Note: Descriptions are shown in the official language in which they were submitted.
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WO 97/44035 PCT/US97/0922S
Title
METHODS OF TREATING HYPERTENSION
Priority Claim
This application claims the benefit of United States
Provisional Patent application 60/018266, filed May 24, 1996.
B~(~k~round of the Invention
Tachykinins are a family of peptides which share a common
amidated carboxy termin~l sequence. Substance P was the first peptide of
this family to be isolated, although its pllrific~tion and the det~rmin~tion
of its primary sequence did not occur until the early 1970's.
Between 1983 and 1984 several groups reported the isol~tion
of two novel m~mm~ n tachykinins, now termed neurokinin A (also
known as substance K, neuromedin L, and neurokinin a), and neurokinin
B (also known as neuromedin K and neurokinin ~). See, J.E. Maggio,
Peptides, 6 (Supplement 3):237-243 (1985) for a review of these
discoveries.
Tachykinins are widely distributed in both the central and
peripheral nervous systems, are released from nerves, and exert a variety
of biological actions, which, in most cases, depend upon activation of
specific receptors expressed on the membrane of target cells. Tachykinins
are also produced by a number of non-neural tissues.
The m~mm~ n tachykinins substance P, neurokinin A, and
neurokinin B act through three major receptor subtypes, denoted as
NK-1, NK-2, and NK-3, respectively. These receptors are present in a
variety of organs.
Substance P is believed inter alia to be involved in the
neurotr~n.~mi.~.~ion of pain sensations, including the pain associated with
migraine headaches and with arthritis. These peptides have also been
implicated in gastrointestinal disorders and diseases of the
gastrointestinal tract such as in~qmm~tory bowel disease. Tachykinins
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WO 97/44035 PCT/US97/09225
have also been implicated as playing a role in numerous other maladies,
as discussed infra.
Tachykinins play a major role in mediating the s~n.~ion
and tr~nsmi.s.~ion of pain or nociception, especially migraine headaches.
see~ e.r., S.L. Shepheard, et al., British Journal of PharmacoloFy. 108
20 (1993); S.M. Moussaoui, et al., European Journal of Ph~rm?,colo~y,
238:421-424 (1993); and W.S. Lee, et al., British Journal of Pharmacolo~y,
112:920-924 (1994).
In view of the wide number of ~linic~l m~l~(lies associated
0 with an excess of tachykinins, the development of tachykinin receptor
antagonists will serve to control these ~linic:~l conditions. The earliest
tachykinin receptor antagonists were peptide derivatives. These
antagonists proved to be of limited ph :~rm$l~eutical utility because of their
metabolic instability.
Recent public~tionq have described novel classes of non-
peptidyl tachykinin receptor antagonists which generally have greater
oral bioav~ hility and metabolic stability than the earlier classes of
tachykinin receptor antagonists. Examples of such newer non-peptidyl
tachykinin receptor antagonists are found in United States Patent
5,491,140, issued February 13, 1996; United States Patent 5,328,927,
issued July 12, 1994; United States Patent 5,360,820, issued November 1,
1994; United States Patent 5,344,830, issued September 6, 1994; United
States Patent 5,331,089, issued July 19, 1994; European Patent
Pllhlic~tion 591,040 A1, published April 6, 1994; Patent Cooperation
Treaty publication WO 94/01402, published January 20, 1994; Patent
Cooperation Treaty publication WO 94/04494, published March 3, 1994;
Patent Cooperation Treaty publication WO 93/011609, published January
21, 1993; Canadian Patent Application 2154116, published January 23,
1996; European Patent Publication 693,489, published January 24, 1996;
and Canadian Patent Application 2151116, published December 11, 1995.
Patent Cooperation Treaty Patent Publication WO 96/11000,
published Ap~il 18, 1996 and European Patent Publication EP 705,600,
published April 10, 1996, describe a synergistic effect on the combination
of a serotonin agonist and a tachykinin receptor antagonist in treating
migraine. United States Patent Application 08/387,056, filed February
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W097/44035 PCT~S97/09225
- 3 -
10, 1995, describes a synergistic effect on the combination of a serotonin
agonist and a tachykinin receptor antagonist in treating a variety of
psychiatric disorders. United States Patent Application 08/408,238, filed
~ March 22, 1995, describes a synergistic effect on the comhin~tion of a
serotonin agonist and a tachykinin receptor antagonist in treating a
variety of types of pain and nociception. United States Patent Application
60/000074, filed June 8, 1995, describes a synergistic effect on the
comhin?.tion of a serotonin agonist and a tachykinin receptor antagonist
in treating the common cold or allergic rhinitis.
0 European Patent Application 0 577 394, published January
5,1994, teaches a series of morpholinyl and thiomorpholinyl tachykinin
receptor antagonists. Patent Cooperation Treaty Patent Application WO
95/18124, published July 6,1995, teaches another series of substituted
morpholines for use as tachykinin receptor antagonists. None of these
references, nor any comhin~tion of them, teach the tachyl~inin receptor
antagonists of the present invention.
Pulmonary hypertension represents a serious, life
threatening spectrum of diseases of multiple etiology. These include
congenital abnormalities of the lung, thorax and diaphragm, congenital or
2 o acquired valvular or myocardial disease, obstructive lung disease, and can
be a complication of autoimmune diseases, vasculitis and collagen based
diseases (Rubin, Chest. 104: 236,1993). Patients with pulmonary
hypertension frequently present with symptoms including dyspnea,
fatigue, syncope, and chest pain, and have increased pulmonary artery
2 5 pressure and demonstrate prominence of the main pulmonary artery,
hilar vessel enlargement and decreased peripheral vessels on chest
radiographs ~ich, Ann. Internal. Med.. 107: 216,1987).
While pulmonary hypertension has multiple etiologies,
primary pulmonary hyperten~ion appears to involve an autoimmune
3 0 component and has been reported as a complication in patients with
mixed connective tissue disease, rheumatoid arthritis, Sjogren's
syndrome, systemic sclerosis and lupus (Sato, Hum. Path., 24: 199,1993).
Primary pulmonary hypertension occurs in females 1.7 times more
frequently than males with the greatest pre(lomin~nce between the third
3 5 and fourth decades of life (Rich, Ann. Internal. Med., 107: 216,1987). The
. , ,, .. .. ,_
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W097/44035 PCTrUS97/09225
increased incidence of primary pulmonary hypertension in women of child
bearing age as well as the ~linic~l observations that the disease can be
exacerbated by pregnancy and oral contraceptives ~ller, Ann. Rheum.
