U.S. patent application number 13/338056 was filed with the patent office on 2012-04-19 for method of using diketopiperazines and composition containing them.
Invention is credited to David Bar-Or, C. Gerald Curtis, Nagaraja K.R. Rao, Greg Thomas.
Application Number | 20120094918 13/338056 |
Document ID | / |
Family ID | 22833957 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120094918 |
Kind Code |
A1 |
Bar-Or; David ; et
al. |
April 19, 2012 |
METHOD OF USING DIKETOPIPERAZINES AND COMPOSITION CONTAINING
THEM
Abstract
The invention provides a method of inhibiting the effects of
platelet activating factor (PAF). For instance, a disease or
condition mediated by PAF (particularly inflammation) can be
treated or platelet aggregation can be inhibited. The invention
also provides a method of inhibiting the production and/or release
of interleukin 8 (IL-8) by cells. The effects of PAF and the
production and/or release of IL-8 are inhibited according to the
invention by a compound of the formula: ##STR00001## wherein
R.sup.1 and R.sup.2 are defined in the application, or a
physiologically-acceptable salt thereof. The invention also
provides pharmaceutical compositions comprising these
compounds.
Inventors: |
Bar-Or; David; (Englewood,
CO) ; Curtis; C. Gerald; (Penylan, GB) ; Rao;
Nagaraja K.R.; (Cardiff, GB) ; Thomas; Greg;
(Highlands Ranch, CO) |
Family ID: |
22833957 |
Appl. No.: |
13/338056 |
Filed: |
December 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10397964 |
Mar 25, 2003 |
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13338056 |
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09922234 |
Aug 2, 2001 |
6555543 |
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10397964 |
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60222849 |
Aug 4, 2000 |
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Current U.S.
Class: |
514/18.7 ;
514/20.8; 514/21.1 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 31/00 20180101; A61P 7/04 20180101; A61P 29/00 20180101; A61P
7/02 20180101; A61P 37/06 20180101; A61K 9/0014 20130101; A61P 1/00
20180101; A61P 11/06 20180101; A61P 25/00 20180101; A61P 31/04
20180101; A61P 13/12 20180101; A61P 31/16 20180101; A61P 37/08
20180101; A61P 19/02 20180101; A61P 21/00 20180101; A61P 27/14
20180101; A61P 27/06 20180101; A61P 11/00 20180101; A61P 9/10
20180101; A61P 7/00 20180101; A61P 35/00 20180101; A61K 31/495
20130101; A61P 1/04 20180101; A61P 17/06 20180101; A61K 31/496
20130101; A61P 43/00 20180101; A61K 9/0073 20130101; A61P 9/00
20180101 |
Class at
Publication: |
514/18.7 ;
514/20.8; 514/21.1 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61P 19/02 20060101 A61P019/02; A61P 27/02 20060101
A61P027/02; A61P 29/00 20060101 A61P029/00; A61P 17/06 20060101
A61P017/06; A61P 37/08 20060101 A61P037/08; A61P 37/06 20060101
A61P037/06 |
Claims
1-28. (canceled)
29. A method for treating a disease or condition selected from the
group consisting of an allergy, arthritis, an autoimmune disease,
ophthalmic inflammation, pain and psoriasis, the method comprising
administering to an animal in need thereof an effective amount of a
compound having the formula: ##STR00006## wherein: R.sup.1 is
--CH.sub.2COR.sup.3, or --CH.sub.2CH.sub.2COR.sup.3; R.sup.2 is the
side chain of an amino acid selected from the group consisting of
alanine, valine, leucine, isoleucine, serine, threonine, aspartic
acid, asparagine, glutamic acid, glutamine, tyrosine and norvaline;
R.sup.3 is --OH, --NH.sub.2, --OR.sup.4, --NHR.sup.4, or
--NR.sup.4R.sup.4; and each R.sup.4 is independently an alkyl,
aryl, alkylaryl, or arylalkyl, or a physiologically-acceptable salt
thereof.
30. The method of claim 29 wherein the disease or condition is an
allergy.
31. The method of claim 29 wherein the disease or condition is
arthritis.
32. The method of claim 29 wherein the disease or condition is an
autoimmune disease.
33. The method of claim 29 wherein the disease or condition is
ophthalmic inflammation.
34. The method of claim 29 wherein the disease or condition is
pain.
35. The method of claim 29 wherein the disease or condition is
psoriasis.
36. The method of claim 29 wherein R.sup.2 is the side chain of
alanine, valine, leucine, isoleucine or norvaline.
37. The method of claim 36 wherein R.sup.2 is the side chain of
alanine.
38. The method of claim 29 wherein R.sup.3 is --OH and R.sup.1 is
--CH.sub.2COOH.
39. The method of claim 36 wherein R.sup.3 is --OH and R.sup.1 is
--CH.sub.2COOH.
40. The method of claim 37 wherein R.sup.3 is --OH and R.sup.1 is
--CH.sub.2COOH.
41. The method of claim 29 wherein R.sup.3 is --NH.sub.2 and
R.sup.1 is --CH.sub.2CONH.sub.2.
42. The method of claim 36 wherein R.sup.3 is --NH.sub.2 and
R.sup.1 is --CH.sub.2CONH.sub.2.
