U.S. patent application number 17/698924 was filed with the patent office on 2022-08-11 for small molecule agonists and antagonists of nr2f6 activity in humans.
This patent application is currently assigned to KCL THERAPEUTICS, INC. The applicant listed for this patent is KCL THERAPEUTICS, INC. Invention is credited to David R. Koos, Harry M. Lander.
Application Number | 20220249436 17/698924 |
Document ID | / |
Family ID | 1000006292355 |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220249436 |
Kind Code |
A1 |
Lander; Harry M. ; et
al. |
August 11, 2022 |
Small Molecule Agonists and Antagonists of NR2F6 Activity in
Humans
Abstract
The present technology is directed to modulators of nuclear
receptor activity, specifically to the modulation of NR2F6 activity
and NR2F6 utilizing compounds, and the immune modulation and
modulation of cancer stem cell activity through administration of
compounds described herein to humans.
Inventors: |
Lander; Harry M.; (La Mesa,
CA) ; Koos; David R.; (La Mesa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KCL THERAPEUTICS, INC |
La Mesa |
CA |
US |
|
|
Assignee: |
KCL THERAPEUTICS, INC
LA MESA
CA
|
Family ID: |
1000006292355 |
Appl. No.: |
17/698924 |
Filed: |
March 18, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15820324 |
Nov 21, 2017 |
11324719 |
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17698924 |
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15652967 |
Jul 18, 2017 |
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15820324 |
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62363588 |
Jul 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/496 20130101; A61P 35/00 20180101; A61K 31/366 20130101;
A61K 35/17 20130101; A61K 31/506 20130101; A61K 31/4706 20130101;
A61K 31/4245 20130101; A61P 37/06 20180101; C07D 311/12 20130101;
A61K 31/5377 20130101; C07D 311/08 20130101 |
International
Class: |
A61K 31/366 20060101
A61K031/366; A61K 31/4706 20060101 A61K031/4706; A61K 31/506
20060101 A61K031/506; A61P 35/00 20060101 A61P035/00; A61K 31/4245
20060101 A61K031/4245; A61K 31/496 20060101 A61K031/496; A61K 45/06
20060101 A61K045/06; A61K 31/5377 20060101 A61K031/5377; A61P 37/06
20060101 A61P037/06; A61K 35/17 20060101 A61K035/17; C07D 311/08
20060101 C07D311/08; C07D 311/12 20060101 C07D311/12 |
Claims
1. A pharmaceutical composition comprising a pharmaceutical
compound in solid form having a structure selected from the group
consisting of Formulas (III) and (VI), or a pharmaceutically
acceptable salt thereof, together with a pharmaceutically
acceptable excipient, diluent, or carrier, wherein in a said
pharmaceutical compound having a structure of Formula (III)
##STR00105## Q is a halogen, an alkyl group, a substituted alkyl
group, a cyclic alkyl group, an aryl group, a substituted aryl
group, a heterocyclic group, an ester, an aldehyde, a ketone, a
carboxylic acid, an amide, an amine, an ether, a thiol, a nitrile,
or NO2, and R1 is a mono-substitution or a di-substitution with H,
a halogen, two halogens, a methyl group, two methyl groups, a butyl
group, a substituted alkyl group comprising at least one oxygen,
two substituted alkyl groups comprising at least one oxygen, a
halogen and an alkyl group, a cyclic alkyl group, an aryl group, a
substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, or a nitrile, and wherein in a said pharmaceutical compound
having a structure of Formula (VI) ##STR00106## any of Q1 and Q2 is
a halogen, an alkyl group, a substituted alkyl group, methoxy, a
cyclic alkyl group, an aryl group, a substituted aryl group, a
heterocyclic group, an ester, an aldehyde, a ketone, a carboxylic
acid, an amide, an amine, an ether, a thiol or a nitrile and R1 is
a mono-substitution or a di-substitution with a halogen, an alkyl
group, two alkyl groups, a substituted alkyl group, two substituted
alkyl groups, a halogen and an alkyl group, a cyclic alkyl group,
an aryl group, a substituted aryl group, a heterocyclic group, an
ester, an aldehyde, a ketone, a carboxylic acid, an amide, an
amine, an ether, or a nitrile.
2. The pharmaceutical composition of claim 1 in an oral dosage
form.
3. The pharmaceutical composition of claim 1, wherein the
pharmaceutical compound having a structure of Formula (III) is
selected from the group consisting of: a compound wherein Q is F,
Cl, or Br, and R1 is 2-F, 3-F, 4-F, 3,5-diF, 3-CI, 4-CI, 3,4-diCI,
4-Br, 3-C1-4-F, 4-Me, 3,4-diMe, 3,5-diMe, 3-MeO, 4-MeO, 3,4-diMeO,
4-t-Bu, 3-C1-4-Me, 3-CH3-4-F, or 3-CN, a compound wherein Q is
MeOOCH2 and R1 is selected from H, 2-F, 3-F, 4-F, 3,5-diF, 3-CI,
4-CI, 3,4-diCI, 4-Br, 3-C1-4-F, 4-Me, 3,4-diMe, 3,5-diMe, 3-MeO,
4-MeO, 3,4-diMeO, 4-t-Bu, 3-C1-4-Me, 3-CH3-4-F, and 3-CN, a
compound wherein Q is MeO and R1 is selected from H, 2-F, 3-F, 4-F,
3,5-diF, 3-CI, 4-CI, 3,4-diCI, 4-Br, 3-CI-4-F, 4-Me, 3,4-diMe,
3,5-diMe, 3-MeO, 4-MeO, 3,4-diMeO, 4-t-Bu, 3-C1-4-Me, 3-CH3-4-F,
and 3-CN, and a compound wherein Q is NO2 and R1 is selected from
H, 2-F, 3-F, 4-F, 3,5-diF, 3-CI, 4-CI, 3,4-diCI, 4-Br, 3-C1-4-F,
4-Me, 3,4-diMe, 3,5-diMe, 3-MeO, 4-MeO, 3,4-diMeO, 4-t-Bu,
3-C1-4-Me, 3-CH3-4-F, and 3-CN.
4. The pharmaceutical composition of claim 3, wherein the
pharmaceutical compound having a structure of Formula (III) is
selected from the group consisting of: Compound C11 wherein Q is
MeOOCH2, R1 is H, Compound C14 wherein Q is MeOOCH2, R1 is 4-F,
Compound C154 wherein Q is Br, R1 is 3,4-diMeO, Compound C155
wherein Q is Cl, R1 is 3,4-diMeO, Compound C157 wherein Q is NO2,
R1 is 3,4-diMeO, Compound C195 wherein Q is Cl, R1 is 3-CN, and
Compound C210 wherein Q is NO2, R1 is H.
5. The pharmaceutical composition of claim 1, wherein the
pharmaceutical compound having a structure of Formula (IV) is
selected from the group consisting of a compound wherein any of Q1
and Q2 is F, Cl, Br, or methoxy (OMe), and R1 is OMe, 3,4-di-Me,
4-Cl, 3,4-di-Cl, 4-OMe, 4-Br, 4-t-Bu, 3-Me-4-F, or 4-F.
6. The pharmaceutical composition of claim 5, wherein the
pharmaceutical compound having a structure of Formula (VI) is
selected from the group consisting of: Compound C289 wherein Q1 is
F, Q2 is F, R1 is 4-OMe, and Compound C290 wherein Q1 is F, Q2 is
F, R1 is 4-Br.
Description
BACKGROUND
[0001] The present technology relates to agonists and antagonists
of nuclear receptor activity, specifically to the modulation of
NR2F6 activity and NR2F6 utilizing compounds, and the immune
modulation and modulation of cancer stem cell activity through
administration of compounds described herein.
[0002] Many drugs administered to treat diseases or conditions are
targeted against differences between a diseased cell and a normal
cell. T cells of the immune system are known to recognize and
interact with specific molecules through receptors (e.g., a T cell
receptor in complex with a CD3 dimer) which, upon recognition or
interaction with these molecules, result in the activation of the T
cell to perform various immune activities. Innate immune cells are
cells of the immune system that are known to be activated by one or
more agents (e.g., allergens, chemicals produced upon injury (e.g.,
opioids and alcohols), polymyxins, crosslinked IgE, crosslinked
complement proteins, cytokines produced by T cells or other immune
cells (e.g., interferon-.gamma.), DAMPs, or PAMPs) that activate
downstream signaling pathway(s) in the innate immune cell and
result in the activation of one or more immune activities of the
innate immune cell.
[0003] Both T cells and innate immune cells play a role in a
mammal's immune defense. For example, the immune activities of an
innate immune cell can protect a mammal against infectious
diseases. The immune activities of a T cell can protect a mammal
against, for example, infectious diseases and cancer.
[0004] Adoptive cell therapy is a method of treatment that includes
harvesting one or more different types of immune cells from a
mammal, culturing and/or manipulating the harvested immune cells ex
vivo, and administering the cultured and/or manipulated immune
cells back to the mammal. The manipulating of a harvested immune
cell ex vivo can include introducing a recombinant nucleic acid
into the immune cell.
[0005] Molecularly targeted therapeutics represent a new approach
to discovering anti-cancer drugs. Using this approach, small
molecules are designed to inhibit directly the very oncogenetic
proteins that are mutated or overexpressed in specific tumor cell
types. By targeting specific molecular defects or conditions found
within tumor cells, this approach can yield therapies tailored to
each tumor's genetic makeup. A complementary strategy involves
searching for genotype-selective anti-tumor agents that become
lethal to tumor cells only in the presence of specific oncoproteins
or only in the absence of specific tumor oppressors. Such
genotype-selective compounds might target oncoproteins directly, or
target other critical proteins involved in oncoprotein-linked
signaling networks.
[0006] The immune system is comprised of activatory and inhibitory
mechanisms that can allow for control of immune responses and
subsequent inhibition of responses after clearance of the immune
target. The central event stimulating immune responses is the
antigen-specific activation of naive CD4.sup.+ T cells subsequent
to binding antigen presenting cell MHC containing antigenic
peptide. The CD4.sup.+ T cell, also known as the "helper T cell,"
helps to coordinate the activation of the adaptive immune response,
playing a role in the stimulation of cytotoxic CD8+ T cells, whose
role includes destroying host cells affected by cancer, viruses,
and intracellular bacteria, as well as stimulating B cell
maturation to eventual plasma cell differentiation. Antibodies can
be critical molecules in clearance of extracellular pathogens such
as various bacteria and parasites.
[0007] Under many circumstances, naive CD4.sup.+ T cells require
two distinct signals to proliferate and differentiate into the
armed effector cells that mediate adaptive immunity. Signal 1 of
this two-signal model is antigen-specific and is generated by
interaction of the TCR with antigenic peptide presented in context
with MHC II antigens. This results in transduction of TCR
intracellular signals leading to production of IL-2 and T cell
activation. Signal 2 is referred to as a "costimulatory" signal
because, while essential, it does not necessarily induce any
functional response in T cells.
[0008] The best characterized costimulatory signal 2 is generated
through the T cell surface molecule CD28. CD28 delivers a
costimulatory signal upon interaction with CD80 or CD86 present on
B cells, macrophages, or dendritic cells. Activation of the TCR in
the presence of costimulatory signals leads to T cell clonal
expansion and initiation of effector functions such as IL-2
production.
[0009] For cancer, immune inhibitory mechanisms, termed "immune
checkpoints," are prematurely activated in order for the tumor to
escape immune attack. Two immune checkpoints exist: a) CTLA-4,
which sends an inhibitory signal to T cells upon binding CD80 or
CD86 on antigen presenting cells; and b) PD-1, which binds to PD-1
ligand on tumor cells, stromal cells, or antigen presenting
cells.
[0010] CTLA-4 is related to CD28, however instead of activating T
cells in a co-stimulatory manner, it leads to inhibition or
co-inhibition of T cells.
[0011] Nuclear receptor subfamily 2, group F, member 6 (NR2F6),
also known as nuclear orphan receptor Ear2, is an orphan member of
the nuclear receptor (NR) superfamily of ligand-activated
receptors, which exhibit a common modular structure and are
involved in various homeostatic functions, but also play a role in
oncogenesis and cancer propagation. Specifically, studies have
shown that members of the NR family regulate development,
reproduction, and metabolism of lipids, drugs and energy. The
importance of this family of proteins in metabolic disease is
exemplified by NR ligands used in the clinic or under exploratory
development for the treatment of diabetes mellitus, dyslipidemia,
hypercholesterolemia, or other metabolic abnormalities.
[0012] Genetic studies in humans and rodents support the notion
that NRs control a wide variety of metabolic processes by
regulating the expression of genes encoding key enzymes,
transporters and other proteins involved in metabolic homeostasis.
Genomic sequence availability has led to the identification of 48
NRs encoded by the human genome and 49 NRs encoded by the mouse
genome.
[0013] The present disclosure is directed to, in certain
embodiments, methods of using small molecule compounds as immune
modulators; as well as to compounds, solid forms and compositions
thereof that are immune modulators and that exhibit desirable
characteristics thereof, as well as to methods of making the
compounds, solid forms and composition thereof.
SUMMARY OF THE DISCLOSED TECHNOLOGY
[0014] In certain embodiments, the present technology is directed
compounds discussed and described herein, which compounds have been
found to modulate the immune system. These compounds can include
any of the following:
##STR00001## ##STR00002## ##STR00003## ##STR00004##
[0015] In various embodiments, in any of the above, moieties R, RA,
RB, R1-R8, X, Q, Q1, Q2, A can be any of the following: C, H, N, O,
S, a halogen, an alkyl group, a substituted alkyl group, a cyclic
alkyl group, an aryl group, a substituted aryl group, a
heterocyclic group, an ester, an aldehyde, a ketone, a carboxylic
acid, an amide, an amine, an ether, a thiol or a nitrile. In
various embodiments, in any of the above, n can be an integer 1, 2,
3, 4, 5 or 6.
[0016] In various embodiments, any of R, RA, RB, R1-R8, X, Q, Q1,
Q2, or A can be any of the following: Me, OMe, Br, N, H, Cl, F or
NO.sub.2. In certain embodiments, any of R, RA, RB, R1-R8, X, Q,
Q1, Q2, or A can be any of the following: 4-Me, 4-OMe, 4-Br,
4-t-Bu, 3,4-di-Me, 4-Cl, 3,4-di-Cl, 3-Cl-4-F, 2-F, 3-Cl,
3-CH.sub.3-4-F, a thiazole, an isothiazole or a dithiolane.
[0017] In various embodiments, any of R1 and R2 can have the values
shown in any of the Figures, for example, FIGS. 1A-1F, FIGS. 3A-3F,
FIGS. 4A-4L, FIGS. 5A and 5B, FIGS. 6A-6F, FIGS. 7A-7O, FIGS.
8A-8M, FIGS. 9A-9J, FIGS. 10A-10J or FIGS. 11A-11G.
[0018] In certain embodiments, the present technology is directed
to a novel compound, any solid form thereof, and any formulation or
composition thereof, that is useful as agonists or antagonists of
nuclear receptor activity, specifically to the modulation of NR2F6
activity and NR2F6 utilizing compounds.
[0019] In certain embodiments, the present technology is directed
to methods of modulating the immune system or modulating cancer
cell activity using compounds that alter activity of NR2F6.
[0020] In certain embodiments, the present technology is directed
to methods of "reprogramming" the immune cells in a patient to
attack tumors or other invasive cells. Such "reprogramming" can
include: (a) extraction of an amount of a patient's cellular
material (including, but not limited to: blood, which itself
includes blood serum, plasma red blood cells, white blood cells and
platelets), (b) isolating specific immune cells from the cellular
material; (c) inhibiting or activating the NR2F6 target in the
extracted immune cells; and (d) re-administering the immune cells
(for example, by injection) to the patient's body.
[0021] In certain embodiments, the present technology is directed
to methods treating or reducing the effect of an autoimmune
response, reaction, disease or disorder, the method comprising any
of the steps discussed herein, or activating the NR2F6 target in
isolated immune cells by binding them with a compound according to
the present technology.
[0022] In certain embodiments, the present technology is directed
to methods of shrinking a tumor, increasing or decreasing activity
of a cell, initiating or inducing an immune response, destroying a
cancer cell, reducing the effect of a disease, alleviating a
symptom of a disease, as well as methods of inducing a cell in a
patient's body to do any of these, the method comprising
administering a compound herein to a tumor, contacting a compound
herein with a cell, or any other steps discussed herein.
[0023] In certain embodiments, the present technology is directed
to a pharmaceutical composition comprising a compound described
herein, with a pharmaceutically acceptable carrier or
excipient.
[0024] The methods herein can, in various embodiments, involve
humans or non-human mammals as subjects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A-1F show certain compounds that have been found to
be effective for the purposes of the present technology.
[0026] FIG. 2A shows a certain compound that has been found to be
effective for the purposes of the present technology. FIG. 2B shows
different domains, or portions of a base compound, that were
substituted with different moieties to ascertain whether these made
a difference in the activity of such compound.
[0027] FIG. 3A shows a certain compound that has been found to be
effective for the purposes of the present technology. FIGS. 3B-3F
show various additional compounds formed from substitution of
different moieties.
[0028] FIGS. 4A-4L show additional compounds that were found to be
useful in accordance with the embodiments herein.
[0029] FIGS. 5A and 5B show additional embodiments of compounds,
along with (in the case of FIG. 5A) different values of moieties R1
and R2.
[0030] FIGS. 6A-6F show additional compounds that were found to be
useful in accordance with the embodiments herein.
[0031] FIGS. 7A-7O show additional compounds that were found to be
useful in accordance with the embodiments herein.
[0032] FIGS. 8A-8M show additional compounds that were found to be
useful in accordance with the embodiments herein.
[0033] FIGS. 9A-9J show additional compounds that were found to be
useful in accordance with the embodiments herein.
[0034] FIGS. 10A-10J show additional compounds that were found to
be useful in accordance with the embodiments herein.
[0035] FIGS. 11A-11G show additional compounds that were found to
be useful in accordance with the embodiments herein.
[0036] FIG. 12 shows a certain compound that has been found to be
effective for the purposes of the present technology; and different
domains, or portions of a base compound, that were substituted with
different moieties to ascertain whether these made a difference in
the activity of such compound.
[0037] FIG. 13A shows a certain compound that has been found to be
effective for the purposes of the present technology. FIGS. 13B-13H
show various additional compounds formed from substitution of
different moieties.
[0038] FIG. 14A shows a certain compound that has been found to be
effective for the purposes of the present technology. FIGS. 14B-14D
show various additional compounds formed from substitution of
different moieties.
[0039] FIGS. 15A-15G show additional compounds that were found to
be useful in accordance with the embodiments herein
[0040] FIGS. 16A and 16B show charts of cytokines release by hPBMC
and cytotox for Compound C11, which was identified as a useful
compound in accordance with the embodiments herein. Results were
repeated in follow set screen from fresh powder. 5 direct analogs
were available (top structure in FIG. 16A).
[0041] FIGS. 17A and 17B show analogues and other related compounds
to Compound C11.
[0042] FIGS. 18A and 18B show charts of cytokines release by hPBMC
and cytotox for Compound 18. For cytokines release and cytotox on
hPBMCs compound was tested at 1.25, 2.5, 5, 10, 25 and 50 uM in
duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293 NR2F6
(full length) compound was tested from 50 uM with dilution step
3.16 in duplicates. Human PBMC were activated by 10 ng/mL PMA+500
ng/mL ionomycin. Data were normalized to controls with (100%) w/o
compounds.
[0043] FIGS. 19A and 19B show the results of Dog's PBMC ELISA and
cytotoxicity experiments. All compounds were tested at 5, 10, 25
and 50 uM in duplicates on activated by 10 ng/mL PMA+500 ng/mL
ionomycin dogs PBMC (1.times.106 cells/mL). Cell culture supernates
were removed and frozen for further ELISA analysis and remained
cells were analyzed. Compounds without cytotox were chosen for
cytokine release inhibition analysis. Compound Z92 was also
analyzed at 5 uM and 10 uM.
[0044] FIGS. 20A and 20B show results of cytokines release by hPBMC
and cytotox for Compound Z95. For cytokines release and cytotox on
hPBMCs compound was tested at 1.25, 2.5, 5, 10, 25 and 50 uM in
duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293 NR2F6
(full length) compound was tested from 50 uM with dilution step
3.16 in duplicates. Human PBMC were activated by 10 ng/mL PMA+500
ng/mL ionomycin. Data were normalized to controls with (100%)
without compounds.
