U.S. patent application number 15/514968 was filed with the patent office on 2017-09-14 for treatments for autoimmune disease.
The applicant listed for this patent is THE PROVOST, FELLOWS, FOUNDATION SCHOLARS, AND THE OTHER MEMBERS OF BOARD,OF THE COLLEGE OF THE HOLY. Invention is credited to Vincent KELLY.
Application Number | 20170258797 15/514968 |
Document ID | / |
Family ID | 51901287 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170258797 |
Kind Code |
A1 |
KELLY; Vincent |
September 14, 2017 |
TREATMENTS FOR AUTOIMMUNE DISEASE
Abstract
The invention relates to a novel approach to the treatment of
autoimmune diseases, particularly multiple sclerosis. In a further
embodiment of the invention there is provided a molecule capable of
acting as substrate for the queuine-insertase enzyme complex and
where said molecule is not a substrate for Hypoxanthine-guanine
phosphoribosyltransferase and said molecule has the effect of
lowering interferon gamma, for use in the treatment of auto-immune
diseases. Preferably the autoimmune disease is multiple
sclerosis.
Inventors: |
KELLY; Vincent; (Drogheda,
IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE PROVOST, FELLOWS, FOUNDATION SCHOLARS, AND THE OTHER MEMBERS OF
BOARD,OF THE COLLEGE OF THE HOLY |
Dublin |
|
IE |
|
|
Family ID: |
51901287 |
Appl. No.: |
15/514968 |
Filed: |
September 29, 2015 |
PCT Filed: |
September 29, 2015 |
PCT NO: |
PCT/EP2015/072488 |
371 Date: |
March 28, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 37/00 20180101; A61P 37/06 20180101; A61P 3/10 20180101; A61P
17/06 20180101; A61P 19/02 20180101; A61K 31/519 20130101; A61P
29/00 20180101; A61P 1/04 20180101; A61P 25/00 20180101; A61P 43/00
20180101; A61K 31/519 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2014 |
GB |
1417165.6 |
Claims
1. A molecule capable of acting as a substrate for an
queuine-insertase enzyme complex to treat autoimmune diseases.
2. The molecule of claim 1, wherein the molecule is not a substate
for Hypoxanthine-guanine phosphoribosyltransferase (HPRT).
3. The molecule of claim 1, wherein the molecule is adapted to
lower interferon gamma production.
4. The molecule of claim 1, wherein said molecule is not a
substrate for HPRT and is adapted to lower interferon gamma
production to treat autoimmune diseases.
5. The molecule of claim 1, wherein the molecule has a displacement
of .gtoreq.50 pmol in a guanine displacement assay.
6. The molecule of claim 1, wherein the molecule yields a Guanine
Monophosphate (GMP) product in amounts .gtoreq.60% of the control
in HPRT assay.
7. The molecule of claim 1, wherein the molecule is adapted to
lower interferon gamma production in wild-type cells at a
concentration of 100 .mu.M.
8. The molecule of claim 1, wherein the autoimmune disease is
selected from the group consisting of rheumatoid arthritis,
ulcerative colitis, psoriasis, diabetes, and inflammatory bowel
disease.
9. The molecule of claim 1, wherein the autoimmune disease is
multiple sclerosis.
10. The molecule of claim 1, further comprising an additional
therapeutic agent.
11. The molecule of claim 10, wherein the additional therapeutic
agent is a medicament for the treatment of multiple sclerosis
damage in patients.
Description
TECHNICAL FIELD
[0001] The invention relates to a novel approach to the treatment
of autoimmune diseases, particularly multiple sclerosis.
BACKGROUND ART
[0002] Multiple sclerosis (MS) is a debilitating disease of the
central nervous system (CNS) in which the body's own immune system
attacks the white matter of the brain and spinal cord. This
triggers inflammation-induced damage to the CNS protective myelin
layer causing demyelination. Loss of myelin exposes neurons to
further attack leading to formation of multiple sclerotic lesions.
This damage disrupts the ability of parts of the nervous system to
communicate, resulting in a wide range of problems including
fatigue, blurred vision, cognitive impairment, and spasticity. Many
patients suffer from the development of irreversible motor
disability. Long term prognosis is poor, within 15 years of disease
onset approximately 50% of patients are unable to walk unassisted
(Polman and Uitdehaag, 2000).
[0003] MS takes several forms, with new symptoms either occurring
in isolated attacks (relapsing forms) or building up over time
(progressive forms).
[0004] There is at present no known cure for multiple sclerosis.
Current treatments attempt to improve function after an attack
and/or prevent subsequent attacks. Medications used to treat MS,
while modestly effective, can have adverse effects and be poorly
tolerated.
[0005] There are a number of injectable front line therapies:
[0006] beta interferon 1a (Avonex) [0007] beta interferon 1a
(Rebif) [0008] beta interferon 1b (Betaferon) [0009] glatiramer
acetate (Copaxone)
[0010] Interferons may produce flu-like symptoms and some people
taking glatiramer experience a post-injection reaction with
flushing, chest tightness, heart palpitations, breathlessness, and
anxiety, which usually lasts less than thirty minutes. More
dangerous but much less common is liver damage. Glatiramer is
associated with skin irritation at the site of injection.
[0011] Additional Therapies Include: [0012] Natalizumab which
reduces the relapse rate more than first-line agents; however, due
to issues of adverse effects such as progressive multifocal
leukoencephalopathy it is a second-line agent reserved for those
who do not respond to other treatments or with severe disease.
[0013] Fingolimod (Gilenya)--licensed in March 2011 for people with
rapidly evolving severe relapsing remitting MS (two or more
relapses a year), and as a second line treatment for people whose
MS remains active despite treatment with one of the beta interferon
drugs. [0014] Dimethyl fumarate (Tecfidera) was licenced by the FDA
in 2013 and is an oral first line therapy for adults with relapsing
remitting forms of MS. [0015] Teriflunomide (Aubagio) was approved
by the FDA in September 2012, is an orally available
immunomodulatory drug for the treatment of relapsing forms of MS
[0016] Mitoxantrone, whose use is limited by severe adverse
effects, systolic dysfunction, infertility, and acute myeloid
leukemia is a third-line option for those who do not respond to
other medications.
[0017] Corticosteroids (or steroids) are sometimes given for a few
days, either in the form of tablets or by intravenous drip. While
there is no evidence that steroids have any effect on the long-term
course of the disease, they can be effective at speeding up
recovery from relapse.
[0018] No treatment has been shown to change the course of primary
progressive MS and as of 2011 only one medication, mitoxantrone,
has been approved for secondary progressive MS. In this population,
tentative evidence supports mitoxantrone moderately slowing the
progression of the disease and decreasing rates of relapse over two
years.
[0019] There is ongoing research looking for more effective,
convenient, and tolerable treatments for relapsing-remitting
MS.
[0020] Monoclonal antibodies have raised high levels of interest.
The CD52 monoclonal antibody alemtuzumab, CD25 monoclonal antibody
daclizumab and CD20 monoclonal antibodies such as rituximab,
ocrelizumab and ofatumumab have all shown some benefit and are
under study as potential treatments. Their use has also been
accompanied by the appearance of potentially dangerous adverse
effects, most importantly opportunistic infections.
[0021] Accordingly, there is a huge unmet medical need for the
treatment of MS.
SUMMARY OF THE INVENTION
[0022] The present invention provides new medicaments for the
treatment of autoimmune diseases, particularly multiple
sclerosis.
[0023] An imbalance of Th cell function and differentiation can
lead to inflammation and autoimmune disease. The activation of
naive CD4+ T cells by antigen causes them to differentiate into
specialized effector T (Teff) cells (Th1, Th2, or Th17) and into
regulatory T (Treg) cells, which suppress the functions of Teff
cells, thereby keeping immune responses in check. In particular,
Th1 and Th17 cells and their signature cytokines IFN-.gamma. and
IL-17 have been shown to play a critical role in the development of
many autoimmune diseases, including multiple sclerosis (MS).
[0024] A novel mechanism for the regulation of this process and
thus treatment of such diseases has been identified. Molecules
which can exploit an enzyme complex made of two proteins, tRNA
guanine transglycosylase (TGT) also known as queuine
tRNA-ribosyltransferase 1, and queuine tRNA-ribosyltransferase
domain containing 1 (QTRTD1), subsequently referred to as the
queuine-insertase enzyme complex, have been shown to have
beneficial effects in models for autoimmune diseases.
