U.S. patent application number 11/846485 was filed with the patent office on 2008-02-21 for drug therapy for celiac sprue.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Kihang Choi, Chaitan Khosla.
Application Number | 20080044401 11/846485 |
Document ID | / |
Family ID | 34619913 |
Filed Date | 2008-02-21 |
United States Patent
Application |
20080044401 |
Kind Code |
A1 |
Khosla; Chaitan ; et
al. |
February 21, 2008 |
DRUG THERAPY FOR CELIAC SPRUE
Abstract
Administering an effective dose of a tTGase inhibitor to a
Celiac or dermatitis herpetiformis patient reduces the toxic
effects of toxic gluten oligopeptides, thereby attenuating or
eliminating the damaging effects of gluten.
Inventors: |
Khosla; Chaitan; (Palo Alto,
CA) ; Choi; Kihang; (Seoul, KR) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
34619913 |
Appl. No.: |
11/846485 |
Filed: |
August 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10716846 |
Nov 18, 2003 |
7265093 |
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11846485 |
Aug 28, 2007 |
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PCT/US03/15343 |
May 14, 2003 |
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10716846 |
Nov 18, 2003 |
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60380761 |
May 14, 2002 |
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60392782 |
Jun 28, 2002 |
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60422933 |
Oct 31, 2002 |
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60428033 |
Nov 20, 2002 |
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Current U.S.
Class: |
424/94.63 ;
514/378; 514/418; 514/789; 548/240 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 43/00 20180101; A61P 29/00 20180101; C07D 261/04 20130101;
A61P 17/04 20180101; A61P 17/02 20180101; A61P 7/06 20180101; A61P
25/16 20180101; A61K 38/005 20130101; A61P 37/00 20180101; A61P
1/00 20180101; A61P 1/12 20180101; A61P 3/00 20180101; A61P 25/00
20180101; A61P 25/28 20180101; A61P 35/00 20180101; A61K 31/42
20130101; C07D 413/12 20130101; A61P 25/14 20180101; A61P 37/08
20180101; A61P 1/14 20180101 |
Class at
Publication: |
424/094.63 ;
514/378; 514/418; 514/789; 548/240 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 31/404 20060101 A61K031/404; A61P 1/00 20060101
A61P001/00; C07D 261/04 20060101 C07D261/04; A61P 17/00 20060101
A61P017/00; A61K 31/42 20060101 A61K031/42 |
Claims
1. A method of treating Celiac Sprue and/or dermatitis
herpetiformis, the method comprising: administering to a patient an
effective dose of a tTGase inhibitor; wherein said tTGase inhibitor
attenuates gluten toxicity in said patient.
2. The method of claim 1, wherein said tTGase inhibitor is or
comprises a dihydroisoxazole moiety or is an analog of isatin.
3. The method of claim 1, wherein said tTGase inhibitor is
administered with a glutenase.
4. The method according to claim 1, wherein said tTGase inhibitor
is administered orally.
5. The method according to claim 1, wherein said tTGase inhibitor
is contained in a formulation that comprises an enteric
coating.
6. A formulation for use in treatment of Celiac Sprue and/or
dermatitis herpetiformis, comprising: an effective dose of a tTGase
inhibitor and a pharmaceutically acceptable excipient.
7. The formulation of claim 6, wherein said tTGase inhibitory
moiety is: ##STR15## wherein R.sub.1 and R.sub.2 are independently
selected from H, alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl,
heteroaryl, alkoxy, alkylthio, arakyl, aralkenyl, halo, haloalkyl,
haloalkoxy, heterocyclyl, and heterocyclylalkyl groups, an amino
acid, a peptide, a peptidomimetic, or a peptidic protecting group;
wherein R.sub.2 can additionally be selected from the group
consisting of LPYPQPQLPY, LPFPQPQLPF--NH.sub.2, LPYPQPQLP,
LPYPQPQLPYPQPQPF, LP-X.sub.2-15, where X.sub.2-15 is a peptide
consisting of any 2-15 amino acid residues followed by a C-terminal
proline; R.sub.3 is selected from F, I, Cl, and Br; n is from 0 to
10; and X is selected from the group consisting of C and NH.
8. The formulation of claim 7, wherein R.sub.1 is selected from the
group consisting of BnO, Me, Cbz, Fmoc, Boc, PQP, Ac--PQP,
PQPQLPYPQP, Ac--PQPQLPFPQP, QLQPFPQP, LQLQPFPQPLPYPQP,
X.sub.2-15--P, where X.sub.2-15 is a peptide consisting of any 2-15
amino acid residues followed by a N-terminal proline.
9. The formulation of claim 7, wherein R.sub.2 is selected from the
group consisting of (S)-Bn, (S)--CO.sub.2Me, (S)-Me, (R)-Bn,
(S)--CH.sub.2CONHBn, (S)-(1H-inol-yl)-methyl,
(S)-(4-hydrohy-phenyl)-methyl, OMe, OtBu, Gly, Gly-NH.sub.2, LPY,
LPF--NH.sub.2.
11. The formulation inhibitor of claim 7, wherein said tTGase
inhibitor is selected from the group consisting of:
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester;
(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-
-carbamoyl]-butyric acid methyl ester;
(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)--
succinamic acid methyl ester;
(S)-2-Benzyloxycarbonylamino-3-phenyl-propionic acid
3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carba-
mic acid benzyl ester;
(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-pr-
opionamide;
{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester;
{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carba-
moyl]-ethyl}-carbamic acid benzyl ester;
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol--
3-yl)-ethyl]-carbamic acid benzyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phe-
nyl-ethyl}-carbamic acid benzyl ester; and
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4--
hydroxy-phenyl)-ethyl]-carbamic acid benzyl ester.
12. The method according to claim 1, wherein said tTGase inhibitor
has the formula: ##STR16## wherein R.sub.1 and R.sub.2 are
independently selected from H, alkyl, alkenyl, cycloalkyl, aryl,
heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl, aralkenyl,
halo, haloalkyl, haloalkoxy, heterocyclyl, and heterocyclylalkyl
groups, an amino acid, a peptide, a peptidomimetic, or a peptidic
protecting group; wherein R.sub.2 can additionally be selected from
the group consisting of LPYPQPQLPY, LPFPQPQLPF--NH.sub.2,
LPYPQPQLP, LPYPQPQLPYPQPQPF, LP--X.sub.2-15, where X.sub.2-15 is a
peptide consisting of any 2-15 amino acid residues followed by a
C-terminal proline; R.sub.3 is selected from F, I, Cl, and Br; n is
from 0 to 10; and X is selected from the group consisting of Q and
NH.
13. The method of claim 12, wherein R.sub.1 is selected from the
group consisting of BnO, Me, Cbz, Fmoc, Boc, PQP, Ac--PQP,
PQPQLPYPQP, Ac--PQPQLPFPQP, QLQPFPQP, LQLQPFPQPLPYPQP,
X.sub.2-15--P, where X.sub.2-15 is a peptide consisting of any 2-15
amino acid residues followed by a N-terminal proline.
14. The method of claim 12, wherein R.sub.2 is selected from the
group consisting of (S)-Bn, (S)--CO.sub.2Me, (S)-Me, (R)-Bn,
(S)--CH.sub.2CONHBn, (S)-(1H-inol-yl)-methyl,
(S)-(4-hydrohy-phenyl)-methyl, OMe, OtBu, Gly, Gly-NH.sub.2, LPY,
LPF--NH.sub.2.
15. The method according to claim 12, wherein said tTGase inhibitor
is selected from the group consisting of:
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester;
(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-
-carbamoyl]-butyric acid methyl ester;
(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)--
succinamic acid methyl ester;
(S)-2-Benzyloxycarbonylamino-3-phenyl-propionic acid
3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carba-
mic acid benzyl ester;
(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-pr-
opionamide;
{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester;
{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carba-
moyl]-ethyl}-carbamic acid benzyl ester;
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol--
3-yl)-ethyl]-carbamic acid benzyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phe-
nyl-ethyl}-carbamic acid benzyl ester; and
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4--
hydroxy-phenyl)-ethyl]-carbamic acid benzyl ester.
16. A tTGase inhibitor of the formula: ##STR17## wherein R.sub.1
and R.sub.2 are independently selected from H, alkyl, alkenyl,
cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio,
arakyl, aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, and
heterocyclylalkyl groups, an amino acid, a peptide, a
peptidomimetic, or a peptidic protecting group; wherein R.sub.2 can
additionally be selected from the group consisting of LPYPQPQLPY,
LPFPQPQLPF--NH.sub.2, LPYPQPQLP, LPYPQPQLPYPQPQPF, LP-X.sub.2-15.
where X.sub.2-15 is a peptide consisting of any 2-15 amino acid
residues followed by a C-terminal proline; R.sub.3 is selected from
F, I, Cl, and Br; n is from 0 to 10; and X is selected from the
group consisting of Q and NH, other than
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester.