43. The method of claim 37 wherein R.sup.3 is --NH.sub.2 and
R.sup.1 is --CH.sub.2CONH.sub.2.
44. The method of claim 29 wherein R.sup.2 is the side chain of
serine, threonine or tyrosine.
45. The method of claim 44 wherein R.sup.2 is the side chain of
tyrosine.
46. The method of claim 44 wherein R.sup.3 is --OH and R.sup.1 is
--CH.sub.2CH.sub.2COOH.
46. The method of claim 45 wherein R.sup.3 is --OH and R.sup.1 is
--CH.sub.2CH.sub.2COOH.
47. The method of claim 44 wherein R.sup.3 is --NH.sub.2 and
R.sup.1 is --CH.sub.2CH.sub.2CONH.sub.2.
47. The method of claim 44 wherein R.sup.3 is --NH.sub.2 and
R.sup.1 is --CH.sub.2CH.sub.2CONH.sub.2.
48. The method of claim 29 wherein R.sup.2 is the side chain of
aspartic acid, asparagine, glutamic acid or glutamine.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/397,964, filed Mar. 25, 2003, which is continuation of
issued U.S. Pat. No. 6,555,543, filed Aug. 2, 2001 and issued Apr.
29, 2003, which claims benefit of provisional application
60/222,849, filed Aug. 4, 2000. The entire disclosure of the prior
applications, are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods of inhibiting the effects
of platelet activating factor using certain diketopiperazines. The
invention also relates to methods of inhibiting the is production
and/or release of interleukin 8 (IL-8) using these
diketopiperazines. Finally, the invention relates to pharmaceutical
compositions comprising the diketopiperazines.
BACKGROUND
[0003] Platelet activating factor (PAF;
1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) is a potent
inflammatory phospholipid mediator with a wide variety of
biological activities. It is generated and released by basophils,
monocytes, macrophages, polymorphonuclear leukocytes, eosinophils,
neutrophils, natural killer lymphocytes, platelets and endothelial
cells, as well as by renal and cardiac tissues under appropriate
immunological and non-immunological stimulation. See PCT
application WO 94/04537. PAF mediates biological responses by
binding to specific PAF receptors found in a wide variety of cells
and tissues. Structure-activity studies on PAF and its analogs
indicate that the ability of PAF to bind to these receptors is
structure specific and stereospecific. See PCT WO 94/04537.
[0004] While PAF mediates essential biological responses, it also
appears to play a role in pathological immune and inflammatory
responses. Many published studies have provided evidence for the
involvement of PAF in diseases, including arthritis, acute
inflammation, asthma, allergic reactions, cardiovascular diseases,
neoplastic diseases, endotoxic shock, pain, psoriasis, ophthalmic
inflammation, ischemia, gastrointestinal ulceration, myocardial
infarction, inflammatory bowel diseases, and acute respiratory
distress syndrome. See PCT application WO 94/04537.
[0005] The involvement of PAF in pathological inflammatory and
immune states has stimulated a substantial research effort to
identify PAF receptor antagonists, and a number of compounds of
diverse chemical structure have been identified as PAF antagonists.
See, e.g., PCT applications WO 94/04537 and WO 96/00212 (and
references cited in these two applications), PCT applications WO
95/18610 and WO 99/49865, U.S. Pat. Nos. 4,940,709, 5,358,938,
5,434,151, 5,463,083, 5,648,486, 5,741,809, 5,792,776, 5,780,503,
5,856,323, Japanese application 63 290868, Shimazaki et al., Chem.
Pharm. Bull., 35(8), 3527-3530 (1987), Shimazaki et al., J. Med.
Chem., 30, 1709-1711 (1987), Yoshida et al., Prog. Biochem.
Pharmacol., 22, 68-80 (1988), Shimazaki et al., Lipids, 26(12),
1175-1178 (1991). Given the significant number of pathological
immune and inflammatory responses that are mediated by PAF, there
remains a need to identify new compounds and compositions is that
inhibit PAF activity.
[0006] Diketopiperazines have been reported to exhibit a variety of
biological activities. See, e.g., U.S. Pat. No. 4,289,759
(immunoregulatory agents), U.S. Pat. No. 4,331,595
(immunoregulatory agents), U.S. Pat. No. 4,940,709 (PAF
antagonists), U.S. Pat. No. 5,700,804 (inhibitors of plasminogen
activator inhibitor), U.S. Pat. No. 5,750,530 (inhibitors of
plasminogen activator inhibitor), U.S. Pat. No. 5,990,112
(inhibitors of metalloproteases), PCT applications WO 97/36888
(inhibitors of farnesyl-protein transferase) and WO 99/40931
(treatment of central nervous system injury), EP application 43219
(immunoregulatory agents), Japanese application 63 290868 (PAF
antagonists), Japanese application 31 76478 (immunosuppressive
agents), Shimazaki et al., Chem. Pharm. Bull., 35(8), 3527-3530
(1987) (PAF antagonists), Shimazaki et al., J. Med. Chem., 30,
1709-1711 (1987) (PAF antagonists), Shimazaki et al., Lipids,
26(12), 1175-1178 (1991) (PAF antagonists), Yoshida et al., Prog.
