U.S. patent application number 12/516151 was filed with the patent office on 2010-07-22 for n-(methyl) -1h-pyrazol-3-amine, n-(methyl)-pyridin-2-amine and n-(methyl)-thiazol-2-amine derivatives for the treatment of diseases associated with amyloid or amyloid-like proteins, like e.g. alzheimer's.
Invention is credited to Maria Pihlgren Bosch, Wolfgang Froestl, Sophie Lohmann, Maria Pilar Lopez Deber, Andreas Muhs, Nampally Sreenivasachary.
Application Number | 20100183513 12/516151 |
Document ID | / |
Family ID | 39092970 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100183513 |
Kind Code |
A1 |
Froestl; Wolfgang ; et
al. |
July 22, 2010 |
N-(METHYL) -1H-PYRAZOL-3-AMINE, N-(METHYL)-PYRIDIN-2-AMINE AND
N-(METHYL)-THIAZOL-2-AMINE DERIVATIVES FOR THE TREATMENT OF
DISEASES ASSOCIATED WITH AMYLOID OR AMYLOID-LIKE PROTEINS, LIKE
E.G. ALZHEIMER'S
Abstract
The present invention relates to novel compounds of formula (II)
that can be employed in the treatment of a group of disorders and
abnormalities associated with amyloid protein, such as Alzheimer's
disease, and of diseases or conditions associated with amyloid-like
proteins. The compounds of the present invention can also be used
in the treatment of ocular diseases associated with pathological
abnormalities/changes in the tissues of the visual system. The
present invention further relates to pharmaceutical compositions
comprising these compounds and to the use of these compounds for
the preparation of medicaments for treating, or preventing diseases
or conditions associated with amyloid and/or amyloid-like proteins.
A method of treating or preventing diseases or conditions
associated with amyloid and/or amyloid-like proteins is also
disclosed. ##STR00001##
Inventors: |
Froestl; Wolfgang;
(Ecublens, CH) ; Sreenivasachary; Nampally;
(Ecublens, CH) ; Lohmann; Sophie; (Ecublens,
CH) ; Lopez Deber; Maria Pilar; (Ecublens, CH)
; Muhs; Andreas; (Pully, CH) ; Bosch; Maria
Pihlgren; (Sulpice, CH) |
Correspondence
Address: |
Johnson & Associates
317A E. Liberty Street
Savannah
GA
31401
US
|
Family ID: |
39092970 |
Appl. No.: |
12/516151 |
Filed: |
November 23, 2007 |
PCT Filed: |
November 23, 2007 |
PCT NO: |
PCT/EP2007/010219 |
371 Date: |
May 22, 2009 |
Current U.S.
Class: |
424/9.1 ;
435/7.1; 514/332; 514/341; 514/370; 546/262; 546/275.4;
548/198 |
Current CPC
Class: |
A61P 9/00 20180101; C07D
231/38 20130101; C07D 213/75 20130101; C07D 277/42 20130101; C07D
401/12 20130101; C07D 403/12 20130101; A61P 27/12 20180101; A61P
27/00 20180101; A61P 27/06 20180101; A61P 21/00 20180101; C07D
213/74 20130101; A61P 43/00 20180101; A61P 25/00 20180101; A61P
25/28 20180101; A61P 25/02 20180101; A61P 25/16 20180101; C07D
417/14 20130101; C07D 405/04 20130101; C07D 409/14 20130101; C07D
417/12 20130101; A61P 3/10 20180101; A61P 35/00 20180101; A61P
27/02 20180101 |
Class at
Publication: |
424/9.1 ;
435/7.1; 514/332; 514/341; 514/370; 546/262; 546/275.4;
548/198 |
International
Class: |
A61K 49/00 20060101
A61K049/00; G01N 33/53 20060101 G01N033/53; A61K 31/444 20060101
A61K031/444; A61K 31/4439 20060101 A61K031/4439; A61K 31/427
20060101 A61K031/427; C07D 401/12 20060101 C07D401/12; C07D 417/14
20060101 C07D417/14; A61P 25/16 20060101 A61P025/16; A61P 25/00
20060101 A61P025/00; A61P 25/28 20060101 A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 2006 |
EP |
06024427.4 |
Claims
1. A compound of the general formula (II) ##STR00067## wherein
independently represents a single bond or a double bond; p is 1, 2
or 3; each linker K is independently C.sub.1-3 alkylene which is
optionally substituted by one or more C.sub.1-4 alkyl groups; each
B is independently a 5- or 6-membered saturated or unsaturated
heterocyclic ring, wherein the heterocyclic ring B is optionally
substituted by one or more substituents selected from C.sub.1-4
alkyl, C.sub.1-4 alkoxy, mono- and di-C.sub.1-4 alkyl amino,
C.sub.3-7 cycloalkyl amino, and 5- or 6-membered saturated
heterocyclyl, or two substituents may be joined to form a
saturated, unsaturated or aromatic 5- to 7-membered ring which is
fused with the heterocyclic ring B, and wherein the heterocyclic
ring B may contain in addition to the units V and W one or more
heteroatoms, selected from N, NR, S and O, wherein R is selected
from H and C.sub.1-4 alkyl; each unit W is independently a H-bond
acceptor; each unit V is independently optional and, if present, is
independently a H-bond donor; R.sup.1 is selected from --H,
-halogen, --C.sub.1-4 alkyl, --NH.sub.2, --NH--C.sub.1-4 alkyl,
--C.sub.1-4 alkylene-NH.sub.2, --C.sub.1-4 alkylene-NH--C.sub.1-4
alkyl, -aryl, -aryl-R.sup.3, --C.sub.1-4 alkylene-aryl, --C.sub.1-4
alkylene-aryl-R.sup.3, -heteroaryl, -heteroaryl-R.sup.3,
--NH--C.sub.1-4 alkylene-aryl, --NH--C.sub.1-4
alkylene-aryl-R.sup.3, --OH and --O--C.sub.1-4 alkyl; and R.sup.3
is C.sub.1-4 alkyl, halogen, OH or O--C.sub.1-4 alkyl; R.sup.2 is
--H, --C.sub.1-4 alkyl, -aryl or a group of the formula
##STR00068## wherein B, V, W and K are as defined above, q is 0 or
1 and r is 0 or 1.
2. The compound of claim 1, wherein the each unit W is
independently N or C.dbd.O.
3. The compound of claim 1, wherein each unit V is NH.
4. The compound of claim 1, wherein each linker K is --CH.sub.2--,
--CH.sub.2CH.sub.2-- or --CH.sub.2CH.sub.2CH.sub.2.
5. The compound of claim 1, wherein each heterocyclic ring B is
independently selected from optionally substituted pyrazolylene,
optionally substituted pyridinylene, optionally substituted
2-pyridinonylene, optionally substituted 2-piperidonylene,
optionally substituted thiazolylene and optionally substituted
isothiazolylene.
6. The compound of claim 1, wherein R.sup.1 is --H, --CH.sub.3,
--NH--C.sub.1-4 alkyl or --CH.sub.2--NH--CH.sub.3.
7. The compound of claim 1, wherein R.sup.2 is H or aryl.
8. A pharmaceutical composition comprising a compound of claim
1.
9-14. (canceled)
15. A method of treating or preventing a disease or condition
associated with an amyloid and/or amyloid-like protein comprising
administering to a subject in need of such treatment an effective
amount of a compound of claim 1.
16. The method of claim 15, wherein the disease is a neurological
disorder selected from Alzheimer's Disease (AD), Lewy body dementia
(LBD), Down's syndrome, hereditary cerebral hemorrhage with
amyloidosis (Dutch type), the Guam Parkinson-Dementia complex mild
cognitive impairment (MCI), progressive supranuclear palsy,
multiple sclerosis, inclusion-body myositis (IBM), Creutzfeld Jacob
disease, Parkinson's disease, HIV-related dementia, amyotropic
lateral sclerosis (ALS), inclusion-body myositis (IBM), adult onset
diabetes, senile cardiac amyloidosis, endocrine tumors, glaucoma,
ocular amyloidoses, primary retinal degeneration, macular
degeneration, optic nerve drusen, optic neuropathy, optic neuritis,
or lattice dystrophy.
17-24. (canceled)
25. A mixture comprising a compound according to claim 1 and at
least one further biologically active compound optionally in
combination with a pharmaceutically acceptable carrier, a diluent,
or an excipient.
26-41. (canceled)
42. A method of collecting data for the diagnosis of an
amyloid-associated disease or condition in a sample or a patient
comprising: (a) bringing a sample or a specific body part or body
area suspected to contain an amyloid protein into contact with a
compound according to claim 1; (b) allowing the compound to bind to
the amyloid protein; (c) detecting the compound bound to the
protein; and (d) optionally correlating the presence or absence of
compound binding with the amyloid protein with the presence or
absence of amyloid protein in the sample or specific body part or
body area.
43. A method of determining the extent of amyloidogenic plaque
burden in a tissue or a body fluid comprising: (a) providing a
sample representative of the tissue or body fluid under
investigation; (b) testing the sample for the presence of amyloid
protein with a compound according to claim 1; (c) determining the
amount of compound bound to the amyloid protein; and (d)
calculating the plaque burden in the tissue or body fluid.
44. (canceled)
45. A method of collecting data for determining a predisposition to
an amyloid-associated disease or condition in a patient comprising
detecting the specific binding of a compound according to claim 1
to an amyloid protein in a sample or in situ which comprises the
steps of: (a) bringing the sample or a specific body part or body
area suspected to contain the amyloid protein into contact with a
compound according to claim 1, which compound specifically binds to
the amyloid protein; (b) allowing the compound to bind to the
amyloid protein to form a compound/protein complex; (c) detecting
the formation of the compound/protein complex; (d) optionally
correlating the presence or absence of the compound/protein complex
with the presence or absence of amyloid protein in the sample or
specific body part or body area; and (e) optionally comparing the
amount of the compound/protein complex to a normal control
value.
46. A method of collecting data for monitoring minimal residual
disease in a patient following treatment with an antibody or a
vaccine composition, wherein the method comprises: (a) bringing a
sample or a specific body part or body area suspected to contain an
amyloid protein into contact with a compound according to claim 1,
which compound specifically binds to the amyloid protein; (b)
allowing the compound to bind to the amyloid protein to form a
compound/protein complex; (c) detecting the formation of the
compound/protein complex; (d) optionally correlating the presence
or absence of the compound/protein complex with the presence or
absence of amyloid protein in the sample or specific body part or
body area; and (e) optionally comparing the amount of the
compound/protein complex to a normal control value.
47. A method of collecting data for predicting responsiveness of a
patient being treated with an antibody or a vaccine composition
comprising: (a) bringing a sample or a specific body part or body
area suspected to contain an amyloid protein into contact with a
compound according to claim 1, which compound specifically binds to
the amyloid protein; (b) allowing the compound to bind to the
amyloid protein to form a compound/protein complex; (c) detecting
the formation of the compound/protein complex; (d) optionally
correlating the presence or absence of the compound/protein complex
with the presence or absence of amyloid protein in the sample or
specific body part or body area; and (e) optionally comparing the
amount of the compound/protein complex to a normal control
value.
48. A test kit for detection and/or diagnosis of an
amyloid-associated disease or condition comprising a compound
according to claim 1.
49-51. (canceled)
52. A method of treating or preventing an ocular disease or
condition associated with a pathological abnormality/change in the
tissue of the visual system, particularly associated with an
amyloid-beta-related pathological abnormality/change in the tissue
of the visual system comprising administering to a subject in need
of such treatment an effective amount of a compound of claim 1.
53. The method of claim 52, wherein the ocular disease or condition
is selected from the group consisting of neuronal degradation,
cortical visual deficits, glaucoma, cataract due to beta-amyloid
deposition, ocular amyloidoses, primary retinal degeneration,
macular degeneration, for example age-related macular degeneration,
optic nerve drusen, optic neuropathy, optic neuritis, and lattice
dystrophy.
54-55. (canceled)
56. The method of claim 16, wherein the macular degeneration is age
related macular degeneration (AMD).
Description
[0001] The present invention relates to novel compounds that can be
employed in the treatment of a group of disorders and abnormalities
associated with amyloid protein, such as Alzheimer's disease, and
of diseases or conditions associated with amyloid-like proteins.
The present invention further relates to pharmaceutical
compositions comprising these compounds and to the use of these
compounds for the preparation of medicaments for the treatment of
diseases or conditions associated with amyloid or amyloid-like
proteins. A method of treating diseases or conditions associated
with amyloid or amyloid-like proteins is also disclosed.
[0002] The compounds of the present invention can also be used in
the treatment of ocular diseases associated with pathological
abnormalities/changes in the tissues of the visual system,
particularly associated with amyloid-beta-related pathological
abnormalities/changes in the tissues of the visual system, such as
neuronal degradation. Said pathological abnormalities may occur,
for example, in different tissues of the eye, such as the visual
cortex leading to cortical visual deficits; the anterior chamber
and the optic nerve leading to glaucoma; the lens leading to
cataract due to beta-amyloid deposition; the vitreous leading to
ocular amyloidoses; the retina leading to primary retinal
degeneration and macular degeneration, for example age-related
macular degeneration; the optic nerve leading to optic nerve
drusen, optic neuropathy and optic neuritis; and the cornea leading
to lattice dystrophy.
[0003] Many diseases of aging are based on or associated with
amyloid or amyloid-like proteins and are characterized, in part, by
the buildup of extracellular deposits of amyloid or amyloid-like
material that contribute to the pathogenesis, as well as the
progression of the disease. These diseases include, but are not
limited to, neurological disorders such as Alzheimer's Disease
(AD), diseases or conditions characterized by a loss of cognitive
memory capacity such as, for example, mild cognitive impairment
(MCI), Lewy body dementia, Down's syndrome, hereditary cerebral
hemorrhage with amyloidosis (Dutch type); the Guam
Parkinson-Dementia complex. Other diseases which are based on or
associated with amyloid-like proteins are progressive supranuclear
palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's
disease, HIV-related dementia, ALS (amyotropic lateral sclerosis),
inclusion-body myositis (IBM), Adult Onset Diabetes; senile cardiac
amyloidosis; endocrine tumors, and other diseases, including
amyloid-associated ocular diseases that target different tissues of
the eye, such as the visual cortex, including cortical visual
deficits; the anterior chamber and the optic nerve, including
glaucoma; the lens, including cataract due to beta-amyloid
deposition; the vitreous, including ocular amyloidoses; the retina,
including primary retinal degenerations and macular degeneration,
in particular age-related macular degeneration; the optic nerve,
including optic nerve drusen, optic neuropathy and optic neuritis;
and the cornea, including lattice dystrophy.
[0004] Although pathogenesis of these diseases may be diverse,
their characteristic deposits often contain many shared molecular
constituents. To a significant degree, this may be attributable to
the local activation of pro-inflammatory pathways thereby leading
to the concurrent deposition of activated complement components,
acute phase reactants, immune modulators, and other inflammatory
mediators.
[0005] Alzheimer's Disease (AD) is a neurological disorder
primarily thought to be caused by amyloid plaques, an accumulation
of abnormal deposit of proteins in the brain. The most frequent
type of amyloid found in the brain of affected individuals is
composed primarily of A.beta. fibrils. Scientific evidence
demonstrates that an increase in the production and accumulation of
beta-amyloid protein in plaques leads to nerve cell death, which
contributes to the development and progression of AD. Loss of nerve
cells in strategic brain areas, in turn, causes reduction in the
neurotransmitters and impairment of memory. The proteins
principally responsible for the plaque build up include amyloid
precursor protein (APP) and two presenilins (presenilin I and
presenilin II). Sequential cleavage of the amyloid precursor
protein (APP), which is constitutively expressed and catabolized in
most cells, by the enzymes .beta. and .gamma. secretase leads to
the release of a 39 to 43 amino acid A.beta. peptide. The
degradation of APPs likely increases their propensity to aggregate
in plaques. It is especially the A.beta.(1-42) fragment that has a
high propensity of building aggregates due to two very hydrophobic
amino acid residues at its C-terminus. The A.beta.(1-42) fragment
is therefore believed to be mainly involved and responsible for the
initiation of neuritic plaque formation in AD and to have,
therefore, a high pathological potential. There is therefore a need
for specific molecules that can target and diffuse amyloid plaque
formation.
[0006] The symptoms of AD manifest slowly and the first symptom may
only be mild forgetfulness. In this stage, individuals may forget
recent events, activities, the names of familiar people or things
and may not be able to solve simple math problems. As the disease
progresses, symptoms are more easily noticed and become serious
enough to cause people with AD or their family members to seek
medical help. Mid-stage symptoms of AD include forgetting how to do
simple tasks such as grooming, and problems develop with speaking,
understanding, reading, or writing. Later stage AD patients may
become anxious or aggressive, may wander away from home and
ultimately need total care.
[0007] Presently, the only definite way to diagnose AD is to
identify plaques and tangles in brain tissue in an autopsy after
death of the individual. Therefore, doctors can only make a
diagnosis of "possible" or "probable" AD while the person is still
alive. Using current methods, physicians can diagnose AD correctly
up to 90 percent of the time using several tools to diagnose
"probable" AD. Physicians ask questions about the person's general
health, past medical problems, and the history of any difficulties
the person has carrying out daily activities. Behavioral tests of
memory, problem solving, attention, counting, and language provide
information on cognitive degeneration and medical tests such as
tests of blood, urine, or spinal fluid, and brain scans can provide
some further information.
[0008] The management of AD consists of medication-based and
non-medication based treatments. Treatments aimed at changing the
underlying course of the disease (delaying or reversing the
progression) have so far been largely unsuccessful. Medicines that
restore the deficit (defect), or malfunctioning, in the chemical
messengers of the nerve cells (neurotransmitters), in particular
the cholinesterase inhibitors (ChEIs) such as tacrine and
rivastigmine, have been shown to improve symptoms. ChEIs impede the
enzymatic degradation of neurotransmitters thereby increasing the
amount of chemical messengers available to transmit the nerve
signals in the brain.
[0009] For some people in the early and middle stages of the
disease, the drugs tacrine (COGNEX.RTM., Morris Plains, N.J.),
donepezil (ARICEPT.RTM., Tokyo, JP), rivastigmine (EXELON.RTM.,
East Hanover, N.J.), or galantamine (REMINYL.RTM., New Brunswick,
N.J.) may help prevent some symptoms from becoming worse for a
limited time. Another drug, memantine (NAMENDA.RTM., New York,
N.Y.), has been approved for treatment of moderate to severe AD.
Medications are also available to address the psychiatric
manifestations of AD. Also, some medicines may help control
behavioral symptoms of AD such as sleeplessness, agitation,
wandering, anxiety, and depression. Treating these symptoms often
makes patients more comfortable and makes their care easier for
caregivers. Unfortunately, despite significant treatment advances
showing that this class of agents is consistently better than a
placebo, the disease continues to progress, and the average effect
on mental functioning has only been modest. Many of the drugs used
in AD medication such as, for example, ChEIs also have side effects
that include gastrointestinal dysfunction, liver toxicity and
weight loss.
[0010] Other diseases that are based on or associated with the
accumulation and deposit of amyloid-like protein are mild cognitive
impairment, Lewy body dementia (LBD), amyotrophic lateral sclerosis
(ALS), inclusion-body myositis (IBM) and macular degeneration, in
particular age-related macular degeneration (AMD).
[0011] Mild cognitive impairment (MCI) is a general term most
commonly defined as a subtle but measurable memory disorder. A
person with MCI experiences memory problems greater than normally
expected with aging, but does not show other symptoms of dementia,
such as impaired judgment or reasoning.
[0012] Lewy body dementia (LBD) is a neurodegenerative disorder
that can occur in persons older than 65 years of age, which
typically causes symptoms of cognitive (thinking) impairment and
abnormal behavioral changes. Symptoms can include cognitive
impairment, neurological signs, sleep disorder, and autonomic
failure. Cognitive impairment is the presenting feature of LBD in
most cases. Patients have recurrent episodes of confusion that
progressively worsen. The fluctuation in cognitive ability is often
associated with shifting degrees of attention and alertness.
Cognitive impairment and fluctuations of thinking may vary over
minutes, hours, or days.
[0013] Amyotrophic lateral sclerosis (ALS) is characterized by
degeneration of upper and lower motor neurons. In some ALS
patients, dementia or aphasia may be present (ALS-D). The dementia
is most commonly a frontotemporal dementia (FTD), and many of these
cases have ubiquitin-positive, tau-negative inclusions in neurons
of the dentate gyrus and superficial layers of the frontal and
temporal lobes.
[0014] Inclusion-body myositis (IBM) is a crippling disease usually
found in people over age 50, in which muscle fibers develop
inflammation and begin to atrophy--but in which the brain is spared
and patients retain their full intellect. Two enzymes involved in
the production of amyloid-.beta. protein were found to be increased
inside the muscle cells of patients with this most common,
progressive muscle disease of older people, in which amyloid-.beta.
is also increased.
[0015] Macular degeneration is a common eye disease that causes
deterioration of the macula, which is the central area of the
retina (the paper-thin tissue at the back of the eye where
light-sensitive cells send visual signals to the brain). Sharp,
clear, `straight ahead` vision is processed by the macula. Damage
to the macula results in the development of blind spots and blurred
or distorted vision. Age-related macular degeneration (AMD) is a
major cause of visual impairment in the United States and for
people over age 65 it is the leading cause of legal blindness among
Caucasians. Approximately 1.8 million Americans age 40 and older
have advanced AMD, and another 7.3 million people with intermediate
AMD are at substantial risk for vision loss. The government
estimates that by 2020 there will be 2.9 million people with
advanced AMD. Victims of AMD are often surprised and frustrated to
find out how little is known about the causes and treatment of this
blinding condition.
[0016] There are two forms of macular degeneration: dry macular
degeneration and wet macular degeneration. The dry form, in which
the cells of the macula slowly begin to break down, is diagnosed in
85 percent of macular degeneration cases. Both eyes are usually
affected by dry AMD, although one eye can lose vision while the
other eye remains unaffected. Drusen, which are yellow deposits
under the retina, are common early signs of dry AMD. The risk of
developing advanced dry AMD or wet AMD increases as the number or
size of the drusen increases. It is possible for dry AMD to advance
and cause loss of vision without turning into the wet form of the
disease; however, it is also possible for early-stage dry AMD to
suddenly change into the wet form.
[0017] The wet form, although it only accounts for 15 percent of
the cases, results in 90 percent of the blindness, and is
considered advanced AMD (there is no early or intermediate stage of
wet AMD). Wet AMD is always preceded by the dry form of the
disease. As the dry form worsens, some people begin to have
abnormal blood vessels growing behind the macula. These vessels are
very fragile and will leak fluid and blood (hence `wet` macular
degeneration), causing rapid damage to the macula.
[0018] The dry form of AMD will initially often cause slightly
blurred vision. The center of vision in particular may then become
blurred and this region grows larger as the disease progresses. No
symptoms may be noticed if only one eye is affected. In wet AMD,
straight lines may appear wavy and central vision loss can occur
rapidly.
[0019] Diagnosis of macular degeneration typically involves a
dilated eye exam, visual acuity test, and a viewing of the back of
the eye using a procedure called fundoscopy to help diagnose AMD,
and--if wet AMD is suspected--fluorescein angiography may also be
performed. If dry AMD reaches the advanced stages, there is no
current treatment to prevent vision loss. However, a specific high
dose formula of antioxidants and zinc may delay or prevent
intermediate AMD from progressing to the advanced stage.
Macugen.RTM. (pegaptanib sodium injection), laser photocoagulation
and photodynamic therapy can control the abnormal blood vessel
growth and bleeding in the macula, which is helpful for some people
who have wet AMD; however, vision that is already lost will not be
restored by these techniques. If vision is already lost, low vision
aids exist that can help improve the quality of life.
[0020] One of the earliest signs of age-related macular
degeneration (AMD) is the accumulation of extracellular deposits
known as drusen between the basal lamina of the retinal pigmented
epithelium (RPE) and Bruch's membrane (BM). Recent studies
conducted by Anderson et al. have confirmed that drusen contains
amyloid beta. (Experimental Eye Research 78 (2004) 243-256).
[0021] Prions cause neurodegenerative diseases such as scrapie in
sheep, bovine spongiform encephalopathy in cattle and
Creutzfeldt-Jacob disease in humans. The only known component of
the particle is the scrapie isoform of the protein, PrPSc. Although
prions multiply, there is no evidence that they contain nucleic
acid. PrPSc is derived from the non-infectious, cellular protein
PrPC by a posttranslational process during which PrPC undergoes a
profound conformational change.
[0022] The scrapie protein PrPSc has a critical role in neuronal
degeneration and during disease development undergoes a three stage
transition as follows: PrPC (normal cellular isoform of
protein)--PrPSc: infectious form (scrapie isoform of
protein)--protein PrP27-30.