17. The tTGase inhibitor of claim 16, wherein R.sub.1 is selected
from the group consisting of BnO, Me, Cbz, Fmoc, Boc, PQP, Ac--PQP,
PQPQLPYPQP, Ac--PQPQLPFPQP, QLQPFPQP, LQLQPFPQPLPYPQP,
X.sub.2-15--P, where X.sub.2-15 is a peptide consisting of any 2-15
amino acid residues followed by a N-terminal proline.
18. The tTGase inhibitor of claim 16, wherein R.sub.2 is selected
from the group consisting of (S)-Bn, (S)--CO.sub.2Me, (S)-Me,
(R)-Bn, (S)--CH.sub.2CONHBn, (S)-(1H-inol-yl)-methyl,
(S)-(4-hydrohy-phenyl)-methyl, OMe, OtBu, Gly, Gly-NH.sub.2, LPY,
LPF--NH.sub.2.
19. The tTGase inhibitor of claim 16, wherein said tTGase inhibitor
is selected from the group consisting of:
(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-
-carbamoyl]-butyric acid methyl ester;
(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)--
succinamic acid methyl ester;
(S)-2-Benzyloxycarbonylamino-3-phenyl-propionic acid
3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carba-
mic acid benzyl ester;
(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-pr-
opionamide;
{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester;
{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carba-
moyl]-ethyl}-carbamic acid benzyl ester;
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol--
3-yl)-ethyl]-carbamic acid benzyl ester;
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phe-
nyl-ethyl}-carbamic acid benzyl ester; and
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4--
hydroxy-phenyl)-ethyl]-carbamic acid benzyl ester.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/716,846, which is a continuation in part of International
Application US03/15343, filed May 14, 2003; which claims priority
to U.S. Provisional Application 60/380,761 filed May 14, 2002; to
U.S. Provisional Application 60/392,782 filed Jun. 28, 2002; and to
U.S. Provisional application No. 60/422,933, filed Oct. 31, 2002,
and to U.S. Provisional Application 60/428,033, filed Nov. 20,
2002, each of which are herein specifically incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] In 1953, it was first recognized that ingestion of gluten, a
common dietary protein present in wheat, barley and rye causes a
disease called Celiac Sprue in sensitive individuals. Gluten is a
complex mixture of glutamine- and proline-rich glutenin and
prolamine molecules and is thought to be responsible for induction
of Celiac Sprue. Ingestion of such proteins by sensitive
individuals produces flattening of the normally luxurious,
rug-like, epithelial lining of the small intestine known to be
responsible for efficient and extensive terminal digestion of
peptides and other nutrients. Other clinical symptoms of Celiac
Sprue include fatigue, chronic diarrhea, malabsorption of
nutrients, weight loss, abdominal distension, anemia, as well as a
substantially enhanced risk for the development of osteoporosis and
intestinal malignancies such as lymphoma and carcinoma. The disease
has an incidence of approximately 1 in 200 in European populations
and is believed to be significantly under diagnosed in other
populations.
[0003] A related disease is dermatitis herpetiformis, which is a
chronic eruption of the skin characterized by clusters of intensely
pruritic vesicles, papules, and urticaria-like lesions. IgA
deposits occur in almost all normal-appearing and perilesional
skin. Asymptomatic gluten-sensitive enteropathy is found in 75 to
90% of patients and in some of their relatives. Onset is usually
gradual. Itching and burning are severe, and scratching often
obscures the primary lesions with eczematization of nearby skin,
leading to an erroneous diagnosis of eczema. Strict adherence to a
gluten-free diet for prolonged periods may control the disease in
some patients, obviating or reducing the requirement for drug
therapy. Dapsone, sulfapyridine, and colchicines are sometimes
prescribed for relief of itching.
[0004] Celiac Sprue (CS) is generally considered to be an
autoimmune disease and the antibodies found in the serum of the
patients support the theory that the disease is immunological in
nature. Antibodies to tissue transglutaminase (tTGase or tTG) and
gliadin appear in almost 100% of the patients with active CS, and
the presence of such antibodies, particularly of the IgA class, has
been used in diagnosis of the disease.
[0005] The large majority of patients express the HLA-DQ2
[DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] molecules.
It is believed that intestinal damage is caused by interactions
between specific gliadin oligopeptides and the HLA-DQ2 or DQ8
antigen, which in turn induce proliferation of T lymphocytes in the
sub-epithelial layers. T helper 1 cells and cytokines apparently
play a major role in a local inflammatory process leading to
villous atrophy of the small intestine.
[0006] At the present time, there is no good therapy for the
disease, except to avoid completely all foods containing gluten.
Although gluten withdrawal has transformed the prognosis for
children and substantially improved it for adults, some people
still die of the disease, mainly adults who had severe disease at
the outset. A leading cause of death is lymphoreticular disease,
especially intestinal lymphoma. It is not known whether a
gluten-free diet diminishes this risk. Apparent clinical remission
is often associated with histologic relapse that is detected only
by review biopsies or by increased EMA titers.
[0007] Gluten is so widely used, for example, in commercial soups,
sauces, ice creams, hot dogs, and other foodstuffs, that patients
need detailed lists of foodstuffs to avoid and expert advice from a
dietitian familiar with celiac disease. Ingesting even small
amounts of gluten may prevent remission or induce relapse.
Supplementary vitamins, minerals, and hematinics may also be
required, depending on deficiency. A few patients respond poorly or
not at all to gluten withdrawal, either because the diagnosis is
incorrect or because the disease is refractory. In the latter case,
oral corticosteroids (e.g., prednisone 10 to 20 mg bid) may induce
response.
[0008] In view of the serious and widespread nature of Celiac Sprue
and the difficulty of removing gluten from the diet, better methods
of treatment are of great interest. In particular, there is a need
for treatment methods that allow the Celiac Sprue individual to eat
gluten-containing foodstuffs without ill effect or at least to
tolerate such foodstuffs in small or moderate quantities without
inducing relapse. The present invention meets this need for better
therapies for Celiac Sprue by providing new drugs and methods and
formulations of new and existing drugs to treat Celiac Sprue.
International Patent Application US03/04743, herein specifically
incorporated by reference, discloses aspects of gluten protease
stability and immunogenicity.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides methods for
treating Celiac Sprue and/or dermatitis herpetiformis and the
symptoms thereof by administration of a tTGase (tissue
transglutaminase) inhibitor to the patient. In one embodiment, the
tTGase inhibitor employed in the method is a small molecule tTGase
inhibitor comprising a 3-halo-4,5-dihydroisoxazole moiety.
[0010] In another aspect, the present invention provides novel
derivative compounds of 3-halo-4,5-dihydroisoxazoles and methods
for treating Celiac Sprue and/or dermatitis herpetiformis by
administering those compounds.
[0011] In one embodiment, the tTGase inhibitor employed in the
method is an analog of isatin (2, 3 diketoindoline).
[0012] In another aspect, the invention provides pharmaceutical
formulations comprising a tTGase inhibitor and a pharmaceutically
acceptable carrier. In one embodiment, the formulation also
comprises one or more glutenases, as described in U.S. Provisional
Application 60/392,782 filed Jun. 28, 2002; and U.S. Provisional
Application 60/428,033, filed Nov. 20, 2002, both of which are
incorporated herein by reference. The invention also provides
methods for the administration of enteric formulations of one or
more tTGase inhibitors to treat Celiac Sprue. In another aspect,
the tTGase inhibitors and/or pharmaceutical formulations of the
present invention are useful in treating disorders where TGases are
a factor in the disease etiology, where such disorders may include
cancer, neurological disorders, wound healing, etc. These
conditions include Alzheimer's and Huntington's diseases, where the
TGases appear to be a factor in the formation of inappropriate
proteinaceous aggregates that may be cytotoxic. In diseases such as
progressive supranuclear palsy, Huntington's, Alzheimer's and
Parkinson's diseases, the aberrant activation of TGases may be
caused by oxidative stress and inflammation.
[0013] These and other aspects and embodiments of the invention and
methods for making and using the invention are described in more
detail in the description of the drawings and the invention, the
examples, the claims, and the drawings that follow.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0014] Celiac Sprue and/or dermatitis herpetiformis are treated by
inhibition of tissue transglutaminase. Methods and compositions are
provided for the administration of one or more tTGase inhibitors to
a patient suffering from Celiac Sprue and/or dermatitis
herpetiformis. The compositions of the invention include
formulations of tTGase inhibitors that comprise an enteric coating
that allows delivery of the agents to the intestine in an active
form; the agents are stabilized to resist digestion or alternative
chemical transformations in acidic stomach conditions. In another
embodiment, food is pretreated or combined with glutenase, or a
glutenase is co-administered (whether in time or in a formulation
of the invention) with a tTGase inhibitor of the invention.
[0015] The subject methods are useful for both prophylactic and
therapeutic purposes. Thus, as used herein, the term "treating" is
used to refer to both prevention of disease, and treatment of a
pre-existing condition. The treatment of ongoing disease, to
stabilize or improve the clinical symptoms of the patient, is a
particularly important benefit provided by the present invention.
