U.S. patent application number 10/871368 was filed with the patent office on 2005-03-03 for methods of using [3.2.0] heterocyclic compounds and analogs thereof.
Invention is credited to Macherla, Venkata Rami Reddy, Neuteboom, Saskia Theodora Cornelia, Palladino, Michael, Potts, Barbara Christine.
Application Number | 20050049294 10/871368 |
Document ID | / |
Family ID | 33567612 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049294 |
Kind Code |
A1 |
Palladino, Michael ; et
al. |
March 3, 2005 |
Methods of using [3.2.0] heterocyclic compounds and analogs
thereof
Abstract
Disclosed are methods of treating cancer, inflammatory
conditions, and/or infectious disease in an animal comprising:
administering to the animal, a therapeutically effective amount of
a heterocyclic compound. The animal is a mammal, preferably a human
or a rodent.
Inventors: |
Palladino, Michael;
(Encinitas, CA) ; Neuteboom, Saskia Theodora
Cornelia; (San Diego, CA) ; Macherla, Venkata Rami
Reddy; (San Diego, CA) ; Potts, Barbara
Christine; (Escondido, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
33567612 |
Appl. No.: |
10/871368 |
Filed: |
June 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60480270 |
Jun 20, 2003 |
|
|
|
60566952 |
Apr 30, 2004 |
|
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Current U.S.
Class: |
514/412 |
Current CPC
Class: |
A61P 33/00 20180101;
Y02A 50/411 20180101; A61P 17/06 20180101; A61P 43/00 20180101;
A61P 31/10 20180101; Y02A 50/30 20180101; A61P 25/28 20180101; A61P
11/06 20180101; A61P 19/02 20180101; Y02A 50/409 20180101; A61P
35/02 20180101; A61P 31/04 20180101; A61K 31/407 20130101; A61P
31/00 20180101; A61P 35/00 20180101; A61P 25/00 20180101; A61P 9/10
20180101; A61P 33/02 20180101; A61P 29/00 20180101; A61P 33/06
20180101 |
Class at
Publication: |
514/412 |
International
Class: |
A61K 031/407 |
Claims
What is claimed is:
1. A method of treating a neoplastic disease in an animal, the
method comprising: administering to the animal, a therapeutically
effective amount of a compound of a formula selected from Formulae
I-V, and pharmaceutically acceptable salts and pro-drug esters
thereof:
2. The method of claim 1, wherein the neoplastic disease is
cancer.
3. The method of claim 2, wherein the cancer is selected from the
group consisting of breast cancer, sarcoma, leukemia, ovarian
cancer, uretal cancer, bladder cancer, prostate cancer, colon
cancer, rectal cancer, stomach cancer, lung cancer, lymphoma,
multiple myeloma, pancreatic cancer, liver cancer, kidney cancer,
endocrine cancer, skin cancer, melanoma, angioma, and brain or
central nervous system (CNS) cancer.
4. The method of claim 3, wherein the cancer is a multiple myeloma,
a colorectal carcinoma, a prostate carcinoma, a breast
adenocarcinoma, a non-small cell lung carcinoma, an ovarian
carcinoma or a melanoma.
5. The method of claim 2, wherein the cancer is a drug resistant
cancer.
6. The method of claim 5, wherein the drug-resistant cancer
displays at least one of the following: elevated levels of the
P-glycoprotein efflux pump, increased expression of the
multidrug-resistance associated protein 1 encoded by MRP1, reduced
drug uptake, alteration of the drug's target or increasing repair
of drug-induced DNA damage, alteration of the apoptotic pathway or
the activation of cytochrome P450 enzymes.
7. The method of claim 5, wherein the drug resistant cancer is a
sarcoma or a leukemia.
8. The method of claim 1, wherein the animal is a mammal.
9. The method of claim 1, wherein the animal is a human.
10. The method of claim 1, wherein the animal is a rodent.
11. The method of claim 1, wherein the the compound is: 84wherein
R.sub.8 is selected from the group consisting of H, F, Cl, Br and
I.
12. The method of claim 1, wherein the compound is: 85wherein
R.sub.8 is selected from the group consisting of H, F, Cl, Br, and
I.
13. The method of claim 1, further comprising the steps of:
identifying a subject that would benefit from administration of an
anticancer agent; performing the method on the subject.
14. A pharmaceutical composition comprising a compound of a formula
selected from Formulae I-V, and pharmaceutically acceptable salts
and pro-drug esters thereof.
15. The pharmaceutical composition of claim 14, further comprising
an anti-microbial agent.
16. A method of inhibiting the growth of a cancer cell, comprising
contacting a cancer cell with a compound of a formula selected from
Formulae I-V, and pharmaceutically acceptable salts and pro-drug
esters thereof.
17. The method of claim 16, wherein the cancer cell is a multiple
myeloma, a colorectal carcinoma, a prostate carcinoma, a breast
adenocarcinoma, a non-small cell lung carcinoma, an ovarian
carcinoma and a melanoma.
18. A method of inhibiting proteasome activity comprising the step
contacting a cell with a compound of a formula selected from
Formulae I-V, and pharmaceutically acceptable salts and pro-drug
esters thereof.
19. A method of inhibiting NF-.kappa.B activation comprising the
step contacting a cell with a compound of a formula selected from
Formulae I-V, and pharmaceutically acceptable salts and pro-drug
esters thereof.
20. A method for treating an inflammatory condition, comprising
administering an effective amount of a compound of a formula
selected from Formulae I-V to a patient in need thereof.
21. The method of claim 20, wherein the inflammatory condition is
selected from the group consisting of rheumatoid arthritis, asthma,
multiple sclerosis, psoriasis, stroke, and myocardial
infarction.
22. A method for treating a microbial illness comprising
administering an effective amount of a compound of a formula
selected from Formulae I-V to a patient in need thereof.
23. The method of claim 22, wherein the microbial illness is caused
by a microbe selected from the group consisting of B. anthracis,
Plasmodium, Leishmania, and Trypanosoma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application No. 60/480,270, filed on
Jun. 20, 2003, entitled USE OF SALINOSPORAMIDE A TO TREAT LeTx
INTOXICATION AND B. ANTHRACIS INFECTION, and to U.S. Provisional
Application No. 60/566,952, filed on Apr. 30, 2004, entitled
METHODS OF USING (3.2.0) HETEROCYCLIC COMPOUNDS AND ANALOGS
THEREOF; the disclosures of both of which are incorporated herein
by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to certain compounds and to
methods for the preparation and the use of certain compounds in the
fields of chemistry and medicine. Embodiments of the invention
disclosed herein relate to methods of using heterocyclic compounds.
In some embodiments, the compounds are used as proteasome
inhibitors. In other embodiments, the compounds are used to treat
inflammation, cancer, and infectious diseases.
[0004] 2. Description of the Related Art
[0005] Cancer is a leading cause of death in the United States.
Despite significant efforts to find new approaches for treating
cancer, the primary treatment options remain surgery, chemotherapy
and radiation therapy, either alone or in combination. Surgery and
radiation therapy, however, are generally useful only for fairly
defined types of cancer, and are of limited use for treating
patients with disseminated disease. Chemotherapy is the method that
is generally useful in treating patients with metastatic cancer or
diffuse cancers such as leukemias. Although chemotherapy can
provide a therapeutic benefit, it often fails to result in cure of
the disease due to the patient's cancer cells becoming resistant to
the chemotherapeutic agent. Due, in part, to the likelihood of
cancer cells becoming resistant to a chemotherapeutic agent, such
agents are commonly used in combination to treat patients.
[0006] Similarly, infectious diseases caused, for example, by
bacteria, fungi and protozoa are becoming increasingly difficult to
treat and cure. For example, more and more bacteria, fungi and
protozoa are developing resistance to current antibiotics and
chemotherapeutic agents. Examples of such microbes include
Bacillus, Leishmania, Plasmodium and Trypanosoma.
[0007] Furthermore, a growing number of diseases and medical
conditions are classified as inflammatory diseases. Such diseases
include conditions such as asthma to cardiovascular diseases. These
diseases continue to affect larger and larger numbers of people
worldwide despite new therapies and medical advances.
[0008] Therefore, a need exists for additional chemotherapeutics,
anti-microbial agents, and anti-inflammatory agents to treat
cancer, inflammatory diseases and infectious disease. A continuing
effort is being made by individual investigators, academia and
companies to identify new, potentially useful chemotherapeutic and
anti-microbial agents.
[0009] Marine-derived natural products are a rich source of
potential new anti-cancer agents and anti-microbial agents. The
oceans are massively complex and house a diverse assemblage of
microbes that occur in environments of extreme variations in
pressure, salinity, and temperature. Marine microorganisms have
therefore developed unique metabolic and physiological capabilities
that not only ensure survival in extreme and varied habitats, but
also offer the potential to produce metabolites that would not be
observed from terrestrial microorganisms (Okami, Y. 1993 J Mar
Biotechnol 1:59). Representative structural classes of such
metabolites include terpenes, peptides, polyketides, and compounds
with mixed biosynthetic origins. Many of these molecules have
demonstrable anti-tumor, anti-bacterial, anti-fungal,
anti-inflammatory or immunosuppressive activities (Bull, A. T. et
al. 2000 Microbiol Mol Biol Rev 64:573; Cragg, G. M. & D. J.
Newman 2002 Trends Pharmacol Sci 23:404; Kerr, R. G. & S. S.
Kerr 1999 Exp Opin Ther Patents 9:1207; Moore, B. S 1999 Nat Prod
Rep 16:653; Faulkner, D. J. 2001 Nat Prod Rep 18:1; Mayer, A. M.
& V. K. Lehmann 2001 Anticancer Res 21:2489), validating the
utility of this source for isolating invaluable therapeutic agents.
Further, the isolation of novel anti-cancer and anti-microbial
agents that represent alternative mechanistic classes to those
currently on the market will help to address resistance concerns,
including any mechanism-based resistance that may have been
engineered into pathogens for bioterrorism purposes.
SUMMARY OF THE INVENTION
[0010] The embodiments disclosed herein generally relate to
chemical compounds, including heterocyclic compounds and analogs
thereof. Some embodiments are directed to the use of compounds as
proteasome inhibitors.
[0011] In other embodiments, the compounds are used to treat
neoplastic diseases, for example, to inhibit the growth of tumors,
cancers and other neoplastic tissues. The methods of treatment
disclosed herein may be employed with any patient suspected of
carrying tumorous growths, cancers, or other neoplastic growths,
either benign or malignant ("tumor" or "tumors" as used herein
encompasses tumors, cancers, disseminated neoplastic cells and
localized neoplastic growths). Examples of such growths include but
are not limited to breast cancers; osteosarcomas, angiosarcomas,
fibrosarcomas and other sarcomas; leukemias; sinus tumors; ovarian,
uretal, bladder, prostate and other genitourinary cancers; colon,
esophageal and stomach cancers and other gastrointestinal cancers;
lung cancers; lymphomas; myelomas; pancreatic cancers; liver
cancers; kidney cancers; endocrine cancers; skin cancers;
melanomas; angiomas; and brain or central nervous system (CNS)
cancers. In general, the tumor or growth to be treated may be any
tumor or cancer, primary or secondary. Certain embodiments relate
to methods of treating neoplastic diseases in animals. The method
can include, for example, administering an effective amount of a
compound to a patient in need thereof. Other embodiments relate to
the use of compounds in the manufacture of a pharmaceutical or
medicament for the treatment of a neoplastic disease. The compounds
can be administered in combination with a chemotherapeutic
agent.
[0012] In still other embodiments, the compounds are used to treat
inflammatory conditions. Certain embodiments relate to methods of
treating inflammatory conditions in animals. The method can
include, for example, administering an effective amount of a
compound to a patient in need thereof. Other embodiments relate to
the use of compounds in the manufacture of a pharmaceutical or
medicament for the treatment of inflammation.
[0013] In certain embodiments, the compounds are used to treat
infectious diseases. The infectious agent can be a microbe, for
example, bacteria, fungi, protozoans, and microscopic algae, or
viruses. Further, the infectious agent can be B. anthracis
(anthrax). In some embodiments the infectious agent is a parasite.
For example, the infectious agent can be Plasmodium, Leishmania,
and Trypanosoma. Certain embodiments relate to methods of treating
infectious agents in animals. The method can include, for example,
administering an effective amount of a compound to a patient in
need thereof. Other embodiments relate to the use of compounds in
the manufacture of a pharmaceutical or medicament for the treatment
of infectious agents.
[0014] Some embodiments relate to uses of a compound having the
structure of Formula I, and pharmaceutically acceptable salts and
pro-drug esters thereof: 1
[0015] wherein the dashed lines represent a single or a double
bond, wherein R1 may be separately selected from the group
consisting of a hydrogen, a halogen, mono-substituted,
poly-substituted or unsubstituted variants of the following
residues: saturated C.sub.1-C.sub.24 alkyl, unsaturated
C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl, acyl,
acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,
alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated
alkyl including polyhalogenated alkyl, where n is equal to 1 or 2,
and if n is equal to 2, then R.sub.1 can be the same or
different;
[0016] wherein R.sub.2, may be selected from the group consisting
of hydrogen, a halogen, mono-substituted, poly-substituted or
unsubstituted variants of the following residues: saturated
C.sub.1-C.sub.24 alkyl, unsaturated C.sub.2-C.sub.24 alkenyl or
C.sub.2-C.sub.24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,
aryloxycarbonyloxy, cycloalkyl, cycloalkenyl (including, for
example, cyclohexylcarbinol), alkoxy, cycloalkoxy, aryl,
heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,
aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl,
cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy,
cyano, and halogenated alkyl including polyhalogenated alkyl;
[0017] wherein R.sub.3 may be selected from the group consisting of
a halogen, mono-substituted, poly-substituted or unsubstituted
variants of the following residues: saturated C.sub.1-C.sub.24
alkyl, unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24
alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,
cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl,
arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,
alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated
alkyl including polyhalogenated alkyl; and wherein each of E.sub.1,
E.sub.2, E.sub.3 and E.sub.4 is a substituted or unsubstituted
heteroatom; in the treatment of cancer, inflammation, and
infectious disease.
[0018] Other embodiments relate to methods of treating a neoplastic
disease in an animal. The methods can include, for example,
administering to the animal, a therapeutically effective amount of
a compound of a formula selected from Formulae I-V, and
pharmaceutically acceptable salts and pro-drug esters thereof.
[0019] Further embodiments relate to pharmaceutical compositions
which include a compound of a formula selected from Formulae I-V.
The pharmaceutical compositions can further include an
anti-microbial agent.
[0020] Still further embodiments relate to methods of inhibiting
the growth of a cancer cell. The methods can include, for example,
contacting a cancer cell with a compound of a formula selected from
Formulae I-V, and pharmaceutically acceptable salts and pro-drug
esters thereof.
[0021] Other embodiments relate to methods of inhibiting proteasome
activity that include the step contacting a cell with a compound of
a formula selected from Formulae I-V, and pharmaceutically
acceptable salts and pro-drug esters thereof.
[0022] Other embodiments relate to methods of inhibiting
NF-.kappa.B activation including the step contacting a cell with a
compound of a formula selected from Formulae I-V, and
pharmaceutically acceptable salts and pro-drug esters thereof.
[0023] Some embodiments relate to methods for treating an
inflammatory condition, including administering an effective amount
of a compound of a formula selected from Formulae I-V to a patient
in need thereof.
[0024] Further embodiments relate to methods for treating a
microbial illness including administering an effective amount of a
compound of a formula selected from Formulae I-V to a patient in
need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings, which are incorporated in and
form part of the specification, merely illustrate certain preferred
embodiments of the present invention. Together with the remainder
of the specification, they are meant to serve to explain preferred
modes of making certain compounds of the invention to those of
skilled in the art. In the drawings:
[0026] FIG. 1 shows the chemical structure of Salinosporamide
A.
[0027] FIG. 2 shows the pan-tropical distribution of the
Salinospora. "X" denotes Salinospora collection sites.
[0028] FIG. 3 shows colonies of Salinospora.
[0029] FIG. 4 shows the typical 16S rDNA sequence of the
Salinospora. Bars represent characteristic signature nucleotides of
the Salinospora that separate them from their nearest
relatives.
[0030] FIG. 5 shows Omuralide, a degradation product of the
microbial metabolite Lactacystin. Also shown is a compound of
Formula II-16, also referred to as Salinosporamide A.
[0031] FIG. 6 illustrates lethal toxin-mediated macrophage
cytotoxicity. NPI-0052 represents the compound of Formula
II-16.
[0032] FIG. 7 depicts the 1H NMR spectrum of a compound having
structure Formula II-20.
[0033] FIG. 8 depicts the 1H NMR spectrum of a compound having
structure Formula II-24C.
[0034] FIG. 9 depicts the 1H NMR spectrum of a compound having
structure Formula II-19.
[0035] FIG. 10 depicts the 1H NMR spectrum of a compound having
structure Formula II-2.
[0036] FIG. 11 depicts the mass spectrum of a compound having
structure Formula II-2.
[0037] FIG. 12 depicts the 1H NMR spectrum of a compound having
structure Formula II-3.
[0038] FIG. 13 depicts the mass spectrum of a compound having
structure Formula II-3.
[0039] FIG. 14 depicts the 1H NMR spectrum of a compound having
structure Formula II-4.
[0040] FIG. 15 depicts the mass spectrum of a compound having
structure Formula II-4.
[0041] FIG. 16 depicts the 1H NMR spectrum of a compound having
structure Formula II-5A.
[0042] FIG. 17 depicts the mass spectrum of a compound having
structure Formula II-5A.
[0043] FIG. 18 depicts the 1H NMR spectrum of a compound having
structure Formula II-5B.
[0044] FIG. 19 depicts the mass spectrum of a compound having
structure Formula II-5B.
[0045] FIG. 20 depicts the 1H NMR spectrum of a compound having
structure Formula IV-3C in DMSO-d.sub.6.
[0046] FIG. 21 depicts the 1H NMR spectrum of a compound having
structure Formula IV-3C in C.sub.6D.sub.6/DMSO-d.sub.6.
[0047] FIG. 22 depicts the 1H NMR spectrum of a compound having
structure Formula II-13C.
[0048] FIG. 23 depicts the 1H NMR spectrum of a compound having
structure Formula II-8C.
[0049] FIG. 24 depicts the 1H NMR spectrum of a compound having
structure Formula II-25.
[0050] FIG. 25 depicts the 1H NMR spectrum of a compound having
structure Formula II-21.
[0051] FIG. 26 depicts the 1H NMR spectrum of a compound having
structure Formula II-22.
[0052] FIG. 27 shows inhibition of the chymotrypsin-like activity
of rabbit muscle proteasomes.
[0053] FIG. 28 shows inhibition of the PGPH activity of rabbit
muscle proteasomes.
[0054] FIG. 29 shows inhibition of the chymotrypsin-like activity
of human erythrocyte proteasomes.
[0055] FIG. 30 shows the effect of II-16 treatment on
chymotrypsin-mediated cleavage of LLVY-AMC substrate.
[0056] FIG. 31 shows NF-.kappa.B/luciferase activity and
cytotoxicity profiles of II-16.
[0057] FIG. 32 shows reduction of I.kappa.B.alpha. degradation and
retention of phosphorylated I.kappa.B.alpha. by II-16 in HEK293
cells (A) and the HEK293 NF-.kappa.B/Luciferase reporter clone
(B).
[0058] FIG. 33 shows accumulation of cell cycle regulatory
proteins, p21 and p27, by II-16 treatment of HEK293 cells (A) and
the HEK293 NF-.kappa.B/Luciferase reporter clone (B).
[0059] FIG. 34 shows activation of Caspase-3 by II-16 in Jurkat
cells.
[0060] FIG. 35 shows PARP cleavage by II-16 in Jurkat cells.
[0061] FIG. 36 shows inhibition of LeTx-induced cytotoxicity by
II-16 in RAW264.7 cells.
[0062] FIG. 37 shows the effects of II-16 treatment on PARP and
Pro-Caspase 3 cleavage in RPMI 8226 and PC-3 cells.
[0063] FIG. 38 shows II-16 treatment of RPMI 8226 results in a
dose-dependent cleavage of PARP and Pro-Caspase 3.
[0064] FIG. 39 shows in vitro proteasome inhibition by II-16, I-17,
and II-18.
[0065] FIG. 40 shows proteasomal activity in PWBL prepared from
II-16 treated mice.
[0066] FIG. 41 shows epoxomicin treatment in the PWBL assay.
[0067] FIG. 42 shows intra-assay comparison.
[0068] FIG. 43 shows decreased plasma TNF levels in mice treated
with LPS.
[0069] FIG. 44 depicts assay results showing the effect of Formula
II-2, Formula II-3 and Formula II-4 on NF-.kappa.B mediated
luciferase activity in HEK293 NF-.kappa.B/Luc Cells.
[0070] FIG. 45 depicts assay results showing the effect of Formula
II-5A and Formula II-5B on NF-.kappa.B mediated luciferase activity
in HEK293 NF-.kappa.B/Luc Cells
[0071] FIG. 46 depicts assay results showing the effect of Formula
II-2, Formula II-3, and Formula II-4 on the chymotrypsin-like
activity of rabbit 20S proteasome.
[0072] FIG. 47 depicts the effect of Formula II-5A and Formula
II-5B on the chymotrypsin-like activity of rabbit 20S
proteasome.
[0073] FIG. 48 depicts the effect of Formulae II-2, II-3, and II-4
against LeTx-mediated cytotoxicity.
[0074] FIG. 49 depicts the 1H NMR spectrum of a compound having
structure Formula II-17.
[0075] FIG. 50 depicts the 1H NMR spectrum of a compound having
structure Formula II-18.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0076] Numerous references are cited herein. The references cited
herein, including the U.S. patents cited herein, are each to be
considered incorporated by reference in their entirety into this
specification.
[0077] Embodiments of the invention include, but are not limited
to, providing a method for the preparation of compounds, including
compounds, for example, those described herein and analogs thereof,
and to providing a method for producing pharmaceutically acceptable
anti-microbial, anti-cancer, and anti-inflammatory compositions,
for example. The methods can include the compositions in relatively
high yield, wherein the compounds and/or their derivatives are
among the active ingredients in these compositions. Other
embodiments relate to providing novel compounds not obtainable by
currently available methods. Furthermore, embodiments relate to
methods of treating cancer, inflammation, and infectious diseases,
particularly those affecting humans. The methods may include, for
example, the step of administering an effective amount of a member
of a class of new compounds. Preferred embodiments relate to the
compounds and methods of making and using such compounds disclosed
herein, but not necessarily in all embodiments of the present
invention, these objectives are met.
[0078] For the compounds described herein, each stereogenic carbon
may be of R or S configuration. Although the specific compounds
exemplified in this application may be depicted in a particular
configuration, compounds having either the opposite stereochemistry
at any given chiral center or mixtures thereof are also envisioned.
When chiral centers are found in the derivatives of this invention,
it is to be understood that the compounds encompasses all possible
stereoisomers.
[0079] Compounds of Formula I
[0080] Some embodiments provide compounds, and methods of producing
a class of compounds, pharmaceutically acceptable salts and
pro-drug esters thereof, wherein the compounds are represented by
Formula I: 2
[0081] In certain embodiments the substituent(s) R.sub.1, R.sub.2,
and R.sub.3 separately may include a hydrogen, a halogen, a
mono-substituted, a poly-substituted or an unsubstituted variant of
the following residues: saturated C.sub.1-C.sub.24 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl
(including for example, cyclohexylcarbinol), cycloalkenyl, alkoxy,
cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy
carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,
phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl,
carboxy, cyano, and halogenated alkyl including polyhalogenated
alkyl. Further, in certain embodiments, each of E.sub.1, E.sub.2,
E.sub.3 and E.sub.4 may be a substituted or unsubstituted
heteroatom, for example, a heteroatom separately selected from the
group consisting of nitrogen, sulfur and oxygen.
[0082] In some embodiments n may be equal to 1 or equal to 2. When
n is equal to 2, the substituents can be the same or can be
different. Furthermore, in some embodiments R.sub.3 is not a
hydrogen.
[0083] Preferably, R.sub.2 may be a formyl. For example, the
compound may have the following structure I-1: 3
[0084] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0085] Preferably, the structure of Formula I-l may have the
following stereochemistry: 4
[0086] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0087] Preferably, R.sub.2 may be a carbinol. For example, the
compound may have the following structure I-2: 5
[0088] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0089] As an example, the structure of Formula I-2 may have the
following stereochemistry: 6
[0090] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0091] As exemplary compound of Formula I may be the compound
having the following structure I-3: 7
[0092] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0093] The compound of Formula I-3 may have the following
stereochemical structure: 8
[0094] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0095] Another exemplary compound Formula I may be the compound
having the following structure I-4: 9
[0096] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0097] Preferably, the compound of Formula I-4 may have the
following stereochemical structure: 10
[0098] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0099] Still a further exemplary compound of Formula I is the
compound having the following structure I-5: 11
[0100] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0101] For example, the compound of Formula I-5 may have the
following stereochemistry: 12
[0102] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0103] In some embodiments, R.sub.2 of Formula I may be, for
example, a 3-methylenecyclohexene. For example, the compound may
have the following structure of Formula I-6: 13
[0104] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0105] Preferably, the compound of Formula I-6 may have the
following stereochemistry: 14
[0106] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0107] In other embodiments, R.sub.2 may be a
cyclohexylalkylamine.
[0108] Also, in other embodiments, R.sub.2 may be a
C-Cyclohexyl-methyleneamine. In others, R.sub.2 may be a
cyclohexanecarbaldehyde O-oxime.
[0109] Furthermore, in some embodiments, R.sub.2 may be a
cycloalkylacyl.
[0110] Compounds of Formula II
[0111] Other embodiments provide compounds, and methods of
producing a class of compounds, pharmaceutically acceptable salts
and pro-drug esters thereof, wherein the compounds are represented
by Formula II: 15
[0112] In certain embodiments the substituent(s) R.sub.1, R.sub.3,
and R.sub.4 separately may include a hydrogen, a halogen, a
mono-substituted, a poly-substituted or an unsubstituted variant of
the following residues: saturated C.sub.1-C.sub.24 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,
alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated
alkyl including polyhalogenated alkyl. Further, in certain
embodiments, each of E.sub.1, E.sub.2, E.sub.3 and E.sub.4 may be a
substituted or unsubstituted heteroatom, for example, a heteroatom
or substituted heteroatom selected from the group consisting of
nitrogen, sulfur and oxygen.
[0113] In some embodiments n may be equal to 1, while in others it
may be equal to 2. When n is equal to 2, the substituents can be
the same or can be different. Furthermore, in some embodiments
R.sub.3 is not a hydrogen. m can be equal to 1 or 2, and when m is
equal to 2, R.sub.4 can be the same or different.
