Note: Descriptions are shown in the official language in which they were submitted.
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This is a divisional application of Canadian Patent
Application Serial No. 610,076, filed on August 31, 1989.
This invention relates to a novel O-substituted
fumagillol derivative, O-chloroacetylcarbamoylfumagillol of
the formula:
(I)
N CH2C1
H
O O
which has angiogenesis inhibitory activity and is effective
in the treatment and prevention of various inflammatory
diseases (rheumatism, psoriasis, etc.), diabetic retinopathy
and cancer, among others.
Angiogenesis is deeply concerned in the course of
manifestation or progress of various diseases, for example
various inflammatory diseases (rheumatism, psoriasis, etc.),
diabetic retinopathy, and cancer. Therefore, to inhibit
angiogenesis is considered to contribute to the treatment and
prevention of such diseases. In fact, several groups of
researchers have so far searched for angiogenesis inhibitory
substances. As examples, there may be mentioned the study by
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Taylor et al.[Taylor, S. et al., Nature,:'.297, 307 (l982)] on
the applicability of protamine and the study by Folkman et al.
[Folkman, J. et al., Science,''2.21, 719 (1983)] on the combined
use of heparin and cortisone. Furthermore, patent applications
have been filed alleging, for example, that ascorbic acid
ethers and related compounds (Japanese Kokai Tokkyo Koho No.
58-131978) and the sulfated polysaccharide DS1152 (Japanese
Kokai Tokkyo Koho No. 63'119500) show angiogenesis inhibitory
activity. However, such substances are not yet fully
satisfactory from the activity viewpoint. The advent of
compounds superior in activity is waited for.
Accordingly, it is an object of the invention to
provide a novel compound having angiogenesis inhibitory
activity.
The present inventors searched for and evaluated
various compounds in an attempt to achieve the above object
and, as a result, found that O-substituted derivatives of
fumagillol, a hydrolyzate of fumagillin so far known as an
antimicrobial and antiprotozoal agent, have potent angiogenesis
inhibitory activity. Based on this finding, they have now
completed the present invention. Thus, the invention relates
to an O-substituted fumagillol derivative, O-chloroacetyl-
carbamoylfumagillol of the formula (I).
The compound of the present invention can be produced
by subjecting fumagillol [Tarbell, D. S. et al., J. Am. Chem.
Soc., 83, 3096 (1961)], which is a hydrolyzate of fumagillin
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produced by microorganisms, to carbamoylation using a carbamoyl-
ating agent in the manner mentioned below.
The carbamoylation reaction for the introduction of
a monosubstituted carbamoyl group is carried out generally by
bringing fumagillol into contact with an isocyanate according
to the following equation:
R5NC0 + fumagillol -~ Compound (I) .
In the above equation, R5 is chloroacetyl. The isocyanate is
used generally in an amount of about 1 to 5 moles per mole of
fumagillol.
This reaction is carried out generally in the
presence of a base. Usable as the base are, for example,
tertiary amines, alkali metal hydrogen carbonates, alkali
metal carbonates, alkali metal hydrides and organometals, such
as diisopropylethylamine, triethylamine, pyridine, N,N-dimethyl-
aminopyridine, sodium hydrogen carbonate, potassium hydrogen
carbonate, sodium and potassium carbonate, sodium and potassium
hydride, butyl lithium, and lithium diisopropylamide. The
level of addition of the base is generally about 1 to 5 moles
per mole of fumagillol.
This reaction is carried out generally in an organic
solvent which will not interfere with the reaction. Usable as
such inert organic solvent are amides, halogenated hydrocarbons,
ethers, esters, nitriles, nitro compounds, ketones and aromatic
hydrocarbons, such as dimethylformamide, dimethylacetamide,
dichloromethane, chloroform, 1,2-dichloroethane, diethyl ether,
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tetrahydrofuran, dioxane, methylacetate, ethylacetate,
isobutylacetate, methylpropionate, acetonitrile,
propionitrile, nitromethane, nitroethane, acetone, methyl
ethyl ketone, benzene and toluene. They may be used either
singly or in the form of a mixture of two or more of them
mixed together in an appropriate ratio. When a tertiary amine
is used as the base, the tertiary amine as such may also serve
as a solvent.
