CA1143726A - Anti-neoplastic use of 1,4-bis (substituted aminoalkyl amino) -anthraquinones - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Method for treating animal neoplasms by administering to a neoplasm-bearing animal host an antineoplastic amount of a 1,4-bis-substituted-9-10-anthracenedione having the formula

Description

11~3~ 6 1 ANTI-NEOPLASTIC USE O~
1,4-BIS (SUBSTITUTED A~IINOALKYL A~IINO)-ANTHRAQUINONES

BACKGROUND OF THE INVENTION

This invention relates to a method of use of 1,4-bis-(substituted aminoalkylamino)-anthraquinones, and more particularly to a use of the same as anti-neoplastic agents against animal neoplasms. 1,4-bis-substituted anthraquinones have long been known in the prior art as dyes.
For example, U.S. Patent No. 2,050,661 teaches a process for preparing 1,4-diaminoan~hraquinone - which is suitable for use for dying cellulose esters and ethers or for coloring fats, oils, waxes and the like. U.S. Patent No. 1,199,176 teaches the use of l,4-bis-(alpha, beta-diphenol-ethylamino) anthraquinone and the like for dying wool and other animal fibers.
- 20 A method for the production of l,4-bis-(substituted aminoalkylamino)-anthraquinones is also known in the art ("The reaction of leuco-quinizarines with alkylenediamines", Greenhalgh and _Iu~hs, J. Chem. Society (C), page 1284, 1968).
Certain naturally occurring substituted anthraquinones, namely, 2,6-bis-substituted and

2,7-bis-substituted anthraquinones, have been found to give indications of possible anti-neoplas-tic inhibitory effects. Generally, these have been reported as inhibitions of DNA polymerases by intercalation between base pairs of DNA double helix. The intercalation theory has been used as a working hypothesis to explain the activity of many anti-cancer drugs including actinomycin D, :, . . . ~

'~3 7~

l daunorubicin, adriamycin, anthramycin, and coralyne.
However, certain of these drugs, even though recognized as very promising and definitely showing good inhibitory activity against leukemia as well : 5 as solid tu~nors, have the disadvantage ~hat they or ~heir metabolites cause severe and irreversible cardio toxicity which cowld be fatal if the accu-mulated dose of these drugs exceeds a limi.ted amount. ~dditionally, they are all naturally produced.
Certain in vivo testing systems and protocols have been developed by the National Cancer Institute for testing compounds to deter-mine their suitability as antineoplastic agents.
These have been reported in Cancer Chemotherapy Reports, Part III, Vol. 3, No. 2, (1972), Deran, ~reenberg, MacDonald, Schumacher and Abbott. These protocols have established standardized screening tests which are generally followed in the field of testing for antineoplastic agents. Three of these systems are particularly significant to the present invention. These are lymphoid leukemia L1210, lymphocytic leukemia P388 and melanotic melanoma B16. All of these neoplasms are found in mice.
Initial screening is usually done with P388 leukemia followed with further screening tests against L1210 leukemia and B16 melanoma if the initial tests appear promising. Generally, good antineoplastic activity shown in these protocols by a percentage increase of mean survival times of the test animals over the control animals is ~- predictive of similar results in human leukemias.
A mean survival time ratio of test over control (with the control group representing 100%) of 125%
; 35 is considered necessary to demonstrate anti-1 neoplastic activity by the substance being tested.
Further detailed description of these protocols is presented hereinbelo~ in the detailed description of the invention.

.

1143~7~t~

1 OB~ECTS OF THE INVENTION
_.

It is a primary object of the invention to provide a compound useful in the treatment of animal neoplasms in an animal neoplasm~bearing host.

Another object of the present invention is to provide compounds effective in the treatment of highly predictive P388 leukemia in mice, the highly predictive L1210 leukemia in mice, and the highly predictive B16 melanoma in mice.
In accordance with the present teachings there is provided a process for producing a substituted anthracene-dione which is represented by the formula CH2)nR

Z NH(CEi2)nRwhich comprises the steps of reacting a dihydroxyleucoquinizarin of the formula Z OH
wherein W and Z are both OH

with an alkylamine or substituted alkylamine of the formula H2N(CH2)nR

.~ ,,.