[0114] E.sub.5 may be, for example, OH, O, OR.sub.10, S, SR.sub.11,
SO.sub.2R.sub.11, NH, NH.sub.2, NOH, NHOH, NR.sub.12, and
NHOR.sub.13, wherein R.sub.10-13 may separately include, for
example, hydrogen, a substituted or unsubstituted of any of the
following: alkyl, an aryl, a heteroaryl, and the like. Also,
R.sub.1 may be CH.sub.2CH.sub.2X, wherein X may be, for example, H,
F, Cl, Br, and I. R.sub.3 may be methyl. Furthermore, R.sub.4 may
include a cyclohexyl. Also, each of E.sub.1, E.sub.3 and E.sub.4
may be O and E.sub.2 may be NH. Preferably, R.sub.1 may be
CH.sub.2CH.sub.2X, wherein X is selected from the group consisting
of H, F, Cl Br, and I; wherein R.sub.4 may include a cyclohexyl;
wherein R.sub.3 may be methyl; and wherein each of E.sub.1, E.sub.3
and E.sub.4 separately may be O and E.sub.2 may be NH.
[0115] For example, an exemplary compound of Formula II has the
following structure II-1: 16
[0116] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0117] Exemplary stereochemistry may be as follows: 17
[0118] In preferred embodiments, the compound of Formula II has any
of the following structures: 18
[0119] The following is exemplary stereochemistry for compounds
having the structures II-2, II-3, and II-4, respectively: 19
[0120] In other embodiments wherein R.sub.4 may include a
7-oxa-bicyclo[4.1.0]hept-2-yl). An exemplary compound of Formula II
is the following structure II-5: 20
[0121] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0122] The following are examples of compounds having the structure
of Formula II-5: 21
[0123] In still further embodiments, at least one R.sub.4 may
include a subsituted or an unsubstituted branched alkyl. For
example, a compound of Formula II may be the following structure
II-6: 22
[0124] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0125] The following is exemplary stereochemistry for a compound
having the structure of Formula II-6: 23
[0126] As another example, the compound of Formula II may be the
following structure II-7: 24
[0127] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0128] The following is exemplary stereochemistry for a compound
having the structure of Formula II-7: 25
[0129] In other embodiments, at least one R.sub.4 may be a
cycloalkyl and E.sub.5 may be an oxygen. An exemplary compound of
Formula II may be the following structure II-8: 26
[0130] R.sub.8 may include, for example, hydrogen (II-8A), fluorine
(II-8B), chlorine (II-8C), bromine (II-8D) and iodine (II-8E).
[0131] The following is exemplary stereochemistry for a compound
having the structure of Formula II-8: 27
[0132] In some embodiments E5 may be an amine oxide, giving rise to
an oxime. An exemplary compound of Formula II has the following
structure II-9: 28
[0133] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine; R may be hydrogen, and a substituted
or unsubstituted alkyl, aryl, or heteroaryl, and the like.
[0134] The following is exemplary stereochemistry for a compound
having the structure of Formula II-9: 29
[0135] A further exemplary compound of Formula II has the following
structure II-10: 30
[0136] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0137] The following is exemplary stereochemistry for a compound
having the structure of Formula II-10: 31
[0138] In some embodiments, E.sub.5 may be NH.sub.2. An exemplary
compound of Formula II has the following structure II-11: 32
[0139] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0140] The following is exemplary stereochemistry for a compound
having the structure of Formula II-11: 33
[0141] In some embodiments, at least one R.sub.4 may include a
cycloalkyl and E.sub.5 may be NH.sub.2. An exemplary compound of
Formula II has the following structure II-12: 34
[0142] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0143] The following is exemplary stereochemistry for a compound
having the structure of Formula II-12: 35
[0144] A further exemplary compound of Formula II has the following
structure II-13: 36
[0145] R.sub.8 may include, for example, hydrogen (II-13A),
fluorine (II-13B), chlorine (II-13C), bromine (II-13D) and iodine
(II-13E).
[0146] The following is exemplary stereochemistry for a compound
having the structure of Formula II-13 : 37
[0147] A still further exemplary compound of Formula II has the
following structure II-14: 38
[0148] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0149] The following is exemplary stereochemistry for a compound
having the structure of Formula II-14: 39
[0150] In some embodiments, the compounds of Formula II, may
include as R.sub.4 at least one cycloalkene, for example.
Furthermore, in some embodiments, the compounds may include a
hydroxy at E.sub.5, for example. A further exemplary compound of
Formula II has the following structure II-15: 40
[0151] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0152] Exemplary stereochemistry may be as follows: 41
[0153] The following is exemplary stereochemistry for compounds
having the structures II-16, II-17, II-18, and II-19, respectively:
42
[0154] The compounds of Formulae II-16, II-17, II-18 and II-19 may
be obtained by fermentation, synthesis, or semi-synthesis and
isolated/purified as set forth below. Furthermore, the compounds of
Formulae II-16, II-17, II-18 and II-19 may be used, and are
referred to, as "starting materials" to make other compounds
described herein.
[0155] In some embodiments, the compounds of Formula II, may
include a methyl group as R.sub.1, for example. A further exemplary
compound, Formula II-20, has the following structure and
stereochemistry: 43
[0156] In some embodiments, the compounds of Formula II, may
include hydroxyethyl as R.sub.1, for example. A further exemplary
compound, Formula II-21, has the following structure and
stereochemistry: 44
[0157] In some embodiments, the hydroxyl group of Formula II-21 may
be esterified such that R.sub.1 may include ethylpropionate, for
example. An exemplary compound, Formula II-22, has the following
structure and stereochemistry: 45
[0158] In some embodiments, the compounds of Formula II may include
an ethyl group as R.sub.3, for example. A further exemplary
compound of Formula II has the following structure II-23: 46
[0159] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine. Exemplary stereochemistry may be as
follows: 47
[0160] In some embodiments, the compounds of Formula II-23 may have
the following structure and stereochemistry, exemplified by Formula
II-24C, where R.sub.8 is chlorine: 48
[0161] In some embodiments, the compounds of Formula II-15 may have
the following stereochemistry, exemplified by the compound of
Formula II-25, where R.sub.8 is chlorine: 49
[0162] Compounds of Formula III
[0163] Other embodiments provide compounds, and methods of
producing a class of compounds, pharmaceutically acceptable salts
and pro-drug esters thereof, wherein the compounds are represented
by Formula III: 50
[0164] In certain embodiments, the substituent(s) R.sub.1
separately may include, for example, a hydrogen, a halogen, a
mono-substituted, a poly-substituted or an unsubstituted variant of
the following residues: saturated C.sub.1-C.sub.24 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio,
arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl
including polyhalogenated alkyl. For example, n can be equal to 1
or 2.
[0165] In certain embodiments, R.sub.4 may be, for example, a
hydrogen, a halogen, a mono-substituted, a poly-substituted or an
unsubstituted variants of the following residues: saturated
C.sub.1-C.sub.24 alkyl, unsaturated C.sub.2-C.sub.24 alkenyl or
C.sub.2-C.sub.24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,
aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy,
aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,
aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,
alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated
alkyl including polyhalogenated alkyl. In some embodiments m can be
equal to 1 or 2, and where m is equal to 2, the substituents can
the same or different. Also, each of E.sub.1, E.sub.2, E.sub.3,
E.sub.4 and E.sub.5 may be, for example, a substituted or
unsubstituted heteroatom. For example, the heteroatom may be
nitrogen, sulfur or oxygen.
[0166] Compounds of Formula IV
[0167] Other embodiments provide compounds, and methods of
producing a class of compounds, pharmaceutically acceptable salts
and pro-drug esters thereof, wherein the compounds are represented
by Formula IV: 51
[0168] In certain embodiments, the substituent(s) R.sub.1 R.sub.3,
and R.sub.5 may separately include a hydrogen, a halogen, a
mono-substituted, a poly-substituted or an unsubstituted variants
of the following residues: saturated C.sub.1-C.sub.24 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio,
arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl
including polyhalogenated alkyl. Also, each of E.sub.1, E.sub.2,
E.sub.3, E.sub.4 and E.sub.5 may be a heteroatom or substituted
heteroatom, for example, nitrogen, sulfur or oxygen. In some
embodiments, R.sub.3 is not a hydrogen. n is equal to 1 or 2. When
n is equal to 2, the substituents can be the same or can be
different. Also, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, or 11.
When m is greater than 1, the substituents can be the same or
different.
[0169] In some embodiments R.sub.5 may give rise to a
di-substituted cyclohexyl. An exemplary compound of Formula IV is
the following structure IV-1, with and without exemplary
stereochemistry: 52
[0170] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine. The substituent(s) R.sub.6 and
R.sub.7 may separately include a hydrogen, a halogen, a
mono-substituted, a poly-substituted or an unsubstituted variants
of the following residues: saturated C.sub.1-C.sub.4 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio,
arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl
including polyhalogenated alkyl. Further, R.sub.6 and R.sub.7 both
may be the same or different.
[0171] For example, an exemplary compound of Formula IV has the
following structure IV-2: 53
[0172] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0173] Exemplary stereochemistry may be as follows: 54
[0174] For example, an exemplary compound of Formula IV has the
following structure IV-3: 55
[0175] R.sub.8 may include, for example, hydrogen (IV-3A), fluorine
(IV-3B), chlorine (IV-3C), bromine (IV-3D) and iodine (WV-3E).
[0176] Exemplary structure and stereochemistry may be as follows:
56
[0177] Additional exemplary structure and stereochemistry may be as
follows: 57
[0178] For example, an exemplary compound of Formula IV has the
following structure IV-4: 58
[0179] R.sub.8 may include, for example, hydrogen, fluorine,
chlorine, bromine and iodine.
[0180] Exemplary stereochemistry may be as follows: 59
[0181] Compounds of Formula V
[0182] Some embodiments provide compounds, and methods of producing
a class of compounds, pharmaceutically acceptable salts and
pro-drug esters thereof, wherein the compounds are represented by
Formula V: 60
[0183] In certain embodiments, the substituent(s) R.sub.1 and
R.sub.5 may separately include a hydrogen, a halogen, a
mono-substituted, a poly-substituted or unsubstituted variants of
the following residues: saturated C.sub.1-C.sub.24 alkyl,
unsaturated C.sub.2-C.sub.24 alkenyl or C.sub.2-C.sub.24 alkynyl,
acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,
cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy
carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,
aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio,
arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkyl
including polyhalogenated alkyl. In certain embodiments, each of
E.sub.1, E.sub.2, E.sub.3, E.sub.4 and E.sub.5 may be a heteroatom
or substituted heteroatom, for example, nitrogen, sulfur or oxygen.
n can be equal to 1 or 2, and when n is equal to 2, the
substituents can be the same or different. Preferably, m may be,
for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. When m is
greater than 1, R.sub.5 may be the same or different.
[0184] Certain embodiments also provide pharmaceutically acceptable
salts and pro-drug esters of the compound of Formulae I-V, and
provide methods of obtaining and purifying such compounds by the
methods disclosed herein.
[0185] The term "pro-drug ester," especially when referring to a
pro-drug ester of the compound of Formula I synthesized by the
methods disclosed herein, refers to a chemical derivative of the
compound that is rapidly transformed in vivo to yield the compound,
for example, by hydrolysis in blood or inside tissues. The term
"pro-drug ester" refers to derivatives of the compounds disclosed
herein formed by the addition of any of several ester-forming
groups that are hydrolyzed under physiological conditions. Examples
of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl,
phthalidyl, indanyl and methoxymethyl, as well as other such groups
known in the art, including a (5-R-2-oxo-1,3-dioxolen-4-yl)me- thyl
group. Other examples of pro-drug ester groups can be found in, for
example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery
Systems", Vol. 14, A.C.S. Symposium Series, American Chemical
Society (1975); and "Bioreversible Carriers in Drug Design: Theory
and Application", edited by E. B. Roche, Pergamon Press: New York,
14-21 (1987) (providing examples of esters useful as prodrugs for
compounds containing carboxyl groups). Each of the above-mentioned
references is hereby incorporated by reference in its entirety.
[0186] The term "pro-drug ester," as used herein, also refers to a
chemical derivative of the compound that is rapidly transformed in
vivo to yield the compound, for example, by hydrolysis in
blood.
[0187] The term "pharmaceutically acceptable salt," as used herein,
and particularly when referring to a pharmaceutically acceptable
salt of a compound, including Formulae I-V, and Formula I-V as
produced and synthesized by the methods disclosed herein, refers to
any pharmaceutically acceptable salts of a compound, and preferably
refers to an acid addition salt of a compound. Preferred examples
of pharmaceutically acceptable salt are the alkali metal salts
(sodium or potassium), the alkaline earth metal salts (calcium or
magnesium), or ammonium salts derived from ammonia or from
pharmaceutically acceptable organic amines, for example
C.sub.1-C.sub.7 alkylamine, cyclohexylamine, triethanolamine,
ethylenediamine or tris-(hydroxymethyl)-aminomethane. With respect
to compounds synthesized by the method of this embodiment that are
basic amines, the preferred examples of pharmaceutically acceptable
salts are acid addition salts of pharmaceutically acceptable
inorganic or organic acids, for example, hydrohalic, sulfuric,
phosphoric acid or aliphatic or aromatic carboxylic or sulfonic
acid, for example acetic, succinic, lactic, malic, tartaric,
citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or
naphthalenesulfonic acid.
[0188] Preferred pharmaceutical compositions disclosed herein
include pharmaceutically acceptable salts and pro-drug esters of
the compound of Formulae I-V obtained and purified by the methods
disclosed herein. Accordingly, if the manufacture of pharmaceutical
formulations involves intimate mixing of the pharmaceutical
excipients and the active ingredient in its salt form, then it is
preferred to use pharmaceutical excipients which are non-basic,
that is, either acidic or neutral excipients.
[0189] It will be also appreciated that the phrase "compounds and
compositions comprising the compound," or any like phrase, is meant
to encompass compounds in any suitable form for pharmaceutical
delivery, as discussed in further detail herein. For example, in
certain embodiments, the compounds or compositions comprising the
same may include a pharmaceutically acceptable salt of the
compound.
[0190] In one embodiment the compounds may be used to treat
microbial diseases, cancer, and inflammation. Disease is meant to
be construed broadly to cover infectious diseases, and also
autoimmune diseases, non-infectious diseases and chronic
conditions. In a preferred embodiment, the disease is caused by a
microbe, such as a bacterium, a fungi, and protozoa, for example.
The methods of use may also include the steps of administering a
compound or composition comprising the compound to an individual
with an infectious disease or cancer. The compound or composition
can be administered in an amount effective to treat the particular
infectious disease, cancer or inflammatory condition.
[0191] The infectious disease may be, for example, one caused by
Bacillus, such as B. anthracis and B. cereus. The infectious
disease may be one caused by a protozoa, for example, a Leishmania,
a Plasmodium or a Trypanosoma. The compound or composition may be
administered with a pharmaceutically acceptable carrier, diluent,
excipient, and the like.
[0192] The cancer may be, for example, a multiple myeloma, a
colorectal carcinoma, a prostate carcinoma, a breast
adenocarcinoma, a non-small cell lung carcinoma, an ovarian
carcinoma, a melanoma, and the like.
[0193] The inflammatory condition may be, for example, rheumatoid
arthritis, asthma, multiple sclerosis, psoriasis, stroke,
myocardial infarction, and the like.
[0194] The term "halogen atom," as used herein, means any one of
the radio-stable atoms of column 7 of the Periodic Table of the
Elements, i.e., fluorine, chlorine, bromine, or iodine, with
bromine and chlorine being preferred.
[0195] The term "alkyl," as used herein, means any unbranched or
branched, substituted or unsubstituted, saturated hydrocarbon, with
C.sub.1-C.sub.6 unbranched, saturated, unsubstituted hydrocarbons
being preferred, with methyl, ethyl, isobutyl, and
tert-butylpropyl, and pentyl being most preferred. Among the
substituted, saturated hydrocarbons, C.sub.1-C.sub.6 mono- and di-
and per-halogen substituted saturated hydrocarbons and
amino-substituted hydrocarbons are preferred, with perfluromethyl,
perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl
being the most preferred.
[0196] The term "substituted" has its ordinary meaning, as found in
numerous contemporary patents from the related art. See, for
example, U.S. Pat. Nos. 6,509,331; 6,506,787; 6,500,825; 5,922,683;
5,886,210; 5,874,443; and 6,350,759; all of which are incorporated
herein in their entireties by reference. Specifically, the
definition of substituted is as broad as that provided in U.S. Pat.
No. 6,509,331, which defines the term "substituted alkyl" such that
it refers to an alkyl group, preferably of from 1 to 10 carbon
atoms, having from 1 to 5 substituents, and preferably 1 to 3
substituents, selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy,
amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo,
thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-substituted
alkyl, --SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl. The other above-listed patents also provide
standard definitions for the term "substituted" that are
well-understood by those of skill in the art.
[0197] The term "cycloalkyl" refers to any non-aromatic hydrocarbon
ring, preferably having five to twelve atoms comprising the ring.
The term "acyl" refers to alkyl or aryl groups derived from an
oxoacid, with an acetyl group being preferred.
[0198] The term "alkenyl," as used herein, means any unbranched or
branched, substituted or unsubstituted, unsaturated hydrocarbon
including polyunsaturated hydrocarbons, with C.sub.1-C.sub.6
unbranched, mono-unsaturated and di-unsaturated, unsubstituted
hydrocarbons being preferred, and mono-unsaturated, di-halogen
substituted hydrocarbons being most preferred. The term
"cycloalkenyl" refers to any non-aromatic hydrocarbon ring,
preferably having five to twelve atoms comprising the ring.
[0199] The terms "aryl," "substituted aryl," "heteroaryl," and
"substituted heteroaryl," as used herein, refer to aromatic
hydrocarbon rings, preferably having five, six, or seven atoms, and
most preferably having six atoms comprising the ring. "Heteroaryl"
and "substituted heteroaryl," refer to aromatic hydrocarbon rings
in which at least one heteroatom, e.g., oxygen, sulfur, or nitrogen
atom, is in the ring along with at least one carbon atom. The term
"heterocycle" or "heterocyclic" refer to any cyclic compound
containing one or more heteroatoms. The substituted aryls,
heterocycles and heteroaryls can be substituted with any
substituent, including those described above and those known in the
art.
[0200] The term "alkoxy" refers to any unbranched, or branched,
substituted or unsubstituted, saturated or unsaturated ether, with
C.sub.1-C.sub.6 unbranched, saturated, unsubstituted ethers being
preferred, with methoxy being preferred, and also with dimethyl,
diethyl, methyl-isobutyl, and methyl-tert-butyl ethers also being
preferred. The term "cycloalkoxy" refers to any non-aromatic
hydrocarbon ring, preferably having five to twelve atoms comprising
the ring. The term "alkoxy carbonyl" refers to any linear,
branched, cyclic, saturated, unsaturated, aliphatic or aromatic
alkoxy attached to a carbonyl group. The examples include
methoxycarbonyl group, ethoxycarbonyl group, propyloxycarbonyl
group, isopropyloxycarbonyl group, butoxycarbonyl group,
sec-butoxycarbonyl group, tert-butoxycarbonyl group,
cyclopentyloxycarbonyl group, cyclohexyloxycarbonyl group,
benzyloxycarbonyl group, allyloxycarbonyl group, phenyloxycarbonyl
group, pyridyloxycarbonyl group, and the like.
[0201] The terms "pure," "purified," "substantially purified," and
"isolated" as used herein refer to the compound of the embodiment
being free of other, dissimilar compounds with which the compound,
if found in its natural state, would be associated in its natural
state. In certain embodiments described as "pure," "purified,"
"substantially purified," or "isolated" herein, the compound may
comprise at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at
least 50% or 75% of the mass, by weight, of a given sample.
[0202] The terms "derivative," "variant," or other similar term
refers to a compound that is an analog of the other compound.
[0203] Certain of the compounds of Formula I-V may be obtained and
purified or may be obtained via semi-synthesis from purified
compounds as set forth herein. Generally, without being limited
thereto, the compounds of Formula II-15, preferably, Formulae
II-16, II-17, II-18 and II-19, may be obtained synthetically or by
fermentation. Exemplary fermentation procedures are provided below.
Futher, the compounds of Formula II-15, preferably, Formulae II-16,
II-17, II-18 and II-19 may be used as starting compounds in order
to obtain/synthesize various of the other compounds described
herein. Exemplary non-limiting syntheses are provided herein.
61
[0204] Formula II-16 is currently produced through a high-yield
saline fermentation (.about.200 mg/L) and modifications of the
conditions has yielded new analogs in the fermentation extracts.
FIG. 1 shows the chemical structure of II-16. Additional analogs
can be generated through directed biosynthesis. Directed
biosynthesis is the modification of a natural product by adding
biosynthetic precursor analogs to the fermentation of producing
microorganisms (Lam, et al., J Antibiot (Tokyo) 44:934 (1991), Lam,
et al., J Antibiot (Tokyo) 54:1 (2001); which is hereby
incorporated by reference in its entirety).
[0205] Exposing the producing culture to analogs of acetic acid,
phenylalanine, valine, butyric acid, shikimic acid, and halogens,
preferably, other than chlorine, can lead to the formation of new
analogs. The new analogs produced can be easily detected in crude
extracts by HPLC and LC-MS. For example, after manipulating the
medium with different concentrations of sodium bromide, a
bromo-analog, Formula II-18, was successfully produced in
shake-flask culture at a titer of 14 mg/L.
[0206] A second approach to generate new analogs is through
biotransformation. Biotransformation reactions are chemical
reactions catalyzed by enzymes or whole cells containing these
enzymes. Zaks, A., Curr Opin Chem Biol 5:130 (2001). Microbial
natural products are ideal substrates for biotransformation
reactions as they are synthesized by a series of enzymatic
reactions inside microbial cells. Riva, S., Curr Opin Chem Biol
5:106 (2001).
[0207] Given the structure of the described compounds, including
those of Formula II-15, for example, the possible biosynthetic
origins are acetyl-CoA, ethylmalonyl-CoA, phenylalanine and
chlorine. Ethylmalonyl-CoA is derived from butyryl-CoA, which can
be derived either from valine or crotonyl-CoA. Liu, et al., Metab
Eng 3:40 (2001). Phenylalanine is derived from shikimic acid.
[0208] Production of Compounds of Formulae II-16, II-17, and
II-18
[0209] The production of compounds of Formulae II-16, II-17, and
II-18 may be carried out by cultivating strain CNB476 in a suitable
nutrient medium under conditions described herein, preferably under
submerged aerobic conditions, until a substantial amount of
compounds are detected in the fermentation; harvesting by
extracting the active components from the fermentation broth with a
suitable solvent; concentrating the solvent containing the desired
components; then subjecting the concentrated material to
chromatographic separation to isolate the compounds from other
metabolites also present in the cultivation medium.
[0210] FIG. 2 shows some collection sites worldwide for the culture
(CNB476), which is also refered to as Salinospora. FIG. 3 shows
colonies of Salinospora. FIG. 4 shows the typical 16S rDNA sequence
of the Salinospora. Bars represent characteristic signature
nucleotides of the Salinospora that separate them from their
nearest relatives.
[0211] The culture (CNB476) was deposited on Jun. 20, 2003 with the
American Type Culture Collection (ATCC) in Rockville, Md. and
assigned the ATCC patent deposition number PTA-5275. The ATCC
deposit meets all of the requirements of the Budapest treaty. The
culture is also maintained at and available from Nereus
Pharmaceutical Culture Collection at 10480 Wateridge Circle, San
Diego, Calif. 92121. In addition to the specific microorganism
described herein, it should be understood that mutants, such as
those produced by the use of chemical or physical mutagens
including X-rays, etc. and organisms whose genetic makeup has been
modified by molecular biology techniques, may also be cultivated to
produce the starting compounds of Formulae II-16, II-17, and
II-18.
[0212] Fermentation of Strain CNB476
[0213] Production of compounds can be achieved at temperature
conducive to satisfactory growth of the producing organism, e.g.
from 16 degree C. to 40 degree C., but it is preferable to conduct
the fermentation at 22 degree C. to 32 degree C. The aqueous medium
can be incubated for a period of time necessary to complete the
production of compounds as monitored by high pressure liquid
chromatography (HPLC), preferably for a period of about 2 to 10
days, on a rotary shaker operating at about 50 rpm to 400 rpm,
preferably at 150 rpm to 250 rpm, for example.
[0214] Growth of the microorganisms may be achieved by one of
ordinary skill of the art by the use of appropriate medium.
Broadly, the sources of carbon include glucose, fructose, mannose,
maltose, galactose, mannitol and glycerol, other sugars and sugar
alcohols, starches and other carbohydrates, or carbohydrate
derivatives such as dextran, cerelose, as well as complex nutrients
such as oat flour, corn meal, millet, corn, and the like. The exact
quantity of the carbon source that is utilized in the medium will
depend in part, upon the other ingredients in the medium, but an
amount of carbohydrate between 0.5 to 25 percent by weight of the
medium can be satisfactorily used, for example. These carbon
sources can be used individually or several such carbon sources may
be combined in the same medium, for example. Certain carbon sources
are preferred as hereinafter set forth.
[0215] The sources of nitrogen include amino acids such as glycine,
arginine, threonine, methionine and the like, ammonium salt, as
well as complex sources such as yeast extracts, corn steep liquors,
distiller solubles, soybean meal, cotttonseed meal, fish meal,
peptone, and the like. The various sources of nitrogen can be used
alone or in combination in amounts ranging from 0.5 to 25 percent
by weight of the medium, for example.
[0216] Among the nutrient inorganic salts, which can be
incorporated in the culture media, are the customary salts capable
of yielding sodium, potassium, magnesium, calcium, phosphate,
sulfate, chloride, carbonate, and like ions. Also included are
trace metals such as cobalt, manganese, iron, molybdenum, zinc,
cadmium, and the like.
[0217] Biological Activity and Uses of Compounds
[0218] Some embodiments relate to methods of treating cancer,
inflammation, and infectious diseases, particularly those affecting
humans. The methods may include, for example, the step of
administering an effective amount of a member of a class of new
compounds. Thus, the compounds disclosed herein may be used to
treat cancer, inflammation, and infectious disease.
[0219] The compounds have various biological activities. For
example, the compounds have chemosensitizing activity,
anti-microbial, anti-inflammation, and anti-cancer activity.
[0220] The compounds have proteasome inhibitory activity. The
proteasome inhibitory activity may, in whole or in part, contribute
to the ability of the compounds to act as anti-cancer,
anti-inflammatory, and anti-microbial agents.
[0221] The proteasome is a multisubunit protease that degrades
intracellular proteins through its chymotrypsin-like, trypsin-like
and peptidylglutamyl-peptide hydrolyzing (PGPH; and also know as
the caspase-like activity) activities. The 26S proteasome contains
a proteolytic core called the 20S proteasome and two 19S regulatory
subunits. The 20S proteasome is responsible for the proteolytic
activity against many substrates including damaged proteins, the
transcription factor NF-.kappa.B and its inhibitor I.kappa.B,
signaling molecules, tumor suppressors and cell cycle regulators.
