U.S. patent application number 10/681457 was filed with the patent office on 2004-08-12 for amido ether substituted imidazoquinolines.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Crooks, Stephen L., Griesgraber, George W., Heppner, Philip D., Merrill, Bryon A..
Application Number | 20040157874 10/681457 |
Document ID | / |
Family ID | 26682656 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157874 |
Kind Code |
A1 |
Crooks, Stephen L. ; et
al. |
August 12, 2004 |
Amido ether substituted imidazoquinolines
Abstract
Imidazoquinoline and tetrahydroimidazoquinoline compounds that
contain ether and amide functionality at the 1-position are useful
as immune response modifiers. The compounds and compositions of the
invention can induce the biosynthesis of various cytokines and are
useful in the treatment of a variety of conditions including viral
diseases and neoplastic diseases.
Inventors: |
Crooks, Stephen L.;
(Mahtomedi, MN) ; Griesgraber, George W.; (Eagan,
MN) ; Heppner, Philip D.; (Woodbury, MN) ;
Merrill, Bryon A.; (River Falls, MN) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
26682656 |
Appl. No.: |
10/681457 |
Filed: |
October 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10681457 |
Oct 7, 2003 |
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10011670 |
Dec 6, 2001 |
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6660747 |
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60254218 |
Dec 8, 2000 |
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Current U.S.
Class: |
514/292 ;
546/82 |
Current CPC
Class: |
A61P 31/12 20180101;
C07D 471/04 20130101; A61P 37/02 20180101 |
Class at
Publication: |
514/292 ;
546/082 |
International
Class: |
A61K 031/4745; C07D
471/02 |
Claims
What is claimed is:
1. A compound of the formula (I): 80wherein: X is --CHR.sub.5--,
--CHR.sub.5-alkyl-, or --CHR.sub.5-alkenyl-; R.sub.1 is selected
from the group consisting of: --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-ar- yl;
--R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
--R.sub.4--CR.sub.3--Z-R.su- b.6-heterocyclyl;
--R.sub.4--CR.sub.3--Z-H; --R.sub.4--NR.sub.7--CR.sub.3--
-R.sub.6-alkyl; --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkenyl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
--R.sub.4--NR.sub.7--CR.sub.- 3--R.sub.6-heteroaryl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl- ; and
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.8; each Z is independently
--NR.sub.5--, --O--, or --S--; R.sub.2 is selected from the group
consisting of: -hydrogen; -alkyl; -alkenyl; -aryl; -heteroaryl;
-heterocyclyl; -alkyl-Y-alkyl; -alkyl-Y-alkenyl; -alkyl-Y-aryl; and
-alkyl or alkenyl substituted by one or more substituents selected
from the group consisting of: --OH; -halogen; --N(R.sub.5).sub.2;
--CO--N(R.sub.5).sub.2; --CO--C.sub.1-10 alkyl; --CO--O--C.sub.1-10
alkyl; --N.sub.3; -aryl; -heteroaryl; -heterocyclyl; --CO-aryl; and
--CO-heteroaryl; each R.sub.3 is .dbd.O or .dbd.S; each R.sub.4 is
independently alkyl or alkenyl, which may be interrupted by one or
more --O-- groups; each R.sub.5 is independently H or C.sub.1-10
alkyl; R.sub.6 is a bond, alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups; R.sub.7 is H, C.sub.1-10
alkyl, or arylalkyl; or R.sub.4 and R.sub.7 can join together to
form a ring; R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and
R.sub.8 can join together to form a ring; each Y is independently
--O-- or --S(O).sub.0-2--; n is 0 to 4; and each R present is
independently selected from the group consisting of C.sub.1-10
alkyl, C.sub.1-10 alkoxy, hydroxy, halogen and trifluoromethyl; or
a pharmaceutically acceptable salt thereof.
2. A compound or salt of claim 1 wherein the heteroaryl is selected
from the group consisting of 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-thiazolyl, and 4-pyrazolyl.
3. A compound or salt of claim 1 wherein X is --CH(alkyl)(alkyl)-
wherein the alkyl groups can be the same or different.
4. A compound or salt of claim 1 wherein X is
--CH.sub.2--CH.sub.2--.
5. A compound or salt of claim 1 wherein X is
--CH(C.sub.2H.sub.5)(CH.sub.- 2)--.
6. A compound or salt of claim 1 wherein R.sub.2 is H.
7. A compound or salt of claim 1 wherein R.sub.2 is alkyl.
8. A compound or salt of claim 1 wherein R.sub.2 is
-alkyl-O-alkyl.
9. A compound of the formula (II) 81wherein: X is --CHR.sub.5--,
--CHR.sub.5-alkyl-, or --CHR.sub.5-alkenyl-; R.sub.1 is selected
from the group consisting of: --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-ar- yl;
--R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
--R.sub.4--CR.sub.3--Z-R.su- b.6-heterocyclyl;
--R.sub.4--CR.sub.3--Z-H; --R.sub.4--NR.sub.7--CR.sub.3--
-R.sub.6-alkyl; --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkenyl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
--R.sub.4--NR.sub.7--CR.sub.- 3--R.sub.6-heteroaryl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl- ; and
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.8; each Z is independently
--NR.sub.5--, --O--, or --S--; R.sub.2 is selected from the group
consisting of: -hydrogen; -alkyl; -alkenyl; -aryl; -heteroaryl;
-heterocyclyl; -alkyl-Y-alkyl; -alkyl-Y-alkenyl; -alkyl-Y-aryl; and
-alkyl or alkenyl substituted by one or more substituents selected
from the group consisting of: --OH; -halogen; --N(R.sub.5).sub.2;
--CO--N(R.sub.5).sub.2; --CO--C.sub.1-10 alkyl; --CO--O--C.sub.1-10
alkyl; --N.sub.3; -aryl; -heteroaryl; -heterocyclyl; --CO-aryl; and
--CO-heteroaryl; each R.sub.3 is .dbd.O or .dbd.S; each R.sub.4 is
independently alkyl or alkenyl, which may be interrupted by one or
more --O-- groups; each R.sub.5 is independently H or C.sub.1-10
alkyl; R.sub.6 is a bond, alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups; R.sub.7 is H, C.sub.1-10
alkyl, arylalkyl; or R.sub.4 and R.sub.7 can join together to form
a ring; R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and R.sub.8
can join together to form a ring; each Y is independently --O-- or
--S(O).sub.0-2--; n is 0 to 4; and each R present is independently
selected from the group consisting of C.sub.1-10 alkyl, C.sub.1-10
alkoxy, hydroxy, halogen, and trifluoromethyl; or a
pharmaceutically acceptable salt thereof.
10. A compound or salt of claim 9 wherein R.sub.2 is H or
alkyl.
11. A compound or salt of claim 9 wherein R.sub.2 is
-alkyl-O-alkyl.
12. A pharmaceutical composition comprising a therapeutically
effective amount of a compound or salt of claim 1 and a
pharmaceutically acceptable carrier.
13. A method of inducing cytokine biosynthesis in an animal
comprising administering a therapeutically effective amount of a
compound or salt of claim 1 to the animal.
14. The method of claim 13 wherein the cytokine is IFN-.alpha..
15. A method of treating a viral disease in an animal comprising
administering a therapeutically effective amount of a compound or
salt of claim 1 to the animal.
16. A method of treating a neoplastic disease in an animal
comprising administering a therapeutically effective amount of a
compound or salt of claim 1 to the animal.
17. A compound of the formula (III): 82wherein: X is --CHR.sub.5--,
--CHR.sub.5-alkyl-, or --CHR.sub.5-alkenyl-; R.sub.1 is selected
from the group consisting of: --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
--R.sub.4--CR.sub.3--Z-R.sub.6-ar- yl;
--R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
--R.sub.4--CR.sub.3--Z-R.su- b.6-heterocyclyl;
--R.sub.4--CR.sub.3--Z-H; --R.sub.4--NR.sub.7--CR.sub.3--
-R.sub.6-alkyl; --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkenyl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
--R.sub.4--NR.sub.7--CR.sub.- 3--R.sub.6-heteroaryl;
--R.sub.4--NR.sub.7CR.sub.3--R.sub.6-heterocyclyl; and
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.8; each Z is independently
--NR.sub.5--, --O-- or --S--; R.sub.2 is selected from the group
consisting of: -hydrogen; -alkyl; -alkenyl; -aryl; -heteroaryl;
-heterocyclyl; -alkyl-Y-alkyl; -alkyl-Y-alkenyl; -alkyl-Y-aryl; and
-alkyl or alkenyl substituted by one or more substituents selected
from the group consisting of: --OH; -halogen; --N(R.sub.5).sub.2;
--CO--N(R.sub.5).sub.2; --CO--C.sub.1-10 alkyl; --CO--O--C.sub.1-10
alkyl; --N.sub.3; -aryl; -heteroaryl; -heterocyclyl; --CO-aryl; and
--CO-heteroaryl; each R.sub.3 is .dbd.O or .dbd.S; each R.sub.4 is
independently alkyl or alkenyl, which may be interrupted by one or
more --O-- groups; each R.sub.5 is independently H or C.sub.1-10
alkyl; R.sub.6 is a bond, or is alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups; R.sub.7 is H, C.sub.1-10
alkyl, or arylalkyl; or R.sub.4 and R.sub.7 can join to form a
ring; R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and R.sub.8 can
join to form a each Y is independently --O-- or --S(O).sub.0-2--; n
is 0 to 4; and each R present is independently selected from the
group consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy,
halogen and trifluoromethyl; or a pharmaceutically acceptable salt
thereof.
18. A compound of the formula (IV): 83wherein X is --CHR.sub.5--,
--CHR.sub.5-alkyl-, or --CHR.sub.5-alkenyl-; R.sub.1 is selected
from the group consisting of: --R.sub.4--CR.sub.3Q-R.sub.6-alkyl;
--R.sub.4--CR.sub.3-Q-R.sub.6-alkenyl;
--R.sub.4--CR.sub.3-Q-R.sub.6-aryl- ;
--R.sub.4--CR.sub.3-Q-R.sub.6-heteroaryl;
--R.sub.4--CR.sub.3-Q-R.sub.6-- heterocyclyl;
--R.sub.4--CR.sub.3-Q-H; --R.sub.4--NR.sub.5--CR.sub.3--R.su-
b.6-alkyl; --R.sub.4--NR.sub.5--CR.sub.3--R.sub.6-alkenyl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
--R.sub.4--NR.sub.7--CR.sub.- 3--R.sub.6-heteroaryl;
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl- ; and
--R.sub.4--NR.sub.7--CR.sub.3--R.sub.8; each Q is independently
--NR.sub.5-- or --O--: each R.sub.3 is .dbd.O or .dbd.S; each
R.sub.4 is independently alkyl or alkenyl, which may be interrupted
by one or more --O-- groups; each R.sub.5 is independently H or
C.sub.1-10 alkyl; R.sub.6 is a bond, alkyl, or alkenyl, which may
be interrupted by one or more --O-- groups; R.sub.7 is H,
C.sub.1-10 alkyl, or arylalkyl; or R.sub.4 and R.sub.7 can join to
form a ring; R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and
R.sub.8 can join to form a ring; n is 0 to 4; and each R present is
independently selected from the group consisting of C.sub.1-10
alkyl, C.sub.1-10 alkoxy, halogen and trifluoromethyl; or a
pharmaceutically acceptable salt thereof.
19. A pharmaceutical composition comprising a therapeutically
effective amount of a compound or salt of claim 9 and a
pharmaceutically acceptable carrier.
20. A method of inducing cytokine biosynthesis in an animal
comprising administering a therapeutically effective amount of a
compound or salt of claim 9 to the animal.
21. The method of claim 20 wherein the cytokine is IFN-.alpha..
22. A method of treating a viral disease in an animal comprising
administering a therapeutically effective amount of a compound or
salt of claim 9 to the animal.
23. A method of treating a neoplastic disease in an animal
comprising administering a therapeutically effective amount of a
compound or salt of claim 9 to the animal.
24. A compound of the formula (V): 84wherein: X is --CHR.sub.5--,
--CHR.sub.5-alkyl-, or --CHR.sub.5-alkenyl-; R.sub.2 is selected
from the group consisting of: -hydrogen; -alkyl; -alkenyl; -aryl;
-heteroaryl; -heterocyclyl; -alkyl-Y-alkyl; -alkyl-Y-alkenyl;
-alkyl-Y-aryl; and -alkyl or alkenyl substituted by one or more
substituents selected from the group consisting of: --OH; -halogen;
--N(R.sub.5).sub.2; --CO--N(R.sub.5).sub.2; --CO--C.sub.1-10 alkyl;
--CO--O--C.sub.1-10 alkyl; --N.sub.3; -aryl; -heteroaryl;
-heterocyclyl; --CO-aryl; and --CO-heteroaryl; each R.sub.4 is
independently alkyl or alkenyl, which may be interrupted by one or
more --O-- groups; R.sub.7 is H, C.sub.1-10 alkyl, or arylalkyl; or
R.sub.4 and R.sub.7 can join to form a ring; each Y is
independently --O-- or --S(O).sub.0-2--; n is 0 to 4; and each R
present is independently selected from the group consisting of
C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy, halogen and
trifluoromethyl; or a pharmaceutically acceptable salt thereof.
Description
[0001] This application claims the benefit of previously filed
Provisional Application Serial No. 60/254,218, filed on Dec. 8,
2000.
FIELD OF THE INVENTION
[0002] This invention relates to imidazoquinoline compounds that
have ether and amido functionality at the 1-position, and to
pharmaceutical compositions containing such compounds. A further
aspect of this invention relates to the use of these compounds as
immunomodulators, for inducing cytokine biosynthesis in animals,
and in the treatment of diseases, including viral and neoplastic
diseases.
BACKGROUND OF THE INVENTION
[0003] The first reliable report on the 1H-imidazo[4,5-c]quinoline
ring system, Backman et al., J. Org. Chem. 15, 1278-1284 (1950)
describes the synthesis of
1-(6-methoxy-8-quinolinyl)-2-methyl-1H-imidazo[4,5-c]quinoli- ne
for possible use as an antimalarial agent. Subsequently, syntheses
of various substituted 1H-imidazo[4,5-c]quinolines Were reported.
For example, Jain et al., J. Med. Chem. 11, pp. 87-92 (1968),
synthesized the compound
1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]quinoline as a possible
anticonvulsant and cardiovascular agent. Also, Baranov et al.,
Chem. Abs. 85, 94362 (1976), have reported several
2-oxoimidazo[4,5-c]quinolines, and Berenyi et al., J. Heterocyclic
Chem. 18, 1537-1540 (1981), have reported certain
2-oxoimidazo[4,5-c]quinolines- .
