U.S. patent application number 10/361603 was filed with the patent office on 2004-06-10 for desleucyl glycopeptide antibiotics and methods of making same.
Invention is credited to Kahne, Daniel, Silva, Domingos J., Walker, Suzanne.
Application Number | 20040110665 10/361603 |
Document ID | / |
Family ID | 22430542 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110665 |
Kind Code |
A1 |
Kahne, Daniel ; et
al. |
June 10, 2004 |
Desleucyl glycopeptide antibiotics and methods of making same
Abstract
Compounds that are analogs of glycopeptide antibiotics are
disclosed. The compounds have the formula
A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.- 6-A.sub.7 wherein
each of the groups A.sub.2 to A.sub.7 is a modified or unmodified
.alpha.-amino acid residue, A.sub.1 is optional and, when present,
is an organic group other than N-substituted leucine, and at least
one of the groups A.sub.1 to A.sub.7 is linked via a glycosidic
bond to one or more glycosidic groups each having one or more sugar
residues, wherein at least one of said sugar residues is modified
to bear at least one hydrophobic substituent. Methods of making
these compounds, compositions including these compounds, and
methods of using the compounds to treat infections in a host are
also disclosed.
Inventors: |
Kahne, Daniel; (Princeton,
NJ) ; Walker, Suzanne; (Princeton, NJ) ;
Silva, Domingos J.; (King of Russia, PA) |
Correspondence
Address: |
KENYON & KENYON
1500 K. Street, N.W.
Washington
DC
20005
US
|
Family ID: |
22430542 |
Appl. No.: |
10/361603 |
Filed: |
February 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10361603 |
Feb 11, 2003 |
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09540761 |
Mar 31, 2000 |
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6518243 |
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60127516 |
Apr 2, 1999 |
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Current U.S.
Class: |
435/6.16 ;
514/3.1; 530/322 |
Current CPC
Class: |
C07K 9/008 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/008 ;
530/322 |
International
Class: |
A61K 038/14; C07K
009/00 |
Claims
What is claimed is:
1. A compound having the formula
A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A- .sub.6-A.sub.7 wherein
each of the groups A.sub.2 to A.sub.7 comprises a modified or
unmodified .alpha.-amino acid residue, A.sub.1 is optional and,
when present, comprises an organic group other than N-substituted
leucine, and at least one of the groups A.sub.1 to A.sub.7 is
linked via a glycosidic bond to one or more glycosidic groups each
having one or more sugar residues, wherein at least one of said
sugar residues is modified to bear at least one hydrophobic
substituent.
2. The compound of claim 1 wherein said glycosidic group is a
disaccharide modified to bear said at least one hydrophobic
substituent.
3. The compound of claim 1 wherein each of the groups A.sub.2,
A.sub.4, A.sub.5, A.sub.6 and A.sub.7 bears an aromatic side chain
and the aromatic side chains of groups A.sub.2 and A.sub.6 are
linked to the aromatic side chain of group A.sub.4 via ether
linkages and the aromatic side chains of groups A.sub.5 and A.sub.7
are linked to each other via a carbon-carbon bond.
4. The compound of claim 3 wherein the group A.sub.4 is linked to a
glycosidic group modified to bear said at least one hydrophobic
substituent.
5. The compound of claim 4 wherein said glycosidic group is a
disaccharide comprising a glucose residue directly bonded to group
A.sub.4 and a vancosamine residue bonded to said glucose
residue.
6. The compound of claim 5 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6- -A.sub.7 is as found in a
compound selected from the group consisting of vancomycin,
eremomycin, chloroeremomycin, and .beta.-avoparcin.
7. The compound of claim 6 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6- -A.sub.7 is as found in
vancomycin.
8. The compound of claim 7 wherein the C.sub.6 position of said
glucose residue is modified to bear at least one substituent other
than hydroxyl.
9. The compound of claim 8 wherein said at least one substituent
other than hydroxyl is a polar substituent.
10. The compound of claim 8 wherein said at least substituent other
than hydroxyl is a hydrophobic substituent.
11. The compound of claim 7 wherein the vancosamine residue in
vancomycin is N-substituted with said at least one hydrophobic
substituent.
12. The compound of claim 7 wherein said glucose residue is
modified to bear at least one substituent other than hydroxyl and
said vancosamine residue is N-substituted with said at least one
hydrophobic substituent.
13. The compound of claim 12 wherein said at least one substituent
other than hydroxyl is a polar substituent.
14. The compound of claim 1 wherein said at least one hydrophobic
substituent is R, OR, NR.sub.1R, SR, SO.sub.2R, C(O)OR, C(O)SR,
C(S)OR, C(S)SR, NR.sub.1C(O)R, C(O)NR.sub.1R, or halo and R is
alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic,
heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; R.sub.1
is hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any
pharmaceutically acceptable salts thereof; and if two or more of
said substituents are present, they can be the same or
different.
15. The compound of claim 1 wherein said organic group is selected
from the group consisting of a modified or unmodified alpha amino
acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl.
16. The compound of claim 1 wherein the group A.sub.7 bears a
terminal carboxyl, ester, thioester, amide, N-substituted amide, or
other carboxylic acid derivative.
17. A method for making a compound of the formula
A.sub.1-A.sub.2-A.sub.3-- A.sub.4-A.sub.5-A.sub.6-A.sub.7 wherein
each of the groups A.sub.2 to A.sub.7 comprises a modified or
unmodified a-amino acid residue, A.sub.1 comprises an organic group
other than N-substituted leucine, and at least one of the groups
A.sub.1 to A.sub.7 is linked via a glycosidic bond to one or more
glycosidic groups each having one or more sugar residues, wherein
at least one of said sugar residues is modified to bear at least
one hydrophobic substituent, said method comprising removing the
N-substituted leucine residue from the compound
N-substituted-leucyl-A.su-
b.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 thereby forming a
compound having a free amino group at A.sub.2; and attaching an
organic group A.sub.1 to the free amino group at A.sub.2.
18. The method of claim 17 wherein the N-substituted leucine
residue is N-methyl leucine.
19. The method of claim 17 wherein said glycosidic group is a
disaccharide modified to bear said at least one hydrophobic
substituent.
20. The method of claim 17 wherein each of the groups A.sub.2,
A.sub.4, A.sub.5, A.sub.6 and A.sub.7 bears an aromatic side chain
and the aromatic side chains of groups A.sub.2 and A.sub.6 are
linked to the aromatic side chain of group A.sub.4 via ether
linkages and the aromatic side chains of groups A.sub.5 and A.sub.7
are linked to each other via a carbon-carbon bond.
21. The method of claim 20 wherein the group A.sub.4 is linked to a
glycosidic group modified to bear said at least one hydrophobic
substituent.
22. The method of claim 21 wherein said glycosidic group is a
disaccharide comprising a glucose residue directly bonded to group
A.sub.4 and a vancosamine residue bonded to said glucose
residue.
23. The method of claim 22 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6- -A.sub.7 is as found in a
compound selected from the group consisting of vancomycin,
eremomycin, chloroeremomycin, and .beta.-avoparcin.
24. The method of claim 23 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6- -A.sub.7 is as found in
vancomycin.
25. The method of claim 24 wherein the C.sub.6 position of said
glucose residue is modified to bear at least one substituent other
than hydroxyl.
26. The method of claim 25 wherein said at least one substituent
other than hydroxyl is a polar substituent.
27. The method of claim 25 wherein said at least substituent other
than hydroxyl is a hydrophobic substituent.
28. The method of claim 24 wherein the vancosamine residue in
vancomycin is N-substituted with said least one hydrophobic
substituent.
29. The method of claim 24 wherein said glucose residue is modified
to bear at least one substituent other than hydroxyl and said
vancosamine residue is N-substituted with said least one
hydrophobic substituent.
30. The method of claim 29 wherein said substituent other than
hydroxyl is a polar substituent.
31. The method of claim 17 wherein said at least one hydrophobic
substituent is R, OR, NR.sub.1R, SR, SO.sub.2R, C(O)OR, C(O)SR,
C(S)OR, C(S)SR, NR.sub.1C(O)R, C(O)NR.sub.1R, or halo and R is
alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic,
heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; R.sub.1
is hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any
pharmaceutically acceptable salts thereof; and if two or more of
said substituents are present, they can be the same or
different.
32. The method of claim 17 wherein said organic group is selected
from the group consisting of a modified or unmodified alpha amino
acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl.
33. The method of claim 17 wherein the group A.sub.7 bears a
terminal carboxyl, ester, thioester, amide, N-substituted amide, or
other carboxylic acid derivative.
34. A method for making a glycopeptide antibiotic having the
formula A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7
wherein A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found
in vancomycin and AI comprises an organic group other than
N-substituted leucine, said method comprising modifying vancomycin
to form a modified vancomycin bearing a hydrophobic substituent at
the vancosamine nitrogen; removing the N-methyl leucine residue
from the modified vancomycin to form a des-N-methyl leucyl modified
vancomycin bearing a free amino group at A.sub.2; and attaching an
organic group A.sub.1 to the amino group at A.sub.2.
35. The method of claim 34 wherein said organic group is selected
from the group consisting of a modified or unmodified alpha amino
acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl.
36. A method for making a glycopeptide antibiotic having the
formula A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7
wherein A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found
in vancomycin and AI comprises an organic group other than
N-substituted leucine, said method comprising modifying vancomycin
to form a first modified vancomycin bearing a substituent other
than hydroxyl at the C.sub.6 position of the glucose attached to
A.sub.4 of vancomycin; modifying said first modified vancomycin to
form a second modified vancomycin bearing a hydrophobic substituent
at the vancosamine nitrogen; removing the N-methyl leucine residue
from said second modified vancomycin to form a des-N-methyl leucyl
second modified vancomycin bearing a free amino group at A.sub.2;
and, attaching an organic group A.sub.1 to the amino group at
A.sub.2.
37. The method of claim 36 wherein said substituent other than
hydroxyl is a polar substituent.
38. The method of claim 36 wherein said organic group is selected
from the group consisting of a modified or unmodified alpha amino
acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl.
39. A method for removing an N-terminal amino acid residue from an
oligopeptide or polypeptide comprising reacting an oligopeptide or
a polypeptide with phenylisothiocyanate in a
pyridine-water-triethylamine solvent medium.
40. The method of claim 39 wherein the reaction is carried out in a
10:10:1 pyridine-water-triethylamine solvent medium.
41. The method of claim 39 wherein the reaction is carried out at a
temperature in the range of from 40-70 C.
42. The method of claim 39 wherein the reaction is carried out for
period of time in the range of from 20-60 minutes.
43. The method of claim 39 wherein the N-terminal amino acid
residue is N-methyl leucine.
44. The method of claim 43 wherein the oligopeptide is selected
from the group consisting of a glycopeptide antibiotic, a
pseudoaglycone and an aglycone.
45. The method of claim 43 wherein the oligopeptide is a
glycopeptide antibiotic or pseudoaglycone in which at least one
glycosidic groups therein is modified to bear at least one
hydrophobic substituent.
46. The method of claim 45 wherein the glycopeptide antibiotic is
vancomycin.
47. The method of claim 46 wherein the disaccharide at A.sub.4 of
vancomycin is modified to bear at least one hydrophobic group.
48. The method of claim 47 wherein the vancosamine nitrogen at
A.sub.4 of vancomycin is modified to bear at least one hydrophobic
group.
49. The method of claim 47 wherein the glucose residue attached
directly to A.sub.4 of vancomycin is modified to bear at least one
substituent other than hydroxyl.
50. The method of claim 49 wherein said at least one hydrophobic
substituent other than hydroxyl is a polar substituent or a
hydrophobic substituent.
51. The method of claim 50 wherein the C.sub.6 position of said
glucose residue attached directly to A.sub.4 of vancomycin is
modified to bear a polar or a hydrophobic substituent.
52. A method of treating an infectious disease in a host comprising
administering to said host an effective amount of a compound of
claim 1 or a pharmaceutically acceptable salt or ester thereof.
53. The method of claim 52 wherein the host is a mammal.
54. The method of claim 53 where the mammal is a human.
55. The method of claim 52 wherein the infectious disease is a
bacterial infection.
56. The method of claim 52 further comprising administering to said
host an additional drug or therapeutic agent in combination with a
compound of claim 1 or a pharmaceutically acceptable salt or ester
thereof.
57. A composition comprising a compound of claim 1 or a
pharmaceutically acceptable salt or ester thereof and a
pharmaceutically acceptable carrier or excipient.
58. The composition of claim 57 further comprising an additional
drug or therapeutic agent.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to glycopeptide compounds
having antibiotic activity, and methods of making glycopeptide
compounds having antibiotic activity.
[0003] 2. Background of the Invention
[0004] Glycopeptide antibiotics are characterized by having at
least one saccharide group chemically bonded to a rigid peptide
structure having a cavity or cleft which acts as a binding site for
the substrate used in bacterial cell wall synthesis. The
glycopeptide antibiotics are further categorized into various
subclasses depending on the identity and interconnections of the
amino acids comprising the peptide backbone and the number and
substitution pattern of the sugar residues in the molecule. The
glycopeptide antibiotics are generally active against Gram-positive
bacteria but relatively ineffective against Gram-negative bacteria.
Most notable among the glycopeptide antibiotics is vancomycin.
Vancomycin is produced by Amycolatopsis orientalis, and is often
referred to as "the drug of last resort" because it is effective
against most multi-drug-resistant gram positive bacteria. However,
in recent years, vancomycin-resistant strains of some bacteria have
emerged.
[0005] The structural formula of vancomycin is shown below and is
characterized by a disaccharide moiety covalently linked to a
heptapeptide structure. The structure of vancomycin places it in a
class of molecules referred to as the "dalbaheptides." [Malabarba
A., et al. (1997a)]. Dalbaheptides in general are characterized by
the presence of seven amino acids linked together by peptide bonds
and held in a rigid conformation by cross-links through the
aromatic substituent groups of at least five of the amino acid
residues. In the heptapeptide structure of vancomycin, which is
commonly referred to as the "aglycone" of vancomycin, the aromatic
side-chains of amino acids 2, 4, and 6 are fused together through
ether linkages. The aromatic side-chains of amino acids 5 and 7 are
joined via a carbon-carbon bond. Amino acids 1 and 3 are N-methyl
leucine and asparagine, respectively. Other naturally-occurring
glycopeptide antibiotics are similar to vancomycin in that they
have the same amino acids 1 through 7 forming the peptide binding
pocket and a glucose residue linked to the aromatic substituent on
amino acid 4 through formation of a bond with a phenolic hydroxyl
group. The glucose residue, in turn, is linked through its vicinal
hydroxyl position to a unique amino sugar, L-vancosamine. Some
glycopeptide antibiotics similar to vancomycin contain additional
glycosidic groups attached to other positions on the peptide (e.g.
chloroeremomycin). Still other glycopeptide antibiotics such as
.beta.-avoparcin are similar to vancomycin in that they contain the
same amino acids at all positions except positions one and three.
.beta.-avoparcin, for example, contains an amino acid containing an
aromatic side chain in place of the asparagine at position three
and does not contain N-methyl leucine at position one.
.beta.-avoparcin contains glycosidic groups at amino acid 4 and at
other positions on the peptide core.
[0006] Vancomycin, chloroeremomycin and .beta.-avoparcin have the
structures as shown below: 1
[0007] Eremomycin has the structure of chloroeremomycin except that
the chlorine substituent on the aromatic group attached to amino
acid 6 is not present in eremomycin.
[0008] The anti-microbial activity of the naturally occurring
glycopeptide antibiotics is believed to be due to their ability to
interfere with biosynthesis of the bacterial cell wall, evidently
by binding to dipeptide termini of uncross-linked peptidoglycan
and/or the disaccharide precursor of peptidoglycan. [Nagarajan R.
(1993)]. NMR evidence has shown that the heptapeptide chain of
vancomycin forms a number of hydrogen bonds with
D-alanyl-D-alanine, the dipeptide that is at the terminus of the
peptide chain attached to the N-acetylmuramic acid unit that is
incorporated into peptidoglycan. [See, e.g., Prowse W., et al.