~.46: 159,1987; and cited in Farhat et al., J PET., 261: 686,1992)
5 suggests a role for estrogen in the disease process. To this extent, Farhat
et al. have demonstrated that estradiol potentiates the vasopressor
response to a thromboxane mimetic in perfused rat lungs (J PET, 261:
686,1992). However, the role of estrogen in pulmonary hypertension is
complex and may be dependent on the etiology of the disease process. In
0 a rat model of pulmonary hypertension induced by injection of
monocrotaline pyrrole (Reindel, Tox. Ap~l. Pharm., 106: 179,1990)
progressive pulmonary hypertension, right ventricular hypertrophy and
interstitial edema around the large airways and blood vessels becomes
apparent, simil~r to the pathology observed in man. Estradiol treatment
15 decreased right ventricular hypertrophy and prevented interstitial edema
in this model (Farhat ~1., Br. J. Pharm., 110: 719,1993) as well as
attenuating the hypoxic vasoconstrictive response in isolated sheep lungs
(Gordon et al., J. Appl. Physiol., 61: 2116,1986).
Current therapy for pulmonary hypertension is inadequate
2 0 and is largely dependent on the use of vasodilators, diuretics, and
anticoagulants ~ubin, DruFs, 43: 37,1992; Palevsky, JAMA. 265:1014,
1991). Vasodilators are effective in only a small subpopulation of patients
with primary pulmonary hypertension and is complicated by systemic
hypotensive responses. Prostacyclin infusion and high dose calcium
2 5 channel blockers are also being used with limited efficacy. Heart-lung
and single lung transplantation have been used on patients which do not
respond to vasodilator therapy, however, due to surgical morbidity and
mortality, this approach is usually limited to those patients who continue
to deteriorate despite aggressive therapy at centers experienced in
3 o mZ~nz~gement of this disease. Patients frequently die of right heart failureand those individuals which have signs of right heart failure have a mean
survival of 6-12 months ~ubin, Dru~, 43: 37,1992).
Therefore, pulmonary hypertensive diseases are
characterized by inadequate therapies, necessity of organ transplantation
3 5 and poor prognosis, and a need exists for new therapies.
CA 022.7.7910 1998 - 1 1 - 23
W O 97/44035 PCTrUS97/09225
Summary of the Invention
This invention provides methods of inhibiting pulmonary
5 hypertensive disease in a m~mm~l which comprise a-lmini.~tering to a
m~mm?ll in need thereof an effective amount of a compound having
activity as a tachykinin receptor antagonist.
Detailed Description and Plerelled F,mhodiments
The terms and abbrevi~tion.~ used in the instant
preparations and examples have their normal me~ning.s unless otherwise
designated. For example "~C" refers to degrees Celsius; "N" refers to
normal or normality; "mmol" refers to millimole or millimoles; "g" refers to
5 gram or grams; "ml" means milliliter or milliliters; "L" means liter or
liters; "M" refers to molar or molarity; "MS" refers to mass spectrometry;
"IR" refers to infrared spectroscopy; and "NMR" refers to nuclear magnetic
resonance spectroscopy.
The term "inhibit" includes its generally accepted meaning
2 0 which includes prohibiting, preventing, restraining, and slowing, stopping
or reversing progression, severity or a resultant symptom. As such, the
present method includes both medical therapeutic and/or prophylactic
1mini.~tration, as appropriate.
Pulmonary hypertensive diseases include all conditions
2 5 characterized by an increase in the blood pressure within the blood
vessels supplying the lungs and can increase the complications associated
with pulmonary embolism, heart failure, valvular disease, chronic lung
diseases and autoimmunity.
- The methods of the present invention employ various
3 0 tachykinin receptors. In recent publications many different groups of
non-peptidyl tachykinin receptor antagonists have been described. The
present invention, however, is not limited to any one of the specific
compound or class of compound. The present invention encompasses any
compound effective as a tachykinin receptor antagonist.
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WO 97/44035 PCT/US97/09225
Patent Cooperation Treaty publication WO 94/01402,
published January 20, 1994, rles~ribes a series of compounds best typified
by the following compound.
~CF3
H3C~N~ ~NH
~N~J~H~ \CF3
O
European Patent Pllblic~tion 591,040 Al, published April 6,
1994 describes a series of compounds typified by the following compound:
CH
H3 C CH3 ~l C 1
Cl
where A iS a ph~rm~(~.eutically acceptable anion.
Patent Cooperation Treaty pllhlic~tion WO 94/04494,
published March 3, 1994, describes a series of compounds typified by the
15 following compound.
H3C~-- C ~
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WO 97/44035 rCT/US97/09225
Patent Cooperation Treaty publication WO 93/01169,
published January 21, 1993, describes a series of compounds typified by
the following compound.
--~CF3
H O'~
~ CF3
Another group of tachykinin receptor antagonists is
characterized by the compound of the formula:
1 o
~N~OCH 3
having the ~le.cign~tion (~)-CP 96345. These compounds and their
syntheses are described in E.J. Warawa, et al., Journal of Me~licinal
Chemistry. 18:357 (1975).
Yet another group of tachykinin receptor antagonists is
characterized by the compound of the formula:
CA 02255910 1998-11-23
WO 97/44035 PCT/US97/09225
NH ~
NJ~J
o~ OCH3
H ~=~
having the ~le.cign~tion RP 67580. These compounds and their syntheses
are described in C. Garret, et al., Proceedin~s of the National Academy of
Sciences (IJSA)~ 88: 10208- 10211 (1991) and the references cited therein.
Patent Cooperation Treaty publication WO 94/07843
describes a series of cyclohexylamine derivatives typified by the following
compound
~.~'\~~ ~ CH~
which are useful as tachykinin receptor antagonists.
Another group of compounds useful as tachykinin receptor
antagonists is typified by the following compound.
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WO 97/44035 PCT/US97109225
CH3
~¢~ NN\>----~
H3 C¦ Br CH3
\~
The synthesis of these compounds is described in European Patent
Application Pllhlic~tion 694,535, published January 31, 1996.
The compound (S)-1-(2-methoxybenzyl)-2-[(4-phenyl-1-
piperazinyl)methyl]-4-(lH-indol-3-ylmethyl)-2-imidazoline and related
compounds are (le.s~rihed in European Patent Pllhlic~t.ion 699,665,
published March 6, 1996. This compound has the following structure.
OCH3
H
N~
The above groups of compounds are only illustrative of the
tachykinin receptor antagonists which are currently under development.
This listing of groups of compounds is not meant to be comprehensive, the
15 methods of the present invention may employ any tachykinin receptor
antagonist and is not limited to any particular class of compound.
