Bis-dioxopiperazine derivatives, process for their preparation, antitumor agents comprising them and compositions containing them

Disclosed are novel bis-dioxopiperazine derivatives and non-toxic salts thereof, process for their preparation, antitumor agents comprising them and compositions containing them. The bis-dioxopiperazine derivatives are represented by the following general formula (I): ##STR1## wherein R represents an alkyl radical having 1 to 17 carbon atoms, a cycloalkyl radical having 3 to 7 carbon atoms, a lower alkyl radical having a substituent or substituents selected from the group consisting of halogeno, carboxy, alkoxycarbonyl having 2 to 5 carbon atoms, lower alkoxy, substituted or unsubstituted phenoxy, naphthyloxy, substituted or unsubstituted phenylthio, substituted or unsubstituted phenyl and naphthyl, a substituted or unsubstituted phenyl lower alkenyl radical, a substituted or unsubstituted phenyl radical, a naphthyl radical, a heterocyclic radical selected from the group consisting of pyridyl, furyl and thienyl, an alkoxy radical having 1 to 8 carbon atoms, a substituted or unsubstituted phenyl lower alkoxy radical, or a substituted or unsubstituted phenoxy radical. A substituent or substituents on R may be selected from the group consisting of lower alkyl, halogeno, lower alkoxy, acyloxy having 2 to 5 carbon atoms, methylenedioxy, carboxy, amino, methanesulfonylamino and nitro. The above-mentioned "lower alkyl", "lower alkoxy" and "lower alkenyl" respectively refer to alkyl having 1 to 4 carbon atoms, alkoxy having 1 to 4 carbon atoms and alkenyl having 2 to 4 carbon atoms.

BACKGROUND OF THE INVENTION 
The present invention relates to novel bis-dioxopiperazine derivatives and 
non-toxic salts thereof, process for their preparation, antitumor agents 
comprising them and compositions containing them. 
Bis-dioxopiperazine derivatives of the present invention are represented by 
the following general formula (I): 
##STR2## 
wherein R represents an alkyl radical having 1 to 17 carbon atoms, a 
cycloalkyl radical having 3 to 7 carbon atoms, a lower alkyl radical 
having a substituent or substituents selected from the group consisting of 
halogeno, carboxy, alkoxycarbonyl having 2 to 5 carbon atoms, lower 
alkoxy, substituted or unsubstituted phenoxy, naphthyloxy, substituted or 
unsubstituted phenylthio, substituted or unsubstituted phenyl and 
naphthyl, a substituted or unsubstituted phenyl lower alkenyl radical, a 
substituted or unsubstituted phenyl radical, a naphthyl radical, a 
heterocyclic radical selected from the group consisting of pyridyl, furyl 
and thienyl, an alkoxy radical having 1 to 8 carbon atoms, a substituted 
or unsubstituted phenyl lower alkoxy radical, or a substituted or 
unsubstituted phenoxy radical. A substituent or substituents on R may be 
selected from the group consisting of lower alkyl, halogeno, lower alkoxy, 
acyloxy having 2 to 5 carbon atoms, methylenedioxy, carboxy, amino, 
methanesulfonylamino and nitro. The above-mentioned "lower alkyl", "lower 
alkoxy" and "lower alkenyl" respectively refer to alkyl having 1 to 4 
carbon atoms, alkoxy having 1 to 4 carbon atoms and alkenyl having 2 to 4 
carbon atoms. 
Several kinds of bis-dioxopiperazine derivatives have been already 
reported. Especially 
1,2-bis(4-morpholinomethyl-3,5-dioxopiperazin-1-yl)ethane is known as an 
analogue of the compound which the present invention concerns and its 
clinical efficacy as an antitumor and radio-potentiative agent was already 
evaluated (see Abstract, 8th International Congress of Pharmacology p441, 
1981). However the known compound had a problem on the preparation because 
it is extremely unstable in the protic polar solvents such as water, lower 
alcohol and so on. 
