NOVEL ANTIBACTERIAL AGENTS

NOUVEAUX AGENTS ANTIBACTERIENS

English Abstract

Novel antibacterial agents that act as multibinding agents are disclosed. The
compounds of the invention comprise from 2-10 ligands covalently connected,
each of said ligands being capable of binding to a transglycosylase enzyme
substrate thereby modulating the biological processes/functions thereof.

French Abstract

La présente invention concerne de nouveaux agents antibactériens qui agissent comme agents multiliaison. Les composés selon l'invention comprennent de 2 à 10 ligands à liaison covalente, chacun desdits ligants étant capable de se lier à un substrat d'enzyme de transglycosylase et, par la même, de moduler les processus/fonctions biologiques dudit substrat.

Claims

158
WHAT IS CLAIMED IS:
1. A multibinding compound of the formula:
(L)p X q
Formula I
in which L is a ligand that may be the same or different at each occurrence;
X is a linker that may be the same or different at each occurrence;
p is an integer of 2-10; and
q is an integer of 1-20,
each of said ligands comprising a ligand domain capable of binding to a
transglycosylase
enzyme substrate;
or a pharmaceutically acceptable salt thereof;
with the proviso that when L is vancomycin, p is 2 and q is 1, and the link is
[C-C], X is
not -NH-Q-NH-, in which Q is (CH2)m, -(CH2)2-NH-(CH2)n NH-(CH2)2-, or
-(CH2)n-S-S-(CH2)n-, where m is an integer of 2-12 and n is an integer of 1-3;
also with the proviso that when L is vancomycin, p is 2 and q is 1, and the
link is [V-V];
X is not alkylene, alkylene interrupted by oxygen, or alkylene interrupted by
phenylene
or 1,4-dioxyphenylene.
2. The compound of claim 1, wherein p is 2 and q is 1.
3. The compound of claim 2, wherein L is an optionally substituted
glycopeptide.
4. The compound of claim 3, wherein the first and second glycopeptides are
selected
from the group consisting of optionally substituted vancomycin, optionally
substituted
N-desmethylvancomycin, and optionally substituted chloroeremomycin, and X
connects
the [C] terminus of the first glycopeptide to the [C] terminus of the second
glycopeptide.
5. The compound of claim 4, wherein X is:
-NH-R6-NH-C(O)-R7-C(O)-NH-R8-NH-



159
in which R6, R7, and R8 are optionally substituted alkylene.
6. The compound of claim 5, wherein both glycopeptides are vancomycin,
R6 and R8 are -CH2-CH2- and R7 is -CH2-CH2-CH2-, namely [C-C]-[pentane-1,5-
dioic
acid bis-[(2-aminoethyl)-amide]-bis-(vancomycin).
7 The compound of claim 5, wherein R6 and R8 are -CH2-CH2- and R7 is
-CH2-CH2-CH2-, said first glycopeptide is unsubstituted vancomycin and said
second
glycopeptide is vancomycin substituted at the [V]-position by alkyl,
alkylamino, or
alkylaminoalkyl.
8. The compound of claim 7, wherein said [V] substitution is n-
decylaminoethyl,
namely [V]-n-decylaminoethyl-(vancomycin) [C-C]-[pentane-1,5-dioic acid bis-
[(2-
aminoethyl)-amide]-(vancomycin).
9. The compound of claim 3, wherein the glycopeptide is chosen from optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin; and X connects the [V] terminus of the first
glycopeptide
to the [V] terminus of the second glycopeptide.
10. The compound of claim 9, wherein X is:
-R6-NH-C(O)-R7-C(O)-NH-R8-
in which R6, R7, and R8 are optionally substituted alkylene.
11. The compound of claim 10, wherein the first and second glycopeptide are
both
vancomycin, R6 and R8 are -CH2-CH2- and R7 is -CH2-CH2-CH2-, namely
[V-V]-pentane-1,5-dioic acid-bis-[(2-ethyl)-amide]-bis-(vancomycin)
12. The compound of claim 10, wherein the glycopeptide is
[C]-3-(dimethylamino)propylamino-vancomycin, R6 and R8 are -CH2-CH2- and R7 is

-CH2-CH2-CH(NH-C(O)-Z)-CH2-CH2-, where Z is 4-biphenyl, namely [V-V]-4-(4-



160
phenylbenzamido)-heptane-1,7-dioic acid-bis[(2-ethyl)amide]-bis-([C]-3-
(dimethylamino)propylamino-vancomycin).
13. The compound of claim 10, wherein the glycopeptide is
[C]-3-(dimethylamino)propylamino-vancomycin, R6 and R8 are -CH2-CH2- and R7 is

-CH2-CH2-CH(NH-C(O)-Z)-, where Z is n-tetradecanoyl, namely [V-V]-4-(4-
(tetradecanoylamido)-heptane-1,7-dioic acid-bis[(2-ethyl)amide]-bis-([C]-3-
(dimethylamino)propylamino-vancomycin).
14. The compound of claim 10, wherein the glycopeptide is
[C]-3-(dimethylamino)propylamino-vancomycin, R6 and R8 are -CH2-CH2- and R' is

1,3-[5-(benzyloxy)phenyl], namely [V-V]-(5-benzyloxy-isophthalic acid) bis [(2-
ethyl)amide]-
bis-([C]-3-(dimethylamino)propylamino-vancomycin).
15. The compound of claim 3, wherein the glycopeptide is chosen from
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin; and X connects the [C] terminus of the first
glycopeptide
to the [V] terminus of the second glycopeptide.
16. The compound of claim 15, wherein X is:
-NH-R6-NH-C(O)-R7-C(O)-NH-R8-
in which R6, R7, and R8 are optionally substituted alkylene.
17. The compound of claim 16, wherein the first and second glycopeptide are
both
vancomycin, and R6, R7, and R8 are all -CH2-CH2-, namely [C-V]-butane-1,4-
dioic acid-
(2-aminoethyl)-amide-(2-ethyl)-amide-bis-(vancomycin).
18. The compound of claim 16, wherein the first glycopeptide is
[V]-n-octylaminoethyl vancomycin, the second glycopeptide is [C]-N-glucosamine-

vancomycin, and R6, R7, and R8 are all -CH2-CH2-, namely -([V] n-octylamino-



161
vancomycin [C-V]-butane-1,4-dioic acid-(2-aminoethyl)-amide-(2-ethyl)-amide).
([C]-N-glucosamine-vancomycin).
19. The compound of claim 16, wherein the first glycopeptide is
[V]-n-decylaminoethyl vancomycin, the second glycopeptide is [C]-N-glucosamine-

vancomycin, and R6, R7, and R7 are all -CH2-CH2-, namely ([V] n-decylamino-
vancomycin)-[C-V]-butane-1,4-dioic acid-(2-aminoethyl)-amide-(2-ethyl)-amide)-
([C]-
N-glucosamine-vancomycin).
20. The compound of claim 16, wherein the first glycopeptide is vancomycin,
the
second glycopeptide is [C]-3-(dimethylamino)propylamino-vancomycin, R6, R7 and
R8
are all -CH2-CH2-, namely (vancomycin)-[C-V]- butane-1,4-dioic acid-(2-
aminoethyl)-
amide-(2-ethyl)-amide-([C]-3-(dimethylamino)propylamino-vancomycin).
21. The compound of claim 15, wherein X is:
-NH-R6-C(O)-NH-R7-
in which R6 and R7 are optionally substituted alkylene.
22. The compound of claim 21, wherein the first glycopeptide is [V]-
decylaminoethyl
vancomycin, the second glycopeptide is [C]-N-glucosamine-vancomycin, R6 is
-CH(NH-C(O)-Z)-CH2, in which Z is N-glucosamine, and R7 is -CH2-CH2-, namely
([V]
n-decylamino-vancomycin)-[C-V]-3-amino-2-(N-glucosaminocarbonylamino)-N-(2-
ethyl)-propionamide-([C]-N-glucosamine-vancomycin).
23. The compound of claim 15, wherein X is:
-NH-R6-
in which R6 is optionally substituted alkylene.
24. The compound of claim 23, wherein the first glycopeptide is
[V]-n-decylaminoethyl-vancomycin, the second glycopeptide is vancomycin, and
R6 is



162
-CH2-CH2-, namely ([V]-n-decylaminoethyl-vancomycin) [C-V]-N-(2-aminoethyl)-
(vancomycin).
25. The compound of claim 23, wherein the first glycopeptide is
[V]-n-decylaminoethyl-vancomycin, the second glycopeptide is vancomycin, and
R6 is
-CH2-CH2-, namely ([V]-n-decylaminoethyl-vancomycin) [C-V]-N-(2-aminoethyl)-
(vancomycin).
26. The compound of claim 23, wherein the first glycopeptide is
[V]-n-decylaminoethyl-vancomycin, the second glycopeptide is [R]-N-methyl-D-
glucamine-
vancomycin, and R6 is -CH2-CH2-, namely ([V]-n-decylaminoethyl-vancomycin)
[C-V]-N-(2-aminoethyl)-([R]-N-methyl-D-glucamine-vancomycin).
27. The compound of claim 23, wherein the first glycopeptide is
[V]-n-decylaminoethyl-vancomycin, the second glycopeptide is [C]-D-glucosamine-
[R]-N-
methyl-D-glucamine-vancomycin, and R6 is -CH2-CH2-, namely
([V]-n-decylaminoethyl-vancomycin) [C-V]-N-(2-aminoethyl)-([C]-D-glucosamine-
[R]-N-
methyl-D-glucamine-vancomycin).
28. The compound of claim 23, wherein the first glycopeptide is
[V]-n-decylaminoethyl-vancomycin, the second glycopeptide is [C]-D-glucosamine-

vancomycin, and R6 is -CH2-CH2-, namely ([V]-n-decylaminoethyl-vancomycin)
[C-V]-N-(2-aminoethyl)-([C]-D-glucosamine-vancomycin).
29. The compound of claim 3, wherein the glycopeptide is chosen from
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin; and X connects the [C] terminus of the first
glycopeptide
to the [N] terminus of the second glycopeptide.



163
30. The compound of claim 29, wherein X is:
-NH-R6-
in which R6 is optionally substituted alkylene.
31. The compound of claim 30, wherein the first glycopeptide is
[V]-n-hexylaminoethyl-vancomycin, the second glycopeptide is
[C]-3-(dimethylamino)propylamino-vancomycin, and R6 is -CH2-CH2-, namely
([V]-n-hexylaminoethyl-vancomycin) [C-N]-N-(2-aminoethyl)-([C]-3-
(dimethylamino)propylamino-vancomycin).
32. The compound of claim 3, wherein the glycopeptide is chosen from
optionally
substituted vancomycin; optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin, and X connects the [N] terminus of the first
glycopeptide
to the [N] terminus of the second glycopeptide.
33. The compound of claim 32, wherein X is optionally substituted aralkyl.
34. The compound of claim 33, wherein both glycopeptides are vancomycin and X
is:
-CH2-Z-O-(CH2)6-O-Z-CH2-
in which Z is 1,4-phenylene; namely [N-N]-hexane-1,6-bis-(phenoxy-4-methyl)
bis-(vancomycin).
35. The compound of claim 3, wherein the glycopeptide is chosen from
optionally
substituted vancomycin; optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin, and X connects the [R] terminus of the first
glycopeptide
to the [R] terminus of the second glycopeptide.
36. The compound of claim 35, wherein X is optionally substituted alkylene.



164
37. The compound of claim 36, wherein both glycopeptides are vancomycin, and
R6
is (CH2)4-, namely [R-R]-n-butylene-bis(vancomycin).
38. The compound of claim 3, wherein the glycopeptide is chosen from
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, and
optionally
substituted chloroeremomycin, and X connects the [N] terminus of the first
glycopeptide
to the [V] terminus of the second glycopeptide.
39. The compound of claim 38, wherein X is:
-NH-R6-NH-C(O)-R7-C(O)-NH-R8-
in which R6, R7, and R8 are optionally substituted alkylene.
40. The compound of claim 39, wherein the first and second glycopeptides are
both
[C]-3-(dimethylamino)propylamino-vancomycin, R6, R8 are both-CH2-CH2-, and R7
is
CH2-CH2-CH2-, namely [N-V]-pentane-1,5-dioic acid bis-[(2-aminoethyl)-amide]
bis-([C]-3-(dimethylamino)propyl-vancomycin).
41. The compound of claim 2, wherein L is an aglycone.
42. The compound of claim 41, wherein the first and second aglycones are both
optionally substituted vancomycin aglycone and X connects the [O] terminus of
the first
aglycone to the [O] terminus of the second aglycone.
43. The compound of claim 42, wherein the first and second aglycones are both
[C]-3-(dimethylamino)propylamino-vancomycin aglycone
44. The compound of claim 42, wherein X is:
-NH-R6-NH-C(O)-R7-C(O)-NH-R8-NH-
in which R6, R7, and R8 are optionally substituted alkylene.



165
45. The compound of claim 44, wherein, R6 and R8 are -CH2-CH2- and R7 is
-CH2-CH2-CH2-, namely [O-O]- pentane-1,5-dioic acid bis-[(2-aminoethyl) amide)-
bis-([C]-3-
(dimethylamino)propylamino-vancomycin aglycone
46. The compound of claim 42, wherein X is optionally substituted alkylene.
47. The compound of claim 46, wherein X is -CH2-CH2-CH2-, namely
[O-O]-n-propylene-bis-([C]-3-(dimethylamino)propylamino-vancomycin aglycone.
48. A method of treating mammals having a disease state that is alleviated by
treatment with an antibacterial agent, comprising administering to a mammal in
need of
such treatment a therapeutically effective amount of a compound of claim 1, or
a
pharmaceutically acceptable salt thereof.
49. The method of claim 48, wherein the disease state is characterized by
resistance
to vancomycin.
50. The method of claim 48, wherein the multibinding compound of claim 1 is a
compound of the formula:
(L)p X q
in which L is an optionally substituted glycopeptide, and X connects the [C]
terminus of
the first glycopeptide to the [V] terminus of the second glycopeptide.
50. The method of claim 48, wherein the compound of formula I is chosen from:
[V]-n-decylaminoethyl-(vancomycin) [C-C]-[pentane-1,5-dioic acid bis-[(2-
aminoethyl)-
amide]-(vancomycin);
[V-V]-4-(4-phenylbenzamido)-heptane-1,7-dioic acid-bis[(2-ethyl)amide]-bis-
([C]-3-
(dimethylamino)propylamino-vancomycin);
[V-V]-4-(4-(tetradecanoylamido)-heptane-1,7-dioic acid-bis[(2-ethyl)amide]-bis-
([C]-3-
(dimethylamino)propylamino-vancomycin);



166
[V-V]-(5-benzyloxy-isophthalic acid) bis [(2-ethyl)amide]-bis-([C]-3-
(dimethylamino)propylamino-vancomycin);
[C-V]-butane-1,4-dioic acid-(2-aminoethyl)-amide-(2-ethyl)-amide-bis-
(vancomycin);
[V] n-octylamino-vancomycin [C-V]-butane-1,4-dioic acid-(2-aminoethyl)-amide-
(2-
ethyl)-amide). ([C]-N-glucosamine-vancomycin);
[V] n-decylamino-vancomycin-[C-V]-butane-1,4-dioic acid-(2-aminoethyl)-amide-
(2-
ethyl)-amide)-([C]-N-glucosamine-vancomycin);
(vancomycin)-[C-V]- butane-1,4-dioic acid-(2-aminoethyl)-amide-(2-ethyl)-amide-
([C]-
3-(dimethylamino)propylamino-vancomycin);
[V] n-decylamino-vancomycin-[C-V]-3-amino-2-(N-glucosaminocarbonylamino)-N-(2-
ethyl)-propionamide-([C]-N-glucosamine-vancomycin);
[V]-n-decylaminoethyl-vancomycin [C-V]-N-(2-aminoethyl)-(vancomycin);
[V]-n-decylaminoethyl-vancomycin [C-V]-N-(2-aminoethyl)-(vancomycin);
[V]-n-decylaminoethyl-vancomycin [C-V]-N-(2-aminoethyl)-([R]-N-methyl-D-
glucamine-vancomycin);
[V]-n-decylaminoethyl-vancomycin [C-V]-N-(2-aminoethyl)-([C]-D-glucosamine-[R]-

N-methyl-D-glucamine-vancomycin);
[V]-n-decylaminoethyl-vancomycin [C-V]-N-(2-aminoethyl)-([C]-D-glucosamine-
vancomycin);
([V]-n-hexylaminoethyl-vancomycin) [C-N]-N-(2-aminoethyl)-([C]-3-
(dimethylamino)propylamino-vancomycin);
[N-N]-hexane-1,6-bis-(phenoxy-4-methyl) bis-(vancomycin);
[R-R]-n-butylene-bis(vancomycin);
[N-V]-pentane-1,5-dioic acid bis-[(2-aminoethyl)-amide] bis-([C]-3-
(dimethylamino)propyl-vancomycin);
[O-O]-n-propylene-bis-([C]-3-(dimethylamino)propylamino-vancomycin aglycone;
and the pharmaceutically acceptable salts thereof.



167
51. A pharmaceutical composition comprising a therapeutically effective amount
of
at least one compound of claim 1, or a pharmaceutically acceptable salt
thereof, admixed
with at least one pharmaceutically acceptable excipient.
52. A process for the preparation of a compound of claim 15, compiling:
a) contacting a compound of the formula:
Image
where the shaded square represents a glycopeptide; the shaded circle
represents a linker,
R is hydrogen or methyl, and R10 is hydrogen, a protecting group, alkyl,
alkylamino, aryl,
heteroaryl, arylalkyl, or heteroarylalkyl;
with a compound of the formula:
Image
where the shaded square, the shaded circle and R, and are as defined above,
and R11 is
hydroxy or -NHR12, where R12 is, alkyl, alkylamino, aryl, heteroaryl,
arylalkyl, or
heteroarylalkyl.
53. The process of claim 52, wherein R is methyl, R10 is n-octylaminoethyl,
R11 is
hydroxy, the shaded square represents vancomycin, the shaded circle in
compound (a)
represents:
-(CH2)2NHOC(CH2)2NHOC(CH2)3CONH(CH2)2CONH(CH2)2CO2Fm
where Fm is 9-fluorenylmethyl, and the shaded circle in compound (b) repesents
(CH2)2.



168
54. The process of claim 53, wherein the reaction is conducted in the presence
of a
tertiary base, PyBOP and HOBT in a polar solvent, at a temperature between
0°-100°C.
55. The process of claim 54, wherein the tertiary base is
diisopropylethylamine, the
polar solvent is N,N-dimethylformamide, and the temperature is 25°C.
56. The compound of claim 16, wherein the first glycopeptide is
N-desmethylvancomycin, the second glycopeptide is
[C]-3-(dimethylamino)propylamino-N-desmethylvancomycin, and R6, R7 and R8
are all -CH2-CH2-, namely (N-desmethylvancomycin)-[C-V]- butane-1,4-dioic
acid'-(2-aminoethyl)-amide-(2-ethyl)-amide-([C]-3-(dimethylamino)propylamino
-N-desmethylvancomycin).

Description

CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
NOVEL ANTIBACTERIAL AGENTS
Field of the Invention
This invention relates to novel antibacterial agents. More particularly, the
invention relates to novel antibacterial agents that act as multibinding
agents. The
multibinding agents of the invention comprise from 2-10 ligands covalently
connected
by a linker or linkers, wherein each of said ligands in their monovalent (i.e.
unlinked)
state have the ability to bind to a cell surface or a precursor used in the
synthesis of the
bacterial cell wall and thereby interfere with the synthesis of the precursor
and the cell
wall. The manner in which the ligands are linked is such that the multibinding
agents so
constructed demonstrate an increased biological and/or therapeutic effect as
compared to
the same number of unlinked ligands available for binding to the ligand
binding site.
The invention also relates to pharmaceutical compositions comprising a
pharmaceutically acceptable excipient and an effective amount of a compound of
the
invention, and to methods of using such compounds and pharmaceutical
compositions
containing them as antibacterial agents.
Still further, the invention also relates to methods of preparing such
compounds.
Back r
A bacterial cell wall consists of linear polysaccharide chains that are cross-
linked
by short peptides. This arrangement confers mechanical support to the cell
wall, and
prevents the bacteria from bursting due to the high internal osmotic pressure.
Cross
linking takes place after lipid-linked disaccharide-pentapeptide constructs
(lipid
intermediate II) are incorporated into linear polysaccharide chains by a
transglycolase
enzyme. The cross-linking reaction is the last step of the synthesis of the
cell wall, and is
catalyzed by an enzyme known as peptidoglycan transpeptidase.
One method by which antibacterial agents exert their antibacterial activity is
by
inhibiting the transglycosylase enzyme, thus interfering with the penultimate
step of the
synthesis of the bacteria cell wall. Although not wishing to be bound by
theory, it is
believed that a glycopeptide, for example vancomycin, binds with high affinity
and
specificity to N-terminal sequences (L-lysyl-D-alanyl-D-alanine in vancomycin-
sensitive
organisms) of peptidoglycan precursors know as lipid intermediate II. By
binding to and


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
2
Osequestering these precursors, vancomycin prevents their utilization by the
cell wall
biosynthesis machinery. In a formal sense, therefore, vancomycin inhibits the
bacterial
transglycosylase that is responsible for adding lipid intermediate II subunits
to growing
peptidoglycan chains. This step preceeds the cross-linking transpeptidation
step which is
inhibited by beta-lactams antibiotics. It is likely that vancomycin also
inhibits
transpeptidation which involves the D-alanyl-D-alanine termini; however, as
this step
occurs subsequent to transglycosylation, inhibition of transpeptidation is not
be directly
observed.
Antibacterial agents have proved to be important weapons in the fight against
pathogenic bacteria. However, an increasing problem with respect to the
effectiveness of
antibacterial agents relates to the emergence of strains of entrococci that
are highly
resistant to such agents; for example, vancomycin-resistant entrococci (VRE),
which are
also multi-drug resistant. It would therefore be highly desirable to find
antibacterial
agents that are active against a broad spectrum of bacteria, in particular
resistant strains
such as VRE. It would be also be advantageous to discover antibacterial agents
that
demonstrate high activity and selectivity toward their targets, and are of low
toxicity.
We have discovered that covalent connection of two or more ligands
(antibacterial agents) by a linker or linkers provides a multibinding agent
that affords an
improved biological effect when compared to the same concentration of unlinked
ligand
(i.e. in its monomeric state), or when compared to a ligand monomer coupled to
the
linker only. That is to say, an improved biological andlor therapeutic effect
of the
multibinding agent is obtained as measured against that achieved by the same
number of
unlinked. ligands available for binding to the ligand binding site of the
peptidoglycan
transglycosylase enzyme substrate.
A preferred ligand is vancomycin. Particularly preferred are vancomycin
bivalent
compounds, which demonstrate greatly enhanced biological effect when compared
to
vancomycin monomer, or vancomycin monomer to which is attached the linking
structure. They are also highly effective when tested against VRE strains.
Although not wishing to be bound by any particular theory or proposed
mechanism of action, it is believed that the surprising activity of the
compounds of the
invention arises from their ability to bind in a multivalent manner with their
target and


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
3
thus lower the energetic costs of binding (i.e. the phenomena of energetically
coupled
binding), which is produced by the optimum positioning of two or more
molecules of a
ligand in relationship to its binding site, i.e., a multivalent interaction.
That is to say, the
compounds act as multibinding agents, in which ligands that are covalently
attached by a
linker or linkers simultaneously (or contemporaneously) bind to multiple
binding sites on
another component, such as an enzyme substrate.
Related Disclosures
Vancomycin derivatives are disclosed in Patent Applications EP 0 802 199, EP 0
801 075, EP 0 667 353, WO 97/28812, WO 97/38702, and in JACS 118, pp 13107-
13108 (1996), JACS 119, pp 12041-12047 (1997), and JACS 116, pp 4573-4590
(1994).
The disclosures of these and other documents referred to throughout this
application
(e.g., in the Pharmacology section of the Detailed Description of the
Invention) are
incorporated herein by reference.
SUMMARY OF THE INVENTION
This invention addresses the above needs by providing novel multibinding
agents. Accordingly, in one aspect, the present invention relates to novel
multibinding
agents;
wherein a multibinding agent comprises 2-10 ligands, which may be the same or
different, covalently connected by a linker or linkers, which may be the same
or
different, each of said ligands comprising a ligand domain capable of binding
to a
transglycosylase enzyme substrate.
The preferred multibinding agents are represented by Formula I:
(L)p(X)a
Formula I
in which L is a ligand that may be the same or different at each occurrence;
X is a linker that may be the same or different at each occurrence;
p is an integer of 2-10; and
q is an integer of 1-20;
or a salt thereof;


CA 02318394 2000-07-12
WO 99142476 PCT/US99/03850
4
wherein each of said ligands comprises a ligand domain capable of binding to a
transglycosylase enzyme substrate, thereby modulating the activity of the
enzyme
substrate. Preferably q is less than p. More preferably p is 2 and q is 1.
Particularly
preferred is the compound of Formula I wherein L at each occurrence represents
optionally substituted vancomycin; and X represents a linker between any
hydroxyl
group, carboxyl group or amino group of the first vancomycin to any hydroxyl
group,
carboxyl group or amino group of the second vancomycin.
In a second aspect, the invention relates to a method of treatment of mammals
having a disease state that is treatable by an antibacterial agent, comprising
administering
a therapeutically effective amount of a novel multibinding agent thereto;
wherein a rnultibinding agent comprises 2-10 ligands, which may be the same or
different, covalently connected by a linker or linkers, which may be the same
or
different, each of said ligands comprising a ligand domain capable of binding
to a
transglycosylase enzyme substrate. The preferred multibinding agent is a
compound of
Formula I, or a mixture of compounds of Formula I.
In a third aspect, the invention relates to a method of treatment of mammals
having a bacterial disease characterized by resistance to vancomycin,
comprising
administering a therapeutically effective amount of a multibinding agent as
defined
above, preferably a compound of Formula I, thereto, or a mixture of
multibinding agents.
In a fourth aspect, the invention relates to a pharmaceutical composition
comprising
a therapeutically effective amount of one or more multibinding agents, or a
pharmaceutically acceptable salt thereof, said multibinding agent comprising 2-
10
ligands, which may be the same or different, covalently connected by a linker
or linkers,
which may be the same or different, each of said ligands comprising a ligand
domain
capable of binding to a transglycosylase enzyme substrate, admixed with at
least one
pharmaceutically acceptable excipient, wherein the multibinding agent is
preferably a
compound of Formula I
In a fifth aspect, the invention relates to processes for preparing the
multibinding
agents of the invention.


