Anti-bacterial 1,4-aminoalkylamino-9H-thioxanthen-9-one derivatives, compositions, and method of use therefor

1,4-Aminoalkylamino-9H-thioxanthen-9-ones have antimicrobial activity and activity against leukemic cells. Methods for their preparation, use and pharmaceutical compositions are described.

BACKGROUND OF THE INVENTION 
Experimental antitumor activity has been reported for thioxanthenones 
notably lucanthone 1a and hycanthone 1b 
##STR1## 
and recently the 7-hydroxy analog of lucanthone has been shown to have in 
vivo P388 activity. 
S. Archer, K. J. Miller, R. Rej, C. Periana, and L. Fricker, J. Med. Chem., 
25, 220 (1982). 
S. Archer, A. H. Zayed, R. Rej, and T. A. Rugino, J. Med. Chem., 26, 1240 
(1983). 
The 4-nitro analog of lucanthone has been described in an earlier paper on 
the preparation of schistosomiasis drugs. 
S. Archer and C. M. Suter, J. Am. Chem. Soc., 74, 4296 (1952). 
In a recent paper, the corresponding amino compound (1c) was prepared and 
shown to be inactive against the P-388 lymphocytic leukemia. 
S. Archer and R. Rej, J. Med. Chem., 25, 328 (1982). 
The corresponding N,N-dimethylaminoethyl and N-hydroxyethylaminoethyl 
analogs of lucanthone were also inactive. 
SUMMARY OF THE INVENTION 
The present invention relates to pharmaceutical compounds having 
antibacterial, antifungal, and antitumor activity, which are represented 
by a compound of the formula 
##STR2## 
and pharmaceutically acceptable acid addition salts thereof, wherein 
R.sub.5 -R.sub.8 each independently represent hydrogen, hydroxy or alkoxy 
of from 1-4 carbon atoms; 
A and B are each independently a straight or branched alkylene chain of 
from 2-5 carbon atoms, optionally substituted with hydroxyl; 
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are H or straight or branched alkyl 
of 1-4 carbon atoms, optionally substituted with hydroxyl and R.sub.1 and 
R.sub.2, or R.sub.3 and R.sub.4 when taken together represent 
##STR3## 
wherein n and m are each an integer from 2-3 and W is a direct bond or O, 
S or NR.sup.9 wherein R.sup.9 is H or straight or branched alkyl of from 
1-4 carbon atoms, optionally substituted with hydroxyl. 
The present invention also includes pharmaceutical compositions for 
treating bacterial, fungal infections or tumors such as lymphocytic 
leukemia in warm-blooded animals comprising an effective amount of a 
compound of formula I in admixture with a pharmaceutically acceptable 
carrier or diluent. 
The present invention further includes a method of treating bacterial or 
fungal infections as well as tumors such as lymphocytic leukemias in 
warm-blooded animals which comprise administering to said warm-blooded 
animal an effective amount of a compound of the formula I in unit dosage 
form such as a pharmaceutical composition as herein defined. 
The present invention also includes novel intermediates of the formula III 
wherein R.sub.1 -R.sub.8 and A are as defined above. These compounds are 
also useful pharmacologically as antitumor agents. 
The compounds of the invention may be prepared conveniently by the 
following reaction sequence 
##STR4## 
The reaction step (1) is carried out as described in S. Archer and C. M. 
Suter, J. Am. Chem. Soc., 74, 4296 (1952). Reduction of the 4-nitro group 
of the compound of the formula II is carried out by known methods using 
catalytic hydrogenation with, for example, hydrogen in the presence of 
Raney nickel catalyst at elevated temperatures in an inert solvent such as 
an alcohol, e.g. ethanol, or with 10% palladium on carbon catalysts in a 
hydrogenator where hydrogen is at a pressure of approximately 40 psi or 1 
atmosphere. 
The reaction step (3) is carried out in the usual way in an inert solvent 
at room or elevated temperatures. Here X is halogen, preferably bromine or 
chlorine, and the reaction may be carried out in the presence of base or 
other appropriate acid acceptor, such as triethylamine, pyridine, and the 
like. 
When --NR.sub.1 R.sub.2 is a secondary amine, i.e., R.sub.1 or R.sub.2 is 
hydrogen, said amine can be appropriately blocked with an amino protecting 
group prior to alkylation, step (3). Blocking groups such as 
t-butyloxycarbonyl or benzyloxycarbonyl can be used which are conveniently 
removed following the alkylation reaction. 
Alternatively when --NR.sub.3 R.sub.4 is a secondary amine and substituted 
by hydroxyl, the alkylation may be carried out with a 
haloalkyleneoxazolidine as, for example, specifically illustrated in 
Example 1. Ring opening of the oxazolidine moiety affords the desired 
hydroxyalkylaminoalkylene group attached to the 4-amino position. 
The compounds of the invention form pharmaceutically acceptable salts with 
both organic and inorganic acids. Examples of suitable acids for salt 
formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, 
malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, 
methanesulfonic, isethionic, lactic, gluconic, glucuronic, sulfamic, 
benzoic, tartaric, pamoic, and the like. The salts are prepared by 
contacting the free base form with an equivalent amount of the desired 
acid in the conventional manner. The free base forms may be regenerated by 
treating the salt form with a base. For example, dilute aqueous base 
solutions may be utilized. Dilute aqueous sodium hydroxide, potassium 
carbonate, ammonia, and sodium bicarbonate solutions are suitable for this 
purpose. The free base forms differ from their respective salt forms 
somewhat in certain physical properties such as solubility in polar 
solvents, but the salts are otherwise equivalent to their respective free 
base forms for purposes of the invention. 
