Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITIONS CONTAINING HIV PROTEASE
INHIBITORS
Technical Field
A liquid pharmaceutical composition providing improved oral
bioavailability is disclosed for compounds which are inhibitors of HIV
protease
(in particular, HIV-1 and HIV-2 protease). In particular, the composition
comprises the HIV protease inhibitor and one or more pharmaceutically
acceptable organic solvents.
This application is a divisional application of Canadian Patent Application
No. 2,178,632, filed January 3, 1995.
Background of the Invention
One measure of the potential usefulness of an oral dosage form of a new
pharmaceutical agent is the bioavailability observed after oral administration
of
the dosage form. Various factors can affect the bioavailability of a drug when
administered orally. These factors include aqueous solubility, drug absorption
throughout the gastrointestinal tract, dosage strength and first pass effect.
Aqueous solubility is one of the most important of these factors. When a drug
has poor aqueous solubility, attempts are often made to identify salts or
other
derivatives of the drug which have improved aqueous solubility. When a salt
or other derivative of the drug is identified which has good aqueous
solubility,
it is generally accepted that an aqueous solution formulation of this alt or
derivative will provide the optimum oral bioavailability. The bioavailability
of
the aqueous oral solution formulation of a drug is then generally used as the
standard or ideal bioavailability against which other oral dosage forms are
measured.
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It has recently been determined that HIV protease inhibiting compounds
are useful for inhibiting HIV protease in vitro and inin vivo and are useful
for
inhibiting an HIV (human immunodeficiency virus) infection and are useful for
treating AIDS (acquired immunodefficiency syndrome). HIV protease inhibiting
compounds typically are characterized by having poor oral bioavailability.
Examples of HIV protease inhibiting compounds include N-(2(R)-
hydroxy-1 (S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-
pyridylmethyl)-2(S)-N'-
(t-butylcarboxamido)-piperazinyl))-pentaneamide and related compounds,
disclosed in European Patent Application No. EP541168, published May 12,
1993;
N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-
L-asparaginyl]aminoJbutyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide and
related compounds, disclosed in U.S. Patent No. 5,196,438, issued March 23,
1993;
[1 S-[1 R*(R*),2S*]}-N~ [3-[[[(1,1-dimethylethyl)amino]carbonyl](2-
methylpropyl)aminoJ-2-hydroxy-1-(phenylmethyl)propyl]-2-[(2-
quinolmylcarbonyl)amino]-butanediamide and related compounds, disclosed in
PCT Patent Application No. W092/08701, published May 29, 1992; and
O -.
HO N"N \ / OH
~ HO OH
and related compounds, disclosed in PCT Patent Application No. W093/07128,
published April 15, 1993.
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It has recently been determined that compounds of the formula I:
CH3
H3C
S ~ N CH3 R2 O H O
~~ N O ~
~NH~ NH~O
'I N H
O R~ SAN
R3
wherein R1 is lower alkyl and R2 and R3 are phenyl are inhibitors of HIV-1 and
HIV-2 protease and are useful for inhibiting HIV protease in vitro and in vivo
and are useful to inhibit HIV (human immunodeficiency virus) infections and,
thus, are useful for the treatment of AIDS (acquired immunodeficiency
syndrome).
In particular, the compound of formula II, has been found to be especially
effective as an inhibitor of HIV-1 and HIV-2 protease.
CH3
H3C
CH3
~N O
'/~ - O
S V V N H H /~'~
O~N
O CH3 Cf
The most preferred compound of formula II is (2S,3S,5S)-5-(N-(N-((N-
Methyl-N-((2-isopropyl-4-thiazolyl)methyl)-amino)carbonyl)valiny!)amino)-2-(N-
((5-thiazolyl)methoxycarbonyl)amino)-1,6-Biphenyl-3-hydroxyhexane
(compound III).
Compound III has an aqueous solubility of approximately 6 micrograms
per milliliter at pH >2. This is considered to be extremely poor aqueous
solubility and, therefore, compound III in the free base form would be
expected
to provide very low oral bioavailability. In fact, the free base form of
compound
III, administered as an unformulated solid in a capsule dosage form, is
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characterized by a bioavailability of less than 2% following a 5 mg/kg oral
dose
in dogs.
Acid addition salts of compound III (for example, bis-hydrochloride, bis-
tosylate, bis-methane sulfonate and the like) have aqueous solubilities of
<0.1
milligrams/milliliter. This is only a slight improvement over the solubility
of.the
free base. This low aqueous solubility would not make practical the
administration of therapeutic amounts of compound III as an aqueous solution.
Furthermore, in view of this low aqueous solubility, it is not surprising that
the
bis-tosylate of compound III, administered as an unformulated solid in a
capsule dosage form, is characterized by a bioavailability of less than 2%
following a 5 mg/kg oral dose in dogs.
In order to have a suitable oral dosage form of compound III, the oral
bioavailability of compound III should be at least 20%. Preferably, the oral
bioavailability of compound III from the dosage form should be greater than
about 40% and, more preferably, greater than about 50%.
While some drugs would be expected to have good solubility in organic
solvents, it would not necessarily follow that oral administration of such an
organic solution would give good bioavailability for the drug. It has been
found
that compound III has good solubility in .pharmaceutically acceptable organic
solvents. In addition, the solubility of compound III in some organic solvents
is
enhanced in the presence of a pharmaceutically acceptable acid.
Unexpectedly, solutions of compound III in pharmaceutically acceptable
organic solvents provide an oral bioavailability of from about 40% to about
100% in dogs.
Disclosure of the Invention
In accordance with the present invention, there is a pharmaceutical com-
position comprising a solution of an HIV protease inhibiting compound
(preferably, a compound of the formula II) in a pharmaceutically acceptable
organic solvent comprising a mixture of (1 ) (a) a solvent selected from
propylene glycol and polyethylene glycol or (b) a solvent selected from
polyoxyethyleneglycerol triricinoleate, polyethylene glycol 40 hydrogenated
castor oil, fractionated coconut oil, polyoxyethylene (20) sorbitan monooleate
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and 2-(2-ethoxyethoxy)ethanol or (c) a mixture thereof and (2) ethanol or
propylene glycol.
A preferred composition of the invention comprises a solution of an HIV
protease inhibiting compound (preferably, a compound of the formula II) in a
pharmaceutically acceptable organic solvent comprising a mixture of
(1 ) propylene glycol or polyoxyethyleneglycerol triricinoleate or a mixture
thereof and (2) ethanol.
The solution composition of the invention can also comprise other
pharmaceutically acceptable organic solvents.
The solution composition of the invention can also comprise from about
0% to about 20% (preferably, from about 0.2% to about 16% and more
preferably from about 0.2% to about 6%, by weight of the total solution) of a
pharmaceutically acceptable acid or a mixture of pharmaceutically acceptable
acids.
The solution composition of the invention can also comprise from about
0% to about 25% (preferably, from about 0% to about 19%, by weight of the
total solution) of water.
The solution composition of the invention can also comprise one or more
pharmaceutically acceptable oils.
The solution of the invention can also comprise pharmaceutically
acceptable sweetening agents and/or pharmaceutically acceptable flavoring
agents (for example, sucrose, aspartame, sorbitol; saccharin sodium and the
like and/or cherry flavor, artificial banana flavor; caramel, chocolate mint
flavor,
grape flavor, wild cherry flavor, raspberry flavor, strawberry flavor, citrus
flavor,
orange flavor, pineapple -flavor, citrus lime flavor, .citrus cream flavor,
cherry
vanilla flavor, creme de menthe flavor and-the like).
In addition, the solution composition of the invention can comprise one or
more pharmaceutically acceptable surfactants.
In addition, the solution composition of the invention can comprise
antioxidants (for example, ascorbic acid, BHA (butylated hydroxyanisole), BHT
(butylated hydroxytoluene), vitamin E, vitamin E PEG 1000 succinate and the
like) for chemical stability. -
The compositions of this invention provide improved oral bioavailability
for compound II when compared to non-formulated compound II (base) or non-
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formulated compound II (acid addition salt), or even when compared to a mixed
aqueous/organic solution (50% water, 20% ethanol, 30% propylene glycol) of
compound II (methansulfonate acid addition salt).
The term "pharmaceutically acceptable organic solvent" as used herein
refers to propylene glycol; polypropylene glycol;.polyethylene glycol (for
example, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol
600, polyethylene glycol 900, polyethylene glycol 540 (all available from
Union
Carbide) and the like); pharmaceutically acceptable alcohols (for example,
ethanol or 2-(2-ethoxyethoxy)ethanol (Transcutol~ , Gattefosse, Westwood, NJ
07675) and the like); polyoxyethylene castor oil derivatives (for example,
polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil
(Cremophor~EL,
BASF Corp.), polyoxyethyleneglycerol oxystearate (Cremophor~RH 40
(polyethyleneglycol 40 hydrogenated castor oil) or Cremophor~RH 60
(polyethyleneglycol 60 hydrogenated castor oil), BASF Corp.) and the like);
fractionated coconut oil (for example, mixed triglycerides with caprylic acid
and
capric acid (Miglyol~812, available from Huls AG, Witten, Germany) and the
like); Tween~ 80; isopropyl palmitate; isopropyl myristate; pharmaceutically
acceptable silicon fluids; and the like.
The term "pharmaceutically acceptable acid" as used herein refers to
(i) an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic
acid and the like, (ii) an organic mono-, di- or tri-carboxylic acid (for
example,
formic acid. acetic acid, adipic acid, alginic acid, citric acid, ascorbic
acid,
aspartic acid, benzoic acid, butyric acid, camphoric acid, gluconic acid,
glucuronic acid, galactaronic acid, glutamic acid, heptanoic acid; hexanoic
acid,
fumaric acid, lactic acid, lactobionic acid, malonic acid; malefic acid,
nicotinic
acid, oxalic acid, pamoic acid, pectinic acid;-3-phenylpropionic acid, picric
acid,
pivalic acid, propionic acid, succinic acid, tartaric acid, undecanoic acid
and the
like) or (iii) a sulfonic acid (for example, benzenesulfonic acid, sodium
bisulfate,
sulfuric acid, camphorsulfonic acid, dodecylsulfonic acid, ethanesulfonic
acid,
methanesulfonic acid, isethionic acid, naphthalenesulfonic acid, p-
toluenesulfonic acid and the like).
The term " pharmaceutically acceptable oil" as used herein refers to
mineral oil or a vegetable oil (for example, safflower oil, peanut oil, olive
oil,
fractionated coconut oil (for example, mixed triglycerides with caprylic acid
and
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capric acid (Miglyol~812, available from Huls AG, Witten, Germany) and the
like).
The term "pharmaceutically acceptable surfactant" as used herein refers
to a pharmaceutically acceptable non-ionic surfactant (for example,
polyethylenepolypropylene glycol; such as Poloxamer~68 (BASF Wyandotte
Corp.) or a mono fatty acid ester of polyoxyethylene (20) sorbitan (for
example,
polyoxyethylene (20) sorbitan monooleate (Tween~ 80), polyoxyethylene (20)
sorbitan monostearate (Tween~ 60), polyoxyethylene (20) sorbitan
monopalmitate (Tween~ 40), polyoxyethylene (20) sorbitan monolaurate
(Tween~ 20) and the like) and the like) or a pharmaceutically acceptable
anionic surfactant (for example, sodium lauryl sulfate and the like).
A preferred composition of the invention comprises an HIV protease
inhibiting compound (preferably, a compound of the formula II) in the amount
of
from about 1 % to about 15% by weight of the total solution (more preferably,
from about 2.5% to about 10% by weight of the total solution) in a
pharmaceutically acceptable organic solvent comprising a mixture of (1 ) (a) a
solvent selected from propylene glycol and polyethylene glycol in the amount
of
from about 10% to about 50% by weight of the total solution or (b) a solvent
selected from polyoxyethyleneglycerol tfiricinoleate, polyethylene glycol 40
hydrogenated castor oil, fractionated coconut oil, polyoxyethylene (20)
sorbitan
monooleate and 2-(2-ethoxyethoxy)ethanol in the amount of from about 5% to
about 35% by weight of the total solution or (c) a mixture thereof and (2)
ethanol
or propylene glycol in the amount of from about 5% to about 45% by weight of
the total solution (more preferably, from about 20% to about 35% by weight of
the total solution).
