U.S. patent application number 11/701983 was filed with the patent office on 2007-10-25 for novel cd40:cd154 binding interruptor compounds and use thereof to treat immunological complications.
Invention is credited to Mary Beth Carter, Yen-Ming Hsu, Satish Jindal, Leonid Kirkovsky, George R. Lenz, YuSheng Liao, Susan Mrose, Huw M. Nash, Gerald W. JR. Shipps, Lihong Sun, David Thomas, Zhongli Zheng.
Application Number | 20070249682 11/701983 |
Document ID | / |
Family ID | 22863773 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070249682 |
Kind Code |
A1 |
Zheng; Zhongli ; et
al. |
October 25, 2007 |
Novel CD40:CD154 binding interruptor compounds and use thereof to
treat immunological complications
Abstract
The present invention relates to novel CD40:CD154 binding
interrupter compounds and use of these compounds and pharmaceutical
compositions comprising them, to treat conditions associated with
inappropriate CD154 activation in a subject. Specifically, this
invention provides compounds which are identified by screening a
library of small molecules for those that are capable of
specifically binding CD154 and interrupting CD40:CD154
interaction.
Inventors: |
Zheng; Zhongli; (Lexington,
MA) ; Carter; Mary Beth; (Arlington, MA) ;
Liao; YuSheng; (Lexington, MA) ; Sun; Lihong;
(Arlington, MA) ; Kirkovsky; Leonid; (San Diego,
CA) ; Mrose; Susan; (Brookline, MA) ; Hsu;
Yen-Ming; (Lexington, MA) ; Thomas; David;
(Houston, TX) ; Shipps; Gerald W. JR.; (Stoneham,
MA) ; Jindal; Satish; (Milton, MA) ; Lenz;
George R.; (Andover, MA) ; Nash; Huw M.;
(Cambridge, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
1211 AVENUE OF THE AMERICAS
NEW YORK
NY
10036-8704
US
|
Family ID: |
22863773 |
Appl. No.: |
11/701983 |
Filed: |
February 2, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10377928 |
Feb 28, 2003 |
7173046 |
|
|
11701983 |
Feb 2, 2007 |
|
|
|
PCT/US01/27361 |
Aug 31, 2001 |
|
|
|
10377928 |
Feb 28, 2003 |
|
|
|
60230055 |
Sep 1, 2000 |
|
|
|
Current U.S.
Class: |
514/332 ;
546/262; 564/80 |
Current CPC
Class: |
C07C 311/47 20130101;
A61P 9/10 20180101; A61P 27/02 20180101; A61P 29/00 20180101; A61P
37/08 20180101; A61P 13/12 20180101; F25B 9/145 20130101; F25B
2309/1424 20130101; C07D 401/14 20130101; F25B 2309/14241 20130101;
A61P 9/02 20180101; A61P 25/28 20180101; F25B 2309/1411 20130101;
A61P 11/06 20180101; A61P 37/06 20180101; A61P 17/06 20180101; A61P
37/00 20180101; A61P 1/04 20180101; F25B 2309/1418 20130101; A61P
1/16 20180101; A61K 31/444 20130101; A61P 11/00 20180101; A61P 9/04
20180101; A61P 19/02 20180101; F25B 9/10 20130101; F25B 2309/1408
20130101; A61P 35/02 20180101 |
Class at
Publication: |
514/332 ;
546/262; 564/080 |
International
Class: |
A61K 31/44 20060101
A61K031/44; A61P 37/08 20060101 A61P037/08; C07C 303/00 20060101
C07C303/00; C07D 213/44 20060101 C07D213/44 |
Claims
1. A compound, which interrupts CD40:CD154 interaction, of the
formula: ##STR143## Formula I, wherein, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently
selected from the group consisting of H, C1-5 alkyl, C1-5
alkyl-aryl, C1-5 alkyl-cycloalkyl, C1-5 alkyl-heteroaryl, C1-5
alkenyl-heterocyclo, cycloalkyl, cycloalkyl-aryl, C1-5
alkenyl-aryl, CR.sup.gR.sup.hCO.sub.2H and
CR.sup.gR.sup.hCO.sub.2alkyl; wherein aryl or heteroaryl are
optionally substituted with one to four substituents selected from
R.sup.d and alkyl is optionally substituted with R.sup.i; or
R.sup.1 and R.sup.2, R.sup.3 and R.sup.4, R.sup.5 and R.sup.6, and
R.sup.7 and R.sup.8 independently form, together with the nitrogen
to which they are attached, a 5-6 membered ring which is optionally
substituted with a heteroatom selected from the group consisting of
nitrogen, sulfur and oxygen, wherein the nitrogen may optionally be
substituted with alkyl or alkyl-aryl and the ring may optionally be
substituted with R.sup.j; R.sup.a, R.sup.b and R.sup.c are
independently selected from the group consisting of H, alkyl and
alkyl-aryl; R.sup.d is selected from the group consisting of
halogen, alkyloxy, NO.sub.2, NH.sub.2, alkyl, SO.sub.2NH.sub.2,
hydroxyl and aryl; R.sup.e and R.sup.f are independently selected
from the group consisting of H, alkyl and cycloalkyl; or
R.sup.eR.sup.f together with the atom to which they are attached
form a 4-7 membered ring containing zero to three heteroatoms
selected from the group consisting of nitrogen, oxygen and sulfur,
wherein R.sup.eR.sup.f together with the atom to which they are
attached may be fused to one or two aromatic rings; R.sup.g and
R.sup.h are independently selected from the group consisting of H,
alkyl, aryl, cycloalkyl, alkyl-aryl and alkyl-cycloalkyl; wherein
alkyl is optionally substituted with R.sup.k; and aryl is
optionally substituted with R.sup.l; or R.sup.gR.sup.h together
with the carbon to which they are attached form a 5-8 membered ring
which may be bicyclic; R.sup.i is selected from the group
consisting of: NR.sup.eR.sup.f, alkyloxy, aryloxy, C(O)aryl, aryl,
OC(O)alkyl, alkylaryloxy and hydroxyl; wherein alkyl is optionally
substituted with halogen and aryl is optionally substituted with
R.sup.d; R.sup.j is selected from the group consisting of
(CH.sub.2).sub.yNR.sup.eR.sup.f, (CH.sub.2).sub.zOH, CO.sub.2H,
C(O)N(alkyl).sub.2, C(O)NH.sub.2, alkylaryl and aryl; wherein aryl
is optionally substituted with one to four substituents selected
from R.sup.d; R.sup.k is selected from the group consisting of
C(O)NH.sub.2, C(O)OH, alkylthio, NH.sub.2, heteroaryl, heteroalkyl,
NHC(NH)NH.sub.2, C1-5 alkylhydroxyl, hydroxyl and alkyloxy; R.sup.l
is selected from the group consisting of hydroxyl, NO.sub.2 and
C1-5 alkyl; U is selected from the group consisting of O,
S(O).sub.y, NR.sup.c, NR.sup.cC(O), NR.sup.cC(O)NR.sup.c; W is
selected from the group consisting of (CR.sup.aR.sup.b).sub.x,
(CR.sup.aR.sup.b).sub.yU(CR.sup.aR.sup.b).sub.y, C2-3 alkynyl, C2-3
alkenyl; each X is independently 0-3; each Y is independently 0-2;
each Z is independently 1-2.
2. A compound, which interrupts CD40:CD154 interaction, of the
formula: ##STR144## R.sup.9 is selected from the group consisting
of C1-5 alkyl-aryl, cycloalkyl and alkenylaryl; wherein the alkyl
is optionally substituted with aryl and the aryl is optionally
substituted with one to four halogens, aryl, NH.sub.2 or NO.sub.2;
R.sup.10 is selected from the group consisting of alkyl and
cycloalkyl; and R.sup.11 is selected from the group consisting of H
and alkyl.
3. A compound, which interrupts CD40:CD154 interaction, of the
formula: ##STR145## wherein, R.sup.9 is selected from the group
consisting of C1-5 alkyl-aryl; wherein alkyl and aryl are
optionally substituted with aryl.
4. The compound according to claim 1, wherein said compound is
derived from S-amines and wherein said compound is selected from
the group consisting of: ##STR146## ##STR147##
5. The compound according to claim 1 or 2, wherein said compound is
derived from S-amines and wherein said compound is selected from
the group consisting of: ##STR148## ##STR149## ##STR150##
##STR151##
6. The compound according to any one of claims 1, 2 or 3, wherein
said compound is derived from S-amines and wherein said compound is
selected from the group consisting of: ##STR152## ##STR153##
7. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound according to any one of claims
1-6.
8. A method for attenuating severity of a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
9. A method for suppressing effects of a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
10. A method for preventing development of a condition associated
with inappropriate CD154 mediated activation in a subject,
comprising the step of administering an effective amount of a
pharmaceutical composition according to claim 7 to the subject.
11. A method for delaying onset of a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
12. A method for inhibiting a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
13. A method for reversing a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
14. A method for treating a condition associated with inappropriate
CD154 mediated activation in a subject, comprising the step of
administering an effective amount of a pharmaceutical composition
according to claim 7 to the subject.
15. A method for preventing a condition associated with
inappropriate CD154 mediated activation in a subject, comprising
the step of administering an effective amount of a pharmaceutical
composition according to claim 7 to the subject.
16. The method according to any one of claims 8-15, wherein the
subject is a primate.
17. The method according claim 16, wherein said primate is a
human.
18. The method according to any one of claims 8-15, wherein the
condition is an unwanted immune response.
19. The method according to any one of claims 8-15, wherein the
condition is an unwanted inflammatory response.
20. The method according to any one of claims 8-15, wherein the
condition is an autoimmune disease.
21. The method according to any one of claims 8-15, wherein the
condition is an allergy.
22. The method according to any one of claims 8-15, wherein the
condition is an inhibitor response to a therapeutic agent.
23. The method according to any one of claims 8-15, wherein the
condition is rejection of a donor organ.
24. The method according to any one of claims 8-15, wherein the
condition is a B cell cancer.
25. The method according to any one of claims 8-15, wherein the
condition is selected from the group consisting of: systemic lupus
erythematosis, lupus nephritis, lupus neuritis, asthma, chronic
obstructive pulmonary disease, bronchitis, emphysema, multiple
sclerosis, uveitis, Alzheimer's disease, traumatic spinal cord
injury, stroke, atherosclerosis, coronary restenosis, ischemic
congestive heart failure, cirrhosis, hepatitis C, diabetic
nephropathy, glomerulonephritis, osteoarthritis, rheumatoid
arthritis, psoriasis, atopic dermatitis, systemic sclerosis,
radiation-induced fibrosis, Crohn's disease, ulcerative colitis,
multiple myeloma and cachexia.
26. A compound, which interrupts CD40:CD154 interaction, of the
formula: ##STR154##
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to novel CD40:CD154 binding
interrupter compounds and use of these compounds and pharmaceutical
compositions comprising them, to treat conditions associated with
inappropriate CD154 activation in a subject. Specifically, this
invention provides compounds which are identified by screening a
library of small molecules for those that are capable of
specifically binding CD154 and interrupting CD40:CD154
interaction.
BACKGROUND OF THE INVENTION
[0002] Data establishing that T cell activation requires both T
cell receptor ("TCR") mediated signals and simultaneously delivered
costimulatory signals have accumulated over the past twenty years.