(1995); Pierce C., et al. (1995); Williams D. et al. (1998)]. The
interaction of vancomycin with peptidoglycan precursors apparently
inhibits or prevents the subsequent transglycosylation and/or
transpeptidation steps of cell wall assembly. Supporting this mode
of action is the fact that vancomycin-resistant strains of bacteria
are found to produce a pentapeptide precursor terminating in a
D-alanyl-D-lactate sequence. It is hypothesized that the reduced
effectiveness of vancomycin against resistant strains is due to
reduced hydrogen bonding interactions between the drug and the
D-alanyl-D-lactate substrate (and possibly repulsive interactions
as well). The affinity of vancomycin for D-alanyl-D-lactate is
estimated to be 2-3 orders of magnitude (4.1 kcal/mol) less than
for D-alanyl-D-alanine. [Walsh C (1993)].
[0009] The sugar residues of vancomycin and other glycopeptide
antibiotics have been shown to affect biological activities.
Structural changes in the sugar residues can produce significant
changes in antibiotic activity. [Malabarba (1997); Nagarajan, R.
(1993)]. It has been proposed that the sugar residues on the
glycopeptide antibiotics may enhance the avidity of these molecules
for surface-bound peptide ligands. At least two different
mechanisms for enhancing avidity have been proposed. [Kannan
(1998); Gerhard (1993); Allen (1997)].
[0010] For example, it has been proposed that the biological
activity of vancomycin, along with that of many other glycopeptide
antibiotics, is enhanced by dimerization [Williams D., et al.
(1993); Gerhard, U., et al., (1993)] facilitated by the saccharide
groups on the convex surface of the molecules. Structural evidence
for dimerization of several different glycopeptides has been
obtained from both NMR and crystallographic studies. It has been
found that there are significant differences in the stability of
the dimers formed in solution by different glycopeptide
antibiotics. [MacKay (1994)]. Dimerization is thought to influence
activity by increasing the avidity of the glycopeptides for
surface-bound D-ala-D-ala peptide ligands [Williams, (1998)]. It is
proposed that the differences in the dimerization constants, due to
different interactions between saccharide groups, may account at
least partially for the differences in biological activity of
different glycopeptide antibiotics which otherwise have very
similar peptide binding pockets and also have similar affinities
for the natural D-ala-D-ala substrate. [Williams (1998)].
[0011] A second mechanism for enhancing activity has been proposed
for the naturally occurring glycopeptide antibiotic teicoplanin and
various semi-synthetic glycopeptides containing hydrophobic
substituents on at least one of the saccharide units. It is
suggested that hydrophobic substituents (a C2 N-acyl group in the
case of teicoplanin) interact with the bacterial membrane, thus
"anchoring" hydrophobically substituted glycopeptides at the
membrane surface. [Beauregard (1995)]. Membrane anchoring is
proposed to enhance activity by localizing the glycopeptide
antibiotic to the membrane where the Lipid II substrates that are
the precursors of peptidoglycan are found. The glycopeptide
antibiotics then bind to the dipeptide termini of these precursors
and prevent transglycosylation and/or transpeptidation.
[0012] It should be noted that teicoplanin is active against some
vancomycin resistant strains. Furthermore, the attachment of
hydrophobic substituents to the vancomycin carbohydrate moiety
confers activity against these and other vancomycin-resistant
bacterial strains. [Nagarajan (1991)]. It has been speculated that
the lipophilic groups on the saccharides, in locating the
antibiotic at the cell surface, help overcome the decreased binding
affinity for D-ala-D-lac in vancomycin resistant
microorganisms.
[0013] It has generally been assumed that peptide binding is
essential for biological activity. In fact, it had been shown that
if the peptide core of vancomycin is damaged by removing the
N-methyl leucine (amino acid 1), the resulting compound loses
affinity for D-ala-D-ala and has no biological activity, even
against sensitive bacterial strains. The lack of biological
activity is presumed to be due to the inability of the compound to
bind D-Ala-D-Ala well.
[0014] Previously, others have explored the possibility of
attaching amino acids other than N-methyl leucine to the amino acid
2 on des-N-methyl leucyl vancomycin. It was found that some amino
acid substitutions produced compounds with comparable activity to
vancomycin; some had worse activity. No useful improvements in
activity have been reported. As far as we know, no substitutions
have ever been made at the A.sub.1 position of any dalbaheptides
wherein A.sub.1 and A.sub.3 are not directly linked by a covalent
bond and wherein there is at least one hydrophobic substituent on
at least one of the sugar moieties attached to at least one of the
amino acids A.sub.2-A.sub.7 Thus, replacing N-methyl leucine at
A.sub.1 on vancomycin with other amino acids did not yield any
compounds having significantly better properties than vancomycin
itself. Hence, it would not be expected that glycopeptides having
at least one hydrophobic substituent attached to a glycosidic group
on any one of amino acids A.sub.2-A.sub.7 and having no A.sub.1
group or an A.sub.1 group other than an N-substituted leucine would
have any antibiotic properties, much less better antibiotic
properties than the precursor glycopeptide compounds.
[0015] Definitions
[0016] A "glycoconjugate" comprises any molecule linked to at least
one carbohydrate of any size. The molecule can be a peptide or
protein, a nucleic acid, a small molecule, a lipid, or another
carbohydrate; it may be of natural or non-natural origin.
[0017] A "glycopeptide" is a glycoconjugate comprising a peptide
linked to at least one carbohydrate.
[0018] A "glycopeptide antibiotic" is a glycopeptide having
antibacterial activity, including, e.g., vancomycin, eremomycin,
chloroeremomycin and .beta.-avoparcin as well as any synthetic and
semi-synthetic derivatives thereof. The term "glycopeptide
antibiotic" is meant to encompass any naturally occurring
antibiotic as well semi-synthetic derivatives thereof.
[0019] An "aglycone" is the result of removing the carbohydrate
residues from a glycopeptide, leaving only a peptide core.
[0020] A "des-N-methyl leucyl aglycone" is the result of removing a
terminal N-methyl leucine residue from an aglycone.
[0021] A "pseudoaglycone" is the result of removing only one of two
sugar residues from a disaccharide residue linked to amino acid
residue A.sub.4 of a glycopeptide. Thus, a pseudoaglycone comprises
an aglycone in which A.sub.4 is linked to a monosaccharide
residue.
[0022] A "des-N-methyl leucyl pseudoaglycone" is the result of
removing a terminal N-methyl leucine residue from an
pseudoaglycone. Thus, a des-N-methyl-leucyl pseudoaglycone is an
aglycone in which A.sub.4 is linked to a monosaccharide residue and
which has a terminal N-methyl leucine residue removed
therefrom.
[0023] A "dalbaheptide" is a glycopeptide containing a heptapeptide
moiety which is held in a rigid conformation by cross-links between
the aromatic substituent groups of at least five of the seven
.alpha.-amino acid residues, including a cross-link comprising a
direct carbon-carbon bond between the aryl substituents of amino
acid residues 5 and 7, and aryl ether cross-links between the
substituents of amino acid residues 2 and 4, and 4 and 6. Amino
acid residues 2 and 4-7 in different dalbaheptides are those found
in the naturally occurring glycopeptide antibiotics. These amino
acid residues differ only in that residues 2 and 6 do not always
have a chlorine substituent on their aromatic rings, and in that
substitution on free hydroxyl or amino groups may be present. Amino
acids residues 1 and 3 may differ substantially in different
dalbaheptides; if both bear aryl substituents, these may be
cross-linked. Molecules having a dalbaheptide structure include,
e.g., the glycopeptide antibiotics mentioned above.
[0024] The term "alkyl" refers to a linear or branched acyclic or
non-aromatic cyclic group having form one to twenty carbon atoms
connected by single or multiple bonds. Thus, the term "alkyl" is
meant to encompass linear or branched acyclic or non-aromatic
groups having one or more carbon-carbon double or triple bonds,
i.e., alkenyl and alkynyl groups. An alkyl group may be substituted
by one or more of halo, hydroxyl, protected hydroxyl, amino,
protected amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, COOH,
aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio,
arylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl,
arylsulfonyl, aroyl, aralkanoyl, heterocyclic, CONH.sub.2,
CONH-alkyl, CON(alkyl).sub.2, COO-aralkyl, COO-aryl or
COO-alkyl.
[0025] The term "aryl" refers to a group derived from a
non-heterocyclic aromatic compound having from six to twenty carbon
atoms and from one to four rings which may be fused or connected by
single bonds. An aryl group may be substituted by one or more of
alkyl, aralkyl, heterocyclic, heterocyclic-alkyl,
heterocyclic-carbonyl, halo, hydroxyl, protected hydroxyl, amino,
protected amino, hydrazino, alkylhydrazino, nitro, cyano, alkoxy,
aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino,
trialkylammonium, alkylthio, arylthio, alkanoyl, alkanoyloxy,
alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl,
COO-alkyl, COO-aralkyl, COO-aryl, CONH.sub.2, CONH-alkyl or
CON(alkyl).sub.2.
[0026] The term "aralkyl" refers to an alkyl group substituted by
an aryl group. Aralkyl may optionally be substituted with one or
more of the groups with which alkyl or aryl may be substituted.
[0027] The term "heterocyclic" refers to a group derived from a
heterocyclic compound having from one to four rings, which may be
fused or connected by single bonds; said compound having from three
to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur
or phosphorus. A heterocyclic group may be substituted by one or
more alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl,
amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano,
alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino,
trialkylamino, alkylthio, arylthio, alkanoyl, alkanoyloxy,
alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl,
COO-alkyl, COO-aralkyl, COO-aryl, CONH.sub.2, CONH-alkyl or
CON(alkyl).sub.2.
[0028] The terms "alkoxy," "aryloxy," and "aralkyloxy," refer to
groups derived from bonding an oxygen atom to an alkyl, aryl or
aralkyl group, respectively. Any alkoxy, aryloxy or aralkyloxy
group may optionally be substituted with one or more of the groups
with which alkyl, aryl or aralkyl may be substituted. The terms
"alkanoyl," "aroyl," and "aralkanoyl" refer to groups derived from
bonding a carbonyl to an alkyl, aryl or aralkyl group,
respectively. Any alkanoyl, aroyl or aralkanoyl group may
optionally be substituted with one or more of the groups with which
alkyl, aryl or aralkyl may be substituted. The terms
"heterocyclic-alkyl" and "heterocyclic-carbonyl" refer to groups
derived from bonding a heterocyclic group to an alkyl or a carbonyl
group, respectively. An heterocyclic-alkyl or heterocyclic-carbonyl
group may optionally be substituted with one or more of the groups
with which heterocyclic or alkyl may be substituted. The term
"heterocyclic-alkyl-carbonyl" refers to a group derived from
bonding a heterocyclic-alkyl group to a carbonyl group. Any
heterocyclic-alkyl-carb- onyl may optionally be substituted with
one or more of the groups with which heterocyclic or alkyl may be
substituted. The term "alkylsulfonyl" refers to a group derived
from bonding an alkyl group to a sulfonyl group. An alkylsulfonyl
group may optionally be substituted with one or more groups with
which alkyl may be substituted. The term "arylsulfonyl" refers to a
group derived from bonding an aryl group to a sulfonyl group. An
arylsulfonyl group may optionally be substituted with one or more
groups with which aryl may be substituted. The term "protected
hydroxyl" refers to a hydroxyl group bonded to a group which is
easily removed to generate the free hydroxyl group by treatment
with acid or base, by reduction or by exposure to light, or by any
other conventional means for removing a protecting group from a
hydroxyl group. The term "protected amino" refers to an amino group
bonded to a group which is easily removed to generate the free
amino group by treatment with acid or base, by reduction or
exposure to light, or by any other conventional means for removing
a protecting group from an amino group.
[0029] A "chemical library" is a synthesized set of compounds
having different structures. The chemical library may be screened
for biological activity to identify individual active compounds of
interest.
[0030] The term "DMF" refers to N,N-dimethylformamide; "THF" refers
to tetrahydrofuran; "TFA" refers to trifluoroacetic acid; "EtOAc"
refers to ethyl acetate; "MeOH" refers to methanol, "MeCN" refers
to acetonitrile; "Tf" refers to the trifluoroacetyl group; "DMSO"
refers to dimethyl sulfoxide; "DIEA" refers to
diisopropylethylamine; "All" in structural formulas refers to the
allyl group; "Fmoc" refers to 9-fluorenylmethyloxycarbonyl; "HOBt"
refers to 1-hydroxybenzotriazole and "Obt" to the
1-oxybenzotriazolyl group; "PyBOP" refers to
benzotriazol-1-yl-oxyatripyrrolidine-phosphonium
hexafluorophosphate; "Su" refers to the succinimidyl group; "HBTU"
refers to O-benzoatriazol-1-yl-N2N3N',N'-tetramethyluronium
hexafluorophosphate; "alloc" refers to allyloxycarbonyl; and "CBZ"
refers to benzyloxycarbonyloxy.
[0031] The term "hydrophobic" as used herein to describe a compound
of the present invention or a substituent thereon, refers to the
tendency of the compound or substituent thereon to lack an affinity
for, to repel or to fail to absorb water, or to be immiscible in
water. The term "hydrophobic" is not mean to exclude compounds or
substituents thereon that are not completely immiscible in
water.
[0032] The term "polar" as used herein to describe a compound of
the present invention or a substituent thereon, refers to the
tendency of the compound or substituent thereon to have an affinity
for, to attract or to absorb water, or to be miscible in water. The
term "polar" is not meant to exclude compounds or substituents
thereon that are not completely miscible in water.
SUMMARY OF THE INVENTION
[0033] Thus, the present invention is directed to compounds of the
formula A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7
wherein each of the groups A.sub.2 to A.sub.7 comprises a modified
or unmodified .alpha.-amino acid residue, A.sub.1 is optional and,
when present, comprises an organic group other than N-substituted
leucine, and at least one of the groups A.sub.2 to A.sub.7 is
linked via a glycosidic bond to one or more glycosidic groups each
having one or more sugar residues, wherein at least one of said
sugar residues is modified to bear at least one hydrophobic
substituent. In preferred compounds of the present invention, the
glycosidic group is a disaccharide modified to bear at least one
hydrophobic substituent. In a preferred embodiment of the present
invention, each of the groups A.sub.2, A.sub.4, A.sub.5, A.sub.6
and A.sub.7 bears an aromatic side chain and the aromatic side
chains of groups A.sub.2 and A.sub.6 are linked to the aromatic
side chain of group A.sub.4 via ether linkages and the aromatic
side chains of groups A.sub.5 and A.sub.7 are linked to each other
via a carbon-carbon bond. In another preferred embodiment of the
invention, the group A.sub.4 bears a glycosidic group. The
glycosidic group is preferably is a disaccharide comprising a
glucose residue directly bonded to group A.sub.4 and a vancosamine
residue bonded to said glucose residue. In preferred compounds of
the present invention, A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub-
.6-A.sub.7 is as found in a compound selected from the group
consisting of vancomycin, eremomycin, chloroeremomycin, and
.beta.-avoparcin, more preferably
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found in
vancomycin. In other preferred compounds of the present invention,
the C.sub.6 position of the glucose residue attached to A.sub.4 is
modified to bear at least one substituent other than hydroxyl, and
more preferably the at least one substituent other than hydroxyl is
a polar substituent or a hydrophobic substituent. In preferred
compounds of the present invention where
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found in
vancomycin, the vancosamine residue in vancomycin is N-substituted
with said at least one hydrophobic substituent. In other preferred
compounds of the present invention wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found in
vancomycin, the glucose residue attached to A.sub.4 of vancomycin
is modified at the C.sub.6 position to bear at least one
substituent other than hydroxyl, preferably a polar substituent,
and said vancosamine residue is N-substituted with said at least
one hydrophobic substituent. The at least one hydrophobic
substituent is preferably selected from R, OR, NR.sub.1R, SR,
SO.sub.2R, C(O)OR, C(O)SR, C(S)OR, C(S)SR, NR.sub.1C(O)R,
C(O)NR.sub.1R, or halo and R is alkyl, aryl, aralkyl, alkanoyl,
aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl,
heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or
arylsulfonyl; R.sub.1 is hydrogen, alkyl, aryl, aralkyl, alkanoyl,
aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl,
heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or
arylsulfonyl; and any pharmaceutically acceptable salts thereof;
and if two or more of said substituents are present, they can be
the same or different. The organic group A.sub.1 in the preferred
compounds of the present invention, is preferably an organic group
selected from the group consisting of a modified or unmodified
alpha amino acid residue other than N-substituted leucine, alkyl,
aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic,
heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl. Also, in the preferred compounds
of the present invention, the A.sub.7 group bears a terminal
carboxyl, ester, thioester, amide, N-substituted amide, or other
carboxylic acid derivative, any of which groups may be substituted
with any of the substituents described herein.