A most preferred class of tachykinin receptor antagonists are
those compounds of the following structure
., ,, ........ , .. ~ .. ., .. , .. .. ,.. . . . ,. . ~
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WO 97/44035 PCT/US97/09225
- 10 -
H o=<~ CH3 R2
CH2 ~
~/Y
where R1 and R2 are independently selected from the group
5 consisting of hydrogen, methyl, methoxy, chloro, and trifluoromethyl, with
the proviso that no more than one of Rl and R2 can be hydrogen; and
Yis
N--O N--N~ N
CH--N~ CH--O , CH {~
N-Ra, or CH-NRbRC
where Ra, Rb, and Rc are independently selected
from the group consisting of hydrogen and C1-
C6 alkyl;
or a pharmaceutically acceptable salt or solvate thereo~ The syntheses of
these compounds are (lesrrihed in Patent Cooperation Treaty Pllhlic~tions
WO 95/14017, published May 26, 1995, and WO 96/01819, published
2 o January 25, 1996. The syntheses of two typical compounds from this class
are detailed in~
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WO 97/44035 PCT/US97/09225
Synthesis of (R)-2-[N-(2-((4-cyclohexyl)piperazin-1-yl)acetyl)amino]-3-(lH-
indol- 3 -yl) - 1- [N - (2 -methoxybenzyl) acetylamino]prop ane
/=\ OCH3
H O CH3
C~2
N
~ ~N ~O
(a) Preparation of (R)-3-(lH-indol-3-yl)-2-(N-
triphenylmethylamino)propanoic acid [N-trityltryptophan]
Tritylation
~OH ~ ~~
H H trityl
Chlorotrimethylsilane (70.0 ml, 0.527 mol) was added at a
moderate rate to a stirred slurry of D-tryptophan (100.0 g, 0.490 mol) in
15 anhydrous methylene chloride (800 ml) under a nitrogen atmosphere.
This mixture was continuously stirred for 4.Z5 hours. Triethylamine
- (147.0 ml, 1.055 mol) was added, followed by the arl(lition of a solution of
triphenylmethyl chloride (147.0 g, 0.552 mol) in methylene chloride (400
ml) using an adllition funnel. The mixture was stirred at room
2 0 temperature, under a nitrogen atmosphere for at least 20 hours. The
reaction was quenched l~y the addition of methanol (500 ml).
The solution was concentrated on a rotary evaporator to near
dryness and the mixture was redissolved in methylene chloride and ethyl
acetate. An aqueous work-up involving a 5% citric acid solution (2X) and
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WO 97/44035 PCT/US97/09225
brine (2X) was then performed. The organic layer was dried over
anhydrous sodium sulfate, f;ltered, and concentrated to dryness on a
rotary evaporator. The solid was dissolved in hot diethyl ether followed
by the addition of hexanes to promote crystallization. By this process
173.6 g (0.389 mol) of analytically pure (R)-3-(lH-indol-3-yl)-2-(N-
triphenylmethylamino)propanoic acid was isolated as a white solid in two
crops giving a total of 79% yield.
FDMS 446 (M+).
H NMR (DMSO-d6) ~ 2.70 (m, lH), 2.83 (m, 2H), 3.35 (m, lH), 6.92-7.20
0 (mj 12H), 7.30-7.41 (m, 8H), 10.83 (s, lH), 11.73 (br s, lH).
Analysis for C30H26N2o2:
Theory: C, 80.69; H, 5.87; N, 6.27.
Found: C, 80.47; H, 5.92; N, 6.10.
(b) Preparation of (R)-3-(lH-indol-3-yl)-N-(2-methoxybenzyV-
2-~-triphenylmethylamino)propanamide
Coupling
~ OH ~ ~ H ~C~
H trityl H trityl R
To a stirred solution of (R)-3-(lH-indol-3-yl)-2-(N-
triphenylmethylamino)propanoic acid (179.8 g, 0.403 mol), 2-
methoxybenzylamine (56.0 ml, 0.429 mol), and hydroxybenzot~ole
hydrate (67.97 g, 0.429 mol) in anhydrous tetrahydrofuran (1.7 L) and
anhydrous N,N-dimethylformamide (500 ml) under a nitrogen
atmosphere at 0~C, were added triethylamine (60.0 ml, 0.430 mol) and 1-
(3-dimethylaminopropyl)-3-ethoxycarbodiimide hydrochloride (82.25 g,
0.429 mol). The mixture was allowed to warm to room temperature under
30 a nitrogen atmosphere for at least 20 hours. The mixture was
concentrated on a rotary evaporator and then redissolved in methylene
chloride and an aqueous work-up of 5% citric acid solution (2X~, saturated
sodium bicarbonate solution (2X), and brine (2X) was performed. The
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WO 97/44035 PCT/US97/09225
- 13 -
organic layer was dried over anhydrous sodium sulfate and concentrated
to dryness on a rotary evaporator. The desired product was then
recrystallized from hot ethyl acetate to yield 215.8 g (0.381 mol, 95%) of
analytically pure material.
5 FDMS 565 ~+).
H NMR (CDCl3) ~ 2.19 (dd, J=6.4 Hz, ~o=14.4 Hz, lH), 2.64 (d, J=6.5 Hz,
lH), 3.19 (dd, J=4.3 Hz, ~=14.4 Hz, lH), 3.49 (m, lH), 3.63 (s, 3H), 3.99
(dd, J=5.4 Hz, ~o=14.2 Hz, lH), 4.25 (dd, J=7. 1 Hz, ~v=14.2 Hz, lH), 6.64
(d, J=2.1 Hz, lH), 6.80 (d, J=8.2 Hz, lH), 6.91 (t, J-7.4 Hz, lH), 7.06-7.38
(m, 21 H), 7.49 (d, J=7.9 Hz, lH), 7.75 (s, lH).
Analysis for C38H3sN3O2:
Theory: C, 80.68; H, 6.24; N, 7.43.
Found: C, 80.65; H, 6.46; N, 7.50.
(c) Preparation of (R)-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)amino] -2-(N-triphenylmethylamino)propane
Reduction of Carbonyl
~ N C~ Rl
2 0 H trityl R2 H trityl R2
RED-AL~ [a 3.4 M, solution of sodium bis(2-
methoxyethoxy)aluminum hydride in toluene] (535 ml, 1.819 mol),
dissolved in anhydrous tetrahy~ofuian (400 ml) was slowly added using
2 5 an addition funnel to a refluxing solution of the acylation product, (R)-3-
(~H-indol-3-yl)-N-(2-methoxybenzyl)-2-(N-
triphenylmethylamino)propanamide (228.6 g, 0.404 mols) produced supra,
in anhydrous tetrahydrofuran (1.0 L) under a nitrogen atmosphere. The
reaction mixture became a purple solution. The reaction was quenched
3 0 after at least 20 hours by the slow addition of excess saturated Rochelle's
salt solution (potassium sodium tartrate tetrahydrate). The organic layer
was isolated, washed with brine (2X), dried over anhydrous sodium
sulfate, filtered, and concentrated to an oil on a rotary evaporator. No
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WO 97/44035 PCT/US97/09225
- 14-
further pllrific~tion was done and the product was used directly in the
next step.