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Definitions
As used herein:
The term "alkyl" refers to a monoradical branched or unbranched saturated
hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-
10 carbon
5 atoms, more preferably 1-6 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-
butyl, secondary butyl, tert-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, 2-
ethyldodecyl,
tetradecyl, and the like, unless otherwise indicated.
The term "substituted alkyl" refers to an alkyl group as defined above having
from 1 to 5 substituents selected from the group consisting of alkoxy,
substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl,
acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,
thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted
thioalkoxy, aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-
aryl, -S02-
heteroaryl, and -NReRb, wherein Ra and Rb may be the same or different and and
are
chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl,
alkynyl, aryl, heteroaryl and heterocyclic.
The term "alkylene" refers to a diradical of a branched or unbranched
saturated
hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-
10 carbon
atoms, more preferably 1-6 carbon atoms. This term is exemplified by groups
such as
methylene
(-CH2-), ethylene (-CH2CH2-), the propylene isomers (e.g., -CH2CH2CH2- and -
CH(CH3)CH2-) and the like.
The term "substituted alkylene" refers to:
(a) an alkylene group as defined above having from 1 to 5 substituents
selected from
the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted
cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino (including, for
example, N-
glucosaminecarbonyl, benzoylamino, biphenylcarbonylamino, and the like),
acyloxy,
amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl,
keto,
thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy,
aryl, aryloxy,


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thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic,
heterocyclooxy,
thioheterocyclooxy, vitro, and -NRaRb, wherein Re and Rb may be the same or
different
and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl,
alkenyl,
cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. Additionally, such
substituted
alkylene groups include those where 2 substituents on the alkylene group are
fused to
form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene
group.
(b) an alkylene group as defined above that is interrupted by 1-20 atoms or
substituents independently chosen from oxygen, sulfur and NRa-, wherein Re is
chosen
from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl,
aryl, heteroaryl and heterocyclic; or
(c) an alkylene group as defined above that has both from 1 to S substituents
as
defined above and is also interrupted by 1-20 atoms as defined above.
Examples of substituted alkylenes are chloromethylene (-CH(Cl)-),
aminoethylene
{-CH(NH2)CH2-), 1-(dodecanoylamino)propylene (-CH[NHC(O)-(CH2)~~-CH3] CH2-),
1-(4-phenylbenzoylamino)pentylene (-CH(NHC(O)-Z] (CH2)4) ,2-carboxypropylene
isomers (-CH2CH(C02H)CH2-), ethoxyethyl (-CH2CH2 O-CH2CH2-), -),
ethylmethylaminoethyl (-CH2CH2 N(CH3) CH2CH2-), 1-ethoxy-2-(2-ethoxy-
ethoxy)ethane (-CH2CH2 O-CH2CH2-O-CH2CH2 O-CH2CH2-), and the like.
The term "alkaryl" or "aralkyl"refers to the groups -alkylene-aryl and -
substituted
alkylene-aryl in which alkylene and aryl are as defined herein. Such alkaryl
groups are
exemplified by benzyl, phenethyl and the like.
The term "alkoxy" refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-,
cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl,
cycloalkenyl, and
alkynyl are as defined herein. Preferred alkoxy groups are alkyl-O- and
include, by way
of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,
sec
butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like
The term "substituted alkoxy" refers to the groups substituted alkyl-O-,
substituted alkenyl-O-, substituted cycloallcyl-O-, substituted cycloalkenyl-O-
, and
substituted alkynyl-O- where substituted alkyl, substituted alkenyl,
substituted
cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined
herein.


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7
The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl,
alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted
alkylene-O-
substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted
alkylene are as
defined herein. Examples of such groups are methylenemethoxy (-CH20CH3),
ethylenemethoxy (-CH2CH20CH3), n-propylene-iso-propoxy (-
CH2CHzCH20CH(CH3~), methylene-t-butoxy (-CH2-O-C(CH3)3) and the like.
The term "alkylthioalkoxy" refers to the group -alkylene-S-alkyl,
alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted
alkylene-S-
substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted
alkylene are as
defined herein. Preferred alkylthioalkoxy groups are alkylene-S-alkyl and
include, by
way of example, methylenethiomethoxy (-CH2SCH3), ethylenethiomethoxy (-
CH2CH2SCH3), n-propylene-iso-thiopropoxy (-CH2CH2CH2SCH(CH3)2), methylene-t-
thiobutoxy (-CH2SC(CH3)3) and the like.
"Alkenyl" refers to a monoradical of a branched or unbranched unsaturated
hydrocarbon preferably having from 2 to 40 carbon atoms, preferably 2-10
carbon atoms,
more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This
term is
further exemplified by such radicals as vinyl, prop-2-enyl, pent-3-enyl, hex-S-
enyl, 5-
ethyldodec-3,6-dienyl, and the like.
The term "substituted alkenyl" refers to an alkenyl group as defined above
having
from 1 to 5 substituents selected from the group consisting of alkoxy,
substituted alkoxy,
acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oXyaminoacyl, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy,
substituted
thioalkoxy, aryl, heteroaryl, heterocyclic, aryloxy, thioaryloxy,
heteroaryloxy,
thioheteroaryloxy, heterocyclooxy, thioheterocyclooxy, nitro, -SO-alkyl, -SO-
substituted
alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-
aryl, -S02-
heteroaryl, and. -NRaRb, wherein Re and Rb may be the same or different and
are chosen
from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl,
aryl, heteroaryl and heterocyclic.
"Alkenylene" refers to a diradical of an unsaturated hydrocarbon, preferably
having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more
preferably 2-6
carbon atoms, and preferably having 1-6 double bonds. This term is further
exemplified


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8
by such radicals as 1,2-ethenyl, 1,3-prop-2-enyl, 1,5-pent-3-enyl, 1,4-hex-5-
enyl, 5-ethyl-
1,12-dodec-3,6-dienyl, and the like.
The term "substituted alkenylene" refers to an alkenylene group as defined
above
having from 1 to S substituents, selected from the group consisting of alkoxy,
substituted
alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,
oxyacylamino,
azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl,
thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl,
heteroaryloxy,
thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, nitro,
and NRBRb,
wherein Ra and Rb may be the same or different and are chosen from hydrogen,
optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,
aryl, heteroaryl
and heterocyclic. Additionally, such substituted alkenylene groups include
those where
2 substituents on the alkenylene group are fused to form one or more
cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
heterocyclic or
heteroaryl groups fused to the alkenylene group.
"Alkynyl" refers to a monoradical of an unsaturated hydrocarbon, preferably
having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more
preferably 2-6
carbon atoms, and preferably having 1-6 triple bonds. This term is further
exemplified
by such radicals as acetylenyl, prop-2-ynyl, pent-3-ynyl, hex-5-ynyl, S-
ethyldodec-3,6-
diynyl, and the like.
The term "substituted alkynyl" refers to an alkynyl group as defined above
having
from 1 to 5 substituents, selected from the group consisting of alkoxy,
substituted alkoxy,
acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy,
substituted
thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy,
thioheteroaryloxy,
heterocyclic, heterocyclooxy, thioheterocycloxy, vitro, -SO-alkyl, -SO-
substituted alkyl,
-SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-
heteroaryl,
S02-heterocyclic, NR8R6, wherein Ra and Rb may be the same or different and
are chosen
from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl,
aryl, heteroaryl and heterocyclic.
"Alkynylene" refers to a diradical of an unsaturated hydrocarbon radical,
preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms,
more


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9
preferably 2-6 carbon atoms, and preferably having 1-6 triple bonds. This term
is further
exemplified by such radicals as 1,3-prop-2-ynyl, 1,5-pent-3-ynyl, 1,4-hex-5-
ynyl, 5-
ethyl-1,12-dodec-3,6-diynyl, and the like.
The term "acyl" refers to the groups -CHO, alkyl-C(O)-, substituted alkyl-C(O)-
,
cycloalkyl-C(O)-, substituted cycloalkyl-C{O)-, cycloalkenyl-C(O)-,
substituted
cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where
alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.
The term "acylamino" refers to the group -C(O)NRR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,
heterocyclic or where
both R groups are joined to form a heterocyclic group (e.g., morpholino)
wherein alkyl,
substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
The term "aminoacyl" refers to the group -NRC(O)R where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic wherein
alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
The term "aminoacyloxy" refers to the group -NRC(O)OR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic wherein
alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-
,
cycloalkyl-C{O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-
C(O)O-, and
heterocyclic-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl,
aryl, heteroaryl, and heterocyclic are as defined herein.
The term "alkylamino" refers to the group -NHRa, where Re is alkyl as defined
above. The term "alkylaminoalkyl" rfers to the group -Rb-NHRe, where Ra is
alkyl as
defined above, and Rb is alkylene as defined above. Examples of
alkylaminoalkyl are n-
decylaminoethyl, 3-(dimethylamino)propyl, and the like.
The term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6
to
20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed
(fused} rings
(e.g., naphthyl or anthryl).
Unless otherwise constrained by the definition for the aryl substituent, such
aryl
groups can optionally be substituted with from 1 to 5 substituents selected
from the


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group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,
alkynyl,
cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted
alkenyl,
substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino,
aminoacyl,
acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano,
halo, vitro,
5 heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy,
oxyacylamino,
thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl,
-SO-
substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted
alkyl, -S02-aryl,
-S02-heteroaryl, trihalomethyl, NRaRb, wherein Ra and Rb may be the same or
different
and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl,
alkenyl,
10 cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. Preferred aryl
substituents
include alkyl, alkoxy, halo, cyano, vitro, trihalomethyl, and thioalkoxy.
The term "aryloxy" refers to the group aryl-O- wherein the aryl group is as
defined above including optionally substituted aryl groups as also defined
above.
The term "arylene" refers to a diradical derived from aryl or substituted aryl
as
defined above, and is exemplified by 1,2-phenylene, 1,3-phenylene, 1,4-
phenylene, 1,2-
naphthylene and the like.
The term "carboxyalkyl" refers to the group "-C(O)Oalkyl" where alkyl is as
defined above.
The term "cycloallcyl" refers to cyclic alkyl groups of from 3 to 20 carbon
atoms
having a single cyclic ring or multiple condensed rings. Such cycloalkyl
groups include,
by way of example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl,
cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and
the like.
The term "substituted cycloalkyl" refers to cycloalkyl groups having from
1 to 5 substituents selected from the group consisting of alkoxy, substituted
alkoxy,
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino,
aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,
heteroaryloxy,
heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, vitro, -SO-alkyl, -SO-

substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted
alkyl, -S02-aryl,
-S02-heteroaryl, and NRaRb, wherein Ra and Rb may be the same or different and
are


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11
chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl,
alkynyl, aryl, heteroaryl and heterocyclic.
The term "cycloalkenyl" refers to cyclic alkenyl groups of from 4 to 20 carbon
atoms having a single cyclic ring or fused rings and at least one point of
internal
unsaturation. Examples of suitable cycloalkenyl groups include, for instance,
cyclobut-
2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
The term "substituted cycloalkenyl" refers to cycloalkenyl groups having from
1
to 5 substituents selected from the group consisting of alkoxy, substituted
alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
acyl,
acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,
cyano,
halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,
thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted
thioalkoxy, aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl, -502-
1 S alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl, and NReRb,
wherein Ra and Rb
may be the same or different and are chosen from hydrogen, optionally
substituted alkyl,
cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
"Haloalkyl" refers to alkyl as defined above substituted by I-4 halo groups as
defined above, which may be the same or different, such as 3-fluorododecyl,
12,12,12-
trifluorododecyl, 2-bromooctyl, -3-bromo-6-chloroheptyl, and the like.
The term "heteroaryl" refers to an aromatic group of from 1 to 15 carbon atoms
and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at
least one ring
(if there is more than one ring).
Unless otherwise constrained by the definition for the heteroaryl substituent,
such
heteroaryl groups can be optionally substituted with 1 to 5 substituents
selected from the
group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,
alkynyl,
cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted
alkenyl,
substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino,
aminoacyl,
acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano,
halo, vitro,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy,
oxyacylamino,


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thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl,
-SO-
substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted
alkyl, -S02-aryl,
-S02-heteroaryl, trihalomethyl, mono-and di-alkylamino, and NRaRb, wherein Ra
and Rb
may be the same or different and are chosen from hydrogen, optionally
substituted alkyl,
cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
Preferred
heteroaryls nclude pyridyl, pyrrolyl and fiuyl.
The term "heteroaryloxy" refers to the group heteroaryl-O-.
The term "heteroarylene" refers to the diradical group derived from heteroaryl
or
substituted heteroaryl as defined above, and is exemplified by the groups 2,6-
pyridylene,
2,4-pyridiylene, 1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,
2,S-
pyridinylene, 1,3-morpholinylene, 2,S-indolenyl, and the like.
The term "heterocycle" or "heterocyclic" refers to a monoradical saturated or
unsaturated group having a single ring or multiple condensed rings, from 1 to
40 carbon
atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms, selected
from
1 S nitrogen, sulfur, phosphorus, and/or oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic
substituent,
such heterocyclic groups can be optionally substituted with 1 to S, and
preferably 1 to 3
substituents, selected from the group consisting of alkoxy, substituted
alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,
acylamino, acyloxy,
amino, aminoacyl, aminoacyloxy, oxyaminoacyl, cyano, halogen, hydroxyl, keto,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryioxy,
thioheterocyclooxy,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,
heteroaryloxy,
heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-

substituted alkyl, -SO-aryl, -SO-heteroaryl, -S02-alkyl, -S02-substituted
alkyl, -S02-aryl,
2S -S02-heteroaryl, and NRaRb, wherein Ra and Rb may be the same or different
and are
chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl,
alkynyl, aryl, heteroaryl and heterocyclic. Such heterocyclic groups can have
a single
ring or multiple condensed rings.
Examples of nitrogen heterocycles and heteroaryls include, but are not limited
to,
pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine,
isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine,


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naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline,
phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine,
phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline,
morpholino,
piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen
containing
heterocycles.
The term "heterocyclics" includes "crown compounds" which refers to a specific
class of heterocyclic compounds having one or more repeating units of the
formula [-
(CH2-)mY-] where m is equal to or greater than 2, and Y at each separate
occurrence can
be O, N, S or P. Examples of crown compounds include [-(CHZ)3-NH-]3, [-((CH2)2-
O)4-
((CH2)2-NH)2] and the like. Typically such crown compounds can have from 4 to
10
heteroatoms and 8 to 40 carbon atoms.
The term "heterocyclooxy" refers to the group heterocyclic-O-.
The term "thioheterocyclooxy" refers to the group heterocyclic-S-.
The term "heterocyclene" refers to the diradical group derived from a
heterocycle
as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-
morpholino and
the like.
The term "oxyacylamino" refers to the group -OC(O)NRR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic wherein
alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined
herein.
The term "thiol" refers to the group -SH.
The term "thioalkoxy" refers to the group -S-alkyl.
The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.
The term "thioaryloxy" refers to the group aryl-S- wherein the aryl group is
as
defined above including optionally substituted aryl groups also defined above.
The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein the
heteroaryl group is as defined above including optionally substituted aryl
groups as also
defined above.
As to any of the above groups which contain one or more substituents, it is
understood, of course, that such groups do not contain any substitution or
substitution
patterns which are sterically impractical and/or synthetically non-feasible.
In addition,


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14
the compounds of this invention include all stereochemical isomers arising
from the
substitution of these compounds.
"Alkyl optionally interrupted by 1-5 atoms chosen from O, S, or N" refers to
alkyl as defined above in which the carbon chain is interrupted by O, S, or
NRa, where Re
is hydrogen, alkyl, aryl, or heteroaryl, all of which may be optionally
substituted. Within
the scope are ethers, sulfides, and amines, for example 1-methoxydecyl, 1-
pentyloxynonane, 1-(2-isopropoxyethoxy)-4-methylnonane, 1-(2-
ethoxyethoxy)dodecyl,
2-(t-butoxy)heptyl, 1-pentylsulfanylnonane, nonylpentylamine, and the like.
"Heteroarylalkyl" refers to heteroaryl as defined above linked to alkyl as
defined
above, for example pyrid-2-ylmethyl, 8-quinolinylpropyl, and the like.
"Ligand" as used herein denotes an antibiotic compound that is a binding
partner
for a transglycosylase enzyme substrate and is bound thereto by
complementarity. The
specific region or regions of the ligand that is {are) recognized by the
enzyme substrate is
designated as the "ligand domain". By virtue of the ligand domain, a ligand
may be
1 S either capable of binding to a transglycosylase enzyme substrate by
itself, or may require
the presence of one or more non-ligand components for binding (e.g. Ca2+,
lVIg2+, or a
water molecule is required for the binding of a ligand domain).
Those skilled in the art will appreciate that portions of the ligand structure
that
are not essential for molecular recognition and binding activity {i.e. that
are not part of
the ligand domain) may be varied substantially, replaced or substituted with
unrelated
structures (for example, with ancillary groups as defined below), and, in some
cases,
omitted entirely without affecting the binding interaction. Accordingly, it
should be
understood that the term ligands is not intended to be limited to compounds
known to be
useful as antibiotics (for example, known drugs). Those skilled in the art
will understand
that the term ligand can equally apply to a molecule that is not normally
recognized as
having useful antibacterial properties, in that ligands that exhibit minimally
useful
properties as monomers can be highly active as multibinding agents, due to the
biological
benefit (increased biological effect) conferred by multivalency. The primary
requirement
for a ligand as defined herein is that it has a ligand domain as defined
above.
A preferred class of ligands are heptapeptides, known as the glycopeptide
antibiotics, characterized by a mufti-ring peptide core, and at least one
sugar attached at


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various sites, of which vancomycin is an important example. Examples of the
glycopeptide class of ligands included in this definition may be found in
"Glycopeptides
Classification, Occurrence, and Discovery", by Raymond C. Rao and Louise W.
Crandall, ("Drugs and the Pharmaceutical Sciences"Volume 63, edited by
Ramakrishnan
5 Nagarajan, published by Marcal Dekker, Inc.), which is hereby incorporated
by
reference. .Disclosed are glycopeptides identified as A477, A35512, A40926,
A41030,
A42867, A47934, A80407, A82846, A83850 ,A84575, A84428, AB-65, Actaplanin,
Actinoidin, Ardacin, Avoparcin, Azureomycin, Balhimycin, Chloroeremomycin,
Chloroorientiein, Chloropolysporin, Decaplanin, N desmethylvancomycin,
Eremomycin,
10 Galacardin, Helvecardin, Izupeptin, Kibdelin, LL-AM374, Mannopeptin,
MM45289,
MM47756, MM47761, MM49721, MM47766, MM55260, MM55266, MM55270,
MM56597, MM56598, OA-7653, Orenticin, Parvodicin, Ristocetin, Ristomycin,
Synmonicin, Teicoplanin, UK-68597, UK-69542, UK-72051, vancomycin, and the
like.
Another preferred class of ligands is the general class of glycopeptides
disclosed above
15 on which the sugar moiety is absent, i.e. the aglycone series of
heptapeptides. For
example, removal of the disaccharide moiety appended to the phenol on
vancomycin (as
shown below as Formula II) by mild hydrolysis gives vancomycin aglycone. A
further
preferred class of ligands are glycopeptides that have been further appended
with
additional saccharide residues, especially aminoglycosides, in a manner
similar to
vancosamine.


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16
"Vancomycin" refers to the antibacterial compound whose structure is
reproduced
below as Formula II.
10
[C
fRl
Formula II
"Optional" or "optionally" means that the subsequently described event or
circumstance may or may not occur, and that the description includes instances
where said
event or circumstance occurs and instances in which it does not. For example,
"optionally
substituted glycopeptide" with respect to a compound of Formula I refers to a
ligand as
defined above in which those positions that are not linked to X may or may not
be
substituted by various groups as defined below. The term also includes those
instances in
which one amino acid of the basic core structure is replaced by another amino
acid, for
example as described in "Preparation and conformational analysis of vancomycin
hexapeptide and aglucovancomycin hexapeptide", by Booth, Paul M.; Williams,
Dudley
fN1


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17
H., Univ. Chem. Lab., Cambridge, UK., J. Chem. Soc., Perkin Trans. 1 (1989),
(12),
2335-9, and "The Edman degradation of vancomycin: preparation of vancomycin
hexapeptide",. Booth, Paul M.; Stone, David J. M.; Williams, Dudley H., Univ.
Chem.
Lab., Cambridge, UK., J. Chem. Soc., Chem. Commun. (1987), (22), 1694-5.
"Optionally substituted vancomycin" with respect to the multibinding agents of
the
invention refers to vancomycin in which the hydroxy group at any position, the
[R] position,
the carboxyl groups at the (C] position, or the amine groups at the [V] or [N]
position that
are not attached to the linker X may or may not be substituted by various
groups. Such
groups include:
Ra, where Ra at each occurrence is chosen from alkyl, alkyl optionally
interrupted by 1-5
atoms chosen from O, S, or NRb-, where Rb is alkyl, aryl, or heteroaryl, all
of which are
optionally substituted, haloalkyl, alkenyl, alkynyl,,alkylamino,
alkylaminoalkyl, cycloalkyl,
alkanoyl, aryl, heteroaryl, heterocyclic, additional saccharide residues,
especially
aminoglycosides, all of which are optionally substituted as defined above;
and:
NRcRd, in which Rc and Rd are independently hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, alkanoyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, or Rc
and Rd when taken
together with the nitrogen to which they are attached represent a heterocyclo
group,
quarternary alkyl and aryl ammonium compounds, pyridinium ions, sulfonium
ions, and the
like, all of which are optionally substituted as defined above. An example of
a preferred [C]
substitution is dimethylaminopropylamino and glucosamino; an example of a
preferred [V]
substitution is alkyl, for example n-decyl, or alkylaminoalkyl, for example n-
decylaminoethyl. "Optionally substituted vancomycin aglycone " with respect to
the
multibinding agents of the invention refers to vancomycin aglycone in which
the hydroxy
group at any position, particularly the hydroxy group at the [O] position, the
[R] position,
the carboxyl groups at the [C] position, or the amine group at the [N]
position, that are not
attached to the linker X may or may not be substituted by various groups Ra as
defined
above.
"Transglycosylase enzyme substrate" as used herein denotes the molecular
target
of the transpeptidase enzyme. The substrate binds to the enzyme and eventually
results
in synthesis of the bacterial cell wall. The action of this enzyme is
inhibited by a ligand
domain that binds to the enzyme substrate. A ligand such as vancomycin binds
to this


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18
substrate and in effect "sequesters" the substrate to prevent its recognition
by the enzyme
and subsequent use in the construction of the bacterial cell wall.
"Multibinding agent" or "multibinding compound" as used herein refers to a
compound that is capable of multivalency as defined below, and which has 2-10
ligands,
which may be the same or different, connected by one or more covalent linker
or linkers,
which may be the same or different, preferably from 1-20. A multibinding agent
provides a biological and/or therapeutic effect greater than the aggregate of
the unlinked
ligands equivalent thereto. That is to say, an improved biological and/or
therapeutic
effect of the multibinding agent is obtained as measured against that achieved
by the
same number of unlinked ligands available for binding to the ligand binding
site of the
transglycosylase enzyme substrate. Examples of increased biological and/or
therapeutic
effect with respect to the target include, for example, increased specificity,
increased
affinity, increased selectivity, increased potency, increased efficacy,
increased
therapeutic index, a change in the duration of action, decreased toxicity,
decreased side
1 S effects, improved bioavailability, improved phanmacokinetics, improved
activity
spectrum, improved ability to kill bacteria, and the like. The multibinding
compounds of
the invention will exhibit one or more of the foregoing effects.
"Potency" as used herein refers to the minimum concentration at which a ligand
is able to achieve a desirable biological or therapeutic effect. The potency
of a ligand is
typically proportional to its affinity for its ligand binding site. In some
cases, the
potency may be non-linearly correlated with its affinity. In comparing the
potency of
two drugs, e.g., a multibinding agent and the aggregate of its unlinked
ligand, the dose-
response curve of each is determined under identical test conditions (e.g., in
an in vitro or
in vivo assay or in an appropriate animal model, such as a human patient. The
finding
that the multibinding agent produces an equivalent biological or therapeutic
effect at a
lower concentration than the aggregate unlinked ligand (e.g., on a per weight,
per mole,
or per ligand basis) is indicative of enhanced potency.
"Univalency" as used herein refers to a single binding interaction between the
ligand domain of one ligand as defined herein with the ligand recognition site
of a
transglycosylase enzyme substrate as defined herein. It should be noted that a
compound
having multiple copies of a ligand (or ligands) exhibits univalency when only
one ligand


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19
of that compound is interacting with a ligand binding site. Examples of
univalent
interactions are depicted below.
where the arrow represents a ligand domain and the indent represents the
ligand
binding site of a transglycosylase enzyme substrate,
"Multivalency" as used herein refers to the concurrent binding of 2 to 10
linked
Iigands (which may be the same or different) and two or more corresponding
ligand
binding sites.
Accordingly, two ligands connected by a linker that bind concurrently to two
ligand binding sites would be considered as a bivalent compound; similarly,
three ligands
thus connected provide a trivalent compound.
It should be understood that all compounds that contain multiple copies of a
ligand attached to a linker (or linkers) do not necessarily exhibit the
phenomena of
multivalency, i.e. that improved biological and/or therapeutic effect of the
multibinding
agent is obtained as measured against that produced by the same number of
unlinked
ligands available for binding to a ligand binding site. For multivalency to
occur, the
ligand domains of the ligands that are connected by a linker have to be
presented to their
"receptors" (i.e. the ligand binding sites) by the linker in a specific manner
in order to
bring about the desired Iigand-orienting result, and thus produce a
multibinding event.
"Selectivity" or "specificity" in general is a measure of the binding
preferences of
a ligand for different receptors and/or different ligands for the same
receptor. The
selectivity of a ligand with respect to its target receptor relative to
another receptor is
given by the ratio of the respective values of Kd (i.e., the dissociation
constants for each
ligand-receptor complex), or in cases where a biological effect is observed
below the Kd,
selectivity is given by the ratio of the respective ECsos (i.e. the
concentrations that
produce SO% of the maximum response for the ligand interacting with the two
distinct


