Note: Descriptions are shown in the official language in which they were submitted.
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1,5-Benzodiazepine Derivatives
This invention relates to novel 1,5-benzodiazepine derivatives, to processes
for
their preparation, to pharmaceutical compositions containing them and to their
use in medicine. More particularly, it relates to compounds which exhibit
agonist
activity for CCK-A receptors.
Cholecystokinin (CCK) is a peptide found in the gastrointestinal tract and the
central nervous system. see A.J. Prange et al., Ann. Reports Med. Chem. 77,
31, 33 (1982), J. A. Williams, Biomed Res. 3, 107 (1982) and V. Mutt,
Gastrointestinal Hormones, G.B.J. Green, Ed., Raven Press, N.Y., 169. CCK
has been implicated inter alia as a physiological satiety hormone involved in
appetite regulation, see Della-Ferra et al, Science, 206, 471 (1979), Saito et
al.,
Nature, 289, 599, (1981 ), G.P. Smith, Eating and Its Disorders, A.J. Stunkard
and
E. Stellar, Eds, Raven Press, New York, 67 (1984), as a regulator of
gallbladder
contraction and pancreatic enzyme secretion, an inhibitor of gastric emptying,
and as a neurotransmitter, see A.J. Prange, supra, J.A. Williams, Biomed Res.,
3,
107 (1982); J.E. Morley, Life Sci. 30, 479, (1982). Gastrin is a peptide
involved in
gastric acid and pepsin secretion in the stomach, see L. Sandvik, et al.,
American
J. Physiology, 260, 6925 (1991 ), C.W. Lin, et al., American J. Physiology,
262,
61113, (1992). CCK and gastrin share structural homology in their C-terminal
tetrapeptide: Trp-Met-Asp-Phe.
Two subtypes of CCK receptors have been identified, designated as CCK-A and
CCK-B, and both have been found in the periphery and central nervous systems.
It has recently been reported that CCK-B receptors are similar to the gastrin
receptor, see Pisegna, J.R., de Weerth, A, Huppi, K, Wank, S.A., Biochem.
Biophys. Res. Commun. 189, 296-303 (1992). CCK-A receptors are located
predominantly in peripheral tissues including the pancreas, gallbladder,
ileum,
pyloric sphincter and vagal afferent nerve fibers; CCK-A receptors are found
to a
lesser extent in the brain, see T.H. Moran, et al., Brain Res., 362, 175-179
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2
(1986), D.R. Hill, et al., Brain Res, 4545, 101, (1988), D.R. Hill, et al.,
Neurosci
Lett., 89, 133, (1988), R.W. Barret, et al., Mol. Pharmacol., 36, 285, (1989),
D.R.
Hill, et al., J. Neurosci, 10, 1070 (1990), V. Dauge et al., Pharmacol Biochem
Behac., 33, 637, (1989), while CCK-B receptors are found predominantly in the
brain, see V.J. Lotti and R.S.L Chang, Proc. Natl. Acad. Sci. U.S.A., 83, 4923
(1986), J.N. Crawley, Trends Pharm. Sci., 88, 232, (1991 ).
CCK agonist activity has been linked to inhibition of food intake in animals
and
thus weight loss, see Della-Fera, et al, supra, K.E. Asin, et al, Intl.
Conference on
Obesity, abstract pp.40 (1990). It has been suggested that CCK acts in the
periphery through vagal fibers and not directly on the brain to produce
satiety,
see Smith, G.P. and Cushin, B.J., Neuroscience Abstr., 4, 180 (1978), Smith,
G.P., Jerome, C., Cushin, B.J., Eterno, R., and Simansky, K.J., Science, 272,
687-689, (1981 ).
U.S. Patent No. 5,646,140 (Sugg, et al.) discloses certain 3-amino 1,5
benzodiazepine compounds which exhibit agonist activity for the CCK-A receptor
thereby enabling them to modulate the hormones gastrin and cholecystokinin
(CCK) in mammals. See in particular, the compound of Example 7. Certain of
these compounds also exhibit antagonist activity at CCK-B receptors.
Briefly, in one aspect, the present invention provides an enantiomerically
enriched compound of Formula (I) or a pharmaceutically acceptable salt or
solvate thereof.
~N w
O~ O
I N N~N ~ COZH
N O IOI I ~
I
w
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The compound of Formula (I) is 3-{3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-
dioxo-5-phenyl-2,3,4,5-tetrahydro-1 H-benzo[b][1,4]diazepine-3-yl]-ureido}
benzoic acid. This compound has a chiral carbon on the benzodiazepine ring.
Applicants have found that the enantiomer which rotates light in the positive
direction, under the conditions described below, is preferred. This enantiomer
which is hereinafter referred to as the (+) enantiomer has the (S)
configuration
according to the Cahn Ingold Prelog convention. Applicants have found that
this
isomer has improved properties over the racemic mixture and is therefore more
suitable than the racemic mixture for the treatment of obesity and other CCK-A
mediated diseases or conditions. As used herein, "enantiomerically enriched"
means that there is more of the (+) enantiomer than the (-) enantiomer as
opposed to the racemic mixture which has equal amounts of each isomer. As
used herein " the compound of this invention" or "the enantiomerically
enriched
compound of this invention", and expressions containing these or similar
phrases,
include pharmaceutically acceptable salts and solvates thereof. The "(+)
enantiomer" refers to the optical rotation of the enantiomer and not to salts
and
solvates thereof. Preferred salts and solvates will be salts and solvates of
the (+)
enantiomer of the compound of Formula (I) regardless of the optical rotation
of
the salt or solvate.