(d) Preparation of (l?v)-3-(lH-indol-3-yl)- 1-[N-(2-
5 methoxybenzyl)-acetylamino]-2-(N-t~iphenylmethylamino)propane
Acylation of Secondary Amine
N. NH 2 N. ¦ H3 C 2
H trityl R H R
trityl
To a stirring solution of (R)-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)amino]-2-(N-triphenylmethylamino)propane (0.404 moV
in anhydrous tetrahydrofuran (1.2 L) under a nitrogen atmosphere at 0~C
was added triethylamine (66.5 ml, 0.477 mol) and acetic anhydride (45.0
ml, 0.477 mol). After 4 hours, the mixture was concentrated on a rotary
evaporator, redissolved in methylene chloride and ethyl acetate, washed
with water (2X) and brine (2X), dried over anhydrous sodium sulfate,
filtered, and concentrated to a solid on a rotary evaporator. The resulting
solid was dissolved in chloroform and loaded onto silica gel 60 (230-400
mesh) and eluted with a 1:1 mixture of ethyl acetate and hexanes. The
product was then crystallized from an ethyl acetate/hexanes mixture. The
resulting product of (R)-3-(lH-indol-3-yl)- 1-[N-(2-
methoxybenzyl)acetylamino]-2-(N-triphenylmethylamino)propane was
crystallized and isolated over three crops giving 208.97 grams (87% yield)
of analytically pure m ate~
- Analysis for C4oH3sN3o2:
Theory: C, 80.91; H, 6.62; N, 7.08.
Found: C, 81.00; H, 6.69; N, 6.94.
(e) Preparation of (R)-2-amino-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)acetylamino~propane
Deprotection
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WO 97/44035 PCT/US97/09225
- 15-
~ ? ~ c
trityl
Formic acid (9.0 ml, 238.540 mmol) was added to a stirring
5 solution of (R)-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-
(N-triphenylmethylamino)propane (14.11 g, 23.763 mmol) in anhydrous
methylene chloride under a nitrogen atmosphere at 0~C. After 4 hours,
the reaction mixture was concentrated to an oil on a rotary evaporator
and redissolved in diethyl ether and 1.0 N hydrochloric acid. The aqueous
10 layer was washed twice with diethyl ether and basified with sodium
hydroxide to a pH greater than 12. The product was extracted out with
methylene chloride (4X). The organic extracts were combined, dried over
anhydrous sodium sulfate, filtered, and concentrated on a rotary
evaporator to a white foam. The compound (R)-2-amino-3-(lH-indol-3-yl)-
1-[N-(2-methoxybenzyl)acetylamino]propane (7.52 g, 21.397 mmols) was
isolated giving a 90% yield. No further p~ c~tion was necessary.
(f) Preparation of (R)-2-amino-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)acetylamino]propane dihydrochloride
trityl
~2 HCl
- A stirring solution of (R)-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)acetylamino]-2-(N-triphenylmethylamino)propane in two
25 volumes of methylene chloride was cooled to between -40~C and -50~C.
Anhydrous hydrogen chloride gas was added at such a rate that the
temperature of the reaction mixture did not exceed 0~C. The reaction
mixture was stirred for 30 minutes to one hour at 0-10~C.
To this reaction mixture was added two volumes of methyl t-
30 butyl ether and the resulting mixture was allowed to stir for 30 minutes
CA 022~910 1998-11-23
WO 97/44035 PCT/US97/09225
- 16 -
to one hour at 0- 10~C. The resulting crystalline solid was removed by
filtration and then washed with methyl t-butyl ether. The reaction
product was dried under vacuum at 50~C. (Yield >98%)
Analysis for C21H2sN3O2 ~ 2 HCl:
Theory: C, 59.44; H, 6.41; N, 9.90.
Found: C, 60.40; H, 6.60; N, 9.99.
(g) Preparation of 2-((4-cyclohexyl)piperazin-1-yl)acetic acid
potassium salt hydrate
Cyclohexylpiperazine (10.0 g, 0.059 mol) was added to ten
volumes of methylene chloride at room temperature. To this mixture was
added sodium hydroxide (36 ml of a 2N solution, 0.072 mol) and
tetrabutylammonium bromide (1.3 g, 0.004 mol). After the addition of the
sodium hydroxide and tetrabutylammonium bromide, methyl
bromoacetate (7.0 ml, 0.073 mol) was added and the reaction mixture was
stirred for four to six hours. The progress of the reaction was monitored
by gas chromatography.
The organic fraction was separated and the aqueous phase
was back-extracted with methylene chloride. The organic phases were
combined and washed twice with deionized water, once with saturated
sodium bicarbonate solution, and then with brine. The organic phase was
dried over magnesium sulfate and the solvents were removed in vacuo to
yield methyl 2-((4-cyclohexyl)piperazin-1-yl)acetate as a yellowish oil.
The title compound was prepared by dissolving the methyl 2-
((4-cyclohexyl)piperazin-1-yl)acetate (10.0 g, 0.042 mol) in ten volumes of
diethyl ether. This solution was cooled to 15~C and then potassium
trimethylsilanoate (5.9 g, 0.044) was added. This mixture was then
stirred for four to six hours. The reaction product was removed by
filtration, washed twice with five volumes of diethyl ether, then washed
twice with five volumes of hexanes, and then dried in a vacuum oven for
12-24 hours at 50~C.
Analysis for Cl2H2lKN202 ~ 1.5 H20:
Theory: C, 49.63; H, 7.98; N, 9.65.
Found: C, 49.54; H, 7.72; N, 9.11.
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WO 97/44035 PCT/US97/09225
(h) Preparation of (R)-2-[N-(2-((4-cyclohexyl)piperazin-1-
yl)acetyl)amino] -3-(lH-indol-3-yl)- 1 - [N-(2-
- methoxybenzyl)acetylamino]propane
- The title compound was prepared by Iirst cooling 2-((4-
cyclohexyl)piperazin-l-yl)acetic acid potassium salt to a temperature
between -8~C and -15~C in 5 volumes of anhydrous methylene chloride.
To this mixture was added isobutylchloroformate at a rate such that the
o temperature did not exceed -8~C. The resulting reaction mixture was
stirred for about 1 hour, the temperature being maintained between -8~C
and - 15~C.
To this mixture was then added (R)-2-amino-3-(lH-indol-3-
yl)-l-[N-(2-methoxybenzyl)acetylamino]propane dihydrochloride at such a
rate that the temperature did not exceed 0~C. Next added to this mixture
was N-methyl morpholine at a rate such that the temperature did not
exceed 0~C. This mixture was then stirred for about 1 hour at a
temperature between -15~C and -8~C.
The reaction was quenched by the addition of 5 volumes of
water. The organic layer was washed once with a saturated sodium
bicarbonate solution. The organic phase was then dried over anhydrous
potassium carbonate and filtered to remove the drying agent. To the
filtrate was then added 2 equivalents of concentrated hydrochloric acid,
followed by 1 volume of isopropyl alcohol. The methylene chloride was
then çx~h~nged with isopropyl alcohol under vacuum by dist~ on.
