U.S. patent application number 15/410874 was filed with the patent office on 2017-07-13 for benzodiazepine bromodomain inhibitor.
The applicant listed for this patent is GlaxoSmithKline LLC. Invention is credited to Romain Luc Marie Gosmini, Olivier Mirguet.
Application Number | 20170197972 15/410874 |
Document ID | / |
Family ID | 42667965 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170197972 |
Kind Code |
A1 |
Gosmini; Romain Luc Marie ;
et al. |
July 13, 2017 |
BENZODIAZEPINE BROMODOMAIN INHIBITOR
Abstract
The present invention relates to a benzodiazepine compound,
processes for its preparation, pharmaceutical compositions
containing such a compound and to its use in therapy.
Inventors: |
Gosmini; Romain Luc Marie;
(Les Ulis, FR) ; Mirguet; Olivier; (Les Ulis,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline LLC |
Wilmington |
DE |
US |
|
|
Family ID: |
42667965 |
Appl. No.: |
15/410874 |
Filed: |
January 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14684659 |
Apr 13, 2015 |
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15410874 |
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13505039 |
Apr 30, 2012 |
9023842 |
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PCT/EP2010/061518 |
Aug 6, 2010 |
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14684659 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 45/06 20130101; A61P 37/00 20180101; A61K 31/551 20130101;
A61P 35/00 20180101; Y02A 50/411 20180101; A61P 37/02 20180101;
C07D 487/04 20130101; A61K 31/5517 20130101; A61P 29/00 20180101;
A61P 43/00 20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 45/06 20060101 A61K045/06; A61K 31/551 20060101
A61K031/551 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2009 |
GB |
0919433.3 |
Jun 22, 2010 |
GB |
1010509.6 |
Claims
1. A compound of formula (I) which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide ##STR00033## or a salt
thereof.
2. A compound of formula (I) which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide ##STR00034## or a
pharmaceutically acceptable salt thereof.
3. A compound of formula (I) which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide ##STR00035##
4. A pharmaceutical composition which comprises a compound of
formula (I) or a pharmaceutically acceptable salt thereof as
defined in claim 2 and one or more pharmaceutically acceptable
carriers, diluents or excipients.
5. A pharmaceutical composition which comprises a compound of
formula (I) as defined in claim 3 and one or more pharmaceutically
acceptable carriers, diluents or excipients.
6. A combination pharmaceutical product comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof as
defined in claim 2 together with one or more other therapeutically
active agents.
7. A compound of formula (I) or a pharmaceutically acceptable salt
thereof as defined in claim 2 for use in therapy.
8. A compound of formula (I) as defined in claim 3 for use in
therapy.
9. A compound of formula (I) or a pharmaceutically acceptable salt
thereof as defined in claim 2, for use in the treatment of diseases
or conditions for which a bromodomain inhibitor is indicated.
10. A compound of formula (I) or a pharmaceutically acceptable salt
thereof for use according to claim 9, wherein the disease or
condition is a chronic autoimmune and/or inflammatory
condition.
11. A compound of formula (I) or a pharmaceutically acceptable salt
thereof for use according to claim 9, wherein the disease or
condition is cancer.
12. The use of a compound of formula (I) or a pharmaceutically
acceptable salt thereof as defined in claim 2, in the manufacture
of a medicament for the treatment of diseases or conditions for
which a bromodomain inhibitor is indicated.
13. A method for treatment of a disease or condition, for which a
bromodomain inhibitor is indicated, in a subject in need thereof
which comprises administering a therapeutically effective amount of
a compound of formula (I) or a pharmaceutically acceptable salt
thereof as defined in claim 2.
14. A method for treatment according to claim 13, wherein the
disease or condition is a chronic autoimmune and/or inflammatory
condition.
15. A method for treatment according to claim 13, wherein the
disease or condition is cancer.
16. A method for treatment according to claim 13, wherein the
subject is a human.
17. A method for inhibiting a bromodomain which comprises
contacting the bromodomain with a compound of formula (I) or a
pharmaceutically acceptable salt thereof as defined in claim 2.
18. A method for treatment according to claim 13, further
comprising administering a therapeutically effective amount of one
or more other therapeutically active agents.
19. A method for treatment according to claim 13, wherein the one
or more other therapeutically active agents is selected from
antibiotics, anti-virals, glucocorticosteroids, muscarinic
antagonists and beta-2 agonists.
20. A method for treatment according to claim 13, wherein the
compound of formula (I) and the one or more other therapeutically
active agents are administered sequentially.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is filed as a continuation application of
U.S. Ser. No. 14/684,659, filed Apr. 13, 2015, which is a
continuation application of U.S. Ser. No. 13/505,039, filed Apr.
30, 2012, which is a National Phase Application of International
Application No. PCT/EP2010/061518 filed Aug. 6, 2010, which claims
priority from GB 0919433.3 filed Nov. 5, 2009 and GB 1010509.6
filed Jun. 22, 2010.
FIELD OF THE INVENTION
[0002] The present invention relates to a benzodiazepine compound,
processes for its preparation, pharmaceutical compositions
containing such a compound and to its use in therapy.
BACKGROUND OF THE INVENTION
[0003] The genomes of eukaryotic organisms are highly organised
within the nucleus of the cell. The long strands of duplex DNA are
wrapped around an octomer of histone proteins (most usually
comprising two copies of histones H2A, H2B H3 and H4) to form a
nuclesome. This basic unit is then further compressed by the
aggregation and folding of nucleosomes to form a highly condensed
chromatin structure. A range of different states of condensation
are possible, and the tightness of this structure varies during the
cell cycle, being most compact during the process of cell division.
Chromatin structure plays a critical role in regulating gene
transcription, which cannot occur efficiently from highly condensed
chromatin. The chromatin structure is controlled by a series of
post translational modifications to histone proteins, notably
histones H3 and H4, and most commonly within the histone tails
which extend beyond the core nucleosome structure. These
modifications include acetylation, methylation, phosphorylation,
ubiquitinylation and SUMOylation. These epigenetic marks are
written and erased by specific enzymes, which place the tags on
specific residues within the histone tail, thereby forming an
epigenetic code, which is then interpreted by the cell to allow
gene specific regulation of chromatin structure and thereby
transcription.
[0004] Histone acetylation is most usually associated with the
activation of gene transcription, as the modification loosens the
interaction of the DNA and the histone octomer by changing the
electrostatics. In addition to this physical change, specific
proteins bind to acetylated lysine residues within histones to read
the epigenetic code. Bromodomains are small (.about.110 amino acid)
distinct domains within proteins that bind to acetylated lysine
resides commonly but not exclusively in the context of histones.
There is a family of around 50 proteins known to contain
bromodomains, and they have a range of functions within the
cell.
[0005] The Bet family of bromodomain containing proteins comprises
4 proteins (BRD2, BRD3, BRD4 and BRD-t) which contain tandem
bromodomains capable of binding to two acetylated lysine resides in
close proximity, increasing the specificity of the interaction.
BRD2 and BRD3 are reported to associate with histones along
actively transcribed genes and may be involved in facilitating
transcriptional elongation (Leroy et al, Mol. Cell. 2008
30(1):51-60), while BRD4 appears to be involved in the recruitment
of the pTEF-B complex to inducible genes, resulting in
phosphorylation of RNA polymerase and increased transcriptional
output (Hargreaves et al, Cell, 2009 138(1): 129-145). It has also
been reported that BRD4 and BRD3 fuse with NUT (nuclear protein in
testis) forming a novel fusion oncogene, BRD4-NUT, in a highly
malignant form of epithelial neoplasia (French et al. Cancer
Research, 2003, 63, 304-307 and French et al. Journal of Clinical
Oncology, 2004, 22 (20), 4135-4139). Data suggests that BRD-NUT
fusion proteins contribute to carcinogensesis (Oncogene, 2008, 27,
2237-2242). BRD-t is uniquely expressed in the testes and ovary.
All family members have been reported to have some function in
controlling or executing aspects of the cell cycle, and have been
shown to remain in complex with chromosomes during cell
division--suggesting a role in the maintenance of epigenetic
memory. In addition some viruses make use of these proteins to
tether their genomes to the host cell chromatin, as part of the
process of viral replication (You et al Cell, 2004
117(3):349-60).
[0006] Japanese patent application JP2008-156311 discloses a
benzimidazole derivative which is said to be a BRD2 bromodomain
binding agent which has utility with respect to virus
infection/proliferation.
[0007] Patent application WO2009/084693A1 discloses a series of
thienotriazolodiazepiene derivatives that are said to inhibit the
binding between an acetylated histone and a bromodomain containg
protein which are said to be useful as anti-cancer agents.
[0008] A compound has been found which inhibits the binding of
bromodomains with its cognate acetylated proteins, more
particularly that inhibits the binding of Bet family bromodomains
to acetylated lysine residues. Such a compound will hereafter be
referred to as a "bromodomain inhibitor".
SUMMARY OF THE INVENTION
[0009] In a first aspect of the present invention, there is
provided a compound of formula (I) or a salt thereof
##STR00001##
[0010] In a second aspect of the present invention, there is
provided a pharmaceutical composition comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof and one
or more pharmaceutically acceptable carriers, diluents or
excipients.
[0011] In a third aspect of the present invention, there is
provided a compound of formula (I), or a pharmaceutically
acceptable salt thereof for use in therapy, in particular in the
treatment of diseases or conditions for which a bromodomain
inhibitor is indicated.
[0012] In a fourth aspect of the present invention, there is
provided a method of treating diseases or conditions for which a
bromodomain inhibitor is indicated in a subject in need thereof
which comprises administering a therapeutically effective amount of
compound of formula (I) or a pharmaceutically acceptable salt
thereof.