[0034] In another aspect, the present invention is also directed to
a method for making compounds of the formula
A.sub.1-A.sub.2-A.sub.3-A.sub.- 4-A.sub.5-A.sub.6-A.sub.7 wherein
each of the groups A.sub.2 to A.sub.7 comprises a modified or
unmodified .alpha.-amino acid residue, A.sub.1 comprises an organic
group other than N-substituted leucine, and at least one of the
groups A.sub.1 to A.sub.7 is linked via a glycosidic bond to one or
more glycosidic groups each having one or more sugar residues,
wherein at least one of said sugar residues is modified to bear at
least one hydrophobic substituent comprising the steps of removing
the N-substituted leucine residue from the compound
N-substituted-leucyl-A.su-
b.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 thereby forming a
compound having a free amino group at A.sub.2; and attaching an
organic group A.sub.1 to the free amino group at A.sub.2, wherein
the hydrophobic substituent and the groups A.sub.1-A.sub.7 are as
described above. Preferably, the N-substituted-leucine residue is
N-methyl leucine. This method is applicable to any of the preferred
compounds as described above.
[0035] In another aspect, the present invention is directed to a
method for making a glycopeptide antibiotic having the formula
A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found in
vancomycin and A.sub.1 comprises an organic group other than
N-substituted leucine, said method comprising modifying vancomycin
to form a modified vancomycin bearing a hydrophobic substituent at
the vancosamine nitrogen; removing the N-methyl leucine residue
from the modified vancomycin to form a des-N-methyl leucyl modified
vancomycin bearing a free amino group at A.sub.2 and attaching an
organic group A.sub.1 to the amino group at A.sub.2, wherein the
hydrophobic substituent and the organic group A.sub.1 are as
defined above.
[0036] In another aspect, the present invention is directed to a
method for making a glycopeptide antibiotic having the formula
A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 wherein
A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 is as found in
vancomycin and A.sub.1 comprises an organic group other than
N-substituted leucine, said method comprising modifying vancomycin
to form a first modified vancomycin bearing a substituent other
than hydroxyl at the C.sub.6 position of the glucose attached to
A.sub.4 of vancomycin; modifying said first modified vancomycin to
form a second modified vancomycin bearing a hydrophobic substituent
at the vancosamine nitrogen; removing the N-methyl leucine residue
from said second modified vancomycin to form a des-N-methyl leucyl
second modified vancomycin bearing a free amino group at A.sub.2;
and, attaching an organic group A.sub.1 to the amino group at
A.sub.2, wherein the hydrophobic substituent and the organic group
A.sub.1 are as described above. It is preferred in this method that
the substituent other than hydroxyl at the C.sub.6 position of the
glucose attached to A.sub.4 of vancomycin is a polar
substituent.
[0037] The present invention is also directed to a method of
treating an infectious disease in a host comprising administering
to said host an effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt or ester thereof.
Preferably the host is a mammalian host, more preferably a human.
The infectious disease is preferably a bacterial infection. The
present invention is also directed to a composition comprising a
compound of the present invention or a pharmaceutically acceptable
salt or ester thereof and a pharmaceutically acceptable carrier or
excipient. The compound of the present invention may be
administered solely or in combination with any other drug or
therapeutic agent.
[0038] The present invention is also directed to a method for
removing an N-terminal amino acid residue from an oligopeptide or
polypeptide comprising reacting an oligopeptide or a polypeptide
with phenylisothiocyanate in a pyridine-water-triethylamine solvent
medium. Preferably, the reaction of the oligopeptide or polypeptide
with phenylisothiocyanate is carried out in a 10:10:1 (volume)
pyridine-water-triethylamine solvent medium. The reaction is
preferably carried out at a temperature in the range of from
40-70.degree. C. and for a period of time in the range of from
20-60 minutes. In preferred methods, the N-terminal amino acid
residue is N-methyl leucine. The preferred oligopeptides are
selected from the group consisting of a glycopeptide antibiotic, a
pseudoaglycone and an aglycone, preferably a glycopeptide
antibiotic or pseudoaglycone in which at least one of the
glycosidic groups therein is modified to bear at least one
hydrophobic substituent. Preferably, the glycopeptide antibiotic is
vancomycin. More preferably, the disaccharide at A.sub.4 of
vancomycin is modified to bear at least one hydrophobic group.
Preferably, the vancosamine nitrogen at A.sub.4 of vancomycin is
modified to bear at least one hydrophobic group. In preferred
methods, the glucose residue attached directly to A.sub.4 of
vancomycin is modified to bear at least one substituent other than
hydroxyl, which is preferably a polar substituent or a hydrophobic
substituent. Where the glucose residue is modified to bear at least
one substituent other than hydroxyl, it is preferred that the
C.sub.6 position of the glucose residue attached directly to
A.sub.4 of vancomycin is modified to bear a polar or a hydrophobic
substituent.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Herein we disclose strategies for finding promising
glycopeptide compounds with good activity against sensitive and
resistant bacterial strains. One strategy involves attaching
substituents to the free amino group of amino acid 2 in
des-N-substituted-leucine glycopeptides and analogs thereof
containing at least one hydrophobic substituent on a glycosidic
group attached to one of the amino acids A.sub.2-A.sub.7. We have
also discovered that where at least one of the glycosidic groups
attached to one of the amino acids A.sub.2-A.sub.7 bears a
hydrophobic substituent, it is not necessary to attach a group to
the free amino group of amino acid A.sub.2 upon removal of the
N-substituted leucine residue in order to produce compounds having
biological activity. Thus, we have found good activity in
des-N-substituted leucine glycopeptide compounds in which at least
one of the glycosidic groups attached to one of the amino acids
A.sub.2-A.sub.7 bears a hydrophobic substituent and in which
A.sub.2 bears a free amino group upon removal of the N-substituted
leucine residue therefrom. We have demonstrated the utility of the
strategy by making a set of compounds, of which several have better
activity against a range of strains than the corresponding
compounds in which A.sub.1 is N-substituted leucine, which is
preferably N-methyl leucine. Some of the substitutions improve
activity against both sensitive and resistant strains relative to
N-methyl leucine; others improve activity more against sensitive
strains than resistant strains; still other improve activity more
against resistant strains than sensitive strains. Thus, it is
possible to manipulate the biological activity in different ways by
choosing appropriate A.sub.1 substituents. It is also possible to
manipulate the biological activity by choosing appropriate
hydrophobic substituents. We also show that the physical properties
of the compounds--e.g., the hydrophobic-hydrophilic balance as
measured by HPLC retention times--are related to both the
hydrophobic substituent, and when present, the specific group
A.sub.1. Having shown that both the physical properties and the
biological activities of glycopeptide derivatives containing
hydrophobic substituents on the sugars are affected by the identity
of the hydrophobic substituent and, when present, the group
A.sub.1, the present invention is thus directed to all compounds of
the general structure A.sub.1-A.sub.2-A.sub.3-A.sub.4-
-A.sub.5-A.sub.6-A.sub.7 wherein the group A.sub.1 is optional and,
when present, is preferably an organic group having from 2-30
carbon atoms, which may contain heteroatoms. The organic group
A.sub.1 may contain more than 30 carbon atoms. The organic group
A.sub.1, when present, is attached to the amino group at A.sub.2.
The organic group A.sub.1 may be linear, branched or cyclic, or
some combination thereof, and may include aliphatic, aromatic,
and/or heterocyclic groups, provided that it is not a leucine or a
modified leucine residue, and provided that it is not directly or
indirectly linked by a covalent bond to amino acid 3. Preferably
the organic group is a modified or unmodified alpha amino acid
residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl,
guanidinyl, carbamoyl, or xanthyl.
[0040] When present, preferred A.sub.1 groups are provided by the
compounds listed in Table I following Example 3. The structural
formulae of these preferred A.sub.1 groups are also provided in
Table I. These are merely preferred A.sub.1 groups, and the present
invention is not to be construed as being limited thereto. In
general, A.sub.1 can be any organic group that can be reacted with
the free amino group at A.sub.2 by, for example, formation of an
amide linkage. Thus, preferred reagents which can be reacted with
the free amino group at A.sub.2 include compounds having a
carboxylic acid group which reacts with the free amino group at
A.sub.2 to form the amide linkage. Such preferred compounds include
those disclosed in Table I.
[0041] In preferred compounds of the present invention, each of the
groups A.sub.2 to A.sub.7 comprises a modified of unmodified
.alpha.-amino acid residue, whereby (i) the group A.sub.1,when
present, is linked to an amino group on the group A.sub.2, (ii)
each of the groups A.sub.2, A.sub.4 and A.sub.6 bears an aromatic
side chain, which aromatic side chains are cross-linked together by
two or more covalent bonds, and (iii) the group A.sub.7 bears a
terminal carboxyl, ester, thioester, amide, N-substituted amide, or
other derivative of a carboxylic acid.
[0042] In the compounds of the present invention, one or more of
the groups A.sub.2 to A.sub.7 is linked via a glycosidic bond to
one or more sugar resides; wherein at least one of said sugar
resides bears at least one hydrophobic substituent wherein the
hydrophobic substituent is preferably selected from R, OR,
NR.sub.1R, SR, SO.sub.2R, C(O)OR, C(O)SR, C(S)OR, C(S)SR,
NR.sub.1C(O)R, C(O)NR.sub.1R, or halo and R is alkyl, aryl,
aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic,
heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; R.sub.1
is hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl,
heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl,
heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any
pharmaceutically acceptable salts thereof; and if two or more of
said substituents are present, they can be the same or
different.
[0043] Modified amino acid residues include amino acid residues
whose aromatic groups have been substituted by halo, alkyl, alkoxy,
alkanoyl, or other groups easily introduced by electrophilic
substitution reactions or by reaction of phenolic hydroxyl groups
with alkylating or acylating agents; and amino acid residues which
have protecting groups or other easily introduced substituents on
their hydroxyl or amino groups including, but not limited to alkyl,
alkanoyl, aroyl, aralkyl, aralkanoyl, carbamoyl, allyloxycarbonyl,
aralkyloxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl,
heterocyclic, heterocyclic-alkyl or heterocyclic-carbonyl
substituents. Examples of preferred protecting groups include
acetyl, allyloxycarbonyl (aloc), CBZ, allyl, benzyl,
p-methoxybenzyl and methyl. Modifications of hydroxyl groups occur
on phenolic hydroxyl groups, benzylic hydroxyl groups, or aliphatic
hydroxyl groups. Other amino acid residues, in addition to A.sub.2,
A.sub.4 and A.sub.6 may be cross-linked through their aromatic acid
substituent groups.
[0044] In the preferred compounds of the present invention, the
residues A.sub.2 to A.sub.7 of the glycopeptides are linked
sequentially by peptide bonds and are cross-linked as in a
dalbaheptide. The preferred glycopeptides have a peptide core in
which the residues are linked as in the glycopeptide antibiotics
vancomycin, eremomycin, chloroeremomycin or .beta.-avoparcin. In
particularly preferred compounds of the present invention, the
structures and interconnections of A.sub.2 to A.sub.7 are those of
vancomycin, i.e., those having the heptapeptide core of vancomycin
with the N-methyl leucine residue removed, subject to the amino
acid modifications and substitutions described herein above.
[0045] The glycopeptide compounds of the present invention contain
at least one glycosidic group attached through a glycosidic bond to
at least one of the amino acid residues A.sub.2 to A.sub.7. In the
preferred compounds of the present invention, a glycosidic group is
linked to residue A.sub.4. This glycosidic group comprises at least
a monosaccharide bearing at least one hydrophobic substituent.
Preferably, the glycosidic group is a disaccharide residue bearing
at least one hydrophobic substituent which disaccharide residue can
be linked to any of the amino acid residues A.sub.2-A.sub.7,
preferably to the amino acid residue A.sub.4. In the particularly
preferred compounds of the present invention, the glycosidic group
attached to A.sub.4 is a disaccharide consisting of a glucose
residue directly attached to the amino acid A.sub.4 and an
N-substituted vancosamine residue attached to the glucose residue.
Preferably, the vancosamine residue is N-substituted with the at
least one hydrophobic substituent. Examples of preferred
hydrophobic substituents which are preferably present as
N-substituents on the vancosamine residues are shown below: 2
[0046] Thus, preferred N-substituents at the vancosamine nitrogen
include, e.g., straight or branched alkyl, aralkyl, alkanoyl,
aralkanoyl and aroyl. Any of these N-substituents may be
substituted with one or more of alkyl, preferably C.sub.4-C.sub.8
alkyl, halo, haloalkyl, aryl, aralkyl, aryloxy, aralkyloxy,
alkaryloxy, alkoxy, preferably C.sub.4-C.sub.8 alkoxy, and
haloalkoxy. Preferred alkoxy substituents on the N-substituent
include, e.g., O-butyl and O-octyl. Where the N-substituent is
alkyl, it is preferred that alkyl has from 8 to 15 carbon atoms.