Based on the attractive biological activities of the known 
bis-dioxopiperazine derivatives, we, the inventors further carried out the 
synthesis studies on these derivatives with the excellent activity and 
pharmaceutically advantageous property. We found that the aforementioned 
bis-dioxopiperazine derivatives of general formula (I) exhibit broader 
spectra of antitumor activities, antimetastatic activity, pharmaceutically 
advantageous stability in the protic polar solvents, and lower toxicity, 
thus accomplishing the present invention. 
DETAILED DESCRIPTION OF THE INVENTION 
Bis-dioxopiperazine derivatives of the present invention are represented by 
the general formula (I) wherein R represents an alkyl radical having 1 to 
17 carbon atoms, a cycloalkyl radical having 3 to 7 carbon atoms, a lower 
alkyl radical having a substituent or substituents selected from the group 
consisting of halogeno, carboxy, alkoxy-carbonyl having 2 to 5 carbon 
atoms, lower alkoxy, substituted or unsubstituted phenoxy, naphthyloxy, 
substituted or unsubstituted phenylthio, substituted or unsubstituted 
phenyl and naphthyl, a substituted or unsubstituted phenyl lower alkenyl 
radical, a substituted or unsubstituted phenyl radical, a naphthyl 
radical, a heterocyclic radical selected from the group consisting of 
pyridyl, furyl and thienyl, an alkoxy radical having 1 to 8 carbon atoms, 
a substituted or unsubstituted phenyl lower alkoxy radical, or a 
substituted or unsubstituted phenoxy radical. A substituent or 
substituents on R may be selected from the group consisting of lower 
alkyl, halogeno, lower alkoxy, acyloxy having 2 to 5 carbon atoms, 
methylenedioxy, carboxy, amino, methanesulfonylamino and nitro. The 
above-mentioned "lower alkyl", "lower alkoxy" and "lower alkenyl" 
respectively refer to alkyl having 1 to 4 carbon atoms, alkoxy having 1 to 
4 carbon atoms and alkenyl having 2 to 4 carbon atoms. The alkyl radical 
having 1 to 17 carbon atoms is selected from the group having a normal or 
branched carbon chain such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 
isobutyl, tert-butyl, pentadecyl, hexadecyl, heptadecyl, and so on. The 
cycloalkyl radical having 3 to 7 carbon atoms is selected from the group 
containing cyclopentyl, cyclohexyl, cycloheptyl and so on. The halogeno 
lower alkyl radical is selected from the group containing chloromethyl, 
bromomethyl, dichlocromethyl, bromoethyl and so on. The carboxy lower 
alkyl radical is selected from the group containing carboxyethyl, 
carboxyproply and so on. The lower alkyl radical substituted with 
alkyloxycarbonyl having 2 to 5 carbon atoms is selected from the group 
containing methoxycarbonylmethyl, methoxycarbonylethyl, 
ethoxycarbonylethyl, methoxycarbonylpropyl and so on. The lower alkoxy 
lower alkyl radical is selected from the group containing methoxymethyl, 
methoxyethyl, ethoxymethyl and so on. The substituted or unsubstituted 
phenoxy lower alkyl radical is selected from the group containing 
phenoxymethyl, phenoxy ethyl, p-chlorophenoxymethyl, 
2,4-dichlorophenoxymethyl, 2,4,5-trichlorophenoxymethyl, 
1-p-chlorophenoxy-1-methylethyl and so on. The naphthyloxy lower alkyl 
radical is selected from the group containing 2-naphthyloxymethyl, 
2-naphthyloxyethyl and so on. The substituted or unsubstituted phenylthio 
lower alkyl radical is selected from the group containing 
phenylthiomethyl, p-bromophenylthiomethyl, p-fluorophenylthiomethyl and so 
on. The substituted or unsubstituted phenyl lower alkyl radical is 
selected from the group containing benzyl, phenethyl, o-chlorobenzyl, 
o-bromobenzyl, o-tolylmethyl, .alpha.-hydroxybenzyl and so on. The 
naphthyl lower alkyl radical is selected from the group containing 
1-naphthylmethyl, 2-naphthylethyl and so on. The substituted or 
unsubstituted phenyl lower alkenyl radical is selected from the group 
containing styryl, cinnamyl, 3,4-dihydroxystyryl, p-methoxystyryl, 
3,4-dimethoxystyryl, 3,4-diacetoxystyryl and so on. The substituted or 
unsubstituted phenyl radical is selected from the group containing phenyl, 
o-chlorophenyl, o-fluorophenyl, p-chlorophenyl, p-bromophenyl, o-tolyl, 
m-tolyl, o-methoxyphenyl, 3,4-dihydroxyphenyl, 3,4-diacetoxyphenyl, 
3,4-methylenedioxyphenyl, p-nitrophenyl, p-cyanophenyl, p-aminophenyl, 
p-methanesulfonylaminophenyl, o-carboxyphenyl and so on. The naphthyl 
radical is selected from the group containing 1-naphthyl, 2-naphthyl and 
so on. The heterocyclic radical is selected from the group containing 
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-thienyl and so on. 
