1) A new potential anticancer agent, 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone (T-2), was synthesized from 1,4,5,8-tetrachloroanthraquinone and characterized. 2) T-2 demonstrated greater cytotoxicity against human breast cancer cells (BOT-2 cell line) than the existing drugs mitoxantrone and doxorubicin in in vitro assays. 3) In in vivo assays using mice with B16 melanoma, T-2 showed an equal life span and tumor response compared to mitoxantrone, but was less toxic.

1. AACR
April 5th San Diego, CA. 2014
1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)amino]anthraquinone as a Potential Anticancer Agent: its synthesis, characterization and anticancer properties
Background
Mitoxantrone (M0) (5,8-dihydroxy-1,4-bis{[2-[(2-
hydroxyethyl)amino]ethyl]amino}-9,10-anthracenedion.HCl has few side
effects except for neutropenia and cardiotoxicity. The cardiotoxicity is
thought to reside with the two ring hydroxyl groups. Without the hydroxyl
groups the anthraquinone has reduced cardiotoxicity but also reduced
cytotoxicity and is used for the treatment of various types of cancer. A
number of 1,4,5,8-tetra-aminoalkylanthraquinone derivatives were
synthesized and it was determined if these compounds had cytotoxic
activity. It was realized that four side chains were possible therefore four
areas of activity. 1,4,5,8 Tetra compounds could be made from 1,4,5,8-
Tetrachloroanthraquinone. Compounds from 1,4,5,8-
tetrachloroanthraquinone were used in applications such as: liquid crystal,
to color gasoline and to absorb infrared radiation. No report of their use as
anti-cancer compounds has been noted.
References
1) Ernst Gutzwiiler, Arylamino Anthraquinone Dystuffs USP 2,688,028, Aug. 31, 1954
2) Greenhalgh et al., The Reaction of Leucoquinizarins with Alkylenediamines J. Chem.
Soc., p. 1284 (1968).
3) K. C. Murdock, R. G. Child, P. F. Fabio, Robert B. Angier, Antitumor Agents. 1. 1,4-Bis[
(aminoalkyl)amino]-9,lO-anthracenediones Journal of Medicinal Chemistry, 1979, Vol.
22, No. 9
4) Nordquist, R. E., Ishmael, D. R., Lovig, E. A., Hyde, D. M., and Hoge, A. F.: The tissue
culture and morphology of human breast tumor cell line BOT-2. Cancer Research, 35-
3100-3105, 1975.
5) Ishmael, D. R., Nordquist, R. E., Bottomley, R. H., and Zieren, J.: Vitamin A Acid
(Retinoic Acid) as an adjuvant to increase Adriamycin cytotoxicity in human breast
cancer cells in vitro. Prevention and Detection of Cancer, Pt. 1, Vol. 1, 727-736, 1977.
