Note: Descriptions are shown in the official language in which they were submitted.
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CALCILYTIC COMPOUNDS
FIELD OF INVENTION
The present invention relates to novel arylalkylamine caicilytic compounds,
pharmaceutical compositions containing these compounds and their use as
calcium receptor
antagonists.
In mammals, extracellular Ca2+ is under rigid homeostatic control and
regulates
various processes such as blood clotting, nerve and muscle excitability, and
proper bone
formation. Extracellular Ca2+ inhibits the secretion of parathyroid hormone
("PTH") from
parathyroid cells, inhibits bone resorption by osteoclasts, and stimulates
secretion of
calcitonin from C-cells. Calcium receptor proteins enable certain specialized
cells to
respond to changes in extracellular Ca2+ concentration.
PTH is the principal endocrine factor regulating Ca2+ homeostasis in the blood
and
extracellular fluids. PTH, by acting on bone and kidney cells, increases the
level of Ca2+
in the blood. This increase in extracellular Ca2+ then acts as a negative
feedback signal,
depressing PTH secretion. The reciprocal relationship between extracellular
Ca2+ and
PTH secretion forms an important mechanism maintaining bodily Ca2+
homeostasis.
Extracellular Ca2+ acts directly on parathyroid cells to regulate PTH
secretion.
The existence of a parathyroid cell surface protein which detects changes in
extracellular
Ca2+ has been confirmed. See Brown et al., Nature X66:574, 1993. In
parathyroid cells,
this protein, the calcium receptor, acts as a receptor for extracellular Ca2+,
detects changes
in the ion concentration of extracellular Ca2+, and initiates a functional
cellular response,
PTH secretion.
Extracellular Ca2+ influences various cell functions, reviewed in Nemeth et
al.,
Cell Calcium 11:319, 1990. For example, extracellular Ca2+ plays a role in
parafollicular
(C-cells) and parathyroid cells. See Nemeth, Cell Calcium 11:323, 1990. The
role of
extracellular Ca2+ on bone osteoclasts has also been studied. See Zaidi,
Bioscience
Reports 10:493, 1990.
Various compounds are known to mimic the effects of extra-cellular Ca2+ on a
calcium receptor molecule. Calcilytics are compounds able to inhibit calcium
receptor
activity, thereby causing a decrease in one or more calcium receptor
activities evoked by
extracellular Ca2+. Calcilytics are useful as lead molecules in the discovery,
development,
design, modification and/or construction of useful calcium modulators which
are- active at
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Ca2+ receptors. Such calcilytics are useful in the treatment of various
disease states
characterized by abnormal levels_of one or more components, e.g., polypeptides
such as
hormones, enzymes or growth factors, the expression and/or secretion of which
is regulated
or affected by activity at one or more Ca2+ receptors. Target diseases or
disorders for
calcilytic compounds include diseases involving abnormal bone and mineral
homeostasis.
Abnormal calcium homeostasis is characterized by one or more of the following
activities: an abnormal increase or decrease in serum calcium; an abnormal
increase or
decrease in urinary excretion of calcium; an abnormal increase or decrease in
bone calcium
levels (for example, as assessed by bone mineral density measurements); an
abnormal
absorption of dietary calcium; an abnormal increase or decrease in the
production and/or
release of messengers which affect serum calcium levels such as PTH and
calcitonin; and
an abnormal change in the response elicited by messengers which affect serum
calcium
levels.
Thus, calcium receptor antagonists offer a unique approach towards the
pharmacotherapy of diseases associated with abnormal bone or mineral
homeostasis, such
as hypoparathyroidism, osteosarcoma, periodontal disease, fracture healing,
osteoarthritis,
rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with
malignancy
and fracture healing, and osteoporosis.
SUMMARY OF THE INVENTION
The present invention comprises arylaikylamine derivatives represented by
Formula (I) hereinbelow and their use as calcium receptor antagonists which
are useful in
the treatment of a variety of diseases associated with abnormal bone or
mineral
homeostasis, including but not limited to hypoparathyroidism, osteosarcoma,
periodontal
disease, fracture healing, osteoarthritis, rheumatoid arthritis, Paget's
disease, humoral
hypercalcemia associated with malignancy and fracture healing, and
osteoporosis.
The present compounds maintain calcium receptor activity while exhibiting
increased stability as compared to other calcium receptor antagonists.
