MOELLER LENNART (SE)
VALLBERG HANS (SE)
BERGMAN JAN (SE)
MOELLER LENNART (SE)
VALLBERG HANS (SE)
| EP0238459A1 | 1987-09-23 |
STN INTERNATIONAL, File CAPLUS, CAPLUS Accession No. 1994:499166, BEHRAVAN G. et al., "The Interaction of Ellipticine Derivatives with Nucleic Acids Studied by Optical and iH-NMR Spectroscopy: Effect of Size of the Heterocyclic Ring System"; & BIOPOLYMERS, (1994), 34(5), 599-609.
| 1. | Use of a compound having the formula I wherein R± represents hydrogen or one or several, preferably 1 to 4, similar or different substituents in the positions 14 and/or 710, selected from halogen, preferably Br, lower alkyl/alkoxy group having not more than 4 carbon atoms, trifluoromethyl group, trichloromethyl group; and in one of the positions 710 R^ can be a hydroxyl group; X is a group _(CH2)nR. |
| 2. | wherein R2 represents a nitrogen containing basic residue such as NH2, NHR4 or NR^Rg wherein R4, R5 and Rg independently are lower alkyl or cycloalkyl and n is an integer of from 1 to 4 and R. |
| 3. | epresents hydrogen, lower alkyl/cycloalkyl group having not more than 4 carbon atoms, and the physiologically acceptable addition products of the compounds with acids and halogen adducts, preferably adducts with iodine, iodine monochloride or iodine monobromide, for preparing a medicament for protecting DNA in the initial phase and/or the promoting phase of carcinogenesis and for preventing oxidative stress in patients with diseases related to free radicals. |
| 4. | 2 Use according to claim 1 for reducing or eliminating the effect of promotors in the promoting phase of carcinogenesis. |
| 5. | 3 Use according to claim 1 or 2 for preparing a medicament to be administrated to persons exposed to mutagenic substances or mutagenic radiation. |
| 6. | Use according to claim 1 for preparing a medicament for administration to persons treated with drugs which per se give rise to tumours. |
| 7. | Use of a compound having the formula I according to any of the previous claims wherein R^ in positions 2 and 3 is methyl and in the other positions hydrogen and wherein X is CH2N(CH3)2 and R is hydrogen. |
| 8. | Use according to any of claims 14 wherein R in positions 1 4 and 7, 8 and 10 is hydrogen, in positions 2 and 3 is methyl and in position 9 is hydroxyl wherein X is CH2N(CH )2 and R is hydrogen. |
The present invention relates to new uses of indolo-[2,3b] guinoxalines of the general formula I
wherein R^ represents hydrogen or one or several, preferably 1 to 4, similar or different substituents in the positions 1-4 and/or 7-10, selected from halogen, preferably Br, lower alkyl/alkoxy group having not more than 4 carbon atoms, trifluoromethyl group, trichloromethyl group; and in one of the positions 7-10 R j can be a hydroxyl group;
X is a group -(CH2) n - 2 wherein R2 represents a nitrogen containing basic residue such as NH2, NHR4 or R^Rg wherein R 4 , R5 and Rg independently are lower alkyl or cycloalkyl and n is an integer of from 1 to 4 and R3 represents hydrogen, lower alkyl/cycloalkyl group having not more than 4 carbon atoms, and the physiologically acceptable addition products of the compounds with acids and halogen adducts, preferably adducts with iodine, iodine monochloride or iodine monobromide, as DNA protecting agent both in the initial phase and in the promoting phase of carcinogenesis and which agent also reduces or eliminates the effect of promotors and which agent has effects
which protect for oxidative stress. Thus, the compounds used according to the present invention activates the defence of the body against carcinogenesis and oxidative stress.
It is known that free radicals (oxidative stress) of different types are associated with a range of diseases such as ischemic or reperfusion injury, thrombosis and embolism, atherosclerosis, allergic/inflammatory conditions such as bronchial asthma and rheumatoid arthritis, diseases caused by ionizing radiation or ultra violet light, conditions related to neurodegenerative diseases for instance Parkinson's disease and Alzheimer's disease, ageing, apoptosis, necrosis and cirrosis, cataract, physical stress, diabetes, autoimmune diseases, intoxications, colitis, hematocrosis, neoplasms and toxicity of antineoplastic or immuno suppressive agents diseases or consequences of viral or bacterial infections and endogeneous or exogeneous chemicals present in air, food, general environmental contamination or lifestyle related exposure. An explanation for these conditions and diseases can be that the endogeneous protecting capacity are not sufficiently active to protect the tissue against radical damage. Lipid peroxidation or DNA-oxidation caused by excess generation of radicals can constitute significant damaging pathways in the above conditions and diseases. The compounds used according to the present invention probably enter into the cell where it activates parts of the genome.
