U.S. patent application number 15/972619 was filed with the patent office on 2018-12-27 for chiral compounds of varying conformational rigidity and methods of synthesis.
The applicant listed for this patent is THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Thomas Kodadek, Glenn C. Micalizio, Mohosin Sarkar.
Application Number | 20180371014 15/972619 |
Document ID | / |
Family ID | 47832581 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371014 |
Kind Code |
A1 |
Micalizio; Glenn C. ; et
al. |
December 27, 2018 |
CHIRAL COMPOUNDS OF VARYING CONFORMATIONAL RIGIDITY AND METHODS OF
SYNTHESIS
Abstract
Synthesis of compounds having varying degrees of conformational
rigidity is obtained via a low cost, high yield and efficient
synthetic reactions. The library of compounds is structurally
diverse, having at least one or more chiral centers and providing
large numbers of compounds having building block diversity and
substantial scaffold diversity. The compounds further provide a
novel method for obtaining candidate therapeutic agents for
prevention, treatment or diagnosis of diseases.
Inventors: |
Micalizio; Glenn C.; (Palm
Beach Gardens, FL) ; Kodadek; Thomas; (Jupiter,
FL) ; Sarkar; Mohosin; (Palm Beach Gardens,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE SCRIPPS RESEARCH INSTITUTE |
La Jolla |
CA |
US |
|
|
Family ID: |
47832581 |
Appl. No.: |
15/972619 |
Filed: |
May 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14343426 |
May 21, 2014 |
9963481 |
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PCT/US2012/054135 |
Sep 7, 2012 |
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15972619 |
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61531810 |
Sep 7, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 57/64 20130101;
C07K 5/0205 20130101; C07B 53/00 20130101; C07C 57/54 20130101;
A61K 38/06 20130101; C07C 57/52 20130101; C07C 51/00 20130101; C07D
405/14 20130101; G01N 33/5008 20130101; A61K 31/19 20130101; C07C
57/60 20130101; C07K 5/08 20130101; C07B 2200/07 20130101; A61K
45/06 20130101; A61K 31/192 20130101; C07K 5/0804 20130101; C07B
2200/11 20130101; G01N 2800/7028 20130101; C07D 405/10
20130101 |
International
Class: |
C07K 5/08 20060101
C07K005/08; A61K 38/06 20060101 A61K038/06; A61K 31/19 20060101
A61K031/19; C07C 57/54 20060101 C07C057/54; C07C 51/00 20060101
C07C051/00; G01N 33/50 20060101 G01N033/50; C07C 57/64 20060101
C07C057/64; C07C 57/60 20060101 C07C057/60; C07C 57/52 20060101
C07C057/52; A61K 45/06 20060101 A61K045/06; C07K 5/02 20060101
C07K005/02; C07K 5/083 20060101 C07K005/083; C07B 53/00 20060101
C07B053/00; C07D 405/10 20060101 C07D405/10; C07D 405/14 20060101
C07D405/14; A61K 31/192 20060101 A61K031/192 |
Claims
1. A method of synthesizing a chiral monomer comprising: obtaining
a stereodefined allylic alcohol via a stereoselective aldol
reaction or a related transformation reaction and proceeding via
stereoselective allylic transposition of the resulting allylic
alcohol; and converting the resulting rearranged product to a
chiral acid having a general structure of Formula I':
##STR00009##
2. The method of claim 1, wherein R.sup.1 and R.sup.2 comprise a
molecular architecture compatible with the synthesis of Formula I'
or introduced after synthesis of a central pentenoic acid of
Formula I'.
3. The method of claim 1, wherein two or more monomers of Formula
I' are optionally oligomerized, the oligomerization of Formula I'
proceeding via a 1- or 2-directional homologation or
functionalization of Formula I'.
4. The method of claim 3, wherein X and Y comprise any molecule
compatible with the oligomerization of Formula I'.
5. The method of claim 2, wherein an oligomer or polymer of Formula
I' comprises homogeneous monomers, heterogeneous monomers or
combinations thereof.
6. A compound comprising a monomer set forth in Formula I:
##STR00010## wherein, X and Y comprise any molecular motifs or
functional groups compatible with oligomerization of two or more
monomers of Formula I.
7. The compound of claim 6, wherein an oligomeric compound
comprises at least two or more monomers represented by Formula I
comprising a chiral center in an R configuration, an S
configuration or multiple combinations thereof.
8. The compound of claim 6, wherein the monomer of Formula I is a
pentenoic amide.
9. The compound of claim 8, wherein the pentenoic amide is a
central N-substituted 5-amino-2, 4-dialkyl-3-pentenoic amide.
10. The compound of claim 6, wherein a monomer of Formula I
comprises substitutions which maintain conformational control about
a .beta., .gamma.-unsaturated carbonyl and minimize allylic
strain.
11. The compound of claim 6, wherein monomers or oligomers of
Formula I comprise substitutions having varying degrees of
flexibility imparted by monomers comprising the backbone.
12. The compound of claim 6, wherein uni- or bidirectional
functionalization of Formula I produces a higher molecular weight
compound comprising Formula I having a structure whereby
conformation is controlled by minimization of A-1,3 strain inherent
to a substituted .beta.,.gamma.-unsaturated carbonyl.
13. The compound of claim 12, wherein the higher molecular weight
compound of Formula I comprises oligomers having repeating units of
Formula I and/or repeating monomers of Formula I having different
substitutions R.sup.1 and R.sup.2, wherein R.sup.1 and R.sup.2
comprise any molecular architecture capable of forming a bond with
the monomers or oligomers of Formula I.
14. The compound of claim 13, wherein R.sup.1 and R.sup.2
independently comprise OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5,
halide, alkyl, linear alkyl, branched alkyl, heteroatom-substituted
alkyl, unsaturated and polyunsaturated linear and branched
hydrocarbons, alkenyl, cycloalkyl, aryl, heteroaryl, heteroaryl,
heterocycloalkyl, heteroatom-substituted unsaturated and
polyunsaturated linear and branched hydrocarbons, cycloalkyl,
heteroatom-substituted cycloalkyl, saturated and unsaturated
heterocycles, substituted cycloalkyl, substituted and unsubstituted
aromatic, substituted and unsubstituted heteroaromatic; R.sup.3
independently comprises H, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, heterocycloalkyl; and,
R.sup.4 independently comprises H, OR.sup.3, alkyl, aryl, or
heteroaryl.
15. The compound of claim 6, wherein X independently comprises
OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5, H, halide, alkyl, alkenyl,
cycloalkyl, aryl, heteroaryl, heteroaryl, heterocycloalkyl; R.sup.3
independently comprises amide, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, heterocycloalkyl; R.sup.4
independently comprises H, OR.sup.4, alkyl, aryl, heteroaryl; *C is
a chiral center [(R) or (S)]; R.sup.1 independently comprises
alkyl, aryl, heteroaryl, alkenyl, OR.sup.4; R.sup.2 independently
comprising alkyl, cycloalkyl, aryl, heteroaryl, alkynyl, alkenyl,
heterocycloalkyl; Y independently comprises a halide, NHR.sup.4,
NR.sup.4R.sup.5, OH, OR.sup.3, or C(O)X.
16. A method of identifying a candidate therapeutic agent,
comprising: screening a library comprising one or more monomers,
oligomers, or polymers of Formula I: ##STR00011## contacting a
biological sample, cell, tissue, or molecule in solution or
attached to a solid or semi-solid support, with a compound of
Formula I; assaying for any desired therapeutic effects; and,
identifying a candidate therapeutic agent.
17. The method of claim 16, wherein for Formula I: X independently
comprises OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5, H, halide, alkyl,
alkenyl, cycloalkyl, aryl, heteroaryl, heteroaryl,
heterocycloalkyl; R.sup.3 independently comprises amide, alkyl,
cycloalkyl, alkenyl, aryl, heteroaryl, NR.sup.4R.sup.5, carboxyl,
heterocycloalkyl; R.sup.4 independently comprises H, OR.sup.4,
alkyl, aryl, heteroaryl; *C is a chiral center [(R) or (S)];
R.sup.1 independently comprises alkyl, aryl, heteroaryl, alkenyl,
OR.sup.4; R.sup.2 independently comprising alkyl, cycloalkyl, aryl,
heteroaryl, alkynyl, alkenyl, heterocycloalkyl; Y independently
comprises a halide, NHR.sup.4, NR.sup.4R.sup.5, OH, OR.sup.3, or
C(O)X.
18. The method of claim 16, wherein desired therapeutic effects
comprise: tumor cell death, inhibition of viral replication,
cytolysis of virally infected cells, modulation of receptors,
modulation of growth factors, modulation of cytokines, modulation
of cellular factors, modulation of immune cells, anti-bacterial
effects, anti-parasitic effects or combinations thereof.
19. A pharmaceutical composition comprising a compound having a
structure of Formula I: ##STR00012##
20. The pharmaceutical composition of claim 19, wherein X and Y
comprise any molecular motif or functional group compatible with
oligomerization of two or more monomers of Formula I.
21. The pharmaceutical composition of claim 19, wherein R.sup.1 and
R.sup.2 comprise any molecular architecture capable of forming a
bond with monomers or oligomers of Formula I.
22. The pharmaceutical composition of claim 21, wherein R.sup.1 and
R.sup.2 independently comprise OR.sup.3, NHR.sup.4,
NR.sup.4R.sup.5, halide, alkyl, linear alkyl, branched alkyl,
heteroatom-substituted alkyl, unsaturated and polyunsaturated
linear and branched hydrocarbons, alkenyl, cycloalkyl, aryl,
heteroaryl, heteroaryl, heterocycloalkyl, heteroatom-substituted
unsaturated and polyunsaturated linear and branched hydrocarbons,
cycloalkyl, heteroatom-substituted cycloalkyl, saturated and
unsaturated heterocycles, substituted cycloalkyl, substituted and
unsubstituted aromatic, substituted and unsubstituted
heteroaromatic; R.sup.3 independently comprises H, alkyl,
cycloalkyl, alkenyl, aryl, heteroaryl, NR.sup.4R.sup.5, carboxyl,
heterocycloalkyl; and, R.sup.4 independently comprises H, OR.sup.3,
alkyl, aryl, or heteroaryl.
23. The pharmaceutical composition of claim 19, wherein X
independently comprises OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5, H,
halide, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heteroaryl,
heterocycloalkyl; R.sup.3 independently comprises amide, alkyl,
cycloalkyl, alkenyl, aryl, heteroaryl, NR.sup.4R.sup.5, carboxyl,
heterocycloalkyl; R.sup.4 independently comprises H, OR.sup.4,
alkyl, aryl, heteroaryl; *C is a chiral center [(R) or (S)];
R.sup.1 independently comprises alkyl, aryl, heteroaryl, alkenyl,
OR.sup.4; R.sup.2 independently comprising alkyl, cycloalkyl, aryl,
heteroaryl, alkynyl, alkenyl, heterocycloalkyl; Y independently
comprises a halide, NHR.sup.4, NR.sup.4R.sup.5, OH, OR.sup.3, or
C(O)X.
24. A compound comprising: ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017##
25. A compound of Formula I comprising a monomer set forth in
Formula I: ##STR00018## wherein, X and Y comprise any molecular
motif or functional group compatible with oligomerization of
monomers of Formula I.; and, one or more monomers of an oligomer of
Formula I are conjugated to a cytotoxic agent or detectable
label.
26. The compound of claim 25, wherein R.sup.1 and R.sup.2 comprise
any molecular architecture capable of forming a bond with monomers
or oligomers of Formula I.
27. The compound of claim 26, wherein R.sup.1 and R.sup.2
independently comprise OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5,
halide, alkyl, linear alkyl, branched alkyl, heteroatom-substituted
alkyl, unsaturated and polyunsaturated linear and branched
hydrocarbons, alkenyl, cycloalkyl, aryl, heteroaryl, heteroaryl,
heterocycloalkyl, heteroatom-substituted unsaturated and
polyunsaturated linear and branched hydrocarbons, cycloalkyl,
heteroatom-substituted cycloalkyl, saturated and unsaturated
heterocycles, substituted cycloalkyl, substituted and unsubstituted
aromatic, substituted and unsubstituted heteroaromatic; R.sup.3
independently comprises H, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, heterocycloalkyl; and,
R.sup.4 independently comprises H, OR.sup.3, alkyl, aryl, or
heteroaryl.
28. The compound of claim 25, wherein X independently comprises
OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5, H, halide, alkyl, alkenyl,
cycloalkyl, aryl, heteroaryl, heteroaryl, heterocycloalkyl; R.sup.3
independently comprises amide, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, heterocycloalkyl; R.sup.4
independently comprises H, OR.sup.4, alkyl, aryl, heteroaryl; *C is
a chiral center [(R) or (S)]; R.sup.1 independently comprises
alkyl, aryl, heteroaryl, alkenyl, OR.sup.4; R.sup.2 independently
comprising alkyl, cycloalkyl, aryl, heteroaryl, alkynyl, alkenyl,
heterocycloalkyl; Y independently comprises a halide, NHR.sup.4,
NR.sup.4R.sup.5, OH, OR.sup.3, or C(O)X.
29. The compound of claim 25, wherein a cytotoxic agent comprises:
a toxin, radioactive molecule, chemotherapeutic agent, an inhibitor
of replication, or combinations thereof.
30. The compound of claim 25, wherein the detectable label
comprises: a fluorescent molecule, radioactive molecule, a metal,
or dye.
31. A method of treating a hematopoietic malignancy in vivo,
comprising: administering to a patient in need thereof an effective
amount of a pharmaceutical composition comprising at least one
compound of claims 6, 24 or 25, wherein the compound is conjugated
to one or more cytotoxic agents and specifically binds to a
hematopoietic malignant cell; and, treating the hematopoietic
malignancy
32. The method of claim 31, wherein the hematopoietic malignancy
comprises: B cell malignancies or neoplasms, chronic
myeloproliferative diseases, myelodysplastic/myeloproliferative
diseases, myelodysplastic syndromes, acute myeloid leukemias, B
cell neoplasms, T-cell and NK-cell neoplasms, Hodgkin's lymphoma,
histiocytic and dendritic cell neoplasms or mastocytosis.
33. The method of claim 32, wherein the B cell malignancy
comprises: B cell chronic lymphocytic leukemia (B-CLL), B cell
lymphomas, aggressive B-cell lymphoma, Hodgkin's disease, B cell
non-Hodgkin's lymphoma (NHL), lymphomas, Waldenstrom's
macroglobulinaemia (lymphoplasmacytic lymphoma or immunocytoma),
central nervous system lymphomas, leukemias, acute lymphoblastic
leukemia (ALL), hairy cell leukemia, chronic myoblastic leukemia),
myelomas, multiple myeloma), small lymphocytic lymphoma, B cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma
of bone, extraosseous plasmacytoma, extra-nodal marginal zone B
cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal
marginal zone B cell lymphoma, follicular lymphoma, mantle cell
lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large
B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma,
B cell proliferations of uncertain malignant potential,
lymphomatoid granulomatosis, and post-transplant
lymphoproliferative disorder.
34. The method of claim 31, whereby the B cell malignancy is
B-CLL.
35. The method of claim 31, further comprising administering to a
patient a chemotherapeutic agent or radiotherapy.
36. A method of diagnosing a hematopoietic malignancy comprising
administering to a patient, or contacting a biological sample in
vitro with a compound of claims 6, 24 or 25, wherein the compound
is conjugated to a detectable label and the compound specifically
binds to a hematopoietic malignant cell in vivo or in vitro; and,
diagnosing a hematopoietic malignancy.
37. The method of claim 36, wherein the hematopoietic malignancy is
B cell chronic lymphocytic leukemia (B-CLL).
38. A method of modulating an immune cell disease or disorder
comprising administering to a patient in need thereof an effective
amount of a pharmaceutical composition comprising at least one
compound of claims 6, 24 or 25, wherein the compound is conjugated
to one or more agents and specifically binds to an immune cell
receptor or ligand and modulates the immune cell mediated disease
or disorder.
39. The method of claim 38, wherein the immune cell disease or
disorder comprises: autoimmune diseases, inflammatory diseases,
transplantation rejection, lymphoproliferative diseases, allergies,
neuroinflammatory diseases and disorders thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority of U.S.
provisional patent application No. 61/531,810 entitled "CHIRAL
COMPOUNDS OF VARYING CONFORMATIONAL RIGIDITY AND METHODS OF
SYNTHESIS" filed Sep. 7, 2011, which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] Embodiments are directed to synthesis of libraries of novel
and structurally diverse chiral compounds having varying degrees of
conformational rigidity. The novel compounds have many uses
including diagnostics, prevention and treatment of diseases or
symptoms thereof.
BACKGROUND
[0003] Oligomerization is the central synthetic strategy by which
nature derives molecules with function. With only a small
collection of monomeric units, and bond-forming processes
compatible with the cellular environment, sequential union
(oligomerization) results in great molecular and functional
diversity. Examples include complex biological polymers like
proteins, nucleic acids, and carbohydrates, as well as small
molecule natural products (i.e. fatty acids, polyketides and
terpenes). The structural diversity of products derived from
oligomerization in nature is clearly vast, resulting in molecules
that have a range of properties and functions. In contrast to
Nature's oligomer-based approach to molecular diversification, the
impressive and elegant laboratory approaches to structural
diversity that define state-of-the-art synthetic solutions
typically embrace strategic and divergent reactivity of complex
organic intermediates (Schreiber, S. L. Target-oriented and
diversity-oriented organic synthesis in drug discovery. Science
287, 1964-1969 (2000); Tan, D. S. Diversity-oriented synthesis:
exploring the intersections between chemistry and biology. Nat.