Such treatment is desirably performed prior to loss of function in
the affected tissues; consequently, the prophylactic therapeutic
benefits provided by the invention are also important. Evidence of
therapeutic effect may be any diminution in the severity of
disease, particularly diminution of the severity of such symptoms
as fatigue, chronic diarrhea, malabsorption of nutrients, weight
loss, abdominal distension, and anemia. Other disease indicia
include the presence of antibodies specific for glutens, antibodies
specific for tissue transglutaminase, the presence of
pro-inflammatory T cells and cytokines, and degradation of the
villus structure of the small intestine. Application of the methods
and compositions of the invention can result in the improvement of
any and all of these disease indicia of Celiac Sprue.
[0016] Patients that can benefit from the present invention include
both adults and children. Children in particular benefit from
prophylactic treatment, as prevention of early exposure to toxic
gluten peptides can prevent development of the disease into its
more severe forms. Children suitable for prophylaxis in accordance
with the methods of the invention can be identified by genetic
testing for predisposition, e.g. by HLA typing; by family history,
and by other methods known in the art. As is known in the art for
other medications, and in accordance with the teachings herein,
dosages of the tTGase inhibitors of the invention can be adjusted
for pediatric use.
[0017] Compounds of interest for inhibition of tTGase include those
having the general formulae: ##STR1##
[0018] where R.sub.1, R.sub.2 and R.sub.3 are independently
selected from H, alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl,
heteroaryl, alkoxy, alkylthio, arakyl, aralkenyl, halo, haloalkyl,
haloalkoxy, heterocyclyl, and heterocyclylalkyl groups. R.sub.1 and
R.sub.2 can also be an amino acid, a peptide, a peptidomimetic, or
a peptidic protecting groups.
[0019] Illustrative R.sub.1 groups include Cbz, Fmoc, and Boc. In
other embodiments of the invention, R.sub.1 is an arylether, aryl,
alkylether or alkyl group, e.g. O-benzyl, benzyl, methyl or
ethyl.
[0020] R.sub.2 groups of interest include OMe, OtBu, Gly, and
Gly-NH.sub.2. In other embodiments, R.sub.2 is selected from the
group consisting of (s)-Bn, (s)-CO.sub.2Me, (s)-Me, (R)-Bn,
(S)--CH.sub.2CONHBn, (S)-(1H-inol-yl)-methyl, and
(S)-(4-hydrohy-phenyl)-methyl.
[0021] R.sub.3 is preferably a halo group, i.e. F, Cl, Br, and
I.
[0022] X.sub.1 and X.sub.2 are selected from the group consisting
of NH, O, and NR.sub.4. where R.sub.4 is a lower alkyl.
[0023] n is a whole number between 0 and 10, usually between 0 and
5, and more usually between 0 and 3.
[0024] The tTGase inhibitory compounds of the invention from the
isoxazoles can be readily prepared using methods known in the art
for other purposes and the teachings herein. Examples of synthetic
routes to these compounds are also described in examples below For
example, Castelhano et al have demonstrated that the
dihydroisoxazole derivative
(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethy-
l}-carbamic acid benzyl ester is an inhibitor of bovine epidermal
transglutaminase (Castelhano et al., Bioorg. Chem. (1988) 16,
335-340). The following general formula for transglutaminase
inhibitors is disclosed in EP0237082: ##STR2##
[0025] Here we identify new compounds within this genus that are
especially effective inhibitors of human tissue transglutaminase,
and may therefore be used to treat Celiac Sprue and/or Dermatitis
Herpetiforms. We also disclose new compounds with comparable
activity.
[0026] Another example of tTGase inhibitors are analogs of the
dioxoindoline isatin. The cyclic .alpha.-keto amide structure of
isatin serves as a good analog of .gamma.-carboxamide group of
tTGase glutamyl substrate. .alpha.-keto amides are widely utilized
as reversible inhibitors of cysteine-dependent proteases and, in a
similar way, the hetetocyclic structure of isatin possesses an
electrophilic carbonyl group which could be recognized by the
enzyme as an analog of the substrate .gamma.-carboxamide carbonyl
group. Using standard procedures known in the art, the aromatic
portion of the isatin structure can be derivatized further to
incorporate additional functional groups into the inhibitors
mimicking the other parts of peptide substrates.
[0027] The illustrative compounds of the invention described above
were tested in a tTGase assay with recombinant human tissue
transglutaminase, which was expressed, purified and assayed as
described (Piper et al., Biochemistry (2001) 41, 386-393).
Competitive inhibition with respect to the Cbz-Gln-Gly substrate
was observed for all substrates; in all cases irreversible
inactivation of the enzyme was also observed.
[0028] To facilitate an appreciation of the invention, the tTGase
inhibitors of the invention have in part been described above with
structures containing variable "R" groups that are defined by
reference to the various organic moieties that can be present at
the indicated position in the structure. Below, brief definitions
are provided for the phrases used to define the organic moieties
listed for each R group.
[0029] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain radical consisting solely of carbon and hydrogen
atoms, containing no unsaturation, having from one to eight carbon
atoms, and which is attached to the rest of the molecule by a
single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl
(iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and
the like. Unless stated otherwise specifically in the
specification, the alkyl radical may be optionally substituted by
hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto,
alkylthio, --N(R.sup.8).sub.2, --C(O)OR.sup.8,
--C(O)N(R.sup.8).sub.2 or --N(R.sup.8)C(O)R.sup.8 where each
R.sup.8 is independently hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkylalkyl, aralkyl or aryl. Unless stated otherwise
specifically in the specification, it is understood that for
radicals, as defined below, that contain a substituted alkyl group
that the substitution can occur on any carbon of the alkyl
group.
[0030] "Alkoxy" refers to a radical of the formula --OR.sub.a where
R.sub.a is an alkyl radical as defined above, e.g., methoxy,
ethoxy, n-propoxy, 1-methylethoxy (iso-propoxy), n-butoxy,
n-pentoxy, 1,1-dimethylethoxy (t-butoxy), and the like. Unless
stated otherwise specifically in the specification, it is
understood that for radicals, as defined below, that contain a
substituted alkoxy group that the substitution can occur on any
carbon of the alkoxy group. The alkyl radical in the alkoxy radical
may be optionally substituted as described above.
[0031] "Alkylthio" refers to a radical of the formula --SR.sub.a
where R.sub.a is an alkyl radical as defined above, e.g.,
methylthio, ethylthio, n-propylthio, 1-methylethylthio
(iso-propylthio), n-butylthio, n-pentylthio, 1,1-dimethylethylthio
(t-butylthio), and the like. Unless stated otherwise specifically
in the specification, it is understood that for radicals, as
defined below, that contain a substituted alkylthio group that the
substitution can occur on any carbon of the alkylthio group. The
alkyl radical in the alkylthio radical may be optionally
substituted as described above.
[0032] "Alkenyl" refers to a straight or branched hydrocarbon chain
radical consisting solely of carbon and hydrogen atoms, containing
at least one double bond, having from two to eight carbon atoms,
and which is attached to the rest of the molecule by a single bond
or a double bond, e.g., ethenyl, prop-1-enyl, but-1-enyl,
pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated
otherwise specifically in the specification, the alkenyl radical
may be optionally substituted by hydroxy, alkoxy, haloalkoxy,
cyano, nitro, mercapto, alkylthio, cycloalkyl, --N(R.sup.8).sub.2,
--C(O)OR.sup.8, --C(O)N(R.sup.8).sub.2 or
--N(R.sup.8)--C(O)--R.sup.8 where each R.sup.8 is independently
hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or
aryl. Unless stated otherwise specifically in the specification, it
is understood that for radicals, as defined below, that contain a
substituted alkenyl group that the substitution can occur on any
carbon of the alkenyl group.
[0033] "Aryl" refers to a phenyl or naphthyl radical. Unless stated
otherwise specifically in the specification, the term "aryl" or the
prefix "ar-" (such as in "aralkyl") is meant to include aryl
radicals optionally substituted by one or more substituents
selected from the group consisting of hydroxy, alkoxy, aryloxy,
haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl,
--N(R.sup.8).sub.2, --C(O)OR.sup.8, --C(O)N(R.sup.8).sub.2 or
--N(R.sup.8)C(O)R.sup.8 where each R.sup.8 is independently
hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or
aryl. The term "aryl" also refers to the compound C.sub.6H.sub.5,
i.e. Bn.
[0034] "Aralkyl" refers to a radical of the formula
--R.sub.aR.sub.b where R.sub.a is an alkyl radical as defined above
and R.sub.b is one or more aryl radicals as defined above, e.g.,
benzyl, diphenylmethyl and the like. The aryl radical(s) may be
optionally substituted as described above.
[0035] "Aralkenyl" refers to a radical of the formula
--R.sub.cR.sub.b where R.sub.c is an alkenyl radical as defined
above and R.sub.b is one or more aryl radicals as defined above,
e.g., 3-phenylprop-1-enyl, and the like. The aryl radical(s) and
the alkenyl radical may be optionally substituted as described
above.