There are three distinct protease activities within the proteasome:
1) chymotrypsin-like; 2) trypsin-like; and the 3) peptidyl glutamyl
peptide hydrolyzing (PGPH) activity.
[0222] As an example, compounds of Formula II-16 were more potent
(EC.sub.50 2 nM) at inhibiting the chymotrypsin-like activity of
rabbit muscle proteasomes than Omuralide (EC.sub.50 52 nM) and also
inhibited the chymotrypsin-like activity of human erythrocyte
derived proteasomes (EC.sub.50 .about.250 pM). FIG. 5 shows
omuralide, which is a degradation product of Lactacystin, and it
shows a compound of Formula II-16. Compounds of Formula II-16
exhibit a significant preference for inhibiting chymotrypsin-like
activity of the proteasome over inhibiting the catalytic activity
of chymotrypsin. Compounds of Formula II-16 also exhibit low nM
trypsin-like inhibitory activity (.about.10 nM), but are less
potent at inhibiting the PGPH activity of the proteasome (EC.sub.50
.about.350 nM).
[0223] Additional studies have characterized the effects of
compounds described herein, including studies of Formula II-16 on
the NF-.kappa.B/I.kappa.B signaling pathway. Treatment of HEK293
cells (human embryonic kidney) with Tumor Necrosis Factor-alpha
(TNF-.alpha.) induces phosphorylation and proteasome-mediated
degradation of I.kappa.B.alpha.: followed by NF-.kappa.B
activation. To confirm proteasome inhibition, HEK293 cells were
pre-treated for 1 hour with compounds of Formula II-16 followed by
TNF-.alpha. stimulation. Treatment with compounds of Formula II-16
promoted the accumulation of phosphorylated I.kappa.B.alpha.
suggesting that the proteasome-mediated I.kappa.B.alpha.
degradation was inhibited.
[0224] Furthermore, a stable HEK293 clone (NF-.kappa.B/Luc 293) was
generated carrying a luciferase reporter gene under the regulation
of 5.times. NF-.kappa.B binding sites. Stimulation of
NF-.kappa.B/Luc 293 cells with TNF-.alpha. increases luciferase
activity as a result of NF-.kappa.B activation while pretreatment
with compounds of Formula II-16 decreases activity. Western blot
analyses demonstrated that compounds of Formula II-16 promoted the
accumulation of phosphorylated-I.kappa.B.alpha- . and decreased the
degradation of total I.kappa.B.alpha. in the NF-.kappa.B/Luc 293
cells. Compounds of Formula II-16 were also shown to increase the
levels of the cell cycle regulatory proteins, p21 and p27.
[0225] Tumor cells may be more sensitive to proteasome inhibitors
than normal cells. Moreover, proteasome inhibition increases the
sensitivity of cancer cells to anticancer agents. The cytotoxic
activity of the compounds described herein, including Formula
II-16, were examined for cytotoxic activity against various cancer
cell lines. Formula II-16 was examined, for example, in the
National Cancer Institute screen of 60 human tumor cell lines.
Formula II-16 exhibited selective cytotoxic activity with a mean
GI.sub.50 value (the concentration to achieve 50% growth
inhibition) of less than 10 nM. The greatest potency was observed
against SK-MEL-28 melanoma and MDA-MB-235 breast cancer cells [both
with LC.sub.50 (the concentration with 50% cell lethality) <10
nM].
[0226] A panel of cell lines including human colorectal (HT-29 and
LoVo), prostate (PC3), breast (MDA-MB-23 1), lung (NCI-H292),
ovarian (OVCAR3), acute T-cell leukemia (Jurkat), murine melanoma
(B16-F10) and normal human fibroblasts (CCD-27sk) was treated with
Salinosporamide A for 48h to assess cytotoxic activity. HT-29,
LoVo, PC3, MDA-MB-231, NCI-H292, OVCAR3, Jurkat, and B16-F10 cells
were sensitive with EC.sub.50 values of 47, 69, 78, 67, 97, 69, 10,
and 33 nM, respectively. In contrast, the EC.sub.50 values for
CCD-27sk cells were 196 nM. Treatment of Jurkat cells with
Salinosporamide A at the approximate EC.sub.50 resulted in
Caspase-3 activation and cleavage of PARP confirming the induction
of apoptosis.
[0227] The anti-anthrax activity of the described compounds was
evaluated using an in vitro LeTx induced cytotoxicity assay. As one
example, the results indicate that Formula II-16 is a potent
inhibitor of LeTx-induced cytotoxicity of murine macrophage-like
RAW264.7 cells. Treatment of RAW264.7 cells with Formula II-16
resulted in a 10-fold increase in the viability of LeTx-treated
cells compared to LeTx treatment alone (average EC.sub.50 of <4
nM).
[0228] Potential Chemosensitizing Effects of Formula II-16
[0229] Additional studies have characterized the effects of the
compounds described herein on the NF-.kappa.B/I.kappa.B signaling
pathway (see the Examples). In unstimulated cells, the
transcription factor nuclear factor-kappa B (NF-?B) resides in the
cytoplasm in an inactive complex with the inhibitory protein
I.kappa.B (inhibitor of NF-.kappa.B). Various stimuli can cause I?B
phosphorylation by I?B kinase, followed by ubiquitination and
degradation by the proteasome. Following the degradation of I?B,
NF-?B translocates to the nucleus and regulates gene expression,
affecting many cellular processes including inhibition of
apoptosis. Chemotherapy agents such as CPT-11 (Irinotecan) can
activate NF-?B in human colon cancer cell lines including LoVo
cells, resulting in a decreased ability of these cells to undergo
apoptosis. Painter, R. B. Cancer Res 38:4445 (1978). Velcadey.TM.
is a dipeptidyl boronic acid that inhibits the chymotrypsin-like
activity of the proteasome (Lightcap, et al., Clin Chem 46:673
(2000), Adams, et al., Cancer Res 59:2615 (1999), Adams, Curr Opin
Oncol 14:628 (2002)) while enhancing the trypsin and PGPH
activities. Recently approved as a proteasome inhibitor,
Velcade.TM., (PS-341; Millennium Pharmaceuticals, Inc.) has been
shown to be directly toxic to cancer cells and also enhance the
cytotoxic activity of CPT-11 in LoVo cells in vitro and in a LoVo
xenograft model by inhibiting I?B degradation by the proteasome.
Blum, et al., Ann Intern Med 80:249 (1974). In addition,
Velcade.TM. was found to inhibit the expression of proangiogenic
chemokines/cytokines Growth Related Oncogene-alpha (GRO-.alpha.)
and Vascular Endothelial Growth Factor (VEGF) in squamous cell
carcinoma, presumably through inhibition of the NF-.kappa.B
pathway. Dick, et al., J Biol Chem 271:7273 (1996). These data
suggest that proteasome inhibition may not only decrease tumor cell
survival and growth, but also angiogenesis.
[0230] Anti-Anthrax Activity
[0231] Another potential application for proteasome inhibitors
comes from recent studies on the biodefense Category A agent B.
anthracis (anthrax). Anthrax spores are inhaled and lodge in the
lungs where they are ingested by macrophages. Within the
macrophage, spores germinate, the organism replicates, resulting
ultimately in killing of the cell. Before killing occurs, however,
infected macrophages migrate to the lymph nodes where, upon death,
they release their contents allowing the organism to enter the
bloodstream, further replicate, and secrete lethal toxins. Hanna,
et al., Proc Natl Acad Sci U S A 90:10198 (1993). Anthrax toxins
are responsible for the symptoms associated with anthrax. Two
proteins that play a key role in the pathogenesis of anthrax are
protective antigen (PA, 83 kDa) and lethal factor (LF, 90 kDa)
which are collectively known as lethal toxin (LeTx). LF has an
enzymatic function, but requires PA to achieve its biological
effect. Neither PA or LF cause death individually; however, when
combined they cause death when injected intravenously in animals.
Kalns, et al., Biochem Biophys Res Commun 297:506 (2002), Kalns, et
al., Biochem Biophys Res Commun 292:41 (2002).
[0232] Protective antigen, the receptor-binding component of
anthrax toxin, is responsible for transporting lethal factor into
the host cell. PA oligomerizes into a ring-shaped heptamer (see
FIG. 6). Each heptamer, bound to its receptor on the surface of a
cell, has the ability to bind up to three molecules of LF. The
complex formed between the PA heptamer and LF is taken into the
cell by receptor-mediated endocytosis. Following endocytosis, LF is
released into the cytosol where it attacks various cellular
targets. Mogridge, et al., Biochemistry 41:1079 (2002), Lacy, et
al., J Biol Chem 277:3006 (2002), Bradley, et al., Nature 414:225
(2001).
[0233] Lethal factor is a zinc dependent metalloprotease, which in
the cytosol can cleave and inactivate signaling proteins of the
mitogen-activated protein kinase kinase family (MAPKK). Duesbery,
et al., Science 280:734 (1998), Bodart, et al., Cell Cycle 1:10
(2002), Vitale, et al., J Appl Microbiol 87:288 (1999), Vitale, et
al., Biochem J 352 Pt 3:739 (2000). Of the seven different known
MAPK kinases, six have been shown to be cleaved by LF. Within the
cell, MAPK kinase pathways transduce various signals involved in
cell death, proliferation, and differentiation making these
proteins highly significant targets. However, certain inhibitors
that prevent LeTx-induced cell death, do not prevent MAPKK cleavage
by LF suggesting that this activity is not sufficient for induction
of cell death. Kim, et al., J Biol Chem 278:7413 (2003), Lin, et
al., Curr Microbiol 33:224 (1996).
[0234] Studies have suggested that inhibition of the proteasome can
prevent LeTx-induced cell death. Tang, et al., Infect Immun 67:3055
(1999). Data have shown that proteasome activity is required for
LeTx-mediated killing of RAW264.7 macrophage-like cells and that
proteasome inhibitors protect RAW264.7 cells from LeTx. Proteasome
inhibition did not block MEK1 cleavage, suggesting the LeTx pathway
is not blocked upstream of MEK1 cleavage in these studies.
Additionally, there is no increase in proteasome activity in cells
treated with LeTx. These data suggested that a novel, potent
proteasome inhibitor like the compounds described herein, may also
prevent LeTx-induced cell death as illustrated in FIG. 6.
[0235] The receptor for PA has been identified and is expressed by
many cell types. Escuyer, et al., Infect Immun 59:3381 (1991).
Lethal toxin is active in a few cell culture lines of macrophages
causing cell death within a few hours. Hanna, et al., Proc Natl
Acad Sci USA 90:10198 (1993), Kim, et al., J Biol Chem 278:7413
(2003), Lin, et al., Curr Microbiol 33:224 (1996). LeTx can induce
both necrosis and apoptosis in mouse macrophage-like RAW264.7 and
J774A.1 cells upon in vitro treatment.
[0236] The results indicate that the compounds described herein act
as a potent inhibitor of LeTx-induced cytotoxicity of murine
macrophage-like RAW264.7 cells. Treatment of RAW264.7 cells with,
for example, compounds of Formula II-16, resulted in a 10-fold
increase in the viability of LeTx-treated cells compared to LeTx
treatment alone (average EC.sub.50 of <4 nM) and therefore
provide a valuable therapy for anthrax infections. Formula II-16,
for example, promoted survival of RAW264.7 macrophage-like cells in
the presence of LeTx indicating that this compound and its
derivatives provide a valuable clinical therapeutic for anthrax
infection.
[0237] Pharmaceutical Compositions
[0238] In one embodiment, the compounds disclosed herein are used
in pharmaceutical compositions. The compounds preferably can be
produced by the methods disclosed herein. The compounds can be
used, for example, in pharmaceutical compositions comprising a
pharmaceutically acceptable carrier prepared for storage and
subsequent administration. Also, embodiments relate to a
pharmaceutically effective amount of the products and compounds
disclosed above in a pharmaceutically acceptable carrier or
diluent. Acceptable carriers or diluents for therapeutic use are
well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985), which is incorporated herein by
reference in its entirety. Preservatives, stabilizers, dyes and
even flavoring agents may be provided in the pharmaceutical
composition. For example, sodium benzoate, ascorbic acid and esters
of p-hydroxybenzoic acid may be added as preservatives. In
addition, antioxidants and suspending agents may be used.
[0239] The compositions, particularly those of Formulae I-V, may be
formulated and used as tablets, capsules, or elixirs for oral
administration; suppositories for rectal administration; sterile
solutions, suspensions for injectable administration; patches for
transdermal administration, and sub-dermal deposits and the like.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, saline, dextrose, mannitol,
lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. If desired, absorption enhancing preparations
(for example, liposomes), may be utilized.
[0240] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or other organic oils such as soybean, grapefruit or almond
oils, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0241] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses. Such formulations can be made using methods known
in the art (see, for example, U.S. Pat. No. 5,733,888 (injectable
compositions); U.S. Pat. No. 5,726,181 (poorly water soluble
compounds); U.S. Pat. No. 5,707,641 (therapeutically active
proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilic agents);
U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S. Pat.
No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676
(particulate medicaments); U.S. Pat. No. 5,654,286 (topical
formulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S.
Pat. No. 5,445,829 (extended release formulations); U.S. Pat. No.
5,653,987 (liquid formulations); U.S. Pat. No. 5,641,515
(controlled release formulations) and U.S. Pat. No. 5,601,845
(spheroid formulations); all of which are incorporated herein by
reference in their entireties.
[0242] Further disclosed herein are various pharmaceutical
compositions well known in the pharmaceutical art for uses that
include intraocular, intranasal, and intraauricular delivery.
Pharmaceutical formulations include aqueous ophthalmic solutions of
the active compounds in water-soluble form, such as eyedrops, or in
gellan gum (Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or
hydrogels (Mayer et al., Ophthalmologica, 210(2):101-3 (1996));
ophthalmic ointments; ophthalmic suspensions, such as
microparticulates, drug-containing small polymeric particles that
are suspended in a liquid carrier medium (Joshi, A. 1994 J Ocul
Pharmacol 10:29-45), lipid-soluble formulations (Alm et al., Prog.
Clin. Biol. Res., 312:447-58 (1989)), and microspheres (Mordenti,
Toxicol. Sci., 52(l):101-6 (1999)); and ocular inserts. All of the
above-mentioned references, are incorporated herein by reference in
their entireties. Such suitable pharmaceutical formulations are
most often and preferably formulated to be sterile, isotonic and
buffered for stability and comfort. Pharmaceutical compositions may
also include drops and sprays often prepared to simulate in many
respects nasal secretions to ensure maintenance of normal ciliary
action. As disclosed in Remington's Pharmaceutical Sciences (Mack
Publishing, 18.sup.th Edition), which is incorporated herein by
reference in its entirety, and well-known to those skilled in the
art, suitable formulations are most often and preferably isotonic,
slightly buffered to maintain a pH of 5.5 to 6.5, and most often
and preferably include anti-microbial preservatives and appropriate
drug stabilizers. Pharmaceutical formulations for intraauricular
delivery include suspensions and ointments for topical application
in the ear. Common solvents for such aural formulations include
glycerin and water.
[0243] When used as an anti-cancer, anti-inflammatory or
anti-microbial compound, for example, the compounds of Formulae I-V
or compositions including Formulae I-V can be administered by
either oral or non-oral pathways. When administered orally, it can
be administered in capsule, tablet, granule, spray, syrup, or other
such form. When administered non-orally, it can be administered as
an aqueous suspension, an oily preparation or the like or as a
drip, suppository, salve, ointment or the like, when administered
via injection, subcutaneously, intraperitoneally, intravenously,
intramuscularly, or the like.
[0244] In one embodiment, the anti-cancer, anti-inflammatory or
anti-microbial can be mixed with additional substances to enhance
their effectiveness. In one embodiment, the anti-microbial is
combined with an additional anti-microbial. In another embodiment,
the anti-microbial is combined with a drug or medicament that is
helpful to a patient that is taking anti-microbials.
[0245] Methods of Administration
[0246] In an alternative embodiment, the disclosed chemical
compounds and the disclosed pharmaceutical compositions are
administered by a particular method as an anti-microbial. Such
methods include, among others, (a) administration though oral
pathways, which administration includes administration in capsule,
tablet, granule, spray, syrup, or other such forms; (b)
administration through non-oral pathways, which administration
includes administration as an aqueous suspension, an oily
preparation or the like or as a drip, suppository, salve, ointment
or the like; administration via injection, subcutaneously,
intraperitoneally, intravenously, intramuscularly, intradermally,
or the like; as well as (c) administration topically, (d)
administration rectally, or (e) administration vaginally, as deemed
appropriate by those of skill in the art for bringing the compound
of the present embodiment into contact with living tissue; and (f)
administration via controlled released formulations, depot
formulations, and infusion pump delivery. As further examples of
such modes of administration and as further disclosure of modes of
administration, disclosed herein are various methods for
administration of the disclosed chemical compounds and
pharmaceutical compositions including modes of administration
through intraocular, intranasal, and intraauricular pathways.
[0247] The pharmaceutically effective amount of the compositions
that include the described compounds, including those of Formulae
I-V, required as a dose will depend on the route of administration,
the type of animal, including human, being treated, and the
physical characteristics of the specific animal under
consideration. The dose can be tailored to achieve a desired
effect, but will depend on such factors as weight, diet, concurrent
medication and other factors which those skilled in the medical
arts will recognize.
[0248] In practicing the methods of the embodiment, the products or
compositions can be used alone or in combination with one another,
or in combination with other therapeutic or diagnostic agents.
These products can be utilized in vivo, ordinarily in a mammal,
preferably in a human, or in vitro. In employing them in vivo, the
products or compositions can be administered to the mammal in a
variety of ways, including parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, vaginally,
nasally or intraperitoneally, employing a variety of dosage forms.
Such methods may also be applied to testing chemical activity in
vivo.
[0249] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0250] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage may range broadly, depending
upon the desired affects and the therapeutic indication. Typically,
dosages may be between about 10 microgram/kg and 100 mg/kg body
weight, preferably between about 100 microgram/kg and 10 mg/kg body
weight. Alternatively dosages may be based and calculated upon the
surface area of the patient, as understood by those of skill in the
art. Administration is preferably oral on a daily or twice daily
basis.
[0251] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. See for example, Fingl et al., in The Pharmacological
Basis of Therapeutics, 1975, which is incorporated herein by
reference in its entirety. It should be noted that the attending
physician would know how to and when to terminate, interrupt, or
adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the disorder of interest will vary with
the severity of the condition to be treated and to the route of
administration. The severity of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary
according to the age, body weight, and response of the individual
patient. A program comparable to that discussed above may be used
in veterinary medicine.
[0252] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
A variety of techniques for formulation and administration may be
found in Remington's Pharmaceutical Sciences, 18th Ed., Mack
Publishing Co., Easton, Pa. (1990), which is incorporated herein by
reference in its entirety. Suitable administration routes may
include oral, rectal, transdermal, vaginal, transmucosal, or
intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections.
[0253] For injection, the agents of the embodiment may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the embodiment into
dosages suitable for systemic administration is within the scope of
the embodiment. With proper choice of carrier and suitable
manufacturing practice, the compositions disclosed herein, in
particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the
embodiment to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated.
[0254] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external micro-environment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0255] Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration may be in the form
of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions may be manufactured in a manner that is itself known,
for example, by means of conventional mixing, dissolving,
granulating, dragee-making, levitating, emulsifying, encapsulating,
entrapping, or lyophilizing processes.
[0256] Compounds disclosed herein can be evaluated for efficacy and
toxicity using known methods. For example, the toxicology of a
particular compound, or of a subset of the compounds, sharing
certain chemical moieties, may be established by determining in
vitro toxicity towards a cell line, such as a mammalian, and
preferably human, cell line. The results of such studies are often
predictive of toxicity in animals, such as mammals, or more
specifically, humans. Alternatively, the toxicity of particular
compounds in an animal model, such as mice, rats, rabbits, dogs or
monkeys, may be determined using known methods. The efficacy of a
particular compound may be established using several art recognized
methods, such as in vitro methods, animal models, or human clinical
trials. Art-recognized in vitro models exist for nearly every class
of condition, including the conditions abated by the compounds
disclosed herein, including cancer, cardiovascular disease, and
various immune dysfunction, and infectious diseases. Similarly,
acceptable animal models may be used to establish efficacy of
chemicals to treat such conditions. When selecting a model to
determine efficacy, the skilled artisan can be guided by the state
of the art to choose an appropriate model, dose, and route of
administration, and regime. Of course, human clinical trials can
also be used to determine the efficacy of a compound in humans.
[0257] When used as an anti-microbial, anti-cancer, or
anti-inflammatory agent, the compounds disclosed herein may be
administered by either oral or a non-oral pathways. When
administered orally, it can be administered in capsule, tablet,
granule, spray, syrup, or other such form. When administered
non-orally, it can be administered as an aqueous suspension, an
oily preparation or the like or as a drip, suppository, salve,
ointment or the like, when administered via injection,
subcutaneously, intraperitoneally, intravenously, intramuscularly,
intradermally, or the like. Controlled release formulations, depot
formulations, and infusion pump delivery are similarly
contemplated.
[0258] The compositions disclosed herein in pharmaceutical
compositions may also comprise a pharmaceutically acceptable
carrier. Such compositions may be prepared for storage and for
subsequent administration. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, such
compositions may be formulated and used as tablets, capsules or
solutions for oral administration; suppositories for rectal or
vaginal administration; sterile solutions or suspensions for
injectable administration. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients include, but are
not limited to, saline, dextrose, mannitol, lactose, lecithin,
albumin, sodium glutamate, cysteine hydrochloride, and the like. In
addition, if desired, the injectable pharmaceutical compositions
may contain minor amounts of nontoxic auxiliary substances, such as
wetting agents, pH buffering agents, and the like. If desired,
absorption enhancing preparations (for example, liposomes), may be
utilized.
[0259] The pharmaceutically effective amount of the composition
required as a dose will depend on the route of administration, the
type of animal being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize.
[0260] The products or compositions of the embodiment, as described
above, may be used alone or in combination with one another, or in
combination with other therapeutic or diagnostic agents. These
products can be utilized in vivo or in vitro. The useful dosages
and the most useful modes of administration will vary depending
upon the age, weight and animal treated, the particular compounds
employed, and the specific use for which these composition or
compositions are employed. The magnitude of a dose in the
management or treatment for a particular disorder will vary with
the severity of the condition to be treated and to the route of
administration, and depending on the disease conditions and their
severity, the compositions may be formulated and administered
either systemically or locally. A variety of techniques for
formulation and administration may be found in Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.
(1990).
[0261] To formulate the compounds of Formulae I-V as an
anti-microbial, an anti-cancer, or an anti-inflammatory agent,
known surface active agents, excipients, smoothing agents,
suspension agents and pharmaceutically acceptable film-forming
substances and coating assistants, and the like may be used.
Preferably alcohols, esters, sulfated aliphatic alcohols, and the
like may be used as surface active agents; sucrose, glucose,
lactose, starch, crystallized cellulose, mannitol, light anhydrous
silicate, magnesium aluminate, magnesium methasilicate aluminate,
synthetic aluminum silicate, calcium carbonate, sodium acid
carbonate, calcium hydrogen phosphate, calcium carboxymethyl
cellulose, and the like may be used as excipients; magnesium
stearate, talc, hardened oil and the like may be used as smoothing
agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be
used as suspension agents or lubricants; cellulose acetate
phthalate as a derivative of a carbohydrate such as cellulose or
sugar, or methylacetate-methacrylate copolymer as a derivative of
polyvinyl may be used as suspension agents; and plasticizers such
as ester phthalates and the like may be used as suspension agents.
In addition to the foregoing preferred ingredients, sweeteners,
fragrances, colorants, preservatives and the like may be added to
the administered formulation of the compound produced by the method
of the embodiment, particularly when the compound is to be
administered orally.
[0262] The compounds and compositions may be orally or non-orally
administered to a human patient in the amount of about 0.001
mg/kg/day to about 10,000 mg/kg/day of the active ingredient, and
more preferably about 0.1 mg/kg/day to about 100 mg/kg/day of the
active ingredient at, preferably, one time per day or, less
preferably, over two to about ten times per day. Alternatively and
also preferably, the compound produced by the method of the
embodiment may preferably be administered in the stated amounts
continuously by, for example, an intravenous drip. Thus, for the
example of a patient weighing 70 kilograms, the preferred daily
dose of the active or anti-infective ingredient would be about 0.07
mg/day to about 700 gm/day, and more preferable, 7 mg/day to about
7 grams/day. Nonetheless, as will be understood by those of skill
in the art, in certain situations it may be necessary to administer
the anti-cancer, anti-inflammatory or the anti-infective compound
of the embodiment in amounts that excess, or even far exceed, the
above-stated, preferred dosage range to effectively and
aggressively treat particularly advanced cancerss or
infections.
[0263] In the case of using the anti-microbial produced by methods
of the embodiment as a biochemical test reagent, the compound
produced by methods of the embodiment inhibits the progression of
the disease when it is dissolved in an organic solvent or hydrous
organic solvent and it is directly applied to any of various
cultured cell systems. Usable organic solvents include, for
example, methanol, methylsulfoxide, and the like. The formulation
can, for example, be a powder, granular or other solid inhibitor,
or a liquid inhibitor prepared using an organic solvent or a
hydrous organic solvent. While a preferred concentration of the
compound produced by the method of the embodiment for use as an
anti-microbial, anticancer or anti-tumor compound is generally in
the range of about 1 to about 100 .mu.g/ml, the most appropriate
use amount varies depending on the type of cultured cell system and
the purpose of use, as will be appreciated by persons of ordinary
skill in the art. Also, in certain applications it may be necessary
or preferred to persons of ordinary skill in the art to use an
amount outside the foregoing range.
[0264] In one embodiment, the method of using a compound as an
anti-microbial, anti-cancer or anti-inflammatory involves
administering an effective amount of -any of the compounds of
Formulae I-V or compositions of those compounds. In a preferred
embodiment, the method involves administering the compound
represented by Formula II, to a patient in need of an
anti-microbial, until the need is effectively reduced or more
preferably removed.
[0265] As will be understood by one of skill in the art, "need" is
not an absolute term and merely implies that the patient can
benefit from the treatment of the anti-microbial, the anti-cancer,
or anti-inflammatory in use. By "patient" what is meant is an
organism that can benefit by the use of an anti-microbial,
anti-cancer or anti-inflammatory agent. For example, any organism
with B. anthracis, Plasmodium, Leishmania, Trypanosoma, and the
like, may benefit from the application of an anti-microbial that
may in turn reduce the amount of microbes present in the patient.