[0045] FIGS. 21A-21D show human and dog results of a cytokine
release experiment--parent compound for dogs and human PBMC, for
Compound D28. All compounds were tested at 5, 10, 25 and 50 uM in
duplicates. Dog PBMC (1.times.106 cells/mL) were activated by 10
ng/mL PMA+500 ng/mL ionomycin. Data were normalized to controls
with (100%) without (0%) PMA+ionomycin activation.
[0046] FIGS. 22A and 22B show results of cytokines release by hPBMC
and cytotox for Compound Z17. For cytokines release and cytotox on
hPBMCs compound was tested at 1.25, 2.5, 5, 10, 25 and 50 uM in
duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293 NR2F6
(full length) compound was tested from 50 uM with dilution step
3.16 in duplicates. Human PBMC were activated by 10 ng/mL PMA+500
ng/mL ionomycin. Data were normalized to controls with (100%)
without compounds.
[0047] FIGS. 23A and 23B show results of cytokines release by hPBMC
and cytotox for Compound Z33. For cytokines release and cytotox on
hPBMCs compound was tested at 1.25, 2.5, 5, 10, 25 and 50 uM in
duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293 NR2F6
(full length) compound was tested from 50 uM with dilution step
3.16 in duplicates. Human PBMC were activated by 10 ng/mL PMA+500
ng/mL ionomycin. Data were normalized to controls with (100%)
without compounds.
[0048] FIG. 24 shows results of a cytokines release by hPBMC for
Compound E56.
[0049] FIGS. 25A and 25B shows results of cytokines release by
hPBMC and cytotox for Compound Z96. For cytokines release and
cytotox on hPBMCs compound was tested at 1.25, 2.5, 5, 10, 25 and
50 uM in duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293
NR2F6 (full length) compound was tested from 50 uM with dilution
step 3.16 in duplicates. Human PBMC were activated by 10 ng/mL
PMA+500 ng/mL ionomycin. Data were normalized to controls without
(100%) compounds.
[0050] FIGS. 26A and 26B show results of cytokine release by hPBMC
and cytotox for Compound Z97.
[0051] FIGS. 27A-27D show NR2F6 and LBD transient transfection,
respectively, for Compound D28. FIGS. 27E and 27F show toxicity of
Compound D28.
[0052] FIGS. 28A-28D show the results of a cytokine release
experiment for dog and human PBMC. All compounds were tested at 5,
10, 25 and 50 uM in duplicates. Dog PBMC (1.times.106 cells/mL)
were activated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were
normalized to controls with (100%)/without (0%) PMA+ionomycin
activation.
[0053] FIGS. 29, 30 31A, 31B and 32 show exemplary methods of
formulating the compounds that have been discussed herein.
[0054] FIGS. 33A and 33B and 34A-34D show NR2F6 and LBD transient
transfection, respectively, for Compound E21.
[0055] FIGS. 35A to 35D show the results of a cytokine release
experiment for dog and human PBMC, for Compound E21.
[0056] FIG. 36 shows additional compounds related to compound E21
that were tested herein.
[0057] FIG. 37 shows additional compounds that were synthesized and
tested according to the present embodiments.
[0058] FIGS. 38A and 38B, 39A-39D and 40A-40D show results of
testing on Compound F1.
[0059] FIG. 41 shows the general SAR strategy for testing Compound
F1 and compounds related to it in structure, by dividing the active
molecule into four domains (Domains A through D), and evaluating
each domain independently to establish SAR trends.
[0060] FIG. 42 shows an exemplary synthesis of a boronate compound,
and the results of other exemplary syntheses of compounds
comprising boronate, and the relative proportions of resultant
compounds.
[0061] FIG. 43 shows methods of synthesis of certain compounds
found to be useful for the embodiments herein.
[0062] FIGS. 44A and 44B and 45A-D show NR2F6 and LBD transient
transfection, respectively, for Compound P1.
[0063] FIGS. 46A and 46B show the results of a cytokine release
experiment for dog and human PBMC, for Compound P1.
[0064] FIGS. 47A and 47B show NR2F6 agonist activity of various
compounds discussed herein.
[0065] FIGS. 48-51 show synthesis of various additional compounds
discussed herein.
[0066] FIGS. 52A and 52B show synthesis of Compounds Z119, Z120,
Z121 and Z123.
[0067] FIG. 53-55 shows synthesis of additional compounds
herein.
[0068] FIGS. 56A and 56B show Nr2F6 agonist activity for various
compounds discussed herein.
[0069] FIGS. 57 and 58 show synthesis of various compounds
discussed herein.
DETAILED DESCRIPTION
[0070] As used herein, "disease" or "disorder" are used
interchangeably and mean a disorder of structure or function in any
living thing (including but not limited to a human, animal, or
plant), especially one that produces specific signs or symptoms or
that affects a specific location and is not simply a direct result
of physical injury.
[0071] As used herein, "mammal" means a warm-blooded vertebrate
animal of a class that is distinguished by the possession of hair
or fur, the secretion of milk by females for the nourishment of the
young, and (typically) the birth of live young. As used herein,
"human" means a person. As used herein, "animal" or "non-human
mammal" means any non-human animal, including but not limited to: a
canine (e.g., a dog), a feline (e.g., a cat), a rodent, an ungulate
(e.g., a cow or ox), an equine (e.g., a horse), or a primate.
[0072] As used herein, "modulator" means a molecule that alters the
basal activity of NR2F6 either positively (activates it or
increases it) or negatively (represses, suppresses or decreases
it). "Modulating" means the act of the modulator, either positive
or negative. A compound of the technology herein can be, in various
embodiments, a modulator of NR2F6, for example, at an effective
concentration or in an effective amount, but not be a modulator of
any other receptor, or not a modulator at any other amount of NR2F6
or any other receptor. This can provide selectivity of effect of a
compound of the technology herein when administered to a patient
for treatment of any disease.
[0073] In certain embodiments, one diastereomer or one enantiomer
of a compound of the present technology can display superior
biological activity compared with the other. When required,
separation of the diastereomeric mixture or the racemic material
can be achieved by HPLC, optionally using a chiral column or by
using a resolving agent such as camphonic chloride for the
resolution of enantiomers. A chiral compound described herein can
also be directly synthesized using a chiral catalyst or a chiral
ligand.
[0074] In certain embodiments, one deuterated or tritiated compound
of the present technology can display superior biological activity
compared with one or more others. When required, separation of the
material can be achieved by one of ordinary skill in the art.
[0075] In certain embodiments, the present technology is directed
to methods of modulating the immune system using compounds that
alter activity of NR2F6.
[0076] In certain embodiments, compounds herein can be utilized for
stimulation of NR2F6 activity, alone, or in combination with, for
example, PKC activation. In certain embodiments, the compounds
herein can be utilized for inhibition of NR2F6 activity, alone or
in combination with, for example, anti-PD1, anti-PDL1 or anti-CTLA4
antibodies.
[0077] In other embodiments, the methods are directed to the
stimulation of NR2F6 for, e.g., induction of immune inhibition, or
stimulation of cellular proliferation without significant induction
of differentiation. Inhibition of NR2F6 can be desirable in
situations where a clinician seeks to augment immune response, or
induce cellular differentiation.
[0078] In other embodiments, inhibition of NR2F6 expression can be
desirable in situations where inhibition of cancer or cancer stem
cells is desired.
[0079] In certain embodiments, activation of NR2F6 expression can
be desirable in situations where inhibition of the immune system is
desired, for example, in connection with autoimmune disorders.
[0080] The interplay between the activation or deactivation of NRs
by different structural classes of endogenous ligands, such as the
steroid and thyroid hormones, lipids, vitamins and other
biochemicals, is an important part of their function. The 48 NR
family members are classified into subgroups based on the
identification of endogenous ligands for each receptor. The
endocrine receptors include the steroid hormone receptors that bind
steroid hormones and the heterodimeric receptors that partner with
the retinoid X receptor and bind thyroid hormones, retinoids, and
vitamin D.
[0081] The identification of specific endogenous ligands for the
endocrine receptors has facilitated the design and development of
selective receptor modulators (SRMs) that exhibit tissue-specific
agonist or antagonist activities and are used for treatment of
hormone-/hormone receptor-dependent diseases. Tamoxifen is one of
many selective estrogen receptor (ER) modulators used in endocrine
therapies for treating ER-positive breast cancer patients.
[0082] Adopted orphan receptors are a subtype of NRs that are
subdivided into groups based on their ligands. The lipid sensor
receptor subtypes and their ligands include retinoid X receptor
(9-cis-retinoic acid), peroxisome proliferator-activated receptors
(PPARs) (fatty acids), liver X receptor (oxysterols), farnesoid X
receptor (bile acids), and pregnane X receptor, which binds
cholesterol derivatives.
[0083] Retinoid X receptors have been found in various cancer stem
cells and methodologies for their utilization, as well as
ligands/synthetic ligands targeting them, have been developed. Any
of these can be utilized by one of skill in the art to practice the
methods of the present technology, which provides compounds useful
for modulating the NR2F6 nuclear receptor. Methods of modulating
PPARs are also amenable to utilization in the context of the
current technology, whose methodologies can, in various
embodiments, be adapted for use with the compounds discussed herein
for treatment of cancer or immune modulation.
[0084] With regard to PPARs, three subtypes of the PPAR family are
PPAR.alpha., PPAR.gamma., and PPAR.delta.. PPAR.gamma. is
abundantly expressed in many cell types, where it regulates lipid
metabolism, glucose homeostasis, tumor progression, and
inflammation. Polyunsaturated fatty acids, eicosanoids,
prostaglandins, and linoleic acid have been identified as
endogenous ligands for PPAR.gamma.. The thiazolidinedione class of
compounds function as high-affinity synthetic agonists for
PPAR.gamma. subsequent to exposure to specific ligands. PPAR.gamma.
forms a heterodimer complex with retinoid X receptor, which then
mediates the target gene expression. In terms of immune modulation,
in certain embodiments, NR2F6 specific compounds can be substituted
for those described for PPAR.
[0085] The enigmatic orphan receptor subtype can include the
constitutive androstane receptor (androstane and many drugs or
xenobiotics), hepatocyte nuclear factor-4, and steroidogenic
factor-1/liver receptor homolog 1 (LRH-1) (phospholipids), retinoid
acid-related orphan receptor (cholesterol and retinoic acids), and
estrogen-related receptor (estrogens). These can be useful in
methods of performing immunotherapy that include NR2F6
modulators.
[0086] The orphan receptors are the third class of NRs. The crystal
structure of the ligand-binding domain of the orphan receptor Nurr1
(NR4A2) shows that several hydrophobic residues protrude into the
ligand-binding pocket, and a typical coactivator-binding site is
lacking, suggesting that some orphan receptors may not bind
ligands.
[0087] Like other NR classes, the orphan receptors play important
roles in cellular homeostasis and diseases including cancer, and
several recent reports document the expression and potential
functions of orphan receptors in different tumors and cancer cell
lines. Breast tumors are routinely classified as ER.sup.+ or
ER.sup.-, and expression of ER has prognostic significance that
influences selection of therapeutic regimens. However, analysis of
ER.sup.+ and ER.sup.- tumors for expression (mRNA) of all 48 NRs
also demonstrated the important prognostic significance of several
orphan receptors. The NR4A (Nur77/TR3, Nurr1, and Nor1) and NR2F6
[v-erbA-related protein (EAR2)] receptors are uniquely
overexpressed in (ER.sup.+ and ER.sup.- combined) tumors. Moreover,
Nur77, EAR2, and chicken ovalbumin upstream promoter transcription
factor II (COUP-TFII) are among a limited group of NRs that are
prognostic for breast cancer classification and histologic grade,
and COUP-TFII expression was a positive prognostic factor for
tamoxifen-treated ER.sup.+ breast cancer patients.
[0088] Examination of lung tumor and nontumor tissue indicated
highly variable NR expression; however, gene combinations and
individual receptors, such as the orphan receptor small heterodimer
partner (SHP, NR0B2), predicted enhanced survival for early-stage
lung cancer patients. Moreover, expression of Nur77 in normal lung
epithelium from patients has been shown to be an indicator for good
prognosis.
[0089] NR profiling of the NCI60 cancer cell panel demonstrated
that relative expression levels of some orphan receptors also
correlated with drug sensitivity. For example, cancer cell
sensitivity to microtubule-disrupting drugs has been found to be
enhanced in cells expressing low levels of NR2F6 and COUP-TFII,
whereas high levels of the orphan receptor tailless (TLX, NR2E1)
correlated with sensitivity to 9-fluoroprednisolone.
[0090] As used herein, the term "NR2F6" means "nuclear receptor
subfamily 2, group F, member 6" or "Ear2." Nuclear receptors are
transcription factors that regulate the expression of specific
target genes, thereby orchestrating a wide array of cellular
processes including cellular activation, development and disease
progression. The nuclear receptor super-family includes receptors
that bind to hormones and orphan receptors with yet undefined
endogenous ligands. As discussed in the present disclosure, NR2F6
can be a target in cancer immunotherapy or autoimmune
suppression.
[0091] The COUP-TF orphan receptors are preferentially expressed in
the central nervous system and organs that depend on the
interaction between mesenchyme and epithelial layers. The three
mammalian COUP-TF family members are NR2F1/Ear3, NR2F2/Arp1 and
NR2F6. The established target genes of said COUP-TF family members
are apolipoproteins and retinoic acid-, peroxisome-, oxytocin-,
estrogen- and vitamin D receptors. By yeast 1-hybrid screen and in
vitro assays with recombinant NR2F6, it was found that the TGACCT
direct-repeat motif is the DNA binding sequence of NR2F6, and that
overexpression of NR2F6 induces repression of the renin gene
transcription in a DNA-binding-specific manner. Wild type
human/animal NR2F6 is known to possess the following nucleotide
sequence: 1 gtgcagcccg tgccccccgc gcgccggggc cgaatgcgcg ccgcgtaggg
tcccccgggc 61 cgagaggggt gcccggaggg aagagcgcgg tgggggcgcc
ccggccccgc tgccctgggg 121 ctatggccat ggtgaccggc ggctggggcg
gccccggcgg cgacacgaac ggcgtggaca 181 aggcgggcgg ctacccgcgc
gcggccgagg acgactcggc ctcgcccccc ggtgccgcca 241 gcgacgccga
gccgggcgac gaggagcggc cggggctgca ggtggactgc gtggtgtgcg 301
gggacaagtc gagcggcaag cattacggtg tcttcacctg cgagggctgc aagagctttt
361 tcaagcgaag catccgccgc aacctcagct acacctgccg gtccaaccgt
gactgccaga 421 tcgaccagca ccaccggaac cagtgccagt actgccgtct
caagaagtgc ttccgggtgg 481 gcatgaggaa ggaggcggtg cagcgcggcc
gcatcccgca ctcgctgcct ggtgccgtgg 541 ccgcctcctc gggcagcccc
ccgggctcgg cgctggcggc agtggcgagc ggcggagacc 601 tcttcccggg
gcagccggtg tccgaactga tcgcgcagct gctgcgcgct gagccctacc 661
ctgcggcggc cggacgcttc ggcgcagggg gcggcgcggc gggcgcggtg ctgggcatcg
721 acaacgtgtg cgagctggcg gcgcggctgc tcttcagcac cgtggagtgg
gcgcgccacg 781 cgcccttctt ccccgagctg ccggtggccg accaggtggc
gctgctgcgc ctgagctgga 841 gcgagctctt cgtgctgaac gcggcgcagg
cggcgctgcc cctgcacacg gcgccgctac 901 tggccgccgc cggcctccac
gccgcgccta tggccgccga gcgcgccgtg gctttcatgg 961 accaggtgcg
cgccttccag gaggaggtgg acaagctggg ccgcctgcag gtcgactcgg 1021
ccgagtatgg ctgcctcaag gccatcgcgc tttcacgcc cgacgcctgt ggcctctcag
1081 acccggccca cgttgagagc ctgcaggaga aggcgcaggt ggccctcacc
gagtatgtgc 1141 gggcgcagta cccgtcccag ccccagcgct tcgggcgcct
gctgctgcgg ctccccgccc 1201 tgcgcgcggt ccctgcctcc ctcatctccc
agctgttctt catgcgcctg gtggggaaga 1261 cgcccattga gacactgatc
agagacatgc tgctgtcggg gagtaccttc aactggccct 1321 acggctcggg
ccagtgacca tgacggggcc acgtgtgctg tggccaggcc tgcagacaga 1381
cctcaaggga cagggaatgc tgaggcctcg aggggcctcc cggggcccag gactctggct
1441 tctctcctca gacttctatt ttttaaagac tgtgaaatgt ttgtcttttc
tgttttttaa 1501 atgatcatga aaccaaaaag agactgatca tccaggcctc
agcctcatcc tccccaggac 1561 ccctgtccag gatggagggt ccaatcctag
gacagccttg ttcctcagca cccctagcat 1621 gaacttgtgg gatggtgggg
ttggcttccc tggcatgatg gacaaaggcc tggcgtcggc 1681 cagaggggct
gctccagtgg gcaggggtag ctagcgtgtg ccaggcagat cctctggaca 1741
cgtaacctat gtcagacact acatgatgac tcaaggccaa taataaagac atttcctacc
1801 tgca, which corresponds to the following amino acid
sequence:
TABLE-US-00001 (SEQ ID NO: 1)
MAMVTGGWGGPGGDTNGVDKAGGYPRAAEDDSASPPGAASDAEPGDEERP
GLQVDCVVCGDKSSGKHYGVFTCEGCKSFFKRSIRRNLSYTCRSNRDCQI
DQHHRNQCQYCRLKKCFRVGMRKEAVQRGRIPHSLPGAVAASSGSPPGSA
LAAVASGGDLFPGQPVSELIAQLLRAEPYPAAAGRFGAGGGAAGAVLGID
NVCELAARLLFSTVEWARHAPFFPELPVADQVALLRLSWSELFVLNAAQA
ALPLHTAPLLAAAGLHAAPMAAERAVAFMDQVRAFQEQVDKLGRLQVDSA
EYGCLKAIALFTPDACGLSDPAHVESLQEKAQVALTEYVRAQYPSQPQRF
GRLLLRLPALRAVPASLISQLFFNIRLVGKTPIETLIRDMLLSGSTFNWP YGSGQ.
[0092] Accordingly, in certain embodiments the present technology
is directed to compounds that bind to a portion or all of an NR2F6
molecule; or any molecule that is, in various embodiments, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% identical to
the amino acid sequence of NR2F6.
[0093] As used herein, the terms "agonist" or "activator" are used
interchangeably and mean a compound or substance capable of fully
or partially stimulating the physiologic activity of one or more
specific receptors. In the context of the present disclosure, an
agonist can therefore stimulate the physiological activity of a
receptor such as NR2F6 upon binding of said compound substance to
said receptor. As further discussed herein, an "agonist" or
"activator" can be used to "activate," "stimulate" or "increase
activity" of a cell.
[0094] In certain embodiments, binding of an "agonist" or
"activator" to a given receptor, e.g., NR2F6, can mimic the action
of an endogenous ligand binding to said receptor. As used herein,
accordingly, the term "agonist" also encompasses partial agonists
or co-agonists or co-activators. In addition, however, an "agonist"
or "activator" of NR2F6 as used herein can also be capable of
stimulating the function of a given receptor, such as NR2F6, by
inducing or enhancing the expression of the nucleic acid molecule
encoding for said receptor. Thus, an agonist or activator of NR2F6
can, in certain embodiments, lead to an increased expression level
of NR2F6 (e.g., increased level of NR2F6 mRNA, NR2F6 protein) which
is reflected in an increased activity of NR2F6. This increased
activity can be measured or detected by the methods herein.
[0095] Accordingly, an activator of NR2F6 in accordance with the
present technology can, in certain embodiments, also encompass
transcriptional activators of NR2F6 expression that are capable of
enhancing NR2F6 function. As mentioned above, "agonist" includes a
partial agonist. "Partial agonists" mean candidate molecules that
behave like agonists, but that, even at high concentrations, cannot
activate NR2F6 to the same extent as a full agonist. As described
below, the increased expression or activity of NR2F6 by an agonist
or activator of NR2F6 can lead to decreased activity (or
expression) of components of the NR2F6-dependent signaling pathway;
in particular the activity of NF-AT and AP-1 can be decreased.
