Note: Descriptions are shown in the official language in which they were submitted.
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Compositions and Methods for the Inhibition of Phospholipase A2
Field of the Invention
The present invention relates to 7 and 8-membered ring dipeptide-derived
nitrogencontaining 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
Phospholipase A2 enzymes (PLA2s) are enzymes that catalyze the hydrolysis of
1o 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 Ca2+-dependent catalytic activity. To
date, 12
mammalian sPLA2s have been identified and classified into 3
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main structural collections: group I/li/V/X, III, and XII.
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.
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.
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
In one aspect, the inventions relates to the use of an effective amount of a
compound in the preparation of a composition for inhibiting the activity of a
phospholipase A2 (PLA2) enzyme, wherein the compound has the formula B-
2
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O
R5 R1
N N/
R4 R2
N
R3 O (B)
wherein R1, R2, R3, R4, and R5 represent, each selected independently from the
other,
a member selected from the group consisting of: a hydrogen atom; an amino acid
side
chain; a (C1-C10) alkyl; a (C2-C10) alkenyl; a (C2-C10) alkynyl; a (C5-C12)
monocyclic or bicyclic aryl; a (C6-C14) monocyclic, bicyclic, or tricyclic
aralkyl; a
monocyclic, bicyclic, or tricyclic (C6-C14) heteroaralkyl; and a (C1-C10)
monocyclic or
bicyclic heteroaryl group having up to 5 heteroatoms selected from the group
consisting of N, 0, 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
1o halogen atom, an NO2, OH, amidine, benzamidine, imidazole, 1,2,3-triazole,
alkoxy,
(C1-C4) alkyl, amino, piperazine, piperidine, dialkylamino, guanidine group,
bis
alkylated or bis acylated guanido group, -C(O)OH, carboxamide, -C(O)-NH-OH,
-P(O)(OH)2, phosphonamidate, sulfhydryl and any combination thereof, wherein
R1
and R2, R3 and R4, or R4 and R5, together with the carbon atoms to which they
are
attached, form a cyclic ring, wherein the compound is effective for inhibiting
PLA2.
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.
In one aspect the present invention relates to methods for synthesizing novel
dipeptide derived heterocyclics of the formula I.
2a
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.III.= Fi
[0010] wherein, W is a member selected from the group consisting of -
C(R5)(R5a)_; _
C(R6)(R6a)-C(R7)(R7a)-; -C(R8)=C(R9)-; -N(R10), and combinations thereof;
[0011] X is a member selected from the group consisting of -N(Ria)C(=Y)N(R4)-;-
OC(=Y)N(R4)-; -N(R'a)C(=Y)O-; -N(Rla)S(=O)N(R4)-; -N(Rla)S(=O)2N(R4)-; -
C(Rla)(R3a)C(=Y)N(R4)-, and combinations thereof;
[0012] Y and Z represent, each independent from the other, a member selected
from the
group consisting of oxygen ("0") and sulfur ("S"); and
[0013] R', Ria > R2 > R3> R3a > R4> Rs> R5a > R6> R6a > R'> R'a > Rg> R9> and
R10 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, 0, 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 NO2, 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, sulthydryl 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
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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] Figure 1. Comparison of the 1,3,5-triazepan-2,6-dione scaffold, B, and
2,5-
diketopiperazines, A.
[0022] Figure 2. a) EtOCOC1, NMM, THF, -20 C, then NaN3 in H20; b) Toluene, 65
C,
then HOSu and pyridine; c) TFA, 30 min; d) DIEA, MeCN; e) PS-DIEA, CH2C12. g =
gem,
refers to the 2-alkyl gem-diamino-derivative of the corresponding amino-acid
according to the
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nomenclature proposed by Chorev and Goodman.
[0023] Figure 3. X-ray crystal structures of representative 1,3,5-triazepan-
2,6-diones 4, 7
and 9.
[0024] Figure 4. a) NaH (4 equiv), RX (4 equiv); b) KF/A12O3 (10 equiv) or NaH
(2 equiv),
RX (1.5 equiv).
[0025] Figure 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] Figure 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
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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:
(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;
(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.
[0031] 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.
[0032] "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, 0 or S.
[0033] "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.
[0034] "Hydroxyl" refers the functional group -OH when it is a substituent in
an organic
compound.