[0013] In a fifth aspect of the present invention, there is
provided the use of a compound of formula (I), or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for the treatment of diseases or conditions for which a
bromodomain inhibitor is indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention relates to a compound of formula (I)
which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide
##STR00002##
[0015] or a salt thereof.
[0016] It will be appreciated that the present invention covers
compounds of formula (I) as the free base and as salts thereof, for
example as a pharmaceutically acceptable salt thereof.
[0017] In one embodiment there is provided a compound which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide.
[0018] Because of their potential use in medicine, salts of the
compounds of formula (I) are desirably pharmaceutically acceptable.
In another embodiment there is provided a compound which is
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3--
a][1,4]benzodiazepin-4-yl]-N-ethylacetamide or a pharmaceutically
acceptable salt thereof.
[0019] Suitable pharmaceutically acceptable salts can include acid
or base addition salts. For a review on suitable salts see Berge et
al., J. Pharm. Sci., 66:1-19, (1977). Typically, a pharmaceutically
acceptable salt may be readily prepared by using a desired acid or
base as appropriate. The resultant salt may precipitate from
solution and be collected by filtration or may be recovered by
evaporation of the solvent.
[0020] A pharmaceutically acceptable base addition salt can be
formed by reaction of a compound of formula (I) with a suitable
inorganic or organic base, (e.g. triethylamine, ethanolamine,
triethanolamine, choline, arginine, lysine or histidine),
optionally in a suitable solvent, to give the base addition salt
which is usually isolated, for example, by crystallisation and
filtration. Pharmaceutically acceptable base salts include ammonium
salts, alkali metal salts such as those of sodium and potassium,
alkaline earth metal salts such as those of calcium and magnesium
and salts with organic bases, including salts of primary, secondary
and tertiary amines, such as isopropylamine, diethylamine,
ethanolamine, trimethylamine, dicyclohexyl amine and
N-methyl-D-glucamine.
[0021] A pharmaceutically acceptable acid addition salt can be
formed by reaction of a compound of formula (I) with a suitable
inorganic or organic acid (such as hydrobromic, hydrochloric,
sulphuric, nitric, phosphoric, succinic, maleic, acetic, propionic,
fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamaic,
aspartic, p-toluenesulfonic, benzenesulfonic, methanesulfonic,
ethanesulfonic, naphthalenesulfonic such as 2-naphthalenesulfonic,
or hexanoic acid), optionally in a suitable solvent such as an
organic solvent, to give the salt which is usually isolated, for
example, by crystallisation and filtration. A pharmaceutically
acceptable acid addition salt of a compound of formula (I) can
comprise or be, for example, a hydrobromide, hydrochloride,
sulfate, nitrate, phosphate, succinate, maleate, acetate,
propionate, fumarate, citrate, tartrate, lactate, benzoate,
salicylate, glutamate, aspartate, p-toluenesulfonate,
benzenesulfonate, methanesulfonate, ethanesulfonate,
naphthalenesulfonate (e.g. 2-naphthalenesulfonate) or hexanoate
salt.
[0022] Other non-pharmaceutically acceptable salts, e.g. formates,
oxalates or trifluoroacetates, may be used, for example, in the
isolation of the compound of formula (I), and are included within
the scope of this invention.
[0023] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the salts of the
compound of formula (I).
[0024] It will be appreciated that many organic compounds can form
complexes with solvents in which they are reacted or from which
they are precipitated or crystallized. These complexes are known as
"solvates". For example, a complex with water is known as a
"hydrate". Solvents with high boiling points and/or capable of
forming hydrogen bonds such as water, xylene, N-methyl
pyrrolidinone, methanol and ethanol may be used to form solvates.
Methods for identification of solvates include, but are not limited
to, NMR and microanalysis. Solvates of the compound of formula (I)
are within the scope of the invention.
[0025] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the solvates of the
compound of formula (I).
[0026] The invention encompasses all prodrugs, of the compound of
formula (I) and pharmaceutically acceptable salts thereof, which
upon administration to the recipient are capable of providing
(directly or indirectly) a compound of formula (I) or a
pharmaceutically acceptable salt thereof, or an active metabolite
or residue thereof.
[0027] Such derivatives are recognizable to those skilled in the
art, without undue experimentation. Nevertheless, reference is made
to the teaching of Burger's Medicinal Chemistry and Drug Discovery,
5.sup.th Edition, Vol 1: Principles and Practice, which is
incorporated herein by reference to the extent of teaching such
derivatives.
[0028] The compound of formula (I) may be in crystalline or
amorphous form. Furthermore, some of the crystalline forms of the
compound of formula (I) may exist as polymorphs, which are included
within the scope of the present invention. Polymorphic forms of the
compound of formula (I) may be characterized and differentiated
using a number of conventional analytical techniques, including,
but not limited to, X-ray powder diffraction (XRPD) patterns,
infrared (IR) spectra, Raman spectra, differential scanning
calorimetry (DSC), thermogravimetric analysis (TGA) and solid state
nuclear magnetic resonance (SSNMR).
[0029] The compound of formula (I) is an individual isomer isolated
such as to be substantially free of the other isomer (i.e.
enantionerically pure) such that less than 10%, particularly less
than about 1%, for example less than about 0.1% of the other
enantiomer is present.
[0030] Separation of isomers may be achieved by conventional
techniques known to those skilled in the art, e.g. by fractional
crystallisation, chromatography or HPLC.
[0031] The compound of formula (I) may exist in one of several
tautomeric forms. It will be understood that the present invention
encompasses all tautomers of the compound of formula (I) whether as
individual tautomers or as mixtures thereof.
[0032] It will be appreciated from the foregoing that included
within the scope of the invention are solvates, isomers and
polymorphic forms of the compound of formula (I) and salts
thereof.
[0033] The compound of formula (I) and pharmaceutically acceptable
salts thereof may be made by a variety of methods, including
standard chemistry. Illustrative general synthetic methods are set
out below and then the specific compound of formula (I) is prepared
in the working Examples. These processes form further aspects of
the present invention.
[0034] The compound of formula (I) may be prepared according to
reaction scheme 1 by reaction of a compound of formula (II) with
EtNH.sub.2 in the presence of HATU or HBTU and DIEA at room
temperature. Alternatively compounds of formula (I) may be prepared
by reacting the compound of formula (II) with oxalyl chloride
followed by addition of EtNH.sub.2 in the presence of
triethylamine.
##STR00003##
[0035] The compound of formula (II) may be prepared according to
reaction Scheme 2. Suitable reaction conditions comprise reacting a
compound of formula (III) with alkaline hydroxide preferably sodium
hydroxide or lithium hydroxide.
##STR00004##
[0036] wherein R represents C.sub.1-6alkyl such as methyl.
[0037] Compounds of formula (III), may be prepared according to
reaction scheme 3 by reacting compounds of formula (IV) with
AcOH.
##STR00005##
[0038] Compounds of formula (IV) may be prepared according to
reaction scheme 4 by reacting compounds of formula (VI) with
hydrazine below 15.degree. C. followed by reaction of the resulting
hydrazone (V) with MeCOCl at 0.degree. C. Generally hydrazone (V)
is used without further purification and is reacted with MeCOCl at,
for example 0.degree. C.
##STR00006##
[0039] Compounds of formula (VI) in which R is C.sub.1-6alkyl (such
as methyl) may be prepared according to reaction scheme 5 from
compounds of formula (VII) by treatment with Lawesson's reagent or
P.sub.4S.sub.10. Suitable reaction conditions comprise reacting
compounds of formula (VIII) with P.sub.4S.sub.10 in
1,2-dichloroethane at, for example 70.degree. C.
##STR00007##
[0040] Compounds of formula (VII) may be prepared according to
reaction scheme 6, by reacting compounds of formula (IX) with an
organic base such as triethylamine followed by reaction of the
resulting amine (VIII) with acetic acid. Generally, amine (VIII) is
used without further purification and is reacted with AcOH at, for
example 60.degree. C.
##STR00008##
[0041] Compounds of formula (IX) may be prepared according to
reaction scheme 7, by reacting compounds of formula (XI) with the
acylchloride (X) derived from protected aspartic acid.
##STR00009##
[0042] Compounds of formula (XI) may be prepared according to
procedures described in Synthesis 1980, 677-688. Acyl chlorides of
formula (X) may be prepared according to procedures described in J.
Org. Chem., 1990, 55, 3068-3074 and J. Chem. Soc. Perkin Trans. 1,
2001, 1673-1695.
[0043] Alternatively the compound of formula (I) may be prepared
according to reaction scheme 8.
##STR00010##
[0044] wherein R represents C.sub.1-4alkyl such as methyl.
[0045] The compound of formula (IIIA) may be prepared according to
reaction scheme 9 by reacting compounds of formula (IVA) with
EtNH.sub.2 in the presence of HATU and DIEA at, for example room
temperature.
##STR00011##
[0046] The compound of formula (IVA) may be prepared according to
reaction scheme 10. Suitable reaction conditions comprise reacting
compounds of formula (VI) with alkaline hydroxide such as sodium
hydroxide.
##STR00012##
[0047] It will be appreciated by those skilled in the art that it
may be advantageous to protect one or more functional groups of the
compounds described in the above processes. Examples of protecting
groups and the means for their removal can be found in T. W. Greene
`Protective Groups in Organic Synthesis` (4th edition, J. Wiley and
Sons, 2006). Suitable amine protecting groups include acyl (e.g.
acetyl, carbamate (e.g. 2',2',2'-trichloroethoxycarbonyl,
benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl),
which may be removed by hydrolysis (e.g. using an acid such as
hydrochloric acid in dioxane or trifluoroacetic acid in
dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl or
benzyloxycarbonyl group or reductive removal of a
2',2',2'-trichloroethoxycarbonyl group using zinc in acetic acid)
as appropriate. Other suitable amine protecting groups include
trifluoroacetyl (--COCF.sub.3) which may be removed by base
catalysed hydrolysis.
