 (s, 2H), 3.50 (m, 4H),
2.51 (m, 4H), 1.47 (s, 9H), 1.41 (t, 3H) 223 513.2 515.14 1.1 (d,
6H), 1.3 (t, 3H), 3.5 (m, 4H), 3.7 (br s, 2H), 4.3 (q, 2H), 4.5 (br
s, 2H), 4.65 (m, 1H), 7.5 (t, 1H), 7.7 (d, 1H), 8.0 (d, 1H), 8.3
(dd, 1H), 8.5 (d, 1H), 9.0 (d, 2H), 9.05 (d, 1H) 224 457.207
459.207 (CD.sub.3OD) 9.54 (s, 1H), 9.28 (s, 1H), 9.22 (d, 1H), 8.97
(d, 1H), 8.93 (d, 1H), 8.85 (br s, 1H), 8.27 (t, 1H), 8.20 (br s,
1H), 7.71 (d, 1H), 4.63 (s, 2H), 4.48 (q, 2H), 4.05 (br s, 4H),
3.44 (br m, 4H), 1.44 (t, 3H) ppm 225 472.2 474.49 (CD.sub.3OD) 9.0
(d, 1H), 8.95 (m, 1H), 8.6 (s, 1H), 8.5 (s, 1H), 8.4 (s, 1H), 8.3
(s, 1H), 7.9 (s, 1H), 7.55 (m, 1H), 4.5 (s, 2H), 3.9 (s, 2H), 3.4
(q, 2H), 3.0 (s, 6H), 1.3 (t, 3H). 226 562.3 564.2 (CD.sub.3OD) 9.0
(d, 1H), 8.9 (m, 1H), 8.6 (s, 1H), 8.5 (s, 1H), 8.3 (s, 1H), 8.25
(s, 1H), 7.9 (s, 1H), 7.55 (m, 1H), 7.3-7.4 (m, 5H), 5.2 (s, 2H),
4.5 (s, 2H), 4.0 (s, 2H), 3.4 (q, 2H), 3.0 (s, 6H), 1.3 (t, 3H) 227
389 391 -- 228 457.3 459.1 -- 229 456.3 458.1 -- 230 433.3 435.1 --
231 487 489 (CD.sub.3OD) 9.30 (s, 2H), 9.04 (d, 2H), 8.89 (d, 1H),
8.11 (d, 1H), 7.54 (t, 1H), 4.46 (q, 2H), 4.02 (m, 2H), 3.59 (m,
2H), 2.89 (m, 2H), 1.44 (t, 2H), 1.13 (t, 3H) ppm. 232 456.3 458.3
(CD.sub.3OD) 9.22 (s, 1H), 9.05 (d, 2H), 8.92 (s, 1H), 8.78 (d,
1H), 8.17 (s, 1H), 8.08 (d, 1H), 7.54 (dd, 1H), 4.42 (s, 2H), 3.,52
(m, 4H), 3.39 (q, 4H), 3.28 (m, 4H), 1.26 (t, 3H) 233 -- 459.1
(CD.sub.3OD) 9.22 (s, 1H), 9.06 (d, 2H), 8.96 (s, 1H), 8.77 (d,
1H), 8.22 (s, 1H), 8.07 (d, 1H), 7.56 (dd, 1H), 4.49 (q, 2H), 4.42
(s, 2H), 3.,52 (m, 4H), 3.29 (m, 4H), 1.45 (t, 3H) 234 -- 537
(CD.sub.3OD) 9.22 (s, 1H), 9.04 (d, 2H), 8.90 (s, 1H), 8.54 (d,
1H), 8.16 (s, 1H), 7.99 (d, 1H), 7.54 (dd, 1H), 4.78 (s, 2H), 4.49
(q, 2H), 3.69 &3.63 (m, 8H), 3.00 (s, 3H), 1.45 (t, 3H) 235
534.2 536.1 (CD.sub.3OD) 9.20 (s, 1H), 9.02 (d, 2H), 8.82 (s, 1H),
8.52 (d, 1H), 8.09 (s, 1H), 7.98 (d, 1H), 7.52 (dd, 1H), 4.78 (s,
2H), 3.69 &3.62 (m, 8H), 3.39 (q, 2H), 3.00 (s, 3H), 1.25 (t,
3H) 236 496.4 498.3 (CD.sub.3OD) 9.28 (s, 1H), 9.06 (br s, 2H),