[0004] Certain 1H-imidazo[4,5-c]quinolin-4-amines and 1- and
2-substituted derivatives thereof were later found to be useful as
antiviral agents, bronchodilators and immunomodulators. These are
described in, inter alia, U.S. Pat. Nos. 4,689,338; 4,698,348;
4,929,624; 5,037,986; 5,268,376; 5,346,905; and 5,389,640, all of
which are incorporated herein by reference.
[0005] There continues to be interest in the imidazoquinoline ring
system.
[0006] Certain 1H-imidazo[4,5-c]naphthyridine-4-amines, 1H-imidazo
[4,5-c]pyridin-4-amines, and 1H-imidazo[4,5-c]quinolin-4-amines
having an ether containing substituent at the 1 position are known.
These are described in U.S. Pat. Nos. 5,268,376; 5,389,640;
5,494,916; and WO 99/29693.
[0007] Despite these attempts to identify compounds that are useful
as immune response modifiers, there is a continuing need for
compounds that have the ability to modulate the immune response, by
induction of cytokine biosynthesis or other mechanisms.
SUMMARY OF THE INVENTION
[0008] We have found a new class of compounds that are useful in
inducing cytokine biosynthesis in animals. Accordingly, this
invention provides imidazo[4, 50c]quinoline-4-amine and
tetrahydroimidazo[4,5-c]quinoline-4-- amine compounds that have an
ether containing substituent at the 1-position. The compounds are
defined by Formulas (I) and(1I), which are defined in more detail
infra. These compounds share the general structural formula: 1
[0009] wherein X, R.sub.1, R.sub.2, and R are as defined herein for
each class of compounds having Formulas (I) and (II).
[0010] The compounds of Formulas (I) and (II) are useful as immune
response modifiers due to their ability to induce cytokine
biosynthesis and otherwise modulate the immune response when
administered to animals. This makes the compounds useful in the
treatment of a variety of conditions such as viral diseases and
tumors that are responsive to such changes in the immune
response.
[0011] The invention further provides pharmaceutical compositions
containing the immune response modifying compounds, and methods of
inducing cytokine biosynthesis in an animal, treating a viral
infection in an animal, and/or treating a neoplastic disease in an
animal by administering a compound of Formula (I) or (II) to the
animal.
[0012] In addition, the invention provides methods of synthesizing
the compounds of the invention and novel intermediates useful in
the synthesis of these compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0013] As mentioned earlier, we have found certain compounds that
induce cytokine biosynthesis and modify the immune response in
animals. Such compounds are represented by Formulas (I) and (II) as
shown below.
[0014] Imidazoquinoline compounds of the invention, which have
ether and amide functionality at the 1-position, are represented by
Formula (I): 2
[0015] wherein: X is --CHR.sub.5--, --CHR.sub.5-alkyl-, or
--CHR.sub.5-alkenyl-;
[0016] R.sub.1 is selected from the group consisting of:
[0017] --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
[0018] --R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
[0019] --R.sub.4--CR.sub.3--Z-R.sub.6-aryl;
[0020] --R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
[0021] --R.sub.4--CR.sub.3--Z-R.sub.6-heterocyclyl;
[0022] --R.sub.4--CR.sub.3--Z-H;
[0023] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkyl;
[0024] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkenyl;
[0025] each R.sub.5 is independently H or C.sub.1-10 alkyl;
[0026] R.sub.6 is a bond, alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups;
[0027] R.sub.7 is H, C.sub.1-10 alkyl, or arylalkyl; or R.sub.4 and
R.sub.7 can join together to form a ring;
[0028] R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and R.sub.8 can
join together to form a ring;
[0029] each Y is independently --O-- or --S(O).sub.0-2--;
[0030] n is 0 to 4; and
[0031] each R present is independently selected from the group
consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy, halogen
and trifluoromethyl;
[0032] or a pharmaceutically acceptable salt thereof.
[0033] The invention also includes tetrahydroimidazoquinoline
compounds that bear an ether and amide containing substituent at
the 1-position. Such tetrahydroimidazoquinoline compounds are
represented by Formula (II): 3
[0034] wherein: X is --CHR.sub.5--, --CHR.sub.5-alkyl-, or
--CHR.sub.5-alkenyl-;
[0035] R.sub.1 is selected from the group consisting of:
[0036] --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
[0037] --R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
[0038] --R.sub.4--CR.sub.3--Z-R.sub.6-aryl;
[0039] --R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
[0040] --R.sub.4--CR.sub.3--Z-R.sub.6-heterocyclyl;
[0041] --R.sub.4--CR.sub.3--Z-H;
[0042] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkyl;
[0043] --R.sub.4--NR.sub.7--R.sub.3--R.sub.6-alkenyl;
[0044] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
[0045] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heteroaryl;
[0046] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl;
[0047] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.8;
[0048] each Z is independently --NR.sub.5--, --O--, or --S--;
[0049] R.sub.2 is selected from the group consisting of:
[0050] -hydrogen;
[0051] -alkyl;
[0052] -alkenyl;
[0053] -aryl;
[0054] -heteroaryl;
[0055] -heterocyclyl;
[0056] -alkyl-Y-alkyl;
[0057] -alkyl-Y-alkenyl;
[0058] -alkyl-Y-aryl; and
[0059] -alkyl or alkenyl substituted by one or more substituents
selected from the group consisting of:
[0060] --OH;
[0061] -halogen;
[0062] --N(R.sub.5).sub.2;
[0063] --CO--N(R.sub.5).sub.2;
[0064] --CO--C.sub.1-10 alkyl;
[0065] --CO--O-.sub.C1-10 alkyl;
[0066] --N.sub.3;
[0067] -aryl;
[0068] -heteroaryl;
[0069] -heterocyclyl;
[0070] --CO-aryl; and
[0071] --CO-heteroaryl;
[0072] each R.sub.3 is .dbd.O or .dbd.S;
[0073] each R.sub.4 is independently alkyl or alkenyl, which may be
interrupted by one or more --O-- groups;
[0074] each R.sub.5 is independently H or C.sub.1-10 alkyl;
[0075] R.sub.6 is a bond, alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups;
[0076] R.sub.7 is H, C.sub.1-10 alkyl, or arylalkyl; or R.sub.4 and
R.sub.7 can join together to form a ring;
[0077] R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and R.sub.8 can
join together to form a ring;
[0078] each Y is independently --O-- or --S(O).sub.0-2--;
[0079] n is 0 to 4; and
[0080] each R present is independently selected from the group
consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy,
halogen, and trifluoromethyl;
[0081] or a pharmaceutically acceptable salt thereof.
[0082] Preparation of the Compounds
[0083] Compounds of the invention can be prepared according to
Reaction Scheme I where R, R.sub.2, R.sub.3, R.sub.4, X, Z and n
are as defined above and R.sub.11 is --R.sub.6-alkyl,
--R.sub.6-aryl, --R.sub.6-heteroaryl or --R.sub.6-heterocyclyl
where R.sub.6 is as defined above.
[0084] In step (1) of Reaction Scheme I a
1H-imidazo[4,5-c]quinolin-1-yl alcohol of Formula X is alkylated
with a halide of Formula XI to provide a
1H-imidazo[4,5-c]quinolin-1-yl ether of Formula XII. The alcohol of
Formula X is reacted with sodium hydride in a suitable solvent such
as N,N-dimethylformamide to form an alkoxide. Alternatively, the
alkoxide can be formed by adding the alcohol to a biphasic mixture
of aqueous 50% sodium hydroxide and an inert solvent such as
dichloromethane in the presence of a phase transfer catalyst such
as benzyltrimethylammonium chloride. The alkoxide is then combined
with the halide. The reaction can be carried out at ambient
temperature. Many compounds of Formula X are known, see for
example, Gerster, U.S. Pat. No. 4,689,338; others can readily be
prepared using known synthetic routes, see for example, Gerster et
al., U.S. Pat. No. 5,605,899 and Gerster, U.S. Pat. No. 5,175,296.
Many halides of Formula XI are commercially available; others can
be readily prepared using known synthetic routes.
[0085] In step (2) of Reaction Scheme I a
1H-imidazo[4,5-c]quinolin-1-yl ether of Formula XII is oxidized to
provide a 1H-imidazo[4,5-c]quinoline-- 5N-oxide of Formula XIII
using a conventional oxidizing agent capable of forming N-oxides.
Preferably a solution of a compound of Formula XII in chloroform is
oxidized using 3-chloroperoxybenzoic acid at ambient
temperature.
[0086] In step (3) of Reaction Scheme I a
1H-imidazo[4,5-c]quinoline-5N-ox- ide of Formula XIII is aminated
to provide a 1H-imidazo[4,5-c]quinolin-4-a- mine of Formula XIV
which is a subgenus of Formula I. Step (3) involves (i) reacting a
compound of Formula XIII with an acylating agent and then (ii)
reacting the product with an aminating agent. Part (i) of step (3)
involves reacting an N-oxide of Formula XIII with an acylating
agent. Suitable acylating agents include alkyl- or arylsulfonyl
chlorides (e.g., benezenesulfonyl chloride, methanesulfonyl
chloride, p-toluenesulfonyl chloride). Arylsulfonyl chlorides are
preferred. Para-toluenesulfonyl chloride is most preferred. Part
(ii) of step (3) involves reacting the product of part (i) with an
excess of an aminating agent. Suitable aminating agents include
ammonia (e.g., in the form of ammonium hydroxide) and ammonium
salts (e.g., ammonium carbonate, ammonium bicarbonate, ammonium
phosphate). Ammonium hydroxide is preferred. The reaction is
preferably carried out by dissolving the N-oxide of Formula XIII in
an inert solvent such as dichloromethane, adding the aminating
agent to the solution, and then slowly adding the acylating agent.
The product or a pharmaceutically acceptable salt thereof can be
isolated using conventional methods.
[0087] Alternatively, step (3) may be carried out by (i) reacting
an N-oxide of Formula XIII with an isocyanate and then (ii)
hydrolyzing the resulting product. Part (i) involves reacting the
N-oxide with an isocyanate wherein the isocyanato group is bonded
to a carbonyl group. Preferred isocyanates include trichloroacetyl
isocyanate and aroyl isocyanates such as benzoyl isocyanate. The
reaction of the isocyanate with the N-oxide is carried out under
substantially anhydrous conditions by adding the isocyanate to a
solution of the N-oxide in an inert solvent such as chloroform or
dichloromethane. Part (ii) involves hydrolysis of the product from
part (i). The hydrolysis can be carried out by conventional methods
such as heating in the presence of water or a lower alkanol
optionally in the presence of a catalyst such as an alkali metal
hydroxide or lower alkoxide. The product or a pharmaceutically
acceptable salt thereof can be isolated using conventional methods.
4
[0088] Compounds of the invention can be prepared according to
Reaction Scheme II where R, R.sub.2, R.sub.4, R.sub.7, R.sub.11, X
and n are as defined above and BOC is tert-butoxycarbonyl.
[0089] In step (1) of Reaction Scheme II the amino group of an
aminoalcohol of Formula XV is protected with a tert-butoxycarbonyl
group. A solution of the aminoalcohol in tetrahydrofuran is treated
with di-tert-butyl dicarbonate in the presence of a base such as
sodium hydroxide. Many aminoalcohols of Formula XV are commercially
available; others can be prepared using known synthetic
methods.
[0090] In step (2) of Reaction Scheme II a protected aminoalcohol
of Formula XVI is converted to an iodide of Formula XVII. Iodine is
added to a solution of triphenylphosphine and imidazole in
dichloromethane; then a solution of a protected aminoalcohol of
Formula XVI in dichloromethane is added. The reaction is carried
out at ambient temperature.
[0091] In step (3) of Reaction Scheme II a
1H-imidazo[4,5-c]quinolin-1-yl alcohol of Formula X is alkylated
with an iodide of Formula XVII to provide a
1H-imidazo[4,5-c]quinolin-1-yl ether of Formula XVIII. The alcohol
of Formula X is reacted with sodium hydride in a suitable solvent
such as N,N-dimethylformamide to form an alkoxide. The iodide is
added to the alkoxide solution at ambient temperature. After the
addition is complete the reaction is stirred at an elevated
temperature (.about.100.degree. C.).
[0092] In step (4) of Reaction Scheme II a
1H-imidazo[4,5-c]quinolin-1-yl ether of Formula XVIII is oxidized
to provide a 1H-imidazo[4,5-c]quinolin- e-5N-oxide of Formula XIX
using a conventional oxidizing agent capable of forming N-oxides.
Preferably a solution of a compound of Formula XVIII in chloroform
is oxidized using 3-chloroperoxybenzoic acid at ambient
temperature.
[0093] In step (5) of Reaction Scheme II a
1H-imidazo[4,5-c]quinoline-5N-o- xide of Formula XIX is aminated to
provide a 1H-imidazo[4,5-c]quinolin4-am- ine of Formula XX. Step
(5) involves (i) reacting a compound of Formula XIX with an
acylating agent and then (ii) reacting the product with an
aminating agent. Part (i) of step (5) involves reacting an N-oxide
of Formula XIX with an acylating agent. Suitable acylating agents
include alkyl- or arylsulfonyl chlorides (e.g., benezenesulfonyl
chloride, methanesulfonyl chloride, p-toluenesulfonyl chloride).
Arylsulfonyl chlorides are preferred. Para-toluenesulfonyl chloride
is most preferred. Part (ii) of step (5) involves reacting the
product of part (i) with an excess of an aminating agent. Suitable
aminating agents include ammonia (e.g., in the form of ammonium
hydroxide) and ammonium salts (e.g., ammonium carbonate, ammonium
bicarbonate, ammonium phosphate). Ammonium hydroxide is preferred.
The reaction is preferably carried out by dissolving the N-oxide of
Formula XIX in an inert solvent such as dichloromethane or
1,2-dichloroethane with heating if necessary, adding the aminating
agent to the solution, and then slowly adding the acylating agent.
Optionally the reaction can be carried out in a sealed pressure
vessel at an elevated temperature (85-100.degree.).
[0094] In step (6) of Reaction Scheme II the protecting group is
removed by hydrolysis under acidic conditions to provide a
1H-imidazo[4,5-c]quinolin-4-amine of Formula XXI. Preferably the
compound of Formula XX is treated with hydrochloric acid/ethanol at
ambient temperature or with gentle heating.
[0095] In step (7) of Reaction Scheme II a
1H-imidazo[4,5-c]quinolin-4-ami- ne of Formula XXI is converted to
an amide of Formula XXII which is a subgenus of Formula I using
conventional synthetic methods. For example, a compound of Formula
XXI can be reacted with an acid chloride of Formula R.sub.11C(O)Cl.