Biochem. Pharmacol., 22, 68-80 (1988) (PAF antagonists), Alvarez et
al., J. Antibiotics, 47(11), 1195-1201 (1994) (inhibitors of
calpain)
[0007] The diketopiperazine composed of aspartic acid and alanine
(3-methyl-2,5-diketopiperazine-6-acetic acid; DA-DKP) is known. It
has been reported to be formed as a result of the degradation of
human albumin stored above 30.degree. C. Chan et al., Eur. J.
Biochem., 227, 524-528 (1995). It is not known to have biological
activity.
SUMMARY OF THE INVENTION
[0008] The invention provides a method of treating a disease or
condition mediated by platelet activating factor. The method
comprises administering to an animal in need thereof an effective
amount of a diketopiperazine of the formula:
##STR00002##
wherein:
[0009] R.sup.1 is --CH.sub.2COR.sup.3, or
--CH.sub.2CH.sub.2COR.sup.3;
[0010] R.sup.2 is the side chain of an amino acid selected from the
group consisting of glycine, alanine, valine, leucine, isoleucine,
serine, threonine, aspartic acid, asparagine, glutamic acid,
glutamine, lysine, hydroxylysine, histidine, arginine,
phenylalanine, tyrosine, tryptophan, thyroxine, cysteine,
methionine, norvaline and ornithine;
[0011] R.sup.3 is --OH, --NH.sub.2--OR.sup.4, --NHR.sup.4, or
--NR.sup.4R.sup.4; and
[0012] each R.sup.4 is independently an alkyl, aryl, alkylaryl, or
arylalkyl; or
[0013] a physiologically-acceptable salt thereof.
[0014] The invention further provides a method of inhibiting
inflammation. The method comprises administering to an animal in
need thereof an effective amount of a compound of formula (1) or a
physiologically-acceptable salt thereof.
[0015] The invention also provides a method of inhibiting
aggregation of platelets. The method comprises contacting the
platelets with an effective amount of a compound of formula (1) or
a physiologically-acceptable salt thereof.
[0016] In addition, the invention provides a method of inhibiting
the production, release or both of interleukin 8 by cells. The
method comprises contacting the cells with an effective amount of a
compound of formula (1) or a physiologically-acceptable salt
thereof.
[0017] The invention further provides a method of inhibiting the
effects of platelet activating factor (PAF). The method comprises
contacting the PAF with an effective amount of a compound of
formula (1) or a physiologically-acceptable salt thereof.
[0018] Finally, the invention provides a pharmaceutical
composition. The composition comprises a
pharmaceutically-acceptable carrier and a compound of formula (1)
or a physiologically-acceptable salt thereof.
DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS
[0019] By "side chain" of an amino acid is meant that portion of
the amino acid attached to the common
##STR00003##
backbone of all of the amino acids listed above. For instance, the
side chain of glycine is --H, the side chain of alanine is
--CH.sub.3, and the side chain of serine is --CH.sub.2OH.
[0020] By "alkyl" is meant a straight-chain or branched-chain alkyl
containing 1-30 carbon atoms, preferably 1-18 carbon atoms. "Lower
alkyl" means a straight-chain or branched chain alkyl containing
1-6 carbon atoms.
[0021] By "aryl" is meant an aromatic group having at least one
aromatic ring (e.g., phenyl).
[0022] By "alkylaryl" is meant a lower alkyl having an aryl having
attached thereto (e.g., --CH.sub.2C.sub.6H.sub.5 or
--CH.sub.3CH(C.sub.6H.sub.5)CH.sub.3).
[0023] By "arylalkyl" is meant an aryl having a lower alkyl having
attached thereto (e.g., --C.sub.6H.sub.4--CH.sub.3).
[0024] "Inhibit" is used herein to mean to reduce (wholly or
partially) or to prevent.
[0025] "Mediated" is used herein to mean caused by, exacerbated by,
or involving.
[0026] "Treat" is used herein to mean to reduce (wholly or
partially) the symptoms of a disease or condition, including curing
the disease or condition, or to prevent the disease or
condition.
[0027] The present invention is based on the discovery that
3-methyl-2,5-diketopiperazine-6-acetic acid (DA-DKP) inhibits PAF
activity. This inhibition appears to be due to the binding of
DA-DKP to both PAF and PAF receptors. It is believed that the
binding of DA-DKP to PAF is due to ion pairing of the carboxyl of
DA-DKP with N.sup.+ on the choline portion of PAF. Thus, other
diketopiperazines comprising one or more carboxyls would be
expected to be effective inhibitors of PAF. Indeed, it is possible
that other non-diketopiperazine compounds comprising carboxyls,
such as poly-aspartic acid or poly-glutamic acid, would also be
effective inhibitors of PAF. The mechanism by which DA-DKP binds to
PAF receptors is not known, but it is hypothesized to be due to the
diketopiperazine ring structure of the DA-DKP and/or the
hydrophobic R.sup.2 side chain of DA-DKP.
[0028] Methods of preparing diketopiperazines are known in the art,
and these methods may possibly be employed to synthesize the
diketopiperazines of formula (1). See, e.g., U.S. Pat. Nos.