NF-AT/AP-1 regulate transcription/expression of further
"downstream" components of the NR2F6-dependent signaling pathway,
such as IL-2, IL-17 and/or IFN-gamma. A decrease in NF-AT/AP-1
activity can result in a decreased transcription of these
"downstream" components (e.g., IL-2, IL-17 and/or IFN-gamma) which
in turn leads to a suppression of an immune response.
[0096] In certain embodiments, an agonist or activator of NR2F6 can
lead to suppression of an immune response. Hence, the use of potent
agonists/activators of NR2F6 can lead to a higher expression or
activity of NR2F6.
[0097] In certain embodiments NR2F6 can be considered its own
"agonist" or "activator." For example, in certain embodiments,
overexpression of NR2F6 can lead to enhanced NR2F6 activity, thus
agonizing NR2F6 function. Accordingly, NR2F6 as defined herein can,
in certain embodiments, be used for the treatment of a disease
related to an augmented immune response.
[0098] For example, NR2F6 can be used in accordance with certain
embodiments of the present technology, wherein NR2F6 is any of the
following: (a) a polypeptide comprising an amino acid encoded by a
nucleic acid molecule having the nucleic acid sequence of NR2F6;
(b) a polypeptide having an amino acid sequence of NR2F6; (c) a
polypeptide encoded by a nucleic acid molecule encoding a peptide
having an NR2F6 amino acid sequence; (d) a polypeptide comprising
an amino acid encoded by a nucleic acid molecule hybridizing to the
complementary strand of nucleic acid molecules as defined in (a) or
(c) and encoding a NR2F6 or a functional fragment thereof; (e) a
polypeptide having at least 60% homology to the polypeptide of any
one of (a) to (d), whereby said polypeptide is a NR2F6 or a
functional fragment thereof; or (f) a polypeptide comprising an
amino acid encoded by a nucleic acid molecule being degenerate as a
result of the genetic code to the nucleotide sequence of a nucleic
acid molecule as defined in (a), (c) and (d). As described herein,
the increase of NR2F6 activity can lead to a decreased activity of
NF-AT/AP-1 (and other components of the NR2F6-dependent signaling
pathway) which in turn can result in a suppressed immune
response.
[0099] An exemplary transfection of CD4.sup.+ T cells with a
construct for the overexpression of NR2F6 is also shown in the
appended examples. As demonstrated therein, overexpression (about
5-fold increase over normal expression level) can lead to a
diminished IL-2 activity/expression and consequently to a reduced
IL-2 amount, resulting in a reduced immune response.
[0100] Therefore, agonists/activators of NR2F6 are useful in the
treatment of diseases where suppression of the immune response is
desired (e.g., diseases with an overstimulated immune response,
such as allergies and multiple sclerosis). As used herein, the term
"overexpression" means that the NR2F6 activity/expression is, in
various embodiments, at least 1-fold, at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at
least 25-fold increased in comparison to a (control) standard value
as defined herein, wherein a value of 25 fold expression level or
greater over normal can be considered as a maximum overexpression
level.
[0101] As used herein, "antagonist" or "inhibitor" are used
interchangeably and mean a compound or substance capable of fully
or partially suppressing or inhibiting the physiologic activity of
one or more specific receptors. In the context of the present
disclosure, an antagonist can therefore suppress the physiological
activity of a receptor upon binding of said compound substance to
said receptor but does not activate the receptor and therefore
blocks the activity of other agonists. As further discussed herein,
an "antagonist" or "inhibitor" can be used to "deactivate,"
"inhibit," "suppress" or "decrease activity" of a cell.
[0102] As used herein, the terms "immune response" or "immune
reaction" are used interchangeably and mean the response or
reaction of the immune system to an antigen. In the case of an
immune response, immune cells are activated in such way that one or
more specific functions of said immune cells can be induced. The
"immune cells" can include, but are not limited to, B cells, T
cells, neutrophils, eosinophils, basophils, mast cells, macrophages
and dendritic cells. In certain embodiments, said "specific
function(s) of activated immune cells" can include, but are not
limited to, secretion of antibodies, presentation of antigen,
proliferation of said immune cells, secretion of cytokines such as
interleukin-2 (IL-2), interleukin-17 (IL-17), interleukin-18
(IL-18), or interferon gamma (IFNgamma), expression of regulatory-,
activation- or adhesion molecules, and the ability to induce
apoptosis or cytolysis.
[0103] As used herein, the term "antigen" means any substance
capable of inducing an immune response. An antigen typically is
associated with a foreign substance (i.e. a "non-self antigen").
However, an own body-derived substance (i.e., a "self antigen") can
also induce an immune response.
[0104] As used herein, accordingly, the term "immune response" also
encompasses autoimmune responses or autoimmune reactions. For
example, in certain embodiments, the technology herein is directed
to a method of treating or reducing the effect of an autoimmune
response, reaction, disease or disorder, the method comprising
activating the NR2F6 target in isolated immune cells by binding
them with a compound according to the present technology.
[0105] As used herein, "treating a cancer," "inhibiting cancer" or
"reducing cancer growth" are used interchangeably and mean
inhibiting or preventing oncogenic activity of cancer cells.
Oncogenic activity can comprise stimulating migration, invasion,
drug resistance, cell survival, anchorage-independent growth,
non-responsiveness to cell death signals, angiogenesis, or
combinations thereof of the cancer cells. In various embodiments,
agents suitable for use in treating a cancer or reducing the growth
rate of a tumor include, but are not limited to, small organic
molecules, peptides, proteins, peptidomimetics, nucleic acids,
antibodies and combinations thereof. In various embodiments, such
agents can be formulated with a pharmaceutically acceptable
carrier, and can be administered: intravenously, orally, buccally,
sublingually, parenterally, by inhalation, by nasal administration,
by insufflation, by topical application, transdermally, by
cutaneous injection, or by local administration. An agent can
additionally be administered in conjunction with one or more
anti-cancer chemotherapeutic agent in an additive or synergistic
manner.
[0106] As used herein, "cancer," "cancer cell," "tumor" and "tumor
cell" are used interchangeably and mean a group of diseases
characterized by uncontrolled, abnormal growth of cells (e.g., a
neoplasia). These can include solid tumor cancer, liquid tumor
cancer and metastatic disease. In some forms of cancer, the cancer
cells can spread locally or through the bloodstream and lymphatic
system to other parts of the body ("metastatic cancer"). As used
herein, "ex vivo activated lymphocytes," "lymphocytes with enhanced
antitumor activity" and "dendritic cell cytokine induced killers"
are used interchangeably and mean composition of cells that have
been activated ex vivo and subsequently reintroduced within the
context of the current disclosure. Although the word "lymphocyte"
is used, this also includes heterogenous cells that have been
expanded during the ex vivo culturing process including dendritic
cells, NKT cells, gamma delta T cells, and various other innate and
adaptive immune cells.
[0107] As used herein, "cancer" means any disease caused by
uncontrolled division or growth of abnormal cells, and any
malignant growth or tumor resultant from such uncontrolled division
or growth. As used herein, "cancer" includes all types of cancer or
neoplasm or malignant tumors found in animals, including leukemias,
carcinomas and sarcomas. Examples of cancers include, but are not
limited to: cancer of the brain, skin (including melanoma), breast,
cervix, head and neck, kidney, lung, non-small cell lung,
mesothelioma, sarcoma, any internal organ (including bladder,
stomach, liver, pancreas, uterus, ovary, prostate, colon) and
Medulloblastoma.
[0108] As used herein, "leukemia" means a broadly progressive,
malignant disease of the hematopoietic organs or systems and is
generally characterized by a distorted proliferation and
development of leukocytes and their precursors in the blood and
bone marrow. Leukemia diseases include, but are not limited to:
acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute
granulocytic leukemia, chronic granulocytic leukemia, acute
promyelocytic leukemia, adult T-cell leukemia, B cell lymphoma,
aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia,
blast cell leukemia, bovine leukemia, chronic myelocytic leukemia,
leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross'
leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell
leukemia, subleukemic leukemia, undifferentiated cell leukemia,
hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic
leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic
leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic
leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid
leukemia, lymphosarcoma cell leukemia, mast cell leukemia,
megakaryocytic leukemia, micromyeloblastic leukemia, monocytic
leukemia, myeloblastic leukemia, myclocytic leukemia, chronic
myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic
leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic
leukemia, and promyelocytic leukemia.
[0109] As used herein, the term "carcinoma" means a malignant new
growth made up of epithelial cells tending to infiltrate the
surrounding tissues, or resist physiological and non-physiological
cell death signals and give rise to metastases. Exemplary
carcinomas include, but are not limited to: acinar carcinoma,
acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,
carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar
carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma
basocellulare, basaloid carcinoma, basosquamous cell carcinoma,
bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic
carcinoma, cerebriform carcinoma, cholangiocellular carcinoma,
chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus
carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma
cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct
carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides,
exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,
gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,
signet-ring cell carcinoma, carcinoma simplex, small-cell
carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle
cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticum,
carcinoma telangiectodes, transitional cell carcinoma, carcinoma
tuberosum, tuberous carcinoma, verrmcous carcinoma, carcinoma
villosum, carcinoma gigantocellulare, glandular carcinoma,
granulosa cell carcinoma, hair-matrix carcinoma, hematoid
carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,
hyalinc carcinoma, hypemephroid carcinoma, infantile embryonal
carcinoma, carcinoma in situ, intraepidermal carcinoma,
intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell
carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma
lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma,
carcinoma medullare, medullary carcinoma, melanotic carcinoma,
carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma
mucocellulare, mucocpidermoid carcinoma, carcinoma mucosum, mucous
carcinoma, carcinoma myxomatodes, nasopharngeal carcinoma, oat cell
carcinoma, carcinoma ossificans, ostcoid carcinoma, papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of
kidney, reserve cell carcinoma, carcinoma sarcomatodes,
schnciderian carcinoma, scirrhous carcinoma, and carcinoma
scroti.
[0110] As used herein, the term, "sarcoma" means a tumor which is
made up of a substance like the embryonic connective tissue and is
generally composed of closely packed cells embedded in a fibrillar,
heterogeneous, or homogeneous substance. Sarcomas include, but are
not limited to: chondrosarcoma, fibrosarcoma, lymphosarcoma,
melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma,
stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic
sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma,
liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal
sarcoma. Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's
sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma,
immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma
of T-cells. Jensen's sarcoma. Kaposi's sarcoma. Kupffer cell
sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma
sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,
serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
Additional exemplary neoplasias include, for example, Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, premalignant
skin lesions, testicular cancer, lymphomas, thyroid cancer,
neuroblastoma, esophageal cancer, genitourinary tract cancer,
malignant hypercalcemia, cervical cancer, endometrial cancer, and
adrenal cortical cancer.
[0111] In some particular embodiments of the present technology,
the cancer treated is a melanoma. As used herein, the term
"melanoma" means a tumor arising from the melanocytic system of the
skin or other organs. Melanomas include, for example,
Harding-Passey melanoma, juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, acral-lentiginous melanoma,
amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma,
S91 melanoma, nodular melanoma subungual melanoma, and superficial
spreading melanoma. As used herein, the term "polypeptide" is used
interchangeably with "peptide," "altered peptide ligand" and
"fluorocarbonated peptides."
[0112] As used herein, the term "pharmaceutically acceptable
carrier" means any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. Except insofar as any conventional
media or agent is incompatible with the active compound, use
thereof in the therapeutic compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions herein.
[0113] As used herein, the terms "T cell" or "T lymphocyte" are
used interchangeably, and mean a cell derived from thymus among
lymphocytes involved in an immune response. In various embodiments
a T cell includes any of: a CD8-positive T cell (cytotoxic T cell:
CTL), a CD4.sup.+ T cell (helper T cell), a suppressor T cell, a
regulatory T cell such as a controlling T cell, an effector cell, a
naive T cell, a memory T cell, an alpha (.alpha.) beta (.beta.) T
cell expressing TCR .alpha. and .beta. chains, and a gamma
(.gamma.) delta (.delta.) T cell expressing TCR .gamma. and .delta.
chains.
[0114] In certain embodiments, the T cell includes a precursor cell
of a T cell in which differentiation into a T cell is directed.
Examples of "cell populations containing T cells" include, in
addition to body fluids such as blood (peripheral blood, umbilical
blood etc.) and bone marrow fluids, cell populations containing
peripheral blood mononuclear cells (PBMC), hematopoietic cells,
hematopoietic stem cells, umbilical blood mononuclear cells etc.,
which have been collected, isolated, purified or induced from the
body fluids.
[0115] Further, a variety of cell populations containing T cells
and derived from hematopoietic cells can be used in connection with
the embodiments of the present technology. These cells may have
been activated by cytokine such as IL-2 in vivo or ex vivo, and can
be collected in any known way, for example, collected from a living
body; obtained via ex vivo culture, for example, a T cell
population obtained by a method herein; or obtained by freeze
preservation.
[0116] As used herein, the term "antibody" means both intact
molecules as well as fragments thereof that include the
antigen-binding site. Whole antibody structure is often given as
H.sub.2L.sub.2 and refers to the fact that antibodies commonly
comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid
chains. Both chains have regions capable of interacting with a
structurally complementary antigenic target. The regions
interacting with the target are referred to as "variable" or "V"
regions and are characterized by differences in amino acid sequence
from antibodies of different antigenic specificity. The variable
regions of either H or L chains contain the amino acid sequences
capable of specifically binding to antigenic targets. Within these
sequences are smaller sequences dubbed "hypervariable" because of
their extreme variability between antibodies of differing
specificity. Such hypervariable regions are also referred to as
"complementarity determining regions" or "CDR" regions. These CDR
regions account for the basic specificity of the antibody for a
particular antigenic determinant structure. The CDRs represent
non-contiguous stretches of amino acids within the variable regions
but, regardless of species, the positional locations of these
critical amino acid sequences within the variable heavy and light
chain regions have been found to have similar locations within the
amino acid sequences of the variable chains. The variable heavy and
light chains of all antibodies each have 3 CDR regions, each
non-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for
the respective light (L) and heavy (H) chains.
[0117] In various embodiments, the antibodies discussed herein can
also be wholly synthetic, wherein the polypeptide chains of the
antibodies are synthesized and, possibly, optimized for binding to
the polypeptides disclosed herein as being receptors. Such
antibodies can be, in various embodiments, chimeric or humanized
antibodies, and can be fully tetrameric in structure, or can be
dimeric and comprise only a single heavy and a single light
chain.
[0118] As used herein, the terms "effective amount" or
"therapeutically effective amount" are used interchangeably and
mean a dosage sufficient to treat, inhibit, or alleviate one or
more symptoms of a disease state being treated or to otherwise
provide a desired pharmacologic or physiologic effect, especially
enhancing T cell response to a selected antigen. The precise dosage
in any given embodiment can vary according to a variety of factors
such as subject-dependent variables (e.g., age, immune system
health, etc.), the disease, and the treatment being
administered.
[0119] As used herein, the terms "individual," "host," "subject"
and "patient" are used interchangeably and mean a mammal,
including, but not limited to, primates, for example, human beings,
as well as rodents, such as mice and rats, and other laboratory
animals or any other animals mentioned herein.
[0120] As used herein, "treat," "treating" or "treatment" means an
alleviation of symptoms associated with a disorder or disease, or
inhibition of further progression or worsening of those symptoms,
or prevention or prophylaxis of the disease or disorder; and
includes: (i) preventing a pathologic condition from occurring
(e.g., prophylaxis); (ii) inhibiting the pathologic condition or
arresting its development (e.g., slowing or stopping proliferation
of cancer cells or tumor growth); (iii) relieving the pathologic
condition; or (iv) diminishing symptoms associated with the
pathologic condition.
[0121] As used herein, the term "treatment regimen" means a
treatment of a disease or a method for achieving a desired
physiological change, such as increased or decreased response of
the immune system to an antigen or immunogen, such as an increase
or decrease in the number or activity of one or more cells, or cell
types, that are involved in such response. In various embodiments
discussed herein, the treatment or method comprises administering
to an animal, such as a mammal, a sufficient amount of one or more
(in certain embodiments two or more) chemical agents or components
of said regimen to effectively treat a disease or to produce said
physiological change. In certain embodiments, the two or more
agents or components are administered together, such as part of the
same composition, or administered separately and independently at
the same time or at different times (i.e., administration of each
agent or component is separated by a finite period of time from one
or more of the agents or components). In certain embodiments,
administration of said one or more agents or components achieves a
result greater than that of any of said agents or components when
administered alone or in isolation.
[0122] As used herein, the terms "anergy" or "unresponsiveness" are
used interchangeably and include unresponsiveness to an immune cell
to stimulation, for example, stimulation by an activation receptor
or cytokine. Anergy can occur due to, for example, exposure to an
immune suppressor or exposure to an antigen in a high dose. Such
anergy is generally antigen-specific, and can continue even after
completion of exposure to a tolerized antigen. For example, the
anergy in a T cell and/or NK cell can be characterized by failure
of production of cytokine, e.g., interleukin (IL)-2. The T cell
anergy and/or NK cell anergy can occur in part when a first signal
(signal via TCR or CD-3) is received in the absence of a second
signal (costimulatory signal) upon exposure of a T cell and/or NK
cell to an antigen.
[0123] As used herein, the terms "enhanced function of a T cell,"
"enhanced cytotoxicity" and "augmented activity" are used
interchangeably and mean that the effector function of the T cell
or NK cell is improved. In certain embodiments, the enhanced
function of the T cell or NK cell can include any of the following:
an improvement in the proliferation rate of the T cell or NK cell,
an increase in the production amount of cytokine, or an improvement
in cytotoxity. Further, in certain embodiments the enhanced
function of the T cell or NK cell includes cancellation or
suppression of tolerance of the T cell or NK cell in the suppressed
state such as the anergy (unresponsive) state, or the rest state,
that is, transfer of the T cell or NK cell from the suppressed
state into the state where the T cell or NK cell responds to
stimulation from the outside.
[0124] As used herein, "expression" means generation of mRNA by
transcription from nucleic acids such as genes, polynucleotides,
and oligonucleotides, or generation of a protein or a polypeptide
by transcription from mRNA. Expression can be detected by any
method including RT-PCR, Northern Blot, or in situ hybridization.
As used herein, "suppression of expression" means a decrease of a
transcription product or a translation product in a significant
amount as compared with the case of no suppression. The suppression
of expression herein shows, in various embodiments, a decrease of a
transcription product or a translation product in amounts of 30% or
more, 50% or more, 70% or more, or 90% or more.
[0125] As used herein, "augmented immune response" means
characterized by a particularly strong response or reaction of the
immune system to the presence of an antigen. Under normal,
non-pathological conditions, immune responses are regulated in a
tightly controlled fashion. Moreover, immune responses are
self-limiting and decline in time after exposure to the antigen. In
case of an "augmented immune response" however, the immune response
can be hypersensitive, i.e., the immune response can cause damage
to the organism's own cells or tissue in presence of an antigen.
Furthermore, in some cases of an "augmented immune response," for
example in auto-immune diseases or disorders or in transplant
rejects (and the like), the immune system can fail to distinguish
between self and non-self substances. As used herein, "disease
related to an augmented immune response" accordingly relates to any
disease or disorder in which an augmented immune response is
etiological for, associated with, secondary to or the resultant of
said disorder.
[0126] In certain embodiments, an augmented immune response can be
determined by directly or indirectly measuring parameters that are
indicative for the magnitude of the immune response or reaction to
an antigen, and comparing the outcome of said measurement with the
outcome of the same test in a physiologically normal subject.
Parameters indicative for the magnitude of the immune
response/reaction can include, but are not limited to: the presence
or quantity of (specific) antibodies; the presence or quantity of
(specific) immune cells; the presence or quantity of (specific)
cytokines; or the presence or quantity of (specific) regulatory-,
activation- or adhesion molecules.