CA 02318394 2000-07-12
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receptors).
The term "ligand recognition site" or "ligand binding site" as used herein
denotes
the site on a transglycosylase enzyme substrate that recognizes a ligand
domain and
provides a binding partner. The ligand binding site may be defined by
monomeric or
5 multimeric structures.
As used herein, the terms "inert organic solvent" or "inert solvent" mean a
solvent
inert under the conditions of the reaction being described in conjunction
therewith
[including, for example, benzene, toluene, acetonitrile, tetrahydrofuran
("THF"),
dimethylformamide ("DMF"), chloroform ("CHC13"), methylene chloride (or
10 dichloromethane or "CH2C 12), diethyl ether, ethyl acetate, acetone,
methylethyl ketone,
methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, and
the IikeJ.
Unless specified to the contrary, the solvents used in the reactions of the
present invention
are inert solvents.
"Pharmaceutically acceptable salt" means those salts which retain the
biological
15 effectiveness and properties of the compounds of Formula I, and which are
not biologically
or otherwise undesirable. The compounds of Formula I are capable of forming
both acid
and base salts by virtue of the presence of amino and carboxyl groups
respectively.
1. Pharmaceutically acceptable base addition salts may be prepared from
inorganic and
organic bases. Salts derived from inorganic bases include, but are not limited
to, the
20 sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts
derived from
organic bases include, but are not limited to, salts of primary, secondary and
tertiary amines,
substituted amines including naturally-occurring substituted amines, and
cyclic amines,
including isopropylamine, trimethyl amine, diethylamine, triethylamine,
tripropylamine,
ethanolamine, 2-dimethyiaminoethanol, tromethamine, lysine, arginine,
histidine, caffeine,
procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-
alkylglucamines,
theobromine, purines, piperazine, piperidine, and N-ethylpiperidine. It should
also be
understood that other carboxylic acid derivatives would be useful in the
practice of this
invention, for example carboxylic acid amides, including carboxamides, lower
alkyl
carboxamides, di(lower alkyl) carboxamides, and the like.
2. Pharmaceutically acceptable acid addition salts may be prepared from
inorganic and
organic acids. Salts derived from inorganic acids include hydrochloric acid,
hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived
from organic acids


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21
include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
malic acid,
malonic acid, succinic acid, malefic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic
acid, salicylic acid and the like.
It should be understood that the compounds of the invention include racemic
ligands
as well as the individual stereoisomers of the ligands, including enantiomers
and non-
racemic mixtures thereof. The scope of the invention as described and claimed
encompasses
the racemic forms of the ligands as well as the individual enantiomers and non-
racemic
mixtures thereof.
The term "treatment" as used herein covers any treatment of a condition or
disease
in an animal, particularly a mammal, more particularly a human, and includes:
(i) preventing the disease or condition from occurring in a subject which may
be
predisposed to the disease but has not yet been diagnosed as having it;
(ii) inhibiting the disease or condition, i.e. arresting its development;
(iii) relieving the disease or condition, i.e. causing regression of the
condition; or.
(iv) relieving the conditions caused by the disease, i.e, symptoms of the
disease.
The term "disease state that is alleviated by treatment with an antibacterial
agent" as
used herein is intended to cover all disease states which are acknowledged in
the art to be
usefully treated with an antibacterial agent in general, and those disease
states which have
been found to be usefully treated by the specific antibacterials of our
invention, including
the compounds of Formula I. Such disease states include, but are not limited
to, treatment of
a mammal afflicted with pathogenic bacteria, in particular staphylococci
(methicillin
sensitive and resistant), streptococci (penicillin sensitive and resistant),
enterococci
(vancomycin sensitive and resistant), and Clostridium docile
The term "therapeutically effective amount" refers to that amount which is
sufficient
to effect treatment, as defined above, when administered to a mammal in need
of such
treatment. The therapeutically effective amount will vary depending on the
subject and
disease state being treated, the severity of the ai~liction and the manner of
administration,
and may be determined routinely by one of ordinary skill in the art.
The term "protecting group" or "blocking group" refers to any group which when
bound to one or more hydroxyl, thiol, amino or carboxyl groups of the
compounds
prevents reactions from occurnng at these groups and which protecting group
can be


CA 02318394 2000-07-12
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22
removed by conventional chemical or enzymatic steps to reestablish the
hydroxyl, thio,
amino or carboxyl group. The particular removable blocking group employed is
not
critical and preferred removable hydroxyl blocking groups include conventional
substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl,
benzylidine, phenacyl,
t-butyl-diphenylsilyl and any other group that can be introduced chemically
onto a
hydroxyl functionality and later selectively removed either by chemical or
enzymatic
methods in mild conditions compatible with the nature of the product.
Protecting groups
are disclosed in more detail in T.W. Greene and P.G.M. Wuts, "Protective
Groups in
Organic Synthesis" 2"d Ed., 1991, John Wiley and Sons, N.Y.
Preferred removable amino blocking groups include conventional substituents
such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),
fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC) and the like, which
can
be removed by conventional conditions compatible with the nature of the
product.
Preferred carboxyl protecting groups include esters such as methyl, ethyl,
propyl,
t-butyl etc. which can be removed by mild conditions compatible with the
nature of the
product.
"Linker" or "linkers" as used herein, identified where appropriate by the
symbol
X, refers to a group or groups that covalently links) from 2-10 ligands (as
defined
herein) in a manner that provides a multibinding agent capable of
multivalency. The
linker is a ligand domain orienting entity that permits attachment of multiple
copies of
ligands (which may be the same or different) thereto. The extent to which
multivalent
binding is realized depends upon the efficiency with which the linker that
joins the
ligands permits the ligand domains to be presented to the ligand recognition
sites (on
enzymes and enzyme substrates). Beyond presenting ligand domains for
multivalent
interactions with multivalent receptors (enzymes or enzyme substrates), the
linker
spatially constrains these interactions to occur within dimensions defined by
the linker.
Thus, the structural features of the linker (valency, geometry, orienting
capabilities, size,
flexibility, chemical composition) are features of multibinding agents that
play an
important role in determining their activities. The term linker, however, does
not include
solid inert supports such as beads, resins, glass particles, rods, fibres, and
the like, but it
should be understood that the multibinding compounds of the invention can be
attached


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23
to a solid support if desired to provide, for example, a material useful for
separation and
purification processes (e.g. affinity chromatography).
The ligands are covalently attached to the linker or linkers using
conventional
chemical techniques, for example reaction between a carboxylic acid and an
amine to
form an amide, an amine and a sulfonyl halide to form a sulfonamide, an
alcohol or
phenol with an alkyl or aryl halide to form an ether, and the like.
The linker (or linkers) is attached to the ligand at a position such that the
ligand
domain is permitted to orient itself appropriately in order to bind to the
ligand binding
site. The term linker embraces everything that is not considered to be part of
the ligand.
The relative orientation in which the ligand domains are displayed derives
from
the particular point or points of attachment of the ligands to the linker, and
on the
framework geometry. The determination of where acceptable substitutions can be
made
on a ligand is typically based on prior knowledge of structure-activity
relationships of the
ligand and/or congeners and/or structural information about ligand-receptor
complexes
(e.g., from X-ray crystallography, NMR). Such positions and the synthetic
methods for
covalent attachment are well known in the art.
Suitable linkers are discussed below.
At present, it is preferred that the multibinding agent is a bivalent
compound, in
which two ligands are covalently linked. For example, a glycopeptide bivalent
compound
may be constructed by linking any hydroxyl group, carboxyl group, "resorcinol"
group [R],
or amino group of a first glycopeptide ligand to any hydroxyl group, carboxyl
group,
"resorcinol" group [R], or amino group of a second glycopeptide. It should be
noted that the
term "hydroxyl group" includes a hydroxyl group obtained by reducing the
carboxyl
group at the [C) position to a hydroxymethyl group, as well as the hydroxyl
groups on
the sugars and on the peptide core structure."
"Increased biological effect" as used herein includes, but is not limited to,
increased affinity, increased selectivity, increased potency, increased
efficacy, increased
duration of action, decreased toxicity, and the like.


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24
The invention relates to multivalent compounds as defined above, comprising
ligands that are binding partners for a transglycosylase enzyme substrate. The
preferred
ligands are optionally substituted glycopeptides, or their corresponding
aglycones.
In the reaction schemes that follow, glycopeptides are depicted in a
simplified form
as a shaded box that shows only the carboxyl terminus, labeled [C], the sugar
amine
terminus (for example, vancosamine), labeled [V], and the "non-sugar" amino
terminus,
labeled [N], as follows:
H O
OH
1
(C(
M
where R is hydrogen (as in N-desmethylvancomycin) or methyl (as in
vancomycin).
It can therefore be seen by way of exemplification that one general class of
multivalent compounds that fall within the scope of the definition of Formula
I include
compounds in which ligands, including optionally substituted ligands, are
connected by one
or more linkers X at the [C-C], [V-V], [N-N], [C-V], [C-N], and [V-N] termini.
Additionally, within the scope of the invention are the aglycone derivatives
of
glycopeptides. In the reaction schemes they are depicted as a shaded triangle
that shows
only the carboxyl terminus, labeled [C], the aglycone hydroxy terminus,
labeled [O], and the
"non-sugar" amino terminus, labeled [N], as follows:
R/
where R is hydrogen (as in N-desmethylvancomycin aglycone) or methyl (as in
vancomycin
aglycone).
It can therefore be seen by way of exemplification that another general class
of
multivalent compounds that fall within the scope of the definition of Formula
I include those


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compounds in which 2-10 ligands are connected by one or more linkers X at the
[C-C], [O-
O], [N-N], [C-O], [C-N], and [O-N] termini.
Also within the scope of the invention are glycopeptides linked via their [C],
[V], or
[N] termini to aglycone derivatives of glycopeptides linked via their [C],
[O], or (N] termini.
5 A third class of compounds falling within the scope of the invention are
those in
which the glycopeptides, or aglycone derivatives thereof, are linked via the
[R] position.
Reaction schemes that exemplify this linking strategy depict the ligands in a
simplified form
as above, i.e. as a shaded box in which the carboxyl terminus is labeled [C],
the
vancosamine amino terminus is labeled [V], and the "non-sugar" amino terminus
is labeled
10 [N], with the addition of the [R] position as a resorcinol derivative, as
below:
lHl
where R is hydrogen or methyl.
As previously described, glycopeptides are generally depicted in a simplified
form
as a shaded box that shows only the glycopeptide carboxyl terminus, labeled
[C], the sugar
amine {vancosamine in vancomycin) terminus, labeled [V], and the "non-sugar"
amino
terminus, labeled [N].
R'
The preferred compounds of the invention are those represented by compounds of
Formula I, (L)~, in which p is 2 and q is 1. The following nomenclature system
will be
used for naming the compounds of the invention and substituted ligands used in
their
preparation, using vancomycin as an example (Formula II), but it should be
understood
that all compounds of the invention may be named following these principles.


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26
Substituted glycopeptides are within the scope of the definition of "ligand",
and
the point of substitution will be indicated as follows. For example,
vancomycin
substituted at the [C] terminus that has the formula:
H O
S R~ N
~~N~
NHz
where R is methyl;
is named [C]-3-(dimethylamino)propylamino-(vancomycin).
It should be noted that although the linkage at the [C] terminus of the
glycopeptide is an amide, the name is intended to indicate the linker only,
i.e. it is named
as an amine, not an amide. In this manner, confusion is avoided as to whether
the
glycopeptide terminal group (in this case [C]) is part of the linker as named.
For bivalent compounds, the positions at which the linking group connects to
two
vancomycin termini will be indicated as [C-C] when the links are joined to the
carboxyl
1 S termini of both vancomycin molecules. Thus, a compound of the structure:
H
R N H'~/~.N~/O . N'R
z HN
z
where R is methyl, represents two glycopeptides (vancomycin in this example)
linked by
a 1,8-diaminooctane group at the [C] positions of the glycopeptide. It will be
named as:
[C-C]-octane-1, 8-diamino-bis(vancomycin)
which indicates that two unsubstituted vancomycin molecules are linked by
NH-(CH2)8_NH-, each NH groups being attached to the carboxy termini of each
2S vancomycin. As above, although the linkage at the [C] terminus of the
glycopeptides is
an amide, the name indicates the linker only, i.e. it is named as a diamine,
not a diamide.
Similarly, [V-V]-heptan-1,7-dioyl-bis-(vancomycin) indicates that two
unsubstituted vancomycin molecules are linked by -C(O)-(CH2)5-C(O)-, both C(O)
groups being attached to the vancosamine (V) termini of each vancomycin, i.e.
a


CA 02318394 2000-07-12
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27
compound of the structure:
H O H
R.N OH HO N.R
N
H
where R is methyl.
In the case where two unsubstituted vancomycin molecules are linked at two
different termini, for example a [C] termini of a first vancomycin to a [V]
termini of a
second vancomycin with a linker of formula -NH-(CH2)6-CO-, i.e., a compound of
the
structure:
R
where R is methyl;
the compound is named [C-V]-1-aminoheptan-7-oyl-bis-(vancomycin).
Where an unsubstituted vancomycin molecule is linked to a vancomycin
molecule that is substituted at the [CJ-position, with a linker of formula -NH-
(CH2)6-CO-
, i.e., a compound of the structure:
R
where R is methyl and R1 is 3-(dimethylamino)propylarnino;
is named (vancomycin) [C-V]-heptan-1-amino-7-oyl-([C]-3-
(dimethylamino)propylamino-vancomycin).
Similarly, (N-N] indicates linking of two vancomycin molecules by the
methylamino termini, [R-R] indicates linking of two vancomycin molecules by
the
"resorcinol" termini. [O-O] indicates that two aglycone molecules are linked
by the


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28
phenolic hydroxy group that remains after hydrolysis of the sugar. [C-V]
indicates that
the [C] terminus of a first vancomycin is linked to the [V] terminus of a
second
vancomycin. Similar nomenclature applies to vancomycin linked [N-V], [C-O], [O-
V],
[C-R], and the like.
When both vancomycins are unsubstituted or identically substituted, the
nomenclature will be as above, i.e. a compound designated as [V-V]-decan-1,10-
dioyl
bis -([C]-butylamino vancomycin) indicates that two vancomycin molecules, both
of
which are substituted at the [C] position by CH3(CH2)3NH- are linked by --C(O)-
(CH2)8-
C(O)-, both C(O) termini being attached to the vancosamine (V) termini of each
vancomycin. However, when both vancomycins are not identically substituted,
for
example a compound in which the first ligand is [C)-dimethylamino-vancomycin,
and a
second ligand is [C]-butylamino vancomycin), linked by
-C(O)-(CH2)g-C(O)-, both C(O) termini being attached to the vancosamine (V)
termini
of each vancomycin, the compound is named:
([C]-dimethylamino-vancomycin) [V-V]-decan-1,10-dioyl-([C]-butylamino
vancomycin).
Similarly, a compound in which the first Iigand is [V]-octylvancomycin, and a
second Iigand is[C]-N-glucosamino-vancomycin, Iinked by
-NH-(CH2)2-NH-C(O)-(CH2)2-C(O)-NH-(CH2)2-
at the [C]-position of the first Iigand to the [V]-position of the second
ligand is named:
([V]-octylvancomycin)-[C-V]-butane-1,4-dioc acid (2-aminoethyl)-amide-(2-
ethyl)-
amide-([C]-N-glucosanuno-vancomycin).
Accordingly, a compound of Formula I wherein p is 2 and q is 1, and the
ligands
L are both vancomycin linked via their carbon [C] termini by:
-NH-(CH2)2 NHC(O)(CH2)3C(O)NH(CH2)2-NH-
is named as
[C-C]-[pentane-1,5-dioic acid bis-[(2-aminoethyl)-amide]-bis-(vancomycin)
A compound of Formula I wherein p is 2 and q is 1, and a first ligand is [V]-n-

decylaminoethylvancomycin, and a second ligand is vancomycin, the ligands
being
linked via their carbon [C] termini by:


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WO 99/42476 PCT/US99/03850
29
-NH-(CH2)2 NHC(O)(CH2)3C(O)NH(CH2)2-NH-
is named as:
([V]-n-decylaminoethylvancornycin)-[C-C]-[pentane-1,5-dioic acid bis-[(2-
aminoethyl)-amide]-(vancomycin)
A compound of Formula I wherein p is 2 and the ligands L are both [V]-3-
dimethyl-aminopropyl vancomycin linked via their carbon [C] termini by:
-NH-{CH2)2 ~C(C){CH2)sC(C)~{CH2)rNH-
is named as:
[C-C]-[pentane-1,5-dioic acid bis-[(2-aminoethyl)-amide]-bis-([V)-3-(dimethyl-
amino)propyl vancomycin)
A compound of Formula I wherein p is 2 and the ligands L are both vancomycin
linked via their vancosoamine [V] termini by:
-(CH2)2 NHC(O){CH2)3C(O)NH(CH2)2-
is named:
[V-V]-pentane-1,5-dioic acid-bis-[(2-ethyl)-amide]-bis-(vancornycin)
A compound of Formula I wherein p is 2 and one ligand L is vancomycin linked
via its carbon [Cj terminus and the second ligand L is vancomycin linked via
its
vancosoamine [VJ terminus by:
-NH-(CH2)2 NHC(O)(CH2)3C(O)NH-(CH2)2-
is named:
[C-V]-pentane-1,5-dioic acid-[(2-aminoethyl)-amide]-[(2-ethyl)-amide]-bis-
(vancomycin).
A compound of Formula I wherein p is 2 and the ligands L are both vancomycin
linked via their methylamino [N] termini by:
-(CH2)2 NHC(O)(CH2)3C(O)NH(CH2)2-
is named:
[N-N]-[pentane-1,5-dioic acid bis-[(2-ethyl)-amide] bis-(vancomycin)


CA 02318394 2000-07-12
WO 99/42476 , PCT/US99/03850
A compound of Formula I wherein p is 2 and the first ligand L is vancomycin
linked via its carbon [C] terminus and the second ligand L is vancomycin
linked via its
methylamino [N] terminus by:
-NH-(CH2)2 NHC(O)(CH2)3C(O)NH(CH2~-
is named:
[C-N]-pentane-1,5-dioic acid-[(2-aminoethyl)-amide]-[(2-ethyl)-amide]-bis-
(vancomycin).
UTILITY
10 The multibinding agents of the invention, including the compounds of
Formula I,
and their pharmaceutically acceptable salts, are useful in medical treatments
and exhibit
biological activity, in particular antibacterial activity, which can be
demonstrated in the tests
described in the Examples. Such tests are well known to those skilled in the
art, and are
referenced and described in the fourth edition of "Antibiotics in Laboratory
Medicine", by
15 Victor Lorian, M.D., published by Williams and Wilkins, which is hereby
incorporated by
reference.
PHARMACEUTICAL COMPOSITIONS
Those compounds of the invention, including the compounds of Formula I, and
their
20 pharmaceutically acceptable salts that are active when given orally can be
formulated as
liquids for example syrups, suspensions or emulsions, tablets, capsules and
lozenges.
A liquid composition will generally consist of a suspension or solution of the
compound or pharmaceutically acceptable salt in a suitable liquid carrier(s),
for example
ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol,
oils or water,
25 with a suspending agent, preservative, surfactant, wetting agent, flavoring
or coloring agent.
Alternatively, a liquid formulation can be prepared from a reconstitutable
powder.
For example a powder containing active compound, suspending agent, sucrose and
a
sweetener can be reconstituted with water to form a suspension; and a syrup
can be prepared
from a powder containing active ingredient, sucrose and a sweetener.
30 A composition in the form of a tablet can be prepared using any suitable
pharmaceutical carriers) routinely used for preparing solid compositions.
Examples of such
carriers include magnesium stearate, starch, lactose, sucrose,
microcrystalline cellulose and


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31
binders, for example polyvinylpyrrolidone. The tablet can also be provided
with a color
film coating, or color included as part of the carrier(s). In addition, active
compound can be
formulated in a controlled release dosage form as a tablet comprising a
hydrophilic or
hydrophobic matrix.
A composition in the form of a capsule can be prepared using routine
encapsulation
procedures, for example by incorporation of active compound and excipients
into a hard
gelatin capsule. Alternatively, a semi-solid matrix of active compound and
high molecular
weight polyethylene glycol can be prepared and filled into a hard gelatin
capsule; or a
solution of active compound in polyethylene glycol or a suspension in edible
oil, for
example liquid paraffin or fractionated coconut oil can be prepared and filled
into a soft
gelatin capsule.
The compounds of the invention, including the compounds of Formula I, and
their
pharmaceutically acceptable salts that are active when given parenterally can
be formulated
for intramuscular, intrathecal, or intravenous administration.
A typical composition for infra-muscular or intrathecal administration will
consist of
a suspension or solution of active ingredient in an oil, for example arachis
oil or sesame oil.
A typical composition for intravenous or intrathecal administration will
consist of a sterile
isotonic aqueous solution containing, for example active ingredient and
dextrose or sodium
chloride, or a mixture of dextrose and sodium chloride. Other examples are
lactated
Ringer's injection, lactated Ringer's plus dextrose injection, Normosol-M and
dextrose,
Isolyte E, acylated Ringer's injection, and the like. Optionally, a co-
solvent, for example
polyethylene glycol, a chelating agent, for example ethylenediamine tetracetic
acid, and an
anti-oxidant, for example, sodium metabisulphite may be included in the
formulation.
Alternatively, the solution can be freeze dried and then reconstituted with a
suitable solvent
just prior to administration.
The compounds of the invention, including the compounds of Formula I, and
their
pharmaceutically acceptable salts which are active on rectal administration
can be
formulated as suppositories. A typical suppository formulation will generally
consist of
active ingredient with a binding and/or lubricating agent such as a gelatin or
cocoa butter or
other tow melting vegetable or synthetic wax or fat.
Compounds of Formula I and their pharmaceutically acceptable salts which are
active on topical administration can be formulated as transdermal
compositions. Such


CA 02318394 2000-07-12
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32
compositions include, for example, a backing, active compound reservoir, a
control
membrane, liner and contact adhesive.
The typical daily dose of a compound of Formula I varies according to
individual needs, the
condition to be treated and with the route of administration. Suitable doses
are in the
general range of from 0.01-100 mg/kg/day, preferably 0.1-50 mg/kg/day. For an
average 70
kg human, this would amount to 0.7mg to 7g per day, or preferably 7mg to 3.Sg
per day.
One of ordinary skill in the art of treating such diseases will be able,
without undue
experimentation and in reliance upon personal knowledge and the disclosure of
this
application, to ascertain a therapeutically effective amount of the compounds
of the
invention, including the compounds of Formula I, for a given disease.
METHODS OF PREPARATION
Linker
The linker (or linkers), when covalently attached to multiple copies of the
ligands, provides a biocompatible, substantially non-immunogenic multibinding
agent.
The biological effects of the multibinding agent are highly sensitive to the
valency,
geometry, composition, size, flexibility or rigidity, the presence or absence
of anionic or
cationic charge, and similar considerations (including hydrophilicity and
hydrophobicity
as discussed below) with respect to the linker. Accordingly, the linker is
preferably
chosen to maximize the desired biological effect. The linker may be
biologically
"neutral", i.e. not itself contribute any biological activity to the compound
of Formula I,
or it may be chosen to enhance the biological effect of the molecule. In
general, the
linker may be chosen from any organic molecule that orients two or more
ligands to the
receptors (enzymes or enzyme substrates), and permits multivalency. In this
regard, the
linker can be considered as a "framework" on which the ligands are arranged in
order to
bring about the desired ligand-orienting result, and thus produce a
multibinding agent.
For example, different orientations can be achieved by including in the
framework groups containing monocyclic or polycyclic groups, including aryl
and
heteroaryl groups, or structures incorporating one or more carbon-carbon
multiple bonds
(i.e., alkenes and alkynes). Other groups can also include oligomers and.
polymers which

CA 02318394 2000-07-12
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33
are branched- or straight-chain species. In preferred embodiments, rigidity is
imparted by
the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl,
heterocycles, etc.). In other
preferred embodiments, the ring is a six-or ten membered ring. In still
further preferred
embodiments, the ring is an aromatic group such as, for example, phenyl or
naphthyl.
Different frameworks can be designed to provide preferred orientations of the
Iigands. Such frameworks may be represented by using an array of dots (as
shown
below) wherein each dot may potentially be an atom, such as C, O, N, S, P, H,
F, Cl, Br,
and F, or the dot may alternatively indicate the absence of an atom at that
position. To
facilitate the understanding of the framework structure, the framework is
illustrated as a
two dimensional array in the following diagram, although clearly the framework
is a
three dimensional array in practice:
...


~~ ~ ~ ~ ~ ~ ~ ~ ~ ....


...


...


...


...



ci I ~ I ~ I ~ I ~ ....


...


....


~ 1 2 3 4 5 8 7 g


Each dot is either an atom, chosen from carbon, hydrogen, oxygen, nitrogen,
sulfur, phosphorus, or halogen, or the dot represents a point in space (i.e.
an absence of
an atom). Only certain atoms on the grid have the ability to act as an
attachment point
for the ligands, namely C, O, N, S, and P.
Atoms can be connected to each other via bonds (single, double, or triple with
acceptable resonance and tautomeric forms), with regard to the usual
constraints of
chemical bonding. Ligands may be attached to the framework via single, double,
or


CA 02318394 2000-07-12
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34
triple bonds (with chemically acceptable tautomeric and resonance forms).
Multiple
ligand groups (2 to 10) can be attached to the framework such that the
minimal, shortest
path distance between adjacent ligand groups does not exceed 100 atoms or 40
angstroms.
The intersection of the framework (linker) and the ligand group, and indeed,
the
framework (linker) itself can have many different bonding patterns. Examples
of
acceptable patterns of three contiguous atom arrangements within the linker
and at the
linker-ligand interface are shown in the following diagram.



CCO OCN SCN PCN
NCO


CCS OCO SCO PCO


CCP NCP OCP SCP PCP



NN


CNO N ONN SN N PNN
N NO


C N P N S ~ P
N N


p - ~ S S
-p ~


ONP SNP pNp


NOC


COO ~ ~ SON PON


EO P ~' _ S P
O
O


C ~ SA P~
D' B'F 'S
'


CSN NSN OSC OSC PSC


CSS NSO OSO SSO



CSP NSP OSP _
';S~F


C
P
P


CPO NPN OPN SPN p
'p
~
C[
-
-


p
Cpp OPS SPS F
a


NPP p'p'~
'


O S P p
P P p'p
P


One skilled in the art would be able to identify bonding patterns that would
produce multivalent compounds. Methods for producing these bonding
arrangements are
described in "Advanced Organic Chemistry, 4~' Edition" by March (Wiley-
Interscience
(New York), 1992). These arrangements are described in the grid of dots shown
in the
Scheme above. All of the possible arrangements for the five most preferred
atoms are
shown. Each. atom has a variety of acceptable oxidation states. The bonding
arrangements underlined are less acceptable and are not preferred.