[0023] Such a cascade of events occurs during the development of
Creutzfeldt-Jacob disease (CJD), Kuru,
Gerstmann-Straussler-Scheinker Syndrome (GSS), fatal familial
insomnia in man, scrapie in sheep and goats, encephalopathy in mink
and bovine spongiform encephalopathy in cattle.
[0024] The cellular non-toxic protein (PrPC) is a sialoglycoprotein
of molecular weight 33000 to 35000 that is expressed predominantly
in neurons. In the diseases mentioned above, PrPC is converted into
an altered form (PrPSc), which is distinguishable from its normal
homologue by its relative resistance to protease digestion. PrPSc
accumulates in the central nervous system of affected animals and
individuals and its protease-resistant core aggregates
extracellularly. Amyloidosis is not a single disease entity but
rather a diverse group of progressive disease processes
characterized by extracellular tissue deposits of a waxy,
starch-like protein called amyloid, which accumulates in one or
more organs or body systems. As the amyloid deposits build up, they
begin to interfere with the normal function of the organ or body
system. There are at least 15 different types of amyloidosis. The
major forms are primary amyloidosis without known antecedent,
secondary amyloidosis following some other condition, and
hereditary amyloidosis.
[0025] Secondary amyloidosis occurs in people who have a chronic
infection or inflammatory disease, such as tuberculosis, a
bacterial infection called familial Mediterranean fever, bone
infections (osteomyelitis), rheumatoid arthritis, inflammation of
the small intestine (granulomatous ileitis), Hodgkin's disease, and
leprosy.
[0026] Glaucoma is a group of diseases of the optic nerve involving
loss of retinal ganglion cells (RGCs) in a characteristic pattern
of optic neuropathy. Glaucoma is often, but not always, accompanied
by an increased eye pressure, which may be a result of blockage of
the circulation of aqueous, or its drainage.
[0027] Although raised intraocular pressure is a significant risk
factor for developing glaucoma, no threshold of intraocular
pressure can be defined which would be determinative for causing
glaucoma.
[0028] The damage may also be caused by poor blood supply to the
vital optic nerve fibers, a weakness in the structure of the nerve,
and/or a problem in the health of the nerve fibers themselves.
[0029] Untreated glaucoma leads to permanent damage of the optic
nerve and resultant visual field loss, which can progress to
blindness.
[0030] RGCs are the nerve cells that transmit visual signals from
the eye to the brain. Caspase-3 and Caspase-8, two major enzymes in
the apoptotic process, are activated in the process leading to
apoptosis of RGCs. Caspase-3 cleaves amyloid precursor protein
(APP) to produce neurotoxic fragments, including Amyloid .beta..
Without the protective effect of APP, Amyloid .beta. accumulation
in the retinal ganglion cell layer results in the death of RGCs and
irreversible loss of vision.
[0031] The different types of glaucomas are classified as
open-angle glaucomas, if the condition is chronic, or closed-angle
glaucomas, if acute glaucoma occurs suddenly. Glaucoma usually
affects both eyes, but the disease can progress more rapidly in one
eye than in the other.
[0032] Chronic open-angle glaucoma (COAG), also known as primary
open angle glaucoma (POAG), is the most common type of glaucoma.
COAG is caused by microscopic blockage in the trabecular meshwork,
which decreases the drainage of the aqueous outflow into the
Schlemm's canal and raises the intraocular pressure (IOP). POAG
usually affects both eyes and is strongly associated with age and a
positive family history. Its frequency increases in elderly people
as the eye drainage mechanism may gradually become clogged with
aging. The increase in intraocular pressure in subjects affected by
chronic open-angle glaucoma is not accompanied by any symptoms
until the loss is felt on the central visual area.
[0033] Acute Angle Closure Glaucoma (AACG) or closed-angle glaucoma
is a relatively rare type of glaucoma characterized by a sudden
increase in intraocular pressure to 35 to 80 mmHg, leading to
severe pain and irreversible loss of vision. The sudden pressure
increase is caused by the closing of the filtering angle and
blockage of the drainage channels. Individuals with narrow angles
have an increased risk for a sudden closure of the angle. AACG
usually occurs monocularly, but the risk exists in both eyes. Age,
cataract and pseudoexfoliation are also risk factors since they are
associated with enlargement of the lens and crowding or narrowing
of the angle. A sudden glaucoma attack may be associated with
severe eye pain and headache, inflamed eye, nausea, vomiting, and
blurry vision.
[0034] Mixed or Combined Mechanism Glaucoma is a mixture or
combination of open and closed angle glaucoma. It affects patients
with acute ACG whose angle opens after laser iridotomy, but who
continue to require medications for IOP control, as well as
patients with POAG or pseudoexfoliative glaucoma who gradually
develop narrowing of the angle.
[0035] Normal tension glaucoma (NTG), also known as low tension
glaucoma (LTG), is characterized by progressive optic nerve damage
and loss of peripheral vision similar to that seen in other types
of glaucoma; however, the intraocular pressure is the normal range
or even below normal.
[0036] Congenital (infantile) glaucoma is a relatively rare,
inherited type of open-angle glaucoma. Insufficient development of
the drainage area results in increased pressure in the eye that can
lead to the loss of vision from optic nerve damage and to an
enlarged eye. Early diagnosis and treatment are critical to
preserve vision in infants and children affected by the
disease.
[0037] Secondary glaucoma may result from an ocular injury,
inflammation in the iris of the eye (iritis), diabetes, cataract,
or use of steroids in steroid-susceptible individuals. Secondary
glaucoma may also be associated with retinal detachment or retinal
vein occlusion or blockage.
[0038] Pigmentary glaucoma is characterized by the detachment of
granules of pigment from the iris. The granules cause blockage of
the drainage system of the eye, leading to elevated intraocular
pressure and damage to the optic nerve.
[0039] Exfoliative glaucoma (pseudoexfoliation) is characterized by
deposits of flaky material on the anterior capsule and in the angle
of the eye. Accumulation of the flaky material blocks the drainage
system and raises the eye pressure.
[0040] Diagnosis of glaucoma may be made using various tests.
Tonometry determines the pressure in the eye by measuring the tone
or firmness of its surface. Several types of tonometers are
available for this test, the most common being the applanation
tonometer. Pachymetry determines the thickness of the cornea which,
in turn, measures intraocular pressure. Gonioscopy allows
examination of the filtering angle and drainage area of the eye.
Gonioscopy can also determine if abnormal blood vessels may be
blocking the drainage of the aqueous fluid out of the eye.
Opthalmoscopy allows examination of the optic nerve and can detect
nerve fiber layer drop or changes in the optic disc, or indentation
(cupping) of this structure, which may be caused by increased
intraocular pressure or axonal drop out. Gonioscopy is also useful
in assessing damage to the nerve from poor blood flow or increased
intraocular pressure. Visual Field testing maps the field of
vision, subjectively, which may detect signs of glaucomatous damage
to the optic nerve. This is represented by specific patterns of
visual field loss. Ocular coherence tomography, an objective
measure of nerve fiber layer loss, is carried out by looking at the
thickness of the optic nerve fiber layer (altered in glaucoma) via
a differential in light transmission through damaged axonal
tissue.
[0041] Optic nerve drusen are globular concretions of protein and
calcium salts which are felt to represent secretions through
congenitally altered vascular structures affecting the axonal nerve
fiber layer. These accumulations occur in the peripapillary nerve
fiber layer and are felt to damage the nerve fiber layer either
directly by compression or indirectly through disruptions of the
vascular supply to the nerve fiber layer. They usually become
visible after the first decade of life in affected individuals.
They occur most often in both eyes but may also affect one eye, and
may cause mild loss of peripheral vision over many years.
[0042] Optic neuropathy is a disease characterized by damage to the
optic nerve caused by demyelination, blockage of blood supply,
nutritional deficiencies, or toxins. Demyelinating optic
neuropathies (see optic neuritis below) are typically caused by an
underlying demyelinating process such as multiple sclerosis.
Blockage of the blood supply, known as ischemic optic neuropathy,
can lead to death or dysfunction of optic nerve cells.
Non-arteritic ischemic optic neuropathy usually occurs in
middle-age people. Risk factors include high blood pressure,
diabetes and atherosclerosis. Arteritic ischemic optic neuropathy
usually occurs in older people following inflammation of the
arteries (arteritis), particularly the temporal artery (temporal
arteritis). Loss of vision may be rapid or develop gradually over 2
to 7 days and the damage may be to one or both eyes. In people with
optic neuropathy caused by exposure to a toxin or to a nutritional
deficiency, both eyes are usually affected.
[0043] About 40% of people with non-arteritic ischemic optic
neuropathy experience spontaneous improvement over time.
Non-arteritic ischemic optic neuropathy is treated by controlling
blood pressure, diabetes and cholesterol levels. Arteritic ischemic
optic neuropathy is treated with high doses of corticosteroids to
prevent loss of vision in the second eye.
[0044] Optic neuritis is associated with mild or severe vision loss
in one or both eyes and may be caused by a systemic demyelinating
process (see above), viral infection, vaccination, meningitis,
syphilis, multiple sclerosis and intraocular inflammation
(uveitis). Eye movement may be painful and vision may deteriorate
with repeat episodes. Diagnosis involves examination of the
reactions of the pupils and determining whether the optic disk is
swollen. Magnetic resonance imaging (MRI) may show evidence of
multiple sclerosis or, rarely, a tumor pressing on the optic nerve,
in which case vision improves once the tumor pressure is relieved.
Most cases of optic neuritis improve over a few months without
treatment. In some cases, treatment with intravenous
corticosteroids may be necessary.
[0045] A cataract is an opacity that develops in the crystalline
lens of the eye or in its envelope. Cataracts typically cause
progressive vision loss and may cause blindness if left untreated.
In the Morgagnian Cataract, the cataract cortex progressively
liquefies to form a milky white fluid and may cause severe
inflammation if the lens capsule ruptures and leaks. If left
untreated, the cataract may also cause phacomorphic glaucoma.
Cataracts may be congenital in nature or caused by genetic factors,
advanced age, long-term ultraviolet exposure, exposure to
radiation, diabetes, eye injury or physical trauma.
[0046] Extra-capsular (ECCE) surgery is the most effective
treatment to treat cataract. In the surgery, the lens is removed,
but the majority of the lens capsule is left intact.
Phacoemulsification, a small incision on the side of the cornea, is
typically used to break up the lens before extraction.
[0047] Ocular amyloidosis is a hereditary disorder associated with
Type I Familial Amyloidotic Polyneuropathy (FAP) and characterized
by abnormal conjunctival vessels, keratoconjunctivitis sicca,
pupillary abnormalities and, in some cases, vitreous opacities and
secondary glaucoma. Type I FAP is associated with mutations in
transthyretin (TTR), a tetrameric plasma protein (prealbumin)
synthesized in the liver, the retinal pigment epithelium2 and
thechoroid plexus of the brain. Different mutations cause
transthyretin to polymerize into a pleated structure of amyloid
fibril, leading to hereditary amyloidosis. The most frequent
mutation is TTR-met303, in which methionine replaces valine at
position 30 in transthyretin.
[0048] Type IV FAP is associated with lattice corneal dystrophy
(LCD). Lattice corneal dystrophy is an inherited, primary, usually
bilateral corneal amyloidosis characterized by the presence of
refractile lattice lines with a double contour in the corneal
stroma. LCD type I (Biber-Haab-Dimmer) is an autosomal dominant,
bilaterally symmetrical corneal disorder characterized by the
presence of numerous translucent fine lattice lines with white dots
and faint haze in the superficial and middle layers of the central
stroma. The symptoms start during the first or second decades of
life, causing a progressive loss of vision. Most patients require a
corneal transplant by 40 years of age. LCD type II is associated
with systemic amyloidosis (Meretoja's syndrome) and is
characterized by the presence of thick lattice lines in the limbus,
central cornea and stroma. Vision is not affected until later in
life. LCD type III affect middle-age people and is characterized by
the presence of thick lattice lines that extend from limbus to
limbus. LCD type III A is characterized by the accumulation of
amyloid deposits in the stroma and the presence of ribbons of
amyloid between the stroma and Bowman's layer, LCD type III A
differs from LCD type III because of the presence of corneal
erosions, the occurrence in whites and the autosomal dominant
inheritance pattern.
[0049] Down's Syndrome (DS) or trisomy 21 is the most common
genetic disorder with an incidence of about 1:700 live births, and
is often associated with various congenital anomalies. The
disorder, which is caused by the presence of an extra chromosome
21, is associated with premature deposits of the plaque-forming
protein amyloid-beta and development of Alzheimer's disease by
middle age. Furthermore, many people affected by DS suffer from
cataracts beginning in childhood and many suffer from congenital
glaucoma. Since the gene for amyloid precursor protein, which is
cleaved to form amyloid beta, is located on the long arm of
chromosome 21 in humans, overexpression of this gene may lead to
increased levels of amyloid precursor protein and amyloid
deposition in Down's syndrome.
[0050] There is no cure for glaucoma. Medications for the treatment
of glaucoma include agents that decrease production of the aqueous
humor in the eye, such as beta blockers (Timoptic, Betoptic),
carbonic anhydrase inhibitors (Trusopt, Azopt), and alpha agonists
(Alphagan, Iopidine), and agents that redirect drainage of the
aqueous humor through a different pathway at the back of the eye,
such as prostaglandin (Xalatan). Laser surgeries include
trabeculoplasty, a procedure that helps the aqueous humor leave the
eye more efficiently. According to the Glaucoma Foundation, nearly
80% of patients respond well enough to the procedure to delay or
avoid further surgery. However, pressure increases again in the
eyes of half of all patients within two years after laser surgery,
according to the National Eye Institute. Incisional surgery is
performed if medication and initial laser treatments are
unsuccessful in reducing pressure within the eye. One type of
surgery, a trabeculectomy, creates an opening in the wall of the
eye so that aqueous humor can drain. However, about one-third of
trabeculectomy patients develop cataracts within five years,
according to the Glaucoma Foundation. If the trabeculectomy fails,
additional incisional procedures include placing a drainage tube
into the eye between the cornea and iris and the use of a laser or
freezing treatment to destroy tissue in the eye that makes aqueous
humor. Surgery may save the remaining vision in the patient, but it
does not improve sight. Vision may actually be worse following
surgery.
[0051] Age-related macular degeneration (AMD) is a major cause of
blindness among Caucasians over age 65. Although much progress has
been made recently in macular degeneration research, there are no
treatments that rescue neuronal cell death that occurs during the
course of the disease. There are also no definitive treatments for
other ocular diseases associated with amyloid beta-related neuronal
degradation, such as cortical visual deficits, optic nerve drusen,
optic neuropathy, optic neuritis, ocular amyloidosis and lattice
dystrophy.
[0052] Amyloid deposits typically contain three components. Amyloid
protein fibrils, which account for about 90% of the amyloid
material, comprise one of several different types of proteins.
These proteins are capable of folding into so-called "beta-pleated"
sheet fibrils, a unique protein configuration which exhibits
binding sites for Congo red resulting in the unique staining
properties of the amyloid protein. In addition, amyloid deposits
are closely associated with the amyloid P (pentagonal) component
(AP), a glycoprotein related to normal serum amyloid P (SAP), and
with sulphated glycosaminoglycans (GAG), complex carbohydrates of
connective tissue.
[0053] One development towards the treatment of Alzheimer's disease
and prion diseases has been the design of molecules that bind the
abnormal .beta.-sheet conformation of A.beta. and PrP,
respectively, thereby preventing aggregation of these molecules.
The .beta.-sheet conformation of peptides is characterized in that
hydrogen bonds are formed in a regular pattern between neighboring
amino acid strands. This arrangement leads to a stable three
dimensional structure. H-bond acceptors (C.dbd.O group) and H-bond
donors (NH group) alternate in naturally occurring .beta.-sheet
peptides with the atoms to be bonded being roughly in one line.
Within each amino acid strand, the distances between neighboring
H-bond donors and H-bond acceptors fall within specific ranges. In
particular, the distance between the H-bond donor (NH group) and
the H-bond acceptor (C.dbd.O group) within one amino acid residue
is from 3.5 to 4.0 .ANG.. The distance between the H-bond acceptor
(C.dbd.O group) of one amino acid residue and the H-bond donor (NH
group) of the following amino acid residue participating in the
inter-strand bonding is from 2.6 to 2.9 .ANG.. In other words, the
distances between neighboring H-bond donors and H-bond acceptors
within one amino acid strand alternate between the following
ranges: [0054] H-bond donor (amino acid 1)-H-bond acceptor (amino
acid 1)=3.5 to 4.0 .ANG.; [0055] H-bond acceptor (amino acid
1)-H-bond donor 2 (amino acid 2)=2.6 to 2.9 .ANG..
[0056] Ligands that are designed to bind n-sheets ideally have an
order of H-bond donors and H-bond acceptors that is complementary
to the order of H-bond donors and H-bond acceptors in the amino
acid strands of the .beta.-sheet.
[0057] In WO 03/095429 and Rzepecki et al., Synthesis (2003) 12,
1815-1826 synthetic molecules are described which are said to bind
the .beta.-conformation of A.beta. or PrP, thereby preventing their
aggregation. To this end, certain molecules were synthesized
containing two or more amino pyrazole moieties linked by carbonyl
group-containing linkers, e.g. "AmpOx" and "Trimer".
##STR00002##
[0058] Some of the molecules described in WO 03/095429 are said to
have an inhibiting effect on the formation of aggregates of A.beta.
in two biophysical assays. According to Rzepecki et al., Synthesis
(2003) 12, 1815-1826 one of the molecules described therein was
able to reduce the aggregation of a recombinant PrP.sup.C in
solution. Physicochemical properties, however, were not
investigated in these studies.
[0059] Physicochemical properties play a key role in the
penetration of the blood-brain barrier by neurotherapeutics.
Factors relevant to the success of CNS drugs have been reviewed (H.
Pajouhesh and G. R. Lenz, NeuroRx.RTM.: J. Am. Soc. Exp. Neurother.
(2005) Vol. 2, 541). These include the partition coefficient
between water and n-octanol (LogP), i.e. basically the
lipophilicity of the compound. Some of the compounds described in
WO 03/095429 and Rzepecki et al., Synthesis (2003) 12, 1815-1826
have an unfavorable calculated LogP and are, therefore, not
expected to pass the blood-brain barrier. In particular, "AmpOx"
has a calculated LogP below zero.
[0060] Other compounds described in the above documents have
properties that make them unsuitable for administration to a
patient due to their deleterious side-effects. For example,
"Trimer" is mutagenic, carcinogenic and metabolically unstable.
[0061] It was an object of the present invention to provide
compounds that can be employed in the treatment of diseases or
conditions associated with amyloid or amyloid-like proteins,
including amyloidosis. The compounds should be able to pass the
blood-brain barrier. Furthermore, they should be pharmaceutically
acceptable, in particular, they should not have mutagenic or
carcinogenic properties or be metabolically unstable.
[0062] A further object of the invention is to provide improved
treatment options for subjects affected by ocular diseases
associated with pathological abnormalities/changes in the tissues
of the visual system, particularly associated with
amyloid-beta-related pathological abnormalities/changes in the
tissues of the visual system, such as, for example, neuronal
degradation. Said pathological abnormalities may occur, for
example, in different tissues of the eye, such as the visual cortex
leading to cortical visual deficits; the anterior chamber and the
optic nerve leading to glaucoma; the lens leading to cataract due
to beta-amyloid deposition; the vitreous leading to ocular
amyloidoses; the retina leading to primary retinal degeneration and
macular degeneration, for example age-related macular degeneration;
the optic nerve leading to optic nerve drusen, optic neuropathy and
optic neuritis; and the cornea leading to lattice dystrophy.
[0063] The present inventors have surprisingly found that these
objects can be achieved by the compounds of the general formula
(II) as described hereinafter.
[0064] Accordingly, the present invention relates to a compound of
general formula (II).
[0065] In a further aspect, the present invention relates to a
pharmaceutical composition comprising a compound of general formula
(II).
[0066] Yet another aspect of the present invention relates to the
use of a compound of general formula (II) for the preparation of a
medicament for the treatment of diseases or conditions associated
with amyloid or amyloid-like proteins, including amyloidosis.
[0067] Also disclosed herein is a method of treating diseases or
conditions associated with amyloid or amyloid-like proteins,
comprising administering to a subject in need of such treatment an
effective amount of a compound of general formula (II).
[0068] Yet another aspect of the present invention relates to the
use of a compound of general formula (I) for the preparation of a
medicament for treating or alleviating the effects of ocular
diseases associated with pathological abnormalities/changes in the
tissues of the visual system.
[0069] Also disclosed herein is a method of treating or alleviating
the effects of ocular diseases associated with pathological
abnormalities/changes in the tissues of the visual system
comprising administering to a subject in need of such treatment an
effective amount of a compound of general formula (I).
[0070] The ocular diseases associated with pathological
abnormalities/changes in the tissues of the visual system are
particularly associated with amyloid-beta-related pathological
abnormalities/changes in the tissues of the visual system, such as,
for example, neuronal degradation. Said pathological abnormalities
may occur, for example, in different tissues of the eye, such as
the visual cortex leading to cortical visual deficits; the anterior
chamber and the optic nerve leading to glaucoma; the lens leading
to cataract due to beta-amyloid deposition; the vitreous leading to
ocular amyloidoses; the retina leading to primary retinal
degeneration and macular degeneration, for example age-related
macular degeneration; the optic nerve leading to optic nerve
drusen, optic neuropathy and optic neuritis; and the cornea leading
to lattice dystrophy.
[0071] In a further aspect the invention relates to a mixture (such
as a pharmaceutical composition) comprising a compound according to
the present invention and optionally at least one further
biologically active compound and/or a pharmaceutically acceptable
carrier and/or a diluent and/or an excipient. The further
biologically active substance can be a known compound used in the
medication of diseases and disorders which are caused by or
associated with amyloid or amyloid-like proteins including
amyloidosis, a group of diseases and disorders associated with
amyloid or amyloid-like protein such as the A.beta. protein
involved in Alzheimer's disease.
[0072] The further biologically active substance or compound may
exert its biological effect by the same or a similar mechanism as
the compound according to the invention or by an unrelated
mechanism of action or by a multiplicity of related and/or
unrelated mechanisms of action.
[0073] A method of collecting data for the diagnosis of an
amyloid-associated disease or condition in a sample or a patient is
also disclosed which comprises: [0074] (a) bringing a sample or a
specific body part or body area suspected to contain an amyloid
protein into contact with a compound according to the present
invention; [0075] (b) allowing the compound to bind to the amyloid
protein; [0076] (c) detecting the compound bound to the protein;
and [0077] (d) optionally correlating the presence or absence of
compound binding with the amyloid protein with the presence or
absence of amyloid protein in the sample or specific body part or
area.
[0078] Another embodiment of the present invention is a method of
determining the extent of amyloidogenic plaque burden in a tissue
and/or a body fluid comprising: [0079] (a) providing a sample
representative of the tissue and/or body fluid under investigation;
[0080] (b) testing the sample for the presence of amyloid protein
with a compound according to the present invention; [0081] (c)
determining the amount of compound bound to the amyloid protein;
and [0082] (d) calculating the plaque burden in the tissue and/or
body fluid.
[0083] A further aspect relates to a method of collecting data for
determining a predisposition to an amyloid-associated disease or
condition in a patient comprising detecting the specific binding of
a compound according to the present invention to an amyloid protein
in a sample or in situ which comprises the steps of: [0084] (a)
bringing the sample or a specific body part or body area suspected
to contain the amyloid protein into contact with a compound
according to the present invention, which compound specifically
binds to the amyloid protein; [0085] (b) allowing the compound to
bind to the amyloid protein to form a compound/protein complex;
[0086] (c) detecting the formation of the compound/protein complex;
[0087] (d) optionally correlating the presence or absence of the
compound/protein complex with the presence or absence of amyloid
protein in the sample or specific body part or area; and [0088] (e)
optionally comparing the amount of the compound/protein complex to
a normal control value.
[0089] Yet another aspect of the present invention is a method of
collecting data for monitoring minimal residual disease in a
patient following treatment with an antibody or a vaccine
composition, wherein the method comprises: [0090] (a) bringing a
sample or a specific body part or body area suspected to contain an
amyloid protein into contact with a compound according to the
present invention, which compound specifically binds to the amyloid
protein; [0091] (b) allowing the compound to bind to the amyloid
protein to form a compound/protein complex; [0092] (c) detecting
the formation of the compound/protein complex; [0093] (d)
optionally correlating the presence or absence of the
compound/protein complex with the presence or absence of amyloid
protein in the sample or specific body part or body area; and
[0094] (e) optionally comparing the amount of the compound/protein
complex to a normal control value.