[0025] Further, those molecules which are selective in avoiding
competing pathways, such as not being substrate for
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) or, more
positively lower interferon gamma levels are particularly
efficacious.
[0026] The present invention provides new medicaments for the
treatment of autoimmune diseases, particularly multiple
sclerosis.
[0027] In a first embodiment it describes a molecule which is
capable of acting as substrate for the human queuine-insertase
enzyme complex for the treatment of autoimmune disease.
[0028] More specifically it describes a molecule capable of acting
as substrate for the queuine-insertase enzyme complex for use in
the treatment of multiple sclerosis.
[0029] In a further embodiment of the invention there is provided a
molecule capable of acting as substrate for the queuine-insertase
enzyme complex and where said molecule is not a substrate for
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) for use in
the treatment of autoimmune diseases. Preferably the autoimmune
disease is multiple sclerosis.
[0030] Additionally described is a molecule capable of acting as
substrate for the queuine-insertase enzyme complex, which also has
the effect of lowering interferon gamma production in immune cell
assays for use in the treatment of autoimmune diseases. Preferably,
the autoimmune disease is multiple sclerosis.
[0031] In a further embodiment of the invention there is provided a
molecule capable of acting as substrate for the queuine-insertase
enzyme complex, where said molecule is not a substrate for HPRT and
where said molecule has the effect of lowering interferon gamma,
for use in the treatment of auto-immune diseases. Preferably the
autoimmune disease is multiple sclerosis.
[0032] For molecules that lower interferon gamma production in
wild-type cells, preferred are those molecules that are able to
lower it at a concentration of 100 .mu.M
[0033] Particularly preferred are those molecules which lower
interferon gamma to .ltoreq.1750 ng/ml
[0034] Most preferred are those molecules which lower interferon
gamma to .ltoreq.1100 ng/ml.
[0035] Alternatively, can test for molecules that lower interferon
gamma production by using cells from animals wherein the TGT gene
has been knocked out, subsequently referred to as TGT-KO cells).
Preferred are those molecules that at a concentration of 100 .mu.M
do not lower it .ltoreq.3,500 ng/ml.
[0036] Particularly preferred are those molecules which do not
lower interferon gamma to .ltoreq.4250 ng/ml
[0037] Most preferred are those molecules which do not lower it
.ltoreq.5000 ng/ml.
[0038] The skilled reader will be aware of methods for determining
whether or not a particular molecule is a substrate for the
queuine-insertase enzyme complex. For example, molecules capable of
acting as a queuine-insertase enzyme complex substrate may be
identified by use of a displacement assay as described below (FIG.
1):
Production of [8-.sup.14C] Guanine Labeled tRNA (tRNA*)
[0039] Components added in the order listed in Table 1. Before
adding the 8-[.sup.14C] guanine solution to the reaction the
solution was neutralised with an equal volume (vol/vol) 0.01 M
NaOH, as the [8-.sup.14C] Guanine is supplied in 0.01 M HCl aqueous
solution. A stock solution of yeast tRNA from Saccharomyces
cerevisiae was made to a concentration of 2 absorbance units (260
nm) in ultrapure nuclease-free water. The recombinant Eschericihia
coli (E. coli) tRNA guanine transglycosylase enzyme (E. coli TGT)
containing an N-terminal polyhistidine tag was produced in BL21
BL21(DE3) tgt::Km.sub.r cells as described previously (Boland et
al., 2009).
TABLE-US-00001 TABLE 1 Components of [8-.sup.14C] Guanine tRNA
labeling reaction Component Volume (.mu.L) Final conc. 1M Tris-HCl
pH 7.5 7.5 50 mM 5M NaCl 0.6 20 mM 1M MgCl.sub.2 0.75 5 mM 1M DTT
0.3 2 mM Yeast tRNA (2 abs/.mu.l) 12.5 25 Abs total in rxn H.sub.2O
to 130 .mu.L E. coli TGT 10 .mu.L 10 .mu.g [8-.sup.14C] guanine 10
.mu.L
[0040] The reaction was incubated for 2 h at 37.degree. C. After
incubation the reaction was made up to 400 .mu.L with buffer. The
reaction mixture was extracted by addition of an of equal volume
(400 .mu.L), of Acid Phenol:chloroform (5:1; pH 4.5) and
centrifuged at 16,000.times. g for 5 min. The upper aqueous phase
was transferred to a new 1.5 mL tube. The radiolabelled tRNA with
[8-.sup.14C] guanine in the third position of the anticodon loop
(tRNA*) was precipitated by the addition of 0.1 volume (40 .mu.L)
of 3 M sodium acetate (aq.) and 2 volumes of ethanol (800 .mu.L)
and incubated overnight at -20.degree. C. The next morning, the
tRNA* was pelleted by centrifugation at 16,000.times. g for 20 min
at 4.degree. C. The pellet was washed with 1 mL of ice-cold 70%
ethanol, without disturbing the pellet. The tRNA* pellet was
resuspended in 20 .mu.L nuclease-free water and the concentration
measured spectrophotometrically at A.sub.260.
[0041] Displacement Assays
[0042] Each reaction was set up in triplicate and incubated for 1 h
at 37.degree. C. Each of the components in the reaction were added
in the order shown in Table 2, with the tRNA* added last to
initiate the reaction. `Compound` refers to the molecules that are
under investigation.
TABLE-US-00002 TABLE 2 Components of [8-.sup.14C] guanine
displacement assays Component Volume (.mu.L) Final Concentration 1M
Tris-HCl pH 7.5 7.5 50 mM 5M NaCl 0.6 20 mM 1M MgCl.sub.2 0.75 5 mM
1M Dithiothreitol 0.3 2 mM Compound (2 mM stock) 15 200 .mu.M
H.sub.2O to 140 Queuine-insertase enzyme 10 2 .mu.g tRNA* 2 Abs
units 1.8 .mu.M
[0043] Preparation of the DEAE columns: Approximately 25 grams of
Whatmann DEAE 52-cellulose resin was weighted into a 50 ml sterile
RNA free tube. A volume of 20 ml of 200 mM Tris-HCl pH7.5 was added
and the tube inverted 5 times and centrifuged at 750.times. g in a
bench top centrifuge to sediment the resin. The supernatant was
removed by pouring and the pH checked. The washing of the resin was
repeated another 4 times until the wash reached a pH of 7.5. The
resin was suspended in a 1:1 slurry with 200 mM Tris-HCl pH7.5 and
loaded into a 1.5 ml spin column (containing a glass fibre filter)
until a 1 ml final bed volume of resin was achieved.
[0044] After incubation the reaction was loaded onto the column and
spun at 0.1.times. g for 10 seconds. The flow-through was collected
and reloaded on the column. This step was repeated 5 times to allow
maximum binding of tRNA. The column was then washed with
8.times.250 .mu.L of Wash Buffer (20 mM Tris-HCl, pH7.5, 10 mM
MgCl.sub.2, 200 mM NaCl) with 10 second spins at 0.1.times. g
between each wash. These loading and wash steps were collected into
scintillation vials. The bound tRNA was then eluted in 4.times.250
.mu.L aliquots in Elution Buffer (20 mM Tris-HCl, pH7.5, 10 mM
MgCl.sub.2, 1M NaCl) and collected into scintillation vials. 10 mL
of Ecoscint A (scintillation cocktail) was added to all vials. The
vials containing the flow-through and washes are counted for
displaced [.sup.14C] guanine.
[0045] In this assay (see FIG. 1), maximum displacement by 200
.mu.M queuine base, the natural substrate of the queuine-insertase
enzyme complex, is 240 pmol [.sup.14C] guanine. Background values
are .ltoreq.10 pmol. Therefore, a displacement of .ltoreq.50 pmol
is considered a positive substrate for TGT.
[0046] Assay for the Ability of Compounds to be used as Substrates
for the Queuine-Insertase Enzyme Complexby Displacing [.sup.14C]
Guanine from Yeast tRNA (FIG. 1).