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WO 99/424?6 PCT/US99/03850
Examples of molecular structures in which the above bonding patterns could be
employed 'as components of the linker are shown below.
0
wO.C~C~ wN.C.C~
N N ~O N
5 0
C. C
wC.C.C~ wC.O~Ci ~C.N~Ci wC.C~C~ wC.N~Ci
II II O
\N~N~ ~O~N~ ~C~N~ \C~C~C' ~ ~ i
O C
0
~ ,S. i O S, i ~ .S, i ~ .S. i
C N ~C~O Ni ~C~O C C C C S
O 0
wC.S.Ci wC.O.Ni ~O~N~ ~C~g~C~ ~C.S~Ci
O
.C. ~ O
wN.O Ni O O \N~~ \N~~ ~C,N~C~
wS~C.O~ wS.C.S~ wN.C~O~ ~
wN~~ wN~~
0
wN,N~ wC.P~Ci ~N,P~Ci w .P~ ~ ' O
p_ O a C
The identification of an appropriate framework geometry for Iigand domain
presentation is an important first step in the construction of a multivalent
binding agent
with enhanced activity. Systematic spatial searching strategies can be used to
aid in the
identification of preferred frameworks through an iterative process. Various
strategies
are known to those skilled in the art of molecular design and can be used for
preparing
the compounds of this invention.
For example, a bivalent compound can comprise ligands (represented as L)

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3b
attached to central core structures such as those shown below.
L L L
L
L ~ I ~ L
I / L
L
L L L~L
L
L
I
L L.O~N~O.L
L
I \ H H
N~L LAN N.
L
L
O OH
It is to be noted that core structures other than those shown here can be used
for
determining the optimal framework display orientation of the ligands. The
process may
require the use of multiple copies of the same central core structure or
combinations of
different types of display cores.


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37
The above-described technique can be extended to trivalent compounds, as
exemplified by structures shown below.
L L L L
L
/ / L / L L
L L L
L
L L~L
L
L~ L L / / L
L~O
L 'O~o~o L
L ~~~ N ~~.L
~L
I N L L'N N~L
L~ \J/ O' -NH
In the same manner, tetravalent and higher order structures can be prepared
L
It can therefore be seen that there is a plethora of possibilities for the
composition of
a linker. Examples of linkers include aliphatic moieties, aromatic moieties,
steroidal
moieties, peptides, and the like. Specific examples are peptides or
polyamides,
hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or
a combination
thereof, and many specific examples of linkers are shown below. However, it
should be
understood that various changes may be made and equivalents may be substituted
without
departing from the true spirit and scope of the invention. For example,
properties of the
linker can be modified by the addition or insertion of ancillary groups into
the linker, for
example, to change solubility of the multibinding agent (in water, fats,
lipids, biological
fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility,
antigenicity, molecular size,
molecular weight, in vivo half life, in vivo distribution, biocompatability,
immunogenicity,
stability, and the like. For example, the introduction of one or more poly or
preferably
oligo(ethylene glycol) (PEG) groups onto the linker enhances hydrophilicity
and water
solubility of the multibinding agent, increases both molecular weight and
molecular size
and, depending on the nature of the unPEGylated linker, may increase the in
vivo retention
time. Further, PEG may decrease antigenicity and potentially enhances the
overall rigidity
of the linker.


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38
Ancillary groups that enhance the water solubility/hydrophilicity of the
linker are
useful in practicing the present invention. Thus, it is within the scope of
the present
invention to use ancillary groups such as, for example, polyethylene glycol),
alcohols,
polyols (e.g., glycerin, glycerol propoxylate, saccharides, including mono-,
oligo- and
polysaccharides, etc.), carboxylates, polycarboxylates (e.g., polyglutamic
acid, polyacrylic
acid, etc.), amines, polyamines (e.g., polylysine, poly(ethyleneimine), etc)
to enhance the
water solubility and/or hydrophilicity of the compounds of Formula I. In
preferred
embodiments, the ancillary group used to improve water
solubility/hydrophilicity will be a
polyether. In particularly preferred embodiments, the ancillary group will be
a
polyethylene glycol).
The incorporation of lipophilic ancillary groups within the structure of the
linker to
enhance the lipophilicity and/or hydrophobicity of the compounds of Formula I
is within the
scope of the present invention. Lipophilic groups of use in practicing the
instant invention
include, but are not limited to, aryl and heteroaryl groups. The aromatic
groups may be
either unsubstituted or substituted with other groups, but are at least
substituted with a group
which allows their covalent attachment to the linker. Other lipophilic groups
of use in
practicing the instant invention include fatty acid derivatives which do not
form bilayers in
aqueous medium until higher concentrations are reached.
Also within the scope of the present invention is the use of ancillary groups
which
result in the compound of Formula I being incorporated into a vesicle such as
a liposome or
a micelle. The term "lipid" refers to any fatty acid derivative that is
capable of forming a
bilayer or micelle such that a hydrophobic portion of the lipid material
orients toward the
bilayer while a hydrophilic portion orients toward the aqueous phase.
Hydrophilic
characteristics derive from the presence of phosphato, carboxylic, sulfato,
amino, sulfhydryl,
nitro, and other like groups. Hydrophobicity could be conferred by the
inclusion of groups
that include, but are not limited to, long chain saturated and unsaturated
aliphatic
hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one
or more
aryl, heteroaryl, cycloalkyl and/or heterocyclic group{s). Preferred lipids
are
phosphoglycerides and sphingolipids, representative examples of which include
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol,
phosphatidic acid, palmitoyloleoyl phosphatidylcholine,
lysophosphatidylcholine,
lysophosphatidyi-ethanolamine, dipalmitoylphosphatidylcholine,


CA 02318394 2000-07-12
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39
dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or
dilinoleoylphosphatidylcholine could be used. Other compounds lacking
phosphorus, such
as sphingolipid and glycosphingolipid families are also within the group
designated as lipid.
Additionally, the amphipathic lipids described above may be mixed with other
lipids
including triglycerides and sterols.
The flexibility of the linker can be reduced by the inclusion of ancillary
groups
which are bulky and/or rigid. The presence of bulky or rigid groups can hinder
free rotation
about bonds in the linker or bonds between the linker and the ancillary
groups) or bonds
between the linker and the functional groups. Rigid groups can include, for
example, those
groups whose conformational lability is restrained by the presence of rings
and/or multiple
bonds, for example, aryl, heteroaryl, cycloalkyl, and/or heterocyclic. Other
groups which
can impart rigidity include polymeric groups. such as oligo- or polyproline
chains.
Rigidity can also be imparted electrostatically. Thus, if the ancillary groups
are
either negatively or positively charged, the similarly charged ancillary
groups will force the
presenter linker into a configuration affording the maximum distance between
each of the
like charges. The energetic cost of bringing the like-charged groups closer to
each other will
tend to hold the linker in a configuration that maintains the separation
between the like-
charged ancillary .groups. Further, ancillary groups bearing opposite charges
will tend to be
attracted to their oppositely charged counterparts and potentially may enter
into both inter-
and intramolecular ionic bonds. This non-covalent bonding mechanism will tend
to hold the
linker into a conformation which allows bonding between the oppositely charged
groups.
The addition of ancillary groups which are charged, or alternatively, bear a
latent charge
which is unmasked, following addition to the linker, by deprotection, a change
in pH,
oxidation, reduction or other mechanisms known to those of skill in the art,
is within the
scope of the present invention.
Rigidity may also be imparted by internal hydrogen bonding, or by hydrophobic
collapse.
Bulky groups can include, for example, large atoms and/or ions (e.g., iodine,
sulfur,
metal ions, etc.) groups containing large atoms, polycyclic groups, including
aromatic
groups, non-aromatic groups and structures incorporating one or more carbon-
carbon
multiple bonds (i.e., alkenes and alkynes). Bulky groups can also include
oligomers and
polymers which are branched- or straight-chain species. Species that are
branched are


CA 02318394 2000-07-12
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expected to increase the rigidity of the structure more per unit molecular
weight gain than
are straight-chain species.
Eliminating or reducing antigenicity of the compounds of Formula I by
judicious
choice of ancillary groups) is within the scope of the present invention. In
certain
applications, the antigenicity of a compound of Formula I may be reduced or
eliminated by
the use of groups such as, for example, polyethylene glycol).
As explained above, the multibinding agents of the invention comprise 2-10
ligands attached to a linker that connects the ligands in such a manner that
they are
presented to the enzyme multivalent receptors for multivalent interactions
with the
10 appropriate receptors (ligand binding site). The linker spatially
constrains these
interactions to occur within dimensions defined by the linker, thus increasing
the
biological effect of the multibinding agent as compared to the same number of
individual
units of the ligand.
The multivalent compounds of the invention, the compounds of Formula I, are
15 represented by the empirical formula (L)P(X)q. This is intended to include
the several
ways in which the ligands can be linked together in order to achieve the
objective of
multivalency, and a more detailed explanation is given below. However, as
previously
noted, the linker can be considered as a framework, and it should be
understood that the
ligands can be attached to this framework at any intermediate point on the
framework,
20 and/or on the termini of the framework. For example, if the linker is a
linear chain, a
bivalent compound can be constructed by attaching two ligands at the two ends
of the
linear chain, or alternatively attaching two ligands at some intermediate atom
along the
chain. The same considerations apply to the compounds of the present invention
containing more than 2 ligands.
25 The simplest (and preferred) multibinding agent is a bivalent compound,
which
can be represented as L-X-L, where L is a ligand and is the same or different,
and X is
the linker. It should be noted that the linker X can be linear or cyclic, or a
combination
of both linear and cyclic constructs, and that the two ligands may be located
at the
termini of the linker or may be attached at some intermediate attachment
point. The
30 same is true for a trivalent compound, which can also be represented in a
linear fashion,
i.e. as a sequence of repeated units L-X-L-X-L, in which L is a ligand and is
the same or


CA 02318394 2000-07-12
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41
different at each occurrence, as can X, or a compound comprising three ligands
attached
to a central core, and thus represented as (L)3X, where the linker X could
include, for
example, an aryl or cycloalkyl group.
The same considerations of geometry apply to the compounds of the present
invention containing 4-10 ligands. For example, a tetravalent compound could
be
represented as
L-X-L-X-L-X-L,or L-X-L-X-L
L
i.e. a branched construct analogous to the isomers of butane (n-butyl, sec-
butyl, tert-
butyl). Alternatively, it could be represented as an aryl or cycloalkyl
derivative as above
with four ligands attached to the core linker. The same principles apply to
the higher
multibinding agents, e.g. pentavalent to decavalent compounds. However, for
multibinding agents attached to a central linker such as benzene, there is the
self evident
constraint that there must be sufficient attachment sites on the linking
moiety to
accommodate the number of ligands present; for example, a benzene ring could
not
accommodate more than six ligands, whereas a saturated and/or mufti-ring
linker
(cyclohexyl, cyclooctyl, biphenyl, etc.) could accommodate a larger number of
ligands.
The formula (L)P(X)q is also intended to represent a cyclic compound of
formula
L~X~L
I I
X~L~X
(-L-X-)", where n is 2-10. For example, where n is 3:
All of the above variations are intended to be within the scope of the
invention as
defined by the Formula I (L)P(X)q.
The preferred linker length will vary depending upon the distance between
adjacent ligand recognition sites, and the geometry, flexibility and
composition of the
linker. The length of the linker will preferably be in the range of about 2-
100
Angstroms, more preferably about 2-SO Angstroms, and even more preferably
about 5-20
Angstroms.


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42
With the foregoing in mind, preferred linkers may be represented by the
following
formula:
-X'-Z-(Y'-Z)m-Y"-Z-X'_
in which:
m is an integer of 0-20;
X' at each separate occurrence is -0-, -S-, -S(O)-, -S(O)2-, NR- (where R is
as defined
below},
-C(O)-, or a covalent bond;
Z at each separate occurrence is alkylene, cycloalkylene, alkenylene,
alkynylene, arylene,
heteroarylene, or a covalent bond;
Y' and Y" at each separate occurrence are
0 0 0
/ \ \ /
, ~,
,
R'
R,\N N/
/ \ -P O
N N ( )2(OR~-0-
R' , R'
p X'
p N ~ -S(O)"-CRR , -S(O),; NR-,
R' ' R' ,
-S-S-, or a covalent bond;
in which:
n is 0, I or 2; and
R, R' and R" at each separate occurrence are chosen from hydrogen, alkyl,
cycloalkyl, alkenyl, cycIoalkenyl, alkynyl, aryl, heteroaryl, and heterocyclo.
Additionally, the linker moiety can be optionally substituted at any atom in
the chain
by alkyl, cycloaikyl, alkenyl, alkynyl, alkoxy, halo, nitro, aryl, heteroaryl,
or heterocyclo.


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43
Preparation of Compounds of Formula I
The preferred compounds of the invention are those in which p is 2 and q is 1,
and for the sake of simplicity the following description is directed toward
the preparation
S of such compounds. However, it should be understood that the same synthetic
principles
can be applied to all compounds within the scope of the invention.
There are many ways in which to prepare the compounds of the invention.
However, there are three basic themes common to their preparation;
1 ) Preparation of a substituted ligand (as defined above) at the [C]
terminus,
the [V] terminus, the [N] terminus, the [O] terminus, and/or the [R] terminus
for use in
coupling reactions;
2) Preparation of a bivalent compound of the invention by coupling two
ligands (the same or different, unsubstituted or substituted) with an
appropriate linker at
the above mentioned termini;
3) Preparation of an intermediate of a ligand having a functional group that
can then be further reacted with an appropriate linker or ligand intermediate
to form a
compound of the invention.
Examples of these reactions are given below. Given that there is a carboxylic
acid terminus [C], a primary amine terminus [V], and a secondary amine
terminus [N]
available on the glycopeptide ligands for linking reactions, one of the most
general
reactions used for this purpose is that between a carboxylic acid and an amine
to form an
amide. For example, as shown in Figure 1, Reactions A and B, a carboxylic
acid, for
example that is available at the [C] terminus, can be reacted directly with an
amine to
form an monoamide, or with a diamine to form a compound of the invention.
However,
it is also possible to react the carboxylic acid in a manner that produces an
intermediate
capable of undergoing further reaction, for example an amino or carboxy
functional
group (Reactions C and D), a hydroxy group, and the like, which can be used to
react
further with an appropriate linker, for example a dicarboxylic acid or
diamine, to form a
compound of the invention.
Other possible reactions include modification of the amine terminus by
reductive
alkylation, formation of an amidine link, and the Like.


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44
It should be understood that the shaded circle in the following Figures and
Reaction Schemes represents, for the sake of simplicity, the "core" of a
linker. That is to
say, a shaded circle showing two attached amino grouips (formula (2))
represents any
diamino compound that can be used in a coupling reaction, for example 1,6-
diaminohexane, 1,2-bis-(aminomethyl)phenyl, 1,4-bis-(aminomethyl)cyclohexane,
and
the like. A complete Iist of commercially available diamines are available on
the ACD
catalogue.
FIGURE 1
H2N_Ri (1) O
O
A~OH A A~H.R
HyN NHZ (2) ~ O
ArOH B A H H ~A + A H NH
Formula I
,PG ~3~ O
Q H C-~-H
Al'OH A N ~PG deprotect
C I-I ~ A~~ NHz
HZN(4)
O bPG O O
A~OH A~ dep~~t
A N
IO D O~ H OPG
where A represents a ligand or substituted ligand to which a carboxylic acid
is attached,
PG is a protecting group, and the shaded circle represents a linker "core". It
will be
apparent to one of skill in the art that secondary amines can also be used in
place of the
primary -NH2 or -NHPG groups shown above.
Similarly, Figure 2 shows reaction of an amine, for example the [V) or [N]
terminus, with a carboxylic acid or dicarboxylic acid to form an amide or
diamide. The
amine can also be reacted to produce a protected intermediate with a second
amino group
or carboxy functional group, which can be reacted further with a dicarboxylic
acid or
diamine in order to produce a compound of the invention.


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4S
It should be noted that in reactions where a carboxylic acid is shown as the
reactant, the equivalent acid chloride can be used in its place.
FIGURE 2
R
A-NHz HO (5) A ~~-Rs
E
O
HO OH (6) O O O O
A-NHz A H H A * A H OH
F
Formula I
HPG ('1)
A-NH H~ O~ ~G deprotect O N
z G p H A-NH Hz
O O
A-NH HO OPG (8) O de rotect O O
z P
S g A PG A- H
where A represents a ligand or substituted ligand to which an amino group is
attached,
PG is a protecting group, and the shaded circle represents a linker "core".
In Figure 3, an amine, for example the [V] or [NJ termini, can be derivatized
by a
reductive alkylation reaction with an aldehyde to form an alkyl substituent,
or reacted
with a dialdehyde under reductive alkylation conditions to give a compound of
the
invention. Reaction with appropriately protected aldehydes or carboxylic acids
gives an
intermediates with an amino or carboxy functional groups (Reactions L and M),
the
products of which can be used to react further with a carboxylic acid or amine
respectively to form an amide.
1S

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46
FIGURE 3
Oy-R9 (9)
A-NHZ H A-N~R9
J H
O
(10)
A-NHZ H H A-NH HN-A + A-
K H
Formula I
_,PG
H (I1) ~PG deprotect
IO A-NHZ L A-NH ~j A-NH NHZ
O
H (12)
O-PG O deprotect O
A NH O-PG A-NH OH
M
1 S Where A represents a ligand or substituted ligand to which an amino group
acid is
attached, PG is a protecting group, and the shaded circle represents a linker
"core".
Figure 4 similarly shows the derivatization of a hydroxy group, for example
the
aglycone hydroxy group designated as [O].

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47
FIGURE 4
BrR~3 (13)
A-OH A-OR~3
N
BrBr (14)
A~OO~A + A~O Br
A-OH
O Formula I
Br ,PG
I U ~ (IS) A-O
,PG deprotect A-O
A-OH ~ ' NH2
P
Br, r p
'-~--~O-PG (16) A- O
A-OH deprotect A-O O
I S Q O PG OH
Where A represents a ligand or substituted ligand to which a hydroxy group is
attached,
PG is a protecting group, and the shaded circle represents a linker "core".
Figure 5 shows the preparation of unsymmetrical compounds of the invention, by
20 reaction of those products described above having amino and carboxyl
functional groups
FIGURE 5
0
H ~A~ A A.
A-NHz + --~.
H
25 Preparation of Startins Materials
The diamines of formula (2) used as coupling reagents in Figure 1 are either
commercially available or are prepared by methods well known in the art. One
example
of such a preparation is shown below in Reaction Scheme 1. It should be noted
that the
following reaction schemes are presented for illustrative purposes, and
accordingly are
30 shown in simplified form.


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48
Accordingly, diamines of Formula (19) may be prepared as shown below in
Reaction Scheme 1.
REACTION SCHEME 1
O ,pG (3) O O
CI CI (I~ HzN H PGNH . . ~ H NHPG deP~~ct
(18)
O O
HzN ~ H NHz
(19)
where PG is a protecting group, preferably t-butyl carbamate, and the shaded
circle
represents a linker core
Preparation of Compounds of Formula f 18)
As illustrated in Reaction Scheme 1, step 1, about two molar equivalents of an
omega-amino carbamic acid ester [formula (3)] reacted with about one molar
equivalent
of a dicarboxylic acid halide, preferably chloride, of formula (17). The
reaction is
1 S conducted in the presence of a hindered base, preferably
diisopropylethylamine, in an
inert solvent, preferably methylene chloride, at a temperature of about 0-
5°C. The
mixture is then allowed to warm to room temperature. When the reaction is
substantially
complete, the compound of formula (18) is isolated and purified by
conventional means.
Preparation of Compounds of Formula (19l
As illustrated in Reaction Scheme 1, step 2, the carbamate protecting group is
hydrolyzed under acid conditions. In general a preferred acid is
trifluoroacetic acid. The
reaction is conducted in an inert solvent, preferably methylene chloride, at
about room
temperature. When the reaction is substantially complete, the compound of
formula (19),
which is a compound of formula (2) is isolated and purified by conventional
means.


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49
The linking groups used as coupling reagents in the figures above are either
commercially available or are prepared by methods well known in the art.
Several
examples of such a preparation are shown below in Reaction Scheme 2-3.
Reaction
Scheme 2 shows the preparation of an aminoaldehyde from the corresponding
aminoalcohol.
REACTION SCHEME 2
H protect H ,PG
oxidize ~ PG
NHZ ~ ~' H
(20)
(21)
(71
1~
where PG is a protecting group, preferably 9-fluorenylmethoxycarbonyl.
Preparation of a Compound of Formula (71
Illustrated in Reaction Scheme 2 is a method for preparing a protected-
I S aminoaldehyde (7) from the corresponding aminoalcohol (20). The
aminoalcohol is
protected by conventional technique, for example, by treatment with 9-
fluorenylmethyl
chloroformate in the presence of base, to yield F-moc protected aminoalcohol
(21).
Oxidation by techniques well known in the art (for example, sulfur
trioxide/pyridine)
yields an aldehyde of formula (7).
20 Similarly, Reaction Scheme 3 shows other methods by which derivatives of
aldehydes are prepared.

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REACTION SCHEME 3
H R'3-Hr (13) HO protect H PG
NHZ ~
~R~3 N~Rm
(93) (94)
oxidize ON~PG
H~''"~ ~R~3
O s
Me0 C ~R (s) O s reduce ERs
Me0 ~R
O NH O H ~ HO H
(91)
(97)
protect N~Rs oxidize O ERs
P H PG
(98) (99)
5 where PG is a protecting group, RS is hydrogen, alkyl, aryl, heteroaryl,
arylalkyl, or
heteroarylalkyl, and R13 is alkyl, arylalkyl, or heteroarylalkyl, ,
Reaction Scheme 4 shows one method by which a [C-C) bivalent compound can
be made. Also produced is a monomer of formula (22).
10 REACTION SCHEME 4
H H H H
OH
HZN NHZ (2) R N N~R R~N N-~ NH
I-1 + H
g
NH2 NHZ H2N NH
Formula I (22)
~C-C/
where R is hydrogen or methyl, and the shaded circle is a linker "core".


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S1
Preparation of a 1C-C1 Linked Compound of Formula I
In general, about two molar equivalents of a glycopeptide, for example
vancomycin, are reacted with about one molar equivalent of the diamine of
general
formula (2) (for example, a diamine of formula (19)), under conventional amide
coupling
conditions. Preferably, a hindered base is employed, preferably
diisopropylethylamine,
in the presence of benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate
(PyBOP) and 1-hydroxybenzotriazole. The reaction is conducted in an inert
polar
solvent, for example N,N dimethylformamide (DMF) or dimethyl sulfoxide (DMSO),
or
preferably a mixture of both, at about room temperature. When the reaction is
substantially complete, the compound of Formula I is isolated and purified by
conventional means, preferably purified by reverse-phase HPLC. Also isolated
is the
monoadduct, a compound of formula (22).
Alternatively, an intermediate of formula (22) may be prepared as shown below
in Reaction Scheme 5, and used to prepare a [C-C] compound of Formula I.
REACTION SCHEME 5
Preparation of a fC-C] Linked Compound of Formula I
FI H [~
R N OH ~~ PG(3) R ~ PG ~~~ R N H NH
C
NHz NHz NHz
(21)
(22)
H
H OH (~ R.N NH O
'BOO
F
NHz HZN
Formnh 1
[C-CJ


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52
where R is hydrogen or methyl, and PG represents a protecting group,
preferably 9-
fluorenylmethoxycarbonyl.
Preparation of a Compound of Formula (21~
S As illustrated in Reaction Scheme 5, step 1, a glycopeptide, for example
vancomycin, is reacted with about 1.1 molar equivalents of a carbamic ester
terminated
by an alkylamino group [formula (3)J. The ester moiety is chosen for ease of
removal
under mild conditions in subsequent reactions, and is preferably 9-
fluorenylmethyl.
Conventional amide coupling conditions are employed, preferably using PyBOP
and 1-
hydroxybenzotriazole. In general, the reaction is conducted in the presence of
a hindered
base, preferably diisopropylethylamine, in an inert polar solvent, preferably
DMF or
DMSO, preferably a mixture of both, at about room temperature. When the
reaction is
substantially complete, the compound of formula (21) is isolated and purified
by
conventional means.
Preparation of Compounds of Formula (,2~2
As illustrated in Reaction Scheme 5, step 2, the compound of formula (21) is
reacted with a mild base to remove the protecting ester group, which also
affords
decarboxylation. In general, the base is preferably piperidine, and the
reaction is
conducted in an inert polar solvent, preferably dimethylformamide, at about
room
temperature for about 10 minutes to one hour. When the reaction is
substantially
complete, the compound of formula (22) is isolated and purified by
conventional means,
preferably using reverse-phase HPLC.
The intermediate of formula (22) may then be converted into a [C-CJ
glycopeptide bivalent compound.
Preparation of a Compound of Formula I
As illustrated in Reaction Scheme 5, step 3, the compound of formula (22) is
reacted with a dicarboxylic acid. In general, about 3 molar equivalents of the
compound
of formula (22) is reacted with about 1 molar equivalent of the dicarboxylic
acid of
formula (6), under conventional amide coupling conditions. Preferably, a
hindered base
is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1


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53
hydroxybenzotriazole. The reaction is conducted in an inert polar solvent,
preferably
DMF, at about room temperature for about 1-3 hours. When the reaction is
substantially
complete, the compound of Formula I is isolated and purified by conventional
means,
preferably by reverse-phase HPLC.
In addition to coupling two ligands of formula (22) with a dicarboxylic acid,
the
[C-C] compounds of the invention can also be prepared by bis reductive
alkylation of
(22) with a dialdehyde.
It should also be understood that although Reaction Scheme 5 shows
unsubstituted glycopeptides as the ligand for reaction, the same reaction is
possible
starting with a glycopeptide substituted (or protected) at the (V] position.
The [V] amino
group can then be further modified in later steps if desired.
Compounds of Formula I wherein the linkage is [C-C] in which the [V] position
is substituted may be prepared from intermediates of formula (25), the
preparation of
which is shown below in Reaction Scheme 6.