The present invention further provides a method for antagonizing calcium
receptors
in an animal, including humans, which comprises administering to an animal in
need
thereof an effective amount of a compound of Formula (I), indicated
hereinbelow.
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DETAILED DESCRIPTION OF THE INVENTION
The compounds of the present invention are selected from Formula (I)
hereinbelow:
/ (R2) n
R~
Formula (I)
wherein;
n is an integer from 0 to 3;
R 1 is selected from the group consisting of R3, and CH2YR3 wherein R3 is C 1
_ 15 aryl or
C 1-20 alkyl and Y is selected from the group consisting of CONH, COO,
CONHNHCO
and CO;
R2 is selected from the group consisting of R3, CONHR3, H, OR3, X, N(R3)2,
CON(R3)2~
COR3, and SR3, wherein R3 is H, C 1 _ 15 aryl or C 1 _20 alkyl.
Preferably, n is 1 and the R2 moiety is beta to the N heteroatom. When n is 2,
the
R2 moiety is alpha or gamma to the N heteroatom.
Preferably, R1 is CH2YR3.
Preferably, Y is CONH or COO.
Preferably, R2 is CONHR3.
As used herein, "alkyl" refers to an optionally substituted hydrocarbon group
joined
by single carbon-carbon bonds and having 1-20 carbon atoms joined together.
The alkyl
hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated.
The
substituents are selected from F, CI, Br, I, N, S and O. Preferably, no more
than three
substituents are present. More preferably, the alkyl has 1-12 carbon atoms and
is
unsubstituted. Preferably, the alkyl group is linear. Preferably, the alkyl
group is saturated.
As used herein, "aryl" refers to an optionally substituted aromatic group with
at
least one ring having a conjugated pi- electron system, containing up to two
conjugated or
fused ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and
biaryl groups, all
of which may be optionally substituted. The substituents are selected from F,
Cl, Br, I, N, S
and O. Preferably, the aryl is either optionally substituted phenyl,or
optionally substituted
2-naphthyl. Preferably, the aryl group is unsubstituted. The most preferred
aryl group is
unsubstituted phenyl.
Preferred compounds useful in the present invention include:
3-Carbamoyl-1-((dodecylcarbamoyt)methyl)pyridinium, chloride
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4,4'-Biphenacylene-bis(pyridiniumbromide),
3-(Dodecylcarbamoyl)-1-methylpyridinium, bromide,
1-({(Dodecyloxy)carbonyl)methyl)-3-(methylcarbamoyl)pyridinium, chloride,
4-Carbamoyl-1-((dodecyloxycarbonyl)methyl)pyridinium, chloride,
3-Carbamoyl-1-((dodecyloxycarbonyl)methyl)pyridinium, chloride,
1-((Decyloxycarbonyl)methyl)3-carbamoyl-pyridinium, chloride,
1-((Decyloxycarbonyl)methyl)-4-carbamoylpyridinium, chloride,
I-((Decylcarbamoyl)methyl)-3-carbamoylpyridinium, chloride,
1-((Dodecylcarbamoyl)methyl)-3-(phenylcarbamoyl)pyridinium, chloride,
1-(Tetradecanoylhydrazinocarbonylmethyl)pyridinium chloride,
I-Methyl-2-(decylcarbamoyl)pyridinium iodide,
2-(4-(4-Dimethylamino)phenyl)-1,3-butadiene-I-yl)-ethylpyridinium perchlorate,
3-Carbamoyl-I-undecylpyridinium bromide, and
1-((Decylcarbamoyl)methyl)-4-carbamoylpyridinium chloride.
More preferred compounds useful in the present invention include:
3-Carbamoyl-i-((dodecylcarbamoy()methyl)pyridinium, chloride,
3-(Dodecylcarbamoyl)-I-methylpyridinium, bromide,
4-Carbamoyl-1-((dodecyloxycarbonyl)methyl)pyridinium, chloride,
3-Carbamoyl-I-((dodecyIoxycarbonyl)methyl)pyridinium, chloride,
1-((Decyloxycarbonyl)methyl)3-carbamoyl-pyridinium, chloride, and
1-((Dodecylcarbamoyl)methyl)-3-(phenylcarbamoyl)pyridinium, chloride.