The compounds of the invention can exist in unsolvated as well as solvated 
forms, including hydrated forms. In general, the solvated forms, with 
pharmaceutically acceptable solvents such as water, ethanol and the like 
are equivalent to the unsolvated forms for purposes of the invention. 
The term halogen is intended to include fluorine, chlorine, bromine, and 
iodine. 
The alkyl and alkoxy groups contemplated by the invention, unless specified 
otherwise, comprise both straight and branched carbon chains of from one 
to about four carbon atoms. Representative of such groups are methyl, 
ethyl, isopropyl, butyl, sec-butyl, isobutyl, t-butyl, methoxy, ethoxy, 
i-propoxy, t-butoxy, and the like. 
The alkylene groups contemplated by the invention, unless specified 
otherwise, comprise both straight and branched carbon chains of from two 
to about 5 carbon atoms. Representative of such groups are ethylene, 
n-propylene, n-butylene, i-propylene, and the like. The preferred alkylene 
groups of the invention have the following structural formulas: 
##STR5## 
Accordingly, a preferred embodiment of the present invention includes a 
compound of the formula I wherein R.sub.5 -R.sub.8 are hydrogen; A, B, 
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are as defined above, and 
pharmaceutically acceptable acid addition salts thereof. 
Another preferred embodiment of the present invention includes a compound 
of the formula I wherein R.sub.5 -R.sub.8 are hydrogen, A and B are each 
independently ethylene or propylene, R.sub.1, R.sub.2, R.sub.3, and 
R.sub.4 are as defined above and pharmaceutically acceptable acid addition 
salts thereof. 
Still another preferred embodiment of the present invention includes a 
compound of the formula I wherein R.sub.5 -R.sub.8 are hydrogen, A and B 
are each independently ethylene or propylene, R.sub.1, R.sub.2, R.sub.3, 
and R.sub.4 are each independently hydrogen, methyl, ethyl, or 
hydroxyethyl and pharmaceutically acceptable acid addition salts thereof. 
Particular embodiments of the present invention include 
1-[[2-(dimethylamino)ethyl]amino]-4-[[2-[(2-hydroxyethyl)amino]-ethyl]amin 
o]-9H-thioxanthen-9-one; 
4-[(2-aminoethyl)amino]-1-[[2-dimethylamino]-ethyl]-9H-thioxanthen-9-one, 
and 
4-[(3-aminopropyl)amino]-1-[[2-dimethylamino)ethyl]amino]-9H-thioxanthen-9 
-one and pharmaceutically acceptable acid addition salts thereof. 
The compounds of the invention are new chemical substances of value as 
pharmacological agents for the treatment of bacterial and fungal 
infections in warm-blooded animals. The antibacterial and antifungal 
activity of representative compounds of the invention was established by 
the screening procedure described below. 
In addition to their usefulness as antibacterial and antifungal agents, 
compounds of the invention display in vitro and in vivo antitumor 
activity. 
1. Preparation of inocula 
(A) Bacteria and yeast: 
The bacterial and yeast isolates are maintained in agar slants or in liquid 
media, hereby designated as inoculum media. The cultures are transferred 
at regular intervals in such media. (See Table for the corresponding 
inoculum media of each culture.) The organisms are generally transferred 
on to agar slants or liquid inoculum media and incubated overnight (18-20 
hours): 37.degree. C. for the bacterial isolates and 28.degree. C. for the 
fungal cultures. 
The microbial cells from the overnight agar slants are then scraped off and 
suspended in saline solution (0.85% NaCl). The microbial concentrations 
are adjusted to a light transmittancy of 20-35%, Junior Coleman 
Spectrophotometer (555M). For the organisms that are maintained in liquid 
media, an aliquot of the culture suspension is simply diluted with saline 
to 20-35% light transmittancy. 
The above microbial suspension serve as inocula for the assay plates. Thus, 
0.16-10 ml (see Table for exact amount) are used to inoculate 100 ml of 
the molten-agar assay medium. 
(B) Mycelial fungi: 
The Penicillium avellaneum is grown for six days, at 28.degree. C., on an 
agar medium. This is to allow sporulation of the culture. The organism is 
then harvested by scraping off the cells from the agar surface (mycelia 
and spores) and suspending them in saline solution containing 0.05% Tween 
80. The suspension is adjusted to a light transmitancy of 20%. One ml of 
this suspension is used to inoculate 100 ml of the molten-agar assay 
medium. 
2. Preparation of assay plates 
Stainless steel frames, 12.3.times.25.3 cm (ID) and glass plates, 
15.3.times.31.7 cm are used to make the test trays. The frames are 
attached to the plates with tape at each end and the inner edges sealed 
with 2% agar. Twenty five ml of inoculated assay medium is spread evenly 
on each tray and allowed to solidify. The trays are covered, inverted, and 
refrigerated until used. 
3. Disking of samples 
The compounds or samples to be tested are dissolved in suitable solvents, 
e.g., alcohols, dimethylsulfoxide, or N,N-dimethylformamide. The samples 
are generally dissolved so that the final concentration of the solvent is 
&lt;10%.* The compounds are tested at different concentrations: 3,000; 1,000; 
500; 100; and 10 mcg/ml. Paper discs (12.7 mm diameter) are placed on the 
agar trays with forceps, then 0.08 ml of the dissolved compound is 
pipetted onto each disc using a 0.2 ml pipette. (*If the compound does not 
stay in solution at &lt;10% alcohol, then the full strength alcohol is used. 
However, the impregnated discs are air-dried before they are laid on to 
the seeded agar plates.) 
4. Interpretation of results 
The disked agar trays are incubated overnight (18-20 hours) at 37.degree. 
C. for the bacterial cultures and 28.degree. C. for the yeasts. The 
Penicillium avellaneium tray is incubated for at least 20-24 hours since 
it is a slower-growing organism. Active compounds show a zone of 
inhibition around the disc. The diameter of the zone is measured in mm. 