Preferably, the (+) enantiomer is at least %90 of the total amount of the
enantiomerically enriched compound. More preferably, the (+) enantiomer is at
least %96 of the total amount of the compound. Most preferably, the (+)
enantiomer is at least %99 of the total amount of the compound.
The (+) enantiomer of the present invention exhibits CCK-A agonist activity
and
can be considered a full cholecystokinin agonist in that it binds to CCK-A
receptors and fully stimulates gallbladder contraction and reduces feeding in
animal paradigms. For example, (+) enantiomer of this invention should be
useful
for the treatment of obesity as well as related pathologies, such as
hypertension,
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gallbladder stasis, and diabetes, indirectly through weight loss and directly
through CCK-A mediated delayed gastric emptying. Moreover, the (+)
enantiomer disclosed herein provides for new approaches for inducing satiety,
providing for appetite regulation and modifying food intake in mammals,
especially humans, to regulate appetite, treat obesity and maintain weight
loss.
Therefore, in a further aspect of the present invention, there is provided
herein a
method for the treatment, in a mammal, including man, of a CCK-A mediated
disease or condition comprising administering to the patient a therapeutically
effective amount of the (+) enantiomer of this invention.
According to another aspect, the present invention provides the use of the
enantiomerically enriched compound of this invention or a pharmaceutically
acceptable salt or solvate thereof for the manufacture of a medicament for the
treatment of CCK-A medicated diseases or conditions.
It will be appreciated by those skilled in the art that reference herein to
treatment
extends to prophylaxis as well as the treatment of established diseases or
symptoms. Moreover, it will be appreciated that the amount of the preferred
enantiomer of the invention required for use in treatment will vary with the
nature
of the condition being treated and the age and the condition of the patient
and will
be ultimately at the discretion of the attendant physician or veterinarian. In
general, however, doses employed for adult human treatment will typically be
in
the range of 0.02 - 5000 mg per day, e.g., 1-1500 mg per day. The desired dose
may conveniently be presented in a single dose or as divided doses
administered
at appropriate intervals, for example as two, three, four or more sub-doses
per
day.
While it is possible that the enantiomerically enriched compound of the
present
invention may be therapeutically administered as the raw chemical, it is
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preferable to present the active ingredient as a pharmaceutical composition.
Accordingly, the present invention further provides for a pharmaceutical
composition comprising the enantiomerically enriched compound of this
invention
together with one or more pharmaceutically acceptable carriers and/or
excipients
5 therefore and, optionally, other therapeutic and/or prophylactic
ingredients. The
carriers) and/or excipients therefor must be "acceptable" in the sense of
being
compatible with the other ingredients of the formulation and not deleterious
to the
recipient thereof.
Formulations of the present invention include those especially formulated for
oral,
buccal, parenteral, implant, topical or rectal administration, however, oral
administration is preferred. For buccal administration; the composition may
take
the form of tablets or lozenges formulated in conventional manner. Tablets and
capsules for oral administration may contain conventional excipients such as
binding agents, (for example, syrup, acacia, gelatin, sorbitol, tragacanth,
mucilage of starch or polyvinylpyrrolidone), fillers (for example, lactose,
sugar,
microcrystalline cellulose, maize-starch, calcium phosphate or sorbitol),
lubricants
(for example, magnesium stearate, stearic acid, talc, polyethylene glycol or
silica), disintegrants (for example, potato starch or sodium starch
glycollate) or
wetting agents, such as sodium lauryl sulphate. The tablets may be coated
according to methods well-known in the art, including enteric coatings.
Alternatively, the preferred enantiomer of the present invention may be
incorporated into oral liquid preparations such as aqueous or oily
suspensions,
solutions, emulsions, syrups or elixirs, for example. Moreover, formulations
containing these the preferred enantiomer may be presented as a dry product
for
constitution with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending agents
such as sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin,
hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or
hydrogenated edible fats; emulsifying agents such as lecithin, sorbitan mono-
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oleate or acacia; non-aqueous vehicles (which may include edible oils) such as
almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl
alcohol;
and preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid.
Such preparations may also be formulated as suppositories, e.g., containing
conventional suppository bases such as cocoa butter or other glycerides.
Additionally, compositions the present invention may be formulated for
parenteral
administration by injection or continuous infusion. Formulations for injection
may
take such forms as suspensions, solutions, or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may be in
powder
form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free
water)
before use.
The composition according to the invention may also be formulated as a depot
preparation. Such long acting formulations may be administered by implantation
(for example, subcutaneously or intramuscularly) or by intramuscular
injection.
Accordingly, the preferred enantiomer of the invention may be formulated with
suitable polymeric or hydrophobic materials (as an emulsion in an acceptable
oil,
for example), ion exchange resins or as sparingly soluble derivatives as a
sparingly soluble salt, for example.
The compositions according to the invention may contain between 0.1 - 99% of
the active ingredient, conveniently from 30 - 95% for tablets and capsules,
and 3
- 50% for liquid preparations.
The (+) enantiomer of this invention can made by first making the racemic
mixture as described in Example 7 in U.S. Patent No. 5,646,140 and then
separating the enantiomers by chiral chromatography.
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Alternatively the (+) enantiomer may be prepared by reaction of the
appropriate
enantiomer, namely the (S)-enantiomer of the amine of formula (II);
N
O
N
NHZ
(II)
N O
with an isocyanate of formula (III; wherein R is a carboxyl protecting group
e.g. t-
butyl), an imidazolide of formula (IV; wherein R is a carboxyl protecting
group e.g.
t-butyl) or an optionally substituted phenyl carbonate of formula (V; wherein
R is a
carboxyl protecting group e.g. t-butyl and R~ is hydrogen or a conventional
phenyl
substituent e.g. N02) followed by removal of the carboxyl protecting group R.