The final volume of isopropyl alcohol was then concentrated
to three volumes by vacuum. The reaction mixture was cooled to 20~C to
25~C and the product was allowed to crystallize for at least one hour. The
desired product was then recovered by filtration and washed with
sllffi~ient isopropyl alcohol to give a colorless filtrate. The crystal cake
was then dried under vacuum at 50~C. MS 560 (M+l+).
lH NMR (CDCl3) o 1.09-1.28 (m, 5H), 1.64 (d, J=10 Hz, lH), 1.80-1.89 (m,
4H), 2.10 (s, 3H), 2.24-2.52 (m, 9H), 2.90 (s, 2H), 2.95 (d, J=7 Hz, lH),
3.02 (d, J=7 Hz, lH), 3.12 (dd, J=5, 14 Hz, lH), 3.77 (s, 3H), 4.01 (dd,
J=10, 14 Hz, lH), 4.49 (ABq, J-17 Hz, 43 Hz, 2H), 4.56 (m, lH), 6.79-6.87
.. ...
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(m, 3H), 7.05-7.24 (m, 4H), 7.34-7.41 (m, 2H), 7.67 (d, J=8 Hz, lH), 8.22
(s, lH).
Analysis for C33H4~NsO3:
Theory: C, 70.81; H, 8.10; N, 12.51.
Found: C, 70.71; H, 8.21; N, 12.42.
... ..
Synthesis of (R)-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-
[N-(2-(4-(piperidin- l-yl)piperidin- l-yl)acetyl)amino]propane
/~\ OCH3
~''X~~
H O CH3
CH
N
\~ N~
(a) Preparation of 2-(4-(piperidin-1-yl)piperidin-1-yl)acetic
acid, potassium salt
4-(Piperidin-1-yl)piperidine (1.20 kg, 7.13 mol) was added to
methylene chloride (12.0 L) under a nitrogen atmosphere.
Tetrabutylammonium bromide (0.150 kg, 0.47 mol) and sodium hydroxide
(1.7 L of a 5 N solution, 8.5 mol) were then added. The reaction mixture
was cooled to 10-15~C and methyl bromoacetate (1.17 kg, 7.65 moV was
added and the resulting mixture was stirred for a minimum of 16 hours.
Deionized water (1.2 L) was then added to the mixture and
the layers separated. The aqueous layer was back-extracted with
methylene chloride (2.4 L). The organic fractions were combined and
2 5 washed with deionized water (3 x 1.2 L), a saturated sodium bicarbonate
solution (1.1 L) and a saturated sodium chloride solution (1.1 L). The
organic fraction was then dried over anhydrous magnesium sulfate and
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concentrated to an oil on a rotary evaporator to yield 1.613 kg (93.5%) of
methyl 2-(4-(piperidin-1-yl)piperidin-1-yl)acetate.
A solution of methyl 2-[4-(piperidin- 1-yl)piperidin- 1-
yl]acetate (2.395 kg, 9.96 mol) in methanol (2.4 L) was added to a solution
of potassium hydroxide (0.662 kg, 10.0 mol ~ 85% purity) in methanol
(10.5 L) under a nitrogen atmosphere. The reaction mixture was heated
to 45-50~C for a minimum of 16 hours.
A solvent exch~nge from methanol to acetone (15.0 L) was
performed on the solution on a rotary evaporator. This solution was
1 o slowly cooled to room temperature over 16 hours. The resulting solids
were filtered, rinsed with acetone (5.0 L) and then dried to yield 2.471 kg
(93.8%) of 2-(4-(piperidin-1-yl)piperidin-1-yl)acetic acid, potassium salt.
MS 265 (M+1)
(b) Preparation of (R)-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)acetylamino]-2-[N-(2-(4-(piperidin- l-yl)piperidin- 1-
yl)acetyl)amino]propane
The title compound was prepared by first a-lmixing (R)-2-
amino-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]propane
dihydrochloride (50.0 g, 0.118 mol) with 100 ml of methylene chloride
under a nitrogen atmosphere.
In a second flask, under a nitrogen atmosphere, 2-(4-
(piperidin-1-yl)piperidin-1-yl)acetic acid potassium salt (62.3 g, 0.236 mol)
was added to 600 ml of methylene chloride. This mixture was cooled to
about -10~C and stirring was continued. To this mixture
isobutylchloroformate (23 ml, 0.177 mol) was added dropwise such that
the temperature of the 2-(4-(piperidin-1-yl)piperidin-1-yl)acetic acid
potassium salt mixture never rose appreciably.
This reaction mixture was stirred at about - 10~C for about
1.5 hours at which time the (R)-2-amino-3-(lH-indol-3-yl)-1-[N-(2-
methoxybenzyl)acetylamino]propane dihydrochloride/methylene chloride
mixture prepared sul~ra was slowly added to the 2-(4-(piperidin-1-
yl)piperidin-1-yl)acetic acidpotassium
salt/isobutylchloroformate/methylene chloride solution. The resulting
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mixture was then stirred for about 1 hour at a temperature between -15~C
and -8~C.
The reaction mixture was removed from the ice bath and
allowed to warm to 15-20~C and the reaction was quenched by the
addition of 200 ml of water. The pH of the solution was adjusted to 2.3-
2.7 by the additon of lN sulfuric acid. The layers were separated and the
aqueous layer was washed with 100 ml of methylene chloride.
The organic fractions were combined and washed with water
(100 ml). The water wash was back extracted with methylene chloride (50
0 ml) and combined with the aqueous fraction from above. Methylene
chloride (500 ml) was added to the combined aqueous layers and the
mixture was stirred at room temperature for 15 minutes as basification
with 2N sodium hydroxide to a final pH of 9.8 to 10.2 was achieved.
The organic and aqueous fractions were separated. The aqueous
fraction was washed with methylene chloride and the methylene chloride
was added to the organic fraction. The organic fraction was then washed
with a mixture of saturated sodium bicarbonate solution (100 ml) and
water (50 ml). The bicarbonate wash was separated from the organic
fraction and back extracted with methylene chloride (50 ml). The back
2 o extraction was combined with the methylene chloride fraction and the
combined fractions were dried over magnesium sulfate. The m~gn~.~ium
sulfate was removed by filtration and the volatiles were removed by
vacuum distillation to yield the title product as a foam. (72.5 g, >g8%
yield). MS 559(M+1)
NMR (DMSO-d6 3:2 mixture of amide rotamers) ~ 1.25-1.70 (m, lOH),
1.77-2.00 (m, 2H), 1.95 (s, 3/5-3H), 2.04 (s, 2/5-3H), 2.10-2.97 (m, 9H),
3.10-3.65 (m, 3H), 3.72 (s, 2/5-3H), 3.74 (s, 3/5-3H), 4.26-4.68 (m, 3H),
6.76-7.12 (m, 6H), 7.13-7.35 (m, 2H), 7.42-7.66 (m, 2H), 10.80 (br s, lH).
Analysis for C33H4sNsO3:
Theory: C, 70.81; H, 8.10; N, 12.51.
Found: C, 70.57; H, 8.05; N, 12.39.
