U.S. patent application number 12/064529 was filed with the patent office on 2009-12-31 for tlr agonists.
This patent application is currently assigned to The Regents of The University of California Office of Technology Transfer. Invention is credited to Dennis A. Carson, Michael Chan, Howard B. Cottam, Suzanne Grimshaw, Kenji Takabayshi, Christina C.N. Wu.
Application Number | 20090324551 12/064529 |
Document ID | / |
Family ID | 37772216 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090324551 |
Kind Code |
A1 |
Carson; Dennis A. ; et
al. |
December 31, 2009 |
TLR AGONISTS
Abstract
The present invention provides for TLR agonist conjugates
(compounds) and compositions, as well as methods of using them. The
compounds of the invention are broad-spectrum, long-lasting, and
non-toxic combination of synthetic immunostimulatory agents, which
are useful for activating the immune system of a mammal, preferably
a human and can help direct the pharmacophore to the receptor
within the endosomes of target cells and enhance the signal
transduction induced by the pharmacophore.
Inventors: |
Carson; Dennis A.; (La
Jolla, CA) ; Takabayshi; Kenji; (Poway, CA) ;
Grimshaw; Suzanne; (Poway, CA) ; Cottam; Howard
B.; (Escondido, CA) ; Chan; Michael; (San
Diego, CA) ; Wu; Christina C.N.; (Escondido,
CA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG & WOESSNER, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
The Regents of The University of
California Office of Technology Transfer
Oakland
CA
|
Family ID: |
37772216 |
Appl. No.: |
12/064529 |
Filed: |
August 21, 2006 |
PCT Filed: |
August 21, 2006 |
PCT NO: |
PCT/US06/32371 |
371 Date: |
February 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60710337 |
Aug 22, 2005 |
|
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Current U.S.
Class: |
424/93.4 ;
424/93.5; 424/93.6; 435/235.1; 435/252.1; 435/252.5; 435/252.8;
514/263.37; 544/276 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 35/00 20180101; A61P 31/20 20180101; C07D 473/34 20130101;
A61P 35/02 20180101; A61P 31/12 20180101; A61P 19/02 20180101; C07D
473/24 20130101; A61P 31/14 20180101; A61P 1/04 20180101; C07D
473/16 20130101; A61P 17/00 20180101; C07D 473/18 20130101 |
Class at
Publication: |
424/93.4 ;
544/276; 514/263.37; 435/252.5; 435/252.1; 435/252.8; 435/235.1;
424/93.5; 424/93.6 |
International
Class: |
A61K 45/00 20060101
A61K045/00; C07D 473/00 20060101 C07D473/00; A61K 31/522 20060101
A61K031/522; C12N 1/20 20060101 C12N001/20; C12N 7/00 20060101
C12N007/00; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The invention described herein was made with government
support under Grant Number 3 UO1 AI056453-01 awarded by the
National Institute of Health. The United States Government has
certain rights in the invention.
Claims
1. A compound having formula (IA): ##STR00008## wherein X.sup.1 is
--O--, --S--, or --NR.sup.c--; wherein R.sup.c is hydrogen,
C.sub.1-10alkyl, or substituted C.sub.1-10alkyl, or R.sup.c and
R.sup.1 taken together with the nitrogen atom can form a
heterocyclic ring or a substituted heterocyclic ring, wherein the
substituents on the alkyl, aryl or heterocyclic groups are hydroxy,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; R.sup.1 is hydrogen,
(C.sub.1-C.sub.10)alkyl, substituted (C.sub.1-C.sub.10)alkyl,
C.sub.6-10aryl, or substituted C.sub.6-10aryl,
C.sub.5-9heterocyclic, substituted C.sub.5-9heterocyclic; wherein
the substituents on the alkyl, aryl or heterocyclic groups are
hydroxy, C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; each R.sup.2 is independently hydrogen,
--OH, (C.sub.1-C.sub.6)alkyl, substituted (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, substituted (C.sub.1-C.sub.6)alkoxy,
--C(O)--(C.sub.1-C.sub.6)alkyl (alkanoyl), substituted
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)--(C.sub.6-C.sub.10)aryl
(aroyl), substituted --C(O)--(C.sub.6-C.sub.10)aryl, --C(O)OH
(carboxyl), --C(O)O(C.sub.1-C.sub.6)alkyl (alkoxycarbonyl),
substituted --C(O)O(C.sub.1-C.sub.6)alkyl, --NR.sup.aR.sup.b,
--C(O)NR.sup.aR.sup.b (carbamoyl), substituted
--C(O)NR.sup.aR.sup.b, halo, nitro, or cyano; wherein the
substituents on the alkyl, aryl or heterocyclic groups are hydroxy,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; each R.sup.a and R.sup.b is independently
hydrogen, (C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.1-C.sub.6)alkoxy, halo(C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, hydroxy(C.sub.1-C.sub.6)alkyl, aryl,
aryl(C.sub.1-C.sub.6)alkyl, Het, Het (C.sub.1-C.sub.6)alkyl, or
(C.sub.1-C.sub.6)alkoxycarbonyl; X.sup.2 is a bond or a linking
group; and R.sup.3 is an auxiliary group; or a pharmaceutically
acceptable salt thereof.
2. The compound of claim 1, wherein X.sup.1 is sulfur atom.
3. The compound of claim 1, wherein X.sup.1 is oxygen atom.
4. The compound of claim 1, wherein X.sup.1 is --NR.sup.c--,
wherein R.sup.c is hydrogen, C.sub.1-6 alkyl or substituted
C.sub.1-6 alkyl; wherein the alkyl substituents are
C.sub.3-6cycloalkyl, hydroxy, C.sub.1-6alkoxy, amino, cyano, or
aryl.
5. The compound of claim 4, wherein X.sup.1 is --NH--.
6. The compound of claim 1, wherein R.sup.1 and R.sup.c taken
together form a heterocyclic ring or a substituted heterocyclic
ring.
7. The compound of claim 6, wherein R.sup.1 and R.sup.c taken
together form a substituted or unsubstituted morpholino,
piperidino, pyrrolidino, or piperazino ring.
8. The compound of claim 1, wherein R.sup.1 is hydrogen,
C.sub.1-4alkyl, or substituted C.sub.1-4alkyl.
9. The compound of claim 8, wherein R.sup.1 is hydrogen,
CH.sub.3--, CH.sub.3--CH.sub.2--, CH.sub.3CH.sub.2CH.sub.2--,
hydroxyC.sub.1-4alkylene, or C.sub.1-4alkoxyC.sub.1-4alkylene.
10. The compound of claim 9, wherein R.sup.1 is hydrogen,
CH.sub.3--, CH.sub.3--CH.sub.2--, CH.sub.3--O--CH.sub.2CH.sub.2--
or CH.sub.3--CH.sub.2--O--CH.sub.2CH.sub.2--.
11. The compound of claim 1, wherein R.sup.2 is hydrogen, halogen,
or C.sub.1-4alkyl.
12. The compound of claim 11, wherein R.sup.2 is hydrogen, chloro,
bromo, CH.sub.3--, or CH.sub.3--CH.sub.2--.
13. The compound of claim 1, wherein the substituents on the alkyl,
aryl or heterocyclic groups are hydroxy, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.1-6alkoxyC.sub.1-6alkylene, C.sub.3-6cycloalkyl, amino,
cyano, halogen, or aryl.
14. The compound of claim 1, wherein X.sup.2 is a bond or a chain
having up to about 24 atoms; wherein the atoms are selected from
the group consisting of carbon, nitrogen, sulfur, non-peroxide
oxygen, and phosphorous.
15. The compound of claim 14, wherein X.sup.2 is a bond or a chain
having from about 4 to about 12 atoms.