Oxidative stress can be chemically, physically or biologically induced. Chemically induced oxidative stress is caused by a compound which gives rise to a tissue damage. Physically induced stress is caused by e.g. 1) radiation, such as radioactive or ionizing radiation or UV radiation; 2) by physical blockage of blood flow. Biologically induced oxidative stress is the defence by the body itself, for instance in asthma, rheomatoid arthritis, diabetes etc, cf. above.
Different conditions such as inflammations, infections, gamma- radiation, UV radiation and deficiency of vitamines/antioxidants
give rise to oxidative stress which leads to the different conditions and diseases stated above. Use of the compounds according to the present invention in preparations to be administrated via different carriers will thus protect the body against oxidative stress and prevent the outburst of different diseases caused by such oxidative stress.
Indolo-[2,3b]-quinoxalines of the general formula I wherein R is a hydroxyl group in one of the positions 7-10 are prepared by means of a process described in the following section. Especially compounds wherein ^ is a hydroxyl group in position 9 show interesting properites for the use according to the present invention.
Substituted indolo-quinoxalines of formula I have previously been demonstrated to possess valuable activity against several types of virus and several of the compounds also have been demonstrated to show a potent anti-cancer effect, cf. our previous patent EP 0 238 459 and US 4.990.510. Furthermore, they have also been shown to be inactive as enzyme inhibitors, cf. Harmenberg et al, Antimicrobial agents and chemotherapy, November 1988, pp 1720- 1724.
Indolo-quinoxaline derivatives hydroxylated in the positions 7, 8, 9 or 10, especially in position 9, are of interest as metabolites or possible metabolites to biologically active quinoxaline derivatives such as lb. Such hydroxylated derivatives possess interesting anti viral and anti cancer properties in addition to their effect as DNA protecting agents and their effects to protect from oxidative stress.
The hydroxylated substances according to the present invention cannot be made by means of the methods described in our above mentioned patents. Thus, reaction of phenylene diamines and methoxyisatin, such as shown in Scheme 1, does not give in- doloquinoxalines but spirocyclic substances which cannot be converted to the desired substances.
Scheme I
Hydroxylation according to Scheme 2 does not function either
Scheme 2
A compound of the general formula II
can be prepared by subjecting a compound of the general formula
to mononitration with one equivalent of potassium nitrate or sodium nitrate in sulfuric acid whereby a compound according to the general formula IV
is obtained, which latter compound by catalytic hydrogenation followed by diazotization with nitrous acid and then conversion of the diazonium ion obtained to the 9-OH group by treatment with a Cu-based catalyst composed of Cu (1*03)2 and Cu 2 0 is converted to the compound of formula II.
This process is illustrated in the following scheme 3
(1a-d) (2a-d)
la R 1= H, R 2 =H lb R-^Me, R 2 =H
1c R^H, R 2 =CH 2 CH 2 NMe 2
Id R 1= Me, R 2 =CH 2 CH 2 NMe 2
(3a-d)
(4a-d)
The preparation of these compounds according to the invention is illustrated in the following examples 1-5.
The compounds used according to the present invention unexpected¬ ly have been found to possess a number of specific properties. Thus, they are not mutagenic or carcinogenic and they do not induce preneoplastic lesions and have not a DNA adduct forming activity. Furthermore, the compounds used in the present invention reduce the spontaneous mutation frequency in the
Salmonella assay.
In addition, the compounds eliminate the mutagenicity of certain mutagens in the test while the mutagenic effect of others are reduced, i.e. they are antimutagens.
Well known chemical substances which are mutagens and which thus initiate the tumour process (carcinogenesis) are polycyclic aromatic hydrocarbons (PAH), nitrated PAH compounds (nitro PAH) e.g. 2-nitrofluorene (NF); nitros- amines; different alkylating agents, food mutagens etc.
Furthermore, both UV radiation and gamma radiation (X-ray) have mutagenic effect and certain metals can catalyze reactions involving formation of DNA-damage.
In the first step in chemical carcinogenesis DNA adducts are formed. A characteristic feature of the compounds used according to the present invention is that they do not form DNA adducts. Furthermore, the compounds used according to the present invention reduce the capability of certain potent mutagens to form DNA adducts while other mutagens are less affected.
As regards the formation of early stages of tumour formation (preneoplastic foci) (in vivo) it has been found that the compounds used according to the present invention do not form any foci and furthermore reduce the capacity of potent carcinogens to form foci.
