U.S. patent application number 14/725096 was filed with the patent office on 2015-09-17 for cyclic glycyl-2-allyl proline improves cognitive performance in impaired animals.
This patent application is currently assigned to Neuren Pharmaceuticals Limited. The applicant listed for this patent is Neuren Pharmaceuticals Limited. Invention is credited to Michael John Bickerdike, Jian Guan.
Application Number | 20150258091 14/725096 |
Document ID | / |
Family ID | 44370076 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150258091 |
Kind Code |
A1 |
Bickerdike; Michael John ;
et al. |
September 17, 2015 |
CYCLIC GLYCYL-2-ALLYL PROLINE IMPROVES COGNITIVE PERFORMANCE IN
IMPAIRED ANIMALS
Abstract
Embodiments of this invention provide methods for therapeutic
use of cyclic G-2-Allyl Proline to treat symptoms of cognitive
impairment associated with developmental disorders as well as
manufacture of medicaments including tablets, capsules, injectable
solutions that are useful for treatment of such conditions.
Inventors: |
Bickerdike; Michael John;
(Auckland, NZ) ; Guan; Jian; (Waltakere,
NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Neuren Pharmaceuticals Limited |
Auckland |
|
NZ |
|
|
Assignee: |
Neuren Pharmaceuticals
Limited
Auckland
NZ
|
Family ID: |
44370076 |
Appl. No.: |
14/725096 |
Filed: |
May 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14314802 |
Jun 25, 2014 |
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14725096 |
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13043215 |
Mar 8, 2011 |
8791117 |
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14314802 |
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12421871 |
Apr 10, 2009 |
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13043215 |
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PCT/US2007/021744 |
Oct 10, 2007 |
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12421871 |
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60851106 |
Oct 11, 2006 |
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Current U.S.
Class: |
514/249 |
Current CPC
Class: |
C07D 487/10 20130101;
A61P 25/28 20180101; C07D 487/04 20130101; A61K 31/4985 20130101;
A61K 31/498 20130101 |
International
Class: |
A61K 31/4985 20060101
A61K031/4985 |
Claims
1. A method for treating a symptom of cognitive impairment in a
mammal in need thereof, comprising: administering a
pharmaceutically effective amount of cyclic Glycyl-2-Allyl Proline
(cG-2-AllylP) to said mammal, said cognitive impairment resulting
from a developmental disorder.
2. The method of claim 1, said cG-2-AllylP comprises an aqueous
solution and one or more pharmaceutically acceptable excipients,
additives, carriers or adjuvants.
3. The method of claim 1, further comprising one or more
excipients, carriers, additives, adjuvants or binders in a tablet
or capsule.
4. The method of claim 1, said cyclic G-2-AllylP is administered
via an oral, intraperitoneal, intravascular, peripheral
circulation, subcutaneous, intraorbital, ophthalmic, intraspinal,
intracisternal, topical, infusion, implant, aerosol, inhalation,
scarification, intraperitoneal, intracapsular, intramuscular,
intranasal, buccal, transdermal, pulmonary, rectal or vaginal.
5. The method of claim 1, said effective amount has a lower limit
of about 0.001 milligrams per kilogram mass (mg/kg) of the animal
and an upper limit of about 100 mg/kg.
6. The method of claim 1, said cognitive impairment is associated
with Autism Spectrum Disorder (ASD), or Neurodevelopmental Disorder
(NDD), or Down's Syndrome.
7. The method of claim 6, said cognitive impairment is associated
with Autistic Disorder, Asperger Syndrome, Childhood Disintegrative
Disorder and Pervasive Developmental Disorder Not Otherwise
Specified (PDD-NOS), and Pathological Demand Avoidance (PDA).
8. The method of claim 6, said cognitive impairment is associated
with Fragile X Syndrome (FXS), Angelman Syndrome, Tuberous
Sclerosis Complex, Phelan McDermid Syndrome, Rett Syndrome, CDKL5
mutations, and X-Linked Infantile Spasm Disorder.
9. The method of claim 1, said symptom being cognitive impairment
or cognitive dysfunction, one or more signs or symptoms of memory
loss, loss of spatial orientation, decreased ability to learn,
decreased ability to form short- or long-term memory, decreased
episodic memory, decreased ability to consolidate memory, decreased
spatial memory, decreased synaptogenesis, decreased synaptic
stability, deficits in executive function, deficits in cognitive
mapping and scene memory, deficits in declarative and relational
memory, decreased rapid acquisition of configural or conjunctive
associations, decreased context-specific encoding and retrieval of
specific events, decreased episodic and/or episodic-like memory,
anxiety, abnormal fear conditioning, abnormal social behaviour,
repetitive behaviour, abnormal nocturnal behavior, seizure
activity, abnormal locomotion, abnormal expression of
Phospho-ERK1/2 and Phospho-Akt, and bradycardia
10. The method of claim 1, said treatment producing an improvement
in a symptom of ASD or NDD as assessed using one or more clinical
tests selected from the group consisting of The Rett Syndrome
Natural History/Clinical Severity Scale, Aberrant Behavior
Checklist Community Edition (ABC), Vineland Adaptive Behavior
Scales, Clinical Global Impression of Severity (CGI-S), Clinical
Global Impression Improvement (CGI-I), the Caregiver Strain
Questionnaire (CSQ), or one or more physiological tests selected
from the group consisting of electroencephalogram (EEG) spike
frequency, overall power in frequency bands of an EEG, hemispheric
coherence of EEG frequencies, stereotypic hand movement, QTc and
heart rate variability (HRV), respiratory irregularities and
coupling of cardiac and respiratory function compared to control
animals not suffering from said disorder.
11. The method of claim 1, said cognitive impairment is caused by a
decrease in glutamate receptors in the granular cell layer (CA1) of
the hippocampus of said mammal.
12. The method of claim 1, said cG-2-AllylP causes an increase in
AMPA receptors in the granular cell layer (CA1) of the hippocampus
of said mammal.
13. The method of claim 1, said cG-2-AllylP causes an increase in
neuronal plasticity caused by said cG-2AllylP in the granule cell
layer (CA1) and the pyramidal cell layer (CA3) regions of said
mammal's hippocampus.
14. The method of claim 1, said cG-2-AllylP causes an increase in
spatial memory in said mammal.
15. The method of claim 1, said cG-2-AllylP causes in increase in
novelty recognition in said mammal.
16. The method of claim 1, said cG-2-AllylP improves glutaminergic
neurotransmission in the hippocampus of said mammal.
17. The method of claim 1, said cG-2-AllylP causes an increase in
the number of pre-synaptic vesicles in the CA1 and C3 regions of
said mammal's hippocampus.
18. The method of claim 1, said cG-2-AllylP causes an increase in
synaptic density in the hippocampus of said mammal.
Description
CLAIM OF PRIORITY
[0001] This application is a Continuation of U.S. Utility
application Ser. No. 14/314,802 filed Jun. 25, 2014, Michael John
Bickerdike and Jian Guan, Inventors, which is a Continuation of
U.S. patent application Ser. No. 13/043,215, filed Mar. 8, 2011,
now U.S. Pat. No. 8,741,117, which is a Continuation-in-Part of
U.S. Utility application Ser. No. 12/421,871, filed Apr. 10, 2009
entitled "Cyclic Glycyl-2-Allyl Proline Improves Cognitive
Performance in Impaired Animals," Michael John Bickerdike and Jian
Guan, inventors (now abandoned), which is a continuation of
PCT/US2007/021744, filed Oct. 10, 2007 (now expired), which claims
priority to U.S. Provisional Patent Application No. 60/851,106,
filed Oct. 11, 2006, entitled "Cyclic Glycyl-2-Allyl Proline
Improves Cognitive Performance in Impaired Animals," Michael John
Bickerdike and Jian Guan, inventors. Each of the above patents and
applications is incorporated herein fully by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel bicyclic compounds
structurally related to diketopiperazines and methods for their
therapeutic use. In particular, this invention relates to the
neuroprotective activity of such compounds. More particularly, this
invention relates to the use of cyclic Glycyl-2-Allyl Proline
("cyclic G-2-AllylP" or "cG-2-AllylP") and pharmaceutical
compositions thereof in the treatment of cognitive disorders.
BACKGROUND
[0003] Cognitive disorders, i.e. impairments of memory and learning
processes, have a significant detrimental effect on the quality of
life of patients affected by it. Clinically recognized cognitive
disorders vary from mild cognitive impairment through to dementias
of varying severity.
[0004] Mild cognitive impairment ("MCI") is a transition stage
between the cognitive changes of normal aging and the more serious
problems caused by Alzheimer's disease. The amnestic subtype of
MCI, which has been linked to development of Alzheimer's disease,
significantly affects memory.
[0005] Dementia is a clinically recognised broad-spectrum syndrome
consisting of degrees of loss of cognitive abilities. Dementia can
be one of many symptoms of various neurological diseases or the
main abnormality associated with the disease, as it is the case in
Alzheimer's disease. (Adams and Victor's, Principle of Neurology,
7.sup.th ed.)
[0006] Most common causes of dementia include: cerebral atrophy
associated with Alzheimer's disease, Lewy-bodies disease,
frontotemporal lobar degeneration, Pick's disease; vascular
narrowing or blockage in the brain (i.e. vascular dementia also
known as multi-infarct dementia); Huntington's disease, Parkinson's
disease; head trauma; HIV infection or Down's syndrome.
[0007] Currently there only several medications that have been
shown to afford at most a modest transient benefit to the patients.
Cholinesterase inhibitors (anticholinesterases), such as donepezil
(Aricept.RTM.), galantamine (Razadyne.RTM., Razadyne ER.RTM.,
Reminyl.RTM., Nivaline.RTM.) and rivastigmine tartrate
(Exelon.RTM.) have been show to be efficacious in mild to moderate
Alzheimer's disease dementia. Exelon.RTM. has recently been
approved for the treatment of mild to moderate dementia associated
with Parkinson's disease. Memantine NMDA receptor antagonists are
the first approved Alzheimer's disease medication acting on the
glutamatergic system (Axura.RTM., Akatinol.RTM., Namenda.RTM.,
Ebixa.RTM.). These drugs however have side effects which in some
cases lead to discontinuation of the therapy.
[0008] With the increase in the life span and general aging of the
population there is a need to develop drugs which could delay or
alleviate the cognitive function in aging patients.
SUMMARY
[0009] We have previously shown in patent application
PCT/US2004/02830 filed Aug. 31, 2004, expressly incorporated herein
fully by reference, that cyclic GP analogues, including but not
limited to cyclic cyclopentyl-G-2-MeP and cyclic-G-2-AllyP are
neuroprotective and neuroregenerative. The inventors have now
discovered that cyclic G-2-AllylP is effective in treatment of
cognitive impairment.
[0010] Thus, one aspect of this invention provides novel cyclic
compounds having the structural formulas and substituents described
below.
##STR00001##
[0011] In some aspects, compounds of Formula 1 include substituents
where:
[0012] X.sup.1 is selected from the group consisting of NR', O and
S;
[0013] X.sup.2 is selected from the group consisting of CH.sub.2,
NR', O and S;
[0014] R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are
independently selected from the group consisting of --H, --OR',
--SR', --NR'R', --NO.sub.2, --CN, --C(O)R', --C(O)OR', --C(O)NR'R',
--C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl,
heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, arylalkyl, substituted arylalkyl,
heteroarylalkyl and substituted heteroarylalkyl; each R' is
independently selected from the group consisting of --H, alkyl,
heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and
heteroarylalkyl; or R and R.sup.5 taken together are
--CH.sub.2--(CH.sub.2).sub.n--CH.sub.2-- where n is an integer from
0-6; or R.sup.2 and R.sup.3 taken together are
--CH.sub.2--(CH.sub.2).sub.n--CH.sub.2-- where n is an integer from
0-6; with the proviso that when R.sup.1=methyl and
R.sup.2=R.sup.3=R.sup.4=H then R.sup.5.noteq.benzyl and; when
R.sup.1=H, at least one of R.sup.2 and R.sup.3.noteq.H.
[0015] In further aspects, this invention provides a compound of
Formula 1 or a pharmaceutically acceptable salt, stereoisomer or
hydrate thereof wherein R.sup.1=allyl,
R.sup.2=R.sup.3=R.sup.4=R.sup.5=H, X.sup.1=NH, X.sup.2=CH.sub.2
(cyclic Glycyl-2-AllylProline).
[0016] In still other aspects, this invention provides
pharmaceutical compositions comprising a pharmaceutically
acceptable excipient and a therapeutically effective amount of
cyclic G-2AllylP.
[0017] In further aspects, this invention provides methods of
treating an animal having a cognitive impairment, comprising
administration to that animal an effective amount of a composition
comprising cyclic G-2-AllylP. In yet further aspects, the animal to
be treated is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] This invention is described with reference to specific
embodiments thereof. Other aspects of this invention can be
appreciated with reference to the drawings, in which:
[0019] FIG. 1A is a graph showing effects of treatment with cyclic
G-2-AllylP on the performance in acquisition phase (days 1-4) of
the Morris Water Maze Test (MWMT) following scopolamine
treatment.
[0020] FIG. 1B is a graph showing effects of treatment with cyclic
G-2-AllylP on the latency to the platform quadrant in the probe
test (day 5) of the MWMT. FIG. 1C is a graph showing the time taken
to find the platform on day 4.sup.th of the acquisition phase for
animals in 3 groups: (1) vehicle-treated, (2) scopolamine and
cG-2AllylP-treated and (3) scopolamine-treated.
[0021] FIG. 2 is a graph showing the difference in time spent on
exploring the familiar vs novel object during the probe test on
days 25 post-treatment. The data points for familiar objects
reflect the average of time spent on exploration of 3 familiar
objects. The data point for novel object recognition is the actual
time spent exploring the novel object.
[0022] FIG. 3 is a graph showing a correlation between the AMPA
glutamate receptor-1 staining of the CA1 region of the hippocampus
and the ratio of time spend on investigation of novel object to
familiar object in testing phase of the NORT on day 24.
[0023] FIG. 4 is a graph showing the effects of cG-2-AllylP (t) on
the density of AMPA GluR1 in CA1 granular cell layer on days 6 and
24 in comparison to vehicle (veh).
