U.S. patent application number 17/466230 was filed with the patent office on 2022-06-02 for pyrrolopyrimidine and pyrrolopyridine derivatives.
The applicant listed for this patent is Galapagos NV. Invention is credited to David AMANTINI, Karl Richard GIBSON, Milan MESIC, Tanja POLJAK, Gordon SAXTY, Ines VUJASINOVIC, David WITTY, Dinko ZIHER.
Application Number | 20220169653 17/466230 |
Document ID | / |
Family ID | 1000006140548 |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220169653 |
Kind Code |
A1 |
AMANTINI; David ; et
al. |
June 2, 2022 |
PYRROLOPYRIMIDINE AND PYRROLOPYRIDINE DERIVATIVES
Abstract
The present invention relates to pyrrolopyrimidine compounds
according to Formula I and their use in the prophylaxis and/or
treatment of pain, inflammatory conditions, cardiovascular
diseases, neurodegenerative diseases, neurological diseases,
complications of type I diabetes, cancer and/or fibrotic diseases.
In a particular aspect, the present compounds are ASK inhibitors,
particularly ASK1 inhibitors. The present invention also provides
methods for the production of a compound of the invention,
pharmaceutical compositions comprising a compound of the invention,
the use of the compounds in the prophylaxis and/or treatment of
pain, inflammatory conditions, cardiovascular diseases,
neurodegenerative diseases, neurological diseases, complications of
type I diabetes, cancer and/or fibrotic diseases. ##STR00001##
Inventors: |
AMANTINI; David;
(Romainville, FR) ; MESIC; Milan; (Zagreb, HR)
; SAXTY; Gordon; (Zagreb, HR) ; POLJAK; Tanja;
(Zagreb, HR) ; VUJASINOVIC; Ines; (Zagreb, HR)
; ZIHER; Dinko; (Zagreb, HR) ; WITTY; David;
(Cambridge, GB) ; GIBSON; Karl Richard; (Kent,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Galapagos NV |
Mechelen |
|
BE |
|
|
Family ID: |
1000006140548 |
Appl. No.: |
17/466230 |
Filed: |
September 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16632202 |
Jan 17, 2020 |
11136325 |
|
|
PCT/GB2018/051983 |
Jul 12, 2018 |
|
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17466230 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/04 20130101;
A61P 25/04 20180101; A61P 11/00 20180101; A61P 1/16 20180101; C07D
471/04 20130101; A61P 19/02 20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61P 1/16 20060101 A61P001/16; A61P 19/02 20060101
A61P019/02; A61P 25/04 20060101 A61P025/04; A61P 11/00 20060101
A61P011/00; C07D 471/04 20060101 C07D471/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2017 |
GB |
1711234.3 |
Claims
1-11. (canceled)
12. A method for prophylaxis and/or treatment of chronic
obstructive pulmonary disease (COPD), inflammatory bowel disease
(IBD), idiopathic pulmonary fibrosis (IPF) and/or diabetic kidney
disease (DKD), comprising administering an effective amount of a
compound according to Formula I: ##STR00038## wherein R.sup.1 is H,
CH.sub.3, F or Cl; X is N, CH or C--CN; and R.sup.2 is CH.sub.3 or
halogen; or a pharmaceutically acceptable salt thereof, to an
individual in need thereof.
13. The method of claim 12, wherein R.sup.1 is F.
14. The method of claim 12, wherein R.sup.2 is CH.sub.3.
15. The method of claim 12, wherein X is CH.
16. The method of claim 12, wherein X is C--CN.
17. The method of claim 12, wherein X is N.
18. The method of claim 12, wherein the compound is:
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide,
2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide,
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]-pyridine-4-carboxamide,
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]pyrid-
ine-4-carboxamide,
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-5-fl-
uoro-pyridine-4-carboxamide,
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide, or
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide.
19. A method for prophylaxis and/or treatment of chronic
obstructive pulmonary disease (COPD), inflammatory bowel disease
(IBD), idiopathic pulmonary fibrosis (IPF) and/or diabetic kidney
disease (DKD), comprising administering a pharmaceutical
composition comprising a compound according to Formula I:
##STR00039## wherein R.sup.1 is H, CH.sub.3, F or Cl; X is N, CH or
C--CN; and R.sup.2 is CH.sub.3 or halogen; or a pharmaceutically
acceptable salt thereof and a pharmaceutically acceptable carrier,
to an individual in need thereof.
20. The method of claim 19, wherein R' is F.
21. The method of claim 19, wherein R.sup.2 is CH.sub.3.
22. The method of claim 19, wherein X is CH.
23. The method of claim 19, wherein X is C--CN.
24. The method of claim 19, wherein X is N.
25. The method of claim 19, wherein the compound is:
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide,
2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide,
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]-pyridine-4-carboxamide,
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]pyrid-
ine-4-carboxamide,
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-5-fl-
uoro-pyridine-4-carboxamide,
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide, or
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to pyrrolopyrimidine compounds
and their use in the prophylaxis and/or treatment of pain,
inflammatory conditions, cardiovascular diseases, neurodegenerative
diseases, neurological diseases, complications of diabetes, cancer
and/or fibrotic diseases. In a particular aspect, the present
compounds are ASK inhibitors, particularly ASK1 inhibitors. The
present invention also provides methods for the production of a
compound of the invention, pharmaceutical compositions comprising a
compound of the invention, the use of the compounds in the
prophylaxis and/or treatment of pain, inflammatory conditions,
cardiovascular diseases, neurodegenerative diseases, neurological
diseases, complications of diabetes, cancer and/or fibrotic
diseases.
BACKGROUND OF THE INVENTION
[0002] Apoptosis signal-regulating kinase (ASK1) is a ubiquitously
expressed Ser/Thr kinase on the mitogen-activated protein kinase
(MAPK) signalling pathway inducing response to stress stimuli
including proinflammatory molecules such as tumor necrosis
factor-.alpha. (TNF-.alpha.) and lipopolysaccharide (LPS),
endoplasmic stress, oxidative stress, genotoxic stress, free
radicals, Fas ligand and calcium overload (Takeda K et al (2008)
Annu Rev Pharacol Toxicol 248 pp 199-225; Nagai H et al (2007) J
Biochem Mol Biol 40 pp 1-6).
[0003] ASK1 is one of a number of MAP kinase kinase kinases
(MAP3Ks) which signal through MAP kinase kinases (MKKs). In the
case of ASK1 signalling, MKK3 and MKK6 activate the p38 pathway and
MKK4 and MKK7 activate the JNK pathway (Davis R J (2000) Cell 103
pp 239-252; Ichijo H et al (1997) Science 275 pp 90-94). Therefore
inhibitors of ASK1 have the potential to suppress signalling
pathways through both p38 and JNK.
[0004] The use of soluble TNF receptor: Fc fusion protein Enbrel
(etanercept) has been shown to be efficacious in the clinic for
inflammatory pain and also in pre-clinical models for neuropathic
pain (Hao S et al (2007) Gene Therapy 14 pp 1010-1016) implying
that TNF-.alpha. is a key mediator in pain response. IL-6 is a key
downstream mediator of TNF-.alpha. signalling and there is clinical
evidence supporting anti-IL-6 therapy as a valid therapeutic
approach for rheumatoid arthritis (Roche has published positive
Phase III results for Actemra/Tocilizumab in May 2008).
[0005] A number of cells that do not have functional ASK1 (isolated
from ASK1 knockout mice, or following gene silencing) are resistant
to TNF-.alpha.c induced apoptosis (Tobiume K, et al (2001) EMBO Rep
2 pp 222-228). ASK1 is therefore pivotal in the TNF-.alpha.c
pathway and supports the hypothesis that disrupting the
TNF-.alpha.c signalling pathway via ASK1 inhibition would lead to
beneficial downstream effects such as relief from pain. There is
strong evidence to link activation of p38 and/or JNK with the
production of pro-inflammatory mediators and subsequent pain
response (Ji R-R and Suter M R (2007) Molecular Pain 3 pp 33-41;
Cheng H T et al (2008) Neuroscience 155 pp 948-958; Ji R-R and Gao
Y-J (2008) Neurosci Lett 437 pp 180-183). As ASK1 activation can
lead to the activation of both p38 and JNK, inhibition of ASK1 has
the potential to be more powerful than p38 inhibitors alone and, as
it is higher up in the signalling cascade, may limit the likelihood
of unwanted liabilities.
[0006] Fibrosis is a wound-healing process in which there is
excessive deposition of extracellular matrix (ECM). ECM is composed
of collagens, noncollagen glycoproteins, matrix bound growth
factors, glycosaminoglycans, proteoglycans and matricellular
proteins, which provide the scaffolding of both the normal and the
fibrotic tissue.
[0007] Non-alcoholic fatty liver disease (NAFLD) is the most common
cause of chronic liver disease in developed countries (Younossi Z
M, et al., Clin Gastroenterol Hepatol 2011; 9: 524-30 and Cohen J
C, et al., Science, 2011; 332: 1519-23). It may be broadly
classified into two categories: non-alcoholic fatty liver (or
simple steatosis) and non-alcoholic steatohepatitis (NASH).
Although previously it was thought that steatosis was largely
nonprogressive while NASH was the progressive form of NAFLD, recent
evidence from serial biopsy studies demonstrates that patients with
steatosis or NASH have an increased risk of subsequent disease
progression to advanced fibrosis and cirrhosis (Singh S, et al,
Clin Gastroenterol Hepatol 2015; 13: 643-54 and McPherson S, et
al., J Hepatol 2015; 62: 1148-55).
[0008] Oxidative stress is known to play a major role in the
activation of hepatic stellate cells (HSCs) in NASH (Bian Z, &
Ma X. Front Physiol 2012; 3: 248 and Koek G H, et al., Clin Chim
Acta 2011; 412: 1297-305), and anti-oxidants not only exert a
preventive effect on hepatocyte injury but may directly contribute
to decreasing fibrogenesis, (Serviddio G et al., Free Radic Biol
Med 2013; 65: 952-68) an effect supported by gene-association
studies where variants affecting cellular anti-oxidant defences
efficacy influence risk of NAFLD fibrosis (Al-Serri A, et al., J
Hepatol 2012; 56: 448-54).
[0009] Apoptosis-signal-regulating kinase 1 (ASK1) is a kinase that
is activated by various stimuli including hyperglycaemia,
TGF-.beta. and ROS (Karnik S, Charlton M R, Li L, et al., The Liver
Meeting 2015, San Francisco, Calif., November 13-17, American
Association for the Study of Liver Diseases, 2015). ASK1 induces
apoptosis, fibrosis and metabolic dysfunction by activating the p38
and JNK1 pathways. The ASK1 pathway has been shown to be activated
in human NASH liver biopsies (Karnik S, The Liver Meeting 2014,
Boston, Mass., November 7-11, American Association for the Study of
Liver Diseases, 2014). Furthermore, in a six month NASH human
clinical study, the ASK1 inhibitor selonsertib has been shown to
lead to a reduction in liver fibrosis stage, progression to
cirrhosis, liver stiffness and liver fat content (Loomba et al. The
liver meeting 2016, Boston, Mass., November 11-15, American
Association for the Study of Liver Diseases, 2016).
[0010] WO 2008/016131 discloses fused heterocyclic ASK1 inhibitors
for use in the treatment of diabetes and inflammatory disease. WO
2004/048565 describes a novel peptide which has ASK1 activity which
may be useful in the treatment of cancer and degenerative diseases.
WO 2009/123986 and WO 2009/027283 both describe ASK1 inhibitors. WO
2008/075172 discloses nicotinamide derivatives as inhibitors of
h-PGDS and their use for treating prostaglandin D2 mediated
diseases, WO 2001/39777 discloses compounds specific to adenosine
A.sub.1 A.sub.2a, and A.sub.3 receptors. EP 2058309 discloses fused
heterocyclic compounds.
[0011] The present invention describes a series of
pyrrolopyrimidine derivatives which are inhibitors of the ASK1
kinase and which may be useful in the prophylaxis and/or treatment
of pain, inflammatory conditions, cardiovascular diseases, neurode
generative diseases, neurological diseases, complications of
diabetes, cancer and/or fibrotic diseases.
SUMMARY OF THE INVENTION
[0012] The present invention is based on the identification of
novel pyrrolopyrimidine and pyrrolopyridine compounds that may be
useful for the prophylaxis and/or treatment of pain and/or fibrotic
diseases. In a particular aspect, the present compounds are ASK
inhibitors, particularly ASK1 inhibitors. The present invention
also provides methods for the production of these compounds,
pharmaceutical compositions comprising these compounds and the use
of the compounds in the prophylaxis and/or treatment of pain,
inflammatory conditions, cardiovascular diseases, neurodegenerative
diseases, neurological diseases, complications of diabetes, cancer
and/or fibrotic diseases.
[0013] Accordingly, in a first aspect of the invention, the
compounds of the invention are provided having a Formula I:
##STR00002##
wherein
[0014] R.sup.1 is H, CH.sub.3, F or Cl;
[0015] X is N, CH or C--CN; and
[0016] R.sup.2 is CH.sub.3 or halogen.
[0017] In a particular aspect, the compounds of the invention may
exhibit selectivity towards the ASK kinase family, in particular
towards ASK1. In a further particular aspect, the compounds of the
invention may show low activity on other kinase enzymes, in
particular JAK2. Such selectivity may result in improved drug
safety and/or reduce off-target associated risks.
[0018] In a further aspect, the present invention provides
pharmaceutical compositions comprising a compound of the invention,
and a pharmaceutical carrier, excipient or diluent. In a particular
aspect, the pharmaceutical composition may additionally comprise
further therapeutically active ingredients suitable for use in
combination with the compounds of the invention. In a more
particular aspect, the further therapeutically active ingredient is
an agent for the prophylaxis and/or treatment of pain and/or
fibrotic diseases.
[0019] Moreover, the compounds of the invention, useful in the
pharmaceutical compositions and treatment methods disclosed herein,
are pharmaceutically acceptable as prepared and used.
[0020] In a further aspect of the invention, this invention
provides a method of treating a mammal, in particular humans,
afflicted with a condition selected from among those listed herein,
and particularly pain and/or fibrotic diseases, which method
comprises administering an effective amount of the pharmaceutical
composition or compounds of the invention as described herein.
[0021] The present invention also provides pharmaceutical
compositions comprising a compound of the invention, and a suitable
pharmaceutical carrier, excipient or diluent for use in medicine.
In a particular aspect, the pharmaceutical composition is for use
in the prophylaxis and/or treatment of pain and/or fibrotic
diseases.
[0022] In a particular aspect, the compounds of the invention are
provided for use in the prophylaxis and/or treatment of pain.
[0023] In additional aspects, this invention provides methods for
synthesizing the compounds of the invention, with representative
synthetic protocols and pathways disclosed later on herein.
[0024] Other objects and advantages will become apparent to those
skilled in the art from a consideration of the ensuing detailed
description.
[0025] It will be appreciated that compounds of the invention may
be metabolized to yield biologically active metabolites.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] The following terms are intended to have the meanings
presented therewith below and are useful in understanding the
description and intended scope of the present invention.
[0027] When describing the invention, which may include compounds,
pharmaceutical compositions containing such compounds and methods
of using such compounds and compositions, the following terms, if
present, have the following meanings unless otherwise indicated. It
should also be understood that when described herein any of the
moieties defined forth below may be substituted with a variety of
substituents, and that the respective definitions are intended to
include such substituted moieties within their scope as set out
below. Unless otherwise stated, the term "substituted" is to be
defined as set out below. It should be further understood that the
terms "groups" and "radicals" can be considered interchangeable
when used herein.
[0028] The articles `a` and `an` may be used herein to refer to one
or to more than one (i.e. at least one) of the grammatical objects
of the article. By way of example `an analogue` means one analogue
or more than one analogue.
[0029] `Amino` refers to the radical --NH.sub.2.
[0030] `Halo` or `halogen` refers to fluoro (F), chloro (Cl), bromo
(Br) and iodo (I). Particular halo groups are either fluoro or
chloro.
[0031] `Pharmaceutically acceptable` means approved or approvable
by a regulatory agency of the Federal or a state government or the
corresponding agency in countries other than the United States, or
that is listed in the U.S. Pharmacopoeia or other generally
recognized pharmacopoeia for use in animals, and more particularly,
in humans.
[0032] `Pharmaceutically acceptable salt` refers to a salt of a
compound of the invention that is pharmaceutically acceptable and
that possesses the desired pharmacological activity of the parent
compound. In particular, such salts are non-toxic may be inorganic
or organic acid addition salts and base addition salts.
Specifically, such salts include: (1) acid addition salts, formed
with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like; or
formed with organic acids such as acetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic
acid, lactic acid, malonic acid, succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluene
sulfonic acid, camphorsulfonic acid,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary
butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic
acid, and the like; or (2) salts formed when an acidic proton
present in the parent compound either is replaced by a metal ion,
e.g. an alkali metal ion, an alkaline earth ion, or an aluminum
ion; or coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, N-methylglucamine and the like.
Salts further include, by way of example only, sodium, potassium,
calcium, magnesium, ammonium, tetraalkylammonium, and the like; and
when the compound contains a basic functionality, salts of
non-toxic organic or inorganic acids, such as hydrochloride,
hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the
like. The term `pharmaceutically acceptable cation` refers to an
acceptable cationic counter-ion of an acidic functional group. Such
cations are exemplified by sodium, potassium, calcium, magnesium,
ammonium, tetraalkylammonium cations, and the like.
[0033] `Pharmaceutically acceptable vehicle` refers to a diluent,
adjuvant, excipient or carrier with which a compound of the
invention is administered.
[0034] `Prodrugs` refers to compounds, including derivatives of the
compounds of the invention, which have cleavable groups and become
by solvolysis or under physiological conditions the compounds of
the invention which are pharmaceutically active in vivo. Such
examples include, but are not limited to, choline ester derivatives
and the like, N-alkylmorpholine esters and the like.
[0035] `Solvate` refers to forms of the compound that are
associated with a solvent, usually by a solvolysis reaction. This
physical association includes hydrogen bonding. Conventional
solvents include water, EtOH, acetic acid and the like. The
compounds of the invention may be prepared e.g. in crystalline form
and may be solvated or hydrated. Suitable solvates include
pharmaceutically acceptable solvates, such as hydrates, and further
include both stoichiometric solvates and non-stoichiometric
solvates. In certain instances the solvate will be capable of
isolation, for example when one or more solvent molecules are
incorporated in the crystal lattice of the crystalline solid.
`Solvate` encompasses both solution-phase and isolable solvates.
Representative solvates include hydrates, ethanolates and
methanolates.
[0036] `Subject` includes humans. The terms `human`, `patient` and
`subject` are used interchangeably herein.
[0037] `Effective amount` means the amount of a compound of the
invention that, when administered to a subject for treating a
disease, is sufficient to effect such treatment for the disease.
The "effective amount" can vary depending on the compound, the
disease and its severity, and the age, weight, etc., of the subject
to be treated.
[0038] `Preventing` or `prevention` refers to a reduction in risk
of acquiring or developing a disease or disorder (i.e. causing at
least one of the clinical symptoms of the disease not to develop in
a subject that may be exposed to a disease-causing agent, or
predisposed to the disease in advance of disease onset).
[0039] The term `prophylaxis` is related to `prevention`, and
refers to a measure or procedure the purpose of which is to
prevent, rather than to treat or cure a disease. Non-limiting
examples of prophylactic measures may include the administration of
vaccines; the administration of low molecular weight heparin to
hospital patients at risk for thrombosis due, for example, to
immobilization; and the administration of an anti-malarial agent
such as chloroquine, in advance of a visit to a geographical region
where malaria is endemic or the risk of contracting malaria is
high.
[0040] `Treating` or `treatment` of any disease or disorder refers,
in one embodiment, to ameliorating the disease or disorder (i.e.
arresting the disease or reducing the manifestation, extent or
severity of at least one of the clinical symptoms thereof). In
another embodiment `treating` or `treatment` refers to ameliorating
at least one physical parameter, which may not be discernible by
the subject. In yet another embodiment, `treating` or `treatment`
refers to modulating the disease or disorder, either physically,
(e.g. stabilization of a discernible symptom), physiologically,
(e.g. stabilization of a physical parameter), or both. In a further
embodiment, "treating" or "treatment" relates to slowing the
progression of the disease.
[0041] As used herein the term `pain` refers to inflammatory pain,
in particular chronic articular pain (e.g. rheumatoid arthritis,
osteoarthritis, rheumatoid spondylitis, gouty arthritis (gout) and
juvenile arthritis) including the property of disease modification
and joint structure preservation; musculoskeletal pain; lower back
and neck pain; sprains and strains; neuropathic pain;
sympathetically maintained pain; myositis; pain associated with
cancer and fibromyalgia; pain associated with migraine; pain
associated with influenza or other viral infections, such as the
common cold; rheumatic fever; pain associated with functional bowel
disorders such as non-ulcer dyspepsia, non-cardiac chest pain and
irritable bowel syndrome; pain associated with myocardial ischemia;
post operative pain; headache; toothache; and dysmenorrhea. More
particularly, the term refers to chronic articular pain. More
particularly the term refers to rheumatoid arthritis,
osteoarthritis, and gouty arthritis (gout).
