U.S. patent application number 17/254854 was filed with the patent office on 2021-09-09 for novel compounds.
The applicant listed for this patent is UCL BUSINESS LTD. Invention is credited to Alexis DENIS, Nerina DODIC, John LIDDLE, David LOMAS, Kate SMITH.
Application Number | 20210276952 17/254854 |
Document ID | / |
Family ID | 1000005613291 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210276952 |
Kind Code |
A1 |
SMITH; Kate ; et
al. |
September 9, 2021 |
NOVEL COMPOUNDS
Abstract
The present invention relates to compounds, compositions,
combinations and medicaments containing said compounds and
processes for their preparation. The invention also relates to the
use of said compounds, combinations, compositions and medicaments,
for example as modulators of alpha I antitrypsin and treating
diseases associated with alpha antitrypsin, particularly liver
diseases.
Inventors: |
SMITH; Kate; (Brentford,
GB) ; DENIS; Alexis; (Brentford, GB) ; DODIC;
Nerina; (Brentford, GB) ; LIDDLE; John;
(Brentford, GB) ; LOMAS; David; (London,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCL BUSINESS LTD |
London |
|
GB |
|
|
Family ID: |
1000005613291 |
Appl. No.: |
17/254854 |
Filed: |
June 21, 2019 |
PCT Filed: |
June 21, 2019 |
PCT NO: |
PCT/GB2019/051761 |
371 Date: |
December 21, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 209/34
20130101 |
International
Class: |
C07D 209/34 20060101
C07D209/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2018 |
GB |
1810290.5 |
May 13, 2019 |
GB |
1906708.1 |
Claims
1. A compound having a molecular weight of 1000 Daltons or less
that is capable of inhibiting .alpha..sub.1-antitrypsin
polymerisation; or a pharmaceutically acceptable solvate, complex,
tautomer, isotopically labelled derivative or prodrug thereof.
2-33. (canceled)
34. The compound of claim 1, wherein the compound is capable of
binding to .alpha..sub.1-antitrypsin by inducing formation of a
cryptic binding site within the .alpha..sub.1-antitrypsin protein
structure, said .alpha..sub.1-antitrypsin comprising the sequence
of SEQ ID NO: 1.
35. The compound of claim 1, wherein the binding site is located
between .beta.-sheet-A and .beta.-sheet-B of said
.alpha..sub.1-antitrypsin, wherein: a) said .beta.-sheet-A
comprises the amino acids corresponding to residues 140-144,
111-121, 181-191, 330-340 and 292-299 of SEQ ID NO: 1; and said
.beta.-sheet-B comprises the amino acids corresponding to residues
228-231, 236-244, 248-256, 369-376, 381-389, and 49-53 of SEQ ID
NO: 1 or b) wherein the binding site is located between amino acid
strands corresponding to each of: (a) residues 191-194 of SEQ ID
NO: 1; (b) residues 288-293 of SEQ ID NO: 1; (c) residues 371-374
of SEQ ID NO: 1; (d) residues 249-253 of SEQ ID NO: 1; and (e)
residues 240-243 of SEQ ID NO: 1; and optionally also (f) residues
338-341 of SEQ ID NO: 1.
36. The compound of claim 35, wherein the binding site comprises
one or more of W194, Y244, L291, P289, F252, K290, I293, L338,
I340, F372 and M374 of SEQ ID NO: 1.
37. The compound of claim 34, wherein: the K.sub.D of the compound
to M-.alpha..sub.1-antitrypsin is less than about 250 nM, said
M-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO:
2; or wherein the K.sub.D of the compound to
Z-.alpha..sub.1-antitrypsin is less than about 25 nM, said
Z-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO:
3; or wherein the K.sub.D of the compound to
Z-.alpha..sub.1-antitrypsin is at least ten times lower than the
K.sub.D of the compound to M-.alpha..sub.1-antitrypsin, said
M-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 2
and said Z-.alpha..sub.1-antitrypsin comprising the sequence of SEQ
ID NO: 3.
38. The compound of claim 1, wherein the compound comprises a
tetravalent moiety of formula (IA) ##STR00041## wherein the
compound is capable of binding to .alpha..sub.1-antitrypsin
comprising the sequence of SEQ ID NO: 1 by hydrogen bond formation
between: (i) hydroxyl group I and L291 of SEQ ID NO: 1; (ii) NH
group II and P289 of SEQ ID NO: 1; and (iii) carbonyl group III and
Y244 of SEQ ID NO: 1.
39. The compound of claim 1, wherein the compound comprises a
divalent moiety of formula (IB) ##STR00042## wherein: the compound
is capable of binding to .alpha..sub.1-antitrypsin comprising the
sequence of SEQ ID NO: 1 by hydrogen bond formation between: (i)
hydroxyl group I and L291 of SEQ ID NO: 1; (ii) NH group II and
P289 of SEQ ID NO: 1; and (iii) carbonyl group III and Y244 of SEQ
ID NO: 1; R.sub.4 is selected from hydrogen, C.sub.1-5 alkyl,
C.sub.2-5 alkenyl, C.sub.2-5 alkynyl and C.sub.1-4 alkoxy; and
R.sub.5 is selected from hydrogen, C.sub.1-5 alkyl, C.sub.2-5
alkenyl, C.sub.2-5 alkynyl and C.sub.1-4 alkoxy.
40. The compound of claim 39, wherein R.sub.5 is hydrogen; and/or
R.sub.4 is C.sub.2-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl or
C.sub.1-3 alkoxy; and/or R.sub.4 is n-propyl.
41. The compound of claim 1, wherein the compound comprises a
monovalent moiety of formula (IC) ##STR00043## wherein: the
compound is capable of binding to .alpha..sub.1-antitrypsin
comprising the sequence of SEQ ID NO: 1 by hydrogen bond formation
between: (i) hydroxyl group I and L291 of SEQ ID NO: 1; (ii) NH
group II and P289 of SEQ ID NO: 1; and (iii) carbonyl group III and
Y244 of SEQ ID NO: 1; R.sub.4 is selected from hydrogen, C.sub.1-5
alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl and C.sub.1-4 alkoxy;
and R.sub.5 is selected from hydrogen, C.sub.1-5 alkyl, C.sub.2-5
alkenyl, C.sub.2-5 alkynyl and C.sub.1-4 alkoxy; and R.sub.6 is a
substituted or unsubstituted aryl or heteroaryl group capable of
stacking with the side chain of W194 of SEQ ID NO: 1.
42. The compound of claim 41, wherein: R.sub.6 is a substituted or
unsubstituted 4-oxindolyl group; or R.sub.6 is a group of formula
R.sub.6' ##STR00044## wherein R.sub.1 is selected from the group
consisting of H, F, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Cl, Br and I.
43. The compound of claim 42, wherein R.sub.1 is selected from the
group consisting of H, F, CH.sub.3, NH.sub.2, OH and Cl; optionally
wherein R.sub.1 is selected from the group consisting of H and
F.
44. The compound of claim 1, wherein the compound has the formula
(ID) ##STR00045## wherein: the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1; R.sub.4 is
selected from hydrogen, C.sub.1-5 alkyl, C.sub.2-5 alkenyl,
C.sub.2-5 alkynyl and C.sub.1-4 alkoxy; R.sub.5 is selected from
hydrogen, C.sub.1-5 alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl and
C.sub.1-4 alkoxy; R.sub.6 is a group of formula R.sub.6'
##STR00046## wherein R.sub.1 is selected from the group consisting
of H, F, CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Cl, Br and I; and R.sub.7 is a substituted or unsubstituted aryl or
heteroaryl group.
45. The compound of claim 44, wherein R.sub.7 is a substituted or
unsubstituted phenyl group; optionally wherein R.sub.7 is a group
of formula R.sub.7' ##STR00047## wherein: R.sub.2 is selected from
the group consisting of CH.sub.3, Cl, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, SH, CN, F, Br and I; and R.sub.3 is selected
from the group consisting of F, Cl, CN, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Br, I and SH.
46. The compound of claim 45, wherein R.sub.2 is selected from the
group consisting of CH.sub.3, Cl, NH.sub.2, OH, SH, CN and F,
optionally wherein R.sub.2 is selected from the group consisting of
CH.sub.3 and Cl; and R.sub.3 is selected from the group consisting
of F, Cl, CN, CH.sub.3, NH.sub.2, OH and SH, optionally wherein
R.sub.3 is selected from the group consisting of F, Cl and CN.
47. The compound of claim 1, wherein the compound has the formula
(I) ##STR00048## wherein R.sub.1 is selected from the group
consisting of H, F, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Cl, Br and I; R.sub.2 is selected from the
group consisting of CH.sub.3, Cl, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, SH, CN, F, Br and I; and R.sub.3 is selected
from the group consisting of F, Cl, CN, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Br, I and SH.
48. The compound of claim 47, wherein R.sub.1 is selected from the
group consisting of H, F, CH.sub.3, NH.sub.2, OH and Cl, optionally
wherein R.sub.1 is selected from the group consisting of H and F;
R.sub.2 is selected from the group consisting of CH.sub.3, Cl,
NH.sub.2, OH, SH, CN and F, optionally wherein R.sub.2 is selected
from the group consisting of CH.sub.3 and Cl; and R.sub.3 is
selected from the group consisting of F, Cl, CN, CH.sub.3,
NH.sub.2, OH and SH, optionally wherein R.sub.3 is selected from
the group consisting of F, Cl and CN.
49. The compound of claim 48, wherein R.sub.1 is H, R.sub.2 is
CH.sub.3 and R.sub.3 is F, or R.sub.1 is H, R.sub.2 is CH.sub.3 and
R.sub.3 is Cl, or R.sub.1 is F, R.sub.2 is Cl and R.sub.3 is CN, or
R.sub.1 is F, R.sub.2 is Cl and R.sub.3 is F.
50. A method for treating of a disease or condition mediated by
.alpha..sub.1-antitrypsinpolymerisation, comprising administering
to a patient in need thereof a compound according to claim 1.
51. The method of claim 50, wherein the disease or condition is
mediated by Z-.alpha..sub.1-antitrypsin polymerisation and the
compound is capable of inhibiting Z-.alpha..sub.1-antitrypsin
polymerisation.
52. A method for identifying a drug candidate compound, the method
comprising: i) contacting the drug candidate compound with
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
to form a complex between the drug candidate compound and said
.alpha..sub.1-antitrypsin; ii) resolving the structure of the
complex; and iii) determining whether, in the complex, the drug
candidate compound is present in a binding site as defined in claim
35; optionally wherein said contacting the drug candidate compound
with said .alpha..sub.1-antitrypsin comprises forming a crystal of
said .alpha..sub.1-antitrypsin and contacting said crystal with
said drug candidate.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compounds, compositions,
combinations and medicaments containing said compounds and
processes for their preparation. The invention also relates to the
use of said compounds, combinations, compositions and medicaments,
for example in the treatment of diseases and conditions associated
with alpha 1 antitrypsin.
BACKGROUND OF THE INVENTION
[0002] Many human genetic disorders are caused by mutations that
impair protein folding and trafficking. The proteins may be
produced in normal amounts but because of their impaired folding
can lead to problems.
[0003] Alpha-1-antitrypsin or aiantitrypsin (A1AT) is a protease
inhibitor belonging to the serpin superfamily. It is a protein made
in hepatocytes and, and to a lesser extent other cells, and
secreted into the blood where it functions to limit enzymatic
activity of key proteases, in particular neutrophil elastase. In
its absence (such as in alpha-1-antitrypsin deficiency) the
activity of key proteases including neutrophil elastase is
unchecked resulting in excessive break down of elastin and
connective tissues. The most common tissue in which this manifests
pathologically is in the lung where the increased degradation of
lung connective tissue commonly results in respiratory
complications such as emphysema or chronic obstructive pulmonary
disease (COPD). More rarely other tissues can also be affected by
an A1AT deficiency/dysfunction such as the skin.
[0004] In many patients alpha-1-antitrypsin deficiency is caused by
mutations including for example an E342K (Glu342Lys) missense
mutation herein referred to as the Z mutant (Z-AT). Mutant Z
alpha-1-antitrypsin forms polymers which accumulate in cells and
can disturb function of the affected tissue. The organ most
commonly affected by a build-up of polymer in alpha-1 antitrypsin
deficiency is the liver, causing damage to the liver and in severe
cases progressing to a requirement for liver transplant. Polymers
of A1AT are also found in other tissues including blood, lungs and
skin. Polymers have been shown to be pro-inflammatory and may
contribute to pathology in tissues where they are found,
particularly the lung and the skin.
[0005] Current therapy for conditions associated with
alpha-1-antitrypsin deficiency is limited to protein replacement
therapy with M-AT (wild type alpha 1 antitrypsin protein) derived
from human plasma, typically dosed on a weekly basis. While such
therapy is effective for lung pathology including emphysema, it has
no effect on liver disease caused by the accumulation of
polymerized Z-AT in the ER of hepatocytes. For the 10-15% of
homozygotes for Z-AT afflicted liver disease including fibrosis,
cirrhosis and hepatocellular carcinoma, liver transplantation is
the only treatment option available. Furthermore, accumulation of
polymerized Z-AT in lung epithelium has a chemoattractant effect on
neurophils, which may cause further destruction of connective lung
tissue. Thus there is a need for therapeutics and methods that
address disease conditions associated with both alpha-1-antitrypsin
deficiency as well as toxic accumulation of
alpha-1-antitrypsin.
[0006] The present inventors have identified compounds which are
capable of modulating alpha 1 antitrypsin, particularly the mutant
Z-AT form, preventing its polymerization in the liver thus being
potentially useful in treating diseases associated with alpha 1
antitrypsin, more particularly with mutant forms of alpha 1
antitrypsin, particularly Z-AT, including diseases of the
liver.
[0007] The present inventors have, in particular, identified
compounds that are capable of binding to .alpha..sub.1-antitrypsin
in such a way as to prevent polymerisation of the protein and
thereby give rise to beneficial therapeutic effects. While
.alpha..sub.1-antitrypsin proteins have previously been
crystallised and their three-dimensional structure studied, the
binding site of the compounds identified by the inventors has not
previously been identified. As discussed in more detail herein, it
is a cryptic binding site formed via interaction between the
protein and the present compounds, the specific location and
structure of which gives rise to the beneficial inhibition of
protein polymerisation. The present inventors have, furthermore,
identified specific structural motifs that contribute to the
creation of the relevant cryptic binding site and binding of the
compounds therein. Thus, the present invention therefore addresses
a longstanding need for the provision of compounds that are capable
of treating diseases or conditions mediated by
.alpha..sub.1-antitrypsin polymerisation.
SUMMARY OF THE INVENTION
[0008] In a first aspect, the present invention provides a
substance for use in a method for treatment of a disease or
condition mediated by .alpha..sub.1-antitrypsin polymerisation,
wherein said substance is: (a) a compound that is capable of
inhibiting .alpha..sub.1-antitrypsin polymerisation; or (b) a
pharmaceutically acceptable solvate, complex, tautomer,
isotopically labelled derivative or prodrug thereof. Preferably,
the disease or condition is mediated by Z-.alpha..sub.1-antitrypsin
polymerisation and the compound is capable of inhibiting
Z-.alpha..sub.1-antitrypsin polymerisation. The substance is
typically a small molecule compound, e.g. a compound that has a
molecular weight of 1000 Daltons or less.
[0009] Preferably, the compound is capable of binding to
.alpha..sub.1-antitrypsin by inducing formation of a cryptic
binding site within the .alpha..sub.1-antitrypsin protein
structure, said .alpha..sub.1-antitrypsin comprising the sequence
of SEQ ID NO: 1. For instance, the binding site may be located
between .beta.-sheet-A and .beta.-sheet-B of said
.alpha..sub.1-antitrypsin, wherein: said .beta.-sheet-A comprises
the amino acids corresponding to residues 140-144, 111-121,
181-191, 330-340 and 292-299 of SEQ ID NO: 1; and said
.beta.-sheet-B comprises the amino acids corresponding to residues
228-231, 236-244, 248-256, 369-376, 381-389, and 49-53 of SEQ ID
NO: 1. More preferably, the binding site is located between amino
acid strands corresponding to each of: (a) residues 191-194 of SEQ
ID NO: 1; (b) residues 288-293 of SEQ ID NO: 1; (c) residues
371-374 of SEQ ID NO: 1; (d) residues 249-253 of SEQ ID NO: 1; and
(e) residues 240-243 of SEQ ID NO: 1; and optionally also (f)
residues 338-341 of SEQ ID NO: 1. The binding site may comprise one
or more of W194, Y244, L291, P289, F252, K290, I293, L338, I340,
F372 and M374 of SEQ ID NO: 1.
[0010] Preferably, the K.sub.D of the compound to
M-.alpha..sub.1-antitrypsin is less than about 250 nM, said
M-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO:
2. Preferably, the K.sub.D of the compound to
Z-.alpha..sub.1-antitrypsin is less than about 25 nM, said
Z-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO:
3. Preferably, the K.sub.D of the compound to
Z-.alpha..sub.1-antitrypsin is at least ten times lower than the
K.sub.D of the compound to M-.alpha..sub.1-antitrypsin, said
M-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 2
and said Z-.alpha..sub.1-antitrypsin comprising the sequence of SEQ
ID NO: 3.
[0011] The compound may comprise a tetravalent moiety of formula
(IA)
##STR00001##
wherein the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1. For instance,
the compound may comprise a divalent moiety of formula (TB)
##STR00002##
wherein: the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1; R.sub.4 is
selected from hydrogen, C.sub.1-5 alkyl, C.sub.2-5 alkenyl,
C.sub.2-5 alkynyl and C.sub.1-4 alkoxy; and R.sub.5 is selected
from hydrogen, C.sub.1-5 alkyl, C.sub.2-5 alkenyl, C.sub.2-5
alkynyl and C.sub.1-4 alkoxy. Optionally R.sub.5 is hydrogen.
Optionally R.sub.4 is C.sub.2-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl or C.sub.1-3 alkoxy. Optionally R.sub.4 is n-propyl.
[0012] More specifically, the compound may comprise a monovalent
moiety of formula (IC)
##STR00003##
[0013] wherein: the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1; R.sub.4 and
R.sub.5 are as defined above; and R.sub.6 is a substituted or
unsubstituted aryl or heteroaryl group capable of stacking with the
side chain of W194 of SEQ ID NO: 1. Optionally, R.sub.6 is a
substituted or unsubstituted 4-oxindolyl group. For instance,
optionally R.sub.6 is a group of formula R.sub.6'
##STR00004##
wherein R.sub.1 is selected from the group consisting of H, F,
CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Cl, Br and I. Optionally R.sub.1 is selected from the group
consisting of H, F, CH.sub.3, NH.sub.2, OH and Cl. For instance
optionally R.sub.1 is selected from the group consisting of H and
F.
[0014] More specifically still, the compound may have the formula
(ID)
##STR00005##
wherein: the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1; R.sub.4 and
R.sub.5 are as defined above; R.sub.6 is as defined above; and
R.sub.7 is a substituted or unsubstituted aryl or heteroaryl
group.
[0015] Optionally R.sub.7 is a substituted or unsubstituted phenyl
group. For instance, optionally R.sub.7 is a group of formula
R.sub.7'
##STR00006##
wherein: R.sub.2 is selected from the group consisting of CH.sub.3,
Cl, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2,
NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH, SH, CN, F, Br and I;
and R.sub.3 is selected from the group consisting of F, Cl, CN,
CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Br, I and SH. Optionally, R.sub.2 is selected from the group
consisting of CH.sub.3, Cl, NH.sub.2, OH, SH, CN and F; and R.sub.3
is selected from the group consisting of F, Cl, CN, CH.sub.3,
NH.sub.2, OH and SH. For instance, optionally R.sub.2 is selected
from the group consisting of CH.sub.3 and Cl; and R.sub.3 is
selected from the group consisting of F, Cl and CN.
[0016] For instance, the compound may have the formula (I)
##STR00007##
wherein: R.sub.1 is selected from the group consisting of H, F,
CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Cl, Br and I; R.sub.2 is selected from the group consisting of
CH.sub.3, Cl, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
SH, CN, F, Br and I; and R.sub.3 is selected from the group
consisting of F, Cl, CN, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Br, I and SH.
[0017] Optionally in formula (I): R.sub.1 is selected from the
group consisting of H, F, CH.sub.3, NH.sub.2, OH and Cl; R.sub.2 is
selected from the group consisting of CH.sub.3, Cl, NH.sub.2, OH,
SH, CN and F; and R.sub.3 is selected from the group consisting of
F, Cl, CN, CH.sub.3, NH.sub.2, OH and SH. For example, optionally:
R.sub.1 is selected from the group consisting of H and F; R.sub.2
is selected from the group consisting of CH.sub.3 and Cl; and
R.sub.3 is selected from the group consisting of F, Cl and CN. For
instance, optionally: R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3
is F; or R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3 is Cl; or
R.sub.1 is F, R.sub.2 is Cl and R.sub.3 is CN; or R.sub.1 is F,
R.sub.2 is Cl and R.sub.3 is F.