4,694,081 and 5,817,751; Smith et al., Bioorg. Med. Chem. Letters,
8, 2369-2374 (1998). However, difficulties may be encountered or
unsatisfactory results may be obtained when using prior art methods
to synthesize diketopiperazines of formula (1) (see co-pending
provisional application 60/223,075, filed on Aug. 4, 2000).
Accordingly, it is highly preferable that the diketopiperazines of
formula (1) be synthesized as described in co-pending provisional
application 60/223,075, the complete disclosure of which is
incorporated herein by reference.
[0029] The synthesis described in provisional application
60/223,075 utilizes standard solution-phase or solid-phase peptide
synthetic methods which are well known in the art. Solid-phase
peptide synthetic methods are preferred.
[0030] The first step of the synthesis described in provisional
application 60/223,075 comprises providing a first amino acid. The
first amino acid is selected from the group consisting of glycine,
alanine, valine, leucine, isoleucine, serine, threonine, aspartic
acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine,
histidine, arginine, phenylalanine, tyrosine, tryptophan,
thyroxine, cysteine, methionine, norvaline and ornithine. These
amino acids, which may be in their D- or L-enantiomeric form, are
commercially available or can be made by methods well known in the
art (see, e.g., Williams, Synthesis Of Optically Active
.alpha.-Amino Acids (Pergammon Press, 1989)). Preferred are
hydrophobic amino acids such as glycine, alanine, valine, leucine,
isoleucine, and phenylalanine. Particularly preferred is
alanine.
[0031] The first amino acid is also preferably protected with one
or more protecting groups to prevent unwanted side reactions during
the synthesis. Such protecting groups, and methods for attaching
and removing them, are well known in the art. See, e.g., Green and
Wuts, Protective Groups In Organic Chemistry (Wiley 1992) and
Grant, Synthetic Peptides: A User's Guide (Freemen 1992).
[0032] The first amino acid is reacted with an aspartic acid
derivative of the following is formula
NH.sub.2CH(CH.sub.2COOR.sup.5)COOH or a glutamic acid derivative of
the following formula NH.sub.2CH(CH.sub.2CH.sub.2COOR.sup.5)COOH,
wherein R.sup.5 is a lower alkyl or alkylaryl. Preferably R.sup.5
is benzyl (--CH.sub.2C.sub.6H.sub.5; Bz). The benzyl group has been
found not only to protect the side-chain carboxyls of these amino
acids, but also to facilitate cyclization of the dipeptide.
Furthermore, the benzyl can be removed from the dipeptide under
neutral conditions which prevents racemization of the chiral center
(carbons bearing the R.sup.1 and R.sup.2 groups).
[0033] The aspartic and glutamic acid derivatives
NH.sub.2CH(CH.sub.2COOR.sup.5)COOH and
NH.sub.2CH(CH.sub.2CH.sub.2COOR.sup.5)COOH are commercially
available or may be prepared by known methods (see, e.g., Bodansky
and Bodansky, The Practice of Peptide Synthesis, pages 63-66 (2nd
ed., Springer-Verlag, 1994). The amino group or a carboxyl group of
the aspartic and glutamic acid derivatives can optionally be
blocked with a standard protecting group (see above) in order to
prevent unwanted side reactions.
[0034] As noted above, the synthesis of the diketopiperazines
preferably utilizes solid-phase peptide synthetic methods. The
first amino acid or the aspartic or glutamic acid derivative is
attached to a solid support through its a carboxyl for solid-phase
synthesis. The solid support may be any solid support which is
compatible with peptide synthesis, such as those described in Grant
and Atherton, Solid Phase Peptide Synthesis: A Practical Approach
(IRL Press 1989). Suitable solid supports are available
commercially or can be prepared by standard methods. See PCT
application WO 96/00391. The solid support may contain linker or
spacer molecules which anchor the first amino acid or the aspartic
acid or glutamic acid derivative to the support surface. A variety
of linkers with different properties are well known in the art.
See, e.g., Grant, Synthetic Peptides: A User's Guide (Freemen 1992)
and PCT application WO 96/00391. The linker will typically include
a functional group to which the first amino acid or the aspartic
acid or glutamic acid derivative is attached.
[0035] Preferably, the first amino acid is attached to the solid
support and, prior to coupling the aspartic acid or glutamic acid
derivative to the first amino acid, the protecting group, if
present, on the a amino group of the bound first amino acid is
removed. The removal of the protecting group of any side-chain
amino groups should be avoided, however, so conditions is must be
chosen to deprotect the a amino group without deprotecting the side
chain amino groups. Suitable deprotection conditions are known in
the art. For example, removal of 9-fluorenylmethyloxycarbonyl may
be performed with 20% to 55% of a secondary amine base, such as
piperidine, in a polar, aprotic solvent, such as dimethylformamide,
methylene chloride or N-methylpyrrolidine. Diisopropyl silane is
preferably added to prevent transesterification during
deprotection, which can be pronounced in large scale
preparations.