Preferably, in the composition='of the invention, the mixture of
pharmaceutically acceptable organic solvents (including ethanol) comprises
from about 50% to about 95% by weight of the total solution. More preferably,
the mixture of pharmaceutically acceptable organic solvents comprises from
about 70% to about 95% by weight of the total solution.
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
42% by weight of the total solution), ethanol (about 32% by weight of the
total
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solution) and water (from about 16% to about 17% by weight of the total
solution).
In the composition of the invention, a preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
32% by weight of the total solution), ethanol (about 37% by weight of the
total
solution) and water (about 15% by weight of the total solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (from
about 44% to about 45% by weight of the total solution),
polyoxyethyleneglycerol triricinoleate (about 20% by weight of the total
solution) and ethanol (from about 24% to about 25% by weight of the total
solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (from
about 39% to about 40% by weight of the total solution),
polyoxyethyleneglycerol triricinoleate (about 20% by weight of the total
solution) and ethanol (about 24% by weight of the total solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
43% by weight of the total solution), ethanol (about 33% by weight of the
total
solution) and water (about 17% by weight of the total solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
36% by weight of the total solution), ~ ethanol (about 36°i° by
weight of the total
solution) and water (about 19% by weight of the total solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
35% by weight of the total solution), ethanol (about 35% by weight of the
total
solution) and water (about 18% by weight of the total solution).
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In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
31 % by weight of the total solution), polyoxyethyleneglycerol triricinoleate
(about 10% by weight of the total solution), ethanol (about 32% by weight of
the
total solution)and water (about 14% by weight of the total solution).
In the composition of the invention, another preferred mixture of
pharmaceutically acceptable solvents is a mixture of propylene glycol (about
46% by weight of the total solution), ethanol (about 21 % by weight of the
total
solution) and polyoxyethyleneglycerol triricinoleate (about 24% by weight of
the total solution).
A preferred pharmaceutically acceptable acid is citric acid.
A most preferred composition of the invention comprises a solution of a
compound of the formula III in the amount of from about 1 % to about 15% by
weight of the total solution (more preferably, from about 2.5% to about 12% by
weight of the total solution) in a pharmaceutically acceptable organic solvent
comprising a mixture of (1 ) propylene glycol or polyoxyethyleneglycerol
triricinoleate or a mixture thereof and (2) ethanol.
In the most preferred composition of the invention, the preferred
pharmaceutically acceptable solvents and acids are as described above for the
preferred composition of the invention. .
An even more preferred composition :of the invention comprises a
solution of'a compound of the formula III inahe amount of from about 5% to
about 8% by weight of the total solution in a pharmaceutically acceptable
organic solvent comprising a mixture of (1 ) propylene glycol in the amount of
from about 34% to about 36% by weight of the total solution, (2) ethanol in
the
amount of from about 34% to about 36% by weight of the total solution and (3)
water in the amount of from about 18% to about 19% by weight of the total
solution. The even more preferred composition also comprises citric acid in
the
amount of from about 0.3% to about 0.4% by weight of the total solution.
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Another even more preferred composition of the invention comprises a
solution of a compound of the formula III in the amount of from about 7% to
about 8% by weight of the total solution in a pharmaceutically acceptable
organic solvent comprising a mixture of (1 ) propylene glycol in the amount of
from about 45% to about 46% by weight of the total solution, (2) ethanol in
the
amount of about 21 % by weight of the total solution and (3)
polyoxyethyleneglycerol triricinoleate in the amount of from about 24% to
about
25% by weight of the total solution. The even more preferred composition also
comprises citric acid in the amount of about 0.5% by weight of the total
solution.
A most highly preferred composition of the invention comprises a
solution of a compound of the formula III in the amount of from about 7% to
about 8% by weight of the total solution in a pharmaceutically acceptable
organic solvent comprising a mixture of (1 ) propylene glycol in the amount of
from about 31 % to about 32% by weight of the total solution, (2) ethanol in
the
amount of about 32% by weight of the total solution, (3)
polyoxyethyleneglycerol triricinoleate in the amount of from about 10% to
about
11 % by weight of the total solution and (4) water in the amount of from about
14% to about 15% by weight of the total solution. The even more preferred
composition also comprises citric acid in the amount of from about 0.2% to
about 0.3% by weight of the total solution.
The compounds of formula I and II contain two or more asymmetric
carbon atoms and thus can exist as pure diastereomers, mixtures of
diastereomers, diastereomeric racemates or mixtures of diastereomeric
racemates. The present invention is intended to include :within its scope all
of
the isomeric forms. The terms "R" and "S" configuration as used herein are as
defined by fUPAC 1974 Recommendations for Section E, Fundamental
Stereochemistry, Pure Appl. Chem. (1976) 45, 13-30.
The preferred isomer of the compound of formula fl is (2S,3S,5S)-5-(N-
(N-((N-Methyl-N-((2-isopropyl-4-thiazolyl)methyl)-amino)carbonyl)-
valinyl)amino)-2-(N-((5-thiazolyl)methoxycarbonyl)amino)-1,6-diphenyi-3-
hydroxyhexane (compound 11l).
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The term "lower alkyl" as used herein refers to straight or branched chain
alkyl radicals containing from 1 to 6 carbon atoms including, but not limited
to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl,, iso-butyl, sec-butyl, n-pentyl,
1-
methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl
and
the like.
In general, the compositions of this invention can be prepared in the
following manner. The ethanol and the pharmaceutically acceptable acid (if
present) and the water (if present) and sweetener (if present) are mixed. To
this
solution is added the propylene glycol or polyethylene glycol and/or
polyoxyethyleneglycerol triricinoleate, polyethylene glycol 40 hydrogenated
castor oil, fractionated coconut oil, polyoxyethylene (20) sorbitan monooleate
or
2-(2-ethoxyethoxy)ethanol until the resulting solution is clear. Compound III
(along with any other solvents, oils or other additives) are added to the
solution
and mixed until the solution is clear. The resulting solution is brought to
the
desired final volume with the addition of propylene glycol or polyethylene
glycol
and/or polyoxyethyleneglycerol triricinoleate, polyethylene glycol 40
hydrogenated castor oil, fractionated coconut oil, polyoxyethylene (20)
sorbitan
monooleate or 2-(2-ethoxyethoxy)ethanol.
Alternatively, the ethanol and pharmaceutically acceptable acid (if
present) are mixed, followed by addition of compound III. To this mixture is
added the propylene glycol or polyethylene glycol ~ and/or
polyoxyethyleneglycerol triricinoleate, polyethylene glycol 40 hydrogenated
castor oil, fractionated coconut oil, polyoxyethylene (20) sorbitan monooleate
or
2-(2-ethoxyethoxy)ethanol until the resulting solution is clear. Then the
water (if
present) and sweetener (if present), along with any other solvents, oils or
other
additives, are added with mixing until the solution is clear. The resulting
solution is brought to the desired final volume with the addition of propylene
glycol or polyethylene glycol and/or polyoxyethyleneglycerol triricinoleate,
polyethylene glycol 40 hydrogenated castor oil, fractionated coconut oil,
polyoxyethylene (20) sorbitan monooleate or 2-(2-ethoxyethoxy)ethanol.
The following examples will serve to further illustrate the invention. In
particular, Examples 5-19 relate specifically to this invention.
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Exam) I~e 1 ~n-formulated cal s~ ulel
An amount of compound III (free base) equivalent to a 5 mg/kg dose was .
placed in hard gelatin capsules (gray, size 0). These capsules were
administered to fasted dogs with 10 ml of water.
,
Fxam Ip a 2 ~(Ca sulel
An amount of compound III (free base) equivalent to a 5 mg/kg dose was
placed in hard gelatin capsules (gray, size 0). These capsules were
administered to non-fasted dogs with ten milliliter of water.
An amount of the bis-tosylate salt of compound III equivalent to a
mg/kg dose of compound III (base equivalent) was filled into hard gelatin
capsules (gray, size 0). These capsules were administered to nonfasted dogs
with ten milliliter of water.
Examg,le 4 lSolutionl
A 5 mg (free base equivalent)/ml solution of the base compound III in
20% ethanol: 30% propylene glycol: dextrose containing 2- molar equivalents
of methane sulfonic acid.
By Weight
Compound III (free base) 0.5
Propylene glycol (Aldrich, reagent)31.9
Ethanol (USP, 200 proof) 16.2
methanesulfonic acid (Aldrich reagent)0.14
Dextrose 3.6
Water for injection (USP) 47.6
A 50 mglml solution of the base compound 11l.
oml o~ nent % Bv We~qht
Compound III (free base) 5:1
Propylene glycol (USP) 42.22
Ethanol (USP, 200 proof, dehydrated)32.21 _
Citric acid (USP, anhydrous, powder)3.93
Water for injection (USP) 16.54
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Citric acid (77.0 g) was mixed with water (300 mL) and stirred until the
solution was clear. Water was added to bring the volume of solution to 400 mL.
Propylene glycol (300 g) and ethanol (800 g) were mixed in a stainless steel
beaker until the solution was clear. To the propylene giycol/ethanol solution
was added compound III (100.4 g) with stirring and mixing was continued until
the solution was clear. To this solution was added the citric acid solution
until
the final volume was 2000 mL.
If a flavoring agent is to be included, it would be added to the solution of
compound III prior to addition of the citric acid solution.
Exam lip a 6
A 25 mg/ml solution of the base compound III.
Component % By Weight
Compound III (free base) 2.6
Propylene glycol (USP) 43.4
Ethanol (USP, 200 proof, dehydrated)33.04
Citric acid (USP, anhydrous, powder)4.0
Water for injection (USP) 16.95
A 50 mg/ml solution of the base compound III.
Component % By Weight
Compound 111 (free base) 5.37
Propylene glycol (USP) 28.85'
Ethanol (USP, 200 proof, dehydrated)43.35
Citric acid (USP, anhydrous, powder)4.3
Water for injection (USP) 18.14
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Exam Ip a 8
A 50 mg/ml solution of the base compound III. ,
Com op nent % By We~ht
Compound III (free base) 5.3
Propylene glycol (USP) 31
Ethanol (USP, 200 proof, dehydrated)42.1
Citric acid (USP, anhydrous, powder)3.8
Water for injection (USP) 16.05
Aspartame 1.60
Wild cherry flavor 0.14
Examlhe 99
A 50 mg/ml solution of the base compound III.
Com on~ent % By Weiqht
Compound III (free base) 5.39
Cremophor~ EL (polyoxyl 35 castor oil, NF) 32.18
Ethanol (USP, 200 proof, dehydrated) 32.3
Miglyol~ 812 ~ 30.13
Exam' In a 10
A 50 mg/ml solution of the base compound III.
Comb o~ nent % B~~t
Compound III (free base) 5.01
Propylene glycol (USP) 44.6
Ethanol (USP, 200 proof; dehydrated)24:53
Citric acid (USP, anhydrous, powder)5.76
Cremophor~ EL (polyoxyl 35 castor 20.1
oil, NF)
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Examlhe 11
A 50 mgiml solution of the base compound III.
Com op vent J Bv Weight
Compound III (free base) 5.04
Propylene glycol (USP) 44.39
Ethanol (USP, 200 proof, dehydrated)24.42
Citric acid (USP, anhydrous, powder)5.82
Cremophor~ EL (polyoxyl 35 castor 20.33
oil, NF)
Citric acid (153.6 g) and ethanol (800 mL) were mixed until the solution
was clear. To this solution was added with mixing polyoxyl 35 castor oil (400
g)
and enough propylene glycol until the solution was clear. Compound III (100 g)
was added with mixing until the solution was clear. Propylene glycol was
added to bring the final volume of the solution to 2000 mL.
Alternatively, citric acid (153.6 g) and ethanol (800 mL) were mixed until
the solution was clear. Compound III (100 g) was added with mixing until the
solution was clear. To this solution was added with mixing polyoxyl 35 castor
oil (400 g) and enough propylene glycol until the solution was clear.