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54
REACTION SCHEME 6
Preparation of a fC-C] Linked Compound of Formula I
H
H~RS W R~ N'R
E
IZN
Formula I
[C-CJ
where R is hydrogen or methyl, PG represents a protecting group, preferably 9-
fluorenylmethoxycarbonyl, and RS is alkyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl,
all of which may be optionally substituted.
Preparation of a Compound Formula (25)
As illustrated in Reaction Scheme 6, step 1, the amino sugar moiety of the
glycopeptide, for example the vancosamine portion of vancomycin, is reacted
with a
protected amino-aldehyde of formula (7) to form a Schiffs base. The ester
moiety is
chosen for ease of removal under mild conditions in subsequent reactions, and
is
preferably 9-fluorenylmethoxycarbonyl (Fmoc). In general, the reaction is
conducted in
an inert polar solvent, preferably N,N-dimethylformamide, in the presence of a
hindered
base, preferably diisopropylethylamine, at about 0-50°C, preferably
about 25°C, for


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WO 99/42476 PCTNS99/03850
about 1-5 hours, preferably about 2 hours. The Schiffs base is further reacted
with a
mild reducing agent. In general, a erotic solvent is added, preferably
methanol, followed
by the reducing agent, preferably sodium cyanoborohydride, and then
trifluoroacetic acid
. The reaction is conducted at about 0-SO°C, preferably about
25°C, for about 1 hour.
When the reaction is substantially complete, the compound of formula (25) is
isolated
and purified by conventional means, preferably purified by reverse-phase HPLC.
It should be noted that other intermediates can be used in place of the
compound of
formula (7), for example a protected acid (to give an amide), or an alkyl or
aryl group can
be appended directly on the [V] amine.
IO
The intermediate of formula (25) may then be converted into a [C-C] vancomycin
bivalent compound in which the [V] position is also substituted.
Preparation of a Compound of Formula (26)
15 As illustrated in Reaction Scheme 6, step 2, the compound of formula (25)
is
reacted with a compound of formula (22), which may be prepared as shown in
Reaction
Scheme 4. In general; conventional amide coupling conditions are employed.
Preferably, a hindered base is used, preferably diisopropylethylamine, in the
presence of
PyBOP and 1-hydroxybenzotriazole. The reaction is conducted in an inert polar
solvent,
20 preferably DMF, at about room temperature for about I-3 hours. When the
reaction is
substantially complete, the compound of formula (26) is isolated and purified
by
conventional means, preferably purified by reverse-phase HPLC.
Preparation of a Compound of Formula (27)
25 As illustrated in Reaction Scheme 6, step 3, the compound of formula (26)
is
deprotected conventionally as shown above, for example in Reaction Scheme 5,
step 2,
to form a compound of formula (27).
Pretiaration of Compounds of Formula I
30 As illustrated in Reaction Scheme 6, step 4, the compound of formula (27)
is
reacted with a compound of formula RS-C02H or RS-COCI, in which RS is alkyl,
aryl,


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56
heteroaryl, arylalkyl, heteroarylalkyl, all of which may be optionally
substituted. In
general, conventional amide coupling conditions are employed. Preferably, a
hindered
base is used, preferably diisopropylethylamine, in the presence of PyBOP and 1-

hydroxybenzotriazole. The reaction is conducted in an inert polar solvent,
preferably
DMF, at about room temperature for about 1-3 hours. When the reaction is
substantially
complete, the compound of Formula I is isolated and purified by conventional
means,
preferably purified by reverse-phase HPLC.
It should be noted that both the compounds of formula (26) and (27) can also
be
considered as compounds of Formula I. Additionally, step 4 can be any reaction
that
gives a substituted derivative of the amine group; for example, reductive
alkylation,
alkylation, and the like.
REACTION SCHEME 7
1 S Preparation of a ~C-C] Linked Compound of Formula I
H
~N NH2 BrBr X14) R~H H NH H C 1'1~R
~NHy O NHp H2N
) Formula I
]C-C]
where R is hydrogen or methyl.
Preparation of a Com,~ound of Formula I
In general, about two molar equivalents of a compound of Formula (22) are
reacted with about one molar equivalent of the dialkylhalide of general
formula (14)
under conventional alkylation conditions, in the presence of a hindered base,
preferably
diisopropylethylamine. The reaction is conducted in an inert polar solvent,
for example
N,N-dimethylformamide (DMF), at about 40-100°C, for about 1-24 hours,
preferably
about 8 hours. When the reaction is substantially complete, the compound of
Formula I


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57
is isolated and purified by conventional means, preferably purified by reverse-
phase
HPLC.
This reaction is, of course, broadly aplicable to any ligand that contains a
free
amore group.
REACTION SCHEME 8
Preparation of a 1V-V] Linked Compound of Formula I
H Rt Rte O H
HO OH ~6~ R N H ~ N,
F O~, O H
NN
H H
Formula I
where R is hydrogen or methyl, PG represents a protecting group, preferably 9-
fluorenylmethoxycarbonyl, and RI is, alkyl, aminoalkyl, alkylaminoalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
Preparation of a Compound of Formulal31
As illustrated in Reaction Scheme 8, step 1, the carboxyl function of the
compound of formula (25) is reacted with an amine of formula (1). In general,
about 1
molar equivalent of the compound of formula (25) is reacted with about 1.5
molar
equivalents of (1) under conventional amide coupling conditions. Preferably, a
hindered
base is employed, preferably diisopropylethylamine, in the presence of PyBOP
and 1-
hydroxybenzotriazole. The reaction is conducted in an inert polar solvent,
preferably


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58
DMF, at about room temperature for about 10 minutes to 2 hours. When the
reaction is
substantially complete, the compound of formula (31) is isolated and purified
by
conventional means, preferably by reverse-phase HPLC.
Preparation of a Compound of Formula (32)
As illustrated in Reaction Scheme 8, step 2, the compound of formula (31) is
deprotected conventionally as shown above, for example in Reaction Scheme 5,
step 2,
to form a compound of formula (32).
The intermediate of formula (32) is then converted into a [V-V] glycopeptide
bivalent compound in which the [C] position is also substituted, as shown
below.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 8, step 3, the compound of formula (32) is
reacted with a dicarboxylic acid (6). In general, about 3 molar equivalents of
the
compound of formula (32) is reacted with about 1 molar equivalent of the
dicarboxylic
acid of formula (6), under conventional amide coupling conditions. Preferably,
a
hindered base is employed, preferably diisopropylethylamine, in the presence
of PyBOP
and 1-hydroxybenzotriazole. The reaction is conducted in an inert polar
solvent,
preferably DMF, at about room temperature for about 1-3 hours. When the
reaction is
substantially complete and the compound of Formula I is isolated and purified
by
conventional means, preferably purified by reverse-phase I-iPLC.
If, instead of preparing a substituted [C] derivative of formula (32), it is
desired to
make a free acid derivative, the compound of formula (25) can be reacted
directly with a
"preactivated" dicarboxylic acid of formula (6), as described below.
An alternative method of preparing compounds of Formula I wherein the linkage
is [V-
V] and the [C]-position is substituted on both glycopeptide units is from
reaction of the
intermediate of formula (36) the preparation of these intermediates is shown
below in
Scheme 9.


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59
REACTION SCHEME 9
Preparation of a fV-V] Linked Compound of Formula I
N Ri N O .R~ N O R~
H HO OPG (8) R deprotect R~ H
H
O
HOPG H OH
(32)
(35) (36)
Foro~la I
rv-v~
in which R is hydrogen or methyl, PG represents a protecting group, preferably
9-
fluorenylmethyl, and where RI, R'' are independently alkyl, aryl, heteroaryl,
arylalkyl,
heteroarylalkyl, all of which may be optionally substituted.
Preparation of Compounds of Formula (36)
As illustrated in Reaction Scheme 9, the compound of formula (32) is reacted
with a monoester of a dicarboxylic acid (8); preferably the monoester is one
easily
hydrolyzed under mild conditions, for example a 9-fluorenylmethyl ester. In
general,
about one molar equivalent of (32) is reacted with one molar equivalent of
(8). The
reaction is carried out under conventional amide coupling conditions.
Preferably, a
hindered base is employed, preferably diisopropylethylamine, in the presence
of PyBOP
and 1 -hydroxybenzotriazole. The reaction is conducted in an inert polar
solvent,
preferably DMF, at about room temperature for about 1-3 hours, preferably
about 80
minutes. When the reaction is substantially complete, the desired product (35)
is isolated
by conventional means, and reacted with a mild base, preferably piperidine, to
remove the
protecting group. The reaction is conducted in an inert polar solvent,
preferably DMF, at


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about room temperature for about 1-3 hours, preferably about 1 hour. When the
reaction
is substantially complete, the compound of formula (36) is isolated and
purified by
conventional means, preferably purified by reverse-phase HPLC.
The intermediate of formula (36) is then converted into a [V-V] bivalent
compound wherein the [C]-position is substituted on both vancomycin subunits
by
reaction with a compound of formula (32') [not necessarily the same as
starting material
(32)].
Preparation of Compounds of Formula I
10 As illustrated in Reaction Scheme 9, step 3, the compound of formula (36)
is
reacted with a compound of formula (32'). In general, about equimolar
equivalents of
the compounds of formula (36) and (32') are reacted under conventional amide
coupling
conditions. Preferably, a hindered base is employed, preferably
diisopropylethylamine,
in the presence of PyBOP and 1-hydroxybenzotriazole. The reaction is conducted
in an
15 inert polar solvent, preferably DMF, at about room temperature for about 30
minutes to 3
hours, preferably about 1 hour. When the reaction is substantially complete,
the
compound of Formula I is isolated and purified by conventional means,
preferably
purified by reverse-phase HPLC.
20 It should be emphasized that the two glycopeptides (represented as shaded
boxes)
can be the same or different (or the same but differently substituted), and
the linking
groups, although always represented by a shaded circle, can be the same or
different at
each occurrence.


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61
REACTION SCHEME 10
Preparation of an [N-N] linked Compound of Formula I
H O O N O R O ~R~
R~N OH ~---NHPG (11) PGHN OH H2N_Rt (1) PGHN N
H
A
NHZ NHZ NHZ
(41)
(42)
R O ~R~ O O
deprotect HZNN ~ HO H
F
NHy
R~~ O R R O
IJ--N~HN--~N H~
O
H2N ~NH2
Formula I
~N-NJ
in which R is hydrogen or methyl, PG represents a protecting group, preferably
9-
fluorenylmethoxycarbonyl, and R' is, alkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, or
a quarternary salt derivative.
Preparation of Compounds of Formula (41}
As illustrated in Reaction Scheme 10, step 1, a glycopeptide, for example
vancomycin, is reacted with a protected aminoaldehyde under conditions
su~cient to
direct the aldehyde to the [Nj-position. The Schiff's base thus formed is
reduced in the
same manner as shown in Reaction Scheme 6, step 1, to form a compound of
formula
(41}.


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62
Preparation of Compounds of Formula (,42)
As illustrated in Reaction Scheme 10, step 2, the compound of formula (41) is
reacted with an amine (1) in a coupling reaction in the same manner as shown
above, for
example in Reaction Scheme 8, step 1, to form an amide of formula (42).
Preparation of Compounds of Formula X43,)
As illustrated in Reaction Scheme 10, step 3, the compound of formula (42) is
deprotected conventionally as shown above, for example in Reaction Scheme 5,
step 2,
to form a compound of formula (43).
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 10, step 4, the compound of formula (43) is
reacted with a dicarboxylic acid in the same manner as shown above, for
example in
Reaction Scheme 8, step 3, to give a compound of Formula I which is isolated
and
purified by conventional means, preferably by reverse-phase I-iPLC.
AGLYCONES
As described above, the invention also encompasses aglycone derivatives of
glycopeptides, for example vancomycin lacking the amino sugar moiety. Such a
compound has a hydroxy group as a point of attachment in place of the [V]
amino group
of the amino sugar. In the following reaction schemes, aglycones are
represented as
described above, i.e. they are depicted as a shaded triangle that shows only
the carboxyl
terminus, the aglycone hydroxy terminus, and the amino terminus.
Reaction Scheme 10 shows the preparation of an aglycone bivalent compound from
a
glycopeptide. The glycopeptide is initially substituted at the [C] position
with an amide,
followed by hydrolysis of the aminosugar, and finally coupling of the phenol
thus
produced.


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63
REACTION SCHEME 11
Preparation of an (O-Ol Linked Compound of Formula I
R
HN O OH H2N R~ (I) ~ O .R' R O
acid ~ ~R~
A --.-. _H
2 OH
(47)
NHR~
Br Hr CIO) R ~ O
o. T ,o
0
R.NH ~'R
Formula I
(o-o~
where R is hydrogen or methyl, and R~ is alkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, all of which may be optionally substituted
Preparation of Compounds of Formula X461
As illustrated in Reaction Scheme 11, step 1, a glycopeptide, for example
vancomycin, is is reacted with an amine (1) in a coupling reaction in the same
manner as
shown above, for example in Reaction Scheme 8, step 1, to form an amide of
formula
(46), which is isolated and purified by conventional means.
Preparation of Compounds of Formula (471
As illustrated in Reaction Scheme 11, step 2, the glycopeptide of formula (46)
is
hydrolysed with a strong acid, preferably at about 50°C, to produce an
aglycone of
formula (47), which is isolated and purified by conventional means.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 11, step 3, two equivalents of the compound
of
formula (47) are reacted with a dihalo compound of formula (10) to give a
compound of
Formula I [O-O]. The reaction is carried out in a polar solvent, preferably
DMF, in the


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presence of a base, preferably potassium carbonate, for about 30 hours. The
compound
of Formula I is isolated and purified by conventional means.
Alternatively, as shown in Reaction Scheme 12, the compound of formula (47)
may be first reacted with a protected haloamine, for example tert-butyl N (2-
bromoethyl)carbamate, to give an aminoether of formula (51), which is then
deprotected
and reacted further with a dicarboxylic acid to give a compound of Formula I.
REACTION SCHEME 12
Preparation of a f0-O] linked Compound of Formula I
R Br R R
~R~ ~-'NHPG (1S) ~ ~.R~ HN N.R
I-I H
deprotect
P I
OH O O
NHPG NHZ
(47) (51) (S2)
$ ,R~ R~ R
H \ H TIH
HO OH
F
O ~ ~ O
Formuh I
[O-O]
where R is hydrogen or methyl, and PG represents a protecting group,
preferably BOC,
and where Rl is alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of
which may be
optionally substituted
Preparation of a Compound of Formula~51 )
As illustrated in Reaction Scheme 12, step 1, an aglycone of formula (47) is
reacted with a protected haloamine, for example tent-butyl N (2-
bromoethyl)carbamate,
under conventional coupling conditions, preferably using potassium carbonate
as a base
in an inert polar solvent, preferably DMF or DMSO, at about room temperature.
When


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the reaction is substantially complete, the compound of formula (51) is
isolated and
purified by conventional means.
Preparation of a Comuound of Formula (52)
As illustrated in Reaction Scheme 12, step 2, the protecting group (carbamate)
is
hydrolyzed under acidic conditions. In general a preferred acid is
trifluoroacetic acid, and
the reaction is conducted in an inert solvent, at about room temperature. When
the
reaction is substantially complete, the compound of formula (52) is isolated
and purified
by conventional means.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 12, step 3, two equivalents of the compound
of
formula (52) are reacted with a dicarboxylic acid compound of formula (6) to
give a
compound of Formula I [O-O]. The reaction is carried out under conventional
amide
I S coupling conditions as detailed above, for example, as in Reaction Scheme
8, step 3, to
give a compound of Formula I
Alternatively, as shown in Reaction Scheme 13, the compound of formula (47)
may be first reacted with a haloester, for example t-butylbromoacetate, to
give a carboxy-
substituted ether, which is deprotected and then reacted further with a
diamine to give a
compound of Formula I.


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REACTION SCHEME 13
Preparation of a ~O-O] Linked Compound of Formula I
O R~ O ~ O .R' R O .R~
~-~--~ N
H BOPG (16) deprotxt ~ H
Q
OH O~ O
OPG OH
(5~
H2 NHZ (2)
B
Formula I
(O-O)
where R is hydrogen or methyl, and PG represents a protecting group, and where
Rl is
alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of which may be
optionally
substituted
Preparation of Compounds of Formula (561
As illustrated in Reaction Scheme 13, step 1, an aglycone of formula (47) is
reacted with a haloester under conventional coupling conditions, preferably
using
potassium carbonate as a base in an inert polar solvent, preferably DMF or
DMSO, at
about room temperature. When the reaction is substantially complete, the
compound of
formula (56) is isolated and purified by conventional means.
Preuaration of a Compound of Formula (57)
As illustrated in Reaction Scheme 13, step 2, the protecting group (carbamate)
is
hydrolyzed under acidic conditions. In general a preferred acid is
trifluoroacetic acid, and
the reaction is conducted in an inert solvent, at about room temperature. When
the


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reaction is substantially complete, the compound of formula (57) is isolated
and purified
by conventional means
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 13, step 3, two equivalents of the compound
of
formula {57) are reacted with a diarnino compound of formula (2) to give a
compound of
Formula I [O-O]. The reaction is carried out under conventional amide coupling
conditions as detailed above. Preferably, a hindered base is employed,
preferably
diisopropylethylamine, in the presence of PyBOP and 1-hydroxybenzotriazole.
The
reaction is conducted in an inert polar solvent, preferably DMF, at about room
temperature for about 30 minutes to 5 hours, preferably about 2 hours. When
the
reaction is substantially complete, the compound of Formula I is isolated and
purified by
conventional means, preferably purified by reverse-phase HPLC.
A new type of chemical modification of glycopeptides is described in a recent
publication from The Institute of New Antibiotics, Russian Academy of Medical
Sciences, B. Pirogovskaya 11, I 19867 Moscow, Russia. The reaction disclosed
therein
utilizes the Mannich reaction to derivatize the glycopeptide at the position
between the
two meta hydroxy groups on the "resorcinol" group (identified above in
Structure II as
the [R] position). This type of reaction can be used to prepare compounds of
the
invention, as shown below in Reaction Scheme 14.
REACTION SCHEME 14
Preparation of a fR-R] linked Compound of Formula I
HZN----NHZ (2)
Formula I
[R-R)
where R is hydrogen or methyl.


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Preparation of a Compound of Formula I
As illustrated in Reaction Scheme 14, a glycopeptide, for example vancomycin,
is
linked at the [R] position. In general, about two molar equivalents of a
glycopeptide, for
example vancomycin, are reacted with about two molar equivalents of formalin
and one
molar equivalent of diamine (2). The reaction is conducted in an inert polar
solvent,
preferably aqueous acetonitrile, for about 18 hours. When the reaction is
substantially
complete, the compound of Formula I is isolated and purified by conventional
means,
preferably purified by reverse-phase HPLC.
Secondary amines can be used in place of the primary amines of the linker of
formula (2) if desired, and the [C) position can be substituted, for example
by NHR~, as
shown previously if preferred.
Alternatively, the Mannich reaction can be used to introduce an amino
sidechain
or an acid sidechain at the [R] position (for example, as shown for the
compound of
formula (82)), which can then be further reacted with an appropriate linker (a
diacid,dialdehyde, dihalo compound) as shown above to provide a compound of
Formula
I.
Compounds of Formula I wherein the link is [C-V] can be prepared as shown
below in Reaction Schemes 15-20.

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REACTION SCHEME 15
Preparation of a fC-Vl Linked Compound of Formula I
R'
H O
R'N OH
preactivate R.
NHPG
(25')
(61)
R
~R
~y--R9 (9)
deprotect
J
Form~la I Formula I
S [C-VI [C-Vj
in which R is hydrogen or methyl, PG represents a protecting group, preferably
9-
fluorenylmethoxycarbonyl, and R9 is, alkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, or
a quarternary salt derivative.


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Preparation of a fC-V] Compound of Formula I
In general, the compounds of (C-V] Formula I are prepared by the procedures of
Reaction Scheme 15 using reactions described previously. However, one
important
difference is that the [C] carboxy group of the compound of formula (25) is
preferably
"preactivated" before reaction with the compound of formula (60), because (60)
also
contains a free carboxy group.
REACTION SCHEME 16
Preparation of a fC-V] Linked Compound of Formula I
N R~ O N Ri N O R~
R H NHPG (7) R 1, ~1 ~R
G
~y--NHPG ~ NHy
(3Z) NHZ
(6~
H
R-N OH
NHZ
Formula I
(GVJ
in which R is hydrogen or methyl, PG represents a protecting group, preferably
9-
fluorenyhnethoxycarbonyl, and Rl is, alkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, or
a quarternary salt derivative.
Preparation of a Compound of Formula (66)
As illustrated in Reaction Scheme 16, the compound of formula (32) is reacted
with an N-protected aminoacid (7) in the same manner as shown above, for
example in
Reaction Scheme 9, step 1, to form a protected amide, which is then
deprotected


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71
conventionally, for example as in Reaction Scheme 5, step 2, to form a
compound of
formula (66).
The compound of formula (66) is then converted into a [C-VJ bivalent compound
of Formula I by reaction with a glycopeptide, for example vancomycin.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 16, step 3, the compound of formula (66) is
reacted with a glycopeptide in a typical coupling reaction, as shown above, to
give a
compound of Formula I [C-V].
Reaction Scheme 17 shows the preparation of [C-V] compounds of Formula I
using techniques described above, i.e. reductive alkylation to provide a
protected amino
"handle", followed by coupling of the [C]-position to a [V] amino substituted
compound,
and deprotection.


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REACTION SCHEME 17
Preparation of a fC-Vl Linked Compound of Formula I
R'
Ii O H O
R-N OH O R R'N OH
N
H~PG
L
NH2 H
N-RZ
PG
Formula I
[C-V/
in which R is hydrogen or methyl, PG represents a protecting group, preferably
9-
fluorenylmethoxycarbonyl, and RI and R2 are independently alkyl, aryl,
heteroaryl,
arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
Compounds of Formula I wherein the linkage is [C-V) may be prepared from
intermediates of formula (72), the preparation of which is shown below in
Reaction
Scheme 18.


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REACTION SCHEME 18
Preparation of a rC-Vl Linked Compound of Formula I
H O Ii O
,T1
NHZ HO OPG ~) R.N H NHPG deprotect
O~-'~O
~NHZ H ~NH2
(71)
H
R.IV
H O
R~N H NH H
O O
Z
2
(72)
Formula I
[C-y
s
in which R is hydrogen or methyl, PG represents a protecting group, for
example 9-
fluorenylmethyl, and Rl is alkyl, alkylamino, alkylaminoalkyl, aryl,
heteroaryl, arylalkyl,
heteroarylalkyl, or a quarternary salt derivative.
Preparation of a Compound of Formula (72)
As illustrated in Reaction Scheme 18, step 1, the compound of formula (22) is
reacted with an acid in the same manner as shown above, for example in
Reaction
Scheme 9, step 1, to form a protected amide, which is then deprotected
conventionally,
for example as in Reaction Scheme 9, step 2, to form a compound of formula
(72).


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74
The compound of formula (72) is then converted into a [C-V] bivalent compound
of Formula I by reaction with a compound of formula (32), prepared as shown
previously, as shown in Reaction Scheme 6.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 18, step 3, the compound of formula (72) is
reacted with a compound of formula (32) in a typical coupling reaction as
shown above,
to give a compound of Formula I [C-V).
The compound of [C-V] Formula I thus prepared is substituted at the second [C]
terminus (by R~NH). The free acid (i.e., the unsubstiututed [C] terminus)can
be
obtained, if desired, by substituting the compound of formula (60) for (32),
and
preactivating the compound of formula (72) as explained above.
Compounds of Formula I wherein the linkage is [C-V] and the [C] terminus of
one vancomycin and the [V] terminus of a second vancomycin are both
substituted may
be prepared from intermediates of formula (77), the preparation of which is
shown below
in Reaction Scheme 19.


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REACTION SCHEME 19
Preparation of a fC-V] Linked Compound of Formula I
H
~H H R9 (9) R.1J ~H HzN- NHz (2) R.1N N NH
H z
B
NHZ J
"..Ry H..R9
(76)
Z
Formula I
~~-~'1
where R is hydrogen or methyl, PG represents a protecting group, preferably 9-
fluorenylmethoxycarbonyl, and R', R9 are independently chosen from hydrogen,
alkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl, and a quarternary salt
derivative.
10 Preparation of a Compound of Formula (76)
As illustrated in Reaction Scheme 19, step 1, a glycopeptide, for example
vancomycin, is reacted with an aldehyde of formula R9CH0, using excess base in
order
to to direct the alkylation to the [V] position. The Schii~''s base thus
formed is reduced in
the same manner as shown in Reaction Scheme 6, step 1, to form a compound of
formula
1 S (76).
Preparation of Compounds of Formula (77)
As illustrated in Reaction Scheme 19, step 2, the compound of formula (76) is
reacted with a diamine (2) to form an amide of formula (77).
20 The compound of formula (77) is then converted into a [C-V] bivalent
compound
of Formula I (in which the [C] terminus of one glycopeptide and the [V]
terminus of a


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76
second glycopeptide are both substituted) by reaction with a compound of
formula (36),
prepared as shown in Reaction Scheme 9.
Preparation of Compounds of Formula I
As illustrated in Reaction Scheme 19, step 3, the compound of formula (77) is
reacted with a compound of formula (36) in a typical coupling reaction as
shown above
to give a compound of Formula I [C-V] in which the [C] terminus of one
glycopeptide
and the [VJ terminus of a second glycopeptide are additionally substituted.
Compounds of Formula I wherein the linkage is [C-V] and the [C] terminus of
one vancomycin and the [V] terminus of a second vancomycin are both
substituted may
also be prepared from intermediates of formula (32), the preparation of which
is shown
below in Reaction Scheme 20.
REACTION SCHEME 20
Preparation of a fC-V] Linked Compound of Formula I
H O
R,N _
NHZ HN NH
+ (38)
M OMe
NHZ
(32)
(22)
Formula I
~C-vl
where R is hydrogen or methyl, and R' is alkyl, aryl, heteroaryl, arylalkyl,
or
heteroarylalkyl, all of which may be optionally substituted.


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77
Preparation of a Compound of Formula I
In general, one molar equivalent of a compound of Formula (32) and one molar
equivalnent of a compound of Formula (22) are reacted with one molar
equivalent of a
bis-imidate of formula (38) under conventional coupling conditions.
Preferably, a
hindered base is employed, preferably diisopropylethylamine. The reaction is
conducted
in an inert polar solvent, for example N,N dimethylformamide (DMF), for about
one
hour. When the reaction is substantially complete, the compound of Formula I
(with a
bis-amidine linker) is isolated and purified by conventional means, preferably
purified by
reverse-phase HPLC.
It should be noted that this reaction, which provides a bis(amidine) linker,
can be
employed in the same manner on any amine shown in the foregoing examples to
give
amidine linkers between all termini of a glycopeptide ([C-C], [V-V], etc)
Compounds of Formula I wherein the linkage is [C-N] may be prepared from
intermediates of formula (43), the preparation of which was shown in Reaction
Scheme
9.
REACTION SCHEME 21
H R O .R~
R ~ H NH H HZN N
O
Z
~NHz ~NHz
(72) (43)
H O R O ~Ri
R N~ NH HN N
H
O
NHz z
2o Formula I
~C-N)
where R is hydrogen or methyl, and R' is alkyl, aryl, heteroaryl, arylalkyl,
or
heteroarylalkyl, all of which may be optionally substituted.


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Preparation of a f C-N1 Linked Compound of Formula I
As illustrated in Reaction Scheme 21, the compound of formula (72) is reacted
with a compound of formula (43) in a typical, coupling reaction as shown
above, to give a
compound of Formula I [C-N).
Compounds of Formula I wherein the linkage is [N-V) may be prepared by
reaction of a compound of formula (36) with a compound of formula (43), as
shown in
Reaction Scheme 22
REACTION SCHEME 22
R R~,
HzNN
Z
NHz
(43)
(36)
R'
R p ~R~,
N N
H
Formula I NH2
where R is hydrogen or methyl, and RI, RI' is alkyl, aryl, heteroaryl,
arylalkyl, or
heteroarylalkyl, all of which may be optionally substituted.