The reaction can be carried out by adding a solution of the acid
chloride in a suitable solvent such as dichloromethane or
1-methyl-2-pyrrolidinone to a solution of a compound of Formula XXI
at ambient temperature. Alternatively, a compound of Formula XXI
can be reacted with an acid of Formula R.sub.11COOH. The reaction
can be carried out at ambient temperature in a solvent such as
dichloromethane or pyridine using a standard coupling reagent such
as 1,3-dicyclohexylcarbodiimide or
1[3-(dimethylamino)propyl]-3-ethylcarbodi- imide. The product or a
pharmaceutically acceptable salt thereof can be isolated using
conventional methods. 5
[0096] Compounds of the invention can be prepared according to
Reaction Scheme III where R, R.sub.2, R.sub.4, R.sub.7, R.sub.11, X
and n are as defined above and BOC is tert-butoxycarbonyl.
[0097] In step (1) of Reaction Scheme III the amino group of an
aminoalcohol of Formula XXIII is protected with a
tert-butoxycarbonyl group. A solution of the aminoalcohol in
tetrahydrofuran is treated with di-tert-butyl dicarbonate in the
presence of a base such as sodium hydroxide. Many aminoalcohols of
Formula XXIII are commercially available; others can be prepared
using known synthetic methods.
[0098] In step (2) of Reaction Scheme III a protected amino alcohol
of Formula XXIV is converted to a methanesulfonate of Formula XXV.
A solution of a compound of Formula XXIV in a suitable solvent such
as dichloromethane is treated with methanesulfonyl chloride in the
presence of a base such as triethylamine. The reaction can be
carried out at a reduced temperature (0.degree.).
[0099] In step (3a) of Reaction Scheme III a methanesulfonate of
Formula XXV is converted to an azide of Formula XXVI. Sodium azide
is added to a solution of a compound of Formula XXV in a suitable
solvent such as N,N-dimethylformamide. The reaction can be carried
out at an elevated temperature (80-100.degree. C.).
[0100] In step (3b) of Reaction Scheme III a compound of Formula
XXVI is alkylated with a halide of Formula Hal-R.sub.7 to provide a
compound of Formula XXVII. In compounds where R.sub.7 is hydrogen
this step is omitted. The compound of Formula XXVI is reacted with
sodium hydride in a suitable solvent such as N,N-dimethylformamide
to form the anion and then combined with the halide. The reaction
can be carried out at ambient temperature.
[0101] In step (4) of Reaction Scheme III an azide of Formula XXVI
or XXVII is reduced to provide an amine of Formula XXVIII.
Preferably, the reduction is carried out using a conventional
heterogeneous hydrogenation catalyst such as palladium on carbon.
The reaction can conveniently be carried out on a Parr apparatus in
a suitable solvent such as methanol or isopropanol.
[0102] In step (5) of Reaction Scheme III a
4-chloro-3-nitroquinoline of Formula XXIX is reacted with an amine
of Formula XXVIII to provide a 3-nitroquinoline of Formula XXX. The
reaction can be carried out by adding an amine of Formula XXVIII to
a solution of a compound of Formula XXIX in a suitable solvent such
as dichloromethane in the presence of a base such as triethylamine.
Many quinolines of Formula XXIX are known compounds or can be
prepared using known synthetic methods, see for example, U.S. Pat.
No. 4,689,338 and references cited therein.
[0103] In step (6) of Reaction Scheme III a 3-nitroquinoline of
Formula XXX is reduced to provide a 3-aminoquinoline of Formula
XXXI. Preferably, the reduction is carried out using a conventional
heterogeneous hydrogenation catalyst such as platinum on carbon.
The reaction can conveniently be carried out on a Parr apparatus in
a suitable solvent such as toluene.
[0104] In step (7) of Reaction Scheme III a compound of Formula
XXXI is reacted with a carboxylic acid or an equivalent thereof to
provide a 1H-imidazo[4,5-c]quinoline of Formula XVIII. Suitable
equivalents to carboxylic acid include orthoesters, and
1,1-dialkoxyalkyl alkanoates. The carboxylic acid or equivalent is
selected such that it will provide the desired R.sub.2 substituent
in a compound of Formula XVIII. For example, triethyl orthoformate
will provide a compound where R.sub.2 is hydrogen and triethyl
orthovalerate will provide a compound where R.sub.2 is butyl. The
reaction can be run in the absence of solvent or in an inert
solvent such as toluene. The reaction is run with sufficient
heating to drive off any alcohol or water formed as a byproduct of
the reaction. Optionally a catalyst such as pyridine hydrochloride
can be included.
[0105] Alternatively, step (7) can be carried out by (i) reacting a
compound of Formula XXXI with an acyl halide of Formula
R.sub.2C(O)Cl or R.sub.2C(O)Br and then (ii) cyclizing. In part (i)
the acyl halide is added to a solution of a compound of Formula
XXXI in an inert solvent such as acetonitrile or dichloromethane.
The reaction can be carried out at ambient temperature or at a
reduced temperature. In part (ii) the product of part (i) is heated
in an alcoholic solvent in the presence of a base. Preferably the
product of part (i) is refluxed in ethanol in the presence of an
excess of triethylamine or heated with methanolic ammonia.
[0106] Steps (8), (9), (10) and (11) are carried out in the same
manner as steps (4), (5), (6) and (7) of Reaction Scheme II. 67
[0107] Compounds of the invention can be prepared according to
Reaction Scheme IV where R, R.sub.1, R.sub.2, X and n are as
defined above
[0108] In Reaction Scheme IV a
4-amino-1H-imidazo[4,5-c]quinolin-1-yl alcohol of Formula XXXII is
alkylated with a halide of Formula XXXIII to provide a
1H-imidazo[4,5-c]quinolin-4-amine of Formula I. The alcohol of
Formula XXXII is reacted with sodium hydride in a suitable solvent
such as N,N-dimethylformamide to form an alkoxide. The halide is
then added to the reaction mixture. The reaction can be carried out
at ambient temperature or with gentle heating (.about.50.degree.
C.) if desired. The product or a pharmaceutically acceptable salt
thereof can be isolated using conventional methods.
[0109] Many compounds of Formula XXXII are known, see for example
Gerster, U.S. Pat. No. 4,689,338 and Gerster et. al., U.S. Pat. No.
5,605,899, the disclosures of which are incorporated by reference
herein; others can readily be prepared using known synthetic
routes, see for example, Andre et. al, U.S. Pat. No. 5,578,727;
Gerster, U.S. Pat. No. 5,175,296; Nikolaides et al., U.S. Pat. No.
5,395,937; and Gerster et. al., U.S. Pat. No. 5,741,908, the
disclosures of which are incorporated by reference herein. Many
halides of Formula XXXIII are commercially available; others can be
readily prepared using known synthetic methods. 8
[0110] Compounds of the invention can be prepared according to
Reaction Scheme V where R, R.sub.2, R.sub.4, R.sub.7, R.sub.11, X
and n are as defined above.
[0111] In step (1) of Reaction Scheme V a
1H-imidazo[4,5-c]quinolin-4-amin- e of Formula XXI is reduced to
provide a 6,7,8,9-tetrahydro-1H-imidazo[4 5-c]quinolin-4-amine of
Formula XXXIV. Preferably the reduction is carried out by
suspending or dissolving a compound of Formula XXI in
trifluoroacetic acid, adding a catalytic amount of platinum (IV)
oxide, and then hydrogenating. The reaction can be conveniently
carried out in a Parr apparatus.
[0112] Step (2) is carried out in the same manner as step (7) of
Reaction Scheme II to provide a
6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinolin-4-ami- ne of Formula
XXXV which is a subgenus of Formula II. The product or a
pharmaceutically acceptable salt thereof can be isolated using
conventional methods. 9
[0113] Compounds of the invention can be prepared according to
Reaction Scheme VI where R, R.sub.1, R.sub.2, X and n are as
defined above.
[0114] In Reaction Scheme VI a
4-amino-6,7,8,9-tetrahydro-1H-imidazo[4,5-c- ]quinolin-1-yl alcohol
of Formula XXXVI is alkylated with a halide of Formula XXXIII to
provide a 6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinolin-- 4-amine of
Formula II. The alcohol of Formula XXXVI is reacted with sodium
hydride in a suitable solvent such as N,N-dimethylformamide to form
an alkoxide. The halide is then added to the reaction mixture. The
reaction can be carried out at ambient temperature or with gentle
heating (.about.50.degree. C.) if desired. The product or a
pharmaceutically acceptable salt thereof can be isolated using
conventional methods.
[0115] Many 6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinolines of
Formula XXXVI are known, see for example, Nikolaides et al., U.S.
Pat. No. 5,352,784; others can be prepared using known synthetic
methods, see for example, Lindstrom, U.S. Pat. No. 5,693,811; the
disclosures of which are incorporated by reference herein. 10
[0116] The invention also provides novel compounds useful as
intermediates in the synthesis of the compounds of Formulas (I) and
(II). These intermediate compounds have the structural Formulas
(III)-(V), described in more detail below.
[0117] One class of intermediate compounds has Formula (III):
11
[0118] wherein: X is --CHR.sub.5--, --CHR.sub.5-alkyl-, or
--CHR.sub.5-alkenyl-;
[0119] R.sub.1 is selected from the group consisting of:
[0120] --R.sub.4--CR.sub.3--Z-R.sub.6-alkyl;
[0121] --R.sub.4--CR.sub.3--Z-R.sub.6-alkenyl;
[0122] --R.sub.4--CR.sub.3--Z-R.sub.6-aryl;
[0123] --R.sub.4--CR.sub.3--Z-R.sub.6-heteroaryl;
[0124] --R.sub.4--CR.sub.3--Z-R.sub.6-heterocyclyl;
[0125] --R.sub.4--CR.sub.3--Z-H;
[0126] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkyl;
[0127] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-alkenyl;
[0128] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
[0129] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heteroaryl;
[0130] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl; and
[0131] --R.sub.4--NR.sub.7--R.sub.3--R.sub.8;
[0132] each Z is independently --NR.sub.5--, --O--, or --S--;
[0133] R.sub.2 is selected from the group consisting of:
[0134] -hydrogen;
[0135] -alkyl;
[0136] -alkenyl;
[0137] -aryl;
[0138] -heteroaryl;
[0139] -heterocyclyl;
[0140] -alkyl-Y-alkyl;
[0141] -alkyl-Y-alkenyl;
[0142] -alkyl-Y-aryl; and
[0143] -alkyl or alkenyl substituted by one or more substituents
selected from the group consisting of:
[0144] --OH;
[0145] -halogen;
[0146] --N(R.sub.5).sub.2;
[0147] --CO--N(R.sub.5).sub.2;
[0148] --CO--C.sub.1-10 alkyl;
[0149] --CO--O--C.sub.1-10 alkyl;
[0150] --N.sub.3;
[0151] -aryl;
[0152] -heteroaryl;
[0153] -heterocyclyl;
[0154] --CO-aryl; and
[0155] --CO-heteroaryl;
[0156] each R.sub.3 is .dbd.O or .dbd.S;
[0157] each R.sub.4 is independently alkyl or alkenyl, which may be
interrupted by one or more --O-- groups;
[0158] each R.sub.5 is independently H or C.sub.1-10 alkyl;
[0159] R.sub.6 is a bond, or is alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups;
[0160] R.sub.7 is H, C.sub.1-10 alkyl, or arylalkyl; or R.sub.4 and
R.sub.7 can join to form a ring;
[0161] R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.7 and R.sub.8 can
join to form a ring;
[0162] each Y is independently --O-- or --S(O).sub.0.2--;
[0163] n is 0 to 4; and
[0164] each R present is independently selected from the group
consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy, halogen
and trifluoromethyl;
[0165] or a pharmaceutically acceptable salt thereof.
[0166] Another class of intermediates is described by formula (IV):
12
[0167] wherein: X is --CHR.sub.5--, --CHR.sub.5-alkyl-, or
--CHR.sub.5-alkenyl-;
[0168] R.sub.1 is selected from the group consisting of:
[0169] --R.sub.4--CR.sub.3-Q-R.sub.6-alkyl;
[0170] --R.sub.4--CR.sub.3-Q-R.sub.6-alkenyl;
[0171] --R.sub.4--CR.sub.3-Q-R.sub.6-aryl;
[0172] --R.sub.4--CR.sub.3-Q-R.sub.6-heteroaryl;
[0173] --R.sub.4--CR.sub.3-Q-R.sub.6-heterocyclyl;
[0174] --R.sub.4--CR.sub.3-Q-H;
[0175] --R.sub.4--NR.sub.5--CR.sub.3--R.sub.6-alkyl;
[0176] --R.sub.4--NR.sub.5--CR.sub.3--R.sub.6-alkenyl;
[0177] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-aryl;
[0178] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heteroaryl;
[0179] --R.sub.4--NR.sub.7--CR.sub.3--R.sub.6-heterocyclyl; and
[0180] --R.sub.4--NR.sub.5--CR.sub.3--R.sub.8;
[0181] each Q is independently --NR.sub.5-- or --O--;
[0182] each R.sub.3 is .dbd.O or .dbd.S;
[0183] each R.sub.4 is independently alkyl or alkenyl, which may be
interrupted by one or more --O-- groups;
[0184] each R.sub.5 is independently H or C.sub.1-10 alkyl;
[0185] R.sub.6 is a bond, alkyl, or alkenyl, which may be
interrupted by one or more --O-- groups;
[0186] R.sub.7 is H, C.sub.1-10 alkyl, or arylalkyl; or R.sub.4 and
R.sub.7 can join to form a ring;
[0187] R.sub.8 is H or C.sub.1-10 alkyl; or R.sub.4 and R.sub.7 can
join to form a ring;
[0188] n is 0 to 4; and
[0189] each R present is independently selected from the group
consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, halogen and
trifluoromethyl;
[0190] or a pharmaceutically acceptable salt thereof.