[0047] Guanine, queuine,
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (shown as compound I) and
7-methylguanine were assessed for their ability to replace guanine
in yeast tRNA. First, yeast tRNA was charged with [8-.sup.14C]
guanine (tRNA*) by the E. coli TGT enzyme. In each reaction 2
A.sub.260 absorbance units of tRNA* was used along with 200 .mu.M
of specified molecule. Reactions were processed on 1 ml DEAE
cellulose columns. Reaction flow-through and washes were collected
and analysed by liquid scintillation counting for presence of
displaced [.sup.14C] guanine.
[0048] Similarly, the skilled reader will be aware of methods
suitable for determining whether a compound is a substrate or
inhibitor of HPRT. For example, molecules that are neither a
substrate nor an inhibitor for HPRT may be identified by use of the
following assay:
[0049] Assay to Evaluate Compounds as Substrates or Inhibitors of
the Hypoxanthine-Guanine Phosphoribosyltransferase Enzyme or `HPRT
Assay` (FIG. 2).
[0050] Preparation and equilibration of column: Dowex-1.times.8,
mesh size 200-400, was weighed out in a sterile 50 ml tube and
washed 3 times with aqueous 1 M HCl to charge the resin. The resin
was then washed 5-6 times with (20 mls each) Milli-Q H.sub.2O until
a neutral pH was reached. Columns (Bio-rad 10 mL plastic columns)
were set up with 1 mL of wet Dowex (wet mix applied, allowed to
settle and more applied until a packed volume of 1 mL was reached).
The packed resin was rinsed with 10 volumes (v/v) milli-Q H.sub.2O
to ensure that a neutral pH and consistent packing was
achieved.
[0051] HPRT Reactions are Set up in 100 .mu.L Volume in Triplicate
with the Following Components:
[0052] 20 mM HEPES (pH 8)
[0053] 10 mM MgCl.sub.2
[0054] 0.1 mM Phosphoribosyl pyrophosphate (PRPP--made fresh on day
of assays)
[0055] 20 .mu.M [8-.sup.14C]-Guanine
[0056] 400 .mu.M Compound to be tested
[0057] The reaction mix was preheated to 37.degree. C. before 10 ng
of recombinant human HPRT enzyme was added. Reactions are allowed
to proceed for 1 h before being stopped by heating for 8 min at
100.degree. C. A control reaction comprising heat-inactivated
enzyme (treated at 100.degree. C. for 8 min prior to adding to the
reaction) was also always included.
[0058] After inactivation, the reactions were cooled to room
temperature (using ice), before being applied to a 1 mL packed
resin column of Dowex-1.times.8, mesh size 200-400. The column was
washed with 5 mL milli-Q H.sub.2O and 10 mL 10 mM HCl, to remove
unbound, unreacted guanine. GMP was eluted from the column with 5
mL of 50 mM HCl into scintillation vials. 15 mL of scintillation
cocktail (Ecoscint A) was added and vials counted to evaluate the
level of conversion of [8-.sup.14C]-guanine to GMP.
[0059] For molecules that are neither a HPRT substrate nor an
inhibitor, the reaction will yield GMP product in amounts
.gtoreq.60% of the control reaction containing only
[8-.sup.14C]-guanine.
[0060] Results for Guanine, Queuine.HCl
(2-Amino-5-[[[(1S,4S,5R)-4,5-dihydroxy-2-cyclopenten-1-yl]amino]methyl]-1-
,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, monohydrochloride) and
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (shown as Compound I on graph) are
shown in FIG. 2.
[0061] The Ability of Molecules to Lower Interferon Gamma may be
Measured with the Following Ex Vivo Assay (FIG. 3):
[0062] Chronic, monophasic EAE was induced in 8-10 week old female
C57BL/6 mice as described by Stromnes and Goverman, (2006). Animals
were transcardially perfused with ice-cold phosphate buffered
saline (PBS) and the spleen was removed.
[0063] Single cell suspensions of spleen were made by extrusion
through a 70 micron nylon mesh and the cell washed in cRPMI medium
(Roswell Park Memorial Institute media containing 10% fetal bovine
serum, 2 mM L-glutamine, 100 units penicillin, 1 mg/ml
streptomycin) by centrifugation at 240.times. g for 5 minutes,
counted and seeded at a final density of 1.times.10.sup.6/mL to
U-bottomed 96 well plates containing medium alone (negative
control) or 100 .mu.l of 50 .mu.g/mL myelin oligodendrocyte
glycoprotein peptide (MOG.sub.33-55). All stock concentrations of
molecules were made up in sterile DMSO and tested in cells at 10
.mu.M and 100 .mu.M concentrations. Molecule administrations did
not exceed 0.5% final DMSO concentration in cells. After 72 h, the
plates were centrifuged at 240.times. g for 3 minutes to allow the
cells to settle down. Supernatants were carefully removed and
assayed for IFN gamma according to manufacturer's protocol supplied
in the eBioscience ELISA kit.
[0064] This test can be used with both wild cells or TGT-KO cells
as described herein
[0065] Suitable molecules for use in the invention include:
[0066]
2-amino-5-(((3-phenylpropyl)amino)methyl)-3,7-dihydro-4H-pyrrolo[2,-
3-d]pyrimidin-4-one
[0067]
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methy-
l)-3-phenylpropan-1-aminium chloride;
[0068]
2-amino-5-((butylamino)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimid-
in-4-one
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)met-
hyl)butan-1-aminium chloride
[0069]
2-amino-5-((hexylamino)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimid-
in-4-one
[0070]
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methy-
l)hexan-1-aminium chloride
[0071] Queuine;
2-amino-5-((((1S,4S,5R)-4,5-dihydroxycyclopent-2-en-1-yl)amino)methyl)-3,-
7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[0072] Queuine HCl
2-Amino-5-[[[(1S,4S,5R)-4,5-dihydroxy-2-cyclopenten-1-yl]amino]methyl]-1,-
7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, monohydrochloride
without being bound by theory, it appears the compounds of the
present invention operate via a new drug pathway. They exploit an
enzyme complex made of two proteins: TGT (tRNA guanine
transglycosylase) and QTRTD1 (queuine tRNA transglycosylase domain
containing 1) herein referred to as the queuine-insertase enzyme
complex. The effect is to decrease populations of effector T cells
(Teff) and/or increase the relative populations of regulatory T
cells (Treg) cells. Treg cells are a part of the immune system that
are intricately involved in the determination of self from non-self
proteins, i.e., protecting proteins of the self from being attacked
by the immune system
[0073] The natural substrate for the queuine-insertase enzyme
complex is queuine, a molecule that cannot be synthesised by
eukaryotic cells. However, it is readily produced by most bacteria,
and in humans queuine must be harvested either from injested food
or the gut microflora. The queuine-insertase enzyme complex inserts
queuine into the anticodon loop of tyrosyl-, histidinyl-,
asparaginyl- and aspartyl-transfer RNA (tRNA of the GUN family;
tRNA.sub.GUN) at the wobble position. The majority of tRNA.sub.GUN
in the body is modified at the wobble position with queuine. It is
notable that queuine is absent or depleted (hypomodified) in the
wobble position of tRNA.sub.GUN isotypes in rapidly proliferating
cells. Earlier work employing 6-thioguanine (6TG) has shown that
use of this alternative substrate for the queuine-insertase enzyme
complex can have a dramatic effect on the murine model of chronic
MS (i.e. Experimental Autoimmune Encephalomyelitis; EAE).
Unfortunately, from a therapeutic perspective, 6TG suffers from the
problem that it is a more potent substrate for HPRT (approx. 10
fold higher than for the queuine-insertase enzyme complex). It is
subsequently incorporated into DNA, making it genotoxic and
unsuitable as a treatment for MS.
[0074] The present invention revolves around molecules able to
exploit the beneficial aspects of 6TG (queuine-insertase dependent
exchange into the wobble position of tRNA.sub.GUN), but which
circumvent the problem associated with 6TGs' main biological
activity (i.e. HPRT activity and subsequent insertion into
DNA).
[0075] FIGS. 4a -4d show that proliferation was reduced to less
than 50% in wild-type T cells upon treatment with
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride at 100 .mu.M concentration (shown as
Compound I in FIGS. 4a-d). Lack of HPRT involvement in the observed
effect was demonstrated by a similar reduction of proliferation of
cells in which the HPRT gene had been knocked out (HPRT KO, FIG.