[0024] FIG. 5 is a graph showing the effects of cG-2AllylP (t) on
the density of AMPA GluR1 in CA1 stratum oriens on day 24 post
treatment.
[0025] FIG. 6 is a graph showing the effect of cG-2-AllylP on the
trend to increase the density of pre-synaptic stain in CA3 region
of the hippocampus at day 24 post-treatment.
[0026] FIG. 7 is a graph showing the effect of cG-2-AllylP on the
trend to increase the density of the pre-synaptic stain in the
stratum oriens of the CA1 region on day 24 post-treatment.
[0027] FIG. 8 is a graph showing the effect of cG-2-AllylP to
increase the density of the pre-synaptic stain in the stratum
radiatum of the CA1 region on day 24 post-treatment.
[0028] FIGS. 9A, B, C are graphs showing the effect of cG-2-AllylP
treatment on the density of the NMDAR-1 in CA1 and CA3.
[0029] FIG. 9A is a graph showing the effect of cG-2-AllylP
treatment on density of the NMDAR-1 in the CA3 region.
[0030] FIG. 9B is a graph showing the effect of cG-2-AllylP
treatment on density of the NMDAR-1 in the CA1 cell layer.
[0031] FIG. 9C is a graph showing the effect of cG-2-AllylP
treatment on density of the NMDAR-1 in the CSA1 region.
[0032] FIG. 10 is a graph showing the effects of cG-2-AllylP on the
density of Krox24 staining in the CA1-2 of the hippocampus.
[0033] FIG. 11 is a graph showing the effects of cG-2-AllyP on the
number of vehicles in a 200 nm.sup.2 square apposing the
post-synaptic density in subregions CA3 and CA1 of the hippocampus
of middle aged rats (n=2 in each group).
[0034] FIG. 12 is a graph showing effects of cyclic G-2-AllylP on
neuronal survival in animals following excitotoxic oxidative
stress.
[0035] FIG. 13 is a graph showing effects of cyclic
cyclopentylG-2-MeP on neuronal survival in animals following
excitotoxic oxidative stress.
[0036] FIG. 14 is a graph showing the neuroprotective effects of
cyclic G-2-AllylP in animals subjected to global brain
ischaemia.
[0037] FIG. 15 is a graph showing effects of different doses of
cyclic G-2-AllylP on neuroprotection in animals subjected to global
brain ischaemia.
DETAILED DESCRIPTION
Definitions
[0038] "Alkenyl" refers to an unsaturated branched, straight chain
or cyclic hydrocarbon radical having at least one carbon-carbon
double bond. The radical may be in either the cis or trans
conformation about the double bond(s). Exemplary alkenyl groups
include allyl, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl,
cyclopentenyl and the like. In some embodiments the alkenyl groups
are (C.sub.2-C.sub.6) alkenyl, and in other embodiments, allyl can
be particularly useful.
[0039] "Alkyl" refers to a saturated branched, straight chain or
cyclic hydrocarbon radical. Exemplary alkyl groups include methyl,
ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, hexyl
and the like. In some embodiments the alkyl groups are
(C.sub.1-C.sub.6) alkyl.
[0040] "Alkynyl" refers to an unsaturated branched, straight chain
or cyclic hydrocarbon radical having at least one carbon-carbon
triple bond. Exemplary alkynyl groups include ethynyl, propynyl,
butynyl, isobutynyl and the like. In some embodiments the alkynyl
group is (C.sub.2-C.sub.6) alkynyl.
[0041] "Aryl" refers to an unsaturated cyclic hydrocarbon radical
with a conjugated z electron system. Exemplary aryl groups include
phenyl, naphthyl and the like. In some embodiments the aryl group
is (C.sub.5-C.sub.20) aryl.
[0042] "Arylalkyl" refers to a straight chain alkyl, alkenyl or
alkynyl group wherein one of the hydrogen atoms bound to the
terminal carbon is replaced with an aryl group. Exemplary arylalkyl
groups include benzyl, naphthylmethyl, benzylidene and the
like.
[0043] "Cognitive Impairment" or "Cognitive Dysfunction" means one
or more signs or symptoms of memory loss, loss of spatial
orientation, decreased ability to learn, decreased ability to form
short- or long-term memory, decreased episodic memory, decreased
ability to consolidate memory, decreased spatial memory, decreased
synaptogenesis, decreased synaptic stability, deficits in cognitive
mapping and scene memory, deficits in declarative and relational
memory, decreased rapid acquisition of configural or conjunctive
associations, decreased context-specific encoding and retrieval of
specific events, decreased episodic and/or episodic-like memory.
Cognitive impairment can be observed in patients having Alzheimer's
disease, Parkinson's disease, Lewy-bodies dementia and other
disorders, as well in aging animals, including humans.
[0044] "Growth factor" refers to an extracellularly active
polypeptide that stimulates a cell to grow or proliferate by
interacting with a receptor on the cell.
[0045] "Heteroalkyl" refers to an alkyl moiety wherein one or more
carbon atoms are replaced with another atom such as N, P, O, S etc.
Exemplary heteroalkyl groups include pyrrolidine, morpholine,
piperidine, piperazine, imidazolidine, pyrazolidine,
tetrahydrofuran, (C.sub.1-C.sub.10) substituted amines,
(C.sub.2-C.sub.6) thioethers and the like.
[0046] "Heteroaryl" refers to an aryl moiety wherein one or more
carbon atoms are replaced with another atom such as N, P, O, S etc.
Exemplary heteroaryl groups include carbazole, furan, imidazole,
indazole, indole, isoquinoline, purine, pyrazine, pyrazole,
pyridazine, pyridine, pyrrole, thiazole, thiophene, triazole and
the like.
[0047] "Injury" includes any acute or chronic damage of an animal
that results in degeneration or death of cells in the nervous
system. Such cells include neuronal cells and non-neuronal cells.
Injury includes stroke, non-hemorrhagic stroke, traumatic brain
injury, perinatal asphyxia associated with fetal distress such as
following abruption, cord occlusion or associated with intrauterine
growth retardation, perinatal asphyxia associated with failure of
adequate resuscitation or respiration, severe CNS insults
associated with near miss drowning, near miss cot death, carbon
monoxide inhalation, ammonia or other gaseous intoxication, cardiac
arrest, coma, meningitis, hypoglycemia and status epilepticus,
episodes of cerebral asphyxia associated with coronary bypass
surgery, hypotensive episodes and hypertensive crises, and cerebral
trauma. It is to be understood that the above examples are by way
of illustration only, and are not intended to be a complete listing
of injuries capable of being treated by the compounds and methods
of this invention.
[0048] A "pharmaceutically acceptable excipient" refers to an
excipient that is useful in preparing a pharmaceutical composition
that is generally safe, non-toxic, and desirable, and includes
excipients that are acceptable for veterinary use as well as for
human pharmaceutical use. Such excipients may be solid, liquid,
semisolid, or, in the case of an aerosol composition, gaseous.
[0049] A "pharmaceutically acceptable salt" refers to a salt that
is pharmaceutically acceptable and has the desired pharmacological
properties. Such salts include salts that may be formed where
acidic protons present in the compounds are capable of reacting
with inorganic or organic bases. Suitable inorganic salts include
those formed with the alkali metals, e.g. sodium and potassium,
magnesium, calcium, and aluminium. Suitable organic salts include
those formed with organic bases such as the amine bases e.g.
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. Such salts also include acid
addition salts formed with inorganic acids (e.g. hydrochloric and
hydrobromic acids) and organic acids (e.g. acetic acid, citric
acid, maleic acid, and the alkane- and arene-sulfonic acids such as
methanesulfonic acid and benzenesulfonic acid). When there are two
acidic groups present, a pharmaceutically acceptable salt may be a
mono-acid mono-salt or a di-acid salt; and similarly where there
are more than two acidic groups present, some or all of such groups
can be present as salts.
[0050] A "protecting group" has the meaning conventionally
associated with it in organic synthesis, i.e. a group that
selectively blocks one or more reactive sites in a multifunctional
compound such that a chemical reaction can be carried out
selectively on another unprotected reactive site and such that the
group can readily be removed after the selective reaction is
complete.
[0051] A "stereoisomer" is a molecule having the structure of
cyclic G-2-Allyl Proline, but having a chiral center. The term
"cyclic G-2-Allyl Proline" includes all stereoisomers.
[0052] "Substituted" refers to where one or more of the hydrogen
atoms on an alkyl, heteroalkyl, alkenyl, alkynyl, aryl, heteroaryl
or arylalkyl radical are independently replaced with another
substituent. Substituents include --R', --OR', --SR', --NR'R',
--NO.sub.2, --CN, --C(O)R', --C(O)OR', --C(O)NR'R', --C(NR')NR'R',
--NR'--C(NR')--OR', --NR'--C(NR')--SR', NR'--C(NR')--NR'R',
trihalomethyl and halogen where each R' is independently --H,
alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl
or heteroarylalkyl.
[0053] A "therapeutically effective amount" means the amount that,
when administered to an animal for treating a disease, is
sufficient to effect treatment for a disease or an injury. A
"therapeutically effective amount" means an amount that decreases
adverse symptoms or findings, promotes desirable symptoms or
findings, and/or treats an underlying disorder, and/or is
curative.
[0054] "Treating" or "treatment" of a disease includes preventing
the disease from occurring in an animal that may be predisposed to
the disease but does not yet experience or exhibit symptoms of the
disease (prophylactic treatment), inhibiting the disease (slowing
or arresting its development), providing relief from the symptoms
or side-effects of the disease (including palliative treatment),
and relieving the disease (causing regression of the disease).
[0055] Implicit hydrogen atoms (such as the hydrogens on the
pyrrole ring, etc.) are omitted from the formulae for clarity, but
should be understood to be present.
Compounds of the Invention
[0056] Certain embodiments of this invention include novel
derivatives of cPG having structures as described below.
##STR00002##
[0057] In certain embodiments, compounds of Formula 1 include
substituents where:
[0058] X.sup.1 is selected from the group consisting of NR', O and
S;
[0059] X.sup.2 is selected from the group consisting of CH.sub.2,
NR', O and S;
[0060] R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are
independently selected from the group consisting of --H, --OR',
--SR', --NR'R', --NO.sub.2, --CN, --C(O)R', --C(O)OR', --C(O)NR'R',
--C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl,
heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, arylalkyl, substituted arylalkyl,
heteroarylalkyl and substituted heteroarylalkyl; each R' is
independently selected from the group consisting of --H, alkyl,
heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and
heteroarylalkyl;
[0061] or R.sup.4 and R.sup.5 taken together are
--CH.sub.2--(CH.sub.2).sub.n--CH.sub.2-- where n is an integer from
0-6;
[0062] or R.sup.2 and R.sup.3 taken together are
--CH.sub.2--(CH.sub.2).sub.n--CH.sub.2-- where n is an integer from
0-6; with the proviso that when R.sup.1=methyl and
R.sup.2=R.sup.3=R.sup.4=H then R.sup.5.noteq.benzyl and; when
R.sup.1=H, at least one of R.sup.2 and R.sup.3.noteq.H.
[0063] In further embodiments, compounds of Formula 1 include
substituents where:
R.sup.1=methyl, R.sup.2=R.sup.3=R.sup.4=R.sup.5=H, X.sup.1=NH,
X.sup.2=CH.sub.2; R.sup.1=allyl, R.sup.2=R.sup.3=R.sup.4=R.sup.5=H,
X.sup.1=NH, X.sup.2=CH.sub.2; R.sup.1=R.sup.2=R.sup.3=H,
R.sup.4=R.sup.5-methyl, X.sup.1=NH, X.sup.2=CH.sub.2;
R.sup.1=R.sup.4=R.sup.5=H, R.sup.2=R.sup.3-methyl, X.sup.1=NH,
X.sup.2=CH.sub.2.
[0064] In other embodiments of the invention, compounds of Formula
1 include substituents where;
R.sup.4 and R.sup.5 taken together are
--CH.sub.2--(CH.sub.2).sub.n--CH.sub.2-- and: R.sup.1=methyl,
R.sup.2=R.sup.3=H, n=0, X.sup.1=NH, X.sup.2=CH.sub.2;
R.sup.1=methyl, R.sup.2=R.sup.3=H, n=2, X.sup.1=NH,
X.sup.2=CH.sub.2; R.sup.1=allyl, R.sup.2=R.sup.3=H, n=0,
X.sup.1=NH, X.sup.2=CH.sub.2; R.sup.1=allyl, R.sup.2=R.sup.3=H,
n=2, X.sup.1=NH, X.sup.2=CH.sub.2. R.sup.1=methyl,
R.sup.2=R.sup.3=H, n=3, X.sup.1=NH, X.sup.2=CH.sub.2;
R.sup.1=allyl, R.sup.2=R.sup.3=H, n=3, X.sup.1=NH,
X.sup.2=CH.sub.2.
[0065] In still other embodiments of the invention, compounds of
Formula 1 include substituents where R.sup.1=methyl or allyl,
R.sup.2=R.sup.3=R.sup.4=H and R.sup.5 is selected from the group
consisting of the side chains of the amino acids: alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, histidine, isolenucine, leucine, lysine, methionine,
proline, serine, threonine, tryptophan, tyrosine, valine,
norvaline, norleucine, citruline, ornithine, homocysteine,
homoserine, alloisoleucine, isovaline, sarcosine and the like.
[0066] In yet further embodiments of the invention, compounds of
Formula 1 include substituents where:
R.sup.1=methyl, R.sup.2=R.sup.3=methyl, R.sup.4=R.sup.5=H,
X.sup.1=NH and X.sup.2=S; R.sup.1=allyl, R.sup.2=R.sup.3=methyl,
R.sup.4=R.sup.5=H, X.sup.1=NH and X.sup.2=S.
[0067] Those with skill in the art will appreciate that the above
structural representations can contain chiral centres, the number
of which will depend on the different substituents. The chirality
may be either R or S at each centre. The structural drawings can
represent only one of the possible tautomeric, conformational
diastereomeric or enantiomeric forms, and it should be understood
that the invention encompasses any tautomeric, conformational
isomeric diastereomeric or enantiomeric form, which exhibits
biological or pharmacological activity as described herein.