For example, antibody production by B lymphocytes in response to
protein antigens requires a specific, costimulatory interaction
with T lymphocytes. This B cell/T cell interaction is mediated
through several receptor-ligand binding events in addition to
engagement of the TCR. See, e.g., Noelle et al. Immunology Today
13: 431-433 (1992). See also Hollenbaugh et al. EMBO J. 11:
4313-4321 (1992). These additional binding events include the
binding of CD40 on B cells to CD154 (CD40L, and also known as gp39,
T-BAM, 5c8 antigen, CD40CR and TRAP) on T cells. Human CD40 is a 50
kilodalton cell surface protein expressed on mature B cells, as
well as macrophages, dendritic cells, fibroblasts and activated
endothelial cells. CD40 belongs to a class of receptors involved in
cell signalling and in programmed cell death, including Fas/CD95
and the tumor necrosis factor (TNF) alpha receptor. Human CD154, a
32 kD type II membrane glycoprotein having homology to TNF alpha,
is a member of the TNF family of receptors and is transiently
expressed primarily on activated T cells. CD40:CD154 binding has
been shown to be required for T cell-dependent antibody responses.
In particular, CD40:CD154 binding provides anti-apoptotic and/or
lymphokine stimulatory signals. See, e.g., Karpusas et al.
Structure 15, 1021-1039 (1995), U.S. patent application Ser. No.
09/180,209 and WO97/00895, the disclosures of all of which are
hereby incorporated by reference.
[0003] The importance of CD40:CD154 binding in promoting T cell
dependent biological responses is underscored by the development of
X-linked hyper-IgM syndrome (X-HIGM) in humans lacking functional
CD154. These individuals have normal or high IgM levels, but fail
to produce IgG, IgA or IgE antibodies. Affected individuals suffer
from recurrent, sometimes severe, bacterial infection (most
commonly Streptococcus pneumoniae, Pneumocystis carinii and
Hemophilus influenzae) and certain unusual parasitic infections, as
well as an increased incidence of lymphomas and abdominal cancers.
These clinical manifestations of disease can be managed through
intravenous immunoglobulin replacement therapy.
[0004] The effects of X-HIGM are simulated in animals rendered
nullizygous for the gene encoding CD154 (knockout animals). Studies
with nullizygotes have confirmed that, while B cells can produce
IgM in the absence of CD40:CD154 binding, they are unable to
undergo isotype switching, or to survive normally and undergo
affinity maturation. In the absence of a functional CD40:CD154
interaction, spleen and lymph node germinal centers do not develop
properly, and the development of memory B cells is impaired. These
defects contribute to a severe reduction or absence of a secondary
(mature) antibody response.
[0005] Individuals with X-HIGM and CD154 nullizygotes also have
defects in cellular immunity. These defects are manifested by an
increased incidence of Pneumocystis carinii, Histoplasma
capsulatum, Cryotococcus neoformans infection, as well as chronic
Giardia lambli infection. Murine nullizygotes are deficient in
their ability to fight Leishmania infection. Many of these
cell-mediated defects are reversible by administration of IL-12 or
IFN-gamma. These data substantiate the view that CD40:CD154 binding
promotes the development of Type I T-helper cell responses. Further
support is derived from the observation that macrophage activation
is defective in CD154-deficient settings, and that administration
of anti-CD154 antibodies to mice diminished their ability to clear
Pneumocystis infection. Blockade of CD40:CD154 binding appears to
reduce the ability of macrophages to produce nitric oxide, which
mediates many of the macrophages' pro-inflammatory activities. It
should be noted, however, that mammals (including humans) who lack
functional CD154 do not develop significant incidences of viral
infection.
[0006] A number of preclinical studies, including those described
in co-pending, commonly assigned PCT patent applications published
as WO98/30241, WO98/30240, WO98/52606, WO98/58669 and WO99/45958,
describe the promise of agents capable of interrupting CD40:CD154
binding as immunomodulating agents. In murine systems, antibodies
to CD154 block primary and secondary immune responses to exogenous
antigens, both in vitro and in vivo. Antibodies to CD154 cause a
reduction in germinal centers in mice and monkeys, consistent with
data on CD154 immunodeficiency. Administration of three doses of
anti-CD154 antibody to lupus-prone mice, age three months,
substantially reduced titers against double-stranded DNA and
nucleosomes, delayed the development of severe nephritis, and
reduced mortality. Moreover, administration of anti-CD154
antibodies to mice age five to seven months with severe nephritis
was shown to stabilize or even reverse renal disease. Anti-CD154
antibodies given concomitantly with small resting allogeneic
lymphocytes permitted unlimited survival of mouse pancreatic islet
allografts. In other animal models, interference with CD40:CD154
binding has been demonstrated to reduce symptoms of autoimmune
disease (e.g., multiple sclerosis, rheumatoid arthritis,
inflammatory bowel disease), graft rejection (e.g., cardiac
allograft, graft-versus-host disease), and mercuric chloride
induced glomerulonephritis, which is mediated by both humoral and
cellular mechanisms.
[0007] Such studies with anti-CD154 antibodies demonstrate the role
of CD154 as a critical target for modulating immune responses.
[0008] These studies establish the utility of CD40:CD154 binding
interrupters as therapeutic agents. As a result, they also suggest
the potential of novel CD40:CD154 binding interrupters.
SUMMARY OF THE INVENTION
[0009] The present invention provides novel compounds, other than
anti-CD154 antibodies and soluble CD40 or CD40 fusion proteins,
that specifically bind CD154 and interrupt CD40:CD154 interaction.
This invention also provides pharmaceutical compositions comprising
these compounds. The invention also provides methods using these
compounds to treat conditions associated with inappropriate CD154
activation.
[0010] The foregoing and other objects, features and advantages of
the present invention, as well as the invention itself, will be
more fully understood from the following description of preferred
embodiments.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The following discussion illustrates and exemplifies the
variety of contexts and circumstances in which the invention can be
practiced, as well as providing specific embodiments of the
invention.
Novel CD40:CD154 Binding Interruptors
[0012] In a preferred embodiment, this invention provides a generic
compound, which binds to CD154 and preferably interrupts CD40:CD154
interaction, represented by the formula (Formula I): ##STR1##
wherein: [0013] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7 and R.sup.8 are independently selected from the
group consisting of H, C1-5 alkyl, C1-5 alkyl-aryl, C1-5
alkyl-cycloalkyl, C1-5 alkyl-heteroaryl, C1-5 alkenyl-heterocyclo,
cycloalkyl, cycloalkyl-aryl, C1-5 alkenyl-aryl,
CR.sup.gR.sup.hCO.sub.2H and CR.sup.gR.sup.hCO.sub.2alkyl; wherein
aryl or heteroaryl are optionally substituted with one to four
substituents selected from R.sup.d and alkyl is optionally
substituted with R.sup.i; or R.sup.1 and R.sup.2, R.sup.3 and
R.sup.4, R.sup.5 and R.sup.6, and R.sup.7 and R.sup.8 independently
form, together with the nitrogen to which they are attached, a 5-6
membered ring which is optionally substituted with a heteroatom
selected from the group consisting of nitrogen, sulfur and oxygen,
wherein the nitrogen may optionally be substituted with alkyl or
alkyl-aryl and the ring may optionally be substituted with R.sup.j;
[0014] R.sup.a, R.sup.b and R.sup.c are independently selected from
the group consisting of H, alkyl and alkyl-aryl; [0015] R.sup.d is
selected from the group consisting of halogen, alkyloxy, NO.sub.2,
NH.sub.2, alkyl, SO.sub.2NH.sub.2, hydroxyl and aryl; [0016]
R.sup.e and R.sup.f are independently selected from the group
consisting of H, alkyl and cycloalkyl; or R.sup.eR.sup.f together
with the atom to which they are attached form a 4-7 membered ring
containing zero to three heteroatoms selected from the group
consisting of nitrogen, oxygen and sulfur, wherein R.sup.eR.sup.f
together with the atom to which they are attached may be fused to
one or two aromatic rings; [0017] R.sup.g and R.sup.h are
independently selected from the group consisting of H, alkyl, aryl,
cycloalkyl, alkyl-aryl and alkyl-cycloalkyl; wherein alkyl is
optionally substituted with R.sup.k; and aryl is optionally
substituted with R.sup.l; or R.sup.gR.sup.h together with the
carbon to which they are attached form a 5-8 membered ring which
may be bicyclic; [0018] R.sup.i is selected from the group
consisting of: NR.sup.eR.sup.f, alkyloxy, aryloxy, C(O)aryl, aryl,
OC(O)alkyl, alkylaryloxy and hydroxyl; wherein alkyl is optionally
substituted with halogen and aryl is optionally substituted with
R.sup.d; [0019] R.sup.j is selected from the group consisting of
(CH.sub.2).sub.yNR.sup.eR.sup.f, (CH.sub.2).sub.zOH, CO.sub.2H,
C(O)N(alkyl).sub.2, C(O)NH.sub.2, alkylaryl and aryl; [0020]
wherein aryl is optionally substituted with one to four
substituents selected from R.sup.d; [0021] R.sup.k is selected from
the group consisting of C(O)NH.sub.2, C(O)OH, alkylthio, NH.sub.2,
heteroaryl, heteroalkyl, NHC(NH)NH.sub.2, C1-5 alkylhydroxyl,
hydroxyl and alkyloxy; [0022] R.sup.1 is selected from the group
consisting of hydroxyl, NO.sub.2 and C1-5 alkyl; [0023] U is
selected from the group consisting of O, S(O).sub.y, NR.sup.c,
NR.sup.cC(O), NR.sup.cCO(O)NR.sup.c; [0024] W is selected from the
group consisting of (CR.sup.aR.sup.b),
(CR.sup.aR.sup.b).sub.yU(CR.sup.aR.sup.b).sub.y, C2-3 alkynyl, C2-3
alkenyl; [0025] each X is independently 0-3; [0026] each Y is
independently 0-2; [0027] each Z is independently 1-2.
[0028] "Alkyl", as well as other groups having the prefix "alk",
such as alkoxy, alkanoyl, means carbon chains which may be linear
or branched or combinations thereof. Examples of alkyl groups
include methyl, ethyl, propyl, isopropyl, butyl, sec and
tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
[0029] "Alkenyl" means carbon chains which contain at least one
carbon-carbon double bond, and which may be linear or branched or
combinations thereof. Examples of alkenyl include vinyl, allyl,
isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl,
2-methyl-2-butenyl, and the like.
[0030] "Alkynyl" means carbon chains which contain at least one
carbon-carbon triple bond, and which may be linear or branched or
combinations thereof. Examples of alkynyl include ethynyl,
propargyl, 3-methyl-1-pentenyl, 2-heptynyl and the like.
"Cycloalkyl" means mono- or bicyclic saturated carbocyclic rings,
each of which have from 3 to 10 carbon atoms. The term also
includes monocyclic rings fused to an aryl group in which the point
of attachment is on the non-aromatic portion. Examples of
cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl,
cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and
the like.
[0031] "Aryl" means mono- or bicyclic aromatic rings containing
only carbon atoms. The term also includes an aryl group fused to a
monocyclic cycloalkyl or monocyclic heterocyclyl group in which the
point of attachment is on the aromatic portion. Examples of an aryl
group include phenyl, naphthyl, indanyl, indenyl,
tetrahydronaphthyl, 2,3 dihydrobenzofuranyl, benzopyranyl,
1,4-benzodioxanyl, and the like.
[0032] "Heteroaryl" means a mono- or bicyclic aromatic ring
containing at least one heteroatom selected from N, O and S, with
each ring containing 5 to 6 atoms. Examples of heteroaryl include
pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl,
oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl,
tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl,
pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl,
benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl, quinolyl,
indolyl, isoquinolyl, and the like.