4b). Lack of HPRT activity has also been confirmed by in vitro
assay employing recombinant HPRT enzyme wherein the molecule is
shown not to be a substrate or inhibitor (FIG. 2). The requirement
for the queuine-insertase enzyme complex is confirmed by the lack
of effect of
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (shown as Compound I) at 100 .mu.M
concentration on on proliferation in cells in which the TGT gene
has been knocked out (TGT KO, FIG. 4d). These data indicate that
the mechanism of action is indeed mediated by the queuine-insertase
enzyme complex, which is further confirmed (FIG. 4c) by the fact
that the double TGT:HRRT knockout is essentially unaffected when
treated with
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (Formula I) at 100 .mu.M
concentration. In vitro analysis of
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (Compound I) using recombinant TGT
enzyme has also shown that the compound is a substrate for TGT
(FIG. 1). The data demonstrates that said compound operates as a
substrate for the queuine-insertase enzyme complex-but is not a
substrate for HPRT.
[0076] The substrate of the queuine-insertase enzyme complex
functions to suppress encepholitogenic effector and memory T cell
populations without impacting the naive population. These same
immune cell populations broadly contribute to the pathogenesis of
all autoimmune diseases.
[0077] The transfer RNA (tRNA) of rapidly proliferating cells is
deficient (hypomodified) in queuine modification; examples include
foetal liver, multiple tumour types and regenerating adult liver.
By contrast, the tRNA of adult, fully differentiated cells contains
high levels of queuine, which cannot be displaced once
incorporated.
[0078] It could be expected that the tRNA of rapidly expanding
immune cells (as occurs in an autoimmune response) are likewise
deficient in the queuine modification. Incorporation of novel
Queuine Insertase substrates selectively into the queuine-deficient
tRNA of immune cells could disrupt proliferation and cytokine
production thereby modulating the immune response.
[0079] Compounds of the present invention find utility in the
treatment of auto-immune conditions, including but not limited to
multiple sclerosis, rheumatoid arthritis, ulcerative colitis,
psoriasis, diabetes and inflammatory bowel disease, including
Crohn's disease; and as agents to suppress transplant
rejection.
[0080] The invention also relates to a method of treating multiple
sclerosis in a mammal, particularly a human, comprising
administering to said mammal an amount of a molecule as defined
herein, or a pharmaceutically acceptable salt or solvate
thereof.
[0081] The term `treatment` is intended to include curing,
reversing, alleviating, palliative and prophylactic treatment of
the condition.
[0082] The invention further relates to molecules of the invention
in combination with other suitable agents, for use in the treatment
multiple sclerosis.
[0083] Patients suffering from multiple sclerosis are commonly
co-administered additional therapeutic agents. For patients
suffering a severe attack, intravenous corticosteroids, such as
methylprednisolone or techniques such as or plasmapheresis may be
coadministered with any treatment.
[0084] The effects of nerve cell damage caused by multiple
sclerosis result in diverse forms of damage to the patient. Nerve
damage can lead to pain, difficulty with control of bladder and
many other issues. For this reason, additional medicaments are
often prescribed patients with multiple sclerosis to help treat the
effects of MS damage. Suitable co-administrants would include:
[0085] For Bladder Problems [0086] botulinum toxin (Botox) [0087]
desmopressin (Desmospray, Desmotabs) [0088] oxybutynin (Ditropan,
Lyrinel) [0089] tolterodine (Detrusitol)
[0090] For Depression [0091] amitriptyline (Triptafen) [0092]
fluoxetine (Prozac) [0093] imipramine (Tofranil) [0094] paroxetine
(Seroxat)
[0095] For Erectile Dysfunction [0096] alprostadil (Caverject,
MUSE, Viridal Duo) [0097] sildenafil citrate (Viagra) [0098]
tadalafil (Cialis) [0099] vardenafil (Levitra)
[0100] For Fatigue [0101] amantadine (Lysovir, Symmetrel) [0102]
modafinil (Provigil)
[0103] For Optic Neuritis [0104] steroids
[0105] For Pain [0106] amitriptyline (Triptafen) [0107]
carbamazepine (Tegretol) [0108] gabapentin (Neurontin) [0109]
ibuprofen [0110] imipramine (Tofranil) [0111] lamotrigine
(Lamictal) [0112] phenytoin (Epanutim) [0113] pregabalin
(Lyrica)
[0114] For Problems with Walking [0115] fampridine (Fampyra)
[0116] For Psuedobulbar Affect [0117] Nuedexta
[0118] For Spasticity and Spasms [0119] baclofen (Lioresal) [0120]
botulinum toxin (Botox) [0121] carbamazepine (Tegretol) [0122]
clonazepam (Rivotril) [0123] dantrolene (Dantrium) [0124] diazepam
(Valium) [0125] gabapentin (Neurontin) [0126] phenol [0127]
Tetrahydrocannabinol and cannabidiol (Sativex) [0128] tizanidine
(Zanaflex)
[0129] For Tremor [0130] clonazepam (Rivotril) [0131]
thalamotomy
[0132] For Trigeminal Neuralgia [0133] carbamazepine (Tegretol)
[0134] gabapentin (Neurontin) [0135] oxcarbazepine (Trileptal)
[0136] phenytoin (Epanutim) [0137] pregabalin (Lyrica)
[0138] Other therapeutic agents are commonly administered to
patients with MS. Other such medicaments are well known to
physicians and others skilled in therapy.
[0139] Such agents may be administered sequentially, simultaneously
or concomitantly.
[0140] The invention also relates to a pharmaceutical composition
comprising a molecule of the present invention and a
pharmaceutically acceptable diluent or carrier.
[0141] Pharmaceutical compositions suitable for the delivery of
compounds of the present invention and methods for their
preparation will be readily apparent to those skilled in the art.
Such compositions and methods for their preparation may be found,
for example, in Remington's Pharmaceutical Sciences, 19th Edition
(Mack Publishing Company, 1995).
[0142] Compounds of formula (I) may be administered orally. Oral
administration may involve swallowing, so that the compound enters
the gastrointestinal tract, or buccal or sublingual administration
may be employed by which the compound enters the blood stream
directly from the mouth. Formulations suitable for oral
administration include solid formulations such as tablets, capsules
containing particulates, liquids, or powders, lozenges (including
liquid-filled), chews, multi- and nano-particulates, gels, solid
solution, liposome, films, ovules, sprays and liquid
formulations.
[0143] Liquid formulations include suspensions, solutions, syrups
and elixirs. Such formulations may be employed as fillers in soft
or hard capsules and typically comprise a carrier, for example,
water, ethanol, polyethylene glycol, propylene glycol,
methylcellulose, or a suitable oil, and one or more emulsifying
agents and/or suspending agents. Liquid formulations may also be
prepared by the reconstitution of a solid, for example, from a
sachet.
[0144] Compounds of formula (I) may also be used in
fast-dissolving, fast-disintegrating dosage forms such as those
described in Expert Opinion in Therapeutic Patents, 11 (6),
981-986, by Liang and Chen (2001).
[0145] For tablet dosage forms, depending on dose, the drug may
make up from 1 weight % to 80 weight % of the dosage form, more
typically from 5 weight % to 60 weight % of the dosage form. In
addition to the drug, tablets generally contain a disintegrant.
Examples of disintegrants include sodium starch glycolate, sodium
carboxymethyl cellulose, calcium carboxymethyl cellulose,
croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl
cellulose, microcrystalline cellulose, lower alkyl-substituted
hydroxypropyl cellulose, starch, pregelatinised starch and sodium
alginate. Generally, the disintegrant will comprise from 1 weight %
to 25 weight %. In one embodiment of the present invention, the
disintegrant will comprise from 5 weight % to 20 weight % of the
dosage form. Binders are generally used to impart cohesive
qualities to a tablet formulation. Suitable binders include
microcrystalline cellulose, gelatin, sugars, polyethylene glycol,
natural and synthetic gums, polyvinylpyrrolidone, pregelatinised
starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose.