[0191] An additional class of intermediate compounds has the
formula (V): 13
[0192] wherein: X is --CHR.sub.5--, --CHR.sub.5-alkyl-, or
--CHR.sub.5-alkenyl-;
[0193] R.sub.2 is selected from the group consisting of:
[0194] -hydrogen;
[0195] -alkyl;
[0196] -alkenyl;
[0197] -aryl;
[0198] -heteroaryl;
[0199] -heterocyclyl;
[0200] -alkyl-Y-alkyl;
[0201] -alkyl-Y-alkenyl;
[0202] -alkyl-Y-aryl; and
[0203] -alkyl or alkenyl substituted by one or more substituents
selected from the group consisting of:
[0204] --OH;
[0205] -halogen;
[0206] --N(R.sub.5).sub.2;
[0207] --CO--N(R.sub.5).sub.2;
[0208] --CO--C.sub.1-10 alkyl;
[0209] --CO--O--C.sub.1-10 alkyl;
[0210] --N.sub.3;
[0211] -aryl;
[0212] -heteroaryl;
[0213] -heterocyclyl;
[0214] --CO-aryl; and
[0215] --CO-heteroaryl;
[0216] each R.sub.4 is independently alkyl or alkenyl, which may be
interrupted by one or more --O-- groups;
[0217] R.sub.7 is H, C.sub.1-10 alkyl,or arylalkyl; or R.sub.4 and
R.sub.7 can join to form a ring;
[0218] each Y is independently --O-- or --S(O).sub.0-2--;
[0219] n is 0 to 4; and
[0220] each R present is independently selected from the group
consisting of C.sub.1-10 alkyl, C.sub.1-10 alkoxy, hydroxy, halogen
and trifluoromethyl;
[0221] or a pharmaceutically acceptable salt thereof.
[0222] As used herein, the terms "alkyl", "alkenyl" and the prefix
"alk-" are inclusive of both straight chain and branched chain
groups and of cyclic groups, i.e. cycloalkyl and cycloalkenyl.
Unless otherwise specified, these groups contain from 1 to 20
carbon atoms, with alkenyl groups containing from 2 to 20 carbon
atoms. Preferred groups have a total of up to 10 carbon atoms.
Cyclic groups can be monocyclic or polycyclic and preferably have
from 3 to 10 ring carbon atoms. Exemplary cyclic groups include
cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl and
adamantyl.
[0223] In addition, the alkyl and alkenyl portions of --X-groups
can be unsubstituted or substituted by one or more substituents,
which substituents are selected from the groups consisting of
alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, arylalkyl,
heteroarylalkyl, and heterocyclylalkyl.
[0224] The term "haloalkyl" is inclusive of groups that
are-substituted by one or more halogen atoms, including
perfluorinated groups. This is also true of groups that include the
prefix "halo-". Examples of suitable haloalkyl groups are
chloromethyl, trifluoromethyl, and the like.
[0225] The term "aryl" as used herein includes carbocyclic aromatic
rings or ring systems. Examples of aryl groups include phenyl,
naphthyl, biphenyl, fluorenyl and indenyl. The term "heteroaryl"
includes aromatic rings or ring systems that contain at least one
ring hetero atom (e.g., O, S, N). Suitable heteroaryl groups
include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl,
indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl,
pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl,
carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl,
quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl,
isothiazolyl, purinyl, quinazolinyl, and so on.
[0226] "Heterocyclyl" includes non-aromatic rings or ring systems
that contain at least one ring hetero atom (e.g., O, S, N) and
includes all of the fully saturated and partially unsaturated
derivatives of the above mentioned heteroaryl groups. Exemplary
heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl,
morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl,
thiazolidinyl, imidazolidinyl, isothiazolidinyl, and the like.
[0227] The aryl, heteroaryl, and heterocyclyl groups can be
unsubstituted or substituted by one or more substituents
independently selected from the group consisting of alkyl, alkoxy,
alkylthio, haloalkyl, haloalkoxy, haloalkylthio, halogen, nitro,
hydroxy, mercapto, cyano, carboxy, formyl, aryl, aryloxy, arylthio,
arylalkoxy, arylalkylthio, heteroaryl, heteroaryloxy,
heteroarylthio, heteroarylalkoxy, heteroarylalkylthio, amino,
alkylamino, dialkylamino, heterocyclyl, heterocycloalkyl,
alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, haloalkylcarbonyl,
haloalkoxycarbonyl, alkylthiocarbonyl, arylcarbonyl,
heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,
arylthiocarbonyl, heteroarylthiocarbonyl, alkanoyloxy,
alkanoylthio, alkanoylamino, aroyloxy, aroylthio, aroylamino,
alkylaminosulfonyl, alkylsulfonyl, arylsulfonyl,
heteroarylsulfonyl, aryldiazinyl, alkylsulfonylamino,
arylsulfonylamino, arylalkylsulfonylamino, alkylcarbonylamino,
alkenylcarbonylamino, arylcarbonylamino, arylalkylcarbonylamino,
heteroarylcarbonylamino, heteroarylalkycarbonylamino,
alkylsulfonylamino, alkenylsulfonylamino, arylsulfonylamino,
arylalkylsulfonylamino, heteroarylsulfonylamino,
heteroarylalkylsulfonylamino, alkylaminocarbonylamino,
alkenylaminocarbonylamino, arylaminocarbonylamino,
arylalkylaminocarbonylamino, heteroarylaminocarbonylamino,
heteroarylalkylaminocarbonylamino and, in the case of heterocyclyl,
oxo. If any other groups are identified as being "substituted" or
"optionally substituted", then those groups can also be substituted
by one or more of the above enumerated substituents.
[0228] Certain substituents are generally preferred. For example,
preferred R.sub.1 groups include
--R.sub.4--CR.sub.3--Z-R.sub.6-alkyl and
--R.sub.4--CR.sub.3--Z-R.sub.6-aryl, wherein the alkyl and aryl
groups can be unsubstituted or substituted; R.sub.3 is preferably
.dbd.O; R.sub.4 is preferably ethylene or n-butylene; and Z is
preferably --NR.sub.5--. Preferably no R substituents are present
(i.e., n is 0). Preferred R.sub.2 groups include alkyl groups
having 1 to 4 carbon atoms (i.e., methyl, ethyl, propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, and cyclopropylmethyl), methoxyethyl,
and ethoxymethyl. For substituted groups such as substituted alkyl
or substituted aryl groups, preferred substituents include halogen,
nitrile, methoxy, trifluoromethyl, and trifluoromethoxy. One or
more of these preferred substituents, if present, can be present in
the compounds of the invention in any combination.
[0229] The invention is inclusive of the compounds described herein
in any of their pharmaceutically acceptable forms, including
isomers (e.g., diastereomers and enantiomers), salts, solvates,
polymorphs, and the like. In particular, if a compound is optically
active, the invention specifically includes each of the compound's
enantiomers as well as racemic mixtures of the enantiomers.
[0230] Pharmaceutical Compositions and Biological Activity
[0231] Pharmaceutical compositions of the invention contain a
therapeutically effective amount of a compound of the invention as
described above in combination with a pharmaceutically acceptable
carrier.
[0232] The term "a therapeutically effective amount" means an
amount of the compound sufficient to induce a therapeutic effect,
such as cytokine induction, antitumor activity, and/or antiviral
activity. Although the exact amount of active compound used in a
pharmaceutical composition of the invention will vary according to
factors known to those of skill in the art, such as the physical
and chemical nature of the compound, the nature of the carrier, and
the intended dosing regimen, it is anticipated that the
compositions of the invention will contain sufficient active
ingredient to provide a dose of about 100 ng/kg to about 50 mg/kg,
preferably about 10 .mu.g/kg to about 5 mg/kg, of the compound to
the subject. Any of the conventional dosage forms may be used, such
as tablets, lozenges, parenteral formulations, syrups, creams,
ointments, aerosol formulations, transdermal patches, transmucosal
patches and the like.
[0233] The compounds of the invention can be administered as the
single therapeutic agent in the treatment regimen, or the compounds
of the invention may be administered in combination with one
another or with other active agents, including additional immune
response modifiers, antivirals, antibiotics, etc.
[0234] The compounds of the invention have been shown to induce the
production of certain cytokines in experiments performed according
to the tests set forth below. These results indicate that the
compounds are useful as immune response modifiers that can modulate
the immune response in a number of different ways, rendering them
useful in the treatment of a variety of disorders.
[0235] Cytokines whose production may be induced by the
administration of compounds according to the invention generally
include interferon-.alpha. (IFN-.alpha.) and/or tumor necrosis
factor-.alpha. (TNF-.alpha.) as well as certain interleukins (IL).
Cytokines whose biosynthesis may be induced by compounds of the
invention include IFN-.alpha., TNF-.alpha., IL-1, IL-6, IL-10 and
IL-12, and a variety of other cytokines. Among other effects, these
and other cytokines can inhibit virus production and tumor cell
growth, making the compounds useful in the treatment of viral
diseases and tumors. Accordingly, the invention provides a method
of inducing cytokine biosynthesis in an animal comprising
administering an effective amount of a compound or composition of
the invention to the animal.
[0236] Certain compounds of the invention have been found to
preferentially induce the expression of IFN-.alpha. in a population
of hematopoietic cells such as PBMCs (peripheral blood mononuclear
cells) containing pDC2 cells (precursor dendritic cell-type 2)
without concomitant production of significant levels of
inflammatory cytokines.
[0237] In addition to the ability to induce the production of
cytokines, the compounds of the invention affect other aspects of
the innate immune response. For example, natural killer cell
activity may be stimulated, an effect that may be due to cytokine
induction. The compounds may also activate macrophages, which in
turn stimulates secretion of nitric oxide and the production of
additional cytokines. Further, the compounds may cause
proliferation and differentiation of B-lymphocytes.
[0238] Compounds of the invention also have an effect on the
acquired immune response. For example, although there is not
believed to be any direct effect on T cells or direct induction of
T cell cytokines, the production of the T helper type 1 (Th1)
cytokine IFN-.gamma. is induced indirectly and the production of
the T helper type 2 (Th2) cytokines IL4, IL-5 and IL-13 are
inhibited upon administration of the compounds. This activity means
that the compounds are useful in the treatment of diseases where
upregulation of the Th1 response and/or downregulation of the Th2
response is desired. In view of the ability of compounds of the
invention to inhibit the Th2 immune response, the compounds are
expected to be useful in the treatment of atopic diseases, e.g.,
atopic dermatitis, asthma, allergy, allergic rhinitis; systemic
lupus erythematosis; as a vaccine adjuvant for cell mediated
immunity; and possibly as a treatment for recurrent fungal diseases
and chlamydia.
[0239] The immune response modifying effects of the compounds make
them useful in the treatment of a wide variety of conditions.
Because of their ability to induce the production of cytokines such
as IFN-.alpha. and/or TNF-.alpha., the compounds are particularly
useful in the treatment of viral diseases and tumors. This
immunomodulating activity suggests that compounds of the invention
are useful in treating diseases such as, but not limited to, viral
diseases including genital warts; common warts; plantar warts;
Hepatitis B; Hepatitis C; Herpes Simplex Virus Type I and Type II;
molluscum contagiosum; varriola major; HIV; CMV; VZV; rhinovirus;
adenovirus; influenza; and para-influenza; intraepithelial
neoplasias such as cervical intraepithelial neoplasia; human
papillomavirus (HPV) and associated neoplasias; fungal diseases,
e.g. candida, aspergillus, and cryptococcal meningitis; neoplastic
diseases, e.g., basal cell carcinoma, hairy cell leukemia, Kaposi's
sarcoma, renal cell carcinoma, squamous cell carcinoma, myelogenous
leukemia, multiple myeloma, melanoma, non-Hodgkin's lymphoma,
cutaneous T-cell lymphoma, and other cancers; parasitic diseases,
e.g. pneumocystis carnii, cryptosporidiosis, histoplasmosis,
toxoplasmosis, trypanosome infection, and leishmaniasis; and
bacterial infections, e.g., tuberculosis, and mycobacterium avium.
Additional diseases or conditions that can be treated using the
compounds of the invention include actinic keratosis; eczema;
eosinophilia; essential thrombocythaemia; leprosy; multiple
sclerosis; Ommen's syndrome; discoid lupus; Bowen's disease;
Bowenoid papulosis; alopecia areata; the inhibition of Keloid
formation after surgery and other types of post-surgical scars. In
addition, these compounds could enhance or stimulate the healing of
wounds, including chronic wounds. The compounds may be useful for
treating the opportunistic infections and tumors that occur after
suppression of cell mediated immunity in, for example, transplant
patients, cancer patients and HIV patients.
[0240] An amount of a compound effective to induce cytokine
biosynthesis is an amount sufficient to cause one or more cell
types, such as monocytes, macrophages, dendritic cells and B-cells
to produce an amount of one or more cytokines such as, for example,
IFN-.alpha., TNF-.alpha., IL-1, IL-6, IL-10 and IL-12 that is
increased over the background level of such cytokines. The precise
amount will vary according to factors known in the art but is
expected to be a dose of about 100 ng/kg to about 50 mg/kg,
preferably about 10 .mu.g/kg to about 5 mg/kg. The invention also
provides a method of treating a viral infection in an animal and a
method of treating a neoplastic disease in an animal comprising
administering an effective amount of a compound or composition of
the invention to the animal. An amount effective to treat or
inhibit a viral infection is an amount that will cause a reduction
in one or more of the manifestations of viral infection, such as
viral lesions, viral load, rate of virus production, and mortality
as compared to untreated control animals. The precise amount will
vary according to factors known in the art but is expected to be a
dose of about 100 ng/kg to about 50 mg/kg, preferably about 10
.mu.g/kg to about 5 mg/kg. An amount of a compound effective to
treat a neoplastic condition is an amount that will cause a
reduction in tumor size or in the number of tumor foci. Again, the
precise amount will vary according to factors known in the art but
is expected to be a dose of about 100 ng/kg to about 50 mg/kg,
preferably about 10 .mu.g/kg to about 5 mg/kg.
[0241] The invention is further described by the following
examples, which are provided for illustration only and are not
intended to be limiting in any way.
[0242] In the examples below some of the compounds were purified
using semi-preparative HPLC. Two different methods were used and
they are described below.
[0243] Method A
[0244] This method used a A-100 Gilson-6 equipped with 900 Series
Intelligent Interface. The semi-prep HPLC fractions were analyzed
by LC-APCI/MS and the appropriate fractions were combined and
lyophilized to provide the trifluoroacetate salt of the desired
compound.
[0245] Column: column Microsorb C18, 21.4.times.250 mm, 8 micron
particle size, 60 .ANG. pore; flow rate: 10 mL/min.; gradient
elution from 2-95% B in 25 min., hold at 95% B for 5 min., where
A=0.1% trifluoroacetic acid/water and B=0.1% trifluoroacetic
acid/acetonitrile; peak detection at 254 nm for triggering fraction
collection.
[0246] Method B
[0247] This method used a Waters Fraction Lynx automated
purification system. The semi-prep HPLC fractions were analyzed
using a Micromass LC-TOFMS and the appropriate fractions were
combined and centrifuge evaporated to provide the trifluoroacetate
salt of the desired compound. The structure was confirmed by
.sup.1H NMR spectroscopy.
[0248] Column: Phenomenex Luna C18(2), 10.times.50 mm, 5 micron
particle size, 100 .ANG. pore; flow rate: 25 mL/min.; gradient
elution from 5-65% B in 4 min., then 65 to 95% B in 0.1 min, then
hold at 95% B for 0.4 min., where A=0.05% trifluoroacetic
acid/water and B=0.05% trifluoroacetic acid/acetonitrile; fraction
collection by mass-selective triggering.