Pharmacology and Utility
[0068] Cyclic Glycyl-2-Allyl proline (cG-2-AllylP) is described in
U.S. Utility application Ser. No. 11/399,974 filed Apr. 7, 2006,
entitled "Cyclic G-2Allyl Proline in Treatment of Parkinson's
Disease," now U.S. Pat. No. 7,776,876, issued Aug. 17, 2010, U.S.
Utility application Ser. No. 10/570,395, filed Mar. 2, 2006
entitled "Neuroprotective Bicyclic Compounds and Methods for Their
Use", PCT International Patent Application No: PCT/US2004/028308,
entitled Neuroprotective Bicyclic Compounds and methods for Their
Use, and U.S. Provisional Patent Application Ser. No. 60/499,956
filed Sep. 3, 2003, entitled "Neuroprotective Bicyclic Compounds
and Methods for Their Use". Each of the above patent applications
and the patent is expressly incorporated herein fully by
reference.
[0069] Certain aspects of this invention include the use of cyclic
G-2-AllylP in treatment of cognitive impairment associated with
aging with neurodegenerative conditions or in situations in which
cognitive impairment is found with no apparent
neurodegeneration.
[0070] Scopolamine is commonly used in animal models of cholinergic
hypofunction associated with Alzheimer's disease. The functional
deficits observed after scopolamine treatment include those found
in human patients with Alzheimer's disease. Thus, scopolamine
treatment is reasonably predictive of cognitive impairment found in
human diseases. Additionally, scopolamine treatment mimics
cognitive dysfunction in humans who do not have neurodegenerative
disorders.
[0071] cG-2-AllylP administered to animals treated with
scopolamine-induced cognitive dysfunction produces clinical
improvement in those animals, similar to the therapeutic
improvement observed in people suffering from cholinergic
hypofunction. For example, cholinergic hypofunction associated with
Alzheimer's disease. Thus, studies of effects of Cyclic G-2-AllylP
scopolamine treated animals are reasonably predictive of effects
observed in human beings suffering from cholinergic
dysfunction.
[0072] Such other agents may be selected from the group consisting
of for example, growth factors and associated derivatives, e.g.,
insulin-like growth factor-I (IGF-I), insulin-like growth factor-II
(IGF-II), the tripeptide GPE, transforming growth factor-.beta.1,
activin, growth hormone, nerve growth factor, growth hormone
binding protein, and/or IGF-binding proteins. Additional compounds
include Glycyl-2-Methyl Prolyl Glutamate and/or other compounds
disclosed in U.S. patent application Ser. No. 10/155,864, now U.S.
Pat. No. 7,041,314, issued May 9, 2006, expressly incorporated
herein fully by reference.
Therapeutic Applications
[0073] Compositions and methods of the invention find use in the
treatment of animals, such as human patients, suffering from
cognitive impairment. Still more generally, the compositions and
methods of the invention find use in the treatment of mammals, such
as human patients, suffering from memory impairment, mild cognitive
impairment, dementia, including dementia including dementias
resulting from cerebral atrophy associated with Alzheimer's
disease, Lewy-bodies disease, frontotemporal lobar degeneration,
Pick's disease; vascular narrowing or blockage in the brain (i.e.
vascular dementia also known as multi-infarct dementia);
Huntington's disease, Parkinson's disease; head trauma; HIV
infection or Down's syndrome.
Pharmaceutical Compositions and Administration
[0074] Cyclic G-2-AllylP can be administered as part of a
medicament or pharmaceutical preparation. This can involve
combining a compound of the invention with any pharmaceutically
appropriate carrier, adjuvant or excipient. The selection of the
carrier, adjuvant or excipient will of course usually be dependent
upon the route of administration to be employed.
[0075] In general, compounds of this invention will be administered
in therapeutically effective amounts by any of the usual modes
known in the art, either singly or in combination with other
conventional therapeutic agents for the disease being treated. A
therapeutically effective amount may vary widely depending on the
disease or injury, its severity, the age and relative health of the
animal being treated, the potency of the compound(s), and other
factors. As anti-apoptotic and anti-necrotic agents,
therapeutically effective amounts of cyclic G-2-AllylP may range
from 0.001 to 100 milligrams per kilogram mass of the animal, with
lower doses such as 0.001 to 0.1 mg/kg being appropriate for
administration through the cerebrospinal fluid, such as by
intracerebroventricular administration, and higher doses such as 1
to 100 mg/kg being appropriate for administration by methods such
as oral, systemic (e.g. transdermal), or parenteral (e.g.
intravenous) administration. A person of ordinary skill in the art
will be able without undue experimentation, having regard to that
skill and this disclosure, to determine a therapeutically effective
amount of a compound of this invention for a given disease or
injury.
[0076] Cyclic G-2-AllylP may be administered peripherally via any
peripheral route known in the art. These can include parenteral
routes for example injection into the peripheral circulation,
subcutaneous, intraorbital, ophthalmic, intraspinal,
intracisternal, topical, infusion (using e.g. slow release devices
or minipumps such as osmotic pumps or skin patches), implant,
aerosol, inhalation, scarification, intraperitoneal, intracapsular,
intramuscular, intranasal, oral, buccal, transdermal, pulmonary,
rectal or vaginal. The compositions can be formulated for
parenteral administration to humans or other mammals in
therapeutically effective amounts (e.g. amounts which eliminate or
reduce the patient's pathological condition) to provide therapy for
the neurological diseases described above.
[0077] Desirably, if possible, when administered as anti-apoptotic
and anti-necrotic agent, cyclic G-2-AllylP can be administered
orally. The amount of a compound of this invention in the
composition may vary widely depending on the type of composition,
size of a unit dosage, kind of excipients, and other factors well
known to those of ordinary skill in the art. In general, the final
composition may comprise from 0.0001 percent by weight (% w) to 10%
w of the compound of this invention, preferably 0.001% w to 1% w,
with the remainder being the excipient or excipients.
[0078] Other convenient administration routes include subcutaneous
injection (e.g. dissolved in a physiologically compatible carrier
such as 0.9% sodium chloride) or direct administration to the CNS.
Using stereotactic devices and accurate maps of an animals' CNS, a
compound may be injected directly into a site of neural damage.
Such routes of administration may be especially desired in
situations in which perfusion of that location is compromised
either by decreased vascular perfusion or by decreased cerebral
spinal fluid (CSF) flow to that area. Examples include
administration by lateral cerebroventricular injection or through a
surgically inserted shunt into the lateral cerebroventricle of the
brain of the patient, intraveneously, direct injection into the
desired location or other routes.
[0079] The effective amount of compound in the CNS may be increased
by administration of a pro-drug form of a compound, which comprises
a compound of the invention and a carrier, where the carrier is
joined to a compound of the invention by a linkage which is
susceptible to cleavage or digestion within the patient. Any
suitable linkage can be employed which will be cleaved or digested
following administration.
[0080] However, there is no intention on the part of the applicants
to exclude other forms of administration.
[0081] In further embodiments of the invention, restoring nerve
function in an animal can comprise administering a therapeutic
amount of cyclic G-2-AllylP in combination with another
neuroprotective agent, selected from, for example, growth factors
and associated derivatives (insulin-like growth factor-I (IGF-I),
insulin-like growth factor-II (IGF-II), transforming growth
factor-.beta.1, activin, growth hormone, nerve growth factor,
growth hormone binding protein, IGF-binding proteins (especially
IGFBP-3), basic fibroblast growth factor, acidic fibroblast growth
factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6,
keratinocyte growth factor, androgen-induced growth factor.
Additional members of the FGF family include, for example, int-2,
fibroblast growth factor homologous factor-1 (FHF-1), FHF-2, FHF-3
and FHF-4, karatinocyte growth factor 2, glial-activating factor,
FGF-10 and FGF-16, ciliary neurotrophic factor, brain derived
growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic
protein 2 (BMP-2), glial-cell line derived neurotrophic factor,
activity-dependant neurotrophic factor, cytokine leukaemia
inhibiting factor, oncostatin M, interleukin), .alpha.-, .beta.-,
.gamma.-, or consensus interferon, and TNF-.alpha.. Other forms of
neuroprotective therapeutic agents include, for example,
clomethiazole; kynurenic acid, Semax, tacrolimus,
L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,
andrenocorticotropin-(4-9) analogue (ORG 2766) and dizolcipine
(MK-801), selegiline; glutamate antagonists such as, NPS1506,
GV1505260, MK-801, GV150526; AMPA antagonists such as
2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX),
LY303070 and LY300164; anti-inflammatory agents directed against
the addressin MAdCAM-1 and/or its integrin .alpha.4 receptors
(.alpha.4.beta.1 and .alpha.4.beta.7), such as anti-MAdCAM-1 mAb
MECA-367 (ATCC accession no. HB-9478).
[0082] Cyclic G-2-AllylP is suitably administered by a
sustained-release system. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or microcapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919; EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman
et al., 1983, Biopolymers: 22: 547-56), poly(2-hydroxyethyl
methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res.: 15:
267), ethylene vinyl acetate (Langer et al., 1981, J. Biomed.
Mater. Res.: 15: 267), or poly-D-(-)-3-hydroxybutyric acid (EP
133,988). Sustained-release compositions also include a liposomally
entrapped compound. Liposomes containing the compound are prepared
by methods known per se: DE 3,218,121, EP 52,322, EP 36,676, EP
88,046, EP 143,949, EP 142,641, Japanese Pat. Appln. 83-118008,
U.S. Pat. Nos. 4,485,045 and 4,544,545, and EP 102,324. Ordinarily,
the liposomes are of the small (from or about 200 to 800 Angstroms)
unilamellar type in which the lipid content is greater than about
30 mol percent cholesterol, the selected proportion being adjusted
for the most efficacious therapy.
[0083] For parenteral administration, in one embodiment cyclic
G-2-AllylP is formulated generally by mixing each at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically, or parenterally,
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation.
[0084] Generally, the formulations are prepared by contacting
cyclic G-2-AllylP uniformly and intimately with liquid carriers or
finely divided solid carriers or both. Then, if necessary, the
product is shaped into the desired formulation. Preferably the
carrier is a parenteral carrier, more preferably a solution that is
isotonic with the blood of the recipient. Examples of such carrier
vehicles include water, saline, Ringer's solution, a buffered
solution, and dextrose solution. Non-aqueous vehicles such as fixed
oils and ethyl oleate are also useful herein.
[0085] A carrier suitably contains minor amounts of additives such
as substances that enhance isotonicity and chemical stability. Such
materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
glycine; amino acids such as glutamic acid, aspartic acid,
histidine, or arginine; monosaccharides, disaccharides, and other
carbohydrates including cellulose or its derivatives, glucose,
mannose, trehalose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as mannitol or sorbitol; counter-ions such as
sodium; non-ionic surfactants such as polysorbates, poloxamers, or
polyethylene glycol (PEG); and/or neutral salts, e.g., NaCl, KCl,
MgCl.sub.2, CaCl.sub.2, etc.
[0086] Cyclic G-2-AllylP is typically formulated in such vehicles
at a pH of from or about 4.5 to 8. It will be understood that use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of salts of the compound. The final
preparation may be a stable liquid or lyophilized solid.
[0087] Formulations of cyclic G-2-AllylP in pharmaceutical
compositions can also include adjuvants. Typical adjuvants which
may be incorporated into tablets, capsules, and the like are a
binder such as acacia, corn starch, or gelatin; an excipient such
as microcrystalline cellulose; a disintegrating agent like corn
starch or alginic acid; a lubricant such as magnesium stearate; a
sweetening agent such as sucrose or lactose; a flavouring agent
such as peppermint, wintergreen, or cherry. When dosage forms are
tablets, cyclic G-2-AllylP compositions can include binders and
optionally, a smooth coating. When the dosage form is a capsule, in
addition to the above materials, it may also contain a liquid
carrier such as a fatty oil. Other materials of various types may
be used as coatings or as modifiers of the physical form of the
dosage unit. A syrup or elixir may contain the active compound, a
sweetener such as sucrose, preservatives like propyl paraben, a
colouring agent, and a flavouring agent such as cherry. Sterile
compositions for injection can be formulated according to
conventional pharmaceutical practice. For example, dissolution or
suspension of the active compound in a vehicle such as water or
naturally occurring vegetable oil like sesame, peanut, or
cottonseed oil or a synthetic fatty vehicle like ethyl oleate or
the like may be desired. buffers, preservatives, antioxidants, and
the like can be incorporated according to accepted pharmaceutical
practice.
[0088] For injection, intraventricular administration and other
invasive routes of administration, cyclic G-2-AllylP must be
sterile. Sterility may be accomplished by any method known in the
art, for example filtration through sterile filtration membranes
(e.g., 0.2 micron membranes). Therapeutic compositions generally
are placed into a container having a sterile access port, for
example, an intravenous solution bag or vial having a stopper able
to be pierced by a hypodermic injection needle.
[0089] A pharmaceutical formulation containing cyclic G-2-AllylP
ordinarily will be stored in unit or multi-dose containers, for
example, in sealed ampoules or vials, as an aqueous solution or as
a lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10 mL vials are filled with 5 mL of
sterile-filtered 1% (w/v) aqueous solution of compound, and the
resulting mixture is lyophilized. The infusion solution is prepared
by reconstituting the lyophilized compound using bacteriostatic
Water-for-Injection. It can be readily appreciated that other
dosage forms and types of preparations can be used, and all are
considered to be part of this invention.
Preparation of the Compounds
[0090] Starting materials and reagents used in preparing cyclic
G-2-AllylP are either available from commercial suppliers such as
Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance,
Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well
known to the person of ordinary skill in the art following
procedures described in such references as Fieser and Fieser's
Reagents for Organic Synthesis, vols 1-17, John Wiley and Sons, New
York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5
and supplements, Elsevier Science Publishers, 1989; Organic
Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991;
March J; Advanced Organic Chemistry, 4.sup.th ed. John Wiley and
Sons, New York, N.Y., 1992; and Larock: Comprehensive Organic
Transformations, VCH Publishers, 1989. In most instances, amino
acids and their esters or amides, and protected amino acids, are
widely commercially available; and the preparation of modified
amino acids and their amides or esters are extensively described in
the chemical and biochemical literature and thus well-known to
persons of ordinary skill in the art.