8.96 (s, 1H), 8.85 (br s, 1H), 8.24 (s, 1H), 8.15 (br s, 1H), 7.56
(br s, 1H), 4.49 (q, 2H), 4.42 (s, 2H), 3.73 (br s, 4H), 3.35 (br
s, 4H), 3.01 (br s, 1H), 1.45 (t, 3H), 1.20 (br s, 2H), 1.00 (br s
2H) 237 496.4 498.3 (CDCl.sub.3) 11.82 (br s, 1H), 8.88 (s, 1H),
8.71 (br s, 2H), 8.48 (s, 1H), 7.89 (d, 1H), 7.79 (s, 1H), 7.42 (d,
1H), 7.05 (br s, 1H), 3.71 (s, 2H), 3.46 (q, 2H), 2.70 & 2.55
(m, 8H), 1.28 (t, 3H), 0.45-0.41 (m, 4H) 238 447.2 449 12.35 (s,
1H); 9.07 (d, 2H); 8.82 (s, 1H); 8.41 (d, 1H); 8.13 (s, 1H); 7.95
(d, 1H); 7.57 (t, 1H); 6.7-5.2 (br. s, 4H); 4.38 (s, 2H); 4.32 (q,
2H); 3.92 (br. d, 4H); 3.32 (br. d, 4H); 1.33 (t, 3H). 239 490.1
492.2 (CD.sub.3OD) 9.21 (s, 1H), 9.05 (d, 2H), 8.92 (s, 1H), 8.54
(d, 1H), 8.17 (s, 1H), 7.97 (d, 1H), 7.55 (dd, 1H), 4.78 (s, 2H),
4.49 (q, 2H), 4.01 (br dd, 2H), 3.74 (br d, 2H), 3.43 (br dd, 2H),
3.27 (br d, 2H), 1.45 (t, 3H) 240 471.3 473 (CD.sub.3OD) 9.28 (s,
1H), 9.05 (br s, 2H), 8.96 (s, 1H), 8.89 (s, 1H), 8.25 (s, 1H),
8.17 (s, 1H), 7.55 (br s, 1H), 4.49 (q, 2H), 4.39 (s, 2H),
3.64-3.13 (m, 8H), 3.00 (s, 3H), 1.45 (t, 3H) 241 -- 531.01 1.3 (t,
3H), 1.55 (d, 3H), 3.3 (br s, 4H), 3.9 (br s, 4H), 4.0 (q, 1H),
4.35 (q, 2H), 4.6 (s, 2H), 7.6 (dd, 1H), 7.8 (d, 1H), 8.0 (s, 1H),
8.4 (d, 1H), 8.42 (s, 1H), 8.7 (s, 1H), 8.8 (d, 1H), 9.15 (s, 1H),
11...2 (br s, 1H) 242 486.2 488.1 (CD.sub.3OD) 9.33 (s, 2H), 8.92
(d, 1H), 8.79 (d, 1H), 8.47 (m, 2H), 8.09 (d, 1H), 7.84 (m, 1H),
4.42 (q, 2H), 4.05 (m, 2H), 3.67 (m, 2H), 3.39 (m, 2H, underneath
solvent peak), 3.00 (m, 2H), 1.42 (t, 3H), 1.28 (d, 6H) ppm 243
488.2 490.05 1.3 (t, 3H), 3.2 (m, 2H), 3.5 (m, 2H), 3.7 (m, 2H),
3.9 (m, 2H), 4.0 (m, 2H), 4.3 (q, 2H), 4.8 (m, 2H), 7.6 (7, 1H),
8.1 (d, 1H), 8.2 (br s, 1H), 8.6 (d, 1H), 8.65 (d, 1H), 8.8 (d,
1H), 9.05 (d, 2H), 11.6 (br s, 1H), 12.7 (br s, 1H) 244 457.21
459.09 1.35 (t, 3H), 2.8 (br s, 3H), 3.2 (m, 2H), 3.4 (m, 2H), 3.55
(m, 2H), 4.3 (m, 2H), 4.35 (q, 2H), 7.6 (t, 1H), 8.1 (d, 1H), 8.3
(br s, 1H), 8.55 (d, 1H), 8.6 (d, 1H), 8.7 (d, 1H), 9.05 (d, 2H),