Tablets may also contain diluents, such as lactose (monohydrate,
spray-dried monohydrate, anhydrous and the like), mannitol,
xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose,
starch and dibasic calcium phosphate dihydrate. Tablets may also
optionally comprise surface active agents, such as sodium lauryl
sulfate and polysorbate 80, and glidants such as silicon dioxide
and talc. When present, surface active agents may comprise from 0.2
weight % to 5 weight % of the tablet, and glidants may comprise
from 0.2 weight % to 1 weight % of the tablet. Tablets also
generally contain lubricants such as magnesium stearate, calcium
stearate, zinc stearate, sodium stearyl fumarate, and mixtures of
magnesium stearate with sodium lauryl sulphate. Lubricants
generally comprise from 0.25 weight % to 10 weight %. In one
embodiment of the present invention, lubricants comprise from 0.5
weight % to 3 weight % of the tablet. Other possible ingredients
include anti-oxidants, colourants, flavouring agents, preservatives
and taste-masking agents.
[0146] Exemplary tablets contain up to about 80% drug, from about
10 weight % to about 90 weight % binder, from about 0 weight % to
about 85 weight % diluent, from about 2 weight % to about 10 weight
% disintegrant, and from about 0.25 weight % to about 10 weight %
lubricant.
[0147] Tablet blends may be compressed directly or by roller to
form tablets. Tablet blends or portions of blends may alternatively
be wet-, dry-, or melt-granulated, melt congealed, or extruded
before tabletting. The final formulation may comprise one or more
layers and may be coated or uncoated; it may even be encapsulated.
Formulations of tablets are discussed in Pharmaceutical Dosage
Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel
Dekker, New York, 1980).
[0148] Consumable oral films for human or veterinary use are
typically pliable water-soluble or water-swellable thin film dosage
forms which may be rapidly dissolving or mucoadhesive and typically
comprise a compound of formula (I), a film-forming polymer, a
binder, a solvent, a humectant, a plasticiser, a stabiliser or
emulsifier, a viscosity-modifying agent and a solvent. Some
components of the formulation may perform more than one function.
The film-forming polymer may be selected from natural
polysaccharides, proteins, or synthetic hydrocolloids and is
typically present in the range 0.01 to 99 weight %, more typically
in the range 30 to 80 weight %. Other possible ingredients include
anti-oxidants, colorants, flavourings and flavour enhancers,
preservatives, salivary stimulating agents, cooling agents,
co-solvents (including oils), emollients, bulking agents,
anti-foaming agents, surfactants and taste-masking agents. Films in
accordance with the invention are typically prepared by evaporative
drying of thin aqueous films coated onto a peelable backing support
or paper. This may be done in a drying oven or tunnel, typically a
combined coater dryer, or by freeze-drying or vacuuming.
[0149] Solid formulations for oral administration may be formulated
to be immediate and/or modified release. Modified release includes
delayed, sustained, pulsed, controlled, targeted and programmed
release. Suitable modified release formulations for the purposes of
the invention are described in U.S. Pat. No. 6,106,864. Details of
other suitable release technologies such as high energy dispersions
and osmotic and coated particles are to be found in Pharmaceutical
Technology On-line, 25(2), 1-14, by Verma et al (2001). The use of
chewing gum to achieve controlled release is described in
WO-A-00/35298.
[0150] Compounds of formula (I) may also be administered directly
into the blood stream, into muscle, or into an internal organ. Such
parenteral administration includes intravenous, intraarterial,
intraperitoneal, intrathecal, intraventricular, intraurethral,
intrasternal, intracranial, intramuscular and subcutaneous
administration. Suitable devices for parenteral administration
include needle (including microneedle) injectors, needle-free
injectors and infusion techniques.
[0151] Compounds of the invention may also be administered
topically to the skin or mucosa, that is, dermally or
transdermally.
[0152] The compounds of formula (I) can also be administered
intranasally or by inhalation, typically in the form of a dry
powder (either alone, as a mixture, for example, in a dry blend
with lactose, or as a mixed component particle, for example, mixed
with phospholipids, such as phosphatidylcholine) from a dry powder
inhaler, as an aerosol spray from a pressurised container, pump,
spray, atomiser (preferably an atomiser using electrohydrodynamics
to produce a fine mist), or nebuliser, with or without the use of a
suitable propellant, such as 1,1,1,2-tetrafluoroethane or
1,1,1,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal
use, the powder may comprise a bioadhesive agent, for example,
chitosan or cyclodextrin. For intranasal use, the powder may
comprise a bioadhesive agent, for example, chitosan or
cyclodextrin.
[0153] The pressurised container, pump, spray, atomizer, or
nebuliser contains a solution or suspension of the compound of
formula (I) comprising, for example, ethanol, aqueous ethanol, or a
suitable alternative agent for dispersing, solubilising, or
extending release of the compound, a propellant as solvent and an
optional surfactant, such as sorbitan trioleate, oleic acid, or an
oligolactic acid.
[0154] Prior to use in a dry powder or suspension formulation, the
drug product is micronised to a size suitable for delivery by
inhalation (typically less than 5 microns). This may be achieved by
any appropriate comminuting method, such as spiral jet milling,
fluid bed jet milling, supercritical fluid processing to form
nanoparticles, high pressure homogenisation, or spray drying.
[0155] Capsules (made, for example, from gelatin or
hydroxypropylmethylcellulose), blisters and cartridges for use in
an inhaler or insufflator may be formulated to contain a powder mix
of the compound of the invention, a suitable powder base such as
lactose or starch and a performance modifier such as l-leucine,
mannitol, or magnesium stearate. The lactose may be anhydrous or in
the form of the monohydrate, preferably the latter. Other suitable
excipients include dextran, glucose, maltose, sorbitol, xylitol,
fructose, sucrose and trehalose.
[0156] A suitable solution formulation for use in an atomiser using
electrohydrodynamics to produce a fine mist may contain from 1
.mu.g to 20 mg of the compound of the invention per actuation and
the actuation volume may vary from 1 .mu.l to 100 .mu.l. A typical
formulation may comprise a compound of formula (I), propylene
glycol, sterile water, ethanol and sodium chloride. Alternative
solvents which may be used instead of propylene glycol include
glycerol and polyethylene glycol.
[0157] Suitable flavours, such as menthol and levomenthol, or
sweeteners, such as saccharin or saccharin sodium, may be added to
those formulations of the invention intended for intranasal
administration. Formulations for intranasal administration may be
formulated to be immediate and/or modified release using, for
example, PGLA. Modified release includes delayed, sustained,
pulsed, controlled, targeted and programmed release.
[0158] Compounds of formula (I) may also be administered directly
to the eye or ear, typically in the form of drops of a micronised
suspension or solution in isotonic, pH-adjusted, sterile
saline.
[0159] Compounds of formula (I) may be combined with soluble
macromolecular entities, such as cyclodextrin and suitable
derivatives thereof or polyethylene glycol-containing polymers, in
order to improve their solubility, dissolution rate, taste,
bioavailability and/or stability when using any of the
aforementioned modes of administration. Drug-cyclodextrin
complexes, for example, are found to be generally useful for most
dosage forms and administration routes. Both inclusion and
non-inclusion complexes may be used. As an alternative to direct
complexation with the drug, the cyclodextrin may be used as an
auxiliary additive, i.e. as a carrier, diluent, or solubiliser.
Most commonly used for these purposes are alpha-, beta- and
gamma-cyclodextrins, examples of which may be found in
international patent publications WO-A-91/11172, WO-A-94/02518 and
WO-A-98/55148
[0160] Experimental
[0161] The synthesis of various molecules suitable for use in the
present invention are described below.
[0162] All starting materials and reagents are commercially
available and were obtained from Aldrich with exception of
2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one which was purchased
from Fluorochem.