As another example, any organism with cancer, such as, a colorectal
carcinoma, a prostate carcinoma, a breast adenocarcinoma, a
non-small cell lung carcinoma, an ovarian carcinoma, multiple
myelomas, a melanoma, and the like, may benefit from the
application of an anti-cancer agent that may in turn reduce the
amount of cancer present in the patient. Furthermore, any organism
with an inflammatory conditions, such as, rheumatoid arthritis,
asthma, multiple sclerosis, psoriasis, stroke, myocardial
infarction, and the like, may benefit from the application of an
anti-inflammatory that may in turn reduce the amount of cells
associated with the inflammatory response present in the patient.
In one embodiment, the patient's health may not require that an
anti-microbial, anti-cancer, or anti-inflammatory be administered,
however, the patient may still obtain some benefit by the reduction
of the level of microbes, cancer cells, or inflammatory cells
present in the patient, and thus be in need. In one embodiment, the
anti-microbial or anti-cancer agent is effective against one type
of microbe or cancer, but not against other types; thus, allowing a
high degree of selectivity in the treatment of the patient. In
other embodiments, the anti-inflammatory may be effective against
inflammatory conditions characterized by different cells associated
with the inflammation. In choosing such an anti-microbial,
anti-cancer or anti-inflammatory agent, the methods and results
disclosed in the Examples may be useful. In an alternative
embodiment, the anti-microbial may be effective against a broad
spectrum of microbes, preferably a broad spectrum of foreign, and,
more preferably, harmful bacteria, to the host organism. In
embodiments, the anti-cancer and/or anti-inflammatory agent may be
effective against a broad spectrum of cancers and inflammatory
conditions/cells/substances. In yet another embodiment, the
anti-microbial is effective against all microbes, even those native
to the host. Examples of microbes that may be targets of
anti-microbials, include, but are not limited to, B. anthracis,
Plasmodium, Leishmania, Trypanosoma, and the like. In still further
embodiments, the anti-cancer agent is effective against a broad
spectrum of cancers or all cancers. Examples of cancers, against
which the compounds may be effective include a colorectal
carcinoma, a prostate carcinoma, a breast adenocarcinoma, a
non-small cell lung carcinoma, an ovarian carcinoma, multiple
myelomas, a melanoma, and the like. Exemplary inflammatory
conditions against which the agents are effective include
rheumatoid arthritis, asthma, multiple sclerosis, psoriasis,
stroke, myocardial infarction, and the like.
[0266] "Therapeutically effective amount," "pharmaceutically
effective amount," or similar term, means that amount of drug or
pharmaceutical agent that will result in a biological or medical
response of a cell, tissue, system, animal, or human that is being
sought. In a preferred embodiment, the medical response is one
sought by a researcher, veterinarian, medical doctor, or other
clinician.
[0267] "Anti-microbial" refers to a compound that reduces the
likelihood of survival of microbes, or blocks or alleviates the
deleterious effects of a microbe. In one embodiment, the likelihood
of survival is determined as a function of an individual microbe;
thus, the anti-microbial will increase the chance that an
individual microbe will die. In one embodiment, the likelihood of
survival is determined as a function of a population of microbes;
thus, the anti-microbial will increase the chances that there will
be a decrease in the population of microbes. In one embodiment,
anti-microbial means antibiotic or other similar term. Such
anti-microbials are capable of blocking the harmful effects,
destroying or suppressing the growth or reproduction of
microorganisms, such as bacteria. For example, such antibacterials
and other anti-microbials are described in Antibiotics,
Chemotherapeutics and Antibacterial Agents for Disease Control (M.
Grayson, editor, 1982), and E. Gale et al., The Molecular Basis of
Antibiotic Action 2d edition (1981). In another embodiment, an
anti-microbial will not change the likelihood of survival, but will
change the chances that the microbes will be harmful to the host in
some way. For instance, if the microbe secretes a substance that is
harmful to the host, the anti-microbial may act upon the microbe to
stop the secretion or may counteract or block the harmful effect.
In one embodiment, an anti-microbial, while, increasing the
likelihood that the microbe(s) will die, is minimally harmful to
the surrounding, non-microbial, cells. In an alternative
embodiment, it is not important how harmful the anti-microbial is
to surrounding, nonmicrobial, cells, as long as it reduces the
likelihood of survival of the microbe.
[0268] "Anti-cancer agent" refers to a compound or composition
including the compound that reduces the likelihood of survival of a
cancer cell. In one embodiment, the likelihood of survival is
determined as a function of an individual cancer cell; thus, the
anti-cancer agent will increase the chance that an individual
cancer cell will die. In one embodiment, the likelihood of survival
is determined as a function of a population of cancer cells; thus,
the anti-cancer agent will increase the chances that there will be
a decrease in the population of cancer cells. In one embodiment,
anti-cancer agent means chemotherapeutic agent or other similar
term.
[0269] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of a neoplastic disease, such as cancer. Examples of
chemotherapeutic agents include alkylating agents, such as a
nitrogen mustard, an ethyleneimine and a methylmelamine, an alkyl
sulfonate, a nitrosourea, and a triazene, folic acid antagonists,
anti-metabolites of nucleic acid metabolism, antibiotics,
pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides,
amines, amino acids, triazol nucleosides, corticosteroids, a
natural product such as a vinca alkaloid, an epipodophyllotoxin, an
antibiotic, an enzyme, a taxane, and a biological response
modifier; miscellaneous agents such as a platinum coordination
complex, an anthracenedione, an anthracycline, a substituted urea,
a methyl hydrazine derivative, or an adrenocortical suppressant; or
a hormone or an antagonist such as an adrenocorticosteroid, a
progestin, an estrogen, an antiestrogen, an androgen, an
antiandrogen, or a gouadotropin-releasing hormone analog. Specific
examples include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine
arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan,
Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan,
Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,
Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,
Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,
Esperamicins, Melphalan, and other related nitrogen mustards. Also
included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors, such as tamoxifen and
onapristone.
[0270] The anti-cancer agent may act directly upon a cancer cell to
kill the cell, induce death of the cell, to prevent division of the
cell, and the like. Alternatively, the anti-cancer agent may
indirectly act upon the cancer cell by limiting nutrient or blood
supply to the cell, for example. Such anti-cancer agents are
capable of destroying or suppressing the growth or reproduction of
cancer cells, such as a colorectal carcinoma, a prostate carcinoma,
a breast adenocarcinoma, a non-small cell lung carcinoma, an
ovarian carcinoma, multiple myelomas, a melanoma, and the like.
[0271] A "neoplastic disease" or a "neoplasm" refers to a cell or a
population of cells, including a tumor or tissue (including cell
suspensions such as bone marrow and fluids such as blood or serum),
that exhibits abnormal growth by cellular proliferation greater
than normal tissue. Neoplasms can be benign or malignant.
[0272] An "inflammatory condition" includes, for example,
conditions such as ischemia, septic shock, autoimmune diseases,
rheumatoid arthritis, inflammatory bowel disease, systemic lupus
eythematosus, multiple sclerosis, asthma, osteoarthritis,
osteoporosis, fibrotic diseases, dermatosis, including psoriasis,
atopic dermatitis and ultraviolet radiation (UV)-induced skin
damage, psoriatic arthritis, alkylosing spondylitis, tissue and
organ rejection, Alzheimer's disease, stroke, atherosclerosis,
restenosis, diabetes, glomerulonephritis, cancer, Hodgkins disease,
cachexia, inflammation associated with infection and certain viral
infections, including acquired immune deficiency syndrome (AIDS),
adult respiratory distress syndrome and Ataxia Telangiestasia.
[0273] In one embodiment, a described compound, preferably a
compound having the Formulae I-V, including those as described
herein, is considered an effective anti-microbial, anti-cancer, or
anti-inflammatory if the compound can influence 10% of the
microbes, cancer cells, or inflammatory cells, for example. In a
more preferred embodiment, the compound is effective if it can
influence 10 to 50% of the microbes, cancer cells, or inflammatory
cells. In an even more preferred embodiment, the compound is
effective if it can influence 50-80% of the microbes, cancer cells,
or inflammatory cells. In an even more preferred embodiment, the
compound is effective if it can influence 80-95% of the microbes,
cancer cells, or inflammatory cells. In an even more preferred
embodiment, the compound is effective if it can influence 95-99% of
the microbes, cancer cells, or inflammatory cells. "Influence" is
defined by the mechanism of action for each compound. Thus, for
example, if a compound prevents the reproduction of microbes, then
influence is a measure of prevention of reproduction. Likewise, if
a compound destroys microbes, then influence is a measure of
microbe death. Also, for example, if a compound prevents the
division of cancer cells, then influence is a measure of prevention
of cancer cell division. Further, for example, if a compound
prevents the proliferation of inflammatory cells, then influence is
a measure of prevention of inflammatory cell proliferation. Not all
mechanisms of action need be at the same percentage of
effectiveness. In an alternative embodiment, a low percentage
effectiveness may be desirable if the lower degree of effectiveness
is offset by other factors, such as the specificity of the
compound, for example. Thus a compound that is only 10% effective,
for example, but displays little in the way of harmful side-effects
to the host, or non-harmful microbes or cells, can still be
considered effective.
[0274] In one embodiment, the compounds described herein are
administered simply to remove microbes, cancer cells or
inflammatory cells, and need not be administered to a patient. For
example, in situations where microbes can present a problem, such
as in food products, the compounds described herein can be
administered directly to the products to reduce the risk of
microbes in the products. Alternatively, the compounds can be used
to reduce the level of microbes present in the surrounding
environment, such working surfaces. As another example, the
compounds can be administered ex vivo to a cell sample, such as a
bone marrow or stem cell transplant to ensure that only
non-cancerous cells are introduced into the recipient. After the
compounds are administered they may optionally be removed. This may
be particularly desirable in situations where work surfaces or food
products may come into contact with other surfaces or organisms
that could risk being harmed by the compounds. In an alternative
embodiment, the compounds may be left in the food products or on
the work surfaces to allow for a more protection. Whether or not
this is an option will depend upon the relative needs of the
situation and the risks associated with the compound, which in part
can be determined as described in the Examples below.
[0275] The following non-limiting examples are meant to describe
the preferred embodiments of the methods. Variations in the details
of the particular methods employed and in the precise chemical
compositions obtained will undoubtedly be appreciated by those of
skill in the art.
EXAMPLES
Example 1
Fermentation of Compound of Formulae II-16 II-20. and II-24C
[0276] Strain CNB476 was grown in a 500-ml flask containing 100 ml
of vegetative medium consisting of the following per liter of
deionized water: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone,
5 g; and synthetic sea salt (Instant Ocean, Aquarium Systems), 30
g. The first seed culture was incubated at 28 degree C. for 3 days
on a rotary shaker operating at 250 rpm. Four ml each of the first
seed culture was inoculated into three 500-ml flasks containing of
100 ml of the vegetative medium. The second seed cultures were
incubated at 28 degree C. and 250 rpm on a rotary shaker for 2
days. Four ml each of the second seed culture was inoculated into
thirty-five 500-ml flasks containing of 100 ml of the vegetative
medium. The third seed cultures were incubated at 28 degree and 250
rpm on a rotary shaker for 2 days. Four ml each of the third seed
culture was inoculated into four hundred 500-ml flasks containing
100 ml of the production medium consisting of the following per
liter of deionized water: starch, 10 g; yeast extract, 4 g; Hy-Soy,
4 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium
carbonate, 1 g; and synthetic sea salt (Instant Ocean, Aquarium
Systems), 30 g. The production cultures were incubated at 28 degree
C. and 250 rpm on roatry shakers for 1 day. Approximately 2 to 3
grams of sterile Amberlite XAD-7 resin were added to the production
cultures. The production cultures were further incubated at 28
degree C. and 250 rpm on rotary shakers for 5 days. The culture
broth was filtered through cheese cloth to recover the Amberlite
XAD-7 resin. The resin was extracted with 2 times 6 liters ethyl
acetate followed by 1 time 1.5 liters ethyl acetate. The combined
extracts were dried in vacuo. The dried extract, containing 3.8
grams the compound of Formula II-16 and lesser quantities of
compounds of formulae II-20 and II-24C, was then processed for the
recovery of the compounds of Formula II-16, II-20 and II-24C.
Example 2
Purification of Compound of Formulae II-16, II-20 and II-24C
[0277] The pure compounds of Formulae II-16, II-20 and II-24C were
obtained by flash chromatography followed by HPLC. Eight grams
crude extract containing 3.8 grams of the compound of Formula II-16
and lesser quantities of II-20 and II-24C was processed by flash
chromatography using Biotage Flash40i system and Flash 40M
cartridge (KP-Sil Silica, 32-63 .mu.m, 90 grams). The flash
chromatography was developed by the following step gradient:
[0278] 1. Hexane (1 L)
[0279] 2. 10% Ethyl acetate in hexane (1 L)
[0280] 3. 20% Ethyl acetate in hexane, first elution (1 L)
[0281] 4. 20% Ethyl acetate in hexane, second elution (1 L)
[0282] 5. 20% Ethyl acetate in hexane, third elution (1 L)
[0283] 6. 25% Ethyl acetate in hexane (1 L)
[0284] 7. 50% Ethyl acetate in hexane (1 L)
[0285] 8. Ethyl acetate (1 L)
[0286] Fractions containing the compound of Formula II-16 in
greater or equal to 70% UV purity by HPLC were pooled and subject
to HPLC purification, as described below, to obtain II-16, along
with II-20 and II-24C, each as pure compounds
1 Column Phenomenex Luna 10u Silica Dimensions 25 cm .times. 21.2
mm ID Flow rate 25 ml/min Detection ELSD Solvent Gradient of 24%
EtOAc/hexane for 19 min, 24% EtOAc/hexane to 100% EtOAc in 1 min,
then 100% EtOAc for 4 min
[0287] The fraction enriched in compound of Formula II-16
(described above; .about.70% pure with respect to II-16) was
dissolved in acetone (60 mg/ml). Aliquots (950 ul) of this solution
were injected onto a normal-phase HPLC column using the conditions
described above. The compound of Formula II-16 eluted at about 14
minutes, and minor compounds II-24C and II-20 eluted at 11 and 23
minutes, respectively. Fractions containing II-16, II-24C, and
II-20 were pooled based on composition of compound present.
Fractions containing the desired compounds were concentrated under
reduced pressure to yield pure compound of Formula II-16, as well
as separate fractions containing II-24C and II-20, which were
further purified as described below.
[0288] Sample containing II-24C (70 mg) was dissolved in
acetonitrile at a concentration of 10 mg/ml, and 500 .mu.l was
loaded on an HPLC column of dimensions 21 mm i.d. by 15 cm length
containing Eclipse XDB-C18 support. The solvent gradient increased
linearly from 15% acetonitrile/85% water to 100% acetonitrile over
23 minutes at a flow rate of 14.5 ml/min. The solvent composition
was held at 100% acetonitrile for 3 minutes before returning to the
starting solvent mixture. Compound II-24C eluted at 19 minutes as a
pure compound under these conditions.
[0289] To obtain pure compound II-20, the enriched samples
generated from the preparative HPLC method described above were
triturated with EtOAc to remove minor lipophilic impurities. The
resulting sample contained compound II-20 in >95% purity.
[0290] Compound of Formula II-16: UV (Acetonitrile/H.sub.2O)
.lambda..sub.max 225(sh) nm. Low Res. Mass: m/z 314 (M+H), 336
(M+Na).
[0291] Compound of Formula II-20: UV (Acetonitrile/H.sub.2O)
.lambda..sub.max 225(sh) nm. Low Res. Mass: m/z 266 (M+H). FIG. 7
depicts the 1 H NMR spectrum of a compound having the structure of
Formula II-20.
[0292] Compound of Formula II-24C: UV (Acetonitrile/H.sub.2O)
.lambda..sub.max 225(sh) nm. Low Res. Mass: m/z 328 (M+H), 350
(M+Na). FIG. 8 depicts the 1H NMR spectrum of a compound having the
structure of Formula II-24C.
Example 3
Fermentation of Compounds of Formulae II-17 and II-18
[0293] Strain CNB476 was grown in a 500-ml flask containing 100 ml
of the first vegetative medium consisting of the following per
liter of deionized water: glucose, 4 g; Bacto tryptone, 3 g; Bacto
casitone, 5 g; and synthetic sea salt (Instant Ocean, Aquarium
Systems), 30 g. The first seed culture was incubated at 28 degree
C. for 3 days on a rotary shaker operating at 250 rpm. Five ml of
the first seed culture was inoculated into a 500-ml flask
containing 100 ml of the second vegetative medium consisting of the
following per liter of deionized water: starch, 10 g; yeast
extract, 4 g; peptone, 2 g; ferric sulfate, 40 mg; potassium
bromide, 100 mg; calcium carbonate, 1 g; and sodium bromide, 30 g.
The second seed cultures were incubated at 28.degree. C. for 7 days
on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gram
of sterile Amberlite XAD-7 resin were added to the second seed
culture. The second seed culture was further incubated at
28.degree. C. for 2 days on a rotary shaker operating at 250 rpm.
Five ml of the second seed culture was inoculated into a 500-ml
flask containing 100 ml of the second vegetative medium. The third
seed culture was incubated at 28.degree. C. for 1 day on a rotary
shaker operating at 250 rpm. Approximately 2 to 3 gram of sterile
Amberlite XAD-7 resin were added to the third seed culture. The
third seed culture was further incubated at 28.degree. C. for 2
days on a rotary shaker operating at 250 rpm. Five ml of the third
culture was inoculated into a 500-ml flask containing 100 ml of the
second vegetative medium. The fourth seed culture was incubated at
28.degree. C. for 1 day on a rotary shaker operating at 250 rpm.
Approximately 2 to 3 gram of sterile Amberlite XAD-7 resin were
added to the fourth seed culture. The fourth seed culture was
further incubated at 28.degree. C. for 1 day on a rotary shaker
operating at 250 rpm. Five ml each of the fourth seed culture was
inoculated into ten 500-ml flasks containing 100 ml of the second
vegetative medium. The fifth seed cultures were incubated at
28.degree. C. for 1 day on a rotary shaker operating at 250 rpm.
Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin were
added to the fifth seed cultures. The fifth seed cultures were
further incubated at 28.degree. C. for 3 days on a rotary shaker
operating at 250 rpm. Four ml each of the fifth seed culture was
inoculated into one hundred and fifty 500-ml flasks containing 100
ml of the production medium having the same composition as the
second vegetative medium. Approximately 2 to 3 grams of sterile
Amberlite XAD-7 resin were also added to the production culture.
The production cultures were incubated at 28.degree. C. for 6 day
on a rotary shaker operating at 250 rpm. The culture broth was
filtered through cheese cloth to recover the Amberlite XAD-7 resin.
The resin was extracted with 2 times 3 liters ethyl acetate
followed by 1 time 1 liter ethyl acetate. The combined extracts
were dried in vacuo. The dried extract, containing 0.42 g of the
compound Formula II-17 and 0.16 gram the compound of Formula II-18,
was then processed for the recovery of the compounds.
Example 4
Purification of Compounds of Formula II-17 and II-18
[0294] The pure compounds of Formula II-17 and II-18 were obtained
by reversed-phase HPLC as described below:
2 Column ACE 5 C18-HL Dimensions 15 cm .times. 21 mm ID Flow rate
14.5 ml/min Detection 214 nm Solvent Gradient of 35%
Acetonitrile/65% H.sub.2O to 90% Acetonitrile/10% H.sub.2O over 15
min
[0295] Crude extract (100 mg) was dissolved in 15 ml of
acetonitrile. Aliquots (900 ul) of this solution were injected onto
a reversed-phase HPLC column using the conditions described above.
Compounds of Formulae II-17 and II-18 eluted at 7.5 and 9 minutes,
respectively. Fractions containing the pure compounds were first
concentrated using nitrogen to remove organic solvent. The
remaining solution was then frozen and lyophilized to dryness.
[0296] Compound of Formula II-17: UV (Acetonitrile/H.sub.2O)
.lambda..sub.max 225(sh) nm. High Res. Mass (APCI): m/z 280.156
(M+H), .DELTA..sub.calc=2.2 ppm, C.sub.15H.sub.22NO.sub.4 FIG. 49
depicts the .sup.1H NMR spectrum of a compound having the structure
of Formula II-17.
[0297] Compound of Formula II-18: UV (Acetonitrile/H.sub.2O)
.lambda..sub.max 225(sh) nm. High Res. Mass (APCI): m/z 358.065
(M+H), .DELTA..sub.calc=-1.9 ppm, C.sub.15H.sub.21NO.sub.4Br. FIG.
50 depicts the .sup.1H NMR spectrum of a compound having the
structure of Formula II-18.
Example 5
Preparation of Compound of Formula II-19 from II-16
[0298] A sample of compound of Formula II-16 (250 mg) was added to
an acetone solution of sodium iodide (1.5 g in 10 ml) and the
resulting mixture stirred for 6 days. The solution was then
filtered through a 0.45 micron syringe filter and injected directly
on a normal phase silica HPLC column (Phenomenex Luna 10 u Silica,
25 cm.times.21.2 mm) in 0.95 ml aliquots. The HPLC conditions for
the separation of compound formula II-19 from unreacted II-16
employed an isocratic HPLC method consisting of 24% ethyl acetate
and 76% hexane, in which the majority of compound II-19 eluted 2.5
minutes before compound II-16. Equivalent fractions from each of 10
injections were pooled to yield 35 mg compound II-19. Compound
II-19: UV (Acetonitrile/H.sub.2O) 225 (sh), 255 (sh) nm; ESMS, m/z
406.0 (M+H); .sup.1H NMR in DMSO-d.sub.6 (see FIG. 9). 62
Example 6
Synthesis of the Compounds of Formulae II-2, II-3, and II-4
[0299] Compounds of Formulae II-2, II-3 and II-4 can be synthesized
from compounds of Formulae II-16, II-17 and II-18, respectively, by
catalytic hydrogenation.
[0300] Exemplary Depiction of Synthesis 63
Example 6A
Catalytic Hydrogenation of Compound of Formula II-16
[0301] Compound of Formula II-16 (10 mg) was dissolved in acetone
(5 mL) in a scintillation vial (20 mL) to which was added the 10%
(w/w) Pd/C (1-2mg) and a magnetic stirrer bar. The reaction mixture
was stirred in a hydrogen atmosphere at room temperature for about
15 hours. The reaction mixture was filtered through a 3 cc silica
column and washed with acetone. The filtrate was filtered again
through 0.2 .mu.m Gelman Acrodisc to remove any traces of catalyst.
The solvent was evaporated off from filtrate under reduced pressure
to yield the compound of Formula II-2 as a pure white powder: UV
(acetonitrile/H.sub.2O): .lambda..sub.max 225 (sh) nm. FIG. 10
depicts the NMR spectrum of the compound of Formula II-2 in
DMSO-d6. FIG. 11 depicts the low resolution mass spectrum of the
compound of Formula II-2: m/z 316 (M+H), 338 (M+Na).
Example 6B
Catalytic Hydrogenation of Compound of Formula II-17
[0302] Compound of Formula II-17 (5 mg) was dissolved in acetone (3
mL) in a scintillation vial (20 mL) to which was added the 10%
(w/w) Pd/C (about lmg) and a magnetic stirrer bar. The reaction
mixture was stirred in a hydrogen atmosphere at room temperature
for about 15 hours. The reaction mixture was filtered through a 0.2
.mu.m Gelman Acrodisc to remove the catalyst. The solvent was
evaporated off from filtrate to yield the compound of Formula II-3
as a white powder which was purified by normal phase HPLC using the
following conditions:
3 Column: Phenomenex Luna 10u Silica Dimensions: 25 cm .times. 21.2
mm ID Flow rate: 14.5 ml/min Detection: ELSD Solvent: 5% to 60%
EtOAc/Hex for 19 min, 60 to 100% EtOAc in 1 min, then 4 min at 100%
EtOAc
[0303] Compound of Formula II-3 eluted at 22.5 min as a pure
compound: UV (acetonitrile/H.sub.2O): .lambda..sub.max 225 (sh) nm.
FIG. 12 depicts the NMR spectrum of the compound of Formula II-3 in
DMSO-d6. FIG. 13 depicts the low resolution mass spectrum of the
compound of Formula II-3: m/z 282 (M+H), 304 (M+Na).
Example 6C
Catalytic Hydrogenation of Compound of Formula II-18
[0304] 3.2 mg of compound of Formula II-18 was dissolved in acetone
(3 mL) in a scintillation vial (20 mL) to which was added the 10%
(w/w) Pd/C (about 1 mg) and a magnetic stirrer bar. The reaction
mixture was stirred in hydrogen atmosphere at room temperature for
about 15 hours. The reaction mixture was filtered through a 0.2
.mu.m Gelman Acrodisc to remove the catalyst. The solvent was
evaporated off from filtrate to yield the compound of Formula II-4
as a white powder which was further purified by normal phase HPLC
using the following conditions:
4 Column: Phenomenex Luna 10u Silica Dimensions: 25 cm .times. 21.2
mm ID Flow rate: 14.5 ml/min Detection: ELSD Solvent: 5% to 80%
EtOAc/Hex for 19 min, 80 to 100% EtOAc in 1 min, then 4 min at 100%
EtOAc
[0305] Compound of Formula II-4 eluted at 16.5 min as a pure
compound: UV (acetonitrile/H.sub.2O): .lambda..sub.max 225 (sh) nm.
FIG. 14 depicts the NMR spectrum of the compound of Formula II-4 in
DMSO-d6. FIG. 15 depicts the low resolution mass of the compound of
Formula II-4: m/z 360 (M+H), 382 (M+Na).
Example 7
Synthesis of the Compounds of Formulae II-5A and II-5B
[0306] Compounds of Formula II-5A and Formula II-5B can be
synthesized from compound of Formula II-16 by epoxidation with
mCPBA.
[0307] Compound of Formula II-16 (101 mg, 0.32 mmole) was dissolved
in methylenechloride (30 mL) in a 100 ml of round bottom flask to
which was added 79 mg (0.46 mmole) of meta-chloroperbenzoic acid
(mCPBA) and a magnetic stir bar. The reaction mixture was stirred
at room temperature for about 18 hours. The reaction mixture was
poured onto a 20 cc silica flash column and eluted with 120 ml of
CH.sub.2Cl.sub.2, 75 ml of 1:1 ethyl acetate/hexane and finally
with 40 ml of 100% ethyl acetate. The 1:1 ethyl acatete/hexane
fractions yield a mixture of diastereomers of epoxyderivatives,
Formula II-5A and II-5B, which were separated by normal phase HPLC
using the following conditions:
5 Column Phenomenex Luna 10u Silica Dimensions 25 cm .times. 21.2
mm ID Flow rate 14.5 ml/min Detection ELSD Solvent 25% to 80%
EtOAc/Hex over 19 min, 80 to 100% EtOAc in 1 min, then 5 min at
100% EtOAc
[0308] Compound Formula II-5A (major product) and II-5B (minor
product) eluted at 21.5 and 19 min, respectively, as pure
compounds. Compound II-5B was further chromatographed on a 3cc
silica flash column to remove traces of chlorobenzoic acid reagent.