[0042] As used herein the term "cardiovascular disease(s)" refers
to diseases affecting the heart or blood vessels or both. In
particular, cardiovascular disease includes arrhythmia (atrial or
ventricular or both); atherosclerosis and its sequelae; angina;
cardiac rhythm disturbances; myocardial ischemia; myocardial
infarction; cardiac or vascular aneurysm; vasculitis, stroke;
peripheral obstructive arteriopathy of a limb, an organ, or a
tissue; reperfusion injury following ischemia of the brain, heart,
kidney or other organ or tissue; shock states associated with a
marked drop in arterial pressure (e.g. endotoxic, surgical,
traumatic shock or septic shock); pulmonary arterial hypertension
(PAH), hypertension, valvular heart disease, heart failure,
abnormal blood pressure; shock; vasoconstriction (including that
associated with migraines); vascular abnormality, varicose therapy,
insufficiency limited to a single organ or tissue, functional or
organic venous insufficiency, cardiac hypertrophy, ventricular
fibrosis, and myocardial remodelling. More particularly, the term
refers to atherosclerosis, pulmonary arterial hypertension, heart
failure, acute coronary syndrome, cardiac hypertrophy, ventricular
fibrosis and myocardial remodeling.
[0043] As used herein the terms `neuropathic pain` or `syndromes
involving neuropathic pain` include both central neuropathic pain
and peripheral neuropathic pain unless the context dictates
otherwise, the terms include: diabetic neuropathy; sciatica;
non-specific lower back pain; multiple sclerosis pain;
fibromyalgia; HIV-related neuropathy; post-herpetic neuralgia;
trigeminal neuralgia; and pain resulting from physical trauma,
amputation, cancer, toxins, chemotherapy induced neuropathy or
chronic inflammatory conditions. Symptoms of neuropathic pain
include spontaneous shooting and lancinating pain, or ongoing,
burning pain. In addition, there is included pain associated with
normally non-painful sensations such as "pins and needles"
(paraesthesias and dysesthesias), increased sensitivity to touch
(hyperesthesia), painful sensation following innocuous stimulation
(dynamic, static or thermal allodynia), increased sensitivity to
noxious stimuli (thermal, cold or mechanical hyperalgesia),
continuing pain sensation after removal of the stimulation
(hyperpathia) or an absence of or deficit in selective sensory
pathways (hypoalgesia).
[0044] As used herein the term `inflammatory condition(s)` refers
to the group of conditions including, rheumatoid arthritis,
osteoarthritis, juvenile idiopathic arthritis, psoriatic arthritis,
ankylosing spondylitis, skin conditions (e.g. sunburn, burns,
eczema, dermatitis, psoriasis), ophthalmic diseases (e.g. glaucoma,
retinitis, retinopathies, uveitis and of acute injury to the eye
tissue (e.g. conjunctivitis)), lung disorders (e.g. allergic airway
disease (e.g. asthma, rhinitis), chronic obstructive pulmonary
disease (COPD), bronchitis, emphysema, respiratory distress
synfrom, pigeon fancier's disease and farmer's lung),
gastrointestinal tract disorders (e.g inflammatory bowel diseases
such as Crohn's disease or ulcerative colitis, aphthous ulcer,
atopic gastritis, gastritis varialoforme, coeliac disease, regional
ileitis, irritable bowel syndrome, gastrointestinal reflux disease,
diarrhoea, and/or constipation), endotoxin-driven disease states
(e.g. complications after bypass surgery or chronic endotoxin
states contributing to e.g. chronic cardiac failure), organ
transplantation and other conditions with an inflammatory component
such as vascular disease, steatohepatitis, migraine, periarteritis
nodosa, thyroiditis, aplastic anaemia, Hodgkin's disease,
sclerodoma, myaesthenia gravis, multiple sclerosis, sorcoidosis,
nephrotic syndrome, Bechet's syndrome, polymyositis, gingivitis,
myocardial ischemia, pyrexia, systemic lupus erythematosus,
polymyositis, tendinitis, bursitis, and Sjogren's syndrome.
Particularly the term refers to rheumatoid arthritis,
osteoarthritis, allergic airway disease (e.g. asthma), chronic
obstructive pulmonary disease (COPD) and inflammatory bowel
diseases. More particularly the term refers to rheumatoid
arthritis, osteoarthritis, chronic obstructive pulmonary disease
(COPD) and inflammatory bowel diseases.
[0045] As used herein the term `asthma` refers to any disorder of
the lungs characterized by variations in pulmonary gas flow
associated with airway constriction of whatever cause (intrinsic,
extrinsic, or both; allergic or non-allergic). The term asthma may
be used with one or more adjectives to indicate the cause.
[0046] As used herein the term `neurodegenerative diseases` refers
to conditions resulting from or including neurodegeneration
including dementia, particularly degenerative dementia (including
senile dementia, Alzheimer's disease, Pick's disease, Huntingdon's
chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS and
motor neuron disease); vascular dementia (including multi-infarct
dementia); as well as dementia associated with intracranial space
occupying lesions; trauma; infections and related conditions
(including HIV infection); peripheral neuropathies, multiple
sclerosis, retinopathies, glaucoma, macular degeneration, cerebral
ischemia and traumatic brain injury and mild cognitive impairment
associated with ageing, particularly Age Associated Memory
Impairment.
[0047] As used herein the term `complications of diabetes` refers
to conditions which are associated with Type I or Type II diabetes,
these conditions include those related to vascular or microvascular
changes e.g. diabetic retinopathy, diabetic microangiopathy,
diabetic nephropathy (also referred to as diabetic kidney disease
(DKD)), macular degeneration, glaucoma, nephrotic syndrome,
diabetic cardiomyopathy, aplastic anaemia, uveitis, Kawasaki
disease and sarcoidosis; as well as disorders of fat metabolism
which may be associated with diabetes or obesity for example
hepatic steatosis. More particularly the term refers to diabetic
retinopathy, diabetic microangiopathy, diabetic nephropathy and
hepatic steatosis.
[0048] As used herein, the term `cancer` refers to a malignant or
benign growth of cells in skin or in body organs, for example but
without limitation, breast, prostate, lung, kidney, pancreas,
stomach or bowel. A cancer tends to infiltrate into adjacent tissue
and spread (metastasise) to distant organs, for example to bone,
liver, lung or the brain. As used herein the term cancer includes
both metastatic tumour cell types (such as but not limited to,
melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and
mastocytoma) and types of tissue carcinoma (such as but not limited
to, colorectal cancer, prostate cancer, small cell lung cancer and
non-small cell lung cancer, breast cancer, pancreatic cancer,
bladder cancer, renal cancer, gastric cancer, glioblastoma, primary
liver cancer, ovarian cancer, prostate cancer and uterine
leiomyosarcoma). In particular, the term `cancer` refers to acute
lymphoblastic leukemia, acute myeloidleukemia, adrenocortical
carcinoma, anal cancer, appendix cancer, astrocytomas, atypical
teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer,
bladder cancer, bone cancer (osteosarcoma and malignant fibrous
histiocytoma), brain stem glioma, brain tumors, brain and spinal
cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma,
cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, colon cancer, colorectal cancer, craniopharyngioma,
cutaneousT-Cell lymphoma, embryonal tumors, endometrial cancer,
ependymoblastoma, ependymoma, esophageal cancer, ewing sarcoma
family of tumors, eye cancer, retinoblastoma, gallbladder cancer,
gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), gastrointestinal stromal
cell tumor, germ cell tumor, glioma, hairy cell leukemia, head and
neck cancer, hepatocellular (liver) cancer, hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, islet cell tumors
(endocrine pancreas), Kaposi sarcoma, kidney cancer, Langerhans
cell histiocytosis, laryngeal cancer, leukemia, Acute lymphoblastic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, hairy cell leukemia, liver cancer,
non-small cell lung cancer, small cell lung cancer, Burkitt
lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin
lymphoma, lymphoma, Waldenstrom macroglobulinemia, medulloblastoma,
medulloepithelioma, melanoma, mesothelioma, mouth cancer, chronic
myelogenous leukemia, myeloid leukemia, multiple myeloma,
asopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma,
non-small cell lung cancer, oral cancer, oropharyngeal cancer,
osteosarcoma, malignant fibrous histiocytoma of bone, ovarian
cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian
low malignant potential tumor, pancreatic cancer, papillomatosis,
parathyroid cancer, penile cancer, pharyngeal cancer, pineal
parenchymal tumors of intermediate differentiation, pineoblastoma
and supratentorial primitive neuroectodermal tumors, pituitary
tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary
blastoma, primary central nervous system lymphoma, prostate cancer,
rectal cancer, renal cell (kidney) cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing sarcoma
family of tumors, sarcoma, kaposi, Sezary syndrome, skin cancer,
small cell Lung cancer, small intestine cancer, soft tissue
sarcoma, squamous cell carcinoma, stomach (gastric) cancer,
supratentorial primitive neuroectodermal tumors, testicular cancer,
throat cancer, thymoma and thymic carcinoma, thyroid cancer,
urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer,
vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. In
another particular embodiment, the term cancer refers to pancreatic
cancer, liver cancer, hepatocellular carcinoma (HCC), breast
cancer, or colon cancer. In particular, it refers to hepatocellular
carcinoma, melanoma, gastric cancer, liposarcoma and cancers caused
by oxidative stresses for example cervical spondylotic
myelopathy.
[0049] As used herein the term `fibrotic diseases` refers to
diseases characterized by excessive scarring due to excessive
production, deposition, and contraction of extracellular matrix,
and those that are associated with the abnormal accumulation of
cells and/or fibronectin and/or collagen and/or increased
fibroblast recruitment and include but are not limited to fibrosis
of individual organs or tissues such as the heart, kidney, liver,
joints, lung, pleural tissue, peritoneal tissue, skin, cornea,
retina, musculoskeletal and digestive tract. In particular, the
term fibrotic diseases refers to idiopathic pulmonary fibrosis
(IPF); cystic fibrosis, other diffuse parenchymal lung diseases of
different etiologies including iatrogenic drug induced fibrosis,
occupational and/or environmental induced fibrosis, granulomatous
diseases (sarcoidosis, hypersensitivity pneumonia), collagen
vascular disease, alveolar proteinosis, Langerhans cell
granulomatosis, lymphangioleiomyomatosis, inherited diseases
(Hermansky Pudlak syndrome, tuberous sclerosis, neurofibromatosis,
metabolic storage disorders, familial interstitial lung disease);
radiation induced fibrosis; chronic obstructive pulmonary disease
(COPD); scleroderma; bleomycin induced pulmonary fibrosis; chronic
asthma; silicosis; asbestos induced pulmonary fibrosis; acute
respiratory distress syndrome (ARDS); kidney fibrosis;
tubulointerstitial fibrosis; glomerular nephritis; focal segmental
glomerular sclerosis; IgA nephropathy; hypertension; Alport
syndrome; gut fibrosis; liver fibrosis; cirrhosis; alcohol induced
liver fibrosis; toxic/drug induced liver fibrosis; hemochromatosis;
nonalcoholic steatohepatitis (NASH); biliary duct injury; primary
biliary cirrhosis; infection induced liver fibrosis; viral induced
liver fibrosis; and autoimmune hepatitis; corneal scarring;
hypertrophic scarring; Dupuytren's disease, keloids, cutaneous
fibrosis; cutaneous scleroderma; systemic sclerosis, spinal cord
injury/fibrosis; myelofibrosis; vascular restenosis;
atherosclerosis; arteriosclerosis; Wegener's granulomatosis;
Peyronie's disease, or chronic lymphocytic. In a particular
embodiment the fibrotic disease is of an individual organ or tissue
such as liver fibrosis, lung fibrosis or kidney fibrosis. In a
particular embodiment, the fibrotic disease is selected from
idiopathic pulmonary fibrosis (IPF), diabetic kidney disease (DKD)
and nonalcoholic steatohepatitis (NASH).
[0050] `Compound(s) of the invention`, and equivalent expressions,
are meant to embrace compounds of the Formula(e) as herein
described, which expression includes the pharmaceutically
acceptable salts, and the solvates, e.g. hydrates, and the solvates
of the pharmaceutically acceptable salts where the context so
permits. Similarly, reference to intermediates, whether or not they
themselves are claimed, is meant to embrace their salts, and
solvates, where the context so permits.
[0051] When ranges are referred to herein, for example but without
limitation, C.sub.1-8 alkyl, the citation of a range should be
considered a representation of each member of said range.
[0052] Other derivatives of the compounds of this invention have
activity in both their acid and acid derivative forms, but in the
acid sensitive form often offers advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism.
[0053] As used herein, the term `isotopic variant` refers to a
compound that contains unnatural proportions of isotopes at one or
more of the atoms that constitute such compound. For example, an
`isotopic variant` of a compound can contain one or more
non-radioactive isotopes, such as for example, deuterium (.sup.2H
or D), carbon-13 (.sup.13C), nitrogen (.sup.15N), or the like. It
will be understood that, in a compound where such isotopic
substitution is made, the following atoms, where present, may vary,
so that for example, any hydrogen may be .sup.2H/D, any carbon may
be .sup.13C, or any nitrogen may be .sup.15N, and that the presence
and placement of such atoms may be determined within the skill of
the art. Likewise, the invention may include the preparation of
isotopic variants with radioisotopes, in the instance for example,
where the resulting compounds may be used for 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. Further, compounds may be prepared that are
substituted with positron emitting isotopes, such as .sup.11C,
.sup.18F, .sup.15O and .sup.13N, and would be useful in Positron
Emission Topography (PET) studies for examining substrate receptor
occupancy.
[0054] Unless indicated otherwise, the description or naming of a
particular compound in the specification and claims is intended to
include both individual enantiomers and mixtures, racemic or
otherwise, thereof. The methods for the determination of
stereochemistry and the separation of stereoisomers are well-known
in the art.
[0055] It will be appreciated that compounds of the invention may
be metabolized to yield biologically active metabolites.
FIGURES
[0056] FIG. 1 shows the effect of Compound 1 at two different doses
given p.o. on the CFA-induced hyperalgesia in the rat model of
Example 3.1.
[0057] FIG. 2 shows the effect of Compound 1 at 10 mg/kg given p.o.
on MIA-induced hyperalgesia in the rat model of Example 3.2.
[0058] FIG. 3 shows the effect of Compound 1 at 5 mg/kg/QD, 15
mg/kg/QD and 15 mg/kg/BID on the expression levels of a panel of
fibrosis related genes in mouse liver samples from the CFAHFD model
of fibrosis (Example 3.10). FIG. 3A: PAI1, FIG. 3B: TIMP1, FIG. 3C:
COL1A1, FIG. 3D: CTGF, FIG. 3E: ACTA2 and FIG. 3F: TGF.beta. Data
are from day 73 and are presented as mean.+-.SEM, *p<0.05,
**p<0.01, ***p<0.001, ****P<0.0001 vs CDAHFD diet+vehicle,
Mann-Whitney test.
[0059] FIG. 4 shows the effect of Compound 1 at 5 mg/kg/QD, 15
mg/kg/QD and 15 mg/kg/BID on the expression levels of a panel of
inflammation related genes in mouse liver samples from the CFAHFD
model of fibrosis (Example 3.10). FIG. 4A: TNF.alpha., FIG. 4B:
IL10, and FIG. 4C: CCL2. Data are from day 73 and are presented as
mean.+-.SEM, *p<0.05, **p<0.01, ***p<0.001,
****P<0.0001 vs CDAHFD diet+vehicle, Mann-Whitney test.
[0060] FIG. 5 shows the effect of Compound 1 at 5 mg/kg/QD, 15
mg/kg/QD and 15 mg/kg/BID on hydroxyproline levels mouse liver
tissue from the CFAHFD model of fibrosis (Example 3.10). Data are
from day 73 and are presented as mean.+-.SEM, *p<0.05,
**p<0.01, ***p<0.001, ****P<0.0001 vs CDAHFD diet+vehicle,
Mann-Whitney test.
[0061] FIG. 6 shows the effect of Compound 1 at 5 mg/kg/QD, 15
mg/kg/QD and 15 mg/kg/BID using Sirius red fibrosis quantification
from the CFAHFD model of fibrosis (Example 3.10). Data are from day
73 and are presented as mean.+-.SEM, *p<0.05, **p<0.01,
***p<0.001, ****P<0.0001 vs CDAHFD diet+vehicle, Mann-Whitney
test.
[0062] FIG. 7 shows the effect of Compound 1 at 5 mg/kg/QD, 15
mg/kg/QD and 15 mg/kg/BID on F4/80 quantification from the CFAHFD
model of fibrosis (Example 3.10). Data are from day 73 and are
presented as mean.+-.SEM, *p<0.05, **p<0.01, ***p<0.001,
****P<0.0001 vs CDAHFD diet+vehicle, Mann-Whitney test.
THE INVENTION
[0063] The present invention is based on the identification of
novel pyrrolopyrimidine and pyrrolopyridine compounds that may be
useful for the prophylaxis and/or treatment of pain, inflammatory
conditions, cardiovascular diseases, neurodegenerative diseases,
neurological diseases, complications of diabetes, cancer and/or
fibrotic diseases. In a particular aspect, the present compounds
are ASK inhibitors, particularly ASK1 inhibitors.
[0064] The present invention also provides methods for the
production of these compounds, pharmaceutical compositions
comprising these compounds and methods for treating pain,
inflammatory conditions, cardiovascular diseases, neurodegenerative
diseases, neurological diseases, complications of diabetes, cancer
and/or fibrotic diseases by administering the compounds of the
invention.
[0065] Accordingly, in a first aspect of the invention, the
compounds of the invention are provided having a Formula I:
##STR00003##
wherein
[0066] R.sup.1 is H, CH.sub.3, F or Cl;
[0067] X is N, CH or C--CN; and
[0068] R.sup.2 is CH.sub.3 or halogen.
[0069] In one embodiment R.sup.1 is F.
[0070] In one embodiment R.sup.1 is F and R.sup.2 is CH.sub.3.
[0071] In one embodiment, X is CH. In an alternative embodiment X
is C--CN. In an alternative embodiment X is N.
[0072] In one embodiment, X is N and R.sup.1 is F. In a particular
embodiment, X is N, R.sup.1 is F and R.sup.2 is CH.sub.3. In a
further embodiment, R.sup.2 is CH.sub.3 or Cl. In a yet further
embodiment, R.sup.2 is CH.sub.3.
[0073] In one embodiment, a compound of the invention is selected
from: [0074]
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-
-piperidyl]pyridine-4-carboxamide (Compound 1), [0075]
2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide (Compound 2), [0076]
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]-pyridine-4-carboxamide (Compound 3), [0077]
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]pyrid-
ine-4-carboxamide (Compound 4), [0078]
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-5-fl-
uoro-pyridine-4-carboxamide (Compound 5), [0079]
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide (Compound 6), and [0080]
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide (Compound 7).
[0081] In one embodiment a compound of the invention is not an
isotopic variant.
[0082] In one aspect a compound of the invention according to any
one of the embodiments herein described is present as the free
base.
[0083] In one aspect a compound of the invention according to any
one of the embodiments herein described is a pharmaceutically
acceptable salt.
[0084] In one aspect a compound of the invention according to any
one of the embodiments herein described is a solvate of the
compound.
[0085] In one aspect a compound of the invention according to any
one of the embodiments herein described is a solvate of a
pharmaceutically acceptable salt of a compound.
[0086] In a specific embodiment, a compound of the invention
according to any one of the embodiments herein displays an improved
activity in an in vitro cellular assay for ASK1 activity as
compared to structurally similar compounds. In a particular
embodiment a compound of the invention displays an improved
activity in the cellular assay for ASK1 activity in peripheral
blood mononuclear cells (PBMCs) as described herein as Example
2.3.
[0087] In an alternative embodiment, a compound of the invention
according to any one of the embodiments herein may show a decreased
induction of Cyp activity. In particular, a compound of the
invention may show a decreased induction of Cyp3A4 activity as
compared to structurally similar compounds.
[0088] In an alternative embodiment, a compound of the invention
according to any one of the embodiments herein may show a decreased
inhibition of Cyp activity. In particular, a compound of the
invention may show a decreased inhibition of Cyp3A4 activity as
compared to structurally similar compounds. More particularly, a
compound of the invention may show an IC.sub.50 for Cyp inhibition
of >10 .mu.M.
[0089] While specified groups for each embodiment have generally
been listed above separately, a compound of the invention includes
one in which several or each embodiment in the above Formula, as
well as other formulae presented herein, is selected from one or
more of particular members or groups designated respectively, for
each variable. Therefore, this invention is intended to include all
combinations of such embodiments within its scope.
[0090] While specified groups for each embodiment have generally
been listed above separately, a compound of the invention may be
one for which one or more variables (for example, R groups) is
selected from one or more embodiments according to any of the
Formula(e) listed above. Therefore, the present invention is
intended to include all combinations of variables from any of the
disclosed embodiments within its scope.
[0091] Alternatively, the exclusion of one or more of the specified
variables from a group or an embodiment, or combinations thereof is
also contemplated by the present invention.
[0092] In certain aspects, the present invention provides prodrugs
and derivatives of the compounds according to the formulae above.
