U.S. patent application number 11/644625 was filed with the patent office on 2008-02-07 for compositions and methods for the inhibition of phospholipase a2.
Invention is credited to Eric Boilard, Gilles Guichard, Gerard Lambeau, Gersande Lena, Pascal Muller, Didier Rognan.
Application Number | 20080031951 11/644625 |
Document ID | / |
Family ID | 39030076 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080031951 |
Kind Code |
A1 |
Guichard; Gilles ; et
al. |
February 7, 2008 |
Compositions and methods for the inhibition of phospholipase A2
Abstract
The present invention relates to various novel substituted
dipeptide derived nitrogen-containing heterocyclic compounds, their
pharmaceutically acceptable salt derivatives, and their methods of
use.
Inventors: |
Guichard; Gilles;
(Wolfishein, FR) ; Lena; Gersande; (Julienas,
FR) ; Muller; Pascal; (Geispolshein, FR) ;
Rognan; Didier; (Bilwistein, FR) ; Boilard; Eric;
(Brookline, MA) ; Lambeau; Gerard; (Lecannet,
FR) |
Correspondence
Address: |
MCCARTER & ENGLISH LLP;CITYPLACE I
185 ASYLUM STREET
HARTFORD
CT
06103
US
|
Family ID: |
39030076 |
Appl. No.: |
11/644625 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60755631 |
Dec 29, 2005 |
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60755632 |
Dec 29, 2005 |
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60755626 |
Dec 29, 2005 |
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Current U.S.
Class: |
424/474 ;
514/169; 514/211.03; 514/424; 540/454; 540/460; 540/461;
548/543 |
Current CPC
Class: |
A61P 29/00 20180101;
Y02A 50/414 20180101; C07D 273/06 20130101; C07D 281/18 20130101;
A61K 31/395 20130101; C07D 273/02 20130101; A61K 31/40 20130101;
Y02A 50/411 20180101; Y02A 50/415 20180101; Y02A 50/30 20180101;
C07D 255/02 20130101; A61P 31/18 20180101; C07D 513/04 20130101;
A61P 33/06 20180101; A61P 31/00 20180101 |
Class at
Publication: |
424/474 ;
514/169; 514/211.03; 514/424; 540/454; 540/460; 540/461;
548/543 |
International
Class: |
A61K 9/28 20060101
A61K009/28; A61K 31/00 20060101 A61K031/00; A61K 31/40 20060101
A61K031/40; C07D 207/00 20060101 C07D207/00; C07D 245/00 20060101
C07D245/00; C07D 267/22 20060101 C07D267/22; C07D 225/04 20060101
C07D225/04; A61P 29/00 20060101 A61P029/00; A61K 31/56 20060101
A61K031/56 |
Claims
1. A compound for inhibiting a PLA2 enzyme comprising the formula
I: ##STR20## or a pharmaceutically acceptable salt thereof,
wherein, W is a member selected from the group consisting of
--C(R.sup.5)(R.sup.5a)--;
--C(R.sup.6)(R.sup.6a)--C(R.sup.7)(R.sup.7a)--;
--C(R.sup.8).dbd.C(R.sup.9)--; --N(R.sup.10), and combinations
thereof; X is a member selected from the group consisting of
--N(R.sup.1a)C(.dbd.Y)N(R.sup.4)--; --OC(.dbd.Y)N(R.sup.4)--;
--N(R.sup.1a)C(.dbd.Y)O--; --N(R.sup.1a)S(.dbd.O)N(R.sup.4)--;
--N(R.sup.1a)S(.dbd.O).sub.2N(R.sup.4)--;
--C(R.sup.1a)(R.sup.3a)C(.dbd.Y)N(R.sup.4)--, and combinations
thereof; Y and Z represent, each independent from the other, a
member selected from the group consisting of oxygen ("O") and
sulfur ("S"); and R.sup.1, R.sup.1a, R.sup.2, R.sup.3, R.sup.3a,
R.sup.4, R.sup.5, R.sup.5a, R.sup.6, R.sup.6a, R.sup.7, R.sup.7a,
R.sup.8, R.sup.9, and R.sup.10 represent, each independent from the
other, a member selected from the group consisting of: a hydrogen
atom; an amino acid side chain; a (C1-C10) alkyl; (C1-C10) alkenyl;
(C1-C10) alkynyl; (C5-C12) monocyclic or bicyclic aryl; (C5-C14)
monocyclic or bicyclic aralkyl; monocyclic or bicyclic (C5-C14)
heteroaralkyl; and (C1-C10) monocyclic or bicyclic heteroaryl group
having up to 5 heteroatoms selected from N, O, S, and P said groups
being able to be non-substituted or substituted by 1 to 6
substituents further selected from the group consisting of: a
halogen atom, an NO.sub.2, OH, amidine, benzamidine, imidazole,
1,2,3-triazole, alkoxy, (C1-C4), amino, piperazine, piperidine,
dialkylamino, guanidine group, bis alkylated or bis acylated
guanido group, carboxylic acid, carboxamide, ester, hydroxamic
acid, phosphinic acid, phosphonate, phosphonamidate, sulfhydryl and
any combination thereof.
2. A therapeutic composition for treating or preventing an
inflammatory condition in an individual comprising an effective
amount of a compound of formula I: ##STR21## or a pharmaceutically
acceptable salt thereof, wherein, W is a member selected from the
group consisting of --C(R.sup.5)(R.sup.5a)--;
--C(R.sup.6)(R.sup.6a)--C(R.sup.7)(R.sup.7a)--;
--C(R.sup.8).dbd.C(R.sup.9)--; --N(R.sup.10), and combinations
thereof; X is a member selected from the group consisting of
--N(R.sup.1a)C(.dbd.Y)N(R.sup.4)--; --OC(.dbd.Y)N(R.sup.4)--;
--N(R.sup.1a)C(.dbd.Y)O--; --N(R.sup.1a)S(.dbd.O)N(R.sup.4)--;
--N(R.sup.1a)S(.dbd.O).sub.2N(R.sup.4)--;
--C(R.sup.1a)(R.sup.3a)C(.dbd.Y)N(R.sup.4)--, and combinations
thereof; Y and Z represent, each independent from the other, a
member selected from the group consisting of oxygen ("O") and
sulfur ("S"); and R.sup.1, R.sup.1a, R.sup.2, R.sup.3, R.sup.3a,
R.sup.4, R.sup.5, R.sup.5a, R.sup.6, R.sup.6a, R.sup.7, R.sup.7a,
R.sup.8, R.sup.9, and R.sup.10 represent, each independent from the
other, a member selected from the group consisting of: a hydrogen
atom; an amino acid side chain; a (C1-C10) alkyl; (C1-C10) alkenyl;
(C1-C10) alkynyl; (C5-C12) monocyclic or bicyclic aryl; (C5-C14)
monocyclic or bicyclic aralkyl; monocyclic or bicyclic (C5-C14)
heteroaralkyl; and (C1-C10) monocyclic or bicyclic heteroaryl group
having up to 5 heteroatoms selected from N, O, S, and P said groups
being able to be non-substituted or substituted by 1 to 6
substituents further selected from the group consisting of: a
halogen atom, an NO.sub.2, OH, amidine, benzamidine, imidazole,
1,2,3-triazole, alkoxy, (C1-C4), amino, piperazine, piperidine,
dialkylamino, guanidine group, bis alkylated or bis acylated
guanido group, carboxylic acid, carboxamide, ester, hydroxamic
acid, phosphinic acid, phosphonate, phosphonamidate, sulfhydryl and
any combination thereof.
3. The therapeutic composition of claim 2, further comprising at
least one of a pharmaceutically acceptable carrier, excipient,
adjuvant, another biologically active agent or a combination
thereof.
4. The therapeutic composition of claim 3, wherein the biologically
active agent comprises at least one of a steroid, non-steroidal
anti-inflammatory, acetylsiacylic acid, a cyclooxygenase inhibitor
or a combination thereof.
5. The therapeutic composition of claim 3, wherein the therapeutic
is present in a pharmaceutically acceptable form.
6. The therapeutic of claim 5, wherein the pharmaceutically
acceptable form comprises a tablet or capsule comprising an enteric
coating.
7. A method for the treatment or prevention of an inflammatory
disease or condition comprising: administering to an individual in
need thereof, an effective amount of the therapeutic of claim
2.
8. The method of claim 7, wherein the therapeutic further comprises
at least one other ingredient selected from the group consisting of
a pharmaceutically acceptable carrier, excipient, adjuvant, another
biologically active agent or a combination thereof.
9. The method of claim 8, wherein the biologically active agent
comprises a steroid, non-steroidal anti-inflammatory,
acetylsiacylic acid, a cyclooxygenase inhibitor or a combination
thereof.
10. The method of claim 7, wherein the therapeutic is administered
in a pharmaceutically acceptable form.
11. The method of claim 10, wherein the pharmaceutically acceptable
form comprises a tablet or capsule comprising an enteric coating
for oral administration.
12. The method of claim 7, wherein the therapeutic is administered
parenterally.
13. The method of claim 7, wherein the inflammatory disease or
condition is selected from the group consisting of tissue injury,
rheumatoid arthritis, osteoarthritis, eczema, lupus, IBD, Crohn's
disease, ARDS, colitis, psoriasis, asthma, cystic fibrosis, AIDS,
stenosis, restenosis, Alzheimer's disease, Parkinson's disease,
cancer, atherosclerosis, arteriosclerosis, and cardiopulmonsary
disease.
14. A kit for treating or preventing a physiological condition
associated with inflammation, said kit comprising a plurality of
containers, wherein at least one of said containers contains the
therapeutic of claim 2 or a pharmaceutically acceptable salt
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Under 35 U.S.C. .sctn.119(e) this application claims the
benefit of U.S. Provisional Patent Ser. No. 60/755,631, filed Dec.
29, 2005, and titled "Compositions and Methods for Synthesizing
Novel Heterocyclic Therapeutics"; U.S. Provisional Patent Ser. No.
60/755,632, filed Dec. 29, 2005, and titled: "Compositions and
Methods for Treatment and Prevention of Disease"; and U.S.
Provisional Patent Application Ser. No. 60/755,626, filed Dec. 29,
2005, and titled "Compositions and Methods for the Inhibition of
Phospholipase A2"; all of which are incorporated herein by
reference in their entirety.