[0091] Starting materials, intermediates, and final products this
invention may be isolated and purified using conventional
techniques, including filtration, distillation, crystallization,
chromatography, and the like. They may be characterized using
conventional methods, including physical constants and spectral
data.
[0092] Cyclic G-2-AllylP is a cyclic dipeptide (bicyclic
2,5-diketopiperazine). In general, cyclic G-2-AllylP may be
prepared by methods such as are already well-known to persons of
ordinary skill in the art of peptide and modified peptide
synthesis, following the reaction schemes set forth in the Figures
following this specification, or by following other methods
well-known to those of ordinary skill in the art of the synthesis
of peptides and analogues. See for example, Bodanzsky: Principles
of Peptide Synthesis, Berlin, New York: Springer-Verlag 1993.
Synthesis of the diketopiperazine compounds of this invention may
be by solution-phase synthesis as discussed in the Examples or via
the solid-phase synthesis method exemplified by Merrifield et al.
1963 J. Amer. Chem. Soc.: 85, 2149-2156. Solid phase synthesis may
be performed using commercial peptide synthesizers, such as the
Applied Biosystems Model 430A, using the protocols established for
the instrument.
[0093] Specific examples of diketopiperazine synthesis can be found
in the Examples following and in, for example, Fischer, 2003, J.
Peptide Science: 9: 9-35 and references therein. A person of
ordinary skill in the art will have no difficulty, taking account
of that skill and the knowledge available, and of this disclosure,
in developing one or more suitable synthetic methods for compounds
of this invention.
[0094] The choice of appropriate protecting groups for the method
chosen (solid-phase or solution-phase), and of appropriate
substrates if solid-phase synthesis is used, will be within the
skill of a person of ordinary skill in the art. Appropriate
protecting groups for peptide synthesis include t-butyloxycarbonyl
(Boc), fluorenylmethyloxycarbonyl (Fmoc), Benzyl (Bzl),
t-amyloxycarbonyl (Aoc), tosyl (Tos), benzyloxycarbonyl (Z or Cbz),
o-bromo-benzyloxycarbonyl (BrZ) and the like. Additional protecting
groups are identified in Goodman M. (ed.), "Synthesis of Peptides
and Peptidomimetics" in Methods of organic chemistry (Houben-Weyl)
(Workbench Edition, E22a,b,c,d,e; 2004; Georg Thieme Verlag,
Stuttgart, New York).
[0095] The choice of coupling agent for the method chosen will also
be within the skill of a person of ordinary skill in the art.
Suitable coupling agents include DCC
(N,N'-Dicyclohexylcarbodiimide), Bop
(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate), PyBop
(Benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate), BopCl (bis(2-oxo-3-oxazolidinyl)phosphinic
chloride), 2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate
(CIP) and the like. Other compounds may be used in the synthesis
e.g. to prevent racemisation, such as HOBt (N-Hydroxybenzotriazole)
and HOAt (1-Hydroxy-7-azabenzotriazole).
[0096] All patent and literature references cited throughout the
specification are expressly incorporated by reference in their
entirety as if each had been separately so incorporated.
EXAMPLES
[0097] The present invention is further illustrated by the
following examples. These examples are offered by way of
illustration only and are not intended to limit the scope of the
invention.
General Methods
[0098] Flash chromatography was performed using Scharlau 60 (40-60
.mu.m mesh) silica gel. Analytical thin layer chromatography was
carried out on 0.20 mm pre-coated silica gel plates (ALUGRAM.RTM.
SIL G/UV.sub.254) and compounds visualized using UV fluorescence,
or heating of plates dipped in potassium permanganate in alkaline
solution.
[0099] Melting points in degrees Celsius (.degree. C.) were
determined on an Electrothermal.RTM. melting point apparatus and
are uncorrected.
[0100] Optical rotations were measured at 20.degree. C. on a Perkin
Elmer 341 polarimeter using 10 cm path length cells and are given
in units of 10.sup.-1 degcm.sup.2g.sup.-1. Samples were prepared in
the solvent indicated at the concentration specified (measured in
g/100 cm.sup.3). IR spectra were recorded on a Perkin Elmer
Spectrum One FT-IR spectrometer. The samples were prepared as thin
films on sodium chloride discs or as solids in potassium bromide
discs. A broad signal indicated by br. The frequencies (u) as
absorption maxima are given in wavenumbers (cm.sup.-1).
[0101] NMR spectra were recorded on a Bruker AVANCE DRX400
(.sup.1H, 400 MHz; .sup.13C, 100 MHz) or a Bruker AVANCE 300
(.sup.1H, 300 MHz; .sup.13C, 75 MHz) spectrometer at ambient
temperatures. For .sup.1H NMR data chemical shifts are described in
parts per million downfield from SiMe.sub.4 and are reported
consecutively as position (.delta..sub.H), relative integral,
multiplicity (s=singlet, d=doublet, t=triplet, dd=doublet of
doublets, m=multiplet, br=broad), coupling constant (J/Hz) and
assignment. For .sup.13C NMR data, chemical shifts are described in
parts per million relative to CDCl.sub.3 and are reported
consecutively as position (.delta..sub.C), degree of hybridization
as determined by DEPT experiments, and assignment. .sup.1H NMR
spectra were referenced internally using SiMe.sub.4 (.delta. 0.00)
or CDCl.sub.3 (.delta. 7.26). .sup.13C NMR spectra were referenced
internally using CDCl.sub.3 (.delta. 77.0). When two sets of peaks
arise in the NMR spectra due to different conformations around the
glycine-proline amide bond, the chemical shift for the minor cis
conformer is marked with an asterisk (*).
[0102] Accurate mass measurements were recorded on a VG-70SE mass
spectrometer. Hexane and dichloromethane were distilled prior to
use. Methanol was dried using magnesium turnings and iodine, and
distilled under nitrogen. Triethylamine was dried over calcium
hydride and distilled under nitrogen.
Example 1
Synthesis of (8aS)-Methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione
(Cyclic G-2MeP)
##STR00003##
[0103]
(2R,5S)-4-Methyl-2-trichloromethyl-1-aza-3-oxabicyclo[3.3.0]octan-4-
-one 9
[0104] n-BuLi (1.31 M, 4.68 cm.sup.3, 6.14 mmol) was added dropwise
to a stirred solution of diisopropylamine (0.86 cm.sup.3, 6.14
mmol) in dry tetrahydrofuran (10 cm.sup.3) at |78.degree. C. under
an atmosphere of nitrogen. The solution was stirred for 5 min,
warmed to 0.degree. C. and stirred for 15 min. The solution was
then added dropwise to a solution of oxazolidinone 8 (1.00 g, 4.09
mmol) in dry tetrahydrofuran (20 cm.sup.3) at -78.degree. C. over
20 min (turned to a dark brown colour), stirred for a further 30
min then iodomethane (0.76 cm.sup.3, 12.3 mmol) was added drop-wise
over 5 min. The solution was warmed to -50.degree. C. over 2 h.
Water (15 cm.sup.3) was added and the solution warmed to room
temperature and extracted with chloroform (3.times.40 cm.sup.3).
The combined organic extracts were dried (MgSO.sub.4), filtered and
evaporated to dryness in vacuo to give a dark brown semi-solid.
Purification of the residue by flash column chromatography (15%
ethyl acetate-hexane) afforded oxazolidinone 9 (0.67 g, 63%) as a
pale yellow solid: mp 55-57.degree. C. (lit., 57-60.degree. C.);
.delta..sub.H (300 MHz, CDCl.sub.3) 1.53 (3H, s, CH.sub.3),
1.72-2.02 (3H, m, Pro.beta.-H and Pro.gamma.-H.sub.2), 2.18-2.26
(1H, m, Pro.beta.-H), 3.15-3.22 (1H, m, Pro.delta.-H), 3.35-3.44
(1H, m, Pro.delta.-H) and 4.99 (1H, s, NCH).
Methyl L-2-methylprolinate hydrochloride 10
[0105] a) Using Acetyl Chloride
[0106] Oxazolidinone 9 (0.60 g, 2.33 mmol) was dissolved in dry
methanol (15 cm.sup.3) under an atmosphere of nitrogen and acetyl
chloride (0.33 cm.sup.3, 4.66 mmol) was added dropwise to the
ice-cooled solution. The solution was heated under reflux for 4.5
h, then the solvent removed under reduced pressure to give a brown
oil which was purified by flash column chromatography (10%
CH.sub.3OH--CH.sub.2Cl.sub.2) affording the hydrochloride 10 (0.2
g, 48%) as a flaky white solid: mp 107-109.degree. C. (lit.,
106-108.degree. C.); .delta..sub.H (300 MHz, CDCl.sub.3) 1.81 (3H,
s, CH.sub.3), 1.93-2.14 (3H, m, Pro.beta.-H.sub.AH.sub.B and
Pro.gamma.-H.sub.2), 2.33-2.39 (1H, m, Pro.beta.-H.sub.AH.sub.B),
3.52-3.56 (2H, m, Pro.delta.-H.sub.2) and 3.82 (3H, s,
CO.sub.2CH.sub.3).
[0107] b) Using Thionyl Chloride
[0108] An ice-cooled solution of oxazolidinone 9 (53 mg, 0.21 mmol)
in dry methanol (1 cm.sup.3) was treated dropwise with thionyl
chloride (0.045 cm.sup.3, 0.62 mmol). The solution was heated under
reflux for 2.5 h, cooled and the solvent removed under reduced
pressure to yield a brown oil. The oil was dissolved in toluene (5
cm.sup.3), concentrated to dryness to remove residual thionyl
chloride and methanol then purified by flash column chromatography
(10% CH.sub.3OH--CH.sub.2Cl.sub.2) to afford the hydrochloride 10
(16 mg, 43%) as a flaky white solid. The .sup.1H NMR assignments
were in agreement with those reported above.
Methyl-N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 12
[0109] Dry triethylamine (0.27 cm.sup.3, 1.96 mmol) was added
dropwise to a solution of hydrochloride 10 (0.11 g, 0.61 mmol) and
N-benzyloxycarbonyl-glycine 11 (98.5%) (0.17 g, 0.79 mmol) in dry
dichloromethane (35 cm.sup.3) under an atmosphere of nitrogen at
room temperature, and the reaction mixture stirred for 10 min.
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) (0.196 g,
0.77 mmol) was added and the resultant colourless solution was
stirred for 20.5 h. The solution was washed successively with 10%
aqueous hydrochloric acid (30 cm.sup.3) and saturated aqueous
sodium hydrogen carbonate (30 cm.sup.3), dried (MgSO.sub.4),
filtered and evaporated to dryness in vacuo. Purification of the
resultant residue by flash column chromatography (50-80% ethyl
acetate-hexane; gradient elution) yielded dipeptide 12 (0.18 g,
92%) as a colourless oil. Amide 12 was shown to exist as a 98:2
trans:cis mixture of conformers by .sup.13C NMR analysis (the ratio
was estimated from the relative intensities of the resonances at
.delta. 20.8 and 23.5 assigned to the Pro.gamma.-C atoms of the
minor and major conformers, respectively): [.alpha.].sub.D -33.0 (c
1.0 in MeOH); .nu..sub.max (film)/cm.sup.-1 3406, 2952, 1732, 1651,
1521, 1434, 1373, 1329, 1310, 1284, 1257, 1220, 1195, 1172, 1135,
1107, 1082, 1052, 1029, 986, 965, 907, 876, 829, 775, 738 and 699;
Ai (300 MHz, CDCl.sub.3) 1.49 (3H, s, CH.sub.3), 1.77-2.11 (4H, m,
Pro.beta.-H.sub.2 and Pro.gamma.-H.sub.2), 3.43-3.48 (2H, m,
Pro.delta.-H.sub.2), 3.61 (3H, s, OCH.sub.3), 3.85-3.89 (2H, m,
Gly.alpha.-H.sub.2), 5.04 (2H, s, PhCH.sub.2), 5.76 (1H, br s,
N--H) and 7.21-7.28 (5H, s, ArH); .delta..sub.C (75 MHz,
CDCl.sub.3) 13.8* (CH.sub.3, Pro.alpha.-CH.sub.3), 21.1 (CH.sub.3,
Pro.alpha.-CH.sub.3), 20.8* (CH.sub.2, Pro.gamma.-C), 23.5
(CH.sub.2, Pro.gamma.-C), 38.0 (CH.sub.2, Pro.beta.-C), 40.8*
(CH.sub.2, Pro.beta.-C), 43.3 (CH.sub.2, Gly.alpha.-C), 45.5*
(CH.sub.2, Gly.alpha.-C), 46.6 (CH.sub.2, Pro.delta.-C), 48.7*
(CH.sub.2, Pro.delta.-C), 51.9* (CH.sub.3, OCH.sub.3), 52.1
(CH.sub.3, OCH.sub.3), 60.0* (quat., Pro.alpha.-C), 66.0 (quat.,
Pro.alpha.-C), 66.3 (CH.sub.2, PhCH.sub.2), 68.6* (CH.sub.2,
PhCH.sub.2), 127.5 (CH, Ph), 127.6 (CH, Ph), 127.9* (CH, Ph), 128.1
(CH, Ph), 128.3* (CH, Ph), 136.2 (quat., Ph), 155.9 (quat.,
NCO.sub.2), 166.0 (quat., Gly-CON), 169.4* (quat., Gly-CON) and
173.6 (quat., CO.sub.2CH.sub.3); m/z (EI+) 334.1535 (M.sup.+.
C.sub.17H.sub.22N.sub.2O.sub.5 requires 334.1529).