The zone diameter of active compounds ranges from a minimum of 13.5 mm to 
as high as 60 mm. The size of the zone diameter generally reflects the 
activity of the compound: the larger the zone the greater the activity. 
TABLE 
______________________________________ 
Inoculum 
Level 
ml/100 Assay 
Culture Number Inoculum Medium 
ml Medium 
______________________________________ 
Aerobacter 
0126 Veal Infusion 
1 Mycin 
aerogenes Broth Agar 
Escherichia 
04863 AM-08 Agar 1 AM-08 
coli 
Bacillus 04555 AM-08 Broth 0.5 AM-08 
subtilis 
Streptococcus 
05045 Folic Acid Assay 
2 AM-09 
faecalis Broth 
Penicillium 
M2988 AM-Agar 1 AM-25 
avellaneum 
______________________________________ 
5. Culture media 
The composition of the various culture media, except for the commercially 
available media, are shown below. The commercial ready-made Veal Infusion 
Medium is obtained from Difco Laboratories, Detroit, Mich., United States. 
Add 1.5% agar to these media for use as agar plates. 
______________________________________ 
AM-08 % 
Glucose 0.2 
Sodium Glutamate 1.04 
KH.sub.2 PO.sub.4 0.03 
Na.sub.2 HPO.sub.4 0.07 
Salts #1.sup.a 1 ml 
Salts #2.sup.b 10 ml 
H.sub.2 O (distilled) 
.sup.a Salts #1 
% .sup.b Salts #2 
% 
MgSO.sub.4 1.0 MnSO.sub.4 1.0 
CaCl.sub.2 5.0 ZnSO.sub.4.7H.sub.2 O 
1.0 
NaCl 5.0 FeSO.sub.4.7H.sub.2 O 
1.0 
CuSO.sub.4.5H.sub.2 O 
0.01 H.sub.2 O (distilled) 
H.sub.2 O (distilled) 
AM-09 
K.sub.2 HPO.sub.4 3.9 gm 
Dextrose 25 gm 
Na-citrate .2 H.sub.2 O 
34.4 gm 
Casein hydrolysate 6.2 gm 
Asparagine 375 mg 
L-tryptophan 125 mg 
Cysteine 312.5 mg 
Glutathione 3.1 mg 
Thiamine HCl 250 g 
Riboflavin 625 g 
Ca pantothenate 500 g 
Nicotinic acid 500 g 
p-aminobenzoic acid 625 g 
Biotin 12.5 g 
Pyridoxine HCl 2.5 g 
Folic Acid 500 g 
NaCl 12.5 g 
MgSO.sub.4 250 g 
FeSO.sub.4 12.5 g 
MnSO.sub.4.H.sub.2 O 125 g 
Tween 80 62.5 mg 
H.sub.2 O (distilled) 1000 ml 
AM-25 % 
Na.sub. 2 HPO.sub.4.H.sub.2 O 
0.35 
KH.sub.2 PO.sub.4 0.05 
Yeast Extract (Difco) 0.5 
Dextrose 1.0 
Distilled Water 
______________________________________ 
Utilizing the above described procedure, the results in Table 1 were 
obtained for representative compounds of the invention. 
In Vitro Antitumor Activity 
A test protocol uses L1210 cells, a murine leukemia cell line, grown in 
RPMI 1640 supplemented with 5% fetal bovine serum and gentamicin (50 
.mu.g/ml). Drug dilutions are prepared in the appropriate solvent and 20 
.mu.l of each dilution are added to 24-well Linbro tissue culture plates, 
followed by the addition of 2.0 ml of cell suspension containing 
3.times.10.sup.4 cells per ml. Solvent and medium controls are included in 
each test. After incubation at 37.degree. C. for three days in 5% 
CO.sub.2, the contents of each well are removed and the cells counted in a 
ZBI Coulter counter. Percent growth are calculated relative to the 
controls and the levels of drug activity are expressed as ID.sub.50 in 
moles per liter. 
In addition to their usefulness as antibiotic and antifungal agents and as 
antileukemic agents, certain of the compounds of the invention display in 
vitro activity against solid tumors when tested by the following 
procedure. 
HCT-8 (human colon adenocarcinoma) cells are trypsinized using 
Trypsin-EDTA. A single cell suspension is achieved by passing the cells 
through a 26 gauge needle with a 20 cc syringe. A cell suspension is 
prepared using RPMI 1640 growth medium (available from Gibco 
Laboratories)+10% fetal calf serum+50 .mu.g/ml garamycin with a cell 
concentration of approximately 30,000 cells/ml. The cell suspension is 
dispensed in Linbro 24-well plates; 1 ml/well. The plates are incubated 
for approximately 48 hrs at 37.degree. C. in a 5% CO.sub.2 atmosphere. At 
this time test compounds are added in the appropriate concentration. Five 
.mu.l of the 200 .mu.g/ml stock solution is added to each well in a 
primary test. Ten .mu.l of the appropriate dilution is added to each well 
for a titration test. The plates are reincubated an additional 60-65 hrs 
at 37.degree. C. in a 5% CO.sub.2 atmosphere. The test is read by lysing 
the cells using a mix of cationic surfactant, glacial acetic acid and 
sodium chloride. Two ml of the lysed cell suspension from each well is 
added to 8 ml of diluent. Each sample is read with a Coulter counter (ZBI 
model). The activity of each sample is measured as a percentage of the 
controls and the data is reported as ID.sub.50, that is the molar quantity 
of drug required to kill 50% of the tumor cells. 