Chem. Biol. 1, 74-84 (2005); Spiegel, D. A. et al. An
oligomer-based approach to skeletal diversity in small-molecule
synthesis. J. Am. Chem. Soc. 128, 14766-14767 (2006); Nielsen, T.
E. et al. Towards the optimal screening collection: a synthesis
strategy. Angew. Chem. Int. Ed. 47, 48-56 (2008)).
SUMMARY
[0004] This Summary is provided to present a summary of the
invention and to briefly indicate the nature and substance of the
invention. It is submitted with the understanding that it will not
be used to interpret or limit the scope or meaning of the
claims.
[0005] Embodiments are directed to synthesis of novel compounds and
methods for synthesizing such compounds.
[0006] In one embodiment, a compound comprises a molecule of
general structure I:
##STR00001##
[0007] Wherein X comprises OR.sup.3, NHR.sup.4, H or halide;
R.sup.3 comprises alkyl, aryl, carboxyl; R.sup.4 comprises H,
OR.sup.3, NR.sup.32, alkyl or aryl; * is a chiral center [(R) or
(S)]; R.sup.1 comprises alkyl, aryl, OR.sup.4; R.sup.2 comprises
alkyl, aryl, halo; Y comprises halide, NHR.sup.4, OH, C(O)X.
[0008] Other aspects are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A-1D are schematics showing natural and synthetic
oligomers, polyketide-derived natural products, and a
polyketide-inspired class of chiral and conformationally rigid
synthetic oligomer. FIG. 1A is a schematic showing a selection of
biological and biopolymer mimetics. FIG. 1B is a schematic showing
a selection of polyketide-derived natural products. FIG. 1C is a
schematic showing the general structure of COPAs--chiral oligomers
of N-substituted 5-amino-2,4-dialkyl-3-pentenoic amides. FIG. 1D is
a schematic showing the structural features that lead to the
rigidification of COPA oligomers.
[0010] FIG. 2: Stereochemistry of COPA backbone has a substantial
impact on skeletal shape and the disposition of side chains in
space. Low energy conformations of a collection of eight N-Me
substituted isomeric COPA tetramers (MMFF calculations--extracted
from a conformer distribution generated in Spartan-08). While
fixing the relative position of the C-terminus (boxed within the
3D-models), each diastereomer is depicted in its predicted low
energy conformation with colored spheres that highlight the
relative position of heteroatoms (green) and alkenes (blue). Not
easily depicted, but even more compelling, is that the conformer
distribution about each low energy conformation is substantially
restricted. In a head-to-head comparison with a polymethylated
peptoid tetramer where >12 conformations can be located within
1.8 kcal/mol of the low energy conformer, by MMFF calculations
COPAs are predicted to be substantially more rigid--in most cases,
only 1-2 conformations were located within 1.8 kcal of the low
energy conformation depicted. While these molecular mechanics
calculations are not thought to predict the solution phase
structure of these simple tetramers, the calculations provide a
uniform mathematical filter to support the unique characteristics
associated with this new class of synthetic oligomer.
[0011] FIGS. 3A-3F are a schematic representation showing the
chemical development of COPA oligomers: From general
oligomerization strategy, asymmetric synthesis and library
construction. FIG. 3A is a schematic showing the "sub-monomer"
style synthesis of peptoids. FIG. 3B is a schematic showing the
asymmetric synthesis of 5-chloro-2,4-dimethyl-3-pentenoic acid 1.
FIG. 3C is a schematic showing the use of 1 in solution phase
oligomerization. FIG. 3D shows a panel of monomers used in library
synthesis. FIG. 3E is a schematic showing general information
regarding resin and linker employed in solid-phase library
synthesis. FIG. 3F is a schematic showing the general structure of
libraries prepared from building blocks depicted in FIG. 3D--COPA
and peptoid tetramers.
[0012] FIGS. 4A-4C are schematic representations showing COPA
library, screening, structure elucidation and validation. FIG. 4A
is an embodiment of a general scheme for on-bead screening of a
COPA library against the DNA binding domain of p53 (p53-DBD,
residues 94 to 312) expressed with an epitope tag FLAG. TENTAGEL
beads bound to p53-DBD protein were visualized under a fluorescent
microscope by treating beads with anti-FLAG primary antibody and
anti-IgG secondary antibody conjugated to Quantum dot emitting red
fluorescent light at 655 nm. FIG. 4B: Sequence elucidation and
identification of a COPA tetramer that binds to the p53-DBD.
Sequence of the COPA tetramer was established by analysis of mass
spectral data derived from ETD-based fragmentation. FIG. 4C shows a
schematic of the fluorescence polarization assay for binding
affinity of fluorescein conjugated COPA tetramer (14a) against
p53-DBD, carbonic anhydrase II (CAH II from bovine erythrocyte),
platelet activating factor acetyl hydrolase (PAFAHIB3), and
bromodomain containing 4 (BRD4) proteins. A COPA tetramer with the
same linker region and different side chains on the amide nitrogens
was used as a control oligomer (co). The binding affinity of COPA
tetramer to p53-DBD was determined as K.sub.D.about.10 .mu.M.
[0013] FIG. 5 is a schematic representation showing the general
structure of a COPA library synthesized from chloropentenoic acids
(R and S configurations) and amine building blocks shown on
right.
[0014] FIG. 6 is a schematic representation of the screening of a
COPA library against CLL monoclonal antibodies (mAbs). (left) COPA
library was first screened against Goat-antihuIgG-Qdot 655 and
against total human IgG to reduce the possibility of false
positives before binding with CLL-mAbs. A total of 70 fluorescent
beads were isolated from initial screening. (right). Positive beads
from initial screening were subjected to binding again with pooled
human IgG. Any fluorescent beads were removed and the rest of the
beads were revalidated for binding with CLL-mAbs. A total of 28
beads with intense red fluorescent color were collected for further
processing.
[0015] FIG. 7 shows a MALDI-TOF spectrum (top) and LTQ-ETD tandem
MS spectrum (bottom) of a positive hit isolated from the screening
of a COPA library against CLL-mAbs.
[0016] FIG. 8 shows the structure of the resynthesized positive
hits isolated from the screening of COPA library against CLL-mAbs.
H0442 and H0478 were obtained from the screening of a second
library (ClAA-COPA-ClA library) against CLL-mAbs 068 and 183.
[0017] FIG. 9 shows the fluorescence polarization assay for the
COPA positive hits. The fluorescein-conjugated COPA compounds (10
nM) were incubated with increasing concentrations (1 nM to 4 .mu.M)
of CLL-mAbs for 1 h at room temperature in the dark and the
fluorescence polarization was measured using Envision Multilabel
Reader (2104) from Perkin Elmer using excitation and emission
wavelengths at 495 nm and 535 nm, respectively.
[0018] FIG. 10 is a schematic representation showing how small
molecules (green triangles) that target the BCR (blue V shape) of
CLL cells could be employed to eradicate these cells selectively.
This would involve the conjugation of the BCR targeting molecules
to a different small molecule that would recruit native antibodies.
Alternatively, the BCR-targeting molecule could be pre-conjugated
to a recombinant antibody.
[0019] FIG. 11 is a schematic representation showing that the
compound of general Formula I may function as a bifunctional
reagent and partake in a variety of mono- and bi-directional
homologation chemistry based on the nature of substituents X and/or
Y wherein either terminus can serve as a nucleophilic or
electrophilic motif. The chloroacid, shown by representative
Formula II was used in sequential amide bond forming reaction and
nucleophilic displacement, wherein Nu is a nucleophile and EI is an
electrophile.
[0020] FIG. 12 is a schematic representation showing an example of
oligomer synthesis, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and
R.sup.5 independently comprise any functionality that does not
interfere with the ability to perform the given chemical
homologation defined by: 1) amide bond formation, and 2)
nucleophilic substitution (displacement of the allylic chloride).
Examples of such functionality include, but is not limited to
alkyl, aryl, heteroaryl, NR.sup.5R.sup.5', OR.sup.5).
DETAILED DESCRIPTION
[0021] Embodiments are directed to novel synthetic compounds and
methods of synthesizing these compounds. The compounds have broad
utility for use in detection and treatment of disease.
[0022] The present invention is described with reference to the
attached figures, wherein like reference numerals are used
throughout the figures to designate similar or equivalent elements.
The figures are not drawn to scale and they are provided merely to
illustrate the invention. Several aspects of the invention are
described below with reference to example applications for
illustration. It should be understood that numerous specific
details, relationships, and methods are set forth to provide a full
understanding of the invention. One having ordinary skill in the
relevant art, however, will readily recognize that the invention
can be practiced without one or more of the specific details or
with other methods. The present invention is not limited by the
illustrated ordering of acts or events, as some acts may occur in
different orders and/or concurrently with other acts or events.
Furthermore, not all illustrated acts or events are required to
implement a methodology in accordance with the present
invention.
[0023] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the relevant art and will not be
interpreted in an idealized or overly formal sense unless expressly
so defined herein.
Definitions
[0024] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. Furthermore, to the extent
that the terms "including", "includes", "having", "has", "with", or
variants thereof are used in either the detailed description and/or
the claims, such terms are intended to be inclusive in a manner
similar to the term "comprising."
[0025] As used herein, the terms "comprising," "comprise" or
"comprised," and variations thereof, in reference to defined or
described elements of an item, composition, apparatus, method,
process, system, etc. are meant to be inclusive or open ended,
permitting additional elements, thereby indicating that the defined
or described item, composition, apparatus, method, process, system,
etc. includes those specified elements--or, as appropriate,
equivalents thereof--and that other elements can be included and
still fall within the scope/definition of the defined item,
composition, apparatus, method, process, system, etc.
[0026] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to
1% of a given value. Alternatively, particularly with respect to
biological systems or processes, the term can mean within an order
of magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
[0027] The terms, "compound" and "compounds" as used herein refers
to a compound encompassed by the generic formulae disclosed herein,
any subgenus of those generic formulae, and any forms of the
compounds within the generic and subgeneric formulae. Unless
specified otherwise, the term further includes the racemates and
stereoisomers, of the compound or compounds.
[0028] As used herein, the term "rigid" refers to a molecule's
propensity to adopt a defined conformation in preference to a
variety of competing conformations. Such "rigidity" can be imparted
by a variety of molecular features that provide a bias in favor of
a local conformation about a molecular skeleton. Within the context
of the invention, rigidification is imparted by allylic strain--a
governing feature of molecular conformation that is well understood
by those skilled in the art (Hoffmann, R. W. Allylic 1,3-Strain as
a Controlling Factor in Stereoselective Transformations, Chem. Rev.
1989, 89, 1841-1860; Hoffmann, R. W. Flexible Molecules with
Defined Shape-Conformational Design, Angew. Chem. Int. Ed., 1992,
31, 1124-1134).
[0029] The term "rigidity" refers to the degree of flexibility of
the molecule and includes the terms "flexible", semi-rigid",
"rigid" and all variations in between. The current invention
provides a means of addressing the conformational rigidity of a
small molecule and related oligomers that is based on introducing
allylic strain in the central molecular motif depicted in FIG.
1D.
[0030] The term "chiral" is used to describe an object that is
nonsuperimposable on its mirror image and therefore has the
property of chirality.
[0031] The term "chirality" refers to the geometric property of a
rigid object (or spatial arrangement of points or atoms) of being
non-superimposable on its mirror image. If the object is
superimposable on its mirror image the object is described as being
achiral.
[0032] The term "chirality axis" refers to an axis about which a
set of ligands is held so that it results in a spatial arrangement
which is not superposable on its mirror image.
[0033] The term "chiral center" refers to an atom holding a set of
ligands in a spatial arrangement, which is not superposable on its
mirror image. A chirality center may be considered a generalized
extension of the concept of the asymmetric carbon atom to central
atoms of any element. Each chiral center (*C) is labeled R or S
according to a system by which its substituents are each designated
a priority according to the Cahn Ingold Prelog priority rules
(CIP), based on atomic number. In some embodiments, the
stereochemistry of the chiral centers (marked by "*C") represents
all possible combinations in terms of relative and absolute
chemistry.
[0034] The term "racemate" as used herein refers to an equimolar
mixture of two optically active components that neutralize the
optical effect of each other and is therefore optically
inactive.
[0035] The term, "enantiomer" refers to one of a pair of optical
isomers containing one or more asymmetric carbons (C*) whose
molecular configurations have left- and right-hand (chiral) forms.
Enantiomers have identical physical properties, except for the
direction of rotation of the plane of polarized light. Enantiomers
have identical chemical properties except toward optically active
reagents.
[0036] The terms "solvate" or "solvates" of a compound refer to
those compounds, where compounds is as defined above, that are
bound to a stoichiometric or non-stoichiometric amount of a
solvent. Solvates of a compound includes solvates of all forms of
the compound. Preferred solvents are volatile, non-toxic, and/or
acceptable for administration to humans in trace amounts. Suitable
solvates include distilled and pyrogen-free water.
[0037] The term "isomer" as used herein refers to one of two or
more molecules having the same number and kind of atoms and hence
the same molecular weight, but differing in chemical structure.
Isomers may differ in the connectivities of the atoms (structural
isomers), or they may have the same atomic connectivities but
differ only in the arrangement or configuration of the atoms in
space (stereoisomers). "Stereoisomer" or "stereoisomers" refer to
compounds that differ in the chirality of one or more
stereocenters. Stereoisomers may include, but are not limited to,
E/Z double bond isomers, enantiomers, and diastereomers. Structural
moieties that, when appropriately substituted, can impart
stereoisomerism include, but are not limited to, olefinic, imine or
oxime double bonds; tetrahedral carbon, sulfur, nitrogen or
phosphorus atoms; and allenic groups. Enantiomers are
non-superimposable mirror images. A mixture of equal parts of the
optical forms of a compound is known as a racemic mixture or
racemate. Diastereomers are stereoisomers that are not mirror
images.
[0038] The term "tautomer" refers to alternate forms of a compound
that differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH--moiety
and a ring .dbd.N-- moiety such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles.
[0039] The term, "electrophile" refers to an ion or atom or
collection of atoms, which may be ionic, having an electrophilic
center, i.e., a center that is electron seeking, capable of
reacting with a nucleophile.
[0040] The term, "nucleophile" refers to an ion or atom or
collection of atoms, which may be ionic, having a nucleophilic
center, i.e., a center that is seeking an electrophilic center or
capable of reacting with an electrophile.
[0041] The term "reactive group" as used herein refers to a group
that is capable of reacting with another chemical group to form a
covalent bond, i.e. is covalently reactive under suitable reaction
conditions, and generally represents a point of attachment for
another substance. The reactive group is a moiety, such as
carboxylic acid that is capable of chemically reacting with a
functional group on a different compound to form a covalent
linkage. Reactive groups generally include nucleophiles,
electrophiles and photoactivatable groups. Exemplary reactive
groups include, but are not limited to, olefins, acetylenes,
alcohols, phenols, ethers, oxides, halides, aldehydes, ketones,
carboxylic acids, esters, amides, cyanates, isocyanates,
thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,
hydrazides, diazo, diazonium, nitro, nitriles, mercaptans,
sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,
sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic
acids isonitriles, amidines, imides, imidates, nitrones,
hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids,
allenes, ortho esters, sulfites, enamines, ynamines, ureas,
pseudoureas, semicarbazides, carbodiimides, carbamates, imines,
azides, azo compounds, azoxy compounds, and nitroso compounds.
Reactive functional groups also include those used to prepare
bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and
the like. Methods to prepare each of these functional groups are
well known in the art and their application to or modification for
a particular purpose is within the ability of one of skill in the
art (see, for example, Sandler and Karo, eds., Organic Functional
Group Preparations, Academic Press, San Diego, 1989).
[0042] An "electrophilic reactive group" refers to a reactive group
as described above that is capable of reaction with a nucleophile.
Exemplary electrophilic reactive groups of the present invention
are halide groups, such as bromide or chloride substituents,
halogens (F, Cl, Br, or I); nitriles (CN); carboxylic esters (COOX)
where X=a good leaving group; carbonyls (CO); carboxyl groups,
-aldehydes (--CHO), acetaldehydes. Good leaving groups are well
known to one of ordinary skill in the art.
[0043] The term "lower" as used herein refers to a group having
between one and six carbons.
[0044] The term "substituted" as used herein refers to substitution
with the named substituent or substituents, multiple degrees of
substitution being allowed unless otherwise stated.
[0045] The term "alkyl" as used herein refers to a straight or
branched chain monovalent or divalent hydrocarbon radical having,
except where specifically indicated otherwise, from one to about
fifty carbon atoms, optionally substituted with substituents
including, but not limited to: halogens, halides, alkylhalides,
lower alkyl, lower alkoxy, lower alkylsulfanyl, lower
alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl, carboxy, carbamoyl optionally
substituted by alkyl, aminosulfonyl optionally substituted by
alkyl, silyloxy optionally substituted by alkoxy, alkyl, or aryl,
silyl optionally substituted by alkoxy, alkyl, or aryl, nitro,
cyano, halogen, or lower perfluoroalkyl, multiple degrees of
substitution being allowed. Such an "alkyl" group may contain one
or more O, S, S(O), or S(O).sub.2 moieties. Examples of"alkyl" as
used herein include, but are not limited to, methyl, ethyl, propyl,
decyl, undecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, decosyl,
tricosyl, tetracosyl, and pentacosyl, n-butyl, t-butyl, n-pentyl,
isobutyl, and isopropyl, and the like. In some embodiments the
alkyl comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon
atoms.