The alkoxy radical having 1 to 8 carbon atoms is selected from the group 
containing methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, 
tert-butoxy, 2-ethylhexyloxy and so on. The substituted or unsubstituted 
phenyl lower alkoxy radical is selected form the group containing 
benzloxy, p-nitrobenzyloxy, o-methoxybenzyloxy, phenethyloxy and so on. 
The substituted or unsubstituted phenoxy radical is selected from the 
group containing phenoxy, o-chlorophenoxy and so on. 
The compounds according to the present invention are for example as 
follows: 
1,2-bis(4-acetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-n-butyryloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-isovaleryloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-pivaloyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-palmitoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-stearoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-cyclohexylcarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-chloroacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-dichloroacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-bromoacetoxymethyl-3,5-dioxopiperazin-1yl)ethane; 
1,2-bis(4-.beta.-carboxypropionyloxymethyl-3,5-dioxopiperazin-1-yl)ethane 
or sodium salts thereof; 
1,2-bis(4-.beta.-methoxycarbonylpropionyloxymethyl-3,5-dioxopiperazin-1-yl) 
ethane; 
1,2-bis(4-methoxyacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-phenoxyacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-p-chlorophenoxyacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis[4-(2,4-dichlorophenoxyacetoxymethyl)-3,5-dioxopiperazin-1-yl]ethane 
; 
1,2-bis[4-(2,4,5-trichlorophenoxyacetoxymethyl)-3,5-dioxopiperazin-1-yl]eth 
ane; 
1,2-bis[4-(2-p-chlorophenoxy-2-methylpropionyloxymethyl)-3,5-dioxopiperazin 
-1-yl]ethane; 
1,2-bis[4-(2-naphthyloxyacetoxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis(4-p-fluorophenylthioacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-p-bromophenylthioacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-phenylacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-chlorophenylacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-bromophenylacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-tolylacetoxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-.alpha.-hydroxy-.alpha.-phenylacetoxymethyl-3,5-dioxopiperazin-1- 
yl)ethane; 
1,2-bis[4-(1-naphthylacetoxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis(4-cinnamoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis[4-(3,4-diacetoxycinnamoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis(4-p-methoxycinnamoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis[4-(3,4-dimethoxycinnamoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis(4-benzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-chlorobenzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-fluorobenzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-toluoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-m-toluoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-o-methoxybenzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis[4-(3,4-diacetoxybenzoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis[4-(3,4-methylenedioxybenzoyloxymethyl)-3,5-dioxopiperazin-1-yl]etha 
ne; 
1,2-bis(4-o-carboxybenzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-p-aminobenzoyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-p-methanesulfonylaminobenzoyloxymethyl-3,5-dioxopiperazin-1-yl)et 
hane; 
1,2-bis[4-(1-naphthoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis[4-(2-pyridylcarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis[4-(3-pyridylcarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis[4-(2-thenoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis[4-(2-furoyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane; 
1,2-bis(4-methoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-ethoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-n-butoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-isobutoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis[4-(2-ethylhexyloxycarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]ethane 
; 
1,2-bis(4-p-nitrobenzyloxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane; 
1,2-bis(4-phenoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane 
The compounds (I) of the present invention may form themselves into acid 
addition salts, e.g., hydrochloride, oxalate, p-toluenesulfonate, acetate 
and so on, as the nontoxic salts. 