The compounds used according to the present invention have been tested as regards the formation of tumours (skin tumours) for mice subjected to strong tumour initiating and strong tumour promotion conditions. In this test model it was found that the compounds tested do not give rise to any tumours and furthermore if a compound according to the present invention was given twice a week the mice did not develop tumours which means that in the presence of a compound according to the present invention neither
the very strong initiating nor the very strong promotion will lead to tumours. Furthermore, if the administration of the compound used according to the present invention is terminated the animals will develop the same amount of tumours as in the positive control. This means that the original DNA injury remains and the tumour promotor which is administrated continously can give rise to a considerable development of tumours. Accordingly a compound used according to the present invention can stop the tumour process caused by potent tumour promoting agents in combination with potent tumour initiators.
As regards toxic effects of the compounds used according to the present invention no such effects have been noted in vivo.
In the promotion phase of carcinogenesis there are also chemical compounds which are known to be very potent promotors. One of the most potent promotors known today is triphorbolester (TPA). When testing a compound used according to the present invention by administration to an animal given TPA it was found that the effect of this promotor was eliminated.
The invention is illustrated by means of the following examples wherein examples 1-6 describe the preparation of intermediates and of compounds used according to the invention and examples 7- 10 describe tests carried out with the compounds according to the invention.
Example 1
2,3-dimethyl-6(2-dimethylaminoethyl)-9-nitro-6H-indolo[2, 3-b]- quinoxaline (2d)
To a solution of 15.9 g (50 πunol) of (Id) in 200 ml concentrated H 2 S0 4 5.06 g (50 mmol) KNO3 were added batchwise so that the temperature did not exceed 10°C. The solution was left with stirring at 5-10 β C in 2h and then was poured onto ice-water. The mixture was made alkaline with 20% KOH-solution. This resulted in a yellow percipitate which was filtered off. The raw product was
recrystallized from EtOH which gave 13.1 g of (2d). Yield: 72% M.p.: 219°C
NMR: δ H (DMSO): 9.1 (1H, s, Ar), 8.6 (1H, d, Ar), 8.1 (1H, s, Ar),
8.1 (1H, d, Ar), 7.9 (1H, s, Ar), 4.6 (2H, t, CH 2 ), 2.8 (2H, t, CH 2 ), 2.5 (3H, s, CH 3 ), 2.5 (3H, s, CH3), 2.2 (6H, s, CH3) ppm. IR: v maχ : 2940, 2760, 1620, 1580, 1510, 1460 br, 1320 br, 1190 br, 1135, 1100, 870, 810, 750, and 680 cm -1 .
Example 2
6-(2-dimethylaminoethyl)-9-nitro-6H-indolo[2,3-b]-quinoxa line
(2c)
(2c) was synthetisized in the same manner as (2d). Yield: 86% (not recrystallized) M.p.: 206-08 β C
NMR: δ H (DMS0): 9.1 (1H, s, Ar), 8.8 (1H, d, Ar), 8.4 (1H, d, Ar),
8.2 (1H, d, Ar), 8.1 (1H, d, Ar), 7.8-8.0 (2H, dt, Ar), 4.7 (2H, t, CH 2 ), 2.8 (2H, t, CH 2 ), 2.2 (6H, s, CH3) ppm.
IR: v maχ : 3060, 2940, 2810, 2770, 1610, 1580, 1510, 1465 br, 1400, 1330 br, 1300, 1245, 1145, 1125, 1105, 1070, 1045, 960, 915, 835, 795, 760, 750, 730, 710 cm "1 .
Example 3
9-amino-2,3-dimethyl-6-(2-dimethylaminoethyl)-6H-indolo[2 ,3-b]- quinoxaline (3d)
A suspension of 3.63 g (10 mmol) of (2d) and 0.36 g 10% Pd/C in 160 ml DMA was left under 2.7 atm hydrogen pressure for 24 h. The product was soluble in DMA. Pd/C was filtered away with celite. The filtrate was poured onto ice-water and pH was increased to basic with 20% KOH, which resulted in a brown precipitate. The precipitate was chromatographied in 20% eoH/CH2Cl2, which resulted in 2.80 g of the product (3d).
Yield: 84% M.P.: 250-51 β C
NMR: δ H (DMSO): 7.9 (1H, s, Ar), 7.8 (1H, s, Ar), 7.5 (1H, s, Ar), 7.4 (1H, d, Ar), 7.1 (1H, d, Ar), 5.1 (2H, br, NH 2 ), 4.4 (2H, t, CH2), 2.7 (2H, t, CH 2 ), 2.5 (3H, s, CH3), 2.5 (3H, s, CH3), 2.2
δ c (DMSO): 145.0 (s), 143.3 (s), 138.7 (s), 138.6 (s), 137.1 (s), 136.0 (s), 135.0 (s), 128.0 (d), 126.5 (d), 119.5 (s), 119.0 (d), 110.6 (d), 105.4 (d), 56.7 (t), 45.2 (q), 38.9 (t), 19.9 (q), 19.6 (q) ppm.