(8aS)-Methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclic
G-2-MeP)
[0110] To a solution of dipeptide 12 (0.167 g, 0.51 mmol) in
methanol (8.0 cm.sup.3) was added 10% Pd on activated charcoal (8.1
mg, 0.076 mmol) and the vessel flushed with hydrogen gas. The
resulting suspension was stirred vigorously under an atmosphere of
hydrogen for 15 h. The mixture was then filtered through a Celite
pad then a short plug of silica gel with methanol, and the solvent
removed under reduced pressure to produce cyclic G-2MeP (83 mg,
98%) as a yellow solid: mp 133-135.degree. C.; [.alpha.].sub.D
-128.1 (c 0.52 in MeOH); .delta..sub.H (300 MHz, CDCl.sub.3) 1.36
(3H, s, CH.sub.3), 1.87-2.01 (3H, m, Pro.beta.-H.sub.AH.sub.B and
Pro.gamma.-H.sub.2), 2.07-2.21 (1H, m, Pro.beta.-H.sub.AH.sub.B),
3.45-3.64 (2H, m, Pro.delta.-H.sub.2), 3.82 (1H, dd, J 17.1 and
4.1, CH.sub.AH.sub.BNH), 3.99 (1H, d, J 17.1, CH.sub.AH.sub.BNH)
and 7.66 (1H, br s, N--H); .delta..sub.C (75 MHz, CDCl.sub.3) 20.2
(CH.sub.2, Pro.gamma.-C), 23.2 (CH.sub.3, Pro.alpha.-CH.sub.3),
35.0 (CH.sub.2, Pro.beta.-C), 44.7 (CH.sub.2, Pro.delta.-C), 45.9
(CH.sub.2, CH.sub.2NH), 63.8 (quat., Pro.alpha.-C), 163.3 (quat.,
NCO) and 173.3 (quat., CONH); m/z (EI+) 168.08986 (M.sup.+.
C.sub.8H.sub.12N.sub.2O.sub.2 requires 168.08988).
Example 2
Synthesis of
(8aS)-Methy-spiro[cyclohexane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1-
,4(2H)-dione (Cyclic cyclohexyl-G-2-MeP)
##STR00004##
[0111] N-Benzyloxycarbonyl-1-aminocyclohexane-1-carboxylic acid
(14)
[0112] To a suspension of 1-aminocyclohexanecarboxylic acid 13
(0.72 g, 5.02 mmol) and sodium carbonate (1.6 g, 15.1 mmol) were
dissolved in water-dioxane (21 cm.sup.3, 3:1) was added benzyl
chloroformate (0.79 cm.sup.3, 5.52 mmol) was added dropwise and the
solution was stirred at room temperature for 19.5 h. The aqueous
layer was washed with diethyl ether (60 cm.sup.3), acidified with 2
M HCl and extracted with ethyl acetate (2.times.60 cm.sup.3). The
organic layers were combined, dried (MgSO.sub.4), filtered and
evaporated under reduced pressure to produce a colourless oil,
which solidified on standing to crude carbamate 14 (1.23 g, 88%) as
a white solid: mp 152-154.degree. C. (lit., 148-150.degree. C.);
.delta..sub.H (400 MHz, CDCl.sub.3) 1.27-1.56 (3H, m,
3.times.cyclohexyl-H), 1.59-1.73 (3H, m, 3.times.cyclohexyl-H),
1.85-1.91 (2H, m, 2.times.cyclopentyl-H), 2.05-2.09 (2H, m,
2.times.cyclopentyl-H), 5.02 (1H, br s, N--H), 5.12 (2H, s,
OCH.sub.2Ph) and 7.27-7.36 (5H, s, Ph); .delta..sub.C (100 MHz,
CDCl.sub.3) 21.1 (CH.sub.2, 2.times.cyclohexyl-C), 25.1 (CH.sub.2,
2.times.cyclohexyl-C), 32.3 (CH.sub.2, cyclohexyl-C), 59.0 (quat.,
1-C), 67.1 (CH.sub.2, OCH.sub.2Ph), 128.1 (CH, Ph), 128.2 (CH, Ph),
128.5 (CH, Ph), 136.1 (quat., Ph), 155.7 (quat., NCO.sub.2) and
178.7 (quat., CO.sub.2H).
Methyl-N-benzyloxycarbonyl-cyclohexyl-glycyl-L-2-methylprolinate
(15)
[0113] Dry triethylamine (0.21 cm.sup.3, 1.5 mmol) was added
dropwise to a solution of hydrochloride 10 (84.0 mg, 0.47 mmol),
carboxylic acid 14 (0.17 g, 0.61 mmol) and
1-hydroxy-7-azabenzotriazole (16 mg, 0.12 mmol) in dry
1,2-dichloroethane (26 cm.sup.3) under an atmosphere of nitrogen at
room temperature, and the reaction mixture stirred for 10 min.
2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (0.13 g,
0.47 mmol) was added and the resultant solution heated under reflux
for 21 h, then washed successively with 10% aqueous hydrochloric
acid (30 cm.sup.3) and saturated aqueous sodium hydrogen carbonate
(30 cm.sup.3), dried (MgSO.sub.4), filtered and evaporated to
dryness in vacuo. Purification of the resultant residue by flash
column chromatography (40-50% ethyl acetate-hexane; gradient
elution) yielded amide 15 (16 mg, 9%) as a white solid. Amide 15
was shown to exist as a 11:1 trans:cis mixture of conformers by
.sup.13C NMR analysis (the ratio was estimated from the relative
intensities of the resonances at .delta. 41.3 and 48.2 assigned to
the Pro.delta.-C atoms of the minor and major conformers,
respectively): mp 219-222.degree. C.; [.alpha.].sub.D -44.9 (c 1.31
in CH.sub.2Cl.sub.2); .nu..sub.max (film)/cm.sup.-1 3239, 2927,
1736, 1707, 1617, 1530, 1450, 1403, 1371, 1281, 1241, 1208, 1194,
1165, 1150, 1132, 1089, 1071, 1028, 984, 912, 796, 749, 739 and
699; .delta..sub.H (400 MHz, CDCl.sub.3) 1.24-2.10 (17H, m,
Pro.alpha.-CH.sub.3, Pro.beta.-H.sub.2, Pro.gamma.-H.sub.2 and
5.times.cyclohexyl-H.sub.2), 3.25-3.48 (1H, br m,
Pro.delta.-H.sub.AH.sub.B), 3.61-3.87 (4H, br m, OCH.sub.3 and
Pro.delta.-H.sub.AH.sub.B), 4.92-5.19 (3H, m, N--H and OCH.sub.2Ph)
and 7.35-7.37 (5H, s, Ph); .delta..sub.C (100 MHz, CDCl.sub.3)
21.26 (CH.sub.2, cyclohexyl-C), 21.33 (CH.sub.2, cyclohexyl-C),
21.7 (CH.sub.3, Pro.alpha.-CH.sub.3), 24.8 (CH.sub.2,
cyclohexyl-C), 25.0 (CH.sub.2, Pro.gamma.-C), 29.4* (CH.sub.2,
cyclohexyl-C), 29.7* (CH.sub.2, cyclohexyl-C), 31.1 (CH.sub.2,
cyclohexyl-C), 31.6 (CH.sub.2, cyclohexyl-C), 31.9* (CH.sub.2,
cyclohexyl-C), 32.2* (CH.sub.2, cyclohexyl-C), 32.8* (CH.sub.2,
cyclohexyl-C), 37.3 (CH.sub.2, Pro.beta.-C), 41.4* (CH.sub.2,
Pro.delta.-C), 48.2 (CH.sub.2, Pro.delta.-C), 52.1 (CH.sub.3,
OCH.sub.3), 59.1 (quat., Gly.alpha.-C), 66.7 (CH.sub.2,
OCH.sub.2Ph), 67.3* (CH.sub.2, OCH.sub.2Ph), 67.4 (quat.,
Pro.alpha.-C), 128.0* (CH, Ph), 128.1* (CH, Ph), 128.3 (CH, Ph),
128.5 (CH, Ph), 128.7 (CH, Ph), 136.6 (quat., Ph), 153.7 (quat.,
NCO.sub.2), 171.0 (quat., Gly-CO) and 174.8 (quat.,
CO.sub.2CH.sub.3); m/z (EI+) 402.2151 (M.sup.+.
C.sub.22H.sub.30N.sub.2O.sub.5 requires 402.2155).
(8aS)-Methyl-spiro[cyclohexane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1-
,4(2H)-dione (Cyclic cyclohexyl-G-2-MeP)
[0114] To a solution of amide 15 (40 mg, 0.01 mmol) in methanol
(3.3 cm.sup.3) was added 10% Pd on activated charcoal (1.6 mg,
0.015 mmol) and the vessel flushed with hydrogen gas. The resulting
suspension was stirred vigorously under an atmosphere of hydrogen
for 61.5 h, then filtered through a Celite.TM. pad with methanol
(15 cm.sup.3). The filtrate was concentrated to dryness under
reduced pressure to produce a yellow semi-solid which was purified
by reverse-phase C18 flash column chromatography (0-10%
CH.sub.3CN/H.sub.2O; gradient elution) to produce cyclic
cyclohexyl-G-2MeP (19 mg, 81%) as a white solid: mp 174-177.degree.
C.; [.alpha.].sub.D -63.8 (c 1.13 in CH.sub.2Cl.sub.2);
.nu..sub.max (film)/cm.sup.-1 3215, 2925, 2854, 1667, 1646, 1463,
1427, 1276, 1232, 1171, 1085, 1014, 900, 868, 818, 783, 726 and
715; .delta..sub.H (400 MHz, CDCl.sub.3) 1.31-1.89 (12H, m,
9.times.cyclohexyl-H and 8a-CH.sub.3), 1.94-2.15 (4H, m, 7-H.sub.2
and 8-H.sub.2), 2.26 (1H, td, J 13.7 and 4.5,
1.times.cyclohexyl-H), 3.44-3.51 (1H, m, 6-H.sub.AH.sub.B),
3.79-3.86 (1H, m, 6-H.sub.AH.sub.B) and 6.40 (1H, br s, N--H);
.delta..sub.C (100 MHz, CDCl.sub.3) 19.5 (CH.sub.2, 7-C), 20.6
(CH.sub.2, cyclohexyl-C), 20.8 (CH.sub.2, cyclohexyl-C), 24.5
(CH.sub.2, cyclohexyl-C), 25.0 (CH.sub.3, 8a-CH.sub.3), 33.7
(CH.sub.2, cyclohexyl-C), 36.3 (CH.sub.2, 8-C), 36.5 (CH.sub.2,
cyclohexyl-C), 44.7 (CH.sub.2, 6-C), 59.5 (quat., 8a-C), 64.0
(quat., 3-C), 168.1 (quat., 4-C) and 171.6 (quat., 1-C); m/z (EI+)
236.15246 (M.sup.+. C.sub.13H.sub.20N.sub.2O.sub.2 requires
236.15248).
Example 3
Synthesis of (8aS)-Allyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione
(Cyclic G-2-AllylP)
##STR00005##
[0115]
(2R,5S)-4-Allyl-2-trichloromethyl-1-aza-3-oxabicyclo[3.3.0]octan-4--
one 17
[0116] n-BuLi (1.31 M, 9.93 cm.sup.3, 13.0 mmol) was added dropwise
to a stirred solution of diisopropylamine (1.82 cm.sup.3, 13.0
mmol) in dry tetrahydrofuran (20 cm.sup.3) at -78.degree. C. under
an atmosphere of nitrogen. The solution was stirred for 5 min,
warmed to 0.degree. C., stirred for 15 min then added dropwise to a
solution of pro-oxazolidinone 16 (2.12 g, 8.68 mmol) in dry
tetrahydrofuran (40 cm.sup.3) at -78.degree. C. over 20 min and the
reaction mixture was stirred for a further 30 min then allyl
bromide (2.25 cm.sup.3, 26.0 mmol) was added dropwise over 5 min.
The solution was warmed slowly to -30.degree. C. over 4 h, quenched
with H.sub.2O (30 cm.sup.3) and the mixture warmed to room
temperature and extracted with chloroform (3.times.80 cm.sup.3).
The combined organic extracts were dried (MgSO.sub.4), filtered and
evaporated to dryness in vacuo to produce a dark brown semi-solid
which was purified by flash column chromatography (10-20% ethyl
acetate-hexane; gradient elution) to produce oxazolidinone 17 (1.48
g, 60%) as an orange oil which solidified at 0.degree. C., for
which the nmr data were in agreement with that reported in the
literature: .delta..sub.H (400 MHz, CDCl.sub.3) 1.58-1.92 (2H, m,
Pro.gamma.-H.sub.2), 1.96-2.14 (2H, m, Pro.beta.-H.sub.2),
2.50-2.63 (2H, m, Pro.delta.-H.sub.2), 3.12-3.23 (2H, m,
CH.sub.2--CH.dbd.CH.sub.2), 4.97 (1H, s, NCH), 5.13-5.18 (2H, m,
CH.dbd.CH.sub.2) and 5.82-5.92 (1H, m, CH.dbd.CH.sub.2);
.delta..sub.C (100 MHz, CDCl.sub.3) 25.1 (CH.sub.2, Pro.gamma.-C),
35.1 (CH.sub.2, Pro.beta.-C), 41.5 (CH.sub.2, Pro.delta.-C), 58.3
(CH.sub.2, CH.sub.2CH.dbd.CH.sub.2), 71.2 (quat., Pro.alpha.-C),
100.4 (quat., CCl.sub.3), 102.3 (CH, NCH), 119.8 (CH.sub.2,
CH.sub.2CH.dbd.CH.sub.2), 131.9 (CH, CH.sub.2CH--CH.sub.2) and
176.1 (quat., C.dbd.O); m/z (CI+) 284.0009 [(M+H).sup.+.
C.sub.10H.sub.13.sup.35Cl.sub.3NO.sub.2 requires 284.0012],
285.9980 [(M+H).sup.+.
C.sub.10H.sub.13.sup.35Cl.sub.2.sup.37ClNO.sub.2 requires
285.9982], 287.9951 [(M+H).sup.+.
C.sub.10H.sub.13.sup.35Cl.sup.37Cl.sub.2NO.sub.2 requires 287.9953]
and 289.9932 [(M+H).sup.+. C.sub.10H.sub.13.sup.37Cl.sub.3NO.sub.2
requires 289.9923].