11.2 (br s, 1H), 12.4 (br s, 1H) 245 529.2 531.1 (CD.sub.3OD) 9.08
(d, 1H), 9.01 (d, 2H), 8.82 (d, 1H), 8.30 (d, 1H), 8.04 (d, 1H),
7.68 (d, 1H), 7.51 (dd, 1H), 4.63 (s, 2H), 4.47 (q, 2H), 4.22 (s,
2H), 3.91 (br s, 4H), 3.49 (br s, 4H), 3.43 (s, 3H), 1.44 (t, 3H)
246 555.3 557.1 (CD.sub.3OD) 9.23 (s, 1H), 9.04 (d, 2H), 8.90 (s,
1H), 8.56 (d, 1H), 8.17 (s, 1H), 8.00 (d, 1H), 7.55 (dd, 1H), 4.77
(s, 2H), 4.49 (q, 2H), 4.12-3.84 (m, 6H), 3.57 (br s, 4H), 3.48 (br
s, 1H), 2.18 (m, 2H), 1.97 (m, 2H), 1.45 (t, 3H) 247 485.1 487
(CD.sub.3OD) 9.34 (s, 2H), 8.93 (d, 1H), 8.80 (d, 1H), 8.48 (m,
2H), 8.10 (d, 1H), 7.85 (t, 1H), 4.88 (s, 2H), 4.44 (q, 2H), 4.21
(s, 2H), 3.86 (m, 2H), 3.79 (m, 2H), 3.09 (s, 3H), 1.42 (t, 3H)
ppm. 248 458.15 460.02 1.23 (t, 3H) 3.17 (m, 4H) 3.65 (m, 4H) 4.34
(q, 2H) 4.77 (s, 2H)7.57 (t, 1H) 8.11 (s, 1H) 8.61 (s, 1H) 9.06 (d,
2H) 9.39 (s, 2H) 9.87 (br s, 2H) 249 472.3 474 (CD.sub.3OD) 9.17
(s, 1H), 9.04 (d, 2H), 8.92 (s, 1H), 8.45 (d, 1H), 8.15 (s, 1H),
7.87 (d, 1H), 7.55 (dd, 1H), 4.68 (s, 2H), 4.69 (q, 2H), 4.11 (dd,
1H), 4.02-3.95 (m, 2H), 3.57 (m, 2H), 3.03 (dd, 1H), 1.44 (t, 3H),
1.26 (d, 3H) 250 499.2 501.3 (CD.sub.3OD) 9.25 (s, 1H), 9.06 (d,
2H), 8.97 (s, 1H), 8.84 (d, 1H), 8.23 (s, 1H), 8.12 (d, 1H), 7.56
(dd, 1H), 4.49 (q, 2H), 4.37 (s, 2H), 3.65-3.12 (m, 9H), 1.46-1.43
(m, 9H) 251 514.9 517 (CD.sub.3OD) 9.27 (s, 1H), 9.05 (d, 2H), 8.95
(s, 1H), 8.86 (d, 1H), 8.25 (s, 1H), 8.16 (d, 1H), 7.56 (dd, 1H),
4.49 (q, 2H), 4.46 (s, 2H), 3.81-3.50 (m, 12 H), 3.44 (s, 3H), 1.45
(t, 3H) 252 527.2 529 (CD.sub.3OD) 9.06 (s, 1H), 8.97 (d, 2H), 8.76
(s, 1H), 8.28 (d, 1H), 8.00 (s, 1H), 7.69 (d, 1H), 7.49 (dd, 1H),
4.65 (s, 2H), 4.45 (q, 2H), 3.99 (br s, 4H), 3.49 (br s, 4H), 2.99
(hept, 1H), 1.44 (t, 3H), 1.14 (d, 6H) 253 502 504 (CD.sub.3OD)
9.08 (d, 2H), 8.73 (s, 1H), 8.51 (s, 1H), 8.38 (dd, 1H), 8.07 (d,
1H), 7.92 (m, 1H), 7.77 (d, 1H), 7.64 (m, 1H), 4.67 (s, 2H), 4.48
(q, 2H), 4.01 (s, 2H), 3.65 (m, 4H), 3.06 (m, 2H), 1.43 (t, 2H),
1.13 (t, 3H) ppm. 254 454.17 456.02 14.52 (s, 1H); 9.22 (s, 2H);