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Preparation of (R)-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-
2-[N-(2-(4-(piperidin- l-yl)piperidin- l-yl)acetyl)amino]propane
dihydrochloride trihydrate
oCH3
~ I
~N~ C=~3
+ > H o=~
N~--N
~ N~N- CH2 ~ CO2 K+ 2 HCl
3 H2O
Under a nitrogen atmosphere 2-(4-(piperidin-1-yl)piperidin-
l-yl)acetic acid, potassium salt (0.75 kg, 2.84 mol) was added to
methylene chloride (7.5 L). The resulting mixture was cooled to -15 to -
8~C and isobutyl chloroformate (0.29 kg, 2.12 mol) was added at such a
rate so as to maintain the temperature of the reaction mixture below -8~C.
After the addition the resulting reaction mixture was stirred for 90
minutes between -15 and -8~C.
The reaction mixture was then cooled to -35~C and solid (R)-
2-amino-3-(lH-indol-3-yl)-l-[N-(2-methoxybenzyVamino]propane
dihydrochloride (0.60 kg, 1.14 mol) was added at such a rate that the
reaction temperature was maintained at less than -20~C. After the
addition, the reaction mixture was stirred for about one hour with the
temperature being maintained between -37~C and -20~C. The reaction
2 0 was quenched by the addition of (i~icmi7ed water (7.5 L). The reaction
mixture was basified to pH 12.8-13.2 by the addition of 5 N sodium
hydroxide. The aqueous fraction was removed and retained. Additional
deionized water (3.75 L) was added to the organic fraction as was
sufficient 5 N sodium hydroxide to re-adjust the pH to 12.8-13.2.
2 5 The two aqueous fractions were combined, back-extracted
with methylene chloride (1.5 L) and then discarded. The organic fractions
were combined and washed with deionized water (4 x 3.5 L). These
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- 22 -
extracts were comhined, back-extracted with methylene chloride (1.5 L),
and then discarded. The two organic layers were combined and washed
with a saturated sodium chloride solution (3.7 L).
The organic fraction was dried over anhydrous magnesium
5 sulfate, filtered, and solvent exchanged from methylene chloride to
acetone (3.75 L) on a rotary evaporator. An aqueous solution of
hydrochloric acid (0.48 L of 6 N solution, 2.88 mol) and seed crystals (2 g)
were added and mixture was stirred for 30-90 minutes. Acetone (13.2 L)
was then added and the slurry stirred for one hour. The resulting solid
was then filtered, washed with acetone (2 x 1.4 L), and dried to yield 633
g (90%) of (R)-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-[N-
(2-(4-(piperidin- 1-yl)piperidin- 1 -yl)acetyl)amino]propane dihydrochloride
trihydrate.
The biological efficacy of a compound believed to be effective
as a tachykinin receptor antagonist may be confirmed by employing an
initial screening assay which rapidly and accurately measured the
binding of the tested compound to known NK- 1 and NK-2 receptor sites.
2 o Assays useful for evaluating tachykinin receptor antagonists are we~l
known in the art. See~ e.~., J. Jukic, et al., Life Sciences, 49:1463-1469
(1991); N. Kucharczyk, et al., Journal of Medicinal Chemistry.
36:1654-1661 (1993); N. Rouissi, et al., Biochemical and Biophysical
Research Communications. 176:894-901 (1991).
NK-1 Receptor Bindin~ Assay
Radioreceptor binding assays were performed using a
derivative of a previously published protocol. D.G. Payan, et al., Journal
o~ ImmunoloFy, 133:3260-3265 (1984). In this assay an aliquot of IM9
cells (1 x 106 cells/tube in RPMI 1604 medium supplemented with 10%
fetal calf serum) was incubated with 20 pM l2~I-labeled substance P in
the presence of increasing competitor concentrations for 45 minutes at
4~C.
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The IM9 cell line is a well-characterized cell line which is
readily available to the public. See. e.F.. Annals of the New York
Academy of Science. 190: 221-234 (1972); Nature (London)~ 251:443-444
(1974); Proceedin~s of the National Academy of Sciences (IJSA)~ 71:84-88
(1974). These cells were routinely cultured in RPMI 1640 supplemented
with 50 ,Lg/ml gent~mi( in sulfate and 10% fetal calf serum.
The reaction was terminated by filtration through a glass
fiber filter harvesting system using filters previously soaked for 20
minutes in 0.1% polyethyl~nimine. Specific binding of labeled substance
0 P was determined in the presence of 20 nM unlabeled ligand.
Many of the compounds employed in the methods of the
present invention are also effective antagonists of the NK-2 receptor.
NK-2 Receptor Bindin~ Assay
The CHO-hNK-2R cells, a CHO-derived cell line transformed
with the human NK-2 receptor, expressing about 400,000 such receptors
per cell, were grown in 75 cm2 flasks or roller bottles in minim~l essential
medium (alpha modi~ication) with 10% fetal bovine serum. The gene
sequence of the human NK-2 receptor is given in N.P. Gerard, et al.,
Journal of Biolo~ir.~l Chemistry. 265:20455-20462 (1990).
For preparation of membranes, 30 confluent roller bottle
cultures were dissociated by washing each roller bottle with 10 ml of
Dulbecco's phosphate buffered saline (PBS) without calcium and
magnesium, followed by addition of 10 ml of enzyme-free cell dissor.i~tion
solution (PBS-based, from Specialty Media, Inc.). After an additional 15
minutes, the dissociated cells were pooled and centrifuged at 1,000 RPM
for 10 minutes in a ~linir.~l centrifuge. Membranes were prepared by
homogeni7.~tion of the cell pellets in 300 ml 50 mM Tris buffer, pH 7.4
with a Tekmar~ homogenizer for 10-15 seconds, followed by
centrifugation at 12,000 RPM (20,000 x g) for 30 minutes using a
Beckm~n JA-14~ rotor. The pellets were washed once using the above
procedure. and the final pellets were resuspended in 100-120 ml 50 mM
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Tris buffer, pH 7.4, and 4 ml aliquots stored frozen at -70~C. The protein
concentration of this preparation was 2 mg/ml.
For the receptor binding assay, one 4-ml ali~uot of the
CH0-hNK-2R membrane preparation was suspended in 40 ml of assay
buffer cont~ining 50 mM Tris, pH 7.4, 3 mM manganese chloride, 0.02%
bovine serum albumin (BSA) and 4 llg/ml chymostatin. A 200 ~11 volume
of the homogenate (40 ,ug protein) was used per sample. The radioactive
ligand was 1125I]iodohistidyl-neurokinin A (New F',ngl~nd Nuclear,
NEX-252), 2200 Ci/mmol. The ligand was prepared in assay buffer at 20
0 nCi per lO0 ~,11; the final concentration in the assay was 20 pM.
Non-specific binding was determined using 1 ~IM eledoisin. Ten
concentrations of eledoisin from 0.1 to 1000 nM were used for a standard
concentration-response curve.