0
O=C=N NON
(III) COzR (IV) C02R
~O
/ O~~ \ C02R
R~
(V)
The reaction conveniently takes place in the presence of a suitable solvent
such
as an ether (e.g. tetrahydrofuran) or a halohydrocarbon (e.g. dichloromethane)
or
nitrite (e.g. acetonitrile) at a temperature in the range of 0-80°C.
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Conveniently the required enantiomer of the amine (II) may be used in the form
of a salt thereof e.g. R-camphorsulphonic acid salt and in this embodiment the
reaction may be carried out in the presence of a base e.g. of a tertiary amine
such as triethylamine.
The hydrolysis of the carboxyl protecting group may be carried out using
conventional procedures. (Protecting groups in Organic Synthesis T. Greene,
Ed,
Wiley Interscience, New York, p168, 1981 ). Thus for example when R is a t-
butyl
group this may be removed by hydrolysis with an appropriate acid such as
hydrochloric acid, trifluoroacetic acid or formic acid using established
procedures.
For example by reaction with hydrochloric acid in a solvent such as 1,4-
dioxane,
or by reaction with formic acid in a solvent such as acetone or aqueous
acetone
and with heating.
The required (S)-enantiomer of the amine of Formula (II);
N \
O 1 O
N
NHz
N O
can be prepared by resolution of the corresponding racemic amine via chiral
HPLC chromatography or through crystallization-induced asymmetric resolution
via the R-camphorsulfonic acid salt.
The racemic amine (II) can be prepared by the method described in Intermediate
11 of US Patent No. 5,646,140.
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Alternatively, the racemic amine (II) may be prepared by concomitant reduction
and hydrogenolysis of the oxime (VI) wherein R2 is an optionally substituted
benzyl group.
N
0
N
N-O R2
N O
(vi)
The reaction is conveniently carried out using a suitable palladium catalyst
e.g.
palladium on carbon e.g. 5% Pd/on charcoal in the presence of hydrogen or
aqueous ammonium formate and in a solvent such as an aqueous alkanol e.g.
ethanol, isopropanol or industrial methylated spirits, or tetrahydrofuran.
Conveniently the reaction is carried out with heating e.g. 40-80° such
as 60°C.
Examples of suitable R2 groups for use in the reaction include benzyt, or
substituted benzyl, such as p-methoxybenzyl, or benzhydryl.
The oxime (VI) may be prepared by reaction of the ortho-phenylene diamine
derivative (VII)
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N
CO
i H2
NH (VII)
NH
Ph
with an activated derivative of the di acid (VIII) wherein R2 is an optionally
substituted benzyl group.
~oR2
N
HO
~OH
O I IO
(VIII)
Conveniently the activated derivative of the di acid (VIII) is the
corresponding
diacyl halide e.g. chloride and this is prepared in situ by reaction of the di
acid
5 (VIII) with an oxalyl halide e.g. oxalyl chloride. The reaction is
conveniently
carried out on an aprotic solvent such as an ester e.g. ethyl acetate,
toluene,
dichloromethane, dimethoxyether or mixtures thereof and in the presence of
dimethylformamide.
10 The di acid (VIII) is conveniently prepared by reaction of a di
alkylketomalonate
e.g. diethyl ketomalonate with the corresponding hydroxylamine R20NH2 in a
solvent such as an alkanol e.g. ethanol or industrial methylated spirits and
in the
presence of a base e.g. pyridine, followed by hydrolysis of the corresponding
di
alkyl-oximino malonate using aqueous sodium hydroxide.
In a further aspect the invention provides a process for preparing the (S)
enantiomer of the compound of formula (I) substantially free of the (R)
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enantiomer thereof from the racemic amine (II) as described above wherein the
racemic amine (II) has been prepared from the oxime (VI) as described above
and more particularly, wherein the oxime (VI) has been prepared from the
compounds (VII) and (VIII).
Isocyanates of Formula (III) may be purchased or prepared by the reaction of
the
corresponding amine (VI) with phosgene or triphosgene in a suitable solvent
such
as methylene chloride. Imidazolides of Formula (IV) can be prepared by
treatment of the corresponding amine (VI) with carbonyl diimidazole in a
suitable
solvent (dichloromethane, ether, tetrahydrofuran) at a temperature ranging
from 0
- 80o C (conveniently at room temperature). The optionally substituted phenyl
carbamates of Formula (V) can be prepared by the reaction of the corresponding
amine (VI) with the optionally substituted phenyl chloroformate in the
presence of
a base (pyridine, triethylamine) in a suitable solvent (dichloromethane) and
at a
temperature of 0 - 50o C. The amines of formula (VI) are either known
compounds and can be prepared by procedures analogous to those used to
prepare the known compounds.
The following examples, which are non-limiting, illustrate the invention.
In the Examples the abbreviations EtOAc = ethyl acetate; MeOH = methanol,
DMF = N, N-dimethylformamide; IPA = isopropyl alcohol; IMS = industrial
methylated spirits.
Intermediate 1
(+]-2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo-[b][1,4]diazepin-
1-yl)-N-isopropyl-N-phenylacetamide camphorsulfonic acid salt
(+/-)-2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo-[b][1,4]diazepin-1-
yl)-N-isopropyl-N-phenylacetamide (10g) and R-camphorsulfonic acid (4.98g)
were stirred in tetrahydrofuran (35m1) and toluene (65m1) to give a solution.
The
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solution was heated to 70°C with formation of a suspension. Water
(0.4m1) was
added followed by a solution of 2-pyridinecarboxaldehyde (0.24g) in toluene
(5m1). The mixture was heated at 70°C for 3h and then cooled to
25°C over 5h
and stirred at 25°C for 16h. The suspension was chilled to 0-5C for
1.5h. The
solid was collected by filtration washing with toluene/tetrahydrofuran (2:1 )
(10m1).