[0095] A method of collecting data for predicting responsiveness of
a patient being treated with an antibody or a vaccine composition
is also described which comprises: [0096] (a) bringing a sample or
a specific body part or body area suspected to contain an amyloid
protein into contact with a compound according to the present
invention, which compound specifically binds to the amyloid
protein; [0097] (b) allowing the compound to bind to the amyloid
protein to form a compound/protein complex; [0098] (c) detecting
the formation of the compound/protein complex; [0099] (d)
optionally correlating the presence or absence of the
compound/protein complex with the presence or absence of amyloid
protein in the sample or specific body part or body area; and
[0100] (e) optionally comparing the amount of the compound/protein
complex to a normal control value.
[0101] A further aspect of the present invention is a test kit for
detection and diagnosis of an amyloid-associated disease or
condition comprising a compound according to the present
invention.
[0102] In a first embodiment the present invention relates to a
compound of the general formula (II)
##STR00003##
independently represents a single bond or a double bond. It is
evident that the selection of the two should lead to a compound
having sufficient stability for pharmaceutical applications.
Therefore, in a first preferred alternative one is a double bond
and the other is a single bond or in a second preferred alternative
both are a single bond. It will be understood that the first
preferred alternative, wherein one is a double bond and the other
is a single bond, includes embodiments, wherein the two are part of
an aromatic system.
[0103] p is 1, 2 or 3.
[0104] Each linker K is independently C.sub.1-3 alkylene which is
optionally substituted by one or more C.sub.1-4 alkyl groups.
Preferably, each linker K is --CH.sub.2--, --CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2--.
[0105] Each B is independently a 5- or 6-membered saturated or
unsaturated heterocyclic ring, wherein the heterocyclic ring B is
optionally substituted by one or more, preferably one or two,
substituents selected from C.sub.1-4 alkyl, C.sub.1-4 alkoxy, mono-
and di-C.sub.1-4 alkyl amino, C.sub.3-7 cycloalkyl amino, and 5- or
6-membered saturated heterocyclyl, or two substituents may be
joined to form a saturated, unsaturated or aromatic 5- to
7-membered ring which is fused with the heterocyclic ring B, and
wherein the heterocyclic ring B may contain in addition to the
units V and W one or more, preferably one or two, heteroatoms,
selected from N, NR, S and O, wherein R is selected from H and
C.sub.1-4 alkyl.
[0106] The 5- or 6-membered saturated heterocyclyl group contains
carbon atoms and 1 or more, preferably 1 or 2, heteroatoms selected
from N, NH, O and S. The nitrogen and sulfur atoms may optionally
be oxidized. The 5- or 6-membered saturated heterocyclyl group may
be attached to its pendant group at any heteroatom or carbon atom.
Examples of the 5- or 6-membered saturated heterocyclyl group
include, but are not limited to, pyrrolidinyl, piperidinyl and
morpholinyl.
[0107] Preferably, each heterocyclic ring B is independently
selected from optionally substituted indoline, optionally
substituted pyrazolylene, optionally substituted pyridinylene,
optionally substituted 2-pyridinonylene, optionally substituted
2-piperidonylene, optionally substituted thiazolylene and
optionally substituted isothiazolylene. More preferably, each
heterocyclic ring B is independently selected from the following
groups:
##STR00004##
[0108] These groups need not be incorporated in the indicated
direction but can also be incorporated in the opposite direction.
E.g.,
##STR00005##
can also be incorporated as
##STR00006##
Preferably, however, they are incorporated in the given
direction.
[0109] R.sup.1 is selected from --H, halogen, --C.sub.1-4 alkyl,
--NH.sub.2, --NH--C.sub.1-4 alkyl, --C.sub.1-4 alkylene-NH.sub.2,
--C.sub.1-4 alkylene-NH--C.sub.1-4 alkyl, -aryl, -aryl-R.sup.3,
--C.sub.1-4 alkylene-aryl, --C.sub.1-4 alkylene-aryl-R.sup.3,
-heteroaryl, -heteroaryl-R.sup.3, --NH--C.sub.1-4 alkylene-aryl,
--NH--C.sub.1-4 alkylene-aryl-R.sup.3, --OH and --O--C.sub.1-4
alkyl. R.sup.1 is preferably --H, --CH.sub.3, --NH--C.sub.1-4 alkyl
or --CH.sub.2NH--CH.sub.3.
[0110] R.sup.3 is C.sub.1-4 alkyl, halogen, OH or O--C.sub.1-4
alkyl.
[0111] R.sup.2 is --H, -aryl, --C.sub.1-4 alkyl or a group of the
formula
##STR00007##
wherein B, V, W and K are as defined above, q is 0 or 1 and r is 0
or 1.
[0112] Preferably, R.sup.2 is H or aryl.
[0113] Each unit W is independently a H-bond acceptor. Preferably,
each unit W is independently N or C.dbd.O.
[0114] Each unit V is independently optional and, if present, is
independently a H-bond donor. Each unit V is preferably NH.
[0115] Aryl is preferably a 5- to 7-membered aryl such as
phenyl.
[0116] Heteroaryl is preferably a 5- to 7-membered heteroaryl
(preferably a 5-membered heteroaryl), which includes at least one
heteroatom selected from O, S or N. Examples are
##STR00008##
[0117] Halogen is preferably F or Cl.
[0118] In one embodiment the compounds of the formula (II) have the
formula (II')
##STR00009##
[0119] independently represents a single bond or a double bond. It
is evident that the selection of the two should lead to a compound
having sufficient stability for pharmaceutical applications.
Therefore, in a first preferred alternative one is a double bond
and the other is a single bond or in a second preferred alternative
both are a single bond. It will be understood that the first
preferred alternative, wherein one is a double bond and the other
is a single bond, includes embodiments, wherein the two are part of
an aromatic system.
[0120] p is 2 or 3.
[0121] Each linker K is independently C.sub.1-3 alkylene which is
optionally substituted by one or more C.sub.1-4 alkyl groups.
Preferably, each linker K is --CH.sub.2-- or
--CH.sub.2CH.sub.2--.
[0122] Each B is independently a 5- or 6-membered heterocyclic
ring, wherein the heterocyclic ring B is optionally substituted by
one or more, preferably one or two, substituents selected from
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, mono and di C.sub.1-4 alkyl
amino, C.sub.3-7 cycloalkyl amino, and 5- or 6-membered saturated
heterocyclyl, or two substituents may be joined to form a
saturated, unsaturated or aromatic 5- to 7-membered ring which is
fused with the heterocyclic ring B, and wherein the heterocyclic
ring B may contain in addition to the units V and W one or more,
preferably one or two, heteroatoms, selected from N, NR, S and O,
wherein R is selected from H and C.sub.1-4 alkyl.
[0123] The 5- or 6-membered saturated heterocyclyl group contains
carbon atoms and 1 or more, preferably 1 or 2, heteroatoms selected
from N, NH, O and S. The nitrogen and sulfur atoms may optionally
be oxidized. The 5- or 6-membered saturated heterocyclyl group may
be attached to its pendant group at any heteroatom or carbon atom.
Examples of the 5- or 6-membered saturated heterocyclyl group
include, but are not limited to, pyrrolidinyl, piperidinyl and
morpholinyl.
[0124] Preferably, each heterocyclic ring B is independently
selected from optionally substituted pyrazolylene, optionally
substituted pyridinylene, optionally substituted 2-pyridinonylene,
optionally substituted 2-piperidonylene, optionally substituted
thiazolylene and optionally substituted isothiazolylene. More
preferably, each heterocyclic ring B is independently selected from
the following groups:
##STR00010##
[0125] R.sup.1 is selected from H, C.sub.1-4 alkyl, NH.sub.2,
NH--C.sub.1-4 alkyl, C.sub.1-4 alkylene-NH.sub.2, C.sub.1-4
alkylene-NH--C.sub.1-4 alkyl, OH and O--C.sub.1-4 alkyl. R.sup.1 is
preferably H, CH.sub.3, NH--CH.sub.3 or CH.sub.2--NH--CH.sub.3.
[0126] R.sup.2 is H, or a group of the formula
##STR00011##
wherein B, V, W and K are as defined above and q is 0 or 1.
[0127] Preferably, R.sup.2 is H.
[0128] Each unit W is independently a H-bond acceptor. Preferably,
each unit W is independently N or C.dbd.O.
[0129] Each unit V is independently optional and, if present, is
independently a H-bond donor. Each unit V is preferably NH.
[0130] Preferred compounds are summarized in the following
table.
TABLE-US-00001 2a ##STR00012## 2c ##STR00013## 2h ##STR00014## 2j
##STR00015## 2k ##STR00016## 2n ##STR00017## 2o ##STR00018## 2p
##STR00019## 2q ##STR00020## 2s ##STR00021## 2t ##STR00022## 2u
##STR00023## 2w ##STR00024## 2y ##STR00025## 2z ##STR00026## 2aa
##STR00027## 2ab ##STR00028## 2ac ##STR00029## 2ad ##STR00030## 2ae
##STR00031## 2af ##STR00032## 2ag ##STR00033## 2ah ##STR00034## 2ai
##STR00035## 2ak ##STR00036## 2al ##STR00037## 2am ##STR00038##
[0131] In the compounds of the present invention H-bond donors and
H-bond acceptors are preferably arranged in a pattern which is
essentially complementary to the pattern of H-bond donors and
H-bond acceptors present in the amino acid strands of .beta.-sheet
structures as set out in the introductory part. In particular, the
distances between neighboring H-bond donors and H-bond acceptors in
the compounds of the present invention are preferably within the
range of 2.6 to 2.9 .ANG. or 3.5 to 4.0 .ANG..
[0132] The distances between neighboring H-bond donors and H-bond
acceptors in the compounds of the present invention can, for
example, directly measured from the Dreiding models of the
compounds. Alternatively, molecular modeling computer programs,
such as MacroModel 7.2, can be used for the distance
determination.
[0133] The compounds of the present invention do not only feature a
H-bond donor/acceptor pattern which promotes their binding to the
amino acid strands of .beta.-sheet structures, they also have
favorable physicochemical properties which facilitate their use as
neurotherapeutics. In particular, their lipophilicity is in a range
which should enhance their penetration of the blood-brain barrier.
Preferably, the calculated partition coefficient (milogP) between
water and n-octanol of the compounds of the present invention is in
the range of from 0 and 4, more preferably from 1 to 3.
[0134] milogP values can be calculated according to the software
available on the world wide web (http://www.molinspiration.com),
provided by P. Ertl of Novartis Pharma AG. A copy of the software
is also available from the applicant.
[0135] In addition to lipophilicity, the polar surface area (PSA)
of a molecule is an important factor for the determination of the
suitability of a compound as a neurotherapeutic (H. Pajouhesh et
al., NeuroRx.RTM.: J. Am. Soc. Exp. Neurother. (2005) Vol. 2, 541).
The PSA is defined as the surface area (.ANG..sup.2) occupied by
nitrogen and oxygen atoms and the polar hydrogens attached to them.
It is strongly reflective of hydrogen bonding capacity and
polarity. While PSA takes into account the three-dimensional
structure of a molecule, topological PSA (TPSA) is based on the
corresponding two-dimensional structure. PSA and TPSA provide
similar results, with TPSA enabling a significantly larger
throughput due to its computationally less intensive
two-dimensional representation. The compounds of the present
invention generally have a TPSA which facilitates penetration of
the blood-brain barrier. Preferably, the TPSA of the compounds of
the present invention is equal to or below 90 .ANG..sup.2.
[0136] TPSA values can be calculated according to the software
available on the world wide web (http://www.molinspiration.com),
provided by P. Ertl of Novartis Pharma AG. A copy of the software
is also available from the applicant.
[0137] The compounds of the general formula (II) can be built
stepwise via formation of peptide bonds and subsequent reduction
with borane dimethylsulfide complex to yield the secondary
amines.
[0138] While it is possible for the compounds of the present
invention to be administered alone, it is preferable to formulate
them into a pharmaceutical composition in accordance with standard
pharmaceutical practice. Thus the invention also provides a
pharmaceutical composition which comprises a therapeutically
effective amount of a compound of formula (II) in admixture with a
pharmaceutically acceptable excipient.
[0139] Pharmaceutically acceptable excipients are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, 15.sup.th Ed., Mack Publishing Co., New
Jersey (1991). The pharmaceutical excipient can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. The excipient must be acceptable in the
sense of being not deleterious to the recipient thereof.
[0140] Pharmaceutically useful excipients that may be used in the
formulation of the pharmaceutical composition of the present
invention may comprise, for example, carriers, vehicles, diluents,
solvents such as monohydric alcohols such as ethanol, isopropanol
and polyhydric alcohols such as glycols and edible oils such as
soybean oil, coconut oil, olive oil, safflower oil cottonseed oil,
oily esters such as ethyl oleate, isopropyl myristate, binders,
adjuvants, solubilizers, thickening agents, stabilizers,
disintegrants, glidants, lubricating agents, buffering agents,
emulsifiers, wetting agents, suspending agents, sweetening agents,
colorants, flavors, coating agents, preservatives, antioxidants,
processing agents, drug delivery modifiers and enhancers such as
calcium phosphate, magnesium state, talc, monosaccharides,
disaccharides, starch, gelatine, cellulose, methylcellulose, sodium
carboxymethyl cellulose, dextrose,
hydroxypropyl-.beta.-cyclodextrin, polyvinylpyrrolidone, low
melting waxes, and ion exchange resins.
[0141] The routes for administration (delivery) of the compounds of
the invention include, but are not limited to, one or more of: oral
(e.g. as a tablet, capsule, or as an ingestible solution), topical,
mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal,
parenteral (e.g. by an injectable form), gastrointestinal,
intraspinal, intraperitoneal, intramuscular, intravenous,
intrauterine, intraocular, intradermal, intracranial,
intratracheal, intravaginal, intracerebroventricular,
intracerebral, subcutaneous, ophthalmic (including intravitreal or
intracameral), transdermal, rectal, buccal, epidural and
sublingual.
[0142] For example, the compounds can be administered orally in the
form of tablets, capsules, ovules, elixirs, solutions or
suspensions, which may contain flavoring or coloring agents, for
immediate-, delayed-, modified-, sustained-, pulsed- or
controlled-release applications.
[0143] The tablets may contain excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine, disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates,
and granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
sucrose, gelatin and acacia. Additionally, lubricating agents such
as magnesium stearate, stearic acid, glyceryl behenate and talc may
be included. Solid compositions of a similar type may also be
employed as fillers in gelatin capsules. Preferred excipients in
this regard include lactose, starch, a cellulose, milk sugar or
high molecular weight polyethylene glycols. For aqueous suspensions
and/or elixirs, the agent may be combined with various sweetening
or flavoring agents, coloring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, ethanol,
propylene glycol and glycerin, and combinations thereof.
[0144] If the compounds of the present invention are administered
parenterally, then examples of such administration include one or
more of: intravenously, intraarterially, intraperitoneally,
intrathecally, intraventricularly, intraurethrally, intrasternally,
intracranially, intramuscularly or subcutaneously administering the
compounds; and/or by using infusion techniques. For parenteral
administration, the compounds are best used in the form of a
sterile aqueous solution which may contain other substances, for
example, enough salts or glucose to make the solution isotonic with
blood. The aqueous solutions should be suitably buffered
(preferably to a pH of from 3 to 9), if necessary. The preparation
of suitable parenteral formulations under sterile conditions is
readily accomplished by standard pharmaceutical techniques well
known to those skilled in the art.
[0145] As indicated, the compounds of the present invention can be
administered intranasally or by inhalation and are conveniently
delivered in the form of a dry powder inhaler or an aerosol spray
presentation from a pressurized container, pump, spray or nebulizer
with the use of a suitable propellant, e.g.
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetra-fluoroethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA134AT) or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurized container, pump, spray or nebulizer
may contain a solution or suspension of the active compound, e.g.
using a mixture of ethanol and the propellant as the solvent, which
may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use
in an inhaler or insufflator may be formulated to contain a powder
mix of the compound and a suitable powder base such as lactose or
starch.
[0146] Alternatively, the compounds of the present invention can be
administered in the form of a suppository or pessary, or it may be
applied topically in the form of a gel, hydrogel, lotion, solution,
cream, ointment or dusting powder. The compounds of the present
invention may also be dermally or transdermally administered, for
example, by the use of a skin patch.
[0147] They may also be administered by the pulmonary or rectal
routes. They may also be administered by the ocular route. For
ophthalmic use, the compounds can be formulated as micronized
suspensions in isotonic, pH adjusted, sterile saline, or,
preferably, as solutions in isotonic, pH adjusted, sterile saline,
optionally in combination with a preservative such as a
benzylalkonium chloride. Alternatively, they may be formulated in
an ointment such as petrolatum.
[0148] For application topically to the skin, the compounds of the
present invention can be formulated as a suitable ointment
containing the active compound suspended or dissolved in, for
example, a mixture with one or more of the following: mineral oil,
liquid petrolatum, white petrolatum, propylene glycol, emulsifying
wax and water. Alternatively, they can be formulated as a suitable
lotion or cream, suspended or dissolved in, for example, a mixture
of one or more of the following: mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and water.
[0149] Typically, a physician will determine the actual dosage
which will be most suitable for an individual subject. The specific
dose level and frequency of dosage for any particular individual
may be varied and will depend upon a variety of factors including
the activity of the specific compound employed, the metabolic
stability and length of action of that compound, the age, body
weight, general health, sex, diet, mode and time of administration,
rate of excretion, drug combination, the severity of the particular
condition, and the individual undergoing therapy.
[0150] A proposed dose of the compounds according to the present
invention for administration to a human (of approximately 70 kg
body weight) is 0.1 mg to 1 g, preferably 1 mg to 500 mg of the
active ingredient per unit dose. The unit dose may be administered,
for example, 1 to 4 times per day. The dose will depend on the
route of administration. It will be appreciated that it may be
necessary to make routine variations to the dosage depending on the
age and weight of the patient as well as the severity of the
condition to be treated. The precise dose and route of
administration will ultimately be at the discretion of the
attendant physician or veterinarian.
[0151] The compounds of the invention may also be used in
combination with other therapeutic agents. When a compound of the
invention is used in combination with a second therapeutic agent
active against the same disease the dose of each compound may
differ from that when the compound is used alone.
[0152] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation. The
individual components of such combinations may be administered
either sequentially or simultaneously in separate or combined
pharmaceutical formulations by any convenient route. When
administration is sequential, either the compound of the invention
or the second therapeutic agent may be administered first. When
administration is simultaneous, the combination may be administered
either in the same or different pharmaceutical composition. When
combined in the same formulation it will be appreciated that the
two compounds must be stable and compatible with each other and the
other components of the formulation. When formulated separately
they may be provided in any convenient formulation, conveniently in
such manner as are known for such compounds in the art.
[0153] The pharmaceutical compositions of the invention can be
produced in a manner known per se to the skilled person as
described, for example, in Remington's Pharmaceutical Sciences,
15.sup.th Ed., Mack Publishing Co., New Jersey (1991).
[0154] Diseases that can be treated with the compounds of the
present invention can be associated with the formation of abnormal
protein structures, in particular abnormal .beta.-sheet structures.
In the context of the present invention, an abnormal protein
structure is a protein structure that arises when a protein or
peptide refolds from the three-dimensional structure, which it
generally adopts in healthy individuals, into a different
three-dimensional structure, which is associated with a
pathological condition. Likewise, an abnormal .beta.-sheet
structure in the context of the present invention is a .beta.-sheet
structure that arises when a protein or peptide refolds from the
three-dimensional structure, which it generally adopts in healthy
individuals, into a .beta.-sheet structure, which is associated
with a pathological condition.
[0155] In particular, in one embodiment diseases that can be
treated with the compounds of the present invention are diseases or
conditions associated with amyloid or amyloid-like proteins.
[0156] This group of diseases and disorders include neurological
disorders such as Alzheimer's Disease (AD), diseases or conditions
characterized by a loss of cognitive memory capacity such as, for
example, mild cognitive impairment (MCI), Lewy body dementia,
Down's syndrome, hereditary cerebral hemorrhage with amyloidosis
(Dutch type); the Guam Parkinson-Dementia complex. Other diseases
which are based on or associated with amyloid-like proteins are
progressive supranuclear palsy, multiple sclerosis; Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM),
Adult Onset Diabetes; senile cardiac amyloidosis; endocrine tumors,
and other diseases, including amyloid-associated ocular diseases
that target different tissues of the eye, such as the visual
cortex, including cortical visual deficits; the anterior chamber
and the optic nerve, including glaucoma; the lens, including
cataract due to beta-amyloid deposition; the vitreous, including
ocular amyloidoses; the retina, including primary retinal
degenerations and macular degeneration, in particular age-related
macular degeneration; the optic nerve, including optic nerve
drusen, optic neuropathy and optic neuritis; and the cornea,
including lattice dystrophy.
[0157] In a preferred embodiment the compounds of the present
invention can be employed for the treatment of Alzheimer's disease,
mild cognitive impairment (MCI), Lewy body dementia (LBD),
amyotropic lateral sclerosis (ALS), inclusion-body myositis (IBM)
and age-related macular degeneration (AMD). In a particulary
preferred embodiment the compounds of the present invention can be
employed for the treatment of Alzheimer's disease.
[0158] The ability of a compound to inhibit the aggregation of
A.beta. can, for example, be determined using fluorescence
correlation spectroscopy as described in Rzepecki et al., J. Biol.
Chem., 2004, 279(46), 47497-47505 or by using the thioflavin T
spectrofluorescence assay.
[0159] In another embodiment the compounds of the present invention
can be used for treating or alleviating the effects of ocular
diseases associated with pathological abnormalities/changes in the
tissues of the visual system, particularly associated with
amyloid-beta-related pathological abnormalities/changes in the
tissues of the visual system, such as, for example, neuronal
degradation. Said pathological abnormalities may occur, for
example, in different tissues of the eye, such as the visual cortex
leading to cortical visual deficits; the anterior chamber and the
optic nerve leading to glaucoma; the lens leading to cataract due
to beta-amyloid deposition; the vitreous leading to ocular
amyloidoses; the retina leading to primary retinal degeneration and
macular degeneration, for example age-related macular degeneration;
the optic nerve leading to optic nerve drusen, optic neuropathy and
optic neuritis; and the cornea leading to lattice dystrophy.
[0160] The compounds according to the present invention can also be
provided in the form of a mixture with at least one further
biologically active compound and/or a pharmaceutically acceptable
carrier and/or a diluent and/or an excipient. The compound and/or
the further biologically active compound are preferably present in
a therapeutically effective amount.
[0161] The nature of the further biologically active compound will
depend on the intended use of the mixture. The further biologically
active substance or compound may exert its biological effect by the
same or a similar mechanism as the compound according to the
invention or by an unrelated mechanism of action or by a
multiplicity of related and/or unrelated mechanisms of action.
[0162] Generally, the further biologically active compound may
include neutron-transmission enhancers, psychotherapeutic drugs,
acetylcholine esterase inhibitors, calcium-channel blockers,
biogenic amines, benzodiazepine tranquillizers, acetylcholine
synthesis, storage or release enhancers, acetylcholine postsynaptic
receptor agonists, monoamine oxidase-A or -B inhibitors,
N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal
anti-inflammatory drugs, antioxidants, and serotonergic receptor
antagonists. In particular, the further biologically active
compound can be selected from the group consisting of a compound
used in the treatment of amyloidoses, compounds against oxidative
stress, anti-apoptotic compounds, metal chelators, inhibitors of
DNA repair such as pirenzepin and metabolites,
3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate
(1,3PDS), .alpha.-secretase activators, .beta.- and
.gamma.-secretase inhibitors, tau proteins, neurotransmitter,
.beta.-sheet breakers, attractants for amyloid beta
clearing/depleting cellular components, inhibitors of N-terminal
truncated amyloid beta including pyroglutamated amyloid beta 3-42,
anti-inflammatory molecules, or cholinesterase inhibitors (ChEIs)
such as tacrine, rivastigmine, donepezil, and/or galantamine, M1
agonists, other drugs including any amyloid or tau modifying drug
and nutritive supplements, an antibody, including any functionally
equivalent antibody or functional parts thereof, an A.beta.
antigenic peptide fragment consisting of a single or repetitive
stretch of a plurality of contiguous amino acid residues from the
N-terminal part of the A.beta. peptide.
[0163] In a further embodiment, the mixtures according to the
invention may comprise niacin or memantine together with a compound
according to the present invention and, optionally, a
pharmaceutically acceptable carrier and/or a diluent and/or an
excipient.