[0002] The present invention is related to U.S. nonprovisional
patent applications "Compositions and Methods for the Treatment and
Prevention of Disease" filed Dec. 22, 2006 (Express Mail No.: EV
902583365 US) and "Compositions and Methods for Synthesizing
Heterocyclic Therapeutic Compounds" filed Dec. 22, 2006 (Express
Mail No.: EV 902583374 US), both of which are incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to 7 and 8-membered ring
dipeptide-derived nitrogen-containing heterocyclic compounds, and
pharmaceutically acceptable salts thereof that are useful for the
inhibition of phospholipase A2 (PLA2). In addition, the invention
relates to compositions useful for the inhibition of a PLA2 enzyme,
treatment or prevention of inflammation or both in an
individual.
BACKGROUND
[0004] Phospholipase A2 enzymes (PLA2s) are enzymes that catalyze
the hydrolysis of phospholipids to release free fatty acids and
lysophospholipids. This catalytic reaction is essential in the
production of lipids involved in various physiological and
pathophysiological processes like prostaglandins, leukotrienes,
thromboxanes, platelet activation factor, lipoxins or
lysophosphatidic acid. PLA2s can be divided into two groups,
intracellular enzymes, including the calcium-dependent group IV
PLA2s, and the calcium-independent group VI PLA2s; and secreted
PLA2s (sPLA2s), which are low molecular weight proteins with a
Ca.sup.2+-dependent catalytic activity. To date, 12 mammalian
sPLA2s have been identified and classified into 3 main structural
collections: group I/II/V/X, III, and XII.
[0005] Although a significant increase in sPLA2 activity is
detected in serum in septic shock, rheumatoid arthritis, acute
pancreatitis, multiple injuries, acute chest syndrome in patients
with sickle cell disease, and in bronchoalveolar lavage (BAL) of
patients with acute respiratory distress syndrome (ARDS), the exact
function of sPLA2s in physio-pathological processes is uncertain.
It seems that the GIIA is very potent in hydrolyzing Gram positive
bacteria membranes and could be involved in the host defense
against micro-organisms. Importantly, elevated concentrations of
hGIIA are found in the eyes, an immune-privileged organ.
[0006] The ever growing body of research implicates PLA2 function
in many important physiological and pathological conditions. As
such, the development of PLA2 inhibitors will be critical to both
the study and further elucidation of PLA2's functional and
pathophysiological roles but also for the development of
pharmaceuticals for the treatment of conditions in which PLA2
function is implicated, for example, inflammatory diseases.
[0007] There is also a need to design and develop therapeutics that
are capable of discriminating between one of PLA2's functional
modes (i.e., inhibition of enzyme activity, and induction of
allosteric changes to the ligand of the M-type receptor), and
alternatively, developing therapeutics that affect both modes to
inhibit all sPLA2s functions.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds and methods for
synthesizing compounds that are efficacious for the treatment
and/or prevention of disease in an individual. In one aspect, the
invention relates to novel dipeptide derived heterocyclic compounds
synthesized using the methods of the invention. The invention also
relates to pharmaceutical compositions comprising effective amounts
of said compounds, and to therapeutic methods comprising their
administration to an individual in need thereof.
[0009] In one aspect the present invention relates to methods for
synthesizing novel dipeptide derived heterocyclics of the formula
I. ##STR1##
[0010] wherein, W is a member selected from the group consisting of
--C(R.sup.5)(R.sup.5a)--;
--C(R.sup.6)(R.sup.6a)--C(R.sup.7)(R.sup.7a)--;
--C(R.sup.8).dbd.C(R.sup.9)--; --N(R.sup.10), and combinations
thereof;
[0011] X is a member selected from the group consisting of
--N(R.sup.1a)C(.dbd.Y)N(R.sup.4)--; --OC(.dbd.Y)N(R.sup.4)--;
--N(R.sup.1a)C(.dbd.Y)O--; --N(R.sup.1a)S(.dbd.O)N(R.sup.4)--;
--N(R.sup.1a)S(.dbd.O).sub.2N(R.sup.4)--;
--C(R.sup.1a)(R.sup.3a)C(.dbd.Y)N(R.sup.4)--, and combinations
thereof;
[0012] Y and Z represent, each independent from the other, a member
selected from the group consisting of oxygen ("O") and sulfur
("S"); and
[0013] R.sup.1, R.sup.1a, R.sup.2, R.sup.3, R.sup.3a, R.sup.4,
R.sup.5, R.sup.5a, R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8,
R.sup.9, and R.sup.10 represent, each independent from the other, a
member selected from the group consisting of: a hydrogen atom; an
amino acid side chain; a (C1-C10) alkyl; (C1-C10) alkenyl; (C1-C10)
alkynyl; (C5-C12) monocyclic or bicyclic aryl; (C5-C14) monocyclic
or bicyclic aralkyl; monocyclic or bicyclic (C5-C14) heteroaralkyl;
and (C1-C10) monocyclic or bicyclic heteroaryl group having up to 5
heteroatoms selected from N, O, S, and P said groups being able to
be non-substituted or substituted by 1 to 6 substituents further
selected from the group consisting of: a halogen atom, an NO.sub.2,
OH, amidine, benzamidine, imidazole, 1,2,3-triazole, alkoxy,
(C1-C4), amino, piperazine, piperidine, dialkylamino, guanidine
group, bis alkylated or bis acylated guanido group, carboxylic
acid, carboxamide, ester, hydroxamic acid, phosphinic acid,
phosphonate, phosphonamidate, sulfhydryl and any combination
thereof.
[0014] In any of the preferred embodiments, the present invention
includes the free base or acid forms, as well as salts thereof, of
the dipeptide derivatived heterocyclics compounds described by the
above formula. The invention also includes the optical isomers,
analogs, and derivatives of the compounds described by the above
formula. In a further embodiment of the invention, mixtures of
enantiomers and/or diastereoisomers, resulting from a single
preparative step, combination, or interconversion are encompassed.
In yet a further embodiment of the invention, the compounds
described by the formula I are included in a pharmaceutically
acceptable form, and optionally include at least one other
ingredient, for example a suitable carrier, excipient, another
pharmaceutically active ingredient or a combination thereof
[0015] The invention also provides prodrug forms of the above
described analogs and derivatives, wherein the prodrug is
metabolized in vivo to produce an analog or derivative as set forth
above. Indeed, some of the above described analogs or derivatives
may be a prodrug for another analog or derivative.
[0016] The term "prodrug" is well understood in the art and
includes compounds that are converted to pharmaceutically active
compounds of the invention in a mammalian system. For example, see
Remington's Pharmaceutical Sciences, 1980, vol. 16, Mack Publishing
Company, Easton, Pa., 61 and 424.
[0017] In another aspect of the invention, compositions containing
the above described compounds are provided. Preferably, the
compositions are formulated to be suitable for pharmaceutical or
agricultural use by the inclusion of appropriate carriers or
excipients.
[0018] In still another aspect of the invention, methods are
provided for the administration of a suitable amount of a
pharmaceutically acceptable form of the compounds described herein,
to a mammal in need thereof, for example a human, for the treatment
and/or prevention of a disease. In one of the embodiments, the
invention comprises methods for inhibiting a PLA2 enzyme.
[0019] In another of the embodiments, the invention comprises
methods for the administration of a suitable amount of a
pharmaceutically acceptable form of the compounds described herein,
to a mammal in need thereof, for the treatment and/or prevention of
inflammatory diseases.
[0020] Additional advantageous features and functionalities
associated with the systems, methods and processes of the present
invention will be apparent from the detailed description which
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Comparison of the 1,3,5-triazepan-2,6-dione
scaffold, B, and 2,5-diketopiperazines, A.
[0022] FIG. 2. a) EtOCOCl, NMM, THF, -20.degree. C., then NaN.sub.3
in H.sub.2O; b) Toluene, 65.degree. C., then HOSu and pyridine; c)
TFA, 30 min; d) DIEA, MeCN; e) PS-DIEA, CH.sub.2Cl.sub.2. g=gem,
refers to the 2-alkyl gem-diamino-derivative of the corresponding
amino-acid according to the nomenclature proposed by Chorev and
Goodman.
[0023] FIG. 3. X-ray crystal structures of representative
1,3,5-triazepan-2,6-diones 4, 7 and 9.
[0024] FIG. 4. a) NaH (4 equiv), RX (4 equiv); b)
KF/Al.sub.2O.sub.3 (10 equiv) or NaH (2 equiv), RX (1.5 equiv).
[0025] FIG. 5. Half Maximum Inhibition Curves (IC50) for inhibitor
molecules compared to Me-IDX. PLA2 enzymes hGX and hGV compared
(Y-axis is percent activity of PLA2; X-axis is concentration of
inhibitor); inhibitor molecule number (e.g., "mol 33#") identifies
the particular compound used from Table I.
[0026] FIG. 6. Half Maximum Inhibition Curves (IC50) for inhibitor
molecules compared to Me-IDX. PLA2 enzymes hGX and hGV compared
(Y-axis is percent activity of PLA2; X-axis is concentration of
inhibitor); inhibitor molecule number (e.g., "mol 33#") identifies
the particular compound used from Table I.
DETAILED DESCRIPTION OF THE INVENTION
[0027] When describing the compounds, compositions and methods of
the invention, the following terms have the following meanings,
unless otherwise indicated.
[0028] "Pharmaceutically acceptable salt" means those salts which
retain the biological effectiveness and properties of the parent
compounds and which are not biologically or otherwise harmful as
the dosage administered. The compounds of this invention are
capable of forming both acid and base salts by virtue of the
presence of amino and carboxy groups respectively. Pharmaceutically
acceptable base addition salts may be prepared from inorganic and
organic bases. Salts derived from inorganic bases include, but are
not limited to, the sodium, potassium, lithium, ammonium, calcium,
and magnesium salts. Salts derived from organic bases include, but
are not limited to, salts of primary, secondary and tertiary
amines, substituted amines including naturally-occurring
substituted amines, and cyclic amines, including isopropylamine,
trimethyl amine, diethylamine, triethylamine, tripropylamine,
ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine,
arginine, histidine, caffeine, procaine, hydrabamine, choline,
betaine, ethylenediamine, glucosamine, N-alkylglucamines,
theobromine, purines, piperazine, piperidine, and
N-ethylpiperidine. It should also be understood that other
carboxylic acid derivatives would be useful in the practice of this
invention, for example carboxylic acid amides, including
carboxamides, lower alkyl carboxamides, di(lower alkyl)
carboxamides, and the like.
[0029] Pharmaceutically acceptable acid addition salts may be
prepared from inorganic and organic acids. Salts derived from
inorganic acids include hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like. Salts
derived from organic acids include acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid
and the like.