[0048] It will be appreciated that in any of the routes described
above, the precise order of the synthetic steps by which the
various groups and moieties are introduced into the molecule may be
varied. It will be within the skill of the practitioner in the art
to ensure that groups or moieties introduced at one stage of the
process will not be affected by subsequent transformations and
reactions, and to select the order of synthetic steps
accordingly.
[0049] Certain intermediate compounds described above are believed
to be novel and therefore form a yet further aspect of the
invention.
[0050] The compound of formula (I) and salts thereof is a
bromodomain inhibitor, and thus is believed to have potential
utility in the treatment of diseases or conditions for which a
bromodomain is indicated.
[0051] The present invention thus provides a compound of formula
(I) or a pharmaceutically acceptable salt thereof for use in
therapy. The compound of formula (I) or pharmaceutically salt
thereof can be for use in the treatment of diseases or conditions
for which a bromodomain inhibitor indicated.
[0052] In one embodiment there is provided a compound of formula
(I) or a pharmaceutically acceptable salt thereof for use in the
treatment of diseases or conditions for which a bromodomain is
indicated. In another embodiment, there is provided a compound or a
pharmaceutically acceptable salt thereof for use in the treatment
of a chronic autoimmune and/or inflammatory condition. In a further
embodiment, there is provided a compound or a pharmaceutically
acceptable salt thereof for use in the treatment of cancer, such as
midline carcinoma.
[0053] In one embodiment there is provided the use of a compound of
formula (I) or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament for the treatment of diseases or
conditions for which a bromodomain inhibitor is indicated. In
another embodiment, there is provided the use of a compound of
formula (I) or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament for the treatment of a chronic
autoimmune and/or inflammatory condition. In a further embodiment,
there is provided the use of a compound of formula (I) or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for the treatment of cancer, such as midline
carcinoma.
[0054] In one embodiment there is provided a method for treatment
of a disease or condition, for which a bromodomain inhibitor is
indicated, in a subject in need thereof which comprises
administering a therapeutically effective amount of compound of
formula (I) or a pharmaceutically acceptable salt thereof. In
another embodiment there is provided a method for treatment of a
chronic autoimmune and/or inflammatory condition, in a subject in
need thereof which comprises administering a therapeutically
effective amount of compound of formula (I) or a pharmaceutically
acceptable salt thereof. In a further embodiment there is provided
a method for treatment of cancer, such as midline carcinoma, in a
subject in need thereof which comprises administering a
therapeutically effective amount of compound of formula (I) or a
pharmaceutically acceptable salt thereof.
[0055] In one embodiment the subject in need thereof is a mammal,
particularly a human. As used herein, the term "effective amount"
means that amount of a drug or pharmaceutical agent that will
elicit the biological or medical response of a tissue, system,
animal or human that is being sought, for instance, by a researcher
or clinician. Furthermore, the term "therapeutically effective
amount" means any amount which, as compared to a corresponding
subject who has not received such amount, results in improved
treatment, healing, prevention, or amelioration of a disease,
disorder, or side effect, or a decrease in the rate of advancement
of a disease or disorder. The term also includes within its scope
amounts effective to enhance normal physiological function.
[0056] Bromodomain inhibitors are believed to be useful in the
treatment of a variety of diseases or conditions related to
systemic or tissue inflammation, inflammatory responses to
infection or hypoxia, cellular activation and proliferation, lipid
metabolism, fibrosis and in the prevention and treatment of viral
infections.
[0057] Bromodomain inhibitors may be useful in the treatment of a
wide variety of chronic autoimmune and inflammatory conditions such
as rheumatoid arthritis, osteoarthritis, acute gout, psoriasis,
systemic lupus erythematosus, multiple sclerosis, inflammatory
bowel disease (Crohn's disease and Ulcerative colitis), asthma,
chronic obstructive airways disease, pneumonitis, myocarditis,
pericarditis, myositis, eczema, dermatitis, alopecia, vitiligo,
bullous skin diseases, nephritis, vasculitis, atherosclerosis,
Alzheimer's disease, depression, retinitis, uveitis, scleritis,
hepatitis, pancreatitis, primary biliary cirrhosis, sclerosing
cholangitis, Addison's disease, hypophysitis, thyroiditis, type I
diabetes and acute rejection of transplanted organs.
[0058] Bromodomain inhibitors may be useful in the treatment of a
wide variety of acute inflammatory conditions such as acute gout,
giant cell arteritis, nephritis including lupus nephritis,
vasculitis with organ involvement such as glomerulonephritis,
vasculitis including giant cell arteritis, Wegener's
granulomatosis, Polyarteritis nodosa, Behcet's disease, Kawasaki
disease, Takayasu's Arteritis and acute rejection of transplanted
organs.
[0059] Bromodomain inhibitors may be useful in the prevention or
treatment of diseases or conditions which involve inflammatory
responses to infections with bacteria, viruses, fungi, parasites or
their toxins, such as sepsis, sepsis syndrome, septic shock,
endotoxaemia, systemic inflammatory response syndrome (SIRS),
multi-organ dysfunction syndrome, toxic shock syndrome, acute lung
injury, ARDS (adult respiratory distress syndrome), acute renal
failure, fulminant hepatitis, burns, acute pancreatitis,
post-surgical syndromes, sarcoidosis, Herxheimer reactions,
encephalitis, myelitis, meningitis, malaria, SIRS associated with
viral infections such as influenza, herpes zoster, herpes simplex
and coronavirus.
[0060] Bromodomain inhibitors may be useful in the prevention or
treatment of conditions associated with ischaemia-reperfusion
injury such as myocardial infarction, cerebro-vascular ischaemia
(stroke), acute coronary syndromes, renal reperfusion injury, organ
transplantation, coronary artery bypass grafting, cardio-pulmonary
bypass procedures and pulmonary, renal, hepatic, gastro-intestinal
or peripheral limb embolism.
[0061] Bromodomain inhibitors may be useful in the treatment of
disorders of lipid metabolism via the regulation of APO-A1 such as
hypercholesterolemia, atherosclerosis and Alzheimer's disease.
[0062] Bromodomain inhibitors may be useful in the treatment of
fibrotic conditions such as idiopathic pulmonary fibrosis, renal
fibrosis, post-operative stricture, keloid formation, scleroderma
and cardiac fibrosis.
[0063] Bromodomain inhibitors may be useful in the prevention and
treatment of viral infections such as herpes virus, human papilloma
virus, adenovirus, poxvirus and other DNA viruses.
[0064] Bromodomain inhibitors may be useful in the treatment of
cancer, including hematological (such as leukaemia), epithelial
including lung, breast and colon carcinomas, midline carcinomas,
mesenchymal, hepatic, renal and neurological tumours.
[0065] Bromodomain inhibitors may be useful in the treatment of
treatment of ophthamological indications such as dry eye.
[0066] In one embodiment the disease or condition for which a
bromodomain inhibitor is indicated is selected from diseases
associated with systemic inflammatory response syndrome, such as
sepsis, burns, pancreatitis, major trauma, haemorrhage and
ischaemia. In this embodiment the bromodomain inhibitor would be
administered at the point of diagnosis to reduce the incidence of:
SIRS, the onset of shock, multi-organ dysfunction syndrome, which
includes the onset of acute lung injury, ARDS, acute renal,
hepatic, cardiac and gastro-intestinal injury and mortality. In
another embodiment the bromodomain inhibitor would be administered
prior to surgical or other procedures associated with a high risk
of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS. In a
particular embodiment the disease or condition for which a
bromodomain inhibitor is indicated is sepsis, sepsis syndrome,
septic shock and/or endotoxaemia. In another embodiment, the
bromodomain inhibitor is indicated for the treatment of acute or
chronic pancreatitis. In another embodiment the bromodomain is
indicated for the treatment of burns.
[0067] In one embodiment the disease or condition for which a
bromodomain inhibitor is indicated is selected from herpes simplex
infections and reactivations, cold sores, herpes zoster infections
and reactivations, chickenpox, shingles, human papilloma virus,
cervical neoplasia, adenovirus infections, including acute
respiratory disease, and poxvirus infections such as cowpox and
smallpox and African swine fever virus. In one particular
embodiment a bromodomain inhibitor is indicated for the treatment
of Human papilloma virus infections of skin or cervical
epithelia.
[0068] The term "diseases or conditions for which a bromodomain
inhibitor is indicated", is intended to include any or all of the
above disease states.
[0069] While it is possible that for use in therapy, a compound of
formula (I) as well as pharmaceutically acceptable salts thereof
may be administered as the raw chemical, it is common to present
the active ingredient as a pharmaceutical composition.
[0070] The present invention therefore provides in a further aspect
a pharmaceutical composition comprising a compound of formula (I)
or a pharmaceutically acceptable salt thereof and one or more
pharmaceutically acceptable carriers, diluents or excipients.
[0071] Thus there is provided a pharmaceutical composition for the
treatment of diseases or conditions in which a bromodomain
inhibitor is indicated comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof.