Process for the preparation of bis-dioxopiperazine derivatives of the 
general formula (I) comprises treatment of the compound shown as formula 
(II): 
##STR3## 
with a reactive derivative such as a chloride or an anhydride of the 
compound shown as general formula (III): 
EQU RCOOH (III) 
wherein R is as defined in the formula (I). 
The chloride of the compound (III) may be selected from the group 
containing acetyl chloride, n-butyryl chloride, isovaleryl chloride, 
pivaloyl chloride, palmitoyl chloride, stearoyl chloride, 
cyclohexanecarbonyl chloride, chloroacetyl chloride, dichloroacetyl 
chloride, bromoacetyl chloride, methoxyacetyl chloride, phenoxyacetyl 
chloride, p-chlorophenoxyacetyl chloride, 2,4-dichlorophenoxyacetyl 
chloride, 2,4,5-trichlorophenoxyacetyl chloride, 
2-p-chlorophenoxy-2-methylpropionyl chloride, 2-naphthyloxyacetyl 
chloride, p-bromophenylthioacetyl chloride, p-fluorophenylthioacetyl 
chloride, o-chlorophenylacetyl chloride, o-bromophenylacetyl chloride, 
.alpha.-chloroacetoxy-.alpha.-phenylacetyl chloride, 1-naphthylacetyl 
chloride, cinnamoyl chloride, 3,4-diacetoxycinnamoyl chloride, 
p-methoxycinnamoyl chloride, 3,4-dimethoxycinnamoyl chloride, benzoyl 
chloride, o-chlorobenzoyl chloride, o-fluorobenzoyl chloride, o-toluoyl 
chloride, m-toluoyl chloride, o-methoxybenzoyl chloride, 
3,4-diacetoxybenzoyl chloride, 3,4-methylenedioxybenzoyl chloride, 
p-benzloxycarbonyl-aminobenzoyl chloride, 2-pyridylcarbonyl chloride, 
3-pyridylcarbonyl chloride, 2-thenoyl chloride, 2-furoyl chloride, methyl 
chloroformate, ethyl chloroformate, n-butyl chloroformate, isobutyl 
chloroformate, 2-ethylhexyl chloroformate, p-nitrobenzyl chloroformate, 
phenyl chloroformate and so on, while the anhydride of the compound (III) 
may be selected from the group containing acetic anhydride, n-butyric 
anhydride, isobutyric anhydride, propionic anhydride, monochloroacetic 
anhydride, succinic anhydride, and so on. 
The compound of the formula (II) which is the starting material in a 
process of the present invention is a novel compound and can be prepared 
easily by reacting 1,2-bis(3,5-dioxopiperazin-1-yl)ethane described in 
British Patent Specification No. 1234935, with formaldehyde. 
In the process of preparing bis-dioxopiperazine derivatives which the 
present invention concerns, at least two equivalent molar amounts of 
chloride or anhydride of the compounds (III) should be used to one molar 
amount of the starting material of the formula (II). 
The reaction temperature may range from -10.degree. to 120.degree. C., 
preferably from 0.degree. to 80.degree. C., and the reaction time may 
range from 1 to 24 hours. 
As for the reaction solvent, an aprotic polar solvent for instance, DMF, 
acetonitrile, chloroform, dichloromethane, ethyl acetate, pyridine, 
lutidine, picoline, or their mixture may be used. The above reaction is 
performed preferably in the presence of the base such as pyridine, 
triethylamine and so on. 
The compounds (I) of the present invention may be prepared by reacting the 
compound of the formula (II) with the compounds of the general formula 
(III) in the presence of the condensing agents such as 
1-methyl-2-chloropyridinium iodide, 2-chloro-3-ethylbenzoxazorium 
tetrafluoroborate, dicyclohexylcarbodiimide and so on. 