16. The compound of claim 14, wherein X.sup.2 is a bond or a chain
having from about 6 to about 9 atoms.
17. The compound of claim 14, wherein X.sup.2 is ##STR00009##
18. The compound of claim 1, wherein the auxiliary group is an
amino acid, a carbohydrate, a peptide, an antigen, a nucleic acid,
a body substance, or a microbe.
19. The compound of claim 18, wherein the peptide, has from 2 to
about 200 amino acid residues.
20. The compound of claim 19, wherein the peptide, has from 10 to
about 200 amino acid residues.
21. The compound of claim 1, wherein the auxiliary group is a
carbohydrate.
22. The compound of claim 18, wherein the nucleic acid is DNA, RNA
or PNA.
23. The compound of claim 18, wherein the body substance is a cell,
lipid, vitamin, or co-factor.
24. The compound of claim 23, wherein the body substance is a cell,
or lipid.
25. The compound of claim 18, wherein the antigen is a microbe.
26. The compound of claim 25, wherein the microbe is a virus,
bacteria, parasite, or fungi.
27. The compound of claim 26, wherein the microbe is a virus or a
bacteria.
28. The compound of claim 27, wherein the bacteria is Bacillus
anthracis (anthrax), Listeria monocytogenes, Francisella
tularensis, or Salmonella.
29. The compound of claim 28, wherein the Salmonella is typhimurium
or enteritidis.
30. The compound of claim 26, wherein the virus is an RNA virus, a
product of the RNA virus, or a DNA virus.
31. The compound of claim 30, wherein the DNA virus is the
Hepatitis B virus.
32. A pharmaceutical composition comprising the compound of claim
1, and a pharmaceutically acceptable carrier.
33. A therapeutic method for preventing or treating a pathological
condition or symptom in a mammal, wherein the activity of TLR
receptors is implicated and agonism of such activity is desired,
comprising administering to a mammal in need of such therapy, an
effective amount of the compound of claim 1.
34. The method of claim 33, wherein the condition or symptom is
cancer, bacterial disease, viral disease, autoimmune disease, or
Crohn's Disease.
35. The method of claim 34, wherein the bacteria is Bacillus
anthracis (anthrax), Listeria monocytogenes, Francisella
tularensis, or Salmonella.
36. The method of claim 35, wherein the Salmonella is typhimurium
or enteritidis.
37. The method of claim 34, wherein the virus is an RNA virus, a
product of the RNA virus, or a DNA virus.
38. The method of claim 37, wherein the DNA virus is the Hepatitis
B virus.
39. The method of claim 34, wherein cancer can be an interferon
sensitive cancer, such as, for example, a leukemia, a lymphoma, a
myeloma, a melanoma, or a renal cancer.
40-41. (canceled)
42. A method to treat cancer, bacterial disease, viral disease,
autoimmune disease, or Crohn's Disease, comprising administering a
composition comprising an effective amount of the compound of claim
1.
43. The method of claim 42, wherein the composition includes a
physiologically acceptable carrier.
44. A compound of formula (II): ##STR00010## wherein X.sup.1 is
--O--, --S--, or --NR.sup.c--; wherein R.sup.c hydrogen,
C.sub.1-10alkyl, or C.sub.1-10alkyl substituted by
C.sub.3-6cycloalkyl, or R.sup.c and R.sup.1 taken together with the
nitrogen atom can form a heterocyclic ring or a substituted
heterocyclic ring, wherein the substituents are hydroxy,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.1-6alkoxyC.sub.1-6alkylene, or cyano; R.sup.1 is
(C.sub.1-C.sub.10)alkyl, substituted (C.sub.1-C.sub.10)alkyl,
C.sub.6-10aryl, or substituted C.sub.6-10aryl,
C.sub.5-9heterocyclic, substituted C.sub.5-9heterocyclic; each
R.sup.2 is independently hydrogen, --OH, (C.sub.1-C.sub.6)alkyl,
substituted (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy,
substituted (C.sub.1-C.sub.6)alkoxy, --C(O)--(C.sub.1-C.sub.6)alkyl
(alkanoyl), substituted --C(O)--(C.sub.1-C.sub.6)alkyl,
--C(O)--(C.sub.6-C.sub.10)aryl (aroyl), substituted
--C(O)--(C.sub.6-C.sub.10)aryl, --C(O)OH (carboxyl),
--C(O)O(C.sub.1-C.sub.6)alkyl (alkoxycarbonyl), substituted
--C(O)O(C.sub.1-C.sub.6)alkyl, --NR.sup.aR.sup.b,
--C(O)NR.sup.aR.sup.b (carbamoyl), substituted
--C(O)NR.sup.aR.sup.b, halo, nitro, or cyano; each R.sup.a and
R.sup.b is independently hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.1-C.sub.6)alkoxy,
halo(C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, hydroxy(C.sub.1-C.sub.6)alkyl, aryl,
aryl(C.sub.1-C.sub.6)alkyl, Het, Het (C.sub.1-C.sub.6)alkyl, or
(C.sub.1-C.sub.6)alkoxycarbonyl; X.sup.2 is a bond or a linking
group; and R.sup.3 is an auxiliary group; n is 1, 2, 3, or 4; m is
1 or 2; q is 1 or 2; or a pharmaceutically acceptable salt
thereof.
45. A compound of formula (III): ##STR00011## wherein X.sup.1 is
--O--, --S--, or --NR.sup.c--; wherein R.sup.c is hydrogen,
C.sub.1-10alkyl, or substituted C.sub.1-10alkyl, or R.sup.c and
R.sup.1 taken together with the nitrogen atom can form a
heterocyclic ring or a substituted heterocyclic ring, wherein the
substituents on the alkyl, aryl or heterocyclic groups are hydroxy,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; R.sup.1 is hydrogen,
(C.sub.1-C.sub.10)alkyl, substituted (C.sub.1-C.sub.10)alkyl,
C.sub.6-10aryl, or substituted C.sub.6-10aryl,
C.sub.5-9heterocyclic, substituted C.sub.5-9heterocyclic; wherein
the substituents on the alkyl, aryl or heterocyclic groups are
hydroxy, C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; each R.sup.2 is independently hydrogen,
--OH, (C.sub.1-C.sub.6)alkyl, substituted (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, substituted (C.sub.1-C.sub.6)alkoxy,
--C(O)--(C.sub.1-C.sub.6)alkyl (alkanoyl), substituted
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)--(C.sub.6-C.sub.10)aryl
(aroyl), substituted --C(O)--(C.sub.6-C.sub.10)aryl, --C(O)OH
(carboxyl), --C(O)O(C.sub.1-C.sub.6)alkyl (alkoxycarbonyl),
substituted --C(O)O(C.sub.1-C.sub.6)alkyl, --NR.sup.aR.sup.b,
--C(O)NR.sup.aR.sup.b (carbamoyl), substituted
--C(O)NR.sup.aR.sup.b, halo, nitro, or cyano; wherein the
substituents on the alkyl, aryl or heterocyclic groups are hydroxy,
C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.3-6-cycloalkyl, C.sub.1-6alkoxyC.sub.1-6alkylene, amino,
cyano, halogen, or aryl; each R.sup.a and R.sup.b is independently
hydrogen, (C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.1-C.sub.6)alkoxy, halo(C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, hydroxy(C.sub.1-C.sub.6)alkyl, aryl,
aryl(C.sub.1-C.sub.6)alkyl, Het, Het (C.sub.1-C.sub.6)alkyl, or
(C.sub.1-C.sub.6)alkoxycarbonyl; X.sup.2 is a bond or a linking
group; and R.sup.3 is an auxiliary group; or a pharmaceutically
acceptable salt thereof.
46. The compound of claim 45 wherein if m is 2, a linker (L) links
each R.sup.3.