IR: v maχ : 3390, 3320, 3200, 2960, 2940, 2920, 2820, 2770, 1580,
1480, 1400, 1330, 1190 br, 870, 810, 720, and 680 cm -1 .
Example 4
2,3-dimethyl-6-(2-dimethylaminoethyl)-9-hydroxy-6H-indolo [2,3-b]- quinoxaline (4d)
660 mg (2 mmol) of (3d) were dissolved in 6 ml 30% H 2 S0 at 15 β C. The solution was cooled to 5°C. A solution of 179 mg (2.6 mmol) of Na 02 in 2 ml H2O was added without increasing the temperature above 5 β C. After 30 minutes urea was added in order to consume unreacted NaNθ2« To the solution at zero degree Centigrade was added a zero degree (4 g Cu(Nθ3)2-3H2θ in 50 ml H2O) and then 286 mg CU2O were added. This resulted in a strong formation of N2~gas. When N2 gas formation stopped the mixture was left in 30 minutes and then pH was increased to weakly alkaline. The mixture was extracted with C^C^* the CH2CI2 phase was dried and roll-evaporated. The raw product formed was chromatographated in 10% MeOH/C^C^ which resulted in 133 mg of the desired product (4d). Yield: 20% M.p.: >260 β C
NMR: δ H (DMSO): 9.5 (1H, s, OH), 8.0 (1H, s, Ar), 7.8 (1H, s, Ar), 7.6 (1H, s, Ar), 7.6 (1H, d, Ar), 7.2 (1H, d, Ar ) , 4.5 (2H, t, CH 2 ), 2.7 (2H, t, CH 2 ), 2.5 (3H, s, CH3 ) , 2.5 (3H, s, CH3 ) , 2.2
δ c (DMSO): 152.0 (s), 138.9 (s), 138.7 (s), 138.3 (s), 137.3 (s), 137.2 (s), 135.3 (s), 128.0 (d), 126.6 (d), 119.5 (d), 111.1 (d), 106.7 (d), 56.8 (t), 45.3 (q), 39.7 (t), 19.9 (q), 19.6 (q) ppm.
IR: v maχ : 3420 (br), 3130 (br), 2940, 2765, 1585, 1490, 1425, 1350, 1240, 1210, 1155, 1025, 1000, 865, and 725 cm -1 .
Example 5 g-Amino-6-(2-dimethyl-aminoethyl)-6H-indolo[2,3-b]-quinoxali ne
Yield: 85% M.p.: 222-223°C
NMR: δ H (DMSO): 8.2 (1H, d, Ar), 8.0 (1H, d, Ar), 7.7 (1H, dd, Ar), 7.6 (1H, dd, Ar), 7.5 (1H, s, Ar), 7.5 (1H, d, Ar), 7.1 (1H, d, Ar), 5.3 (2H, br, NH 2 ), 4.5 (2H, t, CH 2 ), 2.8 (2H, t, CH 2 ),
δ c (DMSO): 145.1 (s), 143.6 (s), 139.7 (s), 139.7 (s), 138.1 (s), 136.2 (s), 128.9 (d), 128.4 (d), 127.2 (d), 125.3 (d), 119.4 (d), 119.2 (s), 110.8 (d), 105.6 (d), 56.2 (t), 44.8 (q), 38.6 (t) ppm.
IR: v maχ : 3405, 3362, 3329, 3054, 2964, 2943, 2920, 2816, 2767, 1582, 1494, 1411, 1324, 1125, 809, 766, 621 and 592 cm "1 .
Example 6
6-(2-dimethylaminoethyl)-9-hydroxy-6H-indolo[2,3-b]-quino xaline
Yield: 18%
M.p.: 269-270 β C
NMR: δ H (DMSO): 9.6 (1H, s, OH), 8.2 (1H, d, Ar), 8.0 (1H, d, Ar),
7.8 (1H, dd, Ar), 7.6-7.7 (2H, m, Ar), 7.6 (1H, d, Ar), 7.2 (1H,
d, Ar), 4.5 (2H, t, CH 2 ) , 2.7 (2H, t, CH 2 ) , 2.2 (6H, s, CH3 ) .