[0018] The present invention also provides a substance as defined
above. Still further, the present invention provides a method for
identifying a drug candidate compound, the method comprising:
contacting the drug candidate compound with
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
to form a complex between the drug candidate compound and said
.alpha..sub.1-antitrypsin; resolving the structure of the complex;
and determining whether, in the complex, the drug candidate
compound is present in a binding site as defined above.
[0019] In still further aspects, the present invention provides the
following embodiments [1] to [11].
[1] a compound that: (a) has the formula (I):
##STR00008##
wherein: R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3 is F; or
R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3 is Cl; or R.sub.1 is
F, R.sub.2 is Cl and R.sub.3 is CN; or R.sub.1 is F, R.sub.2 is Cl
and R.sub.3 is F; or (b) is a pharmaceutically acceptable solvate,
complex, tautomer, isotopically labeled derivative or prodrug
thereof. [2] A compound as defined in [1] for use in therapy. [3] A
compound as defined in [1] for use in the treatment of a disease or
condition mediated by alpha 1 antitrypsin. [4] A pharmaceutical
composition comprising a compound as defined in [1] and one or more
of pharmaceutically acceptable carriers, diluents and excipients.
[5] A method of treating a disease or condition mediated by alpha 1
antitrypsin in a subject comprising administering a therapeutically
effective amount of a compound as defined in [1]. [6] Use of a
compound as defined in [1], in the manufacture of a medicament for
use in treating a disease or condition mediated by alpha 1
antitrypsin. [7] A combination comprising a compound as defined in
[1], and at least one further therapeutic agent. [8] A combination
comprising a compound as defined in [1] and at least one further
therapeutic agent for use in therapy, particularly for treating a
disease or condition mediated by alpha 1 antitrypsin. [9] A
combination comprising a compound as defined in [1] and at least
one further therapeutic agent for use in treating a disease or
condition mediated by alpha 1 antitrypsin. [10] A method of
treating a disease or condition mediated by alpha 1 antitrypsin
comprising administering to a human in need thereof a
therapeutically effective amount of a combination comprising a
compound as defined in [1], and at least one further therapeutic
agent. [11] Use of a combination comprising a compound as defined
in [1] and at least one further therapeutic agent in the
manufacture of a medicament for treating a disease or condition
mediated by alpha 1 antitrypsin.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a representative image of the crystallised complex
formed between a representative compound of the present invention
and an .alpha..sub.1-antitrypsin protein, as described in more
detail in Example 2.
[0021] FIG. 2 is a representative image of a portion of the
crystallised complex formed between a representative compound of
the present invention and an .alpha..sub.1-antitrypsin protein, as
described in more detail in Example 2 (specifically the portion
contains the binding site between the protein and the compound of
the invention).
[0022] FIG. 3 is a schematic, two-dimension visualisation of a
representative compound of the present invention at the binding
site of the protein, and highlighting some of the amino acid
residues that comprise the binding site as well as partial chemical
structures within the protein that form significant intermolecular
interactions (e.g., hydrogen bonds) to the compound (shown as
broken lines), as described in more detail in Example 2.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0023] As used herein, an alkyl group is a straight or branched
saturated hydrocarbon radical. In one example an alkyl group
contains 1 to 5 carbon atoms. Examples of alkyl groups include
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,
tert-butyl, n-pentyl, isopentyl and neopentyl.
[0024] As used herein, an alkenyl group is a straight or branched
hydrocarbon radical that contains one or more (e.g. one) carbon
carbon double bonds. In one example an alkenyl group contains 2 to
5 carbon atoms.
[0025] As used herein, an alkenyl group is a straight or branched
hydrocarbon radical that contains one or more (e.g. one) carbon
carbon triple bonds. In one example an alkynyl group contains 2 to
5 carbon atoms.
[0026] As used herein, an alkoxy group (e.g., a C.sub.1-4 alkoxy
group) is a group of formula --OR in which R is an alkyl (e.g. a
C.sub.1-4 alkyl) group.
[0027] As used herein, an alkylthiol group (e.g., a C.sub.1-4
alkylthiol group) is a group of formula --SR in which R is an alkyl
(e.g. a C.sub.1-4 alkyl) group.
[0028] As used herein, an aryl group is typically a C.sub.6-14
mono-carbocyclic, aromatic ring or poly-carbocyclic ring system,
wherein at least one of the rings therein is aromatic. Preferably
such a group is a C.sub.6-10 mono-carbocyclic, aromatic ring or
bi-carbocyclic ring system, wherein at least one of the rings
therein is aromatic. Examples include phenyl, naphthyl, and
indanyl. Unless expressly indicated otherwise, valency may be
located on any atom of any ring of the aryl group.
[0029] As used herein, a heteroaryl group is typically a 5 to 14
ring atom-containing monocyclic, aromatic ring or polycyclic ring
system, wherein at least one of the rings therein is aromatic. The
heteroaryl group contains at least one (e.g., 1, 2, 3 or 4) ring
heteroatom selected from O, S, N (e.g., forming at least one ring
O, S(O).sub.x (in which x is 0, 1 or 2), N, NH or N.sup.+O.sup.-
moiety), with the other ring atoms being carbon atoms. Preferably
such a group contains 5 to 10 ring atoms. Unless expressly
indicated otherwise, valency may be located on any atom of any ring
of the heteroaryl group.
[0030] Examples of monocyclic heteroaryl groups include thienyl,
furyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl,
oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and
tetrazolyl groups.
[0031] Examples of polycyclic heteroaryl groups include oxindolyl,
benzothienyl, benzofuranyl, benzimidazolyl, benzothiazolyl,
benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benztriazolyl,
indolyl, isoindolyl and indazolyl groups. Oxindolyl includes
2-oxindolyl (i.e. a monovalent group derived from 2-oxindole, also
known as 2-Indolinone) and 3-oxindolyl (i.e., a monovalent group
derived from 3-oxindole, also known as 3-Indolinone).
[0032] In an aryl group or heteroaryl group, at least one (e.g. 1,
2 or 3) carbon ring atom may be substituted by a carbonyl group
(i.e. --C(O)--). For instance, a benzoxazolyl may optionally
comprise --C(O)-- in place of carbon at its 2-position, thereby
giving rise to a monovalent heteroaryl group derived from
benzoxazolone.
[0033] Unless otherwise specified, an aryl or heteroaryl group is
typically unsubstituted. However, where such a group is indicated
to be unsubstituted or substituted, one or more hydrogen atoms are
optionally replaced by deuterium atoms, halogen atoms or hydroxyl,
thiol (--SH), nitro, sulfonic acid, nitrile (--CN), amino (e.g.,
--NR.sub.2 where each R is independently selected from H and
C.sub.1-5 alkyl), alkyl (e.g., C.sub.1-5 alkyl), deuterarated alkyl
(e.g., C.sub.1-5 deuterated alkyl), alkenyl (e.g., C.sub.2-5
alkenyl), alkynyl (e.g., C.sub.2-5 alkynyl), haloalkyl (e.g.,
C.sub.1-5 haloalkyl), haloalkenyl (e.g., C.sub.2-5 haloalkenyl),
alkoxy (e.g., C.sub.1-4 alkoxy), alkylthio (e.g., C.sub.1-4
alkylthio), alkylsulfonyl (e.g., C.sub.1-4 alkylsulfonyl),
haloalkoxy (e.g., C.sub.1-4 haloalkoxy), haloalkylthio (e.g.,
C.sub.1-4 haloalkylthio), haloalkylsulfonyl (e.g., C.sub.1-4
haloalkylsulfonyl), cycloalkyl (e.g., C.sub.3-5 cycloalkyl) or
heterocycloalkyl (e.g., C.sub.3-5 heterocycloalkyl).
[0034] Preferred such substituents are deuterium atoms, fluorine
atoms, chlorine atoms, or hydroxyl, nitro, nitrile (--CN),
--NR.sub.2 (where each R is independently selected from H and
methyl), C.sub.1-5 alkyl, --CD.sub.3, C.sub.2-3 alkenyl, C.sub.2-3
alkynyl, CF.sub.3, CHF.sub.2, CH.sub.2CF.sub.3, C.sub.2F.sub.5,
CF.dbd.CF.sub.2, C.sub.1-3 alkoxy, C.sub.1-3 alkylthio, C.sub.1-3
alkylsulfonyl, OCF.sub.3, SCF.sub.3, SO.sub.2CF.sub.3, cyclopropyl,
cyclobutyl, oxetanyl and azetidinyl.
[0035] Preferably, a substituted aryl or heteroaryl group has from
1 to 5 substituents, more preferably 1 to 3 substituents and most
preferably 1 or 2 substituents. Preferably a substituted aryl or
heteroaryl group carries not more than 2 nitro substituents and not
more than 2 sulfonic acid substituents.
[0036] As used herein, halogen atoms are typically F, Cl, Br or I
atoms, preferably F or Cl atoms.
[0037] For the avoidance of doubt, the terms
.alpha..sub.1-antitrypsin, alpha-1-antitrypsin, alpha 1
antitrypsin, etc., are used interchangeably herein.
[0038] Unless otherwise specified, all references to organic groups
containing one more hydrogen atoms also include their partially or
fully deuterated counterparts. For instance, references herein to
alkyl groups embrace alkyl groups consisting of carbon and hydrogen
atoms, alkyl groups containing carbon atoms and a mixture of
hydrogen and deuterium atoms, and fully deuterated alkyl groups.
Preferably, however, such organic groups are not partially or fully
deuterated except where expressly indicated.
General Information Regarding Compounds of the Invention,
Pharmaceutical Compositions Comprising Such Compounds, and
Combination Therapies
[0039] As used herein, "a compound of the invention" includes
compounds that are capable of inhibiting .alpha..sub.1-antitrypsin
polymerisation and all solvates, complexes, tautomers, polymorphs,
isotope labelled derivatives, stereoisomers and optical isomers of
such compounds.
[0040] As used herein, the term "effective amount" means that
amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal or human
that is being sought, for instance, by a researcher or clinician.
Furthermore, the term "therapeutically effective amount" means any
amount which, as compared to a corresponding subject who has not
received such amount, results in improved treatment, healing,
prevention, or amelioration of a disease, disorder, or side effect,
or a decrease in the rate of advancement of a disease or disorder.
The term also includes within its scope amounts effective to
enhance normal physiological function.
[0041] As used herein, the term "pharmaceutically acceptable"
refers to those compounds, materials, compositions, and dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0042] The compounds of the present invention may be in the form of
a salt.
[0043] Typically, the salts of the present invention are
pharmaceutically acceptable salts. Salts encompassed within the
term "pharmaceutically acceptable salts" refer to non-toxic salts
of the compounds of this invention. For a review on suitable salts
see Berge et al, J. Pharm. Sci. 1977, 66, 1-19.
[0044] The compounds of the invention may exist in solid or liquid
form. In solid form, compound of the invention may exist in a
continuum of solid states ranging from fully amorphous to fully
crystalline. The term `amorphous` refers to a state in which the
material lacks long range order at the molecular level and,
depending upon the temperature, may exhibit the physical properties
of a solid or a liquid. Typically such materials do not give
distinctive X-ray diffraction patterns and, while exhibiting the
properties of a solid, are more formally described as a liquid.
Upon heating, a change from solid to liquid properties occurs which
is characterized by a change of state, typically second order
(`glass transition`). The term `crystalline` refers to a solid
phase in which the material has a regular ordered internal
structure at the molecular level and gives a distinctive X-ray
diffraction pattern with defined peaks. Such materials when heated
sufficiently will also exhibit the properties of a liquid, but the
change from solid to liquid is characterized by a phase change,
typically first order (`melting point`).
[0045] The compound of the present invention may exist in solvated
and unsolvated forms. As used herein, the term "solvate" refers to
a complex of variable stoichiometry formed by a solute (in this
invention, a compound of the invention and a solvent). Such
solvents for the purpose of the invention may not interfere with
the biological activity of the solute. The skilled artisan will
appreciate that pharmaceutically acceptable solvates may be formed
for crystalline compounds wherein solvent molecules are
incorporated into the crystalline lattice during crystallization.
The incorporated solvent molecules may be water molecules or
non-aqueous such as ethanol, isopropanol, DMSO, acetic acid,
ethanolamine, and ethyl acetate molecules. Crystalline lattice
incorporated with water molecules are typically referred to as
"hydrates". Hydrates include stoichiometric hydrates as well as
compositions containing variable amounts of water. The present
invention includes all such solvates.
[0046] The compounds of the invention may have the ability to
crystallize in more than one form, a characteristic, which is known
as polymorphism, and it is understood that such polymorphic forms
("polymorphs") are within the scope of the invention. Polymorphism
generally can occur as a response to changes in temperature or
pressure or both and can also result from variations in the
crystallization process. Polymorphs can be distinguished by various
physical characteristics known in the art such as x-ray diffraction
patterns, solubility and melting point.
[0047] It is also noted that the compounds of the invention may
form tautomers. It is understood that all tautomers and mixtures of
tautomers of the compounds of the present invention are included
within the scope of the compounds of the present invention.
[0048] The invention also includes isotopically-labelled compounds,
which are identical to the compounds of the invention, but for the
fact that one or more atoms are replaced by an atom having an
atomic mass or mass number different from the atomic mass or mass
number most commonly found in nature. Examples of isotopes suitable
for inclusion in the compounds of the invention include isotopes of
hydrogen, such as .sup.2H and .sup.3H, carbon, such as .sup.11C,
.sup.13C and .sup.14C, chlorine, such as .sup.36Cl, fluorine, such
as .sup.18F, iodine, such as .sup.123I and .sup.125I, nitrogen,
such as .sup.13N and .sup.15N, oxygen, such as .sup.15O, .sup.17O
and .sup.18O, phosphorus, such as .sup.32P, and sulphur, such as
.sup.35S.
[0049] Certain isotopically-labelled compounds of the present
invention, for example, those incorporating a radioactive isotope,
are useful in drug and/or substrate tissue distribution studies.
The radioactive isotopes tritium, i.e. .sup.3H, and carbon-14, i.e.
.sup.14C, are particularly useful for this purpose in view of their
ease of incorporation and ready means of detection.
[0050] Substitution with heavier isotopes such as deuterium, i.e.
.sup.2H, may afford certain therapeutic advantages resulting from
greater metabolic stability, for example, increased in vivo
half-life or reduced dosage requirements, and hence may be
preferred in some circumstances.
[0051] Isotopically-labelled compounds of the present invention can
generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described in
the accompanying Examples and Preparations using an appropriate
isotopically-labelled reagents in place of the non-labelled reagent
previously employed.
[0052] In one embodiment the compound of the present invention is
one of:
TABLE-US-00001 Example number structure name 1 ##STR00009##
N-((1S,2R)-1-(3-fluoro-2-methylphenyl)-1-
hydroxypentan-2-yl)-2-oxoindoline-4-carboxamide 2 ##STR00010##
N-((1S,2R)-1-(3-chloro-2-methylphenyl)-1-
hydroxypentan-2-yl)-2-oxoindoline-4-carboxamide 3 ##STR00011##
N-((1S,2R)-1-(2-chloro-3-cyanophenyl)-1-
hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4- carboxamide 4
##STR00012## N-((1S,2R)-1-(2-chloro-3-fluorophenyl)-1-
hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4- carboxamide
[0053] While it is possible that, for use in therapy, the compound
of the invention may be administered as the raw chemical, it is
possible to present the compound of the invention as the active
ingredient in a pharmaceutical composition. Such compositions can
be prepared in a manner well known in the pharmaceutical art and
comprise at least one active compound. Accordingly, the invention
further provides pharmaceutical compositions comprising a compound
of the invention and one or more pharmaceutically acceptable
excipients. The excipient(s) must be acceptable in the sense of
being compatible with the other ingredients of the composition and
not deleterious to the recipient thereof. In accordance with
another aspect of the invention there is also provided a process
for the preparation of a pharmaceutical composition including the
agent, with one or more pharmaceutically acceptable excipients. The
pharmaceutical composition can be for use in the treatment and/or
prophylaxis of any of the conditions described herein.
[0054] Generally, the compound of the invention is administered in
a pharmaceutically effective amount. The amount of the compound
actually administered will typically be determined by a physician,
in the light of the relevant circumstances, including the condition
to be treated, the chosen route of administration, the actual
compound-administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0055] Pharmaceutical compositions may be presented in unit dose
forms containing a predetermined amount of active ingredient per
unit dose. The term "unit dosage forms" refers to physically
discrete units suitable as unitary dosages for human subjects and
other mammals, each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect, in association with a suitable pharmaceutical excipient,
vehicle or carrier. Typical unit dosage forms include prefilled,
premeasured ampules or syringes of the liquid compositions or
pills, tablets, capsules or the like in the case of solid
compositions.
[0056] Preferred unit dosage compositions are those containing a
daily dose or sub-dose, or an appropriate fraction thereof, of an
active ingredient. Such unit doses may therefore be administered
once or more than once a day. Such pharmaceutical compositions may
be prepared by any of the methods well known in the pharmacy
art.
[0057] Pharmaceutical compositions may be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), rectal, inhaled, intranasal,
topical (including buccal, sublingual or transdermal), vaginal or
parenteral (including subcutaneous, intramuscular, intravenous or
intradermal) route. Such compositions may be prepared by any method
known in the art of pharmacy, for example by bringing into
association the active ingredient with the carrier(s) or
excipient(s).
[0058] Pharmaceutical compositions adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions in aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil liquid emulsions.
[0059] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert excipient such
as ethanol, glycerol, water and the like. Powders are prepared by
reducing the compound to a suitable fine size and mixing with a
similarly prepared pharmaceutical excipient such as an edible
carbohydrate, as, for example, starch or mannitol. Flavouring,
preservative, dispersing and colouring agent can also be
present.
[0060] Capsules are made by preparing a powder mixture, as
described above, and filling formed gelatin sheaths. Excipients
including glidants and lubricants such as colloidal silica, talc,
magnesium stearate, calcium stearate or solid polyethylene glycol
can be added to the powder mixture before the filling operation. A
disintegrating or solubilizing agent such as agar-agar, calcium
carbonate or sodium carbonate can also be added to improve the
availability of the medicament when the capsule is ingested.
[0061] Moreover, when desired or necessary, excipients including
suitable binders, glidants, lubricants, sweetening agents,
flavours, disintegrating agents and colouring agents can also be
incorporated into the mixture. Suitable binders include starch,
gelatin, natural sugars such as glucose or beta-lactose, corn
sweeteners, natural and synthetic gums such as acacia, tragacanth
or sodium alginate, carboxymethylcellulose, polyethylene glycol,
waxes and the like. Lubricants used in these dosage forms include
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum and the like. Tablets are
formulated, for example, by preparing a powder mixture, granulating
or slugging, adding a lubricant and disintegrant and pressing into
tablets. A powder mixture is prepared by mixing the compound,
suitably comminuted, with a diluent or base as described above, and
optionally, with a binder such as carboxymethylcellulose, an
aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant
such as paraffin, a resorption accelerator such as a quaternary
salt and/or an absorption agent such as bentonite, kaolin or
dicalcium phosphate. The powder mixture can be granulated by
wetting with a binder such as syrup, starch paste, acadia mucilage
or solutions of cellulosic or polymeric materials and forcing
through a screen. As an alternative to granulating, the powder
mixture can be run through the tablet machine and the result is
imperfectly formed slugs broken into granules. The granules can be
lubricated to prevent sticking to the tablet forming dies by means
of the addition of stearic acid, a stearate salt, talc or mineral
oil. The lubricated mixture is then compressed into tablets. The
compounds of the present invention can also be combined with a free
flowing inert carrier and compressed into tablets directly without
going through the granulating or slugging steps. A clear or opaque
protective coating consisting of a sealing coat of shellac, a
coating of sugar or polymeric material and a polish coating of wax
can be provided. Dyestuffs can be added to these coatings to
distinguish different unit dosages.
[0062] Oral fluids such as solution, suspensions, syrups and
elixirs can be prepared in dosage unit form so that a given
quantity contains a predetermined amount of the compound. Syrups
can be prepared by dissolving the compound in a suitably flavoured
aqueous solution, while elixirs are prepared through the use of a
non-toxic alcoholic vehicle. Suspensions can be formulated by
dispersing the compound in a non-toxic vehicle. Solubilizers and
emulsifiers such as ethoxylated isostearyl alcohols and polyoxy
ethylene sorbitol ethers, preservatives, flavor additive such as
peppermint oil or natural sweeteners or saccharin or other
artificial sweeteners, and the like can also be added.