Prodrugs are derivatives of the compounds of the invention, which
have metabolically cleavable groups and become by solvolysis or
under physiological conditions the compounds of the invention,
which are pharmaceutically active, in vivo. Such examples include,
but are not limited to, choline ester derivatives and the like,
N-alkylmorpholine esters and the like.
[0093] Other derivatives of the compounds of this invention have
activity in both their acid and acid derivative forms, but the acid
sensitive form often offers advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism
(Bundgaard, 1985). Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters prepared by
reaction of the parent acid with a suitable alcohol, or amides
prepared by reaction of the parent acid compound with a substituted
or unsubstituted amine, or acid anhydrides, or mixed anhydrides.
Simple aliphatic or aromatic esters, amides and anhydrides derived
from acidic groups pendant on the compounds of this invention are
preferred prodrugs. In some cases it is desirable to prepare double
ester type prodrugs such as (acyloxy)alkyl esters or
((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, aryl,
C.sub.7-C.sub.12 substituted aryl, and C.sub.7-C.sub.12 arylalkyl
esters of the compounds of the invention.
Pharmaceutical Compositions
[0094] When employed as a pharmaceutical, a compound of the
invention is typically administered in the form of a pharmaceutical
composition. Such compositions can be prepared in a manner well
known in the pharmaceutical art and comprise at least one active
compound of the invention according to Formula I. Generally, a
compound of the invention is administered in a pharmaceutically
effective amount. The amount of compound of the invention 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 of
the invention administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0095] The pharmaceutical compositions of this invention can be
administered by a variety of routes including oral, rectal,
transdermal, subcutaneous, intra-articular, intravenous,
intramuscular, and intranasal. Depending on the intended route of
delivery, a compound of the invention is preferably formulated as
either injectable or oral compositions or as salves, as lotions or
as patches all for transdermal administration.
[0096] The compositions for oral administration can take the form
of bulk liquid solutions or suspensions, or bulk powders. More
commonly, however, the compositions are presented in unit dosage
forms to facilitate accurate dosing. 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. In such compositions, the compound of
the invention according to Formula I is usually a minor component
(from about 0.1 to about 50% by weight or preferably from about 1
to about 40% by weight) with the remainder being various vehicles
or carriers and processing aids helpful for forming the desired
dosing form.
[0097] Liquid forms suitable for oral administration may include a
suitable aqueous or non-aqueous vehicle with buffers, suspending
and dispensing agents, colorants, flavors and the like. Solid forms
may include, for example, any of the following ingredients, or
compound of the inventions of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic
acid, Primogel, or corn starch; a lubricant such as magnesium
stearate; a glidant such as colloidal silicon dioxide; a sweetening
agent such as sucrose or saccharin; or a flavoring agent such as
peppermint or orange flavoring.
[0098] Injectable compositions are typically based upon injectable
sterile saline or phosphate-buffered saline or other injectable
carriers known in the art. As before, the active compound of the
invention according to Formula I in such compositions is typically
a minor component, often being from about 0.05 to 10% by weight
with the remainder being the injectable carrier and the like.
[0099] Transdermal compositions are typically formulated as a
topical ointment or cream containing the active ingredient(s),
generally in an amount ranging from about 0.01 to about 20% by
weight, preferably from about 0.1 to about 20% by weight,
preferably from about 0.1 to about 10% by weight, and more
preferably from about 0.5 to about 15% by weight. When formulated
as an ointment, the active ingredients will typically be combined
with either a paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream
with, for example an oil-in-water cream base. Such transdermal
formulations are well-known in the art and generally include
additional ingredients to enhance the dermal penetration of
stability of the active ingredients or the formulation. All such
known transdermal formulations and ingredients are included within
the scope of this invention.
[0100] A compound of the invention can also be administered by a
transdermal device. Accordingly, transdermal administration can be
accomplished using a patch either of the reservoir or porous
membrane type, or of a solid matrix variety.
[0101] The above-described components for orally administrable,
injectable or topically administrable compositions are merely
representative. Other materials as well as processing techniques
and the like are set forth in Part 8 of Remington's Pharmaceutical
Sciences, 17.sup.th edition, 1985, Mack Publishing Company, Easton,
Pa., which is incorporated herein by reference.
[0102] A compound of the invention can also be administered in
sustained release forms or from sustained release drug delivery
systems. A description of representative sustained release
materials can be found in Remington's Pharmaceutical Sciences.
[0103] The following formulation examples illustrate representative
pharmaceutical compositions that may be prepared in accordance with
this invention. The present invention, however, is not limited to
the following pharmaceutical compositions.
Formulation 1--Tablets
[0104] A compound of the invention according to Formula I may be
admixed as a dry powder with a dry gelatin binder in an approximate
1:2 weight ratio. A minor amount of magnesium stearate may be added
as a lubricant. The mixture may be formed into 240-270 mg tablets
(80-90 mg of active compound of the invention according to Formula
I per tablet) in a tablet press.
Formulation 2--Capsules
[0105] A compound of the invention according to Formula I may be
admixed as a dry powder with a starch diluent in an approximate 1:1
weight ratio. The mixture may be filled into 250 mg capsules (125
mg of active compound of the invention according to Formula I per
capsule).
Formulation 3--Liquid
[0106] A compound of the invention according to Formula I (125 mg),
may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the
resultant mixture may be blended, passed through a No. 10 mesh U.S.
sieve, and then mixed with a previously made solution of
microcrystalline cellulose and sodium carboxymethyl cellulose
(11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color
may be diluted with water and added with stirring. Sufficient water
may then be added with stirring. Further sufficient water may be
then added to produce a total volume of 5 mL.
Formulation 4--Tablets
[0107] A compound of the invention according to Formula I may be
admixed as a dry powder with a dry gelatin binder in an approximate
1:2 weight ratio. A minor amount of magnesium stearate may be added
as a lubricant. The mixture may be formed into 450-900 mg tablets
(150-300 mg of active compound of the invention according to
Formula I) in a tablet press.
Formulation 5--Injection
[0108] A compound of the invention according to Formula I may be
dissolved or suspended in a buffered sterile saline injectable
aqueous medium to a concentration of approximately 5 mg/mL.
Formulation 6--Topical
[0109] Stearyl alcohol (250 g) and a white petrolatum (250 g) may
be melted at about 75.degree. C. and then a mixture of a compound
of the invention according to Formula I (50 g) methylparaben (0.25
g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and
propylene glycol (120 g) dissolved in water (about 370 g) may be
added and the resulting mixture may be stirred until it
congeals.
Methods of Treatment
[0110] In one embodiment, the present invention provides compounds
of the invention, or pharmaceutical compositions comprising a
compound of the invention, for use in medicine. In a particular
embodiment, the present invention provides compounds of the
invention or pharmaceutical compositions comprising a compound of
the invention, for use in the prophylaxis and/or treatment of pain,
inflammatory conditions, cardiovascular diseases, neurodegenerative
diseases, neurological diseases, complications of diabetes, cancer
and/or fibrotic diseases.
[0111] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
pain, inflammatory conditions, cardiovascular diseases,
neurodegenerative diseases, neurological diseases, complications of
diabetes, cancer and/or fibrotic diseases.
[0112] In one embodiment, the present invention provides
pharmaceutical compositions comprising a compound of the invention,
and another therapeutic agent. In a particular embodiment, the
other therapeutic agent is an agent for the prophylaxis and/or
treatment of pain, inflammatory conditions, cardiovascular
diseases, neurodegenerative diseases, neurological diseases,
complications of diabetes, cancer and/or fibrotic diseases.
[0113] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of pain,
inflammatory conditions, cardiovascular diseases, neurodegenerative
diseases, neurological diseases, complications of diabetes, cancer
and/or fibrotic diseases, which methods comprise the administration
of an effective amount of a compound of the invention or one or
more of the pharmaceutical compositions herein described for the
treatment or prophylaxis of said condition.
[0114] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with pain, inflammatory conditions, cardiovascular
diseases, neurodegenerative diseases, neurological diseases,
complications of diabetes, cancer and/or fibrotic diseases, which
methods comprise the administration of an effective amount of a
compound of the invention or one or more of the pharmaceutical
compositions herein described for the treatment or prophylaxis of
said condition.
[0115] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of pain. In a particular embodiment, the pain is selected
from chronic articular pain. In a specific embodiment the chronic
articular pain is the pain of osteoarthritis, rheumatoid arthritis,
rheumatoid spondylitis, gouty arthritis (gout) and/or juvenile
arthritis.
[0116] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
pain. In a particular embodiment, the pain is selected from chronic
articular pain. In a specific embodiment the chronic articular pain
is the pain of osteoarthritis, rheumatoid arthritis, rheumatoid
spondylitis, gouty arthritis (gout) and/or juvenile arthritis.
[0117] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with pain, which methods comprise the administration of
an effective amount of a compound of the invention or one or more
of the pharmaceutical compositions herein described for the
treatment or prophylaxis of said condition. In a particular
embodiment, the pain is selected from chronic articular pain. In a
specific embodiment the chronic articular pain is the pain of
osteoarthritis, rheumatoid arthritis, rheumatoid spondylitis, gouty
arthritis (gout) and/or juvenile arthritis.
[0118] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of inflammatory conditions. In a particular embodiment,
the inflammatory condition is selected from rheumatoid arthritis,
osteoarthritis, chronic obstructive pulmonary disease (COPD) and
inflammatory bowel diseases. More particularly, the inflammatory
condition is osteoarthritis.
[0119] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
inflammatory conditions. In a particular embodiment, the
inflammatory condition is selected from rheumatoid arthritis,
osteoarthritis, chronic obstructive pulmonary disease (COPD) and
inflammatory bowel diseases. More particularly, the inflammatory
condition is osteoarthritis.
In additional method of treatment aspects, this invention provides
methods of prophylaxis and/or treatment of a mammal afflicted with
inflammatory conditions, which methods comprise the administration
of an effective amount of a compound of the invention or one or
more of the pharmaceutical compositions herein described for the
treatment or prophylaxis of said condition. In a particular
embodiment, the inflammatory condition is selected from rheumatoid
arthritis, osteoarthritis, chronic obstructive pulmonary disease
(COPD) and inflammatory bowel diseases. More particularly, the
inflammatory condition is osteoarthritis.
[0120] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of cardiovascular diseases. In a particular embodiment,
the cardiovascular disease is selected from atherosclerosis,
pulmonary arterial hypertension, heart failure, acute coronary
syndrome, cardiac hypertrophy, ventricular fibrosis and myocardial
remodeling.
[0121] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
cardiovascular diseases. In a particular embodiment, the
cardiovascular disease is selected from atherosclerosis, pulmonary
arterial hypertension, heart failure, acute coronary syndrome,
cardiac hypertrophy, ventricular fibrosis and myocardial
remodeling.
[0122] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with a cardiovascular disease, which methods comprise the
administration of an effective amount of a compound of the
invention or one or more of the pharmaceutical compositions herein
described for the treatment or prophylaxis of said disease. In a
particular embodiment, the cardiovascular disease is selected from
atherosclerosis, pulmonary arterial hypertension, heart failure,
acute coronary syndrome, cardiac hypertrophy, ventricular fibrosis
and myocardial remodeling.
[0123] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of neurodegenerative diseases. In a particular
embodiment, the neurodegenerative disease is selected from
degenerative dementia, Alzheimer's disease, multiple sclerosis and
retinopathies.
[0124] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
neurodegenerative diseases. In a particular embodiment, the
neurodegenerative disease is selected from degenerative dementia,
Alzheimer's disease, multiple sclerosis and retinopathies.
[0125] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with a neurodegenerative disease, which methods comprise
the administration of an effective amount of a compound of the
invention or one or more of the pharmaceutical compositions herein
described for the treatment or prophylaxis of said disease. In a
particular embodiment, the neurodegenerative disease is selected
from degenerative dementia, Alzheimer's disease, multiple sclerosis
and retinopathies.
[0126] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of neurological diseases. In a particular embodiment, the
neurological disease is selected from neuropathic pain, dementia,
multiple sclerosis and retinopathies.
[0127] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
neurological diseases. In a particular embodiment, the neurological
disease is selected from neuropathic pain, dementia, multiple
sclerosis and retinopathies.
[0128] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with a neurological disease, which methods comprise the
administration of an effective amount of a compound of the
invention or one or more of the pharmaceutical compositions herein
described for the treatment or prophylaxis of said disease. In a
particular embodiment, the neurological disease is selected from
neuropathic pain, dementia, multiple sclerosis and
retinopathies.
[0129] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of complications of diabetes. In a particular embodiment,
the complication is selected from diabetic retinopathy, diabetic
microangiopathy, diabetic nephropathy and hepatic steatosis.
[0130] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
complications of diabetes. In a particular embodiment, the
complication is selected from diabetic retinopathy, diabetic
microangiopathy, diabetic nephropathy and hepatic steatosis.
[0131] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with complications of diabetes, which methods comprise
the administration of an effective amount of a compound of the
invention or one or more of the pharmaceutical compositions herein
described for the treatment or prophylaxis of said complication. In
a particular embodiment, the complication is selected from diabetic
retinopathy, diabetic microangiopathy, diabetic nephropathy and
hepatic steatosis.
[0132] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of cancer.
[0133] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
cancer.
[0134] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with cancer, which methods comprise the administration of
an effective amount of a compound of the invention or one or more
of the pharmaceutical compositions herein described for the
treatment or prophylaxis of said cancer.
[0135] In one embodiment, the present invention provides compounds
of the invention or pharmaceutical compositions comprising a
compound of the invention, for use in the prophylaxis and/or
treatment of fibrotic diseases. In a particular embodiment the
fibrotic disease is of an individual organ or tissue such as liver
fibrosis, lung fibrosis or kidney fibrosis. In a particular
embodiment, the fibrotic disease is selected from idiopathic
pulmonary fibrosis (IPF), diabetic kidney disease (DKD) and
nonalcoholic steatohepatitis (NASH).
[0136] In another embodiment, the present invention provides
compounds of the invention, or pharmaceutical compositions
comprising a compound of the invention for use in the manufacture
of a medicament for use in the prophylaxis and/or treatment of
fibrotic diseases. In a particular embodiment the fibrotic disease
is of an individual organ or tissue such as liver fibrosis, lung
fibrosis or kidney fibrosis. In a particular embodiment, the
fibrotic disease is selected from idiopathic pulmonary fibrosis
(IPF), diabetic kidney disease (DKD) and nonalcoholic
steatohepatitis (NASH).
[0137] In additional method of treatment aspects, this invention
provides methods of prophylaxis and/or treatment of a mammal
afflicted with a fibrotic disease, which methods comprise the
administration of an effective amount of a compound of the
invention or one or more of the pharmaceutical compositions herein
described for the treatment or prophylaxis of said disease. In a
particular embodiment the fibrotic disease is of an individual
organ or tissue such as liver fibrosis, lung fibrosis or kidney
fibrosis. In a particular embodiment, the fibrotic disease is
selected from idiopathic pulmonary fibrosis (IPF), diabetic kidney
disease (DKD) and nonalcoholic steatohepatitis (NASH).
[0138] Injection dose levels range from about 0.1 mg/kg/h to at
least 10 mg/kg/h, or from about 1 to about 120 h and especially 24
to 96 h. A preloading bolus of from about 0.1 mg/kg to about 10
mg/kg or more may also be administered to achieve adequate steady
state levels. The maximum total dose is not expected to exceed
about 1 g/day for a 40 to 80 kg human patient.
[0139] For the prophylaxis and/or treatment of long-term
conditions, such as degenerative conditions, the regimen for
treatment usually stretches over many months or years so oral
dosing is preferred for patient convenience and tolerance. With
oral dosing, one to four (1-4) regular doses daily, especially one
to three (1-3) regular doses daily, typically one to two (1-2)
regular doses daily, and most typically one (1) regular dose daily
are representative regimens. Alternatively for long lasting effect
drugs, with oral dosing, once every other week, once weekly, and
once a day are representative regimens. In particular, dosage
regimen can be every 1-14 days, more particularly 1-10 days, even
more particularly 1-7 days, and most particularly 1-3 days.
[0140] Using these dosing patterns, each dose provides from about 1
to about 1000 mg of a compound of the invention, with particular
doses each providing from about 10 to about 500 mg and especially
about 30 to about 250 mg.
[0141] Transdermal doses are generally selected to provide similar
or lower blood levels than are achieved using injection doses.
[0142] When used to prevent the onset of a condition, a compound of
the invention will be administered to a patient at risk for
developing the condition, typically on the advice and under the
supervision of a physician, at the dosage levels described above.
Patients at risk for developing a particular condition generally
include those that have a family history of the condition, or those
who have been identified by genetic testing or screening to be
particularly susceptible to developing the condition.
[0143] A compound of the invention can be administered as the sole
active agent or it can be administered in combination with other
therapeutic agents, including other compounds of the invention that
demonstrate the same or a similar therapeutic activity and that are
determined to be safe and efficacious for such combined
administration. In a specific embodiment, co-administration of two
(or more) agents allows for significantly lower doses of each to be
used, thereby reducing the side effects seen.
[0144] In one embodiment, a compound of the invention or a
pharmaceutical composition comprising a compound of the invention
is administered as a medicament. In a specific embodiment, said
pharmaceutical composition additionally comprises a further active
ingredient.
[0145] In one embodiment, a compound of the invention is
co-administered with another therapeutic agent for the treatment
and/or prophylaxis of an inflammatory condition, particular agents
include, but are not limited to, immunoregulatory agents e.g.
azathioprine, corticosteroids (e.g. prednisolone or dexamethasone),
cyclophosphamide, cyclosporin A, tacrolimus, mycophenolate,
mofetil, muromonab-CD3 (OKT3, e.g. Orthocolone.RTM.), ATG, aspirin,
acetaminophen, ibuprofen, naproxen, and piroxicam.
[0146] In one embodiment, a compound of the invention is
co-administered with another therapeutic agent for the treatment
and/or prophylaxis of arthritis (e.g. rheumatoid arthritis),
particular agents include but are not limited to analgesics,
non-steroidal anti-inflammatory drugs (NSAIDS), steroids, synthetic
disease-modifying antirheumatic drugs (DMARDS), (for example but
without limitation methotrexate, leflunomide, sulfasalazine,
Auranofin, sodium aurothiomalate, penicillamine, chloroquine,
hydroxychloroquine, azathioprine, tofacitinib, baricitinib,
fostamatinib, and cyclosporin), and biological DMARDS (for example
but without limitation infliximab, etanercept, adalimumab,
rituximab, and abatacept).
[0147] By co-administration is included any means of delivering two
or more therapeutic agents to the patient as part of the same
treatment regime, as will be apparent to the skilled person. Whilst
the two or more agents may be administered simultaneously in a
single formulation, i.e. as a single pharmaceutical composition,
this is not essential. The agents may be administered in different
formulations and at different times.
Chemical Synthetic Procedures
General
[0148] According to a further aspect of the present invention there
is provided a process for the preparation of compounds of formula
I. The schemes herein are examples of synthetic schemes that may be
used to synthesise the compounds of the invention. In the schemes
herein, reactive groups can be protected with protecting groups and
de-protected according to well established techniques.
[0149] According to a further aspect of the invention there is
provided a process for preparing a compound of formula I as herein
defined which comprises:
(a) reacting a compound of formula II:
##STR00004##
wherein X and R.sub.2 are as defined herein, with a compound of
formula III:
##STR00005##
or a protected derivative thereof, wherein R.sup.1 is as defined
herein; (b) deprotection of a protected derivative of a compound of
formula I; (c) interconversion of a compound of formula I or
protected derivative thereof to a further compound of formula I or
protected derivative thereof; and (d) optional formation of a
pharmaceutically acceptable salt of a compound of formula I.
[0150] Compounds of formulae II and III may be prepared in
accordance with procedures described herein in Schemes 1A, 1B, 1C
and the procedures for preparing Compounds 1 to 7.
[0151] The compound of the invention can be prepared from readily
available starting materials using the following general methods
and procedures. It will be appreciated that where typical or
preferred process conditions (i.e. reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are given,
other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0152] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions. The
choice of a suitable protecting group for a particular functional
group as well as suitable conditions for protection and
deprotection are well known in the art.
[0153] The following methods are presented with details as to the
preparation of a compound of the invention as defined hereinabove
and the comparative examples. A compound of the invention may be
prepared from known or commercially available starting materials
and reagents by one skilled in the art of organic synthesis.
[0154] All reagents are of commercial grade and are used as
received without further purification, unless otherwise stated.
Commercially available anhydrous solvents are used for reactions
conducted under inert atmosphere. Reagent grade solvents are used
in all other cases, unless otherwise specified. Column
chromatography is performed on silica gel 60 (35-70 .mu.m). Thin
layer chromatography is carried out using pre-coated silica gel
60E-254 plates (thickness 0.25 mm) NMR spectra are recorded on
Bruker DPX 300 MHz equipped with a 5 mm BBI probe, Bruker AV400 MHz
equipped with a 5 mm PABBO probe, Bruker DRX 500 MHz equipped with
a 5 mm PABBI probe and Bruker Avance III 600 spectrometer equipped
with a 5 mm RT BBI probe. The samples are recorded at 25.degree. C.
using DMSO-d.sub.6 or CDCl.sub.3 as a solvent, unless otherwise
stated. Chemical shifts (.delta.) for .sup.1H NMR spectra are
reported in parts per million (ppm) relative to tetramethylsilane
(.delta. 0.00) as internal reference.