'7;~16 wherein n is l to 4 and R is H, OH, NHCH2CH2OH, and oxidizing the product with oxygen or air.

Some of the above groups have been found to be especially effective. These include the compounds in Table 1.

W Z

a. OH OH (CH2)2-NH-~CH2)2-OH
b. H H (CH2)2NH(CH2)2OH
c . H H (Cli2 ) 2NHCH2CH3 d. H (CH2)2NH CH3 e. H H (CH2)2NH2 f. H (CH2)2 ~
g. H H (CH2)3NH(CH2)2OH

'7~:6 1 DETAILED DESCRIPTION OF THE I_VENTION

Generally, the method of preparation of the 1,4-bis (substi~uted aminoalkylamino) anthra-qulnones was based on that described byG ~halgh and Hughes, J. Chem. Society (C), Page 128~i, 1968. This involves condensation of leucoquinizarins (II) with an excess amount of the appropriate amines at 50 to 55 C followed by air oxidation of the dihydro intermediates (III) to the desired produc~s (IV).

o OH ~ NHR O NHR

X ~ 2X ~ ~G~ 2 ~

OHNHR HR
II III IV
Since the intermediates III and products IV have clistinct and different UV absorption : in ethanol (III are usually green, with ~ max .. at 465 and 490 nm, whereas the bright blue IV
have ~ max at 580 and 630 nm) the absorption change of the reaction mixture is used to monitor the course of the reaction and to estimate the purity of the proclucts. The products, which are soluble in water, strongly stain shin, fiber, and even plastic material. The intense dark color of solutions of these compounds often affect purification processes.
Oxidation of the dihydro` intermediates III to the aminoanthraquinones IV proceeded readily in most cases and sometimes can even be realized cluring recrystallization of the crude dihydro intermedia~es III or upon standing of III in 1~3'7~6 1 solution. Ilence, attempts to isolate the pure dihydro intermediates III, even under nitrogen, proved to be difficult. For example, attempted isolation of the dihydro intermediate of 1,4-bis (hydroxyethyl-aminoethylamino) anthraquinone gave an 80% yielcl of a crude compound, melting point of about :L30-132C. It was, understandably, still contaminated with the final product. At a higher reaction temperature of about 100C, the product readily cyclized to form compound V.

~ H(CH2)2OI~ (V) NH(CH2)2NH(CH2)20H
For the preparation of many target comPounds, there-fore, the reaction temperature must be kept below 55C, preferably at 50C.
Some difficulties were encountered during the preparation of the dihydro intermediate 1,4-bis (2-aminoethylamino) anthraquinone because of the presence of a primary amine function of the side chains and the insolubility of the inter-mediate in the particular reaction solvents used (ethanol and CH3CN). The dihydro intermediate was eventually obtained but subsequent oxidation by air in CH3CN yielded a high-melting solid, melting point about 308-310C, which was only sparingly soluble in common organic solvents such as ethanol or CHC13. Elemental analysis indicated the presence o~ only three nitrogen atoms in the molecule and suggested VI as one of the possible structures of the product.

,

3'7Z16 O HN

~ (VI) HN

10Apparently compound ~I was formed by an intra-molecular condensation of the two terminal chains with the elimination of NH3. The desired compound e (Table I), melting point of 174-176C, was obtained in 23% yield by repeated recrystalliza-.: 15tions of the dihydro intermediate from CH3CN
. according to the method noted above of Greenhalgh and Hughes.

_XAMPLE 1 Preparation of 1,4-dihydroxy-5,8-bis[[2-_ _ . _ _ _ _ _ _ _ , . _ . _ _ _ ., _ . . _ _ _ _ _ _ . _ . _ _, ., _ . . _ . _ . . . _ . _ _ _ . . _ _ . . . _ _ .
(hydroxy-ethyl)amino]ethyl]amino-9,10-anthracenedione.