[0030] The term "treatment" as used herein includes any treatment
of a condition or disease in an animal, particularly a mammal, more
particularly a human, and includes: [0031] (i) preventing the
disease or condition from occurring in a subject which may be
predisposed to the disease but has not yet been diagnosed as having
it; [0032] (ii) inhibiting the disease or condition, i.e. arresting
its development; relieving the disease or condition, i.e. causing
regression of the condition; or relieving the conditions caused by
the disease, i.e. symptoms of the disease.
[0033] The term "therapeutically effective amount" refers to that
amount which is sufficient to effect treatment, as defined herein,
when administered to a mammal in need of such treatment. The
therapeutically effective amount will vary depending on the subject
and disease state being treated, the severity of the affliction and
the manner of administration, and may be determined routinely by
one of ordinary skill in the art.
[0034] "Heterocycle" refers to a heterocyclic group having from 4
to 9 carbon atoms and at least one heteroatom selected from the
group consisting of N, O or S.
[0035] "Alkyl" refers to a branched or unbranched alkyl group
having 1-6 carbon atoms, a branched or unbranched alkenyl group
having 1-6 carbon atoms, a branched or unbranched alkinyl group
having 1-6 carbon atoms.
[0036] "Hydroxyl" refers the functional group --OH when it is a
substituent in an organic compound.
[0037] "Heterocyclic groups" can be optionally substituted with 1
to 5, 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, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto,
thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy,
aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, --SO-alkyl,
--SO-substituted alkyl, --SO-aryl, --SO-heteroaryl, --SO2-alkyl,
--SO2-substituted alkyl, --SO2-aryl, oxo (.dbd.O), and
--SO2-heteroaryl. Such heterocyclic groups can have a single ring
or multiple condensed rings. Preferred heterocyclics include
morpholino, piperidinyl, and the like.
[0038] Examples of nitrogen heterocycles and heteroaryls include,
but are not limited to, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine,
phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,
phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,
indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like
as well as N-alkoxy-nitrogen containing heterocycles.
[0039] The term "thiol" refers to the group --SH.
[0040] The term "thioalkoxy" refers to the group --S-alkyl.
[0041] "Amino acid" refers to any molecule that contains both amino
and carboxylic acid functional groups, and includes any of the
naturally occurring amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu,
Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, and Val) in D, L, or DL form. The side chains of
naturally occurring amino acids are well known in the art and
include, for example, hydrogen (e.g., as in glycine), alkyl (e.g.,
as in alanine, valine, leucine, isoleucine, proline), substituted
alkyl (e.g., as in threonine, serine, methionine, cysteine,
aspartic acid, asparagine, glutamic acid, glutamine, arginine, and
lysine), alkaryl (e.g., as in phenylalanine and tryptophan),
substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl
(e.g., as in histidine).
[0042] "Amidine" refers to a functional group that has two amine
groups attached to the same carbon atom with one carbon-nitrogen
double bond: HN.dbd.CR'--NH''2.
[0043] "Alkoxyl" refers to an alkyl group linked to oxygen thus:
R--O--, where R is an alkyl.
[0044] "Substituted alkyl" refers to a branched or unbranched
alkyl, alkenyl or alkinyl group having 1-10 carbon atoms and having
substituted by one or more substituents selected from the group
consisting of hydroxyl, mercapto, carbylmercapto, halogen,
carbyloxy, amino, amido, carboxyl, cycloalkyl, sulfo or acyl. These
substituent generic groups having the meanings being identical with
the definitions of the corresponding groups as defined herein.
[0045] "Halogen" refers to fluorine, bromine, chlorine, and iodine
atoms.
[0046] "Acyl" denotes the group --C(O)R.sub.e, where R.sub.e is
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
arylalkyl, substituted arylalkyl, cycloalkyl, substituted
cycloalkyl whereas these generic groups have meanings which are
identical with definitions of the corresponding groups as defined
in this legend.
[0047] "Acloxy" denotes the group --OAc, where Ac is an acyl,
substituted acyl, heteroacyl or substituted heteroacyl whereas
these generic groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0048] "Alkylamino" denotes the group --NR.sub.f R.sub.g, where
R.sub.f and R.sub.g, that are independent of one another, represent
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl or substituted heteroaryl, whereas these generic
substituents have meanings which are identical with definitions of
the corresponding groups defined herein.
[0049] "Aryl" refers to an aromatic carbocyclic group having from 1
to 18 carbon atoms and being composed of at least one aromatic or
multiple condensed rings in which at least one of which being
aromatic.
[0050] "Substituted aryl" refers to an aromatic carbocyclic group
having from 1 to 18 carbon atoms and being composed of at least one
aromatic ring or of multiple condensed rings at least one of which
being aromatic. The ring(s) are optionally substituted with one or
more substituents selected from the group consisting of halogen,
alkyl, hydroxyl, carbylmercapto, alkylamino, carbyloxy, amino,
amido, carboxyl, nitro, mercapto or sulfo, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0051] "Heteroaryl" refers to a heterocyclic group having from 4 to
9 carbon atoms and at least one heteroatom selected from the group
consisting of N, O or S with at least one ring of this group being
aromatic.
[0052] "Substituted heteroaryl" refers to a heterocyclic group
having from 4 to 9 carbon atoms and at least one heteroatom
selected from the group consisting of N, O or S with at least one
ring of this group being aromatic and this group being substituted
with one or more substituents selected from the group consisting of
halogen, alkyl, carbyloxy, carbylmercapto, alkylamino, amido,
carboxyl, hydroxyl, nitro, mercapto or sulfo, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0053] "Carboxyl" denotes the group --C(O)OR.sub.j, where R is
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl or substituted heteroaryl, whereas these generic
substituents have meanings which are identical with definitions of
the corresponding groups defined herein.
[0054] "Cycloalkyl" refers to a monocyclic or polycyclic alkyl
group containing 3 to 15 carbon atoms.
[0055] "Substituted cycloalkyl" refers to a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms and being
substituted by one or more substituents selected from the group
consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulfo, whereas these
generic substituent groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0056] "Heterocycloalkyl" refers to a monocyclic or polycyclic
alkyl group containing 3 to 15 carbon atoms which at least one ring
carbon atom of its cyclic structure being replaced with a
heteroatom selected from the group consisting of N, O, S or P.
[0057] "Substituted heterocycloalkyl" refers to a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms which at
least one ring carbon atom of its cyclic structure being replaced
with a heteroatom selected from the group consisting of N, O, S or
P and the group is containing one or more substituents selected
from the group consisting of halogen, alkyl, substituted alkyl,
carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulfo, whereas
these generic substituent group have meanings which are identical
with definitions of the corresponding groups as defined in this
legend.
[0058] The term "aryl" refers to an unsaturated aromatic
carbocyclic group of from 6 to 20 carbon atoms having a single ring
(e.g., phenyl) or multiple condensed (fused) rings, wherein at
least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl,
fluorenyl, or anthryl). Preferred aryls include phenyl, naphthyl
and the like.
[0059] The term "alkenyl" refers to a monoradical of a branched or
unbranched unsaturated hydrocarbon group preferably having from 2
to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even
more preferably 2 to 6 carbon atoms. Preferred alkenyl groups
include ethenyl (--CH.dbd.CH2), n-propenyl (--CH2CH.dbd.CH2),
iso-propenyl (--C(CH3).dbd.CH2), and the like.
[0060] "Imidazole" refers to a heterocyclic base of the general
formula: C.sub.3H.sub.4N.sub.2.
[0061] "Aralkyl group" refers to, for example, a C1-C6 alkyl group
which is attached to 1 or 2 aromatic hydrocarbon rings having from
6 to 10 carbon atoms and which has a total of 7 to 14 carbon atoms,
such as the benzyl, alpha-naphthylmethyl, indenylmethyl,
diphenylmethyl, 2-phenethyl, 2-alpha-naphthylethyl, 3-phenylpropyl,
3-alpha-naphthylpropyl, phenylbutyl, 4-alpha-naphthylbutyl or
5-phenylpentyl groups.
[0062] "Guanidine" refers generally to the amidine of amidocarbonic
acid and has the general formula of: C(NH.sub.2).sub.3.
[0063] The terms "aralkyl" and "heteroarylalkyl" refer to groups
that comprise both aryl or, respectively, heteroaryl as well as
alkyl and/or heteroalkyl and/or carbocyclic and/or heterocycloalkyl
ring systems according to the above definitions.
[0064] The present invention relates to nitrogen-containing
heterocyclic compounds represented by the general formula I as
follows: ##STR2##
[0065] (I) wherein, W is a member selected from the group
consisting of --C(R.sup.5)(R.sup.5a)--;
--C(R.sup.6)(R.sup.6a)--C(R.sup.7)(R.sup.7a)--;
--C(R.sup.8).dbd.C(R.sup.9)--; --N(R.sup.10), and combinations
thereof;
[0066] X is a member selected from the group consisting of
--N(R.sup.1a)C(.dbd.Y)N(R.sup.4)--; --OC(.dbd.Y)N(R.sup.4)--;
--N(R.sup.1a)C(.dbd.Y)O--; --N(R.sup.1a)S(.dbd.O)N(R.sup.4)--;
--N(R.sup.1a)S(.dbd.O).sub.2N(R.sup.4)--;
--C(R.sup.1a)(R.sup.3a)C(.dbd.Y)N(R.sup.4)--, and combinations
thereof;
[0067] Y and Z represent, each independent from the other, a member
selected from the group consisting of oxygen ("O") and sulfur
("S"); and
[0068] R.sup.1, R.sup.1a, R.sup.2, R.sup.3, R.sup.3a, R.sup.4,
R.sup.5, R.sup.5a, R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8,
R.sup.9, and R.sup.10 represent, each independent from the other, a
member selected from the group consisting of: a hydrogen atom; an
amino acid side chain; a (C1-C10) alkyl; (C1-C10) alkenyl; (C1-C10)
alkynyl; (C5-C12) monocyclic or bicyclic aryl; (C5-C14) monocyclic
or bicyclic aralkyl; monocyclic or bicyclic (C5-C14) heteroaralkyl;
and (C1-C10) monocyclic or bicyclic heteroaryl group having up to 5
heteroatoms selected from N, O, S, and P said groups being able to
be non-substituted or substituted by 1 to 6 substituents further
selected from the group consisting of: a halogen atom, an NO.sub.2,
OH, amidine, benzamidine, imidazole, 1,2,3-triazole, alkoxy,
(C1-C4), amino, piperazine, piperidine, dialkylamino, guanidine
group, bis alkylated or bis acylated guanido group, carboxylic
acid, carboxamide, ester, hydroxamic acid, phosphinic acid,
phosphonate, phosphonamidate, sulfhydryl and any combination
thereof
[0069] The intermediates and the desired compounds in the processes
described can be isolated and purified by purification methods
conventionally used in organic synthetic chemistry, for example,
neutralization, filtration, extraction, washing, drying,
concentration, recrystallization, and various kinds of
chromatography. The intermediates may be subjected to the
subsequent reaction without purification.