[0046] The term "alkenyl," as used herein, denotes a straight
(unbranched) or branched hydrocarbon chain having one or more
double bonds therein where the double bond can be unconjugated or
conjugated to another unsaturated group (e.g., a polyunsaturated
alkenyl) and can be unsubstituted or substituted, with multiple
degrees of substitution being allowed. For example, halides,
alkylhalides, lower alkyl, lower alkoxy, lower alkylsulfanyl, lower
alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl, carboxy, carbamoyl optionally
substituted by alkyl, aminosulfonyl optionally substituted by
alkyl, silyloxy optionally substituted by alkoxy, alkyl, or aryl,
silyl optionally substituted by alkoxy, alkyl, or aryl, nitro,
cyano, halogen, or lower perfluoroalkyl, multiple degrees of
substitution being allowed. Such an "alkenyl" group may contain one
or more O, S, S(O), or S(O).sub.2 moieties. For example, and
without limitation, the alkenyl can be vinyl, allyl, butenyl,
pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl,
2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl,
decenyl, undecenyl, dodecenyl, heptadecenyl, octadecenyl,
nonadecenyl, eicosenyl, heneicosenyl, docosenyl, tricosenyl,
tetracisenyl, pentacosenyl, phytyl, the branched chain isomers
thereof, and polyunsaturated alkenes including
octadec-9,12,-dienyl, octadec-9,12,15-trienyl, and
eicos-5,8,11,14-tetraenyl.
[0047] The term "alkynyl" refers to a hydrocarbon radical having
from about two to about fifty carbons and at least one
carbon-carbon triple bond, optionally substituted with substituents
selected from the group consisting of lower alkyl, lower alkoxy,
lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo,
hydroxy, mercapto, amino optionally substituted by alkyl, carboxy,
carbamoyl optionally substituted by alkyl, aminosulfonyl optionally
substituted by alkyl, silyloxy optionally substituted by alkoxy,
alkyl, or aryl, silyl optionally substituted by alkoxy, alkyl, or
aryl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple
degrees of substitution being allowed. Such an "alkynyl" group may
containing one or more O, S, S(O), or S(O).sub.2 moieties.
[0048] The term "aryl" as used herein refers to an optionally
substituted benzene ring or to an optionally substituted benzene
ring system fused to one or more optionally substituted benzene
rings, with multiple degrees of substitution being allowed.
Substituents include, but are not limited to, lower alkyl, lower
alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by
alkyl, amino sulfonyl optionally substituted by alkyl, acyl, aroyl,
heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl,
silyloxy optionally substituted by alkoxy, alkyl, or aryl, silyl
optionally substituted by alkoxy, alkyl, or aryl, nitro, cyano,
halogen, or lower perfluoroalkyl, multiple degrees of substitution
being allowed. Examples of aryl include, but are not limited to,
phenyl, 2-napthyl, 1-naphthyl, 1-anthracenyl, and the like.
[0049] It should be understood that wherever the terms "alkyl" or
"aryl" or either of their prefix roots appear in a name of a
substituent, they are to be interpreted as including those
limitations given above for alkyl and aryl. Designated numbers of
carbon atoms (e.g., C.sub.1-10) shall refer independently to the
number of carbon atoms in an alkyl, alkenyl or alkynyl or cyclic
alkyl moiety or to the alkyl portion of a larger substituent in
which the term "alkyl" appears as its prefix root.
[0050] The terms "carbamates" or "urethanes" as used herein refer
to a group of organic compounds sharing a common functional group
having the general structure --NR(CO)O--.
[0051] As used herein, "cycloalkyl" (used interchangeably with
"aliphatic cyclic" herein) refers to an alicyclic hydrocarbon group
optionally possessing one or more degrees of unsaturation, having
from about three to about fifty carbon atoms, optionally
substituted with substituents, for example: halogens, halides,
alkylhalides, selected from the group consisting of lower alkyl,
lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl, carboxy, carbamoyl optionally substituted by alkyl,
aminosulfonyl optionally substituted by alkyl, nitro, cyano,
halogen, or lower perfluoroalkyl, multiple degrees of substitution
being allowed. "Cycloalkyl" includes by way of example cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl,
and the like.
[0052] The terms "heterocycle" and "heterocyclic" as used herein
are used interchangeably to refer to a three to about
twelve-membered heterocyclic ring optionally aromatic or possessing
zero, one- or more degrees of unsaturation, containing one or more
heteroatomic substitutions, for example: --S--, --SO--,
--SO.sub.2--, --O--, or --N-- and substituents including, but not
limited to, halogens, halides, alkylhalides lower alkyl, lower
alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl, carboxy, carbamoyl optionally substituted by alkyl,
aminosulfonyl optionally substituted by alkyl, nitro, cyano,
halogen, or lower perfluoroalkyl, multiple degrees of substitution
being allowed. Such a ring optionally may be fused to one or more
of another "heterocyclic," cycloalkyl or aryl ring(s).
[0053] "Cells of the immune system" or "immune cells", is meant to
include any cells of the immune system that may be assayed,
including, but not limited to, B lymphocytes, also called B cells,
T lymphocytes, also called T cells, natural killer (NK) cells,
natural killer T (NK) cells, lymphokine-activated killer (LAK)
cells, monocytes, macrophages, neutrophils, granulocytes, mast
cells, platelets, Langerhan's cells, stem cells, dendritic cells,
peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells,
gene modified immune cells including hybridomas, drug modified
immune cells, antigen presenting cells and derivatives, precursors
or progenitors of the above cell types.
[0054] The term "hematopoietic malignancy" refers to a cancer or
hyperproliferative disorder generated during hematopoiesis
involving cells such as leukocytes, lymphocytes, natural killer
cells, plasma cells, and myeloid cells such as neutrophils and
monocytes. Hematopoietic Malignancies include the diseases listed
in the WHO classification of Human Hematopoietic Malignancies;
Tumors of Hematopoietic and Lymphoid Tissues (Jaffe E. S., Harris
N. L., Stein H., Vardiman J. W. (Eds.) (2001): World Health
Organization Classification of Tumours. Pathology and Genetics of
Tumours of Hematopoietic and Lymphoid Tissues. IARC Press: Lyon)
with the morphology code of the International Classification of
Diseases (ICD-O). Behavior is coded/3 for malignant tumors and/1
for lesions of low or uncertain malignant potential.
[0055] The term "cancer" refers to or describes the physiological
condition in mammals that is typically characterized by unregulated
cell growth. A "tumor" comprises one or more cancerous cells.
Examples of cancer include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g., epithelial squamous cell cancer), lung cancer
including small-cell lung cancer, non-small cell lung cancer
("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the
lung, cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, head and neck cancer, multiple myeloma, acute
myelogenous leukemia, chronic lymphoid leukemia, chronic
myelogenous leukemia, lymphocytic leukemia, myeloid leukemia, oral
cavity and pharynx, non-Hodgkin lymphoma, melanoma, and villous
colon adenoma
[0056] "Inflammatory disorder" as used herein can refer to any
disease, disorder, or syndrome in which an excessive or unregulated
inflammatory response leads to excessive inflammatory symptoms,
host tissue damage, or loss of tissue function. "Inflammatory
disorder" also refers to a pathological state mediated by influx of
leukocytes and/or neutrophil chemotaxis.
[0057] "Inflammation" as used herein refers to a localized,
protective response elicited by injury or destruction of tissues,
which serves to destroy, dilute, or wall off (sequester) both the
injurious agent and the injured tissue. Inflammation is notably
associated with influx of leukocytes and/or neutrophil chemotaxis.
Inflammation can result from infection with pathogenic organisms
and viruses and from noninfectious means such as trauma or
reperfusion following myocardial infarction or stroke, immune
response to foreign antigen, and autoimmune responses. Accordingly,
inflammatory disorders amenable to treatment with compositions
comprising Formula I compounds, encompass disorders associated with
reactions of the specific defense system as well as with reactions
of the nonspecific defense system.
[0058] "Specific defense system" refers to the component of the
immune system that reacts to the presence of specific antigens.
Examples of inflammation resulting from a response of the specific
defense system include the classical response to foreign antigens,
autoimmune diseases, and delayed type hypersensitivity response
mediated by T-cells. Chronic inflammatory diseases, the rejection
of solid transplanted tissue and organs, e.g., kidney and bone
marrow transplants, and graft versus host disease (GVHD), are
further examples of inflammatory reactions of the specific defense
system.
[0059] The term "nonspecific defense system" as used herein refers
to inflammatory disorders that are mediated by leukocytes that are
incapable of immunological memory (e.g., granulocytes, and
macrophages). Examples of inflammation that result, at least in
part, from a reaction of the nonspecific defense system include
inflammation associated with conditions such as adult (acute)
respiratory distress syndrome (ARDS) or multiple organ injury
syndromes; reperfusion injury; acute glomerulonephritis; reactive
arthritis; dermatoses with acute inflammatory components; acute
purulent meningitis or other central nervous system inflammatory
disorders such as stroke; thermal injury; inflammatory bowel
disease; granulocyte transfusion associated syndromes; and
cytokine-induced toxicity.
[0060] "Autoimmune disease" as used herein refers to any group of
disorders in which tissue injury is associated with humoral or
cell-mediated responses to the body's own constituents.
[0061] "Allergic disease" as used herein refers to any symptoms,
tissue damage, or loss of tissue function resulting from allergy.
"Arthritic disease" as used herein refers to any disease that is
characterized by inflammatory lesions of the joints attributable to
a variety of etiologies. "Dermatitis" as used herein refers to any
of a large family of diseases of the skin that are characterized by
inflammation of the skin attributable to a variety of etiologies.
"Transplant rejection" as used herein refers to any immune reaction
directed against grafted tissue, such as organs or cells (e.g.,
bone marrow), characterized by a loss of function of the grafted
and surrounding tissues, pain, swelling, leukocytosis, and
thrombocytopenia. The therapeutic methods of the present invention
include methods for the treatment of disorders associated with
inflammatory cell activation.
[0062] "Inflammatory cell activation" refers to the induction by a
stimulus (including, but not limited to, cytokines, antigens or
auto-antibodies) of a proliferative cellular response, the
production of soluble mediators (including but not limited to
cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive
amines), or cell surface expression of new or increased numbers of
mediators (including, but not limited to, major histocompatability
antigens or cell adhesion molecules) in inflammatory cells
(including but not limited to monocytes, macrophages, T
lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclear
leukocytes such as neutrophils, basophils, and eosinophils), mast
cells, dendritic cells, Langerhan's cells, and endothelial cells).
It will be appreciated by persons skilled in the art that the
activation of one or a combination of these phenotypes in these
cells can contribute to the initiation, perpetuation, or
exacerbation of an inflammatory disorder.
[0063] The term "specifically binds" to a target molecule, such as
for example, an antibody or a polypeptide is a term well understood
in the art, and methods to determine such specific or preferential
binding are also well known in the art. A molecule is said to
exhibit "specific binding" or "preferential binding" if it reacts
or associates more frequently, more rapidly, with greater duration
and/or with greater affinity with a particular cell or substance
than it does with alternative cells or substances. For example, an
antibody "specifically binds" or "preferentially binds" to a target
if it binds with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other substances. It is also
understood by reading this definition that; for example, an
antibody (or moiety or epitope) that specifically or preferentially
binds to a first target may or may not specifically or
preferentially bind to a second target. As such, "specific binding"
or "preferential binding" does not necessarily require (although it
can include) exclusive binding. Generally, but not necessarily,
reference to binding means preferential binding.
[0064] By the term "modulate," it is meant that any of the
mentioned activities, are, e.g., increased, enhanced, increased,
agonized (acts as an agonist), promoted, decreased, reduced,
suppressed blocked, or antagonized (acts as an agonist). Modulation
can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold,
10-fold, 100-fold, etc., over baseline values. Modulation can also
decrease its activity below baseline values. Modulation can also
normalize an activity to a baseline value.
[0065] As used herein, a "pharmaceutically acceptable"
component/carrier etc is one that is suitable for use with humans
and/or animals without undue adverse side effects (such as
toxicity, irritation, and allergic response) commensurate with a
reasonable benefit/risk ratio.
[0066] As used herein, the term "safe and effective amount" refers
to the quantity of a component which is sufficient to yield a
desired therapeutic response without undue adverse side effects
(such as toxicity, irritation, or allergic response) commensurate
with a reasonable benefit/risk ratio when used in the manner of
this invention. By "therapeutically effective amount" is meant an
amount of a compound of the present invention effective to yield
the desired therapeutic response. For example, an amount effective
to delay the growth of or to cause a cancer, either a sarcoma or
lymphoma, or to shrink the cancer or prevent metastasis. The
specific safe and effective amount or therapeutically effective
amount will vary with such factors as the particular condition
being treated, the physical condition of the patient, the type of
mammal or animal being treated, the duration of the treatment, the
nature of concurrent therapy (if any), and the specific
formulations employed and the structure of the compounds or its
derivatives.
[0067] The term "prodrug" refers to any derivative of a compound of
the embodiments that is capable of directly or indirectly providing
a compound of the embodiments or an active metabolite or residue
thereof when administered to a subject. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of the embodiments when such
compounds are administered to a subject (e.g., by allowing an
orally administered compound to be more readily absorbed into the
blood) or which enhance delivery of the parent compound to a
biological compartment (e.g., the brain or lymphatic system)
relative to the parent species. A general overview of prodrugs is
provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery
Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B.
Roche, ed., Bioreversible
[0068] The term "pharmaceutically acceptable salt" refers to
pharmaceutically acceptable salts derived from a variety of organic
and inorganic counter ions well known in the art and include, by
way of example only, sodium, potassium, calcium, magnesium,
ammonium, and tetraalkylammonium, and when the molecule contains a
basic functionality, salts of organic or inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,
and oxalate. Suitable salts include those described in P. Heinrich
Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts
Properties, Selection, and Use; 2002.
[0069] "Diagnostic" or "diagnosed" means identifying the presence
or nature of a pathologic condition. Diagnostic methods differ in
their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay, are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0070] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology or symptoms
of a disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. "Treatment"
may also be specified as palliative care. Those in need of
treatment include those already with the disorder as well as those
in which the disorder is to be prevented. In tumor (e.g., cancer)
treatment, a therapeutic agent may directly decrease the pathology
of tumor cells, or render the tumor cells more susceptible to
treatment by other therapeutic agents, e.g., radiation and/or
chemotherapy. Accordingly, "treating" or "treatment" of a state,
disorder or condition includes: (1) preventing or delaying the
appearance of clinical symptoms of the state, disorder or condition
developing in a human or other mammal that may be afflicted with or
predisposed to the state, disorder or condition but does not yet
experience or display clinical or subclinical symptoms of the
state, disorder or condition; (2) inhibiting the state, disorder or
condition, i.e., arresting, reducing or delaying the development of
the disease or a relapse thereof (in case of maintenance treatment)
or at least one clinical or subclinical symptom thereof; or (3)
relieving the disease, i.e., causing regression of the state,
disorder or condition or at least one of its clinical or
subclinical symptoms. The benefit to an individual to be treated is
either statistically significant or at least perceptible to the
patient or to the physician.
[0071] The terms "patient" or "individual" are used interchangeably
herein, and refers to a mammalian subject to be treated, with human
patients being preferred. In some cases, the methods of the
invention find use in experimental animals, in veterinary
application, and in the development of animal models for disease,
including, but not limited to, rodents including mice, rats, and
hamsters; and primates.
[0072] As used herein, "biological samples" include solid and body
fluid samples. The biological samples used in the present invention
can include cells, protein or membrane extracts of cells, blood or
biological fluids such as ascites fluid or brain fluid (e.g.,
cerebrospinal fluid). Examples of solid biological samples include,
but are not limited to, samples taken from tissues of the central
nervous system, bone, breast, kidney, cervix, endometrium,
head/neck, gallbladder, parotid gland, prostate, pituitary gland,
muscle, esophagus, stomach, small intestine, colon, liver, spleen,
pancreas, thyroid, heart, lung, bladder, adipose, lymph node,
uterus, ovary, adrenal gland, testes, tonsils, thymus and skin, or
samples taken from tumors. Examples of "body fluid samples"
include, but are not limited to blood, serum, semen, prostate
fluid, seminal fluid, urine, feces, saliva, sputum, mucus, bone
marrow, lymph, and tears.
[0073] The term "high-throughput screening" or "HTS" refers to a
method drawing on different technologies and disciplines, for
example, optics, chemistry, biology or image analysis to permit
rapid, highly parallel biological research and drug discovery. HTS
methods are known in the art and they are generally performed in
multiwell plates with automated liquid handling and detection
equipment; however it is also envisioned that the methods of the
invention may be practiced on a microarray or in a microfluidic
system.
[0074] Compositions
[0075] Attempts to emulate Nature's strategy for creation of
structural and functional diversity in a synthetic vein have
resulted in the creation of many interesting compound classes,
including .beta.-peptides, peptoids, and peptide nucleic acids
(FIG. 1A). A substantial value associated with these bio-inspired
oligomers is their compatibility with split-and-pool solid phase
synthesis (Houghten, R. A. Proc. Natl. Acad. Sci. USA, 82,
5131-5135 (1985)), a powerful technology for the creation of large
and diverse chemical libraries. However, a common limitation with
these existing classes of unnatural oligomers is that they lack the
conformational constraints typical of small molecule natural
products, a property that likely limits their affinity for
biological targets due to entropic penalties that result from
assuming a particular bound conformation.