Propylene
glycol was added to bring the final volume of the solution to 2000 mL.
Examihe 1212
A 50 mg/ml solution of the base compound III.
om op nent % By Weight
Compound III (free base) 5.11
Propylene glycol (USP) 24.05
Ethanol (USP, 200 proof, dehydrated)20.29
Cremophor~ EL (polyoxyl 35 castor 35.59
oil, NF)
Miglyol~ 812 5.0
Tween~ 80 9.97
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~xamole 13
A 100 mg/ml solution of the base compound III.
Comp o~ nent % By We'~ht
Compound III (free base) 10.05
Propylene glycol (USP) 39.64
Ethanol (USP, 200 proof, dehydrated)24.3
Cremophor~ EL (polyoxyl 35 castor 20.23
oil, NF)
Citric acid (USP, anhydrous, powder)5.76
~xamp I~ a 14
A 100 mg/ml solution of the base compound III.
~om~ o? nent % By Weight
Compound III (free base) 4.99
Propylene glycol (USP) 43.52
Ethanol (USP, 200 proof, dehydrated)24.2
Cremophor~ EL (polyoxyl 35 castor 19.96
oil, NF)
Citric acid (USP; anhydrous, powder)5.76
Banana flavor 0.5
Strawberry flavor 0.59
Sodium saccharin 0.5
Examl I~ a 15
A 130 mg/ml solution of the base compound III.
Comb onent % Bv Weight
Compound III (free base) 12.0
Propylene glycol (USP) 32.4
Ethanol (USP, 200 proof; dehydrated) 37.0
Citric acid (USP, anhydrous, powder) 3.6
Water for injection (USP) 15.0
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exam Ip a 16
A 50 mg/ml solution of the base compound III.
By Weight
Compound III (free base) 5.125
Propylene glycol (USP) 35.8
Ethanol (USP, 200 proof, dehydrated) 35.75
Citric acid (USP, anhydrous, powder) 0.362
Water for injection (USP) 18.86
Sodium Saccharin (USP, powder, dehydrate)1.02
Wild cherry flavor, artificial 3.0
Flavor enhancer (Prosweet~ Liquid 0.08
"K")
(Prosweet~ Liquid "K" is available
from
Virginia Dare, 882 Third Ave., Brooklyn,
NY)
Dye, Red D&C No. 33 0.01
Examlhe 1717
An 80 mg/ml solution of the base compound III.
By Weight
Compound III (free base) 7.96
Propylene glycol (USP) 34.72
Ethanol (USP, 200 proof, dehydrated) 34.7
Citric acid (USP, anhydrous, powder) 0.35
Water for injection (USP) 18.3
Sodium Saccharin (USP, powder, dehydrate)0.99
Wild cherry flavor, artificial 2.9
Flavor enhancer (Prosweet~ Liquid 0.08
"K")
Dye, Red D&C No. 33 0.01
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Examlhe 18
An 80 mg/ml solution of the base compound III.
~om~ o~ nent % By Weight
Compound III (free base) 7.88
Propylene glycol (USP) 31.31
Ethanol (USP, 200 proof, dehydrated)32.0
Citric acid (USP, anhydrous, powder)0.27
Water for injection (USP) 14.18
Cremophor~ EL 10.4
Sodium Saccharin (USP, powder, dihydrate)0.98
Wild cherry flavor, artificial 2.875
Flavor enhancer (Prosweet~ Liquid 0.08
"K")
Dye, Red D&C No. 33 0.01
The water (14.4 mL) and ethanol (41.2 mL) were mixed and the citric
acid (276 mg) was added with stirring. The sodium saccharin (1.0 g), flavor
enhancer (0.1 mL), propylene glycol (30.7 mL), compound III (8.0 g), red dye
(10.0 mg), and cherry flavor (3.7 mL) were added sequentially with stirring.
The
resulting solution was brought to a final volume of 100 mL by addition of the
Cremophor~ EL. The final solution was stored at 2-8°C until it was
filled into 15
mL vials.
Examl In a 19
An 80 mg/ml solution of the base
compound III.
Comb onent % By Weight
Compound III (free base) 7.57
Propylene glycol (USP) 45.7
Ethanol (USP, 200 proof, dehydrated)21.0
Citric acid (USP, anhydrous, powder)0.51
Cremophor~ EL 24.29
Sodium Saccharin (USP, powder, dihydrate)0.47 _
Flavor enhancer (Prosweet~ Liquid 0.148
"K")
Flavor, chocolate mint 0.37
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The ethanol (28.4 mL) were mixed and the citric acid (545 mg) was
added with stirring. Enough of the propylene glycol was added with stirring to
make the resulting solution clear. The sodium saccharin (500 mg), flavor
enhancer (0.2 mL), compound III (8.0 g), Cremophor~ EL (24.5 mL), and
chocolate mint flavor (0.5 mL) were added sequentially with stirring. The
resulting solution was brought to a final volume of 100 mL by addition of
propylene glycol. The final solution was stored at 2-8°C until it was
filled into 15
mL vials.
The remaining examples provide the preparation of compound III.
Examlhe 2020
~2S.3S.5S)-~N-(~!N-Methyl-N-j!2-isoi r~opyl-4
~hiazo~yl)methyJ, amino bonyl)valinyl amino~(_N~(5
~iazolyl,~ en thoxycarbonyJ~amino -1.6-Biphenyl-3-hydroxyhexane.
A. N-l,(~Benzyl)oxylcarbonyl)-L-~ylalaninal.
A solution of 24.5 ml of anhydrous dimethyl sulfoxide in 870 ml of
anhydrous dichloromethane was cooled under N2 atmosphere to -60~C and
treated over a period of 15 min with 131 ml of a 2 M solution of oxalyl
chloride in
dichloromethane in order that the internal temperature remained below -50~C.
After addition, the solution was stirred at -60~C for 15 min and treated over
a
period of 20 min with a solution of 50 g (0.175 mol) of N-(((benzyl)oxy)-
carbonyl)-L-phenylalaninol in 200 ml of dichloromethane. The resulting
solution was stirred at -60~C for 1 h, then treated over a period of 15 min
with
97 ml of triethylamine in order: that the internal temperature remained below
-50~C. After addition the solution was stirred at -60~C for 15 min, then, with
the
cooling bath in place, was treated rapidly (over a period of 1 min) with a
solution of 163 g of citric acid in 550 ml of water. The resulting-slurry was
stirred
vigorously for 10 min, allowed to warm, diluted to 1 liter with water, and
separated. The organic layer was washed with 700 ml of water followed by a
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mixture of 550 ml of water and 150 ml of saturated aqueous NaHC03, dried
over MgS04, and concentrated in vacuo at 20~C to give the crude desired
compound as a light yellow solid.
B.(2S.3R.4R.5S)-2.5-Bis-(N-(((benzyl~o~r)carbony~amino -3.4-dihvdrox -y 1 6
diphen~exane and 12S.3S:4S.5S1-2.5-Bis-(Nz(l(benz rLlloxv )cr_, arbonvl)amino)
3.4-dihydroxy-1.6-diphenylhexane.
A suspension of 78.5 g of VC13~(tetrahydrofuran)3 and 16 g of zinc dust in
400 ml of dry dichloromethane was stirred under N2 atmosphere for 1 h at
25~C. A solution of 0.175 mol of N-(((benzyl)oxy)carbonyl)-L-phenylalaninal in
200 ml of dichloromethane was then added in one portion, and the resulting
mixture was stirred at ambient temperature under N2 atmosphere for 16 h. The
resulting mixture was added to 500 ml of 1 M aqueous HCI, diluted with 500 ml
of hot chloroform, and shaked vigorously for 2 min. The layers were separated,
and the organic layer was washed with 1 M aqueous HCI and separated.
Filtration of the organic phase provided the crude desired product as a solid
residue. The residue was slurried in 1.25 liters of acetone, treated with 5 ml
of
concentrated H2S04, and stirred for 16 h at ambient temperature. The resulting
mixture was filtered, and the residue (residue A) was washed with 50 ml of
acetone. The combined filtrate was concentrated to a volume of 250 ml, diluted
with 1000 ml of dichloromethane, washed three times with water and
once with saturated brine, dried over MgS04, and concentrated to give a
viscous oil. The oil was taken up in 1000 ml of 1 M HCI in methanol (prepared
from 71 ml of acetyl chloride and 1000 ml of methanol) and stirred at ambient
temperature for 2 h. The resulting precipitate was filtered; washed with
methanol, and air-dried on the filter to Rrovide 26.7 g of the desired
compound
as a white solid. The filtrate was concentrated and filtered to give a second
crop (8.3 g) of (2S,3R,4R,5S)-2,5-bis-(N-(((benzyl)oxy)carbonyl)amino)-3,4-
dihydroxy-1,6-diphenylhexane. 1 H NMR (ds-DMSO) 8 2.59 (dd, J = 13, 5 Hz, 2
H), 2.74 (dd, J = 13, 9 Hz, 2 H), 3.26 (br, 2 H), 4.19 (m, 2 H), 4.54 (m, 2
H), 4.92
(m, 4 H), 6.82 (d, J = 9 Hz, 2 H), 7.0-7.35 (m, 20 H). Mass spectrum: (M + H)+
569.
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Residue A (above, 2.65 g) was suspended in 75 ml of tetrahydrofuran
(THF) and 75 ml of 1 M aqueous HCI and heated at reflux for 24 h. After
concentration of the resulting solution in vacuo, the residue was taken up in
10% methanol in chloroform, washed two times with water, dried over Na2S04,
and concentrated in vacuo to provide (2S;3S,4S,5S)-2,5-bis-(N-
(((benzyl)oxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane as a white
solid. 1H NMR (ds-DMSO) 82.64 (m, 2 H), 3.04 (m; 2 H), 3.49 (m, 2 H), 3.78 (m,
2 H), 4.70 (d, J = 7 Hz, 2 H), 4.93 (AA', 4 H), 7.1-7.4 (m, 20 H). Mass
spectrum:
(M + H)+ = 569.
C. ,~2S.3R.4S.5Sy-3-Acetox~r-2.5-bis-IN ~,((benzyl oxv carbonyllaminoL3
bromo-1.6-diphenvlhexane.
A suspension of 25 g (44 mmol) of (2S,3R,4R,5S)-2,5-bis-(N-
(((benzyl)oxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane in 500 ml of
2:1 dichloromethane/hexane was treated with 23 g of
a-acetoxyisobutyryi bromide. The resulting mixture was stirred at ambient
temperature until the reaction clarified, washed with two 200 ml portions of
saturated aqueous NaHC03, dried over MgS04, and concentrated in vacuo to
give 30.8 g of the crude desired compound. A portion was purified by silica
gel
chromatography using 9:1 dichloromethane:ethyl acetate to provide the pure
desired compound as a white solid. 1H NMR (CDC13) 8 2.21 (s, 3 H), 2:62 (dd, J
= 13, 11 Hz, 1 H), 2.75 (d, J = 7 Hz, 2 H), 2.95 (br d; J = 15 Hz, 1 H), 4.03
(br t, J
= 10 Hz, 1 h), 4.40 (br d, J =10 Hz; 1 H), 4.6-5.0 (m; 6 H), 5.12 (br d, J =
13 Hz, 1
H), 5.33 (br d, J = 11 Hz, 1 H), 7.0-7.4 (m, 10 H). Mass spectrum: (M + NH4)+
_
690, 692.
D. (2S.3R.41~.5S)-2.5-Bis~(N-(,((benz~)oxylcarbonyl)~amino -3.4-a oxv-1.6
diphenylhexane.
A solution of 35.56 g (52.8 mmol) of (2S,3R,4S,5S)-3-acetoxy-2,5-bis-(N-
(((benzyl)oxy)carbonyl)amino)-3-bromo-1,6-diphenylhexane in 375 ml of
dioxane was treated with 255 ml of 1 N aqueous sodium hydroxide and stirred
at ambient temperature for 16 h, during which the desired compound
precipitated. The resulting mixture was filtered, and the residue was washed
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with water and dried to provide 22.23 g (76%) of the desired compound as a
white solid. ~ H NMR (CDC13) S 2.7-2.9 (m, 6 H), 3.9-4.0 (m, 2 H), 4.6-4.7 (m,
2
H), 5.03 (m, 4 H), 7.1-7.4 (m, 10 H).