δ c (DMSO): 152.1 (s), 145.2 (s), 139.8 (s), 139.4 (s), 138.2 (s), 137.7 (s), 128.9 (d), 128.7 (d), 127.3 (d), 125.5 (d), 119.9 (d), 119.1 (s), 111.2 (d), 106.8 (d), 56.7 (t), 45.2 (q), 39.1 (t) ppm.
IR: v maχ : 3127 br, 2964, 2941, 2771, 1586, 1489, 1425, 1261, 1239, 1210, 1119, 1048, 1021, 805 and 756 cm "1 .
Example 7
This example illustrates the mutagenic potential in the bacterial mutagenicity test, "Ames test", with test strain TA100, of B-220 (Figure la) and 9-OH-B-220 (Figure lb). S9 indicates the presence (+S9) or absence (-S9) of a cellular enzymatic fraction capable to metabolize the tested substances. The metabolic activation is a liver fraction containing enzymes needed in metabolism. The results are shown in Figures la and lb. In the Figures rever- tants/plate, an intensity measurement of mutagenic activity, is plotted against μg B-220 and μg 9-OH-B-220.
The results show that the substances are non-mutagenic but also anti-mutagenic (reduces the spontaneous background). The anti- mutagenic potential is similar for B-220 and 9-0H-B-220.
Example 8
The following example illustrates the fact that the compound B- 220 reduces the mutagenicity of potent mutagens in the bacterial mutagenicity test, the "Ames test". In the test the bacterial strain TA 100 was used. In the experiments two strong mutagens were used with and without the addition of B-220. These mutagens were MMS = Methylmethanesulphonate and EMS = Ethylmethanesulpho- nate. The results of the experiments carried out are shown graphically in Figure 2a and 2b. In the Figures revertants/piate, is plotted against microgram MMS and EMS, respectively, plus 70 μg B-220.
From Figure 2a it can be seen that MMS is a strong mutagen but when B-220 is added the mutagenicity disappears.
From Figure 2b it can be seen that EMS is a strong mutagen but needs other routes for DNA repair compared to MMS. The addition of B-220 reduce the mutagenicity but not as much as for MMS.
These tests show that strong mutagens are not able, or reduced in their capacity to generate mutations when B-220 is present. The reduction of mutagenicity is not caused by cell death since tested substances behave differently (mechanistic reason).
Example 9
The in vivo toxicity of B-220 was tested in rats. A portion of the liver was removed (partial hepatecto y, PH) by surgery and the regeneration of liver tissue was studied. The regeneration was measured as liver-somatic index (LSI), in other words the relative liver weight in per cent of the body weight.
AAF (2-acetylaminofluorene) is a very toxic substance which acts as a mito-inhibitor, i.e. inhibits cell division of normal liver cells.
The results are shown in Figure 3 wherein LSI is plotted against days after partial heptatectomy.
As seen from Figure 3 the regeneration of liver tissue in the AAF group was very slow. The control group recieved nothing and there was a rapid regeneration of tissue. In the group administered B- 220 no difference from the controls could be seen. B-220 did not show any toxic effects to rats in vivo measured during very intensive cell division.
From these results it can be seen that B-220 is not toxic in vivo to cells that undergo cell division.
Example 10
This example illustrates a tumor model which is based on in vivo tumor initiation with one dose of dimethylbenzanthracene (DMBA) whereafter a potent tumor promotor triphorbol ester (TPA) was administered twice a week. After a number of weeks the skin tumors can be detected and followed during the study. After approximate 100 days (A) the positive controls reaches the maximum, whereafter the total number declines due to the behavior of the animals. In the group administered B-220 one hour before TPA there were no tumors at all at the same time point. After six months - the normal time for termination in this model - the B- 220 group had developed a very slight increase in tumors. After six months exposure of B-220 + TPA in the B-220 group, B-220 was no longer administered (B). Immediately a rapid increase of tumors was seen. These results are illustrated in Figure 4.
The initial DNA damage was there all the time, but was "silent" in the mice treated with B-220. Not even the very potent tumor promotion did have any effect with B-220 pretreatment. After the termination of B-220 treatment there was a rapid development of tumors indicating that both the initial DNA damage was present and the tumor promotor could catalyze the tumor formation. Tumor initiation was a genotoxic event and tumor promotion was an oxidative stress in this model.
These results show that B-220 is not a carcinogen in it self and further that B-220 can eliminate the effects of tumor initiations by the potent carcinogen DMBA and block tumor promotion by the potent tumor promotor TPA. When B-220 is removed the initial DNA damage can - together with a promotor - generate as much tumors as in the positive control. In other words, B-220 can, in vivo, switch on and off the tumor process.