[0072] The carrier(s), diluent(s) or excipient(s) used in such
pharmaceutical compositions must be acceptable in the sense of
being compatible with the other ingredients of the composition and
not deleterious to the recipient thereof. In accordance with
another aspect of the invention there is also provided a process
for the preparation of a pharmaceutical composition including
admixing a compound of formula (I), or a pharmaceutically
acceptable salt thereof, with one or more pharmaceutically
acceptable carriers, diluents or excipients. The pharmaceutical
composition can be for use in the treatment of any of the
conditions described herein.
[0073] Since the compound of formula (I) and pharmaceutically
acceptable salts thereof are intended for use in pharmaceutical
compositions it will be readily understood that they are each
preferably provided in substantially pure form, for example, at
least 60% pure, more suitably at least 75% pure and preferably at
least 85% pure, especially at least 98% pure (% in a weight for
weight basis).
[0074] Pharmaceutical compositions may be presented in unit dose
forms containing a predetermined amount of active ingredient per
unit dose. Preferred unit dosage compositions are those containing
a daily dose or sub-dose, or an appropriate fraction thereof, of an
active ingredient. Such unit doses may therefore be administered
more than once a day. Preferred unit dosage compositions are those
containing a daily dose or sub-dose (for administration more than
once a day), as herein above recited, or an appropriate fraction
thereof, of an active ingredient.
[0075] Pharmaceutical compositions may be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), rectal, inhaled, intranasal,
topical (including buccal, sublingual or transdermal), vaginal or
parenteral (including subcutaneous, intramuscular, intravenous or
intradermal) route. Such compositions may be prepared by any method
known in the art of pharmacy, for example by bringing into
association the active ingredient with the carrier(s) or
excipient(s).
[0076] In one embodiment the pharmaceutical composition is adapted
for oral administration.
[0077] In one embodiment the pharmaceutical composition is adapted
for parenteral administration, particularly intravenous
administration.
[0078] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the composition isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The compositions may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0079] Pharmaceutical compositions adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions in aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil liquid emulsions.
[0080] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Powders suitable for
incorporating into tablets or capsules may be prepared by reducing
the compound to a suitable fine size (e.g. by micronisation) and
mixing with a similarly prepared pharmaceutical carrier such as an
edible carbohydrate, as, for example, starch or mannitol.
Flavoring, preservative, dispersing and coloring agent can also be
present.
[0081] Capsules may be made by preparing a powder mixture, as
described above, and filling formed gelatin sheaths. Glidants and
lubricants such as colloidal silica, talc, magnesium stearate,
calcium stearate or solid polyethylene glycol can be added to the
powder mixture before the filling operation. A disintegrating or
solubilizing agent such as agar-agar, calcium carbonate or sodium
carbonate can also be added to improve the availability of the
medicament when the capsule is ingested.
[0082] Moreover, when desired or necessary, suitable binders,
glidants, lubricants, sweetening agents, flavours, disintegrating
agents and coloring agents can also be incorporated into the
mixture. Suitable binders include starch, gelatin, natural sugars
such as glucose or beta-lactose, corn sweeteners, natural and
synthetic gums such as acacia, tragacanth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include sodium oleate, sodium
stearate, magnesium stearate, sodium benzoate, sodium acetate,
sodium chloride and the like. Disintegrators include, without
limitation, starch, methyl cellulose, agar, bentonite, xanthan gum
and the like. Tablets are formulated, for example, by preparing a
powder mixture, granulating or slugging, adding a lubricant and
disintegrant and pressing into tablets. A powder mixture is
prepared by mixing the compound, suitably comminuted, with a
diluent or base as described above, and optionally, with a binder
such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl
pyrrolidone, a solution retardant such as paraffin, a resorption
accelerator such as a quaternary salt and/or an absorption agent
such as bentonite, kaolin or dicalcium phosphate. The powder
mixture can be granulated by wetting with a binder such as syrup,
starch paste, acadia mucilage or solutions of cellulosic or
polymeric materials and forcing through a screen. As an alternative
to granulating, the powder mixture can be run through the tablet
machine and the result is imperfectly formed slugs broken into
granules. The granules can be lubricated to prevent sticking to the
tablet forming dies by means of the addition of stearic acid, a
stearate salt, talc or mineral oil. The lubricated mixture is then
compressed into tablets. The compounds of the present invention can
also be combined with a free flowing inert carrier and compressed
into tablets directly without going through the granulating or
slugging steps. A clear or opaque protective coating consisting of
a sealing coat of shellac, a coating of sugar or polymeric material
and a polish coating of wax can be provided. Dyestuffs can be added
to these coatings to distinguish different unit dosages.
[0083] Oral fluids such as solution, syrups and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a non-toxic alcoholic
vehicle. Suspensions can be formulated by dispersing the compound
in a non-toxic vehicle. Solubilizers and emulsifiers such as
ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol
ethers, preservatives, flavor additive such as peppermint oil or
natural sweeteners or saccharin or other artificial sweeteners, and
the like can also be added.
[0084] Where appropriate, dosage unit compositions for oral
administration can be microencapsulated. The formulation can also
be prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax or the
like.
[0085] The compounds of formula (I) and pharmaceutically acceptable
salts thereof can also be administered in the form of liposome
delivery systems, such as small unilamellar vesicles, large
unilamellar vesicles and multilamellar vesicles. Liposomes can be
formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines.
[0086] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils.
[0087] For treatments of the eye or other external tissues, for
example mouth and skin, the compositions are preferably applied as
a topical ointment or cream. When formulated in an ointment, the
active ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base.
[0088] Pharmaceutical compositions adapted for topical
administrations to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent.
[0089] Dosage forms for nasal or inhaled administration may
conveniently be formulated as aerosols, solutions, suspensions,
gels or dry powders.
[0090] For compositions suitable and/or adapted for inhaled
administration, it is preferred that the compound of formula (I) or
pharmaceutically acceptable salt thereof is in a
particle-size-reduced form e.g. obtained by micronisation. The
preferable particle size of the size- reduced (e.g. micronised)
compound or salt is defined by a D50 value of about 0.5 to about 10
microns (for example as measured using laser diffraction).
[0091] Aerosol formulations, e.g. for inhaled administration, can
comprise a solution or fine suspension of the active substance in a
pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol
formulations can be presented in single or multidose quantities in
sterile form in a sealed container, which can take the form of a
cartridge or refill for use with an atomising device or inhaler.
Alternatively the sealed container may be a unitary dispensing
device such as a single dose nasal inhaler or an aerosol dispenser
fitted with a metering valve (metered dose inhaler) which is
intended for disposal once the contents of the container have been
exhausted.
[0092] Where the dosage form comprises an aerosol dispenser, it
preferably contains a suitable propellant under pressure such as
compressed air, carbon dioxide or an organic propellant such as a
hydrofluorocarbon (HFC). Suitable HFC propellants include
1,1,1,2,3,3,3-heptafluoropropane and 1,1,1,2-tetrafluoroethane. The
aerosol dosage forms can also take the form of a pump-atomiser. The
pressurised aerosol may contain a solution or a suspension of the
active compound. This may require the incorporation of additional
excipients e.g. co-solvents and/or surfactants to improve the
dispersion characteristics and homogeneity of suspension
formulations. Solution formulations may also require the addition
of co-solvents such as ethanol.
[0093] For pharmaceutical compositions suitable and/or adapted for
inhaled administration, the pharmaceutical composition may be a dry
powder inhalable composition. Such a composition can comprise a
powder base such as lactose, glucose, trehalose, mannitol or
starch, the compound of formula (I) or a pharmaceutically
acceptable salt thereof (preferably in particle-size-reduced form,
e.g. in micronised form), and optionally a performance modifier
such as L-leucine or another amino acid and/or metals salts of
stearic acid such as magnesium or calcium stearate. Preferably, the
dry powder inhalable composition comprises a dry powder blend of
lactose e.g. lactose monohydrate and the compound of formula (I) or
a pharmaceutically acceptable salt thereof. Such compositions can
be administered to the patient using a suitable device such as the
DISKUS.RTM. device, marketed by GlaxoSmithKline which is for
example described in GB 2242134 A.
[0094] The compound of formula (I) and pharmaceutically acceptable
salts thereof thereof may be formulated as a fluid formulation for
delivery from a fluid dispenser, for example a fluid dispenser
having a dispensing nozzle or dispensing orifice through which a
metered dose of the fluid formulation is dispensed upon the
application of a user-applied force to a pump mechanism of the
fluid dispenser. Such fluid dispensers are generally provided with
a reservoir of multiple metered doses of the fluid formulation, the
doses being dispensable upon sequential pump actuations. The
dispensing nozzle or orifice may be configured for insertion into
the nostrils of the user for spray dispensing of the fluid
formulation into the nasal cavity. A fluid dispenser of the
aforementioned type is described and illustrated in WO2005/044354
A1.
[0095] A therapeutically effective amount of a compound of formula
(I) or pharmaceutically acceptable salt thereof will depend upon a
number of factors including, for example, the age and weight of the
animal, the precise condition requiring treatment and its severity,
the nature of the formulation, and the route of administration, and
will ultimately be at the discretion of the attendant physician or
veterinarian. In the pharmaceutical composition, each dosage unit
for oral or parenteral administration preferably contains from 0.01
to 3000 mg, more preferably 0.5 to 1000 mg, of a compound of
formula (I) or pharmaceutically acceptable salt thereof calculated
as the free base. Each dosage unit for nasal or inhaled
administration preferably contains from 0.001 to 50 mg, more
preferably 0.01 to 5 mg, of a compound of the formula (I) or a
pharmaceutically acceptable salt thereof, calculated as the free
base.