64
[0309] Structural Characterization
[0310] Formula II-5A: UV (Acetonitrile/H.sub.2O) .lambda..sub.max
225 (sh) nm. Low Res. Mass: m/z 330 (M+H), 352 (M+Na). FIGS. 16-17,
respectively depict the 1H NMR spectrum of Formula II-5A and the
mass spectrum of Formula II-5A.
[0311] Formula II-5B: UV (Acetonitrile/H.sub.2O) .lambda..sub.max
225 (sh) nm. Low Res. Mass: m/z 330 (M+H), 352 (M+Na). FIGS. 18-19,
respectively depict the 1H NMR spectrum of II-5B and the mass
spectrum of II-5B.
Example 8
Synthesis of the Compounds of Formulae IV-1 IV-2, IV-3 and IV-4
Synthesis of Diol Derivatives (Formula IV-2)
[0312] Diols may be synthesized by Sharpless dihydroxylation using
AD mix-.alpha. and .beta.: AD mix-.alpha. is a premix of four
reagents, K.sub.2OsO.sub.2(OH).sub.4; K.sub.2CO.sub.3;
K.sub.3Fe(CN).sub.6; (DHQ).sub.2-PHAL
[1,4-bis(9-O-dihydroquinine)phthalazine] and AD mix-.beta. is a
premix of K.sub.2OsO.sub.2(OH).sub.4; K.sub.2CO.sub.3;
K.sub.3Fe(CN).sub.6; (DHQD).sub.2-PHAL
[1,4-bis(9-O-dihydroquinidine)phth- alazine] which are commercially
available from Aldrich. Diol can also be synthesized by acid or
base hydrolysis of epoxy compounds (Formula II-5A and II-5B) which
may be different to that of products obtained in Sharpless
dihydroxylation in their stereochemistry at carbons bearing
hydroxyl groups
[0313] Sharpless Dihydroxylation of Compounds II-16 II-17 and
II-18
[0314] Any of the compounds of Formulae II-16, II-17 and II-18 may
be used as the starting compound. In the example below, compound of
Formula II-16 is used. The starting compound is dissolved in
t-butanol/water in a round bottom flask to which is added AD
mix-.alpha. or .beta. and a magnetic stir bar. The reaction is
monitored by silica TLC as well as mass spectrometer. The pure
diols are obtained by usual workup and purification by flash
chromatography or HPLC. The structures are confirmed by NMR
spectroscopy and mass spectrometry. In this method both hydroxyl
groups are on same side. 65
[0315] Nucleophilic Ring Opening of Epoxy Compounds (11-5):
[0316] The epoxy ring is opened with various nucleophiles like
NaCN, NaN.sub.3, NaOAc, HBr, HCl, etc. to creat various
substituents on the cyclohexane ring, including a hydroxyl
substituent.
[0317] Examples: 66
[0318] The epoxy is opened with HCl to make Formula IV-3: 67
[0319] Compound of Formula II-5A (3.3 mg) was dissolved in
acetonitrile (0.5 ml) in a 1 dram vial to which was added 5% HCl
(500 ul) and a magnetic stir bar. The reaction mixture was stirred
at room temperature for about an hour. The reaction was monitored
by mass spectrometry. The reaction mixture was directly injected on
normal phase HPLC to obtain compound of Formula IV-3C as a pure
compound without any work up. The HPLC conditions used for the
purification were as follows: Phenomenex Luna 10 u Silica column
(25 cm.times.21.2 mm ID) with a solvent gradient of 25% to 80%
EtOAc/Hex over 19 min, 80 to 100% EtOAc in 1 min, then 5 min at
100% EtOAc at a flow rate of 14.5 ml/min. An ELSD was used to
monitor the purification process. Compound of Formula IV-3C eluted
at about 18 min (2.2 mg). Compound of Formula IV-3C: UV
(Acetonitrile/H.sub.2O) .lambda..sub.max 225 (sh) nm; ESMS, m/z 366
(M+H), 388 (M+Na); .sup.1H NMR in DMSO-d.sub.6 (FIG. 20) The
stereochemistry of the compound of Formula IV-3C was determined
based on coupling constants observed in the cyclohexane ring in 1:1
C.sub.6D.sub.6/DMSO-d.sub.6 (FIG. 21) 68
[0320] Reductive ring opening of epoxides (II-5): The compound of
Formula is treated with metalhydrides like BH.sub.3-THF complex to
make compound of Formula IV-4. 69
Example 9
Synthesis of the Compounds of Formulae II-13C and II-8C
[0321] Compound of Formula II-16 (30 mg) was dissolved in
CH.sub.2Cl.sub.2 (6 ml) in a scintillation vial (20 ml) to which
Dess-Martin Periodinane (122 mg) and a magnetic stir bar were
added. The reaction mixture was stirred at room temperature for
about 2 hours. The progress of the reaction was monitored by TLC
(Hex:EtOAc, 6:4) and analytical HPLC. From the reaction mixture,
the solvent volume was reduced to one third, absorbed on silica
gel, poured on top of a 20 cc silica flash column and eluted in 20
ml fractions using a gradient of Hexane/EtOAc from 10 to 100%. The
fraction eluted with 30% EtOAc in Hexane contained a mixture of
rotamers of Formula II-13C in a ratio of 1.5:8.5. The mixture was
further purified by normal phase HPLC using the Phenomenex Luna 10
u Silica column (25 cm.times.21.2 mm ID) with a solvent gradient of
25% to 80% EtOAc/Hex over 19 min, 80 to 100% EtOAc over 1 min,
holding at 100% EtOAc for 5 min, at a flow rate of 14.5 ml/min. An
ELSD was used to monitor the purification process. Compound of
Formula II-13C eluted at 13.0 and 13.2 mins as a mixture of
rotamers with in a ratio of 1.5:8.5 (7 mg). Formula II-13C: UV
(Acetonitrile/H.sub.2O) .lambda..sub.max 226 (sh) & 300 (sh)
nm; ESMS, m/z 312 (M+H).sup.+, 334 (M+Na).sup.+; .sup.1H NMR in
DMSO-d.sub.6 (see FIG. 22).
[0322] The rotamer mixture of Formula II-13C (4 mg) was dissolved
in acetone (1 ml) in a scintillation vial (20 ml) to which a
catalytic amount (0.5 mg) of 10% (w/w) Pd/C and a magnetic stir bar
were added. The reaction mixture was stirred in a hydrogen
atmosphere at room temperature for about 15 hours. The reaction
mixture was filtered through a 0.2 .mu.m Gelman Acrodisc to remove
the catalyst. The solvent was evaporated from the filtrate to yield
compound of Formula II-8C as a colorless gum which was further
purified by normal phase HPLC using a Phenomenex Luna 10 u Silica
column (25 cm.times.21.2 mm ID) with a solvent gradient of 25% to
80% EtOAc/Hex over 19 min, 80 to 100% EtOAc over 1 min, holding at
100% EtOAc for 5 min, at a flow rate of 14.5 ml/min. An ELSD was
used to monitor the purification process. Compound of Formula II-8C
(1 mg) eluted at 13.5 min as a pure compound. Formula II-8C: UV
(Acetonitrile/H.sub.2O) .lambda..sub.max 225 (sh) nm; ESMS, m/z 314
(M+H).sup.+, 336 (M+Na).sup.+; .sup.1H NMR in DMSO-d.sub.6 (See
FIG. 23). 70
Example 10
Synthesis of the Compound of Formulae II-25 from II-13C
[0323] The rotamer mixture of Formula II-13C (5 mg) was dissolved
in dimethoxy ethane (monoglyme; 1.5 ml) in a scintillation vial (20
ml) to which water (15 .mu.l (1% of the final solution
concentration)) and a magnetic stir bar were added. The above
solution was cooled to -78.degree. C. on a dry ice-acetone bath,
and a sodium borohydride solution (3.7 mg of NaBH.sub.4 in 0.5 ml
of monoglyme (created to allow for slow addition)) was added
drop-wise. The reaction mixture was stirred at -78.degree. C. for
about 14 minutes. The reaction mixture was acidified using 2 ml of
4% HCl solution in water and extracted with CH.sub.2Cl.sub.2. The
organic layer was evaporated to yield mixture of compound of
formulae II-25 and II-16 in a 9.5:0.5 ratio as a white solid, which
was further purified by normal phase HPLC using a Phenomenex Luna
10 u Silica column (25 cm.times.21.2 mm ID). The mobile phase was
24% EtOAc/76% Hexane, which was held isocratic for 19 min, followed
by a linear gradient of 24% to 100% EtOAc over 1 min, and held at
100% EtOAc for 3 min; the flow rate was 25 ml/min. An ELSD was used
to monitor the purification process. Compound of formula II-25 (1.5
mg) eluted at 11.64 min as a pure compound. Compound of Formula
II-25: UV (Acetonitrile/H.sub.2O) .lambda..sub.max 225 (sh) nm;
ESMS, m/z 314 (M+H).sup.+, 336 (M+Na).sup.+; .sup.1H NMR in
DMSO-d.sub.6 (see FIG. 24). 71
Example 11
[0324] Synthesis of the Compound of Formulae II-21 from II-19
[0325] Acetone (7.5 ml) was vigorously mixed with 5 N NaOH (3 ml)
and the resulting mixture evaporated to a minimum volume in vacuo.
A sample of 100 l of this solution was mixed with compound of
Formula II-19 (6.2 mg) in acetone (1 ml) and the resulting biphasic
mixture vortexed for 2 minutes. The reaction solution was
immediately subjected to preparative C18 HPLC. Conditions for the
purification involved a linear gradient if 10% acetonitrile/90%
water to 90% acetonitrile/ 10% water over 17 minutes using an Ace
5.mu. C18 HPLC column of dimensions 22 mm id by 150 mm length.
Compound of Formula II-21 eluted at 9.1 minutes under these
conditions to yield 0.55 mg compound. Compound of Formula II-21: UV
(Acetonitrile/H.sub.2O) 225 (sh), ESMS, m/z 296.1 (M+H); .sup.1H
NMR in DMSO-d.sub.6 (see FIG. 25). 72
Example 12
Synthesis of the Compound of Formulae II-22 from II-19
[0326] A sample of 60 mg sodium propionate was added to a solution
of compound of Formula II-19 (5.3 mg) in DMSO (1 ml) and the
mixture sonicated for 5 minutes, though the sodium propionate did
not completely dissolve. After 45 minutes, the solution was
filtered through a 0.45 [ syringe filter and purified directly
using HPLC. Conditions for the purification involved a linear
gradient if 10% acetonitrile/90% water to 90% acetonitrile/ 10%
water over 17 minutes using an Ace 5.mu. C18 HPLC column of
dimensions 22 mm id by 150 mm length. Under these conditions,
compound of Formula II-22 eluted at 12.3 minutes to yield 0.7 mg
compound (15% isolated yield). UV (Acetonitrile/H.sub.2O) 225 (sh),
ESMS, m/z 352.2 (M+H); .sup.1H NMR in DMSO-d.sub.6 (see FIG. 26).
73
Example 13
Oxidation of Secondary Hydroxyl Group in Compounds of Formulae
II-16, II-17 and II-18 and Reaction with Hydroxy or Methoxy
Amines
[0327] Any of the compounds of Formulae II-16, II-17 and II-18 may
be used as the starting compound. The secondary hydroxyl group in
the starting compound is oxidized using either of the following
reagents: pyridinium dichromate (PDC), pyridinium chlorochromate
(PCC), Dess-Martin periodinane or oxalyl chloride (Swern oxidation)
(Ref: Organic Syntheses, collective volumes I-VIII). Preferably,
Dess-Martin periodinane may be used as a reagent for this reaction.
(Ref: Fenteany G. et al. Science, 1995, 268, 726-73). The resulting
keto compound is treated with hydroxylamine or methoxy amine to
generate oximes.
Examples
[0328] 74
Example 14
Reductive Amination of Keto-Derivative
[0329] The keto derivatives, for example Formula II-8 and II-13,
are treated with sodium cyanoborohydride (NaBH.sub.3CN) in the
presence of various bases to yield amine derivatives of the
starting compounds which are subsequently hydrogenated with 10%
Pd/C, H.sub.2 to reduce the double bond in the cyclohexene
ring.
Example
[0330] 75
Example 15
Cyclohexene Ring Opening
[0331] Any compound of Formulae II-16, II-17 and II-18 may be used
as a starting compound. The starting compound is treated with
OsO.sub.4 and NaIO.sub.4 in THF-H.sub.2O solution to yield dial
derivatives which are reduced to alcohol with NaBH.sub.4 in the
same pot.
Example
[0332] 76
Example 16
Dehydration of Alcohol Followed by Aldehyde Formation at
Lactone-Lactam Ring Junction
[0333] A starting compound of any of Formulae II-16, II-17 or 11-18
is treated with mesylchloride in the presence of base to yield a
dehydrated derivative. The resulting dehydrated compound is treated
with OsO.sub.4 and NaIO.sub.4 in THF-H.sub.2O to yield an aldehyde
group at the lactone-lactam ring junction. 77
Example 17
Various Reactions on Aldehyde Derivatives I-1
[0334] Wittig reactions are performed on the aldehyde group using
various phosphorus ylides [e.g.,
(triphenylphosphoranylidene)ethane] to yield an olefin. The double
bond in the side chain is reduced by catalytic hydrogenation.
Example
[0335] 78
[0336] Reductive amination is performed on the aldehyde group using
various bases (eg. NH.sub.3) and sodium cyanoborohydride to yield
amine derivatives. Alternatively, the aldehyde is reduced with
NaBH.sub.4 to form alcohols in the side chain.
Example
[0337] 79
[0338] Organometallic addition reactions to the aldehyde carbonyl,
such as Grignard reactions, may be performed using various alkyl
magnesium bromide or chloride reagents (eg. isopropylmagnesium
bromide, phenylmagnesium bromide) to yield various substituted
secondary alcohols.
Example
[0339] 80
Example 18
In Vitro Biology
[0340] Initial studies of a compound of Formula II-16, which is
also referred to as Salinosporamide A, employed the National Cancer
Institute (NCI) screening panel, which consists of 60 human tumor
cell lines that represent leukemia, melanoma and cancers of the
lung, colon, brain, ovary, breast, prostate and kidney. A detailed
description of the screening procedure can be found at hypertext
transfer protocol (http://)
"dtp.nci.nih.gov/branches/btb/ivclsp.html."
[0341] In brief, each of the 60 human tumor cell lines were grown
in RPMI 1640 medium, supplemented with 5% fetal bovine serum and 2
mM L-glutamine. Cells were plated at their appropriate density in
96-well microtiter plates and incubated at 37.degree. C., 5%
CO.sub.2, 95% air and 100% relative humidity. After 24 hours, 100
pL of various 10-fold serial dilutions of Salinosporamide A were
added to the appropriate wells containing 100 .mu.L of cells,
resulting in a final Salinosporamide A concentration ranging from
10 nM to 100 .mu.M. Cells were incubated for an additional 48 hours
and a sulforhodamine B protein assay was used to estimate cell
viability or growth.
[0342] Three dose response parameters were calculated as
follows:
[0343] GI.sub.50 indicates the concentration that inhibits growth
by 50%.
[0344] TGI indicates the concentration that completely inhibits
growth.
[0345] LC.sub.50 indicates the concentration that is lethal to 50%
of the cells.
[0346] An example of a study evaluating Salinosporamide A in the
NCI screen is shown in Table 1 below.
[0347] Data indicate that the mean GI.sub.50 value of
Salinosporamide A was less than 10 nM. The wide range
(>1000-fold difference) observed in both the mean TGI and mean
LC.sub.50 values for the most sensitive and the most resistant
tumor cell lines illustrates that Salinosporamide A displays good
selectivity and does not appear to be a general toxin. Furthermore,
the mean TGI data suggest that Salinosporamide A shows preferred
specificity towards melanoma and breast cancer cell lines. The
assay was repeated and showed similar results.
[0348] The results of the NCI tumor screen show that
Salinosporamide A: (1) is a potent compound with a mean GI.sub.50
value of <10 nM, and (2) displays good tumor selectivity of more
than 1000-fold difference in both the mean TGI and mean LC.sub.50
values between the most sensitive and resistant tumor cell
lines.
Example 19
Growth Inhibition of Tumor Cell Lines
[0349] B16-F10 (ATCC; CRL-6475), DU 145 (ATCC; HTB-81), HEK293
(ATCC; CRL-1573), HT-29 (ATCC; HTB-38), LoVo (ATCC; CCL-229),
MDA-MB-231 (ATCC; HTB-26), MIA PaCa-2 (ATCC; CRL-1420), NCI-H292
(ATCC; CRL-1848), OVCAR-3 (ATCC, HTB-161), PANC-1 (ATCC; CRL-1469),
PC-3 (ATCC; CRL-1435), RPMI 8226 (ATCC; CCL-155) and U266 (ATCC;
TIB-196) were maintained in appropriate culture media. The cells
were cultured in an incubator at 37 .degree. C. in 5% CO2 and 95%
humidified air.
[0350] For cell growth inhibition assays, B16-F10, DU 145, HEK293,
HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-1,
PC-3, RPMI 8226 and U266 cells were seeded at 1.25.times.10.sup.3,
5.times.10.sup.3, 1.5.times.10.sup.4, 5.times.10.sup.3,
5.times.10.sup.3, 1.times.10.sup.4, 2.times.10.sup.3,
4.times.10.sup.3, 1.times.10.sup.4, 7.5.times.10.sup.3,
5.times.10.sup.3, 2.times.10.sup.4, 2.5.times.10.sup.4 cells/well
respectively in 90 .mu.l complete media into Corning 3904
black-walled, clear-bottom tissue culture plates. 20 mM stock
solutions of Formula II-16 were prepared in 100% DMSO, aliquoted
and stored at -80.degree. C. Formula II-16 was serially diluted and
added in triplicate to the test wells resulting in final
concentrations ranging from of 20 .mu.M to 0.2 .mu.M. The plates
were returned to the incubator for 48 hours. The final
concentration of DMSO was 0.25% in all samples.
[0351] Following 48 hours of drug exposure, 10 .mu.l of 0.2 mg/ml
resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg.sup.2+,
Ca.sup.2+ free phosphate buffered saline was added to each well and
the plates were returned to the incubator for 3-6 hours. Since
living cells metabolize Resazurin, the fluorescence of the
reduction product of Resazurin was measured using a Fusion
microplate fluorometer (Packard Bioscience) with
.lambda..sub.ex=535 nm and .lambda..sub.em=590 nm filters.
Resazurin dye in medium without cells was used to determine the
background, which was subtracted from the data for all experimental
wells. The data were normalized to the average fluorescence of the
cells treated with media +0.25% DMSO (100% cell growth) and
EC.sub.50 values (the drug concentration at which 50% of the
maximal observed growth inhibition is established) were determined
using a standard sigmoidal dose response curve fitting algorithm
(generated by XLfit 3.0, ID Business Solutions Ltd or Prism 3.0,
GraphPad Software Inc).
[0352] The data in Table 2 summarize the growth inhibitory effects
of Formula II-16 against 13 diverse human and mouse tumor cell
lines.
6TABLE 2 Mean EC.sub.50 values of Formula II-16 against various
tumor cell lines EC.sub.50 (nM), Cell line Source mean .+-. SD* n
B16-F10 Mouse, melanoma 47 .+-. 20 12 DU 145 Human, prostate
carcinoma 37 .+-. 10 3 HEK293 Human, embryonic kidney 47 2 HT-29
Human, colorectal adenocarcinoma 40 .+-. 26 5 LoVo Human,
colorectal adenocarcinoma 70 .+-. 8 3 MDA-MB-231 Human, breast
adenocarcinoma 87 .+-. 40 12 MIA PaCa-2 Human, pancreatic carcinoma
46 2 NCI-H292 Human, non small cell lung 66 .+-. 29 12 carcinoma
OVCAR-3 Human, ovarian adenocarcinoma 49 .+-. 31 6 PANC-1 Human,
pancreatic carcinoma 60 2 PC-3 Human, prostate adenocarcinoma 64
.+-. 26 19 RPMI 8226 Human, multiple myeloma 8.6 .+-. 1.9 26 U266
Human, multiple myeloma 4.7 .+-. 0.7 6 *Where n (number of
independent experiments) = 2, the mean value is presented
[0353] The EC.sub.50 values indicate that Formula II-16 was
cytotoxic against B16-F10, DU 145, HEK293, HT-29, LoVo, MDA-MB-231,
MIA PaCa-2, NCI-H292, OVCAR-3, PANC-1, PC-3, RPMI 8226 and U266
cells.
Example 20
In vitro Inhibition of Proteasome Activity by Formulae II-2, II-3,
II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,
II-20, II-21, II-22, II-24C, II-25 and IV-3C
[0354] All the compounds were prepared as 20 mM stock solution in
DMSO and stored in small aliquots at -80.degree. C. Purified rabbit
muscle 20S proteasome was obtained from CalBiochem. To enhance the
chymotrypsin-like activity of the proteasome, the assay buffer (20
mM HEPES, pH7.3, 0.5 mM EDTA, and 0.05% Triton X100) was
supplemented with SDS resulting in a final SDS concentration of
0.035%. The substrate used was suc-LLVY-AMC, a fluorogenic peptide
substrate specifically cleaved by the chymotrypsin-like activity of
the proteasome. Assays were performed at a proteasome concentration
of 1 .mu.g/ml in a final volume of 200 .mu.l in 96-well Costar
microtiter plates. Formula II-2, Formula II-4, Formula II-16,
Formula II-17, Formula II-18, Formula II-19, Formula II-21 and
Formula II-22 were tested as eight-point dose response curves with
final concentrations ranging from 500 nM to 158 pM. Formula II-5A,
Formula II-5B and Formula II-20 were tested at concentrations
ranging from 1 .mu.M to 0.32 nM. Formula II-3 was tested as an
eight-dose response curve with final concentrations ranging from 10
.mu.M to 3.2 nM, while Formula II-8C, Formula II-13C, Formula
II-24C, Formula II-25 and Formula IV-3C were tested with final
concentrations ranging from 20 .mu.M to 6.3nM. The samples were
incubated at 37.degree. C. for five minutes in a temperature
controlled plate reader. During the preincubation step, the
substrate was diluted 25-fold in SDS-containing assay buffer. After
the preincubation period, the reactions were initiated by the
addition of 10 .mu.l of the diluted substrate and the plates were
returned to the plate reader. The final concentration of substrate
in the reactions was 20 .mu.M. All data were collected every five
minutes for more than 1.5 hour and plotted as the mean of
triplicate data points. The EC.sub.50 values (the drug
concentration at which 50% of the maximal relative fluorescence
unit is inhibited) were calculated by Prism (GraphPad Software)
using a sigmoidal dose-response, variable slope model. To evaluate
the activity of the compounds against the caspase-like activity of
the 20S proteasomes, reactions were performed as described above
except that Z-LLE-AMC was used as the peptide substrate. Formulae
II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-17, II-18, II-20,
II-21. II-22, II-24C, II-25 and Formula IV-3C were tested at
concentrations ranging from 20 .mu.M to 6.3 nM. Formula II-2 was
tested at concentrations ranging from 10 .mu.M to 3.2 nM, while
Formula II-16 and Formula II-19 were tested at concentrations
ranging from 5 .mu.M to 1.58 nM. For the evaluation of the
compounds against the trypsin-like activity of the proteasome, the
SDS was omitted from the assay buffer and Boc-LRR-AMC was used as
the peptide substrate. Formula II-20 was tested at concentrations
ranging from 5 .mu.M to 1.6 nM. Formulae II-3, II-8C, II-13C,
II-17, II-21, II-22, II-24C, II-25 and IV-3C were tested at
concentrations ranging from 20 .mu.M to 6.3 nM. For Formulae II-2
and II-5B the concentrations tested ranged from 10 .mu.M to 3.2 nM,
while Formulae II-4, II-5A, II-16, II-18 and II-19 were tested at
concentrations ranging from 1 .mu.M to 0.32 nM.
[0355] Results (mean EC.sub.50 values) are shown in Table 3 and
illustrate that among the tested compounds, Formulae II-5A, II-16,
II-18, II-19, II-20, II-21 and II-22 are the most potent inhibitors
of the chymotrypsin-like activity of the 20S proteasome with
EC.sub.50 values ranging from 2.2 nM to 7 nM. Formulae II-2, II-4,
II-5B and II-17 inhibit the proteasomal chymotrypsin-like activity
with EC.sub.50 values ranging from 14.2 nM to 87 nM, while the
EC.sub.50 value of Formula II-3 is 927 nM. Formula II-24C, II-13C
and IV-3C inhibited the chymotrypsin-like activity with EC.sub.50
values of 2.2 .mu.M, 8.2 .mu.M and 7.8 .mu.M respectively.
EC.sub.50 values for Formulae II-8C and II-25 were greater than 20
.mu.M. Under the conditions tested, Formulae II-2, II-3, II-4,
II-5A, II-5B, II-13C, II-16, II-17, II-18, II-19, II-20, II-21,
II-22 and II-24C were able to inhibit the trypsin-like activity of
the 20S proteasome. Formulae II-4, II-5A, II-16, II-18 and II-19
inhibited the caspase-like activity with EC.sub.50 values ranging
from 250 nM to 744 nM, while Formulae II-2, II-5B, II-17, II-20,
II-21, and II-22 had EC.sub.50 values ranging from 1.2 .mu.M to 3.3
.mu.M.
7TABLE 3 Effects of Formulae II-2, II-3, II-4, II-5A, II-5B, II-8C,
II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C,
II-25 and IV-3C on the various enzymatic activities of purified
rabbit 20S proteasomes EC.sub.50 Values Analog Chymotrypsin-like
Trypsin-like Caspase-like Formula II-2 18 nM 230 nM 1.5 .mu.M
Formula II-3 927 nM 6.6 .mu.M >20 .mu.M Formula II-4 14.2 nM 109
nM 744 nM Formula II-5A 6.5 nM 89 nM 487 nM Formula II-5B 87 nM 739
nM 3.3 .mu.M Formula II-8C* >20 .mu.M >20 .mu.M >20 .mu.M
Formula II-13C 8.2 .mu.M 10.7 .mu.M >20 .mu.M Formula II-16 2.5
nM 21 nM 401 nM Formula II-17 29.5 nM 588 nM 1.2 .mu.M Formula
II-18 2.2 nM 14 nM 250 nM Formula II-19* 3 nM 13 nM 573 nM Formula
II-20* 5 nM 318 nM 1.4 .mu.M Formula II-21* 7 nM 720 nM 2.6 .mu.M
Formula II-22* 5 nM 308 nM 1.3 .mu.M Formula II-24C* 2.2 .mu.M 3.2
.mu.M >20 .mu.M Formula II-25* >20 .mu.M >20 .mu.M >20
.mu.M Formula IV-3C 7.8 .mu.M >20 .mu.M >20 .mu.M *n = 1
Example 21
Salinosporamide A (II-16) Inhibits Chymotrypsin-Like Activity of
Rabbit Muscle 20S Proteasomes
[0356] The effect of Salinosporamide A (11-16) on proteasomes was
examined using a commercially available kit from Calbiochem
(catalog no. 539158), which uses a fluorogenic peptide substrate to
measure the activity of rabbit muscle 20S proteasomes (Calbiochem
20S Proteasome Kit). This peptide substrate is specific for the
chymotrypsin-like enzyme activity of the proteasome.