[0035] "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,
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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, -S02-alkyl, -S02-substituted alkyl, -S02-aryl, oxo (=O),
and
S02-heteroaryl. Such heterocyclic groups can have a single ring or multiple
condensed rings.
Preferred heterocyclics include morpholino, piperidinyl, and the like.
[0036] 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.
[0037] The term "thiol" refers to the group -SH.
[0038] The term "thioalkoxy" refers to the group -S-alkyl.
[0039] "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).
[0040] "Amidine" refers to a functional group that has two amine groups
attached to the
same carbon atom with one carbon-nitrogen double bond: HN=CR'-NH"2.
[0041] "Alkoxyl" refers to an alkyl group linked to oxygen thus: R-O-, where R
is an alkyl.
[0042] "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
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identical with the definitions of the corresponding groups as defined herein.
[0043] "Halogen" refers to fluorine, bromine, chlorine, and iodine atoms.
[0044] "Acyl" denotes the group --C(O)Rej where Re 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.
[0045] "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.
[0046] "Alkylamino" denotes the group --NRf Rg, where Rf and Rg, 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.
[0047] "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.
[0048] "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.
[0049] "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, 0 or S with at
least one ring of this
group being aromatic.
[0050] "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, 0
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
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substituent group have meanings which are identical with definitions of the
corresponding
groups as defined in this legend.
[0051] "Carboxyl" denotes the group --C(O)ORS, 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.
[0052] "Cycloalkyl" refers to a monocyclic or polycyclic alkyl group
containing 3 to 15
carbon atoms.
[0053] "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.
[0054] "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, 0, S or P.
[0055] "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, 0, 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.
[0056] 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.
[0057] 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=CH2), n-propenyl (-CH2CH=CH2), iso-propenyl (-C(CH3)=CH2), and the like.
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[0058] "Imidazole" refers to a heterocyclic base of the general formula:
C3H4N2.
[0059] "Aralkyl group" refers to, for example, a Cl -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.
[0060] "Guanidine" refers generally to the amidine of amidocarbonic acid and
has the
general formula of: C(NH2)3.
[0061] 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.
[0062] The present invention relates to nitrogen-containing heterocyclic
compounds
represented by the general formula I as follows:
III..- F
[0063] (I)wherein, W is a member selected from the group consisting
of -C(R5)(R5a)-; -C(R6)(R6a)-C(R7)(R7a)-; -C(R8)=C(R9)-; -N(R10), and
combinations thereof;
[0064] X is a member selected from the group consisting of -N(Ria)C(=Y)N(R4)-;-
OC(=Y)N(R4)-; -N(R'a)C(=Y)O-; -N(Ria)S(=O)N(R4)-; -N(Ria)S(=0) N(R4)-; -
C(R1a)(R3a)C(=Y)N(R4)-, and combinations thereof;
[0065] Y and Z represent, each independent from the other, a member selected
from the
group consisting of oxygen ("0") and sulfur ("S"); and
[0066] R', Rla > R2 > R3> R3a > R4> Rs> Rya > R6> R6a > R'> R7a > Rg> R9, and
R10 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)
alkenyl; (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
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heteroatoms selected from N, 0, 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 NO2, 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, sulthydryl and any combination thereof.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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
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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.
[0072] 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.
[0073] 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 z mol 4 z mol
3,
respectively.
[0074] 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.
[0075] 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
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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.
[0076] 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
[0077] To date, 12 mammalian sPLA2s have been identified and classified into 3
main
structural collections: group UII/V/X, III, and XII.
[0078] 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.
[0079] 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.
[0080] 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
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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.
[0081] 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.
[0082] 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.
[0083] An indole-derived inhibitor of second generation, called S-
5920/LY31592ONa,
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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.
[0084] 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/LY31592ONa 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.
[0085] 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.
[0086] 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.
[0087] An indole inhibitor called indoxam (IDX) inhibited PGE2 production
induced by
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TGF-a 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 FII/V/X is involved in these processes.
[0088] 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 (IC50 =
130nM and
900nM 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-a
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 FII/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.
[0089] 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.
[0090] 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
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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.
[0091] 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.
[0092] I. Labeling of E.coli membranes with [3H]-oleic acid :
[0093] 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. DH1OB and XL-1 strains seem better than
JM101 strain,
ie they give membranes with less background and are easier to pellet after the
PLA2 assay).