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/710,337 filed Aug. 22, 2005, which
application is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] A great deal has been learned about the molecular basis of
innate recognition of microbial pathogens in the last decade. It is
generally accepted that many somatic cells express a range of
pattern recognition receptors that detect potential pathogens
independently of the adaptive immune system. (See Janeway et al.,
Annu Rev Immunol, 20:197-216 (2002).) These receptors are believed
to interact with microbial components termed pathogen associated
molecular patterns (PAMPs). Examples of PAMPs include
peptidoglycans, lipotechoic acids from gram-positive cell walls,
the sugar mannose (which is common in microbial carbohydrates but
rare in humans), bacterial DNA, double-stranded RNA from viruses,
and glucans from fungal cell walls. PAMPs generally meet certain
criteria that include, (a) their expression by microbes but not
their mammalian hosts, (b) conservation of structure across the
wide range of pathogens, and (c) the capacity to stimulate innate
immunity. Toll-like Receptors (TLRs) have been found to play a
central role in the detection of PAMPs and in the early response to
microbial infections. (See Underhill et al., Curr Opin Immunol,
14:103-110 (2002).) Ten mammalian TLRs and a number of their
agonists have been identified. For example, TLR7 and TLR9 recognize
and respond to imiquimod and immunostimulatory CpG oligonucleotides
(ISS-ODN), respectively. The synthetic immunomodulator R-848
(resiquimod) activates both TLR7 and TLR8. While TLR stimulation
initiates a common signaling cascade (involving the adaptor protein
MyD88, the transcription factor NF-kB, and pro-inflammatory and
effector cytokines), certain cell types tend to produce certain
TLRs. For example, TLR7 and TLR9 are found predominantly on the
internal faces of endosomes in dendritic cells (DCs) and B
lymphocytes (in humans; mouse macrophages express TLR7 and TLR9).
TLR8, on the other hand, is found in human blood monocytes. (See
Hornung et al., J. Immunol, 168:4531-4537 (2002)).
[0004] Interferons (INFs) are also involved in the efficient
induction of an immune response, especially after viral infection
(Brassard et al., J. Leukoc Biol, 71:568-581 (2002).) However, many
viruses produce a variety of proteins that block interferon
production or action at various levels. Antagonism of interferon is
believed to be part of a general strategy to evade innate, as well
as adaptive immunity. (See Levy et al., Cytokine Growth Factor Rev,
12:143-156 (2001).) While TLR agonists (TLR-L) may be sufficiently
active for some methods of treatment, in some instances the
microbial interferon antagonists could mitigate the adjuvant
effects of synthetic TLR-L.
[0005] Accordingly, there exists a need for compounds that augment
TLR-induced signal transduction, i.e., compounds that hinder viral
or bacterial obstruction of interferon production or have the
ability to modulate the innate immune system using the TLR
agonists.
SUMMARY OF THE INVENTION
[0006] The present invention provides for TLR agonist conjugates
(compounds) and compositions, as well as methods of using them. The
compounds of the invention are broad-spectrum, long-lasting, and
non-toxic combination of synthetic immunostimulatory agents, which
are useful for activating the immune system of a mammal, preferably
a human and can help direct the pharmacophore to the receptor
within the endosomes of target cells and enhance the signal
transduction induced by the pharmacophore. The compounds of the
invention include a pharmacophore covalently bound to an auxiliary
group. Accordingly there is provided a compound of the invention
which is a compound of formula (I):
##STR00001##
[0007] wherein X.sup.1 is --O--, --S--, or --NR.sup.c--;
[0008] wherein R.sup.c hydrogen, C.sub.1-10alkyl, or
C.sub.1-10alkyl substituted by C.sub.3-6-cycloalkyl, or R.sup.c and
R.sup.1 taken together with the nitrogen atom can form a
heterocyclic ring or a substituted heterocyclic ring, wherein the
substituents are hydroxy, C.sub.1-6alkyl, hydroxyC.sub.1-6alkylene,
C.sub.1-6alkoxy, C.sub.1-6alkoxyC.sub.1-6alkylene, or cyano;
[0009] R.sup.1 is (C.sub.1-C.sub.10)alkyl, substituted
(C.sub.1-C.sub.10)alkyl, C.sub.6-10aryl, or substituted
C.sub.6-10aryl, C.sub.5-9heterocyclic, substituted
C.sub.5-9heterocyclic;
[0010] each R.sup.2 is independently hydrogen, --OH,
(C.sub.1-C.sub.6)alkyl, substituted (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, substituted (C.sub.1-C.sub.6)alkoxy,
--C(O)--(C.sub.1-C.sub.6)alkyl (alkanoyl), substituted
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)--(C.sub.6-C.sub.10)aryl
(aroyl), substituted --C(O)--(C.sub.6-C.sub.10)aryl, --C(O)OH
(carboxyl), --C(O)O(C.sub.1-C.sub.6)alkyl (alkoxycarbonyl),
substituted --C(O)O(C.sub.1-C.sub.6)alkyl, --NR.sup.aR.sup.b,
--C(O)NR.sup.aR.sup.b (carbamoyl), substituted
--C(O)NR.sup.aR.sup.b, halo, nitro, or cyano;
[0011] each R.sup.a and R.sup.b is independently hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
(C.sub.1-C.sub.6)alkoxy, halo(C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, hydroxy(C.sub.1-C.sub.6)alkyl, aryl,
aryl(C.sub.1-C.sub.6)alkyl, Het, Het (C.sub.1-C.sub.6)alkyl, or
(C.sub.1-C.sub.6)alkoxycarbonyl;
[0012] X.sup.2 is a bond or a linking group; and R.sup.3 is an
auxiliary group;
[0013] n is 1, 2, 3, or 4; m is 1 or 2; q is 1 or 2; or
[0014] a pharmaceutically acceptable salt thereof.
[0015] The auxiliary groups can include organic molecules, composed
of carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous atoms.
These groups are not harmful to body tissues (e.g., they are
non-toxic, and/or do not cause inflammation).
[0016] In addition, the invention also provides a pharmaceutical
composition comprising at least one compound of formula (I), or a
pharmaceutically acceptable salt thereof, in combination with a
pharmaceutically acceptable diluent or carrier.
[0017] In one embodiment, the invention provides a therapeutic
method for preventing or treating a pathological condition or
symptom in a mammal, such as a human, wherein the activity of TLR
agonists is implicated and its action is desired, comprising
administering to a mammal in need of such therapy, an effective
amount of a compound of formula (I), or a pharmaceutically
acceptable salt thereof. Non-limiting examples of pathological
conditions or symptoms that are suitable for treatment include
cancers, treatment for bacterial or viral diseases, treating
autoimmune diseases, and treating Crohn's Disease.
[0018] The compounds of the invention can also be used as or to
prepare vaccines against bacteria, viruses, cancer cells, cancer
specific peptides, enhancers of monoclonal antibodies against
cancer, a CNS stimulant, or for biodefense.
[0019] The invention provides a compound of formula (I) for use in
medical therapy (e.g., for use as an anti-cancer agent, treatment
for bacterial diseases, treatment for viral diseases, such as
hepatitis C and hepatitis B, Crohn's Disease, and as therapeutic
agents for treating immunologic disease). Furthermore, it is
suggested that compounds of formula (I) will prevent carcinogenesis
by hepatitis C and hepatitis B, as well as the use of a compound of
formula (I) for the manufacture of a medicament useful for the
treatment of cancer, viral diseases, Crohn's Disease, and
immunologic disorders in a mammal, such as a human.
[0020] In a specific embodiment, the present invention provides a
method for treating a viral infection in a mammal by administering
a TLR agonist compound of formula (I). The viral infection can be
caused by an RNA virus, a product of the RNA virus that acts as a
TLR agonist and/or a DNA virus. A specific DNA virus for treatment
is the Hepatitis B virus.