[0036] "Alkylene chain" refers to a straight or branched divalent
hydrocarbon chain consisting solely of carbon and hydrogen,
containing no unsaturation and having from one to eight carbon
atoms, e.g., methylene, ethylene, propylene, n-butylene, and the
like. The alkylene chain may be optionally substituted by one or
more substituents selected from the group consisting of aryl, halo,
hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio,
cycloalkyl, --N(R.sup.8).sub.2, --C(O)OR.sup.8,
--C(O)N(R.sup.8).sub.2 or --N(R.sup.8)C(O)R.sup.8 where each
R.sup.8 is independently hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkylalkyl, aralkyl or aryl. The alkylene chain may be
attached to the rest of the molecule through any two carbons within
the chain.
[0037] "Alkenylene chain" refers to a straight or branched divalent
hydrocarbon chain consisting solely of carbon and hydrogen,
containing at least one double bond and having from two to eight
carbon atoms, e.g., ethenylene, prop-1-enylene, but-1-enylene,
pent-1-enylene, hexa-1,4-dienylene, and the like. The alkenylene
chain may be optionally substituted by one or more substituents
selected from the group consisting of aryl, halo, hydroxy, alkoxy,
haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl,
--N(R.sup.8).sub.2, --C(O)OR.sup.8, --C(O)N(R.sup.8).sub.2 or
--N(R.sup.8)C(O)R.sup.8 where each R.sup.8 is independently
hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or
aryl. The alkenylene chain may be attached to the rest of the
molecule through any two carbons within the chain.
[0038] "Cycloalkyl" refers to a stable monovalent monocyclic or
bicyclic hydrocarbon radical consisting solely of carbon and
hydrogen atoms, having from three to ten carbon atoms, and which is
saturated and attached to the rest of the molecule by a single
bond, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
decalinyl and the like. Unless otherwise stated specifically in the
specification, the term "cycloalkyl" is meant to include cycloalkyl
radicals which are optionally substituted by one or more
substituents independently selected from the group consisting of
alkyl, aryl, aralkyl, halo, haloalkyl, hydroxy, alkoxy, haloalkoxy,
cyano, nitro, mercapto, alkylthio, cycloalkyl, --N(R.sup.8).sub.2,
--C(O)OR.sup.8, --C(O)N(R.sup.8).sub.2 or --N(R.sup.8)C(O)R.sup.8
where each R.sup.8 is independently hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkylalkyl, aralkyl or aryl.
[0039] "Cycloalkylalkyl" refers to a radical of the formula
--R.sub.aR.sub.d where R.sub.a is an alkyl radical as defined above
and R.sub.d is a cycloalkyl radical as defined above. The alkyl
radical and the cycloalkyl radical may be optionally substituted as
defined above.
[0040] "Halo" refers to bromo, chloro, fluoro or iodo.
[0041] "Haloalkyl" refers to an alkyl radical, as defined above,
that is substituted by one or more halo radicals, as defined above,
e.g., trifluoromethyl, difluoromethyl, trichloromethyl,
2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl,
3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the
like.
[0042] "Haloalkoxy" refers to a radical of the formula --OR.sub.c
where R.sub.c is an haloalkyl radical as defined above, e.g.,
trifluoromethoxy, difluoromethoxy, trichloromethoxy,
2,2,2-trifluoroethoxy, 1-fluoromethyl-2-fluoroethoxy,
3-bromo-2-fluoropropoxy, 1-bromomethyl-2-bromoethoxy, and the
like.
[0043] "Heterocyclyl" refers to a stable 3- to 15-membered ring
radical which consists of carbon atoms and from one to five
heteroatoms selected from the group consisting of nitrogen, oxygen
and sulfur. For purposes of this invention, the heterocyclyl
radical may be a monocyclic, bicyclic or tricyclic ring system,
which may include fused or bridged ring systems; and the nitrogen,
carbon or sulfur atoms in the heterocyclyl radical may be
optionally oxidized; the nitrogen atom may be optionally
quaternized; and the heterocyclyl radical may be aromatic or
partially or fully saturated. The heterocyclyl radical may not be
attached to the rest of the molecule at any heteroatom atom.
Examples of such heterocyclyl radicals include, but are not limited
to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl,
benzothiadiazolyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,
benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl,
benzothienyl (benzothiophenyl), benzotriazolyl, carbazolyl,
cinnolinyl, decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl,
isothiazolyl, imidazolyl, imidazolinyl, imidazolidinyl,
isothiazolidinyl, indolyl, indazolyl, isoindolyl, indolinyl,
isoindolinyl, indolizinyl, isoxazolyl, isoxazolidinyl, morpholinyl,
naphthyridinyl, oxadiazolyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
2-oxoazepinyl, oxazolyl, oxazolidinyl, oxiranyl, piperidinyl,
piperazinyl, 4-piperidonyl, phenazinyl, phenothiazinyl,
phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl,
pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl, pyrazinyl,
pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl,
quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl,
thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl,
tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl
sulfoxide, and thiamorpholinyl sulfone. Unless stated otherwise
specifically in the specification, the term "heterocyclyl" is meant
to include heterocyclyl radicals as defined above which are
optionally substituted by one or more substituents selected from
the group consisting of alkyl, halo, nitro, cyano, haloalkyl,
haloalkoxy, aryl, heterocyclyl, heterocyclylalkyl, --OR.sup.8,
--R.sup.7--OR.sup.8, --C(O)OR.sup.8, --R.sup.7--(O)OR.sup.8,
--C(O)N(R.sup.8).sub.2, --N(R.sup.8).sub.2,
--R.sup.7--N(R.sup.8).sub.2, and --N(R.sup.8)C(O)R.sup.8 wherein
each R.sup.7 is a straight or branched alkylene or alkenylene chain
and each R.sup.8 is independently hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkylalkyl, aralkyl or aryl.
[0044] "Heterocyclylalkyl" refers to a radical of the formula
--R.sub.aR.sub.e where R.sub.a is an alkyl radical as defined above
and R.sub.e is a heterocyclyl radical as defined above, and if the
heterocyclyl is a nitrogen-containing heterocyclyl, the
heterocyclyl may be attached to the alkyl radical at the nitrogen
atom. The heterocyclyl radical may be optionally substituted as
defined above.
[0045] In the formulas provided herein, molecular variations are
included, which may be based on isosteric replacement. "Isosteric
replacement" refers to the concept of modifying chemicals through
the replacement of single atoms or entire functional groups with
alternatives that have similar size, shape and electro-magnetic
properties, e.g. 0 is the isosteric replacement of S, N, COOH is
the isosteric replacement of tetrazole, F is the isosteric
replacement of H, sulfonate is the isosteric replacement of
phosphate etc.
[0046] As used herein, compounds which are "commercially available"
may be obtained from standard commercial sources including Acros
Organics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis.,
including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton
Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto,
Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester
Pa.), Crescent Chemical Co. (Hauppauge N.Y.), Eastman Organic
Chemicals, Eastman Kodak Company (Rochester N.Y.), Fisher
Scientific Co. (Pittsburgh Pa.), Fisons Chemicals (Leicestershire
UK), Frontier Scientific (Logan Utah), ICN Biomedicals, Inc. (Costa
Mesa Calif.), Key Organics (Cornwall U.K.), Lancaster Synthesis
(Windham N.H.), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish
Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc. (Waterbury
Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co. (Rockford
Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum Quality
Product, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.),
Trans World Chemicals, Inc. (Rockville Md.), Wako Chemicals USA,
Inc. (Richmond Va.), Novabiochem and Argonaut Technology.
[0047] As used herein, "suitable conditions" for carrying out a
synthetic step are explicitly provided herein or may be discerned
by reference to publications directed to methods used in synthetic
organic chemistry. The reference books and treatise set forth above
that detail the synthesis of reactants useful in the preparation of
compounds of the present invention, will also provide suitable
conditions for carrying out a synthetic step according to the
present invention.
[0048] As used herein, "methods known to one of ordinary skill in
the art" may be identified though various reference books and
databases. Suitable reference books and treatise that detail the
synthesis of reactants useful in the preparation of compounds of
the present invention, or provide references to articles that
describe the preparation, include for example, "Synthetic Organic
Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandier et
al., "Organic Functional Group Preparations," 2nd Ed., Academic
Press, New York, 1983; H. O. House, "Modern Synthetic Reactions",
2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.
Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley &
Sons, New York, 1992; J. March, "Advanced Organic Chemistry:
Reactions, Mechanisms and Structure", 4th Ed., Wiley-Interscience,
New York, 1992. Specific and analogous reactants may also be
identified through the indices of known chemicals prepared by the
Chemical Abstract Service of the American Chemical Society, which
are available in most public and university libraries, as well as
through on-line databases (the American Chemical Society,
Washington, D.C., www.acs.org may be contacted for more details).
Chemicals that are known but not commercially available in catalogs
may be prepared by custom chemical synthesis houses, where many of
the standard chemical supply houses (e.g., those listed above)
provide custom synthesis services.
[0049] "Optional" or "optionally" means that the subsequently
described event of circumstances may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances in which it does not. For example, "optionally
substituted aryl" means that the aryl radical may or may not be
substituted and that the description includes both substituted aryl
radicals and aryl radicals having no substitution.