EXAMPLE 1
1-[2-(2-aminoethoxy)ethyl]-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine
[0249] 14
[0250] Part A
[0251] A solution of 2-(2-aminoethoxy)ethanol (29.0 g, 0.276 mol)
in 180 mL of tetrahydrofuran (THF), under N.sub.2, was cooled to
0.degree. C. and treated with 140 mL of 2N NaOH solution. A
solution of di-tert-butyl dicarbonate (60.2 g, 0.276 mol) in 180 mL
of THF was then added dropwise over 1 h to the rapidly stirred
solution. The reaction mixture was then allowed to warm to room
temperature and was stirred an additional 18 h. The THF was then
removed under reduced pressure and the remaining aqueous slurry was
brought to pH 3 by addition of 150 mL of 1M H.sub.2SO.sub.4
solution. This was then extracted with ethyl acetate (300 mL, 100
mL) and the combined organic layers were washed with H.sub.2O
(2.times.) and brine. The organic portion was dried over
Na.sub.2SO.sub.4 and concentrated to give tert-butyl
2-(2-hydroxyethoxy)ethylcarbamate as a colorless oil (47.1 g).
[0252] Part B
[0253] A rapidly stirred solution of tert-butyl
2-(2-hydroxyethoxy)ethylca- rbamate (47.1 g, 0.230 mol) in 1 L of
anhydrous CH.sub.2Cl.sub.2 was cooled to 0.degree. C. under N.sub.2
and treated with triethylamine (48.0 mL, 0.345 mol).
Methanesulfonyl chloride (19.6 mL, 0.253 mol) was then added
dropwise over 30 min. The reaction mixture was then allowed to warm
to room temperature and was stirred an additional 22 h. The
reaction was quenched by addition of 500 mL saturated NaHCO.sub.3
solution and the organic layer was separated. The organic phase was
then washed with H.sub.2O (3.times.500 mL) and brine. The organic
portion was dried over Na.sub.2SO.sub.4 and concentrated to give
2-{2-[(tert-butoxycarbonyl)amin- o]ethoxy}ethyl methanesulfonate as
a brown oil (63.5 g).
[0254] Part C
[0255] A stirred solution of
2-{2-[(tert-butoxycarbonyl)amino]ethoxy}ethyl methanesulfonate
(63.5 g, 0.224 mol) in 400 mL of N,N-dimethylformamide (DMF) was
treated with NaN.sub.3 (16.1 g, 0.247 mol) and the reaction mixture
was heated to 90.degree. C. under N.sub.2. After 5 h, the solution
was cooled to room temperature and treated, with 500 mL of cold
H.sub.2O. The reaction mixture was then extracted with Et.sub.2O
(3.times.300 mL). The combined organic extracts were washed with
H.sub.2O (4.times.100 mL) and brine (2.times.100 mL). The organic
portion was dried over MgSO.sub.4 and concentrated to give 52.0 g
of tert-butyl 2-(2-azidoethoxy)ethylcarbamate as a light brown
oil.
[0256] Part D
[0257] A solution of tert-butyl 2-(2-azidoethoxy)ethylcarbamate
(47.0 g, 0.204 mol) in MeOH was treated with 4 g of 10% Pd on
carbon and shaken under H.sub.2 (3 Kg/cm.sup.2) for 24 h. The
solution was then filtered through a Celite pad and concentrated to
give 35.3 g of crude tert-butyl 2-(2-aminoethoxy)ethylcarbamate as
a colorless liquid that was used without further purification.
[0258] Part E
[0259] A stirred solution of 4-chloro-3-nitroquinoline (31.4 g,
0.151 mol) in 500 mL of anhydrous CH.sub.2Cl.sub.2, under N.sub.2,
was treated with triethylamine (43 mL, 0.308 mol) and tert-butyl
2-(2-aminoethoxy)ethylcar- bamate (0.151 mol). After stirring
overnight, the reaction mixture was washed with H.sub.2O
(2.times.300 mL) and brine (300 mL). The organic portion was dried
over Na.sub.2SO.sub.4 and concentrated to give a bright yellow
solid. Recrystallization from ethyl acetate/hexanes gave 43.6 g of
tert-butyl 2-{2-[(3-nitroquinolin-4-yl)amino]ethoxy}ethylcarbamate
as bright yellow crystals.
[0260] Part F
[0261] A solution of tert-butyl
2-{2-[(3-nitroquinolin-4-yl)amino]ethoxy}e- thylcarbamate (7.52 g,
20.0 mmol) in toluene was treated with 1.5 g of 5% Pt on carbon and
shaken under H2 (3 Kg/cm.sup.2) for 24 h. The solution was then
filtered through a Celite pad and concentrated to give 6.92 g of
crude tert-butyl
2-{2-[(3-aminoquinolin-4-yl)amino]ethoxy}ethylcarbamate as a yellow
syrup.
[0262] Part G
[0263] A solution of tert-butyl
2-{2-[(3-aminoquinolin-4-yl)amino]ethoxy}e- thylcarbamate (3.46 g,
10.0 mmol) in 50 mL of toluene was treated with
triethylorthovalerate (2.5 mL, 14.5 mmol) and the reaction mixture
was heated to reflux. A 25 mg portion of pyridinium hydrochloride
was then added and refluxing was continued for 4 h. The reaction
was then concentrated to dryness under reduced pressure. The
residue was dissolved in 50 mL of CH.sub.2Cl.sub.2 and washed with
saturated NaHCO.sub.3, H.sub.2O and brine. The organic portion was
dried over Na.sub.2SO.sub.4 and concetrated to give a green oil.
The green oil was dissolved in 50 mL of hot MeOH and treated with
activated charcoal. The hot solution was filtered and concentrated
to give 4.12 g of tert-butyl
2-[2-(2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]ethylcarbamate
as a yellow oil.
[0264] Part H
[0265] A solution of tert-butyl
2-[2-(2-butyl-1H-imidazo[4,5-c]quinolin-1--
yl)ethoxy]ethylcarbamate (4.12 g, 10.0 mmol) in 50 mL of
CH.sub.2Cl.sub.2 was treated with 3-chloroperoxybenzoic acid
(MCPBA, 77%, 2.5 g, 11.2 mmol). After stirring for 5 h, the
reaction mixture was treated with saturated NaHCO.sub.3 solution
and the layers were separated. The organic portion was washed with
H.sub.2O and brine then dried over Na.sub.2SO.sub.4 and
concentrated to give 3.68 g of tert-butyl
2-[2-(2-butyl-5-oxido-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]ethylcarbamat-
e as a light brown foam.
[0266] Part I
[0267] A solution of tert-butyl
2-[2-(2-butyl-5-oxido-1H-imidazo[4,5-c]qui-
nolin-1-yl)ethoxy]ethylcarbamate (3.68 g, 8.60 mmol) in 100 mL of
1,2-dichloroethane was heated to 80 .degree. C. and treated with 10
mL of concentrated NH40H solution. To the rapidly stirred solution
was added solid p-toluenesulfonyl chloride (1.87 g, 9.81 mmol) over
a 10 min. period. The reaction mixture was then sealed in a
pressure vessel and heating was continued for 2 h. The reaction
mixture was then cooled and treated with 100 mL of
CH.sub.2Cl.sub.2. The reaction mixture was then washed with
H.sub.2O, 1% Na.sub.2CO.sub.3 solution (3.times.) and brine. The
organic portion was dried over Na.sub.2SO.sub.4 and concentrated to
give 3.68 g of tert-butyl
2-[2-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-
-1-yl)ethoxy]ethylcarbamate as a light brown foam.
[0268] Part J
[0269] Tert-butyl
2-[2-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)eth-
oxy]ethylcarbamate (3.68 g, 8.60 mmol) was suspended in 20 mL of 2M
HCl in EtOH and the mixture was heated to reflux with stirring.
After 3 h, the reaction mixture was concentrated to give a solid.
The solid was triturated with hot EtOH (50 mL) and filtered to give
2.90 g of the product as the hydrochloride salt. The free base was
made by dissolving the hydrochloride salt in 50 mL of H.sub.2O and
treating with 5 mL of concentrated NH.sub.4OH. The aqueous
suspension was extracted with CH.sub.2Cl.sub.2 (3.times.50 mL). The
combined organic layers were dried over Na.sub.2SO.sub.4 and
concentrated to give 1-[2-(2-aminoethoxy)ethyl]-
-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine as a tan powder. MS 328
(M+H).sup.+;
[0270] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.95 (d, J=8.3 Hz,
1 H); 7.83 (d, J=8.4 Hz, 1 H); 7.50 (m, 1 H); 7.30 (m, 1 H); 5.41
(s, 2 H); 4.69 (t, J=5.6 Hz, 2 H); 3.93 (t, J=5.6 Hz, 2 H); 3.39
(t, J=5.1 Hz, 2 H); 2.97 (t, J=7.9 Hz, 2 H); 2.76 (t,J 5.1 Hz, 2
H); 1.89 (m, 2 H); 1.52 (m, 2 H); 1.26 (br s, 2 H); 1.01 (t, J=7.3
Hz, 3 H).
EXAMPLE 2
1-[2-(2-aminoethoxy)ethyl]-1H-imidazo[4,5-c]quinolin-4-amine
[0271] 15
[0272] Part A
[0273] A solution of tert-butyl
2-{2-[(3-aminoquinolin-4-yl)amino]ethoxy)e- thylcarbamate (6.92 g,
20.0 mmol) in 100 mL of toluene was treated with
triethylorthoformate (4.65 mL, 28.0 mmol) and the reaction mixture
was heated to reflux. A 100 mg portion of pyridinium hydrochloride
was then added and refluxing was continued for 2 h. The reaction
was then concentrated to dryness under reduced pressure. The
residue was dissolved in 200 mL of CH.sub.2Cl.sub.2 and washed with
saturated NaHCO.sub.3, H.sub.2O and brine. The organic portion was
dried over Na.sub.2SO.sub.4 and concentrated to give a green oil.
The green oil was dissolved in 200 mL of hot MeOH and treated with
10 g of activated charcoal. The hot solution was filtered and
concentrated to give 5.25 g of tert-butyl
2-[2-(1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]ethylcarbamate as a
light yellow syrup.
[0274] Part B
[0275] A solution of tert-butyl
2-[2-(1H-imidazo[4,5-c]quinolin-1-yl)ethox- y]ethylcarbamate (5.25
g, 14.7 mmol) in 200 mL of CH.sub.2Cl.sub.2 was treated with MCPBA
(77%, 3.63 g, 16.3 mmol). After stirring overnight, the reaction
mixture was treated with saturated NaHCO.sub.3 solution and the
layers were separated. The organic portion was washed with H.sub.2O
and brine then dried over Na.sub.2SO.sub.4 and concentrated to give
4.60 g of tert-butyl
2-[2-(5-oxido-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]ethyl-
carbamate as a light brown foam.
[0276] Part C
[0277] A solution of tert-butyl
2-[2-(5-oxido-1H-imidazo[4,5-c]quinolin-1--
yl)ethoxy]ethylcarbamate (4.60 g, 12.4 mmol) in 150 mL of
1,2-dichloroethane was heated to 80.degree. C. and treated with 10
mL of concentrated NH.sub.4OH solution. To the rapidly stirred
solution was added solid p-toluenesulfonyl chloride (2.71 g, 14.2
mmol) over a 10 min period. The reaction mixture was treated with
an additional 2 mL of concentrated NH4OH solution and then sealed
in a pressure vessel and heating was continued for 3 h. The
reaction mixture was then cooled and treated with 100 mL of
CH.sub.2Cl.sub.2. The reaction mixture was then washed with
H.sub.2O, 1% Na.sub.2CO.sub.3 solution (3.times.) and brine. The
organic portion was dried over Na.sub.2SO.sub.4 and concentrated to
give 4.56 g of tert-butyl
2-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)et-
hoxy]ethylcarbamate as a light brown foam.
[0278] Part D
[0279] Tert-butyl
2-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]ethy-
lcarbamate (4.56 g, 12.3 mmol) was dissolved in 100 mL of EtOH and
treated with 30 mL of 2M HCl in EtOH and the mixture was heated to
reflux with stirring. After 3 h, the reaction mixture was
concentrated to give a solid. The solid was triturated with hot
EtOH (100 mL) and filtered to give the product as the hydrochloride
salt. The free base was made by dissolving the hydrochloride salt
in 50 mL of H.sub.2O and treating with 5 mL of concentrated
NH.sub.4OH. The aqueous suspension was extracted with
CH.sub.2Cl.sub.2 (5.times.50 mL). The combined organic layers were
dried over Na.sub.2SO.sub.4 and concentrated to give 1.35 g of
1-[2-(2-aminoethoxy)ethyl]-1H-imidazo[4,5-c]quinolin-4-amine as a
tan powder.
[0280] MS 272 (M+H).sup.+;
[0281] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.98 (d, J=8.2 Hz,
1 H); 7.88 (s, 1 H); 7.84 (d, J=8.4 Hz, 1 H); 7.54 (m, 1 H); 7.32
(m, 1 H); 5.43 (s, 2 H); 4.74 (t, J=5.2 Hz, 2 H); 3.97 (t, J=5.2
Hz, 2 H); 3.42 (t, J=5.1 Hz, 2 H); 2.78 (t, J=5.1 Hz, 2 H); 1.10
(br s, 2 H).
EXAMPLE 3
1-[2-(2-aminoethoxy)ethyl]-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-4--
amine
[0282] 16
[0283] Part A
[0284] A solution of tert-butyl
2-{2-[(3-aminoquinolin-4-yl)amino]ethoxy}e- thylcarbamate (10.2 g,
29.5 mmol) in 250 mL of anhydrous CH.sub.2Cl.sub.2 was cooled to
0.degree. C. and treated with triethylamine (4.18 mL, 30.0 mmol).
Methoxypropionyl chloride (3.30 mL, 30.3 mmol) was then added
dropwise over 5 min. The reaction was then warmed to room
temperature and stirring was continued for 1 h. The reaction
mixture was then concentrated under reduced pressure to give an
orange solid. This was dissolved in 250 mL of EtOH and 12.5 mL of
triethylamine was added. The mixture was heated to reflux and
stirred under N.sub.2 overnight. The reaction was then concentrated
to dryness under reduced pressure and treated with 300 mL of
Et.sub.2O. The mixture was then filtered and the filtrate was
concentrated under reduced pressure to give a brown solid. The
solid was dissolved in 200 mL of hot MeOH and treated with
activated charcoal. The hot solution was filtered and concentrated
to give 11.1 g of tert-butyl
2-{2-[2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]eth-
oxy}ethylcarbamate as a yellow syrup.