[0127] For a disease to be related to an augmented immune response,
accordingly, said augmented immune response can be detectable
preceding, during or following said disease. In certain
embodiments, the disease related to an augmented immune response is
any of the following: [0128] an acute or chronic transplant
rejection, including septic shock, infections caused by bacteria
including MRSA and viruses; [0129] a dermatological disease, for
example, psoriasis, atopic dermatitis or contact allergy; [0130] T-
and B-cell-mediated inflammatory disease, for example, asthma or
chronic obstructive pulmonary disease (COPD); [0131]
graft-versus-host disease, for example, acute (or fulminant)
graft-versus-host disease or chronic graft-versus-host disease; or
[0132] auto-immune disease, for example, multiple sclerosis,
inflammatory bowel disease, like ulcerative colitis or Behcet's
disease; vasculitis, lupus erythematosus, pemphigus vulgaris,
pemphigus foliaceus, myasthenia gravis, polymyositis, mixed
collective tissue disease (MCTD) rheumatoid arthritis, diabetes
mellitus (whether Type 1 or Type 2), celiac disease, celiac sprue
disease, atherosclerosis, Goodpasture's syndrome, Grave's disease,
autoimmune hepatitis/hepatic autoimmune diseases, autoimmune
thrombocytopenic purpura, granulomatosis (e.g., morbus Wegener),
Sjogren's Syndrome, scleroderma, alopecia areata or autoimmune
hemolytic anemia.
[0133] Immune responses can be exquisitely controlled, requiring
multiple finely tuned levels of activation as well as inactivation
signals. In T lymphocytes among these signaling networks, T cell
receptor (TCR) stimulation activates NF-AT/AP-1, a family of
transcription factors that is of particular importance during
immune cell activation. NF-AT mediates the transcriptional
induction of "cell fate-determining genes," which govern as diverse
outcomes as activation, anergy or apoptosis. Mechanistically, the
rise of intracellular Ca.sup.2+ triggered by antigen binding to the
TCR can lead to the activation of calcineurin's phosphatase
activity. This leads to dephosphorylation of phospho-sites within
the N-terminal regulatory domain on NF-AT and, subsequently,
nuclear import of NF-AT. Upon transient stimuli, however, feedback
inhibition, mediated via GSK3 (glycogen synthase kinase 3), CK1
(casein kinase 1) and DYRK (dual-specificity tyrosine
phosphorylation-regulated kinase) protein kinases can
counter-regulate NF-AT nuclear occupancy by rephosphorylation,
which induces the nuclear export of NF-AT and the abort of immune
activation-associated gene transcription. NF-AT family members are
also subject to regulation in the nucleus through their ability to
directly interact with other transcriptional regulatory factors.
NF-AT requires a protein partner for high-affinity binding at most
DNA sites. NF-AT complexes mostly contain cell type- or cell
lineage-specific protein binding partners. In cardiac, skeletal,
and smooth muscle cells, NF-AT forms complexes with GATA
proteins.
[0134] Accordingly, in certain embodiments the present technology
is directed to agonists or activators of NR2F6 for the treatment of
a disease related to an augmented immune response. In other
embodiments, the present technology is directed to the use of an
agonist or activator of NR2F6 for the preparation of a medicament
for the treatment of a disease related to an augmented immune
response. The utilization of NR2F6 modulating compounds for
alteration of immune response can be utilized by administering in
patients suffering from cancer in which increased efficacy of a
cancer vaccine is desired. In these situations, inhibition of NR2F6
is desirable, optionally in addition to immune stimulation. Thus,
in various embodiments, the compositions herein can comprise any of
the following:
[0135] (a) agonists/activators of NR2F6;
[0136] (b) antagonists/inhibitors of NR2F6;
[0137] (c) agonists/activators of NR2F6 in combination with: (i)
one or more additional immune enhancers (ii) CAR-T cell therapy
(which can reduce side effects); or (iii) autologous cell
therapies, e.g., dendritic cells, anti-PD1 and antiCTLA4
antibodies, PMBC, or umbilical vein cord blood-derived cells.
[0138] (d) antagonists/inhibitors of NR2F6 in combination with: (i)
one or more additional immune suppressants; (ii) CAR-T cell therapy
(which can reduce side effects); or (iii) autologous cell
therapies, e.g., dendritic cells, anti-PD1 and antiCTLA4
antibodies, PMBC, or umbilical vein cord blood-derived cells.
[0139] Accordingly, in certain embodiments, inhibitor compounds of
NR2F6 are administered with a cancer antigen, said cancer antigens
include ROBO-4. In certain embodiments, the antigens can be used to
replace ROBO-4. These can include any of the following: a)
Fos-related antigen 1; b) LCK; c) FAP; d) VEGFR2; e) NA 17; f)
PDGFR-beta; g) PAP; h) MAD-CT-2; i) Tie-2; j) PSA; k) protamine 2;
l) legumain; m) endosialin; n) prostate stem cell antigen; o)
carbonic anhydrase 1X; p) STn; q) Page4; r) proteinase 3; s) GM3
ganglioside; t) tyrosinase; u) MARTI; v) gp100; w) SART3; x) RGS5;
y) SSX2; z) Globol1; aa) Tn; ab) CEA; ac) hCG; ad) PRAME; ac)
XAGE-1; af) AKAP-4; ag) TRP-2; ah) B7H3; ai) sperm fibrous sheath
protein; aj) CYP1B1; ak) HMWMAA; al) sLe(a); am) MAGE A1; an) GD2;
ao) PSMA; ap) mesothelin; aq) fucosyl GM1; ar) GD3; as) sperm
protein 17; at) NY-ESO-1; au) PAX5; av) AFP; aw) polysialic acid;
ax) EpCAM; ay) MAGE-A3; az) mutant p53; ba) ras; bb) mutant ras;
bc) NY-BR1; bd) PAX3; be) HER2/neu; bf) OY-TES1; bg) HPV E6 E7; bh)
PLAC1; bi) hTERT; bj) BORIS; bk) ML-IAP; bl) idiotype of b cell
lymphoma or multiple myeloma; bm) EphA2; bn) EGFRvIII; bo) cyclin
B1; bp) RhoC; bq) androgen receptor; br) surviving; bs) MYCN; bt)
wildtype p53; bu) LMP2; by) ETV6-AML; bw) MUC1; bx) BCR-ABL; by)
ALK; bz) WT1; ca) ERG (TMPRSS2 ETS fusion gene); cb) sarcoma
translocation breakpoint; cc) STEAP; cd) OFA/iLRP; and ce)
Chondroitin sulfate proteoglycan 4 (CSPG4).
[0140] In certain embodiments, the assessment of compounds for
NR2F6 modulating activity is performed utilizing means known in the
art, such as described in U.S. Pat. No. 9,091,696. Compounds useful
for the screening and modification for enhanced NR2F6 modulatory
activity include: CAR Agonists such as 5.beta.-Dihydroprogesterone,
6,7-Dimethylesculetin, Amiodarone, Artemisinin, Benfuracarb,
Carbamazepine, Carvedilol, Chlorpromazine, Chrysin, CITCO,
Clotrimazole, Cyclophosphamide, Cypermethrin, DHEA, Efavirenz,
Ellagic acid, Griseofulvin, Methoxychlor, Mifepristone, Nefazodone,
Nevirapine, Nicardipine, Octicizer, Permethrin, Phenobarbital,
Phenytoin, Reserpine, TCPOBOP, Telmisartan, Tolnaftate,
Troglitazone, Valproic acid. CAR Antagonists such as
3,17.beta.-Estradiol, 3.alpha.-Androstanol, 3.alpha.-Androstenol,
3.beta.-Androstanol, 17-Androstanol, AITC, Ethinyl estradiol,
Meclizine, Nigramide J, Okadaic acid, PK-11195, S-07662, T-0901317.
FXR Agonists such as Bile acids, Cafestol, Chenodeoxycholic acid,
Fexaramine, GW-4064, Obeticholic acid. FXR Antagonists such as
Guggulsterone. LXR Agonists such as 22R-Hydroxycholesterol,
24S-Hydroxycholesterol, 27-Hydroxycholesterol, Cholestenoic acid,
DMHCA, GW-3965, Hypocholamide, T-0901317. PPAR-alpha Agonists such
as 15-HETE, 15-HpETE, Aleglitazar, Aluminum clofibrate, Arachidonic
acid, Bezafibrate, Clofibrate, CP-775146, DHEA, Elafibranor,
Fenofibrate, Gemfibrozil, GW-7647, Leukotriene B4, LG-101506,
LG-100754, Lobeglitazone, Muraglitazar, Oleylethanolamide,
Palmitoylethanolamide, Pemafibrate, Perfluorononanoic acid.
Perfluorooctanoic acid, Pioglitazone, Saroglitazar, Sodelglitazar.
Tesaglitazar, Tetradecylthioacetic acid, Troglitazone, WY-14643.
PPAR-alpha Antagonists such as GW-6471, MK-886. PPAR-delta Agonists
such as 15-HETE, 15-HpETE, Arachidonic acid, Bezafibrate,
Elafibranor, GW-0742, GW-501516, L-165,041, LG-101506, MBX-8025,
Sodelglitazar, Tetradecylthioacetic acid. PPAR-delta Antagonists
such as FH-535, GSK-0660, GSK-3787. PPAR gamma agonists such as
5-Oxo-ETE, 5-Oxo-15-hydroxy-ETE,
15-Deoxy-.DELTA.12,14-prostaglandin J2, 15-HETE, 15-HpETE,
Aleglitazar, Arachidonic acid. Berberine, Bezafibrate, Ciglitazone,
Darglitazone, Edaglitazone, Etalocib, GW-1929, Ibuprofen,
LG-100268, LG-100754, LG-101506, Lobeglitazone, Muraglitazar,
nTZDpa, Perfluorononanoic, acid, Pioglitazone, Prostaglandin J2,
Rosiglitazone, RS5444, Saroglitazar, Sodelglitazar, Telmisartan,
Tesaglitazar, Troglitazone. SSPARMS such as BADGE, EPI-001,
INT-131, MK-0533, 526948. PPAR gamma antagonists such as FH-535,
GW-9662, SR-202, T-0070907. PPAR nonselective agonists such as
Ciprofibrate, Clinofibrate, Clofibride, Englitazone, Etofibrate,
Farglitazar, Netoglitazone, Ronifibrate, Rivoglitazone, Simfibrate.
PXR Agonists such as 5.alpha.-Dihydroprogesterone,
5.beta.-Dihydroprogesterone, 17.alpha.-Hydroxypregnenolone,
17.alpha.-Hydroxyprogesterone, .DELTA.4-Androstenedione,
.DELTA.5-Androstenediol, .DELTA.5-Androstenedione, AA-861,
Allopregnanolone, Alpha-Lipoic acid, Ambrisentan, AMI-193,
Amlodipine besylate, Antimycotics, Artemisinin, Aurothioglucose.
Bile acids, Bithionol, Bosentan, Bumecaine, Cafestol,
Cephaloridine, Cephradine, Chlorpromazine, Ciglitazone,
Clindamycin, Clofenvinfos, Chloroxine, Clotrimazole, Colforsin,
Corticosterone, Cyclophosphamide, Cyproterone acetate, Demecolcine,
Dexamethasone, DHEA, DHEA-S, Dibunate sodium, Diclazuril,
Dicloxacillin, Dimercaprol, Dinaline, Docetaxel, Docusate calcium,
Dodecylbenzenesulfonic acid, Dronabinol, Droxidopa, Eburnamonine,
Ecopipam, Enzacamene, Epothilone B, Erythromycin, Famprofazone,
Febantel, Felodipine, Fenbendazole, Fentanyl, Flucloxacillin,
Fluorometholone, Griscofulvin, Haloprogin, Hetacillin potassium,
Hyperforin (Hypericum perforatum), Indinavir sulfate, Lasalocid
sodium, Levothyroxine, Linolenic acid, LOE-908, Loratadine,
Lovastatin, Meclizine, Methacycline, Methylprednisolone,
Metyrapone, Mevastatin, Mifepristone, Nafcillin, Nicardipine,
Nicotine, Nifedipine, Nilvadipine, Nisoldipine, Norelgestromin,
Omeprazole, Orlistat, Oxatomide, Paclitaxel, Phenobarbital,
Plicamycin, Prednisolone, Pregnanolone, Pregnenolone, Pregnenolone
16.alpha.-carbonitrile, Proadifen, Progesterone. Reserpine, Reverse
triiodothyronine Rifampicin, Rifaximin, Rimexolone, Riodipine,
Ritonavir, Simvastatin, Sirolimus, Spironolactone, Spiroxatrine,
SR-12813, Suberoylanilide, Sulfisoxazole, Suramin, Tacrolimus,
Tenylidone, Terconazole, Testosterone isocaproate, Tetracycline,
Thiamylal sodium, Thiothixene, Thonzonium bromide, Tianeptine,
Troglitazone, Troleandomycin, Tropanyl 3,5-dimethulbenzoate,
Zafirlukast, Zearalanol. PXR Antagonist such as Ketoconazole. RAR
Agonists such as 9CDHRA, 9-cis-Retinoic acid (alitretinoin),
AC-261066, AC-55649, Acitretin, Adapalene, all-trans-Retinoic acid
(tretinoin), AM-580, BMS-493, BMS-753, BMS-961, CD-1530, CD-2314,
CD-437, Ch-55, EC 23, Etretinate, Fenretinide, Isotretinoin,
Palovarotene, Retinoic acid, Retinol (vitamin A), Tamibarotene,
Tazarotene, Tazarotenic acid, TTNPB. RAR Antagonists such as
BMS-195614, BMS-493, CD-2665, ER-50891, LE-135, MM-11253. RXR
Agonists such as 9CDHRA, 9-cis-Retinoic acid (alitretinoin),
all-trans-Retinoic acid (tretinoin), Bexarotene, CD 3254,
Docosahexaenoic acid, Fluorobexarotene, Isotretinoin, LG-100268,
LG-101506, LG-100754, Retinoic acid. Retinol (vitamin A), SR-11237.
RXR Antagonists such as HX-531, HX-630, LG-100754, PA-452,
UVI-3003. TR Agonists such as Dextrothyroxine, GC-1, Levothyroxine,
Liothyronine, Thyroxine, Tiratricol, Triiodothyronine.
[0141] Other compounds useful for modulation of NR2F6 activity
include:
5-tert-butyl-N-[(6-fluoro-4H-1,3-benzodioxin-8-yl)methyl]-2-methylpyrazol-
e-3-carboxamide, ST50775950, ethyl
4-(cyclohexylamino)-2-(3,5-dimethylpyrazol-1-yl)pyrimidine-5-carboxylate,
ethyl
4-(cyclopentylamino)-2-(3,5-dimethylpyrazol-1-yl)pyrimidine-5-carbo-
xylate, AGN-PC-09SAX3, SMR000064686, AGN-PC-0NLTEQ, T6090485,
MLS002548992,
5,6-dimethyl-4-[4-[2-(4-methylphenoxy)ethyl]piperazin-1-yl]thieno[2,3-d]p-
yrimidine, MLS002473459, MLS001030349,
4-(3,4-dihydro-1H-isoquinolin-2-yl)-5H-pyrimido[5,4-b]indole,
4-(3,4-Dihydro-1H-isoquinolin-2-yl)-8-fluoro-5H-pyrimido[5,4-b]indole,
4-[4-(4-methoxyphenyl)piperazino]-5H-pyrimido[5,4-b]indole,
4-[4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl]-7-methoxy-5H-pyrimido[5,-
4-b]indole, SMR000044829,
8-fluoro-N-(3-propan-2-yloxypropyl)-5H-pyrimido[5,4-b]indol-4-amine,
GNF-Pf-1678, MLS003116118,
2-[4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl]-1,3-b-
enzothiazole,
5-methyl-3,6-diphenylpyrazolo[1,5-a]pyrimidin-7-amine,
4-[4-[(4-chlorophenyl)methyl]piperazin-1-yl]-1-[(4-methylphenyl)methyl]py-
razolo[3,4-d]pyrimidine, MLS002632722, MLS002477203, MLS003120814,
AGN-PC-07AHX3, MLS003120821, MLS003120807, MLS003120811,
MLS003120820, ethyl
4-[[1-(2,4-dimethylphenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amino]piper-
idine-1-carboxylate.
N-[2-(3,4-dimethoxyphenyl)ethyl]thieno[2,3-d]pyrimidin-4-amine,
N-[2-(3,4-dimethoxyphenyl)ethyl]-6-methylthieno[2,3-d]pyrimidin-4-amine
hydrochloride, N-(1-phenylethyl)quinazolin-4-amine, AG-F-87638,
ZINC03428816, CHEMBL493153, ST50323391, N-Benzylquinazolin-4-amine,
ST50483228,
N-[4-(2-methyl-1-methylsulfonyl-2,3-dihydroindol-5-yl)-1,3-thiazol-2-v1]--
2-thiophen-2-ylacetamide, F0558-0175, AC1MLRO7,
4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-(3-methylphenyl)-1,3-thiazol-2-a-
mine, AGN-PC-09PPXW, Compound 15Jf, AC1MEEXM, ST50941838,
[2-[(3-carbamoylthiophen-2-yl)amino]-2-oxoethyl]
2-naphthalen-1-ylacetate, F0239-0029, AC1OBZ0O, ST4126227,
1-[(4-bromophenyl)methyl]-2-methylbenzimidazole, SMR000718391.
MLS002694437, Chlormidazole,
2-methyl-1-(2-methylbenzyl)-1H-benzimidazole, MLS003119103,
Ambcb90456311, AGN-PC-04RX4B, MLS001122505, Ambcb81049924,
AGN-PC-04RX7E, Ambcb42757923, MLS001124721,
7-benzyl-4-chloro-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidine,
AGN-PC-04V4GP, MLS000562030, AGN-PC-00YPMB, T5400648, MLS003107990,
AC1NUNJE, MLS002701851, SMR000185185, STK850401,
[(3-bromobenzyl)sulfanyl][(4-fluorophenyl)amino]methylidene,
propanedinitrile, AC1NXBLH, CAS-66-81-9, Cycloheximide, ACTIPHENOL,
MLS001032885, MLS000553012, SMR000285129, MLS000688479,
MLS002702480, GNF-Pf-4659, MLS002702449, T0501-4035, MLS000712179,
AGN-PC-00MQWB, AGN-PC-0NKU3S, T0503-0850, T0501-5798, SMR000212173,
3,3'-Diethylthiazolinocarbocyanine iodide,
2-methyl-3,5-bis(4-methylphenyl)isoxazol-2-ium, MLS000705900,
SMR000211540, AGN-PC-00PL3I, AGN-PC-0NJNZK, SMR000354849,
T0503-1204, MLS000688685, GNF-Pf-4078, T0503-3525, T0503-4982,
T0501-7391, GNF-Pf-3268, TCMDC-125620,
1-[1,1'-Biphenyl]-4-yl-2-(4-imino-1(4H)-pyridinyl)ethanone,
SMR000036350, MLS000080109, MLS000080126, Ambcb40308772,
MLS000733369, Ambcb20390854, MLS000732313, AGN-PC-04RYS6,
Ambcb33735952, AGN-PC-04RYKA, MLS000733096, Ambcb63657849,
MLS001090213, T6132867, MLS003678910, AC1OXF5M, SMR000218920,
MLS000037490, Boc-KS, MLS000734694, AGN-PC-087SDW, ISUPSL100073,
4-{[5,7-bis(trifluoromethyl) benzenol, BAS 07204618, MLS001144057,
MLS001250118, SMR000041809, SMR000635220. MLS003120011, T5546966,
4-chloro-N-(4-chlorobenzyl)-1,3-dimethyl-IH-pyrazole-5-carboxam-
ide,
3-(Toluene-4-sulfonylmethyl)-2,3-dihydro-benzo[4,5]imidazo[2,1-b]thia-
zole, T0508-0735, Carboxyamidotriazole, MLS003116132, F0850-5968,
Verrucarin A 9,10-epoxide, MLS002702133, Ossamycin, MLS002702060,
Dihydrorotenone, SMR000623161, Pyridaben, ASN 09858385, T6069554,
T6302989, SMR000629820, SMR000629835, MLS001028777, MLS001028747,
MLS001028806, SMR000625125, T5403634. T5459762, T5626573, T5337170,
SMR000093473, T6120097,
N-[2-[2-[2,5-dimethyl-1-(thiophen-2-ylmethyl)pyrrol-3-yl]-2-oxoethoxy]phe-
nyl]acetamide, MLS000575323,
N-[4-[2-[2,5-dimethyl-1-(thiophen-2-ylmethyl)pyrrol-3-yl]-2-oxoethyl]sulf-
anylphenyl]acetamide, SMR000274842, T5565081,
6-chloro-N-[3-[(4-methoxyphenyl)sulfamoyl]phenyl]pyridine-3-carboxamide,
N-methyl-N-[(1,3,5-trimethylpyrazol-4-yl)methyl]naphthalene-2-sulfonamide-
, T6099016, T6094971, ASN 04448329, SMR000241542, AGN-PC-03RL0E,
AGN-PC-080KFN, T6151837, AGN-PC-0KIUAY,
N-[4-(4-methylphenyl)-1,3-thiazol-2-yl]-1-thiophen-2-ylsulfonylpiperidine-
-4-carboxamide,
5-(3,5-dimethylpiperidin-1-yl)sulfonyl-N,N-diethyl-3-methyl-1-benzofuran--
2-carboxamide, SMR000124769,
N-(1-benzylpiperidin-4-yl)-1-(5-chloro-2-methylphenyl)sulfonylpiperidine--
4-carboxamide, MLS001095722,
4-ethoxy-N-(pyridin-4-ylmethyl)benzenesulfonamide,
4-chloro-3-ethoxy-N-(pyridin-4-ylmethyl)benzenesulfonamide,
2,4,6-trimethyl-N-(pyridin-4-ylmethyl)benzenesulfonamide, BAS
05598377,
4-bromo-2,5-dimethyl-N-(pyridin-4-ylmethyl)benzenesulfonamide,
MLS000735463, MLS000687652, AGN-PC-093SBW, AG-401/42008258,
5L-526S,
2-[[5-(3-chloro-1-benzothiophen-2-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]aceto-
nitrile,
2-(5-Pyridin-3-yl-[1,3,4]thiadiazol-2-ylsulfanyl)-N-quinolin-4-yl-
-acetamide,
2-[[5-(benzotriazol-1-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]-N-[(4-chlo-
rophenyl)methyl]-N-phenylacetamide,
2-[[5-(benzotriazol-1-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]-N-[(4-fluo-
rophenyl)methyl]-N-phenylacetamide, SR-01000288264,
2-(1-cyclopropyltetrazol-5-yl)sulfanyl-1-[4-[(4-propan-2-ylphenyl)methyl]-
piperazin-1-yl]ethanone,
N-(2,4-difluorophenyl)-4-[5-(trifluoromethyl)pyridin-2-yl]-1,4-diazepane--
1-carbothioamide, T0512-9975,
[[2,7-bis(2-morpholin-4-ylethoxy)fluoren-9-ylidene]amino]thiourea,
MLS001018548, T0507-0244,
4-(4-acetylphenyl)-N-(4-phenoxyphenyl)piperazine-1-carbothioamide,
N-(3-ethoxypropyl)-4-[4-(4-fluorophenyl)-1,3-thiazol-2-yl]piperazine-1-ca-
rbothioamide, (+)-Emetine dihydrochloride hydrate, MLS002302684,
4-(6-chloro-1,3-benzothiazol-2-yl)-N-(2-chloro-6-methylphenyl)-1,4-diazep-
ane-1-carboxamide.