[0021] In another specific embodiment, the present invention
provides a method for treating cancer by administering an effective
amount of a TLR agonist compound of formula (I). The cancer can be
an interferon sensitive cancer, such as, for example, a leukemia, a
lymphoma, a myeloma, a melanoma, or a renal cancer.
[0022] In another specific embodiment, the present invention
provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of a TLR agonist
compound of formula (I) or a pharmaceutically acceptable salt of
such a compound. A specific autoimmune disease is Multiple
Sclerosis, lupus, rheumatoid arthritis and the like.
[0023] In another specific embodiment, the present invention
provides a method of treating Crohn's Disease by administering a
TLR agonist compound of formula (I).
[0024] The TLR agonists can be a homofunctional TLR agonist polymer
and can consist of a TLR-7 agonist or a TLR-8 agonist. The TLR7
agonist can be a 7-thia-8-oxoguanosinyl (TOG) moiety, a
7-deazaguanosinyl (7DG) moiety, a resiquimod moiety, or an
imiquimod moiety. The TLR8 agonist can be a resiquimod moiety. In
another aspect, the TLR agonist is a heterofunctional TLR agonist
polymer. The heterofunctional TLR agonist polymer can include a
TLR-7 agonist and a TLR-8 agonist or a TLR-9 agonist or all three
agonists. The heterofunctional TLR agonist polymer can include a
TLR-8 agonist and a TLR-9 agonist.
[0025] The invention also provides processes and intermediates
disclosed herein that are useful for preparing compounds of formula
(I) or salts thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 is a graphic illustration of the absorption
chromophore (at .about.350 nm) of a compound of formula I
(OVA/IV150 Conjugate).
[0027] FIG. 2 is a graphic illustration of the stimulation of bone
marrow derived dendritic cells (BMDC).
[0028] FIG. 3 illustrates the conjugation of a TLR7 agonist,
UC-1V150, to mouse serum albumin (MSA). The success of conjugation
is indicated by UV spectroscopy. The UC-1V150 to MSA ratio is
approximately 5:1
[0029] FIGS. 4A and B illustrate that the UC-1V150 and MSA
conjugates activate both murine bone marrow-derived macrophages
(4A) and human peripheral blood mononuclear cells (4B). Cells were
incubated with various concentrations of the compound from 0.5 nM
to 10 .mu.M in BMDM or from 0.1 to 10 .mu.M in PBMC. Culture
supernatants were harvested after 24 h and cytokine levels were
analyzed by Luminex.
[0030] FIGS. 5A, 5B, 5C, and 5D illustrate the increased potency
and duration of effect of UC-1V150/MSA. C57BL/6 mice were injected
(i.v.) with (A) 0.1 micromole of SM-360320, a TLR7 ligand, or (B)
equivalent amount of a TLR7 agonist UC-1V150 (aldehyde-modified
SM-360320) or UC-1V150/MSA to 500 .mu.g MSA per mouse. Serum
samples were collected at the indicated time points and cytokine
levels were analyzed by Luminex. MSA=mouse serum albumin. The
effect from the original TLR7 ligand, SM-360320, lasted for only 2
hours whereas UC-1V150/MSA has extended the effect to at least 6
hours.
[0031] FIG. 6 illustrates the effects of UC-1V150 conjugated with
inactivated SIV (6A) or with OVA in combination with ODN (6B).
Myeloid BMDC were incubated for 24 hr with various conditions at
0.1 .mu.g/ml as indicated. IL-12 levels in the cell supernatant
were measured by ELISA.
[0032] FIGS. 7A and 7B illustrates an increased potency of
UC-1V150/MSA. C57BL/6 mice were i.v. injected with 380 mmole of
SM-360320 or UC-1V150, or 500 .mu.g of UC-1V150/MSA (equivalent to
3.8 mmole UC-1V150) per mouse. Serum samples were collected after 2
h and cytokine levels were analyzed by Luminex. To achieve the
similar effect, at least 100-fold higher concentration of either
UC-1V150 or SM-360320 was required as compared to that of
UC-1V150/MSA.
[0033] FIG. 8 is an illustration of the uv spectrum of a
double-conjugate, (OVA/IV150/1043).
[0034] FIG. 9 is an illustration of the induction of IL-12 in BMDC
using OVA/ODN/IV150 conjugates.
[0035] FIG. 10 illustrates direct conjugation of SIV Particles to
the IA compound IV150.
[0036] FIG. 11 illustrates the ability to prepare compounds of the
invention with virus particles attached to a compound having
formula IA and the TLR agonist activity of the compounds.
[0037] FIG. 12 illustrates the molecular areas of specificity for
antibodies raised against the conjugates containing a linker and a
TLR ligand.
[0038] FIGS. 13A and 13B illustrate the distinction between the
four substances applied to the respective lanes on a gel in a
Western plot analysis. In FIG. 13A, the gel membrane was probed
with anti-ovalbumin (anti-OVA) antibody and all lanes gave a
positive band, indicating that OVA was detected in all lanes, as
expected. In FIG. 13B, the gel membrane was probed with the
selective antibody raised to the TLR ligand portion of the
conjugate, and therefore only lane 4 was positive, confirming the
specificity of the antibody for the TLR ligand.
DETAILED DESCRIPTION
[0039] Non-limiting examples of auxiliary groups include side
chains that increase solubility, such as, for example, groups
containing morpholino, piperidino, pyrrolidino, or piperazino rings
and the like; amino acids, polymers of amino acids (proteins or
peptides), e.g., dipeptides or tripeptides, and the like;
carbohydrates (polysaccharides), nucleotides such as, for example,
PNA, RNA and DNA, and the like; polymers of organic materials, such
as, for example, polyethylene glycol, poly-lactide and the like;
monomeric and polymeric lipids; insoluble organic nanoparticles;
non-toxic body substances such as, for example, cells, lipids,
vitamins, co-factors, antigens such as, for example microbes, such
as, for example, viruses, bacteria, fungi, and the like. The
antigens can include inactivated whole organisms, or sub-components
thereof and the like.
[0040] The compounds of the invention can be prepared using
compounds having formula (IA):
##STR00002##
where X is a group that can react to form a bond to the linking
group or can react to form a bond to the auxiliary group. A
specific group of compounds having formula (IA) are disclosed in
U.S. Pat. No. 6,329,381.
[0041] The following definitions are used, unless otherwise
described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy,
alkenyl, alkynyl, etc. denote both straight and branched groups;
but reference to an individual radical such as "propyl" embraces
only the straight chain radical, a branched chain isomer such as
"isopropyl" being specifically referred to. Aryl denotes a phenyl
radical or an ortho-fused bicyclic carbocyclic radical having about
nine to ten ring atoms in which at least one ring is aromatic.
Heteroaryl encompasses a radical attached via a ring carbon of a
monocyclic aromatic ring containing five or six ring atoms
consisting of carbon and one to four heteroatoms each selected from
the group consisting of non-peroxide oxygen, sulfur, and N(X)
wherein X is absent or is H, O, (C.sub.1-C.sub.4)alkyl, phenyl or
benzyl, as well as a radical of an ortho-fused bicyclic heterocycle
of about eight to ten ring atoms derived therefrom, particularly a
benz-derivative or one derived by fusing a propylene, trimethylene,
or tetramethylene diradical thereto.