[0285] Part B
[0286] A solution of tert-butyl
2-{2-[2-(2-methoxyethyl)-1H-imidazo[4,5-c]-
quinolin-1-yl]ethoxy}ethylcarbamate (10.22 g, 24.7 mmol) in 250 mL
of CHCl.sub.3 was treated with MCPBA (77%, 9.12 g, 40.8 mmol).
After stirring 30 min, the reaction mixture was washed with 1 %
Na.sub.2CO.sub.3 solution (2.times.75 mL) and brine. The organic
layer was then dried over Na.sub.2SO.sub.4 and concentrated to give
10.6 g of tert-butyl
2-{2-[2-(2-methoxyethyl)-5-oxido-1H-imidazo[4,5-c]quinolin-1-y-
l]ethoxy}ethylcarbamate as an orange foam that was used without
further purification.
[0287] Part C
[0288] A solution of tert-butyl
2-{2-[2-(2-methoxyethyl)-5-oxido-1H-imidaz-
o[4,5-c]quinolin-1-yl]ethoxy}ethylcarbamate (10.6 g, 24.6 mmol) in
100 mL of 1,2-dichloroethane was heated to 60.degree. C. and
treated with 10 mL of concentrated NH.sub.4OH solution. To the
rapidly stirred solution was added solid p-toluenesulfonyl chloride
(7.05 g, 37.0 mmol) over a 10 min period. The reaction mixture was
treated with an additional 1 mL concentrated NH.sub.4OH solution
and then sealed in a pressure vessel and heating was continued for
2 h. The reaction mixture was then cooled and treated with 100 mL
of CHCl.sub.3. The reaction mixture was then washed with H.sub.2O,
1% Na.sub.2CO.sub.3 solution (2.times.) and brine. The organic
portion was dried over Na.sub.2SO.sub.4 and concentrated to give
10.6 g of tert-butyl
2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]qu- inolin
1-yl]ethoxy}ethylcarbamate as a brown foam.
[0289] Part D
[0290] Tert-butyl
2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinol-
in-1-yl]ethoxy}ethylcarbamate (10.6 g, 24.6 mmol) was treated with
75 mL of 2M HCl in EtOH and the mixture was heated to reflux with
stirring. After 1.5 h, the reaction mixture was cooled and filtered
to give a gummy solid. The solid was washed EtOH and Et.sub.2O and
dried under vacuum to give the hydrochloride salt as a light brown
solid. The free base was made by dissolving the hydrochloride salt
in 50 mL of H.sub.2O and treating with 10% NaOH solution. The
aqueous suspension was then concentrated to dryness and the residue
was treated with CHCl.sub.3. The resulting salts were removed by
filtration and the filtrate was concentrated to give 3.82 g of
1-[2-(2-aminoethoxy)ethyl]-2-(2-methoxyeth-
yl)-1H-imidazo[4,5-c]quinolin-4-amine as a tan powder.
[0291] MS 330 (M+H).sup.+; .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 8.10 (d, J=8.1 Hz, 1 H); 7.66 (d, J=8.2 Hz, 1 H); 7.40 (m,
1 H); 7.25 (m, 1 H); 6.88 (br s, 2 H); 4.78 (t, J=5.4 Hz, 2 H);
3.89 (t, J=4.8 Hz, 2 H); 3.84 (t, J=6.9 Hz, 2 H); 3.54 (t, J=5.4
Hz, 2 H); 3.31 (s, 3 H); 3.23 (t, J=6.6 Hz, 2 H); 2.88 (t, J=5.3
Hz, 2 H).
EXAMPLE 4
N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}-
ethyl)benzamide
[0292] 17
[0293] 1-[2-(2-Aminoethoxy)ethyl]-2-(2-methoxyethyl)-I
H-imidazo[4,5-c]quinolin4-amine (750 mg, 2.28 mmol) was dissolved
in 35 mL of anhydrous CH.sub.2Cl.sub.2 and cooled to 0.degree. C.
under N.sub.2. To the stirred solution were added Et.sub.3N (0.35
mL, 2.50 mmol) and benzoyl chloride (260 .mu.L, 2.28 mmol) and the
reaction was allowed to warm to room temperature over 2.5 h. The
reaction mixture was then quenched by addition of saturated
NaHCO.sub.3 solution (30 mL) and CH.sub.2Cl.sub.2 (30 mL). The
organic layer was separated and washed with H.sub.2O and brine,
dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure
to give a tan foam. Mass spectral analysis showed the presence of
some bis-amide in addition to the desired product. The tan foam was
treated with 1N aqueous HCl solution (50 mL) at 100.degree. C. for
5 h. HPLC analysis showed that all of the bis-amide had been
converted to the desired product. The reaction was cooled to room
temperature and treated with 10% NaOH until the pH.about.11. The
mixture was extracted with CHCl.sub.3 (3.times.30 mL). The combined
organic extracts were washed with H.sub.2O and brine, dried over
Na.sub.2SO.sub.4 and concentrated under reduced pressure to give a
yellow solid. Purification by column chromatography (SiO.sub.2,
5-10% MeOH/CHCl.sub.3) gave 100 mg of
N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quino-
lin-1-yl]ethoxy}ethyl)benzamide as a white powder. m.p.
184-187.degree. C.;
[0294] MS 434 (M+H).sup.+;
[0295] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.40 (m, 1 H);
8.06 (d, J=8.3 Hz, 1 H); 7.76-7.74 (m, 2 H); 7.60 (d, J=7.8 Hz, 1
H); 7.54-7.37 (m, 4 H); 7.19 (t, J=7.3 Hz, 1 H); 6.48 (s, 2 H);
4.79-4.72 (m, 2 H); 3.91-3.84 (m, 2 H); 3.78 (t, J=6.9 Hz, 2 H);
3.48 (t, J=5.5 Hz. 2 H); 3.25 (s, 3 H); 3.20 (t, J=6.3 Hz, 2
H);
[0296] .sup.3C (75 MHz, DMSO-d.sub.6) .delta. 166.7, 152.0, 151.9,
145.2, 134.8, 132.7, 131.4, 128.6, 127.4, 126.7, 121.4, 120.5,
115.1, 70.4, 69.4, 69.2, 58.4, 45.5, 27.6.
EXAMPLE 5
1-[2-(2-aminoethoxy)ethyl]-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-imidaz-
o[4,5-c]quinolin-4-amine
[0297] 18
[0298]
1-[2-(2-Aminoethoxy)ethyl]-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quin-
olin-4-amine (10.0 g, 27.3 mmol) was dissolved in 50 mL of
trifluoroacetic acid and treated with PtO.sub.2 (1.0 g). The
reaction mixture was shaken under H.sub.2 (3 Kg/cm.sup.2). After 4
d, an additional 0.5 g of PtO.sub.2 was added and hydrogenation was
continued for an additional 3 d. The reaction was then filtered
through Celite and concentrated under reduced pressure to give a
brown oil. The yellow oil was dissolved in 200 mL of H.sub.2O then
made basic (pH.about.1) by addition of 10% NaOH solution. This was
then extracted with CHCl.sub.3 (5.times.75 mL) and the combined
organic layers were dried over Na.sub.2SO.sub.4 and concentrated to
give 5.17 g of
1-[2-(2-aminoethoxy)ethyl]-2-(2-methoxyethyl)-6,7,8,9-t-
etrahydro-1H-imidazo[4,5-c]quinolin-4-amine as a tan solid.
[0299] MS 334 (M+H).sup.+;
[0300] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 5.19 (s, 2 H);
4.49 (t, J=5.4 Hz, 2 H); 3.84 (t, J=6.6 Hz, 2 H); 3.71 (t, J=5.4
Hz, 2 H), 3.36 (t, J=5.2 Hz, 2 H); 3.51 (s, 3 H); 3.15 (t, J=6.6
Hz, 2 H); 2.95 (m, 2 H); 2.82 (m, 2 H); 2.76 (t, J=5.1 Hz, 2 H);
1.84 (m, 4 H), 1.47 (br s, 2 H).
EXAMPLE 6
N-(2-{2-[4-amino-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]qu-
inolin-1-yl]ethoxy}ethyl)benzamide
[0301] 19
[0302]
1-[2-(2-Aminoethoxy)ethyl]-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-
-imidazo[4,5-c]quinolin-4-amine (1.00 g, 3.00 mmol) was dissolved
in 30 mL of anhydrous CH.sub.2Cl.sub.2 and cooled to 0.degree. C.
under N.sub.2. To the stirred solution were added Et.sub.3N (0.84
mL, 6.00 mmol) and benzoyl chloride (348 .mu.L, 3.00 mmol) and the
reaction was allowed to warm to room temperature overnight. The
reaction mixture was then quenched by addition of saturated
NaHCO.sub.3 solution (30 mL). The organic layer was separated and
washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and
concentrated under reduced pressure to give a yellow oil. The oil
was dissolved in a minimum amount of hot MeOH and then treated with
Et.sub.2O (50 mL) which caused a white percipitate to form. The
solid was isolated by filtration and dried under vacuum to give 476
mg of
N-(2-{2-[4-amino-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-imida-
zo[4,5-c]quinolin 1-yl]ethoxy}ethyl)benzamide as a white powder.
m.p. 141-143.degree. C.;
[0303] MS 438 (M+H).sup.+;
[0304] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.36 (t, J=5.4
Hz, 1 H); 7.78-7.76 (m, 2 H); 7.54-7.42 (m, 3 H); 5.68 (s, 2 H);
4.43 (t, J=5.4 Hz, 2 H); 3.75-3.69 (m, 4 H); 3.48 (t, J=6.0 Hz, 2
H); 3.37 (t, J=5.5 Hz, 2 H); 3.24 (s, 3 H); 3.07 (t, J=6.9 Hz, 2
H); 2.91 (m, 2 H); 2.63 (m, 2 H); 1.70 (m, 4 H);
[0305] .sup.13C (75 MHz, DMSO-d.sub.6) .delta. 166.7, 151.3, 149.3,
146.2, 138.5, 134.8, 131.4, 128.6, 127.5, 124.9, 105.6, 70.5, 70.5,
69.3, 58.4, 44.6, 32.7, 27.6, 23.8, 23.0, 23.0.
[0306] Anal. Calcd for C.sub.24H.sub.31N.sub.5O.sub.3: % C, 65.88;
% H, 7.14; % N, 16.01. Found: % C, 65.55; % H, 7.15; % N,
15.87.
EXAMPLE 7
2-(2-methoxyethyl)-1-(2-[2-(methylamino)ethoxy]ethyl}-1H-imidazo[4,5-c]qui-
nolin-4-amine
[0307] 20
[0308] Part A
[0309] Sodium hydride (60% oil dispersion, 9.1 g, 228 mmol) was
placed in a round bottom flask and washed with hexanes (3.times.)
under N.sub.2. The dried sodium hydride was treated with 800 mL of
anhydrous THF. A solution of tert-butyl
2-(2-azidoethoxy)ethylcarbamate (41.9 g, 182 mmol) in 200 mL of THF
was then added to the stirred sodium hydride solution over 40 min.
After addition was complete, the reaction was stirred an additional
20 min followed by addition of methyl iodide (13.6 mL, 218 mmol).
After stirring overnight, the reaction was quenched with 300 mL of
saturated NaHCO.sub.3 solution. The reaction mixture was then
treated with 200 mL of H.sub.2O and 1 L of Et.sub.2O. The organic
phase was separated and washed with H.sub.2O and brine. The organic
portion was then dried over MgSO.sub.4 and concentrated under
reduced pressure to give 41.9 g of tert-butyl
2-(2-azidoethoxy)ethyl(methyl)carbamate as a yellow liquid.
[0310] Part B
[0311] A solution tert-butyl
2-(2-azidoethoxy)ethyl(methyl)carbamate (41.9 g, 170 mmol) in 600
mL of MeOH was treated with 2.5 g of 10% Pd on carbon and shaken
under H.sub.2 (3 Kg/cm.sup.2) for 24 h. The solution was then
filtered through a Celite pad and concentrated to give 37.2 g of
crude tert-butyl 2-(2-aminoethoxy)ethyl(methyl)carbamate as a light
yellow liquid.
[0312] Part C
[0313] A stirred solution of 4-chloro-3-nitroquinoline (32.3 g, 155
mmol) in 400 mL of anhydrous CH.sub.2Cl.sub.2, under N.sub.2, was
treated with triethylamine (43.1 mL, 310 mmol) and tert-butyl
2-(2-aminoethoxy)ethyl(m- ethyl)carbamate (37.2 g, 171 mmol). After
stirring overnight, the reaction mixture was washed with H.sub.2O
(2.times.300 mL) and brine (300 mL). The organic portion was dried
over Na.sub.2SO.sub.4 and concentrated to give a brown oil. Column
chromatography (SiO.sub.2, 33% ethyl acetate/hexanes-67% ethyl
acetate/hexanes) gave 46.7 g of tert-butyl
methyl(2-{2-[(3-nitroquinolin-4-yl)amino]ethoxy}ethyl)carbamate as
a yellow solid.
[0314] Part D
[0315] A solution of tert-butyl
methyl(2-{2-[(3-nitroquinolin-4-yl)amino]e- thoxy}ethyl)carbamate
(6.56 g, 16.8 mmol) in 75 mL of toluene was treated with 0.5 g of
5% Pt on carbon and shaken under H.sub.2 (3 Kg/cm.sup.2) for 24 h.
The solution was then filtered through a Celite pad and
concentrated to give 6.8 g of crude tert-butyl
2-{2-[(3-aminoquinolin-4-y- l)amino]ethoxy}ethyl(methyl)carbamate
as an orange syrup which was carried on without further
purification.
[0316] Part E
[0317] A solution of tert-butyl
2-{2-[(3-aminoquinolin-4-yl)amino]ethoxy}e- thyl(methyl)carbamate
(6.05 g, 16.8 mmol) in 200 mL of anhydrous CH.sub.2Cl.sub.2 was
cooled to 0.degree. C. and treated with triethylamine (2.40 mL,
17.2 mmol). Methoxypropionyl chloride (1.72 mL, 17.2 mmol) was then
added dropwise over 5 min. The reaction was then warmed to room
temperature and stirring was continued for 3 h. The reaction
mixture was then concentrated under reduced pressure to give an
orange solid. This was dissolved in 200 mL of EtOH and 7.2 mL of
triethylamine was added. The mixture was heated to reflux and
stirred under N.sub.2 overnight. The reaction was then concentrated
to dryness under reduced pressure and treated with 300 mL of
Et.sub.2O. The mixture was then filtered and the filtrate was
concentrated under reduced pressure to give a brown solid. This was
dissolved in 300 mL of CH.sub.2Cl.sub.2 and washed with H.sub.2O
and brine. The organic portion was dried over Na.sub.2SO.sub.4 and
concentrated under reduced pressure to give a brown oil. The oil
was dissolved in 100 mL of hot MeOH and treated with activated
charcoal. The hot solution was filtered and concentrated to give
7.20 g of tert-butyl 2-{2-[2-(2-methoxyethyl)-1H-imi-
dazo[4,5-c]quinolin-1-yl]ethoxy}ethyl(methyl)carbamate as a yellow
syrup.