Specific preferred N-subsitutents include, but are not limited to
the following:
[0047] 2-naphthylmethyl
[0048] 4-phenylbenzyl
[0049] 1-naphthylmethyl
[0050] 4-phenoxybenzyl;
[0051] 4-benzyloxybenzyl
[0052] 4-trifluoromethoxybenzyl
[0053] 4-allyloxybenzyl
[0054] 4-nonyloxybenzyl;
[0055] 2-methoxy-1-naphthylmethyl
[0056] 4-dodecyloxybenzyl
[0057] 9-phenanthranylmethyl
[0058] 4-decyloxybenzyl
[0059] 9-anthranylmethyl
[0060] 4-[phenylethynyl]4-phenylbenzyl
[0061] 4-methoxy-1-naphthylmethyl
[0062] 1-pyrenylmethyl
[0063] 9-[10-methyl]anthranylmethyl
[0064] 9-[10-chloro]anthranylmethyl
[0065] 2-benzthienylmethyl
[0066] 4-[4-hydroxyphenyl]benzyl
[0067] 4-[4-octylphenyl]benzyl
[0068] 4-[4-pentylphenyl]benzyl
[0069] 4-[4-octyloxyphenyl]benzyl
[0070] 3-pyridylmethyl
[0071] 5-nitro-1-naphthylmethyl
[0072] 4-pyridylmethyl
[0073] 4-quinolylmethyl
[0074] 3-quinolylmethyl
[0075] 4-stilbenzyl
[0076] 2-quinolylmethyl
[0077] 2-pyridylmethyl
[0078] 2-fluorenylmethyl
[0079] 4-phenoxyphenethyl
[0080] 4-[4-pentylcyclohexyl]benzyl
[0081] 4-benzylphenethyl
[0082] 4-[4-biphenyl]benzyl
[0083] 4-trifluoromethylbenzyl
[0084] trans-cinnamyl
[0085] 4-[1-oxa]fluorenylmethyl
[0086] 4-[4-pentoxyphenyl]benzyl
[0087] 4-thiomethylbenzyl
[0088] 2,3-[2-methyl-3.fwdarw.4-t-butylphenyl]]propenyl
[0089] 9-(1-methyl)-acridinylmethyl
[0090] 2-hydroxy-1-naphthylmethyl
[0091] 4-[2-phenyl-6-methoxy]quinoylmethyl
[0092] 4-diphenylmethylbenzyl
[0093] 3,4 cyclohexenylmethyl
[0094] 3,4-methylenedioxylbenzyl
[0095] 3-phenoxybenzyl
[0096] 4-benzylbenzyl
[0097] 3-benzyloxy-6-methoxy benzyl
[0098] 4-benzyloxy-3-methoxybenzyl
[0099] 3,4-dibenzyloxybenzyl
[0100] 4-[4-methoxyphenyl]benzyl
[0101] 4-[3-cyanopropoxy]benzyl
[0102] 3,4-ethylenedioxybenzyl
[0103] 4-[4-nitrophenoxy]benzyl
[0104] 2,3-methylenedioxybenzyl
[0105] 2-benzyloxyphenethyl
[0106] 2-ethoxy-1-naphthylmethyl
[0107] 2-benzylfurylmethyl
[0108] 3-phenoxyphenethyl
[0109] 4-phenoxyphenethyl
[0110] 4-[4-nitrophenyl]benzyl
[0111] 6-methoxy-2-naphthylmethyl
[0112] 3-methyl-5-thienylmethyl
[0113] 5-phenyl-2-thienylmethyl
[0114] 4-benzyloxyphenethyl
[0115] 3-benzyloxyphenethyl
[0116] 4-[2-nitrophenoxy]benzyl
[0117] 5-[4-methoxyphenyl]-2-thienylinethyl
[0118] 4-difluormethoxybenzyl
[0119] 2,3,4,5,6-pentamethylbenzyl
[0120] 5-iodo-2-thienylmethyl
[0121] 4-[2-[2-chloroethoxy]ethoxy]benzyl
[0122] 3,4-dimethylbenzyl
[0123] 3-acetoxybenzyl
[0124] 4-nitrobenzyl
[0125] 4-phenylethynylbenzyl
[0126] 4-[2-chloro-6-fluorobenzyloxy]benzyl
[0127] 4-[3,4-dichlorophenoxy]benzyl
[0128] 4-[3,4-dichlorobenzyloxy]benzyl
[0129] S-[2,3-dihydrobenzfuryl]methyl
[0130] 4-[2-[N,N-diethylamino]ethoxy]benzyl
[0131] 2-bicyclo[2.1.2]heptylmethyl
[0132] 2-hydroxy-5-phenylbenzyl
[0133] 3-[4-chlorophenoxy]benzyl
[0134] 4-[3-chlorophenoxy]-3-nitrobenzyl
[0135] 4-[2-chlorophenoxy]-3-nitrobenzyl
[0136] 3,5-dimethylbenzyl
[0137] 4-[4-ethylphenyl]benzyl
[0138] 3-phenylbenzyl
[0139] 4-[3-fluorophenyl]benzyl
[0140] 4-[4-chlorobenzyloxy]benzyl
[0141] 4-[4-chlorophenoxy]-3-nitrobenzyl
[0142] 4-[4-methylphenoxy]benzyl
[0143] 4-[4-t-butylphenoxy]benzyl
[0144] 4-[4-methylphenyl]benzyl
[0145] 4-[4-methoxyphenoxy]benzyl
[0146] 4-acetoxy-3-methoxybenzyl
[0147] 4-[(2-phenyl)ethyl]benzyl
[0148] 3-[5-phenyl]pyridinylmethyl
[0149] 4-[2-nitrophenyl]benzyl
[0150] 2-[1-hydroxy]fluorenylmethyl
[0151] 4-benzyl-3-methoxybenzyl
[0152] 4-[cyclohexylmethoxy]-3-ethoxybenzyl
[0153] 3-[3,3'-dichlorophenoxy]benzyl
[0154] 4-[4-propylphenyl]benzyl
[0155] 4-thiophenylbenzyl
[0156] 4-[alpha-hydroxybenzyl]benzyl
[0157] 2,2-dinitro-4-thiophenebenzyl
[0158] 3-[3-trifluoromethylphenoxy]benzyl
[0159] 4-[t-butylethynyl]benzyl
[0160] 4-phenoxy-3-methoxy-benzyl
[0161] 4-[3-trifluoromethylphenoxy]-3-nitrobenzyl
[0162] 2-phenylthiobenzyl
[0163] 2-[4-chlorophenyl]-6-benzoxazolemethyl
[0164] 4-[alpha-methoxybenzyl]benzyl
[0165] 4-cyclohexylbenzyl
[0166] 3-[3,4-dichlorophenoxy]benzyl
[0167] acenaphthlenylmethyl
[0168] 4-[1,1,2,2-tetrafluoroethoxy]benzyl
[0169] 4-benzoyloxy-3,3-dimethoxybenzyl
[0170] 3-[cyclohexylmethoxy]benzyl
[0171] 4-cyclohexyloxybenzyl
[0172] 3-[2-quinoylmethoxy]benzyl
[0173] 4-[alpha-ethoxybenzyl]benzyl
[0174] 4-[cyclohexylethoxy]benzyl
[0175] 4-[alpha-propoxybenzyl]benzyl
[0176] 4-[4-methyl-1-piperidino]benzyl
[0177] 2-thiophene-1,2-cyclohexenylmethyl
[0178] 4-[4-nitrobenzyloxy]benzyl
[0179] 3-[4-trifluoromethylphenoxy]benzyl
[0180] 3-benzoyl-2,4-dichlorobenzyl
[0181] 4-[2-[2-thiopropyl]ethoxy]benzyl
[0182] 4-[2-methyl-1-piperidino]benzyl
[0183] 4-hydroxybenzyl
[0184] 4-[2-pyridyl]benzyl
[0185] 4-acetoxybenzyl
[0186] 5,6-benzonorbomylmethyl
[0187] 3-phenylcyclopentylmethyl
[0188] 1-adamantylmethyl
[0189] 3-[cyclohexylmethoxy]-4-methoxybenzyl
[0190] 2-[2-glucosyl]benzyl
[0191] 4-[4-pentoxybiphenyl]benzyl
[0192] 3,4-dihydroxybenzyl
[0193] 4-[4-methylpiperazino]benzyl
[0194] 4-morpholinobenzyl
[0195] 4-[4-chlorophenylsulfonyl]benzyl
[0196] 4-methylsulfonyloxybenzyl
[0197] 4-benzoyloxybenzyl
[0198] 5-phenyl-3-pyridinylmethyl
[0199] 4-[N,N-bis(2-chloroethyl)amino]benzyl
[0200] 3-cyclohexyloxybenzyl
[0201] 4-[2-t-butoxyethoxy]benzyl
[0202] 3,3-dichloro-4-hydroxy-benzyl
[0203] 1,2,3,4,-tetrahydro-9-anthranylmethyl
[0204] 4-cyclohexanoyloxybenzyl
[0205] 4-nonanoyloxybenzyl
[0206] 4-[phenylsulfinyl]benzyl
[0207] 4-anilinobenzyl
[0208] cyclohexylmethyl
[0209] 3-benzoyloxybenzyl
[0210] 3-nonanoyloxybenzyl
[0211] 4-[cyclohexyl]cyclohexylmethyl
[0212] 3-cyclohexanoyloxybenzyl
[0213] 4-[cyclohexanoyloxy]-3,3.fwdarw.dimethoxy]benzyl
[0214] 4-[nonanoyloxy]-3,3.fwdarw.dimethoxy]benzyl
[0215] 1,2,3,4-tetrahydro-6-naphthylmethyl
[0216] 2-hydroxybenzyl
[0217] [2-[6,6-dimethyl-bicyclo>3.1.1]hept-2-enyl]methyl
[0218] 1-cyclohexenyl-4-isopropylmethyl
[0219] 4-[4-methoxyphenyl]butyl
[0220] 4-[[2,3,4,5,6-pentamethyl]phenylsulfonyloxy]benzyl
[0221] 4-[1-pyrrolidinosulfonyl]benzyl
[0222] 3-[4-methoxyphenyl]propyl
[0223] 8-phenyloctyl
[0224] 4-[2,3-dihydroxypropoxy]benzyl
[0225] 4-[N-methylanilino]benzyl
[0226] 2-[2-napthyl]ethyl
[0227] 6-methyl-2-naphthyl methyl
[0228] cis-bicyclo[3.3.0]octane-2-methyl
[0229] 2-tridecynyl
[0230] 4-butyl-2-cyclohexylmethyl
[0231] 4-[(4-fluorobenzoyl)amino]benzyl
[0232] 4-[(3-fluorobenzoyl)amino]benzyl
[0233] 8-phenoxyoctyl
[0234] 6-phenylhexyl
[0235] 10-phenyldecyl
[0236] 8-bromooctyl
[0237] 11-tridecynyl
[0238] 8-[4-methoxyphenoxy]octyl
[0239] 8-[4-phenylphenoxy]octyl
[0240] 8-[4-phenoxyphenoxy]octyl
[0241] 3-[3-trifluoromethylphenoxy]benzyl
[0242] 10-undecenyl
[0243] 4-cyclohexylbutyl
[0244] 4-phenyl-2-fluorobenzyl
[0245] 7-hexadecynyl
[0246] 3-[cyclopentyl]propyl
[0247] 4-[2-methylphenyl]benzyl
[0248] 4-[phenylazo]benzyl
[0249] 4-[4-flurophenyl]benzyl
[0250] 3-nitro-4-[4-nitrophenyl]benzyl
[0251] 3-nitro-4-[2-nitrophenyl]benzyl
[0252] 9-decenyl
[0253] 4-[3,4-dimethoxyphenyl]benzyl
[0254] 4-[4-trifluromethylphenyl]benzyl
[0255] 5-hexenyl
[0256] 4-[2-thienyl]benzyl
[0257] 4-[6-phenylhexyloxy]benzyl
[0258] 9,10-dihydro-2-phenantrene methyl
[0259] 4-[3,4-dimethylphenyl]benzyl
[0260] 4-[4-methylphenyl]-2-methylbenzyl
[0261] 4-[3-phenylpropyloxy]benzyl
[0262] 4-[3-methylphenyl]benzyl
[0263] 4-[4-methylphenyl]-3-methylbenzyl
[0264] 4-[4-pentenyloxy]benzyl
[0265] 4-[1-heptynyl]benzyl
[0266] 3-[4-t-butyl-phenylthio]benzyl
[0267] 4-[4-chlorophenyl]benzyl
[0268] 4-[4-bromophenyl]benzyl
[0269] 4-[4-cyanophenyl]benzyl
[0270] 4-[1-nonynyl]benzyl
[0271] 4-[11-tridecynyloxy]benzyl
[0272] 12-phenyldodecyl
[0273] 6-phenyl-5-hexynyl
[0274] 11-phenyl-10-undecynyl
[0275] 4-[2-methylphenyl]-3-methylbenzyl
[0276] 3-[2-thienyl]-2-thienylmethyl
[0277] 4-[benzyloxymethyl]cyclohexylmethyl
[0278] 4-[4-chlorophenoxy]benzyl
[0279] 4-[benzyl]cyclohexylmethyl
[0280] 4-benzoylbenzyl
[0281] 4-[phenoxymethyl]benzyl
[0282] 4-[4-chlorobenzyl]benzyl
[0283] In another preferred embodiment of the present invention,
the glucose residue attached to A.sub.4 is modified to bear a
substituent, which may be any of the hydrophobic substituents as
described above as well as polar substituents. Thus, the preferred
compounds of the present invention encompass compounds in which the
vancosamine residue is N-substituted with a hydrophobic substituent
and the glucose residue is modified to bear a substituent other
than hydroxyl. Preferably, it is the C.sub.6 position of the
glucose residue that is modified to bear a substituent other than
hydroxyl as described above. Thus, in particularly preferred
compounds of the present invention, the vancosamine residue is
N-substituted with a hydrophobic substituent and the glucose
residue is also modified at the C.sub.6 position to bear a
substituent other than hydroxyl. Where the vancosamine residue is
N-substituted with a hydrophobic substituent and the glucose
residue is also modified to bear a substituent other than hydroxyl,
it is preferred that glucose residue attached to A.sub.4 is
substituted with a polar substituent. Examples of preferred
substituents on the glucose residue, and in particular at the
C.sub.6 position of the glucose residue include, but are not
limited to, mesitylenesulfonyl; 2-thio-6-azathymine;
2-thio-4-hydroxy-6-methylpyrimid- ine;
2-thio-5-amino-1,3,4-thiadiazole;
2-thio-4-amino-3-hydrazino-1,2,4-tr- iazole;
2-thio-4-hydroxy-6-methylpyrimidine; 2-thio-6-azathymine; iodo;
amino; azido; bromo; hydrazino; iminotriphenylphosphoranyl;
S-3-amino-5-mercapto-1,2,4-triazolyl; N-2-quinoxalinyl-Vancosamine.
It is to be understood that the C.sub.6 position can also be
modified to bear any of the hydrophobic substituents as described
above. Thus, where the C.sub.6 position of the glucose residue
attached directly to A.sub.4 is modified to bear a hydrophobic
substituent as described above, it is not necessary that the
vancosamine residue attached to the glucose residue also bear a
hydrophobic substituent as well. However, it is possible that both
the glucose and vancosamine glycosidic groups at the A.sub.4
position can be modified to bear a hydrophobic substituent.
[0284] The invention is not intended to be limited to the
embodiments described above. Thus, beneficial effects of at least
some of the A.sub.1 substituent replacements on a dalbaheptide in
which A.sub.1 is not covalently linked to A.sub.3 would be expected
to apply generally to glycopeptide derivatives containing at least
one hydrophobic group on a sugar covalently bonded to the
peptide.
[0285] The compounds of the present invention can be prepared by
removing the terminal N-substituted leucine residue from a compound
of the formula N-substituted
leucyl-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 wherein at
least one of the glycosidic groups attached to any of
A.sub.2-A.sub.7 bears a hydrophobic substituent to form the
compound
des-N-substituted-leucyl-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7
bearing a free amino group at A.sub.2 and then attaching the group
A.sub.1 to the free amino group at A.sub.2 to form a compound of
the present invention. The N-substituted leucine residue is
preferably N-methyl leucine. The terminal N-substituted leucine
residue can be removed by any conventional process for removing a
terminal amino acid from an oligopeptide or polypeptide. One
conventional method to remove a terminal amino acid is Edman
degradation. This method is described in the literature and can be
readily employed to remove a terminal N-substituted leucine residue
in a process of making the compounds of the present invention. As
applied to the method of forming the des-N-methyl leucyl compounds
of the present invention, Edman degradation involves the reaction
of the amino group of the terminal N-methyl leucine residue with
phenyl isothiocyanate in a suitable solvent. An intermediate
thiourea compound is formed, and the N-methyl leucine residue
splits off from the thiourea as a phenylthiohydantoin, resulting in
the corresponding des-N-methyl leucine compound.
[0286] Thus, the N-methyl leucine residue can be removed from the
compound N-methyl
leucyl-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 by reacting
this compound with phenylisothiocyanate in a suitable organic
solvent, preferably a pyridine-water solvent, more preferably, a
1:1 pyridine-water solvent at a temperature of about 50.degree. C.
This reaction generates the corresponding thiourea, which is then
treated with TFA-CH.sub.2Cl.sub.2 to yield the des-N-methyl leucyl
compound, to which the group A.sub.1 can optionally be attached as
described in more detail below.
[0287] The present inventors have also discovered a modified Edman
degradation procedure by which an N-terminal amino acid residue can
be removed from a polypeptide or an oligopeptide. This procedure
involves reacting the N-terminal amino acid residue on the
polypeptide or oligopeptide with phenylisothiocyanate in a
pyridine-water-triethylamine solvent medium. The
pyridine-water-triethylamine solvent medium preferably comprises
pyridine-water-triethylamine in a ratio of 10:10:1 by volume. The
reaction is conducted for about 20 to 60 minutes, with 60 minutes
preferred. While not wishing to be bound by any particular theory,
it is believed that triethylamine is a key reagent in this modified
Edman degradation protocol. It is believed that the triethylamine
catalyzes the in situ conversion of the initially formed thiourea
to the final product.
[0288] In the context of the present invention, the modified Edman
degradation process described above can be applied to remove the
terminal N-substituted leucine residue from the compound
N-substituted
leucyl-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A.sub.6-A.sub.7 wherein at
least one glycosidic group attached to any of A.sub.2-A.sub.7 bears
a hydrophobic substituent to yield the corresponding desleucyl
compound bearing a free amino group at A.sub.2 which can then be
reacted with the organic group A.sub.1 to yield the compounds of
the present invention. Thus, the compound N-substituted
leucyl-A.sub.2-A.sub.3-A.sub.4-A.sub.5-A- .sub.6-A.sub.7, wherein
the N-substituted leucine residue is preferably N-methyl leucine,
is reacted with phenylisothiocyanate in a
pyridine-water-triethylamine solvent medium, preferably a 10:10:1
pyridine-water-triethylamine solvent medium and at a temperature of
about 50.degree. C. This method yields the corresponding
des-N-methyl leucyl compound in one step in nearly quantitative
yield. The desired des-N-methyl leucine compound can be
precipitated from DMF by adding excess of 20% ethyl acetate-hexane.