[0050] "Pharmaceutically acceptable base addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free acids, which are not biologically or
otherwise undesirable. These salts are prepared from addition of an
inorganic base or an organic base to the free acid. Salts derived
from inorganic bases include, but are not limited to, the sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum salts and the like. Preferred inorganic
salts are the ammonium, sodium, potassium, calcium, and magnesium
salts. Salts derived from organic bases include, but are not
limited to, salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-dimethylaminoethanol,
2-diethylaminoethanol, dicyclohexylamine, lysine, arginine,
histidine, caffeine, procaine, hydrabamine, choline, betaine,
ethylenediamine, glucosamine, methylglucamine, theobromine,
purines, piperazine, piperidine, N-ethylpiperidine, polyamine
resins and the like. Particularly preferred organic bases are
isopropylamine, diethylamine, ethanolamine, trimethylamine,
dicyclohexylamine, choline and caffeine.
[0051] The tTGase inhibitors, or their pharmaceutically acceptable
salts may contain one or more asymmetric centers and may thus give
rise to enantiomers, diastereomers, and other stereoisomeric forms
that may be defined, in terms of absolute stereochemistry, as (R)-
or (S)- or, as (D)- or (L)- for amino acids. The present invention
is meant to include all such possible isomers, as well as, their
racemic and optically pure forms. Optically active (+) and (-),
(R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or chiral reagents, or resolved using conventional
techniques, such as reverse phase HPLC. When the compounds
described herein contain olefinic double bonds or other centers of
geometric asymmetry, and unless specified otherwise, it is intended
that the compounds include both E and Z geometric isomers.
Likewise, all tautomeric forms are also intended to be
included.
[0052] The present invention provides the tTGase inhibitors in a
variety of formulations for therapeutic administration. In one
aspect, the agents are formulated into pharmaceutical compositions
by combination with appropriate, pharmaceutically acceptable
carriers or diluents, and are formulated into preparations in
solid, semi-solid, liquid or gaseous forms, such as tablets,
capsules, powders, granules, ointments, solutions, suppositories,
injections, inhalants, gels, microspheres, and aerosols. As such,
administration of the tTGase inhibitors is achieved in various
ways, although oral administration is a preferred route of
administration. In some formulations, the tTGase inhibitors are
systemic after administration; in others, the inhibitor is
localized by virtue of the formulation, such as the use of an
implant that acts to retain the active dose at the site of
implantation.
[0053] In some pharmaceutical dosage forms, the tTGase inhibitors
are administered in the form of their pharmaceutically acceptable
salts. In some dosage forms, the tTGase inhibitor is used alone,
while in others, the tTGase is used in combination with another
pharmaceutically active compounds. In the latter embodiment, the
other active compound is, in some embodiments, a glutenase that can
cleave or otherwise degrade a toxic gluten oligopeptide, as
described in the Examples below. The following methods and
excipients are merely exemplary and are in no way limiting.
[0054] For oral preparations, the agents are used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and in some
embodiments, with diluents, buffering agents, moistening agents,
preservatives and flavoring agents.
[0055] In one embodiment of the invention, the oral formulations
comprise enteric coatings, so that the active agent is delivered to
the intestinal tract. Enteric formulations are often used to
protect an active ingredient from the strongly acid contents of the
stomach. Such formulations are created by coating a solid dosage
form with a film of a polymer that is insoluble in acid
environments and soluble in basic environments. Exemplary films are
cellulose acetate phthalate, polyvinyl acetate phthalate,
hydroxypropyl methylcellulose phthalate and hydroxypropyl
methylcellulose acetate succinate, methacrylate copolymers, and
cellulose acetate phthalate.
[0056] Other enteric formulations of the tTGase inhibitors of the
invention comprise engineered polymer microspheres made of
biologically erodable polymers, which display strong adhesive
interactions with gastrointestinal mucus and cellular linings, can
traverse both the mucosal absorptive epithelium and the
follicle-associated epithelium covering the lymphoid tissue of
Peyer's patches. The polymers maintain contact with intestinal
epithelium for extended periods of time and actually penetrate it,
through and between cells. See, for example, Mathiowitz et al.
(1997) Nature 386 (6623): 410-414. Drug delivery systems can also
utilize a core of superporous hydrogels (SPH) and SPH composite
(SPHC), as described by Dorkoosh et al. (2001) J Control Release
71(3):307-18.
[0057] In another embodiment, the tTGase inhibitor or formulation
thereof is admixed with food, or used to pre-treat foodstuffs
containing glutens.
[0058] Formulations are typically provided in a unit dosage form,
where the term "unit dosage form," refers to physically discrete
units suitable as unitary dosages for human subjects, each unit
containing a predetermined quantity of tTGase inhibitor calculated
in an amount sufficient to produce the desired effect in
association with a pharmaceutically acceptable diluent, carrier or
vehicle. The specifications for the unit dosage forms of the
present invention depend on the particular complex employed and the
effect to be achieved, and the pharmacodynamics associated with
each complex in the host.
[0059] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0060] Depending on the patient and condition being treated and on
the administration route, the tTGase inhibitor is administered in
dosages of 0.01 mg to 500 mg V/kg body weight per day, e.g. about
100 mg/day for an average person. Dosages are appropriately
adjusted for pediatric formulation. Those of skill will readily
appreciate that dose levels can vary as a function of the specific
inhibitor, the diet of the patient and the gluten content of the
diet, the severity of the symptoms, and the susceptibility of the
subject to side effects. Some of the inhibitors of the invention
are more potent than others. Preferred dosages for a given
inhibitor are readily determinable by those of skill in the art by
a variety of means. A preferred means is to measure the
physiological potency of a given compound.
[0061] The methods of the invention are useful in the treatment of
individuals suffering from Celiac Sprue and/or dermatitis
herpetiformis, by administering an effective dose of a tTGase
inhibitor, through a pharmaceutical formulation, and the like.
Diagnosis of suitable patients may utilize a variety of criteria
known to those of skill in the art. A quantitative increase in
antibodies specific for gliadin, and/or tissue transglutaminase is
indicative of the disease. Family histories and the presence of the
HLA alleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301,
b1*0302)] are indicative of a susceptibility to the disease.
Moreover, as tTG plays an important role in other diseases, such as
Huntington's disease and skin diseases in addition to dermatitis
herpetiformis, a variety of formulated versions of the compounds of
the invention (e.g. topical formulations, intravenous injections)
are useful for the treatment of such medical conditions. These
conditions include Alzheimer's and Huntington's diseases, where the
TGases appear to be a factor in the formation of inappropriate
proteinaceous aggregates that may be cytotoxic. In diseases such as
progressive supranuclear palsy, Huntington's, Alzheimer's and
Parkinson's diseases, the aberrant activation of TGases may be
caused by oxidative stress and inflammation.
[0062] Therapeutic effect is measured in terms of clinical outcome,
or by immunological or biochemical tests. Suppression of the
deleterious T-cell activity can be measured by enumeration of
reactive Th1 cells, by quantitating the release of cytokines at the
sites of lesions, or using other assays for the presence of
autoimmune T cells known in the art. Also both the physician and
patient can identify a reduction in symptoms of a disease.
[0063] Various methods for administration are employed in the
practice of the invention. In one preferred embodiment, oral
administration, for example with meals, is employed. The dosage of
the therapeutic formulation can vary widely, depending upon the
nature of the disease, the frequency of administration, the manner
of administration, the clearance of the agent from the patient, and
the like. The initial dose can be larger, followed by smaller
maintenance doses. The dose can be administered as infrequently as
weekly or biweekly, or more often fractionated into smaller doses
and administered daily, with meals, semi-weekly, and the like, to
maintain an effective dosage level.
[0064] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature), but some experimental errors and
deviations may be present. Unless indicated otherwise, parts are
parts by weight, molecular weight is weight average molecular
weight, temperature is in degrees Centigrade, and pressure is at or
near atmospheric.
EXAMPLE 1
Synthesis of dihydroxyisoxazole Containing tTGase INHIBITORS
[0065] Synthesis of
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester (n=0, X.dbd.NH, R.sub.1=BnO,
R.sub.2.dbd.(S)-Bn, R.sub.3.dbd.Br) (49). N-Cbz-L-Phe (0.30 g, 1.0
mmol) and HOBt (0.15 g, 1.1 eq) were dissolved in 2 mL DMF.
3-Bromo-5-aminomethyl-4,5-dihydroisoazole (0.18 g, 1.0 eq),
prepared following a reported procedure (Rohloff et al. (1992)
Tetrahedron Lett. 33(22):3113-3116), was added to the solution
cooled in an ice bath followed by EDCI (0.23 g, 1.2 eq). The ice
bath was removed and the stirring was continued overnight. The
solution was diluted with ethyl acetate and washed with sat.
NaHCO.sub.3 solution and brine. The organic layer was dried over
MgSO.sub.4 and filtered. The solvent was removed by evaporation and
the residue was purified by SiO.sub.2 chromatography to give the
title compound as a white solid (0.24 g, 52%).