[0318] Part F
[0319] A solution of tert-butyl
2-{2-[2-(2-methoxyethyl)-1H-imidazo[4,5-c]-
quinolin-1-yl]ethoxy}ethyl(methyl)carbamate (7.20 g, 16.8 mmol) in
200 mL of CH.sub.2Cl.sub.2 was treated with MCPBA (77%, 4.32 g,
19.3 mmol). After stirring 6 h, the reaction mixture was treated
with saturated NaHCO.sub.3 solution and the layers were separated.
The organic portion was washed with H.sub.2O and brine then dried
over Na.sub.2SO.sub.4 and concentrated to give 7.05 g of tert-butyl
2-{2-[2-(2-methoxyethyl)-5-oxid-
o-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl(methyl)carbamate as a
light brown solid.
[0320] Part G
[0321] A solution of tert-butyl
2-{2-[2-(2-methoxyethyl)-5-oxido-1H-imidaz-
o[4,5-c]quinolin-1-yl]ethoxy}ethyl(methyl)carbamate (7.05 g, 15.9
mmol) in 100 mL of 1,2-dichloroethane was heated to 80.degree. C.
and treated with 5 mL of concentrated NH.sub.4OH solution. To the
rapidly stirred solution was added solid p-toluenesulfonyl chloride
(3.33 g, 17.5 mmol) over a 10 min period. The reaction mixture was
treated with an additional 5 mL concentrated NH.sub.4OH solution
and then sealed in a pressure vessel and heating was continued for
4 h. The reaction mixture was then cooled and treated with 100 mL
of CH.sub.2Cl.sub.2. The reaction mixture was then washed with
H.sub.2O, 1% Na.sub.2CO.sub.3 solution (3.times.) and brine. The
organic portion was dried over Na.sub.2SO.sub.4 and concentrated to
give 6.50 g of tert-butyl
2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-
-c]quinolin-1-yl]ethoxy}ethyl(methyl)carbamate as a brown oil
[0322] Part H
[0323] Tert-butyl
2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinol-
in-1-yl]ethoxy}ethyl(methyl)carbamate (6.50 g, 14.7 mmol) was
dissolved in 100 mL of EtOH and treated with 20 mL of 2M HCl in
EtOH and the mixture was heated to reflux with stirring. After 6 h,
the reaction mixture was cooled and filtered to give a gummy solid.
The solid was washed with EtOH and Et.sub.2O and dried under vacuum
to give the hydrochloride salt as a light brown powder. The free
base was made by dissolving the hydrochloride salt in 50 mL of
H.sub.2O and treating with 5 mL of concentrated NH.sub.4OH. The
aqueous suspension was extracted with CH.sub.2Cl.sub.2 (5.times.50
mL). The combined organic layers were dried over Na.sub.2SO.sub.4
and concentrated to give 3.93 g of
2-(2-methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-1H-imidazo[4,5-c]qu-
inolin-4-amine as a tan powder.
[0324] MS 344 (M+H).sup.+;
[0325] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.07 (d, J=7.7
Hz, 1 H); 7.62 (dd, J=1.0, 8.3 Hz, 1 H); 7.42 (ddd, J=1.0, 7.1, 8.2
Hz, 1 H); 7.22 (ddd, J=1.1, 7.1, 8.2 Hz, 1 H); 6.49 (s, 2 H); 4.75
(t, J=5.1 Hz, 2 H); 3.83 (t, J=6.8 Hz, 4 H); 3.35 (t, J=5.6 Hz, 2
H); 3.30 (s, 3 H); 3.21 (t, J=6.9 Hz, 2 H); 2.45 (t, J=5.6 Hz, 2
H); 2.12 (s, 3 H).
EXAMPLE 8
N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}-
ethyl)-N-methylbenzamide
[0326] 21
[0327]
2-(2-Methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-1H-imidazo[4,-
5-c]quinolin4-amine (1.00 g, 2.92 mmol) was dissolved in 30 mL of
anhydrous CH.sub.2Cl.sub.2 and cooled to 0.degree. C. under
N.sub.2. To the stirred solution were added Et.sub.3N (0.81 mL,
5.81 mmol) and benzoyl chloride (340 .mu.L, 2.92 mmol) and the
reaction was allowed to warm to room temperature overnight. The
reaction mixture was then quenched by addition of saturated
NaHCO.sub.3 solution (30 mL) and CH.sub.2Cl.sub.2 (30 mL). The
organic layer was separated and washed with H.sub.2O and brine,
dried over Na.sub.2SO.sub.4 and concentrated under reduced
pressure. Purification by column chromatography (SiO.sub.2, 3%
MeOH/CHCl.sub.3 saturated with aqueous NH.sub.4OH) gave the product
as a colorless foam. Crystallization from PrOAc and hexanes gave
540 mg of
N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy-
}ethyl)-N-methylbenzamide as a white powder. m.p. 93.5-97.0.degree.
C.;
[0328] MS 448 (M+H).sup.+;
[0329] .sup.1H NMR (500 MHz, DMSO-d.sub.6, 60.degree. C.) .delta.
8.04 (d, J=7.7 Hz, 1 H); 7.63 (dd, J=0.9, 8.2 Hz, 1 H); 7.42-7.33
(m, 4 H); 7.23-7.19 (m, 3 H); 6.24 (s, 2 H); 4.74 (m, 2 H); 3.86
(m, 2 H); 3.82 (t, J=6.8 Hz, 2 H); 3.51 (m, 2 H); 3.40 (m, 2 H);
3.29 (s, 3 H); 3.18 (t, J=6.7 Hz, 2 H); 2.75 (br s, 3 H);
[0330] .sup.13C NMR (125 MHz, DMSO-d.sub.6, 60.degree. C.) .delta.
152.0, 151.9, 145.3, 137.1, 132.8, 131.3, 129.4, 128.5, 127.0,
126.9, 126.8, 126.6, 121.4, 120.4, 115.3, 70.5, 69.5, 68.8,
58.4,45.5, 27.8.
[0331] Anal. Calcd for C.sub.25H.sub.29N.sub.5O.sub.3: % C, 67.09;
% H, 6.53; % N, 15.65. Found: % C, 67.08; % H, 6.56; % N, 15.58
EXAMPLE 9
2-(2-methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-6,7,8,9-tetrahydro-1-
H-imidazo[4,5-c]quinolin-4-amine
[0332] 22
[0333]
2-(2-Methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-1H-imidazo[4,-
5-c]quinolin4-amine (4.22 g, 12.3 mmol) was dissolved in 25 mL of
trifluoroacetic acid and treated with PtO.sub.2 (0.5 g). The
reaction mixture was shaken under H.sub.2 (3 Kg/cm.sup.2). After 4
d, an additional 0.5 g of PtO.sub.2 was added and hydrogenation was
continued for an additional 3 d. The reaction was then filtered
through Celite and concentrated under reduced pressure to give a
yellow oil. The yellow oil was dissolved in 50 mL of H.sub.2O and
extracted with 50 mL of CHCl.sub.3. The organic portion was removed
and discarded. The aqueous portion was then made basic
(pH.about.12) by addition of 10% NaOH solution. This was then
extracted with CHCl.sub.3 (6.times.50 mL) and the combined organic
layers were dried over Na.sub.2SO.sub.4 and concentrated to a brown
oil. The brown oil was dissolved in 100 mL of hot MeOH and treated
with 1 g of activated charcoal. The hot solution was filtered
through Celite and concentrated to dryness. The resulting gummy
solid was concentrated several times with Et.sub.2O to give 3.19 g
of
2-(2-methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-6,7,8,9-tetrahydro--
1H-imidazo[4,5-c]quinolin-4-amine as an off-white powder.
[0334] MS 348 (M+H).sup.+;
[0335] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.84 (s, 2 H);
4.48 (t, J=5.7 Hz, 2 H); 3.84 (t, J=6.7 Hz, 2 H); 3.70 (t, J=5.7
Hz, 2 H); 3.46 (t, J=5.1 Hz, 2 H); 3.36 (s, 3 H); 3.14 (t, J=6.7
Hz, 2 H); 2.96 (m, 2 H); 2.83 (m, 2 H); 2.65 (t, J=5.1 Hz, 2 H);
2.36 (s, 3 H); 1.85 (m, 4 H).
EXAMPLE 10
N-(2-{2-[4-amino-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]qu-
inolin-1-yl]ethoxy}ethyl)-N-methylbenzamide
[0336] 23
[0337]
2-(2-Methoxyethyl)-1-{2-[2-(methylamino)ethoxy]ethyl}-6,7,8,9-tetra-
hydro-1H-imidazo[4,5-c]quinolin-4-amine (750 mg, 2.16 mmol) was
dissolved in 20 mL of anhydrous CH.sub.2Cl.sub.2 and cooled to
0.degree. C. under N.sub.2. To the stirred solution were added
Et.sub.3N (0.60 mL, 4.32 mmol) and benzoyl chloride (250 .mu.L,
2.16 mmol) and the reaction was allowed to warm to room temperature
overnight. The reaction mixture was then quenched by addition of
saturated NaHCO.sub.3 solution (30 mL) and CH.sub.2Cl.sub.2 (30
mL). The organic layer was separated and washed with H.sub.2O
(3.times.) and brine, dried over Na.sub.2SO.sub.4 and concentrated
under reduced pressure. Purification by column chromatography
(SiO.sub.2, 3% MeOH/CHCl.sub.3 saturated with aqueous NH.sub.4OH)
gave the product as a colorless foam. The foam was concentrated
from iso-propyl alcohol to give an syrup which solidified upon
standing. The solid was dried under vacuum to give the 408 mg of
N-(2-{2-[4-amino-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]q-
uinolin-1-yl]ethoxy}ethyl)-N-methylbenzamide as an off-white
powder.
[0338] m.p 83.0-87.0.degree. C.;
[0339] MS 452 (M+H).sup.+;
[0340] .sup.1H NMR (500 MHz, DMSO-d.sub.6, 60.degree. C.) .delta.
7.37 (m, 3 H); 7.23 (m, 2 H); 5.46 (s, 2 H); 4.43 (m, 2 H); 3.76
(t, J=6.8 Hz, 2 H); 3.68 (m, 2 H); 3.50 (m, 2 H); 3.42 (m, 2 H);
3.27 (s, 3 H); 3.05 (t, J=6.4 Hz, 2 H); 2.92 (m, 2 H); 2.80 (s, 3
H); 2.65 (m, 2 H); 1.74 (m, 4 H);
[0341] .sup.13C NMR (125 MHz, DMSO-d.sub.6, 60.degree. C.) .delta.
150.5, 148.5, 145.8, 137.9, 136.4, 128.7, 127.8, 126.3, 124.5,
105.1, 70.1, 69.8, 68.0, 57.7, 44.0, 32.1, 27.1, 23.2, 22.4,
22.4
[0342] Anal. Calcd for C.sub.25H.sub.33N.sub.5O.sub.3.0.30
C.sub.3H.sub.8O: % C, 66.24; % H, 7.60; % N, 14.91. Found: % C,
65.86; % H, 7.81; % N, 15.10.
EXAMPLE 11
1-{1-[(2-piperidin-4-ylethoxy)methyl]propyl}-1H-imidazo[4,5-clquinolin4-am-
ine
[0343] 24
[0344] Part A
[0345] Using the general method of Part A of Example 1,
4-piperidineethanol (10 g, 77.4 mmol) was reacted with
di-tert-butyl dicarbonate (17.7 g, 81.3 mmol) to provide 13.1 g of
tert-butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate as a clear
oil.
[0346] Part B
[0347] Iodine (7.97 g) was added in three portions to a solution of
imidazole (3.89 g, 57.1 mmol) and triphenylphosphine (14.98 g, 57.1
mmol) in dichloromethane (350 mL). After 5 minutes a solution of
the material from Part A in dichloromethane (70 mL) was added. The
reaction mixture was stirred at ambient temperature overnight. More
iodine (7.97 g) was added and the reaction was stirred at ambient
temperature for 1 hr. The reaction mixture was washed with
saturated sodium thiosulfate (2.times.) and brine, dried over
sodium sulfate, filtered and then concentrated under reduced
pressure to provide an oily residue. The residue was purified by
column chromatography (silica gel eluting with 20% ethyl acetate in
hexanes) to provide 15.52 g of tert-butyl
4-(2-iodoethyl)piperidine-1-carboxylate as a pale yellow oil.
[0348] Part C
[0349] Under a nitrogen atmosphere,
2-(1H-imidazo[4,5-c]quinolin-1-yl)buta- n-1-ol (6.5 g, 26.9 mmol)
was added in three portions to a suspension of sodium hydride (1.4
g of 60%, 35.0 mmol) in anhydrous N,N-dimethylformamide. The
reaction mixture was allowed to stir for 45 minutes by which time
gas evolution had ceased. Tert-butyl
4-(2-iodoethyl)piperidine-1-carboxylate (10.05 g, 29.6 mmol) was
added dropwise over a period of 15 minutes. The reaction mixture
was allowed to stir at ambient temperature for 2.5 hrs; then it was
heated to 100.degree. C. and stirred overnight. Analysis by HPLC
showed that the reaction was about 35% complete. Saturated ammonium
chloride solution was added, the resulting mixture was allowed to
stir for 20 minutes and then it was extracted with ethyl acetate
(2.times.). The ethyl acetate extracts were washed with water
(2.times.) and then with brine, combined, dried over sodium
sulfate, filtered and then concentrated under reduced pressure to
provide a brown oil. The oil was purified by column chromatography
(silica gel eluting sequentially with 30% ethyl acetate in hexanes,
50% ethyl acetate in hexanes, and ethyl acetate) to provide 2.2 g
of tert-butyl
4-{2-[2-(1H-imidazo[4,5-c]quinolin-1-yl)butoxy]ethyl}pipe-
ridine-1-carboxylate.
[0350] Part D
[0351] Using the general method of Example 1 Part H, the material
from Part C was oxidized to provide tert-butyl
4-{2-[2-(5-oxido-1H-imidazo[4,5-
-c]quinolin-1-yl)butoxy]ethyl}piperidine-1-carboxylate as an
oil.