[0076] In a preferred embodiment, a compound comprises the
structure represented by general Formula I:
##STR00002##
[0077] Wherein, the termini X and Y can be suitably reactive to
allow for bidirectional functionalization.
[0078] The compound represented by general Formula I provides a
rigid chiral motif for controlling the three-dimensional
orientation of R.sup.1 and R.sup.2 with respect to each other as
well as COX and CH.sub.2Y. For example, if X.dbd.OH, the carboxylic
acid may be used for amide bond formation with a variety of amines.
Likewise, if Y.dbd.Cl, nucleophilic displacement with primary
amines would deliver secondary allylic amine products. X--C.dbd.O
is more reactive than CH.sub.2Y, such that addition of two
nucleophiles can be controlled and a defined head to tail oligomer
can be synthesized. Thus, COX and CH.sub.2Y are functionalities
that may be used to increase the size of the molecule by suitable
intermolecular reactions at these sites. Without limitation,
R.sup.1 and R.sup.2 comprise, all saturated, partially saturated
and unsaturated hydrocarbons. The saturated hydrocarbons include
all alkanes, that is, linear branched or cyclic structures,
monovalent or polyvalent, substituted, partially substituted or
combinations thereof. Examples of substitutions include, without
limitation, N, O, Si, P, S, NH.sub.2, NH.sub.3, N-oxides, S-oxides
alkyl, aryl, carboxyl, arylalkyl, cycloalkyl, cycloheteroalkyl,
heteroalkyl, heteroaryl or heteroarylalkyl group; and, the like, as
long as the substitutions are compatible with the method of
synthesis and/or can be introduced to the R.sub.1 and R.sub.2
groups post synthesis.
[0079] In a preferred embodiment, compounds of general Formula I
may be employed in oligomerization processes to generate higher
molecular weight species. Such oligomerization chemistry can
proceed by stepwise union of compounds of Formula I (i.e. FIG. 1C
and FIG. 2), or through coupling to other monomeric building
blocks--the nature of the building blocks appended in such fashion
will relate to the precise nature of the compound generalized by
Formula I (i.e. when X.dbd.OH, and Y.dbd.NHR, a range of compounds
including, but not limited to carboxylic acids). In the case of an
oligomerization process that employs the general compound depicted
in Formula I, displacement of an allylic chloride (Y.dbd.Cl) with a
suitable nucleophile (i.e. including but not limited to a primary
amine, hydrazine, OH, etc), followed by acylation with another
molecule of general Formula I (or a different suitable acylating
agent) would deliver complex synthetic oligomers [one of the many
generic structures possible could be depicted as
(XCO--*CHR.sup.1--CH--CR.sup.2CH.sub.2Y).sub.(n+1) wherein n is
equal to or greater than 1, and * is a chiral center [(R) or (S)]--
for oligomers that have a "mixed" backbone (defined by inclusion of
alternative building blocks in addition to compounds of general
Formula I), a simple generic structure is not possible to clearly
depict the great potential of this chemistry to access diverse
molecules--for example,
(XCO--*CHR.sup.1--CH--CR.sup.2CH.sub.2Y).sub.(1)(COCH.sub.2Y).sub.(2)
(XCO--*CHR.sup.1--CH--CR.sup.2CH.sub.2Y).sub.(1) (where
Y.dbd.NR.sup.3) would correspond to a tetramer where the first
residue is composed of compound shown by Formula I, the second and
third residues derive from incorporation of bromoacetic acid, and
the fourth residue derives from another unit of Formula 1].
[0080] While oligomerization of compounds similar to Formula I
(i.e. X.dbd.OH; Y.dbd.C1) is thought to define a particularly
powerful use of its reactivity to generate complex and diverse
libraries of chiral and conformationally restricted molecules of
potential utility as therapeutic agents and diagnostics, or as
components of such agents, other embodiments of the current
invention include the general use of units of Formula 1 as a chiral
scaffold to display chemical information about its core structure.
Here, "chemical information" refers to the nature of the
substituents X, R.sup.1, R.sup.2, and Y, held about the five-carbon
backbone. [X, R.sup.1, R.sup.2, and Y have already been defined].
This backbone defines a readily accessible skeleton to display
building block functionality in defined regions of three
dimensional space based on the minimization of simple non-bonded
steric interactions (i.e. allylic strain).
[0081] The compound of general Formula I may function as a
bifunctional reagent and partake in a variety of mono- and
bi-directional homologation chemistry based on the nature of
substituents X and/or Y wherein either terminus can serve as a
nucleophilic or electrophilic motif. By way of example, this is
illustrated in the general reaction Scheme shown in FIG. 11. This
example is for illustrative purposes only and is not meant to be
limiting or construed as such. The chloroacid, shown by
representative Formula II was used in sequential amide bond forming
reaction and nucleophilic displacement, wherein Nu is a nucleophile
and EI is an electrophile; R.sup.1 and R.sup.2 are as previously
described.
[0082] R.sup.5 independently comprises any functionality that does
not interfere with the ability to perform the given chemical
homologation defined in this case by: 1) amide bond formation, and
2) nucleophilic substitution (displacement of the allylic
chloride). Examples of such functionality include, but are not
limited to alkyl, aryl, heteroaryl, NR.sup.5R.sup.5', OR.sup.5.
[0083] R.sup.6 independently comprises any functionality that does
not interfere with the ability to perform the given chemical
homologation defined in this case by: 1) amide bond formation, and
2) nucleophilic substitution (displacement of the allylic
chloride). Examples of such functionality include, but are not
limited to alkyl, aryl, heteroaryl, NR.sup.5R.sup.6, OR.sup.5).
[0084] R.sup.7 independently comprises any functionality that does
not interfere with the ability to perform the given chemical
homologation defined in this case by: 1) amide bond formation, and
2) nucleophilic substitution (displacement of the allylic
chloride). Examples of such functionality include, but are not
limited to alkyl, aryl, heteroaryl, NR.sup.5R.sup.6, OR.sup.5).
[0085] Due to the ready availability of stereoselective
transformations of the central substituted alkene, a variety of
stereo defined products can be prepared from this starting
material, e.g. hydrogenation, hydroboration, dihydroxylation,
cyclopropanation, epoxidation, etc. Further, the compounds produced
from the homologation of such building blocks, or via other routes
represent a class of compounds with unique and diverse
properties.
[0086] The monomers of Formula I allow for the introduction of a
broad range of substructures, positioned in a defined region of
3-dimensional space with respect to one another.
[0087] In one embodiment, the one or more units comprise
substitutions which are independent of a previous unit's
substitutions. For example in one unit, R.sup.1 can be an alkyl and
in another unit R.sup.1 is an aryl.
[0088] In a preferred embodiment, where molecules of general
Formula I are employed in a controlled oligomerization (to result
in dimers, trimers, tetramers, and higher oligomeric structures),
the local conformational preferences that result from incorporating
these chiral subunits is reminiscent of the motifs commonly
observed in bioactive natural products from polyketide biosynthetic
origin (FIG. 1B). Members of this natural product class often
contain relatively simple stereochemically defined structural
motifs that participate in dictating the overall conformational
preferences of the molecule.
[0089] In a preferred embodiment reflecting the role that
unsaturated stereodefined motifs play in governing the
conformational dynamics of polyketide-derived natural products, a
method of synthesizing oligomers of molecules of general Formula I
has been realized to afford a biomimetic polyketide-inspired
approach to the synthesis of diverse libraries of chiral and
conformationally restricted small molecules. These oligomers,
termed "COPAs" (chiral oligomers of pentoic amides) comprise a
central N-substituted 5-amino 2,4-dialkyl-3-pentenoic amide motif
to provide a chiral environment about each monomeric unit. The
control of conformation resulting from this motif is substantial,
and is based on the minimization of non-bonded steric interactions
about the .alpha.-branched trisubstituted alkene and
.alpha.-branched tertiary amide. As illustrated in FIG. 1D, each of
these structural motifs imparts substantial rigidification, as the
C2 proton is constrained to being roughly in-plane with the
C4-alkyl group, and R.sup.2-amide substituent--defining a rigid
chiral environment at each monomer, where the amide and alkyl
substitution emerging from this core are positioned in
three-dimensional space.
[0090] The combined influence of distinct chiral subunits on the
gross conformational preferences for a COPA oligomer is profound,
and offers a robust strategy to access diverse chiral skeletons
that differentially display building blocks. As illustrated in FIG.
2, analysis of a collection of 8 stereoisomeric but homogeneously
substituted COPA tetramers (all methyl substitution) illustrates
the striking effect that C2 stereochemistry has on the skeletal
structure, and hence three-dimensional orientation of all building
blocks to be installed.
[0091] The synthesis of compounds from the methods described in the
examples section, which follow, are unique and advantageous over
any currently available method. While peptoids, peptides,
.beta.-peptides and all other known synthetic oligomers can be
prepared in great numbers, their skeletons (or core units) are
typically not conformationally biased. In some cases, high
molecular weight oligomers, or designed oligomers that are
rigidified by various macromolecular interactions (i.e. charge
separation, it-stacking, hydrogen-bonding, etc.), are needed to
achieve conformational rigidity. Having to rely on such features
greatly diminishes the potential of such molecular skeletons in
diversity-oriented synthesis and ligand discovery due to a
constraining of the type of substituents or molecular weight
required to achieve rigidification. The advantages of the methods
described herein are that they can be employed to prepare chiral
oligomers of massive number and molecular diversity while doing so
in a manner that rigidifies the core skeleton. The result of this
rigidification is a preferential orientation of diversity elements
(i.e. R.sup.1-R.sup.7) in three-dimensional space, about a skeleton
of relative low molecular weight (in comparison to proteins and
other biological macromolecules). Other advantages include: 1) a
very simple method of synthesis which proceeds in high-yields, 2)
great diversity in building blocks is readily achieved with
available primary amines, 3) compounds are prepared as single
enantiomers, 4) compounds are prepared in diastereomerically pure
form, and 5) compounds have defined conformational preferences
based, in part, by the minimization of non-bonded steric
interactions (allylic strain) that results from the substitution
and stereochemistry of the building block generalized as Formula I.
FIG. 12 is a schematic representation showing an example of
oligomer synthesis, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and
R.sup.5 independently comprise any functionality that does not
interfere with the ability to perform the given chemical
homologation defined by: 1) amide bond formation, and 2)
nucleophilic substitution (displacement of the allylic chloride).
Examples of such functionality include, but is not limited to
alkyl, aryl, heteroaryl, NR.sup.5R.sup.5', OR.sup.5.
[0092] In one embodiment, a method of synthesizing a chiral monomer
comprises obtaining an oxazolidinone and reacting via a
stereoselective aldol reaction with an .alpha.-substituted,
.alpha.,.beta.-unsaturated aldehyde (i.e. including, but not
limited to methacrolein). In the present case, MgBr.sub.2-catalyzed
aldol reaction proceeds to deliver a stereodefined anti-aldol
product (Evans, D. A.; Tedrow, J. S.; Shaw, J. T.; Downey, C. W.
Diastereoselective Magnesium Halide-Catalyzed anti-Aldol Reactions
of Chiral N-Acyloxazolidinones, J. Am. Chem. Soc. 2002, 124,
392-393). The product TMS-ether is converted to a stereodefined
allylic halide by a stereoselective halogenation reaction that
proceeds with allylic transposition (Ravikumar, P. C.; Yao, L.;
Fleming, F. F. Allylic and Allenic Halide Synthesis via NbC15- and
NbBrs-Mediated Alkoxide Rearrangements, J. Org. Chem. 2009, 74,
7294-7299). Finally, hydrolysis of the oxazolidinone proceeds to
deliver the chiral product defined by Formula I'. Here, X.dbd.OH,
and Y.dbd.C1.
##STR00003##
[0093] Based on the reactivity of the compound depicted as Formula
I' above, the method of synthesis described can be employed to
access a range of diverse products related to that described, where
X can independently comprise OR.sup.3, NR.sup.4R.sup.5, H, halide,
alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and the like; R.sup.3
independently comprises amide, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, and the like; R.sup.4
independently comprises H, OR.sup.4, alkyl, aryl, heteroaryl, and
the like; *C is a chiral center [(R) or (S)]; R.sup.1 independently
comprises alkyl, aryl, heterorayl, alkenyl, OR.sup.4, and the like;
R.sup.2 independently comprises alkyl, cycloalkyl, aryl,
heterorayl, alkynyl, alkenyl, and the like. Y independently
comprises a halide, NHR.sup.4, NR.sup.4R.sup.5, OH, OR.sup.3, or
C(O)X. R.sup.4 and R.sup.5 are defined by substituents that are
compatible with the bond forming reactions required to convert the
carboxylic acid-based pentenoic allylic halide to the
functionalized product.
[0094] In another embodiment, R.sup.1 and R.sup.2 independently
comprise OR.sup.3, NHR.sup.4, NR.sup.4R.sup.5, halide, alkyl,
linear alkyl, branched alkyl, heteroatom-substituted (i.e. N, O, S,
halogen, etc.) alkyl, unsaturated and polyunsaturated linear and
branched hydrocarbons, alkenyl, cycloalkyl, aryl, heteroaryl,
heteroaryl, heterocycloalkyl, heteroatom-substituted unsaturated
and polyunsaturated linear and branched hydrocarbons, cycloalkyl,
heteroatom-substituted cycloalkyl, saturated and unsaturated
heterocycles, substituted cycloalkyl, substituted and unsubstituted
aromatic, substituted and unsubstituted heteroaromatic; R.sup.3
independently comprises H, alkyl, cycloalkyl, alkenyl, aryl,
heteroaryl, NR.sup.4R.sup.5, carboxyl, heterocycloalkyl; and,
R.sup.4 independently comprises H, OR.sup.3, alkyl, aryl, or
heteroaryl.
[0095] The optically active monomers comprising the structure of
Formula I' can be employed in oligomerization chemistry as
previously discussed (in solution or on a solid phase). The
products of such oligomerization can be diverse based on
substitution and stereochemistry of the central unsaturated
building block and, in some cases, the nature of the amine building
block (if displacing the allylic chloride is employed as a key
homologation step), as well as the nature of the backbone, which
can incorporate carboxylic acid-based building blocks of different
substitution (i.e. bromoacetic acid in place of the 2-substituted
3-pentenoic acid monomer depicted as Formula 1' where X.dbd.OH, and
Y.dbd.Cl). Various methods including stereoselective hydrogenation,
hydroboration, dihydroxylation, cyclopropanation, epoxidation and
aziridination may be employed to mutate the core skeleton inherent
to Formula 1' to stereodefined motifs that lack the central
alkene.
[0096] Examples of novel compounds of Formula I' which can be
synthesized are shown below. This is not an exhaustive list nor
meant to limit the invention.
##STR00004##
[0097] As an example and not wishing to be bound or limited to any
particular substitutions of compounds of Formula I' as any type of
molecule known or yet to be discovered can be used, such as, for
example, R.sup.1 comprises alkyl, substituted and unsubstituted
aryl, as well as substituted hydrocinammyl. R.sup.2 comprises alkyl
(Me vs. Et).
[0098] Without wishing to be bound by theory, molecules of general
Formula I' are comprised of a chiral halopentenoic acid backbone,
where the nature and stereochemistry of this core influence the
relative three dimensional orientation of substituents R.sup.1 and
R.sup.2 that extend from this backbone. The precise nature of
substituents R.sup.1 and R.sup.2 is broad, includes both known and
yet to be discovered molecules and comprise molecular features that
provide a desired therapeutic, diagnostic, or physical property.
Examples within the scope of this invention include: linear alkyl,
branched alkyl, heteroatom-substituted (i.e. N, O, S, halogen,
etc.) alkyl, unsaturated and polyunsaturated linear and branched
hydrocarbons, heteroatom-substituted (i.e. N, O, S, halogen, etc.)
unsaturated and polyunsaturated linear and branched hydrocarbons,
cycloalkyl, heteroatom-substituted cycloalkyl (i.e. saturated and
unsaturated heterocycles), substituted cycloalkyl, substituted and
unsubstituted aromatic, and substituted and unsubstituted
heteroaromatic. This general description of R.sup.1 and R.sup.2
includes all such molecular architecture, that can be derived from
the method of synthesis or, without being bound by theory, motifs
that can be introduced after completion of a smaller molecular
weight variant of Formula I'.
[0099] In some embodiments, a compound of Formula I comprises:
##STR00005## ##STR00006## ##STR00007## ##STR00008##
[0100] Pharmaceuticals for Diseases or Associated Disorders
Thereof
[0101] In embodiments, the compounds can be used to diagnose and
treat diseases or disorders associated with an immune system
disorder wherein the compounds bind to, for example, immune cell
receptors or ligands. Examples of such diseases or disorders
include without limitation, hematological malignancies and other
cancers, autoimmune diseases, diseases associated with
inflammation, transplantation rejection, allergies, neurological
diseases or disorders, infections, immune cell mediated diseases or
disorders, or combinations thereof.
[0102] In one preferred embodiment, a pharmaceutical composition
comprises one or more compounds of Formula I conjugated to one or
more toxic agents, wherein the conjugate specifically binds to
hematopoietic malignant cells. Hematopoietic malignancies include:
chronic lymphocytic leukemia (CLL), chronic myeloproliferative
diseases, myelodysplastic/myeloproliferative diseases,
myelodysplastic syndromes, acute myeloid leukemias, B cell
neoplasms, T-cell and NK-cell neoplasms, Hodgkin's lymphoma,
histiocytic and dendritic cell neoplasms, mastocytosis and the
like.