[0063] Where appropriate, dosage unit compositions for oral
administration can be microencapsulated. The composition can also
be prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax or the
like. The compounds of the invention may also be administered in
the form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine or phosphatidylcholines.
[0064] Pharmaceutical compositions adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time.
[0065] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils.
[0066] For treatments of the eye or other external tissues, for
example mouth and skin, the compositions are preferably applied as
a topical ointment or cream. When formulated in an ointment, the
active ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base.
[0067] Pharmaceutical compositions adapted for topical
administrations to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent.
[0068] Pharmaceutical compositions adapted for topical
administration in the mouth include lozenges, pastilles and mouth
washes.
[0069] Pharmaceutical compositions adapted for rectal
administration may be presented as suppositories or as enemas.
[0070] Dosage forms for nasal or inhaled administration may
conveniently be formulated as aerosols, solutions, suspensions
drops, gels or dry powders.
[0071] Compositions for intranasal administration include aqueous
compositions administered to the nose by drops or by pressurised
pump. Suitable compositions contain water as the diluent or carrier
for this purpose. Compositions for administration to the lung or
nose may contain one or more excipients, for example one or more
suspending agents, one or more preservatives, one or more
surfactants, one or more tonicity adjusting agents, one or more
co-solvents, and may include components to control the pH of the
composition, for example a buffer system. Further, the compositions
may contain other excipients such as antioxidants, for example
sodium metabisulphite, and taste-masking agents. Compositions may
also be administered to the nose or other regions of the
respiratory tract by nebulisation.
[0072] Intranasal compositions may permit the compound(s) of the
invention to be delivered to all areas of the nasal cavities (the
target tissue) and further, may permit the compound(s) of the
invention to remain in contact with the target tissue for longer
periods of time. A suitable dosing regime for intranasal
compositions would be for the patient to inhale slowly through the
nose subsequent to the nasal cavity being cleared. During
inhalation the composition would be administered to one nostril
while the other is manually compressed. This procedure would then
be repeated for the other nostril. Typically, one or two sprays per
nostril would be administered by the above procedure one, two, or
three times each day, ideally once daily. Of particular interest
are intranasal compositions suitable for once-daily
administration.
[0073] Compositions for administration to the lung or nose may
contain one or more excipients may be protected from microbial or
fungal contamination and growth by inclusion of one or more
preservatives. Examples of pharmaceutically acceptable
anti-microbial agents or preservatives include, but are not limited
to, quaternary ammonium compounds (for example benzalkonium
chloride, benzethonium chloride, cetrimide, cetylpyridinium
chloride, lauralkonium chloride and myristyl picolinium chloride),
mercurial agents (for example phenylmercuric nitrate,
phenylmercuric acetate and thimerosal), alcoholic agents (for
example chlorobutanol, phenylethyl alcohol and benzyl alcohol),
antibacterial esters (for example esters of para-hydroxybenzoic
acid), chelating agents such as disodium edetate (EDTA) and other
anti-microbial agents such as chlorhexidine, chlorocresol, sorbic
acid and its salts (such as potassium sorbate) and polymyxin.
Examples of pharmaceutically acceptable anti-fungal agents or
preservatives include, but are not limited to, sodium benzoate,
sorbic acid, sodium propionate, methylparaben, ethylparaben,
propylparaben and butylparaben. The preservative(s), if included,
may be present in an amount of from 0.001 to 1% (w/w), such as from
0.015% to 0.5% (w/w) based on the total weight of the
composition.
[0074] Compositions (for example wherein at least one compound is
in suspension) may include one or more surfactants which functions
to facilitate dissolution of the medicament particles in the
aqueous phase of the composition. For example, the amount of
surfactant used is an amount which will not cause foaming during
mixing. Examples of pharmaceutically acceptable surfactants include
fatty alcohols, esters and ethers, such as polyoxyethylene (20)
sorbitan monooleate (Polysorbate 80), macrogol ethers, and
poloxamers. The surfactant may be present in an amount of between
about 0.01 to 10% (w/w), such as from 0.01 to 0.75% (w/w), for
example about 0.5% (w/w), based on the total weight of the
composition.
[0075] One or more tonicity-adjusting agent(s) may be included to
achieve tonicity with body fluids e.g. fluids of the nasal cavity,
resulting in reduced levels of irritancy. Examples of
pharmaceutically acceptable tonicity-adjusting agents include, but
are not limited to, sodium chloride, dextrose, xylitol, calcium
chloride, glucose, glycerine and sorbitol. A tonicity-adjusting
agent, if present, may be included in an amount of from 0.1 to 10%
(w/w), such as from 4.5 to 5.5% (w/w), for example about 5.0%
(w/w), based on the total weight of the composition.
[0076] The compositions of the invention may be buffered by the
addition of suitable buffering agents such as sodium citrate,
citric acid, trometamol, phosphates such as disodium phosphate (for
example the dodecahydrate, heptahydrate, dihydrate and anhydrous
forms), or sodium phosphate and mixtures thereof.
[0077] A buffering agent, if present, may be included in an amount
of from 0.1 to 5% (w/w), for example 1 to 3% (w/w) based on the
total weight of the composition.
[0078] Examples of taste-masking agents include sucralose, sucrose,
saccharin or a salt thereof, fructose, dextrose, glycerol, corn
syrup, aspartame, acesulfame-K, xylitol, sorbitol, erythritol,
ammonium glycyrrhizinate, thaumatin, neotame, mannitol, menthol,
eucalyptus oil, camphor, a natural flavouring agent, an artificial
flavouring agent, and combinations thereof.
[0079] One or more co-solvent(s) may be included to aid solubility
of the medicament compound(s) and/or other excipients. Examples of
pharmaceutically acceptable co-solvents include, but are not
limited to, propylene glycol, dipropylene glycol, ethylene glycol,
glycerol, ethanol, polyethylene glycols (for example PEG300 or
PEG400), and methanol. In one embodiment, the co-solvent is
propylene glycol.
[0080] Co-solvent(s), if present, may be included in an amount of
from 0.05 to 30% (w/w), such as from 1 to 25% (w/w), for example
from 1 to 10% (w/w) based on the total weight of the
composition.
[0081] Compositions for inhaled administration include aqueous,
organic or aqueous/organic mixtures, dry powder or crystalline
compositions administered to the respiratory tract by pressurised
pump or inhaler, for example, reservoir dry powder inhalers,
unit-dose dry powder inhalers, pre-metered multi-dose dry powder
inhalers, nasal inhalers or pressurised aerosol inhalers,
nebulisers or insufflators. Suitable compositions contain water as
the diluent or carrier for this purpose and may be provided with
conventional excipients such as buffering agents, tonicity
modifying agents and the like. Aqueous compositions may also be
administered to the nose and other regions of the respiratory tract
by nebulisation. Such compositions may be aqueous solutions or
suspensions or aerosols delivered from pressurised packs, such as a
metered dose inhaler, with the use of a suitable liquefied
propellant.
[0082] Compositions for administration topically to the nose (for
example, for the treatment of rhinitis) or to the lung, include
pressurised aerosol compositions and aqueous compositions delivered
to the nasal cavities by pressurised pump. Compositions which are
non-pressurised and are suitable for administration topically to
the nasal cavity are of particular interest. Suitable compositions
contain water as the diluent or carrier for this purpose. Aqueous
compositions for administration to the lung or nose may be provided
with conventional excipients such as buffering agents,
tonicity-modifying agents and the like. Aqueous compositions may
also be administered to the nose by nebulisation.
[0083] A fluid dispenser may typically be used to deliver a fluid
composition to the nasal cavities. The fluid composition may be
aqueous or non-aqueous, but typically aqueous. Such a fluid
dispenser may have a dispensing nozzle or dispensing orifice
through which a metered dose of the fluid composition is dispensed
upon the application of a user-applied force to a pump mechanism of
the fluid dispenser. Such fluid dispensers are generally provided
with a reservoir of multiple metered doses of the fluid
composition, the doses being dispensable upon sequential pump
actuations. The dispensing nozzle or orifice may be configured for
insertion into the nostrils of the user for spray dispensing of the
fluid composition into the nasal cavity.
[0084] Dry powder compositions for topical delivery to the lung by
inhalation may, for example, be presented in capsules and
cartridges of for example gelatine, or blisters of for example
laminated aluminium foil, for use in an inhaler or insufflator.
Powder blend compositions generally contain a powder mix for
inhalation of the compound of the invention and a suitable powder
base (carrier/diluent/excipient substance) such as mono-, di-, or
polysaccharides (for example lactose or starch). Dry powder
compositions may also include, in addition to the drug and carrier,
a further excipient (for example a ternary agent such as a sugar
ester for example cellobiose octaacetate, calcium stearate, or
magnesium stearate.
[0085] In one embodiment, a composition suitable for inhaled
administration may be incorporated into a plurality of sealed dose
containers provided on medicament pack(s) mounted inside a suitable
inhalation device. The containers may be rupturable, peelable, or
otherwise openable one-at-a-time and the doses of the dry powder
composition administered by inhalation on a mouthpiece of the
inhalation device, as known in the art. The medicament pack may
take a number of different forms, for instance a disk-shape or an
elongate strip.
[0086] A further delivery method for a dry powder inhalable
composition is for metered doses of the composition to be provided
in capsules (one dose per capsule) which are then loaded into an
inhalation device, typically by the patient on demand. The device
has means to rupture, pierce or otherwise open the capsule so that
the dose is able to be entrained into the patient's lung when they
inhale at the device mouthpiece.
[0087] Pressurised aerosol compositions suitable for inhalation can
be either a suspension or a solution and may contain a compound of
the invention or a pharmaceutically acceptable salt thereof and a
suitable propellant such as a fluorocarbon or hydrogen-containing
chlorofluorocarbon or mixtures thereof, particularly
hydrofluoroalkanes, especially 1,1,1,2-tetrafluoroethane,
1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The
aerosol composition may optionally contain additional composition
excipients well known in the art such as surfactants e.g. oleic
acid, lecithin or an oligolactic acid or derivative thereof e.g. as
described in WO 94/21229 and WO 98/34596 (Minnesota Mining and
Manufacturing Company) and co-solvents e.g. ethanol. Pressurised
compositions will generally be retained in a canister (e.g. an
aluminium canister) closed with a valve (e.g. a metering valve) and
fitted into an actuator provided with a mouthpiece.
[0088] Pharmaceutical compositions adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations.
[0089] Pharmaceutical compositions adapted for parental
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the composition isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The compositions may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0090] It should be understood that in addition to the ingredients
particularly mentioned above, the compositions may include other
agents conventional in the art having regard to the type of
formulation in question, for example those suitable for oral
administration may include flavouring agents.
[0091] A therapeutically effective amount of the agent will depend
upon a number of factors including, for example, the age and weight
of the subject, the precise condition requiring treatment and its
severity, the nature of the formulation, and the route of
administration, and will ultimately be at the discretion of the
attendant physician or veterinarian. In particular, the subject to
be treated is a mammal, particularly a human.
[0092] The agent may be administered in a daily dose. This amount
may be given in a single dose per day or more usually in a number
(such as two, three, four, five or six) of sub-doses per day such
that the total daily dose is the same.
[0093] Suitably, the amount of the compound of the invention
administered according to the present invention will be an amount
selected from 0.01 mg to 5 g per day.
[0094] Additionally, the compounds of the invention may be
administered as prodrugs. As used herein, a "prodrug" of a compound
of the invention is a functional derivative of the compound which,
upon administration to a patient, eventually liberates the compound
of the invention in vivo. Administration of a compound of the
invention as a prodrug may enable the skilled artisan to do one or
more of the following: (a) modify the onset of the activity of the
compound in vivo; (b) modify the duration of action of the compound
in vivo; (c) modify the transportation or distribution of the
compound in vivo; (d) modify the solubility of the compound in
vivo; and (e) overcome a side effect or other difficulty
encountered with the compound. Typical functional derivatives used
to prepare prodrugs include modifications of the compound that are
chemically or enzymatically cleavable in vivo. Such modifications,
which include the preparation of phosphates, amides, esters,
thioesters, carbonates, and carbamates, are well known to those
skilled in the art.
[0095] Specific, and strictly non-limiting, examples of prodrugs
within the meaning of the present invention include derivatives of
compounds of formulae (IA), (IB), (IC), (ID) and (I) in which the
hydroxyl moiety is replaced by a reactive moiety that is capable of
degrading in vivo (e.g., by hydrolysis) to yield the hydroxyl
moiety. For avoidance of doubt, the hydroxyl moiety of formulae
(IA), (IB), (IC) and (ID) is that indicated by the label "I" in the
chemical formulae, and the hydroxyl moiety of formula (I) is that
attached to the carbon atom that is between the phenyl ring and the
propyl side group of the compound. Similarly, the present invention
includes derivatives of compounds of formula (II) in which Z is a
reactive moiety that is capable of degrading in vivo (e.g., by
hydrolysis) to yield a hydroxyl moiety. Strictly non-limiting, and
merely representative, examples of such reactive moieties include
moieties of formula OR.sub.z in which R.sub.z has the formula
PO.sub.3H.sub.2, CO(CH.sub.2).sub.nNH.sub.2 and
PO.sub.3H(CH.sub.2).sub.n--NH.sub.2 (where n is integer, e.g. of
from 2 to 5). As those skilled in the art would readily appreciate,
a wealth of other reactive moieties could also be used. In a
compound containing a plurality of hydroxyl groups, it is of course
possible for some or all of the said hydroxyl groups to replace by
such reactive moieties.
[0096] The compounds of the invention may be employed alone or in
combination with other therapeutic agents. The compounds of the
invention and the other pharmaceutically active agent(s) may be
administered together or separately and, when administered
separately, administration may occur simultaneously or
sequentially, in any order. by any convenient route in separate or
combined pharmaceutical compositions.
[0097] The amounts of the compound(s) of the invention and the
other pharmaceutically active agent(s) and the relative timings of
administration will be selected in order to achieve the desired
combined therapeutic effect. The compounds of the present invention
and further therapeutic agent(s) may be employed in combination by
administration simultaneously in a unitary pharmaceutical
composition including both compounds. Alternatively, the
combination may be administered separately in separate
pharmaceutical compositions, each including one of the compounds in
a sequential manner wherein, for example, the compound of the
invention is administered first and the other second and visa
versa. Such sequential administration may be close in time (e.g.
simultaneously) or remote in time. Furthermore, it does not matter
if the compounds are administered in the same dosage form, e.g. one
compound may be administered topically and the other compound may
be administered orally. Suitably, both compounds are administered
orally.
[0098] The combinations may be presented as a combination kit. By
the term "combination kit" "or kit of parts" as used herein is
meant the pharmaceutical composition or compositions that are used
to administer the combination according to the invention. When both
compounds are administered simultaneously, the combination kit can
contain both compounds in a single pharmaceutical composition, such
as a tablet, or in separate pharmaceutical compositions. When the
compounds are not administered simultaneously, the combination kit
will contain each compound in separate pharmaceutical compositions
either in a single package or in separate pharmaceutical
compositions in separate packages.
[0099] The combination kit can also be provided by instruction,
such as dosage and administration instructions. Such dosage and
administration instructions can be of the kind that are provided to
a doctor, for example by a drug product label, or they can be of
the kind that are provided by a doctor, such as instructions to a
patient.
[0100] When the combination is administered separately in a
sequential manner wherein one is administered first and the other
second or vice versa, such sequential administration may be close
in time or remote in time. For example, administration of the other
agent several minutes to several dozen minutes after the
administration of the first agent, and administration of the other
agent several hours to several days after the administration of the
first agent are included, wherein the lapse of time is not limited,
For example, one agent may be administered once a day, and the
other agent may be administered 2 or 3 times a day, or one agent
may be administered once a week, and the other agent may be
administered once a day and the like.
[0101] It will be clear to a person skilled in the art that, where
appropriate, the other therapeutic ingredients(s) may be used in
the form of salts, for example as alkali metal or amine salts or as
acid addition salts, or prodrugs, or as esters, for example lower
alkyl esters, or as solvates, for example hydrates, to optimise the
activity and/or stability and/or physical characteristics, such as
solubility, of the therapeutic ingredient. It will be clear also
that, where appropriate, the therapeutic ingredients may be used in
optically pure form.
[0102] When combined in the same composition it will be appreciated
that the two compounds must be stable and compatible with each
other and the other components of the composition and may be
formulated for administration. When formulated separately they may
be provided in any convenient composition, conveniently, in such a
manner as known for such compounds in the art.
[0103] When the compound of the invention is used in combination
with a second therapeutic agent active against the same disease,
condition or disorder, the dose of each compound may differ from
that when the compound is used alone. Appropriate doses will be
readily appreciated by those skilled in the art.
[0104] In one embodiment the mammal in the methods and uses of the
present invention is a human.
DETAILED DESCRIPTION OF COMPOUNDS OF THE PRESENT INVENTION
[0105] The compound of the present invention is a compound that is
capable of inhibiting .alpha..sub.1-antitrypsin polymerisation.
Typically, the compound is capable of inhibiting
Z-.alpha..sub.1-antitrypsin polymerisation.
[0106] The compound is typically a small molecule compound. As used
herein, a small molecule compound typically has a molecular weight
of 2000 Daltons or less, more usually 1000 Daltons or less and
preferably 800 Daltons or less. More preferably the molecular
weight is 600 or less and most preferably 500 or less. For
instance, the small molecule drug may have a molecular weight of
from 250 to 800, such as from 300 to 600.
[0107] In one aspect of the present invention, the compound that is
capable of inhibiting .alpha..sub.1-antitrypsin polymerisation is a
compound of the general formula (II):
##STR00013##
wherein: [0108] R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are as
defined elsewhere herein (e.g., with respect to general formula
(ID)); [0109] R.sub.8 is hydrogen, deuterium, alkyl (e.g.,
C.sub.1-5 alkyl) or deuterated alkyl (e.g., C.sub.1-5 deuterated
alkyl); [0110] R.sub.9 is hydrogen, deuterium, alkyl (e.g.,
C.sub.1-5 alkyl) or deuterated alkyl (e.g., C.sub.1-5 deuterated
alkyl); [0111] Z is OH, F, --NHCHO, --CH.sub.2F, --CHF.sub.2 or
CF.sub.3; and [0112] Q is --C(.dbd.O)--, --C(.dbd.S)--,
--C(.dbd.NOH)--, --C(.dbd.NNH.sub.2)-- or --S(O).sub.2--.
[0113] Examples of preferred groups R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are as defined elsewhere herein.
[0114] Preferred groups R.sub.8 are hydrogen, deuterium, and
C.sub.1-2 optionally deuterated alkyl. More preferably R.sub.8 is
hydrogen, methyl or ethyl, more preferably still hydrogen or methyl
and most preferably hydrogen.
[0115] Preferred groups R.sub.9 are hydrogen, deuterium, and
C.sub.1-2 optionally deuterated alkyl. More preferably R.sub.9 is
hydrogen, methyl or ethyl, more preferably still hydrogen or methyl
and most preferably hydrogen.
[0116] Z is preferably OH or F, most preferably OH.
[0117] Q is preferably --C(.dbd.O)--, --C(.dbd.S)-- or
--S(O).sub.2--, more preferably --C(.dbd.O)-- or --C(.dbd.S)--, and
most preferably --C(.dbd.O)--.
[0118] In preferred embodiments, the compound of general formula
(II) is a compound of general formula (ID), and more preferably
still a compound of general formula (I), as further defined
elsewhere herein.
Binding of the Compound to .alpha..sub.1-Antitrypsin
[0119] The compound of the present invention is capable of binding
to .alpha..sub.1-antitrypsin. Typically, the compound is capable of
binding to .alpha..sub.1-antitrypsin by via a cryptic binding site
within the .alpha..sub.1-antitrypsin protein structure. A cryptic
binding site as defined herein refers to a binding site that is
absent, occluded, or only transiently accessed in the unbound
protein, but present when the compound is bound to the protein; for
instance, the cryptic binding site may come into existence as a
consequence of contacting the protein with the compound, and/or a
previously only transiently accessed protein configuration
featuring the binding site may become stabilized by the presence of
the protein.