[0066] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.34-7.26 (m,
8H), 7.17 (d, 2H, J=7.6 Hz), 6.19-6.09 (m, 1H), 5.21-5.15 (m, 1H),
5.09 (s, 2H), 4.74-4.60 (m, 1H), 4.41-4.36 (m, 1H), 3.49-3.45 (m,
2H), 3.26-3.12 (m, 1H), 3.07 (d, 2H, J=6.8 Hz), 2.97-2.76 (m,
1H)
[0067] MS (ESI): m/z=460.1 [M+H].sup.+, 482.2 [M+Na].sup.+
##STR3##
Synthesis of
(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-
-carbamoyl]-butyric acid methyl ester (n=2, X.dbd.NH, R.sub.1=BnO,
R.sub.2.dbd.(S)--CO.sub.2Me, R.sub.3.dbd.Br) (50). The title
compound was prepared according to the procedure for compound 49
except using N-Cbz-L-Glu-OMe.
[0068] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.41-7.30 (m,
5H), 6.22-6.12 (m, 1H), 5.63-5.57 (m, 1H), 5.11 (s, 2H), 4.82-4.74
(m, 1H), 4.41-4.33 (m, 1H), 3.75 (s, 3H), 3.54-3.48 (m, 2H),
3.32-3.15 (m, 1H), 3.02-2.88 (m, 1H), 2.34-2.22 (m, 3H), 2.05-1.94
(m, 1H)
[0069] MS (ESI): m/z=456.1 [M+H].sup.+, 478.2 [M+Na].sup.+
##STR4##
[0070] Synthesis of
(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)--
succinamic acid methyl ester (n=1, X.dbd.NH, R.sub.1=BnO,
R.sub.2.dbd.(S)--CO.sub.2Me, R.sub.3.dbd.Br) (51). The title
compound was prepared according to the procedure for compound 49
except using N-Cbz-L-Asp-OMe.
[0071] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.X=7.37-7.30 (m,
5H), 6.00-5.90 (m, 2H), 5.13 (s, 2H), 4.80-4.71 (m, 1H), 4.63-4.58
(m, 1H), 3.76 (s, 3H), 3.54-3.44 (m, 2H), 3.33-3.23 (m, 1H),
2.99-2.70 (m, 3H)
[0072] MS (ESI): m/z=442.1 [M+H].sup.+, 464.2 [M+Na].sup.+
##STR5##
[0073] Synthesis of (S)-2-Benzyloxycarbonylamino-3-phenyl-propionic
acid 3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester (n=0, X.dbd.O,
R.sub.1=BnO, R.sub.2.dbd.(S)-Bn, R.sub.3.dbd.Br) (52). N-Cbz-L-Phe
(0.30 g, 1.0 mmol) was dissolved in the mixture of acetonitrile (6
mL), DIEA (0.18 mL, 1.0 eq) and excess allyl bromide (3 mL). After
the reaction was allowed to proceed overnight, the reaction mixture
was diluted with ethyl acetate, washed with sat. Na.sub.2CO.sub.3
solution and brine, dried over MgSO.sub.4 and concentrated to
provide the ally ester as a clear oil (0.34 g, quant.). The ester
(0.19 g, 0.57 mmol) and dibromoformaldoxime (0.14 g, 1.1 eq) were
dissolved in 3 mL ethyl acetated and NaHCO.sub.3 (0.21 g, 4.3 eq)
was added to the solution. The reaction mixture was stirred
overnight, diluted with ethyl acetated and washed with sat.
NaHCO.sub.3 solution and brine. The organic layer was dried over
MgSO.sub.4 and the solvent was removed by evaporation. The residue
was purified by SiO.sub.2 chromatography to give the title compound
as a white solid (0.15 g, 58%).
[0074] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.35-7.26 (m,
8H), 7.16-7.14 (m, 2H), 5.20-5.05 (m, 3H), 4.85-4.79 (m, 1H),
4.68-4.63 (m, 1H), 4.22-4.15 (m, 2H), 3.27-3.09 (m, 3H), 2.96-2.77
(m, 1H)
[0075] MS (ESI): m/z=461.1 [M+H].sup.+, 483.2 [M+Na].sup.+
##STR6##
[0076] Synthesis of
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carba-
mic acid benzyl ester (n=0, X.dbd.NH, R.sub.1=BnO,
R.sub.2.dbd.(S)-Me, R.sub.3.dbd.Br) (53). The title compound was
prepared according to the procedure for compound 49 except using
N-Cbz-L-Ala.
[0077] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.37-7.34 (b,
5H), 6.68-6.45 (m, 1H), 5.24-5.18 (m, 1H), 5.13 (s, 2H), 4.80-4.76
(m, 1H), 4.26-4.18 (m, 1H), 3.55-3.47 (m, 2H), 3.33-3.19 (m, 1H),
3.05-2.92 (m, 1H), 1.39 (d, 3H, J=7.0 Hz)
[0078] MS (ESI): m/z=384.1 [M+H].sup.+, 406.1 [M+Na].sup.+
##STR7##
[0079] Synthesis of
(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-pr-
opionamide (n=0, X.dbd.NH, R.sub.1=Me, R.sub.2.dbd.(S)-Bn,
R.sub.3.dbd.Br) (54). The title compound was prepared according to
the procedure for compound 49 except using N-Ac-L-Phe.
[0080] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.33-7.18 (m,
5H), 6.14-6.09 (m, 1H), 6.02-5.97 (m, 1H), 4.67-4.59 (m, 2H),
3.49-3.41 (m, 2H), 3.22-3.03 (m, 3H), 2.97-2.70 (m, 1H), 2.00 (s,
3H)
[0081] MS (ESI): m/z=368.1 [M+H].sup.+, 390.2 [M+Na].sup.+
##STR8##
[0082] Synthesis of
{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-eth-
yl}-carbamic acid benzyl ester (n=0, X.dbd.NH, R.sub.1=BnO,
R.sub.2.dbd.(R)-Bn, R.sub.3.dbd.Br) (55). The title compound was
prepared according to the procedure for compound 49 except using
N-Cbz-D-Phe.
[0083] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=7.34-7.26 (m,
8H), 7.17 (d, 2H, J=7.8 Hz), 6.19-6.09 (m, 1H), 5.21-5.15 (m, 1H),
5.09 (s, 2H), 4.74-4.60 (m, 1H), 4.41-4.36 (m, 1H), 3.49-3.45 (m,
2H), 3.26-3.12 (m, 1H), 3.07 (d, 2H, J=7.0 Hz), 2.97-2.76 (m,
1H)
[0084] MS (ESI): m/z=460.1 [M+H].sup.+, 482.2 [M+Na].sup.+
##STR9##
[0085] Synthesis of
{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carba-
moyl]-ethyl}-carbamic acid benzyl ester (n=0, X.dbd.NH,
R.sub.1=BnO, R.sub.2.dbd.(S)--CH.sub.2CONHBn, R.sub.3.dbd.Br) (56).
The title compound was prepared according to the procedure for
compound 49 except using .beta.-benzylamide of N-Cbz-L-Asp
((S)--N-Benzyl-2-benzyloxycarbonylamino-succinamic acid).
[0086] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.=7.38-7.24 (m,
11H), 6.43-6.40 (m, 1H), 6.01-5.99 (m, 1H), 5.14 (s, 2H), 4.80-4.70
(m, 1H), 4.58-4.52 (m, 1H), 4.41 (d, 2H, J=6.4 Hz), 3.57-3.50 (m,
2H), 3.25-3.12 (m, 1H), 3.00-3.94 (m, 2H), 2.62-2.56 (m, 1H)
[0087] MS (ESI): m/z=517.1 [M+H].sup.+, 539.2 [M+Na].sup.+
##STR10##
[0088] Synthesis of
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol--
3-yl)-ethyl]-carbamic acid benzyl ester (n=0, X.dbd.NH,
R.sub.1=BnO, R.sub.2.dbd.(S)-(1H-indol-3-yl)-methyl,
R.sub.3.dbd.Br) (57). The title compound was prepared according to
the procedure for compound 49 except using N-Cbz-L-Trp.
[0089] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.=8.14 (br, 1H),
7.70-7.63 (m, 1H), 7.37-7.31 (m, 6H), 7.22-7.18 (m, 1H), 7.13-7.09
(m, 1H), 7.04-7.02 (m, 1H), 6.15-6.10 (m, 1H), 5.45-5.39 (m, 1H),
5.14-5.06 (m, 2H), 4.59-4.47 (m, 2H), 3.40-3.31 (m, 3H), 3.20-3.14
(m, 1H), 3.11-3.04 (m, 1H), 2.82-2.74 (m, 1H)
[0090] MS (ESI): m/z=499.0 [M+H].sup.+, 521.2 [M+Na].sup.+
##STR11##
[0091] Synthesis of
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phe-
nyl-ethyl}-carbamic acid benzyl ester (n=0, X.dbd.NMe, R.sub.1=BnO,
R.sub.2.dbd.(S)-Bn, R.sub.3.dbd.Br) (58). The title compound was
prepared according to the procedure for compound 52 except using
N-methylallylamine.