[0042] It will be appreciated by those skilled in the art that
compounds of the invention having a chiral center may exist in and
be isolated in optically active and racemic forms. Some compounds
may exhibit polymorphism. It is to be understood that the present
invention encompasses any racemic, optically-active, polymorphic,
or stereoisomeric form, or mixtures thereof, of a compound of the
invention, which possess the useful properties described herein, it
being well known in the art how to prepare optically active forms
(for example, by resolution of the racemic form by
recrystallization techniques, by synthesis from optically-active
starting materials, by chiral synthesis, or by chromatographic
separation using a chiral stationary phase) and how to determine
nicotine agonist activity using the standard tests described
herein, or using other similar tests which are well known in the
art.
[0043] Processes for preparing compounds of formula I or for
preparing intermediates useful for preparing compounds of formula I
are provided as further embodiments of the invention. Intermediates
useful for preparing compounds of formula I are also provided as
further embodiments of the invention.
[0044] In cases where compounds are sufficiently basic or acidic to
form acid or base salts, use of the compounds as salts may be
appropriate. Examples of acceptable salts are organic acid addition
salts formed with acids which form a physiological acceptable
anion, for example, tosylate, methanesulfonate, acetate, citrate,
malonate, tartarate, succinate, benzoate, ascorbate,
.alpha.-ketoglutarate, and .alpha.-glycerophosphate. Suitable
inorganic salts may also be formed, including hydrochloride,
sulfate, nitrate, bicarbonate, and carbonate salts.
[0045] Acceptable salts may be obtained using standard procedures
well known in the art, for example by reacting a sufficiently basic
compound such as an amine with a suitable acid affording a
physiologically acceptable anion. Alkali metal (for example,
sodium, potassium or lithium) or alkaline earth metal (for example
calcium) salts of carboxylic acids can also be made.
[0046] Alkyl includes straight or branched C.sub.1-10 alkyl groups,
e.g., methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl,
1-methylpropyl, 3-methylbutyl, hexyl, and the like.
[0047] Lower alkyl includes straight or branched C.sub.1-6 alkyl
groups, e.g., methyl, ethyl, propyl, 1-methylethyl, butyl,
1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl,
1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl,
1,2-dimethylpropyl, 2,2-dimethylpropyl, and the like.
[0048] The term "alkylene" refers to a divalent straight or
branched hydrocarbon chain (e.g. ethylene
--CH.sub.2--CH.sub.2--).
[0049] C.sub.3-7 Cycloalkyl includes groups such as, cyclopropyl,
cyclopentyl, cyclohexyl, cycloheptyl, and the like, and
alkyl-substituted C.sub.3-7 cycloalkyl group, preferably straight
or branched C.sub.1-6 alkyl group such as methyl, ethyl, propyl,
butyl or pentyl, and C.sub.5-7 cycloalkyl group such as,
cyclopentyl or cyclohexyl, and the like.
[0050] Lower alkoxy includes C.sub.1-6 alkoxy groups, such as
methoxy, ethoxy or propoxy, and the like.
[0051] Lower alkanoyl includes C.sub.1-6 alkanoyl groups, such as
formyl, acetyl, propanoyl, butanoyl, pentanoyl or hexanoyl, and the
like.
[0052] C.sub.7-11 aroyl, includes groups such as benzoyl or
naphthoyl;
[0053] Lower alkoxycarbonyl includes C.sub.2-7 alkoxycarbonyl
groups, such as methoxycarbonyl, ethoxycarbonyl or propoxycarbonyl,
and the like.
[0054] Lower alkylamino group means amino group substituted by
C.sub.1-6 alkyl group, such as, methylamino, ethylamino,
propylamino, butylamino, and the like.
[0055] Di(lower alkyl)amino group means amino group substituted by
the same or different and C.sub.1-6 alkyl group (e.g.
dimethylamino, diethylamino, ethylmethylamino).
[0056] Lower alkylcarbamoyl group means carbamoyl group substituted
by C.sub.1-6 alkyl group (e.g. methylcarbamoyl, ethylcarbamoyl,
propylcarbamoyl, butylcarbamoyl).
[0057] Di(lower alkyl)carbamoyl group means carbamoyl group
substituted by the same or different and C.sub.1-6 alkyl group
(e.g. dimethylcarbamoyl, diethylcarbamoyl,
ethylmethylcarbamoyl).
[0058] Halogen atom means halogen atom such as fluorine atom,
chlorine atom, bromine atom or iodine atom.
[0059] Aryl refers to a C.sub.6-10 monocyclic or fused cyclic aryl
group, such as phenyl, indenyl, or naphthyl, and the like.
[0060] Heterocyclic refers to monocyclic saturated heterocyclic
groups, or unsaturated monocyclic or fused heterocyclic group
containing at least one heteroatom, e.g., 0-3 nitrogen atoms
(--NR.sup.d--), 0-1 oxygen atom (--O--), and 0-1 sulfur atom
(--S--). Non-limiting examples of saturated monocyclic heterocyclic
group includes 5 or 6 membered saturated heterocyclic group, such
as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl,
piperazinyl or pyrazolidinyl. Non-limiting examples of unsaturated
monocyclic heterocyclic group includes 5 or 6 membered unsaturated
heterocyclic group, such as furyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, thienyl, pyridyl or pyrimidinyl. Non-limiting examples
of unsaturated fused heterocyclic groups includes unsaturated
bicyclic heterocyclic group, such as indolyl, isoindolyl, quinolyl,
benzothizolyl, chromanyl, benzofuranyl, and the like.
[0061] R.sup.c and R.sup.1 taken together with the nitrogen atom to
which they are attached can form a heterocyclic ring. Non-limiting
examples of heterocyclic rings include 5 or 6 membered saturated
heterocyclic rings, such as 1-pyrrolidinyl, 4-morpholinyl,
1-piperidyl, 1-piperazinyl or 1-pyrazolidinyl, 5 or 6 membered
unsaturated heterocyclic rings such as 1-imidazolyl, and the
like.
[0062] The alkyl, aryl, heterocyclic groups of R.sup.1 can be
optionally substituted with one or more substituents, wherein the
substituents are the same or different, and include lower alkyl;
cycloalkyl, hydroxyl; hydroxy C.sub.1-6 alkylene, such as
hydroxymethyl, 2-hydroxyethyl or 3-hydroxypropyl; lower alkoxy;
C.sub.1-6 alkoxy C.sub.1-6 alkyl, such as 2-methoxyethyl,
2-ethoxyethyl or 3-methoxypropyl; amino; alkylamino; dialkyl amino;
cyano; nitro; acyl; carboxyl; lower alkoxycarbonyl; halogen;
mercapto; C.sub.1-6 alkylthio, such as, methylthio, ethylthio,
propylthio or butylthio; substituted C.sub.1-6 alkylthio, such as
methoxyethylthio, methylthioethylthio, hydroxyethylthio or
chloroethylthio; aryl; substituted C.sub.6-10 monocyclic or
fused-cyclic aryl, such as 4-hydroxyphenyl, 4-methoxyphenyl,
4-fluorophenyl, 4-chlorophenyl or 3,4-dichlorophenyl; 5-6 membered
unsaturated heterocyclic, such as furyl, pyrrolyl, pyrazolyl,
imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl; and
bicyclic unsaturated heterocyclic, such as indolyl, isoindolyl,
quinolyl, benzothiazolyl, chromanyl, benzofuranyl or
phthalimino.
[0063] The alkyl, aryl, heterocyclic groups of R.sup.2 can be
optionally substituted with one or more substituents, wherein the
substituents are the same or different, and include hydroxyl;
C.sub.1-6 alkoxy, such as methoxy, ethoxy or propoxy; carboxyl;
C.sub.2-7 alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl
or propoxycarbonyl) and halogen.
[0064] The alkyl, aryl, heterocyclic groups of R.sup.c can be
optionally substituted with one or more substituents, wherein the
substituents are the same or different, and include C.sub.3-6
cycloalkyl; hydroxyl; C.sub.1-6 alkoxy; amino; cyano; aryl;
substituted aryl, such as 4-hydroxyphenyl, 4-methoxyphenyl,
4-chlorophenyl or 3,4-dichlorophenyl; nitro and halogen.