9.05 (d, 2H); 8.56 (d, 1H); 8.04 (d, 1H); 7.74 (s, 1H); 7.62 (s,
1H); 7.55 (t, 1H); 6.8-5.6 (br. s); 5.79 (s, 2H); 4.33 (q, 2H);
2.62 (s, 3H); 1.33 (t, 3H). 255 530.17 532.03 1.1 (d, 6H), 1.35 (t,
3H), 2.3 (s, 6H), 3.5 (m, 2H), 3.6 (m, 2H), 3.9 (s, 2H), 4.3 (q,
2H), 4.6 (s, 2H), 4.65 (m, 1H), 7.7 (d, 1H), 7.9 (s, 1H), 8.0 (dt,
1H), 8.3 (s, 1H), 8.4 (dd, 1H), 8.8 (d, 1H), 8.85 (m, 1H), 9.1 (s,
1H) 256 486.23 488.05 1.3 (d, 6H), 1.35 (t, 3H), 3.1 (m, 2H), 3.5
(m, 1H), 3.6 (m, 4H), 4.3 (q, 2H), 4.8 (m, 2H), 7.6 (t, 1H), 7.9
(s, 1H), 8.5 (s, 1H), 8.9 (s, 2H), 9.05 (d, 1H), 10.5 (br s, 1H)
257 499.2 501.1 (CD.sub.3OD) 9.34 (s, 2H), 8.93 (m, 1H), 8.81 (d,
1H), 8.49 (m, 2H), 8.11 (d, 1H), 7.85 (t, 1H), 4.87 (s, 2H), 4.42
(q, 2H), 4.11 (br s, 2H), 3.87 (br m, 4H), 3.71 (m, 3H), 1.48 (d,
6H), 1.42 (t, 3H) ppm. 258 571.3 573.1 (CDCl.sub.3) 12.75 (br s,
1H), 12.29 (s, 1H), 8.94 (d, 2H), 8.92 (s, 1H), 8.60 (s, 1H), 8.05
(s, 1H), 8.04 (d, 1H), 7.59 (d, 1H), 7.25 (dd, 1H), 4.46 (q, 2H),
4.23 (br s, 1H), 3.86 (d, 1H), 3.80 (d, 1H), 3.65 (d, 1H), 3.19
(ddd, 1H), 2.86 (d, 1H), 2.66 (d, 1H) 259 -- 473 (CD.sub.3OD) 9.24
(s, 1H), 9.04 (d, 2H), 8.94 (s, 1H), 8.81 (d, 1H), 8.22 (s, 1H),
8.11 (d, 1H), 7.55 (dd, 1H), 4.49 (q, 2H), 4.42 (s, 2H), 3.77-2.89
(m, 7H), 1.45 (t, 3H), 1.41 (d, 3H) 260 485.3 487.3 (CD.sub.3OD)
9.24 (s, 1H), 9.06 (d, 2H), 8.96 (s, 1H), 8.83 (d, 1H), 8.23 (s,
1H), 8.11 (d, 1H), 7.56 (dd, 1H), 4.49 (q, 2H), 4.39 (s, 2H), 3.76
(m, 2H), 3.40 (d, 2H), 2.80 (d, 2H), 1.45 (t, 3H), 1.40 (d, 6H) 261
485.2 487 (CD.sub.3OD) 9.23 (s, 1H), 9.06 (d, 2H), 8.97 (s, 1H),
8.82 (d, 1H), 8.24 (s, 1H), 8.13 (d, 1H), 7.56 (dd, 1H), 4.74 (d,
2H), 4.49 (q, 2H), 4.25 (d, 1H), 3.80-2.92 (m, 6H), 1.45 (t, 3H),
1.36 (d, 6H) 262 513.2 515 (CD.sub.3OD) 9.10 (s, 1H), 9.02 (d, 2H),
8.85 (s, 1H), 8.31 (d, 1H), 8.06 (s, 1H), 7.68 (d, 1H), 7.52 (dd,
1H), 4.62 (s, 2H), 4.46 (q, 2H), 3.92-3.16 (m, 8 H), 2.48 (q, 2H),
1.86 (m, 2H), 1.44 (t, 3H), 1.14 (t, 3 H) 263 -- 557.3 (CD.sub.3OD)
9.11 (s, 1H), 9.03 (d, 2H), 8.85 (s, 1H), 8.32 (d, 1H), 8.07 (s,