Drying in vacuo at 50°C yielded the title compound as a white solid
(12.6g).
Chromatographic analysis: Eluent: 30%Isopropyl Alcohol, 70% Heptane + 0.05%
Diethylamine; Column: 25cm x4.6mm i.d., Chiralpak AD; Flow rate: 1 ml/minute;
Temperature: 40 degrees C; Detection: UV 230nm; Injection volume: 10 NL;
Sample solution: 0.1 mg/ml; sample in 30% Isopropyl alcohol, 70% Heptane.
Sample solutions were injected immediately after preparation. Retention times:
(+) enantiomer, 8.2 minutes. The unwanted (-) Enantiomer (12.7minutes) was
below limits of detection.
Intermediate 2
3-Nitrobenzoic acid t-butyl ester
Potassium t-butoxide (3.82g) was added to a solution of 3 nitrobenzoyl
chloride
(S.OOg) in anhydrous tetrahydrofuran (70 ml) and stirred under nitrogen for 2
hrs.
The reaction mixture was concentrated in vacuo and partitioned between
dichloromethane and water. After separating the phases, the aqueous layer is
back-extracted with ethyl acetate. The organic layers were combined, dried
over
anhydrous magnesium sulfate, filtered and then concentrated in vacuo. The
crude product was purified on flash grade silica gel using 0-5% gradient of
ethyl
acetate in n-hexane. Fractions containing the product were combined,
concentrated in vacuo, and then dried under high vacuum to provide the title
compound as an oil (3.82g). 1 H NMR (300 MHz, CDC13) b = 1.63 (s, 9H); 7.62
(t,
J=7.9 Hz, 1 H); 8.29-8.41 (m, 2H); 8.78-8.80 (m, 1 H). MS (CI): [M+H]+ = 224.
Intermediate 3
3-Amino-benzoic acid t-butyl ester
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A solution of 3-nitro-benzoic acid t-butyl ester (3.77g), in absolute ethanol
(50 ml)
was combined with palladium on carbon (10 wt%, 0.30 g) and stirred under
atmospheric hydrogen for approximately 3 hrs. The reaction mixture was
filtered
through a pad of diatomaceous earth and then concentrated in vacuo to an oil
which crystallized when dried under high vacuum providing the title compound
as
a tan solid (3.28g). 1 H NMR (300 MHz, CDC13) 8 = 1.58 (s, 9H); 6.79-6.87 (m,
1 H), 7.19 (t, J=8.5 Hz, 1 H); 7.24-7.34 (m, 1 H); 7.38 (d, J=8.0 Hz, 1 H). MS
(CI):
[M+H]+ = 194.
Intermediate 4
3-Isocyano-benzoic acid t-butyl ester
Triphosgene (13.428g) was added to a solution of 3-amino-benzoic acid t-butyl
ester (26.50g) and triethylamine (38.23m1) in anhydrous tetrahydrofuran (600
ml)
at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 2h,
then concentrated in
vacuo to a white solid. The crude product was slurried in hexane (500 ml),
filtered, and the filtrate was concentrated in vacuo to afford the title
compound as
an oil (21.54 g, 71.6%). The crude isocyanate was used without further
purification. 'H NMR (300 MHz, CDC13) 8= 1.59 (s, 9H); 7.23 (bd, J=7.8 Hz, 1
H);
7.36 (t, J=7.8 Hz, 1 H); 7.69 (bs, 1 H); 7.81 (d, J=7.8 Hz, 1 H).
Intermediate 5
(+)3-{3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydro-1H-benzo[b][1,4]diazepine-3-yl]-ureido} benzoic acid t-butyl ester
Method A
Intermediate (I; 66.30g) was slowly added to a solution of 3-Isocyano-benzoic
acid t-butyl ester (21.54g) in anhydrous tetrahydrofuran (750 ml).
Triethylamine
(13.70m1) was added dropwise to the reaction mixture. The resulting reaction
mixture was stirred at ambient temperature overnight. The reaction mixture was
poured into water (3000 ml) to afford a white solid. The solid was collected
by
filtration washing with water (3 X 500 ml). Drying by vacuum filtration
yielded the
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title compound as a white solid (65.01g). The crude title compound was used
without further purification. Chromatographic characterization: Eluent:
30%Isopropyl Alcohol, 70% Heptane + 0.05% Diethylamine. Column: 25cm
x4.6mm i.d., Chiralpak AD; Flow rate: 1 ml/minute; Temperature: 40 degrees C;
Detection: UV 230nm; Injection volume: 10pL; Sample solution: 0.1 mg/ml sample
in 30% Isopropyl alcohol, 70% Heptane. Sample solutions were injected
immediately after preparation. Retention times: (+) enantiomer: 15.6 minutes.
The unwanted (-) enantiomer: (13.3 minutes) was below the limits of detection.
Method B
To a suspension of carbonyl diimidazole (13.2g) in dichloromethane (55m1)
stirring at 20°C was added dropwise a solution of 3-Amino-benzoic acid
t-butyl
ester (15.8g) in dichloromethane (40m1) over 30 mins. The resulting solution
was
stirred at 20°C for 1 hour. To this solution was added a solution of
Intermediate 1
(50g) in dichloromethane (130m1) over 5 minutes. The reaction was quenched by
addition of water (200m1) and stirred for 10 minutes. The phases were
separated
and the organic phase washed with water. The organic phase was concentrated
at atmospheric pressure and 100m1 dichloromethane was removed by distillation.
t-Butyl methyl ether (700m1) was added and the mixture was stirred overnight
at
20°C. The solid was collected by filtration and washed with t-Butyl
methyl ether
(100m1) and dried in vacuo at 45°C to provide the title compound as a
white solid
(42g, 63mmol).