[0036] The reaction between the first amino acid and the aspartic
or glutamic acid derivative takes place under conditions effective
to produce a peptide bond so that a dipeptide is formed. These
conditions are well known in the art. For instance, a coupling
catalyst (such as
2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluroniumtetrafluoroborate,
benzotriazole-1-yl-oxytris(dimethylamino)phosphonium
hexafluorophospate,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexaphosphate,
1-hydroxybenzotriazole, diisopropylamine,
dicyclohexylcarbodiimide), may be used to effect formation of the
dipeptide. Typically, an excess of the coupling catalyst is used,
with quantities ranging from 2 to 10 equivalents or more. Often the
degree of excess is determined with respect to the reactivity of
the chemical species being coupled. Polar, aprotic solvents (such
as dimethylformamide, N-methylpyrollidine, methylene chloride and
dimethylsulfoxide) are preferred. Reaction times may vary from
one-half hour to overnight, and temperatures may vary from room
temperature to reflux.
[0037] Next, if the dipeptide is bound to a solid support, it is
removed from the solid support using standard procedures well known
in the art. The conditions effective to remove the dipeptide from
the solid support will be depend on the solid support and linker
chosen. Generally, the peptide will be removed by acid hydrolysis
using a strong acid, such as trifluoroacetic acid.
[0038] The dipeptide is then cyclized to form a diketopiperazine;
this diketopiperazine will have the side-chain carboxyl of the
aspartic acid or glutamic acid derivative still in the ester form.
Cyclization is accomplished by heating the dipeptide under neutral
conditions. Typically, the dipeptide will be heated at from about
80.degree. C. to about 180.degree. C., preferably at about
120.degree. C. The solvent will be a neutral solvent. For instance,
the solvent may comprise an alcohol (such as butanol, methanol,
ethanol, and higher alcohols, but not phenol) and an azeotropic
co-solvent (such as toluene, benzene, or xylene). Preferably, the
alcohol is butan-2-ol, and the azeotropic co-solvent is toluene.
The heating is continued until the reaction is complete, and such
times can be determined empirically. Typically, the dipeptide will
be cyclized by refluxing it for about 8-24 hours, preferably about
18 hours.
[0039] Finally, the R.sup.5 group is removed from the
diketopiperazine by methods well known in the art for removing
protecting groups (see above). When the R.sup.5 group is benzyl, it
is preferably removed from the diketopiperazine by hydrogenation
using a palladium on carbon (Pd/C) catalyst. The use of strong
acids (mineral acids, such as sulfuric or hydrochloric acids),
strong bases (alkaline bases, such as potassium hydroxide or sodium
hydroxide), and strong reducing agents (e.g., lithium aluminum
hydride) should be avoided, in order to maintain the chirality of
the final compound.
[0040] Once the R.sup.5 group has been removed, the free acid can
be derivatized, if desired, to form standard derivatives, such as
amides and esters. Methods which can be used to convert the free
acid to an amide or ester are well known in the art.
[0041] The physiologically-acceptable salts of the diketopiperzines
of formula (1) may also be used in the practice of the invention.
Physiologically-acceptable salts include conventional non-toxic
salts, such as salts derived from inorganic acids (such as
hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the
like), organic acids (such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, glutamic,
aspartic, benzoic, salicylic, oxalic, ascorbic acid, and the like)
or bases (such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation or organic cations derived
from N,N-dibenzylethylenediamine, D-glucosamine, or
ethylenediamine). The salts are prepared in a conventional manner,
e.g., by neutralizing the free base form of the compound with an
acid.
[0042] A diketopiperazine of formula (1), or a
physiologically-acceptable salt thereof, can is be used to treat a
disease or condition mediated by PAF. To do so, a diketopiperazine
of formula (1), or a physiologically-acceptable salt thereof, is
administered to an animal in need of treatment. Preferably, the
animal is a mammal, such as a rabbit, goat, dog, cat, horse or
human. Effective dosage forms, modes of administration and dosage
amounts for the various compounds of the invention may be
determined empirically, and making such determinations is within
the skill of the art. It is understood by those skilled in the art
that the dosage amount will vary with the particular compound
employed, the disease or condition to be treated, the severity of
the disease or condition, the route(s) of administration, the rate
of excretion of the compound, the duration of the treatment, the
identify of any other drugs being administered to the animal, the
age, size and species of the animal, and like factors known in the
medical and veterinary arts. In general, a suitable daily dose of a
compound of the present invention will be that amount of the
compound which is the lowest dose effective to produce a
therapeutic effect. However, the daily dosage will be determined by
an attending physician or veterinarian within the scope of sound
medical judgment. If desired, the effective daily dose may be
administered as two, three, four, five, six or more sub-doses,
administered separately at appropriate intervals throughout the
day. Administration of the compound should be continued until an
acceptable response is achieved.
[0043] The compounds of the present invention (i.e.,
diketopiperazines of formula (1) and physiologically-acceptable
salts thereof) may be administered to an animal patient for therapy
by any suitable route of administration, including orally, nasally,
rectally, vaginally, parenterally (e.g., intravenously,
intraspinally, intraperitoneally, subcutaneously, or
intramuscularly), intracisternally, transdermally, intracranially,
intracerebrally, and topically (including buccally and
sublingually). The preferred routes of administration are orally
and intravenously.