In Vivo Activity 
The in vivo lymphocytic leukemia P388 test is carried out by the United 
States National Cancer Institute. The animals used are either male or 
female CD.sub.2 F.sub.1 mice. There are six to seven animals per test 
group. The tumor transplant is by intraperitoneal injection of dilute 
ascitic fluid containing cells of lymphocytic leukemia P388. The test 
compounds are administered intraperitoneally in two single doses with a 
four-day interval between doses at various dose levels following tumor 
inoculation. The animals are weighed and survivors are recorded on a 
regular basis for 30 days. A ratio of survival time for treated 
(T)/control (C) animals is calculated. The criterion for efficacy is 
T/C.times.100&gt;125%. The positive control compound in this test is 
1,4-dihydroxy-5,8-[bis[[2-[(2-hydroxyethyl)amino]-ethyl]]amino]-9,10-anthr 
acenedione given at dosages ranging from 12.0 to 0.075 mg/kg. See Cancer 
Chemotherapy Reports, Part 3, 3, 1 (1972) for a comprehensive discussion 
of the protocol. 
Utilizing these procedures, the results in Table 1 were obtained for 
representative compounds of the invention. 
TABLE 1 
__________________________________________________________________________ 
Antitumor, Antibacterial, and Antifungal Data on 
1,4-Aminoalkylamino-9Hthioxanthen-9-ones 
##STR6## 
Inhibitory Zone Diameter 
P388 in vivo 
(Conc. in mg/ml) 
Ex- L1210 in 
HCT-81 
Dose B. S. A. N. 
ample vitro 
in vitro 
(mg/ 
T/C .times. 
E. Sub- 
Fae- 
Vis- 
Cat- 
No. 
NHANR.sub.1 R.sub.2 
NHBNR.sub.3 R.sub.4 
ID.sub.50 (M) 
ID.sub.50 (M) 
kg) 
100 coli 
tilis 
culic 
colacti 
arrhali 
__________________________________________________________________________ 
1 NHCH.sub.2 CH.sub.2 NMe.sub.2 
NHCH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 OH 
6.1 .times. 
-- 25 236 (1)* 
17 19 19 17 21 
10.sup.-7 125 
190 (3000) 
(100) 
(1600) 
(100) 
(100) 
2 NHCH.sub.2 CH.sub.2 NMe.sub.2 
NHCH.sub.2 CH.sub.2 NH.sub.2 
6.0 .times. 
5.9 .times. 
25 212 (1)* 
14 16 14 23 20 
10.sup.-7 
10.sup.-7 
12.5 
187 (1000) 
(100) 
(500) 
(100) 
(100) 
3 NHCH.sub.2 CH.sub.2 NMe.sub.2 
NH(CH.sub.2).sub.3 NH.sub.2 
2.6 .times. 
-- 25 125 0 16 18 14 24 
10.sup.-6 (1000) 
(3000) 
(100) 
(100) 
__________________________________________________________________________ 
*Number of animals surviving 30 days and considered "cured."- 
TABLE 2 
__________________________________________________________________________ 
Antitumor, Antibacterial, and Antifungal Data on 
1,4-Aminoalkylamino-9Hthioxanthen-9-ones 
##STR7## 
P388 in vivo 
Example L1210 in vitro 
HCT-8- in vitro 
Dose Inhibitory Zone Diameter 
(Conc. 
No. NHANR.sub.1 R.sub.2 
X ID.sub.50 (M) 
ID.sub.50 (M) 
(mg/kg) 
T/C .times. 100 
in mg/ml) 
__________________________________________________________________________ 
5 NH(CH.sub.2).sub.3 NMe.sub.2 
H 9.6 .times. 10.sup.-8 
4.6 .times. 10.sup.-7 
50 190 
5 NH(CH.sub.2).sub.2 NMe.sub.2 
7-OMe 
2.5 .times. 10.sup.-7 
1.0 .times. 10.sup.-6 
100 178 
5 NH(CH.sub.2).sub.2 NMe.sub.2 
7-OH 
6.5 .times. 10.sup.-9 
-- -- -- 
5 NH(CH.sub.2).sub.2 NEt.sub.2 
6-OMe 
1.2 .times. 10.sup.-6 
1.1 .times. 10.sup.-6 
100 104 
4 NH(CH.sub.2).sub.2 NMe.sub.2 
H 1.2 .times. 10.sup.-7 
2.7 .times. 10.sup.-7 
-- -- 
5 NH(CH.sub.2).sub.2 NEt.sub.2 
7-OH 
__________________________________________________________________________ 
When being utilized as antibiotic and antifungal agents, the compounds of 
the invention can be prepared and administered in a wide variety of 
topical, oral, and parenteral dosage forms. It will be clear to those 
skilled in the art that the following dosage forms may comprise as the 
active component, either a compound of formula I, certain of the compounds 
of formula II or a corresponding pharmaceutically acceptable salt of one 
of said compounds or a mixture of such compounds and/or salts. 
For preparing pharmaceutical compositions from the compounds described by 
this invention, inert, pharmaceutically acceptable carriers can be either 
solid or liquid. Solid form preparations include powders, tablets, 
dispersible granules, capsules, cachets, and suppositories. A solid 
carrier can be one or more substances which may also act as diluents, 
flavoring agents, solubilizers, lubricants, suspending agents, binders, or 
tablet disintegrating agents; it can also be an encapsulating material. In 
powders, the carrier is a finely divided solid which is in admixture with 
the finely divided active compound. In the tablet the active compound is 
mixed with carrier having the necessary binding properties in suitable 
proportions and compacted in the shape and size desired. The powders and 
tablets preferably contain from 5 or 10 to about 70 percent of the active 
ingredient. Suitable solid carriers are magnesium carbonate, magnesium 
stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, 
tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low 
melting wax, cocoa butter, and the like. The term "preparation" is 
intended to include the formulation of the active compound with 
encapsulating material as carrier providing a capsule in which the active 
component (with or without other carriers) is surrounded by carrier, which 
is thus in association with it. Similarly, cachets are included. Tablets, 
powders, cachets, and capsules can be used as solid dosage forms suitable 
for oral administration. 