[0033] "Heterocyclyl" means mono- or bicyclic saturated rings
containing at least one heteroatom selected from N, S and O, each
of said ring having from 3 to 10 atoms in which the point of
attachment may be carbon or nitrogen. The term also includes
monocyclic heterocycle fused to an aryl or heteroaryl group in
which the point of attachment is on the non-aromatic portion.
Examples of "heterocyclyl" include pyrrolidinyl, piperidinyl,
piperazinyl, imidazolidinyl, 2,3-dihydrofuro(2,3-b) pyridyl,
benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl,
dihydroindolyl, and the like. The term also includes partially
unsaturated monocyclic rings that are not aromatic, such as 2- or 4
pyridones attached through the nitrogen or N-substituted-(1H,3H)
pyrimidine-2,4-diones (N-substituted uracils).
[0034] "Halogen" includes fluorine, chlorine, bromine and
iodine.
[0035] In a more preferred embodiment, this invention provides a
compound that is a subgenera of the compound of Formula (I), said
compound, which binds to CD154 and preferrably interrupts
CD40:CD154 interaction, being represented by the formula (Formula
II): ##STR2## wherein: [0036] R.sup.9 is selected from the group
consisting of C1-5 alkyl-aryl, cycloalkyl and alkenylaryl; wherein
the alkyl is optionally substituted with aryl and the aryl is
optionally substituted with one to four halogens, aryl, NH.sub.2 or
NO.sub.2; [0037] R.sup.10 is selected from the group consisting of
alkyl and cycloalkyl; and [0038] R.sup.11 is selected from the
group consisting of H and alkyl.
[0039] In another more preferred embodiment, this invention
provides a compound that is another subgenera of the compound of
Formula (I) and is a subgenera of the compound of Formula (II),
said compound, which binds to CD154 and preferably interrupts
CD40:CD154 interaction, being represented by the formula (Formula
III): ##STR3## wherein: [0040] R.sup.9 is selected from the group
consisting of C1-5 alkyl-aryl; wherein alkyl and aryl are
optionally substituted with aryl.
[0041] This invention also provides all stereoisomers, including
enantiomers, of each of the compounds above. In a preferred
embodiment, the compounds are derived from S-amines (L amino
acids).
[0042] The most preferred embodiments of this invention are the
twenty-one compounds shown below. All twenty-one compounds are
compounds according to Formula (I). All twenty-one compounds are
derived from S-amines. Seventeen of the twenty-one compounds are
also compounds according to Formula (II); and five of these
seventeen compounds are also compounds according to Formula
(III).
[0043] Compound 1, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 1 being represented by
the formula: ##STR4## wherein: compound 1 is derived from
S-amines.
[0044] Compound 2, which interrupts CD40:CD154 interaction, is a
compound according to Formula (I); compound 2 being represented by
the formula: ##STR5## wherein: compound 2 is derived from
S-amines.
[0045] Compound 3, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 3 being represented by
the formula: ##STR6## wherein: compound 3 is derived from
S-amines.
[0046] Compound 4, which interrupts CD40:CD154 interaction, is a
compound according to Formula (III); compound 4 being represented
by the formula: ##STR7## wherein: compound 4 is derived from
S-amines.
[0047] Compound 5, which interrupts CD40:CD154 interaction, is a
compound according to Formula (III); compound 5 being represented
by the formula: ##STR8## wherein: compound 5 is derived from
S-amines.
[0048] Compound 6, which interrupts CD40:CD154 interaction, is a
compound according to Formula (III); compound 6 being represented
by the formula: ##STR9## wherein: compound 6 is derived from
S-amines.
[0049] Compound 7, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 7 being represented by
the formula: ##STR10## wherein: compound 7 is derived from
S-amines.
[0050] Compound 8, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 8 being represented by
the formula: ##STR11## wherein: compound 8 is derived from
S-amines.
[0051] Compound 9, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); ##STR12## compound 9 being
represented by the formula: wherein: compound 9 is derived from
S-amines.
[0052] Compound 10, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 10 being represented
by the formula: ##STR13## wherein; compound 10 is derived from
S-amines.
[0053] Compound 11, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II), compound 11 being represented
by the formula: ##STR14## wherein: compound 11 is derived from
S-amines.
[0054] Compound 12, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II); compound 12 being represented
by the formula: ##STR15## wherein: compound 12 is derived from
S-amines.
[0055] Compound 13, which interrupts CD40:CD154 interaction, is a
compound according to Formula (III); compound 13 being represented
by the formula: ##STR16## wherein: compound 13 is derived from
S-amines.
[0056] Compound 14, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II), compound 14 being represented
by the formula: ##STR17## wherein: compound 14 is derived from
S-amines.
[0057] Compound 15, which interrupts CD40:CD154 interaction, is a
compound according to Formula (III); compound 15 being represented
by the formula: ##STR18## wherein: compound 15 is derived from
S-amines.
[0058] Compound 16, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II), compound 16 being represented
by the formula: ##STR19## wherein: compound 16 is derived from
S-amines.
[0059] Compound 17, which interrupts CD40:CD154 interaction, is a
compound according to Formula (I), compound 17 being represented by
the formula: ##STR20## wherein: compound 17 is derived from
S-amines.
[0060] Compound 18, which interrupts CD40:CD154 interaction, is a
compound according to Formula (I), compound 18 being represented by
the formula: ##STR21## wherein: compound 18 is derived from
S-amines.
[0061] Compound 19, which interrupts CD40:CD154 interaction, is a
compound according to Formula (I), compound 19 being represented by
the formula: ##STR22## wherein: compound 19 is derived from
S-amines.
[0062] Compound 20, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II), compound 20 being represented
by the formula: ##STR23## wherein: compound 20 is derived from
S-amines.
[0063] Compound 21, which interrupts CD40:CD154 interaction, is a
compound according to Formula (II), compound 21 being represented
by the formula: ##STR24## wherein: compound 21 is derived from
S-amines.
[0064] In a preferred embodiment, this invention provides a
compound of Formula (I), (II) or (III), wherein said compound binds
CD154 specifically, and preferably interrupts CD40:CD154
interaction. Preferably these compounds have an IC.sub.50 for
CD40:CD154 interruption of less than or equal to approximately 310
.mu.M; more preferably less than or equal to approximately 125
.mu.M; and even more preferably less than or equal to approximately
50 .mu.M.
[0065] In a more preferred embodiment, the invention provides a
compound of Formula (I), (II) or (III), wherein said compound binds
CD154 specifically; is a CD40:CD154 binding interrupter, and is not
a TNF-.alpha.:TNFRp60 binding interrupter (i.e., does not bind to
TNF-.alpha. and is not a TNF-.alpha.:TNFRp60 binding interrupter).
Preferably, these compounds have an IC.sub.50 for CD40:CD154
binding interruption of less than approximately 125 .mu.M and an
IC.sub.50 for TNF-.alpha.:TNFRp60 of more 125 .mu.M.
[0066] IC.sub.50, as used herein, is the concentration at which a
compound interrupts the interaction of a protein and its receptor
to 50% interaction in the assay shown in Example 7 (for
interruption of CD40:CD154 interaction) and Example 8 (for
interruption of TNF-.alpha.:TNFRp60 interaction). Examples of such
protein and receptor pairs are CD40:CD154 and
TNF-.alpha.:TNFRp60.
[0067] Compounds 4-6 and 11 were shown to have IC.sub.50 for
CD40:CD154 binding interruption in the A range (less than 50
.mu.M). Compounds 1, 7-10 and 13 were shown to have IC.sub.50 for
CD40:CD154 binding interruption in the B range (50 .mu.M to 125
.mu.M). Compounds 2-3 were shown to have IC.sub.50 for CD40:CD154
binding interruption in the C range (greater than 125 .mu.M). In
addition, compounds 12, 14 and 16-21 are considered to have
IC.sub.50 for CD40:CD154 binding interruption in the B range (50
.mu.M to 125 .mu.M), and compound 15 in the A range (less than 50
.mu.M). Compounds 1-4 were shown to have IC.sub.50 of greater than
125 .mu.M for TNF-.alpha.:TNFRp60 binding interruption. Table I
lists the IC.sub.50 for CD40:CD154 binding interruption and the
mass spectrometry data for these twenty-one compounds.
TABLE-US-00001 TABLE I Compound IC50 Mass Spec * ** 1 B 443.16 ((M
+ 2)/2) 2 C 434.22 ((M + 2)/2) 3 C 434.22 ((M + 2)/2) 4 A 861.4 (M
+ 1) 5 A 469.3 ((M + 2)/2) 6 A 455.23 ((M + 2)/2) 7 B 906.45 (M +
1) 8 B 876.43 (M + 1) 9 B 876 (M + 1) 10 B 953 (M + 1) 11 A 982.24
(M + 1) 12 B 430.42 ((M + 2)/2) 13 B 442.24 ((M + 2)/2) 14 B 470.22
((M + 2)/2) 15 A 438.25 ((M + 2)/2) 16 B 430.29 ((M + 2)/2) 17 B
417 ((M + 2)/2) 18 B 485 ((M + 2)/2) 19 B 457 ((M + 2)/2) 20 B
438.27 ((M + 2)/2) 21 B 418.24 ((M + 2)/2) * M is for molecular
mass. ** The formulas in the parenthesis show what the numbers
represent.
[0068] The chemical syntheses of compounds 4, 7-11 are shown in
Examples 1-6. A person skilled in the art would appreciate that the
other compounds (compounds 1-3, 5-6 and 12-21) may be synthesized
from intermediates and/or products disclosed in Examples 1-6 by
routine methods. The skilled artisan would also appreciate that
other routine methods may be used to synthesize these compounds. It
should be understood that a person skilled in the art can
synthesize any compound of Formula (I), (II) or (III) using
conventional methods.
[0069] A specific assay to determine whether a compound interrupts
CD40:CD154 interaction is shown in Example 7. A specific assay to
determine whether a compound interrupts TNF-.alpha.:TNFRp60
interaction is shown in Example 8. Other well-known assays may be
used for both determinations. One such assay is shown to determine
whether a compound interrupts CD40:CD154 interruption is shown in
Example 9. The interaction of CD40 and CD154 results in
up-regulation of I-CAM1. A CD40:CD154 binding interrupter inhibits
that up-regulation.
Conditions Associated with Inappropriate CD154 Induced Activation
in a Subject
[0070] The CD40:CD154 binding interrupter compounds provided by
this invention can be used to prevent or treat subjects having
conditions associated with inappropriate CD154 induced activation.
Treating a condition associated with inappropriate CD154 induced
activation in a subject includes, inter alia, attenuating severity
of the condition, suppressing effects of the condition, inhibiting
the condition and reversing the condition.
[0071] Examples of conditions associated with inappropriate CD154
mediated activation in a subject, include, inter alia: an unwanted
immune response, an unwanted inflammatory response, an autoimmune
disease, an allergy, an inhibitor response to a therapeutic agent,
rejection of a donor organ and a B cell cancer.
[0072] Examples of conditions associated with inappropriate CD154
mediated activation in a subject, include, inter alia: systemic
lupus erythematosis, lupus nephritis, lupus neuritis, asthma,
chronic obstructive pulmonary disease, bronchitis, emphysema,
multiple sclerosis, uveitis, Alzheimer's disease, traumatic spinal
cord injury, stroke, atherosclerosis, coronary restenosis, ischemic
congestive heart failure, cirrhosis, hepatitis C, diabetic
nephropathy, glomerulonephritis, osteoarthritis, rheumatoid
arthritis, psoriasis, atopic dermatitis, systemic sclerosis,
radiation-induced fibrosis, Crohn's disease, ulcerative colitis,
multiple myeloma and cachexia.