[0164] In still another embodiment of the invention mixtures are
provided that comprise as a further biologically active compound
"atypical antipsychotics" such as, for example clozapine,
ziprasidone, risperidone, aripiprazole or olanzapine for the
treatment of positive and negative psychotic symptoms including
hallucinations, delusions, thought disorders (manifested by marked
incoherence, derailment, tangentiality), and bizarre or
disorganized behavior, as well as anhedonia, flattened affect,
apathy, and social withdrawal, together with a compound according
to the invention and, optionally, a pharmaceutically acceptable
carrier and/or a diluent and/or an excipient.
[0165] Other compounds that can be suitably used in mixtures in
combination with the compound according to the present invention
are, for example, described in WO 2004/058258 (see especially pages
16 and 17) including therapeutic drug targets (pages 36 to 39),
alkanesulfonic acids and alkanolsulfuric acids (pages 39 to 51),
cholinesterase inhibitors (pages 51 to 56), NMDA receptor
antagonists (pages 56 to 58), estrogens (pages 58 to 59),
non-steroidal anti-inflammatory drugs (pages 60 and 61),
antioxidants (pages 61 and 62), peroxisome proliferators-activated
receptor (PPAR) agonists (pages 63 to 67), cholesterol-lowering
agents (pages 68 to 75), amyloid inhibitors (pages 75 to 77),
amyloid formation inhibitors (pages 77 to 78), metal chelators
(pages 78 and 79), anti-psychotics and anti-depressants (pages 80
to 82), nutritional supplements (pages 83 to 89) and compounds
increasing the availability of biologically active substances in
the brain (see pages 89 to 3) and prodrugs (pages 93 and 94), which
document is incorporated herein by reference.
[0166] In one preferred embodiment the further biologically active
compound is an antibody including any functionally equivalent
antibody or functional parts thereof. The antibody can preferably
be monoclonal, chimeric or humanized.
[0167] In a further aspect of the invention, a mixture is provided
comprising in addition to the compound of the invention an antibody
including functional parts thereof, or, more particularly, a
monoclonal antibody including functional parts thereof, which
recognizes and binds to amyloid .beta. (A.beta.), particularly to
the native conformation of amyloid .beta., that is to amyloid
oligomers and fibers, but not to not linearized amyloid
species.
[0168] In particular, said antibodies are capable of inhibiting, in
vitro and in vivo, the aggregation of amyloidogenic monomeric
peptides, specifically .beta.-amyloid monomeric peptides such as,
for example, A.beta. monomeric peptides 1-39; 1-40, 1-41, 1-42, or
1-43, but especially A.beta..sub.1-42 monomeric peptides, into high
molecular polymeric amyloid fibrils or filaments. Through the
inhibition of the aggregation of amyloidogenic monomeric peptides
these antibodies are capable of preventing or slowing down the
formation of amyloid plaques, particularly the amyloid form (1-42),
which is know to become insoluble by change of secondary
conformation and to be the major part of amyloid plaques in brains
of diseased animals or humans.
[0169] In another aspect of the invention, the mixture comprises
antibodies which, upon co-incubation with preformed high molecular
polymeric amyloid fibrils or filaments formed by the aggregation of
amyloid monomeric peptides, specifically .beta.-amyloid monomeric
peptides such as, for example, A.beta. monomeric peptides 1-39;
1-40, 1-41, 1-42, or 1-43, but especially A.beta..sub.1-42
monomeric peptides, are capable of disaggregating said high
molecular polymeric amyloid fibrils or filaments. Through the
disaggregation of amyloidogenic polymeric fibrils or filaments
these antibodies are capable of preventing or slowing down the
formation of amyloid plaques which leads to an alleviation of the
symptoms associated with the disease and a delay or reversal of its
progression.
[0170] In still another aspect of the invention, the mixture
comprises an antibody, but especially a monoclonal antibody or
functional parts thereof, which antibody is bifunctional or
bispecific in that it exhibit both an aggregation inhibition
property as well as a disaggregation property as defined herein
before, particularly paired with a high degree of conformational
sensitivity.
[0171] In one embodiment, the mixture comprises an antibody which
recognizes and binds to a conformational epitope, particularly
conformational epitope which is present in the N-terminal part of
the amyloid .beta. peptide, particularly embedded into the
following core region of the N-terminal part of the amyloid .beta.
peptide:
TABLE-US-00002 Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp- 12
13 14 15 16 17 18 19 20 21 22 23
[0172] Particularly an epitope localized in a region of the
.beta.-amyloid protein between amino acid residue 12 to 24,
particularly between residues 14 to 23, more particularly between
amino acid residues 14 and 20, comprising three distinct
recognition and binding sites which residues are predominantly
involved in the binding of the .beta.-amyloid protein and located
at position 16, 17, and at position 19 and 20, and at position 14,
respectively.
[0173] In a specific embodiment the mixture of the present
invention comprises, in addition to the compound of the invention,
an antibody, particularly a bifunctional antibody, but especially a
monoclonal antibody, particularly a bifunctional monoclonal
antibody, including any functionally equivalent antibody or
functional parts thereof, which antibody has the characteristic
properties of an antibody produced by a hybridoma cell line
selected from the group consisting of FP 12H3, FP 12H3-C2, and FP
12H3-G2 deposited on Dec. 1, 2005 and Dec. 9, 2005, respectively,
as DSM ACC2752, DSM ACC 2750 and DSM ACC2751, respectively, ET 7E3
deposited on Dec. 8, 2005 as DSM ACC2755, and EJ 7H3 deposited on
Dec. 8, 2005 as DSM ACC2756.
[0174] More particularly, the invention relates to an antibody
including any functionally equivalent antibody or functional parts
thereof produced by a hybridoma cell line selected from the group
consisting of FP 12H3, FP 12H3-C2, and FP 12H3-G2 deposited on Dec.
1, 2005 and Dec. 9, 2005, respectively, as DSM ACC2752, DSM ACC
2750 and DSM ACC2751, respectively, ET 7E3 deposited on Dec. 8,
2005 as DSM ACC2755, and EJ 7H3 deposited on Dec. 8, 2005 as DSM
ACC2756.
[0175] The above antibodies are described in the published
international application WO 2007/068412, which is incorporated
herein by reference.
[0176] In a further aspect, the antibody which is comprised in the
mixture according to the invention is a chimeric antibody or a
fragment thereof, or a humanized antibody or a fragment thereof.
These and further antibodies that can be suitably used within the
mixtures according to the present invention are described, for
example, in international application PCT/US2007/073504 filed Jul.
13, 2007.
[0177] If the antibody is a humanized antibody, it preferably
exhibits a light chain and a heavy chain as depicted in SEQ ID No.
2 and SEQ ID No. 4 of International Application No.
PCT/US2007/073504 or exhibits a light chain variable region and a
heavy chain variable region as depicted in SEQ ID No. 1 and SEQ ID
No. 3 of International Application No. PCT/US2007/073504. These
sequences are also shown in the attached sequence listing.
[0178] In still another aspect of the invention, a mixture is
provided which comprises, in addition to the compound according to
the invention and as described herein before, a peptide fragment
from the N-terminal part of the A.beta. peptide, particularly an
A.beta. peptide fragment consisting of a single or repetitive
stretch of between 13 and 15 contiguous amino acid residues from
the N-terminal part of the A.beta. peptide, but particularly an
A.beta. peptide fragment consisting of amino acid residues selected
from the group consisting of residues 1-15, 1-14, and 1-13 from the
N-terminal part of the A.beta. peptide, more particularly of
residue 1-15, including functionally equivalent fragments thereof,
but especially a A.beta. peptide fragment as mentioned herein
before attached to, or incorporated or reconstituted in a carrier
particle/adjuvant such as, for example, a liposome. The peptide
fragment can be comprised in a vaccine composition. In particular,
the peptide antigen is modified by a lipophilic or hydrophobic
moiety, that facilitates insertion into the lipid bilayer of the
liposome carrier/immune adjuvant, particularly by a lipophilic or
hydrophobic moiety which functions as an anchor for the peptide in
the liposome bilayer and has a dimension that leads to the peptide
being positioned and stabilized in close proximity to the liposome
surface.
[0179] In a further embodiment of the invention, the lipophilic or
hydrophobic moiety is a fatty acid, a triglyceride or a
phospholipid, but especially a fatty acid, a triglyceride or a
phospholipid. In particular, the hydrophobic moiety is palmitic
acid and the liposome preparation may in addition contain an
adjuvant such as, for example, lipid A, alum, calcium phosphate,
interleukin 1, and/or microcapsules of polysaccharides and
proteins, but particularly a detoxified lipid A, such as
monophosphoryl or diphosphoryl lipid A, or alum.
[0180] These and further compositions that can be suitably used in
the mixtures of the present invention are described, for example,
in the published international application WO 2007/068411.
[0181] Diagnosis of an amyloid-associated disease or condition or
of a predisposition to an amyloid-associated disease or condition
in a patient may be achieved by detecting the specific binding of a
compound according to the invention to the amyloid protein in a
sample or in situ, which includes bringing the sample or a specific
body part or body area suspected to contain the amyloid antigen
into contact with a compound of the invention which binds the
amyloid protein, allowing the compound of the invention to bind to
the amyloid protein to form a compound/protein complex, detecting
the formation of the compound/protein complex and correlating the
presence or absence of the compound/protein complex with the
presence or absence of amyloid protein in the sample or specific
body part or area, optionally comparing the amount of said
compound/protein complex to a normal control value, wherein an
increase in the amount of said aggregate compared to a normal
control value may indicate that said patient is suffering from or
is at risk of developing an amyloid-associated disease or
condition.
[0182] Monitoring minimal residual disease in a patient following
treatment with a compound or a mixture according to the invention
may be achieved by detecting the specific binding of a compound
according to the invention to the amyloid protein in a sample or in
situ, which includes bringing the sample or a specific body part or
body area suspected to contain the amyloid antigen into contact
with a compound of the invention which binds the amyloid protein,
allowing the compound to bind to the amyloid protein to form an
compound/protein complex, detecting the formation of the
compound/protein complex and correlating the presence or absence of
the compound/protein complex with the presence or absence of
amyloid protein in the sample or specific body part or area,
optionally comparing the amount of said compound/protein complex to
a normal control value, wherein an increase in the amount of said
aggregate compared to a normal control value may indicate that said
patient may still suffer from a minimal residual disease.
[0183] Predicting responsiveness of a patient to a treatment with a
compound or composition or a mixture according to the invention may
be achieved by detecting the specific binding of a compound
according to the invention to the amyloid protein in a sample or in
situ, which includes bringing the sample or a specific body part or
body area suspected to contain the amyloid protein into contact
with a compound of the invention which binds the amyloid protein,
allowing the compound to bind to the amyloid protein to form an
compound/protein complex, detecting the formation of the
compound/protein complex and correlating the presence or absence of
the compound/protein complex with the presence or absence of
amyloid protein in the sample or specific body part or area,
optionally comparing the amount of said compound/protein complex
before and after onset of the treatment, wherein an decrease in the
amount of said aggregate may indicate that said patient has a high
potential of being responsive to the treatment.
[0184] Biological samples that may be used in the diagnosis of an
amyloid-associated disease or condition for diagnosing a
predisposition to an amyloid-associated disease or condition or for
monitoring minimal residual disease in a patient or for predicting
responsiveness of a patient to a treatment with a compound or a
composition or a mixture according to the invention and as
described herein before are, for example, fluids such as serum,
plasma, saliva, gastric secretions, mucus, cerebrospinal fluid,
lymphatic fluid and the like or tissue or cell samples obtained
from an organism such as neural, brain, cardiac or vascular tissue.
For determining the presence or absence of the amyloid protein in a
sample any immunoassay known to those of ordinary skill in the art
(see Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring
Harbor Laboratory, New York, 1988, 555 to 612) may be used such as,
for example, assays which utilize indirect detection methods using
secondary reagents for detection, ELISA's and immunoprecipitation
and agglutination assays. A detailed description of these assays
is, for example, given in WO96/13590 to Maertens and Stuyver, Zrein
et al. (1998) and WO96/29605.
[0185] For in situ diagnosis, the compound or composition or
mixture according to the invention and as described herein before
may be administered to the organism to be diagnosed by methods
known in the art such as, for example, intravenous, intranasal,
intraperitoneal, intracerebral, intraarterial injection such that a
specific binding between the compound according to the invention
and the amyloid antigen may occur. The compound/protein complex may
be detected through a label attached to the compound.
[0186] The immunoassays used in diagnostic applications or in
applications for diagnosing a predisposition to an
amyloid-associated disease or condition or for monitoring minimal
residual disease in a patient or for predicting responsiveness of a
patient to a treatment with a compound or composition or a mixture
according to the invention and as described herein before,
typically rely on labelled antigens, antibodies, or secondary
reagents for detection. These proteins or reagents can be labelled
with compounds generally known to those skilled in the art
including enzymes, radioisotopes, and fluorescent, luminescent and
chromogenic substances including colored particles, such as
colloidal gold and latex beads. Of these, radioactive labelling can
be used for almost all types of assays and with most variations.
Enzyme-conjugated labels are particularly useful when radioactivity
must be avoided or when quick results are needed. Fluorochromes,
although requiring expensive equipment for their use, provide a
very sensitive method of detection. Antibodies useful in these
assays include monoclonal antibodies, polyclonal antibodies, and
affinity purified polyclonal antibodies.
[0187] Alternatively, the compound of the invention may be labelled
indirectly by reaction with labelled substances that have an
affinity for immunoglobulin, such as protein A or G or second
antibodies. The antibody may be conjugated with a second substance
and detected with a labelled third substance having an affinity for
the second substance conjugated to the antibody. For example, the
antibody may be conjugated to biotin and the antibody-biotin
conjugate detected using labelled avidin or streptavidin.
Similarly, the antibody may be conjugated to a hapten and the
antibody-hapten conjugate detected using labelled anti-hapten
antibody.
[0188] Those of ordinary skill in the art will know of these and
other suitable labels which may be employed in accordance with the
present invention. The binding of these labels to antibodies or
fragments thereof can be accomplished using standard techniques
commonly known to those of ordinary skill in the art. Typical
techniques are described by Kennedy, J. H., et al., 1976 (Clin.
Chim. Acta 70:1-31), and Schurs, A. H. W. M., et al. 1977 (Clin.
Chim Acta 81:1-40). Coupling techniques mentioned in the latter are
the glutaraldehyde method, the periodate method, the dimaleimide
method, and others, all of which are incorporated by reference
herein.
[0189] Current immunoassays utilize a double antibody method for
detecting the presence of an analyte, wherein the antibody is
labelled indirectly by reactivity with a second antibody that has
been labelled with a detectable label. The second antibody is
preferably one that binds to antibodies of the animal from which
the monoclonal antibody is derived. In other words, if the
monoclonal antibody is a mouse antibody, then the labelled, second
antibody is an anti-mouse antibody. For the monoclonal antibody to
be used in the assay described below, this label is preferably an
antibody-coated bead, particularly a magnetic bead. For the
polyclonal antibody to be employed in the immunoassay described
herein, the label is preferably a detectable molecule such as a
radioactive, fluorescent or an electrochemiluminescent
substance.
[0190] An alternative double antibody system, often referred to as
fast format systems because they are adapted to rapid
determinations of the presence of an analyte, may also be employed
within the scope of the present invention. The system requires high
affinity between the antibody and the analyte. According to one
embodiment of the present invention, the presence of the amyloid
antigen is determined using a pair of antibodies, each specific for
amyloid antigen. One of said pairs of antibodies is referred to
herein as a "detector antibody" and the other of said pair of
antibodies is referred to herein as a "capture antibody". The
monoclonal antibody can be used as either a capture antibody or a
detector antibody. The monoclonal antibody can also be used as both
capture and detector antibody, together in a single assay. One
embodiment of the present invention thus uses the double antibody
sandwich method for detecting amyloid antigen in a sample of
biological fluid. In this method, the analyte (amyloid antigen) is
sandwiched between the detector antibody and the capture antibody,
the capture antibody being irreversibly immobilized onto a solid
support. The detector antibody would contain a detectable label, in
order to identify the presence of the antibody-analyte sandwich and
thus the presence of the analyte.
[0191] Exemplary solid phase substances include, but are not
limited to, microtiter plates, test tubes of polystyrene, magnetic,
plastic or glass beads and slides which are well known in the field
of radioimmunoassay and enzyme immunoassay. Methods for coupling
antibodies to solid phases are also well known to those skilled in
the art. More recently, a number of porous material such as nylon,
nitrocellulose, cellulose acetate, glass fibers and other porous
polymers have been employed as solid supports.
[0192] The plaque burden in the tissue and/or body fluid (such as
the retinal ganglion cell layer of an animal, particularly a
mammal, but especially a human suffering from an ocular disease
associated with pathological abnormalities/changes in the tissues
of the visual system, particularly associated with
amyloid-beta-related pathological abnormalities/changes in the
tissues of the visual system) can be calculated by methods known in
the art such as that disclosed in Ding, J.-D. et al., "Targeting
age-related macular degeneration with Alzheimer's disease based
immunotherapies: Anti-amyloid-b antibody attenuates pathologies in
an age-related macular degeneration mouse model", Vision Research
(2007), doi:10.1016/j.visres.2007.07.025.
[0193] A compound according to the present invention can also be
incorporated into a test kit for detecting an amyloid protein. The
test kit typically comprises a container holding one or more
compounds according to the present invention and instructions for
using the compound for the purpose of binding to an amyloid protein
to form a compound/protein complex and detecting the formation of
the compound/protein complex such that presence or absence of the
compound/protein complex correlates with the presence or absence of
the amyloid protein.
[0194] The term "test kit" refers in general to any diagnostic kit
known in the art. More specifically, the latter term refers to a
diagnostic kit as described in Zrein et al. (1998).
EXAMPLES
[0195] The present invention is illustrated by the following
non-limiting examples.
Preparation of 2a
5-p-Tolyl-N-((5-p-tolyl-1H-pyrazol-3-yl)methyl)-1H-pyrazol-3-amine
dihydrochloride
##STR00039##
[0197] Acetyl chloride (6.16 mL, 86.60 mmol) was added dropwise to
a suspension of 5-amino-3-(4-methylphenyl)pyrazole (5 g, 28.86
mmol) and potassium carbonate (14 g, 101.03 mmol) in anhydrous MeCN
(100 mL). The reaction mixture was refluxed for 16 hrs. The solvent
was evaporated and the residue was resuspended in CHCl.sub.3, and
washed with 1N HCl, sat. aq. NaHCO.sub.3, H.sub.2O and brine and
dried over Na.sub.2SO.sub.4. The solvent was evaporated and the
crude N-(1-acetyl-5-p-tolyl-1H-pyrazol-3-yl)acetamide was used
without any further purification in the next step.
[0198] Crude N-(1-acetyl-5-p-tolyl-1H-pyrazol-3-yl)acetamide was
dissolved in a mixture of MeOH/THF/H.sub.2O (2:2:1, 150 mL) with 2
drops of 33% ammonia solution. The reaction mixture was stirred for
16 hrs, then the solvents were evaporated and the crude
N-(5-p-tolyl-1H-pyrazol-3-yl)acetamide was used without any further
purification in the next step.
[0199] MS (ESI): m/z: 216.29 [MH.sup.+].
[0200] A mixture of crude N-(5-p-tolyl-1H-pyrazol-3-yl)acetamide
(28.86 mmol), 3,4-dihydro-2H-pyran (6.7 mL, 72.15 mmol) and
trifluoroacetic acid (43 .mu.L, 0.58 mmol) in anhydrous MeCN (60
mL) was refluxed for 16 hrs. The solvent was evaporated and the
residue was resuspended in CH.sub.2Cl.sub.2 (50 mL), washed with
H.sub.2O and brine and dried over Na.sub.2SO.sub.4. The solvent was
evaporated and the crude
N-(5-tolyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)acetamide
was used without any further purification in the next step.
[0201] MS (ESI): m/z: 300.33 [MH.sup.+].
[0202] Crude
N-(5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)acetamide
(28.86 mmol) was dissolved in EtOH/H.sub.2O (2:3, 100 mL) together
with potassium hydroxide (11 g, 202 mmol) and was refluxed for 16
hrs. The reaction mixture was concentrated and then extracted with
CHCl.sub.3. The combined organic layers were washed with brine and
dried over Na.sub.2SO.sub.4. Solvent evaporation and purification
by silica gel column chromatography (PE-EtOAc, 7:3 to 3:7) gave
5-tolyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-amine (2.99 g
regioisomer A and 4.39 g regioisomer B, 99% over 4 steps).
Regioisomer A:
[0203] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.63 (d, J=7.7 Hz,
2H), 7.16 (d, J=8.6 Hz, 2H), 5.81 (s, 1H), 5.38 (dd, J=8.9 Hz,
J=2.6 Hz, 1H), 4.01 (bm, 3H), 3.68 (dt, J=11.5 Hz, J=2.6 Hz, 1H),
2.38 (m, 1H), 2.35 (s, 3H), 2.03-2.14 (m, 2H), 1.62-1.71 (m,
3H).
[0204] MS (ESI): m/z: 258.37 [MH.sup.+].
Regioisomer B:
[0205] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.36 (d, J=8.0 Hz,
2H), 7.24 (d, J=8.0 Hz, 2H), 5.69 (s, 1H), 5.03 (dd, J=10.2 Hz,
J=2.5 Hz, 1H), 4.11 (m, 1H), 3.69 (bs, 2H), 3.54 (dt, J=12.0 Hz,
J=2.5 Hz, 1H), 2.46 (m, 1H), 2.41 (s, 3H), 1.70-1.79 (m, 2H),
1.50-1.59 (m, 3H).
[0206] MS (ESI): m/z: 258.37 [MH.sup.+].
##STR00040##
[0207] 5-p-Tolyl-1H-pyrazole-3-carboxylic acid (250 mg, 1.24 mmol)
and sulfuric acid (120 .mu.L, 1.48 mmol) in methanol (5 mL) were
heated to reflux for 16 hrs. The solvent was evaporated and the
residue was resuspended in CH.sub.2Cl.sub.2. The residue was
filtrated and the filtrate was washed with water and brine and
dried over Na.sub.2SO.sub.4. Solvent was evaporated and the white
solid was combined with the precipitate collected previously.
Methyl 5-p-tolyl-1H-pyrazole-3-carboxylate (224 mg, 84%) was
obtained as a white solid. .sup.1H-NMR (400 MHz, CDCl.sub.3):
(ppm)=7.56 (d, J=7.6 Hz, 2H), 7.22 (d, J=8.0 Hz, 2H), 7.02 (s, 1H),
3.91 (s, 3H), 2.36 (s, 3H).
[0208] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm)=129.61, 125.45,
105.03, 52.02, 21.19.
[0209] A mixture of methyl 5-p-tolyl-1H-pyrazole-3-carboxylate (224
mg, 1.03 mmol), 3,4-dihydro-2H-pyran (190 .mu.L, 2.07 mmol) and
trifluoroacetic acid (2 .mu.L, 0.02 mmol) in anhydrous MeCN (3 mL)
was refluxed for 2 hrs. The solvent was evaporated. The residue was
resuspended in CH.sub.2Cl.sub.2 (50 mL) and was washed with
H.sub.2O and brine. After drying with Na.sub.2SO.sub.4, solvent
evaporation and silica gel column chromatography (PE-EtOAc, 6:4)
methyl
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylate
(363 mg, 68%) was obtained.
[0210] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.40 (d, J=8.0 Hz,
2H), 7.27 (d, J=8.0 Hz, 2H), 6.82 (s, 1H), 5.25 (d, J=10.0 Hz, 1H),
4.11 (m, 1H), 3.92 (s, 3H), 3.57 (t, J=10.8 Hz, 1H), 2.61 (m, 1H),
2.41 (s, 3H), 2.03 (m, 1H), 1.82 (d, J=8.06 Hz, 1H), 1.58-1.52 (m,
3H).
[0211] Methyl
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylate
(363 mg, 0.70 mmol) was dissolved in a mixture of MeOH/THF/H.sub.2O
(1:2:1, 4 mL). Lithium hydroxide was added and the reaction mixture
was stirred for 16 hrs. The reaction mixture was diluted with water
and washed with DCM. The aqueous phase was evaporated.
1-(Tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylic
acid (205 mg, quantitative) was obtained as a white solid which was
used without further purification in the next step.
[0212] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.13 (d, J=6.8 Hz,
2H), 7.04 (d, J=6.8 Hz, 2H), 6.60 (s, 1H), 4.92 (d, J=9.6 Hz, 1H),
3.89 (d, J=8.4 Hz, 1H), 3.35 (t, J=10.8 Hz, 1H), 2.40 (m, 1H), 2.31
(s, 3H), 1.75 (m, 1H), 1.54 (m, 2H), 1.25 (m, 2H).