In this condensation reaction, at least two equivalent molar amount of the 
compound of the general formula (III) should be used to one molar amount 
of the compound of the general formula (II). 
The reaction temperature may range from -30.degree. to 100.degree. C., 
preferably from 0.degree. C. to room temperature and the reaction time may 
range from 1 to 24 hours which depends on reaction temperature. 
As for the reaction solvent, an aprotic polar solvent for instance, DMF, 
acetonitrile, chloroform, dichloromethane, or their mixture may be used. 
Preferably, 4-N,N-dimethylaminopyridine or 4-pyrrolidinopyridine is used 
as catalyst in the above reaction. 
When the compounds (I) of the present invention are prepared, if necessary, 
the functional group of the compound (III) may be protected in usual way. 
In addition, an acid addition salt of the compounds (I) of the present 
invention, for example, hydrochloride, oxalate, p-toluenesulfonate, 
acetate and so on, may be prepared in usual way. 
Antitumor activities of bis-dioxopiperazine derivatives which the present 
invention concerns were verified by the growth inhibition or the increase 
of life span in experimental animals with syngenic tumors such as P388 
lymphocytic leukemia, Lewis lung carcinoma, B-16 melanoma and Colon 
Adenocarcinoma No. 38 as shown below. 
(1) Animal Test on P388 Lymphocytic Leukemia 
The treated group to which the compound of the present invention was 
administered consisted of seven mice, while the control group consisted of 
ten mice. Six weeks old male mice [CDF.sub.1 (BALB/c.times.DBA/2), 25.+-.2 
g of body weight] were employed as host animals. 
Tumor cells (1.0.times.10.sup.6) of P388 lymphocytic leukemia were 
transplanted intraperitoneally into each mouse. The treatment began one 
day after the transplantation and the prescribed dose of each test 
compound was administered intraperitoneally to the mice once a day for 9 
days or on the 1st and 5th day. 
Antitumor activity of the test compound was evaluated by the rate of 
increase in life span (I.L.S.%) which was calculated with the following 
formula. 
##EQU1## 
T: mean survival time of treated mice C: mean survival time of control 
mice 
The results obtained are shown in Table 1A and 1B. 
TABLE 1A 
______________________________________ 
Results of Animal Test on 
P388 Lymphocytic Leukemia 
Daily Dose 
Test .times. Day 
I.L.S. 
Compound (mg/kg) (%) 
______________________________________ 
1,2-bis(4-acetoxymethyl-3,5- 
200 .times. 2 
45 
dioxopiperazin-1-yl)ethane 
60 .times. 9 
118 
20 .times. 9 
63 
1,2-bis(4-pivaloyloxymethyl- 
200 .times. 2 
66 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
53 
1,2-bis(4-chloroacetoxymethyl- 
200 .times. 2 
38 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
54 
1,2-bis(4-p-chlorophenoxyacetoxy- 
200 .times. 2 
94 
methyl-3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
40 
1,2-bis[4-(2,4-dichlorophenoxy- 
200 .times. 2 
72 
acetoxymethyl)-3,5-dioxopiperazin- 
60 .times. 9 
105 
1-yl]ethane 20 .times. 9 
49 
1,2-bis[4-(2,4,5-trichlorophenoxy- 
200 .times. 2 
94 
acetoxymethyl)-3,5-dioxopiperazin- 
20 .times. 9 
65 
1-yl]ethane 
1,2-bis[4-(2-p-chlorophenoxy-2- 
200 .times. 2 
114 
methylpropionyloxymethyl)-3,5- 
20 .times. 9 
63 
dioxopiperazin-1-yl]ethane 
1,2-bis(4-p-fluorophenylthio- 
200 .times. 2 
120 
acetoxymethyl-3,5-dioxopiperazin- 
1-yl)ethane 
1,2-bis(4-p-bromophenylthioacetoxy- 
200 .times. 2 
108 
methyl-3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-phenylacetoxymethyl- 
200 .times. 2 
204 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
143 
1,2-bis(4-o-chlorophenylacetoxy- 
200 .times. 2 
83 
methyl-3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
65 
______________________________________ 
TABLE 1B 
______________________________________ 
Results of Animal Test on 
P388 Lymphocytic Leukemia 
Daily Dose 
Test .times. Day 
I.L.S. 