[0352] Part E
[0353] Ammonium hydroxide solution (20 mL) was added to a solution
of the material from Part D in dichloromethane (20 mL). A solution
of tosyl chloride (0.99 g, 5.2 mmol) in dichloromethane (10 mL) was
added over a period of 5 minutes. Tie resulting biphasic reaction
mixture was allowed to stir overnight. The reaction mixture was
diluted with chloroform and saturated sodium bicarbonate solution.
The layers were separated. The organic layer was dried over sodium
sulfate, filtered and then concentrated under reduced pressure to
provide a brown glass. This material was purified by column
chromatography (silica gel eluting first with 50% ethyl acetate in
hexanes and then with ethyl acetate) to provide 1.0 g of tert-butyl
4-{2-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)butox-
y]ethyl}piperidine-1-carboxylate as pale yellow glassy foam.
[0354] Part F
[0355] Under a nitrogen atmosphere, tert-butyl
4-{2-[2-(4-amino-1H-imidazo-
[4,5-c]quinolin-1-yl)butoxy]ethyl}piperidine-l-carboxylate (1.00 g,
2.1 mmol) and 2N ethanolic hydrochloric acid (10 ml, 20 mmol) were
combined and the solution was stirred at ambient temperature for 14
hours. The solvent was removed in vacuo and the resulting tan solid
was dissolved in water. Saturated aqueous sodium carbonate was
added until the pH reached 10. After extraction with
dichloromethane (3.times.), the organic fractions were combined,
washed with brine, dried (Na.sub.2SO.sub.4), filtered, and the
majority of the solvent was removed in vacuo. Hexane was added to
form a precipitate. Vacuum filtration yielded 0.5 g of
1-{1-[(2-piperidin-4-ylethoxy)methyl]propyl}-1H-imidazo[4,5-c]quinolin-4--
amine as a tan powder.
[0356] .sup.1H-NMR (300MHz, DMSO-d.sub.6): .delta. 8.34 (bs, 1H),
8.19 (d, J=8.49 Hz, 1H), 7.61 (dd, J=8.31, 1.13 Hz, 1H), 7.45-7.39
(m, 1H), 7.25-7.19 (m, 1H), 6.55 (s, 2H), 5.25-5.15 (m, 1H),
4.00-3.80 (m, 2H), 3.5-3.3 (m, 2H), 2.8-2.64 (m, 2H), 2.22-2.11 (m,
2H), 2.09-1.99 (m, 2H), 1.8-1.63 (bs, 1H), 1.37-1.0 (m, 5H),
0.95-0.7 (m, 5H);
[0357] .sup.13C-NMR (75 MHz, DMSO-d.sub.6): .delta. 152.8, 145.8,
140.6, 133.0, 127.8, 127.0, 126.9, 121.3, 121.0, 115.5, 71.8, 68.1,
58.4, 46.1, 36.3, 33.1, 32.7, 24.5, 9.9;
[0358] MS (CI) m/e 368.2459 (368.2450 calcd for
C.sub.21H.sub.30N.sub.5O).
EXAMPLE 12
5-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]-N-methyl-N-phenylpent-
anamide
[0359] 25
[0360] Using the general method of Parts C and D of Example 11,
2-(1H-imidazo[4,5-c]quinolin-1-yl)ethanol (0.63 g, 2.9 mmol) and
5-bromo-N-methyl-N-phenylpentanamide (1.3 g, 4.8 mmol) were
combined to provide 0.24 g of
5-[2-(5-oxido-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]-N--
methyl-N-phenylpentanamide as a colorless oil. The resulting
N-oxide product was dissolved in dichloromethane and
trichloroacetyl isocyanate (0.11 ml) was added dropwise. The
reaction was stirred at room temperature for 2 hours and then the
solvent was removed under vacuum. The resulting oil was dissolved
in methanol and sodium methoxide (0.2 ml, 25% by weight in
methanol) was slowly added. The reaction was maintained overnight
and then concentrated under vacuum. Purification by flash column
chromatography (silica gel, 9:1 ethyl acetate.backslash.methanol)
provided 24 mg of
5-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]-N--
methyl-N-phenylpentanamide as a white solid.
[0361] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.94 (d, J=8.1 Hz,
1H), 7.83 (m, 2H), 7.52 (dt, J=7.7,1.3 Hz, 1H), 7.41-7.28 (m, 4H),
7.12 (d, J=7.8 Hz, 2H), 5.55 (broad s, 2H), 4.65 (t, J=5.3 Hz, 2H),
3.85 (t, J=5.3 Hz, 2H), 3.31 (t, J=6.3 Hz, 2H), 3.24 (s, 3H), 2.02
(m, 2H), 1.56 (m, 2H), 1.40 (m, 2H);
[0362] IR (KBr) 3429, 3104, 2946, 2877, 1646, 1595, 1584, 1532,
1496, 1482, 1398, 1360, 1254, 1121, 749, 705 cm.sup.-1;
[0363] MS (EI) m/e 417.2160 (417.2165 calcd for
C.sub.24H.sub.27N.sub.5O.s- ub.2).
EXAMPLE 13
5-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]-N-butyl-N-phenylpenta-
namide
[0364] 26
[0365] 2-( 1H-Imidazo[4,5-c]quinolin-1-yl)ethanol and
5-bromo-N-butyl-N-phenylpentanamide were combined and treated
according to the general procedure described in Example 12.
Purification by flash column chromatography (silica gel, 98:2 ethyl
acetate.backslash.methanol) provided
5-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy]-N-butyl-N-p-
henylpentamide as a colorless oil.
[0366] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.93 (d, J=8.0 Hz,
1H), 7.87-7.85 (m, 2H), 7.54 (dt, J=7.7,1.1 Hz, 1H), 7.41-7.29 (m,
4H), 7.10 (d, J=7.2 Hz, 2H), 6.20 (broad s, 2H), 4.66 (t, J=5.2 Hz,
2H), 3.85 (t, J=5.2 Hz, 2H), 3.66 (t, J=7.5 Hz, 2H), 3.31 (t, J=6.2
Hz, 2H), 1.96 (t, J=7.2 Hz, 2H), 1.56-1.25 (m, 8H), 0.88 (t, J=7.2
Hz, 3H);
[0367] MS (EI) m/e 459.2631 (459.2634 calcd for
C.sub.27H.sub.33N.sub.5O.s- ub.2).
EXAMPLE 14
Methyl
[2-(4-Amino-1H-imidazo[4,5-c]quinolin-1-yl)butoxy]acetate
[0368] 27
[0369] 2-(4-Amino-1H-imidazo[4,5-c]quinolin-1-yl)butan-1-ol (25 mg,
0.0975 mmol) was placed in a 2 dram (7.4 mL) vial. Sodium hydride
(5 mg of a 60% dispersion in mineral oil, 0.117 mmol) and
N,N-dimethylformamide (1 mL) were added. The vial was placed on a
sonicator for 15 minutes at ambient temperature to allow the
alkoxide to form. Methyl bromoacetate (11 .mu.L, 0.11 i mmol) was
added. The reaction was sonicated at ambient temperature for 1.5
hours. The reaction mixture was analyzed by LC/MS to confirm the
formation of the desired product. The reaction mixture was purified
by semi-preparative HPLC using Method A Mass Measurement (Da.):
Theoretical mass=328.1535, Measured mass=328.1534.
EXAMPLES 15-34
[0370] The compounds in the table below were prepared according to
the synthetic method of step (7) of Reaction Scheme II above using
the following general method.
[0371] The acid chloride (84 .mu.mol) was added to a test tube
containing a solution of
1-[2-(2-aminoethoxy)ethyl]-2-butyl-1H-imidazo[4,5-c]quinoli-
n-4-amine (25 mg, 77 .mu.mol) in dichloromethane (5 mL). The test
tube was capped and then placed on a shaker at ambient temperature
for 20 hr. The solvent was removed by vacuum centrifugation. The
residue was purified by semi-preparative HPLC using Method B
described above. The table below shows the structure of the free
base and the observed accurate mass (M+H).
1 Example Accurate Mass Number Structure of Free Base (obs.) 15 28
396.2404 16 29 412.2717 17 30 424.2717 18 31 432.2407 19 32
438.2748 20 33 446.2560 21 34 450.2318 22 35 452.2116 23 36
457.2369 24 37 457.2333 25 38 460.2693 26 39 462.2529 27 40
462.2502 28 41 467.1952 29 42 472.2708 30 43 476.2659 31 44
482.2568 32 45 500.2246 33 46 500.2252 34 47 516.2217
EXAMPLES 35-51
[0372] The compounds in the table below were prepared according to
the synthetic method of step (7) of Reaction Scheme II above using
the following general method.
[0373] The acid chloride (1.1 eq.) was added to a test tube
containing a solution of
1-{1-[(2-piperidin-4-ylethoxy)methyl)propyl}-1H-imidazo[4,5-c-
]quinolin-4-amine (25 mg) in dichloromethane (5 mL). The test tube
was capped and then placed on a shaker at ambient temperature for
20 hr. The solvent was removed by vacuum centrifugation. The
residue was purified by semi-preparative HPLC using Method B
described above. The table below shows the structure of the free
base and the observed accurate mass (M+H).
2 Example Accurate Mass Number Structure of Free Base (obs.) 35 48
436.2683 36 49 452.3014 37 50 464.3045 38 51 472.2717 39 52
486.2878 40 53 490.2592 41 54 492.2419 42 55 497.2682 43 56
500.3022 44 57 502.2824 45 58 507.2281 46 59 473.2686 47 60
512.3038 48 61 516.2965 49 62 522.2836 50 63 540.2534 51 64
556.2521
EXAMPLES 52-66
[0374] The compounds in the table below were prepared according to
the synthetic method of step (7) of Reaction Scheme II above using
the following general method.
[0375] 1-[2-(2-Aminoethoxy)ethyl]-1H-imidazo[4,5-c]quinolin-4-amine
(20 mg) and 1-methyl-2-pyrrolidinone (5 mL) were combined in a test
tube and sonicated with heating to provide a solution. The acid
chloride (1.1 eq.) was added. The test tube was capped and then
placed on a shaker at ambient temperature for 20 hr. The solvent
was removed by vacuum centrifugation. The residue was purified by
semi-preparative HPLC using Method B described above. The table
below shows the structure of the free base and the observed
accurate mass (M+H).
3 Example Accurate Mass Number Structure of Free Base (obs.) 52 65
356.2093 53 66 376.1783 54 67 382.2260 55 68 390.1949 56 69
396.1503 57 70 401.1707 58 71 404.2105 59 72 406.1855 60 73
406.1861 61 74 411.1320 62 75 420.2047 63 76 426.1912 64 77
444.1628 65 78 444.1655 66 79 460.1608
Cytokine Induction in Human Cells
[0376] An in vitro human blood cell system is used to assess
cytokine induction. Activity is based on the measurement of
interferon and tumor necrosis factor (a) (IFN and TNF,
respectively) secreted into culture media as described by Testerman
et. al. In "Cytokine Induction by the Immunomodulators Imiquimod
and S-27609", Journal of Leukocyte Biology, 58, 365-372 (September,
1995).
[0377] Blood Cell Preparation for Culture
[0378] Whole blood from healthy human donors is collected by
venipuncture into EDTA vacutainer tubes. Peripheral blood
mononuclear cells (PBMCs) are separated from whole blood by density
gradient centrifugation using Histopaque.RTM.-1077. The PBMCs are
washed twice with Hank's Balanced Salts Solution and then are
suspended at 3-4.times.10.sup.6 cells/mL in RPMI complete. The PBMC
suspension is added to 48 well flat bottom sterile tissue culture
plates (Costar, Cambridge, Mass. or Becton Dickinson Labware,
Lincoln Park, N.J.) containing an equal volume of RPMI complete
media containing test compound.
[0379] Compound Preparation
[0380] The compounds are solubilized in dimethyl sulfoxide (DMSO).
The DMSO concentration should not exceed a final concentration of
1% for addition to the culture wells
[0381] Incubation
[0382] The solution of test compound is added to the first well
containing RPMI complete and serial dilutions are made in the
wells. The PBMC suspension is then added to the wells in an equal
volume, bringing the test compound concentrations to the desired
range. The final concentration of PBMC suspension is
1.5-2.times.10.sup.6 cells/mL. The plates are covered with sterile
plastic lids, mixed gently and then incubated for 18 to 24 hours at
37.degree. C. in a 5% carbon dioxide atmosphere.
[0383] Separation
[0384] Following incubation the plates are centrifuged for 5-10
minutes at 1000 rpm (.about.200.times.g) at 4.degree. C. The
cell-free culture supernatant is removed with a sterile
polypropylene pipet and transferred to sterile polypropylene tubes.
Samples are maintained at -30 to -70.degree. C. until analysis. The
samples are analyzed for interferon (a) and for tumor necrosis
factor (a) by ELISA
[0385] Interferon (.alpha.) and Tumor Necrosis Factor (.alpha.)
Analysis by ELISA
[0386] Interferon (.alpha.) concentration is determined by ELISA
using a Human Multi-Species kit from medical Laboratories, New
Brunswick, N.J. Results are expressed in pg/mL.
[0387] Tumor necrosis factor (.alpha.) (TNF) concentration is
determined using ELISA kits available from Genzyme, Cambridge,
Mass.; R&D Systems, Minneapolis, Minn.; or Pharmingen, San
Diego, Calif. Results are expressed in pg/mL.
[0388] The table below lists the lowest concentration found to
induce interferon and the lowest concentration found to induce
tumor necrosis factor for each compound. A "*" indicates that no
induction was seen at any of the tested concentrations; generally
the highest concentration tested was 10 or 30 .mu.M.
4 Cytokine Induction in Human Cells Example Lowest Effective
Concentration (.mu.M) Number Interferon Tumor Necrosis Factor 12
3.33 * 13 10 * 14 0.37 * 15 0.1 1 16 0.1 1 17 1 1 18 1 10 19 1 10
20 0.1 10 21 1 10 22 0.1 10 23 1 10 24 1 10 25 1 10 26 1 10 27 1 10
28 1 10 29 1 10 30 1 10 31 * 10 32 * 10 33 * 10 34 * 10 35 0.1 1 36
1 1 37 1 1 38 1 10 39 1 10 40 1 1 41 0.1 1 42 1 1 43 1 10 44 1 1 45
0.1 1 46 0.1 1 47 1 * 48 0.1 10 49 1 10 50 1 1 51 10 10 52 10 10 53
10 10 54 10 10 55 1 10 56 1 10 57 10 * 58 10 10 59 10 10 60 10 10
61 10 10 62 1 10 63 * 10 64 10 * 65 * * 66 * *
* * * * *