N-(3-chloro-2-methylphenyl)-4-(3-phenyl-1,2,4-thiadiazol-5-yl)-1,4-diazep-
ane-1-carboxamide, MLS000692856, bjm-csc-19, MLS002701991, and
MLS000586514. Additional compounds include 6-formylindolo (3,2-B)
carbazole, 4-hydroxyphenylretinamide, 3,5-Dilodo-L-tyrosine,
Rifampicin, and Z30972355.
[0142] Another aspect of the present disclosure is a pharmaceutical
composition comprising a NR2F6 modulator, such as a NR2F6 inhibitor
or NR2F6 activator, for use in the methods described herein.
Accordingly, in certain embodiments, the present technology
provides a pharmaceutical composition comprising an effective
amount of a NR2F6 inhibitor or NR2F6 activator in admixture with a
pharmaceutically acceptable carrier, excipient or diluent.
[0143] In various embodiments, the pharmaceutical compositions
herein can be used to inhibit NR2F6; or to activate NR2F6.
[0144] In certain embodiments, the pharmaceutical composition is
used to treat a disease or a hematopoietic condition as described
herein. The NR2F6 inhibitors or NR2F6 activators can be formulated
into pharmaceutical compositions for administration to subjects in
a biologically compatible form suitable for administration in
vivo.
[0145] As used herein, "biologically compatible form suitable for
administration in vivo" means a form of the substance to be
administered in which any toxic effects are outweighed by the
therapeutic effects. In various embodiments, the substances herein
can be administered to living organisms including humans, and
animals. Administration of a therapeutically active amount of the
pharmaceutical compositions of the present disclosure is defined as
an amount effective, at dosages and for periods of time necessary
to achieve the desired result. For example, a therapeutically
active amount of a substance can vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of inhibitor to elicit a desired response in the
individual. Dosage regime can be adjusted to provide the optimum
therapeutic response. For example, several divided doses can be
administered daily or the dose can be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
[0146] In various embodiments, the active substance can be
administered by, e.g., injection (subcutaneous, intravenous,
intramuscular, etc.), oral administration, inhalation, intranasal,
transdermal or topical administration (such as topical cream or
ointment, salve, paste or the like), pulmonary, buccal, subdermal,
intradermal, transdermal or parenteral, rectal, subcutaneous,
intravenous, intraurethral, intramuscular, ophthalmic or
suppository administration. Depending on the route of
administration, the active substance can, in certain embodiments,
be coated in a material to protect the compound from the action of
enzymes, acids and other natural conditions which could inactivate
the compound.
[0147] In certain embodiments, the active substance can be
formulated into delayed release formulations such that NR2F6 can be
inhibited or activated for longer periods of time than a
conventional formulation.
[0148] In certain embodiments, a method herein includes the
following steps: (a) extraction of an amount of a patient's
cellular material, including, but not limited to: blood, saliva,
sweat, or any portion of a tumor known or believed to be in a
diseased state; (b) isolating immune cells from the cellular
material; (c) inhibiting or activating the NR2F6 target in the
extracted immune cells; and (d) re-administering the immune cells
(for example, by injection) to the patient's body. This can have
the effect of "reprogramming" the immune cells to attack tumors or
other invasive cells.
[0149] In certain embodiments, other types of a patient's cellular
material can also be extracted. These include, for example, any
part of the blood (blood serum, red blood cells, white blood cells,
plasma, platelets), any other material from the body that includes
the patient's cells (for example, skin, hair, nails, saliva,
cerebrospinal fluid, intracellular fluid, extracellular fluid,
intravascular fluid, interstitial fluid, lymphatic fluid,
transcellular fluid, exudates, lymph, sweat, sebum or serous
fluid). In certain embodiments, the re-administering of the immune
cells to the patient's body can be done by injection, introduction
through the nose or mouth (for example, inhalation), skin or mucous
membranes.
[0150] In certain embodiments, the present technology is directed
to compounds alone or in combination with another medicament. As
set forth herein, compounds herein include stereoisomers
(including, e.g., enantiomers, diastereomers, cis-trans and E-Z
isomers, conformers and atropisomers), tautomers, solvates,
prodrugs, metabolites, pharmaceutically acceptable salts and
mixtures thereof. Compositions containing a compound herein can be
prepared by conventional techniques, and can appear in conventional
forms, for example, oral dosage forms; or any ingestible, inhalable
(e.g., through the mouth, nose or mucosa); or topical applications,
e.g., applicable to the skin, nails, eyes or the like. These can
include, in various embodiments, capsules, tablets, pills, cachets,
dispersible granules, lozenges, aerosols, solutions, powders,
suspensions, emulsions, gels, mousses, foams, drops, lotions,
creams, paste, dragees, suppositories and any application
deliverable to the body of a user.
[0151] In various embodiments, dosages and compounds herein can be
prepared and administered in a wide variety of oral, parenteral,
and topical dosage forms, including, but not limited to, by
injection (e.g., intravenously, intramuscularly, intracutaneously,
subcutaneously, intraduodenally, or intraperitoneally); by
inhalation (e.g., intranasally); or transdermally. In certain
embodiments, multiple routes of administration can be used to
optimize delivery of the compounds herein.
[0152] In various embodiments, the compositions described herein
can be prepared by known methods for the preparation of
pharmaceutically acceptable compositions which can be administered
to subjects, such that an effective quantity of the active
substance is combined in a mixture with a pharmaceutically
acceptable vehicle. On this basis, the compositions can include,
albeit not exclusively, solutions of the substances in association
with one or more pharmaceutically acceptable vehicles or diluents,
and contained in buffered solutions with a suitable pH and
iso-osmotic with the physiological fluids.
[0153] In certain embodiments, a powder or tablet according to a
dosage form herein can contain about 5 to about 75%, about 10 to
about 70%, or about 15 to about 65% of the active compound.
Suitable carriers include, but are not limited to: magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa butter, and the
like.
[0154] In various embodiments, carriers for certain dosages can
include aqueous solutions of dextrose, saline, water, organic
solvents including ethanol, glycerol, propylene glycol, oils
including peanut oil or sesame oil; or polyoxyethylene-block
polymers. Aqueous solutions or suspensions can be made by
dispersing the finely divided active component in water or another
solvent with viscous material, such as natural or synthetic gums,
resins, methylcellulose, sodium carboxymethylcellulose, and other
suspending agents.
[0155] In various embodiments, the compounds or dosages herein can
also be incorporated into liposomes or micelles, or administered
via transdermal pumps or patches.
[0156] Some compounds may have limited solubility in water and
therefore may require a surfactant or other appropriate co-solvent
in the composition. Such co-solvents include: Polysorbate 20, 60,
and 80; Pluronic F-68, F-84, and P-103; cyclodextrin; and polyoxyl
35 castor oil; and in various embodiments, are present in amounts
of about 0.01 to about 10%, about 0.05 to about 5% or about 0.1 to
about 3% by weight.
[0157] In certain embodiments, it may be desirable to increase the
viscosity of the dosage forms herein for ease in dispensing or
delivery. Such viscosity building agents include, for example,
polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy
propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl
cellulose, hydroxy propyl cellulose, chondroitin sulfate and salts
thereof, hyaluronic acid and salts thereof, and combinations of the
foregoing; and in various embodiments, are present in amounts of
about 0.01 to about 10%, about 0.05 to about 5% or about 0.1 to
about 3% by weight.
[0158] The compositions herein can, in certain embodiments,
additionally include components to provide sustained release or
comfort. Such components include, but are not limited to, high
molecular weight, anionic mucomimetic polymers, gelling
polysaccharides, finely-divided drug carrier substrates,
emollients, humectants, moisturizers, essential oils, oils, lipids,
fatty acids, glycerides, extracts of natural ingredients, soaps and
waxes.
Useful Compounds
[0159] The present disclosure includes various compounds that were
found to be modulators of NR2F6 activity and NR2F6 utilizing
compounds, and the immune modulation and modulation of cancer stem
cell activity. Exemplary compounds and methods are shown in the
attached FIGS. 1-58. These compounds were initially found to be
modulators of NR2F6 activity and NR2F6 utilizing compounds, and the
immune modulation and modulation of cancer stem cell activity. In
various embodiments, the compounds comprise one or more of the
following functional groups: a sulfonyl group, a sulfone group
R--S(.dbd.O)2-R' where R and R' are any organic functional groups,
a pyrazine group, any phenyl substituted with one or more halogens
including chlorine or fluorine; or any composition comprising two
or more phenyl constituents. In certain embodiments, a compound
herein can be in amorphous form, crystalline form, or a mixture
thereof; as well as any polymorph or amorphous form, a solvate, a
hydrate or an unsolvated form.
Example 1
[0160] The methodology for screening candidate compounds as NR2F6
agonists was as follows: For primary screening, hit criteria was
ACT %>DMSO control+5*SD (DSO control) at 10 .mu.M, or any
compound with S/B>2. For rescreening, hit criteria was ACT
%>DMSO control+3*SD (DSO control) in each replicate at 10 .mu.M.
For counterscreening, hit criteria was mean ACT<DMSO
control+3*SD (DMSO control) with ER.alpha. transient transfection
in duplicate at 10 .mu.M.
[0161] Table 1 shows screening results from a first set of
compounds.
TABLE-US-00002 TABLE 1 Firefly_ERa, Renilla, Renilla_ERa, Firefly,
cmpd/DMSO cmpd/DMSO cmpd/DMSO cmpd/DMSO Compound I.D. repeat 1
repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 17
2.2 2.4 2.5 2.2 1.3 1.4 1.3 1.0 18 3.8 3.8 2.5 1.9 3.3 4.2 0.8 0.9
19 2.2 1.7 2.3 2.4 1.6 1.7 1.2 1.0 20 2.2 2.1 1.5 1.7 4.4 4.8 1.1
0.9 21 2.4 2.2 1.1 1.0 3.8 3.0 1.1 0.9 22 2.1 2.9 1.5 2.2 1.1 1.3
0.8 1.1 23 3.0 1.9 1.8 1.8 4.6 3.4 1.1 1.2 24 2.0 2.3 1.3 1.6 2.4
2.2 0.9 1.0 25 2.4 1.9 1.3 0.7 3.1 2.1 0.9 1.0 C1 3.4 4.1 1.0 1.0
4.1 1.5 1.3 1.2 C2 2.7 2.2 1.0 0.8 6.0 5.3 1.7 1.6 C3 2.0 2.0 1.8
1.9 1.6 1.2 1.1 1.2 C4 2.5 2.5 1.6 1.7 1.6 1.5 1.1 1.2 C5 2.2 1.7
1.6 2.0 1.3 1.4 1.2 1.0 C6 2.6 1.9 2.2 1.4 1.2 0.9 1.0 1.0 C7 2.1
2.1 0.8 0.5 1.1 1.4 0.8 0.8 C8 2.1 2.8 1.2 1.2 3.4 3.1 1.0 1.1 C9
2.7 1.9 2.5 2.4 2.4 2.5 0.9 1.0 C10 2.1 2.6 1.0 1.3 3.1 1.6 0.7 0.8
C11 13.5 12.6 1.7 1.7 3.8 3.3 1.3 1.2 C16 2.7 2.3 1.0 0.9 3.9 3.2
0.9 1.1
[0162] C1, C7 and C11 were found to have particularly good
activity:
##STR00005##
Additional compounds included the following:
##STR00006## ##STR00007##
[0163] Yet additional compounds tested included Compounds 17, 19,
22 and C3-C6:
##STR00008##
[0164] Compound C1 was found to be particularly promising. FIGS.
16A and 16B show results of cytokins release by hPBMC and cytotox
for Compound C11. For cytokines release and cytotox on hPBMCs, the
Compound was tested at 1.25, 2.5, 5, 10, 25 and 50 uM in
duplicates. For cytotox on HEK293, HEK293 pGL4 and HEK293 NR2F6
(full length) cmpd was tested from 50 uM with dilution step 3.16 in
duplicates.
[0165] The human PBMC were activated by 10 ng/mL PMA+500 ng/mL
ionomycin. Data were normalized to controls with (100%) w/o
compounds.
[0166] Compounds related to Compound C11 were further explored, and
in particular, Compound C11 was substituted with various moieties
to test how this affected its activity.
[0167] In certain embodiments, the present technology is directed
to compounds of Formula (Ia), (Ib) or (Ic):
##STR00009##
wherein any of R, R1 and R2 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0168] In certain embodiments of any of the Formulas (I through
XVII) herein, any of R, RA, RB, R1-R8, X, Q, Q1. Q2, or A can be
any of the following: Me, OMe, Br, N, H, Cl, F or NO.sub.2. In
certain embodiments, any of R, RA, RB, R1-R8, X, Q, Q1, Q2, or A
can be any of the following: 4-Me, 4-OMe, 4-Br, 4-t-Bu, 3,4-di-Me,
4-Cl, 3,4-di-Cl, 3-Cl-4-F, 2-F, 3-Cl, 3-CH.sub.3-4-F, 4-iPr, Ph,
4-MeO--C6H4, 4-tBu, 2, 4-diMe, 2-thienyl, 2-MeO-4-Cl, 4-Cl,
2-furayl, 4-F--C6H4, 2,4-diMeC6H3, 3-Me-4-F or 4-Cl--C6H4.
[0169] Compounds of Formulas I(a) and I(b) were rescreened in
multiple assays to acquire statistical confidence in the results.
Results were repeated in follow set screens from fresh powder.
Analogs obtained are shown in FIG. 17B.
[0170] Four particularly useful compounds that were all based on
C11 (Compounds C12 through C15) were identified as follows:
##STR00010##
[0171] Activity, of the above compounds (C12 through C15) is shown
in Table 2:
TABLE-US-00003 TABLE 2 Firefly, cmpd/DMSO Renilla, cmpd/DMSO
Firefly_pGL, cmpd/DMSO IDNUMBER repeat 1 repeat 2 repeat 1 repeat 2
repeat 1 repeat 2 C12 20.0 16.7 4.5 9.4 2.6 2.9 C11 24.6 21.3 13.9
12.3 3.3 3.3 C13 14.0 12.9 6.4 8.2 3.2 3.2 C14 22.3 20.0 12.1 15.2
2.9 2.6 C15 0.9 1.7 5.8 10.7 1.2 1.7
[0172] Further compounds related to the Compound C11 and the
compound of Formulas (Ia) (Ib) or (Ic) were tested. These include
the following:
##STR00011## ##STR00012## ##STR00013## ##STR00014##
[0173] Further results of the testing of these compounds are shown
in the tables below. Each compound shows results based on the
different identities of the R1 moiety.