The optimal reaction temperature may depend on the
base and solvent and amounts thereof but is generally within
the range of -80~C to 100~C, preferably from 0~C to room
temperature. The reaction time required amounts to about 1
hour to 5 days.
The carbamoylation may also be effected by reacting
fumagillol with a chloroacetyl carbamoyl halide.
The carbamoyl halide is used generally in an amount
of about 1 to 5 moles per mole of fumagillol.
This reaction is carried out generally in the
presence of a base. Usable as the base are those tertiary
amines, alkali metal hydrogen carbonates, alkali metal
carbonates, alkali metal hydrides and organoalkali metals
mentioned above. The level of addition of the base is
generally about 1 to 5 moles per mole of fumagillol.
This reaction is carried out generally in an organic
solvent which will not interfere with the reaction. Usable as
such inert organic solvent are those amides, halogenated
hydrocarbons, ethers, esters, nitriles, nitro compounds,
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ketones and aromatic hydrocarbons mentioned above. They may
be used either singly or in the form of a mixture, in.an
appropriate mixing ratio, of two or more of them. When a
tertiary amine is used as the base, the tertiary amine as such
may also serve as a solvent.
The optimal reaction temperature may vary depending
on the base and solvent and amounts thereof. Generally,
however, the reaction is carried out at a temperature from
about 0~C to a temperature approximately equal to the refluxing
temperature of the reaction medium, preferably from about 25~C
to the refluxing temperature.
_ The O-substituted fumagillol derivative (I) thus
produced can be isolated by ~ se known means of separation
and purification (e. g. chromatography, crystallization) or
by other appropriate means.
The compound (I) has asymmetric centers within the
molecule and accordingly is optically active. The absolute
configuration comes from the starting material fumagillol.
Therefore, it is to be noted that the compound (I) has the
same absolute configuration that fumagillol has.
The compound according to the invention exhibits
angiogenesis inhibitory activity and is useful as a therapeutic
and prophylactic agent for various inflammatory diseases
(rheumatism, psoriasis), diabetic retinopathy, or cancer. It
can be safely administered either orally or nonorally as such
or in the form of pharmaceutical preparations [e. g. tablets,
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capsules (inclusive of soft capsules and microcapsules),
solutions, injections, suppositories] prepared by admixing
with ~ se known pharmaceutically acceptable carriers or
excipients or the like. The dose may vary depending on the
target of administration, route of administration, symptoms
and other factors. Generally, however, in adults, it is used,
for example, at a dose of about 0.1 mg/kg to 40 mg/kg body
weight, preferably about 0.5 mg/kg to 20 mg/kg body weight.
Experimental Example
The product compound (I) obtained in the example
given below was evaluated for angiogenesis inhibitory activity
by the rat cornea micropocket method. The data obtained are
shown in the table given below.
Method of Measurement
Essentially the method of Gimbrone et al. [J.
National Cancer Institute, 52, 4l3-419 (1974)] was followed.
Thus, adult male Sprague-Dawley rats (11 to 16 weeks of age)
were anesthetized with nembutal and locally anesthetized by
instillation of xylocaine eyedrops onto the eyeball. The
cornea was incised to a length of about 2 mm at about 2 mm
inside from the corneal circumference by means of an injection
needle, and a sustained release pellet containing basic fibro-
blast growth factor (bFGF; bovine brain-derived, purified
product; R & D) and a sustained release pellet containing the
test sample were inserted side by side into the incision so
that the bFGF pellet was located on the central side in the
cornea. In the control group, the bFGF pellet and a sample-free
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pellet were inserted into the cornea. After 7 days, the cornea
was observed under a stereoscopic microscope. When the sample
administration resulted in retardation or reduction of
bFGF-induced angiogenesis, the sample was judged to have
inhibitory activity.