[0120] By ".alpha..sub.1-antitrypsin" in the expression "the
compound is capable of binding to .alpha..sub.1-antitrypsin" is
meant a protein that comprises the sequence of SEQ ID NO: 1,
namely
TABLE-US-00002
X.sub.1DPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNST
NIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQ
ELLX.sub.2TLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTV
NFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKW
ERPFEVKDTEEEDFHVDQX.sub.3TTVKVPMMKRLGMFNIQHX.sub.4KKLSSWVLL
MKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPK
LSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLT
IDX.sub.5KGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIX.sub.6QNTKSPLFMG
KVVNPTQK
wherein X.sub.1 is T or E, X.sub.2 is R or H, X.sub.3 is A or V,
X.sub.4 is C or S, X.sub.5 is E or K, and X.sub.6 is D or E.
[0121] For the avoidance of doubt, a compound is a compound of the
invention if it is capable of binding to any such protein, i.e. any
single protein that comprises the sequence of SEQ ID NO: 1 (i.e.,
it is not essential that the compound must bind to all possible
variants embraced by SEQ ID NO: 1, and nor does such binding
preclude the possibility that the compound may also bind to other
proteins, such as those containing amino acid sequences that differ
from SED ID NO: 1).
[0122] Furthermore, also for the avoidance of doubt, all residue
numbers attributed to specific amino acids in the
.alpha..sub.1-antitrypsin protein, for instance in relation to
definition of the binding site of the compounds of the present
invention, are counted from the N-terminus of the sequence of SEQ
ID NO: 1, i.e. X.sub.1 is counted as residue 1, the adjacent D as
residue 2, the adjacent P as residue 3, the adjacent Q as residue
4, the adjacent G as residue 5, and so on. This numbering applies
also when the protein contains additional amino acids at the
N-terminus with respect to SEQ ID NO: 1, i.e. any such additional
"flanking" amino acids do not change the numbering convention, with
X.sub.1 still being counted as residue 1, D as residue 2, P as
residue 3, Q as residue 4, G as residue 5, and so on. Thus, the
numbering is always counted only with reference to the amino acids
of SEQ ID NO: 1 and excludes any additional amino acid residues
present, for instance in any signal peptide or affinity tag and/or
any additional amino acids provided by an expression vector used in
the synthesis of the protein. Consequently in any relevant protein,
X.sub.1 is amino acid 1, X.sub.4 is amino acid 232, X.sub.5 is
amino acid 342, and so on.
[0123] The most common allele of wild-type M-AT is the M1V allele
(estimated at 44-49% of total). In the M1V allele, X.sub.1 is E,
X.sub.2 is R, X.sub.3 is V, X.sub.4 is C, X.sub.5 is E and X.sub.6
is E. The full human M1V protein further comprises a signal peptide
at the N-terminus having the sequence MPSSVSWGILLLAGLCCLVPVSLA.
Thus, the full sequence of the human M1V protein corresponds to the
sequence of SEQ ID NO: 2, namely
TABLE-US-00003 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNK
ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD
EILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEG
LKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDL
VKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMM
KRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHD
IITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLS
GVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNK
PFVFLMIEQNTKSPLFMGKVVNPTQK.
[0124] The Z-.alpha..sub.1-antitrypsin mutant differs from the M1V
allele in that X.sub.5 is K rather than E (i.e. the mutation is
defined as E342K) and X.sub.3(213) is A. Thus, the full sequence of
Z-A1AT corresponds to the sequence of SEQ ID NO: 3, namely
TABLE-US-00004 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNK
ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD
EILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEG
LKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDL
VKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQATTVKVPMM
KRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHD
IITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLS
GVTEEAPLKLSKAVHKAVLTIDKKGTEAAGAMFLEAIPMSIPPEVKFNK
PFVFLMIEQNTKSPLFMGKVVNPTQK.
[0125] For ease of handling (e.g. in generating crystals of
.alpha..sub.1-antitrypsin protein and/or generating complexes of
.alpha..sub.1-antitrypsin protein bound to a compound of the
invention), it may be desirable to mutate cysteine 232 of the
protein to a serine, i.e. to effect the mutation C232S. This is a
conservative mutation and is not located at a position directly
associated with the compound binding site. SEQ ID NO:1 embraces
both proteins in view of its variable amino acid residue
X.sub.4.
[0126] In a preferred embodiment, the compound is capable of
binding to a protein comprising SEQ ID NO: 1 in which X.sub.1 is E,
X.sub.2 is R, X.sub.3 is A, X.sub.4 is C, X.sub.5 is K and X.sub.6
is E, i.e. the sequence has 100% sequence identity to the
corresponding portion of Z-.alpha..sub.1-antitrypsin.
[0127] In another exemplary embodiment, the protein may be a
sequence that comprises SEQ ID NO: 1 and which additionally
comprises an affinity tag (e.g. a hexahistidine affinity tag)
and/or amino acids provided by an expression vector. For instance,
such a protein may be one expressed in E. coli. The full sequence
of one such exemplary protein is that of SEQ ID NO: 4, namely
TABLE-US-00005 MRGSHHHHHHTDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYR
QLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIP
EAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKL
YHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVN
YIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHSKKL
SSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA
SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAV
HKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP LFMGKVVNPTQK.
[0128] This protein, which is used in Example 2 herein, comprises
N-terminal sequence MRGSHHHHHHT having a hexahistidine affinity tag
and amino acids provided by an expression vector used in synthesis,
and in which X.sub.1 is T. This protein further comprises a serine
at position 232 (i.e. X.sub.4 is S) to assist ease of handling.
This protein further comprises glutamate at position 342 (i.e.,
X.sub.5 is E).
[0129] An important finding of the present invention is that the
subject compounds are capable of specific binding to the
.alpha..sub.1-antitrypsin protein in a part of the protein that has
not previously been demonstrated to be a site for drug binding (and
still less one that, upon formation of compound-protein binding, is
capable of inhibiting protein polymerisation).
[0130] More particularly, the said binding site is preferably
located between .beta.-sheet-A and .beta.-sheet-B of said
.alpha..sub.1-antitrypsin. Said .beta.-sheet-A comprises the amino
acids corresponding to one or more of: (i) residues 140-144; (ii)
residues 111-121; (iii) residues 181-191; (iv) residues 330-340;
and (v) residues 292-299 of SEQ ID NO: 1. Preferably said
.beta.-sheet-A comprises the amino acids corresponding to two or
more of, more preferably three or more, more preferably still four
or more, and most preferably all five, of (i) to (v). Said
.beta.-sheet-B comprises the amino acids corresponding to one or
more of: (i') residues 228-231; (ii') residues 236-244; (iii')
residues 248-256; (iv') residues 369-376; (v') residues 381-389;
and (vi') residues 49-53 of SEQ ID NO: 1. Preferably said
.beta.-sheet-B comprises the amino acids corresponding to two or
more of, more preferably three or more, more preferably still four
or more, more preferably still five or more, and most preferably
all six, of (i') to (vi').
[0131] Thus, preferably the binding site is located between
.beta.-sheet-A and .beta.-sheet-B of said
.alpha..sub.1-antitrypsin, wherein: said .beta.-sheet-A comprises
the amino acids corresponding to residues 140-144, 111-121,
181-191, 330-340 and 292-299 of SEQ ID NO: 1; and said 13-sheet-B
comprises the amino acids corresponding to residues 228-231,
236-244, 248-256, 369-376, 381-389, and 49-53 of SEQ ID NO: 1.
[0132] By "located between .beta.-sheet-A and .beta.-sheet-B" is
meant that, when the compound is bound to the
.alpha..sub.1-antitrypsin, at least part (e.g. substantially all or
all) of the compound is accommodated in physical space between said
.beta.-sheet-A and said .beta.-sheet-B. As will be readily
appreciated by those skilled in the art, confirmation of the
location of the binding site of the compound on the protein can be
provided by methods well known in the art, such as by using routine
crystallographic techniques (see, for instance, Example 2
herein).
[0133] More specifically still, the binding site is more preferably
located between amino acid strands corresponding to three or more
of: (a) residues 191-194 of SEQ ID NO: 1; (b) residues 288-293 of
SEQ ID NO: 1; (c) residues 371-374 of SEQ ID NO: 1; (d) residues
249-253 of SEQ ID NO: 1; and (e) residues 240-243 of SEQ ID NO: 1.
It is particularly preferred that the binding site is located
between amino acid strands corresponding to four or more, and most
preferably each of: (a) residues 191-194 of SEQ ID NO: 1; (b)
residues 288-293 of SEQ ID NO: 1; (c) residues 371-374 of SEQ ID
NO: 1; (d) residues 249-253 of SEQ ID NO: 1; and (e) residues
240-243 of SEQ ID NO: 1. It is additionally preferable that the
binding site is also located between amino acid strands
corresponding to (f) residues 338-341 of SEQ ID NO: 1. By "amino
acid strand" is meant a plurality of amino acids residues in the
protein as numbered herein, e.g. an amino acid strand corresponding
residues 191-194 of SEQ ID NO: 1 refers to amino acids 191, 192,
193 and 194 of SEQ ID NO: 1. By "located between" the said amino
acid strands is meant that, when the compound is bound to the
.alpha..sub.1-antitrypsin, at least part (e.g. substantially all or
all) of the compound is accommodated in physical space between the
respective amino acid strands.
[0134] In an exemplary aspect, the binding site comprises one or
more of W194, Y244, L291, P289, F252, K290, I293, L338, I340, F372
and M374 of SEQ ID NO: 1. In this exemplary aspect, the binding
site preferably comprises three or more, more preferably five or
more, more preferably still seven or more, and more preferably nine
or more of W194, Y244, L291, P289, F252, K290, I293, L338, I340,
F372 and M374 of SEQ ID NO: 1. The binding site may, for instance,
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 of W194, Y244,
L291, P289, F252, K290, I293, L338, I340, F372 and M374 of SEQ ID
NO: 1. Particularly preferred amino acids associated with the
binding site are W194, Y244, L291 and P289 and thus preferably the
binding site comprises at least W194, Y244, L291 and P289. As
further described herein, the compound may optimally be capable of
forming non-covalent bonds (including but not limited to hydrogen
bonds) with the functional groups of the relevant amino acid
residues of the protein.
[0135] Preferably the K.sub.D of the compound to
M-.alpha..sub.1-antitrypsin is less than about 250 nM, said
M-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 2
and/or the K.sub.D of the compound to Z-.alpha..sub.1-antitrypsin
is less than about 25 nM, said Z-.alpha..sub.1-antitrypsin
comprising the sequence of SEQ ID NO: 3. Preferably the K.sub.D of
the compound to Z-.alpha..sub.1-antitrypsin is at least ten times
lower than the K.sub.D of the compound to
M-.alpha..sub.1-antitrypsin, said M-.alpha..sub.1-antitrypsin
comprising the sequence of SEQ ID NO: 2 and said
Z-.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO:
3. Methods for measuring K.sub.D are well known in the art and any
such method can be used. One exemplary such method is described in
Example 2. All references herein to K.sub.D are as determined at
room temperature (e.g. at 20.degree. C.).
Preferred Structural Elements Contributing to Protein Binding
[0136] In an exemplary aspect of the present disclosure, the
compound comprises a .beta.-hydroxyamide moiety, i.e. a moiety of
formula --C(OH)(X)--CX.sub.2--NH--C(O)--, where each X
independently represents a substituent (for instance, any specific
substituent as defined elsewhere herein).
[0137] Importantly, it has been found that such a
.beta.-hydroxyamide structural motif substantially contributes to
the ability of the present compounds to bind to
.alpha..sub.1-antitrypsin at the present binding site. In
particular, such a moiety may provide for binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) the .beta.-hydroxyl group
and L291 of SEQ ID NO: 1; (ii) the NH of the carboxamide group and
P289 of SEQ ID NO: 1; and (iii) the carbonyl of the carboxamide
group and Y244 of SEQ ID NO: 1.
[0138] Accordingly, it is preferred that the compound comprises a
tetravalent moiety of formula (IA)
##STR00014##
wherein the compound is capable of binding to
.alpha..sub.1-antitrypsin comprising the sequence of SEQ ID NO: 1
by hydrogen bond formation between: (i) hydroxyl group I and L291
of a SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and
(iii) carbonyl group III and Y244 of SEQ ID NO: 1.
[0139] By tetravalent is meant that the moiety of formula (IA)
contains four points of bonding, i.e. at the positions 1, 2, 3 and
4 indicated below:
##STR00015##
More preferably the compound comprises a divalent moiety of formula
(IB)
##STR00016##
[0140] By divalent is meant that the moiety of formula (IB)
contains two points of bonding, i.e. at the positions 1 and 2
below
##STR00017##
[0141] In formula (IB), as in formula (IA), typically the compound
is capable of binding to .alpha..sub.1-antitrypsin by hydrogen bond
formation between: (i) hydroxyl group I and L291 of SEQ ID NO: 1;
(ii) NH group II and P289 of SEQ ID NO: 1; and (iii) carbonyl group
III and Y244 of SEQ ID NO: 1.
[0142] R.sub.4 and R.sub.5 can be the same or different from one
another. They are independently selected from hydrogen, C.sub.1-5
alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl and C.sub.1-4
alkoxy.
[0143] R.sub.5 is preferably hydrogen, methyl, ethyl, ethenyl,
ethynyl or methoxy. More preferably R.sub.5 is hydrogen, methyl or
ethyl. Most preferably R.sub.5 is hydrogen.
[0144] R.sub.4 is preferably C.sub.2-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl or C.sub.1-3 alkoxy. More preferably R.sub.4 is
n-propyl, --CH.dbd.CH--CH.sub.3, --C--C.dbd.CH.sub.2 or ethoxy.
Most preferably R.sub.4 is n-propyl.
[0145] A particularly preferred combination of R.sub.5 and R.sub.4
is when R.sub.5 is hydrogen and R.sub.4 is n-propyl. The presence
of a single n-propyl substituent at the carbon a to the carboxamide
has been found to be particularly beneficial for enabling the
compound to bind to .alpha..sub.1-antitrypsin at the relevant
binding site.
[0146] More preferably still, the compound comprises a monovalent
moiety of formula (IC)
##STR00018##
[0147] By monovalent is meant that the moiety of formula (IC)
contains one point of bonding, i.e. at the position 1 below
##STR00019##
[0148] In formula (IC), as in formulae (IA) and (IB), typically the
compound is capable of binding to .alpha..sub.1-antitrypsin by
hydrogen bond formation between: (i) hydroxyl group I and L291 of
SEQ ID NO: 1; (ii) NH group II and P289 of SEQ ID NO: 1; and (iii)
carbonyl group III and Y244 of SEQ ID NO: 1. R.sub.4 and R.sub.5
are as defined with reference to formula (TB). R.sub.6 is a
substituted or unsubstituted aryl or heteroaryl group. Typically,
R.sub.6 is capable of stacking with the side chain of W194 of SEQ
ID NO: 1 when the compound is bound to said
.alpha..sub.1-antitrypsin.
[0149] R.sub.6 is preferably substituted or unsubstituted and is:
(a) a C.sub.6-10 aryl group; or (b) a heteroaryl group that
contains 5 to 10 ring atoms. More preferably R.sub.6 is substituted
or unsubstituted and is: (a) a bicarbocyclic aromatic group; or (b)
a bicyclic heteroaryl group.
[0150] When R.sub.6 is an aryl group, particularly preferred groups
include naphthyl and indanyl (in both cases substituted or
unsubstituted and in both cases wherein optionally one carbon ring
atom may be substituted by a carbonyl group), most preferably
indanyl (optionally wherein one carbon ring atom may be substituted
by a carbonyl group).
[0151] When R.sub.6 is a heteroaryl group, preferred groups include
oxindolyl (e.g., 2-oxindolyl or 3-oxindolyl), benzothienyl,
benzofuranyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl,
benzoxazolyl, benzisoxazolyl, benzotriazolyl, indolyl, isoindolyl
and indazolyl (in all cases substituted or unsubstituted and in all
cases wherein optionally one carbon ring atom may be substituted by
a carbonyl group) and more preferred groups include oxindolyl
(e.g., 2-oxindolyl or 3-oxindolyl), benzothienyl, benzofuranyl,
benzimidazolyl, benzothiazolyl, benzoxazolyl and indolyl (in all
cases substituted or unsubstituted and in all cases wherein
optionally one carbon ring atom may be substituted by a carbonyl
group).
[0152] For any aryl or heteroaryl group in which one carbon ring
atom is substituted by a carbonyl group, the resulting ring entity
is --C(O)--. Where the "parent" carbon ring atom forms a double
bond to an adjacent ring atom, the substitution of that carbon ring
atom to --C(O)-- is accompanied by saturation of the said adjacent
ring atom, e.g. by addition of a hydrogen atom thereto or an
optional substituent as defined herein.
[0153] Particularly preferred R.sub.6 groups are substituted or
unsubstituted bicyclic aryl and heteroaryl groups comprising a
six-membered ring fused to a five-membered ring (i.e. containing 9
ring atoms). Of such R.sub.6 groups, preferably the ring atom of
the five-membered ring that is not adjacent to either of the ring
atoms shared by the five-membered ring and the six-membered ring is
a carbon atom bearing a carbonyl group, i.e. it is --C(O)--. For
instance, preferred R.sub.6 groups include the following 6-1 to
6-12:
##STR00020## ##STR00021##
in which one or more H-groups bound to ring atoms (preferably
carbon ring atoms) may be replaced by substituents.
[0154] More preferably still, R.sub.6 is a substituted or
unsubstituted 4-oxindolyl group (i.e. a 4-(2-oxindolyl) group),
which is group 6-1 above.
[0155] Preferably the R.sub.6 group is unsubstituted or substituted
with 1 to 4 substituents, e.g. 1 or 2 substituents. More preferably
the R.sub.6 group is unsubstituted or substituted with 1 or 2
substituents. Most preferably the R.sub.6 group is unsubstituted or
substituted with 1 substituent.
[0156] Optional substituents for R.sub.6 include those defined in
the "Definitions" section herein. Particularly preferred
substituents for R.sub.6 include halogen atoms, C.sub.1-5 alkyl,
--NR.sub.2 (where each R is independently selected from H and
C.sub.1-5 alkyl), and hydroxyl. Still more particularly preferred
substituents include F, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Cl, Br and I and still more preferred
substituents are F, CH.sub.3, NH.sub.2, OH and Cl. Most preferred
substitutes are F and Cl (e.g. F).
[0157] In an exemplary aspect, R.sub.6 is a group of formula
R.sub.6'
##STR00022##
wherein R.sub.1 is selected from the group consisting of H, F,
CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Cl, Br and I. In the formula R.sub.6', R.sub.1 is more preferably
selected from the group consisting of H, F, CH.sub.3, NH.sub.2, OH
and Cl and still more preferably selected from the group consisting
of H and F.
[0158] It is particularly preferred that the compound has the
formula (ID)
##STR00023##
[0159] In formula (ID), as in formulae (IA), (IB) and (IC),
typically the compound is capable of binding to
.alpha..sub.1-antitrypsin by hydrogen bond formation between: (i)
hydroxyl group I and L291 of SEQ ID NO: 1; (ii) NH group II and
P289 of SEQ ID NO: 1; and (iii) carbonyl group III and Y244 of SEQ
ID NO: 1. R.sub.4 and R.sub.5 are as defined with reference to
formula (IB). R.sub.6 is as defined with reference to formula
(IC).
[0160] R.sub.7 in formula (ID) is a substituted or unsubstituted
aryl or heteroaryl group. Preferably R.sub.7 is a substituted or
unsubstituted phenyl, pyridinyl, pyridazinyl, pyrimidinyl pyrazinyl
or triazinyl group. More preferably R.sub.7 is a substituted or
unsubstituted phenyl group.
[0161] R.sub.7 is preferably substituted by 1, 2, 3, 4 or 5
substituents, more preferably 1, 2 or 3 substituents and most
preferably 2 substituents. Optional substituents for R.sub.7
include those defined in the "Definitions" section herein.
Particularly preferred R.sub.7 substituents include halogen atoms
and C.sub.1-5 alkyl, --NR.sub.2 (where each R is independently
selected from H and C.sub.1-5 alkyl), hydroxyl, --SH and --CN
groups.