[0070] The present invention covers all possible isomers including
tautomers and mixtures thereof. Where chiral carbons lend
themselves to two different enantiomers, both enantiomers are
contemplated as well as procedures for separating the two
enantiomers.
[0071] In the case where a salt of a compound is desired and the
compound is produced in the form of the desired salt, it can be
subjected to purification as such. In the case where a compound is
produced in the free state and its salt is desired, the compound is
dissolved or suspended in a suitable organic solvent, followed by
addition of an acid or a base to form a salt.
[0072] The present invention also relates to pharmaceutically
acceptable salts, racemates, and optical isomers thereof of formula
I. The compounds of this invention typically contain one or more
chiral centers. Accordingly, this invention is intended to include
racemic mixtures, diasteromers, enantiomers and mixture enriched in
one or more steroisomer. The scope of the invention as described
and claimed encompasses the racemic forms of the compounds as well
as the individual enantiomers and non-racemic mixtures thereof
[0073] In a further aspect of the invention, methods for the use of
the above described analogs and derivatives, as well as
compositions, are provided. These methods include uses of the
invention's compounds to inhibit a PLA2 enzyme, treat or prevent
human and agricultural diseases and conditions or both. Examples of
human diseases and conditions include, but are not limited to,
inflammation, septic shock, rheumatoid arthritis, acute
pancreatitis, acute chest syndrome in patients with sickle cell
disease, acute respiratory distress syndrome (ARDS), obesity,
obesity-related insulin resistance, hyperalgesia, pulmonary edema,
colitis, ischemia reperfusion, pleurisy, microbial infection,
rheumatoid arthritis, skin inflammation, psoriasis, cancer,
osteoporosis, asthma, autoimmune diseases, HIV, AIDS, rheumatoid
arthritis, systemic lupus erythematosus, Type I insulin-dependent
diabetes, tissue transplantation, malaria, African sleeping
sickness, Chagas disease, toxoplasmosis, psoriasis, restenosis,
inhibition of unwanted hair growth as cosmetic suppression,
hyperparathyroidism, inflammation, treatment of peptic ulcer,
glaucoma, Alzheimer's disease, suppression of atrial tachycardias,
stimulation or inhibition of intestinal motility, Crohn's disease
and other inflammatory bowel diseases, high blood pressure
(vasodilation), stroke, epilepsy, anxiety, neurodegenerative
diseases, hyperalgesic states, protection against hearing loss
(especially cancer chemotherapy induced hearing loss), and
pharmacological manipulation of cocaine reinforcement and craving
in treating cocaine addiction and overdose and other fungal
bacterial, viral, and parasitic diseases.
[0074] In another aspect of the invention, compositions containing
the above described compounds are provided. Preferably, the
compositions are formulated to be suitable for pharmaceutical or
agricultural use by the inclusion of appropriate carriers or
excipients.
[0075] In still another aspect of the invention, methods are
provided for the administration of a suitable amount of a
pharmaceutically acceptable form of the compounds described herein,
to a mammal in need thereof, for example a human, for the treatment
and/or prevention of a disease. In one of the embodiments, the
invention comprises methods for inhibiting a PLA2 enzyme. In
another embodiment, the invention comprises molecules listed in
Table I, which are useful for the inhibition of PLA2. In
particular, molecules 49, 33, 40, 9, 5, 4, and 3 are useful for the
inhibition of group V and group X sPLA2. The molecules demonstrated
the following hierarchy in sPLA2 inhibition: mol 40>mol
33>mol 40; and mol 9>mol 5.apprxeq.mol 4.apprxeq.mol 3,
respectively.
[0076] In another of the embodiments, the invention comprises
methods for the administration of a suitable amount of a
pharmaceutically acceptable form of the compounds described herein,
to a mammal in need thereof, for the treatment and/or prevention of
inflammatory diseases.
[0077] The design and synthesis by combinatorial chemistry
techniques of cyclic/polycyclic molecular frameworks that can
efficiently distribute selected pharmacophores in the 3D space is
an important method to identify small-molecules capable of
modulating biological processes and for dissecting biological
pathways. Molecules incorporating small or medium rings derived
from peptides (e.g. 2,5-diketopiperazines) are of particular
interest owing to the facile access, the chemical and
stereochemical diversity of peptide derivatives, as well as
enhanced diversity resulting from appending operations. To expand
further the skeletal diversity attainable with peptide substrates,
we investigated the synthesis of the densely functionalized (five
points of diversity) dipeptide-derived 1,3,5-triazepan-2,6-dione
scaffold and demonstrated its utility by screening a small
"prospecting" library against the PLA2.
[0078] The description of the embodiments contained herein is given
by way of example and is not limiting on the scope of the present
invention. Additional advantageous features and functionalities
associated with the systems, methods and processes of the present
invention will be apparent from the following examples.
EXAMPLES
[0079] To date, 12 mammalian sPLA2s have been identified and
classified into 3 main structural collections: group I/II/V/X, III,
and XII.
[0080] Although a significant increase in sPLA2 activity is
detected in serum in septic shock, rheumatoid arthritis, acute
pancreatitis, multiple injuries, acute chest syndrome in patients
with sickle cell disease, and in bronchoalveolar lavage (BAL) of
patients with acute respiratory distress syndrome (ARDS), the exact
function of sPLA2s in physio-pathological processes is uncertain.
It seems that the GIIA is very potent in hydrolyzing Gram positive
bacteria membranes and could be involved in the host defense
against micro-organisms. Importantly, elevated concentrations of
hGIIA are found in the eyes, a privileged immune organ.
[0081] The GIB is found at high level in pancreas, has an enhanced
activity toward its substrate in presence of deoxycholate, a
detergent found in bile, and is activated in the intestine by
trypsin. A function for GIB in phospholipid digestion was thus
suggested. Knocking-out the gene coding for this enzyme could not
show its essential role in lipid absorption at first glance, but
feeding mice with a high-fat diet demonstrated GIB-knock-out mice
were less likely to develop obesity and obesity-related insulin
resistance.
[0082] Exogenous addition of GV and GX to various mammalian cell
types leads to the release of arachidonate and eicosanoid
generation, even without activation of the cPLA2. In addition,
zymosan-treated peritoneal macrophages from GV knock-out mice have
reduced prostaglandin E2 (PGE2) and leukotriene C4 (LTC4)
production. Therefore, GV and GX are likely involved in the
generation of eicosanoids under certain conditions. The
physiological roles of GIIC, GIIE, GIIF, GIII, GXIIA and GXIIB have
not yet been clarified, but some evidence suggests that GXII, even
when devoid of any catalytic activity, may be involved in
vertebrate neuronal development.
[0083] Recent evidence suggests that PLA2 proteins not only
hydrolyze phospholipids but may also serve as ligands for different
binding proteins. The best known sPLA2 binding protein is the
M-type receptor (MtR). This receptor was initially cloned as a
transmembrane glycoprotein having common characteristics with the
macrophage mannose receptor, and the more recently cloned receptors
Endo-180 and Dec-205. This receptor has a large extracellular
domain containing a N-terminal cysteine-rich domain, a
fibronectin-like type II domain, eight C-type lectin like domains
(CTLD), a single transmembrane domain and a short cytoplasmic tail.
The M-type receptor can also quickly internalize sPLA2s suggesting
a role in sPLA2 clearance. The identification of a soluble form of
the receptor that can inhibit enzymatic activity upon sPLA2 binding
also agrees with this view. Furthermore, results obtained from gene
targeting of the receptor and other studies using the pancreatic
sPLA2 suggest the M-type receptor acts as a intracellular signaling
molecule through sPLA2 binding, for example, by activating the MAPK
cascade, inducing a proinflammatory phenotype, and upregulating the
cell surface expression of Fas ligand.
[0084] Structure-based strategies to discover group IIA specific
sPLA2 inhibitors led to the identification of indole analogues that
inhibit this sPLA2 with nanomolar affinities. One analogue,
LY311727, was able to inhibit the release of thromboxane A2
triggered by exogenously added hGIIA on guinea pig BAL fluids
containing macrophages, eosinophils and epithelial cells. LY311727
could also inhibit the sPLA2 activity induced by
lipopolysaccharides in a guinea pig model of ARDS. Moreover,
intravenous administration of LY311727 in transgenic mice
overexpressing hGIIA led to a loss of PLA2 catalytic activity in
blood, demonstrating that this inhibitor can be active in vivo, at
least in blood circulation, in these animals. In a murine
toxoplasmosis experimental model, LY311727 injection led to an
earlier mortality, suggesting a protective role of at least one
sPLA2 sensitive to this inhibitor in these mice. In addition,
lumbar intrathecal administration of LY311727 in 3 different
experimental rat models of hyperalgesia attenuated all the
inflammation-related symptoms observed.
[0085] An indole-derived inhibitor of second generation, called
S-5920/LY315920Na, significantly attenuated lung compliance,
pulmonary edema, vascular permeability and lung surfactant
degradation in a rabbit acute lung injury model induced by oleic
acid. Two other inhibitors of sPLA2, the LY333013 (S-3013) and
5-(4-benzyloxyphenyl)-4s-(7-phenylheptanoylamino)-pentanoic acid
protected rats from dextran sulfate- and trinitrobenzene sulfonic
acid-induced colitis. Oral administration of
5-(4-benzyloxyphenyl)-4s-(7-phenylheptanoylamino)-pentanoic acid
also preserved rats intestine from injury following ischemia and
reperfusion.
[0086] Ear edema induced by tetracenoylphorbol-13-acetate in mice
was reduced by YM-26734, a molecule known to be a potent inhibitor
of mGIIA, mGIID, mGIIE, mGV and mGX. This same drug also
significantly decreased the accumulation of exudate fluid and
leukocytes in a carageenin-induced pleurisy rat model. Despite the
promising effects of sPLA2 inhibitors, no significant differences
between the PLA2 inhibitor-treated and the placebo groups were
found when the S-5920/LY315920Na was used in a clinical study
involving humans with sepsis and organ failure. However, because
the GIIA sPLA2 has antibacterial properties, and septic shock is
provoked by a microbial invasion, it is arguable whether it makes
sense to use a sPLA2 inhibitor as a septic shock therapeutic
drug.
[0087] Recently, a clinical trial using another sPLA2 inhibitor,
the orally distributed LY333013, on patients with rheumatoid
arthritis led to significant reduction of the pathology during the
first week of trial, but the benefits were lost thereafter. In this
last report, the authors reported positive impacts on the pathology
when administering the inhibitor intravenously. This same inhibitor
failed to show any benefit on inhaled allergen challenge in
subjects with asthma. It is important to note that the LY333013 was
well tolerated in these patients.