[0103] Examples of histiocytic and dendritic cell neoplasms
include, without limitation: macrophage/histiocytic neoplasm,
histiocytic sarcoma, dendritic cell neoplasms, Langerhan's cell
histiocytosis, Langerhan's cell sarcoma, interdigitating dendritic
cell sarcoma/tumor, follicular dendritic cell sarcoma/tumor,
dendritic cell sarcoma and the like.
[0104] Examples of chronic myeloproliferative diseases include,
without limitation: chronic myelogenous leukemia, chronic
neutrophilic leukemia, chronic eosinophilic
leukemia/hypereosinophilic syndrome, polycythemia, chronic
idiopathic myelofibrosis, thrombocytemia, and the like.
[0105] Examples of myelodysplastic/myeloproliferative diseases
include, without limitation: chronic myelomonocytic leukemia,
atypical chronic myelogenous leukemia, juvenile myelomonocytic
leukemia, and the like.
[0106] Examples of myeloid leukemias or acute myeloid leukemias
include, without limitation: acute myeloid leukemia multilineage
dysplasia, acute myelomonocytic leukemia, acute monoblastic and
monocytic leukemia, acute erythroid leukemia, acute
megakaryoblastic leukemia, acute basophilic leukemia, acute
panmyelosis with myelofibrosis, myeloid sarcoma, and the like.
[0107] Examples of B cell malignancies or neoplasms include,
without limitation: precursor hematopoietic neoplasm, precursor B
lymphoblastic leukemia, lymphoma, mature hematopoietic neoplasm,
chronic lymphocytic leukemia, small lymphocytic lymphoma,
hematopoietic prolymphocytic leukemia, lymphoplasmacytic lymphoma,
splenic marginal zone lymphoma, hairy cell leukemia, plasma cell
myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma,
extranodal marginal zone hematopoietic lymphoma of
mucosa-associated lymphoid tissue (MALT-lymphoma), nodal marginal
zone hematopoietic lymphoma, follicular lymphoma, mantle cell
lymphoma, diffuse large hematopoietic lymphoma, mediastinal
(thymic) large cell lymphoma, intravascular large hematopoietic
lymphoma, primary effusion lymphoma, Burkitt lymphoma, leukemia,
lymphomatoid granulomatosis, post-transplant lymphoproliferative
disorder, pleomorphic and the like.
[0108] Examples of T cell and NK cell neoplasms, include without
limitation: precursor T-cell neoplasms, precursor T lymphoblastic
leukemia, lymphoma, blastic NK cell lymphoma, mature T-cell and
NK-cell neoplasms, T-cell prolymphocytic leukemia, T-cell large
granular lymphocytic leukemia, aggressive NK cell leukemia, adult
T-cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type
lymphoma, enteropathy type T-cell lymphoma, hepatosplenic T-cell
lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis
fungoides, Sezary Syndrome, Primary cutaneous anaplastic large cell
lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell
lymphoma, anaplastic large cell lymphoma, T lymphomatoid papulosis
and the like.
[0109] Examples of Hodgkin lymphomas include without limitation:
nodular lymphocyte predominant Hodgkin lymphoma, classical Hodgkin
lymphoma, nodular sclerosis classical Hodgkin lymphoma,
lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity
classical Hodgkin lymphoma, and the like.
[0110] Examples of histiocytic and dendritic cell neoplasms include
without limitation: macrophage/histiocytic neoplasm, histiocytic
sarcoma, dendritic cell neoplasms, Langerhan's cell histiocytosis,
Langerhan's cell sarcoma, interdigitating dendritic cell
sarcoma/tumor, follicular dendritic cell sarcoma/tumor, dendritic
cell sarcoma, and the like.
[0111] Examples of mastocytosis include without limitation:
cutaneous mastocytosis, indolent systemic mastocytosis, systemic
mastocytosis with associated clonal, hematological non-mast cell
lineage disease, aggressive systemic mastocytosis, mast cell
leukemia, mast cell sarcoma, extracutaneous mastocytoma and the
like.
[0112] In preferred embodiments, the hematopoietic malignancy is B
cell chronic lymphocytic leukemia (B-CLL). B-CLL is an accumulative
disease of slowly proliferating CD5.sup.+ B lymphocytes that
develops in the aging population. Whereas some patients with B-CLL
have an indolent course and die after many years from unrelated
causes, others progress very rapidly and succumb within a few years
from this currently incurable leukemia. Over the past decade,
studies of the structure and function of the B cell antigen
receptor (BCR) used by these leukemic cells have helped redefine
the nature of this disease.
[0113] CD5.sup.+ B lymphocytes in B-CLL patients express low levels
of surface membrane Ig that serves as their receptor for antigen
(BCR). The genetics of this Ig have clinical relevance, as patients
with an Ig that is unmutated in the variable (V) regions have a
significantly worse outcome than those with significant numbers of
mutations in the Ig V region.
[0114] There are several lines of evidence supporting a role for
the Ig molecule in the evolution of B-CLL. Analysis of V region
gene cassette usage has provided inferential evidence that the Ig
molecules on B-CLL cells are not the product of random chance. The
distribution of variable region gene cassettes used by B-CLL clones
differs from that found in normal cells with an increased frequency
of certain V.sub.H genes. Furthermore, the distribution of
mutations among B-CLL cases using these specific V.sub.H genes is
selectively and strikingly biased. For instance, the V.sub.H genes
of about 40% of B-CLL cases contain <2% differences from the
most similar germline gene and about 25% are identical to a
germline V.sub.H counterpart.
[0115] More recently, sets of B-CLL cases with highly similar Ig
molecules have been identified. Unmutated IgG-expressing B-CLL
cases in which the BCR was remarkably similar in structure have
been identified. These Ig molecules used the same V.sub.H, D,
J.sub.H, and in all but one instance the same V.sub.K-J.sub.K.
Furthermore, the HCDR3s were highly similar in sequence and the
LCDR3s were virtually identical with a V.sub.K-J.sub.K junction
contained an invariant, non-templated arginine codon. A larger set
of patients expressing a V.sub.H3-21 chain and a
V.lamda.-3H/J.lamda.3 L chain have been described by Tobin et al.
(Blood. 101(12):4952-7 (2003); Genes Chromosomes Cancer. 2003
August; 37(4):417-20).
[0116] As is known, aggressive forms of B-CLL are correlated with B
cells that have relatively few IgV gene mutations and have
intercellular expression of ZAP-70, and cell surface expression of
CD38 and CD23. These markers are evaluated at first diagnosis to
predict which patients will have an aggressive form of the disease,
in order to determine a course of treatment. Because the B-CLL
cells from patients belonging to identified "sets" with common B
cell receptor genes have low or absent IgV mutations (see, the
Examples section).
[0117] In embodiments, one or more compounds of Formula I
specifically bind to B cell receptors of B-CLL's. The compounds can
be conjugated to a chemotherapeutic or any other toxic agent
providing specific delivery to B-CLL's of the agent. Since the
compounds selectively bind to the malignant cells and not normal
cells, the associated drawbacks of conventional chemotherapy or
radiotherapy are thus avoided.
[0118] The uses of these compounds are not limited to the treatment
of B-CLL but can be used to treat other cancers. In some
embodiments, methods of treating cancer include where the cancer is
breast, ovary, cervix, prostate, testis, genitourinary tract,
esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin,
keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma,
non-small cell lung carcinoma (NSCLC), small cell carcinoma, lung
adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma,
thyroid, follicular carcinoma, undifferentiated carcinoma,
papillary carcinoma, seminoma, melanoma, sarcoma, bladder
carcinoma, liver carcinoma and biliary passages, kidney carcinoma,
pancreatic, myeloid disorders, lymphoma, hairy cells, buccal
cavity, naso-pharyngeal, pharynx, lip, tongue, mouth, small
intestine, colon-rectum, large intestine, rectum, brain and central
nervous system, Hodgkin's, leukemia, bronchus, thyroid, liver and
intrahepatic bile duct, hepatocellular, gastric,
glioma/glioblastoma, endometrial, melanoma, kidney and renal
pelvis, urinary bladder, uterine corpus, uterine cervix, multiple
myeloma, acute myelogenous leukemia, chronic lymphoid leukemia,
chronic myelogenous leukemia, lymphocytic leukemia, myeloid
leukemia, oral cavity and pharynx, non-Hodgkin lymphoma, melanoma,
or villous colon adenoma.
[0119] In other embodiments, the disease or disorder to be treated
are immune cell mediated, such as for example, autoimmune diseases,
transplantation rejection, lymphoproliferative disorders,
neuroinflammatory diseases, inflammatory diseases, and related
disorders thereof. The inflammatory disease can be systemic and
local inflammation, arthritis including rheumatoid arthritis,
inflammation related to immune suppression, organ transplant
rejection, allergies, ulcerative colitis, Crohn's disease,
dermatitis, asthma, systemic lupus erythematosus, Sjogren's
Syndrome, multiple sclerosis, scleroderma/systemic sclerosis,
idiopathic thrombocytopenic purpura (ITP), anti-neutrophil
cytoplasmic antibodies (ANCA) vasculitis, chronic obstructive
pulmonary disease (COPD), psoriasis.
[0120] An inflammatory or inflammation-related disorder is
characterized by a local or systemic, acute or chronic
inflammation. Examples include inflammatory dermatoses (e.g.,
dermatitis, eczema, atopic dermatitis, allergic contact dermatitis,
urticaria, necrotizing vasculitis, cutaneous vasculitis,
hypersensitivity vasculitis, eosinophilic myositis, polymyositis,
dermatomyositis, and eosinophilic fasciitis), inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), acute
respiratory distress syndrome, fulminant hepatitis, pancreatitis,
hypersensitivity lung diseases (e.g., hypersensitivity pneumonitis,
eosinophilic pneumonia, delayed-type hypersensitivity, interstitial
lung disease or ILD, idiopathic pulmonary fibrosis, and ILD
associated with rheumatoid arthritis), asthma, and allergic
rhinitis. Examples also include autoimmune diseases (e.g.,
rheumatoid arthritis, psoriatic arthritis, systemic lupus
erythematosus, myasthenia gravis, juvenile onset diabetes,
glomerulonephritis, autoimmune throiditis, ankylosing spondylitis,
systemic sclerosis, and multiple sclerosis), acute and chronic
inflammatory diseases (e.g., systemic anaphylaxia or
hypersensitivity responses, drug allergies, insect sting allergies,
allograft rejection, and graft-versus-host disease), Sjogren's
syndrome, human immunodeficiency, and virus infection. Examples of
lymphoproliferative disorders include without limitation: Hodgkin's
disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, myeloma, a
monoclonal gammopathy with antibody-mediated neurologic impairment.
Examples of autoimmune diseases include systemic lupus
erythematosus, myasthenia gravis, Grave's disease, type I diabetes
mellitus, autoimmune peripheral neuropathy, and autoimmune
hemolytic anemia.
[0121] In all embodiments, the compounds embodied herein,
conjugated or otherwise can be used in conjunction with
conventional therapies. For example: anti-inflammatory agents,
chemotherapeutics, immune-suppressive drugs, surgery,
radiotherapies and the like. In embodiments, the agent is
conjugated to or linked to one or more compounds of Formula I.
Examples of agents for linking or conjugation to compounds of
Formula I include, without limitation, antibodies, aptamers,
peptides, proteins, glycosylated moieties, receptor molecules,
ligands, natural or synthetic molecules, organic or inorganic
molecules, toxins, chemotherapeutic agents, anti-inflammatory
agents, steroids, hormones, enzymes, nucleic acids, anti-sense
nucleic acids, and the like. In some embodiments, the compounds of
Formula I conjugated to one or more agents are administered to a
patient in conjunction with conventional therapies.
[0122] The agents conjugated to compounds of Formula I embodied
herein, may be any of various therapeutic and diagnostic agents
which are desired to be delivered to a target. Therapeutic agents
which can be included as agents in the delivery system of the
present invention illustratively include but are not limited to
therapeutic compounds such as an analgesic, an anesthetic, an
antibiotic, an anticonvulsant, an antidepressant, an antimicrobial,
an anti-inflammatory, anti-migraine, an antineoplastic, an
antiparasitic, an antitumor agent, an antiviral, an anxiolytic, a
cytostatic, cytokine, a hypnotic, a metastasis inhibitor, a
sedative and a tranquilizer.
[0123] In another preferred embodiment, the molecules are labeled
with a detectable agent, which are administered to a patient for
the in vivo imaging. The specific delivery of the detectable agent
provides a vastly superior means of specific detection of a tumor
or desired target cell and decreases any background noise, allowing
for the early detection and diagnosis of a patient's condition or
disease.
[0124] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer, regardless of mechanism of action. In
embodiments, one or more chemotherapeutic agents are conjugated to
one or more monomers of compounds of Formula I. In other
embodiments, the chemotherapeutic agents are administered in
addition to the compounds embodied herein. Classes of
chemotherapeutic agents include, but are not limited to: alkylating
agents, antimetabolites, spindle poison plant alkaloids,
cytotoxic/antitumor antibiotics, topoisomerase inhibitors,
antibodies, photosensitizers, and kinase inhibitors.
Chemotherapeutic agents include compounds used in "targeted
therapy" and conventional chemotherapy. Examples of
chemotherapeutic agents include: erlotinib (TARCEVA.TM.,
Genentech/OSI Pharm.), docetaxel (TAXOTERE.TM., Sanofi-Aventis),
5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine
(GEMZAR.TM., Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),
cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1),
carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL.TM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab
(HERCEPTIN.TM., Genentech), temozolomide
(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carbox-
-amide, CAS No. 85622-93-1, TEMODAR.TM. TEMODAL.TM., Schering
Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanami-
-ne, NOLVADEX.TM., ISTUBAL.TM., VALODEX.TM.), and doxorubicin
(ADRIAMYClNO.TM.), Akti-1/2, HPPD, and rapamycin.
[0125] More examples of chemotherapeutic agents include:
oxaliplatin (ELOXATIN.TM., Sanofi), bortezomib (VELCADE.TM.,
Millennium Pharm.), sutent (SUNITINIBO.TM., SU11248, Pfizer),
letrozole (FEMARA.TM., Novartis), imatinib mesylate (GLEEVECM,
Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515),
ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca),
SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K
inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK
222584 (Novartis), fulvestrant (FASLODEX.TM., AstraZeneca),
leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE.TM.,
Wyeth), lapatinib (TYKERB.TM.., GSK572016, Glaxo Smith Kline),
lonafarnib (SARASAR.TM., SCH 66336, Schering Plough), sorafenib
(NEXAVAR.TM., BAY43-9006, Bayer Labs), gefitinib (IRESSA.TM.,
AstraZeneca), irinotecan (CAMPTOSAR.TM., CPT-11, Pfizer),
tipifarnib (ZARNESTRA.TM., Johnson & Johnson), ABRAXANE.TM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, II),
vandetanib (rINN, ZD6474, ZACTIMA.TM., AstraZeneca), chloranmbucil,
AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL.TM., Wyeth),
pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA.TM., Telik),
thiotepa and cyclosphosphamide (CYTOXAN.TM., NEOSAR.TM.); alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analog topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogs); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogs, KW-2189 and CB 1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, calicheamicin gammall,
calicheamicin omegall (Angew Chem. Intl. Ed. Engl. (1994)
33:183-186); dynemicin, dynemicin A; bisphosphonates, such as
clodronate; an esperamicin; as well as neocarzinostatin chromophore
and related chromoprotein enediyne antibiotic chromophores),
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSK.TM. polysaccharide complex (JHS Natural Products, Eugene,
Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; vinorelbine; novantrone; teniposide; edatrexate;
daunomycin; aminopterin; capecitabine (XELODAM Roche); ibandronate;
CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine
(DMFO); retinoids such as retinoic acid; and pharmaceutically
acceptable salts, acids and derivatives of any of the above.
[0126] Also included in the definition of"chemotherapeutic agent"
are: (i) anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including NOLVADEX.TM.; tamoxifen citrate), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.TM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.TM. (megestrol acetate),
AROMASIN.TM. (exemestane; Pfizer), formestanie, fadrozole,
RIVISOR.TM. (vorozole), FEMARA.TM. (letrozole; Novartis), and
ARIMIDEX..TM. (anastrozole; AstraZeneca); (iii) anti-androgens such
as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO
2007/044515); (v) lipid kinase inhibitors; (vi) antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in aberrant cell
proliferation, for example, PKC-alpha, Raf and H-Ras, such as
oblimersen (GENASENSE.TM., Genta Inc.); (vii) ribozymes such as
VEGF expression inhibitors (e.g., ANGIOZYME.TM.) and HER2
expression inhibitors; (viii) vaccines such as gene therapy
vaccines, for example, ALLOVECTIN.TM., LEUVECTIN.TM., and
VAXID.TM.; PROLEUKIN.TM. rIL-2; topoisomerase 1 inhibitors such as
LURTOTECAN.TM.; ABARELIX.TM. rmRH; (ix) anti-angiogenic agents such
as bevacizumab (AVASTIN.TM., Genentech); and pharmaceutically
acceptable salts, acids and derivatives of any of the above.