Subjects
[0073] The CD40:CD154 binding interrupter compounds provided by
this invention can be administered for treatment or prophylaxis to
any mammalian subject suffering from or about to suffer a condition
associated with inappropriate CD154 activation. Preferably, the
subject is a primate, more preferably a higher primate, most
preferably a human. In other embodiments, the subject may be a
mammal of commercial importance, or a companion animal or other
animal of value, such as a member of an endangered species. Thus, a
subject may be, inter alia, sheep, horses, cattle, goats, pigs,
dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and
mice.
Route of Administration
[0074] The CD40:CD154 binding interrupter compounds provided by
this invention may be administered in any manner which is medically
acceptable. Depending on the specific circumstances, local or
systemic administration may be desirable. Local administration may
be, for example, by subconjunctival administration. Preferably, the
interrupter compound is administered via an oral, an enteral, or a
parenteral route such as by an intravenous, intraarterial,
subcutaneous, intramuscular, intraorbital, intraventricular,
intraperitoneal, subcapsular, intracranial, intraspinal, topical or
intranasal injection, infusion or inhalation. The interrupter
compound also may be administered by implantation of an infusion
pump, or a biocompatible or bioerodiable sustained release implant,
into the subject.
Dosages and Frequency of Treatment
[0075] Generally, the methods described herein involve
administration of the CD40:CD154 binding interrupter compounds at
desired intervals (e.g., daily, twice weekly, weekly, biweekly,
monthly or at other intervals as deemed appropriate) over at least
a two- or three-week period. The administration schedule is
adjusted as needed to treat the condition associated with
inappropriate CD154 activation in the subject. A particular
treatment regime can be repeated, in the event of a subsequent
episode of illness.
[0076] The amount and frequency of dosing for any particular
CD40:CD154 binding interrupter compound to be administered to a
patient for a given immunological disease associated with
inappropriate CD154 induced activation is within the skills and
clinical judgement of ordinary practitioners of the medical and
pharmaceutical arts. The general dosage and administration regime
may be established by preclinical and clinical trials, which
involve extensive but routine studies to determine the optimal
administration parameters of the compound. Even after such
recommendations are made, the practitioner will often vary these
dosages for different subjects based on a variety of
considerations, such as the individual's age, medical status,
weight, sex, and concurrent treatment with other pharmaceuticals.
Determining the optimal dosage and administration regime for each
synthetic CD40:CD154 binding interrupter compound used is a routine
matter for those of skill in the medical and pharmaceutical
arts.
[0077] Generally, the frequency of dosing may be determined by an
attending physician or similarly skilled practitioner, and might
include periods of greater dosing frequency, such as at daily or
weekly intervals, alternating with periods of less frequent dosing,
such as at monthly or longer intervals.
[0078] To exemplify dosing considerations for a CD40:CD154 binding
interrupter, the following examples of administration strategies
are given for an anti-CD154 mAb. Such illustrative dosing amounts
could easily be adjusted for other types of anti-CD154 compounds
according to the present invention. In general, single dosages of
between about 0.05 and about 50 mg/kg patient body weight are
contemplated, with dosages most frequently in the 1-20 mg/kg range.
For acute treatment, such as before or at the time of
transplantation, or in response to any evidence that graft
rejection is beginning, an effective dose of a representative
antibody (such as mAb 5c8), which may serve as a guideline for the
dosages of a compound according to this invention, ranges from
about 1 mg/kg body weight to about 20 mg/kg body weight,
administered daily for a period of about 1 to 5 days, preferably by
bolus intravenous administration. The same dosage and dosing
schedule may be used in the load phase of a load-maintenance
regimen, with the maintenance phase involving intravenous or
intramuscular administration being patterned on that of an
anti-CD154 mAb in a range of about 0.1 mg/kg body weight to about
20 mg/kg body weight, for a treatment period of anywhere from
weekly to 3 month intervals. Chronic treatment may also be carried
out by a maintenance regimen, patterned on that in which antibodies
are administered by intravenous or intramuscular route, in a range
of about 0.1 mg/kg body weight to about 20 mg/kg body weight, with
interdose intervals ranging from about 1 week to about 3 months. In
addition, chronic treatment may be effected by an intermittent
bolus intravenous regimen, patterned on that in which between about
1.0 mg/kg body weight and about 100 mg/kg body weight of an
anti-CD154 mAb are administered, with the interval between
successive treatments being from 1 to 6 months. For all except the
intermittent bolus regimen, administration may also be by oral,
pulmonary, nasal or subcutaneous routes.
[0079] For treatment, a CD40:CD154 binding interrupter compound can
be formulated in a pharmaceutical or prophylactic composition which
includes, respectively, a pharmaceutically or prophylactically
effective amount of the CD40:CD154 binding interrupter compound
dispersed in a pharmaceutically acceptable carrier. In some
embodiments, the pharmaceutical or prophylactic composition can
also include a pharmaceutically or prophylactically effective
amount of another immunosuppressive or immunomodulatory compound,
including without limitation: an agent that interrupts T cell
costimulatory signaling via CD28 (e.g., CTLA4-Ig), CD80 or CD86; an
agent that interrupts calcineurin signaling (e.g., cyclosporin, a
macrolide such tacrolimus, formerly known as FK506); a
corticosteroid; or an antiproliferative agent (e.g., azathioprine).
Other therapeutically effective compounds suitable for use with the
CD40:CD154 binding interrupter include rapamycin (also known as
sirolimus); mycophenolate mofetil (MMF), mizoribine,
deoxyspergualin, brequinar sodium, leflunomide, azaspirane and the
like.
[0080] Combination therapies according to this invention for
treatment of a condition associated with inappropriate CD154
activation in a subject include the use of a synthetic CD40:CD154
binding interruptor compound together with agents targeted at B
cells, such as anti-CD19, anti-CD28 or anti-CD20 antibody
(unconjugated or radiolabeled), IL-14 antagonists, LJP394 (LaJolla
Pharmaceuticals receptor blocker), IR-1116 (Takeda small molecule)
and anti-Ig idiotype monoclonal antibodies. Alternatively, the
combinations may include T cell/B cell targeted agents, such as
CTLA4Ig, IL-2 antagonists, IL-4 antagonists, IL-6 antagonists,
receptor antagonists, anti-CD80/CD86 monoclonal antibodies, TNF,
LFA1/ICAM antagonists, VLA4/VCAM antagonists, brequinar and IL-2
toxin conjugates (e.g., DAB), prednisone, anti-CD3 mAb (OKT3),
mycophenolate mofetil (MMF), cyclophosphamide, and other
immunosuppressants such as calcineurin signal blockers, including
without limitation, tacrolimus (FK506). Combinations may also
include T cell targeted agents, such as CD4 antagonists, CD2
antagonists and anti-IL-12 antibodies.
[0081] The immunomodulatory compound that may be co-administered
with a synthetic CD40:CD154 binding interruptor compound to a
subject with a condition associated with inappropriate CD154
activation may be an antibody that specifically binds to a protein
selected from the group consisting of CD45, CD2, IL2R, CD4, CD8 and
RANK Fc.
Formulation
[0082] In general, CD40:CD154 binding interrupter compounds of this
invention are suspended, dissolved or dispersed in a
pharmaceutically acceptable carrier or excipient. The resulting
therapeutic composition does not adversely affect the recipient's
homeostasis, particularly electrolyte balance. Thus, an exemplary
carrier comprises normal physiologic saline (0.15 M NaCl, pH 7.0 to
7.4). Other acceptable carriers are well known in the art and are
described, for example, in Remington's Pharmaceutical Sciences,
Gennaro, ed., Mack Publishing Co., 1990. Acceptable carriers can
include biocompatible, inert or bioabsorbable salts, buffering
agents, oligo- or polysaccharides, polymers, viscoelastic
compounds, such as hyaluronic acid, viscosity-improving agents,
preservatives, and the like.
[0083] A CD40:CD154 binding interrupter compound provided by this
invention may be administered in a pharmaceutically effective,
prophylactically effective or therapeutically effective amount,
which is an amount sufficient to produce a detectable, preferably
medically beneficial effect on a subject at risk for or afflicted
with a condition associated with inappropriate CD154 activation.
Medically beneficial effects include preventing, inhibiting,
reversing or attenuating deterioration of, or detectably improving,
the subject's medical condition.
EXAMPLES
[0084] The following examples illustrate preferred embodiments of
this invention related to novel CD40:CD154 binding interrupter
compounds and the use of these compounds to treat or prevent
conditions associated with inappropriate CD154 activation in a
subject. These examples should not be construed as limiting: the
examples are included for the purposes of illustration only.
[0085] The following method describes the preparation of a
mass-coded combinatorial library and the screening technology
applied to obtain CD40:CD154 binding interrupter compounds
disclosed in this invention. This method is advantageous because it
can be used to identify chemical compounds that bind tightly to any
biomolecule of interest, such as CD154. The screening technologies
described can be miniaturized to provide massive parallel screening
capabilities.
[0086] Methods for producing mass-coded combinatorial libraries are
disclosed in PCT patent application WO99/35109, published Jul. 15,
1999, the disclosure of which is hereby incorporated by
reference.
[0087] One method according to WO99/35109 allows identification of
a member or members of a mass-coded combinatorial library which are
ligands for a biomolecule, for example, a protein such as CD154.
This method comprises the steps of: (1) contacting the biomolecule
with the mass-coded molecular library, whereby members of a
mass-coded molecular library which are ligands for the biomolecule
bind to the biomolecule to form biomolecule-ligand complexes and
members of the mass-coded library which are not ligands for the
biomolecule remain unbound; (2) separating the biomolecule-ligand
complexes from the unbound members of the mass-coded molecular
library; (3) dissociating the biomolecule-ligand complexes; and (4)
determining the molecular mass of each ligand to identify the set
of n peripheral moieties present in each ligand.
[0088] The method of WO99/35109 allows rapid production of
mass-coded combinatorial libraries comprising large numbers of
compounds. The mass-coding enables the identification of individual
combinations of scaffold and peripheral moieties by molecular mass.
The resulting libraries also allow the rapid identification of
compounds which are ligands for a given biomolecule, such as
CD154.
[0089] One of the methods of WO99/35109 allows the preparation of a
mass-coded set of compounds, such as a mass-coded combinatorial
library. These compounds are of the general formula X(Y).sub.n,
wherein X is a scaffold, each Y is a peripheral moiety and n is 4.
The term "scaffold", as used in WO99/35109 and herein, refers to a
molecular fragment to which four peripheral moieties are attached
via a covalent bond. The scaffold is a molecular fragment which is
common to each member of the mass-coded set of compounds. The term
"peripheral moiety", as used in WO99/35109 and herein, refers to a
molecular fragment which is bonded to a scaffold. Each member of
the set of mass-coded compounds will include a combination of four
peripheral moieties bonded to the scaffold and this set of
compounds forms a mass coded combinatorial library.
[0090] The term "mass" or "molecular mass", as used in WO99/35109
and herein in relation to combinatorial libraries, refers to the
exact mass of a molecule or collection of chemical moieties in
which each atom is the most abundant naturally occurring isotope
for the particular element. Exact masses and their determination by
mass spectrometry are discussed by Pretsch et al., Tables of
Spectral Data for Structure Determination of Organic Compounds,
second edition, Springer-Verlag (1989), and Holden et al., Pure
Appl. Chem. 55 : 1119-1136 (1983), the contents of each of which
are incorporated herein by reference in their entirety.