[0088] Thus it appears that the path of administration (e.g., oral,
parenteral, enteral, subcutaneous, intravenous, anal, etc . . . )
and the biological system chosen for an inhibitor can affect its
efficacy. For example, BMS-1881162, an inhibitor of both GIIA and
cPLA2, has a very potent anti-inflammatory activity when used as a
topical agent in a mouse model with chronic skin inflammation
induced with repeated exposures to phorbol ester. This same
inhibitor was without effect in psoriatic patients. The use of
labeled BMS-1881162 in volunteers showed almost no discernible
penetration of the drug, probably due to the thicker stratum
corneum in human compared to mouse.
[0089] An indole inhibitor called indoxam (IDX) inhibited PGE2
production induced by TGF-.alpha. and IL-1 in rat gastric
epithelial cells. Me-indoxam (Me-IDX), a derivative of indoxam, is
about 20 fold more potent than LY311727 to inhibit hGIIA. This
indole analogue is suitable for studies on mammalian cells, and not
only it inhibits the enzymatic activity of hGIIA, but also that of
other group I/II/V/X sPLA2s. The fact that IDX and its related
indole compounds affect various inflammatory signals on mammalian
cells and in animal models suggests that at least one sPLA2 from
the group I/II/V/X is involved in these processes.
[0090] Because Me-IDX is known to bind to and to protrude from the
catalytic groove of the sPLA2, it could interfere with sPLA2
interaction to molecules other than phospholipids. In fact, it was
shown that IDX can block the binding of porcine pancreatic group IB
and group X sPLA2 to the mouse cells expressing the M-type receptor
with good efficiency (IC.sub.50=130 nM and 900 nM respectively).
However, it is not known if this observation can be extrapolated to
other sPLA2s in an endogenous context, for example, using sPLA2 and
M-type receptor from the mouse. This research is of high importance
as some pathophysiological disorders may involve sPLA2 binding to
this receptor. Indeed, mice deficient for the M-type receptor are
resistant to endotoxic shock and have lower concentrations of
circulating IL-1 and TNF-.alpha. after LPS treatment when compared
to M-type receptor expressing mice. Nevertheless, the septic shock
induced by injection of lipopolysaccharides in wild-type mice can
be attenuated by indoxam treatment. Recently we found that not only
group IB and group IIA sPLA2s, but also several other mouse sPLA2s
from the group I/II/V/X can bind to the M-type receptor initially
identified with the snake venom sPLA2 OS2, leading to the
hypothesis that one or several sPLA2s may be involved in these
processes, and that the effect of indoxam may be due to either
inhibition of enzymatic activity or of binding to the M-type
receptor.
[0091] Results obtained with analysis of the direct binding
properties of radiolabeled mammalian sPLA2s on cellular membranes
in the presence of Me-IDX, and evaluation of the inhibitory effects
of various other molecules known as inhibitors of sPLA2 strongly
indicate that the effects observed with sPLA2 inhibitors in
different studies may be not only due to the inhibition of the
sPLA2 catalytic activity but also to the modulation of the sPLA2
binding properties to their receptors.
[0092] Interest in designing and evaluating the dipeptide-derived
1,3,5-triazepan-2,6-dione scaffold stems from the remarkable
biological activities exhibited by molecules with diazepine and
triazepine skeletons. In particular seven-membered cyclic ureas
have attracted much attention in recent years with application in
the development of HIV-protease and reverse transcriptase
inhibitors, Factor Xa inhibitors, beta-lactamases inhibitors,
phospholipase C inhibitors, and chemokine receptor antagonists.
[0093] The following studies establish that novel 7 and 8-membered
ring nitrogen containing heterocyclic compounds of the invention
are useful for the inhibition of PLA2, and can be effective for the
treatment and prevention of inflammatory diseases.
[0094] I. Labeling of E. Coli Membranes with [.sup.3H]-Oleic
Acid:
[0095] 1) Prepare a 10 ml overnight preculture from a single colony
of an E. coli strain in Luria Broth (LB) w/ or w/o ampicillin.
DH10B and XL-1 strains seem better than JM101 strain, i.e. they
give membranes with less background and are easier to pellet after
the PLA.sub.2 assay). OD.sub.600 nm of the saturated overnight
preculture is about 2 UDO. Make a 1/5 dilution and measure
OD.sub.600 nm.
[0096] 2) Dilute the preculture in 100 ml of fresh LB to 0.05
UOD.sub.600 nm and add 250 .mu.l of [.sup.3H]-oleic acid (NET289,
NEN, 5 mCi/ml, alcohol solution). Open the vial containing the
radioactive stock solution under the hood and flush the vial with
N.sub.2 before closure. Save a 10 .mu.l aliquot of the culture for
the later quantification of incorporated oleic acid.
[0097] 3) Grow cells for about 5 hours at 37.degree. C. with
vigorous shaking (200-230 rpm) up to 1 UOD.sub.600 nm.
[0098] 4) Spin down the culture for 15 minutes/4,000 rpm/50 ml
falcon tube/RT. Save 50 .mu.l of supernatant for quantification of
incorporated oleic acid. Discard the supernatant and resuspend the
pellet in 50 ml of LB and grow the cells for an additional 30
minutes at 37.degree. C. under shaking (this step allows the
remaining unincorporated labeled oleic acid to get incorporated
into phospholipids).
[0099] 5) Spin down again as above. Save 50 .mu.l of supernatant
for later quantification. Discard the supernatant and resuspend the
pellet in 50 ml of washing buffer.
[0100] 6) Spin down as above. Save 50 .mu.l of supernatant for
later quantification. Discard the supernatant and resuspend the
pellet in 2 ml of washing buffer but WITHOUT BSA. Save a 2 .mu.l
aliquot for counting and transfer the remaining solution in a Corex
glass tube. Put an aluminium foil as a cap and autoclave (20
minutes, 120.degree. C., 1.5 bar). This step can be done
overnight.
[0101] 7) The next day, save another 2 .mu.l aliquot for counting
and transfer the remaining solution into 2 eppendorf tubes. Rinse
the Corex tube with 1 ml of washing buffer and combine with the 2
ml solution.
[0102] 8) Spin down for 1 minute at 14,000 rpm (RT). Save 10 .mu.l
of supernatant for later quantification. Discard the supernatant
and resuspend each pellet in 1.5 ml of washing buffer repeat this
step four more times.
[0103] 9) Resuspend the pellet in 5 ml of washing buffer and count
5 .mu.l for quantification of incorporated oleic acid. Dilute the
solution to 100,000 dpm/.mu.l and make aliquotes of 30 .mu.l.
[0104] 10) Count the different supernatants and calculate the
percentage of radioactivity in step 9 versus the input amount.
Typically, the incorporated radioactivity is more than 30-40% of
the input radioactivity added in step 2.
[0105] II. PLA2 Assay:
[0106] 1) Preparation of Substrate:
[0107] Pipet the required amount of radioactivity (100,000 dpm of
labeled membranes per reaction.times.number of reactions) and
dilute into 1 ml of PLA.sub.2 activity buffer in an eppendorf
tube.
[0108] Spin down for 1 minute at 14,000 rpm (RT). Discard the
supernatant. Carefully resuspend the pellet into 150 .mu.l of
PLA.sub.2 activity buffer and add PLA.sub.2 activity buffer for the
total number of reactions. Store the pool at room temperature (do
not prepare the pool too much in advance).
[0109] 2) PLA2 Assay Reaction:
[0110] A typical reaction is made in an eppendorf tube and consists
of a total volume of 150 .mu.l made with 50 .mu.l PLA2 activity
buffer, a negligible volume of enzyme solution and 100 .mu.l of the
above substrate pool (addition of a quite large volume of substrate
with a multipipette results in enough mixing so that it is not
necessary to vortex after substrate addition).
[0111] Reaction mixtures are incubated for various periods of times
up to 1 hour at 25.degree. C. or 37.degree. C. (Incubations are
routinely performed at RT) and with different amounts of enzyme.
Incubation times and sample volumes are adjusted to ensure
hydrolysis rates within the linear range of enzymatic assays
(typically 10-20% of total substrate hydrolysis). Control
incubations in the absence of added sPLA2 were carried out in
parallel and used to calculate specific hydrolysis.
[0112] 3) Stop the reaction by adding 300 .mu.l of stop buffer.
Spin down the tubes for 3 minutes at 14,000 rpm at room
temperature. Collect and count the supernatant containing released
labeled oleic acid.
[0113] Count also 3 or 4 aliquotes of 100 .mu.l of the substrate
pool to determine the total amount of injected
radioactivity/reaction.
[0114] Note that we routinely considered that the counts in the
above supernatants correspond to free .sup.3H-oleate released from
membrane phospholipids. One may verify that these counts are real
free oleic acid by performing a thin layer chromatography on silica
gel 60 in conditions where free oleic and phospholipid can be
separated.
[0115] Note also that this protocol does not specifically detect
sPLA2 activity, but can also detect the activity of cytosolic
PLA2s.
[0116] Materials:
[0117] DH10B or XL-1 E. coli strain (could be a strain carrying or
not a plasmid); [.sup.3H]-oleic acid (NET289, NEN, 5 mCi/ml in
ethanol); Fraction V Fatty acid free BSA (Sigma #A6003 or A7511);
Fraction V BSA (sigma #A7906); Corex glass tube or equivalent.
Buffers: washing buffer: 0.1 M Tris/HCl pH 8.0, 1 mM EDTA
containing 0.5% Fatty acid free BSA; PLA.sub.2 activity buffer: 0.1
M Tris/HCl pH 8.0, 10 mM CaCl.sub.2, 0.1% BSA; Stop buffer: 0.1 M
EDTA containing 0.2% fatty acid free BSA.
Therapeutic Administration
[0118] One of the embodiments of the present invention includes a
method for inhibiting a PLA2 enzyme. Another of the embodiments of
the present invention includes therapeutic compositions comprising
the compounds of the invention in a pharmaceutically acceptable
form. In still another embodiment, the present invention includes
methods for the treatment and/or prevention of disease, for
example, an inflammatory disease, in a mammal, for example, a
human, comprising administering of an effective amount of a
compound of the invention in a pharmaceutically acceptable form.
The compound of the invention may optionally be administered
together with at least one of a carrier, an excipient, another
biologically active agent or any combination thereof.