Liquid form preparations include solutions, suspensions, and emulsions. As 
an example may be mentioned water or water-propylene glycol solutions for 
parenteral injection. Liquid preparations can also be formulated in 
solution in aqueous polyethylene glycol solution. Aqueous solutions 
suitable for oral use can be prepared by dissolving the active component 
in water and adding suitable colorants, flavors, stabilizing, and 
thickening agents as desired. Aqueous suspensions suitable for oral use 
can be made by dispersing the finely divided active component in water 
with viscous material, i.e., natural or synthetic gums, resins, methyl 
cellulose, sodium carboxymethyl cellulose, and other well-known suspending 
agents. 
Topical preparations include dusting powders, creams, lotions, gels, and 
sprays. These various topical preparations may be formulated by well known 
procedures. See for example Remington's Pharmaceutical Sciences, Chapter 
43, 14th ed. 1970, Mack Publishing Co., Easton Pa., 18042, United States. 
Preferably, the pharmaceutical preparation is in unit dosage form. In such 
form, the preparation is subdivided into unit doses containing appropriate 
quantities of the active component. The unit dosage form can be a packaged 
preparation, the package containing discrete quantities of preparation, 
for example, packeted tablets, capsules, and powders in vials or ampoules. 
The unit dosage form can also be a capsule, cachet, or tablet itself or it 
can be the appropriate number of any of these packaged form. 
The quantity of active compound in a unit dose of preparation may be varied 
or adjusted from 50 mg to 500 mg according to the particular application 
and the potency of the active ingredient. 
In therapeutic use as antibiotic and antifungal agents the compounds 
utilized in the pharmaceutical method of this invention are administered 
at the initial dosage of about 0.1 mg to about 50 mg per kilogram. A dose 
range of about 0.5 mg to about 10 mg per kilogram is preferred. The 
dosages, however, may be varied depending upon the requirements of the 
patient, the severity of the condition being treated, and the compound 
being employed. Determination of the proper dosage for a particular 
situation is within the skill of the art. Generally, treatment is 
initiated with smaller dosages which are less than the optimum dose of the 
compound. Thereafter, the dosage is increased by small increments until 
the optimum effect under the circumstances is reached. For convenience, 
the total daily dosage may be divided and administered in portions during 
the day if desired. 
The active compounds may also be administered parenterally or 
intraperitoneally. Solutions of the active compound as a free base or 
pharmaceutically acceptable salt can be prepared in water suitably mixed 
with a surfactant such as hydroxypropylcellulose. Dispersions can also be 
prepared in glycerol, liquid polyethylene glycols, and mixtures thereof 
and in oils. Under ordinary conditions of storage and use, these 
preparations contain a preservative to prevent the growth of 
microorganisms. 
The pharmaceutical forms suitable for injectable use include sterile 
aqueous solutions or dispersions and sterile powders for the 
extemporaneous preparation of sterile injectable solutions or dispersions. 
In all cases the form must be sterile and must be fluid to the extent that 
easy syringability exists. It must be stable under the conditions of 
manufacture and storage and must be preserved against the contaminating 
action of microorganisms such as bacteria and fungi. The carrier can be a 
solvent or dispersion medium containing, for example, water, ethanol, 
polyol (for example, glycerol, propylene glycol, and liquid polyethylene 
glycol, and the like), suitable mixtures thereof and vegetable oils. The 
proper fluidity can be maintained, for example, by the use of a coating 
such as lecithin, by the maintenance of the required particle size in the 
case of dispersion and by the use of surfactants. The prevention of the 
action of microorganisms can be brought about by various antibacterial and 
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic 
acid, thimerosal, and the like. In many cases, it will be preferable to 
include isotonic agents, for example, sugars or sodium chloride. Prolonged 
absorption of the injectable compositions can be brought about by the use 
in the compositions of agents delaying absorption, for example, aluminum 
monostearate and gelatin. 
Sterile injectable solutions are prepared by incorporating the active 
compound in the required amount in the appropriate solvent with various of 
the other ingredients enumerated above, as required, followed by filtered 
sterilization. Generally, dispersions are prepared by incorporating the 
various sterilized active ingredient into a sterile vehicle which contains 
the basic dispersion medium and the required other ingredients from those 
enumerated above. In the case of the sterile powders for the preparation 
of sterile injectable solutions, the preferred methods of preparation are 
vacuum drying and the freeze-drying technique which yield a powder of the 
active ingredient plus any additional desired ingredient from a previously 
sterile-filtered solution thereof. 
As used herein, "pharmaceutically acceptable carrier" includes any and all 
solvents, dispersion media, coatings, antibacterial and antifungal agents, 
isotonic and absorption delaying agents and the like. The use of such 
media and agents for pharmaceutically active substances is well known in 
the art. Except insofar as any conventional media or agent is incompatible 
with the active ingredient, its use in the therapeutic compositions is 
contemplated. Supplementary active ingredients can also be incorporated 
into the compositions. 
It is especially advantageous to formulate parenteral compositions in 
dosage unit form for ease of administration and uniformity of dosage. 
Dosage unit form as used herein refers to physically discrete units 
suitable as unitary dosages for the mammalian subjects to be treated; each 
unit containing, a predetermined quantity of active material calculated to 
produce the desired therapeutic effect in association with the required 
pharmaceutical carrier. The specification for the novel dosage unit forms 
of the invention are dictated by and directly dependent on (a) the unique 
characteristics of the active material and the particular therapeutic 
effect to be achieved, and (b) the limitation inherent in the art of 
compounding such an active material for the treatment of disease in living 
subjects having a diseased condition in which bodily health is impaired as 
herein disclosed in detail. 