[0162] A particularly preferred R.sub.7 is a phenyl group
substituted with 1, 2, 3, 4 or 5 (preferably at least 2)
substituents. Preferably such a phenyl group comprises at least a
meta-substituent and an ortho-substituent, i.e. it is of formula
R.sub.7''
##STR00024##
wherein R.sub.2 and R.sub.3 are substituents, each R.sub.x is an
independently selected substituent and n is an integer of from 0 to
3. More preferably such a phenyl group has the formula R.sub.7'
##STR00025##
[0163] Preferably in R.sub.7' and R.sub.7'', each of R.sub.2,
R.sub.3 and any R.sub.x is independently selected from halogen
atoms and C.sub.1-5 alkyl, --NR.sub.2 (where each R is
independently selected from H and C.sub.1-5 alkyl), hydroxyl, --SH
and --CN groups. More preferably, R.sub.2 is selected from the
group consisting of CH.sub.3, Cl, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, SH, CN, F, Br and I; and R.sub.3 is selected
from the group consisting of F, Cl, CN, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Br, I and SH. More preferably still R.sub.2
is selected from the group consisting of CH.sub.3, Cl, NH.sub.2,
OH, SH, CN and F; and R.sub.3 is selected from the group consisting
of F, Cl, CN, CH.sub.3, NH.sub.2, OH and SH. Most preferably
R.sub.2 is selected from the group consisting of CH.sub.3 and Cl;
and R.sub.3 is selected from the group consisting of F, Cl and
CN.
[0164] A preferred combination of R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 in formula (ID) is that wherein: [0165] R.sub.4 and R.sub.5
are independently selected from hydrogen, C.sub.1-5 alkyl,
C.sub.2-5 alkenyl, C.sub.2-5 alkynyl and C.sub.1-4 alkoxyl; [0166]
R.sub.6 is a C.sub.6-10 aryl group or a heteroaryl group that
contains 5 to 10 ring atoms, said aryl or heteroaryl group being
unsubstituted or substituted by one or more (e.g. 1 to 5)
substituents selected from halogen atoms and C.sub.1-5 alkyl,
C.sub.1-4 alkoxy, C.sub.1-4 alkylthiol, --NR.sub.2 (where each R is
independently selected from H and C.sub.1-5 alkyl), hydroxyl, thiol
(SH), nitrile (CN), nitro and sulfonic acid groups; and [0167]
R.sub.7 is a phenyl, pyridinyl, pyridazinyl, pyrimidinyl pyrazinyl
or triazinyl group that is unsubstituted or substituted by one or
more (e.g. 1 to 4) substituents selected from halogen atoms and
C.sub.1-5 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 alkylthiol, --NR.sub.2
(where each R is independently selected from H and C.sub.1-5
alkyl), hydroxyl, thiol (SH), nitrile (CN), nitro and sulfonic acid
groups.
[0168] A more preferred combination of R.sub.4, R.sub.5, R.sub.6
and R.sub.7 in formula (ID) is that wherein: [0169] R.sub.4 is
C.sub.2-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl or C.sub.1-3
alkoxy; [0170] R.sub.5 is hydrogen, methyl, ethyl, ethenyl, ethynyl
or methoxy; [0171] R.sub.6 is selected from the following 6-1 to
6-12:
[0171] ##STR00026## ##STR00027## [0172] in which one or more (e.g.
1 to 3) H-groups bound to ring atoms are optionally replaced by
substituents selected from halogen atoms and C.sub.1-5 alkyl,
C.sub.1-4 alkoxy, C.sub.1-4 alkylthiol, --NR.sub.2 (where each R is
independently selected from H and C.sub.1-5 alkyl), hydroxyl, thiol
(SH), nitrile (CN), nitro and sulfonic acid groups; and [0173]
R.sub.7 is a phenyl, pyridinyl, pyridazinyl, pyrimidinyl pyrazinyl
or triazinyl group that is unsubstituted or substituted by one or
more (e.g. 1 to 4) substituents selected from halogen atoms and
C.sub.1-5 alkyl, --NR.sub.2 (where each R is independently selected
from H and C.sub.1-5 alkyl), hydroxyl, --SH and --CN groups.
[0174] A still more preferred combination of R.sub.4, R.sub.5,
R.sub.6 and R.sub.7 in formula (ID) is that wherein: [0175] R.sub.4
is n-propyl, --CH.dbd.CH--CH.sub.3, --C--C.dbd.CH.sub.2 or ethoxy;
[0176] R.sub.5 is hydrogen, methyl or ethyl; [0177] R.sub.6 is a
4-(2-oxindolyl) group that is unsubstituted or substituted by one
or more (e.g. 1 to 3) substituents selected from halogen atoms and
C.sub.1-5 alkyl, --NR.sub.2 (where each R is independently selected
from H and C.sub.1-5 alkyl), and hydroxyl groups; and [0178]
R.sub.7 is a group of formula R.sub.7''
[0178] ##STR00028## [0179] wherein R.sub.2 and R.sub.3 are
substituents, each R.sub.x is an independently selected
substituent, n is an integer of from 0 to 3, and each substituent
is independently selected from halogen atoms and C.sub.1-5 alkyl,
--NR.sub.2 (where each R is independently selected from H and
C.sub.1-5 alkyl), hydroxyl, --SH and --CN groups.
[0180] In one exemplary aspect, the compound has the formula
(I)
##STR00029##
wherein [0181] R.sub.1 is selected from the group consisting of H,
F, CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
Cl, Br and I; [0182] R.sub.2 is selected from the group consisting
of CH.sub.3, Cl, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3,
CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, OH,
SH, CN, F, Br and I; and [0183] R.sub.3 is selected from the group
consisting of F, Cl, CN, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OH, Br, I and SH.
[0184] Preferably in formula (I), R.sub.1 is selected from the
group consisting of H, F, CH.sub.3, NH.sub.2, OH and Cl. Preferably
in formula (I), R.sub.2 is selected from the group consisting of
CH.sub.3, Cl, NH.sub.2, OH, SH, CN and F. Preferably in formula
(I), R.sub.3 is selected from the group consisting of F, Cl, CN,
CH.sub.3, NH.sub.2, OH and SH. Preferably in formula (I), R.sub.1
is selected from the group consisting of H, F, CH.sub.3, NH.sub.2,
OH and Cl; and R.sub.2 is selected from the group consisting of
CH.sub.3, Cl, NH.sub.2, OH, SH, CN and F; and R.sub.3 is selected
from the group consisting of F, Cl, CN, CH.sub.3, NH.sub.2, OH and
SH.
[0185] Particularly preferably in formula (I), R.sub.1 is selected
from the group consisting of H and F. Particularly preferably in
formula (I), R.sub.2 is selected from the group consisting of
CH.sub.3 and Cl. Particularly preferably in formula (I), R.sub.3 is
selected from the group consisting of F, Cl and CN. Particularly
preferably in formula (I), R.sub.1 is selected from the group
consisting of H and F; and R.sub.2 is selected from the group
consisting of CH.sub.3 and Cl; and R.sub.3 is selected from the
group consisting of F, Cl and CN.
[0186] Non-limiting, exemplary compounds of formula (I) are those
in which: (a) R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3 is F;
(b) R.sub.1 is H, R.sub.2 is CH.sub.3 and R.sub.3 is Cl; (c)
R.sub.1 is F, R.sub.2 is Cl and R.sub.3 is CN; and (d) R.sub.1 is
F, R.sub.2 is Cl and R.sub.3 is F.
Therapeutic Indications
[0187] The compounds of the invention may be useful in treating
diseases mediated by alpha 1 antitrypsin, including
alpha-1-antitrypsin deficiency. Diseases mediated by alpha-1
antitrypsin include alpha-1 antitrypsin deficiency, liver
dysfunction, fibrosis, cirrhosis, liver failure and hepatocellular
carcinoma, diseases of lung, dysfunction and inflammation including
asthma, COPD, emphysema and lung cancer, inflammatory conditions of
the skin including dermatitis and pruritus. Usually, diseases
mediated by alpha-1 antitrypsin will be as a result of a mutation
in the alpha-1 antitrypsin gene and conforming to the general class
of diseases termed `serpinopathies`.
[0188] The compounds of this invention may be particularly useful
in the treatment of respiratory disease including COPD and
emphysema.
[0189] More particularly the compounds of the invention maybe
useful in the treatment of disorders due to mutant forms of alpha 1
antitrypsin, including the mutant Z-AT form of alpha 1 antitrypsin,
thus being potentially useful in treating diseases associated with
Z-AT, particularly diseases of the liver including jaundice, liver
failure, liver cirrhosis, autoimmune hepatitis, and hepatocellular
carcinoma.
[0190] Alpha-1 antitrypsin deficiency affects 1 in 2000 people of
Northern European descent, leading to liver and lung disease. Over
100 different mutations have been identified in the SERPINA1 gene
that encodes .alpha..sub.1-antitrypsin but an estimated 95% of
severe deficiency results from the Z-AT form (Glu342Lys). This
amino acid substitution disturbs the folding of
.alpha..sub.1-antitrypsin resulting in the secretion of only
.about.15% mature protein. Most of the remaining protein is
degraded via the ERAD-proteasome pathway, but approximately 15%
forms ordered polymers that accumulate within the endoplasmic
reticulum of hepatocytes. The consequent deficiency of an important
protease inhibitor within the circulation results in insufficient
protection of the lungs from neutrophil elastase, leading to
emphysema. The accumulation of polymers within hepatocytes is
cytotoxic to the cells causing neonatal hepatitis, cirrhosis and
hepatocellular carcinoma. The intrahepatic polymers also sensitise
the liver to damage from environmental insults such as alcohol, fat
or viral hepatitis.
[0191] The inhibition of the target protease--elastase--by
.alpha..sub.1-antitrypsin involves a subversion of the proteolytic
mechanism: following nucleophilic attack on a recognition sequence
in the .alpha..sub.1-antitrypsin reactive centre loop (RCL), the
two proteins become covalently attached through an ester bond.
Rapid insertion of the RCL into central .beta.-sheet A ensues,
converting it from a 5-stranded to 6-stranded configuration. The
concomitant translocation of the protease from one pole of the
serpin to the other, and compression of its active site, prevents
hydrolysis of this normally transient linkage; the two are thereby
irreversibly trapped an inactivated protease-serpin covalent
complex. The Z mutation lies at the head of strand 5 of
.beta.-sheet A. It perturbs the local environment, allowing
.alpha..sub.1-antitrypsin to populate an unstable intermediate
(that has been termed the M* state), in which .beta.-sheet A opens
and the upper part of helix F unwinds. It remains to be established
whether incorporation of the RCL is intermolecular--resulting in a
loop-sheet dimer which extends to form longer polymers--or
intramolecular with a domain-swap of the C-terminal region
providing the inter-subunit linkage for the pathological polymer
that deposits in liver tissue. Other forms of polymerization by
different mutants of .alpha..sub.1-antitrypsin may be possible. The
ability of a destabilised .beta.-sheet A to incorporate the
reactive centre loop is an obligate step in the formation of
polymers. This renders the mechanism of polymerisation closely
related to that of protease inhibition: in both, the reactive
centre loop is a central actor in a thermodynamically-driven
transition between a moderately stable and a hyper-stable
conformation.
[0192] The commonality between the physiological mechanism of
serpin inhibition and the pathological generation of polymer is
also observed within other members of the serpin superfamily and
gives rise to class of related pathologies termed serpinopathies.
These include familial encephalopathy with neuroserpin inclusion
bodies (FENIB) caused by polymerogenic mutations in neuroserpin and
forms of thrombosis caused by polymerisation and deficiency of
antithrombin.
Therapy and Methods of Treatment
[0193] This invention also provides a compound of the invention,
for use in therapy. This invention specifically provides for the
use of a compound of the invention, as an active therapeutic
substance in the treatment of diseases mediated by
alpha-1-antitrypsin.
[0194] The invention also provides for the use of a compound of the
invention in the manufacture of a medicament for use in the
treatment of diseases mediated by alpha-1-antitrypsin.
[0195] In a further aspect there is provided a combination
comprising a compound of the invention and at least one further
therapeutic agent useful in the treatment of diseases mediated by
alpha-1-antitrypsin.
[0196] In a further aspect there is provided a combination
comprising a compound of the invention and at least one further
therapeutic agent useful in the treatment of diseases mediated by
alpha-1-antitrypsin, for use in the treatment of diseases mediated
by alpha-1-antitrypsin.
[0197] In a further aspect there is provided the use of a
combination comprising a compound of the invention and at least one
further therapeutic agent useful in the treatment of COPD in the
manufacture of a medicament for the treatment of diseases mediated
by alpha-1-antitrypsin.
[0198] In a further aspect there is provided a method of treating
COPD comprising administering to a human in need thereof a
therapeutically effective amount of a combination comprising a
compound of the invention and at least one further therapeutic
agent useful in the treatment of diseases mediated by
alpha-1-antitrypsin.
[0199] In a further aspect there is provided a pharmaceutical
composition comprising a combination comprising a compound of the
invention and at least one further therapeutic agent useful in the
treatment of diseases mediated alpha-1-antitrypsin and one or more
of pharmaceutically acceptable excipients.
Respiratory Disease
[0200] For the treatment of respiratory disease, including COPD,
compounds or pharmaceutical compositions of the invention may be
administered together with one or more bronchodilators, or
pharmaceutical compositions thereof. For example, compounds of the
invention may be formulated together with one or more
bronchodilators in a single composition, such as a dry powder for
inhalation. Alternatively, a pharmaceutical composition comprising
a compound of the invention may be administered in conjunction with
a pharmaceutical composition comprising one or more
bronchodilators, either simultaneously or sequentially. In a
further alternative, a composition comprising a compound of the
invention and a bronchodilator may be administered in conjunction
with a pharmaceutical composition comprising a further
bronchodilator. In one embodiment, a pharmaceutical composition
comprising a compound of the invention and a pharmaceutical
composition comprising one or more bronchodilators may each be held
in device suitable for the simultaneous administration of both
compositions via inhalation. In a further embodiment, a
pharmaceutical composition comprising a compound of the invention
together with a bronchodilator and a pharmaceutical composition
comprising a further bronchodilator may each be held in device
suitable for the simultaneous administration of both compositions
via inhalation.
[0201] Suitable bronchodilators for administration together with
compounds of the invention include .beta..sub.2-adrenoreceptor
agonists and anticholinergic agents. Examples of
.beta..sub.2-adrenoreceptor agonists, include, for example,
vilanterol, salmeterol, salbutamol, formoterol, salmefamol,
fenoterol carmoterol, etanterol, naminterol, clenbuterol,
pirbuterol, flerbuterol, reproterol, bambuterol, indacaterol,
terbutaline and salts thereof, for example the xinafoate
(1-hydroxy-2-naphthalenecarboxylate) salt of salmeterol, the
sulphate salt of salbutamol or the fumarate salt of formoterol.
Examples of anticholinergic agents include umeclidinium (for
example as the bromide), ipratropium (for example, as the bromide),
oxitropium (for example, as the bromide) and tiotropium (for
example, as the bromide). In one embodiment, a compound of the
invention may be administered together with a
.beta..sub.2-adrenoreceptor agonist, such as vilanterol, and an
anticholinergic agent, such as, umeclidinium.
Liver Disease
[0202] For the treatment of liver disease the compounds or
pharmaceutical compositions of the invention may be administered
with other therapeutic agents useful in treating these
diseases.
General Synthetic Methods
[0203] Compounds of general formula (I) may be prepared by methods
known in the art of organic synthesis. In all of the methods, it is
well understood that protecting groups for sensitive or reactive
groups may be employed where necessary in accordance with general
principles of chemistry. Protecting groups are manipulated
according to standard methods of organic synthesis (T. W. Green and
P. G. M. Wuts (1999) Protective Groups in Organic Synthesis,
3.sup.rd edition, John Wiley & Sons). These groups are removed
at a convenient stage of the compound synthesis using methods that
are readily apparent to those skilled in the art. The selection of
processes as well as the reaction conditions and order of their
execution shall be consistent with the preparation of compounds of
Formula (I). Other compounds of the invention can also be prepared
using methods routinely known in the art and, where applicable,
with reference to the principles and specific reactions described
herein.
Method for Identifying a Drug Candidate Compound
[0204] The present inventors' discovery that compounds of the
invention inhibit .alpha..sub.1-antitrypsin polymerisation by
binding to the .alpha..sub.1-antitrypsin protein at a particular
binding site also gives rise to an assay method based on
determining the capability of an arbitrary drug candidate compound
to bind to the same site on the protein.
[0205] Thus, the present invention further provides a method for
identifying a drug candidate compound. In the method, the drug
candidate compound is contacted with .alpha..sub.1-antitrypsin to
form a complex between the drug candidate compound and
.alpha..sub.1-antitrypsin. The .alpha..sub.1-antitrypsin is a
protein comprising SEQ ID NO: 1 as defined elsewhere herein (or a
derivative of this sequence with one or more amino acid
substitutions). Preferably the step of contacting the drug
candidate compound with .alpha..sub.1-antitrypsin comprises forming
a crystal of .alpha..sub.1-antitrypsin and contacting said crystal
with said drug candidate.
[0206] Subsequently the method comprises resolving the structure of
the complex, which can be done routinely using crystal structure
resolution methods well known in the art (see also, for instance,
Example 2 of this application).
[0207] Finally, the method comprises determining whether, in the
complex, the drug candidate compound is present in the binding site
as defined herein. If the drug candidate compound is present in the
binding site, then this is indicative of a compound that may be
capable of inhibiting .alpha..sub.1-antitrypsin polymerisation, and
thus having beneficial therapeutic properties, by analogy with the
compounds of the present invention as discussed elsewhere
herein.
EXAMPLES
Example 1
General Methods
[0208] Unless stated otherwise, starting materials were
commercially available. All solvents and commercial reagents were
of laboratory grade and were used as received. Compounds of formula
(I) may be synthesised substantially according to Reaction Scheme 1
from an amide coupling of the corresponding amino alcohol and
corresponding benzoic acid.
##STR00030##
ABBREVIATIONS
[0209] DMSO Dimethyl sulfoxide [0210] HATU
N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-meth-
ylmethanaminium hexafluorophosphate N-oxide [0211] r.t. Room
temperature [0212] Rt Retention time
LCMS Methods
TABLE-US-00006 [0213] Method Description A Column: Acquity UPLC CSH
C18 column (50 mm .times. 2.1 mm i.d. 1.7 .mu.m particle size)
Mobile Phase: A: 0.1% Formic acid in water; B: 0.1% Formic acid in
Acetonitrile Time (min)/%B: 0/3%, 1.5/99.9%, 1.9/99.9%, 2.0/3.0%
Column Temp: 40.degree. C. Flow Rate: 1.0 mL/min B Column: Acquity
UPLC BEH C18 column (50 mm .times. 2.1 mm i.d. 1.7 .mu.m particle
size) Mobile Phase: A: 0.1% v/v ammonia aqueous solution pH 10; B:
Acetonitrile Time (min)/% B: 0/3%, 1.5/99.9%, 1.9/99.9%, 2.0/3.0%
Column Temp: 40.degree. C. Flow Rate: 1.0 mL/min C Column: Acquity
UPLC BEH C18 (50 mm .times. 2.1 mm, 1.7 .mu.m particle size) Mobile
Phase: A: 0.1% Formic acid in water; B: 0.1% Formic acid in
Acetonitrile Time (min)/% B: 0/3.0%, 1.5/100.0%, 1.9/100.0%, 2.0/3%
Column Temp: 40.degree. C. Flow Rate: 1.0 mL/min
Chiral HPLC Method
TABLE-US-00007 [0214] Method Description Chiral Column Chiralpak
AS-H (25 .times. 0.46 cm, 5 um particle size) HPLC Mobile phase:
n-Hexane/(Ethanol + 0.1% isopropylamine) 65/35% v/v Flow rate: 1.0
ml/min
Compound 1
N-[(1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl]-2-oxo-2,3-dih-
ydro-1H-indole-4-carboxamide
##STR00031##
[0216] In a round bottom flask, to a solution of
(1S,2R)-2-amino-1-(3-fluoro-2-methylphenyl)pentan-1-ol
hydrochloride (intermediate 1, 12.9 g, 51.9 mmol),
2-Oxoindoline-4-carboxylic acid (9.2 g, 51.9 mmol) and
triethylamine (10.5 g, 103.9 mmol) in dimethylformamide (100 ml) at
0.degree. C., HATU (19.8 g, 51.9 mmol) was added. The reaction
mixture was stirred at 0.degree. C. for 1 h and then was diluted
with ethyl acetate (200 ml). The solution was washed with a
saturated solution of NaHCO.sub.3 (100 ml) and brine (2.times.100
ml). The organic portion was dried over sodium sulfate, filtered
and evaporated under reduced pressure to obtain crude material
which was purified by flash Chromatography (Biotage Isolera, 340 g
cartridge, Dichloromethane/Acetonitrile 80/20 to 40/60 as eluent)
to afford
N-[(1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl]-2-oxo-2,3-di-
hydro-1H-indole-4-carboxamide as white solid (12.5 g, Y=65.0%).