6) Melvin N. Turetzky, Wayne, and Leon Katz, - dihydroxy .5- butylamino -8- (3-
trimethylamino - propylamino) - anthraquinone methyl sulfate USP 3,281,434 1,4 10/1966
7) David J. Thompson Pleochroic anthraquinone dyes, USP 4,446,047 1984
8) David J. Thompson Pleochroic anthraquinone dyes, USP 4,455,253 1984
9) Tsukasa Ohyama; Shizuo Kuroda; Keisuke Takunia; Hiroshi Aig, Halogenated
anthraquinone and their use as near infrared rays absorbing optical filters USP 5,342,974
1994
10) Gerard Andrew Potter, Laurence Hylton Patterson, Paul Teesdale-Spittle, Zennia
Paniwynk, Anthraquinone Anticancer Drugs, USP 6,465,522, 10/ 15/2002
11) Kim Sang Ho, Yu-Min Chen, Method for Marking Hydrocarbons with Anthraquinones
USP 6,811,575 11/02/2004
Don Richard Ishmael1 and Orn Adalsteinsson2
[1]Medical University of the Americas, Nevis West Indies, NatCel Dev. Okla. City OK [2] International Strategic Cancer Alliance
Method of Synthesis
E-mail ishmaeldr@gmail.com
The 1,4,5,8-Tetrakis-[2-N,N-dimethylaminoethyl)aminoanthraquinone (T-2) was synthesized from 1.4.5.8-tetrachloroanthraquinone by a modification of the methods of
Ohyama et al9. The 1,4,5,8-tetrachloroanthraquinone was reacted with N,N dimethylethylenediamine, at 100 to 140 degrees centigrade in a mixture of sodium acetate, cupric
sulfate in benzyl alcohol for 8 hours. After reaction the solid was collected washed with solvents and dried. The 1,4,5,8-Tetrakis-[2-N,N-dimethylaminoethyl)
aminoanthraquinone (T-2) was dissolved in 15% acetic acid and filtered. The amino compound was then precipitated with 15% sodium hydroxide
solution. The precipitated compound was collected and dried. Melting point was obtained using a Mel-Temp apparatus. The compound was subjected to TLC using
acetone/water/Triethylamine. Samples were sent to Midwest Research Institute for further characterization. The 1,4,5,8-tetrachloroanthraquinone was obtained from
Haiman Chemical Shanghai China. The material for purification was subjected to rigorous extraction by hot sulfuric acid containing 10% fuming sulfuric acid. This removed
impurities. The N,N dimethylethylenediamine was obtained from Aldrich. The 1,4,5,8-Tetrakis-[2-N,N-dimethylaminoethyl)aminoanthraquinone was further purified using
preparative HPLC to 97.8% purity for testing.
1200C
6 hrs.
N,N dimethylethylenediamine
Method for Screening Cytotoxicity In-vitro:
The human breast cancer cell line BOT-2 was used in the cytotoxicity assays. 210l of cells were plated into each well of a
72 well microtiter plate. After confluence of the cells at 24hrs the drug to be tested was added in 70l sol. at the initial
concentration of 1mg/ml. The drug was diluted 1:4 with media in serial fashion. The plates were examined at 24, 48, and
144 hrs for cell kill. The cell concentration giving 100% cell death was recorded. The end-point of this study was cell death
as determined by visual observation of the cells The BOT-2 cell line was established from a patient’s primary cancer in the
mid 1970’s. ER-, Her2neu-, rich in EGFR-1
Method for screening in vivo effect:
Method of screening used was exactly as reported by Murdock2 for mitoxantrone (M-0). The animals used in the testing
were C57BC/6 mice weighing 17g, with 10 animals per test group. The B16 melanoma obtained was homogenized in 10ml
of cold balanced salt solution and 0.5ml aliquot of the homogenate was implanted intraperitoneally into each test mouse at
varying dosages. The compounds are administered intraperitoneally on days 1 through 9 (relative to tumor inoculation).
The survivors were recorded for 60 days. T-2 was compared to M-0 and to untreated controls.