[0119] Suitable routes for administration include oral, rectal,
vassal, topical (including ocular, buccal and sublingual), vaginal
and parental (including subcutaneous, intramuscular, intravitreous,
intravenous, intradermal, intrathecal and epidural). The preferred
route of administration will depend upon the condition of the
patient, the toxicity of the compound and the site of infection,
among other considerations known to the clinician.
[0120] The therapeutic composition of the invention comprises about
1% to about 95% of the active ingredient, single-dose forms of
administration preferably comprising about 20% to about 90% of the
active ingredient and administration forms which are not
single-dose preferably comprising about 5% to about 20% of the
active ingredient. Unit dose forms are, for example, coated
tablets, tablets, ampoules, vials, suppositories or capsules. Other
forms of administration are, for example, ointments, creams,
pastes, foams, tinctures, lipsticks, drops, sprays, dispersions and
the like. Examples are capsules containing from about 0.05 g to
about 1.0 g of the active ingredient.
[0121] The pharmaceutical compositions of the present invention are
prepared in a manner known per se, for example by means of
conventional mixing, granulating, coating, dissolving or
lyophilizing processes.
[0122] Preferably, solutions of the active ingredient, and in
addition also suspensions or dispersions, especially isotonic
aqueous solutions, dispersions or suspensions, are used, it being
possible for these to be prepared before use, for example in the
case of lyophilized compositions which comprise the active
substance by itself or together with a carrier, for example
mannitol. The pharmaceutical compositions can be sterilized and/or
comprise excipients, for example preservatives, stabilizers,
wetting agents and/or emulsifiers, solubilizing agents, salts for
regulating the osmotic pressure and/or buffers, and they are
prepared in a manner known per se, for example by means of
conventional dissolving or lyophilizing processes. The solutions or
suspensions mentioned can comprise viscosity-increasing substances,
such as sodium carboxymethylcellulose, carboxymethylcellulose,
dextran, polyvinylpyrrolidone or gelatin.
[0123] Pharmaceutically acceptable forms include, for example, a
gel, lotion, spray, powder, pill, tablet, controlled release
tablet, sustained release tablet, rate controlling release tablet,
enteric coating, emulsion, liquid, salts, pastes, jellies,
aerosols, ointments, capsules, gel caps, or any other suitable form
that will be obvious to one of ordinary skill in the art.
[0124] Suspensions in oil comprise, as the oily component, the
vegetable, synthetic or semi-synthetic oils customary for injection
purposes. Oils which may be mentioned are, in particular, liquid
fatty acid esters which contain, as the acid component, a
long-chain fatty acid having 8-22, in particular 12-22, carbon
atoms, for example lauric acid, tridecylic acid, myristic acid,
pentadecylic acid, palmitic acid, margaric acid, stearic acid,
arachidinic acid, behenic acid or corresponding unsaturated acids,
for example oleic acid, elaidic acid, euric acid, brasidic acid or
linoleic acid, if appropriate with the addition of antioxidants,
for example vitamin E, beta.-carotene or
3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these
fatty acid esters has not more than 6 carbon atoms and is mono- or
polyhydric, for example mono-, di- or trihydric alcohol, for
example methanol, ethanol, propanol, butanol, or pentanol, or
isomers thereof, but in particular glycol and glycerol. Fatty acid
esters are therefore, for example: ethyl oleate, isopropyl
myristate, isopropyl palmitate, "Labrafil M 2375" (polyoxyethylene
glycerol trioleate from Gattefosee, Paris), "Labrafil M 1944 CS"
(unsaturated polyglycolated glycerides prepared by an alcoholysis
of apricot kernel oil and made up of glycerides and polyethylene
glycol esters; from Gattefosee, Paris), "Labrasol" (saturated
polyglycolated glycerides prepared by an alcoholysis of TCM and
made up of glycerides and polyethylene glycol esters; from
Gattefosee, Paris) and/or "Miglyol 812" (triglyceride of saturated
fatty acids of chain length C.sub.8 to C.sub.12 from Huls A G,
Germany), and in particular vegetable oils, such as cottonseed oil,
almond oil, olive oil, castor oil, sesame oil, soybean oil and, in
particular, groundnut oil.
[0125] The preparation of the injection compositions is carried out
in the customary manner under sterile conditions, as are bottling,
for example in ampoules or vials, and closing of the
containers.
[0126] For example, pharmaceutical compositions for oral use can be
obtained by combining the active ingredient with one or more solid
carriers, if appropriate granulating the resulting mixture, and, if
desired, processing the mixture or granules to tablets or coated
tablet cores, if appropriate by addition of additional
excipients.
[0127] Suitable carriers are, in particular, fillers, such as
sugars, for example lactose, sucrose, mannitol or sorbitol
cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate, or calcium hydrogen phosphate, and
furthermore binders, such as starches, for example maize, wheat,
rice or potato starch, methylcellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or
polyvinyl-pyrrolidine, and/or, if desired, desintegrators, such as
the above mentioned starches, and furthermore carboxymethyl-starch,
cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof,
such as sodium alginate. Additional excipients are, in particular,
flow regulators and lubricants, for example salicylic acid, talc,
stearic acid or salts thereof, such as magnesium stearate or
calcium stearate, and/or polyethylene glycol, or derivatives
thereof.
[0128] Coated tablet cores can be provided with suitable coatings
which, if appropriate, are resistant to gastric juice, the coatings
used being, inter alia, concentrated sugar solutions, which, if
appropriate, comprise gum arabic, talc, polyvinylpyrrolidine,
polyethylene glycol and/or titanium dioxide, coating solutions in
suitable organic solvents or solvent mixtures or, for the
preparation of coatings which are resistant to gastric juice,
solutions of suitable cellulose preparations, such as
acetylcellulose phthalate or hydroxypropylmethylcellulose
phthalate.
[0129] By "controlled release" it is meant for purposes of the
present invention that therapeutically active compound is released
from the preparation at a controlled rate or at a specific site,
for example, the intestine, or both such that therapeutically
beneficial blood levels (but below toxic levels) are maintained
over an extended period of time, e.g., providing a 12 hour or a 24
hour dosage form.
[0130] The term "rate controlling polymer" as used herein includes
hydrophilic polymers, hydrophobic polymers or mixtures of
hydrophilic and/or hydrophobic polymers that are capable of
retarding the release of the compounds in vivo. In addition, many
of the same polymers can be utilized to create an enteric coating
of a drug, drug suspension, or drug matrix. It is within the skill
of those in the art to modify the coating thickness, permeability,
and dissolution characteristics to provide the desired controlled
release profile (e.g., drug release rate and locus) without undue
experimentation.
[0131] Examples of suitable controlled release polymers to be used
in this invention include hydroxyalkylcellulose, such as
hydroxypropylcellulose and hydroxypropylmethylcellulose;
poly(ethylene)oxide; alkylcellulose such as ethycellulose and
methylcellulose; carboxymethylcellulose; hydrophilic cellulose
derivatives; polyethylene glycol; polyvinylpyrrolidone; cellulose
acetate; cellulose acetate butyrate; cellulose acetate phthalate;
cellulose acetate trimellitate; polyvinylacetate phthalate;
hydroxypropylmethylcellulose phthalate;
hydroxypropylmethylcellulose acetate succinate; poly(alkyl
methacrylate); and poly(vinyl acetate). Other suitable hydrophobic
polymers include polymers or copolymers derived from acrylic or
methacrylic acid esters, copolymers of acrylic and methacrylic acid
esters, zein, waxes, shellac and hydrogenated vegetable oils.
[0132] To ensure correct release kinetics, the controlled release
preparation of this invention contains about 5 and 75% by weight,
preferably about 20 and 50% by weight, more preferably about 30 to
45% by weight controlled release polymer(s) and about 1 to 40% by
weight, preferably about 3 to 25% by weight active compounds. The
controlled release preparation according to the invention can
preferably include auxiliary agents, such as diluents, lubricants
and/or melting binders. Preferably, the excipients are selected to
minimize the water content of the preparation. Preferably, the
preparation includes an antioxidant. Suitable diluents include
pharmaceutically acceptable inert fillers such as microcrystalline
cellulose, lactose, dibasic calcium phosphate, saccharides, and/or
mixtures of any of the foregoing. The diluent is suitably a water
soluble diluent. Examples of diluents include microcrystalline
cellulose such as Avicel ph112, Avicel pH101 and Avicel pH102;
lactose such as lactose monohydrate, lactose anhydrous, and
Pharmatose DCL 21; dibasic calcium phosphate such as Emcompress;
mannitol; starch; sorbitol; sucrose; and glucose. Diluents are
carefully selected to match the specific formulation with attention
paid to the compression properties. Suitable lubricants, including
agents that act on the flowability of the powder to be compressed
are, for example, colloidal silicon dioxide such as Aerosil 200;
talc; stearic acid, magnesium stearate, and calcium stearate.
Suitable low temperature melting binders include polyethylene
glycols such as PEG 6000; cetostearyl alcohol; cetyl alcohol;
polyoxyethylene alkyl ethers; polyoxyethylene castor oil
derivatives; polyoxyethylene sorbitan fatty acid esters;
polyoxyethylene stearates; poloxamers; and waxes.
[0133] To improve the stability in the controlled release
preparation, an antioxidant compound can be included. Suitable
antioxidants include sodium metabisulfite; tocopherols such as
alpha, beta, or delta-tocopherol tocopherol esters and
alpha-tocopherol acetate; ascorbic acid or a pharmaceutically
acceptable salt thereof; ascorbyl palmitate; alkyl gallates such as
propyl gallate, Tenox PG, Tenox s-1; sulphites or a
pharmaceutically acceptable salt thereof; BHA; BHT; and
monothioglycerol.
[0134] The controlled release preparation according to the
invention preferably can be manufactured by blending the compounds
with the controlled release polymer(s) and auxiliary excipients
followed by direct compression. Other methods for manufacturing the
preparation include melt granulation. Preferred melt granulation
techniques include melt granulation together with the rate
controlling polymer(s) and diluent(s) followed by compression of
the granules and melt granulation with subsequent blending with the
rate controlling polymer(s) and diluents followed by compression of
the blend. As desired prior to compression, the blend and/or
granulate can be screened and/or mixed with auxiliary agents until
an easily flowable homogeneous mixture is obtained.
[0135] Oral dosage forms of the controlled release preparation
according to the invention can be in the form of tablets, coated
tablets, enterically coated tablets or can be multiparticulate,
such as in the form of pellets or mini-tablets. If desired,
capsules such as hard or soft gelatin capsules, can contain the
multiparticulates. If desired, the multiparticulate oral dosage
forms can comprise a blend of at least two populations of pellets
or mini-tablets having different controlled-release in vitro and/or
in vivo release profiles. If desired, one of the pellet or
mini-tablet populations can comprise immediate release
multiparticulate, such as multiparticulates formed by conventional
means.