[0217] In a round bottom flask, (12.5 g, 33.74 mmol) was suspended
in ethyl acetate (100 ml) at rt. The suspension was heated to
60.degree. C. and ethyl acetate was added until the mixture became
homogeneous (.apprxeq.200 ml of solvent totally). The solution was
left to cool at r.t. overnight and the white precipitate formed was
collected by suction filtration. The solid was stored in oven at
40.degree. C. under vacuum to obtain crystalline
N-[(1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl]-2-oxo-2,3-di-
hydro-1H-indole-4-carboxamide as white solid (10.8 g, >98% ee,
87% recovery).
[0218] LCMS: Rt 0.90 min, MH+371 (Method A)
[0219] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. ppm 0.80 (t,
J=7.34 Hz, 3H), 1.07-1.21 (m, 1H), 1.30-1.43 (m, 1H), 1.45-1.54 (m,
1H), 1.56-1.67 (m, 1H), 2.31 (d, J=1.51 Hz, 3H), 3.46 (s, 2H),
4.06-4.17 (m, 1H), 4.85 (t, J=5.21 Hz, 1H), 5.44 (d, J=4.80 Hz,
1H), 6.89 (d, J=7.68 Hz, 1H), 6.99 (t, J=8.92 Hz, 1H), 7.11-7.25
(m, 3H), 7.28 (d, J=7.68 Hz, 1H), 7.97 (d, J=8.92 Hz, 1H), 10.45
(s, 1H).
[0220] Chiral HPLC: Rt 6.3 min
Intermediate 1
(1S,2R)-2-amino-1-(3-fluoro-2-methylphenyl)pentan-1-ol
hydrochloride
##STR00032##
[0222] In a round bottom flask, tert-butyl
((1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl)carbamate
(intermediate 2, 15.8 g, 50.74 mmol) was dissolved in HCl in
dioxane 4 M (100 ml) at 0.degree. C. The mixture was stirred for 2
h at the same temperature and the solvent was evaporated. The
residue was suspended in 300 ml of diethyl ether and stirred for 1
h. The suspension was filtered to obtain
(1S,2R)-2-amino-1-(3-fluoro-2-methylphenyl)pentan-1-ol
hydrochloride as a white solid (11.9 g, Y=95.0%).
[0223] LCMS: Rt 0.85 min, MH+212 (Method B)
Intermediate 2
tert-butyl
((1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl)carba-
mate
##STR00033##
[0225] (R)-tert-butyl
(1-(3-fluoro-2-methylphenyl)-1-oxopentan-2-yl)carbamate
(intermediate 3, 18.7 g, 60.4 mmol) was dissolved in toluene (150
ml) in a round bottom flask, and isopropanol (100 ml) at r.t. and
aluminum isopropoxide (49.4 g, 241.8 mmol) were added. The solution
was stirred at 50.degree. C. for 16h and then it was poured into a
NH.sub.4Cl sat. solution and extracted with ethyl acetate
(4.times.300 ml). The organic portions were collected, dried over
sodium sulfate and the solvent was removed under reduced pressure
to obtain crude material named as pale yellow oil. This crude
material was purified by flash Chromatography (Biotage Isolera, 340
g cartridge, Cyclohexane/Ethyl Acetate 80/20 to 50/50 as eluent) to
afford tert-butyl
((1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl)carbamate
as a colourless oil (15.8 g, Y=84.0%) LCMS: Rt 1.16 min, MH+312
(Method A)
Intermediate 3
(R)-tert-butyl
(1-(3-fluoro-2-methylphenyl)-1-oxopentan-2-yl)carbamate
##STR00034##
[0227] To a mixture of (3-fluoro-2-methylphenyl)boronic acid
(intermediate 4, 50 g, 0.32 mol) in a round bottom flask,
(R)--S-p-tolyl 2-((tert-butoxycarbonyl)amino)pentanethioate (53.2
g, 0.16 mol), tris(dibenzylideneacetone)dipalladium(0) (7.3 g, 8.0
mmol), and copper(I) thiophene-2-carboxylate (61.0 g, 0.32 mol) in
1,4-Dioxane (500 ml) at r.t., triethyl phosphite (10.6 g, 64.0
mmol) was added. The reaction mixture was stirred at rt for 4h and
then was filtered over a celite pad. The solvent was evaporated
under reduced pressure and the crude was dissolved in ethyl acetate
(500 ml); the solution was washed with water (500 ml) and brine
(2.times.500 ml). The organic portion was dried over sodium
sulfate, filtered and evaporated under reduced pressure to obtain
crude material (100 g) as black oil. A 65 g batch of the crude
material was purified by flash Chromatography (Biotage Isolera,
3.times.340 g cartridge, Cyclohexane/Dichloromethane 60/40 to 0/100
as eluent) to afford (R)-tert-butyl
(1-(3-fluoro-2-methylphenyl)-1-oxopentan-2-yl)carbamate as
colourless oil (18.7 g, 60.4 mmol).
[0228] LCMS: Rt 1.26 min, MH+310 (Method A)
Intermediate 4
(R)--S-p-tolyl 2-((tert-butoxycarbonyl)amino)pentanethioate
##STR00035##
[0230] To a solution of (R)-2-((tert-butoxycarbonyl)amino)pentanoic
acid (intermediate 5, 85.0 g, 0.39 mol) in ethyl acetate (800 ml)
in a round bottom flask, 4-methylbenzene-1-thiol (53.5 g, 0.43 mol)
and 1-Hydroxybenzotriazole (50.0 g, 0.59 mol) were added. The
mixture was cooled to 0.degree. C. and
N,N'-Dicyclohexylcarbodiimide (80.5 g, 0.39 mol) was added. The
reaction mixture was stirred at r.t. for 16h; the white solid
formed was then removed by suction filtration. The filtrate was
washed with a saturated solution of NaHCO3 (700 ml), water (700 ml)
and brine (700 ml). The organic portion was dried over sodium
sulfate, filtered and the solvent was evaporated under reduced
pressure to obtain crude material as a white solid. The crude
material was purified by flash chromatography (Biotage Isolera, 340
g cartridge, Cyclohexane/Dichloromethane 60/40 to 0/100 as eluent)
to afford (R)--S-p-tolyl
2-((tert-butoxycarbonyl)amino)pentanethioate as a white solid
(111.0 g, Y=88.0%).
[0231] LCMS: Rt 1.30 min, MH+324 (Method A)
Intermediate 5
(R)-2-((tert-butoxycarbonyl)amino)pentanoic acid
##STR00036##
[0233] In a round bottom flask, to a suspension of
(2R)-2-aminopentanoic acid (75.0 g, 0.64 mol) was put into a round
bottom flask in water (250 ml) at r.t., sodium carbonate (68.0 g,
0.64 mol) was added. A solution of di-tert-butyl dicarbonate (139.9
g, 0.64 mol) in tetrahydrofuran (400 ml) was added dropwise over
one hour. The reaction mixture was stirred at r.t. for 24h and then
concentrated under reduced pressure (approx. 400 ml). A saturated
solution of citric acid (300 ml) was added until pH reached the
value of .apprxeq.3. The aqueous layer was washed with ethyl
acetate (4.times.500 ml). The organic portions were collected,
dried over magnesium sulfate and evaporated under reduced pressure
to obtain (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid as a
pale yellow oil (137.0 g, Y=98.6%).
[0234] LCMS: Rt 0.82 min, MH+218 (Method A)
Similarly Prepared were:
TABLE-US-00008 Retention Molecular ion + LCMS Example n.degree.
structure name time (min) Identity Method Compound 2 ##STR00037##
N-((1S,2R)-1-(3- chloro-2- methylphenyl)-1- hydroxypentan-2-
yl)-2-oxoindoline- 4-carboxamide 0.93 387 (MH.sup.+) C Compound 3
##STR00038## N-((1S,2R)-1-(2- chloro-3- cyanophenyl)-1-
hydroxypentan-2- yl)-7-fluoro-2- oxoindoline-4- carboxamide 0.82
416 (MH+) C Compound 4 ##STR00039## N-((1S,2R)-1-(2- chloro-3-
fluorophenyl)-1- hydroxypentan-2- yl)-7-fluoro-2- oxoindoline-4-
carboxamide 0.89 409 (MH+) C
[0235] The potencies of the compounds of the invention for
inhibition of Z-alpha-1-antitrypsin (Z-A1AT) polymerisation can be
determined by a time-resolved fluorescence energy transfer
(TR-FRET) assay performed on purified Z-A1AT as described herein.
All compounds of formula (I) have demonstrated inhibitory activity
of Z-A1AT polymerisation in-vitro with a half maximal inhibitory
concentration (IC.sub.50) of less than one micromolar (1 .mu.M).
Mean activity data for the assay described below for compounds
1-4:
TABLE-US-00009 Compound 1 PIC50 8.3 SD = .18, Range 8.0-8.4
Compound 2 PIC50 8.6 SD = .15, Range 8.4-9.9 Compound 3 PIC50 8.5
SD = .1 Range 8.4-8.6 Compound 4 PIC50 8.2 SD = .21, Range
7.8-8.5
Inhibition of Z-Alpha-1-Antitrypsin Polymerisation Assay
Protocol
[0236] Z-alpha-1-antitrypsin was purified from human plasma of
donors homozygous for the Z-A1AT form. The purification was
performed according to a published protocol (Lomas et al.
Biochemistry 1993; 32:500-508) involving ammonium sulphate
precipitation, thiol exchange fractionation, and anion exchange
fractionation.
[0237] Purified Z-A1AT was diluted to 5 nM in PBS buffer with 0.01%
Tween-20 and mixed with different concentrations of inhibitor
compounds dissolved in DMSO, in 384-well assay plates. Following
incubation for 72 hours at 37.degree. C., the amount of Z-A1AT
polymer was detected using a time-resolved fluorescence resonance
energy transfer (TR-FRET) assay format, by adding an antibody
detection mix to the assay plates. The detection mix contained an
A1AT-polymer specific monoclonal antibody (Miranda et al.
Hepatology 2010; 52: 1078-1088), Terbium-labelled anti-mouse
antibody, a polyclonal rabbit anti-A1AT antibody, and an
Alexa488-labelled anti-rabbit antibody. TR-FRET data were
normalised to controls (DMSO vehicle control giving 0% inhibition,
saturating concentrations of an active compound giving 100%
inhibition), and IC50 values were derived by fitting 4-parameter
logistic curves to the normalised data.
Example 2
Introduction
[0238] The aim of this work was to develop a small molecule
corrector of Z .alpha..sub.1-antitrypsin folding able to block the
formation of polymer within the endoplasmic reticulum of
hepatocytes and that was suitable for oral dosing as a potential
treatment for .alpha..sub.1-antitrypsin deficiency. To achieve this
a number of challenges needed to be overcome: (i) the specific form
of the protein representing the drug target is a highly mobile
folding intermediate, located in the endoplasmic reticulum; (ii)
the targeted pharmacology involves prevention of a large
protein-protein interaction and the requirement that a molecule
have drug-like properties to enable oral dosing greatly restricts
suitable chemical space; (iii) as a non-classical drug target,
small molecule binders may well not be well-represented in compound
screening libraries; (iv) the relatively high concentration of
circulating monomeric Z .alpha..sub.1-antitrypsin (.about.5 .mu.M),
even in individuals with severe plasma deficiency, represents a
high affinity sink for compound, restricting its access to the
target in the hepatocyte and requiring high total blood
concentrations of drug to achieve sufficient free drug
concentration and target engagement in the liver.
[0239] Reported herein are the first small molecule drug-like
correctors of Z .alpha..sub.1-antitrypsin folding obtained via
optimisation of hits from an Encoded Library Technology screen,
that are suitable for oral delivery, correct folding in human
patient iPSC-derived hepatocytes and increase circulating Z
.alpha..sub.1-antitrypsin levels in a transgenic mouse model of
.alpha..sub.1-antitrypsin deficiency.
Materials and Methods
[0240] Alpha.sub.1-antitrypsin was purified from the plasma of ZZ
and MM .alpha..sub.1-antitrypsin homozygotes as described in Lomas
et al., Biochemistry. 1993; 32:500-8. Recombinant Cys232Ser
.alpha..sub.1-antitrypsin was expressed and purified as detailed in
Irving et al. Methods Enzymol. 2011; 501:421-66 and Haq et al.
Biosci Rep. 2013; 33(3):e00046.
"Compound 1" is
N-[(1S,2R)-1-(3-fluoro-2-methylphenyl)-1-hydroxypentan-2-yl]-2-oxo-2,3-di-
hydro-1H-indole-4-carboxamide
##STR00040##
[0241] DNA-Encoded Library Technology (ELT) Screen
[0242] An encoded library technology (ELT) screen of approximately
10 million compounds was used to identify small molecules that will
stabilise monomeric Z .alpha..sub.1-antitrypsin at 37 and
41.degree. C. The strongest binders were eluted, identified by
their DNA tag and synthesised.
Antibody-Based Assessment of Polymerisation of Z
.alpha..sub.1-Antitrypsin
[0243] An antibody-based time-resolved fluorescence resonance
energy transfer (TR-FRET) assay was used to monitor the
polymerisation of 5 nM Z .alpha..sub.1-antitrypsin following
incubation with varying concentrations of compounds at 37.degree.
C. for 72 hours. This assay used the 2C1 monoclonal antibody that
is specific to pathological polymers of .alpha..sub.1-antitrypsin
(Miranda et al. Hepatology 2010; 52: 1078-1088), a polyclonal
antibody that binds to all forms of .alpha..sub.1-antitrypsin, an
anti-mouse IgG fluorescence donor (Eu-W1024) and an anti-rabbit IgG
acceptor (APC).
Biochemical Competition Binding Assay for Z
.alpha..sub.1-Antitrypsin and M .alpha..sub.1-Antitrypsin
[0244] A fluorescent derivative of compound 1 was prepared by
derivatizing compound 1 at its ring nitrogen atom with an Alexa
Fluor 488 label via a short organic linker group.
[0245] A serial dilution of test compounds in DMSO was diluted
25-fold with assay buffer, and 2.5 .mu.l of this intermediate
dilution transferred to a black, low-volume 384-well microtitre
plate (Greiner BioOne; product no. 784076). 5 .mu.l of a pre-mixed
solution of assay buffer containing 2.3 nM of the monoclonal
antibody 3Cl 1 binding to antitrypsin, 3 nM of LanthaScreen
Terbium-labeled Anti-mouse IgG which will bind to the 3Cl 1
antibody (Invitrogen), as well as either 2 nM Z-a1AT or 10 nM
M-a1AT were added using a Thermo Multidrop dispenser
(ThermoFisher). Plates were incubated at room temperature for 30
min, then 2.5 .mu.l of ligand solution were added, containing
either 40 nM (for Z .alpha..sub.1-antitrypsin) or 2 .mu.M (for M
.alpha..sub.1-antitrypsin) of the fluorescent derivative of
compound 1, using a Thermo Multidrop dispenser.
[0246] Plates were sealed and incubated overnight at room
temperature, or for 3 days at 4.degree. C. then 6 hours at room
temperature. In the absence of competing compound, energy transfer
can occur between the Terbium- and Alexa488 fluorophores. Compounds
competing with the fluorescent derivative of compound 1 inhibit
this signal. The TR-FRET signal was read on an Envision plate
reader, by excitation of Terbium at 337 nm and detection of
emission at 520 nm and 495 nm.
[0247] Dissociation constants (K.sub.D) for the affinity of
compounds to Z-a1AT or M-a1AT protein were derived from the pIC50
values according to the Cheng-Prusoff equation, taking into account
the concentration of fluorescent ligand used in relation to its
affinity as determined in independent Fluorescence Polarisation and
TR-FRET experiments:
K.sub.D(compound)=10.sup.-pIC50(compound)/(1-([Ligand]/K.sub.D(Ligand)).
Compound Association Experiments
[0248] The intrinsic tryptophan fluorescence of
.alpha..sub.1-antitrypsin is dominated by Trp194 in the breach
region (Tew et al. J Mol Biol. 2001 Nov. 9; 313(5):1161-9) and can
be used to monitor the progress of compound binding. Plasma M and Z
.alpha..sub.1-antitrypsin were diluted to 0.2 mg/ml in 100 .mu.M
PBS, and Compound 1 (or an equivalent volume of DMSO) added to a
concentration of 10 .mu.M. Fluorescence (.lamda..sub.ex=280 nm,
.lamda..sub.em=330 nm) was monitored for at least 2 hours, and the
half-time of change was calculated.
[0249] Kinetic data in stopped flow experiments were fitted to
exponential decay curves with offset and linear slope, to account
for a constant drift in the background, resulting in observed
pseudo-first-order rate constants k(obs):
y=A.sub.0e.sup.-k(obs)t+slopet+offset
where t=time and Ao=amplitude at t=0. The second-order on-rate was
then obtained as the slope when plotting k(obs) values versus
compound concentration. Off-rates were calculated from the on-rates
and the K.sub.D values determined from the binding assays.
Cell Biology
[0250] CHO Tet-On cells that conditionally express Z
.alpha..sub.1-antitrypsin (Ordonez A et al. Hepatology 2013;
57(5):2049-60.) were simultaneously treated with 0.5 .mu.g/mL
oxycycline and various concentrations of compounds for 48 hours in
6 well plates. Cells without doxycycline or treated with 1% v/v
DMSO acted as controls. The cells were lysed and intracellular Z
.alpha..sub.1-antitrypsin polymer was evaluated by ELISA sandwich
with the 2Cl monoclonal antibody (Miranda et al. Hepatology. 2010;
52:1078-88). Newly synthesised .alpha..sub.1-antitrypsin was
evaluated by growing cells induced to express Z
.alpha..sub.1-antitrypsin in DMEM without methionine (Met) and
cysteine (Cys) for 1 h and then labelling with 1.3MBq of
.sup.35S-Met/Cys for 15 min. The cells were then rinsed and
incubated in chase medium containing an excess of unlabeled Met and
Cys for 0, 2 and 6h. After the chase period, the medium was
collected and cells were harvested. .alpha..sub.1-Antitrypsin from
the medium and cell lysates was immunoprecipitated with a
polyclonal .alpha..sub.1-antitrypsin antibody or the 2C.sub.1
monoclonal antibody that detects polymers by splitting each sample
in two equal parts. Radiolabelled proteins were re-suspended in SDS
loading buffer, boiled for 5 min, separated by 10% w/v acrylamide
SDS-PAGE, and quantified by autoradiography.
Immunofluorescence Microscopy
[0251] CHO-K1 cells were incubated with/without doxycycline to
express .alpha..sub.1-antitrypsin and with or without the small
molecule of interest, fixed with 3.7% v/v formaldehyde in PBS for
15 min, permeabilised with 0.1% v/v Triton X-100 in PBS and then
blocked with 10% v/v FBS in PBS for 30 min. Cells were labelled
with a rabbit polyclonal antibody to total
.alpha..sub.1-antitrypsin or the 2Cl monoclonal antibody that
detects polymers followed by Texan red conjugated anti-rabbit or
FITC labelled anti-mouse secondary antibodies respectively. Cells
were imaged using an Axiovert 200 widefield fluorescence microscope
(Carl Zeiss Microimaging Inc., Thornwood, N.Y.).
Thermal Stability Experiments
[0252] The native state stability of serpins on addition of
compounds was investigated by thermal denaturation in the presence
of a 5.times. concentration of SYPRO Orange dye solution (Life
Technologies). A final protein concentration of 0.1 mg/ml and 50 uM
compound suspended in PBS was used (Nettleship et al. Methods Mol
Biol. 2008; 426:299-318). The samples were heated from 25.degree.
C.-95.degree. C. at a rate of 1.degree. C./min in three separate
experiments on an Eppendorph Mastercycler Realplex4 quantitative
real-time PCR instrument, and the fluorescence in the 605.+-.15 nm
bin recorded. The midpoint of denaturation (Tm) is the temperature
at which the first derivative of fluorescence intensity against
temperature reaches a maximum.
[0253] Resistance to heat-induced polymerisation was also
determined using an end-point constant-temperature assay. Plasma
purified M and Z .alpha..sub.1-antitrypsin were diluted to 0.2
mg/ml in PBS with 5% v/v glycerol, incubated with 10 .mu.M Compound
1 (or with DMSO alone) for 2 hours and then heated in 20 .mu.l
aliquots for a further 4 hours in a thermal cycler with a
48-65.degree. C. gradient applied across the plate. Samples were
analysed on a 3-12% w/v acrylamide Bis-Tris Native-PAGE gel (Life
Technologies).
Equilibrium Unfolding
[0254] Bis-ANS is a dye that reports the appearance of the
unfolding intermediate of .alpha..sub.1-antitrypsin (Dafforn et al.