[0065] The heterocyclic ring formed together with R.sup.c and
R.sup.1 and the nitrogen atom to which they are attached can be
optionally substituted with one or more substituents, wherein the
substituents are the same or different, and include C.sub.1-6
alkyl; hydroxy C.sub.1-6 alkylene; C.sub.1-6 alkoxy C.sub.1-6
alkylene; hydroxyl; C.sub.1-6 alkoxy; and cyano.
[0066] The term "amino acid" as used herein, comprises the residues
of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln,
Gly, H is, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, and Val) in D or L form, as well as unnatural amino acids
(e.g. phosphoserine, phosphothreonine, phosphotyrosine,
hydroxyproline, gamma-carboxyglutamate; hippuric acid,
octahydroindole-2-carboxylic acid, statine,
1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,
ornithine, citruline, -methyl-alanine, para-benzoylphenylalanine,
phenylglycine, propargylglycine, sarcosine, and tert-butylglycine).
The term also comprises natural and unnatural amino acids bearing a
conventional amino protecting group (e.g. acetyl or
benzyloxycarbonyl), as well as natural and unnatural amino acids
protected at the carboxy terminus (e.g. as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide; or as an
-methylbenzyl amide). Other suitable amino and carboxy protecting
groups are known to those skilled in the art (See for example, T.
W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York,
1981, and references cited therein). An amino acid can be linked to
the remainder of a compound of formula I through the carboxy
terminus, the amino terminus, or through any other convenient point
of attachment, such as, for example, through the sulfur of
cysteine.
[0067] The term "toll-like receptor" (TLR) refers to a member of a
family of receptors that bind to pathogen associated molecular
patterns (PAMPs) and facilitate an immune response in a mammal. Ten
mammalian TLRs are known, e.g., TLR1-10.
[0068] The term "toll-like receptor agonist" (TLR agonist) refers
to a molecule that binds to a TLR and antagonizes the receptor.
Synthetic TLR agonists are chemical compounds that are designed to
bind to a TLR and activate the receptor. Exemplary novel TLR
agonists provided herein include "TLR-7 agonist" "TLR-8 agonist"
and "TLR-9 agonist."
[0069] The term "nucleic acid" as used herein, refers to DNA, RNA,
single-stranded, double-stranded, or more highly aggregated
hybridization motifs, and any chemical modifications thereof.
Modifications include, but are not limited to, those providing
chemical groups that incorporate additional charge, polarizability,
hydrogen bonding, electrostatic interaction, and fluxionality to
the nucleic acid ligand bases or to the nucleic acid ligand as a
whole. Such modifications include, but are not limited to, peptide
nucleic acids (PNAs), phosphodiester group modifications (e.g.,
phosphorothioates, methylphosphonates), 2'-position sugar
modifications, 5-position pyrimidine modifications, 7-position
purine modifications, 8-position purine modifications, 9-position
purine modifications, modifications at exocyclic amines,
substitution of 4-thiouridine, substitution of 5-bromo or
5-iodo-uracil; backbone modifications, methylations, unusual
base-pairing combinations such as the isobases, isocytidine and
isoguanidine and the like. Nucleic acids can also include
non-natural bases, such as, for example, nitroindole. Modifications
can also include 3' and 5' modifications such as capping with a
BHQ, a fluorophore or another moiety.
[0070] A specific value for X.sup.1 is a sulfur atom, an oxygen
atom or --NR.sup.c--.
[0071] Another specific X.sup.1 is a sulfur atom.
[0072] Another specific X.sup.1 is an oxygen atom.
[0073] Another specific X.sup.1 is --NR.sup.c--.
[0074] Another specific X.sup.1 is --NH--.
[0075] A specific value for R.sup.c is hydrogen, C.sub.1-4 alkyl or
substituted C.sub.1-4 alkyl.
[0076] A specific value for R.sup.1 and R.sup.c taken together is
when they form a heterocyclic ring or a substituted heterocyclic
ring.
[0077] Another specific value for R.sup.1 and R.sup.c taken
together is substituted or unsubstituted morpholino, piperidino,
pyrrolidino, or piperazino ring
[0078] A specific value for R.sup.1 is hydrogen, C.sub.1-4alkyl, or
substituted C.sub.1-4alkyl.
[0079] Another specific R.sup.1 is 2-hydroxyethyl, 3-hydroxypropyl,
4-hydroxybutyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,
methoxymethyl, 2-methoxyethyl, 3-methoxypropyl, ethoxymethyl,
2-ethoxyethyl, methylthiomethyl, 2-methylthioethyl,
3-methylthiopropyl, 2-fluoroethyl, 3-fluoropropyl,
2,2,2-trifluoroethyl, cyanomethyl, 2-cyanoethyl, 3-cyanopropyl,
methoxycarbonylmethyl, 2-methoxycarbonylethyl,
3-methoxycarbonylpropyl, benzyl, phenethyl, 4-pyridylmethyl,
cyclohexylmethyl, 2-thienylmethyl, 4-methoxyphenylmethyl,
4-hydroxyphenylmethyl, 4-fluorophenylmethyl, or
4-chlorophenylmethyl.
[0080] Another specific R.sup.1 is hydrogen, CH.sub.3--,
CH.sub.3--CH.sub.2--, CH.sub.3CH.sub.2CH.sub.2--,
hydroxyC.sub.1-4alkylene, or C.sub.1-4alkoxyC.sub.1-4alkylene.
[0081] Another specific value for R.sup.1 is hydrogen, CH.sub.3--,
CH.sub.3--CH.sub.2--, CH.sub.3--O--CH.sub.2CH.sub.2-- or
CH.sub.3--CH.sub.2-O--CH.sub.2CH.sub.2--.
[0082] A specific value for R.sup.2 is hydrogen, halogen, or
C.sub.1-4alkyl.
[0083] Another specific value for R.sup.2 is hydrogen, chloro,
bromo, CH.sub.3--, or CH.sub.3--CH.sub.2--.
[0084] Specific substituents for substitution on the alkyl, aryl or
heterocyclic groups are hydroxy, C.sub.1-6alkyl,
hydroxyC.sub.1-6alkylene, C.sub.1-6alkoxy,
C.sub.1-6alkoxyC.sub.1-6allylene, C.sub.3-6cycloalkyl, amino,
cyano, halogen, or aryl.
[0085] A specific value for X.sup.2 is a bond or a chain having up
to about 24 atoms; wherein the atoms are selected from the group
consisting of carbon, nitrogen, sulfur, non-peroxide oxygen, and
phosphorous.
[0086] Another specific value for X.sup.2 is a bond or a chain
having from about 4 to about 12 atoms.
[0087] Another specific value for X.sup.2 is a bond or a chain
having from about 6 to about 9 atoms.
[0088] Another specific value for X.sup.2 is
##STR00003##
[0089] Another specific value for X.sup.2 is
##STR00004##
[0090] A specific auxiliary group is an amino acid, a carbohydrate,
a peptide, an antigen, a nucleic acid, a body substance, or a
microbe.
[0091] A specific peptide, has from 2 to about 20 amino acid
residues.
[0092] Another specific peptide, has from 10 to about 20 amino acid
residues.
[0093] A specific auxiliary group is a carbohydrate.
[0094] A specific nucleic acid is DNA, RNA or PNA.
[0095] A specific body substance is a cell, lipid, vitamin, or
co-factor.
[0096] Another specific body substance is a cell or lipid.
[0097] A specific antigen is a microbe.
[0098] A specific microbe is a virus, bacteria, or fungi.