[0213] 5-Tolyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-amine
(compound A, 81 mg, 0.31 mmol) was added to a solution of
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylic
acid (100 mg, 0.34 mmol), 2-chloro-1-methylpyridinium iodide (122
mg, 0.47 mmol) and N,N'-diisopropylethylamine (163 .mu.L, 0.94
mmol) in DCM (10 mL). The reaction mixture was stirred at RT for 16
hrs. The reaction mixture was diluted with water and extracted with
DCM. The organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
by silica gel column chromatography (PE-EtOAc, 9:1) and gave
1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-5-p-
-tolyl-1H-pyrazol-3-yl)-5-p-tolyl-1H-pyrazole-3-carboxamide (70 mg,
43%).
[0214] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=9.37 (s, 0.5H),
9.34 (s, 0.5H), 7.43 (m, 4H), 7.28 (m, 4H), 6.95 (s, 0.5H), 6.94
(s, 0.5H), 6.90 (s, 1H), 5.24 (m, 2H), 4.15 (m, 2H), 3.60 (m, 2H),
2.45 (m, 2H), 2.42 (s, 6H), 1.80 (m, 2H), 1.56 (m, 4H), 1.25 (m,
4H).
[0215]
1-(Tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-5-p-to-
lyl-1H-pyrazol-3-yl)-5-p-tolyl-1H-pyrazole-3-carboxamide (142 mg,
0.27 mmol) was suspended in anhydrous THF (10 mL) and borane
dimethylsulfide complex (177 .mu.L, 1.86 mmol) was added dropwise.
The reaction mixture was stirred under reflux for 16 hrs. The
reaction mixture was then cooled down to 0.degree. C. and MeOH (500
.mu.l) was added. The mixture was then stirred for 10 min.
Concentrated hydrochloric acid (12 N) was added, until a pH <2
was obtained, and the resulting mixture was stirred under reflux
for 16 hrs. The mixture was cooled to room temperature and the
precipitate was filtered and added into an aqueous solution of
sodium hydroxide (1M). The aqueous phase was extracted with DCM
(3.times.10 mL) after drying the organic layers with
Na.sub.2SO.sub.4, the solvent was evaporated. The residue was
purified by chromatography on silica gel column (eluent: EtOAc);
5-p-tolyl-N-((5-p-tolyl-1H-pyrazol-3-yl)methyl)-1H-pyrazol-3-amine
was obtained as a white solid.
[0216]
5-p-Tolyl-N-((5-p-tolyl-1H-pyrazol-3-yl)methyl)-1H-pyrazol-3-amine
(27 mg, 0.078 mmol) was recrystallized in methanolic HCl (3 N, 1
mL). The solid was filtered, washed with Et.sub.2O and dried in
vacuo; white solid. Mp=132.degree. C.
[0217] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.73 (d, J=8.0
Hz, 2H), 7.62 (d, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 7.22 (d,
J=8.0 Hz, 2H), 6.63 (s, 1H), 6.30 (s, 1H), 4.42 (s, 2H), 2.36 (s,
3H), 2.31 (s, 3H).
Preparation of 2c
N.sup.5-Propyl-N.sup.2-((6-((6-(propylamino)pyridin-3-yl)methylamino)pyrid-
in-3-yl)methyl)pyridine-2,5-diamine
##STR00041##
[0219] 2-Chloro-1-methylpyridinium iodide (2.99 g, 10 mmol) and
DIPEA (3 mL) were added to a mixture of methyl 6-aminonicotinate 1
(760 mg, 5 mmol) and 6-bromonicotinic acid 2 (1 g, 5 mmol) in THF
(150 mL). The reaction mixture was stirred at rt (=room
temperature) for 4 days. At the conclusion of the reaction, the
reaction mixture was concentrated to 1/3 of its volume and the
precipitated product was filtered off. The filtrate was
concentrated, diluted with chloroform (100 ml), washed with water
and brine and dried over Na.sub.2SO.sub.4. Evaporation of the
solvent gave a crude yellow product, which crystallized in EtOAc to
give methyl 6-(6-bromonicotinamido)nicotinate 3 as a white solid (1
g, 65.4%). Mp. 234-235.degree. C.
[0220] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=11.5 (s, 1H),
8.96 (d, J=2.0 Hz, 1H), 8.93 (s, 1H), 8.36 (dd, J=2.0 Hz, J=8.0 Hz,
1H), 8.33 (d, J=12 Hz, 1H), 8.28 (dd, J=2.4 Hz, J=8.0 Hz, 1H), 7.83
(d, J=8.4 Hz, 1H), 3.88 (s, 3H).
[0221] MS (ESI): m/z=338 (M+2H)
[0222] KOH pellets (5 g) were added to a suspension of methyl
6-(6-bromonicotinamido) nicotinate 3 (5 g, 14.8 mmol) in methanol
and water (150/50 ml). The reaction mixture was stirred for 4
hours. Then the solvent was evaporated and the pH was adjusted to
below 2. The white solid was filtered off and washed with cold
water and dried under vacuum to give
6-(6-bromonicotinamido)nicotinic acid 4 (1.8 g, 37%). Mp.
270-272.degree. C.
[0223] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=11.52 (s, 1H),
8.95 (s, 1H), 8.91 (s, 1H), 8.29 (m, 3H), 7.83 (d, J=8.0 Hz, 1H).
MS (ESI): m/z=322 (M.sup.+).
[0224] 2-chloro-1-methylpyridinium iodide (2.37 g, 18.6 mmol) was
added to a suspension of 6-(6-bromonicotinamido)nicotinic acid 4 (2
g, 6.21 mmol) and 2-amino-5-bromopyridine 5 (1.07 g, 6.21 mmol) in
THF (100 ml), followed by DIPEA (2.4 g, 18.6 mmol). The reaction
mixture was stirred for 4 days at RT. The suspension was filtered
off and washed with water (25 ml). The filtrate was concentrated,
diluted in chloroform, washed with water and brine, and then dried
over Na.sub.2SO.sub.4. The product precipitated during evaporation
and was dried under vacuum to give
6-bromo-N-(5-(5-bromopyridin-2-ylcarbamoyl)pyridin-2-yl)nicotinamide
6 as a yellow solid (400 mg, 14%). Mp. 245-247.degree. C.
[0225] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=11.1 (br s,
1H), 10.8 (br s, 1H), 9.0 (s, 1H), 8.7 (s, 1H), 8.53 (s, 1H), 8.41
(d, J=8.4 Hz, 1H), 8.30 (d, J=8.8 Hz, 1H), 8.21 (d, J=8.4 Hz, 1H),
8.1 (d, J=9.2 Hz, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.32 (br s, 1H),
6.51 (d, J=8.8 Hz), 3.2 (d, J=6.0 Hz, 4H), 1.57 (q, J=7.2 Hz, 4H),
0.92 (t, J=7.2 Hz, 6H)
[0226] MS (ESI): m/z=478 (M+H).
[0227]
6-Bromo-N-(5-(5-bromopyridin-2-ylcarbamoyl)pyridin-2-yl)nicotinamid-
e 6 (350 mg, 0.73 mmol) was dissolved in neat n-propylamine. The
reaction mixture was heated for 3 days at reflux temperature. Then
the solvent was evaporated to give a crude product, which was
crystallized in EtOAc to give
6-(propylamino)-N-(5-(5-(propylamino)pyridin-2-ylcarbamoyl)pyridin-2-
-yl)nicotinamide 7 (122 mg, 38% yield) as a white solid. Mp.
249-250.degree. C.
[0228] .sup.1H-NMR (400 MHz, D.sub.2O+DMSO-d.sub.6): .delta.=8.95
(d, J=2.0 Hz, 1H), 8.68 (d, J=2.0 Hz, 1H), 8.50 (d, J=2.0 Hz, 1H),
8.36 (dd, J.sub.1=2.2, J.sub.2=8.8 Hz, 1H), 8.24 (d, J=8.8 Hz 1H),
8.15 (d, J=8.8 Hz, 1H), 8.06 (dd, J.sub.1=2.2, J.sub.2=9.2 Hz, 1H),
7.96 (dd, J.sub.1=1.6, J.sub.2=8.8 Hz 1H), 6.51 (d, J=8.8 Hz, 1H),
3.25 (t, J=4.0 Hz, 4H), 1.54 (m, 4H), 0.90 (t, J=7.3 Hz, 6H).
[0229] MS (ESI): m/z=457 (M+Na).
[0230] BMS (129 .mu.L 1.73 mmol) was added to a solution of
6-(propylamino)-N-(5-(5-(propylamino)pyridin-2-ylcarbamoyl)pyridin-2-yl)n-
icotinamide 7 (75 mg, 0.173 mmol) in THF (15 ml). The reaction
mixture was refluxed over night and cooled, then MeOH was added
followed by conc. HCl. The mixture was refluxed again for another
16 hrs. Then the reaction mixture was concentrated, the residue was
diluted in water (5 ml) and the pH was adjusted to 14 using NaOH
solution. The aqueous layer was extracted with chloroform (50
ml.times.3) and the combined organic layers were washed with water
and brine and then dried over Na.sub.2SO.sub.4. Evaporation of the
solvent yielded a residue that was purified on silica gel (2:98,
MeOH:EtOAc) to give a brown sticky material (5 mg, 7%). Then it was
treated with HCl/MeOH to give
N.sup.5-propyl-N.sup.2-((6-((6-(propylamino)pyridin-3-yl)methylamino)pyri-
din-3-yl)methyl)pyridine-2,5-diamine hydrochloride salt 8 (5 mg) as
a gummy material.
[0231] .sup.1H-NMR (400 MHz, D.sub.2O+DMSO-d.sub.6): .delta.=8.48
(s, 3H), 7.88 (d, J=8.8 Hz 3H), 6.33 (d, J=8.8 Hz, 3H), 6.08 (brs,
2H) 5.04 (brs, 2H), 3.42 (m, 2H), 3.27 (m, 2H), 1.63 (m, 4H), 1.03
(m, 6H)
[0232] MS (ESI): m/z=408 (M+3H)
Preparation of 2h
N-Benzyl-5-((5-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00042##
[0234] 2-Chloro-1-methylpyridinium iodide (0.55 g, 2.2 mmol), DIEA
(0.37 g, 2.9 mmol) and 5-p-tolylthiazol-2-amine (0.27 g, 1.44 mmol)
were added to a suspension of 2-bromothiazole-5-carboxylic acid
(0.30 g, 1.44 mmol) in DMF (5 mL). The resulting solution was
stirred at RT till completion of the reaction. The mixture was then
diluted with AcOEt, washed with water, dried with Na.sub.2SO.sub.4
and concentrated. The crude product was purified by column (30%
AcOEt/PE), yielding
2-bromo-N-(5-p-tolylthiazol-2-yl)thiazole-5-carboxamide (250 mg,
46%) as a solid.
[0235] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 8.19 (s, 1H), 7.6
(d, J=8 Hz, 2H), 7.15 (d, J=8 Hz, 2H), 7.04 (s, 1H), 2.31 (s,
3H).
[0236] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 157.89, 149.44,
147.77, 144.33, 143.16, 141.99, 138.10, 137.38, 131.07, 129.32,
125.87, 107.34, 20.93.
[0237] MS (ESI): m/z (%): 379.87 [MH.sup.+].
[0238] Benzylamine (0.143 mL, 1.32 mmol) was added to a solution of
2-bromo-N-(5-p-tolylthiazol-2-yl)thiazole-5-carboxamide (250 mg,
0.66 mmol) in DMF (4 mL). The reaction mixture was stirred at
reflux for 2 hrs. Then it was diluted with AcOEt and washed with
water, dried with Na.sub.2SO.sub.4 and concentrated. The crude
product was purified by precipitation (AcOEt/PE), yielding
2-(benzylamino)-N-(5-p-tolylthiazol-2-yl)thiazole-5-carboxamide
(190 mg, 71%) as a solid.
[0239] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 8.85 (bs, 1H),
8.25 (s, 1H), 7.80 (d, J=8 Hz, 2H), 7.46 (s, 1H), 7.35 (m, 3H),
7.28, (m, 2H), 7.23 (d, J=8 Hz, 2H), 4.53 (s, 1H), 2.33 (s,
3H).
[0240] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 172.44, 158.89,
148.82, 145.74, 145.06, 137.99, 136.75, 131.58, 128.97, 128.15,
127.19, 126.92, 125.46, 118.98, 106.83, 47.44, 20.49.
[0241] MS (ESI): m/z (%): 407.36 [MH.sup.+].
[0242] BMS (0.2 mL, 2.1 mmol) was added to a solution of
2-(benzylamino)-N-(5-p-tolylthiazol-2-yl)thiazole-5-carboxamide
(170 mg, 0.42 mmol) in THF (6 mL) at RT. The resulting solution was
stirred at reflux overnight. The reaction was then quenched with
MeOH (2 mL) and 1N HCl was added until the pH reached 2. After
stirring the reaction mixture at reflux temperature for 12 hrs, the
organic solvents were evaporated and the aqueous solution was
neutralized with 1N NaOH, extracted with CHCl.sub.3, dried with
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
by column (AcOEt), yielding
N-benzyl-5-((5-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine (30
mg, 18%).
[0243] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.59 (d, J=8 Hz,
2H), 7.23 (m, 5H), 7.11 (d, J=8 Hz, 2H), 6.93 (s, 1H), 6.58 (s,
1H), 4.40 (s, 2H), 4.32 (s, 2H), 3.41 (bs, 2H), 2.29 (s, 3H).
[0244] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 197.01, 170.75,
151.05, 137.39, 137.26, 137.03, 131.82, 129.07, 128.48, 127.48,
127.41, 125.75, 121.99, 100.46, 49.30, 41.95, 20.99.
[0245] MS (ESI): m/z: 393.28 [MH.sup.+].
[0246]
N-Benzyl-5-((5-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine (20
mg, 0.05 mmol) was dissolved in methanolic HCl (3 N, 1 mL) and
CHCl.sub.3/AcOEt was added. The precipitated solid was filtered by
decantation and dried in vacuo, yielding
N-benzyl-545-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride as a white solid (11 mg, 10%). Mp. 105-106.degree.
C.
Preparation of 2j
N-Benzyl-5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00043##
[0248] Compound 2j was prepared as described for 2 h starting from
4-p-tolylthiazol-2-amine and 2-bromothiazole-5-carboxylic acid:
(3.5 mg, 59%). Mp. 106-108.degree. C.
[0249] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.61 (d, J=8 Hz,
2H), 7.28 (m, 5H), 7.13 (d, J=8 Hz, 2H), 6.95 (s, 1H), 6.60 (s,
1H), 4.42 (s, 2H), 4.34 (s, 2H), 3.01 (bs, 2H), 2.31 (s, 3H).
[0250] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 170.68, 168.62,
151.11, 138.15, 137.48, 137.23, 131.86, 129.12, 128.55, 127.56,
127.74, 125.80, 122.076, 100.53, 49.33, 40.07, 21.07.
[0251] MS (ESI): m/z (%): 393.29 [MH.sup.+].
Preparation of 2k
N-Benzyl-5-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)thiazol-2-amin-
e dihydrochloride
##STR00044##
[0253] 2-Chloro-1-methylpyridinium iodide (0.84 g, 3.3 mmol), DIPEA
(0.78 mL, 4.4 mmol) and tert-butyl
3-amino-5-(4-fluorophenyl)-1H-pyrazole-1-carboxylate (0.5 g, 2.2
mmol) were added to a solution of 2-bromothiazole-5-carboxylic acid
(0.45 g, 2.2 mmol) in DMF (6 mL). The resulting solution was
stirred at RT till completion of the reaction. Then the mixture was
diluted with AcOEt, washed with water, dried with Na.sub.2SO.sub.4
and concentrated. The crude product was purified by column
(50-%100% AcOEt/PE gradient), yielding tert-butyl
3-(2-bromothiazole-5-carboxamido)-5-(4-fluorophenyl)-1H-pyrazole-1-carbox-
ylate (160 mg, 20%) as a white solid.
[0254] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 11.39 (s, 1 NH),
8.56 (s, 1H), 7.79 (t, J=7 Hz, 2H), 7.31 (t, J=8.6 Hz, 2H), 6.96
(s, 1H), 1.71 (s, 9H).
[0255] Benzylamine (0.1 mL, 0.87 mmol) was added to a solution of
tert-butyl
3-(2-bromothiazole-5-carboxamido)-5-(4-fluorophenyl)-1H-pyrazole-1-carbox-
ylate (160 mg, 0.43 mmol) in dioxane (3 mL). The resulting solution
was stirred at 85.degree. C. for 24 hrs. The solvent was evaporated
and the crude product was purified by column (0-10% MeOH/AcOEt
gradient), yielding
2-(benzylamino)-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)thiazole-5-
-carboxamide (180 mg, 90%) as a solid.
[0256] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.85 (s, 1H), 7.64
(s, 2H), 7.34-7.27 (m, 5H), 7.08 (t, J=8.6 Hz, 2H), 6.71 (s, 1H),
4.51 (s, 2H), 4.39 (s, 2H).
[0257] MS (ESI): m/z: 394.37 [MH.sup.+].
[0258] BMS was added to a solution of
2-(benzylamino)-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)thiazole-5-carboxam-
ide (50 mg, 0.13 mmol) in THF (1 mL). The resulting solution was
stirred at RT overnight. The reaction was then quenched with MeOH
(0.5 mL) and 1N HCl was added until pH=2. After stirring the
reaction mixture at RT for 3 hrs, the organic solvents were
evaporated and the aqueous solution was neutralized with a
NaHCO.sub.3 saturated aqueous solution, extracted with AcOEt, dried
with Na.sub.2SO.sub.4 and concentrated. The crude product was
purified by column (0-2% MeOH/AcOEt gradient), yielding
N-benzyl-5-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)thiazol-2-ami-
ne (59 mg, 98%).
[0259] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm): 7.49
(s, 2H), 7.30-7.27 (m, 5H), 7.03 (t, J=8 Hz, 2H), 6.94 (s, 1H),
5.82 (s, 1H), 4.35 (s, 2H), 4.28 (s, 2H).
[0260] MS (ESI): m/z: 380.34 [MH.sup.+].
[0261]
N-Benzyl-5-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)thiazol-
-2-amine (59 mg, 0.13 mmol) was dissolved in methanolic HCl (3 N, 1
mL) and AcOEt was added. The precipitated solid was filtered by
decantation and dried in vacuo, yielding
N-benzyl-5-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)thiazol-2-ami-
ne dihydrochloride as a yellow solid (14 mg, 25%). Mp.
78-80.degree. C.
Preparation of 2n
N-Methyl-5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00045##
[0263] Compound 2n was prepared as described for 2w, starting from
4-p-tolylthiazol-2-amine and
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde:
(28 mg, 80%). Mp. 114-116.degree. C.
[0264] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.97 (t, J=5.6 Hz,
1H), 7.75 (d, J=8 Hz, 2H), 7.33 (m, 1H), 7.18 (d, J=8 Hz, 2H), 6.99
(s, 1H), 6.96 (s, 1H), 4.46 (d, J=5.6 Hz, 2H), 2.76 (d, J=4.3 Hz,
3H), 2.30 (s, 3H).
[0265] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 169.73, 167.45,
149.76, 137.43, 136.43, 132.15, 128.97, 125.53, 121.96, 100.52,
40.52, 30.62, 20.76.
[0266] MS (ESI): m/z: 317.24 ([MH.sup.+].
Preparation of 2o
N-Methyl-5-((5-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00046##
[0268] Compound 2o was prepared as described for 2w, starting from
5-p-tolylthiazol-2-amine and
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde:
(70 mg, 78%). Mp. not determined (it decomposes above 140.degree.
C.).
[0269] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.97 (t, J=5.6 Hz,
1H), 7.75 (d, J=8 Hz, 2H), 7.33 (m, 1H), 7.19 (d, J=8 Hz, 2H), 7.00
(s, 1H), 6.96 (s, 1H), 4.46 (d, J=5.6 Hz, 2H), 2.75 (d, J=5 Hz,
3H), 2.31 (s, 3H).
[0270] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 170.65, 168.37,
150.68, 138.36, 137.35, 133.08, 129.89, 126.45, 122.89, 101.45,
41.44, 31.55, 21.68.
[0271] MS (ESI): m/z: 317.38 ([MH.sup.+].
Preparation of 2p
N-(Pyridin-2-yl)-5-((thiazol-2-ylamino)methyl)thiazol-2-amine
trihydrochloride
##STR00047##
[0273] DIBAL-H (1M in hexanes, 16.88 mL, 16.88 mmol) was added to a
solution of ethyl 2-bromothiazole-5-carboxylate (2 g, 8.44 mmol) in
CH.sub.2Cl.sub.2 (16 mL) at 0.degree. C. The mixture was stirred to
RT for 6 hrs. After quenching with MeOH (6 mL), Et.sub.2O and a
saturated solution of Rochelle's salt were added, the reaction
mixture was stirred until the two phases were clearly separated.
The organic phase was dried, concentrated and purified by column
chromatography (30%-100% AcOEt/PE gradient), yielding
(2-bromothiazol-5-yl)methanol (1.44 g, 88%) as an oil.
[0274] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.25 (s, 1H), 4.93
(bs, 1H), 4.69 (s, 2H).
[0275] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 144.25, 139.58,
136.89, 57.19.
[0276] MS (ESI): m/z: 193.83 [MH.sup.+].
[0277] MnO.sub.2 (3.8 g, 37.10 mmol) was added to a solution of
(2-bromothiazol-5-yl)methanol (1.44 g, 7.42 mmol) in CHCl.sub.3 (20
mL). The resulting mixture was stirred at RT for 3 days. Then the
solution was filtered through celite and concentrated, yielding
2-bromothiazole-5-carbaldehyde (870 mg, 61%) as a white solid.
[0278] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 9.95 (s, 1H), 8.16
(s, 1H).
[0279] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 180.93, 150.47,
145.48, 142.91.
[0280] MS (ESI): m/z: 192.31 ([MH.sup.+]).
[0281] A solution of 2-bromothiazole-5-carbaldehyde (150 mg, 0.78
mmol) and 2-amino-thiazole (78 mg, 0.78 mmol) in toluene (7 mL) and
3 .ANG. molecules sieves were stirred at reflux overnight. The
strong yellow solution of the corresponding imine was then poured
over NaBH.sub.4 (147 mg, 3.9 mmol) in hot EtOH (50 mL). The
colorless solution was filtered, concentrated and purified by
column chromatography (30% AcOEt/PE) yielding
N-((2-bromothiazol-5-yl)methyl)thiazol-2-amine (110 mg, 51%) as a
solid.
[0282] .sup.1H-NMR (400 MHz, CD.sub.3OD/CDCl.sub.3): (ppm): 7.33
(s, 1H) 6.95 (d, J=3.7 Hz, 1H), 6.40 (d, J=3.7 Hz, 1H), 4.49 (s,
2H), 2.9 (bs, 1H).
[0283] .sup.13C-NMR (100 MHz, CD.sub.3OD/CDCl.sub.3): (ppm):
169.00, 140.64, 139.95, 138.38, 136.27, 107.53, 41.29.
[0284] MS (ESI): m/z: 276.30 ([MH.sup.+]).
[0285] 2-Aminopyridine (0.150 g, 1.6 mmol) was added to a
suspension of NaH (64 mg, 1.6 mmol) in THF (4 mL). The resulting
solution was stirred at 65.degree. C. for 45 min. Then
N-((2-bromothiazol-1-5-yl)methyl)thiazol-2-amine (110 mg, 0.4 mmol)
was added and the reaction mixture was stirred at 65.degree. C.
overnight. The reaction was quenched with water and the reaction
mixture was extracted with AcOEt. The organic phase was dried,
concentrated and purified by column chromatography (in AcOEt).
Precipitation from CHCl.sub.3 was conducted to eliminate excess
2-aminopyridine
N-(Pyridin-2-yl)-5-((thiazol-2-ylamino)methyl)thiazol-2-amine was
obtained as a weakly yellow solid (15 mg, 14%).
[0286] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): (ppm): 11.12 (bs, 1H),
8.24 (d, J=4.4 Hz, 1H), 7.97 (s, 1H), 7.67 (t, J=8 Hz, 1H), 7.26
(s, 1H), 7.05 (d, J=3.2 Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 6.88 (t,
J=5.6 Hz, 1H), 6.64 (d, J=4.4 Hz, 1H), 4.53 (d, J=4.8 Hz, 2H).
[0287] .sup.13C-NMR (100 MHz, DMSO-d.sub.6): (ppm): 168.17, 159.06,
151.41, 146.15, 138.34, 137.51, 135.44, 126.24, 115.52, 110.34,
106.35, 39.96.
[0288] MS (ESI): m/z: 290.32 ([MH.sup.+].