[0091] A scaffold precursor represented by Formula IV, having four
reactive groups which are capable of reacting with a peripheral
moiety precursor reactive group to form a covalent bond was chosen
as a starting material for the synthesis of compounds according to
this invention. That scaffold precursor, used in Scheme I (infra),
is described in K. E. Pryor et. al. Tetrahedron 54: 4107-4124
(1998), the disclosure of which is incorporated by reference
herein. Scaffold precursors V-VI, respectively used in Schemes II
and III (infra) were synthesized according to the reaction scheme
shown by Pryor et. al., followed by additional modification with
conventional chemical protecting techniques. ##STR25##
[0092] A peripheral moiety precursor is a compound which includes a
reactive group which is complementary to the reactive groups of the
scaffold precursor. In addition to the reactive group, a peripheral
moiety precursor can include a wide variety of structural features.
For example, the peripheral moiety precursor can include one or
more functional groups in addition to the reactive group. Any
additional functional group should be appropriately masked or not
interfere with the reaction between the scaffold precursor and the
peripheral moiety precursor. Suitable protecting groups for masking
are known in the art for a variety of functional groups (Greene and
Wuts, Protective Groups in Organic Synthesis, second edition, New
York: John Wiley and Sons (1991), and third edition, New York: John
Wiley and Sons (1999), the disclosures of both of which are
incorporated herein by reference). Particularly useful protecting
groups include t-butyl esters and ethers, acetals, trityl ethers
and amines, acetyl esters, trimethylsilyl ethers and trichloroethyl
ethers and esters. In addition, two peripheral moiety precursors
should not react together under the conditions employed. For
example, a subset of peripheral moiety precursors can include, in
addition to the reactive groups, functionalities selected from
groups spanning a range of charge, hydrophobicity/hydrophilicity,
and sizes. For example, the peripheral moiety precursor can include
a negative charge, a positive charge, a hydrophilic group or a
hydrophobic group.
[0093] For the present purposes, two reactive groups are
complementary if they are capable of reacting together to form a
covalent bond. In a preferred embodiment, the bond forming
reactions occur rapidly under ambient conditions without
substantial formation of side products. Preferably, a given
reactive group will react with a given complementary reactive group
exactly once.
[0094] Complementary electrophilic and nucleophilic groups include
any two groups which react via nucleophilic substitution under
suitable conditions to form a covalent bond. An example of a
suitable electrophilic group is an acid chloride. A suitable
nucleophilic group include primary and secondary amino groups.
[0095] Illustrated below in Table II are examples of suitable
peripheral moiety precursors. Each peripheral moiety precursor
includes a primary or secondary amino group which reacts with the
scaffold precursor to form an amide bond. TABLE-US-00002 TABLE II
BB BB BB ID* MOLSTRUCTURE Mass ID* MOLSTRUCTURE Mass ID*
MOLSTRUCTURE Mass 11 ##STR26## 175.136 61 ##STR27## 107.073 138
##STR28## 155.05 13 ##STR29## 113.12 115 ##STR30## 147.053 140
##STR31## 168.163 15 ##STR32## 127.136 116 ##STR33## 75.032 141
##STR34## 144.163 16 ##STR35## 127.136 117 ##STR36## 131.095 157
##STR37## 116.131 17 ##STR38## 85.0891 118 ##STR39## 131.095 161
##STR40## 179.095 27 ##STR41## 114.116 119 ##STR42## 146.106 201
##STR43## 119.058 36 ##STR44## 75.0684 120 ##STR45## 165.079 204
##STR46## 136.105 45 ##STR47## 87.0684 122 ##STR48## 115.063 214
##STR49## 141.035 50 ##STR50## 220.121 124 ##STR51## 105.043 226
--NH.sub.2 31.0422 52 ##STR52## 154.147 127 ##STR53## 117.079 243
##STR54## 87.1048 245 ##STR55## 102.116 300 ##STR56## 135.105 387
##STR57## 147.105 256 ##STR58## 151.063 301 ##STR59## 137.084 388
##STR60## 147.105 270 ##STR61## 89.0477 307 ##STR62## 165.079 396
##STR63## 151.1 277 ##STR64## 184.984 315 ##STR65## 200.062 419
##STR66## 103.063 283 ##STR67## 179.095 318 ##STR68## 145.11 424
##STR69## 157.11 286 ##STR70## 133.089 323 ##STR71## 151.063 465
##STR72## 111.08 287 ##STR73## 133.089 350 ##STR74## 116.131 466
##STR75## 160.1 288 ##STR76## 137.084 353 ##STR77## 130.147 475
##STR78## 101.084 290 ##STR79## 165.115 370 ##STR80## 127.046 500
##STR81## 185.061 292 ##STR82## 135.105 385 ##STR83## 101.048 514
##STR84## 142.111 533 ##STR85## 114.116 587 ##STR86## 103.063 597
##STR87## 130.111 551 ##STR88## 114.079 588 ##STR89## 182.178 598
##STR90## 266.178 564 NH.sub.3 17.0266 589 ##STR91## 142.147 599
##STR92## 116.095 580 ##STR93## 146.069 590 ##STR94## 174.112 600
##STR95## 183.105 581 ##STR96## 132.054 591 ##STR97## 181.074 601
##STR98## 213.015 582 ##STR99## 155.07 592 ##STR100## 149.051 602
##STR101## 131.095 583 ##STR102## 211.136 593 ##STR103## 156.159
603 ##STR104## 171.126 584 ##STR105## 133.089 594 ##STR106##
157.089 604 ##STR107## 117.079 585 ##STR108## 149.12 595 ##STR109##
149.12 605 ##STR110## 184.121 586 ##STR111## 185.142 596 ##STR112##
164.095 606 ##STR113## 170.142 607 ##STR114## 182.142 617
##STR115## 213.015 634 ##STR116## 210.064 608 ##STR117## 168.163
618 ##STR118## 213.015 635 ##STR119## 157.11 609 ##STR120## 168.126
619 ##STR121## 203.027 636 ##STR122## 209.105 610 ##STR123##
128.131 625 ##STR124## 131.095 638 ##STR125## 157.11 611 ##STR126##
186.083 628 ##STR127## 133.089 639 ##STR128## 131.095 612
##STR129## 172.103 629 ##STR130## 133.089 613 ##STR131## 210.173
630 ##STR132## 129.079 614 ##STR133## 196.194 631 ##STR134##
143.095 615 ##STR135## 154.111 632 ##STR136## 155.095 616
##STR137## 140.131 633 ##STR138## 177.079 *Identification number of
the peripheral moiety precursors.
[0096] The compounds within the combinatorial library set are
mass-coded as a result of the selection of a subset of suitable
peripheral moiety precursors. The mass-coded set of compounds is
synthesized in solution as a combinatorial library.
[0097] In one embodiment, the scaffold precursor is contacted with
all members of the peripheral moiety precursor subset
simultaneously. In general, a scaffold precursor having four
reactive groups will be contacted with at least about 4 molar
equivalents relative to the scaffold precursor of peripheral moiety
precursors from the selected subset. For example, the scaffold
precursor can be contacted with a solution comprising each member
of the subset in approximately equal concentrations. In one
embodiment, the reaction protocol shown below (Scheme I) was used.
##STR139## In another embodiment, the reaction protocol shown below
(Scheme II) was used. ##STR140## In another embodiment, the
reaction protocol shown below (Scheme III) was used. ##STR141##
[0098] Following the reaction of each scaffold precursor reactive
group with a peripheral moiety precursor, any peripheral moiety
having a protected, additional functional group can be deprotected
using methods known in the art.
[0099] The ability to identify individual scaffold plus peripheral
moiety combinations derived from such a mixture is a consequence of
the mass-coding of the library and the ability of mass spectrometry
to identify a molecular mass. This allows the identification of
individual scaffold plus peripheral moiety combinations within the
set which have a particular activity, such as binding to a
particular biomolecule, such as CD154.
[0100] In one embodiment, a method is provided for identifying a
compound or compounds within a mass-coded combinatorial library
which bind to, or are ligands for, a biomolecule, such as a CD154.
The mass-coded combinatorial library can be produced, for example,
by the method disclosed above. The target biomolecule, such as
CD154, is contacted with the mass-coded combinatorial library, and,
if any members of the library are ligands for a target biomolecule,
biomolecule-ligand complexes form. Compounds which do not bind the
target biomolecule are separated from the biomolecule-ligand
complexes. The biomolecule-ligand complexes are dissociated and the
ligands are separated and their molecular masses are determined by
mass spectrometry. Due to the mass coding of the combinatorial
library, a given molecular mass is characteristic of a unique
combination of peripheral moieties or only a small number of such
combinations. Thus, a ligand's molecular mass allows the
determination of its composition.
[0101] Once single ligands are identified by the above-described
process, various levels of analysis can be applied to yield SAR
information and to guide further optimization of the affinity,
specificity and bioactivity of the ligand. For ligands derived from
the same scaffold, three-dimensional molecular modeling can be
employed to identify significant structural features common to the
ligands, thereby generating families of small-molecule ligands that
presumably bind at a common site on the target biomolecule.
[0102] To identify a consensus, highest affinity ligand for a
particular binding site, this analysis should include a ranking of
the members of a given ligand family with respect to their
affinities for the target. This process can provide this
information by identifying both low and high affinity ligands for a
target biomolecule in one experiment.
[0103] Given that each mass-coded set of compounds is synthesized
with a limited number of peripheral moiety precursors, the
described approach can, in certain cases, identify a superior
ligand which combines structural features of molecules synthesized
in separate libraries.
[0104] When possible, the analysis of ligand structural features is
based on information regarding the target biomolecule's structure,
wherein the hypothetical consensus ligand is computationally docked
with the putative binding site. Further computational analysis can
involve a dynamic search of multiple lowest energy conformations,
which allows comparison of high affinity ligands that are derived
from different scaffolds. The end goal is the identification of
both the optimal functionality and the optimal vectorial
presentation of the peripheral moieties that yields the highest
binding affinity/specificity. This may provide the basis for the
synthesis of an improved, second-generation scaffold.
[0105] An advantage of the method discussed above is that it can be
used to identify chemical compounds that bind tightly to any
biomolecule of interest, such as CD154.
Screening a Mass-Coded Combinatorial Library for Novel CD40:CD154
Binding Interruptors
[0106] Methods for identifying compounds in a chemical mixture are
described in the PCT patent application WO00/22649, published Apr.
20, 2000, the disclosure of which is hereby incorporated by
reference. These methods and the methods disclosed in WO99/35109
were used to identify compounds that bind CD154 from a mixture of
compounds (a mass-coded combinatorial library).
[0107] The WO00/22649 application relates generally to Mass
Spectrographic analysis, and more specifically to the
identification of organic compounds in complex mixtures of organic
compounds.
[0108] The mass coded combinatorial library was prepared
by-treating the reactive scaffold precursors (shown in Formulae
IV-VI) with pools of building blocks shown in Table II using the
reaction protocols shown in Schemes I-III.
[0109] The mass-coded combinatorial library was screened for novel
CD40:CD154 binding interrupters according the the methods discussed
herein. The library was first screened for compounds that bind
CD154. Those compounds that interact with CD154 were identified,
isolated and their chemical compositions were verified with mass
spectrometry. The compounds that interact with CD154 can then each
be synthesized on a larger scale following the method described
below.