[0155] Electrospray MS spectra are obtained on Waters Acquity UPLC
with Waters Acquity PDA detector and SQD mass spectrometer. Columns
used: UPLC BEH C18 1.7 .mu.m, 2.1.times.5 mm VanGuard Pre-column
with Acquity UPLC BEH C18 1.7 .mu.m, 2.1.times.50 mm Column or
Acquity UPLC CSH C18 1.7 .mu.m, 2.1.times.50 mm Column. All the
methods are using MeCN/H.sub.2O gradients. MeCN and H.sub.2O
contains either 0.1% Formic Acid or 10 mM NH.sub.4HCO.sub.3.
[0156] For preparative purification HPLC Waters Mass Directed
Autopurification System is used. The system is composed of Waters
Sample Manager 2767, Waters System Fluid Organizer, Waters Binary
Gradient Module 2545, Waters 515 HPLC Pump, Waters Photodiode Array
Detector 2998 and Waters Micromass ZQ MS detector. Software used:
FractionLynx and MassLynx v4.1. General HPLC method parameters:
gradient mobile phase of 0.1% formic acid in H.sub.2O and MeCN or
10 mM NH.sub.4HCO.sub.3 pH=10 and MeCN. Column XBridge 30.times.150
mm, 5 .mu.m. PDA detector settings: wavelength: 210-400 nm,
resolution: 1.2 nm, sampling rate: 1.0 points/sec, filter response:
1. Microwave heating is performed with a Biotage Initiator.
TABLE-US-00001 List of abbreviations used in the experimental
section: Abbreviation Definition .mu.L microliter AUC Area Under
the Curve BAL Broncho-alveolar lavage BALF Broncho-alveolar lavage
fluid BINAP 2,2'-Bis(diphenylphosphino)-1,1'-binaphthalene br. d
Broad doublet Boc tert-Butyloxy-carbonyl br. s Broad singlet BSA
Bovine serum albumine br. t Broad triplet Cat. Catalytic amount
cDNA copy deoxyribonucleic acid Cpd Compound d doublet DavePhos
2-Dicyclohexylphosphino-2'- (N,N-dimethylamino)biphenyl DCM
Dichloromethane DEAD diethyl azodicarboxylate DIPE Diisopropylether
DIPEA N,N-diisopropylethylamine DMA Dimethylacetamide DMAP
4-Dimethylaminopyridine DME Dimethoxyethane DMF
N,N-dimethylformamide DMPU
1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone DMSO
Dimethylsulfoxide dppf 1,1'-Bis(diphenylphosphino)ferrocene EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) EDC.HCl
N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride eq.
Equivalent Et.sub.2O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol
FBS Fetal bovine serum FITC Fluorescein Isothiocyanate g gram h
hour HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium
hexafluorophosphate HOBt Hydroxybenzotriazole HPLC High pressure
liquid chromatography HRP horseradish peroxydase Int Intermediate
JohnPhos (2-Biphenyl)di-tert-butylphosphine kg kilogram L liter
LCMS Liquid Chromatography-Mass Spectrometry LDA Lithium
diisopropylamide LiHMMDS Lithium bis(trimethylsilyl)amide m
multiplet m-CPBA 3-Chloroperbenzoic acid MeCN Acetonitrile MEK
Methyl ethyl ketone MeOH Methanol mg milligram min minute mL
millilitre mmol millimoles MMP Matrix Metallo Proteinase Ms'd Mass
measured by LC-MS Mtd Method MW Molecular weight N.A. Not available
nBuOH n-Butanol Nva Norvaline NMR Nuclear Magnetic Resonance PBF
phosphate buffered formalin PBS Phosphate buffered saline PCR
Polymerase chain reaction Pd(PPh.sub.3).sub.4
Tetrakis(triphenylphosphine)palladium(0) Pd/C Palladium on Carbon
10% Pd.sub.2(dba).sub.3 Tris(dibenzylideneacetone) dipalladium(0)
PdCl.sub.2dppf [1,1'-Bis(diphenylphosphino)ferrocene]
dichloropalladium(II) PdCl.sub.2[P(o-Tol).sub.3].sub.2
Dichlorobis(tri-o-tolylphosphine)palladium(II) Pd(OAc).sub.2
Palladium(II) acetate PEG Polyethylene glycol PEPPSI .TM.-IPr
[1,3-Bis(2,6-Diisopropylphenyl)imidazol-2-
ylidene](3-chloropyridyl)palladium(II) dichloride ppm
part-per-million q quadruplet QrtPCR quantitative real-time PCR QTL
quantitative trait loci r.t. room temperature RNA Ribonucleic acid
Rt retention time RuPhos
2-Dicyclohexylphosphino-2',6'-diisopropoxybiphenyl s singlet sept
septuplet SFC Supercritical fluid chromatography SM Starting
Material Ster Stereochemistry t triplet TBAF Tetra-n-butylammonium
fluoride t-BuOH Tert-butanol TBDPSCl Tert-butyldiphenylsilyl
chloride
Mass-Directed Automated HPLC
[0157] Where applicable, purification by mass-directed automated
HPLC was carried out using the following apparatus and
conditions:
Hardware:
[0158] Waters 2525 Binary Gradient Module
[0159] Waters 515 Makeup Pump
[0160] Waters Pump Control Module
[0161] Waters 2767 Inject Collect
[0162] Waters Column Fluidics Manager
[0163] Waters 2996 Photodiode Array Detector
[0164] Waters ZQ Mass Spectrometer
[0165] Gilson 202 fraction collector
[0166] Gilson Aspec waste collector
Software: Waters MassLynx version 4 SP2 Column: the columns used
were Waters Atlantis, the dimensions of which are 19 mm.times.100
mm (small scale) and 30 mm.times.100 mm (large scale). The
stationary phase particle size is 5 .mu.m.
Acidic Method:
Solvents:
[0167] A: Aqueous solvent=Water+0.1% Formic Acid B: Organic
solvent=Acetonitrile+0.1% Formic Acid Make up
solvent=Methanol:Water 80:20 Needle rinse solvent=Methanol
Methods:
[0168] There were five methods used depending on the analytical
retention time of the compound of interest. Each had a 13.5-minute
runtime, which comprised a 10-minute gradient followed by a 3.5
minute column flush and re-equilibration step. [0169] Large/Small
Scale 1.0-1.5 (HPLC), 0.4-0.6 (UPLC)=5-30% B [0170] Large/Small
Scale 1.5-2.2 (HPLC), 0.6-0.9 (UPLC)=15-55% B [0171] Large/Small
Scale 2.2-2.9 (HPLC), 0.9-1.2 (UPLC)=30-85% B [0172] Large/Small
Scale 2.9-3.6 (HPLC), 1.2-1.4 (UPLC)=50-99% B [0173] Large/Small
Scale 3.6-5.0 (HPLC), 1.4-2.0 (UPLC)=80-99% B (in 6 minutes
followed by 7.5 minutes flush and re-equilibration) Flow rate: all
of the above methods have a flow rate of either 20 mL/min (Small
Scale) or 40 mL/min (Large Scale).
High pH Method:
[0174] Column: the HPLC analysis was conducted on an XBridge C18
column (100 nm.times.19 nm i.d. 5 .mu.m packing diameter) at
ambient temperature
Solvents:
[0175] A: 10 mM Ammonium bicarbonate in water, adjusted to pH 10
with ammonia solution.
B: Acetonitrile.
Methods:
[0176] There were five methods used depending on the analytical
retention time of the compound of interest. They had a 15-minute
runtime, which comprised a 10 minute gradient followed by a 5
minute column flush and re-equilibration step. [0177] Large/Small
Scale 1.0-1.5 (HPLC), 0.4-0.6 (UPLC)=1-30% B [0178] Large/Small
Scale 1.5-2.2 (HPLC), 0.6-0.9 (UPLC)=15-55% B [0179] Large/Small
Scale 2.2-2.9 (HPLC), 0.9-1.2 (UPLC)=30-85% B [0180] Large/Small
Scale 2.9-3.6 (HPLC), 1.2-1.4 (UPLC)=50-99% B [0181] Large/Small
Scale 3.6-5.0 (HPLC), 1.4-2.0 (UPLC)=80-99% B (in 6 minutes
followed by 7.5 minutes flush an re-equilibration) Flow rate: all
of the above methods have a flow rate of either 20 ml/min (small
scale) or 40 ml/min (large scale)
Liquid Chromatography/Mass Spectrometry
[0182] Analysis of the above Examples by Liquid Chromatography/Mass
Spectrometry (LC/MS) was carried out using the apparatus and
conditions indicated in the methods shown below
Liquid Chromatography:
Method Description: Formic Acid Generic Analytical UPLC Open Access
LC/MS
[0183] 2 Minute Method LC/MS System: Acquity UPLC coupled with SQD
mass spectrometer
LC Conditions
[0184] Column: Acquity UPLC BEH C18 (50 mm.times.2.1 mm i.d., 1.7
.mu.m packing diameter Column temperature: 40.degree. C. Solvents:
A=0.1% v/v solution of Formic Acid in Water [0185] B=0.1% v/v
solution of Formic Acid in Acetonitrile
Injection Volume: 2 .mu.l
[0186] The gradient table:
TABLE-US-00002 Time Flow Rate (min) (ml/min) % A % B 0 0.9 97 3 1.5
0.9 0 100 1.9 0.9 0 100 2.0 0.05 97 3
Stop time: 2 min
UV Conditions
[0187] PDA Range: 210 nm-350 nm
[0188] The UV detection was a summed signal from wavelength of 210
nm to 350 nm Acquisition Rate: 40 Hz
MS Conditions
[0189] Ionization Mode: Alternate--scan Positive and Negative
Electrospray (ES.sup.+/ES.sup.-)
Scan Range: 100 to 1000 AMU
Scan Time: 0.15
Inter Scan Delays:
[0190] MS inter-scanan: 0.02 seconds Polarity/Mode switch
inter-scan: 0.02 seconds
Method Description: Ammonium Bicarbonate Generic Analytical UPLC
Open Access LC/MS 2 Minute Method
[0191] LC/MS System: Acquity UPLC coupled with SQD mass
spectrometer
LC Conditions
[0192] Column: Acquity UPLC BEH C18 (50 mm.times.2.1 mm i.d., 1.7
.mu.m packing diameter) Column temperature: 40.degree. C. Solvents:
A=10 mM aqueous solution of NH.sub.4HCO.sub.3 (adjusted to pH 10
with ammonia) [0193] B=Acetonitrile
Injection Volume: 1 .mu.l
[0194] The gradient table:
TABLE-US-00003 Time Flow Rate (min) (mL/min) % A % B 0 0.9 97 3 1.5
0.9 0 100 1.9 0.9 0 100 2.0 0.05 97 3
Stop time: 2 min
UV Conditions
[0195] PDA Range: 210 nm-350 nm The UV detection was a summed
signal from wavelength of 210 nm to 350 nm
Acquisition Rate: 40 Hz
MS Conditions
[0196] Ionization Mode: Alternate--scan Positive and Negative
Electrospray (ES.sup.+/ES.sup.-)
Scan Range: 100 to 1000 AMU
Scan Time: 0.15
Inter Scan Delays:
[0197] MS inter-scanan: 0.02 seconds Polarity/Mode switch
inter-scan: 0.02 seconds
Synthetic Preparation of the Compounds of the Invention
##STR00006##
##STR00007##
##STR00008##
[0198] 1.1 Compound 1:
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide
1.1.1 Step 1: Synthesis of methyl
2-bromo-5-fluoro-pyridine-4-carboxylate
##STR00009##
[0200] To a solution of 2-bromo-5-fluoro-pyridine-4-carboxylic acid
(10.0 g, 45.5 mmol) in methanol (300 mL) cooled to 0.degree. C. was
added thionyl chloride (16.5 mL, 227.3 mmol) dropwise over 30
minutes. The reaction mixture was stirred at ambient temperature
for 24 hours. Toluene (40 mL) was added to the reaction mixture.
After evaporation of methanol, thionyl chloride was distilled out.
The remaining toluene was evaporated on rotary evaporator affording
the crude product, which was dissolved in dichloromethane (30 mL)
evaporated under reduced pressure affording methyl
2-bromo-5-fluoro-pyridine-4-carboxylate (10.3 g). LCMS: MW (calcd):
232.9; MS (ES.sup.+, m/z): 234, 236 (M+H).sup.+.
1.1.2 Alternative Synthetic Procedure
[0201] To a solution of 2-bromo-5-fluoro-pyridine-4-carboxylic acid
(10.0 g, 45.5 mmol) in methanol (35 mL) and toluene (65 mL) cooled
to 0.degree. C. was added (trimethylsilyl)diazomethane (2.0 M
solution in diethyl ether; 45.5 mL, 90.9 mmol) dropwise over 30
minutes. The reaction mixture was stirred at ambient temperature.
After 2 h, the reaction mixture was evaporated under reduced
pressure affording the crude product, which was dissolved in ethyl
acetate (50 mL), washed with water (100 mL) and brine (50 mL)
respectively, filtered through phase separator filter and
evaporated under reduced pressure affording methyl
2-bromo-5-fluoro-pyridine-4-carboxylate (10.46 g). LCMS: MW
(calcd): 232.9; MS (ES.sup.+, m/z): 234, 236 (M+H).sup.+.
1.1.3 Step 2: Synthesis of Methyl
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylate
##STR00010##
[0203] To a solution of methyl
2-bromo-5-fluoro-pyridine-4-carboxylate (10.3 g, 44.0 mmol) in
1,4-dioxane (150 mL) were added tert-butyl carbamate (6.18 g, 52.8
mmol), tris(dibenzylideneacetone)dipalladium(0) (0.86 g, 0.88
mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.02 g,
1.76 mmol) and cesium carbonate (20.08 g, 61.6 mmol). The reaction
mixture was purged with argon, sonicated, caped and left to stir at
90.degree. C. for 24 h. The reaction mixture was cooled, filtrated
through pad of celite and washed with ethyl acetate. Mother liquor
was washed with water (2.times.100 mL) and brine (100 mL) and
evaporated under reduced pressure affording the crude product,
which was triturated from ethyl acetate affording methyl
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylate (8.43
g). LCMS: MW (calcd): 270.1; MS (ES.sup.+, m/z): 215.41
(M+H-56).sup.+.
1.1.4 Alternative Synthetic Procedure (methyl
2-((diphenylmethylene)amino)-5-fluoroisonicotinate
Intermediate)
##STR00011##
[0205] To a degassed and purged with argon suspension of methyl
2-bromo-5-fluoro-pyridine-4-carboxylate (308 mg, 1.32 mmol) in
1,4-dioxane (6 mL) was added benzophenoneimine (0.266 mL, 1.58
mmol), tris(dibenzylideneacetone)dipalladium(0) (24.1 mg, 0.026
mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (30.5 mg,
0.053 mmol) and cesium carbonate (602.0 mg, 0.053 mmol). The
reaction mixture was flushed with argon, sonicated, caped and left
to stir at 100.degree. C. for 16 h. The reaction mixture was
cooled, diluted with ethyl acetate (15 mL), washed with water (15
mL), brine (15 mL), and evaporated under reduced pressure affording
methyl 2-((diphenylmethylene)amino)-5-fluoroisonicotinate (230 mg).
LCMS: MW (calcd): 334.1; MS (ES.sup.+, m/z): 335.34
(M+H).sup.+.
1.1.5 Step 3: Synthesis of
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylic
Acid
##STR00012##
[0207] To a suspension of methyl
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylate (8.43
g, 31.19 mmol) in tetrahydrofuran (100 mL) was added lithium
hydroxide (2.98 g, 124.76 mmol) and water (50 mL). The reaction
mixture was left to stir overnight at ambient temperature. Next day
tetrahydrofuran was evaporated under reduced pressure, the pH of
water layer was adjusted to 4. The formed precipitate was filtered
and coevaporated with toluene (4.times.20 mL) and dried in a vacuum
oven at 40.degree. C. for 5 h affording
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylic acid
(7.79 g). LCMS: MW (calcd): 256.08; MS (ES.sup.+, m/z): 201.4
(M+H-56).sup.+.
1.1.6 Step 4: Synthesis of
4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
##STR00013##
[0209] 5-Bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (10.0 g, 43.0
mmol) was dissolved in dry THF (350 mL) and cooled to -78.degree.
C. under argon atmosphere. n-BuLi (2.5 M in hexane, 38 mL, 94.6
mmol) was added dropwise over one hour. After complete addition,
the solution was stirred for 40 min and iodomethane (4.3 mL, 68.8
mmol) was added. The solution was allowed to slowly reach room
temperature. Water (20 mL) was added and the solvent was removed in
vacuum to yield brown slurry, which was dissolved in water (200 mL)
and extracted with ethyl acetate (3.times.100 mL). Combined
organics were washed with brine (150 mL), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to afford the crude
compound that was triturated from ethyl acetate to yield
4-chloro-5-methyl-7H-pyrrolo[2,3-d] (5.32 g). LCMS: MW (calcd):
167.03; MS (ES.sup.+, m/z): 168.37 (M+H).sup.+.
1.1.7 Step 5: Synthesis of Tert-butyl
N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-carbamate
##STR00014##
[0211] To a solution of
4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (10.0 g, 59.7 mmol)
in 1-methyl-2-pyrrolidinone (40 mL) was added tert-butyl
N-(4-piperidyl)carbamate (17.95 g, 89.6 mmol), and
N,N-diisopropylethylamine (25.2 mL, 149.3 mmol). The reaction
mixture was left to stir at 150.degree. C. for 3 h. The reaction
mixture was cooled and then poured into cold water (300 mL). To the
mixture was added ethyl acetate (40 mL) and was left to stir for 30
min at ambient temperature. The formed precipitate was filtered,
washed with water and diethyl ether and left to dry in vacuum oven
at 40.degree. C. overnight. Drying afforded tert-butyl
N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-carbamate
(19.91 g). LCMS: MW (calcd): 331.2; MS (ES.sup.+, m/z): 332.7
(M+H).sup.+.
1.1.8 Step 6: Synthesis of
1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
##STR00015##
[0213] To a suspension of tert-butyl
N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]carbamate
(38.9 g, 117.3 mmol) in 1,4-dioxane (200 mL) cooled to 0.degree. C.
was added HCl (4M solution in dioxane) (290 mL). The reaction
mixture was stirred at ambient temperature. After 23 h the reaction
mixture was evaporated under reduced pressure, coevaporated with
toluene (200 mL) and dried in vacuum oven at 40.degree. C.
overnight. Drying afforded
1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
(47.68 g) as HCl salt. LCMS: MW (calcd): 231.15; MS (ES.sup.+,
m/z): 232.6 (M+H).sup.+.
1.1.9 Step 7: Synthesis of Tert-butyl
N-[5-fluoro-4-[[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-
carbamoyl]-2-pyridyl]carbamate
##STR00016##
[0215] To a suspension of
2-(tert-butoxycarbonylamino)-5-fluoro-pyridine-4-carboxylic acid
(14.25 g, 55.6 mmol) in N,N-dimethylformamide (130 mL) was added
HATU (19.03 g, 50.1 mmol). The reaction mixture was stirred at
ambient temperature for 20 min upon which
1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine.times.2HCl
(16.92 g, 55.6 mmol) and N,N-diisopropylethylamine (47.56 mL, 278.1
mmol) were added. The reaction mixture was stirred at ambient
temperature. After 3 h reaction was completed. The reaction mixture
was poured into ice cooled water (1.2 L). The formed precipitate
was filtered, then washed with acetonitrile and dried in vacuum
oven at 40.degree. C. for 4 h affording tert-butyl
N-[5-fluoro-4-[[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-
carbamoyl]-2-pyridyl]carbamate (19.26 g). LCMS: MW (calcd): 469.22;
MS (ES.sup.+, m/z): 470.7 (M+H).sup.+.
1.1.10 Step 8: Synthesis of
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide
##STR00017##
[0217] To a suspension of tert-butyl
N-[5-fluoro-4-[[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-
carbamoyl]-2-pyridyl]carbamate (25.76 g, 54.9 mmol) in
dichloromethane (300 mL) cooled to 0.degree. C. was added HCl (4M
solution in dioxane) (130 mL) dropwise. After few minutes gummy
residue was formed. Additional amount of dichloromethane (200 mL)
was added to the reaction mixture. The gummy residue was crushed by
sonification (after 45 min). The reaction mixture was stirred
overnight at ambient temperature. The next day the precipitate was
filtered and washed with water, acetonitrile and methanol and dried
in vacuum oven at 40.degree. C. for 5 h and then overnight at
ambient temperature. Drying afforded
2-amino-5-fluoro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide (13.65 g). LCMS: MW (calcd): 369.17; MS
(ES.sup.+, m/z): 370.77 (M+H).sup.+.