[0044] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical formulation (composition). The
pharmaceutical compositions of the invention comprise a compound or
compounds of is the invention as an active ingredient in admixture
with one or more pharmaceutically-acceptable carriers and,
optionally, with one or more other compounds, drugs or other
materials. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the animal. Pharmaceutically-acceptable carriers are
well known in the art. Regardless of the route of administration
selected, the compounds of the present invention are formulated
into pharmaceutically-acceptable dosage forms by conventional
methods known to those of skill in the art. See, e.g., Remington's
Pharmaceutical Sciences.
[0045] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, powders, granules or as a solution or a suspension in an
aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil
liquid emulsions, or as an elixir or syrup, or as pastilles (using
an inert base, such as gelatin and glycerin, or sucrose and
acacia), and the like, each containing a predetermined amount of a
compound or compounds of the present invention as an active
ingredient. A compound or compounds of the present invention may
also be administered as bolus, electuary or paste.
[0046] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monosterate; (8) absorbents, such as kaolin and bentonite clay; (9)
lubricants, such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0047] A tablet may be made by compression or molding optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0048] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient only, or preferentially, in
a certain portion of the gastrointestinal tract, optionally, in a
delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in microencapsulated form.
[0049] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically-acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0050] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0051] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0052] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or salicylate, and which is
solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound. Formulations of the present invention which
are suitable for vaginal administration also include pessaries,
tampons, creams, gels, pastes, foams or spray formulations
containing such carriers as are known in the art to be
appropriate.
[0053] Dosage forms for the topical or transdermal administration
of compounds of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches, drops and
inhalants. The active ingredient may be mixed under sterile
conditions with a pharmaceutically-acceptable carrier, and with any
buffers, or propellants which may be required.
[0054] The ointments, pastes, creams and gels may contain, in
addition to an active ingredient, excipients, such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0055] Powders and sprays can contain, in addition to an active
ingredient, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
[0056] Transdermal patches have the added advantage of providing
controlled delivery of compounds of the invention to the body. Such
dosage forms can be made by dissolving, dispersing or otherwise
incorporating one or more compounds of the invention in a proper
medium, such as an elastomeric matrix material. Absorption
enhancers can also be used to increase the flux of the compound
across the skin. The rate of such flux can be controlled by either
providing a rate-controlling membrane or dispersing the compound in
a polymer matrix or gel.
[0057] Pharmaceutical formulations include those suitable for
administration by inhalation or insufflation or for nasal or
intraocular administration. For administration to the upper (nasal)
or lower respiratory tract by inhalation, the compounds of the
invention are conveniently delivered from an insufflator, nebulizer
or a pressurized pack or other convenient means of delivering an
aerosol spray. Pressurized packs may comprise a suitable propellant
such as dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
In the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
[0058] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of one or more compounds of the invention
and a suitable powder base, such as lactose or starch. The powder
composition may be presented in unit dosage form in, for example,
capsules or cartridges, or, e.g., gelatin or blister packs from
which the powder may be administered with the aid of an inhalator,
insufflator or a metered-dose inhaler.
[0059] For intranasal administration, compounds of the invention
may be administered by is means of nose drops or a liquid spray,
such as by means of a plastic bottle atomizer or metered-dose
inhaler. Typical of atomizers are the Mistometer (Wintrop) and
Medihaler (Riker).
[0060] Drops, such as eye drops or nose drops, may be formulated
with an aqueous or nonaqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs. Drops can
be delivered by means of a simple eye dropper-capped bottle or by
means of a plastic bottle adapted to deliver liquid contents
dropwise by means of a specially shaped closure.
[0061] Pharmaceutical compositions of this invention suitable for
parenteral administrations comprise one or more compounds of the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, solutes which render the formulation
isotonic with the blood of the intended recipient or suspending or
thickening agents.
[0062] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0063] These compositions may also contain adjuvants such as
wetting agents, emulsifying agents and dispersing agents. It may
also be desirable to include isotonic agents, such as sugars,
sodium chloride, and the like in the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monosterate and gelatin.
[0064] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug is accomplished by
dissolving or suspending the drug in an oil vehicle.
[0065] Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue. The injectable materials can be
sterilized for example, by filtration through a bacterial-retaining
filter.
[0066] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0067] As noted above, PAF has been reported to play a role in a
variety of diseases and conditions. These diseases and conditions
include acute respiratory distress syndrome, allergies, arthritis,
asthma, autoimmune diseases, bronchitis, cardiovascular disease,
Crohn's disease, cystic fibrosis, emphysema, gastrointestinal
ulceration, inflammation, inflammatory bowel disease, ischemia,
multiple organ dysfunction syndrome, myocardial infarction,
neoplastic diseases, ophthalmic inflammation, pain, psoriasis,
respiratory infections, sepsis, shock, and ulcerative colitis. PAF
also mediates platelet aggregation. The diketopiperzines of formula
(1) can be used to treat any of these diseases and conditions and
any other diseases and conditions in which PAF plays a role. The
compounds of the invention can be is given in combination with
other standard therapies for a given disease or condition.