The most preferred compound useful in the present invention is
3-Carbamoyl-1-((dodecylcarbamoyl)methyl)pyridinium, chloride.
The present compounds can also be formulated as pharmaceutically acceptable
salts and
complexes thereof. Pharmaceutically acceptable salts are non-toxic sans in the
amounts
and concentrations at which they are administered.
Pharmaceutically acceptable salts include acid addition salts such as those
containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate,
acetate, citrate,
lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-
toluenesulfonate,
cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be
obtained from
acids such as hydrochloric acid, malefic acid, sulfuric acid, phosphoric acid,
sulfamic acid,
acetic acid, citric acid, lactic acid, tartaric acid, malonic acid,
methanesulfonic acid,
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ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfamic acid,
fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic addition salts such as
those
containing benzathine, chloroprocaine, choline, diethanolamine,
ethylenediamine,
meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium,
ammonium, alkylamine, and zinc, when acidic functional groups, such as
carboxylic acid or
phenol are present.
The present invention provides compounds of Formula (I) above which can be
prepared using standard techniques. An overall strategy for preparing
preferred compounds
described herein can be carried out as described in this section. The examples
which follow
illustrate the synthesis of specific compounds. Using the protocols described
herein as a
model, one of ordinary skill in the art can readily produce other compounds of
the present
invention.
All reagents and solvents were obtained from commercial vendors. Starting
materials (e.g., amines and epoxides) were synthesized using standard
techniques and
procedures.
A general procedure used to synthesize many of the present compounds is as
follows:
The preparation of the compounds described herein either follows previously
reported procedures or was accomplished according to the methods illustrated
below. An
overall strategy for preparing preferred compounds described herein can be
carried out as
described in this section. The examples which follow illustrate the synthesis
of specific
compounds. Using the protocols described herein as a model, one of ordinary
skill in the art
can readily produce other Formula (I) compounds.
All reagents and solvents are obtained from commercial vendors. Starting
materials are
synthesized using standard techniques and procedures:
A general procedure used to synthesize many of the compounds was carried out
as
indicated in Scheme 1 below. A solution of the substituted pyridine (e.g.
nicotinamide, 1
mmol) and excess alkylating agent (e.g. N-dodecyl-2-chloroacetamide, 2 mmol)
in
nitromethane or similar solvent (2 mL) is stirred for 24h at 90-100oC.
Typically upon
cooling, the pyridinium salt precipitates out of solution and the
corresponding salt is
collected.
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chem 1
/ R2 / R2
N N+
R~
a. BrR,, N02CH3, 90-100oC, 24h
With appropriate manipulation and protection of any chemical functionality,
synthesis of the remaining compounds of Formula (I) is accomplished by methods
analogous to those above and to those described in the Experimental section.
In order to use a compound of Formula (I) or a pharmaceutically acceptable
salt
thereof for the treatment of humans and other mammals, it is normally
formulated in
accordance with standard pharmaceutical practice as a pharmaceutical
composition.
The calcilytic compounds can be administered by different routes including
intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical
(transdermal}, or
transmucosal administration. For systemic administration, oral administration
is preferred.
For oral administration, for example, the compounds can be formulated into
conventional
oral dosage forms such as capsules, tablets, and liquid preparations such as
syrups, elixirs,
and concentrated drops.
Alternatively, injection (parenteral administration) may be used, e.g.,
intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the compounds
of the
invention are formulated in liquid solutions, preferably, in physiologically
compatible
buffers or solutions, such as saline solution, Hank's solution, or Ringer's
solution. In
addition, the compounds may be formulated in solid form and redissolved or
suspended
immediately prior to use. Lyophilized forms can also be produced.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, bile salts and fusidic
acid
derivatives. In addition, detergents may be used to facilitate permeation.
Transmucosal
administration, for example, may be through nasal sprays, rectal
suppositories, or vaginal
suppositories.
For topical administration, the compounds of the invention can be formulated
into
ointments, salves, gels, or creams, as is generally known in the art.
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The amounts of various calcilytic compounds to be administered can be
determined
by standard procedures taking into account factors such as the compound ICSO,
ECSO, the
biological half-life of the compound, the age, size and weight of the patient,
and the disease
or disorder associated with the patient. The importance of these and other
factors to be
considered are known to those of ordinary skill in the art.