Compound (mg/kg) (%) 
______________________________________ 
1,2-bis(4-p-methoxycinnamoyloxy- 
200 .times. 2 
36 
methyl-3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
78 
1,2-bis[4-(3,4-diacetoxycinnamoyl- 
200 .times. 2 
51 
oxymethyl)-3,5-dioxopiperazin-1-yl]- 
20 .times. 9 
35 
ethane 
1,2-bis(4-benzoyloxymethyl-3,5- 
200 .times. 2 
107 
dioxopiperazin-1-yl)ethane 
20 .times. 9 
68 
1,2-bis(4-o-chlorobenzoyloxymethyl- 
200 .times. 2 
90 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
55 
1,2-bis(4-o-fluorobenzoyloxymethyl- 
200 .times. 2 
113 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-o-methoxybenzoyloxymethyl- 
200 .times. 2 
99 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
113 
1,2-bis[4-(2-furoyloxymethyl)- 
200 .times. 2 
&gt;200 
3,5-dioxopiperazin-1-yl]ethane 
1,2-bis[4-(2-thenoyloxymethyl)-3,5- 
200 .times. 2 
118 
dioxopiperazin-1-yl]ethane 
1,2-bis(4-methoxycarbonyloxymethyl- 
200 .times. 2 
103 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
75 
1,2-bis(4-ethoxycarbonyloxymethyl- 
200 .times. 2 
45 
3,5-dioxopiperazin-1-yl)ethane 
20 .times. 9 
48 
1,2-bis(4-isobutoxycarbonyloxymethyl- 
200 .times. 2 
138 
3,5-dioxopiperazin-1-yl)ethane 
60 .times. 9 
175 
20 .times. 9 
78 
1,2-bis(4-morpholinomethyl-3,5- 
100 .times. 2 
60 
dioxopiperazin-1-yl)ethane 
20 .times. 9 
90 
(known compound) 
______________________________________ 
Against P388 Lymphocytic leukemia, the compounds of the present invention 
showed excellent I.L.S.(%). 
(2) Animal Test on Lewis Lung Carcinoma 
The treated group to which the compound of the present invention was 
administered consisted of seven mice, while the control group consisted of 
ten mice. Six weeks old male mice [BDF.sub.1 (C57BL/6.times.DBA/2), 
25.+-.2 g of body weight] were employed as host animals. 
Tumor cells (5.times.10.sup.5) of Lewis lung carcinoma were transplanted 
subcutaneously in inguinal region of each mouse. The treatment began one 
day after the transplantation and the prescribed dose of each test 
compound was administered orally to the mice once a day for 8 days. 
On the 20th day after the transplantation, the tumors of all the mice were 
excised and weighed. Antitumor activity of the test compound was evaluated 
by the rate of growth inhibition (G.I.%) which was calculated with the 
following formula. 
##EQU2## 
T: mean tumor weight of treated mice C: mean tumor weight of control mice 
The results obtained are shown in Table 2. 
TABLE 2 
______________________________________ 
Results of Animal Test on Lewis Lung Carcinoma 
Daily Dose 
G.I. 
Test Compound (mg/kg) (%) 
______________________________________ 
1,2-bis(4-acetoxymethyl-3,5-dioxo- 
60 79.6 
piperazin-1-yl)ethane 
1,2-bis(4-chloroacetoxymethyl-3,5- 
50 66.8 
dioxopiperazin-1-yl)ethane 
1,2-bis(4-o-chlorobenzoyloxymethyl- 
40 43.7 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-methoxycarbonyloxymethyl- 
60 57.7 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-isobutoxycarbonyloxymethyl- 
50 54.4 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-morpholinomethyl-3,5- 
60 22.6 
dioxopiperazin-1-yl)ethane 
(known compound) 
______________________________________ 
Against Lewis lung carcinoma, G.I. (%) of the compounds which the present 
invention concerns were superior to that of a comparative compound, i.e. 