[0174] For example, compounds of Formula (Ia) and (Ib) were tested
with different moieties as R1, and the results are shown below in
Table 3:
TABLE-US-00004 TABLE 3 Firefly_pGL, Firefly, cmpd/DMSO Renilla,
cmpd/DMSO cmpd/DMSO ID NUMBER R1 repeat 1 repeat 2 repeat 1 repeat
2 repeat 1 repeat 2 C100 4-CH3 2.0 1.9 1.6 1.3 1.3 1.1 C101 4-OMe
4.5 4.7 1.8 2.0 1.2 1.3 C102 4-Br 13.0 15.3 5.8 6.1 2.3 2.5 C103
4-t-Bu 2.8 1.4 1.9 1.7 1.3 1.4 C104 3,4-di-Me 3.1 4.4 1.7 1.8 2.3
1.2 C105 4-Cl 5.7 4.8 1.7 2.3 1.5 1.8 C106 3,4-di-Cl 1.4 1.3 1.8
5.8 1.0 1.1 C107 3-Cl-4-F 11.0 11.0 8.7 6.5 1.7 2.1 C108 2-F 7.9
7.3 3.0 2.8 1.3 1.5 C109 3-Cl 9.2 10.3 3.4 3.3 3.5 3.4 C110
3-CH3-4-F 7.6 7.2 2.3 2.1 1.7 2.0
[0175] In further embodiments, the present technology is directed
to compounds of Formula (II):
##STR00015##
wherein any of R1 and R2 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0176] In certain embodiments, any of R1 and R2 can be any of the
moieties listed below in Tables 4 and 5. Various compounds of
Formula (II) were tested with different moieties as R1 and R2, and
the results are shown below in Tables 4 and 5:
TABLE-US-00005 TABLE 4 ID Firefly, cmpd/DMSO Renilla, cmpd/DMSO
Firefly_pGL, cmpd/DMSO NUMBER R2 R1 repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C111 phCH2CH2 H 29.8 35.9 29.4 30.8 3.7
3.9 C112 phCH2CH2 4-CH3 11.7 10.1 5.6 5.1 2.5 2.8 C113 phCH2CH2 4-F
5.8 7.0 10.2 10.8 2.2 3.2 C114 phCH2CH2 4-Cl 4.5 4.7 4.0 2.9 2.3
1.6 C115 PhCH2 H 6.8 21.9 4.8 10.9 6.2 7.2 C116 4-CH3C6H4CH2 4-Cl
8.6 9.0 9.8 9.9 2.1 2.0 C117 4-OMeC6H4CH2 4-F 17.3 24.1 28.1 35.6
3.9 4.5 C118 4-F--C6H4CH2 4-F 13.5 17.1 11.8 14.8 2.4 2.7 C119
4-F--C6H4CH2 4-Cl 18.4 16.2 27.8 27.6 2.0 2.2 C120 2-CF3C6H4 4-F
4.5 6.3 15.2 18.6 2.9 3.3 C121 3,4-di-MeO--C6H3 4-CH3 9.0 21.5 18.6
40.1 9.1 8.8 C122 3,4-di-MeO--C6H3 4-Cl 4.4 5.7 14.8 12.8 5.3 4.8
C123 2-MeO--C6H4 H 17.7 16.8 31.9 31.7 8.2 10.6 C124 2-MeO--C6H4
4-F 25.5 29.8 50.0 51.4 6.6 6.7 C123 2-MeO--C6H4 4-Cl 28.0 24.5
30.4 33.8 2.5 2.7 C126 3-MeO--C6H4 4-CH3 45.1 44.8 43.5 42.2 4.8
4.3 C127 3-MeO--C6H4 4-F 36.7 56.2 43.1 43.5 8.3 8.0 C128
3-Cl--C6H4 4-F 1.7 5.2 1.7 10.1 4.4 5.4 C129 4-Cl--C6H4 4-F 0.9 1.2
1.0 0.7 3.3 4.5 C130 3.5-di-MeO--C6H3 H 2.3 3.4 2.2 6.4 6.4 10.2
C131 3.5-di-MeO--C6H3 4-CH3 9.5 15.3 30.8 38.2 4.8 4.7 C132
3.5-di-MeO--C6H3 4-F 1.4 1.4 6.7 2.4 4.3 6.8 C133 3.5-di-MeO--C6H3
4-Cl 2.1 4.3 9.9 12.6 3.2 2.6 C134 2,3-di-MeC6H3 4-CH3 38.1 31.9
21.0 18.9 3.7 5.4 C135 2,3-di-MeC6H3 4-F 5.6 4.9 19.8 18.6 1.9 3.1
C136 2,3-di-MeC6H3 4-Cl 22.1 25.8 12.5 14.9 2.9 3.0
TABLE-US-00006 TABLE 5 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO
cmpd/DMSO cmpd/DMSO NUMBER R2 R1 repeat 1 repeat 2 repeat 1 repeat
2 repeat 1 repeat 2 C159 4-F--C6H4 4-F 0.9 1.2 0.1 0.3 1.8 2.4 C160
2-me-C6H4 4-F 22.9 20.2 9.5 8.2 3.2 2.7 C161 4-Me--C6H4 H 35.4 39.0
28.6 27.7 4.0 5.1 C162 4-Me--C6H4 4-CH3 20.9 20.6 37.9 35.8 3.7 4.1
C163 4-Me--C6H4 4-F 2.0 5.2 6.9 15.8 5.5 5.0 C164 2,4-di-MeC6H3 H
29.3 19.6 13.8 8.9 3.1 2.8 C165 2,4-di-MeC6H3 4-CH3 4.2 4.0 4.7 4.6
1.4 1.4 C166 2,4-di-MeC6H3 4-Cl 3.4 4.2 2.9 2.9 1.4 1.7 C167
3,4-di-MeC6H3 H 1.0 0.7 0.2 0.2 0.9 0.9 C168 3,4-di-MeC6H3 4-CH3
11.8 11.4 14.8 14.0 2.4 1.6 C169 3,4-di-MeC6H3 4-F 8.8 10.6 9.9
10.9 2.0 1.9 C170 3,4-di-MeC6H3 4-Cl 1.7 1.6 0.2 0.3 0.7 1.0 C171
3-MeC6H4 4-CH3 6.4 4.2 15.4 9.8 2.0 2.3 C172 3-MeC6H4 4-F 30.4 32.8
44.9 37.0 3.5 4.7 C173 3-MeC6H4 4-Cl 9.8 6.9 19.8 19.5 2.3 2.4 C174
3,5-di-MeC6H3 H 5.8 14.3 16.0 28.3 4.3 6.0 C175 3,5-di-MeC6H3 4-CH3
13.2 11.9 10.5 12.0 2.5 1.9 C176 3,5-di-MeC6H3 4-F 3.7 9.6 19.0
29.1 3.9 3.8 C177 4-MeOC6H4 H 17.4 8.2 6.7 4.6 1.7 1.3 C178
4-MeOC6H4 4-CH3 15.2 16.9 27.7 28.2 3.5 4.4 C179 4-MeOC6H4 4-F 8.7
10.1 11.9 10.5 2.4 2.5 C180 4-MeOC6H4 4-Cl 34.0 34.5 30.9 30.0 2.5
2.7 C181 2-EtOC6H4 4-F 10.4 13.5 14.6 21.4 2.2 1.7
[0177] In further embodiments, the present technology is direct to
compounds of Formula (III):
##STR00016##
wherein any of Q and R1 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0178] Various compounds of Formula (III) were tested with
different moieties as R1 and Q, and t. These include the
following:
##STR00017## ##STR00018##
[0179] The results of the tests are shown below in Tables 6-8:
TABLE-US-00007 TABLE 6 Firefly, cmpd/DMSO Renilla, cmpd/DMSO
Firefly_pGL, cmpd/DMSO ID NUMBER Q R1 repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C182 Cl 3-F 16.2 17.3 15.1 13.2 2.0 2.1
C183 Br 3-F 1.2 1.2 3.1 2.4 1.1 1.2 C184 Cl 3-MeO 1.1 1.4 1.2 1.8
0.9 1.1 C185 Br 3-MeO 2.6 2.4 0.1 0.3 0.8 1.2 C186 Cl 3,5-diMe 1.0
6.9 0.4 18.8 1.2 1.7 C187 Br 3,5-diMe 0.8 0.7 1.0 0.8 0.9 1.1 C188
Cl 3-Cl-4-Me 9.4 8.7 7.6 6.9 1.8 1.8 C189 Br 3-Cl-4-Me 2.8 2.2 2.7
2.4 1.2 1.1 C190 F 3,5-diMe 2.1 2.5 1.4 1.4 1.3 1.1 C191 Cl 3-Cl
8.6 12.8 35.8 30.6 1.7 2.2 C192 Br 3-Cl 1.8 2.4 9.6 9.5 1.7 2.0
C193 Br 3,5-diF 1.8 1.9 6.5 14.6 1.5 1.8 C194 Cl 3,5-diF 10.6 6.3
7.2 6.2 1.3 1.3 C195 Cl 3-CN 38.7 29.4 22.8 10.1 3.7 5.1 C196 Br
3-CN 3.8 3.4 13.6 18.8 2.3 2.9 C197 Br 4-Me 17.6 14.8 16.1 13.7 2.6
2.5 C198 Br 4-F 1.0 0.8 8.8 5.3 1.2 1.1 C199 Br 4-MeO 19.3 16.1
25.9 23.2 2.2 2.9 C200 Br 4-t-Bu 11.0 8.1 5.8 10.4 1.6 1.9 C201 Cl
H 6.1 5.3 8.8 8.2 1.8 2.4 C202 Cl 4-Me 15.7 14.1 8.3 8.9 2.6 2.3
C203 Cl 4-MeO 9.5 15.5 20.2 22.8 2.1 2.7 C204 Cl 4-Br 5.0 5.8 28.9
27.9 1.5 1.8 C205 Cl 4-t-Bu 6.9 6.0 9.7 8.8 1.5 1.4 C206 MeO H 2.1
1.8 1.1 1.1 1.2 1.4 C207 MeO 4-MeO 2.5 2.8 1.2 0.7 1.5 1.4 C208 MeO
4-Br 0.8 0.9 1.0 1.1 1.1 1.0 C209 MeO 4-t-Bu 3.1 4.2 0.9 1.1 1.3
1.1 C210 NO2 H 22.8 14.3 15.0 9.8 2.4 2.1 C211 NO2 4-Me 5.8 17.7
18.1 18.9 3.3 4.0
TABLE-US-00008 TABLE 7 Firefly, cmpd/DMSO Renilla, cmpd/DMSO
Firefly_pGL, cmpd/DMSO ID NUMBER Q R1 repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C137 NO2 4-F 22.6 23.4 17.6 15.7 2.7 2.9
C138 NO2 4-Br 10.0 16.6 18.3 18.7 2.9 2.8 C139 NO2 4-t-Bu 6.6 6.4
7.0 6.7 1.4 1.8 C140 Cl 4-i-Pr 12.8 12.0 12.4 11.8 2.4 2.6 C141 MeO
4-i-Pr 1.6 1.6 1.2 1.3 1.4 1.1 C142 Br 3,4-diMe 1.2 1.7 1.4 9.8 1.1
1.6 C143 Cl 3,4-diMe 15.2 14.5 19.0 17.7 2.6 2.4 C144 MeO 3,4-diMe
0.9 0.8 3.4 1.1 1.0 0.9 C145 NO2 3,4-diMe 11.2 24.4 20.5 20.5 3.3
3.5 C146 Br 4-Cl 1.5 0.9 0.6 3.2 0.8 1.2 C147 Cl 4-Cl 2.6 2.9 1.8
5.0 1.5 1.4 C148 MeO 4-Cl 1.0 0.9 0.6 1.3 1.1 1.0 C149 NO2 4-Cl 7.8
15.3 15.3 19.7 2.7 2.3 C150 Br 3,4-diCl 8.5 7.9 12.2 11.5 1.7 1.7
C151 Cl 3,4-diCl 3.6 5.5 15.1 15.3 1.1 1.3 C152 OMe 3,4-di-Cl 4.5
3.1 4.4 4.2 1.3 1.1 C153 NO2 3,4-diCl 6.5 9.0 13.6 16.2 1.3 1.6
C154 Br 3,4-diMeO 35.4 35.4 25.6 19.5 4.0 3.8 C155 Cl 3,4-diMeO
37.3 33.3 19.3 15.4 3.5 3.0 C156 MeO 3,4-diMeO 1.0 1.0 0.9 1.0 1.0
1.0 C157 NO2 3,4-diMeO 24.9 20.4 6.9 6.0 2.3 2.1 C158 MeO 3-Cl-4-F
1.5 1.2 2.7 2.4 1.1 1.2
TABLE-US-00009 TABLE 8 Firefly, Renilla, Firefly_pGL, cmpd/DMSO
cmpd/DMSO cmpd/DMSO ID NUMBER Q R1 repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C212 F H 1.7 1.9 1.7 1.7 1.0 1.4 C213 F
4-F 14.0 13.6 9.0 8.1 1.2 1.7 C214 F 4-MeO 1.9 1.9 1.7 1.7 1.0 1.4
C215 F 4-Br 11.6 13.8 14.8 15.1 2.1 2.2 C216 F 4-t-Bu 1.3 1.0 1.3
1.0 1.3 1.2 C217 F 3-CH3-4-F 1.8 1.4 1.8 1.5 0.9 1.4 C218 F 4-Cl
18.2 19.1 11.9 10.2 1.9 2.0 C219 Br 2-F 1.3 2.5 8.7 23.5 2.0 2.3
C220 Cl 2-F 10.4 6.5 8.1 6.7 1.3 1.5 C221 MeO 2-F 1.3 0.9 1.3 1.3
1.4 1.7 C222 MeO 3-Cl 4.5 3.7 2.3 1.8 1.0 1.3 C223 Br 3-CH3-4-F 4.5
5.1 3.7 5.9 1.9 1.4 C224 Cl 3-CH3-4-F 6.6 11.4 18.2 26.9 2.8 3.1
C225 MeO 3-CH3-4-F 1.5 3.1 1.2 1.4 1.3 1.5
[0180] In further embodiments, the present technology is directed
to compounds of Formula (IV):
##STR00019##
wherein any of Q and R1 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0181] Various compounds of Formula (IV) were tested with different
moieties as Q and R1. These included the following:
##STR00020##
[0182] The results are shown below in Table 9:
TABLE-US-00010 TABLE 9 Firefly_pGL, Firefly, cmpd/DMSO Renilla,
cmpd/DMSO cmpd/DMSO IDNUMBER Q R1 repeat 1 repeat 2 repeat 1 repeat
2 repeat 1 repeat 2 C226 OMe 3-OMe 1.1 1.0 0.5 1.1 1.2 1.3 C227 OMe
3,5-di-Me 1.0 1.3 1.1 1.1 1.0 1.1 C228 OMe 3-Cl-4-Me 1.0 1.3 2.2
2.1 1.1 1.0 C229 OMe 3,5-di-F 2.9 1.8 1.5 1.3 1.2 0.8 C230 OEt
3,5-di-F 12.5 8.3 4.5 3.8 1.8 1.5 C231 OMe 4-Me 0.7 0.9 1.3 1.1 1.1
1.0 C232 OMe 4-OMe 1.0 0.9 1.0 1.0 1.0 0.9 C233 OMe 4-Br 2.8 2.1
1.1 1.3 1.3 1.2 C234 OMe 4-t-Bu 2.2 1.8 4.3 2.7 1.0 1.1 C235 OMe H
1.0 1.2 1.0 1.1 1.0 0.9 C236 OEt CH3 4.4 4.9 3.1 3.2 1.6 1.6 C237
OEt 4-F 3.5 2.9 1.5 1.3 1.3 1.3 C238 OEt 4-OMe 1.3 1.4 1.5 1.0 1.3
1.2 C239 OEt 4-Br 1.4 2.2 8.4 17.2 1.2 1.1 C240 OEt 4-t-Bu 1.5 0.9
1.5 1.5 0.9 1.1 C241 OMe 4-i-Pr 2.1 2.7 1.5 1.9 1.2 1.8 C242 OMe
3,4-di-Me 1.1 1.1 0.9 1.1 1.1 1.1 C243 OEt 3,4-di-Me 2.0 3.3 2.3
2.3 1.4 1.9 C244 OEt 4-Cl 9.7 14.1 19.8 18.4 1.7 1.9 C245 OMe
3,4-di-Cl 1.9 1.3 1.7 1.8 1.1 1.3 C246 OEt 3,4-di-Cl 7.4 6.5 7.6
8.4 1.6 1.9 C247 OMe 3,4-di-OMe 2.2 1.6 1.7 1.5 1.3 1.1 C248 OEt
3,4-di-OMe 8.6 4.3 2.8 1.9 1.4 1.4 C249 OMe 2,5-di-OMe 1.4 1.5 1.0
1.1 1.1 1.3 C250 OMe 3-Cl-4-F 1.2 1.8 1.7 1.9 1.1 1.1 C251 OEt
3-Cl-4-F 2.1 1.9 2.4 2.0 1.0 0.9 C252 OEt 4-OEt 3.2 2.4 1.4 1.6 1.4
1.3 C253 OMe 2-F 2.5 2.7 1.5 1.2 1.3 1.2 C254 OEt 2-F 7.6 5.1 2.7
2.5 1.5 1.4 C255 OMe 3-Cl 1.5 1.5 1.2 1.1 1.3 1.0 C256 OEt 3-Cl
11.5 7.9 5.1 3.5 2.0 2.4 C257 OMe 3-Me-4-F 2.1 1.6 1.2 1.1 0.8 1.0
C258 OEt 3-Me-4-F 1.5 1.8 1.3 1.5 1.3 1.5
[0183] In further embodiments, the present technology is directed
to compounds of Formula (V):
##STR00021##
wherein any of A and R1 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0184] Various compounds of Formula (V) were tested with different
moieties as A and R1, and the results are shown below in Table
10:
TABLE-US-00011 TABLE 10 Firefly, cmpd/DMSO Renilla, cmpd/DMSO
Firefly_pGL, cmpd/DMSO ID NUMBER A R repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C259 OMe 3-OMe 1.3 1.9 1.2 1.2 0.9 1.1
C260 OMe 3,5-di-Me 1.0 1.0 1.4 1.3 1.0 1.1 C261 OMe 3-Cl-4-CH3 2.0
1.4 1.7 1.5 0.9 1.2 C262 OMe 3,5-di-F 1.8 2.2 1.4 1.2 1.4 1.3 C263
OMe 3-CN 1.8 1.7 1.4 1.3 1.2 1.0 C264 OMe H 1.1 0.8 1.2 1.2 0.9 1.0
C265 OMe CH3 1.0 1.3 1.4 1.3 1.2 1.1 C266 OMe OMe 0.9 1.2 1.4 1.3
1.0 1.0 C267 OMe 4-Br 1.4 1.7 2.5 1.2 1.2 1.6 C268 OMe 4-t-Bu 2.8
3.1 1.3 2.1 0.9 1.2 C269 OMe 4-i-Pr 3.0 3.0 1.5 1.4 1.2 1.0 C270
OMe 3,4-di-Me 3.2 3.1 2.2 1.8 1.3 1.2 C271 OMe 4-Cl 2.2 1.2 2.3 2.1
1.6 1.6 C272 OMe 3,4-di-Cl 4.5 3.1 4.4 4.2 1.3 1.1 C273 OH H 2.0
1.5 1.6 2.0 1.0 1.2 C274 OMe 3,4-di-OMe 3.3 3.5 1.2 1.5 1.6 1.2
C275 OMe 3-Cl-4-F 2.6 2.5 2.3 1.9 1.2 1.3 C276 OH 3-Cl-4-F 1.2 1.3
2.3 1.9 0.9 1.0 C277 NEt2 4-Me 3.4 2.9 1.5 0.9 1.0 1.3 C278 NEt2
4-Br 2.4 3.0 1.5 1.7 1.0 1.2 C279 NEt2 4-Cl 3.9 2.1 1.4 1.6 0.9 0.9
C280 OMe 2-F 0.6 0.6 1.4 1.4 0.9 1.2 C281 OMe 3-Cl 3.0 4.4 1.8 1.9
1.4 1.3 C282 OMe 3-CH3-4-F 0.7 1.1 1.5 1.7 1.4 2.0
[0185] In further embodiments, the present technology is directed
to compounds of Formula (VI):
##STR00022##
wherein any of Q1, Q2 and R1 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile.
[0186] Various compounds of Formula (VI) were tested with different
moieties as Q1 and Q2. These included the following:
##STR00023## ##STR00024##
[0187] The results are shown below in Table 11:
TABLE-US-00012 TABLE 11 Firefly_pGL, ID Firefly, cmpd/DMSO Renilla,
cmpd/DMSO cmpd/DMSO NUMBER Q1 Q2 R1 repeat 1 repeat 2 repeat 1
repeat 2 repeat 1 repeat 2 C283 Br Br OMe 1.0 1.3 0.1 0.2 1.0 0.9
C284 Br Br 4-i-Pr 2.9 2.5 0.4 0.3 0.9 1.0 C285 Br Br 3,4-di-Me 1.0
1.0 0.1 0.6 1.0 1.2 C286 Br Br 4-C1 2.3 2.4 0.3 0.9 0.8 1.1 C287 Br
Br 3,4-di-C1 3.0 2.6 2.9 2.5 1.1 1.5 C288 F F H 6.8 3.3 4.4 3.3 1.6
1.4 C289 F F OMe 31.3 15.6 13.5 10.0 1.7 1.3 C290 F F 4-Br 13.8
14.6 21.6 17.1 2.0 1.8 C291 F F 4-t-Bu 0.9 0,8 0.1 0.2 0.7 0.7 C292
F F 3-Me-4-F 18.3 14.8 15.6 13.4. 1.7 1.7 C293 F F 4-C1 8.5 11.9
27.8 21.2 2.1 1.7 C294 Br OMe H 16.5 16.5 36.0 27.5 2.4 2.9 C295 Br
OMe 4-F 4.0 4.6 4.3 3.6 1.8 1.6 C296 Br OMe 4-OMe 6.0 7.8 14.9 14.1
2.2 2.0 C297 Br OMe 4-t-Bu 1.5 2.6 2.0 6.9 1.4 1.3 C298 Br OMe
3-Me-4-F 2.2 3,2 1.9 1.9 1.3 1.4
[0188] In certain embodiments, the technology is directed to
compounds of Formulas (VII) or (VIII):
##STR00025##
wherein any of X and R is: C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, and amide, an amine, an
ether, a thiol or a nitrile; and wherein n is an integer 1, 2, 3,
4, 5 or 6. In certain embodiments, the X--R moiety represents a
benzene ring fused to the n-membered ring containing the N
substitution, to create a bicyclic functional group; see, e.g.,
Compounds F41 through F47 below.