E. (2S.3S.5S1-2.5-Bis- N-(,~(benzyl, oxy]carbon~)amino -1.6-di~ohenyl-3
hvdroxyhexane.
A mixture of 39.2 g (71.2 mmol) of (2S,3R,4R,5S)-2,5-bis-(N-
(((benzyl)oxy)carbonyl)amino)-3,4-epoxy-1,6-diphenylhexane in 600 ml of THF
was treated under N2 atmosphere with 13 g (0.36 mol) of sodium borohydride.
The resulting mixture was treated dropwise with 27.7 ml (0.36 mol) of
trifluoroacetic acid. After being stirred for 3.5 h at ambient temperature,
the
resulting mixture was quenched with 1 N aqueous HCI, diluted with water, and
stirred for 16 h. The resulting mixture was filtered. washed with water, and
dried
to provide 22.85 g (58%) of the desired compound as a white solid.
F. ~~2S.3S.5S)-2.5-Diamino-1.6-diphenvl-3-hydroxyhexane.
A suspension of 32 g of the crude resultant compound of Example 20E
and 55.5 g (176 mmol) of barium hydroxide octahydrate in 400 ml of 1,4-
dioxane and 400 ml of water was heated at reflux for 4 h. The resulting
mixture
was filtered, and the residue was rinsed with dioxane. The combined filtrates
were concentrated to a volume of approximately 200 ml and extracted with four
400 ml portions of chloroform. The combined organic layers were dried over
Na2S04, filtered, and concentrated in vacuo. The residue was purified by
silica
gel chromatography using first 2% isopropylamine in chloroform and then 2%
isopropylamine/2% methanol in chloroform to provide 10.1 g (81 %) of the pure
desired compound as a white solid. 1 H NMR (CDC13) 8 1.54 (dt, J = 14, 10 Hz,
1 H), 1.67 (dt, J = 14, 3 Hz, 1 H), 2.50 (dd, J = 13, 8 Hz, 1 H), 2.58 (dd, J
= 13, 8
Hz, 1 H), 2.8 (m, 2 H), 2.91 (dd, J = 13, 5 Hz, 1 H), 3.10 (m, 1 H), 3.72
(ddd, J =
11, 3, 2 Hz, 1 H), 7.1-7.4 (m, 10 H). Mass spectrum:. (M + H)+ = 285.
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G. ~4S 6S 1'S1~1-Amino-2-phenYethyll-4-benzyl-2-ohenvl-3-aza-2-bora-1
oxacyrlohexane.
A solution of 11.28 g (40 mmol) of (2S,3S,5S)-2,5-diamino-1,6-diphenyl-
3-hydroxyhexane and 4.88 g (40 mmol) of phenylboric acid in 1 liter of toluene
was heated at reflux and the water azeotropically removed with the aid of a
Dean Stark trap until the distillate was clear. The solvent was then removed
in
vacuo to provide the crude desired compound which was used immediately
without further purification.
H. Thioformamide.
To a cooled (0°C) 2 L three neck round bottom flask equipped with
an
overhead stirrer charged with a solution of formamide (30.5 mL, 0.76 mol) in 1
L
of diethyl ether was added 89 g (0.19 mol) of phosphorous pentasulfide in
small
portions. The reaction mixture was allowed to warm to ambient temperature,
stirred for 2 h, filtered, and concentrated in vacuo to afford thioformamide
as a
yellow offensive smelling oil which was used without purification.
I. Ethvl2-Chloro-2-formviacetate.
To a three neck 2 L round bottom flask charged with potassium t-
butoxide (0.5 mol, 500 mL of a 1 M solution in THF) and 500 mL of dry THF
cooled to 0°C was added dropwise from an addition funnel a solution of
ethyl
chloroacetate (0.5 mol, 53.5 mL) and ethyl formate ( 0.5 mol, 40.4 mL), in 200
mL of THF over 3 hours. After completion of addition, the reaction mixture was
stirred for 1 hour and allowed to stand overnight. The resulting solid was
diluted with diethyl ether and cooled in an ice bath: Then, the pH was lowered
to approximately 3 using 6N HCI:' The Qrganic phase was separated, and the
aqueous layer was washed 3 times with diethyl ether. The combined ethereal
portions were dried over NaS04, and concentrated in vacuo. The crude
desired compound was stored at -30°C and used without further
purification.
l Ethvl Thiazole-5-carboxylate.
To a round bottom flask was added 250 mL of dry acetone, 7.5 g (0.123
mol) of thioformamide, and 18.54 g (0.123 mol) of ethyl 2-chloro-.2-
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formylacetate. The reaction was heated at reflux for 2 hours. The solvent was
removed in vacuo, and the residue was purified by chromatography (Si02, 6 cm
o.d. column, 100% CHC13, Rf = 0.25) to provide 11.6 g (60%) of the desired
compound as a light yellow oil. NMR (CDC13) b 1.39 (t, J = 7 Hz, 3 H), 4.38
(q, J
= 7 Hz, 2 H), 8.50 (s, 1 H), 8.95 (s, 1 H).
K. 5-(H d~~rmeth_yl)thiazole.
To a precooled (ice bath) three neck 500 mL flask containing lithium
aluminum hydride (76 mmol) in 250 mL of THF was added ethyl thiazole-5-
carboxylate (11.82 g, 75.68 mmol) in 100 mL of THF dropwise over 1.5 hours to
avoid excess foaming. The reaction was stirred for an additional hour, and
treated cautiously with 2.9 mL of water, 2.9 mL of 15% NaOH, and 8.7 mL of
water. The solid salts were filtered, and the filtrate set aside. The crude
salts
were heated at reflux in 100 mL of ethyl acetate for 30 min. The resulting
mixture was filtered, and the two filtrates were combined, dried over Na2S04,
and concentrated in vacuo. The product was purified by silica gel
chromatography eluting sequentially with 0% - 2% - 4% methanol in chloroform,
to provide the desired compound, Rf = 0.3 (4% methanol in chloroform), which
solidified upon standing in 75% yield. NMR (CDC13) 8 4.92 (s, 2 H), 7.78 (s, 1
H), 8.77 (s, 1 H). Mass spectrum: (M + H)+ = 116.
~j5-Thiazolyllmetlyl)~4-nitrol he~nyl)carbonate.
A solution of 3.11 g :(27 mmol) ofi5-(hydroxymethyl)thiazole and excess
N-methyl morpholine in 100 ml of methylene chloride was cooled.to O~C and
treated with 8.2 g (41=mmol) of 4-nitrophenyl chloroformate. After being
stirred
for 1 h, the reaction mixture was diluted with CHC13, washed successively with
1 N HCI, saturated aqueous NaHCOg; and saturated brine, dried over NaS04,
and concentrated in vacuo. The residue was purified by silica gel
chromatography (Si02, 1-2% MeOH/CHC13, Rf=0.5 in 4% MeOH/CHC13) to
yield 5.9 g (78%) of the desired compound as a yellow solid. NMR (CDC13) 8
5.53 (s, 2 H), 7.39 (dt, J = 9, 3 Hz, 2 H), 8.01 (s, 1 H), 8.29 (dt, J = 9, 3
Hz, 2 H),
8.90 (s, 1 H). Mass spectrum: (M + H)+ = 281.
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M. (2S.3S.5S~-5-Amino-2-1N-(j5-thiazolyl)metho~carbonyl)amino)-16
~iphenyl-3-hydroxvhexane and (2S 3S.5S)-2-Amino-5=(N-j(5-thiazolvl)
methoxycarbonyllamino)-1.6-dphenyl-3-hrdroxvhexane.
A solution of 500 mg (1.76 mmol) of (2S,3S,5S)-2,5-diamino-1,6-
diphenyl-3-hydroxyhexane and 480 mg (1.71 mmol) of ((5-thiazolyl)methyl)-(4-
nitrophenyl)carbonate in 20 ml of THF was stirred at ambient temperature for 4
h. After removal of the solvent in vacuo, the residue was purified by silica
get
chromatography using first 2% then 5% methanol in chloroform to provide a
mixture of the two desired compounds. Silica gel chromatography of the
mixture using a gradient of 0 - 1 - 2% methanol in 93:2 isopropylamine:
chloroform provided 110 mg (16%) of (2S,3S,5S)-5-amino-2-(N-((5-thiazolyl)-
methoxycarbonyl)amino)-1,6-Biphenyl-3-hydroxyhexane (Rf 0.48, 96:2:2
chloroform:methanolasopropylamine) and 185 mg (28%) of (2S,3S,5S)-2-
amino-5-(N-((5-thiazolyl)methoxycarbonyl)amino)-1,6-Biphenyl-3-
hydroxyhexane (Rf 0.44, 96:2:2 chloroform:methanol:isopropylamine).
(2S,3S,5S)-5-Amino-2-(N-((5-thiazolyl)methoxycarbonyl)amino)-1,6-diphenyl-
3-hydroxyhexane: NMR (CDC13) b 1.3-1.6 (m, 2 H), 2.40 (dB, J = 14, 8 Hz, 1 H),
2.78 (dB, J = 5 Hz, 1 H), 2.88 (d, J = 7 Hz, 2 H), 3.01 (m, i H), 3.72 (br q,
1 H),
3.81 (br d, J = 10 Hz, 1 H), 5.28 (s, 2 H), 5.34 (br d, J = 9 Hz, 1 H), 7.07
(br d, J =
7 Hz, 2 H), 7.15 - 7.35 (m, 8 H), 7.87 (s, 1 H), 8.80 (s, 1 H). Mass spectrum:
(M
+ H)+ = 426.
(2S,3S,5S)-2-Amino-5-(N-((5-thiazolyl)methoxycarbonyl)amino)-1,6-diphenyl-
3-hydroxyhexane: NMR (CDCI3) 8 1.55 (Bt; J = 14, 8 Hz, 1 H), 1.74 (m, 1 H),
2.44 (dB, J = 15, 1 Hz, 1 H);~2.75 - 3:0 (m, 4 H),~3.44 (m, 1 H), 4.00 (br t,
1 H),
5.28 (m, 3 H), 7.1 - 7.4 (m; 1 O.H), 7.86 (s, 1 H), 8.80 (s, 1 H). Mass
spectrum: (M
+ H)+ = 426.
N. (2S.3S.5S)-5-Amino-2-lN-(~5-thiazo~l)metho~carbon I amino-1 6
dii~henjrl-3-hvdroxyhexane.
A solution of 40 mmol of crude (4S,6S,i'S)-6-(1-amino=2-phenylethyl)-4-
benzyl-2-phenyl-3-aza-2-bora-1-oxacyclohexane in 700 ml of anhydrous THF
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was cooled to -40~C and treated dropwise over a period of 1 h with a solution
of
7.83 g (27.9 mmol) of ((5-thiazolyl)methyl}-(4-nitrophenyl)carbonate in 300 ml
of
dry THF. The resulting solution was allowed to warm to O~C for 3 h, then to
ambient temperature for 16 h. The solvent was removed in vacuo, and the
residue was taken up in 700 ml of ethyl acetate, washed with three 150 ml
portions of 1 N aqueous NaOH and one 150 ml portion: of brine. The organic
phase was dried over Na2S04 and concentrated in vacuo. Purification of the
residue by silica gel chromatography using methanol/chloroform mixtures
provided the desired compound mixed with its regioisomer. A second
chromatography using 1-3% isopropylamine in chloroform provided 5.21 g of
the desired compound which solidified upon standing.
O. 2-Methylpropane-thioamide.
A suspension of 100 g (1.15 mol) of isobutyramide in 4 L of diethyl ether
was stirred vigorously and treated in portions with 51 g (0.115 mol) of P4Sio.
The resulting mixture was stirred at ambient temperature for 2 h, filtered,
and
concentrated in vacuo to provide 94.2 g (80%) of the crude desired compound.