[0110] The compounds were then screened for the ability to inhibit
CD40:CD154 interaction. A person skilled in the art is well aware
of conventional assays to assess whether the compounds designed
according to this invention bind specifically to CD154 and whether
the compounds of this invention interrupt CD40:CD154 interaction.
These assays, for example, detect the extent to which B cells are
activated by activated T cells via the interaction between CD154
and CD40. For example, monitoring of CD23 levels on B cells, or
secretion of immunoglobulins by B cells is indicative of activation
of B cells by activated T cells via the interaction between CD40
and CD154. See, e.g., U.S. Pat. No. 5,474,771. Accordingly,
examples of such assays are: the assay of Example 7, an in vitro
assay for T cell activation of B cells; an in vitro assay for
immunoglobulin production by B cells and an in vivo assay for
inhibition of a humoral immune response.
[0111] Once a compound is discovered by the methods described
above, substitutions may then be made by conventional methods in
some of its atoms or side groups to further improve or modify its
binding and/or inhibition properties. Such substituted chemical
compounds may then be analyzed for binding to CD154 and inhibition
of CD40:CD154 interaction.
Example 1
Chemical Synthesis of Compound 7
[0112] Step 1: A mixture of chelidamic acid monohydrate (1.00 g,
4.97 mmol) and phosphorus pentabromide (8.45 g, 19.6 mmol) was
warmed to 90.degree. C. for 3 hours (h). Chloroform (100 mL) was
added to the warm reaction mixture and the resulting slurry was
filtered. The filtrate was concentrated in vacuo to give a pink
solid. The solid was dissolved in ethanol (vigorous reaction),
stirred for 0.5 h and then concentrated in vacuo to give a yellow
solid. The solid was dissolved in ether (250 mL), washed with
saturated aqueous sodium bicarbonate (50 mL.times.2), dried
(MgSO.sub.4) and concentrated in vacuo to give 1.30 g of a white
solid.
[0113] Step 2: A solution of KOH (0.83 g, 3.31 mmol) in anhydrous
ethanol (40 mL) was added dropwise to a solution of the compound
from step 1 (3.85 g, 12.7 mmol) in anhydrous ethanol (100 mL) over
the course of 3 h. A white precipitate formed. The unstirred slurry
was allowed to sit at room temperature (RT) for 7 h and then at
0.degree. C. overnight. The slurry was filtered and the resulting
white solid dissolved in water (100 mL), cooled to 0.degree. C. and
acidified to pH 1 with 1N HCl. The slurry was filtered and the
resulting white solid dried in vacuo to give 3.00 g white
solid.
[0114] Step 3: Catalytic DMF and 2.0 M oxalyl
chloride/dichloromethane (4.5 mL, 9.0 mmol) were added to a
solution of the compound from step 2 (1.20 g, 4.40 mmol) in
dichloromethane at RT. The reaction solution was stirred until
bubbling ceased and was then concentrated in vacuo. The residue was
dissolved in toluene (20 mL) and added dropwise to a solution of
L-cyclohexylglycine tbutyl ester.HCl (1.20 g, 4.80 mmol) in
pyridine (10 mL) at RT. The reaction was stirred overnight at RT,
diluted with ether, washed with water, dried (MgSO.sub.4) and
concentrated in vacuo to give an oil. The oil was purified via
flash column chromatography (95:5 methylene chloride/ethyl acetate)
to give 1.01 g of a colorless oil.
[0115] Step 4: A solution of the compound from step 3 (0.500 g,
1.07 mmol), bis(pinacolate)diboran (0.300 g, 1.18 mmol),
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride (0.026 g, 0.032 mmol) and potassium acetate (0.315 g,
3.21 mmol) in anhydrous DMF (7 mL) was warmed to 80.degree. C.
under a nitrogen atmosphere for 2 h. The reaction was then cooled
to RT, diluted with ethyl acetate (70 mL), washed with 5% citric
acid (25 mL), 50 sodium bicarbonate (25 mL) and water (25 mL),
dried (MgSO.sub.4) and concentrated in vacuo to give a brown
oil.
[0116] Step 5: 3-Phenyl-1-propylamine (4.0 mL, 28.1 mmol) was added
dropwise to concentrated nitric acid at 0.degree. C. The reaction
was allowed to warm to RT overnight. The reaction was warmed to
50.degree. C. for 2 h, concentrated nitric acid (10 mL) was added
and the reaction was warmed for another 2 h at 50.degree. C. The
reaction was cooled to 0.degree. C. and poured into ice water. The
yellow precipitate was isolated via filtration, washed with water
and air dried to give 1.30 g of a yellow solid. .sup.1H NMR
indicated formation of the para isomer only.
[0117] Step 6: Catalytic DMF and 2.0 M oxalyl
chloride/dichloromethane (4.5 mL, 9.0 mmol) were added to a
solution of the compound from step 2 (1.20 g, 4.40 mmol) in
dichloromethane at RT. The reaction solution was stirred until
bubbling ceased and concentrated in vacuo. The residue was
dissolved in toluene (20 mL) and added dropwise to a solution of
the compound from step 5 (1.20 g, 4.93 mmol) in pyridine (10 mL) at
RT. The reaction was stirred overnight at RT, diluted with ether,
washed with water, dried (MgSO.sub.4) and concentrated in vacuo to
give an oil. The oil was purified via flash column chromatography
(90:10 methylene chloride/ethyl acetate) to give 0.51 g of a yellow
solid.
[0118] Step 7: 2 M aqueous sodium carbonate (2.70 mL, 5.4 mmol) was
added to a solution of the compound from step 4, the compound from
step 6 (0.51 g, 1.17 mmol) and
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride (0.026 g, 0.032 mmol) in anhydrous DMF. The reaction
was warmed to 80.degree. C. overnight. It was then cooled to RT,
diluted with ethyl acetate, washed with water and brine, dried
(MgSO.sub.4) and concentrated in vacuo to give a brown oil.
[0119] Step 8: Lithium hydroxide was added to a solution of the
compound from step 7 in 2:1 THF/water at RT. When all starting
ester was consumed via HPLC analysis, the reaction was diluted with
cold water and washed with ethyl acetate. The aqueous phase was
saturated with ammonium chloride and extracted with THF. The
combined organic phases were dried (MgSO.sub.4) and concentrated in
vacuo. The resulting solid was washed with water and air dried to
give 1.20 g of a dark solid. (approx. 50% desired material)
[0120] Step 9: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (0.80 g,
5.2 mmol), HATU (2.0 g, 5.3 mmol) and diisopropylethyl amine (1.8
mL, 10.3 mmol) were added to a solution of the compound from step 8
(1.2 g) in anhydrous DMF (25 mL) at RT. The reaction was stirred at
RT overnight. The volatiles were removed in vacuo, the residue
dissolved in ethyl acetate and washed with water. The organic phase
was dried (MgSO.sub.4), concentrated in vacuo and purified via
flash column chromatography (100:10:2 ethyl
acetate/methanol/triethyl amine) to give 0.650 g of a brown
solid.
[0121] Step 10: A solution of the compound from step 9 (0.030 g) in
2:1 TFA/methylene chloride (6 mL) was stirred at RT for 3 h. The
reaction was then concentrated in vacuo and purified via reverse
phase HPLC (acetonitrile/water) to give a white solid.
Example 2
Chemical Synthesis of Compound 8
[0122] Step 1: steps 1-9 of EXAMPLE 1 were carried out.
[0123] Step 2: Catalytic 10% Pd/C was added to a solution of the
compound from step 1 (0.100 g) in ethyl acetate. The reaction was
stirred under a hydrogen atmosphere (1 atm) overnight. The reaction
was then centrifuged and the supernatant decanted and concentrated
in vacuo.
[0124] Step 3: A solution of the compound from step 2 (0.020 g) in
2:1 TFA/methylene chloride (6 mL) was stirred at RT for 2 h. The
reaction was then concentrated in vacuo and purified via reverse
phase HPLC (acetonitrile/water) to give an off-white solid.
Example 3
Chemical Synthesis of Compound 4
[0125] Step 1: steps 1 and 2 of EXAMPLE 1 were carried out.
[0126] Step 2: Catalytic DMF and 2.0 M oxalyl
chloride/dichloromethane (3.0 mL, 6.0 mmol) were added to a
solution of the compound from step 1 (2.72 g, 2.64 mmol) in
dichloromethane (20 mL) at RT. The reaction solution was stirred
until bubbling ceased, diluted with toluene (40 mL) and reduced in
volume in vacuo. A solution of 3-phenyl-1-propylamine (1.7 mL, 11.9
mmol) in pyridine (8 mL) was added and the resulting solution was
stirred at RT overnight. The reaction solution was diluted with
ethyl acetate (300 mL), washed with water (150 mL), 1N HCl (150
mL.times.3) and water (150 mL), dried (MgSO.sub.4) and concentrated
in vacuo to give an oil. The oil was dissolved in 1:1 saturated
aqueous lithium hydroxide/THF and stirred overnight at RT. The
reaction was then acidified with 1N HCl, extracted with ethyl
acetate, dried (MgSO.sub.4) and concentrated in vacuo to give 3.456
g of an oil.
[0127] Step 3: A solution of the compound from step 2 (3.456 g,
9.515 mmol), bis(pinacolate)diboran (2.658 g, 10.54 mmol),
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride (1.029 g, 1.26 mmol) and potassium acetate (3.88 g,
39.5 mmol) in anhydrous DMF (60 mL) was warmed to 80.degree. C.
under a nitrogen atmosphere for 2.5 h. The reaction was then cooled
to RT, diluted with ethyl acetate (500 mL) and extracted with 1N
sodium hydroxide (200 mL). The aqueous phase was washed with ethyl
acetate (250 mL.times.2), acidified with concentrated HCl to pH 0
and extracted with ethyl acetate (500 mL.times.2). The combined
organic phases were dried (MgSO.sub.4) and concentrated in vacuo to
give an oil which was slurried with ether. The resultant light
brown solid was filtered and air dried to give 1.48 g of product.
The mother liquor was concentrated in vacuo to give 1.78 g of
product.
[0128] Step 4: L-cyclohexylglycine tbutyl ester.HCl (1.717 g, 6.87
mmol), HATU (2.618 g, 6.89 mmol) and diisopropylethyl amine (4.00
mL, 23.0 mmol) were added to a solution of the compound from
Example 1, step 2 (1.704 g, 6.24 mmol) in anhydrous DMF (10 mL) at
RT. The reaction was stirred at RT for 2.5 h. The reaction was
diluted with saturated aqueous sodium bicarbonate (50 mL) and
extracted with ether (150+100 mL). The organic phases were washed
with water (100+50 mL), dried (MgSO.sub.4) and concentrated in
vacuo to give 2.994 g of a yellow oil.
[0129] Step 5: Saturated aqueous lithium hydroxide (3 mL) was added
to a solution of the compound from step 4 (2.994 g, 6.24 mmol) in
THF (10 mL) at RT. After 1 h, saturated aqueous sodium bicarbonate
(50 mL) was added. The slurry was then cooled to 0.degree. C.,
acidified with 1N HCl and extracted with ethyl acetate (400+100
mL). The combined organic phases were dried (MgSO.sub.4) to give
2.238 g of a light yellow powder.