[0136] If desired, the controlled release matrix tablets or
multiparticulates of this invention can be coated with a controlled
release polymer layer so as to provide additional controlled
release properties. Suitable polymers that can be used to form this
controlled release layer include the rate controlling polymers
listed above.
[0137] As desired, the tablets, pellets or mini-tablets according
to the invention can be provided with a light-protective and/or
cosmetic film coating, for example, film-formers, pigments,
anti-adhesive agents and plasticizers. Such a film former may
consist of fast-dissolving constituents, such as low-viscosity
hydroxypropylmethylcelluose, for example Methocel E5 or D14 or
Pharmacoat 606 (Shin-Etsu). The film coating may also contain
excipients customary in film-coating procedures, such as
light-protective pigments, for example iron oxide, or titanium
dioxide, anti-adhesive agents, for example talc, and also suitable
plasticizers such as PEG 400, PEG 6000, and diethyl phthalate or
triethyl citrate.
[0138] The controlled release polymer of this invention may consist
of a hydrogel matrix. For instance, the compounds can be compressed
into a dosage form containing a rate controlling polymer, such as
HPMC, or mixture of polymers which when wet will swell to form a
hydrogel. The rate of release from this dosage form is controlled
both by diffusion from the swollen tablet mass and by erosion of
the tablet surface over time. The rate of release may be controlled
both by the amount of polymer per tablet and by the inherent
viscosities of the polymers used.
[0139] Dyes or pigments can be admixed to the tablets or coated
tablet coatings, for example for identification or characterization
of different doses of active ingredient.
[0140] Pharmaceutical compositions, which can be used orally, are
also hard capsules of gelatin and soft, closed capsules of gelatin
and a plasticizer, such as glycerol or sorbitol. The hard capsules
can contain the active ingredient in the form of granules, mixed
for example with fillers, such as maize starch, binders and/or
lubricants, such as talc or magnesium stearate, and stabilizers if
appropriate. In soft capsules, the active ingredient is preferably
dissolved or suspended in suitable liquid excipients, such as
greasy oils, paraffin oil or liquid polyethylene glycols or fatty
acid esters of ethylene glycol or propylene glycol, it being
likewise possible to add stabilizers and detergents, for example of
the polyethylene sorbitan fatty acid ester type.
[0141] Other oral forms of administration are, for example, syrups
prepared in the customary manner, which comprise the active
ingredient, for example, in suspended form and in a concentration
of about 5% to 20%, preferably about 10% or in a similar
concentration which results in a suitable individual dose, for
example, when 5 or 10 ml are measured out. Other forms are, for
example, also pulverulent or liquid concentrates for preparing of
shakes, for example in milk. Such concentrates can also be packed
in unit dose quantities.
[0142] Pharmaceutical compositions, which can be used rectally,
are, for example, suppositories that comprise a combination of the
active ingredient with a suppository base. Suitable suppository
bases are, for example, naturally occurring or synthetic
triglycerides, paraffin hydrocarbons, polyethylene glycols or
higher alkanols.
[0143] Compositions which are suitable for parenteral
administration are aqueous solutions of an active ingredient in
water-soluble form, for example of water-soluble salt, or aqueous
injection suspensions, which comprise viscosity-increasing
substances, for example sodium carboxymethylcellulose, sorbitol
and/or dextran, and if appropriate stabilizers. The active
ingredient can also be present here in the form of a lyophilisate,
if appropriate together with excipients, and be dissolved before
parenteral administration by addition of suitable solvents.
Solutions such as are used, for example, for parental
administration can also be used as infusion solutions. Preferred
preservatives are, for example. Antioxidants, such as ascorbic
acid, or microbicides, such as sorbic or benzoic acid.
[0144] Ointments are oil-in-water emulsions, which comprise not
more than 70%, but preferably 20-50% of water or aqueous phase. The
fatty phase consists, in particular, hydrocarbons, for example
vaseline, paraffin oil or hard paraffin's, which preferably
comprise suitable hydroxy compounds, such as fatty alcohol's or
esters thereof, for example cetyl alcohol or wool wax alcohols,
such as wool wax, to improve the water-binding capacity.
Emulsifiers are corresponding lipophilic substances, such as
sorbitan fatty acid esters (Spans), for example sorbitan oleate
and/or sorbitan isostearate. Additives to the aqueous phase are,
for example, humectants, such as polyalcohols, for example
glycerol, propylene glycol, sorbitol and/or polyethylene glycol, or
preservatives and odoriferous substances.
[0145] Fatty ointments are anhydrous and comprise, as the base, in
particular, hydrocarbons, for example paraffin, vaseline or
paraffin oil, and furthermore naturally occurring or semi-synthetic
fats, for example hydrogenated coconut-fatty acid triglycerides,
or, preferably, hydrogenated oils, for example hydrogenated
groundnut or castor oil, and furthermore fatty acid partial esters
of glycerol, for example glycerol mono- and/or distearate, and for
example, the fatty alcohols. They also contain emulsifiers and/or
additives mentioned in connection with the ointments which increase
uptake of water.
[0146] Creams are oil-in-water emulsions, which comprise more than
50% of water. Oily bases used are, in particular, fatty alcohols,
for example lauryl, cetyl or stearyl alcohols, fatty acids, for
example palmitic or stearic acid, liquid to solid waxes, for
example isopropyl myristate, wool wax or beeswax, and/or
hydrocarbons, for example vaseline (petrolatum) or paraffin oil.
Emulsifiers are surface-active substances with predominantly
hydrophilic properties, such as corresponding nonionic emulsifiers,
for example fatty acid esters of polyalcohols or ethyleneoxy
adducts thereof, such as polyglyceric acid fatty acid esters or
polyethylene sorbitan fatty esters (Tweens), and furthermore
polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid
esters, or corresponding ionic emulsifiers, such as alkali metal
salts of fatty alcohol sulfates, for example sodium lauryl sulfate,
sodium cetyl sulfate or sodium stearyl sulfate, which are usually
used in the presence of fatty alcohols, for example cetyl stearyl
alcohol or stearyl alcohol. Additives to the aqueous phase are,
inter alia, agents which prevent the creams from drying out, for
example polyalcohols, such as glycerol, sorbitol, propylene glycol
and/or polyethylene glycols, and furthermore preservatives and
odoriferous substances.
[0147] Pastes are creams and ointments having secretion-absorbing
powder constituents, such as metal oxides, for example titanium
oxide or zinc oxide, and furthermore talc and/or aluminum
silicates, which have the task of binding the moisture or
secretions present.
[0148] Foams are administered from pressurized containers and they
are liquid oil-in-water emulsions present in aerosol for. As the
propellant gases, halogenated hydrocarbons, such as
chlorofluoro-lower alkanes, for example dichlorofluoromethane and
dichlorotetrafluoroethane, or, preferably, non-halogenated gaseous
hydrocarbons, air, N.sub.2 O, or carbon dioxide are used. The oily
phases used are, inter alia, those mentioned above for ointments
and creams, and the additives mentioned there are likewise
used.
[0149] Tinctures and solutions usually comprise an
aqueous-ethanolic base to which, humectants for reducing
evaporation, such as polyalcohols, for example glycerol, glycols
and/or polyethylene glycol, and re-oiling substances, such as fatty
acid esters with lower polyethylene glycols, i.e. lipophilic
substances soluble in the aqueous mixture to substitute the fatty
substances removed from the skin with the ethanol, and, if
necessary, other excipients and additives, are admixed.
[0150] The invention also relates to a process or method for
treatment of the disease states mentioned above. The compounds can
be administered prophylactically or therapeutically as such or in
the form of pharmaceutical compositions, preferably in an amount,
which is effective against the diseases mentioned. With a
warm-blooded animal, for example a human, requiring such treatment,
the compounds are used, in particular, in the form of
pharmaceutical composition. A daily dose of about 0.1 to about 5 g,
preferably 0.5 g to about 2 g, of a compound of the present
invention is administered here for a body weight of about 70
kg.
[0151] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
substitutions, modifications or changes in light thereof will be
suggested to persons skilled in the art and are included within the
spirit and purview of this application and are considered within
the scope of the appended claims. The following examples are given
by way of example of the preferred embodiments, and are in no way
considered to be limiting to the invention. For example, the
relative quantities of the ingredients may be varied to achieve
different desired effects, additional ingredients may be added,
and/or similar ingredients may be substituted for one or more of
the ingredients described. All publications, patents, and patent
applications cited herein are hereby incorporated by reference in
their entirety for all purposes.
Examples of General Synthetic Schemes and Procedures:
Example 1
Synthesis of [1,3,5]Oxadiazepane-2,6-diones (Formula Ia)
[0152] ##STR3##
[0153] General Scheme Synthetic Scheme for Ia. ##STR4##
[0154] a) lobenzene bistrifluoroacetate (IBTFA), THF/H.sub.2O; b)
Boc.sub.2O; c) p-nitrophenylchloroformate, CH.sub.2Cl.sub.2,
Diisopropylethylamine; d) trifluoroacetic acid; e) DIEA, HOBt; f)
NaH, R.sup.3Br.
Example 2
Synthesis of 2-Thioxo-[1,3,5]triazepan-6-ones (Formula Ib)
[0155] ##STR5##
[0156] General Synthetic Scheme for Ib. ##STR6##
[0157] Step a) Dipeptide amide Ib-p1 was dissolved in THF/water
(3:1) and treated with iodobenzene bistrifluoroacetate (1.2 equiv.)
for 3 h, time after which starting material was consumed. Solvents
were removed in vacuo and Et.sub.2O was added. The solid which
formed was collected and washed with Et.sub.2O to yield the
corresponding gem-diamino derivative which was used in the next
step without further purification. Quantitative Yield.
[0158] Step b) bis(benzotriazol-1-yl)methanethione (1 equiv) was
dissolved in CH2Cl2 at rt. The previously synthesized gem-diamino
derivative was added dropwise and the reaction mixture was stirred
for 18 h. Solvent was removed under vacuum and the residue was
redissolved in EtOAc and washed with 5% aqueous sodium carbonate,
water and brine before drying over anhydrous sodium sulphate.
Solvent was removed under vacuum and 1b-p2 was recrystallized from
ethyl acetate.
[0159] Step c) The 1-thiocarbamoylbenzotriazole was treated with
TFA at 0.degree. C. After 30 min, TFA was removed by co-evaporation
with hexane and the TFA salt precipitated by addition of
diethylether. The resulting salt Ib-p3 was collected by filtration
and dried under high vacuum. It was used in the next step without
further purification.