Intermediate 6
Diethyl2-[(benzyloxy)imino]malonate
Di-ethylketomalonate (60g) was added at 20°C to a stirred
suspension of O-
benzylhydroxylamine (57.8g) in IMS (500m1) containing pyridine (30m1). The
reaction was heated at 75°C for 4hr. The reaction was cooled and
solvents
removed under reduced pressure. The residue was partitioned between EtOAc
(500m1) and water (300m1) and the organic layer separated, washed with water
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(250m1) and dried over MgS04. Solvents were evaporated to give the title
compound 95.3g, as a colourless oil (99%th, ca 3%w/w residual EtOAc) which
was used without further purification.
5 1 H NMR (300MHz, CDC13) 7.4 (m, 5H), 5.35 (s, 2H), 4.35 (m, 4H), 1.3 (m,
6H).
Intermediate 7
2-[(benzyloxy)imino]malonic acid
To a solution of Intermediate 6 (40g) in MeOH (80m1) was added 2M NaOH
10 (200m1) over 20 mins. The reaction was stirred at room temperature for 2hr.
MeOH was removed under reduced pressure and the residual solution was
acidified to pH 2 by dropwise addition of conc.HCl (~30m1) while cooling to
maintain the temperature below 35°C. A thick white slurry was formed
which was
diluted with water (50m1) to aid mobility. The solids were collected by
filtration,
15 washed with chilled water (25m1) and dried in vacuo at 55°C to give
the title
compound as a white solid (17g) found to contain ca.10%w/w residual inorganic
salts. Corrected yield - 45%th. Used without further purification.
1 H NMR (300MHz, D20) 7.4 (m, 5H), 5.2 (s, 2H)
Intermediate 8
2-[-3-[(Benzyloxy)imino]-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepinyl)-N-isopropyl-N-phenylacetamide
Oxalyl chloride (38.3g) was added dropwise (~1 hr) to a stirred suspension of
Intermediate 7 (40g, corrected for salt content to 31.4g) in EtOAc (200m1)
containing DMF (0.5m1, 5 mol%). The mixture was stirred at 25°C for 0.5
hour
then filtered through a pad of Dicalite, washing with EtOAc (40m1) to give a
clear
yellow solution. The solution was added (~5mins) to a stirred slurry of N-
isopropyl-N-phenyl-2-(2-phenylaminophenylamino)-acetamide (50g) in EtOAc
(120m1) at 25°C. The mixture was warmed to 60°C and a dark
purple solution
formed. After 1 hr, EtOAc (200m1) was removed by atmospheric distillation. IPA
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(120m1) and water (40m1) were added and the mixture distilled further to
remove
more solvent (80m1). IPA (40m1) and water (40m1) were added and a further
amount of solvent was distilled out (80m1). The reaction mixture was cooled to
25
°C over 1.5hr and the solids collected by filtration. The solids were
washed with
IPA (2 x 120 ml), water (1 x 120m1) and finally IPA again (1 x 40m1) then
dried in
vacuo at 55°C to give the title compound as a salmon pink powder
(56.6g).
1 H NMR (300MHz, CDC13) 2:1 mixture of isomers about the oxime 7.6-6.95 (m.
18H), 6.9 (t 1 H), 5.3 (m, 2H), 4.95 (m, H), 4.65 (d, 0.33H), 4.4 (d, 0.67H),
4.1
(d, 0.67H), 4.0 (d, 0.33H), 0.95 (m, 6H)
Intermediate 9
(~)-2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo-[b][1,4]diazepen-
1-yl)-N-isopropyl-N-phenylacetamide
To a stirred suspension of Intermediate 8 (3g) and ammonium formate (2.08g) in
IMS (30m1) and water (3m1) was added 5% Pd/C (50% w/w water) (0.25g). The
mixture was heated under a nitrogen atmosphere at 60°C overnight. The
hot
reaction mixture was filtered through Dicalite to remove the catalyst. The
catalyst
was washed with hot IMS (60m1) and filtered. The filtrates were concentrated
under reduced pressure to give the title compound as a white solid (2.34g).
Example 1
Chromatographic Resolution of Enantiomers
(+) and (-)-3-{3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-dioxo-5-phenyl
2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-3-yl]-ureido} benzoic acid.
Racemic 3-f3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5
tetrahydro-1 H-benzo[b][1,4]diazepine-3-yl]-ureido) benzoic acid was prepared
as
described for example 7 in U.S. Patent No. 5,646,140 and resolved by chiral
HPLC under the following conditions: a 250 x 4.0 pm (id) column, Sum Diacel
Chiracel OD-R; the eluent was 80:20:0.1:1, 80 parts acetonitrile, 20 parts
water,
0.1 part triethylamine, and 1 part acetic acid; the UV detection wavelength
was
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230nm; the temperature was ambient; the flow rate was 1 ml/min; and the
injection volume was 20u1. Under these conditions the (+) enantiomer had a
retention time of 6.50 minutes and the (-) isomer had a retention time of 3.89
minutes.
Example 2
(+)-3-f 3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydro-1H-benzo[b][1,4]diazepine-3-yl]-ureido} benzoic acid
Method A
(+)-3-f3-[1-(Isopropyl-phenyl-carbamoylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydro-1H-benzo[b][1,4]diazepine-3-yl]-ureido} benzoic acid t-butyl ester
(Intermediate 5: 65.01 g) was stirred in 4N hydrochloric acid in dioxane (280
ml) at
ambient temperature for 6h. The reaction mixture was concentrated in vacuo and
the resulting solid/oil was triturated in water to afford a white solid. The
white
solid was collected by filtration and washed with water (2 X 500 ml). The
crude
product was dissolved in hot acetone (250 ml) and water (275 ml) was added
until the solution became cloudy. Additional acetone (40 ml) was added and the
solution was heated until a clear solution was obtained. The solution was set
aside and allowed to cool. The resulting white solid was collected by
filtration
washed with water (3 X 100 ml) and dried under house vacuum (20-25 in Hg) at
40-50 °C to provide the title compound as a white solid (40.496g).