Preparation 1: 2-octanoylamino-pyrrolo[2,3-d]pyrimidin-4-one
##STR00001##
[0164] A 50 cm.sup.3 round-bottomed flask containing a stirring bar
was charged with 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2.00
g, 13.33 mmol). The flask was fitted with a septum and placed under
an Ar atmosphere. Freshly distilled pyridine (20.00 cm.sup.3) was
added via syringe and the resulting suspension cooled on ice. The
solution was allowed to equilibrate at this temperature (ca. 5 min)
and then octanoyl chloride (6.80 cm.sup.3, 39.99 mmol) was added
dropwise. The resulting suspension was heated at 85.degree. C. for
30 min. After cooling to room temperature 6.5% ethanolic ammonia
(60 cm.sup.3) was added and the resulting suspension stirred at
room temperature overnight. The precipitate of product was removed
via vacuum filtration and washed with ethanol followed by diethyl
ether to yield the desired product (2.56 g, 70%) pure as a yellow
solid, m.p. >300.degree. C. (decomposition). Procedure based on
Akimoto et al. 1986 and Akimoto et al. 1988
[0165] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.86 (3H, t, J 5.1),
1.26 (8H, m), 1.58 (2H, app. quintet), 2.01 (1H, br s, NH), 2.43
(2H, t, J 5.1), 6.40 (1H, d, J 2.0), 7.01 (1H, d, J 2.0), 11.43
(1H, br s, NH), 11.67 (1H, br s, NH)
[0166] HRMS (m/z ESI.sup.-): Found: 275.1517
([M-H].sup.-C.sub.14H.sub.19N.sub.4O.sub.2; Requires: 275.1508)
Preparation 2:
2-octanoylamino-5-((dibenzyl)amino)methyl)-pyrrolo[2,3-d]pyrimidin-4-one
##STR00002##
[0168] A 50 cm.sup.3 reaction vessel containing a stirring bar was
charged with 2-octanoylamino-pyrrolo[2,3-d]pyrimidin-4-one (1.00 g,
3.60 mmol), dibenzylamine (2.00 cm.sup.3, 10.80 mmol), formalin
(349.00 .mu.L, 12.60 mmol) and 80% aqueous acetic acid (36
cm.sup.3). The resulting suspension was heated at 60.degree. C. for
20 h, cooled to room temperature, diluted with 0.5 M HCl (36
cm.sup.3) and stirred at room temperature for 30 min. The mixture
was neutralised with conc. aq. ammonia (36 cm.sup.3) and extracted
with chloroform (3.times.50 cm.sup.3). The organic extracts were
combined, dried (MgSO.sub.4) and evaporated to dryness. The crude
residue was purified by column chromatography (9:1
dichloromethane-MeOH--7:3 dichloromethane-MeOH) to give the desired
compound (1.45 g, 84%) as a yellow powder m.p. >300.degree. C.
(decomposition). Procedure based on Akimoto et al. 1986 and Akimoto
et al. 1988
[0169] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.86 (3H, t, J 7.1),
1.25 (8H, m), 1.57 (2H, m) 2.42 (2H, t, J 7.1), 3.57 (4H, s), 3.76
(2H, s), 6.88 (1H, s), 7.23 (2H, t, J 7.3), 7.31 (4H, app. t), 7.41
(4H, d, J 7.3), 11.34 (1H, s, NH), 11.57 (1H, s, NH), 11.68 (1H, s,
NH)
[0170] HRMS (m/z ESI.sup.+): Found: 486.2863
([M+H].sup.+C.sub.29H.sub.36N.sub.5O.sub.2; requires: 486.2869)
EXAMPLE 1
2-amino-5-(((3-phenylpropyl)amino)methyl)pyrrolo[2,3-d]pyrimidin-4-one
##STR00003##
[0172] A large carousel tube containing a stirring bar was charged
with
2-octanoylamino-5-((dibenzyl)amino)methyl)-pyrrolo[2,3-d]pyrimidin-4-one
(100.0 mg, 0.21 mmol), 3-phenylpropylamine (146.00 .mu.L, 1.03
mmol) and 1:1 THF-methanol (2.00 cm.sup.3). The suspension was
degassed and the reaction vessel sealed. The suspension was heated
at 75.degree. C. for 24 h, cooled to room temperature and treated
with 5 M KOH (146.00 .mu.L) and stirred at room temperature for 65
h. The solution was concentrated in vacuo and the crude residue
purified by column chromatography (9:0.9:0.1
dichloromethane-MeOH-NH.sub.4OH). The resulting solid was washed
with HPLC grade hexane followed by diethyl ether in order to remove
trace impurities. This gave the desired compound (28 mg, 46%) as an
orange powder, m.p. >300.degree. C. (decomposition). Procedure
based on Akimoto et al. 1986 and Akimoto et al. 1988
[0173] .delta..sub.H (600 MHz, DMSO-d.sub.6): 1.24 (1H, br s, NH),
1.66 (2H, app. quintet,), 1.91 (1H, s, NH), 2.44 (2H, t, J 6.9,),
2.57 (2H, t, J 6.9,), 3.59 (2H, s,), 6.15 (2H, br s,), 6.45 (1H,
s,), 7.15 (1H, t, J 7.4,), 7.16 (3H, m), 7.25 (2H, app. t,), 10.70
(1H, br s)
[0174] .delta..sub.C (600 MHz, DMSO-d.sub.6): 30.6, 32.9, 45.2,
47.5, 48.6, 79.2, 98.7 (q), 113.6 (q), 125.5, 128.2, 128.3, 142.3
(q), 152.2 (q), 160.5 (C.dbd.O)
[0175] v.sub.max (film)/cm.sup.-1: 697, 748, 749, 1080, 1420, 1596,
2927
[0176] HRMS (m/z ESI.sup.+): Found: 298.1662
([M+H].sup.+C.sub.16H.sub.20N.sub.5O; Requires: 298.1668)
Preparation 3: 2-chloro-3-oxopropanenitrile
##STR00004##
[0178] In a dry round bottomed flask under a positive pressure of
argon, a suspension of NaOMe (7.14 g, 0.13 mol) in dry THF (90 mL)
was cooled to -5.degree. C. Methyl formate (9 mL, 0.15 mol) was
added dropwise over 1 min by syringe and stirring was continued at
-5.degree. C. for 20 min. Then chloroacetonitrile (8.33 mL, 0.13
mol) was added dropwise via a dropping funnel over 45 min. The
mixture turned from white to yellow and was stirred for a further 2
h at -5.degree. C. at which point the reaction mixture was orange.
The bath was removed and the reaction was allowed to warm up to
room temperature. An aliquot of the reaction mixture was treated
with a drop of concentrated HCl and analysed by TLC which indicated
the presence of the desired product with an R.sub.f=0.45, eluting
with 100% EtOAc. The mixture was cooled to 0.degree. C. and
concentrated HCl (12 mL) was added dropwise during which time the
mixture reaction became cherry-red. The resultant suspension was
filtered through a pad of celite, and the celite was washed with
EtOAc until the filtrate became colourless. The collected filtrates
were concentrated at reduced pressure with the water bath at a
temperature no higher than 40.degree. C. to afford
chloro(formypacetonitrile.sup.1 as a black oil, in quantitative
yield, which was used without further purification. Procedure based
on Brooks 2012.
[0179] .delta..sub.H (400 MHz, CDCl.sub.3) 9.38 (s, 1H).
[0180] .delta..sub.C NMR (400 MHz, DMSO-d.sub.6) b 168.2, 126.6,
67.8.
Preparation 4:
2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
##STR00005##
[0182] 2,4-Diamino-6-hydroxypyrimidine (3.00 g, 24 mmol) was added
to a solution of sodium acetate (6.4 g, 76 mmol) in millipore water
(90 mL) and stirred at 50.degree. C. for 1 hour. While still at
50.degree. C. a solution of crude chloro(formyl)acetonitrile (3.00
g, 32 mmol) in mQ water (44 mL) was added dropwise with a dropping
funnel, during which time the reaction turned beigeand heating
continued for 18 h at 50.degree. C., after which time the reaction
was heated to 100.degree. C. for 3 h. The reaction mixture was
allowed to cool to room temperature and the solid removed by
filtration. The solid was suspended in EtOH and 5M aqueous KOH
solution was added until the solid dissolved. Charcoal was added to
the solution and the mixture stirred for 30 minutes before removal
of the solid by filtration. The pH of the filtrate was adjusted to
pH=6 with concentrated aqueous HCl solution during which time a
precipitate formed and was collected by filtration. In order to
remove the final traces of water from the solid it was dissolved in
a mixture of toluene/methanol 1/1 and then concentrated at reduced
pressure. The resultant solid was dried over P.sub.2O.sub.5 to
afford the desired compound (1.68 g, 9.6 mmol, 40% yield) as beige
solid. Procedure based on Brooks 2012.
[0183] .delta..sub.H (400 MHz, DMSO-d.sub.6) .delta. 11.98 (br s,
1H) 10.74 (br s, 1H), 7.59 (s, 1H), 6.43 (s, 2H).
[0184] .delta..sub.C (100 MHz, DMSO-d.sub.6) .delta. 158.0, 154.3,
152.1, 128.2, 116.4, 99.2, 86.0. HRMS (m/z ESI.sup.-):
C.sub.7H.sub.5N.sub.5O [M-H].sup.- Found 174.0415 Requires:
174.0416.