[0068] PAF has been reported to induce the production and secretion
of interleukin 8 (IL-8) (see discussion in Example 3 below). IL-8
is a pro-inflammatory cytokine which has been reported to play a
role in the pathogenesis of a large number of diseases and
conditions, including acute respiratory distress syndrome,
allergies, arthritis, asthma, autoimmune diseases, bronchitis,
cancer, Crohn's disease, cystic fibrosis, emphysema, endocarditis,
gastritis, inflammatory bowel disease, ischemia reperfusion,
multiple organ dysfunction syndrome, nephritis, pancreatitis,
respiratory viral infections, sepsis, shock, ulcerative colitis,
and other inflammatory disorders. The diketopiperazines of formula
(1) have been found to inhibit the PAF-induced production and/or
release of IL-8. Preliminary data indicate that they also inhibit
the production and/or release of IL-8 in the absence of PAF. In
particular, it has been found that the lipopolysaccharide
(LPS)-induced production and/or release of IL-8 by normal human
bronchial epithelial cells is inhibited (data not shown). Thus, the
diketopiperazines of the invention appear to act by two different
mechanisms and may be used to treat diseases or conditions mediated
by IL-8, as well as PAF.
EXAMPLES
Example 1
Preparation of 3-Methyl-2,5-Diketopiperazine-6-Acetic Acid (5)
[0069] Wang resin having 9-fluorenylmethyloxycarbonyl-protected
alanine (Ala-Fmoc) attached thereto (3 grams (g), 2.52 mmol, 1
equivalent, NovaBiochem) was transferred to a clean round-bottom,
100 mL flask, and a solution of piperidine (12 mL) in
dimethylformamide (DMF; 18 mL) was added to the resin in the flask.
The solution was swirled for 1 hour, and the resin was isolated in
a sintered glass funnel. The resin was washed with DMF (3.times.30
mL) followed by dichloromethane (DCM; 3.times.30 mL) and allowed to
dry under vacuum for 5 minutes.
[0070] The partially-dried resin was transferred into a clean
round-bottom, 100 mL flask, and DMF (10 mL) was added. Then,
Boc-Asp(OBz)OH (3.25 g, 10.07 mmol, 4 equivalents) was added,
followed by diisopropylamine (2.83 mL, 2.04 g, 20.19 mmol, 8
equivalents) and
2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluroniumtetrafluoroborate
(TBTU; 3.24 g, 10.09 mmol, 4 equivalents, Acros). The slurry was
allowed to react under anaerobic conditions over 12 hours. At the
end of this time, the resin showed a negative ninhydrin test,
indicating the completion of the coupling reaction. The resin was
vacuum filtered and washed with DMF (3.times.30 mL) followed by DCM
(3.times.30 mL). The resin was allowed to dry at room temperature
under vacuum for 10 minutes before transferring it into a clean
round-bottom, 100 mL flask.
[0071] Trifluoroacetic acid (TFA; 16.5 mL) was added to the dried
resin and, upon its addition, the resin turned a red color. After
swirling the resin for a further 30 minutes, TFA was removed by
filtration, and the resin was washed with DCM (4.times.20 mL). The
organic components were pooled, and toluene (20 mL) was added. The
combined organic materials were evaporated to dryness under vacuum.
Traces of TFA were removed by the addition of toluene and
evaporation. The process was repeated until all TFA had been
removed. This procedure resulted in a product as a pale yellow oil
whose NMR and mass spectrophotometric data were consistent with the
expected dipeptide benzyl ester whose structure (3) is shown
below.
[0072] The dipeptide 3 was dissolved in butan-2-ol (40 mL) and
diluted with toluene (60 mL). This solution was allowed to reflux
for 24 hours. At the end of this period, the solution was allowed
to cool to room temperature. It was then concentrated on a rotary
evaporator, while maintaining the temperature at 50.degree. C. Upon
concentration, a white solid precipitated, and the precipitate was
removed by filtration. The precipitate was washed with toluene (10
mL) and dried. The residue (0.650 g) gave a negative ninhydrin
test. It was, then, crystallized from hot methanol. The
spectroscopic and analytical results for the crystallized product
confirmed its structure to be the desired compound--Asp-Ala
diketopiperazine-benzyl ester shown below (4).
[0073] This compound (400 mg) was dissolved in methanol (250 mL),
and palladium on carbon catalyst (Pd/C; 10%, 0.4 g) was added
carefully. The flask was purged with hydrogen and kept at a
positive hydrogen pressure. The solution was kept in this
atmosphere for at least 4 hours. The catalyst was removed with a
filtering aid (celite) and washed with methanol. The methanol
washings were combined, and the solvent was removed (yield 200 mg).
Mass spectrometer and NMR analysis showed that the free acid
Asp-Ala diketopiperazine (3-methyl-2,5-diketopiperazine-6-acetic
acid, 5) had formed without any cross contamination.
##STR00004##
Example 2
Preparation of Asp-Ala Diketopiperazine Amide (6)
##STR00005##
[0075] To a solution of 3-methyl-2,5-diketopiperazine-6-acetic acid
(0.151 g, 0.81 mmol, 1 equivalent, preparation described in Example
1, 5) in DMF (2.5 mL) was added carbonyl diimidazole (0.26 g, 1.60
mmol, 2 equivalents, Aldich). After stirring at room temperature
for 1 hour, solid ammonium acetate (0.63 g, 8.17 mmol, 10
equivalents, Aldrich) was added. Stirring at room temperature was
continued overnight, at which time the reaction was partitioned
between water (20 mL) and ethyl acetate (10 mL). The aqueous layer
was washed with a second aliquot of ethyl acetate (10 mL) and then
evaporated to dryness under reduced pressure (61.degree. C.).