1H), 7.69 (d, 1H), 7.52 (dd, 1H), 4.63 (s, 2H), 4.47 (q, 2H),
3.99-3.79 (m, 8 H), 3.50-3.45 (m, 5H), 2.15 (m, 2H), 1.44 (t, 3H)
264 -- 501.1 (CD.sub.3OD) 9.10 (s, 1H), 9.03 (d, 2H), 8.85 (s, 1H),
8.62 (d, 1H), 8.08 (s, 1H), 7.94 (d, 1H), 7.52 (dd, 1H), 4.46 (q,
2H), 4.12 (s, 2H), 3.46-3.01 (m, 10H), 1.80 (m, 2H), 1.43 (t, 3H),
1.04 (t, 3H) 265 -- 531.1 (CD.sub.3OD) 9.21 (s, 1H), 8.96 (d,
2H),
8.82 (s, 1H), 8.59 (d, 1H), 8.12 (s, 1H), 8.06 (d, 1H), 7.48 (dd,
1H), 4.75 (s, 2H), 4.42 (q, 2H), 4.12 (q, 2H), 3.83 (br s, 4H),
3.47 (br s, 4H), 1.38 (t, 3H), 1.23 (t, 3H) 266 543.2 545
(CD.sub.3OD) 9.33 (s, 1H), 8.99 (d, 2H), 8.83 (s, 1H), 8.79 (br s,
1H), 8.18 (s, 1H), 7.54 (br s, 1H), 4.93 (m, 1H), 4.89 (s, 2H),
4.49 (q, 2H), 3.90 (br s, 4H), 3.57 (br s, 4H), 1.45 (t, 3H), 1.29
(d, 6H) 267 516 518 -- 268 557.3 559.2 (CD.sub.3OD) 9.08 (s, 1H),
8.969 (d, 2H), 8.78 (s, 1H), 8.29 (d, 1H), 8.01 (s, 1H), 7.67 (d,
1H), 7.49 (dd, 1H), 4.62 (s, 2H), 4.45 (q, 2H), 3.93 (d, 2H), 3.85
(br s, 4H), 3.46 (br s, 4H), 1.96 (m, 1H), 1.43 (t, 3 H), 0.98 (d,
6 H) 269 553.3 555.1 (CD.sub.3OD) 9.15 (s, 1H), 9.05 (d, 2H), 8.91
(s, 1H), 8.39 (d, 1H), 8.14 (s, 1H), 7.79 (d, 1H), 7.55 (dd, 1H),
4.71 (q, 2H), 4.68 (s, 2H), 4.49 (q, 2H), 3.88 (br s, 4H), 3.49 (br
s, 4H), 1.82 (t, 3H), 1.44 (t, 3 H) 270 557.3 559.2 (CD.sub.3OD)
9.11 (s, 1H), 9.03 (d, 2H), 8.84 (s, 1H), 8.31 (d, 1H), 8.05 (s,
1H), 7.68 (d, 1H), 7.52 (dd, 1H), 4.63 (s, 2H), 4.46 (q, 2H), 3.96
(br s, 4H), 3.52 (br s, 2H), 3.44 (br s, 2H), 2.71 (t, 2H), 2.65
(t, 2H), 1.43 (t, 3 H) 271 573.2 575.2 (CD.sub.3OD) 9.12 (s, 1H),
9.05 (d, 2H), 8.86 (s, 1H), 8.32 (d, 1H), 8.07 (s, 1H), 7.68 (d,
1H), 7.53 (dd, 1H), 4.63 (s, 2H), 4.46 (q, 2H), 4.37 (s, 2H), 4.21
(s, 2H), 3.98 (br s, 4H), 3.52 (br s, 2H), 3.47 (br s, 2H), 1.44
(t, 3 H) 272 500.23 502.05 1.21 (m, 9H) 3.17 (s, 1H) 3.61 (m, 8H)
4.32 (q, 2H) 4.65 (s, 2H)7.56 (t, 1H) 8.10 (s, 1H) 8.58 (s, 1H)
9.07 (d, 2H) 9.30 (s, 2H) 273 476 478 9.29 (s, 2H), 8.66 (s, 1H),
8.02 (s, 1H), 7.95 (m, 2H), 7.59 (m, 1H), 4.76 (s, 2H), 4.30 (m,