The preparation of 1,4-dihydroxy-5, 8-bis[[2-hydroxy ethyl) amino]ethyl]amino-9,10-25 anthracenedione (compound a, Table I) was carried out as follows: To lOg (0.036 mole) of 5,8-dihydroxyleucoquinizarin (commercially available from Bayer AG, 509 Leverkusen, Beyerwerk, West Germany], purified by continuous extraction with dioxane under nitrogen) was added dropwise, under nitrogen with cooling and stirring, 38 g (0.36 mole) of 2-(2-aminoethylamino)ethanol. When a homogeneous paste was obtained, the reaction mix-ture was heated at 50-55C in an oil bath for two .~

1 hours. It was allowed to stir overnight. The mechanical stirring rod was replaced (the stirring rod was rinsed with 4 x 50 ml of ethanol, the ethanol was~-ings were added to the mixture) by a glass sparge tube and dry air (passed through a tube containing Drierite) was bubbled through the reaction mixture (the entire system was under a slightly reduced pressure by connecting the top of the condenser to a water aspirator. This mild oxidation reaction was carried out at 55-60C for approximately 2-3 hours. The color of the syrup gradually changed from purple to a bright blue.
The mixture was then allowed to s~and overnight at room temperature. The resulting dark blue solid was collected by filtration through a sintered glass funnel. The solid product washed with ethanol (2 x 20 ml), petroleum ether (3 x 50 ml) and dried to give 4.6 g (20% yield) of Compound a, m/p/ 158-160C. An analytical sample was prepared by recrystallization of the crude product from a mixture of ethanol and petroleum ether, m.p 160-162CC. ~EtOH 244 (log~4.64), 279 (4.31), 525 m~x (3.70), 620 (4.37) and 660 nm (4.38). Anal.
Calculated for C22H28N~O6 : C, 59.50;
H, 6.34; N, 12.61. Found: C, 59.55; H,6.56;
N.12.33 - l(! -~1~37~

Preparation of 1,4-Bis[[2-(ethylamino) et~y~ no] 9,10-anthracenedione 2 (c~l2)2-NH-cH2-cil3 (VII) (V~ (CH2)2 NH Cll2 CH3 NH-(cH2)2-NH-c~l2-CH3 A mixture of 4 g (o.9165 mole) of 1, 4, 9, 10-tetra-hydroxyanthrancene (leucoquinizarin) - VII and 23 g (0.26 mole) of N-(2-aminoethyl) ethylamine VIII was heated to 50C under N2 for ninety minutes. The mixture was cooled and allowed to stand overnight. To it was added lO0 ml of methanol. A stream of dry air was bubbled through the mixture at 50C for two hours. Blue crystals, which separated from the reaction mixture after standing, were collected by filtra-tion and washed with 10 ml of ethyl acetate followed by 10 ml of hexane. The product was purified by recrystallization from a mixture of chloroform, and heptane to give 3.5 g (56% yield) of IX, m/p/ 118-120C. Anal: Calculated for C22H28N24 2H20:C,63.44;H,7.74; N,13.45 Found C,63.61; H, 7.79; N,13.68.