Amounts admininstered also depend on the routes of admininstration and the
degree of oral bioavailability. For example, for compounds with low oral
bioavailability,
relatively higher doses will have to be admninstered.
Preferably the composition is in unit dosage form. For oral application, for
example, a tablet, or capsule may be administered, for nasal application, a
metered aerosol
dose may be administered, for transdermal application, a topical formulation
or patch may
be admininstered and for transmucosal delivery, a buccal patch may be
administered. In
each case, dosing is such that the patient may administer a single dose.
Each dosage unit for oral administration contains suitably from 0.01 to S00
mg/Kg,
and preferably from 0.1 to 50 mglKg, of a compound of Formula (I) or a
pharmaceutically
acceptable salt thereof, calculated as the free base. The daily dosage for
parenteral, nasal,
oral inhalation, transmucosal or transdermal routes contains contains suitably
from 0.01 mg
to 100 mg/Kg, of a compound of Formula(I). A topical formulation contains
suitably 0.01
to S.0% of a compound of Formula (I). The active ingredient may be
administered from 1
to 6 times per day, preferably once, sufficient to exhibit the desired
activity, as is readily
apparent to one skilled in the art.
As used herein, "modulator" means antagonist.
As used herein, "treatment" of a disease includes, but is not limited to
prevention,
retardation and prophylaxis of the disease.
Diseases and disorders which might be treated or prevented, based upon the
affected cells, include bone and mineral-related diseases or disorders;
hypoparathyroidism;
those of the central nervous system such as seizures, stroke, head trauma,
spinal cord
injury, hypoxia-induced nerve cell damage, such as occurs in cardiac arrest or
neonatal
distress, epilepsy, neurodegenerative diseases such as Alzheimer's disease,
Huntington's
disease and Parkinson's disease, dementia, muscle tension, depression,
anxiety, panic
disorder, obsessive-compulsive disorder, post-traumatic stress disorder,
schizophrenia,
neuroleptic malignant syndrome, and Tourette's syndrome; diseases involving
excess water
reabsorption by the kidney, such as syndrome of inappropriate ADH secretion
(SIADH),
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cirrhosis, congestive heart failure, and nephrosis; hypertension; preventing
and/or
decreasing renal toxicity from cationic antibiotics (e.g., aminoglycoside
antibiotics); gut
motility disorders such as dianrhea and spastic colon; GI ulcer diseases; GI
diseases with
excessive calcium absorption such as sarcoidosis; autoimmune diseases and
organ
transplant rejection; squamous cell carcinoma; and pancreatitis.
In a preferred embodiment of the present invention, the present compounds are
used
to increase serum parathyroid ("PTH") levels. Increasing serum PTH levels can
be helpful
in treating diseases such as hypoparathyroidism, osteosarcoma" periodontal
disease,
fracture, osteoarthritis, rheumatoid arthritis, Paget's disease, humoral
hypercaicemia
malignancy and osteoporosis.
Composition of Formula (I) and their pharmaceutically acceptable salts which
are
active when given orally can be formulated as syrups, tablets, capsules and
lozenges. A
syrup formulation will generally consist of a suspension or solution of the
compound or salt
in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or
water with a
flavoring or coloring agent. Where the composition is in the form of a tablet,
any
pharmaceutical carrier routinely used for preparing solid formulations may be
used.
Examples of such carriers include magnesium stearate, terra alba, talc,
gelatin, acacia,
stearic acid, starch, lactose and sucrose. Where the composition is in the
form of a capsule,
any routine encapsulation is suitable, for example using the aforementioned
carriers in a
hard gelatin capsule shell. Where the composition is in the form of a soft
gelatin shell
capsule any pharmaceutical carrier routinely used for preparing dispersions or
suspensions
may be considered, for example aqueous gums, celluloses, silicates or oils,
and are
incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a
compound
or salt in a sterile aqueous or non-aqueous carrier optionally containing a
parenterally
acceptable oil, for example polyethylene glycol, polyvinylpyrroIidone,
lecithin, arachis oil
or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension
or
emulsion that may be administered as a dry powder or in the form of an aerosol
using a
conventional propellant such as dichIorodifluoromethane or
trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or a
pharmaceutically acceptable salt thereof which is active when administered in
this way,
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with a binding and/or lubricating agent, for example polymeric giycols,
gelatins, cocoa-
butter or other low melting vegetable waxes or fats or their synthetic
analogs.