[0127] Also included in the definition of "chemotherapeutic agent"
are therapeutic antibodies such as alemtuzumab (Campath),
bevacizumab (AVASTIN.TM., Genentech); cetuximab (ERBITUX.TM.,
Imclone); panitumumab (VECTIBIX.TM., Amgen), rituximab
(RITUXAN.TM., Genentech/Biogen Idec), pertuzumab (OMNITARG.TM.,
2C4, Genentech), trastuzumab (HERCEPTIN.TM., Genentech),
tositumomab (Bexxar, Corixia), and the antibody drug conjugate,
gemtuzumab ozogamicin (MYLOTARG.TM., Wyeth).
[0128] Anti-inflammatory agents include NSAID agents. The term
"NSAID" is an acronym for "non-steroidal anti-inflammatory drug"
and is a therapeutic agent with analgesic, antipyretic (lowering an
elevated body temperature and relieving pain without impairing
consciousness) and, in higher doses, with anti-inflammatory effects
(reducing inflammation). The term "non-steroidal" is used to
distinguish these drugs from steroids, which (among a broad range
of other effects) have a similar eicosanoid-depressing,
anti-inflammatory action. As analgesics, NSAIDs are unusual in that
they are non-narcotic. NSAIDs include aspirin, ibuprofen, and
naproxen. NSAIDs are usually indicated for the treatment of acute
or chronic conditions where pain and inflammation are present.
NSAIDs are generally indicated for the symptomatic relief of the
following conditions: rheumatoid arthritis, osteoarthritis,
inflammatory arthropathies (e.g. ankylosing spondylitis, psoriatic
arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic
bone pain, headache and migraine, postoperative pain,
mild-to-moderate pain due to inflammation and tissue injury,
pyrexia, ileus, and renal colic. Most NSAIDs act as non-selective
inhibitors of the enzyme cyclooxygenase, inhibiting both the
cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isoenzymes.
Cyclooxygenase catalyzes the formation of prostaglandins and
thromboxane from arachidonic acid (itself derived from the cellular
phospholipid bilayer by phospholipase A2). Prostaglandins act
(among other things) as messenger molecules in the process of
inflammation. COX-2 inhibitors include celecoxib, etoricoxib,
lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib.
[0129] In one embodiment, the compounds of Formula I are linked to
ligands which comprise: polypeptides such as antibodies or antibody
fragments bearing epitope recognition sites, such as Fab, Fab',
F(ab').sub.2 fragments, Fv fragments, single chain antibodies,
antibody mimetics (such as DARPins, affibody molecules, affilins,
affitins, anticalins, avimers, fynomers, Kunitz domain peptides and
monobodies), peptoids, aptamers and the like. In one embodiment the
first and second ligands are the same type of molecule. In another
embodiment, the first and second ligands are different types of
molecules. In some embodiments, the first or second ligands
comprise: antibodies, antibody fragments, Fv fragments; single
chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments,
humanized antibodies and antibody fragments; camelized antibodies
and antibody fragments, human antibodies and antibody fragments,
monospecific or bispecific antibodies, disulfide stabilized Fv
fragments, scFv tandems ((scFv) fragments), diabodies, tribodies or
tetrabodies, peptoids, peptide or nucleic acid aptamers, antibody
mimetics or combinations thereof. In other embodiments, the first
and second ligands comprise: a polypeptide, antibodies, antibody
fragments, antibody mimetics, single chain antibodies, nucleic
acids, an aptamer, a peptoid or a sugar moiety or combinations
thereof. In certain embodiments, the first and second ligands are
peptide or nucleic acid aptamers. In other embodiments, the first
and second ligands are sugar moieties comprising
glycosaminoglycans, heparan sulfates or chondroitin sulfates.
[0130] Other Uses:
[0131] The synthesized compounds, preferably, oligomers of the
compounds have a multitude of other uses. The methods embodied
herein provide for the synthesis of a plurality of compounds via
steps which do not require expensive equipment or consumables, they
do not require large laboratory spaces or staff, the steps are
efficient in producing high yields of compounds and do not require
expensive starting materials. The end user has the luxury of
designing the compounds in which to build a library so as to assay
for any diagnostic application(s), identification of patients at
risk of developing a condition or disorder, or any therapeutic
effects the synthesized compounds may have. As discussed above, a
library of compounds can be designed to have any desired and
varying degrees of rigidity or flexibility to produce a desired
library. For example, the compounds may comprise R and/or S
configuration chirality and combinations thereof. The compounds can
then be screened in any type of screening assay, for example,
high-throughput screens.
[0132] The compounds can be tested for various effects. For
example, in the case a therapeutic agent is identified as a
candidate for treating cancer, for example, the candidate agent
modulates a tumor gene, follow on tests such as, effects of the
candidate agent on tumor cells and tissues, gene expression,
receptor expression, arresting of cell growth, tumor growth factors
and the like.
[0133] In other embodiments, the compounds can be used in the
synthesis of other compounds, for example, the oligomers can be
used as a rigid backbone, and some of the compounds can be used as
intermediates or starting materials, and the like.
[0134] In other embodiments, the ligands are linked to a detectable
label (detectable molecule), either directly or linked via a
suitable linker. The present invention is not limited to any
particular linker group. Indeed, a variety of linker groups are
contemplated, suitable linkers could comprise, but are not limited
to, alkyl groups, ether, polyether, alkyl amide linker, a peptide
linker, a polypeptide linker, a modified peptide or polypeptide
linker, a peptide nucleic acid (PNA) a Poly(ethylene glycol) (PEG)
linker, a streptavidin-biotin or avidin-biotin linker,
polyaminoacids (e.g. polylysine), functionalized PEG,
polysaccharides, glycosaminoglycans, dendritic polymers PEG-chelant
polymers, oligonucleotide linker, phospholipid derivatives, alkenyl
chains, alkynyl chains, disulfide, or a combination thereof.
[0135] In another embodiment, the detectable label is linked to the
ligand, through a chemical bond, or noncovalently, through ionic,
van der Waals, electrostatic, or hydrogen bonds.
[0136] Fluorophores include any compound, composition or molecule
capable of emitting light in response to irradiation. In many
instances, fluorophores emit light in the visible region of the
spectrum. In other instances, the fluorophores can emit light in
the non-visible regions of the spectrum, such as ultraviolet,
near-ultraviolet, near-infrared, and infrared. For example and
without limitation, examples of fluorophores include: quantum dots;
nanoparticles; fluorescent proteins, such as green fluorescent
protein and yellow fluorescent protein; heme-based proteins or
derivatives thereof; carbocyanine-based chromophores, such as IRDye
800CW, Cy 3, and Cy 5; coumarin-based chromophores, such as
(7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin) (CPM);
fluorine-based chromophores, such as fluorescein, fluorescein
isothiocyanate (FITC); and numerous ALEXA FLUOR.TM. chromophores
and ALEXA FLUOR.TM. bioconjugates, which absorb in the visible and
near-infrared spectra. The emission from the fluorophores can be
detected by any number of methods, including but not limited to,
fluorescence spectroscopy, fluorescence microscopy, fluorimeters,
fluorescent plate readers, infrared scanner analysis, laser
scanning confocal microscopy, automated confocal nanoscanning,
laser spectrophotometers, fluorescent-activated cell sorters
(FACS), image-based analyzers and fluorescent scanners (e.g.,
gel/membrane scanners).
[0137] Chemiluminescent moieties include any compound, composition
or molecule capable of emitting light in response to a chemical
reaction. A bioluminescent compound refers to a naturally occurring
form of a chemiluminescent compound. Examples of chemiluminescent
compounds include: lucigenin, luminol. Examples of bioluminescent
compounds include: luciferins, coelenterazines. The emission from
chemiluminescent compounds can be detected by luminometers or
scanning spectrometers.
[0138] The labeled compounds can be used as diagnostics for both in
vivo and in vitro use. For example, a compound may be identified as
a ligand for a certain receptor which may be up-regulated in a
disease state (e.g. tumor antigens). The compounds can be labeled
with a detectable label in order to detect binding.
[0139] In another preferred embodiment, a candidate compound has a
direct therapeutic effect, that is, without the requirement of any
other modifications. The identified compounds can be then used in
the prevention or treatment of that disease or disorder. For
example, treatment of: inflammatory disease, neuroinflammatory
diseases, cancer, neurological diseases, cardiovascular diseases,
parasitic or bacterial diseases, viral diseases, central nervous
system diseases, brain diseases, etc.
[0140] In some embodiments, the methods are used to identify and
quantify a specific molecule in a sample, for example, for
diagnostic purposes, or monitoring the response to treatment or
metabolism of drugs in vivo, etc. In some embodiments, a method of
quantifying a specific molecule, e.g. a protein in a sample, the
method comprises the steps of: placing the sample containing the
specific target molecule into a receptacle, contacting the sample
with one or more compounds of Formula I wherein the compounds are
conjugated to a detectable label and quantifying the target
molecule. The Examples section details the steps of the types of
assays employed.
[0141] In some embodiments, a method of quantifying a specific
molecule, e.g. a protein in a sample, the method comprises a
Forster Resonance Energy Transfer (FRET), Bioluminescence Resonance
Energy Transfer (BRET), or fluorescence polarization assay.
[0142] In one embodiment, the assay employs Forster Resonance
Energy Transfer or FRET, a process in which a fluorophore ("donor")
that can be excited by light and can transfer the excitation to a
second fluorophore ("acceptor") if and only if they are
sufficiently close, that is, within a distance in the order of 100
.ANG. or less, defined by the Forster radius. Although FRET is used
as an illustrative example, the assays are not limited to FRET
based assays. For example, an assay which uses a bioluminescent
protein, such as luciferase, to excite a proximal fluorophore
(BRET), typically a fluorescent protein (Xu et al. (1999) Proc.
Natl. Acad. Sci. USA 96(1), 151-6). Another assay alternative is a
luminescent oxygen-channeling chemistry (Ullman et al. (1994) Proc.
Natl. Acad. Sci. USA 91(12), 5426-30), wherein a light induced
singlet oxygen generating system transfers the singlet oxygen to a
chemiluminescent system in proximity.
[0143] In one embodiment, the donor and acceptor fluorophores
(detectable label/detectable molecules) are attached to two
distinct compounds of Formula I, for example, varying oligomers of
Formula I that can bind specifically to distinct sites of one and
the same target, for example, B-CLL. When the compounds carrying
the donor and the acceptor fluorophore, respectively, bind to the
same target molecule and in doing so become sufficiently close to
each other, irradiation of the sample at a wavelength that allows
excitation of the donor results in emission of radiation by the
acceptor. Compounds that are not bound to the same target do not
give rise to FRET and therefore need not be removed prior to
measurement of emitted radiation.
[0144] In other embodiments, the assay is an immunoassay. For
example, ELISA's RIA's, Western blots, gels, immunoblots, and the
like. In other examples, the assays, comprise nucleic acid based
assays (e.g. hybridization assays). In embodiments, the assays are
high-throughput screening assays.
[0145] In embodiments, the target is present in a sample
comprising: a liquid, a semi-liquid, a gel, a biological sample, an
intact cell, a permeabilized cell, a disrupted cell, a cell
homogenate, a membrane, or a cellular organelle.
[0146] Pharmaceutical Compositions
[0147] The pharmaceutical compositions of the invention may
contain, for example, more than one specificity. In some examples,
a pharmaceutical composition of the invention, containing one or
more compounds of the invention, is administered in combination
with another useful composition such as an anti-inflammatory agent,
an immunostimulator, a chemotherapeutic agent, an antiviral agent,
or the like. Furthermore, the compositions of the invention may be
administered in combination with a cytotoxic, cytostatic, or
chemotherapeutic agent such as an alkylating agent,
anti-metabolite, mitotic inhibitor or cytotoxic antibiotic, as
described above. In general, the currently available dosage forms
of the known therapeutic agents for use in such combinations will
be suitable.
[0148] Combination therapy (or "co-therapy") includes the
administration of a compound of Formula I, Formula I conjugated to
one or more agents that are administered with a second agent as
part of a specific treatment regimen intended to provide the
beneficial effect from the co-action of these therapeutic agents.
In addition, some compounds of Formula I may be conjugated to one
agent and other compounds of Formula I are conjugated to another
agent and are administered as part of the combination therapy. The
beneficial effect of the combination includes, but is not limited
to, pharmacokinetic or pharmacodynamic co-action resulting from the
combination of the compounds embodied herein and therapeutic
agents. Administration of these the compounds embodied herein and
therapeutic agents in combination typically is carried out over a
defined time period (usually minutes, hours, days or weeks
depending upon the combination selected).
[0149] Combination therapy may, but generally is not, intended to
encompass the administration of two or more of these compounds
embodied herein and therapeutic agents as part of separate
monotherapy regimens that incidentally and arbitrarily result in
the combinations of the present invention. Combination therapy is
intended to embrace administration of these therapeutic compounds
embodied herein, in a sequential manner, that is, wherein each the
compounds embodied herein and therapeutic agent are administered at
a different time, as well as administration of the compounds
embodied herein and therapeutic agents, or at least two of the
compounds embodied herein and, in a substantially simultaneous
manner. Substantially simultaneous administration can be
accomplished, for example, by administering to the subject a single
capsule having a fixed ratio of each of the compounds embodied
herein and therapeutic agent or in multiple, single capsules for
each of the compounds embodied herein and/or therapeutic
agents.
[0150] Sequential or substantially simultaneous administration of
each of the compounds embodied herein and therapeutic agent can be
effected by any appropriate route including, but not limited to,
topical routes, oral routes, intravenous routes, intramuscular
routes, and direct absorption through mucous membrane tissues. The
therapeutic agents can be administered by the same route or by
different routes. For example, a first therapeutic agent of the
combination selected may be administered by injection while the
other therapeutic agents of the combination may be administered
topically.
[0151] Alternatively, for example, all therapeutic agents may be
administered topically or all therapeutic agents may be
administered by injection. The sequence in which the compounds
embodied herein and therapeutic agents are administered is not
narrowly critical unless noted otherwise. Combination therapy also
can embrace the administration of the compounds embodied herein and
therapeutic agents as described above in further combination with
other biologically active ingredients. Where the combination
therapy further comprises a non-drug treatment, the non-drug
treatment may be conducted at any suitable time so long as a
beneficial effect from the co-action of the combination of the
compounds embodied herein and the therapeutic agents and non-drug
treatment is achieved. For example, in appropriate cases, the
beneficial effect is still achieved when the non-drug treatment is
temporally removed from the administration of the compounds
embodied herein and therapeutic agents, perhaps by days or even
weeks.
[0152] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of the active
component(s) of the therapy, dissolved or dispersed in a
pharmaceutically acceptable medium. Pharmaceutically acceptable
media or carriers include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Supplementary active ingredients can also be incorporated into
the therapeutic compositions of the present invention.
[0153] For any compound used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially
from activity assays in cell cultures and/or animals. For example,
a dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined by
activity assays (e.g., the concentration of the test compound,
which achieves a half-maximal inhibition of the proliferation
activity). Such information can be used to more accurately
determine useful doses in humans.
[0154] Toxicity and therapeutic efficacy of the compounds described
herein can be determined by standard pharmaceutical procedures in
experimental animals, e.g., by determining the IC.sub.50 and the
LD.sub.50 (lethal dose causing death in 50% of the tested animals)
for a subject compound. The data obtained from these activity
assays and animal studies can be used in formulating a range of
dosage for use in human.
[0155] The dosage may vary depending upon the dosage form employed
and the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g., Fingl, et
al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.
1). Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain therapeutic effects, termed the minimal effective
concentration (MEC). The MEC will vary for each preparation, but
can be estimated from in vitro and/or in vivo data, e.g., the
concentration necessary to achieve 50-90% inhibition of a
proliferation of certain cells may be ascertained using the assays
described herein. Dosages necessary to achieve the MEC will depend
on individual characteristics and route of administration. HPLC
assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using the MEC value.
Preparations should be administered using a regimen, which
maintains plasma levels above the MEC for 10-90% of the time,
preferable between 30-90% and most preferably 50-90%. Depending on
the severity and responsiveness of the condition to be treated,
dosing can also be a single administration of a slow release
composition described hereinabove, with course of treatment lasting
from several days to several weeks or until cure is effected or
diminution of the disease state is achieved. The amount of a
composition to be administered will, of course, be dependent on the
subject being treated, the severity of the affliction, the manner
of administration, the judgment of the prescribing physician,
etc.
[0156] The preparation of pharmaceutical or pharmacological
compositions will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including eye drops, creams, lotions, salves,
inhalants and the like. The use of sterile formulations, such as
saline-based washes, by surgeons, physicians or health care workers
to treat a particular area in the operating field may also be
particularly useful. Compositions may also be delivered via
microdevice, microparticle or other known methods.
[0157] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0158] In this context, the quantity of active ingredient and
volume of composition to be administered depends on the host animal
to be treated. Precise amounts of active compound required for
administration depend on the judgment of the practitioner and are
peculiar to each individual.
[0159] The pharmaceutical compositions may be sterilized and/or
contain adjuvants, such as preserving, stabilizing, wetting or
emulsifying agents, solution promoters, salts for regulating the
osmotic pressure and/or buffers. In addition, they may also contain
other therapeutically valuable substances. The compositions are
prepared according to conventional mixing, granulating, or coating
methods, and typically contain about 0.1% to 75%, preferably about
1% to 50%, of the active ingredient.
[0160] Liquid, particularly injectable compositions can, for
example, be prepared by dissolving, dispersing, etc. The active
compound is dissolved in or mixed with a pharmaceutically pure
solvent such as, for example, water, saline, aqueous dextrose,
glycerol, ethanol, and the like, to thereby form the injectable
solution or suspension. Additionally, solid forms suitable for
dissolving in liquid prior to injection can be formulated.