[0357] Omuralide was prepared as a 10 mM stock in DMSO and stored
in 5 .mu.L aliquots at -80.degree. C. Salinosporamide A was
prepared as a 25.5 mM solution in DMSO and stored in aliquots at
-80.degree. C. The assay measures the hydrolysis of Suc-LLVY-AMC
into Suc-LLVY and AMC. The released coumarin (AMC) was measured
fluorometrically by using .lambda..sub.ex=390 nm and
.lambda..sub.em=460 nm. The assays were performed in a microtiter
plate (Corning 3904), and followed kinetically with measurements
every five minutes. The instrument used was a Thermo Lab Systems
Fluoroskan, with the incubation chamber set to 37.degree. C. The
assays were performed according to the manufacturer's protocol,
with the following changes. The proteasome was activated as
described with SDS, and held on ice prior to the assay.
Salinosporamide A and Omuralide were serially diluted in assay
buffer to make an 8-point dose-response curve. Ten microliters of
each dose were added in triplicate to the assay plate, and 190
.mu.L of the activated proteasome was added and mixed. The samples
were pre-incubated in the Fluoroskan for 5 minutes at 37.degree. C.
Substrate was added and the kinetics of AMC were followed for one
hour. All data were collected and plotted as the mean of triplicate
data points. The data were normalized to reactions performed in the
absence of Salinosporamide A and modeled in Prism as a sigmoidal
dose-response, variable slope.
[0358] Similar to the results obtained for the in vitro
cytotoxicity (Table 2), Feling, et al., Angew Chem Int Ed Engl
42:355 (2003), the EC.sub.50 values in the 20S proteasome assay
showed that Salinosporamide A was approximately 40-fold more potent
than Omuralide, with an average value of 1.3 nM versus 49 nM,
respectively (FIG. 27). This experiment was repeated and the
average EC.sub.50 in the two assays was determined to be 2 nM for
Salinosporamide A and 52 nM for Omuralide.
[0359] Salinosporamide A is a potent inhibitor of the
chymotrypsin-like activity of the proteasome. The EC.sub.50 values
for cytotoxicity were in the 10-200 nM range suggesting that the
ability of Salinosporamide A to induce cell death was due, at least
in large part, to proteasome inhibition. The data suggest that
Salinosporamide A is a potent small molecule inhibitor of the
proteasome.
Example 22
Salinosporamide A (II- 16) Inhibition of PGPH Activity of Rabbit
Muscle 20S Proteasomes
[0360] Omuralide can inhibit the PGPH activity (also known as the
caspase-like) of the proteasome; therefore, the ability of
Salinosporamide A to inhibit the PGPH activity of purified rabbit
muscle 20S proteasomes was assessed. A commercially available
fluorogenic substrate specific for the PGPH activity was used
instead of the chymotrypsin substrate supplied in the proteasome
assay kit described above.
[0361] Salinosporamide A (II-16) was prepared as a 20 mM solution
in DMSO and stored in small aliquots at -80.degree. C. The
substrate Z-LLE-AMC was prepared as a 20 mM stock solution in DMSO,
stored at -20.degree. C. The source of the proteasomes was the
commercially available kit from Calbiochem (Cat. #539158). As with
the chymotrypsin substrate, the proteasome can cleave Z-LLE-AMC
into Z-LLE and free AMC. The activity can then be determined by
measuring the fluorescence of the released AMC (.lambda..sub.ex=390
nm and .lambda..sub.em=460 nm). The proteasomes were activated with
SDS and held on ice as per manufacturer's recommendation.
Salinosporamide A was diluted in DMSO to generate a 400-fold
concentrated 8-point dilution series. The series was diluted
20-fold with assay buffer and preincubated with the proteasomes as
described for the chymotrypsin-like activity. After addition of
substrate, the samples were incubated at 37.degree. C., and release
of the fluorescent AMC was monitored in a fluorimeter. All data
were collected and plotted as the mean of triplicate points. In
these experiments, the EC.sub.50 was modeled in Prism as normalized
activity, where the amount of AMC released in the absence of
Salinosporamide A represents 100% activity. As before, the model
chosen was a sigmoidal dose-response, with a variable slope.
[0362] Data revealed that Salinosporarnide A inhibited the PGPH
activity in rabbit muscle 20S proteasomes with an EC.sub.5o of 350
nM (FIG. 28). A replicate experiment was performed, which gave a
predicted EC.sub.50 of 610 nM. These results indicate that
Salinosporamide A does block the in vitro PGPH activity of purified
rabbit muscle 20S proteasomes, albeit with lower potency than seen
towards the chymotrypsin-like activity.
Example 23
Inhibition of the Chymotrypsin-Like Activity of Human Erythrocyte
20S Proteasomes
[0363] The ability of Salinosporamide A (II-16) to inhibit the
chymotrypsin-like activity of human erythrocyte 20S proteasomes was
assessed in vitro. The calculated EC.sub.50 values ranged from 45
to 247 pM, and seemed to depend upon the lot of proteasomes tested
(BIOMOL, Cat#SE-221). These data indicate that the inhibitory
effect of Salinosporamide A is not limited to rabbit skeletal
muscle proteasomes.
[0364] Salinosporamide A was prepared as a 20 mM solution in DMSO
and stored in small aliquots at -80.degree. C. The substrate,
suc-LLVY-AMC, was prepared as a 20 mM solution in DMSO and stored
at -20.degree. C. Human erythrocyte 20S proteasomes were obtained
from BIOMOL (Cat. # SE-221). The proteasome can cleave suc-LLVY-AMC
into suc-LLVY and free AMC and the activity can then be determined
by measuring the fluorescence of the released AMC
(.lambda..sub.ex=390 nm and .lambda..sub.em=460 nm ). The
proteasomes were activated by SDS and stored on ice as with the
experiments using rabbit muscle proteasomes. Salinosporamide A was
diluted in DMSO to generate a 400-fold concentrated 8-point
dilution series. The series was then diluted 20-fold with assay
buffer and pre-incubated with proteasomes at 37.degree. C. The
reaction was initiated with substrate, and the release of AMC was
followed in a Fluoroskan microplate fluorimeter. Data were
collected and plotted as the mean of triplicate points. Data were
captured kinetically for 3 hours, and indicated that these
reactions showed linear kinetics in this time regime. The data were
normalized to reactions performed in the absence of Salinosporamide
A and modeled in Prism as a sigmoidal dose-response, variable
slope.
[0365] Replicate experiments performed using human erythrocyte
proteasomes from separate lots resulted in a range of EC.sub.50
values between 45 and 250 pM (FIG. 29 shows a representative
experiment). It has been reported that 20S proteasomes purified
from human erythrocytes are highly heterogeneous in subunit
composition. Claverol, et al., Mol Cell Proteomics 1:567 (2002).
The variability in these experiments may therefore be due to
differences in the composition and activity of the human
erythrocyte proteasome preparations. Regardless, these results
indicate that the in vitro chymotrypsin-like activity of human
erythrocyte 20S proteasomes is sensitive to Salinosporamide A.
Example 24
Salinosporamide A (II- 16) Specificity
[0366] A possible mechanism by which Salinosporamide A inhibits the
proteasome is by the reaction of the .beta.-lactone functionality
of Salinosporamide A with the active site threonine of the
proteasome. This covalent modification of the proteasome would
block the active site, as this residue is essential for the
catalytic activity of the proteasome. Fenteany, et al., J Biol Chem
273:8545 (1998). A structurally related compound, Lactacystin, has
been shown to also inhibit cathepsin A (Ostrowska, et al., Int J
Biochem Cell Biol 32:747 (2000), Kozlowski, et al., Tumour Biol
22:211 (2001), Ostrowska, et al., Biochem Biophys Res Commun
234:729 (1997)) and TPPII (Geier, et al., Science 283:978 (1999))
but not trypsin, chymotrypsin, papain, calpain (Fenteany, et al.,
Science 268:726 (1995)), thrombin, or plasminogen activator (Omura,
et al., J Antibiot (Tokyo) 44:113 (1991)). Similar studies were
initiated to explore the specificity of Salinosporamide A for the
proteasome by evaluating its ability to inhibit the catalytic
activity of a prototypical serine protease, chymotrypsin.
[0367] Salinosporamide A was prepared as a 20 mM solution in DMSO
and stored in small aliquots at -80.degree. C. The substrate,
suc-LLVY-AMC, was prepared as a 20 mM solution in DMSO and stored
at -20.degree. C. Proteolytic cleavage of this substrate by either
proteasomes or chymotrypsin liberates the fluorescent product AMC,
which can be monitored in a fluorimeter (.lambda..sub.ex=390 nm and
.lambda..sub.em=460 nm). Bovine pancreatic chymnotrypsin was
obtained from Sigma (Cat. #C-4129), and prepared as a 5 mg/ml
solution in assay buffer (10 mM HEPES, 0.5 mM EDTA, 0.05% Triton
X-100, pH 7.5) daily. Immediately prior to the assay, the
chymotrypsin was diluted to 1 .mu.g/ml (0.2 .mu.g/well) in assay
buffer and held on ice. Salinosporamide A was diluted in DMSO to
generate an 8-point dose-response curve. The high final
Salinosporamide A concentrations needed to obtain complete
inhibition of chymotrypsin required that the diluted enzyme be
directly added to the compound dilution series. The inclusion of 1%
DMSO (the final concentration of solvent in the test wells) into
the reaction had no significant effect on chymotrypsin activity
towards this substrate. The reactions were pre-incubated for 5
minutes at 37.degree. C. and the reactions were initiated by the
addition of substrate. Data were collected kinetically for one hour
at 37.degree. C. in the Fluoroskan and plotted as the mean of
triplicate data points. The data were normalized to reactions
performed in the absence of Salinosporamide A, and modeled in Prism
as a sigmoidal dose-response, variable slope. Normalized data from
Salinosporamide A inhibition of the chymotrypsin-like activity of
rabbit 20S proteasomes has been included on the same graph.
[0368] The average inhibition observed in two experiments using
Salinosporamide A pretreatment of chymotrypsin was 17.5 .mu.M (FIG.
30 shows a representative experiment). The data indicate that there
is a preference for Salinosporamide A-mediated inhibition of the in
vitro chymotrypsin-like activity of proteasomes over inhibition of
the catalytic activity of chymotrypsin.
[0369] Thus, Salinosporamide A inhibits the chymotrypsin-like and
PGPH activity of the proteasome. Preliminary studies indicate that
Salinosporamide A also inhibits the trypsin-like activity of the
proteasome with an EC.sub.50 value of .about.10 nM (data not
shown).
Example 25
Inhibition of NF-.kappa.B-Mediated Luciferase Activity by Formulae
II-2, II-3, II-4, II-5A. II-5B, II-8C, II-13C, II-16, II-17, II-18,
II-19, II-20, II-21, II-22, II-24C, II-25 and IV-3C; HEK293
NF-.kappa.B/Luciferase Reporter Cell Line
[0370] The HEK293 NF-.kappa.B/luciferase reporter cell line is a
derivative of the human embryonic kidney cell line (ATCC; CRL-
1573) and carries a luciferase reporter gene under the regulation
of 5.times.NF-.kappa.B binding sites. The reporter cell line was
routinely maintained in complete DMEM medium (DMEM plus 10%(v/v)
Fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES and
Penicillin/Streptomycin at 100 IU/ml and 100 .mu.g/ml,
respectively) supplemented with 250 .mu.g/ml G418. When performing
the luciferase assay, the DMEM basal medium was replaced with
phenol-red free DMEM basal medium and the G418 was omitted. The
cells were cultured in an incubator at 37.degree. C. in 5% CO.sub.2
and 95% humidified air.
[0371] For NF-.kappa.B-mediated luciferase assays, HEK293
NF-.kappa.B/luciferase cells were seeded at 1.5.times.10.sup.4
cells/well in 90 .mu.l phenol-red free DMEM complete medium into
Corning 3917 white opaque-bottom tissue culture plates. For Formula
II-2, Formula II-4, Formula II-5A, Formula II-16 and Formula II-18,
a 400 .mu.M starting dilution was made in 100% DMSO and this
dilution was used to generate a 8-point half log dilution series.
This dilution series was further diluted 40.times. in appropriate
culture medium and ten .mu.l aliquots were added to the test wells
in triplicate resulting in final test concentrations ranging from 1
.mu.M to 320 pM. For Formula II-3, Formula II-5B, Formula II-8C,
Formula II-13C, Formula II-17, Formula II-20, Formula II-21,
Formula II-22, Formula II-24C, Formula II-25 and IV-3C, a 8 mM
starting dilution was made in 100% DMSO and the same procedure was
followed as described above resulting in final test concentrations
ranging from 20 .mu.M to 6.3 nM. For Formula II-19, a 127 .mu.M
starting dilution was made in 100% DMSO and the final test
concentrations ranging from 317 nM to 0.1 nM. The plates were
returned to the incubator for 1 hour. After 1 hr pretreatment, 10
.mu.l of a 50 ng/ml TNF-.alpha. solution, prepared in the
phenol-red free DMEM medium was added, and the plates were
incubated for an additional 6 hr. The final concentration of DMSO
was 0.25% in all samples.
[0372] At the end of the TNF-(X stimulation, 100 .mu.l of Steady
Lite HTS luciferase reagent (Packard Bioscience) was added to each
well and the plates were left undisturbed for 10 min at room
temperature before measuring the luciferase activity. The relative
luciferase units (RLU) were measured by using a Fusion microplate
fluorometer (Packard Bioscience). The EC.sub.50 values (the drug
concentration at which 50% of the maximal relative luciferase unit
inhibition is established) were calculated in Prism (GraphPad
Software) using a sigmoidal dose response, variable slope
model.
[0373] Inhibition of NF-.kappa.B Activation by Formulae II-2, II-3,
II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,
II-20, II-21, II-22, II-24C, II-25 and IV-3C
[0374] NF-.kappa.B regulates the expression of a large number of
genes important in inflammation, apoptosis, tumorigenesis, and
autoimmune diseases. In its inactive form, NF-.kappa.B complexes
with I.kappa.B in the cytosol and upon stimulation, I.kappa.B is
phosphorylated, ubiquitinated and subsequently degraded by the
proteasome. The degradation of I.kappa.B leads to the activation of
NF-.kappa.B and its translocation to the nucleus. The effects of
Formula II-2, Formula II-3, Formula II-4, Formula II-5A, Formula
II-5B, Formula II-8C, Formula II-13C, Formula II-16, Formula II-17,
Formula II-18, Formula II-19, Formula II-20, Formula II-21, Formula
II-22, Formula II-24C, Formula II-25 and Formula IV-3C on the
activation of NF-.kappa.B were evaluated by assessing the
NF-.kappa.B-mediated luciferase activity in HEK293 NF-.kappa.B/Luc
cells upon TNF-.alpha. stimulation.
[0375] Pretreatment of NF-.kappa.B/Luc 293 cells with Formula II-2,
Formula II-4, Formula II-5A, Formula II-5B, Formula II-16, Formula
II-17, Formula II-18, Formula II-19, Formula II-20, Formula II-21,
Formula II-22 and Formula II-24C resulted in a dose-dependent
decrease of luciferase activity upon TNF-.alpha. stimulation. The
mean EC.sub.50 values to inhibit NF-.kappa.B-mediated luciferase
activity are shown in Table 4 and demonstrate that compounds of
Formula II-2, Formula II-4, Formula II-5A, Formula Il-5B, Formula
II-16, Formula II-17, Formula II-18, Formula II-19, Formula II-20,
Formula II-21, Formula II-22 and Formula II-24C inhibited
NF-.kappa.B activity in this cell-based assay.
8TABLE 4 Mean EC.sub.50 values of Formulae II-2, II-3, II-4, II-5A,
II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21,
II-22, II-24C, II-25 and IV-3C from NF-.kappa.B-mediated luciferase
reporter gene assay Compound EC.sub.50 (nM) Formula II-2 82 Formula
II-3 >20,000 Formula II-4 77.7 Formula II-5A 31.5 Formula II-5B
270 Formula II-8C* >20,000 Formula II-13C >20,000 Formula
II-16 11.8 Formula II-17 876 Formula II-18 9.5 Formula II-19 8.5
Formula II-20* 154 Formula II-21* 3,172 Formula II-22* 1,046
Formula II-24C* 5,298 Formula II-25* >20,000 Formula IV-3C
>20,000 *n = 1
Example 26
Effect of Salinosporamide A on the NF-.kappa.B Signaling
Pathway
[0376] Experiments were carried out to study the role of
Salinosporamide A in the NF-.kappa.B signaling pathway. A stable
HEK293 clone (NF-.kappa.B/Luc 293) was generated carrying a
luciferase reporter gene under the regulation of
5.times.NF-.kappa.B binding sites. Stimulation of this cell line
with TNF-.alpha. leads to increased luciferase activity as a result
of NF-.kappa.B activation.
[0377] NF-.kappa.B/Luc 293 cells were pre-treated with 8-point
half-log serial dilutions of Salinosporamide A (ranging from 1
.mu.M to 317 pM) for 1 hour followed by a 6 hour stimulation with
TNF-.alpha. (10 ng/mL). NF-.kappa.B inducible luciferase activity
was measured at 6 hours. Viability of NF-.kappa.B/Luc 293 cells,
after treatment with Salinosporamide A for 24 hr, was assessed by
the addition of resazurin dye, as previously described.
[0378] Pretreatment of NF-.kappa.B/Luc 293 cells with
Salinosporamide A resulted in a dose-dependent decrease of
luciferase activity upon TNF-.alpha. stimulation (FIG. 31, right
y-axis). The calculated EC.sub.50 for inhibition of
NF-.kappa.B/luciferase activity was .about.7 nM. A cytotoxicity
assay was simultaneously performed, and showed that this
concentration of Salinosporamide A did not affect cell viability
(FIG. 31, left y-axis). These representative data suggested that
the observed decrease in luciferase activity by Salinosporamide A
treatment was primarily due to an NF-.kappa.B mediated-signaling
event rather than cell death.
Example 27
[0379] In addition to the NF-.kappa.B luciferase reporter gene
assay, the effect of Salinosporamide A on the levels of
phosphorylated-I.kappa.B.alp- ha. and total I.kappa.B.alpha. was
evaluated by western blot. Endogenous protein levels were assessed
in both HEK293 cells and the NF-.kappa.B/Luc 293 reporter
clone.
[0380] Cells were pre-treated for 1 hour with Salinosporamide A at
the indicated concentrations followed by stimulation with 10 ng/mL
of TNF-.alpha. for 30 minutes. Antibodies against total and
phosphorylated forms of I.kappa.B.alpha. were used to determine the
endogenous level of each protein and anti-Tubulin antibody was used
to confirm equal loading of protein.
[0381] As shown in FIG. 32, treatment of both cell lines with
Salinosporamide A at 50 and 500 nM not only reduced the degradation
of total I.kappa.B.alpha. but also retained the
phospho-I.kappa.B.alpha. level when stimulated with TNF-.alpha..
These results strongly support the mechanism of action of
Salinosporamide A as a proteasome inhibitor, which prevents the
degradation of phosphorylated I.kappa.B.alpha. upon TNF-.alpha.
stimulation.
Example 28
Effect of Salinosporamide A on Cell Cycle Regulatory Proteins
[0382] The ubiquitin-proteasome pathway is an essential proteolytic
system involved in cell cycle control by regulating the degradation
of cyclins and cyclin-dependent kinase (Cdk) inhibitors such as p21
and p27. Pagano, et al., Science 269:682 (1995), Kisselev, et al.,
Chem Biol 8:739 (2001), King, et al., Science 274:1652 (1996).
Furthermore, p21 and p27 protein levels are increased in the
presence of proteasome inhibitors. Fukuchi, et al., Biochim Biophys
Acta 1451:206 (1999), Takeuchi, et al., Jpn J Cancer Res 93:774
(2002). Therefore, western blot analysis was performed to evaluate
the effect of Salinosporamide A treatment on endogenous levels of
p21 and p27 using the HEK293 cells and the HEK293
NF-.kappa.B/Luciferase reporter clone.
[0383] The Western blots presented in FIG. 33 were reprobed using
antibodies against p21 and p27 to determine the endogenous level of
each protein and anti-Tubulin antibody was used to confirm equal
loading of protein.
[0384] As shown in FIGS. 33A and 33B, preliminary results indicated
that p21 and p27 protein levels were elevated when both cell lines
were treated with Salinosporamide A at various concentrations. Data
showed that Salinosporamide A acts by inhibiting proteasome
activity thereby preventing the TNF-.alpha. induced activation of
NF-.kappa.B. In addition, this proteasomal inhibition results in
the accumulation of the Cdk inhibitors, p21 and p27, which has been
reported to sensitize cells to apoptosis. Pagano, et al., supra
(1995), King, et al., supra (1996).
Example 29
Activation of Caspase-3 by Salinosporamide A (II-16)
[0385] To address whether Salinosporamide A induces apoptosis, its
effect on the induction of Caspase-3 activity was evaluated using
Jurkat cells (American Type Culture Collection (ATCC) TIB-152,
human acute T cell leukemia).
[0386] Jurkat cells were plated at 2.times.10.sup.6 cells/3 mL per
well in a 6-well plate and incubated at 37.degree. C., 5% (v/v)
C0.sub.2 and 95% (v/v) humidity. Salinosporamide A and Mitoxantrone
(Sigma, St. Louis, Mo. Cat #M6545), were prepared in DMSO at stock
concentrations of 20 mM and 40 mM, respectively. Mitoxantrone is a
chemotherapeutic drug that induces apoptosis in dividing and
non-dividing cells via inhibition of DNA synthesis and repair and
was included as a positive control. Bhalla, et al., Blood 82:3133
(1993). Cells were treated with EC.sub.50 concentrations (Table 5)
and incubated 19 hours prior to assessing of Caspase-3 activity.
Cells treated with 0.25% DMSO served as the negative control. The
cells were collected by centrifugation and the media removed. Cell
pellets were processed for the Caspase-3 activity assay as
described in the manufacturer's protocol (EnzChek Caspase-3 Assay
Kit from Molecular Probes (E-13183; see Appendix G, which form a
part of this application and is also available at hypertext
transfer protocol on the worldwide web at
"probes.com/media/pis/mp13183.pdf.". In brief, cell pellets were
lysed on ice, mixed with the EnzChek Caspase-3 components in a
96-well plate, and then incubated in the dark for 30 minutes prior
to reading fluorescence of cleaved benzyloxycarbonyl-DEVD-AMC using
a Packard Fusion with .lambda..sub.ex=485 nm and
.lambda..sub.em=530 nm filters. Protein concentrations for lysates
were determined using the BCA Protein Assay Kit (Pierce) and these
values were used for normalization.
[0387] Data from representative experiments indicate that
Salinosporamide A treatment of Jurkat cells results in cytotoxicity
and activation of Caspase-3 (Table 5, FIG. 34).
9TABLE 5 EC50 Values of Salinosporamide A and Mitoxantrone
Cytotoxicity against Jurkat Cells Jurkat Cells Compound EC.sub.50
(nM) % max cell kill Salinosporamide A 10 97 Mitoxantrone 50 99
Example 30
PARP Cleavage by Salinosporamide A in Jurkat Cells
[0388] In order to assess the ability of Salinosporamide A to
induce apoptosis in Jurkat cells, cleavage of poly (ADP-ribose)
polymerase (PARP) was monitored. PARP is a 116 kDa nuclear protein
that is one of the main intracellular targets of Caspase-3. Decker,
et al., J Biol Chem 275:9043 (2000), Nicholson, D. W, Nat
Biotechnol 14:297 (1996). The cleavage of PARP generates a stable
89 kDa product, and this process can be monitored by western
blotting. Cleavage of PARP by caspases is a hallmark of apoptosis,
and as such serves as an excellent marker for this process.
[0389] Jurkat cells were maintained in RPMI supplemented with 10%
Fetal Bovine Serum (FBS) at low density (2.times.10.sup.5 cells per
mL) prior to the experiment. Cells were harvested by
centrifugation, and resuspended in media to 1.times.10.sup.6 cells
per 3 mL. Twenty mL of the cell suspension were treated with 100 nM
Salinosporamide A (20 mM DMSO stock stored at -80.degree. C.), and
a 3 mL aliquot removed and placed on ice for the T.sub.0 sample.
Three mL aliquots of the cell suspension plus Salinosporamide A
were placed in 6-well dishes and returned to the incubator. As a
positive control for PARP cleavage, an identical cell suspension
was treated with 350 nM Staurosporine, a known apoptosis inducer
(Sigma S5921, 700 .mu.M DMSO stock stored at -20.degree. C.).
Samples were removed at 2, 4, 6, 8, and 24 hrs in the case of
Salinosporamide A treated cells, and at 4 hrs for the Staurosporine
control. For each time point, the cells were recovered by brief
centrifugation, the cells were washed with 400 .mu.L of PBS, and
the cells pelleted again. After removal of the PBS, the pellets
were stored at -20.degree. C. prior to SDS PAGE. Each cell pellet
was resuspended in 100 .mu.L of NuPAGE sample buffer (Invitrogen
46-5030) and 10 .mu.L of each sample were separated on 10% NuPAGE
BIS-Tris gels (Invitrogen NB302). After electrotransfer to
nitrocellulose, the membrane was probed with a rabbit polyclonal
antibody to PARP (Cell Signaling 9542), followed by goat
anti-rabbit alkaline phosphatase conjugated secondary antibody
(Jackson 11-055-045). Bound antibodies were detected
colorimetrically using BCIP/NBT (Roche 1681451).
[0390] The western blot presented in FIG. 35 shows the cleavage of
PARP within the Jurkat cells in a time-dependent fashion. The
cleaved form (denoted by the asterisk, *) appears in the treated
cells between 2 and 4 hrs after exposure to Salinosporamide A while
the majority of the remaining PARP is cleaved by 24 hrs. The
Staurosporine treated cells (St) show rapid cleavage of PARP with
most of this protein being cleaved within 4 hours. These data
strongly suggest that Salinosporamide A can induce apoptosis in
Jurkat cells.