1 H NMR (DMSO-dg) 81.08 (d, J = 7 Hz, 6 H), 2.78 (heptet, J = 7 Hz, 1 H), 9.06
(br, 1 H), 9.30 (br, 1 H). Mass spectrum: (M + H)+ = 104.
P.P. 4-lChloromethy,-2-isoprowlthiazole h~rdrochloride.
A mixture of 94.0 g (0.91 mol) of 2-methylpropane-thioamide, 115.7 g
(0.91 mol) of 1,3-dichloroacetone, and 109.7 g (0.91 mol) of MgS04 in 1.6
liters
of acetone was heated at reflux for 3:.5 h. The resulting mixture was allowed
to
cool, filtered, and the solvent was removed in vacuo to provide the crude
desired compound as a yellow oil. 1 H NMR (DMSO-d6) 81.32 (d, J = 7 Hz; 6
H), 3.27 (heptet, J = 7 Hz, 1 H), 4.78 (s, 2 H), 7.61 (s, 1 H). Mass spectrum:
(M +
H)+ = 176.
Q. 2-Isopro~,vl-4-~(lN-methvl)amino)meth rLl thiazole.
A solution of 40 g of 4-(chloromethyl)-2-isopropylthiazole hydrochloride
in 100 ml of water was added dropwise with stirring to. 400 ml of 40% aqueous
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methyfamine. The resulting solution was stirred for 1 h, then concentrated in
vacuo. The residue was taken up in chloroform, dried over Na2S04, and
concentrated in vacuo. Purification of the residue by silica gel
chromatography
using 10% methanol in chloroform provided 21.35 g (55%) of the desired
compound. ~ H NMR (DMSO-dg) 81.34 (d, J = 7 Hz, 6 H), 2:56 (s, 3 H), 3.30
(heptet, J = 7 Hz, 1 H), 4.16 (s, 2 H), 7.63 (s, 1 H). Mass spectrum: (M + H)+
171.
R~~4-Nitrophen~,loxvlcarbonvl)-L-valine Methyl Ester.
A solution of 66.1 g (0.328 mol) of 4-nitrophenyl chloroformate in 1.2
liters of CH2C12 was cooled to O~C and treated with L-valine methyl ester
hydrochloride. The resulting mixture was treated slowly, with stirring, with
68.9
ml (0.626 mol) of 4-methylmorpholine. The resulting solution was allowed to
slowly warm to ambient temperature and was stirred overnight. After washing
with 3 portions of 10% aqueous NaHC03, the solution was dried over Na2S04
and concentrated in vacuo. The residue was purified by silica gel
chromatography by eluting with chloroform to provide the desired compound.
~ H NMR (DMSO-ds) b 0.94 (d, J = 7 Hz, 3 H), 0.95 (d, J = 7 Hz, 3 H), 2.12
(octet,
J = 7 Hz, 1 H), 3.69 (s, 3 H), 4.01 (dd, J = 8, 6 Hz, 1 H), 7.41 (dt, J = 9, 3
Hz, 2 H),
8.27 (dt, J = 9, 3 Hz, 2 H), 8.53 (d, J = 8 Hz, 1 H). Mass spectrum: (M +
NH4)+ _
314.
S. N-l!N-Methvl-N- j2-isopro~,yl-4=thiazolyl)meth~ amino)carbonyll-L=valine
Methyl Ester.
A solution of 15.7 g (92 mmol) of 2-isopropyl-4-(((N-methyl)amino)-
methyl)thiazole in 200 ml of THF was combined with a solution of 20.5 g (69
mmol) of N-(((4-nitrophenyi)oxy)carbonyl)-L-valine methyl ester. The resulting
solution was treated with 1.6 g of 4-dimethylaminopyridine and 12.9 ml (92
mmol) of triethylamine, heated at reflux for 2 h, allowed to cool, and
concentrated in vacuo. The residue was taken up in CH2C12, viiashed
extensively with 5% aqueous K2C03, dried over Na2S04, and concentrated in
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vacuo. The resulting product mixture was purified by silica gel chromatography
using chloroform as an eluent to provide 16.3 g (54%) of the desired
compound. 1 H NMR (DMSO-d6) b 0.88 (d, J = 7 Hz, 3 H), 0.92 (d, J = 7 Hz, 3
H), 1.32 (d, J = 7 Hz, 3 H), 2.05 (octet, J = 7 Hz, 1 H), 2.86 (s, 3 H), 3.25
(heptet,
J = 7 Hz, 1 H), 3.61 (s, 3 H), 3.96 (dd, J = 8, 7 Hz, 1 H); 4.44 (AA', 2 H),
6.58 (d, J
= 8 Hz, 1 H), 7.24 (s, 1 H). Mass spectrum: (M + H)+ = 328.
T. N-llN-Methyl-N-((2-isopro~,vl-4-thiazol rLl methyllaminolcarbonvll-L-
valine.
A solution of 1.42 g (4.3 mmol) of the resultant compound of Example
20S in 17 ml of dioxane was treated with 17.3 ml of 0.50 M aqueous LiOH. The
resulting solution was stirred at ambient temperature for 30 min, treated with
8.7
ml of 1 M HCI, and concentrated in vacuo. The residue was taken up in
dichloromethane, washed with water, dried over Na2S04, and concentrated in
vacuo to provide 1.1 g (81 %) of the desired compound. Mass spectrum: (M +
H)* = 314.
U. ~,2S.3S.5S)-~N-(N-((N-Methyl-N-((2-isopropyl-4
~iazolyllmeth~lamino carbon 11~ valinvl)aminol-2-(~f 5
thiazolvllmethoxvcarbonyl amino)-1.6-dighenvl-3-hvdroxvhexane.
A solution of 70 mg (0.223 mmol) of N-((N-methyl-N-((2-isopropyl-4-
thiazolyl)methyl)amino)carbonyl)-L-valine, 79 mg (0.186 mmol) of (2S,3S,5S)-
5-amino-2-(N-((5-thiazolyl)methoxycarbonyl)amino)-1,6-Biphenyl-3-
hydroxyhexane, 30 mg (0.223 mmol) of 1-hydroxybenzotriazole hydrate, and 51
mg (0.266 mmol) of N-ethyl-N'-dimethylaminopropyl carbodiimide in 2 ml of
THF was stirred at ambient temperature for .16 h. The;resuEting solution was
concentrated in vacuo, and the residue was purified by silica gel
chromatography using 97:3 CH2C12:CH30H to provide 100 mg (74%) of the
desired compound (Rf 0.4, 95:5 CH2C12:CH30H) as a solid, mp 61-63~C. Mass
spectrum: (M + H)+ = 721. Anal. Calcd for C37H49N605S2~0.5H20: C, 60.88;
H, 6.77; N, 11.51. Found: C, 60.68; H, 6.53; N, 11.36.
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Exam! la a 2_1 A_
did enylhexane.
To a stirred solution of (2S,3S,5S)-2-(N,N-dibenzylamino)-3-hydroxy-5-
amino-1,6-diphenylhexane (10.0 g, 21.6 mmol) in tetrahydrofuran (200 mL) was
added potasium carbonate (6.0 g, 43.2 mmol) in H20 (200 mL). To this solution
was added di-t-butyldicarbonate (5.64 g, 25.9 mmol) in tetrahydrofuran (10
mL).
The solution which resulted was stirred at room temperature for 3 hours. N,N-
dimethylethylenediamine (1 mL, 8.6 mmol) was added and the reaction mixture
was stirred at room temperature for an additional hour. Ethyl acetate (400 mL)
was added and the organic layer was separated and washed with 5% KH2P04
(2 x 200 mL), water (1 x 200 mL), saturated NaHCOg (2 x 200 mL) and water (1
x 200 mL). The organic solution was then dried over sodium sulfate and
concentrated under reduced pressure to provide the desired product as a light
yellow oil. 300 MHz 1 H NMR (CDCI3) 8 1.40 (s,9H), 1.58 (s, 2H), 2.45-2.85 (m,
4H), 3.05 (m, 1 H), 3.38 (d, 2H), 3.6 (m, 1 H), 3.79 (m, 1 H), 3.87 (d, 2H),
4.35 (s,
1 H), 4.85 (s, broad, 1 H), 7.0-7.38 (m, 20 H).
di~hen~ylhexane.
To a stirred solution of (2S,3S,5S)-2-(N,N-dibenzylamino)-3-hydroxy-5-
(t-butyloxycarbony!amino)-1,6-diphenylhexane (12 g, 21.3 mmol) in methanol
(350 mL) was charged ammonium formate (8.05 g, 128 mmol, 6.0 eq) and
10% palladium on carbon (2.4 g). The solution was stirred under nitrogen at
60 °C for three hours and then at 75 °C for 12 hours. An
additional amount of
ammonium formate (6 g) and 10% palladium on carbon (1.5 g) was added as
well as 1 mL of glacial acetic acid. The reaction was driven to_ completion
within 2 hours at a reflux temperature. The reaction mixture was then cooled
to
room temperature and then filtered through a bed ofCelite.*The filter cake was
* Trade-mark
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washed with methanol (75 mL) and the combined filtrates were concentrated
under reduced pressure. The residue was taken up in 1 N NaOH (300 mL)
and extracted into methylene chloride (2 X 200 mL). The combined organic
layers were washed with brine (250 mL) and dried over sodium sulfate.
Concentration of the solution under reduced pressure provided the desired
product as a light colored oil which slowly crystallized upon standing (5 g).
Further purification of the product could be accomplished by flash
chromatography (silica gel, 5% methanol in methylene chloride). 300 MHz 1 H
NMR (CDC13) 8 1.42 (s, 9H), 1.58 (m, 1 H), 1.70 (m, 1 H), 2.20 (s, broad, 2H),
2.52 (m, 1 H), 2.76-2.95 (m, 4H), 3.50 (m, 1 H), 3.95 (m, 1 H), 4.80 (d,
broad, 1 H),
7.15-7.30 (m, 10H).
Example 22
Alternative Preparation of (2S.3S.5S~-2-Amino-3-hvdrox~~
but~ycarbonvlaminol-1.6-diphenvlhexane.
Example 22A
L5-, S)-2-(t Butyloxycarbon lad)-51N.N-dibenzylamino)-1.6-diphenyl-
4-oxo-2-hexene
To 9.21 gm (20 mmol) of (S)-2-amino-5-(N,N-dibenzylamino)-1,6-
Biphenyl-4-oxo-2-hexene and 0.37 gm (3 mmol) 4-N,N dimethylaminopyridine
in 100 ml of methyl tert-butylether was added via syringe pump a solution
containing 4.80 gm (22 mmol) di-tert-butyl Bicarbonate in the same solvent (25
ml) over a period of 6 h. An additional amount (3 ml) of methyl tert-
butylether
was then added to complete the addition. After stirring at room temperature
for
18 h the reaction mixture was cooled with the aid of an ice water bath. The
resultant solid was collected by suction filtration and washed with cold
(0°C)
methyl tert-butylether and hexane and dried under vacuum to give 9.9 gm of
crude material as a white solid. The material thus isolated was disolved in a
minimal amount of dichloromethane and purified by flash chromatography on
silica gel. Elution of the column with a mixture of hexane-ethyl acetate-
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dichloromethane (8:1:1 ) gave, after concentration of the appropriate
fractions,
8.1 gm (72%) of the desired compound. Mp. 191- 193°C. [a]p -
183.7° (c =
1.05, CHCIg). 1H NMR (CDC13, b): 11.68 (bs, 1 H), 7.05 - 7.47 (m, 20H), 5.28
(s,1 H), 4.27 (d, J=16 Hz, 1 H), 4.02 (d, J=l6Hz, 1 H), 3.58 (m, 4H). 3.40 (m,
1 H),
3.11 (m, 1 H), 2.90 (m, 1 H), 1.48 (s, 9H).
ExamQle 22B
Alternate ~gparation of 15S~-2-l,t-Butylox,~rcarbonvlamino)-5-(N.N
~j~~rlaminQ -1.6-dipherayl-4-oxo-2-hexane
A suspension of (S)-2-amino-5-(N,N-dibenzylamino)-1,6-diphenyl-4-oxo-
2-hexane (100.0 g, 0.217 mol) in 15% ethyl acetate/hexanes (2 liters) under N2
was warmed to about 40°C. The resulting solution was cooled to room
temperature before adding 4.0 g (33 mmol) of N,N-dimethyl-4-aminopyridine
and 49.7 g (0.228 mol) of di-tart-butyl Bicarbonate. The reaction mixture was
allowed to stir overnight at room temperature. (After approximately one hour,
a
white precipitate began to form.)