OD600nm of the saturated overnight preculture is about 2 UDO. Make a 1/5
dilution and
measure OD600nm=
[0094] 2) Dilute the preculture in 100 ml of fresh LB to 0.05 UOD600nm and add
250 gl of
[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 N2 before
closure. Save a l0gl
aliquot of the culture for the later quantification of incorporated oleic
acid.
[0095] 3) Grow cells for about 5 hours at 37 C with vigorous shaking (200-230
rpm) up
tol UOD600nm=
[0096] 4) Spin down the culture for 15 minutes/4,000 rpm/ 50 ml falcon
tube/RT. Save 50 gl
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 C
under shaking (this step allows the remaining unincorporated labeled oleic
acid to get
incorporated into phospholipids).
[0097] 5) Spin down again as above. Save 50 gl of supernatant for later
quantification.
Discard the supernatant and resuspend the pellet in 50 ml of washing buffer.
[0098] 6) Spin down as above. Save 50 gl of supernatant for later
quantification. Discard the
supernatant and resuspend the pellet in 2 ml of washing buffer but WITHOUT
BSA. Save a 2 gl
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 C, 1.5 bar). This step can be
done overnight.
[0099] 7) The next day, save another 2 gl aliquot for counting and transfer
the remaining
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solution into 2 eppendorf tubes. Rinse the Corex tube with 1 ml of washing
buffer and combine
with the 2 ml solution.
[00100] 8) Spin down for 1 minute at 14,000 rpm (RT). Save 10 gl 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.
[00101] 9) Resuspend the pellet in 5 ml of washing buffer and count 5 gl for
quantification of
incorporated oleic acid. Dilute the solution to 100,000 dpm/gl and make
aliquotes of 30 l.
[00102] 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.
II. PLA2 assay :
1) Preparation of substrate:
[00103] Pipet the required amount of radioactivity (100,000 dpm of labeled
membranes per
reaction x number of reactions) and dilute into 1 ml of PLA2 activity buffer
in an eppendorf
tube.
[00104] Spin down for 1 minute at 14,000 rpm (RT). Discard the supernatant.
Carefully
resuspend the pellet into 150 gl of PLA2 activity buffer and add PLA2 activity
buffer for the
total number of reactions. Store the pool at room temperature (do not prepare
the pool too much
in advance).
2) PLA2 assay reaction :
[00105] A typical reaction is made in an eppendorf tube and consists of a
total volume of 150
gl made with 50 gl PLA2 activity buffer, a negligible volume of enzyme
solution and l00gl 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).
[00106] Reaction mixtures are incubated for various periods of times up to 1
hour at 25 C or
37 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
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hydrolysis.
[00107] 3) Stop the reaction by adding 300 gl 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.
[00108] Count also 3 or 4 aliquotes of 100 91 of the substrate pool to
determine the total
amount of injected radioactivity/reaction.
[00109] Note that we routinely considered that the counts in the above
supernatants
correspond to free 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.
[00110] Note also that this protocol does not specifically detect sPLA2
activity, but can also
detect the activity of cytosolic PLA2s.
[00111] Materials :
[00112] DH1OB or XL-1 E.coli strain (could be a strain carrying or not a
plasmid); [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/HC1 pH 8.0, 1 mM EDTA containing 0.5% Fatty acid free BSA;
PLA2
activity buffer : 0.1 M Tris/HC1 pH 8.0, 10 mM CaC12, 0.1% BSA; Stop buffer:
0.1 M EDTA
containing 0.2% fatty acid free BSA.
Therapeutic Administration
[00113] 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.
[00114] Suitable routes for administration include oral, rectal, vassal,
topical (including
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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.
[00115] 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.
[00116] 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.
[00117] 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.
[00118] 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.
[00119] 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,
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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, pentadecyclic acid, palmitic acid, margaric acid, stearic acid,
arachidinic
acid, behenic acid or corresponding unsaturated acids, for example oleic 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-hydroxitoluene. 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
1o 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 C12
from Huls AG, 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.
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.
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.
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
3o binders, such as starches, for example maize, wheat, rice or potato starch,
methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose
and/or
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polyvinyl-pyrrolidine, and/or, if desired, desintegrators, such as the above
mentioned
starches, and furthermore carboxymethyl-starch, cross-linked
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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.
[00123] 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.
[00124] 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.
[00125] 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.
[00126] 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,
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waxes, shellac and hydrogenated vegetable oils.
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
1o 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* phi 12, Avicel* pHIOI 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.
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.
23
*Trade-mark
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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.