[0218] .sup.1H NMR (600 MHz, DMSO-d6): .delta.=1.59-1.70 (m,
J=12.3, 3.7 Hz, 2H), 1.93 (dd, J=12.6, 2.7 Hz, 2H), 2.34 (s, 3H),
3.06 (t, J=11.4 Hz, 2H), 3.94 (d, J=13.2 Hz, 2H), 3.95-4.03 (m,
J=11.2, 11.2, 7.5, 4.2, 4.2 Hz, 1H), 6.02 (s, 2H), 6.51 (d, J=4.8
Hz, 1H), 7.05 (s, 1H), 7.91 (d, J=1.5 Hz, 1H), 8.19 (s, 1H), 8.46
(d, J=7.7 Hz, 1H), 11.51 (br. s., 1H) ppm.
1.2 Compound 2:
(2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piper-
idyl]pyridine-4-carboxamide)
1.2.1 Step 1: Synthesis of Methyl
2-amino-5-methyl-pyridine-4-carboxylate
##STR00018##
[0220] Methyl 2-amino-5-bromo-pyridine-4-carboxylate (200 mg, 0.87
mmol), S-Phos (4.6 mg, 3.2 mmol), tripotassium phosphate (369.3 mg,
1.74 mmol) and diacetoxypalladium (19.5 g, 0.011 mmol) were
combined, degassed and backfilled with N2 and then dissolved in
DMSO (4 ml) and trimethylboroxine (467.5 .mu.L, 3.2 mmol) at rt.
The mixture was then heated to 80.degree. C. and stirred overnight.
After 16 hours the mixture was diluted with EtOAc and washed with
H.sub.2O. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, absorbed onto celite and concentrated in vacuo to afford
crude product which was purified via Biotage purification device on
4 g KP-Sil Interchim column with flowrate 9 ml/min, DCM as solvent
A and DCM:MeOH=20:1 as solvent B. The appropriate fractions have
been collected to afford methyl
2-amino-5-methyl-pyridine-4-carboxylate (167.03 mg). LCMS: MW
(calcd): 166.07; MS (ES.sup.+, m/z): 167.03 (M+H).sup.+.
1.2.2 Step 2: Synthesis of 2-amino-5-methyl-pyridine-4-carboxylic
Acid
##STR00019##
[0222] Methyl 2-amino-5-methyl-pyridine-4-carboxylate (125 mg, 0.75
mmol) was dissolved in MeOH/H.sub.2O (2/2) and NaOH (1M solution,
0.75 .mu.L) was added. The mixture was then heated to 60.degree. C.
and allowed to stir 1 h. After 1 h MeOH was evaporated and aqueous
residue was extracted with EtOAc. Desired product left in aqueous
layer which was lyophilized overnight.
After lyophilisation, mixture was dissolved in acetone, filtered
and mother liquor was evaporated to obtain
2-amino-5-methyl-pyridine-4-carboxylic acid (55 mg). LCMS: MW
(calcd): 152.06; MS (ES.sup.+, m/z): 153.02 (M+H).sup.+.
1.2.3 Step 3: Synthesis
2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide
##STR00020##
[0224] To a solution of 2-amino-5-methyl-pyridine-4-carboxylic acid
(33 mg, 0.22 mmol) in DMF (1 mL) at 0.degree. C. was added EDCxHCl
(54.8 mg, 0.29 mmol) and DIPEA (111.1 .mu.L, 0.64 mmol) and left
stirring for 30 minutes. Then HOBtxH.sub.2O (35.6 mg, 0.26 mmol)
and 1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
(50.2 mg, 0.22 mmol) were added. The reaction was left stirred
overnight at room temperature. Next day reaction mixture was
diluted with water (5 mL) and extracted with EtOAc (3.times.5 mL).
The combined organic layers were washed with brine (10 mL), dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford
crude product which was purified via Biotage purification device on
4 g KP-Sil Interchim column with flow rate 9 ml/min, DCM as weak
solvent and DCM:MeOH:NH.sub.4OH=90:1.5:0.15 as strong solvent. The
appropriate fractions have been collected to afford
2-amino-5-methyl-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide (8 mg). LCMS: MW (calcd): 365.20; MS
(ES.sup.+, m/z): 366.23 (M+H).sup.+. .sup.1H NMR (300 MHz, DMSO-d6)
.delta. 11.51 (br. s., 1H), 8.35 (d, J=8.01 Hz, 1H), 8.17 (s, 1H),
7.75 (s, 1H), 7.05 (s, 1H), 6.35 (s, 1H), 5.81 (s, 2H), 3.95 (m,
3H), 3.05 (t, J=11.41 Hz, 2H), 2.34 (s, 3H), 2.06 (s, 3H), 1.92 (d,
J=9.58 Hz, 2H), 1.56-1.70 (m, 2H) ppm.
1.3: Compound 3:
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]-pyridine-4-carboxamide
1.3.1 Step 1: Synthesis of 3-chloro-1-oxo-pyridine-4-carboxylic
Acid
##STR00021##
[0226] 3-Chloropyridine-4-carboxylic (620 mg, 3.94 mmol) was
dissolved in acetic acid (2 mL) and 30% aq H.sub.2O.sub.2 (6 mL)
was added. The reaction mixture was heated for 38 h at 80.degree.
C. After completion it was concentrated to half of its volume.
Formed crystals were filtered off and dried in vacuum drier to
afford 3-chloro-1-oxo-pyridine-4-carboxylic acid (420 mg). LCMS: MW
(calcd): 172.99; MS (ES.sup.+, m/z): 174.33 (M+H).sup.+.
1.3.2 Step 2: Synthesis of
3-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-1-o-
xo-pyridine-4-carboxamide
##STR00022##
[0228] To a solution of 3-chloro-1-oxo-pyridine-4-carboxylic acid
(52 mg, 0.30 mmol) in DMF (1 mL) at 0.degree. C. was added EDCxHCl
(74.8 mg, 0.39 mmol) and DIPEA (161 .mu.L, 0.87 mmol) and left
stirring for 30 minutes. Then HOBtxH.sub.2O (55.1 mg, 0.36 mmol)
and 1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
(69.4 mg, 0.30 mmol) were added. The reaction was left stirred
overnight at room temperature. Next day reaction mixture was
evaporated to dryness. Oily residue was purified by silicagel
column chromatography (weak eluent: DCM, strong eluent:
DCM:MeOH:NH.sub.4OH=90:5:0.1) to afford
3-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-1-o-
xo-pyridine-4-carboxamide (87 mg). LCMS: MW (calcd): 386.13; MS
(ES.sup.+, m/z): 387.49 (M+H).sup.+.
1.3.3 Step 3: Synthesis of
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide
##STR00023##
[0230] To a solution of
3-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-1-o-
xo-pyridine-4-carboxamide (87 mg, 0.23 mmol) and tert-butylamine
(0.160 mL, 1.53 mmol) in a mixture of chloroform (10 mL) and
benzotrifluoride (1 mL) at 0.degree. C. was added p-toluenesulfonic
anhydride (260 mg, 0.57 mmol) in portions while maintaining
temperature at 5.degree. C. Reaction was completed after 10 min.
Solution of trifluoroacetic acid (5 mL) and reaction mixture was
stirred for 3 h at 80.degree. C. Solvents were reduced under vacuum
and the residue was diluted with dichloromethane and quenched with
40% NaOH to pH 9-10. The aqueous layer was extracted with
dichloromethane (4.times.15 mL). The combined organics were dried
over Na.sub.2SO.sub.4, concentrated and purified by silicagel
column chromatography (weak eluent: DCM, strong eluent:
DCM:MeOH:NH.sub.4OH=90:9:0.5) to give
2-amino-5-chloro-N-[1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperi-
dyl]pyridine-4-carboxamide (9.05 mg). LCMS: MW (calcd): 385.14; MS
(ES.sup.+, m/z): 386.54 (M+H).sup.+. .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 11.52 (br. s., 1H), 8.52 (d, J=7.63 Hz, 1H), 8.20 (s, 1H),
7.91 (d, J=4.88 Hz, 1H), 7.06 (s, 1H), 6.52 (d, J=4.88 Hz, 1H),
6.42 (s, 2H), 3.94 (m, 3H), 3.08 (t, J=11.44 Hz, 2H), 2.34 (s, 3H),
1.95 (d, J=9.77 Hz, 2H), 1.57-1.71 (m, 2H) ppm.
1.4: Compound 4:
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]pyrid-
ine-4-carboxamide
1.4.1 Step 1: Synthesis of
4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine
##STR00024##
[0232] To a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2.0
g, 13.04 mmol) in DCM (80 mL) was added N-chlorosuccinimide (1.7 g,
13.04 mmol). The reaction mixture was refluxed 3 days. The reaction
mixture was dissolved in water and extracted with EtOAc (3.times.50
mL). Combined organic layers were dried over MgSO4, filtered and
evaporated to afford 4,5-dichloro-7H-pyrrolo[2,3-cl]pyrimidine
(1.08 g). LCMS: MW (calcd): 186.97; MS (ES.sup.+, m/z): 187.98
(M+H).sup.+.
1.4.2 Step 2: Synthesis of Tert-butyl
N-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)carbamate
##STR00025##
[0234] Tert-butyl N-(4-piperidyl)carbamate (895.3 mg, 4.47 mmol)
and 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1080 mg, 5.36 mmol)
were dissolved in NMP (8 ml) and DIPEA (2.33 ml, 13.4 mmol) was
added dropwise. The reaction mixture was stirred at room
temperature 3 days. The mixture was dissolved in water and
extracted with EtOAc (3.times.15 mL). Combined organic layers were
dried and concentrated to afford the crude product which was
recrystallized with acetonitrile to afford tert-butyl
N-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)carbamate (1.42 g).
LCMS: MW (calcd): 351.15; MS (ES.sup.+, m/z): 352.22
(M+H).sup.+.
1.4.3 Step 3. Synthesis of
1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
##STR00026##
[0236] To a solution of tert-butyl
N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]carbamate
(2.0 g, 5.69 mmol) in DCM (10 mL) was added trifluoracetic acid
(3.3 mL) and the resulting solution was stirred at rt for 2 h. Upon
completion solvent was evaporated and crude product was put on a
previous conditioned (40 mL MeOH) SCX column (20 g). The column was
washed with MeOH (2.times.40 mL) and then with 7 N NH.sub.3/MeOH
(100 mL). Solvent was evaporated to afford
1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine (1.3
g). LCMS: MW (calcd): 251.09; MS (ES.sup.+, m/z): 252.10
(M+H).sup.+.
1.4.4 Step 4. Synthesis of
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-pyri-
dine-4-carboxamide
##STR00027##
[0238] To a solution of 2-aminopyridine-4-carboxylic acid (200 mg,
1.45 mmol) in DMF (4 mL) at 0.degree. C. was added EDCxHCl (362.3
mg, 1.89 mmol) and DIPEA (732 .mu.L, 4.21 mmol) and left stirring
for 30 minutes. Then HOBtxH.sub.2O (266.4 mg, 1.74 mmol) and
1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
(546.23 mg, 2.17 mmol) were added. The reaction was left stirred
overnight at room temperature, diluted with water (10 mL) and
extracted with EtOAc (3.times.10 mL). The combined organic layers
were washed with brine (20 mL), dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo. Crude was purified via Biotage
purification device on 12 g KP-Sil Interchim column: DCM as weak
solvent and DCM:MeOH:NH.sub.4OH=90:1.5:0.15 as strong solvent. The
appropriate fractions have been collected to afford product which
was triturated with acetonitrile to afford
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-pyri-
dine-4-carboxamide (162.1 mg). LCMS: MW (calcd): 371.13; MS
(ES.sup.+, m/z): 372.18 (M+H).sup.+. .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 12.15 (br. s., 1H), 8.39 (d, J=7.32 Hz, 1H), 8.27 (s, 1H),
7.97 (d, J=4.88 Hz, 1H), 7.49 (s, 1H), 6.77-6.87 (m, 2H), 6.09 (br.
s., 2H), 4.21 (d, J=12.21 Hz, 2H), 4.05 (d, J=7.32 Hz, 1H), 3.10
(t, J=12.36 Hz, 2H), 1.91 (d, J=10.68 Hz, 2H), 1.74 (m, 2H)
ppm.
1.5 Compound 5: Synthesis of
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-5-fl-
uoro-pyridine-4-carboxamide
##STR00028##
[0240] To a solution of
2-(benzhydrylideneamino)-5-fluoro-pyridine-4-carboxylic acid (43.0
mg, 0.13 mmol) in DMF (5 mL) at 0.degree. C. was added EDCxHCl
(32.6 mg, 0.17 mmol) and DIPEA (66.1 .mu.L, 0.38 mmol) and left
stirring for 30 minutes. Then HOBtxH.sub.2O (24.5 mg, 0.16 mmol)
and 1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine
(50.3 mg, 0.20 mmol) were added. The reaction was left stirred
overnight at room temperature. Solvent was evaporated to dryness.
Crude was purified via preparative LC-MS. Appropriate fractions
have been collected and lyophilized overnight to afford
2-amino-N-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidyl]-5-fl-
uoro-pyridine-4-carboxamide (17 mg). LCMS: MW (calcd): 389.12; MS
(ES.sup.+, m/z): 390.39 (M+H).sup.+. .sup.1H NMR (600 MHz, DMSO-d6)
.delta. 12.15 (br. s., 1H), 8.47 (d, J=7.70 Hz, 1H), 8.25 (s, 1H),
7.91 (s, 1H), 7.47 (s, 1H), 6.51 (d, J=4.40 Hz, 1H), 6.02 (s, 2H),
4.14 (d, J=12.65 Hz, 2H), 4.02 (m, 1H), 3.13 (t, J=11.83 Hz, 2H),
1.93 (d, J=10.82 Hz, 2H), 1.67 (m, 2H) ppm.
1.6 Compound 6: Synthesis of
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide
1.6.1. Synthesis of
3-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
##STR00029##
[0242] To a stirred solution of
4-chloro-1H-pyrrolo(3,2-b)pyridine-5-carbonitrile (3 g, 16.89 mmol)
in DMF (20 mL) was added N-bromosuccinimide (3.3 g, 18.582 mmol).
The resulting solution was stirred at room temperature for 20 min.
The reaction mixture was diluted with water (50 mL) and extracted
with ethyl acetate (2.times.25 mL). The combined organic layers
were washed with brine, dried over magnesium sulfate and
concentrated in vacuo. Resulting powder was triturated with EtOAc
and dried to afford
3-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (1.5 g).
LCMS: MW (calcd): 256.49; MS (ES.sup.+, m/z): 257.93
(M+H).sup.+.
1.6.2. Synthesis of
4-chloro-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
##STR00030##
[0244] To a solution of
3-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (1.5 g,
5.85 mmol) in THF (48 mL) at -78.degree. C. under argon was added
butyllithium solution (5.2 mL, 12.87 mmol) and the reaction was
stirred for 20 min. Iodomethane (590 .mu.L, 9.36 mmol) was then
added dropwise and the reaction allowed to warm to room temperature
in the cold bath. The reaction was quenched with water (30 mL) and
pH was adjusted to 7. THF was evaporated and water was added. The
obtained solid was filtered, washed with water and triturated with
EtOAc to afford
4-chloro-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (813
mg). LCMS: MW (calcd): 191.6; MS (ES.sup.+, m/z): 192.05
(M+H).sup.+.
1.6.3. Synthesis of Tert-butyl
N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbama-
te
##STR00031##
[0246] A mixture of
4-chloro-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (813 mg,
4.24 mmol), 4-boc-aminopiperidine (1.27 g, 6.36 mmol) and DIPEA
(1.85 mL, 10.61 mmol) in NMP (5 mL) was stirred at 150.degree. C.
for 3 h and stirring was continued at RT over the weekend. The
reaction mixture was diluted with water (110 mL) and diethyl ether.
The obtained solid was filtered, washed with water and diethyl
ether and dried to afford tert-butyl
N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbama-
te (1.28 g). LCMS: MW (calcd): 355.43; MS (ES.sup.+, m/z): 356.30
(M+H).sup.+.
1.6.4. Synthesis of
4-(4-amino-1-piperidyl)-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
##STR00032##
[0248] To a solution of tert-butyl
N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbama-
te (1.28 g, 3.6 mmol) in DCM (40 mL) was added trifluoroacetic acid
(5.5 mL, 72.03 mmol). The resulting solution was stirred at room
temperature for 1 h. The reaction mixture was loaded onto an SCX
column (20 g, preconditioned with 100 mL of MeOH). MeOH (200 mL)
was passed through the column and the compound was eluted with 7N
NH.sub.3 in MeOH:MeOH=1:4 (300 mL). The filtrate was concentrated
in vacuo to afford
4-(4-amino-1-piperidyl)-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
(1.06 g). LCMS: MW (calcd): 255.32; MS (ES.sup.+, m/z): 256.21
(M+H).sup.+.
1.6.5. Synthesis of
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide
##STR00033##
[0250] To a solution of 2-amino-5-fluoro-pyridine-4-carboxylic acid
(66.0 mg, 0.30 mmol) in DMF (1 mL) at 0.degree. C. was added
EDCxHCl (50.0 mg, 0.26 mmol) and DIPEA (101 .mu.L, 0.58 mmol) and
left stirring for 30 minutes. Then HOBtxH.sub.2O (37.0 mg, 0.24
mmol) and
4-(4-amino-1-piperidyl)-3-methyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
(108.0 mg, 0.30 mmol) were added. The reaction was left stirred
overnight at room temperature. Solvent was evaporated to dryness.
Crude was purified via Biotage purification device on 4 g KP-Sil
Interchim column: DCM as weak solvent and DCM:MeOH=10:1 as strong
solvent. The appropriate fractions have been collected to afford
2-amino-N-[1-(5-cyano-3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl-
]-5-fluoro-pyridine-4-carboxamide (1.27 mg). LCMS: MW (calcd):
393.42; MS (ES.sup.+, m/z): 394.53 (M+H).sup.+. .sup.1H NMR (500
MHz, DMSO-d6) .delta. 11.82 (br. s., 1H), 8.55 (d, J=7.32 Hz, 1H),
8.23 (s, 1H), 7.93 (s, 1H), 7.24 (s, 1H), 6.54 (d, J=4.27 Hz, 1H),
6.04 (br. s., 2H), 3.97 (br. s., 1H), 3.62 (d, J=12.21 Hz, 2H),
3.40 (m, 2H), 2.41 (s, 3H), 1.97 (d, J=11.29 Hz, 2H), 1.72-1.83 (m,
2H) ppm.
Compound 7: Synthesis of
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide
1.7.1. Synthesis of Tert-butyl
N-[1-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate
##STR00034##
[0252] A mixture of 4-chloro-1H-pyrrolo[2,3-b]pyridine (4.0 g,
26.20 mmol), tert-butyl N-(4-piperidyl)carbamate (7.8 g, 39.30
mmol) and DIPEA (11.4 mL, 65.50 mmol) in NMP (28 mL) was stirred at
150.degree. C. for 72 h. The reaction mixture was diluted with
water and extracted with EtOAc (4.times.). Combined organic layers
were dried and concentrated to afford the crude product which was
recrystallized with acetonitrile to afford tert-butyl
N-[1-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate (1.88
g). LCMS: MW (calcd): 316.41; MS (ES.sup.+, m/z): 317.22
(M+H).sup.+.
1.7.2. Synthesis of Tert-butyl
N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate
##STR00035##
[0254] tert-butyl
N-[1-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate (316 mg,
1 mmol) was dissolved in dry DCM (10 ml) and N-chlorosuccinimide
(147 mg, 1.1 mmol) was added. The reaction mixture was left
stirring at 40.degree. C. After 16 h to the reaction mixture was
added water and extracted with DCM (3.times.). Combined organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered thorough
phase separator and evaporated to obtain the crude compound which
was purified via Biotage purification device on 4 g KP-Sil
Interchim column with flowrate 10 ml/min, DCM as solvent A and
DCM:MeOH=7:3 as solvent B. Method: 0-5% B 4 CV; 20% 4 CV; 20-40% B
5 CV; 40% B 5 CV; 40-60% B 4 CV; 60% B 4 CV. Appropriate fractions
were gathered to afford tert-butyl
N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate
(150 mg). LCMS: MW (calcd): 350.85; MS (ES.sup.+, m/z): 351.43
(M+H).sup.+.
1.7.3. Synthesis of
1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)piperidin-4-amine
##STR00036##
[0256] To a mixture of tert-butyl
N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]carbamate
(320 mg, 1.8 mmol) in DCM (10 mL) at 0.degree. C. TFA (1.5 mL) was
added. Reaction mixture was stirred at r.t. for 1 h. After
completion solvent was evaporated and crude product was put on a
previous conditioned (with MeOH) SCX column (5 g). The column was
washed with MeOH (2.times.5 ml) and then with 7 N NH3/MeOH (10 ml)
to afford
1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)piperidin-4-amine
[0257] (108 mg). LCMS: MW (calcd): 250.73; MS (ES.sup.+, m/z):
251.38 (M+H).sup.+.