2H), 3.94-3.68 (m, 4H), 3.50-3.17 (m, 4H), 1.30 (t, 3H) 274 417.1
419 9.1 (s, 2H), 9.0 (s, 2H), 8.4 (s, 1H), 7.8 (s, 1H), 7.5 (t,
1H), 4.1-4.2 (q, 2H), 3.7 (s, 2H), 2.3 (s, 6H), 1.3 (t, 3H). 275
466 468 -- 276 445.1 447 1.33 (t, 3H) 2.06 (m, 1H) 2.26 (m, 1H)
3.88 (m, 4H) 4.33 (q, 2H)5.85 (m, 1H) 6.97 (d, 1H) 7.55 (t, 1H)
7.91 (s, 1H) 8.08 (d, 1H) 8.52 (m, 2H)9.05 (d, 2H) 12.44 (br s, 1H)
277 475.1 477 1.3 (t, 3H), 2.3 (s, 6H), 3.35 (m, 4H), 3.9 (m, 4H),
4.3 (q, 2H), 4.6 (s, 2H), 7.7 (d, 1H), 7.9 (s, 1H), 8.0 (dt, 1H),
8.3 (s, 1H), 8.35 (dd, 1H), 8.75 (d, 1H), 8.8 (br s, 1H), 9.1 (d,
1H), 10.4 (br s, 1H) 278 502.1 504 1.3 (t, 3H), 3.2 (s, 3H), 3.4
(br s, 4H), 3.7 (br s, 4H), 3.8 (br s, 2H), 4.3 (q, 2H), 4.8 (br s,
2H), 7.55 (t, 1H), 8.0 (d, 1H), 8.5 (d, 1H), 9.0 (s, 2H), 9.05 (d,
2H), 11.8 (br s, 1H), 12.6 (br s, 1H) 279 525.2 527 -- 280 567.2
569 (CD.sub.3OD) 9.11 (s, 1H), 9.03 (d, 2H), 8.83 (s, 1H), 8.33 (d,
1H), 8.06 (s, 1H), 7.68 (d, 1H), 7.52 (dd, 1H), 4.63 (s, 2H), 4.46
(q, 2H), 3.94 (m, 4H), 3.59 (q, 2H), 3.47 (m, 4H), 1.44 (t, 3H) 281
458.15 459.98 9.11 (br. s, 2H); 9.09 (s, 1H); 8.82 (d, 1H); 8.78
(br. s, 1H); 8.64 (d, 1H); 8.28 (s, 1H); 8.10 (dd, 1H); 7.88 (s,
1H); 7.52 (dd, 1H); 5.38 (m, 1H); 5.5-4.2 (br. s, 4H); 4.32 (q,
2H); 3.45 (m, 1H); 3.37 (m, 1H); 3.12 (m, 2H); 2.05 (M, 1 282 514.2
516 10.05 (m, 1H); 9.24 (m, 1H); 9.15 (d, 2H); 8.82 (d, 1H); 8.67
(d, 1H); 8.31 (s, 1H); 8.12 (m, 1H); 7.90 (s, 1H); 7.53 (m, 1H);
5.52 (m, 1H); 4.32 (q, 2H); 3.78 (m, 1H); 3.55-2.88 (m, 4H); 2.12
(m, 1H); 2.02 (m, 1H); 1.88 (m, 1H); 1.33 (t 283 519.15 521.02 1.3
(t, 3H), 2.35 (s, 12H), 2.8 (s, 3H), .3.0-3.7 (br s, 10H), 4.3 (q,
2H), 4.7 (br s, 2H), 7.6 (m, 1H), 7.9 (s, 1H), 7.95 (m, 2H), 8.65
(m, 1H), 9.0 (s, 2H) 290 487.1 489 -- 291 -- 490 -- 292 482.1 484
-- 293 483 484.9 -- 294 460.11 461.96 1.15 (t, 3H), 2.3 (s, 9H),
3.25 (m, 2H), 3.5 (m, 4H), 3.8 (m, 4H), 7.5 (br s, 1H), 7.6 (m,
1H), 7.95 (m, 1H), 8.0 (s, 1H), 8.1 (m, 1H), 8.2 (m, 1H), 8.4 (d,
1H), 8.6 (s, 1H), 8.7 (d, 1H), 10.3 (br s, 1H) 295 -- --