[0099] Another specific microbe is a virus or a bacteria.
[0100] Specific bacteria are Bacillus anthracis (anthrax), Listeria
monocytogenes, Francisella tularensis, or Salmonella.
[0101] Specific Salmonella are typhimurium or enteritidis.
[0102] Specific viruses are RNA viruses, a product of the RNA
virus, or a DNA virus.
[0103] A specific DNA virus is the Hepatitis B virus.
[0104] Specific compounds of the invention have the general
formula
IA-L-A.sup.1;
IA-L-(A.sup.1).sub.2;
IA-L-A.sup.1-A.sup.1;
IA-L-A.sup.1-L-A.sup.1;
(IA).sub.2-L-A.sup.1-A.sup.1;
(IA).sub.2-L-A.sup.1-L-A.sup.1;
(IA).sub.2-L-A.sup.1; or
(IA).sub.2-L-(A.sup.1).sub.2;
wherein IA is as disclosed herein; L is absent or is a linking
group; and each A.sup.1 group independently represents an auxiliary
group.
[0105] In one embodiment, the viral infection is caused by a
coronavirus that causes Severe Acute Respiratory Syndrome (SARS), a
Hepatitis B virus, or a Hepatitis C Virus.
[0106] In another embodiment, the viral infection is caused by a
coronavirus that causes Severe Acute Respiratory Syndrome (SARS), a
Hepatitis B virus, or a Hepatitis C Virus.
[0107] Specific cancers that can be treated include melanoma,
superficial bladder cancer, actinic keratoses, intraepithelial
neoplasia, and basal cell skin carcinoma, squamous, and the like.
In addition, the method of the invention includes treatment for a
precancerous condition such as, for example, actinic keratoses or
intraepithelial neoplasia, familial polyposis (polyps), cervical
dysplasia, cervical cancers, superficial bladder cancer, and any
other cancers associated with infection (e.g., lymphoma Karposi's
sarcoma, or leukemia); and the like.
[0108] Non limiting examples of the pathological conditions or
symptoms that can be treated include viral diseases, cancer,
inflammatory diseases of the gastrointestinal tract, brain, skin,
joints, and other tissues.
[0109] The auxiliary groups are believed to enhance the drug
activity of the pharmacophore (compounds of formula (I)) by (a)
helping to direct the pharmacophore to the receptor within the
endosomes of target cells; (b) by enhancing signal transduction
induced by the pharmacophore, by cross-linking the receptor; and/or
(c) the pharmacophore can enhance the response to the auxiliary
group (e.g., immune response). The auxiliary groups should form
generally stable bonds with the pharmacophore, and do not act as
prodrugs.
[0110] The invention includes compositions of a compound of formula
(I) optionally in combination with an inhibitor of inosine
monophosphate dehydrogenase (IMPDH), an enantiomer of such a
compound, a prodrug of such a compound, or a pharmaceutically
acceptable salt of such a compound. As used herein an "IMPDH
inhibitor" refers to an inhibitor of the enzyme inosine
monophosphate dehydrogenase. Currently, three IMPDH inhibitors are
used clinically: ribavirin, mizoribine, and mycophenolate mofetil.
Ribavirin and mizoribine are prodrugs that are phosphorylated
intracellularly to produce IMP analogs (Goldstein et al., Cuff Med
Chem, 6:519-536 (1999)). Viramidine is a prodrug of Ribavirin.
Mycophenolate mofetil is immunosuppressive, and has
gastrointestinal irritative properties that may be attributable to
its enterohepatic recirculation (Papageorgiou C, Mini Rev Med.
Chem., 1:71-77 (2001)). Mizoribine aglycone, a prodrug, is used as
an IMPDH inhibitor. Other non-limiting examples IMPDH inhibitors,
including prodrugs of mizoribine and mizoribine aglycone are known
and are disclosed in published U.S. Patent application No.
20050004144.
[0111] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compounds as salts may be appropriate. Examples of pharmaceutically
acceptable salts are organic acid addition salts formed with acids
which form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate. Suitable inorganic salts may also be
formed, including hydrochloride, sulfate, nitrate, bicarbonate, and
carbonate salts.
[0112] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0113] The compounds (conjugates) of the invention can be prepared
using standard synthetic methods known in the art. A general ester
synthesis is illustrated below:
##STR00005##
##STR00006##
##STR00007##
[0114] Additional examples for preparing specific compounds are
included herein.
[0115] The compounds of formula I can be formulated as
pharmaceutical compositions and administered to a mammalian host,
such as a human patient in a variety of forms adapted to the chosen
route of administration, i.e., orally or parenterally, by
intravenous, intramuscular, topical or subcutaneous routes.
[0116] Thus, the present compounds may be systemically
administered, e.g., orally, in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an assimilable
edible carrier. They may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets, or may be incorporated
directly with the food of the patient's diet. For oral therapeutic
administration, the active compound may be combined with one or
more excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least 0.1% of active compound. The percentage of the compositions
and preparations may, of course, be varied and may conveniently be
between about 2 to about 60% of the weight of a given unit dosage
form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0117] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0118] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0119] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0120] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0121] For topical administration, the present compounds may be
applied in pure form, i.e., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0122] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0123] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0124] Examples of useful dermatological compositions which can be
used to deliver the compounds of formula I to the skin are known to
the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),
Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.
4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0125] Useful dosages of the compounds of formula I can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0126] Generally, the concentration of the compound(s) of formula I
in a liquid composition, such as a lotion, will be from about
0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration
in a semi-solid or solid composition such as a gel or a powder will
be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0127] The amount of the compound, or an active salt or derivative
thereof, required for use in treatment will vary not only with the
particular salt selected but also with the route of administration,
the nature of the condition being treated and the age and condition
of the patient and will be ultimately at the discretion of the
attendant physician or clinician.
[0128] In general, however, a suitable dose will be in the range of
from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75
mg/kg of body weight per day, such as 3 to about 50 mg per kilogram
body weight of the recipient per day, preferably in the range of 6
to 90 mg/kg/day, most preferably in the range of 15 to 60
mg/kg/day.
[0129] The compound is conveniently administered in unit dosage
form; for example, containing 5 to 1000 mg, conveniently 10 to 750
mg, most conveniently, 50 to 500 mg of active ingredient per unit
dosage form.
[0130] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.5 to about 75 .mu.M, preferably, about 1 to 50 .mu.M, most
preferably, about 2 to about 30 .mu.M. This may be achieved, for
example, by the intravenous injection of a 0.05 to 5% solution of
the active ingredient, optionally in saline, or orally administered
as a bolus containing about 1-100 mg of the active ingredient.
Desirable blood levels may be maintained by continuous infusion to
provide about 0.01-5.0 mg/kg/hr or by intermittent infusions
containing about 0.4-15 mg/kg of the active ingredient(s).
[0131] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0132] The ability of a compound of the invention to act as a TLR
agonist may be determined using pharmacological models which are
well known to the art, including the procedures disclosed by Lee et
al.; PNAS, 100 p6646-6651, 2003.
[0133] Processes for preparing compounds of formula (I) are
provided as further embodiments of the invention and are
illustrated by the following procedures in which the meanings of
the generic radicals are as given above unless otherwise
qualified.
EXAMPLES
[0134] General Chemistry. Reagents and solvents were acquired from
Aldrich, Milwaukee, Wis. Uncorrected melting points were determined
on a Laboratory Device MeI-Temp II capillary melting point
apparatus. Proton nuclear magnetic resonance spectra were recorded
on a Varian Unity 500 NMR spectrophotometer at 499.8 MHz or on a
Varian Mercury No spectrophotometer at 400.06 MHz. The chemical
shifts were reported in ppm on the scale from the indicated
reference. Positive and negative ion loop mass spectra were
performed by Department of Chemistry UCSD, San Diego, Calif.