1.7.4. Synthesis of
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide
##STR00037##
[0259] To a solution of 2-amino-5-fluoro-pyridine-4-carboxylic acid
(66.0 mg, 0.43 mmol) in DMF (1 mL) at 0.degree. C. was added
EDCxHCl (70.0 mg, 0.36 mmol) and DIPEA (141 .mu.L, 0.81 mmol) and
left stirring for 30 minutes. Then HOBtxH.sub.2O (52.0 mg, 0.34
mmol) and
1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)piperidin-4-amine (108.0
mg, 0.43 mmol) were added. The reaction was left stirred overnight
at room temperature. Reaction mixture was diluted with water (10
mL) and product was precipitated. Crystals were filtered off and
dried in vacuum drier at 65.degree. C. for 3 h. Filtrate was
extracted with EtOAc (4.times.5 mL). The combined organic layers
were dried over Na.sub.2SO.sub.4, filtered and concentrated in
vacuo to afford the crude. Crude was purified via Biotage
purification device on 4 g KP-Sil Interchim column: DCM as weak
solvent and E1 as strong solvent. The appropriate fractions have
been collected to afford
2-amino-N-[1-(3-chloro-1H-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidyl]-5-fluo-
ro-pyridine-4-carboxamide (7.77 mg). LCMS: MW (calcd): 388.8; MS
(ES.sup.+, m/z): 389.47 (M+H).sup.+. .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 11.81 (br. s., 1H), 8.51 (d, J=7.63 Hz, 1H), 8.06 (d,
J=5.19 Hz, 1H), 7.92 (s, 1H), 7.45 (s, 1H), 6.47-6.64 (m, 2H), 6.03
(s, 2H), 3.94 (m, 1H), 3.50-3.64 (m, 2H), 2.87 (t, J=11.44 Hz, 2H),
1.96 (d, J=10.38 Hz, 2H), 1.72-1.87 (m, 2H) ppm.
Biological Examples
2. In Vitro Assays
2.1 ASK1/2 Biochemical Assays (IMAP Technology)
[0260] IMAP.RTM. technology provides a homogeneous assay applicable
to a wide variety of kinases, phosphatases, and phosphodiesterases
without regard for substrate peptide sequence. The assay is a
simple mix-and-read procedure allowing accurate determination of
enzyme activity. Based on the specific, high-affinity interaction
of phospho groups with trivalent metal-containing nanoparticles
(beads), IMAP is a generic, non-antibody-based platform to assess
kinase, phosphatase, and phosphodiesterase activity. An enzyme
reaction is performed using fluorescently labeled substrate.
Addition of the IMAP Binding System stops the enzyme reaction and
initiates binding of the beads to phosphorylated substrates.
Binding of the substrate to the beads, which correlates to enzyme
activity, can be detected using either FP or TR-FRET as a
readout.
[0261] Apoptosis Signal-regulating Kinase (ASK1), also known as
MAP3K5, is a member of the mitogen-activated protein kinase kinase
kinase (MAP3K) family of kinases. It activates the Jnk and p38
pathways through the MAP2Ks MKK4/7 and MKK3/6 (respectively). This
ASK1 IMAP assay uses the following IMAP peptide: RP7140-T2
(5TAMRA-GTFRAAIRRLAARRR-OH, SEQ ID NO: 1). The assay has been
configured to run at Km for ATP (150 mM) so that it will be
sensitive to ATP-competitive inhibitors. The final assay conditions
are 150 mM ATP, 1 mM DTT, 200 nM peptide and 6 nM ASK1 (total
protein). The binding reagent is made up in 100% A at a final
dilution of 1:200.
Solutions:
[0262] KIT IMAP FP, Cat. No. R8127; Reaction Buffer-Tween (5.times.
conc.), Molecular Devices, Cat. No. R7436; Diluted 1:5 with
H.sub.2O and containing DTT (1 mM from 1M stock); Progressive
Binding Buffer (PBB) A (5.times. conc.), Molecular Devices, Cat.
No. R7282; Progressive Binding Reagent (PB Reagent), Molecular
Devices, Cat. No. R7284; Enzyme Solution (containing 2.times.Ask1
in Reaction buffer); Substrate Solution (containing 2.times.
peptide substrate in Reaction buffer, and 2.times.150 .mu.M ATP in
Reaction buffer); Bead Buffer (Progressive Binding Buffer A diluted
1:5 with H.sub.2O); Bead Solution (Bead Buffer containing 1:200
Progressive Binding Reagent (2.times.))
Experimental
[0263] 100 nL of test compound in 100% DMSO was added to 384-well
plates. For a dose response experiment a 1 in 4 dilution of
compound (25 .mu.M top final concentration) was used. 3 .mu.L of
hASK1 (6 nM), hASK2 (25 nM) or rASK1 (15 nM) was added to each well
except in the control well to which 3 .mu.L buffer was added. 3
.mu.L of substrate solution was added to each well and the plates
were incubated for 4 h at room temperature. After which time 6
.mu.L of IMAP.TM. Bead Solution (Progressive Binding Buffer A+PB
Reagent; final dilution 1:100) was added to each well and spun for
1 minute at 1000 rpm. The plates were then incubated for 2 h at
room temperature after which time the plates were read on a Perkin
Elmer EnVision plate reader.
Data Analysis
[0264] Calculation of IC.sub.50 data, curves and QC analysis was
made by using Excel tools and GraphPadPrism software, v. 5.03.
Briefly, individual concentration-effect curves are generated by
plotting the logarithm of the tested concentration of tested
compounds (X) vs. corresponding percent inhibition values (Y) using
least squares (ordinary) fit. Up to 1/4 points from high/low
controls are allowed to be excluded, but only if values are
>Avg..+-.1*SD. Fitting equation used for IC.sub.50
calculation=log(inhibitor) vs. response-Variable slope (four
parameters). Calculated results: Calc 1: mP value for FP
measurement=1000*(S-G*P)/(S+G*P) where S=<detector 2 or
FP-BodipyTAMRA_Ex531-Em579-optimized(1) channel 2>P=<detector
1 or FP-BodipyTAMRA_Ex531-Em579-optimized(1) channel
1>G=G-factor. Gain=100. Top constrain is used only if top values
are not correctly calculated.
[0265] Minimum constrained if less than -30% I or greater than 30%
I. Maximum constrained if less than 70% I or greater than 130% I.
Z'-Factor Target .gtoreq.0.4. Hill Slope range 0.5 to 5.
[0266] Illustrative compounds of the invention were tested
according to the method of Example 2.1.
[0267] The results are shown below:
TABLE-US-00004 Compound Inhibition (pIC.sub.50) number ASK1/ASK2 1
8.24/7.96 2 8.17/8.08 3 8.05/- 4 8.46/8.19 5 8.43/- 6 8.98/- 7
8.24/-
2.2 ASK1/2 Biochemical Assays (AlphaScreen Technology)
AlphaScreen Technology Description
[0268] ASK1 and ASK2 biochemical activities were also quantified
using AlphaScreen technology which measures the degree of
phosphorylation of a protein substrate (MKK7). AlphaScreen
technology is based on the binding of a substrate to two types of
beads, acceptor and donor. Binding to one bead is through the tag
of the substrate protein. Binding of the second bead is through
phosphospecific binding of antibody to the phosphosite of the
substrate. This forms a sandwich, with the acceptor and donor beads
in close proximity. When the donor beads are excited by light in
the 680 nm range, a singlet oxygen is released and causes emission
of light from the acceptor in the 620 nm range which can be
detected using a suitable plate reader.
ASK1/2 AlphaScreen Assay Description
[0269] The ASK1/2 AlphaScreen assays were enabled by binding of
full length inactive MKK7 protein to glutathione donor beads
through the use of GST-tag. The phosphorylation site on MKK7
(Ser271/Thr275) is then recognised by a phosphospecific antibody.
The phosphospecific antibody is bound to the AlphaScreen acceptor
beads through a Protein-A interaction. Phosphorylation of MKK7 by
ASK1 or ASK2 subsequently facilitates the bringing together of the
donor and acceptor beads into close proximity whereupon the
transfer of the singlet oxygen leads to the generation of the
AlphaScreen signal.
ASK1/2 AlphaScreen Reagents, Conditions and Protocol
[0270] Full length human ASK1 protein with an N-terminal 6His-Avi
tag. [0271] Heterodimer of full length ASK1 protein with an
N-terminal 6His-Avi tag (inactive enzyme where lysine 709 is
replaced with methionine) and full length ASK2 protein with
6His-FLAG tag. [0272] Human MKK7 inactive (Carna Bioscience, Cat.
No. 07-147-10). [0273] Phospho-MKK7 (Ser171/Thr275) antibody (Cell
Signalling Technologies Cat. No. 4171). [0274] Protein A acceptor
beads (Perkin Elmer, Cat No. 6760137). [0275] Glutathione donor
beads (Perkin Elmer, Cat. No. 6765301). [0276] Adenosine
triphosphate, ATP (Promega, Cat No. V915B). [0277] Reaction buffer:
50 mM Hepes, 150 mM NaCl, 10 mM MgCl2, 1 mM CHAPS, pH 7.2. [0278]
Stop buffer: 50 mM Hepes, 150 mM NaCl, 60 mM EDTA, 1 mM CHAPS, pH
7.2.
[0279] The assay was configured to run using the following
conditions (final concentrations): 150 .mu.M ATP; 400 nM MKK7; 0.8
nM ASK1 and 2 nM ASK2.
Experimental
[0280] 100 nL of test compound (starting concentration 6 .mu.M, 11
concentrations with 3 fold serial dilutions) in 100% DMSO was added
to low volume 384 well plates. 2.5 .mu.l of ASK1 or ASK2 was added
to each well except in the control wells to which 2.5 .mu.l buffer
was added. 2.5 .mu.l of MKK7 solution was added to all wells. The
plate was incubated for 60 minutes at room temperature. Protein-A
acceptor beads and phospho-specific antibody were incubated for 30
minutes and then 2.5 .mu.l of Protein-A acceptor
beads/phospho-specific antibody mix (2.5 .mu.g/ml acceptor beads
final concentration and 1/800 antibody final dilution) was added to
each well. The plate was incubated for 30 minutes at room
temperature and then 2.5 .mu.l of GSH donor beads (10 .mu.g/ml
acceptor beads final concentration) was added to each well. The
plate was incubated for a further 60 minutes at room temperature,
following which the plate was read on a Perkin Elmer Enspire plate
reader.
Data Analysis
[0281] Calculation of IC.sub.50 data, curves and QC analysis were
made by using Excel tools and GraphPadPrism software, v. 5.03.
Briefly, individual concentration-effect curves were generated by
plotting the logarithm of the tested concentration of tested
compounds (X) vs. corresponding percent inhibition values (Y) using
least squares (ordinary) fit. Fitting equation used for IC.sub.50
calculation=log(inhibitor) vs. response-Variable slope (four
parameters).
[0282] Illustrative compounds of the invention were tested
according to the method of Example 2.2. The results are shown
below:
TABLE-US-00005 Compound Inhibition (pIC.sub.50) number ASK1/ASK2 1
8.72/7.71 2 8.9/- 3 8.7/7.8 4 9.4/8.2 5 8.8/- 6 8.98/- 7 8.24/-
2.3 ASK1/2 Cellular Assay
[0283] ASK1/2 activity in vitro was assayed by determining the
quantity of phospho-MKK3 protein in H.sub.2O.sub.2-stimulated
PBMCs, using western blot method. MKK3 was shown to be a direct
substrate of ASK1/2 catalyzed phosphorylation in the p38 activation
pathway.
2.3.1 Materials and Assay Conditions
[0284] Lymphoprep (Axis-Shield, Cat. No. 1114545) [0285] Ammonium
chloride lysate buffer pH 7.4 (10.times. concentrated): [0286]
NH.sub.4Cl--final conc. 1.5 M (Kemika, Cat. No. 0137407) [0287]
NaHCO.sub.3-- final conc. 100 mM (Kemika, Cat. No. 1411007) [0288]
Na.sub.2EDTA--final conc. 10 mM (Sigma Aldrich; Cat. No. E-4884)
[0289] RPMI 1640 (Lonza Cat. No. BE12-115F/U1) [0290] FBS (Sigma
Cat. No. F7524, heat inactivated 30'/56.degree. C.) [0291] Dimethyl
sulfoxide (DMSO) (Sigma, Cat. No. D2650) [0292] Cell lysis buffer
(pH 7.4): PBS+1% Triton X-100 (Sigma Aldrich; Cat. No. X100)--10 ml
[0293] Phospho-Stop--1 tablet (Sigma, Cat. No. 4693124001) Protease
Inhibitor--1 tablet (Sigma, Cat. No. 4906837001) [0294] Hydrogen
peroxide solution 3% w/w (Sigma Aldrich, Cat. No. H-6520) [0295]
PBS (Sigma Aldrich, Cat. No. P4417-100TAB) [0296] Microplate, 96
well, pp, v-bottom, clear (Greiner bio-one; Cat. No. 651201) [0297]
Tissue culture plate, 6 well, flat bottom with low evaporation lid
(Falcon, Cat. No. 353224) [0298] 50 mL falcon tube (TPP, Cat. No.
91050) [0299] Pierce BCA Protein Assay Kit (Thermo Scientific, Cat.
No. 23225) [0300] Immulon.RTM. Microtiter.TM. 96-Well Plate 1B
(Thermo Scientific, Cat. No. 3355) [0301] Wes 12-230 kDa Master Kit
(Protein Simple, Cat. No. PS-MK14) [0302] MKK3 (D4C3) Rabbit mAb
(Cell Signaling, Cat. No. 8535) [0303] Phospho-MKK3 (Ser189)/MKK6
(Ser207) (D8E9) Rabbit mAb (Cell Signaling, Cat. No. 12280)
[0304] The assay was configured to run using the following
conditions: Tested compounds concentration range: 1-0.0014 .mu.M (7
three-fold serial dilutions); H.sub.2O.sub.2 concentration: 3
mM
2.3.2 Experimental
[0305] To isolate the PBMCs from buffy coat, the buffy coat was
diluted 1:1 in PBS, then the dilute buffy-coat (25 mL) was
carefully pipetted on top of 20 mL of Lymphoprep in a falcon tube.
The tubes were then spun for 35 min at 400.times.g. The upper
plasma layer is then carefully removed leaving the layer containing
the MNCs. The layer containing the MNCs is then transferred to a
new tube, PBS is added up to a total volume of 50 mL and the tube
is then spun for 10 minutes (200.times.g, 25.degree. C.). The
resulting pellet is then resuspended in 2 mL PBS, the washing step
was repeated. The final pellets were resuspended in ammonium
chloride lysate buffer up to 50 mL, mixed gently and centrifuged
for 10 minutes (200.times.g, 25.degree. C.), resuspended in 2-3 mL
of cell medium and diluted up to 50 mL in cell medium. The cell
density of a 1/20 dilution in cell medium was determined using an
automated cell counter.
[0306] 10.times.10.sup.6 PBMCs were seeded per well in 1 mL of RPMI
1640 cell medium supplemented with 10% FBS in 6-well plates. Seven
sets of three-fold serial dilutions of compounds in DMSO starting
from 1 mM in 96-well v-bottom plate were prepared. The DMSO
compound solutions were diluted 100.times. in the cell medium. 200
.mu.L of 100.times. diluted compound solution were added per well,
control wells contained 200 .mu.L of 1% DMSO prepared in cell
medium. 7.5 mM H.sub.2O.sub.2 in cell medium was prepared and 800
.mu.L was added to each well, except the negative control well to
which 800 .mu.L of cell medium was added. The cells were incubated
for 30 min at 37.degree. C., 5% CO.sub.2, 95% humidity. The cells
were transferred to 2 mL Eppendorf tubes and centrifuged for 10
minutes at 300.times.g at 4.degree. C., they were then washed with
1 mL PBS, the cell pellet was resuspended in 100 .mu.L of lysis
buffer and incubated on ice for 30 minutes. After which time the
cell lysates were centrifuged for 5 minutes at 10000.times.g at
4.degree. C. and the supernatants were then stored at -20.degree.
C.
[0307] Samples were diluted 5.times. in cell lysis buffer and total
protein concentration was determined in 96-well Immulon 1B plate
using BCA Protein Assay Kit following manufacturer's
instructions.
[0308] Western blot assay was performed using Protein Simple Wes
Master Kit and device, following manufacturer's instructions. 0.5
mg/mL of total proteins was loaded per sample; phospho-MKK3 and
MKK3 antibodies were diluted 300.times..
[0309] Western blot results were analysed using Compass 2.7.1
software and phospho-MKK3 quantity was divided by MKK3 quantity for
each sample. Percentages of inhibition were calculated by
normalizing the data to controls using the following equation:
[(Sample-Low control)/(High control-low control)]*100
[0310] IC.sub.50 values of compounds were determined by plotting
percentages of inhibition and logarithm of compound concentrations
using GraphPad Prism software, non-linear regression (curve fit),
log (inhibitor) vs. response--Variable slope (four parameters).
[0311] Illustrative compounds of the invention were tested
according to the method of Example 2.3. The results are shown
below:
TABLE-US-00006 Compound Inhibition number (pIC.sub.50) 1 8.05 2 8.5
3 7.4 4 8.1 5 8.2 6 7.9 7 7.9
2.4. ASK1/2 Human Whole Blood Assay
[0312] ASK1/2 activity in vitro was assayed by determining the
quantity of CCL2 cytokine produced in human whole blood stimulated
with LPS using ELISA. It was described in the literature that CCL2
production was decreased in serum from ASK1 knock-out mice under
nonstimulated and LPS-stimulated conditions.
2.4.1 Materials and assay conditions [0313] RPMI 1640 (Lonza Cat.
No. BE12-115F/U1) [0314] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No.
D2650) [0315] Lipopolysaccharide from E. coli (LPS) (Sigma, Cat.
No. L4391) [0316] Microplate, 96 well, pp, v-bottom, clear
(Greiner; Cat. No. 651201) [0317] Master block 96 well, 2 ml
(Greiner, Cat. No. 780271) [0318] Microplate, 96 well, ps,
u-bottom, clear, with lid (Greiner, Cat. No. 650180) [0319] 50 mL
falcon tube (TPP, Cat. No. 91050) [0320] Immulon 2HB 96-well plate
(Thermo Fisher Scientific, Cat. No. 3455) [0321] Sucrose (Kemika,
Cat. No. 1800408) [0322] Streptavidin-HRP (Calbiochem, Cat. No.
OR03L) [0323] Sulphuric acid (Kemika, Cat. No. 1816501) [0324]
anti-hCCL2 antibody (R&D Systems, Cat. No. MAB679), dissolved
in 1 ml of PBS [0325] anti-hCCL2 detection antibody (R&D
Systems, Cat. No. BAF279), dissolved in 1 ml of PBS [0326]
Recombinant hCCL2 (standard, R&D Systems, Cat. No. 366-6C)
[0327] Wash buffer (PBS+0.05% Tween-20) [0328] PBS (Sigma, Cat. No.
P4417) [0329] Tween-20 (Sigma, Cat. No. P2287) [0330] Substrate for
20 ml: 18 ml H.sub.2O+2 ml 1M sodium acetate+200 .mu.l TMB mix+2.5
.mu.l 30% H.sub.2O.sub.2 [0331] Sodium acetate (Kemika, Cat. No.
1441908) [0332] TMB mix (Sigma, Cat. No. T2885) [0333] Hydrogen
peroxide (Merck, Cat. No. 1.08597) [0334] Coating buffer (15 mM
Na.sub.2CO.sub.3 (xH.sub.2O)+35 mM NaHCO.sub.3) [0335] Sodium
carbonate (Sigma, Cat. No. S-2127) [0336] Sodium bicarbonate
(Kemika, Cat. No. 1411007) [0337] Assay buffer (PBS+0.05%
Tween-20+1% BSA) [0338] Bovine Serum Albumin (BSA) (Sigma, Cat. No.
A2153)
2.4.2 Experimental
[0339] Whole blood was collected on citrate anticoagulant. 100
.mu.L was used for determination of cell count using automated cell
counter.
[0340] 300 .mu.L of whole blood was added to a 2 mL master block
96-well plate. Six three-fold serial dilutions of compounds in DMSO
were prepared starting from 10 mM in 96-well v-bottom plate. 0.6
.mu.L of prepared compounds or 0.6 .mu.L of DMSO (positive and
negative control wells) from v-bottom plate were added to the
master block plate with blood. 2 ng/mL LPS in culture medium
without serum were prepared and 300 .mu.L was added per well to the
master block plate with blood for the test wells. 300 .mu.L of
culture medium was added to the negative control wells. The plate
was incubated with whole blood overnight in CO.sub.2 incubator
(37.degree. C., 5% CO.sub.2, 95% humidity). The plate was then
centrifuged for 7 minutes at 1500.times.g and the supernatants were
transferred to a 96-well u-bottom plate for determination of CCL2
cytokine or the supernatants were stored at -20.degree. C. for
future testing.