(CD.sub.3OD) 9.13 (d, 1H), 9.05 (d, 2H), 8.92 (d, 1H), 8.37 (m,
1H), 8.13 (d, 1H), 7.75 (dt, 1H), 7.55 (t, 1H), 4.70 (s, 2H), 4.49
(q, 2H), 3.62 (m, 4H), 2.47 (m, 4H), 1.44 (t, 3H) ppm
Example 27
Gyrase ATPase Assay
[0203] The ATP hydrolysis activity of DNA gyrase was measured by
coupling the production of ADP through pyruvate kinase/lactate
dehydrogenase to the oxidation of NADH. This method has been
described previously (Tamura and Gellert, 1990, J. Biol. Chem.,
265, 21342).
[0204] ATPase assays are carried out at 30.degree. C. in buffered
solutions containing 100 mM TRIS pH 7.6, 1.5 mM MgCl.sub.2, 150 mM
KCl. The coupling system contains (final concentrations) 2.5 mM
phosphoenol pyruvate, 200 .mu.M nicotinamide adenine dinucleotide
(NADH), 1 mM DTT, 30 ug/ml pyruvate kinase, and 10 ug/ml lactate
dehydrogenase. 40 nanomolar enzyme (374 kDa Gyr A2B2 subunit from
Staphylococcus aureus) and a DMSO solution of the inhibitor to a
final concentration of 4% are added and the reaction mixture is
allowed to incubate for 10 minutes at 30.degree. C. The reaction is
then started by the addition of ATP to a final concentration of 0.9
mM and the rate of NADH disappearance at 340 nanometers is measured
over the course of 10 minutes. The K.sub.i values are determined
from rate versus concentration profiles.
[0205] Compounds of the present invention were found to inhibit
gyrase. In certain embodiments, compounds of the present invention
inhibit gyrase with a K.sub.i value of less than 50 nM in the above
assay.
Example 28
Topo IV ATPase Assay
[0206] The conversion of ATP to ADP by Topo4 enzyme is coupled to
the conversion of NADH to NAD+ and measured by the change in
absorbance at 340 nm. Topo4 is incubated with inhibitor (4% DMSO
final) in buffer for 10 minutes at 30.degree. C. Reaction is
initiated with ATP and rates are monitored continuously for 20
minutes at 30.degree. C. on a Molecular Devices SpectraMAX plate
reader. The inhibition constant, Ki, is determined from plots of
rate vs. [Inhibitor] fit to the Morrison Equation for tight binding
inhibitors.