Analyzed for
purity by chiral chromatography (see chromatographic resolution protocol).
Optical rotation (0.712g in 100mL acetone) [a]p = +84.3. 'H NMR (300 MHz,
DMSO) ~= 0.95 (d, J=7.5 Hz, 3H); 0.97 (d, J=7.2 Hz, 3H); 4.18 (d, J=16.7 Hz,
1 H); 4.48 (d, J=16.7 Hz, 1 H); 4.78 (m, 1 H); 5.02 (d, J=7.8 Hz, 1 H); 6.91
(d, J=7.8
Hz, 1 H); 6.95 (bd, J=8.1 Hz, 1 H); 7.22-7.57 (m, 19H); 8.00 (s, 1 H); 9.34
(s, 1 H);
12.78 (s, 1 H). MS (ES): [M+1] = 606.1; [M+Na] = 628.1; [M-1] = 604.1.
Method B
Intermediate 5 (5g,) was added to a mixture of acetone (15m1) and formic acid
(25m1) at room temperature and the resulting suspension was heated to
55°C
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and stirred for 5h. Water (30m1) was added dropwise to the solution ensuring
the
contents temperature was kept above 50°C. The resultant slurry was
stirred at
55°C for 1 h, cooled to room temperature and stirred overnight. The
slurry was
filtered and washed with water (3x25m1). The residue was added to IMS (50m1)
and the slurry was heated to 45°C and stirred overnight. The slurry was
cooled
to room temperature, filtered and the damp cake was dried in vacuo at
55°C to
the title compound as a white solid (3.40g).
Chiral chromatographic analysis showed that the required product of the
reaction
contained 0.7% of the unwanted (-) enantiomer.
Pharmacy Examples
Oral solution
Active ingredient 0.5-800 mg
Polyethylene glycol 400 NF q.s. to 50 ml
The active ingredient is suspended in Polyethylene glycol 400 and is then
dissolved by sonication to produce the oral solution.
Oral suspension
Active ingredient 0.5-80 mg
Polysorbate 80 NF (Tween 80) 0.02 ml
Sterile Water for Irrigation q.s. to 20m1
The active ingredient is added to a 0.1 % (v/v) Tween 80 solution (20 ml) and
the mixture is then sonicated or shaken to produce the oral suspension.
Tablets
a. Active ingredient 6mg
Lactose anhydrous USP 136.2mg
Sodium Starch Glycolate USP/NF 6mg
Stearic Acid USP/NF 1.5mg
Colloidal Silicon Dioxide USP/NF 0.3mg
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Compression weight 150mg
The active ingredient, lactose, and sodium starch glycolate are sieved through
a
590 micron sieve and blended in a suitable mixer. Stearic acid (screened
through
a 250 micron sieve) and colloidal silicon dioxide are added to and blended
with
the active blend. The blend is compressed into tablets using suitable punches.
b. Active ingredient 6mg
Microcrystalline cellulose USP/NF 136.5mg
Crospovidone USP/NF 6mg
Magnesium stearate USP/NF 1.5mg
Compression weight 150mg
The active ingredient, microcrystallline cellulose and crospovidone are sieved
through a 590 micron sieve and blended in a suitable blender. The magnesium
stearate is screened (through a 250 micron sieve) and blended with the active
blend. The resultant blend is compressed into tablets using suitable tablet
punches.
Capsules
a. Active ingredient 6mg
Microcrystalline cellulose USP/NF 128.25mg
Sodium starch glycolate USP/NF 15mg
Magnesium stearate USP 0.75mg
Fill weight 150mg
The active ingredient, microcrystalline cellulose, and sodium starch glycolate
are
screened through a 590 micron mesh sieve, blended together and lubricated with
magnesium stearate, that has been screened through a 250 micron sieve. The
blend is filled into capsules of a suitable size.
b. Active ingredient 6mg
Lactose monohydrate USP 130.5mg
Povidone USP 6mg
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Crospovidone NF 6mg
Magnesium stearate 1.5mg
Fill weight 150mg
5 The active ingredient and lactose are blended together and granulated with a
solution of Povidone. The wet mass is dried and milled. The magnesium stearate
and Crospovidone are screened through a 250 micron sieve and blended with the
granule. The resultant blend is filled into hard gelatin capsules of a
suitable size.
BIOLOGICAL ASSAYS
The (+) and (-)-enantiomers and the racemic mixture were characterized in the
following assays. The results of these assays are summarized in the table
below.
Guinea Pig Gallbladder Tissue Preparation. Gallbladders were removed from
male Hartley guinea pigs sacrificed with C02 atmosphere. The isolated
gallbladders were cleaned of adherent connective tissue and cut into two rings
from each animal (2-4 mm in length). The rings were suspended in organ
chambers containing a physiological salt solution (118.4 mM NaCI, 4.7 mM KCI,
1.2 mM MgS04, 2.5 mM CaCl2, 1.2 mM KH2P03, 25 mM NaHC03 , 11.1 mM
dextrose). The bathing solution was maintained at 37°C and aerated with
95%
02/5% C02 to maintain pH = 7.4. Tissues were connected via gold chains and
stainless steel mounting wires to isometric force displacement transducers
(Grass, Model FT03 D). Responses were then recorded on a polygraph (Grass,
Model 7E). One tissue from each animal served as a time/solvent control and
did
not receive test compound. Rings were gradually stretched (over a 120-min.
period) to a basal resting tension of 1 gm which was maintained throughout the
experiment. During the basal tension adjustment period, the rings were exposed
to acetylcholine (10-6 M) four times to verify tissue contractility. The
tissues were
then exposed to a submaximal dose of sulfated CCK-8 (Sigma, 3 X 10-9 M).