T-1 T-2 T-3 T-4
Well #
concentration of compound in mg/ml
1:4 dilutions start 1mg/ml
Start mg/ml
20
5
1
0.25
0.0625
0.0156
0.00391
0.000977
0.000244
0.0000610
0.0000152
0.00000384
0.000000954
0.0000002384
0.00000005960
1
2
3
4
5
6
7
8
9
10
11
12
13
T-2,2,2,8-12
100% Cell Death Read at 144 hours
Black Represents All Cells Dead
Colored Bars Represent Living Cells
Black- 100% cell death
Orange T-1 1,4,5,8-terakis[hydroxyethyl(aminoethyl)]aminoanthraquinone
Lt green T-2- 1,4,5,8,-tetrakis-dimethylethylaminoANQ
Sodium acetate, Cupric
sulfate, benzyl alcohol
T Compounds Results of Cytotoxicity Assay
T-8,2,2,2
Green T-3 1,4,5,8,-tetrakis-hydroxyethylaminoANQ
Brown T-8,2,2,2 1 part chloroethylamine and 3 partsN,N dimethylenediamine
Blue T-2,2,2,8-12 T-2,2,2,8 reacted with Bischloroethylamine
Summary of T-2 compound vs bis-compounds
1) Activity of T-2 compound equal to or greater than corresponding bis
compounds
2) Easier synthesis with fewer side reactions
3) Cheaper starting components
4) Potentially Higher yields
5) More functional group options
6) Increased number of active groups sites
7) Potentially easier purification procedures
8) Removal of the para dihydroxyl groups from the cytotoxic 1,4
BisaminoalkylaminoANQ's might reduce the potential for cardiotoxicity
1,4,5,8-Tetra-N,N-dimethylaminoethylaminoanthraquinone (T-2)
Abstract
Compounds based on the base compound 1,4,5,8 tetrahydroxyanthraquinone 1,4-Bis
aminoalkylamino, 1,4-Bis aminoalkyl-5,8-dihydroxy, and 1,4-
hydroxyalkylaminoalkylamino-5,8-dihydroxy anthraquinones have anticancer
properties. One compound of this class mitoxantrone bis-5,8
hydroxyethylaminoethylamino 1,4 dihydroxy anthraquinone is on the market and is
approved by the FDA for the treatment of: acute myelogenous leukemia, various
lymphomas, breast cancer, sarcoma and the non-malignant condition multiple
sclerosis. This drug has a number of drawbacks: (1) for most cancer therapies it is
considered less effective than the anthracyclines, (2) less cardiotoxicity than
anthracyclines it does still has significant cardiotoxicity, (3) rapidly induce multi-drug
resistance factors, (4) significant bone marrow toxicity. compounds of 1,4-Bis
aminoalkylamino class intercalating DNA in the major groove and inhibit of
topoisomerase II enzyme. Numerous attempts have been made to improve the class
of drugs by adding different functional groups. No compound of this class has
exceeded the utility of mitoxantrone. Attempts to make derivatives of leuco 1,4,5,8-
tetrahydroxyanthraquinone replacement of all four hydroxyl groups for the purpose
of additional DNA binding or alkylation has been unsuccessful. 1,4,5,8-
tetrachloroanthraquinone can be used as a base compound producing potential
anticancer activity. 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)
amino]anthraquinone was synthesized and purified. It
demonstrated greater cytotoxicity in the screening assay than mitoxantrone or
adriamycin. Screening was done using the BOT-2 human breast cancer cell line. This
compounds may have an advantage over the Bis-derivatives with: 1) greater
cytotoxicity in screening assay, 2) cheap intermediates, 3) higher yields, 4) increased
number of potential active groups sites 7) removal of the para dihydroxyl groups
from the cytotoxic 1,4 bisaminoalkylaminoanthraquinone might result in reducing
the potential for cardiotoxicity. The 1,4,5,8-Tetrakis-[(2-N,N-dimethylaminoethyl)
amino]anthraquinone was purified to 97.8 purity by HPLC.
Results in General
BOT-2 cells were treated with a cytotoxic compound. The dead cells become rounded, detach and floated. No treatment
controls were run concurrently. Concentration of the compound to give this 100% kill was recorded. At 100% kill there is
never re-growth of the tumor cells. Visual observation gave results identical to the standard MTT cytotoxicity assay.
Results of in vitro Cytotoxicity Assay
Mitoxantrone and adriamycin had equal activity in the BOT-2 cell screen. T-2 activity was greater than either M-0 or for
adriamycin.
Results of in vivo Assay
The mouse data indicated an equal life span and tumor response of T-2 compared with M-0. T-2 was found to
be less toxic. However the survival data for M-0 was much less than reported by Murdock2.
Thanks to Dr. Robert Nordquist for supplying the BOT-2 cell line and to John Nordquist for running the cytotoxicity assays. Also thanks to
Dr. Wei Chen for obtaining the 1,4,5,8-Tetrachloroanthraquinone and running the animal assays
Material presented herein patent #8420861 United States patent office