The principal active ingredient is compounded for convenient and effective 
administration in effective amounts with a suitable 
pharmaceutically-acceptable carrier in dosage unit form as hereinbefore 
disclosed. A unit dosage form can, for example, contain the principal 
active compound in amounts ranging from about 0.1 to about 500 mg, with 
from about 0.5 to about 250 mg being preferred. Expressed in proportions, 
the active compound is generally present in from about 0.1 to about 500 
mg/ml of carrier. In the case of compositions containing supplementary 
active ingredients, the dosages are determined by reference to the usual 
dose and the manner of administration of the said ingredients. The daily 
parenteral doses for mammalian subjects to be treated ranges from 0.1 
mg/kg to 100 mg/kg. The preferred daily dosage range is 0.3 mg/kg to 10 
mg/kg.

The following nonlimiting examples illustrate the inventors' preferred 
methods for preparing the compounds of the invention. 
EXAMPLE 1 
1-[[2-(Dimethylamino)ethyl]amino]-4-[[2-[(2-hydroxyethyl)amino]ethyl]amino] 
-9H-thioxanthen-9-one trihydrochloride 
A mixture of 1-[(2-(dimethylamino)ethyl)amino]-4-aminothio-xanthenone (5.0 
g, 0.016 mole) and 10 g of 3-.beta.-chloroethyloxazolidinone was flushed 
with argon for 0.5 hours and heated in an oil bath for eight hours at 
100.degree.. The reaction mixture was dissolved in 250 ml of CH.sub.2 
Cl.sub.2 and 250 ml of 2NNaOH. The organic layer was extracted with 250 ml 
of 2NNaOH. The organic layers were combined, washed with water, dried over 
MgSO.sub.4 and evaporated to dryness in vacuo. The resultant solid was 
purified by flash chromatography using 410 g of silica gel in a column 
with an outside diameter of 8.5 cm. Elution with CH.sub.2 Cl.sub.2 :MeOH 
(11:1) provided 2.2 g of 3-[2-[[1-[[2- 
dimethyamino)ethyl]amino]-9-oxo-9H-thioxanthen-4-yl]amino]ethyl]-2-oxazoli 
dinone as a red solid. This was dissolved in 20 ml of 2NKOH in MeOH, 10 ml 
of water and 10 ml of tetrahydrofuran, flushed with argon and heated under 
reflux overnight. The organic material was removed in vacuo, 40 ml of 
water was added, and the mixture was extracted with methylene chloride. 
The extracts were washed with water, dried over MgSO.sub.4, and 
concentrated to dryness. The red solid was purified by flash 
chromatography using 180 g of silica gel and a 50 mm column. Elution with 
CH.sub.2 Cl.sub.2.MeOH.Et.sub.3 N (49:49:2) and evaporation provided 14 g 
of product which was dissolved in 40 ml of EtOH and added to a large 
volume of 2-propanol saturated with gaseous HCl. The mixture was warmed 
gently on the steam bath, cooled, and filtered to give 1.6 g of the 
product as a yellow solid, mp 260.degree. (dec). 
Similarly may be prepared: 
1-[[3-(Dimethylamino)propyl]amino]-4-[[2-[2(2-hydroxyethyl)amino]ethyl]amin 
o]-9H-thioxanthen-9-one-from 
1-[[(3-dimethylamino)propyl]amino]-4-aminothioxanthenone. 
1-[[3(-Dimethylamino)propyl]amino]-4-[[2-[2(2-hydroxyethyl)amino]ethyl]amin 
o]-7-methoxy-9H-thioxanthen-9-one from 
1[[(3-dimethylamino)propyl]amino]-7-methoxy-4-aminothioxanthenone. 
1-[[2-(Diethylamino)ethyl]amino]-4-[[2-[(2-hydroxyethyl)amino]ethyl]amino]- 
6-methoxy-9H-thioxanthen-9-one from 
1-[[(2-diethylamino)ethyl]amino]-6-methoxy-4-aminothioxanthenone. 
EXAMPLE 2 
4-[(2-Aminoethyl)amino]-1-[[2-(dimethylamino)ethyl]amino]-9H-thioxanthen-9- 
one, hydrochloride 
A mixture of 4.0 g (0.013 mole) of 
1-[[2-dimethylamino)ethyl]-amino]-4-aminothioxanthenone and 8.0 g (0.039 
mole) of 2-bromoethylamine hydrobromide in 90 ml of ethanol was heated 
under reflux for three days. A further 8 g of 2-bromoethylamine 
hydrobromide and 10 ml of water was added and heating continued for four 
days. The reaction mixture was concentrated in vacuo and 50 ml of ethanol 
was added. The solid was collected, washed with ethanol and dried in vacuo 
at 60.degree. to give 7 g of red solid. This material was dissolved in 200 
ml of water, filtered, and 26 ml of 2NNH.sub.4 OH was added. The mixture 
was extracted with methylene chloride dried over MgSO.sub.4 and 
concentrated in vacuo to a red oil which solidified (4 g). Purification by 
flash chromatography over 240 g of silica gel in a 100 mm column and 
elution with CH.sub.2 Cl.sub.2.MeOH (1:1) to remove starting material and 
then with CH.sub.2 Cl.sub.2 /MeOH/Et.sub.3 N (49:49:2) provided 2.3 g of 
the product. The solid was dissolved in 90 ml of ethanol and treated with 
2-propanol saturated with gaseous hydrogen chloride to provide 2.5 g of 
the product, mp 264.degree. (dec). 