[0096] The compound of formula (I) and pharmaceutically acceptable
salts thereof can be administered in a daily dose (for an adult
patient) of, for example, an oral or parenteral dose of 0.01 mg to
3000 mg per day or 0.5 to 1000 mg per day, or a nasal or inhaled
dose of 0.001 to 50 mg per day or 0.01 to 5 mg per day, of the
compound of formula (I) or a pharmaceutically acceptable salt
thereof, calculated as the free base. This amount may be given in a
single dose per day or more usually in a number (such as two,
three, four, five or six) of sub-doses per day such that the total
daily dose is the same. An effective amount of a pharmaceutically
acceptable salt thereof, may be determined as a proportion of the
effective amount of the compound of formula (I) per se.
[0097] Thus there is provided a pharmaceutical composition
comprising a) 0.01 to 3000 mg of a compound of formula (I) or a
pharmaceutically acceptable salt thereof, and (b) 0.1 to 2 g of one
or more pharamceutically acceptable carriers, diluents and/or
excipients.
[0098] The compound of formula (I) and pharmaceutically acceptable
salts thereof may be employed alone or in combination with other
therapeutic agents. Combination therapies according to the present
invention thus comprise the administration of at least one compound
of formula (I) or a pharmaceutically acceptable salt thereof, and
the use of at least one other pharmaceutically active agent.
Preferably, combination therapies according to the present
invention comprise the administration of at least one compound of
formula (I) or a pharmaceutically acceptable salt thereof, and at
least one other pharmaceutically active agent. The compound of
formula (I) and pharmaceutically acceptable salts thereof, and the
other pharmaceutically active agent(s) may be administered together
in a single pharmaceutical composition or separately and, when
administered separately this may occur simultaneously or
sequentially in any order. The amounts of the compound of formula
(I) and pharmaceutically acceptable salts thereof, and the other
pharmaceutically active agent(s) and the relative timings of
administration will be selected in order to achieve the desired
combined therapeutic effect. Thus in a further aspect, there is
provided a combination comprising a compound of formula (I) or
pharmaceutically acceptable salt thereof and at least one other
pharmaceutically active agent. In one embodiment there is provided
a combination pharmaceutical product comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof together
with one or more other therapeutically active agents.
[0099] Thus in one aspect, the compound of formula (I) and
pharmaceutical compositions according to the invention may be used
in combination with or include one or more other therapeutic
agents, for example selected from antibiotics, anti-virals,
glucocorticosteroids, muscarinic antagonists and beta-2
agonists.
[0100] It will be appreciated that when the compound of formula (I)
and pharmaceutically acceptable sdalt thereof the are administered
in combination with other therapeutic agents normally administered
by the inhaled, intravenous, oral or intranasal route, that the
resultant pharmaceutical composition may be administered by the
same routes. Alternatively the individual components of the
composition may be administered by different routes.
[0101] One embodiment of the invention encompasses combinations
comprising one or two other therapeutic agents.
[0102] It will be clear to a person skilled in the art that, where
appropriate, the other therapeutic ingredient(s) may be used in the
form of salts, for example as alkali metal or amine salts or as
acid addition salts, or prodrugs, or as esters, for example lower
alkyl esters, or as solvates, for example hydrates, to optimise the
activity and/or stability and/or physical characteristics, such as
solubility, of the therapeutic ingredient. It will be clear also
that, where appropriate, the therapeutic ingredients may be used in
optically pure form.
[0103] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical composition and
thus pharmaceutical compositions comprising a combination as
defined above together with a pharmaceutically acceptable diluent
or carrier represent a further aspect of the invention.
[0104] The compound of formula (I) may be prepared by the methods
described below or by similar methods. Thus the following
Intermediates and Examples serve to illustrate the preparation of
the compound of formula (I) and is not to be considered as limiting
the scope of the invention in any way.
General Experimental Details
[0105] All temperatures referred to are in .degree. C.
[0106] Abbreviations
[0107] TLC--thin layer chromatography
[0108] AcOH--acetic acid
[0109] AcCl--acetyl chloride
[0110] PPTS--pyridinium p-toluenesulfonate
[0111] DCM--dichloromethane
[0112] 1,2-DCE--1,2-dichloroethane
[0113] DIC--Diisopropylcarbodiimide
[0114] DIEA--N,N-diisopropylethylamine
[0115] DMF--N,N-dimethylformamide
[0116] DMAP--4-dimethylaminopyridine
[0117] Fmoc--9H-fluoren-9-ylmethyl)oxy]carbonyl
[0118]
HATU--O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate
[0119] HBTU--O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate
[0120] Et.sub.2O--diethyl ether
[0121] EtOAc--ethyl acetate
[0122] i-Pr.sub.2O--di-isopropyl ether
[0123] Config.--absolute configuration
[0124]
Lawesson's--2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2-
,4-disulphide
[0125] Reagent
[0126] MeCN--acetonitrile
[0127] MeOH--methanol
[0128] Rt--retention time
[0129] THF--tetrahydrofuran
[0130] RT--room temperature
[0131] Pd/C--palladium on carbon
[0132] LC/MS refers to analyses by analytical HPLC which were
conducted on two kinds of apparatus: [0133] a) On a Supelcosil
LCABZ+PLUS column (3 .mu.m, 3.3cm.times.4.6 mm ID) eluting with
0.1% HCO.sub.2H and 0.01 M ammonium acetate in water (solvent A),
and 95% acetonitrile and 0.05% HCO.sub.2H in water (solvent B),
using the following elution gradient 0-0.7 minutes 0% B, 0.7-4.2
minutes 0.fwdarw.100% B, 4.2-5.3 minutes 100% B, 5.3-5.5 minutes
100.fwdarw.0% B at a flow rate of 3 mL/minute. The mass spectra
(MS) were recorded on a Fisons VG Platform mass spectrometer using
electrospray positive ionisation [(ES+ve to give [M+H].sup.+ and
[M+NH.sub.4].sup.+ molecular ions] or electrospray negative
ionisation [(ES-ve to give [M-H]- molecular ion] modes. Analytical
data from this apparatus are given with the following format:
[M+H].sup.+ or [M-H].sup.-. [0134] b) On a Chromolith Performance
RP 18 column (100.times.4.6 mm id) eluting with 0.01M ammonium
acetate in water (solvent A) and 100% acetonitrile (solvent B),
using the following elution gradient 0-4 minutes 0.fwdarw.100% B,
4-5 minutes 100% B at a flow rate of 5 mL/minute. The mass spectra
(MS) were recorded on a micromass Platform-LC mass spectrometer
using atmospheric pressure chemical positive ionisation [AP+ve to
give MH.sup.+ molecular ions] or atmospheric pressure chemical
negative ionisation [AP-ve to give (M-H).sup.- molecular ions]
modes. Analytical data from this apparatus are given with the
following format: [M+H]+ or [M-H]- preceded by the acronym APCI to
specify between both mass spectrometry analyses sources.
[0135] LC/HRMS: Analytical HPLC was conducted on a Uptisphere-hsc
column (3 .mu.m 33.times.3 mm id) eluting with 0.01M ammonium
acetate in water (solvent A) and 100% acetonitrile (solvent B),
using the following elution gradient 0-0.5 minutes 5% B, 0.5-3.75
minutes 5.fwdarw.100% B, 3.75-4.5 100% B, 4.5-5 100.fwdarw.5% B,
5-5.5 5% B at a flow rate of 1.3 mL/minute. The mass spectra (MS)
were recorded on a micromass LCT mass spectrometer using
electrospray positive ionisation [ES+ve to give MH.sup.+ molecular
ions] or electrospray negative ionisation [ES-ve to give (M-H)-
molecular ions] modes.
[0136] Mass directed auto-prep HPLC refers to the method where the
material was purified by high performance liquid chromatography on
a HPLCABZ+ 5 .mu.m column (5 cm.times.10 mm i.d.) with 0.1%
HCO.sub.2H in water and 95% MeCN, 5% water (0.5% HCO.sub.2H)
utilising the following gradient elution conditions: 0-1.0 minutes
5% B, 1.0-8.0 minutes 5.fwdarw.30% B, 8.0-8.9 minutes 30% B,
8.9-9.0 minutes 30.fwdarw.95% B, 9.0-9.9 minutes 95% B, 9.9-10
minutes 95.fwdarw.0% B at a flow rate of 8 mL/minute. The Gilson
202-fraction collector was triggered by a VG Platform Mass
Spectrometer on detecting the mass of interest.
[0137] Proton NMR (.sup.1NMR) spectra were recorded at ambient
temperature on a Bruker Avance 300 DPX spectrometer using solvant
as internal standard and proton chemical shifts are expressed in
ppm in the indicated solvent. The following abbreviations are used
for multiplicity of NMR signals: s=singlet, d=doublet, t=triplet,
q=quadruplet, dd=double doublet, m=multiplet.
[0138] TLC (thin layer chromatography) refers to the use of TLC
plates sold by Merck coated with silica gel 60 F254.
EXAMPLE 1
2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a-
][1,4]benzodiazepin-4-yl]-N-ethylacetamide
##STR00013##
[0140] To a solution of
[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetic acid (for a preparation see
Intermediate 1)(16.0 g, 40 mmol) in THF at RT was added DIEA (14
mL, 80 mmol) followed by HATU (30.4 g, 80 mmol). The reaction
mixture was stirred for 3 h at this temperature and ethylamine (40
mL, 2M in THF, 80 mmol) was added. The mixture was stirred for 48 h
before being concentrated under reduced pressure. The crude
material was suspended in water and extracted with DCM. The organic
layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in
vacuo. The crude solid was purified by chromatography on SiO.sub.2
(DCM/MeOH 95/5) and the resulting solid recrystallised in MeCN. The
solid was then dissolved in DCM and precipited with i-Pr.sub.2O to
give the title compound (8 g, 47% yield) as a white solid.