S. aureus Topo4 Buffer:
[0207] 100 mM Tris 7.5, 2 mM MgCl.sub.2, 200 mM K.cndot.Glutamate,
2.5 mM phosphoenol pyruvate, 0.2 mM NADH, 1 mM DTT, 4.25 .mu.g/mL
linearized DNA, 50 .mu.g/mL BSA, 30 .mu.g/mL pyruvate kinase, and
10 .mu.g/mL lactate dehyrodgenase (LDH). E. coli Topo4 Buffer:
[0208] 100 mM Tris 7.5, 6 mM MgCl.sub.2, 20 mM KCl, 2.5 mM
phosphoenol pyruvate, 0.2 mM NADH, 10 mM DTT, 5.25 .mu.g/mL
linearized DNA, 50 .mu.g/mL BSA, 30 .mu.g/mL pyruvate kinase, and
10 .mu.g/mL lactate dehyrodgenase (LDH).
[0209] Compounds of the present invention were found to inhibit
TopoIV. In certain embodiments, compounds of the present invention
inhibit TopoIV with a K.sub.i value of less than 50 nM in the above
assay.
Example 29
Susceptibility Testing in Liquid Media
[0210] Compounds of this invention were also tested for
antimicrobial activity by susceptibility testing in liquid media.
Such assays were performed within the guidelines of the latest
NCCLS document governing such practices: "M7-A5 Methods for
dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically; Approved Standard--Fifth Edition (2000)". Other
publications such as "Antibiotics in Laboratory Medicine" (Edited
by V. Lorian, Publishers Williams and Wilkins, 1996) provide
essential practical techniques in laboratory antibiotic testing.
Essentially, several discrete bacterial colonies of Staphylococcus
aureus (3 to 7) from a freshly streaked plate were transferred to
an appropriate rich broth medium such as MHB, supplemented where
appropriate for the more fastidious organisms. This was grown
overnight to high density followed by a 1 or 2-thousand-fold
dilution to give an inoculation density of between 5.times.10.sup.5
and 5.times.10.sup.6 CFU per mL. Alternatively, the freshly picked
colonies can be incubated at 37.degree. C. for about 4 to 8 hours
until the culture equals or exceeds a turbidity of a 0.5 McFarland
standard (approximately 1.5.times.10.sup.8 cells per mL) and
diluted to give the same CFU per mL as above. In a more convenient
method, the inoculum was prepared using a commercially available
mechanical device (the BBL PROMPT System) that involves touching
five colonies directly with a wand, containing crosshatch grooves
at its bottom, followed by suspension of the bacteria in an
appropriate volume of saline. Dilution to the appropriate inoculum
cell density was made from this cell suspension. The broth used for
testing consists of MHB supplemented with 50 mg per L of Ca.sup.2+
and 25 mg per L of Mg.sup.2+. Standard dilution panels of control
antibiotics were made and stored as in the NCCLS standard M7-A5,
the dilution range typically being in the 128 .mu.g per mL to 0.015
.mu.g per mL (by 2-fold serial dilution). The test compounds were
dissolved and diluted fresh for experimentation on the same day;
the same or similar ranges of concentration as above being used.
The test compounds and controls were dispensed into a multiwell
plate and test bacteria added such that the final inoculation was
approximately 5.times.10.sup.4 CFU per well and the final volume
was 100 .mu.L. The plates were incubated at 35.degree. C. overnight
(16 to 20 hours) and checked by eye for turbidity or quantitated
with a multiwell plate reader. The endpoint minimal inhibitory
concentration (MIC) is the lowest concentration of drug at which
the microorganism tested (Staphylococcus aureus) does not grow.
Such determinations were also compared to the appropriate tables
contained in the above two publications to ensure that the range of
antibacterial activity is within the acceptable range for this
standardized assay.
[0211] Compounds of the present invention were found to have
antimicrobial activity in the above-described S. aureus MIC
assay.
[0212] While we have described a number of embodiments of the
present invention, it is apparent that our basic constructions may
be altered to provide other embodiments which utilize the products
and processes of this invention.
* * * * *