Similarly may be prepared: 
4-[(2Aminoethyl)amino]-1-[[3-(dimethylamino)propyl]amino]-9H-thioxanthen-9- 
one from 1-[[(3-dimethylamino)propyl]amino]-4-amino-thioxanthenone. 
4-[(2-Aminoethyl)amino]-1-[[3-(dimethylamino)propyl]amino]-7-methoxy-9H-thi 
oxanthen-9-one from 
1-[[(3-dimethylamino)propyl]amino]-7-methoxy-4-aminothioxanthenone. 
4-[(2-Aminoethyl)amino]-1-[[2-(diethylamino)ethyl]amino]-6-methoxy-9H-thiox 
anthen-9-one from 
1-[[(2-diethylamino)ethyl]amino]-6-methoxy-4-aminothioxanthenone. 
EXAMPLE 3 
4-[(3-Aminopropyl)amino]-1-[[2-(dimethylamino)ethyl]amino]-9H-thioxanthen-9 
-one, trihydrochloride 
A mixture of 4.0 g (0.013 mole) of 
1-[[2-dimethylamino)ethyl]amino]-4-aminothioxanthenone and 8.5 g (0.039 
mole) of 3-bromopropylamine, hydrobromide in 90 ml of ethanol and 10 ml of 
water was heated under reflux for five days. The mixture was concentrated 
in vacuo and 50 ml of ethanol was added to the residue. The solid was 
collected, washed with cold ethanol and dried in vacuo at 10.degree. to 
give 6.5 g of light orange solid. This material was dissolved in 100 ml of 
water, treated with 26 ml of 2NNH.sub.4 OH and extracted with methylene 
chloride. The extracts were dried over MgSO.sub.4 and concentrated in 
vacuo to give 3.35 g of a dark red solid. Flash chromatography was used 
(200 g silica gel, 60 mm d. column) to provide (CH.sub.2 Cl.sub.2 
:MeOH:Et.sub.3 N) (49:49:2) a dark red solid. The material was dissolved 
in 90 ml of ethanol and treated with 2-propanol saturated with HCl. 
Filtration and drying provided 2.8 g of yellow solid. Recrystallization 
from 200 ml of EtOH-H.sub.2 O (9:1) gave 1.4 g of a yellow solid, mp 
276.degree.-285.degree. (dec). 
Nitro compounds of formula III utilized for the preparation of the 
compounds of formula I are described below. 
EXAMPLE 4 
1-[[2-(dimethylamino)ethyl]amino]-4-nitro-9H-thioxanthen-9-one 
A solution of 9.3 g (0.105 mole) of N,N-dimethylethylenediamine in 35 ml of 
xylene was added dropwise over ten minutes to a stirred mixture of 
1-chloro-4-nitrothioxanthone (24.5 g, 0.084 mole) and K.sub.2 CO.sub.3 
(12.2 g, 0.088 mole) in 350 ml of xylene. The mixture was heated for one 
hour at 70.degree., an additional 1.8 g (0.088 mole) of 
N,N-dimethylethylendiamine was added, and heating was continued for an 
additional hour. The mixture was cooled and the solid was collected, 
washed with cold xylene, petroleum ether, dried, triturated with water, 
and dried in vacuo at 70.degree. to give the product (24.7 g) as a yellow 
solid, mp 177.degree.-179.degree.. 
Treatment of a solution of this material in methanol with excess 
methanesulfonic acid gave the corresponding salt, mp 
242.degree.-246.degree.. 
EXAMPLE 5 
1-[[3-(dimethylamino)propyl]amino]-4-nitro-9H-thioxanthen-9-one 
A solution of 8.5 g (2 equiv) of N,N-dimethylpropylenediamine in 50 ml of 
N,N-dimethylformamide was added in one portion to a suspension of 10 g 
(0.037 mole) of 1-chloro-4-nitrothioxanthone in 100 ml of 
N,N-dimethylformamide. The mixture was stirred at room temperature for 
five hours and then heated on a steam bath for 0.5 hour. The mixture was 
allowed to cool to room temperature and 100 ml of 2-propanol was added. 
Cooling at 0.degree. for two hours gave a solid which was collected, 
washed successively with 2-propanol, water and 2-propanol, and dried in 
vacuo at 60.degree. for 16 hours to afford 10.2 g of the product as a 
yellow solid, mp 105.degree.-108.degree.. 
1-[[2-(Dimethylamino)ethyl]amino]-7-methoxy-4-nitro-9H-thioxanthen-9-one 
was prepared similarly from 8.0 g of 
1-chloro-7-methoxy-4-nitrothioxanthone and 5.6 g of 
N,N-dimethylethylenediamine affording 7.2 g, mp 190.degree.-192.degree.. 
1-[[2-(Dimethylamino)ethyl]amino]-6-methoxy-4-nitro-9H-thioxanthen-9-one 
was prepared similarly from 0.99 g of 
1-chloro-6-methoxy-4-nitrothioxanthone and 0.67 g of 
N,N-diethylethylenediamine affording 0.98 g, mp 144.degree.-145.degree.. 
1-[[2-(Dimethylamino)ethyl]amino]-7-hydroxy-4-nitro-9H-thioxanthen-9-one 
was prepared similarly from 10 g of 1-chloro-7-hydroxy-4-nitrothioxanthone 
and 5.7 g of N,N-dimethylethylenediamine to provide 7.2 g, mp&gt;300.degree.. 
1-[[2-(Diethylamino)ethyl]amino]-7-hydroxy-4-nitro-9H-thioxanthen-9-one was 
prepared similarly from 10 g of 1-chloro-7-hydroxy-4-nitrothioxanthone and 
8.3 g of N,N-diethylethylenediamine to provide 7.3 g as a tan solid which 
was used without further purification. 