Example 31
Anti-Anthrax Activity
[0391] In order to assay for the ability of Salinosporamide A or
other compounds to prevent cell death resulting from LeTx exposure,
RAW264.7 macrophage-like cells and recombinant LF and PA lethal
toxin components were used as an in vitro model system assaying for
cytotoxicity, as described below.
[0392] RAW264.7 cells (ATCC #TIB-71) were adapted to and maintained
in Advanced Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad,
Calif.) supplemented with 5% fetal bovine serum (ADMEM, Mediatech,
Herndon, Va.) at 37.degree. C. in a humidified 5% CO.sub.2
incubator. Cells were plated overnight in ADMEM supplemented with
5% FBS at 37.degree. C. in a humidified 5% CO.sub.2 incubator at a
concentration of 50,000 cells/well in a 96-well plate.
Alternatively, cells cultured in DMEM supplemented with 10% fetal
calf serum were also used and found to be amenable to this assay.
Media was removed the following morning and replaced with
serum-free ADMEM with or without Salinosporamide A or Omuralide at
doses ranging from 1 .mu.M to 0.5 nM for an 8-point dose-response.
The compounds were prepared from a 1 mg/mL DMSO stock solution and
diluted to the final concentration in ADMEM. After a 15 minute
pre-incubation, 200 ng/mL LF or 400 ng/mL PA alone or in
combination (LeTx) were added to cells. Recombinant LF and PA were
obtained from List Biological Laboratories and stored as 1 mg/mL
stock solutions in sterile water containing 1 mg/ml BSA at -80
.degree. C. as described by the manufacturer. Cells were incubated
for 6 hours at 37.degree. C., followed by addition of Resazurin as
previously described. Plates were incubated an additional 6 hours
prior to assessing cell viability by measuring fluorescence. The
data are a summary of three experiments with three to six
replicates per experiment and are expressed as the percent
viability using the DMSO (negative) and the LeTx controls
(positive) to normalize the data using the following equation: %
viability=100*(observed OD-positive control)/(negative
control-positive control).
[0393] The data represented in FIG. 36 indicate that treatment with
Salinosporamide A can prevent LeTx-induced cell death of
macrophage-like RAW264.7 cells in vitro. Treatment of RAW cells
with either LF or PA alone or Salinosporamide A alone resulted in
little reduction in cell viability, whereas treatment with LeTx
resulted in approximately 0.27% cell viability as compared to
controls. Salinosporamide A may enhance macrophage survival by
inhibiting the degradation of specific proteins and decreasing the
synthesis of cytokines, which will ultimately lead to the
inhibition of the lethal effects of anthrax toxins in vivo.
[0394] Although Salinosporamide A treatment alone produced very
modest cytotoxicity at concentrations of 100 nM and above,
treatment with lower, relatively non-toxic levels revealed a marked
increase in RAW 264.7 cell viability in LeTx treated cells (FIG.
36). For example, the Salinosporamide A+LeTx treated group showed
82% cell-viability when pretreated with 12 nM Salinosporamide A,
which was a concentration that showed 96% viability with
Salinosporamide A alone. The average EC.sub.50 for Salinosporamide
A in these studies was 3.6 nM. In contrast, Omuralide showed
relatively little effect on cell viability until concentrations of
1 .mu.M were reached. Even at this high concentration of Omuralide,
only 37% viability was observed indicating that Salinosporamide A
is a more potent inhibitor of LeTx-induced RAW264.7 cell death.
Consistent with these data, Tang et. al., Infect Immun 67:3055
(1999), found that the EC.sub.50 concentrations for MG132 and
Lactacystin (the precursor to Omuralide) in the LeTx assay were 3
.mu.M. Taken together, these data further illustrate that
Salinosporamide A is a more potent inhibitor of LeTx-induced
cytotoxicity than any other compound described to date.
[0395] Salinosporamide A promoted survival of RAW264.7 cells in the
presence of LeTx indicating that this compound or it's derivatives
may be a valuable clinical therapeutic for anthrax. In addition, it
is worth noting that Salinosporamide A is much less cytotoxic on
RAW 264.7 cells than for many tumor cells.
Example 32
Activity of Salinosporamide A Against Multiple Myeloma and Prostate
Cancer Cell Lines
[0396] NF-?B appears to be critical to the growth and resistance to
apoptosis in Multiple Myeloma and has also been reported to be
constitutively active in various prostate cancer cell lines
(Hideshima T et al. 2002, Shimada K et al. 2002 and Palayoor S T et
al. 1999). NF-.kappa.B activity is regulated by the proteasomal
degradation of its inhibitor I.kappa.B.alpha.. Since
Salinosporamide A has been shown to inhibit the proteasome in vitro
and to interfere with the NF-.kappa.B signaling pathway, the
activity of Salinosporamide A against the multiple myeloma cell
line RPMI 8226 and the prostate cancer cell lines PC-3 and DU 145
was evaluated.
[0397] EC.sub.50 values were determined in standard growth
inhibition assays using Resazurin dye and 48 hour of drug exposure.
Results from 2-5 independent experiments (Table 6) show that the
EC.sub.50 values for Salinosporamide A against RPMI 8226 and the
prostate cell lines range from 10-37 nM.
10TABLE 6 EC.sub.50 values of Salinosporamide A (II-16) against
Multiple Myeloma and Prostate Tumor cell lines RPMI 8226 (n = 5) DU
145 (n = 3) EC.sub.50 % EC.sub.50 % PC-3 (n = 2) (nM),
cytotoxicity, (nM), cytotoxicity, EC.sub.50 % Compound mean .+-. SD
mean .+-. SD mean .+-. SD mean .+-. SD (nM) cytotoxicity
Salinosporamide A 10 .+-. 3 94 .+-. 1 37 .+-. 10 75 .+-. 4 31, 25
88, 89
[0398] The ability of Salinosporamide A to induce apoptosis in RPMI
8226 and PC-3 cells was evaluated by monitoring the cleavage of
PARP and Pro-Caspase 3 using western blot analysis. Briefly, PC-3
and RPMI 8226 cells were treated with 100 nM Salinosporamide A
(2345R01) for 0, 8 or 24 hours. Total protein lysates were made and
20 .mu.g of the lysates were then resolved under
reducing/denaturing conditions and blotted onto nitrocellulose. The
blots were then probed with anti-PARP or anti-caspase 3 antibodies
followed by stripping and reprobing with an anti-actin
antibody.
[0399] Results of these experiments illustrate that Salinosporamide
A treatment of RPMI 8226 cells leads to the cleavage of PARP and
Pro-caspase 3 in a time-dependent manner (FIG. 37). RPMI 8226 cells
seem to be more sensitive to Salinosporamide A than PC-3 cells
since the induction of PARP cleavage is already noticeable at 8
hours and complete by 24 hours. In contrast, in PC-3 cells the
cleavage of PARP is noticeable at 24 hours, while the cleavage of
Pro-Caspase 3 is not detected in this experiment (FIG. 37).
[0400] RPMI 8226 cells were used to evaluate the effect of treating
the cells for 8 hours with various concentrations of
Salinosporamide A. Briefly, RPMI 8226 cells were treated with
varying concentrations of Salinosporamide A (2345R01) for 8 hours
and protein lysates were made. 25 .mu.g of the lysates were then
resolved under reducing/denaturing conditions and blotted onto
nitrocellulose. The blots were then probed with anti-PARP or
anti-caspase 3 antibodies followed by stripping and reprobing with
an anti-actin antibody. FIG. 38 demonstrates that Salinosporamide A
induces a dose-dependent cleavage of both PARP and Pro-Caspase
3.
Example 33
Growth Inhibition of Human Multiple Myeloma by Formulae II-2, II-3,
II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,
II-20, and IV-3C; RPMI 8226 and U266 Cells
[0401] The human multiple myeloma cell lines, RPMI 8226 (ATCC;
CCL-155) and U266 (ATCC; TIB-196) were maintained in appropriate
culture media. The cells were cultured in an incubator at
37.degree. C. in 5% CO.sub.2 and 95% humidified air.
[0402] For cell growth inhibition assays, RPMI 8226 cells and U266
were seeded at 2.times.10.sup.4 and 2.5.times.10.sup.4 cells/well
respectively in 90 .mu.l complete media into Corning 3904
black-walled, clear-bottom tissue culture plates. 20 mM stock
solutions of the compounds were prepared in 100% DMSO, aliquoted
and stored at -80.degree. C. The compounds were serially diluted
and added in triplicate to the test wells. The final concentration
range of Formula II-3, II-8C, II-5B, II-13C, II-17, IV-3C and II-20
were from 20 .mu.M to 6.32 nM. The final concentration of Formula
II-16, II-18 and II-19 ranged from 632 nM to 200 pM. The final
concentration range of Formula II-2, II-4 and II-5A were from 2
.mu.M to 632 pM. The final concentration of DMSO was 0.25% in all
samples.
[0403] Following 48 hours of drug exposure, 10 .mu.l of 0.2 mg/ml
resazurin (obtained from Sigmna-Aldrich Chemical Co.) in Mg.sup.2+,
Ca.sup.2+ free phosphate buffered saline was added to each well and
the plates were returned to the incubator for 3-6 hours. Since
living cells metabolize Resazurin, the fluorescence of the
reduction product of Resazurin was measured using a Fusion
microplate fluorometer (Packard Bioscience) with
.lambda..sub.ex=535 nm and .lambda..sub.em=590 nm filters.
Resazurin dye in medium without cells was used to determine the
background, which was subtracted from the data for all experimental
wells. The data were normalized to the average fluorescence of the
cells treated with media +0.25% DMSO (100% cell growth) and
EC.sub.50 values (the drug concentration at which 50% of the
maximal observed growth inhibition is established) were determined
using a standard sigmoidal dose response curve fitting algorithm
(generated by XLfit 3.0, ID Business Solutions Ltd). The data are
summarized in Tables 13 and 15.
Example 34
Salinosporamide A (II-16) Retains Activity Against the Multi-Drug
Resistant Cell Lines MES-SA/Dx5 and HL-60/MX2
[0404] The EC.sub.50 values of Salinosporamide A against the human
uterine sarcoma MES-SA cell line and its multidrug-resistant
derivative MES-SA/Dx5 were determined to evaluate whether
Salinosporamide A retains activity against a cell line
overexpressing the P-glycoprotein efflux pump. Paclitaxel, a known
substrate for the P-glycoprotein pump was included as a
control.
11TABLE 7 EC.sub.50 values of Salinosporamide A against MES-SA and
the drug-resistant derivative MES-SA/Dx5 MES-SA MES-SA/Dx5
EC.sub.50 (nM), % cytotoxicity, EC.sub.50 (nM), % cytotoxicity,
Fold mean mean .+-. SD mean .+-. SD mean .+-. SD change
Salinosporamide A 20 .+-. 5 94 .+-. 1 23 .+-. 1 92 .+-. 2 1.2
Paclitaxel 5 .+-. 2 63 .+-. 7 2040 .+-. 150 78 .+-. 1 408
[0405] Results from these growth inhibition assays (Table 7) show
that, as expected, Paclitaxel did not retain its activity against
MES-SA/Dx5 cells as reflected by the 408 fold increase in the
EC.sub.50 values. EC.sub.50 values for Salinosporamide A against
MES-SA and MES-SA/Dx5 were similar. This illustrates that
Salinosporamide A is able to inhibit the growth of the multi-drug
resistant cell line MES-SA/Dx5 suggesting that Salinosporamide A
does not seem to be a substrate for the P-glycoprotein efflux
pump.
[0406] In addition, Salinosporamide A was evaluated against
HL-60/MX2, the drug resistant derivative of the human leukemia cell
line, HL-60, characterized by having a reduced Topoisomerase II
activity and considered to have atypical multidrug resistance.
EC.sub.50 values for growth inhibition were determined for
Salinosporamide A against the HL-60 and HL-60/MX2. The DNA binding
agent Mitoxantrone was included as a control, as HL-60/MX2 cells
are reported to be resistant to this chemotherapeutic agent (Harker
W. G. et al. 1989).
12TABLE 8 EC.sub.50 values of Salinosporamide A against HL-60 and
the drug resistant derivative HL-60/MX2 HL-60 HL-60/MX2 Fold
EC.sub.50 (nM) % cytotoxicity EC.sub.50 (nM) % cytotoxicity change
Salinosporamide A 27, 30 88, 91 28, 25 84, 89 1.0, 0.8 Mitoxantrone
59, 25 98, 100 1410, 827 98, 99 24, 33
[0407] The data in Table 8 reveals that Salinosporamide A was able
to retain its activity against HL-60/MX2 cells relative to HL-60
cells, indicating that Salinosporamide A is active in cells
expressing reduced Topoisomerase II activity. In contrast,
Mitoxantrone was about 29 fold less active against HL-60/MX2
cells.
Example 35
Salinosporamide A and Several Analogs: Structure Activity
Relationship
[0408] To establish an initial structure activity relationship
(SAR) for Salinosporamide A, a series of Salinosporamide A analogs
were evaluated against the multiple myeloma cell line RPMI 8226.
EC.sub.50 values were determined in standard growth inhibition
assays using Resazurin dye and 48 hour of drug exposure.
[0409] The results of this initial series of SAR (Table 9) indicate
that the addition of a halogen group to the ethyl group seems to
enhance the cytotoxic activity.
13TABLE 9 Initial SAR series of Salinosporamide A EC.sub.50,
Compound .mu.M (mean .+-. % Cytotoxicity No. Molecular Structure
SD) (mean .+-. SD) II-16 81 0.007 .+-. 0.0001 94 .+-. 0 II-17 82
2.6, 2.3 94, 95 II-18 83 0.017, 0.022 94, 94
[0410] Where n>2, mean .+-. standard deviation was
determined
Example 36
In vivo Biology
Maximum Tolerated Dose (MTD) Determination
[0411] In vivo studies were designed to determine the MTD of
Salinosporamide A when administered intravenously to female BALB/c
mice.
[0412] BALB/c mice were weighed and various Salinosporamide A
concentrations (ranging from 0.01 mg/kg to 0.5 mg/kg) were
administered intravenously as a single dose (qdx1) or daily for
five consecutive days (qdx5). Animals were observed daily for
clinical signs and were weighed individually twice weekly until the
end of the experiment (maximum of 14 days after the last day of
dosing). Results are shown in Table 11 and indicate that a single
intravenous Salinosporamide A dose of up to 0.25 mg/kg was
tolerated. When administered daily for five consecutive days,
concentrations of Salinosporamide A up to 0.1 mg/kg were well
tolerated. No behavioral changes were noted during the course of
the experiment.
14TABLE 11 MTD Determination of Salinosporamide A in female BALB/c
Mice Group Dose (mg/kg) Route/Schedule Deaths/Total Days of Death 1
0.5 i.v.; qdx1 3/3 3, 3, 4 2 0.25 i.v.; qdx1 0/3 3 0.1 i.v.; qdx1
0/3 4 0.05 i.v.; qdx1 0/3 5 0.01 i.v.; qdx1 0/3 6 0 i.v.; qdx1 0/3
7 0.5 i.v.; qdx5 3/3 4, 6, 7 8 0.25 i.v.; qdx5 3/3 4, 5, 5 9 0.1
i.v.; qdx5 0/3 10 0.05 i.v.; qdx5 0/3 11 0.01 i.v.; qdx5 0/3 12 0
i.v.; qdx5 0/3
Example 37
Preliminary Assessment of Salinosporamide A Absorption,
Distribution, Metabolism and Elimination (ADME) Characteristics
[0413] Studies to initiate the evaluation of the ADME properties of
Salinosporamide A were performed. These studies consisted of
solubility assessment, LogD.sup.7.4 determination and a preliminary
screen to detect cytochrome P450 enzyme inhibition. Results from
these studies showed an estimated solubility of Salinosporamide A
in PBS (pH 7.4) of 9.6 .mu.M (3 .mu.g/ml) and a LogD.sup.7.4 value
of 2.4. This LogD.sup.7.4 value is within the accepted limits
compatible with drug development (LogD.sup.7.4<5.0) and suggests
oral availability. Results from the preliminary P450 inhibition
screen showed that Salinosporamide A, when tested at 10 .mu.M,
showed no or low inhibition of all P450 isoforms: CYP1A2, CYP2C9
and CYP3A4 were inhibited by 3%, 6% and 6% respectively, while
CYP2D6 and CYP2C19 were inhibited by 19% and 22% respectively.
Example 38
Salinosporamide A and its Effects in vivo on Whole Blood Proteasome
Activity
[0414] Salinosporamide A was previously demonstrated to be a potent
and specific inhibitor of the proteasome in vitro, with an
IC.sub.50 of 2 nM towards the chymotrypsin-like activity of
purified 20S proteasomes. To monitor the activity of
Salinosporamide A in vivo, a rapid and reproducible assay (adapted
from Lightcap et al. 2000) was developed to assess the proteosome
activity in whole blood.
[0415] In brief, frozen whole blood samples were thawed on ice for
one hour, and resuspended in 700 .mu.L of ice cold 5 mM EDTA, pH
8.0 in order to lyse the cells by hypotonic shock. This represents
approximately 2-3 times the volume of the packed whole blood cells.
Lysis was allowed to proceed for one hour, and the cellular debris
was removed by centrifuigation at 14,000.times.g for 10 minutes.
The supernatant (Packed Whole Blood Lysate, PWBL) was transferred
to a fresh tube, and the pellet discarded. Protein concentration of
the PWBL was determined by BCA assay (Pierce) using BSA as a
standard. Approximately 80% of the samples had a total protein
concentration between 800 and 1200 .mu.g/mL.
[0416] Proteasome activity was determined by measuring the
hydrolysis of a fluorogenic substrate specific for the
chymotrypsin-like activity of proteasomes (suc-LLVY-AMC, Bachem
Cat. 1-1395). Control experiments indicated that >98% of the
hydrolysis of this peptide in these extracts is mediated by the
proteasome. Assays were set up by mixing 5 .mu.L of a PWBL from an
animal with 185 .mu.L of assay buffer (20 mM HEPES, 0.5 mM EDTA,
0.05% Triton X-100, 0.05% SDS, pH 7.3) in Costar 3904 plates.
Titration experiments revealed there is a linear relationship
between protein concentration and hydrolysis rate if the protein
concentration in the assay is between 200 and 1000 .mu.g. The
reactions were initiated by the addition of 10 .mu.L of 0.4 mM
suc-LLVY-AMC (prepared by diluting a 10 mM solution of the peptide
in DMSO 1:25 with assay buffer), and incubated in a fluorometer
(Labsystems Fluoroskan) at 37.degree. C. Hydrolysis of the
substrate results in the release of free AMC, which was measured
fluorometrically by using .lambda..sub.ex=390 nm and
.lambda..sub.em=460 nm. The rate of hydrolysis in this system is
linear for at least one hour. The hydrolysis rate of each sample is
then normalized to relative fluorescent units per milligram of
protein (RFU/mg).
[0417] To explore the in vivo activity of Salinosporamide A, male
Swiss-Webster mice (5 per group, 20-25 g in weight) were treated
with various concentrations of Salinosporamide A. Salinosporamide A
was administered intravenously and given its LogD.sup.7.4 value of
2.4, suggestive of oral availability, Salinosporamide A was also
administered orally. Salinosporamide A dosing solutions were
generated immediately prior to administration by dilution of
Salinosporamide A stock solutions (100% DMSO) using 10% solutol
yielding a final concentration of 2% DMSO. The vehicle control
consisted of 2% DMSO in 10% solutol. One group of animals was not
dosed with either vehicle or Salinosporamide A in order to
establish a baseline for proteasome activity. Salinosporamide A or
vehicle was administered at 10 mL/kg and ninety minutes after
administration the animals were anesthetized and blood withdrawn by
cardiac puncture. Packed whole blood cells were collected by
centrifugation, washed with PBS, and re-centrifuged. All samples
were stored at -80.degree. C. prior to the evaluation of the
proteasome activity.
[0418] In order to be certain that the hydrolysis of the substrate
observed in these experiments was due solely to the activity of the
proteasome, dose response experiments on the extracts were
performed using the highly specific proteasomal inhibitor
Epoxomicin. PWBL lysates were diluted 1:40 in assay buffer, and 180
.mu.L were added to Costar 3904 plates. Epoxomicin (Calbochem Cat.
324800) was serially diluted in DMSO to generate an eight point
dose response curve, diluted 1:50 in assay buffer, and 10 .mu.L
added to the diluted PWBL in triplicate. The samples were
preincubated for 5 minutes at 37.degree. C., and the reactions
initiated with substrate as above. The dose response curves were
analyzed in Prism, using a sigmoidal dose response with variable
slope as a model.
[0419] FIG. 40 is a scatter plot displaying the normalized
proteasome activity in PWBL's derived from the individual mice (5
mice per group). In each group, the horizontal bar represents the
mean normalized activity. These data show that Salinosporamide A
causes a profound decrease in proteasomal activity in PWBL, and
that this inhibition is dose dependent. In addition, these data
indicate that Salinosporamide A is active upon oral
administration.
[0420] The specificity of the assay was shown by examining the
effect of a known proteasome inhibitor, Epoxomicin, on hydrolysis
of the peptide substrate. Epoxomicin is a peptide epoxide that has
been shown to highly specific for the proteasome, with no
inhibitory activity towards any other known protease (Meng et al.,
1999). Lysates from a vehicle control and also from animals treated
intravenous (i.v.) with 0.1 mg/kg Salinosporamide A were incubated
with varying concentration of Epoxomicin, and IC.sub.50 values were
determined. Palayoor et al., Oncogene 18:7389-94 (1999). As shown
in FIG. 41, Epoxomicin caused a dose dependent inhibition in the
hydrolysis of the proteasome substrate. The IC.sub.50 obtained in
these experiments matches well with the 10 nM value observed using
purified 20S proteasomes in vitro (not shown). These data also
indicate that the remaining activity towards this substrate in
these lysates prepared from animals treated with 0.1 mg/kg
Salinosporamide A is due to the proteasome, and not some other
protease. The residual activity seen in extracts treated with high
doses of Epoxomicin is less than 2% of the total signal, indicating
that over 98% of the activity observed with suc-LLVY-AMC as a
substrate is due solely to the activity of the proteasomes present
in the PWBL.
[0421] Comparison of intra-run variation in baseline activity and
the ability of Salinosporamide A to inhibit proteasomal activity
was also assessed. In FIG. 42, the results of separate assays run
several weeks apart are shown. Qureshi, et al., J. Immunol. 171(3):
1515-25 (2003). For clarity, only the vehicle control and matching
dose results are shown. While there was some variation in the
proteasomal activity in PWBL derived from individual animals in the
control groups, the overall mean was very similar between the two
groups. The animals treated with Salinosporamide A (0.1 mg/kg i.v.)
also show very similar residual activity and average inhibition.
This suggests that results between assays can be compared with
confidence.
Example 39
Inhibition of in vivo LPS-Induced TNF by Salinosporamide A
[0422] Studies suggest that the proteasome plays a role in the
activation of many signaling molecules, including the transcription
factor NF-.kappa.B via protealytic degradation of the inhibitor of
NF-.kappa.B (I.kappa.B). LPS signaling through the TLR4 receptor
activates NF-.kappa.B and other transcriptional regulators
resulting in the expression of a host of proinflammatory genes like
TNF, IL-6, and IL-1.beta.. The continued expression of
proinflammatory cytokines has been identified as a major factor in
many diseases. Inhibitors of TNF and IL-1.beta. have shown efficacy
in many inflammation models including the LPS murine model, as well
as animal models of rheumatoid arthritis and inflammatory bowel
disease. Recent studies have suggested that inhibition of the
proteasome can prevent LPS-induced TNF secretion (Qureshi et al.,
2003). These data suggest that Salinosporamide A, a novel potent
proteasome inhibitor, may prevent TNF secretion in vivo in the
high-dose LPS murine model.
[0423] To assess the ability of Salinosporamide A to inhibit in
vivo LPS-induced plasma TNF levels in mice, in vivo studies were
initiated at BolderBioPATH, Inc. in Boulder, Colo. The following
methods outline the protocol design for these studies.
[0424] Male Swiss Webster mice (12/group weighing 20-25 g) were
injected with LPS (2 mg/kg) by the i.p. route. Thirty minutes
later, mice were injected i.v. (tail vein) with Salinosporamide A
at 2.5 mg/kg after approximately 5 minutes under a heat lamp.
Ninety minutes after LPS injection, the mice were anesthetized with
Isoflurane and bled by cardiac puncture to obtain plasma. Remaining
blood pellet was then resuspended in 500 .mu.L of PBS to wash away
residual serum proteins and centrifuged again. Supernatant was
removed and blood pellet frozen for analysis of proteasome
inhibition in packed whole blood lysate.
15 TABLE 12 Time Group ID Group n = 0 min +30 min No
injections/baseline 1 5 Saline + solutol vehicle 2 5 saline Saline
+ solutol vehicles 3 5 saline Solutol/DMSO LPS i.p./Vehicle (-30
min) 4 12 LPS LPS i.p./Vehicle (+30 m) 5 12 LPS Solutol/DMSO
saline/Salinosporamide A 6 12 saline (-30 min) 0.25 mg/kg
saline/Salinosporamide A 7 12 saline 0.25 mg/kg (+30 m) 0.25 mg/kg
LPS/Salinosporamide A (-30 min) 8 12 LPS 0.25 mg/kg
LPS/Salinosporamide A (+30 m) 9 12 LPS 0.25 mg/kg 0.25 mg/kg
[0425] Dosing solutions were prepared using a 10 mg/mL
Salinosporamide A stock solution in 100% DMSO. A 10% solutol
solution was prepared by diluting w/w with endotoxin-free water and
a 1:160 dilution was made of the 10 mg/ml Salinosporamide A stock.
Animals were dosed i.v. with 4 ml/kg. A vehicle control solution
was also prepared by making the same 1:160 dilution with 100% DMSO
into 10% solutol solution giving a final concentration of 9.375%
solutol in water and 0.625% DMSO. Measurements of plasma TNF were
performed using the Biosource mTNF Cytoset kit (Biosource Intl.,
Camarillo, Calif.; catalog #CMC3014) according to manufacturer's
instructions. Samples were diluted 1:60 for the assay.
[0426] Data from two independent experiments with at least ten
replicate animals per group indicated that treatment with 0.125 or
0.25 mg/kg Salinosporamide A decreased LPS-induced TNF secretion in
vivo. A representative experiment is shown in FIG. 43. These data
reveal that treatment of animals with 0.25 mg/kg Salinosporamide A
thirty minutes after 2 mg/kg LPS injection resulted in significant
reduction in serum TNF levels. Packed whole blood samples were also
analyzed for ex vivo proteasome inhibition revealing 70.+-.3%
inhibition in animals treated with 0.125 mg/kg and 94.+-.3% in
animals treated with 0.25 mg/kg. No significant differences were
seen in proteasome inhibition in animals treated with or without
LPS. Salinosporamide A reduces LPS-induced plasma TNF levels by
approximately 65% when administered at 0.125 or 0.25 mg/kg i.v.
into mice 30 minutes post-LPS treatment.