The resulting product is then suitably pure for a subsequent
optional step of attaching the group A.sub.1 to the free amino
group at A.sub.2 on the des-N-methyl leucine compound to form a
compound of the present invention.
[0289] Any conventional method can be employed to attach the group
A.sub.1 to the free amino group at A.sub.2 after removal of the
terminal N-substituted leucine residue. Such methods of coupling
amino groups to other organic groups are well known to the
ordinarily skilled chemist. In the preferred compounds of the
present invention the group A.sub.1 is attached to the free amino
group at A.sub.2 by forming an amide linkage. Thus, a carboxylic
acid or other amine-reactive compound can be reacted with the free
amino group at A.sub.2 to form preferred compounds of the present
invention. It is also possible to attach the organic group to the
free amino group at A.sub.2 by reductive alkylation or other common
methods of functionalizing amino groups. It may be desirable in
some cases when attaching the group A.sub.1 to the free amino group
at A.sub.2 to suitably protect free amino groups at other positions
in the intermediate compound so as to selectively attach the
A.sub.1 group to the free amino group at A.sub.2. Such methods of
selectively protecting free amino groups and selectively removing
the protective groups are well known to the ordinarily skilled
chemist. Suitable protecting groups for free amino groups include
9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (CBZ),
tert-butyloxycarbonyl (t-Boc), and allyloxycarbonyl (alloc).
[0290] Preferably, the terminal N-substituted leucine residue is
removed from a dalbaheptide wherein at least one of the groups
A.sub.2-A.sub.7 is linked via a glycosidic bond to one or more
glycosidic groups each having one or more sugar residues and
wherein at least one of the sugar residues is modified to bear at
least one hydrophobic substituent as described above. Thus, for
example, N-methyl leucine can be removed from the dalbaheptide
vancomycin in which the vancosamine residue is N-substituted with a
hydrophobic substituent and which optionally may also be
substituted on the glucose residue, preferably at the C.sub.6
position thereof, with either a hydrophobic or, more preferably, a
polar substituent as described above. Also, N-methyl leucine can be
removed from the dalbaheptide vancomycin in which the C.sub.6
position is modified to bear a hydrophobic substituent and in which
the vancosamine nitrogen optionally also bears a hydrophobic
substituent. The modified des-N methyl leucyl vancomycin can then
be reacted to attach the A.sub.1 substituent to form the compounds
of the present invention.
[0291] An N-substituted vancomycin glycopeptide can be prepared by
attaching a hydrophobic substituent to the amino group on the
vancosamine residue by reductive alkylation or other conventional
methods for functionalizing an amino group. Thus for example, an
aldehyde can be reacted with vancomycin in a suitable organic
solvent, followed by reduction of the aldehyde carbonyl group with
a suitable reducing agent followed by conventional separation and
purification, which may involve recrystallization and/or reverse
phase chromatographic techniques as are well known to the
ordinarily skilled chemist. In some cases it may be desirable to
selectively protect the amino group in the N-methyl leucine residue
prior to the reductive alkylation of the vancosamine nitrogen. Any
amino protecting group may be employed and conventional methods of
removing the amino protecting group may be employed to remove the
protective group after performing the reductive alkylation at the
vancosamine nitrogen. Other methods of coupling free amino groups
to organic substituents can also be employed to attach the
hydrophobic substituent the vancosamine amino group. Thus,
reductive alkylation is merely a preferred method of attaching the
hydrophobic group to this position of the vancosamine residue, and
other methods will be apparent to the person having ordinary skill
in the art.
[0292] As discussed above, in addition to modifying a glycosidic
group to bear at least one hydrophobic substituent as described
above in connection with the vancosamine nitrogen, it may also be
desirable to modify another glycosidic group to bear a hydrophobic
or polar substituent. In fact, any glycosidic group attached to any
of the amino acid residues A.sub.2-A.sub.7 can be modified to bear
a hydrophobic substituent in accordance with the present invention.
Thus, any of the glycosidic groups in, e.g., the glycopeptide
antibiotics vancomycin, eremomycin, chloroeremomycin, and
.beta.-avoparcin can be modified to bear at least one hydrophobic
substituent. Thus, the present invention is not to be construed as
limited to the preferred compounds which comprise a hydrophobic
substituent at the vancosamine nitrogen and, optionally, a
substituent at the C.sub.6 position of the glucose residue directly
attached to amino acid A.sub.4 in vancomycin.
[0293] It is to be understood that where vancomycin is modified to
bear a substituent at the C.sub.6 position of the glucose residue
directly attached to A.sub.4, this substituent may be hydrophobic
or polar, however, it is preferred that where the vancosamine
nitrogen position is modified to bear a hydrophobic substituent,
the C.sub.6 position, when modified, will preferably be modified to
bear a polar substituent. Where the C.sub.6 position of glucose
attached to A.sub.4 of vancomycin is to be modified to bear a
substituent, the following strategy may be employed to introduce a
suitable set of protecting groups onto vancomycin and to
differentiate the C.sub.6 hydroxyl group of the glucose residue on
A.sub.4 of vancomycin from all other hydroxyl groups. This strategy
may be generally employed to modify any selected hydroxyl group of
a glycopeptide antibiotic which need not be limited to vancomycin,
although C.sub.6 modification of the glucose residue attached to
A.sub.4 of vancomycin is preferred. Thus, the following method is
particularly suitable for modification of, e.g., a primary hydroxyl
group on any glycosidic group attached to any of the amino acid
residues A.sub.2-A.sub.7. Thus, while the foregoing method is
described in reference to the primary hydroxyl group at the C.sub.6
position of the glucose residue directly attached to A.sub.4 of
vancomycin, it is to be understood that the synthetic method
described below is applicable to any similarly reactive hydroxyl
group on any glycosidic group attached to any of the amino acid
residues A.sub.2-A.sub.7. A schematic which generally illustrates
the modification of the C.sub.6 position of vancomycin is shown
below:
[0294] Protection of both amines by similar groups requires using
excess acylation reagent while selective protection of the N-methyl
leucine residue is known, allowing selective functionalization of
the vancosamine amine group. See Pavlov et al., J. Antibiotics,
1993, 46, 1731, incorporated herein in its entirety. Selectively
introducing the mesitylenesulfonyl group at the glucose C.sub.6
position differentiates this position from the other hydroxyl
groups and allows further reaction to displace the
mesitylenesulfonyl group, affording may derivatives. A variety of
functional groups are introduced at the glucose C.sub.6 position by
using common methods for nucleophilic displacement of primary
arylsulfonyl groups directly, or by further synthetic modification
of initial displacement products, including azido and iodo groups.
For example, the iodo group is displaced by a variety of
nucleophiles to produce additional C.sub.6 derivatives. A preferred
nucleophile is a thiol compound, especially a heterocyclic thiol.
Modification of an azido group at the C.sub.6 position is
performed, e.g., by reducing the azido group to an amino group,
which in turn is functionalized by means of reductive alkylation,
nucleophilic substitution, or other amino-group reactions well
known to those skilled in the art. In a preferred embodiment of the
invention, an azido group is partially reduced by reaction with a
phosphine compound to produce an iminophosphorane. In a preferred
method of modifying the C.sub.6 position, or other similarly
reactive hydroxyl group on a glycosidic group, the C.sub.6 position
is modified to bear a free amino group by displacing the
mesitylenesulfonyl group with an azido group which is then reduced
to the free amino group. The free amino group at the C.sub.6
position can then be further modified to bear, e.g., a hydrophobic
substituent by reacting the free amino group in a manner similar to
that described above with respect to attaching a hydrophobic
substituent to the vancosamine nitrogen.
[0295] In the process described above, the vancosamine amino group,
the N-methyl leucine amino group and the carboxyl group at A.sub.7
of vancomycin are suitably protected. Then, the C.sub.6 hydroxyl is
substituted with a mesitylenesulfonyl group which, as described
above can be further displaced, e.g., by nucleophilic displacement
to afford many derivatives. While the above method has been
described in connection with attachment of mesitylenesulfonyl group
at the C.sub.6 position, it is to be understood that after suitably
protecting the glycopeptide starting compound, the C.sub.6 hydroxyl
group can be reacted with any compound that will attach a good
leaving group to the C.sub.6 position. The leaving group may then
be displaced by a subsequent reaction, e.g., by nucleophilic
displacement, and further derivatization may then be performed at
the C.sub.6 position yielding many derivatives. The groups
protecting the vancosamine amino group, the N-methyl leucine amino
group and the carboxyl group at A.sub.7 can be removed in a
conventional manner.
[0296] Where the C.sub.6-substituted vancomycin analog is to be
further substituted with a hydrophobic substituent at the
vancosamine nitrogen, the protecting groups at the vancosamine
amino group, at the carboxyl group at A.sub.7 and at the N-methyl
lecuine amino group of the C.sub.6-substituted vancomycin are
removed. The vancosamine nitrogen is then substituted with the
hydrophobic substituent in the manner described above, e.g., by
performing a reductive alkylation reaction. The N-methyl leucine
residue is removed from this compound and the group A.sub.1 is
attached to the free amino group at A.sub.2 as described above. The
resulting compound, may then be further modified at the C.sub.6
position as described above in connection with the C.sub.6
derivatization, thus affording many derivatives. The resultant
product, after separation and purification will thus have a
hydrophobic substituent at the vancosamine nitrogen position and
will have been modified to bear a group other than hydroxyl at the
C.sub.6 position of the glucose residue, and will also have an
organic group other than N-methyl leucine attached to the amino
acid A.sub.2.
[0297] Preferably, the N-substituted leucine residue is removed
from a compound in which one or more of the glycosidic groups
attached to one of the amino acid residues A.sub.2-A.sub.7 is
already modified to bear the at least one hydrophobic substituent.
However, it is also possible to modify the glycosidic group either
prior to or after removal of the N-substituted leucine residue.
Thus, it is possible to attach one or more glycosidic groups onto a
glycopeptide antibiotic, pseudoaglycone or aglycone bearing a
terminal N-substituted leucine residue and then modifying the
glycosidic group to bear the at least one hydrophobic substituent.
Furthermore, the modification of the glycosidic group can also be
conducted either prior to or after attachment of the A.sub.1 group
upon removal of the terminal N-substituted leucine residue.
[0298] The glycosidic group can be attached to any reactive
hydroxyl group in a glycopeptide antibiotic, aglycone or
pseudoaglycone. Preferably the glycosidic group is attached to an
aglycone or to a pseudoaglycone. Where the glycosidic group is
attached to an aglycone, it is preferable to attach a second
glycosidic group to the previously attached glycosidic group, which
results in a disaccharide group attached to one of the amino acid
residues in the aglycone. Preferably, the sequential attachment of
glycosidic groups is performed at the A.sub.4 position of an
aglycone, however, it is to be understood that this method can be
generally applied to any of the amino acid residues forming an
aglycone, pseudoaglycone or glycopeptide antibiotic. The glycosidic
groups can be attached to any of the reactive hydroxyl groups in
glycopeptide antibiotics, aglycones or pseudoaglycones. These
reactive hydroxyl groups are generally phenolic hydroxyl groups,
benzylic hydroxyl groups or aliphatic hydroxyl groups. Thus, a
glycosidic group can be introduced at any of such hydroxyl groups
as desired. Moreover, as discussed above, a glycosidic group can
also be attached to a previously attached glycosidic group, which
results in a disaccharide group attached to one of the amino acid
residues in the aglycone, pseudoaglycone or glycopeptide
antibiotic. Any hydroxyl group on the glycopeptide antibiotic,
aglycone or pseudoaglycone to which a glycosidic group is not
desired to be attached can be suitably protected. Also, the
glycosidic group itself may be suitably protected so that the
desired glycosidic bond to the glycopeptide antibiotic, aglycone or
pseudoaglycone is formed.
[0299] Thus, a glycopeptide antibiotic having a terminal
N-substituted leucine residue can be prepared by (a) selecting: (i)
an aglycone that is soluble in one or more organic solvents, is
derived from a glycopeptide antibiotic, and which aglycone has
exactly one free phenolic hydroxyl group; and (ii) a protected
first glycosyl donor; (b) allowing a non-enzymatic glycosylation
reaction to proceed in an organic solvent such that a first
glycosidic bond is formed, which links said free phenolic hydroxyl
group to the anomeric carbon of the first glycosyl donor to provide
a pseudoaglycone having a protected first glycosyl residue; (c)
selectively removing one protecting group from the first glycosyl
residue to provide a pseudoaglycone bearing exactly one free
hydroxyl group on the first glycosyl residue; (d) selecting a
second protected glycosyl donor; and (e) allowing a non-enzymatic
glycosylation reaction to proceed in an organic solvent such that a
second glycosidic bond is formed which links said free hydroxyl
group on the pseudoaglycone to the anomeric carbon of the second
glycosyl donor. Any of the glycosidic groups on the resultant
compound can be modified to bear the at least one hydrophobic
substituent in accordance with the preferred methods as described
above. Any glycosidic group can be attached to an aglycone,
pseudoaglycone or glycopeptide antibiotic in the foregoing manner.
Thus, it may be desirable to attach a glycosidic group bearing a
free amino group to an aglycone, pseudoaglycone or glycopeptide
antibiotic as described above. A hydrophobic substituent can then
be attached to the free amino group to produce a compound having a
glycosidic group bearing at least one hydrophobic substituent in
accordance with the present invention. Attachment of a glycosidic
group with a free amino group is advantageous because it may avoid
the necessity of functionalizing the glycosidic group to bear an
amino group prior to attaching the hydrophobic substituent
thereto.
[0300] It is apparent that the method described above can be
modified by starting with a pseudoaglycone and then attaching
another glycosidic group thereto. Thus it is to be understood that
the method of suitably protecting and deprotecting hydroxyl groups
can be generally applied to selectively attach a glycosidic group
to any desired hydroxyl group on an aglycone, pseudoaglycone or
glycopeptide antibiotic, any of which may be modified to bear a
hydrophobic substituent on a glycosidic group.
[0301] Where it is desired to attach glycosidic groups to an
aglycone, pseudoaglycone or glycopeptide antibiotic, all reactive
functional groups on any of these starting compounds are suitably
protected. Thus, amine, carboxylic acid, phenolic and benzylic
hydroxyl groups, e.g., need to be protected to avoid their
participation in the reaction that attaches the glycosidic group.
The protecting groups are suitably chosen so as to render the
protected compounds soluble in the reaction medium. The protecting
groups may remain on the final compound, but are preferably removed
by exposure to acidic or basic conditions, catalytic hydrogenation,
or light or other conventional methods for removing protecting
groups. Any conventional protecting groups for the functional
groups mentioned above may be employed. When the aglycone,
pseudoaglycone or glycopeptide antibiotic is or is derived from
vancomycin, it is preferred that the protecting groups are as
follows: carboxybenzyl (CBz) on the amino nitrogen, a benzyl ester
group; benzyl, allyl or methyl phenolic ethers on the phenolic
hydroxyls of A.sub.5 and A.sub.7 , and acetates on the aliphatic
hydroxyls. Removal of the protective groups can be accomplished by
methods well known to the ordinarily skilled organic chemist. Thus,
when it is desired to remove protecting groups from any of the
compounds of this invention, their removal is accomplished using
methods well known to those skilled in the art. The preferred
method for removal of protecting groups is as follows. Aloc groups
on amines, and allyl esters or allyl ethers are removed by using
Pd(O) mediated reactions, e.g., [Ph.sub.3P].sub.2Pd(II)Cl.sub.2 and
Bu.sub.3SnH in 1:1 acetic acid:DMF. Acetate protecting groups are
removed using hydrazine in THF/methanol. The use of protecting
groups to protect any group which might otherwise be reactive under
a particular set of reaction conditions is well known to the
ordinarily skilled artisan. As will be apparent to the ordinarily
skilled artisant, any such conventional protecting groups and the
methodologies employed therewith can be used in the present
invention.
[0302] The suitably protected aglycone, pseudoaglycone or
glycopeptide antibiotic is glycosylated via a non-enzymatic
reaction in an organic solvent with a variety of glycosyl donors,
thereby forming a glycosidic bond between the aglycone,
pseudoaglycone, glycopeptide antibiotic and the glycosyl donor.