1,2-bis(4-morpholinomethyl-3,5-dioxopiperazin-1-yl)ethane. In addition, it 
was confirmed that the compounds of the present invention suppressed the 
metastasis of Lewis lung carcinoma significantly as compared with control 
group. 
(3) Animal Test on B-16 Melanoma 
The treated group to which the compound of the present invention was 
administered consisted of seven mice, while the control group consisted of 
ten mice. Six weeks old male mice [BDF.sub.1 (C57BL/6.times.DBA/2) 25.+-.2 
g of body weight] were employed as host animals. 
Tumor cells (5.times.10.sup.5) of B-16 melanoma were transplanted 
intraperitoneally into each mouse. The treatment began one day after the 
transplantation and the prescribed dose of each test compound was 
administered intraperitoneally to the mice once a day for 8 days. 
Antitumor activity of the test compound was evaluated by the rate of 
increase in life span (I.L.S.%) which was calculated in the same manner as 
used in the animal test on P388 lymphocytic leukemia. The results obtained 
are shown in Table 3. 
TABLE 3 
______________________________________ 
Results of Animal Test on B-16 Melanoma 
Daily Dose 
I.L.S. 
Test Compound (mg/kg) (%) 
______________________________________ 
1,2-bis(4-acetoxymethyl-3,5-dioxo- 
20 39 
piperazin-1-yl)ethane 
1,2-bis(4-chloroacetoxymethyl-3,5- 
20 38 
dioxopiperazin-1-yl)ethane 
1,2-bis[4-(2,4-dichlorophenoxyacetoxy- 
20 35 
methyl)-3,5-dioxopiperazin-1-yl]ethane 
1,2-bis[4-(2-p-chlorophenoxy-2- 
30 86 
methylpropionyloxymethyl)-3,5- 
dioxopiperazin-1-yl]ethane 
1,2-bis(4-p-fluorophenylthio- 
40 83 
acetoxymethyl-3,5-dioxopiperazin- 
1-yl)ethane 
1,2-bis(4-phenylacetoxymethyl-3,5- 
20 83 
dioxopiperazin-1-yl)ethane 
1,2-bis(4-benzoyloxymethyl-3,5- 
20 87 
dioxopiperazin-1-yl)ethane 
1,2-bis(4-o-chlorobenzoyloxymethyl- 
20 65 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis[4-(2-furoyloxymethyl)-3,5- 
20 88 
dioxopiperazin-1-yl]ethane 
1,2-bis(4-methoxycarbonyloxymethyl- 
30 78 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-isobutoxycarbonyloxymethyl- 
20 86 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-morpholinomethyl-3,5- 
20 40 
dioxopiperazin-1-yl)ethane 
(known compound) 
______________________________________ 
Against B-16 melanoma, I.L.S.(%) of the compounds which the present 
invention concerns were nearly equal to or superior to that of the 
comparative compound. 
(4) Animal Test on Colon Adenocarcinoma No. 38 
The treated group to which the compound of the present invention was 
administered consisted of seven mice, while the control group consisted of 
ten mice. Six weeks old male mice [BDF.sub.1 (C57BL/6.times.DBA/2), 
25.+-.2 g of body weight] were employed as host animals. 
Tumor cells (40 mg of fragment) of Colon adenocarcinoma No. 38 were 
transplanted subcutaneously in inguinal region of each mouse. The 
treatment began one day after the transplantation and the prescribed dose 
of each test compound was administered orally to the mice once a day for 8 
days. 
On the 30th day after the transplantation, the tumors of all the mice were 
excised and weighed. Antitumor activity of the test compound was evaluated 
by the rate of growth inhibition (G.I.%) which was calculated in the same 
manner as used in the animal test on Lewis lung carcinoma. The results 
obtained are shown in Table 4. 