[0141] R.sub.f=0.48 (DCM/MeOH: 90/10). Mp>140.degree. C.
(becomes gummy). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
0.53-7.47 (m, 2H), 7.39 (d, J=8.9 Hz, 1H), 7.37-7.31 (m, 2H), 7.20
(dd, J=2.9 and 8.9 Hz, 1H), 6.86 (d, J=2.9 Hz, 1H), 6.40 (m, 1H),
4.62 (m, 1H), 3.80 (s, 3H), 3.51 (dd, J=7.3 and 14.1 Hz, 1H),
3.46-3.21 (m, 3H), 2.62 (s, 3H), 1.19 (t, J=7.3 Hz, 3H). LC/MS: m/z
424 [M(.sup.35Cl)+H].sup.+, Rt 2.33 min.
Intermediate 1:
[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetic acid
##STR00014##
[0143] To a solution of methyl
[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetate (for a preparation see Intermediate
2)(28 g, 68 mmol) in THF (450 mL) at RT was added 1N NaOH (136 mL,
136 mmol). The reaction mixture was stirred at this temperature for
5 h before being cooled down and quenched with 1N HCl (136 mL). THF
was removed under reduced pressure and the aqueous layer was
extracted with DCM. The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude solid was recrystallised in CH.sub.3CN to give the title
compound (23.9 g, 89% yield) as a pale yellow powder. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.55-7.48 (m, 2H), 7.41 (d, J=8.9 Hz,
1H), 7.38-7.31 (m, 2H), 7.22 (dd, J=2.9 and 8.9 Hz, 1H), 6.90 (d,
J=2.9 Hz, 1H), 4.59 (dd, J=6.9 and 6.9 Hz, 1H), 3.81 (s, 3H), 3.70
(dd, J=6.9 and 25.7 Hz, 1H), 3.61 (dd, J=6.9 and 25.7 Hz, 1H), 2.63
(s, 3H). LC/MS: m/z 397 [M(.sup.35Cl)+H].sup.+, Rt 2.11 min.
Intermediate 2: Methyl
[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetate
##STR00015##
[0145] To crude methyl
[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4--
benzodiazepin-3-yl]acetate (for a preparation see Intermediate 3)
(34 g, 79 mmol) was suspended in THF (200 mL) and AcOH (200 mL) was
added at RT. The reaction mixture was stirred at this temperature
overnight before being concentrated to dryness. The residue was
suspended in saturated NaHCO.sub.3 and extracted with DCM. The
organic layer was dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude solid was purified by
chromatography on SiO.sub.2 (DCM/MeOH: 90/10) to give the title
compound (28 g, 86% yield) as a yellow powder.
[0146] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.54-7.47 (m, 2H),
7.40 (d, J=8.8 Hz, 1H), 7.37-7.31 (m, 2H), 7.22 (dd, J=2.8 and 8.8
Hz, 1H), 6.89 (d, J=2.8 Hz, 1H), 4.61 (dd, J=6.4 and 7.8 Hz, 1H),
3.82 (s, 3H), 3.78 (s, 3H), 3.66 (dd, J=7.8 and 16.9 Hz, 1H), 3.60
(dd, J=6.4 and 16.9 Hz, 1H), 2.62 (s, 3H). LC/MS m/z 411
[M(.sup.35Cl)+H].sup.+, Rt 2.88 min.
Intermediate 3: Methyl
[(3S)-2-[2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzo-
diazepin-3-yl]acetate
##STR00016##
[0148] To a suspension of methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzod-
iazepin-3-yl]acetate (for a preparation see Intermediate 4)(30.2 g,
77.7 mmol) in THF (800 mL) at 0.degree. C. was added hydrazine
monohydrate (11.3 mL, 233 mmol) dropwise. The reaction mixture was
stirred for 4 h between 0.degree. C. and 15.degree. C. before being
cooled at 0.degree. C. Et.sub.3N (32.4 mL, 230 mmol) was then added
slowly and AcCl (16.3 mL, 230 mmol) was added dropwise. The mixture
was allowed to warm to RT and stir for 1 h then quenched with water
and concentrated under reduced pressure. The resulting aqueous
layer was then extracted with DCM and the organic layer was dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give
the crude title compound (34 g, 100% yield) which was used without
further purification. LC/MS: m/z 429 [M(.sup.35Cl)+H].sup.+, Rt
2.83 min.
Intermediate 4: Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzod-
iazepin-3-yl]acetate
##STR00017##
[0150] A suspension of P.sub.4S.sub.10 (85.8 g, 190 mmol) and
Na.sub.2CO.sub.3 (20.5 g, 190 mmol) in 1,2-DCE (1.5 L) at RT was
stirred for 1 h before methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiaz-
epin-3-yl]acetate (for a preparation see Intermediate 5) (40 g, 107
mmol) was added. The resulting mixture was stirred at 65.degree. C.
for 4 h before being cooled and filtered. The solid was washed with
DCM and the filtrate washed with sat. NaHCO.sub.3. The organic
layer was dried over Na.sub.2SO.sub.4, filtered and concentrated
under reduced pressure. The title compound was precipitated from a
DCM/i-Pr.sub.2O mixture and filtered. The filtrate was then
concentrated and purified by flash chromatography (DCM/MeOH: 98/2)
to afford another batch of product. The title compound was obtained
combining the two fractions (30.2 g, 73%) as a yellow powder.
LC/MS: m/z 389 [M(.sup.35Cl)+H].sup.+, Rt 3.29 min.
Intermediate 5: Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiaz-
epin-3-yl]acetate
##STR00018##
[0152] To a solution of the crude methyl
N.sup.1-[2[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N.sup.2-{[(9H-f-
luoren-9-ylmethyl)oxy]carbonyl}-L-.alpha.-asparaginate (for a
preparation see Intermediate 6) (assumed 0.2 mol) in DCM (500 mL)
was added Et.sub.3N (500 mL, 3.65 mol) and the resulting mixture
was refluxed for 24 h before being concentrated. The resulting
crude amine was dissolved in 1,2-DCE (1.5 L) and AcOH (104 mL, 1.8
mol) was added carefully. The reaction mixture was then stirred at
60.degree. C. for 2 h before being concentrated in vacuo and
dissolved in DCM. The organic layer was washed with 1N HCl and the
aqueous layer was extracted with DCM (.times.3). The combined
organic layers were washed twice with water, and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude solid was recrystallised in MeCN leading to the title
compound (51 g) as a pale yellow solid. The filtrate could be
concentrated and recrystallised in MeCN to give another 10 g of
Intermediate 9 (total: 61 g, 69% yield based on recovered
Intermediate 12). R.sub.f=0.34 (DCM/MeOH: 95/5). LC/MS m/z 373
[M(.sup.35Cl)+H].sup.+, Rt 2.76 min.
Intermediate 6: Methyl
N.sup.1-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N.sup.2-{[(9H--
fluoren-9-ylmethyl)oxy]carbonyl}-L-.alpha.-asparaginate
##STR00019##
[0154] A mixture of Methyl
N-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-.alpha.-aspartyl
chloride (prepared from J. Org. Chem. 1990, 55, 3068-3074 and J.
Chem. Soc. Perkin Trans. 1 2001, 1673-1695) (221 g, 0.57 mol) and
[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone (for a
preparation see Intermediate 7) (133 g, 0.5 mol) in CHCl.sub.3 (410
mL) was stirred at 60.degree. C. for 1.5 h before being cooled and
concentrated under reduced pressure and used without further
purification. LC/MS: m/z 613 [M(.sup.35Cl)+H].sup.+, Rt=3.89
min.
Intermediate 7:
[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone
##STR00020##
[0156] To a solution of
2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one (for a preparation
see Intermediate 8)(40.0 g, 0.21 mol) in a toluene (560 mL)/ether
(200 mL) mixture at 0.degree. C. was added dropwise a solution of
4-chlorophenylmagnesium bromide (170 mL, 1M in Et.sub.2O, 0.17
mol). The reaction mixture was allowed to warm to RT and stirred
for 1 h before being quenched with 1N HCl. The aqueous layer was
extracted with EtOAc (3.times.) and the combined organics were
washed with brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The crude compound was then
dissolved in EtOH (400 mL) and 6N HCl (160 mL) was added. The
reaction mixture was refluxed for 2 h before being concentrated
under reduced pressure. The resulting solid was filtered and washed
twice with ether before being suspended in EtOAc and neutralised
with 1N NaOH. The aqueous layer was extracted with EtOAc (3.times.)
and the combined organics were washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The title compound was obtained as a yellow solid (39 g, 88% yield)
which was used without further purification.
Intermediate 8: 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one
##STR00021##
[0158] A solution of 5-methoxyanthranilic acid (7.8 g, 46.5 mmol)
was refluxed in acetic anhydride (60 mL) for 2 h15 before being
cooled and concentrated under reduced pressure. The crude residue
was then concentrated twice in the presence of toluene before being
filtered and washed with ether to yield to the title compound (6.8
g, 77% yield) as a beige solid; LC/MS: m/z 192 [M+H].sup.+, Rt 1.69
min.