[0189] Exemplary compounds in accordance with these Formulas
include the following:
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035##
[0190] Further exemplary compounds tested in accordance with the
various Formulas disclosed herein include the following:
##STR00036##
[0191] Tables 12 and 13 show a summary of selected compounds for
CRC analysis.
TABLE-US-00013 TABLE 12 Firefly/Renilla Firefly, Firefly, Renilla
Firefly_pGL, NR2F6 NR2F6 ID cmpd/DMSO cmpd/DMSO cmpd/DMSO stable
(clone stable (clone NUMBER repeat 1 repeat 2 repeat 1 repeat 2
repeat 1 repeat 2 F4) F4)/pGL4 C289 31.3 15.6 13.5 10.0 1.7 1.3 2.0
15.3 C155 37.3 33.3 19.3 15.4 3.5 3.0 2.0 10.9 C157 24.9 20.4 6.9
6.0 2.3 2.1 3.5 10.2 C299 21.5 15.2 5.5 3.8 1.8 1.8 4.0 10.1 C218
18.2 19.1 11.9 10.2 1.9 2.0 1.7 9.5 C177 17.4 8.2 6.7 4.6 1.7 1.3
2.3 8.5 C164 29.3 19.6 13.8 8.9 3.1 2.8 2.1 8.2 C136 22.1 25.8 12.5
14.9 2.9 3.0 1.7 8.1 C134 38.1 31.9 21.0 18.9 3.7 5.4 1.8 7.7 C195
38.7 29.4 22.8 10 1 3.7 5.1 2.1 7.7 C160 22.9 20.2 9.5 8.2 3.2 2.7
2.4 7.2 C11 24.6 21.3 13.9 12.3 3.3 3.3 1.8 7.0 C12 20.0 16.7 4.5
9.4 2.6 2.9 2.6 6.6 C230 12.5 8.3 4.5 3.8 1.8 1.5 2.5 6.5 C202 15.7
14.1 8.3 8.9 2.6 2.3 1.7 6.1 C102 13.0 15.3 5.8 6.1 2.3 1.5 2.4 5.9
C108 7.9 7.3 3.0 2.8 1.3 1.5 2.6 5.5 C248 8.6 4.3 2.8 1.9 1.4 1.4
2.8 4.6 C256 11.5 7.9 5.1 3.5 2.0 2.4 2.3 4.5 C254 7.6 5.1 2.7 2.5
1.5 1.4 2.4 4.4 C13 14.0 12.9 6.4 8.2 3.2 3.2 1.8 4.2 C112 11.7
10.1 5.6 5.1 2.5 2.8 2.0 4.1 C110 7.6 7.2 2.3 2.1 1.7 2.0 3.3 3.9
F312-0003 4.6 4.2 1.1 1.1 1.1 1.3 4.0 3.7 C101 4.5 4.7 1.8 2.0 1.2
1.3 2.4 3.6
TABLE-US-00014 TABLE 13 Firefly/Renilla Firefly, Firefly, Renilla
Firefly_pGL, NR2F6 NR2F6 ID cmpd/DMSO cmpd/DMSO cmpd/DMSO stable
(clone stable (clone NUMBER repeat 1 repeat 2 repeat 1 repeat 2
repeat 1 repeat 2 F4) F4)/pGL4 C222 4.5 3.7 2.3 1.8 1.0 1.3 2.0 3.6
C105 5.7 4.8 1.7 2.3 1.5 1.8 2.6 3.1 C209 3.1 4.2 0.9 1.1 1.3 1.1
3.6 3.1 C109 9.2 10.3 3.4 3.3 3.5 3.4 2.9 2.9 C213 14.0 13.6 9.0
8.1 1.2 1.7 1.6 9.6 C300 9.8 7.4 6.2 4.7 1.7 2.3 1.6 4.3 C154 35.4
35.4 25.6 19.5 4.0 3.8 1.6 9.1 C14 22.3 20.0 12.1 15.2 2.9 2.6 1.5
7.6 C210 22.8 14.3 15.0 9.8 2.4 2.1 1.5 8.2 C107 11.0 11.0 8.7 6.5
1.7 2.1 1.4 5.8 C137 22.6 23.4 17.6 15.7 2.7 2.9 1.4 8.4 C161 35.4
39.0 28.6 27.7 4.0 5.1 1.3 8.1 C194 10.6 6.3 7.2 6.2 1.3 1.3 1.3
6.6 C188 9.4 8.7 7.6 6.9 1.8 1.8 1.2 5.0 C182 16.2 17.3 15.1 13.2
2.0 2.1 1.2 8.2 C200 11.0 8.1 5.8 10.4 1.6 1.9 1.2 5.4 C220 10.4
6.5 8.1 6.7 1.3 1.5 1.1 6.2 C118 13.5 17.1 11.8 14.8 2.4 2.7 1.1
5.9 C292 18.3 14.8 15.6 13.4 1.7 1.7 1.1 9.7 C180 34.0 34.5 30.9
30.0 2.3 2.7 1.1 13.1 C175 13.2 11.9 10.5 12.0 2.5 1.9 1.1 5.7 C111
29.8 35.9 29.4 30.8 3.7 3.9 1.1 8.7 C197 17.6 14.8 16.1 13.7 2.6
2.5 1.1 6.3 C127 36.7 56.2 43.1 43.5 8.3 8.0 1.1 5.7 C126 45.1 44.8
43.5 42.2 4.8 4.3 1.0 9.9
TABLE-US-00015 TABLE 14 Firefly Renilla Compound F4, cmpd/DMSO
(mean) pGL4, cmpd/DMSO (mean) F4, cmpd/DMSO (mean) ID 40 uM 10 uM 2
uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 17 1.3 1.2 1.9 1.3 1.6 1.7 1.0
1.0 0.8 19 0.5 1.0 1.7 0.9 1.3 1.9 0.6 1.0 1.1 22 0.6 1.4 1.3 1.5
1.0 1.6 1.0 1 1 0.8 C1 0.4 0.2 0.5 0.6 0.7 0.7 0.2 0.2 2.1 C3 0.9
1.1 1.5 1.0 1.3 1.1 0.9 0.9 1.0 C4 0.7 0.8 1.1 1.3 0.9 1.0 0.5 0.5
0.6 C5 0.4 0.8 0.9 1.0 1.2 1.3 1.1 0.5 0.7 C6 0.4 0.6 1.4 1.0 1.3
1.3 0.5 0.6 0.7 C301 1.0 1.0 1.2 1.1 1.6 2.4 1.0 0.7 1.1 C302 0.9
0.9 1.2 0.8 1.4 1.8 1.0 1.1 0.7 C303 0.9 0.9 1.2 1.1 1.1 1.1 1.0
0.8 1.0 C7 0.4 0.5 0.6 0.8 0.8 0.9 0.4 0.3 1.0 C11 0.4 1.6 1.7 0.8
1.7 1.1 0.2 1.2 1.1 E12 0.5 0.9 0.7 1.0 0.8 0.9 1.0 0.7 0.7 E53 1.4
1.1 1.0 1.6 1.4 1.5 1.1 0.8 0.9 L1 0.5 0.2 0.9 0.8 0.4 0.8 0.7 0.8
0.8 Z54 0.6 1.6 0.8 0.8 1.9 1.1 0.2 0.1 0.7 Z55 2.0 0.8 1.1 2.3 0.7
0.8 0.1 0.3 0.8 Z56 0.5 2.1 1.2 1.2 8.9 1.2 0.2 0.3 0.9 Z74 0.7 0.2
0.7 0.7 0.6 0.7 0.3 0.2 0.8 Z79 0.1 0.5 0.9 0.8 0.5 0.8 0.2 0.6 0.9
Z81 0.4 0.7 2.4 0.7 0.9 1.4 0.3 0.3 1.1 Z83 0.7 0.6 1.2 0.5 0.9 1.0
0.3 0.8 1.0 Z90 0.4 2.2 0.7 0.7 2.4 0.7 0.1 0.2 0.9 Z91 1.3 1.1 1.0
1.1 1.0 1.0 0.8 1.1 1.1
[0192] Additional compounds were tested, including the
following:
##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041##
[0193] Compounds Z54, Z55 and Z56 were found to have particularly
good activity:
##STR00042##
[0194] Table 15 shows screening results from another set of
compounds.
TABLE-US-00016 TABLE 15 Firefly Renilla F4, cmpd/ ERalpha
transient, F4, cmpd/ ERalpha transient, Compound DMSO (mean)
cmpd/DMSO (mean) DMSO (mean) cmpd/DMSO (mean) ID 40 uM 10 uM 2 uM
40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM Z1 0.8 1.8 1.9
0.9 2.2 2.4 0.8 1.6 1.3 0.7 1.0 1.1 Z2 0.6 1.9 1.4 0.7 1.1 1.6 1.2
1.8 1.2 1.0 1.1 1.2 Z3 1.6 1.1 1.5 1.7 2.6 2.2 0.9 1.4 1.5 0.9 1.0
1.1 Z4 0.8 1.2 1.5 0.8 1.4 1.5 1.4 1.6 1.1 0.8 1.1 1.2 Z5 1.2 0.9
1.5 2.4 2.5 2.4 1.2 0.9 1.0 1.0 1.0 1.0 Z6 1.1 1.4 1.4 1.1 2.2 2.0
0.9 1.1 1.1 0.9 1.1 1.1 Z7 1.0 1.2 0.9 1.5 2.0 1.3 1.1 1.1 1.3 1.0
1.1 1.1 Z8 0.7 4.0 2.6 0.4 2.4 2.2 0.2 9.1 2.7 0.1 1.4 1.4 Z9 1.2
6.7 2.5 0.5 2.7 2.2 0.3 4.9 2.1 0.2 0.8 1.3 Z10 0.7 1.6 5.8 0.5 1.5
4.2 0.3 3.3 3.4 0.2 0.8 1.3 Z11 0.5 1.2 3.5 0.6 1.6 2.1 0.3 2.6 2.9
0.2 0.8 1.4 Z12 0.4 1.8 3.9 0.4 1.3 2.4 0.4 2.7 2.7 0.2 0.7 1.3 Z13
1.6 0.9 1.0 1.1 1.7 1.2 0.4 0.7 0.8 0.4 0.6 0.8 Z14 1.2 0.8 1.2 1.0
1.2 1.0 1.0 0.9 0.8 1.0 1.1 1.1 Z15 0.9 0.5 1.0 1.1 1.1 0.9 1.0 1.1
0.8 0.9 1.0 1.0 Z58 0.8 4.5 0.9 0.8 6.5 1.5 0.8 1.2 1.3 0.7 1.1 1.2
Z17 0.6 6.5 1.5 2.9 9.5 1.7 0.6 1.1 1.2 0.6 1.1 1.3 Z61 0.8 2.5 1.7
0.7 9.1 3.0 0.6 0.5 1.3 0.7 0.8 1.4 Z19 0.7 6.3 1.3 0.7 12.7 1.9
0.7 0.9 1.1 0.6 1.0 1.5 Z67 0.7 0.5 1.3 0.5 1.4 1.4 0.6 1.0 1.2 0.6
1.0 1.1 Z68 1.2 1.1 1.5 0.7 1.5 1.1 0.6 1.0 0.9 0.6 0.9 1.1 Z70 1.0
1.0 1.1 1.5 1.1 0.9 1.3 1.3 0.8 1.2 1.0 1.1 Z71 0.8 1.0 0.8 0.4 1.0
1.4 0.4 0.9 1.1 0.5 1.0 1.0 Z75 0.8 1.2 0.9 0.9 1.9 1.1 0.8 1.0 1.1
0.7 0.9 1.0 Z76 0.8 1.8 0.8 0.7 1.9 1.3 0.5 0.8 1.0 0.4 0.8 1.1 Z78
1.6 1.5 1.4 1.1 2.4 1.5 0.8 1.0 1.1 0.6 0.9 1.0
[0195] Compounds Z8-Z12, Z17and Z19 were found to have particularly
good activity.
##STR00043## ##STR00044##
[0196] Dog's PMBC ELISA and cytotoxicity experiments were performed
on Compound Z92, which also showed good activity. Results are shown
in FIGS. 19A and 19B.
[0197] Another useful compound is Compound Z95:
##STR00045##
[0198] Results of testing done on compound Z95 are shown in Table
16.
TABLE-US-00017 TABLE 16 NR2F6_full (stable, clone F4), cmpd/DMSO
NR2F6_full (transient), cmpd/DMSO Firefly Renilla Firefy Renilla ID
40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM
Z95 3.7 2.2 1.2 0.5 0.5 1.1 2.3 1.3 1.2 0.2 0.4 1.1
[0199] FIGS. 20A and 20B show further results of cytokines release
by hPBMC and cytotox on Compound Z95.
[0200] Table 17 shows screening results from another set of
compounds.
TABLE-US-00018 TABLE 17 Firefly Renilla F4, cmpd/ ERalpha
transient, F4, cmpd/ ERalpha transient, Compound DMSO (mean)
cmpd/DMSO (mean) DMSO (mean) cmpd/DMSO (mean) ID 40 uM 10 uM 2 uM
40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM Z27 0.7 1.3 1.3
0.5 2.1 1.3 0.9 1.2 1.2 0.8 1.2 1.2 Z28 1.3 0.9 1.0 1.3 1.1 1.8 1.2
1.2 1.0 1.0 1.3 1.1 Z29 1.1 1.1 1.3 6.7 1.2 1.0 0.5 0.9 0.8 0.7 1.2
0.8 Z30 1.2 1.1 1.4 1.4 1.1 1.4 0.7 1.1 1.1 1.6 1.1 1.1 Z31 1.1 0.9
0.8 1.6 1.6 0.9 1.0 1.0 1.1 1.0 1.0 1.0 Z32 1.5 0.9 1.8 1.5 0.8 1.8
1.3 1.0 1.0 0.8 1.1 1.0 Z33 6.6 2.0 1.7 8.8 1.8 2.1 0.6 1.0 1.1 0.9
1.5 1.0 Z34 0.7 3.8 1.6 0.9 10.3 2.0 0.6 0.8 1.2 0.7 1.0 1.5 Z35
1.2 1.5 1.9 7.2 2.2 2.6 0.7 2.0 1.1 0.9 1.3 1.1 Z36 0.7 1.8 0.7 5.8
1.6 1.2 0.6 1.8 1.0 0.8 1.6 1.2 Z37 0.7 1.4 1.0 1.2 1.5 1.1 0.5 0.9
1.0 0.7 1.7 1.0 Z38 0.6 0.8 0.5 0.5 0.5 0.9 0.4 0.6 0.7 0.6 0.6 0.9
Z39 0.7 1.0 0.8 0.8 0.5 1.2 0.5 0.5 0.7 0.6 0.6 1.1 Z40 0.8 0.7 0.8
1.0 0.3 1.0 0.5 0.6 0.7 0.6 0.7 1.0 Z41 1.7 1.1 1.0 1.6 1.4 0.9 0.6
0.6 0.7 0.8 1.1 1.0 Z42 0.6 0.9 0.9 1.1 1.8 1.5 0.7 0.9 1.2 0.7 0.9
1.1 Z43 0.8 0.8 1.3 1.6 0.9 1.4 0.7 0.9 1.0 0.9 1.0 1.0 Z44 0.4 0.8
0.8 1.0 1.2 1.2 0.3 1.5 1.7 0.5 0.9 1.1 Z45 0.5 1.2 0.8 0.7 0.6 0.8
1.1 1.2 1.2 1.0 0.8 1.1 Z46 0.6 0.8 0.6 0.6 0.8 0.9 0.6 0.8 0.9 1.0
1.0 0.9 Z47 2.0 1.3 0.9 1.7 1.0 0.9 1.7 1.2 1.1 1.2 1.4 1.2 Z48 0.7
0.5 0.9 0.5 0.3 1.5 0.6 0.6 0.8 0.6 0.8 1.1 Z49 0.6 0.7 0.8 0.4 0.4
1.0 0.8 0.7 1.0 0.8 0.7 1.0 Z50 0.6 0.9 0.8 0.7 0.7 1.3 0.8 2.0 1.6
0.6 0.8 1.0 Z51 0.7 0.6 0.6 0.4 0.8 1.3 0.3 0.7 0.8 0.3 0.9 1.1 Z52
0.7 0.7 0.6 0.7 0.4 0.7 0.6 0.5 0.8 0.7 0.8 1.1 Z53 0.5 0.4 0.8 0.4
0.3 1.0 0.6 0.7 0.7 0.5 0.6 1.0
[0201] Compounds Z33 and Z34 were found to have particularly good
activity:
##STR00046##
[0202] Other useful compounds are Compound D28 and Compound F1:
##STR00047##
[0203] Compound D28 was tested in a cytokine release
experiment--parent compound, dog's and human PMBC. Results are
shown in FIGS. 21A-21D.
[0204] Compound Z17, previously mentioned above, was tested in a
cytokine release experiment. Results are show in in FIGS. 22A and
22B.
[0205] Compound Z33, previously mentioned above, was tested in a
cytokine release experiment. Results are shown in FIGS. 23A and
23B.
[0206] Another compound found to be useful is Compound E56:
##STR00048##
[0207] FIG. 24 shows the results of testing done on Compound
E56.
[0208] Additional compounds found to be useful are Compounds Z96
and Z97:
##STR00049##
[0209] Results of testing done on compounds Z96 and Z97 are shown
in Tables 18 and 19, respectively.
TABLE-US-00019 TABLE 18 NR2F6_full (stable, clone F4), cmpd/DMSO
NR2F6_full (transient), cmpd/DMSO Firefly Renilla Firefy Renilla 40
uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM
Z96 2.6 2.6 2.9 1.2 1,2 1.3 2.4 2.3 2.4 0.9 1.0 1.0
TABLE-US-00020 TABLE 19 NR2F6_full (stable, clone F4), cmpd/DMSO
NR2F6_full (transient), cmpd/DMSO Firefly Renilla Firefy Renilla 40
uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM
Z97 1.5 2.2 2.3 1.2 1.1 1.1 2.3 2.8 2.4 1.1 1.1 1.0
[0210] FIGS. 25A and 25B show further results of testing done on
Compound Z96. FIGS. 26A and 26B show further results of testing
done on Compound Z97.
[0211] Additional compounds found to be useful are Compounds Z93
and Z94:
##STR00050##
[0212] The results of dogs PBMC cytotox for Compounds Z93 and Z94
are included in FIG. 19B, which also shows results for the
following Compounds: Z92, E54, E55 and E53.
[0213] Additional compounds tested included the following:
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
##STR00056##
[0214] Table 20 shows screening results from another set of
compounds.
TABLE-US-00021 TABLE 20 Firefly Renilla F4, cmpd/ ERalpha
transient, F4, cmpd/ ERalpha transient, Compound DMSO (mean)
cmpd/DMSO (mean) DMSO (mean) cmpd/DMSO (mean) ID 40 uM 10 uM 2 uM
40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 26 1.3 1.1 1.0
1.1 1.3 1.0 0.7 0.7 1.0 1.0 1.0 0.9 27 1.0 1.0 1.0 1.2 1.2 1.2 0.8
0.9 0.9 1.2 1.1 1.1 28 0.6 0.9 1.2 0.6 0.5 1.0 0.6 0.8 1.1 0.7 0.8
1.2 29 1.5 1.4 0.9 2.2 1.7 1.2 0.8 0.9 0.8 1.1 1.0 1.1 30 1.8 1.6
1.5 1.3 1.5 1.1 3.4 2.3 1.2 1.0 1.2 1.2 31 0.9 1.0 0.8 1.0 1.2 0.8
1.0 1.0 1.0 1.1 1.1 1.0 32 0.8 1.3 0.8 0.7 1.2 1.2 1.0 1.4 1.2 1.1
1.3 1.2 33 1.1 1.0 0.8 1.4 1.3 1.3 1.4 1.3 1.2 1.3 1.1 1.2 34 0.9
0.8 0.9 1.2 1.2 1.1 0.9 1.1 0.9 1.1 1.1 1.0 35 0.8 1.0 1.1 1.3 0.9
1.0 0.9 0.9 1.1 1.1 1.0 1.0 36 0.9 0.6 1.3 0.4 0.8 1.1 0.5 0.8 1.2
0.3 1.1 1.2 37 1.3 1.1 1.3 1.2 1.4 1.1 1.3 1.2 0.9 1.1 1.1 1.1 38
0.7 0.8 1.2 0.5 0.9 0.8 1.3 1.1 1.1 1.1 1.1 1.2 39 0.9 0.9 1.1 0.9
0.6 0.9 1.0 1.4 1.2 1.2 1.2 1.1 40 0.8 1.0 0.9 0.7 1.0 1.0 1.0 1.3
1.1 1.1 1.2 1.2 41 0.9 0.9 0.8 1.4 1.5 1.5 1.1 1.1 1.1 1.1 1.2 1.2
42 0.9 0.8 1.6 1.0 1.0 2.3 1.1 0.8 0.7 1.0 1.0 0.9 43 0.8 0.9 1.0
1.2 1.4 1.4 1.1 1.1 1.0 1.0 1.0 1.1 44 0.7 1.5 1.3 0.5 1.3 1.5 0.3
1.1 1.1 0.3 1.2 1.3 45 0.7 1.2 3.2 0.6 1.6 1.8 0.2 0.8 1.6 0.2 0.8
1.6
[0215] After several rounds of testing, the following compounds
were found to be particularly optimal.