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Preparation of a (N-V] Linked Compound of Formula I
As illustrated in Reaction Scheme 22, the compound of formula (36) is reacted
with a compound of formula (43), the preparation of which are detailed above,
in a
typical coupling reaction as shown above to give a compound of Formula I (N-
V].
REACTION SCHEME 23
Preparation of a f C-Ri Linked Compound of Formula I
~sl)
(82)
H
R'N OH
__ R9
Z
Formula I
[C-R]
Preparation of a fC-R1 Linked Compound of Formula I
As illustrated in Reaction Scheme 23, the compound of formula (81) is reacted
with a diamine and formaldehyde, using typical reaction conditions for the
Mannich
reaction, to give a compound of formula (82). This amino derivative is then
coupled
with a glycopeptide, in a typical coupling reaction as shown above, to give a
compound
of Formula I [C-R].
Isolation and Purification of the Compounds
Isolation and purification of the compounds and intermediates described herein
can be effected, if desired, by any suitable separation or purification
procedure such as,
for example, filtration, extraction, crystallization, column chromatography,
thin-layer


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chromatography, thick-layer chromatography, preparative low or high-pressure
liquid
chromatography or a combination of these procedures. Specific illustrations of
suitable
separation and isolation procedures can be had by reference to the Examples
hereinbelow. However, other equivalent separation or isolation procedures
could, of
course, also be used.
EXAMPLE 1
Preparation of a Diamine of Formula (2)
(1) Preparation of a Compound of Formula (18)
To a solution of tert-butyl N (2-aminoethyl)carbamate (3) (2.3g, 14.4mmol) and
10 N,N diisopropylethylamine (2.SmL, 14.3mmo1) in lSmL methylene chloride at
0°C was
added glutaryl dichloride (17) (0.6mL, 4.7mmo1) in lSmL methylene chloride
dropwise.
The resulting mixture was allowed to warm to room temperature with stirring
while
adding water ( 1 SmL). The methylene chloride was removed under reduced
pressure and
more water was added (30mL). The resulting suspension was filtered and washed
15 sequentially with 10% potassium hydrogen sulfate, water, saturated sodium
bicarbonate,
and water. The solid was dried under vacuum yielding 1.3g (3.1 mmol, 66%) of
pentanedioic acid bis-[(2-t-butoxycarbonylaminoethyl)amide], a compound of
formula
(18).
20 (2) Preparation of a Compound of Formula (19)
The compound of formula (18) prepared above (1.3g, 3.1 mmol) was suspended
in 15 mL methylene chloride. 1 S mL of trifluoroacetic acid was added at room
temperature giving (with effervescence) a solution that was stirred for 40
minutes, then
evaporated in vacuo. The residue was dissolved in methanol and treated with
3mL 4N
25 hydrogen chloride in dioxane followed by diethyl ether, giving a gum. The
liquids were
decanted and the gum dried under vacuum yielding l .Og (3.4mmol, 110%) of
pentanedioic acid bis-[(2-aminoethyl)arnideJ, a compound of formula (19).


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EXAMPLE 2
Preparation of a Protected Aminoaldehyde of Formula (7)
( 1 ) Preparation of a Compound of Formula (21 )
9-fluorenylmethyl N (2-aminoethyl)carbamate (Fmoc-2-aminoethanol), a compound
of formula (21), was prepared from aminoethanol, a compound of formula (20),
by
conventional techniques (see Example 3, step 2).
(2) Preparation of a Compound of Formula (71
To a mixture of Fmoc-aminoethanol, a compound of formula (21) (37.64g, 133
mmol, 1.0 equiv), TEMPO [2,2,6,6-tetramethyl-1-piperidinyloxy, free radical)
(0.008 M
in CH2C12, 332 mL, 2.66 mmol, 0.02 equiv), KBr (0.5 M in water, 53 mL, 26.6
mmol,
0.2 equiv) and ethyl acetate (1,500 mL), at 0 °C, was added NaOCI (0.35
M, buffered to
pH 8.6 by NaHC03, 760 mL, 266 mmol, 2.0 equiv). A mechanical stirrer was used
to
ensure efficient stirring, and the reaction was monitored by TLC. After 20
min, the two
layers were separated. The aqueous layer was extracted with ethyl acetate (2 x
250 mL),
the combined organic layers were washed with saturated Na2S203, water, and
brine, dried
over Na2S04, filtered and concentrated to about 400 mL. Hexane (1,600 mL) was
added
to give a white precipitate. After filtration, F-moc aminoacetaldehyde (25.2g,
67%), a
compound of formula (7), was collected as a white powder.
EXAMPLE 3A
Preparation of a Protected Aminoaidehyde of Formula (95)
Preparation of a Compound of Formula (931
A solution of aminoethanol (90) (30.5 g, 500 mmol, 30.1 mL) and 1-bromodecane
(13) (27.65 g, 125 mmol, 26 mL) in ethanol was stirred at 65 °C for 4
hr. The solvent
was removed under reduced pressure. The residue was diluted with EtOAc (800
mL) and
the organic solution was washed'with H20 (2 x 200mL), aqueous HCl ( 2 x 200mL,
1.0
M), saturated aqueous NaHC03 (200 mL) and saturated brine (200 mL). The
organic
phase was dried over anhydrous Na2S04, and concentrated under reduced
pressure. The


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82
resulting crude product, 2-{n-decylamino)ethanol (93), was used in the next
reaction
without further purification.
Preparation of a Compound of Formula (94)
A solution of 2-(n-decylamino)ethanol (93) (20.1 g, 100 mmol) in CH2C12 was
treated with DIPEA (diisopropylethylamine, Hunig's base) ( 15.1 g, 120 mmol,
20.9 mL)
at 0 °C, followed by FmocCl (28.5 g, 110 mmol). The resulting reaction
mixture was
stired at 0 °C for 1 hr and was poured into CH2C12 (400 mL). The
mixture was washed
with H20 (3 x 200 mL), aqueous HCl (1 x 200 mL, 1 N), saturated aqueous NaHC03
(200 mL) and brine (200 mL). The organic phase was dried over anhydrous
Na2S04, and
concentrated under reduced pressure. The resulting white solid, N,N-n-decyl-
Fmoc-
aminoethanol (94), was used for the next reaction without further
purification.
Preparation of a Compound of Formula (95)
A solution of N,N n-decyl-Fmoc-aminoethanol (94) ( 12.7 g, 30 mmol) and DIPEA
(15.48 g, 120 mmol, 20.9mL) in CH2Cl2 was treated with a solution of sulfur
trioxide
pyridine complex in DMSO (85 mL) dropwise over 20 min at 0 °C. The
resulting
reaction mixture was stirred at 0 °C for additional 20 min before it
was quenched with
water. The mixture was extracted with CH2Cl2 (5 x 100 mL) and the combined
organic
solutions were concentrated under reduced pressure. The residue was diluted
with EtOAc
(500 mL). The organic solution was washed with H20 (3 x 200 mL), aqueous HCl
(200
mL, 1N), saturated aqueous NaHC03 {200 mL) and saturated brine (200 mL). The
organic phase was dried over anhydrous Na2S04, and concentrated under reduced
pressure. The crude product was purified by column chromatography(25%
EtOAclllexanes) to yield n-decyl Fmoc-aminoaldehyde (95). MS (MH+) calculated
422.2, found 422.2.


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EXAMPLE 3B
Preparation of a Protected- Aminoaldehyde of Formula (99)
Preparation of a Compound of Formula (961
To a solution of decanoyl chloride (5) (2.7mL, l3mmol, 1.Oeq) in methylene
chloride
(20mL) in an ice/acetone bath was added a mixture of glycine methyl ester
hydrochloride
(91) (2.Og, 16 mmol, l.2eq) and DIPEA (S.lmL, 29mmol, 2.2eq) in methylene
chloride
(20mL) dropwise. The reaction was stirred a further 60 min after complete
addition, then
washed with 3N hydrochloric acid (SOmL) twice, followed by saturated sodium
bicarbonate (SOmL). The organics were dried over magnesium sulfate and the
solvents
removed under reduced pressure. n-DecylgIycine, a compound of formula (96)
(3.Og,
l2mmol, 95%) was obtained, and used in the next step without further
purification.
Preparation of a Compound of Formula (97)
Under nitrogen, amide (96) (3.Og, l2mmol, 1.Oeq) was dissolved in anhydrous
tetrahydrofuran (25mL) and cooled in an ice bath. A solution of lithium
aluminum
hydride (1N, 25mL, 25mmo1, 2.Oeq) was added carefully. The resulting solution
was
refluxed under nitrogen overnight, then cooled in an ice bath. SOmL more
tetrahydrofuran was added followed by slow addition of sodium sulfate
decahydrate until
effervescence ceased. The mixture was allowed to warm to room temperature,
filtered,
then concentrated under vacuum. 2-(n-decylamino)ethanol, a compound of formula
(97)
(2.3 g, 1 lmmol, 93%) was obtained, and used without further purification.
Preparation of a Compound of Formula (98)
2-(n-decylamino)ethanol (97) (2.3g, 11 mmol, 1.1 eq) and DIPEA (2.OmL, 11
mmol,
l.leq) were dissolved in methylene chloride (lSmL) and cooled in an ice bath.
9-
Fluorenylmethyl chlorofonmate (2.6g, lOmmol, l.Oeq) in methylene chloride
(lSmL) was
added, the mixture stirred for 30 minutes then washed with 3N hydrochloric
acid (SOmL)
twice and saturated sodium bicarbonate (SOmL). The organics were dried over
magnesium sulfate, and the solvents removed under reduced pressure. F-moc-2-(n-

decylamino)ethanol, a compound of formula {98), (4.6 g, 11 mmol, 108%) was
used
without further purification.


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
84
Preparation of Compound of Formula l99)
F-moc-2-(n-decylamino)ethanol (98) (4.6g, l lmmol, l.Oeq) and DIPEA (7.6mL,
44mmol, 4.Oeq) were dissolved in methylene chloride (30mL) and cooled in an
ice/acetone bath. A solution of sulfur trioxide pyridine complex (6.9g,
43mmol, 4.Oeq)
in dimethyl sulfoxide (30mL) was added, and the solution stirred for 20
minutes.
Crushed ice was added and the mixture partitioned. The organics were washed
with 3N
hydrochloric acid twice, saturated sodium bicarbonate and saturated sodium
chloride,
dried over magnesium chloride, and concentrated under vacuum. F-moc-2-(n-
decylamino)acetaldehyde, a compound of formula (99) (3.4g, 8mmol, 74%) was
used
without further purification.
EXAMPLE 4
Preparation of a fC-Cl Compound of Formula I
( 1 ) Preparation of a Compound of Formula I
At room temperature vancomycin hydrochloride (3.6g, 2.3mmo1) was dissolved
in 36mL of dimethylsulfoxide. To this solution was added pentanedioic acid-bis-
(2-
aminoethyl)amide, a compound of formula (2) (l.Og, ~.4mmo1 suspended in 27mL
N,N
dimethyIformamide) followed by N,N-diisopropylethylamine (2.4mL,13.8mmo1). The
resulting suspension was stirred at room temperature for several hours until
it was mostly
homogenous. Then a solution of PyBOP (1.3g, 2.5 mmol) and 1-
hydroxybenzotriazole
(310mg, 2.3mmo1) in 9mL N,N dimethylformamide was added rapidly dropwise. The
mixture was stirred at room temperature for 1 hour and then added dropwise to
600mL of
acetonitrile, giving a precipitate that was filtered, washed with acetonitrile
,then diethyl
ether, and dried under vacuum. The crude product was purified by reverse-phase
HPLC
(50 minute 2-30% acetonitrile in water containing 0.1% trifluoroacetic acid to
yield [C]-
N N' aminoethyl-glutaric amide (vancomycin) (a compound of formula (22)
contaminated withl-hydroxybenzotriazole, elutes at 29 minutes) and [C-CJ-
[pentane-1,5-
dioic acid bis-((2-aminoethyl)amide]-bis-(vancomycin), a compound of Formula I
(elutes
at 36 minutes) as their respective trifluoroacetic acid salts.


CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
EXAMPLE 5
Preparation of a fC-C]I Compound of Formula I
( 1 ) Preparation of a Compound of Formula,~2l~
At room temperature vancomycin hydrochloride (7.3g, 4.7mmol) was dissolved
5 in 75mL of dimethylsulfoxide. To this solution was added N,N
diisopropylethylamine
(4.lmL, 23.Smmol) followed by 9-fluorenylmethyl N (2-aminoethyl)carbamate
hydrochloride (1.8g, 5.6mmo1). To the resulting solution at room temperature
was added
rapidly dropwise a solution of PyBOP (2.7g, 5.2mmol) and 1-
hydroxybenzotriazole
(630mg, 4.7mmo1) in 75mL 1,3-dimethyl-3,4,5,6-tetrahydro-2(11-pyrimidinone.
The
10 resulting solution was stirred at room temperature for 2 hours, then poured
into 800mL
diethyl ether, giving a gum. The diethyl ether was decanted and the gum washed
with
additional diethyl ether to give [C]-[2-Fmoc-aminoethyl) vancomycin, a
compound of
formula (21).
15 (2) Preparation of a Compound of Formula (22)
T'he compound of formula (2I) prepared above, as a gum, was then taken up in
40mL of N,N dimethylformamide, to which l OmL of piperidine was added and the
solution left to stand at room temperature for 20 minutes. The solution was
then added
dropwise to 450mL of acetonitrile giving a precipitate. Centrifugation was
followed by
20 decantation of the acetonitrile and the residue washed twice with 450mL of
acetonitrile,
once with 450mL of diethyl ether and air dried. The residue was taken up in
water,
acidified to pH<5 with a small amount of 3N hydrochloric acid and purified by
reverse-
phase HPLC using a gradient of 2-30% acetonitrile in water containing 0.1%
trifluoroacetic acid yielding [C)-(2-aminoethyl) vancomycin, a compound of
formula
25 (22).
(3) Preparation of a fC-Cl Linked Compound of Formula I
The compound of formula (22) (400mg, 220p.mo1) and glutaric acid (6) (lOmg,
76~mo1) were dissolved in SmL N,N dimethylformamide and N,N
diisopropylethylamine
30 (140p,L, 800 p,mol) followed by PyBOP (83mg, 160 ~.mol) and 1-
hydroxybenzotriazole
(lOmg, 74 ~mol) in 500 ~L N,N dimethylformamide. The reaction was stirred for
75


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
86
minutes at room temperature then an additional 20mg of PyBOP was added. 75
minutes
later the solution was dripped into 45mL acetonitrile. The resulting
precipitate was
collected by centrifugation, washed with ether, air dried and purified by
reverse-phase
HPLC (50 min 2-30% acetonitrile in water containing 0.1 %trifluoroacetic acid,
elutes at
S 33 min) to give [C-C]-[pentane-1,5-dioic acid bis-[(2-aminoethyl)amide]-bis-
(vancomycin), a compound of Formula I, as its trifluoroacetic salt.
EXAMPLE 6
Preparation of a [C-Cl Com-pound of Formula I
( 1 ) Preparation of a Compound of Formula y25)
To an oven-dried, 1000 mL round bottomed flask, equipped with magnetic
stirring bar, were added vancomycin hydrochloride (34.1 g, 23 mmol, 1 eq), N
Fmoc-
aminoacetaldehyde (7) ( 6.5 g, 23 mmol, 1 eq), DIPEA (8.5 mL, 46 mmol, 2 eq)
and
DMF (340 mL). The mixture was stirred at ambient temperature over 2 hours, and
monitored by HPLC. The reaction became homogenous, and ~90% conversion to the
imine was observed. Methanol (340 mL) and NaCNBH3 (4.3 g, 69 mmol, 3 eq) were
added to the solution, followed by TFA (5.2 mL, 69 mmol, 3eq). Stirring was
continued
for an additional hour at ambient temperature. After the reaction was
complete, methanol
was removed in vacuo. The residue containing the crude product and DMF was
slowly
poured into a 5 L flask and stirred with acetonitrile (3.5 L). A white
precipitate was
formed. The suspension was allowed to settle at ambient temperature and the
supernetant
was decanted. The white solid was filtrated and triturated with ether (2 L).
After
filtration, the crude product (25) was dried under high vacuum overnight.
A 8 x 26 cm column was packed with octadecyl bonded silical gel. The column
was washed with 800 mL of 90% Solvent B [acetonitrile in water , 0.1 % TFA]
and
equilibrated with 800 mL of 10% Solvent B. Crude Fraction A (10 g) was
dissolved in
30% Solvent B (150 mL, containing 2 mL of 3 N HCl) and loaded onto the column.
It
was then flashed with 10%B (800 mL x 2), 40%B (800 mL x 3) and 90%B (800 mL).
The fractions were checked by analytical HPLC. After lyophilization, V-[Fmoc-2-

aminoethyl] vancomycin (25) was obtained as its TFA salt.


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
87
(2) Preparation of a Compound of Formula (26)
At room temperature the compound of formula (25) prepared above (205mg,
0.1 Ommol) and [C]-[glutaryl bis-[(2-aminoethyl)amide] (vancomycin), a
compound of
formula (22), prepared as above (200mg, 0.1 Ommol), were dissolved in 2 mL N,N-

dimethylformamide. To this solution was added N,N-diisopropylethylamine
(126uL,
0.72mmo1), 1-hydroxybenzotriazole (l4mg, O.lOmmol) and PyBOP (59mg, 0.1
lmmol).
The solution was stirred for 2 hours then added dropwise to 45mL acetonitrile
giving a
precipitate that was collected by centrifugation. The precipitate was purified
by reverse-
phase HPLC (50 minutes 10-70% acetonitrile in water containing 0.1 %
trifluoroacetic
acid, elutes at 24 minutes) giving (V]-[2-Fmoc-aminoethyl] [C-C]-pentane-1,5-
dioic acid
bis-[(2-aminoethyl)amide]-bis-(vancomycin), a compound of formula (26), as its
trifluoroacetic acid salt.
(3) Preparation of a Compound of Formula (27)
To a solution of the compound of formula (26) prepared above, in SmL N,N
dimethylformamide was added O.SmL piperidine,. The resulting solution was left
at
room temperature for 30 minutes, then added dropwise to acetonitrile, giving a
precipitate that was collected by centrifugation, washed with ether and air
dried.
Reverse-phase HPLC purification (50 minutes 2-50% acetonitrile in water
containing
0.1 % trifluoroacetic acid, elutes at 24 minutes) yielded ([V]-[2-aminoethyl])
[C-C)-
pentane-1,5-dioic acid bis-[(2-aminoethyl)amide]-bis-(vancomycin), a compound
of
formula (27), as its trifluoroacetic acid salt.
(4) Preparation of a C-C Compound of Formula I in which V is Substituted by n
Decvlamidoethvl
The compound of formula (27) prepared above (lOmg, 2.8~,mol), decanoic acid
(O.Smg, 2.9~mo1) and N,N diisopropylethylamine (2.9p.L, 16.6p,mo1) were
dissolved in
200p,L N,N dirnethylformamide. PyBOP (l.Smg, 2.9p,mol) and
l~hydroxybenzotriazole
(0.4mg, 3.Opmo1) in 20p,L N,N dimethylformamide were added, the solution left
at room
temperature for 30 minutes and then added dropwise to l.SmL of acetonitrile,
giving a
precipitate. The precipitate was collected by centrifugation, washed with
ether and air-


CA 02318394 2000-07-12
WO 99/42476 PCT/US99103850
88
dried. Reverse-phase HPLC purification (60 minutes 10-50% acetonitrile in
water
containing 0.1 % trifluoroacetic acid, elutes at 42 minutes) yielded ([V]-(2-
decylamido)ethyl)vancomycin [C-C]-pentane-1,5-dioic acid bis-[(2-
aminoethyl)amide]-
(vancomycin) a compound of Formula I, as its trifluoroacetic acid salt.
EXAMPLE 7
Preparation of a ~C-Cl Compound of Formula I via Alkvlation
A mixture of [C]-[2-aminoethyl] vancomycin (22) (SOmg, 0.027mmol), a,a'-
dibromo-m-xylene (3.4 mg, 0.013 mmol) and N,N diisopropylethylamine (6.5 mg,
0.050
mmol) in dimethylformamide (0.1 S mL) was heated at 60°C for 8 hours,
cooled to room
temperature for 18 hours and then heated at 60°C for 8 hours. Upon
cooling, the product
was precipitated by the addition of acetonitrile (lSmL) and isolated by
centrifugation.
The solid product was washed with ether ( 1 S mL) and dried in vacuo. Reverse-
phase
preprative HPLC (5-40% acetonitrile in water containing 0.1 % trifluoroacetic
acid over
90 min} gave the [C-C]-m-phenyl-bis[methylaminoethylamino] bis vancomycin as
its
trifluoroacetate salt. MS calculated (MH+) 3082, 3084; found 3084.
Preparation of other [C-Cl Compounds of Formula I
Accordingly, the following [C-C] compounds of Formula I were prepared
following the procedures of Examples 1-7 above. It should be noted that
"Class" denotes
the point of attachment of the linker; Vl, N1, and Rl represent the point of
attachment
with respect to the first glycopeptide; V2, N2, and R2 represent the point of
attachment
with respect to the second glycopeptide.


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
89
. . , , , , ,


. . , , , , ,


N
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0
z - N M




CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850



x x x x x x x x x .~ x x x x



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s


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y


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CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
91



x s x s x x x x x x .~ x x


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CA 02318394 2000-07-12
WO 99/42476 PCT/I3S99/03850
92



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CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
93



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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
94



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~




CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
Preparation of other fC-C] Compounds of Formula I
Similarly, other [C-C] compounds of Formula I are prepared following the
procedures of Examples 1-7 above, including those where the ligand is
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, or
optionally
substituted chloroeremomycin.
EXAMPLE 8
Preparation of fV-Vl Compounds of Formula I in which Position C is Substituted
10 ( 1 ) Preparation of a Compound of Formula (31 )
V-[Fmoc-2-aminoethyl] vancomycin (the compound of formula {25) prepared
above) (300mg, 150~.mo1) was dissolved in N,N dimethylformamide. N,N
diisopropylethylamine (65~L, 370~,mo1) was added followed by 1-
hydroxybenzotriazole
(2Omg, 150~,mo1), 3-(dimethylamino)propylamine (30~L, 240p,mol) and PyBOP
(90mg,
15 170~,umol). The solution was kept at room temperature for 30 minutes, then
dripped into
acetonitrile to give a white precipitate. The precipitate was collected by
centrifugation
and purified by reverse-phase HPLC (50 minutes 10-50% acetonitrile in water
containing
0.1 % trifluoroacetic acid, elutes at 35 minutes) to give [C]-[3-
dimethylamino)propylamino] [V]-[Fmoc-2-aminoethyl] vancomycin a compound of
20 formula (31), as its trifluoroacetic acid salt.
(2) Preparation of a Compound of Formula (321
To a solution of the compound of formula (31) prepared in step 1, in 2mL N,N
dimethyIformamide was added O.SmL piperidine. The solution was left at room
25 temperature for 10 minutes then added dropwise to 1 SmL acetonitrile,
yielding a white
precipitate. The precipitate was collected by centrifugation, washed with
ether, air dried
and purified by reverse-phase HPLC (50 minutes 2-SO% acetonitrile in water
containing
0.1 %trifluoroacetic acid, elutes at 21 minutes) to give [C]-[3-
(dimethylamino)propylamino]-[V]-[2-aminoethyl] vancomycin, a compound of
formula
30 (32) as its trifluoroacetic acid salt (177mg, 92pmo1, 61%).


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
96
(3) Preparation of a Compound of Formula I
To the compound of formula (32) prepared above (20mg, 1 Opmol), was added
glutaric acid (6) (0.7mg, S~,mol) in SOpL N,N dimethylformamide followed by
150~,L
N,N dimethylformamide. To the resulting solution was added PyBOP (5.4mg,
lOp,mol)
and 1-hydroxybenzotriazole (l.4mg, lOp,mol) in SOpL N,N dimethylformamide
followed
by N,N diisopropylethylamine (8.2p.L, 47p,mol). The solution was left at room
temperature overnight, then dripped into 1 SmL acetonitrile. The resulting
precipitate
was collected by centrifugation, washed with ether, air dried and purified by
reverse-
phase HPLC (50 minutes 2-30% acetonitrile in water containing 0.1
%trifluoroacetic
acid, elutes at 26 minutes) to give [V-V]-pentane-1,5-dioic acid bis-[(2-
ethyl)amide]- bis-
([C]-3-dimethylaminopropylamino-{vancomycin)), a [V-V] compound of Formula I
as
its trifluoroacetic acid salt.
EXAMPLE 9
Preparation of a ~V-Vl Compound of Formula I in which Position C is
Substituted
(1) Preparation of a Compound of Formula (36)
To a solution of (C]-(3-dimethylaminopropylamino)-[V]-(2-aminoethyl)
vancomycin, the compound of formula (32) prepared as above (20mg, l0~mo1), and
glutaric acid mono-9-fluorenylmethyl ester (15) (3.2mg, lOpmol) in 200pL N,N
dimethylformamide was added N,N diisopropylethylamine (9.lpL, 52pmo1) followed
by
PyBOP (5.4mg, lOpmol) and 1-hydroxybenzotriazole (l.4mg, lOpmol) in SOuL N,N
dimethylformamide. The resulting solution was kept at room temperature for 80
minutes, then added dropwise to 1 SmL of acetonitrile. It was then dissolved
in SOOuL
N,N dimethylformamide, treated with 150uL piperidine for 1 hour at room
temperature
and added dropwise to lSmL acetonitrile. The resulting precipitate was
collected by
centrifugation, washed with ether, air dried and purified by reverse-phase
HPLC (2-30%
acetonitrile in water containing 0.1 % trifluoroacetic acid) to give [C]-[{3-
(dimethylamino)propylamino]-[V]-pentane-1,5-dioic acid (2-aminoethyl)amide)-
(vancomycin), a compound of formula (36) as its trifluoroacetic acid salt
(13.1 mg, 6.4
~mol, 64%).