[0092] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.=7.34-7.26 (m,
8H), 7.18-7.16 (m, 2H), 5.57-5.56 (m, 1H), 5.12-5.05 (m, 2H),
4.93-4.73 (m, 2H), 3.80-3.67 (m, 1H), 3.36-3.17 (m, 2H), 3.02-3.86
(m, 6H)
[0093] MS (ESI): m/z=474.2 [M+H].sup.+, 496.3 [M+Na].sup.+
##STR12##
[0094] Synthesis of
[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4--
hydroxy-phenyl)-ethyl]-carbamic acid benzyl ester (n=0, X.dbd.NH,
R.sub.1=BnO, R.sub.2.dbd.(S)-(4-hydroxy-phenyl)-methyl,
R.sub.3.dbd.Br) (59). The title compound was prepared according to
the procedure for compound 49 except using N-Cbz-L-Tyr.
[0095] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): .delta.=9.17 (br, 1H),
8.27-8.23 (m, 1H), 7.43-7.40 (m, 1H), 7.32-7.22 (m, 5H), 7.03 (d,
2H, J=7.6 Hz), 6.62 (d, 2H, J=7.6 Hz), 4.93 (s, 2H), 4.68-4.64 (m,
1H), 4.13-4.11 (m, 1H), 3.37-3.19 (m, 3H), 3.05-2.90 (m, 1H),
2.81-2.77 (m, 1H), 2.63-2.58 (m, 1H)
[0096] MS (ESI): m/z=476.1 [M+H].sup.+, 498.2 [M+Na].sup.+
##STR13##
[0097] Synthesis of
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-urea
(X.dbd.NH. X.sub.1.dbd.NH. R.sub.2.dbd., R.sub.3.dbd.Br) (60).
3-Bromo-5-aminomethyl-4,5-dihydroisoazole (20 mg, 0.11 mmol) and
phenyl isocyanate (13 uL, 1.0 eq) were dissolved in the mixture of
THF (0.5 mL) and DMF (0.1 mL). After 30 min of stirring, the
mixture was diluted with ethyl acetate and washed with brine. The
organic layer was dried over Na.sub.2SO.sub.4 and the solvents were
removed by evaporation. The residue was purified by SiO.sub.2
chromatography to give the title compound.
[0098] .sup.1H NMR (acetone-d.sub.6, 400 MHz): .delta.=8.02 (br,
1H), 7.50 (d, 2H), 7.25-7.20 (m, 2H), 6.93 (t, 1H), 6.24 (br, 1H),
4.90-4.86 (m, 1H), 3.56-3.54 (m, 2H), 3.48-3.41 (m, 1H), 3.19-3.13
(m, 1H)
[0099] MS (ESI): m/z=298.0 [M+H].sup.+
[0100] Synthesis of
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2-chloro-5-trifluoromethyl-
-phenyl)-urea (X.sub.1.dbd.NH, X.sub.2.dbd.NH.
R.sub.2=2-chloro-5-trifluoromethyl-phenyl. R.sub.3.dbd.Br) (61).
The title compound was prepared according to the procedure for
compound 60 except using
2-chloro-5-trifluoromethyl-phenylisocyanate.
[0101] .sup.1H NMR (acetone-d.sub.6, 400 MHz): .delta.=8.82 (s,
1H), 8.12 (br, 1H), 7.62 (d, 1H, J=8.0 Hz), 7.30 (d, 1H, J=8.0 Hz),
6.90 (br, 1H), 4.93-4.87 (m, 1H), 3.57-3.54 (m, 2H), 3.49-3.42 (m,
1H), 3.20-3.14 (m, 1H)
[0102] MS (ESI): m/z=400.0 [M+H].sup.+
[0103] Synthesis of
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-chloro-2-trifluoromethyl-
-phenyl)-urea (X.sub.1.dbd.NH, X.sub.2.dbd.NH,
R.sub.2=4-chloro-2-trifluoromethyl-phenyl, R.sub.3.dbd.Br) (62).
The title compound was prepared according to the procedure for
compound 60 except using
4-chloro-2-trifluoromethyl-phenylisocyanate.
[0104] .sup.1H NMR (acetone-d.sub.6, 400 MHz): .delta.=8.20 (d, 1H,
J=7.6 Hz), 7.66 (br, 1H), 7.62-7.60 (m, 2H), 6.82 (br, 1H),
4.89-4.85 (m, 1H), 3.55-3.51 (m, 2H), 3.47-3.40 (m, 1H), 3.18-3.12
(m, 1H)
[0105] MS (ESI): m/z=400.0 [M+H].sup.+
[0106] Synthesis of
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-fluoro-phenyl)-urea
(X.dbd.NH. X.sub.2.dbd.NH. R.sub.2=4-fluoro-phenyl. R.sub.3.dbd.Br)
(63). The title compound was prepared according to the procedure
for compound 60 except using 4-fluoro-phenylisocyanate.
[0107] .sup.1H NMR (acetone-d.sub.6, 200 MHz): .delta.=8.06 (br,
1H), 7.47-7.40 (m, 2H), 6.99-6.90 (m, 2H), 5.94 (br, 1H), 4.82-4.76
(m, 1H), 3.45-3.30 (m, 3H), 3.17-3.04 (m, 1H)
[0108] MS (ESI): m/z=316.0 [M+H].sup.+
[0109] Synthesis of
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2,5-dimethyl-phenyl)-urea
(X.sub.1.dbd.NH, X.sub.2.dbd.NH. R.sub.2=2,5-dimethyl-phenyl.
R.sub.3.dbd.Br) (64). The title compound was prepared according to
the procedure for compound 60 except using
2,5-dimethyl-phenylisocyanate.
[0110] MS (ESI): m/z=326.0 [M+H].sup.+
EXAMPLE 2
Synthesis of Dioxoindole Containing tTGase Inhibitors
[0111] Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid
propylamide. 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl chloride
(0.10 g, 0.41 mmol), prepared by the reaction of the sodium salt of
5-isatinsulfonic acid with POCl.sub.3, was dissolved in 5 mL THF.
This solution was cooled in an ice bath and DIEA (0.14 mL, 2.0 eq)
was added slowly, followed by n-propylamine (35 uL, 1.0 eq).
Stirring was continued for 40 min and the solution was diluted with
ethyl acetate and washed with brine. The organic layer was dried
over Na.sub.2SO.sub.4 and the solvent was removed by evaporation.
The residue was purified by SiO.sub.2 chromatography to give the
title compound (65 mg, 60%).
[0112] .sup.1H NMR (CD.sub.3CN, 400 MHz): .delta.=9.17 (br, 1H),
8.02 (d, 1H, J=8.0 Hz), 7.93 (s, 1H), 7.13 (d, 1H, J=8.0 Hz),
5.62-5.58 (m, 1H), 2.85-2.80 (m, 2H), 1.48-1.42 (m, 2H), 0.85 (t,
3H, J=7.2 Hz)
[0113] MS (ESI): m/z=-267.1 [M-H].sup.-
[0114] Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid
benzylamide. The title compound was prepared from benzyl amine
following the procedure for
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.
[0115] .sup.1H NMR (CD.sub.3CN, 400 MHz): .delta.=9.19 (br, 1H),
7.98 (d, 1H, J=8.4 Hz), 7.85 (s, 1H), 7.31-7.21 (m, 5H), 7.07 (d,
1H, J=8.4 Hz), 6.11 (t, 1H, J=6.3 Hz), 4.11 (d, 2H, J=6.3 Hz)
[0116] MS (ESI): m/z=-315.2 [M-H].sup.-
[0117] [24] Synthesis of
(S)-1-(2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl)-pyrrolidine-2-carboxyl-
ic acid methyl ester. The title compound was prepared from
L-Pro-OMe following the procedure for
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.
[0118] .sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.=8.85 (br, 1H),
8.15-8.11 (m, 2H), 7.11 (d, 1H, J=8.8 Hz), 4.47-4.41 (m, 1H), 3.74
(s, 3H), 3.45-3.39 (m, 2H), 2.20-1.94 (m, 4H)
[0119] MS (ESI): m/z=338.9 [M+H].sup.+
[0120] [28] Synthesis of
(S)-2-(2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonylamino)-3-phenyl-propiona-
mide. The title compound was prepared from L-Phe-NH.sub.2 following
the procedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid
propylamide.
[0121] .sup.1H NMR (CD.sub.3CN, 200 MHz): .delta.=10.70 (br, 1H),
7.78 (d, 1H, J=8.4 Hz), 7.64 (s, 1H), 7.15-7.06 (m, 6H), 6.90 (d,
1H, J=8.4 Hz), 6.79 (br, 1H), 6.08 (br, 1H), 3.98-3.87 (m, 1H),
3.04-2.95 (m, 1H), 2.76-2.64 (m, 1H)
[0122] MS (ESI): m/z=-372.2 [M-H].sup.-
[0123] [32] Synthesis of
(S)--N-(2-Dimethylamino-ethyl)-2-(2,3-dioxo-2,3-dihydro-1H-indole-5-sulfo-
nyl amino)-3-phenyl-propionamide. The title compound was prepared
from L-Phe-NHCH.sub.2CH.sub.2NMe.sub.2 following the procedure for
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.
