Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~3~7~()
THERAP~UTIC COMPOSITIONS WITH A
CYTOST~TIC ACTION AND METHOD
The present invention relates to the use of specific
isocyanurates having three epoxy groups to treat malignant
neoplasias and therapeutic compositions containing the said
isocyanurate with a cytostatic action.
It is known that a number-cfalkylating substances have
a cytostatic or cytotoxic effect. The best known compounds are
derived from so-called nitrogen mustards. It is also known to
use compounds containing at least two epoxide groups in the
molecule as cancerostatics. Such compounds are, for example,
4,4'-bis-(2,3-epoxypropyl)-di-piperidinyl-51,1') and 1,2-15,1~-
diepoxy-4,7,10,13-tetraoxohexadecane. However these diepoxide
compounds bring no substantial improvement in the cytostatic
J treatment and are hardly used. Only for the treatment of tumors,
they are still used occassionally. The limited so~ubility of the
above mentioned compounds also prevents a wide use.
Various protocols for screening cytostats against
animal tumors have been published by Geran, Greenberg, MacDonald,
Schumacher and Abbott in "Cancer Chemotherapy Reports" (Sept.
1972) pages 1 to 87. These procedures will be referred to herein-
after by reference to the page(s) of this report.
1- '~
- ~
11.~37~0
An object of the preserlt inverl~:lorl is to find easily-
soluble, particularly water-soluble compounds with cytostatic or
cytotoxic action, whi.ch are effective against a great number of
malignomas and forms of leukemia. They should have a T/C ratio
(see page 47) of at leaæt 150% in at least one of the following
leukemias: L 1210 and P 388, melanoma B16 and LL-carcinoma
produced in anlmal experiments according to the specifications
of the "Cancer Chemotherapy Reports", pages 7,9,11 and 15.
Another ob~ect of the present invention is the
obtaining of a therapeutic composition with a cytostatic action
consisting essentially of from 0.05% to 5% by weight of an
lsocyanurate having the formula
R" / \ R
CH~C-CH2-N I \o/
O=C C=O
\ /
N R~
CH -C - CH
o
wherein R, R' and R" are members selected from the group consist-
ing of hydrogen and alkyl having from 1 to 4 carbon atoms, and
the remainder to 100% aqueous pharmaceutically acceptable vehicles.
A yet further obJect of the present invention is the
development of a method for the treatment of malignant neoplasias
in warm-blooded animals comprising administering a cytostatically
effective amount of an isocyanurate having the formula
:~.237~(~
I" / \ R~
CH2-C-CH2-N N --CH2-C -CH2
O O=C C=O O
\/
N R'
O
~herein R, R'and R" are members selected from the group consist-
ing of hydrogen and alkyl having from 1 to 4 carbon atoms, to a
warm-blooded animals suffering from a malignant neoplasia.
A still further object of the present invention is
- the development of a method for the destruction of cells of
malignant neoplasias by subjecting said cells to an aqueous solu-
t- tion of an isocyanurate having the formula
C
R" / \ R
CH2-C-CH2-N N _ CH2-C - CH2
O O=C \ C=O O
N R'
CH -C-CH
2 0/ 2
wherein R, R' and R" are members selected from the group consist-
ing of hydrogen and alkyl having from 1 to 4 carbon atoms for a
time sufficient to destroy said cells, said isocyanurate being
employed in such an amount as to have a T/C ratio of at least
150% in at least one of the following: leukemia L 1210,
leukemia P 388, melanoma B16 and I.L~carcinoma produced in animal
experiments.
These and other objects of the invention will become
more apparent as the description thereof proceeds.
--3--
~.237~(3
The problems of the prior art can be overcome and
the above-objects achieved by the development and adMinistration
of therapeutic compositions containing compounds with a cytostatic
action according to ~he general formula (I)
R" C R
/ \
C ~-f CH2 - N ~ CH2 C _ CH
C O=C C=O
N
¦ R' I
\ /
where R, R' and R" are either identical or different alkyl
radicals with 1 to 4 carbon atoms and/or hydrogens. Preferred
are compounds where R, R' and R" are either hydrogen and/or a
methyl group. The compounds are to be used as cytostatic sub-
stances for the treatment of malignant neoplasias. These com-
pounds can be used, if necessary together with conventional
pharmaceutical auxiliary substances and/or vehicles.