Methyl L-2-allylprolinate hydrochloride 18
[0117] An ice-cooled solution of oxazolidinone 17 (0.64 g, 2.24
mmol) in dry methanol (15 cm.sup.3) was treated dropwise with a
solution of acetyl chloride (0.36 cm.sup.3, 5.0 mmol) in methanol
(5 cm.sup.3). The solution was heated under reflux for 24 h, then
cooled and the solvent removed under reduced pressure. The
resultant brown oil was dissolved in toluene (40 cm.sup.3) and
concentrated to dryness to remove residual thionyl chloride and
methanol, then purified by flash column chromatography (5-10%
CH.sub.3OH--CH.sub.2Cl.sub.2; gradient elution) to afford
hydrochloride 18 (0.29 g, 63%) as a green solid for which the NMR
data were in agreement with that reported in the literature:
.delta..sub.H (300 MHz, CDCl.sub.3) 1.72-2.25 (3H, m,
Pro.beta.-H.sub.AH.sub.B and Pray-H.sub.2), 2.32-2.52 (1H, m,
Pro.beta.-H is), 2.72-3.10 (2H, m, Pro.delta.-H.sub.2), 3.31-3.78
(2H, m, CH.sub.2CH.dbd.CH.sub.2), 3.84 (3H, s, CO.sub.2CH.sub.3),
5.20-5.33 (2H, m, CH.dbd.CH.sub.2), 5.75-5.98 (1H, m,
CH.dbd.CH.sub.2) and 8.06 (1H, br s, N--H); m/z (CI+) 170.1183
[(M+H).sup.+. C.sub.9H.sub.16NO.sub.2 requires 170.1181].
Methyl-N-tert-butyloxycarbonyl-glycyl-L-2-allylprolinate 20
[0118] Dry triethylamine (0.28 cm.sup.3, 2.02 mmol) was added
dropwise to a solution of hydrochloride 18 (0.13 g, 0.63 mmol) and
N-tert-butyloxycarbonyl-glycine 19 (0.14 g, 0.82 mmol) in dry
dichloromethane (35 cm.sup.3) under an atmosphere of nitrogen at
room temperature, and the reaction mixture was stirred for 10 min.
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) (0.20 g,
0.80 mmol) was added and the solution stirred for 19.5 h, then
washed successively with 10% aqueous hydrochloric acid (35
cm.sup.3) and saturated aqueous sodium hydrogen carbonate (35
cm.sup.3), dried (MgSO.sub.4), filtered and evaporated to dryness
in vacuo. Purification of the resultant residue by flash column
chromatography (40% ethyl acetate-hexane) yielded dipeptide 20
(0.09 g, 45%) as a light yellow oil: [.alpha.].sub.D +33.8 (c 0.83
in CH.sub.2Cl.sub.2); .nu..sub.max (film)/cm.sup.-1 3419, 3075,
2977, 2930, 2874, 1739, 1715, 1656, 1499, 1434, 1392, 1366, 1332,
1268, 1248, 1212, 1168, 1122, 1051, 1026, 1003, 943, 919, 867, 830,
779, 739, 699 and 679; .delta..sub.H (300 MHz, CDCl.sub.3) 1.42
[9H, s, C(CH.sub.3).sub.3], 1.93-2.08 (4H, m, Pro.beta.-H.sub.2 and
Pro.gamma.-H.sub.2), 2.59-2.67 (1H, m,
CH.sub.AH.sub.BCH--CH.sub.2), 3.09-3.16 (1H, m,
CH.sub.AH.sub.BCH.dbd.CH.sub.2), 3.35-3.44 (1H, m,
Pro.delta.-H.sub.AH.sub.B), 3.56-3.62 (1H, m,
Pro.delta.-H.sub.AH.sub.B), 3.70 (3H, s, OCH.sub.3), 3.89 (2H, d, J
4.2, Gly.alpha.-H.sub.2), 5.06-5.11 (2H, m, CH.dbd.CH.sub.2), 5.42
(1H, br s, Gly-NH) and 5.58-5.72 (1H, m, CH.dbd.CH.sub.2);
.delta..sub.C (75 MHz, CDCl.sub.3) 23.7 (CH.sub.2, Pro.gamma.-C),
28.3 [CH.sub.3, C(CH.sub.3).sub.3], 35.0 (CH.sub.2, Pro.beta.-C),
37.6 (CH.sub.2, CH.sub.2CH.dbd.CH.sub.2), 43.3 (CH.sub.2,
Gly.alpha.-C), 47.5 (CH.sub.2, Pro.delta.-C), 52.5 (CH.sub.3,
OCH.sub.3), 68.8 (quat., Pro.alpha.-C), 79.5 [quat.,
C(CH.sub.3).sub.3], 119.4 (CH.sub.2, CH.dbd.CH.sub.2), 132.9 (CH,
CH.dbd.CH.sub.2), 155.7 (quat., NCO.sub.2), 166.9 (quat., Gly-CON)
and 173.8 (quat., CO.sub.2CH.sub.3); m/z (EI+) 326.1845 (M.sup.+.
C.sub.16H.sub.26N.sub.2O.sub.5 requires 326.1842).
(8aS)-Allyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclic
G-2AllylP)
[0119] To a solution of dipeptide 20 (0.09 g, 0.28 mmol) in
dichloromethane (9 cm.sup.3) at room temperature was added
trifluoroacetic acid (1 cm.sup.3, 0.013 mmol) dropwise and the
reaction mixture was stirred for 1 h under an atmosphere of
nitrogen. The solution was evaporated under reduced pressure to
give a colorless oil which was dissolved in dichloromethane (10
cm.sup.3), dry triethylamine (0.096 cm.sup.3, 0.69 mmol) was added
and the reaction mixture stirred for 4.5 h, after which further
triethylamine (0.096 cm.sup.3, 0.69 mmol) was added. The reaction
mixture was stirred overnight, concentrated to dryness to give a
green oil which was purified by flash column chromatography (10%
CH.sub.3OH--CH.sub.2Cl.sub.2) to produce cyclic G-2AllylP (20 mg,
37%) as an off-white solid: mp 106-109.degree. C.; [.alpha.].sub.D
-102.7 (c 0.95 in CH.sub.2Cl.sub.2); .nu..sub.max
(CH.sub.2Cl.sub.2)/cm.sup.-1 3456, 3226, 2920, 1666, 1454, 1325,
1306, 1299, 1210, 1133, 1109, 1028, 1010, 949, 928, 882, 793, 761
and 733; .delta..sub.H (400 MHz, CDCl.sub.3) 1.92-2.01 (2H, m,
Pro.gamma.-H.sub.2), 2.09-2.16 (2H, m, Pro.beta.-H.sub.2),
2.39-2.56 (2H, m, CH.sub.2CH.sub.2.dbd.CH.sub.2), 3.46-3.53 (1H, m,
Pro.delta.-H.sub.AH.sub.B), 3.78-3.87 (2H, m,
Pro.delta.-H.sub.AH.sub.B and Gly.alpha.-H.sub.AH.sub.B), 4.09 (1H,
d, J 17.2, Gly.alpha.-H.sub.AH.sub.B), 5.16-5.20 (2H, m,
CH.dbd.CH.sub.2), 5.73-5.84 (1H, m, CH--CH.sub.2) and 7.17 (1H, br
s, N--H); .delta..sub.C (100 MHz, CDCl.sub.3) 20.1 (CH.sub.2,
Pro.gamma.-C), 34.1 (CH.sub.2, Pro.beta.-C), 41.7 (CH.sub.2,
CH.sub.2CH.sub.2=CH.sub.2), 44.9 (CH.sub.2, Pro.delta.-C), 46.4
(CH.sub.2, Gly.alpha.-C), 67.2 (quat., Pro.alpha.-C), 120.9
(CH.sub.2, CH.dbd.CH.sub.2), 131.0 (CH, CH.dbd.CH.sub.2), 163.4
(quat., NCO) and 171.7 (quat., CONH); m/z (EI+) 195.1132 (M.sup.+.
C.sub.10H.sub.15N.sub.2O.sub.2 requires 195.1134).
Example 4
Synthesis of
(8aS)-Methyl-spiro[cyclopentane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-
-1,4(2H)-dione (Cyclic Cyclopentyl-G-2-MeP)
##STR00006##
[0120] N-Benzyloxycarbonyl-1-aminocyclopentane-1-carboxylic acid
21
[0121] A solution of benzyl chloroformate (0.290 g, 1.1 mmol) in
dioxane (2.5 cm.sup.3) was added dropwise to a solution of
1-aminocyclopentanecarboxylic acid (Fluka) (0.2 g, 1.54 mmol) and
sodium carbonate (0.490 g, 4.64 mmol) in water (5 cm.sup.3) at
0.degree. C. Stirring was continued at room temperature overnight
and the reaction mixture washed with ether. The aqueous layer was
acidified with 2M hydrochloric acid, extracted with ethyl acetate,
dried (Na.sub.2SO.sub.4), filtered and the solvent removed to
afford carbamate 21 (0.253 g, 62%) as an oil which solidified on
standing. Carbamate 21 was shown to be a 70:30 mixture of
conformers by .sup.1H NMR analysis (the ratio was estimated from
the integration of the resonances at .delta. 5.31 and 7.29-7.40,
assigned to the N--H protons of the major and minor conformers,
respectively): mp 70-80.degree. C. (lit..sup.1 82-86.degree. C.,
ethyl acetate, petroleum ether); .delta..sub.H (400 MHz;
CDCl.sub.3; Me.sub.4Si) 1.83 (4H, br s,
2.times.cyclopentyl-H.sub.2), 2.04 (2H, br s, cyclopentyl-H.sub.2),
2.20-2.40 (2H, m, cyclopentyl-H.sub.2), 5.13 (2H, br s,
OCH.sub.2Ph), 5.31 (0.7H, br s, N--H) and 7.29-7.40 (5.3H, m, Ph
and N--H); .delta..sub.C (100 MHz; CDCl.sub.3) 24.6 (CH.sub.2,
cyclopentyl-C), 37.5 (CH.sub.2, cyclopentyl-C), 66.0 (quat.,
cyclopentyl-C), 66.8 (CH.sub.2, OCH.sub.2Ph), 128.0 (CH, Ph), 128.1
(CH, Ph), 128.4 (CH, Ph), 136.1 (quat, Ph), 155.8 (quat.,
NCO.sub.2) and 179.5 (quat., CO.sub.2H).
Methyl N-benzyloxycarbonyl cyclopentyl-glycyl-L-2-methylprolinate
22
[0122] Dry triethylamine (0.19 cm.sup.3, 1.4 mmol) was added
dropwise to a solution of hydrochloride 10 (78 mg, 0.43 mmol),
carboxylic acid 21 (0.15 g, 0.56 mmol) and
1-hydroxy-7-azabenzotriazole (Acros) (15 mg, 0.11 mmol) in dry
1,2-dichloroethane (24 cm.sup.3) under an atmosphere of nitrogen at
room temperature, and the reaction mixture stirred for 10 min.
2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP)
(Aldrich) (0.12 g, 0.43 mmol) was added and the resultant solution
heated under reflux for 19 h, then washed successively with 10%
aqueous hydrochloric acid (30 cm.sup.3) and saturated aqueous
sodium hydrogen carbonate (30 cm.sup.3), dried (MgSO.sub.4),
filtered and evaporated to dryness in vacuo. Purification of the
resultant residue by flash column chromatography (60% ethyl
acetate-hexane) yielded amide 22 (39 mg, 23%) as a white solid.
Amide 22 was shown to exist as a 3:1 trans:cis mixture of carbamate
conformers by .sup.13C NMR analysis (the ratio was estimated from
the relative intensities of the resonances at .delta. 154.1 and
155.7 assigned to the carbamate carbonyl-C atoms of the major and
minor conformers, respectively): mp 200-203.degree. C.;
[.alpha.].sub.D -54.5 (c 1.52 in CH.sub.2Cl.sub.2); .nu..sub.max
(film)/cm.sup.-1 3432, 3239, 3042, 2953, 1736, 1712, 1627, 1540,
1455, 1417, 1439, 1374, 1282, 1256, 1216, 1194, 1171, 1156, 1136,
1100, 1081, 1042, 1020, 107, 953, 917, 876, 756 and 701; Sa (400
MHz, CDCl.sub.3) 1.33-1.53 (3H, br m, Pro.alpha.-CH.sub.3),
1.62-2.20 (11H, m, Pro.beta.-H.sub.2, Pro.gamma.-H.sub.2 and
7.times.cyclopentyl-H), 2.59-2.71 (1H, br m,
1.times.cyclopentyl-H), 3.31-3.42 (1H, br m,
Pro.delta.-H.sub.AH.sub.B), 3.58-3.79 (4H, br m, OCH.sub.3 and
Pro.delta.-H.sub.AH.sub.B), 4.92-5.17 (3H, in, N--H and
OCH.sub.2Ph) and 7.27-7.42 (5H, s, Ph); .delta..sub.C (100 MHz,
CDCl.sub.3) 21.7 (CH.sub.3, Pro.alpha.-CH.sub.3), 24.1* (CH.sub.2,
cyclopentyl-C), 24.2 (CH.sub.2, cyclopentyl-C), 24.4 (CH.sub.2,
Pro.gamma.-C), 24.5 (CH.sub.2, cyclopentyl-C), 36.4 (CH.sub.2,
cyclopentyl-C), 37.1 (CH.sub.2, cyclopentyl-C), 37.2* (CH.sub.2,
cyclopentyl-C), 37.7 (CH.sub.2, Pro.beta.-C), 38.2* (CH.sub.2,
cyclopentyl-C), 48.5 (CH.sub.2, Pro.delta.-C), 52.1 (CH.sub.3,
OCH.sub.3), 66.6 (CH.sub.2, OCH.sub.2Ph), 66.9 (quat.,
Pro.alpha.-C), 67.2 (quat., Gly.alpha.-C), 127.8 (CH, Ph), 128.2
(CH, Ph), 128.4 (CH, Ph), 136.6 (quat., Ph), 154.1 (quat.,
NCO.sub.2), 155.7* (quat., NCO.sub.2), 170.5 (quat., Gly-CO) and
174.7 (quat., CO.sub.2CH.sub.3); m/z (EI+) 388.1991 (M.sup.+.
C.sub.21H.sub.28N.sub.2O.sub.5 requires 388.1998).