[0341] The day before performing ELISA, the Immulon 2HB plate was
coated with 100 .mu.L per well of 250.times. diluted anti-hCCL2
antibody in coating buffer. The plate was then incubated at
4.degree. C. overnight. The plate was washed three times with 300
.mu.L per well of wash buffer and this was repeated after each
step. 200 .mu.L of blocking (assay buffer+5% sucrose) was added to
each well and incubated for 60 minutes at 37.degree. C. Seven sets
of two-fold serial dilutions of hCCL2 standard in assay buffer were
prepared starting from 2000 pg/mL, in duplicates. Blank wells were
prepared containing only assay buffer. The test samples
(supernatants) were added diluted 10 times in assay buffer to
plate. All final volumes in plate were 100 .mu.L. The plate was
incubated for 60 minutes at 37.degree. C. 100 .mu.L per well of
500.times. diluted anti-hCCL2 detection antibody in assay buffer
was added and the plate was incubated for 45 minutes at 37.degree.
C. 100 .mu.L per well of 50 ng/mL streptavidin-HRP in assay buffer
was added and the plate was incubated for 30 minutes at 37.degree.
C. 100 .mu.L per well of the prepared substrate solution was added
and the plate was incubated at room temperature, protected from
light until blue colour developed in the wells. To stop the colour
development 100 .mu.L per well of 1M sulphuric acid was added. The
plate absorbance was read at 450 nm on a plate reader.
2.4.3 Data Analysis
[0342] ELISA results (absorbance at 450 nm) were analysed using
Microsoft Excel software. Average of blank was subtracted from all
values. Standard curve was plotted with serial dilutions of
standards (pg/mL) on the x-axis versus the corresponding absorbance
values on the y-axis. Amount of cytokine in samples (pg/mL) was
calculated from standard curve. Percentages of inhibition were
calculated by normalizing the data to controls using following
equation:
[(Sample-Low control)/(High control-low control)]*100
[0343] IC.sub.50 values of compounds were determined by plotting
percentages of inhibition and logarithm of compound concentrations
using GraphPad Prism software, non-linear regression (curve fit),
log (inhibitor) vs. response--Variable slope (four parameters).
3. In Vivo Assays
3.1 CFA (Complete Freunds Adjuvant) Induced Hypersensitivity in Rat
Assessed Using Weight Bearing Method
[0344] Intraplantar injection of Complete Freunds adjuvant (CFA)
causes an inflammatory reaction which induces hypersensitivity and
oedema, and mimics some aspects of clinical inflammatory pain.
These effects can be investigated using equipment to measure weight
bearing and plethysmometer.
3.1.1 Weight Bearing
[0345] Naive rats distribute their body weight equally between the
two hind paws. However, when the injected (left) hind paw is
inflamed and/or painful, the weight is re-distributed so that less
weight is put on the affected paw (decrease in weight bearing on
injured paw). Weight bearing through each hind limb was measured
using a rat incapacitance tester (Linton Instruments, UK).
[0346] Rats were placed in the incapacitance tester with the hind
paws on separate sensors and the average force exerted by both hind
limbs was recorded over 4 seconds.
[0347] Base line weight bearing and paw volume readings were taken
and hypersensitivity was induced via injection of CFA (Baseline
weight bearing and paw volume recordings were taken prior to
induction of insult. Inflammatory hypersensitivity was induced by
intraplantar injection of CFA (100 .mu.L of 1 mg/mL solution)
intraplantar to the rats left hind paw.
[0348] Animals (Male, Sprague Dawley Rats (Charles River, UK),
212-260 g) were ranked and randomised to treatment groups according
to the weight bearing CFA window in a Latin square design. Animals
were treated with either Vehicle, Compound 1 10 mg/kg or
Indomethacin 10 mg/kg (10 L1/kg dose volume) 24 hours post CFA.
Weight bearing was measured at 1, 2 and 4 hours post treatment.
Weightbearing (g) readings were taken for both right and left hind
paws and the difference calculated. Data are expressed as %
reversal of the hypersensitivity to pain (mean.+-.s.e.m.). Paw
Volume (mLl) readings were taken for the left hind paws. Data were
expressed as % reversal of the oedema (mean.+-.s.e.m.). The
statistical analysis: was performed with repeated measures ANOVA
followed by Planned comparison test using InVivoStat
(invivostat.co.uk, Clark et al., 2012), p<0.05 considered
significant.
[0349] Compound 1 and Indomethacin (10 mg/kg) significantly
inhibited the hypersensitivity response at all time points tested
post administration.
3.2 MIA Induced Hyperalgesia in the Rat--In Vivo Chronic Pain
Model
[0350] Intra-articular administration of Monosodium Iodoacetate
(MIA) in the ipsilateral knee of Sprague Dawley rats leads to
development of a robust and long-lasting hyperalgesia and allodynia
associated initially with an inflammatory response. The development
of these signs in this animal model are believed to be clinically
relevant; reflecting the symptoms displayed by patients presenting
with chronic inflammatory pain associated with underlying
conditions such as osteoarthritis (OA) or rheumatoid arthritis
(Bove S E et al., Osteoarthritis Cartilage 2003; 11 (11): 821-30;
Fernihough J, et al., Pain 2004; 112 (1-2): 83-93; Kalbhen D A. J
Rheumatol 1987; 14 Spec No: 130-1).
Weight Bearing
[0351] Naive rats distribute their body weight equally between the
two hind paws. However, when the injected (left) hind knee is
inflamed and/or painful, the weight is re-distributed so that less
weight is put on the affected limb (decrease in weight bearing on
injured limb). Weight bearing through each hind limb is measured
using a rat incapacitance tester (Linton Instruments, UK).
[0352] Osteoarthritis (OA) in rats ((Sprague Dawley, male, groups
of 10) was induced via injection of MIA solution (Sigma, 12512), 25
.mu.L of 80 mg/mL, (2 mg) into the knee joint of the left hind leg.
Weight bearing was assessed on Days 3, 5, 7, 9, 12 & 16,
following injection of MIA, for development of chronic pain. At Day
3 weight bearing measurements were taken and animals were ranked
and randomised to treatment groups according to their MIA window in
a Latin square design.
[0353] Animals were treated with Compound 1 10 mg/kg in 0.5%
Methylcellulose or Vehicle (0.5% Methylcellulose) 10 mL/kg p.o. on
day 3 and then daily up to day 16. Weight bearing measurements were
taken 1, 2 and 4 hours post dosing on day 3 and 2 hours post dosing
on days 5, 7, 9, 12 & 16.
[0354] Weight bearing (g) readings were taken for both right and
left hind paws and the difference calculated. Data are expressed as
% ratio ipsilateral/contralateral ((WB left/WB right)*100)
(mean.+-.s.e.m.)
[0355] Calculation: Ipsilateral reading/contralateral
reading.times.100. Naive WB difference-pre dose WB difference was
defined as the MIA window.
[0356] Statistical analysis: Repeated measures ANOVA followed by
Planned comparison test using InVivoStat (invivostat.co.uk),
(p<0.05 considered significant). Data were analysed by comparing
treatment groups to vehicle control group at each time point.
[0357] A significant and marked reversal of hypersensitivity was
seen with Compound 1 when dosed at 10 mg/kg from 1 hours post dose,
until day 12 (9 days post dose). Compound 1 given at 10 mg/kg
showed significant reversal when compared to vehicle at each time
point, comparable to the positive control celecoxib.
3.3 CCl.sub.4 Model of Liver Fibrosis--Protocol 1
[0358] The aim of this study was to assess a model of liver
fibrosis by CCl.sub.4 intoxication. The onset of the disease and
the effect of potential positive references are evaluated by
assessing liver fibrosis by exploring histology, biochemistry,
imaging and gene expression at different time-points. Starkel P.,
Animal models for the study of hepatic fibrosis, Best practice
& Research Clinical Gastroenterology 25: 319-333, 2011
3.3.1 Study Groups:
[0359] The study is performed using 5 week old male BalbC J mice
(Janvier Lab).
TABLE-US-00007 Groups Protocol n* Treatment Sacrifice Sham Olive
oil 10 3 weeks IP twice/ week CCl.sub.4 0.6 mL/kg IP 10 3 weeks
Twice/week Sham Olive oil 10 6 weeks IP twice/ week CCl.sub.4 0.6
mL/kg IP 10 6 weeks Twice/week CCl.sub.4 0.6 mL/kg IP 10 Test
compound 6 weeks Twice/ week 30 mg/kg, b.i.d., p.o. CCl.sub.4 0.6
mL/kg IP 10 Positive control 6 weeks + valproic acid Twice/week
(Valproic acid) ad libitum in drinking water 0.4%
3.3.2 Materials:
[0360] CCl.sub.4: Carbon tetrachloride ACROS 99.8% CAS=56-23-5 Lot
A0293900 [0361] Olive oil, SIGMA 01514-500 mL Lot #BCBQ4885V [0362]
colF (ImmunoChemistry Technologies), ref 6346 Lot 13Y39 Exp May
2017 [0363] stock solution to be diluted in 100 .mu.L of DMSO (6.8
mM) and stored at 4.degree. C. [0364] injection of 1:170 dilution
in ppi water to animal 15 min before imaging (100 .mu.L, sinus
i.v.) [0365] CCl.sub.4 was diluted at 1/2 in olive oil and
administered IP twice per week at 0.6 mL/kg.
3.3.2 Samples and Results
[0366] Blood is collected in serum tubes and samples were
centrifuged at 3000 t/min for 5 min and frozen at -20.degree. C.
for AST, ALT, ALP and total bilirubin evaluations. A sample of the
liver right median lobe is also taken and quickly placed in
Eppendorf tubes (2 mL round bottom) with 1 mL of RNAlater (safe
lock tubes) stored at 4.degree. C.
[0367] The liver is harvested and weighed. Ex vivo imaging of colF
binding in liver is performed immediately (Bruker Xtreme). Livers
are placed into formaldehyde (vial 25 mL-60 cc) for histological
evaluation for aSMA and col I IHC quantification.
[0368] The spleens are weighed for future analysis.
[0369] A panel of 9 fibrosis genes are used for gene expression
analysis.
3.4 CCl.sub.4 Model of Liver Fibrosis--Protocol 2
[0370] The aim of this study was to assess a model of liver
fibrosis by CCl.sub.4 intoxication. The onset of the disease and
the effect of potential positive references are evaluated by
assessing liver fibrosis by exploring histology, biochemistry,
imaging and gene expression at different time-points. Starkel P.,
Animal models for the study of hepatic fibrosis, Best practice
& Research Clinical Gastroenterology 25: 319-333, 2011
3.4.1 Study Groups:
[0371] The study is performed using 8 week old male BALB/c mice
(Charles River, Italy).
TABLE-US-00008 Route and Chal- frequency Treat- lenge/ of ment
Group N IP route Vehicle Treatment treatment Schedule 1 10 Negative
-- -- -- -- 2 10 CCl.sub.4 IP MC 0.5% + Vehicle PO D21-D41 (0.6 1
eq HCl, BID mL/kg) 98.9% 2.times. week dist. water 3 10 D0-D38 MC
0.5% Compound 1 PO 5 mg/kg QD 4 10 MC 0.5% Compound 1 PO 15 mg/kg
QD 5 10 MC 0.5% Compound 1 PO 15 mg/kg BID
3.4.2 Materials:
[0372] CCl.sub.4 (liquid) will be added to olive oil at a
concentration of 0.06 mL/mL. The solution will be administered IP
at the dose of 0.6 mL/kg. Application volume will be 10 mL/kg.
[0373] The test compound will be dissolved/suspended in
Methylcellulose (MC 0.5%).
3.4.3 Samples and Results
[0374] For steady state PK sampling blood will be collected in
He-Lithium tubes to generate plasma. All blood samples will be
processed for plasma by centrifugation (5,000 rpm for 10 minutes at
4.degree. C.) within 30 minutes of collection. Samples are then
stored at -80.degree. C. until analysis.
[0375] Final blood samples will be collected into K.sub.2EDTA micro
tubes by cutting the v. jugularis. All blood samples will be
processed for plasma by centrifugation (3500 rpm for 10 minutes at
4.degree. C.) within 30 minutes of collection. Plasma from each
blood sample stored at -80.degree. C. until analysis.
[0376] The liver is harvested and portions of the left lateral lobe
are stored for RNA gene expression analysis, section of OH proline
measurement, and the rest placed in 10% formalin for
histopathological evaluation.
[0377] A panel of 5 fibrosis genes (Col1, Timp1, Pai1, Snail1,
Acta2) are used for gene expression analysis.
3.5 Methionine and Choline Deficient Model of Steatohepatitis and
Fibrosis
[0378] MCD is a new mouse model of steatohepatitis and fibrosis
induced by a diet: Methionine and Choline-Deficient (MCD). (Wehr et
al 2013 J immunol, 190(10):5226-36; Baeck et al., 2014 Hepatology,
59(3):1060-72; Gautheron et al, 2014 EMBO, 6(8):1062-74).
3.5.1 Study Groups and Dose Regimen:
[0379] C57BL/6 mice from (Janvier Labs (France)) are divided into
groups as set out below, the mice are 8 weeks (>20 grs) at the
initiation of the induction phase
TABLE-US-00009 Frequency & Diet/Groups Project n route Vehicle
Aspect Control diet Ref group 10 BID p.o. PEG200/MC (25/75) + 1 eq
HCl MCD diet Ref group 10 BID p.o. PEG200/MC (25/75) + 1 eq HCl MCD
diet Test 10 BID p.o. PEG200/MC Homogenous compound (25/75)
suspension
3.5.2 Materials and Compounds
[0380] MCD diet w/o choline & Methionine Ref EFTD.90262 MCD
mod. batch 8206423, Ssniff, Soest, Germany-- [0381] Methyl
cellulosis 0.5%, VWR, [0382] PEG 400: P3265, Sigma [0383]
Cryomold.RTM. Standard 25.times.20.times.5 mm (Sakura Finetec,
4557) [0384] O.C.T..TM. Compound cryomold (Sakura Finetec, 4583)
[0385] Formalin for sample preservation--4% buffered with MetOH
3.5.3 Compound Preparation:
[0386] Compounds are dissolved/suspended in appropriate vehicle
(see table above), under agitation. After preparation,
solutions/suspensions are kept at room temperature in dark under
constant magnetic stirring. When dosed 10 mL/kg volume is used and
the concentration of the solution is adjusted according to the
weight of the animal
3.5.4 Protocol:
[0387] The induction phase is 3 weeks. No fasting glucose is given.
Mice are given either the MCD control diet or the MCD diet.
[0388] During the treatment phase the mice are maintained on the
MCD control diet or MCD diet as in induction phase. The mice are
randomly assigned to a treatment group according to their body
weight in order to ensure a homogenous reparation. The mice are
dosed once or twice daily with the test compound during the
evaluation phase
3.6 Prophylactic Bleomycin Induced Pulmonary Fibrosis 14-Day Mice
Model
[0389] The aim of the study is to test the efficacy of a test
compound at three different doses in a 14-day model of bleomycin
induced pulmonary fibrosis in mice.
3.6.1 Animals
[0390] This study is carried out on C5.sup.27 BL/6N male mice,
supplied by Charles River, Italy, which are acclimatized for at
least 5 days in an environment maintained at 22.degree. C., at 55%
relative humidity, with 15-20 air changes per hour under light
cycles of 12 h. Mice pelleted food and water are provided ad
libitum.
[0391] At least one day prior to start of experiment, all animals
are allocated randomly into groups as indicated in the table
below.
[0392] All animal related research is conducted in accordance with
2010/63/EU and National legislation regulating the use of
laboratory animals in scientific research and for other purposes
(Official Gazette 55/13).
3.6.2 Study Groups
TABLE-US-00010 [0393] Treatment schedule Groups Purpose n Dose Days
(Frequency) Route Vehicle 1 control 15 -- D0-D14 (BID) NA NA PBS +
Vehicle 2 control 15 -- D0-D14 (BID) PO PEG/MC BLM + Vehicle 3
control 15 50 D0-D14 (BID) PO 0.1% Natrosol BLM + Pirfenidone mg/kg
4 Active 15 1 D0-D14 (BID) PO PEG400/MC BLM + test mg/kg 0.5% 20/80
(v/v) compound 5 Active 15 3 D0-D14 (BID) PO PEG400/MC BLM + test
mg/kg 0.5% 20/80 (v/v) compound 6 Active 15 10 D0-D14 (BID) PO
PEG400/MC BLM + test mg/kg 0.5% 20/80 (v/v) compound 7 Active 10 10
D0-D7 (BID) PO PEG400/MC BLM + test mg/kg 0.5% 20/80 (v/v) compound
satellite for PK
3.6.3 Materials
[0394] The solvent for the test solutions is prepared by adding 0.5
g of hydroxyethylcellulose (Natrosol) into 500 mL Aqua distillate
(0.1%) under continuous stirring without heating for 5 h on a
magnetic stirrer.
[0395] Anesthetic solution is prepared by adding 1 mL of Narketan
(Narketan 10, Vetoquinol, Bern, Switzerland, 03605877535982) and
0.5 mL of Rompun (Rompun, 2%: Bayer, Leverkusen, Germany) into 9 mL
saline. The resulting solution is administered at 10 mL/kg.
[0396] To prepare a solution for intranasal challenge (i.n.)
challenge, 0.8 mg/mL stock solutions of bleomycin (Bleomycin
sulphate, Enzo Life Sciences, Inc., USA; CAS No. 9041-93-4; Cat.
No. BML-AP302-0010) are thawn and diluted in 330 .mu.L of
saline.
[0397] Prior to i.n administration, mice are anesthetized i.p. with
the anesthetic solution described above.
[0398] Fresh pirfenidone formulation is prepared daily in 0.1%
Natrosol formulations to a final concentration of 5 mg/mL. Before
dosing, animals are weighed and the Pirfenidone amount administered
is adjusted accordingly to individual weights corresponding to 10
mL/kg body weight, twice daily p.o., with 7.5 h interval between
two administrations.
[0399] Finally, test compound solutions are prepared by dissolving
the suitable amount of said test compound in PEG 400 (20% of the
final volume) then MC 0.5% (80% of the final volume) to reach final
concentrations of 1 mg/mL, 0.3 mg/mL and 0.1 mg/mL, thus yielding
compound for doses of 10 mg/kg, 3 mg/kg and 1 mg/kg. Prior to
dosing, animals are weighed and the amount administered adjusted
accordingly to individual weights.
[0400] The application volume of the test doses corresponds to 10
mL/kg body weight, and the test compounds are administered p.o.
twice daily, with 7.5 h interval between two administrations.
3.6.4 Study
[0401] Animals are examined clinically twice daily. List of
clinical signs and parameters are indicated in human endpoints
table. Animals are weighed daily starting from DO.
[0402] On day 14, two hours post dosing with pirfenidone or test
compound, mice are sacrificed by anesthetic overdose.
[0403] The lungs are excised and weighed individually. For all
groups: the whole superior right lung lobe is placed into a
Precellys tube containing silica beads and immediately snap frozen
in liquid nitrogen and subjected to gene expression analysis.
[0404] All remaining lungs are placed into marked bottles
containing 10% buffered formalin for further histopathological
evaluation.
3.6.5 Sample Analysis, Data Processing and Statistical
Evaluation
[0405] Body weight data and lung weight data are processed using MS
Excel. Statistical analysis and graphical presentation are
performed using GraphPad Prism software (version 5.04).
[0406] One-way ANOVA or Mann-Whitney test are employed for lung
weights.
[0407] Two-way ANOVA are employed for body weight changes.
[0408] Differences between groups will be considered statistically
significant when p<0.05.
[0409] For histopathological evaluation, whole lungs (except
sampled superior right lung) are embedded in paraffin and stained
with Mallory's trichrome.
[0410] Pulmonary histological changes are assessed using Matsuse
modification of Ashcroft score (Ashcroft et al., 1988; Matsuse et
al., 1999). Statistical analysis and graphical presentation is
performed using GraphPad Prism software (version 5.04).
Mann-Whitney test is employed.
[0411] Differences between groups will be considered statistically
significant when p<0.05.
TABLE-US-00011 Ashcroft Score 1 Normal lungs (no fibrosis) 2
Minimal fibrotic thickening of alveolar or bronchial walls (network
of fine collagen fibrils) 3 Moderate fibrotic thickening of walls
without obvious damage to lung architecture 4 Fibrosis with damage
of pulmonary structure (coarse fibrous bands or small fibrous
masses, intra-alveolar collagen fibrils) 5 Large fibrous area with
svere distortion of lung structure
3.6.6 PK Analysis--Group 7
3.6.6.1 Protocol
[0412] Animals in group 7 (n=10) are included for PK study only and
are not be subjected to clinical sign scoring.
[0413] These animals are induced with the disease at the start of
treatment at day 0 and are sequentially sacrificed on day 7 at 1 h,
3 h, 6 h, 8 h, 24 h after the first administration of test
compound.
[0414] A blood sample (50 .mu.L) is collected from the tail vein
into Li-heparin anticoagulant tubes for each time point and kept on
ice until separation. Within maximum 30 min after collection, blood
samples are centrifuged at 2000 g for 10 min at 4.degree. C. and
the resulting plasma samples are aliquoted into polypropylene tubes
(1.times.25 .mu.L). The samples are stored frozen at -20.degree. C.
until analysis.
[0415] The lung tissue is collected at sacrifice after blood
sampling for each animal, then weighed and placed into
polypropylene tubes prior to freezing. The samples are stored
frozen at -80.degree. C. until analysis.
3.6.6.2 Plasma Concentration and Pharmacokinetic Analysis
[0416] Plasma and lung concentrations are measured via LC-MS/MS.