[0159] Preparation of Reference Compound for Use in Biological
Assays
[0160] Experimental details of LC-MS methods A and B as referred to
herein are as follows:
[0161] LC/MS (Method A) was conducted on a Supelcosil LCABZ+PLUS
column (3 .mu.m, 3.3cm.times.4.6 mm ID) eluting with 0.1%
HCO.sub.2H and 0.01 M ammonium acetate in water (solvent A), and
95% acetonitrile and 0.05% HCO.sub.2H in water (solvent B), using
the following elution gradient 0-0.7 minutes 0% B, 0.7-4.2 minutes
0.fwdarw.100% B, 4.2-5.3 minutes 100% B, 5.3-5.5 minutes
100.fwdarw.0% B at a flow rate of 3 mL/minute. The mass spectra
(MS) were recorded on a Fisons VG Platform mass spectrometer using
electrospray positive ionisation [(ES+ve to give [M+H].sup.+ and
[M+NH.sub.4].sup.+ molecular ions] or electrospray negative
ionisation [(ES-ve to give [M-H]- molecular ion] modes. Analytical
data from this apparatus are given with the following format:
[M+H].sup.+ or [M-H].sup.-.
[0162] LC/MS (Method B) was conducted on an Sunfire C18 column (30
mm.times.4.6 mm i.d. 3.5 .mu.m packing diameter) at 30 degrees
centigrade, eluting with 0.1% v/v solution of Trifluoroacetic Acid
in Water (Solvent A) and 0.1% v/v solution of Trifluoroacetic Acid
in Acetonitrile (Solvent B) using the following elution gradient
0-0.1 min 3% B, 0.1-4.2 min 3-100% B, 4.2-4.8 min 100% B, 4.8-4.9
min 100-3% B, 4.9-5.0 min 3% B at a flow rate of 3 ml/min. The UV
detection was an averaged signal from wavelength of 210 nm to 350
nm and mass spectra were recorded on a mass spectrometer using
positive electrospray ionization. Ionisation data was rounded to
the nearest integer.
[0163] LC/HRMS: Analytical HPLC was conducted on a Uptisphere-hsc
column (3 .mu.m 33.times.3 mm id) eluting with 0.01M ammonium
acetate in water (solvent A) and 100% acetonitrile (solvent B),
using the following elution gradient 0-0.5 minutes 5% B, 0.5-3.75
minutes 5.fwdarw.100% B, 3.75-4.5 100% B, 4.5-5 100.fwdarw.5% B,
5-5.5 5% B at a flow rate of 1.3 mL/minute. The mass spectra (MS)
were recorded on a micromass LCT mass spectrometer using
electrospray positive ionisation [ES+ve to give MH.sup.+ molecular
ions] or electrospray negative ionisation [ES-ve to give (M-H)-
molecular ions] modes.
[0164] TLC (thin layer chromatography) refers to the use of TLC
plates sold by Merck coated with silica gel 60 F254.
[0165] Silica chromatography techniques include either automated
(Flashmaster or Biotage SP4) techniques or manual chromatography on
pre-packed cartridges (SPE) or manually-packed flash columns.
Reference Compound A:
2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one
##STR00022##
[0167] A solution of 5-methoxyanthranilic acid (Lancaster) (41.8 g,
0.25 mol) was refluxed in acetic anhydride (230 mL) for 3.5 h
before being concentrated under reduced pressure. The crude
compound was then concentrated twice in the presence of toluene
before being filtered and washed twice with ether to yield to the
title compound (33.7 g, 71% yield) as a brown solid; LC/MS (Method
A): m/z 192 [M+H].sup.+, Rt 1.69 min.
Reference Compound B:
[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone
##STR00023##
[0169] To a solution of
2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one (for a preparation
see Reference compound A) (40.0 g, 0.21 mol) in a toluene/ether
(2/1) mixture (760 mL) at 0.degree. C. was added dropwise a
solution of 4-chlorophenylmagnesium bromide (170 mL, 1M in
Et.sub.2O, 0.17 mol). The reaction mixture was allowed to warm to
room temperature and stirred for 1 h before being quenched with 1N
HCl (200 mL). The aqueous layer was extracted with EtOAc
(3.times.150 mL) and the combined organics were washed with brine
(100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated
under reduced pressure. The crude compound was then dissolved in
EtOH (400 mL) and 6N HCl (160 mL) was added. The reaction mixture
was refluxed for 2 h before being concentrated to one-third in
volume. The resulting solid was filtered and washed twice with
ether before being suspended in EtOAc and neutralised with 1N NaOH.
The aqueous layer was extracted with EtOAc (3.times.150 mL) and the
combined organics were washed with brine (150 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The title compound was obtained as a yellow solid (39 g, 88%
yield); LC/MS (Method A): m/z 262 [M+H]+, Rt 2.57 min.
Reference Compound C: Methyl
N.sup.1[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N.sup.2-{[(9H-f-
luoren-9-ylmethyl)oxy]carbonyl}-L-.alpha.-asparaginate
##STR00024##
[0171] Methyl
N-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-.alpha.-aspartyl
chloride (Int. J. Peptide Protein Res. 1992, 40, 13-18) (93 g, 0.24
mol) was dissolved in CHCl.sub.3 (270 mL) and
[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone (for a
preparation see Reference compound B) (53 g, 0.2 mol) was added.
The resulting mixture was stirred at 60.degree. C. for 1 h before
being cooled and concentrated at 60% in volume. Ether was added at
0.degree. C. and the resulting precipitate was filtered and
discarded. The filtrate was concentrated under reduced pressure and
used without further purification.
Reference Compound D: Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiaz-
epin-3-yl]acetate
##STR00025##
[0173] To a solution of Methyl
N1-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N2-{[(9H-fluoren-9--
ylmethyl)oxy]carbonyl}-L-.alpha.-asparaginate (for a preparation
see Reference compound C) (assumed 0.2 mol) in DCM (500 mL) was
added Et.sub.3N (500 mL, 3.65 mol) and the resulting mixture was
refluxed for 24 h before being concentrated. The resulting crude
amine was dissolved in 1,2-DCE (1.5 L) and AcOH (104 mL, 1.8 mol)
was added carefully. The reaction mixture was then stirred at
60.degree. C. for 2 h before being concentrated in vacuo and
dissolved in DCM. The organic layer was washed with 1N HCl and the
aqueous layer was extracted with DCM (.times.3). The combined
organic layers were washed twice with water, and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude solid was recrystallised in MeCN leading to the title
compound (51 g) as a pale yellow solid. The filtrate could be
concentrated and recrystallised in MeCN to give to another 10 g of
the desired product R.sub.f=0.34 (DCM/MeOH: 95/5). HRMS (M+H).sup.+
calculated for C.sub.19H.sub.18.sup.35ClN.sub.2O.sub.4 373.0955;
found 373.0957.
Reference Compound E: Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzod-
iazepin-3-yl]acetate
##STR00026##
[0175] A suspension of P.sub.4S.sub.10 (36.1 g, 81.1 mmol) and
Na.sub.2CO.sub.3 (8.6 g, 81.1 mmol) in 1,2-DCE (700 mL) at room
temperature was stirred for 2 h before Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiaz-
epin-3-yl]acetate (for a preparation see Reference compound D)
(16.8 g, 45.1 mmol) was added. The resulting mixture was stirred at
70.degree. C. for 2 h before being cooled and filtered. The solid
was washed twice with DCM and the filtrate washed with sat.
NaHCO.sub.3 and brine. The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude product was purified by flash-chromatography on silica
gel (DCM/MeOH: 99/1) to afford the title compound (17.2 g, 98%
yield) as a yellowish solid. LC/MS (Method A): m/z 389
[M(.sup.35Cl)+H].sup.+, Rt 2.64 min
[0176] HRMS (M+H).sup.+ calculated for C.sub.19H
.sub.18.sup.35ClN.sub.2O.sub.3S 389.0727; found 389.0714.
Reference Compound F: Methyl
[(3S)-2-[2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzo-
diazepin-3-yl]acetate
##STR00027##
[0178] To a suspension of Methyl
[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzod-
iazepin-3-yl]acetate (for a preparation see Reference compound E
(9.0 g, 23.2 mmol) in THF (300 mL) at 0.degree. C. was added
hydrazine monohydrate (3.4 mL, 69.6 mmol) dropwise. The reaction
mixture was stirred for 5 h between 5.degree. C. and 15.degree. C.
before being cooled at 0.degree. C. Et.sub.3N (9.7 mL, 69.6 mmol)
was then added slowly and acetyl chloride (7.95 mL, 69.6 mmol) was
added dropwise. The mixture was then allowed to warm to room
temperature for 16 h before being concentrated under reduced
pressure. The crude product was dissolved in DCM and washed with
water. The organic layer was dried over Na.sub.2SO.sub.4, filtered
and concentrated in vacuo to give the crude title compound (9.7 g,
98% yield) which was used without further purification.
R.sub.f=0.49 (DCM/MeOH: 90/10).
Reference Compound G: Methyl
[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetate
##STR00028##
[0180] The crude Methyl
[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4--
benzodiazepin-3-yl]acetate (for a preparation see Reference
compound F) (assumed 9.7 g) was suspended in THF (100 ml) and AcOH
(60 mL) was added at room temperature. The reaction mixture was
stirred at this temperature for 2 days before being concentrated
under reduced pressure. The crude solid was triturated in
i-Pr.sub.2O and filtered to give the title compound (8.7 g, 91%
over 3 steps) as an off-white solid.
[0181] HRMS (M+H).sup.+ calculated for
C.sub.21H.sub.20ClN.sub.4O.sub.3 411.1229; found 411.1245.
Reference Compound H:
[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetic acid
##STR00029##
[0183] To a solution of Methyl
[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetate (for a preparation see Reference
compound G)(7.4 g, 18.1 mmol) in THF (130 mL) at room temperature
was added 1N NaOH (36.2 mL, 36.2 mmol). The reaction mixture was
stirred at this temperature for 5 h before being quenched with 1N
HCl (36.2 mL) and concentrated in vacuo. Water is then added and
the aqueous layer was extracted with DCM (.times.3) and the
combined organic layers were dried over Na.sub.2SO.sub.4, filtered
and concentrated under reduced pressure to give the title compound
(7 g, 98% yield) as a pale yellow solid.