(8aS)-Methyl-spiro[cyclopentane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]--
1,4(2H)-dione (Cyclic cyclopentyl-G-2-MeP)
[0123] To a solution of amide 22 (54 mg, 0.14 mmol) in methanol
(4.6 cm.sup.3) was added 10% Pd on activated charcoal (2.2 mg,
0.021 mmol) and the vessel flushed with hydrogen gas. The resulting
suspension was stirred vigorously under an atmosphere of hydrogen
for 17 h, then filtered through a Celite.TM. pad with methanol (15
cm.sup.3). The filtrate was concentrated to dryness under reduced
pressure to give a yellow semi-solid which was purified by
reverse-phase C18 flash column chromatography (0-10%
CH.sub.3CN/H.sub.2O; gradient elution) to produce cyclic
cyclopentyl-G-2MeP (20 mg, 65%) as a yellow solid: mp
160-163.degree. C.; [.alpha.].sub.D-97.9 (c 1.61 in
CH.sub.2Cl.sub.2); .nu..sub.max (film)/cm.sup.-1 3429, 2956, 2928,
2856, 1667, 1643, 1463, 1432, 1373, 1339, 1254, 1224, 1175, 1086,
1048, 976, 835, 774 and 730; .delta..sub.H (300 MHz, CDCl.sub.3)
1.47 (3H, br s, 8a-CH.sub.3), 1.56-2.19 (11H, m, 8-H.sub.2,
7-H.sub.2 and 7.times.cyclopentyl), 2.58-2.67 (1H, br m,
1.times.cyclopentyl), 3.48-3.56 (1H, m, 6-H.sub.AH.sub.B),
3.72-3.82 (1H, m, 6-H.sub.AH.sub.B) and 6.56 (1H, br s, N--H);
.delta..sub.C (75 MHz, CDCl.sub.3) 19.9 (CH.sub.2, 7-C), 24.6
(CH.sub.2, cyclopentyl), 24.92 (CH.sub.3, 8a-CH.sub.3), 24.93
(CH.sub.2, cyclopentyl), 36.0 (CH.sub.2, 8-C), 38.7 (CH.sub.2,
cyclopentyl), 41.9 (CH.sub.2, cyclopentyl), 44.8 (CH.sub.2, 6-C),
64.3 (quat., 8a-C), 66.8 (quat., 3-C), 168.3 (quat., 4-C) and 172.2
(quat., 1-C); m/z (EI+) 222.1369 (M+.
C.sub.12H.sub.18N.sub.2O.sub.2 requires 222.1368).
In Vivo Testing
[0124] The following pharmacological studies demonstrate efficacy
of cyclic G-2AllylP in attenuation of cognitive impairment. They
are not intended to be limiting, and other compositions and methods
of this invention can be developed without undue experimentation.
All of those compositions and methods are considered to be part of
this invention. All the following experiments were carried out
using protocols developed under guidelines approved by the
University of Auckland Animal Ethics Committee.
[0125] Efficacy of nootropic drugs can be conveniently tested using
models of cholinergic hypofunction. Cholinergic hypofunction has
been shown to contribute to dementia-related cognitive decline and
remains a target of therapeutic intervention for Alzheimer's
disease (Hunter 2004). The cholinergic hypofunction model is also
applicable to other conditions. For example, it has been shown that
scopolamine-induced cholinergic hypofunction can selectively impair
the recognition accuracy of disgust and anger facial expressions
rendering the of scopolamine on emotion-recognition similar to
those found in Huntington's disease patients (Kamboy 2006).
Scopolamine has been commonly used to induce cholinergic
hypofunction, and is a well-known model for human Alzheimer's
disease, aging and other disorders of cognitive function (Liskowsky
et al, Int. J. Dev. Neurosci, 24(2-3):149-156 (2006), Lindner et
al., Psychopharmacology (Berl.) September 27 (2006), Bouger et al.,
Eur. Neuropsychopharmacol 15(3):331-346 (2005), Ebert et al, Eur.
J. Clin. Invest., 28(11):944-949 (1998), Barker et al, Int. J.
Geriatr. Psychiatry, 13(4):244-247 (1998), G. Smith, Brain Res.
471(2):103-118 (1998), Flood et al, Behav. Neural. Biol.
45(2):169-184 (1986)).
Example 5
Morris Water Maze (MWM) Model of Learning and Memory Used to Assess
Effects of Cyclic G-2-AllylP on Cognitive Function
[0126] The purpose of the study was to investigate cyclic G-2AllylP
in modes of cognitive deficit and affective state (anxiety).
Methods
[0127] The first part of the study involved acute testing of the
cG-2AllylP in the Morris Water Maze memory model. The MWM test is
one of the most frequently used tests for assessing spatial memory
in rats and is well recognized to accurately predict effects of
disease and treatment on spatial memory generally. Therefore, the
MWM test reflects effects of disease and treatment in human
subjects.
[0128] The standard procedure for MWM was followed. We used a
circular swimming pool (80 cm depth.times.150 cm diameter) filled
with opaque water, with the temperature maintained at 20.degree. C.
A platform was hidden 1 cm below the water surface, with a white
flag (10 cm.times.10 cm) located either 20 cm above the platform
for the visual cue and at 3 o'clock position in relation to the
starting location for a spatial cue. On days 1-4 of the experiment
rats underwent memory acquisition trials with 6 trials (60 seconds
each) in each day of testing (habituation phase). Latency to reach
the platform was recorded and the daily reduction of average
latency was used to measure the capability to learn where the
hidden platform was.
[0129] On day 5 of the experiment normal, non-aged Wistar rats were
split into groups to receive either saline (n=28) or scopolamine
(0.5 mg/kg, i.p., n=27) to induce memory deficit. Scopolamine was
administered half an hour before the probe test commenced.
[0130] 10 min following the scopolamine treatment, the cyclic
G-2AllylP was administered orally at 30 mg/kg (n=31) with
vehicle-treated animals administered the diluent by oral gavage
using an identical treatment protocol (n-24).
[0131] Acute effects of cG-2allylP were then tested in animals with
scopolamine-induced memory impairment and in age-matched control
animals with no memory impairment to determine any direct
pharmacological effect on memory processing. Experimental groups
are detailed in the Table 1 below.
TABLE-US-00001 TABLE 1 Animals Used to Test Effects of cG-2-AllylP
on Memory Scopolamine Vehicle Vehicle N = 12 N = 12 cG-2AllylP N =
15 N = 16
[0132] On day 5, the probe MWM test was performed with the platform
removed. There were 6 trials, each of maximum duration of 60 s, at
least 5 min rest between trials). The amount of time the rats spend
swimming near the platform provided a measure of how much they
relied on visual and spatial cue to locate the platform, as opposed
to using a non-spatial strategy. Data was collected and analysed
using Any-maze (v4.2) software.
[0133] The data generated from behavioural tests was analysed using
one-way ANOVA for determining the difference between the
aged-groups. Two-way ANOVA was used for examining the progress of
behavioral results with the time points treated as dependent
factors. GraphPad Prism version 3.02 was used for data
analysis.
Results
[0134] Treatment with scopolamine significantly impaired
acquisition of spatial memory in treated animals (time to platform
approximately 208% of control on day 4). Cyclic G-2AllylP (30
mg/kg; daily) significantly reversed the cognitive impairment
induced by scopolamine (FIGS. 1A, 1B, 1C).
Example 6
cG-2-AllylP Improves Synaptic Plasticity and Aging-Related Memory
Loss Methods
[0135] Aged rats (male Wistar rats, 18-20 months old) were divided
into four groups: two vehicle-treated (groups 1 and 3) and two
G-2-AllylP treated (groups 2 and 4) (all groups n=6-8). Cyclic
G-2-AllylP was synthesised by the Department of Medicinal Chemistry
and dissolved in normal saline before the treatment. On day 1 a
single dose of cyclic G-2-AllylP was given centrally (20 ng/animal,
i.c.v.) to the animals in groups 2 and 4; saline was administered
to groups 1 and 3. The memory tests using Novel Object Recognition
Test started either on day 3 (groups 1 and 2) or 24 (groups 3 and
4) after the treatment. On the completion of the NORT, the rats
were killed with an overdose of sodium pentobarbital and were
perfused transcardially with normal saline followed by 10%
formalin. Tissues collected at day 7 in groups 1 and 2, and at day
28 from groups 3 and 4. The brains were kept in the same fixative
for a minimum of 2 days before being processed using a standard
paraffin embedding procedure. Briefly, small blocks
(10.times.10.times.3 mm) of tissue were fixed for up to 24 hrs. The
blocks were then infiltrated and embedded with paraffin and cut in
ribbons and mounted on slides. Slides were then stored until
immunostaining was commenced.
[0136] Synaptogenesis in brain tissue was examined using
immunohistochemical staining.
Novel Object Recognition Test (NORT)
[0137] Exploratory activity is a typical learning behaviour
displayed by animals including humans and rats in novel
environments. Exploratory activity decreases over time when the
novel becomes familiar and the habituation occurs. In familiar
environments, exploratory activity can be reactivated by
introducing a novel object. The increase in exploring behaviour
once the environment is altered following a habituation provides a
measure of the memory for the familiarity and the recognition of
the novelty.
[0138] In this Example, we carried out two NORTs, one at days 3-6
and the other at 24-27 days. The rats were allowed to familiarise
themselves with the testing arena (90.times.60.times.40 cm) in the
first day of NORT. In the following two days of each test, four
novel objects were placed into the testing arena and the rats had 2
trials each day (each of 15 min duration and 2 hours apart). The
time spent on exploring the objects was reduced once the animal
tested learned about the objects (training phase). In the last day
(day 4 of each test), one familiar object was replaced by a novel
object before the second trial (test 6, testing phase). The average
time spent on exploring the 3 familiar objects and the time spent
on exploration of the novel object was used as a measure for the
memory of familiarity and the novelty recognition.
Effects of cG-2-AllylP on Expression of NMDA Receptors, AMPA
Receptors, rKrox-24 and Synaptophysin mRNA in the Hippocampus
[0139] It is accepted that the hippocampal formations in humans and
animals play a crucial role in a number of memory types (Morris et
al. 2006 Europ. J. Neurosci. 23, 2829). The specific functionality
remains under dispute, but there is an understanding that
hippocampus plays a key role in the automatic encoding and initial
storage of attended experiences (episodic memory formation), memory
consolidation and novelty detection. The first aspect, encoding and
short-term storage of memories, is dependant on the synaptic
plasticity and synaptic transmission, both of which are linked to
glutaminergic neurotransmission.
[0140] Glutaminergic transmission is facilitated by two types of
glutamate receptors: N-methyl-D-aspartate receptors (NMDAR) and
a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid receptors
AMPA or non-NMDA receptors.
[0141] APMA receptor subunit GluR1 is a post-synaptic receptor and
has been commonly used for memory measurement. GluR1 is believed to
mediate calcium influx, and has a vital function in synaptic
plasticity related to learning. It has been previously suggested
(Hayashi et al., 2000) that incorporation of GluR1 into synapses
might be important for long-term potentiation (LTP), which is
essential for learning and memory.
[0142] It had been demonstrated that NMDA receptor subunit NR1 is
crucial for formation of spatial memory. In knock-out models where
the R1 subunit of the NMDA receptor in the pyramidal cells of the
CA1 region was selectively knocked-out, the long-term potentiation
was shown to be abolished (Tsien 1996).
[0143] Synaptophysin is a presynaptic vesicle protein. Its
quantitative detection is established as a molecular marker of
synaptic density.
[0144] The neuronal transcript factor Krox24 staining is used as a
marker for neuronal plasticity. The protein products of the Krox24
family (as well as by brain-derived neurotrophic factor, BDNF) have
recently linked with stabilizing synaptic modifications occurring
during NMDA-receptor-mediated hippocampal LTP and LTD. (Dragunow.
2006. Behaviour genetics. 23; 293).
Immunohistochemical Staninhg
[0145] Conventional deparaffinisation and rehydration techniques
were used to allow the water-based buffers and antibodies to
penetrate the tissue slices. Antigen retrieval was used only prior
to AMPA receptor (GluR1) staining, i.e. the slides were placed in
boiling citrate buffer and allowed to cool.
The following antibodies were used: [0146] i) primary rabbit
antibody to NMDA NR1 subunit, at 1:200 concentration in buffer,
incubated for 48 hrs (Chemicon--AB1516), followed by Sigma
fluorescent secondary antibody (alexaFluor 594), at 1:200 dilution,
incubated for 24 hours at 40 C. [0147] ii) primary antibody to AMPA
GluR1 subunit, at 1:50 concentration in buffer, incubated for 48
hrs (Chemicon--AB1504) followed by 3,3'-diaminobenzidine (DAB) at
1:200 dilution, incubated for 24 hours at 40 C. [0148] iii) Primary
antibody to mSynaptophysin (Sigma--S5768), at 1:200 concentration
in buffer, followed by DAB at 1:200 dilution, incubated for 24
hours at 40 C). [0149] iv) Primary antibody to rKrox-24 (Santa
Cruz--catalogue number SC-189) at 1:200 concentration in the
buffer, followed by anti-rabbit secondary antibody at concentration
of 1:200 dilution, incubated for 24 hours at 40 C. (e) Antibodies
were detected using light microscopy.
Results
NORT
[0150] A trend to improve the novelty recognition in the groups
treated with cG-2AllylP was observed after 27 days (FIG. 2), but
not 6 days after the treatment (no figure). We conclude that the
cG-2AllyP treatment improved novelty recognition in the
drug-treated animals at 27 days.
AMPA Glutamate Receptor-1 Staining
[0151] Hippocampal slices from regions CA1 (granular cell layer,
strata oriens and radiatum) and CA3 (pyramidal cell layer) were
stained for AMPA receptors GluR1.
[0152] In CA3 there was no change in the number of receptors in
each region on either day 7 or 28. There was however a significant
increase in the number of AMPA receptors in CA1 (granular cell
layer) (FIG. 4) and CA1 stratum oriens (FIG. 5) and on day 28.
[0153] That histological change was correlated with the improved
performance in the novel object recognition test. The improved
memory (FIG. 2) was correlated to the elevated AMPA glutamate
receptor-1 (FIG. 3). We concluded that cG-2AllylP improved
glutaminergic neuro-transmission (GluR1) at post-synaptic
level.
[0154] We observed that cG-2-AllylP treatment resulted in a long
term increase in GluR1 staining on the post-synapses and increased
the density of pre-synaptic vesicles. As the majority of vesicles
in the hippocampus are glutamic vesicles, we concluded that the
long term memory improvement was associated with increased glutamic
neurotransmission.
Synaptophysin Staining
[0155] We subsequently analysed effect of cG-2AllyP on the levels
of synaptophysin staining in CA3 and CA1 regions of the
hippocampus.