Elemental analyses were performed by NuMega Resonance Labs, San
Diego, Calif. Column chromatography was conducted on E Merck silica
gel (230-400 mesh) with the indicated solvent system. Analytical
thin layer chromatography (TLC) was conducted on silica gel 60
F-254 plates (EM Reagents).
Example 1
Preparation of 4-(2,6-dichloropurin-9-ylmethyl)benzonitrile
[0135] 2,6-dichloro-9H-purine (16 mmol) is dissolved in DMF (50 mL)
and potassium carbonate (50 mmol) is added.
.alpha.-Bromo-p-tolunitrile (22 mmol) is then added and the mixture
is stirred at ambient temperature for 16 h. After filtration to
remove insoluble inorganic salts, the filtrate is poured into water
(1500 mL) and extracted with ethyl acetate (2.times.400 mL), dried
over magnesium sulfate and evaporated to yield a residue which is
subjected to flash silica gel chromatography using 1:2:10 ethyl
acetate/acetone/hexanes. Yield 3.33 g (69%). UV, NMR and MS were
consistent with structure assignment.
Example 2
Preparation of 4-(6-amino-2-chloropurin-9-ylmethylbenzonitrile
[0136] The product of example 1 (1.9 g) is placed in a steel
reaction vessel and methanolic ammonia (80 mL, 7 N) is added. The
sealed vessel is heated at 60.degree. C. for 12 h, cooled in ice
and the solid product filtered off. Yield 1.09 g. UV, NMR and MS
were consistent with assigned structure.
Example 3
Preparation of
4-[6-amino-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile
[0137] Sodium salt of 2-methoxyethanol is generated by dissolving
sodium metal (81 mg) in 2-methoxyethanol (30 mL) with heat. To this
solution is added the product of example 2 (1.0 g) dissolved in
methoxyethanol (300 mL, with heat). The reaction mixture is heated
for 8 h at 115.degree. C. bath temperature, concentrated in vacuo
to near dryness and the residue partitioned between ethyl acetate
and water. Flash silica gel chromatography of the organic layer
using 5% methanol in dichloromethane gave 763 mg product. NMR is
consistent with structure assignment.
Example 4
Preparation of
4-[6-amino-8-bromo-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile
[0138] The product of example 3 (700 mg) is dissolved in
dichloromethane (400 mL) and bromine (7 mL) is added dropwise The
mixture is stirred overnight at room temperature and extracted with
aqueous sodium thiosulfate (2 L of 0.1 M) solution and then with
aqueous sodium bicarbonate (500 mL, saturated). The residue from
the organic layer is chromatographed on silica gel using 3%
methanol in dichloromethane) to yield 460 mg of bromo product. NMR,
UV and MS are consistent with structure assignment.
Example 5
Preparation of
4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile
[0139] Sodium methoxide is generated by reaction of sodium metal
(81 mg) in dry methanol (30 mL). The product of example 4 (700 mg)
is dissolved in dry dimethoxyethane and the temperature raised to
100.degree. C. After overnight reaction, the mixture is
concentrated in vacuo and the residue is chromatographed on silica
using 5% methanol in dichloromethane. Yield 120 mg. NMR is
consistent with structure assignment.
Example 6
Preparation of Lithium N,N'-(dimethylethylenediamino)aluminum
hydride
[0140] This reducing agent used to convert the nitrile to the
aldehyde function is prepared essentially as described in Bull.
Korean Chem. Soc. (2002), 23(12), 1697-1698. A 0.5 M solution in
dry THF is prepared.
Example 7
Preparation of
4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde
[0141] The product of example 5 (100 mg) is dissolved in dry THF (3
mL) and cooled to 0.degree. C. under argon. The reagent generated
in example 6 (0.72 mL) is added to the reaction flask and the
mixture is stirred at 0-5.degree. C. for 1 h and then quenched by
addition of 3 M HCl. The mixture is then extracted with ethyl
acetate and then dichloromethane and concentrated in vacuo to yield
85 mg. NMR is consistent with structure assignment.
Example 8
Preparation of
4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde
(1V150)
[0142] The product of example 7 (800 mg) is combined with sodium
iodide (504 mg) and acetonitrile (40 mL), and then
chlorotrimethylsilane (0.5 mL) is slowly added. The mixture is
heated at 70.degree. C. for 3.5 h, cooled and filtered. The solid
product is washed with water, then ether to yield 406 mg. NMR, UV,
MS are consistent with structure assignment. This material is
suitable for conjugation reactions between linkers and auxiliary
groups.
Example 9
Preparation of methyl
4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate.
(Procedure as described by Jayachitra, et al., Synth. Comm., (2003)
33(19), 3461-3466.)
[0143] The product of example 5 (1 mmol) is dissolved in dry
methanol (5 mL) and freshly distilled BF.sub.3 etherate (4 mmol) is
added to the solution. The resulting mixture is refluxed under
argon for 20 h. The solvent is removed in vacuo and the residue is
taken up in dichloromethane (10 mL) and extracted with dilute
aqueous sodium bicarbonate (2.times.10 mL) and the organic layer is
dried over magnesium sulfate. After evaporation the product is
purified by silica gel column chromatography using 5% methanol in
dichloromethane to yield 0.8 mmol.
Example 10
Preparation of
4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoic
acid
[0144] The product of example 9 (100 mg) is combined with sodium
iodide (63 mg) and acetonitrile (10 mL), and then
chlorotrimethylsilane (120 mL) is slowly added. The mixture is
heated at 70.degree. C. for 6 h, cooled and filtered. The solid
product is washed with water, then ether to yield 51 mg.
Example 11
Preparation of 2,5-dioxopyrrolidin-1-yl
4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate
[0145] The product of example 10 (2 mmol) is dissolved in
dichloromethane or dioxane (10 mL) and EDC (2 mmol) is added. To
this solution is added N-hydroxysuccinimide (2 mmol) and resulting
mixture is stirred at room temperature for 1 h. The mixture is
taken to dryness in vacuo and the crude product is purified by
silica gel chromatography to yield 2 mmol of product that is
suitable for conjugation reactions involving primary amines.
Example 12
Conjugation of IV150 to Mouse Serum Albumin (MSA)
[0146] Modification of MSA with SANH: 200 .mu.l of MSA (25 mg/ml)
was mixed with 100 .mu.l of conjugation buffer (1M NaPi, pH=7.2)
and 690 .mu.l of PBS. 844%1 g of SANH in 10 .mu.l of DFM (40-fold
molar excess to MSA) was added to protein solution (Final
concentration of MSA in reaction mixture is 5 mg/ml). After gentle
mixing reaction was proceeded at room temperature for 2 hr. To
remove excess of SANH the reaction mixture was loaded on NAP-10
column equilibrated with PBS and modified MSA was eluted with 1.5
ml of PBS.
[0147] Attachment of IV150 to MSA modified with SANH: 460 .mu.g of
IV150 dissolved in 10 .mu.l of DMF was added to MSA modified with
SANH and the reaction mixture was incubated at RT overnight. To
remove excess of IV150 the reaction mixture was firstly
concentrated to 1 ml using micro-spin column (Millipore: BIOMAX 5K)
and loaded on NAP-10 column as mentioned above.
[0148] The abbreviations used herein have their conventional
meaning within the chemical and biological arts. All publications,
patents, and patent documents cited in the specification are
incorporated by reference herein, as though individually
incorporated by reference. In the case of any inconsistencies, the
present disclosure, including any definitions therein will prevail.
The invention has been described with reference to various specific
and preferred embodiments and techniques. However, it should be
understood that many variations and modifications may be made while
remaining within the spirit and scope of the invention.
* * * * *