1.1~37~6 1 EXAMPLE -[II
Preparation of 1,4-~Bis[[2-methylamino) ethyl]amino]-9 10 anthracenedione :, 1l ~ H- (CH2) 2-NH-CH3 (VII) ~ 12N- (C~12) 2-NilC1-13 O NH - (C~2)2- NH-CH3 ( X ) (XI ) Twenty-two grams (0.29 mole) of N- (2-aminoethyl) methylamino X was added dropwise into 10.0 g (0.045 mole) of VII under nitrogen with stirring in 15 min. After the addition was complete, the mixture was stirred for an additional 30 min., then heated at 50-55C for three hours. It was then cooled and 100 ml of ethanol was added. Dry air was bubbled through the mixture at 45-50C for three hours. It was allowed to stand overnight and to it was added 40 ml of ethanol and 20 ml of Skelly solve F
(petroleum ether, bp 35-60C). The mixture was stirred for thirty minutes and the solid product was collected by filtration. It was washed successively with ethanol (2 x 5 ml) and Skelly solve F (2 x 50 ml) and dried to give 8.2 g (52% yield) of XI, mp 120C. An analytical sample was prepared by recrystallization of 1.2 g of XI
from 50 ml of chloroform and 100 ml of Skelly solve F mp 119-121C. Anal. Calculated for ~1~37~6 1 C20H24N4o2 1/2H2O: C,66.46; H,7.06; N,15 50.
Found: C, 66.29;H,7.11; N, 15.31 EXAMPLE IV
P eparation of 1,4-bis[2-aminoethyl)amino]
-9,l0-anthracenedione _ __ 1' IH (CH2)2 NH2 VII ~ ~2~-(C~2)2-NH2 NH-(CH2)2-NH2 (XII) (XIII) A mixture of 14.5 g (0.06 mole) of VII
and 150 ml of ethylenediamine XII was heated under N2 for one hour at 50C. Dry air was bubbled through the mixture at 50C for forty-five minutes.
The mixture, after standing overnight, deposited XIII which was collected by filtration, washed successively with acetonitrile (3 x 30 ml) and diethyl ether (2 x 50 ml) and dried to give 17 g of crude XIII, mp 165-168C.
Its ultraviolet absorption spectrum showed the presence of dihydro derivatives. Three recrystallizations from acetonitrile gave 5.9 g (23% yield) of purified XIII, mp 174-176C. Anal.
ed for C18H20N4O2: C, 66.65; H,6.21;
N, l7.27. Found: C,66.45; H 6.36; N, 1.7.10.
The antineoplastic activity of the various compounds was determined in in vivo testing following the protocols developed by the National Cancer Institute reported by Deran et al in 3t7;~6 1 Cancer Chemotherapy Reports, part 3, Vol. 3, No. 2, (1972). These specific test systems were lymphoid leukemia 1,1210, lymphocytic leukemia P388 and Melano~ic melanoma B16. The key test result noted is the mean survival time ratio of test animals over control animals which is hereinafter noted as percentage increase of life span of test over control (percent II.S). As noted above, a percent ILS of 125% is considered to show good anti-neoplastic activity.
Activity against lymphocytic leukemia P388is tested by implanting ascitic fluid in BDFl mice.
Treatment with the compound begins twenty-four hours after the implant. As noted abo-ve, the results are expressed as a percent increase in life span or survival time. The innoculum site for screening is ip ancl the compound is administered ip daily for nine days. The implant size is 0.1 ml of diluted ascitic fluid containing 106 cells. The number of survivors of the test group on day 6 is checked.
All deaths of test animals after day 6 are not con-sidered to be due solely to drug toxicity. Testing ends on day 30. The mean animal weight difference between the test group and the control group is computed between days 1 and 5. At the completion of testing the percent ILS is computed for all test groups with greater than 65% survivors on day 5.
A percent ILS value of less than 85% indicates a toxic test, a percent ILS of greater than 125% is considered to demonstrate antineoplastic activity Any survivor of the test group on the day of evalua-tion (day 30) is recorded as a cure.