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
97
(2) Preparation of a Compound of Formula (32')
To a solution of the compound of formula (25) prepared as described in Example
6 (30mg, 1 Spmol) in 300p,L of N,N dimethylformamide was added dodecylamine
(4.3mg, 23p.mo1) 20p,L N,N dimethylformamide followed by N,N
diisopropylethylamine
(67p,L, 38p,mo1) then 1-hydroxybenzotriazole (20mg, ISOp,mol) and PyBOP (90mg,
170pmo1) in 40pL N,N dimethylformamide. The solution was kept at room
temperature
for an hour, then dripped into lSmL of acetonitrile. The resulting precipitate
was
collected by centrifugation, washed with ether and air dried. It was then
dissolved in
1mL N,N dimethylformamide, treated with 200p,L piperidine for 10 minutes, then
dripped into I SmL of acetonitrile. The resulting precipitate was collected by
centrifugation, washed with ether, air dried and purified by reverse-phase
HPLC (50
minutes 10-70% acetonitrile in water containing 0.1 % trifluoroacetic acid,
elutes at 38
minutes) to give (C)-n-dodecylamino [V]-pentane-1,5-dioic acid (2-
aminoethyl)amide)-
vancomycin, a compound of formula (32') as its trifluoroacetic acid salt
(22.Smg,
l2pmol, 77%).
(3) Preparation of a Compound of Formula I
The compound of formula (36) (10.8mg, 5.3pmo1) and the compound of formula
(32') (lO.Omg, 5.3pmo1) and N,N diisopropylethylamine (6.SpL, 37p,mo1),
prepared as
above, were dissolved in 200p,L of N,N dimethylformamide. A solution of PyBOP
(2.8mg, 5.4p.mo1) and I-hydroxybenzotriazole (0.7mg, 5.2p,mo1) in 20uL ofN,N
dimethylformamide was added and the resulting solution left for 1 hour at room
temperature. PyBOP (0.7mg, 1.3p,mo1) and 1-hydroxybenzotriazole (0.2mg, 1
.3pmo1)
in Sp,L of N,Ndimethylformamide was added. The solution was left for 1 hour
and
dripped into 1 SmL acetonitrile. The resulting precipitate was collected by
centrifugation, washed with ether, air dried and purified by reverse-phase
HPLC (10-
50% acetonitrile in water containing 0.1 %trifluoroacetic acid) to give [C]-(3-

dimethylaminopropylamino)-[V-V]- pentane-I,5-dioic acid bis-[(2-
aminoethyl)amide]-[
C']-(n-dodecylamino)-bis-(vancomycin), a compound of Formula I as its
trifluoroacetic
acid salt.


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
98
Preparation of other fV-V] Compounds of Formula I
Following the procedures of Examples 8 and 9, the following [V-V] compounds
of Formula I were prepared. It should be noted that "Class" denotes the point
of
attachment of the linker; C 1, N 1, and R 1 represent the point of attachment
with respect to
the first glycopeptide; C2, N2, and R2 represent the point of attachment with
respect to
the second glycopeptide.


CA 02318394 2000-07-12
WO 99141476 PCTNS99/03850
99
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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
101



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CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
102



,


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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
103
Preparation of other f V-Vl Com,~ounds of Formula I
Similarly, other [V-V] compounds of Formula I are prepared following the
procedures of Examples 8 and 9 above, including those where the ligand is
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, or
optionally
S substituted chloroeremomycin.
EXAMPLE 10
Preparation of an fN-Nl Compound of Formula I
{ 1 ) Preparation of a Compound of Formula (411
Vancomycin hydrochloride (4.00 g, 2.60mmol) was suspended in 40 mL of 1,3-
dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone and heated to 70°C for
15 minutes. N
(9-fluorenylmethoxycarbonyl)-aminoacetaldehyde (7) (720 mg, 2.6 mmol) was
added
and the mixture was heated at 70°C for one hour. Sodium
cyanoborohydride (160 mg,
2.5 mmol) in 2 mL methanol was added and the mixture was heated at 70°C
for 2 hours,
then cooled to room temperature. The reaction solution was added dropwise to
20mL of
acetonitrile, giving a precipitate that was collected by centrifugation. The
precipitate was
purified by reverse-phase HPLC on a Ranin C 18 Dynamax column (2.5 cm x 25 cm,
8~,m particle size), at 10 mL/min flow rate using 0.045% TFA in water as
buffer A and
0.045% TFA in acetonitrile as buffer B (HPLC gradient of 10-70% B over 90
minutes),
which yielded [N]-[Fmoc] -2 aminoethylvancomycin, a compound of formula (41)
as its
trifluroacetate salt. MS calculated: MH+, 1715; Found, 1715.
(2) Preparation of a Compound of Formula (42)
The compound of formula (41) obtained above (291 mg, 150 ~,mol) was
dissolved in 3mL of DMF. 3-(dimethylamino)propylamine (1) (28.3 ~,L, 225 ~mol)
was
added, followed by the addition of PyBOP (85.8 mg, 165 ~,mol), HOBt (20.3 mg,
1 SO
pmol) and Hunig's base {65.0 pL, 375 pmol). The reaction solution was stirred
for one
hour then). The reaction solution was stirred for 1 hour and then added
dropwise to
20mL of acetonitrile giving a precipitate, which was collected by
centrifugation, to give


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
104
compound of formula [C]-3-(dimethylamino)propylamino)-[N]-[Fmoc]-2-
aminoethylvancomycin (42) as a white solid. MS calculated: MH+, 1800; Found,
1800.
(3) Preparation of a Compound of Formula l43)
The compound of formula (42) obtained above was dissolved in 1 mL of DMF,
and 100 p,L of piperidine added to the solution. The solution was allowed to
stand at
room temperature for 30 minutes and the course of the reaction was followed by
mass
spectroscopy. The reaction solution was added dropwise to 20mL of
acetonitrile, giving
a precipitate that was collected by centrifugation. The precipitate was
purified by
reverse-phase HPLC on a Ranin C 18 Dynamax column (2.5 cm x 25 cm, 8pm
particle
size), at 10 mL/min flow rate using 0.045% TFA in water as buffer A and 0.045%
TFA
in acetonitrile as buffer B (HPLC gradient of 2-50% B over 90 minutes), which
yielded
[C]-3-(dimethylamino)propylamino)-[NJ-2-aminoethylvancomycin, a compound of
formula (43), as its trifluroacetate salt. MS calculated: MH+, 1578; Found,
1578.
Preparation of a Compound of Formula I
The compound of formula (43) prepared above (20.0 mg, 12.7 pmol) was
dissolved in S00 wL of DMF. H02C-(CH2)2-NH-C(O)-(CH2)2-NH-C(O)-(CH2)3-C(O)-
NH-(CH2)2-C(O)-NH-(CH2~-C02H (6) (2.64 mg, 6.34 p,mol) was added, followed by
PyBOP (8.24 mg, 15.8 pmol), HOBt (2.13 mg, 15.8 p,mol) and Hunig's base (8.8
p,l,
51.0 p,mol). The reaction solution as added dropwise to 20mL of acetonitrile,
giving a
precipitate that was collected by centrifugation. The precipitate was purified
by reverse-
phase HPLC on a R.anin C 18 Dynamax column (2.5 cm x 25 cm, 8p,m particle
size), at
10 mL/min flow rate using 0.045% TFA in water as buffer A and 0.045% TFA in
acetonitrile as buffer B (HPLC gradient of 2-50% B over 90 minutes), which
yielded a
[N-N] Linked compound of Formula I as its trifluoroacetate salt. MS
calculated: MH+,
3533; Found, 3533.
Preparation of other fN-N] Compounds of Formula I
Following the procedures of Example 10, the following [N-N] compounds of
Formula I were prepared. It should be noted that "Class" denotes the point of
attachment


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
105
of the linker; C 1, V 1, and R 1 represent the point of attachment with
respect to the first
glycopeptide; C2, V2, and R2 represent the point of attachment with respect to
the
second glycopeptide.


CA 02318394 2000-07-12
WO 99142476 PCT/LJS99/03850
106



.,
x ,


N
> x


..
> x



0


O



O


U


z


z


U O


N
x
~?


N
c



x
~.
U~


~N


,'', N
.



~a


8 z


v x


0
x


V




CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
107
Preparation of other fN-N) Compounds of Formula I
Following the procedures of Example 10, other [N-NJ compounds of Formula I
are prepared, including those where the ligand is optionally substituted
vancomycin,
optionally substituted N-desmethylvancomycin, or optionally substituted
S chloroeremomycin.
EXAMPLE 11
Preparation of an Aelvcone f0-O] Compound of Formula I
(1) Preparation of a Compound of Formula f47)
Vancomycin hydrochloride hydrate (lOg, fi.4mmo1) was dissolved in 100mL of
dimethyl sulfoxide (DMSO) and 3-(dimethylamino)propylamine (3.2mL, 26 mmol)
was
added. PyBOP (3.3g, 6.4mmol) and 1-hydroxybenzotriazole (HOBT, 0.9g, 6.4mmo1)
dissolved in 100mL N,N dimethylformamide (DMF) was added dropwise at room
temperature. The reaction was stirred for one hour and dripped into
acetonitrile to give a
white precipitate, which was filtered and washed with acetonitrile, ether and
dried under
vacuum to give a syrup of crude [C]-3-(dimethylamino)propyl amino vancomycin
(46).
A portion of this syrup was dissolved in 1 OOmL trifluoroacetic acid (TFA),
heated at
323K for 2 hours, cooled to room temperature and added dropwise to ether,
resulting in a
green precipitate. The precipitate was collected by filtration, dried under
vacuum and
purified by reverse-phase HPLC (2-50% acetonitrile in water containing 0.1 %
TFA) to
give [C]-3-(dimethylamino)propyl amino vancomycin aglycone, a compound of
formula
(47), as its TFA salt.
SUBSTITUTE SHEET (RULE 26j


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
108
(2) Preparation of an [O-O] Compound of Formula I
[C]-3-(dimethylamino)propyl amino vancomycin aglycone trifluoroacetate
(32mg, 22umol) was dissolved in 320uL DMF and potassium carbonate (35mg,
250umo1) was added. The suspension was stirred for 30 minutes at room
temperature
then 1,3-dibromopropane (0.9uL, 9umol) was added in 20uL DMF. The suspension
was
stirred for 36 hours and diluted with 1mL 10% acetic acid in water. Reverse-
phase
HPLC purification yielded [O-O]-1,3-propane-bis([C]-3-
(dimethylamino)propylamino-
vancomycin) (as its trifluoroacetate salt.
EXAMPLE 12
Preparation of an Aelvcone j0-O] Compound of Formula I
(1) Preparation of a Compound of Formula y52)
[C]-3-(dimethylamino)propylamide vancomycin aglycone (47), as its
trifluoroacetate salt (SOOmg, 340umo1) was dissolved in SmL DMF and potassium
carbonate (SOOmg, 3.6mmo1) was added. The mixture was stirred for 15 minutes
at room
temperature then tert-butyl N (2-bromoethyl)carbamate (77mg, 340umo1) was
added.
The mixture was stirred at room temperature for 24 hours, then tent-butyl N (2-

bromoethyl)carbamate {70mg, 310umol) was added. The mixture was stirred at
room
temperature for 7 hours then dripped into ether giving a precipitate that was
collected by
centrifugation, washed with acetonitrile and dissolved in S:I:2 water/acetic
acid/acetonitrile. This solution was purified by reverse-phase HPLC giving
vancomycin
[C]-dimethylaminopropylamide [O]-2-(N t-BOC-amino)ethoxy aglycone as the
trifluoroacetate salt, which was treated with 1mL TFA for 30 minutes at room
temperature. Reverse-phase HPLC purification yielded [C]-3-
(dimethylamino)propylamino [O]-(2-aminoethyl) vancomycin aglycone (52) as the
trifluoroacetate salt.
SUBSTITUTE SHEET (RULE 28j


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
109
(2) Preparation of a Compound of Formula I
Succinic acid (0.18mg, l.Sp,mo1), HATU (1.2 mg, 3p,mo1) and HOAT (0.4mg,
3umo1) were dissolved in 40p,L DMF at room temperature. N,N
diisopropylethylamine
(O.Sp,L, 3p.mol) was added, the solution left for 10 minutes, then added to a
mixture of
S vancomycin [C]-dimethylaminopropylamide [O]-(2-aminoethoxy) aglycone (52) as
its
trifluoroacetate salt (Smg,3p,mol) and N,N diisopropylethylamine (2,2p,L,
l2p,mol) in
100p,L DMF.. This mixture was left at room temperature for a further 20
minutes, then
dripped into acetonitrile giving a white precipitate, which was collected by
centrifugation. Reverse-phase HPLC gave [O-O]-butane-1,4-dioic bis-[(2-
ethyl)amide]
[C,C'])-bis-([C]-3-(dimethylamino)propylamino)-bis-(vancomycin aglycone), a
compound of Formula I, as its trifluoroacetate salt.
EXAMPLE 13
Preparation of an Aelycone [O-O] Compound of Formula I
(1) Preparation of a Compound of Formula (57)
A compound of formula (47) prepared as shown in Example 12 above (500mg,
340umol) was dissolved in SmL DMF and potassium carbonate (SOOmg, 3.6mmol) was
added. The mixture was stirred for 1 S minutes at room temperature then tert-
butyl
bromoacetate (12) (46pL, 310pmol) was added. The mixture was stirred at room
temperature for 1 hour, then dripped into ether giving a precipitate that was
collected by
centrifugation, washed with acetonitrile and dissolved in 10:1 water/acetic
acid. This
solution was purified by reverse-phase HPLC giving (in order of elution)
recovered
starting material, a product formed by alkylation on the dimethylamino group,
followed
by desired product (i.e. the compound of formula (56), its BOC derivative),
which was
treated with 1mL TFA for 20 minutes at room temperature. Reverse-phase HPLC
purification yielded [C]-3-(dimethylamino)propylamino [O]-(2-carboxymethyl)
vaizcomycin aglycone, the compound of formula (57), as its trifluoroacetate
salt.
SUBSTITUTE SHEET (RULE 26)


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
110
(2) Preparation of a Compound of Formula I
At room temperature the compound of formula (57), prepared as shown above
(7mg, Sumol), was dissolved in 160p.L DMF, N,N diisopropylethylamine (3.Sp,L,
20p,mol) and 1,3-diaminopropane (2) (0.2p.L, 2.Sp.mo1) were added followed by
PyBOP (2.6mg, Spmol) and HOBT (0.7mg, Sp,mol) in 20uL DMF. The mixture was
left for 110 minutes then dripped into acetonitrile giving a white
precipitate. The
precipitate was collected by centrifugation and purified by reverse-phase HPLC
giving
[O-O]-1,3-bis-(2-acetylamino)propane-bis-([C]-3-(dimethylamino)propylamino)-
(vancomycin aglycone), a compound of Formula I, as its trifluoroacetate salt.
(4) Preparation of other Compounds of Formula I
Accordingly, following the procedures of Examples 11-13, other aglycone [O-O]
compounds of Formula I were prepared.
SUBSTITUTE SHEET (RULE 26)


CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
111
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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
112
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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
113
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x x x x x x x x x


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CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03$50
114
Preparation of other (O-O] Compounds of Formula I
Following the procedures of Example 11-13, other [O-O] compounds of Formula
I are prepared, including those where the ligand is optionally substituted
vancomycin,
optionally substituted N-desmethylvancomycin, or optionally substituted
chloroeremomycin.
EXAMPLE 14
Preparation of a fR-Rl Compound of Formula I via Scheme 13
Preparation of a Compound of Formula I
To SO% aqueous acetonitrile (4.0 mL) was added sequentially 37% formalin
(lSuL, 0.20mmo1), 1,4-diaminobutane (8.8mg, O.lOmmol), N,IV
diisopropylethylamine
(174uL, l.Ommol) and vancomycin hydrochloride (300mg, 0.20mmol). After
stirring
18h, the product was precipitated by the addition of acetonitrile (45mL). The
solid was
isolated by centrifugation, dried in vacuo, and purified by reverse-phase HPLC
(5-20%B
over 45min at a flow rate of 50 mL.min). Fractions containing the desired
product were
identified by mass spectrometry, pooled, and lyophilized to give [R-R]-1,4-bis-

(methylamino)butane-bis-(vancomycin), a white powder. MS calculated (MH+,
3008;
found, 3008).


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N
z x x x x


x x x x x


N
x x x x


..
x x x x


x


U
v


z


x


V~ Y


x
0 o z o



O



O U



~r


U



Y


O ~


V O O


N
x



x


z
N


N
x


U


Y



O
v



x


U


x



x x


z z
z o


r ~ _
N



Y Y .U Y
.


~ z



U


a


o4 0. AG e4
G G ~ i


a o ; c
;


d
z


.~ N m





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Preparation of other (R-R,~ Compounds of Formula I
Following the procedures of Example 14, other [R-R] compounds of Formula I
are prepared, including those where the ligand is optionally substituted
vancomycin,
optionally substituted N-desmethylvancomycin, or optionally substituted
chloroeremomycin.
EXAMPLE 1 S
Preparation of a (C-V] Compound of Formula I
Preparation of a Compound of Formula (607
[V]-2-[Fmoc-aminoethyl]vancomycin(compound of formula 25) (263mg,
0.135mmo1) was dissolved in a mixture of DMF (2.OmL) and piperidine (I.OmL).
After
45 minutes, the product was isolated by the addition of acetonitrile (45mL)
followed by
centrifugation. The resulting solid was washed with ether (45mL) followed by
1 S centrifugation. The crude product was dried in vacuo and purified by
reverse phase
preprative HPLC (S-15% acetonitrile in water containing 0.1% trifluoroacetic
acid over
30 min). The appropriate fractions were combined and lyophylized to give jV]-2-

(aminoethyl)vancomycin (a compound of formula (60)) (200mg) as its
trifluoroacetate
salt. MS calculated (MH+) 1493; found 1494.
Preparation of a Compound of Formula t62)
A 0.010 M solution of PyBOP and HOBt in DMF (9.OSmL, 0.0905mmo1 each)
was added to [V]-2-[Fmoc-aminoethyl]vancomycin (a compound of formula 25')
(176mg, 0.0905mmol) followed by diisopropylethylamine (12.8mg, O.lOmmol).
After
stirnng for 15 minutes, another portion of diisopropylethylamine (12.8mg, 0.1
Ommol)
was added. After a further 5 minutes, the compound of formula (60) obtained
above
(166mg, 0.0905mmo1) and diisopropylethylamine ( 35mg 0.27mmo1) in DMF (l.SmL)
were added. The reaction mixture was stirred for 30 minutes, at which time the
product
of formula (61) was isolated by the addition of acetonitrile (905mL) followed
by
centrifugation. The resulting solid was dried in vacuo and then dissolved in
DMF (4mL)
and piperidine (l.OmL). After 10 minutes, the product was isolated by the
addition of


CA 02318394 2000-07-12
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117
acetonitrile (90mL) followed by centrifugation. The resulting solid was washed
with
ether (90mL) followed by centrifugation. The solid was dried in vacuo and
purified by
reverse phase preprative HPLC (5-15% acetonitrile in water containing 0.1%
trifluoroacetic acid over 20 min). The appropriate fractions were combined and
lyophylized to give namely ([V]-aminoethyl-vancomycin) [C-V]-N-(2-aminoethyl)
(vancomycin)- the product of formula 62 ( 173mg) as its trifluoroacetate salt.
MS
calculated (MH+) 2966 found 2966.
Preparation of a Compound of Formula I [C-V]
The compound of formula (62) obtained above (300mg, 0.085mmo1) was
dissolved in DMF (3.OmL) and then decanal was added (13.3mg, 0.085mmo1). After
stirring for 30 minutes, methanol (3.OmL} was added followed by 1.0 M sodium
cyanoborohydride in methanol (0.085mL, 0.085 mmol). After 1 hour, the product
was
precipitated by the addition of ether (45mL) followed by centrifugation. The
resulting
solid was dried in vacuo and purified by reverse phase preprative HPLC (15-35%
acetonitrile in water containing 0.1% trifluoroacetic acid over 35 min). The
appropriate
fractions were combined and lyophylized to give ([VJ-n-decylaminoethyl-
vancomycin)
[C-V)-N-(2-aminoethyl)-(vancomycin), a compound of formula I (126mg) as its
trifluoroacetate salt. MS calculated (MH+) 3106; found 3107.
EXAMPLE 16
Preparation of a [C-V] Compound of Formula I
Preparation of a Compound of Formula (65)
To a stirred solution of
[C]-(3-(dimethylamino)propylamine)-[V]-2-[Fmoc-aminoethyl]vancomycin (500
mg, 0.26 mmol, 1.0 equivalent) and succinic acid mono-(2-aminoethylamide
(109 mg, 0.29 mmol, 1.1 eq) in 6 ml DMF was added PyBOP (151 mg, 0.29 mmol),
HOBT (35 mg, 0.26 mmol), and finally diisopropylethylamine (120 microliters,
0.65 mmol, 2.5 eq). This was allowed to stir at room temperature for twenty
minutes, at which time the electrospray mass spectrogram of the crude


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118
reaction mixture indicated complete conversion to product (M+ = 1940). This
reaction was poured into 60 ml of acetonitrile; the resulting precipitate
was collected by centrifugation, redissolved in 6 ml DMF, and treated with 1
ml piperidine. This reaction was stirred for 20 minutes then checked by
HPLC and MS which showed complete conversion to the desired product (M+ _
1718) 6. The reaction mixture was poured into 60 ml acetonitrile and the
resulting precipitate was collected by centrifugation. The product was
purified by reversed phase HPLC to provide pure material as the TFA salt.
Preparation of a Compound of Formula I [C-V]
To a stirred solution of 100 mg of the compound obtained as above (0.058
mmol) in 1.0 ml DMSO was added 104 mg (0.070 mmol) of vancomycin. The
resulting suspension was stirred until a clear solution was obtained. To
this solution was then added 1.0 ml DMF, 37 mg (0.070 mmol) PyBOP, 8 mg
(0.058 mmol) HOBT, and diisopropylethylamine (27 microliters, 0.145 mmol).
The reaction was stirred for one hour, then added to I 5 ml acetonitrile.
The resulting precipitate was collected by centrifugation, then purified by
reversed phase HPLC to afford (vancomycin)-[C-V)-butane-1,4-dioic
acid'-(2-aminoethyl)-amide-(2-ethyl)-amide-([C)-3-(dimethylamino)propylamino
-vancomycin), (M+ = 3150).
EXAMPLE 17
Preparation of Formula I (C-V)
The compound of formula (68) (61 mg, 30 pmol), PyBOP (16 mg, 31 pmol) and
HOBt (5 mg, 31 p,mol) were dissolved in 1 mL of DMF. To the solution were
added
compound (66) (60 mg, 28 p,mol) in I mL of DMF and Hunig's base (15 mL, 86
p,mol).
The reaction was stirred at room temperature for 1 hour. Piperidine (0.5 mL)
was added
to the solution and the reaction was continued for additional 1 S min. The
reaction
solution was added to 45 mL of ether, giving a precipitate which was collected
by
centrifugation. The precipitate was dried on vacuum and purified by reverse
phase HPLC
(80 min 5-45%acetonitrile in water containing 0.1 % trifluoroacetic acid,
compound


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eluted at 50 min), yielding ([V]-decylaminoethylvancomycin)-[C-V]- butane-1,4-
dioic
acid-(2-aminoethyl)-amide-(2-ethyl)-amide-([C]-N-glucosamino-vancomycin), a
compound of formula I (61 mg, 55%) as its trifluroacetate salt. MS calculated,
MH+
3409; Found, 3409.
(In this case, Rl= glucosamine, R2= decyl)
EXAMPLE 18
Preparation of a fC-Vl Compound of Formula I in which Position fCl is
Substituted
(1 ) Preparation of a Compound of Formula (72)
To a solution of [C]-2-aminoethyl vancomycin (22) (40.0 mg, 26.8 ~,mol) in
DMF (2.0 mL) was added a compound of the formula:
H02C-
CH2CH2NHOCCH2CH2NHOCCH2CH2CH2CONHCH2CH2CONHCH2CH2C02Fm (15)
(Fm refers to Fluorenylmethyl) (20.0 mg, 26.8 p,mol), followed by PyBOP (20.9
mg,
40.2 ~.mol), HOBt (5.40 mg, 40.2 ~.mol), and Hunig's base (23.3 p,L, 134
~,mol). The
reaction solution was stirred for 1 hour and then added dropwise to 20mL of
acetonitrile
giving a precipitate, which was collected by centrifugation. The crude
precipitate was
dried in air, yielding a compound having a MS calculated: MH+, 2068; Found,
2068.
The compound (71) was used in the next step without further purification by
dissolving
in 1mL of DMF, and I OO~L of piperidine added to the solution. The solution
was
allowed to stand at room temperature for 30 minutes, following the course of
the reaction
by mass spectroscopy. The reaction solution was added dropwise to 20mL of
acetonitrile, giving a precipitate that was collected by centrifugation. The
precipitate was
purified by reverse-phase HPLC on a Rarlin C18 Dynamax column (2.5 cm x 25 cm,
8?m
particle size), at 10 mL/min flow rate using 0.045% TFA in water as buffer A
and
0.045% TFA in acetonitrile as buffer B (HPLC gradient of 2-SO% B over 90
minutes).
The desired product was identified by mass spectroscopy using an API 300
electrospray
mass spectrometer and afterwards lyophilized to a white powder to afford
compound (72)
as a white powder. MS calculated: MH+, 1890; Found, 1890.


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(3) Preparation of a Compound of Formula I
The compound of formula (72) prepared above (10.0 mg, 4.80 pmol) was
dissolved in 500 p,L of DMF. [C]-(3-dimethylaminopropylamino)-[VJ-2-
(aminoethyl)
vancomycin, a compound of formula (32) (7.20 mg, 4.80 pmol) was added to the
solution, followed by PyBOP (2.50 mg, 4.8umol), HOBt (0.65 mg, 4.80 p,mol) and
Hunig's base (6.70 pl, 38.4 pmol). The reaction solution was added dropwise to
20mL
of acetonitrile, giving a precipitate that was collected by centrifugation.
The precipitate
was purified by reverse-phase HPLC on a Ranin C 18 Dynamax column (2.5 cm x 25
cm,
8p,m particle size), at 10 mL/min flow rate using 0.045% TFA in water as
buffer A and
0.045% TFA in acetonitrile as buffer B (HPLC gradient of 2-50% B over 90
minutes).
The desired product was identified by mass spectroscopy using an API 300
electrospray
mass spectrometer. The compound of Formula I was obtained as a white powder.
MS
calculated: MH+, 3448; Found, 3448.
EXAMPLE 19
Preparation of a fC-V) Compound of Formula I
( 1 ) Preparation of a Compound of Formula X761
Vancomycin hydrochloride (5.0 g, 3.2 mmol) was suspended in 40mL of 1,3-
dimethyl-3,4,5,6-tetrahydro-2-( 1 H)-pyrimidinone and heated to 70°C
for 15 minutes. 4-
Butoxybenzaldehyde (9){570 mg, 3.2 mmol) was added and the mixture heated at
70°C
for 1 hour. Sodium cyanoborohydride (241 mg, 3.8 mmol) in 2 mL methanol was
added
and the mixture was heated at 70°C for 2 hours, then cooled to room
temperature. The
reaction solution was added dropwise to 20mL of acetonitrile, giving a
precipitate that
was collected by centrifugation. The precipitate was purified by reverse-phase
HPLC on
a Ranin C18 Dynamax column (2.5 cm x 25 cm, 8pm particle size), at 10 mL/min
flow
rate using 0.045% TFA in water as buffer A and 0.045% TFA in acetonitrile as
buffer B
(HPLC gradient of 10-70% B over 90 minutes), yielding a compound of formula
(76) as
its trifluroacetate salt. MS calculated: MH+, 1610; Found, 1610.