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After obtaining a stable response, the tissues were washed out 3 times rapidly
and every 5 to 10 minutes for 1 hour to reestablish a stable baseline.
Agonist EC50's. Compounds were dissolved in dimethylsulfoxide (DMSO) then
diluted with water and assayed via a cumulative concentration-response curve
to
test compound (10-11 to 3 X 10-5 M) followed by a concentration-response curve
to sulfated CCK-8 (10-10 to 10-6 M) in the presence of the highest
concentration
of the test compound. As a final test, acetylcholine (1 mM) was added to
induce
maximal contraction. A minimum of three determinations of activity were made
for each test compound.
Establishment of stable CCK receptor bearing cell lines. The cDNA clones
for the human CCK-A18 or CCK-B19 receptors were ligated into pcDNA1-Neo
vector from Invitrogen Corp (San Diego, CA) for direct transfection. DNA was
prepared by the alkaline lysis method and transfected into CHO-K1 cells (ATCC,
Rockville, MD) using the Lipofectin reagent24 (Gibco BRL, Gaithersberg, MD).
Stable transfectants were initially selected by the use of Geneticin (Gibco
BRL)
and receptor bearing resistant cells were enriched by fluorescence-activated
cell
sorting based on binding of Fluorescein-Gly-[(NIe28,31]-CCK-8. Clonal lines
were subsequently established by the limiting dilution method.
Cell Membrane Preparation. CHO-K1 cells stably transfected with human
CCK-A or CCK-B receptor cDNA were grown at 37oC under a humidified
atmosphere (95% 02/5% C02) in Ham's F12 medium supplemented with 5%
heat inactivated fetal bovine serum. The cells were passaged twice weekly and
grown to a density of 2-4 million cells/mL. The cells were collected by
centrifugation (600 X g, 15 min, 4oC) and resuspended in buffer (20 mL, pH
7.4)
containing TrisHCl (25 mM), EDTA (5 mM), EGTA (5 mM), phenyl sulfonyl
fluoride (0.1 mM) and soybean trypsin inhibitor (100 pg/mL). Cells were
disrupted with a motorized glass teflon homogenizer (25 strokes) and the
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homogenate was centrifuged at low speed (600 X g, 10 min, 4oC). The
supernatent was collected and centrifuged at high speed (500,000 X g, 15 min
4oC) to pellet the particulate fraction. The low speed pellet was processed
three
additional times. High-speed particulate fractions were combined and
resuspended in buffer (1-5 mg protein/mL) and frozen at -80oC. Protein
concentration was determined according to manufacturer's directions using
BioRad reagent and bovine serum albumin as standard.
Receptor Binding Assays. 1251_golton Hunter CCK-8 (Amersham, 2000
Ci/mmol) was dissolved in binding buffer (pH 7.4, 100,000 cpm/25 ~,L)
containing
HEPES (20 mM), NaCI (118 mM), KCI (5 mM), MgCl2 (5 mM) and EGTA (1 mM).
Nonspecific binding was determined with MK-32920 (10 ~M, CCK-A) or L-
365,26021 (10 ~.M, CCK-B). Test compounds were dissolved in DMSO at a
stock concentration of 100 times the final assay concentration and diluted to
appropriate concentrations with binding buffer. Binding assays were performed
in triplicate using 96-well plates to which the following were added
sequentially:
test compound (25 ~L), 1251_golton Hunter CCK-8 (25 ~.L), buffer (pH 7.4, 150
~L) and receptor preparation (50 ~L). The final concentration of DMSO was 1
in all assay wells. After 3 hours at 30oC, the incubation was terminated by
rapid
filtration of the mixture onto glass filters (Whatman GF/B) with subsequent
washing to remove unbound ligand. Bound radioactivity was quantified by
gamma counting.
Intracellular Calcium Measurements: CHO-K1 cells stably transfected with
hCCK-A or hCCK-B receptors were grown on glass coverslips to 75-90%
confluency. The cells were loaded for 50 minutes in serum-free culture medium
containing 5 mM FURA2-AM and 2.5 mM probenecid. A JASCO CAF-102
calcium analyzer was used to measure changes in intracellular calcium
concentration by standard ratiometric techniques using excitation wavelengths
of
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340 nm and 380 nm. Cells were perfused with increasing concentrations of CCK-
8 (n = 2) or compounds (n = 2) until a plateau in the 340/380 ratio was
achieved.
A washout/recovery period of at least 10 minutes was allowed between
successive stimulations. The maximal response was normalized to the maximal
response induced by CCK-8. EC50's were calculated at the concentration
required to induce half-maximal response. In addition to the agonist
concentration-response curves, the CHO-K1 cells expressing the human CCK-B
receptor were perfused for 1 hour with three concentrations of compounds (10-
$,
10-', 10-6 M, n = 2), then a concentration response curves were acquired for
CCK-8 (10-'2 to 10-6M).