[0160] Step d) The TFA salt Ib-p3 was dissolved in MeCN and
diisopropylethylamine (2.5 equiv) was then added and the reaction
mixture was stirred for 24 h. Solvent was removed in vacuum and the
residue was redissolved in EtOAc, washed with 5% aqueous sodium
carbonate, 1M HCl, water, and brine before drying over anhydrous
sodium sulphate. Solvent was removed in vacuum and cyclic Ib-1 was
purified by recrystallization from CH.sub.2Cl.sub.2/diisopropyl
ether.
Example 3
Synthesis of 4-Benzyl-6-methyl-[1,3,6]oxadiazocane-2,5-dione
(Formula Ib-1)
[0161] ##STR7##
[0162] General Synthetic Scheme for Ib-1. ##STR8##
[0163] Step a) Dipeptide amide Ib-p1 was dissolved in THF/water
(3:1) and treated with iodobenzene bistrifluoroacetate (1.2 equiv.)
for 3 h, time after which starting material was consumed. Solvents
were removed in vacuo and Et.sub.2O was added. The solid which
formed was collected and washed with Et.sub.2O to yield the
corresponding gem-diamino derivative which was used in the next
step without further purification. Quantitative Yield.
[0164] Step b) bis(benzotriazol-1-yl)methanethione (1 equiv) was
dissolved in CH2Cl2 at rt. The previously synthesized gem-diamino
derivative was added dropwise and the reaction mixture was stirred
for 18 h. Solvent was removed under vacuum and the residue was
redissolved in EtOAc and washed with 5% aqueous sodium carbonate,
water and brine before drying over anhydrous sodium sulphate.
Solvent was removed under vacuum and 1b-p2 was recrystallized from
ethyl acetate.
[0165] Step c) The 1-thiocarbamoylbenzotriazole was treated with
TFA at 0.degree. C. After 30 min, TFA was removed by co-evaporation
with hexane and the TFA salt precipitated by addition of
diethylether. The resulting salt Ib-p3 was collected by filtration
and dried under high vacuum. It was used in the next step without
further purification.
[0166] Step d) The TFA salt Ib-p3 was dissolved in MeCN and
diisopropylethylamine (2.5 equiv) was then added and the reaction
mixture was stirred for 24 h. Solvent was removed in vacuum and the
residue was redissolved in EtOAc, washed with 5% aqueous sodium
carbonate, 1M HCl, water, and brine before drying over anhydrous
sodium sulphate. Solvent was removed in vacuum and cyclic Ib-1 was
purified by recrystallization from CH.sub.2Cl.sub.2/diisopropyl
ether.
Example 4
Synthesis of 2-Thioxo-[1,3,5]oxadiazepan-6-ones (Formula Ic)
[0167] ##STR9##
[0168] General Synthetic Scheme for Ic. ##STR10##
[0169] a) lobenzene bistrifluoroacetate (IBTFA), THF/H.sub.2O; b)
bis(benzotriazolyl)methanethione, CH.sub.2Cl.sub.2; c)
trifluoroacetic acid; d) diisopropylethylamine, MeCN, NaH.
Example 5
Synthesis of [1,3,6]Oxadiaxocane-2,5-diones (Formula Id)
[0170] ##STR11##
[0171] General Synthetic Scheme for Id. ##STR12##
[0172] a) para-nitrophenyl chloroformate (2 eq), pyridine (1,1 eq),
CH2Cl2, TA overnight; b) TFA, TA 30 minutes; c) DIEA (2,6 eq),
HOBt, (1 eq), MeCN, TA 3 jours.
Example 6
Synthesis of 4-Benzyl-6-methyl-[1,3,6]oxadiazocane-2,5-dione
(Formula Id-1)
[0173] ##STR13##
[0174] General Synthetic Scheme for Id-1. ##STR14##
[0175] a) para-nitrophenyl chloroformate (2 eq), pyridine (1,1 eq),
CH.sub.2Cl.sub.2, TA overnight; b) TFA, TA 30 minutes; c) DIEA (2,6
eq), HOBt (1 eq), MeCN, TA 3 jours.
[0176] 1) Synthesis of p-nitrophenyl carbonate precursor Id-p2
##STR15##
[0177] The starting dipeptide alcohol Id-p1 (300 mg, 0.93 mmol, 1
eq) is dissolved in 5 mL CH.sub.2Cl.sub.2 and 82 .mu.L pyridine
(1.02 mmol, 1.1 eq). A solution of 4-nitrophenyl chloroformate
(0.37 g, 1.86 mmol, 2 eq) in 2 mL.
[0178] After stirring for 24 h, the reaction mixture is diluted
with 15 mL CH.sub.2Cl.sub.2, and washed with 1N NaHCO.sub.3 The
organic phase is dried on Na.sub.2SO.sub.4, concentrated and
purified by flash chromatography (eluant 1:2 AE/cyclohexane) to
yield pure carbonate Id-p2 with 59% yield. HPLC tR 14.1 (gradient
30-100% B, 20 min.)
[0179] .sup.1H NMR (300 MHz, CDCl3) .delta. 8.3 (m, 2H, arom-H
.alpha.-NO2), 7.39 (m, 2H, arom-H .beta.-NO2), 7.24 (m, 5H,
arom-H), 5.34 (m, J=10.55 Hz, 1H NH), 4.85 (q, J=14.9, 7.9 Hz, 1H
.alpha.-NH), 4.31 4.14 (dd, J=9.97, 5.1 Hz, 2H .alpha.-O), 3.77
3.54 (dd, J=14.5, 5.2 Hz, 2H .alpha.-NMe), 2.98 (m, 2H
.alpha.-Phe), 2.79 (s, 3H NMe), 1.43 (s, 9H Boc).
[0180] .sup.13C NMR (100 MHz, CDCl3) .delta. 171.8 (CO amide),
154.8 (CO carbamate), 154.5 (CO carbonate), 151.6 (C arom
.alpha.-NO2), 144.8 (C arom .delta.-NO2), 135.5 (C arom Phe), 128.8
(2CH Phe), 128.7 (2CH Phe), 127.8 (CH-Phe), 124.7 (CH arom), 121.1
(CH-arom), 79.3 (C Boc), 66.0 (CH2 .alpha.-O), 50.9 (CH
.alpha.-NH), 46.4 47.0 (CH2 .alpha.-N), 39.4 (CH2 Phe), 35.8 33.6
(CH3 NMe), 27.7 (3 CH3 Boc).
[0181] 2) Cyclization to Id-p1
[0182] p-Nitrophenyl carbonate Id-p2 is treated with
trifluoroacetic acid for 30'. Addition of ether gave the
corresponding TFA salt which precipitated as a white solide. It was
filtered and used in the next step without further purification.
The TFA salt (220 mg, 0.44 mmol, 1 eq) dissolved in MeCN (10 mL)
was added slowly to a solution of Diisopropylethylamine (194 .mu.L,
1.14 mmol, 2.6 eq) and hydroxybenzotriazole (HOBt) (60 mg, 0.44
mmol, 1 eq) in 25 mL MeCN. The reaction mixture was stirred for 3
days and concentrated in vacuo. CH.sub.2Cl.sub.2 is then added and
the organic phase was washed with 1N NaHCO3, brine, dried over
Na2SO4 and concentrated in vacuo. The residue (110 mg) was then
purified by silica gel chromatography.
[0183] [CHCl.sub.3/MeOH/AcOH (20:0.5:0.1) then puis CHCl.sub.3/MeOH
[20:1]) to afford 42 mg of Id-1.
[0184] HPLC t.sub.R (Id-1) 5.88 (gradient 30-100% B, 20 min)
[0185] HRMS (ESI) calculated for C.sub.13H.sub.16N.sub.2O.sub.3
249.1234, found 249.1230.
[0186] .sup.1H NMR Id-1 (300 MHz, CDCl.sub.3) .delta. 7.25 (m, 5H,
arom-H), 6.10 (d, H.sup.4), 4.75 (dd, J=8.9, 7.4 Hz, H.sup.5), 4.20
(m, 2H.sup.3), 4.15 (m, H.sup.2), 3.28 (dd, J=14.0, 7.6 Hz,
1H.sup.6), 3.17 (m, H.sup.2'), 3.02 (dd, 1H.sup.6), 3.0 (s,
3H.sup.1).
[0187] .sup.13C NMR Id-1 (100 MHz, CDCl.sub.3) .delta. 172.3 (CO
amide), 157.7 (CO carbonate), 136.9 (C-arom), 129.3 (2CH arom),
128.6 (2CH arom), 126.8 (CH arom), 69.6 (CH.sub.2 .alpha.-O), 54.0
(CH .alpha.-N), 52.9 (CH.sub.2 .alpha.-N), 36.6 (CH.sub.3 Me), 35.7
(CH.sub.2 Phe).
Example 7
Synthesis of 1,1-Dioxo-1
.lamda..sup.6-[1,2,5,8]thiatriazocan-4-ones (Formula If)
[0188] ##STR16##
[0189] General Synthetic Scheme for If. ##STR17##
[0190] i) (a) TFA; (b) NaHCO.sub.3 satured, DCM; ii) Burgess
reagent (2,5 eq), THF, 70.degree. C. for two hours.
Example 8
Synthesis of
10-methyl-6,6,11-trioxo-8,9,10,11,11a,12-hexahydro-5H-6.lamda..sup.6-thia-
-5a,7,10-triaza-cycloocta[b]naphthalene-7-carboxylic acid methyl
ester (Formula If-1)
[0191] ##STR18##
[0192] General Synthetic Scheme for If-1. ##STR19##
[0193] i) The N-Boc protected dipeptide alcohol was treated with
TFA for 30 minutes at 0.degree. C. The TFA was removed under vacuum
and the residue was dissolved in AcOEt. Saturated NaHCO3 was added
under stirring and after 10 minutes the organic phase was dried
with Na2SO4 and concentrated under vacuum to give If-p1.
[0194] ii) Compound 1f-p1 (175 mg, 0.75 mmol, 1 eq), is dissolved
in 10 mL anhydrous THF and Burgess reagent (534 mg, 2.24 mmol, 2.5
eq) is added. The solution is then heated under reflux at from
about 70.degree. C. to about 90.degree. C. for 2 days. The reaction
mixture is then poured into a solution of saturated NH.sub.4Cl (40
mL). The mixture is extracted with CH2Cl2 and the organic phase is
washed with H.sub.2O, dried over Na.sub.2SO.sub.4 and concentrated
under vacuum. The crude mixture is then purified by silica gel
chromatography (CHCl.sub.3/MeOH/AcOH (18:1:0.2) to yield If-1.
* * * * *