[0161] Effective doses of the compositions of the present
invention, for the treatment of the above described diseases, vary
depending upon may different factors, including means of
administration, physiological state of the patient, whether the
patient is human or an animal, other medications administered, and
whether treatment is prophylactic or therapeutic. Usually, the
patient is a human, but in certain embodiments, a patient is an
animal, particularly an animal selected from a mammalian species
including rat, rabbit, bovine, ovine, porcine, canine, feline,
murine, equine, and primate.
[0162] The compounds can be administered on multiple occasions,
wherein intervals between single dosages can be daily, weekly,
monthly, or yearly. Alternatively, one or more of the compounds of
the invention can be administered as a sustained-release
formulation, in which case less frequent administration is
required. Dosage and frequency may vary depending on the half-life
of the compounds of the invention. In therapeutic applications, a
relatively high dosage at relatively short intervals is sometimes
required until progression of the disease is reduced or terminated,
and sometimes until the patient shows partial or complete
amelioration of symptoms of the disease. Thereafter, the patient
can be administered a prophylactic regime.
[0163] Administration of a pharmaceutical composition of the
compounds described herein can be carried out via a variety of
routes including, but are not limited to, oral, topical, pulmonary,
rectal, subcutaneous, intradermal, intranasal, intracranial,
intramuscular, intraocular, or intra-articular injection and the
like. One or more compounds described herein can optionally be
administered in combination with other biological or chemical
agents that are at least partly effective in treatment of a
disease.
[0164] As noted above, the compounds described herein may be
administered for example, but are not limited to, orally,
topically, pulmonary, rectally, subcutaneously, intradermally,
intranasally, intracranially, intramuscularly, intraocularly, or
intra-arterially and the like. The carrier or excipient or
excipient mixture can be a solvent or a dispersive medium
containing for example, but are not limited to, various polar or
non-polar solvents, suitable mixtures thereof, or oils. As used
herein "carrier" or "excipient" means a pharmaceutically acceptable
carrier or excipient and includes any and all solvents, dispersive
agents or media, coating(s), antimicrobial agents,
iso/hypo/hypertonic agents, absorption-modifying agents, and the
like. The use of such substances and the agents for
pharmaceutically active substances is well known in the art.
Moreover, other or supplementary active ingredients can also be
incorporated into the final composition.
[0165] Administration of the compounds embodied herein, by
intravenous formulation is well known in the pharmaceutical
industry. An intravenous formulation should possess certain
qualities aside from being just a composition in which the
compounds embodied herein are soluble. For example, the formulation
should promote the overall stability of the active ingredient(s),
also, the manufacture of the formulation should be cost effective.
All of these factors ultimately determine the overall success and
usefulness of an intravenous formulation.
[0166] Other accessory additives that may be included in
pharmaceutical formulations of compounds of the present invention
as follow: solvents: ethanol, glycerol, propylene glycol;
stabilizers: ethylene diamine tetraacetic acid (EDTA), citric acid;
antimicrobial preservatives: benzyl alcohol, methyl paraben, propyl
paraben; buffering agents: citric acid/sodium citrate, potassium
hydrogen tartrate, sodium hydrogen tartrate, acetic acid/sodium
acetate, maleic acid/sodium maleate, sodium hydrogen phthalate,
phosphoric acid/potassium dihydrogen phosphate, phosphoric
acid/disodium hydrogen phosphate; and tonicity modifiers: sodium
chloride, mannitol, dextrose.
[0167] The presence of a buffer may be necessary to maintain the
aqueous pH in the range of from about 4 to about 8 and more
preferably in a range of from about 4 to about 6. The buffer system
is generally a mixture of a weak acid and a soluble salt thereof,
e.g., sodium citrate/citric acid; or the monocation or dication
salt of a dibasic acid, e.g., potassium hydrogen tartrate; sodium
hydrogen tartrate, phosphoric acid/potassium dihydrogen phosphate,
and phosphoric acid/disodium hydrogen phosphate.
[0168] The compositions can be formulated in an oral unit dosage
form. The term "unit dosage forms" refers to physically discrete
units suitable as unitary dosages for a patient, each unit
containing a predetermined quantity of active material calculated
to produce the desired therapeutic effect, in association with a
suitable pharmaceutical excipient.
[0169] Kits and Methods
[0170] The present invention further provides systems and kits
(e.g., commercial therapeutic, diagnostic, or research products,
reaction mixtures, etc.) that contain one or more or all components
sufficient, necessary, or useful to practice any of the methods
described herein. These systems and kits may include buffers,
detection/imaging components, positive/negative control reagents,
instructions, software, hardware, packaging, or other desired
components.
[0171] The kits provide useful tools for diagnosis, treatment of
patients, screening of compounds for therapeutic potential and the
like, and contain one or more compounds of Formula I. In some
embodiments, the kits comprise the compounds of Formula I and one
or more detectible labels or therapeutic agents. In other
embodiments, the kits comprise the compounds of Formula I
conjugated to linked to one or more detectible labels or
therapeutic agents. The kits can be packaged in any suitable manner
to aid research, clinical, and testing labs, typically with the
various parts, in a suitable container along with instructions for
use.
[0172] In certain embodiments, the kits may further comprise, where
necessary, agents for reducing the background interference in a
test, positive and negative control reagents, apparatus for
conducting a test, and the like.
[0173] In certain embodiments of the methods and kits provided
herein, solid phase supports are used for purifying proteins,
labeling samples or carrying out the solid phase assays. Examples
of solid phases suitable for carrying out the methods disclosed
herein include beads, particles, colloids, single surfaces, tubes,
multiwell plates, microtiter plates, slides, membranes, gels and
electrodes. When the solid phase is a particulate material (e.g.,
beads), it is, in one embodiment, distributed in the wells of
multi-well plates to allow for parallel processing of the solid
phase supports.
[0174] Methods and kits disclosed herein may be carried out in
numerous formats known in the art. In certain embodiments, the
methods provided herein are carried out using solid-phase assay
formats. In certain embodiments, the methods provided herein are
carried out in a well of a plate with a plurality of wells, such as
a multi-well plate or a multi-domain multi-well plate. The use of
multi-well assay plates allows for the parallel processing and
analysis of multiple samples distributed in multiple wells of a
plate. Multi-well assay plates (also known as microplates or
microtiter plates) can take a variety of forms, sizes and shapes
(e.g., round- or flat-bottom multi-well plates). Exemplary
multi-well plate formats that can be used in the methods provided
herein include those found on 96-well plates (12.times.8 array of
wells), 384-well plates (24.times.16 array of wells), 1536-well
plate (48.times.32 array of well), 3456-well plates and 9600-well
plates. Other formats that may be used in the methods provided
herein include, but are not limited to, single or multi-well plates
comprising a plurality of domains, cuvettes, microarrays etc.. In
certain embodiments, the plates are black-wall, black-bottom
plates. In certain embodiments, the plates are black-wall,
white-bottom plates. In certain embodiments, the plates have black
walls and clear bottoms in order to allow bottom reading of the
fluorescence signals. In certain embodiments, the plates are chosen
with minimal and uniform intrinsic fluorescence intensity within
the range utilized in the method to avoid interference with the
FRET signals.
[0175] The methods provided herein, when carried out in
standardized plate formats can take advantage of readily available
equipment for storing and moving these plates as well as readily
available equipment for rapidly dispensing liquids in and out of
the plates (e.g., robotic dispenser, multi-well and multi-channel
pipettes, plate washers and the like).
[0176] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of this disclosure,
may make modifications and improvements within the spirit and scope
of the invention.
[0177] All documents mentioned herein are incorporated herein by
reference. All publications and patent documents cited in this
application are incorporated by reference for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted. By their citation of various
references in this document, Applicants do not admit any particular
reference is "prior art" to their invention.
Examples
[0178] While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. The following
non-limiting examples are illustrative of the invention.
Example 1: A Biomimetic and Polyketide-Inspired Approach to Small
Molecule Ligand Discovery
[0179] Development of Approach:
[0180] Synthetically, COPAs have been designed to be accessible
using a "sub-monomer" route akin to that employed in peptoid
synthesis (FIG. 3A). This would allow simple primary amines, of
which hundreds are commercially available, to be employed as one of
the diversity elements in split-and-pool library synthesis.
Furthermore, this design was optimistically thought to be
compatible with MS-MS analysis, where the compound derived from a
single bead would be sufficient to decode the precise structure of
oligomer present. As such, one could avoid employing an encoding
strategy to elucidate hit-structure.
[0181] To affect this strategy, a practical and scalable synthesis
of both antipodes of chloropentenoic acids like 1 was required
(see, FIG. 3B). Ideally, this synthesis would also be capable of
delivering future analogs of 1 with diverse C2 and C4 substitution.
Therefore, a convergent synthetic pathway was targeted to
facilitate future analog generation that could avoid
chromatographic purification at any step, and that would proceed
from readily available starting materials, limit the use of air and
moisture sensitive reagents, and deliver the chiral monomers with
high levels of stereochemical fidelity. The solution to this
problem is depicted in FIG. 3B.
[0182] Results:
[0183] Synthesis of the propionyl oxazolidinone 4 was accomplished
without the requirement of a highly reactive base (Ho, G-J et al.
J. Org. Chem. 60, 2271-2273 (1995)) and subsequent stereoselective
aldol reaction with methacrolein (Nielsen, P. E. Chem. Biodivers.
7, 786-804 (2010)) was achieved under reaction conditions that do
not require pre-generation of a metal enolate (Evans, D. A et al.
J. Am. Chem. Soc. 124, 392-393 (2002)). Isolated by simple
extraction, the TMS-ether 7 was converted to the stereodefined
allylic chloride 8 by Nb-mediated stereoselective halogenation
(E:Z.gtoreq.20:1) (Ravikumar, P. C. et al. J Org. Chem. 74,
7294-7299 (2009)). While seemingly difficult to accomplish in a
highly selective fashion, hydrolysis of the imide proceeded
uneventfully (without significant hydrolysis of the allylic
chloride) and delivered 1 in .gtoreq.95% ee and 41% overall yield.
Notably, this synthesis procedure delivers optically active 1 in
acceptable yield and purity through a four-step sequence that does
not require a single chromatographic operation. As an indication of
the robust nature of this sequence, 10 g of 4 was converted to ca.
4 g of 1 routinely.
[0184] As depicted in FIG. 3B, solution phase amide bond formation
with a simple secondary amine (via the mixed anhydride) proceeded
effectively, in this case delivering the chloroamide 10 in 94%
yield. Unlike related coupling reactions for the synthesis of
peptides, no evidence was found for epimerization of the
potentially labile .alpha.-stereocenter of 10. Subsequent coupling
with benzylamine proceeded in a similarly straightforward manner,
delivering aminoamide 11 in 82% yield. This two-step sequence
validates the central steps of the proposed oligomerization of 1
and confirms that chiral chloroacids of this and related structures
can be functionalized in a manner related to a-bromoacetic acid in
peptoid synthesis.
[0185] Subsequent homologation of 11 with either enantiomer of 1
leads to the production of the corresponding dimers 12 and 13 with
similarly high levels of efficiency, indicating that double
asymmetric relationships between amine 11 and acid 1 have little
impact on chemical efficiency for this bond construction.
[0186] Moving forward to explore the utility of COPA oligomers as a
potential source of protein ligands, a library of tetramers was
prepared by split-and-pool methods. To be compatible with the
on-bead screening platform (Xiao, X et al. J. Comb. Chem. 9,
592-600 (2007)), 160m TENTAGEL beads were selected, that were
functionalized with a tetrameric polyamide (FIG. 3D). Targeting a
library of 160,000 members, ten primary amines and two pentenoic
acids were employed, as depicted in FIG. 3E. Since structural
elucidation was to be conducted by mass spectrometry, a heavy atom
label (CD.sub.3 at C2) was employed to correlate differences in the
mass of fragment ions with absolute stereochemistry at that center.
Alongside these efforts, a library of peptoid tetramers was
prepared with the same amines used for the COPA library (FIG. 3E)
in an effort to establish a baseline for comparison between these
two synthetic oligomer platforms. MALDI mass spectra revealed a
single strong peak for the COPAs released from several individual
beads chosen randomly from the library, indicating that each bead
displays predominantly a single compound and that each synthetic
step proceeded in high yield.
[0187] Having established the high quality of the library, it was
screened against the DNA-binding domain of p53, an important
transcription factor that regulates a variety of genes involved in
cell cycle control and apoptosis. More than half of human cancers
express inactive p53 due to the presence of missense mutations in
the DNA-binding domain that destabilize the folding of the protein
(Levine, A J. et al. Nat. Rev. Cancer, 9, 749-758 (2009)). There is
considerable interest in the identification of "chemical
chaperones" whose binding to p53 might stabilize the wild-type,
functional, folded conformation (Brown, C. J., et al. Nat. Rev.
Cancer, 9, 862-873 (2009)). Since transcription factors are
generally considered to be extremely challenging targets for small
molecules (Cochran, A G. Chem. & Biol., 7, R85-R94 (2000)), p53
recognition was considered as a stringent test of the utility of
this new class of compounds.
[0188] Purified, bacterially expressed, FLAG-tagged p53 DBD (10
.mu.M) was incubated with the bead-displayed COPA library in the
presence of high levels of competitor proteins to suppress
non-specific binding events. The beads were then washed and treated
with anti-FLAG antibody followed, after another washing step, by
anti-IgG antibodies conjugated to red quantum dots. The beads were
then examined under a low power fluorescent microscope. Several
beads with a strong red halo surrounding them, indicating binding
of the quantum dot via the p53-FLAG/anti-FLAG antibody/anti-IgG-QD
sandwich complex, were observed (FIG. 4A). These, as well as some
beads with weaker staining, were picked using a micropipette. In
all, 22 beads were collected. Six of these putative hits proved to
be ligands for either anti-FLAG antibody or the secondary
antibody-conjugated quantum dots. The same experiment was done with
the peptoid library. In this case, no obvious "hits" with strong
red halos were observed, but several more weakly fluorescent beads
were picked. The beads were separated in wells of a microtiter
plate and released from the bead via CNBr-mediated cleavage of a
methionine residue in the linker.
[0189] While strong molecular ion peaks were observed in the MALDI
mass spectrum for the COPAs, well-defined fragments were not
produced in the MS/MS spectrum. Therefore, these molecules were
sequenced via tandem ESI mass spectrometry using electron transfer
dissociation (ETD) as the fragmentation method (Syka, J. E. P., et
al. Proc. Natl. Acad. Sci. USA, 101, 9528-9533 (2004)). This proved
reasonably successful and the sequences of 8 of the 16 COPA hits
could be determined unequivocally. These eight molecules were
re-synthesized with a fluorescein tag and tested for binding to p53
by fluorescence anisotropy. Two of the eight COPA molecules showed
clear binding to p53. The best of these, compound 14 bound to the
p53 DBD with a K.sub.D of approximately 10 .mu.M, but did not bind
detectably (K.sub.D>500 .mu.M) to two control proteins (FIG.
4C). COPA 14 constitutes the first non-covalent (Lambert, J. M. et
al. Cancer Cell, 15, 376-388 (2009)) small molecule ligand for the
wild-type (Boekler, F. M. et al. Proc. Natl. Acad. Sci. USA, 105,
10360-10365 (2008)) p53 DNA-binding domain. The addition of an
oligonucleotide that binds p53 tightly did not disrupt the p53-COPA
complex, indicating that the synthetic ligand does not recognize
the DNA-binding surface of the p53 core domain. The same set of
experiments was carried out for the peptoids collected as possible
hits in the screening experiment. Not surprisingly, given the low
intensity of QD fluorescence observed on the beads, none of the
peptoids exhibited binding to the p53 DNA-binding domain
(K.sub.Ds>500 .mu.M). This is interesting in that the COPA and
peptoid libraries contained exactly the same amine-derived side
chains. This experiment is consistent with the proposition that
conformationally constrained COPAs may be superior to peptoids as a
source of protein ligands.
[0190] In summary, a new class of natural product-inspired
oligomeric compounds have been developed that promise to be a
valuable source of protein ligands. COPAs are unusual amongst
synthetic small molecule oligomers in that they employ concepts
long utilized in organic synthesis for acyclic stereo control, such
as minimization of A1,3 interactions, to impose significant
conformational constraints on the main chain, and subsequent
disposition of all main chain substituents in three-dimensional
space. In essence, this results in a practical chemical solution to
diversity-oriented library construction that couples building block
diversity to substantial scaffold diversity (i.e. in the case of a
tetrameric COPA that employs antipodes of a common pentenoic acid,
16 distinct and relatively inflexible scaffolds; FIG. 2). This is
noteworthy, because the desirability of scaffold diversity in
natural product-like libraries has been well documented (Clemons,
P. A. et al. Proc. Natl. Acad. Sci. USA 107, 18787), and current
solutions to this problem require careful synthesis planning to
accomplish strategic and divergent reactivity of complex organic
intermediates in a library synthesis (Morton, D. et al. Angew.
Chem. Int. Ed. 48, 104-109 (2009); Luo, T. J. Am. Chem. Soc. 131,
5667-5674 (2009); Uchida, T. et al. Org. Lett. 11, 1559-1562
(2009)). Further compounding the virtues of COPAs as a chemical
foundation to discovery-oriented science, the synthetic strategy
described here is completely compatible with split-and-pool solid
phase synthesis, making very large libraries readily accessible.