Typical dermal and transdermal formulations comprise a conventional aqueous or
non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in
the form of a
medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet,
capsule or
metered aerosol dose, so that the patient may administer a single dose.
No unacceptable toxological effects are expected when compounds of the present
invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by
the
following tests:
(I) Calcium Receptor Inhibitor Assay
Calcilytic activity was measured by determining the ICgO of the test compound
for
blocking increases of intracellular Ca2+ elicited by extracellular Ca2+ in HEK
293 4.0-7
cells stably expressing the human calcium receptor. HEK 293 4.0-7 cells were
constructed
as described by Rogers et al., J. Bone Miner. Res. 10 Suppl. I:S483, 1995
(hereby
incorporated by reference herein). Intracellular Ca2+ increases were elicited
by increasing
extracellular Ca2+ from 1 to 1.75 mM. Intracellular Ca2+ was measured using
fluo-3,
a fluorescent calcium indicator.
The procedure was as follows:
1. Cells were maintained in T-I50 flasks in selection media (DMEM supplemented
with 10% fetal bovine serum and 200 ug/mL hygromycin B), under 5% C02:95% air
at
37 °C and were grown up to 90% confluency.
2. The medium was decanted and the cell monolayer was washed twice with
phosphate-buffered saline (PBS) kept at 37 °C. After the second wash, 6
mL of 0.02%
EDTA in PBS was added and incubated for 4 minutes at 37 °C. Following
the incubation,
cells were dispersed by gentle agitation.
3. Cells from 2 or 3 flasks were pooled and pelleted (100 x g). The cellular
pellet
was resuspended in 10-IS mL of SPF-PCB+ and pelleted again by centrifugation.
This
washing was done twice.
Sulfate- and phosphate-free parathyroid cell buffer (SPF-PCB) contains 20 mM
Na-Hepes, pH 7.4, 126 mM NaCI, 5 mM KCI, and 1 mM MgCl2. SPF-PCB was made up
and stored at 4 °C. On the day of use, SPF-PCB was supplemented with 1
mglmL of
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D-glucose and 1 mM CaCl2 and then split into two fractions. To one fraction,
bovine
serum albumin (BSA; fraction V, ICN) was added at 5 mg/mL (SPF-PCB+). This
buffer
was used for washing, loading and maintaining the cells. The BSA-free fraction
was used
for diluting the cells in the cuvette for measurements of fluorescence.
4. The pellet was resuspended in 10 mL of SPF-PCB+ containing 2.2 uM fluo-3
(Molecular Probes) and incubated at room temperature for 35 minutes.
S. Following the incubation period, the cells were pelleted by centrifugation.
The
resulting pellet was washed with SPF-PCB+. After this washing, cells were
resuspended in
SPF-PCB+ at a density of 1-2 x 106 cells/mL.
6. For recording fluorescent signals, 300 uL of cell suspension were diluted
in 1.2
mL of SPF buffer containing I mM CaCl2 and 1 mg/mL of D-glucose. Measurements
of
fluorescence were performed at 37 °C with constant stirring using a
spectrofluorimeter.
Excitation and emission wavelengths were measured at 485 and 535 nm,
respectively. To
calibrate fluorescence signals, digitonin (5 mg/mL in ethanol) was added to
obtain Fmax
and the apparent Fmin was determined by adding Tris-EGTA (2.5 M Tris-Base, 0.3
M
EGTA). The concentration of intracellular calcium was calculated using the
following
equation:
Intracellular calcium = (F-Fmin~max) x Kd; where Kd = 400 nM.
7. To determine the potential calcilytic activity of test compounds, cells
were
incubated with test compound (or vehicle as a control) for 90 seconds before
increasing the
concentration of extracellular Ca2+ from 1 to 2mM. Calcilytic compounds were
detected
by their ability to block, in a concentration-dependent manner, increases in
the
concentration of intracellular Ca2+ elicited by extracellular Ca2+ .
In general, those compounds having lower IC50 values in the Calcium Receptor
Inhibitor Assay are more preferred compounds. Compounds having an IC50 greater
than
SO uM were considered to be inactive. Preferred compounds are those having an
ICgO of
lOuM or lower, more preferred compounds have an IC50 of 1 uM, and most
preferred
compounds have an ICgO of O.IuM or lower.