:
~3~7~6 1 Activity against lymphoid leukemia L1210 is tested by implanting ascitic fluid into BDFl mice. Treatment begins twenty-four hours after the implant. The results are expressed as a per-centage of increased life span of the test animals over the control animals. The innoculum site is ip and the coMpound is administered ip. The implant size is 0.1 ml of diluted ascitic fluid containing 105 cells. As with P388, the toxicity check date is day 6 and the experiment is run for - thirty days. Mean animal weight is also checked between days 1 and 5 and the percent ILS is : computed at the end of the experiment. Any survivors of the test group on day thirty are considered to be cures.
Activity against melanotic melanoma B16 is tested by implanting a tumor homogenate ip in BDFl mice. The treatment begins twenty-four hours after the ip implant and the results are expressed as a increased percentage of life span of the test animals over the control animals. The drug is administered ip daily for nine days. To prepare the homogenate, 1 gram of tumor is mixed with 10 ml of cold balanced salt solution and homogenized.
0.5 ml oE the tumor homogenate is implanted ip.
The check date for compound toxicity is day 5 and the experiment runs until day 60.
All dosages are in milligrams per kilogram of body weight of test animal per in- t -jection. The mean survival time of the test animals is computed and ratioed with the mean survival time of the control animals and the result is expressed as a percentage (%ILS). Other specific information regarding the animal selec-tion and animal care, randomization of animals in l the testing, specific preparation and administra-tion of test materials, selection of doses, pro-pagation of tumor lines, tumor quality control, specific techniques of tumor transplantation, and the e~act calculational method for test evaluation can be found in the above noted reference to Deran et al, which is incorporated herein by reference.

ANTINEOPLASTIC TESTING EXAMPLES
lO In addition to the compounds listed in Table 1, the following compounds with noted structure are presented:

TABLE II
. l~ . . z R
h. H H (CH2)2NI~(CH,,)2CH3 i. H H (CH2)2 ~
i. l~ ( 2)2 (CH2)2NH(cH2)2oH
k. H H (CH2)2S(CH2)2 OH
1. H (CH2)50H
~, m. H ~ - OCH3 The following results were obtained testing for activity against lymphocytic leul~emia P3&&:

3'7~6 ACTIVITY A~AINST_P388 Compound Dose Survi~al Wt. Diff. %ILS Cures a - _m:~ /kg gm 280 5 / 6 1 5/6 -0.6 277 3/6 0.5 6/6 -2.0 299 4/6 0.25 6/6 -2.3 280 2/6 0.12 6/6 -1.4 200 0.06 6/6 1.7 208 b 32 6/6 -5.9 81 16 6/6 -3.9 275 3/6 8 6/6 -2.0 276 4/6

4 6/6 0 275 3/6 c 25 11/12 -4.8 132 1/12 12.5 5/6 -2.8 168 6.25 6/6 -2.2 149 3.13 6/6 -2.3 142 d 12.5 6/6 -1.9 200 e 25 6/6 -2.6 215 12.5 6/6 -1.1 174 6.25 6/6 -1.1 159 2 3.13 6/6 -019 107 1.56 6/6 -1.6 137 0.78 6/6 -1.3 150 f 50 10/12 -217 ].34 12/ 12 - 1.0 137 .~ 12.5 12/12 1.1 117 6.25 6/6 -0.4 125 100 11/12 -2.6 98 1/12 ] ~/12 -2.1 133 11/12 -2.1 130 12.5 6/6 1.4 117 6.25 6/6 0.9 101 h 100 6/6 -2.6 124 6/6 -1.7 125 6/6 -0.8 118 i 100 12/12 -4.5 1~
~0 12/12 -2. ~ 110 6/6 -3.6 10

5~ ~

~f~'7~'6 1 Compound ~)ose Survival Wt. Diff. %ILS Cures j 10~0~ -15/18 -2g6m 120 18/18 -1.3 112 18/18 -1.6 110 k 100 6/6 -2.2 94

6/6 -1.4 86 6/6 -1.6 95 1 100 6/6 -4.0 88 6/6 -1.5 78 6/6 -1.4 88 m 400 6/6 -2.9 91 200 6/6 -3.2 101 100 6/6 -1.7 86 Certain of these compounds were tested against Melanotic Melanoma B16 and Lymphoid Leukemia L1210, the data being presented in Tables IV
and V respectively:

TABLE IV

2~
Compound Dose %ILS Cures e 16 281 8/10 a 1 503 7/10 0.5 466 ~/10 TABLE V

Compound Dose %ILS
b 125 272 12.5 227 6.25 156 ~ ~ ~ 3~7~ ~

1 As can be seen from the data in Table III, all oE the 1,4-bis(substituted aminoalkylamino) anthraquinones showed at least some antineoplastie activity against P388. A %II.S of greater than 125 is considered good antineoplastic activity.
Some of the results are espeeially noteworthy. Namely, with the absence of the second amino group (compounds k, 1, and m) no activity was evidenced. Apparently, the nitrogen atom in center of the side ehain plays an im-portant role in antileukemie aetivity. No aetivity is noted when this nitrogen atom is replaeed by a methylene unit (even though eompound 1 retains the same ehain length) or by a sulphur atom. The insertion of an additional ethyl amino unit into the side ehain as in eompound j, drastieally reduces the activity below the marginal level.
The distanee between the nitrogen atoms appears to have signifieanee whieh is apparent in the eom-parision between the results of eompound g andcompownd b. ~urther importance of the nitrogen atom in the center of the side chain is demon-strated by ehanging the original seeondary amino function to a tertiary amino function. Compound i is onl.y marginally aetive when eompared with eompound b. The activity of eompound f is slight-ly thereabove probably due to the presenee of the binding terminal. On the other hand, eompound e, whieh eontains primary amino groups at the end of both side chains, still remains good, although not as high as compound b.
Surprisingly excellent antileukemie aetivity is obtained in eompound a (note the extremely low dosage levels both for aetivity against P388 and activity against B16). This 1~37Z6 1 compound is more soluble in water than compound b and appears to require less than one-tenth of the optimum dose of compound b to produce good activity.
Co~pound e additionally shows excellent activity against B16 with a high rate of cures (survivors at the end of day 30 of the test group.) It should now be apparent that the objects initially set forth have been successfully achieved. Moreover, while there is shown and described present examples of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

: 20 .

,.,

Claims (14)

WHAT IS CLAIMED IS:

1. A process for producing a substituted anthracene-dione represented by the formula:
comprising the steps of reacting a dihydroxyleucoquinizarin having the formula:

wherein W and Z are both OH
with an alkylamine or substituted alkylamine having the formula:

H2N(CH2)nR
where n is 1-4 and R is H, OH, NHCH2CH2OH, and oxidizing the product with oxygen or air.

2. The process set forth in claim 1, wherein n is 1 and R is OH.

3. The process set forth in claim 1, wherein n is 3 and R is H.

4. The process set forth in claim 1, wherein n is 3 and R is OH.

5. The process set forth in claim 1, wherein n is 4 and R is H.

6. The process set forth in claim 1, wherein n is 4 and R is OH.

7. The process set forth in claim 1, wherein n is 2 and R is NHCH2CH2OH.

8. A substituted anthracenedione having the formula wherein W and Z are both OH and R is H, OH and NHCH2CH2OH
and n is 1 to 4 whenever prepared or produced by the process of claim 1 or by any obvious chemical equivalent thereof.

9. The anthracenedione of claim 8 wherein R is OH and n is 1 whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

10. The anthracenedione of claim 8 wherein R is H and n is 3 whenever prepared or produced by the process of claim 3 or by any obvious chemical equivalent thereof.

11. The anthracenedione of claim 8 wherein R is OH and n is 3 whenever prepared or produced by the process of claim 4 or by any obvious chemical equivalent thereof.

12. The anthracenedione of claim 8 wherein R is H and n is 4 whenever prepared or produced by the process of claim 5 or by any obvious chemical equivalent thereof.

13. The anthracenedione of claim 8 wherein R is OH and n is 4 whenever prepared or produced by the process of claim 6 or by any obvious chemical equivalent thereof.

14. The anthracenedione of claim 8 wherein R is NHCH2CH2OH and n is 2 whenever prepared or produced by the process of claim 7 or by any obvious chemical equivalent thereof.