TABLE 4 
______________________________________ 
Results of Animal Test on 
Colon Adenocarcinoma No. 38 
Daily Dose 
G.I. 
Test Compound (mg/kg) (%) 
______________________________________ 
1,2-bis(4-acetoxymethyl-3,5-dioxo- 
60 68.0 
piperazin-1-yl)ethane 
1,2-bis(4-isobutoxycarbonyloxymethyl- 
60 68.8 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-morpholinomethyl-3,5- 
60 72.8 
dioxopiperazin-1-yl)ethane 
(known compound) 
______________________________________ 
Against Colon adenocarcinoma No. 38, G.I.(%) of the compound which the 
present invention concerns were nearly equal to that of the comparative 
compound. 
The toxicities of bis-dioxopiperazine derivatives which the present 
invention concerns were examined by the following tests. 
(5) Acute Toxicity Test 
The test group to which the compound of the present invention was 
administered consisted of ten mice. Five weeks old male mice (ddY, 23.+-.2 
g of body weight) were employed as test animals. 
These animals were intraperitoneally given the test compound which was 
suspended in the saline solution containing carboxymethyl cellulose (CMC) 
by 0.5% and were observed for 14 days successively, and LD.sub.50 value of 
acute toxicity was determined by Litchfield-Wilcoxon method. The results 
obtained are shown in Table 5. 
TABLE 5 
______________________________________ 
Results of the Acute Toxicity Test 
LD.sub.50 (mg/kg) 
Test Compound i.p. p.o. 
______________________________________ 
1,2-bis(4-acetoxymethyl-3,5- 
&gt;1000 &gt;2000 
dioxopiperazin-1-yl)ethane 
1,2-bis(4-methoxycarbonyloxymethyl- 
about 500 
&gt;2000 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-isobutoxycarbonyloxymethyl- 
&gt;850 &gt;1000 
3,5-dioxopiperazin-1-yl)ethane 
1,2-bis(4-morpholinomethyl-3,5- 
280 &gt;1000 
dioxopiperazin-1-yl)ethane 
(known compound) 
______________________________________ 
These data show that the acute toxicity of the compound which the present 
invention concerns is remarkably lower than that of the comparative 
compound. 
The following descriptions are given for the administration routes, 
pharmaceutical forms and doses when bis-dioxopiperazine derivatives of the 
present invention are applied to human. 
The compounds of the present invention may be administered orally in forms 
such as tablets, powders, granules, capsules, syrups and so on. They may 
be also administered parenterally in forms such as injections which may 
include dissolvable freeze-drying form, suppositories and so on. 
In the preparation of these forms, pharmaceutically acceptable diluent 
bases, binders, disintegrators, lubricants, suspensions, emulsifiers, 
antiseptics, stabilizers and dispersing agents, for example, lactose, 
sucrose, starch, dextrin, crystalline cellulose, kaolin, calcium 
carbonate, talc, magnesium stearate, distilled water and physiological 
saline solution may be used. 
Although the daily doses of these compounds may be varied according to the 
conditions, ages and weights of the subjects to be treated, the daily 
doses to adult humans may normally fall within the range of 50 to 3000 mg, 
preferably 500 to 1000 mg, and may be divided into two or three portions. 
From the above description, the compounds of the present invention are not 
only expected useful as antitumor agents and radio-potentiative agents 
similarly to the known compund of 
1,2-bis(4-moropholinomethyl-3,5-dioxopiperazin-1-yl)ethane, but also 
expected to have a broader antitumor spectrum and more excellent antitumor 
activity from the results of aforementioned antitumor tests as well as to 
have lower toxicity than that of the comparative compound. In addition, 
differently from the comparative compound, the compounds (I) exhibit 
pharmaceutically advantageous stability to protonic polar solvents. Thus, 
they have wider pharmaceutical usages as antitumor agents. 
The preparation methods of the compound (I) claimed in the present 
invention consist of relatively simple steps in high yields and are 
suitable for industrial production.