(II) Calcium Recet~tor Binding A~sav
HEK 293 4.0-7 cells stably transfected with the Human Parathyroid Calcium
Receptor("HuPCaR") were scaled up in T180 tissue culture flasks. Plasma
membrane is
obtained by polytron homogenization or glass Bouncing in buffer (SOmM Tris-HCl
pH 7.4,
1 mM EDTA, 3mM MgCl2) in the presence of a protease inhibitor cocktail
containing 1 uM
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Leupeptin, 0.04 uM Pepstatin, and 1 mM PMSF. Aliquoted membrane was snap
frozen and
stored at -80oC. 3H labeled compound was radiolabeled to a radiospecific
activity of
8lCi/mmole and was aliquoted and stored in liquid nitrogen for radiochemical
stability.
A typical reaction mixture contains 2 nM 3H compound ((R,R)-N-4'-Methoxy-t-3-
3'-methyl-1'-ethylphenyl-1-(1-naphthyl)ethylamine), 4-10 ug membrane in
homogenization
buffer containing 0.1 % gelatin and 10% EtOH in a reaction volume of 0.5 mL.
Incubation
is performed in 12 x 75 polyethylene tubes in an ice water bath. To each tube
25 uL of test
sample in 100% EtOH is added, followed by 400 uL of cold incubation buffer,
and 25 uL of
20 nM 3H-compound in 100% EtOH. The binding reaction is initiated by the
addition of
50 uL of 80-200 ug/mL HEK 293 4.0-7 membrane diluted in incubation buffer, and
allowed
to incubate at 4oC for 30 min. Wash buffer is 50 mM Tris-HCl containing 0.1 %
PEI.
Nonspecific binding is determined by the addition of 100-fold excess of
unlabeled
homologous ligand, and is generally 30% of total binding. The binding reaction
is
terminated by rapid filtration onto 1 % PEI pretreated GF/C filters using a
Brandel
Harvestor. Filters are placed in scintillation fluid and radioactivity
assessed by liquid
scintillation counting.
The following examples are illustrative, but not limiting of the embodiments
of the
present invention.
EXAMPLE 1
2-lDecvlcarbamoyl -1-methylpyridinium iodide
a) N-Dec~picolinamid~
A mixture of picolic acid ( 1.0 g, 8.1 mmole), decylamine ( 1.28 g, 8.1
mmole),
HOBT.H20 ( 1.31 g, 9.7 mmole), diisopropylethylamine (2.10 g, 16.2 mmole), and
EDC
(2.89 g, 9.7 mmole) in dried acetonitrile ( 15 mL) was stirred at room
temperature for 14h.
The mixture was concentrated, taken up in H20, extracted with ethyl acetate
(3x). The
combined organic extracts were washed with saturated NaHC03, brine, dried over
MgS04,
filtered, and concentrated to afford an light brown oil 2.00 g, 94%. 1H-NMR
(250 MHz,
CDCl3) d 0.88 (t, J = 4.3 Hz, 3H); 1.40 (m, 14H); 1.65 (m, 2H); 3.45 (q, J =
4.3 Hz, 2H);
7.40 (ddd, J = 1.1, 4.8, 9.6 Hz, 1H); 7.85 (ddd, J = I.1, 4.8, 9.6 Hz, 1H);
8,10 (s, 1H); .8 Hz,
I H); 8.54 (dd, J = 0.95 Hz, 2.91 Hz, 1 H).
b) 2-(N-Decvlcarboxamidyl)-1-methvlpyridinium iodide
A mixture of N-decylpicolinamide (0.41 g, 1.5 mmole) and methyl iodide (1.02
g,
7.8 mmole) in anhydrous p-dioxane (5 mL) was refluxed for 12h. The mixture was
cooled
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and concentrated to dryness. The residue was triturated with acetone/ether to
afford white
solid (0.11 g, 17%). 1 H-NMR (250 MHz, DMSO-d6): d 0.88 (t, J = 4.3 Hz, 3H);
1.45 (m,
14H); 1.65 (m, 2H); 3.40 (q, J = 4.3 Hz, 2H); 4.30 (s, 3H); 8.20 (d, J = 4.8
Hz, 1 H); 8.25 (t,
J = 4.8 Hz, 8.70 (t, J = 4.8 Hz, 1 H); 9.10 (d, J = 4.8 Hz, 1 H); 9.25 (s, 1
H). IR ( KBR, cm- I ):
3430, 3209, 3050, 2900, 1673, 1623, 1552, 1461, 1455, 1316, 1286. Anal. Calcd
for
C 17H29IN20Ø50 H20: C, 49.40; H, 7.32; N, 6.78); Found: C, 49.51; H, 7.11;
N, 6.85.