All samples and standards were added to the incubation in
10 ~LI dimethylslllfoxi-l~ (DMSO) for screening (single dose) or in 5 ~1
DMSO for ICso determin~tions. The order of additions for incubation was
190 or 195 ~1 assay buffer, 200 111 homogenate, 10 or 5 ~1 s~ le in
DMSO, 100 ~1 radioactive ligand. The samples were incubated 1 hr at
room temperature and then filtered on a cell harvester through filters
2 o which had been presoaked for two hours in 50 mM Tris buffer, pH 7.7,
containing 0.5% BSA. The filter was washed 3 times with approximately
3 ml of cold 50 mM Tris buffer, pH 7.7. The filter circles were then
punched into 12 x 75 mm polysly~ e tubes and counted in a gamma
counter.
Upon a showing that a compound has activity as a
tachykinin receptor antagonist, assays may be used to demonstrate the
ability of such a compound to control or treat hypertension. Typical such
assays are described below.
In Vivo Assays
Assay 1
The procedure as set out in Farhat, et al., J PET, 261: 686
(1992) (herein incorporated by reference) is carried out. Four to thirty
3 5 rats are sacrificed. The lungs are exsanguinated by perfusion via the
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hepatic pulmonary vein. The pulmonary artery is cannulated as is the
trachea to maintain ventilation and the pulmonary cannula is connected
to the perfusion line and the whole ventilated lung is removed and
suspended in a perfusion chamber. The effects of vasoconstrictor
5 substances on perfusion pressure of the isolated perfused lung is
- measured using a Statham pressure transducer. The increase in
perfusion pressure (vasoconstriction) induced by thromboxane mimetics in
the presence of estradiol is det~rmined and the ability to block the
thromboxane effects with a trst compound or the estradiol potentiation of
0 the thromboxane effects will be determined.
Activity of compounds having activity as tachykinin receptor
antagonists is illustrated by a reduction in pulmonary perfusion pressure
increase following thromboxane mimetic stim~ t.ion
15 Assay 2
Between five and fifty rats are ~rlmini.~tered a single IV dose
of monocrotaline pyrrole (3.5 mg/kg) and pulmonary disease is evaluated
by histopathology, accumulation of fluorescein conjugated dextran in
bronchial alveolar lavage fluid (as a measurement of pulmonary edema),
2 0 and measurements of pulmonary artery pressure using a Standtham
P23ID pressure transducer (:Reindel et al., Tox and Applid. Pharm, 106:
179-200 (1990), incorporated herein by reference. A test compound is
allmini~tered and the effect on the rats are evaluated.
Activity of compounds having activity as tachykinin receptor
2 5 antagonists is illustrated by a reduction in uptake of fluorescein
conjugated dextran from bronchial alveolar lavage fluids of ~nim~
treated with a compound of formula 1, indicating a reduction in
pulmonary edema. Rat lungs will also be removed from thorax, perfused
with modified Karnovskys fixitive and processed for histopathology. A
3 o reduction in t~irkening of the arterial walls in treated rats is evidence for
the protective role of compounds of formula 1 as is a decrease in
pulmonary artery pressure.
While it is possible to aflmini.~ter a compound employed in
3 5 the methods of this invention directly without any formulation, the
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compounds are usually ~llmini.qtered in the form of ph~rm~ceutical
compositions comprising a ph~rm~ceutically acceptable excipient and at
least one active ingredient. These compositions can be a-lmini.~tered by a
variety of routes including oral, rectal, trans(lerm~l, subcutaneous,
intravenous, intramuscular, and intranasal. Many of the compounds
employed in the methods of this invention are effective as both injectable
and oral compositions. Such compositions are prepared in a manner well
known in the pharmaceutical art and comprise at least one active
compound. See. e.~., REMINGTON'S PHARMACEUTICAL SCIENCES, (16th
0 ed. 1980).
In m~king the compositions employed in the present
invention the active ingredient is usually mixed with an excipient, diluted
by an excipient or enclosed within such a carrier which can be in the form
of a capsule, sachet, paper or other container. When the excipient serves
as a diluent, it can be a solid, semi-solid, or liquid material, which acts as
a vehicle, carrier or medium for the active ingredient. Thus, the
composition.~ can be in the form of tablets, pills, powders, lozenges,
sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,
aerosols (as a solid or in a liquid medium), ointments cont~inin~ for
2 o example up to 10% by weight of the active compound, soft and hard
gelatin capsules, suppositories, sterile injectable solutions, and sterile
packaged powders.
In preparing a fôrmulation, it may be necessary to mill the
active compound to provide the appropriate particle size prior to
2 5 combining with the other ingredients. If the active compound is
substantially insoluble, it or-lin~rily is milled to a particle size of less than
200 mesh. If the active compound is substantially water soluble, the
particle size is normally adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
3 0 Some examples of sllit~hle excipients include lactose,
dextrose, sucrose, sorbitol, m~nnitol, starches, gum ~c~ci~, calcium
phosphate, ~1gin~tes, tragacanth, gelatin, calcium silicate,
microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup,
and methyl cellulose. The formulations can additionally include:
3 5 lubricating agents such as talc, magnesium stearate, and mineral oil;
CA 022~910 1998-11-23
WO 97/44035 PCT/US97/09225
wetting agents; emulsifying and suspending agents; preserving agents
such as methyl- and propylhydroxybenzoates; sweetening agents; and
flavoring agents. The compositions of the invention can be formulated so
~ as to provide quick, sustained or delayed release of the active ingredient
5 after a(lmini.~tration to the patient by employing procedures known in the
art.
The compositions are preferably formulated in a unit dosage
form, each dosage cont~ining from about 0.05 to about 100 mg, more
usually about 1.0 to about 30 mg, of the active ingredient. The term "unit
10 dosage form" refers to physically discrete units suitable as unitary
dosages for human subjects and other m~mm~ , each unit cont~ining a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable ph~rm~qceutical
excipient.
The active compounds are generally effective over a wide
dosage range. For examples, dosages per day normally fall within the
range of about 0.01 to about 30 mg/kg of body weight. In the treatment of
adult humans, the range of about 0.1 to about 15 mg/kg/day, in single or
divided dose, is especially preferred. However, it will be understood that
2 0 the amount of the compound actually ~3(1mini.~tered will be determined by
a physician, in the light of the relevant circumstances, including the
condition to be treated, the chosen route of atlmini.~tration, the actual
compound or compounds a(lmini~tered, the age, weight, and response of
the individual patient, and the severity of the patient's symptoms, and
2 5 therefore the above dosage ranges are not intended to limit the scope of
the invention in any way. In some instances dosage levels below the
lower limit of the aforesaid range may be more than adequate, while in
other cases still larger doses may be employed without causing any
harmful side effect, provided that such larger doses are first divided into
3 0 several smaller doses for a(lmini~tration throughout the day.