Preparation 5:
4,7-Dihydro-4-oxo-2-[(triphenylmethyl)amino]-3H-pyrrolo[2,3-d]pyrimidine--
5-carbonitrile
##STR00006##
[0186] In a dry round bottomed flask under an atmosphere of argon,
trityl chloride (1.20 g, 4.28 mmol) was added to a solution of
2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(0.50 g, 2.85 mmol) in dry pyridine (29 mL). The mixture reaction
was heated at 90.degree. C. for 48 h. The reaction mixture was
concentrated under reduced pressure then absorbed on silica gel and
purified by flash chromatography on silica gel eluting with
dichloromethane/MeOH with a gradient starting at 2% of MeOH and
rising to 10%. The desired compound was obtained as a brown solid
(0.63 g, 1.5 mmol, 53% yield).
[0187] Procedure based on Olgen 2008.
[0188] .delta..sub.H (400 MHz, DMSO-d.sub.6) .delta. 11.80 (br s,
1H); 10.64 (br s, 1H), 7.56 (s, 1H), 7.41(s, 1H), 7.29-7.28 (m,
12H), 7.23-7.17 (m, 3H), 5.73 (s, 1H).
[0189] HRMS (m/z ESI.sup.+): C.sub.26H.sub.18N.sub.5O
[M-H].sup.+Found 416.1514 Requires: 416.1511.
Preparation 6:
4,7-Dihydro-4-oxo-2-[(triphenylmethypamino]-3H-pyrrolo[2,3-d]pyrimidine-5-
-carboxaldehyde
##STR00007##
[0191] HMDS (6 mmol, 1.3 mL) was added to a mixture of
4,7-dihydro-4-oxo-2-[(triphenylmethyl)amino]-3H-pyrrolo[2,3-d]pyrimidine--
5-carbonitrile (1.30 g, 3 mmol) with ammonium sulphate (397 mg, 0.3
mmol) in dry toluene (8 mL) in a round bottomed flask. A reflux
condenser was fitted, and the flask was heated at reflux
temperature overnight. The mixture was cooled to room temperature
and concentrated under reduced pressure. Under a positive pressure
of argon, the crude reaction mixture was solubilised in dry
dichloromethane (8 mL) and cooled to -78.degree. C. At this
temperature, DiBAL-H (4.5 mL, 1 M in dichloromethane, 4.5 mmol) was
added dropwise. After 2 hours, analysis by TLC (EtOAc 100%)
indicated that some starting material remained. So, a further 2 mL
DiBAL-H solution was added dropwise. After 1 hour, the reaction was
complete and a mixture of H.sub.2O/AcOH (9/1, 3.5 mL) was added at
-78.degree. C. The reaction mixture was allowed to warm to room
temperature slowly. A mixture of EtOAc/H.sub.2O (1/1, 300 mL) was
added to the reaction mixture and stirring continued at room
temperature for 2 hours. The layers were separated and the organic
layer was washed with brine and the aqueous layers were extracted
with EtOAc. The combined organic fractions were dried over
MgSO.sub.4, filtered and concentrated at reduced pressure. The
crude reaction product was filtered through a pad of silica gel
eluting with EtOAc to afford a yellow solid (1.01 g, 2.38 mmol,
76%). Procedure based on Brooks 2010 and Brooks 2012.
[0192] .delta..sub.H (400 MHz, DMSO-d.sub.6) .delta. 11.82 (s, 1H),
10.63 (s, 1H), 9.99 (s, 1H), 7.54 (s, 1H), 7.31-7.27 (m, 13H),
7.23-7.19 (m, 3H).
[0193] HRMS (m/z ESI.sup.-): C.sub.26H.sub.18N.sub.4O.sub.2
[M-H].sup.- Found 419.1508 Requires: 419.1508.
Preparation 7:
5-((3-phenylpropylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidi-
n-4(7H)-one
##STR00008##
[0195] General procedure A: To a suspension of
N-((4-oxo-2-(tritylamino)-4,7-dihydro-3H-pyrrolo[2,3-capyrimidin-5-yl)met-
hyl)formamide (200.0 mg, 0.48 mmol) and sodium sulphate (5.0 mg) in
methanol (5 cm.sup.3) under an argon atmosphere was added
3-phenylpropylamine (74.00 .mu.L, 0.52 mmol) and the resulting
suspension stirred at room temperature for 2 h. Sodium borohydride
(55.00 mg, 1.43 mmol) was then added and the reaction mixture
stirred at room temperature for a further 1 h. Water (5 cm.sup.3)
was added and the resulting suspension stirred for 10 min before
being extracted with dichloromethane (3.times.5 cm.sup.3). The
combined organic layers were dried (MgSO.sub.4) and concentrated in
vacuo to yield the crude product which was purified by flash
chromatography (9:1 dichloromethane-MeOH) to yield the desired
compound as a white solid (210 mg, 41.8%), m.p. >300.degree. C.
(decomposition). Procedure based on Brooks 2010 and Brooks
2012.
[0196] .sup.1H (400 MHz, DMSO-d.sub.6): 1.73 (2H, quintet, J 7.8),
2.56 (4H, m), 3.74 (1H, s), 6.42 (1H, s, H-6), 7.19 (20H, m), 7.45
(1H, bs, NH), 10.78 (1H, bs, NH) .sup.13C (400 MHz, DMSO-d.sub.6):
31.7, 32.8, 45.4, 47.8, 70.4 (q), 99.7 (q), 114.9, 117.6 (q),
125.9, 126.0, 126.9, 128.0, 128.6, 129.0, 142.6 (q), 145.4 (q),
150.0 (q), 150.4 (q), 159.7 (C.dbd.O)
[0197] HRMS (m/z-ESI.sup.+): Found: 540.2757
[M+H].sup.+C.sub.35H.sub.34N.sub.5O Requires: 540.2765)
[0198] v.sub.max/cm.sup.-1: 1542, 1611, 1670, 2868, 2951
EXAMPLE 2
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-ph-
enylpropan-1-aminium chloride
##STR00009##
[0200] General procedure B: A 5 cm.sup.3 reaction vessel containing
a stirring bar was charged with
5-((3-phenylpropylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidi-
n-4(7H)-one (210.0 mg, 0.39 mmol) and 1.25 M methanolic HCl (3
cm.sup.3). The resulting solution was stirred at room temperature
for 16 h. The precipitated product was removed by vacuum filtration
and washed with dichloromethane to yield the desired compound as a
white powder (84 mg, 68%), m.p. >300.degree. C. (decomposition).
Procedure based on Brooks 2010 and Brooks 2012.
[0201] .delta..sub.H (600 MHz, DMSO-d.sub.6): 1.90 (2H, app.
quintet), 2.63 (2H, t, J 7.8), 2.90 (2H, m), 4.13 (2H, t, J 5.2),
6.57 (2H, bs), 6.80 (1H, d, J 2.3), 7.16 (3H, m), 7.26 (2H, t, J
7.0), 9.11 (2H, bs), 11.05 (1H, m, NH), 11.31 (1H, broad doublet,
NH)
[0202] .delta..sub.C (125 MHz, DMSO-d.sub.6): 27.6, 32.1, 42.9,
45.6, 48.9, 98.6, 108.7 (q), 117.9 (q),126.4, 128.6, 128.7, 140.9
(q), 152.9 (q), 160.5 (C.dbd.O)
[0203] HRMS (m/z ESI.sup.+): Found: 298.1662
(M.sup.+C.sub.16H.sub.20N.sub.5O Requires: 298.1664)
[0204] v.sub.max (film)/cm.sup.-1: 1456, 1625, 2443, 2713, 2756,
2873, 2933, 3184
Preparation 8
5-((3-butylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-
-one
##STR00010##
[0206] Prepared as per general procedure A using
N-((4-oxo-2-(tritylamino)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)met-
hyl)formamide (200.00 mg, 0.48 mmol), n-butylamine (95.00 .mu.L,
0.95 mmol) and NaBH.sub.4 (55 mg, 1.43 mmol) to yield the desired
product as a white powder (200 mg, 88%), m.p. >300.degree. C.
(decomposition).