Traces of DMF were removed by further co-evaporations with water
and then toluene to give a white solid (362 mg). This was taken up
into a minimum volume of methanol in DCM (20:80 v/v). The solvent
eluted was fractionated, and the appropriate fractions were pooled
and evaporated under reduced pressure (40.degree. C.) to give a
white solid. The product was then recrystallized from methanol to
given the desired product (0.116 g, 76% yield, 6).
Example 3
Inhibition of Release of IL-8
[0076] Interleukin 8 (IL-8) is a pro-inflammatory cytokine and a
potent chemoattractant and activator of neutrophils. It has also
been reported to be a chemoattractant and activator of
T-lymphocytes and eosinophils. IL-8 is produced by immune cells
(including lymphocytes, neutrophils, monocytes and macrophages),
fibroblasts and epithelial cells. Reports indicate an important
role for IL-8 in the pathogenesis of respiratory viral infections,
asthma, bronchitis, emphysema, cystic fibrosis, acute respiratory
distress syndrome, sepsis, multiple organ dysfunction syndrome, and
other inflammatory disorders.
[0077] It has been reported that PAF induces the transcription and
secretion of IL-8 in human lung fibroblasts. Roth et al., J. Exp.
Med., 184, 191-201 (1996). It has also been reported that PAF
enhances the production of IL-8 by human mononuclear cells in
response to lipopolysaccharide (LPS), but that PAF alone only
weakly induces the production of IL-8 by these cells. Arbabi et
al., Archives Surgery, 134, 1348-1353 (1999). These authors
hypothesize that PAF "primes" the innate immune system to produce
enhanced amounts of proinflammatory mediators in response to a
second inflammatory stimulus that otherwise would have been
insufficient to trigger an inflammatory response. They further
speculate that if this priming is generalized, it may become
harmful. In such a case, the second stimulus, which would be
considered minor by the unprimed innate immune system, would induce
an aggressive, diffuse, and nonfocused release of inflammatory
mediators, possibly leading to multiple organ dysfunction
syndrome.
[0078] NHBE 6122 normal human bronchial epithelial cells
(Clonetics, San Diego, Calif.) were added to a 24-well tissue
culture plate (Falcon, now BD Biosciences, Franklin Lakes, N.J.) at
20,000 cells/well and allowed to adhere overnight (16-18 hours) in
BEGM (bronchial epithelial growth medium; Clonetics) containing
epinephrine (complete medium) at 37.degree. C. and 5% CO.sub.2.
After adhering, the cells were washed twice with BEGM medium
without epinephrine. They were then incubated in complete medium or
in complete medium containing 20 .mu.M
3-methyl-2,5-diketopiperazine-6-acetic acid (DA-DKP; preparation
described in Example 1, 5; stock solution made in HEPES buffered
saline (HBSS; Clonetics) at 4 mM for 20 minutes at 37.degree. C.
and 5% CO.sub.2. Platelet activating factor (PAF; Sigma, St. Louis,
Mo.) dissolved in dimethylsulfoxide (DMSO; tissue culture grade;
Sigma, St. Louis, Mo.) was then added to a final concentration of
100 nM or 500 nM, and the cells were incubated for an additional 6
hours at 37.degree. C. and 5% CO.sub.2. Medium containing DMSO and
HBSS was used as a control.
[0079] The concentration of IL-8 in cell supernatants was
determined by an ELISA using human IL-8 matched pair antibodies
(Endogen, Cambridge, Mass.). The ELISA was performed using an ELISA
kit from Endogen, Cambridge, Mass. according to the manufacturer's
instructions with the following exceptions: (1) coating antibody at
1 .mu.g/ml; (2) detecting antibody 30 ng/ml; StrepAvidin HRP
diluted 1:32,000.
[0080] The results are presented in Tables 1-3 below. As can be
seen, IL-8 secretion induced by PAF in NHBE 6122 cells was
inhibited by the pre-incubation of the cells with DA-DKP. It is
hypothesized that the DA-DKP binds to PAF, the PAF receptor, or
both, blocking the signal to produce (release) IL-8.
TABLE-US-00001 TABLE 1 IL-8 (pg/ml) SEM DMSO 729.88 8.46 HBSS
809.62 198.23 AA-DKP (20 .mu.M) 803.11 67.18 PAF (100 nM) 1094.68
103.21 PAF + AA-DKP 714.91 88.95
TABLE-US-00002 TABLE 2 IL-8 (pg/ml) SEM DMSO 602.99 73.48 HBSS
581.86 64.36 AA-DKP (20 .mu.M) 837.84 100.73 PAF (500 nM) 887.87
112.56 PAF + AA-DKP 542.5 37.17
TABLE-US-00003 TABLE 3* IL-8 (pg/ml) SEM DMSO 209.79 13.24 HBSS
233.08 5.79 AA-DKP (20 .mu.M) 184.86 34.73 PAF (100 nM) 355.36
11.28 PAF + AA-DKP 201.93 20.64 *For Table 3, cells were split to
give 5,000 cells/well four days prior to the experiment and were
allowed to grow to 70% confluence.
* * * * *