Samples are prepared for LC-MS/MS analysis via protein
precipitation. The plasma concentrations measured below the lower
limit of quantification (LLOQ) are reported as below the limit of
quantification (BLQ).
[0417] The test compound concentrations in plasma are expressed in
ng/mL.
[0418] Mean plasma concentrations are calculated. For mean
calculation, the concentrations below the LLOQ are set to zero.
Therefore, mean values may be BLQ. Standard deviation (SD),
standard error of the mean (SE) and coefficient of variation (CV,
%) are tabulated when at least three plasma concentration values
are above the LLOQ.
[0419] Non-compartmental analysis on individual plasma
concentrations is performed using Phoenix.TM. WinNonlin.RTM. 6.3
(Pharsight Corporation) to determine at least, the following
pharmacokinetic parameters:
[0420] Maximum plasma concentration, Cmax (.mu.g/mL) with the
corresponding time, tmax (h),
[0421] Area under the plasma concentration versus time curve up to
the last quantifiable concentration AUC.sub.0-t or up to 24 h
AUC.sub.0-24 h (.mu.gh/mL) (if compound is quantifiable up to 24 h
postdose), and/or up to infinity AUC.sub.0-.infin., (.mu.gh/mL) is
calculated according to the linear up/log down trapezoidal rule.
Partial AUC may be calculated if deemed necessary. Concentrations
below the limit of quantification (BLQ) are set to zero. No AUC is
calculated if there are less than three quantifiable time points.
AUC0-.infin. is considered if % AUCextra <20%.
[0422] Apparent terminal elimination half-life, t1/2 (h) is only
reported if three or more time points, excluding tmax is used for
linear regression, and if the adjusted R.sup.2>0.80.
[0423] Normalized AUC and Cmax Dose.
[0424] Mean pharmacokinetic parameters are calculated. Standard
deviation (SD) and coefficient of variation (CV, %) are tabulated
if at least three values are available.
3.7 CIA Model
3.7.1 Materials
[0425] Complete Freund's adjuvant (CFA) and incomplete Freund's
adjuvant (IFA) are purchased from Difco. Bovine collagen type II
(CII), lipopolysaccharide (LPS), and Enbrel are obtained from
Chondrex (L'Isle-d'Abeau, France), Sigma (P4252, L'Isle-d'Abeau,
France), Wyeth (25 mg injectable syringe, France) Acros Organics
(Palo Alto, Calif.), respectively. All other reagents used are of
reagent grade and all solvents are of analytical grade.
3.7.2 Animals
[0426] Dark Agouti rats (male, 7-8 weeks old) are obtained from
Harlan Laboratories (Maison-Alfort, France). Rats are kept on a 12
h light/dark cycle (0700-1900). Temperature is maintained at
22.degree. C., and food and water are provided ad libitum.
3.7.3 Collagen Induced Arthritis (CIA)
[0427] One day before the experiment, CII solution (2 mg/mL) is
prepared with 0.05 M acetic acid and stored at 4.degree. C. Just
before the immunization, equal volumes of adjuvant (IFA) and CII
are mixed by a homogenizer in a pre-cooled glass bottle in an ice
water bath. Extra adjuvant and prolonged homogenization may be
required if an emulsion is not formed. 0.2 mL of the emulsion is
injected intradermally at the base of the tail of each rat on day
1, a second booster intradermal injection (CII solution at 2 mg/mL
in CFA 0.1 mL saline) is performed on day 9. This immunization
method is modified from published methods (Jou et al. 2005; Sims et
al. 2004).
3.7.4 Study Design
[0428] The therapeutic effects of the compounds are tested in the
rat CIA model. Rats are randomly divided into equal groups and each
group contains 10 rats. All rats are immunized on day 1 and boosted
on day 9. Therapeutic dosing lasted from day 16 to day 30. The
negative control group is treated with vehicle and the positive
control group with Enbrel (10 mg/kg, 3.times./week, s.c.). A
compound of interest is typically tested at 4 doses, e.g., 0.3, 1,
3, and 10 mg/kg, p.o.
3.7.5 Clinical Assessment of Arthritis
[0429] Arthritis is scored according to literature-described
methods (Khachigian 2006 Nat Protoc. 2006; 1(5):2512-6; Lin et al.
2007, Br J Pharmacol, 150, pp 862-872; Nishida et al. 2004,
Arthritis and Rheumatism, 50(10), pp 3365-3376). The swelling of
each of the four paws is ranked with the arthritic score as
follows: 0-no symptoms; 1-mild, but definite redness and swelling
of one type of joint such as the ankle or wrist, or apparent
redness and swelling limited to individual digits, regardless of
the number of affected digits; 2-moderate redness and swelling of
two or more types of joints; 3-severe redness and swelling of the
entire paw including digits; 4-maximally inflamed limb with
involvement of multiple joints (maximum cumulative clinical
arthritis score 16 per animal) (Nishida et al. 2004).
[0430] To permit the meta-analysis of multiple studies the clinical
score values may be normalised as follows:
[0431] AUC of clinical score (AUC score): The area under the curve
(AUC) from day 1 to day 14 is calculated for each individual rat.
The AUC of each animal is divided by the average AUC obtained for
the vehicle in the study from which the data on that animal is
obtained and multiplied by 100 (i.e., the AUC is expressed as a
percentage of the average vehicle AUC per study).
[0432] Clinical score increase from day 1 to day 14 (End point
score): The clinical score difference for each animal is divided by
the average clinical score difference obtained for the vehicle in
the study from which the data on that animal is obtained and
multiplied by 100 (i.e., the difference is expressed as a
percentage of the average clinical score difference for the vehicle
per study).
3.7.6 Change in Body Weight (%) after Onset of Arthritis
[0433] Clinically, body weight loss is associated with arthritis
(Rall & Roubenoff 2004 Rheumatology (Oxford); 43(10):1219-23;
Shelton et al. 2005 Pain; 116(1-2):8-16; Walsmith et al. 2004 J
Rheumatol.; 31(1):23-9). Hence, changes in body weight after onset
of arthritis can be used as a non-specific endpoint to evaluate the
effect of therapeutics in the rat model. The change in body weight
(%) after onset of arthritis is calculated as follows:
Mice : Body .times. .times. Weight .times. .times. ( week .times.
.times. 6 ) - Body .times. .times. Weight .times. .times. ( week
.times. .times. 5 ) Body .times. .times. Weight .times. .times. (
week .times. .times. 5 ) * 100 .times. % ##EQU00001## Rats : Body
.times. .times. Weight .times. .times. ( week .times. .times. 4 ) -
Body .times. .times. Weight .times. .times. ( week .times. .times.
3 ) Body .times. .times. Weight .times. .times. ( week .times.
.times. 3 ) * 100 .times. % ##EQU00001.2##
3.7.7 Radiology
[0434] X-ray photos are taken of the hind paws of each individual
animal A random blind identity number is assigned to each of the
photos, and the severity of bone erosion is ranked by two
independent scorers with the radiological Larsen's score system as
follows: 0--normal with intact bony outlines and normal joint
space; 1--slight abnormality with any one or two of the exterior
metatarsal bones showing slight bone erosion; 2--definite early
abnormality with any three to five of the exterior metatarsal bones
showing bone erosion; 3--medium destructive abnormality with all
the exterior metatarsal bones as well as any one or two of the
interior metatarsal bones showing definite bone erosions; 4--severe
destructive abnormality with all the metatarsal bones showing
definite bone erosion and at least one of the inner metatarsal
joints completely eroded leaving some bony joint outlines partly
preserved; 5--mutilating abnormality without bony outlines. This
scoring system is a modification from literature protocols (Bush et
al. 2002, Arthritis and Rheumatism, 46(3), 802-805; Jou et al.
2005, Arthritis Rheum, 52(1), 339-44; Salvemini et al. 2001,
Arthritis Rheum, 44(12), 2909-21; Sims et al. 2004, Arthritis
Rheum, 50(7), 2338-46).
3.7.8 Histology
[0435] After radiological analysis, the hind paws of mice are fixed
in 10% phosphate-buffered formalin (pH 7.4), decalcified with rapid
bone decalcifier for fine histology (EUROBIO, Les Ulis, France) and
embedded in paraffin. To ensure extensive evaluation of the
arthritic joints, at least four serial sections (5 .mu.m thick) are
cut and each series of sections are 100 .mu.m in between. The
sections are stained with hematoxylin and eosin (H&E).
Histologic examinations for synovial inflammation and bone and
cartilage damage are performed double blind. In each paw, four
parameters are assessed using a four-point scale. The parameters
are cell infiltration, pannus severity, cartilage erosion and bone
erosion. Scoring is performed as follows: 1-normal, 2-mild,
3-moderate, 4-marked. The four scores are summed together and
represented as an additional score, namely the `RA total
score`.
3.7.9 Micro-Computed Tomography (.mu.CT) Analysis of Calcaneus
(Heel Bone)
[0436] Bone degradation observed in RA occurs especially at the
cortical bone and can be revealed by .mu.CT analysis (Oste et al.
2007; Sims et al. 2004). After scanning and 3D volume
reconstruction of the calcaneus bone, bone degradation is measured
as the number of discrete objects present per slide, isolated in
silico perpendicular to the longitudinal axis of the bone. The more
the bone is degraded, the more discrete objects are measured. One
thousand slices, evenly distributed along the calcaneus (spaced by
about 10.8 .mu.m), are analyzed.
3.7.10 Steady State PK
[0437] At day 7 or later, blood samples are collected at the
retro-orbital sinus with lithium heparin as anti-coagulant at the
following time points: predose, 1, 3 and 6 h. Whole blood samples
are centrifuged and the resulting plasma samples are stored at
-20.degree. C. pending analysis. Plasma concentrations of each test
compound are determined by an LC-MS/MS method in which the mass
spectrometer is operated in positive electrospray mode.
Pharmacokinetic parameters are calculated using WinNonlin.RTM.
(Pharsight.RTM., United States) and it is assumed that the predose
plasma levels are equal to the 24 h plasma levels.
3.8 MAB Model
[0438] The MAB model allows a rapid assessment of the modulation of
an RA-like inflammatory response by therapeutics Khachigian, 2006.
DBA/J mice are injected i.v. with a cocktail of mAbs directed
against collagen II. One day later, compound treatment is
initiated. Three days later, mice receive an i.p. LPS injection (50
.mu.g/mouse), resulting in a fast onset of inflammation. Compound
treatment is continued until 10 days after the mAb injection.
Inflammation is read by measuring paw swelling and recording the
clinical score of each paw. The cumulative clinical arthritis score
of four limbs is presented to show the severity of inflammation. A
scoring system is applied to each limb using a scale of 0-4, with 4
being the most severe inflammation. [0439] 0 Symptom free [0440] 1
Mild, but definite redness and swelling of one type of joint such
as the ankle or wrist, or apparent redness and swelling limited to
individual digits, regardless of the number of affected digits
[0441] 2 Moderate redness and swelling of two or more types of
joints [0442] 3 Severe redness and swelling of the entire paw
including digits [0443] 4 Maximally inflamed limb with involvement
of multiple joints
3.9 In Vivo Menisectomized (MNX) Rat Model
3.9.1 In Vivo Efficacy in the Rat MNX Model
[0444] In vivo efficacy was studied in a female Lewis
meniscectomised rat (MNX) model. The MNX rat model is a
well-validated disease model of osteoarthritis (Bendele, 2001, J
Musculoskel Neuron Interact, 1(4), 377-85; Janusz et al., 2002,
Osteoarthr Cartilage, 10, 785-91; Pritzker et al., 2006, Osteoarthr
Cartilage, 14, 13-29).
3.9.2 Surgery and Dosing
[0445] Osteoarthritis is induced by meniscectomy at day 0 (DO) in
the right leg of each rat by a transection of the medial collateral
ligament and 4 mm of ligament are removed. Internal part of the
meniscus is transected vertically into two flaps which are pushed
to the front and the back of the synovial cavity. Sham animals
undergo only anaesthesia, skin and muscle incision then suture. On
day 1, rats are randomly assigned to a treatment group (n=20 per
group) according to their body weight, in order to have a
homogenous distribution. From C2 to D21, rats are dosed per os (po)
once daily (qd) or twice a day (bid) with compounds formulated in
methylcellulose (MC) 0.5% or in HP.beta.CD 10% pH3.0.
3.9.3 Steady-State PK Determination (ssPK)
[0446] After at least 7 days of treatment, blood is sampled at 4
time points post administration: 0, 1, 3 and 6 h (and assuming 24 h
is equal to the pre-dose sample), in order to determine
steady-state plasma exposure.
3.9.4 Histology
[0447] At sacrifice, the right tibia of each rat is collected and
processed for histological analysis. After 48 h of fixation in 4%
formaldehyde, tibias are decalcified in Osteosoft for 7 days, and
cut into 2 half parts prior to embedding face to face in paraffin.
Five series of sections are cut at 200 .mu.m intervals, covering
about 1.5 mm of the middle part of the bone. One series of slides
is stained with Safranin 0 and light green for morphological
evaluation and OARSI scoring. The other series of slides are
mounted with DAPI for chondrocyte density measurement.
[0448] The extent of cartilage injury reflecting osteoarthritis in
the tibial plateau is evaluated and scored using the OARSI method
based on the grading and the staging of cartilage lesion (Pritzker
et al, 2006). The OARSI scoring is assessed in a blinded manner by
two different readers. For each tibia, one score is attributed as
the median of the OARSI score of the 5 sections.
[0449] For statistical analysis, medians of groups are compared
with a stratified Kruskal-Wallis test followed by Dunnett multiple
comparison post hoc test.
[0450] Significance levels: ns: not statistically significant;
*p<0.05; **p<0.01; ***p<0.001 versus MNX-vehicle.
Statistical analyses are done on all groups of the studies.
3.10--Diet-Induced Mouse Model of Non-Alcoholic Steatohepatitis
(NASH)
[0451] This model uses a choline-deficient, L-amino acid-defined,
high-fat diet (CDAHFD) consisting of 60 kcal % fat and 0.1%
methionine to induce NASH.
3.10.1 Study Groups and Dose Regimen:
[0452] C57/BL6 mice (Charles River) are divided into groups as set
out below, the mice are 8 weeks (>20 grs) at the initiation of
the induction phase.
TABLE-US-00012 Diet/Groups Project N Dose Frequency & route
Vehicle Control Vehicle 10 n/a BID p.o. MC 0.5% + 1 eq diet HCl,
98.9 % dist. water CDAHFD Vehicle 10 n/a BID p.o. MC 0.5% + 1 eq
diet HCl, 98.9 % dist. water CDAHFD Compound 1 10 5 mg/kg QD p.o.
MC 0.5% diet CDAHFD Compound 1 10 15 mg/kg QD p.o. MC 0.5% diet
CDAHFD Compound 1 10 15 mg/kg BID p.o. MC 0.5% diet
3.10.2 Materials and Compounds
[0453] CDAHFD; Research Diets Inc., Ref. No. A06071302 [0454]
Control diet: normal diet (VRF 1, P), Special Diets Services
3.10.3 Treatment Protocol:
Induction Phase: for 4 Weeks
[0455] In order to induce a non-alcoholic steatohepatitis (NASH),
animals will be fed with choline-deficient, L-amino acid-defined,
high-fat diet (CDAHFD).
Treatment Phase: for 6 Weeks
[0456] Mice were randomly assigned to a treatment group and treated
according to schedule presented in the Table above until the
evaluation phase.
3.10.4--Sampling
[0457] Steady state PK sampling--Blood will be collected in K2-EDTA
tubes to generate plasma. All blood samples will be processed for
plasma by centrifugation (5,000 rpm for 10 minutes at 4.degree. C.)
within 30 minutes of collection. Plasma from each blood sample will
be quickly frozen in liquid nitrogen and stored in a freezer
maintained at -80.degree. C. until analysis.
[0458] Final blood sample--Blood will be collected in tubes for
serum preparation containing protease inhibitors. All blood samples
will be processed by centrifugation (3,500 rpm for 15 minutes at
4.degree. C.). Aliquots of obtained serum samples will be stored
frozen at -80.degree. C. until further analysis.
[0459] Tissue samples: The liver is harvested and portions of the
left lateral lobe are stored for RNA gene expression analysis, TG
assay, sectioned for OH proline measurement, and the rest placed in
10% formalin for histopathological evaluation. Histopathological
evaluation includes sections stained with Sirius red to evaluate
extent of fibrosis, and sections stained with F4/80 to assess
macrophage accumulation.
[0460] A panel of 6 fibrosis genes (Col1A1, Timp1, Pai1, CTGF,
TGF.beta. and Acta2), inflammation genes (TNF.alpha., IL10 and
CCL2) and 2 house keeping genes are used for gene expression
analysis.
3.10.5 Results
[0461] Compound 1 when tested in this model showed a significant
effect on at least the highest doses tested on the expression
levels of Pai1, TIMP1, CTGF, and TGF.beta. in the fibrosis gene
panel, (FIG. 3) and on all three genes in the inflammation panel
(FIG. 4). Additionally it showed a significant effect on
hydroxyproline levels at 15 mg/kg/QD and 15 mg/kg/BID (FIG. 5), a
significant effect on the Sirius red fibrosis quantification at 15
mg/kg/QD and 15 mg/kg/BID (FIG. 6), and a significant effect on the
F4/80 quantification at 15 mg/kg/BID (FIG. 7).
CYP Inhibition
[0462] The inhibitory potential of a test compound for human
cytochrome P450 isoenzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) is
assessed using cDNA-expressed human cytochrome P450 isoenzymes and
non-fluorescent substrates which are metabolized to fluorescent
metabolites.
[0463] Compounds are tested at 3.3 and 10 .mu.M, with a final DMSO
concentration of 0.3%. Compounds are incubated for 15 min with
enzyme before the cofactor-substrate mix is added. Final reaction
concentrations in cofactor mix for the CYP3A4 (BD Biosciences,
456202), CYP2C9 (BD Biosciences, 456258), CYP2C19 (BD Biosciences,
456259) and CYP1A2 (BD Biosciences, 456203) assays are: 0.4 U/mL
glucose-6-phophate-dehydrogenase (G6PDH, Roche, 10165875001), 3.3
mM MgCl.sub.2 (Sigma, M2670), 3.3 mM D-glucose-6-phosphate (Sigma,
G7879) and 1.3 mM NADP+ (Sigma, N0505). For CYP2D6 (BD Biosciences,
456217), final reaction concentrations in the assay are 0.4 U/ml
G6PDH, 0.41 mM MgCl.sub.2, 0.41 mM D-glucose-6-phosphate and 8.2
.mu.M NADP+. The concentrations of enzyme and substrate are
reported in 0. After an incubation period, the reaction is stopped
by adding a stop solution. For experiments with DBF as substrate, a
2 N NaOH stop solution is used, while for all other substrates the
stop solution is 80% MeCN/20% 0.5 M Tris base.
[0464] Fluorescence is read either immediately (for CEC, AMMC,
BFC), or after 20 min (for CYP2C9 and CYP3A4 using DBF as
substrate) on a PerkinElmer EnVision.RTM. reader at the appropriate
excitation and emission wavelength (cf. 0).
[0465] The percentage inhibition of CYP by the test compound is
then calculated by normalizing the data to blank samples: 100%
inhibition is the blank sample stopped before addition of the
enzyme/substrate mix, and 0% inhibition is the blank sample stopped
after the enzymatic reaction has occurred (50 min).
TABLE-US-00013 CYP3A4 CYP3A4 CYP2C19 CYP2C9 CYP1A2 CYP2D6 Substrate
(.mu.M) DBF 1 -- -- 0.5 -- -- CEC -- -- 35 -- 4 -- AMMC -- -- -- --
-- 0.5 BFC -- 120 -- -- -- -- Phosphate 200 90 25 25 25 25 buffer
pH 7.4 (mM) Enzyme 1 1.5 6 2 1.5 3 (pmol/well) Incubation time 50
50 50 50 50 50 (min) Positive control ketoconazole ketoconazole
fluvoxainine sulfaphenazole fluvoxamine quinidine Excitation 485
400 400 485 400 380 wavelength (nm) Emission 530 530 460 530 460
460 wavelength (nm) AMMC: aminoethyl-7-methoxy-4-methylcoumarin
BFC: 7-benzyloxy-4-trifluoromethylcoumarin CEC:
3-cyano-7-ethoxycoumarin DBF: dibenzylfluorescein
5 Time-Dependent CYP3A4 Inhibition
[0466] Time-dependent CYP3A4 inhibition by the compounds, assessed
in pooled HLM, is determined via IC.sub.50 determination according
to Grimm et al. Drug Metabolism and Disposition 2009, 37, 1355-1370
and the draft FDA Guidance for Industry (Drug Interaction
Studies--Study Design, Data Analysis, Implications for Dosing, and
Labeling Recommendations), 2006,
http://www.fda.gov/cder/guidance/index.htm. Testosterone is used as
probe substrate and troleandomycin is used as positive control.
Sequence CWU 1
1
1115PRTArtificialSynthetic Peptide 1Gly Thr Phe Arg Ala Ala Ile Arg
Arg Leu Ala Ala Arg Arg Arg1 5 10 15
* * * * *
References