[0207] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.84 (3H, t, J 7.3),
1.30 (2H, app. sextet), 1.51 (2H, app. quintet), 2.82 (2H, t, J
7.3), 4.01 (2H, s), 6.62 (1H, s), 7.24 (15H, m), 7.57 (1H, bs),
11.07 (1H, bs)
[0208] .delta..sub.C (400 MHz, DMSO-d.sub.6): 13.9, 19.6, 28.0,
42.9, 46.0, 70.6 (q), 99.4 (q), 108.9, 118.0 (q), 127.0, 128.1,
129.0, 145.2 (q), 150.5 (q), 150.6 (q), 160.3 (C=0)
[0209] v.sub.max (film)/cm.sup.-1: 1545, 1613, 1672, 2870, 2956
[0210] HRMS (m/z ESI.sup.+): Found: 478.2600
([M+H].sup.+C.sub.30H.sub.32N.sub.5O; Requires: 478.2607)
EXAMPLE 3
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)butan-
-1-aminium chloride
##STR00011##
[0212] Prepared as per general procedure B using
5-((butylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-o-
ne (108 mg, 0.39 mmol) and 1.25 M methanolic HCl (3 cm.sup.3) to
yield the desired product as a white powder (72 mg, 67%), m.p.
>300.degree. C. (decomposition).
[0213] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.86 (3H, t, J 7.4),
1.31 (2H, app. sextet), 1.57 (2H, app. quintet), 2.90 (2H, m), 4.12
(2H, t, J 5.3), 6.49 (2H, bs), 6.81 (1H, s), 9.01 (2H, bs), 10.98
(2H, bs), 11.29 (1H, bs)
[0214] .delta..sub.C (400 MHz, DMSO-d.sub.6): 18.7, 24.7, 32.7,
47.4, 50.6, 103.6 (q), 114.0, 123.2 (q), 153.3 (q), 157.6 (q),
164.7 (C.dbd.O)
[0215] HRMS (m/z ESI.sup.+): Found: 236.1518
(M+C.sub.11H.sub.18N.sub.15O Requires: 236.1511)
[0216] v.sub.max (film)/cm.sup.-1: 1456, 1625, 1668, 2443, 2713,
2756, 2873, 2933, 3184
Preparation 9
5-((3-hexylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-
-one
##STR00012##
[0218] Prepared as per general procedure A using
N-((4-oxo-2-(tritylamino)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)met-
hyl)formamide (200.0 mg, 0.48 mmol), n-hexylamine (125.00 4, 0.95
mmol) and sodium borohydride (55 mg, 1.43 mmol) to yield the
desired product as a white powder (200 mg, 83.0%), m.p.
>300.degree. C. (decomposition).
[0219] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.82 (3H, t, J 7.4),
1.20 (6H, m), 1.34 (2H, app. quintet), 2.40 (2H, t, J 7.4), 3.60
(2H, s), 6.30 (1H, s), 7.23 (15H, m), 7.37 (1H, bs), 10.62 (1H,
bs)
[0220] .delta..sub.C (400 MHz, DMSO-d.sub.6): 14.4, 22.5, 26.8,
31.6, 29.6, 45.2, 48.3, 70.4 (q), 99.7 (q), 115.2, 116.7 (q),
127.0, 128.1, 129.0, 145.4 (q), 150.1 (q), 150.5 (q), 159.7
(C.dbd.O)
[0221] v.sub.max (film)/cm.sup.-1: 1552, 1648, 1734, 2856, 2928
[0222] HRMS (m/z ESI.sup.-): Found: 504.2769
([M-H].sup.-C.sub.32H.sub.34N.sub.5O; Requires: 504.2763)
Example 4
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)hexan-
-1-aminium chloride
##STR00013##
[0224] Prepared as per general procedure B using
5-((hexylamino)methyl)-2-(tritylamino)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-o-
ne (190 mg, 0.39 mmol) and 1.25 M methanolic HCl (3 cm.sup.3) to
yield the desired product as a white powder (70 mg, 64.8%), m.p.
>300.degree. C.
[0225] .delta..sub.H (400 MHz, DMSO-d.sub.6): 0.83 (3H, t, J 7.2),
1.25 (6H, m), 1.59 (2H, app. sextet, J 7.2), 2.87 (2H, m), 4.11
(2H, t, J 5.3), 6.71 (2H, bs), 6.84 (1H, d, J 3.6), 9.11 (2H, bs),
11.25 (1H, bs), 11.46 (1H, bs)
[0226] .delta..sub.C (400 MHz, DMSO-d.sub.6): 14.3, 22.3, 25.8,
25.9, 31.1, 42.6, 46.1, 98.9 (q), 109.3 (q), 118.5, 148.1 (q),
152.8 (q), 159.8 (C.dbd.O)
[0227] v.sub.max (film)/cm.sup.-1: 1578, 1625, 1669, 2429, 2712,
2861, 2930, 2957, 3266
[0228] HRMS (m/z ESI.sup.+: Found: 264.1830
([M+H].sup.+C.sub.13H.sub.22N.sub.5O Requires: 264.1824)
[0229] All the examples described herein are TGT substrates.
[0230] All the examples described herein are not inhibitors or
substrates for HPRT
[0231] To assess the potential of these compounds in vivo, a
chronic monophasic EAE disease in mice was induced before treatment
with the new chemical entity (NCE). EAE Disease was induced in 8-10
week old female mice (C57BL/6) by sub-cutaneous (s.c.) injection of
200 .mu.l emulsion containing 150 .mu.g MOG.sub.33-55 peptide
(Genscript) in Complete Freund's Adjuvant (CFA; containing 5 mg/ml
heat-inactivated Mycobacterium tuberculosis). On the same day, mice
were administered 500 ng Pertussis Toxin (Kaketsuken, Japan)
intraperitoneally (i.p.) and again two days later. Disease severity
was recorded every 24 hours: 0-Normal; 1-flaccid tail;
2-imparied/wobbly gait; 3-complete hind limb weakness; 4-hind limb
and forelimb paralysis; 5-moribund state/dead. Protocol is based on
the Nature Protocols for Active induction of experimental allergic
encephalomyelitis, which includes the scoring methodology:
[0232] Stromnes I M, Goverman J M (2006) Active induction of
experimental allergic encephalomyelitis. Nat Protoc.
1(4):1810-9.
[0233] FIG. 5 shows the results of the in vivo testing for molecule
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-cl]pyrimidin-5-yl)methyl)-3--
phenylpropan-1-aminium chloride (shown as Compound I) . The EAE
score relates to an assessment of disease progression with respect
to issues like tail paralysis and limb paralysis, a higher score is
a worse condition. A score of 1 indicates decreased tail tone, a
score of 2 indicates hind leg weakness (paraparesis), a score of 3
indicates hind limb paralysis and/or incontinence. Note that the
untreated animals have a continuous and progressive worsening of
disease. In contrast, the animals treated with
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-cl]pyrimidin-5-yl)methyl)-3--
phenylpropan-1-aminium chloride (shown as Compound I) exhibit rapid
(within 24 hrs) reversal of symptoms at the highest dosage given
(30 mg/kg, i.p.) and animals were scored as disease free after 4
daily treatments. At lower doses animals were slower to respond but
in all cases disease progression was halted and reversed.
[0234] FIG. 6 shows that in agreement with the observed disability
scores, treated animals showed a dramatic return to normal weight
gain, reaching a weight level 96.6% that of controls (19.9 grams
versus 20.6 grams), at 21 days post inoculation (dpi). This
contrasts with the sustained drop in body weight of non-treated EAE
diseased animals to a level 86.4% that of controls at 21 dpi (17.8
grams versus 20.6 grams). Motor coordination and hind-limb strength
were also evaluated by the ability of mice to cross a horizontal
bar (FIG. 7). Non-diseased animals crossed the bar with an average
time of 4.3.+-.0.57 seconds. From 9 dpi, EAE mice showed a rapid
deterioration in performance and were subsequently unable to
maintain a grip on the apparatus. Strikingly,
N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-3-p-
henylpropan-1-aminium chloride (shown as Compound I) treatment
fully restored the performance of EAE diseased mice to control
levels within 4 treatments.
[0235] In addition to the data shown in FIGS. 5, 6 and 7, all the
molecules described herein were tested in the chronic EAE model and
found to show substantial improvement up to and including reversal
of symptoms to a disease free state in subjects.
* * * * *