Moreover, the highly practical and scalable synthesis of either
optically pure antipode of building blocks (i.e. 1), combined with
the use of simple primary amines as the source of side chain
diversity, allows the synthesis of potentially millions of
compounds at a modest cost. Finally, COPA libraries synthesized on
hydrophilic TENTAGEL beads can be employed in a variety of
inexpensive, yet powerful, binding screens (Xiao, X. et al., J.
Comb. Chem. 9, 592-600 (2007); Reddy, M. M. et al. Cell, 144, in
press (2011); Udugamasooriya, D. G. et al., J. Am. Chem. Soc. 130,
5744-5752 (2008)) hence eliminating the requirement of substantial
infrastructure to maintain diverse compound collections for use in
traditional high-throughput screening. Further developments of this
technology will explore the power of this chemistry in combination
with available on-bead screening technologies as a platform for the
discovery of bioactive synthetic molecules.
Example 2: COPA Library Design and Synthesis
[0191] The COPA library was synthesized on TENTAGEL resin (1.0 g,
.about.520,000 beads/g, 0.55 mmol/g loading capacity) from Rapp
Polymere GmbH, Germany. The approximate total number of beads used
to synthesize the library was 520000, which was enough to get at
least one copy of each library member (total number of
beads/library diversity, 520000/405000=1.28). A COPA library was
synthesized on resin following the established
one-bead-one-compound split and pool technique (Lam K. S. et al.
Nature 354, 82-84, (1991)). Fifteen amines (as shown in FIG. 5)
were used at four positions. With COPA (S) and (R) isomers at three
different positions, the total diversity of the library was 405000.
A polyamide linker (FIG. 5) was synthesized first on TENTAGEL MB
NH.sub.2 resin (160 m) following standard solid-phase peptide
synthesis and microwave assisted submonomer protocols (Zuckermann,
R. N., et al., J. Am. Chem. Soc. 114, 10646-10647 (1992); Olivos,
H. J., Alluri, P. G., Reddy, M. M., Salony, D. & Kodadek, T.
Org Lett 4, 4057-4059, (2002)). To avoid the artifacts of the
MALDI-TOF MS, MALDI matrix, and tandem MS/MS with low molar mass
compounds a longer linker was designed. Met residue was used to
allow the cleavage of the compound from TENTAGEL.TM. resin by CNBr
treatment. A positively charged amino side chain was used to
increase the ionization mobility in mass spectrometry. The amine,
4-bromophenylethylamine, was used to help differentiate the sets of
signals derived from N-terminal product ions which appeared as
singlet and the C-terminal product ions which appeared as doublet
due the isotopic pattern of bromine present in the fragments.
[0192] TENTAGEL resins (1.0 g, 0.55 mmol) were swelled in anhydrous
DMF for 1 h. The beads were treated with 5 equiv of HOBt (1.04 g,
2.75 mmol), 5 equiv of HBTU (1.04 g, 2.75 mmol), 5 equiv of
N-methyl morpholine (277.7 .mu.L, 2.75 mmol) and 5 equiv of
Fmoc-Met-(OH) (1.02 g, 2.75 mmol) with gentle shaking at room
temperature for 3 h in a 50 mL glass reaction vessel from
ChemGlass. The beads were washed thoroughly with DMF. The Fmoc
group was removed by treating resin with 20% piperidine in DMF for
20 min (2.times.). The beads were thoroughly washed with DMF. The
rest of the linker peptoid was synthesized using microwave assisted
solid phase sub-monomer methods for peptoid synthesis using
boc-diaminoethane, 4-Br-pehenthylamine, methoxyethylamine and
methoxy propylamine.
[0193] For library synthesis TENTAGEL.TM. beads with linker were
treated with 20 mL of 1 M bromoacetic acid and 1 M diisopropyl
carbodiimide and microwave for 15 sec twice (10% power level)
before splitting them equally to distribute into 15 reaction
vessels for amine displacement. Each of the vessels were treated
with one of 15 amines (1 M in DMF, 2 mL) and subjected to microwave
for 15 sec twice (10% power level). The beads were washed with DMF
(5.times.) and split equally to distribute into 2 reaction vessels
and subjected to the coupling of (R)- or (S)-COPA isomers. Each
vessel with 500 mg of resins was treated with 7 equiv of
diisopropylcarbodiimide (1.93 mmol), 5 equiv of HOAt (1.38 mmol)
and 5 equiv of (R)- (1.38 mmol) or (S)- (1.38 mmol) in 10.0 mL of
anhydrous DMF. The coupling was carried out at 37.degree. C. for 3
h with gentle shaking. Beads were thoroughly washed with DMF and
pooled together and mixed well before subjecting them to amine
displacements. The beads were split equally to distribute into 15
reaction vessels (0.037 mmol in each vessel) and each of which was
treated with 2.0 mL of 1 M solution of one of 15 amines. The
reaction was carried out at 37.degree. C. for 3 h with gentle
shaking. The beads were thoroughly washed with DMF pooled together
and mixed thoroughly. The acylation reaction with COPA (R) and (S),
and displacement reaction with 15 amines were repeated two more
times to synthesize 4-mer COPA library. The side chain protecting
groups were removed by treating pooled resins with 20 mL of 94%
trifluoroacetic acid (TFA), 2% triisopropylsilane (TIS), 2%
thioanisole and 2% water with gentle shaking for 2 h at room
temperature. The resins were washed with DCM thoroughly, dried
under vacuum and stored at -20.degree. C.
[0194] Expression and purification of recombinant CLL monoclonal
antibodies: Purified CLL monoclonal antibodies used in screening
were obtained from professor Nicholas Chiorazzi's (The Feinstein
Institute for Medical Research, North Shore-Long Island Jewish
Health System, Manhasset, N.Y.). The heavy chain and light chain
were cloned separately and both plasmids were transiently
transformed with 293A HEK (human embryonic kidney) fibroblasts
(Catera R., et al., Mol. Med. 14 (11-12) 665-674 (2008)). The cells
were cultured in DMEM supplemented with 10% ultra-low IgG FCS
(GIBCO) and co-transfected with 12.5 g of IgH and IgL chain
encoding plasmid DNA by calcium phosphate precipitation. 8-12h
after transfection cells were washed with serum-free DMEM and
thereafter cultured in DMEM supplemented with 1% Nutridoma SP
(Roche). Supernatants were collected after 8 days of culture. For
self-reactivity HEp-2 ELISAs and IFAs antibodies were purified on
protein G SEPHAROSE.TM. (Amersham Pharmacia Biosciences). CLL mAbs
used in the screening of COPA libraries are listed below.
TABLE-US-00001 IgH CLL-mAb subset IgHV GenBank 14 9 1-69 AF021951
068 6 1-69 AY553640 169 NA 3-33 AY055480 183 4 4-34 AF021948
[0195] Library Screening:
[0196] The TENTAGEL resins displaying COPA library (1.0 g) were
swollen in DMF for 1 h, washed with DMF (5.times.), washed
extensively with .sub.ddH.sub.2O and incubated with water overnight
at room temperature (rt). The resins were washed with 1.times.PBST
(5.times.) and equilibrated with 1.times.PBST for 4 h at rt. The
resins were incubated with blocking buffer (1% BSA in Starting
Block PBS Blocking Buffer from Thermoscientific) for 2 h at rt and
washed with 1.times.PBST (3.times.) before subjecting them to
3-stage screening processes as shown in FIG. 6. First prescreen was
carried out using secondary antibody (Goat anti-human-IgG
conjugated to Quantum dot 655 from Life Technologies). A 1 to 250
dilution of the antibody was incubated with the resin in
1.times.PBST containing 1% BSA for 1 h at rt. Resins were
visualized under a fluorescence microscope (Olympus BX-51 equipped
with a 10.times.DAPI filter) and any bead that emitted red
fluorescence light was removed. All non-fluorescent beads were
pooled together and washed with 1.times.PBST and incubated with
total human-IgG (Invitrogen, 5 mg/mL stock) with 1 to 250 dilution
in 1.times.PBST for 1 h at rt. Any unbound human-IgG (huIgG) was
removed by washing resins with 1.times.PBST (3.times.) and the
resins were treated with Goat anti-human-IgG conjugated to Quantum
dot 655 (1 to 250 dilution). Beads emitting red fluorescence light
were removed and all non-fluorescent beads pooled together and
washed with 1.times.PBST.
[0197] The pre-screened resin beads were treated with 1% SDS at
90.degree. C. for 10 min to remove any bound proteins, washed with
water (10.times.) and 1.times.PBST (5.times.) and incubated in
1.times.PBST for 4 h at rt. To minimize nonspecific binding beads
were then washed with 1.times.PBST (3.times.) and equilibrated in
Starting Block PB S Blocking Buffer (from Thermoscientific)
containing 1% BSA for 2 h at rt. The resins were washed with
1.times.PBST (3.times.) and incubated with recombinant CLL (chronic
lymphocytic leukemia) monoclonal antibodies (CLL-mAbs) with a
concentration of 150 nM in 1.times.PBST containing 1% BSA. Three
CLL-mAbs (CLL-14, CLL-169 and CLL-183) were used (150 nM each) for
binding with beads for 1 h at rt. Any unbound CLL-mAbs were removed
by washing resins with 1.times.PBST (3.times.) and the beads were
incubated with Goat anti-human-IgG conjugated to Quantum dot 655 (1
to 250 dilution) for 1 h at rt. The beads were washed with
1.times.PBST and visualized under a fluorescence microscope for any
bead emitting red fluorescent light. Seventy positive beads with
intense red fluorescent color were isolated manually, washed with
1.times.PBST, treated with 1% SDS for 10 min at 90.degree. C. to
remove any bound protein, washed with .sub.ddH.sub.2O (10),
1.times.PBST and incubated in 1vPBST for 4 h. All seventy positive
beads were subjected to another round of binding with total
human-IgG and CLL-mAbs, respectively, following methods described
above. A total of 28 beads with intense fluorescent color were
collected as positive hits.
[0198] A second 4-mer COPA-ClAA library (1.sup.st two positions
with chloroacetic acid 3.sup.rd position with COPA and 4.sup.th
position with chloroacetic acid, with same 15 amine building blocks
as shown in FIG. 5) was screened following the way described above
against CLL-mAbs 068 and 183.
[0199] Sequence Identification: All positive beads were pooled
together and treated with 1% SDS for 10 min at 90.degree. C. and
washed with .sub.ddH.sub.2O (10.times.), DMF (5.times.) and DCM
(5.times.) and dried under vacuum. The beads were isolated and
placed in individual wells of a 96-well plate. The compounds on the
beads were released by treating each bead with 20 .mu.L of CNBr
solution (50 mg CNBr in 5:4:1
CH.sub.3CN:CH.sub.3COOH:H.sub.2O).
[0200] Mass of the each compound was determined by MALDI-TOF mass
spectrometry (MALDI-MS 4800Plus from Applied Biosystems) and the
sequence of the unknown COPA was determined from sequence specific
product ions obtained by electron transfer dissociation (ETD) of
the [M+2H].sup.2+ and [M+3H].sup.3+ precursor ions using
Electrospray Ionization (ESI) Linear Ion Trap mass spectrometer
(LTQ-ETD; ThermoFinnigan, San Jose, Calif.). The sequences of 24
out of 28 COPA compounds were decoded unequivocally. A
representative MALDI-TOF spectrum and ETD spectrum are shown in
FIG. 7.
[0201] Resynthesis of Positive Hits:
[0202] A total of 12 positive hits from 1.sup.st library (screened
against CLL-mAbs 14, 169 and 183) and 2 positive hits from 2.sup.nd
library (screened against CLL-mAb 068 and 183) were resynthesized
with a fluorophore tag for the validation of binding by
fluorescence polarization assay. Each of the compounds was
synthesized on 50 mg of Rink-amide resin (0.75 mmol loading
capacity, from NovaBiochem) with a linker (as shown in FIG. 8)
containing an Fmoc-protected cysteine. Fmoc group was removed by
treating resin with 20% piperidine in DMF for 20 min twice. The
resins were washed with DMF thoroughly and incubated in 0.2 M DTT
(dithiothreitol) in DMF for 10 min in order to reduce the disulfide
bonds that could be formed by oxidation of Cys side chain. The
beads were washed with DMF thoroughly to remove the DTT completely
and treated with 1.3 equiv Fluorescein-5-maleimide (F5M,
Thermoscientific) in 2 mL DMF for 4 h at rt in the dark. The beads
were washed with DMF to remove the unreacted F5M and DCM thoroughly
and dried under vacuum. To remove the fluorophore-tagged compounds
Rink resins were treated with 94% TFA, 2% TIS, 2% thioanisole and
2% water for 1 h at room temperature. The cleavage cocktail was
removed from the compounds by using an argon flow and by using a
high capacity Savant SpeedVac system (Explorer 2000) from
Thermoelectron Corp. COPA compounds were then purified on a
WATERS-1525 Binary HPLC system (equipped with Waters 1525 binary
HPLC pumps and a 2487 dual .lamda. absorbance detector) with
CH.sub.3CN:H.sub.2O gradient (5 to 80% acetonitrile over 30 min
run) using Vydac C18 Reverse-phase preparative column. The
fractions containing the F5M-conjugated compounds (confirmed by
MALDI-TOF) were pooled together and lyophilized using Virtis
Benchtop K (Model 4KBTZL) Lyophilizer.
[0203] Fluorescence Polarization (FP) Assay:
[0204] The FP assay was performed by titrating
fluorophore-conjugated COPA compounds (10 nM) with increasing
concentrations of CLL-mAbs (from 1 nM to 4 .mu.M) in 1.times.PBS
(pH 7.4) in a 10 .mu.L total volume in a 384-well plate (from
Greiner Bio-one, 784076). The compounds were incubated with
CLL-mAbs for 1 h in the dark at rt before measuring the
fluorescence polarization using Envision Multilabel Reader (2104)
from Perkin Elmer using excitation and emission wavelengths at 495
nm and 535 nm, respectively. The K.sub.D value was determined by
fitting the curve in a non-linear regression method using equation,
y=[x/(K.sub.D+x)](y.sub.max-y.sub.min)+y.sub.min, using GraphPad
Prism 5.0 (GraphPad Software, San diego Calif.). COPA oligomers
those were detected to show binding affinities (FIG. 9) with
CLL-mAbs are listed in the table below.
TABLE-US-00002 mAbs COPA hits CLL183 HH01 HH05 HH06 H0442 CLL14
HH02 HH031 HH041 HH06 CLL068 H0601 CLL169 none
Example 3: COPA Ligands
[0205] The COPA ligands for the CLL antibodies may be useful in two
ways: diagnostic and therapeutic.
[0206] For diagnostic purposes, the molecules will be immobilized
on an appropriate analytical platform, such as an ELISA plate and
pass either CLL or control sera over it. The amount of antibody
retained will be measured by subsequent incubation with a labeled
secondary antibody. This will allow for the measuring of the levels
of antibodies that bind to a particular COPA and this may provide a
serum test for CLL. Since different CLL patients can have different
antibodies, a multiplexed ELISA-like assay for this purpose can be
developed in which peptoids against a variety of different CLL
antibodies are employed. At present the inventors have ligands
against three of them and will work to obtain ligands to the other
.apprxeq.12 major types of CLL antibodies present in patients. The
idea would be that if any one of COPAs captured significant amounts
of antibodies, then the diagnosis would be CLL.
[0207] COPAs can be attached to ELISA plates by a variety of
methods. It is possible that they could be physisorbed to the
plate, though this might compromise their binding to the antibody.
Derivatives with a C-terminal cysteine could be attached covalently
and specifically to maleimide-activated plates. Finally, the COPA
could be conjugated to a carrier protein such as bovine serum
albumin (BSA) and this small molecule-protein conjugate could be
physically absorbed to the plastic plate.
[0208] There are a variety of other analytical platforms that the
COPAs could be mounted on to create multiplexed assays, such as
Luminex beads (Luminexcorp, Austin Tex.) or an Aushon plate (Aushon
BioSystems, Inc., Billerica, Mass.).
[0209] As an alternative diagnostic modality, a
fluorescently-labeled COPA molecule could be mixed with blood cells
from case or control patients and then fluorescence activated cell
sorting (FACS) analysis could be employed to determine the level of
reactive B cells in the blood.
[0210] With respect to therapeutic value, these compounds could be
used as "magic bullets" to deliver toxic cargo to the CLL cells
that display the B cell receptor (BCR) corresponding to the
antibody on their surface (see FIG. 10). There are several ways
this could be done. One is to conjugate the BCR-binding COPA to a
small molecule that would recruit native antibodies to the CLL B
cells (see FIG. 10). These antibodies would then be expected to
recruit the killing machinery of the immune system, such as
complement, natural killer cells and macrophages. This would result
in the selective eradication of the CLL cells without undue
toxicity to non-target cells. Dinitrophenol (DNP) derivatives have
been used previously as the native antibody-recruiting molecules
and DNP conjugates containing small molecules that bind selectively
to cancer cells have been shown to mediate selective cell killing
ex vivo (Murelli R. P., et al., Journal of the American Chemical
Society 131, 17090-17092 (2009)). In a similar vein, the
BCR-binding COPA molecule could be tethered to a recombinant
antibody (see, for example, Rader C. et al., Proc Natl Acad Sci USA
100, 5396-5400 (2003)) and this preformed conjugate could be used
as the therapeutic.
* * * * *