[0289]
N-(Pyridin-2-yl)-5-((thiazol-2-ylamino)methyl)thiazol-2-amine (15
mg, 0.05 mmol) was dissolved in methanolic HCl (3 N, 1 mL) and
Et.sub.2O was added. The precipitated solid was filtered by
decantation and dried in vacuum, yielding
N-(pyridin-2-yl)-5-((thiazol-2-ylamino)methyl)thiazol-2-amine
trihydrochloride (14 mg, 2%, 95.5% pure by HPLC). Mp. decomposition
>145.degree. C.
Preparation of 2q
N-Benzyl-5-((5-fluoropyridin-2-ylamino)methyl)pyridin-2-amine
##STR00048##
[0291] 2-Chloro-1-methylpyridinium iodide (2.2 g, 9.8 mmol) and
DIPEA (1 ml) were added to a mixture of 5-fluoropyridin-2-amine 1
(554 mg, 4.9 mmol) and 6-bromonicotinic acid 2 (1 g, 4.9 mmol) in
THF. The reaction mixture was stirred at rt for 2 days. At the
conclusion of the reaction, the reaction mixture was concentrated
to 1/3 of its volume and the precipitated product was filtered off.
The filtrate was concentrated, diluted with chloroform (100 ml),
washed with water and brine and then dried over Na.sub.2SO.sub.4.
Evaporation of the solvent gave a crude yellow product, which
crystallized in EtOAc to give
6-bromo-N-(5-fluoropyridin-2-yl)nicotinamide 3 (1.05 g, 71%) as a
white solid. Mp. 173-174.degree. C.
[0292] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=11.27 (s, 1H),
8.94 (s, 1H), 8.43 (s, 1H), 8.27 (d, J=8.0 Hz, 1H), 8.2 (d, J=12
Hz, 1H), 7.83 (m, 2H).
[0293] MS (ESI): m/z=(297, M+H).
[0294] The 6-bromo-N-(5-fluoropyridin-2-yl)nicotinamide 3 (500 mg,
1.68 mmol) was dissolved in benzyl amine (2 ml), and heated at
140.degree. C. for 48 h. Then the reaction mixture was
concentrated. The product was recrystallized from ethyl acetate and
pet-ether to give
6-(benzylamino)-N-(5-fluoropyridin-2-yl)nicotinamide 4 (500 mg,
92%) as a white solid.
[0295] Mp. 167-168.degree. C.
[0296] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=10.5 (s, 1H),
8.69 (s, 1H), 8.36 (s, 1H), 8.17 (s, 1H), 7.79 (s, 1H) 7.24-7.70
(m, 7H), 6.57 (d, J=8.0 Hz, 1H) 4.18 (s, 2H).
[0297] MS (ESI): m/z=(323, M+H)
[0298] BMS (400 .mu.L) was added to a solution of
6-(benzylamino)-N-(5-fluoropyridin-2-yl)nicotinamide 4 in THF (20
mL). The reaction mixture was refluxed for 16 hrs. Then the
reaction mixture was cooled to RT. MeOH (2 mL), followed by conc.
HCl were added. The reaction mixture was refluxed for 8 hrs,
concentrated and diluted with water (2 mL). The pH was adjusted to
14 and the reaction mixture was extracted with chloroform (50
mL.times.2). All organic phases were washed with brine and dried
over Na.sub.2SO.sub.4. Evaporation of the solvent gave a crude
product which was purified on silica gel (1:1, EtOAc: Pet-ether) to
give N-benzyl-5-((5-fluoropyridin-2-ylamino)methyl)pyridin-2-amine
5. This was treated with HCl/MeOH to give the corresponding salt
(50 mg 13%) as a white solid. Mp. 166-168.degree. C.
[0299] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=8.11 (d, J=1.6,
1H) 7.99 (d, J=2.8, 1H) 7.46 (dd, J=2.4, J=8.4 Hz, 1H) 7.33-7.37
(m, 3H), 7.28 (m, 3H), 7.20 (dt, J=3.2, J=8.0 Hz, 1H) 6.35-6.40 (m,
2H) 4.96 (brs, 1H) 4.61 (s, 1H), 4.53 (d, J=6.0 Hz, 2H), 4.34 (d,
J=5.2 Hz 2H)
[0300] MS (ESI): m/z=(309, M+H)
Preparation of 2s
##STR00049##
[0302] To a suspension of
5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylic
acid 1 (400 mg, 1.37 mmol) and 2-chloro-1-methylpyridinium iodide
(524 mg, 2.0 mmol) in THF (20 ml), DIPEA (0.46 ml) followed by
6-bromopyridin-2-amine 2 (237 mg, 1.37 mmol) were added. The
suspension was stirred for 72 hrs. Then the reaction mixture was
concentrated, diluted in water (10 ml) and extracted with
dichloromethane (50 ml.times.2). All organic phases were washed
with water and brine, dried over Na.sub.2SO.sub.4 and concentrated.
The residue was purified on a silica gel column (EtOAc: pet-ether
1:4) to give
N-(6-bromopyridin-2-yl)-5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1-
H-pyrazole-3-carboxamide 3 (180 mg) as a white solid. Mp.
151-152.degree. C.
[0303] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=9.53 (s, 1H),
8.31 (d, J=8.0 Hz, 1H), 7.83 (dd, J=5.2, J=8.8 Hz, 2H), 7.64 (t,
J=8.0 Hz, 2H), 7.30 (m, 2H) 7.13 (t, J=9.2 Hz, 2H), 6.04 (dd, J=2.4
Hz, J=10 Hz, 1H), 4.26 (d, J=11.0 Hz, 1H), 3.86 (t, J=11.6 Hz, 1H),
2.52-2.60 (m, 1H), 2.11 2.16 (m, 2H), 1.27-1.57 (m, 2H).
[0304]
N-(6-Bromopyridin-2-yl)-5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-
-yl)-1H-pyrazole-3-carboxamide 3 (300 mg, 0.67 mmol) was dissolved
in benzylamine (3 ml). The reaction mixture was heated for 24 hrs
at 130.degree. C. Then the reaction mixture was purified on a
silica gel column (1:5 EtOAc:Pet-ether) to give
N-(6-(benzylamino)pyridin-2-yl)-5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-
-2-yl)-1H-pyrazole-3-carboxamide 4 as a white solid (187 mg, 59%).
Mp. 139-140.degree. C.
[0305] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.79 (brs, 2H),
7.18-7.39 (m, 8H), 7.09 (t, J=7.6 Hz, 2H), 6.94 (s, 1H), 6.84 (d,
J=8.0 Hz, 1H), 6.43 (d, J=8.0 Hz, 1H), 5.98 (d, J=9.6 Hz, 1H), 4.64
(s, 2H), 3.96 (d, J=10.8 Hz, 1H), 3.54 (t, J=10.4 Hz, 1H), 2.54 (m,
1H), 2.02-2.10 (m, 2H), 1.56-1.67 (m, 3H).
[0306] Boran dimethylsufide (50 .mu.L) was added to a solution of
N-(6-(benzylamino)pyridin-2-yl)-5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-
-2-yl)-1H-pyrazole-3-carboxamide 4 (180 mg, 0.38 mmol) in THF (10
ml). The reaction mixture was refluxed over night and cooled. MeOH
was added, followed by conc. HCl and the reaction mixture was
heated for 5 hrs. Then the reaction mixture was concentrated in
vacuum, diluted with water (5 ml) and extracted with chloroform (50
ml.times.2). The combined organic layers were washed with water and
brine, then dried over Na.sub.2SO.sub.4 and concentrated under
reduced pressure. The crude product was purified on silica gel (50%
EtOAc: pet-ether) to give
N.sup.2-benzyl-N.sup.6-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)methyl)pyridin-
e-2,6-diamine 5 (20 mg) as an oily material, which was treated with
methanolic hydrochloric acid for 2 hrs. The solvent was evaporated
under reduced pressure to give a gummy material (20 mg).
[0307] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.75 (dd, J=5.6
Hz, J=8.4 Hz, 2H), 7.28-7.37 (m, 8H), 7.11 (t, J=8.8 Hz, 2H), 6.44
(s, 1H), 3.93 (s, 2H), 3.87 (s, 2H).
[0308] MS (ESI): m/z=374 (M+H)
Preparation of 2t
N-Benzyl-6-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-amin-
e
##STR00050##
[0310] 2-Chloro-1-methylpyridinium iodide (950 mg, 3.7 mmol) and
DIPEA (0.7 mL) were added to a solution of 6-bromopicolinic acid 2
(500 mg, 2.47 mmol) in dry DCM/DMF (30/5 mL). The reaction mixture
was stirred for 1 hr. tert-Butyl
3-amino-5-(4-fluorophenyl)-1H-pyrazole-1-carboxylate 1 was added
(650 mg, 2.47 mmol). The resulting yellow reaction mixture was
stirred for 3 days. Then the reaction mixture was concentrated
under reduced pressure, the residue was diluted in chloroform and
washed with water and brine, and then dried over Na.sub.2SO.sub.4.
The resulting crude mixture was purified on silica gel (EtOAc:
pet-ether, 1:3) to give
6-bromo-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide 3 (450
mg, 50%) as a white solid.
[0311] Mp. 245-247.degree. C.
[0312] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=10.3 (s, 1H),
8.15 (d, J=7.6 Hz, 1H), 8.02 (t, J=7.6 Hz, 1H), 7.95 (d, J=8.0 Hz,
1H), 7.82 (dd, J=5.2 Hz, J=8.0 Hz, 2H), 7.32 (t, J=8.8 Hz 1H), 7.05
(s, 1H).
[0313] MS (ESI): m/z=361 (M+H).
[0314] 6-Bromo-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide 3
(200 mg, 0.55 mmol) was dissolved in neat benzylamine (5 ml). The
reaction mixture was heated for 24 hrs. Then the solvent was
evaporated and the residue was purified on silica gel (EtOAc:
pet-ether, 1:4) to give
6-(benzylamino)-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide
4 (180 mg, 84%). Mp. 185-187.degree. C.
[0315] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 10.1 (brs, 1H),
7.81 (d, J=5.6, J=8.4 Hz, 2H), 7.60 (t, J=7.6 2H), 7.44 (d, J=7.2
Hz 2H), 7.28-7.36 (m, 5H), 7.23 (t, J=7.2 Hz 1H), 7.02 (brs, 1H),
6.79 (d, J=8.4 Hz 1H), 4.58 (s, 2H).
[0316] MS (ESI): m/z=388 (M+H).
[0317] Boron dimethylsulfide (50 mg, 0.66 mmol) was added to a
solution of
6-(benzylamino)-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide
4 (150 mg, 0.37 mmol). The reaction mixture was refluxed for 16
hrs. After cooling the reaction mixture to RT MeOH (2 mL), followed
by conc. HCl were added. The reaction mixture was refluxed for
another 12 hrs. The reaction mixture was concentrated and diluted
with water (4 ml) and the pH was adjusted to 12 using NaOH
solution. The reaction mixture was extracted with chloroform
(40.times.3 mL). All organic layers were then washed with water and
brine, then dried over Na.sub.2SO.sub.4, and the crude product was
purified on silica gel column (100% EtOAc) to give an oily material
(40 mg, 27%), which was then treated with HCl/MeOH to give
N-benzyl-6-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-ami-
ne hydrochloride salt 6 (20 mg, 39%).
[0318] Mp. 133-134.degree. C.
[0319] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=8.88 (br, 1H)
7.82 (brs, 2H), 7.40 (m, 5H), 6.99 (d, J=5.2 Hz 1H), 6.87 (brs,
1H), 6.2 (brs, 1H), 5.76 (brs, 1H), 4.69 (brs, 2H), 4.52 (brs,
2H).
[0320] MS (ESI): m/z=374 (M+H).
Preparation of 2u
Preparation of
3-(2,3-dihydrothiophen-2-yl)-N-((5-(4-fluorophenyl)-1H-pyrazol-3-yl)methy-
l)-1H-pyrazol-5-amine (7)
##STR00051##
[0322] 3-4 Drops of conc. H.sub.2SO.sub.4 were added to a solution
of 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid 1 in EtOH. The
reaction mixture was refluxed for 2 days. The reaction mixture was
concentrated and diluted with chloroform (100 ml) and washed with
saturated NaHCO.sub.3 solution, water and brine, and dried over
Na.sub.2SO.sub.4. Evaporation of the solvent under vacuum gave
ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate 2 as a brown
solid (1.05 g, 93%).
[0323] Mp. 148-150.degree. C.
[0324] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.77 (dd, J=5.6
Hz, J=8.4 Hz, 2H), 7.13 (t, J=8.4 Hz, 2H), 7.06 (s, 1H), 4.41 (q,
J=7.2 Hz, 2H), 1.40 (t, J=7.2 Hz, 3H).
[0325] .sup.13C-NMR (100 MHz, CDCl.sub.3): .delta. 164.5, 162.1,
160.6, 127.9, 127.8, 116.3, 116.1, 105.8, 61.8, 14.6.
[0326] MS (ESI): m/z=235 (M+H).
[0327] DHP (20.4 mmol, 1.7 ml) was added to a solution of ethyl
5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate 2 (800 mg, 3.4 mmol)
in dry THF (50 ml), followed by a catalytic amount of TFA (20
.mu.L). Then the reaction mixture was refluxed for 2 days. The
reaction mixture was concentrated, diluted with chloroform (100
ml), washed with water and brine, and dried over Na.sub.2SO.sub.4.
Purification of the crude product on silica gel (Pet-ether:EtOAc,
9:1) gave ethyl
5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylate
3 (600 mg, 55.5%) as a white solid. Mp. 95-96.degree. C.
[0328] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.84 (dd, J=5.6,
J=8.4 Hz, 2H), 7.15 (s, 1H), 7.10 (t, J=8.8 Hz, 2H), 6.33 (dd,
J=2.4, J=9.6 Hz, 1H), 4.40 (q, J=7.2 Hz 2H), 4.09-4.16 (m, 2H),
3.75-3.80 (m, 1H), 2.56-2.61 (m, 1H), 2.15 (m, 1H), 2.0 (d, J=12.8
Hz, 1H), 1.56-1.78 (m, 2H), 1.42 (t, J=7.2 Hz, 3H).
[0329] LiOH (120 mg, 5 mmol) was added to a solution of ethyl
5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylate
3 (1.1 g 3.4 mmol) in THF/H.sub.2O (1:1, 5 ml). The reaction
mixture was stirred over night. The clear solution was
concentrated, and suspended in chloroform (25 ml), The solid was
filtered off and dried under vacuum to give
5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carbox-
ylic acid 4 (987 mg, 99%) as a white solid. Mp. 188-190.degree.
C.
[0330] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.88 (dd, J=5.6
Hz, J=8.8 Hz, 2H), 7.20 (t, J=8.8 Hz, 2H), 6.82 (d, J=8.8 Hz, 1H),
6.71 (s, 1H), 3.90 (d, J=11.2 Hz, 1H), 3.55 (m, 1H), 2.31 (m, 1H),
2.02 (d, J=12 Hz, 1H), 1.76 (d, J=12.8 Hz, 1H), 1.52-1.65 (m,
2H).
[0331] MS (ESI): m/z=291 (M.sup.+).
[0332] 2-Chloro-1-methylpyridinium iodide (650 mg, 2.5 mmol) was
added to a solution of
5-(4-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylic
acid 4 (500 mg, 1.72 mmol) in THF/DMF (20/2 ml), followed by DIPEA
(0.5 ml). The reaction mixture was stirred for 30 min, then
tert-butyl 3-amino-5-(thiophen-2-yl)-1H-pyrazole-1-carboxylate 5
(456 mg, 1.72 mmol) was added. The reaction mixture was stirred for
2 days. The reaction mixture was concentrated, diluted with
chloroform (100 ml) and washed with water and brine, dried over
Na.sub.2SO.sub.4 and the solvent was evaporated. The crude product
was dried well and dissolved in THF (10 ml), subsequently BMS (0.3
ml) was added. The reaction mixture was heated for overnight and
cooled, then MeOH and conc. HCl were added. The reaction mixture
was again heated for another 5 hrs. The reaction mixture was
cooled, concentrated and diluted with water (5 ml), the pH was
adjusted to 14 with NaOH pellets, and extracted with chloroform (50
ml.times.2). All organic layers were washed with water and brine,
dried over Na.sub.2SO.sub.4 and the solvent was evaporated. The
crude product was purified on a silica gel column (4:1, EtOAc:
pet-ether) to give
N-((5-(4-fluorophenyl)-1H-pyrazol-3-yl)methyl)-5-(thiophen-2-yl)-1H-pyraz-
ol-3-amine 7 (66 mg, 11% overall). Mp. 128-130.degree. C.
[0333] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=4.76 (s, 2H),
6.33 (s, 2H), 7.02-7.06 (m, 3H), 7.28-7.50 (m, 2H), 7.73-7.74 (m,
2H).
[0334] MS (ESI): m/z=340 (M+H)
Preparation of 2w
N-Methyl-5-((thiazol-2-ylamino)methyl)thiazole-2-amine
dihydrochloride
##STR00052##
[0336] TFA (22 .mu.L, 0.3 mmol) and DHP (3.9 mL, 43.5 mmol) were
added to a suspension of ethyl 2-aminothiazole-5-carboxylate (5 g,
29 mmol) in CH.sub.3CN (40 mL) at RT. The resulting mixture was
stirred at reflux overnight. The reaction mixture was concentrated
and dissolved in AcOEt/PE and kept at 4 C overnight. The resultant
white solid was filtrated and washed with PE, yielding ethyl
2-(tetrahydro-2H-pyran-2-ylamino)thiazole-5-carboxylate (4.73 g,
64%).
[0337] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.85 (s, 1H), 6.69
(bs, 1H), 4.75 (t, J=3.5 Hz, 1H), 4.29 (q, J=4.5 Hz, 2H), 3.97 (m,
1H), 3.60 (m, 1H), 1.96 (m, 2H), 1.58 (m, 4H), 1.34 (t, J=4.5 Hz,
3H).
[0338] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 173.26, 161.98,
145.98, 117.41, 83.63, 65.19, 60.82, 30.39, 24.88, 21.62,
14.33.
[0339] MS (ESI): m/z (%): 257.31 ([MH.sup.+], 100%)
[0340] Ethyl
2-(tetrahydro-2H-pyran-2-ylamino)thiazole-5-carboxylate (4.73 g,
18.5 mmol) was added to a suspension of NaH (740 mg, 18.5 mmol) in
THF (20 mL) at 0.degree. C., followed by methyl iodide (1.15 mL,
18.5 mmol). The resulting mixture was heated at reflux during 3
hrs. After quenching with water and extracting with AcOEt, the
crude product was purified by column chromatography (50% AcOEt/PE),
yielding ethyl
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carboxylate
(2.02 g, 40%) as an oil.
[0341] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.89 (s, 1H), 5.29
(t, J=3.8 Hz, 1H), 4.29 (q, J=4.5 Hz, 2H), 4.07 (d, J=7.3 Hz, 1H),
3.65 (td, J=7 Hz, 2 Hz, 1H), 3.09 (s, 3H), 1.96 (m, 1H), 1.75-1.55
(m, 5H), 1.34 (t, J=4.5 Hz, 3H).
[0342] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 174.19, 162.02,
147.40, 116.83, 87.06, 68.37, 60.61, 32.47, 28.69, 24.97, 23.03,
14.30.
[0343] MS (ESI): m/z (%): 271.38 ([MH.sup.+], 100%).
[0344] LiAlH.sub.4 (425 mg, 11.20 mmol) was added to a solution of
ethyl
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carboxylate
(2.02 g, 7.47 mmol) in THF (20 mL) at 0.degree. C. in small
portions. After stirring at 0.degree. C. for 30 min, the reaction
was quenched slowly by H.sub.2O (1 mL), 5% NaOH (3 mL) and again
H.sub.2O (5 mL). Then AcOEt was added. The reaction mixture was
dried with Na.sub.2SO.sub.4 and filtered through celite. The
filtrate was concentrated and purified by column chromatography
(30%-50% AcOEt/PE gradient), yielding
(2-(methyl-(tetrahydro-2H-pyran-2-yl)amino)thiazol-5-yl)methanol
(1.5 g, 93%) as an oil.
[0345] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.00 (s, 1H), 5.16
(m, 1H), 4.65 (s, 2H), 4.06 (d, J=7 Hz, 1H), 3.63 (td, J=7.3 Hz, 2
Hz, 1H), 3.46 (s, 1H), 3.04 (s, 3H), 1.95-1.52 (m, 6H).
[0346] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 171.68, 137.27,
126.45, 87.64, 68.308, 57.61, 32.34, 28.91, 25.11, 23.30.
[0347] MS (ESI): m/z (%): 229.29 ([MH.sup.+], 100%).
[0348] MnO.sub.2 (3 g, 34.82 mmol) was added to a solution of
(2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazol-5-yl)methanol
(1.59 g, 6.96 mmol) in CHCl.sub.3 (50 mL). The resulting mixture
was stirred at RT for 2 days. Then the solution was filtered
through celite and concentrated, yielding
2-(methyl-(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde
(1.5 g, 96%) as a yellow oil.
[0349] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 9.70 (s, 1H), 7.88
(s, 1H), 5.35 (t, J=3.8 Hz, 1H), 4.08 (d, J=7 Hz, 1H), 3.65 (td,
J=7.3 Hz, 2 Hz, 1H), 3.12 (s, 3H), 1.97-1.55 (m, 6H).
[0350] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 180.21, 175.18,
152.36, 128.35, 86.66, 67.92, 32.38, 28.11, 24.44, 22.44.
[0351] MS (ESI): m/z (%): 227.31 ([MH.sup.+], 100%).
[0352] A solution of
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde
(250 mg, 1.10 mmol) and 2-aminothiazole (110 mg, 1.10 mmol) in
toluene (11 mL) and molecular sieves 3 .ANG. were stirred at reflux
overnight. The strongly yellow solution of the corresponding imine
was then poured over NaBH.sub.4 (212 mg, 5.61 mmol) in hot EtOH (80
mL). The colorless solution was filtered, concentrated and purified
by column chromatography yielding
N-methyl-N-(tetrahydro-2H-pyran-2-yl)-5-((thiazol-2-ylamino)meth-
yl)thiazol-2-amine (50 mg, 17%) as a solid.
[0353] .sup.1H-NMR (400 MHz, CD.sub.3OD/CDCl.sub.3): (ppm): 7.11
(d, J=2.5 Hz, 2H), 6.50 (d, J=2.3 Hz, 1H), 5.73 (bs, 1H), 5.14 (dd,
J=5.8 Hz, 1.8 Hz, 1H), 4.50 (s, 2H), 4.03 (d, J=7.3 Hz, 1H), 3.61
(td, J=7.0 Hz, 2 Hz, 1H), 3.02 (s, 3H), 1.97-1.52 (m, 6H).
[0354] .sup.13C-NMR (100 MHz, CD.sub.3OD/CDCl.sub.3): (ppm):
171.31, 169.33, 138.83, 138.08, 122.57, 106.98, 87.54, 68.32,
42.34, 32.31, 28.89, 25.11, 23.28.
[0355] MS (ESI): m/z (%): 311.35 ([MH.sup.+], 100%).
[0356] A solution of
N-methyl-N-(tetrahydro-2H-pyran-2-yl)-5-((thiazol-2-ylamino)methyl)thiazo-
l-2-amine (50 mg, 0.16 mmol) in 10% TFA in CHCl.sub.3 (5 mL) was
stirred at RT overnight. After evaporating the solvent, the residue
was dissolved in MeOH, neutralized with Na.sub.2CO.sub.3 and
extracted with CHCl.sub.3. The organic phase was dried,
concentrated and purified by column chromatography yielding
N-methyl-5-((thiazol-2-ylamino)methyl)thiazol-2-amine (28 mg, 77%)
as a solid.
[0357] .sup.1H-NMR (400 MHz, CD.sub.3OD/CDCl.sub.3): (ppm): 7.02
(d, J=2.3 Hz, 1H), 6.92 (s, 1H), 6.44 (d, J=2.3 Hz, 1H), 4.38 (s,
2H), 3.37 (bs, 2H), 2.84 (s, 3H).
[0358] .sup.13C-NMR (100 MHz, CD.sub.3OD/CDCl.sub.3): (ppm):
171.95, 169.74, 138.18, 137.23, 121.48, 106.87, 42.08, 31.6.
[0359] MS (ESI): m/z (%): 227.29 ([MH.sup.+], 100%).
[0360] N-Methyl-5-((thiazol-2-ylamino)methyl)thiazol-2-amine (28
mg, 0.12 mmol) was dissolved in methanolic HCl (3 N, 1 mL) and
Et.sub.2O was added. The precipitated solid was filtered by
decantation and dried in vacuo, yielding
N-methyl-5-((thiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride as a white solid (29 mg, 79%). Mp. 128-130.degree.