[0130] Step 6: 2 M aqueous sodium carbonate (7.55 mL, 15.1 mmol)
was added to a solution of the compound from step 3 (0.820 g, 2.00
mmol), the compound from 5 (0.883 g, 2.00 mmol) and
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride (0.267 g, 0.327 mmol) in anhydrous DMF (10 mL). The
reaction was warmed to 85.degree. C. for 2.5 h. It was then cooled
to RT, diluted with ethyl acetate (400 mL), and extracted with
water (150 mL). The aqueous phase was washed with ethyl acetate
(100 mL) and acidified with 1N HCl. The resulting slurry was
extracted with ethyl acetate (400+100 mL), the combined organic
phases were dried (MgSO.sub.4) and concentrated in vacuo to give a
white solid. The white solid was slurried with ether, filtered and
air dried to give 0.932 g of product. Concentration in vacuo of the
mother liquor produced another 0.359 g of desired product.
[0131] Step 7: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (0.200 mL,
1.23 mmol), HATU (0.434 g, 1.14 mmol) and diisopropylethyl amine
(0.300 mL, 1.72 mmol) were added to a solution of the compound from
step 6 (0.359 g, 0.557 mmol) in anhydrous DMF (3 mL) at RT. The
reaction was stirred at RT for 3 h. The reaction was diluted with
ethyl acetate (300 mL), washed with saturated aqueous sodium
carbonate (50 mL.times.2), dried (MgSO.sub.4) and concentrated in
vacuo to give 0.675 g of an oil.
[0132] Step 8: TFA (4 mL) and water (0.100 mL) was added to a
solution of the compound from step 7 (0.675 g) in acetonitrile (3.0
mL). After 4 h, the reaction was concentrated in vacuo and purified
via reverse phase HPLC (acetonitrile/water) to give 0.123 g of
product.
Example 4
Chemical Synthesis of Compound 10
[0133] Step 1: steps 1 and 2 of EXAMPLE 1 were carried out.
[0134] Step 2: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (5.3 g,
34.4 mmol), HATU (17.5 g, 46.0 mmol) and diisopropylethyl amine (17
mL, 97.6 mmol) were added to a solution of the compound from step 1
(8.50 g, 31.1 mmol) in anhydrous DMF (100 mL) at RT. The reaction
was stirred at RT for 0.75 h. The reaction was diluted with ethyl
acetate, washed with saturated aqueous sodium bicarbonate and
water, dried (MgSO.sub.4) and concentrated in vacuo to give 13 g of
the desired product.
[0135] Step 3: The compound from step 2 (6.5 g, 15.8 mmol) was
dissolved in 1:1 THF/saturated aqueous lithium hydroxide (50 mL) at
RT. After 2 h, the reaction was concentrated in vacuo, acidified
with saturated ammonium chloride and concentrated in vacuo. The
resulting solid was slurried in THF and filtered. The filtrate was
concentrated in vacuo to give the desired product.
[0136] Step 4: L-cyclohexylglycine tbutyl ester.HCl (3.75 g, 15.0
mmol), HATU (7.5 g, 19.7 mmol) and diisopropylethyl amine (10 mL,
57 mmol) were added to a solution of the compound from step 3 in
anhydrous DMF (200 mL) at RT. The reaction was stirred at RT for
0.5 h. The reaction was diluted with ethyl acetate, washed with
saturated sodium bicarbonate and water, dried (MgSO.sub.4) and
concentrated in vacuo to give the desired product.
[0137] Step 5: A solution of the compound from step 2 (6.5 g, 15.8
mmol), bis(pinacolate)diboran (4.4 g, 17.3 mmol),
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride (0.388 g, 0.475 mmol) and potassium acetate (4.7 g,
47.9 mmol) in anhydrous DMF (100 mL) was warmed to 80.degree. C.
under a nitrogen atmosphere for 2 h. The reaction was then cooled
to RT, diluted with ethyl acetate, washed with 5% citric acid and
5% sodium bicarbonate, dried (MgSO.sub.4) and concentrated in vacuo
to give a brown oil.
[0138] Step 6: 2 M aqueous sodium carbonate (40 mL, 80 mmol) was
added to a solution of the compound from step 4, the compound from
step 5 and
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II).cndot.methyl-
ene chloride in anhydrous DMF (100 mL). The reaction was warmed to
80.degree. C. overnight. It was then cooled to RT, acidified with
saturated ammonium chloride and extracted with THF (10.times.). The
combined organic phases were dried (MgSO.sub.4) and concentrated in
vacuo to give the carboxylic acid. One half of the material was
used as crude material and half was purified via reverse phase HPLC
(acetonitrile/water).
[0139] Step 7: Diethylcyanomethylphosphonate (2.7 mL, 16.7 mmol)
was added dropwise to a slurry of NaH/mineral oil (0.730 g, 19.0
mmol) in anhydrous THF (10 mL) at RT. After the initial bubbling
ceased, the reaction was warmed to reflux for 0.25 h. A solution of
3-nitrobenzophenone (1.90 g, 8.36 mmol) in anhydrous THF (20 mL)
was added dropwise and the reaction warmed at reflux for 3 h. After
cooling to RT, the reaction was poured into citric acid solution
and extracted with ethyl acetate (2.times.). The combined organic
phases were washed with water, dried (MgSO.sub.4) and concentrated
in vacuo. The residue was purified via flash column chromatography
(methylene chloride) to give 1.81 g of an oil.
[0140] Step 8: A slurry of 10% Pd/C (0.500 g) and the compound from
step 7 (1.81 g) in ethyl acetate (50 mL) was subjected to a
hydrogen atmosphere (60 psi) at RT. After 18 h the reaction slurry
was filtered and concentrated in vacuo to give 1.50 g of the
desired product.
[0141] Step 9: 1M Borane tetrahydrofuran complex/THF (28 mL, 28
mmol) was added to a solution of the compound from step 8 (1.50 g)
in anhydrous THF. The reaction was warmed to reflux for 3 days,
cooled to RT and quenched with methanol. After the initial bubbling
had ceased, more methanol (15 mL) was added and the reaction warmed
to reflux for 0.5 h. The reaction was concentrated in vacuo,
diluted with methanol, filtered and the filtrate concentrated in
vacuo to give the desired product.
[0142] Step 10: The compound from step 9 (0.127 g, 0.561 mmol),
HATU (0.110 g, 0.289 mmol) and diisopropylethyl amine (excess) were
added to a solution of the compound from step 6 in anhydrous DMF (5
mL) at RT. The reaction was stirred at RT for 0.5 h. The reaction
was diluted with ethyl acetate, washed with saturated sodium
bicarbonate and water, dried (MgSO.sub.4), concentrated in vacuo
and purified via reverse phase HPLC to give 0.005 of the desired
product.
[0143] Step 11: A solution of the compound from step 10 in 1:1
TFA/methylene chloride was stirred at RT for 2 h, concentrated in
vacuo and purified via reverse phase HPLC to give 0.0015 g of the
desired product.
Example 5
Chemical Synthesis of Compound 11
[0144] Step 1: 4-nitrodiphenylmethane (1.00 g, 4.69 mmol) was
slowly added to a slurry of potassium t-butoxide (1.05 g, 9.36
mmol) in anhydrous DMF (20 mL) at RT. After 5 min. t-butyl
bromoacetate (1.0 mL, 6.77 mmol) was added and the reaction stirred
overnight at RT. The reaction was diluted with ether, washed with
5% citric acid solution and water, dried (MgSO.sub.4) and
concentrated in vacuo to give an oil. The oil was purified via
flash column chromatography (60:40 methylene chloride/hexanes) to
give a colorless solid.
[0145] Step 2: The compound from step 1 was dissolved in 1:1
TFA/methylene chloride (20 mL) at RT. After 0.5 h the reaction
solution was concentrated in vacuo. The residue was dissolved in
anhydrous methylene chloride, DMF (1 drop) and 2M oxalyl
chloride/methylene chloride (10 mL, 20 mmol) was added. After 0.5 h
bubbling ceased, and the reaction was concentrated in vacuo. The
residue was dissolved in toluene (25 mL) and ammonia gas was
bubbled into the reaction for 0.5 h. The reaction was diluted with
ethyl acetate, washed with sodium bicarbonate solution, dried
(MgSO.sub.4) and concentrated in vacuo to give 0.560 g of a
colorless oil.
[0146] Step 3: 1M Borane tetrahydrofuran complex/THF (10 mL, 10
mmol) was added to a solution of the compound from step 2 (0.560 g)
in anhydrous THF (10 mL). The reaction was warmed to reflux
overnight, cooled to RT and quenched with methanol. After the
initial bubbling had ceased, more methanol (10 mL) was added and
the reaction warmed to reflux for 0.5 h. The reaction was
concentrated in vacuo, and purified via flash column chromatography
(80:20:2 ethyl acetate/methanol/triethylamine) to give 0.130 g of a
light yellow oil.
[0147] Step 4: Diisopropylethylamine (0.200 mL, 1.12 mmol) was
added to a solution of the compound from step 3 (0.130 g, 0.507
mmol), the compound from Example 4, step 6 (0.250 g, 0.313 mml) and
HATU (0.200, 0.526 mmol) in anhydrous DMF (10 mL) at RT., After 0.5
h the reaction was concentrated in vacuo, dissolved in ethyl
acetate, washed with saturated sodium bicarbonate and water, dried
(MgSO.sub.4), concentrated in vacuo and purified via reverse phase
HPLC (acetonitrile/water) to give 0.140 g of an off-white
powder.
[0148] Step 5: A solution of the compound from step 4 (0.010 g) in
1:1 TFA/methylene chloride (4 mL) was stirred at RT for 2 h. The
reaction was then concentrated in vacuo and purified via reverse
phase HPLC (acetonitrile/water) to give a white solid.
Example 6
Chemical Synthesis of Compound 9
[0149] Step 1: 2M (trimethylsilyl)diazomethane/hexanes (0.100 mL,
0.200 mmol) was added to a solution of the compound from Example 3,
step 8 (0.020 g, 0.023 mmol) in acetonitrile (0.5 mL). The reaction
was stirred at RT for 0.25 h, concentrated in vacuo and purified
via reverse phase HPLC (acetonitrile/water) to give a white
solid.
Example 7
Binding Assay to Assess whether a compound Binds CD154 and Inhibits
CD40:CD154 Interaction
[0150] 1) Ninety-six well plates of Nunc Maxisorp were coated with
100 .mu.l of 5-10 .mu.g/ml human CD40-Ig produced in Pichia cells,
(in PBS pH 7.2) per well, covered with plate sealers (Costar, Cat.
No. 3095) and stored overnight at 4.degree. C.
[0151] 2) The CD40-Ig-coated plates were shook out and blotted
dry.
[0152] 3) The CD40-Ig-coated plates were blocked with 300 .mu.l per
well of PBS (0.05% Tween-20, 1% BSA, pH 7.2) at RT for one hour or
in the refrigerator for up to 3 weeks.
[0153] 4) The CD40-Ig-coated plates were washed 3.times. with
PBS+0.05% Tween-20 pH 7.4 with plate washer at RT.
[0154] 5) The plates were optionally blotted dry.
[0155] 6) Primary Screening of compounds: [0156] a. Compounds were
titrated in 100 mM Hepes+0.005% BSA pH 7.2 in Titertube (Bio-Rad
Laboratories) and 50 .mu.l of titrated compounds was then added
into each well of the plate. IC50 parameters: [0157] One 7 point
curve per compound was generated where each point was run in
triplicate. A 2.times. dilution series was used to generate the
curve. [0158] Top concentration in assay of test compound was 125
.mu.M. [0159] b. Fifty .mu.l/well of 0.07-1.6 .mu.g/ml biotin-CD154
in 100 mM Hepes+0.005% BSA (pH 7.2) was added per well. Final
concentration of biotin-CD154 was 0.035-0.08 .mu.g/ml. It was mixed
with compound in each well in the plate. [0160] c. The plates were
covered with plate sealer and incubated for one-hour at RT.