Example 40
Potential in vitro Chemosensitizing Effects of Salinosporamide
A
[0427] Chemotherapy agents such as CPT-11 (Irinotecan) can activate
the transcription factor nuclear factor-kappa B (NF-?B) in human
colon cancer cell lines including LoVo cells, resulting in a
decreased ability of these cells to undergo apoptosis. Cusack, et
al., Cancer Res 61:3535 (2001). In unstimulated cells, NF-?B
resides in the cytoplasm in an inactive complex with the inhibitory
protein I.kappa.B (inhibitor of NF-.kappa.B). Various stimuli can
cause I.kappa.B phosphorylation by I.kappa.B kinase, followed by
ubiquitination and degradation of I.kappa.B by the proteasome.
Following the degradation of I.kappa.B, NF-.kappa.B translocates to
the nucleus and regulates gene expression, affecting many cellular
processes, including upregulation of survival genes thereby
inhibiting apoptosis.
[0428] The recently approved proteasome inhibitor, Velcade.TM.
(PS-341; Millennium Pharmaceuticals, Inc.), is directly toxic to
cancer cells and can also enhance the cytotoxic activity of CPT-11
against LoVo cells in vitro and in a LoVo xenograft model by
inhibiting proteasome induced degradation of I?B. Adams, J., Eur J
Haematol 70:265 (2003). In addition, Velcade.TM. was found to
inhibit the expression of proangiogenic chemokines/cytokines
GRO-.alpha. and VEGF in squamous cell carcinoma, presumably through
inhibition of the NF-.kappa.B pathway. Sunwoo, et al., Clin Cancer
Res 7:1419 (2001). The data indicate that proteasome inhibition may
not only decrease tumor cell survival and growth, but also
angiogenesis.
Example 41
Growth Inhibition of Colon, Prostate, Breast, Lung, Ovarian,
Multiple Myeloma and Melanoma
[0429] Human colon adenocarcinoma (HT-29; HTB-38), prostate
adenocarcinoma (PC-3; CRL-1435), breast adenocarcinoma (MDA-MB-231;
HTB-26), non-small cell lung carcinoma (NCI-H292; CRL-1848),
ovarian adenocarcinoma (OVCAR-3; HTB-161), multiple myeloma (RPMI
8226; CCL-155), multiple myeloma (U266; TIB-196) and mouse melanoma
(B16-F10; CRL-6475) cells were all purchased from ATCC and
maintained in appropriate culture media. The cells were cultured in
an incubator at 37.degree. C. in 5% CO.sub.2 and 95% humidified
air.
[0430] For cell growth inhibition assays, HT-29, PC-3, MDA-MB-231,
NCI-H292, OVCAR-3 and B16-F10 cells were seeded at
5.times.10.sup.3, 5.times.10.sup.3, 1.times.10.sup.4,
4.times.10.sup.3, 1.times.10.sup.4 and 1.25.times.10.sup.3 cells/
well respectively in 90 .mu.l complete media into 96 well (Corning;
3904) black-walled, clear-bottom tissue culture plates and the
plates were incubated overnight to allow cells to establish and
enter log phase growth. RPMI 8226 and U266 cells were seeded at
2.times.10.sup.4 and 2.5.times.10.sup.4 cells/well respectively in
90 .mu.l complete media into 96 well plates on the day of the
assay. 20 mM stock solutions of the compounds were prepared in 100%
DMSO and stored at -80.degree. C. The compounds were serially
diluted and added in triplicate to the test wells. Concentrations
ranging from 6.32 .mu.M to 632 .mu.M were tested for II-2 and II-4.
II-3 and II-17 were tested at concentrations ranging from 20 .mu.M
to 6.32 nM. Formula II-18 and II-19 were tested at concentrations
ranging from 2 .mu.M to 200 .mu.M. Formula II-5A and Formula II-5B
were tested at final concentrations ranging from 2 .mu.M to 632
.mu.M and 20 .mu.M to 6.32 nM respectively. The plates were
returned to the incubator for 48 hours. The final concentration of
DMSO was 0.25% in all samples.
[0431] Following 48 hours of drug exposure, 10 .mu.l of 0.2 mg/ml
resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg.sup.2+,
Ca.sup.2+ free phosphate buffered saline was added to each well and
the plates were returned to the incubator for 3-6 hours. Since
living cells metabolize Resazurin, the fluorescence of the
reduction product of Resazurin was measured using a Fusion
microplate fluorometer (Packard Bioscience) with
.lambda..sub.ex=535 nm and .lambda..sub.em=590 nm filters.
Resazurin dye in medium without cells was used to determine the
background, which was subtracted from the data for all experimental
wells. The data were normalized to the average fluorescence of the
cells treated with media +0.25% DMSO (100% cell growth) and
EC.sub.50 values (the drug concentration at which 50% of the
maximal observed growth inhibition is established) were determined
using a standard sigmoidal dose response curve fitting algorithm
(XLfit 3.0, ID Business Solutions Ltd). Where the maximum
inhibition of cell growth was less than 50%, an EC.sub.50 value was
not determined.
[0432] The data in Table 13 summarize the growth inhibitory effects
of Formulae II-2, II-3, II-5A, II-5B, II-17, II-18 and II-19
against the human colorectal carcinoma, HT-29, human prostate
carcinoma, PC-3, human breast adenocarcinoma, MDA-MB-231, human
non-small cell lung carcinoma, NCI-H292, human ovarian carcinoma,
OVCAR-3, human multiple myelomas, RPMI 8226 and U266 and murine
melanoma B16-F10 cell lines.
16TABLE 13 EC.sub.50 values of Formulae II-2, II-3, II-4, II-5A,
II-5B, II-17, II-18 and II-19 against various tumor cell lines
EC.sub.50 (nM)* Cell line II-2 II-3 II-4 II-5A II-5B II-17 II-18
II-19 HT-29 129 .+-. 21 >20000 132 .+-. 36 85 1070 >20000 18
.+-. 7.8 13 PC-3 284 .+-. 110 >20000 204 .+-. 49 97 1330
>20000 35 .+-. 5.6 27 MDA-MB-231 121 .+-. 23 >20000 114 .+-.
4 66 1040 5900 .+-. 601 16 .+-. 2.8 17 NCI-H292 322 >20000 192
90 >20000 >20000 29 31 395 >20000 213 >20000 41 OVCAR-3
188 >20000 >6320 NT NT >20000 >2000 NT 251 >6320
>20000 >2000 RPMI 8226 49 >20000 57 36 326 6200 6.3 5.9 45
>20000 51 29 328 3500 6.3 7.1 U266 39 >20000 39 10 118 1620
4.2 3.2 32 >20000 34 9 111 1710 4.2 3.4 B16-F10 194 >20000
163 NT NT 10500 19 NT 180 >20000 175 10300 36 *Where n = 3, mean
.+-. standard deviation is presented; NT = not tested
[0433] The EC.sub.50 values indicate that the Formulae II-2, II-4
and II-18 were cytotoxic against the HT-29, PC-3, MDA-MB-231,
NCI-H292, RPMI 8226, U266 and B16-F10 tumor cell lines. II-2 was
also cytotoxic against the OVCAR-3 tumor cells. Formula II-17 was
cytotoxic against MDA-MB-23 1, RPMI 8226, U266 and B16-F10 tumor
cell lines. Formulae II-5A, II-5B and II-19 were cytotoxic against
HT-29, PC-3, MDA-MB-231, RPMI 8226 and U266 tumor cells. Formula
II-5A and II-19 were also cytotoxic against NCI-H292 tumor
cells.
[0434] The data in Table 15 summarize the growth inhibitory effects
of Formulae II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16,
II-17, II-18, II-19, IV-3C and Formula II-20 against the human
multiple myeloma cell lines, RPMI 8226 and U266.
17TABLE 15 Mean EC.sub.50 values of Formulae II-2, II-3, II-4,
II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, IV-3C and
Formula II-20 against RPMI 8226 and U266 cells RPMI 8226 U266
Compound EC.sub.50 (nM) EC.sub.50 (nM) Formula II-17 4800 1670
Formula II-16 7.0 4.1 Formula II-18 6.3 4.2 Formula II-2 47 36
Formula II-3 >20000 >20000 Formula II-4 54 36 Formula II-5A
33 10 Formula II-5B 327 115 Formula II-8C >20000 >20000
Formula II-13C >20000 >20000 Formula II-19 6.5 3.3 Formula
IV-3C >20000 8020 Formula II-20* 10500 3810 *n = 1
[0435] The EC.sub.50 values indicate that Formulae II-2, II-4,
II-5A, II-5B, II-16, II-17, II-18, II-19 and II-20 were cytotoxic
against RPMI 8226 and U266 cells. Formula IV-3C was cytotoxic
against U266 cells
Example 42
Growth Inhibition of MES-SA, MES-SA/Dx5, HL-60 and HL-60/MX2 Tumor
Cell Lines
[0436] Human uterine sarcoma (MES-SA; CRL-1976), its multidrug
resistant derivative (MES-SA/Dx5; CRL-1977), human acute
promyelocytic leukemia cells (HL-60; CCL-240) and its multidrug
resistant derivative (HL-60/MX2; CRL-2257) were purchased from ATCC
and maintained in appropriate culture media. The cells were
cultured in an incubator at 37.degree. C. in 5% CO.sub.2 and 95%
humidified air.
[0437] For cell growth inhibition assays, MES-SA and MES-SA/Dx5
cells were both seeded at 3.times.10.sup.3 cells/ well in 90 .mu.l
complete media into 96 well (Corning; 3904) black-walled,
clear-bottom tissue culture plates and the plates were incubated
overnight to allow cells to establish and enter log phase growth.
HL-60 and HL-60/MX2 cells were both seeded at 5.times.10.sup.4
cells/ well in 90 .mu.l complete media into 96 well plates on the
day of compound addition. 20 mM stock solutions of the compounds
were prepared in 100% DMSO and stored at -80.degree. C. The
compounds were serially diluted and added in triplicate to the test
wells. Concentrations ranging from 6.32 .mu.M to 2 nM were tested
for II-2 and II-4. II-3 and II-17 were tested at concentrations
ranging from 20 .mu.M to 6.32 nM. Compound II-18 was tested at
concentrations ranging from 2 .mu.M to 632 pM. The plates were
returned to the incubator for 48 hours. The final concentration of
DMSO was 0.25% in all samples.
[0438] Following 48 hours of drug exposure, 10 .mu.l of 0.2 mg/ml
resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg.sup.2+,
Ca.sup.2+ free phosphate buffered saline was added to each well and
the plates were returned to the incubator for 3-6 hours. Since
living cells metabolize Resazurin, the fluorescence of the
reduction product of Resazurin was measured using a Fusion
microplate fluorometer (Packard Bioscience) with
.lambda..sub.ex=535 nm and .lambda..sub.em=590 nm filters.
Resazurin dye in medium without cells was used to determine the
background, which was subtracted from the data for all experimental
wells. The data were normalized to the average fluorescence of the
cells treated with media +0.25% DMSO (100% cell growth) and
EC.sub.50 values (the drug concentration at which 50% of the
maximal observed growth inhibition is established) were determined
using a standard sigmoidal dose response curve fitting algorithm
(XLfit 3.0, ID Business Solutions Ltd). Where the maximum
inhibition of cell growth was less than 50%, an EC.sub.50 value was
not determined.
[0439] The multidrug resistant MES-SA/Dx5 tumor cell line was
derived from the human uterine sarcoma MES-SA tumor cell line and
expresses elevated P-Glycoprotein (P-gp), an ATP dependent efflux
pump. The data in Table 16 summarize the growth inhibitory effects
of Formulae II-2, II-3, II-4, II-17 and II-18 against MES-SA and
its multidrug resistant derivative MES-SA/Dx5. Paclitaxel, a known
substrate of the P-gp pump was included as a control.
18TABLE 16 EC.sub.50 values of Formulae II-2, II-3, II-4, II-17 and
II-18 against MES-SA and MES-SA/Dx5 tumor cell lines EC.sub.50 (nM)
Fold Compound MES-SA MES-SA/Dx5 change* II-2 193 220 1.0 155 138
II-3 >20000 >20000 NA >20000 >20000 II-4 163 178 0.9
140 93 II-17 9230 9450 0.8 12900 7530 II-18 22 32 1.2 17 14
Paclitaxel 5.6 2930 798 4.6 5210 *Fold change = the ratio of
EC.sub.50 values (MES-SA/Dx5:MES-SA)
[0440] The EC.sub.50 values indicate that II-2, II-4, II-17 and
II-18 have cytotoxic activity against both MES-SA and MES-SA/Dx5
tumor cell lines. The multidrug resistant phenotype was confirmed
by the observation that Paclitaxel was .about.800 times less active
against the resistant MES-SA/Dx5 cells.
[0441] HL-60/MX2 is a multidrug resistant tumor cell line derived
from the human promyelocytic leukemia cell line, HL-60 and
expresses reduced topoisomerase II activity. The data presented in
Table 17 summarize the growth inhibitory effects of Formulae II-2,
II-3, II-4, II-17 and II-18 against HL-60 and its multidrug
resistant derivative HL-60/MX2. Mitoxantrone, the topoisomerase II
targeting agent was included as a control.
19TABLE 17 EC.sub.50 values of Formulae II-2, II-3, II-4, II-17 and
II-18 against HL-60 and HL-60/MX2 tumor cell lines EC.sub.50 (nM)
Fold Compound HL-60 HL-60/MX2 change* II-2 237 142 0.7 176 133 II-3
>20000 >20000 NA >20000 >20000 II-4 143 103 0.8 111 97
II-17 >20000 >20000 NA II-18 27 19 0.7 23 18 Mitoxantrone 42
1340 30.6 40 1170 *Fold change = the ratio of EC.sub.50 values
(HL-60/MX2:HL-60)
[0442] The EC.sub.50 values indicate that II-2, II-4 and II-18
retained cytotoxic activity against both HL-60 and HL-60/MX2 tumor
cell lines. The multidrug resistant phenotype was confirmed by the
observation that Mitoxantrone was .about.30 times less active
against the resistant HL-60/MX2 cells.
Example 43
Inhibition of NF-.kappa.B-Mediated Luciferase Activity: HEK293
NF-.kappa.B/Luciferase Reporter Cell Line
[0443] The HEK293 NF-.kappa.B/luciferase reporter cell line is a
derivative of the human embryonic kidney cell line (ATCC; CRL-1573)
and carries a luciferase reporter gene under the regulation of
5.times.NF-.kappa.B binding sites. The reporter cell line was
routinely maintained in complete DMEM medium (DMEM plus 10%(v/v)
Fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES and
Penicillin/Streptomycin at 100 IU/ml and 100 .mu.g/ml,
respectively) supplemented with 250 .mu.g/ml G418. When performing
the luciferase assay, the DMEM basal medium was replaced with
phenol-red free DMEM basal medium and the G418 was omitted. The
cells were cultured in an incubator at 37.degree. C. in 5% CO2 and
95% humidified air.
[0444] For NF-.kappa.B-mediated luciferase assays, HEK293
NF-.kappa.B/luciferase cells were seeded at 1.5.times.10.sup.4
cells/well in 90 .mu.l phenol-red free DMEM complete medium into
Corning 3917 white opaque-bottom tissue culture plates. For Formula
II-2, Formula II-4 and Formula II-5A, a 400 .mu.M starting dilution
was made in 100% DMSO and this dilution was used to generate a
8-point half log dilution series. This dilution series was further
diluted 40.times. in appropriate culture medium and ten .mu.l
aliquots were added to the test wells in triplicate resulting in
final test concentrations ranging from 1 .mu.M to 320 pM. For
Formula II-3 and Formula II-5B, a 8 mM starting dilution was made
in 100% DMSO and the same procedure was followed as described above
resulting in final test concentrations ranging from 20 .mu.M to 6.3
nM. The plates were returned to the incubator for 1 hour. After 1
hr pretreatment, 10 .mu.l of a 50 ng/ml TNF-.alpha. solution,
prepared in the phenol-red free DMEM medium was added, and the
plates were incubated for an additional 6 hr. The final
concentration of DMSO was 0.25% in all samples.
[0445] At the end of the TNF-.alpha. stimulation, 100 .mu.l of
Steady Lite HTS luciferase reagent (Packard Bioscience) was added
to each well and the plates were left undisturbed for 10 min at
room temperature before measuring the luciferase activity. The
relative luciferase units (RLU) were measured by using a Fusion
microplate fluorometer (Packard Bioscience). The EC.sub.50 values
(the drug concentration at which 50% of the maximal relative
luciferase unit inhibition is established) were calculated in Prism
(GraphPad Software) using a sigmoidal dose response, variable slope
model.
[0446] NF-.kappa.B regulates the expression of a large number of
genes important in inflammation, apoptosis, tumorigenesis, and
autoimmune diseases. Thus compounds capable of modulating or
affecting NF-.kappa.B activity are useful in treating diseases
related to inflammation, cancer, and autoimmune diseases, for
example. In its inactive form, NF-.kappa.B complexes with I.kappa.B
in the cytosol and upon stimulation, I.kappa.B is phosphorylated,
ubiquitinated and subsequently degraded by the proteasome. The
degradation of I.kappa.B leads to the activation of NF-.kappa.B and
its translocation to the nucleus. The effects of Formula II-2,
Formula II-3, Formula II-4, Formula II-5A and Formula II-5B on the
activation of NF-.kappa.B was evaluated by assessing the
NF-.kappa.B-mediated luciferase activity in HEK293 NF-.kappa.B/Luc
cells upon TNF-.alpha. stimulation.
[0447] Results from a representative experiment evaluating Formula
II-2, Formula II-3 and Formula II-4 (FIG. 44) revealed that
pretreatment with Formula II-2 and Formula II-4 resulted in a
dose-dependent decrease of luciferase activity in NF-.kappa.B/Luc
293 cells upon TNF-.alpha. stimulation. The calculated EC.sub.50 to
inhibit NF-.kappa.B inducible luciferase activity in this
experiment was 73 nM for Formula II-2, while EC.sub.50 value for
Formula II-4 was 67 nM. Similar data were observed in a replicate
experiment.
[0448] Results from a representative experiment evaluating Formula
II-5A and Formula II-5B are shown in FIG. 45 and illustrate that
Formula II-5A and Formula II-5B inhibit NF-.kappa.B inducible
luciferase activity with EC.sub.50 values of 30 nM and 261 nM
respectively. Similar data were observed in a replicate
experiment.
Example 44
In vitro Inhibition of Proteasome Activity by Formula II-2, Formula
II-3, Formula II-4, Formula II-5A and Formula II-5B
[0449] Formula II-2, Formula II-3, Formula II-4, Formula II-5A and
Formula II-5B were prepared as 20 mM stock solution in DMSO and
stored in small aliquots at -80.degree. C. Purified rabbit muscle
20S proteasome was obtained from CalBiochem. To enhance the
chymotrypsin-like activity of the proteasome, the assay buffer (20
mM HEPES, pH7.3, 0.5 mM EDTA, and 0.05% Triton X100) was
supplemented with SDS resulting in a final SDS concentration of
0.035%. The substrate used was sucLLVY-AMC, a fluorogenic peptide
substrate specifically cleaved by the chymotrypsin-like activity of
the proteasome. Assays were performed at a proteasome concentration
of 1 .mu.g/ml in a final volume of 200 .mu.l in 96-well Costar
microtiter plates. Formula II-2 and Formula II-4 were tested as
eight-point dose response curves with final concentrations ranging
from 500 nM to 0.16 nM, while Formula II-3 was tested with final
concentrations ranging from 10 .mu.M to 3.2 nM. Formula II-5A and
Formula II-5B were tested with final concentrations ranging from 1
.mu.M to 0.32 nM. The samples were incubated at 37.degree. C. for
five minutes in a temperature controlled plate reader. During the
preincubation step, the substrate was diluted 25-fold in assay
buffer supplemented with 0.035% SDS. After the preincubation
period, the reactions were initiated by the addition of 10 .mu.l of
the diluted substrate and the plates were returned to the plate
reader. The final concentration of substrate in the reactions was
20 .mu.M. All data were collected every five minutes for more than
1.5 hour and plotted as the mean of triplicate data points. The
EC.sub.50 values (the drug concentration at which 50% of the
maximal relative fluorescence unit is inhibited) were calculated by
Prism (GraphPad Software) using a sigmoidal dose-response, variable
slope model.
[0450] Results from a representative experiment evaluating Formula
II-2, Formula II-3 and Formula II-4 are shown in FIG. 46 and
illustrate that Formula II-2 and Formula II-4 inhibit the
chymotrypsin-like activity of the proteasome with EC.sub.50 values
of 18.5 nM and 15 nM respectively. Formula II-3 is active in this
assay with an EC.sub.50 value of 890 nM. Similar results were
obtained from an independent experiment.
[0451] Results from a representative experiment evaluating Formula
II-5A and Formula II-5B are shown in FIG. 47 and illustrate that
Formula II-5A and Formula II-5B inhibit the chymotrypsin-like
activity of the proteasome with EC.sub.50 values of 6 nM and 88 nM
respectively. Similar results were obtained in an independent
experiment.
Example 45
Inhibition of Anthrax Lethal Toxin
[0452] Anthrax toxin is responsible for the symptoms associated
with anthrax. In this disease, B. anthracis spores are inhaled and
lodge in the lungs where they are ingested by macrophages. Within
the macrophage, spores germinate, replicate, resulting in killing
of the cell. Before killing occurs, however, infected macrophages
migrate to the lymph nodes where, upon death, they release their
contents, allowing the organism to enter the bloodsteam, further
replicate, and secrete lethal toxins.
[0453] Two proteins called protective antigen (PA 83 kDa) and
lethal factor (LF, 90 kDa), play a key role in the pathogenesis of
anthrax. These proteins are collectively known as lethal toxin
(LeTx). When combined, PA and LF cause death when injected
intravenously in animals. Lethal toxin is also active in a few cell
culture lines of macrophages causing cell death within a few hours.
LeTx can induce both necrosis and apoptosis in mouse
macrophage-like RAW264.7 cells upon in vitro treatment.
[0454] In vitro Cell-Based Assay for Inhibitors of Lethal
Toxin-Mediated Cytotoxicity
[0455] RAW264.7 cells (obtained from the American Type Culture
Collection) were adapted to and maintained in RPMI-1640 medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1%
Penicillin/Streptomycin (complete medium) at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. For the assay, cells were plated
overnight in complete medium at a concentration of 50,000
cells/well in a 96-well plate. Media was removed the following day
and replaced with serum-free complete medium with or without
varying concentrations of Formulae II-2, II-3, II-4, II-5A, II-5B,
II-13C, II-17, II-18 and IV-3C starting at 330 nM and diluting at
1/2 log intervals for an 8-point dose-response. After a 45 minute
preincubation, 1 .mu.g/ml LF and 1 .mu.g/ml PA alone or in
combination (LF:PA, also termed lethal toxin (LeTx)) were added to
cells. Recombinant LF and PA were obtained from List Biological
Laboratories. Additional plates with no LeTx added were included as
a control. Cells were then incubated for six hours followed by
addition of 0.02 mg/ml resazurin dye (Molecular Probes, Eugene,
Oreg.) prepared in Mg++, Ca++ free PBS (Mediatech, Herndon, Va.).
Plates were then incubated an additional 1.5 hours prior to the
assessment of cell viability. Since resazurin is metabolized by
living cells, cytotoxicity or cell viability can be assessed by
measuring fluorescence using 530 excitation and 590 emission
filters. Data are expressed as the percent viability as compared to
a DMSO alone control (high) and the LeTx alone control (low) using
the following equation: Percent viability=100*((Measured OD-low
control)/(high control-low control)).
[0456] Inhibition of Anthrax Lethal Toxin-mediated Cytotoxicity in
RAW 264.7 Cells
[0457] Data in FIG. 48 summarize the effects of Formula II-2,
Formula II-3 and Formula II-4 against LeTx-mediated cytotoxicity of
the RAW 264.7 murine macrophage-like cell line. Treatment of RAW
264.7 cells with Formula II-2 and Formula II-4 resulted in an
increase in the viability of LeTx treated cells with EC.sub.50
values of 14 nM (FIG. 48). The EC.sub.50 values for Formula II-3
for LeTx protection was not be determined at the concentrations
tested (EC.sub.50>330 nM, the maximum concentration evaluated).
Data in Table 18 show the effects of Formulae II-5A, II-5B, II-13C,
II-17, II-18 and IV-3C against LeTx-mediated cytotoxicity of the
RAW 264.7 murine macrophage-like cell line. Treatment of RAW 264.7
cells with Formula II-5A and II-18 showed an increase in the
viability of LeTx treated RAW 264.7 cells with EC.sub.50 values of
3 nM and 4 nM respectively. Treatment with Formula II-17 and
Formula II-5B resulted in an increase in the viability of LeTx
treated cells with EC.sub.50 values of 42 nM and 45 nM
respectively. The EC.sub.50 values for Formulae II-13C and IV-3C
for LeTx protection could not be determined at the concentrations
tested (EC.sub.50>330 nM, the maximum concentration
evaluated).
20TABLE 18 EC.sub.50 values for inhibition of RAW 264.7 cell
cytotoxicity mediated by anthrax lethal toxin Compound EC.sub.50
(nM) Formula II-17 42 Formula II-18 4 Formula II-5A 3 Formula II-5B
45 Formula II-13C >330 nM Formula IV-3C >330 nM
Example 46
Formulation to be Administered Orally or the Like
[0458] A mixture obtained by thoroughly blending 1 g of a compound
obtained and purified by the method of the embodiment, 98 g of
lactose and 1 g of hydroxypropyl cellulose is formed into granules
by any conventional method. The granules are thoroughly dried and
sifted to obtain a granule preparation suitable for packaging in
bottles or by heat sealing. The resultant granule preparations are
orally administered at between approximately 100 ml/day to
approximately 1000 ml/day, depending on the symptoms, as deemed
appropriate by those of ordinary skill in the art of treating
cancerous tumors in humans.
[0459] The examples described above are set forth solely to assist
in the understanding of the embodiments. Thus, those skilled in the
art will appreciate that the methods may provide derivatives of
compounds.
[0460] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and procedures described herein are presently
representative of preferred embodiments and are exemplary and are
not intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention.
[0461] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
embodiments disclosed herein without departing from the scope and
spirit of the invention.
[0462] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0463] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions indicates the exclusion of
equivalents of the features shown and described or portions
thereof. It is recognized that various modifications are possible
within the scope of the invention. Thus, it should be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be falling within the scope of the
invention, which is limited only by the following claims.
* * * * *