C.
Preparation of 2y
N.sup.2-((6-(benzylamino)pyridin-3-yl)methyl)pyridine-2,6-diamine
##STR00053##
[0362] 2-Chloro-1-methylpyridinium iodide (2.18 g, 9.8 mmol) and
DIPEA (2.1 ml 11.5 mmol) were added to a mixture of 6-bromo
pyridin-2-amine 1 (1 g, 5.7 mmol) and 6-bromonicotinic acid 2 (1.16
g, 5.7 mmol) in THF. The reaction mixture was stirred at RT for 2
days. At the conclusion of the reaction, the reaction mixture was
concentrated, diluted with chloroform (150 ml) and washed with
water and brine, and then dried over Na.sub.2SO.sub.4. Evaporation
of the solvent gave a crude yellow product, which crystallized in
EtOAc to give 6-bromo-N-(6-bromopyridin-2-yl)nicotinamide 3 (1.1 g,
54%) as a white solid.
[0363] Mp. 171-173.degree. C.
[0364] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.94 (d, J=2.0
Hz, 1H), 8.26 (dd, J=2.4, J=8.0 Hz, 2H), 7.80-7.84 (m, 2H), 7.45
(d, J=7.6 Hz, 1H).
[0365] MS (ESI): m/z=(357 M+H)
6-Bromo-N-(6-bromopyridin-2-yl)nicotinamide (650 mg, 1.8 mmol) 3
was dissolved in benzylamine (2 ml) and was heated at 140.degree.
C. for 24 hrs. Then the reaction mixture was concentrated, and the
product was recrystallized from ethyl acetate and pet-ether to give
6-(benzylamino)-N-(6-(benzylamino)pyridine-2-yl)nicotinamide 4 (450
mg, 62%) as a white solid. Mp. 132-133.degree. C.
[0366] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.72 (bs, 1H),
8.54 (s, 1H), 7.86 (s, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.61 (s, 1H)
7.23-7.32 (m, 1H), 6.53 (d, J=8.8 Hz 1H), 4.53 (s, 2H), 4.44 (s,
2H).
[0367] MS (ESI): m/z=410 (M+H).
[0368] BMS (2.2 mmol, 165 .mu.L) (400 .mu.L) was added to a
solution of 6-bromo-N-(6-bromopyridin-2-yl)nicotinamide 4 (300 mg,
0.73 mmol) in THF (20 ml). The reaction mixture was refluxed for 16
hrs. Then the reaction mixture was cooled to RT, MeOH (2 mL) was
added, followed by conc. HCl. and the reaction mixture was refluxed
for 8 hrs. The reaction mixture was concentrated and diluted with
water (2 ml), the pH adjusted to 14 and the reaction mixture was
extracted with chloroform (50 ml.times.2). The combined organic
phases were washed with brine, dried over Na.sub.2SO.sub.4.
Evaporation of the solvent gave a crude product that was purified
on silica gel (1:1, EtOAc: pet-ether) to give a white solid which
was treated with MeOH/HCl to give
N.sup.2-((6-(benzylamino)pyridin-3-yl)methyl)pyridine-2,6-diamine
5, hydrochloride salt (50 mg, 49%) as a white solid. Mp.
261-262.degree. C.
[0369] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=9.62 (brs, 1H),
8.1 (s, 1H), 7.86 (brs, 1H), 7.54 (m, 2H), 7.28-7.37 (m, 7H), 6.89
(brs, 1H), 4.61 (brs, 2H), 4.13 (brs, 2H), 4.06 (brs, 2H).
[0370] MS (ESI): m/z=304 (M+H).
Preparation of 2z
N-(Pyridin-2-yl)-5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-amine
trihydrochloride
##STR00054##
[0372] Compound 2z was prepared as described for 2p starting from
2-bromothiazole-5-carbaldehyde and 4-p-tolylthiazol-2-amine: (29
mg, 38%). Mp. 169-170.degree. C.
[0373] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): (ppm): 11.13 (s, 1H),
8.19 (d, J=4.83 Hz, 1H), 8.09 (t, J=6.4 Hz, 1H), 7.79 (d, J=8 Hz,
2H), 7.67 (t, J=8 Hz, 1H), 7.32 (s, 1H), 7.20 (d, J=8 Hz, 2H), 7.05
(s, 1H), 7.02 (s, 1H), 6.88 (t, J=6.4 Hz, 1H), 4.60 (d, J=4.8 Hz,
2H), 2.33 (s, 3H).
[0374] MS (ESI): m/z (%): 380.34 ([MH.sup.+], 100%).
Preparation of 2aa
4-(4-Chlorophenyl)-N-((2-(methylamino)thiazol-5-yl)methyl)thiazol-2-amine
dihydrochloride
##STR00055##
[0376] Compound 2aa was prepared as described for 2w starting from
2-(methyl-(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde
and 4-(4-chloro phenyl)thiazol-2-amine: (30 mg, 77%). Mp.
122-123.degree. C.
[0377] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.65 (d, J=8 Hz,
1H), 7.27 (d, J=8 Hz, 2H), 6.93 (s, 1
[0378] H), 6.63 (s, 1H), 4.43 (s, 2H), 2.8 (s, 3H).
[0379] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 172.03, 168.77,
149.84, 136.63, 133.19, 133.11, 128.52, 127.13, 121.69, 101.67,
41.84, 31.65.
[0380] MS (ESI): m/z (%): 337.38 ([MH.sup.+], 100%).
Preparation of 2ab
N.sup.5-Propyl-N.sup.2-((6-(propylamino)pyridin-3-yl)methyl)pyridine-2,5-d-
iamine
##STR00056##
[0382] DIPEA (5.5 ml, 29 mmol) and 2-chloro-1-methylpyridinium
iodide (8 g, 26 mmol) were added to a solution of
5-bromopyridin-2-amine 1 (2.56 g, 14.8 mmol) and 6-bromonicotinic
acid (3 g, 14.8 mmol) 2 in THF (150 ml). The reaction mixture was
stirred at RT for 4 days. Then the precipitate was filtered off.
The filtrate was concentrated and dissolved in chloroform (250 ml),
washed with water and brine and dried over Na.sub.2SO.sub.4. The
solvent was evaporated to give a crude product, which was
recrystallized from ethyl acetate to give
6-bromo-N-(5-bromopyridin-2-yl)nicotinamide 3 (4.3 g, 81%) as a
white solid. Mp. 204-205.degree. C.
[0383] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=11.33 (s, 1H),
8.93 (s, 1H), 8.54 (s, 1H), 8.25 (d, J=8.8 Hz 1H), 8.17 (d, J=8.8
Hz 1H), 8.11 (d, J=6.4 Hz, 1H), 7.82 (d, J=8.4 Hz 1H).
[0384] MS (ESI): m/z=357 (M.sup.+)
[0385] A solution of 6-bromo-N-(5-bromopyridin-2-yl)nicotinamide 3
(1.1 g, 3.0 mmol) in n-propylamine (neat, 5 ml) was heated at
80.degree. C. for 2 days. Then the solvent was evaporated to give
6-(propylamino)-N-(5-(propylamino)pyridin-2-yl)nicotinamide 4 as a
white solid (880 mg, 91%). Mp. 134-135.degree. C.
[0386] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=10.6 (s, 1H),
8.69 (d, J=2.4 Hz, 1H), 8.47 (d, J=2.4 Hz, 1H), 8.16 (d, J=8.8 Hz,
1H), 8.03 (dd, J1=2.4, J2=8.8 Hz, 1H), 7.97 (d, J=8.8 Hz, 1H), 7.67
(brs, 2H), 7.30 (t, J=5.2 Hz, 1H), 6.5 (d, J=8.8 Hz, 1H), 3.27 (q,
J=6.8 Hz, 2H), 2.74 (t, J=7.2 Hz, 2H), 1.54 (m, 4H), 0.91 (t, J=5.6
Hz, 6H).
[0387] MS (ESI): m/z=335 (M+Na)
[0388] BMS (215 .mu.L) was added to a solution of 4 (180 mg, 0.575
mmol) in THF (10 ml). The reaction mixture was stirred at reflux
temperature over night. The reaction mixture was cooled to RT. Then
methanol (2 ml) was added slowly followed by conc. HCl (3 mL). The
reaction mixture was refluxed for another 5 hrs. Then the reaction
mixture was cooled, concentrated under reduced pressure, and
diluted with cold water and the pH was adjusted to 14 with KOH
pellets and extracted with chloroform (50 ml.times.3). The organic
phase was washed with brine solution and dried over
Na.sub.2SO.sub.4. The solvent was evaporated to give a crude
product, which was purified on a silica gel column (EtOAc: PE,
80:20) to give the product (10 mg, 6.3%). Mp. 129-130.degree.
C.
[0389] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=8.14 (d, J=1.6
Hz, 1H), 8.0 (s, 1H), 6.42 (d, J=8.8 Hz, 1H), 6.32 (d, J=8.8 Hz,
1H), 5.0 (br s, 1H), 4.9 (br s, 1H), 4.33 (d, J=5.2 Hz, 2H), 3.25
(q, J=6.4 Hz, 4H), 1.66 (q, J=7.2 Hz, 4H), 1.01 (t, J=7.2 Hz
6H).
[0390] MS (ESI): m/z=321 (M+Na).
Preparation of 2ac
Preparation of
N-benzyl-6-((5-(thiophen-2-yl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-amin-
e (5)
##STR00057##
[0392] 2-Chloro-1-methylpyridinium iodide (580 mL, 2.2 mmol) and
DIPEA (0.4 mL) were added to a solution of 6-bromopicolinic acid 2
(304 mg, 1.5 mmol) in a mixture of dry DCM/DMF (20/2 mL). The
reaction mixture was stirred for 1 hr before tent-butyl
3-amino-5-(thiophen-2-yl)-1H-pyrazole-1-carboxylate 1 (400 mg, 1.5
mmol) was added. The resulting yellow reaction mixture was stirred
for 3 days. Then the reaction mixture was concentrated under
reduced pressure. The residue was diluted in chloroform and washed
with water and brine, and dried over Na.sub.2SO.sub.4. After
evaporation of the solvent, the crude product was purified on a
silica gel column (EtOAc: pet-ether, 1:1) to give
6-bromo-N-(5-(thiophen-2-yl)-1H-pyrazol-3-yl)picolinamide 3, (307
mg, 58%).
[0393] MS (ESI): m/z=349 (M+H) 350.8 (M+2H)
[0394] The
6-bromo-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide 3 (307
mg, 0.88 mmol) was dissolved in neat benzylamine (3 mL) without
further characterization. The reaction mixture was heated at
130.degree. C. for 24 hrs. Then the solvent was evaporated and the
residue was purified on silica gel (EtOAc: pet-ether, 1:4) to give
6-(benzylamino)-N-(5-(thiophen-2-yl)-1H-pyrazol-3-yl)picolinamide 4
as a sticky solid (200 mg, 58%).
[0395] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=10.1 (s, 1H),
7.59 (m, 2H), 7.33 (m, 5H), 7.09 (s, 1H), 6.64 (d, J=8.0 Hz, 1H),
5.11 (brs, 1H), 4.60 (s, 2H).
[0396] MS (ESI): m/z=376 (M+H).
[0397] Borane dimethylsulfide (85 .mu.L, 1.06 mmol) was added to a
solution of
6-(benzylamino)-N-(5-(4-fluorophenyl)-1H-pyrazol-3-yl)picolinamide
4 (200 mg, 0.53 mmol). The reaction mixture was refluxed for 16
hrs. The reaction mixture was cooled to RT and MeOH (2 mL) followed
by conc. HCl (2 mL) were added and the reaction mixture was
refluxed for another 4 hrs. The reaction mixture was concentrated
and diluted with water (4 ml) and the pH was adjusted to 14 using
NaOH solution and subsequently the reaction mixture was extracted
with chloroform (40 ml.times.3). All organic layers were then
washed with water and brine, dried over Na.sub.2SO.sub.4. After
evaporating the solvent, the crude product was recrystallized from
EtOAc and pet-ether to give
N-benzyl-645-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-amine-
, (80 mg, 41%). Mp. 117-118.degree. C.
[0398] 20 mg of the composition were then treated with HCl/MeOH to
give 5,
N-benzyl-6-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-ami-
ne hydrochloride salt. Mp. 120-122.degree. C.
[0399] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.23-7.42 (m, 8H)
7.06 (d, J=1.6 Hz, 1H), 6.63 (d, J=6.8 Hz, 1H), 6.30 (d, J=8.0 Hz,
1H), 5.79 (s, 1H), 5.11 (brs, 1H), 4.74 (brs, 1H), 4.52 (brs, 2H),
4.30 (s, 2H).
[0400] MS (ESI): m/z=362 (M+H)
Preparation of 2ad
N-Propyl-5-((5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-ylamino)methy-
l)thiazol-2-amine trihydrochloride
##STR00058##
[0402] DMAP (35 mg, 0.29 mmol), Et.sub.3N (16 mL, 116 mmol) and
di-tert-butyl dicarbonate (13 mL, 58 mmol) were added to a solution
of ethyl 2-aminothiazole-5-carboxylate (10 g, 58.1 mmol) in THF
(100 mL). The resulting solution was stirred at RT until completion
of the reaction. The solvents were evaporated and the crude product
was purified by precipitation with PE, yielding ethyl
2-(tert-butoxycarbonylamino)thiazole-5-carboxylate (14.2 g, 90%) as
a white solid.
[0403] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 8.03 (s, 1H), 4.32
(q, J=7.2 Hz, 2H), 1.597 (s, 9H), 1.35 (t, J=6.8 Hz, 3H).
[0404] MS (ESI): m/z (%): 273.37 [MH.sup.+].
[0405] A solution of ethyl
2-(tert-butoxycarbonylamino)thiazole-5-carboxylate (5 g, 18.36
mmol) and KOH (10.3 g, 184 mmol) in EtOH/H.sub.2O (1:1) (60 mL) was
stirred at RT for 12 hrs. The solution was acidified with 1N HCl
and the precipitate was filtered and dried, yielding
2-(tert-butoxycarbonylamino)thiazole-5-carboxylic acid (3.84 g,
86%) as a white solid.
[0406] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): (ppm): 12.0 (bs, 1H),
7.94 (s, 1H), 1.5 (s, 9H).
[0407] MS (ESI): m/z (%): 245.35 [MH.sup.+].
[0408] 2-Chloro-1-methylpyridinium iodide (0.47 g, 1.84 mmol), DIEA
(0.44 mL, 2.5 mmol) and 4-p-tolylthiazol-2-amine (0.24 g, 1.23
mmol) were added to a solution of
2-(tert-butoxycarbonylamino)thiazole-5-carboxylic acid (0.30 g,
1.23 mmol) in DMF (5 mL). The resulting solution was stirred at RT
until completion of the reaction. Then the reaction mixture was
diluted with AcOEt, washed with water, dried with Na.sub.2SO.sub.4
and concentrated. The crude product was purified by column (100%
AcOEt), yielding tert-butyl
5-(4-p-tolylthiazol-2-ylcarbamoyl)thiazol-2-ylcarbamate (80 mg,
17%) as a solid.
[0409] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 10.8 (bs, 2H),
7.76 (s, 1H), 7.61 (d, J=8 Hz, 2H), 7.15 (d, J=8 Hz, 2H), 7.09 (s,
1H), 2.35 (s, 3H), 1.42 (s, 9H).
[0410] MS (ESI): m/z (%): 417.32 [MH.sup.+].
[0411] TFA (0.100 mL) was added to a solution of tert-butyl
5-(4-p-tolylthiazol-2-ylcarbamoyl)thiazol-2-ylcarbamate (80 mg,
0.19 mmol) in CHCl.sub.3 (1 mL). The resulting solution was stirred
at RT overnight. Then the solvents were evaporated and the crude
product was dissolved in H.sub.2O. After neutralization with
NaHCO.sub.3 saturated aqueous solution, it was extracted with
AcOEt, dried and concentrated. Purification by column
chromatography (100% AcOEt) yielded
2-amino-N-(4-p-tolylthiazol-2-yl)thiazole-5-carboxamide (40 mg,
67%) as a white solid.
[0412] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm): 7.67
(s, 1H), 7.47 (d, J=8 Hz, 2H), 6.97 (d, J=8 Hz, 2H), 6.87 (s, 1H),
2.13 (s, 3H).
[0413] .sup.13C-NMR (100 MHz, CDCl.sub.3/CD.sub.3OD): (ppm):
173.92, 159.45, 158.36, 149.58, 143.45, 137.53, 131.35, 129.00,
125.59, 119.89, 106.58, 20.60.
[0414] MS (ESI): m/z (%): 317.33 [MH.sup.+].
[0415] 2-Chloro-1-methylpyridinium iodide (0.048 g, 0.18 mmol),
DIEA (0.045 mL, 0.25 mmol) and
2-amino-N-(4-p-tolylthiazol-2-yl)thiazole-5-carboxamide (0.04 g,
0.126 mmol) were added to a solution of
2-bromothiazole-5-carboxylic acid (26 mg, 0.126 mmol) in DMF (1
mL). The resulting solution was stirred at RT until completion of
the reaction. Then a solid was precipitated with AcOEt and the
solid was separated by centrifugation, yielding
2-bromo-N-(5-(4-p-tolylthiazol-2-ylcarbamoyl)thiazol-2-yl)thiazole-5-carb-
oxamide (20 mg, 30%) as a solid that was used in the following step
without further purification.
[0416] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): (ppm): 8.65 (s, 1H),
7.95 (s, 1H), 7.82 (d, J=8 Hz, 2H), 7.61 (s, 1H), 7.25 (d, J=8 Hz,
2H), 2.33 (s, 3H).
[0417] A solution of
2-bromo-N-(5-(4-p-tolylthiazol-2-ylcarbamoyl)thiazol-2-yl)thiazole-5-carb-
oxamide (15 mg, 0.03 mmol) in neat propylamine (0.5 mL) was stirred
at reflux for 24 hrs. The solvent was evaporated and the crude
product was purified by column chromatography (0%-5% MeOH/AcOEt)
yielding
2-(propylamino)-N-(5-(4-p-tolylthiazol-2-ylcarbamoyl)-thiazol-2-yl)thiazo-
le-5-carboxamide (15 mg, 17%).
[0418] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm): 8.12
(s, 1H), 7.86 (s, 1H), 7.56 (d, J=8 Hz, 2H), 7.08 (d, J=8 Hz, 2H),
6.98 (s, 1H), 3.63 (bs, 1H), 3.13 (t, J=6.4 Hz, 2H), 2.23 (s, 3H),
1.55 (q, J=6.4 Hz, 2H), 0.86 (t, J=6.4 Hz, 3H).
[0419] MS (ESI): m/z (%): 485.33 [MH.sup.+].
[0420] BMS (0.015 mL, 0.15 mmol) was added to a solution of
2-(propylamino)-N-(5-(4-p-tolylthiazol-2-ylcarbamoyl)thiazol-2-yl)thiazol-
e-5-carboxamide (15 mg, 0.03 mmol) in THF (1 mL) at RT. The
resulting solution was stirred at RT overnight. The reaction was
then quenched with MeOH (0.5 mL). 1N HCl was added until pH=2.
After stirring the reaction mixture at RT for 12 hrs, the organic
solvents were evaporated and the aqueous solution was neutralized
with a NaHCO.sub.3 saturated aqueous solution, extracted with
AcOEt, dried with Na.sub.2SO.sub.4 and concentrated. The crude
product was purified by column chromatography (0%-5% MeOH/AcOEt),
yielding
N-propyl-5-((5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-ylamino)meth-
yl)thiazol-2-amine (5 mg, 32%).
[0421] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm): 7.68
(d, J=8 Hz, 2H), 7.17 (d, J=8 Hz, 2H), 7.05 (s, 1H), 6.96 (s, 1H),
6.66 (s, 1H), 4.52 (s, 2H), 4.42 (s, 2H), 3.41 (bs, 1H), 3.15 (t,
J=6.4 Hz, 2H), 2.34 (s, 3H), 1.62 (m, 2H), 0.96 (t, J=6.4 Hz,
3H).
[0422] MS (ESI): m/z (%): 457.29 [MH.sup.+].
[0423]
N-Propyl-5-((5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-ylamin-
o)methyl)-thiazol-2-amine (5 mg, 0.01 mmol) was dissolved in
methanolic HCl (3 N, 0.5 mL) and Et.sub.2O was added. The
precipitated solid was filtered by decantation and dried in vacuo,
yielding
N-propyl-5-((5-((4-p-tolylthiazol-2-ylamino)methyl)thiazol-2-ylamino)meth-
yl)thiazol-2-amine trihydrochloride as a white solid (1.6 mg,
32%).
[0424] Mp. >135.degree. C. decomposition.
Preparation of 2ae
4-Benzyl-N-((2-(benzylamino)thiazol-5-yl)methyl)thiazol-2-amine
dihydrochloride
##STR00059##
[0426] Compound 2ae was prepared as described for 2 h, starting
from 4-benzylthiazol-2-amine and 2-bromothiazole-5-carboxylic acid:
(10 mg, 29%). Mp. 78-79.degree. C.
[0427] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm):
7.34-7.20 (m, 10H), 6.99 (s, 1H), 5.99 (s, 1H), 4.42 (s, 4H), 3.88
(s, 2H).
[0428] MS (ESI): m/z (%): 393.30 ([MH.sup.+], 100%).
Preparation of 2af
4-Benzyl-N-((2-(methylamino)thiazol-5-yl)methyl)thiazol-2-amine
dihydrochloride
##STR00060##
[0430] Compound 2af was prepared as described for 2w starting from
4-benzylthiazol-2-amine and
2-(methyl(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde:
(50 mg, 46%). Mp. 126-127.degree. C.
[0431] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.19-7.13 (m, 5H),
6.85 (s, 1H), 5.87 (s, 1H), 4.28 (s, 2H), 3.81 (s, 2H), 3.76 (s,
2H), 2.78 (s, 2H).
[0432] MS (ESI): m/z (%): 317.30 ([MH.sup.+], 100%).
Preparation of 2ag
5-Benzyl-N-((2-(benzylamino)thiazol-5-yl)methyl)thiazol-2-amine
dihydrochloride
##STR00061##
[0434] Compound 2ag was prepared as described for 2 h, starting
from 5-benzylthiazol-2-amine and 2-bromothiazole-5-carboxylic acid:
(25 mg, 69%). Mp. 96-97.degree. C.
[0435] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): (ppm):
7.27-7.14 (m, 10H), 6.88 (s, 1H), 6.73 (s, 1H), 4.33 (s, 2H), 4.29
(s, 2H), 3.88 (s, 2H).
[0436] MS (ESI): m/z (%): 393.30 ([MH.sup.+], 100%).
Preparation of 2ah
5-Benzyl-N-((2-(methylamino)thiazol-5-yl)methyl)thiazol-2-amine
dihydrochloride
##STR00062##
[0438] Compound 2ah was prepared as described for 2w starting from
5-benzylthiazol-2-amine and
2-(methyl-(tetrahydro-2H-pyran-2-yl)amino)thiazole-5-carbaldehyde
(55 mg, 69%). Mp. 76-77.degree. C.
[0439] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.20-7.13 (m, 5H),
6.84 (s, 1H), 6.71 (s, 1H), 4.28 (s, 2H), 3.85 (s, 4H), 2.78 (s,
3H).
[0440] .sup.13C-NMR (100 MHz, CDCl.sub.3): (ppm): 171.79, 168.97,
139.47, 136.87, 134.74, 128.30, 128.08, 126.35, 125.83, 121.50,
41.65, 33.01, 31.38.
[0441] MS (ESI): m/z (%): 317.30 ([MH.sup.+], 100%).
Preparation of 2ai
5-p-Tolyl-N-((5-((5-p-tolyl-1H-pyrazol-3-yl)methylamino)-1H-pyrazol-3-yl)m-
ethyl)-1H-pyrazol-3-amine trihydrochloride
1. Synthesis of
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazol-3-amine
[0442] 1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazol-3-amine
was prepared as described for compound 2a. [0443]
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylic
acid was prepared as described previously for 2a.
2. Synthesis of
5-p-tolyl-N-((5-((5-p-tolyl-1H-pyrazol-3-yl)methylamino)-1H-pyrazol-3-yl)-
methyl)-1H-pyrazol-3-amine trihydrochloride
##STR00063##
[0445] 5-Nitro-3-pyrazole-carboxylic acid (8 g, 50.90 mmol) and
sulfuric acid (5 mL, 1.48 mmol) in methanol (200 mL) were heated to
reflux for 4 hrs. The solvent was evaporated and the residue was
resuspended in CH.sub.2Cl.sub.2 and washed with water and brine and
dried Na.sub.2SO.sub.4. Methyl 5-nitro-1H-pyrazole-3-carboxylate
(7.5 g, 86%) was obtained as a white solid.