[0156] In all tested areas there was either a significant increase
(CA3) or a clear trend towards (CA1--strata oriens and radiatum)
the increase in the density of synaptophysin staining at 28 days
post-treatment. That increase is a marker of increased synaptic
plasticity and a clear indication of synaptogenesis which is a most
likely cause of the improvement in the performance of the treated
groups in applied memory tests.
NMDA Receptor-1 Staining
[0157] While there is a significant improvement in AMPA receptors
post-treatment, the changes in the NMDA receptors are not so
pronounced (FIGS. 9A, 9B and 9C).
Krox24 Staining
[0158] We analysed the density of the Krox24 staining in the CA1-2
regions of the hippocampus. We observed a trend towards the
increased density in treatment group in comparison to the vehicle
treated group. We conclude that the Krox24 staining results
positively correlate with improved memory function (FIG. 10).
Example 7
cG-2-AllylP Increases the Number in Pre-Synaptic Vesicles in the
Hippocampus of Middle Aged Rats
[0159] Four middle aged Wistar male rats (12 months) were divided
into two groups: one vehicle-treated (n=2) and one
cG-2-AllylP-treated (n=2). The rats were treated subcutaneously
with 3 mg/kg/day of either saline or cG-2-AllylP for 7 days. On day
21 of the experiment the animals were sacrificed and the
hippocampal tissue was harvested. Semi-thin sections of the tissue
were fixed with OsO.sub.4 and embedded in resin. CA1 stratum oriens
and CA3 sections were then sliced into ultra-thin, 80 nm slices and
stained with uranyl acetate and lead citrate. Approximately 50
synapses per animal were analysed, synapse type classified and
vesicle density was measured using AnalySIS.RTM..
[0160] Transmission electron microscopy was used to count the total
number of vesicles on the slides. The average density was
calculated by measuring the total area (using AxioVision software)
and using the number of vesicles. We followed the protocol in
Yoshida et al. 97, Journal of Neurochemistry to calculate the
vesicle density in a 200 nm.times.200 nm square apposing the
post-synaptic density (PSD).
Results
[0161] The number of pre-synaptic vesicles in the CA1 and C3
subregions of the hippocampus was increased after cG-2-AllylP
treatment (3 mg/kg/day.times.7 days, s.c.) compared to the vehicle
treated animals at 21 days after the treatment (FIG. 11).
[0162] We conclude from these studies that scopolamine treatment
can decrease cognitive function in animals, and that these changes
can mimic cognitive impairment in human beings with one or more of
a variety of neurological conditions. Additionally, we conclude
that cG-2-AllylP can improve cognitive function in
scopolamine-treated animals and in animals with normal
aging-related cognitive impairment. Further, we conclude that
cG-2-AllylP can increase synaptogenesis, increase AMPA receptors,
increase neural plasticity, can stabilize synaptic modifications
and can increase novelty recognition.
[0163] These studies therefore support the use of cG-2-AllylP as an
effective pharmacological agent to treat a variety of cognitive
impairments in animals including humans suffering from Alzheimer's
disease, Parkinson's disease, and other chronic neural disorders,
as well as cognitive impairment associated with aging.
In Vitro and In Vivo Testing
[0164] The following pharmacological studies demonstrate
neuroprotective features of this invention. They are not intended
to be limiting, and other compositions and methods of this
invention can be developed without undue experimentation. All of
those compositions and methods are considered to be part of this
invention. All the following experiments were carried out using
protocols developed under guidelines approved by the University of
Auckland Animal Ethics Committee.
Example 8
Effects of Cyclic G-2-AllylP and Cyclic cyclopentyl-G-2MeP on
Cerebellar Cell Explants
[0165] To determine the effects of cG-2-AllylP and cyclic
cyclopentyl-G-2-MeP on neuronal cells in vitro, a series of studies
was carried out using cerebellar explants from adult rats. In vitro
systems are suitable for studying neuronal proliferation, neurite
growth, formation of nerve bundles, and effects of toxins on neural
cells, effects that parallel effects observed in vivo. Thus,
results of studies using in vitro cerebellar explants are
predictive of effects of interventions in vivo.
[0166] In a first series of studies, effects of glutamate on
cerebellar explants were determined. At physiological
concentrations, glutamate is a neurotransmitter in the CNS of
mammals, including humans. However, at sufficiently high
concentrations, glutamate is neurotoxic, resulting in neuronal cell
death. Because glutamate is a naturally occurring neurotransmitter
in the CNS of mammals, including humans, and because glutamate
neurotoxicity is recognized in the art as reflective of
neurotoxicity in general, and including cell death and
degeneration, it is a valuable tool useful for identifying and
characterizing agents effective in treatment of neurodegeneration
and neural cell death.
Materials and Methods
[0167] Cover slips were placed into a large Petri dish and washed
in 70% alcohol for 5 minutes, then washed with Millipore H.sub.2O.
The cover slips were air dried, and coated with Poly-D-Lysine (1
mg/ml stock solution in PBS, 90-100 .mu.l) for 2 hours at
34.degree. C.
Extraction of Cerebellar Tissue
[0168] Postnatal day 8 Wistar rats were used for the study. The
rats were sacrificed and placed in ice for 1 minute, decapitated
and the cerebellum removed and placed on ice. Cerebellum tissue was
placed in 1 ml of 0.65% glucose-supplemented PBS (10 .mu.l 65%
stock D (+)glucose/1 ml PBS) in a large Petri dish, chopped up into
smaller sections and triturated with a 1 ml insulin syringe via a
23 G (0.4 mm) needle, and then squirted back into the glucose
solution in the large Petri dish. The tissue was sieved through
(125 .mu.m pore size gauze) and centrifuged (2 minutes at 60 g)
twice to exchange the medium into serum-free BSA-supplemented START
V medium (Biochrom, Germany). The second centrifugation step was
done with 1 ml of START V medium. The microexplants were
reconstituted into 500 .mu.l of START V medium and put on ice.
Cultivation of Cerebellar Cells
[0169] Two hours after PDL-coating, the slides were washed with
Millipore H.sub.2O and air dried. Each slide was placed into a
small Petri dish (diameter: 35 mm) and 40 .mu.l of START V/cell
suspension was added. The tissue was incubated for 2 hours at
34.degree. C. (settlement period). START V-medium (1 ml) was then
added to the Petri dish and cultivated at 34.degree. C. in the
presence of 5% CO.sub.2 in air at 100% humidity for 48 hours.
Drug Application
[0170] For the study, certain explant cultures were exposed to
vehicle (PBS) only. In the first study (Study 1) 10 .mu.l of toxin
1 (L-glutamate--100 mM in Millipore water, final concentration: 1
mM) and 10 .mu.l of toxin 2 (3-nitropropionic acid--50 mM--pH 7--in
Millipore water, final concentration: 0.5 mM) was applied
simultaneously with the drug to be tested (10 mM stock solution
prepared in PBS and diluted to final concentrations between 1-100
nM). In each case, the drugs were left in contact with the explants
for the duration of the study.
Methods for Determining Drug Effects After explants were exposed to
drugs for the study period, cells were then rinsed in PBS and then
fixed in increasing concentrations of paraformaldehyde (500 .mu.l
of 0.4% PFA was applied; then 1.2% PFA; then 3% PFA and finally 4%
PFA (each fixation step: 2-3 minutes). Finally, the microexplants
were rinsed in PBS.
[0171] Neurons in the explants were then evaluated for morphology
(presence of neurites) and counted as live cells per microscopic
field. Four fields displaying highest cell density were counted per
cover slip and the data presented as mean.+-.standard error of the
mean (SEM); n=4 each. Statistical significance was evaluated by
using the non-paired Student's t-test.
Results
Cyclic G-2-AllylP
[0172] The results of the study are shown in FIG. 12. Glutamate
treatment (1 mM; filled bar) resulted in about an 85% loss of
cerebellar neurons having neurites compared to vehicle-treated
controls (open bar). In contrast, cG-2AllylP significantly
increased the numbers of cells having neurites in a dose-dependent
manner when administered simultaneously with glutamate (shaded
bars). Treatment with low doses of cG-2AllyP (100 pm to 10 nm)
showed a significant decrease in glutamate-induced
neurotoxicity.
Cyclic cyclopentyl-G-2-MeP
[0173] The results of the study are shown in FIG. 13. Cyclic
cyclopentyl-G-2MeP significantly increased the number of cells
having neurites when simultaneously administered with glutamate
(light shaded bars). Treatment with low doses of cyclic
cyclopentyl-G-2MeP showed a significant decrease in
glutamate-induced neurotoxicity.
Conclusions
[0174] Both cG-2-AllylP and cyclic cyclopentyl-G-2-MeP
independently decreased or prevented glutamate-induced
neurotoxicity, indicating that both drugs are neuroprotective and
can be used to inhibit neuronal degeneration or cell death.
Example 9
Effects of cG-2-AllylP on Hypoxic-Ischemic Injury I
Materials and Methods
[0175] To determine whether cG-2AllylP might prevent neuronal
injury in response to stroke, cardiac arterial bypass graft surgery
(CABG) or other hypoxic insults, a series of studies were carried
out in rats that had been exposed to hypoxic-ischemic injury (HI).
Adult rats (Wistar, 280-310 g, male) were used. The modified Levine
model preparation and experimental procedures were used (Rice et
al, 1981, Ann. Neurol.: 9: 131-141; Guan et al J., 1993, Cereb.
Blood Flow Metab.: 13(4): 609-16). These procedures in brief,
consist of an HI injury induced by unilateral carotid artery
ligation followed by inhalational asphyxia in the animals with an
implanted lateral ventricular cannula. A guide cannula was
stereotaxically placed on the top of the dura 1.5 mm to the right
of the mid-line and 7.5 mm anterior to the interaural zero plane
under halothane anaesthesia. The right carotid artery was double
ligated two days after the cannulation. After 1 hour recovery from
the anaesthesia, each of the rats were placed in an incubator where
the humidity (90.+-.5%) and temperature (31.degree..+-.0.5.degree.
C.) were controlled for another hour, then exposed to hypoxia (6%
oxygen) for 10 min. The animals were kept in the incubator for an
additional 2 hours before treatment.
[0176] Nine pairs of rats were treated intracerebral ventricularly
(icy) with either cG-2AllylP (2 ng) or its vehicle (normal saline)
2 hours after hypoxic-ischemic insult. Rats in each group were
simultaneously infused with cG-2-AllylP or its vehicle under light
anaesthesia (1.5% halothane) 2 hours after the insult. A total
volume of 20 .mu.l was infused (icy) over 20 minutes by a
micro-infusion pump.
[0177] Histological examination was performed on rats 5 days after
the hypoxic-ischemic injury. The rats were killed with an overdose
of sodium pentobarbital and were perfused transcardially with
normal saline followed by 10% formalin. The brains were kept in the
same fixative for a minimum of 2 days before being processed using
a standard paraffin imbedding procedure.
[0178] Coronal sections 8 .mu.m in thickness were cut from the
striatum, cerebral cortex and hippocampus and were stained with
thionin and acid fuchsin. The histological outcome was assessed at
three levels: (1) the mid level of the striatum, (2) where the
completed hippocampus first appeared and (3) the level where the
ventral horn of the hippocampus just appears. The severity of
tissue damage was scored in the striatum, cortex and the CA1-2,
CA3, CA4 and dentate gyrus of the hippocampus. Tissue damage was
identified as neuronal loss (acidophilic (red) cytoplasm and
contracted nuclei), pan-necrosis and cellular reactions. Tissue
damage was scored using the following scoring system: 0: tissue
showed no tissue damage, 1: <5% tissue was damaged, 2: <50%
tissue was damaged, 3: >50% tissue was damaged and 4: >95%
tissue was damaged.
Results and Conclusion
[0179] The results of this study are shown in FIG. 14. FIG. 14
shows that hypoxic-ischemic injury (left bars of each set) resulted
in significant damage scores in each of the areas of the brain
studied. FIG. 14 also shows that central administration of a
relatively low dose of cG-2-AllylP (right bars of each set; 2 ng)
significantly reduced the tissue damage in each brain region
examined compared to the vehicle treated group (p<0.001).
[0180] It can be seen that cG-2-AllylP can be neuroprotective
against neural damage caused by hypoxic-ischemic injury, even when
administered after hypoxic-ischemic injury. This surprising finding
indicates that cG-2-AllylP is a useful agent to treat a variety of
conditions characterized by neural degeneration or cell death.
Example 10
Effects of cG-2-AllylP on Hypoxic-Ischemic Injury II
Materials and Methods
[0181] Materials and methods described in Example 9 were used and
the number of treatment groups was increased. Rats were divided
into 5 treatment groups treated intracerebral ventricularly (icv)
with one of 4 doses of cG-2-AllylP or with its vehicle (normal
saline) 2 hours after hypoxic-ischemic insult (1: n-10, 2 ng, 2:
n=9, 4 ng; 3: n=9, 20 ng; 4: n=10, 100 ng; and 5: n=9,
vehicle).
[0182] FIG. 15 shows hypoxia alone (vehicle) produces neuronal
damage scores in all areas of the brain studied. In animals treated
with cG-2-AllylP, hypoxia had less effect, even though the agent
was administered after the hypoxic/ischemic injury. The
neuroprotective effect was observed for all doses of cG-2-AllylP,
except for the highest dose (100 ng) administered to the striatum.
However, in all other sites and with all other doses, cG-2-AllylP
lessened the neural damage effects of hypoxia/ischemia. Moreover,
cG-2-AllylP had an increased efficacy in brain regions that
experienced progressive injury associated with delayed cell death,
such as that associated with apoptosis. In brain regions such as
the dentate gyms and the cerebral cortex, that are more resistant
to HI injury, the progression of injury is known to be slower and
more severe than in the brain regions that are more sensitive to HI
injury such as the striatum and the CA1-2, CA3 and CA4 sub-regions
of the hippocampus. This result shows that cG-2allylP can be
beneficial in treatment of chronic neurological disorders.
[0183] This invention is described with reference to specific
embodiments thereof. Other features and embodiments of this
invention can be produced by those of skill in the art without
undue experimentation and a reasonably likelihood of success. All
of those embodiments are considered to be part of this
invention.
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