Reference Compound I: 1,1-dimethylethyl
[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4-
,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate
##STR00030##
[0185] A mixture of
[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a]-
[1,4]benzodiazepin-4-yl]acetic acid (for a preparation see
Reference compound H) (1.0 g, 2.5 mmol), HATU (1.9 g, 5 mmol) and
DIPEA (0.88 ml, 5 mmol) was stirred for 80 minutes at room
temperature, to this was added 1,1-dimethylethyl
(4-aminobutyl)carbamate (1.05 ml, 5.0 mmol, available from
Aldrich). The reaction mixture was stirred at room temperature for
2 h before it was concentrated. The residue was taken up in
dichloromethane and washed with 1N HCl. The aqueous layer was
extracted with dichloromethane twice. Organic layer was washed with
1N sodium hydroxide, followed by a saturated solution of sodium
chloride, dried over sodium sulphate and concentrated. The residue
was purified by flash-chromatography on silica using
dichloromethane/methanol 95/5 to give the title compound as a
yellow solid (1.2 g). LC/MS (Method A): rt=3.04 min.
Reference Compound J:
N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1-
,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamide
trifluoroacetate
##STR00031##
[0187] To a solution of 1,1-dimethylethyl
[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4-
,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate (for a
preparation see Reference compound H) (0.2 g, 0.34 mmol) in
dichloromethane (3 ml) was added trifluoroacetic acid (0.053 ml,
0.68 mmol) dropwise at 0.degree. C. The reaction mixture was
stirred for 3 h from 0.degree. C. to room temperature. The reaction
mixture was concentrated to dryness to afford the title compound as
a hygroscopic yellow oil (200 mg)
[0188] LC/MS (Method A): rt=2.33 min.
[0189] HRMS (M+H).sup.+ calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.2 481.2119; found 481.2162.
Reference Compound K: Mixture of 5- and 6-isomers of Alexa Fluor
488-N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4-
H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamide
##STR00032##
[0191]
N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-
-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamide
trifluoroacetate (for a preparation see Reference compound J)(7.65
mg, 0.013 mmol) was dissolved in N,N-Dimethylformamide (DMF) (300
.mu.l) and added to Alexa Fluor 488 carboxylic acid succinimidyl
ester (5 mg, 7.77 .mu.mol, mixture of 5 and 6 isomers, available
from Invitrogen, product number A-20100) in an Eppendorf centrifuge
tube. Hunig's base (7.0 .mu.l, 0.040 mmol) was added and the
mixture vortex mixed overnight. After 18 h the reaction mixture was
evaporated to dryness and the residue redissolved in DMSO/water
(50%, <1ml total), applied to a preparative Phenomenex Jupiter
C18 column and eluted with a gradient of 95% A: 5% B to 100% B
(A=0.1% trifluoroacetic acid in water, B=0.1% TFA/90%
acetonitrile/10% water) at a flow rate of 10 m/min over 150
minutes. Impure fractions were combined and re-purified using the
same system. Fractions were combined and evaporated to yield the
title product (2.8 mg) as a mixture of the 2 regioisomers shown.
LC/MS (Method B): MH+=999, rt=1.88 min.
Biological Test Methods
[0192] Fluorescence Anisotropy Binding Assay
[0193] The binding of the compound of formula (I) to Bromodomain 2,
3 and 4 was assessed using a Fluorescence Anisotropy Binding
Assay.
[0194] The Bromodomain protein, fluorescent ligand (Reference
compound K see above) and a variable concentration of test compound
are incubated together to reach thermodynamic equilibrium under
conditions such that in the absence of test compound the
fluorescent ligand is significantly (>50%) bound and in the
presence of a sufficient concentration of a potent inhibitor the
anisotropy of the unbound fluorescent ligand is measurably
different from the bound value.
[0195] All data was normalized to the mean of 16 high and 16 low
control wells on each plate. A four parameter curve fit of the
following form was then applied:
y=a+((b-a)/(1+(10 x/10 c) d)
[0196] Where `a` is the minimum, `b` is the Hill slope, `c` is the
pIC50 and is the maximum.
[0197] Recombinant Human Bromodomains (Bromodomain 2 (1-473),
Bromodomain 3 (1-435) and Bromodomain 4 (1-477)) were expressed in
E.coli cells (in pET15b vector) with a six-His tag at the
N-terminal. The His-tagged Bromodomain was extracted from E.coli
cells using 0.1 mg/ml lysozyme and sonication. The Bromodomain was
then purified by affinity chromatography on a HisTRAP HP column,
eluting with a linear 10-500mM Imidazole gradient, over 20 Cv.
Further purification was completed by Superdex 200 prep grade size
exclusion column. Purified protein was stored at -80C in 20mM HEPES
pH 7.5 and 100 mM NaCl.
[0198] Protocol for Bromodomain 2: All components were dissolved in
buffer composition of 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM
CHAPS with final concentrations of Bromodomain 2, 75 nM,
fluorescent ligand 5 nM. 10 .mu.l of this reaction mixture was
added using a micro multidrop to wells containing 100 nl of various
concentrations of test compound or DMSO vehicle (1% final) in
Greiner 384 well Black low volume microtitre plate and equilibrated
in dark 60 mins at room temperature. Fluorescence anisotropy was
read in Envision (.lamda.ex=485 nm, .lamda.EM=530 nm; Dichroic -505
nM).
[0199] Protocol for Bromodomain 3: All components were dissolved in
buffer of composition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM
CHAPS with final concentrations of Bromodomains 3 75 nM,
fluorescent ligand 5 nM. 10 .mu.l of this reaction mixture was
added using a micro multidrop to wells containing 100 nl of various
concentrations of test compound or DMSO vehicle (1% final) in
Greiner 384 well Black low volume microtitre plate and equilibrated
in dark 60 mins at room temperature. Fluorescence anisotropy was
read in Envision (.lamda.ex=485 nm, .lamda.EM=530 nm; Dichroic -505
nM).
[0200] Protocol for Bromodomain 4: All components were dissolved in
buffer of composition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM
CHAPS with final concentrations of Bromodomain 4 75 nM, fluorescent
ligand 5 nM. 10 .mu.l of this reaction mixture was added using a
micro multidrop to wells containing 100 nl of various
concentrations of test compound or DMSO vehicle (1% final) in
Greiner 384 well Black low volume microtitre plate and equilibrated
in dark 60 mins at room temperature. Fluorescence anisotropy was
read in Envision (.lamda.ex=485 nm, .lamda.EM=530 nm; Dichroic -505
nM).
[0201] Example 1 had a pIC50.gtoreq.6.0 in each of the BRD2, BRD3
and BRD4 assays described above.
[0202] LPS Stimulated Whole Blood Measuring TNF.alpha. Levels
Assay
[0203] Activation of monocytic cells by agonists of toll-like
receptors such as bacterial lipopolysaccharide (LPS) results in
production of key inflammatory mediators including TNF.alpha.. Such
pathways are widely considered to be central to the pathophysiology
of a range of auto-immune and inflammatory disorders.
[0204] Compounds to be tested are diluted to give a range of
appropriate concentrations and 1 .mu.l of the dilution stocks is
added to wells of a 96 plate. Following addition of whole blood
(130 ul) the plates are incubated at 37 degrees (5% CO2) for 30 min
before the addition of 10 ul of 2.8 ug/ml LPS, diluted in complete
RPMI 1640 (final concentration=200 ng/ml), to give a total volume
of 140 ul per well. After further incubation for 24 hours at 37
degrees, 140 ul of PBS are added to each well. The plates are
sealed, shaken for 10 minutes and then centrifuged (2500
rpm.times.10 min). 100 ul of the supernatant are removed and
TNF.alpha. levels assayed by immunoassay (typically by MesoScale
Discovery technology) either immediately or following storage at
-20 degrees. Dose response curves for each compound was generated
from the data and an 1050 value was calculated.
[0205] Example 1 was found to have a pIC50 >6.0 in the above
assay.
[0206] These data demonstrate that Example 1 tested in the above
assay inhibited the production of the key inflammatory mediator
TNF.alpha.. This suggests that such a compound has a strong
anti-inflammatory profile, which is likely to translate into
clinical benefit in inflammatory disorders.
[0207] In Vivo Mouse Endotoxemia Model
[0208] High doses of Endotoxin (bacterial lipopolysaccharide)
administered to animals produce a profound shock syndrome including
a strong inflammatory response, dysregulation of cardiovascular
function, organ failure and ultimately mortality. This pattern of
response is very similar to human sepsis and septic shock, where
the body's response to a significant bacterial infection can be
similarly life threatening.
[0209] To test the compound of formula (I) and pharmaceutically
acceptable salts thereof groups of eight Balb/c male mice were
given a lethal dose of 15 mg/kg LPS by intraperitoneal injection.
Ninety minutes later, animals were dosed intravenously with vehicle
(20% cyclodextrin 1% ethanol in apyrogen water) or compound (10
mg/kg). The survival of animals was monitored at 4 days.
[0210] Numbers of animals surviving at 4 days (summed across
multiple repeat experiments)
TABLE-US-00001 Vehicle 4/66 (6%) Example 1 24/56 (52%)
[0211] These data demonstrate that Example 1 tested in the above
model gave rise to a significant animal survival effect following
intravenous administration. This suggests that the compound of
formula (I) has the potential for a profound effect on inflammatory
responses in humans.
[0212] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
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