The suspension was filtered and the precipitate was washed with hexanes to
afford the desired product as colorless crystals. TLC: 25% ethyl
acetate/hexanes Rf 0.38.
A solution of the product .of Example 22A (5 g, 8.9mmol) in
dichloromethane (1 OOmI) and 1,4-dioxolane (100m1) was cooled to between
-10° and -15° C and treated dropwise with 1 M BH3THF (26.7m1,
26.7mmol).
The solution was stirred at this temperature for 3 hr. The clear solution was
quenched with excess methanol (20m1) and stirred at room temperature for
30 min. The solvent was removed in vacuo.
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The resulting white foam was dissolved in THF (75m1) and cooled to
-40° C. A solution of LAH (9m1, 1 M in THF, 9mmol) was added dropwise.
After
min. the solution was quenched with water followed by dilute aqueous HCI.
The organics were removed and the aqueous layer extracted with ethyl acetate
(3 x 20 ml). The combined organics were washed (saturated aqueous
bicarbonate followed by brine), dried (Na2S04), filtered and evaporated to
afford 4.9 g (99%) of the desired product as a white foam.
Alternatively, the white foam resulting from the BH3THF reaction step
was dissolved in MeOH (45m1), cooled to +3 °C and treated portionwise
with
KBH4 (1.44 g, 26.7 mmol). After addition of the last portion of KBH4 the
reaction was stirred for an additional 4 hours at +4 to +5 °C. The
solution was
concentrated by 1 /2 the volume in vacuo, diluted with 1 /1 hexane-EtOAc (70
ml)
and quenched (with cooling, maintain temp. <30 °C) by adding a 10 %
solution
of KHS04 to pH = about 5. NaOH (15 % aqueous) was added to pH = 12 - 13.
The insoluble salts were removed by filtration, and the filter cake washed 3
times with 7 ml 1/1 hexane/EtOAc. The filtrate and washes were transferred to
a
separatory funnel, diluted with 15 ml hexane and 15 ml H20. The organics
were removed and the aqueous layer was extracted once with 20 ml (1 /1 )
hexane-EtOAc. The combined organics were washed (saturated brine), dried
(Na2S04), filtered, and evaporated to afford 5.2 g of the desired product
which
was used without further purification in subsequent reactions.
Rf 0.5 (25% EtOAc/hexane) ~ H NMR (CDC13) b 7.37-7.10 (m 20H); 6.78 (br. s,
1 H); 4.62 (d, 1 H); 4.50 (s, 1 H); 4.18 (dd, 1 H); 3.9 (d, 2H); 3.65 (dd,
2H); 3.40 (d,
2H); 3.00 (m, 2H); 2.77 (m, 1 H); 1.39 (s, 9H). MS (El) m/e565 (M+H).
Examihe 22D
~2S. 3S. 5Sl-2-Amino-3-hydroxy-~t-butyloxycarbonylamin~-1.6
iohenylhexane.
A solution of the product from Example 22C (150 gm, 250 mmol)
dissolved in absolute EtOH (2 liters) was treated with 10 % Pd/C (l8gm, pre-
wetted), followed by addition of ammonium formate (78.6 gms, 1.25 moles)
dissolved in H20 (200m1). The resulting mixture was stirred at reflux for 2.5
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hours. The mixture was cooled to room temperature and filtered through a pad
of infusorial earth (20g). The filter cake was washed 3 times with EtOH (70m1
each). The filtrate was concentrated in vacuo. The residue was dissolved into
EtOAc (1 L) and washed (1 N NaOH, followed by H20, followed by brine), dried
(Na2S04), filtered and concentrated in vacuo. to a constant weight of 95 gms.
(99.2 % of theory). The light yellow solid (91.5 gm of the 95 gm) was slurried
in
hot heptane (600 ml) (steam bath) and treated with isopropanol (45m1), and
swirled to effect solution. The solution was allowed to slowly coot to room
temperature over 3 hours, kept at room temperature for 2 more hours and
filtered. The filter cake was washed 10 times with 9/1 hexane-isopropanol
(30m1 each) to give the desired product as an off-white finely crystalline
solid
which was dried to constant weight of 57.5 gm.
The crude product (20 gm) was recrystallized from hot 140 ml heptane/
17 ml isopropanol. After letting the solution cool slowly to room temperature,
the mixture was let stand at room temperature for 2 hours and then filtered.
The
filter cake was rinsed (5 X 15 ml (8/1 ) heptane/isopropanol) and dried to a
constant weight of 18.5 gm.
Alternative Preparation of (2S.3S.5S)-5-(~N-(!N-Methy~(2-isopropyl-4
thiazolyl methyl)amino)carbonyl)valinyl)amino)-2-(N-((5
~yl)methoxycarbonv~amino)-1.6-Biphenyl-3-hydroxvhexane
~2S.3S.5S)-5-(t-B~yloxycarbonylamino)-2-~(N-((5
thiazolvllmethoxvcarbonvllaminol-1.6-dic~henvl-3-hvdroxvhexane
The product of Example 22D (6.0g , 15.6 mmoles) was dissolved in 60
mL of DMF under nitrogen atmosphere. To this stirred solution at room
temperature was added 5-(p-nitrophenyloxycarbonyloxymethyl)thiazole (4.67g ,
15.6 mmole) and the resulting solution was stirred for 4 h. The solvent was
removed under reduced pressure by rotary evaporation and the residue
dissolved in 150 mL EtOAc. This solution was washed with 5 x 75 mL 1 N
NaOH solution, 100 mL brine, dried over Na2S04. The solvent was removed
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to afford 8.02 g of a slightly yellowish oil. This material was crystallized
from 30
mL EtOAc and 40 mL hexane to afford 6.53g (80%) of the desired product as a
white solid. mp 118-120 ~C H 1NMR (CDC13) b 8.79 (s, 1 H), 7.83 (s, 1 H), 7.30-
7.15 (m, 8H), 7.08 (m, 2H), 5.23 (s, 2H), 5.14 (d, 1 H, J = 9 Hz), 4.52 (m, 1
H),
3.92-3.72 (m, 3H), 3.65 (m; 1 H), 2.85 (d-apparent, 2H, J = 7.5 Hz), 2.72 (d-
apparent, 2H, J = 7 Hz), 1.61 (m, 2H), 1.38 (s, 9H). CIMS m/z (526) (M + H)+,
543 (M + 18)+..
m
The product of Example 23A (6.43g, 12.23 mmoles) was dissolved in 25
mL dioxane at room temperature under nitrogen atmosphere. To this stirred
solution was added 20.25 mL of 4N HCI in dioxane, and after approximately 10
min a thick precipitate formed. An additional 10 mL of dioxane was added to
loosen up the slurry. This mixture was stirred for 1 h and then filtered. The
filter
cake of the product bis-HCI salt was washed with 20 mL dioxane, air dried, and
then dissolved in 175 mL water. To this solution was added 175 mL ethyl
acetate and the two phase mixture rapidly stirred. The pH of this mixture was
adjusted to pH = 10 by the dropwise addition of 3N NaOH to the rapidly stirred
mixture. The organic layer was isolated, washed with brine (150 mL), and dried
over Na2S04. The solvent was removed to afford 5.18g (99%) of the desired
product as a clear oil. H~ NMR (CDC13) 8 8.81 (s, 1 H), 7.87 (s, 1 H), 7.35-
7.05
(m, 10 H), 5.33 (d, 1 H, J = 9.3 Hz), 5.28 (m,2H), 3.81 (m, 1 H), 3.72 (m, 1
H), 3.01
(m, 1 H), 2.88 (m, 2H), 2.78 (dd, 1 H, J = :13.5, 5.1 Hz), 2.39 (dd, 1 H. J =
9.0, 4.5
Hz), 1.57-1.30 (m, 2H). CIMS m/z 426 (Ms+ H)+.
j2S.3S.5S1-5-l~~(N-Methyl-N-f (2-is ~ropyl-4
thiazolyl_)methyrl amino)carbo~,yl)valiipyl amino)-~N_((~
thiazolvllmethoxvcarbonvllaminol-1.6-diohenvl-3-hvdroxvhPxanP
N-((N-Methyl-N-((2-isopropyl-4-thiazolyl)methyl)amino)carbonyl)-L-
valine (4.13g, 13.18 mmole) and hydroxybenztriazole (2.23g, 16.48 mmoles)
were dissolved in 70 mL THF and then dicyclohexyl-carbodiimide( 2.71 g, 13.18
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mmoles) was added in one portion to the stirred solution under nitrogen
atmosphere. This mixture was stirred for 4h at room temperature and then
filtered to remove dicyclohexylurea precipitate. (2S,3S,5S)-5-Amino-2-(N-((5-
thiazolyl)-methoxycarbonyl)amino)-1,6-diphenyl-3-hydroxyhexane (5.1 g, 11.99
mmoles) was dissolved in 100 mL THF under nitrogen atmosphere. To this
stirred solution was added the filtrate of HOST-active ester and the resulting
solution was stirred at room temperature for 4 h; and the solvent removed via
rotary evaporation. The residue was dissolved in 150 mL ethyl acetate and
washed with 2 x 100 mL 1 N NaOH, 100 mL brine, 100 mL of 1 % w/w aqueous
KHS04 and the solvent was removed by rotary evaporation to afford a residue.
The residue was dissolved in 175 mL 1 N HCL, and the solution filtered to
remove the small quantity of dicyclohexylurea. The filtrate solution was added
to 175 mL ethyl acetate and the two phase mixture rapidly mixed. The pH of
this rapidly stirred mixture was adjusted to pH = 7 by dropwise addition of
cold
3N NaOH. The organic layer was isolated, washed with 100 mL brine, dried
over Na2S04, filtered, and the solvent was removed to afford 8.6 g of a
colorless foam. This material was crystallized from 42 mL EtOAc and 21 mL
hexane to give 7.85g of the desired product as a white solid. mp = 122-123 ~C.
CIMS m/z 721 (M + H) +.
Exam
I
Alterna tivePreparationof (2S.3S.5S)-5-Amino-2-(~(5-
thiazollyl~methoxycarbonvl],aminoj-1.6-di enyl-3-hydroxyhexane
Alternative A
The product of Example 17F (9.5 g, 33.4 mmol) and phenylboronic acid
(4.1 g, 33.6 mmol) were combined in toluene (150 mL) and refluxed for 2.5
hours with azeotropic water removal (Dean-Stark trap). Toluene (100 mL) was
distilled out at atmospheric pressure, then the remaining toluene was removed
under vacuum, to provide a yellow syrup which was dissolved in DMF (50 mL)
and cooled to -60 °C. A solution of 5-(p-nitrophenyloxycarbonyloxy-
methyl)thiazole (9.5 g, 33.5 mmol) in DMF (50 mL) was added over 45 minutes.
The resulting mixture was stirred for 8 hours at -55~5 °C, then 14
hours at
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-25°C, then was allowed to warm to room temperature. The reaction
mixture
was diluted with 1 N HCI (250 mL) and washed with CH2C12 (2 x 80 mL). The
combined organic layers were back-extracted with 1 N HCI (60 mL). The
combined aqueous HC1 layers were cooled in an ice-bath to 2 °C, and
conc.
(37%) HCL (30 mL) was added over 5 minutes. The desired product (bis HCI
salt) began to precipitate within 30 minutes. The slurry was stirred 3 hours
at 2-
°C, then the product (bis HCI salt) was collected by filtration and
dried in a
vacuum oven at 55-60 °C. Yield 11.4 g (68%).