##STR00057## ##STR00058##
[0216] Table 21 shows results of testing on Compound D28.
TABLE-US-00022 TABLE 21 Firefly, DMSO Renilla, Firefly, cmpd/DMSO
Firefly, cmpd mean mean cmpd/DMSO Renilla, DMSO repeat repeat
repeat repeat plate plate repeat repeat mean mean 1 2 mean 1 2 1 2
1 2 plate1 plate2 14.1 11.7 12.8 784 644 56 55 0.7 0.5 3237
2930
[0217] FIGS. 27A and 27B show NR2F6 and LBD transient transfection,
respectively, for Compound D28. Higher concentrations were excluded
due to lower signal (tox effect). FIGS. 27C and 27D show NR2F6 and
LBD transient transfection at different concentrations for
different compounds. 9 compounds were tested on LBD transfected
cells (40, 10, 2 and 0.5 .mu.M, 4 replicates). FIGS. 27E and 27F
show toxicity of Compound D28. 9 compounds were tested for
cytotoxicity on LBD transfected cells (40, 10, 2 and 0.5 .mu.M, 4
replicates). Tox effect was found to cause lower signal compared to
DMSO. Cytotoxicity normalized to DMSO is shown in FIG. 27F (0%
cytotoxicity corresponds to DMSO signal, 100%--zero signal).
[0218] FIGS. 28A-D show the results of a cytokine release
experiment for dog and human PBMC. All compounds were tested at 5,
10, 25 and 50 uM in duplicates.
[0219] Dog PBMC (1.times.106 cells/mL) were activated by 10 ng/mL
PMA+500 ng/mL ionomycin. Data were normalized to controls with
(100%)/without (0%) PMA+ionomycin activation.
[0220] In further embodiments, the present technology is directed
to compounds of Formula (IX):
##STR00059##
wherein R.sub.A is C, H, N, O, S, a halogen, an alkyl group, a
substituted alkyl group, a cyclic alkyl group, an aryl group, a
substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile. For example, exemplary but
non-limiting compounds are shown below:
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065##
[0221] In further embodiments, the present technology is directed
to compounds of Formula (X):
##STR00066##
wherein R.sub.B is C, H, N, O, S, a halogen, an alkyl group, a
substituted alkyl group, a cyclic alkyl group, an aryl group, a
substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile. For example, exemplary but
non-limiting compounds are shown below:
##STR00067## ##STR00068## ##STR00069## ##STR00070##
[0222] In further embodiments, the present technology is directed
to compounds of Formula (XI):
##STR00071##
wherein any of R.sub.1-R.sub.5 are C, H, N, O, S, a halogen, an
alkyl group, a substituted alkyl group, a cyclic alkyl group, an
aryl group, a substituted aryl group, a heterocyclic group, an
ester, an aldehyde, a ketone, a carboxylic acid, an amide, an
amine, an ether, a thiol or a nitrile. For example, exemplary but
non-limiting compounds are shown below:
##STR00072## ##STR00073## ##STR00074##
[0223] In various other embodiments, the structure of the Compounds
found to be useful have other variations. For example, in certain
embodiments, the present technology is directed to compounds of
Formula (XII):
##STR00075##
wherein any of R.sub.1 and R.sub.8 is C, H, N, O, S, a halogen, an
alkyl group, a substituted alkyl group, a cyclic alkyl group, an
aryl group, a substituted aryl group, a heterocyclic group, an
ester, an aldehyde, a ketone, a carboxylic acid, an amide, an
amine, an ether, or a thiol. Exemplary but non-limiting compounds
are shown below:
##STR00076## ##STR00077##
[0224] FIGS. 29-32 show exemplary methods of formulating the
compounds that have been discussed herein.
[0225] FIG. 31A shows an exemplary synthesis of compounds including
the following: Compound Z17, Compound Z61, Compound Z19, Compound
Z70, Compound Z71, Compound Z67, Compound Z76, Compound Z75,
Compound Z78, Compound Z68, Compound Z27, Compound Z79, Compound
Z64, Compound Z69, Compound Z74, Compound Z154, Compound Z80,
Compound Z155, Compound Z156, Compound Z157, Compound Z158,
Compound Z159.
[0226] Another compound developed herein, and found to have
desirable activity, is Compound E21:
##STR00078##
[0227] Table 22 shows results of testing on Compound E21.
TABLE-US-00023 TABLE 22 Firefly, DMSO Renilla, Firefly, cmpd/DMSO
Firefly, cmpd mean mean cmpd/DMSO Renilla, DMSO repeat repeat
repeat repeat plate plate repeat repeat mean mean 1 2 mean 1 2 1 2
1 2 plate1 plate2 9.5 5.6 7.3 526 306 56 55 0.6 0.8 3237 2930
[0228] FIGS. 33A and 33B show NR2F6 and LBD transient transfection
for Compound E21. Higher concentrations were excluded due to lower
signal (tox) effect.
[0229] FIGS. 34A and 34B show NR2F6 LBD transient transfection for
Compound E21. 9 compounds were tested on LBD transfected cells (40,
10, 2 and 0.5 pM, 4 replicates). FIGS. 34C and 34D show NR2F6 and
LBD transient transfection at different concentrations for
different compounds. 9 compounds were tested on LBD transfected
cells (40, 10, 2 and 0.5 pM, 4 replicates). Tox effect was found to
cause lower signal compared to DMSO. Cytotoxicity normalized to
DMSO is shown in FIG. 34D (0% cytotoxicity corresponds to DMSO
signal, 100%--zero signal). All compounds were tested at 5, 10, 25
and 50 uM in duplicates. Dog PBMC (1.times.106 cells/mL) were
activated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized
to controls with (100%)/without (0%) PMA+ionomycin activation.
[0230] As shown in FIG. 36, related compounds were generated from
Compound E21 and tested for activity.
[0231] Additional related compounds found to have desirable
activity include the following:
##STR00079## ##STR00080##
[0232] Table 23 shows the screening results of certain of the above
compounds.
TABLE-US-00024 TABLE 23 Firefly Compound ID NR2F6 full length, F4
cmpd/ Concentration, cmpd/DMSO (mean) DMSO (mean) mM 40 10 2 40 10
2 Screening Compound E4 1.0 1.0 1.1 0.8 0.9 0.9 Results Compound E8
0.9 1.2 1.2 0.7 1.0 1.0 Compound E10 1.1 1.5 1.3 0.8 1.0 1.1
Compound E15 1.1 0.8 1.7 0.8 1.1 1.3 Compound E57 0.9 1.2 1.3 1.3
1.3 1.0 Compound E24 1.1 1.1 1.0 0.7 0.9 1.0 Compound E41 0.9 1.0
1.1 0.4 0.7 0.9 Compound E52 1.4 1.3 1.0 0.7 0.9 0.7
[0233] Additional compounds synthesized and found to have desirable
activity include the following:
##STR00081## ##STR00082##
[0234] FIG. 37 shows exemplary methods of formulating the compounds
that have been discussed herein; specifically, exemplary synthesis
of compounds including the following: Compound Z160, Compound Z161,
Compound Z162, Compound Z163.
[0235] As discussed above, Compound F1 was of particular interest
herein:
##STR00083##
[0236] Table 24 shows results of testing on Compound F1.
TABLE-US-00025 TABLE 24 Firefly, DMSO Renilla, Firefly, cmpd/DMSO
Firefly, cmpd mean mean cmpd/DMSO Renilla, DMSO repeat repeat
repeat repeat plate plate repeat repeat mean mean 1 2 mean 1 2 1 2
1 2 plate 1 plate2 5.6 4.6 5.1 310 252 56 55 1.1 1.2 3237 2930
[0237] FIGS. 38A and 38B show NR2F6 and LBD transient transfection,
respectively, for Compound F1.
[0238] FIGS. 39A and 39B show NR2F6 LBD transient transfection for
Compound F1. 9 compounds were tested on LBD transfected cells (40,
10, 2 and 0.5 .mu.M, 4 replicates). FIGS. 39C and 39D show toxicity
of NR2F6 LBD transient transfection. 9 compounds were tested for
cytotoxicity on LBD transfected cells (40, 10, 2 and 0.5 .mu.M, 4
replicates). Tox effect causes lower signal compared to DMSO.
Cytotoxicity normalized to DMSO is shown in FIG. 39D (0%
cytotoxicity corresponds to DMSO signal, 100%--zero signal).
[0239] FIGS. 40A-D show the results of cytokine release experiment
for dogs and human PBMC. All compounds were tested at 5, 10, 25 and
50 uM in duplicates.
[0240] Dog PBMC (1.times.106 cells/mL) were activated by 10 ng/mL
PMA+500 ng/mL ionomycin. Data were normalized to controls with
(100%)/without (0%) PMA+ionomycin activation.
[0241] FIG. 41 shows the general SAR strategy for testing Compound
F1 and compounds related to it in structure. Formally, the active
molecule was divided into four domains (Domains A through D). Each
domain was evaluated independently to establish SAR trends.
Combinations of optimized domains evaluated additive or synergistic
effect. 4 related analogs were available.
[0242] For example, compounds were tested with varying values of
Domain A. Exemplary compounds found to be useful are listed as
follows:
##STR00084## ##STR00085##
[0243] In certain embodiments, a compound herein has Formula
(XIII):
##STR00086##
wherein n is an integer 1, 2, or 3, and R is any other moiety
mentioned in the present disclosure (e.g., C, H, N, O, S, a
halogen, an alkyl group, a substituted alkyl group, a cyclic alkyl
group, an aryl group, a substituted aryl group, a heterocyclic
group, an ester, an aldehyde, a ketone, a carboxylic acid, an
amide, an amine, an ether, a thiol or a nitrile).
[0244] Similarly, compounds were tested with varying values of
Domain C. Exemplary compounds found to be useful are listed as
follows:
##STR00087## ##STR00088##
[0245] In certain embodiments, a compound herein has Formula
(XIV):
##STR00089##
wherein X is C, H, N, O, S, a halogen, an alkyl group, a
substituted alkyl group, a cyclic alkyl group, an aryl group, a
substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol, a nitrile or any other moiety mentioned herein.
[0246] In other embodiments, the present technology is directed to
compounds comprising a boronate, and synthesis of such compounds.
For example, FIG. 42 shows an exemplary synthesis of a boronate
compound. In various embodiments, the synthesis achieved a yield of
at least about 95%, at least about 90% and at least about 85%; with
at least about 85% purity. In various embodiments, a regioisomer
was present in the yield, in amounts of about 10 to about 20%, or
about 12 to about 18%. In various embodiments, the regioisomers
could be separated.
[0247] FIG. 42 shows the results of other exemplary syntheses of
compounds comprising boronate, and the relative proportions of
resultant compounds.
[0248] Another compound found to have good activity is Compound
P1:
##STR00090##
[0249] Table 25 shows results of testing on Compound P1.
TABLE-US-00026 TABLE 25 Firefly, DMSO Renilla, Firefly, cmpd/DMSO
Firefly, cmpd mean mean cmpd/DMSO Renilla, DMSO repeat repeat
repeat repeat plate plate repeat repeat mean mean 1 2 mean 1 2 1 2
1 2 plate 1 plate2 4.0 3.0 3.5 236 166 59 56 1.0 0.9 3030 3118
[0250] FIGS. 44A and 44B show NRdF6 and LBD transient transfection
of Compound P1.
[0251] FIGS. 45A-D show NR2F6 LBD transient transfection for 9
different compounds, including Compound P1.
[0252] FIGS. 46A and 46B show the results of the cytokine release
experiment with dogs PBMC.
[0253] FIGS. 47A and 47B show NR2F6 agonist activity and NR2F6
agonist activity (Renilla signal) for 7 compounds along with a DMSO
control. The conclusion is that Compound Z92 shown similar slight
activity (.about.3 times firefly signal over DMSO level) at 10 uM
and 50 uM. Compound E53 increases firefly activity in 6 times at 10
uM and appeared to show strong cytotox effect (great decreasing
both renilla and firefly activity). Both Compounds Z92 and E53 will
be tested on greater concentration range for confirmation on both
cell line with double stable transfection (clone F1-pGL4) and cell
line with transient transfection.
[0254] In certain embodiments, the present technology is directed
to compounds of Formula (XV):
##STR00091##
wherein R is C, H, N, O, S, an alkyl group, a substituted alkyl
group, a cyclic alkyl group, an aryl group, a substituted aryl
group, a heterocyclic group, an ester, an aldehyde, a ketone, a
carboxylic acid, an amide, an amine, an ether, a thiol or a
nitrile. In certain embodiments, R is H or an alkyl group.
[0255] In certain embodiments, the present technology is directed
to compounds of Formula (XVI):
##STR00092##
wherein any of R1 and R2 are C, H, N, O, S, a halogen, an alkyl
group, a substituted alkyl group, a cyclic alkyl group, an aryl
group, a substituted aryl group, a heterocyclic group, an ester, an
aldehyde, a ketone, a carboxylic acid, an amide, an amine, an
ether, a thiol or a nitrile. In certain embodiments, either or both
of R1 and R2 are H, alkyl, phenyl, piperidine, or pyrrolidine.
[0256] Exemplary compounds include the following:
##STR00093## ##STR00094##
[0257] Table 26 shows activity results for two exemplary compounds,
Compound Z95 and Compound Z113.
TABLE-US-00027 TABLE 26 ID Firefly, cmpd/DMSO (mean) F4, cmpd/DMSO
(mean) Concentration 40 10 2 40 10 2 Z95 2.3 1.3 1.2 3.7 2.2 1.2
Z113 0.9 1.1 1.3 1.2 1.5 1.2
[0258] FIGS. 48-51 show embodiments of a synthetic methods of
formulating a compound according to the present technology.
[0259] Further compounds found to have good activity include the
following:
##STR00095## ##STR00096##
[0260] FIGS. 52-55 illustrate syntheses of various compounds
discussed herein.
[0261] FIGS. 56A and 56B show HTS activity confirmation for various
compounds. Compound E53 shown similar slight activity (.about.3
times firefly signal over DMSO level) at 10 uM and 50 uM. 8010-3060
increase firefly activity in 6 times at 10 uM and It seems it shown
strong cytotox effect (great decreasing both renilla and firefly
activity). Both Compound Z92 and Compound E53 will be further
tested on greater concentration range for confirmation on both cell
line with double stable transfection (clone F1-pGL4) and cell line
with transient transfection.
[0262] Further compounds found to have good activity include the
following:
##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101##
##STR00102## ##STR00103## ##STR00104##
[0263] FIGS. 57 and 58 show syntheses of exemplary compositions
found to be useful.
[0264] Table 27 shows the activity of various compounds discussed
herein.
TABLE-US-00028 TABLE 27 Firefly, Renilla, cmpd/DMSO cmpd/DMSO ID
(mean) (mean) Z134 1.1 0.9 Z135 1.0 1.0 Z152 1.6 1.0 Z153 2.0 1.1
Z150 1.0 0.9 Z151 1.1 1.0 Z149 1.5 1.0 Z139 1.5 1.1 Z140 1.2 1.0
Z141 1.8 0.9 Z142 1.1 0.9 Z97 2.1 1.0 Z113 1.0 0.9
[0265] The present technology is directed to a composition
according to any of the compounds described herein, substantially
free of impurities. As used herein, the term "substantially free of
impurities" means that the compound contains no significant amount
of extraneous matter, including starting materials, residual
solvents, or any other impurities that may result from the
preparation or isolation of the compounds herein. In various
embodiments, at least about 95%, at least about 97% or at least
about 98% by weight of a compound herein is present in a dosage
form herein.
[0266] Although the present technology has been described in
relation to particular embodiments thereof, these embodiments and
examples are merely exemplary and not intended to be limiting. Many
other variations and modifications and other uses will become
apparent to those skilled in the art. The present technology
should, therefore, not be limited by the specific disclosure
herein, and can be embodied in other forms not explicitly described
here, without departing from the spirit thereof.
Sequence CWU 1
1
111803DNAHomo Sapiens 1gtgcagcccg tgccccccgc gcgccggggc cgaatgcgcg
ccgcgtaggg tcccccgggc 60cgagaggggt gcccggaggg aagagcgcgg tgggggcgcc
ccggccccgc tgccctgggg 120ctatggccat ggtgaccggc ggctggggcg
gccccggcgg cgacacgaac ggcgtggaca 180aggcgggcgg ctacccgcgc
gcggccgagg acgactcggc ctcgcccccc ggtgccgcca 240gcgacgccga
gccgggcgac gaggagcggc cggggctgca ggtggactgc gtggtgtgcg
300gggacaagtc gagcggcaag cattacggtg tcttcacctg cgagggctgc
aagagctttt 360tcaagcgaag catccgccgc aacctcagct acacctgccg
gtccaaccgt gactgccaga 420tcgaccagca ccaccggaac cagtgccagt
actgccgtct caagaagtgc ttccgggtgg 480gcatgaggaa ggaggcggtg
cagcgcggcc gcatcccgca ctcgctgcct ggtgccgtgg 540ccgcctcctc
gggcagcccc ccgggctcgg cgctggcggc agtggcgagc ggcggagacc
600tcttcccggg gcagccggtg tccgaactga tcgcgcagct gctgcgcgct
gagccctacc 660ctgcggcggc cggacgcttc ggcgcagggg gcggcgcggc
gggcgcggtg ctgggcatcg 720acaacgtgtg cgagctggcg gcgcggctgc
tcttcagcac cgtggagtgg gcgcgccacg 780cgcccttctt ccccgagctg
ccggtggccg accaggtggc gctgctgcgc ctgagctgga 840gcgagctctt
cgtgctgaac gcggcgcagg cggcgctgcc cctgcacacg gcgccgctac
900tggccgccgc cggcctccac gccgcgccta tggccgccga gcgcgccgtg
gctttcatgg 960accaggtgcg cgccttccag gaggaggtgg acaagctggg
ccgcctgcag gtcgactcgg 1020ccgagtatgg ctgcctcaag gccatcgcgc
tttcacgccc gacgcctgtg gcctctcaga 1080cccggcccac gttgagagcc
tgcaggagaa ggcgcaggtg gccctcaccg agtatgtgcg 1140ggcgcagtac
ccgtcccagc cccagcgctt cgggcgcctg ctgctgcggc tccccgccct
1200gcgcgcggtc cctgcctccc tcatctccca gctgttcttc atgcgcctgg
tggggaagac 1260gcccattgag acactgatca gagacatgct gctgtcgggg
agtaccttca actggcccta 1320cggctcgggc cagtgaccat gacggggcca
cgtgtgctgt ggccaggcct gcagacagac 1380ctcaagggac agggaatgct
gaggcctcga ggggcctccc ggggcccagg actctggctt 1440ctctcctcag
acttctattt tttaaagact gtgaaatgtt tgtcttttct gttttttaaa
1500tgatcatgaa accaaaaaga gactgatcat ccaggcctca gcctcatcct
ccccaggacc 1560cctgtccagg atggagggtc caatcctagg acagccttgt
tcctcagcac ccctagcatg 1620aacttgtggg atggtggggt tggcttccct
ggcatgatgg acaaaggcct ggcgtcggcc 1680agaggggctg ctccagtggg
caggggtagc tagcgtgtgc caggcagatc ctctggacac 1740gtaacctatg
tcagacacta catgatgact caaggccaat aataaagaca tttcctacct 1800gca
1803
* * * * *