The invention therefore relates to a therapeutic
composition with a cytostatic action consisting essentially of
from 0.5% to 5% by weight ofan isocyanurate having the formula
--4--
~.23~4(~
R" C R
~~ 7 ¦ o
o . o=c C=O
\ N
CH -C _CH
2 \ / 2
O
wherein R, R' and R" are members selected from the group consist-
ing of hydrogen and alkyl having from 1 to 4 carbon atoms and
the remainder to 100% aquff~ pha~ceutically accepta~e vehicles; as
well as the method for the treatment of malignant neoplasias and
cells of malignant neoplasias employing said isocyanurates.
These glycidyl compounds containing an isocyanurate
! ring are principally known substances. They have already been
synthetized in more or less pure form and are used for the produc-
tion of cross-linked plastics. While the preferred compounds,
in which R, R' and R" are hydrogen~ are characterized by an
astonishingly good solubility in water for this type of compound,
or in hydrophilic water-miscible solvents, nobody has ever thought
of using them in hydrous or aqueous hydrophili.c solvents,
particularly for pharmaceutical purposes or in drugs.
If the three substituents R, R' and R" are identical,
two substances are obtained which are diastereomeric. The same
holds true if there is no alkyl radical present, and the sub-
stituent is hydrogen. These compounds where R, R' and R" are
hydrogen are then called ~-triglycidyl isocyanurate or ~trigl.y-
cidyl isocyanurate (see Angew. Chemie., (1968), pages 851/2).
Their production is described i.n U.S. ~atent No. 3,300,490 and in
Il.S. Patent No. 3,33~,509. These two compounds are readi:Ly
~.Z3740
obtainable by reacting cyanuric acid with excess epichlorohydri~
and dehydrochlorination with formation of the oxirane ring The
pure products (a and ~) can be obtained from the crude productby
fractionating crystalllzation, for example, from methanol,
methylene chloride, ethylene glycol monomethyl ether, ethylene
glycol monoethyl ether, etc. The so-called a-form has, in the
pure form, a melting point of 106C while the ~-form has a
melting point of 158C. Though a separation is generally not
necessary for technical purposes, the therapeutic effectiveness
of the two isomers was investigated separately in the present
case. Due to the different solubility in water or in mixture
with the above-mentioned solvents, a clear separation is readily
possible. Pure products show an epoxide oxygen content which
is between 16% and 16.2% by weight. Naturally the enantisomers
can also be obtained in more or less pure form from the diasto-
meric a- and ~-triglyceridyl isocyanurate, and the effectiveness
can be further increased in some cases.
The preparation of tri-(2-methylglycidyl) isocyanurate
is described in the published German patent application De-OS
1954531.
For use as cancerostatics, the active substances
should be administered by means of suitable vehicles. In the
present case, the use of water, if necessary together with com-
patible glycol ethers, like ethylene glycol monomethylether or
butylene glycol methylether or propylene glycol methylether were
found to be of advantage, particularly if the active substance
is administered parenterally. In oral administration, conven-
tional pharmaceutical auxiliary substances and vehicles can be
l~.Z3740
used, provided they show a corresponding compatibility ~Jith the
glycidyl compounds. Ordinarily, the glycidyl compounds are
employed in amounts of from 0.05% to 5% by weight in the therapeu-
tical compositions. In this range they are soluble in water,
which is unexpected for thts type of compound.
In animal experiments, the use of freshly prepared
a~ueous solutions administered ~ntraperitoneally proved advisable.
The maximum daily dose of ~- or ~-triglycidyl isocyanurate in
mice can be 100 mg~kg of mouse. Pronounced toxic effects appear
only at higher doses. The optimum dose was found to be in many
cases to be 30 mg to 60 mg/kg of mouse per day during an applica-
tion period of 1 to 9 days.
The above-mentioned compounds are effective against
various forms of leukemia and malignant neoplasmas, like lung
cancer, cancer of the colon, melanomas, ependymoblastomas and
sarcomas. A clear superiority over cyclophosphamide and fluorur-
acil was found in many cases.
Naturally it is also possible in addition to using
~-triglycidyl isocyanurate and ~-triglycidyl isocyanurates as
cancerostatics to employ the other compounds of formula I, where
R, R' and R" represent at least partly a methyl group. As far as
the corresponding solubility exists, the alkyl group R, R' and R"
can naturally contain more carbon atoms.
A combination therapy with other alkylating sub-
stances, like derivatives of nitrogen mustard or fluoruracil
is naturally also possible.
The following are examples of the invention without
being limitative in any respect.