Second crop recovery:
The HCI mother liquors were stirred with ethyl acetate (190 mL) and
neutralized to pH 9-10 with aqueous K2C03 (200-300 g of 25% w/w K2C03
was required). The ethyl acetate Layer was concentrated under vacuum to an
oil which was redissolved in 1 N HCI (90 mL) and washed with methylene
chloride (45 mL). The aqueous layer was cooled to 2 °C. Conc. (37%) HCI
(9.0
mL) was added to precipitate a second crop. After stirring for 1-3 hours at 2-
5
°C, the solid was collected by filtration and dried in a vacuum oven at
55-60 °C.
Yield 2.1 g (12.6%).
Neutralization of Bis HCI Salt:
The bis HCI salt (10.66 g, 21.4 mmol, mixture of first and second crops)
was stirred with CH2C12 (110 mL) and 5% aqueous NaHC03 (110 mL) until all
solids dissolved (2 hours). The aqueous Layer was separated and extracted
with another 50 mL CH2C12. The combined organic extracts were dried with
Na2S04 (10 g), filtered and concentrated under vacuum at <_40 °C to
an oil.
The oil was dried on a vacuum pump to give the title compound as a yellow
foam, 9.1 g (100 %).
Alternative B
The product of Example 17F (15.0 g, 0.053 mole) was dissolved in DMF
(75 mL). Triisopropylborate (24.4 mL, 0.105 mole) was added and stirred at
ambient temperature for approximately 1.5 hours. The solution was cooled to
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-10°C and a solution of 5-(p-nitorphenyloxycarbonyloxymethyl)thiazole
(15.0 g,
0.054 mole) in DMF (75 mL) was added over 80 minutes. The reaction was
stirred for approximately 1 hour at -10 °C, then was diluted with
methylene
chloride (250 mL) and quenched with a mixture of triethanolamine (24.8 g) and
5% aqueous sodium bicarbonate (300 mL). The biphasic mixture was stirred
for 1 hour, then the layers were separated and the aqueous was extracted with
another portion of methylene chloride (50 mL). The combined organic layers
were extracted with 1 N HCI (1 x 390 mL, then 1 x 95 mL). The acid layers were
combined, cooled in an ice-bath, and further acidified with conc. HCI (50 mL)
which produced a white slurry of product. The slurry was stirred for
approximately 1 hour at 2°C. The desired product bis HCI salt) was
collected
by filtration and dried at 55 °C in a vacuum oven. Yield 18.5 g (70%).
xamlhe 2525
Alterna_tivP Preparation of (2S.3S.5S)-~N-lN-(lN-Methyl-N-((2-iso~ro,~yJ-4
thiazoly[ methyl)amino carbonyl)valinyl)amino)-2-(N-((5
thiazoly~methoxycarbonyl)aminoLl .6-dilahenyf-3-hydroxyhexane
To a solution of the product of Example 24 (9.1 g, 21.4 mmol), HOST (3.6
g, 23.5 mmol) and N-((N-Methyl-N-((2-isopropyl-4-thiazolyl)methyl)amino)-
carbonyl)-L-valine (7.37 g, 23.5 mmol) in THF (170 mL) was added DCC (4.85
g, 23.5 mmol). The solution was stirred at ambient temperature for 16 hours
(DCU precipitates). THF was removed under vacuum and the resulting paste
was stirred with cold 1 N HCI (106 mL at 5 °C) for 3 hours to dissolve
the the
crude product. The DCU was removed by filtration and the filter cake was
washed with 1 N HCI (30 mL). KH2P04 (3.2 g) was dissolved in the combined
HCI filtrates. The solution was mixed with ethyl acetate (80 mL) and
neutralized
to pH 7 with aqueous NaOH (60.3 g of 10% w/w NaOH). The aqueous layer
was extracted with another 25 mL ethyl acetate and the combined ethyl acetate
extracts were washed with aqueous NaHC03 (2 x 37 mL of 5% w/w NaHC03).
The organic layer was dried with Na2S04 (13 g), filtered, and concentrated
under vacuum at s45 °C. The residue was dissolved in a 1:1 ethyl
acetate/heptane mixture (200 mL) at 70 °C. The solution was allowed to
cool
slowly and stirred overnight at room temperature to provide a thick slurry.
The
product was collected by filtration and washed with 1:1 ethyl acetatelheptane
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(20 mL). The product was dried briefly at 55 °C in a vacuum oven to
obtain an
approximate weight prior to the second crystallization (12.85 g, 83%).
A second crystallization from 144 mL of 2:1 ethyl acetatelheptane (dissolved
at
~70 °C, then stirred at room temperature 12 hours) produced a thick
slurry of
fine white solid. The product was collected by filtration and washed with 15
mL
2:1 ethyl acetate/heptane, then dried in a vacuum oven at 55 °C for 2
days to
give the desired product. Yield 11.9 g (77%).
~Qle 26
Alternate Prer~aration of~l5-Thiazolyl)methyl)=j4-nitrophenvl)carbonate
~xami Ip a 26A
2-Amino-5-~ethoxycarbonyl)thiazole Hydrochloride
To a -10 °C solution of potassium tert-butoxide (110 g, 0.98 mol)
in THF
(1.9 L) was added a solution of ethyl chloroacetate (100 mL, 0.934 mol) and
ethyl formate (75 mL, 0.928 mol) in THF (400 mL) dropwise over 2 hours, with
good mechanical stirring. The thick solution was stirred another 2 hours at
ca.
-1 °C then the reaction was quenched by addition of a solution of NaCI
(150 g)
in 1 N HCL (750 mL). The mixture was allowed to warm to 20 °C and the
lower
aqueous layer (containing some precipitated salt) was separated. The organic
layer was stripped under vacuum on a rotary evaporator. The oil was
redissolved in 500 mL ethyl acetate, dried with 75 g Na2S04 for 1 hour,
filtered
and concentrated under vacuum (40-50 °C bath~temperature) to an oil.
The
resulting crude chloroaldehyde (161 g) and thiourea (70 g, 0.92 mol) were
dissolved in THF (2 L) and warmed to gentle reflux (60 °C). The
thiourea
dissolved during warming, and within 20 minutes, product precipitated from
solution. After 100 minutes the suspension was allowed to cool to room
temperature, then was cooled in an ice-bath for 1 hour. The product was
collected on a fritted Buchner funnel and washed with 2 x 100 mL cold THF,
then dried overnight in a vacuum oven at 50 °C. Yield: 122 g of title
compound
as a tan-colored solid, m.p. 182-185 °C (dec.). 1 H NMR (DMSO-d6) b
7.86 (s,
1 H), 4.19 (q, 2H), 1.21 (t, 3H). 13C NMR (DMSO-dg) 8 171.9, 160.4, 140.4,
114.4, 61.1, 14.2.
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Examlhe 26B
2-Amino-5-lethoxycarbonyl)thiazole
To a -10 °C solution of potassium tert-butoxide (150 g, 1.3 mol)
in THF
(1.35 L) was added a solution of ethyl chloroacetate (139 mL, 1.3 mol) and
ethyl
formate (103 mL, 1.27 mol) in THF (150 mL) dropwise over 75 minutes, with
good mechanical stirring. A THF rinse (25 mL) was added over 5 minutes. The
thick solution was stirred another 3 hours at ca. -5 to 0 °C, then the
reaction was
quenched by addition of a solution of NaCI (240 g) and conc. HCI (90 mL) in
water (960 mL). The mixture was allowed to warm to 15 °C and the lower
aqueous layer was discarded. Thiourea (97 g, 1.27 mol) was dissolved in the
crude THF solution of chloroaldehyde. The solution was warmed to 65 °C
and
refluxed for 1 hour, then cooled to 30 °C. Addition of a solution of
K2C03 (88g,
0.64 mol) in 1500 mL water produced two layers (aqueous pH=7). The THF
was removed under vacuum at _<45 °C, causing the product to precipitate
as a
yellow solid. The slurry was cooled to 15 °C, and the product was
collected on
a fritted Buchner funnel and washed with 3 x 200 mL water, then dried 24 hours
in a vacuum oven at 55 °C to provide 151 g of title compound as a
yellow solid,
m.p. 155-158 °C. 1 H NMR (DMSO-ds) b 7.8 (br s, 2H, NH2), 7.62 (s, 1
H), 4.13
(q, 2H), 1.18 (t, 3H). 13C NMR (DMSO-dg) 8 173.4, 161.3, 147.9, 114.5, 60.1,
14.3.
Examlhe 26C .
~( thoxy ac rbonylythiazole
A solution of 2-amino-5-(ethoxycarbonyl)thiazole (50 g, 0.29 mmol) in a
mixture of DMF (83 mL) and THF (317 mL) was added dropwise over 87
minutes to a stirred 41 °C solution of isoamyl nitrite (59 mL, 0.44
mol) in DMF
(130 mL). A maximum temperature of 60 °C was observed during the
exothermic addition. After another 40 minutes the THF was removed under
vacuum at 45 °C. The concentrated DMF solution was cooled to 25
°C and
diluted with toluene (420 mL) and water (440 mL). The toluene layer was
extracted with 3 x 120 mL water, then dried with Na2S04 (50 g)-for 1 hour.
After
filtration the toluene layer was stripped on a rotary evaporater at 50
°C bath
temperature, then on a vacuum pump at 21 °C. The crude residue
containing
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the title compound weighed 65.6 g. This material was used directly in the next
step. A sample of similarly prepared material was purified by column
chromatography to give a yellow oil. ~ H NMR (CDC13) b 8.95 (s, 1 H), 8.51 (s,
1 H), 4.39 (q, 2H), 1.40 (t, 3H). 13C NMR (CDC13) 8 161.0, 157.9, 148.6,
129.8,
61.6, 14.1.
To a slurry of lithium aluminum hydride (9.0 g) in THF (633 mL) was
added a solution of crude 5-(ethoxycarbonyl)thiazole (65.6 g) in THF (540 mL)
over 95 minutes at 0-5 °C. After an additional 25 minutes, the reaction
was
quenched at 5 °C by sequential addition of water (8.1 mL), 15% NaOH
(8.1 mL),
and water (24.3 mL). After drying with Na2S04 (44 g) for 2 hours, the slurry
was
filtered, and the filter cake was washed with 100 mL THF. The combined
filtrates were concentrated under vacuum at 45 °C to a brown oil (39
g). The oil
was fractionally distilled through a short-path apparatus. The product
fractions
distilled at 97-104 °C vapor temperature at 3-5 mm, providing 20.5 g of
the title
compound as a turbid orange oil. ~ H NMR (CDC13) b 8.74 (s, 1 H), 7.72 (s, 1
H),
4.89 (s, 2H), 3.4 (br s, 1 H, OH). 13C NMR (CDC13) 8 153.4, 140.0, 139.5,
56.6.
y -a Nitrol~hen,yoxycarbonyloxvmethy~thiazole H~rdrochloride
Distilled 5-(hydroxymethyl)thiazole (14.1 g, 123 mmol) and triethylamine
(17.9 mL; 129 mmol) were dissolved.in ethyl acetate (141 mL) and cooled to
-1 °C (ice/salt bath). A solution of 4-nitrophenyl chloroformate (26.0
g, 129
mmol) dissolved in ethyl acetate (106 mL) was added dropwise over 50
minutes at an internal temperature of 0-4 °C. An ethyl acetate flask
rinse (20
mL) was also added. Salts precipitated from solution throughout the addition.
The yellow mixture was stirred another 1 hour 45 minutes at 0-2 °C,
then a
solution of dilute HCI (3.1 g, 31 mmol of cone. NCI in 103 mL water) was added
at once. The mixture was stirred for 0.5 hours while warming to 15 °C,
then
stirring was stopped. The organic layer was washed twice with aqueous 5%
K2C03 solution (2 x 70 mL), then dried with Na2S04 (30 g). After filtration
the
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solution was concentrated under vacuum on a rotary evaporater (bath
temperature of 41 °C) to a brown oil (38g). The crude 5-(p-
nitrophenyoxycarbonyloxymethyl)-thiazole was dissolved in ethyl acetate (282
mL), then cooled in an ice bath to 2 °C. Dry HCI gas (7.1 g, 195 mmol)
was
bubbled in slowly over 50 minutes (temperature 2-4 °C). After stirring
for
another 1 hour 45 minutes at 2-4°C, the solid precipitate was collected
on a
sintered glass funnel under a nitrogen blanket and the flask was washed out
with 50 mL cold ethyl acetate which was used to rinse the filter cake. The
cake
was dried on the funnel under strong nitrogen purge for 15 minutes then dried
in a vacuum oven at 50 °C with a nitrogen purge to provide 29.05 g of
the title
compound as tan powder, m.p. 131-135 °C (dec.). 1 H NMR (DMSO-d6) 8
9.21
(d, 1 H), 8.27 (m, 2H), 8.06 (d, 1 H), 7.52 (m. 2H), 5.54 (s, 2H). 13C NMR
(DMSO-d6) 8 157.3, 155.2, 151.8, 145.3, 143.7, 131.9, 125.5, 122.7, 62.1.