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 multi particulate, 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
multi particulate 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 multi particulate, such as multiparticulates formed by conventional
means.
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.
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.
24
*Trade-mark
CA 02635354 2011-02-25
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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.
24a
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WO 2007/074169 PCT/EP2006/070256
[00134] 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.
[00135] 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.
[00136] 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.
[00137] 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.
[00138] 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.
[00139] 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,
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
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.
[00140] 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.
[00141] 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.
[00142] 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.
26
CA 02635354 2011-02-25
12016-3
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, N2 0, 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.
Tinctures and solutions usually comprise an aqueous-ethanolic base to which,
humectants for reducing evaporation, such as polyalcohols, for example
glycerol,
1o 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.
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.
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.
Examples of General Synthetic Schemes and Procedures:
27
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
[00147] Example 1: Synthesis of[ 1,3,5]Oxadiazepane-2,6-diones (Formula Ia)
(Ia)
[00148] General Scheme synthetic scheme for Ia.
R1 R2 0 R1 R2
Bn.0
~Y-?~ NNH2 Bn_ONYN'Boc
O R5 O R5
1 c, d
0 0
R3 N) -0 f X0 02N 0I R1 R2 e
R5~ 1 5 ~ N rR1 OAO N~NH3+
N O RN IS
R2 R2 0 0 R5
[00149] a) lobenzene bistrifluoroacetate (IBTFA), THF/H20; b) Boc20; c) p-
nitrophenylchloroformate, CH2C12, Diisopropylethylamine; d) trifluoroacetic
acid; e) DIEA,
HOBt; f) NaH, R3Br.
[00150] Example 2: Synthesis of 2-Thioxo-[ 1,3,5]triazepan-6-ones (Formula lb)
(Ib)
[00151] General synthetic scheme for lb.
R1 R2 0 R1 R2 N=N
H
a, b Boc,N_'_Y N` I N 2 Boc,N IN NUN
II
R O R5 R O R5 S
C
S
XNH R1 R2 H N=N
HN R1 d N IN N
R5~ CIF )0 2R~ 0 R
N S S
R2 O
28
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
[00152] Step a) Dipeptide amide lb-pl was dissolved in THE/water (3 :1) and
treated with
iodobenzene bistrifluoroacetate (1.2 equiv.) for 3h, time after which starting
material was
consumed. Solvents were removed in vacuo and Et20 was added. The solid which
formed was
collected and washed with Et20 to yield the corresponding gem-diamino
derivative which was
used in the next step without further purification. Quantitative Yield.
[00153] Step b) bis(benzotriazol-l-yl)methanethione (1 equiv) was dissolved in
CH202 at rt.
The previously synthesized gem-diamino derivative was added dropwise and the
reaction
mixture was stirred for 18h. 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 lb-p2 was
recrystallized
from ethyl acetate.
[00154] Step c) The 1-thiocarbamoylbenzotriazole was treated with TFA at 0 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.
[00155] 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 24h. Solvent
was removed in
vacuum and the residue was redissolved in EtOAc, washed with 5% aqueous sodium
carbonate,
1M HC1, water, and brine before drying over anhydrous sodium sulphate. Solvent
was removed
in vacuum and cyclic lb-1 was purified by recrystallization from
CH2C12/diisopropyl ether.
[00156] Example 3: Synthesis of 4-Benzyl-6-methyl-[1,3,6]oxadiazocane-2,5-
dione (Formula
lb- 1)
IJH
H-~(lb-1)
[00157] General synthetic scheme for lb-1.
29
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
N
O H N-
Boc-N N` ~NH a b Boc-N NvN N
z
H O R5 H O S
lb-p1 lb-p2
Ic _
S
N
HN NH d CF3000 H3N NvNU
N II
O S
O
1-b l
[00158] Step a) Dipeptide amide lb-pl was dissolved in THE/water (3 :1) and
treated with
iodobenzene bistrifluoroacetate (1.2 equiv.) for 3h, time after which starting
material was
consumed. Solvents were removed in vacuo and Et20 was added. The solid which
formed was
collected and washed with Et20 to yield the corresponding gem-diamino
derivative which was
used in the next step without further purification. Quantitative Yield.
[00159] Step b) bis(benzotriazol-l-yl)methanethione (1 equiv) was dissolved in
CH2C12 at rt.