Preferably, the glycosyl donors are activated monosaccharide
anomeric sulfoxides which are fuctionalized at the 6 position or
elsewhere. These sulfoxide donors are differentially protected so
as to allow for selective deprotection of a single hydroxyl after
formation of the glycosidic bond. The single hydroxyl can then be
the reactive site for forming another glycosidic bond with a
glycosidic group. Suitable protecting groups to allow for this
selective deprotection include, but are not limited to, the
2,2-dimethyl acetoacetate group, the 4-azidobutyryl group and any
other groups which can be removed in the presence of other
protecting groups.
[0303] Glycosidic groups can also be removed from a glycopeptide
antibiotic or pseudoaglycone. Thus, a glycosidic group can be
removed from a glycopeptide antibiotic by (a) selecting a
glycopeptide antibiotic that is soluble in one or more organic
solvents; (b) contacting the glycopeptide antibiotic with a Lewis
acid, and allowing a degradation reaction to proceed such that a
sugar residue is removed, producing a pseudoaglycone having exactly
one free hydroxyl group on a remaining sugar residue of the
pseudoaglycone; a glycosidic group can then optionally be attached
to the free hydroxyl group on the pseudoaglycone by the subsequent
steps of (c) selecting a protected glycosyl donor; and (d) allowing
a non-enzymatic glycosylation reaction to proceed in an organic
solvent such that a glycosidic bond is formed which links the free
hydroxyl group of the remaining sugar residue on the pseudoaglycone
to the anomeric carbon of the glycosyl donor. Thus, the foregoing
method can be applied to removal of a glycosidic group, e.g., from
a glycopeptide antibiotic having a disaccharide attached to
A.sub.4. The glycopeptide antibiotic bearing a disaccharide at
A.sub.4 is treated with a Lewis acid in an organic solvent to
remove a sugar residue from the disaccharide group. The Lewis acid
is preferably boron trifluoride, preferably as a complex with
diethyl ether. When the glycopeptide antibiotic having a
disaccharide group at A.sub.4 is vancomycin, it is preferred that
allyloxycarbonyl (aloc) groups are present on the amines of A.sub.1
and the vancosamine residue, acetates on the aliphatic hydroxyl
groups, allyl phenyl ethers on the phenolic hydroxyls, and an allyl
or o-nitrobenzyl ester on the A.sub.7 terminal carboxyl, where a
solid-phase synthesis is employed, the o-nitrobenzyl ester is
preferred. A degradation reaction then proceeds which remove a
glycosidic group to produce a pseudoaglycone in which all reactive
functional groups (amine, carboxylic acid, phenols, and benzylic
alcohols) are suitably protected except for a hydroxyl group on the
remaining glycosidic group attached to residue A.sub.4 which is
where another glycosidic group can optionally be attached.
[0304] Pharmaceutical formulations of the compounds of the present
invention are also a part of the present invention, as well as the
use of the compounds and formulations thereof to treat infectious
diseases in mammals, preferably humans, comprising administering an
amount of the compound of the present invention or a
pharmaceutically acceptable salt or ester thereof to a mammal, the
amount being effective to treat the infectious disease.
[0305] Thus, the compounds of the present invention, or
pharmaceutically acceptable salts or esters thereof can be
formulated for any conventional means of delivery, including oral
or parenteral delivery for the therapeutical or prophylactic
treatment of infectious diseases, preferably bacterial diseases.
The bacterial diseases which may be therapeutically or
prophylactically treated with the compounds and/or formulations of
the present invention include those caused by Gram-positive and/or
Gram-negative microorganisms.
[0306] The compounds of the present invention may be administered
separately or in combination with any other drug or therapeutic
agent. Examples of other therapeutic agents and/or drugs that can
be administered with the compounds and/or formulations of the
present invention include, but are not limited to, beta lactam
antibiotics, such as penems, penams, cephems, carbapenems,
oxacephems, carbacephems, and monobactams, or other antibiotics
such as cycloserine and fosfomycin. The other therapeutic agent
need not be an antibiotic.
[0307] The compounds and/or formulations are administered to the
mammal in a therapeutically effective amount, which amount is
effective to treat, prevent, mitigate and/or alleviate the
infectious disease. Thus, the compound of the present invention can
be administered to the mammal, preferably a human, in an amount
ranging from about 0.5 to about 2 grams per day. The compounds
and/or formulations of the present invention can be administered in
a single daily dosage or in multiple doses per day. Other periodic
treatment protocols may also be adopted. Thus, the treatment
protocol may require administration over extended periods of time,
e.g., for several days or for from about one to six weeks. The
therapeutically effective amounts of the compound of the invention
discussed above are merely exemplary. Thus, the amount per
administered dose or the total amount administered will depend on
such factors as the nature and severity of the infection, the age
and general health of the patient, the tolerance of the patient to
the compounds and/or formulations of the present invention and the
microorganism or microorganisms involved in the infection.
[0308] In the pharmaceutical formulations of the present invention,
the compound can be admixed with any conventional pharmaceutical
carriers and/or excipients and can be formulated for immediate or
sustained release. Other time-release profiles, such as
combinations of immediate and sustained release are also possible.
Thus, the compound of the present invention can be admixed with
conventional pharmaceutical carriers and excipients and used in the
form of tablets, capsules, caplets, elixirs, suspensions, syrups,
wafers and the like. The compounds of the present invention can
also be formulated for topical administration. Typical excipients
and/or carriers include, but are not limited to corn starch,
gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,
mannitol, dicalcium phosphate, sodium chloride, and alginic acid.
Disintegrators commonly used in the formulations of this invention
include croscarmellose sodium, microcrystalline cellulose, corn
starch, sodium starch glycolate and alginic acid. Tablet binders
that can be included are acacia, methylcellulose,
polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose,
starch and ethylcellulose. Lubricants that can be used include
magnesium stearate or other metallic stearates, stearic acid,
silicone fluid, talc, waxes, oils and colloidal silica. Flavoring
agents such as peppermint, oil of wintergreen, cherry flavoring or
the like can also be used. It may also be desirable to add a
coloring agent to make the dosage form more esthetic in appearance
or to help identify the product. Tablets may be coated to
facilitate swallowing or to modify release of the active compound,
or some combination of these.
[0309] The compounds and/or formulations can also be administered
intravenously or intramuscularly. For intravenous (IV) use, a
water-soluble form of the compound is preferably dissolved in one
of the commonly used intravenous fluids, and administered by
infusion. Such fluids as, for example, physiological saline,
Ringer's solution or 5% dextrose can be used. For intramuscular
preparations, a sterile formulation of a suitable salt or ester
form of the compound of the present invention, for example the
hydrochloride salt form can be dissolved and administered in a
pharmaceutical diluent such as water-for-injection, physiological
saline or 5% glucose solution. A suitable insoluble form of the
compound may be prepared and administered as a suspension in an
aqueous base or a pharmaceutically acceptable oil base, such as an
ester of a long chain fatty acid such as ethyl oleate.
[0310] For oral use, a sterile formulation of a suitable salt or
ester form of the compound, for example, the hydrochloric acid
salt, formulated in a diluent such as distilled or deionized water
is particularly useful. Alternatively, the unit dosage form of the
compound can be a solution of the compound, preferably in its salt
or ester from, in a suitable diluent in sterile, hermetically
sealed ampoules.
EXAMPLES
[0311] The present invention will now be described with reference
to the specific examples below, to which the present invention is
not to be construed as limited to.
Example 1
p-chlorobiphenyl vancomycin
[0312] The structural formula of p-chlorobiphenyl vancomycin is
shown below:
[0313] To a solution of vancomycin hydrochloride (20 mg; 13
.mu.moles) in 1.5 mL DMF was added diisopropylethylamine (11 .mu.L,
65 .mu.moles) and 4,4'-chlorobiphenylaldehyde (280 .mu.L of a 10
mg/mL solution in DMF; 13 .mu.moles). The reaction mixture was
stirred at 60.degree. C. for half an hour. Sodium cyanoborohydride
(77 .mu.L of a 0.5M solution in DMF) was added, and the system was
stirred at 60.degree. C. for 1 hour.
[0314] The reaction mixture was cooled to room temperature and
diluted with 25 mL of ethyl ether. The precipitate was collected
and purified by reverse phase HPLC:
[0315] HPLC Conditions for Product Analysis:
1 Column: Phenomenex C18 column, 21.2 .times. 250 mm Flow: 8 mL/min
Mobile Phase: B: acetonitrile 4: 10 mM ammonium acetate, pH 5.2
Program: 0 min 30% B 0.1 min 30% B 25 min 55% B (linear gradient)
35 min 90% B (linear gradient) 35.5 min 90% B (linear gradient) 45
min 30% B
Example 2
Des-leucyl p-chlorobiphenyl vancomycin (TS-518)
[0316] The structural formula of des-leucyl-p-chlorobiphenyl
vancomycin is shown below:
[0317] Under an argon atmosphere, p-chlorobiphenyl vancomycin (130
mg, 7.8 .mu.moles) was dissolved in 3.4 mL of a 10:10:1 mixture of
freshly distilled pyridine, distilled water and triethylamine
(99%). Sonication was used to promoted total dissolution. To the
colorless solution was added phenyl thioisocyanate (11 .mu.L, 90.1
.mu.moles), and the system was kept at 50.degree. C. for 20
minutes. The slightly yellow solution was transferred to a
separatory funnel, diluted with 10 mL of 10:10:1
pyridine-water-triethylamine solution, and washed with 10 mLof 10%
ethyl acetate-hexane. The top layer was discarded. The yellowish
bottom layer was transferred to a round-bottom flask, diluted with
5 mL of 2-butanol and concentrated to dryness. The residue was
azeotroped twice with toluene. The resulting solid was dissolved in
minimal amount of DMF (3 mL) and the product was precipitated by
adding a large volume of 50% ethyl acetate-hexane (40 mL). The
precipitate was collected by filtration, washed with methylene
chloride (3.times.10 mL) and dried under vacuum to afford a nearly
quantitative yield of des-leucyl p-chlorobiphenyl vancomycin
(off-white solid). The product may be further purified by flash
chromatography on silica gel (3:3:2 ethyl
acetate-ethanol-water).
[0318] HPLC Conditions for Product Analysis:
2 Column: Nucleosil 4 C18 100 A (250 .times. 4.6 mm) Flow: 0.75
mL/min Mobile Phase: B: acetonitrile 5: 10 mM ammonium acetate, pH
5.2 Program: 0 min 25% B 0.1 min 25% B 20 min 40% B (linear
gradient) 30 min 90% B (linear gradient) 30.1 min 25% B (linear
gradient) 40 min 25% B Retention time 11.6 min. of product:
Example 3
[0319] Compounds were typically prepared in batches of 48. To each
of 48 test tubes was added the appropriate carboxylic acid (0.77
mmoles). Bis-(6-carboxy-HOBT)-N-(2-aminoethyl)-aminomethyl
polystyrene resin (1.56 mmole/g; purchased from NovaBiochem; 1.19
g) was suspended in 28 ml of amino-free DMF using mild, yet
thorough, stirring. An aliquot of the suspension (500 .mu.l) was
added to each test tube, followed by 500 .mu.L of amino-free DMF
and 100 uL of a solution of 1,3-diisopropyl-carbodiimid- e in DMF
(prepared by adding 750 .mu.oL of 1,3-diisopropyl-carbodiimide to 5
mL of amine-free DMF). The test tubes were shaken on an orbital
shaker at 350 rpm for one hour. The supernatant was removed by
filtration and discarded. The resin was washed with 2 mL of
amine-free DMF (6.times.).
[0320] Des-(N-methyl-leucyl)-p-chlorobiphenyl-vancomycin (1.5 g)
was dissolved in 50 mL of amine-Free DMF using sonication. An
aliquot of this solution (1 mL) was added to each test tube,
followed by 1 mL of amine-Free DMF. The test tubes were shaken on
an orbital shaker at 350 rpm for one hour. The supernatant of each
reaction mixture was transferred to the well of a labeled 48-well
plate. The resin was washed with 1 mL of amine-free DMF (2.times.)
and the washings were combined with the supernatant. In the cases
where the carboxylic acid contained an Fmoc group, a 20% solution
of piperidine in DMF (1 mL) was added to the corresponding
well.
[0321] The plates were then dried in a centrifugal evaporator. The
residues were treated with 5 mL of DMSO (molecular biology grade)
and sonicated until total dissolution. The resulting solutions were
used as such for analytical analysis and biological screening.
[0322] In the structural formulae in Table I below, "X" designates
the --COOH group in the compound that is reacted with the free
amino group at A.sub.2, forming the new amide linkage.
[0323] The antibacterial activity of each of the compounds against
the bacterial strains E. faceium (ATCC 49624), S. epidermidis (ATCC
12228), S. aureus (ATCC 29213), E. faecalis (CL 4877) and E.
faecalis (ATCC 292121) was tested. Each of the compounds was
screened in a 96 will agar array format. Antibacterial activity was
referenced to the zones of inhibition observed for
p-chlorobiphenylvancomycin. The MIC's of p-chlorobiphenylvancomycin
against resistant isolates were approximately 6 ug/ml. A zone score
of 2 was assigned when the zone of inhibition for a given compound
was equal to the zone generated by the delivery of a 1 mg/ml stock
solution of p-chlorobiphenylvancomycin. A zone score of 1 was
assigned if the area of the zone was 25% of the area of the zone
generated by a 1 mg/ml stock solution of
p-chlorobiphenylvancomycin. Likewise a zone score of 3 was assigned
if the zone size was 4 times the size of the zone generated by a 1
mg/ml stock solution of p-chlorobiphenylvancomycin. Similarly, a
zone score of 4 was assigned if the zone size was 16 times the size
of the zone generated by a 1 mg/ml stock solution of
p-chlorobiphenylvancomycin and a zone score of 5 was assigned if
the zone size was 64 times the size of the zone generated by a 1
mg/ml stock solution of p-chlorobiphenylvancomycin. The screening
data for each of the compounds is presented in Table I, below.