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Formulation Preparation 1
Hard gelatin capsules cont~ining the following ingredients
are prepared:
Quantity
In~redient (m ~/capsule)
Active Ingredient(s) 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin
15 capsules in 340 mg quantities.
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Formulation Preparation 2
A tablet formula is prepared using the ingredients below:
Quantity
In~redient (m~/tablet)
Active Ingredient(s) 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
The components are blended and compressed to form tablets,
each weighing 240 mg.
Formulation Preparation 3
2 o A dry powder inhaler formulation is prepared cont~ining the
following components:
Tneredient W~ ht %
Active Ingredient(s) 5
Lactose 95
The active mixture is mixed with the lactose and the mixture
is added to a dry powder inh~ling appliance.
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Formulation Preparation 4
Tablets, each cont~ining 30 mg of active ingredient, are
prepared as follows:
-~~ Quantity
Tn~redient (m~/tablet)
Active Ingredient(s) 30.0 mg
o Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone
(as 10% solution in water) 4.0 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1.0 ~n F
Total 120 mg
2 5 The active ingredient, starch and cellulose are passed
through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of
polyvinylpyrrolidone is mixed with the resultant powders, which are then
passed through a 16 mesh U.S. sieve. The granules so produced are dried
at 50-60~C and passed through a 16 mesh U.S. sieve. The sodium
3 o carboxymethyl starch, magnesium stearate, and talc, previously passed
through a No. 30 mesh U.S. sieve, are then added to the granules which,
after mi~ing, are compressed on a tablet m~hine to yield tablets each
w~i ghin g 120 mg.
3 5 Formulation Preparation 5
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WO 97144035 PCTIUS97/09225
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Capsules, each cont~ining 40 mg of medicament are made as
follows:
Quantity
Tn~redient (m~/car~sule)
Active Ingredient(s) 40.0 mg
Starch 109.0 mg
Magnesium stearate 1.0 mF
Total 150.0 mg
The active ingredient, cellulose, starch, and magnesium
stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled
into hard gelatin capsules in 150 mg quantities.
Formulation Preparation 6
Suppositories, each cont~ining 25 mg of active ingredient are
made as follows:
TT~redient Amount
2 5 Active Ingredient(s) 25 mg
Saturated fatty acid glycerides to 2,000 mg
The active ingredient(s) is passed through a No. 60 mesh
3 0 U.S. sieve and suspended in the saturated fatty acid glycerides previously
melted using the minimum heat necess2ry. The mixture is then poured
into a suppository mold of nomin~l 2.0 g capacity and allowed to cool.
.
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Formulation Preparation 7
Suspensions, each cont~ining 50 mg of medicament per 5.0
5 ml dose are made as follows:
Tn~redient Amount
Active Ingredient(s) 50.0 mg
Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (11%)
Microcrystalline cellulose (89%) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v.
Purified water to 5.0 ml
The medicament, sucrose and xanthan gum are blended,
passed through a No. 10 mesh U.S. sieve, and then mixed with a
2 5 previously made solution of the microcrystal~ine cellulose and sodium
carboxymethyl cellulose in water. The sodium benzoate, flavor, and color
are diluted with some of the water and added with stirring. Sllffir.ient
water is then added to produce the required volume.
.
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Formulation Preparation 8
Capsules, each cont~ining 15 mg of medicament, are made as
5 follows:
Quantity
InFredient(mFlcapsule)
Active Ingredient(s)15.0 mg
Starch 407.0 mg
Magnesium stearate 3.0 mF
Total 425.0 mg
The active ingredient(s), cellulose, starch, and magnesium
stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled
into hard gelatin capsules in 425 mg quantities.
Formulation Preparation 9
An intravenous form~tion may be prepared as follows:
InFre~ nt Quantity
Active Ingredient(s) 250.0 mg
Isotonic saline 1000 ml
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Formulation PreparatioII 10
A topical formulation may be prepared as follows:
Tn Fredient Quantity
Active Ingredient(s) 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 g
The white soft paraffin is heated until molten. The liquid paraffin and
15 emulsifying wax are incorporated and stirred until dissolved. The active
ingredient is added and stirring is continued until dispersed. The
mixture is then cooled until solid.
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FormulationPre~aration 11
Sublingual or buccal tablets, each cont~ining lO mg of active
~ ingredient, may be prepared as follows:
Quantity
Tn~redient Per Tablet
Active Ingredient(s) 10.0 mg
0 Glycerol 2 10.5 mg
Water 143.0 mg
Sodium Citrate 4.5 mg
Polyvinyl Alcohol 26.5 mg
Polyvinylpyrrolidone 15.5 m F
Total 410.0 mg
The glycerol, water, sodium citrate, polyvinyl alcohol, and
polyvinylpyrrolidone are admixed together by continuous stirring and
maint~ining the temperature at about 9O~C. VVhen the polymers have
gone into solution, the solution is cooled to about 50-55~C and the
2 5 medicament is slowly admixed. The homogenous mixture is poured into
forms made of an inert material to produce a drug-cont~ining diffusion
matrix having a thickness of about 2-4 mm. This diffusion m~trix is then
cut to form individual tablets having the appropriate size.
3 0 Another preferred formulation employed in the methods of
the present invention employs transrlerm~l delivery devices ("patches").
Such trans~rm~l patches may be used to provide continuous or
discontinuous infusion of the compounds of the present invention in
controlled amounts. The construction and use of transd~rm~l patches for
3 5 the delivery of ph ~rm ~ceutical agents is well known in the art. See~ e.~.,
CA 022~910 1998-11-23
WO 97/44035 PCT/US97/09225
- 36 -
U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by
reference. Such patches may be constructed for continuous, pulsatile, or
on (lem7~nd delivery of ph~rm~reutical agents.
Frequently, it will be desirable or necessary to introduce the
5 ph~rm~ceutical composition to the brain, either directly or indirectly.
Direct techniques usually involve placement of a drug delivery catheter
into the host's ventricular system to bypass the blood-brain barrier. One
such implantable delivery system, used for the transport of biological
factors to specific anatomical regions of the body, is described in U.S.
Patent 5,011,472, issued April 30, 1991, which is herein incorporated by
reference.
Indirect techniques, which are generally plefelled, usually
involve form~ ng the compositions to provide for drug latentiation by
the conversion of hydrophilic drugs into lipid-soluble drugs or prodrugs.
15 Latentiation is generally achieved through blocking of the hydroxy,
carbonyl, sulfate, and p~imary amine groups present on the drug to
render the drug more lipid soluble and amenable to transportation across
the blood-brain barrier. Altematively, the delivery of hydrophilic drugs
may be enhanced by intra-arterial infusion of hypertonic solutions which
2 o can transiently open the blood-brain barrier.
The type of formulation employed for the a-lmini.~tration of
the compounds employed in the methods of the present invention may be
dictated by the particular compounds employed, the type of
2 5 pharmacokinetic profile desired from the route of a-lmini~tration and the
compound(s), and the state of the patient.