~.237'~V
F.XA~ S
The ~ollowlng example~ were carried out accord-lng to
the test specifications of t~e Natural Cancer Institute, Bethesda,
Maryland, 20014LI, published in "Cancer Chemotherapy Reports" part
3, Sept. 72, Vol. 3, No. 2. The active substance used was either
~-triglycidyl isocyanature (mp: 106C) or ~-triglycidyl isocyanu-
rate (mp: 158C), both with 16.1% epoxide-oxygen content (for pre-
paration see U.S. Patent No. 3,337,509). The preparation of tri-
(2-methylglycidyl) isocyanurate is described in the pub~lshed
German Offenlegungsschrift 19 54 531, but on repeating the pro-
cedure there was only obtained a product which did not show the
expected lnfrared spectrum. Therefore, the desired compound was
prepared by reaction of the potassium salt of cyanuric acid and
methallylchloride followed by the epoxidation of the trimethallyl
compound intermediate.
a) A mixture of 86 gm of potassium salt of cyanuric acid and
95 gm of methallylchloride in 391 gm of acetonitrile was
heated in an autoclave under nitrogen pressure at i50C for
a period of 3 hours. After cooling, the mixture was
filtered and all volatile material was distilled off in
vacuo at 0.1 mm pressure. The crude material was dissolved
in cyclohexene and, after evaporation~ recrystallized from
the same solvent. The purified material had a melting
point from 84 to 85C and a iodine number of 264
(calculated: 261,3).
b) 20 gm of the material received as described before was
dissolved in 300 gm of C~l2C12 and treated with 15,85 gm of
m-chloroperbenzoic acid. The mixture was allowed to stand
for 70 hours in a refrigerator at 4C. Thereafter~ the
precipitate of 3-chlorobenzoic acid was filtered off. T~e
37~
organic solution of the epoxide recovered was washed with
an aqueous solut~on o~ sodium carbonate (10% by weight),
an~ water for several times and dried with anhydrous sodium
sulfate. When no more peracid was present the CH2C1~ was
distilled off in vacuo. The residue was recrystallized
using diethylether. Yield: 12.9 gm of white crystals
having a m.p. of 69 to 74C (55.7% of the theory). Epoxide
content: 13.7% by weight (calculated: 14.1%).
The infrared spectrum gives typically strong isocyanurate absorp-
tions at 1700 and 1455 cm . H-NMR spectroscopy in CDC13
(reference TMS) sho~s the protons of the CH3 groups at 1.~ ppm.
The two protons on C-atom 1 of the 2-methylglycidyl groups give
- an AB system at Ll.1 ppm , the two protons on C-atom 3 an AB system
with a smaller coupling constant at 2.6 ppm. The structure is
further confirmed by 3C-NMR and mass spectroscopy.
All aqueous 1% injection solutions were prepared fresh
~ust prior to administration. The triglycidy] isocyanurate is
also referred to by its initials TGI.
EXAMPLE 1
In mice a P 388 (leukemia) tumor was emplanted i.p.
with 10 cells/mouse according to protocol 1.200 (page 9). The
untreated animals has a mean survival time (m.s.t.) of 10.5 days.
After nine days of i.p. treatment with 100 mg/kg of
~-TGI per day, the mean life expectancy increased to 285%, com-
pared to the control group (T/C, extension rate). Half of the
treated mice lived longer than L~o days and must be considered as
cured.
If only 50 mg/kg of ~TGI per day were administered
i.p. for 9 days, the corresponding values were 200~ T/C and 17%cured.
~.237'~0
The correspondlng value with ~-TGr f`or ~00 rng/kg o~ mouse per
day were 228% T/C anc~ for 50 mg/kg per day 179% T/C. Healing
was observed in 17% of the test animals.
Comparison test
A leukemia form of P 388, which was resistent to
cyclophosphamide (NSC 26271), was emplanted in groups of mice and
treated with ~-TGI or cyclophosphamide. Each group considered
of 10 mice.
With daily doses of 40 mg/kg of ~-TGI for 1 to 9
days, all mice lived for 60 days (T/C 478%). On the other hand,
those treated with 180 mg/kg of mouse of cyclophosphamide did
not live even for 30 days (T/C 150%).
EXAMPLE 2
Example 1 was repeated with a dose of 25 mg/kg per
i day for 9 days and the following observation were made: ~-TGI
showed a T/C of 196% and ~-TGI a T/C of 174%.
EXAMPLE 3
Leukemia L 1210 was produced in mice according to
protocol 1.100 (pagé 7) by i.p. administration of 0.1 ml diluted
stimulating solution corresponding to 105 cells. In the control
group the mean survival time was 8 days (m.s.t.) A) One group
(6 mice) received from the first to the 9th day 50 mg/kg of mouse
per day of ~TGI i.p. The mean survival time rose to 23.8 days
corresponding to a T/C of 297%. 3 mice lived for 30 days, that is,
a healing rate of about 50% was achieved.