3TABLE I Side Chains attached to Free Amino Group at A.sub.2 of
des-leucyl-p- chlorobiphenyl vancomycin and Biological Screening
Results E. faecium S. epidermidis S. aureus E. faecalis E. faecalis
Cmpd. ATCC ATCC ATCC CL ATCC No. Reagent Name 49624 12228 29213
4877 29212 1 3 ALA 2 4 4 4 1 2 4 ASN(TRT) 1 1 1 2 1 3 5 ASP(OTBU) 1
3 2 2 1 4 6 CHA 1 3 2 2 1 5 7 CIS 2 3 3 2 1 6 8 CYC(MMT) 1 1 1 2 1
7 9 PHE 1 3 2 2 1 8 10 SAR 3 4 4 2 2 9 11 SER(TRT) 1 1 1 1 1 10 12
THI 2 3 2 3 1 11 13 THR(TRT) 1 1 1 2 1 12 14 TRP 1 2 2 4 1 13 15
CYS(TRT) 1 1 1 1 1 14 16 D-CYS(TRT) 1 1 1 2 1 15 17 D-MET 4 5 5 2 4
16 18 D-PHE 3 5 4 3 3 17 19 D-SER(TBU) 5 5 5 5 5 18 20 2 3 2 3 1 19
21 TYR(TBU) 1 3 2 3 1 20 22 VAL 2 4 3 4 1 21 23 L-(+)-LACTIC ACID 1
2 2 2 1 22 24 MYR-GLY 1 2 2 3 1 23 25 (R,S)-2-CARBOXY MORPHOLINE 1
2 2 2 1 24 26 4-PIPERAZIN-1-YL ACETIC ACID 2 2 3 4 1 25 27 D-TRP 2
4 2 2 2 26 28 GLU(OBZL) 1 3 2 2 1 27 29 GLN(TRT 1 1 1 3 1 28 30 GLY
2 4 3 2 2 29 31 HIS(TRT) 1 1 1 1 1 30 32 HYP(TBU) 2 4 3 5 1 31 33
(3S,4S)-4-AMINO- 3-HYDROXY-6- METHYLTHIO- HEXANOIC ACID 1 2 2 1 1
32 34 3-AMINO-1- CARBOXY METHYL- PYRIDIN-2-ONE 2 3 2 2 1 33 35
4-(2- AMINOETHYL)-1- CARBOXY- METHYL- PIPERAZINE DIHYDRO CHLORIDE 2
3 2 2 1 34 36 2-CARBOXY METHYL- PIPERAZINE 2 3 4 5 1 35 37 ALA-ALA
1 2 2 2 1 36 38 ALA-GLY 2 4 4 5 1 37 39 ILE 1 3 3 5 1 38 40 LEU 2 4
2 1 1 39 41 LYS(DDE) 1 3 2 2 1 40 42 LYS(MTT) 1 1 1 2 1 41 43 MET 2
4 3 2 1 42 44 NLE 2 4 4 5 1 43 45 ALLO-THR 2 3 4 2 1 44 46
ASN(GLCNAC(AC) 3-BETA-D) 1 1 1 1 1 45 47 ASN(TMOB) 1 2 2 2 1 46 48
BETA, BETA- DIMETHYL-D- CYS(ACM) 4 5 5 2 4 47 49 BETA-ALA 2 4 3 2 1
48 50 CYS(2-HYDROXY ETHYL) 1 2 2 2 1 49 51 CYS(ACM) 1 4 2 2 1 50 52
CYS(ME) 2 4 1 2 1 51 53 D-ALA 4 5 5 2 4 52 54 D-ALLO-THR 3 4 4 1 1
53 55 D-ASN 1 2 4 2 1 54 56 D-CIS-HYP 1 2 2 2 1 55 57 D-CYS(ACM) 3
4 4 2 1 56 58 D-DPR(DDE) 2 4 3 3 1 57 59 D-GLN 2 4 3 3 2 58 60
D-HIS 1 1 1 2 1 59 61 D-ISO ASPARAGINE 1 2 2 2 1 60 62 DL-ISOSER 1
1 1 2 1 61 63 D-LYS (CARBAMYL) 4 5 5 3 4 62 64 D-ORN (CARBAMYL) 4 5
4 4 4 63 65 D-SER 3 4 2 2 1 64 66 D-THR 4 5 4 2 2 65 67 GAMMA-ABU 1
2 2 2 1 66 68 GLN(TMOB) 1 2 2 2 1 67 69 GLY-GLY-GLY 1 2 2 4 1 68 70
GLY-GLY 2 3 3 5 1 69 71 GLY-PRO-HYP 1 1 1 2 1 70 72 GLY-VAL 1 1 1 2
1 71 73 HIS 1 1 1 2 1 72 74 HYP 2 2 3 2 1 73 75 L-ASPARAGINE 1 1 1
1 1 74 76 L-ISO ASPARAGINE 1 2 2 1 1 75 77 L-LYS(BIOTIN) 2 2 1 2 2
76 78 LYS(AC) 2 2 1 1 1 77 79 LYS(BIOTINYL- EPSILON- AMINOCAPROYL)
1 1 1 5 1 78 80 LYS(CARBAMYL) 1 2 2 1 1 79 81 LYS(FOR) 2 3 2 5 1 80
82 LYS(ME)3 CHLORIDE 2 4 2 5 1 81 83 MET(O) 2 2 3 5 1 82 84 MET(O2)
2 3 3 4 1 83 85 ORN(PYRAZINLY CARBONYL) 2 3 2 4 1 84 86 PEN(ACM 2 2
2 1 1 85 87 PRO-GLY 2 2 2 2 1 86 88 PRO-PRO 1 2 2 2 1 87 89 SER(AC)
1 2 2 2 1 88 90 SER 2 2 2 2 1 89 91 THR 2 2 1 2 1 90 92 N-ALPA-L-
ARGININE 1 1 2 2 1 91 93 N-ALPHA-L- GLUTAMINE 1 2 3 5 1 92 94
N-ALPHA-N-BETA- ALOC-L-DIAMINO PROPIONIC ACID 1 3 3 2 1 93 95
N-ALPH-N- GAMMA-ALLOC- L-DIAMINO BUTYRIC ACID 1 2 2 3 1 94 96
(2-CARBOXY ETHYL) DIMETHYL SULFONIUM CHLORIDE 1 2 2 2 1 95 97
(3-ACETYL-2- METHYL-5-OXO-2- ACETIC ACID 1 1 1 3 1 96 98
(S)-(-)-4-OXO-2- AZETIDINE CARBOXYLIC ACID 1 1 1 2 1 97 99
[3-METHYOXY CARBONYL)-2- METHYL-5-OXO-2- PYRROLIN-4- YL]ACETIC ACID
1 1 1 1 1 98 100 1-(AMINO CARBONYL)-1- CYCLOPROPANE CARBOXYLIC ACID
1 1 2 2 1 99 101 1-ACETYL PIPERIDINE-4- CARBOXYLIC ACID 1 2 3 1 1
100 102 2-(2-METHOXY ETHOXY)ACETIC ACID 1 1 2 3 1 101 103
2-[2-(2-METHOXY ETHOXY) ETHOXY]ACETIC ACID 1 1 1 2 1 102 104
2-ACETAMIDO ACRYLIC ACID 1 1 1 2 1 103 105 2-PYRAZINE CARBOXYLIC
ACID 1 1 1 2 1 104 106 3,4-DIACETAMIDO BENZOIC ACID 1 1 2 2 1 105
107 3-AMINO PYRAZINE-2- CARBOXYLIC ACID 1 1 1 2 1 106 108 3-HYDROXY
PROPIONIC ACID 1 2 2 2 1 107 109 4-ACETAMIDO BUTYRIC ACID 1 1 2 2 1
108 110 4-(NITRO BENZOYL- GLYCYL- GLYCINE 1 1 2 4 1 109 111
5-AMINOOROTIC ACID 1 1 2 1 1 110 112 AC-ALA 1 1 2 1 1 111 113
AC-ARG 1 1 1 2 1 112 114 AC-D-ALA 1 1 2 4 1 113 115 AC-D-ASN 1 1 1
2 1 114 116 AC-DL-LYS(AC) 1 1 1 2 1 115 117 AC-D-MET 1 1 2 2 1 116
118 AC-D-PRO 1 2 2 2 1 117 119 ACETOXYACETIC ACID 1 2 2 1 1 118 120
ACETYL-DL- CARNITINE HYDROCHLORIDE 1 2 2 4 1 119 121 ACETYL-L-
CARNITINE- HYDROCHLORIDE 1 2 2 2 1 120 122 AC-GLY-GLY 1 2 2 2 1 121
123 AC-HYP 1 1 2 2 1 122 124 AC-LEU-GLY 1 1 1 1 1 123 125
AC-LYS(AC) 1 1 2 1 1 124 126 AC-MET(O) 1 1 2 4 1 125 127 AC-THR 1 1
1 2 1 126 128 ALLANTOIC ACID 1 1 1 2 1 127 129 ARABIC ACID 1 1 1 2
1 128 130 ARABINIC ACID 1 1 2 1 1 129 131 BETAINE HYDROCHLORIDE 1 2
1 2 1 130 132 BICINE 1 1 2 2 1 131 133 BOC-ALA-GLY- GLY 1 1 2 2 1
132 134 BOC-ALA-GLY- SAR 1 1 2 2 1 133 135 BOC-ASN 1 1 1 2 1 134
136 BOC-ASP-NH2 1 2 3 2 1 135 137 BOC-D-ASN 1 2 2 3 1 136 138
BOC-D-GLN 1 2 1 4 1 137 139 BOC-GLU-NH2 1 1 2 2 1 138 140
BOC-GLY-ARG 1 1 1 2 1 139 141 BOC-GLY-GLY- GLY 1 1 2 2 1 140 142
BOC-GLY-GLY 1 1 1 2 1 141 143 BOC-L-GLUTAMINE 1 1 2 2 1 142 144
BOC-MET(O) 2 1 2 2 1 143 145 BOC-MET(O2) 1 1 2 1 1 144 146
CACOTHELINE 1 1 1 2 1 145 147 CREATINE 1 1 1 2 1 146 148
D-(-)-QUINIC ACID 1 1 1 2 1 147 149 D-(+)- GALACTURONIC ACID
MONOHYDRATE 1 1 1 2 1 148 150 D-ALPHA- GALACTURONIC ACID 1 1 1 2 1
149 151 D-CARNITINE HYDROCHLORIDE 2 1 1 2 1 150 152 D-GLUCURONIC
ACID 1 1 1 1 1 151 153 DL-CARNITINE HYDROCHLORIDE 2 2 4 2 1 152 154
DL-GLYCERIC ACID 1 1 1 2 1 153 155 DL-PYRO GLUTAMIC ACID 1 1 1 1 1
154 156 D-PYRO GLUTAMIC ACID 1 1 1 1 1 155 157 D-SACCHARIC ACID
1,4- LACTONE 1 1 1 2 1 156 158 D-SACCHARIC ACID 3,5- LACTONE 1 1 1
1 1 157 159 GLUCONIC ACID 1 1 2 2 1 158 160 GLYCOLIC ACID 1 2 2 2 1
159 161 GLYOXYLIC ACID 1 1 1 2 1 160 162 GUANIDOACETIC ACID 1 1 1 1
1 161 163 HIPPURYL-GLY- GLY-OH 1 1 1 2 1 162 164 HYDANTOIC ACID 1 1
1 2 1 163 165 HYDANTOIN-5- ACETIC ACID 1 1 2 1 1 164 166
LACTOBIONIC ACID 1 1 1 1 1 165 167 L-ARGININIC ACID 1 1 1 2 1 166
168 L-BETA-IMIDAZO LELACTIC ACID 1 1 1 1 1 167 169 L-CARNITINE
HYDROCHLORIDE 1 1 1 1 1 168 170 L-DIHYDRO OROTIC ACID 1 1 1 2 1 169
171 L-PYRO GLUTAMIC ACID 1 1 1 2 1 170 172 MALEAMIC ACID 1 2 1 2 1
171 173 METHANE SULFONYL ACETIC ACID 1 1 1 2 1 172 174 N-(ACETO
ACETYL) GLYCINE 1 1 1 2 1 173 175 N,N-DIMETHYL SUCCINAMIC ACID 1 1
1 1 1 174 176 N-ACETYL-DL- ALANINE 1 1 1 2 1 175 177 N-ACETYL-DL-
METHIONINE 1 2 2 1 1 176 178 N-ACETYL-DL- PROLINE 1 2 2 2 1 177 179
N-ACETYL-DL- PROPARGYL- GLYCINE 1 1 2 2 1 178 180 N-ACETYL-DL
SERINE 1 1 1 2 1 179 181 N-ACETYL GLYCINE 1 1 1 2 1 180 182
N-ACETYL-L- GLUTAMINE 1 1 1 1 1 181 183 N-ACETYL-L- HISTIDINE 1 2 2
2 1 182 184 N-ACETYL-L- METHIONINE 1 2 1 2 1 183 185 N-ACETYL-L-
PROLINE 1 2 2 2 1 184 186 N-ALPHA- ACETYL-L- ARGININE DIHYDRATE 1 1
1 2 1 185 187 N-ALPHA- ACETYL-L- ASPARAGINE 1 1 1 1 1 186 188
BETA-GUANIDINO PROPIONIC ACID 1 1 1 1 1 187 189 N-ALPHA-
CARBAMYL-L- ARGININE 1 1 2 2 1 188 190 N-ALPHA- CARBOETHOXY-
L-ASPARAGINE 1 1 2 2 1 189 191 N-CARBOMOYL MALEAMIC ACID 1 1 2 2 1
190 192 N-CARBAMYL- ALPHA-AMINO- ISOBUTYRIC ACID 1 1 2 3 1 191 193
N-CARBAMYL-DL- ALPHA-AMINO-N- BUTYRIC ACID 1 1 1 3 1 192 194
N-CARBAMYL-L- HISTIDINE HYDROCHLORIDE 1 1 1 2 1 193 195 N-FORMYL
GLYCINE 1 2 3 2 1 194 196 N-FORMYL-L- ALANINE 1 1 2 2 1 195 197
N-FORMYL-L- HISTIDINE 1 2 2 2 1 196 198 N-FORMYL-L- METHIONINE 1 1
2 3 1 197 199 NICOTINURIC ACID 1 1 2 2 1 198 200 OROTIC ACID 1 2 2
3 1 199 201 OXALYL MONOGUANYL HYDRAZIDE 1 1 2 2 1 200 202 OXAMIC
ACID 1 1 2 2 1 201 203 SHIKIMIC ACID 1 1 2 2 1 202 204 SUCCINAMIC
ACID 1 2 2 2 1 203 205 SUCCINIC ACID 2,2-DIMETHYL HYDRAZIDE 1 1 2 1
1 204 206 SUCCINIC SEMIALDEHYDE 1 1 2 2 1 205 207 SULFOACETIC ACID
1 1 2 2 1 206 208 DL-2-UREIDO PROPIONIC ACID 1 2 2 2 1 207 209
THYMINE-1- ACETIC ACID 1 2 4 3 1 208 210 URACIL-5- CARBOXYLIC ACID
1 1 2 2 1 209 211 Z-ALA-GLY-GLY 1 2 2 2 1 210 212 Z-BETA-ALA-GLY-
GLY 1 2 2 2 1 211 213 Z-GLN-GLY 1 2 2 2 1 212 214 Z-GLY-GLN 1 2 2 2
1 213 215 Z-GLY-GLY-ALA 1 2 2 2 1 214 216 Z-GLY-GLY-GLY- GLY (SEQ
ID NO. 1) 1 1 2 2 1 215 217 Z-GLY-GLY-GLY 1 1 2 2 1 216 218
DELTA-VAL 1 1 2 1 1 217 219 D-ARG 2 3 3 2 1 218 220 ACPC 1 1 2 1 1
219 221 DELTA-ABU 1 1 2 2 1 220 222 ALA 2 4 4 1 1 221 223 3-UREIDO
PROPIONIC ACID 1 1 2 2 1 222 224 CARBOXY METHOXYL AMINE HEMIHYDRO
CHLORIDE 1 1 2 2 1 223 225 BOC-GLN-GLN-OH 1 1 2 2 1 224 226
(-)-2-OXO-4 THIAZOLIDINE- CARBOXYLIC ACID 1 2 2 2 1 225 227
(ETHYLTHIO) ACETIC ACID 1 3 4 2 1 226 228 (METHYLTHIO) ACETIC ACID
1 2 4 1 1 227 229 (R)-(-)-5-OXO-2- TETRAHYDRO FURAN CARBOXYLIC ACID
1 2 2 1 1 228 230 1H-TETRAZOLE-1- ACETIC ACID 1 2 1 2 1 229 231
1-METHYL PYRROLE-2- CARBOXYLIC ACID 1 2 2 2 1 230 232 2-(4-CHLORO
PHENYLTHIO) NICOTINIC ACID 1 1 2 2 1 231 233 2-(TRIFLUORO METHYL)
PROPENOIC ACID 1 1 2 2 1 232 234 2,2-BIS(HYDROXY METHYL) PROPIONIC
ACID 1 1 2 2 1 233 235 2,4,5-TRICHLORO PHENOXY ACETIC ACID 1 1 2 2
1 234 236 2,4-DICHLORO PHENYLACETIC ACID 1 2 2 2 1 235 237
2,4-DIHYDROXY BENZOIC ACID 1 1 1 1 1 236 238 2,4-DIMETHOXY BENZOIC
ACID 1 1 2 2 1 237 239 2-AMINO NICOTINIC ACID 1 2 4 2 1 238 240
2-FLUORO BENZOIC ACID 1 2 2 1 1 239 241 2-FURAN GLYOXYLIC ACID 1 1
2 1 1 240 242 2-FUROIC ACID 1 2 2 1 1 241 243 2-HYDROXY NICOTINIC
ACID 1 1 1 2 1 242 244 2-KETOBUTYRIC ACID 1 1 1 2 1 243 245
2-METHYL PYRAZINE-5- CARBOXYLIC ACID 1 1 2 2 1 244 246 3-(2-FURYL)
ACRYLIC ACID 1 2 2 3 1 245 247 3-(3,4- DIMETHOXYL PHENYL) PROPIONIC
ACID 1 2 2 2 1 246 248 3-(PHENYL SULFONYL) PROPIONIC ACID 2 4 5 3 1