J Biol Chem. 1999; 274:9548-55.). In a microplate, 10 .mu.l
aliquots of M and Z .alpha..sub.1-antitrypsin at 0.5 mg/ml with 100
.mu.M bis-ANS and 100 .mu.M Compound 1 (or an equivalent volume of
DMSO) were rapidly mixed with various concentrations of guanidine
hydrochloride giving a final concentration of 0-6 M denaturant in
PBS. Fluorescence intensity measurements of bis-ANS
(.lamda..sub.ex=385 nm, .lamda..sub.em=490 nm) were recorded once a
stable signal was achieved, and the values scaled to lie between 0
and 1.0.
Rapid Refolding
[0255] Refolding of .alpha..sub.1-antitrypsin in vitro is not fully
reversible, due to loss of material from amorphous aggregation,
polymerisation and misfolding. These contributions cannot be
readily deconvoluted by bulk spectroscopic methods and therefore
refolding was evaluated by non-denaturing PAGE. M and Z
.alpha..sub.1-antitrypsin at 9.6 mg/ml were denatured in 6 M urea
and 15 mM sodium phosphate pH 8.0 for 4 hours at room temperature,
and 1.25 .mu.l was snap refolded into 250 .mu.l PBS containing 0-50
.mu.M Compound 1 and a normalised concentration of 5% v/v DMSO.
After incubation for 1 hour at room temperature samples were
analysed by Coomassie-stained 3-12% w/v acrylamide Bis-Tris Native
PAGE gel (Life Technologies).
Crystallography
[0256] Crystals of recombinant Cys232Ser .alpha..sub.1-antitrypsin
were grown using the hanging drop vapour diffusion method by
combining 1 .mu.l of 12.1 mg/ml protein with 1 .mu.l of reservoir
buffer and equilibrating against reservoir buffer, which comprised
0.1 M MES pH 6.0 buffer with 20-22.25% w/v PEG 1500. A crystal that
formed in 0.1M MES pH 6.0 with 22.25% w/v PEG 1500 was transferred
into a buffer containing 0.09M MES pH6.0, 18% PEG1500, 13.5%
glycerol, 5% d6 DMSO and 25 mM of the compound for incubation at
20.degree. C. for 24 hours. The crystals were snap-frozen in liquid
nitrogen and data was collected at the Diamond synchrotron 103
beamline.
[0257] Specifically, the .alpha..sub.1-antitrypsin protein used for
the crystallisation experiments corresponded to the M1V variant of
M-AT with the following modifications: (a) Cys232 is mutated to Ser
(for ease of handling, obviating the need for reducing agents
during different assays; the substitution is a conservative one);
(b) the signal peptide component (MPSSVSWGILLLAGLCCLVPVSLA) and
Glu1 at the N-terminus of mammalian M-AT are not present at the
N-terminus of the protein used; (c) the N-terminus of the protein
used has a hexahistidine affinity tag for ease of purification and
a few amino acids provided by the expression vector, the sequence
thereof being MRGSHHHHHHT.
[0258] The full sequence of the protein used was thus:
TABLE-US-00010 MRGSHHHHHHTDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYR
QLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIP
EAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKL
YHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVN
YIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHSKKL
SSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA
SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAV
HKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP LFMGKVVNPTQK;
wherein the underlined portion corresponds to SEQ ID NO: 1.
Results
Identification of Compound 1 Through Encoded Library Technology
Screening, Structure Guided Drug Design and Cellular Profiling
[0259] Z .alpha..sub.1-antitrypsin is a conformationally dynamic
molecule that polymerises from a near-native conformation late in
the folding pathway and therefore represents a non-classical target
for drug discovery. Thus it was reasoned that hit finding may
benefit from exploration of broad and minimally biased chemical
space. An Encoded Library Technology (ELT) screen was used to
search for binders to Z .alpha..sub.1-antitrypsin. Glycosylated Z
.alpha..sub.1-antitrypsin, purified from the plasma of Z
.alpha..sub.1-antitrypsin homozygotes, was used since this
represents the disease-relevant human pathophysiological drug
target. ELT selections were performed by immobilising Z
.alpha..sub.1-antitrypsin on an antibody-based affinity resin for
incubation with DNA-encoded compound libraries, with secondary
screening by thermal shift experiments.
[0260] A single lead series of chiral hydroxy-carboxamides was
identified from the ELT screen that demonstrated functional
activity at blocking polymerisation in the TR-FRET immunoassay.
Optimisation of the initial hit followed a structure-based design
approach, exploiting knowledge from crystal structures of small
molecule ligands complexed with .alpha..sub.1-antitrypsin. The
central hydroxy carboxamide was found to facilitate binding to Z
.alpha..sub.1-antitrypsin. An alkyl side chain (at the carbon
adjacent to the N atom of the carboxamide) was also found to be
beneficial, particularly a propyl side chain. Medicinal chemistry
development focused on modifications such as the aromatic groups at
the terminal ends of the compound. This resulted in the discovery
of Compound 1.
Compound 1 is a Potent Inhibitor of Polymerisation In Vitro and in
Cell Models of Disease
[0261] Compound 1 binds to Z .alpha..sub.1-antitrypsin with a high
affinity (K.sub.D) of 1.5 nM as determined by a competition binding
assay with a fluorescently labelled derivative; a similar value was
obtained by microscale thermophoresis. The binding demonstrates
selectivity with at least 50-fold lower affinity for
plasma-purified wild-type M .alpha..sub.1-antitrypsin.
[0262] The shape of the curves and native mass spectrometry were
consistent with a single compound binding site. No binding of the
fluorescent derivative to polymers of the Z variant could be
observed, indicating loss of the binding pocket upon polymerisation
and a resulting marked conformational selectivity. The rate of
interaction of the compound with the target could be monitored
through changes in intrinsic tryptophan fluorescence; this property
was used to determine the second-order association rates for
Compound 1 binding to Z (4.1.times.10.sup.4 M.sup.-1 s.sup.-1) and
M .alpha..sub.1-antitrypsin (2.1.times.10.sup.2 M.sup.-1 s.sup.-1).
From these values, first-order dissociation rates were calculated
for Z (6.1.times.10.sup.-5 s.sup.-1) and M
.alpha..sub.1-antitrypsin (1.6.times.10.sup.-5 s.sup.-1) and found
to be of the same order of magnitude. Therefore, the selectivity of
the compound for Z over M .alpha..sub.1-antitrypsin is dominated by
the difference in the rate of association rather than
dissociation.
[0263] Compound 1 was found to ablate polymerisation in vitro of
plasma-derived Z .alpha..sub.1-antitrypsin in a dose-dependent
manner with a measured pIC50 of 8.3, near the tight binding limit
of the TR-FRET assay. Its ability to block Z
.alpha..sub.1-antitrypsin polymerisation in the ER during folding
was assessed by adding Compound 1 to CHO-TET-ON-Z-A1AT cells
(Ordonez et al. Hepatology. 2013; 57(5):2049-60) with simultaneous
induction of Z .alpha..sub.1-antitrypsin expression using
doxycycline. In comparison with controls, Compound 1 completely
blocked the intracellular formation of Z .alpha..sub.1-antitrypsin
polymers, as measured by staining with the 2C1
anti-.alpha..sub.1-antitrypsin polymer monoclonal antibody
(pIC50=6.3). To investigate whether in addition to blocking
polymerisation, Compound 1 was also able to increase secretion of Z
.alpha..sub.1-antitrypsin, Z .alpha..sub.1-antitrypsin expression
was induced in CHO-TET-ON-Z-A1AT cells, Compound 1 was added, and
the total Z .alpha..sub.1-antitrypsin in the supernatant was
measured by immunoassay after 6 hours. Compound 1 increased
secreted levels approximately 3-fold compared to vehicle control
with a pEC50 of 6.3. Similar potency between the effects on
secretion and polymerisation was observed for other compounds of
the invention, supporting the hypothesis that these effects are
caused by the same pharmacological mode of action.
[0264] In order to confirm that the potency and efficacy of
Compound 1 in CHO-TET-ON-Z-A1AT was a reasonable estimate of what
could be expected in hepatocytes with endogenous levels of
expression, the effect of Compound 1 on the secretion and
polymerisation of Z .alpha..sub.1-antitrypsin was measured in
iPSC-derived hepatocytes with the ZZ .alpha..sub.1-antitrypsin
genotype (Yusa et al. Nature. 2011; 478:391-4). Compound 1
inhibited polymerisation and increased secretion with a similar
potency in iPSC-hepatocytes as in CHO-TET-ON-Z-A1AT cells, with a
pIC50 of 6.4 and pEC50 of 6.5 respectively, and with a similar
efficacy, inducing an approximately 3-fold increase of secreted
levels of Z .alpha..sub.1-antitrypsin.
[0265] Since all individuals with the .alpha..sub.1-antitrypsin Z
allele exhibit polymer in their livers but only a relatively small
fraction develop clinical liver disease it is likely
.alpha..sub.1-antitrypsin liver disease in adults is a two-hit
process whereby the generation of Z .alpha..sub.1-antitrypsin
polymer in the liver sensitises the liver to a secondary insult
such as alcohol, drug or liver fat. To investigate the ability of
Compound 1 to protect cells from sensitisation to a secondary
toxin, Z .alpha..sub.1-antitrypsin expression was induced in
CHO-TET-ON-Z-A1AT cells in the presence or absence of 10 .mu.M
Compound 1 before exposure to varying concentrations of the ER
stressor tunicamycin. In a cell viability assay, cells expressing
wild-type M .alpha..sub.1-antitrypsin were less sensitive to
tunicamycin than cells expressing Z .alpha..sub.1-antitrypsin.
Compound 1 completely abrogated polymer formation in Z
.alpha..sub.1-antitrypsin expressing cells and restored sensitivity
of .alpha..sub.1-antitrypsin expressing cells to that of the
wild-type control cells.
Compound 1 Binds to a Novel Cryptic Binding Site More Frequently
Sampled by the Z Variant
[0266] High resolution crystal structures of
.alpha..sub.1-antitrypsin complexed with Compound 1 were generated
by soaking compound into apo .alpha..sub.1-antitrypsin crystals
(Table 1).
TABLE-US-00011 TABLE 1 Data collection and refinement statistics
Compound 1 Data collection Space group C2 Cell dimensions a, b, c
(.ANG.) 113.9, 39.6, 90.5 .alpha., .beta., .gamma. ( ) 90.0, 105.0,
90.0 Resolution (.ANG.) 55.05-1.76 (1.85-1.76) R.sub.merge (%)
0.025(0.398) CC-half 0.999(0.842) I/.sigma.I 19.6(2.4) Completeness
(%) 99.2(99.2) Multiplicity 3.3(3.3) Refinement Resolution (.ANG.)
55.05-1.76(1.80-1.76)* No. reflections 36959(2858)
R.sub.work/R.sub.free 18.7 (27.8)/22.2 (32.1) No. atoms Protein
2868 Water 289 B-factors Protein 46.8 Water 59.2 R.m.s. deviations
Bond lengths (.ANG.) 0.05 Bond angles ( ) 1.03 *Values in
parentheses are for highest-resolution shell.
[0267] The structures reveal that interaction with the compounds
induces the formation of a cryptic binding site not evident in apo
structures, at the top of .beta.-sheet-A behind strand 5. This
region is referred to as the `breach` as it is the point at which
the reactive centre loop first inserts during protease inhibition
(Whisstock et al. J Mol Biol. 2000; 295(3):651-65), and includes
the site of the E342K Z-mutation.
[0268] Representative images of the identified binding site are
shown in FIGS. 1 to 3. FIG. 1 shows an overview of the protein
bound to Compound 1 (residues 342-356 are disordered and hence
missing from the representation). FIG. 2 provides a more detailed
view of the binding site including some of the proximate residues,
with hydrogen bonds shown as broken lines. FIG. 3 is a schematic,
two-dimension visualisation of Compound 1 at the binding site of
the protein, and highlighting some of the amino acid residues that
comprise the binding site as well as partial chemical structures
within the protein that form significant intermolecular
interactions (e.g., hydrogen bonds) to Compound 1 (shown as broken
lines).
[0269] In more detail, the structure of .alpha..sub.1-antitrypsin
complexed with Compound 1 reveals that the oxindolyl ring stacks
with the side chain of Trp194 whilst the carbonyl group forms a
hydrogen bond with Trp194 mainchain (see FIG. 2 and FIG. 3),
consistent with the change in intrinsic tryptophan fluorescence
induced by binding. The phenyl ring and the propyl chain occupy two
highly hydrophobic pockets. Hydrogen bonds are formed between the
Compound 1 hydroxyl group and the Leu291 backbone, the amide
nitrogen hydrogen and the backbone carbonyl oxygen of Pro289, and
between the amide carbonyl and the Tyr244 hydroxyl group (see FIG.
2 and FIG. 3). As a consequence, there is displacement of residues
189 to 195, 290 to 295 and 336 to 342, of strand 5A with a
corresponding loss of electron density for the proximal hinge of
the reactive centre loop, an .about.170.degree. rotation of the
Trp194 side-chain and 3.8A shift in the Tyr244 hydroxyl group and
displacement of residues 196-203. A comparison of the Compound
1-bound structure with the apo form shows an overall 0.64A
root-mean-square deviation for 335 residue pairs.
[0270] Specifically, therefore, Compound 1 was found to be capable
of binding to the .alpha..sub.1-antitrypsin at a cryptic binding
site as defined elsewhere herein. For completeness, it is noted
that the hexahistidine affinity tag and amino acids provided by the
expression vector in the protein used were not apparent in the
electron density profile of the binding site and were not therefore
associated with the binding of compound 1. Similarly, the mutated
Ser232 (replacing Cys232 in the wild-type protein) was not at a
position directly associated with the compound binding site.
Compound 1 Interferes with the Transition to the
Polymerisation-Prone Intermediate M* by Stabilising .beta.-Sheet
A
[0271] Polymerisation of .alpha..sub.1-antitrypsin occurs from a
transient intermediate state known as M*, that is readily populated
by the Z variant. One of the hallmarks of M* is its recognition by
environment-sensitive fluorescent reporter dyes. Thermal shift
assays that make use of the dye SYPRO Orange report the stability
of the protein native state against heat-induced unfolding. A
series of experiments, performed using different temperature
gradients in the presence and absence of 50 .mu.M Compound 1,
demonstrated a marked increase in the transition midpoint
temperature; in .alpha..sub.1-antitrypsin this is indicative of a
native state stabilised against the transition to M*.
Correspondingly, in a constant-temperature experiment, oligomers
were generated at higher temperatures in the presence of the
compound than in its absence when visualised by non-denaturing
PAGE. Native state stability can also be probed by equilibrium
unfolding using chemical denaturants, where a peak in bis-ANS
fluorescence corresponds with a maximally populated unfolding
intermediate. The measured profiles showed that for guanidinium
hydrochloride-induced unfolding this point occurs at 1.8 M
denaturant in the presence of 50 .mu.M Compound 1, reflecting an
increase in native state stability with respect to its absence (1.2
M).
[0272] Mutations that interfere with the opening of .beta.-sheet A
or that perturb its interaction with the N-terminal portion of the
reactive centre loop alter the ability of serpins to inhibit target
proteases. The stoichiometry of inhibition (SI) was determined for
M and Z .alpha..sub.1-antitrypsin in discontinuous experiments
against a model target protease, chymotrypsin. The pre-incubation
of both variants with Compound 1 led to a >98% loss of protease
inhibitory activity. Resolution of the products of the interaction
by SDS-PAGE showed full cleavage of the reactive centre loop;
therefore, this is not a consequence of the protease recognition
site in the reactive centre loop becoming inaccessible to the
enzyme. These data are consistent with a mechanism in which the
compound stabilises .beta.-sheet A against conformational change
that mediates both inhibitory activity and pathological misfolding.
To investigate whether this activity was consistent with action as
a chemical chaperone, M and Z .alpha..sub.1-antitrypsin were
unfolded in vitro into 6 M guanidine hydrochloride, and rapidly
refolded by snap dilution into denaturant-free buffer in the
presence or absence of Compound 1. Electrophoresis of the products
by non-denaturing PAGE showed an anodally-shifted migration for the
Z variant in the absence of compound--consistent with a misfolded
byproduct of M* (Ekeowa et al. Proc Natl Acad Sci USA. 2010;
107(40):17146-51)--which was corrected at stoichiometric
concentrations and above.
Characterisation of Drug-Like Properties of Compound 1
[0273] In order to investigate suitability of Compound 1 for
progression into in vivo studies and the potential for taking
forward as a clinical candidate for testing in human, Compound 1
was profiled against a panel of in vitro assays for off-targets
considered predictive of potential safety liabilities. Compound 1
was inactive against the majority of these targets with a low level
of activity close to the lower limit of sensitivity in 16 assays.
The measured potency of these marginal activities were at least
10-fold below the cellular activity of Compound 1 at blocking Z
.alpha..sub.1-antitrypsin polymerisation (pEC50 6.3). Loss of
inhibitory activity represents a means by which possible off-target
engagement with other serpins can be evaluated. However in contrast
to .alpha..sub.1-antitrypsin upon incubation with 50 .mu.M Compound
1 the inhibitory activity of the homologues antithrombin,
neuroserpin and antichymotrypsin was not affected.
[0274] Since Compound 1 exhibited a good level of selectivity over
the off-target panel and other serpins the in vitro and in vivo PK
properties of the molecule were explored with a view to exploring
target engagement in vivo. Compound 1 has a measured ChromLogD
(pH7.4) of 3.8, low binding to human serum albumin of 84.2% and
good solubility of amorphous drug substance in FaSSIF.
[0275] Compound 1 had in vitro ADME properties suitable for in vivo
evaluation of the pre-clinical and clinical pharmacology of the
molecule. Permeability was high and in vitro clearance in
cryopreserved hepatocytes was low in human and dog (0.3.+-.0.05 and
<0.65 mL/min/g respectively) and moderate to high in mouse and
rat (4.6 and 7.4.+-.0.7 mL/min/kg respectively). Mean exposure of
Compound 1 in blood in the male CD-1 mouse increased with dose
following single PO administration at 10, 30 or 100 mg/kg (mean
dose-normalised C.sub.max 58.+-.112, 113.+-.27 and 113.+-.27;
DNAUC.sub.inf 202.+-.101, 294.+-.47 and 403.+-.246,
respectively).
Compound 1 Increases Secretion of Z .alpha..sub.1-Antitrypsin in a
Transgenic Mouse Model of .alpha..sub.1-Antitrypsin Deficiency
[0276] In order to investigate the therapeutic potential of
Compound 1 in Z .alpha..sub.1-antitrypsin deficiency, the compound
was tested in a transgenic mouse model engineered with a random
insertion of the human Z .alpha..sub.1-antitrypsin gene (Teckman et
al. Am J Physiol Gastrointest Liver Physiol. 2004; 286:G851-G62).
To determine a suitable dose regimen for Compound 1 in these
studies the total and unbound exposure in transgenic mice was
estimated in an in silico model. This was based on the observed PK
parameters from wild-type mice and incorporating a circulating high
affinity sink for drug, representing the mean 5 .mu.M total Z
.alpha..sub.1-antitrypsin in the blood of the mice at baseline with
an affinity for drug of 1.5 nM. Based on this modelling an oral
dosing regimen of 100 mg/kg three times a day was predicted to
maintain the free drug concentration above the EC50 measured for
secretion of Z .alpha..sub.1-antitrypsin (.about.300 nM) for the
duration of the dosing period. In contrast, unbound concentrations
of Compound 1 after 30 and 10 mg/kg three times a day would be
expected to be well below the EC50 for secretion for most of the
dosing period.
[0277] To explore the PK-PD relationship of Compound 1, Z
.alpha..sub.1-antitrypsin transgenic animals were dosed with 100,
30 or 10 mg/kg Compound 1 three times a day and on day 6 blood and
liver were harvested at 3 hr (.about.C.sub.max) and 8 hr
(C.sub.min) after the dose for the measurement of total and free
drug in both tissues. Blood was also harvested for the measurement
of monomeric Z .alpha..sub.1-antitrypsin in plasma. Total
concentrations of Compound 1 were determined by specific LC-MS/MS
assay and the free unbound drug in both tissues was determined
using equilibrium dialysis and used to derive unbound
concentrations. Free and total blood concentrations were consistent
with the predictions and confirmed that the C.sub.min levels of the
free drug concentration was at or above 300 nM for the majority of
the dosing period following 100 mg/kg dosing, whereas 30 mg/kg and
10 mg/kg doses resulted in free drug levels in blood significantly
below the cellular EC50 concentrations for a large part of the
dosing period. Both free and total drug concentrations of Compound
1 at the targeted site of action in the liver were equivalent to
those in blood.