[0446] .sup.1H-NMR (400 MHz, CDCl.sub.3): b (ppm)=7.40 (s, 1H),
4.00 (s, 3H).
[0447] A mixture of methyl 5-nitro-1H-pyrazole-3-carboxylate (7.5
g, 43.8 mmol), 3,4-dihydro-2H-pyran (8 mL, 87.7 mmol) and
trifluoroacetic acid (65 .mu.L, 0.9 mmol) in anhydrous MeCN (100
mL) was refluxed for 16 hrs. The solvent was evaporated and the
residue was resuspended in CH.sub.2Cl.sub.2 (50 mL) and washed with
H.sub.2O and brine. After drying with Na.sub.2SO.sub.4, solvent
evaporation and silica gel column chromatography (PE-EtOAc, 9:1)
methyl
1-(tetrahydro-2H-pyran-2-yl)-5-nitro-1H-pyrazole-3-carboxylate
(3.71 mg, 33%) was obtained.
[0448] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.41 (s, 1H), 6.35
(dd, J=9.2 Hz, J=2.4 Hz, 1H), 4.01 (d, J=9.6 Hz, 1H), 3.94 (s, 3H),
3.73 (t, J=8.0 Hz, 1H), 2.43 (m, 1H), 2.13 (m, 1H), 2.01 (m, 1H),
1.74-1.62 (m, 3H).
[0449] Methyl
1-(tetrahydro-2H-pyran-2-yl)-5-nitro-1H-pyrazole-3-carboxylate (500
mg, 0.70 mmol) was dissolved in a mixture of MeOH/THF/H.sub.2O
(1:2:1, 20 mL). Lithium hydroxide (56.3 mg, 2.35 mmol) was added
and the reaction mixture was stirred for 16 hrs. The reaction
mixture was diluted with water and washed with DCM. The aqueous
phase was evaporated which gave
1-(tetrahydro-2H-pyran-2-yl)-5-nitro-1H-pyrazole-3-carboxylic acid
(300 mg, 64%) as a white solid which was used without further
purification in the next step.
[0450] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=7.22 (s, 1H), 6.61
(dd, J=9.2 Hz, J=2.4 Hz, 1H), 3.94 (d, J=11.2 Hz, 1H), 3.65 (t,
J=10.4 Hz, 1H), 2.35 (m, 1H), 2.09 (m, 1H), 1.92 (m, 1H), 1.71-1.54
(m, 3H).
[0451] 5-Tolyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-amine (97
mg, 0.38 mmol) was added to a solution of
1-(tetrahydro-2H-pyran-2-yl)-5-nitro-1H-pyrazole-3-carboxylic acid
(100 mg, 0.41 mmol), 2-chloro-1-methylpyridinium iodide (145 mg,
0.56 mmol) and N,N'-diisopropylethylamine (193 .mu.L, 1.13 mmol) in
DCM (10 mL). The reaction mixture was stirred at room temperature
for 16 hrs. The reaction was diluted with water and extracted with
DCM. The organic layer was washed with brine, dried with
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
by silica gel column chromatography (PE-EtOAc, 8:2) and gave
5-nitro-1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-5-p--
tolyl-1H-pyrazol-3-yl)-1H-pyrazole-3-carboxamide (60 mg, 33%).
[0452] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=9.50 (s, 0.5H),
9.39 (s, 0.5H), 7.43 (d, J=6.4 Hz, 2H), 7.38 (s, 0.5H), 7.34 (s,
0.5H), 7.28 (d, J=8.0 Hz, 2H), 6.87 (s, 1H), 6.16 (dd, J=10.0 Hz,
J=2.4 Hz, 1H), 6.14 (dd, J=10.0 Hz, J=2.0 Hz, 1H), 5.17 (d, J=10.4
Hz, 1H), 5.16 (d, J=8.4 Hz, 1H), 3.80 (m, 2H), 3.60 (m, 2H), 2.42
(s, 3H), 2.13 (m, 2H), 1.78-1.56 (m, 8H).
[0453] Methanol (5 mL) and Pd/C were added to a solution of
5-nitro-1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-5-p--
tolyl-1H-pyrazol-3-yl)-1H-pyrazole-3-carboxamide (183 mg, 0.38
mmol) in THF. The flask was then evacuated and filled with
hydrogen. The reaction mixture was stirred for 16 hrs. The catalyst
was filtered on celite and the solution was concentrated and dried
to give
5-amino-1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-5-p--
tolyl-1H-pyrazol-3-yl)-1H-pyrazole-3-carboxamide (170 mg,
quantitative).
[0454] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=8.94 (s, 0.5H),
8.83 (s, 0.5H), 7.43 (d, J=6.8 Hz, 2H), 7.28 (d, J=8.0 Hz, 2H),
6.95 (s, 1H), 6.41 (s, 1H, 1H), 6.06 (m, 2H), 5.17 (d, J=10.0 Hz,
2H), 3.73 (m, 2H), 3.59 (t, J=11.2 Hz, 2H), 2.42 (s, 3H), 2.05 (m,
2H), 1.80-1.54 (m, 8H).
[0455] MS (ESI): m/z: 451.32 [MH.sup.+].
[0456]
5-Amino-1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl-
)-5-p-tolyl-1H-pyrazol-3-yl)-1H-pyrazole-3-carboxamide (102 mg,
0.23 mmol) was added to a solution of
1-(tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazole-3-carboxylic
acid (59 mg, 0.20 mmol), 2-chloro-1-methylpyridinium iodide (79 mg,
0.31 mmol) and N,N'-diisopropylethylamine (105 .mu.L, 0.62 mmol) in
DCM (10 mL). The reaction mixture was stirred at room temperature
for 16 hrs. The reaction mixture was diluted with water and
extracted with DCM. The organic layer was washed with brine, dried
with Na.sub.2SO.sub.4 and concentrated. The crude product was
purified by silica gel column chromatography (PE-EtOAc, 7:3) and
gave
1-(tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-3-(1-(tetrah-
ydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazol-3-ylcarbamoyl)-1H-pyrazol-5-yl)-5-
-p-tolyl-1H-pyrazole-3-carboxamide (25 mg, 15%).
[0457] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm)=9.42 (s, 1H), 9.40
(s, 1H), 7.43 (m, 4H), 7.37 (s, 0.5H, 0.5H), 7.28 (m, 4H), 6.93 (s,
1H), 6.88 (s, 1H), 5.20 (m, 3H), 4.14 (m, 3H), 3.77 (t, J=11.2 Hz,
1H), 3.62 (t, J=11.6 Hz, 1H), 3.59 (t, J=10.0 Hz, 1H), 2.43 (s,
6H), 2.06 (m, 3H), 1.74 (m, 3H), 1.54 (m, 6H), 1.24 (m, 6H).
[0458] MS (ESI): m/z: 719.39 [MH.sup.+].
[0459]
1-(Tetrahydro-2H-pyran-2-yl)-N-(1-(tetrahydro-2H-pyran-2-yl)-3-(1-(-
tetrahydro-2H-pyran-2-yl)-5-p-tolyl-1H-pyrazol-3-ylcarbamoyl)-1H-pyrazol-5-
-yl)-5-p-tolyl-1H-pyrazole-3-carboxamide (28 mg, 0.04 mmol) was
suspended in anhydrous THF (500 .mu.L) and borane dimethylsulfide
complex (26 .mu.L, 0.27 mmol) was added dropwise. The reaction
mixture was stirred under reflux for 16 hrs. The reaction mixture
was then cooled down to 0.degree. C. and MeOH (50 .mu.L) was added
and the mixture was stirred for 10 min. Concentrated hydrochloric
acid (12 N) was added until pH <2 was obtained The resulting
mixture was stirred at 50.degree. C. for 16 hrs.
[0460] The mixture was cooled to room temperature and the solvent
was evaporated. The residue was resuspended in THF and the
precipitate was filtrated and washed with cold THF.
5-p-Tolyl-N-((5-((5-p-tolyl-1H-pyrazol-3-yl)methylamino)-1H-pyrazol-3-yl)-
methyl)-1H-pyrazol-3-amine was obtained as a white solid.
[0461]
5-p-Tolyl-N-((5-((5-p-tolyl-1H-pyrazol-3-yl)methylamino)-1H-pyrazol-
-3-yl)methyl)-1H-pyrazol-3-amine (10 mg, 0.023 mmol) was
recrystallized in methanolic HCl (3 N, 0.5 mL). The solid was
filtered, washed with Et.sub.2O and dried in vacuo to give a white
solid. Mp.=167.degree. C.
[0462] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=7.68 (d, J=8.0
Hz, 2H), 7.60 (d, J=8.0 Hz, 2H), 7.29 (d, J=8.0 Hz, 2H), 7.21 (d,
J=8.0 Hz, 2H), 6.57 (s, 1H), 6.23 (s, 1H), 5.80 (s, 1H), 4.40 (s,
2H), 4.34 (s, 2H), 2.34 (s, 3H), 2.31 (s, 3H).
[0463] MS (ESI): m/z: 439.36 [MH.sup.+].
Preparation of 2ak
N-Benzyl-5-((4-(4-chlorophenyl)thiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00064##
[0465] Compound 2ak was prepared as described for 2 h starting from
4-(4-chlorophenyl)thiazol-2-amine and 2-bromothiazole-5-carboxylic
acid (31 mg, 49%). Mp. 105-106.degree. C.
[0466] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.64 (d, J=8 Hz,
2H), 7.27-7.24 (m, 7H), 6.91 (s, 1H), 6.63 (s, 1H), 4.42 (s, 2H),
4.33 (s, 1H), 4.21 (bs, 4H).
[0467] MS (ESI): m/z (%): 413.35 ([MH.sup.+], 100%).
Preparation of 2al
N-Benzyl-5-((4-(thiophen-2-yl)thiazol-2-ylamino)methyl)thiazol-2-amine
dihydrochloride
##STR00065##
[0469] Compound 2al was prepared as described for 2 h, starting
from 4-(thiophen-2-yl)thiazol-2-amine and
2-bromothiazole-5-carboxylic acid (6 mg, 50%); mp 97-99.degree.
C.
[0470] .sup.1H-NMR (400 MHz, CDCl.sub.3): (ppm): 7.29-7.25 (m, 6H),
7.17 (d, J=4.8 Hz, 1H), 6.98 (d, J=5.2 Hz, 2H), 6.56 (s, 1H), 4.42
(s, 2H), 4.36 (s, 2H).
[0471] MS (ESI): m/z (%): 385.38 ([MH.sup.+], 100%).
Preparation of 2am
N-Butyl-6-((5-(4-fluorophenyl)-1H-pyrazol-3-ylamino)methyl)pyridin-2-amine
##STR00066##
[0473] Compound 2am was synthesized as described for 2s (59%). Mp.
93-94.degree. C.
[0474] .sup.1H-NMR (500 MHz, CDCl.sub.3): .delta.=7.57 (dd, J=7.0
Hz, J=11 Hz 2H), 7.40 (t, J=10.5 Hz, 1H), 7.05 (t, 10.5 Hz, 2H),
6.58 (d, J=9.0 Hz, 1H), 6.58 (d, J=9.0, 1H), 6.26 (d, J=10.5 Hz,
1H), 5.81 (s, 1H), 4.72 (brs, 1H), 4.29 (s, 2H) 3.22 (m, 2H), 1.60
(m, 2H), 1.43 (m, 2H), 0.95 (t, J=9.5 Hz 3H).
[0475] MS (ESI): m/z=340 (M+H)
Example 1
[0476] The milogP and TPSA values of various compounds are
indicated in table 1. milogP and TPSA values were calculated
according to the software available on the world wide web
(http://www.molinspiration.com), provided by P. Ertl of Novartis
Pharma AG.
TABLE-US-00003 TABLE 1 Compound milogP TPSA 2a 4.56 69.39 2c 3.40
86.78 2h 5.16 49.84 2j 4.92 49.84 2k 4.08 65.63 2n 3.52 49.84 2o
3.77 49.84 2p 2.63 62.73 2q 2.99 49.84 2s 4.35 65.63 2t 3.96 65.63
2u 3.61 69.39 2w 1.40 49.84 2y 2.48 75.86 2z 4.75 62.73 2aa 3.75
49.84 2ab 3.04 61.86 2ac 3.58 65.63 2ad 4.95 74.76 2ae 4.99 49.84
2af 3.60 49.84 2ag 4.99 49.84 2ah 3.60 49.84 2ai 4.56 110.10 2aj
4.44 61.87 2ak 5.15 49.84 2al 4.25 49.84 2am 4.00 65.63
Example 2
[0477] A number of the small molecules were tested for their
capacity to inhibit the aggregation of amyloid beta (A.beta.) 1-42
peptide using a thioflavin T spectrofluorescence assay.
Preparation of (A.beta.) Peptide Film
[0478] A.beta.1-42 lyophilized powder (Bachem) was reconstituted in
hexafluoroisopropanol (HFIP) to 1 mM. The peptide solution was
sonicated for 15 min at room temperature, agitated overnight, and
aliquots were placed into non-siliconized microcentrifuge tubes.
The HFIP was then evaporated under a stream of argon. The resulting
peptide film was dried under vacuum for 10 min, tightly sealed and
stored at -80.degree. C. until used.
Inhibition of A.beta.1-42 Aggregation
[0479] To assay for the small molecule-mediated inhibition of
A.beta.1-42 aggregation, the small molecules were dissolved before
each experiment in anhydrous dimethyl sulfoxide (DMSO,
Sigma-Aldrich) to reach a concentration of 7.4 mM. A.beta.1-42
peptide film was dissolved in DMSO to reach 400 .mu.M. Assay
solution in PBS buffer was prepared in non-siliconized incubation
tubes to reach the following concentrations: 330 .mu.M small
molecule, 33 .mu.M A.beta.1-42, 10 .mu.M thioflavin T (ThT), and
12.8% DMSO. Therefore, the final molar ratio of small molecule to
A.beta.1-42 was 10:1. A positive control without a small molecule
was prepared to measure maximum RFU. A negative control without
A.beta.1-42 was prepared for each small molecule. 3-Aminopyrazole
trimer (Trimer) was tested in all assays to ascertain
reproducibility between independent experiments. The solutions were
incubated for 24 hrs at 37.degree. C., and the spectrofluorescence
(relative fluorescence units; RFU) was read in six replicates in
black 384-well assay plates (Perkin-Elmer) on a Perkin-Elmer
FluoroCount spectrofluorometer. Inhibition of aggregation is
expressed as mean % inhibition or .+-.1 standard deviation (SD)
according to the following equation:
% inhibition = ( RFU of positive control - RFU of negative control
) - ( RFU of sample with A .beta. 1- 42 - RFU of sample without A
.beta. 1 - 42 ) ( RFU of positive control - RFU of negative control
) .times. 100 ##EQU00001##
[0480] Cut-off criterium for the selection of functional molecules
was defined at 50% inhibition capacity.
Results
[0481] The small molecules were tested for their capacity to
inhibit aggregation of A.beta.1-42 in the ThT assay. The results
for the molecules are summarized in the following table. All the
small molecules synthesized inhibited the aggregation of
A.beta.1-42 in the ThT assay to some extent and a number of the
molecules tested demonstrated an inhibition capacity over 50%.
TABLE-US-00004 TABLE Inhibition of A.beta.1-42 aggregation and
disaggregation of preformed A.beta.1-42 fibers by small molecules
Compound % inhibition 2a* 65.3 .+-. 2.6 2c 77.0 .+-. 5.3 2h 31.9
.+-. 10.1 2j 70.7 .+-. 6.8 2k 81.2 .+-. 7.3 2n 27.8 .+-. 9.9 2o
39.6 .+-. 2.8 2p 43.6 .+-. 2.1 2q 45.0 .+-. 8.3 2s* 55.1 .+-. 6.4
2t 86.8 .+-. 2.9 2u 55.3 .+-. 1.0 2w 13.9 .+-. 12.9 2y 57.0 .+-.
15.9 2z 58.7 .+-. 3.0 2aa 41.5 .+-. 9.6 2ab 1.7 .+-. 11 2ac 78.5
.+-. 1.3 2ad 82.1 .+-. 2.2 2ae 51.2 .+-. 1.5 2af 41.3 .+-. 9.2 2ag
34.2 .+-. 1.6 2ah 35.7 .+-. 5.6 2ai 84.8 .+-. 0.0 2ak 37.2 .+-.
14.8 2al 66.1 .+-. 15.4 2am 68..2 .+-. 9.7 *Fluorescent compound in
absence of Amyloid .beta.1-42
[0482] The small molecules were evaluated for their capacity to
mediate inhibition of A.beta.1-42 aggregation at a 10:1 small
molecule to A.beta.1-42 molar ratio. The results are expressed as
mean.+-.standard deviation of two independent experiments.
Example 3
FCS-Assay with 5 nM Oregon Green Labelled All-Peptide
[0483] In order to analyse for the disaggregating properties of the
compounds, preformed aggregates were used, which were induced
straight before the FCS-measurement by diluting 500 nM DMSO-stock
solutions of Oregon Green labelled A.beta.1-42 1:1 with deionized
water. The final concentration of Oregon Green labelled
A.beta.-peptide was 5 nM in 1.times.PBS and 3% DMSO. In order to
improve the reproducibility of the measurements of the
concentration dependencies of all samples were prepared
fourfold.
TABLE-US-00005 TABLE FCS-Measurements: percentage of the "number of
peaks" value obtained for the control reaction without added
compound. Compound 200 nM 100 nM 2t 67.2 nd 2c 51.2 29.4 nd: not
done
TABLE-US-00006 TABLE FCS-Measurements: percentage of the "peaks
.times. height" value obtained for the control reaction without
added compound. Compound 200 nM 100 nM 2t 53.3 nd 2c 45.1 26.6 nd:
not done
Example 4
Effect of a Compound of the Invention on Cultured Retinal Ganglion
Cell (RGC) Apoptosis
[0484] To assess the in vitro capacity of a compound of the
invention to reduce retinal ganglion cell (RGC) death related to
ocular diseases associated with pathological abnormalities/changes
in the tissues of the visual system, particularly associated with
amyloid-beta-related pathological abnormalities/changes in the
tissues of the visual system, such as, for example, neuronal
degradation, cultured RGCs from rats and mice are used.
[0485] To isolate the cells, at sacrifice the animals are
anesthesized, their eyes are removed and the retina is dissected
and incubated in 2 mg/ml papain solution for 25 minutes at
37.degree. C. to break down the extracellular matrix. At the end of
treatment, the cells are washed three times with RCG medium in the
presence of a protease inhibitor to stop the papain action. The
tissue is then triturated by passing it quickly up and down through
a Pasteur pipette until the cells are dispersed. A commercially
available Coulter counter is used to determine cell density in the
cell suspension, before culturing the cells in 95% air/5% CO.sub.2
at 37.degree. C.
[0486] In order to mimic the damage from ocular diseases associated
with pathological abnormalities/changes in the tissues of the
visual system, particularly associated with amyloid-beta-related
pathological abnormalities/changes in the tissues of the visual
system, such as, for example, neuronal degradation, and assess the
preventive effect of a compound of the invention, the cells are
incubated with L-glutamate for three days in the presence or
absence of a compound of the invention. Cells cultured in buffer
alone serve as control.
[0487] To determine RGC survival, at the end of the incubation
period the cells are fixed with 3.7% formaldehyde in phosphate
buffered saline (PBS) at room temperature for 30 minutes, rinsed
three times in PBS and incubated for 1 hour in PBS containing RGC
specific markers Thy1.1 or NF-L antibody. The antibody is then
removed by washing and the cells are incubated for 30 minutes with
fluorescence-labeled secondary antibodies goat anti-mouse IgG, goat
anti-rabbit IgG or rabbit anti-goat IgG. At the end of the
incubation, the cells are washed, stained for 5 minutes with DAPI
solution and rinsed. Surviving RGCs are counted by fluorescence
microscopy.
Example 5
Effect of a Compound of the Invention on Retinal Ganglion Cell
(RGC) Apoptosis In Vivo
[0488] To assess the in vivo capacity of a compound of the
invention to reduce retinal ganglion cell (RGC) death in
individuals affected by ocular diseases associated with
pathological abnormalities/changes in the tissues of the visual
system, particularly associated with amyloid-beta-related
pathological abnormalities/changes in the tissues of the visual
system, such as, for example, neuronal degradation, rats and mice
are used for a 2 to a 16 week long induced intra-ocular pressure
(TOP) study. Retinal ganglion cell death is measured at the end of
the study by both in vivo imaging and histological endpoint
analysis.
[0489] In order to mimic the increase in intra-ocular pressure
associated with certain ocular diseases associated with
pathological abnormalities/changes in the tissues of the visual
system, particularly associated with amyloid-beta-related
pathological abnormalities/changes in the tissues of the visual
system, such as, for example, neuronal degradation, glaucoma in
particular, the animals are first anesthetized with intraperitoneal
ketamine (75 mg/kg) and xylazine (5 mg/kg) and topical proparacaine
1% eye drops. Two alternative methods are then used to artificially
elevate IOP in one eye (unilaterally) in rats and mice. In the
first method, the anesthetized animals receive laser-induced injury
to the trabecular meshwork by treating the aqueous outflow area
with a 532-nm diode laser at the slit lamp perpendicular to the
trabeculae and parallel to the iris. The animals receive an initial
treatment of 40 to 50 spots of 50-.mu.m size, 0.4 W, and 0.6 second
duration. In the second method to artificially increase IOP, the
anesthesized animals receive a 50 .mu.l injection of hypertonic
saline solution into the episcleral veins in one eye using a
microneedle with a force just sufficient to blanch the vein.
[0490] To measure IOP, a commercially available handheld tonomer
(Tonopen XL-VET) is used. The measurements are taken while the
animals are under anesthesia as the average of 10 readings
immediately before laser treatment, 1 day after, and then weekly
for the duration of the experiment. If, at an interval of one week,
the difference in the IOP between the two eyes of the animals is
less than 6 mm Hg, the animals are not further included in the
study.
[0491] In order to evaluate the preventive effect of a compound of
the invention on RGC apoptosis, half of the animals receiving the
IOP-inducing treatment receive an intravitreal or intravenous
injection of a compound of the invention at the time of IOP
elevation. Half of the animals serve as control. The number of RGCs
is measured by both in vivo imaging and histological endpoint
analysis at 2, 4, 8 and 16 weeks after induced elevation of IOP.
Analysis of RGCs undergoing apoptosis in vivo is performed by the
DARC method. The DARC method consists in administering
intravitreally fluorophore-conjugated Annexin 5, which specifically
binds to apoptotic cells, to the animals and visualizing the RCGs
undergoing apoptosis in vivo. If necessary, this method may be used
in conjunction with backlabelling of the optic nerve from the SCN
to identify live RCGs which no longer possess an intact axon and
have lost connectivity with their targets.
[0492] In addition, in order to measure the total number of RCGs,
endpoint histological analysis of the retina and optic nerve is
performed at sacrifice. The retinas of the animals are fixed in 4%
paraformaldehyde and stained in sections or whole mount using the
RGC specific markers, such as Thy1.1, NF-L and SMI 32, as well as
antibodies specific for cells undergoing apoptosis. In each of
these methods, the total number of RGCs is measured at 2, 4, and 8,
and 16 weeks after surgical elevation of IOP.
[0493] To measure the number of RGC axons remaining in the optic
nerve after IOP elevation, the optic nerves of the animals are
dissected and the nerves are fixed in 4% paraformaldehyde,
sectioned, and stained with toluidine blue for analysis.
Sequence CWU 1
1
41112PRTArtificial SequenceArtificial Humanized C2 HuVK 1 Variable
Light Chain 1Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val Tyr Ser 20 25 30Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln
Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
1102219PRTArtificial SequenceArtificial Humanized C2 Light Chain
2Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5
10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr
Ser 20 25 30Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 2153112PRTArtificial SequenceArtificial Humanized C2 HuVH
AF 4 Variable Heavy Chain 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Leu Val 35 40 45Ala Ser Ile Asn Ser Asn Gly
Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105
1104439PRTArtificial SequenceArtificial Humanized C2 Heavy Chain
4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Leu Val 35 40 45Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 100 105 110Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 115 120 125Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser145 150 155
160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr 180 185 190Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro 210 215 220Glu Phe Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys225 230 235 240Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 245 250 255Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 260 265 270Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 275 280
285Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
290 295 300Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu305 310 315 320Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 325 330 335Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 340 345 350Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 355 360 365Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 370 375 380Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser385 390 395
400Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
405 410 415Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 420 425 430Leu Ser Leu Ser Leu Gly Lys 435
* * * * *
References