The sustained release pellets were prepared in the
following manner. An ethylene-vinyl acetate copolymer (Takeda
Chemical Industries) was dissolved in dichloromethane to a
concentration of 8%. A 3-ul portion of the solution was air-
dried on a glass dish, an aqueous solution of bFGF (250 ng)
was then placed thereon and air-dried and, finally 3 ~1 of the
above ethylene-vinyl acetate copolymer solution was placed
further thereon and air-dried to give a laminate consisting of
two copolymer layers and a bFGF layer sandwiched therebetween.
This sandwich sheet was made round into a bFGF pellet. The
test sample pellets were prepared by dissolving each sample in
ethanol in a concentration of 20 ~rg/2 ul, mixing the solution
(2 N1) with 6 ul of an ethylene-vinyl acetate copolymer
solution, air-drying the mixed solution on a glass dish and
making the thus-obtained sheet round.
Table - Angiogenesis Inhibitory Activity
Compound Inhibition Rate Judgement
(I) 6/6 +
Judged after 7 days.
In the above table, the inhibition rate is the ratio
of the number of rats in which angiogenesis inhibitory
activity was observed to the number of rats tested.
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Example
The following example is further illustrative of the
present invention but is by no means limitative of the scope
of the invention.
In the following example, elution in column chromato-
graphy(the eluent being given in the parentheses) was performed
under TLC (thin layer chromatography) observation. In TLC
observation, Merck Kieselgel 60F250 (70-230 mesh) was used
for preparing TLC plates and the solvent used as the eluent in
column chromatography was used also as the developing solvent.
For detection, a UV detector or colouration with phosphomolybdic
acid, for example, was used. Merck Kieselgel 60 (70-230 mesh)
was used also as the column Backing silica gel. Each NMR
spectrum is a proton NMR (1H-NMR) spectrum measured on a
Varian model Gemini 200 NMR spectrometer with tetramethyl-
silane as an internal or external standard and reported in
terms of d values in ppm.
In the example, the following abbreviations are
used:
s: singlet; br: broad; d: doublet; dd: double
doublet; ddd: doublet doublet doublet; t: triplet; q:
quartet; m: multiplet; ABq: AB quartet; J: coupling
constant; Hz: hertz; CDC13: deuteriochloroform; d6-DMSO:
deuterated dimethyl sulfoxide; ~: ~ by weight.
In the following example, the term "room temperature"
means about 15-25~C. The melting point and temperature data
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are all given on the Celsius scale.
Example
0-Chloroacetylcarbamoylfumagillol
O
0
.,
' ~~OCH 3
OCONHCOCH2 Cl
To a solution of fumagillol (314 mg) in dichloro-
methane (5 ml) was added dropwise chloroacetyl isocyanate
(160 mg) under ice cooling, followed by addition of dimethyl-
aminopyridine (130 mg). The mixture was stirred at 0~C for
2 hours. To this reaction mixture was added water and the
mixture was extracted with dichloromethane. The organic layer
was washed with saturated aqueous sodium chloride solution
and dried over anhydrous magnesium sulfate. The solvent was
distilled off under reduced pressure and the residue was
subjected to silica gel column chromatography. The eluate
obtained with a mixture of n-hexane and ethyl acetate (3:1)
was concentrated under reduced pressure to give colourless,
powdery O-chloroacetylcarbamoylfumagillol (318 mg) (71$ yield).
1H-NMR (CDC13) 6: 1.10 (1H, m), 1.21 (3H, s), 1.66
(3H, s), 1.75 (3H, s), 1.93 (1H, d, J=11.4 Hz), 1.8-2.5 (5H,
m), 2.57 (1H, d, J=4.2 Hz), 2.58 (1H, m), 2.99 (1H, d, J=4.2
Hz), 3.47 (3H, s), 3.68 (1H, dd, J=l1.4 Hz, J=2.8 Hz), 4.44
(2H, s), 5.20 (1H, m), 5.61 (1H, m), 8.33 (1H, br s).
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