[0124] .sup.1H NMR (CD.sub.3CN, 400 MHz): .delta.=7.84 (d, 1H,
J=8.0 Hz), 7.69 (s, 1H), 7.22-7.12 (m, 6H), 6.98 (d, 1H, J=8.0 Hz),
6.76 (br, 1H), 3.96-3.93 (m, 1H), 3.10-3.02 (m, 2H), 3.00-2.95 (m,
1H), 2.78-2.72 (m, 1H), 2.22-2.17 (m, 2H), 2.15 (s, 6H)
[0125] MS (ESI): m/z=445.2 [M+H].sup.+
[0126] Synthesis of 6-Bromo-7-methyl-1H-indole-2,3-dione. Chloral
alcoholate (0.43 g, 1.05 eq) and Na.sub.2SO.sub.4 (2.84 g, 20 mmol)
were dissolved in 10 mL water. 3-Bromo-2-methylaniline (0.33 g,
1.77 mmol) was added to the solution followed by 0.16 mL conc. HCl
aqueous solution and NH.sub.2OH.HCl (0.38 g, 3.0 eq). The mixture
was refluxed for 15 min and stirring was continued for additional 1
hr at RT. The precipitate was collected by filtration, washed with
water and dried under vacuum. This precipitate was dissolved in 1
mL H.sub.2SO.sub.4 and the solution was heated (80.degree. C.) for
15 min. After cooling down to RT, the mixture was poured into
ice-water mixture and the precipitate was collected, washed with
water and dried under vacuum to give the title compound (0.26 g,
61%).
[0127] .sup.1H NMR (CD.sub.3CN, 200 MHz): .delta.=9.02 (BR, 1 h),
7.38 (d, 1H, J=7.8 Hz), 7.30 (d, 1H, J=7.8 Hz), 2.30 (s, 3H)
[0128] MS (ESI): m/z=-238.2 [M-H].sup.-
[0129] Synthesis of 7-Methyl-6-phenyl-1H-indole-2,3-dione.
6-Bromo-7-methyl-1H-indole-2,3-dione (100 mg, 0.38 mmol) and
phenylboronic acid (53 mg, 1.1 eq) were dissolved in 10 mL DME.
Pd(PPh.sub.3).sub.4 (22 mg, 0.05 eq) were added followed by
NaHCO.sub.3 (65 mg, 2.0 eq) dissolved in 10 mL water. The mixture
was refluxed for 2.5 hr and the organic solvent was removed by
evaporation. The mixture was extracted with ethyl acetate and the
combined organic layers were dried over Na.sub.2SO.sub.4 and
purified by SiO.sub.2 chromatography to give the title compound (50
mg, 51%).
[0130] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.=8.53 (br, 1H),
7.52 (d, 1H, J=7.6 Hz), 7.49-7.43 (m, 3H), 7.31 (d, 2H, J=6.4 Hz),
7.03 (d, 1H, J=7.6 Hz), 2.16 (s, 3H) MS (ESI): m/z=-236.3
[M-H].sup.-
[0131] Inhibition of tTG. tTG (9 .mu.M) was inactivated in 200 mM
MOPS, pH=7.1, 5 mM CaCl.sub.2, 1 mM ETDA at 30.degree. C.
containing 0-600 .mu.M Pro-Gln-Pro-Aci-Leu-Pro-Tyr. Every 20
minutes a 40 .mu.l aliquot was removed and residual tTG activity
was assayed in 0.5 ml reaction containing 200 mM MOPS, pH=7.1, 5 mM
CaCl.sub.2, 1 mM ETDA, 10 mM .alpha.-ketoglutarate, 180 U/ml
glutamate dehydrogenase (Biozyme laboratories) at 30.degree. C. for
20 minutes by measuring the decrease of absorption at 340 nm.
Residual activity was corrected by the corresponding uninhibited
tTG reaction (0 .mu.M inhibitor) and fitted to an exponential
decay. Kinetic parameters were obtained by double-reciprocal
plotting of the apparent second-order inactivation constant or, for
isatin analogs, by fitting the data for reversible inhibitors to a
standard Michaelis Menten equation with a competitive inhibition
constant. The results of these inhibition experiments are shown in
Tables 1 and 2 below. TABLE-US-00001 TABLE 1 Tissue
transglutaminase inhibition by dihydroisoxazoles K.sub.l k.sub.inh
k.sub.inh/K.sub.l Tested Compound (M) (min.sup.-1)
(min.sup.-1M.sup.-1) {(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.73
.times. 10.sup.-3 1.4 1900
ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamic acid benzyl ester
(49) (S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5- 1.6 .times.
10.sup.-3 0.32 200 dihydro-isoxazol-5-ylmethyl)-carbamoyl]-butyric
acid methyl ester (50) )
(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5- 0.87 .times. 10.sup.-3
0.43 490 dihydro-isoxazol-5-ylmethyl)-succinamic acid methyl ester
(51) (S)-2-Benzyloxycarbonylamino-3-phenyl- 1.3 .times. 10.sup.-3
0.32 230 propionic acid 3-bromo-4,5-dihydro-isoxazol-5- ylmethyl
ester (52) {(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.91 .times.
10.sup.-3 0.41 450 ylmethyl)-carbamoyl]-ethyl}-carbamic acid benzyl
ester (53) (S)-2-Acetylamino-N-(3-bromo-4,5-dihydro- 2.7 .times.
10.sup.-3 0.60 220 isoxazol-5-ylmethyl)-3-phenyl-propionamide (54)
{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.31 .times. 10.sup.-3
0.29 940 ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamic acid benzyl
ester (55) {(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro- 0.24
.times. 10.sup.-3 0.54 2300
isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamic acid benzyl ester
(56) [(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.31 .times.
10.sup.-3 0.78 2500 ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl]-
carbamic acid benzyl ester (57)
{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.26 .times. 10.sup.-3
0.19 730 ylmethyl)-methyl-carbamoyl]-2-phenyl-ethyl}- carbamic acid
benzyl ester (58) [(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.42
.times. 10.sup.-3 0.86 2000
ylmethyl)-methyl-carbamoyl]-2-(4-hydroxy- phenyl)-ethyl]-carbamic
acid benzyl ester (59)
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 1.1 .times.
10.sup.-3 0.89 810 phenyl-urea (60)
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 0.91 .times.
10.sup.-3 0.95 1000 (2-chloro-5-trifluoromethyl-phenyl)-urea (61)
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 1.3 .times.
10.sup.-3 1.1 850 (4-chloro-2-trifluoromethyl-phenyl)-urea (62)
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 1.3 .times.
10.sup.-3 1.0 770 (4-fluoro-phenyl)-urea (63)
1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 0.96 .times.
10.sup.-3 0.97 1000 (2,5-dimethyl-phenyl)-urea (64)
[0132] TABLE-US-00002 TABLE 2 Tissue transglutaminase inhibition by
Istatin derivatives ##STR14## Tested compound (R) K.sub.I (M) R1 =
R2 = R3 = H 8.6 .times. 10.sup.-4 R2 = R3 = H, R1 = NO.sub.2 4.8
.times. 10.sup.-5 R2 = R3 = H; R1 = I 2.2 .times. 10.sup.-5 R2 = R3
= H; R1 = F 1.8 .times. 10.sup.-5 R1 = R2 = H; R3 = Ph 4 .times.
10.sup.-4 R1 = R3 = H; R2 = Ph 3.5 .times. 10.sup.-4
[0133] The above results demonstrate that the compounds tested have
tTGase inhibitory activity.
[0134] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0135] The present invention has been described in terms of
particular embodiments found or proposed by the present inventor to
comprise preferred modes for the practice of the invention. It will
be appreciated by those of skill in the art that, in light of the
present disclosure, numerous modifications and changes can be made
in the particular embodiments exemplified without departing from
the intended scope of the invention. Moreover, due to biological
functional equivalency considerations, changes can be made in
protein structure without affecting the biological action in kind
or amount. All such modifications are intended to be included
within the scope of the appended claims.
Sequence CWU 1
1
8 1 10 PRT Artificial Sequence synthetic polypeptides 1 Leu Pro Tyr
Pro Gln Pro Gln Leu Pro Tyr 1 5 10 2 10 PRT Artificial Sequence
synthetic polypeptides VARIANT (10)...(10) Phenylalanine with NH2
attached 2 Leu Pro Phe Pro Gln Pro Gln Leu Pro Phe 1 5 10 3 9 PRT
Artificial Sequence synthetic polypeptides 3 Leu Pro Tyr Pro Gln
Pro Gln Leu Pro 1 5 4 16 PRT Artificial Sequence synthetic
polypeptides 4 Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro
Gln Pro Phe 1 5 10 15 5 10 PRT Artificial Sequence synthetic
polypeptides 5 Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro 1 5 10 6 10
PRT Artificial Sequence synthetic polypeptides ACETYLATION
(1)...(1) 6 Pro Gln Pro Gln Leu Pro Phe Pro Gln Pro 1 5 10 7 8 PRT
Artificial Sequence synthetic polypeptides 7 Gln Leu Gln Pro Phe
Pro Gln Pro 1 5 8 15 PRT Artificial Sequence synthetic polypeptides
8 Leu Gln Leu Gln Pro Phe Pro Gln Pro Leu Pro Tyr Pro Gln Pro 1 5
10 15
* * * * *
References