The previously synthesized gem-diamino derivative was added dropwise and the
reaction
mixture was stirred for 18h. 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 lb-p2 was
recrystallized
from ethyl acetate.
[00160] Step c) The 1-thiocarbamoylbenzotriazole was treated with TFA at 0 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.
[00161] 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 24h. Solvent
was removed in
vacuum and the residue was redissolved in EtOAc, washed with 5% aqueous sodium
carbonate,
1M HC1, water, and brine before drying over anhydrous sodium sulphate. Solvent
was removed
in vacuum and cyclic lb-1 was purified by recrystallization from
CH2C12/diisopropyl ether.
[00162] Example 4: Synthesis of 2-Thioxo-[ 1,3,5 ]oxadiazepan-6 -ones (Formula
Ic)
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
r "
(Ic)
[00163] General synthetic scheme for Ic.
R1 R2 0 R1 R2 N=N
TBDMS,O) /N` kNH b TBDMS,O NYNUN
]'~ ~" z II
O R5 O R5 S
S IC
,111,LO R1 R2 H N=N
R5-~R1 HO(NYNUN
N O O IR5 ISI
Rz
[00164] a) lobenzene bistrifluoroacetate (IBTFA), THF/H20; b)
bis(benzotriazolyl)methanethione, CH2C12; c) trifluoroacetic acid; d)
diisopropylethylamine,
MeCN,NaH.
[00165] Example 5: Synthesis of [1,3,6]Oxadiaxocane-2,5-diones (Formula Id)
F4
FR , (Id)
[00166] General synthetic scheme for Id.
H ~Ft 0
a H0 hf
YCH OYO-a
R1 RF R7 R1 RF R7 O
O R
0 O
b H3
~~Fe ~ O c O~ R1 +
CF3000 R1 O ~O
NOz ~N
w R2
31
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WO 2007/074169 PCT/EP2006/070256
[00167] a) para-nitrophenyl chloroformate (2 eq), pyridine (1,1 eq), CH2C12,
TA overnight;
b) TFA, TA 30 minutes; c) DIEA (2,6 eq), HOBt, (1 eq), MeCN, TA 3 jours.
[00168] Example 6: Synthesis of 4-Benzyl-6-methyl-[1,3,6]oxadiazocane-2,5-
dione (Formula
Id-1)
(Id-1)
[00169] General synthetic scheme for Id-1.
N O a H O
Boc' N"OH Bocce Nfl, Nz-,~G
II
Bn Id-p1 Bn Id-p2 O NO2
H
b,c N
O
~N O I Id-1
[00170] a) para-nitrophenyl chloroformate (2 eq), pyridine (1,1 eq), CH2C12,
TA overnight; b)
TFA, TA 30 minutes; c)DIEA (2,6 eq), HOBt (1 eq), MeCN, TA 3 jours.
[00171] 1) Synthesis ofp-nitrophenyl carbonate precursor Id-p2
O
Boc'N N'-1OYO H O )::;IN02
(Id-p2)
[00172] The starting dipeptide alcohol Id-pl (300 mg, 0.93 mmol, 1 eq) is
dissolved in 5 mL
CH2C12 and 82 L pyridine (1.02 mmol, 1.1 eq). A solution of 4-nitrophenyl
chloroformate (0.37
g, 1.86 mmol, 2 eq) in 2mL.
[00173] After stirring for 24h, the reaction mixture is diluted with 15 mL
CH2C12, and washed
with IN NaHCO3 The organic phase is dried on Na2SO4, concentrated and purified
by flash
chromatography (eluant 1:2 AE/cyclohexane) to yield pure carbonate Id-p2 with
59% yield.
32
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WO 2007/074169 PCT/EP2006/070256
HPLC tR 14.1 (gradient 30-100%B, 20 min.)
[00174] 'H NMR (300 MHz, CDC13) 6 8.3 (m, 2H, arom-H u.-N02),7.39 (m, 2H, arom-
H (3-
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 U.-
NH), 4.31 4.14 (dd, J= 9.97, 5.1 Hz, 2H u.-O), 3.77 3.54 (dd, J= 14.5, 5.2 Hz,
2H a-NMe), 2.98
(m, 2H a-Phe), 2.79 (s, 3H NMe), 1.43 (s, 9H Boc).