[0278] Recent work has shown that a significant fraction of the
total Z .alpha..sub.1-antitrypsin in the circulation is in the
polymeric conformation (Tan et al. Eur Respir J. 2014;
43(5):1501-4). Since there are no antibodies specific for monomeric
Z .alpha..sub.1-antitrypsin to directly determine its
concentration, a deconvolution method was developed based on
immunoassays with antibodies for either total or polymeric
.alpha..sub.1-antitrypsin, and calibration curves with purified
monomeric and polymeric Z .alpha..sub.1-antitrypsin. Monomeric Z
.alpha..sub.1-antitrypsin was measured in plasma samples following
6 days of dosing and levels were normalised to each animals'
pre-dose control levels to account for the natural variation of Z
.alpha..sub.1-antitrypsin between animals. 100 mg/kg Compound 1
resulted in a 6- to 7-fold increase in circulating monomeric Z
.alpha..sub.1-antitrypsin levels demonstrating robust target
engagement in the liver. 30 mg/kg and 10 mg/kg groups also gave
significant, dose-dependent increases in circulating Z
.alpha..sub.1-antitrypsin despite free concentrations being below
the cellular EC50 for secretion for much or all of the dosing
period. Total drug levels and changes in Z
.alpha..sub.1-antitrypsin following 3 days of dosing were
indistinguishable to those following 6 days of dosing.
[0279] Taken together these results confirm target engagement in
the liver following oral dosing of Compound 1 and demonstrate that
circulating levels of Z .alpha..sub.1-antitrypsin can be increased
by a therapeutically relevant magnitude in a model
.alpha..sub.1-antitrypsin deficiency. The increase of circulating Z
.alpha..sub.1-antitrypsin at lower doses may suggest that achieving
sustained free drug levels above the cellular EC50 for secretion
may not be required in vivo to significantly increase Z
.alpha..sub.1-antitrypsin levels.
[0280] Since Compound 1 blocks polymer formation in cells the
effect of dosing Compound 1 on liver polymer levels was explored.
It has recently been shown that Z .alpha..sub.1-antitrypsin
polymers formed in CHO-TET-ON-Z-A1AT and ZZ-iPSC-hepatocytes are
cleared from cells with a t1/2 of between 8 and 48 hours depending
on whether they partition to the soluble or insoluble fractions.
Since the compound does not bind Z .alpha..sub.1-antitrypsin
polymer, an effect on total liver polymer levels will be dependent
on the rate at which the liver can clear the polymer already
present and the rate at which polymer continues to accumulate in
animals not treated with drug. Compound 1 was therefore dosed at
100 mg/kg three times a day as above for 20 days. On days 15 and 21
of dosing, changes of monomeric Z .alpha..sub.1-antitrypsin plasma
samples were determined as above, and increases of 7- to 8-fold
over the pre-dosing baseline levels were observed, similar to the
effect in animals dosed for 3 or 6 days, consistent with sustained
target engagement through the dosing period. Liver polymer levels
were investigated by staining with 2C1 anti-polymer monoclonal
antibody and were scored blinded either by a pathologist or by
quantification using an algorithm to measure all areas of positive
staining. There was no difference observed in total liver polymer
load via manual or quantitative scoring. Aged Z
.alpha..sub.1-antitrypsin mice exhibit dense polymeric inclusions
that are not observed in livers of individuals with
.alpha..sub.1-antitrypsin deficiency.
[0281] To investigate whether there is a subpopulation of polymer
in the liver that was responding to compound over the time frame of
the experiment, areas of staining were divided into low, medium and
high intensity regions and scored separately for response to
compound. No effect of compound was discernible in any of these
subdivided regions. Given that polymer formation is completely
blocked by Compound 1 in vitro it was concluded that longer term
dosing will be required to affect total polymer burden in Z
.alpha..sub.1-antitrypsin transgenic mice. However, it remains to
be determined whether the mouse liver has the capacity to clear Z
.alpha..sub.1-antitrypsin polymer following the chronic insult of
generating Z .alpha..sub.1-antitrypsin in the liver throughout life
or indeed whether this is required to elicit a benefit to the
sensitivity to secondary triggers of liver dysfunction.
DISCUSSION
[0282] The co-crystal structures with .alpha..sub.1-antitrypsin
provide insight into the mode of action by which Compound 1
inhibits Z .alpha..sub.1-antitrypsin polymerisation. Whilst the
mechanism that results in pathological liver polymer formation has
not been established, the obligate and central role of the reactive
centre loop in this process is well-accepted (see (a) Lomas et al.
Nature. 1992; 357:605-7; (b) Ekeowa et al. Proc Natl Acad Sci USA.
2010; 107(40):17146-5; and (c) Yamasaki et al. EMBO Rep. 2011;
12(10):1011-7). All extant models are based on the insertion of an
extra .beta.-strand derived from the reactive centre loop between
strands 3 and 5 of the A-sheet, facilitated by the destabilising
effect of the Z mutation on this structural element. The co-crystal
structure of .alpha..sub.1-antitrypsin and Compound 1 suggests that
the compound may ameliorate the effect of the E342K Z-mutation due
to: (i) optimisation of hydrophobic packing in the breach region;
(ii) formation of hydrogen bonds with buried polar atoms; and also
(iii) displacement of the backbone at the top of strand 5A into a
configuration less compatible with partial loop insertion, which is
suggested to be an early step in loop-sheet polymerisation (Gooptu
et al. Proc Natl Acad Sci (USA). 2000; 97(1):67-72.) and an
obligate one in C-terminal polymerisation (Yamasaki et al. EMBO
Rep. 2011; 12(10):1011-7.). The displacement evident at the top of
strand 5A is consistent with the association rate-driven preference
for the Z variant, which destabilises this region with respect to
the wild-type protein. In the crystal structures the side-chain at
342 is oriented towards the solvent, and does not directly interact
with Compound 1; thus the key parameter driving compound preference
is likely to be an increased availability of the cryptic pocket.
The pocket, once formed, appears to be structurally equivalent in
both variants as reflected by a similar rate of dissociation. This
mode of action is also compatible with the lack of binding to
polymers, in which partial or complete insertion of the reactive
centre loop completes a beta-hairpin turn that would occlude the
compound binding site.
[0283] Upon Compound 1 binding within the breach region, there is a
marked stabilisation of the .alpha..sub.1-antitrypsin native state
against the conformational change associated with M* intermediate
formation. This in turn prevents the formation of polymers.
Precedent for this general mechanism can be found in a tool
monoclonal antibody that exerted a similar effect on
.alpha..sub.1-antitrypsin (Ordonez et al. FASEB J. 2015;
29:2667-78; Motamedi-Shad et al. Biochem J. 2016;
473(19):3269-90).
[0284] The lack of serpin activity in the lower airways leading to
digestion of the lung parenchyma by locally-released neutrophil
elastase contributes to the emphysema observed in individuals with
.alpha..sub.1-antitrypsin deficiency since rare patients who are
completely null for .alpha..sub.1-antitrypsin develop pan-lobular
emphysema (Fregonese et al. Respir Med. 2008; 102(6):876-84.) and
.alpha..sub.1-antitrypsin replacement therapy has demonstrated some
benefit on disease progression (Chapman et al. Lancet. 2015;
386(9991):360-8.). Compound 1 increases Z .alpha..sub.1-antitrypsin
secretion by correcting the folding defect and increases plasma
concentrations of the serpin up to 8-fold in a transgenic mouse
model of .alpha..sub.1-antitrypsin deficiency
[0285] Since Compound 1 inhibits the serpin activity of
.alpha..sub.1-antitrypsin whilst being bound to the protein it
would not be expected to significantly increase serpin activity
during the dosing period, indeed Compound 1 would inhibit the
serpin activity of residual .alpha..sub.1-antitrypsin in patients
and would be expected to block activity of replacement therapy
should this be co-administered with Compound 1. However, the
half-life of monomeric Z .alpha..sub.1-antitrypsin in human is
believed to be .about.3-5 days whereas drug would be expected to be
cleared with a t1/2 of a few hours after dosing raising the
possibility of a pulsatile dosing regimen that would lead to
increased, active serpin. The definition of how long exposure to
drug would be required to significantly elevate levels, to deplete
the inflammatory polymer in the lungs and the washout period
between doses to retain serpin activity requires further study.
Similarly, the risk-benefit of blocking residual serpin activity
during the dosing period will need to be assessed; however, the
slow development of the lung disease in individuals with
.alpha..sub.1-antitrypsin deficiency over many decades suggests
that acute effects associated with inhibition of serpin activity
are unlikely. .alpha..sub.1-antitrypsin polymers have been
identified in the lungs of COPD patients with two normal
.alpha..sub.1-antitrypsin alleles, suggesting that
.alpha..sub.1-antitrypsin polymers may be a prevalent feature of
the inflammation in COPD, independent of .alpha..sub.1-antitrypsin
genotype (Bazzan et al. Chest. 2018; 154:607-16).
[0286] Compound 1 blocks Z .alpha..sub.1-antitrypsin polymerisation
in cell free media and in the ER of cells and newly formed polymer
is cleared by cells via secretion, proteasomal degradation or
autophagy with a half-life of 8-48 hours. However, no reduction in
Z .alpha..sub.1-antitrypsin polymers was observed in the liver
after 20 days of dosing Compound 1 in a Z .alpha..sub.1-antitrypsin
transgenic mouse despite a 7-fold increase in circulating levels
within 3 days of dosing which was maintained throughout the
experiment, indicating robust target engagement. The likely
explanation for this is that the polymers that build up in the
livers of the transgenic mice are not cleared as readily as those
that can be generated in model systems such as CHO cells and
iPSC-hepatocytes over a few days. Whilst this likely results from
chronicity of insult, the molecular or cellular mechanism of this
is unclear but cellular aging and senescence may be a contributor
to this effect. Previous studies with the autophagy activator
Carbamazapine have demonstrated pronounced effects on liver polymer
in Z .alpha..sub.1-antitrypsin transgenic mice following 2 weeks of
dosing suggesting that the liver does retain the ability to clear
polymer following chronic insult of Z .alpha..sub.1-antitrypsin
polymer accumulation (Hidvegi et al. Science. 2010; 329:229-32.).
In contrast, RNAi approaches that inhibit Z
.alpha..sub.1-antitrypsin expression and polymer formation have
reported decreases in Z .alpha..sub.1-antitrypsin in transgenic
mouse liver following 12-33 weeks of treatment, albeit without
reports of data at earlier timepoints (Guo et al. J Clin Invest
2014; 124(1):251-61). Together these data suggest that the liver
retains the ability to clear polymer following chronic insult and
that it is likely that Compound 1 will need to be dosed to
transgenic Z .alpha..sub.1-antitrypsin mice for significantly
longer than 20 days to demonstrate an effect on total liver polymer
levels. It remains to be seen whether the built-up polymer is
actually toxic to the cells and needs to be cleared from the liver
in order to have some functional benefit or whether the accumulated
polymer inclusions are actually inert and shutting off production
of polymer is sufficient to restore the functioning of the ER and
health of the cells (Ordonez et al. Hepatology. 2013;
57(5):2049-60; Dickens et al. FASEB J. 2016; 30(12):4083-97).
[0287] The observed steady state total and free compound levels of
Compound 1 in the transgenic Z .alpha..sub.1-antitrypsin mouse were
predicted well by the in silico PK model built on in vitro
metabolic clearance data and plasma protein binding data, in vivo
PK data from wild type mice and a term comprising a 5 .mu.M
circulating sink for drug with an affinity of 1.5 nM, representing
the Z .alpha..sub.1-antitrypsin within blood. The target free drug
concentration has been selected based on the observed potency in
the in vitro secretion assays in which the total drug approximates
to the free drug in the assay. However, the increase in Z
.alpha..sub.1-antitrypsin in the circulation at the lower doses of
Compound 1 is surprising given that systemic free drug levels are
between the EC10-EC20 and EC20-EC30 for the duration of the dosing
period for 10 and 30 mg/kg doses respectively. The reason for this
is unclear however it is possible that the target engagement in
vivo is greater than predicted from the potency in the in vitro
cellular assays. Alternatively, it is possible that the first pass
effect of drug reaching the liver immediately after absorption
delivers some efficacy over that predicted from modelling the
compound concentration at steady state levels. Together these data
suggest potential upsides for the required compound exposure to
deliver efficacy in the clinic.
Novel Mechanism for Treating a Conformational Disease
[0288] The compounds of the invention are believed to stabilise
monomeric Z .alpha..sub.1-antitrypsin by binding at the head of
strand 5 of .beta.-sheet A thereby negating the local effect of the
Z mutation (Lomas et al. Biochemistry. 1993; 32:500-8).
Specifically they displace strand 5 towards strand 3 and so
`correct` the local perturbation induced by a lysine residue at
position 342. The binding site of Compound 1 has not been
identified in previous crystal structures of
.alpha..sub.1-antitrypsin (Elliott et al. Protein Science. 2000;
9:1274-81.). However their effect on blocking the transition of
fully folded Z .alpha..sub.1-antitrypsin to polymerogenic
intermediate, and their efficacy in cell and animal models of
disease, suggests that they are acting on Z
.alpha..sub.1-antitrypsin in a near-native or native conformation.
This implies that polymers form from a near-native or native
conformation, rather than a more extended intermediate, and that
the small molecules prevent the monomer linking to form a polymer.
This small molecule correction of the Z mutation provides a novel
strategy to treat the liver disease in individuals with
.alpha..sub.1-antitrypsin deficiency.
Sequence CWU 1
1
61394PRTArtificial
Sequencealpha1-antitrypsinMISC_FEATURE(1)..(1)Xaa is T or
EMISC_FEATURE(101)..(101)Xaa is R or HMISC_FEATURE(213)..(213)Xaa
is A or VMISC_FEATURE(232)..(232)Xaa is C or
SMISC_FEATURE(342)..(342)Xaa is E or KMISC_FEATURE(376)..(376)Xaa
is D or E 1Xaa Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser
His His1 5 10 15Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn
Leu Ala Glu 20 25 30Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln
Ser Asn Ser Thr 35 40 45Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr
Ala Phe Ala Met Leu 50 55 60Ser Leu Gly Thr Lys Ala Asp Thr His Asp
Glu Ile Leu Glu Gly Leu65 70 75 80Asn Phe Asn Leu Thr Glu Ile Pro
Glu Ala Gln Ile His Glu Gly Phe 85 90 95Gln Glu Leu Leu Xaa Thr Leu
Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110Thr Thr Gly Asn Gly
Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125Lys Phe Leu
Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140Val
Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr145 150
155 160Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu
Leu 165 170 175Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe
Phe Lys Gly 180 185 190Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr
Glu Glu Glu Asp Phe 195 200 205His Val Asp Gln Xaa Thr Thr Val Lys
Val Pro Met Met Lys Arg Leu 210 215 220Gly Met Phe Asn Ile Gln His
Xaa Lys Lys Leu Ser Ser Trp Val Leu225 230 235 240Leu Met Lys Tyr
Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255Glu Gly
Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265
270Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu
275 280 285Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val
Leu Gly 290 295 300Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala
Asp Leu Ser Gly305 310 315 320Val Thr Glu Glu Ala Pro Leu Lys Leu
Ser Lys Ala Val His Lys Ala 325 330 335Val Leu Thr Ile Asp Xaa Lys
Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350Leu Glu Ala Ile Pro
Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365Pro Phe Val
Phe Leu Met Ile Xaa Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380Met
Gly Lys Val Val Asn Pro Thr Gln Lys385 3902418PRTHomo sapiens 2Met
Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys1 5 10
15Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala
20 25 30Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe
Asn 35 40 45Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr
Arg Gln 50 55 60Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser
Pro Val Ser65 70 75 80Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly
Thr Lys Ala Asp Thr 85 90 95His Asp Glu Ile Leu Glu Gly Leu Asn Phe
Asn Leu Thr Glu Ile Pro 100 105 110Glu Ala Gln Ile His Glu Gly Phe
Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125Gln Pro Asp Ser Gln Leu
Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140Ser Glu Gly Leu
Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys145 150 155 160Leu
Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170
175Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys
180 185 190Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe
Ala Leu 195 200 205Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg
Pro Phe Glu Val 210 215 220Lys Asp Thr Glu Glu Glu Asp Phe His Val
Asp Gln Val Thr Thr Val225 230 235 240Lys Val Pro Met Met Lys Arg
Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255Lys Lys Leu Ser Ser
Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270Thr Ala Ile
Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285Asn
Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295
300Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly
Thr305 310 315 320Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile
Thr Lys Val Phe 325 330 335Ser Asn Gly Ala Asp Leu Ser Gly Val Thr
Glu Glu Ala Pro Leu Lys 340 345 350Leu Ser Lys Ala Val His Lys Ala
Val Leu Thr Ile Asp Glu Lys Gly 355 360 365Thr Glu Ala Ala Gly Ala
Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380Pro Pro Glu Val
Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu385 390 395 400Gln
Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410
415Gln Lys3418PRTArtificial Sequencefull sequence of Z-A1AT 3Met
Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys1 5 10
15Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala
20 25 30Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe
Asn 35 40 45Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr
Arg Gln 50 55 60Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser
Pro Val Ser65 70 75 80Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly
Thr Lys Ala Asp Thr 85 90 95His Asp Glu Ile Leu Glu Gly Leu Asn Phe
Asn Leu Thr Glu Ile Pro 100 105 110Glu Ala Gln Ile His Glu Gly Phe
Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125Gln Pro Asp Ser Gln Leu
Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140Ser Glu Gly Leu
Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys145 150 155 160Leu
Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170
175Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys
180 185 190Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe
Ala Leu 195 200 205Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg
Pro Phe Glu Val 210 215 220Lys Asp Thr Glu Glu Glu Asp Phe His Val
Asp Gln Ala Thr Thr Val225 230 235 240Lys Val Pro Met Met Lys Arg
Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255Lys Lys Leu Ser Ser
Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270Thr Ala Ile
Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285Asn
Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295
300Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly
Thr305 310 315 320Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile
Thr Lys Val Phe 325 330 335Ser Asn Gly Ala Asp Leu Ser Gly Val Thr
Glu Glu Ala Pro Leu Lys 340 345 350Leu Ser Lys Ala Val His Lys Ala
Val Leu Thr Ile Asp Lys Lys Gly 355 360 365Thr Glu Ala Ala Gly Ala
Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380Pro Pro Glu Val
Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu385 390 395 400Gln
Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410
415Gln Lys4404PRTArtificial SequenceSEQ ID NO 1 comprising an
affinity tag 4Met Arg Gly Ser His His His His His His Thr Asp Pro
Gln Gly Asp1 5 10 15Ala Ala Gln Lys Thr Asp Thr Ser His His Asp Gln
Asp His Pro Thr 20 25 30Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe
Ala Phe Ser Leu Tyr 35 40 45Arg Gln Leu Ala His Gln Ser Asn Ser Thr
Asn Ile Phe Phe Ser Pro 50 55 60Val Ser Ile Ala Thr Ala Phe Ala Met
Leu Ser Leu Gly Thr Lys Ala65 70 75 80Asp Thr His Asp Glu Ile Leu
Glu Gly Leu Asn Phe Asn Leu Thr Glu 85 90 95Ile Pro Glu Ala Gln Ile
His Glu Gly Phe Gln Glu Leu Leu Arg Thr 100 105 110Leu Asn Gln Pro
Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu 115 120 125Phe Leu
Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val 130 135
140Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp
Thr145 150 155 160Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu
Lys Gly Thr Gln 165 170 175Gly Lys Ile Val Asp Leu Val Lys Glu Leu
Asp Arg Asp Thr Val Phe 180 185 190Ala Leu Val Asn Tyr Ile Phe Phe
Lys Gly Lys Trp Glu Arg Pro Phe 195 200 205Glu Val Lys Asp Thr Glu
Glu Glu Asp Phe His Val Asp Gln Val Thr 210 215 220Thr Val Lys Val
Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln225 230 235 240His
Ser Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly 245 250
255Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His
260 265 270Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu
Glu Asn 275 280 285Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys
Leu Ser Ile Thr 290 295 300Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly
Gln Leu Gly Ile Thr Lys305 310 315 320Val Phe Ser Asn Gly Ala Asp
Leu Ser Gly Val Thr Glu Glu Ala Pro 325 330 335Leu Lys Leu Ser Lys
Ala Val His Lys Ala Val Leu Thr Ile Asp Glu 340 345 350Lys Gly Thr
Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met 355 360 365Ser
Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met 370 375
380Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val
Asn385 390 395 400Pro Thr Gln Lys524PRTArtificial SequenceSignal
peptide 5Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly
Leu Cys1 5 10 15Cys Leu Val Pro Val Ser Leu Ala 20611PRTArtificial
Sequencehexahistidine affinity tag and expression vector amino
acids 6Met Arg Gly Ser His His His His His His Thr1 5 10
* * * * *