Exam lip a 26F
5-~ -ip Nitrol ha enoxycarbonyloxymethyl)thiazole
5-(p-Nitrophenoxycarbonyloxymethyl)thiazole hydrochloride
(3.0 g) was slurried in ethyl acetate (30 mL) and cooled to 10-15 °C. A
solution of 5% aqueous potassium carbonate (30 mL) was added with rapid
stirring. After 15 minutes, stirring was stopped and the aqueous layer was
separated. The organic layer was dried with Na2S04 (3 g), filtered, and
solvent was distilled under vacuum to give 2.49 g of the title compound as a
brown syrup which slowly solidified, m.p. 62-64 °C. 1H NMR (CDC13) 8
8.90 (d,
1 H), 8.29 (m, 2H), 8.01 (d, 1 H), 7.39 (m, 2H), 5.52 (s, 2H). ~3C NMR (CDC13)
8
155.4, 155.2, 152.2, 145.4, 144.9, 130.6, 125.3, 121.6, 61.9.
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Example 27
Alternative Preparation of N-((N-Methyl-N ~(2-isopropyl-4
thiazoly~methyl)amino carbonyl)-L-valine
Example 27A
To a 1 liter three neck round bottom flask equipped with mechanical
stirrer, nitrogen atmosphere, condenser, thermocouple and 15 ° C water
bath
was charged (26.0 g, 0.298 mots) isobutyramide followed by ( 19.9 g, 0.045
mols) phosphorous pentasulfide and 375 mls THF. This solution was stirred at
20 ~ 5 ° C for 3 hours, then was warmed to 60 °C and stirred an
additional 3
hours. The THF was removed under vacuum with a 50 ° C bath temperature
to
afford a yellow oil. This oil was neutralized with a solution of 5 g NaOH, 10
g
NaCI and 90 g water. Next the product was extracted into EtOAc (2 X 250 mls)
and the combined organics reduced under vacuum to an oil. The oil was
dissolved in 50 mls THF and again the solvent was removed under vacuum to
give the desired product as a yellow oil. (yield approx. 27 grams, 88%).
Example 27B
2-Isopropyl-4-((~N-methyl)amino methxllthiazole
The thioisobutyramide resulting from Example 24A was dissolved in 70
mls THF and added slowly to a solution of (34.1 g , .27 mots) 1,3-
dichloracetone
in 40 mls THF. A 10 ml rinse of THF was used to completely transfer the
thioamide. The reaction wass carried out in a 250 ml flask with mechanical
stirring under nitrogen atmosphere. The reaction temperature was maintained
below 25 ° C during addition Hrith ar15 ~ 5 ° C bath: The bath
was kept in place
for 1 hour after which it was removed and the reaction stirred for 18 hours.
Next
this stirred chloromethyl-thiazole solution was added to 376 mls (4.37 mols)
40 % aqueous methylamine solution at 15 ° C in a 1 liter flask. The
temperature
was maintained below 25 ° C during addition. After half an hour the
bath was
removed and the reaction stirred for 3 hours at ambient temperature. The
solvent was removed under vacuum with a 50 ° C bath to an end volume of
310
mls. The residue was then basified with 50 g 10 % NaOH to pH 12 and
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extracted into methylene chloride (2 X 160 mls). The combined organics were
then washed with 1 X 150 g of 20 % ammonium chloride followed by 1 X 90 g of
20 % ammonium chloride. The combined aqueous washes were then back
extracted with 150 mls methylene chloride. The combined product methylene
chloride layers were then extracted with 100 g of a solution of 25 g conc. NCI
and 75 g water. This acidic product solution was then washed with 135 mls
methylene chloride. Next the acidic product solution was cooled, then
neutralized with 100 g 20 % NaOH solution. The product was extracted from
this mixture with methylene chloride (2 X 135 mls). The solvent was removed
under vacuum to afford the desired product as an amber oil. (yield approx. 28
grams)
~,xamlhe 27C
N-~(~J,-Methyl-N-(l2-isoo, ro~~yl-4-thiazoly)methyl)amino)carbonyl)-L-valine
Methyl. Ester
Into a 500 ml 3-neck round bottom flask equipped with mechanical
stirrer, nitrogen atmosphere, thermocouple, heating mantle and condensor was
charged the product of Example 27B (28.1 g, .165 mols), phenoxycarbonyl-(L)-
valine (41.5 g, .165 mol) and 155 mf toluene. This solution was warmed to
reflux (110 °C) and stirred for three hours, then cooled to 20~
5° C and washed
with 2 X 69 ml 10 % citric acid followed by 1 X 69 ml water, 1 X 116 mls 4
sodium hydroxide, 1 X 58 ml 4 % sodium hydroxide and finally 1 X 58 ml water.
The organic product solution was then treated with 3 grams of activated carbon
at reflux for 15 minutes , filtered through infusorial earth to remove carbon,
and
the carbon/infusorial earth cake was washed with 25 ml hot toluene. Next the
solvent was removed to afford a brown oil which solidifed upon cooling. This
brown solid was dissolved with warming in 31 ml EtOAc and 257 ml heptane at
60~5° C. This solution was slowly cooled to 25 °C, stirred 12
hours, cooled
further to 0°C, and stirred 3 hours. The crystals were collected by
filtration and
washed with 50 ml 1:9 EtOAc/Heptane. The solid was dried in a 50°C
vacuum
oven for 12 hours to afford 41.5 grams of the desired product as a tan-colored
solid (76.9%). _
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~jN-Methvl-N=jf,2-isoQro~yl-4-thiazolyl)methyl amino)carbonyl)-L-valine
To a one liter three neck flask was charged the product of Example 27C
(50 g, 0.153 mol), lithium hydroxide monohydrate (13 g, 0.310 mol), 200 ml THF
and 190 ml water. This hazy solution was stirred for 2 hours. The reaction was
quenched with a solution of cons. NCI (32.4 g, 0.329 mol) in 65 mL water, the
THF was removed under vacuum and the product extracted into methylene
chloride (3 X 210 ml). (NOTE: If necessary, the pH of the aqueous layer should
be adjusted to maintain pH 1-4 during the extractions.) The combined organics
were then dried with 50 g sodium sulfate, filtered with a 150 ml methyle~e
chloride rinse of the sodium sulfate, and the solvent was removed under
vacuum. The product was dissolved in 450 ml THF and again the solvent was
removed. Next the product was dissolved in 475 ml THF containing 0.12 g
butylated hydroxytoluene (BHT) for storage. If desired, the solvent can be
removed under vacuum and the residual syrup dried in a vacuum oven at 55
°C
to provide a glassy solid.
The above process for the preparation of compound 111 is disclosed in
PCT Patent Application No. W094!14436, published July 7, 1994.
Protocol For Oral Bioavailability Studies
Pr9tocol A j~olid dosag~form~
Dogs (beagle dogs, mixed sexes, weighing 7-14 kg) were fasted
overnight prior to dosing, but were permitted water ad libitum. Each dog
received a 0.5 mg/kg subcutaneous dose of histamine approximately 30
minutes prior to dosing. Each dog received a single solid dosage form
corresponding to a 5 mg/kg dose of the drug. The dose was followed by
approximately 10 milliliters of water. Blood samples.were obtained from each
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animal prior to dosing at 0.25, 0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10 and 12 hours
after
drug administration. The plasma was separated from the red cells by
centrifugation and frozen (-30°C) until analysis. Concentrations of
parent drug
were determined by reverse phase HPLC with low wavelength UV detection
following liquid-liquid extraction of the plasma samples. The parent drug area
under the curve was calculated by the trapezoidal method over the time course
of the study. The absolute bioavailability of each test composition was
calculated by comparing the area under the curve after oral dosing to that
obtained from a single intravenous dose. Each capsule or capsule composition
was evaluated in a group containing six dogs; the values reported are averages
for each group of dogs. The average bioavailability data for the compositions
of
the Examples is shown in Table I.
Protocol B (,liquid dosage forms
Dogs (beagle dogs, mixed sexes, weighing 7-14 kg) were fasted
overnight prior to dosing, but were permitted water ad libitum. Each dog
received a 0.5 mg/kg subcutaneous dose of histamine approximately 30
minutes prior to dosing. A 5 mg/kg dose, measured in a disposable syringe,
was placed in the back of the throat of each animal. The dose was followed by
approximately 10 milliliters of water. Blood samples were obtained from each
animal prior to dosing at 0.25, 0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10 and 12 hours
after
drug administration. The plasma was separated from the red cells by
centrifugation and frozen (-30°C) until analysis. Concentrations of
parent drug
were determined by reverse phase HPLC with low wavelength UV detection
following liquid-liquid extraction of the plasma samples. The parent drug area
under the curve was calculated by the trapezoidal method over the time course
of the study. The absolute bioavailability of each test composition was
calculated by comparing the area under the curve after oral dosing to that
obtained from a single intravenous dose. Each liquid composition was
evaluated in a group containing six dogs; the values reported are averages for
each group of dogs. The average bioavailability data for the compositions of
the Examples is shown in Table I.
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Protocol C ll~uid dosage form. followed by milkl
The animals were selected and pretreated with histamine as described
in Protocol B above. The liquid dosage form (5 mg/kg dose), measured in a
plastic syringe, was instilled into the back of the throat of each animal. A
10 mL
aliquot of milk was also administered into the back of the throat of each dog.
The liquid dosage form/milk combination was chased with an additional aliquot
of water as described in Protocol B above. The sampling times, sample
preparation and data analysis were as described in Protocol B.
Protocol D ~I~id dosage form with milkl
The animals were selected and pretreated with histamine as described
in Protocol B above. The liquid dosage form (5 mg/kg dose) was diluted ten
fold with milk. The diluted dosage was measured in a plastic syringe and
instilled into the back of the throat of each animal. The liquid dosage form
was
chased with an additional aliquot of water as described in Protocol B above.
The sampling times, sample preparation and data analysis were as described
in Protocol B.
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TABLE 1
Mean
Exam Ip a % Bioavailabilitv Protocol
No.
Example 1 0.0 A
Example 2 0.0 A
Example 3 2.5 A
Example 4 37.4 B
Example 5 56.7 D
Example 6 72.7 B
Example 7 42.1 C
Example 8 62.0 D
Example 9 53.7 D
Example 10 78.9 C
Example 11 100.0 C
Example 12 58.3 B
Example 13 89.7 C
Example 14 65.1 C
Example 16 87.2 C
Example 17 78.2 C
Example 18 66.4 C
Example 19 100.0 C
This data indicates that solution formulations provided significantly better
bioavailability than non-formulated compound 111.
Compounds I, II and III are inhibitors of HIV-1 and HIV-2 protease. They
are useful for inhibiting an HIV infection and treating AIDS in humans. Total
daily dose of compound 1, II or III administered to a human in single or
divided
doses may be in amounts, for example, from 0.001 to 1000 mg/kg body weight
daily but more usually 0.1 to 50 mg/kg body weight daily. Dosage unit
compositions may contain such amounts of submultiples thereof to make up the
daily dose. It will be understood, however, that the specific dose level for
any
particular patient will depend upon a variety of factors including the age,
body
weight, general health, sex, diet, time of administration, rate of excretion,
drugs
administered in combination and the severity of the particular disease
undergoing therapy.
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The foregoing is merely illustrative of the invention and is not intended to
limit the invention to the disclosed compounds, methods and compositions.
Variations and changes which are obvious to one skilled in the art are
intended
to be within the scope and nature of the invention which are defined in the
appended claims.