[00175] 13C NMR (100 MHz, CDC13) 6 171.8 (CO amide), 154.8 (CO carbamate),
154.5 (CO
carbonate), 151.6 (C arom u.-N02), 144.8 (C arom 6-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 u.-O), 50.9 (CH u.-NH), 46.4 47.0 (CH2 u.-N), 39.4 (CH2 Phe), 35.8 33.6
(CH3 NMe), 27.7
(3 CH3 Boc).
[00176] 2) Cyclization to Id-pl
[00177] 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 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.
CH2C12 is then
added and the organic phase was washed with IN NaHCO3, brine, dried over
Na2SO4 and
concentrated in vacuo. The residue (110 mg) was then purified by silica gel
chromatography.
[00178] [CHC13/MeOH/AcOH (20:0.5:0.1) then puis CHC13/MeOH [20:1]) to afford
42 mg of
Id-1.
[00179] HPLC tR (Id-1) 5.88 (gradient 30-100%B, 20 min)
[00180] HRMS (ESI) calculated for C13H16N203 249.1234, found 249.1230.
[00181] 'H NMR Id-1 (300 MHz, CDC13) 6 7.25 (m, 5H, arom-H), 6.10 (d, H4),
4.75 (dd, J=
8.9, 7.4 Hz, H5), 4.20 (m, 2H3), 4.15 (m, H2), 3.28 (dd, J= 14.0, 7.6 Hz,
1H6), 3.17 (m, H2'), 3.02
(dd, 1H6), 3.0 (s, 3H').
[00182] 13C NMR Id-1 (100 MHz, CDC13) 6 172.3 (CO amide), 157.7 (CO
carbonate), 136.9
(C-arom), 129.3 (2CH arom), 128.6 (2CH arom), 126.8 (CH arom), 69.6 (CH2 a-O),
54.0 (CH a-
N), 52.9 (CH2 a-N), 36.6 (CH3 Me), 35.7 (CH2 Phe).
[00183] Example 7: Synthesis of 1,1-Dioxo-1 k6 -[1,2,5,8] thiatriazocan-4 -
ones (Formula If)
33
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WO 2007/074169 PCT/EP2006/070256
F
F (If)
[00184] General synthetic scheme for If.
R4 0 R6 0 0 R4
Boc'N\,NJ /OH Me02C-N~s-N R1
R1 1 R2R"7 7
R ~N O
R6 R2
OO O
NEt3 S,NAOMe If
0 0
Burgess reagent
[00185] i) (a) TFA; (b) NaHCO3 satured, DCM; ii) Burgess reagent (2,5 eq),
THF, 70 C for
two hours.
[00186] Example 8: Synthesis of 10-methyl-6,6,11-trioxo-8,9,10,11,11 a, 12-
hexahydro-5H-
6? 6-thia-5a,7,10-triaza-cycloocta[b]naphthalene-7-carboxylic acid methyl
ester (Formula If-1)
(If-1)
[00187] General synthetic scheme for If-l.
R O
ii
XNN0H O`O
Me02C-N,S-N
~N O
R =Boc If-p1
R = H If-p2
If-1
O
NEt OSO ' OMe (i) (a) TFA, 0 C, 30min ; (b) NaHCO3 sature,
8 e DCM ; (ii) Burgess reagent (2,5 eq), THF, 70 C
Burgess reagent 2h.
[00188] i) The N-Boc protected dipeptide alcohol was treated with TFA for 30
minutes at
34
CA 02635354 2008-06-27
WO 2007/074169 PCT/EP2006/070256
0 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
Na2S04 and concentrated under vacuum to give If-pl.
[00189] ii) Compound if--pl (175 mg, 0.75 mmol, 1 eq), is dissolved in 10 mL
anhydrous
THE and Burgess reagent (534 mg, 2.24 mmol, 2.5 eq) is added. The solution is
then heated
under reflux at from about 70 C to about 90 C for 2 days. The reaction mixture
is then poured
into a solution of saturated NH4C1 (40 mL). The mixture is extracted with
CH2C12 and the
organic phase is washed with H20, dried over Na2SO4 and concentrated under
vacuum. The
crude mixture is then purified by silica gel chromatography (CHC13/MeOH/AcOH
(18:1:0.2) to
yield If-1.