The requisite 1-chloro-7-methoxy-4-nitrothioxanthone was prepared as 
follows: A mixture of 118.5 g (0.35 mole) of 
2-(5-chloro-2-nitrophenylthio)-5-methoxybenzoic acid and 131 ml of thionyl 
chloride in 600 ml of toluene was heated under reflux for 1.5 hours. The 
mixture was concentrated in vacuo, and the resulting solid was dissolved 
in 950 ml of nitrobenzene with heating and then cooled to 0.degree. in an 
ice bath. Aluminum chloride (44.2 g) was added in portions over 0.5 hour 
with cooling, and the mixture was then allowed to come to room temperature 
and stirred overnight. The mixture was poured into 5 l of cold water. As 
much of the water layer as possible was decanted. The residue was 
triturated twice with 1 l portions of methanol and then twice with 2 l 
portions of methanol. The oil was triturated with 4 l of methanol and a 
solid resulted. This was washed with methanol and dried in vacuo at 
75.degree. to give 72.7 g of the product as a brownish-yellow solid, mp 
235.degree.-240.degree.. 
The requisite 2-(5-chloro-2-nitrophenylthio)-5-methoxybenzoic acid was 
prepared as follows. A solution of 93.5 g (0.56 mole) of 
2-amino-5-methoxybenzoic acid and 38.6 g (0.56 mole) of sodium nitrite in 
700 ml of water containing 44 ml of 50% aqueous sodium hydroxide (1.05 
mole) was purged with nitrogen and added, under nitrogen, dropwise to a 
stirred mixture of 175 ml of concentrated hydrochloric acid and 230 g of 
ice cooled in an ice-salt bath to -10.degree.. The mixture was allowed to 
stir at 0.degree. for one hour, neutralized to pH 5.1 with potassium 
acetate, and transferred to an addition funnel using nitrogen pressure. 
The addition funnel was attached to a 5 l flask containing a solution of 
potassium ethyl xanthate (259 g, 1.6 mole) in 940 ml of water heated to 
80.degree., and the diazonium salt was added in a thin stream over one 
hour, maintaining the temperature at 75.degree.-80.degree.. To the hot 
reaction mixture was added 97.5 g (0.56 mole) of sodium dithionite, and 
the mixture was then allowed to cool to 20.degree. and acidified to pH 2.4 
under nitrogen with concentrated hydrochloric acid (200 ml). Methylene 
chloride (1.2 l) was added, the mixture was stirred vigorously and the 
insoluble material removed by filtration. Maintaining the solutions under 
nitrogen as much as possible, the methylene chloride layer was separated 
and the aqueous layer was extracted again with methylene chloride (1.2 l). 
The combined extracts were dried over MgSO.sub.4, concentrated in vacuo at 
50.degree., and the crude thiol was warmed with 470 ml of ethanol and 
added under nitrogen to a solution of 107.5 g of 2,4-dichloronitrobenzene 
in sodium ethoxide (prepared from 25.8 g of sodium and 1.1 l of ethanol). 
The mixture was heated under reflux under nitrogen for one hour, cooled, 
and the solvent was removed in vacuo. The residue was taken up in 1 l of 
water and 1 l of ether. The ether layer was removed, and the aqueous 
layer washed two more times with 1 l portions of ether. The aqueous layer 
was acidified with concentrated hydrochloric acid (50 ml), and the solid 
was collected, washed with water and dried in vacuo. The solid (99.2 g) 
was recrystallized from 2.3 l of toluene to give 53.8 g of product, mp 
178.degree.-183.degree., which was sufficiently pure for use in the next 
step. 
The requisite 1-chloro-6-methoxy-4-nitrothioxanthone was prepared as 
follows: To a prewashed (pentane) suspension of NaH in 500 ml of 
tetrahydrofuran cooled to 0.degree. was added dropwise over about 25 
minutes a solution of 35.4 g of 2,6dichloro-5-nitrobenzoic acid in 200 ml 
of tetrahydrofuran. After about five minutes a solution of 20 g of 
m-methoxybenzenethiol in 50 ml of tetrahydrofuran was added, and the 
mixture was stirred for 20 hours as it gradually came to ambient 
temperature. The mixture was treated with a 10% aqueous hydrochloric acid 
solution, extracted into ethyl acetate, dried, and the solvent was removed 
in vacuo. The orange gum was purified by flash chromatography on SiO.sub.2 
with a 90:5:0.5 mixture of methylene chloride:methanol:acetic acid. The 
resulting oil was sufficiently pure for use in the next step. A solution 
of this material (24.8 g) in 120 ml of trifluoroacetic acid was treated 
with 60 ml of trifluoroacetic anhydride and allowed to stir at room 
temperature for 30 minutes. The solution was diluted with acetonitrile, 
and the precipitate was collected and triturated with boiling acetonitrile 
to give 15.8 g of the product, mp 254.degree.-258.degree.. 
The requisite 1-chloro-7-hydroxy-4-nitrothioxanthone was prepared as 
follows: To a suspension of 5.0 g of 
1-chloro-7-methoxy-4-nitro-thioxanthone in 50 ml of methylene chloride was 
added 20 ml of boron tribromide in methylene chloride (1M). The mixture 
was stirred for two days, and an additional 20 ml of boron tribromide was 
added. The mixture was stirred for three hours at room temperature and 
heated under reflux for two hours. An additional 20 ml of boron tribromide 
was added and the mix was heated under reflux for two hours. The mixture 
was cooled in an ice bath and 50 ml of methanol was added carefully. The 
solution was heated under reflux for one hour, and the solvent was removed 
in vacuo. The residue was boiled in about 50 ml of ethanol, collected, and 
retreated with hot ethanol to provide the product as an orange powder, mp 
240.degree.-254.degree. (dec).