Another group received from the first to the 9th day
either 50 or 100 mg/kg of ~TGI per day, i.p. The mean survival
time was 16.3 or 25.2 days respectively corresponding to a T/C
of 203~ or 315% respective]y.
374(~
B) The influence of the treatment plan on the therapeutic effect
of ~~TGI in turnor L 1210 after 30 days can be seen from the
following Table I.
TABLE I
Dependence of the effectiven_ss on the treatment plan with ~-TGI_
Dose per day Surviving / entire group
in mg/kg Leukemic control group
mice (not emplanted with
leukemia)
50 mg for 5 x i.p. 5/10 7/8
(lst to 5th day)
40 mg for 9 x i.p. 8/10 8/8
(lst to 9th day)
EXAMPLE 4
t 1/10 homogenate melanoma B 16 were administered i.p.
according to protocol 1.300 (page 11) at a rate of 0.5 ml per
mouse.
The control group had a mean survival time of 15.8
(m.s.t.)
Treated groups received from the first to the 9th day
various amounts of a-TGI i.p. The following Table II shows the
mean survival time and T/C in dependence on the daily dose of
active substance.
TABLE II
Effect of a-TGI as a function of the dose
mg~-TGI/kg mouse/day m.s.t. (days) T/C
50 37.0 234%
25 36.o 227%
12.5 29.8 188%
~ 23740
EXAMP~,~ 5
According to protocol 1.400 (page 13) cells of a
1 mm3 piece of lung cancer (Lewis) were implanted s.c. in mice.
40 mg/kg of -TGI and 90 mg/kg of ~-TGI respectively were
administered daily i.p. to each mouse from the first to the 11th
day.
The inhibition of metastases was 92~ for a-TGI and
87% for ~-TGI compared to the control group after 23 days.
EXAMPLE 6
In a test group of mice, 1 mm3 of a tissue of ependy-
moblastoma was implanted intracerebrally. The mean survi~al time
in the control group was 19.3 days.
In a treatment with 40 mg/kg a-TGI daily for 9 days,
a T/C of 165% was achieved.
e EXAMPLE 7
Sarcoma 180 was produced by i.p. administration of
106 cells/mouse. The untreated control group had a mean survival
time of 20.2 days.
For the treatment, 30 mg/kg of mouse of a-TGI were
administered i.p. on a daily basis from 1 to 9 days. A T/C of
183% was observed.
EXAMPLE 8
A) Groups of 10 mice were implanted with about 1 mm3 of colon
carcinoma 38, s.c. The mean tumor weight in the untreated control
group after 20 days was 512 mg/mouse.
One group received on the 2nd and 9th day 50 mg/kg of
mouse daily of ~,-TGI, and another group addjtional received the
same amount on the 16th day. 1'he rnean tumor weight was 153 mg and
183 mg respectively per mouse.
3~4V
B) A carclnoma was produced by i.p. implantation (1 mm colon
carcinoma 26) in groups of 10 mice each. The treatment consisted
in the i.p. admlnistration of ~-TGI. The following Table III
shows the results as a function of the amount administered on the
1st, 5th and 9th day.
TABLE III
mg/kg of mouse
25 mg i.p. 25 days all mice survived
60 days 6 out of 10 mice survived
50 mg i.p. 60 days 10 mice still alive
100 mg i.p. 49 days all mice still alive
60 days 4 mice still alive
A positive comparison by treatment with methyl CCNU (NSC-95441)
10 mg~kg of mouse i.p. showed that only 2 out of 10 mice were still alive on
the 60th day (mean survival time 55 days)
EXAMPLE 9
In mice a P 388 (leukemia) tumor was emplanted i.p. with 10
cells/mouse according to protocol 1.200 (page 9). The untrea~ed animals have
a mean survival time (m.s.t.) of 10 days.
After nine days of i.p. treatment with 100 mg/kg of tri-(2-methyl-
glycidyl) isocyanurate per day, the mean life expectancy increased to 170%, com-
pared to the control g~oup (T/C, extension rate).
If only 50 mg/kg of tri-(2-methylglycidyl) isocyanurate per day
were administered i.p. for 9 days, the corresponding value was 150% T/C.
The preceding specific embodiments are illustrative of the pract-
ice of the invention. It is to be understood however, that other expedients
known to those skilled in the art, or disclosed herein, may be employed without
departing from the spirit of the invention or the scope Or the appended claims.