CA 02635354 2009-08-04
TABLE I
Structure Mol Formula MoiWei ExactMass ClogP IogP
N
1 C12H15N3O2 233,2694 233.1164 1.13 0.47
HH 2 C12H15N3O2 233,2694 233.1164 1.13 0.47
3 C19H21N3O2 323,3938 323.1634 3.36 2.44
4 C18H25N3O4 347,4132 347.1845 3,24 1.11
C19H21N3O2 323,3938323.1634 3.07 2.48
p
o H
6 C14H17N3O2 259,3072 259.1321 1.31 0.78
~11H -
7 C14H17N3O2 259,3072 259.1321 1.31 0.78
8 C26H27N3O2 413,5182 413.2103 5.80 4.41
35a
CA 02635354 2009-08-04
Structure Mol Formula MolWel ExactMass CIogP IogP
9 C24H35N306 461,557 461.2526
C40H39N304 625,7658 625.2941 9.17 7.62
11 C14H19N302 261,323 261.1477 1.13 0.03
12 C10H17N302 211,2632 211.1321 1.25 0.08
13 C131-115N302 245,28 245.1164 1.07 0.50
14 C21H23N303 365,431 365.1739 2.94 2.23
/emu
M(, 15 C9H17N302 199,2522 199.1321 1.13 0.03
cop 16 C331-133N302 503,6426 503.2573 7.74 6.41
35b
CA 02635354 2009-08-04
Structure Mol Formula MolWel ExactMass ClogP logP
17 C21H224404 394,4292 394.1641
O
18 C11H19N302 225,29 225.1477 1.36 0.34
M N
19 C8H15N302 185,2254 85.1164 0.47 -0.32
20 C15H17N302 271,318 271.1321 1.46 0.72
21 C14H17N303 275,3066 275.1270 0.34 0.13
22 C20H34N604 422,5264 422.2642 3.37 0.16
23 C6H12N402 172,1864 172.0960 -1.24 -2.44
24 C14H17N304 291,306 291.1219 1.70 -0.03
35c
CA 02635354 2009-08-04
Structure Mol Formula MoiWel ExactMass CIogP logP
O,{y
~M fry
~~~III 25 C7H14N4O2 186,213 186.1117 -1.65 -2.33
26 C 19H25CIN4O 408,8839 408.1564 2.58 1.66
27 C18H19N302 309,367 309.1477 2.49 1.78
28 C21 H21 N3O4 379,4146 379.1532 2.69 1.72
29 C16H17N302 283,3292 283.1321 2.30 1.47
30 C17H20N4O6 376,368 376.1383 -0.6 -1.79
31 C13H15N3O2 245,2804 245.1164 1.28 0.41
32 C11H13N3O2 219,2426219.1008 0.85 0.19
35d
CA 02635354 2009-08-04
Structure Mol Formula MolWei ExactMass CIogP IogP
-~~ ql~ 33 C14H25N304 299,3692 299.1845 2.58 0.32
34 C15H21N302 275,3498 275.1634 2.69 1.65
35 C16H19N306 349,342 349.1274 2.63 -0.52
36 C20H27N304 373,451 373.2002 3.42 1.42
37 C26H37N306 487,5948 487.2682 5.88 2.06
38 C21H23N302 349,4316 349.1790 3.54 2.75
39 C28H29N302 439,556 439.2260 5.98 4.72
40 C22H27N304 397,473 397.2002 4.41 2.11
35e
CA 02635354 2009-08-04
Structure MCI Formula MolWei ExactMass CIogP IogP
*Y-( 41 C19H25N3O4 359,4242 359.1845 3.60 1.05
42 C17H23N3O4 333,3864 333.1689 3.11 0.83
43 C24H29N3O4 423,5108 423.2158 4.71 2.42
44 C18H26N4O3 W.4284346,2005 2.38 0.43
45 C15H19N3O4 305,3328 305.1376 1.95 0.24
p,~N
46 C14H19N3O3 277,3224 277.1426 1.247 0.19
47 C14H17N3O3 275,3066 275.1270 1.4848 -0.29
48 C20H27N3O4 373,451 373.2002 3.5758 1.80
35f
CA 02635354 2009-08-04
Structure Mot Formula MolWei ExactMass CIogP IogP
49 C16H19N3O4 317,3438 317.1376 1.8463 0.66
50 C14H18N4O4 306,320 306.1328 0.5532 -0.51
51 C20H29N3O4 375,4668 375.2158 3.7939 1.68
52 C16H21N3O4 319,3596 319.3556 1.9053 0.55
53 C16H22N4O3 318,374 318.1692 1.1233 -0.1
54 C14H18N4O3 290,3212 290.1379 0.8796 -0.68
35g