EXAMPLE 2
3-Carbamoyl-1-f(dodecylcarbamoyl)methyllpyridinium. chloride
a) N-Dodecyl-2-chloroacetamide
To a stirred, cooled mixture of n-decylamine ( 13.93 g, 88.5426 mmole), and
triethylamine (8.96 g, 88.5426 mmole) in dried THF (100mL) was added
chloroacetyl
chloride ( 10.0 g, 88.5426 mmole) dropwise. After stirring at O~C in 1 h, the
mixture was
quenched by H20, and extracted with EtOAc (3x). The organic extracts were
washed with
brine, dried over MgS04, filtered, and concentrated to give a light brown
solid which was
triturated in pentane to afford an off white solid (15.52 g, 75%). 1H-NMR (250
MHz,
CDCl3) d : 6.50 (s, 1H), 3.89 (s, 2H), 3.30 (q, J = 6.6 Hz, 2H), 1.56 (m, 2H),
1.30 (m, 18H),
0.88 (t, J = 6.6 Hz, 3H).
b) 3-Carbamoyl-1-f(dodecylcarbamoyllmethvllp~idinium, chloride
A mixture of nicotinamide (0.77 g, 6.3 mmole), and N-dodecyl-2-chloroacetamide
(2.9 g, 12.6 mmole) in nitromethane ( l5 mL) was refluxed for 24h. The mixture
was
cooled, and the solid thus obtained was filtered and triturated in
acetone/EtOAc to give a
light yellow solid (0.83 g, 37%). MS (ES) m/e 348.2 (M+) 1H-NMR (400 MHz, DMSO-
D-
6) d: 9.40 (s, IH), 9.03 (d, J =7.lHz, 1H), 8.98 (d, J =7.3Hz, 1H), 8.60(bs,
2H, NH2), 8.22
(dd, J=7. 3, 7.1 Hz, 1H), 8.1 (bs, 1H, NH), 5.2 (s, 2H), 3.05- 3.11 (m, 2H),
1.39-1.48 (m,
2H),2.16-2.25 (m, 18H), 0.82 (t, J=6.5 Hz, 3H).
Formulations for pharmaceutical use incorporating compounds of the present
invention can be prepared in various forms and with numerous excipients.
Examples of
such formulations are given below.
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CA 02285556 1999-10-04
WO 98/44925 PCT/US98/06618
EXAMPLE 3
Inhalant Formulation _
A compound of Formula (I) ( 1 mg to 100 mg) is aerosolized from a metered dose
inhaier to deliver the desired amount of drug per use.
EXAMPLE 4
Tablet Formulation
Tablets/Ineredients Per Tablet
1. Active ingredient 40 mg
(Cpd of Form. (I))
2. Corn Starch 20 mg
3. Alginic acid 20 mg
4. Sodium Alginate 20 mg
5. Mg stearate 1.3 mg
Procedure for tablet formulation~
Ingredients l, 2, 3 and 4 are blended in a suitable mixer/blender. Sufficient
water is
added portion-wise to the blend with careful mixing after each addition until
the mass is of
a consistency to permit its conversion to wet granules. The wet mass is
converted to
granules by passing it through an oscillating granulator using a No. 8 mesh
{2.3$ mm)
screen. The wet granules are then dried in an oven at 140°F
(60°C) until dry. The dry
granules are lubricated with ingredient No. 5, and the lubricated granules are
compressed on
a suitable tablet press.
EXAMPLE 5
Parenteral Formulation
A pharmaceutical composition for parenteral administration is prepared by
dissolving an appropriate amount of a compound of Formula (I) in polyethylene
glycol with
heating. This solution is then diluted with water for injections (to 100 ml).
The solution is
then rendered sterile by filtration through a 0.22 micron membrane filter and
sealed in
sterile containers.
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