247 249 3-(TRIFLUORO METHYL) BENZOIC ACID 1 2 3 2 1 248 250
3-(TRIFLUORO METHYLTHIO) BENZOIC ACID 1 1 2 2 1 249 251
3,3,3-TRIFLUORO PROPIONIC ACID 1 3 4 2 1 250 252 3,4,5- TRIACETOXY
BENZOIC ACID 1 2 3 1 1 251 253 3,4-DICHLORO CINNAMIC ACID 1 1 1 2 1
252 254 3,4-DIFLUORO PHENYLACETIC ACID 1 2 4 1 1 253 255
3,4-DIMETHOXY BENZOIC ACID 1 2 2 2 1 254 256 3,5-BIS (TRIFLUORO
METHYL) BENZOIC ACID 1 1 2 2 1 255 257 3,5-DIHYDROXY BENZOIC ACID 1
1 2 2 1 256 258 3-AMINO-1,2,4- TRIAZOLE-5- CARBOXYLIC ACID 1 1 2 2
1 257 259 3-AMINOBENZOIC ACID 1 2 2 2 1 258 260 3-ETHOXY PROPIONIC
ACID 1 2 2 2 1 259 261 3-FLUORO BENZOIC ACID 1 2 3 1 1 260 262
3-FUROIC ACID 1 2 3 1 1 261 263 3-HYDROXY BENZOIC ACID 1 2 2 2 1
262 264 3-HYDROXY BUTYRIC ACID 1 2 2 2 1 263 265 3-METHYL
THIOPROPIONIC ACID 1 4 5 1 1 264 266 4-(METHYL SULFONYL) BENZOIC
ACID 1 2 2 0 1 265 267 4-ACETAMIDO BENZOIC ACID 1 1 2 2 1 266 268
4-ACETOXY BENZOIC ACID 1 2 2 2 1 267 269 4-ACETYL BENZOIC ACID 2 2
4 1 1 268 270 4-AMINO BENZOIC ACID 1 1 1 2 1 269 271 4-CARBOXY
BENZENE SULFONAMIDE 1 2 2 2 1 270 272 4-FLUORO BENZOIC ACID 1 2 2 2
1 271 273 4-HYDROXY BENZOIC ACID 1 2 2 4 1 272 274 4-IMIDAZOLE
CARBOXYLIC ACID 1 2 2 2 1 273 275 4-METHOXY CINNAMIC ACID 1 2 3 3 1
274 276 4-AMINO SALICYLIC ACID 1 2 2 2 1 275 277 5-METHYL
ISOXAZOLE-4- CARBOXYLIC ACID 1 2 2 2 1 276 278 6-HYDROXY NICOTINIC
ACID 1 2 3 1 1 277 279 6-HYDROXY PICOLINIC ACID 1 2 3 2 1 278 280
6-OXOHEPTANOIC ACID 1 1 2 2 1 279 281 ACETIC ACID 1 2 3 2 1 280 282
ANTHRANILIC ACID 1 2 3 2 1 281 283 BENZOTRIAZOLE- 5-CARBOXYLIC ACID
1 2 2 2 1 282 284 COUMALIC ACID 1 1 2 2 1 283 285 PYRAZINE-2,3-
DICARBOXYLIC ACID MONOAMIDE 1 2 2 4 1 284 286 D-DESTHIO BIOTIN 1 2
3 2 1 285 287 DIFLUOROACETIC ACID 1 2 2 2 1 286 288 DL-2-HYDROXY-
N-BUTYRIC ACID 1 2 2 2 1 287 289 ETHOXY ACETIC ACID 1 2 2 2 1 288
290 FUMARIC ACID MONETHYL ESTER 1 2 4 2 1 289 291 GLYOXYLIC ACID
SEMICARBAZONE 1 2 2 1 1 290 292 HEPTAFLUORO BUTYRIC ACID 1 2 2 2 1
291 293 INDOLE-2- CARBOXYLIC ACID 1 2 2 2 1 292 294 ISONICOTINIC
ACID 1 2 4 2 1 293 295 ITACONIC ACID MONOMETHYL ESTER 1 2 2 2 1 294
296 LACTIC ACID 1 2 3 2 1 295 297 LEVULINIC ACID 1 2 4 2 1 296 298
MALEIC ACID MONOETHYL ESTER 1 2 3 2 1 297 299 MALEIC ACID
MONOMETHYL ESTER 1 1 2 2 1 298 300 2,3-DIHYDRO-3- OXOPYRIDAZINE-
6-CARBOXYLIC ACID 1 1 2 2 1 299 301 MAYBRIDGE BTB 09316 1 2 2 4 1
300 302 MAYBRIDGE KM 01502 1 2 3 2 1 301 303 MAYBRIDGE KM 06000 1 2
2 2 1 302 304 3-(4-CHLORO BENZENE SULPHONYL) BUTYRIC ACID 1 3 4 3 1
303 305 METHOXY ACETIC ACID 1 2 4 4 1 304 306 MONO-METHYL GLUTARATE
1 2 4 3 1 305 307 MONO-METHYL SUCCINATE 1 2 4 3 1 306 308
N-[3-(2-FURYL ACRYLOYL)]- GLYCINE 1 2 3 2 1 307 309 NICOTINIC ACID
1 3 4 2 1 308 310 NICOTINIC ACID N-OXIDE 1 2 4 2 1 309 311 N0METHYL
MALEAMIC ACID 1 2 3 2 1 310 312 PENTAFLUORO PHENYLACETIC ACID 1 2 4
2 1 311 313 PERFLUORO PENTANOIC ACID 1 1 2 1 1 312 314 PICOLINIC
ACID 1 2 2 2 1 313 315 PICOLINIC ACID N-OXIDE 1 2 2 2 1 314 316
PYRUVIC ACID 1 2 2 2 1 315 317 SALICYLIC ACID 1 1 1 2 1 316 318
TETRAHYDRO-2- FUROIC ACID 1 2 2 3 1 317 319 TETRAHYDRO-3- FUROIC
ACID 1 3 4 2 1 318 320 THIOPHENE-2- ACETIC ACID 1 2 4 3 1 319 321
THIOPHENE-2- CARBOXYLIC ACID 1 2 3 2 1 320 322 THIOPHENE-3- ACETIC
ACID 1 2 3 2 1 321 323 UROCANIC ACID 1 2 2 2 1 322 324 HSE(ME) 1 2
2 2 1 323 325 L-THREONINE MONOHYDRATE 1 2 2 2 1 324 326
N-ACETYL-DL- HISTIDINE HYDRATE 1 2 2 2 1 325 327 N-FORMYL-DL-
ALANINE 1 2 3 2 1 326 328 N-FORMYL-DL- METHIONINE 1 2 2 2 1 327 329
OXAMIC ACID 1 2 3 3 1
[0324] In Table II, below, MIC (minimum inhibitory concentration)
values of certain compounds of the present invention are provided
for the bacterial strains E. faecium ATCC 49624, E. faecium CL
4931, E. faecalis ATCC 29212, E. Faecalis CL 4877, S aureus ATCC
29213, and S. Aureus ATCC 33591. The minimum inhibitory
concentrations (MIC) of test compounds were determined using
bacteria grown in brain heart infusion media (BHI) supplemented
with 0.1% casamino acids. Logarithmically growing cells were
diluted to approximately 5.times.10.sup.5 CFU/ml and subjected to
test compounds solubilized and serially diluted in DMSO. A 5% final
DMSO concentration had no affect on cell viability or killing.
After 18 hours at 37.degree. C., the OD.sub.600 was determined by
reading the ninety-six well microtiter plates on a microplate
reader. For a given concentration, an MIC determination was made
if:
[OD.sub.600 Control-OD.sub.600 Test Conc.]/[OD.sub.600
Control-OD.sub.600 Media].times.100.gtoreq.90%
4TABLE II MIC Values of Compounds of the Invention Against Selected
Baterial Strains 330 E. faecium E. faecalis S. aureus Cmpd ATCC CL
ATCC CL ATCC ATCC No. R1 R2 Reagent Name 49624 4931 29212 4877
29213 33591 0 OH 331 Vancomycin (Vancosamine sugar is unsubstituted
in this compound only.) 1.25 >250 3.12 >250 1.25 2.5 1 OH 332
L-ALA 0.78 25 1.56 25 0.78 0.78 7 OH 333 L-PHE 0.12 6.25 0.78 6.25
0.39 0.39 11 OH 334 L-THR(TRT) 3.12 6.25 3.12 3.12 3.12 3.12 12 OH
335 L-TRP 0.39 6.25 1.56 3.12 0.78 0.78 15 OH 336 D-MET 0.12 12.5
0.25 12.5 0.25 0.12 16 OH 337 D-PHE 0.12 12.5 0.25 12.5 0.25 0.25
17 OH 338 D-SER(TBU) 0.062 6.25 0.25 3.91 0.062 0.12 18 OH 339
D-THR(TBU) 0.25 25 1.56 25 0.78 0.78 20 OH 340 L-VAL 0.25 12.5 0.78
3.12 0.25 0.39 21 OH 341 L-(+)-LACTIC ACID 3.12 25 6.25 25 1.56
3.12 24 OH 342 PIPERAZIN-1-YL ACETIC ACID HYDRATE 1.56 25 1.56 6.25
0.78 0.78 25 OH 343 D-TRP 0.25 12.5 0.39 6.25 0.25 0.25 26 OH 344
L-GLU(OBZL) 1.56 >25 1.56 12.5 0.78 0.78 30 OH 345 L-HYP(TBU)
0.78 25 3.12 25 1.56 1.56 34 OH 346 4-CARBOXY METHYL- PIPERAZINE
0.78 25 3.12 6.25 0.78 1.56 36 OH 347 L-ALA-GLY 0.12 12.5 0.78 6.25
0.39 0.39 37 OH 348 L-ILE 0.25 12.5 0.78 3.12 0.39 0.39 38 OH 349
L-LEU 0.78 12.5 1.56 12.5 0.78 0.78 41 OH 350 L-MET 0.25 12.5 0.78
12.5 0.39 0.39 42 OH 351 L-NLE 0.39 12.5 0.78 6.25 0.39 0.39 56 OH
352 D-DPR(DDE) 0.12 12.5 0.78 12.5 0.25 0.25 56-3 OH 353 D-DPR(DDE)
Deprotect/Rearrange 0.12 6.25 0.25 6.25 0.25 0.25 61 OH 354 D-LYS
(CARBAMYL) 0.12 12.5 0.25 12.5 0.12 0.12 62 OH 355 D-ORN (CARBAMYL)
0.12 12.5 0.25 12.5 0.25 0.25 63 OH 356 D-SER 0.78 25 1.56 12.5
0.78 0.78 64 OH 357 D-THR 0.39 25 1.56 12.5 0.78 0.78 67 OH 358
GLY-GLY-GLY 0.78 12.5 1.56 6.25 0.78 0.78 68 OH 359 GLY-GLY 0.39
12.5 1.56 6.25 0.78 0.78 75 OH 360 L-LYS(BIOTIN) 0.39 25 3.12 25
1.56 1.56 79 OH 361 L-LYS(FOR) 0.78 25 3.12 26 1.56 1.56 81 OH 362
L-MET(O) 0.78 25 1.56 25 0.78 1.56 82 OH 363 L-MET(O2) 0.39 25 1.56
12.5 0.78 0.78 83 OH 364 L-ORN(PYRAZINYL CARBONYL) 0.25 25 0.78
12.5 0.39 0.78 88 OH 365 L-SER 0.78 12.5 1.56 25 0.78 1.56 89 OH
366 L-THR 0.78 >25 3.12 12.5 1.56 1.56 91 OH 367 N-ALPHA-L-
GLUTAMINE 0.78 25 1.56 12.5 0.78 0.78 100 OH 368 2-(2-METHOXY
ETHOXY)ACETIC ACID 0.78 6.25 1.56 6.25 0.25 0.78 108 OH 369
4-NITROBENZOYL- GLYCYL-GLYCINE 1.56 >25 3.12 >25 0.78 0.78
135 OH 370 BOC-D-ASN 0.39 >25 3.12 12.5 1.56 1.56 136 OH 371
BOC-D-GLN 0.78 >25 1.56 12.5 0.78 0.78 188 OH 372 N-ALPHA-
CARBOETHOXY-L- ASPARAGINE 0.062 6.25 0.39 3.12 0.39 0.39 257 OH 373
3-AMINOBENZOIC ACID 0.39 25 1.56 12.5 0.78 0.78 287 OH 374
ETHOXYACETIC ACID 1.56 25 3.12 12.5 1.56 3.12 303 OH 375
METHOXYACETIC ACID 1.56 25 3.12 12.5 1.56 1.56 328 OH 376 D-ME-VAL
0.12 12.5 0.39 12.5 0.25 0.25 329 OH 377 D-ME-LEU 0.031 6.25 0.12
6.25 0.25 0.12 330 OH 378 L-ME-ILE 0.12 12.5 0.78 3.12 0.25 0.25
331 OH 379 L-ME-SER(BZL) 0.39 12.5 1.56 6.25 0.78 0.78 332 OH 380
L-CYS(BZL) 1.56 25 3.12 12.5 1.56 1.56 333 OH 381 L-CYS(TBU) 1.56
25 3.12 25 1.56 1.56 334 OH 382 D-CYS(TBU) 0.12 25 0.78 12.5 0.25
0.25 335 OH 383 D-ILE 0.12 12.5 0.39 12.5 0.12 0.25 336 OH 384
D-LEU 0.12 6.25 0.25 12.5 0.25 0.25 337 OH 385 D-NVA 0.062 12.5
0.25 12.5 0.12 0.12 338 OH 386 D-SER(BZL) 0.25 12.5 1.56 25 1.56
0.39 339 OH 387 D-VAL 0.12 12.5 0.25 12.5 0.25 0.25 340 OH 388
L-ME-VAL 0.39 12.5 1.56 25 0.39 0.39 341 OH 389 L-NVA 0.78 12.5
1.56 25 0.78 0.78 342 OH 390 L-SER(BZL) 0.39 25 1.56 12.5 0.78 0.78
343 OH 391 L-SER(TBU) 0.39 25 3.12 12.5 1.56 0.78 344 OH 392
L-THR(TBU) 0.78 25 3.12 12.5 1.56 1.56 345 OH 393 L-CYS(STBU) 0.39
25 3.12 12.5 0.78 0.78 517 OH 394 TS5017 (Cl-Biphenyl Vancomycin)
0.062 12.5 0.25 12.5 0.25 0.25 518 OH X--H TS5018 (Des-Lecyl 1.56
12.5 3.12 25 3.12 6.25 Cl-Biphenyl Vanc) 519 I 395 6'-Deoxy-6'-Iodo
0.78 >25 3.12 >25 1.56 3.12 520 I 396 6'-Deoxy-6'-Iodo-
ChloroBiphenyl 0.12 12.5 0.78 12.5 0.78 0.78 521 NH2 397
6'-Deoxy-6'-Amino TS1017 0.12 12.5 0.78 6.25 0.78 0.78 522 OH X--H
Des-Lecyl >25 >25 >25 >25 >25 >25 Vancomycin 523
NH2 398 6'-Deoxy-6'-Amino TS5017 0.12 6.25 0.25 6.25 0.78 0.39 524
OH 399 D-SER(ET) 0.25 25 1.56 12.5 0.39 0.39 525 OH 400 D-SER(ME)
0.25 25 1.56 25 0.39 0.39 526 OH 401 N-Methy-L- SER(TBU) 0.25 25
0.78 6.25 0.39 0.39 527 OH 402 N-Methyl-D-SER(ET) 0.25 25 1.56 12.5
0.39 0.39 528 OH 403 D-SER(ISOPROPYL) 0.12 25 0.78 12.5 0.25 0.25
529 OH 404 L-SER(ISOPROPYL) 0.12 25 0.78 12.5 0.25 0.25 554 OH 405
L-HIS 0.39 12.5 3.12 25 1.56 0.78 555 OH 406 D-HIS 0.39 12.5 1.56
12.5 0.78 0.39 556 OH 407 D-GLY-(2-Pyridyl) 0.39 25 0.78 25 0.78
0.39 557 OH 408 L-Phe(3-Nitro-2- Hydroxy) 0.39 25 1.56 25 1.56
1.56
[0325]
Sequence CWU 1
1
1 1 10 PRT Artificial sequence Synthetic Peptide sequence designed
to provide antibiotic activity 1 Gly Gly Gly Gly Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10
* * * * *