Anorexia Assays: Male Long-Evans rats (225-300 g) were conditioned for two
weeks to consume a palatable liquid diet (Bio-Serve F1657, Frenchtown, NJ)
after a 2 hr fast. On pretreatment day, rats were fasted (100 min) and
injected IP
with drug vehicle (propylene glycol, PG, 1 mUkg) and an oral preload of saline
(0.9% NaCI, 8 mL/kg). Liquid diet access was provided 20 min later and
consumption was measured at 30, 90 and 180 min. To qualify for the drug
treatment study, rats had to consume at least 8 mL of liquid diet within the
first 30
minutes on the pretreatment day. The next day, following the 100 min
deprivation, rats (8 - 10 animals per dose) were treated IP or PO with vehicle
(PG, 1 mL/kg) or various doses (0.01 to 10 ~,mol/kg) of test compound
dissolved
in PG (1 mUkg), immediately followed by the saline oral preload. Food access
was again provided 20 min later and food intake was measured at 30, 90 and 180
min. All food intake data were normalized for each rat to the respective
values
from the pretreatment day. Potency was determined at 30 min and efficacy at
the
min, 1 ~mol/kg dose.
Mouse gallbladder emptying assay: Makovec, F.; Bani, M.; Cereda, R.;
Chiste, R.; Pacini, M. A.; Revel, L.; Rovati, L. C. Antispasmodic Activity on
the
30 Gallbladder of the Mouse of CR1409 (Lorglumide), a Potent Antagonist of
Peripheral Chlolecystokinin. Pharmacol. Res. Commun. 1987, 19, 41-51.
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PApp values: A measurement of intestinal permeation determined using the in
vitro assay of Artursson P. and Karlson J. 1991, Biochem. Biophys Res.
Common.175, 880-885 (Correlation between oral drug absorption in humans and
apparent drug permeability co-efficients in human intestinal epithelical (CACO-
2)
cells).
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Pharmacological Comparison of Enantiomers & Racemate
Assay (+) (-) Racemate
Enantiomer Enantiomer
In vitro
GPGB ECSO (nM) 9.3 63 40
HCCK-A IC5o (nM) 148 191 123
HCCK-A EC5o (nM) 150 298 252
HCCK-B K; (nM) 3.2 22.3 10
HCCK-B ECSO(nM) Antagonist Antagonist Antagonist
Selectivity Ratio 46 8.6 12.3
PApp (x 10-' cmlsec)1.6 0.7
in vivo
Rat Anorexia
EDSO IP (~.mol/kg)0.034 0.48 0.06
EDSO PO (~.mol/kg)1.1 Inactive 2.0
Mouse Gallbladder
Emptying Assay
EDSO IP (~mol/kg)Ø002 0.012 0.007
EDSO PO (~mollkg) 0.007 Inactive' 0.055
1. Selectivity ratio = IC50 (hCCK-A)/Ki (hCCK-B)
5 2. Inactive up to 10 ~,mol/kg
3. No significant reduction in feeding observed with doses up to
10Nmol/kg.
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Three unexpected biological activities distinguish the (+) enantiomer from the
(-)
enantiomer and the racemate. Two of these activities relate to enhanced CCK-A
efficacy, which should improve the beneficial activity of this enantiomer. The
third
relates to the CCK-B antagonist activity of the (+) enantiomer which should
prove
beneficial through decreased toxicity.
The (+) enantiomer was four-fold more potent than the racemate in the in vitro
isolated guinea pig gallbladder test ("GPGB"). The (+) enantiomer was eight-
fold
more potent than the racemate in the mouse gallbladder emptying assay (oral
dosing). This increased potency is expected to be beneficial in the treatment
of
gallbladder stasis and in the treatment of obesity, since gallbladder stasis
is a
critical problem with rapid weight loss.
Anorectic agents are intended for chronic use and thus it is essential that
they
possess minimal risk for toxicity. The primary toxicity associated with the
use of
cholecystokinin is concomitant CCK-B receptor agonist activity. Activation of
the
CCK-B receptor is primarily associated with increased anxiety and increased
gastric acid secretion. The utility of CCK-B antagonists have been explored
for
both the development of anxiolytic agents and anti-ulcer agents. See, for
example, Lowe, J, "Cholecystokinin-B Receptor Antagonists" in Exp. Opin. Ther.
Patents, 5(3), pp 231-237 (1995).
The predominant CCK receptor subtype in the rodent pancreas is the CCK-A
subtype and activation of this subtype induces pancreatic hyperstimulation and
hypertrophy in rodents. Both of these activities are considered to be
undesirable.
Recently, the tissue distribution of CCK receptors in human tissues has been
reported. Surprisingly, the predominate receptor subtype in human pancreas is
the CCK-B receptor subtype. See, for example, Wank, S. A., "Cholecystokinin
Receptors" in American Journal of Physiology - Gastrointestinal & Liver
Physiology, 32(5):, pp 6628-6646, (1995). Thus, in humans, activation of the
CCK-B receptor (CCK-B agonist activity) could induce increased anxiety and
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gastric acid secretion, as well as pancreatic hyperstimulation and hypertrophy
with long term use.
In order to decrease the risk of undesirable in vivo CCK-B agonist activity,
the
preferred compound should have affinity for the CCK-B receptor and have
measurable CCK-B antagonist activity in in vitro assays. Both enantiomers and
the racemate are CCK-B antagonists. Although all three compositions have
similar human CCK-A receptor affinities (1C50) and efficacies (EC50), the (+)
enantiomer has the highest CCK-B receptor affinity (1C50) and selectivity (46-
fold). Thus, the (+) enantiomer is preferred both in terms of CCK-A potency
and
efficacy, as well as in terms of the minimal potential for CCK-B induced toxic
side
effects.
The (+) enantiomer of the invention is essentially non-toxic at
therapeutically
useful doses. For example, in single dose oral studies the maximum non-lethal
dose was found to be greater than 2000mg/kg in the rat and 1000mg/kg for male
mice and 500mg/kg for female mice.