[0161] 7) The plates were washed 3.times. with plate washer with
PBS+0.05% Tween-20 (pH 7.4) at RT.
[0162] 8) The plates were optionally blotted dry.
[0163] 9) 100 .mu.l of a 1/300,000 dilution of HRP-Strep/Avidin
(Jackson Immuno Research Code3 #016-030-084) was added to each
well. Dilution buffer was: PBS+0.05% Tween-20. The plates were
covered with plate sealer and incubated for one hour at room
temperature.
[0164] 10) The plates were washed with plate washer 3.times. at
RT.
[0165] 11) The plates were optionally blotted dry.
[0166] 12) 100 .mu.l/well of TMB(1-component) (Kirkegarrd &
Perry Labs, Prod #50-76-04) was added to each well and incubated at
RT about 10 min.
[0167] 13) 100 .mu.l/well of 0.18M H.sub.2SO.sub.4 was added to
stop reaction in the plates.
[0168] 14) The plates were read within 30 minutes of stopping the
reaction at 450 nM on a microplate reader (Molecular Devices
Co.).
[0169] Compounds that bound to CD154 and blocked CD40:CD154
interaction prevented CD154 from binding to immobilized CD40-Ig and
gave a low absorbance reading.
Controls:
[0170] 1. Total reaction: Reaction well contained 50 .mu.l of 100
mM Hepes/0.005% BSA (no test compound) [0171] 2. Positive control:
Reaction well contained 10 .mu.l of 1 mg/ml 5c8 (anti-CD154 mAb)
and 50 .mu.l of 100 mM Hepes/0.005% BSA. [0172] 3. Blank control:
Reaction well contained 100 .mu.l 100 mM Hepes/0.005% BSA. [0173]
4. Compound control: Titration series in triplicate of
BIO-002108-00.
[0174] BIO-002108-00 is one of the derivatives of suramin
(CalBiochem catalog number 574625). It has been demonstrated that
BIO-002108-00, as well as other derivatives of suramin, is a
CD40:CD154 binding interrupter. BIO-002108-00 has an IC50 in the A
range (less than 50 .mu.M) for CD40:CD154 binding interruption as
determined by this assay. BIO-002108-00 has an IC50 of greater than
125 .mu.M (average value) for TNF-.alpha.:TNFRp60 binding
interruption. BIO-002108-00 has the following structure:
##STR142##
[0175] It was synthesized by the following method:
Step 1
[0176] 2M (Trimethylsilyl)diazomethane/hexanes (50 mL, 100 mmol)
was added dropwise to a solution of tech.
[0177] 3-amino-2-naphthoic acid (3 g, 12.8 mmol) in methanol (300
mL) at RT. A grey precipitate was formed. The reaction was
centrifuged, the supernatant was decanted, concentrated in vacuo
and purified via flash column chromatography (methylene chloride)
to give 1.78 g of a yellow crystalline solid.
Step 2
[0178] A solution of the compound from step 1 (0.205 g, 1.02 mmol)
and 3-nitrobenzenesulfonyl chloride (0.250 g, 1.13 mmol) in
pyridine (5 mL) was stirred at RT for 0.5 h. The reaction was then
poured into 1N aqueous hydrochloric acid (100 mL) and extracted
with methylene chloride (2.times.100 mL). The combined organic
phases were washed with 1N aqueous hydrochloric acid (100 mL) and
water (100 mL), dried (MgSO.sub.4) and concentrated in vacuo to
give 0.380 g of a reddish syrup.
Step 3
[0179] 10% Pd/C (0.500 g) was added to a solution of the compound
from step 2 (0.380 g) in 1:1 ethyl acetate/methanol (50 mL). The
reaction was stirred under a hydrogen atmosphere (1 atm) for 16 h.
The reaction was centrifuged, the supernatant decanted,
concentrated in vacuo and purified via flash column chromatography
(95:5 methylene chloride/ethyl acetate) to give 0.278 g of a white
solid.
Step 4
[0180] A solution of the compound from step 3 (0.060 g, 0.169 mmol)
and 1,1'-carbonyldiimidazole (0.030 g, 0.185 mmol) in anhydrous THF
(1 mL) was refluxed overnight. The reaction was then cooled to RT,
concentrated in vacuo and purified via reverse phase HPLC
(acetonitrile/water) to give 0.018 g of a white solid.
Step 5
[0181] Lithium hydroxide (0.100 g, 2.28 mmol) was added to a
solution of the compound from step 4 (0.018 g) in 2:1:1
THF/methanol/water (20 mL). After stirring overnight at RT, the
reaction was poured into 1N aqueous hydrochloric acid (25 mL) and
extracted with ethyl acetate (2.times.50 mL). The combined organic
phases were dried (MgSO.sub.4) and purified via reverse phase HPLC
(acetonitrile/water) to give 0.001 g white solid. MS (ESP-) 709.09
(M-1)
Example 8
Binding Assay to Assess whether a Compound Binds to TNF-.alpha. and
Inhibits TNF-.alpha./TNFRp60 Interaction
[0182] 1) Ninety-six well plates of Nunc Maxisorp were coated with
100 .mu.l/well of 0.6 ug/ml tumor necrosis factor receptor p60
(TNFRp60) (Browning, J. L.; Douglas, I.; Ngam-ek, A.; Bourdon, P.
R.; Ehrenfels, B. N.; Miatkowski, K.; Zafari, M.; Yampaglia, A. M.;
Lawton, P.; Meier, W.; Benjamin, C. P.; Hession, C. J. Immunol.
1995, 154, 33-46) in PBS, pH 7.2, covered with plate sealers
(Costar, Cat. No. 3095) and stored overnight at 4.degree. C.
[0183] 2) The coated plates were shook out and blotted dry.
[0184] 3) TNFRp60-coated plates were blocked with 300 .mu.l per
well of PBS (0.05% Tween-20, 1% BSA, made from media prep) at
4.degree. C. for one hour. Plates were stored for 3 weeks at
4.degree. C.
[0185] 4) The plates were washed 3.times. with PBS+0.05% Tween-20)
by plate washer at room temperature.
[0186] 5) The plates were optionally blotted dry.
[0187] 6) Primary Screening of compounds
[0188] Compounds were titrated in 100 mM Hepes+0.005% BSA, pH 7.2
in Titertube (Bio-Rad Laboratories) and then 50 .mu.l of titrated
compounds was added into each well of the plate.
[0189] IC50 parameters: One 7 point curve per compound was
generated where each point was run in triplicate. A 2.times.
dilution series was used to generate the curve. The top
concentration in assay of test compound was 125 .mu.M.
[0190] 50 .mu.l/well of 8 ng/ml Biotin-TNF-.alpha. (R&D System)
in 100 mM Hepes+0.005% BSA, pH 7.2 were added. The final
concentration of Biotin-TNF.alpha. was 4 ng/ml. It was mixed with
compound in each well in the plate. The plates were covered with
sealer and incubated one hour at RT.
[0191] 7) The plates were washed 3.times. by plate washer with
PBS+0.05% Tween-20 at RT.
[0192] 8) The plates were optionally blotted dry.
[0193] 9) 100 .mu.l of Streptavidin-HRP (diluted at 1:300,000 in
PBS+0.05% Tween-20) was added to each well. The plates were covered
with sealer and incubated one hour at RT.
[0194] 10) The plates were washed by plate washer 3.times. at
RT.
[0195] 11) The plates were optionally blotted dry.
[0196] 12) 100 .mu.l/well of TMB(1-component) (Kirkegarrd &
Perry Labs, Prod #50-76-04) were added at RT and incubated about 10
min in dark environment.
[0197] 13) 100 .mu.l/well of 0.18M H.sub.2SO.sub.4 was added to
stop the reaction.
[0198] 14) The plates were read within 30 minutes of adding stop
reagent at 450 nM by microplate reader (Molecular Devices Co.)
Controls:
[0199] Total reaction: Reaction well contained 50 .mu.l of 100 mM
Hepes/0.005% BSA (no test compound). [0200] Positive control:
Reaction well contained 10 .mu.l of 1.5 mg/ml anti-TNF-.alpha. and
50 ul of 100 mM Hepes/0.005% BSA. [0201] Blank control: Reaction
well contains 100 .mu.l 100 mM Hepes/0.005% BSA. [0202] Compound
control: Titration series in triplicate of BIO-002108-00.
[0203] Compounds that blocked TNF-.alpha./TNFRp60 interaction
prevented TNF-.alpha. from binding to immobilized TNFRp60 and gave
a low absorbance reading.
[0204] The above-described assay may also be carried out using
TNFRp60-Fc in place of TNFRp60.
[0205] In addition to the foregoing assays (Examples 7-8), the
following binding assay may be used to assess activity of compound
according to this invention.
Example 9
ICAM Assay
[0206] 1. Set up wells containing: [0207] Different concentrations
of compounds in 50 uL of RPMI (10% serum, 4 mM glutamine,
pen/strep) [0208] 0.5 ug of CD40 ligand in 50 uL [0209]
2.times.10.sup.5 2G6 cells in 100 uL
[0210] Set up groups of cells only [0211] Cells+compound [0212]
Cells+ligand [0213] Cells+ligand+10 ug of 5c8 mAb
[0214] Each group should have a final volume in the well of 200 uL.
If the concentration of DMSO>0.22%, there should be that
control+ligand. [0215] b. Incubate the plates at 37.degree. C. for
18 hours in a CO.sub.2 incubator. [0216] c. Add 40 uL of lysis
buffer (part of sICAM-1 kit from Endogen EH-5400) to the wells and
pipet up and down. [0217] d. Shake for M hour on rocker platform RT
at 150 RPMs/min. [0218] e. Make a 1:1 dilution (40 uL of lysate+40
uL of diluent from sICAM-1 kit from Endogen EH-5400) in Falcon
microtest flexible assay plates. [0219] f. Pipet 25 uL of this
mixture into ELISA kit plate (sICAM-1 precoated plate, Endogen)
Also, pipet 25 uL of standards on the plate. [0220] g. Add 75 uL of
HRP- conjugated Anti-ICAM-1 antibody (from sICAM-1 kit from Endogen
EH-5400) to all wells except the blank. [0221] h. Cover the plate
and incubate 2 hrs at RT on shaking platform 150 RPM/min. [0222] i.
Wash plate using T-Cell protocol on plate washer. The wash buffer
is PBS+0.05% tween-20. [0223] j. Dissolve 1 tablet of OPD per 5 mL
of substrate buffer (from sICAM-1 kit from Endogen EH-5400, use 15
mL/plate). [0224] k. Pipet 100 uL into each well. [0225] l.
Incubate uncovered for 30 minutes at RT. [0226] m. Add 50 uL of 2N
H.sub.2SO.sub.4 to stop the, reaction. [0227] n. Read absorbance at
490 nM. [0228] Positive control: Cells Plus ligand [0229] Negative
control: Cells only, no ligand [0230] Experimental control: Cells
plus ligand plus 10 ug of 5c8 mAb. Equivalents
[0231] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative of, rather than limiting on, the
invention disclosed herein. All changes which come within the
meaning and range of equivalency of the claims are intended to be
embraced therein.
* * * * *