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2. Preparation of a compound of Formula (77)
The compound of formula (76) prepared above (82 mg, 45.0 ~.mol) was dissolved
in 2mL of DMF. Ethylene diamine (2) (13.4 mg, 22.3 ~.mol) was added followed
by
PyBOP (28.0 mg, 54.0 ~,mol), HOBt (7.2 mg, 54.0 p,mol) and Hunig's base (63.0
~L,
360 pmol). The reaction solution was added dropwise to 20mL of acetonitrile,
giving a
precipitate that was collected by centrifugation. The precipitate was purified
by reverse-
phase HPLC on a Ranin C 18 Dynamax column (2.5 cm x 25 cm, 8p,m particle
size), at
mL/min flow rate using 0.045% TFA in water as buffer A and 0.045% TFA in
acetonitrile as buffer B (HPLC gradient of 2-50% B over 90 minutes), yielding
a
10 compound of formula (77) as its trifluroacetate salt. MS calculated: MH+,
1654; Found,
1654.
3. Preparation of a C-V Compound of Formula I in which one V position is
substituted
The compound of formula (77) prepared above (9.7 mg, 4.9 ~,lnol) was dissolved
in 500 ~L of DMF. The compound of formula (36) (10.0 mg, 4.9 ~,mol) was added
to
the solution, followed by PyBOP (3.06 mg, 5.9 ~,mol), HOBt (0.80 mg, 5.9 ~mol)
and
Hunig's base (6.7 ~L, 38.4 ~,mol). The reaction solution was added dropwise to
20mL of
acetonitrile, giving a precipitate that was collected by centrifugation. The
precipitate was
purified by reverse-phase HPLC on a Ranin C 18 Dynamax column (2.5 cm x 25 cm,
8~,m particle size), at 10 mL/min flow rate using 0.045% TFA in water as
buffer A and
0.045% TFA in acetonitrile as buffer B (HPLC gradient of 2-50% B over 90
minutes),
yielding a compound of Formula I as its trifluroacetate salt. MS calculated:
MH+, 3312;
Found, 3312.
EXAMPLE 20
Preparation of a fC-V] Compound of Formula I
To a solution of [C)-3-(dimethylaminopropylamido)- [V]-(2-
aminoethyl)vancomycin tetra-TFA salt (32) (30 mg, 0.015 mmol) and [C)-(2-
aminoethylamido)vancomycin di-TFA salt (27 mg, 0.015 mmol) in DMF (0.20 mL)
was
added a solution of dimethyl suberimidate dihydrochloride (4.1 mg, 0.01 S
mmol) and
diisopropylethylamine (3.9 mg, volume, 0.030 mmol) in DMF (0.41 mL). After one


CA 02318394 2000-07-12
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hour, the reaction was judged complete by mass spectrometry. The solution was
added
to acetonitrile ( l4mL), and the resultant precipitate isolated by
centrifugation,
resuspended in ether (l4mL) and isolated again by centrifugation. After drying
in vacuo,
the precipate was purified by reverse-phase HPLC on a Ranin C 18 Dynamax
column (2.5
cm x 25 cm, 8~, particle size) at 16 mL/min flow rate using 0.045%TFA in water
as
buffer A and 0.045%TFA in acetonitrile as buffer B (HPLC gradient of 5-20 %B
over
60 minutes). Fractions were analyzed by mass spectrometry and analytical HPLC,
and
those containg the title product were combined and lyophylized to give a [C-V)
linked
compound of Formula I as a white powder ( 9mg, 15%). MH+ calculated: 3205.2
found:
3205.5
(4) Pret~aration of other Compounds of Formula I
Accordingly, following the procedures of Examples 1 S-20 other [C-V]
compounds of Formula I were prepared (see Table below).


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123



.,
a


N
U U U U U U


z z z z z z


.e .: .; ,c


U U U U U U


_
x x x x x x


V Z Z Z Z Z



a
x s
'


U o,
v


Q N
N


N LV
~


U


x x ~ X13 ~3
Y


x x x x x x



U


z x x x x x x


x


z


0
..


U


:a


U x


x x x x
x x z z z


o x o 0 0 0


z ~ fi ~ ~?


N ~ . H
r
N


U N


Y Y


O O O O O p
V


x x x x


x ~ x
z z~


z z z z


x x~ ~ x x


U U U U V
~


a x xx x x Y x


Z Z,U Z Z Z


> > > > > >


U U U U U U U
h


d
-, N t~1ef V p




CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
124



" .r -= "
x x U U U U U V


V
~ z z z U U


z z z z
" z z


a - ~
a


Y Y Y Y U U U V V x x
U


Y ~' Y Y U
z z z z o z z z


z z z z


x V '>.
~


a a,
c a


a s O 5' .e s . .:
'


Uv o. ~ Y 4v V N,o ~ V
~ ~ ' P


N NN z QN N NN ~~ C~~7
o ~ N II
N


"~ "v x x"~i N y~ N ~ N
x~ xs ." a ~ ~'


"
x x ~?3 ~3 YU ~?3~ ~3 ~3~:~~~3 ~'3 ~3
~


x


x x x x x x x x x x x x


N ~


NN
cg


_
N


x"~~Im
x x x x x x a


x x x ~?3 x x
N


z z z z


N
N



z z z z v v v x x


v v
O O O O ~ x x Y


x x Y
~? ~? ~? ~ ~ ~ ~ ~ z z z


x" x" x x" 0 0 0 0 0 0 0


R ~ ~ '~ '''
..


.C "


x" x" x" x" x x x ~ " x x
~ ~


O O ~ X Y Y X .Va..
~ ~


.. .. O O O O O O O


z z z ~ x


z z z z x x
z z


z z
" "


x Y Y U U V x
Y U


x ~ Y Y Y . ~ U
z z z z~ z z z z z r.


, ~ z z
. ,


> > > >
U U U U U


U U V ~j ~j V V


~ eo a - ~ ~ ~~ '~ . :
00




CA 02318394 2000-07-12
WO 99/42476 PCT/US99103850
125



U U U U U U U x x x


U U U
z z z z z z z


, z z z
x x x x x ~ x ~ x x


Y Y v Y Y Y Y a Y
v


Y
z z o ~z z z z z


, z z z o 0



x ~ ~ x


U yL' _o U
~ a t Y N
s a


a H ,
s ~, x ~ a .Y O p
c. c


U v n, Y H ~ U
'~ a
~


U N ~ ~ ~ ~ z z z z
N ' N tJ ~
o


L '~ N N N N Z' "' .~. .= ~
L ~ ~' ~ a N
N


x ~3 Y ~?'~3v~3 S~3 ~3 ~


~ x Y ~U
~


x x x x x x x x x x x x x


m x x x x x x x x x x x x



N
x"a x~



"


xN xN x x x x x x


x x x x ~ Y ~''Y ~ Y U
~ ~


z~ z, z z z z z z z z z z z
3



> > > > > > > > > > > > >
U U U


U U U U U U U U U U


N N N N N N N N N ~ O .~


N tn M




CA 02318394 2000-07-12
WO 99/42476 PCTlUS99/03850
126



N
U U N ~ N N N U x
U U U V~ U U


z


, z z z z z z z


_ ,
~


U U U V U U U U
Y ~' Y


z x z x x x x x x x x x x x


0 0 o z o z z z z z z z


' ~ , ,
N N
x x x x"


>, ~ ,~ Y Y Y .U.i.
T


U N ~ O O O
V


r
x x x x x


:c x x
z z n z


z
N


x x x N x ~ 'a -a
~ ~ ,~ ~ x x x


Y Y ~ 'Us'UYU YU YU x x 'U
3 '


x s x x


x x x x x x x x x x x x x x


N
:O


N N o
v


_o


x ,~
x x x x x x x x x x x v ~
~


x x 3 aN


x ~ N
V U U


x


% z z


a


Y '' o o x


~? U
Y


x ~ z x -a"~ ~z


,
Y U O Z U U O


O x ~ U


z ~ c~ O O '
, .r


z _ ~ " ~ x


~ z z


, ,
'a N N -~ '~ N ~ H U_ U x
'~ 'a U


x _


x x x x x x ~ x x


z z z x z z z z z ~z z ~ z z


, Z
,


U V C > > > > > > > > > >


) U U U U U U U U U U U


m M r'~nM t~~f M eon ~ ~ M


~ V




CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
127



"


x x ac x x x x


U U U U
z z


z z z z z ~ ~? ~a o



N ~ N


Y Y ?' Y Y x


Y Y Y ~ Y Y Y
~'


xv x
z z z z o z z z z z z


Y3 o z


x x x x"
U ~


N U Cj
Y s


. N
O O p x " "
= a


x x
s U


z z~' ~


z z z z z
N n
N


n


U U


x x x YU Y3 YU Y Y~3 x x x


, , x x


x x x x x x x x x x x x x x x


t~ a


N N


N ~
~


x a
~


~ x x x x x x x x x x x x x x
natV


-"a



x x Y Y


z z x xz,
x


x o 0 0


Y


N
N


U U U U U x x


p Y Y Y Y Y U U


U ~ z O O O O O


s O O ~ ~ V


a cT ~ ~ z z z z
.
'



U ~ ~ ~ ~ .C '~ '~ N N
~


N N N N
x U U U U U U U U U U


U U U V
x x x "~'Y Y


x x x Y Y Y Y Y
z z z z ~z z z ~ z


z , z, z z z z



> > > > > > > > > > > > > > >


U U U U U U U U U U U U U U U


v N v, vN,~ ~ h ~ n ~ '


v v ~ ~
, , ,




CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
128



.= ~ ~ ~ .: ~ :


U U U U U U . U U U U U
U


U
z z z z z z z z z z z z ~z z z


, z
~


x~? x x x x~ x
V U U U U U U U U U


U U U U U
Y'N Y Y Y Y Y Y Y Y Y Y Y~'~


Z p z z Z x x
U


z z z 2 z Z Z
U


V ~ Y ~ ~ U U


N R ~ ~ -~ N


U V x x ,
.
x


U x
Y ~ Y Y Y Y Y a
~
a


z z z z z z



N N
U U U U U U U


U U
x x Y x x Y Y Y Y Y Y Y x



x x x x x x x x x x x m x x x x x


x x x x x x x x x x x x x x x x x


N x ~ N N ~ IV ~ ~ ~ N ~ ~ N


U U U U U U U U U U U U U U U U U
Y Y Y Y


Y Y Y Y Y Y Y Y Y Y Y Y
x x ~ ~


z z, z, z z z z z z z z ~ z z z
,


,


> > > > > > > > > > > > > > > > >
U U


U U U U U U U U U U U U U V U


~o ~o c~ ~ v, W n ao ~ n n ~ ~ ~ '~
vo vo o ~o


t




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WO 99/42476 PCT/US99/03850
129



.:
U U


V U U U U V U
z z


z z x z z z z


U U U U U
x ~ x x x x ~ "


a x z
x Y Y Y Y Y


x x x x Y Y Y V


z z z z z z z z z z z z z z


x x" z z


= ~ ~ ~
x


o
a x o,a o~ o... x x xs


U U
U Y ~ C~ C~ Y Z
x x x x x a
xv x~ ~


z xx x x x~ x
z z z~ z~ ;~ z~ z~


z ~
"C~~ x U x N
U


.. x U U U x ~
~ ~ x a


Y V Y YZ YZ Y~ Y~ x x V
,Z


x x U


x x x x x x x x x x x x x x


x x x x x x x x x x x x x x


a


z ~ Y


x z


z


0 0


x x
" x


~ x x


x x x
v "
x


z x" x


o z ~ ~ o


U U ~ _
~


U _


z z z
a


"' ~ ~ _ O O
O a


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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
146
Preparation of other fC-V1 Compounds of Formula I
Following the procedures of Examples 14-20, other [C-V] compounds of Formula
I are prepared, including those where the ligand is optionally substituted
vancomycin,
optionally substituted N-desmethylvancomycin, or optionally substituted
chloroeremomycin.
EXAMPLE 21
Preparation of a fC-N] Compound of Formula I
Preparation of a Compound of Formula I
The compound of formula (43) prepared in Example 9 (6.20 mg, 3.14 ~,mol) was
dissolved in 500 ~,L of DMF. The compound of formula (72) prepared in Example
14
(4.15 mg, 3.14 ~.mol) was added to the solution, followed by PyBOP (2.44 mg,
4.8
~,mol), HOBt( 0.65 mg, 4.8 ~,mol) and Hunig's base (6.7 ~L, 38.4 ~,mol)..).
The
reaction solution was added dropwise to 20mL of acetonitrile, giving a
precipitate that
was collected by centrifugation. The precipitate was purified by reverse-phase
HPLC on
a Ranin C18 Dynamax column (2.5 cm x 25 cm, 8~,m particle size), at 10 mL/min
flow
rate using 0.045% TFA in water as buffer A and 0.045% TFA in acetonitrile as
buffer B
(HPLC gradient of 2-50% B over 90 minutes), which yielded a compound of
Formula I
as its trifluroacetate salt. MS calculated: MH+, 3533; Found, 3533.
EXAMPLE 23
Preparation of a fC-R] Compound of Formula I
(1) To 50% aqueous acetonitrile (5.0 mL) was added diaminoethane (120mg,
2.Ommo1), 37% formalin (32uL, 0.42mmo1) and [C]-dimethylaminopropylamide
vancomycin (744mg, 0.40mmo1). After stirring for 4h, the product was
precipated by the
additon of acetonitrile (40mL). The solid was isolated by centrifugation, then
washed
with ether (40mL). The resulting solid was dried in vacuo, and purified by
reverse-phase
HPLC (S-30% acetonitrile in water containing 0.1% trifluoroacetic acid over
40min at a
flow rate of SOmI/min). Fractions containing the desired product were
identified by mass


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147
spectrometry, pooled, and lyophilized to give [R]-N-(aminoethyl)aminomethyl
[C]-
dimethylaminopropylamide vancomycin (448mg) as a white powder. MS calculated
(MH+), 1604; found, 1604.
(2) To a solution of vancomycin hydrochloride (34 mg, 0.023 mmol) and [R]-(N-
aminoethyl)aminomethyl-vancomycin-[C]-dimethylaminopropylamide in
dimethyiformamide ( 1.0 mL) and dimethylsulfoxide ( 1.0 mL) was added
sequentially
N,N-diisopropylethylamine ( l4uL, 0.078 mmol), and a solution of PyBOP and
HOBt in
DMF (0.26 mL of a solution 0.1 M in each, 0.026 mmol each). After 2 hours, the
product
was precipitated by the addition of acetonitrile (40mL) and isolated by
centrifugation.
The solid product was washed with ether (40 mL) and dried in vacuo. Reverse
phase
preprative HPLC (2-20% acetonitrile in water containing 0.1 % trifluoroacetic
acid over
90 min) gave the title product as its trifluoroacetate salt. MS calculated
(MH+) 3036;
found 3036.


CA 02318394 2000-07-12
WO 99/42476 PCTNS99/03850
148
, , , ,


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a , , , ,


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x x x x


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z z z z


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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
149



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x


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z x



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CA 02318394 2000-07-12
WO 99/42476 PCT/US99/03850
150



..
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a


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x x x x


v


z z


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CA 02318394 2000-07-12
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151
Preparation of other Compounds of Formula I
Following the procedures of Example 21-22, other [C-N], [C-R], and [N-V]
compounds of Formula I are prepared, including those where the ligand is
optionally
substituted vancomycin, optionally substituted N-desmethylvancomycin, or
optionally
substituted chloroeremomycin.
EXAMPLE 24
This example illustrates the preparation of a representative pharmaceutical
formulation containing a compound of Formula I, e.g., [C-C]-pentanedioic acid
bis-[(2-
aminoethyl)amide]-bis-(vancomycin).
An oral suspension is prepared having the following composition.
Ingredients
1 S Active Compound 1.0
g


Fumaric acid 0.5
g


Sodium chloride 2.0
g


Methyl paraben 0.1
g


Granulated sugar 25.5
g


Sorbitol (70% solution) 12.85
g


Veegum K (Vanderbilt Co.) 1.0
g


Flavoring 0.035
ml


Colorings 0.5
mg


Distilled water q.s. to 100 ml


_____


Other compounds of Formula I can be used as the active compound in the
preparation of the orally administrable formulations of this example.


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EXAMPLE 25
This example illustrates the preparation of a representative pharmaceutical
formulation containing an active compound of Formula I, e.g. [C-C]-
pentanedioic acid bis-
[(2-aminoethyl)amide]-bis-(vancomycin).
An injectable preparation buffered to a pH of 4 is prepared having the
following
composition:
Ingredients
Active Compound 0.2 g
Sodium Acetate Buffer Solution (0.4 M) 2.0 ml
HCL (1N) q.s. to pH 4
Water (distilled, sterile) q.s. to 20 ml
Other compounds of Formula I can be used as the active compound in the
preparation of the injectable formulations of this example.
EXAMPLE 26
This example illustrates the preparation of a representative pharmaceutical
formulation for injection containing an active compound of Formula I, e.g. [C-
C]
pentanedioic acid bis-[(2-aminoethyl)amide]-bis-(vancomycin).
A reconstituted solution is prepared by adding 20 ml of sterile water to lg of
the
compound of Formula I. Before use, the solution is then diluted with 200 ml of
an
intravenous fluid that is compatible with the compound of Formula I. Such
fluids are chosen
from 5% dextrose solution, 0.9% sodium chloride, or a mixture of 5% dextrose
and 0.9%
sodium chloride. Other examples are lactated Ringer's injection, lactated
Ringer's plus 5%
dextrose injection, Normosol-M and 5% dextrose, Isolyte E, and acylated
Ringer's injection
Other compounds of Formula I can be used as the active compound in the
preparation of the injectable formulations of this example.


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153
EXAMPLE 27
This example illustrates the preparation of a representative pharmaceutical
formulation for topical application containing a compound of Formula I, e.g.,
[C-C)-
pentanedioic acid bis-[(2-aminoethyl)amide)-bis-(vancomycin).
Ingredients grams
Active compound 0.2-10


Span 60 2


Tween 60 2


Mineral oil


Petrolatum 10


Methyl paraben 0.15


Propyl paraben 0.05


BHA (butylated hydroxy anisole) 0.01


Water q.s. to 100


All of the above ingredients, except water, are combined and heated to
60°C with
stirring. A sufficient quantity of water at 60°C is then added with
vigorous stirring to
emulsify the ingredients, and water then added q.s. 100 g.
Other compounds of Formula I can be used as the active compound in the
preparation of topical formulations of this example.
EXAMPLE 28
This example illustrates the preparation of a representative pharmaceutical
formulation containing a compound of Formula I, e.g., [C-CJ-pentanedioic acid
bis-[(2-
aminoethyl)amide]-bis-(vancomycin).
A suppository totalling 2.5 grams is prepared having the following
composition:
Ingredients
Active Compound 500 mg
Witepsol H-15* balance


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154
(*triglycerides of saturated vegetable fatty acid; a product of Riches-Nelson,
Inc., New
York, N.Y.)
Other compounds of Formula I can be used as the active compound in the
preparation of the suppository formulations of this example.
EXAMPLE 29
DETERMINATION OF ANTIBACTERIAL ACTIVITY
In Vitro Determination of Antibacterial Activity
Bacterial strains were obtained from either American Type Tissue Culture
Collection (ATCC), Stanford University Hospital (SU), Kaiser Permanente
Regional
Laboratory in Berkeley (KPB), Massachusetts General Hospital (MGH), the
Centers for
Disease Control (CDC), the San Francisco Veterans' Administration Hospital
(SFVA) or the
University of California San Francisco Hospital (UCSF). Vancomycin resistant
enterococci
were phenotyped as Van A or Van B based on their sensitivity to teicoplanin.
Some
vancomycin resistant enterococci that had been genotyped as Van A, Van B, Van
Cl or Van
C2 were obtained from the Mayo Clinic.
Minimal inhibitory concentrations (MICs) were measured in a microdilution
broth
procedure under NCCLS guidelines. Routinely the compounds were serially
diluted into
Mueller-Hinton broth in 96-well microtiter plates. Overnight cultures of
bacterial strains
were diluted based on absorbance at 600 nm so that the final concentration in
each well was
5 x 105 cfu/ml. Plates were returned to a 35oC incubator. The following day
(or 24 hours
in the case of Enterococci strains), MICs were determined by visual inspection
of the plates.
Strains routinely tested in the initial screen included methicillin-sensitive
Staphylococcus
aureus (MSSA), methicillin-resistant Staphylococcus aureus, methicillin-
sensistive
Staphylococcus epidermidis (MSSE), methicillin-resistant Staphylococcus
epidermidis
(MRSE), vancomycin sensitive Enterococcus faecium (VSE Fm), vancomycin
sensitive
Enterococcus faecalis (VSE Fs), vancomycin resistant Enterococcus faecium also
resistant
to teicoplanin (VRE Fm Van A), vancomycin resistant Enterococcus faecium
sensistive to
teicoplanin (VRE Fm Van B), vancomycin resistant Enterococcus faecalis also
resistant to
teicoplanin (VRE Fs Van A), vancomycin resistant Enterococcus faecalis
sensitive to
teicoplanin (VRE Fs Van B), Enterococcus gallinarium of the Van A genotype
(VRE Gm


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Van A)" Enterococcus gallinarium of the Van C-1 genotype (VRE Gm Van C-1),
Enterococcus casseliflavus of the Van C-2 genotype (VItE Cs Van C-2), ),
Enterococcus
flavescens of the Van C-2 genotype (VRE Fv Van C-2), and penicillin-sensitive
Streptococcus pneumoniae (PSSP) and penicillin-resistant Streptococcus
pneumoniae
S (PSRP). Because of the inability of PSSP and PSRP to grow well in Mueller-
Hinton broth,
MICs with those strains were determined using either TSA broth supplemented
with
defibrinated blood or blood agar plates. Compounds which had significant
activity against
the strains mentioned above were then tested for MIC values in a larger panel
of clinical
isolates including the species listed above as well as non-spectated coagulase
negative
Staphylococcus both sensitive and resistant to methicillin. (MS-CNS and MR-
CNS). In
addition, they are tested for MICs against gram negative organisms, such as
Escherichia colt
and Pseudomonas aeruginosa.
Determination of Kill Time
Experiments to determine the time required to kill the bacteria were conducted
as
described in Lorian. These experiments were conducted normally with both
Staphylococcus
and Enterococcus strains.
Briefly, several colonies were selected from an agar plate and grown at 35o C
under
constant agitation until it achieved a turbidity of approximately 1.5 and 10g
CFU/ml. The
sample was then diluted to about 6 x 106 CFUImI and incubation at 35oC under
constant
agitation was continued. At various times aliquots were removed and five ten-
fold serial
dilutions were performed. The spread plate method was used to determine the
number of
colony forming units (CFUs).
The compounds of Formula I were active in the above tests.
In Yivo Detenmination of Antibacterial Activity
1) Acute tolerability studies in mice
In these studies, the compounds of Formula I were administered either
intravenously or
subcutaneously and observed for 5-15 minutes. If there were no adverse
effects, the dose
was increased in a second group of mice. This dose incrementation continued
until
mortality occurred, or the dose was maximized. Generally, dosing began at 20
mg/kg and
increased by 20 mg/kg each time until the maximum tolerated dose (MTD) is
achieved.


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2) Bioavailabilitv studies in mice
Mice were administered the compound of Formula I either intravenously or
subcutaneously at a therapeutic dose (in general, approximately 50 mg/kg).
Groups of
animals were placed in metabolic cages so that urine and feces could be
collected for
analysis. Groups of animals (n=3) were sacrificed at various times (10 min, 1
hour and 4
hours). Blood was collected by cardiac puncture and the following organs were
harvested-
lung, liver, heart, brain, kidney, and spleen. Tissues were weighed and
prepared for HPLC
analysis. HPLC analysis on the tissue homogenates and fluids was used to
determine the
concentration of the compound of Formula I present. Metabolic products
resulting from
changes to the compound of Formula were also determined at this juncture.
3) Mouse septecemia model.
In this model, an appropriately virulent strain of bacteria (most commonly S.
aureus, or
E. Faecalis or E. Faecium) was administered to mice (N=5 to 10 mice per group)
intraperitoneally. The bacteria was combined with hog gastric mucin to enhance
virulence.
The dose of bacteria (normally 105-10~) was that sufficient to induce
mortality in all of the
mice over a three day period. One hour after the bacteria was administered,
the compound
of Formula I was administered in a single dose either IV or subcutaneously.
Each~dose was
administered to groups of 5 to I 0 mice, at doses that typically ranged from a
maximum of
about 20 mg/kg to a minimum of less than 1 mg/kg. A positive control (normally
vancomycin with vancomycin sensitive strains) was administered in each
experiment. The
dose at which approximately 50% of the animals are saved was calculated from
the results.
4) Neutropenic thigh model.
In this model antibacterial activity of the compound of Formula I was
evaluated against
an appropriately virulent strain of bacteria (most commonly S. aureus, or E.
Faecalis or E.
Faecium, sensitive or resistant to vancomycin). Mice were initially rendered
neutropenic by
administration of cyclophosphamide at 200 mg/kg on day 0 and day 2. On day 4
they were
infected in the left anterior thigh by an IM injection of a single dose of
bacteria. The mice
were then administered the compound of Formula I one hour after the bacteria
and at various
later times (normally 1, 2.5, 4 and 24 hours) the mice were sacrificed (3 per
time point) and


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157
the thigh excised, homogenized and the number of CFUs (colony forming units)
were
determined by plating. Blood was also plated to determine the CFUs in the
blood.
5) Pharmacokinetic studies
The rate at which the compound of Formula I was removed from the blood can be
determined in either rats or mice. In rats, the test animals were cannulated
in the jugular
vein. The compound of Formula I was administered via tail vein injection, and
at various
time points (normally 5, I 5, 30,60 minutes and 2,4,6 and 24 hours) blood was
withdrawn
from the cannula. In mice, the compound of Formula I was also administered via
tail vein
injection, and at various time points. Blood was normally obtained by cardiac
puncture.
The concentration of the remaining compound of Formula I was determined by
HPLC.
The compounds of Formula I were active in the above tests, and
demonstrated a broad spectrum of activity.
While the present invention has been described with reference to the specific
embodiments thereof, it should be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spirit and
scope of the invention. In addition, many modifications may be made to adapt a
particular
situation, material, composition of matter, process, process step or steps, to
the objective,
spirit and scope of the present invention. All such modifications are intended
to be within
the scope of the claims appended hereto.